Nitric oxide synthase and cGMP activity in the salivary glands of the American dog tick Dermacentor variabilis.

We colocalized nitric oxide synthase (NOS) activity in epithelial cells that surround the salivary gland duct in female Dermacentor variabilis with NADPH diaphorase histochemistry and immunohistochemistry using a polyclonal anti-endothelial NOS. Using size-exclusion chromatography, a fraction with a molecular mass of about 185 kDa that had diaphorase activity was eluted from tick salivary gland homogenate. This fraction converted arginine to citrulline with the production of nitric oxide (NO), which was detected by using electron spin resonance spectroscopy. The complete activity of the diaphorase fraction was dependent on NADPH, FAD, tetrahydrobiopterin, calmodulin, (CaM), and Ca(2+), but was not dependent on dithiothreitol. The arginine analog N(G)-monomethyl-L-arginine inhibited the activity of this fraction. NO and arginine activated soluble guanylate cyclase to produce cGMP in dopamine-stimulated isolated salivary glands. Dopamine-stimulated isolated salivary glands treated with tick saline containing either EDTA, the NOS inhibitor N(G)-nitro-L-arginine methyl ester, or the calcium/CaM binding inhibitor W-7 showed no increase in cGMP. The NO donor sodium nitroprusside significantly increased cGMP levels in unstimulated isolated salivary glands. A possible function for NO in salivation by this ixodid tick is discussed.

Preganglionic endings from nucleus of Edinger-Westphal in pigeon ciliary ganglion contain neuronal nitric oxide synthase.

The avian ciliary ganglion (CG) controls choroidal blood flow by its choroidal neurons, and pupil constriction and accommodation by its ciliary neurons. It was previously reported that both choroidal and ciliary neurons label positively for NADPH diaphorase (NADPHd), a marker for nitric oxide synthase (NOS). To assess if this labeling is preganglionic or postganglionic and to determine if it is attributable to neuronal NOS (nNOS), we studied pigeon CG using NADPHd histochemistry and nNOS immunohistochemistry (IHC). Short-duration staining times by NADPHd histochemistry yielded intense labeling of structures that appeared to be the cap-like endings on ciliary neurons and the boutonal endings on choroidal neurons that arise from the nucleus of Edinger-Westphal (EW), and light or no postganglionic perikaryal staining. The light postganglionic staining that was observed tended to be localized to ciliary neurons. Consistent with this, NADPHd+ nerve fibers were observed in the postganglionic ciliary nerves but rarely in the postganglionic choroidal nerves. These same staining times yielded robust staining of neurons in the orbital pterygopalatine microganglia network, which are known to be nNOS+. Diffuse staining of CG perikarya was observed with longer staining durations, and this staining tended to mask the preganglionic labeling. Preganglionic NADPHd+ staining in CG with short staining times was blocked by the NOS inhibitors iodonium diphenyl (IDP) and dichlorophenol-indophenol (DPIP), but the diffuse postganglionic staining observed with the longer staining times was not completely blocked. Labeling of CG sections for substance P (SP) by IHC (which labels EW-originating preganglionic endings in CG) and subsequently for NADPHd confirmed that NADPHd was localized to preganglionic endings on CG neurons. Immunohistochemical double labeling for nNOS and SP or enkephalin further confirmed that nNOS is found in boutonal and cap-like endings in the CG. Two studies were then carried out to demonstrate that the nNOS+ preganglionic endings in CG arise from EW. First, NADPHd+ and nNOS+ neurons were observed in EW in pigeons treated with colchicine to enhance perikaryal labeling. Second, NADPHd+ and nNOS+ preganglionic endings were eliminated from CG ipsilateral to an EW lesion. These various results indicate that NOS is present in EW-arising preganglionic endings on choroidal and ciliary neurons in avian CG. NOS also appears to be found in some ciliary neurons, but its presence in choroidal neurons is currently uncertain.

Cellular localization of huntingtin in striatal and cortical neurons in rats: lack of correlation with neuronal vulnerability in Huntington's disease.

