Surfactant releases internal calcium stores in neutrophils by G protein-activated pathway.

Pulmonary surfactant with surfactant-associated proteins (PS+SAP) decreases pulmonary inflammation by suppressing neutrophil activation. We have observed that PS+SAP inserts channels into artificial membranes, depolarizes neutrophils, and depresses calcium influx and function in stimulated neutrophils. We hypothesize that PS+SAP suppresses neutrophil activation by depletion of internal Ca(++) stores and that PS+SAP induces depletion through release of Ca(++) stores and through inhibition of Ca(++) influx. Our model predicts that PS+SAP releases Ca(++) stores through insertion of channels, depolarization of neutrophils, and activation of a G protein-dependent pathway. If the model of channel insertion and membrane depolarization is accurate, then gramicidin-a channel protein with properties similar to those of PS+SAP-is expected to mimic these effects. Human neutrophils were monitored for [Ca(++)] responses after exposure to one of two different PS+SAP preparations, a PS-SAP preparation, gramicidin alone, and gramicidin reconstituted with phospholipid (PLG). [Ca(++)] responses were reexamined following preexposure to inhibitors of internal Ca(++) release or the G protein pathway. We observed that (i) 1% PS+SAP-but not PS-SAP-causes transient increase of neutrophil [Ca(++)] within seconds of exposure; (ii) 1% PLG-but not gramicidin alone-closely mimics the effect of PS+SAP on Ca(++) response; (iii) PS+SAP and PLG equally depolarize neutrophils; (iv) direct inhibition of internal Ca(++) stores releases or of G protein activation suppresses Ca(++) responses to PS+SAP and PLG; and (v) preexposure to either PS+SAP or PLG inhibits Ca(++) influx following fMLP stimulation. We conclude that PS+SAP independently depolarizes neutrophils, releases Ca(++) from internal stores by a G protein-mediated pathway, and alters subsequent neutrophil response to physiologic stimulants by depleting internal Ca(++) stores and by inhibiting Ca(++) influx during subsequent fMLP activation. The mimicking of these results by PLG supports the hypothesis that PS+SAP initiates depolarization via channel insertion into neutrophil plasma membrane.

Linkage of body mass index to chromosome 20 in Utah pedigrees.

Several linkage studies have hinted at the existence of an obesity predisposition locus on chromosome 20, but none of these studies has produced conclusive results. Therefore, we analyzed 48 genetic markers on chromosome 20 for linkage to severe obesity (BMI> or =35) in 103 extended Utah pedigrees (1,711 individuals), all of which had strong aggregation of severe obesity. A simple dominant model produced a maximum multipoint heterogeneity LOD score of 3.5 at D20S438 (55.1 cM). Two additional analyses were performed. First, a one-gene, two-mutation model (with one dominant mutation and one recessive mutation) increased the LOD score to 4.2. Second, a two-locus model (with one locus dominant and one recessive) generated a multipoint LOD score of 4.9. We conclude that one or more severe obesity predisposing genes lie within an interval of approx. 10 cM on chromosome 20. This study generated significant LOD scores which confirm suggestive linkage reports from previous studies. In addition, our analyses suggest that the predisposing gene(s) is localized very near the chromosome 20 centromere.

Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probes.

A total of 65 epidemiologically unrelated tetracycline-resistant isolates of the six Salmonella enterica subsp. enterica (Salm.) serovars Dublin, Choleraesuis, Typhimurium, Enteritidis, Hadar and Saintpaul were investigated for the presence of tetracycline resistance genes. For this, specific gene probes of the tetracycline resistance genes (tet) of the hybridization classes A, B, C, D, E and G were constructed by cloning PCR-amplified internal segments of the respective tet structural genes. These gene probes were sequenced and used in hybridization experiments with plasmid DNA or endonuclease digested whole cell DNA as targets. Only tet(A) genes were detected on plasmids in all Salm. Dublin isolates as well as in single isolates of Salm. Choleraesuis and Salm. Typhimurium. Genes of the hybridization classes B, C, D and G, but also in some cases those of class A, were located in the chromosomal DNA of the corresponding Salmonella isolates. Restriction fragment length polymorphisms (RFLPs) of tet gene carrying fragments were detected in chromosomally tetracycline-resistant isolates. These RFLPs might represent valuable additional tools for the identification and characterization of tetracycline-resistant Salmonella isolates.

