Effect of dexamethasone on development of in vitro–produced bovine embryos

Studies in somatic cells have shown that glucocorticoids such as dexamethasone (DEX) may trigger or prevent apoptosis depending on the cell type in culture. Because the dysregulation of apoptosis may lower in vitro embryo production efficiency, we sought to investigate the effects of supplementing IVC medium with DEX (0.1 μg/mL) on embryo morphology, development kinetics, and apoptosis rates of in vitro–produced bovine preimplantation embryos. Embryo morphology was graded on Day 7, and development rates were assessed on Days 4 and 7 of IVC. Apoptosis was evaluated via annexin/propidium iodide staining under fluorescence microscopy where a cell labeled with annexin, propidium iodide, or both would be considered apoptotic. An embryo was counted in the apoptosis rates, if it displayed at least one such labeled cell. Although DEX supplementation did not reduce apoptosis rates, it had a positive impact on developmental kinetics and cell number both on Days 4 and 7 of embryo culture. Presumably, such effect resulted from increased cell proliferation rather than a direct inhibition of apoptosis. Further studies may evaluate the mechanisms by which glucocorticoids may affect embryo development, as DEX supplementation could become a tool to improve in vitro embryo yield in mammalian species.     

Evaluation of sperm subpopulation structure in relation to in vitro sperm–oocyte interaction of frozen-thawed semen from Holstein bulls

The present study examined the relationship between the relative amount of high motile sperm and sperm–oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.     

Expression pattern of glucose metabolism genes in relation to development rate of buffalo (Bubalus bubalis) oocytes and in vitro–produced embryos

The expression pattern of glucose metabolism genes (hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase [G6PDH], lactate dehydrogenase [LDH], and pyruvate dehydrogenase [PDH]) were studied in buffalo in vitro–matured oocytes and in vitro–produced embryos cultured under different glucose concentrations (0 mM, 1.5 mM, 5.6 mM, and 10 mM) during in vitro maturation of oocytes and culture of IVF produced embryos. The expression of the genes varied significantly over the cleavage stages under different glucose concentrations. Developmental rate of embryos was highest under a constant glucose level (5.6 mM) throughout during maturation of oocytes and embryo culture. Expression pattern of glucose metabolism genes under optimum glucose level (5.6 mM) indicated that glycolysis is the major pathway of glucose metabolism during oocyte maturation and early embryonic stages (pre-maternal to zygotic transition [MZT]) and shifts to oxidative phosphorylation during post-MZT stages in buffalo embryos. Higher glucose level (10 mM) caused abrupt changes in gene expression and resulted in shifting toward anaerobic metabolism of glucose during post-MZT stages. This resulted in decreased development rate of embryos during post-MZT stages. High expression of LDH and PDH in the control groups (0 mM glucose) indicated that in absence of glucose, embryos try to use available pyruvate and lactate sources, but succumb to handle the post-MZT energy requirement, resulting to poor development rate. Expression pattern of G6PDH during oocyte maturation as well early embryonic development was found predictive of quality and development competence of oocytes/ embryos.     

The influence of reduced glutathione in fertilization medium on the fertility of in vitro–matured C57BL/6 mouse oocytes

It is well known that IVM oocytes show a decreased potential for fertility and development compared with in vivo–matured oocytes. In this study, we added reduced glutathione (GSH) to the fertilization medium during IVF to investigate its effect on the fertility and early embryo development of IVM oocytes. The fertilization rate for IVM oocytes and fresh sperm increased with the addition of GSH (0, 1.0, and 2.0 mM: 51%, 76%, and 70%). Moreover, the addition of GSH to the fertilization medium also improved the developmental potential compared with the control sample (0 mM). In addition, we performed IVF using IVM oocytes and frozen/thawed sperm that had been cryopreserved in a mouse bank. Results indicated a marked increase in the fertilization rate when 1.0 mM GSH was added to the fertilization medium compared with when no GSM was used (0.0 mM GSH: 2% (3/195); 1.0 mM GSH: 33% (156/468)). Furthermore, the fertilization rate improved dramatically via zona drilling using laser equipment (52%: 267/516), whereas normal offspring were obtainsed after transferring embryos created via IVF using IVM oocytes and frozen/thawed sperm. This is the first report in which offspring have been obtained via IVF using IVM oocytes and frozen/thawed sperm.     

