Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Early events in the cellular formation of proparathyroid hormone

Early events in the cellular synthesis and subsequent transfer into membrane-limited compartments of pre-proparathyroid hormone (pre-proPTH) and proparathyroid hormone (proPTH) were investigated by electrophoretic analyses of newly synthesized proteins in subcellular fractions of parthyroid gland slices pulse-labeled for 0.5-5 min with [(35)S] methionine. During these short times of incubation, both pre-proPTH and proPTH were confined to the microsomal fraction. Labeled pre-proPTH and proPTH were detected in a 30-s interval between 0.5 and 1.0 min of incubation. The radioactivity in proPTH became relatively constant between 3 and 5 min, whereas the radioactivity in ProPTH increased markedly over this period. When corrected for the known content of methionine in the prohormone and the prohormone, we found four times as much radiolabeled prohormone as prehormone between 0.5 and 1.0 min of synthesis. Sequestration of labeled prohomrone into endoplasmic reticulum compartments was shown by treatment of the microsomal fraction with chymotrypsin and trypsin, which resulted in the degradation of the prehormone but not of the prohormones. Approximately 50 percent of pre-prohormone and 25 percent of prohormone were released from the microsomes by their extraction with 1.0 M KCl, whereas 80-90 percent of both was released by treatment with Triton X-100. These results in intact cells support the signal hypothesis proposed by Blobel and his co-workers in studies utilizing cell-free systems, inasmuch as the results indicate transfer of prohormone into the cisternal space of the rough endoplasmic reticulum concomitant with the growth of the nascent polypeptide chain. Appearance of membrane-sequestered proPTH takes place without entry of pre-proPTH into the cisternal space, suggesting that proteolytic removal of the leader peptide occurs during transfer of the polypeptide through the lipid bilayer. Further evidence in support of this process is that pre-proPTH is only partly extracted from the microsomes by treatment with 1.0 M KCl, suggesting that a substantial fraction of the nascent pre-proPTH is integrally inserted into the membranes before it is cleaved to form proPTH.     

Phylogeny of some Fusarium species, as determined by large-subunit rRNA sequence comparison

Fifty-two strains from eight species of Fusarium were analyzed by rapid rRNA sequencing. Two highly variable stretches (138 and 214 nucleotides) of the 5' end of the 28S-like rRNA molecule were sequenced. Such stretches permit evaluation of the divergence between closely related species and even between varieties within a species. The phylogenetic tree computed from the number of nucleotide differences shows seven Fusarium species to be more closely related to one another than the eighth species, F. nivale, is to them. On the basis of these data, we discuss both the phylogenetic value of taxonomical criteria and the impact of our findings on the demarcation of the genus Fusarium. We conclude that this method is suitable for establishing a precise phylogeny between closely related species within a genus.      

Polymerase chain reaction amplification of two polymorphic simple repeat sequences within the von Willebrand factor gene: application to family studies in von Willebrand disease

Summary We have used the polymerase chain reaction to amplify two variable number of tandem repeats (VNTRs) within a region of repetitive DNA located in intron 40 of the von Willebrand factor (vWf) gene. Heterozygosity for VNTR I was observed in 30 out of 39 normal unrelated individuals tested (77%), and for VNTR II in 29 out of 44 (66%) similar individuals. Family studies were carried out on 11 kindreds with von Willebrand disease (vWD). Ten of these families were found to be informative for one or other of the VNTRs or for a combination of data from both VNTRs. This method can be used for antenatal diagnosis and for carrier diagnosis in recessive forms of vWD. It is also useful for tracking the gene associated with vWD in type I families where there may be one or more individuals with a phenotypically uncertain diagnosis.     

A prospective cytogenetic study of 36 cases of DiGeorge syndrome

Cytogenetic analysis was carried out in a prospective series of 36 children with DiGeorge syndrome. High-resolution banding (> 850 bands/haploid set) was achieved in 30 cases. Monosomy 22q11.21-->q11.23 was found in 9 of these 30 cases. In each of these cases monosomy 22q11.21-->q11.23 resulted from an interstitial deletion and not from a translocation. No other chromosome abnormalities were seen.     

Replication of association between ELAVL4 and Parkinson disease: the GenePD study

Genetic variants in embryonic lethal, abnormal vision, Drosophila-like 4 (ELAVL4) have been reported to be associated with onset age of Parkinson disease (PD) or risk for PD affection in Caucasian populations. In the current study we genotyped three single nucleotide polymorphisms in ELAVL4 in a Caucasian study sample consisting of 712 PD patients and 312 unrelated controls from the GenePD study. The minor allele of rs967582 was associated with increased risk of PD (odds ratio = 1.46, nominal P value = 0.011) in the GenePD population. The minor allele of rs967582 was also the risk allele for PD affection or earlier onset age in the previously studied populations. This replication of association with rs967582 in a third cohort further implicates ELAVL4 as a PD susceptibility gene.     

Staining of intracellular deposits of uranium in cultured murine macrophages

In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2- pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylene-diaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.