The effect of antiretroviral treatment of different durations in primary HIV infection.

OBJECTIVES: To compare immunological, virological and clinical outcomes in persons initiating combination antiretroviral therapy (cART of different durations within 6 months of seroconversion (early treated) with those who deferred therapy (deferred group). DESIGN: CD4 cell and HIV-RNA measurements for 'early treated' individuals following treatment cessation were compared with the corresponding ART-free period for the 'deferred' group using piecewise linear mixed models. Individuals identified during primary HIV infection were included if they seroconverted from 1st January 1996 and were at least 15 years of age at seroconversion. Those with at least 2 CD4 less than 350 cells/microl or AIDS within the first 6 months following seroconversion were excluded. RESULTS: Of 348 'early treated' patients, 147 stopped cART following treatment for at least 6 (n = 38), more than 6-12 (n = 40) or more than 12 months (n = 69). CD4 cell loss was steeper for the first 6 months following cART cessation, but subsequent loss rate was similar to the 'deferred' group (n = 675, P = 0.26). Although those treated for more than 12 months appeared to maintain higher CD4 cell counts following cART cessation, those treated for 12 months or less had CD4 cell counts 6 months after cessation comparable to those in the 'deferred' group. There was no difference in HIV-RNA set points between the 'early' and 'deferred' groups (P = 0.57). AIDS rates were similar but death rates, mainly due to non-AIDS causes, were higher in the 'deferred' group (P = 0.05). CONCLUSION: Transient cART, initiated within 6 months of seroconversion, seems to have no effect on viral load set point and limited beneficial effect on CD4 cell levels in individuals treated for more than 12 months. Its long-term effects remain inconclusive and need further investigation.

Injection of partially purified estrogen receptor protein from Xenopus liver nuclei into oocytes activates the silent vitellogenin locus.

Injection of extracts from Xenopus liver nuclei that are enriched 2000 times in estradiol receptor into Xenopus oocytes induces transcription of the silent vitellogenin locus, which is activated in liver by estradiol, but not of the albumin locus, which is active in liver but suppressed by high levels of estradiol. Transcription initiates within the 5'-end region of the gene we have studied and probably continues into the 3' third. The activation seems to be very efficient, but most of the primary transcripts are probably rapidly and inaccurately processed. New proteins are also made and secreted by the oocytes.

The transcriptional repressor REST determines the cell-specific expression of the human MAPK8IP1 gene encoding IB1 (JIP-1).

Islet-brain 1 (IB1) is the human and rat homologue of JIP-1, a scaffold protein interacting with the c-Jun amino-terminal kinase (JNK). IB1 expression is mostly restricted to the endocrine pancreas and to the central nervous system. Herein, we explored the transcriptional mechanism responsible for this preferential islet and neuronal expression of IB1. A 731-bp fragment of the 5' regulatory region of the human MAPK8IP1 gene was isolated from a human BAC library and cloned upstream of a luciferase reporter gene. This construct drove high transcriptional activity in both insulin-secreting and neuron-like cells but not in unrelated cell lines. Sequence analysis of this promoter region revealed the presence of a neuron-restrictive silencer element (NRSE) known to bind repressor zinc finger protein REST. This factor is not expressed in insulin-secreting and neuron-like cells. By mobility shift assay, we confirmed that REST binds to the NRSE present in the IB1 promoter. Once transiently transfected in beta-cell lines, the expression vector encoding REST repressed IB1 transcriptional activity. The introduction of a mutated NRSE in the 5' regulating region of the IB1 gene abolished the repression activity driven by REST in insulin-secreting beta cells and relieved the low transcriptional activity of IB1 observed in unrelated cells. Moreover, transfection in non-beta and nonneuronal cell lines of an expression vector encoding REST lacking its transcriptional repression domain relieved IB1 promoter activity. Last, the REST-mediated repression of IB1 could be abolished by trichostatin A, indicating that deacetylase activity is required to allow REST repression. Taken together, these data establish a critical role for REST in the control of the tissue-specific expression of the human IB1 gene.

Evidence from glut2-null mice that glucose is a critical physiological regulator of feeding.