Melphalan,alone or conjugated to an FSH-β peptide,kills murine testicular cells in vitro and transiently suppresses murine spermatogenesis in vivo

New approaches to sterilizing male animals are needed to control captive and wild animal populations. We sought to develop a nonsurgical method of permanent sterilization for male animals by administering the gonadotoxicant melphalan conjugated to peptides derived from the β-chain of FSHβ. We hypothesized that conjugating melphalan to FSHβ peptides would magnify the gonadotoxic effects of melphalan while minimizing systemic toxicity. The ability of conjugates of melphalan and FSHβ peptides to kill murine testicular cells was first tested in vitro in a three-dimensional testicular cell coculture system. In this system, melphalan caused considerable cell death as measured both by increases in lactate dehydrogenase concentrations in the culture supernatant and direct visualization of the cultures. Of the conjugates tested, melphalan conjugated to a 20-amino acid peptide derived from human FSHβ consisting of amino acids 33 to 53 (FSHβ (33–53)-melphalan) was very potent, with cell cytotoxicity and lactate dehydrogenase release roughly one-half that of melphalan. The effects of melphalan and FSHβ (33–53)-melphalan on spermatogenesis were then tested in vivo in mature C56Bl/6 male mice. Four weeks after intraperitoneal injection, all mice treated with either FSHβ (33–53)-melphalan or melphalan had approximately 75% reductions in testicular spermatid counts compared with control animals. Testicular histology revealed significant reduction in mature spermatids and spermatocytes in most tubules. However, 12 weeks after the injection, testicular spermatid counts and histology were similar to controls, except in one animal receiving FSHβ (33–53)-melphalan that had no apparent spermatogenesis. We conclude that melphalan and FSHβ (33–53)-melphalan are potent gonadotoxicants in male mice resulting in marked suppression of spermatogenesis 4 weeks after a single intraperitoneal injection. However, this effect is transient in most mice as spermatogenesis is similar to control animals 12 weeks after drug administration. Melphalan or FSHβ (33–53)-melphalan may be useful for the temporary control of fertility in male animals, but additional research will be needed to develop a single dose method of permanent sterilization for male animals.     

Induction of osteoclastogenesis in an in vitro model of Gaucher disease is mediated by T cells via TNF-α

Gaucher disease is a lysosomal storage disorder caused by deficiency of glucocerebrosidase enzymatic activity leading to accumulation of its substrate glucocerebrosidase mainly in macrophages. Skeletal disorder of Gaucher disease is the major cause of morbidity and is highly refractory to enzyme replacement therapy. However, pathological mechanisms of bone alterations in Gaucher disease are still poorly understood. We hypothesized that cellular alteration in Gaucher disease produces a proinflammatory milieu leading to bone destruction through enhancement of monocyte differentiation to osteoclasts and osteoclasts resorption activity. Against this background we decided to investigate in an in vitro chemical model of Gaucher disease, the capacity of secreted soluble mediators to induce osteoclastogenesis, and the mechanism responsible for this phenomena. We demonstrated that soluble factors produced by CBE-treated PBMC induced differentiation of osteoclasts precursors into mature and active osteoclasts that express chitotriosidase and secrete proinflammatory cytokines. We also showed a role of TNF-α in promoting osteoclastogenesis in Gaucher disease chemical model. To analyze the biological relevance of T cells in osteoclastogenesis of Gaucher disease, we investigated this process in T cell-depleted PBMC cultures. The findings suggest that T cells play a role in osteoclast formation in Gaucher disease. In conclusion, our data suggests that in vitro GCASE deficiency, along with concomitant glucosylceramide accumulation, generates a state of osteoclastogenesis mediated in part by pro-resorptive cytokines, especially TNF-α. Moreover, T cells are involved in osteoclastogenesis in Gaucher disease chemical model.     

