Acta biochimica Polonica

1. L-Asparaginase has been isolated from aerobically grown Escherichia coli 055:B5 and purified about 140-fold in a three-step procedure involving acidification to pH 4.5, ammonium sulphate fractionation and column chromatography on DEAE-Sephadex A-50. The activity of the preparation is 140 U/mg protein. 2. The enzyme acts within a broad pH range (pH 5-9) and is affected neither by PCMB, N-ethylmaleimide nor metal ions. 3. Molecular weight of the isolated asparaginase is 130 000.

The influence of nine newly synthesized uracil acyclonucleosides, and 36 derivatives of 1,2,3,4-tetrahydroisoquinoline on the activity of enzymes catalysing dTMP and dGMP synthesis, on the content of dTTP and dGTP in acid soluble fraction and on the incorporation of [14C]dThd and [14C ]dGuo into DNA in tumour homogenates was studied. The influence of the compounds was studied in the cytosol from intraoperatively excised human tumours - neurofibrosarcoma and ovarian cancer. It was shown that dTMP and dGMP synthesis is inhibited competitively by 34.1+/-4.0% in both types of tumours by 0.2 mM 1-N-(3'-hydroxypropyl)-6-methyluracil (1) and 0.2 mM 1-N-(3'-hydroxypropyl)- 5,6- tetramethyleneuracil (2). The mentioned acyclonucleosides reduced the content of dTTP and dGTP in the acid soluble fraction of tumours (59.7+/-3.1% of control). 1-(4-chlorophenyl)-6,7-dihydroxy- 1,2,3,4-tetrahydroisoquinoline (3), 1-(2,3-dichlorophenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (4) and 1-(3-methoxyphenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (5) at 0.2 mM concentration caused a mixed type inhibition of the synthesis of dTMP and dGMP by, on average, 33.2+/-4.4%, and reduced the content of dTTP and dGTP in the acid soluble fraction (52.6+/-3.7% of control) but were active only in the cytosol of neurofibrosarcoma. While acyclonucleosides undergo phosphorylation in the cytosol by cellular kinases, with their triphosphates being active acyclonucleoside metabolites, active 1,3,4,5-tetrahydroisoquinoline derivatives (compounds not containing a deoxyribose moiety), cannot be phosphorylated. ACN and THI derivatives which inhibit dThd and dCyd kinase activities, inhibit also the incorporation of [14C]dThd and [14C]dGuo (ACN - 50.2+/-2.7%, THI - 53.4+/-3.9% of incorporation inhibition) into tumour DNA. The obtained results point to the mechanism of uracil acyclonucleosides and 1,2,3,4-tetrahydroisoquinoline biological activity consisting in inhibiting the synthesis of DNA components.

Acetylation with acetic anhydride of methyl 5-amino-1H-[1,2,4]triazole-3-carboxylate, one of the hetareneamino acids, was studied using HPLC, H NMR, FTIR and GC-MS. The compound has a significantly decreased susceptibility to acetylation compared to 5-amino-1H-[1,2,4]triazole itself. Two isomeric diacetylated products were found.

In the presented study the ribavirin-TP--an established inhibitor of the NTPase activity of the superfamily NTPase/helicases II--was investigated as an inhibitor of the unwinding activity of the hepatitis C virus (HCV) NTPase/helicase. The kinetics of the reaction revealed that ribavirin-TP reduces the turnover number of the helicase reaction by a mechanism that does not correspond to that of the inhibition of the NTPase activity. Our results suggest that derivatives of ribavirin-TP with enhanced stability towards hydrolytic attack may be effective inhibitors of the enzyme.

1,25-dihydroxyvitamin D(3) has quite significant anticancer properties, but its strong calcemic effect in principle excludes it as a potential anticancer drug. Currently, a lot of effort is being devoted to develop potent anticancer analogs of 1,25-dihydroxyvitamin D(3) that would not induce hypercalcemia during therapy. In this work, the free binding energy of the VDR receptor with 1,25-dihydroxyvitamin D(3) and its three potent analogs (EB 1089, KH 1060 and RO 25-9022) is calculated and compared with each other. With this approach, we could estimate the relative binding affinity of the most potent analog, RO 25-9022, and also revealed a quite distinct mechanism of its interaction with VDR.

