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Analysis of human sodium/iodide symporter, thyroid transcription factor-1, and paired-box-protein-8 gene expression in benign thyroid diseases.

The ability to concentrate iodide, a fundamental property of normally functioning thyroid tissue, is altered in various thyroid diseases. Given the critical role of the Na+/I- symporter (NIS) in controlling iodide access to the thyroid gland, altered expression of NIS may be responsible, at least in part, for an enhanced or diminished capacity to concentrate iodide. In this study, we used Northern blot analysis, a newly established quantitative polymerase chain reaction (PCR) assay and in addition hNIS-directed immunohistochemical analysis to assess the levels of hNIS mRNA and protein expression in various localized and diffuse benign thyroid abnormalities, including Graves' disease (GD), scintigraphically cold solitary benign thyroid nodule (CBTN), nontoxic multinodular goiter (NMNG), solitary autonomously functioning thyroid nodule (AFTN), and mild diffuse iodine deficiency goiter (IDG). In addition, in view of the recent identification of putative binding sites for the transcription factors thyroid transcription factor-1 (TTF-1) and human paired-box-protein-8 (Pax-8) in the human NIS gene promoter, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to assess in these same samples the levels of TTF-1 and Pax-8 gene expression. Northern blot analysis revealed high levels of hNIS gene expression in thyroid specimens derived from patients with GD and AFTN. In contrast, levels of hNIS mRNA expression were moderate in NMNG, low in diffuse IDG, and very low in CBTN. Quantitative RT-PCR analysis of hNIS mRNA transcripts revealed variable but generally low levels of hNIS gene expression in IDG and NMNG, and undetectable or very low levels of hNIS mRNA in all scintigraphically CBTN studied. In contrast, markedly elevated levels of hNIS mRNA transcripts were detected in active GD (up to 17-fold) and AFTN (up to 25-fold). Immunohistochemical analysis revealed abundant hNIS protein expression by thyroid follicular cells in GD, moderate and heterogeneous levels in NMNG, and very low levels in CBTN. hNIS mRNA levels were correlated with TTF-1 and Pax-8 gene expression in GD and, to a lesser degree, in AFTN, NMNG, and IDG, but not in CBTN. In general, hNIS gene expression was more closely correlated with TTF-1 as compared to Pax-8 gene expression. In conclusion, the abundance of hNIS mRNA and protein expression in a broad range of benign thyroid pathologies correlated well with their functional state as assessed by thyroid scintigraphy. In addition to TTF-1 and Pax-8, other transcription factors and enhancer elements may contribute to regulation of NIS gene promoter activity.

Carrier Proteins↗

Heterogeneity of the cold-insoluble globulin of human plasma (CIg), a circulating cell surface protein.

The cold-insoluble globulin of human plasma (CIg), a circulating cell surface protein, exists in multiple molecular forms. Most molecules are found as two chain (MR approximately 220 000 per chain) disulfide-bridged dimeric units but several minor components of smaller size have also been identified; based upon their migration rates in dodecyl sulfate gel electrophoretic experiments, the smaller molecules characterized in this study range in molecular size from 235 000 to 146 000. The component of molecular weight 235 000 apparently represents a two chain disulfide-bridged derivative of larger parent molecules (one chain of 220 000 plus a smaller remnant), whereas smaller CIg components appear to be single chain proteins. These observations plus electrophoretic analyses of samples of plasmic digests of CIg indicate that the interchain disulfide bridging in the two chain molecule is located in a segment within approx. 175 residues of the NH2- or COOH-terminus.

Dithiothreitol↗

Detection of eubacteria in interstitial cystitis by 16S rDNA amplification.

