CD15 monoclonal antibodies react with a phosphotyrosine-containing protein on the surface of human neutrophils

mAb are useful as probes in the study of the roles of cell-surface components in neutrophil function. Many mAb that bind to human neutrophils react with the oligosaccharide lacto-N-fucopentaose III (CD15 antibodies). These antibodies, as well as several other widely used mAb reactive with human neutrophils, were employed to detect phosphoproteins present on these cells. Immunoprecipitation and subsequent gel electrophoresis of proteins from neutrophils labeled with [gamma-32P]ATP revealed a 170 to 190-kDa phosphoprotein specifically reactive with CD15 antibodies. No phosphoproteins were immunoprecipitated by CD11 or CD18 mAb. Phosphoamino acid analysis of the 170- to 190-kDa protein showed that it contained predominantly phosphotyrosine and a low level of phosphoserine. Recently, it was shown that this phosphoprotein is one of the major substrates of ecto-protein kinase activity on human neutrophils. The roles for the 170- to 190-kDa phosphoprotein and the ecto-protein kinase in neutrophil function remain to be determined.     

Identification and characterization of a porcine parvovirus nonstructural polypeptide.

Sera from porcine parvovirus (PPV)-infected swine fetuses immunoprecipitated and 84- to 86-kilodalton polypeptide in addition to the A and B virion structural proteins. This polypeptide, designated NS-1, was present in PPV-infected cell lysates but not in purified virions. Partial proteolysis mapping revealed that NS-1 was not related to the A and B viral structural proteins. All three proteins in infected cells were phosphorylated at serine residues, and NS-1 also contained phosphothreonine. From pulse-labeling experiments with either 32Pi or [35S]methionine, NS-1 was found to first appear 5 to 7 h postinfection, whereas the viral structural polypeptides were first synthesized 9 to 11 h postinfection. Pulse-chase experiments revealed that NS-1 initially appeared as an 84-kilodalton protein and was subsequently structurally modified to forms of slower electrophoretic mobilities. The time of appearance of NS-1 after virus infection coincided with the initiation of viral DNA synthesis, suggesting that this polypeptide (and the modified forms thereof) may be involved in PPV replication.     

KBSH parvovirus: comparison with porcine parvovirus.

We compared the molecular, antigenic, and pathogenic properties of KBSH parvovirus to those of porcine parvovirus (PPV) isolate NADL-8. KBSH, propagated in swine testes cells in culture, possessed two major capsid polypeptides of 83 and 64 kilodaltons that were similar in size to those of PPV. KBSH-infected cells also contained an 86-kilodalton nonstructural polypeptide that was identical in size to the PPV nonstructural polypeptide (NS-1). The KBSH polypeptides were structurally similar but not identical to the corresponding PPV polypeptides, as revealed by partial proteolysis mapping. Viral replicative-form DNA from KBSH-infected cells was similar in size to PPV replicative-form DNA and exhibited similar but not identical restriction endonuclease cleavage patterns to that of PPV replicative-form DNA. Antigenically, the two viruses were also very closely related. By using heterologous and homologous antisera, the two viruses were indistinguishable in hemagglutination inhibition and immunoprecipitation assays. However, pathogenically these viruses were dramatically different. NADL-8 caused fetal death when injected into swine fetuses in utero and viremia and high persisting antibody titers when administered orally to weaning-age swine. KBSH-inoculated fetuses were normal in appearance, and pigs orally exposed to KBSH failed to establish viremia and demonstrated only transient antibody titers. Thus, KBSH appears to be a PPV that is very closely related to a highly pathogenic PPV isolate, yet is itself nonpathogenic in swine. This reduced pathogenic potential of KBSH may be attributable to its poor ability to replicate in swine.     

Extrapolating and interpolating surfaces in depth

Random-dot stereograms were generated with a blank area placed in part of the right-hand image so making a patchwork of monocular and binocular areas. The perceived depth and shape of the monocular region, where depth was not explicitly marked, depended in part on the depth and surface orientation of adjacent binocular areas. Thus a monocular rectangle flanked by two binocular rectangles which were placed in different fronto-parallel planes was seen as a sloping surface spanning the depth between the binocular regions, and, under some conditions, the gradient of a sloping binocular plane extended into a neighbouring monocular area. Division of the monocular region into two by textural discontinuities or discontinuities of motion sometimes altered the shape of the extrapolated surface. Often, though, the shape was unchanged by such discontinuities implying that both two- and three-dimensional features are used to segment a scene into separate surfaces. Pictorial cues also contribute to the shape and apparent depth of the monocular surface. For instance, when subjects viewed a display consisting of portions of a cube of which two ends were shown stereoscopically and one side monocularly, the monocular side was seen in three dimensions filling the gap between the ends. When stereo cues were pitted against pictorial cues, sometimes pictorial cues and sometimes stereo cues dominated, and sometimes the surface contained sharp discontinuities enabling both to be accommodated.     

Small-field, binocular neurons in the superficial layers of the frog optic tectum

Binocular neurons with receptive fields about 5 degrees across were recorded just beneath the pia. Most of them responded to dark stimuli in the lower half of their receptive field and to light stimuli above. There was almost no vertical disparity between the left and right fields and the modal value of the horizontal disparity of the population of cells was 1.7 degrees. Because frogs do not verge their eyes it is possible to calculate at what distance the receptive fields through the two eyes are superimposed. This calculation suggests that the neurons are tuned to detect features in the external world about 50 cm away. This is too far for the neurons to be involved in the frog's everyday distance vision. It is more likely that they are concerned with assessing the vertical position of a horizontal surface.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Expression strategy of a phlebovirus: biogenesis of proteins from the Rift Valley fever virus M segment.

The middle (M) RNA segment of Rift Valley fever virus (RVFV) encodes four proteins: the major viral glycoproteins G2 and G1, a 14-kilodalton (kDa) protein, and a 78-kDa protein. These proteins are derived from a single large open reading frame (ORF) present in the virus-complementary M-segment mRNA. We used recombinant vaccinia viruses in which sequences representing the M-segment ORF were engineered as a surrogate system to study phlebovirus protein expression. To investigate the translational initiation codon requirements for synthesis of these proteins, we constructed a series of vaccinia virus recombinants containing specific sequence changes which eliminated select ATG codons found in the region of the ORF preceding the mature glycoprotein-coding sequences (the preglycoprotein region). Examination of phleboviral proteins synthesized in cells infected with these vaccinia virus recombinants clearly showed that the first ATG of the ORF was required for the production of the 78-kDa protein, while synthesis of the 14-kDa protein was absolutely dependent on the second in-phase ATG codon. Efficient biosynthesis of glycoprotein G2 was shown to depend on one or more ATG codons within the preglycoprotein region, but not the first one of the ORF. Synthesis of about one-half of the total glycoprotein G1 was affected by the amino acid changes that eliminated ATG codons, while production of the remainder appeared to be independent of all ATG codons in the preglycoprotein region. These data indicated that the means for glycoprotein G1 biosynthesis was distinct from those of the other three M-segment gene products. The results presented herein suggest that a surprisingly complex expression strategy is employed by the RVFV M segment. Although the full nature of the mechanisms involved in the biogenesis of the four RVFV M-segment proteins remains unclear, it does involve the use of at least two (ATG codons 1 and 2), and likely more, distinct translation start sites within the same ORF to produce its complete complement of gene products.     

Deep-level diagnostic value of the rDNA-ITS region

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.