Extended structural variation of a pentanucleotide repeat in the GSTP1 gene: characterisation in a normal population and in thyroid and gastric tumours

The promoter region of the human GSTP1 gene contains a polymorphic short tandem repeat (STR) locus consisting of pentanucleotide repeat units (ATAAA). In this work we report the existence of a total of 26 alleles in a Caucasian population. While differences in size (ranging from one to five base pairs) were responsible for the major variation, in five size-defined classes, two alternative sequences were found. Automatic fragment sizing and sequencing analysis revealed that this polymorphism is of a highly complex nature in contrast with previous reports. A genetic population study was carried out on a random sample from Portugal showing no deviation from Hardy-Weinberg equilibrium. Somatic instability studies were also performed on gastric and thyroid tumours using this STR: no instability was detected in thyroid tumour tissues when compared with their normal counterpart but in gastric tumour tissues microsatellite instability (MSI) was detected in 9.6% of the cases and loss of heterozygosity (LOH) also in 9.6% of the cases studied. The results obtained with GSTP1 in gastric cancer were compared with previously reported data on MSI using BAT-26 and several dinucleotide repeat markers.     

Novel autosomal recessive progressive hyperpigmentation syndrome

We present a family of Iraqui origin with three siblings affected by a novel type of progressive hyperpigmentation syndrome. The generalized initially diffuse, later disseminated hyperpigmentation started in early infancy and increased during childhood. It also affected palms and soles, and the face but spared the cheeks. Additional features were dry, itchy and sunlight sensitive skin, dystrophy of toe nails, hair loss, and myopia, but normal sweat glands. Light and electron microscopy showed signs of pigment incontinence and compound melanosomes as well as fibrillar bodies. The occurrence of this entity in affected siblings from a consanguineous mating suggests autosomal recessive inheritance. Extensive review of the literature showed no previous report with this distinct combination of clinical and microscopic findings.     

A novel 5q35.3 subtelomeric deletion syndrome

We observed a novel 3.5 Mb 5q subtelomeric deletion in a 3-year-old girl with developmental delay, hypotonia and multiple minor anomalies. Comparison of her phenotype with the few published patients with terminal 5q35 deletions revealed several overlapping features, but also showed remarkable differences such as shortness of stature versus macrosomia. After the report of 5q35.3 microdeletions in Sotos syndrome we integrated the published BACs into the public draft sequence and exactly mapped the deletion size in our patient by FISH analysis with 15 BAC probes. We demonstrated that the deletion in our patient is immediately adjacent to the reported Sotos syndrome deletion site. Subtracting the symptoms of Sotos syndrome from the published patients with larger 5q35.3 deletions allowed us to delineate a distinct phenotype of prenatal lymphedema with increased nuchal translucency, pronounced muscular hypotonia and delay of reaching motor milestones, but speech development within normal limits, wide fontanels, failure to thrive with postnatal short stature, and multiple minor anomalies such as mildly bell-shaped chest, minor congenital heart disease, and a distinct facial gestalt, associated with the novel 3.5 Mb cryptic deletion. We further showed in our patient that the deletion of the LCT(4) synthase gene results in a reduction of cysteinyl leukotriene synthesis to about 65% compared to normal values. The prenatal nuchal lymphedema associated with this deletion syndrome my be related to the deletion of the FLT4 gene causing autosomal dominant primary lymphedema and contributes to the differential diagnosis of increased fetal nuchal translucency.     

Effect of losartan on sodium appetite of hypothyroid rats subjected to water and sodium depletion and water, sodium and food deprivation

