Purpose – To provide the author's opinions about key issues in strategy and the future to the readership. Design/methodology/approach – Opinion column. Findings – Discussion of the difficulty of reconciling different personality types in the strategy process. Research limitations/implications – Speculative, and based not on rigorous research but on the author's experience of planning engagements across a wide variety of private and public enterprises. Practical implications – Warns strategists about the difficulties of getting disparate personality types to work together. Originality/value – Expresses several opinions that the author believes have not been expressed (or adequately appreciated) elsewhere.
When relatively large amounts of 57Co B12 were added to serum in vitro (for most persons, over 300 pg. of 57Co B12 per milliliter of serum), some of the labeled vitamin was bound to a fraction eluted from the DEAE-cellulose column with 0.1M sodium phosphate buffer, pH 5.8. This fraction differed from transcobalamin I (TCI) and transcobalamin II (TCII) in molecular size as judged by gel filtration on Sephadex G-200 column, mobility in polyacrylamide gel electrophoresis, and plasma clearance rate in normal persons. The yield of this fraction depended on the amount of 57Co B12 added to serum.
E. coli-gomez (Varela and co-workers, 1946) and E. coli 0111:B4 (Kauffmann and Dupont, 1950) are identical, and at the same time have the Salmonella somatic antigen XXXV. It is interesting that this supposedly pathogenic coliform organism has a major somatic antigen of the Salmonella group.
E. Coli 014 cell walls contain classical specific somatic O antigen and another polysaccharide antigen, the common antigen, which crossreacts with many other Enterobacteriaceae. We have tested the ability of E. Coli 014 to inhibit hemagglutination of A, B, or (H) O human RBC (red blood cells), and we have demonstrated that E. Coli 014 also contains A and B but no (H) O blood-group-active polysaccharide antigens. E. Coli 014 also cross-reacts extensively with E. Coli 086, which is also blood-group active. Since many other Enterobacteriaceae contain common and human blood-group-active antigens, it was thought that only common antigen activity might relate to cross-reactions with blood group antigens. E. Coli 014 specific somatic and common antigen fractions both contain B-like and probably A-like bacterial antigen. A survey of 33 patients with urinary infection (none with E. Coli 014) and 12 normal persons tested against E. Coli 014 indicated that infected persons less often recognized E. Coli 014 A-like antigen than controls, but both groups recognized B-like bacterial antigen equally well. Further studies of this apparent tolerance to A-like bacterial antigen may elucidate selective deficiency in resistance of some human subjects to gram-negative infection.
Hypergastrinemia is known to cause hyperplasia of the gastric mucosa, especially in gastric enterochromaffinlike (ECL) cells. In some clinical conditions causing hypergastrinemia, such as long-term gastric-acid inhibition and gastric-mucosa atrophy, hyperplastic ECL cells may develop into gastric carcinoid tumors. A newly developed gastrin-receptor antagonist, S-0509, has been reported to block gastrin-induced stimulation of gastric-acid secretion. We therefore investigated whether S-0509 inhibits the omeprazole- and gastrin-stimulated hyperproliferation of gastric mucosa, especially of ECL cells. Daily administration of omeprazole and gastrin in male Sprague-Dawley rats induced marked hypergastrinemia and increased proliferation of gastric-mucosa cells. The numbers of ECL cells and of ECL cells producing messenger RNA for regenerating gene, a potent growth factor for gastric-mucosa cells, were also augmented by long-term administration of omeprazole and gastrin. Coadministration of S-0509 with omeprazole or gastrin almost completely inhibited the omeprazole- and gastrin-induced changes in gastric mucosa, including mucosal thickening and ECL hyperplasia. S-0509 did not induce gastric-mucosa atrophy, even when administered for as long as 4 weeks. In summary, we have found that a newly developed gastrin receptor antagonist, S-0509, inhibits omeprazole- and gastrin-induced mucosal hyperplasia, especially ECL-cell hyperplasia, in rats.
From a series of 1,089 bronchospirometric examinations several groups of patients were selected which, on the basis of clinical and roentgenographic findings, were judged to have unilateral pulmonary involvement of particular anatomic type. In 170 cases with unilateral parenchymal disease split function studies showed a slight reduction of respiratory participation of the involved lung with oxygen uptake, resting ventilation, and vital capacity almost equally affected. The degree of impairment was less severe than anticipated from the appearance of the chest roentgenogram. A group of 127 patients with unilateral pleural complications showed a striking diminution of function of the involved lung. Oxygen uptake was most defective, vital capacity was less affected, and ventilation least. In some patients minute ventilation was normal or even increased in spite of severely impaired oxygen uptake. The degree of impairment was more severe than anticipated from the appearance of the chest roentgenogram. Bronchial disease was responsible for an abnormal distribution of function only when a major bronchus was completely or almost completely occluded. Obstruction of one branch of the pulmonary artery had little effect on ventilation or vital capacity but oxygen uptake was severely impaired on the affected side. Bronchospirometric findings in various clinical syndromes and after different types of medical and surgical treatment are discussed in the light of the more general functional patterns. The loss of function after subtotal pulmonary resection was, on the average, equal to that after thoracoplasty of similar extent. The occurrence of pleural complications following resection made the outcome more variable and hence less predictable than the results after collapse. Patients were studied before and after surgery designed to improve pulmonary function. Decortication caused marked increase in function if the collapsed lung was not involved by pre-existing parenchymal disease. Some recovery was found following resection of pulmonary cysts. The functional improvement after bronchial reconstruction was encouraging. The results of autonomic nerve resection for the treatment of bronchial asthma or pulmonary emphysema were not good.
