Analysis of the genetic variability of the 1st (CCC/ACC,P52T) and the 10th exons (bp 1012–1704) of the TSH receptor gene in Graves’ disease

We determined the genetic variability of the 1st (CCC/ACC, P52T polymorphic variant) and 10th exons (bp 1012–1704) of the TSH receptor (TSHR) gene in Graves’ disease. A total of 101 Graves’ patients and 163 control subjects were screened. The A253 mutant allele was carried by nine patients with Graves’ disease (8.91%) and 13 control subjects (7.98%) in heterozygous genotype. No significant difference in the frequency of the mutant allele was found between Graves’ patients and control subjects. These results provide evidence that the A253 polymorphism has no genetic relevance in Graves’ disease. Moreover, the DNA nucleotide sequence of 693 bp of the 10th exon (bp 1012–1704) of the TSHR gene was determined in 15 Graves’ patients. Six patients were homozygous for the wild‐type allele and nine were heterozygous for the mutant allele at the 253rd nucleotide of the first exon. No polymorphism was found in the DNA sequences obtained from leukocytes of Graves’ patients, similarly to the sequences obtained from the nine control subjects. None of the nine patients carrying the A253 polymorphism in the 1st exon of the TSHR had polymorphism in the examined part of the 10th exon, including two additional patients whose thyroid tissue was directly analysed. In all likelihood, the polymorphisms of the examined regions of either the 1st or the 10th exon of the THSR gene do not contribute to the genetic susceptibility to Graves’ disease.     

Differentiation of Rhizomucor Species by Carbon Source Utilization and Isoenzyme Analysis

Nineteen Rhizomucor miehei and Rhizomucor pusillus isolates were assayed for their ability to utilize 87 various substrates as a single carbon source. Besides a difference in sucrose utilization, distinctive differences were found in the utilization of glycine, phenylalanine, and β-alanine. Five isoenzyme systems also proved useful for the determination of markers of distinctive value at a species level. Data were used to obtain information about the genetic polymorphism of these species: a high degree of variability was found among the R. pusillus isolates, whereas the group of R. miehei isolates was more homogeneous genetically.     

Distribution and pharmacological characterization of somatostatin receptor binding sites in the sheep brain

Somatostatin binding sites have been localized and quantified in the sheep brain using ¹²⁵I-Tyr₀-DTrp₈-somatostatin, by quantitative high resolution light microscopic autoradiography. Sections were analyzed by densitometry on radioautographic film, and subsequently on slides coated with photoemulsion. Specific somatostatin binding sites were concentrated in the medial habenula, superior colliculus, dorsal motor nucleus of the vagus nerve, inferior olive, spinal trigeminal nucleus, and cerebellum. In competition experiments, octreotide, a sst2/sst3/sst5 selective agonist only partially displaced ¹²⁵I-Tyr₀-DTrp₈-somatostatin in the three cerebellar layers while it was fully active as compared to somatostatin 14 and 28 in the deeper layers of the parietal cortex. Moderate to low somatostatin receptor densities were present in the mesencephalic periaqueductal gray, dorsal raphe, thalamic paraventricular nucleus, interpeduncular nucleus, pineal gland, dorsal tegmental, dorsolateral tegmental and parabrachial nuclei, nucleus of the solitary tract. The distribution of somatostatin binding sites generally correlates with the data obtained on slides dipped in photoemulsion which provided better resolution and more precise localization. In most of the labeled areas, ¹²⁵I-Tyr₀-DTrp₈-somatostatin receptor binding was distributed between both neuropil and perikarya. Perikarya bearing ¹²⁵I-Tyr₀-DTrp₈-somatostatin receptors were observed in areas which did not display detectable binding sites on film such as the preoptic-anterior hypothalamic complex and arcuate nucleus and in the locus coeruleus. In conclusion, the distribution of ¹²⁵I-Tyr₀-DTrp₈-somatostatin binding sites in sheep brain is very reminiscent of other mammals being closer to the human than to rodents.     

Prostanoid response in the kidney of hypertensive subjects as part of renal counteraction to gallopamil-induced blood pressure decrease

The purpose of the study was to characterize the renal TxA2, PGI2 and PGF2 alpha release in response to arterial blood pressure (BP) fall induced by systemic and intrarenal vasorelaxation in subjects with essential hypertension. Significantly enhanced TxB2- and PGF2 alpha excretion and no change in ratio TxB2/6-keto-PGF1 alpha were found in urine in hypertensive patients after administration of the Ca++ entry blocker gallopamil, used to induce BP fall. This response was associated with significant PRA elevation in peripheral venous samples. In in vitro experiments, direct and indirect effects of gallopamil on renal tissue could be distinguished. Gallopamil resulted in significant diminution of TxA2 production and a decrease in TxB2/6-keto PGF1 alpha ratio in incubated rat kidney slices. This model was also used to test biochemically the effect of reflex sympathetic activation on prostanoid generation in kidney. It was concluded that this mechanism was only one among the indirect mechanisms by which gallopamil could induce that renal prostanoid response in hypertensive subjects. The response in urinary TxB2- and PGF2 alpha excretion was found to be significantly related to the changes in sodium reabsorption. These results suggested, that the increase in renal TxA2 and PGF2 alpha production in response to systemic and intrarenal "vasodilation" induced by gallopamil in hypertensive subjects can be interpreted as part of counteraction of the kidneys to BP fall.     

