Proteomic screening and identification of differentially distributed membrane proteins in Escherichia coli

Bacteria show asymmetric subcellular distribution of many proteins involved in diverse cellular processes such as chemotaxis, motility, actin polymerization, chromosome partitioning and cell division. In many cases, the specific subcellular localization of these proteins is critical for proper regulation and function. Although cellular organization of the bacterial cell clearly plays an important role in cell physiology, systematic studies to uncover asymmetrically distributed proteins have not been reported previously. In this study, we undertook a proteomics approach to uncover polar membrane proteins in Escherichia coli. We identified membrane proteins enriched in E. coli minicells using a combination of two-dimensional electrophoresis and mass spectrometry. Among a total of 173 membrane protein spots that were consistently detected, 36 spots were enriched in minicell membranes, whereas 15 spots were more abundant in rod cell membranes. The minicell-enriched proteins included the inner membrane proteins MCPs, AtpA, AtpB, YiaF and AcrA, the membrane-associated FtsZ protein and the outer membrane proteins YbhC, OmpW, Tsx, Pal, FadL, OmpT and BtuB. We immunolocalized two of the minicell-enriched proteins, OmpW and YiaF, and showed that OmpW is a bona fide polar protein whereas YiaF displays a patchy membrane distribution with a polar and septal bias.     

Variation of toxigenic Vibrio cholerae O1 in the aquatic environment of Bangladesh and its correlation with the clinical strains

The diversity of toxigenic V. cholerae O1 in the aquatic environment of Bangladesh is not known. A total of 18 environmental and 18 clinical strains of toxigenic V. cholerae O1 were isolated simultaneously from four different geographical areas and tested for variation by the pulsed-field gel electrophoresis method. Environmental strains showed diversified profiles and one of the profiles was common to some environmental strains and most clinical strains. It appears that one clone has an advantage over others to cause disease. These findings suggest that the study of the molecular ecology of V. cholerae O1 in relation to its environmental reservoir is important in identifying virulent strains that cause disease.     

Interaction of aldehydes with collagen: effect on thermal, enzymatic and conformational stability

Stabilization of type I rat tail tendon (RTT) collagen by various aldehydes, viz. formaldehyde, gluteraldehyde, glyoxal and crotanaldehyde was studied to understand the effect of each on the thermal, enzymatic and conformational stability of collagen. The aldehydes have been found to increase the heat stability of rat tail tendon collagen fibres from 62 to 77-86 degrees C. The increase in thermal stability was found to be in a species dependent manner. The variation in the thermal stability of collagen brought about by aldehydes was in the order of formaldehyde > gluteraldehyde > glyoxal > crotanaldehdye. The aldehydes also impart a high degree of stability to collagen against the activity of the degrading enzyme, collagenase. The order of enzymatic stability brought about by aldehydes follows the same trend as the thermal stability brought about by them. This shows that the number of cross-links formed influence both the thermal and enzymatic stability in the similar manner. The effect of various aldehydes on the secondary structure of collagen was studied using circular dichroism and it was found that the aldehydes lead to changes in the amplitude of the circular dichroic (CD) spectrum but did not alter the triple helical conformation of collagen. The secondary structure of collagen is not significantly altered on interaction with different aldehydes.     

Fluorescence resonance energy transfer from tryptophan to a chromium(III) complex accompanied by non-specific cleavage of albumin: a step forward towards the development of a novel photoprotease

A chromium(III) complex, transdiaqua [N, N'-propylenebis(salicylideneimino)chromium(III)]perchlorate ([Cr(salprn)(H2O)(2)]ClO(4)) in the presence of sodium azide and upon photoexcitation was found to bring about non-selective cleavage of bovine serum albumin (BSA). Electron paramagnetic resonance (EPR) evidence has been obtained for the formation of a Cr(V) species upon photolysis of a solution containing the chromium(III) complex and sodium azide. This Cr(V) species non-selectively cleaves BSA. The fluorescence excitation spectrum of BSA-[Cr(salprn)(H2O)(2)]+ adduct showed a band at lambda(max)(ex) nm due to charge transfer transition of the chromium(III) complex as well as a prominent band at 290 nm attributable to tryptophan absorption. This indicated an efficient Forster type fluorescence energy transfer (FRET) from the tryptophan residues to the chromium(III) complex indicating that the Cr(III) complex binds in the vicinity of the tryptophan residue.     

A Schiff base complex of chromium(III): an efficient inhibitor for the pathogenic and invasive potential of Shigella dysenteriae

A Schiff base complex of chromium(III), transdiaqua[N,N'ethylenebis (salicylideneimine)chromium(III)]perchlorate, [Cr(salen)(OH(2))(2)](+), was found to have an inhibitory effect on the growth of Shigella dysenteriae. The chromium(III) complex was found to cure (remove) the invasive plasmid and thereby render the microbe more sensitive to the tested antibiotics. The loss in the catalytic activity of the isolated endo-alpha-N-acetyl galactosaminidase on mucin as a substrate was also observed in the presence of [Cr(salen)(OH(2))(2)](+). This suggests that [Cr(salen)(OH(2))(2)](+) is toxic to the microbe and could make the microbe non-pathogenic and non-invasive, thus establishing its role in microbiological applications to reduce the toxic potentials of a microbe.     

