The effect of phorbol esters on human erythrocyte morphological discocyte-echinocyte transitions

12-O-Tetradecanoylphorbol-13-acetate (TPA) (100 nM) when incubated with human erythrocytes under conditions of ATP depletion, delayed the onset of the morphological transition from discocytes to echinocytes so that at 2 h, when control incubations were estimated to contain 65% echinocytes, those treated with TPA contained 23% echinocytes. TPA did not alter the subsequent rate of the transition which was complete by 3 h in control cells and 5 h in TPA-treated cells. Addition of 100 nM TPA to ATP-depleted erythrocytes at 2.5 h (greater than 80% echinocytes) for 0.5 h at 37 degrees C resulted in 17% reversal to a discocyte morphology, but as the time of incubation under conditions of ATP depletion was extended, the level of the reversal fell. TPA had no significant effect on the fall in ATP concentrations over the time course of the experiments (5 h). Preincubation of discocytes with TPA for 10 min also prevented, by approx. 50%, the echinocytosis induced by the calcium (0.2 mM) loading of discocytes using 5 microM A23187. TPA was unable to reverse the echinocyte morphology of calcium-loaded cells back to discocytes. The less potent tumour promotor 4-phorbol-12,13-didecanoate had no effect on this discocyte-echinocyte transition. Incubation of discocytes with the diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) (1-10 microM) had complex effects on morphology, and the ATP-induced morphological transition, ranging from stomatocyte formation to echinocyte formation, depending upon the concentration of the agent and the time of incubation.     

Expression of matrix metalloproteinase activity in idiopathic dilated cardiomyopathy: A marker of cardiac dilatation

Background: Idiopathic dilated cardiomyopathy (DCM), ventricular systolic dysfunction and chamber dilatation are accompanied by architectural remodeling, wall thinning and cardiac myocyte slippage. Recent work has demonstrated an association between collagen degradation and an increased expression of matrix metalloproteinases (MMPs). Accordingly, we have sought to correlate (a) collagen degradation with MMP elevations and, (b) assay the neutralizing potential of a known inhibitor of MMP, tetracycline on MMPs in DCM. Methods: Assessment of LV volume and shape by 2-D echocardiography was performed. Light microscopic assessment of histopathology in picrosirius red stained biopsy samples of 11 DCM patients and six post-transplant patients was performed. Zymographic estimation of MMP activity and influence of tetracycline on MMP activity was assessed. Results: Small amount of interstitial collagen was noted in the control group, whereas in the DCM hearts, chamber dilatation was associated with areas of scanty myocyte necrosis, islands of excess collagen, and focal areas of absent or scanty collagen with intact myocytes. In cardiomyopathic tissue, collagenase activity was markedly elevated at 63% compared with 8% in post-transplant tissue. Tetracycline at a concentration of 285 ± 10 μM (IC50) inhibited collagenase activity by 50% in cardiomyopathic tissue. Conclusions: Areas of focal interstitial collagen accumulation were accompanied by collagen fiber lysis and increased collagenase activity in dilated cardiomyopathy. This enhanced collagenolytic activity found in endomyocardial biopsy tissue was inhibited by tetracycline. The non-antibiotic property of tetracycline may be of potential value in the prevention of ventricular dilatation in idiopathic dilated cardiomyopathy. (Mol Cell Biochem 264: 183–191, 2004)     

Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma

Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull‐down, and OptiPrep^TM density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep^TM density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.     

Production of ethanol by raw starch hydrolysis and fermentation of damaged grains of wheat and sorghum

The simultaneous saccharification and fermentation was used to produce ethanol from raw starch of damaged quality wheat and sorghum grains by utilising crude amylase preparation from B. subtilis VB2 and an amylolytic yeast strain S. cerevisiae VSJ4. Various concentrations of damaged wheat and sorghum starch from 10% to 30%W/V were used and 25% was found to be optimum for damaged wheat and sorghum starch yielding 4.40%V/V and 3.50%V/V ethanol respectively. Whereas 25% raw starch of fine quality wheat and sorghum grains gave an yield of 5.60%V/V and 5.00%V/V respectively. The process was carried out at 35v°C, 5.8 pH and 200 rpm for 4 days.     

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis

In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3′ end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.     