This study compared bone remodeling, bone mineralization, and parathyroid hormone secretion in rats treated with ethane-1-hydroxy-1,1-diphosphonate (EHDP) or dichloromethylene diphosphonate (Cl2MDP). The results were compared with findings in rachitic and control rats. In addition, calcification was induced in the skin by administration of lead acetate intravenously and polymyxin B subcutaneously; the efficacies of EHDP and Cl2MDP in preventing the calcific plaque were compared. Both the serum biochemical values and the morphologic findings indicate that Cl2MDP and EHDP influence bone and mineral metabolism differently. Even with the relatively large doses used in this study, Cl2MDP had a minor or no effect while EHDP had a marked effect that, in general, is undersirable. EHDP caused increase in circulating immunoreactive parathyroid hormone to levels greater than those in rachitic rats. Both diphosphonates appear to have a similar effect on the ectopic mineralization produced by calciphylaxis, suggesting that Cl2MDP may be the agent of choice in this problem because the undersirable side-effects would be avoided.
To determine the mechanism responsible for serum phosphorus elevation during therapy with disodium etidronate (ethane-1-hydroxy-1,1-diphosphonate, EHDPTM), 30 mg. per kilogram EHDPTM was administered daily, orally, in 4 divided doses to 10 adult male volunteers age 22 to 45 for 11 or 12 days. Clearances of calcium, phosphorus, and creatinine were estimated in each of 4 one hour periods on 2 different days before, and on 2 days at the end of therapy while still on the drug. Intravenous bovine parathyroid hormone (PTH) was given at the beginning of the experiment on the second day in each case. Diffusible calcium and phosphorus clearances were determined on 5 of these subjects. The response to 40 units (5 subjec or 200 units (5 subjects) bovine PTH during treatment with EHDPTM30 mg. per kilogram for 11 or 12 days was compared to the response during the control period without treatment. Urine cyclic AMP excretion was also compared in each case. EHDPTM caused a significant rise in total and diffusible serum phosphorus levels with nonsignificant differences between them. Phosphorus clearance was decreased (P < 0.01) but basal phosphorous excretion was unchanged by EHDPTM therapy. The phosphaturia and urine cyclic AMP excretion in response to intravenous bovine PTH, 40 units (5 subjects) or 200 units (5 subjects) was unaltered by EHDPTM therapy. The per cent change in phosphorus clearance in response to intravenous bovine PTH was also unaltered by treatment with EHDPTM. Urine calcium excretion was decreased for 2 hours following bovine PTH injection, both with and without treatment with EHDPTM. The phosphaturic response to 200 units bovine PTH was slightly greater than the response to 40 units, however, the urinary cyclic AMP excretion in response to 200 units bovine PTH was about ten-fold higher than the response to 40 units. It was concluded that EHDPTM causes hyperphosphatemia by increasing tubular reabsorption of phosphorus without causing inhibition of parathyroid hormone action on renal tubules. It is assumed that a mechanism (s) other than PTH exists for control of renal handling of phosphorus and that EHDPTM affects it (them).
This investigation was performed to compare the entry of [3H]water and [14C]PEG into samples of salivary mucus from patients with CF and normal subjects. A solution containing both radioisotopes was added to samples of salivary mucus and mixed, and entry of each into mucus was determined by their dilution in the supernatant at 1, 2, 4, and 24 hr. [3H]water rapidly entered and equilibrated with the water content of both normal and CF mucus. [14C]PEG entered mucus more slowly than [3H]water and by 24 hr had entered at a ratio of only 0.794 +/- 0.108 and 0.766 +/- 0.039 of the water space accessible to [3H]water in normal and CF mucus, respectively. The mechanisms responsible for the partial exclusion of the large polyethylene glycol molecule were not explored but may reflect steric restriction of access to a portion of mucus water. Thus water entry and solute exclusion, both determinants of permeability, were unaltered in CF mucus, which fact provides evidence against the presence of a generalized defect of mucus permeability in this disease. The techniques developed for this study may be adaptable to the study of the permeability properties of mucus from other sources and other gels.
Spontaneously hypertensive rats (SHR) have several abnormalities of calcium metabolism compared with normotensive control Wistar-Kyoto (WKY) rats. Previously the vitamin D metabolite 1,25-dihydroxycholecalciferol (1,25[OH]2D3) was found to be inappropriately low in SHR in view of their ionized hypocalcemia and hyperparathyroidism. We examined the responses of plasma 1,25(OH)2D3 to several known stimuli. Baseline plasma 1,25(OH)2D3 levels tended to be lower in SHR than WKY rats (51.5 +/- 4.3 vs. 82.3 +/- 14.1 pg/ml, P = 0.06). Infusion of a pharmacologic dose of parathyroid hormone (8 U/hr over a period of 17 hours) resulted in a plasma 1,25(OH)2D3 level of 504 +/- 77 pg/ml in SHR vs. 1016 +/- 211 pg/ml in WKY rats (P less than 0.03). Cyclic adenosine monophosphate infusion (1 mumol/hr/100 gm over a period of 17 hours) in thyroparathyroidectomized animals resulted in a 1,25(OH)2D3 level of 121 +/- 24 pg/ml in SHR vs. 557 +/- 26 pg/ml in WKY rats (P less than 0.01). After dietary phosphorus depletion for 3 weeks, SHR also had lower 1,25(OH)2D3 levels than WKY rats (83 +/- 13 vs. 300 +/- 42 pg/ml, P less than 0.001) even though a comparable degree of hypophosphatemia was achieved. Thus, the response of plasma 1,25(OH)2D3 levels to several known stimuli is submaximal in SHR as compared with WKY rats, suggesting defective synthesis or enhanced metabolic clearance of this hormone.
