A mutant rat major histocompatibility haplotype showing a large deletion of class I sequences

The LEW.1LM1 inbred rat strain, which has been derived from a (LEW×LEW.1W) F₂ hybrid, carries a major histocompatibility (RT1) haplotype which is distinct from that of the LEW strain (RT1 ¹) in that certainRT1.C region-determined class I antigens are not expressed. Here we show that this phenotypic defect is due to genomic deletion of about 100 kb of theRT1.C region. Certain deleted DNA fragments have been cloned from the wild-type DNA into the EMBL4 vector. Five clones have been characterized and are shown to possess different restriction maps and to each carry a single stretch of class I cross-hybridizing sequences. Probes derived from the non-class I coding part of two clones detect fragments which are present in the wild-type but absent from thelm1 mutant. The type of deletion described here in the rat is discussed in the context ofH-2D/Q deletions in the mouse.     

Evidence for a new pathway in the bacterial degradation of 4-fluorobenzoate

Six bacterial strains able to use 4-fluorobenzoic acid as their sole source of carbon and energy were isolated by selective enrichment from various water and soil samples from the Stuttgart area. According to their responses in biochemical and morphological tests, the organisms were assigned to the genera Alcaligenes, Pseudomonas, and Aureobacterium. To elucidate the degradation pathway of 4-fluorobenzoate, metabolic intermediates were identified. Five gram-negative isolates degraded this substrate via 4-fluorocatechol, as described in previous studies. In growth experiments, these strains excreted 50 to 90% of the fluoride from fluorobenzoate. Alcaligenes sp. strains RHO21 and RHO22 used all three isomers of monofluorobenzoate. Alcaligenes sp. strain RHO22 also grew on 4-chlorobenzoate. Aureobacterium sp. strain RHO25 transiently excreted 4-hydroxybenzoate into the culture medium during growth on 4-fluorobenzoate, and stoichiometric amounts of fluoride were released. In cell extracts from this strain, the enzymes for the conversion of 4-fluorobenzoate, 4-hydroxybenzoate, and 3,4-dihydroxybenzoate could be detected. All these enzymes were inducible by 4-fluorobenzoate. These data suggest a new pathway for the degradation of 4-fluorobenzoate by Aureobacterium sp. strain RHO25 via 4-hydroxybenzoate and 3,4-dihydroxybenzoate.     

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5''-disrupted myb loci.

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.     

Electroneutral, HCO3(-)-independent, pH gradient-dependent uphill transport of Cl- by ileal brush-border membrane vesicles. Possible role in the pathogenesis of chloridorrhea.

By applying a rapid filtration technique to isolated brush border membrane vesicles from guinea pig ileum, 36Cl uptake was quantified in the presence and absence of electrical, pH and alkali-metal ion gradients. A mixture of 20 mM-Hepes and 40 mM-citric acid, adjusted to the desired pH with Tris base, was found to be the most suitable buffer. Malate and Mes could be used to replace the citrate, but succinate, acetate and maleate proved to be unsuitable. In the absence of a pH gradient (pHout:pHin = 7.5:7.5), Cl- uptake increased slightly when an inside-positive membrane potential was applied, but uphill transport was never observed. A pH gradient (pHout:pHin = 5.0:7.5) induced both a 400% increase in the initial Cl- influx rate and a long-lasting (20 to 300 s) overshoot, indicating that a proton gradient can furnish the driving force for uphill Cl- transport. Under pH gradient conditions, initial Cl- entry rates had the following characteristics. (1) They were unaffected by cis-Na+ and/or -K+, indicating the absence of Cl-/K+, Cl-/Na+ or Cl-/K+/Na+ symport activity. (2) Inhibition by 20-100 mM-trans-Na+ and/or -K+ occurred, independent of the existence of an ion gradient. (3) Cl- entry was practically unaffected by short-circuiting the membrane potential with equilibrated potassium and valinomycin. (4) Carbonyl cyanide m-chlorophenylhydrazone was strongly inhibitory and so, to a lesser extent, was 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid [(SITS)], independent of the sign and size of the membrane potential. (5) Cl- entry was negligibly increased (less than 30%) by either trans-Cl- or -HCO3-, indicating the absence of an obligatory Cl-/anion antiport activity. In contrast, the height of the overshoot at 60 s was increased by trans-Cl-, indicating time-dependent inhibition of 36Cl efflux. That competitive inhibition of 36Cl fluxes by anions is involved here is supported by initial influx rate experiments demonstrating: (1) the saturability of Cl- influx, which was found to exhibit Michaelis-Menten kinetics; and (2) competitive inhibition of influx by cis-Cl- and -Br-. Quantitatively, the conclusion is warranted that over 85% of the total initial Cl- uptake energized by a pH gradient involves an electroneutral Cl-/H+ symporter or its physicochemical equivalent, a Cl-/OH- antiporter, exhibiting little Cl- uniport and either Cl-/Cl- or Cl-/HCO3- antiport activities.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bradykinin and thrombin effects on polyphosphoinositide hydrolysis and prostacyclin production in endothelial cells.

