Complete genomic sequence of a dengue type 2 virus from the French West Indies.

Severe forms of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome, were not prominent in the Americas until the epidemic of Cuba in 1981. Since that time, they have spread to other countries in Central and South America, correlating with the spread of dengue type 2 viruses related to Southeast Asian strains. We report here the complete genomic sequence of a dengue type 2 virus isolated during the epidemic in La Martinique in 1998. This constitutes the first complete genetic characterization of a dengue virus strain from French West Indies, and also the first molecular identification in this region of a dengue 2 strain phylogenetically related to the emerging American type 2 dengue viruses.

Complete nucleotide sequence and phylogeny of an American strain of yellow fever virus, TRINID79A.

Yellow fever was presumably imported to the Americas from West-Africa from the 16th to the 19th century. American and African genotypes of the virus are distinguishable, indicating separate evolution in different vector/host cycles. The complete nucleotide sequence of the yellow fever virus strain TRINID79A, isolated in Trinidad in 1979, has been established. It exhibits extensive homology with those of current West-African strains and attenuated strain FNV. However, a unique deletion of the 3' non-coding region (NCR) of the viral RNA has been identified. It indicates that RYF1 and RYF2 repeated sequences of the 3' NCR are not necessary to the replication of the virus.

[Molecular epidemiology of yellow fever]. / Epidémiologie moléculaire de la fièvre jaune.

Although the causative agent has long been identified and a safe, effective vaccine is available, yellow fever still poses a threat in South America and tropical areas of Africa where it mainly affects young people. The presentation of yellow fever has changed relatively little since description of the first outbreaks, but study has entered a new age thanks to development of more accurate techniques for characterization of viral strains. Comparison of the RNA sequences of virus from different geographical regions has demonstrated the existence of several stable genotypes which have been designated as topotypes. These topotypes have yet to be correlated with different viral activity but their recognition has allowed better epidemiologic surveillance of the disease. Study of the viral genome has also allowed improvement of diagnostic techniques, identification of factors influencing virulence, and enhancement of understanding of viral mutations. Ultimately knowledge of the processes underlying viral evolution could assist development of effective preventive strategies.

High-resolution solution structure of the inhibitor-free catalytic fragment of human fibroblast collagenase determined by multidimensional NMR.

The high-resolution solution structure of the inhibitor-free catalytic fragment of human fibroblast collagenase (MMP-1), a protein of 18.7 kDa, which is a member of the matrix metalloproteinase family, has been determined using three-dimensional heteronuclear NMR spectroscopy. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 3333 experimental NMR restraints, consisting of 2409 approximate interproton distance restraints, 84 distance restraints for 42 backbone hydrogen bonds, 426 torsion angle restraints, 125 3JNH alpha restraints, 153 C alpha restraints, and 136 C beta restraints. The atomic rms distribution about the mean coordinate positions for the 30 structures for residues 7-137 and 145-163 is 0.42 +/- 0.04 A for the backbone atoms, 0.80 +/- 0.04 A for all atoms, and 0.50 +/- 0.03 A for all atoms excluding disordered side chains. The overall structure of MMP-1 is composed of a beta-sheet consisting of five beta-strands in a mixed parallel and anti-parallel arrangement and three alpha-helices. A best-fit superposition of the NMR structure of inhibitor-free MMP-1 with the 1.56 A resolution X-ray structure by Spurlino et al. [Spurlino, J. C., Smallwood, A. M., Carlton, D. D., Banks, T. M., Vavra, K. J., Johnson, J. S., Cook, E. R., Falvo, J., and Wahl, R. C., et al. (1994) Proteins: Struct., Funct., Genet. 19, 98-109] complexed with a hydroxamate inhibitor yields a backbone atomic rms difference of 1.22 A. The majority of differences between the NMR and X-ray structure occur in the vicinity of the active site for MMP-1. This includes an increase in mobility for residues 138-144 and a displacement for the Ca(2+)-loop (residues 74-80). Distinct differences were observed for side-chain torsion angles, in particular, the chi 1 for N80 is -60 degrees in the NMR structure compared to 180 degrees in the X-ray. This results in the side chain of N80 occupying and partially blocking access to the active site of MMP-1.

