Biomedical subjects

J W Dennis

Publications and source records attributed to J W Dennis.

At least 19 recordsLinked to original sources

PubMed2000

Decreased UDP-GlcNAc levels abrogate proliferation control in EMeg32-deficient cells.

The hexosamine pathway provides UDP-N:-acetylhexosamine donor substrates used in cytosolic and Golgi-mediated glycosylation of proteins and for formation of glycosylphosphatidylinositol (GPI) anchors, which tether proteins to the outer plasma membrane. We have recently identified the murine glucosamine-6-phosphate (GlcN6P) acetyltransferase, EMeg32, as a developmentally regulated enzyme on the route to UDP-N:-acetylglucosamine (UDP-GlcNAc). Here we describe embryos and cells that have the EMeg32 gene inactivated by homologous recombination. Homozygous mutant embryos die at around embryonic day (E) 7.5 with a general proliferative delay of development. In vitro differentiated EMeg32(-/-) ES cells show reduced proliferation. Mouse embryonic fibroblasts (MEFs) deficient for EMeg32 exhibit defects in proliferation and adhesiveness, which could be complemented by stable re-expression of EMeg32 or by nutritional restoration of intracellular UDP-GlcNAc levels. Reduced UDP-GlcNAc levels predominantly translated into decreased O-GlcNAc modifications of cytosolic and nuclear proteins. Interestingly, growth-impaired EMeg32(-/-) MEFs withstand a number of apoptotic stimuli and express activated PKB/AKT. Thus, EMeg32-dependent UDP-GlcNAc levels influence cell cycle progression and susceptibility to apoptotic stimuli.

Acetyltransferases↗

PubMed2000

A homogeneous cell-based assay to identify N-linked carbohydrate processing inhibitors.

Malignant transformation is accompanied by altered cell surface glycosylation. N-Linked oligosaccharides carrying beta1-6GlcNAc branches are associated with tumor invasion and metastasis. Therefore, compounds that can enter cells and block biosynthesis of beta1-6GlcNAc-branched glycans without overt cytotoxicity are potential anticancer agents. We have developed a homogeneous cell-based assay for detection of such compounds. The method enables identification of agents that block beta1-6GlcNAc-branched glycan expression after incubation for 16-20 h with MDAY-D2 tumor cells, thereby protecting the cells from the subsequent addition of leukoagglutinin, a cytotoxic plant lectin. We observed that MDAY-D2 cell number is directly proportional to the level of endogenous alkaline phosphatase activity measured spectrophotometrically in cultures after the addition of substrate. The alkaline phosphatase assay was capable of detecting as few as 1,500 cells. The assay was readily adapted for high-throughput screening as reagent costs are low and no cell harvesting and washing steps are required. Under high-throughput operating conditions, the coefficient of variation for controls was found to be 4.2%. The results suggest that measurement of alkaline phosphatase in this cell assay format may be adapted for wider applications in high-throughput screenings for compounds that relieve cells from other growth inhibitors.

Alkaline Phosphatase↗

PubMed2000

Sak kinase gene structure and transcriptional regulation.

The Sak gene encodes a serine/threonine kinase, which is a member of the Polo family of mitotic regulators. Sak transcripts are present in S/G2/M phase cells, and in proliferating cell layers of the mouse embryo and adult tissues. In this report, we have characterized the murine Sak gene structure, the Sak chromosomal location, and identified the promoter. The murine Sak gene is located on the proximal arm of mouse chromosome 13, as determined by RFLP analysis. The murine gene comprises 15 coding exons spanning 16kb of genomic sequence, and encodes two alternately spliced transcripts. Sak-a, the predominant transcript, is encoded by 15 exons, while early termination of transcription and alternative splicing at exons 5 and 6 results in Sak-b. This truncated transcript encodes the complete kinase domain and a carboxyl end translated from 147bp of sequence contiguous with exon 5. Human Sak-a (Stk18) cDNA is reported to contain an insertion of sequence corresponding to the mouse Sak-b tail. Primer extension analysis of murine Sak revealed one major transcription start site at position -303bp relative to the start of translation. A genomic fragment of 3.5kb located 5' of the Sak transcriptional start drives expression of a luciferase-reporter gene in CHO and GC1-SPG cells in an orientation-dependent fashion. Using various Sak promoter/luciferase constructs, the core promoter region required for expression was located within 400bp of the message Cap site, and sequence further 5' strongly suppressed transcription.

