Primary cultures of rat pancreatic acinar cells in serum-free medium

Summary Rat pancreatic acinar cells were isolated and cultured in Ham's F12 medium with 15% bovine calf serum. Caerulein, insulin, somatostatin, and dexamethasone (DEX) had no effect on intracellular or secreted amylase in these cultured cells. A serum-free medium, using Waymouth's MB 752/1 supplemented with albumin, epidermal growth factor (EGF), DEX, and HEPES, was then developed to avoid serum factors that might mask hormonal effects. In this SF medium, pancreatic acinar, cells maintained the morphological and ultrastructural characteristics of freshly isolated cells and secreted amylase in response to the secretagogue, carbamyl choline. Insulin, at a concentration of 1 μg/ml, significantly increased intracellular and secreted amylase activity after 3 d. This model cell system can be used to study the regulation of the synthesis of amylase and other pancreatic enzymes in vitro.     

Mobilization of a Sym plasmid from a fast-growing cowpea Rhizobium strain.

A large Sym plasmid from a fast-growing cowpea Rhizobium species was made mobilizable by cointegration with plasmid pSUP1011, which carries the oriT region of RP4. This mobilizable Sym plasmid was transferred to a number of Rhizobium strains, in which nodulation and nitrogen fixation functions for symbiosis with plants of the cowpea group were expressed.     

Isolation and identification of 1 alpha,25-dihydroxy-24-oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3

Three new in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3 were isolated from the serum of dogs given large doses (two doses of 1.5 mg/dog) of 1 alpha,25-dihydroxyvitamin D3. The metabolites were isolated and purified by methanol-chloroform extraction and a series of chromatographic procedures. By cochromatography on a high-performance liquid chromatograph, ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry, and specific chemical reactions, the metabolites were identified as 1 alpha,25-dihydroxy-24- oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3. According to these procedures, the total amounts of the isolated metabolites were as follows: 1 alpha,25-dihydroxyvitamin D3, 23.6 micrograms; 1 alpha,25-dihydroxy-24- oxovitamin D3, 1.8 micrograms; 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 9.2 micrograms; 1 alpha,24(R),25-trihydroxyvitamin D3, 15.4 micrograms; 1 alpha,24(S),25-trihydroxyvitamin D3, 1.0 microgram. With recovery corrections, the serum levels of each metabolite were approximately 49 ng/mL for 1 alpha,25-dihydroxyvitamin D3, 3.7 ng/mL for 1 alpha,25-dihydroxy-24- oxovitamin D3, 19 ng/mL for 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 32 ng/mL for 1 alpha,24(R),25-trihydroxyvitamin D3, and 2.1 ng/mL for 1 alpha,24(S),25-trihydroxyvitamin D3.     

Distribution of calcium during interphase and mitosis as observed by ion microscopy

The ion microscope, based on secondary ion mass spectrometry, has been used to demonstrate the distribution of calcium in the root tip cells of two plant species, Allium cepa and Vicia faba. Interphase nuclei showed higher intensities of calcium than cytoplasm, while nucleoli exhibited higher calcium intensities than the rest of the nucleoplasm. The chromosomes showed high intensities of calcium at all stages of mitosis. Calcium was also detected in the cell plate and phragmoplast region of dividing cells. It appears that during prophase calcium concentrates in the condensing chromosomes, and during telophase it is transferred to nucleoli. These observations suggest that chromosomes may serve as a reservoir of calcium during mitosis.     

Monoclonal antibodies to plant plasma-membrane antigens

Murine monoclonal antibodies to membrane antigens were generated by immunization with a crude cellular membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. From a panel of thirteen monoclonal antibodies, seven were found to be directed against antigens present on the plasma-membrane by immunofluorescence visualization of antibody binding to the surface of isolated protoplasts. The corresponding set of plasma-membrane antigen(s) were present in root, shoot and leaf tissue and some but not all of these antigens were of wide species distribution, being found in Nicotiana tabacum L., N. plumbaginifolia L., Glycine max L., Phaseolus vulgaris L. and Triticum aestivum L. Topologically specific labeling of intact protoplasts with a monoclonal antibody reactive with an epitope present on the plasma-membrane specifically labeled a membrane fraction which equilibrated at a density of 1.14 kg/l following centrifugation in a sucrose gradient. In addition to use as biochemical markers for fractionation and molecular characterization of plasma-membranes, these monoclonal antibodies provide the basis for new selection tools in plant cell and gene manipulations.     

