Metal-binding stoichiometry and selectivity of the copper chaperone CopZ from Enterococcus hirae.

We studied the interaction of several metal ions with the copper chaperone from Enterococcus hirae (EhCopZ). We show that the stoichiometry of the protein-metal complex varies with the experimental conditions used. At high concentration of the protein in a noncoordinating buffer, a dimer, (EhCopZ)2-metal, was formed. The presence of a potentially coordinating molecule L in the solution leads to the formation of a monomeric ternary complex, EhCopZ-Cu-L, where L can be a buffer or a coordinating molecule (glutathione, tris(2-carboxyethyl)phosphine). This was demonstrated in the presence of glutathione by electrospray ionization MS. The presence of a tyrosine close to the metal-binding site allowed us to follow the binding of cadmium to EhCopZ by fluorescence spectroscopy and to determine the corresponding dissociation constant (Kd = 30 nm). Competition experiments were performed with mercury, copper and cobalt, and the corresponding dissociation constants were calculated. A high preference for copper was found, with an upper limit for the dissociation constant of 10-12 m. These results confirm the capacity of EhCopZ to bind copper at very low concentrations in living cells and may provide new clues in the determination of the mechanism of the uptake and transport of copper by the chaperone EhCopZ.     

Uses and limitations of monoclonal antibodies to Giardia lamblia-specific 66-kDa copro-antigen in copro-immunodiagnosis of giardiasis

Abstract Antigen-capture enzyme-linked immunosorbent assay for the detection of Giardia lamblia -specific antigen in stool eluates from clinical subjects employing monoclonal antibody directed at 66-kDa G. lamblia copro-antigen has been evaluated. The G. lamblia copro-antigen was detected in 67% (31 of the 46 cases) of stool eluates from clinical cases, while none of the stool eluates from subjects with other intestinal parasites or from apparently healthy individuals, had detectable levels of G. lamblia copro-antigen. Monoclonal antibodies secreted by clones B4C5 and D3F4 recognised the periodate-sensitive and -insensitive epitopes of 66-kDa G. lamblia specific copro-antigen, respectively. Eight (73%) of the 11 symptomatic cases of giardiasis had trypsin-/periodate-sensitive epitopes of 66-kDa copro-antigen while 9 (92%) of 11 of the symptomatic cases and asymptomatic G. lamblia cyst carriers had trypsin-sensitive periodate-insensitive G. lamblia specific copro-antigen. The data tend to suggest that detection of periodate-insensitive epitopes of G. lamblia copro-antigen would indicate the presence of the parasite while the detection of periodate sensitive epitopes of G. lamblia copro-antigen would suggest symptomatic active giardial infection.     

Glutamine synthetase induction in chick embryo retina monolayers

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially single cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4--6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4 degrees C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.     

Cyclical changes in the pituitary gonadotrophs (GTH cells) in relation to the testicular cycle in Puntius sarana (Hamilton)

The pituitary gland of Puntius sarana is of the leptobasic type and has three subdivisions--a proximal pars distalis (PPD), a rostral pars distalis (RPD) and a pars intermedia (PI) arranged vertically one below the other. The gonadotrophs (GTH cells) of the PPD were identified by their PAS, ATh, AF, aniline blue and alcian blue positivity and by their immunoreactivity to ovine anti-LH beta-subunit, anti-salmon GTH and anti-silver carp GTH. The testes of Puntius sarana have been classified as the "unrestricted type". The morphological, histological and histometric findings indicate that the testing passes through four distinct stages--preparatory, fully mature, spawning and spent. Spermatogenesis begins in January and continues up to July. The spawning season lasts from late July until October. Cyclical changes in the gonadotrophs correlate well with changes in the testes. The histometric data show that the GTH cells attain maximum values (size and number) during the breeding season. The peak values for testicular weight, lobule diameter, the gonadosomatic index (GSI) and the sperm concentration in the testes, and the number, size and amount of secretory material in the cytoplasm of the gonadotrophs, coincide with the time of maximum environmental temperature and the longest days.     

Isolation of a minireplicon of the virulence plasmid pXO2 of Bacillus anthracis and characterization of the plasmid-encoded RepS replication protein

A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis, Bacillus cereus, and Bacillus subtilis. The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5' and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.     

Living with a large predator: Assessing the root causes of Human–brown bear conflict and their spatial patterns in Lahaul valley,Himachal Pradesh

Brown bear‐mediated conflicts have caused immense economic loss to the local people living across the distribution range. In India, limited knowledge is available on the Himalayan brown bear (HBB), making human–brown bear conflict (HBC) mitigation more challenging. In this study, we studied HBC in the Lahaul valley using a semi‐structured questionnaire survey by interviewing 398 respondents from 37 villages. About 64.8% of respondents reported conflict in two major groups—crop damage (30.6%) and livestock depredations (6.2%), while 28% reported both. Conflict incidences were relatively high in summer and frequently occurred in areas closer to the forest (<500 m) and between the elevations range of 2700 m to 3000 m above sea level (asl). The dependency of locals on forest resources (70%) for their livelihood makes them vulnerable to HBC. The “upper lower” class respondents were most impacted among the various socioeconomic classes. Two of the four clusters were identified as HBC hot spots in Lahaul valley using SaTscan analysis. We also obtained high HBC in cluster II with a 14.35 km radius. We found that anthropogenic food provisioning for HBB, livestock grazing in bear habitats, and poor knowledge of animal behavior among the communities were the major causes of HBC. We suggest horticulture crop waste management, controlled and supervised grazing, ecotourism, the constitution of community watch groups, and others to mitigate HBC. We also recommend notifying a few HBB abundant sites in the valley as protected areas for the long‐term viability of the HBB in the landscape.