Biomedical subjects
Publications and source records attributed to A Datta.
Nuclear magnetic resonance measurements indicate that hypericin exists in the same "normal" tautomeric form irrespective of whether the solvent is dimethyl sulfoxide or tetrahydrofuran. This result is discussed in the context of previous experimental and theoretical work. It is concluded that solvent perturbations cannot induce tautomerization in hypericin.
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The aim of this study was to establish whether or not to cement the hemiarthroplasty for displaced intracapsular femoral neck fractures in the elderly. Consecutive patients treated by hemiarthroplasty in adjacent hospitals were reviewed. The same monoblock prosthesis was used; in hospital A they were uncemented (121 patients), and in hospital B they were cemented (123 patients). Notes were reviewed retrospectively. Surviving patients (50 and 56 respectively) were assessed prospectively for pain and functional ability using validated scoring systems. Follow-up was 32-36 months. Patient demographics were similar. Fewer of the cemented group had been revised or were awaiting revision ( P=0.036). There was no difference in general complication or mortality rates. There was a highly statistically significant greater deterioration in pain ( P=0.003), walking ability ( P=0.002), use of walking aids ( P=0.003) and activities of daily living ( P=0.009) in the uncemented group. Our findings support the use of cemented hemiarthroplasty in the elderly.
N-Acetyl-D-glucosamine specific cell-associated hemagglutinin (HA)/lectin, previously purified from a strain of Vibrio cholerae O1, had been established as an adhesin molecule of V. cholerae O1 cells. This communication records the isolation and purification of the glycoprotein receptor of the N-acetyl-D-glucosamine specific HA of the V. cholerae O1 strain from chicken erythrocyte membranes. The most salient feature of this study is that the pretreatment of partially purified glycoprotein with purified HA could completely inhibit the hemagglutinating activity of the V. cholerae O1 strain with chicken erythrocytes.
The oxalate decarboxylase (OXDC) gene from Collybia velutipes is overexpressed as an active form in Schizosaccharomyces pombe. The recombinant enzyme shows similar pH optima and stability, while substrate kinetic analysis shows a ninefold decrease in K(m) value with respect to native OXDC. Most of the expressed protein was present in periplasm and remained firmly bound to cell-wall materials. However, 20% of enzyme expressed was secreted out into the medium suggesting the presence of a secretion signal (C. velutipes) in the oxalate decarboxylase gene. This secretion signal is associated with the N-terminal of OXDC as is evident by secretion of nonsecretory genes AmA1 and beta-galactosidase. An expression vector using this signal is constructed for expression and secretion of heterologous proteins in S. pombe.
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DDB has been implicated in DNA repair as well as transcription. Mutations in DDB have been correlated with the repair-deficiency disease, xeroderma pigmentosum group E (XP-E). The XP-E cells exhibit deficiencies in global genomic repair, suggesting a role for DDB in that process. DDB also possesses a transcription stimulatory activity. We showed that DDB could function as a transcriptional partner of E2F1. But the mechanism by which DDB stimulates E2F-regulated transcription or carry out its DNA repair function is not understood. To investigate the mechanisms, we looked for nuclear proteins that interact with DDB. Here we show that DDB associates with the CBP/p300 family of proteins, in vivo and in vitro. We suggest that DDB participates in global genomic repair by recruiting CBP/p300 to the damaged-chromatin. It is possible that the histone acetyltransferase activities of the CBP/p300 proteins induce chromatin remodeling at the damaged-sites to allow recruitment of the repair complexes. The observation offers insights into both transcription and repair functions of DDB.
Four new D-secopaclitaxel analogues were synthesized from paclitaxel. The key step of the synthesis involved the opening of the D-ring by Jones oxidation. Two of the compounds had been predicted to be nearly as active as paclitaxel in a minireceptor model of the binding site on tubulin, but all were biologically inactive in an in vitro cytotoxic assay and a tubulin assembly assay. The biological results identify a weakness in our predictive minireceptor model and suggest a corrective remedy in which additional amino acids are needed to accommodate ligand-protein steric effects around the oxetane ring. These changes to the model lead to correct predictions of the bioactivity. Conformational analysis and dynamics simulations of the compounds showed that the 4-acetyl substituent is as important as the oxetane in determining the A ring conformation.
Cancer cells show increased genome rearrangements, although it is unclear what defects cause these rearrangements. Mutations in Saccharomyces cerevisiae RFC5, DPB11, MEC1, DDC2 MEC3, RAD53, CHK1, PDS1, and DUN1 increased the rate of genome rearrangements up to 200-fold whereas mutations in RAD9, RAD17, RAD24, BUB3, and MAD3 had little effect. The rearrangements were primarily deletion of a portion of a chromosome arm along with TEL1-dependent addition of a new telomere. tel1 mutations increased the proportion of translocations observed, and in some cases showed synergistic interactions when combined with mutations that increased the genome rearrangement rate. These data suggest that one role of S phase checkpoint functions in normal cells is to suppress spontaneous genome rearrangements resulting from DNA replication errors.
