Biological activities and chemical composition of a cytotoxin of Klebsiella oxytoca.

A low-molecular-mass cytotoxin produced by Klebsiella oxytoca isolated previously from patients with antibiotic-associated haemorrhagic enterocolitis was purified, and its biological and chemical properties were elucidated. The toxin inhibited the syntheses of DNA and RNA by HEp-2 cells dose-dependently, whereas protein synthesis was only slightly inhibited, as measured by the incorporation of radioactive precursors. When synchronously cultured HEp-2 cells were examined in the presence of cytotoxin, inhibition of DNA synthesis occurred promptly within 5 h, but cell-rounding, the earliest visible morphological change, was not observed until 6 h after exposure. The intracellular levels of ATP decreased with an approximately similar time course. These results suggest that cytotoxicity toward HEp-2 cells is primarily due to the inhibitory effect of the cytotoxin on nucleic acid synthesis, possibly on DNA synthesis. Cell rounding and cell death were induced even in the absence of the cytotoxin after incubation with the cytotoxin for 6 h. The cytotoxin was heat-labile, cytotoxic activity decreasing to 50% of the initial level on heating at 70 degrees C for 20 min. Plasmids were extracted from three strains of K. oxytoca producing the cytotoxin and analysed by agarose gel electrophoresis. Two strains possessed plasmids of different sizes, but one strain possessed no plasmid, indicating that the cytotoxin is probably chromosomally encoded. Analysis by NMR and FAB-mass-spectrometry revealed that the molecular mass of the cytotoxin should be 217.1062 Da (exact mass), its molecular formula being C8H15O4N3.


The effect of AMP on the NAD-dependent glutamate dehydrogenase during activation and morphogenesis in the cellular slime moulds.

In extracts from vegetative Dictyostelium discoideum V12 the basal NAD-dependent glutamate dehydrogenase (NAD-GDH) activity was low, but it increased on standing at 4 degrees C. When 0.1 mM-AMP was included in the assay mix, enzyme activity was stimulated nearly 30-fold. As the extract was allowed to age, the enzyme rapidly lost its ability to be stimulated by AMP. The response of NAD-GDH to AMP was also dependent on the stage of morphogenesis. The ratios of NAD-GDH activity assayed with and without AMP (+AMP/-AMP ratios) in freshly prepared extracts from cells at 0, 4, 8 and 12 h of development were similar, but declined later in morphogenesis. The +AMP/-AMP ratio decreased sharply during activation at 4 degrees C in extracts from cells at 0, 4, 16 and 20 h of development. By contrast, extracts from cells starved for 8 and 12 h remained more responsive to AMP throughout activation. Analysis of Western blots showed that vegetative NAD-GDH did not undergo any detectable proteolytic cleavage during 96 h of activation at 4 degrees C. Also, no change in molecular mass appeared to take place within the cells until culmination (20-24 h), when some breakdown products appeared. Activation of NAD-GDH also occurred in D. discoideum strains NC4 and AX3, and in D. mucoroides. In addition, the enzyme from these four strains was stimulated by AMP and the +AMP/-AMP ratio declined with similar kinetics during activation. The enzyme from Polysphondylium violaceum was not activated on standing, but it was stimulated by AMP. The effect of activation of NAD-GDH is discussed in relation to a postulated catabolic role for this enzyme.


Long-chain alcohol and aldehyde dehydrogenase activities in Acinetobacter calcoaceticus strain HO1-N.

Three alcohol dehydrogenases have been identified in Acinetobacter calcoaceticus sp. strain HO1-N: an NAD(+)-dependent enzyme and two NADP(+)-dependent enzymes. One of the NADP(+)-dependent alcohol dehydrogenases was partially purified and was specific for long-chain substrates. With tetradecanol as substrate an apparent Km value of 5.2 microM was calculated. This enzyme has a pI of 4.5 and a molecular mass of 144 kDa. All three alcohol dehydrogenases were constitutively expressed. Three aldehyde dehydrogenases were also identified: an NAD(+)-dependent enzyme, an NADP(+)-dependent enzyme and one which was nucleotide independent. The NAD(+)-dependent enzyme represented only 2% of the total activity and was not studied further. The NADP(+)-dependent enzyme was strongly induced by growth of cells on alkanes and was associated with hydrocarbon vesicles. With tetradecanal as substrate an apparent Km value of 0.2 microM was calculated. The nucleotide-independent aldehyde dehydrogenase could use either Würster's Blue or phenazine methosulphate (PMS) as an artificial electron acceptor. This enzyme represents approximately 80% of the total long-chain aldehyde oxidizing activity within the cell when the enzymes were induced by growing the cells on hexadecane. It is particulate but can be solubilized using Triton X-100. The enzyme has an apparent Km of 0.36 mM for decanal.


Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.


Defective interfering L RNA segments of tomato spotted wilt virus retain both virus genome termini and have extensive internal deletions.

Defective interfering (DI) RNA molecules derived from the genomic L RNA segment of tomato spotted wilt virus (TSWV) were generated during sequential passage of the virus at high multiplicity. Characterization of DI RNAs from four distinct isolates by Northern blot analysis and sequence determination revealed that both the 5' and 3' genomic termini were retained in these molecules. Each DI RNA contained a single internal deletion of approximately 60% to 80% of the L RNA segment. All DI RNAs studied maintain an open reading frame (ORF) which suggests that these defective molecules should be translatable by ribosomes. Detection of only defective molecules with ORFs indicates either that association with ribosomes or translation is a prerequisite for the selection and maintenance of replicating DI RNAs, or that the truncated proteins produced play a role in their selection or replication. Analysis of the junction sites in the DI RNAs showed that short nucleotide sequences are repeated, one at the release and another at the reinitiation point on the L RNA. One of these is lost during the generation of the DI molecules. The presence of repeated sequences at the junction sites seems to be unique for tospovirus DI L RNAs; they have not been described for other DI systems of either positive- or negative-strand RNA viruses. A model for TSWV DI RNA generation is proposed in which the viral polymerase can 'jump' across the internal sequences from one secondary structure to another containing the repeated sequences, during the replication of the viral complementary L RNA segment.


Cross-reacting and heterospecific monoclonal antibodies produced against arabis mosaic nepovirus.

Monoclonal antibodies (MAbs) were produced against arabis mosaic nepovirus (AMV). A hybridoma screening procedure was applied which involved the testing of culture supernatants, before the hybridomas were cloned to single cell lines, for their reaction with eight nepoviruses [AMV, cherry leafroll virus (CLRV), grapevine fanleaf virus (GFLV), peach rosette mosaic virus, raspberry ringspot virus (RRSV), tobacco ringspot virus, tomato black ring virus (TBRV) and tomato ringspot virus]. In addition to AMV-specific MAbs, this screening technique has allowed the selection of two cross-reacting MAbs: one reacting with AMV and GFLV, and one reacting with AMV and RRSV. This is the first report of MAbs cross-reacting with these nepoviruses. In addition, five heterospecific MAbs (HS-MAbs) could be selected: two reacting with RRSV, two with CLRV and one with TBRV. The usefulness of the screening technique that was applied for the selection of cross-reacting MAbs and HS-MAbs, and the potential use of such antibodies are discussed.


Complete nucleotide sequence and genetic organization of papaya ringspot virus RNA.

The complete nucleotide sequence of the RNA genome of papaya ringspot virus (PRSV) was determined from four overlapping cDNA clones and by direct sequencing of viral RNA. The genomic RNA is 10326 nucleotides in length, excluding the poly(A) tract, and contains one large open reading frame that starts at nucleotide positions 86 to 88 and ends at positions 10118 to 10120, encoding a polyprotein of 3344 amino acids. The highly conserved sequence AAAUAAAANANCUCAACACAACAUA at the 5' end of the RNA of PRSV and those of the other five reported potyviruses shows 80% similarity, suggesting that this region may play a common important role for potyvirus replication. Two cleavage sites of the polyprotein were determined by amino acid sequencing of the N termini of helper component (HC-Pro, amorphous inclusion) and cylindrical inclusion (CI) proteins. Other cleavage sites were predicted by analogy with the other potyviruses. The genetic organization of PRSV is similar to that of the other potyviruses except that the first protein processed from the N terminus of the polyprotein (NT protein) has an M(r) of 63K, 18K to 34K larger than those of the other potyviruses. The cleavage site for liberating the N terminus of the HC-Pro protein was found at the same location down-stream from the consensus sequence FI(V)VRG as that reported for tobacco vein mottling virus. The NT protein of potyviruses is the most variable and may be considered important for identification of individual potyviruses. The most conserved protein of potyviruses appears to be the NIb protein, the putative polymerase for the replication of the potyviral RNA. The genetic organization of PRSV RNA is tentatively proposed to be VPg-5' leader-63K NT-52K HC-Pro-46K-72K CI-6K-48K NIa-59K NIb-35K coat protein-3' non-coding region-poly(A) tract.


Nucleotide sequence responsible for the synthesis of a truncated coat protein of brome mosaic virus strain ATCC66.

The ATCC66 strain of brome mosaic virus (BMV) (propagated at Kyoto University) contained two types of coat protein in its virion whereas the Russian strain of BMV has been known to contain a single coat protein; both strains have two initiation codons for coat protein in the same reading frame at the 5'-proximal end of the gene in RNA 4. Comparative studies on the nucleotide sequences of the ATCC66 and Russian strains of BMV demonstrated that in the ATCC66 strain, two adjacent adenine residues were absent from RNA 3 in the leader sequences of the coat protein gene just a few nucleotides 5' to the first initiation codon of the coat protein gene. Using biologically active cDNA clones of BMV RNA of the ATCC66 strain, we inserted two adjacent adenine residues into the cDNA of RNA 3 to obtain an RNA 3 transcript which has the same nucleotide sequence as the Russian strain in the non-coding leader sequence of the coat protein gene. Barley protoplasts inoculated with this RNA 3 transcript together with RNA 1 and 2 produced a single coat protein. To obtain further insight into the mechanism of translation of the BMV coat protein, we constructed several types of RNA 4 by changing the sequence surrounding the first AUG codon in the coat protein gene and analysed the in vitro translation products of the mutant RNA 4. The results confirmed that the absence of the two adjacent adenine residues was responsible for the production of two types of coat protein in the ATCC66 strain. The deletion of the two adjacent adenine residues in ATCC66 resulted in a base substitution of A with U three nucleotides 5' to the first AUG in the coat protein gene. The base substitution reduced translational activity from the first AUG codon and concomitantly increased translational activity from the second AUG codon from which a truncated coat protein was translated.


