Cloning and characterization of retinol dehydrogenase transcripts expressed in human epidermal keratinocytes.

The normal growth and differentiation of the epidermis require an adequate supply of vitamin A. The active form of vitamin A for normal epidermal homeostasis is retinoic acid (RA). Retinoic acid controls the expression of retinoid-responsive genes via interactions of the retinoic acid/nuclear receptor complexes at specific DNA sequences in their control regions. The message conveyed by RA is likely modulated by the concentration of the ligand available for binding to the receptors. Following the uptake of plasma retinol, epidermal keratinocytes synthesize retinoic acid via two sequential reactions with retinaldehyde as an intermediate. Several retinol dehydrogenase (RDH) enzymes, members of the short-chain dehydrogenase/reductase (SDR) gene superfamily, catalyze the first and rate-limiting step that generates retinaldehyde from retinol bound to cellular retinol-binding protein (holo-CRBP). However, little is known about these enzymes and their genes in the epidermal cells. Our work describes the first member of the RDH family found in epidermis. We show that this gene is expressed predominantly in the differentiating spinous layers and that it is under positive, feed-forward regulation by retinoic acid. It encodes a protein that, using NAD+ as a preferred cofactor, utilizes free and CRBP-bound all-trans-retinol and steroids as substrates.     

Cimaterol-induced growth in rats: growth pattern and biochemical characteristics

Sixty Sprague-Dawley female rats weighing 170 g were used to investigate the effect of cimaterol on the growth pattern and biochemical characteristics of skeletal muscles. Cimaterol (CIM) was mixed in a powdered rat chow at 10 ppm. A significant (P less than .01) improvement in the weight gain of CIM group was observed during the first 8 days of the 49-day trial period, followed by no significant improvement thereafter. When CIM was withdrawn from the diet, treated rats lost weight, eventually regressing to the same weight as control rats after 28 days. CIM-fed rats showed 28.6% (P less than .01) and 12.9% (P less than .05) increases in plantaris muscle weight over the control at 8 and 14 d, respectively. DNA concentration significantly (P less than .05) decreased, whereas RNA concentration increased at 3 d (31%) and 8 d (12.7%), but decreased at 14 d compared to the control. The increase in RNA concentration preceded the significant improvement of muscle weight in the CIM group. In the soleus muscle, there was no significant difference in muscle weight or DNA concentration between the control and CIM groups. Cimaterol-feeding significantly (P less than .05) increased the size of both type I and II fibers. The size increase of type II fibers in plantaris was twice that of type I fibers in soleus (34.2% vs 17.5%). This may explain why the plantaris muscle (type II predominant) showed a greater weight increase than the soleus muscle (type I predominant).     

Glucose-6-phosphate dehydrogenase deficiency in Sicily. Incidence, biochemical characteristics and clinical implications

This report deals with the incidence, type and clinical implications of G6PD deficiency in Sicily. Of 3347 male subjects examined, 56 were deficient in G6PD. They were distributed throughout the island. The G6PD levels in RBC were almost zero; in leukocytes, platelets and saliva they were found to be 26%, 18% and 16%, respectively, of controls. The Michaelis constant for NADP and G6PD was lower than for controls. Conversely, the utilization of the analogous Ga16P and 2dG6P was higher. The thermostability of the enzyme was lower and the pH optima (6.5 and 9.5) were different from the controls. An identical electrophoretic pattern was found both in normal and deficient subjects. This pattern is superimposable on that described as Mediterranean variant. The analysis among 270 subjects admitted to our Clinic with hemolysis due to G6PD deficiency demonstrated that the most frequent disease is favism, followed by neonatal jaundice, while hemolysis due to drugs is very rare. Ingestion of fresh fava beans was the most frequent cause of favism, but cases occurred after breast feeding and inhalation of pollen.     

Long-chain L-3-hydroxyacyl-coenzyme a dehydrogenase deficiency: a molecular and biochemical review

Since the first report of long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase deficiency a little more than a decade ago, its phenotypic and genotypic heterogeneity in individuals homozygous for the enzyme defect has become more and more evident. Even more interesting is its association with pregnancy-specific disorders, including preeclampsia, HELLP syndrome (hemolysis, elevated liver enzymes, low platelets), hyperemesis gravidarum, acute fatty liver of pregnancy, and maternal floor infarct of the placenta. In this review we discuss the biochemical and molecular basis, clinical features, diagnosis, and management of long-chain L-3-hydroxyacyl-coenzyme A dehydrogenase deficiency.     