We have used synchrotron x rays to study three different liquids near solid-liquid interfaces. For either ultrathin (45-90 A) or thick ( approximately 5000 A) liquid films on silicon substrates, we find (on the basis of diffraction peaks or specular reflectivity data) that the molecules form 3-6 layers at the interface, with plane spacings close to the molecular dimensions. Rough surfaces and/or impurities reduce the density oscillation amplitudes. Making the liquid film very thin does not observably enhance the effect, which implies that layering is present even at an isolated interface (i.e., in a semi-infinite liquid). On the other hand, predeposited impurities diffuse away from the interface more easily if the liquid films are thick. The liquids studied are nonconducting, nonpolar, and nonreactive; the molecules are roughly spherical; and our substrate surface has no lateral structure. Thus our observations should apply to any liquid near a hard wall.
The fibroin protein isolated from the posterior silkgland of the tropical Saturniid silkworm Antheraea mylitta, was solubilized in lithium dodecyl sulfate and purified by gel filtration. The major fraction from gel filtration was analyzed by SDS-PAGE under non-reducing and reducing conditions. One major protein band of ca 395 kDa was obtained under non-reducing conditions and a doublet band of approximately 197 kDa under reducing conditions. The appearance of a single spot in two-dimensional electrophoresis confirmed the purity of the protein indicating that it may be a homodimeric protein of two similar sized polypeptides. Amino acid composition analysis showed that, like other Saturniid fibroins, it is rich in glycine, alanine and serine amino acids. N-terminal amino acid sequence shows significant homology with other Antheraea species. The enzymatic deglycosylation analysis indicates that the fibroin protein is glycosylated and the oligosaccharides are O-linked to the protein backbone by N-acetylgalactoseamine moiety which conforms to a Core 1 mucin-type glycosylation pattern.
Fibroin gene expression during the larval developmental stages of the Saturniid silkworm, Antheraea mylitta, was analyzed. Northern blot analysis of larval silk gland total RNA using the fibroin gene as a probe showed that fibroin is expressed in the intermoult stages and repressed during the moulting stages. Abundance of fibroin transcripts gradually increased from the third to fifth intermoult stage, reaching a peak in the fifth intermoult. Transcripts declined during the early spinning stage. Western blot analysis of fibroin protein production with anti-fibroin antibody confirmed the differential fibroin expression, in accordance with fibroin mRNA synthesis. Dot blot hybridization of genomic DNA isolated from each larval developmental stage with the labelled fibroin gene showed that at the genomic level, the relative concentration of the fibroin gene was constant throughout the developmental stages. Our data confirm that fibroin gene expression in A. mylitta, like in B. mori, is transcriptionally controlled and shows differential temporal variations.
Three series of furfural derivatives, namely N-O-furfural oxime ethers, furfural Schiff bases (furfurylidene anilines), and furfural chalcones, have been synthesized and evaluated for nitrification inhibition activity in laboratory incubation studies in typic Ustocrept soil. Furfural oxime ethers and furfural Schiff bases showed potential activity, but furfural chalcones were only mildly active. N-O-ethyl furfural oxime among the oxime ethers, and furfurylidine-4-chloroaniline among the furfural Schiff bases, performed the best. These two compounds showed more than 50% nitrification inhibition on the 45th day at 5% dose as compared to 73% inhibition by nitrapyrin. Activity of furfural oxime ethers decreased with an increase in carbon atoms in the N-O-alkyl side chain. Introduction of a chlorine atom in the phenyl ring of furfurylidene anilines increased the persistence of their activity. N-O-Ethyl furfural oxime and furfurylidine-4-chloroaniline coated urea performed at par with their application in solution form. Ethyl and N-O-isopropyl oxime, as well as chloro- and nitro- substituted Schiff bases, did not reveal any phytotoxicity (adverse effect on germination) on chickpea seeds (Cicer arietinum) even at the highest dose (40 ppm, soil basis).
The Escherichia coli gene recQ was identified as a RecF recombination pathway gene. The gene SGS1, encoding the only RecQ-like DNA helicase in Saccharomyces cerevisiae, was identified by mutations that suppress the top3 slow-growth phenotype. Relatively little is known about the function of Sgs1p because single mutations in SGS1 do not generally cause strong phenotypes. Mutations in genes encoding RecQ-like DNA helicases such as the Bloom and Werner syndrome genes, BLM and WRN, have been suggested to cause increased genome instability. But the exact DNA metabolic defect that might underlie such genome instability has remained unclear. To better understand the cellular role of the RecQ-like DNA helicases, sgs1 mutations were analyzed for their effect on genome rearrangements. Mutations in SGS1 increased the rate of accumulating gross chromosomal rearrangements (GCRs), including translocations and deletions containing extended regions of imperfect homology at their breakpoints. sgs1 mutations also increased the rate of recombination between DNA sequences that had 91% sequence homology. Epistasis analysis showed that Sgs1p is redundant with DNA mismatch repair (MMR) for suppressing GCRs and for suppressing recombination between divergent DNA sequences. This suggests that defects in the suppression of rearrangements involving divergent, repeated sequences may underlie the genome instability seen in BLM and WRN patients and in cancer cases associated with defects in these genes.
