Showing papers by "Southern Illinois University School of Medicine published in 1998"

Germ Cell Genotype Controls Cell Cycle during Spermatogenesis in the Rat

Abstract: Spermatogenesis is one of the most productive self-renewing systems in the body: on the order of 10(7) spermatozoa are produced daily per gram of testis tissue. In each mammalian species, the time required for completion of the process is unique and unalterable. Because the process is supported by somatic Sertoli cells, it has generally been thought that cell-cell interaction between germ and Sertoli cells controls the duration of cell cycles and cellular organization. We have used the newly developed technique of spermatogonial transplantation to examine which cell type(s) determines the rate at which germ cells proceed through spermatogenesis. Rat germ cells were transplanted into a mouse testis, and the mouse was killed 12.9-13 days after administration of a single dose of [3H]thymidine. The most advanced rat cell type labeled was the pachytene spermatocyte at stages VI-VIII of the spermatogenic cycle. In animals given only rat cells, some endogenous spermatogenesis of the mouse recovered. The most advanced labeled mouse cell types in recipients killed 12.9-13 days after administration of a single dose of [3H]thymidine were meiotic cells or young spermatids, which is consistent with a spermatogenic cycle length comparable to the 8.6 days reported for the mouse. The same results were obtained if a mixture of rat and mouse cells were transplanted. There existed two separate timing regimens for germ cell development in the recipient mouse testis; one of rat and one of mouse duration. Rat germ cells that were supported by mouse Sertoli cells always differentiated with cell cycle timing characteristic of the rat and generated the spermatogenic structural pattern of the rat, demonstrating that the cell differentiation process of spermatogenesis is regulated by germ cells alone.

Testicular degeneration in Bclw -deficient mice

Abstract: To identify genes required for mammalian spermatogenesis, we screened lines of mutant mice created using a retroviral genetrap system1 for male infertility. Homozygous ROSA41 male mice exhibit sterility associated with progressive testicular degeneration. Germ-cell defects are first observed at 19 days post-natal (p19). Spermatogenesis is blocked during late spermiogenesis in young adults. Gradual depletion of all stages of germ cells results in a Sertoli-cell-only phenotype by approximately six months of age. Subsequently, almost all Sertoli cells are lost from the seminiferous tubules and the Leydig cell population is reduced. Molecular analysis indicates that the gene mutated is Bclw, a death-protecting member of the Bcl2 family. The mutant allele of Bclw in ROSA41 does not produce a Bclw polypeptide. Expression of Bclw in the testis appears to be restricted to elongating spermatids and Sertoli cells. Potential roles for Bclw in testicular function are discussed.

Cardiac Sympathetic Dysinnervation in Diabetes Implications for Enhanced Cardiovascular Risk

Abstract: Background—Regional cardiac sympathetic hyperactivity predisposes to malignant arrhythmias in nondiabetic cardiac disease. Conversely, however, cardiac sympathetic denervation predicts increased morbidity and mortality in severe diabetic autonomic neuropathy (DAN). To unite these divergent observations, we propose that in diabetes regional cardiac denervation may elsewhere induce regional sympathetic hyperactivity, which may in turn act as a focus for chemical and electrical instability. Therefore, the aim of this study was to explore regional changes in sympathetic neuronal density and tone in diabetic patients with and without DAN. Methods and Results—PET using the sympathetic neurotransmitter analogue 11C-labeled hydroxyephedrine ([11C]-HED) was used to characterize left ventricular sympathetic innervation in diabetic patients by assessing regional disturbances in myocardial tracer retention and washout. The subject groups comprised 10 diabetic subjects without DAN, 10 diabetic subjects with mild DAN, ...