The effect of vitamin D3 on the activity of different forms of alkaline phosphatase as well as on the ability of their subunits to form phosphorylated intermediates in the presence of [gamma-32P]ATP was studied. In experiments with rats fed a diet enriched in vitamin D3 phosphatase F3 activity was doubled and the radioactivity of the phosphorylated intermediate of the F3 subunit was greatly enhanced. In contrast, in the case of phosphatase F1 the enzymic activity and the radioactivity of F1 subunit phosphorylated intermediate remained unchanged. In rats fed a vitamin D3-deficient diet, the phosphatase F3 activity was greatly reduced and there were only traces of radioactivity of F3 subunit phosphorylated intermediate.

Inflammatory response has been recognized as a central feature in the development and progression of atherosclerosis, and VSMCs (Vascular Smooth Muscle Cells) - the main cellular component of media, play an important role in this process. Many reports indicate that the biologically active vitamin D metabolite - 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3) = calcitriol), besides its well established role in calcium homeostasis, plays an essential role in the regulation of the inflammation process. The aim of this study was to determine the regulatory effects of calcitriol, applied at two supra-physiological doses (10 nM and 100 nM), in VSMC culture. Secretion of the pro-inflammatory cytokines, IL-6 and TNF-α, was significantly attenuated in calcitriol-treated VSMC culture, but the level of anti-inflammatory TGF-β was generally unchanged. Since in advanced atherosclerosis lesions several cell types, including VSMCs, overproduce the HSP70 chaperone protein, we also checked the effects of calcitriol on its synthesis. The presence of 1,25(OH)(2)D(3) did not affect HSP70 synthesis under physiological conditions but the synthesis of HSP70 in VSMCs exposed to heat shock was significantly inhibited by calcitriol (=100 nM). We observed that 1,25(OH)(2)D(3) induced SOD 1 activity, stimulated the expression of IκB-α, and did not influence the level of NF-κB-p65 in VSMCs. The results of our study suggest that 1,25(OH)(2)D(3) may serve as a natural anti-inflammatory agent and may therefore play a beneficial role in the physiology of VSMC in some contexts of atherosclerosis.

Organic extracts of six urine samples from children treated with nitisinone, a medicine against tyrosinemia type I, were investigated by (1)H and (19)F NMR spectroscopy. The presence of unchanged 2-[2-nitro-4-(trifluoromethyl)benzoyl]cyclohexane-1,3-dione (NTBC), 6-hydroxy-2-[2-nitro-4-(trifluoromethyl)benzoyl]cyclohexane-1,3-dione (NTBC-OH) and 2-nitro-4-trifluoromethylbenzoic acid (NTFA) as well as a few other unidentified compounds containing CF(3) group was documented.

In the face of shortage of fossil fuel supplies and climate warming triggered by excessive carbon dioxide emission, alternative resources for chemical industry have gained considerable attention. Renewable resources and their derivatives are of particular interest. Glycerol, which constitutes one of the by-products during biodiesel production, is such a substrate. Thus, generated excess glycerol may become an environmental problem, since it cannot be disposed of in the environment. The most promising products obtained from glycerol are polyols, including 1,3-propanediol, an important substrate in the production of synthetic materials, e.g. polyurethanes, unsaturated polyesters, and epoxy resins. Glycerol can be used as a carbon and energy source for microbial growth in industrial microbiology to produce 1,3-propanediol. This paper is a review of metabolic pathways of native producers and E. coli with the acquired ability to produce the diol via genetic manipulations. Culture conditions during 1,3-PDO production and genetic modifications of E. coli used in order to increase efficiency of glycerol bioconversion are also described in this paper.

