Enhancement of adenovirus-mediated gene transfer to human bone marrow cells

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.     

Effect of ergotamine and ergonovine on plasma concentrations of thyroid hormones and cortisol in cattle

Plasma samples from two experiments were processed to determine whether ergot alkaloids associated with endophyte-infected tall fescue altered peripheral thyroxine (T4), triiodothyronine (T3), or cortisol concentrations in cattle. In Exp. 1, seven Angus steers (294 kg) received i.v. bolus injections of saline (SAL), ergonovine maleate (7 mg; EM), or ergotamine tartrate (7 mg; ET) at weekly intervals, and they received all treatments during the study. Blood was sampled every 15 min for 5 h, and treatments were given after h 1. Mean ambient temperature was 34 degrees C. Treatment x time affected plasma concentrations of T3 (P < .05) and of cortisol (P < .001) but not that of T4 (P > .2). Plasma T3 concentrations were not affected by SAL, whereas concentrations increased (P < .01) after either EM or ET treatment. Plasma cortisol concentrations were not altered by SAL or EM, but they were increased (P < .001) by ET treatment. In Exp. 2, six Holstein cows (499 kg) nursing calves received a bolus i.v. injection of SAL, EM (9.5 mg), or ET (9.5 mg) per estrous cycle, and all treatments were given over three cycles. Blood was sampled every 20 min for 5 h; treatments were given after h 1. Mean ambient temperature was 26 degrees C. Treatment x time affected T3 (P = .08) and cortisol (P < .001) and tended to influence (P = .16) T4 concentrations. Plasma T3, T4, and cortisol concentrations were not influenced by SAL treatment. Plasma T3 was higher (P < or = .01) after EM or ET treatment compared with pretreatment concentrations. Concentrations of T4 during the 4 h after EM and ET were increased (P < .001) compared with pretreatment. Plasma cortisol concentrations were not altered by EM but were increased (P < .001) by ET. Ergot alkaloids implicated as contributing agents to fescue toxicosis alter plasma concentrations of hormones important to metabolic and thermoregulatory functions in cattle.     

Clinical utility of the polymerase chain reaction in the diagnosis of extrapulmonary tuberculosis

This study examines the diagnostic utility of the polymerase chain reaction (PCR) in 156 patients (five human immunodeficiency virus (HIV) seropositive) suspected of extrapulmonary tuberculosis. The results of PCR in 226 samples from 11 different sites were compared with the results of microscopy and culture. Positive culture results were predicted in 86% of samples by PCR but in only 31% by microscopy. Specificity of PCR was 92%. In cases with culture-proven tuberculosis, PCR identified all 11 microscopy positive cases and 19 of 24 (79%) of the microscopy-negative cases. In four patients, PCR excluded the diagnosis of tuberculosis in microscopy-positive samples, which were later shown to contain mycobacteria other than Mycobacterium tuberculosis or laboratory contaminants. In 20 patients (microscopy, PCR and culture negative) a trial of antituberculous drugs was given, but patients showed no improvement and treatment was stopped. In 17 patients, all culture negative (in nine PCR was positive, three of whom also had positive microscopy) the diagnosis was probable tuberculosis based on clinical findings and response to treatment. This polymerase chain reaction has a much higher sensitivity than microscopy and can facilitate therapeutic decisions for those with suspected extrapulmonary tuberculosis.     

GCase and LIMP2 Abnormalities in the Liver of Niemann Pick Type C Mice

The lysosomal storage disease Niemann–Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1^−/− mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1^−/− liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1^−/− liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1^−/− liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1^−/− hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes.     

