In order to study $\beta$-adrenergic regulation of Na$\sp+$ transport in heart, a method was developed to directly measure intracellular free-Na$\sp+$ concentration ($\rm\lbrack Na\sp+\rbrack\sb{i}$) in ventricular myocytes freshly isolated from adult rabbit heart. The approach consisted of injecting the Na$\sp+$-sensitive fluorescent indicator SBFI by iontophoresis, and the simultaneous measurement of transmembrane ion current through the use of a single electrode voltage-clamp technique. Using this approach, steady-state $\rm\lbrack Na\sp+\rbrack\sb{i}$ was found to be 6.4 $\pm$ 1.8 mM in myocytes superfused at 37$\sp\circ$C with modified Tyrode's solution containing 2 mM Ca$\sp{2+}$. Decreasing Ca$\sp{2+}$ concentration to 0.8 mM caused an increase of $\rm\lbrack Na\sp+\rbrack\sb{i}$ to 13.0 $\pm$ 1.4 mM. The $\beta$-adrenergic agonist isoproterenol (ISO), when applied at a concentration of 0.5 $\mu$M, failed to cause any detectable change of steady-state $\rm\lbrack Na\sp+\rbrack\sb{i}$ or transmembrane current. To test whether this lack of effect also occurred at higher $\rm\lbrack Na\sp+\rbrack\sb{i}$, cells were exposed to the cation-selective ionophore gramicidin (10 nM), which induced a slow but continuous increase in $\rm\lbrack Na\sp+\rbrack\sb{i}.$ Under those conditions, ISO caused a decrease or even reversed the gramicidin-induced $\rm\lbrack Na\sp+\rbrack\sb{i}$ rise, while inducing an outward shift of the transmembrane current. These relative changes induced by ISO were not significantly affected by the removal of extracellular Ca$\sp{2+}$; $-$8.0 $\pm$ 2.5 mM/min for the ISO-induced changes in $\rm\lbrack Na\sp+\rbrack\sb{i}$ rate of rise, and 58 $\pm$ 17 pA for the transmembrane current. Under those conditions, strophanthidin (100 $\mu$M) completely inhibited the effects of ISO. Washout of intracellular Ca$\sp{2+}$ by prolonged preincubation of the myocytes in solutions containing 0 mM Ca$\sp{2+}$, 0.5 mM EGTA and 25 $\mu$M BAPTA-AM caused an important attenuation of the effects of ISO. (Abstract shortened by UMI.)

The long-term objective of this study was to prepare isolated brown adipose tissue (BAT) cells which contain either a high or a low level of thyroxine 5$\sp\prime$-deiodinase (T5$\sp\prime$D) activity for future use in the study of the role of this enzyme, which produces the thermogenically active thyroid hormone, 3,5,3$\sp\prime$-triiodothyronine (T$\sb3$), in the regulation of metabolic processes in BAT cells. Cold acclimation was used to increase T5$\sp\prime$D activity in BAT, since it is known to have this effect in rats, hamsters and mice. However, the guinea pig was chosen as experimental animal because this is known to be the only species for which cells isolated from BAT of the cold acclimated animal have a much greater thermogenic response to noradrenaline (NA) than cells from BAT of the warm acclimated animal. T5$\sp\prime$D had not hitherto been studied in BAT of the guinea pig. (Abstract shortened by UMI.)

With the aim of increasing our plant knowledge of Frankia, we have investigated the structure of the ribosomal RNA genes from Frankia sp. strain AcN14a. A genomic library was constructed in EMBL3 and two types of clones, each containing a different set of rRNA genes, were identified by restriction analysis. The rRNA genes of Frankia AcN14a have been compared to those of E. Coli and other eubacterial species. Frankia AcN14a rRNA genes show over 98% idenity to those of Frankia ORS020606 and usually show over 80% identity to other actinomycete rRNA sequences. The mature 16S, 23S and 5S rRNAs are predicted to contain 1509, 3105 and 120 nucleotides, repectively. Thus, Frankia 16S rRNA is smaller than that of E. coli whereas the 23S rRNA of Frankia is longer than it E. coli counterpart. In spite of these size differences, all three rRNAs could be folded into secondary structures similar to the E. coli models and most tertiary interactions proposed for E. coli rRNAs could be formed in Frankia AcN14a 16S and 23S rRNAs. Finally, the potential usefulness of rRNA RFLP analysis for the categorization of Frankia isolates has been investigated. Hybridization of both 16S and 23S rDNA specific probes to total DNA digested with BamH1, revealed sufficient polymorphism to allow classification of twenty-six isolates into six groups (A to F). In addition, eight other isolates exhibit unique patterns. (Abstract shortened by UMI.)

