Resolution of pea legumin subunits by high-performance liquid chromatography

Pea legumin was dissociated into its component subunits by 6 M urea: these were subsequently fractionated by FPLC using a combination of Mono P, Mono Q, and Mono S columns. The resolution and speed of separation were greatly improved in comparison with previous fractionations. Twelve discrete fractions were obtained and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six "normal" legumin subunits (Mr 60,000) were identified as well as some "large" (Mr 66,000) and "small" (Mr 44,000) subunits. A few polypeptides of unknown origin were also observed. Four subunits were purified to homogeneity as adjudged by electrophoresis and HPLC and in sufficient yields to permit further studies. Anomalous electrophoretic behavior of the legumin subunits was also observed.     

Insulin release from a cloned hamster B-cell line (HIT-T15). The effects of glucose, amino acids, sulphonylureas and colchicine

Insulin release from statically incubated HIT-T15 cells was maximally stimulated by glucose, L-arginine and L-leucine. L-arginine stimulated insulin release in the absence of glucose. Glucose induced insulin release was potentiated by the addition of L-leucine, L-arginine and the two in combination. Both glibenclamide and chlorpropamide stimulated insulin release from HIT-T15 cells. Glibenclamide was the more potent and equivalent in insulinotrophic action to 7.5 mmol/l glucose. Only chlorpropamide significantly potentiated glucose induced insulin release. Perifused HIT-T15 cells produced a reproducible biphasic insulin response to glucose challenge which was characterised by a pronounced and sustained first phase and a reduced second phase. The stimulation of phase I by glibenclamide alone and the inhibition of phase II of glucose induced insulin release by colchicine suggested the presence of a readily available pool of insulin granules which was not rapidly restored by insulin biosynthesis and granule margination.     

Induced recombination between duplicated neo genes stably integrated in the genome of CHO cells

Homologous recombination between 2 truncated neo genes stably integrated in the genome of Chinese hamster ovary (CHO) cells was studied. A vector containing a functional gpt gene and 2 tandemly arranged G418 resistance (neo) gene fragments with about 400 bp of sequence homology was transfected into CHO cells. Clonal cell lines were established from transfected cultures and the spontaneous frequency of G418-resistant revertants was found to range between 1 x 10(-4) and 5 x 10(-4). The ability of the alkylating agents MMS and HN2 to induce recombination of the transfected neo genes was studied in 2 of the cell lines. After treatment with MMS at doses that reduced survival to 10% of the control these cell lines showed a dose-dependent increase in the frequency of G418-resistant revertants. No effect was observed after treatment with HN2. All G418-resistant subclones contained a new restriction fragment indicating that a whole neo gene had been formed by rearrangement in pairs of truncated neo genes. Hence, this system can be used to study molecular mechanisms and chemical inducibility of homologous recombination in mammalian cells.     

Autoimmune syndrome after induction of neonatal tolerance to alloantigens: effects of in vivo treatment with anti-T cell subset monoclonal antibodies

BALB/c (H-2d) mice rendered tolerant to h-2b alloantigens by neonatal injection of semiallogeneic (C57BL/6 X BALB/c)F1 spleen cells develop autoimmune features due to an abnormal activation of persisting F1 donor B cells. The role of T cells in this autoimmune syndrome was studied by in vivo treatment of tolerant mice with anti-L3T4(GK-1.5) or anti-Ly-2 (H-35-17.2) monoclonal antibodies. The treatment of tolerant mice from day 2 to day 21 of life with anti-L3T4 MAb completely prevented the occurrence of circulating immune complexes of anti-ssDNA anti-Sm and anti-hapten (FITC) IgG antibodies as well as the glomerular deposition of Ig that were usually seen in untreated tolerant mice. This effect persisted for at least 6 wk after stopping this treatment. When the injections of anti-L3T4 MAb were delayed until day 15 of life, a very significant decrease of the autoimmune manifestations was still observed. Treatment of tolerant mice with anti-Ly-2 MAb during the same period had no effects on the autoimmune disease as compared with untreated tolerant mice. No effects on the maintenance of tolerance vs H-2b alloantigens were observed after treatment with anti-L3T4 MAb, as followed by the decrease of CTL and CTL-p alloreactivity and by the persistence of F1 donor B cells, indicated by the presence of Ig bearing the Ighb donor allotype. These results suggest the existence of interactions between L3T4+ T cells and persisting autoreactive B cells from F1 donor origin in the development of the autoimmune syndrome after neonatal induction of transplantation tolerance.     

