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M Yu

Publications and source records attributed to M Yu.

At least 253 records · Page 14Linked to original sources
PubMed1996

Ovine 11 beta-hydroxysteroid dehydrogenase type 2 gene predicts a protein distinct from that deduced by the cloned kidney cDNA at the C-terminus.

The gene encoding ovine 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) was cloned and characterized. This gene consists of five exons and is greater than 4 kb in length. It contains an open reading frame of 1215 bp, which encodes a protein of 404 amino acids with a predicted MW of 44 kDa. The deduced ovine 11 beta-HSD2 protein displays over 78% sequence identity to those of the human, rabbit, rat, and mouse. However, this differs from the published sequence of ovine kidney 11 beta-HSD2 cDNA which predicts a protein of 427 amino acids. Sequence alignment indicated that this discrepancy is attributed to two single nucleotide omissions in the published cDNA sequence which resulted in a shift in the open reading frame at the codon for residue 358. Therefore, the present results have provided conclusive evidence that the primary structure of 11 beta-HSD2 protein is well conserved between the sheep and the other four mammals. Moreover, Northern blot analysis of total RNA samples from 15 peripheral tissues and seven brain regions of the mature fetal sheep revealed that the expression of 11 beta-HSD2 gene is highly tissue-specific in that it is only expressed in the kidney and adrenal gland, and at a much lower abundance in the testis, colon and placenta. The cloning of the sheep 11 beta-HSD2 gene should facilitate future studies on the regulation of 11 beta-HSD2 gene expression during fetal development in a mammalian model.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

Cloning and transcription factor-binding sites of the human c-rel proto-oncogene promoter.

We report here the cloning, sequencing, functional analysis and DNase I footprinting of the human c-rel promoter region. The results revealed an 824-bp BsaAI-StuI minimal promoter region with a large number of NF-kappa B, Ap2 and Sp1-binding sites, some of them variants of known consensus sequences. This is the first promoter in the Rel/NF-kappa B/I kappa B family to be subjected to a detailed footprinting analysis for the binding of transcription activator proteins. Our finding of 14 Ap2-binding sites may indicate why the human c-rel promoter, unlike the chicken c-rel promoter, has a strong function and is highly responsive to phorbol esters. The presence of five NF-kappa B and six Sp1-binding sites in turn adds to growing evidence that, in mammals, the promoter of the Rel/NF-kappa B/I kappa B family may utilize multiple NF-kappa B- and Sp1-binding sites for their interactive regulation. Furthermore, there are putative binding sites for the PU.1 and Oct 1/2 transcription activator proteins, also present in the mouse c-rel promoter, which may help explain the preferential transcription of the c-rel gene in B- and T-lymphoid cells.

Amino Acid Sequence↗
PubMed1996

A predictable sequential determinant spreading cascade invariably accompanies progression of experimental autoimmune encephalomyelitis: a basis for peptide-specific therapy after onset of clinical disease.

The development of autoimmune disease is accompanied by the acquired recognition of new self-determinants, a process commonly referred to as determinant spreading. In this study, we addressed the question of whether determinant spreading is pathogenic for progression of chronic-relapsing experimental autoimmune encephalomyelitis (EAE), a disease with many similarities to multiple sclerosis (MS). Our approach involved a systematic epitope mapping of responses to myelin proteolipid protein (PLP) as well as assaying responses to known encephalitogenic determinants of myelin basic protein (MBP 87-89) and myelin oligodendrocyte glycoprotein (MOG 92-106) at various times after induction of EAE in (SWR X SJL)F1 mice immunized with PLP 139-151. We found that the order in which new determinants are recognized during the course of disease follows a predictable sequential pattern. At monthly intervals after immunization with p139-151, responses to PLP 249-273, MBP 87-99, and PLP 137-198 were sequentially accumulated in al mice examined. Three lines of evidence showed that determinant spreading is pathogenic for disease progression: (a) spreading determinants mediate passive transfer of acute EAE in naive (SWR X SJL)F1 recipients; (b) an invariant relationship exists between the development of relapse/progression and the spreading of recognition to new immunodominant encephalitogenic determinants; and (c) after EAE onset, the induction of peptide-specific tolerance to spreading but not to nonspreading encephalitogenic determinants prevents subsequent progression of EAE. Thus, the predictability of acquired self-determinant recognition provides a basis for sequential determinant-specific therapeutic intervention after onset of the autoimmune disease process.

