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Biomedical subjects

Y Zhou

Publications and source records attributed to Y Zhou.

At least 739 records · Page 41Linked to original sources
PubMed1995

Mutational analysis of PC1 (SPC3) in PC12 cells. 66-kDa PC1 is fully functional.

The proteinase mPC1, a neuroendocrine member of the mammalian family of subtilisin-like enzymes, has previously been shown to be converted to a carboxyl-terminally truncated 66-kDa form during transport through the secretory pathway. The cleavage site and the function of this carboxyl-terminal truncation event are unknown. We have performed site-directed mutagenesis of two paried basic sites in the mPC1 carboxyl-terminal tail and expressed these constructs in PC12 cells, a rat pheochromocytoma known to lack endogenous PC1. We found that the most likely site for the truncation event was at Arg590-Arg591 since mutation of this site to Lys-His prevented processing of 87-kDa PC1. A PC1 mutant carboxyl-terminally truncated at this site and expressed in PC12 cells was efficiently routed to the secretory pathway and stored in secretory granules, indicating that the carboxyl-terminal extension is not required for sorting of this enzyme. The function of the various PC1 constructs was assessed by analyzing proneurotensin cleavage to various forms. The carboxyl-terminally truncated PC1 mutant was found to perform most of the cleavages of this precursor as well as wild-type PC1; however, the blockade mutant processed proneurotensin much less efficiently. Differences between the site preferences of the various enzymes were noted. Our results support the notion that carboxyl-terminal processing of PC1 serves to regulate PC1 activity.

Animals↗
PubMed1995

Free radical formation in autopsy samples of Alzheimer and control cortex.

The formation of free radicals in homogenates of frontal cortex from brains taken at autopsy and verified histologically to be from five patients with Alzheimer's disease or from six age matched normal controls, was measured by electron paramagnetic resonance spectroscopy. During incubation at 37 degrees C, the formation of free radicals by Alzheimer's samples was 22% higher (P < 0.05) than controls. Following incubation in the presence of ferrous sulfate (200 microM), samples of Alzheimer's frontal cortex produced nearly 50% more free radicals than did controls (P < 0.01). Although these post mortem in vitro observations are consistent with an increased free radical burden in tissue from patients with Alzheimer's disease, that comparable differences exist in vivo between Alzheimer's patients and non-demented people remains to be demonstrated.

Aged↗
PubMed1995

A comparison of delays in the treatment of cancer with radiation in Canada and the United States.

PURPOSE: Waiting lists for medical care in Canada have been used as an argument against the single-payer option for health care reform in the United States, but there have been no direct comparisons of access to care in these two health care systems. The objective of this study was to compare how long cancer patients wait for radiotherapy in Canada and the USA. METHODS AND MATERIALS: Heads of radiation oncology at all cancer centers listed by the International Union Against Cancer (UICC) in Canada and the United States were sent a questionnaire that asked how long their patients waited for radiotherapy, and how long they though it was acceptable for patients to wait, in six clinical situations. RESULTS: Ninety-two of 97 eligible centers responded (95%). Median waiting times to start of radiotherapy were as follows: carcinoma of the larynx (T2,N0,M0), 29 days in Canada, 10 days in the USA; carcinoma of the lung (Stage IIIb, squamous), 34 days in Canada, 9 days in the USA; carcinoma of the prostate (Stage B2), 40 days in Canada, 11 days in the USA; carcinoma of the breast (T2,N0,M0) referred 1 month after lumpectomy, 43 days in Canada, 10 days in the USA; carcinoma of the prostate with painful bone metastases, 17 days in Canada, 5 days in the USA; carcinoma of the prostate with spinal cord compression, < 1 day in Canada, < 1 day in the USA. The differences in waiting times between Canada and the USA were all statistically significant (p < 0.0001), except in the case of emergency treatment for cord compression. The majority of radiation oncologists in both Canada and the USA regarded the delays reported by Canadian departments as medically unacceptable. CONCLUSIONS: Patients almost everywhere in Canada wait longer for radiotherapy than they do almost anywhere in the United States.

Aged↗
PubMed1995

Processing of mouse proglucagon by recombinant prohormone convertase 1 and immunopurified prohormone convertase 2 in vitro.