A Candida albicans mutant with mutations in the N-acetylglucosamine (GlcNAc) catabolic pathway gene cluster, including the GlcNAc-6-phosphate deacetylase (DAC1), glucosamine-6-phosphate deaminase (NAG1), and GlcNAc kinase (HXK1) genes, was not able to grow on amino sugars, exhibited highly attenuated virulence in a murine systemic candidiasis model, and was less adherent to human buccal epithelial cells in vitro. No germ tubes were formed by the mutant after induction with GlcNAc, but the mutant exhibited hyperfilamentation under stress-induced filamentation conditions. In addition, the GlcNAc catabolic pathway played a vital role in determining the colony phenotype. Our results imply that this pathway is very important because of its diverse links with pathways involved in virulence and morphogenesis of the organism.
The hepatitis B virus (HBV) X protein (HBx) is critical for the life cycle of the virus. HBx associates with several host cell proteins including the DDB1 subunit of the damaged-DNA binding protein DDB. Recent studies on the X protein encoded by the woodchuck hepadnavirus have provided correlative evidence indicating that the interaction with DDB1 is important for establishment of infection by the virus. In addition, the interaction with DDB1 has been implicated in the nuclear localization of HBx. Because the DDB2 subunit of DDB is required for the nuclear accumulation of DDB1, we investigated the role of DDB2 in the nuclear accumulation of HBx. Here we show that expression of DDB2 increases the nuclear levels of HBx. Several C-terminal deletion mutants of DDB2 that fail to bind DDB1 are able to associate with HBx, suggesting that DDB2 may associate with HBx independently of binding to DDB1. We also show that DDB2 enhances the nuclear accumulation of HBx independently of binding to DDB1, since a mutant that does not bind DDB1 is able to enhance the nuclear accumulation of HBx. HBV infection is associated with liver pathogenesis. We show that the nuclear levels of DDB1 and DDB2 are tightly regulated in hepatocytes. Studies with regenerating mouse liver indicate that during late G1 phase the nuclear levels of both subunits of DDB are transiently increased, followed by a sharp decrease in S phase. Taken together, these results suggest that DDB1 and DDB2 would participate in the nuclear functions of HBx effectively only during the late-G1 phase of the cell cycle.
Adhesion of cells to extracellular matrix is mediated by integrin family receptors. The process of receptor-ligand binding is dependent on metabolic energy and is regulated by intracellular signals, termed inside-out signals. The strength of the initial alpha5beta1-mediated adhesion of v-src-transformed chicken embryo fibroblasts (v-srcCEF) was similar to that of normal CEF. A chemically cross-linked fibronectin substrate was able to restore cell spreading and the ability of v-srcCEF to assemble a fibronectin matrix. Over time, v-srcCEF showed decreased adhesion due to the reduction of alpha5beta1-fibronectin bonds consequent on the reduction of substrate-bound fibronectin due to the secretion of proteases by v-srcCEF. Excess synthesis of hyaluronic acid by v-srcCEF also reduced the alpha5beta1-fibronectin bonds and contributed to cell detachment at later times in culture. Thus, the adhesion defects were not due to a failure of alpha5beta1 function and adhesion of the v-srcCEF was alpha5beta1 dependent. Integrin-mediated adhesion also produces signals that affect cell proliferation and cell differentiation. An early consequence of these "outside-in" signals was the phosphorylation of FAK Y397 in direct proportion to the number of alpha5beta1-fibronectin bonds formed. In contrast, v-srcCEF had an increased level of phosphorylation on five different tyrosines in FAK, and none of these phosphorylation levels were sensitive to the number of alpha5beta1-fibronectin bonds. In the absence of serum, CEF proliferation was sensitive to changes in alpha5beta1-mediated adhesion levels. Transformation by v-src increased the serum-free proliferation rate and made it insensitive to alpha5beta1-mediated adhesion. Thus, the v-srcCEF were insensitive to the normal outside-in signals from alpha5beta1 integrin.
The excited-state intramolecular H-atom transfer of hypericin (Hyp) was investigated as a function of pH in monodispersed reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane/water and in complexes with Tb3+ under conditions in which one of the two carbonyl groups of Hyp is incapable of accepting a hydrogen atom. The results of pump-probe transient absorption experiments provide no evidence for a concerted H-atom transfer mechanism.