Animals↗

PubMed2000

Regulation of human monocyte proMMP-9 production by fetuin, an endogenous TGF-beta antagonist.

Members of the matrix metalloproteinase family of enzymes degrade specific components of the extracellular matrix. MMP-9 is a type IV/V collagenase necessary for normal osteogenesis and is increased in inflammatory and neoplastic conditions. In such destructive diseases as emphysema and arthritis, and in epithelial cancers, MMP-9 is produced by cells of the monocyte lineage. Fetuin, a prominent serum glycoprotein, binds to and inactivates TGF-beta family members and through this mechanism regulates osteogenesis (Binkert et al., 1999, J Biol Chem 274:28514-28520.). We studied the effects of TGF-beta1 and fetuin on proMMP-9 release by the human monocyte line THP-1. Exogenous TGF-beta1 stimulated proMMP-9 release by THP-1 cells, with half-maximal stimulation at approximately 0.01 ng/ml TGF-beta1. Human fetuin (0.5-2 microM) partially inhibited this stimulation. Human fetuin alone stimulated THP-1 monocyte proMMP-9 release, with half maximal stimulation at approximately 0.25 microM fetuin. Neutralizing antibody specific for TGF-beta1 also stimulated proMMP-9 release, suggesting that endogenously-derived TGF-beta1 has an inhibitory effect. In freshly isolated human peripheral blood monocytes, fetuin stimulated proMMP-9 release with a dose-response curve similar to that observed in THP-1 cells. Human fetuin also activated proMMP-9 present in THP-1 conditioned medium. Taken together, these data suggest that under physiological conditions, fetuin facilitates matrix degradation by monocyte-derived MMP-9, both by opposing the autocrine inhibitory effect of endogenous TGF-beta1 on proMMP-9 release, and by activating proMMP-9 in the pericellular milieu. Conversely, fetuin may limit the stimulation of monocyte proMMP-9 release by high levels of exogenous TGF-beta1. Such modulation could prove important under pathological conditions where TGF-beta1 derived from paracrine sources is abundant, such as in epithelial malignancies.

Collagenases↗

PubMed2000

Suppression of tumor growth and metastasis in Mgat5-deficient mice.

Golgi beta1,6N-acetylglucosaminyltransferase V (MGAT5) is required in the biosynthesis of beta1,6GlcNAc-branched N-linked glycans attached to cell surface and secreted glycoproteins. Amounts of MGAT5 glycan products are commonly increased in malignancies, and correlate with disease progression. To study the functions of these N-glycans in development and disease, we generated mice deficient in Mgat5 by targeted gene mutation. These Mgat5-/- mice lacked Mgat5 products and appeared normal, but differed in their responses to certain extrinsic conditions. Mammary tumor growth and metastases induced by the polyomavirus middle T oncogene was considerably less in Mgat5-/- mice than in transgenic littermates expressing Mgat5. Furthermore, Mgat5 glycan products stimulated membrane ruffling and phosphatidylinositol 3 kinase-protein kinase B activation, fueling a positive feedback loop that amplified oncogene signaling and tumor growth in vivo. Our results indicate that inhibitors of MGAT5 might be useful in the treatment of malignancies by targeting their dependency on focal adhesion signaling for growth and metastasis.

Animals↗

PubMed2000

Continued experience with physical examination alone for evaluation and management of penetrating zone 2 neck injuries: results of 145 cases.