Microstamp patterns of biomolecules for high-resolution neuronal networks

Abstract: A microstamping technique has been developed for high-resolution patterning of proteins on glass substrates for the localisation of neurons and their axons and dendrites. The patterning process uses a microfabricated polydimethylsiloxane stamp with micrometer length features to transfer multiple types of biomolecules to silanederivatised substrates, using glutaraldehyde as a homobifunctional linker. To test the efficacy of the procedure, substrates are compared in which poly-d-lysine (PDL) was physisorbed and patterned by photoresist with those stamped with PDL. Fluorescein isothiocyanate labelled poly-I-lysine was used to verify the presence and uniformity of the patterns on the glass substrates. As a biological assay, B104 neuroblastoma cells were plated on stamped and physisorbed glass coverslips. Pattern compliance was determined as the percentage of cells on the pattern 8h after plating. Results indicate that the stamping and photoresist patterning procedure are equivalent. Substrates stamped with PDL had an average pattern compliance of 52.6±4.4%, compared to 54.6±8.1% for physisorbed substrates. Measures of background avoidance were also equivalent. As the procedure permits successive stamping of multiple proteins, each with its own micropattern, it should be very useful for defining complex substrates to assist in cell patterning and other cell guidance studies.

Gait in patients with cerebellar ataxia

Abstract: The gait pattern in 10 patients with cerebellar degenerations was studied and the results were compared with 10 matched normal subjects, seeking the principal patterns in this disorder. Gait at natural speed was studied in a biomechanics laboratory using a video-based kinematic data acquisition system for measuring body movements. Patients showed a reduced step and stride length with a trend to reduced cadence. Heel off time, toe off time, and time of peak flexion of the knee in swing were all delayed. Range of motion of ankle, knee, and hip were all reduced, but only ankle range of motion reached significance. Multijoint coordination was impaired, as indicated by a relatively greater delay of plantar flexion of the ankle compared with flexion of the knee and a relatively late knee flexion compared with hip flexion at the onset of swing. The patients also showed increased variability of almost all measures. Although some of the deviations from normal were simply the result of slowness of walking, the gait pattern of patients with cerebellar degeneration shows incoordination similar to that previously described for their multijoint limb motion.

Multicenter radioimmunoscintigraphic evaluation of patients with prostate carcinoma using indium‐111 capromab pendetide

Abstract: BACKGROUND Optimum therapy for prostate carcinoma patients requires accurate staging, but computed tomography (CT) and magnetic resonance imaging (MRI) have limitations as methods for detecting soft tissue metastases. In this study, radioimmunoscintigraphy (RIS) was evaluated for its ability to identify sites of metastatic disease in lymph nodes. METHODS RIS was evaluated in 51 prostate carcinoma patients at high risk for metastatic disease. An intravenous infusion of indium-111 capromab pendetide was given, followed by nuclear medicine imaging on two separate dates. Bilateral, open pelvic lymph node dissection was performed with additional exploration and biopsy of scan positive extraprostatic regions. Histologic evaluation of removed tissue confirmed the accuracy of RIS. In addition, results were compared with other standard methods for diagnosing patients prior to surgery. RESULTS Nineteen patients (37%) had evidence of lymph node involvement with RIS. Fifteen of the 19 positive patients had pathologic evidence of cancer in the biopsied lymph nodes. Sensitivity, specificity, accuracy, and the positive predictive value for detection of extraprostatic disease were 75%, 86%, 81%, and 79%, respectively. CT, MRI, and ultrasound of the pelvis demonstrated a combined accuracy of only 48% in detecting lymph node disease. Twenty-five previously undetected sites were deemed positive with RIS. Fourteen of these were biopsy-proven tumor sites, seven were probable tumor sites, and four were assumed to be false-positive. CONCLUSIONS RIS had an impact on patient management through its detection of occult disease in more than 50% of prostate carcinoma patients studied, and it provided information concerning the likelihood that lymph node metastases would be found during surgery. Cancer 1998;83:739-747. © 1998 American Cancer Society.

Oxidative stress increases A1 adenosine receptor expression by activating nuclear factor kappa B.

