Molecular evolutionary dynamics of cytochrome b in strepsirrhine primates: the phylogenetic significance of third-position transversions

DNA sequences of the complete cytochrome b gene are shown to contain robust phylogenetic signal for the strepsirrhine primates (i.e., lemurs and lorises). The phylogeny derived from these data conforms to other molecular studies of strepsirrhine relationships despite the fact that uncorrected nucleotide distances are high for nearly all intrastrepsirrhine comparisons, with most in the 15%-20% range. Cytochrome b sequences support the hypothesis that Malagasy lemuriforms and Afro-Asian lorisiforms each comprise clades that share a sister- group relationship. A study (Adkins and Honeycutt 1994) of the cytochrome c oxidase subunit II (COII) gene placed one Malagasy primate (Daubentonia) at the base of the strepsirrhine clade, thereby suggesting a diphyletic Lemuriformes. The reanalysis of COII third- position transversions, either alone or in combination with cytochrome b third-position transversions, however, yields a tree that is congruent with phylogenetic hypotheses derived from cytochrome b and other genetic data sets.      

Identification of changes in gene loci potentially associated with cervical cancer using NotI microarrays

The investigation of the cancer-associated structural and epigenetic changes in cell genome is a major approach for understanding mechanisms of cancerogenesis. To investigate these genome changes, novel technique of microarrays comprising NotI-linking genome clones was developed. Twenty eight samples from patients with cervical cancer were analyzed using NotI microarrays of human chromosome 3. Deletions, amplifications and methylation were detected for 109 out of 182 NotI clones with different frequency. Notably, 17 NotI-linking clones showed genomic changes in more than 35% of tumor samples investigated, which suggests involvement of genes associated with these clones in development of cervical cancer.     

An In-Depth Comparison of Latent HIV-1 Reactivation in Multiple Cell Model Systems and Resting CD4+ T Cells from Aviremic Patients

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.     

LRRC3B gene is frequently epigenetically inactivated in several epithelial malignancies and inhibits cell growth and replication

Chromosome 3 specific NotI microarrays containing 180 NotI linking clones associated with 188 genes were hybridized to NotI representation probes prepared using matched tumor/normal samples from major epithelial cancers: breast (47 pairs), lung (40 pairs) cervical (43 pairs), kidney (34 pairs of clear cell renal cell carcinoma), colon (24 pairs), ovarian (25 pairs) and prostate (18 pairs). In all tested primary tumors (compared to normal controls) methylation and/or deletions was found. For the first time we showed that the gene LRRC3B was frequently methylated and/or deleted in breast carcinoma - 32% of samples, cervical - 35%, lung - 40%, renal - 35%, ovarian - 28%, colon - 33% and prostate cancer - 44%. To check these results bisulfite sequencing using cloned PCR products with representative two breast, one cervical, two renal, two ovarian and two colon cancer samples was performed. In all cases methylation was confirmed. Expression analysis using RT-qPCR showed that LRRC3B is strongly down-regulated at the latest stages of RCC and ovarian cancers. In addition we showed that LRRC3B exhibit strong cell growth inhibiting activity (more than 95%) in colony formation experiments in vitro in KRC/Y renal cell carcinoma line. All these data suggest that LRRC3B gene could be involved in the process of carcinogenesis as a tumor suppressor gene.     

Interactions of neural glycosaminoglycans and proteoglycans with protein ligands: assessment of selectivity, heterogeneity and the participation of core proteins in binding

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8- fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400- fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.      

Size and mortality rates of Glossina pallidipes in the semi-arid zone of southwestern Kenya

Seasonal changes in the mean size of tsetse, Glossina pallidipes Austen, as indicated by wing vein length, were monitored during 1983-86 at Nguruman, southwestern Kenya. Changes in size of nulliparous females and wing fray category 1 males were shown to be correlated with the relative humidity 2 months before they were captured. Soil temperature when flies were in the pupal stage had much less effect. Size dependent mortality was demonstrated, with the mean size of flies emerging from pupae significantly less than that of field-caught flies. This mortality must occur at emergence, since there was no evidence of size-dependent mortality once the flies became available to the trap. Size was correlated with density-independent mortality acting on the parent population 2 months previously. It might therefore be possible to use size as an index of the intensity of such mortality. This could be useful when assessing the level of additional mortality required to suppress tsetse populations.     

Nuclear DNA sequences from late Pleistocene megafauna

We report the retrieval and characterization of multi- and single-copy nuclear DNA sequences from Alaskan and Siberian mammoths (Mammuthus primigenius). In addition, a nuclear copy of a mitochondrial gene was recovered. Furthermore, a 13,000-year-old ground sloth and a 33,000- year-old cave bear yielded multicopy nuclear DNA sequences. Thus, multicopy and single-copy genes can be analyzed from Pleistocene faunal remains. The results also show that under some circumstances, nucleotide sequence differences between alleles found within one individual can be distinguished from DNA sequence variation caused by postmortem DNA damage. The nuclear sequences retrieved from the mammoths suggest that mammoths were more similar to Asian elephants than to African elephants.