Assignments, secondary structure and dynamics of the inhibitor-free catalytic fragment of human fibroblast collagenase.

Fibroblast collagenase (MMP-1), a 169-residue protein with a molecular mass of 18.7 kDa, is a matrix metalloproteinase which has been associated with pathologies such as arthritis and cancer. The assignments of the 1H, 15N, 13CO and 13C resonances, determination of the secondary structure and analysis of 15N relaxation data of the inhibitor-free catalytic fragment of recombinant human fibroblast collagenase (MMP-1) are presented. It is shown that MMP-1 is composed of a beta-sheet consisting of five beta-strands in a mixed parallel and antiparallel arrangement (residues 13-19, 48-53, 59-65, 82-85 and 94-99) and three alpha-helices (residues 27-43, 112-124 and 150-160). This is nearly identical to the secondary structure determined from the refined X-ray crystal structures of inhibited MMP-1. The major difference observed between the NMR solution structure of inhibitor-free MMP-1 and the X-ray structures of inhibited MMP-1 is the dynamics of the active site. The 2D 15N-1H HSQC spectra, the lack of information in the 15N-edited NOESY spectra, and the generalized order parameters (S2) determined from 15N T1, T2 and NOE data suggest a slow conformational exchange for residues comprising the active site (helix B, zinc ligated histidines and the nearby loop region) and a high mobility for residues Pro138-Gly144 in the vicinity of the active site for inhibitor-free collagenase. In contrast to the X-ray structures, only the slow conformational exchange is lost in the presence of an inhibitor.

Homogeneity of yellow fever virus strains isolated during an epidemic and a post-epidemic period in West Africa.

Three strains of yellow fever virus (YFV) were isolated in 1982 in The Ivory Coast, one from a human case and two from Aedes luteocephalus, during and subsequent to an epidemic. The complete genomic sequence of the human strain was determined and compared to that of the 1927 Asibi strain of YFV. The divergence observed was on average of 8.3%, ranging from 5.5 to 11.7% in the coding region. The transitions to transversions ratio was 5.9. Most mutations (84.3%) occurred on the third position of the codons, with synonymous mutations representing 92.5%. However, when partial sequences representing 60% of each genome were compared, homology between the three Ivory Coast strains was greater than 99%. These results demonstrate the homogeneity of the virus strains circulating in different hosts and vectors in a limited geographical region and validate the concept of topotype in viral quasi-species.

[Current status of dengue]. / Actualité de la dengue.

Dengue has become a major public health problem in intertropical areas where an estimated 60 million new cases and 30,000 deaths occur annually. The causative agent is transmitted by the bite of Aedes mosquitoes which are the main and perhaps only reservoir of the disease. In addition to increasing incidence clinical manifestations of dengue have changed over the last 40 years. An increasing number of reported cases involve hemorrhage, shock, and other severe complications especially in Southeast Asia, northern regions of South America, and the Caribbean. Some of the same factors responsible for these changes are probably implicated in the development of other emerging viral diseases.

Therapy of migraine by modulating dopamine hypersensitivity: its effect on mood and pain.

Clinical and pharmacological evidences support the hypothesis of a hypersensitivity of dopamine (DA) receptors in migraine patients. The aim of our single-blind and placebo-controlled study is to evaluate the presence of this hypersensitivity and to desensitize the DA system with apomorphine, a classical DA receptor agonist. We studied six patients suffering from migraine without aura resistant to the prophylactic treatment and with high frequency of attacks. In these patients, we first tested individual sensitivity by a single i.m. low dose of apomorphine to assess the lowest effective dose to be administered s.c. in continuous infusion without side-effects. We used the response of growth hormone (GH) to apomorphine as a marker of DA2-receptor function. Clinical evaluation, blood pressure and heart rate were recorded before placebo or apomorphine administration and monitored every 15 min for 1 h after each injection. The treatment consisted of the continuous subcutaneous infusion of apomorphine for three weeks at the dosage previously assessed by the test. No difference in blood pressure and heart rate was found during test and treatment. The treatment was effective in reducing the frequency and the severity of migraine attacks.

