Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and-insensitive (IisCaP) nonmitochondrial Ca2+ pools in rat pancreatic acinar cells

Summary We have measured Ca²⁺ uptake and Ca²⁺ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca²⁺ uptake into cells was monitored with a Ca²⁺ electrode, whereas Ca²⁺ uptake into membrane vesicles was measured with⁴⁵Ca²⁺. Using inhibitors of known action, such as the H⁺ ATPase inhibitors NBD-Cl and NEM, the Ca²⁺ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP₃) and its analog inositol 1,4,5-trisphosphorothioate (IPS₃), we could functionally differentiate two non-mitochondrial Ca²⁺ pools. Ca²⁺ uptake into the IP₃-sensitive Ca²⁺ pool (IsCaP) occurs by a MgATP-dependent Ca²⁺ uptake mechanism that exchanges Ca²⁺ for H⁺ ions. In the absence of ATP Ca²⁺ uptake can occur to some extent at the expense of an H⁺ gradient that is established by a vacuolar-type MgATP-dependent H⁺ pump present in the same organelle. The other Ca²⁺ pool takes up Ca²⁺ by a vanadate-sensitive Ca²⁺ ATPase and is insensitive to IP₃ (IisCaP). The IsCaP is filled at higher Ca²⁺ concentrations (10^–6 mol/liter) which may occur during stimulation. The low steady-state [Ca²⁺] of 10^–7 mol/liter is adjusted by the IisCaP.It is speculated that both Ca²⁺ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.     

Structural gene isolation and prepeptide sequence of gallidermin, a new lanthionine containing antibiotic

Peptide antibiotics containing lanthionine and 3-methyllanthionine bridges, named lantibiotics are of increasing interest. A new lantibiotic, gallidermin, has been isolated from Staphyloccus gallinarum. Here we report the isolation of its structural gene which we name gdmA. In all lantibiotics so far studied genetically, three peptides can be formally distinguished: (i) the primary translation product, which we call the prepeptide; (ii) the propeptide lacking the leader sequence and (iii) the mature lantibiotic. Unlike the plasmid-coded epidermin, gdmA is located on the chromosome. The gdmA locus codes for a 52 amino acid residue prepeptide, consisting of an alpha-helical leader sequence of hydrophilic character, which is separated from the C-terminus (propeptide) by a characteristic proteolytic processing site (Pro-2 Arg-1 Ile1). Although pro-gallidermin differs from pro-epidermin (a recently isolated lantibiotic) only by a single amino acid residue exchange. Leu instead of Ile, the N-terminus of the prepeptide differs by an additional two exchanges.     

Immunological and chemical characterization of hamster brain polyribosomes-cytomatrix complexes

We have studied the cytoskeletal nature of a brain subcellular fraction previously shown to contain polyribosomes. We have identified the major proteins of this fraction by electrophoretic comparison to a standard cytoskeletal fraction and by immunodetection. These methods have shown the presence of actin, glial fibrillary acidic protein, and neurofilament triplet proteins. We have also studied the effect of various ions and nonionic detergents on the stability of this structure. It was stable in presence of Triton X-100 up to 2% but disrupted by 200 mM K⁺ acetate.Abbreviations CMT cytomatrix - CSK cytoskeleton - DOC sodium deoxycholate - DTT dithiothreitol - EGTA ethylenglycolbis (-Ether)-N,N-N-N-Tetraacetic Acid - GFAP glial fibrillary acidic protein - PR polyribosome - PRCMC polyribosomes-cytomatrix complex     

The effect of diabetes mellitus on aortic prostanoid synthesis and serum cholesterol levels in the rat fed a high cholesterol diet

The effect of diabetes mellitus on serum cholesterol and aortic microsomal prostanoid synthesis was studied in cholesterol fed male Lewis rats. Normal, diabetic and diabetic rats treated with pancreatic islets were divided into three diet subgroups, control diet, control +2% cholesterol for 8 weeks and control +2% cholesterol diet for 16 weeks. Serum glucose levels were elevated three-fold in the diabetic group compared to normal. Treatment with islets restored serum glucose to normal levels in diabetic rats. The 2% cholesterol diet did not significantly alter serum glucose levels in any of the groups. Body weights in the diabetic group were significantly lower than normal or diabetic rats treated with islets. Feeding 2% cholesterol for 16 weeks significantly increased weight in normal and islet treated diabetic rats but not in the diabetic group. Aortic microsomal prostanoid synthesis was similar in all experimental groups with 6-keto-PGF1 alpha (PGI2 metabolite) being the major product synthesized in all groups. Aortic microsomal prostanoid levels were not altered by the 2% cholesterol diet. Serum cholesterol levels increased 14-fold in the diabetic group which returned to the normal level in the diabetic animals treated with islets. These data show that diabetes does not alter aortic microsomal prostanoid levels in the rat. However, diabetes significantly increased serum cholesterol levels which were reversed by islet transplantation.     

