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Biomedical subjects

D Wen

Publications and source records attributed to D Wen.

At least 55 records · Page 3Linked to original sources

Open fracture of the tibia in children.

The records of eighty-three children who had had an open fracture of the tibial metaphysis or diaphysis between January 1983 and July, 1993 were studied retrospectively. The average duration of follow-up was fourteen months (range, two to seventy-five months). There were twenty-four grade-I, forty grade-II, thirteen grade-IIIA, six grade-IIIB, and no grade-IIIC fractures, according to the classification scheme of Gustilo et al. Sixty patients (72 per cent) had sustained the fracture when they were struck by an automobile, and forty-eight patients (58 per cent) had other associated major injuries. All fractures were treated with irrigation and débridement, and antibiotics were administered parenterally for a minimum of forty-eight hours. Thirty-two patients were managed with immobilization in a cast only; forty, with transcutaneous fixation with an average of two Steinmann pins followed by immobilization in a cast; nine, with external fixation; one, with open reduction and internal fixation with two screws and two pins; and one, with delayed intramedullary nailing. Fifty-seven wounds were closed primarily (forty-four, over a Penrose drain, and thirteen, without a drain), ten were treated with delayed closure, four were allowed to heal by secondary intention, seven were covered with a soft-tissue flap, and five were treated with skin-grafting (a split-thickness skin graft was used for four, and a split-thickness and a full-thickness skin graft were used for one). The average time to union was fifteen weeks (range, five to sixty-one weeks), with the fracture healing by sixteen weeks in sixty-four patients (77 per cent). Eighteen patients (22 per cent) had delayed union, and only one patient (1 per cent) had non-union. Secondary procedures were necessary to achieve union in only two patients. Two patients had a superficial wound infection, and no patient had osteomyelitis. One patient, who had been managed with external fixation, had a pin-track infection; none of the patients who had had transcutaneous fixation had a pin-track infection. Two patients had a compartment syndrome, and two patients had a transient stretch injury of a nerve (the peroneal nerve in one and the sciatic nerve in the other). Four fractures healed with an angulatory deformity of more than 10 degrees in any plane. Five patients had overgrowth of the limb of one centimeter or more. Physeal arrest did not occur in any patient. We concluded that treatment of unstable open fractures of the tibia in children with débridement and transcutaneous fixation followed by immobilization in a cast leads to good anatomical and functional results. We prefer this technique to external fixation, which is associated with several potential complications. Loose closure of a clean open wound over a Penrose drain is effective and can be safely utilized in selected children.

Adolescent↗

Detection of neu differentiation factor with a biospecific affinity sensor during chromatography.

A technique using a biospecific affinity sensor, BIAcore, was applied to monitor and determine mammalian cell-derived neu differentiation factor (NDF) in column fractions during chromatography. Specific purified polyclonal antibody against Escherichia coli-derived NDF was chemically bound to the surface of BIAcore sensor chips and the derivatized sensor chips were used to detect the specific binding of NDF. The measurement of NDF at very low levels can be assessed by injecting small volumes of the crude media or column fractions into the BIAcore sensor containing antibody-bound sensor chips. This automated procedure performed under computer programming control allows direct measurement of multiple NDF samples in a short period of time and provides excellent quantitative data, which is not possible using other related methods such as Western blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stimulatory activity assay on receptor autophosphorylation.

Animals↗

Neu differentiation factor inhibits EGF binding. A model for trans-regulation within the ErbB family of receptor tyrosine kinases.

Neu differentiation factor (NDF, or heregulin) and epidermal growth factor (EGF) are structurally related proteins that bind to distinct members of the ErbB family of receptor tyrosine kinases. Here we show that NDF inhibits EGF binding in a cell type-specific manner. The inhibitory effect is distinct from previously characterized mechanisms that involve protein kinase C and receptor internalization because it occurred at 4 degrees C and displayed reversibility. The extent of inhibition correlated with both receptor saturation and affinity of different NDF isoforms, and it was abolished upon overexpression of either EGF receptor or ErbB-2. Binding kinetics and equilibrium analyses indicated that NDF reduced the affinity, rather than the number, of EGF receptors, through an acceleration of the rate of ligand dissociation and deceleration of the association rate. On the basis of co-immunoprecipitation of EGF and NDF receptors, we attribute the inhibitory effect to the formation of receptor heterodimers. According to this model, EGF binding to NDF-occupied heterodimers is partially blocked. This model of negative trans-regulation within the ErbB family is relevant to other subgroups of receptor tyrosine kinases and may have physiological implications.