Stimulation of specific immune responses to simian immunodeficiency virus using chimeric hepatitis B core antigen particles.

Subunit approaches to vaccines against viral diseases have resulted in the development of a number of methods for presentation of defined epitopes to the immune system. We have exploited a highly immunogenic presentation system based on hepatitis B core antigen (HBcAg) particles to produce a number of candidate vaccines against simian immunodeficiency virus (SIV). Recombinant particles have been produced in bacteria which carry multiple copies of defined or predicted neutralizing epitopes of SIV at a number of different sites within the particle. In parallel, a number of synthetic peptide-based SIV vaccines have been produced based on homology to reported neutralizing epitopes in human immunodeficiency virus. Although potent immune responses were elicited against both particulate and peptide forms of the antigen, neutralizing antibodies were not induced as judged by available assays.


Synthesis and processing of the haemagglutinin-esterase glycoprotein of bovine coronavirus encoded in the E3 region of adenovirus.

The haemagglutinin-esterase gene (HE) of bovine coronavirus (BCV) encodes a major viral membrane glycoprotein that elicits BCV-neutralizing antibodies. The BCV HE gene was cloned into a human adenovirus serotype 5 (Ad5) transfer vector in place of early transcription region 3, and a helper-independent recombinant virus was constructed by rescue of the transcription unit by homologous in vivo recombination between the vector and Ad5 genomic DNA. The BCV HE polypeptide expressed by this recombinant Ad was characterized in vivo and in vitro. A 65K polypeptide was identified using an anti-BCV antibody in both human (293) and bovine (MDBK) cells infected with the recombinant Ad. In the absence of a reducing agent, migration of the 65K polypeptide was shifted to 130K, indicating that the recombinant HE polypeptide existed in a dimeric form. The HE polypeptide was glycosylated, as demonstrated by labelling with [3H]glucosamine, and was immunoreactive with three distinct groups of conformation-specific anti-HE monoclonal antibodies (MAbs). Cells infected with recombinant Ad expressing BCV HE exhibited both haemadsorption activity and acetylesterase activity. In addition, the anti-HE group A MAbs HC10-5 and KD9-40 inhibited both the haemadsorption activity and esterase activity of the recombinant HE polypeptide, suggesting that the antigenic domain responsible for BCV neutralization may overlap (or is closely associated with) the domain(s) responsible for haemagglutination and/or acetylesterase activities. When mice were inoculated intraperitoneally with live recombinant Ad, a significant level of BCV-neutralizing HE-specific antibody was induced. These results indicate that the recombinant Ad replicates and directs the synthesis of the BCV HE polypeptide in vivo.


Nucleotide sequence of the tick-borne orthomyxo-like Dhori/India/1313/61 virus membrane protein gene.

The complete nucleotide sequence of the sixth largest segment of ssRNA (RNA-6) of the tick-borne orthomyxo-like Dhori/India/1313/61 virus was determined by using cloned cDNA derived from infected cell mRNA and dideoxynucleotide sequencing of viral RNA. RNA-6 contains 962 nucleotides and is predicted to encode a protein of 270 amino acids with an M(r) of 30,498 in its first open reading frame (ORF). This protein is likely to represent the viral membrane (M1) protein, based on its predicted M(r) of 29,000 (estimated by PAGE), its relatively high abundance in infected cells and amino acid composition analysis. In addition, a second ORF was found which overlaps the M1 protein gene sequence by 327 nucleotides. This additional reading frame, in the +3 frame, potentially can encode a protein of 141 amino acids. However, S1 nuclease analysis of RNA-6 mRNA from infected cells indicated that there was only a single abundant RNA species corresponding in size to the full-length genomic RNA (0.96 kb). Further studies are needed to determine whether expression of the second ORF occurs and how that expression might arise.


A monoclonal antibody recognizes a human cell surface glycoprotein involved in measles virus binding.

Measles virus (MV) has a very limited host range, humans being the only natural reservoir of the virus. This restriction may be due to the absence of an MV receptor on the surface of non-primate cells. We have studied the MV-binding ability of several cell lines and attempted to characterize the receptor by studying the binding of 35S-labelled MV and by a rosette formation technique. We confirmed that all the human cell lines examined (HeLa, Raji and Jurkat) bound MV and that the murine cell lines (BW and L) did not. The glycoprotein nature of the receptor activity was demonstrated by the fact that it could be removed from the cell membrane using proteolytic enzymes and by its failure to be re-expressed in the presence of a protein synthesis inhibitor or an N-glycosylation inhibitor. A monoclonal antibody isolated after immunization of mice with Raji cells specifically inhibited MV binding and infection of human cells, and recognized human and simian but not murine cells. Depending on the cell line (HeLa, Raji, Jurkat or Vero), this antibody immunoprecipitated one or two glycoproteins with apparent M(r)s of 57K and/or 67K from human and simian cells, but not from murine cells.


Differential effect of DNA supercoiling on transcription of adenovirus genes in vitro.

We examined the effect of DNA template topology on the transcription of immediate early (E1a), early (E1b) and late (pIX) adenovirus genes in vitro. Transcription in whole cell extracts was measured by quantitative hybridization to end-labelled DNA and protection of hybrids from S1 nuclease digestion. Two- to fourfold more E1a RNA was synthesized from supercoiled, compared to linear, DNA templates. Similarly, transcription of the E1b gene was stimulated three- to sevenfold when the template was supercoiled. In contrast, RNA synthesis from the late pIX gene was found to be independent of DNA topology. These results show that DNA topology affects transcription in a promoter-specific manner.


Sequence analysis of the membrane protein gene of human coronavirus OC43 and evidence for O-glycosylation.

The gene encoding the membrane (M) protein of the OC43 strain of human coronavirus (HCV-OC43) was amplified by a reverse transcription-polymerase chain reaction of viral RNA with HCV-OC43- and bovine coronavirus (BCV)-specific primers. The nucleotide sequence of the cloned 1.5 kb fragment revealed an open reading frame (ORF) of 690 nucleotides which was identified as the M protein gene from its homology to BCV. This ORF encodes a protein of 230 amino acids with an M(r) of 26416. The gene is preceded by the motif UCCAAAC, analogous to the consensus coronavirus transcription initiation sequence. The M protein of HCV-OC43 shows features typical of all coronavirus M proteins studied: a hydrophilic, presumably external N terminus including about 10% of the protein, and a potential N-glycosylation site followed by three major hydrophobic transmembrane domains. The amino acid sequence of the M protein of HCV-OC43 has 94% identity with that of the Mebus strain of BCV, and also contains six potential O-glycosylation sites in the exposed N-terminal domain. Indeed, the glycosylation of the M protein was not inhibited in the presence of tunicamycin, which is indicative of O-glycosylation, as previously reported for BCV and murine hepatitis virus. Virions released from tunicamycin-treated cells contained the M glycoprotein but were devoid of both peplomer (S) and haemagglutinin-esterase (HE) proteins. Thus, inhibition of the N-glycosylation of the S and HE structural proteins prevented their incorporation into progeny virions, an indication that they are dispensable for virion morphogenesis, unlike the M protein.


Antigenic and genetic characterization of the haemagglutinins of recent cocirculating strains of influenza B virus.

The antigenic and genetic characteristics of the haemagglutinins of influenza type B viruses isolated since 1988 during periods of both widespread activity (1990/1991) and sporadic activity (1989/1990) were examined using microneutralization tests and direct RNA sequencing. During 1989/1990, influenza B viruses representative of two distinct lineages antigenically and genetically related to either B/Victoria/2/87 or B/Yamagata/16/88 were isolated, and a minor drift variant of B/Yamagata/16/88, B/Hong Kong/22/89, was identified. In 1990/1991, B/Hong Kong/22/89- or B/Yamagata/16/88-like viruses accounted for the majority of the influenza virus isolates in most countries. Sequence analysis of the HA1 domains of representative viruses confirmed the continued existence of two main lineages among recent strains of influenza B virus and identified unique amino acid changes that could account for the altered antigenic reactivity of some variants. Sequence analysis of the HA2 domains of some of the recent influenza B viruses allowed for a comparison of the evolutionary rates and patterns between the HA1 and HA2 domains.


Geminivirus replication proteins are related to prokaryotic plasmid rolling circle DNA replication initiator proteins.

It is demonstrated, by means of computer-assisted analysis, that C1 protein involved in the replication of geminivirus DNA is related to the rolling circle replication initiator proteins of eubacterial plasmids, particularly the plasmids of the pMV158 family. Three sequence motifs conserved in the geminivirus and plasmid replication proteins were delineated, one of them encompassing the Tyr residue that presumably forms a covalent linkage to DNA. These findings are compatible with the results of recent analyses of geminivirus replicative intermediates suggesting a rolling circle mechanism for geminivirus DNA replication. It is hypothesized that C1 protein initiates the rolling circle replication of geminivirus DNA by nicking a specific site in the virus-sense DNA and covalently linking to the 5' side of the nick. The putative rolling circle replication initiator domain comprises the N-terminal portion of C1, whereas its C-terminal part is a putative helicase domain. By analogy with prokaryotic systems, it is speculated that the replication initiator domain and the helicase domain function coordinately. The possibility of the origin of geminiviruses from prokaryotic circular ssDNA replicons is discussed.


Sequence analysis of the 3'-terminal halves of RNA 1 of two strains of barley mild mosaic virus.

DNA complementary to the 3'-terminal halves of RNA 1 of two strains of barley mild mosaic virus (BaMMV) from Japan, BaMMV-Ka1 and BaMMV-Na1, was cloned and sequenced. The sequences start within a single long open reading frame (ORF), and are followed by 337 and 338 3' non-coding nucleotides, for BaMMV-Ka1 and BaMMV-Na1 respectively. The two strains have 88% nucleotide identity in the ORFs and 92% identity in the non-coding regions. The putative ORF products contain the capsid proteins at the C termini, as indicated by amino acid sequence analysis, and two putative non-structural proteins are arranged in the same manner as in RNA 1 of barley yellow mosaic virus (BaYMV). The deduced capsid proteins of BaMMV-Ka1 and BaMMV-Na1 each contain 251 amino acids and have 94% sequence identity, which is compatible with their close serological relationship. Most of the sequence differences between the two capsid proteins are found in the N-terminal region, and might explain their serological differences. Significant sequence similarities of the capsid proteins of the two BaMMV strains (37 and 35% respectively) with that of BaYMV, and their marginal similarities (21 to 26%) to the capsid proteins of aphid-borne or mite-borne potyviruses support the classification of BaMMV and BaYMV as distinct members of the same virus group, which is separate from the group(s) containing aphidborne or mite-borne potyviruses.


