Direct fermentation of cassava starch to ethanol by mixed cultures of Endomycopsis fibuligera and Zymomonas mobilis: Synergism and limitations

Summary A mixed culture of Endomycopsis fibuligera NRRL 76 and Zymomonas mobilis ZM4 could directly and more efficiently ferment cassava starch (22.5% w/v) to ethanol (10.5% v/v) than the monocultures. The combination of culture filtrate of E.fibuligera containing amylases and Z.mobilis simultaneously saccharified and fermented the cassava starch to ethanol equally well. Glucoamylase (0.01%) added to the fermenting medium improved ethanol (13.2% v/v) production by the above mixed culture to almost the theoretical level (98%) indicating that this enzyme is a rate-limiting factor in E.fibuligera. Z. mobilis alone converted the enzymehydrolyzed starch only to almost theoretical level (98%).     

Deciphering the Probiotic Potential of Bacillus amyloliquefaciens COFCAU_P1 Isolated from the Intestine of Labeo rohita Through In Vitro and Genetic Assessment

Probiotics and Antimicrobial Proteins - In this study, a bacterial strain COFCAU_P1, isolated from the digestive tract of a freshwater teleost rohu (Labeo rohita), was identified as Bacillus...     

Identification and Quantification of AKT Isoforms and Phosphoforms in Breast Cancer Using a Novel Nanofluidic Immunoassay

Breast cancer subtype-specific molecular variations can dramatically affect patient responses to existing therapies. It is thought that differentially phosphorylated protein isoforms might be a useful prognostic biomarker of drug response in the clinic. However, the accurate detection and quantitative analysis of cancer-related protein isoforms and phospho-isoforms in tumors are limited by current technologies. Using a novel, fully automated nanocapillary electrophoresis immunoassay (NanoPro^TM 1000) designed to separate protein molecules based on their isoelectric point, we developed a reliable and highly sensitive assay for the detection and quantitation of AKT isoforms and phosphoforms in breast cancer. This assay enabled the measurement of activated AKT1/2/3 in breast cancer cells using protein produced from as few as 56 cells. Importantly, we were able to assign an identity for the phosphorylated S473 phosphoform of AKT1, the major form of activated AKT involved in multiple cancers, including breast, and a current focus in clinical trials for targeted intervention. The ability of our AKT assay to detect and measure AKT phosphorylation from very low amounts of total protein will allow the accurate evaluation of patient response to drugs targeting activated PI3K-AKT using scarce clinical specimens. Moreover, the capacity of this assay to detect and measure all three AKT isoforms using one single pan-specific antibody enables the study of the multiple and variable roles that these isoforms play in AKT tumorigenesis.Activation of the PI3K-AKT signaling pathway is one of the most common events in cancer (1, 2). Pathway activation can confer a number of advantages to the cancer cells, including enhanced proliferation and survival (1, 2). Multiple mechanisms exist by which the pathway may become activated, including amplification or activation of receptor tyrosine kinases (e.g. ERBB2 in breast and EGFR in lung tumors), mutation of the catalytic or regulatory subunits of PI3K (e.g. PIK3CA in colorectal and breast tumors), loss of the negative regulator PTEN (e.g. mutation in prostate and melanoma), and gain of function of AKT (e.g. amplification or mutation in breast and pancreatic tumors) (reviewed in Refs. 1 and 2).AKT represents a central node in the PI3K signaling cascade (3). AKT is recruited to the cell membrane via its pleckstrin homology domain when PI3K phosphorylates PIP₂ to form PIP₃ (4, 5). Following recruitment, AKT is phosphorylated by PDK1 and the rictor-mTOR complex, resulting in conformational changes and activation of the protein (5–8). Multiple studies have shown that the phosphorylation of AKT leads to the phosphorylation and activation of downstream effectors of the signaling pathway, such as mTOR complex 1 and S6K (reviewed in Ref. 1). The central role of this pathway in cancer is further underscored by the efforts of multiple pharmaceutical companies that have developed inhibitors against AKT as potential anti-oncogenic therapeutics (9).Despite the importance of AKT in growth and survival signaling in cancer, there are surprisingly few data that address the specific roles played in growth and survival by the multiple AKT family members (AKT-1, -2, and -3) and different phosphorylation and putative phosphorylation sites that can potentially activate the protein. Western blot analysis has been the foundation of most AKT studies, but in many cases pan-AKT antibodies have been employed that fail to distinguish between the different AKT isoforms. Recent siRNA silencing studies have indicated distinct functions for different AKT family members within a cell (10, 11). Moreover, there is evidence in breast cancer that the three isoforms exhibit different localizations and therefore must have at least partially distinct functions (12). Similarly, evidence is mounting for multiple phosphorylation sites in AKT beyond the two most studied phosphorylation events (Thr-308 and Ser-473) (5–8). Phosphorylation at serine and threonine residues at Thr-72 and Ser-246 may be required for the activation or regulation of kinase activity (13). The functional significance of constitutive phosphorylation of Ser-124 and Thr-450 is still unknown (14). Finally, there is evidence that phosphorylation of tyrosine residues at Tyr-315 and Tyr-326 is required for full kinase activity (15).Analysis of such phospho- and isoform-specific activation often requires complicated in-depth analyses using large quantities of proteins, purified recombinant protein, immunoprecipitation, incorporation of ³²P isotopes, and/or mass spectroscopy, which makes such studies more difficult to perform and not easily adaptable to clinical specimens. Thus, better methods are required for the accurate assessment of both phosphoform and isoform usage in cells with an activated PI3K-AKT pathway and the effects of pathway inhibitors using relatively small amounts of starting material. We describe here the development of such an assay using nanocapillary-based isoelectric focusing (16). This approach allows the separation of AKT into distinct peaks that correspond to different iso- and phosphoforms using a small amount of starting material and a single pan-specific antibody. This approach should allow for more accurate determinations of isoform usage in different cell types, as well as of changes in phosphorylation states in response to pathway inhibition, including in clinical specimens.     

