Highly efficient transduction of repopulating bone marrow cells using rapidly concentrated polymer-complexed retrovirus

Using the cationic polymer, Polybrene, and the anionic polymer, chondroitin sulfate C, we concentrated recombinant retrovirus pseudotyped with an ecotropic envelope, which is susceptible to inactivation by high-speed concentration methods. To evaluate gene marking, murine bone marrow was harvested from C3H mice, transduced with polymer-concentrated GFP virus, and transplanted into lethally irradiated recipients. Total gene marking in mice averaged 30-35% at 8 weeks post-transplant and transgene expression remained stable for over 16 weeks. Using the polymer concentration method, a second retroviral vector encoding the drug resistant variant of dihydrofolate reductase (L22Y-DHFR) was concentrated and tested. Approximately 40% of transduced murine bone marrow progenitor cells were protected against trimetrexate concentrations that completely eliminated the growth of non-modified cells. These results show that anionic and cationic polymers can be combined to rapidly concentrate viruses that are normally difficult to concentrate, and the concentrated virus efficiently transduces hematopoietic stem cells.     

Correlation Between Physicochemical Properties of Soil and Presence of <Emphasis Type="Italic">Leptospira</Emphasis>

Leptospirosis is an important global public health problem. Favourable environmental factors are influencing the survival of leptospires in soil, which is an important link in the transmission cycle. The present study was designed to understand the correlation between various soil nutrients and presence of Leptospira in soil samples of different regions of Andaman and Nicobar Islands. The study revealed a significant positive relationship between presence of Leptospira and concentration of iron, manganese and copper in soil. Presence of iron, manganese and copper in the soil may influence the survival and transmission of leptospirosis.     

Pharmacokinetics and pharmacodynamics in copper deficiency I

The effect of nutritional copper (Cu) deficiency on the antiinflammatory activity and pharmacokinetics of aspirin (ASA) was investigated in rats. Male, weanling Sprague-Dawley rats were fed either a Cu-deficient (CuD) or Cu-sufficient (CuS) diet for 49–50 d. The antiinflammatory activity of ASA was studied using the carrageenan-induced paw edema (CPE) test. ANOVA analyses of edema volumes at 2, 3, 4, 5, and 21 h postcarrageenan indicated significant differences between groups. The percent inhibition of edema due to ASA treatment in CuS was lower than that in CuD rats at 5 h, AUC_5h, and AUC_21h. ASA was found to be significantly more effective in inhibiting the CPE in CuD rats when compared to the CuS rats. Thus, we hypothesized that the increase in ASA's antiinflammatory activity in CuD rats was a result of a decrement in its elimination during nutritional Cu deficiency. The elimination of ASA in CuD and CuS rats was studied using an iv dose of 200 mg/kg. Concentrations of ASA and salicylic acid (SA) were determined in blood; whereas the concentrations of SA, salicylic phenol-glucuronide (SPG), and salicyluric acid (SUA) were determined in urine by HPLC. The results of the pharmacokinetic analyses from blood and urinary data indicated no significant differences in the disposition of ASA between CuD and CuS rats. For instance, the total body clearance for ASA (mean±SD, mL/min/kg) was 37.9±9.4 and 38.5±13.9 (p>0.05); and the volume of distribution (Vd) for ASA (mean±SD, mL/kg) was 385.5±110.3 and 397.1.1±137.9 (p>0.05) for CuD and CuS groups, respectively. Thus, contrary to our hypothesis, the enhanced antiinflammatory activity of ASA in CuD rats does not appear to be mediated via a decrement in the elimination of the drug. In addition, plasma ASA-esterase activity was found to be independent of Cu nutritional status.     

Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo^−/−) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.     

Murine Glial Cells Regenerate NAD, After Peroxide-Induced Depletion, Using Either Nicotinic Acid, Nicotinamide, or Quinolinic Acid as Substrates

Abstract: The potential for regeneration of intracellular pyridine nucleotide levels from different precursors, after peroxide-induced NAD depletion, in cultured glial cells was investigated. Cultured murine glial cells showed a decrease in intracellular NAD levels of >40% after treatment with H₂O₂ (100 µ M ). Removal of the H₂O₂ followed by a 2-h incubation did not result in NAD recovery in the absence of precursors. However, NAD levels increased significantly in these cells after the following substrate additions, at minimum effective concentrations of 1 m M for quinolinic acid (QUIN), 500 µ M for nicotinamide, and 2 µ M for nicotinic acid. The regeneration of significant amounts of NAD from nicotinic acid at doses 250 and 500 times lower than either nicotinamide or QUIN indicates a preferred route for NAD biosynthesis in glial cells in vitro, probably via nicotinic acid phosphoribosylation.     