PURPOSE: Our preliminary experience with physical examination alone in the evaluation of penetrating zone 2 neck injuries for vascular trauma was previously reported in 28 patients over a 2-year period (1991-1993). The purpose of the current study was to examine the results of this approach in a much larger group of patients over an 8-year period. METHODS: The medical records for all patients admitted to our level I trauma center (all of them entered into our prospective protocol) between December 1991 and April 1999 with penetrating zone 2 neck trauma were reviewed for their initial presentation and any documented vascular injury. RESULTS: A total of 145 patients made up the study group; in 30 of these patients, the penetrating trajectory also traversed zone 1 or 3. Thirty-one patients (21%) had hard signs of vascular injury (active bleeding, expanding hematoma, bruit/thrill, pulse deficit, central neurologic deficit) and were taken immediately to the operating room; 28 (90%) of these 30 patients had either major arterial or venous injuries requiring operative repair (the false-positive rate for physical examination thus being 10%). Of the 114 patients with no hard signs, 23 underwent arteriography because of proximity of the injury to the vertebral arteries or because the trajectory included another zone. Of these 23 arteriograms, three showed abnormalities, but only one required operative repair. This case had no complications relating to the initial delay. The remaining 91 patients with no hard signs were observed without imaging or surgery for a minimum of 23 hours, and none had any evidence of vascular injury during hospitalization or during the initial 2-week follow-up period (1/114; false-negative rate for physical examination, 0.9%). CONCLUSIONS: This series confirms the earlier report indicating that patients with zone 2 penetrating neck wounds can be safely and accurately evaluated by physical examination alone to confirm or exclude vascular injury. The missed-injury rate is 0.7% (1/145) with this approach, which is comparable to arteriography in accuracy but less costly and noninvasive. Long-term follow-up is needed to confirm this management option.

Adult↗

PubMed2000

Biogenesis of multilamellar bodies via autophagy.

Transfection of Mv1Lu mink lung type II alveolar cells with beta1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the beta1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella.

Animals↗

PubMed2000

Minimal catalytic domain of N-acetylglucosaminyltransferase V.

UDP-GlcNAc: Manalpha1-6Manbeta-R beta1-6 N-acetylglucosaminyltransferase V (EC 2.4.1.155, GlcNAc-TV) is a Golgi enzyme that substitutes the trimannosyl core in the biosynthetic pathway for complex-type N-linked glycans. GlcNAc-TV activity is regulated by oncogenes frequently activated in cancer cells ( ras, src, and her2/neu ) and by activators of T lymphocytes. Overexpression of GlcNAc-TV in epithelial cells results in morphological transformation, while tumor cell mutants selected for loss of GlcNAc-TV products show diminished malignant potential in mice. In this report, we have expressed and characterized a series of N- and C-terminal deletions of GlcNAc-TV. Portions of GlcNAc-TV sequence were fused at the N-terminal domain to IgG-binding domains of staphylococcal Protein A and expressed in CHOP cells. The secreted fusion proteins were purified by IgG Sepharose affinity chromatography and assayed for enzyme activities. The peptide sequence S(213-740)of GlcNAc-TV was determined to be essential for the catalytic activity, the remaining amino acids comprising a 183 amino acid stem region, a 17 amino acid transmembrane domain and a 12 amino acid cytosolic moiety. Further deletion of 5 amino acids to produce peptide R(218-740)reduced enzyme activity by 20-fold. Similar K(m)and V(max)values for donor and acceptor were observed for peptide S(213-740), the minimal catalytic domain, and peptide Q(39-740), which also included the stem region. Truncation of five amino acids from the C-terminus also resulted in a 20-fold loss of catalytic activity. Secondary structure predictions suggest a high frequency of turns in the stem region, and more contiguous stretches of alpha-helix found in the catalytic domain.

Amino Acid Sequence↗

PubMed1999

Glycoprotein glycosylation and cancer progression.