Structure-function studies of the bacteriophage T4 DNA polymerase. Isolation of a novel suppressor mutant

We describe here our first attempt in using suppressor mutations to study structure-function relationships of the bacteriophage T4 DNA polymerase. One intragenic suppressor mutation, J5(43) degrees, was isolated that suppresses the temperature sensitivity but not the mutator activity of tsM19, a DNA polymerase mutant. Thus, the substituted amino acid induced by the tsM19 lesion decreases DNA polymerase fidelity, even if the temperature sensitivity has been corrected by a second amino acid substitution in the DNA polymerase polypeptide. The isolation, mapping and characterization of the J5(43) degrees mutation as well as the purification and characterization of the tsM19-J5(43) degrees mutant DNA polymerase are presented. The suppressor isolation procedure has general applicability for the selection of suppressor mutations of other T4 DNA polymerase mutator mutants.     

Infective Factors in Exacerbations of Bronchitis and Asthma

Infections of the respiratory tract were studied in a group of families each containing a patient with chronic bronchitis or with asthma. A wide variety of infective agents may be associated with exacerbations in susceptible subjects, but the types of organism to which patients are most at risk differ according to the family structure. Exacerbations in the susceptible subject are more likely to be related to viral infections when the family contains children than when it does not.Two patients with asthma experienced frequent respiratory infections, many of which provoked attacks of asthma.     

Effects of residency training in family medicine v. internship training on professional attitudes and practice patterns.

OBJECTIVES: To determine whether the professional attitudes and practice patterns of physicians with residency training in family medicine differ from those of generalists with internship training. DESIGN: Mail survey conducted in 1985-86. SETTING: Province of Quebec. PARTICIPANTS: A stratified random sample of French-speaking family and general practitioners who graduated after 1972 (325 physicians with residency training and 304 with internship training) (response rate 82%). MAIN RESULTS: Physicians with residency training were 3 years younger on average than those with internship training, were more likely to be female (38% v. 18%, p less than 0.001) and were more likely to work on a salaried basis in CLSCs (public community health centres) (36% v. 14%, p less than 0.001). Even after these confounding factors were controlled for, physicians with residency training seemed to be more sensitive to the psychosocial aspects of patient care and tended to attach more importance to informing patients about useful materials and resources concerning their health problems. They were not, however, more likely to value health counselling or integrate it in medical practice. CONCLUSION: Our findings provide some evidence that the new requirement that physicians complete a residency in family medicine to obtain medical licensure in general practice in Quebec may foster a more patient-centred approach to health care.     

Colpohistologic correlations. A computerized study

Data from 300 colposcopic cases were computerized to study the correlations between the colposcopic observations and the histologic and other data. Colposcopy was shown to be most useful in women under 35 years of age. Use of oral contraceptives (the Pill) seemed to relate to a higher level of successful colposcopic visualization and may even prevent the need for conization, based on a higher incidence of negative endocervical curettages. Although 14.5% of the cases were colposcopically undergraded and 17% overgraded when compared to the histopathology, the histologic predictability of the colposcopic observation of white epithelium and vascular changes (punctation and mosaicism) was excellent. Colposcopy remains an essential technique in the appraisal and management of cervical intraepithelial neoplasia.     

δ-Aminolevulinate dehydratase from Raphanus sativus cotyledons

Phytochrome induces δ-aminolevulinate dehydratase (ALAD) activity in radish seedling cotyledons under continuous far red light. Analysis of the enzymatic activity in etioplasts vs total activity shows a constant ALAD level in these organelles (10 %) in etiolated seedlings. In far red irradiated seedlings, the percentage of enzyme detected into etioplasts increases up to 45 % of the total. Comparative kinetic studies of ALAD activity detected in the cytoplasm and the etioplasts indicate an increase in both compartments with a maximum value reached respectively at 96 and 120 hr from sowing. Treatment with cycloheximide shows a very fast abolition of cytoplasmic ALAD activity which is always correlated to an etioplast decrease with a time shift of ca 24 hr. Erythromycin acts only on the cytoplasmic level of ALAD, and only for far red irradiated seedlings, with an increase of activity twice the level detected in untreated ones. This unexpected effect is discussed.