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Biomedical subjects

J Sun

Publications and source records attributed to J Sun.

At least 523 records · Page 29Linked to original sources
PubMed1996

Identification of the epitope for monoclonal antibody 4B1 which uncouples lactose and proton translocation in the lactose permease of Escherichia coli.

Monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic surface of the lactose permease of Escherichia coli, uncoupling lactose and H+ translocation in a manner indicating that it blocks deprotonation [Carrasco, N., Viitanen, P., Herzlinger, D., & Kaback, H. R. (1984) Biochemistry 23, 3681; Herzlinger, D., Viitanen, P., Carrasco, N., & Kaback, H. R. (1984) Biochemistry 23, 3688]. In this paper, 4B1 binding to purified lactose permease is shown to exhibit a KD of about 5 x 10(-10) M by surface plasmon resonance. Furthermore, the combined use of mutants containing 6 contiguous His residues in each periplasmic loop in the permease and Cys-scanning mutagenesis in conjunction with chemical labeling demonstrates that 4B1 binds specifically to the periplasmic loop between helices VII and VIII and that Phe247 and Gly254 are the primary determinants. Remarkably, although 4B1 binding uncouples lactose and H+ translocation, none of the amino acid residues in periplasmic loops, particularly Phe247 or Gly254, play an important role in the transport mechanism. Moreover, binding of avidin to biotinylated Glu255-->Cys in the loop containing the epitope has no effect on transport activity. Therefore, the uncoupling effect of 4B1 involves highly specific interactions which in all likelihood exert a torsional effect on the loop, resulting in a conformational change in helix VII and/or VIII that alters the pKas of residues involved in lactose-coupled H+ translocation.

Amino Acid Sequence↗
PubMed1996

Heme oxygenase (HO-1): His-132 stabilizes a distal water ligand and assists catalysis.

His-25 and His-132 are the primary candidates for the proximal heme iron ligand in heme oxygenase isozyme-1 (HO-1). The unambiguous spectroscopic demonstration that His-25 is the proximal iron ligand leaves the role of His-132 uncertain. Absorption and resonance Raman spectroscopy are used here to establish that mutation of His-132 to an alanine, glycine, or serine does not alter the histidine-iron bond, but results in the loss of the water molecule coordinated to the distal side of the iron in the wild-type enzyme-substrate complex. The His-132 mutations also (a) destabilize the ferrous-O2 complex with respect to autoxidation, which should result in partial uncoupling of NADPH consumption from heme oxidation, and (b) decrease the affinity of the enzyme for heme. The catalytic activity of the protein is decreased but not suppressed by these mutations: the H132G and H132A mutants retain 40-50% and the H132S mutant 20% of the activity of the wild-type protein. His-132, however, is required for catalytic turnover of the protein with H2O2. These results place His-132 close to the iron on the distal side of the heme pocket and indicate that His-132 facilitates, but is not absolutely required for, the catalytic turnover of HO-1.

Heme Oxygenase (Decyclizing)↗
PubMed1996

Early computation of shape and reflectance in the visual system.

A compelling sense of three-dimensional shape may be conveyed by the photograph of an object. Cues such as contour, shading, perspective and occlusion, to name a few, contribute to this percept. Psychophysical experiments suggest that certain aspects of three-dimensional shape are computed rapidly and in parallel by the visual system. Here we report that reflectance is also computed rapidly; moreover, it is the apparent reflectance, rather than brightness or perceptual three-dimensional shape, that is the primary basis for discrimination during the early stages of visual processing.

Humans↗
PubMed1996

Efficient and sustained transgene expression in mature rat oligodendrocytes in primary culture.

