Biomedical subjects

B Chen

Publications and source records attributed to B Chen.

At least 145 records · Page 8Linked to original sources

PubMed2000

Inhibition of the interferon-gamma/signal transducers and activators of transcription (STAT) pathway by hypermethylation at a STAT-binding site in the p21WAF1 promoter region.

Expression of the cyclin-dependent kinase inhibitor p21WAF1 can be up-regulated by activation of signal transducers and activators of transcription (STAT) proteins in response to IFN-gamma. In this study, we examined CpG methylation at the p21WAF1 promoter region in rhabdomyosarcomas (RMSs) using Southern blot analysis with the methylation-sensitive restriction enzyme HpaII. Sis-inducible element (SIE)-1, a STAT-responsive element located upstream of the p21 WAF1 CpG island, was completely methylated at an internal CpG in 13 of 26 (50%) primary RMS tumors and 2 of 5 RMS cell lines. In contrast, all normal tissues examined showed a partial methylation pattern at SIE-1. Complete methylation within SIE-1 strongly correlated with decreased p21WAF1 mRNA expression in RMS. We further studied the effects of SIE-1 hypermethylation on p21WAF1 induction by STAT activation. CpG methylation within SIE-1 significantly inhibited binding of activated STAT1 in electrophoretic mobility shift assays and abrogated STAT-mediated transcription activation in response to IFN-gamma in luciferase reporter gene assays. Activation of STAT1 in response to IFN-gamma resulted in increased p21WAF1 expression and growth suppression in RMS cells containing unmethylated SIE-1 but failed to induce p21WAF1 or growth inhibition in RD and A673 cells, both of which were completely methylated within SIE-1. However, demethylation at SIE-1, induced by a demethylating agent 5-aza-2'-deoxycytidine, reactivated p21WAF1 expression and restored the responsiveness to IFN-gamma in RD cells. Our results indicate a mechanism by which altered DNA methylation in the p21 WAF1 promoter region, by precluding STAT1 binding to SIE-1, directly inhibits the p21WAF1 induction and cell growth regulation through the IFN-gamma/STAT signaling pathway in RMS cells.

Antimetabolites, Antineoplastic↗

PubMed2000

The Missing Link: Early Methane ("T") Dwarfs in the Sloan Digital Sky Survey.

We report the discovery of three cool brown dwarfs that fall in the effective temperature gap between the latest L dwarfs currently known, with no methane absorption bands in the 1-2.5 µm range, and the previously known methane (T) dwarfs, whose spectra are dominated by methane and water. The newly discovered objects were detected as very red objects in the Sloan Digital Sky Survey imaging data and have JHK colors between the red L dwarfs and the blue Gl 229B-like T dwarfs. They show both CO and CH(4) absorption in their near-infrared spectra in addition to H(2)O, with weaker CH(4) absorption features in the H and K bands than those in all other methane dwarfs reported to date. Due to the presence of CH(4) in these bands, we propose that these objects are early T dwarfs. The three form part of the brown dwarf spectral sequence and fill in the large gap in the overall spectral sequence from the hottest main-sequence stars to the coolest methane dwarfs currently known.

Journal Article↗

PubMed2000

Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo.

Do changes in neuronal structure underlie cortical plasticity? Here we used time-lapse two-photon microscopy of pyramidal neurons in layer 2/3 of developing rat barrel cortex to image the structural dynamics of dendritic spines and filopodia. We found that these protrusions were highly motile: spines and filopodia appeared, disappeared or changed shape over tens of minutes. To test whether sensory experience drives this motility we trimmed whiskers one to three days before imaging. Sensory deprivation markedly (approximately 40%) reduced protrusive motility in deprived regions of the barrel cortex during a critical period around postnatal days (P)11-13, but had no effect in younger (P8-10) or older (P14-16) animals. Unexpectedly, whisker trimming did not change the density, length or shape of spines and filopodia. However, sensory deprivation during the critical period degraded the tuning of layer 2/3 receptive fields. Thus sensory experience drives structural plasticity in dendrites, which may underlie the reorganization of neural circuits.

