Biomedical subjects

D Chang

Publications and source records attributed to D Chang.

At least 19 recordsLinked to original sources

PubMed1999

Second-look surgery after cytoreduction and intraperitoneal chemotherapy for peritoneal carcinomatosis from colorectal cancer: analysis of prognostic features.

Assuming that peritoneal carcinomatosis is a local/regional dissemination of disease, a treatment strategy utilizing cytoreductive surgery and intraperitoneal chemotherapy was developed to treat colon cancer. In an attempt to improve knowledge of the mechanisms controlling abdominal and pelvic recurrences and for better selection of patients for reoperation, we studied those patients who had a second-look surgery following cytoreduction for peritoneal carcinomatosis from colorectal cancer. A group of 18 patients with symptoms and signs of recurrent peritoneal carcinomatosis were treated with reoperative surgery after definitive cytoreduction and intraperitoneal chemotherapy. An analysis of clinical features of these patients was performed using survival as an endpoint for evaluation of prognosis. The data suggest that the clinical features to be used to select patients for a second-look procedure after prior cytoreduction were the completeness of resection at the time of initial cytoreduction (p = 0.04) and the completeness of resection at the time of the second look (p = 0.066). In addition, a limited extent of peritoneal carcinomatosis distribution found at the time of the second look predicted a favorable result. A new objective assessment of peritoneal carcinomatosis, the peritoneal cancer index, was found to be of help during patient selection (p = 0.066). We concluded that second-look surgery with potential curative intent should be considered in patients who had a complete initial cytoreduction and those in whom total removal of the recurrence is judged possible at the time of the second look. At the time of abdominal exploration, a limited distribution and volume of peritoneal carcinomatosis as defined by the peritoneal cancer index should be considered. Palliative debulking procedures should be used to alleviate symptoms in other patients.

Adult↗

PubMed1999

The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells.

The full-length major capsid protein, VP1, of the human polyomavirus JC virus was cloned and expressed in Escherichia coli. VP1 protein expressed in E. coli self-assembled into capsid-like particles and caused haemagglutination of human O-type red blood cells. Caesium chloride density-gradient centrifugation analysis revealed that the capsid-like particles consisted of virion-like pseudovirion and empty capsid-like pseudocapsid populations. The morphology of pseudo-virion and pseudocapsid particles was observed under the electron microscope. The pseudovirions contained DNA and RNA molecules but the pseudocapsids did not contain any nucleic acid, as analysed by DNA extraction. DNA-binding activity of VP1 was also demonstrated by the South-Western probing method in vitro. Furthermore, pseudocapsids were able to deliver exogenous DNA into human foetal kidney epithelial cells. These results indicate that recombinant JC virus VP1 is able to self-assemble into capsid-like particles and to package DNA in the absence of the minor capsid proteins, VP2 and VP3. This prokaryotic assembly system may facilitate the investigation of maturation mechanism(s) of polyomaviruses. Furthermore, capsid-like particles of JC virus VP1 generated in E. coli potentially could be used as a human gene transfer vector.

Capsid↗

PubMed1998

Synthesis and antitumour effect of the melanogenesis-based antimelanoma agent N-propionyl-4-S-cysteaminylphenol.

Chemotherapy of malignant melanoma is still a great challenge, as no effective drugs are available. The development of melanogenesis-based drugs is a promising area of research because melanogenesis is a unique biochemical pathway operating only in melanoma cells (and their normal counterparts) so that the tumour can be targeted. We have been using cysteinylphenol, a sulphur-containing analogue of tyrosine, and derivatives for that purpose. N-Acetyl-4-S-cysteaminylphenol was found to have the best antimelanoma effect in cell culture systems and in mice bearing B16 melanoma tumours. It also caused depigmentation of the skin, suggesting the possibility of use as a hypopigmenting agent. To improve the efficiency of the drug, we thought of replacing the acetyl group in N-acetyl-4-S-cysteaminylphenol with a propionyl group in the hope that increased hydrophobicity would increase the cellular uptake of the drug. N-Propionyl-4-S-cysteaminylphenol was synthesized by condensing 4-hydroxythiophenol with 2-ethyl-2-oxazoline. The drug showed both cytostatic and cytocidal effects in a human melanotic melanoma cell line. The drug was found to be a good depigmenting agent for the black hair follicles of C57 black mice when given s.c. for 14 days. A 10-day treatment with N-propionyl-4-S-cysteaminylphenol at 300 mg/kg body weight reduced the growth rate of B16 melanoma s.c. tumours in mice by 36%. The propionyl derivative was found to increase the life span of mice bearing melanoma more effectively than did the acetyl derivative.

