Toward development of optimum specimen designs and modeling of in-plane uniaxial compression testing of aluminum alloy 2024 and AISI 1008 steel sheet material.

Tension-compression testing is commonly conducted to understand and predict springback during a stamping process. However, large strains are generally difficult to achieve during the in-plane compression portion of the test. Proper specimen design and control of frictional forces are necessary for obtaining large strains. This paper describes extensive finite element analyses (FEA) and optimization studies (Phase 1) that were conducted to calibrate the model test assembly for three different buckling modes obtained in uniaxial compression tests of aluminum alloy 2024 and American Iron and Steel Institute (AISI) 1008 steel specimens. In addition to obtaining these three buckling modes correctly, calibrated FEA model predicted forces matched measured forces reasonably well. Also, a good agreement between computed and measured stress-strain data was demonstrated for one compression experiment. In the Phase 2 optimization study, optimum specimen geometries will be developed by using these verified, optimum FEA model test assemblies in three types of compression buckling experiments.

Multivariate statistical analysis of heavy metals in soils of a Pb-Zn mining area, India.

Surface soil samples collected from a Pb and Zn mining area in India were subjected to multi-elemental analysis by using inductively coupled plasma-atomic emission spectrometry. Multivariate statistical methods such as principal component analysis and cluster analysis, coupled with correlation coefficient analysis, were used to analyze the data and to apportion the possible sources of elements in soils of a metal mining area. Soils in this area have elevated heavy metal concentrations especially Pb, Zn, Mn, Cu, As, and Tl. Using principal component (PC) analysis, six components were extracted, out of which two PCs explaining 50.12% of total variance are more important. The first principal component with a high contribution of Ag, As, Be, Cd, Co, Cu, Mg, Mn, Ni, Pb, and Zn was deemed to be technogenic/anthropogenic component, and the second principal component, with high loadings for the five discerning variables (Al, Be, Cr, K, Li), was considered as lithogenic component. The third component having strong loadings of Ba, Ca, K, and Na is supposed to have a mixed origin (lithogenic as well as technogenic). Electrical conductivity and total organic matter were not correlated with any element and also have a strong loading in the fifth component which is probably the biomass and ions present in these soils. The findings of the principal component analysis were also substantiated by the cluster analysis. The present study would not only enhance our knowledge regarding the soil pollution status in the study area but would also provide us information to manage the sources of these elements in the study area.

Associations of cadmium, zinc, and lead in soils from a lead and zinc mining area as studied by single and sequential extractions.

An exploratory study of the area surrounding a historical Pb-Zn mining and smelting area in Zawar, India, detected significant contamination of the terrestrial environment by heavy metals. Soils (n=87) were analyzed for pH, EC, total organic matter (TOM), Pb, Zn, Mn, and Cd levels. The statistical analysis indicated that the frequency distribution of the analyzed parameters for these soils was not normal. The median concentrations of metals in surface soils were: Pb 420.21 µ g/g, Zn 870.25 µ g/g, Mn 696.70 µ g/g, and Cd 2.09 µ g/g. Zn concentrations were significantly correlated with Cd (r=0.867), indicating that levels of Cd are dependent on Zn. However, pH, electrical conductivity and total organic matter were not correlated significantly with Cd, Pb, Zn, and Mn. To assess the potential mobility of Cd, Pb, and Zn in soils, single (EDTA) as well as sequential extraction scheme (modified BCR) were applied to representative (n=23) soil samples. The amount of Cd, Pb, and Zn extracted by EDTA and their total concentrations showed linear positive correlation, which are statistically significant (r values for Cd, Pb, and Zn being 0.901, 0.971, and 0.795, respectively, and P values being <0.001). The correlation coefficients indicate a strong relation between EDTA-extractable metal and total metal. These results appear to justify the use of 'total' metal contents as a useful preliminary indicator of areas where the risks of metal excess or deficiency are high. The EDTA extractability was maximum for Cd followed by Pb and Zn in soils from all the locations. As indicated by single extraction, the apparent mobility and potential bioavailability of metals in soils followed the order: Cd ≥ Pb > > Zn. Soil samples were sequentially extracted (modified BCR) so that solid pools of Cd, Zn, and Pb could be partitioned into four operationally defined fractions viz. acid-soluble, reducible, oxidizable, and residual. Cadmium was present appreciably (39.41%) in the acid-soluble fraction and zinc was predominantly associated (32.42%) with residual fraction. Pb (66.86%) and Zn (30.44%) were present mainly in the reducible fraction. Assuming that the mobility and bioavailability are related to solubility of geochemical forms of metals and decrease in the order of extraction, the apparent mobility and potential metal bioavailability for these contaminated soil samples is Cd > Zn > Pb.