Animals↗

PubMed2000

[Assessment on acute toxicity of combined pesticides].

The acute oral toxicity (LD50) of two pesticides combined from 3 categories of insecticides, i.e. organophosphates(OPs), pyrethroids and carbamates, was evaluated by Harris method on equal toxicity doses. The OPs compounds studied included: methylparathion, omethoate, methamidophos, phoxim, dichlorvos, profenofos, isocarbophos and malathion; the pyrethroids: alpha-cypermethrin, deltamethrin, fenvalerate and fenpropathrin; the carbamates: methomyl, isoprocarb and metolcarb. The mixtures of two OPs on the combination of dichlorvos plus omethoate and methamidophos plus profenofos showed additive effects, but methylparathion plus phoxim showed antagonistic effect. Most of the combination of OPs with pyrethroids showed synergistic effects, such as the mixtures of phoxim plus deltamethrin, phoxim plus alpha-cypermethrin, methlyparathion plus alpha-cypermethrin and ioscoarbphos plus fenpropthrin, with the exception of two mixtures showed additive effects: phoxim plus fenvalerate resulting in 1.5 times higher toxicity than expected, while dichlorvos plus deltamethrin resulting in less toxicity than expected. The effects of the mixtures of OPs and carbamates: phoxim plus methomyl and methamidophos plus metolcarb, were all additive, but the mixtures of he methylparathion plus methomyl showed antagonistic effect and malathion plus isoprocarb were synergistic. The biochemical mechanisms of changed toxicity of pesticides mixtures were discussed based on their toxicokinetics and toxicodynamics. It was concluded that the combined effects of insecticide mixtures were additive for OPs plus OPs or plus carbamates in most cased, but synergistic for OPs plus pyrethroids.

Animals↗

PubMed2000

Mechanisms of hepatic very low density lipoprotein overproduction in insulin resistance. Evidence for enhanced lipoprotein assembly, reduced intracellular ApoB degradation, and increased microsomal triglyceride transfer protein in a fructose-fed hamster model.

A novel animal model of insulin resistance, the fructose-fed Syrian golden hamster, was employed to investigate the mechanisms mediating the overproduction of very low density lipoprotein (VLDL) in the insulin resistant state. Fructose feeding for a 2-week period induced significant hypertriglyceridemia and hyperinsulinemia, and the development of whole body insulin resistance was documented using the euglycemic-hyperinsulinemic clamp technique. In vivo Triton WR-1339 studies showed evidence of VLDL-apoB overproduction in the fructose-fed hamster. Fructose feeding induced a significant increase in cellular synthesis and secretion of total triglyceride (TG) as well as VLDL-TG by primary hamster hepatocytes. Increased TG secretion was accompanied by a 4.6-fold increase in VLDL-apoB secretion. Enhanced stability of nascent apoB in fructose-fed hepatocytes was evident in intact cells as well as in a permeabilized cell system. Analysis of newly formed lipoprotein particles in hepatic microsomes revealed significant differences in the pattern and density of lipoproteins, with hepatocytes derived from fructose-fed hamsters having higher levels of luminal lipoproteins at a density of VLDL versus controls. Immunoblot analysis of the intracellular mass of microsomal triglyceride transfer protein, a key enzyme involved in VLDL assembly, showed a striking 2.1-fold elevation in hepatocytes derived from fructose-fed versus control hamsters. Direct incubation of hamster hepatocytes with various concentrations of fructose failed to show any direct stimulation of its intracellular stability or extracellular secretion, further supporting the notion that the apoB overproduction in the fructose-fed hamster may be related to the fructose-induced insulin resistance in this animal model. In summary, hepatic VLDL-apoB overproduction in fructose-fed hamsters appears to result from increased intracellular stability of nascent apoB and an enhanced expression of MTP, which act to facilitate the assembly and secretion of apoB-containing lipoprotein particles.

Animals↗

PubMed2000

Differences between family physicians and patients in their knowledge and attitudes regarding traditional chinese medicine.

