Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Interferon-gamma (IFN-gamma) produced by lesional T cell clones is critical for the induction into G1 of the cell cycle by psoriatic keratinocyte stem cells; however, direct data demonstrating psoriatic lesional T cell subset IFN-gamma expression, and quantitation at a single cell level to calculate in vivo proportions, are lacking. In this study, using flow cytometry of freshly isolated normal and psoriatic lesional T cells from keratome biopsies, we found elevated CD3+, CD4+, and CD8+ T cells in all compartments of psoriatic skin, compared with normals. Using Brefeldin A to induce short-term intracellular accumulation of IFN-gamma in T cells capable of IFN-gamma production, we found that 90% of psoriatic patients have IFN-gamma-producing T cells at a greater proportion of their CD3+ cells than normals, with a mean of 16%+/-3%, as compared with 4%+/-2% in normal epidermis (p = 0.01). Expressed as density in the tissue, the IFN-gamma+ CD3+ cell number in psoriatic epidermis was 97+/-22 per mm2 surface area, as compared with 4.4+/-1.8 per mm2 of normal epidermis (p = 0.002). Thus, the total number of IFN-gamma+CD3+ T cells in the skin of a patient with 20% involvement is estimated to be 3.9 x 10(8). CD4+ and CD8+ IFN-gamma+ T cells were both elevated in psoriatic epidermis (p = 0.04 and p = 0.008, respectively) relative to normal skin. In the dermis, only 44% of patients demonstrated a higher percentage of IFN-gamma-producing T cells than did normals (p = 0.1), possibly indicating dilution, in some patients, by fresh infiltrating T cells. Interleukin-4 was not found by a combination of flow cytometry, reverse transcriptase-polymerase chain reaction, western blot, and immunoprecipitation. In conclusion, a significant portion of lesional T cells in psoriasis are IFN-gamma producing, without interleukin-4. The increased numbers of both IFN-gamma+CD4+ and IFN-gamma+CD8+ T cells indicate that both CD4+ and CD8+ IFN-gamma+ T cells are present in appropriate anatomic locations to sustain the lesional pathology.
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PMID:Identification and quantitation of interferon-gamma producing T cells in psoriatic lesions: localization to both CD4+ and CD8+ subsets. 985 19

Methotrexate is widely used in the treatment of severe psoriasis. However, little is currently known about the mechanisms underlying its therapeutic activity in the skin. Methotrexate has been shown to be carried into cells through the reduced folate carrier (RFC-1). The recent cloning and characterization of the human gene encoding this transmembranal carrier enabled us to investigate RFC-1 gene expression in human skin. Biopsies were obtained from the skin of healthy and psoriatic volunteers. RNA extracted from these biopsies was analyzed by the reverse transcriptase-polymerase chain reaction technique. While RFC-1 gene expression was barely detectable in the uninvolved skin of psoriatic patients and in the skin of healthy volunteers, high levels of RFC-1 transcripts were found in biopsies obtained from psoriatic plaques. To further investigate this pattern of gene expression, we studied skin biopsies by in situ hybridization with a labeled antisense riboprobe specific for the RFC-1 gene. The RFC-1 gene was found to be weakly expressed in the epidermis, in biopsies obtained from the skin of healthy subjects as well as in those from the uninvolved skin of psoriatic patients. In contrast, in biopsies obtained from psoriatic plaques, high levels of RFC-1 gene transcripts were found mostly in the spinous layer of the epidermis. These results suggest the existence of a specific methotrexate carrier in the human epidermis, and may bear relevance to the cutaneous manifestations of methotrexate toxicity.
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PMID:Reduced folate carrier (RFC-1) gene expression in normal and psoriatic skin. 987 34