Amino Acid Sequence↗
PubMed1996

BTag: a novel six-residue epitope tag for surveillance and purification of recombinant proteins.

Epitope tagging (Eta) is becoming an increasingly useful technique in molecular biology and biotechnology for the detection, characterisation and purification of recombinant proteins (re-proteins). Here we describe a novel Eta system composed of two different monoclonal antibodies (mAb; D11 and F10) and a 6-amino-acid Eta (Gln-Tyr-Pro-Ala-Leu-Thr or QYPALT). This Eta was derived from a highly conserved region of the major core protein, VP7, of bluetongue (BT) viruses, hence the name BTag. BTag is unique among current tagging systems in its lack of charge and the fact the tag sequence can be placed and detected in any region of a re-protein. Other useful features of BTag include its small size and its recognition by two different mAb. Using the BTag system, more than 30 re-proteins have been produced from a variety of host organisms, and the antigenicity of the tag sequence was maintained in all of the proteins tested to date. Our result demonstrated that BTag could be superior to other existing Eta systems for certain applications.

Amino Acid Sequence↗
PubMed1996

Evaluation of hydroxyl radical-scavenging property of garlic.

Garlic has been reported to provide protection against hypercholesterolemic atherosclerosis and ischemia-reperfusion-induced arrhythmias and infarction. Oxygen free radicals (OFRs) have been implicated as causative factors in these diseases and antioxidants have been shown to be effective against these conditions. The effectiveness of garlic in these disease states could be due to its ability to scavenge OFRs. However, the OFR-scavenging activity of garlic is not known. Also it is not known if its activity is affected by cooking. We therefore investigated, using high pressure liquid chromatography, the ability of garlic extract (heated or unheated) to scavenge exogenously generated hydroxyl radical (.OH). .OH was generated by photolysis of H2O2 (1.2-10 mumoles/ml) with ultraviolet (UV) light and was trapped with salicylic acid (500 nmoles/ml). H2O2 produced .OH in a concentration-dependent manner as estimated by .OH adduct products 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. Garlic extract (5-100 microliters/ml) produced an inhibition (30-100%) of 2,3-DHBA and 2,5-DHBA generated by photolysis of H2O2 (5.00 pmoles/ml) in a concentration-dependent manner. Its activity is reduced by 10% approximately when heated to 100 degrees C for 20, 40 or 60 min. The extent of reduction in activity was similar for the three heating periods. Garlic extract prevented the .OH-induced formation of malondialdehyde in the rabbit liver homogenate in a concentration-dependent manner. It alone did not affect the MDA levels in the absence of .OH. These results indicate that garlic extract is a powerful scavenger of .OH and that heating reduces its activity slightly.

Animals↗
PubMed1996

Evolution of stomach lysozyme: the pig lysozyme gene.