Immunohistochemistry and single-cell RT-PCR were used to characterize the localization of huntingtin and/or its mRNA in the major types of striatal neurons and in corticostriatal projection neurons in rats. Single-label immunohistochemical studies revealed that striatum contains scattered large neurons rich in huntingtin and more numerous medium-sized neurons moderate in huntingtin. Double-label immunohistochemical studies showed that the large huntingtin-rich striatal neurons include nearly all cholinergic interneurons and some parvalbuminergic interneurons. Somatostatinergic striatal interneurons, which are medium in size, rarely contained huntingtin. Calbindin immunolabeling showed that the vast majority of the medium-sized striatal neurons that contain huntingtin are projection neurons, but only approximately 65% of calbindin-labeled projection neurons (localized to the matrix compartment of striatum) were labeled for huntingtin. Calbindin-containing projection neurons of the matrix compartment and calbindin-negative projection neurons of the striatal patch compartment contained huntingtin with comparable frequency. Single-cell RT-PCR confirmed that striatal cholinergic interneurons contain huntingtin, but only approximately 65% of projection neurons contained detectable huntingtin message. The finding that huntingtin is not consistently found in striatal projection neurons [which die in Huntington's disease (HD)] but is abundant in striatal cholinergic interneurons (which survive in Huntington's disease) suggests that the mutation in huntingtin that causes HD may not directly kill neurons. In contrast to the heterogeneous expression of huntingtin in the different striatal neuron types, we found all corticostriatal neurons to be rich in huntingtin protein and mRNA. One possibility raised by our findings is that the HD mutation may render corticostriatal neurons destructive rather than render striatal neurons vulnerable.

Vascular endothelial growth factor increases release of gelatinase A and decreases release of tissue inhibitor of metalloproteinases by microvascular endothelial cells in vitro.

The present study was designed to determine the influences of vascular endothelial growth factor (VEGF) on cell proliferation and the release of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) from human dermal microvascular endothelial cells. Treatment of cultures with 10 ng/ml or more of VEGF significantly increased cell proliferation. The effect of VEGF treatment on the levels of specific MMPs and TIMPs in the media was subsequently examined in cultures that were treated with 10 ng/ml VEGF. Zymography and Western blot analyses demonstrated that gelatinase A levels in the media were increased by VEGF treatment. Collagenase was detected by Western blots in both VEGF-treated and untreated culture media, but the levels were not significantly increased by the VEGF treatment. An ELISA assay confirmed that VEGF treatment significantly increased gelatinase A levels but did not significantly increase collagenase levels. Western blot and ELISA data showed that VEGF treatment significantly decreased TIMP-1 and TIMP-2 levels compared to untreated cultures. The data suggest that VEGF may modulate endothelial cell-derived MMP activity by: (1) increasing the abundance of gelatinase A; (2) disinhibiting gelatinase A by decreasing the abundance of TIMP-2; and (3) disinhibiting preexisting collagenase by reducing levels of TIMP-1. These actions could contribute to the ability of VEGF to promote endothelial cell invasion of new territory.

Evidence of a myoepithelial cell in tick salivary glands.

Type III acini from feeding female Dermacentor variabilis varied in size during in vitro and in vivo fluid production. As the type III acinus enlarged, its lumen enlarged and the adlumenal cell became thinner. As the acinus contracted, its lumen became smaller while the adlumenal cell became wider. Actin was demonstrated in salivary glands using an immunoblot technique. Actin was localized in the adlumenal cells of type III acini with fluorescent microscopy using rhodamine-phalloidin and with electron microscopy using heavy meromyosin to decorate actin filaments. Pre-treatment of salivary glands with cytochalasin D abolished fluorescence in adlumenal cells subsequently treated with rhodamine-phalloidin. These results support the hypothesis that the adlumenal cell in type III acini functions as a myoepithelial cell.

Fluid secretion by isolated tick salivary glands dependent on an intact cytoskeleton.

Isolated salivary glands from female Dermacentor variabilis (Say) were pre-treated with either cytochalasin D or nocodazol, followed by stimulation with dopamine. Glands pre-treated with 1 microM-cytochalasin D did not secrete fluid; pre-treatment with 1 nM-cytochalasin D did not significantly reduce fluid secretion. Glands pre-treated with 1 microM-nocodazol had a significant reduction in fluid secretion (P = 0.003); 1 nM-nocodazol did not significantly affect fluid secretion. Ligation of the main salivary duct and pre-treatment with 1 microM-cytochalasin D significantly increased gland weight compared to the dopamine stimulated controls (P = 0.0412). Cytochalasin D-treated type III acini had a significantly larger mean diameter compared to the dopamine control (P = 0.0047). Glands treated with 1 mM-verapamil plus 10 microM-dopamine exhibited a significant decrease in fluid secretion (P = 0.017), and when ligated, had a significantly decreased weight compared to the controls (P = 0.0028).

The effect of glycoprotein IIb-IIIa receptor occupancy on the cytoskeleton of resting and activated platelets.

