The generation of a new organism by the union of gametes, dubbed fertilization, is a unifying theme across nearly all living species. Concurrently, divergencies amongst mechanisms of this event exist even within creatures of the same genus, and arguably, within species. The following presents an examination of the mechanisms of fertilization and the subsequent regulation of intracellular calcium ([Ca2+]i) signaling in the eggs of bos taurus (domestic cattle) with specific attention to both conserved and divergent mechanisms from that of the more commonly studied mus musculus (common mouse). Through a multiparametric approach consisting of Ca2+ imagery, protein analysis, molecular mutation, and assisted reproductive technologies we examined first the cytologic nature of the release of the sperm-borne egg-activating factor, referred to as sperm factor, during fertilization. We followed by ascertaining the functional properties and minimal requirements of the type I 1,4,5-inositol trisphosphate receptor (IP3R-1) in bovine eggs during fertilization. Finally, we examined the [Ca2+]i oscillation-inducing ability and cellular localization of the purported sperm factor molecule, phospholipase C&zgr; (PLC&zgr;), during egg activation, with close attention to species specific dynamics of calcium release and molecular determinants of functionality. Our data show that first, although bull sperm contain a cytosolic sperm factor, it is insufficient to trigger [Ca2+]i oscillations in bovine eggs without sperm-egg fusion first occurring. Second, bovine eggs possess a virtually redundant amount of IP3R-1, capable of mounting near-normal Ca2+ responses with only a 20-30% complement of IP3R-1. Third, bovine PLC&zgr; demonstrates an attenuated enzymatic activity compared to PLC&zgr; from other species, however, remains cytoplasmic as bovine zygotes reach interphase, and appears capable of continued IP 3 production as determined both by calcium imagery and quantification of IP3R-1. Additionally, unique sequence elements of bovine PLC&zgr; may be responsible for this decreased enzymatic activity. Lastly, we have determined that bovine eggs possess a unique mechanism to down regulate, but not terminate, interphase stage [Ca2+]i oscillations through alteration of IP3R-1 Ca2+ conductivity, independent of IP3R-1 numbers. The culmination of these results exemplifies how mammals have evolved to adapt components of the same system to achieve the unique requirements of the various respective species.^

Remarkably little is known about nitrogenous excretion in freshwater invertebrates. In the current study, the nitrogen excretion mechanism in the carnivorous ribbon leech, Nephelopsis obscura, was investigated. Based on gene expression analysis and Ussing chamber experiments, the skin was identified as a major site of ammonia excretion. Pharmacological experiments and enzyme assays suggested an ammonia excretion mechanism that involves the V-ATPase, Na+/K+-ATPase and carbonic anhydrase, but not necessarily a functional microtubule system. Most importantly, functional expression studies of the identified Rh-protein cloned from leech skin (NoRhp) revealed an ammonia transport capability of this protein when expressed in yeast. Exposure to high environmental ammonia (HEA) caused a new adjustment of body ammonia, accompanied with a decrease in NoRhp and Na+/K+-ATPase mRNA levels, but unaltered ammonia excretion rates. The results of this study showed many similarities to the ammonia excretion mechanisms proposed in the gills of freshwater fish. / May 2015