Glycosylation of glycoproteins and glycolipids is one of many molecular changes that accompany malignant transformation. GlcNAc-branched N-glycans and terminal Lewis antigen sequences have been observed to increase in some cancers, and to correlate with poor prognosis. Herein, we review evidence that beta1, 6GlcNAc-branching of N-glycans contributes directly to cancer progression, and we consider possible functions for the glycans. Mgat5 encodes N-acetylglucosaminyltransferase V (GlcNAc-TV), the Golgi enzyme required in the biosynthesis of beta1,6GlcNAc-branched N-glycans. Mgat5 expression is regulated by RAS-RAF-MAPK, a signaling pathway commonly activated in tumor cells. Ectopic expression of GlcNAc-TV in epithelial cells results in morphological transformation and tumor growth in mice, and over expression in carcinoma cells has been shown to induce metastatic spread. Ectopic expression of GlcNAc-TIII, an enzyme that competes with GlcNAc-TV for acceptor, suppresses metastasis in B16 melanoma cells. Furthermore, breast cancer progression and metastasis induced by a viral oncogene expressed in transgenic mice is markedly suppressed in a GlcNAc-TV-deficient background. Mgat5 gene expression and beta1, 6GlcNAc-branching of N-glycans are associated with cell motility, a required phenotype of malignant cells.

Amino Sugars↗

PubMed1999

Overexpression of the alpha2,6-sialyltransferase, ST6Gal I, in a low metastatic variant of a murine lymphoblastoid cell line is associated with appearance of a unique ST6Gal I mRNA.

Multiple mRNA isoforms are generated from Siat1, the gene encoding ST6Gal I (beta-galactoside alpha2,6-sialyltransferase, SiaT-1, ST6N, alpha2,6ST). These isoforms, transcriptionally initiated from a number of physically distinct promoter regions, differ only in the 5'-most untranslated region and share an identical ST6Gal I coding region. W16 cells, a spontaneous mutant from MDAY-D2, the highly metastatic murine lymphoid tumor cell line, is considerably less metastatic and exhibits significantly slower tumor growth characteristics [R. Takano, E. Muchmore, and J. W. Dennis (1994) Glycobiology 4, 665-674]. Takano et al. further reported that ST6Gal I mRNA in W16 is elevated 40-fold compared to the parental cells. Here, by means of 5'-RACE analysis, we demonstrate a heretofore undocumented ST6Gal I mRNA form expressed in W16 cells. This ST6Gal I mRNA contains a novel 5'-most untranslated region with 96% sequence similarity to the retroviral-like transposable element, intracisternal particle A (IAP). This observation suggests the notion that elevated ST6Gal I expression in W16 cells is the result of DNA rearrangement in the Siat1 locus. Atypical transcriptional activation of Siat1 is the result of this IAP transposition.

Animals↗

PubMed1999

Regulation of osteogenesis by fetuin.

Osteoporosis is a common problem of aging and results from a failure of homeostatic mechanisms to regulate osteogenesis and mineralization. Bovine and human forms of fetuin glycoprotein bind to the transforming growth factor (TGF)-beta/BMP (bone morphogenic protein) cytokines and block their osteogenic activity in cell culture assays (Demetriou, M., Binkert, C., Sukhu, B., Tenenbaum, H. C., and Dennis, J. W. (1996) J. Biol. Chem. 271, 12755-12761). Fetuin is a prominent serum glycoprotein and a major noncollagenous component of mineralized bone in mammals. In this study, we show that recombinant fetuin and native serum protein have similar potency as inhibitors of osteogenesis in dexamethasone-treated rat bone marrow cell cultures (dex-RBMC). Recombinant bovine fetuin also bound to TGF-beta1 and BMP-2 in vitro with kinetics similar to native fetuin. Although TGF-beta1 is required for osteogenesis in dex-RBMC, the cytokine also inhibited osteogenesis at concentrations >/=10 pM. Titration of fetuin or anti-TGF-beta1 antibodies into the bone marrow cultures in the presence of 10 pM TGF-beta1 restored osteogenesis, whereas titrations of the same reagents into cultures with 0.3 pM added TGF-beta1 were inhibitory, confirming the biphasic nature of the TGF-beta1 response. Suppression of osteogenesis by both TGF-beta1 and the antagonist proteins required their presence within the first 6 days of culture, well before mineralization at 10-12 days. Northern analysis showed that both fetuin and high dose TGF-beta1 suppressed expression of the bone-associated transcripts alkaline phosphatase, osteopontin, collagen type I, and bone sialoprotein. The suppression of osteogenesis by fetuin and by high dose TGF-beta1 was accompanied by the differentiation of an alternate cell lineage with adipocyte characteristics. In summary, the biphasic osteogenic response to TGF-beta1 suggests that overlapping gradients of TGF-beta/BMP cytokines and fetuin regulate osteogenesis in remodeling bone.

