Replacing β-mercaptoethanol in RNA extractions

RNA extractions are potentially compromised in terms of both yield and quality by ribonucleases (RNases). The pungent and toxic reducing agent β-mercaptoethanol (β-ME), therefore, is commonly added to the biospecimen’s lysis buffer to aid in RNase deactivation. Using different tissue types (liver tissue, kidney tissue, and cell pellets), extraction kits (RNeasy Mini Kit, Illustra RNA Spin Mini Kit, and PureLink Mini Kit), RNA quality assays (RNA integrity numbers [RINs] and quantitative real-time polymerase chain reaction [qRT–PCR]), yield assessments, and in vitro functional RNase assays (RNaseAlert Kit), we demonstrate that β-ME should be replaced by the less toxic dithiothreitol (DTT) alternative.     

Mitochondrial β-amyloid in Alzheimer's disease

It is well established that the intracellular accumulation of Aβ (amyloid β-peptide) is associated with AD (Alzheimer's disease) and that this accumulation is toxic to neurons. The precise mechanism by which this toxicity occurs is not well understood; however, identifying the causes of this toxicity is an essential step towards developing treatments for AD. One intracellular location where the accumulation of Aβ can have a major effect is within mitochondria, where mitochondrial proteins have been identified that act as binding sites for Aβ, and when binding occurs, a toxic response results. At one of these identified sites, an enzyme known as ABAD (amyloid-binding alcohol dehydrogenase), we have identified changes in gene expression in the brain cortex, following Aβ accumulation within mitochondria. Specifically, we have identified two proteins that are up-regulated not only in the brains of transgenic animal models of AD but also in those of human sufferers. The increased expression of these proteins demonstrates the complex and counteracting pathways that are activated in AD. Previous studies have identified approximate contact sites between ABAD and Aβ; on basis of these observations, we have shown that by using a modified peptide approach it is possible to reverse the expression of these two proteins in living transgenic animals and also to recover mitochondrial and behavioural deficits. This indicates that the ABAD-Aβ interaction is potentially an interesting target for therapeutic intervention. To explore this further we used a fluorescing substrate mimic to measure the activity of ABAD within living cells, and in addition we have identified chemical fragments that bind to ABAD, using a thermal shift assay.     

Biosynthesis of β-glucans in fungi

Glucans are the most abundant polysaccharides present in fungi. The present review provides updated information on the structure and synthesis of -glucans in fungal cells. Synthesis of these polymers made up of B1,3 chains with a variable degree of B1,6 branching involves several reactions: initiation, chain elongation and branching, of which the most studied one is the elongation step. This reaction, catalyzed by the so-called glucan synthetases, utilizes UDPG as sugar donor. Properties of glucan synthetases are extremely variable depending on the fungal species, and their developmental stage. Because of the importance of these polysaccharides it is anticipated that comprehension of their mechanism of synthesis, is important for the understanding of cell wall assembly and cell growth and morphogenesis, as well as for the design of specific antifungal drugs.Abreviations UDPG uridine-diphospho-glucose - GDPG guanosine-diphospho-glucose - ADPG adenosine-diphospho-glucose - MW molecular weight - mic minimal inhibitory concentration - d.p. degree of polymerization - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Exogenous seeding of cerebral β-amyloid deposition in βAPP-transgenic rats

Deposition of the amyloid-β (Aβ) peptide in senile plaques and cerebral Aβ angiopathy (CAA) can be stimulated in Aβ-precursor protein (APP)-transgenic mice by the intracerebral injection of dilute brain extracts containing aggregated Aβ seeds. Growing evidence implicates a prion-like mechanism of corruptive protein templating in this phenomenon, in which aggregated Aβ itself is the seed. Unlike prion disease, which can be induced de novo in animals that are unlikely to spontaneously develop the disease, previous experiments with Aβ seeding have employed animal models that, as they age, eventually will generate Aβ lesions in the absence of seeding. In the present study, we first established that a transgenic rat model expressing human APP (APP21 line) does not manifest endogenous deposits of Aβ within the course of its median lifespan (30?months). Next, we injected 3-month-old APP21 rats intrahippocampally with dilute Alzheimer brain extracts containing aggregated Aβ. After a 9-month incubation period, these rats had developed senile plaques and CAA in the injected hippocampus, whereas control rats remained free of such lesions. These findings underscore the co-dependence of agent and host in governing seeded protein aggregation, and show that cerebral Aβ-amyloidosis can be induced even in animals that are relatively refractory to the spontaneous origination of parenchymal and vascular deposits of Aβ.     

