The effect of antiserum quality on strain specificity assessment of foot and mouth disease virus by the neutralization reaction

The factors affecting the virus strain specificity of antibody to foot an mouth disease virus prepared by a variety of protocols in several species were evaluated by neutralization tests. The time at which the serum was taken, the antigen dose given, whether or not revaccination had occurred and the animal species in which the sera were prepared, did not appear to affect the strain specificity of serum prepared to inactivated antigens when measured in neutralization tests, probably because of the restricted nature of the antigenic site involved. However, variation was observed with convalescent animal sera or sera from animals which had received trypsin cleaved virus were used. For these reasons banks of reference antisera are prepared as pooled sera using one or two inoculations of inactivated antigen.     

The fine structure of HNK-1 (Leu7) positive cells

Summary The ultrastructural characteristics of HNK-1 (Leu7) positive cells, visualized with a peroxidase labelled anti-mouse IgM serum, were analysed. Our investigation demonstrates: 1) the majority of Leu7 positive cells has a low nuclear/cytoplasmic ratio (N/C), an irregular outline, a well developed smooth endoplasmic reticulum, parallel tubular arrays (PTA) and electron dense granules; 2) the minority of Leu7 positive cells has a high N/C, regular profiles and lacks electron dense granules. The presence of two distinct ultrastructural patterns within Leu7 positive cells may represent: 1) the expression of subsequent stages of cell differentiation; 2) two distinct Leu7 positive cell subpopulations.     

Efficient direct chromosome analyses and enzyme determinations from chorionic villi samples in the first trimester of pregnancy

Chorionic villi were obtained by an aspiration technique which proved to be the best of four alternative procedures. We report in detail the series of experiments which led to (1) successful, rapidly growing cell cultures practically free of maternal cell contamination (the use of hormone-supplemented Chang medium greatly increased the growth rate); (2) an efficient direct method to obtain high quality metaphases from the Langhans cells of the cytotrophoblast tissue and with which the fetal karyotype is defined within a few hours of chorionic villi sampling; and (3) successful testing for the activity of eight enzymes directly from the villi samples, thus showing that this material is suitable for a rapid, direct diagnosis of the related metabolic diseases.     

Optokinetic visual detection in the rat visual centres. A [14C]-2-deoxy-D-glucose study

Following forty-five min of binocular optokinetic stimulation (OKS) the autoradiographic maps of [14C]-2-deoxy-D-glucose (2DG) assumption of Long-Evans brain reveal clearly different patterns of optical density within visual centres. The most superficial layers of superior colliculus (SC) and a pretectal area including the nucleus of the optic tract (NOT) appear symmetrically, strongly darker than other visual structures such as lateral geniculate nucleus (LGN) and visual cortex (VC). Whereas the lack of metabolic increase at LGN and VC levels entirely confirms the non-involvement of the geniculo-cortical path in mediating the optomotor response following OKS in Rodents, it is postulated that the symmetrical increase of 2DG uptake even upon unidirectional OKS found even at pretectal level may represent a commissural transfer of visual information between homologous pretectal areas like the nuclei of the optic tract.     

Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus.

DNA polymerase alpha, delta and epsilon can be isolated simultaneously from calf thymus. DNA polymerase delta was purified to apparent homogeneity by a four-column procedure including DEAE-Sephacel, phenyl-Sepharose, phosphocellulose, and hydroxylapatite, yielding two polypeptides of 125 and 48 kDa, respectively. On hydroxylapatite DNA polymerase delta can completely be separated from DNA polymerase epsilon. By KCl DNA polymerase delta is eluted first, while addition of potassium phosphate elutes DNA polymerase epsilon. DNA polymerases delta and epsilon could be distinguished from DNA polymerase alpha by their (i) resistance to the monoclonal antibody SJK 132-20, (ii) relative resistance to N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate, (iii) presence of a 3'----5' exonuclease, (iv) polypeptide composition, (v) template requirements, (vi) processivities on the homopolymer poly(dA)/oligo(dT12-18), and (vii) lack of primase. The following differences of DNA polymerase delta to DNA polymerase epsilon were evident: (i) the independence of DNA polymerase epsilon to proliferating cell nuclear antigen for processivity, (ii) utilization of deoxy- and ribonucleotide primers, (iii) template requirements in the absence of proliferating cell nuclear antigen, (iv) mode of elution from hydroxylapatite, and (v) sensitivity to d2TTP and to dimethyl sulfoxide. Both enzymes contain a 3'----5' exonuclease, but are devoid of endonuclease, RNase H, DNA helicase, DNA dependent ATPase, DNA primase, and poly(ADP-ribose) polymerase. DNA polymerase delta is 100-150 fold dependent on proliferating cell nuclear antigen for activity and processivity on poly(dA)/oligo(dT12-18) at base ratios between 1:1 to 100:1. The activity of DNA polymerase delta requires an acidic pH of 6.5 and is also found on poly(dT)/oligo(dA12-18) and on poly(dT)/oligo(A12-18) but not on 10 other templates tested. All three DNA polymerases can be classified according to the revised nomenclature for eukaryotic DNA polymerases (Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618).     

The substrate specificity of protein kinases which phosphorylate the alpha subunit of eukaryotic initiation factor 2.

