The central question of my dissertation is does cellular transformation increase the occurrence of gene amplification. I used a model system in which cellular transformation could be controlled by shifting the temperature. Rat-1 was the parental cell line and was not transformed at any temperature. The LA24 cell line carries an avian sarcoma virus with a temperature sensitive mutation in the v-src gene. These cells are transformed at 35°C, but become nontransformed at 40°C. Measurements of cell transformation included (1) growth on plastic, (2) growth in soft agar and (3) v-src RNA and protein expression. My first experiments were to examine the frequency and mechanisms of MTX and colchicine resistant variants. The frequency in generating MTX resistant clones was the same in transformed and nontransformed LA24 cells. However, dihydrofolate reductase gene amplification was the preferred mechanism of MTX resistance in transformed LA24 clones. The frequency in generating colchicine resistant clones was no different in transformed and nontransformed LA24 cells. P-glycoprotein gene amplification was also the preferred mechanism of colchicine resistance in transformed LA24 clones. To directly study the mutation rate of colchicine resistant variants on transformed and nontransformed LA24 cells, Luria-Delbruck fluctuation analysis was performed. Colchicine resistant variants were derived from a single cell that were grown to a population size of 10⁵ cells; they were then selected with colchicine. The data showed no increase in the mutation rate of transformed LA24 cells compared to untransformed cells upon selection in colchicine. In summary, I have shown that v-src transformation in Rat-1 fibroblasts causes a high frequency of P-glycoprotein gene amplification when selected with colchicine, and a high frequency of dihydrofolate reductase gene amplification when selected with MTX. Even though gene amplification is enhanced by v-src transformation, the frequency of generating colchicine and MTX variants is not related to cellular transformation state.

The purposes of this study were to evaluate and compare the independent and combined effects of arginine/lysine (AL) supplementation and resistance training (RT) on glucose tolerance and golmerular filtration rate, and to determine whether or not alterations were associated with changes in selected hormonal parameters. The study involved 30 physically active college males, ages 20-30 years, randomly assigned to one of four groups: Placebo/Control (P/C, n = 7), P/RT (n = 8), AL/C (n = 7), or AL/RT (n = 8). During the 10-week program, exercise subjects participated in a progressive resistance training program stressing all major muscle groups. An arginine/lysine supplement at a dosage of 132 mg/kg fat-free body (FFB) or placebo was administered to controls and training groups. Oral glucose tolerance (OGT) tests were performed on each subject before and after the 10-week intervention in order to evaluate resting levels of plasma insulin-like growth factor-1 (IGF-1), as well as resting levels and responses of glucose, insulin and glucagon for 180 minutes following an oral glucose challenge. Glomerular filtration rate (GFR) was determined from creatinine clearance (C(Cr)) as calculated from plasma creatinine, urine creatinine and urine flow. Significant increases in strength, and fat-free body (FFB) weight were seen in both resistance trained groups compared to controls, while supplement status had no apparent effect. Glucose tolerance parameters which significantly increased following the 10-week intervention included resting insulin for P/RT and glucagon area under the curve (AUC) for P/C, AL/C, and P/RT. While IGF-1 did not significantly increase within groups, a significant post-treatment difference was seen between P/RT (0.93 ± 0.10 U/ml) and AL/RT (0.60 ± 0.08 u/ml); percent carbohydrate in diet and absolute change in FFB were significant predictors of the absolute change in IGF-1, accounting for 22.0% (p < 0.01) and 20.8% (p < 0.01) of the variability, respectively. It was concluded that AL supplementation for 10 weeks had no significant effect on strength, FFB, OGT or GFR, while RT increased both strength and FFB with no significant effect on OGT or GFR.