The acquisition of an efficient stomach lysozyme is associated with the success of the ruminants. Advanced ruminants, such as cow, sheep, and deer, have approximately 10 lysozyme genes, some of which are expressed and function in the stomach and some which are expressed and function in nonstomach tissues (e.g., trachea or kidney). The pig possesses a single conventional lysozyme c gene that is expressed in both stomach and nonstomach tissues, and in this respect is similar to what was thought to exist in the early artiodactyl, before the acquisition of the ruminant lifestyle. To better understand the genetic events that occurred early in the origin and evolution of stomach lysozyme, we have isolated and characterized the pig lysozyme gene. The pig lysozyme gene is similar in size to that of other mammalian species, and both stomach and nonstomach expression utilize the same promoter. All the duplications of the ruminant lysozyme gene occurred after the divergence of the pig lineage from the lineage leading to the advanced ruminants. Comparison of the nucleotide sequence of the coding region of mature stomach lysozymes from advanced ruminants and pig revealed no change in the rate of synonymous substitutions. Comparison of the numbers of nonsynonymous and synonymous substitutions provides evidence for positive selection along the early ruminant lineage. These results indicate that changes in selective pressure, and not mutation rate, account for the changes in rates of stomach lysozyme evolution.

Alleles↗
PubMed1996

Functional expression of the dihydrofolate reductase domain of Leishmania major dihydrofolate reductase-thymidylate synthase bifunctional protein.

The dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase from Leishmania major has been subcloned and expressed as a soluble protein in Escherichia coli strain PA414 harboring plasmid pLMDHFR. Homogeneous L. major DHFR was obtained by chromatography on methotrexate-Sepharose followed by DE52. The purified enzyme migrated as a single 25-kDa protein on SDS-PAGE. The native molecular weight was determined to be 26 kDa, indicating that the isolated domain is a monomer. N-terminal sequence analysis revealed that serine, the second amino acid in the coding sequence, was the N-terminal amino acid of the protein. The enzyme showed a pH optimum similar to that of the bifunctional protein. For purified DHFR, the Km values were <1.0 microM for H2folate and <1.0 microM for NADPH. The kcat of the most active DHFR preparation was 5 s-1. The Km and kcat values were similar to those of the bifunctional enzyme.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of vertebrate lysozyme genes.

Lysozyme genes have been model genes in molecular genetics. The chicken lysozyme c gene was among the first genes to be isolated and characterized, but since then, many other members of the lysozyme gene family have been isolated and characterized. Of all the members of the gene family, the conventional lysozyme c gene has been the most extensively studied at the molecular level. General properties of members of the lysozyme gene family are that they are relatively small genes of less than 10 kilobases in length, and are made up of four exons and three introns. There has been a long history of gene duplication events within the lysozyme gene family, and in several cases, eg., stomach lysozymes, this has led to the evolution of novel biological functions. Initially the structure of the lysozyme c gene appeared to support the exon theory of genes, but the recent characterization of additional lysozymes shows that the predictions of this theory are not supported. Lysozyme genes continue to yield new insights into the molecular processes moulding the vertebrate genome.

Animals↗
PubMed1996

Strong sequence conservation of African swine fever virus p72 protein provides the molecular basis for its antigenic stability.

The major capsid protein p72 of African swine fever virus (ASFV) has long been considered an important immunodominant antigen for serologic diagnosis. Here we describe the cloning and sequence analysis of two p72-coding genes from ASFV strains Uganda (UGA) and Dominican Republic-2 (DR2). Sequence comparison of these genes, together with those from two other ASFV strains (BA71V and E70), demonstrated that the p72 proteins are highly conserved (97.8% to 100% amino acid sequence identity) in strains isolated from different parts of the world. These results support previous observations indicating that p72 is antigenically stable, and provide a useful molecular basis for further development of ASFV serologic tests using this important antigenic molecule.

African Swine Fever↗
PubMed1996

Amplifications of oncogene erbB-2 and chromosome 20q in breast cancer determined by differentially competitive polymerase chain reaction.

