Telomerase is activated in the prostate and seminal vesicles of the castrated rat

Telomeres, the repetitive non-coding DNA sequences found at the ends of all eukaryotic chromosomes, shorten with each cell division. It has been proposed that telomere shortening may be the counting element of a mitotic clock that keeps track of cell divisions; with shortening to a critical length acting as a senescence signal underlying cellular aging. The enzyme telomerase functions to maintain telomere length, thus allowing unlimited cell division, and has been associated with cellular immortalization and cancer. Stem cells have large, perhaps unlimited, replicative capacities. Since these cells are potentially immortal, we reasoned that they might posses active telomerase. We therefore assayed for telomerase activity in the stem cell enriched pools of the androgen-depleted sex accessory tissues in the castrated male rat. Following castration, the ventral prostate and seminal vesicles of the rat involute, losing approximately 90% of their cells by 21 days. These residual glands persist, and are enriched for stem cells, being capable of fully regenerating these glands if testosterone is re-introduced into the animal. We assayed telomerase activity in extracts from normal, involuted, and regenerating ventral prostate and seminal vesicles. Normal glands were found to be telomerase negative, whereas telomerase activity appeared as these glands involuted following castration. Conversely, telomerase activity disappeared during testosterone-induced regeneration of these residual glands. These results provide strong evidence for the ability of androgen to negatively-regulate telomerase activity in stem cell populations of the rat ventral prostate and seminal vesicles. and represent the first in vivo model system for the modulation of telomerase activity.     

Screening of Australian medicinal plants for antiviral activity

The nanoencapsulation of a model protein drug, bovine serum albumin (BSA), using gelatin as the matrix material is reported. Nanoencapsulation was conducted using a modified water-in-oil (w/o) emulsion method, which is emulsifier-free and simple. The nanoencapsulation product, BSA-containing gelatin nanoparticles, is characterized in terms of nanoparticle morphology, size and size distribution, water content, and in vitro protein release. The BSA-containing gelatin nanoparticles obtained from this nanoencapsulation process are nearly spherical and have a log-normal size distribution. The average diameter of the BSA-containing gelatin nanoparticles is approximately 840 nm. They can absorb 51-72% of water. In vitro release experiments demonstrate that BSA has been successfully encapsulated in, and can be released from the gelatin nanoparticles. The release of BSA from the gelatin nanoparticulate matrix follows a diffusion-controlled release mechanism. It is found that temperature affects both the water content and the BSA release rate of the gelatin nanoparticles.     

Uptake and intracellular trafficking of asialoglycoprotein-polylysine-DNA complexes in isolated rat hepatocytes

Receptor-mediated gene delivery has been reported for a number of different receptor systems although the intracellular fate of such systems has not been systematically investigated. In this study, we have determined the fate of a commonly used asialoglycoprotein (ASGP)-dependent DNA delivery system in isolated rat hepatocytes. ASPG-polylysine (PLL296) was ionically complexed with pSV-CAT DNA at a molar ratio of 10:1. The resulting complex inhibited 125I-ASGP binding to rat hepatocytes but ASGP only partially inhibited the binding of complex. The ASGP-independent binding was due to the interaction of the PLL component of the complex with plasma membranes and could be minimised by replacing PLL296 with PLL19. Following internalisation, ASGP was cleaved from the complex and translocated to the lysosomes where it was degraded. The DNA, however, remained in an intracellular compartment that cosedimented with plasma membranes in Percoll density gradients. This study shows first that hepatocytes do not process DNA internalised as ASGP complexes in a manner similar to ASGP itself, and second that the differential sorting of the two cleaved molecules leads to a rapid intracellular compartmentalisation of the DNA. Controlled release from this compartment may be a means for prolonged gene expression in gene therapy protocols.     

Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA

Methylation analysis of individual cytosines in genomic DNA can be determined quantitatively by bisulphite treatment and PCR amplification of the target DNA sequence, followed by restriction enzyme digestion or sequencing. Methylated and unmethylated molecules, however, have different sequences after bisulphite conversion. For some sequences this can result in bias during the PCR amplification leading to an inaccurate estimate of methylation. PCR bias is sequence dependent and often strand-specific. This study presents a simple method for detection and measurement of PCR bias for any set of primers, and investigates parameters for overcoming PCR bias.     

Receptive field changes after strokelike cortical ablation: a role for activation dynamics

The reorganization of neural activity that takes place after stroke is of paramount importance in producing functional recovery. Experimental stroke models have suggested that this reorganization may have two phases, but physiology alone cannot fully resolve what causes each phase. Computer modeling suggests that these phases might involve an initial change in dynamics occurring immediately, followed by synaptic plasticity. We combined physiological recording from macaque middle temporal cortex (area MT) with a neural network computer model to examine this first phase of altered cortical function after a small, experimentally induced cortical lesion. Major receptive field (RF) changes seen in the first few days postlesion included both expansion and contraction of receptive fields. Although only expansion could be reproduced in an initial model, addition of inhibitory interneuron loss in a ring around the primary ablation, suggested by immunohistochemical examination, permitted contraction to be replicated as well. We therefore predict that this immunochemical observation reflects an immediate extension of the lesion rather than a late response. Additionally our model successfully predicted a correlation between increased firing rate and RF size. Our model suggests that activation dynamics alone, without anatomic remodeling, can cause the large receptive field changes that allow the rapid behavioral recovery seen after middle temporal lesions.     

Control of vertical eye alignment in three-dimensional space

A target that is nearer to one eye than the other subtends a larger visual angle in the closer eye. Consequently, when making saccades between vertically separated targets that are closer to one eye, there is a vertical retinal disparity that must be overcome by a change in the relative alignment of the eyes. We recorded eye movements in three normal subjects and showed that in such viewing circumstances subjects made unequal vertical saccades that led to a rapid change (peak velocity up to 30 deg/sec) in vertical eye alignment. On average, 81% of the required change in alignment occurred within the saccade for downward movements and 47% for upward movements. Such unequal vertical saccades occurred independently of immediate disparity cues; saccades remained unequal when refixing to the remembered locations of the vertically-oriented targets, or even when the natural vertical disparity was nullified by a prism. On the other hand, when subjects wore the nullifying prism in front of the inferior visual field of the left eye for 8-20 hr, they showed a decrease in saccade disconjugacy (to 12-35% of the preadaptation value) to targets closer to the left eye in the inferior but not in the superior visual field. We suggest that the brain develops a three-dimensional map (horizontal, vertical, depth) for vertical saccade yoking, which is under adaptive control, and which is used to preprogram automatically the relative excursions of the eyes during vertical saccades as a function of the current and the desired point of regard.     

A longitudinal study of adolescents' attitudes about assistance in the development of moral values

A questionnaire-based study of 2,863 adolescents (ages 12 to 22 years), with data collected over 15 years, measured the extent to which informants believed that elementary, secondary, and college-age young people should receive guidance on moral issues from the family, school, clergy, peers, and/or the individual himself or herself. Averaging the ratings of all respondents over the first three categories yielded an overall authority score of 1.85, where a score of 2.00 represented a belief in moderate influence. Results showed a minimal difference in the ratings between genders, among different age groups, and across the different calendar years of questionnaire administration. Much more substantial differences existed among the actual influences investigated and across the ages of those to whom the influence was to be directed.