Triketone herbicides are inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD), a key enzyme of the tyrosine transformation pathway, common for plants and animals. One of these herbicides, 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC), is so selective and efficient that it can be applied as a medicine in a hereditary metabolic disease - tyrosinemia type I. In this paper the available information concerning the molecular mechanism of HPPD inhibition by NTBC, originating from experimental investigations as well as theoretical modeling, has been collected. It is supplemented by results of additional theoretical DFT and/or MP2 calculations of the energetic effects of individual elementary molecular transformations. All these data are discussed and a consistent picture of HPPD inhibition by NTBC is proposed.

Study presented here demonstrates the ability of three newly isolated strains, obtained from environmental probes (manure, bottom sediment, and food waste) and identified as Clostridium bifermentans, Clostridium butyricum, and Hafnia alvei, to synthesize 1,3-propanediol (1,3-PD), organic acids (such as lactic, acetic, fumaric, succinic, and butyric acids), and ethanol from glycerol. The production of 1,3-PD as well as the glycerol pathways in C. bifermentans and H. alvei cells have not been investigated and described yet by others. Moreover, there is no data in the available literature on the products of glycerol utilization by H. alvei and there is only some incoherent data (mainly from the first half of the twentieth century) about the ability of C. bifermentans to carry out glycerol degradation. Additionally, this study presents complete hypothetical glycerol pathways and the basic fermentation kinetic parameters (such as yield and productivity) for both strains as well as for the newly isolated C. butyricum strain.

Twenty nine environmental samples were screened for the presence of anaerobic microorganisms fermenting glycerol with 1,3-propanediol as a final product. Seven samples were then selected for the next step of our research and eight bacteria strains were cultured anaerobically. Seven of them produced 1,3-propanediol with a yield of 0.47-0.58. Six of the the isolated microorganisms were then classified as Clostridium butyricum (four strains), C. lituseburense (one strain), and C. sartagoforme (one strain). We suggest that of all these strains C. butyricum 2CR371.5 is the best 1,3-propanediol producer as producing no lactate as a by-product and growing well on a glycerol-containing medium.

1,3-propanediol is used as a monomer in the production of some polymers e.g. polytrimethylene terephthalate used in the production of carpets and textile fibers and in the thermoplastics engineering. However, the traditional chemical synthesis is expensive, generates some toxic intermediates and requires a reduction step under high hydrogen pressure. Biological production of 1,3-propanediol could be an attractive alternative to the traditional chemical methods. Moreover, crude glycerol which is a by-product of biodiesel production, can be used. We constructed a recombinant Escherichia coli strain producing 1,3-propanediol from glycerol by introducing genes of the dha operon from Clostridium butyricum 2CR371.5, a strain from our collection of environmental samples and strains. The E. coli strain produced 3.7 g of 1,3-propanediol per one litre of culture with the yield of 0.3 g per 1 g of glycerol consumed.

Inhibition of jack bean activity by 2,5-dichloro-1,4-benzoquinone (DCBQ) was studied in phosphate buffer, pH 7.0. It was found that DCBQ acted as a strong, time and concentration dependent inactivator of urease. Under the experimental conditions obeyed the terms of pseudo-first-order reaction, urease was totally inactivated. Application of Wilson-Kitz method proved that the urease-DCBQ interaction followed a simple bimolecular process and the presence of intermediate complex was undetectable. The determined second order rate constant of the inactivation was 0.053 (μM min)(-1). Thiols such as l-cysteine, glutathione and dithiothreitol (DTT) protected urease from inhibition by DCBQ but DCBQ-modified urease did not regain its activity after DTT application. The thiol protective studies indicated an essential role of urease thiol(s) in the inhibition. The irreversibility of the inactivation showed that the process was a result of a direct modification of urease thiol(s) by DCBQ (DCBQ chlorine(s) substitution). The decomposition of DCBQ in aqueous solution at natural light exposure was monitored by visible spectrophotometry, determination of the total reducing capacity (Folin-Ciocalteu method) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging ability. The DCBQ conversion resulted in a decrease of the inhibition power and was well correlated with the increase of the total reducing capacity and DPPH scavenging ability. These findings were attributed to DCBQ transformation by photolysis and the hydrolysis effect was found to be negligible.