Heavy metal uptake and its effect on macronutrients, chlorophyll, protein, and peroxidase activity of Paspalum distichum grown on sludge-dosed soils. Heavy metal uptake and its effect on P. distichum.

This study assessed the heavy metal (Cr, Mn, Ni, Cu, Zn, and Pb) uptake and its effect on biochemical parameters in Paspalum distichum, a wetland plant. Sludge collected from Bhalswa waste dump, New Delhi, was used as heavy metal source and dosed in different proportions viz. 20%, 40%, 60%, and 80% to the garden soil. The plants accumulated metals mostly in belowground organs. The metal accumulation followed the order: Cr>Mn>Cu>Zn>Ni>Pb. The range of heavy metal concentration in tissue of belowground organs after 180 days of growth was 1,778.65-4,288.01 ppm Cr, 828.11-1,360 ppm Mn, 236.52-330.07 ppm Ni, 155.79-282.35 ppm Cu, 27.05-91.16 ppm Zn, and 27.09-50.87 ppm Pb. The biochemical parameters viz. chlorophyll and protein contents and peroxidase (POD) activity exhibited no considerable adverse effect indicating the plants' tolerance towards heavy metals. The high POD activity and synthesis of new protein bands at high sludge-dosed plants were also in support of this fact.

Bronchial hyperreactivity associated with tracheal gangliosides.

Gangliosides, which are membrane constituents of animal cells, may be altered under various conditions that cause change in metabolism. In this study, gangliosides from tracheal and lung tissues were extracted and measured as a function of bronchial hyperreactivity in a guinea pig model of bronchial asthma. When plotted logarithmically, the data showed that tracheal gangliosides decreased with an increase in airway reactivity index, suggesting a linear relation between them. Differential analysis of tracheal gangliosides in the acute stage of bronchial hyperreactivity indicates accumulation of polysialogangliosides. These data support the hypothesis that tracheal gangliosides are intimately involved in the development of bronchial asthma.

Isolated chromaffin cells from adrenal medulla contain primarily monoamine oxidase B.

Cultured chromaffin cells from bovine adrenal medulla were found to contain primarily the B form of monoamine oxidase. This monoamine oxidase B enzyme was somewhat distinct from B enzymes from other sources, in that noradrenaline was a much poorer substrate than serotonin. Nonetheless, studies with selective inhibitors of the A form (clorgyline) and the B form [(-)-deprenyl] confirmed that chromaffin cell monoamine oxidase was the B form. The observation that chromaffin cell monoamine oxidase has poor affinity for catecholamines is consistent with physiological needs that require the cell to synthesize and store large amounts of catecholamines.

Requirement of protein kinase type I for cAMP-mediated up-regulation of lipid-linked oligosaccharide for asparagine-linked protein glycosylation.

Glycan chains of asparagine-linked (N-linked) glycoproteins play a significant role in protein structure and function, as well as in angiogenesis an essential process for breast or other solid tumor growth. Non-availability of these chains causes incorrect folding of glycoproteins and leads to programmed cell death (i.e., apoptosis) through unfolded protein response (UPR) signaling. Cells actively processing cAMP signals modulate the glycan chain biosynthesis by PKA. Glycosylation of cellular proteins in a PKA type I-deficient CHO mutant 10248 was much reduced when compared with the wild type CHO 10001. The rate of LLO biosynthesis is similar in both cell types but quantitatively it is low in the mutant. Pulse-chase experiments indicated that the t(1/2) for LLO-turnover in CHO 10248 was twice as high as that of the wild type. This correlated with the reduced DPMS activity. The Km for GDP-mannose for the DPMS activity was 3-4 folds higher in the mutant than that of the wild type with or without exogenously added Dol-P. The kcat of DPMS was also reduced in the mutant. In vitro phosphorylation of microsomes from the CHO 10248 by PKA, on the other hand, restored the DPMS activity to the normal level. The LLO biosynthesis also improved significantly in MR1, a revertant of the CHO 10248. The turnover of LLO in MR1 and the glycoprotein profile were also at par with the wild type. Therefore, we conclude that PKA type I plays an important role in modulating the protein N-glycosylation in cAMP responsive cells.