Differential effects of mitochondrial heat shock protein 60 and related molecular chaperones to prevent intracellular beta-amyloid-induced inhibition of complex IV and limit apoptosis

Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been reported in Alzheimer disease tissue and in cultured cells that overexpress amyloid precursor protein. Mitochondrial dysfunction contributes to neurodegeneration in Alzheimer disease partly through formation of reactive oxygen species and the release of sequestered molecules that initiate programmed cell death pathways. The heat shock proteins (HSP) are cytoprotective against a number of stressors, including accumulations of misfolded proteins and reactive oxygen species. We reported on the property of Hsp70 to protect cultured neurons from cell death caused by intraneuronal beta-amyloid. Here we demonstrate that Hsp60, Hsp70, and Hsp90 both alone and in combination provide differential protection against intracellular beta-amyloid stress through the maintenance of mitochondrial oxidative phosphorylation and functionality of tricarboxylic acid cycle enzymes. Notably, beta-amyloid was found to selectively inhibit complex IV activity, an effect selectively neutralized by Hsp60. The combined effect of HSPs was to reduce the free radical burden, preserve ATP generation, decrease cytochrome c release, and prevent caspase-9 activation, all important mediators of beta-amyloid-induced neuronal dysfunction and death.     

Parkin protects against mitochondrial toxins and beta-amyloid accumulation in skeletal muscle cells

Mutations in the ubiquitin ligase-encoding Parkin gene have been implicated in the pathogenesis of autosomal recessive Parkinson disease. Outside of the central nervous system, Parkin is prominently expressed in skeletal muscle. We have found accumulations of Parkin protein in skeletal muscle biopsies taken from patients with inclusion body myositis, a degenerative disorder in which intramyofiber accumulations of the beta-amyloid peptide are pathognomonic. In comparing primary cultures of skeletal muscle derived from parkin knock-out and wild-type mice, we have found the absence of parkin to result in greater sensitivity to mitochondrial stressors rotenone and carbonyl cyanide 3-chlorophenylhydrazone, without any alteration in sensitivity to calcium ionophore or hydrogen peroxide. Utilizing viral expression constructs coding for the Alzheimer disease and inclusion body myositis-linked beta-amyloid precursor protein and for its metabolic byproducts A beta42 and C100, we found that parkin knock-out muscle cells are also more sensitive to the toxic effects of intracellular A beta. We also constructed a lentiviral system to overexpress wild-type Parkin and have shown that boosting the levels of parkin expression in normal skeletal muscle cultures provides substantial protection against both mitochondrial toxins and overexpressed beta-amyloid. Correspondingly, exogenous Parkin significantly lowered A beta levels. These data support the hypothesis that in myocytes parkin has dual properties in the maintenance of skeletal muscle mitochondrial homeostasis and in the regulation of A beta levels.     

Influence of imaging on touch imprint cytology of breast lesions

Purpose: Touch imprint cytology (TIC) facilitates rapid diagnosis of breast diseases in women attending triple assessment clinics. Some pathologists, in our centre, feel that pathological interpretation of TIC slides is contentious when the lesions are radiologically indeterminate (R3), as these can lead to potentially higher false positive or false negative cytology results. We hypothesised that: ‘(R3) lesions are more likely to have higher false positive or false negative TIC and/or be inadequate for TIC assessment’. In other words, ‘imaging influences cytological classification especially when indeterminate’. Methods: Review of the data collected in our centre between December 2003 and July 2005. All patients who attended the one stop symptomatic breast clinic and had a TIC performed following an ultrasound (US) guided core biopsy (CB) were included. Demographic, radiological, cytological and core biopsy grading data were collected. Cytology grading was correlated with radiology classification to assess our hypothesis. Results: A total of 248 patients underwent 254 CB/TIC. The average patient's age of the group was 54 years (range of 29–95). On TIC, 186 (73%) were deemed malignant, 23(9%) benign while 33(13%) were inadequate for assessment. There was no false positive or false negative TIC. There was good correlation between TIC and CB results (p < 0.0001). Thirty-three cases were inadequate (C1) for cytology assessment, of these16 (48.5%) were indeterminate on imaging. R3 lesions were 6 times more prone to have C1 cytology (p < 0.0001). Conclusion: Touch imprint cytology is a reliable and efficient method in running a one stop breast clinic, with the backup of full tissue diagnosis. Careful selection of cases that would benefit from this technique is highly recommended as a significant number of radiologically indeterminate lesions are likely to be insufficient for cytological assessment. Further prospective trials are required to assess this further. Until then the diagnosis in this sub-group should depend on core biopsy.