Nucleoside exchange catalysed by the cytoplasmic 5''-nucleotidase.

The inhibition of the cytoplasmic 5'-nucleotidase (EC 3.1.3.5) by its product, inosine, was studied with a partially purified preparation of the enzyme from rat liver. Inhibition of Pi production was found to be due to exchange of the inosine moiety between inosine and IMP. Exchange was not catalysed by reversal of the hydrolytic reaction, suggesting, instead, the mediation of an enzyme-phosphate intermediate. Two models for the catalytic mechanism are proposed and rate equations for the dependence of Pi production on inosine concentration are derived. The experimentally determined dependence was consistent with a mechanism in which hydrolysis of the enzyme-phosphate intermediate occurred only when it was unoccupied by inosine. This conclusion suggests that inosine analogues that cannot participate in exchange should inhibit the enzyme. Such inhibitors might be useful in defining the enzyme's physiological role or as pharmacological agents to decrease breakdown of purine nucleotides. The possibility that nucleoside exchange provides an alternative route for the phosphorylation of mutagenic or cytotoxic nucleoside analogues should also be considered.     

Partial purification of the maturation-promoting factor MPF from unfertilized eggs of Xenopus laevis

A 200-fold purification of the maturation-promoting factor or MPF from unfertilized eggs of Xenopus laevis is reported for the first time. Purification was achieved by three successive column chromatographies on hydroxyapatite, trisacryl blue and L-arginine-agarose. The presence of MPF was assessed by the usual maturation criteria after injections of test material into immature stage VI unstimulated X. laevis oocytes: the precocious appearance of the maturation spot (within 45-120 min), the germinal vesicle breakdown, the presence of the first polar body and the second metaphase spindle. Purification was monitored by the decrease of the minimal amount of protein injected in a constant volume (50 nl) required to induce 50% frequency of germinal vesicle breakdown. This amount decreased from 500 ng in the crude extract to 2.5 ng in the 200-fold purified material. Analysis by SDS-PAGE of the crude extract showed about 40 Coomassie-blue-stained polypeptides with molecular masses ranging from 300 kDa to 20 kDa, whereas in the 200-fold purified MPF only 5 stained polypeptides were revealed, with molecular masses of 62, 53, 49, 39 and 37 kDa. In vitro phosphorylations for the detection of kinase activities for endogenous and exogenous substrates were monitored by analysis of autoradiograms of SDS-PAGE, after treatment of fractions with [gamma-32P]ATP. Only inactive fractions eluted from columns ahead of MPF, and fractions containing MPF activity were tested. Phosphorylation of numerous stained polypeptides was demonstrated in the crude MPF extract and exogenous substrates such as phosvitin, casein and histone type II-AS were also strongly phosphorylated. In the MPF fraction, purified on hydroxyapatite, a polypeptide of 53 kDa was more highly and specifically phosphorylated and the presence of kinase activities was observed for the above three exogenous substrates. In the 100-fold and 200-fold purified MPF, phosphorylation of endogenous substrates could not be shown and kinase activities for the above three substrates were drastically decreased as compared with the crude and purified MPF obtained after hydroxyapatite column chromatography. However, neither endogenous phosphorylations nor kinase activities with the above exogenous substrates could be shown in inactive fractions eluted ahead of MPF at the different purification steps. Some characteristics of the purified material are also described in this paper.     

Objective test for food sensitivity in asthmatic children: increased bronchial reactivity after cola drinks.

Ten asthmatic children with a history of cough and wheeze after drinking a cola drink performed histamine inhalation tests before and 30 minutes after a drink of Pepsi-Cola, soda water, and water on three separate study days. There was no significant change in baseline peak expiratory flow after any of the three drinks. Sensitivity to histamine was increased after the cola drink (p less than 0.005) but was not significantly different after soda water or water. The detection of change in sensitivity to histamine appears to be a simple and effective method of testing for food sensitivity in asthma.     

Growth of acetotrophic,methane-producing bacteria in a pH auxostat

Three acetotrophicMethanosarcina species, which included marine, nonmarine, and thermophilic strains, were grown on acetate in a 10-liter pH auxostat. Specific growth rates and molar growth yields were constant throughout growth. Cell yields were up to 18-fold greater than previously reported. These properties of the pH auxostat indicate that it is a preferred culture method for the biochemical study of methanogenesis from acetate.     

Analysis of the antigenic and immunogenic properties of protein M of the influenza virus by an immunoenzyme method

A test system permitting the detection of influenza virus protein M at a concentration of 0.1-0.5 ng/ml in ELISA has been developed. The use of this system made it possible to detect influenza viruses A and B directly in crude virus-containing material and clinical samples obtained from influenza patients. During the outbreak of influenza in the spring of 1983 ELISA was successfully used for the rapid diagnosis of influenza, and some of its advantages in comparison with the conventional immunofluorescence test were thus demonstrated. To overcome difficulties arising from the low immunogenic potency of protein M, in the process of obtaining diagnostic sera and ascitic fluids the animals were immunized with the conjugate of protein M and polyelectrolite, which ensured considerable activation of humoral immune response.     

Identification of N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver DNA after treatment with the carcinogens, N,N-dimethylnitrosamine or 1,2-dimethylhydrazine

Human Tamm-Horsfall urinary glycoprotein from an individual of the blood group Sd(a+) phenotype was tritium-labelled by treatment with galactose oxidase and sodium boro[3H]hydride and was then digested with endo-beta-galactosidase. A series of dialysable, labelled fragments was released from which a pentasaccharide was isolated that strongly inhibited the agglutination of Sd(a+) red cells by human anti-Sda serum and hence contained the Sda determinant structure. Reduction, methylation analysis and sequential exo-glycosidase digestion established the structure of the pentasaccharide as: GalNAc beta(1 leads to 4)[NeuAc(2 leads to 3)]Gal beta(1 leads to 4)GlcNAc beta(1 leads to 3)Gal     

The activity of jaw elevator muscles during peanut chewing in patients with temporomandibular joint and muscle pain dysfunction syndrome

A synchronized system of EMG and jaw motion tracking device was used to observe some chewing parameters of jaw elevator muscles in 15 patients with temporomandibular joint and muscle pain dysfunction syndrome (TMJ) and 15 normal subjects. Duration of tooth contact (DTC), duration of muscle contraction before tooth contact (DMC), total duration of muscle contraction (DTM) and velocity of jaw movement during peanut chewing were observed. Symptoms of the TMJ patients included pain and tenderness at joints and muscles, and limitation and clicking at joints during jaw movements. It was found that the TMJ patients needed more numerous breaking off strokes before trituration at the occlusal level. There was a longer DMC in the earlier trituration period and TMJ patients had longer DMC than in normals. No difference was found between right and left side chewing or between temporalis and masseter muscles. DTM in the TMJ group was only slightly longer than in normals and the difference between early and late chewing periods was statistically not significant. DTC was only slightly shorter in the TMJ group while the difference between early and late chewing periods in both groups was significant. The average and maximum closing velocities were significantly lower in the TMJ group in both right and left chewing. The difference in the opening phase was not as significant. It was concluded that DMC and jaw closing velocity are more sensitive parameters than DTM and DTC on the diagnosis of TMJ dysfunction with or without occlusal interference. DTM and DTC are parameters more closely related to the influence of occlusal factors.(ABSTRACT TRUNCATED AT 250 WORDS)