Expression of blood clotting factor VIII:C gene in capillary endothelial cells.

The essential role of Factor VIII:C (FVIII:C, anti-hemophilia factor A) as a cofactor for Factor IXa-dependent activation of Factor X has been established. In this paper, we describe that capillary endothelial cells from bovine adrenal medulla express active FVIII:C gene. Accumulation of FVIII:C in conditioned media from an 8-day-old culture is approximately twice as high as that stored in the cell when immunoprecipitated FVIII:C was analyzed for its ability to convert Factor X to Factor Xa. Analysis of [35S]methionine-labeled and immunoprecipitated FVIII:C from cells or conditioned media on SDS-PAGE under fully denatured conditions indicated that the newly synthesized FVIII:C consists of heavy chain of M(r) 200,000 and light chain of M(r) 46,000. The secreted FVIII:C in the non-reduced condition however, has a molecular weight of 270,000 which suggests that in native protein, the heavy and light chains are held together by S-S bonds. Furthermore, susceptibility of the immunoprecipitated FVIII:C to N-glycanase digestion establishes that the endothelial cells derived FVIII:C contains asparagine-linked carbohydrate side chains.

N-linked protein glycosylation in the rat parotid gland during aging.

N-Linked protein glycosylation was examined in vitro in dispersed rat parotid acinar cells from young adult (3-6 months) and aged (22-24 months) rats. A small decrease in general protein production was observed with cells from aged animals (approximately 20% lower incorporation of [14C]leucine into 10% CCl3 COOH insoluble protein during continuous pulse labeling). Incorporation of [3H]mannose into N-linked glycoproteins by aged cells was further reduced (approximately 35%). Similarly microsomal membranes from parotid glands of aged animals showed approximately 50% reduction in the synthesis of mannosylphosphoryl dolichol, a key intermediate in the dolichol pathway of protein N-glycosylation. Man-P-Dol synthase, the microsomal enzyme responsible for production of this saccharide-lipid, displayed no change in apparent Km for GDP-mannose when preparations from aged animals were utilized, but did show approximately 50% reduction in Vmax. Following beta-adrenoreceptor activation, cells from both young adult and aged glands showed increased N-linked protein glycosylation almost to the same extent (approximately 2-fold). The data suggested that in aged rat parotid cells there is a basal reduction of activity in the pathway responsible for asparagine-linked protein glycosylation, but that following exocytotic stimuli this pathway responds in a manner comparable to cells from young adult glands.

Changes in T and B blood lymphocytes after splenectomy.

The blood lymphocytes of 37 splenectomised patients were analysed by means of T and B lymphocyte surface markers. Sixteen patients had had a splenectomy for non-haematological and 21 for haematological reasons. The results show that 15 had normal numbers of T and B cells; decreased T cells were found in two patients, raised B cells in seven, raised T and B cells in eight, and raised T cells in five patients. Increased numbers of 'null' cells were observed in some patients, especially in those with raised B cells. Follow-up studies indicate that raised levels of T and B cells can be established by one to three months post-splenectomy and may persist, although in some patients the cells fall to normal levels. The lymphocyte proliferative response to phytohaemagglutinin and Concanavalin A in vitro was normal in eight out of nine patients with raised T cells and was depressed in one patient, possibly due to an intrinsic cell defect.

Susceptibility of Mycobacterium leprae to the bactericidal activity of mouse peritoneal macrophages and to hydrogen peroxide.

Macrophages from athymic nude mice were infected in vitro with Mycobacterium leprae to study the intracellular fate of this organism. Using the proportional bactericidal test, we have shown that the viability of M. leprae declines rapidly within these macrophages, although results of clearance experiments demonstrate that live and killed organisms are cleared at comparable rates. We have also shown that M. leprae is susceptible to the bactericidal effects of hydrogen peroxide and we suggest that hydrogen peroxide generated by macrophages is responsible for the killing of intracellular M. leprae.

A recent approach to the study of dolichyl monophosphate topology in the rough endoplasmic reticulum.

Amphomycin though withdrawn as an antibiotic against the gram-positive bacterial infection, can certainly serve as an excellent tool for determination of the topology of Dol-P in the endoplasmic reticulum membranes which has been otherwise impossible.

dl-17 beta-hydroxy-8 alpha-androst-4-en-3-one (8-isotestosterone).

