Muscle Rad expression and human metabolism: potential role of the novel Ras-related GTPase in energy expenditure and body composition

Ras associated with diabetes (Rad), a new ras-related GTPase, was recently identified by subtractive cloning as an mRNA in skeletal muscle that is overexpressed in NIDDM. To better understand its metabolic significance, we measured skeletal muscle Rad expression in well-characterized insulin sensitive (IS) and insulin resistant (IR) subjects with normal glucose tolerance and in untreated NIDDM patients. We found no differences in expression of Rad mRNA levels among IS, IR, and NIDDM groups using a ribonuclease protection assay (0.22 +/- 0.06, 0.13 +/- 0.01, and 0.16 +/- 0.02 relative units, respectively; NS) and no differences in Rad protein expression using a specific anti-peptide Rad antibody (1.05 +/- 0.18, 1.14 +/- 0.08, and 1.08 +/- 0.21 units/mg protein, respectively; NS). However, Rad protein levels were positively correlated with BMI (r = 0.43, P = 0.03) and percentage body fat (r = 0.55, P < 0.005), two independent measures of obesity, and negatively correlated with resting metabolic rate (r = 0.49, P = 0.01). In multiple regression analyses, percentage body fat and resting metabolic rate independently accounted for 30 and 10% of individual variability in muscle Rad protein expression. In conclusion, Rad expression in skeletal muscle is not altered as a function of insulin resistance or NIDDM in humans. However, these data, for the first time, implicate a role for Rad in regulating body composition and energy expenditure and provide a framework for studies designed to elucidate Rad's cellular functions.     

Bronchoscopic evaluation of pulmonary infiltrates following bone marrow transplantation

Mutants of human prothymosin alpha with impaired ability to inhibit yeast Saccharomyces cerevisiae. cerevisiae cell growth were characterized. Two types of prothymosin alpha-inactivating mutations were observed. Mutations that belong to the first type compromised the nuclear entry of prothymosin alpha by affecting its nuclear localization signal. Analysis of subcellular distribution of GFP-prothymosin alpha fusions revealed a bipartite nuclear localization signal that is both necessary and sufficient for nuclear import of the protein in human cells. Mutations of the second type abrogated the inhibitory action of prothymosin alpha through an unknown mechanism, without influencing the nuclear import of the protein.     

Analysis of insulin-like growth factors (IGF)-I, and -II, type II IGF receptor and IGF-binding protein-2 mRNA and peptide levels in normal and nephrectomized rat kidney

Immunocytochemistry, in situ hybridization, and radioimmunoassay were employed to examine the cellular distribution of mRNAs and proteins for IGF-I, II, IGF-II/M6P receptor, IGFBP2 as well as the levels of IGF-I and II in normal and unilaterally nephrectomized (Nx) adult rat kidneys. A similar distribution of immunoreactive IGF-I, and -II as well as IGF-II/M6P receptor was found in the principal cells of the cortical collecting duct and in all cells of the inner medullary collecting duct. In addition, immunostainable IGF-I and IGF-II/M6P receptor were noted in some inner medullary loops of Henle, while IGFBP2 was seen in the collecting ducts and loops of Henle of the inner medullar and the renal vasculature of all animals. By comparison, in situ hybridization revealed IGF-I mRNA only in the medullary thick ascending limbs while IGF-II mRNA was localized to the wall of the renal microvasculature in all kidneys. IGFBP2 mRNA was localized to the renal corpuscle and to inner medullary interstitial cells of all kidneys. These data suggest that renal IGF-I and IGFBP2 are synthesized at upstream sites along the nephron and then transported downstream for interaction with IGF receptors. Following nephrectomy, the renal levels of IGF-I peptide and mRNA were elevated at both 5 and 33 days post-nephrectomy, supporting a potential functional role for IGF-I in stimulating the structural and functional recovery in compensatory hypertrophy.     

