Protein kinase C (PKC) activity and PKC messenger RNAs in human pituitary adenomas.

Protein kinase C (PKC) is involved in the differentiation and growth regulation of a variety of tissues including anterior pituitary gland cells. To determine the distribution of PKC in different types of adenomas, PKC activity was analyzed in human pituitary tumors and the effects of hypothalamic hormone stimulation on PKC activity were examined in cultured adenoma cells. Gonadotroph (LH/FSH) and null cell adenomas had significantly higher levels of particulate, soluble, and total PKC activity compared with growth hormone (GH) adenomas (P < 0.05). Chronic stimulation of null cell adenomas with gonadotropin hormone-releasing hormone or of one GH adenoma with GH-releasing hormone for 7 days did not significantly alter total PKC activity in pituitary cells cultured in serum-free medium. Localization of the calcium-dependent PKC isozymes (alpha, beta and gamma) by immunohistochemistry and in situ hybridization revealed predominantly PKC alpha in all adenomas and variable expression of PKC beta and gamma in some tumors. When the calcium-independent PKC isozymes (delta, epsilon, and zeta) were localized by in situ hybridization, normal and neoplastic pituitaries expressed abundant mRNA for PKC epsilon, whereas some tumors and one normal pituitary had a few cells positive for PKC zeta mRNA as evaluated by grain density and the number of cells labeled. These results indicate that there is a variable distribution of PKC mRNA isozymes in human pituitary adenomas and that normal pituitaries and pituitary adenoma cells express the mRNA for both the calcium-dependent and some of the calcium-independent PKC isozymes. Chronic treatment with the hypothalamic gonadotropin hormone-releasing hormone and GH-releasing hormone, which increased LH/FSH and GH secretion, respectively, did not increase PKC activity in cultured adenoma cells. The presence of calcium-dependent and calcium-independent PKC isozymes in normal and neoplastic pituitary cells indicates that PKC probably plays a major role in signal transduction in the human pituitary adenomas examined in this study.     

Rat posterior parietal cortex: topography of corticocortical and thalamic connections

Anatomical and functional findings support the contention that there is a distinct posterior parietal cortical area (PPC) in the rat, situated between the rostrally adjacent hindlimb sensorimotor area and the caudally adjacent secondary visual areas. The PPC is distinguished from these areas by receiving thalamic afferents from the lateral dorsal (LD), lateral posterior (LP), and posterior (Po) nuclei, in the absence of input from the ventrobasal complex (VB) or dorsal lateral geniculate (DLG) nuclei. Behavioral studies have demonstrated that PPC is involved in spatial orientation and directed attention. In the present study we used fluorescent retrograde axonal tracers primarily to investigate the cortical connections of PPC, in order to determine the organization of the circuitry by which PPC is likely to participate in these functions, and also to determine how the topography of its thalamic connections differs from that of neighboring cortical areas. The cortical connections of PPC involve the ventrolateral (VLO) and medial (MO) orbital areas, medial agranular cortex (area Fr2), portions of somatic sensory areas Par1 and Par2, secondary visual areas Oc2M and Oc2L, auditory area Tel, and retrosplenial cortex. The secondary visual areas Oc2L and Oc2M have cortical connections which are similar to those of PPC, but are restricted within orbital cortex to area VLO, and within area Fr2 to its caudal portion, and do not involve auditory area Te1. The cortical connections of hindlimb cortex are largely restricted to somatic sensory and motor areas. Retrosplenial cortex, which is medially adjacent to PPC, has cortical connections that are prominent with visual cortex, do not involve somatic sensory or auditory cortex, and include the presubiculum. We conclude that PPC is distinguished by its pattern of cortical connections with the somatic sensory, auditory and visual areas, and with areas Fr2, and VLO/MO, in addition to its exclusive thalamic connectivity with LD, LP and Po. Because recent behavioral studies indicate that PPC, Fr2 and VLO are involved in directed attention and spatial learning, we suggest that the interconnections among these three cortical areas represent a major component of the circuitry for these functions in rats.     

Biological activities of monoclonal antibodies to Mycoplasma pneumoniae membrane glycolipids.

A purified preparation of membranes was obtained by using a unique method of treating Mycoplasma pneumoniae with the ATPase inhibitor, diethylstilbestrol. This method was shown to yield highly purified membranes with little or no cytoplasmic contamination. These membranes were used to immunize mice for subsequent productions of monoclonal antibodies (MAbs). Hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay with whole-cell M. pneumoniae and lipid extract antigens. Four stable MAbs were obtained and characterized. MAb CP3-46F5 reacted with a protein of a molecular weight of approximately 52,000 as determined by Western blot (immunoblot). MAbs CP3-50C2, CP3-53C5, and CP3-53C8 did not react with any antigens on Western blots but did bind to at least 10 distinct glycolipid bands as determined by orcinol staining on thin-layer chromatograms of M. pneumoniae lipid extracts. The MAbs did not react with similarly prepared lipid extracts from Mycoplasma genitalium, Mycoplasma neurolyticum, and Mycoplasma gallisepticum. These MAbs did not inhibit M. pneumoniae metabolism or attachment to WiDr cell cultures. The anti-glycolipid MAbs recognize determinants specific to M. pneumoniae, unlike polyclonal hyperimmune sera against M. pneumoniae, which cross-react with lipid extracts of M. genitalium.     