A new method of measuring gene copy number in small samples of DNA was used to measure amplification of the erbB-2 gene and of chromosome 20q in breast cancer. This method, termed 'differentially competitive polymerase chain reaction' (DC-PCR) combines the advantages of two other techniques for measuring amplification by PCR, namely differential PCR and competitive PCR. The DC-PCR methodology was evaluated for sensitivity and specificity by comparing amplification of erbB-2 measured by DC-PCR with that obtained by fluorescence in situ hybridization (FISH) for 42 cases or Southern blotting and/or slot blot analysis for 34 cases. There was over 90 percent concordance with both FISH and Southern blotting and/or slot blot analysis. DC-PCR was used to further characterize the newly described amplicon at chromosome 20q. By analyzing DNA from 10 breast cancer cell lines at 7 different loci, we identified a potential common region of amplification of approximately 5 centimorgans at chromosome 20q13 bordered by loci D20S52 and RMC20C100-S1. One hundred and seventeen cases of primary breast cancer were evaluated for amplification at these two loci. Amplification at one or more loci, defined as > 1.5 fold higher copy number than that of normal DNA, was found in 25 cases (21%). Sixteen cases were amplified at only one of the two probes (12 cases for RMC20C001-S1 and 4 cases for D20S52), suggesting that the target gene lies between the two markers or that there are two independent target genes within a small chromosome region.

Blotting, Southern↗
PubMed1996

Differences in PTH (1-84) release in response to ambient calcium concentrations of parathyroid adenoma fragments and dispersed parathyroid adenoma cells in culture.

In a previous study we observed that during perfusion of normal human parathyroid tissue, the release of PTH (1-84) was modulated by ambient extracellular calcium (Ca++) and lithium (Li+) concentrations in the media and preliminary studies indicated that this stimulus-response coupling was absent in human parathyroid adenoma fragments. The present study compares the responsiveness of parathyroid adenoma fragments and isolated parathyroid adenoma cells from the same adenoma and their response to Ca++ changes and Li+ presence in culture media. The data indicate that parathyroid adenoma tissue fragments fail to respond to ambient changes in Ca++ and Li+. In contrast, dispersed parathyroid cells preparations responded with a significant increase of PTH (1-84) release (50%) under the influence of low ambient calcium concentrations. Six of the dispersed cell preparations also responded with a 45% decrease in PTH release under the influence of a high Ca concentration in the medium. Isolated parathyroid cells obtained from the same adenoma's did not respond to the presence of Li++ in the medium. These data suggest tat human parathyroid adenoma tissue functions autonomously and is not sensitive to calcium regulation in the tissue configuration as opposed to the isolated cell suspensions. The nature of this difference remains elusive.

Adenoma↗
PubMed1996

Interferon-beta inhibits progression of relapsing-remitting experimental autoimmune encephalomyelitis.

The results of two phase III clinical trials have recently shown that interferon-beta (IFNbeta) is effective in the treatment of relapsing-remitting multiple sclerosis (RRMS). Treatment with IFNbeta results in a significant decrease in the rate of clinical relapse and a marked delay in progression to disability compared to placebo-treated control patients. In the present study, we demonstrate similar therapeutic effects after treating (SWR X SJL)F1 mice with IFNbeta at the onset of clinical signs of experimental autoimmune encephalomyelitis (EAE), a disease animal model widely used in MS studies. EAE was actively induced by immunization of (SWR X SJL)F1 mice with the immunodominant encephalitogenic peptide 139-151 of myelin proteolipid protein (PLP). In blinded testing, mice treated with IFNbeta at EAE onset showed a delay in progression to clinical disability as determined by marked improvement with time in mean clinical score, significant delay in onset of relapse, and significant decrease in exacerbation frequency compared to placebo-treated control mice. The therapeutic effect of IFNbeta was accompanied by a significant inhibition of delayed-type hypersensitivity (DTH) but not proliferation in response to the priming PLP 139-151. In addition, IFNbeta treatment resulted in an overall decrease in severity of both inflammation and demyelination in the central nervous system. These results mimic in an autoimmune animal model the effectiveness of IFNbeta treatment observed in MS. Moreover, our study suggests that anti-viral properties of IFNbeta are not essential for producing therapeutic effects in autoimmune demyelinating disease, and that the efficacy of IFNbeta in the treatment of MS may be due to inhibition of autoreactivity.

