Structure and genomic sequence of the myotonic dystrophy (DM kinase) gene

The mutation causing myotonic dystrophy (DM) has recently beenidentified as an unstable CTG trinucleotide repeat located inthe 3' untranslated region of a gene encoding for a proteinwith putative serine-threonine protein kinase activity. In thisreport we present the genomic sequences of the human and murineDM kinase gene. A comparison of these sequences with each otherand with known cDNA sequences from both species, led us to predicta translation initiation codon, as well as determine the organizationof the DM kinase gene. Several polymorphisms within the humanDM kinase gene have been identified, and PCR assays to detecttwo of these are described. The complete sequence and characterizationof the structure of the DM kinase gene, as well as the identificationof novel polymorphisms within the gene, represent an importantstep in a further understanding of the genetics of myotonicdystrophy and the molecular biology of the gene.     

Influence of deposition parameters on chemical properties of calcium phosphate coatings prepared by using electrostatic spray deposition

The electrostatic spray deposition (ESD) technique offers the possibility of depositing calcium phosphate (CaP) coatings onto various substrate materials with defined chemical and morphological properties. The relationship between physical, apparatus-related deposition parameters, and the chemical characteristics of ESD coatings was investigated by means of X-ray diffraction, Fourier transform infrared spectroscopy, and energy dispersive spectroscopy to be able to deposit CaP coatings with tailored chemical properties. The results showed that the chemical characteristics of CaP coatings, deposited with use of the ESD technique, were strongly dependent on the deposition temperature, the nozzle-to-substrate distance, the liquid flow rate, and the geometry of the spraying nozzle. By investigating the influence of the deposition temperature, information could be obtained on the formation mechanism of CaP coatings-and specifically the biologically interesting carbonate apatite phase-using the ESD technique. CaP coatings were not formed merely because of solvent evaporation; a chemical reaction was needed to synthesize the coatings. This reaction involved thermal decomposition of the organic solvent butyl carbitol into carbonate ions via formation of intermediate oxalate ions. The amount of carbonate incorporation, and consequently, the Ca/P ratios of the deposited coatings, was shown 1) to decrease with increasing nozzle-to-substrate distance, 2) to decrease with increasing liquid flow rate, and 3) to decrease by making use of a novel two-component nozzle geometry.     

In vivo magnetic resonance imaging explorative study of ectopic bone formation in the rat

In animal studies of tissue engineering of bone, histology remains the standard for assessing bone formation. As longitudinal studies with this method are feasible only at the cost of large numbers of animals, we looked for an alternative. Therefore, demineralized bone matrix (DBM) and inactivated demineralized bone matrix (iDBM) implants were subcutaneously implanted in a rat. At 1, 3, 5, and 7 weeks postimplantation soft X-ray and magnetic resonance imaging (MRI) were done to monitor bone formation in the implants. At 7 weeks, the animal was killed and the implants were retrieved for histology. Our results showed that in vivo MRI is well suited to assess bone formation larger than 0.5 mm in diameter and to monitor the complete three-dimensional shape of the newly formed bone noninvasively and longitudinally. The MRI results matched well with the histology results obtained at 7 weeks. In contrast, X-ray imaging appeared inappropriate to monitor the bone formation process in DBM.     

Autoradiographic localisation of NMDA, quisqualate and kainic acid receptors in human spinal cord.

The phencyclidine (PCP) binding site of the N-methyl-D-aspartate receptor, the kainic acid (KA) receptor and the quisqualate (QA) receptor were visualised, using autoradiography in the human spinal cord and the distributions compared with that of benzodiazepine (BDZ) receptors and substance P (SP). All of the receptor types, and SP, were concentrated in lamina II of the dorsal horn, consistent with physiological data indicating that glutamate is a neurotransmitter of primary afferent terminals in the spinal cord.     

Effect of hypertriglyceridemia on endotoxin responsiveness in humans.

Triglyceride-rich lipoproteins can inhibit endotoxin activity in vitro and in rodents. We sought to determine whether Intralipid, a triglyceride-rich fat emulsion which in contact with plasma functions similarly to endogenous lipoproteins, can alter the human response to endotoxin. Intralipid inhibited endotoxin-induced cytokine production in human whole blood in vitro in a dose-dependent manner, with maximal inhibition (up to 70%) being achieved at a concentration of 10 g/liter. In healthy men, a bolus intravenous injection of endotoxin (lot EC-5; 20 U/kg of body weight) was given midway through a 4-h infusion (125 ml/h) of either 5% glucose (n = 5) or 20% Intralipid (n = 5). The infusion of Intralipid led to an increase in triglyceride levels in serum from 95 +/- 16 to 818 +/- 135 mg/dl prior to endotoxin administration, i.e., levels that importantly reduced cytokine production in endotoxin-stimulated whole blood. However, in vivo hypertriglyceridemia did not influence inflammatory responses to endotoxin (fever, release of tumor necrosis factor and soluble tumor necrosis factor receptors, and leukocytosis) or even potentiated endotoxin responses (release of interleukins 6 and 8 and neutrophil degranulation). Hypertriglyceridemia does not inhibit the in vivo responses to endotoxin in humans.     

