MRI of the cervical spinal cord predicts respiratory dysfunction in ALS.

For patients with amyotrophic lateral sclerosis (ALS), the primary therapeutic goal is to minimize morbidity. Non-invasive ventilation improves survival. We aim to assess whether Magnetic Resonance Imaging (MRI) of the cervical spinal cord predicts the progression of respiratory disorders in ALS. Brain and spinal MRI was repeatedly performed in the SOD1G86R mouse model, in 40 patients and in healthy controls. Atrophy, iron overload, white matter diffusivity and neuronal loss were assessed. In Superoxide Dismutase-1 (SOD1) mice, iron accumulation appeared in the cervical spinal cord at symptom onset but disappeared with disease progression (after the onset of atrophy). In ALS patients, the volumes of the motor cortex and the medulla oblongata were already abnormally low at the time of diagnosis. Baseline diffusivity in the internal capsule was predictive of functional handicap. The decrease in cervical spinal cord volume from diagnosis to 3 months was predictive of the change in slow vital capacity at 12 months. MRI revealed marked abnormalities at the time of ALS diagnosis. Early atrophy of the cervical spinal cord may predict the progression of respiratory disorders, and so may be of value in patient care and as a primary endpoint in pilot neuroprotection studies.

Hippocampal Deformations and Entorhinal Cortex Atrophy as an Anatomical Signature of Long-Term Cognitive Impairment: from the MCAO Rat Model to the Stroke Patient.

Stroke patients have an elevated risk of developing long-term cognitive disorders or dementia. The latter is often associated with atrophy of the medial temporal lobe. However, it is not clear whether hippocampal and entorhinal cortex atrophy is the sole predictor of long-term post-stroke dementia. We hypothesized that hippocampal deformation (rather than atrophy) is a predictive marker of long-term post-stroke dementia on a rat model and tested this hypothesis in a prospective cohort of stroke patients.Male Wistar rats were subjected to transient middle cerebral artery occlusion and assessed 6 months later. Ninety initially dementia-free patients having suffered a first-ever ischemic stroke were prospectively included in a clinical study. In the rat model, significant impairments in hippocampus-dependent memories were observed. MRI studies did not reveal significant atrophy of the hippocampus volume, but significant deformations were indeed observed-particularly on the ipsilateral side. There, the neuronal surface area was significantly lower in ischemic rats and was associated with a lower tissue density and a markedly thinner entorhinal cortex. At 6 months post-stroke, 49 of the 90 patients displayed cognitive impairment (males 55.10%). Shape analysis revealed marked deformations of their left hippocampus, a significantly lower entorhinal cortex surface area, and a wider rhinal sulcus but no hippocampal atrophy. Hence, hippocampal deformations and entorhinal cortex atrophy were associated with long-term impaired cognitive abilities in a stroke rat model and in stroke patients. When combined with existing biomarkers, these markers might constitute sensitive new tools for the early prediction of post-stroke dementia.

Continuous cerebroventricular administration of dopamine: A new treatment for severe dyskinesia in Parkinson's disease?

In Parkinson's disease (PD) depletion of dopamine in the nigro-striatal pathway is a main pathological hallmark that requires continuous and focal restoration. Current predominant treatment with intermittent oral administration of its precursor, Levodopa (l-dopa), remains the gold standard but pharmacological drawbacks trigger motor fluctuations and dyskinesia. Continuous intracerebroventricular (i.c.v.) administration of dopamine previously failed as a therapy because of an inability to resolve the accelerated dopamine oxidation and tachyphylaxia. We aim to overcome prior challenges by demonstrating treatment feasibility and efficacy of continuous i.c.v. of dopamine close to the striatum. Dopamine prepared either anaerobically (A-dopamine) or aerobically (O-dopamine) in the presence or absence of a conservator (sodium metabisulfite, SMBS) was assessed upon acute MPTP and chronic 6-OHDA lesioning and compared to peripheral l-dopa treatment. A-dopamine restored motor function and induced a dose dependent increase of nigro-striatal tyrosine hydroxylase positive neurons in mice after 7days of MPTP insult that was not evident with either O-dopamine or l-dopa. In the 6-OHDA rat model, continuous circadian i.c.v. injection of A-dopamine over 30days also improved motor activity without occurrence of tachyphylaxia. This safety profile was highly favorable as A-dopamine did not induce dyskinesia or behavioral sensitization as observed with peripheral l-dopa treatment. Indicative of a new therapeutic strategy for patients suffering from l-dopa related complications with dyskinesia, continuous i.c.v. of A-dopamine has greater efficacy in mediating motor impairment over a large therapeutic index without inducing dyskinesia and tachyphylaxia.

