Effects of suprachiasmatic transplants on circadian rhythms of neuroendocrine function in golden hamsters

A number of kinetic measurements of peptide dissociation from class II MHC-peptide complexes provide compelling evidence for the existence of conformational isomers in solution. There is evidence that T-lymphocytes can distinguish such isomers. However, virtually nothing is known about the structure of these isomers. Accordingly, we have investigated a water-soluble version of the murine class II MHC molecule I-Ek complexed with an antigenic peptide derived from pigeon cytochrome c residues 89-104 (PCC) by 19F-NMR. Two fluorine labels were placed on the PCC peptide; one fluorine label was placed at a MHC contact site, the other at a position involved in T-cell receptor (TCR) recognition. Introduction of these labels did not alter the observed kinetics of the PCC/I-Ek complex. The NMR data show two conformational isomers of this immunogenic complex. The presence of conformational isomers at a TCR contact site suggests that these structures may be recognized differently by the TCR. The agreement between the dissociation kinetics and the 19F-NMR data demonstrate that kinetic heterogeneity is correlated with structural counterparts observed by NMR. Dissociations in the presence of dimethyl sulfoxide were used to show that the rate of interconversion of these conformational isomers at pH 7.0 is low, with a lifetime on the order of hours or more. Modification of a peptide residue of PCC occupying the minor MHC binding pocket P6 alters the 19F-NMR spectra of both labels. This demonstrates that distant changes of amino acid residues can influence the conformation of the whole antigenic peptide inside the MHC binding cleft.     

Effects of a colostrum replacement product derived from serum on immunoglobulin G absorption by calves

Calves are born hypogammaglobulinemic and rely on immunoglobulin (Ig) from colostrum to obtain passive immunity. Previous research has indicated that colostrum supplements derived from milk are less effective than is maternal colostrum in providing adequate IgG to neonatal calves. Our objective was to determine the absorption of IgG by newborn calves fed a USDA food-grade colostrum supplement derived from bovine serum or fed pooled maternal colostrum. Holstein calves (n = 20; 10 bulls) were removed from the dam within 1 h of birth and were housed in individual stalls for the 24-h study. Calves were fed 2 L of colostrum or colostrum replacer at 1.5 and 13.5 h (+/- 0.1 h). Calves were blocked by colostrum pool, and replacer was fed to provide equal intakes of IgG within blocks. Jugular blood was collected at 1 and 24 h (+/- 0.1 h) for analysis of IgG by radial immunodiffusion. At 24 h, calves were injected with 1.5 ml of Evans blue dye to estimate plasma volume. Mean plasma IgG at 24 h of age was 7.3 +/- 0.4 g/L and was affected by an interaction of block and treatment. Apparent efficiency of IgG absorption of 24 h was reduced when 750 g of the colostrum replacement product were fed but was increased when 266 g of colostrum replacement product were fed. Mean plasma volume was unaffected by treatment and was 3.5 +/- 0.2 L or 9.1% of BW. These data indicate that efficiency of IgG absorption from the colostrum replacement product may be affected by amount of material fed. Proteins other than IgG in the colostrum replacement product might have reduced the efficiency of IgG absorption.     

Control of tuberculosis by community health workers in Bangladesh

BACKGROUND: Tuberculosis remains a major public-health problem in Bangladesh, despite national efforts to improve case identification and treatment compliance. In 1984, BRAC (formerly the Bangladesh Rural Advancement Committee), a national, non-governmental organisation, began an experimental tuberculosis-control programme in one thana (subdistrict). Community health workers screened villagers for chronic cough and collected sputum samples for acid-fast bacillus (AFB) microscopy (phase one). Positive patients received 12 months of directly observed therapy. Phase two (1992-94) included another nine thanas and, in phase three (1995), eight more thanas were included. From 1995, the treatment was an 8-month oral regimen. METHODS: In 1995-96, we analysed all programme data from 1992 to 1995. First we analysed phases two (12-month therapy) and three (8-month therapy) separately for proportion cured, died, treatment, failed, defaulted, migrated, and referred. Second, we did a cross-sectional survey of tuberculosis cases in more than 9000 randomly selected households in two phase-two thanas and one non-programme thana, and analysed the follow-up of all patients treated in the programme thanas. FINDINGS: In the phase-two analysis, 3497 (90%) of 3886 cases identified had accepted 12-month treatment. In phase three, all of 1741 identified cases accepted the 8-month regimen. 2833 (81.0%) and 1496 (85.9%) in phases two and three, respectively, were cured; 336 (9.6%) and 133 (7.6%) died. The relapse rate 2 or more years after treatment was discontinued was higher than the early relapse rate. The drop-out rate was 3.1%. In the cross-sectional survey, the prevalence of tuberculosis in the two programme thanas was half of that in the comparison thana, where only government services were available (0.07 vs 0.15 per 100 [corrected]). INTERPRETATION: The BRAC tuberculosis-control programme has successfully achieved high rates of case detection and treatment compliance, with a cure rate of at least 85% and a drop-out rate of 3.1%. The prevalence survey suggested that at least half of all existing cases had been detected by the programme.     

