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Individual smooth muscle cells respond in a dose dependent manner to extracellular ATP with an immediate initial transient rise followed by oscillations in (Ca$\sp{2+}$) $\sb{\rm i}$ that are asynchronous from cell to cell. There is convincing pharmacological evidence that two receptors, the putative P$\sb{\rm 2u}$ and P$\sb{\rm 2g}$ nucleotide receptors mediate the cellular response to extracellular nucleotides. At low ATP concentrations, cells initially respond with a measurable lag time that is a function of ligand concentration. Electrophysiological experiments also provide evidence that ATP produces an initial transient and oscillations of Cl$\sp-$ inward current with a time course similar to the (Ca$\sp{2+}$) $\sb{\rm i}$ transient and oscillations. This Cl$\sp-$ current is activated only in the presence of both receptor occupation and a (Ca$\sp{2+}$) $\sb{\rm i}$ rise. While the initial transient rise in (Ca$\sp{2+}$) $\sb{\rm i}$ rapidly desensitizes, the subsequent oscillations can persist in the continued presence of extracellular ATP and Ca$\sp{2+}$. Further evidence indicates that the oscillations are controlled by a different mechanism than the initial transient. First, the EC$\sb{50}$ for the oscillations is 20 fold higher than that for the initial transient (2.6 and O.11 $\mu$M, respectively). Second, the initial transient appears to have access to a separate intracellular Ca$\sp{2+}$ pool from the one shared by both phenomena. The (Ca$\sp{2+}$) $\sb{\rm i}$ oscillations are shown to be spatially nonuniform, with (Ca$\sp{2+}$) $\sb{\rm i}$ propagating from multiple initiation sites as both cytoplasmic (Ca$\sp{2+}$) $\sb{\rm i}$ waves and gradients. Furthermore, an individual cell can display separate oscillations of discrete frequencies that draw upon Ca$\sp{2+}$ pools that are independently regulated. Adenosine, a metabolite of ATP, increases the fraction of cells that oscillate without an initial transient. ATP stimulates a rise in arachidonic acid production and secretion in a time- and dose-dependent manner. Pretreatment with indomethacin, an inhibitor of PGE$\sb2$ production from arachidonic acid (AA), abolished the ATP-mediated oscillations. Forskolin, an activator of adenylyl cyclase which causes a rise in cellular (cAMP), supports oscillations in ATP-stimulated cells that have previously responded with an initial transient followed by a high noisy plateau. These findings indicate that (Ca$\sp{2+}$) $\sb{\rm i}$ oscillations are regulated by the cAMP second messenger signal cascade.

Simian virus 40 (SV40) infections of semipermissive human cells and permissive monkey cells were compared. Viral specific events were examined on per cell basis using immunocytochemistry to detect viral antigen-producing cells and in situ hybridization to identify viral DNA-replicating cells. The SV40 infection aborted before T-antigen expression in many cells of each of the human cell lines examined. This was further investigated by in situ hybridizations to probe for early SV40 message. The results showed that the same number of cells synthesize early message as produce T antigen indicating that the block to T-antigen expression is prior to transcription. In all but one of the human cell lines studied, most of the T-antigen-producing cells replicated viral DNA. However, in the A172 line of human glial cells only a small percentage of the T-antigen-expressing cells replicated viral DNA. Different structural and functional classes of T antigen have been characterized using anti-T monoclonal antibodies. Therefore, infected A172 cells were examined with a panel of ten anti-T monoclonal antibodies to determine if viral DNA replication might correlate with the expression of a particular epitope of T antigen. One anti-T monoclonal, PAb 100, did specifically recognize that subset of A172 cells which replicated SV40 DNA. The percentage of PAb 100-reactive A172 cells was dramatically increased by the DNA synthesis inhibitors hydroxyurea and aphidicolin. Removal of the hydroxyurea was followed by an increase in the percentages of cells replicating viral DNA corresponding to the increased percentage reactive with PAb 100. The pattern of SV40 infection in A172 cells was not altered by infection with viable viral mutants containing lesions in the small t protein, the agnoprotein, or the enhancer region. Additional studies on carrier systems of SV40-infected A172 and rhesus cells are also described.