In order to evaluate the characteristics and efficiency of gene transfer in primary cultures of oligodendrocytes, four different techniques including particle bombardment (Accell gene gun), cationic liposome-mediated transfection (lipofection), calcium phosphate co-precipitation and retroviral infection were compared using the LacZ and luciferase reporter genes. Highly purified postnatal adult rat oligodendrocytes were obtained by sequential immunopanning, plated in culture, and transfected using various reporter and promoter genes. The most efficient expression of LacZ and luciferase genes was found with particle mediated gene delivery. The transgene expression level obtained with gene gun delivery was at least two- to 100-fold greater than three other tested gene transfer methods. Comparison of the relative strength of four viral and two cellular promoters in these primary oligodendrocytes cultures demonstrated that the CMV promoter was the strongest. Using a human growth hormone (hGH) reporter gene, a long-term transgene expression pattern in primary oligodendrocytes was demonstrated to be sustained in culture for the entire experimental period (4 weeks) after particle-mediated gene transfer. These results demonstrate that expression of a foreign gene can be effectively achieved in primary cultures of adult oligodendrocytes, especially by using the particle bombardment method. The results also suggest that the current ex vivo gene transfer system may be used to manipulate oligodendrocytes for future application in gene therapy studies.

Adenoviridae↗
PubMed1996

Quantitative analysis of glomeruli lesions in patients with mesangial proliferative glomerulonephritis.

By using computer imaging analysis system combined with light microscopy, the glomeruli lesions on biopsy specimens sections were quantitatively analysed. The pathological changes of mesangial proliferative glomerulonephritis (MsPGN) in children were quantitatively evaluated and the correlation between the mesangial matrix area (MA) and some clinical data were also analysed. The results indicated that the levels of various glomerular parameters in MsPGN group were higher than those of normal controls. No correlation was found between MA and 24 h urinary protein excretion, but a negative correlation was revealed between MA and estimated GFR. MA was also correlated with the duration of MsPGN recovery. It was suggested that the quantitative analysis of glomerular parameters by computer is a reproducible methods. The parameter of MA may be used for evaluation of the renal function, determination of the duration of therapy and evaluation of prognosis of MsPGN in children.

Adolescent↗
PubMed1996

Endarterectomy for calcified porcelain aorta associated with aortic valve stenosis.

BACKGROUND: A calcified porcelain aorta may complicate aortic valve insertion and require an alternative, more complex method for valve replacement. The reason for this is that sutures cannot be inserted through the calcific plates in the annulus and ascending aorta. METHODS: In 6 patients with an average age of 73.8 years (range, 65 to 81 years), we performed the simpler procedure of aortic endarterectomy of the calcific plates with the aortic valve replacement. We realized that there may be an increased risk of postoperative complications, particularly stroke. The calcific plates were fractured to allow debridement of the calcium. In addition, an end-arterectomy was performed of the left main coronary ostium in 2 patients, and 5 patients also had coronary artery bypass grafting performed. RESULTS: All 6 patients underwent successful operations without major complications. On follow-up, echocardiography or computed tomographic scans in 3 patients have not shown dilation of the ascending aorta. CONCLUSION: Endarterectomy of the aorta may be an option in the management of patients with calcification of the aorta.

Aged↗
PubMed1996

Cerebral protection during moderate hypothermic circulatory arrest: histopathology and magnetic resonance spectroscopy of brain energetics and intracellular pH in pigs.

OBJECTIVE: We evaluated the effect of antegrade and retrograde brain perfusion during moderate hypothermic circulatory arrest at 28 degrees C. METHODS: Phosphorus 31-magnetic resonance spectroscopy was used to follow brain energy metabolites and intracellular pH in pigs during 2 hours of ischemia and 1 hour of reperfusion. Histopathologic analysis of brain tissue fixed at the end of the experimental protocol was performed. Fourteen pigs were divided into two experimental groups subjected to antegrade (n = 6) or retrograde (n = 8) brain perfusion. Anesthesia (n = 8) and hypothermic cardiopulmonary bypass groups (15 degrees C, n = 8) served as control subjects. In the antegrade and retrograde brain perfusion groups, the initial bypass flow rate was 60 to 100 ml x kg(-1) x min(-1). In the antegrade group, the brain was perfused through the carotid arteries at a flow rate of 180 to 210 ml x min(-1) during circulatory arrest at 28 degrees C. In the retrograde group, the brain was perfused through the superior vena cava at a flow rate of 300 to 500 ml x min(-1) during circulatory arrest at 28 degrees C. RESULTS: The intracellular pH was 7.1 +/- 0.1 and 7.2 +/- 0.1 in the anesthesia and hypothermic bypass groups, respectively. Brain intracellular pH and high-energy metabolites (adenosine triphosphate, phosphocreatine) did not change during the course of the 3.5-hour study. In the antegrade group, adenosine triphosphate and intracellular pH were unchanged throughout the protocol. In the retrograde perfusion group, the intracellular pH level decreased to 6.4 +/- 0.1, and adenosine triphosphate and phosphocreatine levels decreased within the first 30 minutes of circulatory arrest and remained at low levels until the end of reperfusion. High-energy phosphates did not return to their initial levels during reperfusion. Histopathologic analysis of nine regions of the brain showed good preservation of cell structure in the anesthesia, hypothermic bypass, and antegrade perfusion groups. The retrograde perfusion group showed changes in all the regions examined. CONCLUSIONS: The study shows that moderate hypothermic circulatory arrest at 28 degrees C with antegrade brain perfusion during circulatory arrest protects the brain but that retrograde cerebral perfusion at 28 degrees C does not protect the brain.

