Insulin Sensitivity in African‐American and White Women: Association With Inflammation

Whether the contribution of inflammation to risk for chronic metabolic disease differs with ethnicity is not known. The objective of this study was to determine: (i) whether ethnic differences exist in markers of inflammation and (ii) whether lower insulin sensitivity among African Americans vs. whites is due to greater inflammatory status. Subjects were African‐American (n = 108) and white (n = 105) women, BMI 27–30 kg/m². Insulin sensitivity was assessed with intravenous glucose tolerance test and minimal modeling; fat distribution with computed tomography; body composition with dual‐energy X‐ray absorptiometry; markers of inflammation (tumor necrosis factor (TNF)‐α, soluble tumor necrosis factor receptor (sTNFR)‐1, sTNFR‐2, C‐reactive protein (CRP), and interleukin (IL)‐6) with enzyme‐linked immunosorbent assay (ELISA). Whites had greater intra‐abdominal adipose tissue (IAAT), insulin sensitivity, and concentrations of TNF‐α, sTNFR‐1, and sTNFR‐2 than African Americans. Greater TNF‐α in whites vs. African Americans was attributed to greater IAAT in whites. Among whites, but not African Americans, CRP was independently and inversely associated with insulin sensitivity, after adjusting for IAAT (r = ?0.29 P < 0.05, and r = ?0.13 P = 0.53, respectively). Insulin sensitivity remained lower in African Americans after adjusting for CRP (P < 0.001). In conclusion, greater IAAT among whites may be associated with greater inflammation. Insulin sensitivity was lower among African Americans, independent of obesity, fat distribution, and inflammation.     

Stellate cell apoptosis by a soluble mediator from immortalized human hepatocytes

Activated hepatic stellate cells (HSCs) are the major source of extracellular matrix in fibrosis and cirrhosis. In this study, we have investigated the role of hepatitis C virus (HCV) core protein induced immortalized human hepatocytes (IHH) on HSC growth. Preferential growth of IHH and apoptosis of activated human hepatic stellate cells (LX2) were observed upon coculture of these two cell types in a dual chamber or in the presence of conditioned medium (CM) from IHH. CM did not display a growth inhibitory role on other hepatic (Huh-7, HepG2, Hep3B and THLE) and non-hepatic (HeLa, MCF-7, and BHK) epithelial cells, indicating that the soluble mediator from IHH does not have a generalized effect on cell lines examined in our study. Further studies suggested that CM from IHH increased the expression of TRAIL receptors on LX2 cell surface, and induced apoptosis by a caspase dependent mechanism. Peptide mass fingerprinting of the purified soluble mediator from CM suggested that gelsolin fragments may play a role in apoptosis of LX2 cells. Taken together, our results suggested that a soluble mediator secreted from immortalized human hepatocytes plays an important role in hepatic stellate cell growth regulation.     

Oxidative refolding of lysozyme in trifluoroethanol (TFE) and ethylene glycol: interfering role of preexisting alpha-helical structure and intermolecular hydrophobic interactions

We investigated the effect of aldosterone on Src kinase. In the kidney cell line, M-1 aldosterone leads to a >2-fold transient activation of Src kinase seen as early as 2 min after aldosterone administration. Maximal Src kinase activation was measured at an aldosterone concentration of 1 nM. In parallel to activation, autophosphorylation at Tyr-416 of Src kinase increased. Src kinase activation was blocked by spironolactone. Aldosterone led to increased association of Src with HSP84. Furthermore, rapamycin blocked aldosterone-induced Src activation. We conclude that Src activation by aldosterone is mediated through the mineralocorticoid receptor and HSP84.     