Whole cells of T. hyodysenteriae serotypes 1-7, avirulent T. hyodysenteriae serotypes 1 and 2, and 5 strains of T. innocens were chemically extracted to selectively remove a lipopolysaccharide-like substance (LPSLS). The different LPSLS were analyzed electrophoretically, immunologically, and chemically. SDS-PAGE demonstrated migratory differences that were unique for individual serotypes/strains. Additional differences were observed during attenuation which resulted in reduced mobility of upper molecular weight components. Western blotting with hyper-immunized rabbit serum (HRS) against serotype 1, 2, and 6 whole cell bacterins produced homologous and heterologous reactions with serotype specific LPSLS. Rabbit antisera to serotypes 3, 4, 5, and 7 whole cell bacterins produced only homologous reaction to the LPSLS. Convalescent-phase swine sera (CSS) against serotype 1 disease showed only homologous reaction to the LPSLS. Convalescent-phase swine sera against serotype 2 produced both homologous and heterologous reaction to the LPSLS. Antigenicity was recognized by HRS and CSS to the hydrophobic and hydrophilic components of acid hydrolyzed LPSLS. Thin layer chromatography (TLC) of the hydrophobic portion demonstrated the presence of a spot with an Rf of 0.18 that correlates with nonpathogenicity. Gas chromatography (GC) analysis of the carbohydrate content of the LPSLS revealed the presence of glucose, galactose, N-acetyl neuraminic acid, N-acetyl glucosamine, and N-acetyl galactosamine. 2-keto-3-deoxyoctulonate (KDO) and L-glycero-D-manno-heptose were absent. The LPSLS from T. hyodysenteriae was found to have low anticomplement activity in comparison with LPS. The LPSLS from T. innocens had one half the activity of LPS. T. hyodysenteriae LPSLS demonstrated comparable activity to LPS in inducing Ia expression by macrophages. The LPSLS from T. innocens was unable to stimulate expression of a molecules on macrophages.

It is generally accepted that the endocrine basis of integumental color change in vertebrates is due to pituitary secretion of alpha-melanocyte stimulating hormone (α-MSH). However, physiological levels of α-MSH has not been reported for several species that have historically been the models of choice for investigating changes in pigmentation. The goal of this research was to quantitatively report levels of circulating α-MSH in two important genera: leopard frogs (Rana pipiens and R. chiricahuensis) and the green anole (Anolis carolinensis). Histology of the ventral skin of R. chiricahuensis was also examined due to its unusual melanization and ability to change color. Effects of background color and low temperature on α-MSH levels were examined by radioimmunoassay. In the species examined, black background color induced dark skin color and high levels of α-MSH. Rana chiricahuensis exhibited much greater plasma α-MSH than the other species. Anolis and R. chiricahuensis darkened in response to low temperature, but R. pipiens did not. Cold-induced darkening was associated with increased plasma α-MSH, but not to the extent observed during background adaptation. Ventral skin of R. chiricahuensis, in vivo, darkened at low temperatures, and the skin histology revealed numerous large dermal melanophores dispersed among iridophores. These ventral dermal melanophores are active components of R. chiricahuensis physiological color change.

The rat liver peripheral-type benzodiazepine receptor (PBR) was characterized by ligand binding with [³H]Ro5-4864 and by photolabeling using [³H]PK 14105. The native liver receptor could be solubilized with digitonin. The Mr of the native photolabeled receptor was 170 kDa while a single Mr 19 kDa protein was identified under denaturing conditions. Radioligand binding to rat liver subcellular fractions showed protoporphyrin IX had a 6.2-fold greater affinity for [³H]Ro5-4864 binding sites in mitochondria than in microsomes. Although heterogeneity of the rat liver benzodiazepine binding site, but not the isoquinoline binding site, was observed, a single 19 kDa protein band was identified by photolabeling with an isoquinoline ligand. Bovine and rat PBR have a similar tissue and subcellular distribution, but are pharmacologically and biochemically distinct. The bovine PBR had low affinity for Ro5-4864 and diazepam while [³H]PK 11195 binding was insensitive to modification of histidine residues. The native Mr the receptor was 200 kDa by gel filtration. Photolabeling identified a 17 kDa protein from both rat and bovine adrenal mitochondria under denaturing conditions. An affinity matrix was constructed to purify the native components of the PBR from both species, but the PBR could not be eluted from the matrix. To compare receptor components, the cDNA encoding the rat isoquinoline binding protein was used to screen a fetal calf adrenal cDNA library. A 822 base pair bovine cDNA was identified that encoded a polypeptide of 169 amino acids which had 78% positional identity to the rat protein and was 97% similar after accounting for conserved replacements. Comparison of the amino acid sequences indicates the rat and bovine proteins are homologs and the species differences in ligand binding may not be due to differences in the primary sequence of the isoquionoline binding proteins. Our results indicate the common characteristics of PBR is their ability to bind isoquinoline ligands, not benzodiazepine ligands, with high affinity. A conserved 17-19 kDa protein is required for demonstration of this receptor. A new nomenclature is presented which designates these receptors as τ (tau) receptors.