Plasmid-encoded tetracycline resistance in Salmonella enterica subsp. enterica serovars choleraesuis and typhimurium: identification of complete and truncated Tn1721 elements.

During routine screening of Salmonella enterica subsp., S. enterica isolates of animal origin for plasmid-encoded tetracycline resistance, two tetracycline resistance plasmids, the 50 kbp plasmid pGFT3 of Salmonella choleraesuis and the 9.5 kbp plasmid pGFT4 of Salmonella typhimurium var. Copenhagen DT002, were detected. The respective tetracycline resistance genes (tet) were identified by hybridization and PCR analysis to belong to hybridization class A. Conjugation experiments identified plasmid pGFT3 as a conjugative plasmid. Molecular analysis of the tet(A) gene area and the flanking regions identified a complete Tn1721-like transposon on plasmid pGFT3 and a truncated Tn1721-like element on plasmid pGFT4. The complete Tn1721-like element was integrated into a transposase reading frame of a truncated Tn3 transposon also located on plasmid pGFT3. The truncated Tn1721-like element of plasmid pGFT4 lacked the entire transposase part. This Tn1721-relic was integrated in an unknown reading frame which on amino acid level showed homology to the Rop protein of Escherichia coli. A model for the deletion of the transposase part was developed on the basis of the sequences present at the termini of the truncated Tn1721-like element.

Molecular characterization of Salmonella enterica subsp. enterica serovar Typhimurium DT009 isolates: differentiation of the live vaccine strain Zoosaloral from field isolates.

A total of 25 epidemiologically unrelated Salmonella enterica subsp. enterica (S.) serovar Typhimurium DT009 isolates from various human and animal sources, the original S. Typhimurium DT009 Zoosaloral live vaccine strain and two Zoosaloral strains reisolated from vaccinated chickens were investigated by various molecular typing methods (I) to determine the most suitable method or combination of methods for the differentiation of DT009 field isolates and (II) to investigate which molecular methods are suitable to differentiate the Zoosaloral live vaccine strain from field isolates of the same phage type. Based on the results of plasmid profile analysis, IS200 typing and macrorestriction analysis with XbaI, SpeI and BlnI, the 28 S. Typhimurium DT009 isolates were assigned to 16 different genomic groups, one of which was exclusively represented by the original and the reisolated Zoosaloral strains. IS200 typing was the most discriminatory single method for the differentiation of the DT009 isolates followed by plasmid profile analysis and BlnI-macrorestriction analysis. The Zoosaloral vaccine strain differed from the DT009 field isolates by its unique HindIII-fragment pattern of the virulence plasmid and by its unique SpeI-macrorestriction pattern.

Tetracycline resistance in Salmonella enterica subsp. enterica serovar Dublin.

The 47-kbp plasmid pGFT1 from Salmonella enterica subsp. enterica serovar Dublin mediated tetracycline resistance via a tet(A) gene located on an integrated copy of a Tn1721-analogous transposon. The integration site of the transposon was located within the reading frame of a fip gene. Plasmid pGFT1 was shown to be conjugative and to be able to replicate and express tetracycline resistance in Escherichia coli.

Mitochondrial glutamate dehydrogenase from Leishmania tarentolae is a guide RNA-binding protein.

To identify specific proteins interacting with guide RNAs (gRNAs) in mitochondrial ribonucleoprotein complexes from Leishmania tarentolae, fractionated and unfractionated mitochondrial extracts were subjected to UV cross-linking with added labeled gRNA and also with [alpha-32P]UTP-labeled endogenous RNA. An abundant 110-kDa protein (p110) localized in the T-V complex, which sediments in glycerol gradients at the leading edge of the 10S terminal uridylyltransferase peak, was found to interact with both types of labeled RNAs. The p110 protein was gel isolated and subjected to microsequence analysis, and the gene was cloned. The sequence revealed significant similarity with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised against a recombinant fragment of the p110 gene and was used to demonstrate a stable and specific gRNA-binding activity by coimmunoprecipitation and competitive gel shift analyses. Complex formation was strongly inhibited by competition with poly(U) or by deletion or substitution of the gRNA 3' oligo(U) tail. Also, addition of a 3' oligo(U) tail to an unrelated transcript was sufficient for p110 binding. Both the gRNA-binding activity of the p110 protein and in vitro gRNA-independent and gRNA-dependent uridine insertion activities in the mitochondrial extract were inhibited by high concentrations of dinucleotides.

Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract.

Two polypeptides of 50 and 45 kDa were adenylated by incubation of a mitochondrial extract from Leishmania tarentolae with [alpha-32P]ATP. These proteins were components of a complex that sedimented at 20S in glycerol gradients and migrated as a single band of approximately 1800 kDa in a native gel. The facts that RNA ligase activity cosedimented at 20S and that the ATP-labeled p45 and p50 polypeptides were deadenylated upon incubation with a ligatable RNA substrate suggested that these proteins may represent charged intermediates of a mitochondrial RNA ligase. Hybridization of native gel blots with guide RNA (gRNA) probes showed the presence of gRNA in the previously identified T-IV complexes that sedimented in glycerol at 10S and contained terminal uridylyl transferase (TUTase) activity, and also in a previously unidentified class of heterodisperse complexes that sedimented throughout the gradient. gRNAs were not detected in the p45 + p50-containing 1800 kDa complex. The heterodisperse gRNA-containing complexes were sensitive to incubation at 27 degrees C and appear to represent complexes of T-IV subunits with mRNA. Polyclonal antiserum to a 70 kDa protein that purified with terminal uridylyl transferase activity was generated, and the antiserum was used to show that this p70 polypeptide was a component of both the T-IV and the heterodisperse gRNA-containing complexes. We propose that the p45 + p50-containing 1800 kDa complex and the p70 + gRNA-containing heterodisperse complexes interact in the editing process. Further characterization of these various complexes should increase our knowledge of the biochemical mechanisms involved in RNA editing.

Uridine insertion into preedited mRNA by a mitochondrial extract from Leishmania tarentolae: stereochemical evidence for the enzyme cascade model.

An RNA editing-like internal uridine (U) incorporation activity (G. C. Frech, N. Bakalara, L Simpson, and A. M. Simpson, EMBO J. 14:178-187, 1995) and a 3'-terminal U addition activity (N. Bakalara, A. M. Simpson, and L. Simpson, J. Biol. Chem. 264:18679-18686, 1989) have been previously described by using a mitochondrial extract from Leishmania tarentolae. Chiral phosphorothioates were used to investigate the stereoconfiguration requirements and the stereochemical course of these nucleotidyl transfer reactions. The extract utilizes (SP)-alpha-S-UTP for both 3' and internal U incorporation into substrate RNA. The internal as well as the 3' incorporation of (SP)-alpha-S-UTP proceeds via inversion of the stereoconfiguration. Furthermore, internal U incorporation does not occur at sites containing thiophosphodiesters of the RP configuration. Our results are compatible with an enzyme cascade model for this in vitro U insertion activity involving sequential endonuclease and uridylyl transferase directly from UTP and RNA ligase steps and are incompatible with models involving the transfer of U residues from the 3' ends of guide RNAs.

In vitro RNA editing-like activity in a mitochondrial extract from Leishmania tarentolae.

A mitochondrial extract from Leishmania tarentolae directs the incorporation of uridylate (U) residues within the pre-edited domain of synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has several characteristics of an in vitro RNA editing activity, but no direct evidence for involvement of guide RNAs was obtained. Inhibition by micrococcal nuclease suggests a requirement for some type of endogenous RNA. The limitation of internal U-incorporation to the pre-edited region in the CYb mRNA and the inhibition by deletion or substitution of both mRNA anchor sequences for CYb gRNA-I and -II could be consistent either with a gRNA-mediated process or a secondary structure-mediated process. A low level of incorporation of [alpha-32P]CTP occurs at the same sites as UTP. Internal U-incorporation activity is selectively inhibited by heterologous RNAs, suggesting an involvement of low affinity RNA-binding proteins which can be competed by the added RNA.

