Culture medium optimization for improved puerarin production by cell suspension cultures of Pueraria tuberosa (Roxb. ex Willd.) DC.

The effects of carbon, nitrogen, phosphate, and copper on cell growth and production of the isoflavone puerarin by suspension cultures of Pueraria tuberosa (Roxb. ex. Willd.) DC were investigated. Among the various sugars evaluated (glucose, galactose, fructose, maltose, and sucrose), use of sucrose in the medium led to the maximum accumulation of puerarin. A sucrose-feeding strategy in which additional sucrose was added to the flasks 15?d into the culture cycle stimulated both cell biomass and puerarin production. The maximum production of puerarin was obtained when a concentration balance of 20:60?mM NH ₄ ⁺ /NO ₃ ^? was used as the nitrogen source. Alteration in the concentration balance of nitrogen components (NH ₄ ⁺ /NO ₃ ^? 60:20?mM) or the use of either NH ₄ ⁺ or NO ₃ ^? alone decreased biomass production and puerarin accumulation compared with the control culture (NH ₄ ⁺ /NO ₃ ^? 20:20?mM). High amounts of phosphate (2.5 and 5?mM) in the medium inhibited puerarin production whereas 0.625?mM phosphate promoted puerarin production (68.3???g/g DW on day?25). An increase in Cu²⁺ concentration from 0.025 to 0.05?mg/l in the P. tuberosa cell culture medium resulted in a 2.2-fold increase in puerarin production (up to 141???g/g DW on day?25) but reduced cell culture biomass.     

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.     

Identification of psoriatic arthritis mediators in synovial fluid by quantitative mass spectrometry

Background

Synovial fluid (SF) is a dynamic reservoir for proteins originating from the synovial membrane, cartilage, and plasma, and may therefore reflect the pathophysiological conditions that give rise to arthritis. Our goal was to identify and quantify protein mediators of psoriatic arthritis (PsA) in SF.

Methods

Age and gender-matched pooled SF samples from 10 PsA and 10 controls [early osteoarthritis (OA)], were subjected to label-free quantitative proteomics using liquid chromatography coupled to mass spectrometry (LC-MS/MS), to identify differentially expressed proteins based on the ratios of the extracted ion current of each protein between the two groups. Pathway analysis and public database searches were conducted to ensure these proteins held relevance to PsA. Multiplexed selected reaction monitoring (SRM) assays were then utilized to confirm the elevated proteins in the discovery samples and in an independent set of samples from patients with PsA and controls.

Results

We determined that 137 proteins were differentially expressed between PsA and control SF, and 44 were upregulated. The pathways associated with these proteins were acute-phase response signalling, granulocyte adhesion and diapedesis, and production of nitric oxide and reactive oxygen species in macrophages. The expression of 12 proteins was subsequently quantified using SRM assays.

Conclusions

Our in-depth proteomic analysis of the PSA SF proteome identified 12 proteins which were significantly elevated in PsA SF compared to early OA SF. These proteins may be linked to the pathogenesis of PsA, as well serve as putative biomarkers and/or therapeutic targets for this disease.     

QCT/FEA predictions of femoral stiffness are strongly affected by boundary condition modeling

Quantitative computed tomography-based finite element models of proximal femora must be validated with cadaveric experiments before using them to assess fracture risk in osteoporotic patients. During validation, it is essential to carefully assess whether the boundary condition (BC) modeling matches the experimental conditions. This study evaluated proximal femur stiffness results predicted by six different BC methods on a sample of 30 cadaveric femora and compared the predictions with experimental data. The average stiffness varied by 280% among the six BCs. Compared with experimental data, the predictions ranged from overestimating the average stiffness by 65% to underestimating it by 41%. In addition, we found that the BC that distributed the load to the contact surfaces similar to the expected contact mechanics predictions had the best agreement with experimental stiffness. We concluded that BC modeling introduced large variations in proximal femora stiffness predictions.     

Oxidative stress in zinc-induced dopaminergic neurodegeneration: implications of superoxide dismutase and heme oxygenase-1

The study was undertaken to investigate the effect of zinc (Zn) on glutathione S-transferase (GST) and superoxide dismutases (SOD) activities and on the expressions of cytosolic Cu, Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2), γ-glutamyl cysteine synthetase (γ-GCS) and heme oxygenase-1 (HO-1) in the nigrostriatal tissue of rats. Additionally, Zn-induced alterations in the neurobehavioral parameters, lipid peroxidation (LPO), striatal dopamine and its metabolites and tyrosine hydroxylase (TH) protein expression were measured to assess their correlations with the oxidative stress. Zn exposure reduced the locomotor activity, rotarod performance, striatal dopamine and its metabolites and TH protein expression. LPO, total SOD, SOD1 and SOD2 activities were increased while GST and catalase were reduced in a dose and time dependent manner. Expressions of SOD1 and HO-1 were increased while no change was observed in SOD2 and γ-GCS expressions. The results obtained suggest that Zn-induced augmentation of total SOD, SOD1, SOD2 and HO-1 was associated with increased oxidative stress and neurodegenerative indexes indicating the involvement of both cytosolic and mitochondrial machinery in Zn-induced oxidative stress leading to dopaminergic neurodegeneration.     