Prostacyclin (PGI2) production by thrombin- and bradykinin-stimulated bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) was related to the receptor-linked activation of inositide hydrolysis. Bradykinin caused a rapid and transient 3-fold increase in the formation of inositol polyphosphates in BAEC. The increase in InsP3 reflected changes mainly in the Ins(1,4,5)P3 isomer. Thrombin was less effective than bradykinin in increasing InsP3 levels and appeared to only minimally stimulate the production of PGI2 in BAEC. In HUVEC, thrombin caused a 5-fold elevation of Ins(1,4,5)P3, closely related to a rise in PGI2 production. However, bradykinin did not affect inositol phosphates and PGI2 production in HUVEC. Other inositol phosphates were also assessed to obtain information on putative metabolism of Ins(1,4,5)P3. The present study supports the notion that formation of Ins(1,4,5)P3 is linked to an increase in PGI2 production in endothelial cells and furthermore provides evidence for a large degree of heterogeneity in the responses of BAEC and HUVEC to thrombin and bradykinin.     

Berenil-induced undercondensation in human heterochromatin

The aromatic diamidine berenil specifically inhibits the condensation of a subset of constitutive heterochromatin in human lymphocyte cultures. In the normal male chromosome complement, only the quinacrine-brilliant Y heterochromatin exhibits distinct undercondensation. The optimal culture conditions for inhibiting heterochromatin condensation are achieved when berenil is added at a final concentration of 150 micrograms/ml 24 h before cell harvest. Various examples of the use of berenil in the analysis of chromosome rearrangements involving quinacrine-brilliant heterochromatin are presented. A variant, giant-satellited chromosome 22 was found to respond to berenil treatment, although its enlarged and quinacrine-bright short-arm region did not contain Y heterochromatin. Southern blot analysis and chromosome in situ hybridization suggested that most chromosome 22 variants do not stem from Y; acrocentric translocations. The experimentally undercondensed Y heterochromatin is characterized by moderate C-band labeling, bright quinacrine fluorescence, and specific silver staining. At the ultrastructural level, undercondensation is associated with loosely packed, mutliply folded chromatin fibers with a diameter of approximately 250 A and organized probably as loops.     

Mammalian sex-chromosome evolution: a conserved homoeologous segment on the X and Y chromosomes in primates

In a representative sample of primate species, including simians (Catarrhini and Platyrrhini) and prosimians (Lemuriformes and Lorisiformes), high-resolution, early replication banding revealed a homoeologous early replicating segment at the ends of both sex chromosomes. The DXYZ2 element, a repeated sequence specific for the human pseudoautosomal region, is conserved in the genomes of all primate species studies and is specifically localized in the distal early replicating segments of the X and Y chromosomes. Thus, cytogenetic and molecular evidence is presented of a highly conserved sex-chromosomal segment in primates. The pseudoautosomal behavior of this segment is discussed.     

Histochemical and immunological demonstration of purple acid phosphatase in human and bovine alveolar macrophages

A tartrate-resistant purple acid phosphatase was localized in human and bovine alveolar macrophages by enzyme- and immuno-histochemistry using an antibody to bovine spleen purple phosphatase. The enzyme could be detected in human and bovine lung tissues as well as on cytospin preparations of alveolar macrophage suspensions from bronchoalveolar lavages. The immunological identity of human and bovine purple phosphatases from alveolar macrophages was demonstrated by Western blot analysis of material separated by polyacrylamide gel electrophoresis. A possible significance of the purple phosphatase as a marker enzyme of activated cells of the mononuclear phagocyte system is discussed.