Background: With patients turning more to alternative medicine, most physicians are still conservative in their views toward these practices. It is important to know whether there is a difference between physicians and patients regarding knowledge and attitudes about alternative medicine. This study focused on information regarding a specific type of alternative medicine, traditional Chinese medicine (TCM). Methods: Two questionnaires-one for physicians, one for patients-were designed. Two clinics were chosen as sites where patients could fill out the questionnaires while they were waiting for treatment. A total of 85 patient questionnaires were collected. Forty-four completed questionnaires were returned from family physicians in Cincinnati area. Data analyiss included t-test, analysis of variance, and logistic regression. Results: Generally, physicians and patients had the same knowledge level on TCM. Physicians, however, had more knowledge on basic concepts, whereas patients knew more about practice, such as the specific conditions that could be treated by TCM. A difference between physicians and patients on attitudes toward TCM was found, with patients having a more positive attitude. More than 60% of the patients in this study had visited alternative practitioners during the year prior to this study. The inform rate was higher for the patients visiting physicians who practiced alternative treatment. Conclusions: There are differences between physicians and patients regarding their knowledge and attitudes about TCM.

Journal Article↗

PubMed2000

Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer.

The collection of small aliquots of bone marrow (BM), followed by ex vivo expansion for autologous transplantation may be less morbid, and more cost-effective, than typical BM or blood stem cell harvesting. Passive elimination of contaminating tumor cells during expansion could reduce reinoculation risks. Nineteen breast cancer patients underwent autotransplants exclusively using ex vivo expanded small aliquot BM cells (900-1200 x 10(6)). BM was expanded in media containing recombinant flt3 ligand, erythropoietin, and PIXY321, using stromal-based perfusion bioreactors for 12 days, and infused after high-dose chemotherapy. Correlations between cell dose and engraftment times were determined, and immunocytochemical tumor cell assays were performed before and after expansion. The median volume of BM expanded was 36.7 mL (range 15.8-87.0). Engraftment of neutrophils greater than 500/microL and platelets greater than 20,000/microL were 16 (13-24) and 24 (19-45) days, respectively; 1 patient had delayed platelet engraftment, even after infusion of back-up BM. Hematopoiesis is maintained at 24 months, despite posttransplant radiotherapy in 18 of the 19 patients. Transplanted CD34(+)/Lin(-) (lineage negative) cell dose correlated with neutrophil and platelet engraftment, with patients receiving greater than 2.0 x 10(5) CD34(+)/Lin(-) cells per kilogram, engrafting by day 28. Tumor cells were observed in 1 of the 19 patients before expansion, and in none of the 19 patients after expansion. It is feasible to perform autotransplants solely with BM cells grown ex vivo in perfusion bioreactors from a small aliquot. Engraftment times are similar to those of a typical 1000 to 1500 mL BM autotransplant. If verified, this procedure could reduce the risk of tumor cell reinoculation with autotransplants and may be valuable in settings in which small stem cell doses are available, eg, cord blood transplants. (Blood. 2000;95:2169-2174)

Adult↗

PubMed2000

Photodynamic therapy efficacy and tissue distribution of hypericin in a mouse P388 lymphoma tumor model.

The phototherapeutic properties and tissue distribution of hypericin were investigated in DBA/2 mice bearing subcutaneously transplanted P388 lymphoma cells. The efficacy of the photodynamic therapy (PDT) 2 h after administration of hypericin (2, 5, or 20 mg/kg, i.p., 120 J/cm2, 595 nm) was substantially greater than the efficacy after a 24 h interval. PDT with Photofrin (5 mg/kg, i.p., 24-h interval, 120 J/cm2, 630 nm) showed no significant antitumoral effect. The hypericin uptake in some tissues was measured after administration of hypericin (5 or 20 mg/kg, i.p.) up to 168 h. A comparison of the distribution data and the PDT efficacy at various intervals suggests that the plasma concentration of hypericin, and to a lesser extent the tumor uptake, determines the tumor response to PDT with hypericin.

