[Show abstract][Hide abstract] ABSTRACT: Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with (125)I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC(3)(5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to Ostrinia nubilalis, we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.
Applied and Environmental Microbiology 05/2006; 72(4):2594-600. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pore-forming activity of Cry1Ab, Cry1Fa and Cry1Ca toxins and their interaction with leucine transport mediated by the K(+)/leucine cotransporter were studied in brush border membrane vesicles (BBMVs) isolated from the midgut of Ostrinia nubilalis and Sesamia nonagrioides. In both species, as in other Lepidoptera, leucine uptake by BBMVs can take place in the absence of cations, but it can also be driven by a K(+) gradient. Experiments with the voltage-sensitive fluorescent dye 3,3'-diethylthiacarbocyanine iodide proved that Cry1Ab, a Bacillus thuringiensis toxin active in vivo, enhanced the membrane permeability to potassium in O. nubilalis BBMVs. This result is in agreement with similar effects observed in S. nonagrioides BBMV incubated with various Cry1 toxins active in vivo. The effect of the above toxins was tested on the initial rate of 0.1 mM: leucine influx. Instead of an increase in leucine influx, a reduction was observed with the Cry1 toxins active in vivo. Cry1Ab and Cry1Fa, but not the inactive toxin Cry1Da, inhibited in a dose-dependent manner leucine uptake both in the absence and in the presence of a K(+) gradient, a clear indication that their effect is independent of the channel formed by the toxins and that this effect is exerted directly on the amino acid transport system.
Journal of Membrane Biology 02/2006; 214(3):157-64. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Brush border membrane vesicles (BBMV) from insects midgut can be successfully used to study several membrane phenomena, including nutrient absorption, ions permeability and insecticides mode of action. Midgut BBMV, purified from Musca domestica whole larvae, were used for the functional characterization of leucine transport. The amino acid uptake was accelerated in the presence of sodium or potassium and increased significantly when the extravesicular pH was 5.0, in agreement with the luminal pH in vivo. Radiolabelled leucine uptake was significantly reduced by an excess of cold leucine, histidine, serine and glycine, suggesting that the amino acid transporter is a broad scope carrier that does not recognize proline, glutamine and the dibasic amino acids lysine and arginine.Midgut BBMV were also obtained from homogenization of M. domestica and Bactrocera oleae adults. The final preparations showed a high enrichment in the specific activity of the BBM marker enzymes aminopeptidase N and γ-glutamyl transpeptidase, and were poorly contaminated by basolateral membranes, as indicated by the low specific activities of their marker enzyme Na+/K+ ATPase. Electron microscopy of B. oleae BBM fraction showed the presence of closed vesicles. Similar SDS-PAGE patterns, with numerous distinct bands, were detected for both B. oleae and M. domestica BBMV.
[Show abstract][Hide abstract] ABSTRACT: It is well documented that in the model system Aphidius ervi Haliday (Hymenoptera, Braconidae)/Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) host regulation by the parasitoid larva induces in the aphid haemolymph major changes of the titer of nutritional compounds such as proteins, acylglycerols and free amino acids, in order to meet the stage-specific demands of the developing larva. Since little is known about how the larva absorbs these mobilized nutritional resources, nutrient absorption by larval stages of A. ervi was studied. In 2nd instar larvae, leucine was ten-fold accumulated in the haemocoel, and tyrosine and glutamine two-fold. Glucose and fructose were readily absorbed and fructose was extensively metabolized by larval tissues. In 3rd instars, the presence of a number of larvae that did not ingest the incubation medium enabled us to determine the respective amounts of substrate absorbed by the epidermis and the midgut. An accumulation of leucine in the haemocoel was observed only when midgut cells were involved in absorption, while the amino acid concentration within body fluids never exceeded that of the incubation medium when the uptake was performed only by epidermal cells. The immunofluorescence analysis, the mutual inhibition exerted on labeled glucose or fructose uptakes by a 100-fold excess of the sugars and the strong inhibition of uptakes induced by 0.2mM cytochalasin B support the expression of facilitative GLUT2-like transporters in the apical and basal cell membranes of midgut epithelial cells. Taken together, these results prove that both midgut and epidermis are involved in nutrient absorption throughout the parasitoid development, that GLUT2 transporters are responsible for glucose and fructose uptakes and that the chemical gradient that favors the passive influx of the two sugars is maintained by their conversion to other substrates.
