New type of interaction between the SARAH domain of the tumour suppressor RASSF1A and its mitotic kinase Aurora A.

The tumour suppressor protein RASSF1A is phosphorylated by Aurora A kinase, thereby impairing its tumour suppressor function. Consequently, inhibiting the interaction between Aurora A and RASSF1A may be used for anti-tumour therapy. We used recombinant variants of RASSF1A to map the sites of interaction with Aurora A. The phosphorylation kinetics of three truncated RASSF1A variants has been analysed. Compared to the RASSF1A form lacking the 120 residue long N-terminal part, the Km value of the phosphorylation is increased from 10 to 45 µM upon additional deletion of the C-terminal SARAH domain. On the other hand, deletion of the flexible loop (Δ177-197) that precedes the phosphorylation site/s (T202/S203) results in a reduction of the kcat value from about 40 to 7 min-1. Direct physical interaction between the isolated SARAH domain and Aurora A was revealed by SPR. These data demonstrate that the SARAH domain of RASSF1A is involved in the binding to Aurora A kinase. Structural modelling confirms that a novel complex is feasible between the SARAH domain and the kinase domain of Aurora A. In addition, a regulatory role of the loop in the catalytic phosphorylation reaction has been demonstrated both experimentally and by structural modelling.

Matrix metalloproteinase activity assays: Importance of zymography.

INTRODUCTION: Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of degrading extracellular matrix, including the basement membrane. MMPs are associated with various physiological processes such as morphogenesis, angiogenesis, and tissue repair. Moreover, due to the novel non-matrix related intra- and extracellular targets of MMPs, dysregulation of MMP activity has been implicated in a number of acute and chronic pathological processes, such as arthritis, acute myocardial infarction, chronic heart failure, chronic obstructive pulmonary disease, inflammation, and cancer metastasis. MMPs are considered as viable drug targets in the therapy of the above diseases. METHODS: For the development of selective MMP inhibitor molecules, reliable methods are necessary for target validation and lead development. Here, we discuss the major methods used for MMP assays, focusing on substrate zymography. We highlight some problems frequently encountered during sample preparations, electrophoresis, and data analysis of zymograms. RESULTS AND DISCUSSION: Zymography is a widely used technique to study extracellular matrix-degrading enzymes, such as MMPs, from tissue extracts, cell cultures, serum or urine. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and therefore helps to understand the widespread role of MMPs in different pathologies and cellular pathways.

Ghrelin-induced sleep responses in ad libitum fed and food-restricted rats.

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor and a well-characterized food intake regulatory peptide. Hypothalamic ghrelin-, neuropeptide Y (NPY)-, and orexin-containing neurons form a feeding regulatory circuit. Orexins and NPY are also implicated in sleep-wake regulation. Sleep responses and motor activity after central administration of 0.2, 1, or 5 microg ghrelin in free-feeding rats as well as in feeding-restricted rats (1 microg dose) were determined. Food and water intake and behavioral responses after the light onset injection of saline or 1 microg ghrelin were also recorded. Light onset injection of ghrelin suppressed non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) for 2 h. In the first hour, ghrelin induced increases in behavioral activity including feeding, exploring, and grooming and stimulated food and water intake. Ghrelin administration at dark onset also elicited NREMS and REMS suppression in hours 1 and 2, but the effect was not as marked as that, which occurred in the light period. In hours 3-12, a secondary NREMS increase was observed after some doses of ghrelin. In the feeding-restricted rats, ghrelin suppressed NREMS in hours 1 and 2 and REMS in hours 3-12. Data are consistent with the notion that ghrelin has a role in the integration of feeding, metabolism, and sleep regulation.

Rhythms of ghrelin, leptin, and sleep in rats: effects of the normal diurnal cycle, restricted feeding, and sleep deprivation.