OBJECTIVE: To determine what role non-culturable microorganisms play in the etiology of interstitial cystitis (IC). MATERIALS AND METHODS: Thirty patients fulfilling NIH criteria for the diagnosis of interstitial cystitis and sixteen control patients with culture negative urine gave written informed consent and underwent bladder biopsy. Polymerase chain reaction (PCR) using two sets of universal primers for bacterial 16S rDNA was performed on urine from the cystoscope and on a cold cup bladder biopsy specimen. Of the PCR positive bladder biopsies, three patients with interstitial cystitis and three controls were randomly selected and cloned. Ten clones from each were sequenced and putative taxonomic assignments made. RESULTS: 12/26 (46%) IC and 5/12 (42%) control urine specimens and 16/30 (53%) and 9/15 (60%) bladder biopsies were PCR positive, respectively. The bacterial populations in the two patient groups tested appeared to be different based upon analysis of the 16S rRNA sequences. CONCLUSIONS: Both IC and control patients had non-culturable bacteria in their bladders. A random sampling of the two populations revealed that the bacterial populations are different, suggesting a possible link between one or more bacterial species and IC.

Bacteria↗

Unique features of the hemoglobin system of the Antarctic notothenioid fish Gobionotothen gibberifrons.

The hemolysate of the Antarctic teleost Gobionotothen gibberifrons (family Nototheniidae) contains two hemoglobins (Hb 1 and Hb 2). The concentration of Hb 2 (15-20% of the total hemoglobin content) is higher than that found in most cold-adapted Notothenioidei. Unlike the other Antarctic species so far examined having two hemoglobins, Hb 1 and Hb 2 do not have globin chains in common. Therefore this hemoglobin system is made of four globins (two alpha- and two beta-chains). The complete amino-acid sequence of the two hemoglobins (Hb 1, alpha2(1)beta2(1); Hb 2, alpha2(2)beta2(2)) has been established. The two hemoglobins have different functional properties. Hb 2 has lower oxygen affinity than Hb 1, and higher sensitivity to the modulatory effect of organophosphates. They also differ thermodynamically, as shown by the effects on the oxygen-binding properties brought about by temperature variations. The oxygen-transport system of G. gibberifrons, with two functionally distinct hemoglobins, suggests that the two components may have distinct physiological roles, in relation with life style and the environmental conditions which the fish may have to face. The unique features of the oxygen-transport system of this species are reflected in the phylogeny of the hemoglobin amino-acid sequences, which are intermediate between those of other fish of the family Nototheniidae and of species of the more advanced family Bathydraconidae.

Allosteric Regulation↗

Lipases in human milk: effect of gestational age and length of lactation on enzyme activity.

Human milk contains two lipases, bile salt-stimulated lipase (BSSL) and lipoprotein lipase (LPL). In the mammary gland, LPL provides long-chain fatty acid for milk fat synthesis. LPL has no known function in milk, but has been implicated in milk fat hydrolysis during cold storage. BSSL may have an important role in infant fat digestion. The aims of the present studies were to assess (1) the methodological validity of using whole milk to analyze BSSL activity, (2) the longitudinal variation of BSSL and LPL activity in the milk of mothers delivering premature and full-term infants, and (3) the stability of BSSL and LPL activity during cold storage. Diluted whole milk and purified BSSL were shown to have similar characteristics. LPL activity was equally stable at -20 and -70 degrees C, whereas BSSL activity was higher in milks stored at -70 than at -20 degrees C (38.8 +/- 0.88 vs 33.3 +/- 0.87 U/ml milk, respectively; 1U = 1 mumol free fatty acid release/min). Levels of BSSL activity in preterm and term milk were similar. LPL activity tended to be higher in term milk. Overall, BSSL activity showed significant longitudinal variation, being highest at 1 and 3 weeks of lactation (43.2 +/- 0.04 and 42.6 +/- 1.03 U/ml milk, respectively). For LPL, the longitudinal pattern of activity depended upon the length of pregnancy. Implications for infant nutrition and mammary gland biology are discussed.

Female↗

Interleukin-10 gene expression in acute virus-induced asthma.

