Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome)

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.     

Correlation of protein expression, Gleason score and DNA ploidy in prostate cancer

The prognosis of prostate cancer correlates with tumor differentiation. Gleason score and DNA ploidy are two prognostic factors that correlate with prognosis. We analyzed differences in protein expression in prostate cancer of high and low aggressiveness according to these measures. From 35 prostatectomy specimens, 29 cancer samples and 10 benign samples were harvested by scraping cells from cut surfaces. DNA ploidy was assessed by image cytometry. Protein preparations from cell suspensions were examined by 2-DE. Protein spots that differed quantitatively between sample groups were identified by MS fingerprinting of tryptic fragments and MS/MS sequence analysis. We found 39 protein spots with expression levels that were raised or lowered in correlation with Gleason score and/or DNA ploidy pattern (31 overexpressed in high-malignant cancer, 8 underexpressed). Of these, 30 were identified by MS. Among overexpressed proteins were heat-shock, structural and membrane proteins and enzymes involved in gene silencing, protein synthesis/degradation, mitochondrial protein import (metaxin 2), detoxification (GST-pi) and energy metabolism. Stroma-associated proteins were generally underexpressed. The protein expression of prostate cancer correlates with tumor differentiation. Potential prognostic markers may be found among proteins that are differentially expressed and the clinical value of these should be validated.     

Carbon monoxide and hydrogen sulfide: gaseous messengers in cerebrovascular circulation.

This review focuses on two gaseous cellular messenger molecules, CO and H2S, that are involved in cerebrovascular flow regulation. CO is a dilatory mediator in active hyperemia, autoregulation, hypoxic dilation, and counteracting vasoconstriction. It is produced from heme by a constitutively expressed enzyme [heme oxygenase (HO)-2] expressed highly in the brain and by an inducible enzyme (HO-1). CO production is regulated by controlling substrate availability, HO-2 catalytic activity, and HO-1 expression. CO dilates arterioles by binding to heme that is bound to large-conductance Ca2+-activated K+ channels. This binding elevates channel Ca2+ sensitivity, that increases coupling of Ca2+ sparks to large-conductance Ca2+-activated K+ channel openings and, thereby, hyperpolarizes the vascular smooth muscle. In addition to dilating blood vessels, CO can either inhibit or accentuate vascular cell proliferation and apoptosis, depending on conditions. H2S may also function as a cerebrovascular dilator. It is produced in vascular smooth muscle cells by hydrolysis of l-cysteine catalyzed by cystathione gamma-lyase (CSE). H2S dilates arterioles at physiologically relevant concentrations via activation of ATP-sensitive K+ channels. In addition to dilating blood vessels, H2S promotes apoptosis of vascular smooth muscle cells and inhibits proliferation-associated vascular remodeling. Thus both CO and H2S modulate the function and the structure of circulatory system. Both the HO-CO and CSE-H2S systems have potential to interact with NO and prostanoids in the cerebral circulation. Much of the physiology and biochemistry of HO-CO and CSE-H2S in the cerebral circulation remains open for exploration.     

Development of a highly reproducible three-dimensional organotypic model of the oral mucosa

In this report we describe the development of a standardized three-dimensional (3D) system of the human oral mucosa based on an immortalized human oral keratinocyte cell line (OKF6/TERT-2). The procedure takes approximately 2-3 weeks to complete and includes three main stages: preparation of collagen-embedded fibroblasts, addition of the mucosal component and airlifting of cultures to ensure adequate differentiation/stratification. This procedure results in a multilayer epithelial structure in which layers are organized similarly to the cells in native oral mucosa. Specifically, this model system consists of a stratum basale, having one layer of columnar to round cells, a relatively flattened stratum spinosum and stratum granulosum, and a non-keratinizing stratum corneum. This 3D system resembles the commercially available system based on the cell line TR146 (SkinEthic), with the exception that our model system does not contain dyskeratotic changes and has a submucosal component, and thus better represents the normal human mucosa and submucosa.     

Fungal metabolic model for tyrosinemia type 3: molecular characterization of a gene encoding a 4-hydroxy-phenyl pyruvate dioxygenase from Aspergillus nidulans

Mutations in the human HPD gene (encoding 4-hydroxyphenylpyruvic acid dioxygenase) cause hereditary tyrosinemia type 3 (HT3). We deleted the Aspergillus nidulans homologue (hpdA). We showed that the mutant strain is not able to grow in the presence of phenylalanine and that it accumulates increased concentrations of tyrosine and 4-hydroxyphenylpyruvic acid, mimicking the human HT3 phenotype.     

Cell population indexes of spermatogenic yield and testicular sperm reserves in adult jaguars (Panthera onca)

The intrinsic yield of spermatogenesis and supporting capacity of Sertoli cells are the desirable indicators of sperm production in a species. The objective of the present study was to quantify intrinsic yield and the Sertoli cell index in the spermatogenic process and estimate testicular sperm reserves by histological assessment of fragments obtained by testicular biopsy of five adult jaguars in captivity. The testicular fragments were fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 μm thickness. In the seminiferous epithelium of the jaguar, 9.2 primary spermatocytes in pre-leptotene were produced by “A” spermatogonia. During the meiotic divisions only 3.2 spermatids were produced by a primary spermatocyte. The general spermatogenic yield of the jaguar was about 23.4 cells and each Sertoli cell was able to maintain about 19.2 germ cells, 11 of them were round spermatids. In each seminiferous epithelium cycle about 166 million spermatozoa were produced by each gram of testicular tissue. In adult jaguars, the general spermatogenic yield was similar to the yield observed in pumas, greater than that observed for the domestic cat, but less compared to most domestic animals.