Mark is an accomplished biomedical research scientist and veterinary physician who has earned three post-baccalaureate degrees including two doctorates in Veterinary Medicine and Biochemistry/Cell Biology with numerous peer-reviewed professional publications to his credit. Working as a top award-winning Sales Executive with a major Fortune 200 company, he consults on nanotechnology needs for biological and material scientists employed with academic/biotech/government research labs including the National Institutes of Health (NIH), National Cancer Institute (NCI), National Institute of Standards & Technology (NIST), Department of Defense (U.S. Army, Navy & Air Force), Federal Bureau of Investigation (FBI), Department of Homeland Security (DHS), Bureau of Alcohol, Tobacco, Firearms & Explosives (ATF), Food & Drug Administration (FDA), U.S. Department of Agriculture (USDA), National Aeronautics & Space Administration (NASA), Johns Hopkins Applied Physics Laboratory (APL), Howard Hughes Medical Institute (HHMI), Centers for Disease Control (CDC) and the Smithsonian Institution. He is a member of the American Institute of Aeronautics & Astronautics, the University of Maryland NanoCenter and the World Veterinary Association. After joining the Civil Air Patrol (U.S. Air Force auxiliary) in 1975, he quickly rose thru the ranks to become a cadet officer in 1976, received his solo aviation wings in 1977, selected for IACE in 1978 and capped his CAP cadet career with the achievement of the General Carl A. Spaatz Award in 1981 for which he received a congratulatory letter from former president Richard M. Nixon and met with Paul E. Garber (First Curator of the National Air & Space Museum). Capt. Kukucka is currently serving on Maryland Wing HQ staff as the state Director of Professional Development and is also active in the Glenn L. Martin Composite Squadron where he serves as the unit’s Aerospace Education Officer and Medical Officer in addition to being a commercial instrument-rated pilot. http://independent.academia.edu/MarkAKukuckaDVMPhD
Abstract: Leydig cells were isolated from adult male guinea-pig testes using a multi-step procedure involving enzymatic dissociation and Percoll-gradient centrifugation. The following description is the first account of a successful isolation of adolescent guinea-pig Leydig cells. The enriched Leydig-cell preparation routinely isolated from six intact testicles yielded approximately 5.0 x 10(6) +/- 0.7 x 10(6) (+/- SEM) Leydig cells with a viability of 98.0 +/- 0.4% as determined using the trypan-blue exclusion method. The purity of the isolated cell population as assessed by 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) staining averaged 82.5 +/- 0.8%. Under light microscopy, guinea-pig Leydig cells were polyhedral in shape with a large prominent nucleus and a distinct nucleolus. The acidophilic cytoplasm contained numerous lipid-filled vesicles. Ultrastructurally, guinea-pig Leydig cells displayed an eccentrically located ovoid nucleus with dark-staining peripheral heterochromatin. Large quantities of mitochondria, smooth endoplasmic reticulum and particulate-laden lipid droplets were also evident. The steroidogenic potential of the isolated Leydig cells was verified using a maximally stimulating dose of ovine LH (100 ng ml-1) and human CG (200 mIU ml-1). Leydig cells incubated in a shaking (120 cycles min-1) water bath for 3 h at 37 degrees C in capped polypropylene microcentrifuge tubes produced 233 +/- 21 ng and 223 +/- 18 ng testosterone per 1 x 10(6) cells when maximally stimulated with oLH or hCG, respectively. The inclusion of low (1-5 microM) levels of sodium ascorbate during culture enhanced significantly Leydig-cell viability vs. control values.
Abstract: A specific high-performance liquid chromatographic (HPLC) method is described for the reliable quantitation of oxytocin using culture media supernatants. The procedure employs solid-phase extraction, antibody-based immunoaffinity purification and isocratic HPLC with dual channel coulometric detection (ED). The lower limit of detection for this cyclic nonapeptide was 40 pg (40 fmol). Due to its relative simplicity, specificity and precision, the HPLC-ED of oxytocin is an accurate and attractive alternative to many existing quantitative methods.
Abstract: In recent years, several metabolic roles have been proposed for vitamin C. Recent information suggests a strong causal relationship between high endogenous levels of ascorbic acid and changes in normal reproductive biology. Using highly enriched populations of guinea pig Leydig cells, we have found that elevated levels (50 to 500 microM) of ascorbate significantly (P < 0.01) depressed testosterone production in a dose-dependent manner while low levels (0 to 10 microM) were without effect. Leydig cells incubated under hypoxic (3% oxygen) culture conditions produced significantly (P < 0.01) more testosterone than similar cells cultured under normoxic (19% oxygen) conditions. The results of this study suggest that high concentrations of ascorbate and normoxic culture conditions suppress testosterone production in isolated Leydig cells. Thus, it would seem that there exists a delicate balance between normal metabolic requirements for vitamin C and excessive ascorbate levels that might alter normal gonadal reproductive events.
Abstract: Utilization of highly enriched preparations of steroidogenic Leydig cells have proven invaluable for studying the direct effects of various hormones and agents on Leydig cell function in vitro. However, recent work indicates that isolated Leydig cells are often subjected to oxygen (O2) toxicity when cultured at ambient (19%) oxygen concentrations. Because intracellular antioxidants play an important role in protecting cells against oxygen toxicity, we have investigated the intracellular antioxidant defense system of isolated Leydig cells. The cellular levels of several antioxidants including catalase, glucose-6-phosphate dehydrogenase (G-6-PDH), superoxide dismutase (SOD) of the Cu/Zn & Mn variety, glutathione peroxidase, glutathione reductase and total glutathione were quantitated using enriched populations of Leydig cells isolated from adult male guinea pig testes. Compared to whole testicular homogenates, Leydig cells contained significantly (P < 0.01) less G-6-PDH, total SOD, glutathione reductase and total glutathione, but significantly (P < 0.001) more glutathione peroxidase. Compared to hepatic values previously reported in the guinea pig, Leydig cells contain nearly 400 times less catalase, about 14 times less glutathione peroxidase and almost 11 times less glutathione reductase. Since G-6-PDH and glutathione reductase are both necessary to regenerate reduced glutathione (GSH) which couples with glutathione peroxidase to breakdown hydrogen peroxide (H2O2) under normal conditions, it is plausible that the oxygen toxicity observed in isolated Leydig cells is due to the intracellular accumulation of H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Highly purified populations of guinea pig Leydig cells were incubated with a maximally stimulating dose of 100 ng/mL LH for 24 h in the presence of increasing concentrations of sodium ascorbate. Sample supernatants were extracted, concentrated under vacuum, and reconstituted with acidified absolute ethanol. Samples were analyzed for oxytocin using high-performance liquid chromatography with electrochemical detection and known concentrations of an authentic oxytocin standard. Leydig cells stimulated with 0, 25, and 50 microM ascorbate produced and secreted 40.1 +/- 1.23, 77.4 +/- 13.8, 74.2 +/- 26.3 pg of an oxytocin-like peptide, respectively, per 1 x 10(6) cells. These results indicate that guinea pig Leydig cells are capable of producing an oxytocin-like peptide de novo and that low concentrations of ascorbate stimulate the production of this peptide in Leydig cells cultured in vitro.
