Simian immunodeficiency virus SIVagm dynamics in African green monkeys

The mechanisms underlying the lack of disease progression in natural simian immunodeficiency virus (SIV) hosts are still poorly understood. To test the hypothesis that SIV-infected African green monkeys (AGMs) avoid AIDS due to virus replication occurring in long-lived infected cells, we infected six animals with SIVagm and treated them with potent antiretroviral therapy [ART; 9-R-(2-phosphonomethoxypropyl) adenine (tenofovir) and beta-2,3-dideoxy-3-thia-5-fluorocytidine (emtricitabine)]. All AGMs showed a rapid decay of plasma viremia that became undetectable 36 h after ART initiation. A significant decrease of viral load was observed in peripheral blood mononuclear cells and intestine. Mathematical modeling of viremia decay post-ART indicates a half-life of productively infected cells ranging from 4 to 9.5 h, i.e., faster than previously reported for human immunodeficiency virus and SIV. ART induced a slight but significant increase in peripheral CD4(+) T-cell counts but no significant changes in CD4(+) T-cell levels in lymph nodes and intestine. Similarly, ART did not significantly change the levels of cell proliferation, activation, and apoptosis, already low in AGMs chronically infected with SIVagm. Collectively, these results indicate that, in SIVagm-infected AGMs, the bulk of virus replication is sustained by short-lived cells; therefore, differences in disease outcome between SIVmac infection of macaques and SIVagm infection of AGMs are unlikely due to intrinsic differences in the in vivo cytopathicities between the two viruses.     

Cutting edge: Experimentally induced immune activation in natural hosts of simian immunodeficiency virus induces significant increases in viral replication and CD4+ T cell depletion

Chronically SIVagm-infected African green monkeys (AGMs) have a remarkably stable nonpathogenic disease course, with levels of immune activation in chronic SIVagm infection similar to those observed in uninfected monkeys and with stable viral loads for long periods of time. In vivo administration of LPS or an IL-2/diphtheria toxin fusion protein (Ontak) to chronically SIVagm-infected AGMs triggered increases in immune activation and subsequently of viral replication and depletion of intestinal CD4(+) T cells. Our study indicates that circulating microbial products can increase viral replication by inducing immune activation and increasing the number of viral target cells, thus demonstrating that immune activation and T cell proliferation are key factors in AIDS pathogenesis.     

Translation of heterologous mRNAs by chloroplast stroma components

In vitro translation of exogenous mRNAs has been difficult to achieve using chloroplast components. An in vitro protein synthesis system is described, based on a pea ( Pisum sativum L, cv. Spring) chloroplast stroma 30000 g supernatant, which was capable of translating polyuridylie acid and MS2 phage RNA into corresponding proteins. The kinetics of label incorporation and fluorograms of unique translation products are presented. The unusual steps which may have contributed to successful translation include the following: the removal of exogenous unlabeled amino acids from the incubation mixture, ultrafiltration of the stromal supernatant and the removal of thylakoid-bound polyribosomes. The system could be further developed to study translational control of gene expression in the chloroplasts.     

Prevalence of the 35delG mutation in the GJB2 gene of patients with nonsyndromic hearing loss from Croatia

The aim of this study was to investigate the allelic frequency of 35delG mutation in patients with recessive, nonsyndromic hearing loss (NSHL) compared to normal hearing individuals in the Croatian population. For this purpose, we analyzed 27 unrelated individuals with nonsyndromic hearing loss and 342 healthy individuals. The method we used is based on the principle of polymerase chain reaction (PCR)-mediated, site-directed mutagenesis, followed by a BsiYI digestion. Among patients with NSHL, the 35delG mutation was found on 51.85% alleles. Carrier frequency among healthy control individuals was 1 in 68.4 (1.5%). The patients, found to be wild-type, either in heterozygous or homozygous form, were further tested by direct sequencing. Among them, two different mutations were observed, W24X and 313del14. Relatively high prevalence of 35delG mutation among patients with NSHL indicate that it is an important cause of NSHL in Croatia. Early diagnosis by identification of the 35delG mutation would greatly improve genetic counseling, as well as treatment and management of deafness in Croatia.     

