Ras transformation results in an elevated level of cyclin D1 and acceleration of G1 progression in NIH 3T3 cells.

Ectopic overexpression of v-H-Ras protein in NIH 3T3 cells resulted in cellular transformation and an acceleration of G1 progression of these cells. A shortened G1 phase was found to be associated with an increased level of cyclin D1 but not cyclin E protein. Using an antisense blocking method, reduced synthesis of cyclin D1 in v-H-Ras transformants resulted in a slower G1 progression rate of these cells. Although constitutive overexpression of cyclin D1 in NIH 3T3 cells accelerated G1 progression, cells remained untransformed. Furthermore, inhibition of cyclin D1 synthesis greatly impaired the soft-agar cloning efficiency of v-H-Ras transformants. These results suggest that increased expression of cyclin D1 is necessary but not sufficient for the transforming activity of v-H-Ras. Similar effect on cell cycle progression was also observed in Raf-transformed cells. In addition to cyclin D1, cyclin E protein was found to be elevated in Src transformants. This may account for the further shortening of the G1 phase of these cells. Activation of an additional Ras-independent pathway was suggested to be responsible for the further acceleration of the G1 phase in Src transformants.     

GI双突变体GIK253RA198C的构建及其性质分析

以双引物法对葡萄糖异构酶（GI）基因进行定点突变,将突变体基因于大肠杆菌中表达,获得了GI双点突变体GIK253RA198C.研究K253R和A198C双点突变对GI的结构和性质的作用,结果表明GIK253RA198C的热稳定性明显下降,最适反应温度降低5℃.文章从结构和机制上解释了为何同是K253R突变,对SM33 GI和密苏里游动放线菌GI的热稳定性产生不同的影响,认为这是由于Lys253在两种GI结构的位置上存在微小差异,从而使引入的Arg对亚基间的相互作用产生了相反效应所引起.     

Strength, skeletal muscle composition, and enzyme activity in multiple sclerosis

Kent-Braun, J. A., A. V. Ng, M. Castro, M. W. Weiner, D. Gelinas, G. A. Dudley, and R. G. Miller. Strength, skeletal musclecomposition and enzyme activity in multiple sclerosis. J. Appl. Physiol. 83(6):1998-2004, 1997.This study examined functional, biochemical, andmorphological characteristics of skeletal muscle in nine multiplesclerosis (MS) patients and eight healthy controls in an effort toascertain whether intramuscular adaptations could account for excessivefatigue in this disease. Analyses of biopsies of the tibialis anteriormuscle showed that there were fewer type I fibers (66 ± 6 vs. 76 ± 6%), and that fibers of all types were smaller (average26%) and had lower succinic dehydrogenase (SDH; average40%) and SDH/-glycerol-phosphate dehydrogenase (GPDH) butnot GPDH activities in MS vs. control subjects, suggesting that musclein this disease is smaller and relies more on anaerobic thanaerobic-oxidative energy supply than does muscle of healthyindividuals. Maximal voluntary isometric force fordorsiflexion was associated with both average fiber cross-sectionalarea (r = 0.71, P = 0.005) and muscle fat-free cross-sectional area by magnetic resonance imaging(r = 0.80, P < 0.001). Physical activity,assessed by accelerometer, was associated with average fiber SDH/GPDH(r = 0.78, P = 0.008). There was a tendency forsymptomatic fatigue to be inversely associated with average fiber SDHactivity (r = 0.57,P = 0.068). The results of thisstudy suggest that the inherent characteristics of skeletal musclefibers per se and of skeletal muscle as a whole are altered in thedirection of disuse in MS. They also suggest that changes in skeletalmuscle in MS may significantly affect function.

     

Mutations in the X-linked E1 alpha subunit of pyruvate dehydrogenase: exon skipping, insertion of duplicate sequence, and missense mutations leading to the deficiency of the pyruvate dehydrogenase complex.

Human pyruvate dehydrogenase (PDH)-complex deficiency is an inborn error of metabolism that is extremely heterogeneous in its presentation and clinical course. In a study of 14 patients (7 females and 7 males), we have found a mutation in the coding region of the E1 alpha gene in all 14 patients. Two female patients had the same 7-bp deletion at nt 927; another female patient had a 3-bp deletion at nt 931. Another female patient was found to have a deletion of exon 6 in her cDNA. Two other female patients were found to have insertions, one of 13 bp at nt 981 and one of 46 bp at nucleotide 1078. Two male patients were found to have a 4-bp insertion at nucleotide 1163. The remaining six patients all had missense mutations. A male patient and a female patient both had an A1133G mutation. The other missense mutations were C214T, C615A, and C787G (two patients). Five of these mutations are novel mutations, five have been previously reported in other patients, and two were published observations in other patients in an E1 alpha-mutation summary. In the four cases where parent DNA was available, only one mother was found to be a carrier of the same mutation as her child.     

