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Biomedical subjects

P Zhang

Publications and source records attributed to P Zhang.

At least 91 records · Page 5Linked to original sources

Does human lactoferrin in the milk of transgenic mice deliver iron to suckling neonates?

Lactoferrin is an iron-binding glycoprotein abundantly present in human milk, and has been postulated both to increase and to decrease intestinal iron absorption. To examine this problem, the interaction of milk iron with pup hemoglobin was studied in controls and in transgenic mice overexpressing human lactoferrin in their milk (2 lines expressing 12 mg/mL and 4 mg/mL, respectively). At day 14 of gestation, pregnant mice were switched from a diet of commercial chow containing iron at 300 mg/kg to diets containing 5, 15, or 50mg iron/kg; controls continued on chow. Nontransgenic pups were cross-fostered to transgenic dams to ensure that any results found in the pups were the effect of milk components. The hemoglobin level in the blood of 10-day-old suckling neonates was measured and calculated as total hemoglobin per pup. The total hemoglobin levels were lower in the pups receiving milk high in human lactoferrin, but the difference reached significance (P < 0.02) only at the highest level of dietary iron. Our findings do not support the hypothesis that lactoferrin functions as an intestinal iron scavenger, at least at high doses.

Animals↗

Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces.

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10(-6) M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.

Animals↗

The centromere structure in Robertsonian wheat-rye translocation chromosomes indicates that centric breakage-fusion can occur at different positions within the primary constriction.

Univalent chromosomes at meiotic metaphase I have a tendency to misdivide at the centromeres. Fusion of the misdivision products may produce Robertsonian translocations. The fine structure of the centromeres in Robertsonian wheat-rye translocation chromosomes was analyzed by fluorescence in situ hybridization (FISH) using two centromere-specific DNA clones: pRCS1, derived from rice, and pAWRC1, derived from rye. Clone pRCS1 hybridizes to the centromeres of all grasses including wheat and rye, whereas clone pAWRC1 is rye specific and hybridizes only to the centromeres of rye. Four of the six wheat-rye translocations derived from a single centric misdivision event (1st generation translocations) had hybrid centromeres, with approximately half of the centromere derived from rye and half from wheat. In the two other 1st generation translocations, the entire centromere was derived from rye. Among eight reconstructed wheat and rye chromosomes that originated from two consecutive centric misdivision-fusion events (2nd generation translocations), T1BS.1BL (derived from T1BS.1RL and T1RS.1BL) and one of three T2BS.2BL (derived from T2RS.2BL and T2BS.2RL) had hybrid centromeres. T1RS.1RL (derived from T1BS.1RL and T1RS.1BL), two of three T2BS.2BL, and all three T2RS.2RL (derived from T2RS.2BL and T2BS.2RL) had rye centromeres. All three 3rd generation translocations had hybrid centromeres with approximately half of the centromere derived from rye. There were no indications that the composite structure of the centromere in these chromosomes affected their behavior in mitosis or meiosis. These observations support the notion of a compound structure of the centromere in higher organisms, and indicate that during the centric breakage-fusion event, centromere breakage may occur in different positions along the segment of the chromosome that interacts with the spindle fibers. Normal behavior of the 1st, 2nd, and 3rd generation centric translocations in mitosis and meiosis indicates that, at least in wheat and rye, centromeres are not chromosome specific.

Cell Line↗

Cytometric methods to analyze ionizing-radiation effects.

Four cytometric assays for the assessment of radiation-induced DNA damage in individual cells are presented. Two of these, the alkaline and neutral comet assays, are useful for the detection of DNA damage due to very low radiation doses and promise to be useful for the quantitation of genomic damage after clinically or environmentally relevant exposures. The other two, the halo and halo-comet assays, reveal aspects of chromatin structure in the presence of DNA damage that reflect differences in intrinsic cellular radiosensitivity. Further development of these assays used alone, or in combination, should eventually lead to the definition of readily measurable cytometric parameters that will be useful as predictive markers for cellular responses to DNA damaging agents.