Expression of the murine retinol dehydrogenase 10 (Rdh10) gene correlates with many sites of retinoid signalling during embryogenesis and organ differentiation.

Retinoic acid acts as a signalling molecule regulating many developmental events in vertebrates. As this molecule directly influences gene expression by activating nuclear receptors, its patterns of synthesis have to be tightly regulated, and it is well established that at least three retinaldehyde dehydrogenases (RALDHs) are involved in such tissue-specific synthesis. Whereas embryos from oviparous species can obtain retinaldehyde by metabolizing carotenoids stored in the yolk, placental embryos rely on retinol transferred from the maternal circulation. Here, we show that the gene encoding one of the murine retinol dehydrogenases, Rdh10, is expressed according to complex profiles both during early embryogenesis and organ differentiation. Many of its expression sites correlate with regions of active retinoid signalling and Raldh gene expression, especially with Raldh2 in the early presomitic and somitic mesoderm, retrocardiac and posterior branchial arch region, or later in the pleural mesothelium and kidney cortical region. Rdh10 also shows cell-type and/or regional specificity during development of the palate, teeth, and olfactory system. During limb bud development, it may participate in retinoic acid production in proximal/posterior cells, and eventually in interdigital mesenchyme. These data implicate the retinol to retinaldehyde conversion as the first step in the tissue-specific regulation of retinoic acid synthesis, at least in mammalian embryos.     

Biomechanical and biochemical characteristics of a human fibroblast-produced and remodeled matrix

We report on a culture method for the rapid production of a strong and thick natural matrix by human cells for tissue engineering applications. Dermal fibroblasts were cultured for three weeks at high density on porous substrates in serum-containing or chemically defined media. The mechanical and biochemical properties of the resulting cell-derived matrix (CDM) were compared to those of standard fibroblast-populated collagen and fibrin gels and native human skin. We found that the ultimate tensile strength of CDM cultured in our chemically defined media (313+/-8.7 kPa) is significantly greater than for collagen gels (168+/-39.3 kPa), fibrin gels (133+/-8.0 kPa) and CDM cultured with serum (223+/-9.0 kPa), but less than native skin (713+/-55.2 kPa). In addition to the biomechanics, this *CDM is also biochemically more similar to native skin than the collagen and fibrin gels in terms of all parameters measured. As *CDM is produced by human cells in a chemically defined culture medium and is mechanically robust, it may be a viable living tissue equivalent for many connective tissue replacement applications requiring initial mechanical stability yet a high degree of biocompatibility.     

Lobular distribution pattern of lactate dehydrogenase and 6-phosphogluconate dehydrogenase activity in rat liver

Lactate dehydrogenase (LDH) and 6-phosphogluconate dehydrogenase (6-PGDH) activities were measured in lobular areas expanding between 3 portal tracts and an efferent central vein in the livers of male Wistar rats, using a Lowry technique. The maximum of LDH activity was found in a nearly uniform broad area in the lobular periphery. From that area values decreased along periportal/septal-->perivenous gradients, but only slightly within that area along the periportal-->septal axis of the vascular septum. Maximum values of 6-PGDH activity were present in an intermediate area close to the central vein demonstrating a rather inhomogeneous distribution pattern without a clear definition of zonal limits. Our data on the distribution pattern of LDH are in agreement with the concept of the metabolic lobulus and are supported by a recent evaluation of the vascular architecture in rat liver. The lobular distribution pattern of 6-PGDH cannot be interpreted without doubt in accordance with that concept.     