Genetic relationship between β-galactosidase and β-fucosidase in the mouse

Evidence for the identity of β-galactosidase and β-fucosidase enzymes in the house mouse was obtained by examination of the enzyme activities in animals from different crosses between C57BL/Kl mice with high galactosidase and fucosidase activities and DBA/2/Kl mice with low activities. There is a strong correlation between the activities of these two enzymes in different tissues of F₂ animals. A comparison of the fractionation properties of β-galactosidase and β-fucosidase showed that the two activities had a parallel distribution and identical thermostability. These data suggest that the same enzyme catalyzes the hydrolysis of both substrates.     

Roles of β-catenin in somitogenesis in rat embryos

Summary We studied the roles of β-catenin in somitogenesis using immunostaining and antisense experiments in rat embryos. High levels of β-catenin appeared transiently in the developing rat somites. Initially, β-catenin accumulation was observed in the core cells of presomitic cell aggregates and then in the lumen of epithelial vesicles. Subsequently, it was confined to the dermomyotomes and their lumen and then the myotomes. High levels of cyclin D1 were observed in the core cells, in the lumen of epithelial vesicles, in myotomes, and in mesenchymal sclerotomes. When embryos were cultured in medium supplemented with β-catenin antisense oligodeoxynucleotide (ODN), the accumulation of β-catenin, but not of cyclin D1, in the nascent somites and dermomyotomes was suppressed, while the number of somites was the same as that observed in control embryos. The number of myosin-positive somites and the amount of myosin per somite in embryos treated with the antisense ODN were lower than those in controls. These results suggested that β-catenin promotes development of myotomal cells during somitogenesis. The function of β-catenin in the development of myotomes may not be correlated to cyclin D1.     

Alterations in glycosaminoglycan metabolism in β-aminopropionitrile-treated chick embryos

1. Na(2) (35)SO(4), [1-(14)C]glucosamine and [1-(14)C]acetate were used as precursors of the sulphated glycosaminoglycans to study the biochemical effect of beta-aminopropionitrile in chick embryos. The incorporation of all three precursors was decreased in the treated embryos between days 7 and 10 of embryonic development. No inhibition of incorporation of these precursors occurred between days 16 and 20 of embryonic development at the dosages of beta-aminopropionitrile used. 2. beta-Aminopropionitrile treatment also decreased the amount of N-acetylhexosamines in the chick embryo and decreased the percentage of the hexosamine esterified by nucleotides. Respiration was decreased by homogenates prepared from treated embryos. Likewise, UDP-xylosyl- and UDP-galactosyl-transferase activities were decreased in treated embryos and cartilage from embryos and growing chicks. 3. The data suggest that beta-aminopropionitrile, in addition to the known lathyrogenic activity, either is or gives rise to a potent metabolic poison that interferes with basic cellular metabolism. The results are consistent with a decreased rate of ATP generation as an explanation for the decrease in glycosaminoglycan synthesis.     

Deprotonation of β-cyclodextrin in alkaline solutions

Variable pH ¹³C NMR and ¹H NMR spectroscopic studies of the β-cyclodextrin (β-CD) in alkaline aqueous solutions revealed that β-CD does not deprotonate at pH < 12.0. Further increase in solution pH results in the deprotonation of OH-groups adjacent to C-2 and C-3 carbon atoms of β-CD glucopyranose units, whereas the deprotonation of OH-groups adjacent to C-6 carbon atoms is expressed less markedly. The pK_a values for β-CD OH-groups adjacent to C-2 and C-3 carbon atoms are rather close, pK_a1,2 being 13.5 ± 0.2 (22.5 °C).     

Nutrient toxicity in pancreatic β-cell dysfunction

Nutrients, such as glucose and fatty acids, have a dual effect on pancreatic beta-cell function. Acute administration of high glucose concentrations to pancreatic beta-cells stimulates insulin secretion. In addition, short term exposure of this cell type to dietary fatty acids potentiates glucose-induced insulin release. On the other hand, long-term exposure to these nutrients causes impaired insulin secretion, characterized by elevated exocytosis at low concentrations of glucose and no response when glucose increases in the extracellular medium. In addition, other phenotypic changes are observed in these conditions. One major step in linking these phenotypic changes to the diabetic pathology has been the recognition of both glucose and fatty acids as key modulators of beta-cell gene expression. This could explain the adaptative response of the cell to sustained nutrient concentration. Once this phase is exhausted, the beta-cell becomes progressively unresponsive to glucose and this alteration is accompanied by the irreversible induction of apoptotic programs. The aim of this review is to present actual data concerning the development of the toxicity to the main nutrients glucose and fatty acids in the pancreatic beta-cell and to find a possible link to the development of type 2 diabetes.     

