Deletion of Mycobacterium tuberculosis sigma factor E results in delayed time to death with bacterial persistence in the lungs of aerosol-infected mice

The stress-induced extracytoplasmic sigma factor E (SigE) of Mycobacterium tuberculosis shows increased expression after heat shock, sodium dodecyl sulfate treatment, and oxidative stress, as well as after phagocytosis in macrophages. We report that deletion of sigE results in delayed lethality in mice without a significant reduction of bacterial numbers in lungs.     

Dietary restriction: effects of short-term fasting on protein uptake and cell death/proliferation in the rat liver

Dietary restriction (DR) is known to prolong life in laboratory animals. Intermittent (alternate-day) fasting or short-term repeated fasting has also been reported to increase the life span of animals. In the present study, we investigated the changes or induction of abnormalities of protein metabolism in rats during fasting, and measured asialoglycoprotein uptake and cell death/proliferation in the liver of rats receiving fasting and refeeding. In the results, liver weight decreased significantly after 48 h of fasting and increased during the refeeding period, returning to the pre-fasting level by 12 h of refeeding. Cell death, determined by single stranded DNA (ssDNA) staining method, increased during the fasting period, and returned to the pre-fasting level during the refeeding period. Cell proliferation, determined using antibodies (Ab) against proliferating cell nuclear antigen, decreased during the fasting period, and increased during the refeeding period. Changes in cell death and cell proliferation were inversely related. However, there was no significant difference in asialoglycoprotein uptake by the whole liver between the ad libitum (AL)-fed rats and 48 h fasted rats. Thus, neither the changes in liver weight nor cell death/proliferation affected asialoglycoprotein uptake on a living body. These results suggest that episodes of 48 h fasting do not induce protein metabolism abnormalities in the liver.     

Reduced-intensity unrelated cord blood transplantation for patients with advanced malignant lymphoma.

We report the results of reduced-intensity unrelated cord blood transplantation (RI-UCBT) in patients with advanced malignant lymphoma. Twenty patients (median age, 46.5 years; range, 27-66 years) underwent RI-UCBT with a preparative regimen consisting of fludarabine 125 mg/m2 , melphalan 80 mg/m 2 , and 4 Gy of total body irradiation. The median infused total cell dose was 2.75 x 10(7)/kg (range, 2.3-3.4 x 10(7)/kg). Graft-versus-host disease (GVHD) prophylaxis was composed of cyclosporine or tacrolimus alone. Fifteen patients achieved primary neutrophil engraftment after a median of 20 days. Eight patients developed grade II to IV acute GVHD, and 2 developed chronic GVHD. Of the 16 patients with evaluable disease, 10 achieved a complete response. Primary disease recurred in 1 patient, and transplant-related mortality within 100 days occurred in 8 of 20 patients. The estimated 1-year probability of progression-free survival was 50%. These data suggest that RI-UCBT is a feasible option for patients with refractory lymphoma who lack an HLA-matched donor.     

The Validity of the Neale and Kendler Model-Fitting Approach in Examining the Etiology of Comorbidity

Given that knowledge regarding the etiology of comorbidity between disorders can have a significant impact on research regarding the classification, treatment, and etiology of the disorders, the ability to reject incorrect hypotheses regarding the causes of comorbidity is very important. A simulation study was conducted to assess the validity of the Neale and Kendler (1995) model-fitting approach in examining the etiology of comorbidity between two disorders. First, data were simulated under the assumptions of the 13 alternative comorbidity models described by Neale and Kendler. Second, model-fitting analyses testing the comorbidity models were conducted on the simulated datasets. Thirteen sets of data with varying model parameters were simulated to test Neale and Kendler's assertion that their model-fitting approach is appropriate across a range of potential prevalences and degrees of familiality. The validity of the model-fitting approach in examining unselected twin data and a combination of selected family data and unselected family data was explored. The model-fitting approach successfully discriminated several classes of comorbidity models, although discrimination between models within classes of related models was less accurate. Results suggest that the model-fitting approach can be a useful tool in examining the etiology of the comorbidity between disorders if the caveats of the present study's results are considered carefully. As predicted by Neale and Kendler, variations in the disorder prevalences and familial correlations did not affect the validity of their model-fitting approach, but affected the power to discriminate the correct model. As suggested by Neale and Kendler, the model-fitting approach can be applied to both unselected and selected data and to both twin and family data.     

Segregation of a supernumerary del(15) marker chromosome in sperm

Supernumerary marker chromosomes (SMC) can be associated with both normal and abnormal phenotypes. In addition, SMC are found at higher frequency in males with infertility. We identified a SMC, characterized as a del(15)(q11.2) chromosome, in a phenotypically normal male. Using fluorescence in situ hybridization (FISH), we examined the segregation of the del(15) chromosome in sperm from this patient. Only 6.23% of sperm nuclei showed disomy using a chromosome 15 alpha-satellite FISH probe, instead of the expected 50%. In addition, FISH analysis showed no increase for non-disjunction of chromosome 18, excluding an interchromosomal effect for this chromosome. The significant decrease in sperm bearing the del(15) may be due to tissue-specific mosaicism or a result of some form of selection against the del(15) during spermatogenesis. This finding provides a basis for the observation that SMC(15) are less likely to be inherited from a paternal carrier.     