Fertility in estrus-cycling and noncycling virgin heifers and suckled beef cows after induced ovulation

A procedure was developed to either induce or synchronize ovulation in heifers and suckled cows. Beef females were assigned to two breeding programs: 1) two injections of prostaglandin F2alpha (PGF2alpha) given 14 d apart to synchronize estrus (PGF2alpha control; n = 179), with inseminations 12 to 16 h after detected estrus or at 80 h in the absence of estrus, or 2) two injections of PGF2alpha (d -14 and 0) plus 100 microg of GnRH on d -7 when 6 mg of norgestomet was implanted (PGF2alpha/NORG/GnRH treatment; n = 173). Implants were removed 24 h after the second PGF2alpha injection (d +1) and females were inseminated 12 to 16 h after detected estrus until 54 h after PGF2alpha. The remaining cattle were given a second 100-microg GnRH injection 54 h after PGF2alpha and inseminated 18 to 20 h later. Percentages of noncycling females with subsequently elevated progesterone (P4) on d 0 or +1 were not different between treatment groups (20.4 vs 25%), but conception rate was greater (P < .05) in noncycling treated females than in noncycling controls (55 vs 12.8%). Conception rates in cycling (59.2%) and noncycling (62.2%) treated females were similar to those in cycling controls (56.2%) but greater (P = .06) than those in noncycling controls (26.5%). Conception rates in treated females inseminated 12 to 16 h after detected estrus (63.1%) or at one fixed time (58.3%) were similar to those in controls inseminated 12 to 16 h after detected estrus (68.7%). This treatment procedure produced fertility after one timed insemination that was equal to controls inseminated after detected estrus and induced equally fertile ovulations in noncycling heifers and cows.     

G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 2. Role of protein kinase C-delta in the regulation of eicosanoid production

PGI2 generation by the vessel wall is an agonist for cyclic-AMP-dependent cholesteryl ester hydrolysis. The process of enhanced PGI2 synthesis is stimulated, in part, by G-protein-coupled receptor ligands. Cellular cholesterol enrichment has been hypothesized to alter G-protein-mediated PGI2 synthesis. In the studies reported herein, cells generated PGI2 in response to AlF4-, GTPgammaS, and ATP in a dose-dependent manner. G-protein agonists stimulated eicosanoid production principally by activating phospholipase A2, but not phospholipase C. This is in contrast to PDGF, which stimulated phospholipase A2 and PLCgamma activities. Galphai subunits mediate G-protein agonist-induced PGI2 synthesis, since ATP- and PDGF-induced PGI2 synthesis was inhibited by pertussis toxin. Although cholesterol enrichment reduced arachidonic acid- and PDGF-induced PGI2 synthesis, cholesterol enrichment enhanced PGI2 release in response to AlF4-, GTPgammaS, and ATP. The enhancement of PGI2 release in cholesterol-enriched cells was augmented by mevalonate, which inhibits the ability of cholesterol enrichment to reduce membrane-associated G-protein subunits. Since cholesterol enrichment inhibited PDGF and AlF4--induced MAP kinase activity [Pomerantz, K., Lander, H. M., Summers, B., Robishaw, J. D., Balcueva, E. A., & Hajjar, D. P. (1997) Biochemistry 36, 9523-9531] (the major mechanism by which phospholipase A2 is activated), these results suggest that cholesterol enrichment induces other alternative signaling pathways leading to phospholipase A2 activation. A PKC-dependent pathway is described herein that is involved in enhanced eicosanoid production in cholesterol-enriched cells. This conclusion is supported by two observations: (1) G-protein-linked PGI2 production is inhibited by calphostin, and (2) cholesterol enrichment augments the specific translocation of the delta-isoform of PKC from the cytosol to the plasma membrane following treatment of cells with phorbol ester. These data support the concept that, in cells possessing normal levels of cholesterol, MAP-kinase-dependent pathways mediate eicosanoid synthesis in response to G-protein activation; however, under conditions of high cellular cholesterol levels, augmented G-protein-linked eicosanoid production results from enhanced PKCdelta activity.     

Incidence of acute appendicitis confirmed by histopathologic diagnosis

In this column, the Joint Commission on Accreditation of Healthcare Organizations (JCAHO) addresses the issue of competence, how it is documented and the areas in which staff must demonstrate competence. Readers are urged to send in any questions they may have and JCAHO staff will answer them.