Biomedical subjects

J M Chen

Publications and source records attributed to J M Chen.

At least 19 recordsLinked to original sources

PubMed1997

Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase.

Legumain is a cysteine endopeptidase that shows strict specificity for hydrolysis of asparaginyl bonds. The enzyme belongs to peptidase family C13, and is thus unrelated to the better known cysteine peptidases of the papain family, C1 (Rawlings, N. D., and Barrett, A. J. (1994) Methods Enzymol. 244, 461-486). To date, legumain has been described only from plants and a blood fluke, Schistosoma mansoni. We now show that legumain is present in mammals. We have cloned and sequenced human legumain and part of pig legumain. We have also purified legumain to homogeneity (2200-fold, 8% yield) from pig kidney. The mammalian sequences are clearly homologous with legumains from non-mammalian species. Pig legumain is a glycoprotein of about 34 kDa, decreasing to 31 kDa on deglycosylation. It is an asparaginyl endopeptidase, hydrolyzing Z-Ala-Ala-Asn-7-(4-methyl)coumarylamide and benzoyl-Asn-p-nitroanilide. Maximal activity is seen at pH 5.8 under normal assay conditions, and the enzyme is irreversibly denatured at pH 7 and above. Mammalian legumain is a cysteine endopeptidase, inhibited by iodoacetamide and maleimides, but unaffected by compound E64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane). It is inhibited by ovocystatin (cystatin from chicken egg white) and human cystatin C with Ki values < 5 nM. We discuss the significance of the discovery of a cysteine endopeptidase of a new family and distinctive specificity in man and other mammals.

Amino Acid Sequence↗

PubMed1997

Conformation-dependent phosphorylation of p53.

Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 ((His)p53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and beta subunit of casein kinase II (CKIIbeta) as well as by kinases present in whole cell extracts (WCEs). We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. We further demonstrate JNK interaction with GST-p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length (His)p53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53wt) undergoes better phosphorylation by CKIIbeta and by WCE kinases than mutant forms of p53 at amino acid 249 (p53(249)) or 273 (p53(273)). Moreover, shifting the kinase reaction's temperature from 37 degrees C to 18 degrees C reduces the phosphorylation of mutant p53 to a greater extent than of p53wt. Comparing truncated forms of p53 revealed that the ability of CKIIbeta, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97-155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region we have identified residues 97-117 that increase p53 phosphorylation by CKIIbeta while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53(249) exhibits significant changes to the conformation of p53 within amino acids 97-117. In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase.

Adenosine Triphosphate↗

PubMed1996

Demonstration of a glycoprotein derived from the 24p3 gene in mouse uterine luminal fluid.

A glycoprotein in mouse uterine luminal fluid was purified to homogeneity via a series of purification steps involving Sephadex G-100 chromatography, Sephadex G-50 chromatography and HPLC on a reverse-phase C18 column, in that order. Automated Edman degradation was unable to determine the N-terminal residue of the glycoprotein and the partial sequences determined from its trypsin digests were found to be identical with the protein sequence deduced from 24p3 cDNA. The core protein and the total amount of carbohydrate together gave a molecular mass of 25.8 kDa. Results from the characterization of the glycopeptide bond indicated the presence of N-linked carbohydrate but no O-linked carbohydrate in the protein, which has two potential sites for N-linked carbohydrate at Asn81 and Asn85, as deduced from analysis of the primary structure. The core protein was shown to have a molecular mass equal to that of the putative protein deduced from cDNA, suggesting that this protein may contain no signal peptide. Results of Northern-blot analysis for various tissues of adult mice revealed that the 24p3 gene was expressed in lung, spleen, uterus, vagina and epididymis.

Acute-Phase Proteins↗

PubMed1996

Prediction of the three-dimensional structure of the rap-1A protein from its homology to the ras-gene-encoded p21 protein.

rap-1A, an anti-oncogene-encoded protein, is a ras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32-47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A at ras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure of ras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 A from that of ras-p21. The effector binding domains from residues 32-47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115-126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.