Hindlimb un weighting by tail-cast suspension markedly alters the carbohydrate metabolism of rat soleus muscle. Due to reduced contractile activity, muscle glycogen concentrations increase dramatically. When normal weight-bearing function is restored (reloading), a triphasic response characterizes the return of glycogen to control levels. Between 15 min and 2 h of reloading, muscle glycogen concentrations decrease as a consequence of increased fractional activity of glycogen phosphorylase. From 2 to 4 h, phosphorylase activity declined and an elevated activity of glycogen synthase led to increased glycogen levels. Further increases of glycogen up to 24 h did not correlate with enzyme activities, thereby suggesting a transient uncoupling of the inverse relationship between glycogen concentrations and synthase activity. Between 24 and 72 h of reloading, glycogen decreased to control values, possibly initiated by high phosphorylase activity at 24 h. Further studies concerning the B-adrenergic response of carbohydrate metabolism showed that isoproterenol inhibition of glucose uptake and the mechanism by which isoproterenol inhibits skeletal muscle glucose uptake were similar in unweighted and weight-bearing soleus muscle. In contrast, isoproterenol effects on glycogen metabolism were increased in unweighted, but not denervated, soleus. Increased response of cAMP accumulation to isoproterenol but not to forskolin, which directly activates adenylate cyclase, suggested a receptor-mediated alteration in B-adrenergic response. Greater Badrenergic binding capacity per milligram muscle in unweighted soleus confirmed this hypothesis. Since the number of B-receptors in the muscle did not change following unweighting, this suggests that increased receptor concentration in unweighted muscle is due to a preferential loss of structural proteins and not receptor up-regulation. Conversely, denervation did not alter the number of receptors per milligram muscle, but reduced the total number of receptors in the muscle. These findings support a parallel loss of receptor and non-receptor protein during denervation. Since membrane receptors are degraded in lysosomes, contrasting B-adrenergic responses and binding capacities provided a novel means for showing marked differences in lysosomal proteolysis between unweighted and denervated muscle. Results from these studies indicate that while both unweighting and denervation induce muscle atrophy, mechanisms of proteolysis and hormonal responses in these two models of reduced use are markedly different.

