Introduction: Listening to the Voice of the C/S/X: Consumer/Survivor/Expatient

People who are receiving services for professionally diagnosed psychological disabilities often are not consulted about the nature of those services, or their willingness to participate in them. This issue of the journal presents the autobiographical accounts of four such people, followed by commentaries about those accounts by three professional service-givers. This collection emphasizes the need to obtain informed consent for any psychological services that are offered, for ethical, humane, and professional reasons.     

Evaluation of the A/Seal/Mass/1/80 virus in squirrel monkeys

An influenza A virus isolated from seals [A/Seal/Mass/1/80 (H7N7)] and an isolate of this virus obtained from a human conjunctiva were evaluated for replication and virulence in squirrel monkeys. When the seal virus was administered intratracheally, it replicated in lungs and nasopharynges and induced illness almost to the same extent that a human influenza A virus [A/Udorn/72 (H3N2)] did. In one monkey that died of pneumonia, the seal virus was recovered from spleen, liver, and muscle as well as lung. After conjunctival administration in monkeys, the seal virus replicated to a peak titer in the conjunctivae 30-fold greater than that attained by the human virus, but this difference was not statistically significant. In contrast, the seal virus replicated less well than the human virus in the tracheae and nasopharynges when administered by the conjunctival route. These results indicate that the seal virus can replicate efficiently in primates, that it can spread systemically, and that it might differ from human virus in being able to replicate slightly better in primate conjunctival tissue.     

Sequence of the nucleoprotein gene of influenza A/parrot/Ulster/73

The nucleotide sequence of the nucleoprotein (NP) gene of the avian influenza A virus strain A/parrot/Ulster/73 (H7N1) has been determined. The gene (RNA segment 5) consists of 1565 bases. The only large open reading frame of the complementary RNA codes for a protein of 498 amino acids. A comparison of its sequence with that of three other influenza virus NPs shows that the NP of the parrot Ulster strain, although closely related to the NP of the other avian strain (A/FPV/Rostock/34), is definitely more closely related genetically to the NPs of the two human influenza strains, A/PR/8/34 and A/NT/60/68 than that of FPV. This raises the question how far the NP gene can cross the species barrier by reassortment and become adapted by mutation to the new host.     

An evaluation of the genetic stability and pathogenicity of the Russian cold-adapted influenza A donor strains A/Leningrad/134/17/57 and A/Leningrad/134/47/57 in ferrets

The influenza A components of live attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47), and virulent epidemic strains. The lesions responsible for attenuation within the six internal genes of each donor strain have been sequenced and described, but relatively little is known as to their stability before and after passage in susceptible hosts. In the work reported in this paper, RT-PCR restriction analysis and limited sequencing of individual genes were used to evaluate the stability of lesions in stocks of the both donor strains after passage in ferrets, which have been used widely as susceptible hosts for assessment of the virulence of influenza strains. Len/47 was shown to possess expected lesions by RT-PCR and restriction analysis. Substitution at position 1066 of the NP gene, which has been previously reported to be unique to Len/47 [Klimov et al., Virology 186 (1992) 795], was also shown to be present in all clones of Len/17. This change was confirmed by limited sequence analysis and was shown to be retained in progeny viruses isolated from the lungs and turbinates of inoculated ferrets. Two other changes in the PB2 and PB1 genes that were present in Len/47 were detected by limited sequence analysis alone. Further previously unreported minor changes were shown to be present for Len/17 and Len/47, but not both, and their significance is unknown. Limited replication of each donor strain occurred in ferrets and minimal clinical signs and histopathology were present. By contrast, the parental strain Len/57 and the recent epidemic strain A/Sydney/6/97 induced clinical signs and histopathology that were typical of influenza disease.     

Nucleotide sequence of RNA segment 3 of the avian influenza A/FPV/Rostock/34 and its comparison with the corresponding segment of human strains A/PR/8/34 and A/NT/60/68

The nucleotide sequence of RNA segment 3 of A/FPV/Rostock/34 (H7N1), an avian strain of influenza A virus, has been determined from a cloned DNA copy. Segment 3 codes for the PA polypeptide and the sequence specifies an acidic polypeptide of 716 amino acid residues. Comparison of the sequence with the corresponding segment of two human strains A/PR/8/34 and A/NT/60/68 indicates significant divergence of the avian sequence from the human sequences at the nucleotide level. At the amino acid level there is considerably greater homology between the avian and human strains. This presumably reflects a constraint on divergence of the PA polypeptide imposed by a common functional requirement of PA in all influenza virus strains.     