The alpha subunit of eukaryotic protein synthesis initiation factor (eIF-2 alpha) is phosphorylated at a single serine residue (Ser51) by two distinct and well-characterized protein kinase, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI). The sequence adjacent to Ser51 is rich in basic residues (Ser51-Arg-Arg-Arg-Ile-Arg) suggesting that they may be important in the substrate specificity of the two kinases, as is the case for several other protein kinases. A number of proteins and synthetic peptides containing clusters of basic residues were tested as substrates for HCR and dsI. Both kinases were able to phosphorylate histones and protamines ar multiple sites as judged by two-dimensional mapping of the tryptic phosphopeptides. These data also showed that the specificities of the two kinases were different from one another and from the specificities of two other protein kinases which recognise basic residues, cAMP-dependent protein kinase and protein kinase C. In histones, HCR phosphorylated only serine residues while dsI phosphorylated serine and threonine. Based on phosphoamino acid analyses and gel filtration of tryptic fragments, dsI was capable of phosphorylating both 'sites' in clupeine Y1 and salmine A1, whereas HCR acted only on the N-terminal cluster of serines in these protamines. The specificities of HCR and dsI were further studied using synthetic peptides with differing configurations of basic residues. Both kinases phosphorylated peptides containing C-terminal clusters of arginines on the 'target' serine residue, provided that they were present at positions +3 and/or +4 relative to Ser51. However, peptides containing only N-terminal basic residues were poor and very poor substrates for dsI and HCR, respectively. These findings are consistent with the disposition of basic residues near the phosphorylation site in eIF-2 alpha and show that the specificities of HCR and dsI differ from other protein kinases whose specificities have been studied.     

A mouse CDC25-like product enhances the formation of the active GTP complex of human ras p21 and Saccharomyces cerevisiae RAS2 proteins.

GDP-dissociation stimulators (GDSs) are the key element for the regeneration of the active state of ras proteins, but despite intensive investigations, little is so far known about their functional and structural properties, particularly in mammals. A growing number of genes from various organisms have been postulated to encode GDSs on the basis of sequence similarity with the Saccharomyces cerevisiae CDC25 gene, whose product acts as a GDS of RAS proteins. However, except for CDC25 and the related SDC25 C-domain, no biochemical evidence of ras GDS activity for these CDC25-like proteins has yet been available. We show that the product of a recently isolated mouse CDC25-like gene (CDC25Mm) can strongly enhance (more than 1000 times) the GDP release from both human c-Ha-ras p21 and yeast RAS2 in vitro. As a consequence, the CDC25Mm induces a rapid formation of the biologically active Ras.GTP complex. This GDS is much more active on the GDP than on the GTP complex and has a narrow substrate specificity, since it was found to be inactive on several ras-like proteins. The mouse GDS can efficiently substitute for yeast CDC25 in an in vitro adenylylcyclase assay on RAS2 cdc25 yeast membranes. Our results show that a cloned GDP to GTP exchange factor of mammalian ras belongs to the novel family of CDC25-like proteins.     

Enzymatic synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid in a membrane reactor

Summary Dehydrocholic acid (3,7,12-trioxo-5-cholanic acid) (0.5% concentration) was completely and selectively reduced to 12-ketoursodeoxycholic acid (3, 7-dihydroxy-12-oxo- 5-cholanic acid) in a membrane reactor by means of 3-hydroxysteroid dehydrogenase and 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with the glucose-glucose dehydrogenase system.     

Isolation of a plant glycoprotein involved with control of intercellular recognition

A recognition molecule was isolated from stigmas of S-allele genotype S₂S₂ of Brassica oleracea var. capitata L. After Sephadex chromatography, it eluted as a single symmetrical peak during diethylaminoethane-cellulose chromatography. A high degree of purity was affirmed by: sedimentation as a single peak during ultracentrifugation through 5 to 20% sucrose gradients; elution as a single peak from Sephadex G-100; visualization as a single band which stains with Coomassie blue and periodic acid Schiff reagent after electrophoresis on polyacrylamide gels. Other criteria supporting the conclusion that it is a glycoprotein are: (a) the highly purified preparation is anthrone-positive and has a Lowry protein to anthrone-positive carbohydrate ratio of 1.3; (b) the preparation contains arabinose, galactose, glucose, and mannose, although it is not precipitated by concanavalin A; (c) the immunological properties of the molecule are lost following protease treatment, and it has a molecular weight of 90,000 by Sephadex gel-filtration analysis and 54,500 by velocity sedimentation analysis.     

Effect of 6-(p-hydroxyphenylazo)-uracil on the homologous and heterologous transduction processes in Bacillus subtilis.

We have studied the effect of 6-(p-hydroxyphenylazo)-uracil on the recombination processes that operate in the homologous and heterologous transduction mediated by PBS1 and SP10 phages of Bacillus subtilis. The results obtained demonstrate that the process of heterologous genetic exchange is sensitive to this compound, whereas the homologous process is not. The present data, along with those of our previous work (U. Canosi, A. G. Siccardi, A. Falaschi, and G. Mazza, J. Bacteriol. 126:108--121, 1976), suggest that the DNA polymerase III is involved in the recombination process that operates in transformation and heterologous transduction, whereas homologous transduction follows a partially independent pathway not involving this protein.