Animals↗

PubMed1998

Ovotestes in B6-XXSxr sex-reversed mice.

The sex-reversed mutation Sxr results in XX males. In the absence of any other mutations, testis differentiation in XXSxr fetuses is essentially normal and only one report of an XXSxr fetus with ovotestes is in the literature. We report that 84% (21/25) of 13 days postcoitum XXSxr fetuses on the B6 inbred genomic background have ovotestes. Ovotestes were found in fetuses from both Sxra and Sxrb variants. Examination of fetuses older than 13 dpc suggests that the presence of ovotestes is transient in most fetuses. However, one overt hermaphrodite was identified after birth. The development of ovotestes is associated with the inbred background and is exacerbated by the dominant spotting oncogene allele KitW-42J. We propose that spreading of X-inactivation into the Sxr region resulting in loss of Sry expression is more extensive in B6-Sxr strains.

Animals↗

PubMed1998

Protein changes observed in pacing-induced heart failure using two-dimensional electrophoresis.

Rapid ventricular pacing in dogs results in a low output cardiomyopathic state which is similar to idiopathic dilated cardiomyopathy in man. However, the pathophysiological mechanisms which cause this failure following pacing are unknown. Five dogs underwent rapid ventricular pacing. Hearts were stimulated at 245 beats per min (bpm) for four weeks and then reduced to 190 bpm to stabilize the failure. Six unoperated dogs were used as controls. This paper compares the two-dimensional gel electrophoresis (2-DE) protein patterns of left ventricular samples from the paced myocardium with the control dogs. Changes in protein expression were analyzed qualitatively and semi-quantitatively. In the paced dog samples 69 protein spots were significantly altered of which 42 were decreased and 27 were elevated. One qualitative change was observed: elongation factor Tu was present only the control hearts. Of these proteins, 20 have been identified by a combination of N-terminal protein microsequencing, peptide mass profiling by mass spectrometry, amino acid compositional analysis, and by comparison with databases of canine and human ventricular proteins. Ten of these are associated with mitochondria and energy production, including: pyruvate dehydrogenase E1 component, isocitrate dehydrogenase subunit alpha, HSP60 and HSP70, creatine kinase M and fatty acid binding protein. The cytoskeletal protein desmin was detected in reduced quantities and a spot corresponding to a fragment of desmin was increased. These results indicate that the development of heart failure in the paced dog involves alterations in mitochondrial energy production, the cytoskeleton and calcium activation.

Animals↗

PubMed1998

Hyperthermic intraperitoneal doxorubicin: pharmacokinetics, metabolism, and tissue distribution in a rat model.

