Structure of the N-terminal fragment of topoisomerase V reveals a new family of topoisomerases

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting no conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.     

Long term excessive Zn-supplementation promotes metabolic syndrome-X in Wistar rats fed sucrose and fat rich semisynthetic diet

During the last two decades Zinc (Zn) as a micronutrient is being used indiscriminately in agricultural and husbandry practices and also in baby foods and multivitamin supplements with a view that Zn is non-toxic and promotes linear growth and body weight in the consumers. The long-term effect of increasing Zn load in the body has not been worked out so far. In this study, three groups of rats were fed on a semi-synthetic diet containing 20 mg (control, group-I), 40 mg (group-II) and 80 mg Zn /kg (group-III) diet respectively for 6 months. The results revealed that the gain in body weight increased in rats in Zn-concentration dependent manner. The urine examined on weekly basis showed glucosuria in group-II on week 10 and in group-III on week 8 and thereafter. The arterial blood pressure was significantly higher in group-II and III than their control counter parts on monthly basis. Histochemical examination of skin revealed an increase in the number of adipocytes filled with triglycerides making a subcutaneous fatty tissue thicker in group-II and group-III than that of control group. The blood profile after 180 days of dietary treatment, displayed a significant rise in glucose, total lipids, cholesterol, triglycerides, LDL-cholesterol, VLDL-cholesterol, insulin, cortisol and aldosterone whereas HDL-cholesterol, T3, T4 and TSH showed a reduction in their levels in the blood serum. The tissue metal status showed an increase of Zn, Cu and Mg in the serum, a rise in Zn in liver, hair and abdominal muscles and fall in Cu and Mg concentrations in liver, hair and abdominal muscles. This data suggest that Zn in excess in diet when fed for longer periods of time induces metabolic syndrome-X.     

Three New Species of Brevitobrilus (Nematoda) with a Discussion on Relationships within the Genus

Three new species of the genus BrevitobriusTsalolikhin, 1981 are described. Brevitobrilus glandulatus n. sp. is characterized by conspicuous sphincter between pars dilatata and uterus; two pairs of vaginal glands; spicules having elliptical capitula with small proximal stiffening piece; proximally-arcuate gubernaculum; S3 and S4 smaller than other supplements; S6 out of spicular range and 57-60 micropapillae. Brevitobrilus dimorphicus n. sp. is diagnosed by sexual dimorphism in labial sensilla and amphids; thick-walled rectum with a diverticulum protruding into intestinal lumen and males with boat-shaped spicules and S6 occasionally slightly smaller than other supplements. Brevitobrilus allahabadensis n. sp. possesses large amphids of 28-33% of corresponding labial diameter in both sexes; vagina and uterus with muscular, plicate walls; well developed sphincter between vas deferens and ejaculatory duct; capitulate spicules with sloping ventral and angular dorsal walls; S3, S4 and S6 smaller than other supplements, S6 close to cloaca and 28-37 micropapillae. The relationships of the species of genus Brevitobrilus have been assessed using morphological characters subjected to parsimony and a non cladistic key to identification of species is given.     

Structure–activity relationship (SAR) of parthenin analogues with pro-apoptotic activity: Development of novel anti-cancer leads

Analogues of parthenin were synthesized by substitutions at different reaction centres to establish a structure–activity relationship (SAR). Some of the molecules have displayed significant cytotoxicity in human cervical carcinoma (HeLa) and human myeloid leukemia (HL-60) cells. A few of the compounds also induced apoptosis in HL-60 cells measured in terms of sub-Go/G1 DNA fraction. Also one of the lead molecules has been shown to be the inhibitor of both telomerase and topoisomerase-II.     

Structural transition from dimeric to tetrameric i‐motif,caused by the presence of TAA at the 3′‐end of human telomeric C‐rich sequence

Widely dispersed in genomic DNA, the tandem C‐rich repetitive stretches may fold below physiological pH, into i‐motif structures, stabilized by C·C⁺ pairing. Herein, structural status of a 9‐mer stretch d(CCCTAACCC), [the truncated double repeat of human telomeric sequence], and its extended version, comprising of additional ? TAA segment at the 3′‐end, representing the complete double repeat d(CCCTAACCCTAA), has been investigated. The pH dependent monophasic UV‐melting, Gel and CD data suggested that while the truncated version adopts a bimolecular i‐motif structure, its complete double repeat (12‐mer) sequence exists in two (bimolecular and tetramolecular) forms. A model is proposed for the tetramolecular i‐motif with conventional C · C⁺ base pairs, additionally stabilized by asymmetric A · A base pairs at the ?3′ TAA flanking ends and Watson–Crick A · T hydrogen bonding between intervening bases on antiparallel strands. Expanding the known topologies of DNA i‐motifs, such atypical geometries of i‐motifs may have implications in their recognition by proteins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 150–160, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Constitutive Endocytic Recycling and Protein Kinase C-mediated Lysosomal Degradation Control KATP Channel Surface Density

Pancreatic ATP-sensitive potassium (K_ATP) channels control insulin secretion by coupling the excitability of the pancreatic β-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed K_ATP channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that K_ATP channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases K_ATP channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic β-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native K_ATP channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic β-cells, we propose that PKC-regulated K_ATP channel trafficking may play a role in the regulation of insulin secretion.     

