Microfluorimetric imaging study of the mechanism of activation of the Na+/H+ antiport by muscarinic agonist in rat mandibular acinar cells.

The mechanism of regulation of intracellular pH (pHi) in dispersed acini from the rat mandibular salivary gland has been studied with a microfluorimetric imaging method and the pH probe 2',7'-bis(2-carboxyethyl)-5(and -6)-carboxyfluorescein. The pHi in the TRIS/HEPES-buffered standard solution was 7.29 +/- 0.01. Addition of 1 mumol/l acetylcholine (ACh) or ionomycin caused a sustained increase in the pHi. These agents decreased pHi in the absence of external Na+ or in the presence of amiloride. The rate of pHi recovery from an acid load after NH+4 prepulse was a linear function of pHi and increased as pHi became more acidic. Addition of ACh shifted the relationship towards a more alkaline pHi range. The increase in pHi induced by ACh or ionomycin was not inhibited by the protein kinase C inhibitors staurosporine (10 nM) and 1-(5-isoquinolinesulfonyl)-1-methylpiperazine (50 mumol/l). Addition of 0.1-1 mumol/l phorbol 12-myristate 13-acetate (TPA) had little effect on pHi within 10 min; however, exposure to TPA for 120 min resulted in a significant rise in pHi. In Ca(2+)-free solution with 50 mumol/l 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the ACh-induced rise in both pHi and cytosolic Ca2+ concentration was suppressed. ACh and ionomycin caused an increment of amiloride-sensitive acid output into the extracellular fluid, while 20 mumol/l 1-oleoyl-2-acetylglycerol had little effect on it. It was concluded that (a) stimulation with ACh activated the Na+/H+ antiport in the plasma membrane, (b) ACh also stimulated the intracellular acid production but acid extrusion by the Na+/H+ antiport prevented the cell from intracellular acidification, and (c) the major route of signal transduction for the ACh-induced activation of the Na+/H+ antiport was independent of protein kinase C but was dependent on the rise in cytosolic Ca2+ concentration. The implication of the cytosolic acidification and cell volume change in pHi regulation is discussed.

Simultaneous determination of GTP cyclohydrolase activity and biopterin content in pheochromocytoma PC12h cells.

A method to measure the GTP cyclohydrolase and biopterin (BP) content at the same time in the sonicate of pheochromocytoma PC12h cells was described. The cells cultured in a 5-cm dish were sonicated with 0.4 to 0.5 ml of 50 mM Tris-HCl, pH 7.8, and a 50 microliters aliquot was used for one assay. After the GTP cyclohydrolase reaction, dihydroneopterin triphosphate, the reaction product, was oxidized and dephosphorylated to form neopterin (NP); and the pterins in the reaction mixture were then analyzed by high-performance liquid chromatography (HPLC). Mainly three peaks of pterins were detected in the eluate from the HPLC column: they were NP, BP, and 2-amino-4-hydroxypteridine (AHP). The amount of NP was a measure of the enzyme activity. On the other hand, a significant part of the biopterin contained in the enzyme sample was converted to AHP during the GTP cyclohydrolase reaction. Therefore, the sum amount of BP plus AHP was a measure of total BP content in the enzyme sample (sonicate of PC12h cells). The method is convenient and useful to study the actions and action mechanisms of various biologically active substances on the BP level of pheochromocytoma cells in culture.

The predominance of human papillomavirus type 16 in vulvar neoplasia.

Southern transfer analysis for human papillomavirus genomic sequences was conducted on 152 vulvar and vaginal tissue specimens obtained from 86 patients. Histopathologic diagnoses included condyloma acuminatum, intraepithelial neoplasia, and invasive cancer. In six patients, lesions of more than one pathologic type were identified. Vaginal lesions constituted less than 5% of tissues examined. Distribution of lesions was as follows: condyloma, 93 lesions from 57 patients; intraepithelial neoplasia, 47 lesions from 29 patients; and invasive carcinoma, 12 lesions from six patients. Seventy-five percent of the patients were white. The mean age of the patients increased from 25 years for condyloma to 38 years for vulvar intraepithelial neoplasia III to 56 years for invasive cancer. A viral diagnosis was made in 81% of condylomas, 84% of vulvar intraepithelial neoplasia III, and 58% of invasive carcinomas. Distribution of viral types differed markedly for the various histopathologies. Types 6/11 accounted for 77% of condylomas and 0% of vulvar intraepithelial neoplasia III. Type 16 was recovered from 12% of condylomas and 81% of vulvar intraepithelial neoplasia III. Type 18 was identified in a small proportion in both categories; type 31 was seen in a few vulvar intraepithelial neoplasia III lesions. In invasive carcinomas, type 16 was the predominantly identified virus. Papillomavirus type 16 emerges as the dominant oncogenic virus in vulvar neoplasms. Its presence in a large percentage of condylomas raises the issue of an "atypical condyloma" as a precursor of neoplasia.

Human papillomaviruses in women with a history of abnormal Papanicolaou smears and in their male partners.