Diversity of arbuscular mycorrhizal fungi in the rhizosphere of three host plants in the farming–pastoral zone, north China

The spatial diversity and distribution of AM fungi were investigated in three plots located in farming–pastoral zone, north China. The rhizospheres of Caragana korshinskii, Artemisia sphaerocephala and Salix psammophila were sampled and thirty AM fungal species belonging to five genera were isolated. The study demonstrated that AM fungal diversity and distribution differed significantly among the three host plants and the three studied plots. Spore density of AM fungi ranged between 2 and 22 spores per g^?1 of soil and species richness between 8 and 23. Correlation coefficient analysis demonstrated that spore density was significantly and positively correlated with soil organic matter and available N (P?<?0.01). Species richness was significantly and positively correlated with soil organic matter and available P (P?<?0.01), but significantly and negatively correlated with soil pH (P?<?0.01). Finally, the Shannon–Weiner index was significantly and positively correlated with soil organic matter (P?<?0.05). In this farming–pastoral zone, Glomus reticulatum and G.melanosporum may be more adaptable to the arid conditions than other AM fungal species. This research into AM fungal diversity may lead to exploitation of AM fungi for the mitigation of soil erosion and desertification using mycorrhizal plants, such as C.korshinskii, A.sphaerocephala and S. psammophila. The results of this study support the conclusion that diversity and distribution of AM fungi might be useful to monitor desertification and soil degradation.     

Effect of arbuscular mycorrhizal fungi, organic fertilizer and soil sterilization on maize growth ☆

The effects of inoculation with arbuscular mycorrhizal (AM) fungi, organic fertilizer (F) applications, and soil sterilization on maize growth were evaluated in a pot experiment. The experiment was in a completely randomized factorial design (2 × 4 × 2) with six replicates for each treatment. There were two soil treatments (sterilized soil, SS and unsterilized soil, US), four organic fertilizer treatments (0.0, 0.5, 1.0 and 2.0 g kg^-1 soil), and two AM fungi treatments (inoculation with Glomus mosseae, +AM and uninoculated control, －AM). Inoculated plants generally had greater AM colonization, plant height, dry weight and phosphorus (P) uptake than uninoculated controls, and these parameters were significantly increased as the organic fertilizer application increased up to 0.5 g kg^-1 but decreased or had no significant effect compared to the uninoculated plants at the highest fertilizer rate (2.0 g kg^-1). Plant growth, P uptake and AM colonization of root system were significantly higher in sterilized soil compared to the unsterilized control. Our results indicated that the inoculation of AM fungi in field soil with optimal organic fertilizer application greatly improved maize growth and nutrient uptake, and the effect was greater under sterilized soil condition.     

Effect of temporary meiosis block during prematuration of bovine cumulus–oocyte complexes on pregnancy rates in a commercial setting for in vitro embryo production

Ovum pick up (OPU) associated with in vitro production (IVP) of embryos has been shown as an important tool in cattle breeding to increase the number of descendants from animals of high genetic value. In herds maintained distant from the laboratory, collecting cumulus–oocyte complexes (COCs) and transporting them to the laboratory may take several hours and decrease COCs viability, representing a challenge for commercial settings. In this study, a prematuration culture to induce temporary meiosis block was evaluated in a commercial scale IVP setting as a strategy to transport bovine OPU-derived COCs from Nelore and Brangus donors. Effects on embryo yield and pregnancy rates were assessed. Viable COCs from each donor were destined to one of the experimental groups (control, blocks 1 and 2). Control group COCs were placed in cryotubes with 1 mL TCM199–HEPES. In block groups (1 and 2), COCs were placed in cryotubes with 300 μL TCM 199 + 12 μM butyrolactone I (block medium). All groups were gassed and kept in a thermos bottle for 4 hours at 36 °C. Next, COCs in the control group were transferred to IVM medium and block 1 group to block medium, and cultured for 22 hours and 15 hours, respectively, at 38.5 °C and 5% CO₂ in air. Block 2 COCs were kept in the cryotubes and in the thermos bottle for another 15 hours at 36 °C to simulate long-term transport conditions. After meiosis block in prematuration culture, blocks 1 and 2 COCs were matured in vitro for 22 hours as for the control group. After IVM, COCs in all groups were submitted to IVF and IVC, and blastocyst rates were evaluated on day 7. Embryos were transferred and pregnancy rates evaluated at 60 days of gestation. The mean total number of COCs retrieved by OPU did not differ between Nelore and Brangus donors (16.8 and 17.2, respectively, P > 0.05), but Nelore donors produced more viable COCs than Brangus (10.1 and 7.6, respectively, P < 0.05) and more embryos/cow (3.8 and 2.7, respectively, P < 0.05). Blastocyst rates were similar for control (40.2% and 36.7%), block 1 (37.3% and 34.5%), and block 2 groups (34.7% and 33.6%) for Nelore and Brangus cattle, respectively (P > 0.05). Pregnancy rates did not differ regardless of breed or treatment (36.7%, P > 0.05). In conclusion, temporary meiosis block during prematuration culture did not affect embryo development or pregnancy rates; therefore, this strategy may be used to transport bovine COCs in a commercial IVP setting.     