Animals↗

PubMed2000

Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius.

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO(2) using formate dehydrogenase (FDH). PDH was produced by growth of T. intermedius ATCC 33205 or by growth of recombinant Escherichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Using heat-dried T. intermedius as a source of PDH and heat-dried Candida boidinii SC13822 as a source of FDH,98%, but production of T. intermedius could not be scaled up. Using heat-dried recombinant E. coli as a source of PDH and heat-dried Candida boidinii 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermoactinomyces98% ee.

Journal Article↗

PubMed2000

Expression of emmprin by oral squamous cell carcinoma.

A transmembrane glycoprotein recently identified on some tumor cells, extracellular matrix metalloproteinase inducer (EMMPRIN), has been shown to induce metalloproteinase (MMP) production by peritumor fibroblasts (PTF). We examined biopsy specimens of normal human oral mucosa and oral squamous cell carcinoma (SCC) for expression of EMMPRIN. In normal mucosa, EMMPRIN was expressed at the cell membrane throughout the epithelium with a slight enhancement along the basal cell layer. In oral SCC, EMMPRIN was expressed at the cell membrane throughout the entire lesion. Immunofluorescence microscopy localized EMMPRIN to the cell membrane in a highly invasive oral SCC cell line in agreement with our in vivo observations. Function-blocking antibodies to EMMPRIN significantly inhibited oral SCC cell migration on tenascin-C (TN-C) and fibronectin as well as invasion through a reconstituted basement membrane (RBM). We previously showed that soluble factors from SCC cells and PTF are required for deposition of a TN-C matrix. To determine whether EMMPRIN may modulate the release or expression of these soluble factors, we again used function-blocking antibodies. Antibodies to EMMPRIN completely inhibited the organization of TN-C matrices and partially reduced the deposition of FN matrices by oral SCC cell /PTF co-cultures. In addition, antibodies to EMMPRIN perturbed the expression of MMP-2. Moreover, antibodies to MMP-2 perturbed oral SCC cell invasion of an RBM by approx. 75%. Our results demonstrate that EMMPRIN is highly expressed in oral SCC, facilitates tumor cell motility, and mediates TN-C matrix deposition. Taken together, these results suggest that EMMPRIN may help regulate oral squamous cell carcinoma invasion.

Antigens, CD↗

PubMed2000

Detection of TT virus DNA in patients with liver disease and recipients of liver transplant.

TT virus (TTV) is transfusion-transmissible but its involvement in post-transfusion hepatitis is uncertain. To investigate the potential association of TTV with liver diseases, the prevalence of TTV DNA was tested by semi-nested PCR in 113 carriers of hepatitis C virus (HCV), 10 patients with acute liver failure, 11 patients with cryptogenic cirrhosis and 200 control blood donors. Thirty-seven of these patients underwent liver transplantation and were tested pre- and post-transplantation. TTV DNA was semi-quantified in serial samples from seven patients with unexplained post-transplant hepatitis. TTV genotyping was performed on samples from 28 patients by sequence analysis. The prevalence of TTV DNA in blood donors was 1.5% and 17% in HCV infected haemophiliacs. In patients with acute or chronic liver disease or hepatitis, 6 to 27% prevalence was observed. After liver transplantation, the prevalence of TTV DNA increased from 16 to 46% (P < 0.01). In patients who developed unexplained hepatitis post-transplantation, TTV viraemia did not parallel ALT levels. TTV DNA either increased in titre or became detectable shortly after transplantation, suggesting that either TTV was transfusion-transmitted, or, more likely, that immunosuppression caused a recurrence of low level or undetectable TTV viraemia. TTV had considerable genomic diversity in the N22 region, corresponding to at least 4 genotypes. Genotype 2 was found in 14/28 patients.

Adult↗

PubMed2000

A study of post-traumatic shingles as a work related injury.