Amino Acid Sequence↗

PubMed1999

Protein glycosylation in development and disease.

N- and O-linked glycan structures of cell surface and secreted glycoproteins serve a variety of functions related to cell-cell communication in systems affecting development and disease. The more sophisticated N-glycan biosynthesis pathway of metazoans diverges from that of yeast with the appearance of the medial-Golgi beta-N-acetylglucosaminyltransferases (GlcNAc-Ts). Tissue-specific regulation of medial- and trans-Golgi glycosyltransferases contribute structural diversity to glycoproteins in metazoans, and this can affect their molecular properties including localization, half-life, and biological activity. Null mutations in glycosyltransferase genes positioned later in the biosynthetic pathway disrupt expression of smaller subsets of glycan structures and are progressively milder in phenotype. In this review, we examine data on targeted mutations affecting glycosylation in mice and congenital mutations in man, with a view to understanding the molecular functions of glycan structures as modulators of glycoprotein activity. Finally, pathology associated with the expression of GlcNAc-Ts in cancer and diabetes-induced cardiac hypertrophy suggest that inhibitors of these enzymes may have therapeutic value.

Animals↗

PubMed1999

A solid-phase glycosyltransferase assay for high-throughput screening in drug discovery research.

Glycosyltransferases mediate changes in glycosylation patterns which, in turn, may affect the function of glycoproteins and/or glycolipids and, further downstream, processes of development, differentiation, transformation and cell-cell recognition. Such enzymes, therefore, represent valid targets for drug discovery. We have developed a solid-phase glycosyltransferase assay for use in a robotic high-throughput format. Carbohydrate acceptors coupled covalently to polyacrylamide are coated onto 96-well plastic plates. The glycosyltransferase reaction is performed with recombinant enzymes and radiolabeled sugar-nucleotide donor at 37 degrees C, followed by washing, addition of scintillation counting fluid, and measurement of radioactivity using a 96-well beta-counter. Glycopolymer construction and coating of the plastic plates, enzyme and substrate concentrations, and linearity with time were optimized using recombinant Core 2 beta1-6-N-acetylglucosaminyltransferase (Core 2 GlcNAc-T). This enzyme catalyzes a rate-limiting reaction for expression of polylactosamine and the selectin ligand sialyl-Lewis(x) in O-glycans. A glycopolymer acceptor for beta1-6-N-acetylglucosaminyltransferase V was also designed and shown to be effective in the solid-phase assay. In a high-throughput screen of a microbial extract library, the coefficient of variance for positive controls was 9.4%, and high concordance for hit validation was observed between the Core 2 GlcNAc-T solid-phase assay and a standard solution-phase assay. The solid-phase assay format, which can be adapted for a variety of glycosyltransferase enzymes, allowed a 5-6 fold increase in throughput compared to the corresponding solution-phase assay.

Acrylic Resins↗

PubMed1999

Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice.

