Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae.

To further our studies of protein sorting and biogenesis of the lysosome-like vacuole in yeast, we have isolated spontaneous mutations in 11 new VPL complementation groups, as well as additional alleles of the eight previously described VPL genes. These mutants were identified by selecting for cells that mislocalize vacuolar proteins to the cell surface. Morphological examination of the vpl mutants indicated that most contain vacuoles of normal appearance; however, some of the mutants generally lack a large vacuole, and instead accumulate smaller organelles. Of the 19 VPL complementation groups, 12 were found to be identical to 12 of 33 VPT complementation groups identified in a separate study. Moreover, the end1 mutant and all of the previously reported pep mutants, with the exception of pep4, were found to exhibit a profound vacuolar protein sorting defect, and complementation tests between the PEP, VPL VPT and END1 groups demonstrated that there are extensive overlaps between these groups. Collectively, mutants in these four collections define 49 complementation groups required to deliver or retain soluble vacuolar enzymes, including carboxypeptidase Y (CPY) and proteinase A. We have also isolated 462 new mutants that lack normal levels of vacuolar CPY activity. Among these latter mutants, only pep4 mutants were found to be specifically defective in vacuolar zymogen activation. We conclude that there is a large number of gene products required for sorting or retention of vacuolar proteins in yeast, and only a single gene, PEP4, that is essential for activation of CPY and other vacuolar zymogens.     

Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Summary The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 μM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of inducation may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA. This work was conducted while the authors were with the Biological Carcinogenesis Program, Frederick Cancer Research Facility, Frederick, MD 21701, and was supported under Contract NO1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, MD 20205.     

Physiological profile of world-class high-altitude climbers

The functional characteristics of six world-class high-altitude mountaineers were assessed 2-12 mo after the last high-altitude climb. Each climber on one or several occasions had reached altitudes of 8,500 m or above without supplementary O2. Static and dynamic lung volumes and right and left echocardiographic measurements were found to be within normal limits of sedentary controls (SC). Muscle fiber distribution was 70% type I, 22% type IIa, and 7% type IIb. Mean muscle fiber cross-sectional area was significantly smaller than that of SC (-15%) and of long-distance runners (LDR, -51%). The number of capillaries per unit cross-sectional area was significantly greater than that of SC (+ 40%). Total mitochondrial volume was not significantly different from that of SC, but its subsarcolemmal component was equal to that of LDR. Average maximal O2 consumption was 60 +/- 6 ml X kg-1 X min-1, which is between the values of SC and LDR. Average maximal anaerobic power was 28 +/- 2.5 W X kg-1, which is equal to that of SC and 40% lower that that of competitive high jumpers. All subjects were characterized by resting hyperventilation both in normoxia and in moderate (inspired O2 partial pressure = 77 Torr) hypoxia resulting in higher oxyhemoglobin saturation levels in hypoxia. The ventilatory response to four tidal volumes of pure O2 was similar to that of SC. It is concluded that elite high-altitude climbers do not have physiological adaptations to high altitude that justify their unique performance.     

Endurance training in humans: aerobic capacity and structure of skeletal muscle

The adaptation of muscle structure, power output, and mass-specific rate of maximal O2 consumption (VO2max/Mb) with endurance training on bicycle ergometers was studied for five male and five female subjects. Biopsies of vastus lateralis muscle and VO2max determinations were made at the start and end of 6 wk of training. The power output maintained on the ergometer daily for 30 min was adjusted to achieve a heart rate exceeding 85% of the maximum for two-thirds of the training session. It is proposed that the observed preferential proliferation of subsarcolemmal vs. interfibrillar mitochondria and the increase in intracellular lipid deposits are two possible mechanisms by which muscle cells adapt to an increased use of fat as a fuel. The relative increase of VO2max/Mb (14%) with training was found to be smaller by more than twofold than the relative increase in maximal maintained power (33%) and the relative change in the volume density of total mitochondria (+40%). However, the calculated VO2 required at an efficiency of 0.25 to produce the observed mass-specific increase in maximal maintained power matched the actual increase in VO2max/Mb (8.0 and 6.5 ml O2 X min-1 X kg-1, respectively). These results indicate that despite disparate relative changes the absolute change in aerobic capacity at the local level (maintained power) can account for the increase in aerobic capacity observed at the general level (VO2max).     

