Gene sequence tags from Plasmodium falciparum genomic DNA fragments prepared by the "genease" activity of mung bean nuclease.

A genes-first approach to genome sequencing is described which efficiently generates gene sequence tags from genomic DNA. Mung bean nuclease (EC 3.1.30.1) cleaves the genomic DNA of many organisms before and after genes and within some introns. Analysis of gene sequence tags prepared from mung bean nuclease-digested Plasmodium falciparum DNA demonstrates that this method has several advantages over the popular cDNA expressed sequence tag approach. To date, 673 sequence tags containing over 215 kb of sequence have been generated from 400 clones. Sixty clones (15%) have significant similarity to sequences in the protein and translated nucleic acid data bases. These represent 51 unique genes, of which only 5 encode previously known P. falciparum proteins. The identified proteins include those expressed in erythrocytic, exoerythrocytic, and gametocytic stages of the parasite. Thirty percent of clones identified appear to carry complete coding regions. The spacer DNA separating genes is rarely cloned. These gene sequence tags will form a useful data base from which to initiate projects to develop new therapeutics, vaccines, and strategies to control human malaria.     

Eleven novel mutations in the factor VIII gene from Brazilian hemophilia A patients

The molecular characterization of the mutations in hemophilia A patients is hampered by the large size of the factor VIII gene and the great heterogeneity of mutations. In this study, we have performed a protocol involving multiplex polymerase chain reaction in which 19 exons were amplified in four different combinations followed by nonradioactive single-strand conformational polymorphism (SSCP) to screen for mutations. Southern blotting was used to detect inversion of the factor VIII gene resulting from recombination between copies of the gene A (F8A) located in intron 22 of the factor VIII gene and two copies close telomeric region of X chromosome. Forty-two hemophilia A patients (21 with severe and 21 with mild-to-moderate disease) were studied. The inversion of factor VIII occurred in 13 of 21 patients affected by severe hemophilia A. One patient showed a large extra band in addition to the three bands observed after Southern blotting with the F8A probe. An abnormal electrophoretic pattern of SSCP was detected in 85% and 50% of the patients affected by mild-to-moderate and severe disease, respectively. Sixteen different mutations were identified. Eleven mutations were novel and comprised 9 point mutations and 2 small deletions. This study shows that the methodology used is safe and rapid and has potential for detecting almost all of the genetic defects of the studied hemophilia A patients.     

Endothelin-mediated remodeling in aortas of diabetic rats

BACKGROUND: Smooth muscle cells proliferation and extracellular matrix (ECM) protein deposition are key features of diabetic macroangiopathy. In the present study, we have studied the role of endothelin(A) (ET(A)) receptor, the predominant receptor on smooth muscle cells, in diabetes-induced vascular hypertrophy and remodeling. METHODS: Streptozotocin-induced diabetic rats were administrated a selective ET(A) receptor antagonist, TBC3214, for 26 weeks. Following treatment, aortas were harvested and subjected to gene expression and morphometric analyses. We quantified fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) expression as indicators of increased ECM protein synthesis. ET-1, ET-3, transforming growth factor-beta1 (TGF-beta1) and angiotensinogen mRNA levels were measured to elucidate genes involved in FN expression. We have investigated an embryonic splice variant of FN, oncofetal FN, and nonmuscle myosin heavy chain (SMemb) as vascular remodeling indicators. RESULTS: Our results show that diabetes leads to upregulation of FN, PAI-1, ET-1, ET-3, TGF-beta1 and angiotensinogen mRNA levels in association with increased medial thickness. Immunohistochemical analyses revealed concurrent protein level changes. Diabetes also upregulated oncofetal FN and SMemb mRNA levels. Treatment with TBC3214 attenuated the mRNA levels of several genes and prevented increased medial thickness. CONCLUSIONS: These results indicate that diabetes-induced vascular hypertrophy and remodeling is associated with reexpression of embryonic forms of FN and myosin heavy chain. Such changes are ET-dependent and may be mediated via TGF-beta1 and angiotensin.     