N-(trifluoromethylphenyl)-2-cyano-3-hydroxy-crotonic acid amide (A77 1726), the physiologically active metabolite of leflunomide, has been described to exert antiproliferative effects in vitro and anti-inflammatory actions in several animal models. Currently, its use is being evaluated in clinical trials in psoriasis, which is characterized by epidermal hyperproliferation and infiltration of inflammatory cells. We studied the effects of A77 1726 on growth and gene expression in cultured epidermal cells by 5-bromo-2'-deoxy-uridine (BrdU) incorporation, reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot hybridizations and flow cytometry. A77 1726 inhibited epidermal proliferation at concentrations above 5 microM after 24 hr. However, the cells were still fully viable at a concentration of 100 microM. The drug caused a dose-dependent reduction in the mRNA level of the type A receptor for the proinflammatory cytokine interleukin-8 (IL-8-RA) and, in contrast, induced gene expression of the receptor for the anti-inflammatory cytokine IL-10 (IL-10R) at the mRNA and protein levels. In addition, the mRNA and protein levels of the p53 gene, which is a negative cell cycle regulator, were up-regulated by A77 1726. These data suggest that A77 1726 exerts its anti-inflammatory action via the modulation of epidermal gene expression.
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PMID:Differential modulation of pro- and anti-inflammatory cytokine receptors by N-(4-trifluoromethylphenyl)-2-cyano-3-hydroxy-crotonic acid amide (A77 1726), the physiologically active metabolite of the novel immunomodulator leflunomide. 1007 46

Activator protein-2 is an important transcription factor for the activation of a number of genes. Here we report the induction of activator protein-2 in response to inflammatory cytokines such as interleukin-6 in keratinocytes. Immunoblotting and semiquantitative reverse transcriptase-polymerase chain reaction assays using normal human keratinocytes revealed that interleukin-6 caused a time- and concentration-dependent induction of activator protein-2 mRNA and protein. The increase of activator protein-2 mRNA was detected at 30 min after stimulation and that of activator protein-2 protein was at 2 h. Their levels were lower than the control levels at 24 h. The interleukin-6-dependent induction of activator protein-2 mRNA was completely blocked by adding actinomycin D, whereas it was approximately 50% affected by cycloheximide. Co-incubation with neutralizing antibodies against various inflammatory cytokines resulted in inhibition of the interleukin-6-dependent activator protein-2 induction at varying degrees, indicating an involvement of various cytokines in the activator protein-2 induction. The activator protein-2 induction was observed in keratinocytes derived from lesional skins with psoriasis or squamous cell carcinoma, and the high levels of activator protein-2 were histochemically detected in these lesions. Furthermore, a gel mobility shift assay using the nuclear extracts from interleukin-6-treated cells showed that interleukin-6 induced the functional activator protein-2 protein for the gene activation. These findings suggest a possible regulation mechanism of activator protein-2 through a complex cytokine system, which is conceivably the initial reaction leading to skin inflammation, and resultant keratinocyte growth and carcinogenesis.
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PMID:Induction of transcription factor AP-2 by inflammatory cytokines in human keratinocytes. 1050 47

The present study was designed to investigate by using enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction methods whether photochemotherapy (PUVA) or ultraviolet (UV) B treatment affects C3 production by interferon (IFN)-gamma-stimulated keratinocytes cultured in serum-free medium. The results showed that PUVA and UVA reduced C3 production by IFN-gamma-stimulated epidermal keratinocytes dose-dependently, although the effect of PUVA was stronger than that of UVA alone. Interestingly, UVB induced an enhancement of C3 production at doses ranging from 10 to 50 mJ cm-2. This phenomenon was found at both the protein and mRNA levels. In every experiment, changes in C3 mRNA levels preceded those in its protein levels. Reduced C3 production at higher doses of 75 and 100 mJ cm-2 were probably due to cytotoxic effects of UVB. In our experimental system, PUVA, UVA or UVB treatment did not affect C3 production without IFN-gamma stimulation. Our results suggest that a reduction in C3 production by PUVA treatment may in part explain the efficacy of PUVA in the treatment of inflammatory dermatoses such as psoriasis, while the results of the UVB experiments may partially explain the proinflammatory nature of UVB.
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PMID:Ultraviolet B radiation exerts enhancing effects on the production of a complement component, C3, by interferon-gamma-stimulated cultured human epidermal keratinocytes, in contrast to photochemotherapy and ultraviolet A radiation that show suppressive effects. 1079 15