BACKGROUND: The cytotoxic effect of several anticancer agents, including doxorubicin, can be enhanced by hyperthermia. The purpose of this study was to evaluate the effect of hyperthermia on the pharmacokinetics, metabolism, and tissue distribution of intraperitoneal (i.p.) doxorubicin in a rodent model. METHODS: Doxorubicin was given i.p. to 20 Sprague-Dawley rats at a dose of 2 mg/kg over 60 min. Rats were randomized into two groups according to the temperature of the peritoneal perfusate: group NT received normothermic (37 degrees C) i.p. doxorubicin; group HT received hyperthermic (43 degrees C) i.p. doxorubicin. During the course of i.p. chemotherapy, peritoneal fluid and blood were sampled every 10 min. At the end of the procedure, rats were sacrificed and tissue samples (liver, spleen, small bowel, omentum, bladder, diaphragm, abdominal wall, heart) were collected. Concentrations of doxorubicin and its aglycone metabolites were determined in peritoneal fluid, plasma, and tissues by HPLC. RESULTS: No significant differences in areas under the curve (AUC) of peritoneal fluid doxorubicin and plasma doxorubicin were found between group NT and group HT. AUC ratios (AUC peritoneal fluid/AUC blood) were 87.9 for group NT and 82.9 for group HT. Group HT exhibited increased doxorubicin concentrations for all intraabdominal tissues. These differences were significant for spleen (P = 0.03), small bowel (P = 0.03), and omentum (P = 0.03). Doxorubicin aglycone was detected in plasma of both groups within the first 10 min of the procedure. There was a significant (P < 0.001) increase in plasma aglycone AUC for group HT when compared with group NT. Group HT exhibited increased aglycone concentration for all tissues. This difference was significant for liver (P < 0.001) and bladder (P < 0.001). CONCLUSION: Hyperthermia did not affect significantly the pharmacokinetics of i.p. doxorubicin. Tissue concentrations of doxorubicin in small bowel, omentum, and spleen were significantly increased when the drug was administered by hyperthermic i.p. perfusion. Hyperthermia increased significantly the doxorubicin aglycone concentrations in plasma, liver, and bladder.

Animals↗

PubMed1998

Effects of cisapride on QT interval in children.

Recent reports of torsade de pointes and heart block associated with prolonged QT interval in children receiving cisapride raise questions about its safety. We prospectively examined the effects of cisapride on the QT interval in children. Electrocardiography was performed on 30 children before and after cisapride was administered. An additional 71 children underwent electrocardiography only after starting cisapride. The incidence of a corrected QT (QTc) interval > 440 msec or a marked abnormality in T wave morphology was determined in all 101 children. Cisapride significantly lengthened the QTc with a mean increase of 15.5 +/- 4.6 msec (mean +/- SEM, p = 0.002 in the 30 children with baseline electrocardiographs. Twelve of the 101 patients were found to have a QTc > 440 msec, and one had a new prominent notched T wave in all leads. In these 13 (13%) patients with repolarization abnormalities, other factors that might contribute to a long QT were noted in 11 (85%) patients. We conclude that cisapride use in children is associated with a modest increase in QT interval. The incidence of QTc > 440 msec is low. Most children with long QTc have other factors that could compound the effects of cisapride.

Child↗

PubMed1998

Localization of hepatitis delta virus RNA in the nucleus of human cells.

Hepatitis delta virus (HDV) is a human pathogen that can greatly increase the severity of liver damage caused by an hepatitis B infection. HDV contains a circular, single-stranded RNA genome that encodes a unique protein, the delta antigen. Two forms of the delta antigen, deltaAg-S and deltaAg-L, are derived from a single open reading frame by RNA editing. Here we analyze the subcellular distribution of HDV RNA and its spatial relationship to known intranuclear structures. The human hepatoma cell line Huh7 was stably transfected with wild-type HDV cDNA and the viral RNAs were localized by in situ hybridization and fluorescence confocal microscopy. HDV RNA is detected throughout the nucleoplasm, with additional concentration in focal structures closely associated with nuclear speckles or clusters of interchromatin granules. Both the smaller form of the delta antigen (deltaAg-S), which is required for HDV genomic replication, and the larger form of the delta antigen (deltaAg-L), which represses replication, co-localize with delta RNA throughout the nucleoplasm and in the foci. However, the foci do not incorporate bromo-UTP and do not concentrate either RNA polymerase II or cleavage and polyadenylation factors required for viral RNA synthesis and 3' end processing, respectively. Thus, it is unlikely that the delta foci represent major sites of viral transcription or replication. In conclusion, the data show that viral RNA-protein complexes accumulate in structures closely associated with interchromatin granules, a subnuclear domain highly enriched in small nuclear ribonucleoproteins, poly(A+) RNA, and RNA splicing protein factors. This implies a specific compartmentalization of ribonucleoproteins in the nucleus.