The effect of 2 X 10(-10) to 2 X 10(-7) mol/L 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) and 10(-9) to 10(-6) mol/L 13-cis-retinoic acid on in vitro differentiation and proliferation of marrow cells from patients with myelodysplastic syndrome (MDS) was assessed. Cells from 17 patients were studied by the semisolid technique, and cells from seven patients by both liquid and semisolid cultures. After incubation in liquid culture with 1,25(OH)2D3, in six of seven patients evaluated an increasing number of myeloid cells (185% to 470%) acquired the morphologic appearance of mature monocyte-macrophages, and a decrease in the number of immature myeloid cells (26% to 75%) was observed. Phagocytosis and killing of Candida albicans by monocyte-macrophages incubated with 1,25(OH)2D3 were normal and similar to those processes in untreated cells. 1,25(OH)2D3 increased the percentage of monocytes that phagocytosed C. albicans in three patients. Thirteen of 17 patients showed reduced myeloid cloning, and eight showed increased cluster formation. Cloning efficiency was significantly lower in patients with refractory anemia with excess of blasts and chronic myelomonocytic leukemia. Concentrations of 2 X 10(-9) to 2 X 10(-8) mol/L 1,25(OH)2D3 and 10(-8) to 10(-7) mol/L retinoic acid had a stimulatory effect on myeloid colony growth in five of the six patients with sideroblastic and refractory anemia, but in only two of the 11 patients with refractory anemia with excess of blasts and chronic myelomonocytic leukemia. The results indicate that the differentiation pattern of myeloid precursor cells from a subset of patients with MDS was altered by exposure to 1,25(OH)2D3.
We observed that inhibitors of RNA synthesis, actinomycin D and cordycepin, stimulate chick duodenal alk Pase activity by means which are additive to the stimulaation by the biologically active metabolite of vitamin D3, 1,12(OH)2D3, and its analogue, 1 alpha OH D3. The protein synthesis inhibitor, cycloheximide, inhibited basal alk Pase activity and blocked its stimulation by actinomycin D and 1,25(OH)2D3. Both actinomycin D and cycloheximide blocked the ability of 1,25(OH)2D3 and 1 alpha OHD3 to raise serum calcium levels. The paradoxical stimulation of duodenal alk Pase activity by RNA synthesis inhibitors additive to the stimulation by 1,25(OH)2D3 suggests that alk Pase activity is controlled by mechanisms other than gene activation. (J LAB CLIN MED 94:88, 1979.)
The conditions requisite for biological expression of the renal tubular actions of 1,25 DHCC were investigated in the vitamin D-depleted, TPTX rat. When infused alone, 1 U/hr 1,25 DHCC for 6 hr had no effect on UpV, UNaV or UcaV, nor was there any alteration in the excretion of AMP. However, the infusion of this dose of 1,25 DHCC along with an amount of purified PTH (0.2 U/hr) which itself has no biological effect in this model system resulted in a reduction in UpV from 7.11 ± 0.87 to 3.41 ± 0.92 μg/min (p < 0.001) and in UNaV from 0.35 ± 0.06 to 0.28 ± 0.06 μEq/min (p < 0.02) and a decline in ucAMPV from 35.8 ± 6.5 to 28.0 ± 6.9 pmol/min (p < 0.05). The infusion of 0.011 mU/min VP resulted in no change in electrolyte excretion, but an antidiuresis did occur: V fell from 0.021 ± 0.001 to 0.007 ± 0.002 ml/min (p < 0.001). When VP administration and 1,25 DHCC infusion were combined, an antiphosphaturia developed (UpV fell from 8.73 ± 1.48 to 5.18 ± 1.13 μg/min, p < 0.01), associated with an antidiuresis (V declined from 0.018 ± 0.000 to 0.007 ± 0.002 ml/min, p < 0.001) and a numerical rise in ucAMPV (from 25.6 ± 9.9 to 54.8 ± 17.8 pmol/min) which did not quite reach statistical significance (p > 0.05). We conclude from these experimental observations that both PTH and VP may act as "permissive" substances for the antiphosphaturic effects of 1,25 DHCC to become manifest. It is postulated that the metabolite can affect reabsorption at more than one nephron transport site or can influence transport which is mediated by more than one mechanism.
Anephric patients have undetectable plasma concentrations of 1,25-(OH)2-D, but when anephric patients are treated with DHT2 (Hytakerol), their plasma contains a substance that is co-purified with and displaces authentic 3H-1,25(OH)2-D3 from its intestinal cytoplasmic receptor. The concentration of this substance in the plasma of anephric patients taking DHT2 is proportional to the administered daily dose of DHT2 per kilogram body weight. When DHT2 therapy is discontinued, the substance disappears from plasma with an average t 1/2 of about 8 days.