Amino Acid Sequence↗
PubMed1996

Application of linker-ligation-PCR for construction of phage display epitope libraries.

An efficient method for construction of random epitope libraries using filamentous phage is described. Random DNA fragments generated by DNase I digestion were blunt ended by T4 DNA polymerase and ligated with a 12-mer linker, followed by PCR amplification using the same oligonucleotide linker as primers. The results showed that only the ligated product containing linker sequences on both ends was specifically amplified. When the linker ligated-PCR (LL-PCR) product was used for the construction of phage display epitope libraries, the total number of independent clones in the libraries was increased by 100 to 1000 fold in comparison to that obtained for libraries constructed using other methods. In addition, the LL-PCR strategy also increased the probability of isolating recombined DNA fragments which are derived by random in-frame ligation of two or more discontinuous peptide-coding sequences before being inserted into the display vector. Such randomly recombined DNA fragments might be useful in defining conformational epitopes.

African Swine Fever Virus↗
PubMed1996

Hypertonic saline improves brain resuscitation in a pediatric model of head injury and hemorrhagic shock.

INTRODUCTION: Brain injury accompanied by hypovolemic shock is a frequent cause of death in multiply injured children. Hypertonic saline (HTS) has been shown to return hemodynamics to normal in adult models, without increasing intracranial pressure (ICP) as seen with crystalloids. To assess fluid resuscitation, the authors evaluated HTS versus lactated Ringer's solution (LR) with respect to hemodynamics and cerebrovascular hemoglobin oxygen saturation (Sco2) in anesthetized, head-injured, 1-month-old piglets. METHODS: Group 1 (n = 6) was studied for 3.5 hours after a cryogenic brain injury and no shock. Groups 2 and 3 had cryogenic brain injury followed by hemorrhagic shock, in which mean arterial pressure (MAP) was reduced to 40 to 50 mm Hg and maintained for 30 minutes. Group 2 (n = 5) was then resuscitated with 1 mL of 7.5% HTS per 1 mL of blood loss. Group 3 (n = 6) was resuscitated with 3 mL of LR per 1 mL of blood loss. Sco2 was determined by near-infrared spectroscopy in the injured region of the brain. All data were analyzed using analysis of variance with repeated measures. RESULTS: MAP, ICP, temperature, serum sodium, and cardiac output (CO) were similar in all groups during baseline and between groups 2 and 3 during shock. After resuscitation, MAP, CO, and core temperature were similar in all three groups, and serum sodium was increased in the HTS group (by 29%). Sco2 increased transiently after cryogenic injury in all groups, then gradually decreased to below baseline. After shock, Sco2 decreased precipitously in group 2 and 3. After resuscitation, Sco2 was different in the two resuscitation groups, increasing in the HTS group, above baseline values, but remaining below baseline values in the LR group (P < .002). ICP was lowered by HTS resuscitation and increased by LR resuscitation (P < .002) CONCLUSION: In our model of head injury and shock, resuscitation with either HTS or LR restored MAP and CO to control levels. However, during shock, the injured brain was severely deoxygenated, and administration of HTS restored cerebral oxygenation whereas LR did not, reflecting improved cerebral resuscitation by HTS without elevating ICP. The data suggest that HTS is a better resuscitation fluid than LR in head-injured children with hemorrhagic shock.

Animals↗
PubMed1996

Pattern of 11 beta-hydroxysteroid dehydrogenase type 1 messenger ribonucleic acid expression in the ovine uterus during the estrous cycle and pregnancy.