Progress and recent trends in biotechnological methods for leather processing

Global environmental regulations are changing the leather-processing industry. Pre-tanning and tanning processes contribute 80-90% of the total pollution in the industry and generate noxious gases, such as hydrogen sulfide, as well as solid wastes, such as lime and chrome sludge. The use of enzyme-based products is currently being explored for many areas of leather making. Furthermore, enzymes are gaining increasing importance in the de-hairing process, eliminating the need for sodium sulfide. This review discusses emerging novel biotechnological methods used in leather processing. One significant achievement is the development of a bioprocess-based de-hairing and fiber-opening methodology to reduce toxic waste.     

Post pressure hyperemia in the rat

In prior studies in man, we have demonstrated that pressure-induced hyperemia lasts for prolonged periods as compared to the short-term hyperemia created by proximal arterial occlusion. We have analyzed this phenomenon in our well-studied rat model of skin blood flow. Skin blood flow was measured using laser Doppler techniques in Wistar Kyoto rats at the back, a nutritively perfused site, and at the plantar surface of the paw, where arteriovenous anastomotic perfusion dominates. A customized pressure feedback control device was used to vary applied pressures. At the back, pressures in excess of 80 mmHg resulted in occlusion, whereas at the paw 150 mmHg was required. The peak hyperemic flow after release of pressure was comparable to that elicited by proximal arterial occlusion with a blood pressure cuff. However, the post pressure hyperemia peak descended to a plateau value, which was 50-100% greater than baseline and continued for up to 20 min while the peak following proximal arterial occlusion returned to baseline within 4 min. At the back, post pressure hyperemia reached a maximum after application of 100 mmHg pressure. The application of higher pressures than required for occlusion produced no greater hyperemic response. At the paw, maximum post pressure hyperemia occurred at 100 mmHg, although this pressure level was not totally occlusive. Higher pressures resulted in no greater hyperemia. At the back, 10 min of occlusion produced a maximal peak value whereas 1 min was sufficient at the paw. The application of pressure to a heated probe with subsequent release, produced a hyperemic response. Normalized to baseline blood flow, there was no difference between the hyperemic responses at basal skin temperature and at 44 degrees C. There is a prolonged hyperemic response following local pressure occlusion compared to a much shorter period following proximal ischemic occlusion. One can presume two different mechanisms, one related to ischemia and the other a separate pressure related phenomenon. The thermal vasodilatory response is additive, not synergistic with the post pressure hyperemia we have demonstrated. This finding suggests that different mechanisms are involved in thermal vasodilation and post pressure hyperemia.     

Investigation of various factors affecting encapsulation on the In-Cap automatic capsule-filling machine

The purpose of this study was to determine the factors that influence fill weight and weight variability of capsules produced on the In-Cap and to assess any differences in terms of capsule defects between gelatin and HPMC (Quali-V) shells. The In-Cap is an automatic tamping type capsule-filling machine and the low output of ≈3000 capsules/hour makes it ideal for early formulation development and phase I/IIa clinical supplies manufacture. Four commonly used excipients (Avicel PH101, Avicel PH302, A-Tab, and Prosolv HD90) and a poorly flowing drug blend were encapsulated at various pin settings and powder bed heights. The average fill weight and coefficient of weight variation were determined. The percentage of defective capsules formed during encapsulation was calculated. Results of the study showed that pin setting was critical for controlling the fill weight and the weight variation. The order of pin setting with pin 1 (closer to the powder chute) set to a relatively higher position and pin 4 (before ejection) set to a lower position was found to give higher fill weights with relatively lower weight variability. The powder bed height influenced the fill weight for poorly flowing powders. The capsule machine speed did not appear to significantly influence the fill weight. The fill weight and weight variation were found to depend on the flow property of the material. A large percentage of defective capsules was obtained using HPMC shell size #00. Some of the commonly observed defects included split caps and improperly closed filled capsules. In general, appropriate selection of pin settings and bed height can reduce the weight variability seen, especially with poorly flowing high-dose formulations.     

LOC3D: annotate sub-cellular localization for protein structures

LOC3D (http://cubic.bioc.columbia.edu/db/LOC3d/) is both a weekly-updated database and a web server for predictions of sub-cellular localization for eukaryotic proteins of known three-dimensional (3D) structure. Localization is predicted using four different methods: (i) PredictNLS, prediction of nuclear proteins through nuclear localization signals; (ii) LOChom, inferring localization through sequence homology; (iii) LOCkey, inferring localization through automatic text analysis of SWISS-PROT keywords; and (iv) LOC3Dini, ab initio prediction through a system of neural networks and vector support machines. The final prediction is based on the method that predicts localization with the highest confidence. The LOC3D database currently contains predictions for >8700 eukaryotic protein chains taken from the Protein Data Bank (PDB). The web server can be used to predict sub-cellular localization for proteins for which only a predicted structure is available from threading servers. This makes the resource of particular interest to structural genomics initiatives.