TNF-alpha and IL-1beta inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells.

AIMS: Joint inflammation leads to bone erosion in rheumatoid arthritis (RA), whereas it induces new bone formation in spondyloarthropathies (SpAs). Our aims were to clarify the effects of tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) on osteoblast differentiation and mineralization in human mesenchymal stem cells (MSCs). MAIN METHODS: In MSCs, expression of osteoblast markers was assessed by real-time PCR and ELISA. Activity of tissue-nonspecific alkaline phosphatase (TNAP) and mineralization were determined by the method of Lowry and alizarin red staining respectively. Involvement of RUNX2 in cytokine effects was investigated in osteoblast-like cells transfected with a dominant negative construct. KEY FINDINGS: TNF-alpha (from 0.1 to 10 ng/ml) and IL-1beta (from 0.1 to 1 ng/ml) stimulated TNAP activity and mineralization in MSCs. Addition of 50 ng/ml of IL-1 receptor antagonist in TNF-alpha-treated cultures did not reverse TNF-alpha effects, indicating that IL-1 was not involved in TNF-alpha-stimulated TNAP activity. Both TNF-alpha and IL-1beta decreased RUNX2 expression and osteocalcin secretion, suggesting that RUNX2 was not involved in mineralization. This hypothesis was confirmed in osteoblast-like cells expressing a dominant negative RUNX2, in which TNAP expression and activity were not reduced. Finally, since mineralization may merely rely on increased TNAP activity in a collagen-rich tissue, we investigated cytokine effects on collagen expression, and observed that cytokines decreased collagen expression in osteoblasts from MSC cultures. SIGNIFICANCE: The different effects of cytokines on TNAP activity and collagen expression may therefore help explain why inflammation decreases bone formation in RA whereas it induces ectopic ossification from collagen-rich entheses during SpAs.

Gene expression in normotopic and heterotopic human bone: increased level of SP7 mRNA in pathological tissue.

Head injury-induced heterotopic ossification (HO) develops at vicinity of joints and in severe cases requires surgical intervention. Our previous study demonstrated high mRNA levels of osteocalcin (OC), type 1 collagen (COL1), osteonectin and RUNX2/CBFA1 in osteocytes and lining osteoblasts from non-evolutive HO compared to equivalent healthy cells from the proximal femoral shaft of patients receiving prosthesis. This allowed a first molecular characterisation of this pathological bone. The aims of this study is to extend the analysis to 10 more genes and determine those involved in the high OC mRNA level observed in pathological bone samples. RNAs were prepared from normotopic and heterotopic human bone samples digested by collagenase. After cDNA synthesis, mRNA levels were determined by real-time PCR and normalised using beta actin and glyceraldehyde-3-phosphate dehydrogenase. OSTERIX/SP7 expression was observed for the first time in human adult bone biopsies. In HO samples higher levels of SP7 (four- to sevenfold increase) and 1alpha,25-dihydroxy vitamin D(3) receptor (VDR) (two- to threefold increase) were observed compared to control samples. Moreover, SP7 level was correlated to OC and RUNX2 levels. In control samples, OC and SP7 levels were correlated. Our study further confirms that the involvement of SP7 in bone physiology is not only limited to the developmental step. Moreover, our results support the hypothesis that in HO the high level of OC expression could be due not only to an increase in RUNX2, but also in SP7 or VDR or to an imbalance in their respective activities.

Transgenic mice overexpressing alpha2A-adrenoceptors in pancreatic beta-cells show altered regulation of glucose homeostasis.

