Ti plasmid-encoded genes responsible for catabolism of the crown gall opine mannopine by Agrobacterium tumefaciens are homologs of the T-region genes responsible for synthesis of this opine by the plant tumor

Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source. When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP. Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP. DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right. Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors. MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2'. MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis. These results indicate that the moc and mas genes evolved from a common origin. MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4. MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively. Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates. We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.     

The effect of dark-rearing, strobe-rearing and acute visual cortex removal on the visual responses in the superficial superior colliculus of the guinea-pig

The interaction of zinc and vitamin A in rats receiving a regional diet of Manaus, supplemented with vitamin A, zinc and zinc and vitamin A was studied. The regional diet was elaborated according to data of Shrimpton and Giugliano (6), for families receiving less than two minimum salaries. The biological test to study the interaction was based on the depletion of zinc and vitamin A in rats in the period of lactation, and a period of repletion where supplements of zinc (0.82 mg%) and vitamin A (94.2 micrograms %) were given, either separately or together, according to the recommendations of the Committee on Laboratory Animal Diets (7). From the results, it was concluded that there was an interaction of these nutrients in terms of mobilization of hepatic vitamin A. Although the regional diet of Manaus did not meet the zinc RDA, the amount present was enough to utilize the available vitamin A. Although the amount of zinc present in the diet, as determined by parameters of bioavilability, such as growth, concentration in organs and zinc-dependent enzymes, was adequately used by the animals, probably due to promoting factors in the diet. The Manaus regional diet needs to be supplemented with vitamin A in order to maintain the hepatic reserves, and with zinc, to maintain the normal levels of vitamin A in plasma.     

A portable magnetometer

A portable magnetometer to determine magnetic field anomalies is described. The block diagram of the magnetometer and its specifications are presented.     

Time utilisation pattern of staff of two primary health centres in Ballavgarh, Haryana

BACKGROUND: All National health programmes are implemented through the Primary Health Centre staff. Targets for the year 2000 A.D. have been fixed for different programmes. Some programmes are getting more emphasis, perhaps at the cost of others. The study area has already achieved most of the targets set for 2000 A.D. Studying the time utilisation pattern of the workers of these PHCs can give valuable information for planning of working of other PHCs. OBJECTIVE: To study the time utilisation pattern of the staff of the two PHCs run by Centre for Community Medicine, AIIMS. METHODS: The multipurpose workers (MPWs) and the health assistants (HAs) were accompanied by investigators and information collected regarding their utilisation of time in the field. The Medical Officers were asked to maintain a diary from which this information was collected RESULTS: The MPWs spend about 3.3 minutes in each house. Child care (immunisation, Vit. A and folifer distribution) is the main activity being carried out by both male as well as female worker. Other important activities for male worker are: family welfare (18%), malaria work (11%) and collection of vital statistics (10%). For the female worker Antenatal care (25%) and family welfare (20%) were other important activities. For the HAs also child care was an important activity. However for the male HA malaria related work was the most important. The Medical Officer spends about 60% of this time in administrative and supervisory work. CONCLUSIONS: Immunisation programme is getting the maximum input from workers, which is reflected in > 90% coverage of all vaccines. Family Welfare and Tuberculosis activity are not getting the emphasis which they deserve. Some rethinking about the strategy is essential if all round progress in achieving the targets for the year 2000 A.D. is to be made.     

Molecular characterization of N-methyl-D-aspartate receptors expressed in mammalian cells yields evidence for the coexistence of three subunit types within a discrete receptor molecule

The N-methyl-D-aspartate R1 (NMDA R1), NMDA R2A, and NMDA R2C subunits were expressed transiently in double or triple combinations in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult mouse forebrain and cerebellum. Under conditions established for maximal expression, cotransfection of the NMDA R1 and R2C subunits yielded a protein detected immunologically with a molecular size of 780,000-850,000 daltons. No cell death was observed in the transfected cells, and the KD for [3H]MK801 binding to the NMDA R1/R2C receptor was 346 +/- 158 nM. This was in contrast to a value of KD = 22 +/- 9 nM found for native cerebellar receptors. Co-transfection with NMDA R1/R2A/R2C subunits with a DNA ratio, 1:3:3, resulted in the expression of a protein with a size similar to the NMDA R1/R2C combination, but the affinity of [3H]MK801 was now 22 +/- 5 nM, and the percentage cell death post-transfection was 89 +/- 17%. Immunoprecipitation assays of detergent-solubilized transfected cells with NMDA R1 subunit-specific antibodies co-precipitated the NMDA R2A and NMDA R2C subunits in 1/2A and 1/2C transfections, respectively. Similarly, immunoprecipitations with either NMDA R1 or NMDA R2C subunit-specific antibodies co-precipitated the NMDA R2A subunit in the R1/2A/2C triple transfections. These results show that the three NMDA receptor subunit types can co-assemble following their co-expression in mammalian cells with a pharmacological profile that is similar to that found for adult cerebellar NMDA receptors.     