Liposomes and Liposome-like Vesicles for Drug and DNA Delivery to Mitochondria

Mitochondrial research is presently one of the fastest growing disciplines in biomedicine. Since the early 1990s, it has become increasingly evident that mitochondrial dysfunction contributes to a large variety of human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity, and diabetes to ischemia-reperfusion injury and cancer. Most remarkably, mitochondria, the “power house” of the cell, have also become accepted as the “motor of cell death” reflecting their recognized key role during apoptosis. Based on these recent exciting developments in mitochondrial research, increasing pharmacological efforts have been made leading to the emergence of “Mitochondrial Medicine” as a whole new field of biomedical research. The identification of molecular mitochondrial drug targets in combination with the development of methods for selectively delivering biologically active molecules to the site of mitochondria will eventually launch a multitude of new therapies for the treatment of mitochondria-related diseases, which are based either on the selective protection, repair, or eradication of cells. Yet, while tremendous efforts are being undertaken to identify new mitochondrial drugs and drug targets, the development of mitochondria-specific drug carrier systems is lagging behind. To ensure a high efficiency of current and future mitochondrial therapeutics, colloidal vectors, i.e., delivery systems, need to be developed able to selectively transport biologically active molecules to and into mitochondria within living human cells. Here we review ongoing efforts in our laboratory directed toward the development of different phospholipid- and non-phospholipid-based mitochondriotropic drug carrier systems.     

Insulin adsorption onto zinc oxide nanoparticle mediates conformational rearrangement into amyloid-prone structure with enhanced cytotoxic propensity

Background

Injection localized amyloidosis is one of the most prevalent disorders in type II diabetes mellitus (TIIDM) patients relying on insulin injections. Previous studies have reported that nanoparticles can play a role in the amyloidogenic process of proteins. Hence, the present study deals with the effect of zinc oxide nanoparticles (ZnONP) on the amyloidogenicity and cytotoxicity of insulin.