The involvement of angiotensin AT1 receptors in sodium appetite was studied in hypothyroid rats treated with the angiotensin II antagonist losartan. Losartan was administered chronically by the oral route or acutely by the subcutaneous route after water and sodium depletion or water, sodium and food deprivation. Three days after addition of losartan to the food at the dose of 1.0 mg x g(-1), the rats significantly reduced (P < 0.02) their spontaneous intake of 1.8% NaCl. Increasing the dose of losartan to 2.0 and 4.0 mg x g(-1) did not reduce NaCl intake; in contrast, the intensity of the sodium appetite gradually returned to previous levels. The simultaneous administration of captopril, an angiotensin converting enzyme inhibitor, and losartan significantly increased (P < 0.05) NaCl intake and after captopril removal NaCl intake returned to the levels observed with losartan treatment alone. The administration of losartan 4 days after the beginning of captopril treatment significantly reduced (P < 0.0001) NaCl intake. Following acute administration of losartan, water- and sodium-depleted rats significantly reduced their NaCl and water intake (P < 0.001). The administration of losartan also induced a significant reduction in NaCl and water intake in water, NaCl and food-deprived rats (P < 0.0001 and P < 0.001, respectively). The present results show that chronic treatment with oral losartan inhibited spontaneous sodium appetite in hypothyroid rats. Continuation of treatment rendered rats resistant to the blockade of AT1 receptors. Water and sodium depletion and water, NaCl and food deprivation induced sodium appetite, which in the short term depends on cerebral angiotensinergic activity mediated by the activation of AT1 receptors.     

Myocardial ANP expression in Pompe's disease: An immunohistochemical study

The expression of atrial natriuretic peptide (ANP) was analyzed in the atrial and ventricular myocardium in three cases of Pompe's disease (glycogen storage disease of the myocardium), using an immunoperoxidase technique. The cytoplasm of almost all atrial myocytes and some subendocardial myocytes from the right and left ventricles were ANP-positive, excluding the typical central vacuole, which was occupied by glycogen. Ventricular ANP expression was usually more prominent in left ventricular samples, and its distribution was similar to that described in dilated, hypertrophic, restrictive, or ischemic heart disease; however, the enlargement of the myocytes in Pompe's disease is not caused by hypertrophy. We conclude that the atrial myocytes in Pompe's disease maintain ANP expression, despite severe cytoplasmic vacuolization. These results suggest that ventricular ANP expression may be related to mechanical stimuli, such as the increase in wall stress, and not directly related to myocyte hypertrophy.     

Hypoxia-activated Ca2+ currents in pacemaker neurones of rat rostral ventrolateral medulla in vitro.

We examined the effects of brief periods of hypoxia or application of cyanide on the discharge and membrane properties of medullary pacemaker neurones in slices of the rostral ventrolateral reticular nucleus (RVL) of the medulla oblongata of rats. Stable intracellular recordings were obtained from seventy-nine neurones within the RVL which exhibited spontaneous rhythmic discharge in the absence of excitatory postsynaptic potentials (EPSPs). The membrane potential cycles of these neurones could be reset with an evoked spike without eliciting EPSPs or inhibitory postsynaptic potentials and hence met criteria of RVL pacemaker neurones. Hypoxia, produced by reducing O2 from 95 to 20% for 40 s or exposure to cyanide (30-300 microM for 40 s), reversibly increased neuronal discharge 1.6-fold (20% O2) or 2.6-fold (300 microM cyanide), respectively, in association with membrane depolarization and a significant fall in membrane resistance. The membrane responses to hypoxia and cyanide were observed in the presence of tetrodotoxin (TTX) at a concentration (10 microM) which eliminated spontaneous spikes or spikes evoked by intracellular depolarization. When recorded at a holding potential of -70 mV by single-electrode voltage clamp, hypoxia or cyanide (300 microM) elicited inward currents of 0.44 +/- 0.06 and 0.58 +/- 0.08 nA, respectively, which are attenuated by reducing the concentration of extracellular Ca2+ ions, and abolished by 2 mM CoCl2 and 100 microM NiCl2, but not affected by 50 microM CdCl2, replacement of 83% extracellular Na+, or adenosine deaminase (2U ml-1). We conclude that hypoxia and cyanide directly excite RVL pacemaker neurones in vitro by a common mechanism: activation of Ca2+ channel conductance.     

The role of the solitary and paramedian reticular nuclei in mediating cardiovascular reflex responses from carotid baro- and chemoreceptors