Carcinoma, Hepatocellular↗

PubMed1998

Metabolic engineering as therapy for inborn errors of metabolism--development of mice with phenylalanine hydroxylase expression in muscle.

Treatment of many inherited liver enzyme deficiencies requires the removal of toxic intermediate metabolites from the blood of affected individuals. We propose that circulating toxins can be adequately cleared and disease phenotype influenced by enzyme expressed in tissues other than the liver. Phenylalanine hydroxylase (PAH) activity was constitutively expressed in skeletal and cardiac muscle of transgenic mice which carried the PAH cDNA under the transcriptional control of the mouse muscle creatine kinase promoter. Muscle PAH-expressing mice were bred to liver PAH-deficient, hyperphenylalaninemic mice to yield progeny that lack PAH activity in liver but express PAH in muscle. These mice exhibited hyperphenylalaninemia at baseline, but serum phenylalanine levels decreased significantly when the mice were supplemented with tetrahydrobiopterin (BH4), a required cofactor for PAH. This is the first demonstration that a liver-specific enzyme, when expressed in a heterologous tissue and supplied with necessary cofactors, can effectively clear toxic metabolites from the circulation of individuals with inherited enzyme deficiency. This result suggests that gene therapy targeted to heterologous tissues, such as muscle, will be effective in the treatment of selected inborn errors of metabolism.

Animals↗

PubMed1998

Characterization of two distinct monoclonal antibodies specific for glial cell line-derived neurotrophic factor.

Here we report the generation and characterization of two distinct monoclonal antibodies, G-90 and B-1531, specific to glial cell line-derived neurotrophic factor (GDNF). ELISA results confirmed that G-90 and B-1531 both recognize GDNF. Western blots showed that G-90 recognized only the GDNF dimer, whereas B-1531 recognized both the monomer and dimer. Peptide competition ELISA (PCE) and BIAcore data suggested that G-90 and B-1531 recognize different epitopes: PCE confirmed that B-1531 binds to NH2-terminal peptides between amino acids 18 and 37, whereas G-90 does not; BIAcore data showed that B-1531 binds to the NH2 terminus of GDNF, whereas G-90 does not. G-90, in a concentration-dependent manner, completely neutralized the GDNF-induced increases of choline acetyltransferase in cultured motoneuron and of dopamine uptake and morphological differentiation in dopaminergic neuron cultures. B-1531 had no neutralizing effects. GDNF-induced Ret autophosphorylation in NGR-38 cells was completely neutralized by G-90, whereas B-1531 had a moderate effect. These data show that G-90 and B-1531 are specific antibodies to GDNF. The data also suggest that the NH2 terminus of GDNF is not critical for activity. Partial inhibition of Ret phosphorylation is insufficient to down-regulate GDNF-induced biological activity.

Animals↗

PubMed1998

Prospective randomized trial of early postoperative intraperitoneal chemotherapy as an adjuvant to resectable gastric cancer.