This thesis presents the results of my investigation of the process of cell-mediated cytotoxicity and in particular the role of the microtubules and intermediate filaments in MTOC/GA orientation. Immunofluorescence microscopy was used to determine if the orientation of the MTOC is accompanied by the posttranslational modification of tubulin. My results indicate that, if a more stable subset of microtubules is formed during the orientation of the MTOC towards the TC, it does not persist long enough for the posttranslational modification of tubulin to occur. Immunofluorescence microscopy also demonstrated that conjugation results in the formation of filamentous vimentin aggregates in CTLs similar to those induced by microtubule disassembling drugs. However, intermediate filament aggregates are not formed in all conjugated CTLs and there is no evidence to suggest that their formation is required for lytic activity. A hyperthermia treatment of 42$\sp\circ$C for 30 min results in a marked inhibition, followed by a transient recovery of cytolytic activity that peaks within 6 h. The close correlation observed between the loss and recovery of microtubule organization and the loss and recovery of cytolytic activity suggest that the initial inhibitory effect of hyperthermia on cytolytic activity is due to the disruption of MTOC-microtubule organization. The microtubules of taxol-treated cells remain associated with the centrosome, but the characteristic radial array is replaced by large bundles. This microtubule organization has no effect on centrosome organization, orientation of the MTOC, nor on cytolytic ability. The results show that the normal radial organization of microtubules is not required for the functional polarization of CTL, but suggest that this process is dependent upon centrosome organization. It is proposed that 50% of the cytolytic activity of the effector cells used in this study is due to the synthesis and secretion of a lytic protein subsequent to TC binding and that the remaining activity is occurring via a protein synthesis-independent apoptotic pathway. To what extent the recovery of cytolytic activity is dependent upon the recovery of each of the pathways could not be determined. The final results presented suggest that the long-term loss in cytolytic activity may be due to hyperthermia-induced effector cell death. (Abstract shortened by UMI.)

The effects of dl-$\alpha$-tocopherol and dl-$\alpha$-tocopheryl succinate on neuroblastoma, N1E 115, cells were studied. Tocopherol had no growth-arresting properties, whereas its succinate ester derivative inhibited growth at concentrations $\ge$20 $\mu$M. The succinate derivative was taken up somewhat more readily than free tocopherol; however, for any equal uptake of both forms of vitamin E, only the succinate derivative could affect growth. Tocopheryl succinate was taken up without marked conversion to tocopherol. The data point to the functionality of the carboxyl group of the succinate derivative as a basis for the difference in potency of the two forms of vitamin E. The succinate derivative of vitamin E was effective in growth inhibition of neuroblastoma, although the mechanism of action appears to involve cytotoxicity rather than induction of differentiation. The effects of retinoic acid on the morphology, the lipid metabolism and protein kinase activities of two related human neuroblastoma cell lines, SK-N-SH-F (SH-F) and SK-N-SH-N (SH-N) were studied. Pulse-chase experiments with both cell lines showed that turnover of arachidonic acid in lipid classes was most rapid in phosphatidylinositol, followed by phosphatidylethanolamine and phosphatidylcholine, and very low in phosphatidylserine. The redistribution of the arachidonic acid label following the chase was found to be markedly different in these variant cell lines. In SH-F the decrease of label in phospholipids was accompanied by an increase in the labelling of triglyceride. In SH-N, this redistribution of label in triglyceride was not observed. Following a six day exposure to retinoic acid, increased arachidonic acid turnover was observed in phosphatidylcholine and phosphatidylserine in SH-F and SH-N cell lines. The effect of retinoic acid on components of signal transduction pathways was investigated. Marked, but opposite, changes in protein kinase C activity were observed in both cell lines following retinoic acid treatment. SH-F cells displayed a substantial increase in protein kinase C activity; and SH-N cells, exhibited a decrease in protein kinase C activity. (Abstract shortened by UMI.)

Protein phosphorylation is a mechanism of modulating protein activity which plays a central role in a wide variety of cellular functions. The ready reversibility of the phosphotransfer reaction makes it well-suited for rapid cellular responses, such as the responses to signals from extracellular stimuli. Members of the protein kinase family have been identified as components of cellular signal tranduction networks, and in fact many protein kinases mediate signals controlling cell growth, differentiation, and development. The work carried out in this thesis describes the cloning and characterization of the TIK kinase, a novel member of the protein kinase family. The TIK kinase was isolated and cloned from murine pre-B cells on the basis of its immunoreactivity with antibodies to phosphotyrosine, and was therefore originally thought to be a protein tyrosine kinase. Characterization of the in vitro and in vivo activity of this kinase, however, revealed that the TIK kinase possesses only serine and threonine phosphorylating ability. A kinase deficient TIK protein, constructed by site-directed mutagenesis, is not immunoreactive with the antiphosphotyrosine antibodies. These observations indicate the phosphoserine and/or phosphothreonine residues may be assuming a conformation within the context of this protein which mimicks the epitope(s) recognized by antiphosphotyrosine antibodies. The gene encoding the TIK kinase is transcribed to produce three distinct messenger RNA (mRNA) transcripts in all murine tissues examined, and in a number of murine leukemic cell lines. In the murine lymphocytic leukemia line L1210, however, three truncated mRNA molecules are produced in addition to the three normal transcripts detected in other tissues. Evidence is presented here that one allele of the TIK gene has undergone a genomic rearrangement in this cell line, and appears to giving rise to the abnormal pattern of mRNA expression observed. A cDNA corresponding to the smallest truncated mRNA transcript encoding the TIK kinase in the L1210 cell line has been obtained, and this cDNA encodes a mutant TIK protein lacking kinase activity. I postulate that the TIK kinase plays some role in the control of growth and differentiation during lymphocyte development, and that the mutation of the TIK gene has contributed to the initiation or maintenance of transformation in the L1210 cell line.