Animals↗

Comparison of methods for monitoring bacterial transport in the subsurface.

The purpose of this study was to compare in a laboratory experiment, a suite of methods developed to track viable bacteria during field transport experiments. The criteria for development and selection of these methods included: (1) the ability to track bacteria within the environment from which they were isolated; (2) the lack of any effect upon the viability or the transport characteristics of the strain; (3) low detection limits; (4) a quantification range that covered several orders of magnitude; and (5) an analytical cost and turnover time commensurate with the analysis of several thousands of samples in a few months. The approaches developed included: enumeration of bacteria labeled with a vital fluorescent stain (CFDA/SE) using microplate spectrofluorometry, flow cytometry, and ferrographic (immunomagnetic) capture; enumeration of highly (13)C-enriched bacteria using combustion-IRMS; and quantitative PCR. These methods were compared to direct microscopic enumeration and plate counts during a bacterial transport experiment performed in an intact sediment core and designed to simulate the field experiment. Four of the seven methods had equivalent recoveries for the breakthrough of a pulse of bacteria eluting from a 50-cm long sediment core, and all of the methods detected the arrival of cells in the effluent prior to the conservative tracer. Combustion IRMS and ferrographic enumeration had the lowest quantification limits (approximately 2 to 20 cells/ml), whereas microplate spectrofluorometry had the highest quantification limit (approximately 10(5) cells/ml). These methods have the potential for numerous applications beyond tracking bacteria injected into the subsurface.

Bacteria↗

Asbestos fiber burdens in lung tissues of Hong Kong Chinese with and without lung cancer.

To investigate the asbestos exposure level in non-occupational population and its relation to lung cancer, pulmonary asbestos fiber counts were observed with a modified alkali digestion method, in lung tissues from 65 random surgically treated lung cancer patients (42 males and 23 females) and that obtained from 107 random autopsy cases who died of either accidental death or acute myocardial infarction (44 males and 63 females) during 1987-1988. The patients and cases were all Hong Kong Chinese with no definite history of occupational exposure to asbestos. The non-lung cancer cases (NLCC) were divided into three groups based on different age ranges: 10 approximately, 40 approximately and 70 approximately years. The results showed that the medians of uncoated fiber counts (0, 32 and 52 fibers per mg dry tissue [f/mgDT] in the different age groups, respectively) increased in an age-dependent manner (P<0.01). However, the medians of coated fiber (asbestos body) counts were all 0 f/mgDT, with no statistically significant difference among the groups, showing a very low level of asbestos body burden in NLCC. In each age group, no difference in those counts between males and females was observed (P>0.05). Moreover, those counts in male lung cancer cases (medians of 62.5 and 17.5 f/mgDT, respectively) were greater than that in the control (32.5 and 0 f/mgDT, respectively) (P<0.01, P<0.05, respectively), however, regarding the female groups there was no statistically significant difference from the control. The results suggest that there is an environmental exposure to asbestos in Hong Kong Chinese, the level of which is, however, generally not sufficient to induce asbestos body. Environmental asbestos exposure in Hong Kong males may be one of the carcinogenic factors leading to lung cancer.

Accidents↗

ATP stimulated cyclic AMP formation in bovine chromaffin cells is enhanced by neuropeptide Y.

ATP increases cAMP formation in bovine chromaffin cells, EC(50) = 7.1 x 10(-6) M. NPY, EC(50) = 4.1 x 10(-8) M, increases the efficacy of ATP (1.5-2 fold). Inclusion of the selective Y1 receptor antagonist 1229U91 produced a decrease in NPY potency (EC(50) = 2.7 x 10(-7) M). PTX pretreatment did not abolish either the effect of ATP nor the enhancement by NPY. NPY could also enhance the ability of angiotensin and bradykinin to increase cAMP formation. The selective phospholipase C inhibitor, U73122, and the selective protein kinase C inhibitors, bisindolylmaleimide I and RO-31-8425, were effective inhibitors of the enhancing effect of NPY.

Adenosine Triphosphate↗

DNA strand breakage and oxygen tension: effects of beta-carotene, alpha-tocopherol and ascorbic acid.