Breast Neoplasms↗

ErbB-3 mediates differential mitogenic effects of NDF/heregulin isoforms on mouse keratinocytes.

The family of Neu differentiation factors (NDFs, or heregulins) includes a dozen secreted glycoproteins, whose receptor binding domain displays two variants, alpha and beta, and they bind to two receptor tyrosine kinases, ErbB-3 and ErbB-4. Certain isoforms were reported to induce growth-arrest and differentiation of mammary tumor cells, while other breast cancer cell lines responded mitogenically. The present study addressed the biologic effects of various NDF isoforms on normal EGF-dependent epithelial cells, Balb/MK keratinocytes, that can undergo either proliferation or differentiation. We found that beta isoforms of NDF induced a mitogenic effect, that was significantly smaller than the maximal response to EGF. By contrast with NDF-beta, NDF-alpha isoforms exerted almost no mitogenic effect, but they were sufficient to maintain keratinocytes in culture. Consistent with their higher mitogenic potency, NDF-beta isoforms bound to Balb/MK cells with higher affinity (Kd = 2.2 nM) than alpha isoforms, however both groups shared their receptor, that we identified as ErbB-3. No transcript of ErbB-4 was detectable in the keratinocytes, but these cells express multiple NDF mRNAs and also ErbB-2. We conclude that different isoforms of NDF induce distinct growth regulatory effects on cultured keratinocytes, through direct interaction with ErbB-3.

Animals↗

Hyperactive autocrine loop mediated by a NDF-related factor in neoplastic hamster embryo fibroblasts expressing an activated cph oncogene.

We described previously the characterization of a novel oncogene, cph, activated in primary Syrian hamster embryo fibroblasts by exposure to 3-methylcholanthrene (Velasco et al., Oncogene 9:2065-2069, 1994). The present report describes the participation in the neoplastic conversion of cph-expressing (81C39) hamster fibroblasts of a hyperactive autocrine loop involving a neu differentiation factor [NDF]-like protein. The tyrosine phosphorylation of the p185erbB-2 receptor in the human breast carcinoma MDA-MB-453 cells was stimulated by conditioned medium from neoplastic 81C39 cells. The extent of this stimulatory effect was much greater than that induced by conditioned medium from normal 84-3 hamster cells. The p185erbB-2 tyrosine phosphorylation-stimulating activity was partially blocked by the heparin analogue pentosan polysulfate [PPS], a known antagonist of p185erbB-2 ligands, and was partially purified from 81C39 conditioned medium by heparin-Sepharose chromatography. The level of p185erbB-2 tyrosine phosphorylation-stimulating activity in the heparin-Sepharose fractions correlated directly with their content in NDF-like protein as immunodetected with an anti-rat NDF antibody. Consistently, the steady-state level of NDF-related mRNA was found to be four times greater in neoplastic 81C39 cells than in normal 84-3 cells. However, the levels of erbB-2 mRNA were similar in both cell types, while the expression of erbB-4 mRNA was upregulated in the neoplastic fibroblasts. The ability of 81C39 conditioned medium to stimulate protein tyrosine phosphorylation and to induce other PPS-sensitive growth responses on 81C39 cells themselves suggested the involvement of an autocrine loop in their neoplastic conversion. The participation of a NDF-related factor in this autocrine loop was confirmed by the ability of an anti-NDF antibody to block the mitogenic activity present in their own conditioned medium. The involvement of the cph oncogene in the upregulation of NDF-related expression was evidenced when cph-transformed NIH3T3 fibroblasts showed elevated levels of NDF-related mRNA, and their conditioned medium induced tyrosine phosphorylation on MDA-MB-453 cells, reproducing the effect of the medium from 81C39 hamster cells.

3T3 Cells↗

Nonequilibrium antifreeze peptides and the recrystallization of ice.

Evidence is presented that the nonequilibrium antifreeze peptide (AFP) from winter flounder has a special ability to inhibit recrystallization in ice only when an appreciable amount of liquid is present, as is the case when the system contains salts and the temperature is not too low. In this circumstance the AFP binds to the ice surface at the ice-solution interfaces in grain boundaries, preventing migration of the solution and effectively immobilizing the boundaries. In the absence of liquid, recrystallization inhibition appears to be a common property of many peptides. This is consistent with the view that the special effects of AFPs require a structural fit onto ice, and therefore require the AFP molecules to have the mobility to achieve that fit. Since the concentration of salt required to induce the special recrystallization inhibition effects of AFPs is lower (< 10 mM) than that found normally in physiological fluids, AFPs could play a role in the survival of organisms by preventing damage due to recrystallization. The proposition that mobility is needed for AFP molecules to produce their special influence upon ice growth argues against any special effects of AFPs in devitrification.