OBJECTIVE: Surgeons have postulated on numerous occasions that cancer resection may participate in the dissemination of a malignancy. This randomized trial sought to determine whether a large volume of chemotherapy solution used perioperatively to flood the peritoneal cavity could eliminate microscopic residual disease and thereby improve survival of patients with gastric cancer. SUMMARY BACKGROUND DATA: Surgical treatment failures in patients with gastric cancer are confined to the abdomen in most patients. Resection site and peritoneal surface spread, along with liver metastases, are the most common areas of recurrence. Survival and quality of life of patients with gastric cancer would be improved if disease progression at these anatomic sites was reduced. METHODS: In a prospective randomized trial of 248 patients, intraperitoneal mitomycin C on day 1 and intraperitoneal 5-fluorouracil on days 2 through 5 were administered after gastric cancer resection. Patients who were thought to have stage II or stage III disease were randomized after resection to surgery alone versus surgery plus early postoperative intraperitoneal chemotherapy. After final pathologic examinations, there were 39 patients with stage I, 50 with stage II 95 with stage III, and 64 with resected stage IV cancer. RESULTS: The 5-year survival of the surgery-only group was 29.3%, and the surgery-plus-intraperitoneal chemotherapy group was 38.7% (p = 0.219). In a subset analysis, the patients with stage I, stage II, and stage IV disease showed no statistically significant difference in survival. The 5-year survival rate of patients with stage III disease who underwent surgery only was 18.4% versus a survival rate of 49.1% for patients who underwent surgery plus intraperitoneal chemotherapy (p = 0.011). CONCLUSIONS: In a subset analysis, patients with stage III gastric cancer have shown a statistically significant improvement in survival when treated with perioperative intraperitoneal chemotherapy. Further studies in patients with gastric cancer with surgically directed chemotherapy are suggested.

Adult↗

PubMed1998

Morbidity and mortality of early postoperative intraperitoneal chemotherapy as adjuvant therapy for gastric cancer.

Intraperitoneal chemotherapy (IC) is emerging as a valuable adjuvant therapeutic modality in patients with gastric cancer. The purpose of this study was to assess morbidity and mortality of early postoperative IC (EPIC) in gastric cancer patients. Two hundred forty-eight gastric cancer patients thought to have resectable cancer were randomized intraoperatively to receive EPIC with mitomycin C on postoperative day 1 and 5-fluorouracil on postoperative days 2 to 5 versus surgery only. Sixty-four patients who were stage IV at histopathologic examination remain in the analysis. Morbidity and mortality were compared using Fisher's exact test. All patients completed the therapy. In the study group, overall morbidity was higher than in the control group (28.8% versus 20.3%, respectively), although the difference was not significant (P = 0.121). Intra-abdominal sepsis without anastomotic leak (P = 0.008) and bleeding (P = 0.002) occurred significantly more often in the study group. Also, 37.6 per cent of patients who received EPIC experienced a variety of minor complications attributable to EPIC. Postoperative mortality was higher in the study group (5.6%) than in controls (0.8%), but not significantly (P = 0.299). Patients treated with EPIC stayed in the hospital an average of 4 days longer (P = 0.002); in patients with morbidity, however, there was no difference with the control group. A period analysis of the morbidity demonstrated that it followed the pattern of a learning curve. Surgery with EPIC tended to increase the postoperative morbidity and mortality. The therapy-associated risk must be justified by a significant improvement in survival of treated patients with stage III disease. Selective application of perioperative IC may be indicated.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1997

Differential down-regulation of CD28 by B7-1 and B7-2 engagement.

Physiologically relevant full activation of T cells requires signal transduction through the T cell receptor and additional costimulatory cell surface molecules. Best understood of these costimulatory interactions are those between CD28 and its ligands B7-1 (CD80) and B7-2 (CD86). While B7-1 and B7-2 bind the same receptors (CD28 and CTLA-4), they share only 25% sequence homology, are expressed at different times during immune responses, and in some systems have been shown to differentially affect T cell cytokine expression. Although CD28 is an activation antigen, its expression is down-regulated after engagement by B7-1. Here we show that B7-2 engagement is considerably less effective at down-regulating CD28, which indicates a differential effect of these two CD28 ligands on activated T cells.

Animals↗

PubMed1997

Effect of three elution buffers on the recovery and structure of monoclonal antibodies.