Osteoclast formation in vitro from bone marrow mononuclear cells in osteoclast-free bone

Recent evidence points to the fact that osteoclasts are derived from mononuclear cells of hematopoietic bone marrow. In this study we have examined the formation of osteoclasts from mononuclear cells in vitro. The mononuclear cells were isolated after 7 days from cultures of mouse bone marrow cells. The isolated cells were co-cultured with osteoclast-free, fetal-mouse calvaria. After 10 to 14 days of co-culture, multinucleated cells which have all the characteristics of osteoclasts were found in juxtaposition to seams of woven bone. These data strongly suggest that bone marrow mononuclear cells, when suitably induced, can give rise to osteoclasts in vitro.     

The effect of alphaMSH analogues on rat bones

Melanocortin is the downstream mediator of leptin signaling and absence of leptin signaling in ob/ob and db/db mice revealed the enhancement of bone formation through the central regulation. While alpha-melanocyte-stimulating hormone (alphaMSH) inhibits the secretion of interleukin-1alpha and tumor necrosis factor-alpha from the inflammatory cells, alphaMSH can also enhance clonal expansion of pro B cells linked to stimulation of osteoclastogenesis. Therefore, we tested the effect of melanocortin on bones. alphaMSH analogues [(6)His]alphaMSH-ND and [(6)Asn]alphaMSH-ND were synthesized and the radio-ligand receptor binding- and cyclic AMP generating activity were analyzed in China Hamster Ovary cell line over- expressing melanocortin receptors. The EC(50) of [(6)His]alphaMSH-ND measured from melanocortin-1, 3, 4 and 5 receptors were 0.008 +/- 0.0045, 1.523 +/- 0.707, 0.780 +/- 0.405, and 250.320 +/- 42.234 nM, respectively, and the EC(50) of [(6)Asn]alphaMSH-ND were 16.8 +/- 6.94, 271.8 +/- 21.95, 8.0 +/- 1.21, and 1132.5 +/- 635.46 nM, respectively. Four weeks after the subcutaneous injection of the analogues, the body weights in the [(6)His]alphaMSH-ND and the [(6)Asn]alphaMSH-ND treated groups (346.0 +/- 20.63 g vs. 350.0 +/- 13.57 g) were lower than that of the vehicle treated group (375.8 +/- 17.31 g, p < 0.05). There was no difference in the total femoral BMD measured by dual x-ray absorptiometry among the three groups. Among the three groups, there were no differences in the total numbers of crystal violet positive- or alkaline phosphatase positive colonies, in the expression of Receptor Activator of Nuclear Factor Kappa-B ligand on the tibia and the total number of multinucleated osteoclast-like cells differentiated from primary cultured bone marrow cells. From the above results, no evidence of bone gain or loss was found after treatment of the alphaMSH analogues peripherally.     

Activation of SiO2-supported zirconocene catalysts by common trialkylaluminiums

A modified SiO₂ was prepared by reacting SiO₂ with Cl₂Si(CH₃)₂ in toluene, on which methylaluminoxane (MAO) was supported to obtain a catalyst precursor. The mixture of the precursor and Cp₂ZrCl₂ (Cp: cyclopentadienyl) gave polyethylene in a high yield even by using common trialkylaluminiums as cocatalyst. Surprisingly, the MAO-free catalyst system composed of the modified SiO₂ and Cp₂ZrCl₂ was also found to be activated by common trialkylaluminiums.     

Serial cloning of pigs by somatic cell nuclear transfer: restoration of phenotypic normality during serial cloning.

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.     

Controlled elimination of intracellular H(2)O(2): regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification

The predominant enzymes responsible for elimination of hydrogen peroxide (H(2)O(2)) in cells are peroxiredoxins (Prxs), catalase, and glutathione peroxidases (GPxs). Evidence suggests that catalytic activities of certain isoforms of these H(2)O(2)-eliminating enzymes are extensively regulated via posttranslational modification. Prx I and Prx II become inactivated when phosphorylated on Thr(90) by cyclin B-dependent kinase Cdc2. In addition, the active-site cysteine of Prx I-IV undergoes a reversible sulfinylation (oxidation to cysteine sulfinic acid) in cells. Desulfinylation (reduction to cysteine) is achieved by a novel enzyme named sulfiredoxin. c-Abl and Arg nonreceptor protein tyrosine kinases associate with catalase in cells treated with H(2)O(2) by mechanisms involving the SH3 domains of the kinases and the Pro(293)PheAsnPro motif of catalase and activate catalase by phosphorylating it on Tyr(231) and Tyr(386). Similarily, GPx1 is activated by c-Abl- and Arg-mediated phosphorylation. The tyrosine phosphorylation is critical for ubiquitination-dependent degradation of catalase.