A new procedure for assaying the phagocytosis of isolated rat rod outer segments (ROS) by cultured rat pigment epithelial (PE) cells has been developed. Utilizing an ROS antiserum and the technique of indirect immunofluorescence, ROS which are attached to the external surfaces of the PE cells can be distinguished from those which have already been ingested by the cells. With this assay procedure, large numbers of PE cells can be visualized for study. Most importantly, the procedure makes it possible to separate and to quantitate both ROS attachment and ingestion. This makes it feasible to study the effects of a variety of metabolites on the recognition and attachment phases of phagocytosis, as well as on the ingestion phase. / The Royal College of Surgeons (RCS) strain of rat has been extensively studied in recent years because it has an hereditary retinal dystrophy in which there is a defect in the phagocytosis of shed ROS material by the PE cells. Numerous in vivo and in vitro studies have been unable to localize the step in the phagocytic process which is affected by the genetic mutation. Using cultured, dystrophic PE cells, and the assay procedure described above, it was possible to show that the recognition and attachment phases of phagocytosis are normal in these dystrophic cells. However, the ingestion phase is deficient. After 4 hours of incubation, normal rat PE cells ingested about 80% of the ROS which had attached to the cells. In contrast, after this incubation period, less than 20% of the ROS which had bound to the dystrophic rat PE cells had been ingested. / Since actin is the main component of the microfilaments which surround an attached particle during its ingestion by a phagocytic cell, it seemed likely that actin might be the defective component in the ingestion process for dystrophic PE cells. Utilizing actin antibodies and the technique of indirect immunofluorescence, normal PE cells were shown to have an extensive network of actin filaments. These filament arrangements did not change when the PE cells were challenged with isolated ROS. This was true whether the ROS were externally bound, in the process of being ingested, or completely internalized. However, a localized concentration of actin was seen to take place at many sites of ROS attachment and ingestion. The arrangement of actin filaments in the dystrophic PE cells appeared normal. Additionally, actin was seen to accumulate at the few sites of ROS ingestion. Thus it appears that actin, a contractile protein important for the ingestion phase of phagocytosis, functions normally in the dystrophic rat PE cell. However, the ingestion phase of phagocytosis becomes activated at very few sites of ROS attachment. / The failure of ingestion to take place at all but a few sites of ROS attachment in the dystrophic PE cells raises the possibility that these cells are deficient in those plasma membrane receptor sites which normally mediate ROS ingestion. However, the phagocytic defect in these PE cells may be due to a failure of actin to accumulate and/or form the microfilaments necessary for pseudopod extension. If this is found to be true, then the defect may be in one of the proteins which regulates the intracellular pool of actin, or in the signal necessary to cause actin accumulation and filament formation at sites of ROS attachment. / Source: Dissertation Abstracts International, Volume: 42-01, Section: B, page: 0034. / Thesis (Ph.D.)--The Florida State University, 1981.

A segment of DNA from the African clawed frog (Xenopus laevis) was recently cloned into a bacterial plasmid and subsequently microinjected into unfertilized eggs of X. laevis by Watanabe and Taylor (1980). The recombinant plasmid was found to replicate with increased efficiency in unfertilized eggs than the vector itself. To further investigate the possible role of this segment of DNA ('Xori') in replication, I subcloned fragments of Xori into the bacterial plasmid pBR322. The recombinant plasmids obtained were microinjected into X. laevis eggs and their replication in vivo was studied. Comparison of the efficiencies of replication of the different recombinants and the vector confirmed that specific DNA sequences are required for the initiation of DNA replication in eukaryotes. Furthermore, Xori was found to have a sequence structure similar to that of the Alu family of mammalian repetitive sequences, some of which have been shown to be transcribed in vitro. The plasmids containing fragments of Xori were then microinjected into oocyte nuclei of X. laevis to determine if they could be transcribed. Preliminary results indicated that the recombinant plasmids were more efficiently transcribed than the vector, thus suggesting the possibility of the Alu type sequences functioning as eukaryotic origins of DNA replication. / Source: Dissertation Abstracts International, Volume: 43-04, Section: B, page: 0947. / Thesis (Ph.D.)--The Florida State University, 1982.