Journal of Insect Physiology 12/2005; 51(11):1183-92. · 2.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the last decade, the study of peptide and protein absorption by the insect gut has received increasing attention because of the considerable impact this information may have on the development of new delivery strategies for insecticide macromolecules targeting haemocoelic receptors. Available experimental evidence in vivo suggests that, in insects, peptides and proteins can cross the intestinal barrier reaching the haemocoel, but the functional bases of this absorption pathway have not yet been thoroughly investigated. The current knowledge of the mechanisms involved in protein and polypeptide absorption in animals derives from the extensive studies performed in mammalian polarised epithelial cells, where the transcellular transport of proteins by transcytosis has been demonstrated. In this process, proteins are internalised at one pole of the cell and transported by cytoplasmic vesicular traffic to the opposite plasma membrane domain, where they are released with unchanged biological activity. Here we report data on albumin translocation across the isolated midgut of Bombyx mori caterpillars perfused in vitro. The functional properties of the transepithelial transport of this protein are described and, since absorption prevails over secretion, its lumen-to-haemolymph flux is characterised. Low-temperature incubations nearly abolish the transepithelial transport, while the peculiar physiological features of the larval midgut, i.e. the high lumen positive transepithelial voltage and the luminal alkaline pH, do not affect the flux. The obtained results indicate that albumin crosses B. mori larval midgut by transcytosis.
Journal of Insect Physiology 09/2005; 51(8):933-40. · 2.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aphidius ervi Haliday (Hymenoptera, Braconidae) is an endophagous parasitoid of several aphid species of economic importance, widely used in biological control. The definition of a suitable artificial diet for in vitro mass production of this parasitoid is still an unresolved issue that, to be properly addressed, requires a deeper understanding both of its nutritional needs and of the functional properties of the larval epithelia involved in nutrient absorption. The experimental evidence presented in this paper unequivocally demonstrates that the uptake of sugars and amino acids takes place through the body surface of the larval stages of A. ervi. These nutrients are efficiently absorbed by the larval epidermis, but the transport rate progressively declines over time. The epidermis exhibits a cross-reactivity to antibodies raised against the mammalian facilitative glucose transporter GLUT2 and the sodium cotransporter SGLT1. The analysis of sugar transport sensitivity to specific inhibitors indicates the involvement of GLUT2-like transporters, while a role for SGLT1-like transporters is not supported. The peculiar pathways of nutrient absorption in A. ervi larvae further corroborate the general idea that the pre-imaginal stages of endophagous koinobiont Hymenoptera, like Metazoan parasites, show a high degree of physiological integration with their hosts.
Journal of Insect Physiology 01/2004; 49(12):1115-24. · 2.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have identified three methyl esters that have a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids in the silkworm Bombyx mori. L-Leucine methyl ester, the most powerful activator, determined a large dose-dependent, K(+)-independent increase of leucine uptake into midgut brush border membrane vesicles. Kinetic experiments revealed non-essential mixed-type activation, with K(a) values of 27+/-2 and 47+/-8 microM in the presence and in the absence of K(+), respectively. The activation increased K(m) twofold, and V(max) up to 18-fold depending upon the experimental conditions. Leucine uptake mediated by the amino acid uniport appears to be unaffected by the activator.
[Show abstract][Hide abstract] ABSTRACT: A number of methyl and ethyl esters of naturally occurring amino acids exert a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids along the midgut of the silkworm Bombyx mori. L-Leucine methyl ester (Leu-OMe), one of the most effective activators, induces a large increase of the initial rate of leucine uptake in midgut brush border membrane vesicles (BBMV) from the anterior-middle (AM) region, and a small effect in BBMV from the posterior (P) region. Nonetheless, the methyl ester causes in both regions a relevant K(+)-, Deltapsi- and pH-independent increase of the intravesicular accumulation of the amino acid. The activation by Leu-OMe proves that amino acid absorption can be modulated all along the B. mori larval midgut and that the AM region, where the ability to transport and concentrate the substrate is very low, is more susceptible than the P region. Leucine uptake in AM-BMMV can be activated by amino acid methyl esters with definite structural requisites, with the following order of potency: L-leucine>L-phenylglycine>L-methionine>L-phenylalanine>L-norleucinez.Gt;L-isoleucine. The activation is stereospecific and occurs also with some ethyl esters (e.g. leucine and phenylalanine). No activation was observed with esters of amino acids with short hydrophobic or polar side-chains. The activation mechanism here described plays a fundamental role in larval growth since silkworms reared on artificial diets supplemented with leucine or methionine methyl esters reach maximum body weight 12-18 h before control larvae and spin cocoons with a larger shell weight. This novel regulatory mechanism of an amino acid transport protein appears to be widespread among lepidopteran larvae.
