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The function of the pars tuberalis as a mediator of the action of melatonin remains elusive. As a direct method of assessing the potential role of secretory proteins, ovine pars tuberalis cells have been cultured and radiolabelled with 35S-methionine, and the accumulation of specific radioactive products in the medium, measured after separation by SDS-PAGE and fluorography. The synthesis and secretion of a number of labelled proteins are increased by forskolin (1 microM) and inhibited dose dependently by melatonin (IC50, 300 pM), although consistently a 72-kD protein (p72), is the most intensely labelled of these. Thus, 72 acts as a useful marker of cellular activity for melatonin, whereas prolactin (p23) provides a melatonin non-responsive marker in ovine pars tuberalis cell cultures. The synthesis and secretion of p72 and other melatonin-sensitive proteins is regulated through the cyclic AMP/protein kinase A second-messenger pathway, as analogues of cyclic AMP mimic the action of forskolin, yet 1,9-dideoxyforskolin, a forskolin analogue that is not active on adenylate cyclase, has no effect. However, the phorbol ester, phorbol-12,13-myristate acetate, also regulates the synthesis and secretion of the same profile of proteins as forskolin indicating a potential role for protein kinase C, which occurs through an independent rather than a synergistic pathway. The differential effects of nocadazole (1 microM) and extracellular calcium depletion upon p72 and prolactin secretion indicates that p72 is secreted by a calcium and microtubule independent pathway, in contrast to prolactin. These observations in conjunction with the absence of dense-core storage vesicles in melatonin-responsive cells of the ovine PT are consistent with constitutive secretion of p72 from the latter and regulated secretion of prolactin from melatonin non-responsive cells. Using immunoprecipitation de novo synthesis and secretion of either LH or LH-like proteins from ovine pars tuberalis cells could not be detected under the conditions used. The absence of 125I-(Des-Gly10[D-Ala6]-LHRH-ethylamide) binding over most, but not all, of the ovine pars tuberalis supports the contention that the majority of the cells of the ovine pars tuberalis are not gonadotrophs. These results provide further support for the unique function for the pars tuberalis.

Functional receptors for melatonin have been localized and characterized on the pars tuberalis (PT) of a number of mammalian species, but the cell-type responsive to melatonin is unknown. The ultrastructure of the ovine pars tuberalis has been examined and these findings correlated with the functional response of the gland to melatonin. This study revealed that two secretory cell types predominate in the ovine PT, which differ in the abundance of dense-core granules. The most abundant of the cells are either agranular or very sparsely granulated and represent 90% of the total population, with the remaining 10% being composed of cells with abundant dense-core vesicles. Few follicular cells were observed. This ratio of secretory cell-types persisted in primary culture, with the two types non-separable by Percoll gradient centrifugation. Using forskolin, as a non-specific stimulant of adenylate cyclase, melatonin was shown to inhibit the formation of cyclic AMP by 80–90% in cells both before and after Percoll centrifugation. The results demonstrate that the agranular secretory cells of the ovine pars tuberalis are the melatonin responsive cell-type of this gland.

A dense network of neuropeptide Y (NPY)-like immunoreactive (NPY-LI) fibres was revealed in the ovine pineal gland at the light microscope level. The dorsal and peripheral regions of the gland contained the most dense concentration of NPY-LI fibres with relatively few fibres in the mid-region and almost none in the pineal stalk. The effect of NPY in conjunction with isoproterenol (ISO) on cyclic AMP (cAMP) accumulation and noradrenaline (NA) on melatonin synthesis was investigated using in vitro techniques. NPY had no effect on the stimulation of cAMP or melatonin synthesis by the adrenergic agonists.