DNA damage is involved in carcinogenesis, aging and other degenerative diseases. The relationship between DNA strand breakage and beta-carotene (0.1-1.6 microM) was examined under different O(2) tensions and with other antioxidants: alpha-tocopherol (5-80 microM), ascorbic acid (10-160 microM) and mixtures of these antioxidants. Supercoiled plasmid DNA pBR322 was incubated with 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) to induce DNA strand breaks in the presence of antioxidants under 15, 150, and 760 torr of O(2) tension. Under 15 torr of O(2) tension, beta-carotene, alpha-tocopherol, ascorbic acid and mixtures of these antioxidants provided a dose-dependent protection against AAPH-induced DNA strand breaks. The best protection was achieved in the mixture of antioxidants. Under 150 torr of oxygen tension, the antioxidant effect of beta-carotene was diminished at > or = 0.8 microM. A prooxidant effect was found at 0.8 > or = microM beta-carotene, producing more single- and double-strand breaks. alpha-Tocopherol and ascorbic acid exhibited dose-dependent antioxidant effects at 150 torr of oxygen tension. Under 760 torr of O(2) tension, the prooxidant effect of 0.8 microM beta-carotene was significant, causing supercoiled DNA to completely breakdown to circular and linear forms. In addition, 760 torr of O(2) tension attenuated the antioxidant effects of alpha-tocopherol and ascorbic acid. Thus, beta-carotene causes concentration-dependent DNA breakdown at high O(2) tension. The protection of DNA from the prooxidant effects of beta-carotene afforded by alpha-tocopherol and/or ascorbic acid was limited at high O(2) tension.

Antioxidants↗

Services provided to nonpregnant women during general medical and gynecologic examinations in the United States.

OBJECTIVES: To estimate the number of visits coded as general medical or gynecologic examinations (GMGEs) per 100 nonpregnant women aged > or = 18 years per year, to examine the content of the services provided during those examinations, to examine the degree of agreement between the patient's stated reason for visits and the diagnosis coded by physicians for GMGEs, and to examine the extent of preventive services delivered during GMGE visits and non-GMGE visits. METHODS: We analyzed data from two national surveys: the 1997 National Ambulatory Medical Care Survey and 1997 National Hospital Ambulatory Medical Care Survey. In addition, we obtained the number of women aged > or = 18 years in the United States from the Bureau of Census and the number of women who were pregnant from the National Center for Health Statistics. RESULTS: The number of visits coded as GMGEs by physicians was estimated to be < 21 visits per 100 nonpregnant women aged > or = 18 years per year. The leading four services provided during a GMGE were blood pressure check, breast examination, pelvic examination, and Papanicolaou (Pap) test. Of the visits coded as GMGEs by physicians, 82% of patients stated that their reason for visit was a GMGE. Only 35% of the visits that were initially considered to be GMGEs by patients were classified as such by physicians. For the visits initially considered to be GMGEs by patients, women were more likely to receive preventive services if the visits were also classified as GMGEs by physicians. Of Pap tests provided to nonpregnant women aged > or = 18 years, 44% were provided during these visits that were classified as non-GMGE visits by both physicians and by patients. CONCLUSIONS: GMGEs were important means of delivering preventive services to nonpregnant women, but not many women had GMGE visits. Evaluation of the acceptability and cost effectiveness of GMGEs to deliver preventive services to different populations is needed.

Adult↗

Antioxidant and prooxidant roles for beta-carotene, alpha-tocopherol and ascorbic acid in human lung cells.

