Comparison between surrogate indexes of insulin sensitivity and resistance and hyperinsulinemic euglycemic clamp estimates in mice

Insulin resistance contributes to the pathophysiology of diabetes, obesity, and their cardiovascular complications. Mouse models of these human diseases are useful for gaining insight into pathophysiological mechanisms. The reference standard for measuring insulin sensitivity in both humans and animals is the euglycemic glucose clamp. Many studies have compared surrogate indexes of insulin sensitivity and resistance with glucose clamp estimates in humans. However, regulation of metabolic physiology in humans and rodents differs and comparisons between surrogate indexes and the glucose clamp have not been directly evaluated in rodents previously. Therefore, in the present study, we compared glucose clamp-derived measures of insulin sensitivity (GIR and SI(Clamp)) with surrogate indexes, including quantitative insulin-sensitivity check index (QUICKI), homeostasis model assessment (HOMA), 1/HOMA, log(HOMA), and 1/fasting insulin, using data from 87 mice with a wide range of insulin sensitivities. We evaluated simple linear correlations and performed calibration model analyses to evaluate the predictive accuracy of each surrogate. All surrogate indexes tested were modestly correlated with both GIR and SI(Clamp). However, a stronger correlation between body weight per se and both GIR and SI(Clamp) was noted. Calibration analyses of surrogate indexes adjusted for body weight demonstrated improved predictive accuracy for GIR [e.g., R = 0.68, for QUICKI and log(HOMA)]. We conclude that linear correlations of surrogate indexes with clamp data and predictive accuracy of surrogate indexes in mice are not as substantial as in humans. This may reflect intrinsic differences between human and rodent physiology as well as increased technical difficulties in performing glucose clamps in mice.     

DNA Clasping by Mycobacterial HU: The C-Terminal Region of HupB Mediates Increased Specificity of DNA Binding

Background

HU a small, basic, histone like protein is a major component of the bacterial nucleoid. E. coli has two subunits of HU coded by hupA and hupB genes whereas Mycobacterium tuberculosis (Mtb) has only one subunit of HU coded by ORF Rv2986c (hupB gene). One noticeable feature regarding Mtb HupB, based on sequence alignment of HU orthologs from different bacteria, was that HupB_Mtb bears at its C-terminal end, a highly basic extension and this prompted an examination of its role in Mtb HupB function.

Methodology/Principal Findings

With this objective two clones of Mtb HupB were generated; one expressing full length HupB protein (HupB_Mtb) and another which expresses only the N terminal region (first 95 amino acid) of hupB (HupB_MtbN). Gel retardation assays revealed that HupB_MtbN is almost like E. coli HU (heat stable nucleoid protein) in terms of its DNA binding, with a binding constant (K_d) for linear dsDNA greater than 1000 nM, a value comparable to that obtained for the HUαα and HUαβ forms. However CTR (C-terminal Region) of HupB_Mtb imparts greater specificity in DNA binding. HupB_Mtb protein binds more strongly to supercoiled plasmid DNA than to linear DNA, also this binding is very stable as it provides DNase I protection even up to 5 minutes. Similar results were obtained when the abilities of both proteins to mediate protection against DNA strand cleavage by hydroxyl radicals generated by the Fenton''s reaction, were compared. It was also observed that both the proteins have DNA binding preference for A:T rich DNA which may occur at the regulatory regions of ORFs and the oriC region of Mtb.

Conclusions/Significance

These data thus point that HupB_Mtb may participate in chromosome organization in-vivo, it may also play a passive, possibly an architectural role.     

Primitive neuroectodermal tumor of the kidney. A report of two cases diagnosed by fine needle aspiration cytology

BACKGROUND: Primitive neurocetodermal tumors (PNETs) constitute a family of neoplasms of presumed neuroectrodermal origin most often presenting as bone or soft tissue masses. There are very few reported cases of PNET of the kidney and none diagnosed by fine needle aspiration cytology (FNAC), to the best of our knowledge, in the world literature. We present two cases of renal PNET diagnosed on cytology. CASES: Two patients with renal masses were diagnosed as having PNET on FNAC. Cytologically the tumors showed a dispersed population of malignant small round cells with focal rosette formation and perivascular arrangement of tumor cells. Immunohistochemistry on the cell blocks in both cases showed strong membrane positivity for CD99 (MIC2). Cytogenetic studies in both cases showed the characteristic t(11;22)(q24;q12) translocation, with additional chromosomal abnormalities in case 2. CONCLUSION: PNET of the kidney is a distinct entity and can be diagnosed on fine needle aspiration smears and confirmed with immunohistochemistry and cytogenetic studies. A diagnosis of PNET must be included in the differential diagnosis of renal masses in adolescents and young adults.     