Improvement of catalytic properties of lipase from Arthrobacter sp. by encapsulation in hydrophobic sol–gel materials

In this work, lipase from Arthrobacter sp. was immobilized by sol–gel encapsulation to improve its catalytic properties. Various silanizing agents including vinyl-trimethoxy silane, octyl-trimethoxy silane, γ-(methacryloxypropyl)-trimethoxy silane (MAPTMS) and tetraethoxysilane (TEOS) were chosen as the precursors. Among them, MAPTMS was for the first time utilized to encapsulate lipases, and the prepared enzyme by copolymerization of MAPTMS and TEOS exhibited the highest activity in both the hydrolysis of p-nitrophenyl palmitate and the asymmetric acylation of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one. The effects of various immobilization parameters were investigated. Under the optimum conditions of MAPTMS/TEOS = 1/1 (mol/mol), water/silane molar ratio (R value) = 20 and lipase loading = 0.01 g/mL sol, the total activity of the immobilized enzyme reached up to 13.6-fold of the free form. Moreover, the encapsulated lipase exhibited higher thermal stability than the free form and retained 54% of the original activity after uses for 60 d. Enantioselectivity of enzyme was also improved with an E value of 150 after encapsulation from 85 for the free form.     

Effects of docosahexaenoic acid on in vitro amyloid beta peptide 25–35 fibrillation

Amyloid β peptide_25–35 (Aβ_25–35) encompasses one of the neurotoxic domains of full length Aβ_1–40/42, the major proteinaceous component of amyloid deposits in Alzheimer's disease (AD). We investigated the effect of docosahexaenoic acid (DHA, 22:6, n-3), an essential brain polyunsaturated fatty acid, on the in vitro fibrillation of Aβ_25–35 and found that it significantly reduced the degree of fibrillation, as shown by a decrease in the intensity of both the thioflavin T and green fluorescence in confocal microscopy. Transmission electron microscopy revealed that DHA-incubated samples were virtually devoid of structured fibrils but had an amorphous-like consistency, whereas the controls contained structured fibers of various widths and lengths. The in vitro fibrillation of Aβ_25–35 appeared to be pH-dependent, with the strongest effect seen at pH 5.0. DHA inhibited fibrillation at all pHs, with the strongest effect at pH 7.4. It also significantly decreased the levels of Aβ_25–35 oligomers. Nonreductive gradient gel electrophoresis revealed that the molecular size of the oligomers of Aβ_25–35 was 10 kDa (equivalent to decamers of Aβ_25–35) and that DHA dose-dependently reduced these decamers. These results suggest that DHA decreases the in vitro fibrillation of Aβ_25–35 by inhibiting the oligomeric amyloid species and, therefore, Aβ_25–35-related neurotoxicity or behavioral impairment could be restrained by DHA.     