The corpus luteum is an endocrine gland which forms in the ovary each reproductive cycle, secretes progesterone, and regresses if pregnancy does not occur. An understanding of the factors and mechanisms that determine the function and lifespan of the corpus luteum is fundamental to the understanding of the mechanisms that cause luteal dysfunction. Prostaglandin F₂(α)(PGF₂(α)) is the primary lutelytic agent in ewes and appears to initiate luteal regression by altering cytosolic-free calcium ([Ca²⁺]ᵢ) and stimulating calcium-dependent intracellular pathways. The primary focus of this dissertation was to investigate the roles of PGF₂(α) and calcium in the regulation of progesterone secretion in the ovine corpus luteum. In fura-2 loaded large cells, PGF₂(α) (0.5 μM) induced a rapid transient increase in [Ca²⁺]ᵢ followed by a sustained elevation of [Ca²⁺]ᵢ. The transient nature of the [Ca²⁺]ᵢ increase was due, at least in part, to the ability of PGF₂(α) to stimulate (p < 0.05)⁴⁵Ca²⁺ efflux. PGF₂(α) did not alter [Ca²⁺]ᵢ in small cells. The PGF₂(α)-induced calcium transient was modified by incubation of large cells in conditions known to alter calcium homeostasis. The transient was attenuated by incubation of large cells in Ca²⁺-free medium (±EGTA). These results suggest that PGF₂(α) induces release of calcium from intracellular calcium pools. However, pre-incubation (2 min) of large cells with 1mM LaCl₃ eliminated the PGF₂(α)-induced calcium transient, suggesting a role of extracellular calcium. Two different results were observed in this study regarding the role of calcium in the regulation of progesterone secretion. First, the inhibitory effect of PGF₂(α) on secretion of progesterone was reduced under conditions that reduced the magnitude of the PGF₂(α)-induced calcium transient. Second, a sustained elevation or reduction in [Ca²⁺]ᵢ level also reduced basal progesterone secretion in large cells. Thus, both phases of the PGF₂(α)-induced [Ca²⁺]ᵢ response, transient increase and sustained elevation, appear to be linked to the inhibitory action of PGF₂(α) on progesterone secretion. Finally, this study provides evidence to suggest that large and small cells differ in their ability to regulate calcium homeostasis.

An iron-binding 77 kilodalton glycoprotein was isolated from hemolymph of the adult sphinx moth, Manduca sexta. This protein bound a single ferric ion both in vivo and in vitro and had a secondary structure similar to that of human serum transferrin and human lactoferrin, as judged by CD spectra. Antiserum generated against this protein was used to screen a fifth instar, day four, larval fat body cDNA library. A 2.0 kilobase clone was isolated and used to probe a northern blot of both total and poly(A)⁺ RNA from fat body, revealing a message of 2.3 kilobases. The message is expressed throughout the fourth instar, fifth instar, wandering, pupal and adult stages. The 2.0 kilobase clone selected an mRNA which, when translated in vitro, produced an immunoprecipitable 77 kDa protein. The 2.0 kb clone was used as a probe to further screen the cDNA library, resulting in the isolation of three full-length clones. The complete nucleotide sequence of one 2183 base pair cDNA insert was determined. The deduced amino acid sequence contained an 18 amino acid signal sequence which, when cleaved, resulted in a mature protein sequence of 663 amino acids with a calculated molecular weight of 73,436. The first 34 residues of the mature protein were identical to those determined by Edman degradation of the intact protein. The sequence contained four consensus N-linked glycosylation sites (Asn-X-Thr/Ser). The sequence was used to search the National Biomedical Research Foundation protein database. The proteins exhibiting the greatest similarity were human serum transferrin, chicken ovotransferrin, human lactoferrin and human melanotransferrin. When the five sequences were aligned using a multiple alignment program, the insect protein contained approximately 27% identical residues when compared to each of the other transferrins. The greatest areas of similarity were around the iron binding sites. Moreover, 23 of the 24 cysteine residues in the insect protein occupied identical positions as compared to the other transferrins, indicating a similar overall tertiary structure. The insect protein also exhibited some internal homology between the N-terminal and C-terminal halves of the molecule. Ligands capable of binding an iron atom were present in the N-terminal half, but most were lacking in the C-terminal half. Based upon sequence comparisons and other structural and functional data, we believe that we have isolated and sequenced an invertebrate transferrin, the first such molecule to have its entire sequence determined.