Long-term reduction of vasopressin excretion induced by the central injection of an immunoconjugate (antibody to vasopressin linked to ricin a chain)

We have previously demonstrated that vasopressin-producing neurons are the target of monoclonal antibodies to vasopressin microinjected into the brain tissue. At the same time, this central microinjection of vasopressin-monoclonal antibody into the supraoptic nuclei produced hydro-osmotic disorders mimicking the effects of a central diabetes insipidus. In order to investigate the increase in both duration and amplitude of the biological effects seen after the injection of vasopressin-monoclonal antibody, an immunoconjugate was constructed with the vasopressin-monoclonal antibody IgG1k isotype and the cytotoxic part of the ricin molecule, the ricin A chain. The biological parameters, such as diuresis and urine osmolality which are directly regulated by vasopressin, and vasopressin excretion, were measured after the central injection of this immunotoxin/immunoconjugate. The consequences of immunotoxin injection were also studied when immunotoxin was co-injected with monensin (50 nM) which has been shown to decrease the intracellular degradation of immunotoxin, and plasma complement, which has been shown to increase the neuronal uptake of immunotoxin. Single injection of immunotoxin near the hypothalamic supraoptic nuclei significantly increased diuresis and decreased vasopressin excretion. However, these effects were only transient and disappeared 24 h later. Four successive injections of immunotoxin (one per day) with monensin induced a decrease of vasopressin excretion which was still observed after a resting period of four days after the fourth injection. The long-term reduction of vasopressin excretion was induced in rats receiving four successive injections of a mixture consisting of immunotoxin with monensin and plasma complement. In such experiments, the vasopressin content of urine remained low (55% under the baseline value), two weeks after the fourth injection of immunotoxin. At the same time, the diuresis was increased (80% above the baseline value) and urine osmolality lowered (45% under the baseline value). When non-specific IgG replaced specific antibody, vasopressin excretion, diuresis as well as urine osmolality were unchanged.

The results of this study demonstrated that the use of a specific immunotoxin results in a local interference with the vasopressinergic neurons and induces a long-term reduction of vasopressin secretion.     

Functional and phenotypical studies of the Leu-4 (CD3)+, Leu-1 (CD5)- T lymphocyte.

A small T cell subpopulation expressing the phenotype Leu-5(CD2)+, Leu-4(CD3)+, Leu-1 (CD5)- can be found in peripheral blood and bone marrow of normal individuals. When these cells were sorted out by three colour immunofluorescence cell sorting and tested in limiting dilution assays, they were found to have lower frequencies of proliferating (9.0 +/- 5.6 times, n = 7) and of IL-2 producing cells (11.5 +/- 5.0 times n = 5), and a higher frequency of cytotoxic cells (3.1 +/- 2.6 times, n = 2) than T lymphocytes expressing the three markers. In peripheral blood lymphocytes, 1/3 of the CD3+, CD5- cells were positive for Leu-2a (CD8) while virtually all were negative for Leu-3a (CD4). Four colour flow cytometric analysis revealed a small subset of T cells positive for CD3 and negative for CD5, CD4 and CD8. Approximately 75% of the CD3+, CD5- cells were negative for Leu-7 and CD16 simultaneously. These results shed a light on the phenotype of T cells that escape killing by CD5 and complement in T cell depleted bone marrow and may explain why fewer residual T cells in the depleted marrow are detected by limiting dilution assays than by flow cytometric analysis.     

Surveillance of community-acquired viral infections due to respiratory viruses in Rhone-Alpes (France) during winter 1994 to 1995.

Nasal swab from patients with acute flu-like illness were evaluated for the presence of respiratory viruses in the Rhone-Alpes region of France from 1 October 1994 through 2 May 1995. The relative frequencies and seasonal distributions of the specific viruses were assessed. In addition, virus type was correlated with specific clinical signs and symptoms. During the study, 962 samples were collected by 75 medical practitioners participating in the Groupe Regional d'Observation de la Grippe surveillance network. One or more viruses were detected from 348 samples (36.1%), including 108 respiratory syncytial virus (RSV), 64 influenza virus A type H3N2, 47 influenza virus B, 64 coronavirus, 35 rhinovirus, 22 adenovirus, 5 enterovirus, and 3 parainfluenza-fluenza strains. There were 16 mixed infections. RSV infections peaked in the early winter, and influenza viruses A and B infections peaked during the late winter and early spring. There were two peaks of coronavirus infections (late fall and late winter). Other viruses were detected at lower levels throughout the study period. Patients from whom adenovirus was isolated were significantly more likely to have a fever of > 39.5 degrees C than were patients with other detectable viruses (P < 0.001). Furthermore, there was a significant correlation between influenza and cough (P < 0.01) and RSV and bronchiolitis (P < .001). Thus, the current study defined the overall and relative frequencies of respiratory virus detection from nasal swab specimens in patients with an acute flu-like illness in the Rhone-Alpes region of France during a 7-month period. Correlation with clinical signs and symptoms and provisional conclusions regarding seasonality were also determined.