Journal of insect physiology 06/2002; 48(5):585-592. · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present work describes Aphidius ervi Haliday (Hymenoptera, Braconidae) larval anatomy and development, focusing on time-related changes of body structure and cell ultrastructure, especially of the epithelial layers involved in nutrient absorption. Newly hatched 1st instar larvae of A. ervi are characterised by gut absence and a compact cluster of cells makes up their body. As the parasitoid larva develops, the central undifferentiated cell mass becomes hollowed out, leading to the formation of gut anlage. This suggests that absorption of nutrients at that stage may take place through the body surface, as more directly demonstrated by the occurrence on the epidermis of proteins associated with transepithelial transport, such as Na+/K+-ATPase and alkaline phosphatase (ALP). Second instar larvae show the presence of the gut with a well-differentiated brush border and a peritrophic membrane. Gut cells are filled by masses of glycogen granules and lipid droplets. The tracheal system starts to be visible. The haemocoel becomes evident in late 2nd instar, and contains large silk glands. Mature 3rd instar larvae are typically hymenopteriform. The midgut accounts for most of the body volume and is actively involved in nutrient absorption, as indicated by the well developed brush border and by the presence of Na+/K+-ATPase and ALP on the basolateral and luminal membrane respectively. At this stage, large lipid droplets have gradually replaced the cellular glycogen stores in the midgut cells. The tracheae are completely differentiated, but their internal lumen still contains fibrillar material, suggesting that they are not functional as long as host fluids bath the parasitoid larva. In late 3rd instar larvae, silk glands, structurally similar to Malpighian tubules, show a very intense vesicular traffic toward the internal lumen, which, eventually, results in being filled by secretion products, suggesting the possible recycling of metabolic waste products during mummy formation.
Arthropod structure & development 11/2001; · 1.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the larval midgut of Bombyx mori a K(+)-dependent transporter for leucine and amino acids with a hydrophobic side chain is responsible for the absorption of most essential amino acids. We investigated if a modulation of its activity occurred as a result of starvation or after hormonal treatments. We measured amino acid uptake in brush border membrane vesicles (BBMV) purified from the anterior-middle (AM) and posterior (P) regions of the midgut in fifth instar larvae. Silkworms were either starved or topically treated with low dosages of fenoxycarb, a molecule often used as a juvenile hormone mimic. The maximal uptake value of K(+)-driven leucine transport was increased in BBMV of AM- and P-midgut regions of starved larvae. The initial uptake rates of serine and glutamine, two amino acids transported by the same cotransporter as leucine, were also increased. Leucine kinetics proved that V(max) was the kinetic parameter modified by starvation in both midgut regions. Topical applications of fenoxycarb at a dose of 2.5 fg/larva immediately after the fourth ecdysis, induced an increase of leucine initial uptake rates and of intravesicular accumulation of leucine in both AM- and P-BBMV. Kinetic analysis of leucine uptake indicated again that V(max) was increased in BBMV from both midgut regions in treated larvae.
Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology 07/2001; 129(2-3):665-72. · 2.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transport pathways for dibasic amino acids were investigated in brush border membrane vesicles (BBMV) from the anterior-middle (AM) and posterior (P) regions of Bombyx mori midgut. In the absence of K(+), a low-affinity saturable transport of arginine in both AM- and P-BBMV (K(m) 1.01 mM, V(max) 4.07 nmol/7s/mg protein and K(m) 1.38 mM, V(max) 2.26 nmol/7s/mg protein, respectively) was detected. Arginine influx was dependent on the membrane electrical potential (Deltapsi) and increased raising the alkalinity of the external medium from pH 7.2 to 10.6. Competition experiments indicated the following order of substrate affinity: arginine, homoarginine, N(G)-monomethylarginine, N(G)-nitroarginine>lysine>ornithine>cysteine>methionine. Leucine, valine and BCH (2-amino-2-norbornanecarboxylic acid) did not inhibit arginine influx. In the presence of external K(+), the influx of arginine as a function of arginine concentration fitted to a complex saturation kinetics compatible with both a low-affinity and a high-affinity component. The latter (K(m) 0.035 mM, V(max) 2.54 nmol/7s/mg protein) was fully characterized. The influx rate had an optimum at pH 8.8, was strongly affected by Deltapsi and was homogeneous along the midgut. The substrate affinity rank was: homoarginine>arginine, N(G)-monomethylarginine>cysteine, lysine>N(G)-nitroarginine>ornithine>methionine. Leucine and amino acids with a hydrophobic side chain were not accepted. This system is also operative in the absence of potassium, with the same order of specificity but a very low activity. Lysine influx is mediated by two more transport systems, the leucine uniport and the K(+)/leucine symport specific for amino acids with a hydrophobic side chain that recognizes lysine at extravesicular pH values (pH(out)) exceeding 9. Both the uniport and the symport differ from the cationic transport systems so far identified in mammals because they are unaffected by N-ethylmaleimide, have no significant affinity for neutral amino acids in the presence of the cation and show a striking difference in their optimum pH.