Elevated levels of glycocojugates, commonly observed in the myocardium of diabetic animals and patients, are postulated to contribute to the myocardial dysfunction in diabetes. Previously, we reported that UDP-GlcNAc: Galbeta1-3GalNAcalphaRbeta1-6-N-acetylglucosaminyltransferas e (core 2 GlcNAc-T), a developmentally regulated enzyme of O-linked glycans biosynthesis pathway, is specifically increased in the heart of diabetic animals and is regulated by hyperglycemia and insulin. In this study, transgenic mice overexpressing core 2 GlcNAc-T with severe increase in cardiac core 2 GlcNAc-T activities were normal at birth but showed progressive and significant cardiac hypertrophy at 6 months of age. The heart of transgenic mice showed elevation of sialylated O-glycan and increases of c-fos gene expression and AP-1 activity, which are characteristics of cardiac stress. Furthermore, transfection of PC12 cells with core 2 GlcNAc-T also induced c-fos promoter activation, mitogen activated-protein kinase (MAPK) phosphorylation, Trk receptor glycosylation, and cell differentiation. These results suggested a novel role for core 2 GlcNAc-T in the development of diabetic cardiomyopathy and modulation of the MAP kinase pathway in the heart.-Koya, D., Dennis, J. W., Warren, C. E., Takahara, N., Schoen, F. J., Nishio, Y., Nakajima, T., Lipes, M. A., King, G. L. Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice.

Animals↗

PubMed1998

A three-year follow-up on standard versus thin wall ePTFE grafts for hemodialysis.

PURPOSE: Expanded polytetraflouroethylene (ePTFE) grafts are the most popular prosthetic grafts for hemodialysis patients in whom autogenous fistulas cannot be constructed. Long-term studies to study the durability and complication rate of the different wall configurations of ePTFE grafts have not been carried out. The primary, secondary, and cumulative patency and other complications between standard thickness (STD) and thin wall (THN) 6 mm stretch ePTFE grafts (WL Gore & Assoc, Flagstaff, AZ) was prospectively evaluated. METHODS: From September 1993 to August 1995, 108 patients receiving new grafts were randomized into 2 groups: those receiving STD grafts (n = 56) or those receiving THN (n = 52) grafts. Data prospectively collected included day of first access, primary patency, interventions required, and long-term results. Infections, pseudoaneurysms, and mortality were also documented. Student's unpaired t-test was used to compare the 2 groups, and log-rank life tables were constructed and compared. RESULTS: Mean follow-up examination time was 38.1 +/- 0.8 months for STD grafts and 35.1 +/- 1.0 months for THN grafts (P<.03). Longer patency was noted in the STD group of grafts (18.2 months for STD vs. 12.1 months for THN). Biographical data and complications, including pseudoaneurysm (6% vs. 5%), infection (2% vs. 3%), and mortality (22% vs. 19%), between STD and THN groups were not different statistically. Mean primary (18.2 months vs. 12.1 months), secondary (20.9 months vs. 13.7 months), and cumulative patency times (22.2 months vs. 15.2 months) for the STD group were significantly more than those for the THN group (P<.000 by log rank of life tables). Other complications were not different between groups. CONCLUSION: Standard thickness ePTFE is the graft of choice when placing ePTFE arteriovenous grafts for hemodialysis.

Adolescent↗

PubMed1998

The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time.

The increased polylactosamine glycosylation of LAMP-2 in MDCK cells cultured for 1 day relative to cells cultured for 3 days has been correlated with its slower rate of Golgi transit (Nabi and Rodriguez-Boulan, 1993, Mol. Biol. Cell., 4, 627-635). To determine if the differential polylactosamine glycosylation of LAMP-2 is a consequence of glycosyltransferase expression levels, the activities of beta1-6GlcNAc-TV, beta1-3GlcNAc-T(i), beta1-2GlcNAc-TI, beta1, 4Gal-T, alpha2-6sialyl-T, and alpha2-3sialyl-T were assayed and no significant differences in the activities of these enzymes in 1 and 3 day cell extracts were detected. During MDCK epithelial polarization, the Golgi apparatus undergoes morphological changes and apiconuclear Golgi networks were more evident in 3 day cells. Treatment with nocodazole disrupted Golgi networks and generated numerous Golgi clusters in both 1 day and 3 day cells. In the presence of nocodazole the differential migration of LAMP-2 in 1 and 3 day MDCK cells was maintained and could be eliminated by treatment with endo-beta-galactosidase, indicating that gross Golgi morphology did not influence the extent of LAMP-2 polylactosamine glycosylation. Nocodazole treatment did, however, result in the faster migration of LAMP-2 which was not due to modification of core N-glycans as the precursor form of the glycoprotein migrated with an identical molecular size. Following incubation at 20 degrees C, which prevents the exit of proteins from the trans-Golgi network, the molecular size of LAMP-2 increased to a similar extent in both 1 and 3 day MDCK cells. Extending the time of incubation at 20 degrees C did not influence the size of LAMP-2, demonstrating that its glycosylation is modified not by its retention within the Golgi but rather by its equivalent slower Golgi passage at the lower temperature in both 1 and 3 day cells. An identical effect was observed in nocodazole treated cells, demonstrating that Golgi residence time determines the extent of LAMP-2 polylactosamine glycosylation, even in isolated Golgi clusters.

