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.     

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.     

Molecular evidence for the expression of nicotinic acetylcholine receptor alpha-chain in mouse thymus.

The presence and structure of nicotinic acetylcholine receptor (nAChR) in the thymus has been a subject of interest for many years because of its possible role in the pathogenesis of the autoimmune disease myasthenia gravis. Using the polymerase chain reaction with primers specific for the alpha-chain of nAChR (nAChR-alpha), an 880-bp homologous band was found after amplification of cDNA prepared from mouse thymus, thymic medullary and cortical epithelial cell lines, but not from thymocytes or kidney. Sequencing of the polymerase chain reaction product from the thymus and thymic medullary and cortical epithelial lines showed identity with skeletal muscle nAChR-alpha over the region examined. This region includes the domains of the molecule on which B cell and T cell autoantigenic targets have been described. No evidence was found in mouse tissue for the exon 3A, which has been described in human muscle and the human rhabdomyosarcoma cell line TE671. Our results provide evidence at the RNA level for the expression of the nAChR-alpha on stromal cells but not on thymocytes in normal murine thymus and are consistent with a role for intrathymic autoantigen expression in the pathogenesis of myasthenia gravis.     

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     

Restriction fragment length polymorphisms of horse class II MHC genes observed using various human alpha-and beta-chain cDNA probes

Genomic DNA isolated from 20 horses was digested with up to six restriction endonucleases and subjected to southern blot hybridization analysis using various human class II alpha- and beta-chain cDNA probes. A high degree of restriction fragment length polymorphism (RFLP) was found for the DQ alpha, DP beta, DQ beta and DR beta probes, about 20 polymorphic bands being detected for each. DR alpha showed 2-4 polymorphic bands, whereas no evidence for DP alpha-like genes was found. A number of correlations of RFLPs with individual alloantisera were apparent.     

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)     

The histology and calcification of regenerating scales in the blackspotted topminnow, Fundulus olivaceus (Storer)

The regenerating scale and tissues comprising the scale pocket of Fundulus olivaceus were examined microscopically at specific intervals. Scale removal resulted in a thickening of the epidermis which persisted through the early stages of regeneration. This thickening was due in part to the appearance of columnar basal cells which divided producing cells that became mucous cells and squamous cells. The scale regenerated as a relatively large plate of bone which first appeared between layers of scleroblasts on the floor of the scale pocket and then grew producing circuli and radii. By the fourth day of regeneration, calcium was observed in the cytoplasm of the scleroblasts and at randomly distributed foci in the osseous portion of the scale. The osseous layer was completely calcified by 15 days of regeneration.     

Reproductive hormones and bone mineral density in women runners.

We examined the relationships among reproductive hormone concentrations and bone mineral density (BMD) in 43 women runners classified as eumenorrheic (n = 24), oligomenorrheic (n = 8), or amenorrheic (n = 11). Results were compared with a eumenorrheic nonrunner control group (n = 11). Serum 17 beta-estradiol, progesterone, and dehydroepiandrosterone sulfate concentrations were determined in daily blood samples for 21 days, and integrated concentrations (areas under the curve) were calculated. BMD was assessed at the lumbar spine and proximal femur by dual-photon absorptiometry. As expected, 17 beta-estradiol, progesterone, and lumbar spine BMD were higher in the control and eumenorrheic runner groups than in the oligomenorrheic and amenorrheic runner groups (P less than 0.05). Progesterone concentration was significantly correlated with lumbar spine BMD in the eumenorrheic runners (r = 0.61). None of the steroid hormones was significantly related to BMD in the oligomenorrheic/amenorrheic group. The present data suggest that circulating levels of gonadal steroid hormones affect axial BMD in eumenorrheic runners.     

Oxidative degradation of thymine with O2 promoted by L-ascorbic acid and Cu(II) ion

Oxidation of thymine with O2 was promoted by copper(I) ion generated from reaction of L-ascorbic acid (AA) with copper (II) ion. The main oxidation products were thymine glycol (TG) and N-formyl-N'-pyruvylurea (FPU). At higher concentration of O2, formation of FPU was favored, while TG was dominant at higher Cu(II) ion and lower O2 concentrations. Reaction mechanism involving hydroxy thyminyl radical was proposed.