Cytogenetic characterization of 20 lymphoblastoid lines derived from human individuals differing in bleomycin sensitivity

Summary Forty lymphoblastoid (lymphoid) lines were established from 42 volunteer blood donors, including healthy individuals and patients with head and neck carcinomas. Each peripheral blood sample was split into two portions, one for the establishment of a lymphoid line and the other for short-term culture, which was used to estimate bleomycin sensitivity by cytogenetic procedures. Twenty lymphoid lines were selected at random to compare bleomycin sensitivity with data obtained from short-term lymphocyte cultures. In each set, bleomycin sensitivity of lymphoid cells was similar to that of the lymphocytes. The lymphoid lines, which can be propagated for an unlimited supply of relatively homogeneous cellular material, will be useful for a variety of future investigations. This investigation was supported by grants from the John S. Dunn Foundation, Houston, TX, the Esther Knispel Fund administered by The University of Texas M. D. Anderson Cancer Center, Houston, TX, and Department of Health and Human Services PHS grant DE 07007.     

Characterization of K+ Channels in the Basolateral Membrane of Rat Tracheal Epithelia

To study K⁺ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml) in the symmetrical high K⁺ (145 mm) Ringer solution. During measurement of the macroscopic K⁺ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K⁺ currents: one is an inwardly rectifying K⁺ current and the other is a slowly activating outwardly rectifying K⁺ current. The inwardly rectifying K⁺ current is inhibited by Ba²⁺. The slowly activating K⁺ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of I _SK channel. RT-PCR analysis revealed the presence of I _SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba²⁺ only had minimal affect on short-circuit current (I _sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I _SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia. Received: 1 March 1996/Revised: 5 August 1996     

Regional Reductions of Transketolase in Thiamine-Deficient Rat Brain

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.     

Production of a polysaccharide, methylan, in Methylobacterium organophilum by controlling ammonium ion

Summary Cell growth increased proportionally to the initial concentration of ammonium ion, however, methylan production was significantly inhibited at the high concentration of ammonium ion. The control of ammonium ion within the desired level(usually 0.45 g/l) was needed to reduce the inhibition. Methylan production was increased to 12.5 g/l by maintaining ammonium ion below 0.15 g/l.     

A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.     

New biologically active hybrid bacteriocins constructed by combining regions from various pediocin-like bacteriocins: the C-terminal region is important for determining specificity.

The pediocin-like bacteriocins, produced by lactic acid bacteria, are bactericidal polypeptides with very similar primary structures. Peptide synthesis followed by reverse-phase and ion-exchange chromatographies yielded biologically active pediocin-like bacteriocins in amounts and with a purity sufficient for characterizing their structure and mode of action. Despite similar primary structures, the pediocin-like bacteriocins, i.e., pediocin PA-1, sakacin P, curvacin A, and leucocin A, differed in their relative toxicities against various bacterial strains. On the basis of the primary structures, the polypeptides of these bacteriocins were divided into two modules: the relatively hydrophilic and well conserved N-terminal region, and the somewhat more diverse and hydrophobic C-terminal region. By peptide synthesis, four new biologically active hybrid bacteriocins were constructed by interchanging corresponding modules from various pediocin-like bacteriocins. All of the new hybrid bacteriocin constructs had bactericidal activity. The relative sensitivity of different bacterial strains to a hybrid bacteriocin was similar to that to the bacteriocin from which the C-terminal module was derived and quite different from that to the bacteriocin from which the N-terminal was derived. Thus, the C-terminal part of the pediocin-like bacteriocins is an important determinant of the target cell specificity. The synthetic bacteriocins were more stable than natural isolates, presumably as a result of the absence of contaminating proteases. However, some of the synthetic bacteriocins lost activity, but this was detectable only after months of storage. Mass spectrometry suggested that this instability was due to oxidation of methionine residues, resulting in a 10- to 100-fold reduction in activity.     

RELATIONSHIPS AMONG MORPHOLOGICAL STATUS, STEROID HORMONES, AND POST-THAWING VIABILITY OF FROZEN SPERMATOZOA OF MALE MINKE WHALES (BALAENOPTERA ACUTOROSTRATA)

Spermatozoa from 21 mature minke whales ( Balaenoptera acutorostrata ) taken in the Antarctic Ocean for Japanese research were recovered from vasa deferentia, diluted 1:9 in a Tris-based diluent, and frozen at - 80°C on board the vessel. After a period ranging from 45 to 125 d, the samples were transferred to liquid nitrogen and transported to the laboratory. After thawing at 37°C the motility (percentage of motile spermatozoa), vitality (proportion of live spermatozoa), and sperm concentration were determined for each sample. These values were tested for correlations with morphological measurements (body size, body weight, testis weight) and serum concentrations of progesterone (Pd), estradiol-17β (E2), and testosterone (T). Ten of 21 samples had motile spermatozoa (2%-40%). Although no motile spermatozoa were observed in 1.1 samples, all sperm samples were examined by eosinnigrosin staining and showed vitality levels of 3%44%. It was found that the motility (Y = 0.54) and vitality (r = 0.53) of the spermatozoa were significantly (P < 0.01) correlated with the E₂ levels (8.50 ± 1.80 pg/ml). Serum T levels (0.07 ± 0.02 ngml) were significantly correlated with the E₂ levels (r = 0.58, P < 0.01>, but sperm concentrations were not correlated with either Ea or T levels. The present study demonstrates that spermatozoa of minke whales can be successfully cryopreserved.     

The carboxylate type siderophore rhizoferrin and its analogs produced by directed fermentation

Summary Rhizoferrin is a novel carboxylate-type siderophore which has recently been isolated fromRhizopus microsporus and other fungi of the Mucorales (Zygomycetes). The present investigation shows that a variety of rhizoferrin analogs can be produced by directed fermentation. Thus both the diaminobutane backbone and the citric acid side chains of rhizoferrin have been substituted by diamine and citric acid analogs added to the culture medium. The new ligands as well as their iron complexes have been characterized by physicochemical methods. Conditions of precursor incorporation and implications for the biosynthesis of the new siderophores are discussed.     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.