Effect of high temperature heat treatment on intergranular corrosion of AlMgSi(Cu) model alloy

Copper containing 6000-series aluminium alloys may become susceptible to intergranular corrosion (IGC) as a result of improper thermomechanical processing. Effect of cooling rate after solution heat treatment on the corrosion behaviour of a model AlMgSi(Cu) alloy of nominal composition (wt%) 0.6 Mg, 0.6 Si, 0.2 Fe, 0.2 Mn and 0.1 Cu was investigated. Slow cooling rates were simulated by isothermal treatment for predetermined times in lower temperature baths immediately after solution heat treatment. Treatment for 10-100 s at temperatures below 400 °C introduced susceptibility to IGC. Longer heat treatment at the same temperatures introduced susceptibility to pitting. A corrosion resistant time zone was found between the zones of IGC and pitting at temperatures lower than 350 °C. Quenching in water after solution heat treatment prevented IGC. IGC was related to microgalvanic coupling between the noble Q-phase (Al₄Mg₈Si₇Cu₂) grain boundary precipitates and the adjacent depleted zone. Pitting was attributed to coarse particles in the matrix. Possible mechanisms causing the corrosion resistant intermediate zone are discussed. The results indicate possible methods for obtaining increased corrosion resistance of similar alloys by proper thermal processing.     

Biomimetic Polymer Nanostructures by Injection Molding

The nanometer scale topography of self‐assembling structural protein complexes in animals is believed to induce favorable cell responses. An important example of such nanostructured biological complexes is fibrillar collagen that possesses a cross‐striation structure with a periodicity of 69 nm and a peak‐to‐valley distance of 4–6 nm. Bovine collagen type I was assembled into fibrillar structures in vitro and sedimented onto solid supports. Their structural motif was transferred into a nickel replica by physical vapor deposition of a small‐grained metal layer followed by galvanic plating. The resulting inverted nickel structure was found to faithfully present most of the micrometer and nanometer scale topography of the biological original. This nickel replica was used as a die for the injection molding of a range of different thermoplastic polymers. Total injection molding cycle times were in the range of 30–45 seconds. One of the polymer materials investigated, polyethylene, displayed poor replication of the biological nanotopographical motif. However, the majority of the polymers showed very high replication fidelity as witnessed by their ability to replicate the cross‐striation features of less than 5 nm height difference. The latter group of materials includes poly(propylene), poly(methyl methacrylate), poly(L ‐lactic acid), polycaprolactone, and a copolymer of cyclic and linear olefins (COC). This work suggests that the current limiting factor for the injection molding of nanometer scale topography in thermoplastic polymers lies with the grain size of the initial metal coating of the mold rather than the polymers themselves.

     

In vitro effects of urease inhibitors on rate of urea hydrolysis in soils

We studied the effect of urease inhibitors on the urea hydrolysis in some Sundanese soils belonging to the orders of Vertisol and Entisol. The hydrolysis showed a lag period of about 3 days and its rate (Y) per unit time (t) could be described by a two constants exponential equation of the general form Y = K₁t^K ₂. Statistical analysis showed that the intercept K₁ (rate of urea hydrolysis) was significantly affected by soil type rather than treatment. It seems that K₁ is associated with the soils' initial urease activity as it closely correlates with the Michaelis constant (km).The gradient, K₂, being significantly affected by soil type as well as treatment is probably associated with the induced urease activity with time and it, therefore, varied with variations in soils and treatments. Of the so-called urease inhibitors used in this study Ca(OH)₂, p-benzoquinone (PBQ) and orthophosphoric acid (OP) only PBQ reduced urea hydrolysis while the other chemicals have effects possibly related to modifying the soil pH. Inhibitor treated soils had substantial amounts of unacounted for N which was believed to be present, presumably, in the form of carbamate.Contribution from the Department of Biochemistry and Soil Science, Faculty of Agriculture, Shambat, Sudan.     

Effect of artificial aging on intergranular corrosion of extruded AlMgSi alloy with small Cu content

The effect of artificial aging parameters on the corrosion performance of air cooled AlMgSi(Cu) model alloy extrusions was investigated. Accelerated corrosion test revealed that the extrusions were highly susceptible to intergranular corrosion (IGC) in the naturally aged condition. However, IGC susceptibility was reduced, and finally eliminated, by artificial aging. Overaging introduced slight pitting susceptibility. EDS X-ray mapping in FE-TEM revealed Mg₂Si and Q-phase (Al₄Cu₂Mg₈Si₇) grain boundary precipitates and a continuous Cu-enriched grain boundary film. IGC susceptibility was related to the Cu-enriched grain boundary film. Increased IGC resistance was caused by coarsening of the grain boundary film by aging. Pitting susceptibility by over aging evolved due to coarsening of the Q-phase particles in the grain bodies.