RATIONALE: Virus-induced asthma is characterized by marked neutrophil influx and eosinophil degranulation, suggesting a mode of immunopathogenesis different from that of allergen-induced asthma. OBJECTIVES: This study compared induced sputum cytokine responses in subjects with severe asthma exacerbation and respiratory virus infection with those of patients with stable asthma, healthy control subjects, and virus-infected nonasthmatic subjects. METHODS: Subject infection status and pulmonary history were established on the basis of common cold and asthma questionnaires, and lung function and atopy tests were performed. Respiratory virus infection was diagnosed by cell culture and direct polymerase chain reaction, using induced sputum. The induced sputum cellular profile was examined and cytokine gene expression was assessed by quantitative real-time polymerase chain reaction. RESULTS: A respiratory virus was detected in 78% of subjects with acute asthma. Specific viruses detected were rhinovirus (83%), influenza (15%), enterovirus (4%), and respiratory syncytial virus (2%). Virus-infected subjects with acute asthma or no asthma had increased RANTES (regulated on activation, normal T cell expressed and secreted) and macrophage inflammatory protein-1alpha messenger RNAs compared with other groups. Interleukin (IL)-10 mRNA was significantly increased in virus-infected acute asthma and reduced on recovery from acute asthma. IL-5, eotaxin, and IL-8 mRNA transcripts were similar across groups. CONCLUSIONS: Asthma exacerbation triggered by respiratory virus infection is characterized by increased IL-10 gene expression that may explain the suppressed eosinophil influx in acute asthma. Airway neutrophilia due to respiratory virus infection is associated with chemokine gene expression involving RANTES and macrophage inflammatory protein-1alpha.

Acute Disease↗

Absence of p16/MTS1 gene mutations in human prostate cancer.

The tumor suppressor gene p16/MTS1, located on chromosome 9p21, is a cell cycle regulatory gene which is frequently altered in human cancers. The role of this gene in prostate cancer is unknown. To determine the frequency of deletions and point mutations of p16/MTS1 in human prostate cancer, we examined 18 cancer and matched benign and hyperplastic tissue specimens. Deletions of p16/MTS1 were detected by semi-quantitative multiplex polymerase chain reaction in which a portion of exon 2 of the p16/MTS1 gene and a control marker, the glyceraldehyde 3-phosphate dehydrogenase gene, were amplified simultaneously. 'Cold' single-stranded conformational polymorphism (SSCP) analysis was performed to examine exons 1 and 2 of the p16/MTS1 gene for point mutations. Our data indicate no evidence for intragenic homozygous deletion in the prostate tumors. One prostate tumor and matched benign tissue showed mobility shifts. Direct DNA sequencing of the SSCP positive samples showed a G --> A transition in codon 140 which would result in an amino acid change from alanine to threonine. Our results indicate that deletions and point mutations in the p16/MTS1 gene are rare and do not play a major role in human prostate carcinogenesis.

Aged↗

Rhinovirus viremia in children with respiratory infections.

RATIONALE: Viremia has been implicated in many viral infections; however, viremia due to rhinovirus (RV; rhinoviremia) has been considered not to occur in normal individuals. OBJECTIVE: To evaluate whether RV enters the bloodstream and identify the possible risk factors. METHODS: Nasopharyngeal washes (NPWs) of 221 children with respiratory infections were examined for the presence of RV by reverse transcription-polymerase chain reaction. Blood from 88 children, whose NPW was RV-positive, and 31 of RV-negative control subjects was subsequently examined for the presence of RV in the blood by semi-nested reverse transcription-polymerase chain reaction. Rhinoviremia was then correlated with clinical characteristics of the disease. RESULTS: RV was detected in the blood of 10 out of 88 NPW RV-positive cases (11.4%): 7 of 28 children with asthma exacerbations (25.0%), 2 of 26 with common cold (7.7%), 1 of 25 with bronchiolitis (4.0%), and 0 of 9 with pneumonia (0%). All NPW RV-negative cases were negative in the blood. The proportion of rhinoviremia in children with asthma exacerbation was significantly higher compared with children suffering from the other diseases (25 vs. 5%, p = 0.01). Significant risk factors were: sampling <or= 24 hours from symptom initiation, personal history of asthma, and male sex. Age, fever, family, and personal history of atopy did not affect the presence of RV in the blood. CONCLUSIONS: Viremia may occur during RV respiratory infections in normal children and is rather common in the early course of acute asthma exacerbations, suggesting that rhinoviremia may be involved in asthma exacerbation pathogenesis.