Characterization of two classes of ribonucleoprotein complexes possibly involved in RNA editing from Leishmania tarentolae mitochondria.

The molecular mechanism of RNA editing in trypanosomatid mitochondria is an unsolved problem. We show that two classes of ribonucleoprotein complexes exist in a mitochondrial extract from Leishmania tarentolae and appear to be involved in RNA editing. The 'G' class of RNP complexes consists of 170-300 A particles which contain guide RNAs and proteins, show little terminal uridylyl transferase (TUTase) activity and exhibit an in vitro RNA editing-like activity. The 'T' class consists of approximately six RNP complexes, the endogenous RNA of which can be self-labeled with [alpha-32P]UTP. The most abundant T complex, T-IV, is visualized by electron microscopy as 80-140 A particles. This complex exhibits TUTase activity in the native gel and contains guide RNAs. Both G and T complexes are possibly involved with RNA editing in vivo. These results are a starting point for the analysis of the biochemistry of RNA editing.

Distinct spatial and temporal expression patterns of K+ channel mRNAs from different subfamilies.

Different types of K+ channels play important roles in many aspects of excitability. The isolation of cDNA clones from Drosophila, Aplysia, Xenopus, and mammals points to a large multigene family with several distinct members encoding K+ channels with unique electrophysiological and pharmacological properties. Given the pivotal role K+ channels play in the fine tuning of electrical properties of excitable tissues, we studied the spatial and temporal basis of K+ channel diversity. We report the isolation of two putative K+ channels that define two new subfamilies based upon amino acid sequence similarities with other known K+ channels. Northern blot and in situ hybridization studies revealed differences in the spatial and temporal expression patterns for these two new clones along with mRNAs from other K+ channel subfamilies. Two of the K+ channels studied are predominantly expressed in the brain. One of the "brain-specific" K+ channels is first expressed after about 2 weeks of postnatal cerebellar development and remains at levels about 10-fold higher in the cerebellum than in the rest of the brain.

Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor.

Thyrotropin-releasing hormone (TRH) is an important extracellular regulatory molecule that functions as a releasing factor in the anterior pituitary gland and as a neurotransmitter/neuromodulator in the central and peripheral nervous systems. Binding sites for TRH are present in these tissues, but the TRH receptor (TRH-R) has not been purified from any source. Using Xenopus laevis oocytes in an expression cloning strategy, we have isolated a cDNA clone that encodes the mouse pituitary TRH-R. This conclusion is based on the following evidence. Injection of sense RNA transcribed in vitro from this cDNA into Xenopus oocytes leads to expression of cell-surface receptors that bind TRH and the competitive antagonist chlordiazepoxide with appropriate affinities and that elicit electrophysiological responses to TRH with the appropriate concentration dependency. Antisense RNA inhibits the TRH response in Xenopus oocytes injected with RNA isolated from normal rat anterior pituitary glands. Finally, transfection of COS-1 cells with this cDNA leads to expression of receptors that bind TRH and chlordiazepoxide with appropriate affinities and that transduce TRH stimulation of inositol phosphate formation. The 3.8-kilobase mouse TRH-R cDNA encodes a protein of 393 amino acids that shows similarities to other guanine nucleotide-binding regulatory protein-coupled receptors.

Alteration and restoration of K+ channel function by deletions at the N- and C-termini.

Voltage-dependent ion channels are thought to consist of a highly conserved repeated core of six transmembrane segments, flanked by more variable cytoplasmic domains. Significant functional differences exist among related types of K+ channels. These differences have been attributed to the variable domains, most prominently the N- and C-termini. We have therefore investigated the functional importance of both termini for the delayed rectifier K+ channel from rat brain encoded by the drk1 gene. This channel has an unusually long C-terminus. Deletions in either terminus affected both activation and inactivation, in some cases profoundly. Unexpectedly, more extensive deletions in both termini restored gating. We could therefore define a core region only slightly longer than the six transmembrane segments that is sufficient for the formation of channels with the kinetics of a delayed rectifier.

A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning.