Antibodies are routinely purified by acid/salt elution from antigen affinity columns. The antibodies recovered with this procedure are active, but the recovery of protein is often low. We investigated the effect of acid and other denaturing or chaotropic solvents on the conformation of monoclonal antibodies (mAbs) made against the extracellular region of Her2 receptor (sHer2) derived from Chinese hamster ovary cells. The mAb remain almost completely folded in the 0.1 M glycine, pH 2.9, commonly used for elution, with the beta-sheet secondary structure intact, and only very small changes detected in the environment of the tryptophans. In 7 M urea, 50 mM NaAc pH 4.0, the antibody was partially unfolded, with the Trp environment further perturbed and some of the beta-sheet structure converted to disordered structure. In 6 M guanidine HCl, 50 mM NaAc, pH 4.0, the antibody is completely unfolded, with no secondary or tertiary structure present. The antibodies exposed to glycine or urea were refolded by dialysis into phosphate-buffered saline (PBS), while the guanidine HCl-denatured antibodies were refolded by dialysis into 7 M urea, pH 4.0, followed by dialysis into PBS. The refolded antibodies were capable of forming antigen-antibody complexes which could be isolated by gel filtration chromatography. Two different mAbs were subjected to immunoaffinity chromatography on sHer2-Sepharose. mAb86 was eluted by 0.1 M Gly, pH 2.9, while mAb52 was eluted with the 7 M urea, 50 mM NaAc, pH 4.0. The isolated antibodies were refolded by dialysis into PBS, analyzed for their ability to recognize native sHer2 by immunoprecipitation, and denatured sHer2 by Western blot analysis. Both preparations recognized the native protein, but precipitated slightly different forms of sHer2, indicating that they might recognize different epitopes. The mAb52 is a more sensitive reagent for Western blot analysis. Thus, this procedure can be used to recover antibodies which would not be recovered with glycine as the only eluate. It is also possible that the antibodies can be fractionated by the different eluants into populations which can be used for different applications.

Animals↗

PubMed1997

Fractionation and characterization of polyclonal antibodies using three progressively more chaotropic solvents.

In the previous paper we described the effect of several different solvents on the structure of antibodies and demonstrated that 0.1 M glycine, pH 2.9, 7 M urea, pH 4.0, and 6 M guanidine-HCl, pH 4.0, unfold the antibodies to different degrees. Antibodies can be refolded from all of these solvents by dialysis. Polyclonal antibodies (pAbs) are a mixture of antibodies which recognize and bind different epitopes on the same antigen, with the strength of the antigen-antibody binding varying with each subpopulation. When rabbit antisera to the extracellular domain of Her2 receptor (sHer2), derived from Chinese hamster ovary cells, was applied to an antigen column, bound pAbs were recovered with a step-wise elution of 0.1 M glycine, pH 2.9 (44% of the total recovered pAb), 7 M urea, pH 4.0 (29%), and 6 M guanidine-HCl, pH 4.0 (27%), with baseline resolution between them. Fluorescence spectra of the pAbs confirmed that the 0. 1 M glycine pH 2.9 sample had near-native structure, the pAbs in 7 M urea, pH 4.0, were partially unfolded, and the pAbs in the 6 M guanidine-HCl, pH 4.0, were totally unfolded. The glycine- or urea-eluted sample was refolded by dialysis into PBS, while the guanidine-HCl-eluted sample was first dialyzed into the 7 M urea pH 4.0 buffer and then into PBS. The refolded material from glycine or urea had native-like spectra, while the spectrum of the protein refolded from 6 M guanidine-HCl was slightly perturbed. All three of these subpopulations of pAbs formed antigen-antibody complexes which could be isolated by gel-filtration chromatography, precipitated sHer2 during immunoprecipitation, and recognized sHer2 in Western blots. The guanidine-HCl-eluted material was most sensitive for Western blotting. Identical results were obtained with pAbs applied either in the batch mode or to the top of the column, indicating that antibody aggregation which may occur when applied from the top of the column is not responsible for the distribution of pAbs into different subpopulations. These results indicate that the sequential use of these three increasingly chaotropic solvents to elute antibodies results in both increased recovery of antibodies and fractionation of pAbs into subpopulations with potentially different antigen binding characteristics.

Animals↗

PubMed1997

Notch activity influences the alphabeta versus gammadelta T cell lineage decision.