Experiments were conducted to determine the antioxidant and prooxidant effects of beta-carotene, alpha-tocopherol and ascorbic acid on human lung cells at different oxygen (O(2)) tensions. Free radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), was used to induce the cellular damage associated with lipid peroxidation, protein oxidation and DNA breaks. Under hypoxic conditions (0 torr O(2) tension) all compounds produced a concentration-dependent antioxidant effect. Mixtures of the three compounds exhibited greater protective affects than any individual compound. At 143 torr O(2) tension, all compounds exhibited concentration-dependent protective effects against AAPH-induced cellular lipid, protein and DNA damage. At 722 torr O(2) tension, cells exhibited a consistent increase in lipid peroxidation (isoprostane formation), protein oxidation (carbonyl formation) and DNA damage (p53 protein accumulation). beta-Carotene (1.5 microM) produced a prooxidant effect by promoting 12% isoprostane formation. Protein oxidation and DNA damage at 722 torr O(2) tension was not increased by beta-carotene; however, the antioxidant effect of beta-carotene was attenuated. The antioxidant effects of alpha-tocopherol, ascorbic acid, and mixtures of the three antioxidant compounds also were reduced by the high O(2) conditions. These results partially substantiate the hypothesis that the antioxidant and prooxidant effects of beta-carotene are dependent on O(2) tension and concentration of beta-carotene. Such findings may partially explain why selected populations, such as smokers, respond adversely when supplemented with beta-carotene.

Antioxidants↗

beta-Carotene: interactions with alpha-tocopherol and ascorbic acid in microsomal lipid peroxidation.

beta-Carotene, alpha-tocopherol, and ascorbic acid were tested for their ability to inhibit, enhance, or react synergistically with O(2) (15, 150, 760 torr) and, 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) or 1,1'-azobis (cyclohexane-carbonitrile) (ACCN) in isolated rat liver microsomes. beta-Carotene did not protect against lipid peroxidation, i.e., malondialdehyde (MDA) formation, in microsomal samples incubated at 37 degrees C with aqueous soluble AAPH at all added beta-carotene concentrations and oxygen tensions. More MDA (16%, p < 0.001) was produced at 15 torr of O(2,) and 160 nmol/mg protein of beta-carotene compared to respective vehicle control. Individually, alpha-tocopherol and ascorbic acid exhibited antioxidant protection (ascorbic acid &z.Gt; alpha-tocopherol); however, a mixture of both compounds was no more protective than ascorbic acid alone. beta-Carotene demonstrated a concentration-dependent antioxidant affect at 15 torr O(2) (p < 0.01); but a prooxidant effect at higher O(2) at 150 and 760 torr (>57%, p < 0.001) by lipid-soluble ACCN. alpha-Tocopherol exhibited concentration-dependent inhibitory effects on microsomal MDA formation at all oxygen tensions, but was most effective under 150 torr. Ascorbic acid demonstrated a concentration-dependent antioxidant effect only at 150 torr. ACCN-induced lipid peroxidation was no greater for the combination of the three compounds than ascorbic acid added alone. Thus, antioxidant or prooxidant activities for beta-carotene, alpha-tocopherol, and ascorbic acid in microsomal suspensions are related to O(2) tension, solubility, antioxidant concentrations and are governed by complex interactions. Differences between AAPH- and ACCN-induced lipid peroxidation are related to differences in lipid solubility.

Journal Article↗

Genomic DNA hybridizes with the same rate constant on the QCM biosensor as in homogeneous solution.

Hybridization rates of sheared, genomic E. coli DNA in 0.14 M, pH 6.7 phosphate buffer at 65 degrees C were determined by: (1) observing the rate of absorbance decrease at 260 nm due to self-hybridization in solution; and (2) measurement of the rate of mass increase caused by hybridization between DNA in solution and DNA photografted to polystyrene. The latter measurement was done using a quartz crystal microbalance (QCM). In both the spectrophotometric and QCM experiments the probe was identical to the target, as both were taken from the same sample of sheared E. coli DNA. In the QCM measurements, viscoelastic effects were made negligible by drying the biopolymer layer on the QCM's surface before taking the frequency readings. Our purpose was to explore the effect of immobilizing DNA on its hybridization rate constant. A second-order constant of 2.32 +/- 0.09 x 10(-6) ml microg(-1) s(-1), n = 14, for hybridization in solution was obtained spectrophotometrically, while the QCM experiment gave a constant of 2.2 +/- 0.3 x 10(-6) ml microg(-1) s(-1), n = 6. These values are not statistically different. The reaction half-lives for the spectrophotometric and QCM experiments were 6.5 h and 13 min, respectively. The shorter half-life on the QCM can be explained solely by the much greater reactant concentration in the QCM experiment. About 25% of the DNA was inactivated by the attachment reaction. After correcting for this, the surface-attached DNA hybridized with the same rate constant as DNA free in solution. Therefore, it is concluded that, in these specific experiments with genomic DNA, the immobilized regions must have been short compared to the length of the molecules. The data demonstrate the high hybridization rate obtainable when nucleic acids are hybridized in a thin-film, micro-volume reaction on a non-porous surface.