Temporal variability in abundance and biomass of ciliates and copepods in the eutrophicated part of Kaštela Bay (Middle Adriatic Sea)

Seasonal variations in abundance and carbon biomass of ciliated protozoa and micrometazoa were studied from May 1998 to November 1999 in the eutrophicated area of Katela Bay (Middle Adriatic Sea). Ciliates showed peaks in spring and autumn, primarily due to changes in the abundance and biomass of tintinnines, which participated in total ciliate abundance and biomass with 40.48 and 60.02%, respectively. The highest tintinnine density was 4,278 ind. l^–1, while their average biomass varied from 0.611 to 26.557 gC l^–1 . Maximal average density and biomass of non-loricates were 1,430 ind. l^–1 and 3.925 gC l^–1, respectively. The micrometazoa community was dominated by copepod nauplii, especially during the summer and autumn. The copepod biomass ranged between 3.47 and 26.75 gC l^–1 . High abundance and biomass values of the investigated zooplankton groups point to an important role of these organisms in the secondary production in the Bay, indicating that they may be (1) a crucial factor in controlling the populations of nano-/pico-phytoplankton and heterotrophic nanoflagellates, and (2) a significant prey for larger micrometazoans.     

Unique long terminal repeat and surface glycoprotein gene sequences of feline leukemia virus as determinants of disease outcome

The outcome of feline leukemia virus (FeLV) infection in nature is variable, including malignant, proliferative, and degenerative disorders. The determinants of disease outcome are not well understood but are thought to include viral, host, and environmental factors. In particular, genetic variations in the FeLV long terminal repeat (LTR) and SU gene have been linked to disease outcome. FeLV-945 was previously identified as a natural isolate predominant in non-T-cell neoplastic and nonneoplastic diseases in a geographic cohort. The FeLV-945 LTR was shown to contain unique repeat elements, including a 21-bp triplication downstream of the enhancer. The FeLV-945 SU gene was shown to encode mutational changes in functional domains of the protein. The present study details the outcomes of infection with recombinant FeLVs in which the LTR and envelope (env) gene of FeLV-945, or the LTR only, was substituted for homologous sequences in a horizontally transmissible prototype isolate, FeLV-A/61E. The results showed that the FeLV-945 LTR determined the kinetics of disease. Substitution of the FeLV-945 LTR into FeLV-A/61E resulted in a significantly more rapid disease onset but did not alter the tumorigenic spectrum. In contrast, substitution of both the FeLV-945 LTR and env gene changed the disease outcome entirely. Further, the impact of FeLV-945 env on the disease outcome was dependent on the route of inoculation. Since the TM genes of FeLV-945 and FeLV-A/61E are nearly identical but the SU genes differ significantly, FeLV-945 SU is implicated in the outcome. These findings identify the FeLV-945 LTR and SU gene as determinants of disease.     

Lateral diffusion of CO<Subscript>2</Subscript> in leaves of the crassulacean acid metabolism plant<Emphasis Type="Italic"> Kalanchoë daigremontiana</Emphasis> Hamet et Perrier

Dynamic patchiness of photosystem II (PSII) activity in leaves of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier, which was independent of stomatal control and was observed during both the day/night cycle and circadian endogenous oscillations of CAM, was previously explained by lateral CO₂ diffusion and CO₂ signalling in the leaves [Rascher et al. (2001) Proc Natl Acad Sci USA 98:11801–11805; Rascher and Lüttge (2002) Plant Biol 4:671–681]. The aim here was to actually demonstrate the importance of lateral CO₂ diffusion and its effects on localized PSII activity. Covering small sections of entire leaves with silicone grease was used for local exclusion of a contribution of atmospheric CO₂ to internal CO₂ via transport through stomata. A setup for combined measurement of gas exchange and chlorophyll fluorescence imaging was used for recording photosynthetic activity with a spatiotemporal resolution. When remobilization of malic acid from vacuolar storage and its decarboxylation in the CAM cycle caused increasing internal CO₂ concentrations sustaining high PSII activity behind closed stomata, PSII activity was also increased in adjacent leaf sections where vacuolar malic acid accumulation was minimal as a result of preventing external CO₂ supply due to leaf-surface greasing, and where therefore CO₂ could only be supplied by diffusion from the neighbouring malic acid-remobilizing leaf tissue. This demonstrates lateral CO₂ diffusion and its effect on local photosynthetic activity.     