BACKGROUND: After chicken pox, the herpes varicella-zoster (HVZ) virus may remain dormant in the dorsal root ganglion until later reactivation causes shingles, characterized by painful dysesthesias and cutaneous vesicular eruptions along a unilateral dermatome. Shingles as a work-related injury has not been previously addressed in the medical literature. Case History We present a 50-year old female hospital employee who, while working, sustained an acute, traumatic hyperextension injury to her right wrist, hand, and fingers. Although she initially responded to treatment for flexor tendinitis, she suddenly developed shingles in the right C5-C6 dermatomes. She was treated with famcyclovir and her skin lesions resolved, but post-herpetic neuralgia persisted. CONCLUSIONS: It was felt that her shingles was causally related to her occupational injury since trauma (previously reported to precipitate shingles) was her only risk factor and the timing and location of the lesions corresponded closely to the occupational injury. In addition to appropriately diagnosing and treating their patients, workers' compensation physicians often must determine if a particular condition was caused by the original work-related incident. Clinicians who treat trauma patients and injured workers should be aware of post-traumatic shingles and understand the causal relationship of this uncommon but clinically important phenomenon.

Accidents, Occupational↗

PubMed2000

Adrenergic stimulation regulates Na(+)/Ca(2+)Exchanger expression in rat cardiac myocytes.

The Na/Ca exchanger protein encoded by the NCX1 gene provides the predominant mechanism for calcium efflux during cardiac relaxation. Because beta -adrenergic stimulation increases expression of Ca(2+)channels (Ca(2+)influx) in cardiac myocytes, we tested the hypothesis that isoproterenol would concomitantly augment expression of NCX1. Four hour treatment of neonatal myocytes with isoproterenol significantly increased NCX1 gene and protein expression, and increased the rate of transcript initiation. Alpha-adrenergic stimulation significantly decreases NCX1 mRNA levels. Calcium transient measurements revealed that for cells that had been pretreated with isoproterenol there was a faster relaxation rate of the Ca(2+)transient in the presence of thapsigargin, indicating an enhanced rate of intracellular Ca(2+)removal. We conclude that effectors that increase calcium channel expression in neonatal myocytes also augments NCX1 gene and protein expression over a similar time course, and that this is due to enhanced NCX1 transcription. The regulation of expression of NCX1 by adrenergic pathways may play an important role in regulation of excitation-contraction coupling in cardiac myocytes.

Adrenergic alpha-Agonists↗

PubMed2000

Transcriptional regulation of L-type calcium channel expression in cardiac myocytes.

The L-type calcium channel is a heteromultimeric protein complex, which is expressed in the cardiac sarcolemma. Although post-translational regulation of its subunits by protein kinase A (PKA) has been widely reported, little is known about molecular processes that regulate expression of calcium channel subunits (alpha(1C), alpha(2)- delta, and beta(2A)subunits). Previous studies from our group demonstrate that the steady-state mRNA level of the alpha(1C)unit is increased by treatment of myocytes with beta -adrenergic agonists. The current study is designed to determine whether the mRNA levels for all subunits of the L-type calcium channel are coordinately controlled by a beta -adrenergic agonist, and whether this occurs predominantly through control of rate of transcription. Nuclear run-on assays were used to determine the transcription initiation rate of these genes in cultured neonatal rat cardiac myocytes. In isoproterenol (10(-7)m)-treated myocytes, transcription of genes encoding the alpha(1C), alpha(2)- delta, and beta(2A)subunits was enhanced. The increases in transcription initiation rate for alpha(1C), alpha(2)- delta, and beta(2A)subunits genes were 404%, 367%, and 240% of control, respectively. Pretreatment with the beta -adrenergic antagonist propranolol (10(-5)m) or PKA inhibitor H-89 (10(-6)m) blocked the effects of isoproterenol, while either drug alone did not affect the gene transcription rate significantly. Steady state mRNA levels of the subunits increased following isoproterenol treatment. These results suggest that beta -adrenergic stimulation and the PKA signaling pathway play an important role in transcriptional regulation of the L-type calcium channel in myocyte. The expression of all the subunits of this ion channel is under coordinate transcriptional control.

