A productivity bottleneck in the Baltic herring (Clupea harengus membras): Early life-history processes and recruitment variability.

Exogenous anomalies induced by contemporary climate change may severely impact dynamics of early life stages of fish. Here, we modelled how growth rate and abundance of postflexion larvae, and recruitment of Baltic spring-spawning herring (Clupea harengus membras) in the Pärnu Bay, Gulf of Riga (GoR) may respond to shifting climate variables. Higher larval growth rates were aligned with later seasonal emergence of yolk-sac larvae, while lower abundance of postflexion larvae occurred in years of earlier seasonal seawater warming. Cooler temperatures (<16 °C) in spring expanded the optimal thermal window for first-feeding herring larvae, attributable to the absence of early seasonal water temperature warming. Higher recruitment levels emerged in years of seasonally delayed warming and were associated with higher abundance of postflexion larvae. In recent decades, the trend towards earlier warming of the Baltic Sea in spring threatens to create a bottleneck to successful recruitment of herring. The existing paradigm that abundant Baltic herring year-classes occur only in the years following mild winters no longer stands as environmental conditions undergo rapid change. The relative contribution of Pärnu Bay larval nursery areas to recruitment has diminished as the suitable thermal window has been dramatically reduced in recent decades. Evolving thermal dynamics in the GoR have developed relatively recently and in future present a bottleneck for herring production.

Feeding ecology of pelagic fish species in the Gulf of Riga (Baltic Sea): the importance of changes in the zooplankton community.

The feeding ecology of four pelagic fish species was studied in relation to their prey availability in the Gulf of Riga (Baltic Sea) during the summer 1999-2006. The zooplankton community was dominated by the cladoceran Bosmina longispina, rotifers Keratella cochlearis and K. quadrata and the copepod Eurytemora affinis, with the highest interannual variability in abundance recorded for B. longispina. The last influenced the diet of adult sprat Sprattus sprattus, juvenile smelt Osmerus eperlanus and three-spined stickleback Gasterosteus aculeatus as these were strongly selecting for B. longispina. The fish feeding activity did not match the abundance dynamics of their preferred prey, suggesting that fishes may switch to consume other prey in case the preferred diet was limited. A considerable dietary overlap indicated high potential competition between pelagic fish species. While herring Clupea harengus membras and G. aculeatus were relying on very different food, the diets of young O. eperlanus and G. aculeatus were very similar. Interannual variability in zooplankton composition and abundance significantly affected the diet composition of fishes, but those changes were insufficient to exert a consistent influence upon fish feeding activity and total amounts of zooplankton consumed.

Levels of PCDD/F and dioxin-like PCB in Baltic fish of different age and gender.

Concentrations of polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF) and polychlorinated biphenyls (PCB) in Baltic Sea fish like herring (Clupea harengus membras), sprat (Sprattus sprattus balticus), perch (Perca fluviatilis), pikeperch (Sander lucioperca) and flounder (Platichthys flesus trachurus) collected from four areas of the Estonian coastal waters are reported. All samples are studied for their relationship between the length (cm) and wet weight (g); length (cm) and age (years); lipid content and dry matter. The level of PCDD/F and PCB concentrations in younger 1-5 years old Baltic herring and sprat collected in 2002-2005 from the eastern and central parts of the Gulf of Finland, Gulf of Riga and Open Baltic Sea (Central Baltic) is related to the fish age and compared with those found in the 1990s. In addition, PCDD/F and PCB concentrations of different age groups herring, sprat, perch, pikeperch and flounder collected in 2003-2004 from the Lake Peipsi, Gulf of Finland, Gulf of Riga and Open Baltic Sea are related also to their age. Consequently, it was manifested that in older Baltic fish the concentrations of PCDD/F and PCB were higher than in the younger age groups. By the help of principle component analysis (PCA) the effect of gender on the concentrations of PCDD/F for the juvenile Baltic herring and sprat collected in 2004-2005 is investigated for the first time. It was summarized that the biological factor age plays a large role for the contamination of the fish with important toxic organohalogenated compounds such as PCDD/F.

Neutrophil and monocyte adhesion molecules in bronchopulmonary dysplasia, and effects of corticosteroids.