Adenosine Triphosphate↗
PubMed1996

The VH and CH immunoglobulin genes of swine: implications for repertoire development.

Swine have the largest number of IgG subclass genes of all species so far studied but have a single gene for IgA which occurs in two allelic forms that differ in hinge length. Swine also have constant region genes for C mu and C epsilon, but lack a gene homologous to that which encodes IgD in rodents and primates, despite the otherwise high degree of sequence similarity of all other swine CH genes with those of humans. Swine have < 20 VH genes, a single JH and perhaps a limited number of DH segments. Newborn piglets show preferential VH and DH usage and may use gene conversion as a mechanism for expanding their antibody repertoire. Despite the close similarity of their Ig gene sequences to humans, swine belong to the group of animals that includes rabbits, chickens and cattle when classified on the basis of B cell development. This group, unlike rodents and humans, have a single VH family, use hindgut follicles early in life (rather than bone marrow throughout life) to diversify their antibody repertoire and probably all use gene conversion. It is proposed that IgD may serve a function in repertoire development in rodents and humans which is unnecessary in the chicken-lagomorph-artiodactyl group. The diversity of immunoglobulins and immunoglobulin genes among species justifies the quest of veterinary immunologists to define the system for their species of interest rather than making extrapolations from mouse and human immune systems.

Animals↗
PubMed1996

Disruption of CD40-CD40 ligand interactions results in an enhanced susceptibility to Leishmania amazonensis infection.

To study the role of CD40 ligand (CD40L) in the host immune responses against intracellular pathogens, we infected CD40L knockout (CD40L-/-) mice with Leishmania amazonensis. Although wild-type mice were susceptible to infection and developed progressive ulcerative lesions, tissue parasite burdens in CD40L-/- mice were significantly higher. This heightened susceptibility to infection was associated with an impaired T cell and macrophage activation and altered inflammatory response, as reflected by low levels of IFN gamma, lymphotoxin-tumor necrosis factor (LT-TNF), and nitric oxide (NO) production. Furthermore, CD40L-/- mice failed to generate a protective immune response after immunization. These results indicate an essential role of cognate CD40-CD40L interactions in the generation of cellular immune responses against an intracellular parasite.

Animals↗
PubMed1996

Molecular characterization of VDJ transcripts from a newborn piglet.

The sequences of 42 transcripts, expressed with IgM, IgG and IgA and cloned from the mesenteric lymph node of a newborn piglet, are described. Forty transcripts used either DHA and DHB and their FR4 were identical to the single swine germline JH. The low frequency of somatic mutation made it possible to identify 35/41 as originating from five putative germline VH genes, of which VHA, VHB and VHC accounted for > 85%. The remaining six transcripts were hybrids of these five germline genes. The most 3' functional VH gene (VH2 = VHB) was the only one exclusively expressed with IgM although VHA, of unknown location in the genome, accounted for half of all transcripts. Junctional diversity in CDR3 was extensive and asymmetrical, in that D-J joining contributed more diversity than V-DJ joining. Reading frame II was used twice as frequently as frame III and the CDR3 generated using the former would have a higher expected R/S ratio. This study indicated that the expressed VH repertoire of the newborn piglet is restricted and nearly germline although junctional diversity is mature and better developed than in fetal mice. The hybrid clones suggest that swine compensate for their < 20 VH genes and single JH by using somatic gene conversion. There was no evidence for exclusive or preferential expression of the most 3' VH gene as occurs in chickens and rabbits respectively, and switching to downstream constant regions probably occurs in utero, even in the theoretical absence of environmental antigens and maternal regulatory molecules. Preferential VHA expression is probably a selection phenomenon.