In many bacteria and most eukaryotes, some cytosine residues are modified following DNA replication by enzymatic methylation at the 5' position. This modification does not affect base pairing, but it can cause changes in DNA-protein interactions and helix stability. Examination of a large number of gene sequences indicates a strong, though not absolute, correlation between undermethylation of some sites and gene expression. To determine whether DNA methylation can cause loss of expression potential I and others have initiated studies using genes methylated in vitro at sites likely to be modified in cells, and introduced into suitable test systems. My original goal was to methylate SV4C DNA containing a cloned mammalian gene with the Hpa II methylase, and to analyse transcription of the modified gene during a lytic infection of monkey cells. However, I showed that SV40 DNA loses any in vitro methylation during such an infection. Finally, a plasmid, pTK, containing the thymidine kinase gene from the Herpes simplex type I virus was methylated in vitro and injected into Xenopus laevis oocytes. Unmethylated pTK DNA is transcribed into mRNA and processed into functional thymidine kinase. Using S(,1) nuclease and thymidine kinase activity assays, I have shown that methylated pTK DNA is also efficient in mRNA and protein production when the plasmid is injected at a high concentration. However, preliminary studies indicate that low concentrations of methylated injected pTK DNA may be far less efficiently transcribed. This indicates that methylation of Hpa II sites within the Herpes tk gene probably has some effect on its transcription. Similar results have been reported for the tk gene methylated and assayed in a tk('-) cell line. Much more drastic effects have been reported following investigation of adenovirus early genes and SV40 DNA methylated at Hpa II sites / and tested in the Xenopus system, and of rabbit globin genes methylated at all CG sites. The relatively small depression of synthesis produced in the tk gene may be due to the lack of Hpa II sites in the 5' region of the gene. Whether sites important to transcription are methylated in vivo remains to be confirmed. / Source: Dissertation Abstracts International, Volume: 45-04, Section: B, page: 1090. / Thesis (Ph.D.)--The Florida State University, 1984.

The cell surface localization and structural diversity of neuronal glycoproteins has resulted in the proposal that these molecules are important in cell recognition processes related to axon growth. Accordingly, alterations in cell surface glycoproteins, particularly those binding the mannose-specific lectin Concanavalin A, have been documented in developing neurons. However, a paucity of information has been provided concerning the molecular nature of these lectin receptors. This study investigated the properties, including lectin affinity, of axonally transported glycoproteins and their carbohydrate chains in regenerating garfish olfactory nerve. / Results of this study show that regeneration-related axon growth is accompanied by a two-fold increase in the proportion of low molecular weight (MW) carbohydrate chains associated with glycoproteins. When Con A affinity of the total glycopeptide fraction is assessed, the greatest proportional increase in Con A binding occurs in the low MW fraction. An analysis of the composition of these low MW Con A-binding molecules reveals a typical mannose-rich structure. This study also indicates that the proportion of radioactivity in low MW Con A-binding glycopeptides exhibits increases over intact nerve at all post-operative times analyzed, but to a significantly greater extent at earlier regeneration times. This result may indicate a participation by these molecules in axon-environmental interactions during axon growth. Consistent with this view, these mannose-rich glycopeptides are enriched 15-fold during axon growth in an axonal subcellular fraction that may resemble the cell surface coat or glycocalyx. / The analysis of intact glycoprotein molecules in garfish olfactory nerve reveals regeneration-dependent increases in the transport of glycoproteins with MWs of 180-200k, 105-115k and 80-90k daltons and a diminution in the transport of a 150-160k component. Con A fractionation of intact glycoproteins shows that in regenerating nerve an augmented proportion of glycoprotein radioactivity binds the lectin, with the largest growth-correlated increases occurring in molecules with MWs of 180-200k and 80-115k daltons. It can also be assumed that these Con A-binding glycoproteins probably contain a significant fraction of the mannose-rich carbohydrate chains described above. / Source: Dissertation Abstracts International, Volume: 43-07, Section: B, page: 2084. / Thesis (Ph.D.)--The Florida State University, 1982.