Biosensing Techniques↗

Chromosome healing by addition of telomeric repeats in wheat occurs during the first mitotic divisions of the sporophyte and is a gradual process.

Alien gametocidal chromosomes cause extensive chromosome breakage prior to S-phase in the first mitotic division of gametophytes lacking the alien chromosome. The broken chromosomes may be healed either by addition of telomeric repeats in the gametophyte or undergo fusions to form dicentric or translocation chromosomes. We show that dicentric chromosomes undergo breakage fusion-bridge (BFB) cycles in the first few mitotic divisions of the sporophyte, are partially healed before the germ line differentiation regimen, and are healed completely in the ensuing gametophytic stage. The gametocidal factor on chromosome 4Mg of Aegilops geniculata was used to induce dicentrics involving the satellite chromosomes1B and 6B of wheat, Triticum aestivum. The dicentrics 1BS x 1BL-2AL x 2AS and 6BS x 6BL-4BL x 4BS initiated BFB cycles that ceased 2 to 4 weeks after seed germination. At the end of the BFB cycles, we observed deficient 1B and 6B chromosomes with breakpoints in proximal regions of the 1BL and 6BL arms. The process of chromosome healing was analyzed in root tip meristems, at meiotic metaphase I, and in the derived progenies by fluorescence in-situ hybridization analysis using a telomeric probe pAtT4. The results show that chromosome healing in wheat occurs during very early mitotic divisions in the sporophyte by de-novo addition of telomeric repeats and is a gradual process. Broken chromosome ends have to pass through several cell divisions in the sporophyte to acquire the full telomeric repeat length.

Cell Differentiation↗

Dissection of abscisic acid signal transduction pathways in barley aleurone layers.

Abscisic acid (ABA) induces genes that are highly expressed during late embryogenesis, but suppresses gibberellin (GA)-responsive genes essential for seed germination and seedling growth. Promoter elements necessary and sufficient for ABA up- and down-regulation of gene expression have been previously defined in barley aleurone layers. We have studied the effect of a protein phosphatase 2C, ABI1, an ABA-inducible protein kinase, PKABA1, and a transcription factor, VP1, on ABA action in a barley aleurone transient expression system. The observations have allowed us to dissect ABA signal transduction pathways leading to either induction or suppression of gene expression. The ABA induction of embryogenesis genes is highly inhibited in the presence of a mutated protein phosphatase 2C, encoded by the abi1-1 dominant mutant gene that is known to block ABA responses in Arabidopsis. However, the abi1-1 gene product has no effect on the ABA suppression of a GA-responsive alpha-amylase gene. On the other hand, PKABA1 suppresses the expression of alpha-amylase genes, but has little effect on ABA up-regulated genes. Therefore, it appears that ABA induction and suppression follow two separate signal transduction pathways with the former inhibited by ABI1 and the latter modulated by PKABA1. The presence of VP1 enhances the ABA induction of late embryogenesis genes, but also suppresses germination specific genes. A schematic model based on these observations is presented to explain the effect of these regulatory proteins on ABA-mediated gene expression.

Abscisic Acid↗

Identification of human, mouse and rat PPP1R14A, protein phosphatase-1 inhibitor subunit 14A, & mapping human PPP1R14A to chromosome 19q13.13-q13.2.