CD59 blocks not only the insertion of C9 into MAC but inhibits ion channel formation by homologous C5b-8 as well as C5b-9

Activation of the complement system on the cell surface results in the insertion of pore forming membrane attack complexes (MAC, C5b-9). In order to protect themselves from the complement attack, the cells express several regulatory molecules, including the terminal complex regulator CD59 that inhibits assembly of the large MACs by inhibiting the insertion of additional C9 molecules into the C5b-9 complex. Using the whole cell patch clamp method, we were able to measure accumulation of homologous MACs in the membrane of CD59⁻ human B-cells, which formed non-selective ion channels with a total conductance of 360 ± 24 pS as measured at the beginning of the steady-state phase of the inward currents. C5b-8 and small-size MAC (MAC containing only a single C9) can also form ion channels. Nevertheless, in CD59⁺ human B-cells in spite of small-size MAC formation, an ion current could not be detected. In addition, restoring CD59 to the membrane of the CD59⁻ cells inhibited the serum-evoked inward current. The ion channels formed by the small-size MAC were therefore sealed, indicating that CD59 directly interfered with the pore formation of C5b-8 as well as that of small-size C5b-9. These results offer an explanation as to why CD59-expressing cells are not leaky in spite of a buildup of homologous C5b-8 and small-size MAC. Our experiments also confirmed that ion channel inhibition by CD59 is subject to homologous restriction and that CD59 cannot block the conductivity of MAC when generated by xenogenic (rabbit) serum.     

Differentiation of Rhizomucor species on the basis of their different sensitivities to lovastatin

The opportunistic pathogens Rhizomucor pusillus and Rhizomucor miehei may be agents of frequently fatal mycotic diseases. In the present study, the susceptibilities of 27 clinical and environmental isolates of R. miehei and R. pusillus to lovastatin under different culturing conditions were investigated. Most of the R. miehei strains grew at lovastatin concentrations as high as 64 to 128 microg/ml. In contrast, the inhibitory effect of lovastatin on all of the R. pusillus strains was evident at lovastatin concentrations as low as 1 to 2 microg/ml. A simple and reliable method for species-level differentiation, based on the significantly higher sensitivity of R. pusillus to lovastatin than that of R. miehei, was elaborated. According this, on malt extract agar containing 6 mug of lovastatin/ml, R. pusillus is not able to produce colonies, while R. miehei will form compact colonies.     

Preconditioning-specific reduction of c-fos expression in hippocampal granule and pyramidal but not other forebrain neurons of ischemic brain: a quantitative immunohistochemical study

To specify targets for an ischemic preconditioning paradigm (ischemic tolerance), c-fos expressions in ischemic (induced by 10 min bilateral carotid-occlusions subsequent to coagulation of vertebral arteries) and preconditioned rats (treated for 4 min carotid-occlusions 72 h before ischemia) were compared in 12 forebrain areas/nuclei. Fos immunostaining was applied to serial sections of the forebrain and the density (cell number/area measured) of Fos-immunopositive (Fos+) neurons, as well as their percentile changes were determined in five hippocampal and seven extrahippocampal areas/nuclei of ischemic and preconditioned rats. The ratio of counts found in ischemic over control animals showed several fold increase of Fos+ cells in the three layers (granule cell, molecular and polymorphic) of the dentate gyrus, CA3 and CA1 pyramidal neurons, as well as in thalamic and hypothalamic nuclei and limbic cortical areas. In contrast, preconditioning did not alter c-fos expressions significantly in the extrahippocampal brain areas investigated. These results strengthen the hypothesis that the hippocampal and dentate neurons are more susceptible to ischemic tolerance than cells in other brain regions. In fact stress-response and induction of ischemic tolerance in different forebrain areas can be distinguished.     

High co-prevalence of genogroup 1 TT virus and human papillomavirus is associated with poor clinical outcome of laryngeal carcinoma

BACKGROUND: The aetiology and factors leading to the progression of laryngeal cancer are still unclear. Although human papillomavirus (HPV) has been suggested to play a role, reports concerning the effect of HPV infection on tumour development are controversial. Recently, transfusion transmitted virus (TTV) was suggested to play a role in certain infections as a causative or coinfecting agent. AIMS: To investigate whether the development and progression of laryngeal squamous cell carcinoma is associated with coinfection with TTV and HPV. METHODS: The prevalence of TTV and HPV was investigated using the polymerase chain reaction in tissue samples from 40 healthy individuals, 10 patients with recurrent papillomatosis, five patients with papillomatosis with malignant transformation, and 25 patients with laryngeal carcinoma. The obtained prevalence data were compared and analysed statistically. RESULTS: In the 11 patients with carcinoma who had metastasis or relapse there was a high rate of coinfection with genogroup 1 TTV and HPV (eight of 11), whereas in the 14 without tumour progression no coinfection was found. Coinfection was associated with significantly lower tumour free survival in patients with carcinoma (p < 0.001). Furthermore, four of five patients who had papillomatosis with malignant transformation were coinfected with genogroup 1 TTV and HPV. CONCLUSIONS: Although the nature of cooperation between HPV and TTV needs to be investigated further, coinfection with genogroup 1 TTV and HPV appears to be associated with poor clinical outcome in laryngeal cancer.     

Additional human papillomavirus types detected by the hybrid capture tube test among samples from women with cytological and colposcopical atypia

The type specificity of the human papillomavirus (HPV) Hybrid Capture Tube (HCT) test was evaluated by using typing with PCR (MY09-MY11)-restriction fragment length polymorphism (RFLP) and sequencing. All samples HCT test positive for only low-risk HPV (n = 15) or only high-risk HPV (n = 102) were confirmed, whereas 9 of 12 HCT test double-positive samples contained only high-risk HPV types as determined by PCR-RFLP. Several high-risk HPV types (HPV-53, -58, -62, -66, -CP8304, and -MM4) not included in the HCT test were indeed detected, indicating a broader detection range with retained distinction between low-risk and high-risk HPV types.