To determine the relationships among plasma ghrelin and leptin concentrations and hypothalamic ghrelin contents, and sleep, cortical brain temperature (Tcrt), and feeding, we determined these parameters in rats in three experimental conditions: in free-feeding rats with normal diurnal rhythms, in rats with feeding restricted to the 12-h light period (RF), and in rats subjected to 5-h of sleep deprivation (SD) at the beginning of the light cycle. Plasma ghrelin and leptin displayed diurnal rhythms with the ghrelin peak preceding and the leptin peak following the major daily feeding peak in hour 1 after dark onset. RF reversed the diurnal rhythm of these hormones and the rhythm of rapid-eye-movement sleep (REMS) and significantly altered the rhythm of Tcrt. In contrast, the duration and intensity of non-REMS (NREMS) were hardly responsive to RF. SD failed to change leptin concentrations, but it promptly stimulated plasma ghrelin and induced eating. SD elicited biphasic variations in the hypothalamic ghrelin contents. SD increased plasma corticosterone, but corticosterone did not seem to influence either leptin or ghrelin. The results suggest a strong relationship between feeding and the diurnal rhythm of leptin and that feeding also fundamentally modulates the diurnal rhythm of ghrelin. The variations in hypothalamic ghrelin contents might be associated with sleep-wake activity in rats, but, unlike the previous observations in humans, obvious links could not be detected between sleep and the diurnal rhythms of plasma concentrations of either ghrelin or leptin in the rat.

Fos-immunoreactivity in the hypothalamus: dependency on the diurnal rhythm, sleep, gender, and estrogen.

Diurnal variations and sleep deprivation-induced changes in the number of Fos-immunoreactive (Fos-IR) neurons in various hypothalamic/preoptic nuclei were studied in the rat. The nuclei implicated in sleep regulation, the ventrolateral preoptic (VLPO), median preoptic (MnPO), and suprachiasmatic (SCN, dorsomedial subdivision) nuclei, displayed maximum c-fos expression in the rest (light) period. Sleep deprivation (S.D.) suppressed Fos-IR in the dorsomedial subdivision of SCN but failed to alter Fos in the VLPO. Fos-IR increased in the VLPO during recovery after S.D. A nocturnal rise in Fos expression was detected in the arcuate (ARC), anterodorsal preoptic (ADP) and anteroventral periventricular (AVPV) nuclei whereas the lateroanterior hypothalamic nucleus (LA) and the ventrolateral subdivision of SCN did not display diurnal variations. S.D. stimulated Fos expression in the ARC, ADP, and LA. Statistically significant, albeit modest, differences were noted in the number of Fos-IR cells between males and cycling female (estrus/diestrus) in the VLPO, MnPO, ARC, LA, and AVPV, and the female ADP did not display diurnal variations. Ovariectomy (OVX) was followed by marked reduction in Fos expression in the VLPO, SCN, and AVPV, and the diurnal rhythm decreased in the VLPO, and vanished in the dorsomedial SCN, and AVP. Estrogen administration to OVX female rats stimulated Fos expression in most nuclei, and the lost diurnal variations reoccurred. In contrast, castration of male rats had little effect on Fos expression (slight rises in diurnal Fos in the ARC and ventrolateral SCN). The results suggest that Fos expression is highly estrogen-dependent in many hypothalamic nuclei including those that have been implicated in sleep regulation.

Ultrastructural comparison of "alveolar" and "solid" areas in bronchioloalveolar carcinoma.

The purpose of this study was to compare the ultrastructural features of bronchioloalveolar carcinomas, contrasting the well-differentiated alveolar component and the poorly-differentiated solid component in the same tumor. We studied 7 cases of non-mucinous bronchioloalveolar carcinomas by electron microscopy. Two of these cases showed lamellar bodies in both the alveolar and solid components and the remaining 5 cases revealed Clara cell granules in both components. We conclude that the neoplastic cells in bronchioloalveolar carcinoma retain their ultrastructural phenotypes after becoming invasive carcinoma with loss of alveolar differentiation.

Sleep of transgenic mice producing excess rat growth hormone.