AIMS/HYPOTHESIS: To study the role of the human alpha2A-adrenoceptor in the regulation of insulin secretion and the maintenance of glucose homeostasis in transgenic mice overexpressing this receptor in pancreatic beta cells. METHODS: A human insulin promoter/human alpha2C10-adrenoceptor chimeric gene was microinjected into mouse embryos and transgenic mice were obtained. RESULTS: Analysis by RT-PCR showed that the expression of the transgene was restricted to pancreatic islets. Study of the binding of the alpha2-antagonist [3H]RX821002 to membrane preparations showed that islets from transgenic mice had ninefold higher alpha2-adrenoceptor density than those from controls. Immunohistological analysis showed, however, no change in the number or size of islets between control and transgenic mice. Transgenic animals had normal glycaemia and insulinaemia in basal conditions but greater hyperglycaemic and hypoinsulinaemic responses after injection of the alpha2-agonist, UK14304. The lower blood insulin concentration detected in transgenic mice was a reflection of a stronger inhibitory effect of the alpha2-agonist on glucose-stimulated insulin secretion in transgenic islets than in controls. Furthermore, transgenic mice did not have lower glycaemia to basal values after an intraperitoneal glucose tolerance test. This defect was abolished by treatment with the alpha2-adrenoceptor antagonist, RX821002. CONCLUSION/INTERPRETATION: These results provide evidence in vivo that overexpression of alpha2-adrenoceptors in beta cells can lead to impaired insulin secretion and glucose intolerance.

Alpha(2) adrenoceptors regulate proliferation of human intestinal epithelial cells.

BACKGROUND AND AIMS: Previous studies on rodents have suggested that catecholamines stimulate proliferation of the intestinal epithelium through activation of alpha(2) adrenoceptors located on crypt cells. The occurrence of this effect awaits demonstration in humans and the molecular mechanisms involved have not yet been elucidated. Here, we examined the effect of alpha(2) agonists on a clone of Caco2 cells expressing the human alpha(2A) adrenoceptor. METHODS: Cells were transfected with a bicistronic plasmid containing the alpha2C10 and neomycin phosphotransferase genes. G418 resistant clones were assayed for receptor expression using radioligand binding. Receptor functionality was assessed by testing its ability to couple Gi proteins and to inhibit cAMP production. Mitogen activated protein kinase (MAPK) phosphorylation was followed by western blot, and cell proliferation was estimated by measuring protein and DNA content. RESULTS: Permanent transfection of Caco2 cells allowed us to obtain a clone (Caco2-3B) expressing alpha(2A) adrenoceptors at a density similar to that found in normal human intestinal epithelium. Caco2-3B retained morphological features and brush border enzyme expression characteristic of enterocytic differentiation. The receptor was coupled to Gi2/Gi3 proteins and its stimulation caused marked diminution of forskolin induced cAMP production. Treatment of Caco2-3B with UK14304 (alpha(2) agonist) induced a rapid increase in the phosphorylation state of MAPK, extracellular regulated protein kinase 1 (Erk1), and 2 (Erk2). This event was totally abolished in pertussis toxin treated cells and in the presence of kinase inhibitors (genistein or PD98059). It was unaffected by protein kinase C downregulation but correlated with a transient increase in Shc tyrosine phosphorylation. Finally, sustained exposure of Caco2-3B to UK14304 resulted in modest but significant acceleration of cell proliferation. None of these effects was observed in the parental cell line Caco2. CONCLUSION: The results obtained in the present study support a regulatory role for alpha(2) adrenoceptors in intestinal cell proliferation.

Transgenic mice overexpressing insulin-like growth factor-II in beta cells develop type 2 diabetes.

During embryonic development, insulin-like growth factor-II (IGF-II) participates in the regulation of islet growth and differentiation. We generated transgenic mice (C57BL6/SJL) expressing IGF-II in beta cells under control of the rat Insulin I promoter in order to study the role of islet hyperplasia and hyperinsulinemia in the development of type 2 diabetes. In contrast to islets from control mice, islets from transgenic mice displayed high levels of IGF-II mRNA and protein. Pancreases from transgenic mice showed an increase in beta-cell mass (about 3-fold) and in insulin mRNA levels. However, the organization of cells within transgenic islets was disrupted, with glucagon-producing cells randomly distributed throughout the core. We also observed enhanced glucose-stimulated insulin secretion and glucose utilization in islets from transgenic mice. These mice displayed hyperinsulinemia, mild hyperglycemia, and altered glucose and insulin tolerance tests, and about 30% of these animals developed overt diabetes when fed a high-fat diet. Furthermore, transgenic mice obtained from the N1 backcross to C57KsJ mice showed high islet hyperplasia and insulin resistance, but they also developed fatty liver and obesity. These results indicate that local overexpression of IGF-II in islets might lead to type 2 diabetes and that islet hyperplasia and hypersecretion of insulin might occur early in the pathogenesis of this disease.

Evidence from transgenic mice that interferon-beta may be involved in the onset of diabetes mellitus.