A zucchini yellow mosaic virus coat protein gene mutation restores aphid transmissibility but has no effect on multiplication.

An aphid-transmissible (AT) and two non-aphid-transmissible (NAT) isolates of zucchini yellow mosaic virus (ZYMV) were studied. The predicted amino acid sequences of the coat protein (CP) of the three virus isolates were analysed and compared. The NAT isolates differed from the AT isolate in having a Thr instead of an Ala residue at position 10 in the conserved Asp-Ala-Gly triplet in the N-terminal region of CP. Aphid transmissibility was restored in a progeny virus derived from an infectious clone of the ZYMV-NAT isolate in which Thr was changed back to Ala by site-directed mutagenesis. However this mutation did not have any effect on the multiplication rate in squash, which was significantly higher than that of the AT isolate. The involvement of this mutation in aphid transmission and virus multiplication is discussed.


The nucleotide sequence of RNA-2 of raspberry ringspot nepovirus.

The nucleotide sequence of raspberry ringspot nepovirus (RRV) RNA-2 consists of 3928 nucleotides and a poly(A) tract at the 3' end. RNA-2 contains one open reading frame which encodes a polypeptide of M(r) 123508 (123K). Edman degradation located the N terminus of the coat protein 514 residues from the C-terminal end of the 123K protein, which suggests that the coat protein is released from the polyprotein by cleavage of a C-A bond. The RRV coat protein has some sequence similarities with the coat proteins of other nepoviruses, but is no more like any one nepovirus than another. In contrast, the portion of the 123K protein to the N-terminal side of the coat protein is similar in sequence to the corresponding parts of the polyproteins of tomato black ring and grapevine chrome mosaic nepoviruses, though not to those of other nepoviruses.


Measles virus from a long-term persistently infected human T lymphoblastoid cell line, in contrast to the cytocidal parental virus, establishes an immediate persistence in the original cell line.

To investigate the mechanisms of measles virus (MV) establishment and maintenance of persistence in lymphoid cells, we have established a long-term persistent infection with MV, Edmonston strain, in the human T lymphoblastoid cell line MOLT4, which has been in continuous culture for over 8 years. In this culture, designated MOMP1, more than 98% of cells display viral antigens. The MOMP1 culture is immune to superinfection with MV and is not cured by anti-MV antibodies. No evidence of defective interfering particles was obtained. The persistently infected culture releases an infectious virus showing a miniplaque and thermoresistant modified phenotype that, unlike the parental virus Edmonston strain which produces a lytic infection with extensive cell fusion, establishes an immediate persistence in MOLT4 cells with neither significant loss of cell viability nor cell fusion. This suggests that the modification in the virus suffices to maintain the state of persistence without requiring a coevolution of the host cell during the infection, as has been reported in other persistent virus infections.


Measles virus gene expression in lytic and persistent infections of a human lymphoblastoid cell line.

MOMP1 is a measles virus (MV) long-term steady-state persistently infected culture of the human T lymphoblastoid cell line MOLT4. The analysis of MV gene expression revealed that in MOMP1 cells, the major MV proteins, haemagglutinin (H), phosphoprotein (P), nucleoprotein, fusion (F) and matrix (M), are present and the fusion precursor (F0) is cleaved into F2 and F1 peptides. H and F2 proteins are glycosylated in both lytic and persistent MOLT4 infections. All major proteins are underexpressed in the persistently infected cultures in comparison to the lytically infected cells. However a relatively greater reduction was observed for H, M and P proteins. Pulse-chase labelling experiments indicated that this underexpression of H, M and P proteins was not due to selective degradation of these proteins in the persistent infection (p.i.). The relative amounts of the major monocistronic and dicistronic mRNAs for MV proteins, with the exception of P mRNA, was not altered in the p.i. with respect to lytically infected MOLT4 cells, suggesting that the defective expression of H and M proteins was not due to a restriction in the transcription of their mRNAs. In contrast, the mRNA for P protein, the most abundant MV mRNA in these lytically infected T lymphoid cells, is markedly underexpressed in the homologous p.i. Thus the underexpression of P protein in p.i. could be due to a decreased availability of P mRNA. This unbalanced underexpression of MV proteins may impair the cell fusion and c.p.e. of MV and facilitate viral persistence in human lymphoid cells.


Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies.

Bacterial fusion proteins containing portions of the Bunyamwera virus L protein were used as immunogens to prepare antisera in rabbits. Of five fusion proteins injected into rabbits, three yielded sera that reacted with the Bunyamwera virus L protein, detected by Western blotting or immunoprecipitation. Two of these antisera were specific for either the amino- or carboxy-terminal regions of the L protein. The specificity of these antisera was confirmed by their pattern of reactivity with full-length and truncated forms of the L protein. Plasmids containing the L gene cDNA under control of a bacteriophage T7 promoter were transfected into CV-1 cells which had previously been infected with a recombinant vaccinia virus, vTF7-3, that expresses T7 RNA polymerase. Antigenically authentic L protein was expressed. Using a nucleocapsid transfection assay developed previously, we showed that the transiently expressed L protein had RNA synthesis activity. Site-specific mutations were made in the L cDNA-containing plasmid to change certain amino acids in the putative polymerase domain of the L protein. The effects of these amino acid substitutions on the RNA synthesis activity of the L protein were monitored using the nucleocapsid transfection assay. These experiments showed that residues strictly conserved between the L proteins of different viruses in the family Bunyaviridae were obligatorily required for activity, whereas non-conserved residues could be substituted without abolishing RNA synthesis capability. Our results provide direct evidence for the functional significance of particular amino acids in the polymerase domain of a negative-strand virus RNA polymerase.


Organization of Germiston bunyavirus M open reading frame and physicochemical properties of the envelope glycoproteins.

We describe the construction of plasmids which express fusion proteins representing various regions of Germiston virus M polyprotein. The fusion proteins were purified and inoculated into rabbits to produce antisera. The N- and C-terminal regions of the polyprotein induced specific antibodies which reacted with glycoproteins G2 and G1, respectively, and the intermediate region induced antibodies against the NSM polypeptide. This enabled us to determine the gene order: G2-NSM-G1. Glycoproteins G1 and G2 form the spikes on the surface of the virion. We attempted to determine the structural organization of the glycoproteins by using a membrane-permeable cross-linking reagent, dimethyl suberimidate, but were unable to demonstrate that G1 and/or G2 form oligomeric structures. We analysed the glycoproteins further and showed that, like peripheral membrane proteins, the G2 and NSM proteins are almost completely extracted into the aqueous phase of detergent Triton X114-treated cellular extracts, whereas glycoprotein G1 is distributed in almost equal proportions between the aqueous and the detergent fractions. This indicates that G1 is a membrane-associated protein, but its presence in the aqueous phase suggests that it is less hydrophobic than a typical membrane protein. We have also characterized the intracellular transport of the envelope glycoproteins from the endoplasmic reticulum to the Golgi complex. Pulse-chase labelling followed by immunoprecipitation and treatment with endoglycosidase H (endo H) showed that both G1 and G2 are transported from the endoplasmic reticulum to the Golgi complex. Conversion to the endo H-resistant form is a rather slow process which takes more than 2 h. The mature G1 and G2 proteins present in the virion particle contain almost completely endo-H-resistant glycans.


Non-random reassortment between the tripartite RNA genomes of La Crosse and snowshoe hare viruses.

The process of reassortment between the tripartite RNA genomes (segments designated L, M and S) of snowshoe hare and La Crosse bunyaviruses (Bunyaviridae) has been investigated by polymerase chain reaction analysis of greater than 250 progeny recovered at 72 h post-infection from dual wild-type virus infections involving high multiplicities (approximately 5) of each virus in a BHK-21 cell line. Statistical analysis of the data indicated that RNA segment reassortment was not random, and for these two viruses the data appeared to fit the hypothesis that there was a preference for homologous L-M and M-S associations among the progeny formed.


Human T cell leukaemia virus type 1 p21X mRNA: constitutive expression in peripheral blood mononuclear cells of patients with adult T cell leukaemia.

Although the p21X protein of human T cell leukaemia virus type 1 (HTLV-1) is generally thought to be expressed from a doubly spliced mRNA transcript (tax/rex mRNA) that encodes the p40tax, p27rex and p21X proteins, we have shown previously that a novel, alternatively spliced mRNA transcript (p21X mRNA) is responsible for p21X production in HTLV-1-infected cell lines. In the present study, we analysed expression of p21X mRNA and tax/rex mRNA in uncultured and cultured peripheral blood mononuclear cells (PBMCs) from eight patients with adult T cell leukaemia by using a quantitative polymerase chain reaction coupled to reverse transcription. The results demonstrated that the expression of p21X mRNA occurs constitutively in all uncultured and cultured PBMCs, whereas the expression of tax/rex mRNA is inducible in the cultured PBMCs, as described previously. In uncultured and cultured PBMCs from the one specimen in which p21X mRNA was highly expressed, the p21X protein was detectable by Western blotting. On the other hand, p27rex protein was detectable only after cultivation. These findings indicate that p21X mRNA is constitutively expressed in vivo and is responsible for production of p21X protein.


The cloning, sequencing and expression of a major antigenic region from the feline calicivirus capsid protein.

RNA purified from the feline calicivirus (FCV) F9 vaccine strain was used to prepare a cDNA library in the expression vector lambda gt11. The library was screened for expression of FCV antigen using a rabbit antiserum prepared against purified FCV. A 330 bp cDNA clone was identified and used as a probe to obtain a larger overlapping clone of 1369 bp. Comparative sequence analysis with the CFI and F4 strains showed that the clones were derived from the 3' open reading frame encoding the capsid protein. The region encoded by the 330 bp clone was shown to be variable in the three strains compared, and therefore the probable location of major antigenic variation. This clone was expressed in a bacterial system and antiserum to the recombinant protein was used in immunoblots to confirm that this clone was derived from the gene encoding the capsid protein. From these immunoblots, several other capsid-related polypeptides were identified. Comparison with immunoblots using post-vaccination cat sera showed the antibody response in the cat was directed mainly against the capsid protein. Antiserum to the recombinant protein was shown to be effective in neutralizing the infectivity of FCV, indicating that at least one major neutralizing epitope had been cloned.