Alicyclic β-amino acids in Medicinal Chemistry

Summary. The structural element of alicyclic β-amino acids shows some remarkable biological effects: For some 5- and 6-membered β-amino acids a unique anti fungal activity has been observed, 7-membered β-amino acid derivatives have been investigated for neurological disorders. The application of 5-, 6- and 7-membered alicyclic β-amino acids in Medicinal Chemistry will be reported.     

Involvement of β-arrestins in cancer progression

β-arrestins, including β-arrestin1 and β-arrestin2, are ubiquitous cytosolic proteins which localize in the cytoplasm and plasma membrane, initially be regarded as an potential character in G protein-coupled receptors (GPCR) desensitization, sequestration, and internalization. Besides, recent many studies increasingly revealed that β-arrestins served widely as versatile adapter proteins for scaffolding many intracellular signaling networks to modulate the strength and duration of signaling by diverse types of receptors and downstream kinases. As we known, the biologic and clinical behaviors of many tumors are largely determined by multiple molecular signal pathways. More recently, accumulating evidences established that β-arrestins got widely involved in many cancer developmental signaling events which responsible for tumor viability and metastasis, suggesting an impressive role of β-arrestins in tumor progression. Because of the regulation and biological output of β-arrestins is so complex, the role of β-arrestins in cancer development still remains enigmatic. However, the further understanding with the clinical prognosis and oncogenic potential of β-arrestins might facilitate the identification of diagnosis biomarkers and development of drug targets in cancer. In this article, we reviewed a comprehensive summary of the β-arrestins-mediated functions in human cancers.     

Proton-coupled β-galactoside translocation in non-metabolizingEscherichia coli

Acid-base and electrogenic processes coupled to the flux of -galactosides into non-metabolizing cells ofEscherichia coli have been studied.When -glactoside was added to non-metabolizing suspensions ofE. coli, the pH of the suspension medium increased, indicating that the -galactoside travelled in with acid equivalents. When the cells were made permeable to K⁺ ions, this inflow of acid equivalents was accompanied by an equal outflow of K⁺ ions, indicating that each acid equivalent carried one positive charge across the membrane, and corresponded to an H⁺ ion going in or an OH^– ion coming out. The effective movement of H⁺ ions, caused either by a pH difference or by an electrical potential difference across the membrane of the cells, was specifically facilitated by the presence of -galactoside. These effects of -galactoside were abolished by N-ethyl maleimide, which is known to inhibit the specific -galactoside translocation.The possible involvement of a Na⁺--galactoside symporter was ruled out by showing that the galactoside-induced inflow of acid was practically independent of Na⁺ ion concentration in the range 0.05–50.0 mM, and that Na⁺ ions did not flow into the bacteria under the influence of a -galactoside concentration gradient.It is concluded that the -galactoside translocation inE. coli is probably mediated by a -galactoside-H⁺ symporter or by a -galactoside/OH^– antiporter.Abbreviations ATPase adenosine triphosphatase - FCCP carbonylcyanidep-trifluoromethoxyphenylhydrazone - NEM N-ethyl maleimide - TMG methyl--D-thiogalactoside - H ₀ ⁺ quantity of H⁺ ions entering unit volume of the outer aqueous phase; pH₀, the pH of the outer aqueous phase. The same conventions are used for potassium (K) and sodium (Na)     

Induction of β-N-acetylhexosaminidase in Aspergillus oryzae

Summary Extracellular -N-acetylhexosaminidase in basic specific activity 1.5 U/mg protein was induced 15 – 35 times (up to 50 U/mg protein) by mixture of chitooligomers (crude chitin hydrolysate), 10 – 20 times (20 – 30 U/mg protein) by N-acetylglucosamine, and 10 times (14 U/mg protein) by chitosan in Aspergillus oryzae. Addition of NaCl (15 – 23 g/l) to the cultivation medium enhanced the induction in 10 – 20 %.     

Alarming β-lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae

Resistance to β-lactams and other antibiotics in the Enterobacteriaceae is frequently associated with plasmidic resistance determinants that are easily transferred among species. β-Lactamase-mediated resistance is increasingly associated with plasmid-encoded extended-spectrum β-lactamases (ESBLs) and carbapenemases, specifically the CTX-M family of ESBLs, the KPC family of serine carbapenemases, and the VIM, IMP, and NDM-1 metallo-β-lactamases. Although clonal dispersion of resistant isolates was seen initially, more diverse genetic platforms are being observed as variations of mobile elements are transferred worldwide. These enzymes are now appearing in multiple combinations of ESBLs and carbapenemases, thereby conferring resistance to virtually all β-lactam antibiotics.     