AIMS: To study a longitudinal change in the expression of adhesion molecules CD11b, CD18, and CD62L on neutrophils and monocytes in very low birth weight babies who develop respiratory distress syndrome, to compare these levels between bronchopulmonary dysplasia (BPD) and non-BPD infants, and to assess the effect of corticosteroid treatment on these adhesion molecules. METHODS: Of 40 eligible neonates, 11 neonates were oxygen dependent at 36 weeks (BPD 36 weeks), 16 infants were oxygen dependent at 28 days, but not at 36 weeks (BPD d28), and 13 infants did not develop BPD. Seventeen neonates received a six day course of steroid treatment. Expression of CD11b, CD18, and CD62L was measured on neutrophils and monocytes in arterial blood on days 1, 3, 7, 14, 21, and 28, and before and 2-3 days after initiation of dexamethasone treatment by flow cytometry. RESULTS: CD18 expression on neutrophils and monocytes and CD62L on neutrophils, measured as mean fluorescent intensity, was significantly decreased in BPD neonates compared to non-BPD neonates on days 1-28. Dexamethasone treatment significantly decreased CD11b, CD18, and CD62L expression on neutrophils, and CD11b and CD18L expression on monocytes. CONCLUSIONS: Decreased CD18 expression on neutrophils and monocytes, and decreased CD62L expression on neutrophils, measured as mean fluorescent intensity during the first four weeks of life in micropremies may be risk factors and early predictors of BPD. Dexamethasone use was associated with decreased expression of CD11b, CD18, and CD62L.

Respiratory burst activity in bronchopulmonary dysplasia and changes with dexamethasone.

The first objective of this study was to evaluate longitudinal changes in respiratory burst activity in circulating neutrophils and monocytes in infants of less than 30 weeks of gestation with respiratory distress syndrome (RDS), and to examine differences in neonates who subsequently developed bronchopulmonary dysplasia (BPD) compared with those neonates who did not. The second objective was to investigate the effects of dexamethasone on respiratory burst activity in neutrophils and monocytes. We measured burst activity on neutrophils and monocytes in fresh heparinized blood in response to E. coli, N-formyl-met-leu-phe (fMLP), and phorbol 12-myristate 13-acetate stimulation on days 3, 7, 14, and 21 of life, before and 2-3 days after initiating a 6-day course of dexamethasone treatment. Infants with RDS participating in the study were followed until discharge, and were classified as non-BPD and either 1) BPD d28, reflecting their oxygen requirement at day of life 28, or 2) BPD 36 weeks, reflecting oxygen dependence at 36 weeks' corrected gestational age. The diagnosis of BPD was supported by radiological changes of BPD. The percentage of activated neutrophils producing a respiratory burst increased in all premature infants with increasing postnatal days during the first 28 days of life, when the physiological stimulus E. coli was used as an activator (P < 0.02). There was no significant difference in respiratory burst activity measured either as percent activation or as mean fluorescence intensity between non-BPD and BPD infants after adjusting for the difference in weight and gestational age between the two groups. The treatment of premature infants with dexamethasone was associated with decreased activation of neutrophils (P < 0.005) when E. coli was used as a stimulus. In conclusion, a significant increase in neutrophil respiratory burst activity occurs during the first month of life in very low birth weight infants. Greater pulmonary damage in BPD cannot be attributed to reduced burst activity in either neutrophils or monocytes. Dexamethasone treatment was associated with decreased neutrophil respiratory burst activity.

Cloning of differentially expressed genes in an HIV-1 resistant T cell clone by rapid subtraction hybridization, RaSH.

An HIV-1 resistant T cell clone R1c2 has been generated that carries mutant, latent HIV-1 in a minority of the cell population. Resistant cells express HIV-1 receptors CD4 and CXCR4 and display resistance to infection by wild type (wt) HIV-1 at the level of virus transcription. To begin to define the repertoire of genes modulated in R1c2 cells that correlate with and potentially control expression of the HIV-1 resistance phenotype we have employed a rapid subtraction hybridization (RaSH) technique. For this approach, cDNA libraries were prepared from double-stranded cDNAs that were enzymatically digested into small fragments, ligated to adapters, PCR amplified followed by incubation of tester and driver PCR fragments. The RaSH scheme resulted in the cloning of genes displaying differential expression between HIV-1 resistant (R1c2) and susceptible (SupT1) cells, including known genes and those not described in current DNA databases. Analysis of the pattern of expression of the differentially expressed genes documented eleven genes with enhanced (HR clones) and six genes with reduced (HS clones) expression in HIV-1 resistant versus HIV-1 susceptible T-cell clones.