Sequence comparison between the phosphoprotein mRNAs of human and bovine respiratory syncytial viruses identifies a divergent domain in the predicted protein.

The nucleotide and deduced amino acid sequences of the phosphoprotein (P) mRNA of bovine respiratory syncytial virus (BRSV) strain A51908 have been determined. The P mRNA is 860 nucleotides long with a single large open reading frame and the encoded polypeptide is 241 amino acids long. Comparison with the corresponding sequences of human respiratory syncytial virus (HRSV) subgroups A and B revealed 72 to 74% identity at the nucleotide level, and 81% at the amino acid level. The P protein contains a single divergent domain (37% amino acid identity) flanked by highly conserved domains (87% amino acid identity). The 3' end non-coding region is 47 nucleotides shorter than the corresponding region of HRSV. Comparison of the P mRNA sequences of two strains of BRSV (A51908 and FS-1) showed that there was extensive sequence identity at both the nucleotide (97%) and amino acid (97.9%) levels.


Expression of human endogenous retroviral sequences in peripheral blood mononuclear cells of healthy individuals.

The polymerase chain reaction was used to detect expression of retroviral sequences with oligonucleotide primers derived from conserved regions of the retroviral genome. Four primer pairs derived from gag and one from pol were used in amplification of reverse-transcribed total RNA prepared from peripheral blood mononuclear cells of seven blood donors. The amplification pattern was the same from each of the seven samples. Sequencing of cloned amplification products revealed that at least three subclasses of sequences related to the human endogenous retroviruses (HERV) RTVL-H, HERV-E and HERV-K, are expressed in peripheral blood mononuclear cells of healthy individuals. This has not been previously reported.


Two novel viruses associated with severe disease symptoms of the green stinkbug Nezara viridula.

Two viruses were isolated from green stinkbugs (Nezara viridula) with severe disease symptoms. These viruses have been named N. viridula virus type 1 (NVV-1) and NVV-2 according to their relative sedimentation coefficients. NVV-1 is a small picorna-like virus with a diameter of 29 nm, a buoyant density in CsCl of 1.34 g/ml and a sedimentation coefficient of 153S. NVV-1 particles contain a 9.4 kb ssRNA segment and have three coat proteins of M(r)s 32,100, 31,500 and 30,700. NVV-2 sediments as two components on sucrose gradients; the top 104S component consists almost entirely of 41 nm empty capsids and the faster sedimenting 177S component consists of intact 39 nm spherical particles. NVV-2 particles have a buoyant density in CsCl of 1.39 g/ml and consist of one major protein of M(r) 73,800 and at least two minor proteins of M(r)s 13,500 and 16,500. Only one dsRNA segment of 6.2 kb was identified. The properties of NVV-2 are similar to those of the Totiviridae. Individual stinkbugs were infected with either NVV-1 or NVV-2, or with a mixture of the two viruses. Re-infection of virus-free stinkbugs with the mixture resulted in typical disease symptoms. Both viruses were vertically transmitted through the eggs and insects were infected by surface contamination of their food source.


The nucleotide sequence of red clover mottle virus bottom component RNA.

The complete nucleotide sequence of the bottom component RNA (B RNA) of red clover mottle virus strain S has been determined. The sequence consists of 6033 nucleotides and contains a single long open reading frame sufficient to encode a protein of M(r) 210,258. The proteolytic processing sites within this protein have been deduced by comparison of its sequence with that of the B RNA-encoded protein of cowpea mosaic virus. Comparison of the amino acid sequences of the individual proteins confirms that the two viruses have a similar genome organization.


A comparative study of the RNA-2 nucleotide sequences of two sweet clover necrotic mosaic virus strains.

The nucleotide sequences of the RNA-2s of two strains of sweet clover necrotic mosaic virus (SCNMV-38 and -59) have been determined. The RNA-2s of SCNMV-38 and -59 consist of 1446 and 1449 nucleotides, respectively, and both contain one major open reading frame (ORF) which potentially can encode polypeptides of 326 amino acid residues (about 36.5K), designated SC38P2 and SC59P2, respectively. The nucleotide sequences of SCNMV-38 and -59 RNA-2s show 93.2% similarity, and the amino acid sequences of SC38P2 and SC59P2 are 91.7% identical, although the identical nucleotides and amino acids are not distributed uniformly in RNA-2 and the encoded proteins. Two highly conserved regions (from positions 23 to 221 and 297 to 326) and a relatively divergent region (from positions 222 to 296) are found in the P2 proteins of these strains. A similar pattern is apparent on comparison of the nucleotide and deduced amino acid sequences of RNA-2 of these SCNMV strains with those of the Australian and Czechoslovakian isolates of red clover necrotic mosaic virus.


Phytohemagglutinin and concanavalin A activate hepatitis B virus in peripheral blood mononuclear cells of patients with chronic hepatitis B virus infection.

Peripheral blood mononuclear cells (PBMC) from 25 patients with chronic hepatitis B were tested for the presence of free monomeric hepatitis B virus (HBV) DNA migrating as a single 3.2 Kb band by Southern blot analysis. The PBMC were cultured for 7 days in the presence of phytohemagglutinin (PHA) or concanavalin A (ConA) both of which yielded a proliferative response. By contrast, both bacterial lipopolysaccharide (LPS) and interleukin 2 (IL2) failed to do so. Dot blot assays were used to monitor HBV DNA level increase within PBMC. Following mitogen exposure HBV DNA levels increased above pre-stimulation levels in 19/25 PHA cultures, 6/15 ConA cultures, 1/15 LPS cultures, and 1/15 IL2 cultures. In 15 patients, Southern blot analysis was carried out before and after PHA exposure. In 13/15 cases, a single 3.2 Kb band was observed in unstimulated cultures as well as in PHA cultures even though PHA induced a HBV DNA increase. One case exhibited bands migrating faster than the 3.2 Kb signal, compatible with replicating intermediates and one case provided evidence of viral concatemers within PBMC after PHA stimulation. No HBV DNA was detected in the culture supernatants. The increase of HBV DNA level in PBMC induced by mitogen was strongly associated with an increase in HBV DNA expression (HBV RNA and HBs antigen). These studies indicate that HBV DNA present in human PBMC does represent a potential reservoir for infection with endogenous reactivation following PBMC activation.


Enteric non-A, non-B hepatitis: epidemics, animal transmission, and hepatitis E virus detection by the polymerase chain reaction.

We studied epidemics of viral hepatitis occurring at three different places in India. One was a combined epidemic due to hepatitis E virus (HEV) and hepatitis A virus (HAV) infections. In this epidemic, HAV affected children below 10 years of age, whereas HEV infected the young adult population. HEV was transmitted to rhesus monkeys (Macaca mulata) and confirmed by the polymerase chain reaction (PCR) on bile from the animals. Fecal material from acutely infected patients in one of the epidemics was also found positive for HEV RNA by PCR. This may help in confirming the nature of future epidemics. The bile and liver from experimental animals can be used as a source of material for further virological and molecular biological studies of HEV.


Acute sporadic hepatitis E in children living in Cairo, Egypt.

Seventy-three pediatric patients with acute hepatitis and 19 control patients without liver disease living in Cairo, Egypt, were evaluated with a newly developed Western blot assay for IgM antibody to hepatitis E virus (IgM anti-HEV). The mean age of acute hepatitis patients was 6.4 years (range, 1-13 years); 56% were male. Among the 73 acute cases, hepatitis A was diagnosed in 30 (41%), possible acute hepatitis B in three (4%), hepatitis E in nine (12%), and by exclusion, non-A, non-B hepatitis in 29 (40%). Two additional acute cases were positive for both IgM anti-HAV and IgM anti-HEV. None of the 19 control subjects had IgM anti-HEV. Parenteral risk factors were associated with cases of non-A, non-B hepatitis but were not associated with acute hepatitis E. Contact with a family member with jaundice was associated with acute hepatitis A. In contrast to prior epidemics of enterically-transmitted non-A, non-B hepatitis, HEV was found to be a common cause of acute hepatitis in a pediatric population. This study provides additional evidence that HEV may be a frequent cause of acute sporadic hepatitis among children living in some developing countries.


Concurrent hepatitis C virus and hepatitis delta virus superinfection in patients with chronic hepatitis B virus infection.

Since hepatitis C virus (HCV) and hepatitis delta virus (HDV) are transmitted by the same routes as hepatitis B virus (HBV), simultaneous or concurrent HCV and HDV infection in patients with chronic HBV infection may occur. To test this hypothesis and to examine the clinicohistological and immunopathological presentations of such multiple hepatitis virus infections, acute and/or convalescent serum specimens from 86 patients with acute HDV superinfection were tested by enzyme immunoassay for antibodies to HCV. Of the 86 patients, 18 (20.9%) were associated with HCV infection. Although patients with early mortality cannot be evaluated by the HCV markers used in this study, the results showed that the clinical and histologic features were similar except that patients with HCV infection were older than those without HCV infection (P less than 0.01). Immunopathological studies carried out within 2 months after the onset of acute HDV superinfection demonstrated that hepatitis B core antigen (HBcAg) was not detected in any patient and HDV antigen was detected in 18.2% of the patients with HCV infection whereas HBcAg and HDAg were found in 7% and 65.1%, respectively, of those without HCV coinfection (P less than 0.02). It is concluded that concurrent HCV and HDV superinfections can and do occur in patients with chronic HBV infection. In these triple viral infections, HCV may even transiently suppress HDV and HBV.


Dideoxyinosine for chronic hepatitis B infection.

Six patients positive for both human immunodeficiency virus (HIV) and hepatitis B were studied to assess the effect of dideoxyinosine (DDI) on hepatitis B virus (HBV) replication. Two patients died during the follow-up period and four had at least 8 weeks of therapy. One patient demonstrated HBV DNA suppression and became transiently negative. In the remaining five patients, there was no appreciable change in HBV DNA levels during DDI therapy. DDI was well tolerated in all patients, the only significant side effect being diarrhoea. It is concluded that DDI has no notable antiviral effect in patients with chronic HBV infection when coinfected with HIV.