Algorithms↗

PubMed1996

Conformational effects in the p53 protein of mutations induced during chemical carcinogenesis: molecular dynamic and immunologic analyses.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Vinyl chloride, a known human carcinogen that induces the rare sentinel neoplasm angiosarcoma of the liver, has been associated with specific A-->T transversions at the first base of codons 249 and 255 of the p53 gene. These mutations result in an Arg-->Trp amino acid substitution at residue 249 and an Ile-->Phe amino acid substitution at residue 255 in a highly conserved region in the DNA-binding core domain of the p53 protein. To determine the effects of these substitutions on the three-dimensional structure of the p53 protein, we have performed molecular dynamics calculations on this core domain of the wild-type and the Trp-249 and Phe-255 mutants to compute the average structures of each of the three forms. Comparisons of the computed average structures show that both mutants differ substantially from the wild-type structure in certain common, discrete regions. One of these regions (residues 204-217) contains the epitope for the monoclonal antibody PAb240, which is concealed in the wild-type structure but accessible in both mutant structures. In order to confirm this conformational shift, tumor tissue and serum from vinyl chloride-exposed individuals with angiosarcomas of the liver were examined by immunohistochemistry and enzyme-linked immunosorbent assay. Individuals with tumors that contained the p53 mutations were found to have detectable mutant p53 protein in their tumor tissue and serum, whereas individuals with tumors without mutations and normal controls did not.

Antibodies, Monoclonal↗

PubMed1996

Computed three-dimensional structures for the ras-binding domain of the raf-p74 protein complexed with ras-p21 and with its suppressor protein, rap-1A.

The three-dimensional structures of the ras-p21 protein and its protein inhibitor, rap-1A, have been computed bound to the ras-binding domain, RBD (residues 55-131), of the raf-p74 protein, a critical target protein of ras-p21 in the ras-induced mitogenic signal transduction pathway. The coordinates of RBD have been reconstructed from the stereoview of an X-ray crystal structure of this domain bound to rap-1A and have been subjected to energy minimization. The energy-minimized structures of both ras-p21 and rap-1A, obtained in previous studies, have been docked against RBD, using the stereo figure of the RBD-rap-1A complex, based on a six-step procedure. The final energy-minimized structure of rap-1A-RBD is identical to the X-ray crystal structure. Comparison of the ras-p21- and rap-1A-RBD complexes reveals differences in the structures of effector domains of ras-p21 and rap-1a, including residues 32-47, a domain that directly interacts with RBD, 60-66, 96-110, involved in the interaction of ras-p21 with jun kinase (JNK) and jun protein, and 115-126, involved in the interaction of p21 with JNK. The structure of the RBD remained the same in both complexes with the exception of small deviations in its beta-2 binding loop (residues 63-71) and residues 89-91, also involved in binding to rap-1A. The results suggest that the binding of these two proteins to RBD may allow them to interact with other cellular target proteins such as JNK and jun.

GTP-Binding Proteins↗

PubMed1996

Experience with right ventricular assist devices for perioperative right-sided circulatory failure.

BACKGROUND: Right-sided circulatory failure remains a significant source of morbidity and mortality for both cardiac transplant and left ventricular assist device recipients. METHODS: We reviewed our experience with 11 patients who required a right ventricular assist device (RVAD) after either orthotopic heart transplantation or left ventricular assist device implantation. Variables analyzed included total time of RVAD support, hemodynamic and hematologic parameters, and parameters of end-organ perfusion. These were assessed at five time points: (1) at least 2 weeks before RVAD implantation, (2) intraoperatively just before RVAD insertion, (3) while on RVAD support, and, for those who survived, (4) just before RVAD explantation, and (5) off RVAD support. Survival was assessed as the ability to be weaned successfully from RVAD support. Urine output and serum transaminase levels were recorded throughout the period of RVAD support. RESULTS: Five patients received an ABIOMED 5000 BVS RVAD, and 6 received a Bio-Medicus centrifugal pump. Nine patients in the study underwent orthotopic heart transplantation and had development of right-sided circulatory failure from 0 to 96 hours after donor organ insertion, and 2 patients underwent left ventricular assist device implantation 12 to 48 hours before RVAD support. The mean time of RVAD support for survivors was 133.6 +/- 33.6 hours (range, 107 to 190 hours). Six patients were successfully separated from RVAD support, and 5 patients died while on RVAD support. Causes of death included sepsis (2), biventricular failure (2), and coagulopathy (1). Continuous arteriovenous hemodialysis was employed in 3 of 6 survivors and 1 of 5 nonsurvivors. CONCLUSIONS: Right ventricular assist devices work most effectively if implanted early enough to avoid significant, potentially irreversible end-organ injury. We liberally employ continuous arteriovenous hemodialysis, minimize the use of heparin immediately postoperatively, keep patients sedated, and continue RVAD support until the patient displays signs of hemodynamic and end-organ recovery as heralded by (1) a decrease in central venous pressure and, more importantly, a decrease in pulmonary artery diastolic pressure, (2) an increase in urine output, and (3) a decrease in serum transaminase levels.