The mouse tumor cell line alpha TC1-6 was used as a model system to examine the post-translational processing of proglucagon. Determination of the mouse preproglucagon cDNA sequence and comparison with the published sequences of rat and human preproglucagons revealed nucleic acid homologies of 89.1 and 84%, respectively, and amino acid homologies of 94 and 89.4%, respectively. Immunohistochemical analyses with antibodies directed against PC2 and glucagon colocalized both the enzyme and substrate within the same secretory granules. PC1 was also immunolocalized in secretory granules. Cells were metabolically labeled with [3H]tryptophan, and extracts were analyzed by reverse-phase high pressure liquid chromatography. Radioactive peptides with retention times identical to those of synthetic peptide standards were recovered and subjected to peptide mapping to verify their identities. To determine the potential role of PC1 and PC2 in proglucagon processing, 3H-labeled proglucagon was incubated in vitro with recombinant PC1 and/or immunopurified PC2. Both enzymes cleaved proglucagon to yield the major proglucagon fragment, glicentin, and oxyntomodulin, whereas only PC1 released glucagon-like peptide-I from the major proglucagon fragment. Neither PC1 nor PC2 processed glucagon from proglucagon in vitro. These results suggest a potential role for PC1 and/or PC2 in cleaving several of the normal products, excluding glucagon, from the mouse proglucagon precursor.

Amino Acid Sequence↗
PubMed1995

Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.

Human placental trophoblasts lie at the maternal-fetal interface, a position in which they could play an important role in maternal tolerance of the fetal semi-allograft. Central to this hypothesis is their unusual MHC class I expression: they suppress class Ia production while expressing HLA-G, a class Ib molecule. We investigated human trophoblast HLA-G protein production in vivo and in vitro. We first used a synthetic peptide corresponding to the variable sequence of the alpha 1 domain to produce mAbs that recognized HLA-G. Ab specificity was demonstrated by immunoaffinity purification of a single protein with the same molecular mass (38 kDa) as HLA-G from choriocarcinoma cells. Use of these Abs to stain tissue sections of the maternal-fetal interface containing cytotrophoblasts in all stages of differentiation showed that HLA-G is expressed only by cytotrophoblasts that invade the uterus. Our previous in vitro studies showed that when early-gestation cytotrophoblast stem cells are cultured, they differentiate rapidly along the invasive pathway, as demonstrated by their expression of stage-specific markers. Here we show they also up-regulate HLA-G production. Cytotrophoblasts from term placentas, which have reduced invasive capacity in vitro, also had decreased ability to up-regulate HLA-G protein expression. We detected high levels of HLA-G mRNA in cytotrophoblasts isolated from first- and second-trimester placentas, but only trace amounts in term cells. Taken together, these results suggest that HLA-G production is a critical component of cytotrophoblast differentiation along the invasive pathway.

Amino Acid Sequence↗
PubMed1995

Blood transfusions and immunophenotypic alterations of lymphocyte subsets in sickle cell anemia. The Transfusion Safety Study Group.

Transfusions purportedly induce dysfunction of cell-mediated immunity in sickle cell anemia (SCA). We studied hematologic and lymphocytic indices in 173 human immunodeficiency virus (HIV)-negative subjects with SCA and 131 black controls. Children aged 1 to 7 years with SCA had leukocyte counts and percentages of granulocytes, monocytes, natural killer cells, and T-cell markers (CD2+CD11b+, CD4+CD26+, CD4+CD29+) that were significantly higher than those for control children. Percent total lymphocytes was decreased for this age group, but the total number of lymphocytes and T and B cell counts were similar to controls. Platelets were not increased. Adolescents (aged 8 to 17 years) and adults (aged > or = 18 years) with SCA had increased total leukocytes and monocytes and lymphocytes counts that remained level instead of decreasing, as did comparably aged controls. Lymphocyte subsets typically increased in count, but their percentage remained similar to children. The exception was CD56+ cell counts, which were increased in adolescents and adults. No lymphocytic subset change suggested impaired cellular immunity, and none could be related to transfusion. Prophylactically transfused patients had higher granulocyte counts, but these may arise from the complications of SCA itself.

Adolescent↗
PubMed1995

Interleukin-4 suppresses the expression of macrophage NADPH oxidase heavy chain subunit (gp91-phox).

