Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.

We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.     

Conformational changes in fragments D and double-D from human fibrin(ogen) upon binding the peptide ligand Gly-His-Arg-Pro-amide

The structure of fragment double-D from human fibrin has been solved in the presence and absence of the peptide ligands that simulate the two knobs exposed by the removal of fibrinopeptides A and B, respectively. All told, six crystal structures have been determined, three of which are reported here for the first time: namely, fragments D and double-D with the peptide GHRPam alone and double-D in the absence of any peptide ligand. Comparison of the structures has revealed a series of conformational changes that are brought about by the various knob-hole interactions. Of greatest interest is a moveable "flap" of two negatively charged amino acids (Glubeta397 and Aspbeta398) whose side chains are pinned back to the coiled coil with a calcium atom bridge until GHRPam occupies the beta-chain pocket. Additionally, in the absence of the peptide ligand GPRPam, GHRPam binds to the gamma-chain pocket, a new calcium-binding site being formed concomitantly.     

Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India

Background

Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.

Methods/Principal Findings

In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients’ sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05).

Conclusion

The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.     

Follicular and endocrinological turnover associated with GnRH induced follicular wave synchronization in Indian crossbred cows

The goal of this study was to record the hormonal and follicular turnover in Jersey crossbred cows when subjected for follicular wave synchronization using GnRH. Six healthy, non-lactating and regularly cycling Jersey crossbred cows (5-6 y) were used for the study. In the control group, the follicular wave pattern was ultrasonographically investigated in 18 cycles (3 cycles/cow). In the treatment group, GnRH analogue (buserelin acetate 10 μg im) was administered on Day 6 of the cycle and follicular wave pattern was studied in 12 cycles (2 cycles/animal). Follicular population was categorized based on their diameter Class I, ≤5 mm; Class II, >5-<9 mm; Class III, ≥9 mm) and the number of follicles in each category was determined on Day 6, Day 8 and Day 10. Plasma FSH and progesterone concentrations were estimated in both control and treatment groups. Out of 18 estrous cycles studied, 14 cycles (77.8%), three cycles (16.7%) and one cycle (5.6%) exhibited three-, two- and four-follicular waves per cycle, respectively. It was evident that the DF of Wave I established its dominance and was in the growing phase by Day 6 of the estrous cycle in all the normally cycling crossbred cows. The DF ovulated in all the animals (100%) in the mean interval of 27.7 ± 0.2 h after GnRH administration. A synchronized homogenous group of follicles emerged two days after GnRH injection (Day of 8.0 ± 0.0) in all the animals (100%). The combination of LH surge induced ovulation of DF (abrupt termination of Wave I) and FSH surge stimulated homogenous recruitment of Class I follicles, led to a synchronized emergence of follicular wave. All the GnRH treated cows had three follicular waves because of early emergence and short period of dominance of Wave II DF.     

X-ray characterisation of an additional binding site in lysozyme

Bromophenol red (BPR) binds to lysozyme and inhibits its activity against bacterial cell walls, but not against the polysaccharide component of peptidoglycan. The binding site of BPR in the enzyme has been characterised by X-ray analysis of the complex at 5.5A resolution. The new binding site, which is outside the cleft close to subsite F, is presumably involved in interactions with the peptide component of peptidoglycan, in the action of lysozyme against bacterial cell walls.     

Structural transformations in protein crystals caused by controlled dehydration

Recent experiments in this laboratory on structural transformations caused by controlled dehydration of protein crystals have been reviewed. X-ray diffraction patterns of the following crystals have been examined under varying conditions of environmental humidity in the relative humidity range of 100-75%: a new crystal form of bovine pancreatic ribonuclease A grown from acetone solution in tris buffer (I), the well-known monoclinic form of the protein grown from aqueous ethanol (II), the same form grown from a solution of 2-methyl pentan-2,4-diol in phosphate buffer (III), tetragonal (IV), orthorhombic (V), monoclinic (VI) and triclinic (VII) hen egg white lysozyme, porcine 2 Zn insulin (VIII), porcine 4 Zn insulin (IX) and the crystals of concanavalin A(X). I, II, IV, V and VI undergo one or more transformations as evidenced by discontinuous changes in the unit cell dimensions, the diffraction pattern and the solvent content. Such water-mediated transformations do not appear to occur in the remaining crystals in the relative humidity range explored. The relative humidity at which the transformation occurs is reduced when 2-methyl pentan-2,4-diol is present in the mother liquor. The transformations are affected by the crystal structure but not by the amount of solvent in the crystals. The X-ray investigations reviewed here and other related investigations emphasize the probable importance of water-mediated transformations in exploring hydration of proteins and conformational transitions in them.     

Computer automated motion analysis of crossbred bull spermatozoa and its relationship with in vitro fertility in zona-free hamster oocytes

The objective of this study was to determine the effective relationship between different motion characteristics of bull spermatozoa assessed by computer assisted semen analyzer (CASA) and in vitro fertilization percentage in zona-free hamster oocytes. A total of 64 frozen semen samples from 16 different crossbred bulls (Bos taurusxBos indicus) with four ejaculates from each bull were taken for analysis. Various motion characteristics of spermatozoa like progressive motility, path velocity, progressive velocity, beat cross frequency, straightness and linearity were recorded. Hypo-osmotic swelling test and sperm penetration bioassay were conducted to assess the membrane integrity and fertilization percentage of spermatozoa respectively. Significant positive correlation (P<0.01) was found between fertilization percentage and progressive motility (r=0.791) and between velocity parameters (VAP; r=0.612 and VSL; r=0.625) and fertilization percentage. Among different CASA variables, progressive motility alone contributed to 62.6% variation in the fertilization percentage. The velocity measurements (VAP and VSL) together with progressive motility and %HOS spermatozoa contributed to 66.1% of variation in fertilization percentage which was found to be significant (P<0.05).