The interaction of D-fructose-1,6-bisphosphate 1-phosphohydrolase (Fru-P2-ase, EC 3.1.3.11), with bovine serum albumin (BSA) results in the fluorescence quenching of BSA. BSA increases fluorescence anisotropy of Fru-P2-ase modified with o-phthaldialdehyde. A program in Fortran, to simulate the experimental titration curves of BSA with Fru-P2-ase and o-phthaldialdehyde modified Fru-P2-ase with BSA, was written. For fluorescence quenching experiments the best fit was obtained for a model where one subunit of native Fru-P2-ase binds up two molecules of BSA. The determined dissociation constants at 5, 15, 25 and 35 degrees C were 2.2, 1.6, 0.83 and 0.03 microM, respectively.

Denaturation of fructose-1,6-bisphosphatase (Fru-P2-ase, EC 3.1.3.11) by urea and renaturation of denatured enzyme has been investigated. Denaturation lowers the specific activity of the enzyme but even at 8 M urea concentration in the presence of sucrose the activity of the enzyme is detectable. Centrifugation of the enzyme in a sucrose density gradient at 4 M urea reveals one peak of protein corresponding to a dimer. Denaturation increases intensity of intrinsic fluorescence of Fru-P2-ase and causes a red shift of fluorescence peak of the thioisoindole derivative of the enzyme. Renaturation of the denatured enzyme followed as the reappearance of enzymatic activity in the presence and absence of bovine serum albumin (BSA) is characterised by first order kinetics, k = 1.78 X 10(-3) s-1. The presence of BSA does not affect the rate of renaturation but perceptibly increases the recovery of enzymatic activity. A 100% recovery of Fru-P2-ase activity is observed at 0.5 micrograms/mL concentration of the enzyme and 2 mg/mL of BSA.

Phosphorylated fructose-1,6-bisphosphatase (FBPase) was isolated from rabbit muscle in an SDS/PAGE homogeneous form. Its dephosphorylation with alkaline phosphatase revealed 2.8 moles of inorganic phosphate per mole of FBPase. The phosphorylated FBPase (P-FBPase) differs from the dephosphorylated enzyme in terms of its kinetic properties like K(m) and k(cat), which are two times higher for the phosphorylated FBPase, and in the affinity for aldolase, which is three times lower for the dephosphorylated enzyme. Dephosphorylated FBPase can be a substrate for protein kinase A and the amount of phosphate incorporated per FBPase monomer can reach 2-3 molecules. Since interaction of muscle aldolase with muscle FBPase results in desensitisation of the latter toward AMP inhibition (Rakus & Dzugaj, 2000, Biochem. Biophys. Res. Commun. 275, 611-616), phosphorylation may be considered as a way of muscle FBPase activity regulation.

Structure of aldolase, its interaction with nucleotides, the path of enzyme reaction and the scheme of range of conformational changes of this enzyme are presented. Retrospectives and perspectives of aldolase topography investigations are included.

The method is described for separation and purification by chromatography on Biogel 1.5 m of subnucleosomal nucleoprotein particles obtained by extensive digestion of calf thymus nuclei with micrococcal nuclease.

The crystal structure of rat transthyretin (rTTR) complex with 3,5,3',5'-tetraiodothyroacetic acid (T4Ac) was determined at 1.8 A resolution with low temperature synchrotron data collected at CHESS. The structure was refined to R = 0.207 and Rfree = 0.24 with the use of 8-1.8 A data. The additional 8000 reflections from the incomplete 2.1-1.8 data shell, included in the refinement, reduced the Rfree index by 1.3%. Structure comparison with the model refined against the complete 8-2.1 A data revealed no differences in the ligand orientation and the conformation of the polypeptide chain in the core regions. However, the high-resolution data included in the refinement improved the model in the flexible regions poorly defined with the lower resolution data. Also additional sixteen water molecules were found in the difference map calculated with the extended data. The structure revealed both forward and reverse binding of tetraiodothyroacetic acid in one binding site and two modes of forward ligand binding in the second site, with the phenolic iodine atoms occupying different sets of the halogen binding pockets.