The production of superoxide anion by NADPH oxidase is a principal nonspecific bactericidal activity of macrophages and neutrophils in host defense. However, exuberant production of superoxide anion also damages host tissues. Cloning and DNA sequencing of the 91 kDa subunit (gp91-phox) open reading frame indicated a high degree of sequence conservation, greater than 90% in nucleotide and amino acid sequences, between the porcine and human cDNAs. We show in pigs that interleukin-4 (IL-4), a T lymphocyte cytokine which plays a major role in mediating antibody responses to pathogens, suppresses superoxide anion production in macrophages by specifically reducing the level of mRNA encoding gp91-phox. Messenger RNA levels are suppressed approx. 70% within 4 h and persist for 24 h without any change in the rate of mRNA turnover. Nuclear run-on analysis showed that IL-4 did not alter the rate of gp91-phox gene transcription under conditions in which IL-1 beta transcription was inhibited. These results indicate that IL-4 suppresses the inflammatory response of macrophages by mechanisms that include post-transcriptional regulation of the 91 kDa catalytic subunit of NADPH oxidase, and transcriptional regulation of inflammatory cytokine expression.

Animals↗
PubMed1995

Identification of a 68 kDa protein species as a specific DNA-binding component of the H3abp complex interacting with the histone H3.2 G1/S regulatory domain.

The hamster histone H3.2 promoter contains a protein binding site (referred to as site X) required for G1/S transcriptional activation. We report here that nuclear extracts prepared from serum synchronized cells at various stages of the cell cycle show a biphasic increase in the H3.2 specific complex, H3abp, binding to site X. An increase in binding activity occurs as cells first enter the cell cycle and later at the G1/S border. The H3.2 specific binding activity is enhanced by Mg2+ and Ca2+ in vitro, but is inhibited by Zn2+. Site X resembles a Jun/AP-1 site, but previously it has been shown that the H3abp complex is immunologically distinct from the characterized AP-1 proteins. Here, we identify the size of the hamster nuclear protein(s) that bind specifically to the H3abp site by ultra-violet crosslinking and renaturation of specific protein bands following gel electrophoresis. In addition, we purify H3abp by affinity chromatography and show that the purified H3abp has a different methylation interference profile from AP-1. Our results indicate that a protein species around 68 kDa is the major DNA binding component of the H3abp complex and it binds specifically to the histone promoter site required for G1/S regulation.

Animals↗
PubMed1995

Requirement of tyrosine- and serine/threonine kinases in the transcriptional activation of the mammalian grp78/BiP promoter by thapsigargin.

Depletion of endoplasmic reticulum (ER) Ca2+ store by thapsigargin (Tg) in mammalian cells induces a set of ER protein genes known as the glucose-regulated proteins. Recently, IRE1p, a transmembrane protein postulated to have a serine/threonine kinase activity, has been identified as required for the induction of ER resident proteins genes in Saccharomyces cerevisiae. To investigate whether IRE1p can stimulate mammalian grp transcription, a stable Chinese hamster ovary cell line containing amplified copies of IRE1p has been created. The IRE1p expressing transfectants exhibited a modest (2-fold) enhancement of both the basal and Tg induced level of grp78 and grp94, two coordinately regulated grp genes. Using okadaic acid as a specific inhibitor for the endogenous serine/threonine protein phosphatase activities, a mild (2-fold) stimulative effect was observed for Tg induction of grp78 transcription. The okadaic acid potentiating effect requires a 50-base pair region in the vicinity of the grp78 TATA element. In contrast, the transcriptional activation of grp78 by Tg is almost totally eliminated by genistein, a tyrosine kinase inhibitor. The grp core, the C3 and C1 elements which are major Tg response elements of the rat grp78 promoter, are also major targets of the inhibitive effects of genistein.

3T3 Cells↗
PubMed1995

Identification of two positive transcriptional elements within the 91-base pair promoter for mouse testis angiotensin converting enzyme (testis ACE).

Testis angiotensin-converting enzyme (testis ACE) is an isozyme of ACE only expressed by male germ cells during spermiogenesis. It is the result of a strong sperm-specific promoter found within the 12th intron of the somatic ACE gene. Previous studies have localized the boundaries of the mouse testis ACE promoter as being from -91 to -9, relative to the transcriptional start site, and have suggested two important DNA regulatory elements starting at positions -55 and -32. DNA constructs were made in which these motifs were either eliminated or substituted. Each construct was tested for its ability to promote transcription in vitro, using a rat testis nuclear extract. Disruption of either motif reduced in vitro transcription to about 30% of control levels, while mutations of both elements abolished transcription. Two sites were selected inside each motif and altered by point mutation. Each of four constructs, containing a mutation at -51, -48, -30, or -28, transcribed at 29% or less the efficiency of the parent construct. The DNA element at -55, TGAGGTCA, is homologous to a consensus cyclic AMP response element. The motif at -32, TCTTAT, is located at a position analogous to a TATA box. Substitution of the -32 motif with a consensus TATA box sequence, TATAAA, stimulated transcriptional activity about 3-fold. As measured by gel mobility shift, oligonucleotides encompassing the -32 motif and the consensus TATA box formed different DNA-protein complexes. However, the -32 motif oligonucleotide was recognized by nuclear proteins prepared from either liver or testis nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Use of a new fluorescent probe, seminaphthofluorescein-calcein, for determination of intracellular pH by simultaneous dual-emission imaging laser scanning confocal microscopy.