In a continuous series of experiments dealing with the dynamics of the olfactory neuroepithelium, this study was undertaken to explore the survival and turnover rate of olfactory neurons before and after removal of their target. The total number of receptor cells in the mouse olfactory epithelium was estimated and its correlation with age was determined. There is a 64% increase in total number of neurons from one to four months of age followed by a 28% decrease in a period from four to thirteen months of age. / Autoradiographic studies were conducted to describe neuronal renewal and turnover in young adult animals. The results indicate that the percentage of labeled basal cells reached its maximum of 95.5% at 24 hours after the injection of tritiated thymidine. Ten days after injection, labeled basal cells percentage dropped to 47.6% simultaneously with an increase in the percentage of labeled neurons reading 48% of the total labeled cells. By 30 days post injection the percentage of labeled basal cells declined to 8.7%. 74% of the total labeled mature neurons have already disappeared by 55 days postinjection. / Neonatal and young adult mice which underwent unilateral partial bulbectomy were injected with tritiated thymidine 60 days after surgery and then were sacrificed at different time intervals. On the operated side the morphological observations have shown that the olfactory neuroepithelium is thinner than the normal due to a reduction of 11% and 48% in the neuronal population of neonatal and adult bulbectomized mice respectively. Autoradiographic observations indicate that neonatal animals sacrificed 24 hours postinjection had 46.3% more labeled basal cells in the experimental side than in the controls. The labeled basal cells disappeared from the experimental side at a faster rate than those in the control side. / Labeled sensory cell percentage rose from zero percent at 24 hours to 45.8% of the total labeled elements in the operated side at 7 days postinjection while those of the control side comprised only 30.7%. By 15 days after injection, the percentage of labeled neurons in the operated side declined to 36.4% while those of the control still increasing reaching a maximum of 45.8% of the total labeled elements indicating a shorter lifespan of regenerating neurons. / Source: Dissertation Abstracts International, Volume: 44-02, Section: B, page: 0385. / Thesis (Ph.D.)--The Florida State University, 1983.

Phospholipid, ester-linked fatty acids (PLFA) can be used as measures of the viable microbial biomass and community structure in complex environments. Methodology is described, reviewed and tested for the specific analysis of PLFA with minimal artifacts or contamination. In addition, a hexane/isopropanol extraction system is shown to be equivalent to the standard chloroform/methanol system allowing lipid analyses to be done off Nuclepore filters and other materials that will dissolve in chloroform. The reproducibility of PLFA profiles for prokaryotic communities was also examined. A homogeneous, estuarine benthic microbial community was sub-divided randomly to give identical microcosms. These microcosms were nutrient supplemented and incubated either aerobically or anaerobically. The PLFA profiles of independent microcosms treated identically had less variance than those treated differently such that statistically significant differences for treatment effect could be described. The PLFA changes during long-term starvation in Vibrio cholerae were also examined. A progressive, reproducible, and significant increase in the PLFA trans/cis ratio was seen as well as an increase in the cyclopropyl PLFA. These changes may be an adaptation to starvation-survival. There is, however, no known de novo biosynthetic pathway for trans-monounsaturated PLFA. To prove that one exists, Pseudomonas atlantica, which has three cis/trans PLFA pairs, was incubated with ('14)C-acetate. The PLFA isomers were physically separated, the trans content was directly verified by FT-IR, and the specific activity of the trans PLFA was shown to be equivalent to the saturated PLFA verifying de novo synthesis of trans-monounsaturated PLFA from acetate. / Source: Dissertation Abstracts International, Volume: 47-07, Section: B, page: 2723. / Thesis (Ph.D.)--The Florida State University, 1986.