19-Nor-1,25-dihydroxyvitamin D(2) (19-norD(2)) a less calcemic and phosphatemic analog of 1,25-dihydroxyvitamin D (1,25[OH](2)D(3)), is approved for the treatment of secondary hyperparathyroidism in patients with kidney failure. We have previously demonstrated that 19-norD(2) is less active than 1,25(OH)(2)D(3) in stimulating bone resorption. In this study, we compared the potencies of 19-norD(2) and 1,25(OH)(2)D(3) in stimulating net calcium and phosphate absorption in the intestine. Mineral balance was assessed in normal rats during the last 4 days of a 14-day treatment with various daily doses of 19-norD(2) or 1,25(OH)(2)D(3). Calcium absorption increased from 16.5% +/- 7.8% in vehicle-treated rats to 27.5% +/- 7.2% in rats given 10 ng/day 1,25(OH)(2)D(3) and to 21.6% +/- 3.9%, 26.2% +/- 5.5%, and 27.4% +/- 5.1% in rats treated with 10, 50, and 100 ng/day 19-norD(2), respectively. Thus comparable stimulation of calcium transport was attained with 10 ng 1,25(OH)(2)D(3) and 100 ng 19-norD(2). Similar results were obtained for phosphate absorption, with an increase from 28.2% +/- 5.5% in vehicle-treated rats to 40.2% +/- 4.7% in rats given 10 ng/day 1,25(OH)(2)D(3) and to 32.9% +/- 2.2%, 36.2% +/- 4.5%, and 36.8% +/- 3.8% in rats given 10, 50, and 100 ng/day 19-norD(2), respectively. Vitamin D compounds are believed to increase calcium absorption by inducing a calcium channel (epithelial calcium transporter or calcium transporter-1 [CaT1]) on the luminal membrane, a calcium-binding protein (Calbindin D9k) in the cytosol, and a calcium pump (plasma membrane calcium adenosine triphosphatase-1 [PMCA1]) on the basolateral membrane. Northern-blot analysis of intestinal ribonucleic acid of vitamin D-deficient rats given seven daily injections of vehicle or 100 ng 1,25(OH)(2)D(3) or 19-norD(2) revealed that 19-norD(2) was less potent than 1,25(OH)(2)D(3) in stimulating expression of CaT1, Calbindin D9k and PMCA1. In summary, the reduced calcemic and phosphatemic activities of 19-norD(2) can be attributed to lower potency in stimulating intestinal calcium and phosphate absorption.
It has been previously found that activation of human lymphocytes in vitro causes the expression of receptors for the hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and that 1,25(OH)2D3 has immunoregulatory properties including the ability to inhibit interleukin-2, to suppress lymphocyte proliferation, and to inhibit antibody production. In the present study we found that 13 of a group of 17 (76%) seropositive patients with rheumatoid arthritis had lymphocytes that possessed 1,25(OH)2D3 receptors (without activation in vitro) compared with only three of 17 (18%) normal individuals. The biochemical characteristics of the 1,25(OH)2D3 receptor, including affinity, sedimentation coefficient, and DNA-binding properties in the rheumatoid arthritis lymphocytes were indistinguishable from those established for this receptor in the classic target tissue of the hormone. This finding raises the possibility that 1,25(OH)2D3, acting through its receptor, might play a previously unsuspected role on lymphocytes of patients with rheumatoid arthritis.
Decreased production of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) by the kidney may be responsible for vitamin D resistance in renal failure and contribute to renal osteodystrophy. The present studies investigate the effect of reduced renal mass and reduced renal function on conversion of 3H-25-hydroxycholecalciferol to 3H-1,25-(OH)2D3 in vitamin D-deficient rats. Renal mass was reduced by partial or total nephrectomy. Renal function was reduced by ureteral ligation. At comparable degrees of renal function, reducing renal mass decreased 1,25-(OH)2D3 formation approximately proportional to the amount of renal tissue removed. Ureteral ligation without reduction of renal mass also reduced production within the first 24 hours. This may have been caused by the marked hyperphosphatemia which occurred with ureteral ligation. When partial nephrectomy and ureteral ligation were done together the results were additive. The decreased 1,25-(OH)2D3 formation produced by unilateral nephrectomy was not improved 4 weeks after surgery, although renal function had returned to normal.
One hundren seventy-eight azotemic patients, 114 on hemodialysis, had measurements of total serum ALP, and definition of isoenzyme patterns on acrylamide gel electrophoresis. In addition, bone histomorphometry was defined in all of the patients by means of transiliac bone biopsies. Serial estimations over 2 years were carried out on several patients, including some being treated with vitamin D2, 1alphaOHD3, and 1,25(OH)2D3. (1) In both nondialyzed and dialyzed patients, serum ALP showed a significant positive correlation with osteitis fibrosa due to secondary hyperparathyroidism irrespective of the presence or absence of concurrent osteomalacia. Increases in the bone isoenzyme were largely responsible for the rise in total ALP. (2) A higher incidence of osteomalacia (p less than 0.001) was observed in patients on hemodialysis in Newcastle Upon Tyne. In hemodialyzed patients where osteomalacia was accompanied by either no secondary hyperparathyroidism (21 patients) or minimal secondary hyperparathyroidism (14 patients), serum ALP remained within normal limits, giving no indication of the existing osteomalacic bone disease. Isoenzyme studies revealed a high prevalence of the intestinal type and also varied combinations of hepatic, intestinal, and bone types. (3) Good response to vitamin D depended on the presence of significant amounts of the bone isoenzyme. Azotemic osteodystrophy characterized by a raised serum ALP and a prominent bone isoenzyme predicted a good response to vitamin D, and the decrease in serum ALP following vitamin D was the result of a reduction in the bone isoenzyme. Patients with symptomatic dialysis osteomalacic bone disease, accompanied by normal total serum ALP and no elevation of the bone isoenzyme, responded less well.
