Rat spermatogenesis in vitro traced by quantitative flow cytometry

In vitro differentiation of germ cells in rat seminiferous tubule segments at stages II-III of the epithelial cycle was studied. DNA flow cytometry was used for quantitation of absolute cell numbers from the cultured tubule segments that were compared to freshly isolated stages of the cycle, as identified by transillumination stereomicroscopy of the seminiferous tubules and phase-contrast microscopy of live cell squashes. Spermatogonia and spermatocytes from stages II-III showed normal morphological differentiation during 7 days in vitro. Round spermatids differentiated to Step 7 of spermiogenesis but Step 16 spermatids failed to develop. Acid phosphatase activity in the spermatogenic cells changed normally during the culture. As compared with freshly isolated control tubule segments, 35% of round spermatids and 42% of pachytene spermatocytes were present in culture after 7 days. The cell numbers recovered from defined stages by DNA flow cytometry were close to those found in morphometric studies. Flow cytometry is an efficient quantitation method for cells liberated from seminiferous epithelium. Spermatogonia, spermatocytes, and early spermatids are able to differentiate in vitro, but spermatids approaching the elongation (acrosome) phase, and particularly the maturation phase, fail to differentiate under present culture conditions.     

Sequential Expression of Nucleoproteins during Rat Spermiogenesis

Transition proteins and protamines are highly basic sperm-specific nuclear proteins that serve to compact the DNA during late spermiogenesis. To understand their sequential role in this function, transition protein 1 (TP1), transition protein 2 (TP2), and protamine 1 (P1) were assayed by polyacrylamide gel electrophoresis in pools of microdissected, staged seminiferous tubule segments in the rat. The results were compared with immunocytochemical analyses of squash preparations from accurately identified stages of the epithelial cycle. TP2 was the first to appear as a faint band at stages IX–XI, followed by high levels at stages XII–XIV of the cycle. TP1 showed a low expression at stage XII of the cycle and peaked at stages XIII–I, whereas protamine 1 first appeared at stage I of the cycle and remained high throughout the rest of spermiogenesis. Immunocytochemical analyses and Western blots largely confirmed these results: TP2 in steps 9–14, TP1 in steps 12–15, and P1 from late step 11 to step 19 of spermiogenesis. We propose that TP2 is the first nucleoprotein that replaces histones from the spermatid nucleus, and its appearance is associated with the onset of nuclear elongation. TP1 shows up along with the compaction of the chromatin. The two transition proteins seem to have distinct roles during transformation of the nuclei and compaction of spermatid DNA.     

Effect of drought on proteins and isoenzymes in rice during germination

Two drought tolerant varieties TKM-1 and TKM-2 and two drought susceptible varieties Jaya and Improved Sabarmati of rice were studied for soluble protein pattern and isoenzymes of malate dehydrogenase, glutamate dehydrogenase, esterase and peroxidase during germination at different water stress. MDH, GDH and esterase patterns were not affected, but the soluble proteins were changed. Peroxidase isoenzyme pattern from drought tolerant and susceptible varieties showed characteristic differences. The intensity of bands with higher electrophoretic mobility decreased in Jaya and Improved Sabarmati while in TKM-1 and TKM-2 the intensity of these bands did not change much after 72 hr water stress. In shoots of Jaya and Improved Sabarmati, the activity of the peroxidase isoenzymes decreased more than in TKM-1 and TKM-2 shoots with increase in water stress.     

Characterization of the testicular cell types present in the rat by in vivo 31P magnetic resonance spectroscopy.

Testes of vitamin A-deficient Wistar rats before and after vitamin A replacement, of rats irradiated in utero, and of control rats were investigated by in vivo 31P magnetic resonance (MR) spectroscopy. The testicular phosphomonoester/ATP (PM/ATP) ratio ranged from 0.79 +/- 0.05 for testes that contained only interstitial tissue and Sertoli cells to 1.64 +/- 0.04 for testes in which spermatocytes were the most advanced cell types present. When new generations of spermatids entered the seminiferous epithelium, this ratio decreased. The testicular phosphodiester/ATP (PD/ATP) ratio amounted to 0.16 +/- 0.06 for testes in which Sertoli cells, spermatogonia, or spermatocytes were the most advanced cell type present. When new generations of spermatids entered the seminiferous epithelium, the PD/ATP ratio rapidly increased and finally reached a value of 0.71 +/- 0.06 for fully developed testes. Taken together, specific patterns of the PM/ATP ratio, the PD/ATP ratio, and pH were obtained that were correlated to the presence of spermatogonia, spermatocytes, round spermatids, and elongated spermatids or to the absence of spermatogenic cells. Hence, a good impression of the status of the seminiferous epithelium in the rat can be obtained by in vivo 31P MR spectroscopy.     

