Testosterone regulates granzyme K expression in rat testes.

OBJECTIVE: Testosterone depletion induces increased germ cell apoptosis in testes. However, limited studies exist on genes that regulate the germ cell apoptosis. Granzymes (GZM) are serine proteases that induce apoptosis in various tissues. Multiple granzymes, including GZMA, GZMB and GZMN, are present in testes. Th us, we investigated which granzyme may be testosterone responsive and possibly may have a role in germ cell apoptosis aft er testosterone depletion. METHODS: Ethylene dimethane sulfonate (EDS), a toxicant that selectively ablates the Leydig cells, was injected into rats to withdraw the testosterone. The testosterone depletion effects after 7 days post-EDS were verified by replacing the testosterone exogenously into EDS-treated rats. Serum or testicular testosterone was measured by radioimmunoassay. Using qPCR, mRNAs of granzyme variants in testes were quantified. The germ cell apoptosis was identified by TUNEL assay and the localization of GZMK was by immunohistochemistry. RESULTS: EDS treatment eliminated the Leydig cells and depleted serum and testicular testosterone. At 7 days post-EDS, testis weights were reduced 18% with increased germ cell apoptosis plus elevation GZMK expression. GZMK was not associated with TUNEL-positive cells, but was localized to stripped cytoplasm of spermatids. In addition, apoptotic round spermatids were observed in the caput epididymis. CONCLUSIONS: GZMK expression in testes is testosterone dependent. GZMK is located adjacent to germ cells in seminiferous tubules and the presence of apoptotic round spermatids in the epididymis suggest its role in the degradation of microtubules in ectoplasmic specializations. Thus, overexpression of GZMK may indirectly regulate germ cell apoptosis by premature release of round spermatids from seminiferous tubule lumen.

Moderate level exposure to magnetic nanodots encased in tunable poly(ethylene glycol) analouge biopolymer shell do not deleteriously affect neurite outgrowth.

Recently, huge interest has been generated in investigating the possible therapeutic use of tunable magnetic nanostructures to overcome the existing challenges to treat central nervous system damage related conditions. However, several issues (e.g., biocompatibility or remote controlled actuation for multi-modal therapeutics) limit the use of conventional magnetic nanoparticles for biomedical applications. To address many of these shortcomings, we have synthesized a monodisperse nanoscale system consisting highly water dispersible magnetic nanodots encased in a remotely tunable polyethylene glycol analouge biopolymer shell. The monodisperse nature of the nanospheres, their response to external magnetic field and volumetric transition near physiological temperatures are very attractive, especially for drug delivery systems where triggered release is necessary. To further analyze the potential for combinatorial therapeutics for central nervous system damage related conditions, we have explored the efficiency of the uptake of nanospheres into pheochromocytoma cell line 12 (PC12) cells and assessed several additional measures of neurite outgrowth. We find that nanospheres were readily incorporated into the cytosolic compartment within 3 hours and did not alter the morphology of cellular processes compared to cells not exposed to nanospheres. Quantification of neurite outgrowth did not reveal any significant differences in neurite initiation or elongation between cells treated with moderate level nanomagnet exposure compared to control cultures under similar conditions. Thus, this study reports an attractive nano-scale system with great potential to deliver therapeutics to precise locations within the nervous system for axonal outgrowth and guidance.

Fixation temperature affects DNA integrity in the testis as measured by the TUNEL assay.

Mature rat testes and liver were fixed with Bouin's fluid (BF) or modified Davidson's fixative (mDF) at room temperature (23 °C) or 4 °C, and DNA integrity was examined by the TUNEL assay. When testes were fixed in BF, TUNEL-stained cells were more prevalent than when fixation occurred in mDF. Independent of fixative, TUNEL-staining was higher when testes were fixed at room temperature relative to 4 °C. Significant effects were present for fixative and temperature of fixation, but not their interaction. Relative to BF, mDF also provided for lower TUNEL-staining in liver, but staining was not affected by fixation temperature. Since the TUNEL assay depends on the detection of fragmented DNA strands, harsh fixatives that induce breaks in the DNA can introduce substantial artifacts. Such potential artifacts are especially prevalent in a tissue such as testes with its ongoing division and differentiation activities. Therefore, the current findings lead the authors to conclude that fixation of mature testes in mDF at 4 °C minimizes generation of false TUNEL-positive cells.

Role of engineered nanocarriers for axon regeneration and guidance: current status and future trends.

There are approximately 1.5 million people who experience traumatic injuries to the brain and 265,000 who experience traumatic injuries to the spinal cord each year in the United States. Currently, there are few effective treatments for central nervous system (CNS) injuries because the CNS is refractory to axonal regeneration and relatively inaccessible to many pharmacological treatments. Smart, remotely tunable, multifunctional micro- and nanocarriers hold promise for delivering treatments to the CNS and targeting specific neurons to enhance axon regeneration and synaptogenesis. Furthermore, assessing the efficacy of treatments could be enhanced by biocompatible nanovectors designed for imaging in vivo. Recent developments in nanoengineering offer promising alternatives for designing biocompatible micro- and nanovectors, including magnetic nanostructures, carbon nanotubes, and quantum dot-based systems for controlled release of therapeutic and diagnostic agents to targeted CNS cells. This review highlights recent achievements in the development of smart nanostructures to overcome the existing challenges for treating CNS injuries.

Alternating Magnetic Field Controlled, Multifunctional Nano-Reservoirs: Intracellular Uptake and Improved Biocompatibility.

Biocompatible magnetic nanoparticles hold great therapeutic potential, but conventional particles can be toxic. Here, we report the synthesis and alternating magnetic field dependent actuation of a remotely controllable, multifunctional nano-scale system and its marked biocompatibility with mammalian cells. Monodisperse, magnetic nanospheres based on thermo-sensitive polymer network poly(ethylene glycol) ethyl ether methacrylate-co-poly(ethylene glycol) methyl ether methacrylate were synthesized using free radical polymerization. Synthesized nanospheres have oscillating magnetic field induced thermo-reversible behavior; exhibiting desirable characteristics comparable to the widely used poly-N-isopropylacrylamide-based systems in shrinkage plus a broader volumetric transition range. Remote heating and model drug release were characterized for different field strengths. Nanospheres containing nanoparticles up to an iron concentration of 6 mM were readily taken up by neuron-like PC12 pheochromocytoma cells and had reduced toxicity compared to other surface modified magnetic nanocarriers. Furthermore, nanosphere exposure did not inhibit the extension of cellular processes (neurite outgrowth) even at high iron concentrations (6 mM), indicating minimal negative effects in cellular systems. Excellent intracellular uptake and enhanced biocompatibility coupled with the lack of deleterious effects on neurite outgrowth and prior Food and Drug Administration (FDA) approval of PEG-based carriers suggest increased therapeutic potential of this system for manipulating axon regeneration following nervous system injury.