This study evaluates the role of vitamin D metabolites in the genesis of the skeletal resistance to the calcemic action of PTH in uremia. The changes in serum calcium after infusion of 2 U of PTE per kilogram per hour for 8 hr were evaluated in thyroparathyroidectomized dogs before and after 1 and 3 days of acute uremia produced by bilateral nephrectomy. The animals received vitamin D metabolites during the 3 days of uremia. Supplementation of 0.68 microgram/day 1,25(OH)2D3 and 24R,25(OH)2D3 restored the calcemic response to PTE to normal. This is in contrast to only partial correction of the response to PTE by 1,25(OH)2D3 alone. Administration of 1.36 microgram/day 24R,25(OH)2D3 did not improve the calcemic response to PTE. The results indicate that (1) both 1,25(OH)2D3 and 24R,25(OH)2D3 are necessary for the complete reversal of the impaired calcemic response to PTE, (2) this effect is not due to the increase in the amount of the dihydroxylated compounds of vitamin D, since equivalent amounts of these compounds in the form of 24R,25(OH)2D3 alone had no effect, and (3) the better effect of the combination of 1,25(OH)2D3 and 24R,25(OH)2D3 is most probably due to an interaction between these two metabolites of vitamin D permitting an intact calcemic action of PTH.
Impairment in the stimulation of renal production of 1,25-dihydroxyvitamin D[1,25 (OH)2D] by parathyroid hormone (PTH) occurs in diabetes. Renal response to PTH in terms of 25-hydroxyvitamin D-1-hydroxylase (1-OHase) stimulation involves increased cyclic adenosine monophosphate (cAMP) production, increased cAMP-dependent protein kinase activity, and dephosphorylation of renal ferredoxin (renoredoxin). To identify the step where diabetes might impair PTH stimulation of 1-OHase, we studied the effects of PTH on 1,25(OH)2D production, cAMP content, cAMP-dependent protein kinase activity, and the phosphorylation state of renoredoxin by using renal slices from diabetic and nondiabetic rats. PTH and forskolin significantly stimulated 1,25(OH)2D production in renal slices from nondiabetic animals but not from diabetic animals. PTH-stimulated cAMP production and cAMP-dependent protein kinase activity in renal slices were not altered by diabetes. However, diabetes significantly impaired the capacity of PTH to dephosphorylate renoredoxin and to increase the activity of the 1-OHase enzyme complex. These results suggest that the decreased capacity of PTH to stimulate 1-OHase activity in diabetic animals may reflect the decreased capacity of PTH to alter the phosphorylation state of renoredoxin in these animals.
Within 6 hr of the intravenous administration of radiolabeled 1,25(OH)2D3 to five normal vitamin D-replete human subjects, 15.6% of the injected dose appeared in bile as polar metabolites of 1,25(OH)2D3. Of the injected dose, 27% and 7.5% appeared in the feces and urine, respectively, at 24 hr. In another two subjects, biliary radioactivity was sampled at two jejunal sites separated by a distance of 40 cm; a 24.8% decrease in radioactivity over this segment of bowel was noted. These data demonstrate that products of 1,25(OH)2D3 are excreted in normal human bile. Furthermore, these products are reabsorbed as such or as free 1,25(OH)2D3 in the intestine and re-excreted as polar products in bile. Our data suggest that there is an enterohepatic circulation of the products of 1,25(OH)2D3 in normal man.
We examined the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in both hypercalcemic and hypocalcemic rat models and the effect of exogenous 25-hydroxyvitamin D3 (25(OH)D3) on serum and tissue aluminum (Al) burdens. Rats fed a 0.2% Al diet received daily subcutaneous injections of either 1,25(OH)2D3 (80.9 ng/kg, n = 5 and 809 ng/kg, n = 8), 25 (OH)D3 (809 ng/kg, n = 4, and 8090 ng/kg, n = 8) or propylene glycol vehicle for 18 days. Rats given 809 ng/kg of 1,25(OH)2D3 were hypercalcemic and when compared with pair-fed controls had higher serum (33.1 vs. 14.3 μg/L, P < 0.01), bone (21.2 vs. 13.2 μg/gm, P < 0,01), and kidney (6.5 vs. 2.0 μg/gm, P < 0.01) but not brain (1.2 vs. 1.5 μg/gm) or liver (0.9 vs. 0.8 μg/gm dry tissue) Al concentration. The lower dose of 1,25(OH)2D3 had no effect on serum or tissue Al. Treatment with 25(OH)D3 did hot increase serum Ca and Al or tissue Al concentration. To dissociate a specific effect of exogenous 1,25(OH)2D3 from the concurrent hypercalcemia, endogenous production of 1,25(OH)2D3 was stimulated. Animals were fed a low Ca diet until hypocalcemia developed and were then divided into four groups: one given low Ca (n = 7) for 21 days, one given low Ca plus 0.2% Al (n = 7) for 21 days, one returned to a normal Ca diet (n = 4) for 30 days, and one returned to a normal Ca diet for 9 days and continued with a normal diet plus 0.2% Al (n = 5) for 21 days. Hypocalcemic rats fed the Al diet, when compared with hypocalcemic controls, had higher serum (143.6 vs. 31.8 μg/L, p < 0.01), bone (16.0 vs. 2.9 μg/gm, P < 0.01), and kidney (8.2 vs. 2.8 μg/gm, P < 0.005) but not brain (3.4 vs. 2.3 μg/gm) or liver (3.8 vs. 2.3 μg/gm) Al concentrations. Serum, bone, and kidney Al concentration was also significantly higher than that in normocalcemic rats fed the Al diet. These results indicate that pharmacologic doses of 1,25(OH)2D3 and dietary hypocalcemia enhance gastrointestinal Al absorption and serum, kidney, and bone Al concentration.