Heritability of C-Reactive Protein and Association with Apolipoprotein E Genotypes in Japanese Americans

Numerous studies have demonstrated that increased C‐reactive protein (CRP) levels predict coronary heart disease, stroke, peripheral vascular disease, and diabetes, and are associated with features of the metabolic syndrome. Only three previous studies have investigated the heritability of CRP levels, primarily in samples of Caucasian families.The purpose of the present study was to estimate the magnitude of genetic influences on CRP levels, and to examine potential associations between variation in the APOE gene and CRP levels, using a sample of 562 individual Japanese Americans from 68 extended kindreds. In general, correlation coefficients between first‐degree relatives for CRP were approximately 0.2, and spouse correlations did not differ from zero, consistent with genetic influences. Heritability estimates were approximately 0.3 (p < 0.01), even with adjustment for factors known to influence CRP levels. A significant relationship was seen between unadjusted CRP levels and APOE genotypes (p = 0.02), with the highest mean CRP level among ?2 carriers (1.20 mg/L), and nearly the same mean levels among ?3/?3 subjects and ?4 carriers (0.72 and 0.74 mg/L, respectively). However, this relationship was diminished with adjustment for covariates (p = 0.07). These results demonstrate the presence of both genetic and environmental effects on CRP levels among Asian Americans, and additional studies are needed to determine if the APOE gene contributes to these genetic influences.     

Detection of Borrelia burgdorferi in human blood and urine using the polymerase chain reaction.

We investigated the use of the polymerase chain reaction (PCR) to detect Borrelia burgdorferi strain B-31 in human blood and urine experimentally inoculated with 5 and 1 borreliae/cm3, respectively, and to biotinylate a DNA probe specific for B. burgdorferi in the dot blot and Southern blot assays. When the blood and urine samples were subjected to PCR, a 370-bp amplified product was consistently visible on agarose gel electrophoresis after 30 and 45 cycles, respectively. The total human genomic DNA extracted from a 1-cm3 sample of inoculated blood was approximately 6.25 micrograms, and the total amount of B. burgdorferi DNA was estimated to be 0.01 pg/6.25 micrograms of the human DNA. For PCR, 2.5 micrograms of human DNA which contained the equivalent of 0.004 pg of borrelia DNA (approximately two borreliae) were used for enzymatic amplification. When 1/20 or 1/10 of the PCR-amplified products were used either for dot blot or Southern blot hybridization, the accessible copies of amplified B. burgdorferi DNA were sufficient for detectable hybridization to occur. PCR amplification of B. burgdorferi DNA in clinical specimens followed by dot blot hybridization may be a valuable adjunct or alternative to current but inadequate laboratory methods for the diagnosis of Lyme disease.     

Demonstration of human immunodeficiency virus by colorimetric in situ hybridization: a rapid technique for formalin-fixed paraffin-embedded material

A colorimetric method of in situ hybridization has been developed for the rapid detection of human immunodeficiency virus (HIV) in formalin-fixed paraffin-embedded material. Following optimization of digestion conditions, biotin-labeled DNA probes are detected with an alkaline phosphatase conjugate. The method is verified using fixed paraffin-embedded cell blocks of HIV-infected and uninfected lymphocyte cell cultures. Hybridization specifically detects both viral RNA and proviral DNA. Formalin fixation for intervals up to 21 d did not significantly hamper the signal under the appropriate digestion conditions; however, Trump's fixation for even 12 h greatly reduced the intensity of the hybridization. This technique for in situ hybridization is amenable to automation, provides results within 6 h, and results in good morphologic preservation. A key feature of the technique is the use of human placental DNA as an endogenous positive control to optimize the empirically determined conditions for protein digestion.     

Mycoplasma pneumoniae attachment: competitive inhibition by mycoplasmal binding component and by sialic acid-containing glycoconjugates.