Acute Disease↗

Evidence for increased proteolysis in intact beta thalassemia erythroid cells.

Much excess alpha chain is synthesized, but little accumulates in the erythroid cells of patients with homozygous beta thalassemia. To determine if the proteases known to exist in erythroid cells play a role in the destruction or alteration of any of this excess alpha chain, thalassemic and nonthalassemic erythroid cells were incubated for 90 minutes with 3H-leucine. The cells were then washed, and incubated twice for 15 minutes in 100 volumes of cold leucine-rich media, a procedure which eliminates almost all intracellular TCA soluble radioactivity. After these incubations levels of TCA soluble and TCA precipitable radioactivity in the cell lysates were determined, and the cells incubated for 120 minutes more in two volumes of leucine-rich media. At the end of this incubation, total TCA soluble and precipitable radioactivity was again determined in the cell lysate, and also in the two hour incubation media. The total increase in TCA soluble radioactivity in the cells and their media was divided by the 0 time TCA precipitable radioactivity, to determine the percent proteolysis labelled globin chains. In five control patients percent proteolysis ranged from 0 to 3.10 (mean = 1.50); in four severe and three mild thalassemia patients percent proteolysis ranged from 5.80 to 14.1 (mean = 11.0). The difference between the control and thalassemic groups was significant at a p of less than 0.001. This data is the first direct evidence that more proteolysis takes place in intact thalassemic cells than in non-thalassemic cells.

Adolescent↗

Kinetics of Ii synthesis, processing, and turnover in n-butyrate-treated Burkitt's lymphoma cell lines which express or do not express class II antigens and in hairy leukemic cells.

We have sought to understand the role of the electrophoretically invariant chain (Ii) in class II antigen functions, particularly in certain transformed cells in which we have previously demonstrated hyperexpression of Ii. Molecular structures and relative kinetics of Ii synthesis, processing and turnover were compared in paired, Ia+ and Ia- Burkitt's lymphoma (BL) cell lines and in hairy cell leukemia (HCL) cells. Cells were metabolically labeled with [35S] methionine for 15 min (with or without a cold methionine chase to 3 hr) or were continuously labeled for 3 hr. One- and two-dimensional gel electrophoresis resolved immunoprecipitates formed with a) a heteroantiserum to purified class II antigen (demonstrating alpha and beta chains and Ii associated with that complex), b) a heteroantiserum to hairy cell leukemia (HCL) membranes (demonstrating principally the dominant, basic form of Ii molecules, class I antigens, and some additional proteins), and c) a monoclonal antibody to human Ii. Treatment of Ia+ Jijoye and its daughter, Ia- P3HR-1, BL cells with 4 mM butyrate for 48 hr enhanced the synthesis of the dominant, basic form of Ii but did not affect apparent turnover rates of that pool of Ii chains in either cell line. In Ia+ Jijoye cells but not in Ia- P3HR-1 cells Ii was terminally processed to more acidic, sialic acid-derivatized forms. Butyrate treatment did not alter the relative turnover rate of terminally processed Ii in Jijoye cells. The level of the dominant, basic form of Ii in HCL cells equaled that in butyrate-treated Jijoye cells, and relative turnover rates of this terminally unprocessed Ii pool were similar in HCL and Jijoye cells. However, HCL Ia-associated Ii was not terminally processed, as was Ia-associated Ii in Jijoye cells. The expression of Ia auxiliary proteins, p41 and p25, was also enhanced in Jijoye cells by butyrate treatment and was prominent in HCL cells. From these experiments, we may hypothesize the following. In lymphoblastoid cells, two pathways for Ii turnover could exist. One is through association with Ia complexes and progressive terminal processing of carbohydrate side chains and a second is not associated with Ia or, apparently, with such processing. Because Ii is not found to be terminally processed in the absence of class II antigen, Ia might be considered to direct the processing of a subset of Ii towards some function (rather than vice versa).(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, Differentiation, B-Lymphocyte↗