A number of cytokines have been shown to alter the function of pancreatic beta-cells and thus might be involved in the development of type 1 diabetes. Interferon-beta (IFN-beta) expression is induced in epithelial cells by several viruses, and it has been detected in islets of type 1 diabetic patients. Here we show that treatment of isolated mouse islets with this cytokine was able to alter insulin secretion in vitro. To study whether IFN-beta alters beta-cell function in vivo and leads to diabetes, we have developed transgenic mice (C57BL6/SJL) expressing IFN-beta in beta-cells. These mice showed functional alterations in islets and impaired glucose-stimulated insulin secretion. Transgenic animals presented mild hyperglycemia, hypoinsulinemia, hypertriglyceridemia, and altered glucose tolerance test, all features of a prediabetic state. However, they developed overt diabetes, with lymphocytic infiltration of the islets, when treated with low doses of streptozotocin, which did not induce diabetes in control mice. In addition, about 9% of the transgenic mice obtained from the N3 back-cross to outbred albino CD-1 mice spontaneously developed severe hyperglycemia and hypoinsulinemia and showed mononuclear infiltration of the islets. These results suggest that IFN-beta may be involved in the onset of type 1 diabetes when combined with either an additional factor or a susceptible genetic background.

Molecular cloning, sequencing and functional study of the promoter region of the human alpha2C4-adrenergic receptor gene.

Screening of a human foetal brain genomic DNA library allowed us to isolate an EcoRI-EcoRI fragment containing 6 kb of the 5'-flanking region, the open reading frame and 4 kb of the 3'-flanking region of the alpha2C4 gene. Analysis of the sequenced region (4850 bp) revealed that the first 900 bp 5' to the start codon are very rich in GC (84%), contain several Sp1-binding sites and lack a consensus TATA box. The 5'- and 3'-ends of the alpha2C4 transcript were determined by RNase-protection assays carried out with a series of antisense probes. The data obtained with cellular RNA from HepG2 cells demonstrated that transcription is initiated 891 bases upstream of the translation-start site and that the polyadenylation site is located 550 bases downstream of the stop codon. These results are consistent with the existence of a non-conventional TATA box (TTAGAAA) and the presence of a unique polyadenylation signal (AATAAA). They also fit with the size of alpha2C4-RNA found by Northern-blot analysis (2.9 kb). The transcriptional activity of the alpha2C4 promoter region was investigated by transfecting several cell types with chimaeric constructs containing various fragments of the 5'-non-coding region and luciferase as a reporter gene. The activity of the construct containing the entire 5'-non-coding region appeared to depend on the host cell. Removal of the 5'-untranslated region resulted in loss of cell specificity and a concomitant increase in luciferase activity. Transfection of HepG2 and SK-N-MC cells with constructs deleted of additional 5'-flanking fragments permitted the definition of a minimal 200 bp promoter fragment containing the pseudo-TATA box and two putative SP1-binding sites.

Regulation of alpha 2A-adrenergic receptor expression in the human colon carcinoma cell line HT29: SCFA-induced enterocytic differentiation results in an inhibition of alpha 2C10 gene transcription.

Previous studies on the intestinal epithelium from various species have shown that the number of alpha 2-adrenergic receptors in immature cells from the crypts is several-fold higher than in mature cells from the villi, thus suggesting an inverse relationship between enterocytic differentiation and the expression of this inhibitory receptor. The receptor density along the surface-crypt axis of the human colonic mucosa is correlated with the amount of alpha 2C10 mRNA; however, the mechanisms underlying this regulation remain unknown. The human colonic adenocarcinoma cell line HT29, which expresses the alpha 2A-adrenergic receptor and is able to undergo enterocytic differentiation, is a suitable model with which to investigate this question in vitro. In this study, we explored the effects of short chain fatty acids (SCFAs), differentiating agents normally present in the colon lumen, on alpha 2-adrenergic receptor expression. Exposure of HT29 cells to butyrate and propionate, but not acetate, resulted in a large diminution of [3H]RX821002 binding sites. The reduction of alpha 2-adrenergic receptor number induced by butyrate or propionate was due to decreased amounts of alpha 2C10 mRNA and was associated with an increase of alkaline phosphatase activity, which reflected the emergence of a more differentiated phenotype. The changes in alpha 2C10 mRNA level induced by both SCFAs were dose-dependent, rapid, and reversible and resulted from a diminution in the transcription rate of the alpha 2C10 gene. Finally, these effects were mimicked by trichostatin A, indicating that they are triggered primarily through inhibition of histone deacetylases. The present findings demonstrate that decrease of alpha 2-adrenergic receptor expression is a very early event of the HT29 cell differentiation process. They also suggest that SCFAs, which originate from bacterial fermentation of dietary fibers, may play a role in the regulation of the alpha 2-adrenergic receptivity of colonic mucosa in vivo.

