Biochemical association of poly(ADP-ribose) polymerase-1 and its apoptotic peptide fragments with DNA polymerase beta

We have characterized the biochemical association of two DNA damage-dependent enzymes, poly(ADP-ribose) polymerase-1 (PARP-1) [EC 2.4.2.30] and DNA polymerase beta (pol beta) [2.7.7.7]. We reproducibly observed that pol beta is an efficient covalent target for ADP-ribose polymers under standard conditions of enzymatically catalyzed ADP-ribosylation of betaNAD+ as a substrate. The efficiency of poly(ADP-ribosyl)ation increased as a function of the pol beta and betaNAD+ concentrations. To further characterize the molecular interactions between these two unique polymerases, we also subjected human recombinant PARP-1 to peptide-specific enzymatic degradation with either caspase-3 or caspase-7 in vitro. This proteolytic treatment, commonly referred to as 'a hallmark of apoptosis', generated the two physiologically relevant peptide fragments of PARP-1, e.g., a 24-kDa amino-terminus and an 89-kDa carboxy-terminal domain. Interestingly, co-incubation of the two peptide fragments of PARP-1 with full-length pol beta resulted in their domain-specific molecular association as determined by co-immunoprecipitation and reciprocal immunoblotting. Therefore, our data strongly suggest that, once PARP-1 is proteolyzed by either caspase-3 or caspase-7 during cell death, the specific association of its apoptotic fragments with DNA repair enzymes, such as pol beta, may serve a regulatory molecular role in the execution phase of apoptosis.     

Design of bivalent ligands using hydrogen bond linkers: synthesis and evaluation of inhibitors for human beta-tryptase

We exploit the concept of using hydrogen bonds to link multiple ligands for maintaining simultaneous interactions with polyvalent binding sites. This approach is demonstrated by the syntheses and evaluation of pseudo-bivalent ligands as potent inhibitors of human β-tryptase.     

Catecholamines act via a beta-adrenergic receptor to maintain fetal heart rate and survival

Mice lacking catecholamines die before birth, some with cardiovascular abnormalities. To investigate the role of catecholamines in development, embryonic day 12.5 (E12.5) fetuses were cultured and heart rate monitored. Under optimal oxygenation, wild-type and catecholamine-deficient fetuses had the same initial heart rate (200-220 beats/min), which decreased by 15% in wild-type fetuses during 50 min of culture. During the same culture period, catecholamine-deficient fetuses dropped their heart rate by 35%. Hypoxia reduced heart rate of wild-type fetuses by 35-40% in culture and by 20% in utero, assessed by echocardiography. However, catecholamine-deficient fetuses exhibited greater hypoxia-induced bradycardia, reducing their heart rate by 70-75% in culture. Isoproterenol, a beta-adrenergic receptor (beta-AR) agonist, reversed this extreme bradycardia, restoring the rate of catecholamine-deficient fetuses to that of nonmutant siblings. Moreover, isoproterenol rescued 100% of catecholamine-deficient pups to birth in a dose-dependent, stereo-specific manner when administered in the dam's drinking water. An alpha-AR agonist was without effect. When wild-type fetuses were cultured with adrenoreceptor antagonists to create pharmacological nulls, blockade of alpha-ARs with 10 microM phentolamine or beta-ARs with 10 microM bupranolol alone or in combination did not reduce heart rate under optimal oxygenation. However, when combined with hypoxia, beta-AR blockade reduced heart rate by 35%. In contrast, the muscarinic blocker atropine and the alpha-AR antagonist phentolamine had no effect. These data suggest that beta-ARs mediate survival in vivo and regulate heart rate in culture. We hypothesize that norepinephrine, acting through beta-ARs, maintains fetal heart rate during periods of transient hypoxia that occur throughout gestation, and that catecholamine-deficient fetuses die because they cannot withstand hypoxia-induced bradycardia.     

Protective effect of Pongamia pinnata flowers against cisplatin and gentamicin induced nephrotoxicity in rats

Ethanolic extract of flowers of Pongamia pinnata was studied for its protective effect against cisplatin and gentamicin induced renal injury in rats. When the extract (300 & 600 mg kg(-1)) was administered orally for 10 days following cisplatin (5 mg kg(-1) i.p.) on day 5, toxicity of cisplatin, as measured by loss of body weight, elevated blood urea and serum creatinine declined significantly. Similarly in gentamicin (40 mg kg(-1) s.c.) induced renal injury, the extract (600 mg kg(-1)) normalized the raised blood urea and serum creatinine levels. Reversal of cisplatin and gentamicin renal cell damage as induced by tubular necrosis ie, marked congestion of the glomeruli with glomerular atrophy, degeneration of tubular epithelial cells with casts in the tubular lumen and infiltration of inflammatory cells in the interstitium was confirmed on histopathological examination. In the preventive regimen, co-administration of the extract with gentamicin significantly prevented the renal injury both functionally and histologically. Ethanolic extract of flowers had a marked nitric oxide free radical scavenging effect, suggesting an antioxidative property. Two flavonoids, known for their antioxidant activity viz. kaempferol and 3, 5, 6, 7, 8-pentamethoxy flavone were isolated from the extract. The results suggested that the flowers of Pongamia pinnata had a protective effect against cisplatin and gentamicin induced renal injury through antioxidant property.     

Oxidative stress during vitamin A-induced abnormal tail regeneration in the tadpoles of Polypedates maculatus

Vitamin A and its derivatives inhibit normal tail regeneration in amphibians. The most remarkable effect is the development of limbs at the cut end of the tail in anurans. Prior to ectopic limb development, there is an abnormal tail regeneration in the treated tadpoles. The purpose of the present study was to compare oxidative stress condition in the regenerated tail of normal and vitamin A (10I U/ml, 72 h) treated tadpoles. The present findings show a hyper-oxidative stress condition in the regenerated tail of the vitamin A-treated tadpoles of the Indian jumping frog, Polypedates maculatus (Anura: Rhacophoridae).     

Development of cloning vectors and transformation methods for<Emphasis Type="Italic"> Amycolatopsis</Emphasis>

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA-rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains. Electronic Publication     

Structural polymorphism of the major capsid protein of rotavirus

Rotaviruses are important human pathogens with a triple-layered icosahedral capsid. The major capsid protein VP6 is shown here to self-assemble into spherical or helical particles mainly depending upon pH. Assembly is inhibited either by low pH (<3.0) or by a high concentration (>100 mM) of divalent cations (Ca(2+) and Zn(2+)). The structures of two types of helical tubes were determined by electron cryomicroscopy and image analysis to a resolution of 2.0 and 2.5 nm. In both reconstructions, the molecular envelope of VP6 fits the atomic model determined by X-ray crystallography remarkably well. The 3-fold symmetry of the VP6 trimer, being incompatible with the helical symmetry, is broken at the level of the trimer contacts. One type of contact is maintained within all VP6 particles (tubes and virus), strongly suggesting that VP6 assemblies arise from different packings of a unique dimer of trimers. Our data show that the protonation state and thus the charge distribution are important switches governing the assembly of macromolecular assemblies.