Abstract: The A1 adenosine receptor (A1AR) contributes to the cytoprotective action of adenosine under conditions known to generate reactive oxygen species (ROS). Pharmacological manipulation of A1AR expression has been shown to modulate this cytoprotective role. In this study, we provide evidence that ROS generated could increase the expression of the A1AR and thereby offset the detrimental effects of ROS. Incubation of DDT1MF-2 smooth muscle cells with ROS-generating chemotherapeutic agents, such as cisplatin (2.5 μm) or H2O2 (10 μm), elicited an increase in A1AR expression within 24 hr. The induction by H2O2 was reduced by the ROS scavenger catalase but not superoxide dismutase. Inhibition of nuclear factor κB (NFκB) by pyrrolidine dithiocarbamate (200 μm), dexamethasone (100 nm), or genistein (1 μm) abrogated the cisplatin-mediated increase in A1AR. Cisplatin promoted rapid translocation of NFκB (but not AP-1) to the nucleus, as detected by electrophoretic mobility shift assays and by Western blotting. A putative NFκB sequence in the A1AR promoter effectively competed with labeled κB probe for binding in nuclear preparations derived from DDT1MF-2 cells. Transient transfection of DDT1MF-2 cells with the A1AR promoter coupled to firefly luciferase reporter gene led to cisplatin-inducible and pyrrolidine dithiocarbamate-sensitive luciferase activity, suggesting the presence of functional NFκB binding site(s) in the A1AR promoter sequence. Treatment of cells with (R)-phenylisopropyladenosine (1 μm), an agonist of the A1AR, reduced cisplatin-mediated lipid peroxidation, which was reversed after blockade of the A1AR. These data suggest that ROS can increase the expression of the A1AR by activating NFκB regulatory site(s) on this gene and thereby enhance the cytoprotective role of adenosine.

Neuroendocrine and reproductive functions in male mice with targeted disruption of the prolactin gene.

Abstract: Mice with a targeted disruption (knock-out) of the PRL gene (PRL-KO) were used to study the physiological role of PRL in the control of male neuroendocrine functions related to reproduction. Compared with normal males, PRL-KO mice had significant reductions in median eminence dopamine content, plasma LH levels, LH and FSH secretion in vitro (per mg pituitary), and weights of seminal vesicles and ventral prostate. PRL was not detectable in incubation medium with pituitaries from PRL-KO mice. No alterations were detected in PRL-KO mice in median eminence norepinephrine, plasma testosterone levels, or testosterone release (per mg testis) in vitro with or without LH. No differences were detected in PRL-KO vs. normal male mice in the interval from housing with normal female mice until conception, rate of pregnancy, or the number of live pups per litter. Pituitary weight in PRL-KO mice was increased (1.78 +/- 0.22 vs. 3.35 +/- 0.20 mg; P < 0.001), presumably due to reduced feedback inhibition and hypertrophy and/or hyperplasia of nonfunctional lactotrophs. These results indicate that the absence of PRL reduces pituitary LH release, attenuates median eminence dopaminergic activity, and affects the growth of seminal vesicles and ventral prostate. Although it was previously shown that PRL can repair the reproductive defect in male pituitary dwarf mice, our current results imply that the PRL deficiency alone is not sufficient to cause male infertility, although there are obvious alterations in reproductive neuroendocrine function in PRL-KO males.

Protection by ebselen against cisplatin-induced nephrotoxicity: antioxidant system.

Abstract: This study was designed to investigate the cisplatin-induced alteration in renal antioxidant system and the nephroprotection with ebselen. Male Wistar rats were injected with (1) vehicle control; (2) cisplatin; (3) ebselen; and (4) cisplatin plus ebselen. Rats were sacrificed three days post-treatment and plasma as well as kidney were isolated and analyzed. Plasma creatinine increased 598% following cisplatin administration alone which decreased by 158% with ebselen pretreatment. Cisplatin-treated rats showed a depletion of renal glutathione (GSH) levels (52% of control), while cisplatin plus ebselen injected rats had GSH values close to the controls. Antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities decreased 38, 75 and 62% of control, respectively, and malondialdehyde (MDA) levels increased 174% of control following cisplatin administration, which were restored to control levels after ebselen treatment. The renal platinum level did not significantly change with ebselen pretreatment. This study suggests that the protection offered by ebselen against cisplatin-induced nephrotoxicity is partly related to the sparing of antioxidant system.