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), serves as the receptor for fibrinogen. This study examined what effect GPIIb-IIIa receptor occupancy had on the cytoskeleton of resting and activated platelets. Triton X-100-insoluble residues (cytoskeletons) were isolated from resting washed platelets incubated with either 500 microM RGDS or 500 microM RGES and examined for protein content. RGDS did not increase the amount of GPIIb-IIIa associated with the cytoskeletal residues which sedimented at either 15,800 x g or 100,000 x g. To determine the effect of receptor occupancy on the formation of the activated platelet cytoskeleton, stirred and nonstirred RGDS-treated platelets in plasma were activated with ADP. Triton X-100-insoluble residues were isolated and examined for both protein content and retention of GPIIb-IIIa. Further, morphological studies were performed on the RGDS-ADP-stimulated platelets. The results of this study suggest that 1) RGDS peptide receptor occupancy does not lead to GPIIb-IIIa linkage to the cytoskeleton, 2) ADP-stimulated platelet shape change, polymerization of actin, and association of myosin with the cytoskeleton are unaffected by RGDS peptide receptor occupancy. 3) RGDS inhibits an aggregation-dependent incorporation of ABP, alpha-actinin, talin, and GPIIb-IIIa into the Triton-insoluble residue.

Hemocytic encapsulation of implants in the tick Dermacentor variabilis.

Implants of Epon, inserted in Dermacentor variabilis (Say) through incisions in the cuticle, were encapsulated by hemocytes. We followed this process at intervals of 1,3,6, 12 and 24 h, and every 24 h thereafter up to 120 h. Degranulation of Type 1 granulocytes and coagulation of hemolymph were first seen at 1 h after implantation and were the earliest evidence of encapsulation. By 3 h after implantation, the degranulation and disintegration of granulocytes had formed a matrix at the Epon surface. From 6 h until encapsulation was completed, plasmatocytes and granulocytes continued to respond to degranulation and formed multiple cell layers around the Epon implant. The capsule was complete at 72 h after implantation. Completion was marked by decreasing degranulation, migration of hemocytes from the outermost layers of the capsule, and by the appearance of loosely attached hemocytes on the outer surface of the capsule. The most common junctional complex observed was gap junctions.

Onset of vitellogenin production and vitellogenesis, and their relationship to changes in the midgut epithelium and oocytes in the tick Dermacentor variabilis.

In Dermacentor variabilis (Say), the onset of vitellogenin production and vitellogenesis (uptake of vitellogenin into oocytes) began during the rapid-engorgement feeding period. Mating was required for both vitellogenin production and vitellogenesis to complete the tick's life cycle. Complete immunological identity, as measured by Ouchterlony's double diffusion test, existed between vitellogenin from the fat body, midgut and hemolymph, and vitellin from the ovaries and eggs. Antivitellin antibody did not react with host hemoglobin nor with fat body, midgut, and ovary extracts from feeding females prior to rapid engorgement, feeding unmated females, or unfed or fed males. Some unmated females fed for 13 days and then hand-detached from the host eventually began oviposition after going through a preoviposition period. In these ticks, organ extracts from the midgut, fat body and ovary reacted with antivitellin antibody. The presence or absence of presumed vitellogenic cells in the midgut and yolk bodies in oocytes corresponded with the presence or absence of vitellogenin and vitellogenesis as measured by Ouchterlony's test. Presumed vitellogenic cells increased in size during the preoviposition period. These cells reached their greatest size during the time when the most eggs were being produced, and then declined in size toward the end of oviposition. Vitellogenin was deposited directly into developing yolk bodies in oocytes and was not processed through lysosomes. Feeding was the process that initiated the formation of eggshell cuticle. Detachment from the host was required for the initiation of oviposition.

Comparison of microbiologic characteristics of pathogenic and saprophytic coagulase-negative staphylococci from patients on continuous ambulatory peritoneal dialysis.

Twenty-one microbiologically documented episodes of coagulase-negative staphylococcal peritonitis occurred in 21 continuous ambulatory peritoneal dialysis patients. All strains involved in these infections were tested for antimicrobial susceptibility and in vitro adherence assays. Twenty of the strains were species identified using two commercially available systems. For comparison, 20 saprophytic strains of coagulase-negative staphylococci obtained from the nares and axillae of 10 uninfected, peritoneal dialysis patients were included for in vitro characterization. Staphylococcus epidermidis was the species most often identified for both clinical and saprophytic strains. Eighteen of the 21 (86%) clinical strains were resistant to penicillin G. Methicillin resistance, which was present in five clinical strains, was not found in saprophytic strains. Adherence assay determinations showed marked differences between clinical versus colonization strains, with the clinical isolates significantly more adherent (p less than 0.025) than colonization strains. Electron microscopic examination of silastic catheter segments incubated with a strain of S. epidermidis in used and unused dialysis fluids demonstrated marked differences in attachment of bacteria to catheter material.