A culture substratum appropriate for brain cells is a chondroitin sulfate glycosaminoglycan in serum

When cells dissociated from the neonatal rat brains are plated on a poly-lysine-coated surface in a serum-free medium, they display a strange morphology: a dark and extended cell body. Preincubation of the surface with fetal bovine serum was found to inhibit the appearance of this strange contraction of the basal cell sheets in a dose-dependent manner. This finding indicated the presence of a factor(s) in the serum, which might be an appropriate substratum for prolonged survival of brain neurons. In the current study, this factor was highly purified through DEAE ion-exchange chromatography followed by gel filtration. The factor was eluted from a Superose column at fractions corresponding to a molecular weight greater than 1000 kDa. By SDS-PAGE analysis, these fractions were found to contain a major band (≥1000 kDa) positive for alcian blue and few minor bands faintly stainable with Coomassie blue. The activity of the purified sample, inducing the morphological change in cells, was diminished by incubation with chondroitinase ABC. Neither heparitinase II, hyaluronidase, nor trypsin modified the activity. An authentic chondroitin sulfate (type B) mimicked the serum action on the morphology of brain cells in early stages of culture. Taking these findings together, it is suggested that the factor in serum beneficial for the attachment of brain cells is composed of a chondroitin sulfate with a Mr greater than 1000 kDa. Cortical cells dissociated from the neonatal rat brain attached well to the purified factor-coated surface and displayed a healthy morphology: an optically-reflective cell body with thick neurites for at least 3 days in the absence of serum.     

Measurement of amyloid formation by turbidity assay—seeing through the cloud

Detection of amyloid growth is commonly carried out by measurement of solution turbidity, a low-cost assay procedure based on the intrinsic light scattering properties of the protein aggregate. Here, we review the biophysical chemistry associated with the turbidimetric assay methodology, exploring the reviewed literature using a series of pedagogical kinetic simulations. In turn, these simulations are used to interrogate the literature concerned with in vitro drug screening and the assessment of amyloid aggregation mechanisms.     

A gene cluster for the synthesis of serotype g-specific polysaccharide antigen in Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is an important pathogen related to aggressively progressive periodontal breakdown in adolescents and adults. The species can be divided into six serotypes (a–f) according to their surface carbohydrate antigens. Recently, a new serotype g of A. actinomycetemcomitans was proposed. The aim of the present study was to sequence the gene cluster associated with the biosynthesis of the serotype g-specific polysaccharide antigen and develop serotype-specific primers for PCR assay to identify serotype g strains of A. actinomycetemcomitans. The serotype-specific polysaccharide (SSPS) gene cluster of the NUM-Aa 4039 strain contained 21 genes in 21,842-bp nucleotides. The similarity of the SSPS gene cluster sequence was 96.7 % compared with that of the serotype e strain. Seventeen serotype g genes showed more than 90 % homology both in nucleotide and amino acids to the serotype e strain. Three additional genes with 1,579 bp in NUM-Aa 4039 were inserted into the corresponding ORF13 of the serotype e strain. The serotype g-specific primers were designed from the insertion region of NUM-Aa 4039. Serotypes of the a–f strains were not amplified by serotype-specific g primers; only NUM-Aa 4039 showed an amplicon band. The NUM-Aa 4039 strain was three genes in the SSPS gene cluster different from those of serotype e strain. The specific primers derived from these different regions are useful for identification and distribution of serotype g strain among A. actinomycetemcomitans from clinical samples.     

Selectivity of Mitis Salivarius agar and a new selective medium for oral streptococci in dogs

An evaluation on the applicability of Mitis Salivarius agar (MS) medium, commonly used for the detection of oral streptococci in human and animals, to dog specimens and the development of a new selective medium for isolating streptococci from the canine oral cavity are described. Oral samples from dogs were cultured on MS medium under anaerobic conditions. The predominant facultative anaerobic bacteria on MS plates were gram-negative rods. Selectivity of streptococci on MS medium was 21.2%. A new selective medium, designated MS-CAN-AE, was developed for the isolation of streptococci from the canine oral cavity. The average growth recovery of laboratory and clinically isolated strains of streptococci on MS-CAN-AE medium was 84.1% of that on MS medium. Gram-positive rods and gram-negative rods and cocci rarely grew on the MS-CAN-AE. The selectivity of MS-CAN-AE was 95.0% for clinical samples. MS-CAN-AE medium will be helpful for investigations of streptococci in the canine oral cavity.     

Examination of drug resistance activity of human TAP-like (ABCB9) expressed in yeast

A half-type ABC transporter, human TAP-like (hTAPL) tagged with histidine cluster, was expressed in budding yeast protease-deficient strain BJ5457, and the effect of expression for resistance to peptide compounds including antibiotics and proteinase inhibitor was examined. Among these compounds, the yeast expressing hTAPL exhibits high sensitivity to valinomycin, a monovalent cation ionophore. A mutation in Walker A motif, which lost ATP-binding activity of hTAPL, eliminated the enhanced sensitivity to valinomycin. These findings suggest that the transport activity of hTAPL is important for conferring high valinomycin-sensitive phenotype to yeast.     

Identification and characterization of Arabidopsis gibberellin receptors

Three gibberellin (GA) receptor genes (AtGID1a, AtGID1b and AtGID1c), each an ortholog of the rice GA receptor gene (OsGID1), were cloned from Arabidopsis, and the characteristics of their recombinant proteins were examined. The GA-binding activities of the three recombinant proteins were confirmed by an in vitro assay. Biochemical analyses revealed similar ligand selectivity among the recombinants, and all recombinants showed higher affinity to GA(4) than to other GAs. AtGID1b was unique in its binding affinity to GA(4) and in its pH dependence when compared with the other two, by only showing binding in a narrow pH range (pH 6.4-7.5) with 10-fold higher affinity (apparent K(d) for GA(4) = 3 x 10(-8) m) than AtGID1a and AtGID1c. A two-hybrid yeast system only showed in vivo interaction in the presence of GA(4) between each AtGID1 and the Arabidopsis DELLA proteins (AtDELLAs), negative regulators of GA signaling. For this interaction with AtDELLAs, AtGID1b required only one-tenth of the amount of GA(4) that was necessary for interaction between the other AtGID1s and AtDELLAs, reflecting its lower K(d) value. AtDELLA boosted the GA-binding activity of AtGID1 in vitro, which suggests the formation of a complex between AtDELLA and AtGID1-GA that binds AtGID1 to GA more tightly. The expression of each AtGID1 clone in the rice gid1-1 mutant rescued the GA-insensitive dwarf phenotype. These results demonstrate that all three AtGID1s functioned as GA receptors in Arabidopsis.