Sequence of the hemagglutinin gene from influenza virus A/Seal/Mass/1/80

A double-strand DNA copy of the influenza virus A/Seal/Mass/1/80 (H7N7) [seal] hemagglutinin (HA) gene was cloned into the plasmid pAT153/PvuII/8 and sequenced to deduce the primary amino acid sequence. The gene is 1731 nucleotides long and codes for a protein of 560 amino acids with a nonglycosylated molecular weight of 62098 Da. The deduced amino acid sequence displays similarities to all other sequenced hemagglutinins by retaining six of seven potential glycosylation sites, showing conversation in the number and position of cysteine residues, conservation in the fusion and anchor peptides, and conservation in the putative receptor site of the molecule. However, three features of the primary amino acid sequence could be distinguished from the H7 amino acid sequence of A/fowl plague/Rostock/34 (FPV), another avian H7 influenza virus which does not produce disease in mammals. First, the seal HA sequence has three fewer amino acids in the connecting peptide region of the HA than FPV. This lack of multiple basic amino acids in the connecting peptide is similar to that found in avirulent H7 avian strains and to mammalian serotypes H1, H2, and H3. Second, the seal HA has gained four additional proline residues, all in HA1, as compared to FPV. These residues may alter the tertiary structure of the HA and ultimately contribute to the biological features of this virus. Third, the seal HA has lost a potential carbohydrate attachment site at residue 149 which lies at the tip of the HA structure. The loss of this carbohydrate could alter the seal HAs interaction with host cell receptors.     

A comparison of the biochemical properties of the human diaphorase (DIA3) isozymes determined by the common alleles DIA13, DIA23 and DIA33

(1) Various buffer systems for the starch gel electrophoresis of human diaphorase isozymes have been explored. Electrophoresis in a Tris/Borate system at pH 8.6 which includes 70 micron NADH in the gel and cathodal electrode buffers, provides good resolution of the six DIA3 phenotypes previously resolved by isoelectric focusing. (2) The variant genes DIA13, DIA23 and DIA33 occur with frequencies of about 0.76, 0.23 and 0.01 respectively in the English population. (3) The isozymes determined by the least common gene, DIA33, are markedly different from the isozymes determined by DIA13 and DIA23 in their relatively low heat stability, high affinity for Blue Sepharose and slow anodal electrophoretic mobility in buffer systems containing borate. The DIA3 1 and DIA3 2 isozymes are similar to one another in these characteristics.     

Localisation of the temperature-sensitive defect in the nucleoprotein of an influenza A/FPV/Rostock/34 virus

The nucleotide sequences of the nucleoprotein (NP) genes of fowl plague virus (FPV) and of a temperature-sensitive (ts) mutant (ts81) derived therefrom have been determined. The ts81-NP nucleotide sequence possesses a single nucleotide substitution in comparison to the wild type. This causes an amino acid exchange at position 332 of the NP. An alanine in the wild type-NP is substituted by a threonine in ts81-NP. This substitution leads to a significant difference in the secondary structure prediction. Although this mutation is located within the karyophilic region of the NP, the accumulation of the NP in ts81-infected cells is not significantly affected at 40 degrees C. Therefore, we assume that the cooperation with one of the polymerase proteins (P) is interfered with at 40 degrees C, leading to the loss of viral vRNA or replicative cRNA synthesis. The comparison of the FPV-NP nucleotide sequence to a previously published sequence of the same strain (Tomley and Roditi, 1984) highlights ten nucleotide differences, four of them leading to amino acid substitutions.     