The molecular conformation of dl-8-isotestosterone has been determined crystallographically. Crystals of the title compound belong to the space group P21/c with a = 11.449(4), b = 10.962(4), c = 25.860(5) A, beta = 100.95(4)o, with two molecules in the asymmetric unit. The structure has been refined to a final R value of 0.052 for 2227 reflections. Unlike testosterone, which is a flat molecule, its 8-isomer has a folded conformation. The conformations of the ring-B in the two crystallographically independent molecules (A and B) corresponded to the twist form and differ significantly from one another.

Mannosylphosphodolichol synthase activity is associated with a 32 kDa phosphoprotein.

Failure of actinomycin D to block the activation of Dol-P-Man synthase in isoproterenol-treated capillary endothelial cells, supported that isoproterenol effect was not mediated by active transcription of the Dol-P-Man synthase gene during a short-term beta-adrenoreceptor stimulation. Instead, it was a net effect of protein phosphorylation by cAMP-dependent protein kinase. Using antibody as a probe we have now demonstrated that Dol-P-Man synthase activity is associated with a 32 kDa ER phosphoprotein.

Monoclonal antibody to amphomycin. A tool to study the topography of dolichol monophosphate in the membrane.

Understanding the topographical orientation of dolichol monophosphate (Dol-P) in the membrane of the endoplasmic reticulum (ER) is of utmost importance for studying the regulation of asparagine-linked protein glycosylation in eukaryotic cells. This was practically impossible due to the nonavailability of a suitable probe. Recent studies on the specific interaction between a lipopeptide, amphomycin, and Dol-P, provided an insight to develop a monospecific antibody to amphomycin which could recognize the amphomycin-Dol-P complex in order to detect Dol-P immunocytochemically in the ER membrane. We report herein the successful production of a monoclonal antibody to amphomycin. The antibody belongs to the IgG+IgM subclasses and is specific for amphomycin when analyzed by the enzyme-linked immunoassay and immunoblot procedures. The antibody recognizes with equal potency both the native amphomycin and also mild acid-hydrolyzed amphomycin from which N-terminal fatty acylated aspartic acid has been removed. Preincubation of amphomycin with the antibody partially reduced the inhibitory action of amphomycin on dolichol phosphate mannosyltransferase (EC 2.4.1.83). Furthermore, exposure of capillary endothelial cells to amphomycin, followed by the monoclonal antibody to amphomycin, followed sequentially by staining with FITC-conjugated goat anti-mouse IgG and examination under a fluorescent microscope gives intense fluorescence at the perinuclear region of the cell with a structure reminiscent of the ER.

Modulation of oligosaccharide processing in an exocrine secretory glycoprotein of rat parotid cells by beta-adrenoreceptor activation.

Such stimulation of rat parotid acinar cells in vitro modulated the rate of processing of N-linked oligosaccharides in a high-molecular weight (220 kdalton) secretory glycoprotein. Conversion of polymannose-type oligosaccharides to complex-type oligosaccharides was evaluated by sensitivity to endoglucosaminidase H and alpha-mannosidase, and with a specific inhibitor of glucosidases I/II. Oligosaccharide maturation in the 220 kdalton glycoprotein required one-third to half less time in cells exposed to the beta-adrenergic agonist isoproterenol than in controls.

Tunicamycin inhibits capillary endothelial cell proliferation by inducing apoptosis. Targeting dolichol-pathway for generation of new anti-angiogenic therapeutics.

Bovine adrenal medulla microvascular endothelial cells used in this study undergo cellular proliferation and differentiation upon culturing in vitro as observed both by light and scanning electron microscopy. Cells also respond to the growth promoting activity of serum and basic fibroblast growth factor (FGF2). Flow cytometric analysis of a synchronized culture established that cells take 68 hours to complete one cell cycle spending 36 hours in the G1 phase, 8 hours in the S phase, and 24 hours in the G2 + M phase when cultured in EMEM containing 2% heat-inactivated fetal bovine serum (FBS). At 10% serum, or in the presence of FGF2 (10 ng/ml-100 ng/ml) length of the cell cycle is reduced to 56 hours due to shortening of the G1 phase by 12 hours. Tunicamycin (a glucosamine-containing pyrimidine nucleotide), and an inhibitor of glucosaminyl-1-phosphate (GlcNAc 1-P) transferase, the first step of Glc3Man9GlcNAc2-PP-Dol (OSL) biosynthesis is found to inhibit the endothelial cells proliferation by inducing apoptosis as observed by flow cytometry and DNA laddering. Cell shrinkage, compaction of nuclei, membrane fragmentation, etc., typical of apoptotic response are frequently seen by light microscopy in the presence of tunicamycin. Scanning electron microscopy also exhibited a considerable amount of cell surface blebbing. Accumulation of an immunopositive cell specific asparagine-linked (N-linked) glycoprotein, Factor VIII:C in the absence of Glc3Man9GlcNAc2-PP-Dol in tunicamycin treated cells has been proposed as an apoptotic triggering mechanism under the current experimental conditions.