Post-Operative Hypertension after Total Knee Arthroplasty and the Effects on Transfusion Rates

Transfusions are a cause of significant patient morbidity as well as expense. Anesthesia literature has examined controlled intraoperative hypotension as a means for reducing blood loss and transfusions. Our hypothesis is that inversely increased blood pressure post-operatively would then lead to increased blood loss and transfusions.We examined 105 consecutive patients who underwent TKA. We found a significant odds ratio of 1.123 for pre-operative hematocrit. For post-operative blood pressure, we calculated an insignificant odds ratio of 1.007, proving no relationship between post-operative blood pressure and transfusions.This is the first study to examine increased post-operative blood pressure''s contribution to transfusion rates. Although we confirmed that low pre-operative hematocrit contributes to increased transfusions, we did not find a relationship between post-operative blood pressure and transfusions.     

Antimicrobial protein from Streptomyces fulvissimus inhibitory to methicillin resistant Staphylococcus aureus

Fermented culture of Streptomyces fulvissimus was found to secrete an antibacterial protein inhibitory to Micrococcus luteus, Bacillus subtilis, Bacillus cereus and methicillin resistant Staphylococcus aureus (MRSA) strains. The extracellular protein from the fermented culture on concentration revealed a high molecular weight peptide of 63kDa on SDS-PAGE gel and the region on gel displayed inhibitory activity against methicillin resistant Staphylococcus aureus. Bioactivity of the extra cellular protein was non-sensitive to proteinase K, alpha chymotrypsin, protease, EDTA (ethylene diamine tetra acetic acid), PMSF (phenyl methyl sulfonyl fluoride) and DMSO (dimethyl sulfoxide) but partially susceptible to amylase and heat. Glycoprotein nature of the proteinaceous compound was confirmed by periodic acid schiffs (PAS) staining. The secretary protein of S. fulvissimus demonstrated a significant activity against MRSA strain. It could be an important source for developing new drugs to control multidrug resistant gram positive bacteria.     

Time-Lapse Microscopy of Streptomyces coelicolor Growth and Sporulation

Bacteria from the genus Streptomyces are among the most complex of all prokaryotes; not only do they grow as a complex mycelium, they also differentiate to form aerial hyphae before developing further to form spore chains. This developmental heterogeneity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. As a result, in order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy. Through the use of time-lapse microscopy we have demonstrated that Streptomyces coelicolor germ tubes undergo a temporary arrest in their growth when in close proximity to sibling extension sites. Following germination, in this system, hyphae extended at a rate of ~20 μm h⁻¹, which was not significantly different from the rate at which the apical ring of the cytokinetic protein FtsZ progressed along extending hyphae through a spiraling movement. Although we were able to generate movies for streptomycete sporulation, we were unable to do so for either the erection of aerial hyphae or the early stages of sporulation. Despite this, it was possible to demonstrate an arrest of aerial hyphal development that we suggest is through the depolymerization of FtsZ-enhanced green fluorescent protein (GFP). Consequently, the imaging system reported here provides a system that allows the dynamic movement of GFP-tagged proteins involved in growth and development of S. coelicolor to be tracked and their role in cytokinesis to be characterized during the streptomycete life cycle.     

Immune response to leishmania: paradox rather than paradigm

The leishmaniases are a group of diseases caused by protozoan parasites of the genus Leishmania. Various Leishmania species can cause human infection, producing a spectrum of clinical manifestations. It is estimated that 350 million people are at risk, with a global yearly incidence of 1-1.5 million for cutaneous and 500,000 for visceral leishmaniasis (VL). VL is a major cause of morbidity and mortality in East Africa and the Indian subcontinent. Coinfection with HIV enhances the risk of the disease. The only control measure currently available in India is case detection and treatment with antimonial drugs, which are expensive, not always available and cannot be self-administered. Newer drugs like oral miltefosine have not become widely available. Vector and reservoir control is difficult due to the elusive nature of the vector and the diversity of the animal reservoir. A detailed knowledge of immune response to the parasite would help in designing prophylactic and therapeutic strategies against this infection.