Quantitative determination of hemoglobin and cytochemical staining for peroxidase using 3,3',5,5'-tetramethylbenzidine dihydrochloride, a safe substitute for benzidine.

The quantitative determination of hemoglobin employing 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB-d) is described. This agent can also be used for staining myeloperoxidase-containing granules in granulocytes. TMB-d is at least as sensitive a reagent as benzidine but, unlike benzidine and diaminobenzidine, it is not a carcinogen.     

Biphasic uptake of iron-transferrin complex by L1210 murine leukemia cells and rat reticulocytes

The kinetics of the cellular uptake of iron-transferrin complex was studied in L1210 murine leukemia cells and rat reticulocytes using ¹²⁵I-transferrin. Saturation of transferrin with iron was necessary for optimal uptake. Following the incubation of cells with the radiolabeled complex a biphasic pattern of uptake was observed. The initial phase was rapid and relatively temperature-independent and was not altered by ethylamine, an inhibitor of transglutaminase activity which is necessary for receptor-mediated endocytosis. This phase was considered to result from receptor-ligand interaction which could be reversed to a great degree by replacement with unlabeled transferrin. A plateau was then reached, indicating a saturation of receptors. After 30 min a second phase of uptake was indicated by the second rise in the curve. This phase was slow, relatively temperature-dependent and could be abolished by ethylamine. It was interpreted as evidence of internalization of the ligand. Analysis of the data from competition studies with unlabeled transferrin indicated that the first phase might itself comprise a reversible and an irreversible step with a ratio of 5 to 1.4 for bound transferrin. Thus, the cellular uptake of iron-transferrin complex may consist of a reversible ligand-receptor interaction. Conformational changes may render this interaction irreversible and the internalization of the ligand may then follow.     

Transendothelial transport of macromolecules: the concept of tissue-blood barriers.

In addition to its many functions in biosynthesis, growth, coagulation and rheology, vascular endothelium is anatomically interposed between the vascular space and the tissue fluid. Recent evidence indicates that it mediates cellular and molecular exchange between these compartments. The exchange can occur through differentiated microdomains of endothelium such as fenestrae. These areas are differentiated with regard to surface charge, protein distribution within the lipid bilayer, membrane fluidity and other features. The exchange is also affected by certain characteristics of the molecule to be transported: molecular size, charge, shape and its carbohydrate content. Proportionately, the largest volume of exchange occurs across the endothelial cytoplasm by vesicular transport systems. Two systems are particularly in evidence; (a) receptor-mediated transcytosis which is specific, and (b) fluid-phase endocytosis. The molecule may become modified in transit and the modification may be of essence in determining its target point and its subsequent metabolism. While most of these modifications involve the carbohydrate moiety of the glycoproteins, glycosylation of non-glycoproteins such as albumin, may also be of physiological significance in transendothelial transport. By virtue of its transport potential, albumin can thus affect the transport of other substances. Recent advances in the molecular transport function of endothelium have been reviewed in the context of its physiological and clinical significance. The basis for the concept of a generalized tissue-blood barrier has been offered.     

A mammalian cell variant in which 3-aminobenzamide does not potentiate the cytotoxicity of dimethyl sulphate

Variants of mouse leukaemia L1210 cells have been isolated in which cytotoxicity to dimethyl sulphate is not fully potentiated by ADP-ribosyl transferase inhibitor 3-aminobenzamide, as occurs in normal L1210 cells. These variants were selected after mutagenesis by growing the cells in dimethyl sulphate and 3-aminobenzamide. The characterisation of one of these variants is described. Variant 3 cells repair low doses of DNA damage in the presence of ADP-ribosyl transferase inhibitors. The Vmax of the ADP-ribosyl transferase enzyme in these cells is only increased 35% compared to normal wild-type L1210 cells. The basal DNA ligase I activity is increased 66% above wild-type whereas DNA ligase II activity appears to be unchanged. The most striking observation, however, is that the DNA ligase II activity is not increased after dimethyl sulphate treatment as occurs in wild-type L1210 cells. It seems that by increasing DNA ligase I levels these cells can survive DNA damage in the presence of 3-aminobenzamide. This variant (mutant) provides genetic evidence for our previously published hypothesis that (ADP-ribose)n biosynthesis is required for efficient DNA repair after DNA damage by monofunctional alkylating agents, because ADP-ribosyl transferase activity regulates DNA ligase activity. This variant is the first mammalian cell reported in which DNA ligase activity is altered, as far as we are aware. In yeast, a DNA ligase mutant has a cell division cycle (cdc) phenotype. Presumably, DNA ligase is essential for DNA synthesis, repair and recombination. The present variant provides further evidence that in mammalian cells, DNA ligase II activity is related to ADP-ribosyl transferase activity.     

