Haematinic effect of Hygrophila spinosa T. Anderson on experimental rodents

Ethanolic extract of the aerial parts of H. spinosa a semiwoody herb was examined on male albino rats for certain haematological changes. The extract (100 & 200 mg/kg, po) significantly increased the haemoglobin, haematocrit, RBC and total WBC, as compared with vehicle treated control rat haemogram. In anemic male albino rats, the extract significantly increased haemoglobin, haematocrit and RBC count. Serum iron and serum total iron binding capacity were significantly decreased in H. spinosa extract treated anemic rats as compared with those in the vehicle treated anemic control rats. These findings demonstrated the haematinic effect of H. spinosa extract on experimental animals.     

Isolation,purification and partial chemical characterization of a lethal factor from common indian toad (Bufo melanostictus,Schneider) skin extract

Indian toad (Bufo melanostictus, Schneider) skin extract (TSE) is pharmacologically potent and probably contains several bioactive compounds [Das et. al., Indian J Pharmacol, 28 (1996) 72]. A lethal factor was isolated and purified by neutral alumina column chromatography followed by HPLC. Spectroscopic (UV, IR, FAB-MASS) study indicated that the lethal factor (TSE-LF) was a 254 Da long chain compound with carbonyl, hydroxyl and ester as functional groups. LD50 of TSE-LF was found to be 3.5 mg/kg (iv). Biological study showed that TSE-LF possesses hypotensive, cardiotoxic, neurotoxic activity and produced death by apnoea in experimental animal. Cyproheptadine antagonised TSE-LF induced contraction of isolated smooth muscle indicating involvement of histamine/serotonin receptors. TSE-LF induced neurotoxic action on chick biventer cervices was mediated through Ca2+ ion.     

RecA-mediated rescue of Escherichia coli strains with replication forks arrested at the terminus

The recombinational rescue of chromosome replication was investigated in Escherichia coli strains with the unidirectional origin oriR1, from the plasmid R1, integrated within oriC in clockwise (intR1(CW)) or counterclockwise (intR1(CC)) orientations. Only the intR1(CC) strain, with replication forks arrested at the terminus, required RecA for survival. Unlike the strains with RecA-dependent replication known so far, the intR1(CC) strain did not require RecBCD, RecF, RecG, RecJ, RuvAB, or SOS activation for viability. The overall levels of degradation of replicating chromosomes caused by inactivation of RecA were similar in oriC and intR1(CC) strains. In the intR1(CC) strain, RecA was also needed to maintain the integrity of the chromosome when the unidirectional replication forks were blocked at the terminus. This was consistent with suppression of the RecA dependence of the intR1(CC) strain by inactivating Tus, the protein needed to block replication forks at Ter sites. Thus, RecA is essential during asymmetric chromosome replication for the stable maintenance of the forks arrested at the terminus and for their eventual passage across the termination barrier(s) independently of the SOS and some of the major recombination pathways.     

Polymerized liposome assemblies: bifunctional macromolecular selectin inhibitors mimicking physiological selectin ligands

Monomeric sialyl Lewis(X) (sLe(x)) and sLe(x)-like oligosaccharides are minimal structures capable of supporting selectin binding in vitro. However, their weak binding interactions do not correlate with the high-affinity binding interactions witnessed in vivo. The polyvalent display of carbohydrate groups found on cell surface glycoprotein structures may contribute to the enhanced binding strength of selectin-mediated adhesion. Detailed biochemical analyses of physiological selectin ligands have revealed a complicated composition of molecules that bind to the selectins in vivo and suggest that there are other requirements for tight binding beyond simple carbohydrate multimerization. In an effort to mimic the high-affinity binding, polyvalent scaffolds that contain multicomponent displays of selectin-binding ligands have been synthesized. Here, we demonstrate that the presentation of additional anionic functional groups in the form of sulfate esters, on a polymerized liposome surface containing a multimeric array of sLe(x)-like oligosaccharides, generates a highly potent, bifunctional macromolecular assembly. This assembly inhibits L-, E-, and P-selectin binding to GlyCAM-1, a physiological ligand better than sLe(x)-like liposomes without additional anionic charge. These multivalent arrays are 4 orders of magnitude better than the monovalent carbohydrate. Liposomes displaying 3'-sulfo Lewis(X)-like oligosaccharides, on the other hand, show slight loss of binding with introduction of additional anionic functional groups for E- and P-selectin and negligible change for L-selectin. The ability to rapidly and systematically vary the composition of these assemblies is a distinguishing feature of this methodology and may be applied to the study of other systems where composite binding determinants are important for high-affinity binding.     

Characterization of herpes simplex virus-containing organelles by subcellular fractionation: role for organelle acidification in assembly of infectious particles

The cytoplasmic compartments occupied by exocytosing herpes simplex virus (HSV) are poorly defined. It is unclear which organelles contain the majority of trafficking virions and which are occupied by virions on a productive rather than defective assembly pathway. These problems are compounded by the fact that HSV-infected cells produce virus continuously over many hours. All stages in viral assembly and export therefore coexist, making it impossible to determine the sequence of events and their kinetics. To address these problems, we have established assays to monitor the presence of capsids and enveloped virions in cell extracts and prepared HSV-containing organelles from normally infected cells and from cells undergoing a single synchronized wave of viral egress. We find that, in both cases, HSV particles exit the nucleus and accumulate in organelles which cofractionate with the trans-Golgi network (TGN) and endosomes. In addition to carrying enveloped infectious virions in their lumen, HSV-bearing organelles also displayed nonenveloped capsids attached to their cytoplasmic surface. Neutralization of organellar pH by chloroquine or bafilomycin A resulted in the accumulation of noninfectious enveloped particles. We conclude that the organelles of the TGN/endocytic network play a key role in the assembly and trafficking of infectious HSV.     