Insights into the oligomeric states, conformational changes, and helicase activities of SV40 large tumor antigen

The large T (LT) antigen encoded by SV40 virus is a multi-domain, multi-functional protein that can not only transform cells but can also function as an efficient molecular machine to unwind duplex DNA for DNA replication. Here we report our findings on the oligomeric forms, domain interactions, and ATPase and helicase activities of various LT constructs. For the LT constructs that hexamerize, only two oligomeric forms, hexameric and monomeric, were detected in the absence of ATP/ADP. However, the presence of ATP/ADP stabilizes LT in the hexameric form. The LT constructs lacking the N- and C-terminal domains, but still retaining hexamerization ability, have ATPase as well as helicase activities at a level comparable to the full-length LT, suggesting the importance of hexamerization for these activities. The domain structures and the possible interactions between different LT fragments were probed with limited protease (trypsin) digestion. Such protease digestion generated a distinct pattern in the presence and absence of ATP/ADP and Mg(2+). The most C-terminal fragment (residues 628-708, containing the host-range domain), which was thought to be completely unstructured, was somewhat trypsin-resistant despite the presence of multiple Arg and Lys, possibly due to a rather structured C terminus. Furthermore, the N- and C-terminal fragments cleaved by trypsin were associated with other parts of the molecule, suggesting the interdomain interactions for the fragments at both ends.     

Behavioral observations of adolescent Holstein heifers cloned from adult somatic cells

Cloning using somatic cells offers many potential applications in biomedicine and basic research. The objective of this study was to test whether clones from the same genotype can be used as models to study the genetic influences of behavior. Specifically, several aspects of the behavior of four prepubertal heifers cloned from somatic cells of a 13-year-old Holstein cow along with age-matched control heifers were compared to determine whether juvenile clones from an aged adult behave similarly to their age-matched controls, and whether clones with identical genetic makeup exhibit any behavioral trends. Behavioral observations or behavior challenge tests were conducted to compare the following traits: vocalization, play behavior, movement frequencies, grooming, curiosity, and companion preference, as well as dominance and aggressiveness. From play behavior, movements and vocalization, we observed that these four juvenile clones of an aged genetic donor did not show behavioral indications of aging and were similar to their counterparts of comparable chronological age except that they tended to play less than controls. Behavioral trends were also observed in the clones that indicated that they exhibited higher levels of curiosity, more grooming activities and were more aggressive and dominant than controls. Furthermore, these four clones preferred each other or the donor as companions, which may indicate genetic kin recognition.     

Glycogen synthase kinase 3beta-mediated apoptosis of primary cortical astrocytes involves inhibition of nuclear factor kappaB signaling

Recent studies have revealed a positive correlation between astrocyte apoptosis and rapid disease progression in persons with neurodegenerative diseases. Glycogen synthase kinase 3beta (GSK-3beta) is a molecular regulator of cell fate in the central nervous system and a target of the phosphatidylinositol 3-kinase (PI-3K) pathway. We have therefore examined the role of the PI-3K pathway, and of GSK-3beta, in regulating astrocyte survival. Our studies indicate that inhibition of PI-3K leads to apoptosis in primary cortical astrocytes. Furthermore, overexpression of a constitutively active GSK-3beta mutant (S9A) is sufficient to cause astrocyte apoptosis, whereas an enzymatically inactive GSK-3beta mutant (K85M) has no effect. In light of reports on the interplay between GSK-3beta and nuclear factor kappaB (NF-kappaB), and because of the antiapoptotic activity of NF-kappaB, we examined the effect of GSK-3beta overexpression on NF-kappaB activation. These experiments revealed strong inhibition of NF-kappaB activation in astrocytes upon overexpression of the S9A, but not the K85M, mutant of GSK-3beta. This was accompanied by stabilization of the NF-kappaB-inhibitory protein, IkappaBalpha and down-regulation of IkappaB kinase (IKK) activity. These findings therefore implicate GSK-3beta as a regulator of NF-kappaB activation in astrocytes and suggest that the pro-apoptotic effects of GSK-3beta may be mediated at least in part through the inhibition of NF-kappaB pathway.