Fatal non-transport injuries in Nairobi, Kenya

Records from the office of the Registrar of Births and Deaths in Nairobi, Kenya, were studied with the aim of determining the magnitude of fatalities due to injuries sustained in the living environment. This information covered the period between 1986 and 1990. Data were collected over a one month period from 3rd July 1991 to 9th August 1991. The information which was collected from the death certificates included type of injury resulting in death, age and sex of the victim. The results from a total of 944 records revealed that males suffered more deaths than females (M:F ratio was 2.67:1). The most commonly occurring type of injury resulting in death was burns (22.5%). This was followed by drowning (18.1%), head injuries (18%) and suicide by hanging (12%). Stab wounds and poisoning (excluding food poisoning) each accounted for 6% of the total deaths, inhalation of vomit (5.2%) and crush injuries due to falling from a height (3.8%). Bullet wounds, asphyxia due to choking, abortion and electrocution each contributed less than 3% of total deaths. The age bracket with the highest number of deaths was between 20 years to 39 year's (51.4%) while infants and children 0-4 years alone contributed 16% of the total deaths. Since non-transport fatalities are common in all age groups, health education programmes must target both children and the adult population.     

Childhood absence epilepsy with tonic-clonic seizures and electroencephalogram 3-4-Hz spike and multispike-slow wave complexes: linkage to chromosome 8q24

Childhood absence epilepsy (CAE), a common form of idiopathic generalized epilepsy, accounts for 5%-15% of childhood epilepsies. To map the chromosomal locus of persisting CAE, we studied the clinical and electroencephalographic traits of 78 members of a five-generation family from Bombay, India. The model-free affected-pedigree member method was used during initial screening with chromosome 6p, 8q, and 1p microsatellites, and only individuals with absence seizures and/or electroencephalogram 3-4-Hz spike- and multispike-slow wave complexes were considered to be affected. Significant P values of .00000-.02 for several markers on 8q were obtained. Two-point linkage analysis, assuming autosomal dominant inheritance with 50% penetrance, yielded a maximum LOD score (Zmax) of 3.6 for D8S502. No other locus in the genome achieved a significant Zmax. For five smaller multiplex families, summed Zmax was 2.4 for D8S537 and 1.7 for D8S1761. Haplotypes composed of the same 8q24 microsatellites segregated with affected members of the large family from India and with all five smaller families. Recombinations positioned the CAE gene in a 3.2-cM interval.     

Chemical modification and chemical cross-linking for protein/enzyme stabilization

The developmental competence of bovine follicular oocytes that had been meiotically arrested with the phosphokinase inhibitor 6-dimethylaminopurine (6-DMAP) was studied. After 24 h in vitro culture with 2 mM 6-DMAP, 85 +/- 12% of the oocytes were at the germinal vesicle stage compared to 97 +/- 3% at the start of culture (P > 0.05). After release of the 6-DMAP inhibition, followed by 24 h IVM, 82 +/- 18% were at MII stage, compared with 93 +/- 7% in the control group (P > 0.05). The 6-DMAP oocytes displayed a much higher frequency of abnormal MII configurations than the control oocytes (67% vs 23%; P < 0.0001). In addition spontaneous oocyte activation was more frequent than among control oocytes (5% vs 0.3%; P 0.0006). After IVF of 6-DMAP oocytes, normal fertilization was lower (76 +/- 8% vs 89 +/- 7%; P < 0.01), oocyte activation higher (11 +/- 5% vs 2 +/- 2%; P < 0.01), and polyspermy slightly but not significantly higher (8 +/- 7% vs 4 +/- 4%; P > 0.05), compared with the control group. Cleavage was lower (61 +/- 13% vs 81 +/- 6%; P < 0.001), as well as day 8 blastocyst formation (17 +/- 7% vs 36 +/- 8%; P < 0.001). The MII kinetics was different for 6-DMAP and control oocytes. Maximum MII levels were reached at 22 h IVM in both groups, but 50% MII was reached at 17 h in 6-DMAP oocytes, compared to 20 h in control oocytes. Ultrastructure of MII oocytes was similar in the two groups, but in 6-DMAP oocytes the ooplasmic vesicle pattern at GV was at a more advanced stage than in control oocytes. In conclusion, 6-DMAP exposure of GV oocytes prior to IVM induce asynchronous cytoplasmic maturation, leading to aberrant MII kinetics. Thus, at the time of insemination a smaller cohort of oocytes will be at the optimal stage for normal fertilization and subsequent blastocyst development.     