Functional and biochemical characteristics of a murine interleukin 2 receptor-inducing factor

High density (resting) murine Lyt-2+ T cells exposed in vitro to the ligand concanavalin A (Con A) remain interleukin 2 (IL 2) unresponsive, i.e. do not express functional IL 2 receptors, unless reconstituted with accessory cells. This finding provides a bio-assay to define functional and biochemical characteristics of an IL 2 receptor-inducing factor (RIF). RIF bioactivity as secreted from the macrophage cell line P388-D1 is associated with a trypsin-sensitive protein of 44 kDa which does not need to be glycosylated and which binds to and can be eluted from hydroxylapatite and phenyl-Sepharose. While both RIF and IL 1 are produced by accessory cells the lymphokines separate from each other according to functional and biochemical criteria. Either accessory cells, RIF or the protein kinase C activator phorbol myristate acetate can substitute for each other and are equally active for the induction of IL 2 responsiveness in high-density Lyt-2+ T cells exposed to Con A. To explain these results we conclude that in the mitogen system used, induction of IL 2 responsiveness (activation) represents a two-step event in which first cross-linking of cell surface structures by the ligand Con A excites the responder T cells, which subsequently respond to the accessory cell product RIF.     

Clinical and biochemical outcomes of patients with medium-chain acyl-CoA dehydrogenase deficiency

BackgroundMedium-Chain Acyl-CoA Dehydrogenase (MCAD) deficiency is a fatty acid oxidation disorder that can have variable clinical severity. There is still limited information on its clinical presentation and longitudinal history by genotype, and effectiveness of newborn screening (NBS).MethodsRetrospective data were collected from 90 patients (44 female, 46 male) to compare biochemical data with clinical outcomes. The frequency of adverse events (number of hypoglycemia-related ER visits and admissions) was assessed by genotype (homozygosity or not for the common pathogenic variant, p.Lys329Glu, in the ACADM gene), and method of diagnosis (NBS vs. clinical).ResultsMCAD deficiency in Utah was more frequent compared to the United States average (1: 9266 versus 1:17,759 newborns). With age, C8-carnitine did not change significantly whereas C2-carnitine decreased (p < .001), possibly reflecting reduced carnitine supplementation typically seen with age. Children with MCAD deficiency had normal growth. p.Lys329Glu homozygotes had higher NBS C8-carnitine (23.4 ± 19.6 vs. 6.6 ± 3.0 μmol/L) and lifetime plasma C8-carnitine levels (6.2 ± 5 vs. 3.6 ± 1.9 μmol/L) compared to patients with at least one other pathogenic variant (p < .001 for both) and higher transaminases compared to compound heterozygotes (ALT 41.9 ± 6.2 vs. 31.5 ± 3.7 U/L, AST 63.9 ± 5.8 vs. 45.7 ± 1.8 U/L, p < .05 for both). On average, p.Lys329Glu homozygotes had more hypoglycemic events than compound heterozygotes (1.44 versus 0.49 events/patient) as did patients diagnosed clinically compared to those diagnosed by NBS (2.15 versus 0.62 events/patient), though these differences were not statistically significant. Neonatal death was observed before results of newborn screening were available in one patient homozygous for the common p.Lys329Glu pathogenic variant, but severe neonatal complications (hypoglycemia, cardiac arrhythmia) were also seen in patients with other mutations. No irreversible complications were observed after diagnosis in any patient with MCAD deficiency.DiscussionHomozygosity for the common ACADM p.Lys329Glu pathogenic variant was associated with increased levels of C8-carnitine and transaminases. Newborn screening provides the opportunity to reduce morbidity and post-neonatal mortality in all patients with MCAD deficiency, regardless of genotype.     

表皮干细胞的生物学特性及应用

背景：尽早去除创面坏死组织、重建皮肤结构和功能,是大面积深度烧伤治疗的关键和最终目标。表皮干细胞作为皮肤组织特异性干细胞,拥有无可置疑的潜能。 目的：总结表皮干细胞的生物学特性及应用现状。 方法：应用计算机检索2000-01/2010-10 PubMed数据库相关文章,检索词“epidermal stem cells,basic study”,并限定文章语言种类为English。同时计算机检索2005-01/2010-10万方数据库相关文章,检索词为“表皮干细胞”,并限定文章语言种类为中文。共检索到文献271篇,最终纳入符合标准的文献38篇。 结果与结论：表皮干细胞是具有多向分化潜能、自我更新能力和高度增殖能力的细胞,具有慢周期性和对基底膜的黏附能力,其增殖分化受到壁龛及Wnt信号通路、MAPK、c-Myc、Notch信号传导通路、细胞因子等的调控。目前尚无公认的特异性标志物,可用于创面修复、基因治疗、组织工程领域。随着表皮干细胞研究的深入,将为皮肤基础研究、创面功能修复和皮肤遗传病的治疗提供新途径。     