Immune responses induced by intranasal imiquimod and implications for therapeutics in rhinovirus infections

Notwithstanding the progress recently made in immunology and virology, there is yet no effective, specific treatment for the common cold. Symptomatic treatment is minimally effective. An anecdotal report of rapid clearing of the common cold of recent onset after intranasal application of imiquimod in several subjects by one of the authors, made us test the hypothesis that this treatment works through the secretion of interferon by the nasal mucosa. We decided to do an animal study in primates (Indian Macaca Mulata): 5 treatment and 3 control animals were used. Imiquimod or placebo was massaged into the nares of the animals and periodic samples of post-nasal fluid were taken and measurements for Interferon alpha (IFNalpha) and Tumor Necrosis Factor alpha (TNFalpha) were made by ELISA methods, and kinetic studies. mRNA IFNalpha was also isolated and analyzed by quantitative competitive RT-PCR. The internal standard was constructed to be complementary to and compete with oligonucleotide primers and for amplification of target sequences. One intranasal application of imiquimod rapidly (1-4 Hours) induced high levels of mRNA for IFNalpha, and minimal levels in the control animals. Rapid induction of INFalpha, and proportional increase of TNFalpha sustained for 4 and 6 hours respectively were noted. No adverse reactions to treatment were found in macaques during this short period of intranasal imiquimod usage (except in one macaque with a short period of lacrimation). No animal had cytotoxic effects when examined at 6 hr, 12 hr, 24 hr or 48 hr, except one animal, which had an episode of lacrimation for 6 hr post treatment. Thus both safety and efficacy of short treatment with imiquimod is proven in this animal model. Proof of principle for intranasal treatment of the common cold with imiquimod is shown. We think that this work will encourage a number of double blind clinical trials to confirm the effectiveness of the intranasal treatment of the common cold with imiquimod.     

A High-Resolution Genetic Map of the Familial Mediterranean Fever Candidate Region Allows Identification of Haplotype-Sharing among Ethnic Groups

Familial Mediterranean fever (FMF) is a recessive disorder of inflammation caused by mutations in a gene (designatedMEFV) on chromosome 16p13.3. We have recently constructed a 1-Mb cosmid contig that includes the FMF critical region. Here we show genotype data for 12 markers from our physical map, including 5 newly identified microsatellites, in FMF families. Intrafamilial recombinations placedMEFVin the ∼285 kb betweenD16S468/D16S3070andD16S3376.We observed significant linkage disequilibrium in the North African Jewish population, and historical recombinants in the founder haplotype placedMEFVbetweenD16S3082andD16S3373(∼200 kb). In smaller panels of Iraqi Jewish, Arab, and Armenian families, there were significant allelic associations only forD16S3370andD16S2617among the Armenians. A sizable minority of Iraqi Jewish and Armenian carrier chromosomes appeared to be derived from the North African Jewish ancestral haplotype. We observed a unique FMF haplotype common to Iraqi Jews, Arabs, and Armenians and two other haplotypes restricted to either the Iraqi Jewish or the Armenian population. These data support the view that a few major mutations account for a large percentage of the cases of FMF and suggest that some of these mutations arose before the affected Middle Eastern populations diverged from one another.     

The use of Fmoc-Lys(Pac)-OH and penicillin G acylase in the preparation of novel semisynthetic insulin analogs.

In this paper, we present the detailed synthetic protocol and characterization of Fmoc-Lys(Pac)-OH, its use for the preparation of octapeptides H-Gly-Phe-Tyr-N-MePhe-Thr-Lys(Pac)-Pro-Thr-OH and H-Gly-Phe-Phe-His-Thr-Pro-Lys(Pac)-Thr-OH by solid-phase synthesis, trypsin-catalyzed condensation of these octapeptides with desoctapeptide(B23-B30)-insulin, and penicillin G acylase catalyzed cleavage of phenylacetyl (Pac) group from Nepsilon-amino group of lysine to give novel insulin analogs [TyrB25, N-MePheB26,LysB28,ProB29]-insulin and [HisB26]-insulin. These new analogs display 4 and 78% binding affinity respectively to insulin receptor in rat adipose membranes.