Transfer of amino acids and nitrate from the roots into the xylem of Ricinus communis seedlings

The loading of amino acids and nitrate into the xylem was investigated by collection and analysis of root-pressure exudate from the cut hypocotyl stumps of seedlings of Ricinus communis L. Glutamine was found to be the dominant amino acid in the exudate and also to be the amino acid which is transferred to the xylem most rapidly and accumulated to the greatest extent. The comparison between uptake and xylem loading showed significant differences in specificity between these two transport reactions, indicating a different set of transport systems. Nitrate is transferred to the xylem at a higher relative rate than any amino acid despite the great nitrate-storage capacity of the root system. Thus the supply of nitrate to Ricinus plants leads to enhanced nitrogen allocation to the shoots.     

Isolation of a pathogenic clone of mouse mammary tumor virus.

Exogenous mouse mammary tumor virus (MMTV) was cloned from a GR mammary tumor. Clone lambda GRT39 contained a full-length integrated MMTV(GR) provirus and both 5' and 3' host flanking DNA. The lambda GRT39 provirus had no apparent structural changes associated with cloning and retained the exogenous MMTV gag gene poison sequence. When introduced into rat mammary adenocarcinoma LA7 cells, the lambda GRT39 provirus was fully expressed. lambda GRT39-transfected LA7 cells made MMTV RNA, had gp52 SU protein on the cell surface, and produced B-type retrovirus particles characteristic of MMTV. Mammary tumors developed in hormone-stimulated BALB/c females injected with MMTV from lambda GRT39-transfected LA7 cells [MMTV (lambda GRT39)]. The tumors had new, clonally integrated copies of the MMTV(lambda GRT39) provirus and were expressing MMTV antigen. These data indicate that the lambda GRT39 provirus is biologically active and pathogenic.     

d-Ribulokinase from Klebsiella pneumoniae for continuous production of d-(−)-ribulose-5-phosphate

Summary The production of d-ribulose-5-phosphate in an enzyme membrane reactor was examined. Phosphoryl transfer from ATP to d-ribulose was catalysed by d-ribulokinase isolated from Klebsiella pneumoniae. For production of d-ribulose-5-phosphate the phosphoryl donor ATP was used either in stoichiometric or in catalytic amounts. Using catalytic amounts of ATP requires a second enzyme, e.g. pyruvate kinase, to regenerate ATP. The kinetic parameters for d-ribulokinase and pyruvate kinase were determined to calculate the performance of an enzyme membrane reactor for continuous production of d-ribulose-5-phosphate. Both processes operated for more than 200 h. Regardless of whether ATP was used in catalytic or stoichiometric amounts, about the same production parameters were determined. In continuous production space/time yields of 117 g (with ATP regeneration) and 103 g (without ATP regeneration) of d-ribulose-5-phosphate 1^–1 per day were reached.Offprint requests to: D. Gygax     

Continuous microbial production ofl-leucine with cell retention

Summary Continuous production ofl-leucine was carried out withCorynebacterium glutamicum, strain ATCC 13032 starting from-ketoisocaproic acid as the precursor, glucose as the carbon source and ammonium sulphate as the nitrogen source, with biotin in a mineral medium. By means of cross-flow microfiltration or centrifugal separation for cell retention in continuous fermentation an increase in cell density was achieved and the product solution was obtained cell-free. The cells were concentrated to over 70 g cell dry mass/1. In experiments of up to 42 days, conversion rates of 85%–99% andl-leucine yields of 85%–93% were achieved. With a substrate residence time of 3.6 h, 114 mmol/1l-leucine was produced with a space-time yield of 97 g/1 per day. A scale-up of the fermentation volume from 4 to 1001 provided comparable results.     

Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanicallyinduced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.     

Localization of calcium and microfilament changes in mechanically stressed cells

We combined fluorescence labeling, digital image processing, and micromanipulation to investigate the intracellular events induced by inflicting a mechanical stress on rat basophilic leukemia cells. Our findings were as follows:

1. Most cells displayed a localized calcium rise in response to micropipet aspiration. This represented an average threefold increase as compared to resting level, and it was observed during the first 10 s following aspiration. A slow return to initial level occurred within about 3 min. Further, this calcium rise involved a mobilization of intracellular stores, since it was not prevented by adding a calcium chelator into the extracellular medium.
2. All micropipet-aspirated cells displayed a local accumulation of microfilaments, with a preferential localization in the cell protrusions or near the pipet tips.
3. No absolute correlation was found between the localization of calcium rise and cytoskeletal accumulation.
4. Cell deformability was decreased when intracellular calcium was maintained at a constant (high or low) level with ionomycin and/or EGTA.

It is concluded that cells have a general ability to respond to mechanical stimulation by a coordinated set of events. More parameters must be studied before the mechanisms of cell shape regulation are fully understood.