Amino Acid Sequence↗
PubMed1996

The influence of surface slope on human gait characteristics: a study of urban pedestrians walking on an inclined surface.

Human locomotion on inclined surfaces has been little studied. This work investigated uphill and downhill walking of urban pedestrians on slopes of up to 9 degrees. During a 3-month period, the gait characteristics of 1200 female and 1200 male pedestrians were recorded as they traversed a ramp of naturally varying slope at Sydney's Circular Quay. Walking speed, cadence and step length were determined for each subject, and average population gait parameters, for each ramp angle, were also calculated. The most significant finding was that the pedestrians' step length was decreased during ramp descent. Since theoretical analysis concludes that a reduction in step length produces a reduction in the friction demand, a shortening of the stride length is probably a means of counteracting the higher friction demand that would otherwise be required at heel strike during downhill walking.

Adolescent↗
PubMed1996

The swine Ig heavy chain locus has a single JH and no identifiable IgD.

Sequence analysis of 84 V region cDNAs expressed with IgM, IgG and IgA from both adult and newborn swine suggested that their JH segments had been derived from the same germline JH. Only a single hybridizing JH segment could be identified in genomic DNA, in a JH-C mu cosmid and in a Sacl fragment of the cosmid extending 5 kb 5' of the EnhH. The single germline JH segment mapped 6 kb 5' to C mu. This JH had a sequence identical to 40 of 42 JH segments expressed in a newborn piglet and 25 of 42 expressed by adult swine. None of the 19 JH segments which varied from the germline sequence were identical to each other and half of the nucleotide changes were silent. No cosmid DNA hybridizing with heterologous probes for C delta could be found within 20 kb 3' of C mu and C delta could not be cloned from genomic or cDNA libraries. A conserved IgD fragment could be amplified from human, mouse and rat genomic DNA but not from rabbit, swine or cattle. We hypothesize that heavy chain organization and constituency in homeothermic vertebrates is correlated with the site of secondary antibody repertoire development and the mechanism(s) used.

Animals↗
PubMed1996

Preclinical trial of a bioartificial liver support system in a porcine fulminant hepatic failure model.

BACKGROUND: This study describes the pre-clinical trials of an extracorporeal bioartificial liver support system (BALSS). It includes the biochemical changes which occur in the plasma and blood of pigs with devascularized livers when the plasma is treated in a BALSS, and the testing of the system for presence or absence of infective agents, pyrogens and for toxicity. METHODS: Hepatic cells were prepared from littermate juvenile white landrace pigs with a double-step collagenase digest technique. The cell preparations were incubated with collagen-coated dextran microspheres (CDM) for 3 h and the medium was tested to determine cellular metabolic activity. Incubation continued for a further 20 h during which the hepatic cells attach to the CDM. The CDM-attached cells were inoculated into a hollow fibre bioreactor which was part of an extracorporeal liver support system. RESULTS: Hepatic cell content of the bioreactor was 6 x 10(9) +/- 3 x 10(8) cells, equivalent to those present in half a pig's liver. The system was tested in a controlled trial with the plasma of pigs with fulminant hepatic failure (FHF) due to devascularized livers. When plasma from FHF pigs was circulated through the device there was significantly less of an increase in the accumulation of ammonia, lactate and most amino acids when hepatic cells were included in the circuit compared with those in control experiments when they were excluded. Similar changes occurred in procine blood. There were few infections diagnosed and an absence of pyrogens, endotoxins and toxicity in the bioreactor contents or in the terminating reservoir or animal blood samples. CONCLUSIONS: We believe that the results, demonstrating function of the porcine hepatic cells in the circuit, together with low risks, justify a clinical trial of use of the BALSS in Australia.

Amino Acids↗
PubMed1996

Modulation of integrin expression in endometrial stromal cells in vitro.