In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation

A novel nonmyeloablative conditioning regimen was investigated in 44 patients with hematologic malignancies. The median patient age was 41 years. Many of the patients had high-risk features, including 19 patients with a previous failed transplant. Recipient conditioning consisted of CAMPATH-1H, 20 mg/day on days -8 to -4; fludarabine, 30 mg/m(2) on days -7 to -3; and melphalan, 140 mg/m(2) on day -2. Thirty-six recipients received unmanipulated granculocyte colony-stimulating factor-mobilized peripheral blood stem cells from HLA-identical siblings, and 8 received unmanipulated marrow from matched unrelated donors. GVHD prophylaxis was with cyclosporine A alone for 38 patients and cyclosporine A plus methotrexate for 6 sibling recipients. Forty-two of the 43 evaluable patients had sustained engraftment. Results of chimerism analysis using microsatellite polymerase chain reaction indicate that 18 of 31 patients studied were full-donor chimeras while the other patients were mixed chimeras in one or more lineages. At a median follow-up of 9 months (range 3 to 29 months), 33 patients remain alive in complete remission or with no evidence of disease progression. Seven patients relapsed or progressed post-transplantation, and 4 of them subsequently died. Four patients died of regimen-related complications. There were no cases of grades III-IV acute GVHD. Only 2 patients developed grade II acute GVHD, and only 1 had chronic GVHD. The estimated probability of nonrelapse mortality was 11%. Although longer follow-up is needed to establish the long-term remission rates, this study demonstrates that this nonmyeloablative preparative regimen is associated with durable engraftment, minimal toxicity, and low incidence of GVHD.     

Regulation of brain m calpain Ca2+ sensitivity by mixtures of membrane lipids: Activation at intracellular Ca2+ level

Combinations of certain phospholipids and gangliosides increase the specific activity of m calpain and can activate m calpain at 1 to 10 μM Ca²⁺ concentration. However, this level of calcium is still greater than the normal intracellular calcium level. We have used combinations of lipids to demonstrate the m calpain activity at the physiological Ca²⁺ level. GD1a (100 μM) and cerebroside (Cerb; 750 μM; 1:7.5) mixture was the most effective. At 0.5 μM to 1.0 Ca²⁺ concentrations, 15–20% of the maximal activity was detected for the purified myelin and cytosolic m calpains. Other combinations were GD1a (100 μM), GM1 (100 μM), Cerb (750 μM), sulfatide (Sulf; 750 μM), and phosphatidylinositol (PI; 300 μM) at a ratio of 1:1:7.5:7.5:3, respectively. These lipid mixtures stimulated calpain activity at three- to tenfold less calcium concentration than control. The other mixtures, including GD1a:Sulf (1:9) > GD1a:PI (1:4) > PI:Sulf (1:5) > Cerb:Sulf (1:5) and PI:Cerb (1:2.5), also stimulated calpain activity at 1.0 μM Ca²⁺ concentration. Triton X-100, oxidized glutathione (GSSG), and calpain activator did not affect the Ca²⁺ requirement. Liposomes containing GD1a, Cerb, and m calpain also showed recognizable calpain activity at a significantly reduced Ca²⁺ concentration (0.4 μM), confirming the glycolipid-mediated enzyme modulation. These studies indicate that specific lipid mixtures can stimulate m calpain activity at an intracellular level of Ca²⁺. © 1996 Wiley-Liss, Inc.     

Practical aspects of electrophoretic deposition to produce commercially viable supercapacitor energy storage electrodes

Electrophoretic deposition (EPD) is a highly convenient and demonstrated industrial operation for the manufacture of surface coatings. Recent years are seeing increasing evidence in using this technique to produce energy storage electrodes (notably for lithium-ion batteries, solid-state devices, supercapacitors, and flow batteries), but their advancement for industrialisation remains unclear. Using activated carbon (AC) as an exemplary supercapacitor material, this study reports the practical aspects of porous energy storage electrodes produced by the EPD technique. Practical electrodes with commercially viable parameters are shown, specifically high density active material (in excess of 9.8 mg cm⁻²) and very thick coating layer (about 168 μm). Research investigations including colloidal electrolyte formulations, electrode deposition parameters and cell performance testing are reported. Materials and electrode properties were studied by various charactersisation tools. Prototype A7 sized pouch cells were assembled and tested to show evidence of practical EPD electrodes in a commercial cell format. Electrochemical performance of EPD over slurry casting is presented. In short, this research shows the successful production of practical EPD electrodes for electrochemical energy storage, which is directly relevant for scale-up industrial adoption and can be applied as a platform electrode manufacturing technology for any battery and supercapacitor materials.

Electrophoretic deposition (EPD) is a highly convenient and demonstrated industrial operation for coatings manufacture. It is now suitable for the production of practical energy storage electrodes for batteries, capacitors & solid-state devices.     

Prolonged intraperitoneal dwell decreases ultrafiltration coefficient in rabbits

In rabbits undergoing peritoneal dialysis, hypertonic (6% dextrose) dialysis solution increased the net ultrafiltration rate (UF) from 233 to 462 microL/kg/min, which was not proportional to the increment in the osmotic gradient, so the ultrafiltration coefficient decreased. As intraperitoneal dwell of hypertonic dialysate was prolonged, the gross and net UFs and ultrafiltration coefficients decreased, and the UF per dextrose absorption declined. The decrement in UF was multifactorial, including a component of fluid and solute stagnation, increasing the distance over which osmotic forces must exert their effects. Excessively hypertonic dialysis fluid should be used only briefly to achieve ultrafiltration efficiently and to avoid the high dextrose loading.