Adolescent↗

PubMed1996

Multivariate analysis of factors affecting waiting time to heart transplantation.

BACKGROUND: The growing clinical success of cardiac transplantation has resulted in a dramatic increase in the number of patients referred and subsequently listed for cardiac transplantation. Paradoxically, in the presence of a limited donor organ pool, such expansion has increased both the waiting time for transplantation and the number of patients dying while on the waiting list. METHODS: We performed univariate and multivariate analyses of the waiting times of 301 patients listed for transplantation using a Cox proportional hazards model to evaluate the simultaneous effect of multiple variables on the waiting time of heart transplant candidates. Variables considered included age, sex, race, blood type, weight at listing, United Network for Organ Sharing (UNOS) status at listing, UNOS status at transplantation, and proportion of time on the waiting list as UNOS status 1. RESULTS: The mean waiting time for patients ultimately having transplantation was 170.2 +/- 206.0 days; the median waiting time was 103.5 days. Age, sex, weight, blood type, and percent of time as UNOS status 1 all had a significant impact on waiting time in the univariate analysis. By multivariate analysis, proportion of time as UNOS status 1, lower weight at listing, and blood type AB were all highly associated as predictors of a shorter waiting time. Weight at listing represented a continuous variable whose risk ratio for a shorter waiting time correlated in such a way that the risk of a longer waiting time increased 2.3 per 22.5-kg (50-pound) increase in weight. Blood types A and B, although associated with a shorter waiting time, correlated less strongly than the other three variables. CONCLUSIONS: Our findings from this multivariate analysis demonstrate that UNOS status, blood type, and weight were the variables that most strongly affected overall waiting time for transplantation. It is our hope to define more accurately a group of patients with both a high likelihood of a long waiting time and a prohibitive risk of death while on the waiting list, who therefore may benefit from surgical alternatives to transplantation.

ABO Blood-Group System↗

PubMed1996

Responses to primary and a booster dose of acellular, component, and whole-cell pertussis vaccines initiated at 2 months of age.

A second generation acellular pertussis vaccine (component pertussis vaccine) containing purified pertussis toxin (PT) and filamentous hemagglutinin (FHA) was tested for its immunogenicity and safety in 2-month-old infants in comparison with first-generation acellular and whole-cell pertussis vaccines. At the ages of 2, 4, 6, and 18 months, respectively, 350 subjects were inoculated one dose of pertussis vaccine, which was combined with diphtheria and tetanus toxoids. Both acellular and component vaccines elicited significantly much fewer local and systemic reactions than whole-cell vaccine did. Besides, although not reaching statistical significance, the component vaccine was less reactogenic than the acellular vaccine. After each dose of the primary immunization, antibodies against PT and FHA were much higher in acellular and component pertussis vaccinees than in whole-cell vaccinees. However, at 18 months of age, just before the booster dose, both anti-PT and anti-FHA declined very close to, or even lower than, the prevaccination levels in all three groups and then responded rapidly to a booster dose to attain high levels. The booster responses were also significantly higher (P < 0.01) in acellular and component groups than in whole-cell group. Component and acellular vaccines induced similar levels of anti-FHA but the former induced higher anti-PT than the latter (P < 0.01). Our results indicate that both in primary immunization and as a booster, acellular and component pertussis vaccines are much more immunogenic for PT and FHA and much less reactogenic than whole-cell vaccine. However, the persistence of anti-PT and anti-FHA was not as good as one can expect from other protein antigens without giving a booster dose. A long-term follow-up of the vaccinees has been underway to understand the persistence of these antibodies after the first booster.

