Recycling cell surface glycoproteins undergo limited oligosaccharide reprocessing in LEC1 mutant Chinese hamster ovary cells

The ability of particular cell surface glycoproteins to recycle and become exposed to individual Golgi enzymes has been demonstrated. This study was designed to determine whether endocytic trafficking includes significant reentry into the overall oligosaccharide processing pathway. The Lec1 mutant of Chinese hamster ovary (CHO) cells lack N - acetylglucosaminyltransferase I (GlcNAc-TI) activity resulting in surface expression of incompletely processed Man5GlcNAc2 N -linked oligosaccharides. An oligosaccharide tracer was created by exoglycosylation of cell surface glycoproteins with purified porcine GlcNAc-TI and UDP-[3H]GlcNAc. Upon reculturing, all cell surface glycoproteins that acquired [3H]GlcNAc were acted upon by intracellular mannosidase II, the next enzyme in the Golgi processing pathway of complex N -linked oligosaccharides (t1/2= 3-4 h). That all radiolabeled cell surface glycoproteins were included in this endocytic pathway indicates a common intracellular compartment into which endocytosed cell surface glycoproteins return. Significantly, no evidence was found for continued oligosaccharide processing consistent with transit through the latter cisternae of the Golgi apparatus. These data indicate that, although recycling plasma membrane glycoproteins can be reexposed to individual Golgi-derived enzymes, significant reentry into the overall contiguous processing pathway is not evident.      

The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N- glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.      

Injection tube differentiation in gun cells of a haptoglossa species which infects nematodes

The gun cells which develop from germinating cysts in Haptoglossa produce a specialized infection apparatus, the injection tube. Upon eversion this tube fires a missile-like projectile which penetrates the host cuticle and then forms an infective sporidium within the body cavity of the nematode host. The temporal assembly of this complex cell organelle has been determined by serial-section reconstructions of maturing gun cells in a previously undescribed Haptoglossa species. The differentiation of the partially walled inverted injection tube is an unusual example of internal tube growth, in which membrane and wall assembly are temporally separated. There is no evidence that the shape of this inverted tube, which coils around the nucleus until it doubles back on itself, is dictated by the disposition of cytoplasmic microtubules. However, actin-like material was associated with the delimiting membrane of the differentiating tube, particularly in the regions of extension. From these studies it seems likely that the "head and buttress" structures previously depicted as the barbed tip of the "harpoon-like" penetration missile are part of a separate, structurally complex system which we suggest locks the "missile" into position in the invaginated injection tube. From this detailed account of cell architecture, models for the likely mechanism of infection cell firing are discussed, and unresolved questions relating to the cell biology and biochemistry of these complex organelles are highlighted. Copyright 1998 Academic Press.     

Replication process of the parvovirus H-1. VIII. Partial denaturation mapping and localization of the replication origin of H-1 replicative-form DNA with electron microscopy.

Partial denaturation mapping, restriction endonuclease digestion, and electron microscopy were used to determine which end of the linear duplex replicative-form (RF) DNA molecule contains the origin of RF replication for the parvovirus H-1. This origin was localized within approximately 300 base pairs of the arbitrarily designated right end of the RF DNA, in the EcoRI or HaeII-A fragment. Based on denaturation behavior in formamide, the right end was also found to have a relatively high guanine plus cytosine content, whereas the region adjacent to the left terminus of the RF DNA molecule was adenine plus thymine rich.     

Improving the specificity of high-throughput ortholog prediction

Background  

Orthologs (genes that have diverged after a speciation event) tend to have similar function, and so their prediction has become an important component of comparative genomics and genome annotation. The gold standard phylogenetic analysis approach of comparing available organismal phylogeny to gene phylogeny is not easily automated for genome-wide analysis; therefore, ortholog prediction for large genome-scale datasets is typically performed using a reciprocal-best-BLAST-hits (RBH) approach. One problem with RBH is that it will incorrectly predict a paralog as an ortholog when incomplete genome sequences or gene loss is involved. In addition, there is an increasing interest in identifying orthologs most likely to have retained similar function.     

The effects of hair density of beef cattle on Haematobia irritans horn fly populations

Abstract We show the relationships that exist between the amount of hair and quantity of sebum on cattle skin and the population density of the horn fly, Haematobia irritans. Brahman and Chianina steers had means of 2390 and 1587 hairs per cm², respectively, significantly more than the mean number of hairs on Angus, Brahman x Angus Crossbred, Charolais, and Red Poll steers. The Chianina steers had > 30% more sebum present on their skin and hair (0.58g/929cm²) than the Angus, Charolais, and Red Poll steers at the Beef Cattle Research Station Savoy, Arkansas. The Brahman steers had a significantly greater amount of sebum present on the skin (1.51 g/ 929 cm²) than the Crossbred and purebred Angus steers (0.55 and 0.25g/929cm², respectively) at the South Central Family Farms Research Centre Booneville, Arkansas. The Brahman and Chianina steers had means of 61.9 and 17.0 horn flies per steer, respectively, during the fly season, whereas the Angus, Crossbred, Charolais and Red Poll steers had fly season means that ranged from 76.9 to 265.8 flies per steer. Regression analysis showed that an increase of 100 hairs per cm², was associated with a reduction of 11 horn flies in the Angus II, 5 in Angus I, 20 in Charolais, 37 in Red Poll, and 0.4 in Chianina steers at the Savoy Station and a reduction of 6.6 horn flies for the Angus, Brahman, and Crossbred steers at the Booneville Centre. Regardless of cattle breed, an increase of 1.0 g of sebum per 929 cm² output by the steer was associated with 478.5 additional hairs per cm² on the animal. Each increase of 0.25 g of sebum per 929 cm² resulted in a decrease of 9.2 horn flies per steer. We conclude that some of the factors responsible for fly-resistance in cattle are hair density and the corresponding amount of sebum present on cattle skin and hair.     

The effects of selection for size in cattle on horn fly population density

Abstract. Statistically significant differences were observed in the population density of the horn fly, Haematobia irritans irritans L., on Angus cows having significantly different frame sizes. Angus cows, averaging <112.5 cm in height at the hip, had significantly lower numbers of horn flies than Angus cows that measured 112.5–117.5 cm, 117.5–120 cm, 120–126 cm and >126 cm in height at the hip. The Angus I cows(126 cm). The estimated heritability (h²) of horn fly resistance was 0.43 ± 0.07 and 0.95 ± 0.31 for 1989 and 1990, respectively. Horn fly counts on the Angus I herd (<112.5 cm in height) was 118.1 (probable breeding value, PBV = -20.69) to 165 horn flies per cow (PBV = 26.9 flies per cow in 1989) and from 75.9 (PBV = -29.1) to 134.5 (PBV = 29.5) flies per cow in 1990. Angus I bulls had PBV = -23.7 to 13.4 and from-26.5 to 14.75 in 1989 and 1990, respectively. The Angus II cows had horn fly counts that ranged from 159.6 (PBV of-23.5) to 208.1 (PBV of 25) per cow in 1989 and from 232.3 (PBV of-56.2) to 378.7 (PBV of 90) per cow in 1990. Angus II bulls had PBVs that ranged from-17.1 to 18.9 in 1989 and from -28.1 to 48.8 in 1990. The Angus I cows had significantly (P < 0.0001) lower numbers of hom flies (mean of 63.8 horn flies per m²) than the small, medium or large Angus II cows (mean of 129.4, 149.6 and 145.5 hom flies per m², respectively). The data indicated that some specific factor(s) associated with cow size contribute(s) to innate resistance of cattle to the horn fly.