The accumulating evidence supports the idea that increases in the intracellular free calcium concentration ((Ca$\sp{2+}$) $\sb{\rm i}$) regulate chromosome-to-pole motion. To directly test this hypothesis I have iontophoretically injected Ca$\sp{2+}$, Ca$\sp{2+}$ buffers (EGTA, Br$\sb2$-BAPTA) or nucleotides into Tradescantia stamen hair cells during anaphase and measured their effect on (Ca$\sp{2+}$) $\sb{\rm i}$ and on chromosome motion. The results show that: (1) elevations of the (Ca$\sp{2+}$) $\sb{\rm i}$ within a limited physiological window (0.8-1.0 $\mu$M) accelerate motion two-fold. (2) the elevation of the (Ca$\sp{2+}$) $\sb{\rm i}$ above this window (2-10 $\mu$M) or below the physiological resting level ($<$0.05 $\mu$M) decelerates or stops motion. (3) GTP$\gamma$S, and to a lesser extent GTP, enhance motion without causing a sufficient increase in (Ca$\sp{2+}$) $\sb{\rm i}$. Further studies of the role of Ca$\sp{2+}$ and related agents in regulating chromosome motion requires a visualization of spindle microtubules (MTs). To achieve this goal I have adapted the technique of fluorescent analogue cytochemistry to plant cells. In this method carboxyfluorescein (CF) labeled brain tubulin is injected into dividing cells and acts as a reporter molecule for the MT structures into which it becomes incorporated. Direct observation of MT dynamics is then carried out with confocal laser scanning microscopy (CLSM). The results show that brain tubulin incorporates into plant MTs, and is utilized in the preprophase band, the perinuclear sheath at late prophase, the kinetochore fibers (k-fibers) during prometaphase, metaphase and anaphase, the interzone spindle during anaphase, and finally the phragmoplast during late anaphase and telophase. All of these MT-containing structures and, notably, their transitions from one to another, have been observed in single live cells progressing through mitosis and cytokinesis. To explore further the mechanism by which Ca$\sp{2+}$ and GTP$\gamma$S regulate chromosome motion I have directly observed the effect of these agents on the structure of CF-tubulin labeled kinetochore MTs (kMTs) during anaphase. Quantitative analysis of the changes in the fluorescence in k-fibers/background (ratio) shows that: (1) Ca$\sp{2+}$ levels (2-10 $\mu$M) known to inhibit motion, cause extensive degradation of kMT structure (70-90% decrease in ratio). (2) Ca$\sp{2+}$ levels (0.8-1.0 $\mu$M) known to accelerate motion, however, cause only a modest level of kMT depolymerization (30-50% decrease in ratio) in which the k-fibers appear less distinct, and the overall spindle fluorescence is more diffuse. (3) Ca$\sp{2+}$ buffers, which transiently block motion, have no detectable effect on spindle structure. (4) GTP$\gamma$S, which enhances motion also has no discernible effect on spindle structure. I propose that both MT depolymerization and a MT motor are involved in the control of anaphase chromosome motion.

Upon activation of the acetylcholine receptor (muscarinic or nicotinic), chromaffin cells secrete catecholamine, nucleotides and neuropeptides. The secretory granules contain 150 mM ATP. Once this ATP is secreted to the extracellular environment it can regulate the cell by a feedback mechanism. Our laboratory has shown that ATP can enhance the catecholamine secretion evoked by the nicotinic receptor or high K+ and that this effect can be blocked by pretreatment with cholera toxin. Exposure of the cells to ATP prior to stimulation causes inhibition of catecholamine secretion. This inhibitory effect can be prevented by pretreatment with pertussis toxin. The goal of this thesis is to study the signalling pathways of the ATP-evoked effects. I have identified the bacterial toxin-sensitive G-proteins coupled to the ATP receptors and their effectors. By using isolated membranes or intact cells, we found that the ATP receptors were coupled to G$\sb{\rm s}$, G$\sb{\rm i}$ and G$\sb{\rm o}.$ The roles of calcium influx and its release from internal stores have been assessed. ATP can evoke calcium entry and release from internal stores. ATP$\gamma$S, an analogue which mimics the ATP enhancement of secretion, induced IP3 turnover. ATP$\gamma$S + DMPP evoked an increase in IP3 turnover that was greatly potentiated by pretreatment with cholera toxin. ADP, which inhibits secretion, inhibited IP3 turnover. This inhibition was prevented by pretreatment with pertussis toxin. The enhancement and inhibition of phospholipase C evoked by correlated with the effects on secretion. These effects on the activity of phospholipase C might reflect changes in calcium influx evoked by ATP. Part of the ATP-evoked effects on secretion can be explained by the effects of this nucleotide on chromaffin cell calcium currents. Upon addition of ATP, or ADP, which mimics the ATP inhibitory effects, 97% showed a decrease in their long-lasting calcium current. This inhibition of the calcium currents was prevented by pretreatment with pertussis toxin. In 20% of the cells tested, stimulation with ATP caused an increase in the inward current and an enhancement of the long-lasting currents. These currents desensitized to control current in about 30 seconds. Pretreatment with cholera toxin slowed down the rate of desensitization of both enhanced currents.