The presence of DNA sequences of the Lucké herpesvirus in normal and neoplastic kidney tissue of Rana pipiens.

The Lucké tumour herpesvirus (LTV) is the aetiological agent of the Lucké renal adenocarcinoma of the northern leopard frog, Rana pipiens. LTV virions can be detected by electron microscopy in renal adenocarcinomata after prolonged exposure to low temperature. Tumours maintained at warm temperatures do not contain viral particles. To gain insight into the processes of viral infection, replication and oncogenesis, evidence was sought for the presence of LTV DNA in warm renal tumours and normal renal tissue. The polymerase chain reaction was used to amplify a Hind III restriction fragment of LTV DNA in tissue homogenates. LTV DNA was detected in a significant percentage of normal kidneys as well as in "virus-free" warm tumours and virus-containing cold tumours.

Adenocarcinoma↗

Adaptive changes in TEF-1 gene expression during cold acclimation in the medaka.

How animals adaptively respond to a cold or hot environment has been questioned for a long time. Recently, with the aid of microarray analysis, various temperature-sensitive genes have been identified in several species. However, a definitive hypothesis regarding the mechanism of adaptation has not been proposed. In the present study, we surveyed, in medaka (Oryzias latipes), genes for which the level of expression changes depending on the surrounding temperature. A messenger RNA differential display of medaka muscle total RNA revealed one such gene encoding transcription enhancer factor-1 (TEF-1). In medaka muscle, the TEF-1 gene produces two splicing variants, TEF-1A and TEF-1B mRNAs. During cold acclimation, the mRNA level of TEF-1A decreased, whereas that of TEF-1B increased. We also found that three putative downstream genes of TEF-1, two for myosin heavy chain (MyHC) and one for troponin T (TnT), a specific group of muscle proteins, were transcribed in a temperature-dependent manner. These results suggest that the transcription of MyHC and/or TnT is regulated by TEF-1 and that these molecules participate in muscle reconstruction during temperature adaptation in fish.

Acclimatization↗

Antarctic fish hemoglobins: evidence for adaptive evolution at subzero temperature.

Notothenioids represent a large group of marine teleosts that are mostly endemic to the Antarctic Ocean. In this environment, the low metabolic demand and the high oxygen concentration reduce the need for hemoglobin(s) [Hb(s)]. The extreme condition is represented by the icefish (Channichthyidae, Notothenioidei), the only vertebrates that lack Hb. We obtained the nucleotide sequence coding for the beta-globin chain of the single major Hb form in six red-blooded notothenioids. These included Gymnodraco acuticeps, one of the closest species to the Hb-less icefish, which is also the only known fish having a single Hb without Bohr effect. This species shows a higher rate of nonsynonymous substitutions (KA), in contrast with the homogeneity of synonymous substitution (KS) rates, and KA/KS ratios significantly greater than one in the majority of comparisons. These results are suggestive of positive selection, diversifying the single major Hb toward specialized functions. A single Hb that is free to diversify means that its role in routine oxygen transport can be reduced in the presence of a combination of physiological, ecological, and environmental factors. Although a reduced "routine" function for Hb, as is apparent in G. acuticeps, might, indeed, evoke the lack of Hb in icefish, evidence of diversifying selection reported here is at variance with the hypothesis of a simple trend from a single Hb toward the Hb-less condition.