His296 of Zymomonas mobilis levansucrase (EC 2.4.1.10) is crucial for the catalysis of the transfructosylation reaction. The three-dimensional structures of levansucrases revealed the His296 is involved in the substrate recognition and binding. In this study, nine mutants were created by site-directed mutagenesis, in which His296 was substituted with amino acids of different polarity, charge and length. The substitutions of His296 with Arg or Trp retained partial hydrolysis and transfructosylation activities. The positively charged Lys substitution resulted in a 2.5-fold increase of sucrose hydrolysis. Substitutions with short (Cys or Ser), negatively charged (Glu) or polar (Tyr) amino acids virtually abolished both the activities. Analysis of transfructosylation products indicated that the mutants synthesized different oligosaccharides, suggesting that amino acid substitutions of His296 strongly affected both the enzyme activity and transfructosylation products.

The activities of extracellular signal-regulated kinases (ERK1/ERK2) is required for proliferation of several types of cells. The performed analysis showed stimulation of ERK's by fetal calf serum (FCS) or fibronectin in the C3H 10T1/2 cell cultures at logarithmic phase of growth. The ERKs activity was not stimulated in confluent cells. This could not be accounted for a partial down regulation of ERK since its level was stable in both types of cells regardless of their density and kind of stimulation. Searching for ERK up-stream elements we studied the integrin receptor gene transcript by RT-PCR and focal adhesion kinase (FAK) by Western blotting and phosphorylation assays. It was found that FCS and fibronectin stimulated phosphorylating activity of FAK in the cells at the logarithmic phase of growth, but were inefficient in the confluent cells. RT-PCR showed the presence of alpha5 and beta1 integrin transcripts, and p125FAK was at the same level regardless of the type of stimulation. These data indicate that the ability of FAK to be activated plays an important role in ERK regulation and, in consequence in proliferation and growth inhibition during confluence.

1. No essential differences were found in the activities of tricarboxylic acid-cycle enzymes in the newly isolated facultative methylotroph Pseudomonas J26 and obligate methylotroph Methylomonas Pl1. 2-Oxoglutarate dehydrogenase and succinate dehydrogenase were absent in Methylomonas Pl1; in Pseudomonas J26 the functioning of the cycle was imparied only on the methanol medium. Citrate synthase of both organisms showed low sensitivity to 2-oxoglutarate, NADH and ATP. 2. In both methylotrophs, methanol dehydrogenase was inhibited non-competitively by ATP: the activity was reduced by half by ATP at a concentration of 5 mM. 3. Concentration of ATP in the log-phase cultures of Methylomonas Pl1 was about twice as high as in Pseudomonas J26 (4.7 and 1.7 mumol/g dry wt., respectively). 4. Differences between the energy state of Methylomonas Pl1 and Pseudomonas J26 might be due to the higher ability of the former to oxidize methanol and/or lower energy requirement for C1 assimilation by the hexulose pathway in the obligate methylotroph.

We have analyzed the DNA fragment localized about 11 to 17.5 kb upstream of the chicken alpha-globin gene domain (the fragment was designed as alpha-0). The nucleotide sequence of its 3.3 kb-long 5' part was established and interactions with nuclear matrix proteins were studied. The DNA region localized about 16 kb upstream of the embryonic pi-globin gene showed high affinity to nuclear matrices in vitro. Two palindromes and a cluster of inverted repeats were co-localized in the same region. The whole 6.6 kb alpha-0 fragment decreased the activity of linked CAT reporter gene when transfected into chicken erythroblastoid cells.

