Surveillance of acute respiratory virus infections in general practice

The ordinary postal system provides a simple method for the transportation of specimens for virus isolation to the laboratory. This was used to carry out an epidemiological survey, in general practice, of respiratory viruses in patients with acute respiratory disease. Three distinct outbreaks of myxovirus infections were recognized and most other types of respiratory viruses were isolated sporadically. After the initial “pilot” phase of the study an isolation rate of 24% was obtained over the months from October 1968 to June 1969 (20% isolations from adults'' specimens and 29% from those of children).     

Concurrent targeting of nitrosative stress-PARP pathway corrects functional, behavioral and biochemical deficits in experimental diabetic neuropathy

Peroxynitrite mediated nitrosative stress, an indisputable initiator of DNA damage and overactivation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated after sensing DNA damage, are two crucial pathogenetic mechanisms in diabetic neuropathy. The intent of the present study was to investigate the effect of combination of a peroxynitrite decomposition catalyst (PDC), FeTMPyP and a PARP inhibitor, 4-ANI against diabetic peripheral neuropathy. The end points of evaluation of the study included motor nerve conduction velocity (MNCV) and nerve blood flow (NBF) for evaluating nerve functions; thermal hyperalgesia and mechanical allodynia for assessing nociceptive alterations, malondialdehyde and peroxynitrite levels to detect oxidative stress-nitrosative stress; NAD concentration in sciatic nerve to assess overactivation of PARP. Additionally immunohistochemical studies for nitrotyrosine and Poly(ADP-ribose) (PAR) was also performed. Treatment with the combination of FeTMPyP and 4-ANI led to significant improvement in nerve functions and pain parameters and also attenuated the oxidative-nitrosative stress markers. Further, the combination also reduced the overactivation of PARP as evident from increased NAD levels and decreased PAR immunopositivity in sciatic nerve microsections. Thus, it can be concluded that treatment with the combination of a PDC and PARP inhibitor attenuates alteration in peripheral nerves in diabetic neuropathy (DN).     

AraCyc: a biochemical pathway database for Arabidopsis

AraCyc is a database containing biochemical pathways of Arabidopsis, developed at The Arabidopsis Information Resource (http://www.arabidopsis.org). The aim of AraCyc is to represent Arabidopsis metabolism as completely as possible with a user-friendly Web-based interface. It presently features more than 170 pathways that include information on compounds, intermediates, cofactors, reactions, genes, proteins, and protein subcellular locations. The database uses Pathway Tools software, which allows the users to visualize a bird's eye view of all pathways in the database down to the individual chemical structures of the compounds. The database was built using Pathway Tools' Pathologic module with MetaCyc, a collection of pathways from more than 150 species, as a reference database. This initial build was manually refined and annotated. More than 20 plant-specific pathways, including carotenoid, brassinosteroid, and gibberellin biosyntheses have been added from the literature. A list of more than 40 plant pathways will be added in the coming months. The quality of the initial, automatic build of the database was compared with the manually improved version, and with EcoCyc, an Escherichia coli database using the same software system that has been manually annotated for many years. In addition, a Perl interface, PerlCyc, was developed that allows programmers to access Pathway Tools databases from the popular Perl language. AraCyc is available at the tools section of The Arabidopsis Information Resource Web site (http://www.arabidopsis.org/tools/aracyc).     

Antiviral immunity following smallpox virus infection: a case-control study

Outbreaks of smallpox (i.e., caused by variola virus) resulted in up to 30% mortality, but those who survived smallpox infection were regarded as immune for life. Early studies described the levels of neutralizing antibodies induced after infection, but smallpox was eradicated before contemporary methods for quantifying T-cell memory were developed. To better understand the levels and duration of immunity after smallpox infection, we performed a case-control study comparing antiviral CD4(+) and CD8(+) T-cell responses and neutralizing antibody levels of 24 smallpox survivors with the antiviral immunity observed in 60 smallpox-vaccinated (i.e., vaccinia virus-immune) control subjects. We found that the duration of immunity following smallpox infection was remarkably similar to that observed after smallpox vaccination, with antiviral T-cell responses that declined slowly over time and antiviral antibody responses that remained stable for decades after recovery from infection. These results indicate that severe, potentially life-threatening disease is not required for the development of sustainable long-term immunity. This study shows that the levels of immunity induced following smallpox vaccination are comparable in magnitude to that achieved through natural variola virus infection, and this may explain the notable success of vaccination in eradicating smallpox, one of the world's most lethal diseases.     