The present study was designed to examine the pattern and cellular localization of 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) gene expression in the ovine uterus during pregnancy and at 3 mo postpartum. High levels of 11 beta-HSD1 mRNA were detected in the endometrium from Days 60 to 143 (term = 145 days), and the levels did not change significantly during that time. The level of 11 beta-HSD1 mRNA in the endometrium was always much higher than that in the myometrium, in which the mRNA was not readily detectable throughout pregnancy; at 3 mo postpartum, 11 beta-HSD1 mRNA became undetectable in both endometrium and myometrium. Within the endometrium, intense immunoreactive 11 beta-HSD1 was localized exclusively to the luminal epithelium, and the intensity of 11 beta-HSD1 immunostaining closely followed the level of 11 beta-HSD1 mRNA. To determine whether the level of endometrial 11 beta-HSD1 mRNA was related to the status of ovarian function, tissues from non-pregnant animals at different stages of their reproductive cycle were also examined. It was found that 11 beta-HSD1 mRNA was undetectable in the endometrium of cycling animals up to Day 9 of the estrous cycle but was detectable thereafter. Taken together, these results demonstrate that within the ovine uterus the endometrium is always the dominant site of 11 beta-HSD1 gene expression in relation to the myometrium. Furthermore, the expression of 11 beta-HSD1 mRNA in the endometrium is closely related to the status of the reproductive cycle. The mRNA for 11 beta-HSD1 is highly expressed only during pregnancy and in non-pregnant animals during the late luteal phase. Since circulating levels of progesterone are elevated during both of these periods, the present findings suggest a progesterone effect on uterine 11 beta-HSD1 gene expression.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

Validation of a flow-differential technique for the recording of splenic blood-volume changes to vasoactive agents.

Splenic blood-volume changes to norepinephrine were determined in the anesthetized cat by recording the integral of the difference between splenic arterial flow and splenic venous flow (flow-differential technique) with ultrasonic transit-time flowmeters, and the changes recorded with this technique were compared with those recorded with an independent technique, the gravimetric method. Norepinephrine (0.003-0.3 microgram/min/kg of body weight) induced dose-related decreases in splenic volume, and the values recorded by the flow-differential technique were not significantly different from those recorded with the gravimetric technique. The values recorded by the two methods were highly correlated (r = 0.86). The slope of the line plotting the relation between the two methods was close to unity (0.97 +/- 0.12), and the intercept of the line was close to zero (-0.46 +/- 1.6 ml). The results suggest that the recording of the flow differential between splenic arterial flow and splenic venous flow with ultrasonic transit time flowmeters is a valid method for the recording of splenic blood-volume changes to vasoactive agents.

Animals↗
PubMed1996

The relationship of oxygen consumption measured by indirect calorimetry to oxygen delivery in critically ill patients.

OBJECTIVE: The existence of oxygen supply dependency, defined as oxygen consumption (VO2) limited by oxygen delivery (DO2), is still questioned. This study examined the relationship between VO2 and DO2 in two groups of critically ill surgical patients 50 years and older in the first 24 hours of resuscitation after pulmonary artery catheter insertion. Group 1 patients had systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, and adult respiratory distress syndrome (ARDS). Group 2 patients had hemorrhagic shock. METHODOLOGY: Study methodology included (1) augmenting DO2 with fluids, blood, and vasopressors, (2) measuring VO2 by indirect calorimetry to avoid the problem of mathematical coupling with DO2 calculation, and (3) analyzing data during steady states of temperature, sedation, paralyzing agents, and vasopressors. RESULTS: Six to 18 measurements collected on all study patients during a period within the first 24 hours were analyzed using a linear regression analysis. Statistical significance was set at p < or = 0.05. Seven of nine patients in group 1 demonstrated positive, statistically significant relationships between VO2 and DO2. Of six patients in group 2, one patient demonstrated a positive, significant relationship of VO2 and DO2, three demonstrated inverse relationships, and two patients did not show a DO2/VO2 relationship. Supply dependency did not exist in all patients but was present in seven out of nine patients with systemic inflammatory response syndrome, sepsis, severe sepsis, septic shock, and adult respiratory distress syndrome in the first 24 hours of treatment.

Aged↗