Degradation of angiogenic mediators might be an underlying cause of chronic wounds. To test this hypothesis, we evaluated the expression and integrity of vascular endothelial growth factor, a potent angiogenic mediator, and its receptors, Flt-1 and KDR, in chronic venous leg ulcerations. Immunohisto- chemical, in situ hybridization, and semiquantitative reverse transcriptase polymerase chain reaction analyses all indicate that expression of vascular endothelial growth factor is elevated in ulcerative tissue, with vascular endothelial growth factor mRNA being especially pronounced in the hyperplastic epithelium of the wound margin. Flt-1 and KDR protein and mRNA were detected in the papillary vessels in close vicinity to the lesional epithelium of chronic wounds. Although increased expression of vascular endothelial growth factor protein was detected in the epidermis, the intensity of this staining was weak compared with the epidermal staining in psoriatic lesions and compared with the strong vascular endothelial growth factor mRNA signal in chronic wounds and psoriasis. To analyze whether this apparent decrease in immunoreactivity could be the result of degradation of vascular endothelial growth factor by proteolytic activities from the wound environment, we examined the stability of recombinant vascular endothelial growth factor in wound fluid from chronic leg ulcers. As demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, incubation of rVEGF165 with chronic, but not acute, wound fluid resulted in rapid proteolytic degradation of rVEGF165. Protease inhibitor studies indicate that serine proteases, such as plasmin, are involved in this degradation. Together, our data show that, although vascular endothelial growth factor expression is elevated in chronic wounds, increased proteolytic activity in this environment results in its degradation, which may contribute to an impaired wound healing response.
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PMID:Expression and proteolysis of vascular endothelial growth factor is increased in chronic wounds. 1088 1

In psoriasis an etiopathogenetic vicious circle is nowadays hypothesized that the disease is triggered by skin-specific autoantigen structures, the expression and accessibility of which are positively correlated with the intensity of the hyperproliferation and inflammation in the epidermopapillary compartment driven by autoreactive T cells. Despite the close microanatomical relation between skin and mucosa, clinicians have always been intrigued by the observation that psoriatic affection of the mucosa, if at all existing, is only seen as very rare events in the lips and tongue sparing buccopharyngeal sites. This prompted us to establish an experimental model system comparing psoriatic-involved skin and peritonsillar mucosa from tonsillectomies by a reverse transcriptase-polymerase chain reaction/differential display strategy. Among more than 60 cDNA species to be displayed in psoriasis, but missing in peritonsillar mucosa, one species was identified as coding for the RNA polymerase IIA seventh subunit (hsRPB7 gene) as a most critical factor for DNA to RNA transcription. Immunohistochemistry showed a hitherto unknown, distinctive pattern of hsRPB7 expression that was 1) tissue type-dependent with a surplus in skin keratinocytes and a near absence in peritonsillar mucosa, 2) tightly regulated by the keratinocyte differentiation process with a sharp suprabasal up-regulation in contrast to a basal down-regulation, and 3) substantially augmented in psoriatic-involved skin as compared to normal and psoriatic uninvolved skin. Keratinocytes of actinic keratoses also showed a strong hsRPB7 expression that however did not strictly spare the basal cell layer presumably reflecting the disturbed intraepidermal stratification because of the premalignant status of these precancerous lesions.
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PMID:Suprabasal overexpression of the hsRPB7 gene in psoriatic epidermis as identified by a reverse transcriptase-polymerase chain reaction differential display model comparing psoriasis plaque tissue with peritonsillar mucosa. 1115 73