Results of mutation analysis in exon 11 of the CF gene have been presented. Using the SSCP technique 18 mutations (of four different types) were detected in cystic fibrosis patients of Polish origin. Thus, we were able to detect in exon 11 about 10% of all CF mutations occurring in the affected population examined.

Aggregation of Abeta peptides is a seminal event in Alzheimer's disease. Detailed understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Here comparative conformational and aggregation studies using CD spectroscopy and thioflavine T fluorescence assay are presented. As a model peptide, the 11-28 fragment of Abeta was used. This model peptide is known to contain the core region responsible for Abeta aggregation. The structural and aggregational behaviour of the peptide was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21-23 (A21G, E22K, E22G, E22Q and D23N). In HFIP (hexafluoro-2-propanol), a strong alpha-helix inducer, the CD spectra revealed an unexpectedly high amount of beta-sheet conformation. The aggregation process of Abeta(11-28) variants provoked by water addition to HFIP was found to be consistent with a model of an alpha-helix-containing intermediate. The aggregation propensity of all Abeta(11-28) variants was also compared and discussed.

Synthesis of polyphenylalanine and polylysine in the E. coli MRE 600 and Q 13 cell-free systems was inhibited by erythromycin A and cyclic 11, 12-erythromycin A carbonate to a similar or identical extent. Both compounds inhibited translation of phage f2 RNA in the E. coli Q13 cell-free system. Neither antibiotic affected binding of initiator tRNA or phage f2 RNA to E. coli ribosomes, and neither inhibited translation of BMV RNA in the wheat-germ cell-free system.

The location and nature of the linkage between peptidoglycan and oligoglucans in the cell wall of Mesorhizobium loti HAMBI 1148 have been defined by the analysis of nitrous acid deamination of peptidoglycan glucosaminyl residues. The MurNH(2)-Glc(n) fraction was obtained after converting deaminoacylated and N-deacetylated muramyl residues in the cell wall preparation to lactam forms which were stable during subsequent deamination, followed by reduction and opening of the lactams. GC/MS analysis of this material, subjected to partial hydrolysis and reduction or to methanolysis followed by peracetylation, confirmed the presence of glucosyl residues glycosidically attached to muramic acid. The MALDI-TOF spectroscopic analysis of the deaminated material also revealed the presence of [M-H](-) or [M+Na-2H](-) ions representing fragments containing muramic acid with one to three linked glucose residues. The analysis of fully methylated neutral oligosaccharides released from the peptidoglycan with lysozyme followed by borohydride reduction showed the presence of di- and trisaccharides lacking the reducing end.

We have analysed the heteroplasmy level in 11 individuals from 3 families harbouring the mitochondrial 11778A mutation responsible for Leber hereditary optic neuropathy using last cycle hot PCR. The mutation level exceeded 90% both in affected and in unaffected individuals. We also checked whether any of the families belonged to the J haplogroup of mitochondrial DNA and obtained a negative result.

The aim was to investigate different genotypes and haplotypes of methylenetetrahydrofolate reductase (MTHFR-677, -1298) and plasma concentration of total homocysteine (tHcy) in Macedonian patients with occlusive artery disease (OAD) and deep venous thrombosis (DVT). Investigated groups consists of 80 healthy, 74 patients with OAD, and 63 patients with DVT. Plasma tHcy was measured with Microplate Enzyme Immunoassay. Identification of MTHFR genotypes and haplotypes was done with CVD StripAssay. The probability level (P-value) was evaluated by the Student's t-test. Plasma concentration of tHcy in CC and CT genotypes of MTHFR C677T was significantly increased in patients with OAD and in patients with DVT. Plasma concentration of tHcy in AC genotype of MTHFR A1298C was increased in patients with OAD and in patients with DVT. Plasma concentration of tHcy was significantly increased in AA genotype of patients with OAD, but not in patients with DVT. We found a significant increase of plasma tHcy in patients with OAD in comparison with healthy respondents for normal:heterozygote (CC:AC), heterozygote:normal (CT:AA), and heterozygote:heterozygote (CT:AC) haplotypes. Plasma concentration of tHcy in patients with DVT in comparison with healthy respondents was significantly increased for normal:normal (CC:AA), normal heterozygote (CC:AC), and heterozygote:heterozygote (CT:AC) haplotypes. We conclude that MTHFR C677T and MTHFR A1289C genotypes and haplotypes are connected with tHcy plasma levels in Macedonian patients with OAD and DVT.

