NMR studies of muscle glycogen synthesis in insulin-resistant offspring of parents with non-insulin-dependent diabetes mellitus immediately after glycogen-depleting exercise.

To examine the impact of insulin resistance on the insulin-dependent and insulin-independent portions of muscle glycogen synthesis during recovery from exercise, we studied eight young, lean, normoglycemic insulin-resistant (IR) offspring of individuals with non-insulin-dependent diabetes mellitus and eight age-weight matched control (CON) subjects after plantar flexion exercise that lowered muscle glycogen to approximately 25% of resting concentration. After approximately 20 min of exercise, intramuscular glucose 6-phosphate and glycogen were simultaneously monitored with 31P and 13C NMR spectroscopies. The postexercise rate of glycogen resynthesis was nonlinear. Glycogen synthesis rates during the initial insulin independent portion (0-1 hr of recovery) were similar in the two groups (IR, 15.5 +/- 1.3 mM/hr and CON, 15.8 +/- 1.7 mM/hr); however, over the next 4 hr, insulin-dependent glycogen synthesis was significantly reduced in the IR group [IR, 0.1 +/- 0.5 mM/hr and CON, 2.9 +/- 0.2 mM/hr; (P < or = 0.001)]. After exercise there was an initial rise in glucose 6-phosphate concentrations that returned to baseline after the first hour of recovery in both groups. In summary, we found that following muscle glycogen-depleting exercise, IR offspring of parents with non-insulin-dependent diabetes mellitus had (i) normal rates of muscle glycogen synthesis during the insulin-independent phase of recovery from exercise and (ii) severely diminished rates of muscle glycogen synthesis during the subsequent recovery period (2-5 hr), which has previously been shown to be insulin-dependent in normal CON subjects. These data provide evidence that exercise and insulin stimulate muscle glycogen synthesis in humans by different mechanisms and that in the IR subjects the early response to stimulation by exercise is normal.

Two mutant prion proteins expressed in cultured cells acquire biochemical properties reminiscent of the scrapie isoform.

Prion diseases are a group of fatal neurodegenerative disorders that are unique in being infectious, genetic, and sporadic in origin. Infectious cases are caused by prions, which are composed primarily of PrPSc, a posttranslationally modified isoform of the normal cellular prion protein PrPC. Inherited cases are linked to insertional or point mutations in the host gene encoding PrPC. To investigate the molecular mechanisms underlying inherited prion diseases, we have constructed stably transfected Chinese hamster ovary cells that express mouse PrPs homologous to two human PrPs associated with familial Creutzfeldt-Jakob disease. One mouse PrP molecule carries a Glu-->Lys substitution at codon 199, and the other carries an insertion of six additional octapeptide repeats between codons 51 and 90. We find that both of these mutant PrPs display several biochemical hallmarks of PrPSc when synthesized in cell culture. Unlike wild-type PrP, the mutant proteins are detergent insoluble and are relatively resistant to digestion by proteinase K, yielding an N-terminally truncated core fragment of 27-30 kDa. Pulse-chase labeling experiments demonstrate that these properties are acquired posttranslationally, and are accompanied by increased metabolic stability of the protein. Our results provide the first evidence that a molecule with properties reminiscent of PrPSc can be generated de novo in cultured cells.

Using ubiquitin to follow the metabolic fate of a protein.

We describe a method that can be used to produce equimolar amounts of two or more specific proteins in a cell. In this approach, termed the ubiquitin/protein/reference (UPR) technique, a reference protein and a protein of interest are synthesized as a polyprotein separated by a ubiquitin moiety. This tripartite fusion is cleaved, cotranslationally or nearly so, by ubiquitin-specific processing proteases after the last residue of ubiquitin, producing equimolar amounts of the protein of interest and the reference protein bearing a C-terminal ubiquitin moiety. In applications such as pulse-chase analysis, the UPR technique can compensate for the scatter of immunoprecipitation yields, sample volumes, and other sources of sample-to-sample variation. In particular, this method allows a direct comparison of proteins' metabolic stabilities from the pulse data alone. We used UPR to examine the N-end rule (a relation between the in vivo half-life of a protein and the identity of its N-terminal residue) in L cells, a mouse cell line. The increased accuracy afforded by the UPR technique underscores insufficiency of the current "half-life" terminology, because in vivo degradation of many proteins deviates from first-order kinetics. We consider this problem and discuss other applications of UPR.

