Quantitative in situ hybridization of 3H-labeled complementary deoxyribonucleic acid (cDNA) to the messenger ribonucleic acid of thyroglobulin in human thyroid tissues.

Thyroglobulin (Tgb) mRNA content was studied in human thyroid tissues using liquid hybridization and in situ hybridization. Liquid hybridization revealed no differences in mRNA content, except in the case of colloid adenoma in which a lower amount of Tgb mRNA was found. Conditions for quantitative in situ hybridization of [3H]DNA complementary to the mRNA of Tgb are described. In situ hybridization allowed correlation of the morpho-functional state of the follicles and their content of Tgb mRNA.

Production of rat Spot14 protein in Escherichia coli.

Hormonal, nutritional and developmental factors modulate, in rat lipogenic tissues, the transcription of the mRNA coding for a protein of unknown function, called Spot14 (S14). The corresponding protein has never been purified from tissues. In this paper, we describe the production of S14 in Escherichia coli. In the absence of available antibodies (Ab) directed against S14 protein, our strategy was to produce this protein by constructing two different recombinant expression vectors. The first recombinant plasmid produced a S14::protein A fusion which was easily purified and then rabbit Ab could be raised against it. The second expression vector directly produced S14. This expression was demonstrated by specific binding of polyclonal Ab directed against the fusion protein. These Ab also recognized a rat-liver protein sharing characteristics of S14.

[Amyotrophy and neuronal depopulation of cerebral origin]. / Amyotrophies et dépopulations neuronales d'origine encéphalique

Amyotrophy of cerebral origin was analyzed in 12 hemiplegics by means of comparative histological, histoenzymological, histographic and biochemical analysis of biopsies carried out in symmetrical zones. In the 6 cases in which atrophy predominated on the paralyzed side, it occurred early, the deficiency affecting the upper limb in particular and the sensory disturbance being irregular. Attempted numeration of the motor units remaining, according to the "incremential" method of stimulation, suggests a numerical reduction on the hemipelgic side. Despite reservations and general criticisms of this method, its comparative value, although approximate, must be acknowledged. However, although amyotrophy presumes a transsynaptic change in trophic function to have taken place in the peripheral neurone, neuronal depopulation--if one accepts it--cannot be other than functional.

[Identifying hepatitis C virus by the gene amplification technique in the saliva of patients for whom the search is simultaneously negative in their serum: 9 cases]. / Découverte du virus d'hépatite C par technique d'amplification génomique dans la salive de patients pour qui la recherche est simultanément négative dans leur sérum: 9 cas.

RNA-PCR for Hepatitis C virus was positive in saliva sample of 9 patients, while it was negative in their serum. Clinical and biochemical results of these patients are presented. Saliva detection of HCV is useful for diagnostic of hepatitis, sicca syndrome.

[Micro-determination of hemoglobin by measuring the peroxidase activity]. / Microdosage de l'hémoglobine par mesure de l'activité peroxydasique

A simple and rapid method is described for the determination of serum hemoglobin. Free hemoglobin shows no peroxidase activity but human hemoglobin-human haptoglobin complex presents a peroxidase activity which permits hemoglobin determination. It is shown that a good source of haptoglobin is constituted by pools of non-hemolyzed human serums.

Oligonucleotide screening of beta thalassemia mutations in the south east of France.

France is a non-endemic region for beta thalassemia. In this country, the sporadic cases of Cooley's disease encountered affect almost constantly subjects of Mediterranean origin. In this report, we have screened, using oligonucleotide probes, the distribution of the main beta thalassemia mutations present in the population of South-eastern France whose origins lie in the mixing of several Mediterranean ethnic groups. Among 105 beta thalassemia chromosomes, we have observed a limited number of alleles, since, by using oligonucleotide probes for six mutations, we have characterized the molecular defect in 90% of the chromosomes. The four main mutations were found in more than 85% of the chromosomes and the others in about 5%. The distribution of the beta thalassemia mutations within the various ethnic groups was determined.

Presence of the NHE3 isoform of the Na+/H+ exchanger in human gallbladder.

1. In man and in various animal species, absorption of NaCl from bile by the gallbladder mucosa is associated with luminal proton secretion. A similar absorption of NaCl in small intestine, colon and renal tubule is related, at least in part, to the presence of the NHE3 isoform of the Na+/H+ exchanger. This work was designed to find out whether NHE3 is also present in human gallbladder. 2. At surgery, 100-200 mg of the gallbladder wall was obtained from patients treated by cholecystectomy for gallstones. After isolation of the mucosa, total RNA was extracted and submitted to reverse transcription-polymerase chain reaction with two primers: 5'-AAGCCICTGGTGCAGTGGCTGAAGG-3' and 5'-GGAGTCCTTIAAGTCGGCIAAGCTGGGC-3', designed to amplify a sequence of 645 bp of rabbit NHE3 mRNA (642 bp in man). RNA from human liver and from rabbit heart, neither of which contain NHE3, and human ileal RNA, which does contain NHE3, were used as controls. 3. RNA extracted from the mucosal moiety of the gallbladder wall gave an amplification product of about 645 nucleotides. Controls gave the expected negative or positive results. Sequencing of the amplified RNA showed it was almost identical to previously determined sequences of NHE3 in other human tissues. 4. It is concluded that the mucosa of human gallbladder contains the mRNA of NHE3 isoform. This isoform could therefore play a role in sodium absorption from bile.

