Developmentally regulated expression of persyn, a member of the synuclein family, in skin

Synucleins constitute a group of unique, evolutionarily conserved proteins that are expressed predominantly in neurons of the central and peripheral nervous system. Although the normal cellular functions of synucleins are not clear, these proteins have been implicated in various neurodegenerative conditions in humans. We found that persyn, a recently characterized member of the synuclein family, is expressed not only in the nervous system but also in the stratum granulosum of the epidermis of neonatal and adult mice. This finding together with our recent observations that persyn influences neurofilament network integrity in sensory neurons raises the possibility that persyn in skin could be involved in modulation of the keratin network.     

Differential intracellular regulation of cortical GABA(A) and spinal glycine receptors in cultured neurons

The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.     

Tamoxifen downregulates TGF-beta production in keloid fibroblasts

BACKGROUND: It has recently been suggested that primary lactase deficiency might have been selected for by malaria, as occurred for beta-thalassaemia and glucose 6-phosphate dehydrogenase deficiency. However, recently we have found that the prevalence of primary lactase deficiency in the area of Sassari (Northern Sardinia), where, in the past, there was intermediate malarial endemicity, is comparable to that observed in the adult population from other areas of Southern Italy where malaria was less endemic. AIMS: To address the problem further, we have determined the prevalence of primary lactase deficiency, glucose 6-phosphate dehydrogenase deficiency deficiency and beta-thalassaemia trait in the populations of three Sardinian villages which differ in altitude above sea-level, socioeconomic features, history of endemic malaria and prevalence of b-thalassaemia and glucose 6-phosphate dehydrogenase deficiency. SUBJECTS: We tested 138 adult males: 53 were from Fonni (a non-malarial mountain village, with a strong pastoral tradition), 38 from Lodé (a village with a similar pastoral tradition, but high malarial endemicity in the past) and 47 from Terralba (a lowland fishing village with an agricultural tradition and heavy malarial morbidity and mortality). METHODS: A blood sample was obtained in all subjects for determination of HbA2 and glucose 6-phosphate dehydrogenase activity. Lactase deficiency was assessed by measuring breath hydrogen production after oral administration of lactose (50 g), by gas-chromatography. RESULTS: The frequencies of glucose 6-phosphate dehydrogenase deficiency and of beta-thalassaemia trait in the non-malarial village of Fonni were strikingly low, compared to frequencies found in the two villages (Terralba and Lodé) with a very high past malarial morbidity. In contrast, there was no significant difference in the prevalence of lactase deficiency in the three groups of subjects from the three villages. CONCLUSIONS: These data obtained in Northern Sardinia do not support the hypothesis of a selection of primary lactase deficiency by malaria. For definitive conclusions, however, the malaria hypothesis should be tested in other parts of the world.     

Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole activity

Human P450 2C19 is selective for 4'-hydroxylation of S-mephenytoin and 5-hydroxylation of omeprazole, while the structurally homologous P450 2C9 has low activity toward these substrates. To identify the critical amino acids that determine the specificity of human amino acids that determine the specificity of human P450 2C19, we constructed chimeras of p450 2C9 replacing various proposed substrate binding sites (SRS) with those of P450 2C19 and then replaced individual residues of P450 2C19 and then replaced individual residues of P450 2C9 by site-directed mutagenesis. The 339 NH2-terminal amino acid residues (SRS-1-SRS-4) and amino acids 160-383 (SRS-2-SRS-5) of P450 2C19 conferred omeprazole 5-hydroxylase activity to P450 2C9. In contract, the COOH terminus of P450 2C19 (residues 340-490 including SRS-5 and SRS-6), residues 228-339 (SRS-3 and SRS-4) and residues 292-383 (part of SRS-4 and SRS-5) conferred only modest increases in activity. A single mutation Ile99 --> His increased omeprazole 5-hydroxylase to approximately 51% of that of P450 2C19. A chimera spanning residues 160-227 of P450 2C19 also exhibited omeprazole 5-hydroxylase activity which was dramatically enhanced by the mutation Ile99 --> His. A combination of two mutations, Ile99 --> His and Ser200 --> Pro, converted P450 2C9 to an enzyme with a turnover number of omeprazole 5-hyrdroxylation, which resembled that of P450 /c19. Mutation of Pro221 --> Thr enhanced this activity. Residue 99 is within SRS-1, but amino acids 220 and 221 are in the F-G loop and outside any known SRS. Mutation of these three amino acids did not confer significant S-mephenytoin 4'-hydroxylase activity to P450 2C9, although chimeras containing SRS-1-SRS-4 and SRS-2-SRS-5 of P450 2C19 exhibited activity toward this substrate. Our results thus indicate that amino acids 99, 220, and 221 are key residues that determine the specificity of P450 2C19 for omeprazole.     