Ectomycorrhizal fungi increase the vitality of Norway spruce seedlings under the pressure of Heterobasidion root rot in vitro but may increase susceptibility to foliar necrotrophs

We tested if root colonisation by ectomycorrhizal fungi (EMF) could alter the susceptibility of Norway spruce (Picea abies) seedlings to root rot infection or necrotic foliar pathogens. Firstly, spruce seedlings were inoculated by various EMF and challenged with Heterobasidion isolates in triaxenix tubes. The ascomycete EMF Meliniomyces bicolor, that had showed strong antagonistic properties towards root rot causing Heterobasidion in vitro, protected spruce seedlings effectively against root rot. Secondly, spruce seedlings, inoculated with M. bicolor or the forest humus, were subjected to necrotrophic foliar pathogens in conventional forest nursery conditions on peat substrates. Botrytis cinerea infection after winter was mild and the level of needle damage was independent of substrate and EMF colonisation. Needle damage severity caused by Gremminiella abietina was high in seedlings grown in substrates with high nutrient availability as well as in seedlings with well-established EMF communities. These results show that albeit M. bicolor is able to protect spruce seedlings against Heterobasidion root rot in axenic cultures it fails to induce systemic protection against foliar pathogens. We also point out that unsterile inoculum sources, such as the forest humus, should not be considered for use in greenhouse conditions as they might predispose seedlings to unintended needle damages.     

Ultra structure analysis of cell–cell interactions between pericytes and neutrophils in vitro

Neutrophils’ adhesion to the endothelium during inflammatory is a well-known processes. In contrast the interaction of neutrophils with cells of the neurovascular unit after they have been transmigrated into the brain is less clear. Recently, lymphocyte function-associated antigen-1 (LFA-1) dependent subendothelial crawling of neutrophils has been observed in vivo. This is mediated by intracellular adhesion molecule-1 (ICAM-1), which is expressed on the cell surface of pericytes. In our work we demonstrated in vitro a cell–cell interaction between porcine brain capillary pericytes (PBCPs) and neutrophils, with further characterization of the initial contact between these cells. PBCPs increase ICAM-1 protein expression in response to the cytokine tumor necrosis factor-alpha (TNF-α). Furthermore, an increase in neutrophil adhesion to PBCPs was determined by immunofluorescence staining. By means of scanning force microscopy (SFM), we could additionally show that pericytes as well as neutrophils form cell extensions towards the neighboring cell. Interestingly, these extensions differ for different cell types.     

Rapid degradation of lindane (��-hexachlorocyclohexane) at low temperature by Sphingobium strains

Bioremediation of anthropogenic organic pollutants in cold climates is often limited by lower microbial or enzyme activity induced by low temperature. The present study addressed this issue through the degradation of ??-hexachlorocyclohexane (??-HCH) by three Sphingobium strains (S. indicum B90A, S. japonicum UT26 and S. francense Sp+) under low temperature (4 °C). After 5 days incubation at 4 °C, 79.7% and 43.8% of 5 and 25 mg L⁻¹ of ??-HCH added were degraded, respectively by the inoculation of 1.75 × 10⁷ cells mL⁻¹ of S. indicum B90A. An increase in inoculum concentration to 1.72 × 10⁸ cells mL⁻¹ significantly increased the degradation to 98.1 ± 1.7% of 5 mg L⁻¹ within 24 h. Further, S. indicum B90A and S. japonicum UT26 can rapidly degrade ??-HCH at 4 °C, while the degradation capability of S. francense Sp+ is relatively low. At 4 °C, ??-HCH is transformed to extremely low amounts of 1,2,4-trichlorobenzene (1,2,4-TCB) and 2,5-dichlorophenol (2,5-DCP) by S. indicum B90A, but most of ??-HCH were transformed to 2,5-Dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) by S. japonicum UT26. These results revealed that haloalkane dehalogenases in some Sphingobium species are very active at temperature as low as 4 °C and S. indicum B90A might be a good candidate for developing novel bioremediation techniques for cold regions to decontaminate ??-HCH from soils/waters.     