Development of Germ Cell Transplants in Mice

Abstract: Development of spermatogonial transplants was studied by using 5- to 6-wk-old histocompatible mice as cell donors and sterile (W-locus) mice as recipients. Groups of animals transplanted with germ cell suspensions were killed at 10 min, 9 h, 24 h, 1 wk, 1 mo, 2 mo, and 3 mo along with age-matched "start" and "end" W-locus controls. Weight of testes increased significantly at 24 h through 3 mo after germ cell transplantation, suggesting that the infused cells quickly stimulated organ function. Small clones of young spermatocytes were evident at 1 mo and sperm at 2 mo. The percentage of tubular profiles containing active spermatogenesis originating from spermatogonia increased with time (0.8% at 1 mo, 8.9% at 2 mo, and 28.2% at 3 mo). Most transplanted germ cells were eliminated from the seminiferous epithelium through phagocytosis by Sertoli cells that occurred primarily before 1 wk, although some pachytene cells were able to proceed through meiosis by 1 wk. A variety of abnormal features are described that characterize developing spermatogenesis in the transplanted testis. Spermatogenesis improved quantitatively and qualitatively with time although released sperm were frequently engulfed by intratubular macrophages and Sertoli cells. A quantitative analysis of spermatogenesis from transplanted germ cells will serve as a basis for improving spermatogonial transplant efficiency.

The endocrine system: an overview.

Abstract: A plethora of hormones regulate many of the body's functions, including growth and development, metabolism, electrolyte balances, and reproduction. Numerous glands throughout the body produce hormones. The hypothalamus produces several releasing and inhibiting hormones that act on the pituitary gland, stimulating the release of pituitary hormones. Of the pituitary hormones, several act on other glands located in various regions of the body, whereas other pituitary hormones directly affect their target organs. Other hormone-producing glands throughout the body include the adrenal glands, which primarily produce cortisol; the gonads (i.e., ovaries and testes), which produce sex hormones; the thyroid, which produces thyroid hormone; the parathyroid, which produces parathyroid hormone; and the pancreas, which produces insulin and glucagon. Many of these hormones are part of regulatory hormonal cascades involving a hypothalamic hormone, one or more pituitary hormones, and one or more target gland hormones.

Mechanism of Nicotine-Induced Relaxation in the Porcine Basilar Artery

Abstract: The present experiment was designed to examine possible influence of adrenergic nerves on nicotine-induced neurogenic vasodilation in porcine basilar arteries denuded of endothelium. Nicotine and transmural nerve stimulation (TNS) induced relaxation of basilar arteries. Tetrodotoxin (TTX) abolished the relaxation elicited by TNS, but only partially blocked that induced by nicotine. Relaxation induced by both nicotine and TNS was abolished by N-nitro-l-arginine. The N-nitro-l-arginine inhibition of both TNS- and nicotine-induced relaxation was reversed byl-arginine but not by d-arginine. Hexamethonium abolished the relaxation induced by nicotine, but did not affect that elicited by TNS. Relaxation induced by nicotine was diminished by guanethidine, which did not affect the relaxation induced by TNS, suggesting that guanethidine blockade of nicotine-induced relaxation is not due to its local anesthetic effect. Results from histochemical studies indicated that catecholamine fluorescence and NADPH-diaphorase fibers were not appreciably affected by guanethidine. Following incubation with 6-hydroxydopamine for 1 hr, the catecholamine fluorescence fibers in the basilar arteries completely disappeared, although the NADPH-diaphorase fibers were not affected. In these adrenergically denervated arteries, nicotine-induced relaxation was abolished, while the TNS-elicited relaxation was not affected. Furthermore, norepinephrine-induced relaxation in basilar arteries was blocked by N-nitro-l-arginine, but was not affected by N-nitro-d-arginine or hexamethonium. These results suggest that in porcine cerebral arteries nicotine-induced nitric oxide-mediated relaxation is dependent on an intact adrenergic innervation. Nicotine appears to act on nicotinic receptors on the presynaptic adrenergic nerve terminals to release norepinephrine or a related substance, which then stimulates release of nitric oxide from the neighboring nitric oxidergic nerves. The TNS-elicited nitric oxide-mediated relaxation, however, is resulted from direct depolarization of nitric oxidergic nerves.