Amino Sugars↗

PubMed1998

Validation of nonoperative management of occult vascular injuries and accuracy of physical examination alone in penetrating extremity trauma: 5- to 10-year follow-up.

PURPOSE: To establish by long-term follow-up the safety and efficacy of nonoperative management of clinically occult arterial injuries and the use of physical examination (PE) alone in determining treatment of penetrating extremity trauma (PET). METHODS: Two groups of patients were studied: (1) all patients with PET, arterial abnormalities on arteriograms, and no hard signs of vascular injury treated nonoperatively from 1986 to 1989; and (2) all patients with PET to the extremities managed by PE alone from 1989 to 1991. Telephone contact, PE, and duplex ultrasonography (US) were attempted in all group 1 patients, and telephone interviews were attempted in all group 2 patients. RESULTS: Group 1 had 43 patients with 44 clinically occult penetrating injuries to extremity arteries. Arteriography identified 21 intimal flaps/irregularities, 19 narrowings, 2 pseudoaneurysms, and 1 arteriovenous fistula. Four patients (9%) had clinical deterioration within 1 month and required surgery, with good results. Twenty-three of the other 39 patients (58%) were able to be contacted, and 17 (43%) with 18 injuries underwent PE and US. All were asymptomatic and had normal PE, and one had mild residual narrowing on US. The other 6 patients (four in prison, two out of state) reported no symptoms of vascular insufficiency and never sought medical attention for vascular problems. Mean follow-up was 9.1 years (range, 8.6-11.1 years). Group 2 had 287 patients (309 injuries) with PET treated by observation based on PE alone. Four patients (1.3%) required delayed surgery within the first week, and 78 with 90 injuries (29%) were able to be contacted. No patient reported any vascular symptoms or ever saw a physician for vascular problems. Mean follow-up was 5.4 years (range, 2.2-6.0 years). CONCLUSION: This is the first long-term follow-up of nonoperative management of clinically occult arterial injuries of the extremities and the use of PE alone in the initial management of PET. The results show these approaches to be safe, effective, and now a proven standard of care.

Adult↗

PubMed1998

The Internet and education in surgery.

The purpose of this review is to explain the developing role of the Internet and the World Wide Web (WWW) in promoting education in surgery. Internet sites relevant to surgery are appearing rapidly. Remote literature searches can query for surgery trials and results. Societies are using the WWW for transmission and review of publication materials. News groups interactively discuss current developments and trends. Surgeons are using personal and institutional sites to advertise services. Conventional slide shows migrate to the WWW for convenient downloading for surgeons and patients. Multimedia capabilities of the WWW expand the depth of information transmission, enabling education emanating from remote sites with narration and video depiction of procedures. These sophisticated tools can be demonstrated today with real online applications. One site facilitates surgical education using the WWW for program information, symposium coordination, links to regional subspecialty societies, residency cataloging, patient question and answer forums, and multimedia procedure descriptions. The principles of WWW communication used in this website can adapt to meet any educational need. The specialty of surgery is well suited to incorporation of online multimedia education over the Internet to follow new developments in our field.

Audiovisual Aids↗