Stimulation of growth and glucose catabolite enzymes by succinate in some thermophilic fungi

Thermophilic Humicola lanuginosa, Penicillium duponti, Sporotrichum thermophile and Mucor pusillus required succinate in addition to glucose for optimal growth. The requirement for succinate was concentration-dependent and the concentration needed for one half of the maximal growth was 6.14 mM. In the presence of succinate, glucose utilization from the medium was markedly increased and this was associated with increased levels of the enzymes of the glycolytic and Krebs cycle pathways. Addition of succinate to cultures growing in glucose at any stage of growth stimulated the growth with the resulting rate of growth remaining high if the addition was made within 3 days of inoculation. Cycloheximide (71.4 M) prevented the succinate-mediated derepression of the enzymes suggesting that succinate may remove the catabolite repression in the presence of glucose.A preliminary part of this work was presented at the 17th annual meeting of the Association of Microbiologists of India at Manipal (India) held from Dec. 13 to 15, 1976     

A new moderately repetitive DNA sequence family of novel organization.

In cloning adenovirus homologous sequences, from a human cosmid library, we identified a moderately repetitive DNA sequence family consisting of tandem arrays of 2.5 kb members. A member was sequenced and several non-adjacent, 15-20 bp G-C rich segments with homology to the left side of adenovirus were discovered. The copy number of 400 members is highly conserved among humans. Southern blots of partial digests of human DNA have verified the tandem array of the sequence family. The chromosomal location was defined by somatic cell genetics and in situ hybridization. Tandem arrays are found only on chromosomes 4 (4q31) and 19 (q13.1-q13.5). Homologous repetitive sequences are found in DNA of other primates but not in cat or mouse. Thus we have identified a new family of moderately repetitive DNA sequences, unique because of its organization in clustered tandem arrays, its length, its chromosomal location, and its lack of homology to other moderately repetitive sequence families.     

Occurrence of novel Cu2+-dependent sialic acid-specific lectin,on the outer surface of mature caprine spermatozoa

Effects of several bivalent metal ions on the autoagglutination event in mature caprine epididymal sperm cells have been investigated using a chemically defined medium. This study demonstrates for the first time that Copper (Cu²⁺) ion (300 μM) has high specificity for autoagglutination of mature cauda-epididymal sperm. Head-to-head interaction of the male gametes is responsible for this event. Studies on the effect of various sugars reveal that the autoagglutinated cells can be dissociated specifically with neutralized sialic acid (50 mM), which also inhibits the sperm cell autoagglutination phenomenon. Blood serum protein fetuin, that contains terminal sialic acid residue, showed high efficacy for inhibiting this autoagglutination event at 4 μM concentration. However, asialofetuin is not capable of inhibiting this Cu²⁺-dependent cellular event. Mature sperm cells bound with caprine erythrocytes at their head region in presence of Cu²⁺ ion. The purified sperm membrane fraction isolated by aqueous two phase polymer method showed high efficacy to agglutinate erythrocytes. These sperm-erythrocyte interactions as well as sperm membrane induced haemagglutination were strongly blocked by neutralized sialic acid (50 mM). The results confirm the occurrence of unique Cu²⁺ dependent, sialic acid-specific lectin on the outer surface of a mammalian cell using caprine sperm as the model. The observed Cu²⁺-mediated cellular autoagglutination is caused by the interaction of the cell surface lectin with the lectin receptor on the surface of the neighboring homologous cell.     

α-phenyl-tert-butyl-nitrone inhibits free radical release in brain concussion

Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (·OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systematically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (α_N = 15.4 and α^β_H = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of α-phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF),6significantly (P< 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI.     