Three-dimensional architecture and gating mechanism of a K+ channel studied by EPR spectroscopy

The transmembrane organization of a potassium channel from Streptomyces lividans has been studied using site-directed spin labeling techniques and electron paramagnetic resonance spectroscopy. In the tetrameric channel complex, two alpha-helices were identified per monomer and assigned to the amino acid sequence. Probe mobility and accessibility data clearly establish that the first helix (TM1) is located in the perimeter of the channel, showing extensive protein-lipid contacts, while the second helix (TM2) is closer to the four-fold symmetric axis of the channel, lining the intracellular vestibule. A large conformational change in the C-terminal end of TM2 was measured when comparing conditions that favor either the open or closed states. The present data suggest that the diameter of the internal vestibule increases with channel opening.     

The choice of the optimal schedules in the vaccination procedure against influenza in elderly subjects

The paper describes a clinical case of the cerebral hyperperfusion syndrome, a rare complication of carotid endarterectomy. The syndrome appeared as the generalized convulsive syndrome in the patient in the early postoperative period. In the context of clinical observation, the results of analysis of the literature are presented and the pathogenesis, diagnosis, therapy, and prevention of the cerebral hyperperfusion syndrome considered.     

Rhesus thymic/liver xenografts in severe combined immunodeficient mice: immunologic reconstitution and intrathymic infection with simian immunodeficiency virus

By serving as host recipients of xenografts from both humans and animals, severe combined immunodeficient (SCID) mice have become valuable to many laboratories interested in examining the pathophysiology of different diseases. To gain insight into the usefulness of the SCID mutation in retrovirus research, rhesus monkey fetal hematolymphoid tissues (liver and thymus) were used to construct a SCID-rhesus chimeric mouse (SCID-rh) and were engrafted in the renal capsule. The size and maturation of the thymic engrafts were monitored grossly, histologically, and immunologically. SCID mice were tolerant to rhesus tissues, and thymic engrafts contained thymocytes at different stages of maturation and differentiation that had morphologic features similar to age-matched rhesus thymus. Mature single positive CD2+, CD4+, and CD8+ T lymphocytes that were phenotypically similar to rhesus T lymphocytes were present at low levels (2% to 5%) in the peripheral blood and at moderately higher levels (7% to 15%) in the spleens of SCID-rh mice obtained between 12 and 15 weeks after thymus/liver engraftment. Within 3 weeks after engraftment, > 85% of the thymocytes in the thymic engrafts were immature double positive CD4+CD8+ T cells. The highest number of positive cells were seen in thymic engrafts obtained at 12 to 18 weeks. During these weeks, > 90% of the cells were double positive (CD2+CD4+, CD2+CD8+, and CD4+CD8+). After infection of the engrafted thymus tissue with simian immonodeficiency virus (SIVmac239), PCR analysis revealed successful viral infection of engrafts at 2 and 4 weeks after infection. No significant histopathologic and flow cytometric changes were observed in the thymic engrafts at 2 and 4 weeks after infection. An unrelated lesion of thymic lymphomas involving the SCID host thymus was seen in 12% of the mice. The data presented herein suggest that the SCID-rh is a valuable model for specific studies related to thymus-retrovirus interaction and that it could be used for further studies. The results are discussed in relation to current knowledge of thymus involvement during simian and human immunodeficiency virus infection.     

Preparation of hydrogel microspheres by coacervation of aqueous polyphosphazene solutions

A new method of preparing ionically cross-linked polyphosphazene hydrogel microspheres which enables effective control over microsphere size distribution has been developed. The synthesized microspheres can be used in protein delivery and, especially, as vaccine delivery vehicles. A new technique utilizes the ability of aqueous solutions of poly[di(carboxylatophenoxy)phosphazene] (PCPP) to form coacervate microdroplets upon addition of sodium chloride. These microdroplets are then stabilized via polymer cross-linking with calcium ions. It was demonstrated that the method enables efficient microencapsulation of proteins. It was also shown that microsphere size can be controlled through the manipulation of microencapsulation conditions. The process is simple, highly reproducible and generates microspheres with a narrower microsphere size distribution, compared to the previous technologies.