Influence of calcium/calmodulin on budding of Ebola VLPs: implications for the involvement of the Ras/Raf/MEK/ERK pathway

The VP40 matrix protein of Ebola virus is able to bud from mammalian cells as a virus-like particle (VLP). Interactions between L-domain motifs of VP40 and host proteins such as Tsg101 and Nedd4 serve to facilitate budding of VP40 VLPs. Since intracellular levels of calcium are known to influence localization and function of host proteins involved in virus budding, we sought to determine, whether alterations of calcium or calmodulin levels in cells would affect budding of VP40 VLPs. VP40 VLP release was assessed in cells treated with BAPTA/AM, a calcium ion chelator, or with ionomycin, a calcium ionophore. In addition, VLP budding was assessed in cells treated with W7, W13, or TFP; all calmodulin antagonists. Results from these experiments indicated that: (i) budding of VP40 VLPs was reduced in a dose-dependent manner in the presence of BAPTA/AM, and slightly enhanced in the presence of ionomycin, (ii) VP40 VLP budding was reduced in a dose-dependent manner in the presence of W7, whereas VP40 VLP budding was unaffected in the presence of cyclosporine-A, (iii) budding of VSV-WT and a VSV recombinant (M40 virus) possessing the L-domains of Ebola VP40 was inhibited in the presence of W7, W13, or TFP, (iv) inhibition of virus budding by W7, W13, and TFP appears to be L-domain independent, and (v) the mechanism of calcium/calmodulin-mediated inhibition of Ebola VLP budding may involve the Ras/Raf/MEK/ERK signaling pathway.     

Role of the Ek Molecule in the Generation of Suppressor T Cells in Response to LDHB

The role of the Ek (E alpha kE beta k) molecule in the generation of suppressor T (Ts) cells specific for lactate dehydrogenase B (LDHB) was studied using different approaches. First, lymph node cells from LDHB-primed B10.A(2R) (AkEk) nonresponder mice were shown to suppress the LDHB-specific and Ak-restricted proliferative response of T cells from the congenic responder strain B10.A(4R), which does not express E molecules (AkEo). Similarly, lymph node cells from primed CBA (AkEk) mice suppressed the anti-LDHB response of Lyt-1+Lyt-2-T cells (depleted of Lyt-2-bearing Ts cells) from the same mice. Second, in vitro priming of 2R (AkEk) T cells with LDHB-pulsed 4R (AkEo) antigen-presenting cells (APC) generated T-cell proliferation but not suppression. Third, nonresponder 2R mice were turned into responders by injecting them with LDHB-pulsed 4R APC or monoclonal Ia.m7 antibody that blocks the Ek molecule. The data demonstrate that expression of Ek molecules by the APC is necessary to generate LDHB-specific Ts cells, which in turn prevent the proliferation of Lyt-1+Lyt-2- (probably helper) cells recognizing the same antigen in the context of the Ak molecule.     

Immunochemical Detection of the VKIV Subgroup

A Bence Jones protein (BJ Juv) with a rather unusal N-terminal amino acid sequence has been found. An antiserum specific for this protein was developed and used to screen normal sera, M-component sera, Bence Jones protein containing urines, and isolated kappa chains. Out of the sera tested only 2 of the 66 macroglobulinemia sera reacted strongly. N-terminal sequence analysis of the isolated kappa chains showed that the amino acid sequence was nearly identical with that found for BJ Juv and that of the V_KIV subgroup.     

Piperacillin/tazobactam/amikacin versus piperacillin/amikacin/teicoplanin in the empirical treatment of neutropenic patients

A prospective randomized trial was performed to compare the efficacy of a regimen containing a glycopeptide versus one containing a beta-lactamase inhibitor in the treatment of febrile episodes in neutropenic patients. Fifty-eight patients received piperacillin/amikacin/teicoplanin (group 1) and 56 received piperacillin/amikacin/tazobactam (group 2). In the case of persistence of fever without microbiological documentation of the cause, teicoplanin was also given empirically in group 2 on day 4, and amphotericin B in both groups on day 6. In 114 evaluable febrile episodes, the rate of success without modification of therapy was 60 % in patients on piperacillin/amikacin/teicoplanin and 41 % in patients on piperacillin/amikacin/tazobactam (p<0.03). Eleven of 34 patients in the latter group who failed to improve eventually responded upon addition of teicoplanin. Ten and nine patients in group 1 and group 2 respectively required the addition of amphotericin B for definite improvement. There were 14 episodes of gram-positive septicemia in each group: the response rate was 100 % in group 1 and 43 % in group 2. Three episodes of gram-negative breakthrough septicemia occurred in group 1 versus no cases in group 2 (p=0.1). Three deaths occurred in each group. Piperacillin/amikacin/tazobactam may be as efficacious as piperacillin/amikacin/teicoplanin in the treatment of febrile neutropenic patients provided the regimen is modified (usually by addition of teicoplanin) in unresponsive cases.     