Entrapment of β-galactosidase in polyvinylalcohol hydrogel

β-Galactosidase isolated from Aspergillus oryzae was immobilized in lens-shaped polyvinylalcohol capsules (with activity 25 U g⁻¹) giving 32% of its original activity. Immobilization did not change the pH optimum (4.5) of lactose hydrolysis. The relative enzyme activity during product inhibition testing was, in average, 10% higher for immobilized enzyme. No decrease of activity was observed after 35 repeated batch runs and during 530 h of continuous hydrolysis of lactose (10%, w/v) at 45°C. The immobilized enzyme was stable for 14 months without any change of activity during the storage at 4°C and pH 4.5.     

Fungal β-glucosidase expression in Saccharomyces cerevisiae

Recombinant Saccharomyces cerevisiae strains expressing β-glucosidases from Thermoascus aurantiacus (Tabgl1) and Phanerochaete chrysosporium (PcbglB and Pccbgl1) were constructed and compared to S. cerevisiae Y294[SFI], previously identified as the best β-glucosidase-producing strain. The PcbglB was also intracellularly expressed in combination with the lac12 lactose permease of Kluyveromyces lactis in S. cerevisiae Y294[PcbglB?+?Lac12]. The recombinant extracellular β-glucosidases indicated maximum activity in the pH range 4-5 and temperature optima varying from 50 to 75?°C. The S.?cerevisiae Y294[Pccbgl1] strain performed best under aerobic and anaerobic conditions, producing 2.6 times more β-glucosidase activity than S. cerevisiae Y294[SFI] and an ethanol concentration of 4.8?g?l(-1) after 24?h of cultivation on cellobiose as sole carbohydrate source. S. cerevisiae Y294[Tabgl1] was unable to grow on cellobiose (liquid medium), whereas S. cerevisiae Y294[PcbglB?+?Lac12] exhibited limited growth.     

Engineering lower inhibitor affinities in β-d-xylosidase

β-d-Xylosidase catalyzes hydrolysis of xylooligosaccharides to d-xylose residues. The enzyme, SXA from Selenomonas ruminantium, is the most active catalyst known for the reaction; however, its activity is inhibited by d-xylose and d-glucose (K _i values of ～10^?2?M). Higher K _i’s could enhance enzyme performance in lignocellulose saccharification processes for bioethanol production. We report here the development of a two-tier high-throughput screen where the 1° screen selects for activity (active/inactive screen) and the 2° screen selects for a higher K _i(d-xylose) and its subsequent use in screening ～5,900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXA-C3, K _i(d-xylose) is threefold and K _i(d-glucose) is twofold that of wild-type SXA. C3 contains four amino acid mutations, and one of these, W145G, is responsible for most of the lost affinity for the monosaccharides. Experiments that probe the active site with ligands that bind only to subsite ?1 or subsite +1 indicate that the changed affinity stems from changed affinity for d-xylose in subsite +1 and not in subsite ?1 of the two-subsite active site. Trp145 is 6 Å from the active site, and its side chain contacts three active-site residues, two in subsite +1 and one in subsite ?1.     

Molecular characterization of β-thalassemia in Azerbaijan

We have analyzed the -thalassemia mutations in 99 chromosomes of 49 adults with -thalassemia major and of one with Hb S--thalassemia, who are regular patients at a large hematology clinic in Bakü, Azerbaijan. A total of 20 different mutants were identified; three [frameshift at codon 8 (-AA); IVS-II-I (GA); IVS-I-110 (GA)] were present in about two-thirds of all chromosomes. Most alleles are the same as found in Mediterranean populations; a few have an Asian origin or come from Kurdistan, Lebanon, Saudi Arabia, or a black population. One mutant [frameshift at codons 82/ 83 (-G)] might be specific for the Azerbaijanian population. Nearly all patients were transfused, which made quantitation of Hb F impossible; high^G values were present in the Hb F of those patients whose -thalassemia chromosome carried the C T mutation at position — 158 in the promoter of the ^G-globin gene.     

Phytohormone-regulated β-amylase gene expression in rice

The expression of -amylase genes in rice (Oryza sativa) and its regulation by phytohormones gibberellic acid (GA) and abscisic acid (ABA) were examined. Upon germination -amylase is synthesizedde novo in aleurone cells and (GA) is not required. Exogenous addition of GA does not enhance the -amylase activity, while ABA inhibits the -amylase activity, mRNA accumulation, and the germination of rice seeds. GA can reverse ABA inhibition of -amylase expression, but not ABA inhibition of seed germination. Such regulation represents a new interaction of ABA and GA.