Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands

N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-giycosideby the action of CMPN-acetylneuramlnic acid (CMP-Neu5Ac) hydroxylase.This enzyme is a soluble cytochrome bs-dependent monooxygenaseand has been purified to apparent homogeneity from pig submandibularglands by precipitation with N-cetyN,N,N-trimethylam-moniumbromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose,Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12.This procedure resulted in an 8960-fold purification of thehydroxylase with a recovery of 0.8%. The molecular mass of thisprotein was shown to be 65 kDa on SDS-PAGE and 60 kDa as determinedby gel filtration on Superose S.12, which suggests that theenzyme is a monomer. The purified CMP-Neu5Ac hydroxylase isactivated by FeSO₄ and inhibited by iron-binding reagents suchas o-phenanthroline, KCN, Tiron and ferro-zine. An apparentKm of 11 µM was determined for the substrate CMP-Neu5Acusing purified hydroxylase in the presence of Triton X-100-solubilizedmicrosomes. In a reconstituted system consisting of purifiedhydroxylase, cytochrome b₅, cytochrome b₅ reductase and catalase,an apparent K_m of 3 µM was measured. The apparent K_mforcytochrome b₅ in this system was 0.24 µM. Immunizationof a rabbit with enriched and purified hydroxylase led to anantiserum that inhibited CMP-Neu5Ac hydroxylase activity andreacted with the purified 65 kDa protein on a Western blot afterSDS-PAGE. Antibodies specific for this 65 kDa protein were isolatedand showed a strong reaction with the purified CMP-Neu5Ac hydroxylasefrom mouse liver after immunoblotting. Initial experiments withthis monospecific antibody suggest that the activity of thehydroxylase in a particular tissue correlates with the amountof immuno-reactive protein. cytochrome b₅ N-glcoloylneuraminic acid hydroxylase pig submandibular gland mucin sialic acid     

A Neurofilament-Associated Kinase Phosphorylates Only a Subset of Sites in the Tail of Chicken Midsize Neurofilament Protein

Although neurofilaments are among the most highly phosphorylated proteins extant, relatively little is known about the kinases involved in their phosphorylation. The majority of the phosphates present on the two higher-molecular-mass neurofilament subunits are added to multiply repeated sequence motifs in the tail. We have examined the specificity of a neurofilament-associated kinase (NFAK) partially purified from chicken spinal cord that selectively phosphorylates the middle-molecular-mass neurofilament subunit, NF-M. Two-dimensional phosphopeptide mapping of ³²P-labeled NF-M shows that, in vitro, NFAK phosphorylates a subset of peptides phosphorylated in vivo in cultured neurons. The absence of a complete complement of labeled phosphopeptides following in vitro phosphorylation, compared with phosphorylation in vivo, is not due to a lack of availability of phosphorylation sites because the same maps are obtained when enzymatically dephosphorylated NF-M is used as an in vitro substrate. Phosphopeptide maps from in vitro-phosphorylated NF-M and those from a recombinant fusion protein containing only a segment of the tail piece of chicken NF-M reveal identical labeled peptides. The fusion protein lacks a segment containing 17 KXX(S/T)P putative phosphorylation sites contained in the tail of chicken NF-M but contains a segment that includes four KSPs and a KSD site also present in the intact tail. These results suggest (a) that NFAK mediates the phosphorylation of some, but not all, potential phosphorylation sites within the tail of NF-M and (b) that multiple kinases are necessary for complete phosphorylation of the NF-M tail.     

QUANTITATIVE GENETICS OF RESPONSE TO COMPETITORS IN NEMOPHILA MENZIESII: A GREENHOUSE STUDY

To determine the potential for adaptation to a local biotic environment, we examined the magnitude and nature of genetic variation in response to neighboring plants within a natural population of the native California annual, Nemophila menziesii. A total of 22 plants from a natural population were crossed in three reciprocal factorials. The progeny were grown in a greenhouse in nine treatments that varied in conspecific density and in the density of a naturally co-occurring grass species, Bromus diandrus. Increasing the density of each species significantly reduced individual survival, fruit number, and dry weight. Among survivors, we found small to moderate heritability of dry weight within treatments. Additive genetic correlations (r_A) of dry weight between competitive regimes were generally large and positive. In no case were they significantly different from 1, as expected under the null hypothesis that the relative performance of the genotypes under consideration is the same in all environments. On the basis of these results, we cannot conclude that the structure of genetic covariation within this population would promote genetic differentiation in response to locally varying conditions of density of these two species. Aspects of the experiment that may have compromised our ability to detect r_A differing from 1 are discussed.     

The inheritance of vegetative growth traits in strawberries (Fragaria x ananassa) grown at low temperatures and their relationship to field productivity

The genetic relationship between vegetative growth at low temperatures and productivity was investigated for strawberries grown in controlled and field environments. Genotypes from 20 biparental crosses were grown in controlled environments with 11°, 14°, and 17 °C days, 11 °C nights, and 11-h daylength to simulate a range of winter growing conditions expected in mediterranean environments. Individual plants were scored for two initial runner traits and eight vegetative growth traits. Significant main effects of temperature and cross were detected for all growth chamber traits, and conservative estimates of the broad sense heritability (h²) for these traits were 0.10–0.28. None of the temperature x cross interaction effects were significant, suggesting that genetic potential for vegetative growth and vigor is expressed similarly at low and optimal growing temperatures. Highly significant genetic correlations were detected between many growth chamber trait pairs, indicating pleiotropic effects for the genes that condition these traits. Complementary field trials were established, and individual plants were scored for traits that describe yield, production pattern, and plant size. Significant negative genetic correlations were detected between traits that describe growth in the chambers and early production in the field trials, but genetic correlations between chamber growth traits and mid-season or total production were significantly positive and occasionally large. Several of the yield and field growth variables were genetically correlated to initial runner plant traits, suggesting that indirect selection using traits scored in the nursery can be used to improve yield and modify production pattern in the field.     

Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins.

Cross-linking of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins on T cells can trigger cell activation. We and others have shown an association between GPI-anchored proteins and the protein tyrosine kinases (PTKs) p56lck and p59fyn, suggesting a pathway for signaling through GPI-anchored proteins. Studies of decay-accelerating factor (DAF) or CD59 in either the C32 cell line or the HeLa cell line transfected with PTK cDNA demonstrated that the GPI-anchored proteins associated noncovalently with p56lck and p59fyn but not with p60src. Nonmyristylated versions of p56lck and p59fyn also failed to associate with the GPI-anchored proteins. Mutational analysis of the PTK demonstrated that the association with the GPI-anchored proteins mapped to the unique amino-terminal domains of the PTK. A chimeric PTK consisting of the 10 amino-terminal residues of p56lck or p59fyn replacing the corresponding amino acids in p60src was sufficient for association with DAF, but the converse constructs containing the first 10 amino acids of p60src plus the remainder of p56lck or p59fyn did not associate with DAF. Mutation of cysteine to serine at positions 3 and 6 in p59fyn or positions 3 and 5 in p56lck abolished the association of these kinases with DAF. Mutation of serine to cysteine at positions 3 and 6 in p60src conferred on p60src the ability to associate with DAF. Direct labeling with [3H]palmitate demonstrated palmitylation of this amino-terminal cysteine motif in p56lck. Thus, palmitylation of the amino-terminal cysteine residue(s) together with myristylation of the amino-terminal glycine residue defines important motifs for the association of PTKs with GPI-anchored proteins.     

THE COMPARISON OF QUANTITATIVE GENETIC PARAMETERS BETWEEN POPULATIONS

A statistical method for comparing matrices of genetic variation and covariation between groups (e.g., species, populations, a single population grown in distinct environments) is proposed. This maximum-likelihood method provides a test of the overall null hypothesis that two covariance component matrices are identical. Moreover, when the overall null hypothesis is rejected, the method provides a framework for isolating the particular components that differ significantly between the groups. Simulation studies reveal that discouragingly large experiments are necessary to obtain acceptable power for comparing genetic covariance component matrices. For example, even in cases of a single trait measured on 900 individuals in a nested design of 100 sires and three dams per sire in each population, the power was only about 0.5 when additive genetic variance differed by a factor of 2.5. Nevertheless, this flexible method makes valid comparison of covariance component matrices possible.     

Are the spatial patterns of weeds scale-invariant?

In previous empirical and modelling studies of rare species and weeds, evidence of fractal behaviour has been found. We propose that weeds in modern agricultural systems may be managed close to critical population dynamic thresholds, below which their rates of increase will be negative and where scale-invariance may be expected as a consequence. We collected detailed spatial data on five contrasting species over a period of three years in a primarily arable field. Counts in 20×20 cm contiguous quadrats, 225,000 in 1998 and 84,375 thereafter, could be re-structured into a wide range of larger quadrat sizes. These were analysed using three methods based on correlation sum, incidence and conditional incidence. We found non-trivial scale invariance for species occurring at low mean densities and where they were strongly aggregated. The fact that the scale-invariance was not found for widespread species occurring at higher densities suggests that the scaling in agricultural weed populations may, indeed, be related to critical phenomena.     

An alternate model of thyroid cytology: practical not perfect

A sample of 384 thyroid cytology specimens prepared by cytospin over a 2.5-year period was classified by original report into inadequate, non-neoplastic and suspicious of neoplasia or worse. This was then compared with subsequent histology. The resulting data showed an inadequacy rate of 33%, a sensitivity of 55%, a specificity of 59%, a positive predictive value of 64% and a negative predictive value of 93%. On review of the cytology, in knowledge of the subsequent histology, the maximum achievable results were determined to have a positive predictive value of 79% and a negative predictive value of 97%. No clinically significant adverse event was detected.     

Simplified methods for monitoring petroleum‐contaminated ground water and soil vapor

Portable meters and simplified gas Chromatographic (GC) techniques were investigated for monitoring volatile hydrocarbon (HC), CO₂, and O₂, concentrations in groundwater, exhaust gases, and soil vapor during in situ remediation using soil vapor extraction (SVE) and air sparging (AS). Results of groundwater samples analyzed in‐house using a headspace technique compared well to split samples analyzed by a certified analytical laboratory (r² = 0.94). SVE exhaust gas HC and CO₂ concentrations measured using a GT201 portable HC/O₂ meter and a RA‐411A meter (GasTech), respectively, were highly correlated with in‐house laboratory GC analyses (r² = 0.91). O₂ concentrations fell in a small range and meter analyses were not well correlated with laboratory analyses. Results of soil gas monitoring were not as well correlated as those for exhaust gases for HC, CO₂, or O₂, perhaps due to environmental conditions such as changes in relative humidity or the wider range of soil gas values. Overall, the meters were good indicators of vapor contamination, they greatly simplified estimates of total HC mass removal, and they allowed estimates of the biological contribution to contaminant removal during the remediation process.