Prolonged infection of peripheral blood lymphocytes by Vif-negative HIV type 1 induces resistance to productive HIV type 1 infection through soluble factors.

The auxiliary protein Vif is essential for productive HIV-1 infection of primary lymphocytes and macrophages. Vif is required for the synthesis of infectious progeny virus and infection of peripheral blood lymphocytes (PBLs) by Vif-negative HIV-1 was thought to be confined to a single cycle. Here we define conditions for the maintenance of Vif-negative HIV-1 in PBLs during multiple rounds of viral infection. PBLs were infected with Vif-negative HIV-1 and then were serially cocultivated with uninfected PBLs. As determined by measurement of viral DNA, viral burdens declined but then rebounded and reached 1 copy per 30 cells after 7 weeks of culture. Viral core antigen p24 levels dropped and remained below detection limits after three cocultivations with no observed cytotoxicity. Viral RNA was also undetectable in cocultivated cells. The incapacitating deletion in vif was maintained during cocultivation as shown by the size of the vif amplicon. The presence of viral DNA in the absence of viral p24 RNA or protein suggested that the cells were capable of control of HIV-1 expression. This regulatory capacity was confirmed by the demonstration of resistance of PBLs or isolated CD4-positive cells to expression of exogenous wild-type R5 or X4 HIV-1. Resistant PBLs were susceptible to fusion with HIV-1 envelope-expressing cells and to reverse transcription of incoming viral DNA, indicating that the block to replication of exogenous virus was imposed after viral entry and DNA synthesis. Using a dual-chamber apparatus, we demonstrated that resistant Vif-negative HIV-1-infected PBLs secrete soluble factors that confer resistance on naive cells. These findings indicate that Vif-negative HIV-1 infection of primary CD4-positive lymphocytes results in maintenance of unexpressed virus and induces the production of soluble factors conferring resistance to wild-type HIV-1 replication on uninfected cells.

Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.

Synthesis of full-length viral DNA in CD4-positive membrane vesicles exposed to HIV-1. A model for studies of early stages of the hiv-1 life cycle.

CD4-positive membrane vesicles (MV) were isolated under isotonic conditions from human T lymphoblastoid cells MT-2 and CEM and tested for their ability to support reverse transcription of viral RNA upon exposure to human immunodeficiency virus, type 1 (HIV-1). MV contained cytoplasms as confirmed by the presence of mitochondrial DNA but were devoid of chromosomal DNA. Virus binding and vesicle lysis assays revealed that 4-19% (depending upon virus dose) of MV-bound HIV-1 entered the vesicles. HIV-1 internalized in MV was able to initiate and complete viral DNA synthesis as determined by the detection of products of reverse transcription using polymerase chain reaction amplification of viral DNA using regions present in early (strong stop) transcripts and full-length double-stranded molecules. Viral DNA was undetectable in MV exposed to HIV-1 at 0 degrees C, in MV exposed to UV-inactivated virus at 37 degrees C, or after exposure to intact virus at 37 degrees C in the presence of reverse transcriptase inhibitors 2',3'-dideoxycytidine and a tetrahydroimidazo[4,5,1-jk](1,4)-benzodiazepin-2-(1H)-thione derivative, indicating that viral DNA detected in HIV-1-exposed MV was synthesized de novo. Kinetic studies revealed that HIV-1 DNA synthesis in MV was very rapid; full-length viral DNA was detected within 15 min of exposure at 37 degrees C, and the DNA levels increased 90-fold after 1 h and declined thereafter. Strong stop viral DNA was 10-fold more abundant than full-length DNA after 1 h at 37 degrees C, indicating that 10% of input viral genomes are fully transcribed in MV within this time frame. This system preserves the critical features of intact CD4-bearing cells to permit studies of HIV-1 entry, uncoating, and reverse transcription of viral RNA.