Health risk assessment due to exposure of arsenic contamination in drinking water of district Shiekhupura,Punjab, Pakistan

Abstract

The present study was conducted to assess the magnitude and health impacts of As in drinking water. Drinking water samples (n?=?60) were collected from twenty different sites of Shiekhupura District (Pakistan). Health risk assessment through average daily dose (ADD), hazard indices (HI), hazard quotient (HQ), carcinogenic risk (CR), and cancer indices (CI) for dermal and oral exposure were determined. Results revealed that As concentration ranged from 2 to 900?µg?L^?1 in water samples, which was significantly greater than the safe limit of As (10?µg?L^?1) in water. Health risk assessment of As showed that ADD (1.07E?02–9.85E?04), HQ (1.06E+01–9.85E+00), and CR (1.60E?02–9.85E?04) for oral exposure and ADD (1.03E?05–9.69E?06), HQ (1.19E?02–7.96E?03), and CR (1.11E?05–8.98E?05) for dermal exposure which were exceeded the toxic risk index value. Comparison of the two exposure pathways indicated that the oral exposure is much higher risk than the dermal contact. Both values of HI and CI were greater than WHO limit. It is concluded that residents of study area are at higher risk of As induced diseases and carcinogenicity.     

Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth

The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand-alone unit operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.     

Wolbachia host shifts: routes,mechanisms, constraints and evolutionary consequences

Wolbachia is one of the most abundant endosymbionts on earth, with a wide distribution especially in arthropods. Effective maternal transmission and the induction of various phenotypes in their hosts are two key features of this bacterium. Here, we review our current understanding of another central aspect of Wolbachia's success: their ability to switch from one host species to another. We build on the proposal that Wolbachia host shifts occur in four main steps: (i) physical transfer to a new species; (ii) proliferation within that host; (iii) successful maternal transmission; and (iv) spread within the host species. Host shift can fail at each of these steps, and the likelihood of ultimate success is influenced by many factors. Some stem from traits of Wolbachia (different strains have different abilities for host switching), others on host features such as genetic resemblance (e.g. host shifting is likely to be easier between closely related species), ecological connections (the donor and recipient host need to interact), or the resident microbiota. Host shifts have enabled Wolbachia to reach its enormous current incidence and global distribution among arthropods in an epidemiological process shaped by loss and acquisition events across host species. The ability of Wolbachia to transfer between species also forms the basis of ongoing endeavours to control pests and disease vectors, following artificial introduction into uninfected hosts such as mosquitoes. Throughout, we emphasise the many knowledge gaps in our understanding of Wolbachia host shifts, and question the effectiveness of current methodology to detect these events. We conclude by discussing an apparent paradox: how can Wolbachia maintain its ability to undergo host shifts given that its biology seems dominated by vertical transmission?     

Design of Metasurface-Based Multi-layer THz Filters Utilizing Optimization Algorithm with Distinct Fitness Function Definitions

In this paper, an evolutionary optimization procedure is presented to generate band-pass metasurface-based filters in terahertz regime. As a measure of novelty, pass-band, transition, and out-band characteristics are investigated separately, all of which result in different metasurfaces for filtering applications. The presented approach is defined based on random hill climbing algorithm, regarding the established link between Matlab and HFSS software. A metasurface-based filter with specific properties is considered as the main problem to be solved by the optimization method. Moreover, the fuzzy theory, mean square method, and weighting coefficient procedure are considered to define an efficient fitness function evaluation approach. Also, a step-by-step procedure is used to generate desired structures with a great note of efficiency. The final generated structure has magnificent characteristics including sharp transitions together with transmittance around 0.68 and less than 0.04 at pass-band and out-band regions, respectively. Also, the generated metasurface benefits from wide bandwidth (65%) and great compactness compared to other previous works.