Prevalence of serum antibodies against lymphocytic choriomeningitis virus in selected populations from two U.S. cities.

An ELISA was developed for measuring serum antibodies against the arenavirus lymphocytic choriomeningitis virus (LCMV) and a closely related isolate termed callitrichid hepatitis virus (CHV). The ELISA was used to test sera from healthy adults and from hepatitis patients. In Birmingham, Alabama, the seropositivity rate for healthy black women was 5.1% (7/138), and the rate for patients with all types of hepatitis or cirrhosis was 4.3% (2/46). In San Antonio, Texas, the seropositivity rate among a clinical series of patients with non-A, non-B hepatitis was 0 (0/20), and the rate among persons rejected from blood donation because of high serum alanine aminotransferase levels was 2.4% (2/82). These results indicate that infection with LCMV or CHV is common in Birmingham but that infection is not associated with hepatitis.


Sexual transmission of human T-lymphotropic virus type I in Peruvian prostitutes.

The epidemiology of HTLV-I infection in female prostitutes was studied in a survey of 395 prostitutes from Callao, Peru (the port city of Lima), 72 prostitutes from Iquitos, Peru (another port city on the Amazon River), and 510 prenatal clinic patients from Lima. Prostitutes reported a mean of 8.8 years (range, 1-39 years) of active prostitution and a mean of 205 sexual contacts during the month prior to the study. The percentage of prostitutes with HTLV-I antibody (21.8%) was significantly higher than patients attending a prenatal clinic (3.1%; P less than .0001). The prevalence of HTLV-I antibody increased steadily with age in prostitutes, but no age trend was noted in prenatal patients. By multiple logistic regression analysis, an independent association was found between HTLV-I seropositivity and a history of prostitution in Callao, age, and positive syphilis serology when all 977 study subjects were evaluated. When prostitutes alone were analyzed, the number of years of exposure as a practicing prostitute was associated with HTLV-I seropositivity after controlling for age. These data indicate a greatly increased risk of HTLV-I infection in prostitutes in Callao, Peru, and suggest an association between sexual activity and HTLV-I transmission.


HIV-2 infections in a rural Senegalese community.

In a community study in rural Senegal, 22 human immunodeficiency virus type-2 (HIV-2) seropositive cases and 64 matched controls were examined clinically and evaluated immunologically. The presence of clinical signs was highly correlated with HIV-2 seropositivity: 9 anti-HIV-2 positive patients and 5 controls presented with clinical signs (odd ratio [OR] = 8.2, confidence limits [CL] 2-35). The main symptom associated with HIV-2 seropositivity was a chronic cough (OR = 18.5, CL 1.8-899). The presence of diarrhoea was not significant (OR = 3.1, CL 0.3-3.5). The total number of CD8 cells, CD4/CD8 ratio, beta 2 microglobulin, and IgG level discriminated between seropositive and seronegative individuals (P less than 0.05). When the anti-HIV-2 positives were grouped as 13 healthy and 9 sick people, red blood cells, lymphocytes, T lymphocytes, CD4 cells, and beta 2 microglobulin differed significantly. Clinical symptoms were associated with immunodepression: 5 of 14 sick people had less than 500 CD4/microliters vs. 1 of 72 healthy persons. This study at the community level emphasizes the clinical and immunological impact of HIV-2 infection. Even if it presents with a longer incubation period than HIV-1, this virus is a major threat to public health.


Dependency conflict, marital threat, and alcohol consumption in a middle-aged sample.

The hypothesis that dependency conflict is associated with higher levels of alcohol consumption when dependency needs are threatened or thwarted was tested with a sample of 672 middle-aged, married adults with college-age children. The subjects' current level of alcohol consumption was predicted based on the present level of threat to the marital relationship (assessed by reports from several family members) and on indices of dependency need and inhibition of dependent behavior estimated from sibship size, sibship density, and sibling position. A multiple regression analysis yielded a significant two-way interaction (p less than .05) between marital threat and subject sex, and a significant three-way interaction of dependency need, inhibition of dependent behavior, and marital threat. High marital threat was associated with higher levels of alcohol consumption in men and slightly lower levels of alcohol consumption in women. Additionally, when dependency need was high, alcohol consumption was generally low, except when both inhibition of dependent behavior and marital threat were high. However, when dependency need was low, the highest alcohol consumption score occurred when marital threat was low and inhibition was high.


Using a personal robot to teach young children.

Seventy-five preschool children were instructed about birds by a human teacher, a moving personal robot, a stationary personal robot, and a tape recorder. How much the children learned and how much attention the children paid were compared for each type of instruction. The children learned when they were taught by the human teacher and when they were taught by the animated and the stationary robots. The children paid more attention to the live teacher and to the moving robot than they did to the stationary robot or to the tape recorder. The difference between the amount of attention the children paid to the animated robot and the amount of attention they paid to the human teacher was not statistically significant.


Adjustment problems experienced by children during cross-cultural orientation: a pilot study.

A study was undertaken to investigate the adjustment problems of children aged 8-10 years when they relocated from their native countries to Vienna, Austria. There was a moderate correlation between anxiety and adjustment to changes in the physical and material environment and a significant correlation between anxiety and changes in social support systems. Adjustment to changes in parental behavior and changes in language demands both showed a low negative correlation with anxiety. Children who had spent the least time in Vienna showed greater evidence of anxiety and stress than did children who had lived in the city for at least 1 year.


Popular and rejected children's social reasoning: linking social status and social knowledge.

The relationship between children's social status/sex and their moral judgements was examined. Sixty-four second- and third-grade children (33 boys, 31 girls) who were identified as popular or rejected by peer sociometric measures were shown pictures of children engaged in moral and second-order transgressions. The children were asked to rate each event on (a) the degree of seriousness for other and self, (b) the amount of punishment for other and self, and (c) rule alterability. The children were also asked for justification of the transgressions (why they thought the transgressions were wrong). The popular and rejected children differentiated between moral and second-order transgressions based upon criterion ratings and justifications. Differences emerged between the popular and the rejected children's ratings and justifications for moral transgressions, suggesting that children's moral judgements are related to social experiences associated with peer acceptance and rejection.


Mothers with hostile, Type A predisposing child-rearing practices.

The authors examined characteristics of Finnish mothers (N = 924) who use hostile child-rearing practices (i.e., they ignore the child, are punitive and irritable, and perceive the child as a burden), practices that have been shown (Räikkönen & Keltikangas-Järvinen, 1992) to predispose children to Type A behavior. The results of this study indicate that two factors--Type A behavior in the mothers and the mothers' sociodemographic background (low occupational status, low educational level and young age)--increase the probability of the mothers' treating their children in a hostile manner. Also, the mothers of boys in this study reported more hostile child-rearing practices than the mothers of girls did.


Guidelines for developing, evaluating, and revising the classification of personality disorders.

The authors suggest ways to improve the classification of personality disorders by changing the way the classification is developed, evaluated, and modified. Specific proposals are intended to establish a system that can be evaluated and revised so that it successively approximates a valid system. The importance of stating the principles underlying the classification is emphasized. These principles include: the conception of personality disorder underlying each diagnosis, the structure used to define diagnoses, the nature of diagnostic items, and the model for organizing diagnostic decisions. A distinction is drawn among the theoretical, measurement, and diagnostic models underlying each diagnosis. The substantive aspects of the classification are discussed in terms of the evidence required to validate diagnostic concepts. It is argued that the first step in developing a classification is to ensure the content validity of diagnostic concepts because this is a prerequisite for other components of validity. Evaluation and revision of the classification are discussed in terms of the importance of convergent and discriminant evidence. It is recommended that the classification be evaluated and revised using criteria derived from the theoretical and measurement models associated with each diagnosis. It is also recommended that the classification be evaluated in terms of the degree to which diagnostic constructs are consistent across clinicians, different sets of diagnostic exemplars, and different samples of patients. Realization of these aims should provide a classification that may be verified, modified, or disproved in the scientific tradition.


Gender roles and alcohol abuse. Costs of noncaring for future physicians.

Psychiatric epidemiological research has focused disproportionate attention on traditionally female disorders such as depression. This paper shifts epidemiological gears to elaborate and test an etiological model of a traditionally male disorder, that of alcohol abuse. We argue that social-relational deficits (narcissistic orientations) lead to abuse of alcohol for stress reduction purposes, given interpersonally oriented stressors and the incapacity to form social supports. The model was tested in the context of training for a traditionally male occupation encompassing both social-relational demands and limited social supports. A cohort of medical students was surveyed from medical school entrance through a portion of clinical training. Time 1 social-relational deficits were predictive of time 3 alcohol abuse, partially as a function of social support deficits and, to a lesser extent, patient care-related stressors. Moreover, there was an initial gender difference in social-relational deficits and a trend-level gender difference in alcohol abuse that disappeared when social-relational deficits were held constant. By time 3, women did not differ from men in social-relational deficits or alcohol abuse.


Manifestation of depressive symptoms among adolescents. A comparison of Mexican Americans with the majority and other minority populations.

The purpose of this research was to investigate differential manifestation of depressive symptomatology among adolescents from diverse ethnocultural groups. Data from a national survey of persons 12 to 17 years of age (N = 2200) were analyzed, comparing responses of Anglo, African, Mexican, and other Hispanic Americans with a 12-item version of the Center for Epidemiologic Studies Depression Scale. The results indicated minimal differences in terms of item response and internal consistency-reliability among the four adolescent groups. However, there were differences in patterns of item endorsement, as indicated by principal component factor analysis. Anglo- and African Americans exhibited similar factor structure, represented by negative affect, positive affect, and psychosomatic symptoms. The two Hispanic groups also exhibited a three-dimensional pattern, but there was a tendency among Hispanic adolescents for somatic symptoms and negative affect symptoms to cluster together. This pattern may indicate a more prominent role of somatic complaints in the presentation of depression among Mexican Americans and other Hispanics.


Changes in ego defenses with recovery from depression.

