Comparison of five tandem repeat loci between humans and chimpanzees.

Five tandem repeat loci were studied in humans and chimpanzees using VNTR probes derived from human DNA. Shared alleles were found at three loci and were often the modal allele in one species but never in both. There was no difference in the mean number of alleles per locus. However, these species exhibited substantially different levels of gene diversity, with chimpanzees monomorphic at two loci. Evidence of reduced variability in chimpanzees corroborates earlier comparisons using isozymes and plasma proteins. Molecular mechanisms, population dynamics, or both may be responsible for these differences. Equal numbers of alleles per locus may reflect high mutation rates. By one test, chimpanzees were out of equilibrium at one locus, which may reflect a typing error or population substructure. The long divergence time, and the high probability of backward mutations, precludes accurate estimation of genetic distance between these species.     

Increase in nitrate reductase activity in the presence of sucrose in bean leaf segments

The supply of sucrose to leaf segments from light-grown bean seedlings caused a substantial increase in substrate inducibility of in vivo and in vitro nitrate reductase activity but only a small increase in total protein. Cycloheximide and chloramphenicol inhibited the increase in enzyme activity by nitrate and sucrose. The in vivo decline in enzyme activity in nitrate-induced leaf segments in light and dark was protected by sucrose and nitrate. The supply of NADH also protected the decline in enzyme activity, but only in the light. In vitro stability of the extracted enzyme was, however, unaffected by sucrose. The size of the metabolic nitrate pool was also enhanced by sucrose. The experiments demonstrate that sucrose has a stimulatory effect on activity or in vivo stability ' of nitrate reductase in bean leaf segments, which is perhaps mediated through increased NADH level and/or mobilization of nitrate to the metabolic pool.     

Identification of a PstI polymorphism in the p21Cip1/Waf1 cyclin-dependent kinase inhibitor gene

A PstI polymorphism in the 3 flanking region of the p21^CiP1/Waf1 cyclin-dependent kinase inhibitor gene is described. DNA sequencing analysis identified a CT base substitution in the 3 flanking region of the gene. This substitution leads to the destruction of a PstI site and results in a biallelic DNA polymorphism. This restriction fragment length polymorphism (RFLP) provides the first known genetic marker for this cell cycle regulatory gene.     

Known data on the therapeutic use of Azadiracta indica (neem) for type 2 diabetes mellitus

There has been growing interest for the therapeutic use of traditional herbs in the management of diabetes mellitus (DM) and its complications. Data shows the hypoglycemic activity of Azadiracta indica in diabetes. Therefore, it is of interest to document known data on the therapeutic use of Azadiracta indica (neem) for type 2 diabetes mellitus (T2DM).     

A comparison of nonlethal sampling methods for amphibian gut microbiome analyses

Noninvasive sampling methods for studying intestinal microbiomes are widely applied in studies of endangered species and in those conducting temporal monitoring during manipulative experiments. Although existing studies show that noninvasive sampling methods among different taxa vary in their accuracy, no studies have yet been published comparing nonlethal sampling methods in adult amphibians. In this study, we compare microbiomes from two noninvasive sample types (faeces and cloacal swabs) to that of the large intestine in adult cane toads, Rhinella marina. We use 16S rRNA gene sequencing to investigate how microbial communities change along the digestive tract and which nonlethal sampling method better represents large intestinal microbiota. We found that cane toads' intestinal microbiota was dominated by Bacteroidetes, Proteobacteria and Firmicutes and, interestingly, we also saw a high proportion of Fusobacteria, which has previously been associated with marine species and changes in frog immunity. The large and small intestine of cane toads had a similar microbial composition, but the large intestine showed higher diversity. Our results indicate that cloacal swabs were more similar to large intestine samples than were faecal samples, and small intestine samples were significantly different from both nonlethal sample types. Our study provides valuable information for future investigations of the cane toad gut microbiome and validates the use of cloacal swabs as a nonlethal method to study changes in the large intestine microbiome. These data provide insights for future studies requiring nonlethal sampling of amphibian gut microbiota.     