Chromosome assignment, biochemical and immunological studies on a human aldehyde dehydrogenase, ALDH3

The biochemical properties of ALDH isozymes have been examined in human tissues and one set, designated ALDH3, has been studied in detail. These components occur at highest levels in lung and stomach, but were not expressed in fetal tissues, or in blood, hair roots and fibroblasts. The ALDH3 isozymes show optimal activity with benzaldehyde and can use either NAD or NADP as cofactor. Antiserum against a partially purified ALDH3, from stomach, selectively precipitates this isozyme from human tissues and selectively recognizes an homologous component in the rat. Human and rodent ALDH3 were not immunoprecipitated by anti-ALDH1 or anti-ALDH2 antisera. High levels of expression were found in human-rodent hybrids, constructed using rat hepatoma cells, and these hybrids were used to assign the human ALDH3 gene to chromosome 17.     

Equine biochemical multiple acyl-CoA dehydrogenase deficiency (MADD) as a cause of rhabdomyolysis

Two horses (a 7-year-old Groninger warmblood gelding and a six-month-old Trakehner mare) with pathologically confirmed rhabdomyolysis were diagnosed as suffering from multiple acyl-CoA dehydrogenase deficiency (MADD). This disorder has not been recognised in animals before. Clinical signs of both horses were a stiff, insecure gait, myoglobinuria, and finally recumbency. Urine, plasma, and muscle tissues were investigated. Analysis of plasma showed hyperglycemia, lactic acidemia, increased activity of muscle enzymes (ASAT, LDH, CK), and impaired kidney function (increased urea and creatinine). The most remarkable findings of organic acids in urine of both horses were increased lactic acid, ethylmalonic acid (EMA), 2-methylsuccinic acid, butyrylglycine (iso)valerylglycine, and hexanoylglycine. EMA was also increased in plasma of both animals. Furthermore, the profile of acylcarnitines in plasma from both animals showed a substantial elevation of C4-, C5-, C6-, C8-, and C5-DC-carnitine. Concentrations of acylcarnitines in urine of both animals revealed increased excretions of C2-, C3-, C4-, C5-, C6-, C5-OH-, C8-, C10:1-, C10-, and C5-DC-carnitine. In addition, concentrations of free carnitine were also increased. Quantitative biochemical measurement of enzyme activities in muscle tissue showed deficiencies of short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and isovaleryl-CoA dehydrogenase (IVD) also indicating MADD. Histology revealed extensive rhabdomyolysis with microvesicular lipidosis predominantly in type 1 muscle fibers and mitochondrial damage. However, the ETF and ETF-QO activities were within normal limits indicating the metabolic disorder to be acquired rather than inherited. To our knowledge, these are the first cases of biochemical MADD reported in equine medicine.     

Expression of epidermal growth factors and epidermal growth factor receptor in normal cycling human ovaries

Immunolocalization of transforming growth factor- (TGF), epidermalgrowth factor (EGF), cripto-1, amphireg-ulin and epidermal growthfactor receptor (EGFR ) was studied in 51 premenopausal humanovaries at various phases of the menstrual cycle. Localizationof mRNA for TGF and EGF was also studied by in-situ hybridization.Immunoreactive TGF was observed predominantly in theca cellsin 12 of 33 antral follicles in the follicular phase (6/14 dominantfollicles, and 6/19 non-dominant) but not in any of the 18 folliclesin the luteal phase or in primordial and pre-antral follicles.TGF immunoreactivity was present predominantly in the luteinizedgranulosa cells in 13 of 15 corpora lutea in the luteal phase,which are considered to be active in steroidogenesis, but notin any of the regressed corpora lutea. Accumulation of TGF mRNAhybridization signal was observed only in the theca cells inthe follicles and luteinized theca cells in the ovaries thatwere immunohistochemically positive for TGF. EGFR immunoreactivitywas detected in 24 of 33 antral follicles in the follicularphase and in two of 18 follicles in the luteal phase but notin any of the corpora lutea. Immunoreactive EGF, cripto-1 andamphiregulin or EGF mRNA was not detected in any follicles,corpora lutea, or the stroma cells examined. These results indicatethat, of the epidermal growth factors examined in this study,TGF is locally synthesized in normal cycling human ovaries andTGF may be synthesized in theca cells and act on the granulosacells in a paracrine fashion through the EGFR in ovarian follicles. EGF family/human/immunohistochemistry/in-situ hybridization/ovary     