This study aimed to determine whether patterns of ego defense change with short-term treatment of psychiatric illness. The subjects were 37 inpatients and outpatients with a DSM-III-R diagnosis of major depressive disorder being treated using standard clinical methods. Ego defenses before and 7 to 9 weeks after commencement of treatment were measured using a shortened version of the Defense Style Questionnaire. There was a significant decline in the use of immature defenses with symptomatic recovery, but no change in the neurotic or mature defenses. Patients with additional axis I diagnoses and/or abnormal personality traits (N = 15) used more neurotic defenses than their counterparts with major depression alone (N = 22), but this pattern did not change with time. The study demonstrates the short-term mutability of immature defenses in relation to an episode of psychiatric illness and provides empirical support for the concept of temporary regression in the context of psychiatric illness episodes.


Observational measurement of symptoms responsive to treatment of major depressive disorder in children and adolescents.

Observational methods were used to determine whether depressive symptoms in 20 inpatients with major depressive disorder could be observed to change during the course of treatment with antidepressant medication. Four consecutive weekly observation sessions were done using the Emotional Disorders Rating Scale, which collects information about symptoms of depression, mania, anxiety, hostility, and irritability. Only those scales indexing depressive symptoms evidenced change. Significant change was observed between the third and fourth weeks of hospitalization, thereby replicating the findings reported in the adult literature regarding depressive symptoms' responsiveness to antidepressants.


Season of birth and neuropsychological impairment in schizophrenia.

Repeated studies suggest a relationship between winter birth and increased incidence of schizophrenia. Furthermore, there may be seasonal fluctuations in schizophrenia risk factors (e.g., influenza epidemics) and the severity of biological anomalies (e.g., enlarged cerebral ventricles in neuroimaging studies). In order to assess whether winter-born schizophrenics show greater neuropsychological impairment, 112 males meeting Research Diagnostic criteria for schizophrenia were administered the Luria-Nebraska Neuropsychological Battery, a thorough measure of higher cortical functioning deficit. Sixty-four of these 112 patients were also administered the Wechsler Adult Intelligence Scale-Revised, the Benton Visual Retention Test, and the Rey Auditory Verbal Learning Test. Despite the use of several definitions of winter and nonwinter birth, there was no evidence of elevated rates of neuropsychological dysfunction among winter-born patients on any measure. The current study contains certain limitations (e.g., variable medication status at testing), but the results suggest no strong season of birth relationship with neuropsychological impairment in a reasonably large, research-diagnosed sample of schizophrenic patients.


Psychophysiological processes during insight-oriented therapy. Further investigations into nonlinear psychodynamics.

Experimental evidence shows that many physiological processes abide by nonlinear dynamics and evidence chaos. Novel conceptual models postulate chaotic phenomena in psychological processes as well. The challenge in empirically testing these models is to develop measures that are sufficiently precise to permit nonlinear dynamical analyses and that also say something meaningful about psychological phenomena. Toward this end, we examined the spontaneously occurring autonomic activity of a patient during psychotherapy. Phase portraits were constructed of the patient's heart rate data for each of several therapy sessions. The flows within these phase spaces were visually segmented and assigned to one of four previously defined trajectory classifications. These trajectories were quantitatively analyzed, and the corresponding clinical material was assessed qualitatively. The trajectories in the physiological phase space were recognizable, recurrent, and robust; they appeared coupled to ongoing psychological processes. Possible refinements in this typology are discussed and suggestions are made for potentially using such analyses to track and navigate among psychophysiological states.


Psychiatric diagnoses of abusive mothers. A preliminary report.

The Structured Clinical Interview for DSM-III-R diagnoses of 54 mothers who had maltreated their children were compared with those of 37 controls. The maltreatment group showed a significantly greater incidence of both current and past diagnoses. Maltreating mothers exhibited a significantly greater incidence of current mood disorder, alcohol abuse, and personality disorder than did controls. The results indicate that past abuse of cocaine, alcohol, other substances and past mood disorders were significantly more prevalent among the maltreatment sample than among controls. Mothers who had maltreated their children were significantly more likely to have histories of posttraumatic stress disorder than were controls.


Glucose metabolism in psychiatric disorders: how can we facilitate comparisons among studies?

Positron emission tomography (PET) offers a possibility to study brain function and its relationship to psychiatric disorders. Clinical studies have demonstrated that several psychiatric diseases are coupled with changes in brain glucose metabolism. Schizophrenia seems to involve a lower metabolism in wide areas of the brain--both cortical and subcortical structures. Depression probably involves dysfunction of the metabolism in dorsolateral prefrontal cortex. Obsessive compulsive disorder, panic disorder, anorexia nervosa and the experience of anxiety may involve increased metabolic rates. The results from the different studies do not allow quantitative comparisons or detailed analyses because of large differences in experimental and clinical methodology. The term Good Clinical PET Practice (GCPP) is suggested to encourage standardization in clinical investigations. GCPP includes standardization of both experimental factors (lumped constant, arterialization, purity of tracer, regions of interest, relative rates) and clinical factors (state of the subject, wakefulness, anxiety, gender, course of the disease) in PET performance.


Operators and scales: diagnostic and rating issues in psychiatric PET research.

In psychiatric research that for various reasons has to restrict itself to a limited number of subjects, such as studies involving expensive positron emission tomography techniques, issues concerning the parsimonious description of patients gain in importance. The number of descriptive variables must be optimally small. This paper offers a conceptual back-ground for the choice of operators in operational diagnostic systems designed to delimit pathological types, and of rating scales designed to measure syndromal severity in a dimensional way. A practical suggestion in five tenets for the organization of clinical research of this kind is presented.


The FDG model and its application in clinical PET studies.

The FDG method, as it is applied in clinical PET studies is reviewed. The influence of different implementations of the method and instrumental inaccuracies on the values of cerebral metabolic rate of glucose is discussed. For the comparison of the results between different groups standardized procedures are recommended.


On the influence of spatial resolution and of the size and form of regions of interest on the measurement of regional cerebral metabolic rates by positron emission tomography.

Factors that affect the accuracy of the positron emission tomographic (PET) quantification of cerebral metabolic rates include the spatial resolution of the employed imaging device and the method used for extraction of regional metabolic values from the PET data set. The present article reviews (i) how and to what extent these two factors are presumed to influence the measurement of absolute values of cerebral metabolic rates and their ratios, and (ii) whether and how these factors may affect comparisons of regional metabolic rates between groups of subjects.


The role of anatomic information in quantifying functional neuroimaging data.

When using modern neuroimaging tools, such as CT, PET, SPECT, MRI and MEG, in brain research and brain diagnostics, there is a common need for including external anatomical information into the interpretation and analysis of data. This information may be used to aid the interpretation of structures in images from low resolution imaging tools. With high resolution tools it can help to identify resolved structures. It can also facilitate the merging of data from different modalities, or from different individuals. The anatomical information is often given as regions of interests (ROIs), which may be manually created from an anatomy rich image or automatically created from a standard template collection or from an atlas data base. Automatic methods will lead to a substantial reduction in bias and in size of the systematic errors. Functional ROIs can correspondingly be derived from functional images (usually PET or SPECT). Different aspects of these processes are discussed in the report.


The dorsolateral prefrontal cortex, schizophrenia and PET.

Central neurophysiology can be measured with PET. These measurements are providing insights into the regional abnormalities associated with schizophrenia. Cohorts of schizophrenic subjects have been studied cross-sectionally in attempts to identify common regional deficits. More recently the advent of fast dynamic measurements of regional cerebral blood flow have allowed rapid serial measurements in the same subject in different brain states (activation studies). These complementary approaches are based upon, and are interpreted with reference to, a number of methodological considerations and underlying hypotheses. The key hypotheses underpinning cross-sectional and activation studies are discussed within the framework of the lesion model and functional anatomy models of brain function. This brief review of some assumptions, ideas and methodological constraints is illustrated with empirical data implicating the dorsolateral prefrontal cortex in schizophrenic symptoms.


Back to basics.

The basic strength of the AMCA family is the wide range of expertise and "can do" attitude of its diverse membership. The AMCA is currently experiencing problems with federal legislation and fiscal stability because we have not fully utilized member strengths to reevaluate the association's direction and set new goals to address change. However, the AMCA Board of Directors and many committee chairpersons met, reevaluated direction and clarified goals, and initiated major changes to meet the challenges facing the association and its members. Members of the AMCA are encouraged to contribute their individual talents to ensure that the association prosper and serve the needs of the mosquito and vector control family.


Patrick Manson and the discovery age of vector biology.

There have been few scientists who have had a greater impact on the history of vector biology than Sir Patrick Manson (1844-1922). By demonstrating that mosquitoes became infected with microfilariae in the process of taking a blood meal, he became the first to prove an association between insects and pathogens causing human and animal diseases. He also contributed substantially to the discovery of mosquito transmission of malaria parasites and was a principal force behind the founding of the London School of Tropical Medicine and the Royal Society of Tropical Medicine and Hygiene. Manson's career is reviewed in historical context as well as in relation to modern concepts of vector biology.


Influence of temperature and larval nutrition on the diapause inducing photoperiod of Aedes albopictus.

Photoperiod induced dormancy for 14 North American strains of Aedes albopictus were determined at 21, 26 and 29 degrees C. Strains tested at 21 degrees C and intermediate temperatures of 25-27 degrees C demonstrated clear photoperiodic responses whereas temperatures of 29 degrees C and above, greatly reduced or negated diapause incidence. A suboptimal larval diet increased the percentage diapause in eggs laid by resulting adults. This larval diet was also associated with a slight increase in critical photoperiod.


Evaluation of methoprene (Altosid XR) sustained-release briquets for control of culex mosquitoes in urban catch basins.

A sustained-release, briquet formulation of methoprene (Altosid XR), applied at a rate of one briquet per catch basin in Saginaw, Michigan, provided ca. 70% reduction in emergence of Culex pipiens and Cx. restuans adults, compared with nontreated catch basins, during a period of 15 wk in the summer of 1990. In a parallel study using one briquet per 10.5 liter bucket, there was 99% reduction in adult emergence of these species for a period of 12 weeks. The difference between catch basins and buckets may be attributable to water movement through the catch basins with each rainfall, causing a dilution of methoprene through time. However, both studies indicated that the briquets released methoprene for 12-15 wk, suggesting that this formulation may offer season-long control of Culex mosquitoes from urban catch basins in Michigan, with a single treatment of insecticide.