A role for glucose in the control of feeding has been proposed, but its precise physiological importance is unknown. Here, we evaluated feeding behavior in glut2-null mice, which express a transgenic glucose transporter in their beta-cells to rescue insulin secretion (ripglut1;glut2-/- mice). We showed that in the absence of GLUT2, daily food intake was increased and feeding initiation and termination following a fasting period were abnormal. This was accompanied by suppressed regulation of hypothalamic orexigenic and anorexigenic neuropeptides expression during the fast-to-refed transition. In these conditions, however, there was normal regulation of the circulating levels of insulin, leptin, or glucose but a loss of regulation of plasma ghrelin concentrations. To evaluate whether the abnormal feeding behavior was due to suppressed glucose sensing, we evaluated feeding in response to intraperitoneal or intracerebroventricular glucose or 2-deoxy-D-glucose injections. We showed that in GLUT2-null mice, feeding was no longer inhibited by glucose or activated by 2-deoxy-D-glucose injections and the regulation of hypothalamic neuropeptide expression by intracerebroventricular glucose administration was lost. Together, these data demonstrate that absence of GLUT2 suppressed the function of central glucose sensors, which control feeding probably by regulating the hypothalamic melanocortin pathway. Furthermore, inactivation of these glucose sensors causes overeating.

Infrequent expression of the MAGE gene family in uveal melanomas.

It has previously been reported that MAGE-1, -2, -3 and -4 genes are expressed in human cancers including cutaneous melanoma. MAGE-1 and MAGE-3 represent targets for specific immunotherapy because they encode peptide antigens which are recognised by cytotoxic T lymphocytes (CTL) when presented by HLA class I molecules, and pilot clinical trials with these peptides are currently in progress. It is likely that other members of the MAGE gene family may also encode antigens recognised by CTL. Uveal melanomas, like cutaneous melanomas, arise from melanocytes that are derived from the neural crest. To determine if uveal melanoma patients would be suitable for MAGE-peptide immunotherapy, the expression of MAGE-1, -2, -3 and -4 genes was assessed by reverse transcription followed by polymerase chain reaction (RT-PCR) amplification and ethidium bromide staining. Expression of MAGE genes was not detected in any of 27 primary tumours. Either MAGE-1 or MAGE-4 was expressed in only 2 of 26 metastatic samples, but expression of MAGE-2 or -3 was not detected. Our data suggest that, unlike cutaneous melanomas, uveal melanomas may not be suitable candidates for MAGE-peptide immunotherapy.

PRPF31 alternative splicing and expression in human retina

PURPOSE: To provide a mechanistic link between mutations in PRPF31, and essential and ubiquitously expressed gene, and retinitis pigmentosa, a disorder restricted to the eye. METHODS: We investigated the existence of retina-specific PRPF31 isoforms and the expression of this gene in human retina and other tissues, as well as in cultured human cell lines. PRPF31 transcripts were examined by RT-PCR, quantitative PCR, cloning and sequencing. RESULTS: Database searching revealed the presence of a retina-specific PRPF31 isoform in mouse. However, this isoform could not be experimentally identified in transcripts from human retina or from a human whole eye. Nevertheless, four different PRPF31 isoforms, that were common to all analyzed tissues and cell lines, were isolated. Three of these harbored the full-length PRPF31 coding sequence, whereas the fourth was very short and probably non-coding. The amount of PRPF31 mRNA was previously found to be lower in patients with mutations in this gene than in healthy individuals, making it likely that retinal cells are more sensitive to variation in PRPF31 expression. However, quantitative PCR experiments revealed that PRPF31 mRNA levels in human retina were comparable to those detected in other tissues. CONCLUSIONS: Our results show that the retina-restricted phenotype caused by PRPF31 mutations cannot be explained by the presence of tissue-specific isoforms, or by differential expression of PRPF31 in the retina. As a consequence, the etiology of PRPF31-associated retinitis pigmentosa likely relies on other, probably more subtle molecular mechanisms.

The mif gene is transcriptionally regulated by glucose in insulin-secreting cells.

Macrophage migration inhibitory factor (MIF) is an important regulator of glucose homeostasis. In pancreatic beta-cells, MIF expression is regulated by glucose and its secretion potentiates the glucose-induced insulin secretion. The molecular mechanisms by which glucose mediates its effect on MIF expression are not elucidated. Herein, we report that incubating the differentiated insulin-secreting cell line INS-1 in high glucose concentration increases MIF transcriptional activity as well as the reporter gene activity driven by the -1033 to +63 bp fragment of the MIF promoter. A minimal region located between -187 and -98 bp of this promoter sequence contributes both to basal activity and glucose-responsiveness of the gene. Within this promoter region, two cis-binding sequences were identified by mobility shift assays and footprinting experiments. Both cis-elements interact with nuclear proteins expressed specifically in insulin-secreting cells. In conclusion, we identified a minimal region of the MIF promoter which contributes to the glucose stimulation of the mif gene in insulin-secreting cells.