Molecular and biochemical studies of virus infections in two filamentous brown algae

The filamentous marine brown algae Ectocarpus siliculosus and Feldmannia simplex are infected by host specific DNA-viruses. Under Percoll isolation, Ectocarpus siliculosus-virus (EsV)-particles maintained their infective potential.The EsV has a circular genome of dsDNA with a size of 320 kb. A restriction map has been established. The gene of a major coat protein (gp1) was detected in a genomic library and partly sequenced. Using gpl- sequences for polymerase chain reaction (PCR) amplification analysis, EsV specific sequences could be detected in various symptom-free, clonal cultures of Ectocarpus. The PCR was also used to follow the passage of the virus genome during the meiosis of hosts. A monospecific antibody against recombinant gpl was used for immunostaining and infection experiments.The Feldmannia simplex-virus (FlexV-1) has a circular genome with a size of 220 kb and a 43% G+C content. FsV-DNA contains methylated bases. 5-methylcytosine (5 mC) makes up 12% of the total cytosines.     

MedicCyc: a biochemical pathway database for Medicago truncatula

MOTIVATION: There is an imperative need to integrate functional genomics data to obtain a more comprehensive systems-biology view of the results. We believe that this is best achieved through the visualization of data within the biological context of metabolic pathways. Accordingly, metabolic pathway reconstruction was used to predict the metabolic composition for Medicago truncatula and these pathways were engineered to enable the correlated visualization of integrated functional genomics data. Results: Metabolic pathway reconstruction was used to generate a pathway database for M. truncatula (MedicCyc), which currently features more than 250 pathways with related genes, enzymes and metabolites. MedicCyc was assembled from more than 225,000 M. truncatula ESTs (MtGI Release 8.0) and available genomic sequences using the Pathway Tools software and the MetaCyc database. The predicted pathways in MedicCyc were verified through comparison with other plant databases such as AraCyc and RiceCyc. The comparison with other plant databases provided crucial information concerning enzymes still missing from the ongoing, but currently incomplete M. truncatula genome sequencing project. MedicCyc was further manually curated to remove non-plant pathways, and Medicago-specific pathways including isoflavonoid, lignin and triterpene saponin biosynthesis were modified or added based upon available literature and in-house expertise. Additional metabolites identified in metabolic profiling experiments were also used for pathway predictions. Once the metabolic reconstruction was completed, MedicCyc was engineered to visualize M. truncatula functional genomics datasets within the biological context of metabolic pathways. Availability: freely accessible at http://www.noble.org/MedicCyc/     

Antiviral effect of ribonuclease conjugated oligodeoxynucleotides targeting the IRES RNA of the hepatitis C virus

Hepatitis C virus (HCV) translation initiation is mediated by a highly structured and conserved RNA, termed the Internal Ribosome Entry Site (IRES), located at the 5′-end of its single stranded RNA genome. It is a key target for the development of new antiviral compounds. Here we made use of the recently developed HCV cell culture system to test the antiviral activity of artificial ribonucleases consisting of imidazole(s) linked to antisense oligodeoxynucleotides targeting the HCV IRES. Results from the cell culture model indicate that the naked antisense oligodeoxynucleotide displayed an efficient antiviral activity. Despite the increased activity observed with the addition of imidazole moieties when tested with the cell-free system, it appears that these improvements were not reproduced in the cellular model.     

Inhibiting alternative pathway complement activation by targeting the factor D exosite

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.     

Antiviral activity of human immunodeficiency virus type 1 Gag-specific cytotoxic T lymphocyte targeting is not necessarily intrinsically superior to envelope targeting

Across several cohorts, human immunodeficiency virus type 1 (HIV-1) Gag- and Env-specific CD8(+) T lymphocyte (CTL) responses have demonstrated inverse and positive correlations, respectively, to viremia. The mechanism has been proposed to be superior antiviral activity of Gag-specific CTLs in general. Addressing this hypothesis, we created two HIV-1 constructs with an epitope translocated from Gag (SLYNTVATL, SL9) to Env, thereby switching the protein source of the epitope. A virus expressing SL9 in Env was similar to the original virus in susceptibility to SL9-specific CTLS. This finding suggests that Env targeting is not intrinsically inferior to Gag targeting for CTL antiviral activity.     