The production of ATP is of the utmost importance to cell survival. To maintain energy homeostasis, cells regulate mitochondrial content through the control of degradative and synthetic processes. Mitochondrial biogenesis is primarily controlled through a small number of transcriptional regulators, primarily nuclear respiratory factor-1 (NRF-1), NRF-2, and peroxisome proliferator-activated receptors (PPARs). DNA-binding proteins regulate genes encoding the machinery of oxidative phosphorylation. In addition to these DNA-binding proteins, the coactivator PPAR gamma coactivator-1 alpha (PGC-1α) is central to control of mitochondrial genes, so much so that it has been dubbed a “master controller” of energy homeostasis in mammalian muscle tissues. Though well studied in mammals, previous studies suggest that this NRF-1-PGC1α axis may be disrupted in fish. The response to treatments such as temperature and diet cause reciprocal effects on NRF-1 and PGC-1α. A serine-rich insertion into the NRF-1 binding domain of PGC-1α most likely disrupts this interaction. In this study I looked at the ability for the goldfish PGC-1α gene to interact with the PGC-1α binding domain of NRF-1. I have found that goldfish PGC-1α does not physically bind NRF-1, which would suggest that the PGC-1α-NRF-1 axis in fish is disrupted. To further explore the role of PGC-1α in fish we looked at the role of AMP-activated kinase (AMPK) to phosphorylate goldfish, zebrafish, and human PGC-1α. The results from this analysis show that AMPK in a zebrafish embryonic cell line (ZEB2J) have their AMPK activated by the AMPK activator AICAR. This response was shown to be both dose and time dependent. Transcript data was generated looking at typical AMPK responsive targets in the mammalian system. The target of ramapamycin (TOR) gene responded with a decrease as is expected in mammals. Hexokinase 2 (HK2), PGC-1α, and NRF-1 all decreased which is opposite of the typical mammalian response. COX7C a downstream target of the PGC-1α-NRF-1 axis did not respond to treatment. Indicating a disruption in the AMPK- PGC-1α-NRF-1 pathway. / Thesis (Master, Biology) -- Queen's University, 2013-09-06 01:37:05.537

No description available.

Ten male student volunteers (mean age, 27.1 years) participated in this investigation to determine if exercise and cold stress could differentially effect coagulation and fibrinolysis. Euglobulin lysis times (ELT) and partial thromboplastin times (PTT) were used to assess fibrinolytic activity and coagulability, respectively. Cold and neutral environmental conditions were established at 5(DEGREES)C and 28(DEGREES)C. Exercise consisted of pedaling a bicycle ergometer at 300 kgm(.)min('-1) for five minutes followed by ten minutes at 900 kgm(.)min('-1). The testing schedule was as follows: / rest exercise / Session A (Cold): PC CR CE / rest exercise / Session B (Neutral): PN NR NE / Time (mins) 60 15 / ELT, PTT, and hematocrit (HCT) was assessed at PC, pretest-cold; CR, cold-rest; CE, cold-exercise; PN, pretest-neutral; NR, neutral- rest; and NE, neutral-exercise. Mean weighted skin temperatures (T(,sk)) and rectal temperatures (T(,re)) were assessed at 15-minute(' ) intervals throughout.(' )T(,sk) dropped continuously during cold exposure (CR, CE) but was relatively unaffected under neutral conditions. T(,re) response to cold was more complex, exhibiting an initial increase followed by a decrease with continued exposure. T(,re) increased in response to exercise at 28(DEGREES)C but exhibited no change at 5(DEGREES)C. ELT was reduced to 74, 62, and 44% of pretest values for CR, NE, and CE, respectively. No difference was found to exist between groups for PTT. HCT was increased to 107, 107, and 111% of pre-test values for CR, NE, and CE, respectively. These data suggest that coagulation and fibrinolysis may be affected differentially in response to exercise, a result that speaks in favor of exercise-induced fibrinolysis as a prophylaxis for atherosclerosis. / Source: Dissertation Abstracts International, Volume: 43-02, Section: B, page: 0350. / Thesis (Ph.D.)--The Florida State University, 1982.