[Show abstract][Hide abstract] ABSTRACT: Topical or oral applications of fenoxycarb (FC) to fifth instar larvae of Bombyx mori were performed immediately after the fourth ecdysis. A clear dose-response relationship in the frass-failure assay was recorded after 48 h for topically treated silkworms, whereas the oral treatment caused the maximal decrease of frass production even at the lowest dose (2.5 fg/larva). Preincubation of midgut brush border membrane vesicles from untreated larvae with variable amounts of FC reduced leucine uptake in vesicles from the anterior-middle midgut region, suggesting a toxic effect on the plasma membrane, absent in vesicles from the posterior region. The topical application of 2.5 fg of FC in vivo caused a significant increase of K+-dependent leucine uptake and accumulation into membrane vesicles of both the anterior-middle and posterior midgut regions, whereas application of 2.5 μg reduced leucine transport. Conversely, the forced ingestion of the same doses always caused an increase in leucine uptake and accumulation in both midgut regions. The brush border membrane composition is affected by both oral and topical treatment, but membranes from the posterior midgut region are less affected, especially by the oral treatment. Regardless of the considered gut region, the analysis of the fatty acid composition of the membrane revealed the presence of an unidentified component (X), which decreases following FC treatment. Interestingly, after treatment, the increases of saturated or unsaturated fatty acids is balanced by the loss of the X component. The results presented here suggest a multiple mode of action of fenoxycarb, involving a hormonal-like stimulation of leucine absorption with low-dose topical applications or as a long-term result of oral applications and a modification of brush border membrane lipid composition, which both affect amino acid absorption in the larval midgut.
[Show abstract][Hide abstract] ABSTRACT: The epithelial cells of the integument of body, arms and tentacles of Sepia officinalis present on their apical membrane a well-organised brush border and show the morphological and histochemical characteristics of a typical absorptive epithelium. The ability of the integument to absorb amino acids was investigated both in the arms incubated in vitro and in a purified preparation of brush border membrane vesicles (BBMV). Autoradiographic pictures of the integument after incubation of the arms in sea-water with or without sodium, showed that proline intake was Na+-dependent, whereas leucine intake appeared to be a largely cation-independent process. Time course experiments of labelled leucine, proline and lysine uptakes in BBMV evidenced that these amino acids are accumulated within the vesicles in the presence of an inwardly directed sodium gradient. The sodium-driven accumulation proves that cationic and neutral amino acids are taken up by the apical membrane of the epithelium of Sepia integument through a secondary active mechanism. For leucine, a 90% inhibition of the uptake was recorded in the presence of a large excess of the substrate. In agreement with the autoradiography results, an analysis of the cation specificity transport in BBMV showed that leucine uptake had a low cation specificity, whereas lysine and proline uptakes were Na+-dependent. An excess of lysine and proline, which share with alanine two different transport systems in the gill epithelium of marine bivalves, reduced eucine uptake. The possible role of the absorptive ability of the integument in a carnivorous mollusc is discussed.
Tissue and Cell 11/2000; 32(5):389-98. · 1.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: L-leucine uptake into membrane vesicles from Bombyx mori larval midgut was tested for inhibition by 55 compounds, which included sugars, N-methylated, alpha-, beta-, gamma-, delta-, epsilon-amino acids, primary amines, alpha-amino alcohols, monocarboxylic organic acids and alpha-ketoacids. Based on cis-inhibition experiments performed at the high pH (10.8) characteristic of the midgut luminal content in vivo, we find that the carrier binding site interacts with molecules which possess a well-defined set of structural features. Amino acids are preferentially accepted as anions and the ideal inhibitor must have an hydrophobic region and a polar head constituted by a chiral carbon atom bearing two hydrophilic groups, a deprotonated amino-group and a dissociated carboxylic group. Binding is reduced if one of the two hydrophilic groups is removed. Lowering the pH to less alkaline value (8.8) only affects the affinity of delta- and epsilon-amino acids, which are excluded from binding because of their positively charged side-chain. Modifications of the potassium electrochemical gradient increased the affinity constant values of the molecules, but have little effect on the rank of specificity. Physiological implications of the data reported are discussed.