The effects of chronic excess of growth hormone (GH) on sleep-wake activity was determined in giant transgenic mice in which the metallothionein-1 promoter stimulates the expression of rat GH (MT-rGH mice) and in their normal littermates. In the MT-rGH mice, the time spent in spontaneous non-rapid eye movement sleep (NREMS) was enhanced moderately, and rapid eye movement sleep (REMS) time increased greatly during the light period. After a 12-h sleep deprivation, the MT-rGH mice continued to sleep more than the normal mice, but there were no differences in the increments in NREMS, REMS, and electroencephalogram (EEG) slow-wave activity (SWA) during NREMS between the two groups. Injection of the somatostatin analog octreotide elicited a prompt sleep suppression followed by increases in SWA during NREMS in normal mice. These changes were attenuated in the MT-rGH mice. The decreased responsiveness to octreotide is explained by a chronic suppression of hypothalamic GH-releasing hormone in the MT-rGH mice. Enhancements in spontaneous REMS are attributed to the REMS-promoting activity of GH. The increases in spontaneous NREMS are, however, not consistent with our current understanding of the role of somatotropic hormones in sleep regulation. Metabolic, neurotransmitter, or hormonal changes associated with chronic GH excess may indirectly influence sleep.

The somatostatin analog, octreotide, causes accumulation of growth hormone-releasing hormone and depletion of angiotensin in the rat hypothalamus.

Rats were injected intracerebroventricularly with the somatostatin analog, octreotide (OCT; 0.1 microg) or vehicle, and hypothalamic contents of growth hormone-releasing hormone (GHRH), angiotensin II, and vasopressin were determined 10 min, 1, 3 and 6 h post-injection. OCT elicited an immediate release of angiotensin II (10 min) and a rise in GHRH content (1 h) followed by gradual (1-6 h) depletion of accumulated GHRH. Hypothalamic vasopressin was not altered but decreases in pituitary vasopressin occurred 10 min post-injection. The OCT-induced alterations in GHRH may explain previously reported changes in sleep whereas angiotensin may mediate OCT-induced drinking, vasopressin secretion and rises in blood pressure via sst2 somatostatin receptors.

A better enzyme to cope with cold. Comparative flexibility studies on psychrotrophic, mesophilic, and thermophilic IPMDHs.

3-isopropylmalate dehydrogenase (IPMDH) from the psychrotrophic bacterium Vibrio sp. I5 has been expressed in Escherichia coli and purified. This cold-adapted enzyme is highly homologous with IPMDHs from other organisms, including mesophilic E. coli and thermophilic Thermus thermophilus bacteria. Its molecular properties are similar to these counterparts. Whereas the E. coli and T. thermophilus enzymes are hardly active at room temperature, the Vibrio IPMDH has reasonable activity below room temperature. The thermal stabilities, conformational flexibilities (hydrogen-deuterium exchange), and kinetic parameters of these enzymes were compared. The temperature dependence of the catalytic parameters of the three enzymes show similar but shifted profiles. The Vibrio IPMDH is a much better enzyme at 25 degrees C than its counterparts. With decreasing temperature i.e. with decreasing conformational flexibility, the specific activity reduces, as well; however, in the case of the Vibrio enzyme, the residual activity is still high enough for normal physiological operation of the organism. The cold-adaptation strategy in this case is achieved by creation of an extremely efficient enzyme, which has reduced but still sufficient activity at low temperature.

Octreotide-induced drinking, vasopressin, and pressure responses: role of central angiotensin and ACh.

The involvement of central angiotensinergic and cholinergic mechanisms in the effects of the intracerebroventricularly injected somatostatin analog octreotide (Oct) on drinking, blood pressure, and vasopressin secretion in the rat was investigated. Intracerebroventricular Oct elicited prompt drinking lasting for 10 min. Water consumption depended on the dose of Oct (0.01, 0.1, and 0. 4 microgram). The drinking response to Oct was inhibited by pretreatments with the intracerebroventricularly injected angiotensin-converting enzyme inhibitor captopril, the AT(1)/AT(2) angiotensin receptor antagonist saralasin, the selective AT(1) receptor antagonist losartan, or the muscarinic cholinergic receptor antagonist atropine. The dipsogenic effect of Oct was not altered by prior subcutaneous injection of naloxone. Oct stimulated vasopressin secretion and enhanced blood pressure. These responses were also blocked by pretreatments with captopril or atropine. Previous reports indicate that the central angiotensinergic and cholinergic mechanisms stimulate drinking and vasopressin secretion independently. We suggest that somatostatin acting on sst2 or sst5 receptors modulates central angiotensinergic and cholinergic mechanisms involved in the regulation of fluid balance.