Characterization of a Nuclear Deformed Epidermal Autoregulatory Factor-1 (DEAF-1)-Related (NUDR) Transcriptional Regulator Protein

Abstract: A monkey kidney cDNA that encodes a nuclear regulatory factor was identified by expression and affinity binding to a synthetic retinoic acid response element (RARE) and was used to isolate human placental and rat germ cell cDNAs by hybridization. The cDNAs encode a 59-kDa protein [nuclear DEAF-1-related (NUDR)] which shows sequence similarity to the Drosophila Deformed epidermal autoregulatory factor-1 (DEAF-1), a nonhomeodomain cofactor of embryonic Deformed gene expression. Similarities to other proteins indicate five functional domains in NUDR including an alanine-rich region prevalent in developmental transcription factors, a domain found in the promyelocytic leukemia-associated SP100 proteins, and a zinc finger homology domain associated with the AML1/MTG8 oncoprotein. Although NUDR mRNA displayed a wide tissue distribution in rats, elevated levels of protein were only observed in testicular germ cells, developing fetus, and transformed cell lines. Nuclear localization of NUDR was demonstrated by immunocytochemistry and by a green fluorescent protein-NUDR fusion protein. Site-directed mutagenesis of a nuclear localization signal resulted in cytoplasmic localization of the protein and eliminated NUDR-dependent transcriptional activation. Recombinant NUDR protein showed affinity for the RARE in mobility shifts; however it was efficiently displaced by retinoic acid receptor (RAR)/retinoid X receptor (RXR) complexes. In transient transfections, NUDR produced up to 26-fold inductions of a human proenkephalin promoter-reporter plasmid, with minimal effects on the promoters for prodynorphin or thymidine kinase. Placement of a RARE on the proenkephalin promoter increased NUDR-dependent activation to 41-fold, but this RARE-dependent increase was not transferable to a thymidine kinase promoter. Recombinant NUDR protein showed minimal binding affinity for proenkephalin promoter sequences, but was able to select DNA sequences from a random oligonucleotide library that had similar core-binding motifs (TTCG) as those recognized by DEAF-1. This motif is also present between the half-sites of several endogenous RAREs. The derived consensus- binding motif recognized by NUDR (TTCGGGNNTTTCCGG) was confirmed by mobility shift and deoxyribonuclease I (DNase I) protection assays; however, the consensus sequence was also unable to confer NUDR-dependent transcriptional activation to the thymidine kinase promoter. Our data suggests that NUDR may activate transcription independently of promoter binding, perhaps through protein-protein interaction with basal transcription factors, or by activation of secondary factors. The sequence and functional similarities between NUDR and DEAF-1 suggest that NUDR may also act as a cofactor to regulate the transcription of genes during fetal development or differentiation of testicular cells.

Tremor in ostensibly normal elderly people

Abstract: Tremor in ostensibly normal people, aged 70-91, was assessed clinically and electrophysiologically with the goal of estimating the prevalence of abnormal tremor. Fifty men and 50 women, mean age 76.0 +/- 4.7 yrs, were recruited through advertisements for healthy volunteers (n = 50 "biased" control subjects) and from the spouses of patients referred to us for dementia or Parkinson's disease (n = 50 "unbiased" control subjects). All participants were interviewed and examined by the author. Tremor was assessed quantitatively with rating scales, triaxial accelerometry, electromyography, a digitizing tablet, and spectral analysis. Twenty-three people (23%) were judged clinically to have mildly abnormal tremor resembling mild essential tremor. Twelve people with abnormal tremor belonged to the biased group and 11 were in the unbiased group. The clinical diagnosis of abnormal hand tremor correlated well with the presence of motor unit entrainment in the forearm EMG and with writing or drawing tremor that was measurable with a digitizing tablet. Only 10 of 23 people with abnormal tremor were aware of their tremor, and none had been diagnosed previously by a physician. Nine of 77 people (11.7%) with normal tremor had a parent or sibling with possible essential tremor, and five of the 23 people (21.7%) with abnormal tremor had this family history. Mild undiagnosed tremor, resembling essential tremor, is common in this age group.

Microcirculatory studies of frostbite injury.