Attachment of Mycoplasma pneumoniae to human WiDr cell culture monolayers was examined by using radiolabeled M. pneumoniae. The amount of attachment was proportional to the density of the WiDr cells and to the concentration of M. pneumoniae in the assay. Saturation of the monolayers was achieved with 40 micrograms of virulent strain M129 per assay, whereas binding of avirulent strain B176 was 70% less than that of strain M129. A competitive attachment inhibition assay was used to measure specific binding component activity. Attachment was inhibited when WiDr cells were pretreated with unlabeled virulent strain M129, whereas avirulent noncytadsorbing strain B176 did not inhibit attachment as well as the virulent strain. A protein-rich extract prepared from virulent, cytadsorbing strains of M. pneumoniae also inhibited attachment. The amount of inhibition was dependent on the amount of extract used, and units for binding component activity in the extract were calculated from the competitive attachment inhibition assays. The competitive attachment inhibition assay was also used to investigate the nature of the receptor site on the WiDr cells. Attachment was inhibited when the radiolabeled M. pneumoniae suspensions were pretreated with human sialoglycoproteins, such as orosomucoid and ceruloplasmin, and bovine gangliosides. These findings support the present concept that the mammalian receptor site for M. pneumoniae is a sialic acid-containing glycoprotein.     

FMLP-induced enzyme release from neutrophils: a role for intracellular calcium

The ability of the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) to stimulate beta-glucuronidase release and 45Ca2+ release from rabbit neutrophils was studied. FMLP stimulated enzyme release from cytochalasin B-treated cells either in the presence or the absence of extracellular calcium. Depletion of cell calcium, by exposure to either ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid or the calcium ionophore A23187, blocked the ability of FMLP to stimulate enzyme release and 45Ca2+ release in the absence of extracellular calcium. The ability of A23187 to lower the 45Ca2+ content of neutrophils, to block FMLP-stimulated 45Ca2+ release, and to inhibit FMLP-stimulated enzyme release in the absence of calcium was dose dependent over the same concentration range (10(-8) to 10(-6) M A23187) for all three actions. In contrast, FMLP stimulated enzyme release from A23187-treated cells, provided that extracellular calcium was present. This secretory response was normal as judged by cell ultrastructure and FMLP dose-response relationships. It is concluded that A23187 depletes a pool of intracellular calcium usually released by FMLP and that release of calcium from this pool is necessary for initiation of enzyme secretion in the absence of extracellular calcium.     

Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization

Acidophilic pituitary adenomas commonly produce growth hormone (GH) or prolactin (PRL), according to studies employing immunohistochemical and ultrastructural methods. To examine this question, in situ hybridization with oligonucleotide probes was done on routinely processed tissues received in the pathology laboratory to analyze for the presence of GH and PRL messenger RNA (mRNA) in 4 normal pituitaries, 10 prolactinomas, and 16 GH-secreting adenomas. Most acidophilic cells in normal pituitaries expressed either GH or PRL hormone and the respective mRNAs, but GH mRNA and PRL hormone were also detected in some of the same cells. Patients with a clinical diagnosis of prolactinoma had cells with only PRL mRNA in their tumors, while most (14 of 16) patients with a clinical diagnosis of acromegaly or gigantism had both GH and PRL mRNAs in their tumors. The GH adenomas varied in these studies. In situ hybridization was helpful in characterizing the adenoma from a patient with acromegaly who had immunoreactive PRL, but no immunoreactive GH in the resected tumor; in situ hybridization analysis revealed mRNAs for both GH and PRL in the same tumor cells. Our findings indicate that pituitary adenomas from patients with acromegaly commonly express PRL mRNA. It is concluded that in situ hybridization provides new information about the clinical biology and the histopathologic classification of pituitary adenomas.     

Levels and specificity of antibody in bronchoalveolar lavage (BAL) and serum in an animal model of trimellitic anhydride-induced lung injury

A study was undertaken to characterize the antibody response in rats exposed to trimellitic anhydride (TMA) by inhalation. Total antibody levels directed to trimellitic rat serum albumin (TM-RSA) from TMA-exposed rats were assayed by an ammonium sulfate technique. Total antibody levels in bronchoalveolar lavage (BAL) and the matched serum were compared by correction for the albumin content of each. An ELISA was developed to detect IgG, IgA, and IgM directed toward TM-RSA in BAL and serum and to compare class-specific antibody levels in BAL and serum by normalizing for albumin content. The specificity of the rat IgG response was determined by ELISA inhibition with TM-RSA and TM-human serum albumin (TM-HSA) and compared with reciprocal inhibition studies with serum from TMA-exposed workers. The levels of total antibody in BAL were three to 15 times greater than the levels found in the matched serum pair. IgG, IgA, and IgM antibodies were detected in the BAL and the serum of TMA-exposed rats but not in control rats. In each of the four rats tested, all antibody classes were present in equal or greater amounts in the BAL than in the serum. Complete inhibition of the rat IgG binding in ELISA was observed when TM-RSA or TM-HSA were added as inhibitors. Human IgG was inhibited in ELISA only by TM-HSA. In an animal model of human lung disease, the levels of total antibody as well as class-specific antibodies directed against TM-RSA were greater in BAL than in serum.(ABSTRACT TRUNCATED AT 250 WORDS)