Antibodies, Bacterial↗

PubMed1996

In vitro evaluation of neonatal human immunity against the pig.

The critical shortage of organ donors has greatly limited the number of clinical allotransplantations. This is particularly true for neonatal patients, for whom xenotransplantation could provide an alternative therapeutic option to allotransplantation. The role of neonatal infant immunity in xenotransplantation is not, however, clearly understood. We examined both the proliferative responses of human neonatal lymphocytes to pig aortic endothelial cells and serum levels of neonatal natural antipig xenoantibody. Neonatal human lymphocytes and serum were isolated from umbilical cord blood. Adult human lymphocytes and serum were used as controls. A one-way xenogeneic mixed lymphocyte-endothelial cell reaction was performed, and lymphocyte proliferation was measured by tritiated thymidine uptake. Neonatal human lymphocytes recognized and proliferated in response to pig aortic endothelial cells (mean 63,926 +/- 26,054 counts per minute). The level of xenogeneic mixed lymphocyte-endothelial cell reaction of neonatal lymphocytes was significantly lower (p < 0.004) than that of adult human lymphocytes (mean 122,444 +/- 33,132 counts per minute). An enzyme-linked immunosorbent assay was performed to determine the binding of natural immunoglobulin M and G antibodies to pig endothelial cells. Whole-cell enzyme-linked immunosorbent assay demonstrated neonatal human serum to contain very low binding levels of natural antipig immunoglobulin M xenoantibody compared with adult serum. Like adult serum, neonatal human serum contained natural antipig immunoglobulin G xenoantibody. Neonatal serum was not cytotoxic to pig endothelial cells, suggesting that immunoglobulin G was not the predominant xenoreactive antibody. To assess whether neonatal pig endothelial cells also expressed xenoantigens, adult and neonatal cultured pig endothelial cells were examined by enzyme-linked immunosorbent assay with adult human serum. Adult human natural immunoglobulin M xenoantibody recognized not only adult pig endothelial cell xenoantigens but also neonatal pig endothelial cell xenoantigens. The binding levels of adult natural antipig immunoglobulin M xenoantibodies to adult and neonatal pig endothelial cells were similar, suggesting that neonatal pig aortic endothelial cells express xenoantigens. The findings of low binding levels of cytotoxic antipig immunoglobulin M xenoantibody and low levels of lymphocyte xenoreactivity to pig endothelial cells in human neonates suggests that pig organs may eventually be a suitable source of xenografts for human neonates.

Adult↗

PubMed1996

Inhibition of hepatitis B surface antigen secretion on human hepatoma cells. Components from Rubia cordifolia.

The antiviral activity in the roots of Rubia cordifolia was examined, and three naphthohydroquinones, furomollugin (1), mollugin (2), and rubilactone (3), were isolated from it. Compounds 1 and 2 strongly suppressed the secretion of hepatitis B surface antigen (HBsAg), both with IC50 = 2.0 micrograms/mL, in human hepatoma Hep3B cells while having little effect on the viability of the cells. Evaluation of structurally related derivatives of 1 and 2 revealed that a 6-hydroxy group and a pyran or furan ring contribute to this suppressive effect.

Animals↗

PubMed1996

Oncogenic amino acid substitutions in the inhibitory rap-1A protein cause it to adopt a ras-p21-like conformation as computed using molecular dynamics.