We have studied the effect of 1,25-dihydroxyvitamin D3 (1,25, (OH)2D3) on mesangial cell growth. Previous studies have shown that the monocyte-macrophage is the principal effector cell in immune-mediated nephritis; this cell infiltrates the glomerular mesangium, and its products may have important effects on the physiology of the mesangial cell. One of the substances produced by the activated macrophage is 1,25,(OH)2D3. We have investigated the effect of 1,25,(OH)2D3 on mesangial cell growth and found that this vitamin D metabolite suppresses the proliferation of mouse mesangial cells as assessed by mesangial cell tritiated thymidine uptake and by cell counts; this substance also antagonizes the mitogenic effect of epidermal growth factor on mesangial cell growth. By comparison, the vitamin D metabolite 25 hydroxyvitamin D3 has no significant suppressive effect on the proliferation of mesangial cells. It has also been possible to demonstrate that 1,25,(OH)2D3 could suppress the growth of mesangial cells that had been committed to proliferate by the prior addition of epidermal growth factor. The results of these studies are relevant to our understanding of the pathogenesis of the cellular abnormalities that occur in immune-mediated nephritis, and especially in subjects who have concurrent hypertension, because a segment of subjects with hypertension have demonstrable abnormalities in the levels of circulating 1,25,(OH)2D3.
We measured in vitro 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) production by kidney proximal tubules prepared by Percoll density centrifugation from male and female rats. 1,25(OH)2D3 in tubule extracts was determined by a sensitive and specific radioreceptor assay. Ingestion of diets adequate in vitamin D3 and containing either normal calcium (1.2% Ca, NC), reduced calcium (0.6% Ca, RCD) or low calcium (0.002% Ca, LCD) increased 1,25(OH)2D3 net synthesis (for male rats, NC vs. RCD vs. LCD 1.8 +/- 0.1 SEM vs. 9 +/- 2 vs. 17 +/- 2 pmol/mg protein/20 min; P less than 0.05 for all comparisons). At either level of reduced calcium intake, tubules from male rats produced more 1,25(OH)2D3 than tubules from females. Serum 1,25(OH)2D3 and tubule cyclic adenosine monophosphate (cAMP) content rose in parallel with progressive dietary calcium restriction, and males had higher circulating 1,25(OH)2D3 and tubule cAMP content than females at each level of reduced calcium intake. L-Epinephrine (10(-4) mol/L), in vitro, increased tubule accumulation of 1,25(OH)2D3 and cAMP. Yohimbine, and alpha 2-receptor antagonist, blocked this response, whereas prazosin was without effect. Increased 1,25(OH)2D3 net synthesis by tubules from male vs. female rats partly explains the higher serum levels and enhanced mineral conservation demonstrated previously in male rats. Preparation of proximal tubules from vitamin D-replete rats permits studies in vitro of 1,25(OH)2D3 production and regulation under more physiologic conditions in which parathyroid hormone, inorganic phosphorus, and calcium may be varied independently.
Parathyroid hormone and calcitonin, both endocrine modulators of calcium homeostasis, may influence blood rheology. Parathyroid hormone is known to reduce erythrocyte survival, leading to anemia. Calcitonin has been found to have some vascular effects. We have analyzed the Influence of parathyroid hormone (10(-7) to 10(-10) mol/L), calcitonin (10(-6) to 10(-12) mol/L), 1,25(OH)2 cholecalciferol (10(-7) to 10(-10) mol/L), additional calcium in plasma (+1 and 2 mmol/L), and the calcium lonophore A23187 (50 micromol/L) on erythrocyte morphology and blood viscosity at high shear rate (94 s(-1)) and low shear rate (0.1 s(-1)) in vitro. The loading of erythrocytes with calcium by the ionophore A23187 produced a marked echinocytic shape transformation, an increased blood viscosity at high shear rate caused by decreased deformability of these cells, and a decreased viscosity at low shear rate caused by decreased aggregation of echinocytes. In contrast, increasing plasma calcium concentrations, parathyroid hormone, calcitonin, and 1,25(OH)2 vitamin D3 had no effect on erythrocyte morphology and blood viscosity. We conclude that an increase in intraerythrocytic calcium leads to severe echinocytosis and altered blood viscosity. The endocrine modulators of calcium homeostasis--namely, parathyroid hormone, calcitonin, and 1,25(OH)2 vitamin D3--apparently do not influence intraerythrocytic calcium to a significant degree and have, therefore, no influence on cell morphology and blood viscosity.
The effect of tissue age on in vitro incorporation of glycerol-1,3-C14 into phospholipids and glycerides by polyvinyl sponge granulomas has been studied by utilizing tissue slices 7 to 450 days of age. Similar studies were carried out with open wound granulomas 12 to 14 days of age. Maximum incorporation of isotope occurred with 14 day sponge granulomas, decreased progressively during the period of greatest cellularity (25 to 42 days) and was minimal in the oldest granulomas (360 to 450 days). The specific activities of phosphatidylcholine and phosphatidylethanolamine were highest in 14 day tissue, and decreased in parallel with the specific activity of the total tissue phosphatides. That of monophosphoinositide increased independently, reaching its maximum in 26 day granulomas. Alterations in the tissue composition of individual phospholipids occurred in granulomas 7 and 14 days of age. The distribution of radioactivity in neutral lipids and phospholipids also changed as a function of tissue age. Approximately 90 per cent of isotope incorporation in neutral lipids was found in the triglycerides as compared to the monoglycerides plus diglycerides. No transfer of radioactivity into sterols or sphingomyelin was detected. In situ formation of lipid by cells of experimentally induced connective tissues was directly influenced by granulation tissue age.
