Human papillomavirus type 16 DMA in an odontogenic keratocyst

DNA was extracted from an odontogenic keratocyst and assayed for the presence of human papilloma virus (HPV) type 16 DNA sequences using high stringency Southern blot hybridization. HPV type 16 homologous DNA sequences were detected at a copy number of 50-100 genome copies per diploid cell. The oral HPV DNA was not identical to the prototype HPV 16 when cleaved with the restriction enzyme Pst-I, since it appeared to lack the Pst-I C fragment (L2/L1 ORFs) and contained "off-sized" high molecular weight fragments suggestive of integration events into the host cell chromosome.     

Accelerated Differentiation in Response to Retinoic Acid After Retrovirally Mediated Gene Transfer of GAP-43 into Mouse Neuroblastoma Cells

Although substantial evidence exists for the involvement of growth-associated protein-43 (GAP-43) in neuronal development and regeneration, the precise role of this protein in neurite outgrowth is currently debated. To investigate the role of GAP-43 in the initiation of neurite outgrowth, we transfected a full-length cDNA coding for GAP-43 into a mouse neuroblastoma cell line (Neuro-2a) which can be differentiated to a neuronal phenotype using retinoic acid (RA). We show that the consequent overexpression of GAP-43 results in a change in the basic morphology of these cells, but is not in itself sufficient to induce the extension of neurites. However, overexpression of GAP-43 results in a marked acceleration of neurite formation in response to RA. We propose that while GAP-43 does not trigger the initiation of neurite extension, its expression is rate-limiting for neurite outgrowth in response to differentiation agents such as RA.     

Dysregulation of the Hypothalamus-Pituitary-Adrenal Axis in Male and Female, Genetically Obese (ob/ob) Mice

The autosomal, recessive obesity of ob/ob mice is associated with hypercorticosteronemia and amelioration of most symptoms of obesity following adrenalectomy. Increased adrenocorticotropic hormone (ACTH) secretion has been hypothesized on the basis of several reports of higher pituitary ACTH content in ob/ob mice compared to lean littermates. However, the only measurement of ACTH blood concentration found lower levels in ob/ob mice than in leans suggesting that hypercorticosteronemia might result solely from an enhanced adrenal response to ACTH and also suggesting that the ob/ob's elevated pituitary ACTH content might be due to decreased ACTH secretion rather than increased ACTH synthesis. In our study, basal serum ACTH levels were higher in ob/ob males and females compared to sex-matched lean littermates. Anterior pituitary ACTH synthesis was also elevated as indicated by increased content of ACTH and proopiomelanocortin mRNA in obese mice of both sexes; however hypothalamic corticotropin-releasing factor content was not different in lean and obese mice. Basal serum ACTH and corticosterone (CS) levels showed normal circadian rhythm in both phenotypes and sexes, but the circadian increase in CS level was much greater in obese mice than in leans despite equal serum ACTH increases in the two phenotypes. Ether stress at both peak and trough of the circadian rhythm also stimulated much larger serum CS increases in obese mice even though ACTH increases were again equal in the two phenotypes. Taken together, these results strongly indicate that ob/ob mice have increased synthesis and secretion of pituitary ACTH despite the presence of chronically elevated serum CS. This hyperactivity of the hypothalamo-pituitary-adrenal axis appears to be most pronounced in ob/ob females since pituitary ACTH content was equal in obese males and females despite much higher circulating CS levels in the females. Furthermore, the results also indicate an enhanced response to ACTH by the adrenal cortex of the obese mouse. Thus, ob/ob mice exhibit abnormal hypothalamo-pituitary-adrenal axis function with hyperactivity occurring at the level of pituitary ACTH synthesis/secretion as well as at the level of adrenocortical response to ACTH.     