1. With dye-filled micro-electrodes single neurones in the medulla of anaesthetized paralysed cats were identified which: (a) fired rhythmically in synchrony with or were modulated by the cardiac cycle, and which ceased firing with occlusion of the ipsilateral common carotid artery (carotid sinus baroreceptor neurones); (b) were excited by stimulation of carotid body chemoreceptors by close intra-arterial injection of lobeline into the thyroid artery (carotid body chemoreceptor neurones).2. Twelve carotid baroreceptor neurones were identified, in thirty-three cats, nine of which were localized in the intermediate area of the nucleus of the solitary tract (NTS) within 1 mm ahead of or behind the obex; three units were located either in the parahypoglossal area or the dorsal portion of the paramedian reticular nucleus (PRN).3. Of the twenty-one carotid chemoreceptor neurones which were identified, thirteen were localized in the NTS, three in the parahypoglossal area and four in the dorsal PRN.4. Bilateral lesions of the paramedian reticular area of medulla destroying the PRN, abolished or reversed the depressor response to electrical stimulation of myelinated fibres of the carotid sinus nerve (CSN), attenuated the depressor response to carotid sinus stretch and augmented the pressor response to chemoreceptor stimulation by lobeline. Such lesions did not significantly alter the reflex heart rate responses.5. Small lesions of the NTS within an area 1 mm rostral to the obex abolished all reflex blood pressure and heart rate responses to electrical stimulation of the CSN or natural stimulation of carotid baro- or chemoreceptors.6. Baroreceptors and chemoreceptors of the CSN project both to the intermediate zone of the NTS and to more medial areas of the medulla, particularly the dorsal PRN and parahypoglossal area.7. The PRN serves to mediate the reflex depressor, but not cardio-vagal, response from myelinated baroreceptors and buffers the pressor responses from chemoreceptors; it may serve as an important area integrating cardiovascular activity descending from forebrain, brain stem and cerebellum with baroreceptor reflexes.8. Cardiovascular reflex responses arising from non-myelinated baroreceptors and all chemoreceptors are mediated by neurones in the intermediate area of the NTS.     

Immunohistochemical study of the expression of MUC5AC and MUC6 in breast carcinomas and adjacent breast tissues

AIM: To study the protein expression patterns of MUC5AC and MUC6 in normal and diseased breast tissues and to compare their expression with that of a mucin (MUC1) normally expressed in mammary tissues. METHODS: Formalin fixed, paraffin wax embedded tissue from 69 cases of invasive breast carcinoma and surrounding breast tissue was studied immunohistochemically with monoclonal antibodies against MUC1 (SM3), MUCSAC (CLH2), and MUC6 (CLH6), using the avidin-biotin-peroxidase method. RESULTS: MUC5AC was detected in five of 68 cases of invasive carcinoma including one of three cases of pure colloid carcinoma. MUCSAC expression in the adjacent normal breast epithelium was present in one of 29 cases and in one of two cases of ductal carcinoma in situ. None of 15 cases of ductal hyperplasia without atypia was positive for MUCSAC. MUC6 was present in 15 of 65 cases of invasive carcinoma, in four of 29 cases of normal adjacent epithelium, two of 15 cases of ductal hyperplasia without atypia, and one of two cases of ductal carcinoma in situ. MUC1 immunoreactivity detected by the SM3 antibody was present in 50 of the 67 cases of invasive carcinoma, but expression was also detected in benign epithelium. All invasive carcinomas expressing MUCSAC were positive for MUC1 and four were positive for MUC6. No significant association was found between the expression of these mucins and tumour size, histological grade, node status, oestrogen receptor status, p53 positivity, or c-ErbB-2 overexpression. CONCLUSIONS: This study documents the expression of two different mucins (MUCSAC and MUC6) not described as being expressed by normal breast tissues in a minority of breast carcinomas, as well as in normal and hyperplastic epithelium. Although the role of mucins in malignant transformation and the progression of breast cancer is not well understood, in some cases, there is probably an upregulation of several genes that encode distinct mucin proteins.     

Receptor-selective analogs demonstrate NPY/PYY receptor heterogeneity in rat brain.

Neuropeptide Y (NPY) receptors are heterogeneous, consisting of at least two subclasses, Y1 and Y2. We sought evidence for differential expression of NPY receptor subtypes in the rat brain. Tissue was incubated with 125I-peptide YY (PYY) which labels NPY and PYY binding sites. The Y1-selective agonist, p[Pro34]NPY, and the Y2-selective agonist, pNPY 13-36, were used as displacing ligands. Autoradiographic analyses of regional receptor binding demonstrated heterogeneity across brain regions. We conclude that Y1- and Y2-receptors may be independently expressed in the brain. While the predominate NPY/PYY receptor subtype in the brain is Y2, there are also Y1-receptors in some brain regions such as the superficial layers of the parietal cortex.