The choice between the alphabeta or gammadelta T cell fates is influenced by the production of functional, in-frame rearrangements of the TCR genes, but the mechanism that controls the lineage choice is not known. Here, we show that T cells that are heterozygous for a mutation of the Notch1 gene are more likely to develop as gammadelta T cells than as alphabeta T cells, implying that reduced Notch activity favors the gammadelta T cell fate over the alphabeta T cell fate. A constitutively activated form of Notch produces a reciprocal phenotype and induces thymocytes that have functional gammadeltaTCR gene rearrangements to adopt the alphabeta T cell fate. Our data indicate that Notch acts together with the newly formed T cell antigen receptor to direct the alphabeta versus gammadelta T cell lineage decision.

Animals↗

PubMed1997

Effects of L-arginine and N omega-nitro-L-arginine methyl ester on cardiac perfusion and function after 1-day cold preservation of isolated hearts.

BACKGROUND: Coronary flow responses to endothelium-dependent (acetylcholine [ACh] or 5-hydroxytryptamine [5-HT]) and endothelium-independent (adenosine [ADE] or nitroprusside [NP]) vasodilators may be altered before and after 1-day hypothermia during the perfusion of arginine vasopressin (AVP), D-arginine (D-ARG), L-arginine (L-ARG), or nitro-L-arginine methyl ester (L-NAME). METHODS AND RESULTS: Four groups of guinea pig hearts (37.5 degrees C [warm]) were perfused for 6 hours with AVP, L-ARG, L-NAME, or nothing (control). Five heart groups (cold) were perfused with AVP, D-ARG, L-ARG, L-NAME, or nothing (control), but after 2 hours they were perfused at low flow for 22 hours at 3.7 degrees C and again for 3 hours at 37.5 degrees C. ADE, butanedione monoxime, and NP were given for cardioprotection before, during, and after hypothermia. In warm groups, L-ARG did not alter basal flow or ADE, ACh, 5-HT, or NP responses, whereas L-NAME and AVP reduced basal flow and the ADE response, abolished ACh and 5-HT responses, and increased the NP response. In cold groups after hypothermia. L-ARG did not alter basal flow, but L-NAME, AVP, D-ARG, and control reduced flow. In the postcold L-ARG group, ACh increased peak flow, but NP did not increase flow in other cold groups. Effluent L-ARG and L-CIT in the cold control group fell from 64 +/- 9 and 9 +/- 1 micrograms/L at 1 hour to 36 +/- 5 and 5 +/- 1 micrograms/L at 25 hours, respectively. Left ventricular pressure and cardiac efficiency improved more in the postcold L-ARG group than in the postcold D-ARG, AVP, and L-NAME groups. CONCLUSIONS: Endogenous effluent levels of L-ARG and L-CIT decrease after 24 hours in isolated hearts, whereas perfusion of L-ARG improves cardiac performance, basal coronary flow, and vasodilator responses. In contrast, L-NAME, L-ARG, and AVP limit flow and performance but maintain a partial vasodilatory response to NP. Sustained release of NO may account for improved performance after L-ARG after hypothermia.

Animals↗

PubMed1997

Mapping of the active site of recombinant human erythropoietin.

Recombinant human erythropoietin (rHuEPO) variants have been constructed to identify amino acid residues important for biological activity. Immunoassays were used to determine the effect of each mutation on rHuEPO folding. With this strategy, we could distinguish between mutations that affected bioactivity directly and those that affected bioactivity because the mutation altered rHuEPO conformation. Four regions were found to be important for bioactivity: amino acids 11 to 15, 44 to 51, 100 to 108, and 147 to 151. EPO variants could be divided into two groups according to the differential effects on EPO receptor binding activity and in vitro biologic activity. This suggests that rHuEPO has two separate receptor binding sites. Mutations in basic residues reduced the biologic activity, whereas mutations in acidic residues did not. This suggests that electrostatic interactions between rHuEPO and the human EPO receptor may involve positive charges on rHuEPO.

Amino Acid Sequence↗