Role of the NHE3 isoform of the Na+/H+ exchanger in sodium absorption by the rabbit gallbladder.

The absorption of water and electrolytes by the gallbladder seems to be largely dependent upon a Na+/H+ exchange at the apical membrane of the gallbladder epithelium. To find out if the exchanger involved is the NHE3 isoform, as in other absorbing epithelia, two studies were performed using the rabbit gallbladder. First, we studied 22Na absorption in Ussing chambers with Krebs buffer as a control solution, and in the presence of amiloride (100, 200 or 1000 microM), ethyl-isopropyl-amiloride (EIPA, 1 or 5 microM), or the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 1 microM). A net mucosal-to-serosal Na+ flux was observed with control buffer. No inhibition of this net flux was observed with 5 microM EIPA, and the IC50 for amiloride was found to be 200 microM. PMA induced a reduction of absorption by 30% that was prevented by incubation with calphostin C. Resistance to amiloride and EIPA, and inhibition by PMA are consistent with the involvement of the NHE3 isoform. The second study involved reverse-transcriptase polymerase chain reaction (RT-PCR) of total gallbladder RNA, with two primers designed to amplify a 645-base-pair fragment from NHE3 mRNA. A cDNA fragment of the expected size was actually obtained from gallbladder RNA, while RT-PCR of RNA from the liver, which does not contain NHE3, gave negative results. A sequence of 492 nucleotides of the amplified product was determined, which was almost superimposable onto the known sequence of the corresponding fragment of rabbit NHE3. It is concluded that, in rabbit gallbladder, neutral NaCl absorption is, at least in part, dependent on the NHE3 isoform of the Na+/H+ exchanger.

[Plasma lipid fractions in insulin-dependent diabetic patients. Effects of short-term control of glycaemia (author's transl)]. / Fractions lipidiques plasmatiques du diabétique insulino dépendant. Effet du contrôle glycémique à court terme.

In order to elicitate a possible influence of short-term control of glycaemia on circulating plasma lipid fractions, the authors have endeavoured to find out: (a) whether there was a correlation between these fractions and glycosyl-haemoglobin (Hb A1) which indicates previous glycaemic balance, and (b) whether the various lipid fractions were modified by absolute control of glycaemia during a 24-hour application of artificial pancreas. They found that HbA1 correlated positively with total cholesterol and VLDL + LDL cholesterol, but not with HDL cholesterol. After 24 hours on artificial pancreas there was a significant decrease in total blood cholesterol without changes in blood triglycerides. The decrease was homogenous and concerned cholesterol concentrations in both low and high density lipoproteins. The authors conclude that the increased risk of atherosclerosis in insulin-dependent patients is in-related to a decrease in HDL cholesterol.

Cloning of four DNA fragments complementary to human thyroglobulin messenger RNA.

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA . mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.

Quantification of thyroglobulin messenger RNA by in situ hybridization in differentiated thyroid cancers. Difference between well-differentiated and moderately differentiated histologic types.

Thyroglobulin messenger RNA (mRNA) was located and quantified in tissue sections of differentiated human thyroid cancers by in situ hybridization using cloned complementary DNA probes. The cells of the well-differentiated follicular and papillary forms contained similar levels of thyroglobulin mRNA, corresponding to about 2000 copies per cell. In contrast, cells of moderately differentiated thyroid cancers contained about two to three times less thyroglobulin mRNA. It was also found that thyroglobulin mRNA was present almost exclusively in polyribosomes under the form of heavy polyribosomes actively synthesizing thyroglobulin. It is suggested that in situ hybridization method allows localization of specific mRNA in differentiated thyroid cancers and correlation with the level of differentiation of the cells.

Integrity of the NS5A (amino acid 2209 to 2248) region in hepatitis C virus 1b patients non-responsive to interferon therapy.