Human papillomavirus infection of the genital tract was identified by the filter in situ hybridization test. Exfoliated cervical cells were tested separately for the prevalence of human papillomavirus 6/11 and 16/18. Human papillomavirus deoxyribonucleic acid (DNA) was identified in 70 and 92% of specimens of U.S. and West German women, respectively, who showed concurrent cytologic and colposcopic abnormalities, and in 50 and 54% of women, respectively, who showed neither cytologic nor colposcopic abnormalities at the time of examination. In the cytologic categories of condyloma, mild to moderate dysplasia (cervical intraepithelial neoplasia I/II), and severe dysplasia-carcinoma in situ (cervical intraepithelial neoplasia III), the overall DNA detection rate of human papillomavirus 6/11 and 16/18 varied between 75 and 83%; but human papillomavirus 16/18 was recovered relatively more frequently from the more severe lesions. Forty-eight West German women were monitored cytologically over a period of three to 24 months; progression to carcinoma in situ (cervical intraepithelial neoplasia III) was correlated with initial isolation of human papillomavirus 16/18. The vagina and vestibule were found to be frequent sites of human papillomavirus infection with the same virus type as in the cervix. In an investigation of male partners of 40 human papillomavirus-positive women, human papillomavirus was identified in exfoliated cells from 26; in 19 instances, the males harbored the same human papillomavirus types as their female partners.

Effects of starvation and immobilization on imino acids in mouse brain and peripheral tissues.

Effects of starvation and immobilization on the concentration of pipecolic acid and proline in mouse brain regions, liver, heart, kidney and blood plasma were analyzed. Pipecolic acid concentration in mouse brain and liver was increased after 24 or 48 h starvation, while proline concentration was not affected. Significant increases in levels of pipecolic acid were also observed in the rhombencephalon, liver and heart after 3 h immobilization. Proline in the blood plasma and kidney was decreased, while that in liver was increased, by the immobilization. Thus, the effect of such stress on concentration of pipecolic acid differed from that seen with proline. The possible involvement of pipecolic acid in synaptic mechanisms in the central nervous system and/or in pathogenesis of the diseases related to abnormal pipecolic acid metabolism should be given attention.

Identification of genital tract papillomaviruses HPV-6 and HPV-16 in warts of the oral cavity.

Warty lesions of the oral cavity were examined for etiologic association with genital tract papillomaviruses HPV-6, HPV-11, and HPV-16. DNAs extracted from ten oral biopsies were screened for HPV genomic sequences by Southern transfer hybridization with 32P-labeled viral DNA probes. Nonstringent hybridization with an HPV-6 probe revealed papillomavirus DNA sequences in four of seven tissues with histologic evidence of papillomatosis, in none of two tissues without histologic evidence of papillomatosis, and in one tissue that was not examined by histology. Stringent hybridization tests with HPV-6 and HPV-16 probes identified the genome in one tissue as being HPV-16, in a second tissue as being HPV-6 subtype a, and in a third tissue as HPV-6 (subtype unidentified); papillomavirus DNA sequences in two tissues are as yet not identified. An additional case of HPV-6 or HPV-11 related oral cavity lesion was diagnosed by in situ hybridization of paraffin sections with a 35S-labeled, mixed HPV-6 + HPV-11 probe. The hybridization in the positive section was extensive and confined to epithelial nuclei. The oral lesions associated with genital tract papillomaviruses were asymptomatic, multiple or single, and were located in different parts of the oral cavity, for example, on the gingivae, on the tongue, on the lip, on the tonsillar pillar, and on the floor of the mouth.

Specific identification of human papillomavirus type in cervical smears and paraffin sections by in situ hybridization with radioactive probes: a preliminary communication.

Cervical Papanicolaou smears and paraffin sections of biopsy specimens obtained from women attending dysplasia clinics were examined for viral DNA sequences by in situ hybridization technique using 35S-labeled cloned recombinant DNA probes of human papillomavirus (HPV) types 6, 11, and 16. These and one unrelated DNA probe complementary to measles virus RNA were labeled by nick translation using either one or two 35S-labeled nucleotides. The radiolabeled probes were reduced in size with DNase to 60-160 nucleotides. Paraffin sections and cervical smears were collected on pretreated slides, hybridized with the probes under stringent or nonstringent conditions for 50 h, and autoradiographed. Additional cervical specimens from the same women were examined for the presence of genus-specific papillomavirus capsid antigen by the immunoperoxidase technique. Preliminary results may be summarized as follows. The infecting virus could be identified in smears as well as in sections. Viral DNA sequences were detected only when there were condylomatous cells in the specimen and in only a proportion of the condylomatous cells. Even under stringent conditions, some specimens reacted with both HPV-6 and HPV-11. None of the specimens hybridized with HPV-16 or with the unrelated probe. In some instances, the cells did not hybridize with any of the three probes even when duplicate specimens contained frankly condylomatous, capsid antigen-positive cells. In situ hybridization of Papanicolaou smears or of tissue sections is a practical method for diagnosis and follow-up of specific papillomavirus infection using routinely collected material.