Physiological investigations by image analysis

Cataract formation in diabetic lenses has been attributed to polyol-osmotic pressure-generated influx of water. The ensuing swelling in the form of pocket and lake accumulations cause light scattering. The authors tested whether clear lenses of diabetic patients show different hydration properties than age matched normal lenses. Normal and diabetic human lenses were investigated for their nonfreezable water content by differential scanning calorimetry. The total water content of the lens sections were studied by thermogravimetric analysis. Non-cataractous diabetic lenses in all three regions showed a higher total water content than normal lenses. The nonfreezable water content, seems to increase with age in diabetic lenses and decrease with age in normal human lenses. Thus, hydration changes in human diabetic lenses precede cataract formation. While syneresis, the release of bound water into the bulk, is part of the normal aging process, it appears to occur in the younger diabetics only. In older diabetics syneresis is halted or even reversed. This may be due to the glycation of lens proteins in diabetic patients which tends to immobilize water and therefore, reverse the syneresis due to aging.     

Selective activation of Galphao by D2L dopamine receptors in NS20Y neuroblastoma cells

D2L dopamine receptor activation results in rapid inhibition and delayed heterologous sensitization of adenylate cyclase in several host cell types. The D2L dopamine receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibition and sensitization in a neuronal cell environment and to identify the particular G-proteins involved. Acute activation of D2L receptors with the selective D2 agonist quinpirole inhibited forskolin-stimulated cAMP accumulation, whereas prolonged incubation (2 hr) with quinpirole resulted in heterologous sensitization (more than twofold) of forskolin-stimulated cAMP accumulation in NS20Y-D2L cells. To unambiguously identify the pertussis toxin (PTX)-sensitive G-proteins responsible for inhibition and sensitization, we used viral-mediated gene delivery to assess the ability of genetically engineered PTX-resistant G-proteins (Galphai1*, Galphai2*, Galphai3*, and Galphao*) to rescue both responses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibition of GTPgammaS-stimulated adenylate cyclase, respectively. To assess the specificity of D2L-Galpha coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-resistant G-protein alpha subunits and treated with PTX, and quinpirole-induced responses were measured. Infection of NS20Y-D2L cells with HSV-Galphao* rescued both inhibition and sensitization in PTX-treated cells, whereas infection with HSV-Galphai1*, HSV-Galphai2*, or HSV-Galphai3* failed to rescue either response. In summary, the current study provides strong evidence that the D2L dopamine receptor couples to Galphao in neuronal cells, and that this coupling is responsible for both the acute and subacute effects of D2 receptor activation on adenylate cyclase activity.     

Crystal structure of tryptophanase

The X-ray structure of tryptophanase (Tnase) reveals the interactions responsible for binding of the pyridoxal 5'-phosphate (PLP) and atomic details of the K+ binding site essential for catalysis. The structure of holo Tnase from Proteus vulgaris (space group P2(1)2(1)2(1) with a = 115.0 A, b = 118.2 A, c = 153.7 A) has been determined at 2.1 A resolution by molecular replacement using tyrosine phenol-lyase (TPL) coordinates. The final model of Tnase, refined to an R-factor of 18.7%, (Rfree = 22.8%) suggests that the PLP-enzyme from observed in the structure is a ketoenamine. PLP is bound in a cleft formed by both the small and large domains of one subunit and the large domain of the adjacent subunit in the so-called "catalytic" dimer. The K+ cations are located on the interface of the subunits in the dimer. The structure of the catalytic dimer and mode of PLP binding in Tnase resemble those found in aspartate amino-transferase, TPL, omega-amino acid pyruvate aminotransferase, dialkylglycine decarboxylase (DGD), cystathionine beta-lyase and ornithine decarboxylase. No structural similarity has been detected between Tnase and the beta 2 dimer of tryptophan synthase which catalyses the same beta-replacement reaction. The single monovalent cation binding site of Tnase is similar to that of TPL, but differs from either of those in DGD.     

Non-natural aglycones of glycopeptide antibiotics of the vancomycin group. Synthesis and antibacterial activity

A new approach for the modification of the heptapeptide core of glycopeptide antibiotics was proposed based on the replacement of amino acid residues in positions 1 and 3 in teicoplanin aglycone and in position 1 in the eremomycin aglycone. Six novel nonnatural aglycones of the vancomycin type were obtained. Compounds derived from the teicoplanin aglycone exhibited in vitro activity against Gram-positive bacteria, and two of them were also active against the vancomycin-resistant enterococci.