Adaptation, Physiological↗

In situ demonstration of improvement of liver mitochondria function by melatonin after cold ischemia.

In a previous investigation, reperfusion with a melatonin-containing medium was demonstrated to enhance bile production and tissue ATP levels in rat livers, cold-preserved with University of Wisconsin (UW) or Celsior solutions, with respect to melatonin-free reperfusion; lipid peroxidation products in the perfusate were not influenced by the indole. This was ascribed to an increased efficiency of the hepatocyte mitochondria induced by melatonin. Reactive oxygen species (ROS) normally leak from the electron transfer chain in mitochondria and excessive ROS production is presumed to mediate ischemia-reperfusion (I/R) damage. A histochemical reaction was used to demonstrate ROS on the same model. Compared to the lobular zonation of ROS in control livers, the stained area of cold-preserved livers reperfused without melatonin was restricted to a narrow portal region, in keeping with the much lower ATP content. When reperfusion was performed with melatonin, the liver morphology was improved and the ROS reaction in hepatocytes more intense, though not reaching the control liver pattern. Sinusoidal cells were poorly-stained in both cases. In conclusion, with this different approach, melatonin was confirmed to improve mitochondrial performance and to discriminate parenchymal from sinusoidal cell behavior. Our observations confirm that melatonin mitigates I/R injury and support its potential in liver transplantation.

Adenosine↗

Cold-stable microtubules from Antarctic fishes contain unique alpha tubulins.

The cytoplasmic microtubules of Antarctic fishes assemble from their tubulin subunits at physiological body temperatures in the range -2 to +2 degrees C. Our objective is to determine the structural features that enhance the assembly of Antarctic fish tubulins at low temperatures. Here we compare the structures of tubulin subunits from three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus), from three temperate fishes (the dogfish shark Mustelus canis, the channel catfish Ictalurus punctatus, and the goosefish Lophius americanus), and from a mammal (the cow Bos taurus). When reduced, carboxymethylated, and examined by polyacrylamide gel electrophoresis, multiple alpha chains were observed in tubulins from the Antarctic fishes, the catfish, and the goosefish; dogfish and bovine alpha tubulins migrated as single components on this gel system. Prominent in the Antarctic fish tubulins was an alpha variant that migrated more rapidly than the bovine alpha chain; smaller amounts of a rapidly migrating alpha chain were also present in catfish and goosefish tubulins. The beta tubulins of the fishes, with the exception of the goosefish, resolved into major and minor variants with mobilities similar to those of beta 1 and beta 2 tubulins from bovine brain. Peptide mapping demonstrated that the alpha tubulins of Antarctic fishes were similar in structure, yet differed from the alpha chains of the dogfish and the cow (which, in turn, were similar to each other). In contrast, the beta tubulins from these organisms gave peptide patterns of near identity. Finally, the alpha chains of native tubulins from N. coriiceps neglecta and the cow differed in the sensitivity of their C-terminal domains to digestion by subtilisin. These results demonstrate that the alpha tubulins of Antarctic fishes (but not their beta chains) differ structurally from those of temperate fishes and a mammal.

Animals↗

Mouse models for disorders of mitochondrial fatty acid beta-oxidation.

Mitochondrial beta-oxidation of fatty acids is vital for energy production in periods of fasting and other metabolic stress. Human patients have been identified with inherited disorders of mitochondrial beta-oxidation of fatty acids with enzyme deficiencies identified at many of the steps in this pathway. Although these patients exhibit a range of disease processes, Reye-like illness (hypoketotic-hypoglycemia, hyperammonemia and fatty liver) and cardiomyopathy are common findings. There have been several mouse models developed to aid in the study of these disease conditions. The characterized mouse models include inherited deficiencies of very long-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, mitochondrial trifunctional protein-alpha, and medium-/short-chain hydroxyacyl-CoA dehydrogenase. Mouse mutants developed, but presently incompletely characterized as models, include carnitine palmitoyltransferase-1a and medium-chain acyl-CoA dehydrogenase deficiencies. In general, the mouse models of disorders of mitochondrial fatty acid beta-oxidation have shown clinical signs that include Reye-like syndrome and cardiomyopathy, and many are cold intolerant. It is expected that these mouse models will provide vital contributions in understanding the mechanisms of disease pathogenesis of fatty acid oxidation disorders and the development of appropriate treatments and supportive care.

