Avidin related protein 2 shows unique structural and functional features among the avidin protein family

Background  

The chicken avidin gene family consists of avidin and several avidin related genes (AVRs). Of these gene products, avidin is the best characterized and is known for its extremely high affinity for D-biotin, a property that is utilized in numerous modern life science applications. Recently, the AVR genes have been expressed as recombinant proteins, which have shown different biotin-binding properties as compared to avidin.     

Regional Cerebral Glucose Metabolism and Blood Flow During the Silent Phase of Methylmercury Neurotoxicity in Rats

Methylmercuric chloride was given to rats in a neurotoxic dose regimen (six daily doses of 8 mg kg-1 p.o.). During the silent (asymptomatic) phase of intoxication, the rates of cerebral glucose influx and cerebral glucose phosphorylation were measured simultaneously using 2-deoxyglucose. Regional cerebral blood flow was also measured using iodoantipyrine. The unidirectional flux of glucose into brain was not affected by methylmercury, and differences in the rates of glucose phosphorylation from region to region remained coupled to the regional cerebral blood flow. However, the blood flow was reduced throughout the brain, an observation suggesting that the operational level of metabolically regulated blood flow had been reset. Thus, in spite of a generalised reduction in blood flow, there was no indication of impaired cerebral glucose supply or utilization during the silent phase of methylmercury intoxication.     

The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

Plakoglobin (PG) is an armadillo protein that associates with both classic and desmosomal cadherins, but is primarily concentrated in mature desmosomes in epithelia. While reduced levels of PG have been reported in localized and hormone refractory prostate tumors, the functional significance of these changes is unknown. Here we report that PG expression is reduced in samples of a prostate tumor tissue array and inversely correlated with advancing tumor potential in 7 PCa cell lines. Ectopically expressed PG enhanced intercellular adhesive strength, and attenuated the motility and invasion of aggressive cell lines, whereas silencing PG in less tumorigenic cells had the opposite effect. PG also regulated cell-substrate adhesion and motility through extracellular matrix (ECM)-dependent inhibition of Src kinase, suggesting that PG's effects were not due solely to increased intercellular adhesion. PG silencing resulted in elevated levels of the ECM protein vitronectin (VN), and exposing PG-expressing cells to VN induced Src activity. Furthermore, increased VN levels and Src activation correlated with diminished expression of PG in patient tissues. Thus, PG may inhibit Src by keeping VN low. Our results suggest that loss of intercellular adhesion due to reduced PG expression might be exacerbated by activation of Src through a PG-dependent mechanism. Furthermore, PG down-regulation during PCa progression could contribute to the known VN-dependent promotion of PCa invasion and metastasis, demonstrating a novel functional interaction between desmosomal cell-cell adhesion and cell-substrate adhesion signaling axes in prostate cancer.     

Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)₂. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.     

A reassessment of decreased amino acid accumulation by ehrlich ascites tumor cells in the presence of metabolic inhibitors

This study was undertaken to examine the mechanism by which metabolic inhibition reduces amino acid active transport in ehrlich ascites tumor cells. At 37 degrees C the metabolic inhibitor combination 0.1 mM 2,4-dinitrophenol (DNP) + 10 mM 2- deoxy-D-glucose (DOG) reduced the cell ATP concentration to 0.10- 0.15 mM in less than 5 min. This inhibition was associated with a 20.6 percent +/- 6.4 percent (SD) decrease in the initial influx of α-aminoisobutyric acid (AIB), and a two- to fourfold increase in the unidirectional efflux. These effects could be dissociated from changes in cell Na(+) or K(+) concentrations. Cells incubated to the steady state in 1.0-1.5 mM AIB showed an increased steady-state flux in the presence of DNP + DOG. Steady- state fluxes were consistent with trans-inhibition of AIB influx and trans-stimulation of efflux in control cells, but trans- stimulation of both fluxes in inhibited cells. In spite of the reduction of the cell ATP concentration to less than 0.15 mM and greatly reduced transmembrane concentration gradients of Na(+) and K(+), cells incubated to the steady state in the presence of the inhibitors still established an AIB distribution ration 13.8 +/- 2.6. The results are interpreted to indicate that a component of the reduction of AIB transport produced by metabolic inhibition is attributable to other actions in addition to the reduction of cation concentration gradients. Reduction of cell ATP alone is not responsible for the effects of metabolic inhibition, and both the transmembrane voltage and direct coupling to substrate oxidation via plasma-membrane-bound enzymes must be considered as possible energy sources for amino acid active transport.     

Patterns of MADS-box gene expression mark flower-type development in Gerbera hybrida (Asteraceae)

Background  

The inflorescence of the cut-flower crop Gerbera hybrida (Asteraceae) consists of two principal flower types, ray and disc, which form a tightly packed head, or capitulum. Despite great interest in plant morphological evolution and the tractability of the gerbera system, very little is known regarding genetic mechanisms involved in flower type specification. Here, we provide comparative staging of ray and disc flower development and microarray screening for differentially expressed genes, accomplished via microdissection of hundreds of coordinately developing flower primordia.     

A pilot study on the relation between irisin single-nucleotide polymorphism and risk of myocardial infarction

BackgroundMyocardial infarction (MI) is the major cause of death and disability worldwide. Many recent studies revealed the relationship between circulating irisin levels, endothelial dysfunctions and subclinical atherosclerosis in adult patients.ObjectivesThe aim of this study was to investigate the distribution of Irisin gene single nucleotide polymorphism in patients with MI and its association with other clinical and laboratory variables in these patients.Patients and methodsThis study was carried out in 100 patients with MI, and 100 healthy subjects served as controls. All studied subjects underwent laboratory investigations, including measurement of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c) high-density lipoprotein cholesterol (HDL-c), creatinine kinase-MB (CK-MB), troponin I (TnI) and genotyping of rs 3480 and rs726344 of Irisin genes using the TaqMan Allelic Discrimination assay technique.ResultsThere was a significant difference of Irisin genotypes in patients when compared to controls. By estimating odd ratio (OR) an association was found between G allele of rs 3480 and A allele of rs726344with increase the risk of developing myocardial infarction by 4.03 and 3.47 fold respectively. GG of rs 3480 carriers had significantly increased Troponin I and triglyceride levels, while GA carriers of rs726344 had significantly increased CKMB, Total cholesterol, LDLc, HDLc, troponin I and triglyceride levels compared with other genotypes.ConclusionG allele of rs 3480 and A allele of rs726344can considered as genetic risk factors for MI; these findings could have an impact on preventive strategy for myocardial infarction.     

Vacuolar reticulum in oomycete hyphal tips: An additional component of the Ca2+Regulatory system?

Cultures of Achlya sp., Phytophthora cinnamomi, Saprolegnia diclina, S. ferax, and S. parasitica, treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae of S. ferax. The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca2+ sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization. Copyright 1997 Academic Press. Copyright 1997 Academic Press