Ipriflavone inhibits osteoclast differentiation in parathyroid transplanted parietal bone of rats

Summary Ipriflavone, a synthetic isoflavone-derived flavonoid, was shown to have inhibitory effect on bone resorption. In order to study its mechanism of action directly on bone, 46 female Wistar rats were divided into six groups and medicated orally for 25 days as follows: groups 1 and 2 were given 1% carboxymethylcellulose solution (vehicle), groups 3, 4, 5, and 6 were administered ipriflavone at doses of 0.178, 0.356, 0.712, and 1.424 mmol/kg/day (suspended in vehicle), respectively. On the 22nd day, parathyroid glands, taken from donor rats, were transplanted in contact with the outer surface of the periosteum of both the right and the left parietal bones of rats from groups 2, 3, 4, 5, and 6. The group 1 rats underwent sham operation. Bone histomorphometry, performed on the ectocranial periosteum of parietal bones, showed that absolute erosion boundary, absolute eroded area, absolute erosion depth, number of tartrate-resistant acid phosphatase (TRAP)-positive polinucleated osteoclasts, and number of TRAP-positive mononucleated cells decreased in ipriflavone-treated rats compared with group 2 rats. The reduction was roughly proportional to the increase of drug dosage and reached statistical significance in rats of groups 5 and 6. The same parameters were extremely low in group 1 rats. Mineral apposition rate did not differ in any of the groups. Significant increase of serum calcium and significant decrease of serum phosphate were found in group 2 rats compared with group 1 rats, whereas no differences from controls were detected in ipriflavone-treated animals.The results demonstrate that ipriflavone has a direct inhibitory effect upon bone resorption, probably by reducing recruitment or differentiation of osteoclasts, rather than by inhibiting the resorption activity of differentiated osteoclasts. Ipriflavone also seems to exert a protective action against parathyroid hormone (PTH) diffusion from the site of parathyroid gland transplantation.     

The renal cytochrome P-450 arachidonic acid system

In addition to cyclooxygenase and lipoxygenase, arachidonic acid (AA) is metabolized by the cytochrome P-450 monooxygenase system. The kidney is one of the major extrahepatic tissues that display cytochrome P-450 enzyme activities, in particular the cortex, specifically the proximal tubule demonstrate the highest concentration. AA is metabolized by the renal cytochrome P-450 epoxygenase and /-1 hydroxylases to epoxyeicosatrienoic acids and /-1 alcohols (20- and 19-mono-hydroxyeicosatetraenoic acids), respectively. These metabolites possess a broad spectrum of biological and renal effects which include: vasodilation, vasoconstriction, inhibition and stimulation of Na⁺–K⁺-ATPase, inhibition of ion transport mechanisms, natriuresis, inhibition of renin release and stimulation of cell growth. These metabolites are endogenous constituents of the kidney and are present in urine with increasing concentration under pathological conditions such as pregnancy-induced hypertension. The cytochrome P-450-dependent metabolism of AA is specifically localized to the proximal tubule and exhibits developmental changes, i.e., renal production of metabolites is very low in the fetus, newborn and up to 3 weeks of age, after which a remarkable increase in enzyme activities is observed. These characteristics call attention to the importance of this enzyme system in producing cellular mediators for regulating renal function in normal and diseased states.     

Evaluation of cellular viability by quantitative autoradiographic study of myocardial uptake of a fatty acid analogue in isoproterenol-induced focal rat heart necrosis

Previous studies led us to hypothesize that a fatty acid analogue, 15-p-iodophenyl--methyl pentadecanoic acid (IMPPA or BMIPP), which is taken up but not quickly metabolized by heart cells, would be a more suitable tracer of cellular viability than thallium-201. Biodistribution studies of 1-¹⁴C-IMPPA in conscious, freely moving rats showed that the concentration ratio of radioactivity in the heart with respect to the blood was about 8 for at least 60 min after intravenous administration, permitting its use as a putative tracer in these conscious, freely moving rats. Thereafter, the myocardial uptake of¹⁴C-IMPPA was studied in isoproterenol-treated rats (daily treatment for 10 days in order to induce cardiac hypertrophy and necrotic foci) with respect to control ones. Comparison of myocardial localizations by quantitative autoradiography of the uptake of²⁰¹Tl and¹⁴C-IMPPA with that of triphenyltetrazolium chloride (TTC) staining enabled comparative evaluation of nutritional blood flow, localization and uptake of¹⁴C-IMPPA and necrotic foci size. Distributions of¹⁴C-IMPPA and²⁰¹1 T1 in control rats' hearts were homogeneous, like TTC staining. In infarcted hearts, areas of decreased¹⁴C-IMPPA uptake were nearly the same (100%±5%) as those unstained by TTC. These areas were larger than those showing a decrease in thallium uptake (about 70%±5% of the total scar size). Therefore, IMPPA seems to be a more accurate and sensitive indicator of necrosis localization compared with thallium. It may be a useful agent for assessment of myocardial viability by single photon emission tomography (SPET) imaging.     