Antibody to hog brain tubulin was raised in rabbits and used to stain microtubules within fertilized eggs of the sea urchins Arbacia punctulata and Lytechinus variegatus. The unfertilized egg was found to be devoid of microtubules until 3 min post-insemination, at which time numerous microtubules appear, all of which emanate from the sperm centioles. These microtubules radiate throughout much of the egg's volume by 8 min and appear to bind to and apply a pulling force to the egg nucleus. As a result the egg nucleus moves to the sperm aster in a fashion very reminiscent of mitotic chromosome motion. These observations may help to resolve the long-standing controversy over the mechanism of pronuclear movement. / Since the sea urchin egg contains no microtubules until after sperm-egg fusion it seemed that this might be an excellent system for studying the control of microtubule assembly. Eggs can be artificially activated in the absence of sperm at a number of control points and it was hoped that comparison of the microtubule configurations of eggs activated by different treatments might allow the identification of the cause of microtubule assembly. By this method it was found that neither the Ca('++) transient nor the presence of centrioles can be said to control the number of microtubules developed by the egg, although Ca('++) and centrioles profoundly effect the behavior of microtubules in vitro. Surprisingly, blocking the shift in cytoplasmic pH which accompanies fertilization prevented the appearance of microtubules, even though the direction of the pH shift is away from the optimum pH for microtubule assembly in vitro. To assist in these experiments a spectroscopic method of intracellular pH determination was adapted to sea urchin eggs and found to work extremely well with these cells. This method was used to show that pH can be clamped at 6.8-6.9 either by incubation in choline-substituted sea water (to block Na('+)-H('+) antiport) or by incubation in 10('-2) M Na acetate. Either treatment prevented the formation of microtubules after fertilization. / There are recent suggestions that microtubules in animal cells are associated at all times with centrioles. In the sea urchin egg centrioles are not present and yet the cell is capable of assembling large numbers of microtubules after metabolic derepression. However, the behavior of the microtubule-containing structures in activated eggs is quite different from that seen in fertilized eggs (which contain centrioles), suggesting that centrioles have a minimal effect on the extent of microtubule assembly but are important in regulating the behavior of microtubule-containing structures. / Briefly, the major contributions of the work reported in this monograph are: (i) The establishment of the structure of the apparatus which moves the pronuclei at fertilization and the identification of this apparatus as a favorable system for studies on the mechanism by which microtubule-containing structures generate force, one of the major unsolved problems in cell biology, (ii) The finding that prior to fertilization the egg contains no microtubules, and the identification of the change in cytoplasmic pH as being important in making egg cytoplasm conducive to microtubule assembly, and (iii) The use of artificially activated eggs to show that at least in these cells centrioles do not play a major role in the control of microtubule assembly. / Source: Dissertation Abstracts International, Volume: 42-06, Section: B, page: 2179. / Thesis (Ph.D.)--The Florida State University, 1981.

Source: Dissertation Abstracts International, Volume: 41-01, Section: B, page: 0037. / Thesis (Ph.D.)--The Florida State University, 1980.