Expression pattern and functional characteristics of two novel splice variants of the two-pore-domain potassium channel TREK-2

Two novel alternatively spliced isoforms of the human two-pore-domain potassium channel TREK-2 were isolated from cDNA libraries of human kidney and fetal brain. The cDNAs of 2438 base pairs (bp) (TREK-2b) and 2559 bp (TREK-2c) encode proteins of 508 amino acids each. RT-PCR showed that TREK-2b is strongly expressed in kidney (primarily in the proximal tubule) and pancreas, whereas TREK-2c is abundantly expressed in brain. In situ hybridization revealed a very distinct expression pattern of TREK-2c in rat brain which partially overlapped with that of TREK-1. Expression of TREK-2b and TREK-2c in human embryonic kidney (HEK) 293 cells showed that their single-channel characteristics were similar. The slope conductance at negative potentials was 163 ± 5 pS for TREK-2b and 179 ± 17 pS for TREK-2c. The mean open and closed times of TREK-2b at −84 mV were 133 ± 16 and 109 ± 11 μs, respectively. Application of forskolin decreased the whole-cell current carried by TREK-2b and TREK-2c. The sensitivity to forskolin was abolished by mutating a protein kinase A phosphorylation site at position 364 of TREK-2c (construct S364A ) . Activation of protein kinase C (PKC) by application of phorbol-12-myristate-13-acetate (PMA) also reduced whole-cell current. However, removal of the putative TREK-2b-specific PKC phosphorylation site (construct T7A) did not affect inhibition by PMA. Our results suggest that alternative splicing of TREK-2 contributes to the diversity of two-pore-domain K⁺ channels.     

Expression of a stage-specific lipophosphoglycan in Leishmania major amastigotes.

Amastigotes of Leishmania major were isolated from infected mice and radiolabeled for 2 h with [3H]galactose. An acidic [3H]glycoconjugate was extracted from a dilipidated residue fraction with the solvent water/ethanol/diethylether/pyridine/NH4OH (15:15:5:1:0.017). The radioactivity labeled glycoconjugate was found to possess the following characteristics that were similar to the lipophosphoglycan extractable from promastigotes: (i) migrated as a broad band upon electrophoresis on SDS polyacrylamide gels; (ii) deaminated with nitrous acid; and (iii) hydrolyzed with phosphatidylinositol-specific phospholipase C. Furthermore, analysis of the aqueous soluble material released by the latter enzyme revealed a negatively-charged [3H]polysaccharide intermediate in size compared to the analogous portions of LPG isolated from non-infective and metacyclic promastigotes. Most importantly, the [3H]polysaccharide was found to contain phosphate and was susceptible to mild acid hydrolysis, establishing that the intact molecule is a lipophosphoglycan. A structural difference, however, was found in the major, mild acid-generated fragment of the amastigote phosphoglycan, which was larger in size and not as anionic as the analogous fragment from the promastigote phosphoglycans. These results indicate that the amastigotes do express a lipophosphoglycan, but that it is structurally distinct from its promastigote counterparts.     

Aspartylglucosaminuria: Unique biochemical and ultrastructural characteristics

The observation of vacuolated lymphocytes in a coarsely featured two year old female with hepatosplenomegaly, mitral insufficiency, and mild psychomotor retardation led to the first diagnosed case of aspartylglucosaminuria in the United States. Although physical characteristics and bone roentgenograms were consistent with a mucopolysaccharide disorder, analysis of the urine showed no mucopolysaccharide elevation. The chromatographic, enzymatic, and ultrastructural studies confirming the diagnosis are presented.