We extended our previous studies of the selectivity and mechanism of action as an enzyme inhibitor of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an antitumor drug now widely used to inactivate glutathione reductase (GSSG-R) experimentally. In contrast to other enzymes examined so far, lipoamide dehydrogenase (LSSLNH2-D) was, like its genetic relative GSSG-R, also strongly inhibited by BCNU. The drug concentration needed to inactivate GSSG-R and LSSLNH2-D was much smaller than that affecting the least resistant of five other flavoenzymes tested. When oxidized, both GSSG-R and LSSLNH2-D were resistant to BCNU, and to be effective, the drug had to interact directly with enzyme protein reduced by its specific pyridine nucleotide. In intact human erythrocytes, GSSG-R was mostly reduced and LSSLNH2-D activity undetectable. The partial genetic homology of GSSG-R and LSSLNH2-D and their special sensitivity to BCNU provided a unique opportunity to define more exactly the site of drug-enzyme interaction through comparative coenzyme studies combined with direct and reciprocal substrate competition experiments. The results, together with earlier data on the prevention of BCNU inhibition by cysteine, indicate that the nitrosourea achieves its relative selectivity against the two related flavoenzymes by interacting with at least one of the two reduced cysteinyls located within their oxidoredox active site. For GSSG-R, the attacked cysteinyl is most probably Cys-58.
Human platelets exposed in vitro to increasing amounts of BCNU rapidly develop a progressive, relatively selective, and almost complete deficiency of GSSG-R activity. Several other enzymes are not inhibited when intact platelets are exposed to the nitrosourea; lipoamide dehydrogenase was investigated because of the remarkable similarity of the structure of its active site with that of GSSG-R. BCNU inhibits lipoamide dehydrogenase and GSSG-R only when they are in the reduced state; in the intact platelet, lipoamide dehydrogenase (unlike GSSG-R) is oxidized and is therefore unaffected. This is the first documentation of lipoamide dehydrogenase activity in platelets. After BCNU exposure, there is a reduced release of 14C-serotonin in response to collagen; the cells become incapable of aggregating in response to even large doses of epinephrine, ADP, collagen, or arachidonic acid, with loss of both primary and secondary waves of aggregation. At higher doses of BCNU, there is also a diminished PF-3 activity of intact platelets; sonication of drug-treated platelets normalizes coagulant activity. The drug-induced functional abnormalities occur despite preservation of the number of platelets, their electron microscopic appearance, and their capacity to take up 14C-serotonin. BCNU induced GSSG-R deficiency precedes the development of the earliest evidence of platelet dysfunction, and almost all of the enzyme's activity must be abolished before any functional abnormality becomes detectable. A small fraction of GSSG-R activity is essential for platelet function, and BCNU provides a powerful new tool to investigate the role of the enzymatic reduction of glutathione in platelet physiology and pathology.
The effects of acellular hemoglobin-based oxygen carriers in preclinical models of sepsis and endotoxemia have been inconclusive with regard to outcomes reported for survival. In the present study, mice were infused with 1 gm/kg of recombinant human hemoglobin, rHb1.1, and the effects on mortality and systemic tumor necrosis factor (TNF) and interleukin-6 (IL-6) levels were determined by using both lethal and sublethal bolus endotoxin challenge. Pretreatment of mice with rHb1.1 and challenge with 20 mg/kg of lipopolysaccharide (LPS) at an LD100 resulted in a 100% mortality rate by 20 hours, whereas the same mortality rate with the vehicle or 5% albumin groups occurred at 50 hours. Mice challenged with lower LPS concentrations of 10 and 2.5 mg/kg, corresponding to LD15 and LD0, respectively, had 100% and 17% mortality rates in the rHb group and 17% and 0% mortality rates in the vehicle-treated animals. These doses of LPS resulted in maximal increases in systemic TNF, and there were only modest differences between the rHb and the vehicle groups at LPS challenge doses of 2.5 and 20 mg/kg, whereas no difference was observed at the 10 mg/kg concentration. At LPS concentrations below 10 microg/kg, the increases in circulating TNF were dose dependent and no differences were observed in serum TNF levels between the rHb1.1 and vehicle groups. In addition, there were generally no differences in IL-6 levels between the experimental groups, although at 10 mg/kg LPS, a twofold increase in plasma IL-6 levels over those in the controls was observed in the rHb1.1-treated animals. Infusion of rHb1.1 alone did not induce any increase in circulating IL-6 or TNF. These data demonstrate that endotoxin exacerbation, although apparent, was observed only at the highest doses of LPS and that at lower concentrations, there were no differences in the extent of cytokine elevation or in survival rate when rHb1.1-, albumin-, or vehicle-pretreated animals were compared.