Studies on the blocking action of human Kv3.4 inactivation peptide variants in the mouse cloned Kv1.1 K+ channel.

1. Whole-cell patch clamp recordings were made from Chinese hamster ovary (CHO) cells stably expressing homomeric mouse Kv1.1 (delayed rectifier K+; mKv1.1) channels. The effects of internal application of a number of different peptides, based on part of the amino terminal sequence of the human Kv3.4 channel subunit (hKv3.4), were examined in order to determine their influence on N-type inactivation. 2. For the native hKv3.4 peptide, the association rate constant (kon) increased with membrane depolarization, whilst the dissociation rate constant (koff) had little dependence on voltage. This resulted in the apparent dissociation constant (KD) of the hKv3.4 peptide tending to increase with depolarization. 3. In general, kon increased and apparent KD decreased with positive charge of the hKv3.4 peptide variants; in peptides lacking a hydrophobic amino terminal this correlation was not maintained. In contrast, the rate of dissociation of the variant peptides (koff) was independent of net charge. 4. The blocking activity of the hKv3.4 peptide was not dependent on a disulphide bridge between cysteine residues C6 and C24 and the presence of cysteine residues in the hKv3.4 peptide was not a prerequisite for rapid inactivation. All cysteine-substituted variants, especially at C6, showed a more rapid recovery from inactivation than the hKv3.4 peptide. Substitutions at C24, and not C6, reduced kon. 5. The present results concerning the action of the mammalian hKv3.4 channel inactivation particle on mKv1.1 channels complement earlier models for the invertebrate Shaker K+ channel. It is proposed that the hydrophobic amino terminal region of the hKv3.4 inactivation peptide blocks the channel pore, whilst the adjacent positively charged region interacts with negative charges on the channel protein.

Partial genomic sequence determination of yellow fever virus strain associated with a recent epidemic in Gabon.

A limited epidemic, with important mortality among the initial human cases, occured in a forest region in the north of Gabon by the end of 1994. It was identified as yellow fever according to first serological and reverse transcription/polymerase chain reaction (RT-PCR) results, but no virus was isolated. We received 37 sera from people who lived in the same region and presented symptoms of the disease during the period of concern. In ten of them, we were able to detect and identify yellow fever virus (YFV) RNA by RT-PCR and endonuclease digestion of the RT-PCR products. Nucleotide (nt) sequence of two regions (242 and 161 nt long) of YFV RNA was determined for three sera. As it differed from that of the Asibi strain of YFV, the presence of a new topotype (strain) of YFV is presumed.

Dopamine hypersensitivity in migraine: role in apomorphine syncope.

There is some evidence supporting a potential role of hypersensitivity of the dopaminergic system in the pathogenesis of migraine. In this case report, we describe a syncopal episode in a patient with migraine without aura after the administration of a very low dose of apomorphine, a classical agonist of dopaminergic receptors. The absence of cardiovascular risk factors in this patient suggests that the clinical event might have been caused by hypersensitivity of the dopaminergic system.

Immunogenicity of recombinant influenza virus haemagglutinin carrying peptides from the envelope protein of human immunodeficiency virus type 1.

Haemagglutinin (HA), the major surface glycoprotein of influenza virus, is a potent immunogen against which viral neutralizing antibodies are directed. Studies of the three-dimensional structure of HA have identified major antigenic sites on the molecule. We have exploited HA as a carrier for small antigenic regions (epitopes) of the HIV-1 envelope (env) glycoprotein. Using recombinant DNA techniques, the epitopes were inserted in-frame into a known antigenic site of HA to produce HA-epitope chimeras. Guinea-pigs and mice immunized with these chimeras in combination with adjuvant generated significant immune responses against the carrier HA and also produced epitope-specific antibodies that recognized the native whole HIV-1 env. One of the chimeras which contained a V3-loop sequence of HIV-1 env elicited neutralizing antibodies against the homologous strain of HIV-1. The antibodies against HA and the inserted epitopes remained at high levels for up to 72 weeks. Remarkably, these responses were generated with low doses of immunogens containing only nanogram quantities of the inserted epitopes. These results suggest the utility of HA as a carrier to allow selective antibody induction against foreign epitopes, and offer a new approach for vaccine development as well as for the production of monospecific antibodies useful in diagnostics and research.