Human adipocytes express alpha 2-adrenergic receptor of the alpha 2A-subtype only: pharmacological and genetic evidence.

In the present study we have reinvestigated the subtype of alpha 2-adrenoceptors expressed in human adipocytes (from subcutaneous and internal fat deposits) by means of radioligand binding using subtype-selective antagonists, and RNase mapping using a set of specific probes prepared from human alpha 2-adrenoceptors genes (alpha 2C2, alpha 2C4 and alpha 2C10). Comparison of the pharmacological properties of the human adipocyte alpha 2-adrenoceptors with those of the different human adrenoceptors expressed in COS-7 cells demonstrated that: i) human adipocyte alpha 2-adrenoceptors displays a KD for [3H]RX821002 and [3H]MK912 identical to that found in COS-7 cells transfected with the alpha 2C10 gene; ii) yohimbine and oxymetazoline is 1,000-fold more potent than prazosin to inhibit [3H]antagonist binding. RNase protection assays on cellular RNA prepared from the three fat deposits showed the presence of substantial amounts of alpha 2C10 transcripts: in contrast, mRNAs from alpha 2C2 and alpha 2C4 genes were undetectable. Altogether these results definitively establish that human adipocytes express only one alpha 2-adrenoceptor which is of the alpha 2A-subtype and encoded by the alpha 2C10 gene.

Alpha 2-adrenergic stimulation promotes preadipocyte proliferation. Involvement of mitogen-activated protein kinases.

Primary cultures of rat preadipocytes were shown to express alpha 2- and beta-adrenoreceptors when maintained in serum-deprived medium. alpha 2-Adrenoreceptor stimulation led to an increase in cell number, whereas beta-adrenoreceptor stimulation was without effect. On 3T3-F442A clones stably transfected with the human alpha 2-C10 gene and expressing a physiologically related number of alpha 2-adrenoreceptors to overexpression, the proliferative effect of alpha 2-adrenoreceptor agonist UK14304 was proportional to the level of alpha 2-adrenoreceptor expressed in individual clones and required alpha-2 adrenoreceptor stimulation. Analysis of the signaling pathway linked to alpha 2-adrenoreceptor activation showed that the mitogenic effect was adenylyl cyclase- and protein kinase C-independent. It was pertussis toxin-sensitive, implying the involvement of pertussis toxin-sensitive G proteins. The activation of the mitogen-activated protein kinase pathway was increased after alpha 2-adrenoreceptor stimulation in both 3T3-F442 clones and rat preadipocytes and inhibited by pertussis toxin treatment. So, catecholamines are involved in the control of white preadipocyte proliferation through the alpha 2-adrenoreceptor activation, linked to the mitogen-activated protein kinase pathway.

Further characterization of human alpha 2-adrenoceptor subtypes: [3H]RX821002 binding and definition of additional selective drugs.

The characteristics of [3H]RX821002 binding to the different human alpha 2-adrenoceptor subtypes were studied on membranes from COS-7 cells transfected with the genes: alpha 2C2, alpha 2C4 and alpha 2C10. Saturation experiments indicated that the radioligand labels the three adrenoceptors with high affinity. A difference was however observed between the subtypes. The affinity of [3H]RX821002 for alpha 2C10-adrenoceptors (KD = 1.41 +/- 0.15 nM) was 3-fold higher than for alpha 2C4-adrenoceptors (KD = 4.42 +/- 0.63 nM) and 7-fold higher than for alpha 2C2-adrenoceptors (KD = 10.2 +/- 0.9 nM). Inhibition experiments with a series of 17 competitors confirmed that prazosin, oxymetazoline, WB4101, ARC239, corynanthine and chlorpromazine are subtype-selective drugs. They also demonstrated that BRL44408 and guanfacine are selective for the alpha 2C10-receptor, whereas BRL41992 and imiloxan are selective for the alpha 2C2. Given that these two latter drugs were previously shown to be specific for the alpha 2B pharmacological subtype originally defined in neonatal rat lung, these results confirm that the alpha 2C2 gene encodes for the human homolog of this receptor subtype. It is concluded that the combined use of [3H]RX821002 and of these new selective drugs may be useful for the identification of the alpha 2-adrenoceptor subtypes in human tissues.