Three novel cDNAs encoding putative protein phosphatase-1 inhibitory protein (PPP1R14A) were isolated from human, mouse and rat. The human, mouse and rat PPP1R14A proteins were all of 147 amino acids sharing about 90% sequence identity to porcine CPI17, an inhibitor of protein phosphatase-1 holoenzyme. The protein sequence 33RHARVTVK40, which is important for the inhibitory potency of porcine CPI17, was conserved in mammalian PPP1R14A. Northern blot analysis showed that human PPP1R14A was highly expressed as a 600 bp transcript in heart, prostate, testis, ovary, colon, small intestine and pancreas, lowly in brain, placenta, skeletal muscle and spleen and lung. The distribution of mouse and rat PPP1R14A was more specific and different from that of human PPP1R14A, abundant in lung, moderate/abundant in testis, moderate in brain and low in heart. In addition, we mapped human PPP1R14A to chromosome 19q13.13-q13.2 by radiation hybrid mapping, and determined that the human PPP1R14A gene spanned a 5.1-kb region and consisted of four exons and three introns.

Amino Acid Sequence↗

Activation of Arp2/3 complex-mediated actin polymerization by cortactin.

Cortactin, a filamentous actin (F-actin)-associated protein and prominent substrate of Src, is implicated in progression of breast tumours through gene amplification at chromosome 11q13. However, the function of cortactin remains obscure. Here we show that cortactin co-localizes with the Arp2/3 complex, a de novo actin nucleator, at dynamic particulate structures enriched with actin filaments. Cortactin binds directly to the Arp2/3 complex and activates it to promote nucleation of actin filaments. The interaction of cortactin with the Arp2/3 complex occurs at an amino-terminal domain that is rich in acidic amino acids. Mutations in a conserved amino-acid sequence of DDW abolish both the interaction with the Arp2/3 complex and complex activation. The N-terminal domain is not only essential but also sufficient to target cortactin to actin-enriched patches within cells. Interestingly, the ability of cortactin to activate the Arp2/3 complex depends on an activity for F-actin binding, which is almost 20-fold higher than that of the Arp2/3 complex. Our data indicate a new mechanism for activation of actin polymerization involving an enhanced interaction between the Arp2/3 complex and actin filaments.

3T3 Cells↗

Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia.

The transcription factor C/EBPalpha (for CCAAT/enhancer binding protein-alpha; encoded by the gene CEBPA) is crucial for the differentiation of granulocytes. Conditional expression of C/EBPalpha triggers neutrophilic differentiation, and no mature granulocytes are observed in Cebpa-mutant mice. Here we identify heterozygous mutations in CEBPA in ten patients with acute myeloid leukemia (AML). We found that five mutations in the amino terminus truncate the full-length protein, but did not affect a 30-kD protein initiated further downstream. The mutant proteins block wild-type C/EBPalpha DNA binding and transactivation of granulocyte target genes in a dominant-negative manner, and fails to induce granulocytic differentiation. Ours is the first report of CEBPA mutations in human neoplasia, and such mutations are likely to induce the differentiation block found in AML.

Base Sequence↗

Three-dimensional reconstruction of dynamin in the constricted state.

Members of the dynamin family of GTPases have unique structural properties that might reveal a general mechanochemical basis for membrane constriction. Receptor-mediated endocytosis, caveolae internalization and certain trafficking events in the Golgi all require dynamin for vesiculation. The dynamin-related protein Drp1 (Dlp1) has been implicated in mitochondria fission and a plant dynamin-like protein phragmoplastin is involved in the vesicular events leading to cell wall formation. A common theme among these proteins is their ability to self-assemble into spirals and their localization to areas of membrane fission. Here we present the first three-dimensional structure of dynamin at a resolution of approximately 20 A, determined from cryo-electron micrographs of tubular crystals in the constricted state. The map reveals a T-shaped dimer consisting of three prominent densities: leg, stalk and head. The structure suggests that the dense stalk and head regions rearrange when GTP is added, a rearrangement that generates a force on the underlying lipid bilayer and thereby leads to membrane constriction. These results indicate that dynamin is a force-generating 'contrictase'.

Animals↗