Adrenergic beta-Agonists↗

PubMed2000

Development of a fungus-specific PCR assay for detecting low-level fungi in an indoor environment.

A fungus-specific PCR assay using only one primer set has been developed for detecting indoor fungi. Four fungal primer sets, NS3/NS4, NS5/NS6, FF1/FR1 and FF2/FR1, were tested with DNA from humans, rats, mice, bacteria, pollens and six commonly found fungal species (Alternaria chamydospora, Aspergillus flavus, Candida famata, Cladosporium fermentans, Penicillium chrycoIgenum and Stachybotrys chartarum). Results indicated that, although all four primer sets could amplify the fungal DNA, only FF2/FR1 demonstrated no cross-amplification with non-fungal DNA. In addition, these amplified fragments were sequenced to ensure that they indeed matched known fungal DNA sequences. Furthermore, besides the tested fungi, eighteen more genera of fungal sequences were examined and found to match the FF2/FR1. Here, the method of bead-beating was identified as the most effective way for spore breakage and fungal DNA release. The PCR amplification efficiency and potential inhibition were examined using different process solutions and preparation procedures. It was found that, when using 20% nutrient media and homogenization-first procedure, a higher amplification efficiency with less inhibition was achieved. Although positive bands were observed at 0.2 fungal spore/reaction using the homogenization-first procedure, the sensitivity of this assay would be two fungal spores/reaction for environmental samples.

Air Pollution, Indoor↗

PubMed2000

The feedback circuit connecting the superior colliculus and central mesencephalic reticular formation: a direct morphological demonstration.

The central mesencephalic reticular formation (cMRF) has been distinguished from the surrounding reticular formation due to its involvement in the control of saccades. A role in saccade function has been proposed for this region based on electrical-stimulation experiments, its neuronal activity, and its pattern of connections. The present study was undertaken in an attempt to further characterize the location of the central mesencephalic reticular formation by anatomical methods and to examine its connections with the superior colliculus at the neuronal level. Biotinylated dextran amine (BDA) was injected into the superior colliculus of two cynomolgus monkeys (Macaca fascicularis). This resulted in the retrograde labeling of a large number of neurons in a restricted area of the mesencephalic reticular formation. They were distributed bilaterally, with an ipsilateral predominance, forming a cellular band in the ventral half of the midbrain reticular formation that was 2.7 mm in its rostrocaudal extent. Its rostral pole lay dorsolateral to the red nucleus and ventrolateral to, but not immediately adjacent to, the interstitial nucleus of Cajal. The cell band was widest caudally, where it occupied an area of approximately 2.7 mm wide and 2 mm in depth. Labeled neurons displayed a wide variety of multipolar somatic shapes and sizes, with long, slightly tapering, sparsely branched dendrites. Tectal terminal arbors were also labeled within the mesencephalic reticular formation. They were concentrated bilaterally, with an ipsilateral predominance, in the same areas that contained retrogradely labeled neurons. Numerous, primarily en passant labeled boutons of various sizes and shapes were seen in close association with both labeled and unlabeled neurons. They formed axosomatic and, more commonly, axodendritic relationships with labeled neurons. The extensive relationship of labeled terminals and labeled cells suggests the existence of a strong interconnection between the deeper layers of the colliculus and the central mesencephalic reticular formation neurons projecting back to the tectum. The bidirectional neural circuit directly demonstrated in this study presumably provides an anatomical substrate for feedback modification of gaze signals generated in the colliculus. However, the presence of tectal terminals around unlabeled reticular neurons suggests that the collicular signal may also be fed forward to the downstream targets of the central mesencephalic reticular formation.

Animals↗

PubMed2000

Strong immunostaining for myogenin in rhabdomyosarcoma is significantly associated with tumors of the alveolar subclass.