Nucleotide sequence of the avian influenza A/Mallard/NY/6750/78 virus polymerase genes

The avian influenza A/Mallard/NY/6750/78 virus is currently being evaluated as a donor of attenuating genes in the construction of live avian-human influenza A reassortant virus vaccines for use in humans. We determined the nucleotide sequences of the three polymerase gene segments of this virus. This completes the nucleotide sequence of the six transferrable genes of the avian donor virus. Comparison of the nucleotide and deduced amino acid sequences of the non-glycoprotein genes of the avian A/Mallard/78 virus with representative avian and human influenza A viruses suggests that the PB1 gene of H2N2 subtype human influenza A viruses may have been derived from a non-human, possibly avian influenza A virus by genetic reassortment. In addition, several regions of conserved amino acids with potential functional significance were identified in the deduced amino acid sequences of the polymerase proteins.     

Primary structure of the hemagglutinin gene in strains of the influenza virus A/Leningrad/624/86 and A/Leningrad/621/86 serologic subtype H1N1

A molecular analysis was made of genomes of influenza A (H1N1) virus strains, the causative agents of an epidemic in Leningrad, 1986. The primary structure of hemagglutinin gene of two of these strains, A/Leningrad/624/86 and A/Leningrad/621/86, was established, as well as partial primary structure of PB1 gene of certain current strains of the A (H1N1) subtype. A hypothesis of a "shift" of PB1 gene in 1950-1957 is suggested.     

Analysis of the antigenic structure of influenza A/USSR/90/77 virus hemagglutinin

Evolution of the antigenic structure of influenza virus hemagglutinin with the antigenic formula HA1 was studied by the determination of the capacity for interaction with monoclonal antibody and aminoacid substitutions in the protein. Consecutive changes in protein epitopes were found in isolates accumulating from the time of isolation of H1 influenza viruses in 1977.     

Determination of the rpoB gene sequences of Bartonella henselae and Bartonella quintana for phylogenic analysis

Using the Genome Walker™ procedure, which allows PCR amplification of genomic DNA using a single gene-specific primer and direct automated sequencing methodology, we obtained the nucleotide sequence of the RNA polymerase β subunit (rpoB) from Bartonella henselae and Bartonella quintana. A phylogenetic tree constructed from these data and other rpoB sequences available in GenBank is, in part, consistent with those previously derived from 16S rRNA gene sequences and confirms the position of Bartonella within the α subdivision of Proteobacteria. In fact, this analysis showed that rpoB data are similar to 16S rRNA data for the α, β and γ subdivisions of Proteobacteria. In contrast, concerning other bacteria included in our study, the topologies of phylogenetic trees were different. Based on the bootstrap values derived from rpoB phylogenic analysis, we believe that this molecule should contribute to better understanding the evolutionary process.     

Monitoring the self-assembly of chitosan/glutaraldehyde/cysteamine/Au-colloid and the binding of human serum albumin with hesperidin

A new method for monitoring, in real-time, the self-assembly of chitosan/glutaraldehyde/cysteamine (CGC) on the gold surface and the immobilization of Au-colloid on CGC membrane with piezoelectric quartz crystal impedance (PQCI) are firstly proposed. Cyclic voltammogram and electrochemical impedance spectroscopy were also use to investigate the formation of Au-colloid/CGC. The viscosity-average molecular weight of chitosan used was firstly estimated as 16.8 x 10(5) with piezoelectric quartz crystal (PQC) sensor. On the basis of the analysis of the multi-dimensional information provided by PQCI, two stages existed in chitosan drying course: the frequency shift of the first stage was controlled by viscoelastic of the liquid, while the total frequency shift was due to mass change. The cross-link ratio of glutaraldehyde with chitosan was about 0.13, while for glutaraldehyde with cysteamine was about 0.217. PQCI also showed that the Au-colloid immobilization is a first-order reaction, while the HSA immobilization is a sum of two exponential functions, e.g., adsorption and re-arrangement. The association of the immobilized HSA with the purified hesperidin was monitored, and the association constant was estimated as 3.42 ml mg(-1) by Scatchard analysis.