Genetic variability and function of the long terminal repeat from syncytium-inducing and non-syncytium-inducing human immunodeficiency virus type 1.

We analyzed sequence variability and function of the long terminal repeat (LTR) from syncytium-inducing (SI) and non-syncytium-inducing (NSI) HIV-1. Twenty LTR DNA clones were obtained by polymerase chain reaction amplification and molecular cloning from short-term cultures of SI and NSI viruses from an AIDS patient and two asymptomatic individuals, respectively. All the LTR clones tested contained multiple nucleotide changes (mostly G-to-A transitions), compared to the subtype B consensus sequence, which were clustered within the negative regulatory element, including NF-AT, USF, and TCF-1 alpha binding sites. The core promoter/TAR region sequences were highly conserved. The basal and Tat-mediated transcriptional activities of selected LTR clones tested were 0.1 to 1 and 0.2 to 0.5 times that of the control, respectively, regardless of the SI or NSI origin of the clones. Phylogenetic analysis revealed interi-solate sequence divergence in the LTR that was similar but not identical to previously analyzed vif sequences from the same samples. In particular, the inter-isolate distances from reference sequences differed for the LTR and vif. This raises the possibility that recombination occurred between corresponding LTR and vif loci of the quasi-species present in the isolates described here.

Interference to human immunodeficiency virus type 1 infection in the absence of downmodulation of the principal virus receptor, CD4.

It is thought that interference during human immunodeficiency virus type 1 (HIV-1) infection is established by downmodulation of the principal virus receptor, CD4. Here we present evidence to the contrary. At various times after primary infection, we superinfected T cells in vitro by exposure to a genetically distinct viral clone or to a virus carrying the chloramphenicol acetyltransferase gene. Replication of each virus strain was determined by restriction enzyme analysis of total cellular DNA, by PCR amplification of viral DNA, or by assay of cell extracts for chloramphenicol acetyltransferase activity. We found that efficient viral interference is established within 24 h of infection at a multiplicity of infection of 1. At that time, expression of viral structural proteins was low and infected cells displayed undiminished levels of surface CD4 and were fully susceptible to virus binding and fusion. Superinfection by either cell-free HIV-1 or cocultivation was blocked. Cells resistant to superinfection by HIV-1 remained susceptible to Moloney murine leukemia and vaccinia viruses. No interference was observed 4 h after primary infection or in cells infected with either UV-inactivated HIV-1 or a mutant virus defective in virus-cell fusion activity, indicating that binding of primary virus to CD4 is insufficient to prevent superinfection. The minimum viral requirements for this interference are that HIV-1 must be able to enter cells and synthesize viral DNA; Tat-mediated transcription is dispensable. Our results support the existence of a novel pathway to interference to HIV-1 infection, which we term postentry interference, which blocks superinfection during intracellular phases of the virus life cycle.

Aberrant Gag protein composition of a human immunodeficiency virus type 1 vif mutant produced in primary lymphocytes.

Productive, spreading infection of peripheral blood lymphocytes (PBL) with human immunodeficiency virus type 1 (HIV-1) requires the viral protein Vif. To study the requirement for vif in this system, we infected PBL with a phenotypically complemented HIV-1 clone mutated in vif. Progeny virus was produced which was noninfectious in PBL but replicated in SupT1 cells. Analysis of metabolically labeled proteins of sedimentable extracellular particles made in PBL by radioimmunoprecipitation with either serum from a patient with AIDS or a monoclonal antibody reactive with HIV-1 Gag proteins revealed that vif-negative but not wild-type particles carry higher levels of p55, p41, and p38 Gag-specific proteins compared with those of p24. Similar results were obtained with sucrose-purified virions. Our data indicate that vif plays a role in Gag protein processing or in incorporation of processed Gag products into mature virions. The presence of unprocessed precursor Gag polyprotein (Pr55gag) and other Gag processing intermediates in PBL-derived vif-negative extracellular particles may contribute to the reduced infectivity of this virus.