Mitochondrial mechanism of oxidative stress and systemic hypertension in hyperhomocysteinemia

Formation of homocysteine (Hcy) is the constitutive process of gene methylation. Hcy is primarily synthesized by de-methylation of methionine, in which s-adenosyl-methionine (SAM) is converted to s-adenosyl-homocysteine (SAH) by methyltransferase (MT). SAH is then hydrolyzed to Hcy and adenosine by SAH-hydrolase (SAHH). The accumulation of Hcy leads to increased cellular oxidative stress in which mitochondrial thioredoxin, and peroxiredoxin are decreased and NADH oxidase activity is increased. In this process, Ca2+-dependent mitochondrial nitric oxide synthase (mtNOS) and calpain are induced which lead to cytoskeletal de-arrangement and cellular remodeling. This process generates peroxinitrite and nitrotyrosine in contractile proteins which causes vascular dysfunction. Chronic exposure to Hcy instigates endothelial and vascular dysfunction and increases vascular resistance causing systemic hypertension. To compensate, the heart increases its load which creates adverse cardiac remodeling in which the elastin/collagen ratio is reduced, causing cardiac stiffness and diastolic heart failure in hyperhomocysteinemia.     

Frequently asked questions about in vivo chlorophyll fluorescence: practical issues

The aim of this educational review is to provide practical information on the hardware, methodology, and the hands on application of chlorophyll (Chl) a fluorescence technology. We present the paper in a question and answer format like frequently asked questions. Although nearly all information on the application of Chl a fluorescence can be found in the literature, it is not always easily accessible. This paper is primarily aimed at scientists who have some experience with the application of Chl a fluorescence but are still in the process of discovering what it all means and how it can be used. Topics discussed are (among other things) the kind of information that can be obtained using different fluorescence techniques, the interpretation of Chl a fluorescence signals, specific applications of these techniques, and practical advice on different subjects, such as on the length of dark adaptation before measurement of the Chl a fluorescence transient. The paper also provides the physiological background for some of the applied procedures. It also serves as a source of reference for experienced scientists.     

Bioaerosols from municipal and animal wastes: background and contemporary issues

Global population increases, coupled with intensive animal and livestock production practices, have resulted in the generation, accumulation, and disposal of large amounts of wastes around the world. Aerosolization of microbial pathogens, endotoxins, odors, and dust particles is an inevitable consequence of the generation and handling of waste material. Bioaerosols can be a source of microbial pathogens, endotoxins, and other allergens. Given the close proximity of population centers to concentrated animal-rearing operations and municipal treatment facilities in many parts of the world, there is concern regarding the occupational and public health impacts associated with the exposure to bioaerosols from municipal and animal wastes. Major advances have been made in our understanding of bioaerosol characteristics, identifying the hazards, and identifying possible human and animal health links with aerosolized pathogens and allergens. However, significant knowledge and technology gaps still exist. These include a lack of clear understanding of the fate and transport of bioaerosols, especially within the open environment, an inability to accurately predict the health risks associated with bioaerosolized pathogens, and a lack of standardized bioaerosol sampling protocols, and efficient samplers. This review synthesizes the information related to bioaerosols and addresses the contemporary issues associated with bioaerosols from municipal and animal wastes, with a focus on pathogens.     

Increasing copper alters cellular elemental composition (Mo and P) of marine diatom

The elemental composition (surface adsorbed and internalized fraction of Cu, Mo and P) in marine phytoplankton was first examined in cultures of the diatom Phaeodactylum tricornutum which were exposed to various levels of Cu concentrations ranging from 0.25 to 16 μmol/L with equivalent free [Cu²⁺] concentrations of 0.4–26 nmol/L. We observed an acceleration of algal growth rates (20–40%) with increasing ambient Cu levels, as well as slightly increased levels of internalized Cu in cells (2–13 × 10^?18 mol/cell) although cellular Cu mostly accumulated onto the cell surface (>50% of the total: intracellular + surface adsorbed). In particular, we documented for the first time that the elemental composition (Mo and P) in algal cells varies dynamically in response to increased Cu levels: (1) Cellular P, predominantly in the intracellular compartment (>95%), shows with a net consumption as indicated by a gradual decrease with increasing [Cu²⁺] (120→50 × 10^?15 mol P/cell) probably due to the fact that P, a backbone bioelement, is largely required in forming biological compartments such as cell membranes; and (2) cellular Mo, predominantly encountered in the intracellular compartment, showed up to tenfold increase in concentration in the cultures exposed to Cu, with a peak accumulation of 1.1 × 10^?18 mol Mo/cell occurring in the culture exposed to [Cu²⁺] at 3.7 nmol/L. Such a net cellular Mo accumulation suggests that Mo might be specifically required in biological processes, probably playing a counteracting role against Cu.