Rhabdomyosarcomas are a heterogeneous group of tumors with respect to their molecular basis, degree of differentiation, histology, and clinical behavior. Because of the wide variation of tumor morphology, it is often difficult to distinguish between the distinct subtypes of rhabdomyosarcomas. By using cryosections of tumor specimens and immunohistochemistry, in the present study we show that strong expression of myogenin in rhabdomyosarcoma is associated with alveolar histology (P = <0.0001, Fisher's exact test). Although staining for myogenin was observed in 22 of 26 rhabdomyosarcomas, all alveolar rhabdomyosarcomas (nine of nine) showed high levels of staining for myogenin, as defined by the frequency and intensity of staining of the tumor cells. The staining pattern suggests that the tumor cells are clonally derived from myogenin-positive progenitor cells. In contrast, most embryonal rhabdomyosarcomas (13 of 15) were either negative or showed a low level of staining for myogenin. In these tumors a larger proportion of tumor cells were distinctly negative for myogenin. Six of seven alveolar rhabdomyosarcomas that strongly stained for myogenin were also positive for Pax3-7/Forkhead (FKHR) by polymerase chain reaction/reverse transcriptase-polymerase chain reaction. One of two embryonal rhabdomyosarcomas that strongly stained for myogenin was retrospectively found to be positive for Pax3/FKHR transcripts. Quantitative analysis for myogenin by Western blotting using a smaller subset of rhabdomyosarcomas revealed that in general there was a good correlation between immunohistochemical staining and Western blotting (P = 0.01, Pearson Correlation), although the former technique was more sensitive for detecting tumors with low levels of the protein. On average, alveolar rhabdomyosarcomas expressed at least threefold more myogenin than embryonal rhabdomyosarcomas. Our data show that staining for myogenin will be a simple, rapid, and accurate adjunct for distinguishing between alveolar and embryonal rhabdomyosarcomas. We propose that embryonal rhabdomyosarcomas result from an early block in myogenesis, before the expression of myogenin. In contrast, we propose that alveolar rhabdomyosarcomas either originate from a late block in myogenesis (after expression of myogenin) or that the pathological mechanisms involved in these neoplasms also induce strong expression of this protein.

Antibodies, Monoclonal↗

PubMed2000

Promotion of selective cell attachment by the RGD sequence in dentine matrix protein 1.

Dentine matrix protein 1 (DMP1) is an important component of the non-collagenous extracellular matrix of developing teeth and bones. Functions of DMP1 other than a putative role in the initiation of mineralization are largely unknown. A first report on the DNA and deduced amino acid sequence showed that DMP1 has a single Arg-Gly-Asp (RGD) sequence. Here, whether the RGD sequence functions as a cell-attachment domain was tested. Using site-directed mutagenesis, two mutant recombinant DMP1 proteins with specific alterations at the RGD site were created. In the first mutant protein the RGD sequence was altered to a RGE (RGE) sequence; in the second the RGD domain was deleted (DEL). Mutated proteins were confirmed to be DMP1 by partial protein sequencing and dot-blot analysis with an anti-DMP1 antibody. Attachment of RPC-C2A (dental pulp cells), MC3T3-E1 (calvarial cells) or CHO (Chinese hamster ovary cells) to non-tissue-culture plastic coated with either DMP1, RGE or DEL proteins was compared. Bovine serum albumin and fibronectin served as negative and positive controls, respectively. The RGD-containing native DMP1 protein effectively allowed cell attachment and spreading. The RGE and DEL proteins with the altered and deleted RGD sites were significantly less effective in promoting cell attachment than the recombinant DMP1. Both RPC-C2A pulp cells and MC3T3-E1 cells showed similar reductions in attachment to mutated proteins. Treatment of RPC-C2A cells with a RGD-containing peptide prior to plating on DMP1-coated chambers abolished DMP1-mediated cell attachment. In contrast to RPC-C2A and MC3T3-E1cells, CHO cells, which normally do not express DMP1, failed to attach to DMP1. These data demonstrate that DMP1 promotes cell attachment through the RGD domain and that the attachment is cell- and tissue-specific. A basis for these observations is proposed using computer-generated models of the polypeptides within the DMP1 protein containing the RGD, RGE or DEL sequences.

3T3 Cells↗