Prevalence of active hepatitis C virus infections among general public of Lahore,Pakistan

Background

To find out the prevalence of active hepatitis C virus (HCV) infections among general public in Lahore city, since data concerning the prevalence of active HCV in this city is currently unavailable.

Methods

Blood samples were collected randomly from individuals visiting different clinical laboratories in Lahore. Serum was separated and processed by nested PCR qualitative assay for the detection of HCV RNA. The samples were categorized into different age groups on the basis of pre-test questionnaires in order to record the age-wise differences regarding the prevalence of active HCV. Data were analyzed statistically using Chi-Square test.

Results

Out of the 4246 blood samples analyzed in this study, 210 were confirmed to be positive for active HCV infection. Gender-wise active HCV prevalence revealed no significant difference [OR =?1.10 CI =?(0.83-1.46), p >?0.05]. However, among the age groups the highest prevalence was observed in the age groups 20–29 (7.7%) and 30–39 years (6.4%) with odds of prevalence of 14.8% (OR =?2.48, CI =?(1.40-4.38), p <?0.05) and 10.3% (OR =?2.03, CI =?(1.10-3.71), respectively. In age groups above 40 years (40–49, 50–59 and >59 years), a decrease in levels of active HCV prevalence was observed.

Conclusions

Among tested samples, 4.9% of the subjects were confirmed to harbour active HCV infections and the “middle aged” population in Lahore was found to be at a higher risk of the HCV ailments compared to both their younger and older peers.

     

Gluconeogenesis: An ancient biochemical pathway with a new twist

Synthesis of sugars from simple carbon sources is critical for survival of animals under limited nutrient availability. Thus, sugar-synthesizing enzymes should be present across the entire metazoan spectrum. Here, we explore the evolution of glucose and trehalose synthesis using a phylogenetic analysis of enzymes specific for the two pathways. Our analysis reveals that the production of trehalose is the more ancestral biochemical process, found in single cell organisms and primitive metazoans, but also in insects. The gluconeogenic-specific enzyme glucose-6-phosphatase (G6Pase) first appears in Cnidaria, but is also present in Echinodermata, Mollusca and Vertebrata. Intriguingly, some species of nematodes and arthropods possess the genes for both pathways. Moreover, expression data from Drosophila suggests that G6Pase and, hence, gluconeogenesis, initially had a neuronal function. We speculate that in insects—and possibly in some vertebrates—gluconeogenesis may be used as a means of neuronal signaling.     

A biochemical pathway for a cellular behaviour: pHi, phosphorylcreatine shuttles, and sperm motility

The transmission of electrical impulses in nerve and muscle cells depends fundamentally on the operation of specific ion channels in their membranes. Recent technical advances in electrical recording from cell membranes have permitted the analysis of the properties of single ion channels and the measurement of gating currents. The results have revealed considerable complexities, in particular in the operation of voltage-gated sodium channels, and in the relationships between the several open and closed states of the channels. An important new development is the cloning and analysis of the structural genes for the acetylcholine receptor and sodium channel protein, which promises to yield fresh insights into the functioning of these proteins.     

Metabolism of D-mannose in Aerobacter aerogenes: evidence for a cyclic pathway

Kamel, M. Y. (Michigan State University, East Lansing), and R. L. Anderson. Metabolism of d-mannose in Aerobacter aerogenes: evidence for a cyclic pathway. J. Bacteriol. 92:1689-1697. 1966.-Evidence is presented which suggests a cyclic pathway for the constitutive utilization of d-mannose in extracts of Aerobacter aerogenes PRL-R3. d-Mannose is phosphorylated with d-glucose-6-phosphate to yield d-mannose-6-phosphate and d-glucose. d-Glucose-6-phosphate may be regenerated by isomerization of d-mannose-6-phosphate through d-fructose-6-phosphate, or by phosphorylation of d-glucose with adenosine-5'-triphosphate. The pathway involves the participation of four constitutive enzymes: d-glucose-6-phosphate isomerase, d-mannose-6-phosphate isomerase, a stereospecific d-glucokinase, and a phosphotransferase which phosphorylates d-mannose with d-glucose-6-phosphate, acetyl phosphate, or carbamyl phosphate. The absence of d-mannokinase (adenosine-5'-triphosphate:d-mannose phosphotransferase) activity in extracts of this organism suggests that the pathway may be of functional significance. Also, the pathway accounts for an apparent 2-epimerization of d-mannose to d-glucose that was observed in extracts.     

