Glycogen stability and glycogen phosphorylase activities in isolated skeletal muscles from rat and toad

There is increasing evidence that endogenous glycogen depletion may affect excitation–contraction (E–C) coupling events in vertebrate skeletal muscle. One approach employed in physiological investigations of E–C coupling involves the use of mechanically skinned, single fibre preparations obtained from tissues stored under paraffin oil, at room temperature (RT: 20–24°C) and 4°C for several hours. In the present study, we examined the effect of these storage conditions on the glycogen content in three muscles frequently used in research on E–C coupling: rat extensor digitorum longus (EDL) and soleus (SOL) and toad iliofibularis (IF). Glycogen content was determined fluorometrically in homogenates prepared from whole muscles, stored under paraffin oil for up to 6 h at RT or 4°C. Control muscles and muscles stored for 0.5 and 6 h were also analysed for total phosphorylase (Phos_total) and phosphorylase a (Phos a) activities. No significant change was observed in the glycogen content of EDL and SOL muscles stored at RT for 0.5 h. In rat muscles stored at RT for longer than 0.5 h, the glycogen content decreased to 67.6% (EDL) and 78.7% (SOL) of controls after 3 h and 25.3% (EDL) and 37.4% (SOL) after 6 h. Rat muscles stored at 4°C retained 79.0% (EDL) and 92.5% (SOL) of glycogen after 3 h and 75.2% (EDL) and 61.1% (SOL) after 6 h. The glycogen content of IF muscles stored at RT or 4°C for 6 h was not significantly different from controls. Phos_total was unchanged in all muscles over the 6 h period, at both temperatures. Phos a was also unchanged in the toad IF muscles, but in rat muscles it decreased rapidly, particularly in EDL (4.1-fold after 0.5 h at RT). Taken together these results indicate that storage under paraffin oil for up to 6 h at RT or 4°C is accompanied by minimal glycogen loss in toad IF muscles and by a time- and temperature-dependent glycogen loss in EDL and SOL muscles of the rat.     

Mycobacterium avium invades the intestinal mucosa primarily by interacting with enterocytes

Previous studies have demonstrated that Mycobacterium avium can invade intestinal epithelial cells both in vitro and in vivo. When given to mice orally, M. avium preferentially interacts with the intestinal mucosa at the terminal ileum. We evaluated the mechanism(s) of M. avium binding and invasion of the intestinal mucosa using three different systems: (i) electron microscopy following administration of M. avium into an intestinal loop in mice, (ii) quantitative comparison of the bacterial load in Peyer's patch areas of the terminal ileum versus areas that do not contain Peyer's patches, and (iii) investigation of the ability of M. avium to cause disseminated infection following oral administration using B-cell-deficient mice, lacking Peyer's patches, in comparison with C57BL/6 black mice. By all approaches, M. avium was found to invade the intestinal mucosa by interacting primarily with enterocytes and not with M cells.     

Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture.

The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures. E. equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks. The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology. Homologous equine antibodies and human anti-HGE convalescent serum recognized E. equi grown in tick cell culture. Electron microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates. The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.     

Immunohistochemical study of nasopharyngeal carcinoma using monoclonal keratin antibodies

Nasopharyngeal carcinoma (NPC) provides a unique opportunity to evaluate distinctive epidemiologic features and a possible etiologic relationship with Epstein-Barr virus (EBV) in human malignancy. The lack of a uniformly accepted pathologic classification for NPC has limited the application of this data, although the World Health Organization (WHO) developed a classification that may solve this problem. Monoclonal keratin antibodies were used for staining of NPC for evaluation of its assistance in diagnosis and classification. In the present immunohistochemical study, monoclonal keratin antibodies, designated AE1, AE2, and AE3, and a polyclonal keratin antibody (RAK) were used for study of the presence of keratin in 121 cases of NPC obtained from China and the United States. AE1 monoclonal antibody, which recognizes keratin protein classes 56.5K, 50K, and 40K, was shown to be the most sensitive and specific for NPC tumor cells among the keratin antibodies studied. In addition, some different keratin expression patterns could be identified between different kinds of epithelium and different tumor groups, with possible relevance to the histogenesis of the histologic subtypes of NPC.     

Hydrogenase activity in catalase-positive strains of Campylobacter spp.

A rapid hydrogenase assay has been developed which may be useful in separating the species Campylobacter jejuni and C. coli from the subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. This assay employs the impermeant redox dye benzyl viologen, and positive determinations can be made within 20 min. All strains of C. jejuni and C. coli were found to be strongly hydrogenase positive. All strains of C. fetus subsp. fetus and C. fetus subsp. venerealis were negative for hydrogenase when the assay was performed at a benzyl viologen concentration of 2 mM and an incubation temperature of 30 degrees C. Some strains of C. fetus had low levels of hydrogenase as determined with cell extracts but were hydrogenase negative by the benzyl viologen assay. Since there are few rapid diagnostic tests available for screening Campylobacter isolates, we hope that the rapid hydrogenase assay will prove useful.     

Functional MRI of visuospatial working memory in schizotypal personality disorder: a region-of-interest analysis

BACKGROUND: Functional MRI studies have begun to identify neural networks implicated in visuo-spatial working memory in healthy volunteers and patients with schizophrenia. The study of schizotypal personality disorder (SPD) provides regional analysis in unmedicated patients in the schizophrenia spectrum. METHOD: Unmedicated patients with SPD by DSM-IV criteria and normal controls were assessed with fMRI while performing a visuo-spatial working-memory task. It required the subjects to retain the location of three dots located on the circumference of an imaginary circle and then respond to a query display in which one dot was presented and the subject required to press a button to indicate whether the probe dot location was previously displayed. Subject groups did not differ significantly in spatial memory scores. The exact Talairach and Tournoux coordinates of brain areas previously reported to show activation with spatial memory tasks were assessed. RESULTS: The majority of these locations showed BOLD response activation significantly less in patients during the memory retention period, including the left ventral prefrontal cortex, superior frontal gyrus, intraparietal cortex and posterior inferior gyrus. Regions in the right middle prefrontal and prestriate cortex showed greater activation at a trend level for patients with SPD than for normal controls. In addition, we replicated the findings of increased activation with the task in healthy volunteers in the premotor areas, ventral prefrontal cortex and parietal cortex. CONCLUSIONS: SPD patients show decreased activation compared to healthy volunteers in key frontal regions and we also provided a partial replication of findings reported in healthy subjects.