A Ca$\sp{2+}$-binding protein has been purified and characterized from Blattella germanica eggs. This protein has biochemical features in common with calmodulin. These common features include a relatively low molecular weight of $\sim$19 KDa, thermal stability, an acidic pI of 4.0, a low specific absorbance (E$\sb{\rm 277nm}\sp{1\%}$ = 2.8), an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of 1 mM Ca$\sp{2+},$ and calcium-dependent binding to the calmodulin antagonist W-7. These features, considered together with activation of calmodulin-dependent phosphodiesterase in a Ca$\sp{2+}$-dependent manner and cross-reactivity with anti-bovine brain calmodulin antibody, are sufficient to define this protein as bone fide calmodulin. A rabbit antibody specific for the B. germanica calmodulin cross-reacts with bovine brain calmodulin. The calmodulin levels during oogenesis and embryogenesis were estimated by densitometric analysis of immunoblots using anti-Blattella germanica egg calmodulin antibody as a probe. A high concentration of calmodulin is present in vitellogenic follicles and early embryonic eggs (about 15 ng calmodulin/$\mu$g protein). During oogenesis calmodulin accumulates in the oocyte throughout the yolk deposition phase. During embryogenesis calmodulin is present at uniformly high levels until vitellin utilization starts, then it is undetectable until the pharate larval stage at the end of embryogenesis. $\sp{14}$C-labeled egg calmodulin in a gel-overlay technique binds to vitellin, the major yolk protein of B. germanica eggs. The calmodulin-binding site of vitellin is located on the 95 KDa subunit before degradation, and on the 53 KDa fragment after 95 KDa subunit breakdown. There is sufficient calmodulin to bind stoichiometrically (1:1) with the vitellin trimer. Circumstantial evidence suggests that this CaM is derived from outside the oocyte. In vitro experiments with ($\sp{35}$S) -methionine showed that the highly abundant calmodulin accumulating in vitellogenic follicles was not synthesized by the oocyte. On the other hand, isolated follicle cells rapidly synthesize large amounts of calmodulin. No calmodulin is detectable in serum. These facts suggest that calmodulin produced by follicle cells is most likely the source of calmodulin in the vitellogenic oocyte. Indirect immunofluorescent staining with anti-egg calmodulin demonstrated that in early- and mid-vitellogenic follicles calmodulin is localized in the cytoplasm of follicle cells and the cytoplasmic compartment surrounding yolk granules but not inside yolk granules. Immunofluorescence was most intense in the cortex of the oocyte and outside the membranes of yolk granules. Transport of calmodulin into the cytoplasmic compartment of the oocyte is not via binding to vitellogenenin.