BACKGROUND/AIMS: In hepatitis C virus-1b, it has been suggested that an amino acid stretch (aa 2209-2248) of the carboxy terminal half of the non-structural 5A (NS5A) region participates in the response to interferon treatment. We tested the hypothesis that absence of mutations in the NS5A (aa 2209-2248) sequence is required for interferon resistance. We also investigated the importance of different HCV-1b isolates in interferon response in France. METHODS: We determined the NS5A sequences of 70 patients with chronic hepatitis C before IFN therapy and then compared them with HCV-J prototype sequence. The isolates were determined by NS5B sequencing, the "gold standard" method for genotyping and subtyping. Pre-therapeutic viral load was also measured. RESULTS: No sustained virological response was observed in the patients without amino acid substitutions in the NS5A (aa 2209-2248) sequence, and in the patients with HCV-J isolates. Viral load was significantly higher in the patients with no amino acid substitutions in the NS5A (aa 2209-2248) sequence. CONCLUSIONS: In HCV-lb infected patients, an HCV-J strain with no amino acid substitution in the NS5A (aa 2209 2248) region indicates a poor prognosis for response to IFN therapy. The low interferon response rate in HCV-lb infection in Europe is probably not due to a difference between isolates.

Persistent hepatitis C virus RNA replication in haemophiliacs: role of co-infection with human immunodeficiency virus.

In order to evaluate the evolution of transfusional hepatitis C in haemophiliacs, we performed a retrospective study of ALT levels and HCV viraemia with a RNA PCR assay in 57 patients. We found that the vast majority of HCV-infected patients remained viraemic (43/57 = 75%) and higher ALT levels correlated with HCV viraemia. Although indicators of the transfusional viral load (age, severity of haemophilia) and HBV co-infection did not correlate with HCV RNA replication, HIV seropositivity was strongly associated with persistence of HCV viraemia (23/25 = 92% in HIV-positive versus 20/32 = 62% in HIV-negative patients), without any correlation with CD4 counts. Genotyping of HCV in the 43 viraemic patients shows more frequent genotype 1 in the HIV-seropositive group (14/23) than in the seronegative group (6/20). Our data emphasize that besides the role of the immunodeficiency status, the genotypes of HCV might be involved in the differences observed in terms of HCV RNA replication between the HIV-seropositive and seronegative haemophiliacs.

Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA.

BACKGROUND/AIMS: Both HCV RNA viral load and HCV genotype have been described as important predicting factors determining the response to interferon in chronic hepatitis C. To investigate whether processing and storage conditions might influence the stability and could alter the concentration of the HCV RNA in serum, quantification of HCV RNA was performed by branched DNA assay. METHODS: We studied serum samples obtained from seven patients with histologically proven chronic hepatitis C. These were subjected to the following physical conditions: (1) immediate quantification, (2) storage at room temperature for 5 days, (3) storage at 4 degrees C for 5 days, (4) storage at -20 degrees C for 5 days, (5) storage at -80 degrees C for 5 days, (6) five freeze-thaw cycles, (7) blood unspun for 4 h at room temperature then centrifuged and stored at -80 degrees C for 5 days, (8) storage at 4 degrees C for 6 months, (9) storage at -20 degrees C for 6 months, (10) storage at -80 degrees C for 6 months. RESULTS: A loss of 100% HCV RNA titers was observed after storage at RT for 5 days and then storage at 4 degrees C for 6 months. A surprising decrease of HCV RNA titer (15.6%) was observed in sera stored for 5 days at -20 degrees C. Five freeze-thaw cycles resulted in a 16% decrease of the HCV RNA level. When centrifugation was performed after a 4 h delay at room temperature, a significant loss of HCV RNA titers of 29.5% was observed. Long-term stability (6 months) was observed at -80 degrees C with a slight loss of about of 10% HCV RNA titers, but a significant decrease in HCV RNA of 23% was observed at -20 degrees C. The reproducibility of the bDNA assay on five patient samples was performed eight times in duplicate and showed an average coefficient of variation of 9.1%. CONCLUSIONS: These data confirm the importance of storage and handling in measuring the amount of HCV RNA in clinical samples.

Mother-to-infant transmission of hepatitis C virus: molecular evidence of superinfection by homologous virus in children.

BACKGROUND/AIM: Vertical transmission of hepatitis C virus (HCV) is well established but its incidence is low. To assess the molecular evidence of mother-to-infant transmission or intrafamilial transmission of HCV, the NS5 B region and the hypervariable region 1 (HVR1) of the E2/NS1 region of the HCV genome from each member of a family were investigated. METHODS: A 35-year-old mother with chronic hepatitis C virus infection and her four infected boys were studied. The same HCV 1a genotype was found in all five. Phylogenetic analysis was done by the neighbor-joining, the maximum likelihood, and the maximum parsimony methods. RESULTS: Comparison of the phylogenetic trees in the NS5B and HVR1 regions showed that the sequences in the children were more closely related to the population of variants of their own mother than to any genotype la sequence available in the databases. However, four HVR1 clones from two brothers (E2 and E3) had a strong homology, but were significantly divergent from the variants of the mother. CONCLUSIONS: These results suggest that a cluster of HCV strains exists in the family and that E3 could have been superinfected by E2 HCV strains and reciprocally. In conclusion, phylogenetic analysis through variable regions of the genome suggests that at least two modes of transmission are involved in this family: perinatal and horizontal.