A new pH indicator, seminaphthofluorescein (SNAFL)-calcein acetoxymethyl ester, was used for intracellular pH (pHi) measurement in living MDCK cells with a laser scanning confocal microscope (LSCM) equipped with an Argon/Krypton laser and dual-excitation and dual-emission (FITC/Texas Red) filter set. SNAFL-calcein excitation maxima are approximately 492/540 nm (acid/base) and emission maxima are approximately 535/625 nm (acid/base) with a pKa value at approximately 7.0. The absorption/emission spectra of SNAFL-calcein indicate that the ratio of emission intensities of its basic/acidic forms is pH dependent. With an Argon/Krypton LSCM, we were able to monitor the acidic and basic forms of this dye simultaneously using dual-excitation (488/568 nm) and dual-emission (525-614 nm/> or = 615 nm) wavelengths (lambda s). The simultaneous dual-excitation/emission LSCM system allows for efficient recording of pHi dynamics (time resolution approximately 1 sec) in living cells. We have analyzed emission stability of the dye at different temperatures (22 degrees C and 37 degrees C) and constant pH, and at the same temperature (22 degrees C) but various pHs (6.6, 7.0, and 7.4). Bleaching rate is slightly higher at 37 degree C than that at 22 degrees C. The basic form of the dye (lambda Em approximately 625 nm) has a slightly higher bleaching rate than the acidic form (lambda Em approximately 535 nm) in standard culture medium (pH 7.3) at either 22 degree C or 37 degrees C. The pHi in MDCK cells calculated from ratio images (535 nm/625 nm) was 7.19 +/- 0.03 (mean +/- SEM, n = 20). Calibration experiments show that the useful pH range of SNAFL-calcein appears to be between 6.2 and 7.8, as the dye is difficult to calibrate outside this pH range.

Animals↗
PubMed1995

Microcomputer system for automatic identification of the Cryptococcus neoformans and its clinical application.

In this study, microcomputer image processing and pattern recognition technology, and the knowledge of morphology and optical characteristics of Cryptococcus neoformans were used for identification of Cryptococcus neoformans. Four groups of mice were lethally infected with standard strain, Wuhan strain, American B-2643 strain and Var. Shanghainesis of the Cryptococcus neoformans. The samples collected included mice brain, lung, kidney, liver, small intestine tissue and were observed under a light microscope. More than 600 images of the fungus were input into a microcomputer. A system of computer for automatic identification of the Cryptococcus neoformans was developed. The technique involved image preprocessing, image segmenting, coding of line-length on the edge, curve fitting, extracting of image feature, building of image library and feature data bank etc.. And then, 768 images of the clinical samples and other fungus samples whose morphological features tend to be confused with Cryptococcus neoformans were input into microcomputer and subjected to automatic identification. The Cryptococcus neoformans was accurately identified within 15 min, and the consistency rate with results of routine culture was 98%.

Animals↗
PubMed1995

Concussion-induced retrograde amnesia in rats.

Three experiments were conducted to investigate the characteristics of retrograde amnesia (RA) induced by concussion in rats. In Experiment 1, rats receiving experimental concussion shortly after training in a single punishment trial exhibited severe forgetting on a retention test 48 h later. In the second experiment, rats receiving a concussion within 6 h after training showed severe RA, while those receiving concussion one day to five days after training exhibited progressively weaker amnesia. In Experiment 3, amnesic animals in one group received pretest noncontingent foot shock as a reminder treatment. This pretest cue significantly increased the cross-through latency, thus indicating a reduction in the memory deficit resulting from concussion. These results suggest that experimental concussion can be an effective method to induce retrograde memory loss in rats; that the RA caused by concussion is time-dependent; and that concussion-induced RA can be alleviated by a pretest cue indicating that the underlying mechanism of concussion-induced RA is more likely to be a retrieval deficit than a consolidation failure.

Amnesia, Retrograde↗