Transmission of La Crosse virus by four strains of Aedes albopictus to and from the eastern chipmunk (Tamias striatus).

Eastern chipmunks were successfully infected with La Crosse virus by bites of 3 New World strains of Aedes albopictus infected orally or transovarially. The virus was subsequently passed from the chipmunks to Ae. albopictus, POTOSI strain, and Ae. triseriatus. The chipmunks developed viremias of 1-4 days duration and antibody titers were similar in intensity and duration to those reported in chipmunks infected by Ae. triseriatus. After feeding on viremic chipmunks, Ae. albopictus became infected and transmitted La Crosse virus at rates similar to the native vector, Ae. triseriatus. Aedes albopictus transmitted La Crosse virus transovarially to first gonotrophic cycle offspring.


Effect of ULV malathion on automotive paint finishes.

The relationship between malathion droplet size (VMD) and degree of damage to 1990, 1K and 2K General Motors paint standards was investigated in the laboratory and field. Laboratory tests indicated a positive correlation between malathion droplet VMD and damage spot size. Laboratory settling chamber tests revealed that size-thresholds of droplets too small to cause visible damage averaged 8 and 11 mu on washed 1K and 2K paints, respectively. Field tests indicated malathion caused no visible damage to 1K or 2K paint panels under routine operating conditions, although droplet sizes (VMD) sampled on the automobile surface averaged 10.2 +/- 4.5 and 11.7 +/- 5.7 mu. Microscopic damage was found on paint panels placed on the hood, roof, trunk and doors of the automobile when parked parallel or perpendicular to the course of the spray truck and when driven through the spray of a stationary spray truck.


Comparative efficacy of aphid extracts and some juvenoids against the development of mosquitoes.

Comparative efficacy of natural juvenile hormones extracted from Aphis craccivora and A. gosypii and 5 juvenoids, i.e., methoprene, Neporex, OMS 3007, OMS 3019 and DPE-28 on the development of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus have been evaluated. OMS 3007, OMS 3019, DPE-28 and Neporex show species specific related activities, while methoprene and aphid extracts do not show such activity against these mosquito species. Treatment of mosquito eggs with an EC50 dose of these compounds caused mortality while an EC90 dose completely ceased adult emergence. The fecundity and fertility rates of mosquitoes emerged shows significant reduction (P less than 0.001) when treated with EC50 doses of all the compounds.


Detection of human antibodies against Plasmodium falciparum antigens in blood meals of anopheline mosquitoes.

Human IgG antibodies against Plasmodium falciparum asexual stages, gametocytes and sporozoites were detected by indirect fluorescent antibody (IFA) techniques in the blood meals of Anopheles gambiae s.l. from a malaria-endemic area of western Kenya. Field-collected mosquitoes, which had been stored dry for over 2 years, were screened first for human IgG by ELISA. In 141 blood meal samples from human-fed mosquitoes, the prevalence of stage-specific antibodies was 87.9% for asexual-stage parasites, 78.0% for gametocytes, and 87.9% for sporozoites. There were no differences in the prevalence of stage-specific antibodies for mosquitoes collected from 2 sites, before and after the long rainy season of 1988. The detection of specific human antibodies in mosquito blood meals by IFA, or by more efficient methods, may provide alternative approaches for large-scale, epidemiologic studies of malaria and other vector-borne diseases.


Evaluation of different methods of catching anopheline mosquitoes in western Venezuela.

During a longitudinal study of vector biology and malaria transmission in western Venezuela, adult mosquitoes were collected by different methods and their efficiency was compared with human landing catches. CDC light traps, a double-net, a calf-baited trap and collection of resting mosquitoes on vegetation were tested. These methods did not prove to be effective substitutes for human landing catches.


Effect of Nosema algerae on the house fly Musca domestica (Diptera: Muscidae).

Larvae of Musca domestica were exposed to spores of Nosema algerae on the surface of their diet. Infective concentrations (IC50 and IC90) for the larvae were 3.6 x 10(4) and 1.6 x 10(6) spores/cm2, respectively. The disease appeared to cause no larval mortality, but the longevity of adult females was reduced. At 30 days post-infection, there were at least 1 x 10(7) spores per fly in all dosage groups. At lower dosages, the development of spores was delayed and fewer spores were produced.


Lethal effects of ivermectin on Anopheles quadrimaculatus.

Female Anopheles quadrimaculatus adults were blood fed on 15 mixed breed dogs 4 h after the dogs were given oral dosages of ivermectin. Dogs were divided into 5 treatment groups of 3 dogs each, at 10, 500, 1,000, 2,500 micrograms/kg, and untreated. Additionally, An. quadrimaculatus were fed on lambskin-membranes containing blood drawn from one dog in each treatment group. Mosquitoes were allowed to feed on the dogs or the lambskin-membranes and were observed for death at 24 and 48 h post-feeding. Greater than 90% mortality was recorded in all ivermectin treatment groups except at the 24 h post-feeding period with the 10 micrograms/kg dog dose blood fed through the lambskin-membrane (65.4% mortality). The highest 2 dosages produced 100% mosquito mortality at 48 h post-feeding from either a dog or the in vitro system using a lambskin-membrane.


Seasonal incidence and horizontal distribution patterns of oviposition by Aedes aegypti in an urban environment in Trinidad, west Indies.

The oviposition patterns of Aedes aegypti in ovitraps placed along 5 horizontal transects were monitored weekly for 52 wk (January to December 1988) in St. Joseph, Trinidad. Of the 2,550 ovitraps exposed, 270 were lost and 1,177 (52%) out of 2,280 were used by gravid females, containing 38,118 eggs. During the dry season 43% (16,265 eggs) of the eggs were collected whereas during the wet season 57% (21,853 eggs) were collected. Ovitraps exposed under eaves, under houses and in the open yard attracted similar oviposition occurrences and proportions of eggs. Inspections of the 52 properties within St. Joseph revealed that 17 (House Index = 32.7%) harbored Ae. aegypti pre-adult stages. The container index was 5.5%. This study revealed no evidence of behavioral changes in the container preferences of Ae. aegypti in St. Joseph, Trinidad, after 12 years of treating with fenthion and temephos.


Sublethal effects of larval methoprene exposure on adult mosquito longevity.

Larvae of Aedes aegypti were exposed to sublethal concentrations of the insect growth regulator, methoprene, and the glycogen content of pupae and surviving adults was compared and effects on adult longevity determined. The glycogen reserves in both male and female Ae. aegypti pupae were significantly reduced as a result of methoprene exposure. The longevity of adult females was also significantly reduced, but exposure affected neither the longevity nor the glycogen content of adult males. Adult sugar feeding increased the amount of glycogen in both treated and control females. The reduced longevity of adult females from larval methoprene treatment appeared not to be directly related to reduced glycogen, but rather reflected neuroendocrine abnormalities induced by this juvenile hormone analogue.


Ecology of Anopheles pulcherrimus in Baluchistan, Iran.

Studies were conducted on the ecology of Anopheles pulcherrimus over a period of 20 months in the village of Zeineddini, Sistan and Baluchistan Province, southeastern Iran. The species was active throughout the year with 2 peaks of activity, April-May and August-September. Light traps captured the highest number of An. pulcherrimus females (65%) as compared to cattle bait collections (19.3%), pyrethrum space spray catches (14.2%), pit shelter (1%) and human bait collections (0.6%). However, 95% of the females captured in light traps were unfed or freshly fed females as opposed to about only 44% of those collected in pyrethrum space spray catches and pit shelter collections. The species was mainly exophilic as shown by the gravid/fed ratio of 0.4 obtained in outlet window traps.


Photoperiod and the relationship between wing length and body weight in Anopheles quadrimaculatus.

The effect of photoperiod on wing length, body weight, and relationship between wing length and body weight was investigated in the mosquito species Anopheles quadrimaculatus. Individuals reared under a short photoperiod (8 h light: 16 h dark) had longer wings and larger weights than did those reared under a long photoperiod (16 h light: 8 h dark). Covariance analysis showed that photoperiod and wing length interacted so that photoperiod did not have a uniform effect on body weight at all wing lengths. At small mosquito sizes, body weight was higher in short than in long-photoperiod individuals of the same wing length, but at large mosquito sizes, body weight was higher in long than in short-photoperiod individuals of the same wing length. Thus, among smaller mosquitoes of this species, wings were disproportionately longer in long-photoperiod individuals, but among larger mosquitoes, wings were disproportionately longer in short-photoperiod individuals. These results, together with previous studies, suggest that photoperiod and temperature have similar effects on the developing insect.


Mosquito vector control and biology in Latin America--a second symposium.

The second Spanish language symposium presented by the American Mosquito Control Association (AMCA) was held as part of the 58th Annual Meeting in Corpus Christi, TX in March 1992. The principal objective, as it was for the 1991 symposium, was to increase and stimulate greater participation in the AMCA by vector control specialists and public health workers from Latin America. This publication includes summaries of 25 individual presentations that were given in Spanish. The symposium included the following topics: biology and chemical control of Aedes aegypti and anopheline vectors of malaria; field and laboratory studies of biological control agents for Aedes aegypti; community participation in the prevention of dengue; and other various aspects of the biology of other medically important arthropods (e.g., Simulium ochraceum, Lutzomyia and Culicoides).


First record of Aedes albopictus establishment in Italy.

In September 1991, the first breeding populations of Aedes albopictus were discovered in Veneto Region, Italy. Larvae were collected in a wide variety of peridomestic containers and in used tires. Attempts were made to eradicate this species from infested areas. No larvae or adults of Ae. albopictus were found from October 1991 onwards. It is not yet assessed if this was because of the control measures taken or due to the low fall temperatures and the short critical photoperiod prevailing in the area (L:D, 12:45). Since tire casings are believed to be the primary mode of introduction and dispersal of Ae. albopictus, an investigation of tire retreading operations was initiated to determine the source and mode of introduction of Ae. albopictus into Italy.


Aedes albopictus and other mosquitoes imported in tires into Durban, South Africa.

The results of surveillance for immature stages of mosquitoes in samples of wet tires in 5 consignments imported into Durban from Japan is reported. Three of these consignments contained tires with immatures of Aedes aegypti, Ae. albopictus and Uranotaenia n. novobscura. The proportions of all the wet tires calculated to contain mosquitoes were 189/1,488 (13%), 20/813 (2.5%) and 13/1,032 (1.3%) which emphasizes the need for effective mosquito control measures to prevent Ae. albopictus escaping from such tires and establishing itself in Durban.