rap-1A is a membrane-bound G-protein in the ras superfamily that, like the ras-p21 protein, is activated by binding GTP in place of GDP. When activated, however, this protein inhibits the action of ras-p21, which is to induce mitogenesis in cells A chimeric protein containing RAS-p21 residues 1-65 and rap-1A residues 66-184 becomes ras-p21-like in its activity. The critical changes in sequence that result in this transformation are G26N, 127H, E30D, K31E, and E45V. All of these substitutions occur in or around a critical effector domain of p21 that is involved in interacting with GTPase activating protein (GAP), raf-p74 protein and inositol-3-hydroxy kinase. Using molecular dynamics, we have computed the average low energy structures for each of the three proteins, ras-p21, rap-1A and mutant rap1A, called rap-M, that contains these critical amino acid substitutions. We find that rap-M more closely superimposes on ras-p21 (rms deviation 1.9 A) than on wild-type rap-1A (rms deviation 3.4 A). In particular, the amino terminal domains (residues 3-59) of both RAS-p21 and rap-M are superimposable while they deviate when the average structures of these two proteins are superimposed on that of wild-type rap-1A. We have identified Pro 34 as a critical residue which may determine if the protein transforms cells or inhibits cell transformation. In addition, we have found that ras-p21 and rap-M proteins are superimposable in the region 96-110 except at Asp 105. The 96-110 domain of ras-p21 has been found to be involved in the binding of this protein to the nuclear transcription protein, jun and its kinase, jun kinase, JNK. Both segments differ in structure from that of the rap-1A segment at Asp 108, implicating this residue as also being important in determining the activity of the protein. Overall, the oncogenic substitutions introduced into the rap-1A protein cause it to adopt a conformation that is very similar to that of ras-p21 rather than wild-type rap-1A.

Amino Acid Sequence↗

PubMed1996

Computer model of a bovine type I collagen microfibril.

Collagens are a family of structural proteins of the extracellular matrix. The fibril-forming collagens are the major structural proteins of skin, cartilage, bone, blood vessel walls and internal organs. In addition to biological function, the collagens provide natural structural frameworks that are utilized in the medical, food and leather industries. Many schemes for the organization of type I collagen into triple helices, microfibrils and fibrils have been proposed during the past 30 years. Here, the development of a molecular model of a bovine type I collagen "Smith' microfibril is described. In cross-section, this model exhibits a symmetrical, pentagonal grouping of five triple helices. The model comprises 15 polypeptide chains having 315 residues each. This model is large enough to allow a comparison of its gross structural features with images of stained collagen obtained by electron microscopy, yet small enough to be manipulated on a minicomputer or work-station. The model is useful for (among others) studies of structure-function relationships in collagen, exploring folding pathways, predicting the efficacy of potential crosslinking agents or chemical modifications, and designing synthetic collagen-like materials or modifications for specific applications.

Amino Acid Sequence↗

PubMed1996

Surgical anatomy of the epitympanic space.

A clear understanding of the surgical anatomy of the epitympanic space is crucial in modern otologic surgery. The tortuous facial nerve within the tight confines of this region presents important surgical challenges, especially when normal anatomy is obscured by disease. Microdissections of 20 human temporal bones were carried out with particular emphasis on the spatial relations of the facial nerve with various temporal bone landmarks. Measurements were made through a standard transmastoid approach to ensure clinical relevance. A high margin of safety can be achieved by observing consistent landmarks and relations in dealing with the facial nerve. We demonstrated the limitations of the transmastoid approach, particularly in accessing the labyrinthine segment of the facial nerve.

Ear, Inner↗

PubMed1996

Topical gentamicin-induced hearing loss: a mitochondrial ribosomal RNA study of genetic susceptibility.

Mitochondrial ribosomal RNA mutation has been shown to predispose affected individuals to aminoglycoside-induced hearing loss (AIHL). An A-to-G nucleotide substitution at the 1555 position within the 12S ribosomal RNA gene has been identified with a maternally inherited pattern in affected Asian pedigrees. The aim of this study was to identify the frequency of this DNA polymorphism in subjects who appear to show hypersensitivity to topical (middle ear) application of aminoglycoside. In this pilot study, 10 subjects with AIHL were recruited. Eight underwent vestibular ablative therapy with middle ear instillation of gentamicin for disabling vertigo, and two were treated with topical antibiotic drops for otitis media in the presence of a tympanic perforation. DNA samples were extracted, and polymerase chain reaction (PCR) technique was used for gene amplification and purification, searching for 1555 A-to-G substitution in the 12S ribosomal RNA gene. None of the subjects demonstrated this specific mutation.

Adult↗

PubMed1995

Reversal of chronic ventricular dilation in patients with end-stage cardiomyopathy by prolonged mechanical unloading.