Binding of human syndecan to extracellular matrix proteins.

We have isolated a cell surface proteoglycan from a human mammary cell line (HBL-100). This proteoglycan was found to be a human equivalent to mouse syndecan, because (i) it has identical biochemical properties with murine syndecan, including size, charge, buoyant density, and glycosaminoglycan composition, (ii) its core protein has identical size with murine syndecan as studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iii) the core protein is detected with anti-peptide antibody for the cytoplasmic domain of syndecan. HBL-100 cells also showed high expression of syndecan mRNAs, when probed with mouse syndecan cDNA. The ectodomain of the human syndecan revealed binding to type I collagen fibrils and fibronectin but not to laminin, duplicating the binding properties of murine syndecan. Very interestingly, syndecan did not bind to vitronectin, which is known to contain a heparin binding domain and is one of the major adhesive factors of serum for cultured cells. Syndecans are known to change their glycosaminoglycan composition yielding tissue-type specific polymorphic forms of syndecan (Sanderson, R., and Bernfield, M. (1988) Proc. Natl. Acad. Sci. U. S.A. 85, 9562-9566). The members of this family may thus represent a collection of structurally related matrix receptors that could differ in their interactions due to variation of the ectodomain glycosylation.     

Nanotechnology a novel approach to enhance crop productivity

Nanobiotechnology provides novel set of tools to manipulate and enhance crop production using nanoparticles, nanofibres, nanoemulsions, and nanocapsules. Nanomaterials provide a platform to deliver agrochemicals and various macromolecules needed for plant growth enhancement and resistance to stresses. Smart delivery of agrochemicals increases the yield by optimizing water and nutrient conditions. Another added advantage is controlled release and site-directed delivery of agrochemicals. Further enhancement in quality and quantity in agriculture can be achieved by nanoparticle-mediated gene transformation and delivery of macromolecules that induces gene expression in plants. Various types of nanomaterials have been tested so far and the results have been promising in terms of productivity and quality enhancement.     

Impact of Cardiovascular Calcifications on the Detrimental Effect of Continued Smoking on Cardiovascular Risk in Male Lung Cancer Screening Participants

Background

Current smokers have an increased cardiovascular disease (CVD) risk compared to ex-smokers due to reversible as well as irreversible effects of smoking. We investigated if current smokers remain to have an increased CVD risk compared to ex-smokers in subjects with a long and intense smoking history. We in addition studied if the effect of smoking continuation on CVD risk is independent of or modified by the presence of cardiovascular calcifications.

Methods

The cohort used comprised a sample of 3559 male lung cancer screening trial participants. We conducted a case-cohort study using all CVD cases and a random sample of 10% (n = 341) from the baseline cohort (subcohort). A weighted Cox proportional hazards model was used to estimate the hazard ratios for current smoking status in relation to CVD events.

Results

During a median follow-up of 2.6 years (max. 3.7 years), 263 fatal and non-fatal cardiovascular events (cases) were identified. Age, packyears and cardiovascular calcification adjusted hazard ratio of current smokers compared to former smokers was 1.33 (95% confidence interval 1.00–1.77). In additional analyses that incorporated multiplicative interaction terms, neither coronary nor aortic calcifications modified the association between smoking status and cardiovascular risk (P = 0.08).

Conclusions

Current smokers have an increased CVD risk compared to former smokers even in subjects with a long and intense smoking history. Smoking exerts its hazardous effects on CVD risk by pathways partly independent of cardiovascular calcifications.     

Delivery systems for gene therapy

The structure of DNA was unraveled by Watson and Crick in 1953, and two decades later Arber, Nathans and Smith discovered DNA restriction enzymes, which led to the rapid growth in the field of recombinant DNA technology. From expressing cloned genes in bacteria to expressing foreign DNA in transgenic animals, DNA is now slated to be used as a therapeutic agent to replace defective genes in patients suffering from genetic disorders or to kill tumor cells in cancer patients. Gene therapy provides modern medicine with new perspectives that were unthinkable two decades ago. Progress in molecular biology and especially, molecular medicine is now changing the basics of clinical medicine. A variety of viral and non-viral possibilities are available for basic and clinical research. This review summarizes the delivery routes and methods for gene transfer used in gene therapy.