To reveal nucleic acid localization in mitochondria, we designed molecular beacon fluorescent probes against: i) the light strand complementary to ND5 mitochondrial DNA (mtDNA) gene (annealing also to corresponding mRNA); ii) displacement (D) loop 7S DNA (annealing also to parallel heavy strand mtDNA and corresponding light strand transcript); iii) the proximal D-loop heavy strand displaced by the light strand promoter minor RNA. Confocal microscopy demonstrated ND5 probe spreading (less for other probes) in mitochondrial reticulum tubules but upon RNase A treatment all probes contoured mtDNA nucleoid localization. DNase I spread the signal over mitochondrial tubules. Future applications are discussed.

UNC-13 protein participates in regulating neurotransmitter release. In Drosophila melanogaster, proteasomal degradation controls UNC-13 levels at synapses. Function of the amino-terminal region of a 207 kDa form of Caenorhabditis elegans UNC-13 is unknown. Yeast two-hybrid and secondary yeast assays identified an F-box protein that interacts with this amino-terminal region. As F-box proteins bind proteins targeted for proteasomal degradation, this protein may participate in degrading a subset of UNC-13 proteins, suggesting that different forms of UNC-13 are regulated differently. Yeast assays also identified an exonuclease, a predicted splicing factor, and a protein with coiled-coil domains, indicating that UNC-13 may affect RNA function.

S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. In the present study, we examined the potential role of S100A4 in metastasis in breast cancer and its relation with matrix metalloproteinase-13 (MMP-13). Analysis of 100 breast cancer specimens including 50 with and 50 without lymph node metastasis showed a significant upregulation of S100A4 and MMP-13 expression in metastatic breast cancer tissues. Positive immunoreactivity for S100A4 was associated with MMP-13 expression. Overexpression of S100A4 in the MDA-MB-231 breast cancer cell line upregulated MMP13 expression leading to increased cell migration and angiogenesis. SiRNA-mediated silencing of S100A4 downregulated MMP13 expression and suppressed cell migration and angiogenesis. Moreover, neutralization of MMP-13 activity with a specific antibody blocked cell migration and angiogenesis in MDA-MB-231/S100A4 cells. In vivo siRNA silencing of S100A4 significantly inhibited lung metastasis in transgenic mice. The present results suggest that the S100A4 gene may control the invasive potential of human breast cancer cells by modulating MMP-13 levels, thus regulating metastasis and angiogenesis in breast tumors. S100A4 could therefore be of value as a biomarker of breast cancer progression and a novel therapeutic target for human breast cancer treatment.

Carotenoids and their metabolites are essential factors for the maintenance of important life processes such as photosynthesis. Animals cannot synthesize carotenoids de novo, they must obtain them via their food. In order to make intensive animal husbandry possible and maintain human and animal health synthetic nature identical carotenoids are presently commercially available at the multi-tonnes scale per year. Synthetically accessible (13)C enriched carotenoids are essential to apply isotope sensitive techniques to obtain information at the atomic level without perturbation about the role of carotenoids in photosynthesis, nutrition, vision, animal development, etc. Simple highly (13)C enriched C(1), C(2) and C(3) building blocks are commercially available via 99% (13)CO. The synthetic routes for the preparation of the (13)C enriched building blocks starting from the commercially available systems are discussed first. Then, how these building blocks are used for the synthesis of the various (13)C enriched carotenoids and apocarotenoids are reviewed next. The synthetic Schemes that resulted in (13)C enriched β-carotene, spheroidene, β-cryptoxanthin, canthaxanthin, astaxanthin, (3R,3'R)-zeaxanthin and (3R,3'R,6'R)-lutein are described. The Schemes that are reviewed can also be used to synthetically access any carotenoid and apocarotenoid in any (13)C isotopically enriched form up to the unitarily enriched form.

Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism (SNP) at -1306, which disrupts a Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with the T allele. In the present study, we investigate whether this MMP-2 SNP is associated with susceptibility to breast cancer in the Saudi population. Ninety breast cancer patients and 92 age matched controls were included in this study. TaqMan Allele Discrimination assay and DNA sequencing techniques were used for genotyping. The results showed that, the frequency of MMP-2 CC wild genotype was lower in breast cancer patients when compared with healthy controls (0.65 versus 0.79). The homozygous CC (OR=2, χ(2)=5.36, p=0.02) and heterozygous CT (OR=1.98, χ(2)=4.1, p=0.04) showing significantly high risk of breast cancer in the investigated group. In conclusion our data suggest that the MMP-2 C(-1306)T polymorphism may be associated with increased breast cancer risk in the Saudi population.

Plasminogen activator inhibitor 1 (PAI-1) content in colorectal cancer tissue extracts may be of strong prognostic value: high levels of PAI-1 in tumours predict poor prognosis. The gene encoding PAI-1 is highly polymorphic and PAI-1 gene variability could contribute to the level of PAI-1 biosynthesis. In the present work the distribution of genotypes and frequency of alleles of the 1334G/A polymorphism in 92 subjects with colorectal cancer in samples of cancer tissue and distant mucosa samples as well as in blood were investigated. Blood samples age matched healthy individuals (n = 110) served as control. The 1334G/A polymorphism was determined by PCR amplification using allele specific primers. No differences in the genotype distributions and allele frequencies between blood, distant mucosa samples and cancer tissue were detected. However, the distribution of the genotypes of the 1334G/A polymorphism in patients differed significantly (P <0.05) from those predicted by the Hardy-Weinberg equilibrium. There were significant differences in the frequencies of alleles between the colorectal cancer subjects and controls (P <0.05). The results support the hypothesis that the 1334G/A polymorphism may be associated with the incidence of colorectal cancer.

Our previous study showed the efficacy of lactoferrin-monophosphoryl lipid A isolated from Hafnia alvei LPS complex (LF-MPL(H.a.)) as an adjuvant in stimulation of humoral and cellular immune response in mice to conventional antigens and a lower pyrogenicity of the complex as compared with MPL(H.a.) alone. In the present investigation we demonstrated that LF-MPL(H.a.) complex enhanced the immunity of BALB/c mice immunized with Plesiomonas shigelloides CNCTC 138/92 bacterial vaccine, against P. shigelloides infection. The adjuvant effect was evidenced by a significant increase of the antigen-specific serum IgG, IgG(2a), and IgG(1) and elevation of antigen-specific serum IgA concentrations. In addition, application of the adjuvant facilitated better clearance of the bacteria in spleens and livers of infected mice when compared with MPL(H.a.) alone. These features of the new adjuvant may predispose it for vaccination protocols in humans.

Two-dimensional infrared correlation spectroscopy (2D-IR) was applied to the study of urea- and heat-induced unfolding denaturation of sarcoplasmic reticulum Ca(2+)-ATPase (SR ATPase). Urea at 2-3 M causes reversible loss of SR ATPase activity, while higher concentrations induce irreversible denaturation. Heat-induced denaturation is a non-two-state process, with an "intermediate state" (at t approximately 45 degrees C) characterized by the presence of protein monomers, instead of the native oligomers. 2D-IR reveals that urea denaturation causes loss of the structural transition to the "intermediate state". Whenever the urea effect can be reversed, the transition to the "intermediate state" is re-established.