Characterization and distribution of alpha 2-adrenergic receptors in the human intestinal mucosa.

The subtype and the expression of the alpha 2-adrenergic receptor were investigated in the normal mucosa from human intestine by means of radioligand binding, RNase mapping, and measurement of adenylate cyclase activity. The study of the binding of the alpha 2-adrenergic antagonist, [3H]RX821002, to epithelial cell membranes indicated the existence of a single class of noninteracting sites displaying a high affinity for the radioligand (Kd = 1.1 +/- 0.5 nM). The rank order of potency of antagonists to inhibit [3H]RX821002 binding (RX821002 > yohimbine = rauwolscine > phentolamine approximately idazoxan >> chlorpromazine > prazosin) suggested that the receptor is of the alpha 2A subtype. A conclusion which is confirmed by the fact that only alpha 2C10 transcripts were found in the human intestine mucosa. Competition curves with (-)-norepinephrine demonstrated that 60% of the receptor population exhibited high affinity for agonists. This high-affinity state was abolished by the addition of GTP plus Na+ or by prior treatment of the membranes with pertussis toxin indicating it corresponded to G protein-coupled receptors. [32P]ADP-ribosylation and immunoblotting experiments identified two pertussis toxin-sensitive G proteins corresponding to Gi2 and Gi3. The study of the distribution of the receptor indicated that (a) the proximal colon is the intestine segment exhibiting the highest receptor density and (b) the receptor is predominantly expressed in crypts and is preferentially located in the basolateral membrane of the polarized cell. The distribution of the receptor along the crypt-surface axis of the colon mucosa can be correlated with a higher level of alpha 2C10-specific mRNA and a higher efficiency of UK14304 to inhibit adenylate cyclase in crypt cells.

Androgenic regulation of adipocyte alpha 2-adrenoceptor expression in male and female Syrian hamsters: proposed transcriptional mechanism.

Adaptation of male hamsters to short daily (SD) photoperiod induced a reduction of the adipocyte alpha 2-adrenoceptor (alpha 2-AR) expression which was related to a sexual involution and could be reversed by testosterone administration. In the present paper, the possible mechanisms of such a physiological regulation are explored. The effect of testosterone on the adipocyte alpha 2-AR was rapid, dose-dependent, occurred at the physiological plasma concentrations of androgen, and was mimicked by dihydrotestosterone, but not by 17 beta-estradiol, progesterone, hydrocortisol, insulin, or T3. Adaptation of female hamsters to SD photoperiod also induced a sexual involution, but no modification of the adipocyte alpha 2-AR number was observed. Administration of testosterone induced a large up-regulation of the adipocyte alpha 2-AR. Testosterone was also able to up-regulate the adipocyte alpha 2-AR in male hamsters adapted to long day photoperiod whatever their age (6-, 12-, and 25-week old). Adaptation to SD photoperiod did not modify the adipocyte adenylyl cyclase activity (basal, forskolin-stimulated, GppNHp-inhibited). Conversely, UK14304-mediated inhibition of the adenylyl cyclase was suppressed in SD photoperiod and recovered after testosterone treatment. Administration of testosterone in young male hamsters adapted to long day photoperiod induced an increase in the amount of the alpha 2-AR messenger RNA, which coincided with the increase in the alpha 2-AR maximal number. The existence of a specific regulation of the adipocyte alpha 2-AR by the androgens, probably via a transcriptional mechanism, can be hypothesized. This regulation, which occurs in both male and female hamsters, appears to be physiologically relevant.

Uracil-DNA glycosylase affects mismatch repair efficiency in transformation and bisulfite-induced mutagenesis in Streptococcus pneumoniae.