BACKGROUND: Ventricular dilation, indexed by marked shifts toward larger volumes of the end-diastolic pressure-volume relation (EDPVR), has been considered to represent an irreversible aspect of ventricular remodeling in end-stage heart failure. However, we hypothesized that such dilation could be reversed with sufficient hemodynamic unloading, such as can be provided by a left ventricular assist device (LVAD). METHODS AND RESULTS: The EDPVRs of hearts from seven patients with end-stage idiopathic cardiomyopathy and comparable baseline hemodynamics were measured ex vivo at the time of cardiac transplantation; these were compared with EDPVRs from three normal human hearts that were technically unsuitable for transplantation. Four of the patients received optimal medical therapy; three of the patients, who deteriorated on optimal therapy, underwent LVAD support for approximately 4 months. Compared with the normal hearts, EDPVRs of hearts from medically treated patients were shifted toward markedly larger volumes. In contrast, EDPVRs of hearts from LVAD patients were similar to those of normal hearts. CONCLUSIONS: Chronic hemodynamic unloading of sufficient magnitude and duration can result in reversal of chamber enlargement and normalization of cardiac structure as indexed by the EDPVR, both important aspects of remodeling, even in the most advanced stages of heart failure.

Adult↗

PubMed1995

Newborn baboon serum lacks natural anti-pig xenoantibody.

Discordant xenotransplantation represents an attractive alternative to allotransplantation in light of the shortage of donor organs currently available for cardiac allotransplantation. Unfortunately, discordant xenotransplantation is still limited by hyper-acute rejection, a process thought to be mediated by natural anti-xenodonor antibody. Based on data that cytotoxic natural xenoantibodies are IgM in nature, we postulated that natural xenoantibodies may be absent from newborn serum. Baboon sera were collected from infant baboons. Pooled adult baboon sera were used as controls. A whole cell ELISA was performed to determine the binding of xenoantibodies to pig aortic endothelial cells and pig lymphocytes. The cytotoxicity of both adult and newborn baboon sera to pig aortic endothelial cells was measured by a MTT (3-(4,5-dimethyl-thiazoyl-2-y) 2,5 diphenyl-tetrazolium bromide) assay. Newborn baboon sera demonstrated very low levels of binding of natural IgM xenoantibodies to pig endothelial cells and lymphocytes, whereas natural IgM xenoantibodies from adult baboon sera bound significantly to both pig aortic endothelial cells and lymphocytes. IgG natural antibodies in both adult and newborn sera bound to pig endothelial cells and pig lymphocytes. The MTT assay demonstrated high levels of cytotoxicity to pig endothelial cells from adult baboon sera and very low levels of cytotoxicity from newborn baboon sera. In this study, newborn baboon sera were demonstrated to be free of natural IgM xenoantibodies to pig endothelial cells and lymphocytes. Although natural anti-pig IgG antibodies were present in newborn sera, newborn baboon sera lack cytotoxicity to pig target cells. These findings suggest that IgM is the more important xenoantibody and that hyperacute rejection of discordant cardiac xenografts may be avoidable in the newborn.

Aging↗

PubMed1995

Immunolocalization of thimet oligopeptidase in chicken embryonic fibroblasts.

An antiserum was raised against chicken thimet oligopeptidase (TOP), and specific antibodies were isolated by immunoadsorption. The specificity of the antibodies was confirmed by immunoblot analysis with the cell extract of cultured chicken fibroblasts. Subcellular localization of TOP in normal and Rous sarcoma virus-transformed chicken embryonic fibroblasts was investigated by indirect immunofluorescence staining with affinity-purified, monospecific anti-TOP antibodies. In addition to the diffuse reaction expected for a cytosolic enzyme, a punctate, cytoplasmic localization was observed. The appearance of the organelles containing TOP did not correspond to that expected for secondary lysosomes, mitochondria, endoplasmic reticulum, or Golgi apparatus, but did resemble that of endosomes. When cells were allowed to internalize Lucifer yellow before fixation, some of the organelles containing TOP were observed to contain Lucifer yellow. Moreover, those organelles containing TOP were positively immunolabeled with the anti-transferrin receptor antibody. We conclude that part of the TOP in chicken embryonic fibroblasts is located in endosomes and discuss the implications of this.

Animals↗