Hypertension increases connexin43 in a tissue-specific manner.

BACKGROUND: Connexin43 (Cx43), a membrane protein involved in the control of cell-to-cell communication, is thought to play a role in the contractility of the vascular wall and in the electrical coupling of cardiac myocytes. The aim of this study was to investigate the effects of experimental hypertension on Cx43 expression in rat aorta and heart. METHODS AND RESULTS: Rats were made hypertensive after one renal artery was clipped (two kidney, one-clip renal model) or after the administration of deoxycorticosterone and salt (DOCA-salt model). After 4 weeks, all rats showed a similar increase in intra-arterial mean blood pressure and in the thickness of both the aortic wall and the heart. Northern blot analysis of aorta mRNA and immunolabeling for Cx43 showed that hypertensive rats expressed twice as much Cx43 in aorta as the control animals. In contrast, no difference in Cx43 mRNA or in the immunolabeled protein was observed in heart. CONCLUSIONS: The results show that rats exhibiting a similar degree of blood pressure elevation, as the result of different mechanisms, feature a comparable increase in Cx43 gene expression, which was observed in the aortic but not in the cardiac muscle. These data suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Renin and angiotensin II receptor gene expression in kidneys of renal hypertensive rats.

The aim of this investigation was to examine the interrelation between renal mRNA levels of renin and angiotensin II receptor type 1 (AT1) in a renin-dependent form of experimental hypertension. Rats were studied 4 weeks after unilateral renal artery clipping. Mean blood pressure and plasma renin activity were significantly higher in the hypertensive rats (n = 10 206 +/- mm Hg and 72.4 +/- 20.9 ng/mL-1/h-1, respectively) than in sham-operated controls (n = 10, 136 +/- 3 mm Hg and 3.3 +/- 0.5 ng/mL-1/h, respectively). Northern blot analysis of polyA+ RNA obtained from the kidneys of renal hypertensive rats showed increased levels of renin mRNA in the clipped kidney, whereas a decrease was observed in the unclipped kidney. Plasma renin activity was directly correlated with renin mRNA expression of the poststenotic kidney (r = .94, P < .01). AT1 mRNA expression was lower in both kidneys of the hypertensive rats. This downregulation was specific for the AT1A subtype since the renal expression of the AT1B subtype remained normal in hypertensive rats. The downregulation of the renal AT1A receptor may be due to high circulating angiotensin II levels. This is supported by the significant inverse correlation (r = .71, P < .01) between plasma renin activity and AT1A mRNA expression measured in the clipped kidney of the hypertensive rats.

A 338-bp proximal fragment of the glucose transporter type 2 (GLUT2) promoter drives reporter gene expression in the pancreatic islets of transgenic mice.

The high Km glucose transporter GLUT2 is a membrane protein expressed in tissues involved in maintaining glucose homeostasis, and in cells where glucose-sensing is necessary. In many experimental models of diabetes, GLUT2 gene expression is decreased in pancreatic beta-cells, which could lead to a loss of glucose-induced insulin secretion. In order to identify factors involved in pancreatic beta-cell specific expression of GLUT2, we have recently cloned the murine GLUT2 promoter and identified cis-elements within the 338-bp of the proximal promoter capable of binding islet-specific trans-acting factors. Furthermore, in transient transfection studies, this 338-bp fragment could efficiently drive the expression of the chloramphenicol acetyl transferase (CAT) gene in cell lines derived from the endocrine pancreas, but displayed no promoter activity in non-pancreatic cells. In this report, we tested the cell-specific expression of a CAT reporter gene driven by a short (338 bp) and a larger (1311 bp) fragment of the GLUT2 promoter in transgenic mice. We generated ten transgenic lines that integrated one of the constructs. CAT mRNA expression in transgenic tissues was assessed using the RNAse protection assay and the quantitative reverse transcribed polymerase chain reaction (RT-PCR). Overall CAT mRNA expression for both constructs was low compared to endogenous GLUT2 mRNA levels but the reporter transcript could be detected in all animals in the pancreatic islets and the liver, and in a few transgenic lines in the kidney and the small intestine. The CAT protein was also present in Langerhans islets and in the liver for both constructs by immunocytochemistry. These findings suggest that the proximal 338 bp of the murine GLUT2 promoter contain cis-elements required for the islet-specific expression of GLUT2.

n-3 PUFAs modulate T-cell activation via protein kinase C-alpha and -epsilon and the NF-kappaB signaling pathway.