Pathogen-specific CD8 T cell responses are directly inhibited by IL-10

Regulation of CD8 T cell expansion and contraction is essential for successful immune defense against intracellular pathogens. IL-10 is a regulatory cytokine that can restrict T cell responses by inhibiting APC functions. IL-10, however, can also have direct effects on T cells. Although blockade or genetic deletion of IL-10 enhances T cell-mediated resistance to infections, the extent to which IL-10 limits in vivo APC function or T cell activation/proliferation remains unknown. Herein, we demonstrate that primary and memory CD8 T cell responses following Listeria monocytogenes infection are enhanced by the absence of IL-10. Surface expression of the IL-10R is transiently up-regulated on CD8 T cells following activation, suggesting that activated T cells can respond to IL-10 directly. Consistent with this notion, CD8 T cells lacking IL-10R2 underwent greater expansion than wild-type T cells upon L. monocytogenes infection. The absence of IL-10R2 on APCs, in contrast, did not enhance T cell responses following infection. Our studies demonstrate that IL-10 produced during bacterial infection directly limits expansion of pathogen-specific CD8 T cells and reveal an extrinsic regulatory mechanism that modulates the magnitude of memory T cell responses.     

Deletions in chromosome 4 differentially associated with the development of cervical cancer: evidence of slit2 as a candidate tumor suppressor gene

The aim of this study was to locate the candidate tumor suppressor genes (TSGs) loci in the chromosomal 4p15-16, 4q22-23 and 4q34-35 regions associated with the development of uterine cervical carcinoma (CA-CX). Deletion mapping of the regions by microsatellite markers identified six discrete areas with high frequency of deletions, viz. 4p16.2 (D1: 40%), 4p15.31 (D2: 35–38%), 4p15.2 (D3: 37–40%), 4q22.2 (D4: 34%), 4q34.2-34.3 (D5: 37–59%) and 4q35.1 (D6: 40–50%). Significant correlation was noted among the deleted regions D1, D2 and D3. The deletions in D1, D2, D5 and D6 regions are suggested to be associated with the cervical intraepithelial neoplasia (CIN), and deletions in the D2, D3, D5 and D6 regions seems to be associated with progression of CA-CX. The deletions in the D2 and D6 regions showed significant prognostic implications (P = 0.001; 0.02). The expression of the candidate TSG SLIT2 mapped to D2 region gradually reduced from normal cervix uteri →CIN → CA-CX. SLIT2 promoter hypermethylation was seen in 28% CIN samples and significantly increased with tumor progression (P = 0.04). Significant correlation was seen between SLIT2 deletion and its promoter methylation (P = 0.001), indicating that both these phenomena could occur simultaneously to inactivate this gene. Immunohistochemical analysis showed reduced expression of SLIT2 in cervical lesions and CA-CX cell lines. Although no mutation was detected in the SLIT2 promoter region (−432 to + 55 bp), CC and AA haplotypes were seen in −227 and −195 positions, respectively. Thus, it indicates that inactivation of SLIT2-ROBO1 signaling pathway may have an important role in CA-CX development.     

Interaction among bovine serum albumin (BSA) molecules in the presence of anions: a small-angle neutron scattering study

Journal of Biological Physics - Protein–protein interaction in solution strongly depends on dissolved ions and solution pH. Interaction among globular protein (bovine serum albumin, BSA),...     

A critical life-supporting role for cystathionine γ-lyase in the absence of dietary cysteine supply

This study examined the important relationship between cystathionine γ-lyase (CSE) functionality and cysteine supply for normal growth and life span. Mice with a targeted deletion of the CSE gene (CSE-KO) were fed a cysteine-limited diet and their growth and survival patterns as well as levels of cysteine, homocysteine, glutathione, and hydrogen sulfide (H2S) were measured. CSE-KO mice fed a cysteine-limited diet exhibited growth retardation; decreased levels of cysteine, glutathione, and H2S; and increased plasma homocysteine level. However, histological examinations of liver did not reveal any abnormality and plasma levels of aspartate aminotransferase, alanine aminotransferase, and albumin were normal in these animals. No CSE-KO mice survived after 12 weeks of feeding with the cysteine-limited diet. Supplementation of H2S to the CSE-KO mice failed to reverse the aforementioned abnormalities. On the other hand, supplementation of cysteine in the drinking water of the CSE-KO mice significantly increased plasma cysteine and glutathione levels. This eventually led to an increase in body weight and rescued the animals from death. In conclusion, CSE is critical for cysteine biosynthesis through the transsulfuration pathway and the combination of CSE deficiency and lack of dietary cysteine supply would threaten life sustainability.