Amino Acid Sequence↗

Expression of messenger RNA for amphiregulin, heregulin, and cripto-1, three new members of the epidermal growth factor family, in human breast carcinomas.

The expression of amphiregulin (AR), heregulin (HRG), and cripto-1 (CR-1) mRNA transcripts was assessed in 60 human primary breast carcinoma. AR and HRG transcripts were expressed respectively in 58% and 25% of the carcinomas as measured by Northern blot analysis. CR-1 mRNA was found in 77% of the carcinomas using Reverse Transcriptase-PCR analysis. Coexpression of two or three of these peptides was observed in several specimens. There was no significant association between AR, HRG, and CR-1 expression and nodal status, EGF receptor, or c-erbB-2 protooncogene expression in these tumors. However, a significant association between AR expression and estrogen receptor positivity was observed.

Amphiregulin↗

Neu differentiation factor upregulates epidermal migration and integrin expression in excisional wounds.

Neu differentiation factor (NDF) is a 44-kD glycoprotein which was isolated from ras-transformed rat fibroblasts and indirectly induces tyrosine phosphorylation of the HER-2/neu receptor via binding to either the HER-3 or HER-4 receptor. NDF contains a receptor binding epidermal growth factor (EGF)-like domain and is a member of the EGF family. There are multiple different isoforms of NDF which arise by alternative splicing of a single gene. To date, in vivo biologic activities have not been demonstrated for any NDF isoform. Since NDF, HER-2/neu, and HER-3 are present in skin, and other EGF family members can influence wound keratinocytes in vivo, we investigated whether NDF would stimulate epidermal migration and proliferation in a rabbit ear model of excisional wound repair. In this model, recombinant human NDF-alpha 2 (rhNDF-alpha 2), applied once at the time of wounding, induced a highly significant increase in both epidermal migration and epidermal thickness at doses ranging from 4 to 40 micrograms/cm2. In contrast, rhNDF-alpha 1, rhNDF-beta 1, and rhNDF-beta 2 had no apparent biologic effects in this model. rhNDF-alpha 2 also induced increased neoepidermal expression of alpha 5 and alpha 6 integrins, two of the earliest integrins to appear during epidermal migration. In addition, rhNDF-alpha 2-treated wounds exhibited increased neoepidermal expression of cytokeratin 10 and filaggrin, both epidermal differentiation markers. NDF alpha isoforms were expressed in dermal fibroblasts of wounded and unwounded skin, while both HER-2/neu and HER-3 were expressed in unwounded epidermis and dermal adnexa. In wounds, HER-2/neu expression was markedly decreased in the wound neoepidermis while neoepidermal HER-3 expression was markedly upregulated. Taken together, these results suggest that endogenous NDF-alpha 2 may function as a paracrine mediator directing initial epidermal migration during cutaneous tissue repair.

Animals↗

[Constituents of the petroleum ether and ethyl acetate extract fractions from Dracaena cochinensis (Lour.) S.C. Chen].

Six constituents have been isolated from the petroleum ether and ethyl acetate extract fractions of Dracaena cochinensis. Their structures have been identified as 1,2,4,5-tetrachloro-3,6- dimethoxy-benzene,docosyl alcohol, octadecyl acetate, eicosyl acetate, resveratrol and 4',7-dihydroxy-flavone on the basis of physical, chemical and spectral deta. Of these compounds 1,2,4,5-tetrachloro-3,6-dimethoxy-benzene is a new one.

Chlorobenzenes↗

[The significance of high-density lipoprotein subfractions and triglycerides in predicting coronary artery disease].

Serum Lipid profiles, including high-density lipoprotein (HDL) and its subfractions HDL2 and HDL3 were obtained in 78 cases undergoing coronary angiography. Coronary artery disease (CAD) was present in 51 patients and absent in 27. We used a scoring methold of CAD to reflect the extent or narrowing of coronary artery. The largest difference between the two groups was observed in HDL2 Cholesterol with a mean of 0.37 mmol/L in patients with CAD as compared with 0.49 mmol/L in normal subjects (P < 0.001) and in TG (1.85 mmol/L vs 1.16 mmol/L) as well. Smaller difference was found in TC (5.9 mmol/L vs 4.87 mmol/L, P < 0.05). No singnificant difference was found in serum HDL (P > 0.05). Multivariate regression analysis revealed that the most powerful indenpendent variable associated with the extent of CAD was HDL2 cholesterol and triglyceride. The score of CAD was significantly correlated with HDL2-c (r = 0.32 P < 0.01) and TG (r = 0.34 P < 0.01). It is shown that high serum level of TG (> 1.6 mmol/L) and low serum level of HDLC2 (< 0.37 mmol/L) were the strengest predictors of presence and extent of CAD.