Voltage-activated potassium channels play an important part in the control of excitability in nerve and muscle. Different K+ channels are involved in establishing the resting potential, determining the duration of action potentials, modulation of transmitter release, and in rhythmic firing patterns and delayed excitation. Using in vitro transcripts made from a directional complementary DNA library we have isolated, by expression cloning in Xenopus oocytes, a novel K+-channel gene (drk1). Functionally, drk1 encodes channels that are K+ selective and belong to the delayed rectifier class of channels, rather than the A-type class encoded by the Shaker gene of Drosophila. The channels show sigmoidal voltage-dependent activation and do not inactivate within 500 ms. Structurally, drk1 encodes an amino-acid sequence which is more closely related to the Drosophila Shab gene than to the Shaker gene.

Construction of directional cDNA libraries enriched for full-length inserts in a transcription-competent vector.

We have designed a simple procedure for the construction of directional cDNA libraries enriched for full-length inserts in a transcription-competent cloning vector. An oligonucleotide, its 5' end starting with a heteropolymeric sequence encoding the rare restriction sites for NotI and SfiI, followed by 50 dT residues, is used to prime first-strand synthesis on size-selected mRNA. After second-strand synthesis and EcoRI linker addition, the cDNA is double digested with EcoRI and NotI, or with EcoRI and SfiI, to generate DNA fragments with asymmetric ends that can be directionally cloned. The cDNA fragments are enriched for "full length" by size selection and ligated into a phage lambda vector containing the T3 and T7 RNA polymerase promoters. These cDNA libraries can directly be used for in vitro synthesis of sense or antisense RNA.

No functional gamma-chain transcripts detected in an alloreactive cytotoxic T-cell clone.

Three groups of genes that undergo rearrangements during T-cell maturation have been isolated from T cells. Two of them encode the alpha- and beta-subunits of the T-cell antigen receptor and are shared between antigen-specific, major histocompatibility (MHC) class I-restricted cytotoxic T cells and antigen-specific, MHC class II-restricted helper T cells. The third group of genes, called gamma, is preferentially transcribed in cytotoxic T cells. This led to the hypothesis that the unidentified gamma-gene products could be part of a putative T-cell receptor responsible for MHC class I recognition. We report here on the isolation of three different types of gamma-gene transcripts of an alloreactive cytotoxic T-cell clone (3F9). Two are derived from two rearrangements that have occurred at the same locus (V gamma 10.8A to J gamma 10.5 and transcribed with C gamma 10.5), while the third involves a new V gamma-gene segment that is joined to J gamma 13.4 and transcribed with C gamma 13.4. All these rearrangements are abortive and lead to the formation of non-functional gamma-chain genes because the proper translational reading frame is not maintained. Because the second copy of the C gamma 13.4 gene segment is deleted and as C gamma 7.5 is considered to be a pseudogene and has not undergone any rearrangements in 3F9, we conclude that the alloreactive cytotoxic T-cell clone 3F9 does not contain a functional transcript of a known gamma-chain gene.

Sodium acetate as a preservative in protein hydrolysate solutions.

The inhibitory effect of sodium acetate on microorganism growth in protein hydrolysate solutions was studied. Solutions of 5% protein hydrolysate and 5% dextrose in water (seven parts) and 50% dextose in water (three parts) containing 0, 30, 50 and 90 mEq/liter of sodium acetate were inoculated with Staphylococcus aureus, Escherichia coli, Candida albicans and Pseudomonas aeruginosa. The number of colony-forming units in the solutions after inoculation was compared with that after incubation for 24 hours at 37 C. Sodium acetate inhibited growth of S aureus and E coli. Growth of P aeruginosa was inhibited in protein hydrolysate solutions with and without sodium acetate; inhibition could not be attributed solely to sodium acetate and may have been releated to pH of the solutions (4.7 to 5.4). Growth of C albicans was not inhibited by sodium acetate. Sodium acetate reduced growth of some common contaminants of protein hydrolysates. Sodium acetate is known to reduce metabolic acidosis, a reported complication of parenteral nutrient therapy and a possible predisposing factor in C albicans sepsis. Addition of sodium acetate to protein hydrolysate solutions should be considered seriously.