We elucidated the mechanisms of action of two n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in Jurkat T-cells. Both DHA and EPA were principally incorporated into phospholipids in the following order: phosphatidylcholine < phosphatidylethanolamine < phosphatidylinositol/phosphatidylserine. Furthermore, two isoforms of phospholipase A(2) (i.e., calcium-dependent and calcium-independent) were implicated in the release of DHA and EPA, respectively, during activation of these cells. The two fatty acids inhibited the phorbol 12-myristate 13-acetate (PMA)-induced plasma membrane translocation of protein kinase C (PKC)-alpha and -epsilon. The two n-3 PUFAs also inhibited the nuclear translocation of nuclear factor kappaB (NF-kappaB) and the transcription of the interleukin-2 (IL-2) gene in PMA-activated Jurkat T-cells. Together, these results demonstrate that DHA and EPA, being released by two isoforms of phospholipase A(2), modulate IL-2 gene expression by exerting their action on two PKC isoforms and NF-kappaB in Jurkat T-cells.

Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.

Expression of T cell receptor genes in the thymus: localization of transcripts in situ and comparison of mature and immature subsets.

We have analyzed the expression of T cell receptor (TcR) genes in the thymus using in situ RNA hybridizations with probes to the constant regions of the TcR alpha, beta, gamma and delta chains. Localization of transcripts revealed low TcR alpha mRNA levels in the thymus cortex and very low levels in the subcapsular region. In contrast, TcR beta message was very abundant in the cortex. TcR gamma or delta mRNA+ thymocytes showed a scattered, predominantly cortical localization. In contrast to gamma, TcR delta transcripts were abundant in the subcapsular region. Control experiments with sorted TcR alpha/beta or gamma/delta cells revealed a detection efficiency of 75%-85% for the respective TcR mRNA and data on TcR gene expression in mature, CD3+ thymocytes were consistent with previous reports. The analysis of immature, CD3- thymocyte subsets, however, revealed a virtual absence of TcR alpha transcripts and an unexpectedly high proportion of cells (14%-29%) expressing the gene for the TcR delta chain. The data are discussed in view of current models of lineage relationships in the thymus.

Cross-species GacA-controlled induction of antibiosis in pseudomonads.

Signal extracts prepared from culture supernatants of Pseudomonas fluorescens CHA0 and Pseudomonas aeruginosa PAO stimulated GacA-dependent expression of small RNAs and hence of antibiotic compounds in both hosts. Pseudomonas corrugata LMG2172 and P. fluorescens SBW25 also produced signal molecules stimulating GacA-controlled antibiotic synthesis in strain CHA0, illustrating a novel, N-acyl-homoserine lactone-independent type of interspecies communication.

Fate of linear and supercoiled multinucleosomic templates during transcription.

Electron microscopy was used to monitor the fate of reconstituted nucleosome cores during in vitro transcription of long linear and supercoiled multinucleosomic templates by the prokaryotic T7 RNA polymerase and the eukaryotic RNA polymerase II. Transcription by T7 RNA polymerase disrupted the nucleosomal configuration in the transcribed region, while nucleosomes were preserved upstream of the transcription initiation site and in front of the polymerase. Nucleosome disruption was independent of the topology of the template, linear or supercoiled, and of the presence or absence of nucleosome positioning sequences in the transcribed region. In contrast, the nucleosomal configuration was preserved during transcription from the vitellogenin B1 promoter with RNA polymerase II in a rat liver total nuclear extract. However, the persistence of nucleosomes on the template was not RNA polymerase II-specific, but was dependent on another activity present in the nuclear extract. This was demonstrated by addition of the extract to the T7 RNA polymerase transcription reaction, which resulted in retention of the nucleosomal configuration. This nuclear activity, also found in HeLa cell nuclei, is heat sensitive and could not be substituted by nucleoplasmin, chromatin assembly factor (CAF-I) or a combination thereof. Altogether, these results identify a novel nuclear activity, called herein transcription-dependent chromatin stabilizing activity I or TCSA-I, which may be involved in a nucleosome transfer mechanism during transcription.