Oestradiol-17 beta modulates PAF-evoked phospholipase D activity but not inositide-lipid hydrolysis in human endometrial cell line, HEC-1B

The skeleton of a middle-aged adult male, found in the Late Epigravettian necropolis of Arene Candide cave (Italy) and dated to the XII millennium B.C., exhibits abnormal changes, including bowing deformities, stunted growth, enthesopathies, and increased bone density. The pattern of the observed changes is less consistent with diagnoses of metaphyseal chondrodysplasias, hypophosphatasia, dietary rickets, or diffuse idiopathic skeletal hyperostosis (DISH) than with X-linked hypophosphatemic rickets, which is the most likely etiological factor. This diagnosis may explain other abnormalities (exceptional elongation of the skull and bilateral absence of the lesser trochanter) displayed by other individuals from the same site.     

Magnetic resonance imaging of the pelvic floor in the postpartum patient

Magnetic resonance imaging (MRI) was used to assess anatomical changes in the pelvic floor after childbirth. Six women underwent serial MRI examination within 30 hours and at 1 week, 2 weeks, 6 weeks and 6 months after delivery; 8 additional women were studied only within 30 hours of delivery. T-1 and T-2-weighted images of the pelvis in the transverse and sagittal planes with a 1.5-T MR imager were obtained. In the sagittal section we assessed the urethrovesical angle, urethral length, distance from the symphysis to the proximal and distal vagina, vaginal length, width and length of the sphincters, and the presence of sphincter defects. Axial sections were assessed for sphincter defects for the distance between the symphysis and midurethra, vagina and rectum. Only one parameter (distance between symphysis and distal vagina) changed significantly over time, without a clear trend in direction. Interobserver variation was reasonable (<15%) except for anal canal length, urethral length and distance between symphysis and anus. There were no significant correlations between birthweight and MRI parameters. There was a non-significant association (P = 0.09) between the sole combined sphincter defect and rectal injury, but not with episiotomy or parity. We concluded that it is feasible to determine multiple measurements on MR images to evaluate structures of the pelvic floor.     

Functional expression of the cystic fibrosis transmembrane conductance regulator in yeast

Recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) has been produced in a Saccharomyces cerevisiae expression system used previously to produce transport ATPases with high yields. The arrangement of the bases in the region immediately upstream from the ATG start codon of the CFTR is extremely important for high expression levels. The maximal CFTR expression level is about 5-10% of that in Sf9 insect cells as judged by comparison of immunoblots. Upon sucrose gradient centrifugation, the majority of the CFTR is found in a light vesicle fraction separated from the yeast plasma membrane in a heavier fraction. It thus appears that most of expressed CFTR is not directed to the plasma membrane in this system. CFTR expressed in yeast has the same mobility (ca. 140 kDa) as recombinant CFTR produced in Sf9 cells in a high resolution SDS-PAGE gel before and after N-glycosidase F treatment, suggesting that it is not glycosylated. The channel function of the expressed CFTR was measured by an isotope flux assay in isolated yeast membrane vesicles and single channel recording following reconstitution into planar lipid bilayers. In the isotope flux assay, protein kinase A (PKA) increased the rate of 125I- uptake by about 30% in membrane vesicles containing the CFTR, but not in control membranes. The single channel recordings showed that a PKA-activated small conductance anion channel (8 pS) with a linear I-V relationship was present in the CFTR membranes, but not in control membranes. These results show that the human CFTR has been expressed in functional form in yeast. With the reasonably high yield and the ability to grow massive quantities of yeast at low cost, this CFTR expression system may provide a valuable new source of starting material for purification of large quantities of the CFTR for biochemical studies.