Chlorophyll-protein and polypeptide composition of Mn-deficient sugar beet thylakoids

The chlorophyll-protein and polypeptide composition of manganese deficient and control sugar beet thylakoids was examined using three different detergent-electrophoresis systems. On a per chlorophyll basis, manganese deficiency reduced the amounts of CPa complex (separated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis), and CP 47 and CP 43 complexes (separated by octylglucoside/SDS-polyacrylamide gel electrophoresis) without decreasing the amounts of light harvesting complexes. Lithium dodecylsulfate/Triton X-100 polyacrylamide gel electrophoresis showed that manganese deficiency decreased several thylakoid polypeptides, including a chlorophyll b containing 30 kilodalton chlorophyll-protein complex, but did not decrease the amounts of 28 and 29 kilodalton light-harvesting chlorophyll b-containing polypeptides.     

Variation in photosynthetic electron transport capacity in vivo and its effects on the light modulation of ribulose bisphosphate carboxylase

Photosynthetic electron transport capacity was varied in vivo in sugar beets using iron deficiency, and its effects on the light modulation of ribulose bisphosphate carboxylase (RuBPCase) studied. Three treatment groups corresponding to decreasing amounts of thylakoids per leaf area were examined: iron sufficient (control), moderately iron-stressed, and severely iron-stressed. Reduction in electron transport capacity in vivo was correlated with a substantial decrease in the level of RuBPCase activation, even at saturating irradiances. These results indicate a direct relationship between RuBPCase activation and photosynthetic electron transport. In addition, our data suggest that the activation of RuBPCase could not solely account for the increases in the photosynthetic rate at high irradiances; RuBPCase reached maximal activation at irradiances well below light saturation for net photosynthesis.Abbreviations Chl chlorophyll - FeCN ferricyanide - FBPase fructose 1,6-bisphosphatase - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose 1,5-bisphosphate carboxylase - SBPase sedoheptulose 1,7-bisphosphatase     

Tripartite sequences within and 3'' to the sea urchin H2A histone gene display properties associated with a transcriptional termination process.

We have defined a DNA sequence that behaves as an RNA polymerase II termination signal by using the human HeLa cell transient expression system. Surprisingly, this sequence is tripartite, including part of the coding region of the sea urchin H2A histone gene together with two separate sequences in the 3' flanking region of the gene. We demonstrate that this signal functions both in its normal gene environment and also when placed within the human alpha-globin gene. However, we have failed to detect a discrete 3' terminus. Rather, our data indicate the presence of an extremely heterogeneous series of nonpolyadenylated RNAs. These heterogeneous nonpolyadenylated RNAs are stable when transcribed from the intact histone gene but are highly unstable within the human alpha-globin gene. This provides evidence for the role of poly(A) in the stability of mRNA.     

Absence of female-specific protein in the hemolymph of stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae)

Changes, during the reproductive cycle, in fat body, hemolymph, and ovarian proteins of the stable fly Stomoxys calcitrans were characterized quantitatively and qualitatively using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein content of all three tissues increased after blood feeding. Fat body protein increased first, followed by hemolymph and ovarian proteins. SDS-PAGE failed to identify vitellogenin in both female hemolymph and fat body samples. No single protein or group of proteins predominated at any stage of the reproductive cycle. Comparisons between male and female stable fly hemolymph and fat body proteins failed to detect female-specific proteins. Female-specific proteins, however, were detected in the hemolymph of four other species of Diptera.