[Show abstract][Hide abstract] ABSTRACT: The K(+)-dependent symporter for leucine and other neutral amino acids expressed along the midgut of the silkworm Bombyx mori operates with best efficiency in the presence of a steep pH gradient across the brush-border membrane, with external alkaline pH values up to 11, and an electrical potential difference (delta psi) of approximately 200 mV. Careful determinations of leucine kinetics as a function of external amino acid concentrations between 50 and 1,000 microM, performed with brush-border membrane vesicles (BBMV) obtained from the middle and posterior midgut regions, revealed that the kinetic parameter affected by the presence of a delta pH was the maximal rate of transport. The addition of delta psi caused a further marked increase of the translocation rate. At nonsaturating leucine concentrations in the solution bathing the external side of the brush-border membrane, leucine accumulation within BBMV and midgut cells was not only driven by the gradient of the driver cation K+ and delta psi but occurred also in the absence of K+. The ability of the symporter to translocate the substrate in its binary form allows the intracellular accumulation of leucine in the absence of K+, provided that a pH gradient, with alkaline outside, is present. The mechanisms involved in this accumulation are discussed.
The American journal of physiology 06/1998; 274(5 Pt 2):R1361-71. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the kinetics of leucine influx as a function of external substrate concentration between 0.03 and 16 mM in brush-border membrane vesicles (BBMV) prepared from the middle region of Bombyx mori larval midgut. A detailed kinetic analysis of leucine uptake led to the identification, in parallel with the K(+)-dependent symporter for neutral amino acids, of a K(+)-independent, low-affinity, high-capacity system. The parameter values of the Michaelis constant (7.12 mM) and maximal rate of transport (4.48 nmol.7 s-1.mg protein-1) were not influenced by an external alkaline pH nor by a transmembrane electrical potential difference. The uniporter is poorly specific, as it displayed the following rank of preference: Leu, His, Val, Ile, Phe, Ser > Lys, Arg, Gln > Pro, 2-amino-2-norbornane-carboxylic acid, Ala, Gly. The kinetic analysis performed in BBMV prepared from the posterior midgut portion indicates that the low-affinity, high-capacity uniporter is present along the entire length of the silkworm larval midgut with similar expression and functional properties.
The American journal of physiology 06/1998; 274(5 Pt 2):R1372-5. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have examined the type of inhibition exerted by an activated preparation of the Bacillus thuringiensis delta-endotoxin CrylAa on K+-dependent leucine transport into midgut brush border membrane vesicles or epithelial cells of the isolated midgut from Bombyx mori to study its possible interaction with the amino acid symporter. K+ permeability and the cation-dependent amino acid translocation into brush border membrane vesicles were evaluated by monitoring the fluorescence of the voltage-sensitive cyanine dye 3, 3'-dipropylthiadicarbocyanine iodide. The symporter ability to accept Na+ instead of K+ was exploited and the dissipation of an imposed inside-negative potential (K+ gradient in>out and valinomycin) was registered in the presence of a Na+ gradient (out>in) and of the amino acid. The fluorescence quenching dissipated more rapidly when the amino acid was present. Preincubation of brush border membrane vesicles with CrylAa caused a significant decrease of the amino acid-dependent recovery of fluorescence, whereas K+ permeability was sparely affected. In the isolated midgut, CrylAa inhibits leucine uptake as well as the transepithelial electrical potential difference. The strong inhibition exerted by the delta-endotoxin was observed also in the absence of potassium and the transepithelial electrical potential difference. The results obtained strongly suggest a direct interaction of CrylAa delta-endotoxin with the K+/amino acid symporter.
Journal of Membrane Biology 10/1997; 159(3):209-17. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of topical applications of the commercial insecticide Insegar (25% fenoxycarb) on the fifth instar larvae of Bombyx mori fed on an artificial diet were investigated. Clear dose-response relationships were established with respect to frass production as well as to the percentage of dauer larvae, whereas less evident was the correlation to the growth rate and the percentage of pupation. The Insegar concentration, giving 50% of dauer larvae, was 60 pg per larva, but effects on the parameters tested were evident also with concentrations as low as 100 fg per larva. Apparently, this insect growth regulator (IGR) causes a reduced intake of food as well as a reduction in growth rate. The absorption of amino acids carried out by the K+-dependent symporter in the anterior-middle and the posterior regions of the midgut was evaluated in vitro from leucine uptake into brush border membrane vesicles obtained from control and treated larvae. IGR in a dose of 10 fg per larva causes a significant, albeit small, increase of amino acid intake in both midgut regions. At higher doses, no effect of Insegar on leucine uptake is evident in the posterior midgut, whereas a significant decrease of the uptake in the anterior-middle region is apparent at the dose of 10 μg per larva.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 01/1996;