Cultures of Chlamydomonas reinhardtii (1 $\times$ 10$\sp6$ cells/mL) were incubated with 5.0 $\mu$g/mL of the insecticide fenitrothion, (FEN), (O,O-dimethyl-O-(3-methyl-4-nitrophenyl)phosphorothioate). The rate of abiotic fenitrothion degradation in Gorman and Levign growth medium (pH = 6.8, T = 21 $\sp\circ$C, Vita Lite$\sp\circler$ 40 w/m$\sp2$), was enhanced 9-fold in the light relative to the dark under abiotic conditions (T$\sb{1/2}$ values of 145 h (light) and 54 days (dark)). Endogenous metabolism by C. reinhardtii produced significant amounts of 3-methyl-4-nitrophenol (nitrocresol), demethyl fenitrothion, formyl fenitrothion, hydroxymethyl fenitrothion, and carboxyfenitrothion within the cells. This biodegradation was slower in the dark than in the light. Studies of algal biodegradation of fenitrothion in the presence of 5.0 $\mu$g/mL fenitrothion plus 23.2 $\mu$g/mL phenobarbital, (a cytochrome P$\sb{450}$ monooxygenase inducer), demonstrated a 10 fold increase in the levels of intracellular hydroxymethyl fenitrothion relative to control algal cultures. It was concluded from this study that degradation of fenitrothion by metabolic activity of cytochrome P$\sb{450}$ monooxygenase may be a significant determinant in fenitrothion degradation in C. reinhardtii cultures. The amounts of formyl fenitrothion, carboxy fenitrothion and, carboxyfenitrooxon were demonstrated to depend on the availability of intracellular hydroxymethyl fenitrothion pool produced by algal PSMO activity, while NC and DSM levels were attributable to other cellular enzymes. (Abstract shortened by UMI).

The relationship between Sertoli cell lipid inclusions and spermatogenic activity was investigated using light microscopy and morphometric analysis of the lipid droplet content of Sertoli cells and spermatids from the seasonal breeding mink. Tissue was obtained and analyzed each month during a twelve month period to establish the presence of seasonal and stage-related cycles of lipid droplet content in these cells. It is concluded that the amount of lipid inclusions in Sertoli cells varies with the degree of spermatogenic activity being lowest after the release of mature spermatids in the lumen during the active spermatogenic phase and at the time of maximal testicular regression. Furthermore, cholesterol esters are a component of these lipid inclusions and the seasonal changes in lipid inclusions of Sertoli cells are reflected by seasonal changes in the amount of Sertoli cell cholesterol esters. (Abstract shortened by UMI.)

No description available.

The total cellular ([T-DDP]), intracellular ultrafilterable ([F-DDP]) and precipitable cellular bound ([B-DDP]) cisplatin concentrations were all compared in the HTB56 human lung adenocarcinoma call line and its E43/0.7 variant that has acquired cisplatin resistance. After a 20-minute exposure to 509muM cisplatin, uptake was terminated by spinning cell suspensions in a microcentrifuge at 14000 rpm for 10s. Ultrafiltration with a 500 molecular weight cut-off separated cellular free from bound cisplatin. Fragmentation by sonication and micro-centrifugal spinning precipitated cellular bound cisplatin. Flow cytometry was used to measure the intracellular pH (pHi) of the HTB56 cell line, the E8/0.7 cell lines, the OV2008 cell line and its C13 resistant variant, as well as of the A2780 and its A2780/CP resistant variant. The DNA-bound DDP and Protein-bound DDP ([P-DDP]) were also compared when equal [T-DDP] was achieved for both sensitive and resistant lung cancer cells by exposing them to two pairs of DDP concentrations, i.e., 509 vs 911 muM DDP, and 111 vs 666 muM DDP respectively. After one-hour drug exposure in these equal loading experiments, uptake was terminated and cells were scraped. Platinum was assayed by flameless atomic absorption spectrophotometry. (Abstract shortened by UMI.)