Primary cultures of ovine pars tuberalis (PT) cells of the pituitary were established to investigate the mode of action of melatonin. The heterogeneous population of cells was shown to bind the radioligand 2-[125I]-melatonin over 72 h in culture, although there was a progressive decline in specific binding with time. In cells cultured for 24 h, forskolin (1 μmol/l) was found to stimulate a 12-fold increase in cyclic AMP accumulation. This response could be inhibited by melatonin in a dose-dependent manner, with an IC50 of approx. 6 pmol/l. However, melatonin did not inhibit basal levels of cyclic AMP. In homogenates of ovine PT, forskolin stimulated a dose-dependent increase in cyclic AMP, although the magnitude of this response was found to be lower than that observed in cells. This response was not inhibited by either 10 nmol or 1 μmol melatonin/l, and was also unaffected by GTP. These results provide the first evidence that the melatonin-binding site on ovine PT, recently characterized using the radioligand 2-[125I]-melatonin, functions as a physiological receptor.

The effect of guanine nucleotides and related analogues on the binding of 2-[125I]-melatonin to membranes prepared from ovine pars tuberalis was studied. Dose-dependent inhibition of 2-[125I]-melatonin binding was observed, with an order of potency of GTP gamma S much greater than Gpp(NH)p greater than GTP = GDP. GMP, cyclic GMP and ATP had negligible effects. Analysis of saturable binding revealed that GTP gamma S (1 microM) promoted an apparent reduction in receptor density of about 50%, without a concomitant change in receptor affinity. These results are consistent with a melatonin receptor existing in an equilibrium between high- and low-affinity states, with GTP and related analogues able to cause a shift in the equilibrium in favour of the lower-affinity form. The sensitivity of 2-[125I]-melatonin binding to guanine nucleotides implies the presence of a melatonin receptor on the ovine pars tuberalis, the action of which is mediated via a G protein.

A high-affinity, discretely localized melatonin receptor has been characterized and mapped within the brain and pituitary of the Syrian hamster using the high specific activity ligand [(125)|]iodomelatonin and a combination of in vitro autoradiography and membrane homogenate receptor assays. Specific binding of radioligand was found in regions of the epithalamus and hypothalamus in the brain and the pars tuberalis of the pituitary. Excitatory amino-acid lesions destroyed [(125)|]iodomelatonin binding within the brain, demonstrating that binding sites are located on neurons. Analysis of [(125)|]iodomelatonin binding to membrane homogenates of the pars tuberalis revealed a linear relationship between specific ligand binding and the amount of tissue. The time-course of specific binding at 37 degrees C reached equilibrium after 30 min and remained stable thereafter. The addition of increasing concentrations of [(125)|]iodomelatonin alone and in the presence of 1 muM melatonin showed that specific binding reached equilibrium at 80 to 100 pM. Analysis of the saturation isotherm using a one-site binding model was consistent with a single receptor site with a K(d) of 29.3 (+/-5.9 SEM) pM and B(max) of 2.54 (+/-0.19 SEM) fmol/mg protein.

The kinetics of radiochromate-labeled autologous blood lymphocytes were studied in ten cases of chronic lymphocytic leukemia (CLL). The labeled cells equilibrated with an enlarged recirculating lymphocyte pool (RLP) distributed between the blood, the spleen, and the bone marrow. The size of the RLP, estimated from dilution of the labeled cells, was better correlated with the degree of splenomegaly and marrow infiltration than was the blood lymphocyte count. The marrow fraction of the RLP turned over more slowly than the splenic pool. The half-life of the labeled cells in the RLP varied in individual cases from one to 11 days. Lymphocyte life span was not related either to the blood lymphocyte count or to the size of the RLP. The mean half-life of 3.8 days was significantly shorter than the normal lymphocyte half-life of 18 days. We conclude that CLL is characterized by overproduction of a strain of abnormally short-lived lymphocytes.

The kinetics of radiochromate-labeled autologous blood lymphocytes were studied in normal subjects. The labeled cells equilibrated within hours with a recirculating lymphocyte pool (RLP) distributed between the blood, the spleen, and the bone marrow. The RLP was found to be about twice the size of the blood lymphocyte pool contained within it and turned over through the blood about 12 times daily. The labeled cells had an average half life in the RLP of 18 days.

