Use of precision cut human liver slices for studying the metabolism and genotoxic potential of xenobiotics by means of the 32P-postlabelling technique: steps towards method validation using testosterone and 2-aminofluorene

In the present study, a new in vitro model combining the short-term incubation of precision-cut human liver slices with DNA-adduct analysis by the ³²P-postlabelling technique is proposed for investigation of the genotoxic potential of xenobiotics. For method validation, the metabolic turnover of testosterone (TES) and the DNA-adduct inducing potential of 2-aminofluorene (2-AF) were used. Precision-cut human liver slices were prepared from a total of 12 human liver samples which were freshly obtained as parts of resectates from liver surgery. The slices were incubated as submersion cultures with TES and 2-AF for up to 6 h in 12-well tissue culture plates at concentrations of 10-50 and 0.06-28 μM, respectively. Slices recovered from the slicing procedure in the 4 °C cold Krebs-Henseleit buffer as indicated by intracellular potassium concentrations which increased for 2 h and then remained stable until the end of the incubation. TES was extensively metabolized by human liver slices with a similar metabolite pattern as observed in vivo. Almost 90% of the metabolites were conjugates. Major phase-I metabolites were androstendione, 6β-OH-androstendione, 6β-OH-TES, and 15β-OHTES. After incubation with 2-AF, substance related DNA-adducts were detected which increased dose-dependently from 12 to 1146 adducts per 10⁹ nucleotides. The adduct pattern consisted of one major adduct spot, A, representing 80-90% of the total adduct level and up to four minor adduct spots, B-E. In summary, the present data demonstrate that precision-cut liver slices are a valuable alternative in vitro system for DNA-adduct determination to screen chemicals for potential genotoxicity in humans.     

Exploring Coal Biodesulfurization Potential of a Novel Organic Sulfur Metabolizing Rhodococcus spp. (Eu-32) – A Case Study

Coal is one of the most abundant nonrenewable fossil fuels, in Pakistan. However, in general, the quality of coal is too low to offset the practical, economic, and regulatory barriers to its utilization. High sulfur content comes up as one of the bottlenecks in productive usage of indigenous coal. Biotechnology can emerge as a panacea for upgrading the huge reserves of high sulfur coal. In current study, the sulfur removal potential of Rhodococcus spp. (Eu-32) was investigated using coal from Dukki, Baluchistan, Pakistan. Biodesulfurization process was optimized for various parameters and maximum decrease of 40% and 60% in total and organic sulfur contents, respectively were achieved in 15 days. The Langmuir and Brunauer–Emmett–Teller (BET) surface areas of the biotreated coal were increased by 20 and 16 times, respectively. Scanning electron microscope showed higher tendency of attachment of bacterial cells to the coal particles. Our results revealed that Eu-32 could remove significant amounts of organic sulfur from coal and could be used in the pre-combustion operations with appropriate arrangements.     

Physical associations of microglia and the vascular blood-brain barrier and their importance in development,health, and disease

Brain endothelial cells (BEC) of the vascular blood-brain barrier (BBB) interact with many different cell types in the brain, including microglia, the brain's resident immune cells. Physical associations of microglia with the BBB and the importance of these interactions in health and disease are an emerging area of study and likely involved in neuroimmune communication. In this mini-review, we consider how microglia and the BBB are intrinsically linked in the developing brain, discuss possible mechanisms that attract microglia to the vasculature in healthy physiological conditions, and examine the known microglial-vascular associated changes in systemic infection and various disease states. Our findings shed light on the complexities of microglial-vascular interactions and highlight the contributions of microglia to the functions of the neurovascular unit.     

Determination of oxytetracycline hydrochloride in milk and egg white samples using Ru(bipy)32+–Ce(SO4)2 chemiluminescence

A novel, rapid and sensitive chemiluminescence (CL) method for the determination of oxytetracycline hydrochloride (OTCH) is described in this paper. The presented method was based on the fact that OTCH could immensely enhance the CL of the reaction of cerium sulfate and tris(2,2‐bipyridyl) ruthenium (II) in acidic medium. Under optimal experimental conditions, CL intensity was favorably linear for OTCH in the range 5.0 × 10^?7 to 5.0 × 10^?5 g/ml, with a detection limit of 1.5 × 10^?7 g/ml (S/N = 3). The relative standard detection was 4.76% for 5.0 × 10^?6 g/ml (n = 11). This method was successfully applied to the analysis of OTCH in milk and egg white samples. According to the results of the kinetic curves for OTCH in the Ru(bipy)₃²⁺–Ce(SO₄)₂ CL system, together with CL and ultraviolet (UV)–visible spectra, the possible mechanism of the CL reaction is discussed briefly.     