Characterization of non-planar peptide groups in protein crystal structures

Peptide groups are generally assumed to be planar in protein structure, due to 'rigid' partial double bond character of peptide bonds, thus the value of peptide torsion angle omega should be restricted to 180 degrees for the usual trans form of peptide unit. However, on analyzing the ultra-high resolution protein crystal database, we find that in some cases, omega deviates >10 degrees from its usual value of 180 degrees, indicating significant non-planarity of peptide groups. Moreover, the non-planarity for most of the amino acids is found to be 'biased' towards values of omega smaller than 180 degrees. Similar trend for to is confirmed by the neutron diffraction data for proteins. The neutron diffraction database also reveals that non-planar peptide groups are generally correlated to 'pyramidal' structure of the peptide-nitrogen bonds. Consequently, the hydrogen atom of peptide group deviates from its planar position, as measured by the 'improper' torsion angle theta. Thus, we find that both the angles omega and theta point towards a significant amount of non-planarity of peptide groups, which cannot be ignored. The role of peptide nonplanarity in protein function is, however, not yet clear.     

Expanded turn conformations: characterization and sequence-structure correspondence in alpha-turns with implications in helix folding

Like the beta-turns, which are characterized by a limiting distance between residues two positions apart (i, i+3), a distance criterion (involving residues at positions i and i+4) is used here to identify alpha-turns from a database of known protein structures. At least 15 classes of alpha-turns have been enumerated based on the location in the phi,psi space of the three central residues (i+1 to i+3)-one of the major being the class AAA, where the residues occupy the conventional helical backbone torsion angles. However, moving towards the C-terminal end of the turn, there is a shift in the phi,psi angles towards more negative phi, such that the electrostatic repulsion between two consecutive carbonyl oxygen atoms is reduced. Except for the last position (i+4), there is not much similarity in residue composition at different positions of hydrogen and non-hydrogen bonded AAA turns. The presence or absence of Pro at i+1 position of alpha- and beta-turns has a bearing on whether the turn is hydrogen-bonded or without a hydrogen bond. In the tertiary structure, alpha-turns are more likely to be found in beta-hairpin loops. The residue composition at the beginning of the hydrogen bonded AAA alpha-turn has similarity with type I beta-turn and N-terminal positions of helices, but the last position matches with the C-terminal capping position of helices, suggesting that the existence of a "helix cap signal" at i+4 position prevents alpha-turns from growing into helices. Our results also provide new insights into alpha-helix nucleation and folding.     

Lack of Apparent Neurological Abnormalities in Rabbits Sensitized by Gangliosides

Two very high titer polyclonal antibodies against two ganglioside antigens, GM1 and GD1a, have been raised in New Zealand white rabbits using a homogeneous suspension of the highly purified antigens in Keyhole Limpet Hemocyanin and Freunds adjuvant. The antisera were prepared over a period of 6 months with repeated injections of the ganglioside suspension, followed by an intravenous injection of the purified ganglioside solution, and collecting the serum (approximately 50 ml) at defined time intervals. The GM1-antibody, thus prepared, showed a cross reactivity toward GD1b and asialo-GM1 (GA1), while the GD1a-antibody reacted with GD1a, GM1 and GA1 and GD1b as determined by immuno-overlay and ELISA methods. The titer for GM1 antiserum, determined by ELISA, was greater than 1/10,000 dilution while the titer for GD1a antibody was greater than 1/5,000 dilution. No neurological or behavioral abnormality was observed during the period of antiserum production. To evaluate any likely pathological damage caused by such a high titer ganglioside-antibody, autopsy of CNS as well PNS tissues from the rabbits were carried out after the final bleeding. No obvious pathological changes, including demyelination, were noted in any of the four rabbits. These observations cast doubt as to the direct effect of anti-ganglioside antibody induced neurological and pathological disorders.Special issue dedicated to Lawrence F. Eng.     

CoPS: Comprehensive Peptide Signature database

We present the development of a Comprehensive database of 12 076 invariant Peptide Signatures (CoPS) derived from 52 bacterial genomes with a minimum occurrence in at least seven organisms. These peptides were observed in functionally similar proteins and are distributed over nearly 1250 different functional proteins. The database provides function, structure and occurrence in biochemical pathways of the proteins containing these signature peptides. It houses additional information on the signature peptides, such as identical match in other motif/pattern (e.g. PROSITE, BLOCKS, PRINTS and Pfam) databases and the database of interacting proteins, human proteome and mutation effect on these signature peptides. There is a wide applicability of this database in the identification of critical functional residues in proteins. The database also facilitates the identification of folding nucleus/structural determinants in proteins and functional assignment to yet unknown proteins. We demonstrate functional assignment to 2605 hypothetical proteins in bacterial genomes and 112 unknown proteins in human using this database. AVAILABILITY: The database can be freely accessed through the following URL: http://203.195.151.46/copsv2/index.html or http://203.90.127.70/copsv2/index.html     

Topoisomerases of kinetoplastid parasites as potential chemotherapeutic targets

The protozoan parasites Trypanosoma, Leishmania and Crithidia, which belong to the order kinetoplastidae, emerge from the most ancient eukaryotic lineages. The diversity found in the life cycle of these organisms must be directed by genetic events, wherein topoisomerases play an important role in cellular processes affecting the topology and organization of intracellular DNA. Topoisomerases are valuable as potential drug targets because they have indispensable function in cell biology. This review summarizes what is known about topoisomerase genes and proteins of kinetoplastid parasites and the roles of these enzymes as targets for therapeutic agents.