Energy metabolism of medium-chain triglycerides versus carbohydrates during exercise

Medium-chain triglycerides (MCT) are known to be rapidly digested and oxidized. Their potential value as a source of dietary energy during exercise was compared with that of maltodextrins (MD). Twelve subjects exercised for 1 h on a bicycle ergometer (60% VO2 max), 1 h after the test meal (1MJ). The metabolism of MCT was followed using 1-13C-octanoate (Oc) as tracer and U-13C-glucose (G) was added to the 13C-naturally enriched MD. After MCT ingestion no insulin peak was observed with some accumulation of ketone bodies (KB), blood levels not exceeding 1 mM. Total losses of KB during exercise in urine, sweat and as breath acetone were small (less than 0.2 mmol X h-1). Hence, the influence of KB loss and storage on gas exchange data was negligible. The partition of fat and carbohydrate utilization during exercise as obtained by indirect calorimetry was practically the same after the MCT and the CHO meals. Oxidation over the 2-h period was 30% of dose for Oc and 45% for G. Glycogen decrements in the Vastus lateralis muscle were equal. It appears that with normal carbohydrate stores, a single meal of MCT or CHO did not alter the contribution of carbohydrates during 1 h of high submaximal exercise. The moderate ketonemia after MCT, despite substantial oxidation of this fat, led to no difference in muscle glycogen sparing between the diets.     

Identification of indolepyruvic acid as an intermediate of rebeccamycin biosynthesis

Summary Experimental evidence is presented to demonstrate that indolepyruvic acid is an intermediate in the rebeccamycin biosynthetic pathway. [3-¹⁴C]Indolepyruvic acid was prepared and efficiently incorporated (8%) into rebeccamycin bySaccharothrix aerocolonigenes.     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.     

Improved resolution with one-dimensional polyacrylamide gel electrophoresis: myofibrillar proteins from typed single fibers of human muscle

Standard procedures for one-dimensional discontinuous sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and silver staining were modified to give more effective separation and an improved resolution of human skeletal muscle proteins. In this system, an electrophoresis buffer composed of 100 mM L-isoleucine, 25 mM Tris base, and 0.1% SDS was used. The separating gel consisted of 16% acrylamide with N,N'-methylenebisacrylamide as a crosslinker (1:23), 0.4% SDS, 1.5 M Tris-HCl, pH 8.8. By the present procedure, the slow and the fast forms of myosin light chains (LCs, LCf) and other contractile proteins from human muscle could be better separated. The silver stain is based on a combination of methods previously described. The modified method requires a small fragment of a single fiber to observe as few as 10 ng of myofibrillar muscle proteins. The described simplifications made it possible to assay and compare up to 40 single fibers in the same electrophoretic run. Improved separation of other proteins migrating at basic pH could be achieved by a similar approach.     

Effects of parathyroid hormone and calcitonin on adenylate cyclase in murine mononuclear phagocytes

Peritoneal mononuclear phagocytes elicited by thioglycollate demonstrate responsiveness to parathyroid hormone (PTH) and calcitonin (CT) which differs from that seen in the normal resident population. PTH causes a twofold stimulation of adenylate cyclase activity in elicited cells but inhibits this activity in resident cells. CT causes a greater stimulation of adenylate cyclase in elicited than in resident cells. Both CT and PTH cause an increase in cyclic AMP accumulation in cultures of elicited mononuclear phagocytes. These results indicate that cells of the mononuclear phagocyte lineage have functional receptors for both PTH and CT. This is the first biochemical evidence to support the hypothesis that mononuclear phagocytes are precursors of the bone resorbing osteoclast.