Attenuated protein kinase C activity and translocation in Alzheimer's disease brain.

Protein kinase C (PKC) activity and its redistribution were determined in postmortem Alzheimer's disease (AD) and age-matched control brains. Cytosolic and membrane-associated PKC activities were lower in frontal and temporal cortices and hippocampi of AD brains. Increased concentrations of phosphatidyl-L-serine, Ca2+ or phorbol 12-myristate, 13-acetate only weakly increased enzyme activity in AD tissues. Redistribution of cytosolic PKC to the membranous fraction was elicited in control brain slices by 162 nM PMA in the presence of K+ (65 mM). This redistribution of the enzyme was markedly reduced in AD brain slices. In contrast, the immunoreactivity of the alpha- and gamma-PKC isozymes were elevated in cortical tissue from AD subjects. No changes were noted in beta-PKC immunoreactivity. These results suggest that the reduced PKC activity and the attenuated translocation of the enzyme in AD brain tissue may be attributed to down regulation of PKC or to alteration in PKC protein. The increase in PKC immunoreactivity may be a reflection of an altered susceptibility to proteolysis or a compensatory response secondary to the loss in enzyme activity.

Propionyl carnitine in stable effort angina.

The aim of this study was to investigate the anti-ischemic activity of propionyl carnitine (PC) in 18 informed, volunteer male patients, aged 37-70, suffering from a typical stable effort angina. The study design was randomized, balanced, crossover, and double blinded. The study lasted 75 days. In the first 15 days of washout the patients performed two maximal symptom-limited bicycle tests to verify the repeatability of the parameters examined. Then one group received PC for 30 days 500 mg three times a day, and the other group received placebo (PL) three times a day. At the end of 30 days the groups exchanged treatments. At the end of each period, 2 hours after the last oral administration, the patients performed a maximal symptom-limited bicycle exercise test with increased loads of 10 watts/min. No significant differences were observed between the two tests performed during the wash-out period, for a 1 mm ST-segment depression time, for the time to the end of exercise, and for the rate x pressure product at the same experimental time. The oral administration of PC in coronary patients increased both the 1 mm ST-segment depression time and the time to the end of exercise. Furthermore, the drug reduced the ischemic depression of ST at maximal common work and at maximal work. After PC, the rate x pressure product was not significantly different in relation to placebo at submaximal and maximal exercise. Thus PC seems to have an antiischemiclike effect, probably related to its metabolic activity.

Dephosphorylation of B-50 in synaptic plasma membranes.

Synaptic plasma membranes from rat brain cortex possess intrinsic ability to dephosphorylate the endogenous protein B-50. At low concentrations of [gamma-32P]ATP, B-50 phosphorylation in synaptic membranes is maximal at 30 seconds, followed by dephosphorylation for an additional 60 minutes. The dephosphorylation of 32P-labeled B-50 is not sensitive to the protease inhibitor leupeptin and not correlated with a loss of the B-50 content of synaptic membranes as measured with immunoblot analysis. Dephosphorylation of membrane-associated B-50 is stimulated to a small extent by Mg2+ but not by Ca2+. Heat-stable protein phosphatase inhibitors prevent dephosphorylation of 32P-labeled B-50. Dephosphorylation of B-50 in synaptic membranes is stimulated by ATP, ADP, or adenosine 5'-O-thiotriphosphate, but not by adenine, adenosine, other adenine or guanine nucleotides, nonhydrolyzable analogs of ATP or GTP, nor by adenosine 5'-O-(2-thiodiphosphate). B-50, phosphorylated by exogenous protein kinase C and purified to homogeneity, has been used as a substrate to follow the purification of B-50 phosphatase activity. B-50 phosphatase activity can be solubilized from synaptic membranes with 0.5% Triton X-100 and 75 mM KCl. Chromatography of the extract on DEAE-cellulose yields enhanced B-50 phosphatase activity.