Genetic and phytochemical analysis of the in vitro regenerated Pilosocereus robinii by ISSR,SDS–PAGE and HPLC

Pilosocereus robinii is a rare species which is experiencing sudden population collapse. Identifying and developing effective conservation and management strategies to halt the forestall extinction of this species is crucial. The present study was conducted to assess the best conditions for in vitro propagation of this plant in regard to its morphogenic, genetic as well as the chemical potentials. A successful in vitro propagation system of P. robinii has been developed. MS hormone-free medium induced the best root morphogenic potential. The plants were acclimatized in the greenhouse at 100% survival rate. Besides, the somaclonal variations between the in vitro raised plants were analyzed using PCR-ISSR markers and SDS–PAGE protein, where the regenerated explants on MS medium supplemented with TDZ were the highest in inducing new specific marker bands. Sh6 ISSR primer showed the highest polymorphism value, 81.8% with 33 total amplified fragments, while Sh3 ISSR primer showed the lowest value with polymorphic percentage of 14.3%. Furthermore, SDS–PAGE protein analysis showed no variation in protein pattern of the studied treatments. On the other side, HPLC analysis of the in vitro plantlets extracts has shown that 2iP based treatments were the highest in organic acids accumulation, while the phenolic constituents' accumulation was found to reach its peak in the BA based treatments.     

Direct and indirect influences of arbuscular mycorrhizal fungi on phosphorus uptake by two root hemiparasitic Pedicularis species: do the fungal partners matter at low colonization levels?

Background and Aims

Because most parasitic plants do not form mycorrhizal associations, the nutritional roles of arbuscular mycorrhizal (AM) fungi in them have hardly been tested. Some facultative root hemiparasitic Pedicularis species form AM associations and hence are ideal for testing both direct and indirect effects of AM fungi on their nutrient acquisition. The aim of this study was to test the influence of AM inoculation on phosphorus (P) uptake by Pedicularis rex and P. tricolor.

Methods

³²P labelling was used in compartmented pots to assess the contribution of the AM pathway and the influence of AM inoculation on P uptake from a host plant into the root hemiparasites. Laboratory isolates of fungal species (Glomus mosseae and G. intraradices) and the host species (Hordeum vulgare ‘Fleet’) to which the two Pedicularis species showed obvious responses in haustorium formation and growth in previous studies were used.

Key Results

The AM colonization of both Pedicularis spp. was low (<15 % root length) and only a very small proportion of total plant P (<1 %) was delivered from the soil via the AM fungus. In a separate experiment, inoculation with AM fungi strongly interfered with P acquisition by both Pedicularis species from their host barley, almost certainly because the numbers of haustoria formed by the parasite were significantly reduced in AM plants.

Conclusions

Roles of AM fungi in nutrient acquisition by root parasitic plants were quantitatively demonstrated for the first time. Evidence was obtained for a novel mechanism of preventing root parasitic plants from overexploiting host resources through AM fungal-induced suppression of the absorptive structures in the parasites.     

Exploration of structure–function relationships in Escherichia coli cystathionine γ-synthase and cystathionine β-lyase via chimeric constructs and site-specific substitutions

Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL) share a common structure and several active-site residues, but catalyze distinct side-chain rearrangements in the two-step transsulfuration pathway that converts cysteine to homocysteine, the precursor of methionine. A series of 12 chimeric variants of Escherichia coli CGS (eCGS) and CBL (eCBL) was constructed to probe the roles of two structurally distinct, ~ 25-residue segments situated in proximity to the amino and carboxy termini and located at the entrance of the active-site. In vivo complementation of methionine-auxotrophic E. coli strains, lacking the genes encoding eCGS and eCBL, demonstrated that exchange of the targeted regions impairs the activity of the resulting enzymes, but does not produce a corresponding interchange of reaction specificity. In keeping with the in vivo results, the catalytic efficiency of the native reactions is reduced by at least 95-fold, and α,β versus α,γ-elimination specificity is not modified. The midpoint of thermal denaturation monitored by circular dichroism, ranges between 59 and 80 °C, compared to 66 °C for the two wild-type enzymes, indicating that the chimeric enzymes adopt a stable folded structure and that the observed reductions in catalytic efficiency are due to reorganization of the active site. Alanine-substitution variants of residues S32 and S33, as well as K42 of eCBL, situated in proximity to and within, respectively, the targeted amino-terminal region were also investigated to explore their role as determinants of reaction specificity via positioning of key active-site residues. The catalytic efficiency of the S32A, S33A and the K42A site-directed variants of eCBL is reduced by less than 10-fold, demonstrating that, while these residues may participate in positioning S339, which tethers the catalytic base, their role is minor.