Putative receptor binding sites on alphaviruses as visualized by cryoelectron microscopy.

The structures of Sindbis virus and Ross River virus complexed with Fab fragments from monoclonal antibodies have been determined from cryoelectron micrographs. Both antibodies chosen for this study bind to regions of the virions that have been implicated in cell-receptor recognition and recognize epitopes on the E2 glycoprotein. The two structures show that the Fab fragments bind to the outermost tip of the trimeric envelope spike protein. Hence, the same region of both the Sindbis virus and Ross River virus envelope spike is composed of E2 and is involved in recognition of the cellular receptor.

Toward the therapeutic editing of mutated RNA sequences.

If RNA editing could be rationally directed to mutated RNA sequences, genetic diseases caused by certain base substitutions could be treated. Here we use a synthetic complementary RNA oligonucleotide to direct the correction of a premature stop codon mutation in dystrophin RNA. The complementary RNA oligonucleotide was hybridized to a premature stop codon and the hybrid was treated with nuclear extracts containing the cellular enzyme double-stranded RNA adenosine deaminase. When the treated RNAs were translated in vitro, a dramatic increase in expression of a downstream luciferase coding region was observed. The cDNA sequence data are consistent with deamination of the adenosine in the UAG stop codon to inosine by double-stranded RNA adenosine deaminase. Injection of oligonucleotide-mRNA hybrids into Xenopus embryos also resulted in an increase in luciferase expression. These experiments demonstrate the principle of therapeutic RNA editing.

Interactions between quantitative trait loci in soybean in which trait variation at one locus is conditional upon a specific allele at another.

A large recombinant inbred population of soybean has been characterized for 220 restriction fragment-length polymorphism (RFLP) markers. Values for agronomic traits also have been measured. Quantitative trait loci (QTL) for height, yield, and maturity were located by their linkage to RFLP markers. QTL controlling large amounts of trait variation were analyzed for the dependence of trait variation on particular alleles at a second locus by comparing cumulative distributions of the trait for each genotype (four genotypes per pair of loci). Interesting pairs of loci were analyzed statistically with maximum likelihood and Monte Carlo comparison of additive and epistatic models. For each locus affecting height, variation was conditional upon the presence of a particular allele at a second unlinked locus that itself explained little or no trait variation. The results show that interactions between QTL are frequent and control large effects. Interactions distinguished between different QTL in a single linkage group and between QTL that affect different traits closely linked to one RFLP marker--i.e., distinguished between pleiotropy and closely linked genes. The implications for the evolution of inbreeding plants and for the construction of agronomic breeding strategies are discussed.

Diminished degradation of yeast cytochrome c by interactions with its physiological partners.

The level and structure of yeast iso-1-cytochrome c and iso-2-cytochrome c, encoded by the nuclear genes CYC1 and CYC7, respectively, are normally not altered in rho- mutants, which completely lack the cytochromes a.a3 subunits and cytochrome b that are encoded by mitochondrial DNA. In contrast, iso-cytochromes c containing the amino acid change Thr-78-->Ile (T78I) were observed at the normal or near-normal wild-type level in rho+ strains but were completely absent in rho- mutants. We have demonstrated with the "global" suppressor mutation Asn-52-->Ile and by pulse-chase labeling that the T78I iso-1-cytochrome c undergoes rapid cellular degradation in rho- mutants. Furthermore, specific mutations revealed that the deficiency of T78I iso-1 cytochrome c can be caused by the lack of cytochrome a.a3 or cytochrome c1, but not by the lack of cytochrome b. Thus, this and certain other, but not all, labile forms of cytochrome c are protected from degradation by the interaction with its physiological partners.