Pilumnus sayi (Crustacea, Brachyura, Xanthidae) occupied heads, i.e., colonies, of Schizoporella pungens (Bryozoa, Cheilostomata) on seagrass beds located along a barrier shoals in the northern Gulf of Mexico. In a collection from 23 m('2), 94 adult crabs were found among 369 colonies. Sex ratio for adults was significantly skewed at 1:1.85 males to females. Schizoporella heads were significantly clumped according to nearest neighbor analyses, and were renewable within 6 1/2 months. Heads differed in size, presence or absence of cavities, and state of attachment to the substrate. Adult crabs were seen foraging on and around heads. Observed predators included the wrasse Halichoeres radiatus and the box crab Hepatus epheliticus. / Laboratory experiments tested predictions of consumer behavior deduced from characteristics of the heads and possible selective pressures for this association, i.e., predation, trophic pressures, and sexual selection. Adult P. sayi preferred heads larger than a 17 ml threshold, attached heads, and heads with cavities, contrary to predictions derived from trophic pressures and consistent with those from predation. Crab preferences for clumped heads was sex dependent, thus implicating sexual selection, used in a broad sense pertaining to mate acquisition. Movement to heads was quick and direct as expected from predatory pressures; and males moved between heads more often than did females, again implicating sexual selection. Crab size was not a factor in either movement or preferences when crabs were tested individually. However, larger crabs dominated smaller crabs in agonistic contests for individual heads, regardless of the sex or residency of participants. An ethogram of 6 modal action patterns (MAPs) and 5 non-MAP acts was compiled, and the structure of contests was analyzed. Escalation of violence was likely to be initiated by either crab, but was more readily continued by the larger one. In group experiments, the location of larger and smaller males was independent of either the location of females or the spatial pattern of heads. However, in group experiments, larger males moved between heads significantly more often than did smaller males, which moved at the same frequency as females. / Ecological patterns of head use by P. sayi were consistent with expectations derived from consumer behavior and population descriptions. The number of adult crabs equaled the number of preferred heads. Adults occupied significantly more heads with cavities than heads without cavities. They also occupied significantly larger than average heads, but not necessarily heads larger than their preferred threshold. Consequently, there was a slight but significant correlation between crab size and the size of heads occupied. Furthermore, males and females did not differ in the types of heads occupied. Adults occupied heads individually except for occasional double occupancy by a male and female. In such cases, the male tended to be larger. Further spatial pattern among crags was revealed by an analysis that incorporated head patterns; among adults, there were fewer heterosexual nearest-neighbor pairs than expected by chance alone. / The combination of behavioral experiments and ecological sampling suggested that a relative shortage of this resource existed for the population of P. sayi under study. Furthermore, behavior of crabs in experimental situations and their spatial pattern in nature was consistent with resource defense polygyny, although a capacity for male dominance polygyny was indicated. Behavioral scaling that incorporates resource structure and risk of predation is hypothesized as a determinant of this crab's mating system. / Source: Dissertation Abstracts International, Volume: 41-03, Section: B, page: 0786. / Thesis (Ph.D.)--The Florida State University, 1980.

More than 450 ventricles, isolated from 51 species of bivalve molluscs, were surveyed for their responses to the molluscan cardioactive neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH(,2)). The responses were diverse, ranging from excitation to inhibition, but over half of the species were only excited by the peptide, and only two were exclusively inhibited. Oysters were notably insensitive. FMRFamide also caused catch contractures of the anterior byssus retractor muscle (ABRM) of Geukensia demissa which were indistinguishable from those of acetylcholine (ACh). Nevertheless, their mechanisms of action differed. FMRFamide contractures were less Na-dependent, although extracellular Na was necessary for catch. FMRFamide caused a larger influx of Ca than did ACh, but, in Ca-free ASW, FMRFamide also had access to intracellular Ca that could not be released by ACh. Neither agent affected cyclic nucleotide levels in the muscle. The specificity of the FMRFamide response in the ABRM and two hearts (Macrocallista nimbosa which is excited by the peptide, and Lampsilis claibornensis which is inhibited) were examined in detail. The C-terminal amide and Arg('3) residue are critical to FMRFamide-like activity on all three preparations; extensions or substitutions at the N-terminal are less deleterious. The three preparations differ in specificity, however. Lampsilis hearts are the least restrictive, especially with respect to the Arg('3) residue. The ABRMs are consistently more sensitive to analogs with extensions at the N-terminal, but the increase in sensitivity is relatively small compared to other preparations. The effects of FMRFamide on cAMP levels in hearts of Lampsilis claibornensis were also examined. FMRFamide inhibition is accompanied by a dose-dependent and time-dependent increase in cAMP. But the increase follows the change in mechanical activity, indicating that / cAMP does not cause the early inhibitory effects of the peptide. It is not clear whether the cAMP is elevated as a direct result of FMRFamide-receptor interaction, or whether it is elevated indirectly, as a result of the inhibition. In either case, it might serve a homeostatic function. / Source: Dissertation Abstracts International, Volume: 42-07, Section: B, page: 2661. / Thesis (Ph.D.)--The Florida State University, 1981.