Coexistence of GABA and glutamate in mossy fiber terminals of the primate hippocampus: an ultrastructural study.

One of the links in the trisynaptic circuit of the hippocampus is the synapse between the mossy fibre terminals of dentate granule cells and CA3 pyramidal cells of Ammon's horn. This synapse has been physiologically characterized as excitatory, and there is pharmacological and immunohistochemical evidence that mossy fibre terminals utilize glutamate as a neurotransmitter. This study demonstrates the presence of GABA-immunoreactivity in mossy fibre axons and terminals of the monkey at the electron microscopic level. We combined Golgi impregnation to identify CA3 pyramidal neurones, with postembedding immunocytochemistry to characterize the inputs to the identified cells. GABA immunoreactivity was present in mossy fibre terminals that made synaptic contact with complex embedded spines of identified Golgi-impregnated CA3 pyramidal neurones. GABA immunoreactivity could be demonstrated in serial sections of the same mossy fibre terminals by using 3 different antisera raised against GABA. In serial sections, the mossy fibre terminals were shown to be immunoreactive for both glutamate and GABA. In contrast, glutamate immunoreactivity but not GABA immunoreactivity was found in other terminals that did not have the morphological characteristics of mossy fibre terminals. GABA immunoreactivity in mossy fibre terminals was also demonstrated in a human surgical specimen of hippocampus. The coexistence of an "excitatory" amino acid and of an "inhibitory" amino acid in the same "excitatory" nerve terminal raises the possibility of corelease of the two transmitters, suggesting that the control of hippocampal neural activity is more complex than hitherto suspected.     

Creatine kinase isoenzyme (CK-BB) in combination to prostatic acid phosphatase measured by RIA in the diagnosis of prostatic cancer

Summary Creatine kinase isoenzyme (CK-BB) measured by mass was used to determine its value in the early diagnosis of prostatic cancer. Sera of patients with prostatic carcinoma of various stages (treated and untreated) were compared to normal male sera and sera of patients with benign hyperplasia of the prostate (BPH) with respect to CK-BB. The sera were simultaneously tested for PAP content. The sensitivity of the CK-BB-RIA was 1.63+/-0.08 g/l and reproducibility in the higher and lower concentration range 7.6% and 10.5%, respectively. CK-BB alone or in combination with PAP is no marker for early detection of prostatic cancer. In individual cases changes occurred similar to those found with a malignant growth of the prostate.     

Chemical modification of membrane proteins by brominated taurodehydrocholate in isolated hepatocytes; relationship to the uptake of cholate and of phalloidin and to the sensitivity of hepatocytes to phalloidin

Summary In vitro treatment of isolated rat hepatocytes with brominated taurodehydrocholic acid (BTC) reduced their sensitivity against phalloidin and inhibited the uptake of phalloidin as well as of cholate in an irreversible and concentration dependent manner. BTC was taken up itself by liver cells; this process was inhibited by 4,4-diisothiocyano 2,2-stilbene disulfonate (DIDS).When hepatocytes were incubated with ³⁵S-BTC their plasma membranes contained five labeled protein species with molecular weights of 67,000, 49,000, 38,000, 32,000 and 24,000 as shown by SDS-electrophoresis. No marked difference was observed when isolated plasma membranes from livers were directly treated with the affinity label. DIDS suppressed covalent binding of ³⁵S-BTC to membrane components drastically. Incubation of phalloidin insensitive AS-30D ascites hepatoma cells with ³⁵S-BTC did not result in a chemical modification of the above five proteins. This agrees with an earlier observation that hepatoma cells are unable to take up phalloidin and bile acids (Petzinger et al. 1979; Rufeger and Grundmann 1977; Kroker et al. 1978).Abbreviations used BTC brominated taurodehydrocholic acid - ³⁵S-BTC ³⁵S labeled brominated taurodehydrocholic acid - DIDS 4,4-diisothiocyano 2,2-stilbene disulfonate - [³H]DMP [³H]demethylphalloin This work was supported by the Deutsche Forschungsgemeinschaft