A comparison of the structure of isolated and in situ chromatin fibers was performed using starfish (Patiria miniata) sperm nuclei. The simple protein content consisting of five major histones was found. A DNA repeat length of 222bp was calculated. Compact chromatin fibers solubilized from detergent isolated nuclei showed diameters of 38 to 44nm by electron microscopy. Chromatin fibers observed in whole cells and in mildly treated nuclear preparations revealed fiber diameters of approximately 24nm when embedded in epoxy resin. Investigation of the chromatin isolation process uncovered a mechanism where fiber integrity was lost during nuclear isolation. Following nuclease digestion and chromatin release, fiber-like structures reformed in solution. The relationship of the isolated "fibers" to the native structures is not known. Nuclear permeability has been found to be a factor in both release of cleaved chromatin from nuclei and inward diffusion of nuclease. Permeability to various sized dextrans was consistent with a mechanism where cut chromatin exits nuclei as nucleosomal chains and aggregates in solution to form the isolated chromatin state. Attempts to isolate detergent free nuclei revealed that completely isolated nuclei were susceptible to the loss of chromatin fiber integrity suggesting that the medium was incapable of maintaining fiber structure. Other buffer compositions did not improve stability of in situ fibers underscoring a general lack of understanding of the nuclear environment.

Lymphatic filariasis is a human parasitic disease which causes great physical suffering, while also having a substantial economic impact on the peoples of the tropics and sub-tropics. In order to collect accurate epidemiological data which are crucial for designing effective control programs, reliable diagnostic and taxonomic information is needed. The studies described here were designed to obtain DNA sequence data from specific coding and non-coding loci of the parasite genome, which could augment existing morphological, biochemical, and ecological data, to test hypotheses concerning diagnostic and taxonomic classification. Highly repeated DNA elements, members of the Hha I repeat family, were cloned, sequenced and the data employed in a phylogenetic analysis of species, sub-species and distinct geographic isolates of human filarial parasites from the genus Brugia. Comparative analysis reveals that although the 322 base pair (bp) repeat sequences between Brugia pahangi and Brugia malayi are nearly 90% identical overall, there is a small 70 bp region which contains enough divergence to clearly distinguish between these two major species. Nucleotide differences in this and other regions were exploited to draw distinctions between repeats cloned from B. timori, B. patei and various geographic isolates of B. malayi which differ in biological characteristics such as host range, vector preference and periodicity. In addition to the Hha I repeats, the gene encoding a prominent, stage-specific surface antigen from the animal parasite, Brugia pahangi was also cloned and sequenced. Homologous sequences were obtained from the related human pathogen Brugia malayi and a comparative analysis initiated. The results show that the protein coding, flanking and intervening sequences are highly conserved between the two species. In addition to its utility as a taxonomic and phylogenetic tool, the highly conserved nature of this protein sequence makes it a potential candidate for recombinant vaccine development.

The object of my dissertation research was to evaluate the effects of mitogens on ionic currents in A431 cells. the tools used were fluorescence imaging of a membrane potential sensitive dye, di-8-ANEPPS, and whole cell patch-clamp electrophysiology. Imaging studies revealed that application of 100 $\mu$M ATP caused $>$95% of the cells to hyperpolarize on the order of 30 mV. This hyperpolarization lasted for the duration of agonist presence, and was independent of extracellular calcium. Whole-cell electrophysiology was used to determine the ionic nature of the hyperpolarization. At a holding potential of $-$60 mV, ATP caused an inward current after a 4 second latency. This latency indicated a second messenger accumulation. Removal of extracellular calcium served to reduce the average magnitude of the inward current. By making various substitutions of both the intracellular and extracellular solutions, it was determined that chloride efflux made up the majority of the ATP-evoked inward current. At more depolarized holding potentials, ATP caused a transient inward current followed by a prolonged outward current. The outward current was determined to be carried by potassium, and was sensitive to charybdotoxin, which is known to block Ca$\sp{2+}$ -activated K$\sp{+}$ channels. The source of calcium was determined to be intracellular calcium pools. Inclusion of 5 mM BAPTA in the recording electrode abolished ATP activated currents. The role of G-proteins was demonstrated by the ability of GDP$\beta$S in the dialysis electrode to abolish ATP mediated currents. A dose-response study of the ATP effect revealed that at lower concentrations of ATP, fewer cells responded and that the latency prior to response was increased. The magnitude of the response, however, was independent of ATP concentration. My hypothesis for this "all or none" regenerative phenomenon is that a second messenger cascade causes the release of calcium internal stores. This calcium acts in a positive feedback loop to increase permeability of the plasma membrane to external calcium. The sustained rise in cytosolic calcium allows for the activation of multiple calcium dependent ion channels, including Ca$\sp{2+}$-activated K$\sp{+}$ channels, which cause an overall membrane hyperpolarization in A431 cells.