Integrins, a class of cell adhesion molecules found on virtually all cells, display dynamic temporal and spatial patterns of expression in the endometrium during the menstrual cycle and in early pregnancy. To study integrin regulation, we measured the expression of eight different integrin subunits on cultured human endometrial stromal cells obtained from proliferative phase endometrium, using immunofluorescence and flow cytometry. Treatment with estrogen and progesterone induced hormonal changes of decidualization but did not alter the expression of any of the integrins. It is presently unknown whether steroid hormones other than estrogen or progesterone affect integrin expression. In contrast, treatment with several growth factors and cytokines resulted in specific alterations of integrin levels. Epidermal growth factor, transforming growth factor-alpha, and transforming growth factor-beta 1 induced expression of the alpha 1 beta 1 collagen/laminin receptor. There also was a trend towards decreased expression of the alpha 6 subunit in response to interleukin-1 alpha, interleukin-1 beta, and tumor necrosis factor-alpha. The expression of alpha 1 beta 1 was accompanied by increased adhesion to collagen but there was no change in the binding to fibronectin and vitronectin. Our findings suggest that some aspects of decidualization may be regulated by steroid hormones, whereas others, such as integrin expression, are regulated by cytokines or growth factors, possibly of trophoblast origin. Integrins are likely to play a role in the interaction between trophoblast and endometrium.

Adult↗
PubMed1996

Molecular markers of ionizing radiation-induced gene mutations in mammalian cells.

We have isolated independent Chinese hamster ovary (CHO) cell mutants at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus from untreated, 60Co gamma-ray-exposed, and 212Bi alpha-exposed cells and identified the molecular changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. Both the parental CHO-K1 cells and the X-ray-sensitive mutant xrs-5 cells were studied. The radiosensitive xrs-5 cells are defective in DNA double-strand break rejoining ability and in V(D)J recombination, which can be complemented by Ku protein. Of the 71 spontaneous CHO-K1 hprt mutants analyzed, 78% showed no change in exon number or size, 20% showed loss of one to eight exons (partial deletion), and 3% showed loss of all nine hprt exons (total deletion). Exposure of CHO-K1 cells to 6 Gy of gamma rays, which reduced survival levels to 10%, produced a high deletion spectrum with 45% of the 20 mutants analyzed showing a loss of one to eight exons and 30% showing total deletion. Exposure to an equitoxic dose of alpha radiation from 212Bi, a 220Rn daughter, resulted in a spectrum similar to the gamma-ray spectrum in that 75% of the 49 mutants analyzed were deletions. To alpha radiation, however, tended to produce larger intragenic deletions than gamma radiation. Of the 92 spontaneous xrs-5 mutants analyzed for deletions, 43% showed a loss of one to eight exons and 14% showed total deletion. This suggests that, in certain regions of the hprt gene, base alterations can be converted into large deletions and alteration in the Ku protein complex can influence this type of mutational process. Exposure to alpha radiation (10% survival) to xrs-5 cells resulted in a deletion spectrum similar to that seen in CHO-K1 cells. Of the 49 mutants analyzed, 43% showed on change in exon number or size, 16% showed a loss of one to eight exons, and 41% showed total deletion. While the defect in xrs-5 cells has a profound effect on spontaneous mutant spectra, this defect does not appear to affect alpha-induced mutation spectra.

Alpha Particles↗
PubMed1996

Gene gun delivery of mRNA in situ results in efficient transgene expression and genetic immunization.

The use of mRNA to transfer genetic information into mammalian somatic cells in vivo or ex vivo may be advantageous in a number of gene therapy protocols. Success in utilizing in vivo RNA delivery for transgene expression has been extremely limited, partially due to RNA instability and to the lack of an efficient intracellular delivery mechanism applicable to a wide variety of tissue or organ systems. We report here that a particle-mediated gene delivery technology can be used to effectively deliver RNA molecules into a number of mammalian somatic tissue types. Expression from RNA transcripts of three reporter genes, firefly luciferase, human growth hormone and human alpha-1 antitrypsin, was detected in monolayer and suspension cell cultures bombarded in vitro, and in in vivo bombarded rat liver tissues, and mouse liver and epidermal tissues. Gene gun treatment of mouse epidermis in vivo with human alpha-1 antitrypsin messenger RNA elicited a strong, consistent antibody response which showed an increased titer with subsequent boosts. Results from this study point to future opportunities of applying RNA delivery techniques for transgenic studies, genetic vaccination, and gene therapy.

Animals↗