Anthrax biosensor, protective antigen ion channel asymmetric blockade

The significant threat posed by biological agents (e.g. anthrax, tetanus, botulinum, and diphtheria toxins) (Inglesby, T. V., O'Toole, T., Henderson, D. A., Bartlett, J. G., Ascher, M. S., Eitzen, E., Friedlander, A. M., Gerberding, J., Hauer, J., Hughes, J., McDade, J., Osterholm, M. T., Parker, G., Perl, T. M., Russell, P. K., and Tonat, K. (2002) J. Am. Med. Assoc. 287, 2236-2252) requires innovative technologies and approaches to understand the mechanisms of toxin action and to develop better therapies. Anthrax toxins are formed from three proteins secreted by fully virulent Bacillus anthracis, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). Here we present electrophysiological measurements demonstrating that full-length LF and EF convert the current-voltage relationship of the heptameric PA63 ion channel from slightly nonlinear to highly rectifying and diode-like at pH 6.6. This effect provides a novel method for characterizing functional toxin interactions. The method confirms that a previously well characterized PA63 monoclonal antibody, which neutralizes anthrax lethal toxin in animals in vivo and in vitro, prevents the binding of LF to the PA63 pore. The technique can also detect the presence of anthrax lethal toxin complex from plasma of infected animals. The latter two results suggest the potential application of PA63 nanopore-based biosensors in anthrax therapeutics and diagnostics.     

Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods

The cyanobacterial circadian clock can be reconstituted in vitro by mixing recombinant KaiA, KaiB and KaiC proteins with ATP, producing KaiC phosphorylation and dephosphorylation cycles that have a regular rhythm with a ca. 24-h period and are temperature-compensated. KaiA and KaiB are modulators of KaiC phosphorylation, whereby KaiB antagonizes KaiA's action. Here, we present a complete crystallographic model of the Synechococcus elongatus KaiC hexamer that includes previously unresolved portions of the C-terminal regions, and a negative-stain electron microscopy study of S. elongatus and Thermosynechococcus elongatus BP-1 KaiA-KaiC complexes. Site-directed mutagenesis in combination with EM reveals that KaiA binds exclusively to the CII half of the KaiC hexamer. The EM-based model of the KaiA-KaiC complex reveals protein-protein interactions at two sites: the known interaction of the flexible C-terminal KaiC peptide with KaiA, and a second postulated interaction between the apical region of KaiA and the ATP binding cleft on KaiC. This model brings KaiA mutation sites that alter clock period or abolish rhythmicity into contact with KaiC and suggests how KaiA might regulate KaiC phosphorylation.     

Structural, bioinformatic, and in vivo analyses of two Treponema pallidum lipoproteins reveal a unique TRAP transporter

Treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite ATP-independent periplasmic transporter (TRAP-T). TRAP-Ts typically employ a periplasmic substrate-binding protein (SBP) to deliver the cognate ligand to the transmembrane symporter. Herein, we demonstrate that the genes encoding the putative TRAP-T components from T. pallidum, tp0957 (the SBP), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. We determined the crystal structure of recombinant Tp0956; the protein is trimeric and perforated by a pore. Part of Tp0956 forms an assembly similar to those of "tetratricopeptide repeat" (TPR) motifs. The crystal structure of recombinant Tp0957 was also determined; like the SBPs of other TRAP-Ts, there are two lobes separated by a cleft. In these other SBPs, the cleft binds a negatively charged ligand. However, the cleft of Tp0957 has a strikingly hydrophobic chemical composition, indicating that its ligand may be substantially different and likely hydrophobic. Analytical ultracentrifugation of the recombinant versions of Tp0956 and Tp0957 established that these proteins associate avidly. This unprecedented interaction was confirmed for the native molecules using in vivo cross-linking experiments. Finally, bioinformatic analyses suggested that this transporter exemplifies a new subfamily of TPATs (TPR-protein-associated TRAP-Ts) that require the action of a TPR-containing accessory protein for the periplasmic transport of a potentially hydrophobic ligand(s).     

Differentiation in calli originated from isolated protoplasts of rice (Oryza sativa L.) through plating technique

Summary Protoplasts from mesophyll cells and callus cells of rice (Oryza sativa L.) have been isolated by enzyme treatment involving 2% pectinase followed by 3% cellulase at pH 5.4 in 0.45 M mannitol (viable protoplasts from mesophyll cells in 50–60% yield, 60–70% yield of viable protoplasts from callus cells through treatment with the mixture of the above mentioned enzymes at the same concentration). Our completely defined medium is the combination of three established media (Table 1). Culture conditions are: soft agar in petri dishes at 26° C, where they regenerated cell walls after 24 h. The first cell division was observed after 4 days in culture for callus protoplasts and after 5 days in culture for mesophyll protoplasts. Cell division continues thereafter, and after 4 weeks of culture small white calli were visible in the petri dishes. The type of plant material (whitish leaf sheaths) and cell density are important factors for the efficiency of colony formation (30% plating efficiency). Healthy root formation through transfer to suitable medium is up to now the morphogenetic reaction of the calli.Work carried out at Molecular Cytogenetics Research Unit, Deptt, of Genetics and Plant Breeding, Banaras Hindu University, Varanasi, India