Angiogenesis: characterization of a cellular model.

Endothelial cell proliferation and differentiation into blood capillaries (i.e., angiogenesis) are essential for growth and development, wound healing, osetogenesis, etc. But abnormal angiogenesis during tumor progression could lead to serious consequences. Angiogenesis is a complex biochemical process, and is often difficult to study the molecular mechanism in vivo due to interference by multitude of factors. Here, I present a non-transformed capillary endothelial cell line as a model which has been extensively characterized morphologically and biochemically to study the fundamentals of the angiogenic process. Studies completed in our laboratory also evidenced that expression of Glc3Man9GlcNAc2-PP-Dol is intricately connected with the balance between the cellular proliferation and apoptosis during angiogenesis.

Is asparagine-linked protein glycosylation an obligatory requirement for angiogenesis?

Dependence of protein N-glycosylation on capillary endothelial cell proliferation has been studied. Amphomycin, a potent N-glycosylation inhibitor, inhibited capillary endothelial cell proliferation in a dose-dependent manner. beta-Agonist isoproterenol as well as other intracellular cAMP enhancing agents, viz. cholera toxin, prostaglandin E1 and 8Br-cAMP, also enhanced capillary endothelial cell proliferation. In addition to cell proliferation, isoproterenol also enhanced protein glycosylation in these cells. Isoproterenol effect was mediated by beta-adrenoreceptors, as it got reduced on pre-treatment of cells with either atenolol or ICI 118, 551 or propranolol. Furthermore, isoproterenol stimulation of protein glycosylation by exogenous dolichyl monophosphate and its inhibition by tunicamycin (GlcNAc-1P transferase inhibitor) supported the concept that isoproterenol specifically stimulated protein N-glycosylation event(s) in the cell.

Comparison of two sequential extraction procedures for heavy metal partitioning in mine tailings.

This study aimed at elucidating the fractionation of Cd, Zn and Pb in mine tailings and evaluating the extraction selectivity of different extractants--and thus the efficiency of two sequential extraction procedures. For the purpose of figuring out the applicability of different sequential extraction procedures (SEP's) in Cd, Zn and Pb fractionation in mine tailings, two SEP's (i) modified BCR and (ii) Tessier's scheme were applied to mine tailings (new as well as old). To compare the results obtained by two methods having different number of fractions (four and five respectively), the fractions were rearranged into four "equivalent fractions" defined as acid soluble, reducible, oxidisable and residual. Systematic variations in the "reducible" fraction, i.e. the content of metal in the reducible fractions obtained by Tessier's procedure were, in general, higher than those obtained by BCR scheme. This may be attributed to stronger conditions used in Tessier's scheme to extract this fraction. This shows that the fractions are only empirically (operationally) defined and are not totally exclusive to the specified mineral phase. Percentages of Cd, Zn and Pb extracted in the organic fraction by modified BCR scheme were always higher than those obtained by Tessier's scheme. Based on the fractionation results by both the schemes, it appears that as compared to Cd and Zn, Pb has lower extraction yield in old tailings as compared to new tailings in organic/sulphides bound fraction. This can be explained by the formation of secondary Pb minerals of low solubility, formed by the oxidation of sulphides in old tailings as a result of weathering. Speciation data indicate that Cd has the greatest affinity in the acid soluble fraction, Pb is associated with the reducible fraction and Zn is incorporated into the residual fraction (crystalline silicate matrices) irrespective of the solid matrix type. The metal associations in the oxidizable fraction appear to be matrix specific, i.e., Zn dominates this fraction in new tailings and Pb in old tailings.