Key characters for identifying Aedes bahamensis and Aedes albopictus in North America, north of Mexico.

Aedes bahamensis, a species recently introduced into southern Florida represents the first member of the subgenus Howardina to be found in the United States. Its separation from all other Nearctic Aedes is the subject of this work, integrating it into the North American mosquito keys (Darsie and Ward 1981). The key revisions presented are expanded to include the other exotic species now found in the United States, Aedes albopictus.


Chromium picolinate increases membrane fluidity and rate of insulin internalization.

The effects of chromium chloride, chromium nicotinate, and chromium picolinate on insulin internalization in cultured rat skeletal muscle cells was examined. Insulin internalization was markedly increased in cells cultured in a medium that contained chromium picolinate and the increased internalization rate was accompanied by a marked increase in the uptake of both glucose and leucine. The effect was specific for chromium picolinate since neither zinc picolinate nor any of the other forms of chromium tested was effective. The increased insulin internalization rate may result from an increase in membrane fluidity since chromium picolinate and to a lesser extent, chromium nicotinate, increased the membrane fluidity of synthetic liposomal membranes.


Synthesis, characterization, and antibacterial activity of transition metal complexes with 5-hydroxy-7,4'-dimethoxyflavone.

Complexes of CuII, NiII, CoII, ZnII, FeIII, CrIII, CdII, and MnII with the natural product 5-hydroxy-7,4'-dimethoxyflavone have been synthesized and the probable structures of these complexes have been proposed on the basis of elemental analyses, molecular weight determination, magnetic moments, and electronic and IR spectral data. The presence of coordinated and crystal water molecules was demonstrated by thermal studies. The antibacterial activity of the ligand and all the complexes has been determined on gram positive and gram negative bacteria.


Palladium(II) salt and complexes of spermidine with a six-member chelate ring. Synthesis, characterization, and initial DNA-binding and antitumor studies.

By reaction of spermidine trihydrochloride with K2PdCl4 and PdCl2 at different pH's, we have synthesized the [sperH3]2[PdCl4]3 (I), [PdCl2(sperH)]2[PdCl4] (II), and [(PdCl2)3(sper)2] (III) compounds. The structure of these compounds was studied by IR and 1H NMR; complex II was analyzed by x-ray diffraction. In this complex the spermidine is attached to the PdCl2 group forming a six-member chelate ring with a protonated terminal amine group. The crystal of [PdCl2(sperH)]2[PdCl4] x 2H2O (II) is monoclinic, P2(1)/n, with a = 7.023(1) A, b = 12.662(1) A, c = 18.435(3) A, and beta = 99.95(1) degrees, Z = 4, R = 0.051, and Rw = 0.058 on the basis of 2690 independent reflections. We have compared the antitumor activity in vitro against the isolated human breast carcinoma MDA-MB 468 cell line of compounds I, II, and III with that of cis-diamminedichloroplatinum(II), cis-DDP. The results show that compounds III and III have values of ID50 similar (0.74 microgram/ml) or even lower (0.56 microgram/ml) than cis-DDP (0.80 microgram/ml). We also observed that compounds I, II, and III have the ability to induce conformational changes in covalently closed circular (ccc) form of the pUC8 plasmid DNA. Compounds II and III also induce conformational changes in the open circular (oc) form of this plasmid.


Evidence that the loss of the voltage-dependent Mg2+ block at the N-methyl-D-aspartate receptor underlies receptor activation during inhibition of neuronal metabolism.

In this study, the importance of the Mg2+ blockade of the N-methyl-D-aspartate (NMDA) receptor during metabolic stress was examined in embryonic day 13 chick retina. Retina exposed to mild conditions of metabolic stress (i.e., blockade of glycolysis with 1 mM iodoacetate for 30 min) underwent acute histological somal and neuritic swelling and an increase in gamma-aminobutyric acid (GABA) release into the medium. These acute signs of metabolic stress were eliminated by NMDA antagonists present during pharmacological blockade of glycolysis, occurred in the absence of a net increase in extracellular glutamate or aspartate, and were not affected by the presence or absence of Ca2+ in the incubation medium. One possible explanation for the activation of NMDA receptors in the absence of an increase in extracellular ligand is that NMDA sensitivity during metabolic stress may be governed at the receptor level. Depolarization of membrane potential during metabolic stress may result in the loss of the Mg2+ blockade from the NMDA receptor channel, resulting in an increased potency for glutamate. To test this, the dose-response characteristics for NMDA, glutamate, and kainate in the presence or absence of extracellular Mg2+ and the effects of Mg2+ on metabolic inhibition were examined. The potency for NMDA- or glutamate-mediated acute toxicity was enhanced two- to fivefold in the absence of Mg2+. Omission of Mg2+ greatly decreased the minimal concentration of agonist needed to produce acute excitotoxicity; 25 versus 5 microM for NMDA and 300 versus 10 microM for glutamate in 1.2 or zero Mg2+, respectively. Elevating external Mg2+ to 20 mM completely protected against NMDA-mediated acute toxic effects. In contrast, varying external Mg2+ had no effect on kainate-induced toxicity. Acute toxicity caused by inhibition of metabolism was not potentiated in the absence of Mg2+ but was attenuated by elevating extracellular Mg2+. The protective effect of Mg2+ during metabolic inhibition was not additive with NMDA antagonists, suggesting that the action of Mg2+ was at the level of the NMDA receptor. These findings are consistent with the hypothesis that the Mg2+ block is lifted during metabolic inhibition and may be the primary event resulting in NMDA receptor activation.


Correlation between electroencephalogram isoelectric time and hippocampal norepinephrine levels, measured by microdialysis, during ischemia in rats.

It is suggested that norepinephrine (NE) plays a role during transient forebrain ischemia. NE may have a protective action against neuronal cell death in the hippocampus, or it may be one of the causes of injurious ischemic effects. We used the microdialysis technique to study extracellular NE levels in the rat hippocampus before, during, and after 30 min of transient incomplete forebrain ischemia (induced by four-vessel occlusion) to describe the time course of NE in this condition. There was a maximal increase (fivefold) in extracellular NE after 10 min of reflow only when the electroencephalogram was isoelectric. NE levels returned to baseline 40 min after release of the carotid clamps and remained constant for the next 80 min. Thus there appears to be a transient NE overflow in the hippocampus during ischemia, closely related to the complete loss of brain electrical activity.


Effect of N,N'-dicyclohexylcarbodiimide on acetylcholine release from Torpedo synaptosomes and proteoliposomes reconstituted with the proteolipid mediatophore.

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.


Lesion-induced changes in the production of newly synthesized and secreted apo-E and other molecules are independent of the concomitant recruitment of blood-borne macrophages into injured peripheral nerves.

Peripheral nerve injury produces Wallerian degeneration characterized by a change in the composition of resident nonneuronal cells: macrophages are recruited from the circulation to join Schwann, fibroblast, and endothelial cells. At the same time, the nonneuronal cell population exhibits, as a whole, alterations in synthesis and secretion of diffusible molecules, some of which are instrumental in nerve repair mechanisms. In this study, we determined whether changes in the production of secreted molecules depend on the concomitant modification in cell composition. Therefore, we studied the secretion of newly synthesized molecules by defined cell populations of intact nerves, intact nerve explants undergoing in vitro axonal degeneration, in vivo degenerating nerves, and recruited cells. Nerves were incubated in serum-free, [35S]methionine-containing media. Secreted, radioactively labeled proteins were precipitated from the medium and analyzed by gel electrophoresis. Reduced production of 43-, 46-, and 48-kDa proteins and increased production of 33-34-, 37-, 49-, 59-, and 67-kDa proteins were detected in in situ degenerating nerves. High-density ultracentrifugation and immunoblot analysis revealed that the 33-34-kDa protein is apolipoprotein-E (apo-E). Similar alterations in the production of these molecules were detected in intact nerve explants from which blood-borne cells were excluded. Apo-E, 37-, 49-, 59-, and 67-kDa proteins were also produced in frozen nerves that lacked the intact nerve nonneuronal cell population. Instead, these preparations contained blood-borne cells, primarily macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of the irreversible inhibition of high-affinity choline transport produced by hemicholinium mustard.

The inhibition of high-affinity choline transport by hemicholinium mustard (HCM), an alkylating analogue of hemicholinium-3, was examined in rat brain synaptosomes and guinea pig myenteric plexus. In synaptosomes, 50% high-affinity choline transport inhibition occurs with an HCM concentration of 104 nM (4-min incubation). A 10-min preincubation with 10 microM HCM results in essentially complete (greater than 95%) inactivation that persists after washing. Low-affinity choline transport in synaptosomes is unaffected by HCM inhibition at all concentrations examined (1-50 microM). Time course experiments indicate that the maximum irreversible inhibition (58%) seen after a 1-min preincubation with 500 nM HCM decreases to 46% inhibition after a 15-min preincubation; however, analysis of variance reveals that this difference is not significant. HCM inhibition of acetylcholine release from myenteric plexus-longitudinal muscle preparations persists for at least 2 h after removal of drug from the incubation bath; this inactivation can be prevented by coincubation with a high choline concentration during treatment with the mustard. In contrast, inhibition produced by the parent compound hemicholinium-3 is largely reversed by washing in both preparations examined. The observed potency and selectivity of HCM suggest its usefulness as a covalent probe for high-affinity choline transport.


Beta-amyloid precursor protein isoforms in various rat brain regions and during brain development.

To address the question of the possible functions of different Alzheimer's disease beta-amyloid precursor protein (beta-APP) isoforms in the brain, we studied their expression at different times during postnatal rat brain development and in various regions of the adult rat brain. Polyclonal antibodies directed to two peptide antigens were used. The majority of all beta-APP forms was found to be soluble as revealed by western blot analysis. The highest level of most beta-APP forms was reached in the second postnatal week, which is the time of brain maturation and completion of synaptic connections. Strikingly high concentrations of the Kunitz protease inhibitor-containing beta-APP were present in the adult olfactory bulb, where continuous synaptogenesis occurs in the adult animal. These findings support the idea of an involvement of beta-APPs in the processes of cell differentiation and, probably, in the establishment of synaptic contacts.