Protein-protein interaction conferring stability to an extracellular acetyl (xylan) esterase produced by Termitomyces clypeatus

Acetyl esterase (AE) activity present in the culture filtrate of Termitomyces clypeatus was separated into lower molar mass (LMM) and higher molar mass (HMM) protein fractions during BioGel P-200 gel chromatography. AE was purified as a 30 kDa nonglycosylated protein from LMM fractions by CM-Sepharose ion exchange chromatography and HPGPLC. Although the HMM fraction had a number of enzyme activities (sucrase, beta-xylosidase, beta-glucosidase, and alpha-L-arabinofuranosidase) other than AE, protein present in the fraction was eluted as a single protein peak in HPGPLC and gave a single band in native PAGE. The fraction, subsequently purified by DEAE-Sephadex chromatography, was a SDS-PAGE homogeneous 80 kDa glycoprotein, but with both AE and cellobiase activities. The aggregate dissociated during ConA-Sepharose chromatography and 30 kDa AE and 56 kDa glycosylated cellobiase were purified separately. The dissociation caused significant loss of cellobiase activity but not that of AE. AE purified from both HMM and LMM fractions was characterized to be the same enzyme in terms of molar masses, pI (7.3), and other physicochemical properties. AE as an aggregate with cellobiase showed higher thermostability, temperature optimum, and resistance toward chemical denaturants than those of purified AE. Compared to cellobiase purified earlier from the same fungus, the enzyme present with AE in the aggregate also showed higher catalytic activity, thermostability, and temperature optimum. The study indicated that the formation of such SDS-resistant enzyme aggregate was associated with significant changes in the physicochemical properties of the enzymes, mainly toward improvement of rigidity of enzymes, and sometimes with the improvement of catalytic activity.     

P21-activated kinase-1 phosphorylates and transactivates estrogen receptor-alpha and promotes hyperplasia in mammary epithelium

Stimulation of p21-activated kinase-1 (Pak1) induces cytoskeleton reorganization and signaling pathways in mammary cancer cells. Here, we show that inhibition of Pak1 kinase activity by a dominant-negative fragment or by short interference RNA markedly reduced the estrogen receptor-alpha (ER) transactivation functions. To understand the role of Pak1 in mammary glands, we developed a murine model expressing constitutively active Thr423 glutamic acid Pak1 driven by the beta-lactoglobulin promoter. We show that mammary glands from these mice developed widespread hyperplasia associated with apocrine metaplasia and lobuloalveolar hyperdevelopment during lactation. Mammary tissues with active Pak1 also exhibited an increased activation of mitogen-activated protein kinase and stimulated transactivation functions of the ER and expression of endogenous ER target genes. Furthermore, Pak1 directly phosphorylated the activation function-2 domain of the ER at the N-terminal residue Ser305, and its mutation to Ala (S305A) abolished the Pak1-mediated phosphorylation and transactivation functions of the ER, while its mutation to glutamic acid (S305E) promoted transactivation activity of ER. These findings reveal a novel role for the Pak1-ER pathway in promoting hyperplasia in mammary epithelium.     

Analgesia in phasic and tonic pain tests in a pharmacological model of autotomy

Self-mutilation or self-injurious behaviour is a well known behavioural disorder in humans. The proposition that this behaviour in animals is a response to chronic pain of peripheral nerve injury has been met with controversy. In the present study a pharmacological model, which produces no sensory or motor loss was used to study how autotomy is related to pain. In a group of rats autotomy was induced by amphetamine in phenoxybenzamine and reserpine treated animals. The pain tests, both phasic and tonic were then performed. The results of this study showed that a total analgesia was produced in both phasic and tonic pain tests, in animals that exhibited autotomy. Injection of naloxone in these animals prevented autotomy. A correlation between autotomy and no pain is suggested in this pharmacological model of autotomy.     

Dirhodium(II) tetrakis[N,N-dimethyl-2-pyrrolidone-5(S)-carboxamide]. Structural effects on enantioselection in metal carbene transformations

