Conformational dynamics of DnaB helicase upon DNA and nucleotide binding: analysis by intrinsic tryptophan fluorescence quenching

DnaB helicase of E. coli unwinds duplex DNA in the replication fork using the energy of ATP hydrolysis. We have analyzed structural and conformational changes in the DnaB protein in various nucleotides and DNA bound intermediate states by fluorescence quenching analysis of intrinsic fluorescence of native tryptophan (Trp) residues in DnaB. Fluorescence quenching analysis indicated that Trp48 in domain alpha is in a hydrophobic environment and resistant to fluorescence quenchers such as potassium iodide (KI). In domain beta, Trp294 was found to be in a partially hydrophobic environment, whereas Trp456 in domain gamma appeared to be in the least hydrophobic environment. Binding of oligonucleotides to DnaB helicase resulted in a significant attenuation of the fluorescence quenching profile, indicating a change in conformation. ATPgammaS or ATP binding appeared to lead to a conformation in which Trp residues had a higher degree of solvent exposure and fluorescence quenching. However, the most dramatic increase of Trp fluorescence quenching was observed with ADP binding with a possible conformational relaxation. Site-specific Trp --> Cys mutants of DnaB helicase demonstrated that conformational change upon ADP binding could be attributed exclusively to a conformational transition in the alpha domain leading to an increase in the solvent exposure of Trp48. However, formation of DnaB.ATPgammaS.DNA ternary complex led to a conformation with a fluorescence quenching profile similar to that observed with DnaB alone. The DnaB.ADP.DNA ternary complex produced a quenching curve similar to that of DnaB.ADP complex pointing to a change in conformation due to ATP hydrolysis. There are at least four identifiable structural/conformational states of DnaB helicase that are likely important in the helicase activity. The noncatalytic alpha domain in the N-terminus appeared to undergo the most significant conformational changes during nucleotide binding and hydrolysis. This is the first reported elucidation of the putative role of domain alpha, which is essential for DNA helicase action. We have correlated these results with partial structural models of alpha, beta, and gamma domains     

Global patterns in human mitochondrial DNA and Y-chromosome variation caused by spatial instability of the local cultural processes

Because of the widespread phenomenon of patrilocality, it is hypothesized that Y-chromosome variants tend to be more localized geographically than those of mitochondrial DNA (mtDNA). Empirical evidence confirmatory to this hypothesis was subsequently provided among certain patrilocal and matrilocal groups of Thailand, which conforms to the isolation by distance mode of gene diffusion. However, we expect intuitively that the patterns of genetic variability may not be consistent with the above hypothesis among populations with different social norms governing the institution of marriage, particularly among those that adhere to strict endogamy rules. We test the universality of this hypothesis by analyzing Y-chromosome and mtDNA data in three different sets of Indian populations that follow endogamy rules to varying degrees. Our analysis of the Indian patrilocal and the matrilocal groups is not confirmatory to the sex-specific variation observed among the tribes of Thailand. Our results indicate spatial instability of the impact of different cultural processes on the genetic variability, resulting in the lack of universality of the hypothesized pattern of greater Y-chromosome variation when compared to that of mtDNA among the patrilocal populations.     

The stimulation of collagenase production in rabbit articular chondrocytes by interleukin-1 is increased by collagens

Osteoarthritis is characterized by a loss of articular cartilage due at least in part to the action of degradative enzymes secreted by chondrocytes. We have investigated the effect of type II collagen from cartilage and interleukin 1 on collagenase production in cultures of rabbit articular chondrocytes. Interleukin 1 alone stimulated the chondrocytes to secrete collagenase but this response was increased as much as fivefold by the addition of rabbit type II collagen. Bovine type II and chick type I collagens were also stimulatory. The native form of the collagens was not required since denatured collagens and purified chick type II alpha chains were effective. The observed effects of collagens and interleukin 1 may contribute to the progressive nature of osteoarthritis.     

Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier

Cardiac triacylglycerol (TG) catabolism critically depends on the TG hydrolytic activity of adipose triglyceride lipase (ATGL). Perilipin 5 (Plin5) is expressed in cardiac muscle (CM) and has been shown to interact with ATGL and its coactivator comparative gene identification-58 (CGI-58). Furthermore, ectopic Plin5 expression increases cellular TG content and Plin5-deficient mice exhibit reduced cardiac TG levels. In this study we show that mice with cardiac muscle-specific overexpression of perilipin 5 (CM-Plin5) massively accumulate TG in CM, which is accompanied by moderately reduced fatty acid (FA) oxidizing gene expression levels. Cardiac lipid droplet (LD) preparations from CM of CM-Plin5 mice showed reduced ATGL- and hormone-sensitive lipase-mediated TG mobilization implying that Plin5 overexpression restricts cardiac lipolysis via the formation of a lipolytic barrier. To test this hypothesis, we analyzed TG hydrolytic activities in preparations of Plin5-, ATGL-, and CGI-58-transfected cells. In vitro ATGL-mediated TG hydrolysis of an artificial micellar TG substrate was not inhibited by the presence of Plin5, whereas Plin5-coated LDs were resistant toward ATGL-mediated TG catabolism. These findings strongly suggest that Plin5 functions as a lipolytic barrier to protect the cardiac TG pool from uncontrolled TG mobilization and the excessive release of free FAs.     

Leishmanicidal and Anticandidal Activity of Constituents of Indian Edible Mushroom Astraeus hygrometricus

Two new lanostane-type triterpenes, 1 and 2, were isolated from Astraeus hygrometricus. The structures were established by IR, (1) H- and (13) C-NMR, MS, and X-ray crystallographic experiments. The triterpenes exhibited excellent in vitro toxicities against Candida albicans, comparable to standard antifungal antibiotics. The triterpene 2 significantly inhibited the growth of Leishmania donovani promastigotes in vitro. The triterpene skeleton may be considered a template structure in search for new compounds with anticandidal and leishmanicidal activity.     

Towards a better production of bacterial exopolysaccharides by controlling genetic as well as physico-chemical parameters

Bacterial extracellular polymeric substances, which are basically bacterial metabolites, have currently become a subject of great concern of modern day microbiologists and biotechnologists. Among these metabolites, bacterial exopolysaccharides or EPS, in particular, have gained a significant importance. EPS are formed by the bacteria in their late exponential or stationary phase of growth under special situations for specific purposes. They take part in the formation of bacterial biofilms. There is a great diversity in the types of EPS. Strikingly enough, a same species of bacterium can produce different types of EPS under different situations. The importance of EPS is largely because of their different applications in various industries. Now that the bacterial EPS has got the potentiality to become an upcoming tool in various futuristic applications of human benefit, the focus currently develops towards how better they can be produced in the laboratory by promoting the favorable factors for their production. While studying with different EPS forming bacteria, both the intrinsic factors like genetic configuration of the bacteria and the extrinsic factors like culture conditions under the influence of different physico-chemical parameters in order to maximize the EPS production have been taken into consideration. Both the factors have proved their worth. Hence, towards a better outcome for EPS production, it is indicated that a genetic manipulation of the bacteria should be synchronized with a proper selection of its culture condition by controlling different physico-chemical parameters.

     

PsbY is required for prevention of photodamage to photosystem II in a PsbM-lacking mutant of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The PsbM (3.9 kDa) and PsbY (4.2 kDa) proteins are membrane-spanning, single-helix, subunits associated with the chlorophyll-binding CP47 pre-complex of photosystem II (PSII). Removal of PsbM resulted in accumulation of PSII pre-assembly complexes and impaired electron transfer between the primary (Q_A) and secondary (Q_B) plastoquinone electron acceptors of PSII indicating that the Q_B-binding site and bicarbonate binding to the non-heme iron were altered in this strain. Removal of PsbY alone had only a minor impact on PSII activity but deleting PsbY in the ΔPsbM background led to additional modification of the acceptor side resulting in ΔPsbM:ΔPsbY cells being susceptible to photodamage and this required protein synthesis for recovery. Addition of bicarbonate was able to compensate for the light-induced damage in ΔPsbM:ΔPsbY cells potentially re-occupying the modified bicarbonate-binding site in the ΔPsbM:ΔPsbY strain and complementation of ΔPsbM:ΔPsbY cells with the psbY gene restored the ΔPsbM phenotype.     

Selenium salts and chromosome damage

Sodium selenite and sodium selenate, fed by gavaging to age-matched male Swiss albino mice and observed after 24 h following a colchicine-fixative-air drying-Giemsa schedule, were found to induce chromosome breaks and spindle disturbances in bone marrow cells. The four concentrations used were fractions of LD₅₀ and the effects were directly proportionate to the concentration of the chemical. Sodium selenite induced a slightly higher frequency of chromosomal aberrations than sodium selenate.