Starfish oocyte maturation: evidence for a cyclic AMP-dependent inhibitory pathway

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Cyclic AMP seems to play a negative role in maturation since 1-MeAde triggers a decrease of the oocyte cAMP concentration and since intracellular microinjections of cAMP delay or inhibit maturation. Cyclic GMP is also inhibitory but other nucleotides such as cCMP, cIMP, and cUMP are inactive. The involvement of cAMP and cGMP in the control of oocyte maturation has been further investigated by the use of the stereoisomers of the phosphodiesterase-stable adenosine and guanosine 3',5'-phosphorothioates (cAMPS and cGMPS). The Sp isomers of cAMPS and cGMPS respectively activate cAMP-dependent protein kinase and cGMP-dependent kinase, while the Rp isomers inhibit the kinases. Extracellular addition of these cAMPS and cGMPS isomers has no effect on the oocytes. Intracellular microinjection of the kinase-activating (Sp)-cAMPS and (Sp)-cGMPS delays or inhibits 1-MeAde-induced maturation in a concentration-dependent manner (I50, 30 and 300 microM, respectively). Microinjections of (Rp)-cAMPS and (Rp)-cGMPS have no inhibitory effects and neither trigger nor facilitate maturation. Using various analogs, we found that the delaying or inhibiting effect is restricted to the compounds activating cAMP-dependent kinase, while the compounds inactive on or inhibiting the kinase have no effects on maturation. The inhibitory effect of (Sp)-cAMPS can be reversed by comicroinjection of the heat-stable inhibitor of cAMP-dependent protein kinase, by comicroinjection of the antagonist (Rp)-cAMPS, or by an increase in the 1-MeAde concentration. The negative effects of (Sp)-cAMPS or (Sp)-cGMPS are observed only when these isomers are microinjected during the hormone-dependent period. These results suggest that a cAMP-dependent inhibitory pathway participates in the maintenance of the prophase arrest of oocytes and that 1-MeAde acts both by inhibiting this negative pathway (dis-inhibitory pathway) and by stimulating a parallel activatory pathway leading to oocyte maturation. The generality of this mechanism is discussed.     

Precise hit: adeno-associated virus in gene targeting

Vectors based on the adeno-associated virus (AAV) have attracted much attention as potent gene-delivery vehicles, mainly because of the persistence of this non-pathogenic virus in the host cell and its sustainable therapeutic gene expression. However, virus infection can be accompanied by potentially mutagenic random vector integration into the genome. A novel approach to AAV-mediated gene therapy based on gene targeting through homologous recombination allows efficient, high-fidelity, non-mutagenic gene repair in a host cell.     

Antiviral compounds modulate elasticity,strength and material fatigue of a virus capsid framework

This study investigates whether the biochemical and antiviral effects of organic compounds that bind different sites in the mature human immunodeficiency virus capsid may be related to the modulation of different mechanical properties of the protein lattice from which the capsid is built. Mechanical force was used as a probe to quantify, in atomic force microscopy experiments at physiological pH and ionic strength, ligand-mediated changes in capsid lattice elasticity, breathing, strength against local dislocation by mechanical stress, and resistance to material fatigue. The results indicate that the effects of the tested compounds on assembly or biochemical stability can be linked, from a physics-based perspective, to their interference with the mechanical behavior of the viral capsid framework. The antivirals CAP-1 and CAI-55 increased the intrinsic elasticity and breathing of the capsid protein lattice and may entropically decrease the probability of the capsid protein to assemble into a functionally competent conformation. Antiviral PF74 increased the resistance of the capsid protein lattice to disruption by mechanical stress and material fatigue and may enthalpically strengthen the basal capsid lattice against breakage and disintegration. This study provides proof of concept that the interrogation of the mechanical properties of the nanostructured protein material that makes a virus capsid may provide fundamental insights into the biophysical action of capsid-binding antiviral agents. The implications for drug design by specifically targeting the biomechanics of viruses are discussed.