Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Human neoplastic submandibular intercalated duct cells express an acinar phenotype when cultured on a basement membrane matrix

Abstract. Culture of the human neoplastic submandibular gland intercalated duct cell line, HSG, on the basement membrane extract Matrigel induces dramatic morphologic changes and cytodifferentiation. Transmission electron microscopy demonstrated an acinar cell phenotype with polarized cells containing a well-developed Golgi apparatus, multiple microvilli-like projections from the apical surfaces into a lumenal-like area, and numerous granule-like organelles. Amylase, an acinar cell marker, was detected by both immunocytochemical and Northern blot analyses. A 50% reduction in [³H]thymidine incorporation by cells cultured on Matrigel, as compared to cells cultured on tissue culture plates, confirmed the differentiated phenotype of the cells. Multiple components of Matrigel appear to contribute to the morphologic differentiation of the HSG cells since antibodies to both laminin and collagen IV, as well as the lamininderived bioactive peptide containing SIKVAV, have potent inhibitory effects on HSG cell organization on Matrigel. Collectively, these data indicate that culture of HSG cells on Matrigel is a useful model to study salivary gland acinar development.     

In vivo 3-D distributions of electric fields in pig skin with rectangular pulse electrical current stimulation (RPECS)

We developed stimulating and detecting electrodes. We experimentally examined three dimensional (3-D) distributions of electric fields in living pig skin under and around the stimulating electrodes with the detecting electrodes and rectangular pulsed electrical current stimulation (RPECS). We verified our previous physical assumption, E ≈ I / (A σ_dz), in the skin under the electrode, where E, I, A and σ_dz respectively represent the electric field, the externally imposed peak current, the cross sectional area of the stimulating electrode and the perpendicular conductivity of the skin. Pulses were 30 mA, 140 μs and 128 pulses per second (pps). These parameters were previously used in our laboratory to enhance cutaneous regeneration, in vivo, with RPECS. © 1996 Wiley-Liss, Inc.     

Purification,partial characterization,and localization of Sak57, an acidic intermediate filament keratin present in rat spermatocytes,spermatids, and sperm

We have purified a 57 kDa protein (designated Sak57, for spermatogenic cell/sperm-associated keratin) from sodium dodecyl sulfate-β-mercaptoethanol(SDS-βME)-dissociated outer dense fibers isolated from rat sperm tails. Internal protein sequence analysis of Sak57 yielded two 15-mer and 10-mer fragments with 70–100% homology to human, rat, and mouse keratins and corresponding to the 1A and 2A regions of the α-helical rod domain of keratins. A multiple antigenic peptide (MAP) was constructed using the 10-mer amino acid sequence KAQYEDIAQK (corresponding to the 2A region) and used as antigen for the production of polyclonal antibodies in rabbit. Anti-MAP sera were used for further analysis of the biochemical characteristics of Sak57 in testis and sperm tails using chromatofocusing, immunobloting, and [³²P]orthophosphate-labeling. We have found that rat testis displays two immunoreactive proteins: a soluble 83 kDa protein with pl range 5.9–6.3, regarded as a precursor, and both detergent-insoluble and soluble 57 kDa protein with pl range 5.0–5.9, corresponding to the mature form Sak57. The testicular soluble form was phosphorylated. Rat sperm tail samples displayed only the Sak57 detergent-insoluble form and its pl was more acidic (4.7–4.8). Whole-mount electron microscopy of negatively stained preparations of sperm-derived Sak57 resuspended in SDS-βME revealed a rod-shaped pattern. A decrease in the concentration of SDS-βME resulted in the side-by-side aggregation of rod-shaped Sak57 forming thick bundles. Indirect immunofluorescence was used to determine the localization of Sak57 in isolated outer dense fibers, epididymal sperm, spermatids, and pachytene spermatocytes. Confocal laser scanning microscopy was used to analyze the three-dimensional arrangement of Sak57 in pachytene spermatocytes. Isolated outer dense fiber and sperm tails displayed an immunoreactive product in the form of linear clusters. In elongating spermatids (steps 10–11), Sak57 immunoreactivity was predominant in the head region whereas pachytene spermatocytes displayed a cortical cytoplasmic distribution. Results of this study demonstrate that Sak57 has the characteristics of a keratin intermediate filament and is present during meiotic and postmeiotic stages of spermatogenesis. © 1996 Wiley-Liss, Inc.     