Abstract: Frostbite represents a spectrum of injury ranging from irreversible cellular destruction to reversible changes seen after rewarming. These changes include increases in tissue edema, circulatory stasis, and progressive thrombosis leading to further tissue necrosis. For this reason, it is often difficult at the time of surgical debridement to determine the extent of frostbite injury. This delayed tissue injury is similar to that seen in muscle during ischemia/reperfusion injury. Muscle that initially appears viable on reperfusion may subsequently necrose due to collapse of the microcirculation. Adherent neutrophils have been specifically cited as important components in ischemia/reperfusion injury and have also been suggested to play a role in frostbite injury. We have used an intravital microscopic muscle preparation to study microcirculatory changes carefully in frostbite injury during rewarming. The right gracilis muscle of male Wistar rats is dissected free from its primary vascular pedicle and the rat is positioned on a specially constructed microsurgical stage. Temperature changes of the muscle are recorded. The prepared axial pattern flap is transilluminated with a microscope and projected on a video screen, allowing measurement of arteriolar diameters and changes in the numbers of stuck and rolling neutrophils before frostbite, during rewarming, and for several hours later. Cold silicone oil is used to freeze the muscle to -5 ± 2°C in 2 to 3 minutes and to hold this temperature for 5 minutes. The muscle is rewarmed with 42°C normal saline placed directly on the muscle surface. Baseline vessel diameter and leukocyte counts in 100-mm segments of the microvasculature are recorded as well as at 5, 15, and 30 minutes, and at 1, 2, and 3 hours postrewarming of frozen muscle. Observations from our initial 11 animals show that reperfusion of the muscle following freezing is varied temporally and spatially , with circulation to most vascular segments restored 5 to 10 minutes after rewarming. In 9 of 11 animals we observed the shedding of white clots in small arterioles and venules occurring as soon as 5 minutes after thawing. In some instances shedding continued for as long as 1 hour after rewarming. Microvascular hemorrhage was widespread 1 hour following the thaw, but there was no significant increase in neutrophil adherence observed until 3 hours following rewarming. The exact nature of the vascular injury and the composition of the white clots are now being determined from ultrastructural studies. Blood flow in microcirculation stops during freezing, but small-vessel perfusion returns immediately on thawing. This suggests that the vascular architecture is maintained during the freezing and thawing. Unlike ischemia/reperfusion injury, neutrophil adhesion plays a smaller role in the early response to frostbite injury. The early microcirculatory observations seen after rewarming suggest progressive and severe perturbations in platelet function and fibrin formation that are significantly different from ischemia/reperfusion injury.

Stimulation of angiogenesis to improve the viability of prefabricated flaps.

Abstract: The cutaneous area in a prefabricated myocutaneous flap surviving after elevation is dependent on the rate and amount of vascular ingrowth that occurs from the underlying muscle. Two modalities, basic fibroblast growth factor and hyperbaric oxygen, were used separately and together in a prefabricated myocutaneous flap animal model to improve flap survival. The semimembranous muscle, based on the saphenous vessels of 40 female Wistar rats weighing between 250 and 325 grams, was tunneled under the ipsilateral abdominal skin and sutured in place. A 3 x 5-cm silicone sheet was placed beneath the muscle flap, and the ipsilateral epigastric vessels were ligated. Four groups of 10 animals each received one of the following treatment regimes: a 1-ml normal saline infusion into the saphenous arterial pedicle, a 1-ml infusion of basic fibroblast growth factor (1.0 microg/gm of muscle), a 1-ml normal saline infusion and 14 hyperbaric oxygen treatments, or a 1-ml basic fibroblast growth factor infusion and 14 hyperbaric oxygen treatments. After 1 week, the muscle, still based on the saphenous vessels, was elevated with a 3 x 5-cm abdominal skin paddle. The flap was sutured back in place, leaving the silicone sheet intact. The surviving area of each flap was measured 1 week later after it had demarcated into viable and necrotic regions. Laser Doppler skin perfusion measurements were taken before and after flap elevation and before animal euthanasia. Sixteen flaps, 4 in each group, were examined histologically for vascularity by means of hematoxylin and eosin staining. There was a statistically significant increase in flap survival area when either basic fibroblast growth factor or hyperbaric oxygen was used alone. Further improvement was noted with combination therapy. Histology confirmed improved vascularity in the basic fibroblast growth factor and hyperbaric oxygen-treated flaps. This study shows a significant and reliable increase in the area of prefabricated myocutaneous flap survival using either basic fibroblast growth factor or hyperbaric oxygen. There is a further complementary effect when these two modalities are combined, leading to near complete flap survival through improved vascularity.