Acyl-CoA Dehydrogenase↗

Inhibition of cold insolubility of an IgA cryoglobulin by decanedicarboxylic acid and related compounds.

Cold insolubility of a serum IgA cryoimmunoglobulin was found to be inhibited by the addition of 1.5 mM sodium decanedicarboxylate in vitro. The patient with the cryoglobulin had advanced multiple myeloma complicated by severe hyperviscosity that caused lethargy and episodic loss of consciousness. Decanedicarboxylic acid administered orally resulted in transient relief of symptoms and the loss of cryoprecipitability of the paraprotein. Further in vitro studies revealed that sodium salts of long-chain monocarboxylic acids with a minimum of eight carbons, and dicarboxylic acids with a minimum of 12 carbons inhibited cryoprecipitation. Salts of short-chain carboxylic acids, by contrast, enhanced cryoprecipitation. Sodium phenolate and sodium salts of benzoic acid, 2,4-DNP, phenylpropionic acid, and salicylic acid were also inhibitory. These latter compounds, which have a ring structure, did not cause precipitation at any concentration. It was demonstrated that the presence of a free carboxylic group was required for these activities; conversion of carboxylic acid to amide resulted in the loss of both the inhibitory and cryoprecipitation-enhancing effects. Normal plasma, or plasma from five other patients who had IgG, IgM, or mixed-type cryoglobulinemia, were not affected by any of these compounds. It is suggested that in selected cases of hyperviscosity syndrome associated with cryoglobulinemia, some of these compounds, especially monocarboxylic acids with appropriate chain lengths, or those with a ring structure, may have therapeutic applications.

Acetamides↗

Identification of genes regulated by changing salinity in the deep-sea bacterium Shewanella sp. WP3 using RNA arbitrarily primed PCR.

The differential gene transcription of a deep-sea bacterium Shewanella sp. WP3 in response to changing salinity was analyzed by RNA fingerprinting using arbitrarily primed PCR (RAP-PCR). Ninety primer sets were used to scan two different RNA pools derived from cultures of 1% and 7% NaCl concentrations. Forty-three putative differential-expressed fragments were identified, cloned, and sequenced. Six out of the 43 fragments were confirmed to be truly differentially transcribed in terms of changing salinity. The deduced amino acid sequences of the six gene fragments showed highest identities (66-96%) with ribosomal protein L24, ATP binding protein, and chaperon protein HscA of Shewanella oneidensis MR-1 (Y6, Y9, and Y29); isocitrate lyase of Pseudomonas aeruginosa (Y15); peptidylprolyl cis-trans isomerase of Shewanella sp. SIB1 (Y21), glutamine synthetase of Shewanella violacea (Y25), respectively. Four genes (Y6, Y15, Y21, and Y25) were up regulated in 7% NaCl, while the other two (Y9 and Y29) contained more abundant transcripts in 1% NaCl. The data suggested that strategies involved in controlling protein synthesis, protein folding and/or trafficking, glutamate concentration, fatty acid metabolism, and substance transporting were used for salt adaptation in Shewanella sp. WP3. The expression patterns of the six genes in response to transient stress shocks including salt shock (3% NaCl shift to 12%), cold shock (15 degrees C shift to 0 degrees C), and high-hydrostatic pressure shock (0.1 MPa shift to 50 MPa) were further examined. Y29 encoding the putative HscA chaperon protein was indicated to be involved in adaptation of all the stresses tested.

Cold Temperature↗