Malonyl CoA decarboxylase deficiency: C to T transition in intron 2 of the MCD gene.

Malonyl CoA decarboxylase (MCD) is an enzyme involved in the metabolism of fatty acids synthesis. Based on reports of MCD deficiency, this enzyme is particular important in muscle and brain metabolism. Mutations in the MCD gene result in a deficiency of MCD activity, that lead to psychomotor retardation, cardiomyopathy and neonatal death. To date however, only a few patients have been reported with defects in MCD. We report here studies of a patient with MCD deficiency, who presented with hypotonia, cardiomyopathy and psychomotor retardation. DNA sequencing of MCD revealed a homozygous intronic mutation, specifically a -5 C to T transition near the acceptor site for exon 3. RT-PCR amplification of exons 2 and 3 revealed that although mRNA from a normal control sample yielded one major DNA band, the mutant mRNA sample resulted in two distinct DNA fragments. Sequencing of the patient's two RT-PCR products revealed that the larger molecular weight fragments contained exons 2 and 3 as well as the intervening intronic sequence. The smaller size band from the patient contained the properly spliced exons, similar to the normal control. Western blotting analysis of the expressed protein showed only a faint band in the patient sample in contrast to a robust band in the control. In addition, the enzyme activity of the mutant protein was lower than that of the control protein. The data indicate that homozygous mutation in intron 2 disrupt normal splicing of the gene, leading to lower expression of the MCD protein and MCD deficiency.

Detection and cloning of epidermal zinc-alpha 2-glycoprotein cDNA and expression in normal human skin and in tumors.

Zinc-alpha 2-glycoprotein (Zn alpha 2gp) is almost ubiquitous in body fluids, and its antibody labels the corresponding secretory epithelia. We have found that Zn alpha 2gp is also expressed in human epidermis. We cloned the Zn alpha 2gp cDNA by screening our cDNA library, derived from epidermal keratinocytes, with a probe for prostate Zn alpha 2gp. It had complete nucleic acid sequence homology with that from prostate, including the signal peptide. Just as Zn alpha 2gp expression is higher in more differentiated breast tumors, so in skin tumors the highest mRNA levels occurred in the normal controls, the lowest in basal cell carcinomas (the least differentiated epidermal tumor type), and intermediate levels in squamous cell carcinomas and Merkel cell carcinomas. A similar increase in Zn alpha 2gp gene expression with differentiation was observed when epidermal keratinocytes were cultured in media that varied in cellular maturation potential.

Oral famciclovir for suppression of recurrent genital herpes simplex virus infection in women. A multicenter, double-blind, placebo-controlled trial. Collaborative Famciclovir Genital Herpes Research Group.

OBJECTIVE: To evaluate the efficacy and safety of oral famciclovir in the suppression of genital herpes. METHODS: In this randomized, double-blind, placebo-controlled trial that was performed at 11 university and 9 private ambulatory care referral centers, 375 women who were 18 years of age or older and had a history of 6 or more episodes of genital herpes during 12 of the last 24 months in the absence of suppressive therapy were treated for 4 months with oral famciclovir, 125 mg once daily or twice daily, 250 mg once daily or twice daily, 500 mg once daily, or placebo. The primary outcome measures included the time to first clinically and virologically confirmed recurrences, and safety as measured by clinical laboratory tests and adverse experiences. RESULTS: The median time to first recurrence was 82 days in the placebo group, 114 days in those receiving famciclovir, 125 mg once daily, and more than 120 days in the other treatment groups. When compared with placebo recipients, the time to the first clinical recurrence was significantly prolonged in subjects who received famciclovir, 125 mg twice daily (hazard ratio, 1.8; 95% confidence interval, 1.0-3.0; P = .03), and in those who received famciclovir, 250 mg twice daily (hazard ratio, 3.6; 95% confidence interval, 1.9-6.9; P < .001). Treatment was well tolerated, and there was no evidence of emergence of resistance during or after suppressive famciclovir therapy. CONCLUSIONS: Oral famciclovir, 250 mg, given twice daily for 4 months is an effective, well-tolerated treatment for the suppression of genital herpes in women with frequent recurrences, but single daily doses produced less complete suppression of genital herpes.

Induction of blister-causing antibodies by a recombinant full-length, but not the extracellular, domain of the pemphigus vulgaris antigen (desmoglein 3).

Pemphigus vulgaris (PV) is mediated by autoantibodies to desmoglein 3, the pemphigus vulgaris antigen (PVA). PVA and an extracellular domain of PVA-Ig fusion protein (PV-Ig) can completely adsorb the blister-causing Abs from PV patient sera, suggesting that the extracellular segment of PVA might be sufficient to induce pathogenic Abs. To test this, we immunized rabbits with either PVA or its extracellular domain (EPVA) expressed in insect cells in our laboratory. When Igs were passively transferred from these rabbits into neonatal mice, anti-PVA, but not the anti-EPVA, induced blisters. To understand the basis for their differential pathogenic effects, we examined the properties of these sera. Both sera showed comparable ELISA titers and indirect immunofluorescence reactivity against monkey esophagus, a source of native PVA. Moreover, EPVA, like PVA adsorbed blister-causing Abs from sera of PV patients and rabbits immunized with PVA. In contrast, when IgG preparations were incubated with fura-2-AM (acetyloxymethyl ester)-loaded human keratinocytes in culture, only IgG from anti-PVA serum induced intracellular calcium mobilization. These data showed that PVA but not EPVA can elicit Abs that induced blisters in neonatal mice and mediate intracellular signaling through calcium mobilization.