The generalized mismatch repair system of Streptococcus pneumoniae (the Hex system) can eliminate base pair mismatches arising in heteroduplex DNA during transformation or by DNA polymerase errors during replication. Mismatch repair is most likely initiated at nicks or gaps. The present work was started to examine the hypothesis that strand discontinuities arising after removal of uracil by uracil DNA-glycosylase (Ung) can be utilised as strand discrimination signals. We show that mismatch repair efficiency is enhanced 3- to 6-fold when using uracil-containing DNA as donor in transformation. In order to assess the contribution of Ung to nascent strand discrimination for postreplication mismatch repair, we developed a positive selection procedure to isolate S. pneumoniae Ung- mutants. We succeeded in isolating Ung- mutants using this procedure based on chromosomal integration of uracil-containing hybrid DNA molecules. Cloning and characterization of the ung gene was achieved. Comparison of spontaneous mutation rates in strains either proficient or deficient in mismatch and/or uracil repair gave no support to the hypothesis that Ung plays a major role in targeting the Hex system to neosynthesized DNA strands. However Ung activity is responsible for the increased efficiency of mismatch repair observed in transformation with uracil-containing DNA. In addition Ung is involved in repair of bisulfite-treated transforming DNA.

Osmotic induction of gene osmC expression in Escherichia coli K12.

osmC, an osmotically inducible gene of Escherichia coli, was physically mapped on the bacterial chromosome, cloned on multicopy plasmids, and its product, OsmC, was identified as a 14 kDa protein in maxicells. The DNA sequence of the gene and its upstream region were determined. The sequence of an osmC-phoA gene fusion confirmed the osmC reading frame. A deletion of osmC from the E. coli chromosome was constructed by gene replacement, demonstrating that it is not an essential gene. The osmCp promoter region was subcloned and a lac operon fusion transcribed under osmCp control was constructed. The expression of this operon fusion demonstrated that osmC regulation occurs at the transcriptional level. S1 nuclease protection experiments and deletion analysis identified two overlapping promoters with transcription start sites separated by ten nucleotides. All the sequences necessary for osmotic regulation of both promoters are located within a 137 base-pair DNA fragment extending from position -95 to +42 with respect to the putative osmC translation start. Two deletions were obtained that abolish the functioning of the upstream promoter. Yet, under our experimental conditions, the subsequent expression of the osmC-lacZ fusion was equivalent to that obtained from the tandem promoters. Mutations leading to constitutive expression of osmC were selected. Two independent mutations were obtained, both affected osmZ, the gene encoding the histone-like protein H1.

Regulation of the alpha 2A-adrenergic receptor in the HT29 cell line. Effects of insulin and growth factors.

The density of the alpha 2A-adrenergic receptor in the HT29 cell line, a human colonic adenocarcinoma, increases when the cells are placed in fetal calf serum (FCS)-free culture medium and decreases again, in a concentration-dependent manner, when they are re-exposed to FCS. In an attempt to identify the FCS components responsible for this phenomenon, we examined the effect of insulin and of various growth factors on receptor expression. Incubation of HT29 cells with insulin resulted in a time- and dose-dependent lowering of the alpha 2-adrenergic receptor number. The decrease of [3H] RX821002 binding sites after a 48-h period of treatment reached 70-75% with 170 nM insulin, and a half-maximal effect was observed at 2.6 nM. This value is in agreement with the EC50 of the hormone for stimulating the glycolytic activity of HT29 cells (8 nM) and is sufficiently low to indicate that the decrease of alpha 2-adrenergic receptor number is mediated through stimulation of insulin receptors. Direct quantification of [3H] UK14304 binding sites and the study of the inhibition of [3H]RX821002 binding by (-)-epinephrine indicated that the degree of receptor coupling to Gi protein was not affected when the receptor number was decreased by insulin treatment. The reduction in receptor number did result in an attenuation of the inhibitory effect of UK14304 on forskolin-induced cAMP accumulation in a manner which was consistent with the existence of a large population of spare receptors in untreated cells. The action of insulin is not due to an accelerated rate of receptor degradation and can be mimicked by other growth factors (epidermal growth factor and insulin-like growth factors I and II) acting through stimulation of tyrosine kinase receptors. RNase mapping experiments with a 0.35-kilobase riboprobe prepared from the human alpha 2 C10-adrenergic receptor gene demonstrated that the decrease of receptor number induced by the different treatments is a reflection of changes occurring at the level of its mRNA. The use of cycloheximide indicated that the effect of insulin on alpha 2-adrenergic receptor mRNA does not require protein synthesis. The half-life of the alpha 2-adrenergic receptor mRNA measured after the addition of actinomycin D was unchanged by insulin which suggests that a decrease in the transcription rate is the predominant factor responsible for the observed regulation of receptor expression.