Genetic and biochemical evidences reveal novel insights into the mechanism underlying <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Sae2-mediated abrogation of DNA replication stress

In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in double-strand break (DSB) repair, replication stress and telomere length maintenance. Another protein linked to DSB repair is Sae2, which regulates MRX persistence at DSBs. However, very little is known about its role in DNA replication stress and repair. Here, we reveal a crucial role for Sae2 in DNA replication stress. We show that different mutant alleles of SAE2 cause hypersensitivity to genotoxic agents, and when combined with Δmre11 or nuclease-defective mre11 mutant alleles, the double mutants are considerably more sensitive suggesting that the sae2 mutations synergize with mre11 mutations. Biochemical studies demonstrate that Sae2 exists as a dimer in solution, associates preferentially with single-stranded and branched DNA structures, exhibits structure-specific endonuclease activity and cleaves these substrates from the 5′ end. Furthermore, we show that the nuclease activity is indeed intrinsic to Sae2. Interestingly, sae2^G270D protein possesses DNA-binding activity, but lacks detectable nuclease activity. Altogether, our data suggest a direct role for Sae2 nuclease activity in processing of the DNA structures that arise during replication and DNA damage and provide insights into the mechanism underlying Mre11-Sae2-mediated abrogation of replication stress-related defects in S. cerevisiae.     

Characterization and comparison of the soluble and membrane-bound cyclic AMP-dependent protein kinase from swine kidney

The catalytic subunits of adenosine 3′:5′ monophosphate-dependent protein kinase (ATP:protein transferase, E.C. 2.7.1.1.37) from the soluble and membrane fractions of swine kidney were purified to homogeneity by a new procedure and their structural, kinetic and immunological properties were compared. The specific activities of the purified enzymes were 2.35 and 2.6 µmol/min/mg of protein, with histone as the substrate. Both preparations contained a single polypeptide chain, and only one band was observed upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of both enzymes determined by gel electrophoresis was 42 000 ± 1000, and sedimentation equilibrium yielded a value of 41000 ± 800. Analysis by sedimentation velocity showed the presence of a single peak with and S_20,w of 3.1 ± 0.2 for each preparation. The amino acid compositions are very similar, and each enzyme contains about one residue of cysteine which is essential for enzymatic activity. ATP and Mg²⁺ protect both enzymes from inhibition by thiol specific reagents to the same extent. The catalytic subunits have similar apparent K m's for protein substrates. The enzymes exhibit single completely confluent precipitin lines when examined by immunodiffusion and the particulate catalytic subunit competitively displaces the soluble ¹²⁵I-catalytic subunit in homologous radioimmunoassays. The soluble and particulate ¹²⁵I-catalytic subunits bind to the regulatory subunits in the washed plasma membranes with attendent loss of kinase activity, which could be reversed by cyclic AMP. The results of experiments with kidney cortex slices treated with parathyroid hormone, epinephrine or dibutyryl cyclic AMP showed the translocation of phosphotransferase activity from the cytosol to the particulate membrane fraction. Taken collectively, these observations suggest that only one form of the catalytic subunit of cyclic AMP-dependent protein kinase is present in swine kidney, and that it may exchange between the cytosol and membrane fractions in response to specific physiological signals.     

Molecular and Functional Characterization of RecD,a Novel Member of the SF1 Family of Helicases,from Mycobacterium tuberculosis

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5′ overhangs relative to the 3′ overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having ≥18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3′ overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5′ overhangs, it could also catalyze significant unwinding of substrates containing 3′ overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5′ → 3′ and weak 3′ → 5′ unwinding activities. The extent of unwinding of Y-shaped DNA structures was ∼3-fold lower compared with duplexes with 5′ overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.     

Homologous pairing between nucleosome cores on a linear duplex DNA and nucleoprotein filaments of RecA protein-single stranded DNA.

The ability of E coli recA protein to promote homologous pairing with linear duplex DNA bound to HU protein (Nucleosome cores) was found to be differentially affected. The formation of paranemic joint molecules was not affected whereas the formation of plectomic joint molecules was inhibited from the start of the reaction. The formation of paranemic joint molecules between nucleoprotein filaments of recA protein-circular single stranded DNA and closed circular duplex DNA is believed to generate positive supercoiling in the duplex DNA. We found that the positively superhelical duplex DNA was inert in the formation of joint molecules but could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. These observations initiate an understanding of the structural features of E coli chromosome such as DNA supercoiling and nucleosome-like structures in homologous recombination.     

Nature of the thiamin-binding protein from chicken egg yolk.

A simple, rapid and efficient procedure for the purification of thiamin-binding protein from chicken egg yolk was developed. The method involved removal, by exclusion, of lipoproteins from DEAE-cellulose and subsequent elution of water-soluble proteins held on the ion-exchanger with 1 M-NaCl, followed by treatment of the eluted protein fraction with an aqueous suspension of dextran/charcoal to generate apoprotein from the holoprotein. The resultant protein fraction was subjected to bioaffinity chromatography on thiamin pyrophosphate--AE (aminoethyl)-Sepharose. The protein eluted specifically with 10 microM-thiamin at pH 7.0, was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, had a mol.wt. of 38 000 +/- 2000 and was not a glycoprotein. The purified thiamin-binding protein specifically interacted with riboflavin-binding protein with no detectable deleterious affect on its (14C)thiamin-binding capacity. The protein bound [14C]thiamin with a molar ratio of 1.0, with dissociation constant (Kd) 0.41 microM. This protein-ligand interaction was inhibited by thiamin analogues and antagonists. The absorption spectrum of the protein in the presence of thiamin exhibited significant hypochromism at the 278 nm band, indicating the involvement of aromatic amino acid residues of the protein, during its binding to the ligand. The protein cross-reacted with the monospecific antiserum to egg-white thiamin-binding protein, showing thereby that thiamin-binding proteins present in chicken egg yolk and white are the products of the same structural gene.