In the assay for glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase, it is shown that hemolysis with saponin or digitonin gives somewhat higher values than those obtained with hemolysis by water. These hemolytic agents apparently free the enzymes from the stroma while hemolysis in water accomplishes this only to a partial and variable degree. This fact leads to a simplified assay in which washed red cells are pipetted into a saponin solution in the cell of the ultraviolet spectrophotometer and combined reagents (which can be stored frozen) are added. The generation of NADPH, followed at 340 mμ, is linear for several minutes. The stoichiometry of the two reactions has been carefully rechecked, and it is concluded that the only valid measurement of glucose-6-phosphate dehydrogenase activity is obtained by subtracting the activity with 6PG as substrate from the activity with G6P and 6PG as combined substrate. The second reaction is roughly the speed of the first reaction but varies from individual to individual.
Earlier we found a high percentage of subnormal total glutathione (G(T)) levels in blood from elderly subjects and patients with chronic diseases. These findings suggested a hypothesis that high levels of G(T) in the blood occur in old persons who are in excellent physical and mental health. To this end, we recruited 87 white women who ranged in age from 60 to 103 years and reported that they felt healthy. Their health was verified with physical examinations, clinical chemistry profiles, psychosocial assessments, and blood G(T) determinations. This evaluation was performed in three waves over a 5-year period. The values were compared with those from representative individuals in this region and with normal national data. The results verified that these healthy subjects were in top physical and mental health. We also found that subjects of all ages had very high blood G(T) levels in waves I and II but only normal levels in wave III. These findings confirm that high blood G(T) concentrations and excellent physical and mental health are characteristics of long-lived women.
In the plasma, lysophosphatidylcholine (LPC) is formed by the action of lecithin-cholesterol acyltransferase (LCAT) when a fatty acid is removed from plasma phosphatidylcholine (PC) and transferred to cholesterol. To determine whether plasma LPC might also be generated by the hydrolysis of hepatic PC, we assessed phospholipid production by the isolated perfused rat liver. Bile duct-cannulated livers were perfused with bile salt and a recirculating, lipid-free medium containing albumin. We found that LPC accumulated in the perfusate to a greater extent than any other phospholipid, exceeding the accumulation of PC (the second most prevalent phospholipid) twofold. We further found that perfusate LPC was not formed by hydrolysis of PC in the perfusate and was not dependent on the presence of infused bile salt. LPC that accumulated in the perfusate was highly unsaturated and markedly dissimilar to the more saturated LPC that results from the activity of LCAT. Results thus indicate that the isolated liver directly secretes LPC, which is presumably generated from hydrolysis of hepatic PC. Because plasma LPC is to a great extent unsaturated in the live rat, these findings suggest that direct hepatic secretion is a quantitatively important source of plasma LPC.
Postoperative hemorrhage remains a serious complication in cardiopulmonary bypass (CPB) surgery. In our study, alternative anticoagulation with a new low molecular weight (LMW) heparinoid (Org 10172) was compared with a standardized heparin regimen. A preliminary dose-finding study indicated the minimal effective heparinoid dose to be 260 anti-Xa U/kg body weight, which was comparable to the standardized heparin regime, as revealed by similar plasma anti-Xa values. The following randomized open pilot study in 12 mongrel dogs undergoing CPB showed the heparinoid to be as effective as heparin, with an additional advantageous decrease in postoperative blood loss in the Org 10172 group. Our randomized blind study in 16 mongrel dogs undergoing CPB was performed to confirm previous results. Both antithrombotic agents were effective in the prevention of clot formation within the extracorporeal circuit. Hematocrit values and erythrocyte and platelet counts showed no significant intergroup differences. Post-CPB leukocyte counts revealed a significantly more rapid increase in the group given heparinoid (P less than 0.05). In the group given heparin, the expected prolongations of both the thrombin time (TT) and activated partial thromboplastin time (APTT) were noted, whereas in the group given heparinoid, only a transient peak prolongation of the TT after dose administration was revealed, and no significant prolongation of the APTT. Mean anti-Xa plasma levels were similar during CPB, showing a rapid decrease in the group given heparin on protamine administration, as did the APTT. Assessment of the operating field indicated an elevated intraoperative blood loss in the group given heparin. Postoperative blood loss measured over a period of 2.5 hours after closure of the thorax was significantly lower in the group given heparinoid than in the heparinized animals (625 +/- 100.0 ml, mean +/- SD, and 806 +/- 178.2 ml, respectively; P less than 0.05). Our observations suggest that the LMW heparinoid Org 10172 has an increased benefit/risk ratio over standard heparin and is effective in CPB in dogs. Additional investigations in humans should verify the possibility of use of this substance as an alternative means of anticoagulation during CPB in patients in whom heparin is relatively contraindicated.
Plasma concentrations of corticotropin (ACTH), growth hormone (GH), and 11-OHCS (hydroxycorticosteroid) were determined every 15 minutes in 7 patients during major surgery. GH and 11-OHCS were determined by radioimmunoassay and fluorometry, respectively. ACTH was measured by radioimmunoassay using an antiserum reactive to 1 to 24 amino acid sequence of ACTH molecule, with which the data were consistent with the known deviation of pituitary-adrenal function in patients with pituitary-adrenal dysfunction. Plasma ACTH concentration fluctuates rather rapidly during surgical operation. The first peak was noticed between 15 and 45 minutes after incision. The maximum ACTH concentration of these patients ranged from 110 to 1,000 pg. per milliliter with a mean of 596 pg. per milliliter. Plasma 11-OHCS could not fully reflect ACTH concentration in plasma. The first peak of GH appeared 15 to 45 minutes behind that of ACTH in 6 of 7 patients examined; the secretion patterns of these hormones were quite different, suggesting the dichotomy in the regulatory mechanism of these hormones during surgical stress.