The control of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion by a hypothalamic releasing peptide (GnRH) and the modulation of FSH and LH secretion by ovarian steroids has been demonstrated. There is increasing evidence that another ovarian factor, gonadostatin (GnS) may also contribute significantly to the control of gonadotropin (FSH and LH) secretion. GnS appears to contribute to the negative feedback on gonadotropin secretion, particularly FSH. The function of a separate factor in the negative feedback control of FSH may be to control, ultimately the number of follicles that develop and ovulate. / Examination of ovarian steroid feedback at physiological concentrations, demonstrated that steroids alone do not provide complete feedback control of FSH secretion. GnS is thought to be the additional factor that completes the negative feedback control of FSH release. Administration of GnS in the form of porcine follicular fluid (pFF) together with physiological levels of estradiol resulted in basal levels of serum FSH. / Steroids exert positive as well as negative feedback control of gonadotropin secretion. Basal and surge release of LH could be fully controlled with estradiol and progesterone alone (replaced to mimic normal changing levels). In contrast, neither basal nor surge secretion of FSH were controlled with steroids. The addition of GnS to a cyclic steroid regimen provided complete control of FSH secretion (both basal and surge) and suggests that GnS may play a significant role in the control of FSH secretion throughout the estrous cycle. / The response of dispersed pituitary cells to estradiol (with or without GnRH) is an enhancement of gonadotropin secretion, in contrast to suppression of gonadotropin secretion seen in vivo. The use of a perfused pituitary cell system allowed the cells to be pulsed with GnRH thereby producing the conditions thought to occur in vivo. Under conditions of pulsed GnRH stimulation the secretion of gonadotropins was dramatically suppressed by gonadostatin and to a small extent by estradiol. / These studies have suggested that gonadostatin may play a significant role in the negative feedback of FSH and to a lesser extent LH secretion. / Source: Dissertation Abstracts International, Volume: 42-12, Section: B, page: 4701. / Thesis (Ph.D.)--The Florida State University, 1982.

The contribution of protein as a substrate source during exercise has recently been suggested to be 5-20% instead of 1-2%. Isolated studies have observed female and male responses but never simultaneously in the study. It was the purpose of this study to observe protein utilization in women and men during the same experimental stress. Five trained women and men, who were matched by their aerobic capacity (42-50 ml/kg/min) underwent a discontinuous VO(,2)max test to determine 70% and 60% VO(,2)max values. The subjects returned to the lab for a resting, baseline session and two exercise series consisting of a one-hour (1 h) 70% VO(,2)max muscle glycogen depletion ride and a 1h 60% VO(,2)max data collection ride. Subjects alternated between a carbohydrate loaded diet (CHO-l) and a carbohydrate depleted diet (CHO-d). Urine samples were collected pre-post exercise. Serum samples were obtained pre-, mid-, and post-exercise. Sweat was collected in a gauze pad on the upper arm, chest, and abdomen during three 15-minute intervals. Urea nitrogen in the urine, serum, and sweat was used as an indicator of protein utilization and 3-methylhistidine as an indicator of skeletal muscle degradation. There were no significant gender differences with respect to urea nitrogen and 3-methylhistidine. The CHO-d diet significantly increased the concentration of urea nitrogen in the serum (p < .05) and the urea nitrogen excretion rate in the sweat and the urine (p < .05) in comparison with CHO-l and baseline. Three-methylhistidine excretion in the urine was not significantly altered by the diets. The established range of protein contribution in this study was between .80-1.36%. The results of this study indicate that women and men respond similarly with respect to protein contribution to energy production when introduced to the same relative exercise stress. / Source: Dissertation Abstracts International, Volume: 45-04, Section: B, page: 1123. / Thesis (Ph.D.)--The Florida State University, 1984.

The purpose of this investigation was to determine the biochemical and physiological responses of the ischemic diabetic rat heart as left ventricular peak systolic pressure declined to 75% and 50% of baseline. In addition, this study assessed the influence of iodoacetate in non-diabetic and diabetic animals. The results show that diabetic and iodoacetate-treated hearts reached the selected declines in pressure significantly faster (p < .05) than non-diabetic hearts. Significantly lower (p < .05) myocardial ATP levels were found in the diabetic and iodoacetate-treated rat hearts indicating that the relative lack of anaerobically-derived ATP hastened the reduction in pressure. Myocardial glycogen utilization was significantly greater (p < .05) in diabetic hearts. However, intramuscular lactate indicative of anaerobic ATP production remained relatively lower than non-diabetic values. The results strongly suggest that the relative decrease in anaerobically-derived ATP is responsible for the rapid decline in left ventricular peak systolic pressure associated with the diabetic rat heart. / Source: Dissertation Abstracts International, Volume: 44-06, Section: B, page: 1732. / Thesis (Ph.D.)--The Florida State University, 1983.