Central administration of the somatostatin analog octreotide induces captopril-insensitive sleep responses.

The effects of intracerebroventricular injections of the long-lasting somatostatin analog octreotide (Oct) were studied on sleep and behavior in rats. Pyrogen-free physiological saline and Oct (0.001, 0.01, 0.1 microgram) or vehicle were administered at light onset, and the electroencephalogram (EEG), motor activity, and cortical brain temperature were recorded during the 12-h light period. Plasma growth hormone (GH) concentrations were measured in samples taken at 30-min intervals after Oct. Oct (0.01 and 0.1 microgram) suppressed non-rapid eye movement sleep (NREMS) for 1-2 h. NREMS intensity (delta EEG activity during NREMS) dose dependently increased in hour 3 postinjection and thereafter (0.1 microgram). Plasma GH concentrations were suppressed after Oct (0.01 and 0.1 microgram), but pulses of GH secretions occurred 90-120 min postinjection in each rat. Oct (0.1 microgram) enhanced behavioral activity, including prompt drinking followed by grooming, scratching, and feeding. Intracerebroventricular injection of the angiotensin-converting enzyme inhibitor captopril (30 microgram, 10 min before Oct), blocked these behavioral responses but not the Oct-induced sleep alterations. The changes in sleep after intracerebroventricular Oct suggest an intracerebral action site and might result from Oct-induced variations in the sleep-promoting activity of GH-releasing hormone.

A unique association of renal carcinoma with sarcoid reaction in the kidney.

Sarcoid reactions have been described in association with lymphomas and rarely with other solid tumors. We describe a patient with renal papillary adenocarcinoma and prominent sarcoid-like granulomatous infiltration of the ipsilateral and most likely the contralateral kidney. There was no evidence of extrarenal granulomas. This is the first description of impairment in renal function in a patient with renal carcinoma and with sarcoid reaction to this tumor isolated in the kidney.

Association of intracranial meningioma with arteriovenous malformation.

Simultaneous occurrence of a cerebral arteriovenous malformation and a primary brain tumor is rare. A case of a left occipital meningioma and a right parietotemporal arteriovenous malformation is reported. Clinical, radiological, and postmortem findings are described. Thirty previous reports of arteriovenous malformations associated with primary brain tumors are reviewed. In 18 cases the two lesions were intermixed or in close proximity. This spatial relationship between the lesions suggests more than a coincidental association. Several hypotheses are proposed to explain common causal connections.

Auditory and somatosensory evoked potentials in a case of "locked-in" syndrome: a clinical and pathological study.

Brainstem auditory evoked potentials were bilaterally normal, and somatosensory evoked potentials were unilaterally abnormal in a patient with a large pontine infarct causing a "locked-in" syndrome. In the post mortem examination, the lesion extended unilaterally into the pontine tegmentum, partially involving the left medial lemniscus. The P14 potential was absent and the N20 potential was diminished in amplitude with right median nerve stimulation. The origin of the P14 potential has been debated in the literature. This case provides evidence for the P14 generator being located at the pontine level, in relation to a lemniscal area above the decussation of the somatosensory pathway. Evoked potentials can help to determine the tegmental extension of the pontine infarcts in the "locked-in" syndrome, especially in patients unable to cooperate with clinical examination.

The role of H-2 in apoferritin-induced murine immune complex glomerulonephritis.