The FTA test was developed at a time when immunofluorescence procedures were not well-defined. Through technique control and research, a modification of the FTA test, the FTA-ABS, has attained a position as one of the leading treponemal tests to confirm the reagin tests for syphilis. In this review of the FTA-ABS test, attention has been focused on reagent development, with the anticipation that reagent standardization may soon become a reality. The T. pallidum antigen obtained by extracting infected rabbit testicular tissue has evolved from a preparation in which the treponemes remained in the initial extracting fluid to a reagent that can be free of rabbit tissue and globulin. These washed antigen preparations improve visibility of the treponemes on the microscope slide, reduce background fluorescence, and reduce or prevent from occurring nonspecific reactions that are a result of tissue and globulin components. Both washed and nonwashed antigens are available commercially, and, to date, little differentiation has appeared on the product label. The predominant immunoglobulin that reacts with T. pallidum in the indirect fluorescent antibody tests appears to be IgG. This is the major immunoglobulin detected in the FTA-ABS test. IgM, although increased in early syphilis, is also increased in other clinical conditions. Several reports suggest that adult IgM detection in the present FTA-ABS test would be nonspecific. Until specific IgM antibody in adult syphilis can be detected without a risk to test specificity, the conjugate for the FTA-ABS test should continue to be an anti-IgG reagent. Class-specific, anti-IgG reagents are more expensive than other reagents; however, their use may eliminate the problem of nonspecificity resulting from IgM detection. Additionally, micromethods can be used to reduce cost, and this possibility should be investigated. The sorbent that contains an antigen to the Reiter treponeme may or may not specifically absorb the reactivity that occurs in normal sera; certainly, there are questionable aspects about this reagent. Group antibodies not related to Reiter treponemes may be responsible for some nonspecific reactivity; additionally, antiglobulin factors have been reported to participate in the reaction. Antigens free of rabbit serum factors and class-specific, antiimmunoglobulin reagents are available, and may lead to a better understanding of nonspecific reactions. These reagents should allow resolution of the possible multiplicity of reactivity. In this interim period, the sorbent, with its possible nonspecific nature, appears to maintain a biological balance between natural or group and immune antibodies when used to detect IgG antibody.

THERE are problems associated with the diagnosis of gonorrhea in the asymptomatic female. For many years, the sulfonamides have had little effect in inhibiting the gonococcus, and surveillance studies indicate ever-increasing resistance to penicillin, streptomycin, and oxytetracycline (1, 2). For this reason, it is expedient to obtain accurate laboratory diagnosis rapidly in primary cases and to indicate later whether treatment with antibiotics has been successful. The conventional smear stained by Gram's method is of value in early infections when typical intracellular diplococci may be present in material taken from genital orifices. These organisms are more likely to be found in the male than in the female by this direct examination. As the infection advances or in chronic cases where few organisms are present, the value of the smear decreases (3). Because the genitourinary tract harbors staphylococci, streptococci, various rod types, and JIrim,eae or other species of Neisseria which resemble the gonococcus, information obtained from smears may not be reliable. When the examination is not performed in a venereal disease or a reference laboratory, uncertainty may be compounded because many diagnostic laboratories receive relatively few requests to identify Neisseria gonorrhoeae as compared with tlhe number of such specimens submitted to specialized laboratories. In an effort to broaden tlhe examiination, many workers also culture various exudates on chocola.te agar which well supports the growth of the gonococcus. But due to overgrowtlh, the presence of numerous bacteria that show positive oxidase reactions, and variations in colonial morphology among gonococcal varieties, correct identification of organisms may be difficult (4, 5). In recent years immunofluorescent techniques for identifying gono,cocci have bee.n used successfully (6). Thayer and Martin (7) have developed a selective medium (vanicomycin, colistimethate, nystatin medium) for the isolation and cultivation of N. gonorrhoeae. This medium, hereafter referred to a.s VCN, was prepared in the Ohio Department of Health's laboratory and was used in this study. The purpose of this paper is to report our experience using VCN as a transport medium and to demonstrate the higher percentage of positive results obtained by combining immunofluorescence with culture procedures. This study began in March 1966 and ended in January 1968. The proce.dure described in the study is now being followed routinely at the Ohio Department of Health laboratories.