Cooperative Interception of Neuronal Apoptosis by BCL-2 and BAG-1 Expression: Prevention of Caspase Activation and Reduced Production of Reactive Oxygen Species

Abstract: Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D- and cycloheximide-sensitive caspase (ICE-like) activity. The peptide inhibitor of caspase activity, N -acetyl-Asp-Glu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone in preventing NGF withdrawal-induced apoptosis, suggesting an important role for caspase-3 (CPP32)-like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert -butyl-α-phenylnitrone, and the antioxidant, N -acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL-2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone.     

Cloning and Expression of a Chitinase Gene from Sanguibacter sp. C4

The chitinase Chi58 is an extracellular chitinase produced by Sanguibacter sp.strain C4. The gene-specific PCR primers were used to detect the presence of the chiA gene in strain C4. A chiA fragment (chiA-F) was amplified from the C4 genomic DNA and was used to blast-search the related sequences from the GenBank dadabase. By alignment and selection of the highly conserved regions of the homologous sequences, two pairs of primers were designed to amplify the open reading frame (ORF) of the chitinase from strain C4 by nested PCR. The results revealed that the Chi58 ORF consisted of 1 692 nucleotides encoding a protein of 563 amino acid residues. The molecular weight of the mature protein was predicted to be 58.544 kDa. The Chi58 ORF was a modular enzyme composed of a signal peptide sequence, a polycystic kidney disease I domain, and a glycosyl hydrolase family 18 domain. The chitinase of C4 exhibited a high level of similarity to the chitinase A of Serratia (88.9%-99.6%) at the amino acid sequence level. The Chi58 gene was cloned into the expression vector pET32a to construct the recombinant plasmid pChi58 and was expressed in E. coli BL-21 (DE3) cells with IPTG induction. The molecular weight of the Trx-Chi58 fusion protein was estimated to be 81.1 kDa by SDS-PAGE.     

Induction of heat shock proteins in cerebral cortical cultures by celastrol

Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis (ALS) are ‘protein misfolding disorders’ of the mature nervous system that are characterized by the accumulation of protein aggregates and selective cell loss. Different brain regions are impacted, with Alzheimer’s affecting cells in the cerebral cortex, Parkinson’s targeting dopaminergic cells in the substantia nigra and ALS causing degeneration of cells in the spinal cord. These diseases differ widely in frequency in the human population. Alzheimer’s is more frequent than Parkinson’s and ALS. Heat shock proteins (Hsps) are ‘protein repair agents’ that provide a line of defense against misfolded, aggregation-prone proteins. We have suggested that differing levels of constitutively expressed Hsps (Hsc70 and Hsp27) in neural cell populations confer a variable buffering capacity against ‘protein misfolding disorders’ that correlates with the relative frequencies of these neurodegenerative diseases. The high relative frequency of Alzheimer’s may due to low levels of Hsc70 and Hsp27 in affected cell populations that results in a reduced defense capacity against protein misfolding. Here, we demonstrate that celastrol, but not classical heat shock treatment, is effective in inducing a set of neuroprotective Hsps in cultures derived from cerebral cortices, including Hsp70, Hsp27 and Hsp32. This set of Hsps is induced by celastrol at ‘days in vitro’ (DIV) 13 when cultured cortical cells reached maturity. The inducibility of a set of neuroprotective Hsps in mature cortical cultures at DIV13 suggests that celastrol is a potential agent to counter Alzheimer’s disease, a neurodegenerative ‘protein misfolding disorder’ of the adult brain that targets cells in the cerebral cortex.     

Hypomania: a transcultural perspective

This study examined the transcultural robustness of a screening instrument for hypomania, the Hypomania Checklist-32, first revised version (HCL-32 R1). It was carried out in 2606 patients from twelve countries in five geographic regions (Northern, Southern and Eastern Europe, South America and East Asia). In addition, GAMIAN Europe contributed data from its members. Exploratory and confirmatory factor analyses were used to examine the transregional stability of the measurement properties of the HCL-32 R1, including the influence of sex and age as covariates. Across cultures, a two-factor structure was confirmed: the first factor (F1) reflected the more positive aspects of hypomania (being more active, elated, self-confident, and cogni-tively enhanced); the second factor (F2) reflected the more negative aspects (being irritable, impulsive, careless, more substance use). The measurement properties of the HCL-32 R1 were largely invariant across cultures. Only few items showed transcultural differences in their relation to hypomania as measured by the test. F2 was higher among men and in more severe manic syndromes; F1 was highest in North and East Europe and lowest in South America. The scores decreased slightly with age. The frequency of the 32 items showed remarkable similarities across geographic areas, with two excep-tions: South Europeans had lower symptom frequencies in general and East Europeans higher rates of substance use. These findings support the interna-tional applicability of the HCL-32 R1 as a screening instrument for hypomania.