Despite the various responses of human skin to female sex hormones, cellular and subcellular targets and the mechanisms of action of estrogen and progesterone in human skin are not well understood. The detection of estrogen receptor (ER) and progesterone receptor (PR) in the skin is of great importance to understand the effect of estrogen and progesterone. In primary cultures of human keratinocytes, expression of ER and PR was monitored by immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). Paraffin embedded skin tissues were stained with monoclonal antibodies to human ER and PR by immunohistochemistry. Cultured human keratinocytes expressed cytoplasmic PR protein and PR mRNA transcripts. By contrast, ER was detected only at the mRNA level. Suprabasal keratinocytes from samples of pruritic urticarial papules, plaques of pregnancy (PUPPP) and psoriasis were stained positively only for PR, while those from samples of erythema nodosum were negative for both ER and PR. Lesional epidermis of PUPPP showed positive PR immunoreactivity, while nonlesional epidermis did not. No other cells in the normal human skin were stained with ER and PR. The present study suggests that by expressing PR human keratinocytes act as targets for progesterone action.
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PMID:Expression of progesterone receptor in human keratinocytes. 1119 91

Ceramides are the most abundant lipids constituting the intercellular matrix of the skin stratum corneum and their critical role in skin homeostasis has been extensively documented. Their concentration in the skin highly depends on the rate of availability of the enzymes involved in ceramide generation. The aim of this study was to investigate whether the concentration of prosaposin was altered in the skin of patients with psoriasis vulgaris. Prosaposin, the precursor of saposins (sphingolipid activator proteins), was measured in lesional and nonlesional skin of psoriatic patients and in normal skin from surgical patients, both at the mRNA and at the protein level. Densitometric analysis of reverse transcriptase-polymerase chain reaction bands separated by gel-electrophoresis showed a progressive decrease of prosaposin mRNA expression in nonlesional and lesional psoriatic skin, being substantially decreased in lesional psoriatic skin compared with normal control skin. Immunohistochemical analysis showed a significant decrease of prosaposin level in the stratum corneum of psoriatic lesional skin (both in active-type and in chronic-type plaque) compared with nonlesional and with normal skin (p < 0.01), and in psoriatic nonlesional skin compared with normal control (p < 0.05). Immunolocalization of sphingomyelinase in lesional and nonlesional psoriatic skin showed a decrease in the level of this enzyme in the stratum corneum of psoriatic lesional, compared with nonlesional skin. These results support the concept that disturbance of epidermal barrier function caused by derangement in ceramide generation can be crucial for the development of psoriatic skin diseases.
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PMID:The level of prosaposin is decreased in the skin of patients with psoriasis vulgaris. 1123 13

Zidovudine (AZT) is a thymidine analogue which inhibits retroviral reverse transcriptase, terminates DNA chain synthesis, and thus inhibits viral replication. The clinical benefit of AZT patients with advanced HIV disease was established in a phase II placebo-controlled study. Administered at dosages of 500 mg per day, AZT benefits asymptomatic patients with CD4 counts below 500, slows progression to AIDS, and increases survival. It has therefore become the major antiviral drug for treating HIV infection. Psoriasis is a common papulosquamous disease affecting 1-2% of the general population. It may also be the initial symptom of HIV infection, potentially very severe and difficult to treat with conventional therapy in such patients. The presence of psoriasis contributes significantly to AIDS morbidity and may be even more disabling than Kaposi's sarcoma. Researchers have, however, reported in the Archives of Dermatology the complete clearing of psoriasis in two patients several weeks after they began AZT therapy for AIDS. 33% of patients had a complete clearing of disease symptoms, while 90% had a partial improvement. A more than 75% reduction in body surface involvement was observed in 47% of patients. Improvement during AZT therapy was correlated with the presence of antigenemia and an increase in mean white blood cell count. While other forms of therapy for severe psoriasis have resulted in profound immunosuppression in some patients with AIDS, may activate HIV, or may be poorly tolerated in patients, AZT therapy seems safe and effective for ameliorating or clearing psoriasis in HIV-infected individuals at dosages of 1200 mg per day. The effectiveness of the drug at lower doses has not been established. Information is also lacking on what effect the early administration of AZT in asymptomatic HIV patients may have on the eventual expression of psoriasis.
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PMID:Zidovudine improves psoriasis in HIV-positive males. 1217 18


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