Yersinia pseudotuberculosis in China

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence-associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.     

Exfoliative cytology in the evaluation of interferon treatment of cervical intraepithelial neoplasia

Local interferon injection in four patients with cervical intraepithelial neoplasia (CIN) regularly elicited progressive regression of the lesions. The response was observed with exfoliative cytology after each injection, guided by colposcopic examination. The cytologic changes showed a cytocidal effect mainly on the dyskaryotic cells, preceded by cellular degeneration not unlike that of nonspecific inflammation and accompanied by an increase in neutrophil infiltration. The cytologic response was closely correlated with partial or complete clinical regression based on the absence of viable or degenerated dyskaryotic cells in the cervical smears. Three patients showed complete clinical regression after treatment. One patient showed recurrent viable dyskaryotic cells when the dosage was reduced, and treatment was suspended temporarily although her lesion had regressed completely after five injections. Clinical recurrence was noted one week after viable dyskaryotic cells reappeared in her smears. These observations suggest that cytology may be a useful means of monitoring interferon treatment in CIN.     

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons : II. Developmental aspects

Dissociated sympathetic neurons from the neonatal rat, grown in cell culture in the virtual absence of other cell types, can develop many of the properties expected of differentiated adrenergic neurons including the ability to synthesize and accumulate catecholamines (CA)². However, in the presence of high concentrations of appropriately conditioned medium (CM), the cultures develop the ability to synthesize and accumulate acetylcholine (ACh); correspondingly, their ability to synthesize CA decreases. In this paper several developmental aspects of the CM effect are described. The time course of development of cultures grown with or without CM was followed using synthesis and accumulation of [³H]CA from [³H]tyrosine and production of [³H]ACh from [³H]choline as assays for adrenergic and cholinergic differentiation. The ability to produce CA or ACh developed along parallel time courses in the two sets of cultures, rising primarily during the second week in vitro and reaching a plateau during the fourth week. When CM was used as a cholinergic developmental signal, the sympathetic neurons showed a decreasing response to addition of CM as they matured adrenergically; addition of CM during the third or fourth 10 days in vitro was not as effective in inducing ACh production as addition during the first or second 10 days. Similarly, removal of CM at various times from cultures previously grown in CM showed that the cholinergic induction caused by CM was not easily reversible in older cultures. Thus, as with the adrenergic decision, the cholinergic decision becomes less reversible as the phenotype becomes fully expressed.     

SIALYLTRANSFERASES IN RAT BRAIN: REACTION KINETICS, PRODUCT ANALYSES, AND MULTIPLICITIES OF ENZYME SPECIES

Abstract— The incorporation of NeuNAc from CMP-NeuNAc into endogenous glycolipids and glyco-proteins, and exogenously added GM_1a (monosialoganglioside) and desialylated fetuin (DS-fetuin) was studied with particulate preparations from 11 to 15 day old rat cerebra. The apparent +K++_m values of the enzyme systems for the different substrates, assayed with 0.5 mg enzyme protein, were: CMP-NeuNAc, 0.13 mm (same with endogenous and exogenous glycolipid and glycoprotein substrates); GM_1a, 0.20 mm ; DS-fetuin, 0.15 mm (or 1.2 mm in terms of acceptor sites). The activities, expressed as nmoles NeuNAc incorporated per 0.5 mg enzyme protein per 30 min incubation at 37°C and pH 6.3, were 0.094, 0.039, 0.17 and 0.64 with the endogenous glycolipids, endogenous glycoproteins, exogenous GM_1a and exogenous DS-fetuin, respectively. Incorporation into endogenous glycolipids was mainly in GM₃, while exogenously added GM_1a was converted to GD_1a. Incorporation into endogenous glycoproteins yields about 20 sialoglycopolypeptides on SDS-polyacrylamide gel electrophoresis. Neura-minidase pretreatment of the particulate enzyme preparation decreased sialylation of the higher molecule weight polypeptides but increased sialylation of the lower molecule weight species. The sialyltransferase activity with the endogenous glycolipid substrates was more heat resistant than the activities with exogenous GM_1a. Since more than 60% of the endogenous glycolipid activity was due to the conversion of lactosylceramide to GM₃, the sialyltransferase responsible for this reaction appears to be different from the one that acts on GM_1a. This was supported by the observation that exogenously added GM_1a did not diminish the incorporation of NeuNAc into endogenous lactosylceramide. These two glycolipid sialyltransferase activities were distinguishable from the glycoprotein sialyltransferase activity since exogenous DS-fetuin did not compete with either the endogenous or the exogenous glycolipids for CMP-NeuNAc.