The role of the major histocompatibility complex in the development of apoferritin induced immune complex glomerulonephritis was studied in H-2 congenic B10 mice. The glomerular lesions varied strikingly among the three different strains studied. The B10 (H-2b) mice had minimal mesangial expansion or no lesions at all. The B10.BR (H-2k) mice had mesangial expansion and proliferative glomerulonephritis without crescents or interstitial mononuclear cell infiltration. In contrast, the B10.D2 (H-2d) mice had necrotizing glomerulonephritis with crescents and an interstitial mononuclear cell infiltrate. Immunofluorescence and electron microscopy demonstrated only minimal mesangial deposits in B10 (H-2b) mice, predominantly mesangial deposition in the B10.BR (H-2k) mice, and mesangial and subepithelial immune complex deposits in B10.D2 (H-2d) mice. These morphologic differences correlated with functional abnormalities. Only the B10.D2 (H-2d) mice developed proteinuria, hematuria, and elevated blood urea nitrogen. They also had the most elevated antiapoferritin IgG levels. These experiments demonstrate that differences in the pathologic lesions and susceptibility to immune complex glomerulonephritis can be seen in animals that differ only at the H-2 locus. This model will lend itself to the study of the mechanisms by which the major histocompatibility complex influences the development of immune complex glomerulonephritis.

Peanut agglutinin binding as a histochemical tool for diagnosis of eosinophilic granuloma.

Histochemical detection of peanut agglutinin binding performed on tissue sections in 11 cases of eosinophilic granuloma of bone revealed simultaneous appearance of the reaction product both at the cell surface and within the cytoplasm (in the Golgi area) of a vast majority of Langerhans' cells. Based on this unique characteristic of Langerhans' cells, peanut agglutinin binding assay may represent a simple and inexpensive tool for morphologic differentiation of eosinophilic granuloma or other types of histiocytosis X from other monocytic and histiocytic lesions in equivocal cases.

Choroid plexus as a barrier to immunoglobulin delivery into cerebrospinal fluid.

The concentration of gamma globulins is greatly increased in the cerebrospinal fluid (CSF) during inflammatory and degenerative disorders of the central nervous system (CNS). The mechanism by which immunoglobulins enter the CSF under normal conditions is unknown. The extent of participation of the blood-brain barrier in protein delivery to the CSF is unclear, although the choroid plexus is known to have primary responsibility for the formation and movement of certain proteins into the CSF. To investigate the role of the choroid plexus in immunoglobulin delivery to the CSF, the authors evaluated rat brain tissue by light and electron microscopic immunohistochemical technique using the peroxidase technique of immunoglobulin (Ig)G and IgA detection. Peanut agglutinin was used to identify macrophages, cells known to have important immune functions and which have been reported as a normal component of the choroid plexus. Antisera to IgG' and IgA demonstrated diffuse surface staining of the choroidal epithelial cells with light and electron microscopy; the cytoplasm and nuclei did not contain immunoglobulins. Macrophages were not present in the choroid plexus, in contrast to previous reports. The results demonstrate that immunoglobulins do not enter the CSF via the choroid plexus, unlike other proteins in similar concentrations in the CSF. In addition, macrophages are shown to be an insignificant component of the plexus, thereby further diminishing the likelihood of participation-of the choroid plexus in the regulation of immunoglobulin entry into the CNS under normal conditions.

Postprandial hyperinsulinemia in patients with mild essential hypertension.

Glucose tolerance tests and diurnal profiles of glucose, insulin, free fatty acids, serum triglycerides, total and high-density lipoprotein cholesterol levels were performed in 8 male patients with mild essential hypertension as well as in 20 normotensive subjects. Although glucose tolerance and postprandial glucose levels appeared equal in both groups, the insulin response after a glucose load and after each meal was significantly increased in hypertensive subjects as compared with the controls (p less than 0.01). The levels of free fatty acids were higher in the postabsorptive phase of patients with hypertension in comparison to normotensive subjects, but decreased markedly when plasma insulin levels rose after meals. In both subject groups serum triglyceride levels showed the typical postprandial increase. Total and high-density lipoprotein cholesterol levels showed neither diurnal variations nor differences between hypertensive subjects and normotensive controls. Postprandial hyperinsulinemia in patients with mild essential hypertension possibly may provoke lipid accumulation in the arterial wall and therefore may be a relevant risk factor for atherosclerosis in these subjects.