This study investigated the distribution and composition of glycoconjugates axonally transported to subcellular fractions of goldfish optic tectum, specifically the SPM, SV and soluble fractions. In addition this study also assessed the effectiveness of using different solvents for the extraction of intact axonally transported glycoconjugates. / The subcellular distribution of radioactivity in the goldfish optic tectum 12 hrs and 24 hrs postintraocular injection of ('35)S-sulfate or ('3)H-fucose, respectively, indicated that the RSA of both SV and SPM was greater than those of other membranous fractions. A small portion of transported label was associated with the soluble fraction. At 36 hrs PIO injection of ('35)SO(,4), the RSA of the SV and SPM increased by 18% and 9% respectively, whereas the RSA of the soluble fraction decreased by 16%. / Compositional analysis of the SPM glycoconjugates demonstrated that fucosylated molecules had a broad MW distribution mainly below 100k daltons, whereas sulfated molecules had MWs > 100k daltons. In contrast to SPM, transported sulfated and fucosylated GC of the SV fraction demonstrated an MW distribution primarily below 100k daltons. / Anion exchange chromatography resulted in the separation of 3 populations of transported soluble GC. Those molecules eluting with increasing ionic strength from the anion exchange column showed a progressive increase in MW as seen by gel filtration. The late eluting peak of highly acidic molecules contained in addition to sulfated glycopeptides all of the soluble glycosaminoglycans indicating the presence of transport labeled proteoglycans. / To determine conditions which would lead to solubilization of membrane-affiliated AT GC, pooled optic tecta were sequentially homogenized in different solvents 12 hrs PIO injection of ('35)SO(,4). Approximately 20% of the recovered transported label was soluble in buffered sucrose, 12% was extracted with sucrose containing 1.0 M NaCl, 34% was extracted with 4 M guanidine and 11% remained unextracted. The sucrose and salt extracted mainly GP while guanidine extracts and unextracted residue were enriched in PGs. / Source: Dissertation Abstracts International, Volume: 44-09, Section: B, page: 2672. / Thesis (Ph.D.)--The Florida State University, 1983.

These studies involve the identification, partial purification and physiological characterization of a prolactin inhibitory activity (PIA) of uterine origin. A dispersed anterior pituitary cell culture system was validated and employed as a bioassay for the PIA. This bioassay coupled with hormone radioimmunoassays and protein assays allowed for the assessment of the amount and potency of the PIA. Initial experiments demonstrated that prolactin (PRL) release from pituitary cells exposed to uterine extract for a 24 h period is inhibited in a dose-dependent manner. Accompanying this inhibition is a dose-dependent increase in intracellular PRL. Hormonal specificity of PIA is suggested by the fact that neither follicle-stimulating hormone nor luteinizing hormone were affected by uterine extract. Estimation of PIA in the uterus and similarly prepared extracts of gut, cardiac muscle and diaphragm reveal that PIA is restricted to uterine tissue. Pharmacological studies using specific receptor blockers indicate that the PIA is neither dopamine nor GABA. PIA is neither proteolytic nor cytotoxic since incubation with standard rat PRL did not decrease the amount of immunoreactive PRL and following uterine extract treatment pituitary cells secrete elevated levels of PRL. / Isolation and culturing of uterine epithelial, stromal and myometrial cells reveal that the PIA is confined to secretions of the epithelial layer. PIA is greater in sera from ovariectomized rats than in sera from ovariectomized-hysterectomized rats. Decrease of PIA in the circulation of hysterectomized rats suggests that the activity is hormonal in nature. / Isolation and purification of PIA involved acid extraction, heat fractionation, gel permeation chromatography, isocratic and reverse-phase high pressure liquid chromatography. These methods followed by amino acid analysis indicate that the PIA is a 63 amino acid polypeptide with an apparent MW of approximately 6900. / In summary, the uterus may function as an endocrine organ by secreting into circulation a non-dopaminergic, non-GABAergic low MW peptide which specifically suppresses PRL release from the anterior pituitary gland. / Source: Dissertation Abstracts International, Volume: 46-01, Section: B, page: 0077. / Thesis (Ph.D.)--The Florida State University, 1984.