Human immunodeficiency virus envelope glycoprotein 120 alters sleep and induces cytokine mRNA expression in rats [published errata appear in Am J Physiol 1996 Aug;271(2 Pt 2):section R following table of contents and 1996 Dec;271(6 Pt 3):section R following table of contents].

Sleep is altered during the course of viral infection, including that in which the human immunodeficiency virus (HIV) is the etiologic agent. Alterations in the sleep of HIV-infected individuals occur early in the course of infection, prior to the onset of AIDS. The mechanisms for such alterations in sleep are not known. The HIV envelope glycoprotein 120 (gp120) induces the synthesis and secretion of cytokines that enhance [e.g., interleukin (IL)-1 and tumor necrosis factor] and suppress (e.g., IL-10 and IL-1 receptor antagonist) sleep. We used a well-defined rat model to test the hypothesis that the HIV gp120 alters sleep. Recombinant HIV-1IIIB gp120 was injected intracerebroventricularly (20- 500 ng) into rats prior to dark onset. Sleep-wake behavior was not altered after the 20-ng dose, whereas both non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) were initially enhanced and subsequently suppressed after the 100-ng dose. NREMS was enhanced for 8 h after the 500-ng dose; REMS was not affected by this dose. Brain temperature was not altered by any of the gp120 doses used in this study. In addition, mRNA expression for IL-1 beta and IL-10 was induced in the hypothalamus by gp120; this brain region is crucial for the regulation of sleep. These new data support the hypothesis that altered cytokine concentrations within the central nervous system play a pivotal role in the complex alterations in sleep observed during HIV infection.

Gene expression of markers associated with proliferation and differentiation in human keratinocytes cultured from epidermis and from buccal mucosa.

Normal keratinocytes from epidermis and from buccal mucosa underwent dissimilar stages of differentiation in the same culture medium and responded differently to changes in the composition of the medium. Manifestations of these variations were examined in terms of the expression at the mRNA level (as measured by reverse transcriptase-polymerase chain reaction) of three regulatory genes (cdc2, c-myc, and p53) and five that encode structural proteins (keratins K5, K10 and K13, involucrin, and filaggrin), in three growth-medium formulations. The culture conditions enhanced or retarded maturation; the observed alterations in gene expression correlated with these changes. Except for the proliferation genes, the non-keratinizing buccal mucosa generally responded more weakly than the orthokeratotic epidermis to culture-medium supplementation favouring differentiation. Gene expression in cultured keratinocytes reflected their ability to differentiate in vivo; genes were expressed even when the corresponding protein was not seen in vitro. Although keratin K10 is not prevalent in the buccal mucosa nor keratin K13 in the epidermis, the genes for both were found to be expressed in both tissues.

Presence of interleukin-10 transcripts in human pituitary and hypothalamus.

1. Recent data have shown that interleukin-10 (IL-10) is expressed and acts in mouse pituitary tumor cells and freshly isolated mouse pituitaries. 2. In this study, we show that poly(A+) RNA derived from normal human pituitary and hypothalamus expresses IL-10 message. 3. The majority of transcripts was likely from the pituitary and hypothalamus, and not from lymphocytes in the pituitary and hypothalamic vasculature, since both IL-10 and interferon-gamma mRNA levels, compared to equivalent amounts of RNA from peripheral blood lymphocytes, were much lower. 4. These results indicate that IL-10 may function in human neuroendocrine process as it does in the murine system, thus serving as an important signal molecule for bidirectional communication between the neuroendocrine and the immune systems.

Cytokines modulate terminal differentiation and the expression of predesquamin in cultured keratinocytes.

Predesquamin is a glycoprotein that we have isolated from human epidermis. Its monospecific polyclonal antibody immunolocalizes to the lower stratum corneum. We have previously found that it is not expressed in primary keratinocyte cultures unless both interferon-gamma and serum are added to the culture media. We now show similar results with interleukin-1 alpha substituted for interferon-gamma (but not with retinoic acid). Predesquamin is expressed in media supplemented with both cytokines (with or without serum), accompanied by the observation of larger outer squames and a more mature pattern of desquamation. As both cytokines are naturally present in the epidermis, this suggests that they contribute to desquamation in vivo. The predesquamin expression is demonstrated by Western blots of cell extracts with the specific antibody. Corresponding morphological changes are illustrated by phase-contrast microscopy.