The repair hypothesis argues that natural genetic transformation in Bacillus subtilis evolved as a system for recombinational repair of damages in the recipient cell's genome. Cell survivorship and the apparent transformation frequency were examined in a rec+ strain as well as in two repair-deficient mutant strains. One of the mutant strains YB1005 was defective in its ability to carry out excision repair, the second mutant strain YB1260 was deficient in its ability to elicit an SOS-like response to DNA damage or following the onset of competence. Cells were subjected to two experimental treatments that differ in the order of administration of damage and transforming donor DNA. In one treatment cells were exposed to UV radiation or damage first and then allowed to undergo recombination with transforming donor DNA (UV-DNA). In the second treatment the cells were transformed first and then subjected to UV radiation (DNA-UV). In order to more closely simulate the effects of transformation in natural populations the YB886 rec+ strain was transformed using damaged and undamaged donor DNA. In nature donor DNA is likely to come from neighboring cells exposed to similar levels of damage. Experimental results show a qualitative difference in the relationship between the survival of transformed and total cells in the two treatments. The density of competent cells increases, relative to noncompetent cells with increasing UV dose, when cells are transformed after damage. There was also a consistent difference between the UV-DNA and DNA-UV treatments in the relationship between the apparent transformation frequency and UV dose. The apparent transformation frequency increases with increasing UV dose when the cells are damaged prior to transformation. The results persist in the two mutant strains examined, therefore the greater increase in the apparent transformation frequency does not depend upon excision repair or inducible SOS-like repair. In addition, the apparent benefit of transformation remains even when the donor DNA is derived from damage cells. All of the results reported here are consistent with the repair hypothesis for the evolution of natural transformation in Bacillus subtilis. They provide empirical support for the hypothesis that the evolutionary function of competence is to bring DNA into the cell for use as template in recombinational repair of DNA damage.

Platelet activating factor (PAF) is a potent mediator in a variety of inflammatory events. Determining whether PAF participates in the bronchial hyperresponsiveness characteristic of asthma is the long term obj ecti ve for which the studies described here represent an initial step. PAF is a potent agonist that causes contraction of guinea pig peripheral lung strips. To determine if specific receptor sites for PAF could be demonstrated in guinea pig lung membranes (GPLM), direct radioligand binding studies were performed with [³H]C₁₆-PAF (l-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) and the PAF antagonists [³H]WEB 2086 and [³H]RP52770. Binding parameters were compared to those from rabbit platelet membranes (RPM). These studies demonstrated specific binding sites for [³H] C₁₆-PAF of high affinity in GPLM with a Kd of 3 nM,• and in RPM with a K(d) of 1 nM. [³H]C₁₆-PAF identified receptor densities in GPLM of 200 fmol/mg protein and in RPM of 1922 fmol/mg protein. In both tissue preparations binding of inhibited to the same maximum degree by C₁₆-PAF, C₁₈-PAF, WEB 2086, and RP52770, all with pseudo-Hill coefficients of unity. The PAF antagonist [³H]WEB 2086 identified a receptor density similar to that of [³H]C₁₆-PAF. The binding of [³H]WEB 2086 was inhibited to the same degree by C₁₆-PAF, C₁₈-PAF, WEB 2086 and RP52770, indicating WEB 2086 and PAF interact at the same receptor sites in both GPLM and RPM. Although inhibition curves for antagonists yielded pseudo-Hill coefficients of unity, inhibition by agonists yielded shallow inhibition curves suggesting two types or states for the PAF receptor. The PAF antagonist [³H]RP52770 was found to be an unsuitable ligand because it labeled a much larger density of binding sites (1200 fmol/mg protein in GPLM, and 10105 fmol/mg protein in RPM) and was inhibited to little or no extent by C₁₆-PAF, C₁₈-PAF, WEB 2086 or lyso-C₁₆-PAF . studies of signal transduction suggest that the binding affinity of the agonists C₁₆-PAF and C₁₈-PAF (but not for the antagonist WEB 2086) is regulated by GTPgamroa- S and Na⁺, providing indirect evidence that the PAF receptor in both tissue preparations is coupled to a guanine nucleotide regulatory protein. However, agonist binding retained shallow inhibition curves indicating heterogeneity of sites with respect to this regulation. Binding affinity for the agonists was not affected by cholera toxin or pertussis toxin. These results indicate PAF receptors in lung tissue could not be distinguished from those in RPM, however, both tissues appear to show heterogeneity of binding indicating the existence of receptor subtypes or states.