Adult↗

Amino-acid sequence of a cooperative, dimeric myoglobin from the gastropod mollusc, Buccinum undatum L.

The complete amino-acid sequence of a dimeric myoglobin from the radular mussel of the gastropod mollusc, Buccinum undatum L. has been determined. The globin, which shows cooperative binding of oxygen, contains 146 amino acids, is N-terminal aminoacetylated, and has histidine residues at position 65 and 97, corresponding to the heme-binding histidines seen in mammalian myoglobins. It shows about 75% and 50% homology, respectively, with the dimeric molluscan myoglobins from Busycon canaliculatum and Cerithidea rhizophorarum, the former of which also shows weak cooperatively, but much less similarity to other species of myoglobin and hemoglobin.

Amino Acid Sequence↗

ErbB-3 and ErbB-4 function as the respective low and high affinity receptors of all Neu differentiation factor/heregulin isoforms.

Neu differentiation factor (NDF or heregulin) elevates tyrosine phosphorylation of the ErbB-2 receptor tyrosine kinase, and it was, therefore, thought to function as a ligand of this receptor. However, several lines of evidence raised the possibility that the interaction between NDF and ErbB-2 involves another molecule, which belongs to the family of epidermal growth factor receptors. To address this question we constructed soluble chimeric proteins between alkaline phosphatase and the extracellular domains of ErbB-2 and either ErbB-3 or ErbB-4, two newly recognized members of the epidermal growth factor receptor family. Using the soluble proteins we found that beta isoforms of NDF specifically bind to the ErbB-3 and ErbB-4 receptors but not to the soluble ErbB-2 protein. When ectopically expressed in monkey fibroblasts, the full-length ErbB-3 and ErbB-4 receptors conferred specific binding to NDF. In these cells ErbB-3 displayed lower ligand binding affinity than ErbB-4, but like the latter receptor it preferred to bind the beta isoform over the alpha class of NDFs. These results indicate that both ErbB-3 and ErbB-4 function as physiological receptors of all NDF isoforms and suggest that a still unknown ligand of ErbB-2 exists.

Amino Acid Sequence↗

Brain neurons and glial cells express Neu differentiation factor/heregulin: a survival factor for astrocytes.

Neu differentiation factor (NDF, also called heregulin) was isolated from mesenchymal cells on the basis of its ability to elevate phosphorylation of ErbB proteins. Earlier in situ hybridization analysis showed that NDF was transcribed predominantly in the central nervous system during embryonic development. To gain insights into the role of NDF in brain we analyzed its distribution by immunohistochemistry and in situ hybridization. Late-gestation (day 17) rat embryos displayed high NDF immunoreactivity in both motor (e.g., putamen) and limbic (e.g., septum) regions. Lower levels of the factor were exhibited by adult brain, except for the cerebellum, where NDF expression was increased postnatally. Both neurons and glial cells were identified by immunohistochemistry as NDF-producing cells (e.g., pyramidal neurons in the cerebral cortex and glial cells in the corpus callosum). By establishment of primary cultures of rat brain cells we confirmed that NDF was expressed in neurons as well as in astrocytes. In addition, by using such primary cultures we observed that NDF treatment exerted only a limited mitogenic effect, which was accompanied by significant acceleration of astrocyte maturation. Furthermore, long-term incubation with the factor specifically protected astrocytes from apoptosis, implying that NDF functions in brain as a survival and maturation factor for astrocytes.

Animals↗

Erythropoietin structure-function relationships. Identification of functionally important domains.

In order to delineate functionally important domains in erythropoietin (Epo), we have prepared and tested a series of amino acid replacements at 51 conserved sites predicted to be on the surface of the molecule. Alanine replacements permitted preservation of alpha-helical structure. Wild type and mutant Epo cDNAs were transiently expressed at high levels in COS1 and COS7 cells. The biological activity of wild type and mutant Epos was assayed in three Epo-responsive cell types: primary murine erythroid spleen cells, the murine HCD57 erythroleukemia cell line, and the human UT7-EPO leukemia cell line. When Arg14 on predicted Helix A was replaced by Ala, biological activity was substantially reduced, whereas replacement with Glu resulted in total loss of specific bioactivity. In a similar manner, the mutein Arg103-->Ala in Helix C was completely lacking in biological activity, whereas both Ser104-->Ala and Leu108-->Ala had decreased bioactivity. In Helix D, the mutein Gly151-->Ala had markedly decreased bioactivity, whereas that of the adjacent Lys152-->Ala mutein was moderately impaired. In contrast, Ala replacements at three nearby sites on Helix D (147, 146, and 143) resulted in muteins with increased bioactivity. In conclusion, our mutagenesis experiments have identified functionally important domains on the surface of the Epo molecule, at sites comparable with those established for other cytokines.