The transmission dynamics of gonorrhoea: modelling the reported behaviour of infected patients from Newark, New Jersey.

A survey of the sexual behaviour of gonorrhoea patients in Newark was undertaken to evaluate parameters within a model of gonorrhoea transmission. Modelling work aimed to explain observed epidemiological patterns and to explore the potential impact of interventions. Reported behaviours, along with values derived from the literature, were used within a standard deterministic model of gonorrhoea transmission, where the population was stratified according to sex and rates of sex-partner change. The behaviours reported, particularly among women, are insufficient by themselves to explain the continued existence of gonorrhoea within the population. The majority of symptomatic patients seek treatment within a few days, and report that they do not have unprotected sex while symptomatic. The proportion of patients with low numbers of sex partners suggests that sexual mixing between people categorized according to sexual behaviour is near random. To explain the persistence of gonorrhoea, there must be some patients who, when infected, do not seek care in public clinics. In addition, gonorrhoea incidence in the model is sensitive to change, such that very small reductions in risk behaviour could lead to its elimination. This does not accord with the observed failure of many interventions to eliminate infection, suggesting that the modelled infection is too sensitive to change. The model, which has been influential in gonorrhoea epidemiology, is not consistent with the observed epidemiology of gonorrhoea in populations. Alternative models need to explore the observed stability of gonorrhoea before robust modelling conclusions can be drawn on how best to control infection. However, the current results do highlight the potential importance of asymptomatic infections and infections in those who are diseased and do not attend public health services. Screening and contact-tracing to identify asymptomatic infections in both men and women will be more effective in reaching those who maintain the infection within the community rather than simply treating symptomatic cases.     

Loss of caveolin-1 polarity impedes endothelial cell polarization and directional movement

The ability of a cell to move requires the asymmetrical organization of cellular activities. To investigate polarized cellular activity in moving endothelial cells, human endothelial cells were incubated in a Dunn chamber to allow migration toward vascular endothelial growth factor. Immunofluorescent staining with a specific antibody against caveolin-1 revealed that caveolin-1 was concentrated at the rear of moving cells. Similarly, monolayer scraping to induce random cell walk resulted in relocation of caveolin-1 to the cell rear. These results suggest that posterior polarization of caveolin-1 is a common feature both for chemotaxis and chemokinesis. Dual immunofluorescent labeling showed that, during cell spreading, caveolin-1 was compacted in the cell center and excluded from nascent focal contacts along the circular lamellipodium, as revealed by integrin beta1 and FAK staining. When cells were migrating, integrin beta1 and FAK appeared at polarized lamellipodia, whereas caveolin-1 was found at the posterior of moving cells. Notably, wherever caveolin-1 was polarized, there was a conspicuous absence of lamellipod protrusion. Transmission electron microscopy showed that caveolae, similar to their marker caveolin-1, were located at the cell center during cell spreading or at the cell rear during cell migration. In contrast to its unphosphorylated form, tyrosine-phosphorylated caveolin-1, upon fibronectin stimulation, was associated with the focal complex molecule phosphopaxillin along the lamellipodia of moving cells. Thus, unphosphorylated and phosphorylated caveolin-1 were located at opposite poles during cell migration. Importantly, loss of caveolin-1 polarity by targeted down-regulation of the protein prevented cell polarization and directional movement. Our present results suggest a potential role of caveolin polarity in lamellipod extension and cell migration.