Melatonin Distribution Reveals Clues to Its Biological Significance in Basal Metazoans

Although nearly ubiquitous in nature, the precise biological significance of endogenous melatonin is poorly understood in phylogenetically basal taxa. In the present work, we describe insights into the functional role of melatonin at the most “basal” level of metazoan evolution. Hitherto unknown morphological determinants of melatonin distribution were evaluated in Nematostella vectensis by detecting melatonin immunoreactivity and examining the spatial gene expression patterns of putative melatonin biosynthetic and receptor elements that are located at opposing ends of the melatonin signaling pathway. Immuno-melatonin profiling indicated an elaborate interaction with reproductive tissues, reinforcing previous conjectures of a melatonin-responsive component in anthozoan reproduction. In situ hybridization (ISH) to putative melatonin receptor elements highlighted the possibility that the bioregulatory effects of melatonin in anthozoan reproduction may be mediated by interactions with membrane receptors, as in higher vertebrates. Another intriguing finding of the present study pertains to the prevalence of melatonin in centralized nervous structures. This pattern may be of great significance given that it 1) identifies an ancestral association between melatonin and key neuronal components and 2) potentially implies that certain effects of melatonin in basal species may be spread widely by regionalized nerve centers.     

Differential Expression of Antioxidant Enzymes During Degradation of Azo Dye Reactive black 8 in Hairy roots of Physalis minima L.

The enzymes involved in the protection of plant metabolism in presence of azo dye was characterized by studying activities of the role of antioxidant enzymes in the hairy roots (HRs) of Physalis minima L. during degradation of an azo dye, Reactive Black 8 (RB8). When the HRs were exposed to RB8 (30 mg L^?1), a nine fold increase in SOD activity was observed after 24 h, while 22 and 50 fold increase in activity was observed for POX and APX respectively after 72 h, whereas there was no significant change in activity of CAT. The activation of different antioxidant enzymes at different time intervals under dye stress suggests the synchronized functioning of antioxidant machinery to protect the HRs from oxidative damage. FTIR analysis confirmed the degradation of dye and the non-toxic nature of metabolites formed after dye degradation was confirmed by phytotoxicity study.     

A thermodynamic overview of naturally occurring intramolecular DNA quadruplexes

Loop length and its composition are important for the structural and functional versatility of quadruplexes. To date studies on the loops have mainly concerned model sequences compared with naturally occurring quadruplex sequences which have diverse loop lengths and compositions. Herein, we have characterized 36 quadruplex-forming sequences from the promoter regions of various proto-oncogenes using CD, UV and native gel electrophoresis. We examined folding topologies and determined the thermodynamic profile for quadruplexes varying in total loop length (5–18 bases) and composition. We found that naturally occurring quadruplexes have variable thermodynamic stabilities (ΔG₃₇) ranging from −1.7 to −15.6 kcal/mol. Overall, our results suggest that both loop length and its composition affect quadruplex structure and thermodynamics, thus making it difficult to draw generalized correlations between loop length and thermodynamic stability. Additionally, we compared the thermodynamic stability of quadruplexes and their respective duplexes to understand quadruplex–duplex competition. Our findings invoke a discussion on whether biological function is associated with quadruplexes with lower thermodynamic stability which undergo facile formation and disruption, or by quadruplexes with high thermodynamic stability.     

Effect of loop length variation on quadruplex-Watson Crick duplex competition

The effect of loop length on quadruplex stability has been studied when the G-rich strand is present along with its complementary C-rich strand, thereby resulting in competition between quadruplex and duplex structures. Using model sequences with loop lengths varying from T to T5, we carried out extensive FRET to discover the influence of loop length on the quadruplex-Watson Crick duplex competition. The binding data show an increase in the binding affinity of quadruplexes towards their complementary strands upon increasing the loop length. Our kinetic data reveal that unfolding of the quadruplex in presence of a complementary strand involves a contribution from a predominant slow and a small population of fast opening conformer. The contribution from the fast opening conformer increases upon increasing the loop length leading to faster duplex formation. FCS data show an increase in the interconversion between the quadruplex conformers in presence of the complementary strand, which shifts the equilibrium towards the fast opening conformer with an increase in loop length. The relative free-energy difference (Delta DeltaG(o)) between the duplex and quadruplex indicates that an increase in loop length favors duplex formation and out competes the quadruplex.