Amino Acid Sequence↗

NDF/heregulin stimulates the phosphorylation of Her3/erbB3.

Her3/erbB3 has been identified as a third member of the epidermal growth factor receptor (EGFR) family [(1989) Proc. Natl. Acad. Sci. USA 86, 9193-9197; (1990) Proc. Natl. Acad. Sci. USA 87, 4905-4909]. The natural ligand for Her3 has not been identified. Although recently NDF has been proposed as a specific ligand for Her4 [(1993) Nature 366, 473-475; (1993) J. Biol. Chem. 268, 18407-18410], we report here that Her3 was phosphorylated on tyrosine not only in three breast carcinoma cell lines, MDAMB453, MDAMB468 and SKBR3, but also in Her3-transfected CHO cells in response to NDF stimulation. In further studies, cells were reacted with 125I-labeled NDF and then chemically crosslinked. Immunoprecipitation with anti-Her3 revealed a dense high Mw band, greater than 400 kDa. The results suggest that NDF may be a ligand of Her3 and induces receptor hetero-oligomerization.

Animals↗

Structural and functional aspects of the multiplicity of Neu differentiation factors.

We used molecular cloning and functional analyses to extend the family of Neu differentiation factors (NDFs) and to explore the biochemical activity of different NDF isoforms. Exhaustive cloning revealed the existence of six distinct fibroblastic pro-NDFs, whose basic transmembrane structure includes an immunoglobulin-like motif and an epidermal growth factor (EGF)-like domain. Structural variation is confined to three domains: the C-terminal portion of the EGF-like domain (isoforms alpha and beta), the adjacent juxtamembrane stretch (isoforms 1 to 4), and the variable-length cytoplasmic domain (isoforms a, b, and c). Only certain combinations of the variable domains exist, and they display partial tissue specificity in their expression: pro-NDF-alpha 2 is the predominant form in mesenchymal cells, whereas pro-NDF-beta 1 is the major neuronal isoform. Only the transmembrane isoforms were glycosylated and secreted as biologically active 44-kDa glycoproteins, implying that the transmembrane domain functions as an internal signal peptide. Extensive glycosylation precedes proteolytic cleavage of pro-NDF but has no effect on receptor binding. By contrast, the EGF-like domain fully retains receptor binding activity when expressed separately, but its beta-type C terminus displays higher affinity than alpha-type NDFs. Likewise, structural heterogeneity of the cytoplasmic tails may determine isoform-specific rate of pro-NDF processing. Taken together, these results suggest that different NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions.

Amino Acid Sequence↗

A fluorescence-conjugated immunobinding assay for the detection of P-selectin on platelets.

P-selectin (GMP-140 or PADGEM) is translocated to the plasma membrane of platelets after platelet activation. P-selectin, therefore, may be a potential marker for evaluating platelet activation. A fluorescence-conjugated immunobinding assay (FCIBA) has been developed to detect specifically P-selectin on platelets. Platelets were isolated from fresh blood by centrifugation and stimulated with various doses of ADP before being fixed with 1% of paraformaldehyde. Fixed platelets were incubated with fluorescence-conjugated anti-P-selectin monoclonal antibody in the wells of fluoricon microtiter plates, and the fluorescence intensity was read on a fluorescence concentration analyzer. Once platelets were fixed, the procedures were completed in < 2 hours. The intra-assay coefficient of variation (CV) was 6.97% (n = 40), the time-based interassay CV was 8.11% (n = 16), and the sample-based inter-assay CV was 6.17% (n = 16). The FCIBA had an excellent correlation (r = 0.936, p < 0.001) with flow cytometry in the measurement of expressed P-selectin in platelets of 20 normal donors. Translocation of P-selectin in plasma-suspended platelets in response to increasing doses of adenosine diphosphate (ADP) occurred in a dose-dependent manner and correlated positively with ADP-induced platelet aggregation in terms of both stimulating doses of ADP (r = 0.99, p < 0.01) and time intervals (r = 0.92, p < 0.05). The findings show that FCIBA is a fast and convenient assay with good precision for the determination of P-selectin expression of human platelets.

Adenosine Diphosphate↗