The preparation and structural characterization of dirhodium(II) tetrakis[N,N-dimethyl-2-pyrrolidone-5(S)-carb- oxamide], Rh₂(5S-DMAP)₄, a new sterically-demanding catalyst for enantioselective metal carbene transformations, is described. The pyrrolidone ligands are arrayed around the dirhodium(II) core with two oxygen and two nitrogen donor atoms, each oriented cis, bound to each octahedral rhodium. The crystal structure of this compound has been determined to be that of Rh₂(5S-DMAP)₄(CH₃CN)₂·CH₃CN·6H₂O: space group P2₁2₁2₁ with cell constants a= 12.685(4), b=15.050(3), c=24.035(4) Å; V=4588.5(1.9) ų, Z=4, R=0.0316, Rh---Rh DISTANCE =2 4538(5) Å. Decreased activity for diazodecomposition catalyzed by Rh₂(5S-DMAP)₄ is observed, and enantiocontrol for cyclopropanation and carbon-hydrogen insertion is lower than expected by analogy to the corresponding di- rhodium(II) tetrakis[methyl 2-pyrrolidone-5(S)-carboxylate], Rh₂(5S-MEPY)₄ Electronic stabilization of the in- termediate metal carbene is absent in reactions catalyzed by Rh₂(5S-DMAP)₄.     

Identification of a Strain of Peanut Chlorotic Streak Virus Causing Chlorotic Vein Banding Disease of Groundnut in India

A virus disease characterized by chlorotic vein banding, chlorotic line pattern along the margins or midrib of mature leaflets and chlorotic spots/rings was observed on commercial groundnut crops in Rayalaseema area of Andhra Pradesh with an incidence from 1% to nearly 60%. The virus was transmitted by mechanical inoculation in extracts prepared with 0.01 M potassium phosphate butter, pH 8.0 to 21 species from the Chenopodiaceae, Cruciferae, Leguminosae and Solanaceae, Chenopodium quinoa was found to be a good local lesion host. The virus was neither seed-transmitted through 1591 groundnut seeds nor aphid-transmitted by Aphis craccivora, Myzus persicae and Rhopalosiphum maidis either in non-persistent or semi-persistent manner. The virus remained infective in buffered tobacco leaf sap at a dilution of 10^?5; in a 10^?1 dilution of buffered sap the virus was infective for 2–3 days at 22–29°C or when heated to 65°C for 10 min but not to 70°C. Clarification treatments with organic solvents with 10% chloroform was least damaging. The virus was purified from Nicotiana rustica leaves. Purified virus contained isometric particles of 51 nm in diameter with an electron dense core of 22 nm and two major polypeptides of 76 kDa and 36 kDa. A polyclonal antiserum to this virus was produced. In agar gel double diffusion, enzyme-linked immunosorbent assay and in electro-blot immunoassay rests the virus was related to peanut chlorotic streak virus and not to cauliflower mosaic, figwort mosaic and soybean chlorotic mottle viruses.     

Maturation of rat spermatozoa: ESR spin labeling studies.

The ability of spermatozoa to reduce nitroxide spin--TEMPO has been used as a parameter to understand maturation, capacitation and calcium uptake of sperm obtained from Holstman strain rats. The rate of spin label reduction by sperm follows the trend--caput greater than cauda greater than corpus. With the increase in age, the electron donating capability shows first a gradual increase, for 60- to 85-day-old rats, peaking at 85 days (corresponding to puberty) and leveling off after 92 days. Calcium uptake takes place in two phases which corresponds to accumulation of and activation by calcium. The presence of polyclonal antibody which is known to cause agglutination, does not adversely affect the sperm activity.     

Replacement of phe(8) in substance P by tyr (Tyr(8)-SP) alters the conformation of the peptide in DMSO, water, and lipid bilayers.

The conformation of [Tyr(8)]SP (Y8SP) in dimethylsulfoxide (DMSO), water, and dipalmitoyl phosphatidylcholine (DPPC) bilayers has been investigated by two-dimensional nmr and molecular dynamics simulations. Molecular modeling of the conformation of Y8SP by incorporating nuclear Overhauser effects as distance restraints shows wide differences in its conformation in the three media. In DMSO, the main structural features are gamma-bends along with a nonspecific bend around Gln(6)-Phe(7)-Tyr(8). The random coil structure seen in water is transformed into a beta-turn around the segment Gln(5)-Gln(6)-Phe(7)-Tyr(8) when Y8SP is incorporated into DPPC bilayers. The lower biological activity of Y8SP compared to the native peptide (SP) has been attributed to the absence of any helix like structure at the central residues, a feature shown to be an important prerequisite for SP and SP agonists to bind to the neurokinin 1 tachykinin receptor.