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Biomedical subjects

J Mao

Publications and source records attributed to J Mao.

At least 37 records · Page 2Linked to original sources
PubMed2001

Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.

To understand how the Wnt coreceptor LRP-5 is involved in transducing the canonical Wnt signals, we identified Axin as a protein that interacts with the intracellular domain of LRP-5. LRP-5, when expressed in fibroblast cells, showed no effect on the canonical Wnt signaling pathway by itself, but acted synergistically with Wnt. In contrast, LRP-5 mutants lacking the extracellular domain functioned as constitutively active forms that bind Axin and that induce LEF-1 activation by destabilizing Axin and stabilizing beta-catenin. Addition of Wnt caused the translocation of Axin to the membrane and enhanced the interaction between Axin and LRP-5. In addition, the LRP-5 sequences involved in interactions with Axin are required for LEF-1 activation. Thus, we conclude that the binding of Axin to LRP-5 is an important part of the Wnt signal transduction pathway.

3T3 Cells↗
PubMed2001

Fourier-transform Raman and infrared spectroscopic analysis of dipyrrinones and mesobilirubins.

The Fourier-transform Raman (FT-Raman), infrared (FT-IR), and UV-visible absorption spectra of four dipyrrinones and two mesobilirubins have been investigated in the solid state and in CH2Cl2 solutions. A detailed spectral analysis, assignment and discussion of these spectra are presented. The bands at 1735-1738, 1691-1707 and 1359-1377 cm(-1) which were assigned to the stretching vibrations of the C-O-C and C-O-H and symmetric deformation of C-H bonds, respectively, can act as a marker to distinguish the compounds of this class. The striking differences between the spectra of the compounds suggest that mesobilirubin XIIIalpha is tending to adopt as ridge-tile conformation, rather than linear conformation.

Bilirubin↗
PubMed2001

Identification and initial characterization of 5000 expressed sequenced tags (ESTs) each from adult human normal and osteoarthritic cartilage cDNA libraries.

OBJECTIVE: To prepare, sequence and analyse adult human cartilage cDNA libraries to study the gene expression pattern between normal and osteoarthritic cartilage. METHODS: Poly A(+)RNA from adult human normal and osteoarthritic articular cartilage was isolated and used to prepare cDNA libraries. Approximately 5000 ESTs from each library were sequenced and analysed using bioinformatic tools. The expression of select genes was confirmed by Northern blot and in situ hybridization analysis. RESULTS: Multiple gene families including several classical cartilage matrix protein encoding genes were identified. Approximately 28-40% of the genes sequenced from these libraries were novel, while half of the genes encoded known proteins and 4-6% of the genes encoded novel homologs of known proteins. Several known genes, whose expression has not been reported previously in cartilage, were also identified. We have confirmed the cartilage expression of three known (CTGF, CTGF-L and clusterin) and two novel homologs of known genes (PCPE-2 and Gal-Nac transferase) by Northern blot and in situ hybridization analysis. CONCLUSION: This is the first report of the preparation and sequencing of cDNA libraries from adult human normal and osteoarthritic articular cartilage. Further analysis of genes identified from these libraries may provide molecular targets for diagnosis and/or treatment of osteoarthritis (OA).

Adult↗
PubMed2001

Feed restriction and insulin treatment affect subsequent luteal function in the immediate postovulatory period in pigs: progesterone production in vitro and messenger ribonucleic acid expression for key steroidogenic enzymes.

Progesterone production and release in vitro, and mRNA expression for key steroidogenic enzymes, were studied in luteal tissue recovered in the immediate postovulatory period from cyclic gilts allocated to one of three treatments: moderate feed restriction during the first (RH) or second week of the estrous cycle, with (HR+I) or without (HR) concomitant injections of long-acting insulin. Time of feed restriction affected neither progesterone production or release, nor mRNA expression for several key steroidogenic enzymes. However, luteal tissue from RH but not from HR gilts responded to LH stimulation by increasing progesterone production and release (P: < 0.05). Insulin treatment increased progesterone production and release, restored luteal tissue responsiveness to LH, up-regulated steroidogenic enzyme mRNA expression, and down-regulated the tissue inhibitor of metalloproteinase-I mRNA expression in HR+I compared with HR gilts (P: < 0.05). In vitro progesterone production and gene expression were affected by time of tissue collection after ovulation in RH and HR gilts but not in HR+I gilts, and were correlated with temporal changes in oviductal and peripheral plasma progesterone concentrations. Inherent differences in luteal function therefore appear to mediate latent effects of nutrition and insulin treatment on circulating progesterone concentrations in the critical postovulatory period in gilts.

3-Hydroxysteroid Dehydrogenases↗
PubMed2001

Spinal cord neuroplasticity following repeated opioid exposure and its relation to pathological pain.

Convincing evidence has accumulated that indicates neuroplastic changes within the spinal cord in response to repeated exposure to opioids. Such neuroplastic changes occur at both cellular and intracellular levels. It has been generally acknowledged that the activation of N-methyl-D-aspartate (NMDA) receptors plays a pivotal role in the development of neuroplastic changes following repeated opioid exposure. Intracellular cascades can also be activated subsequent to NMDA receptor activation. In particular, protein kinase C has been shown to be a key intracellular element that contributes to the behavioral manifestation of neuroplastic changes. Moreover, interactions between NMDA and opioid receptors can lead to potentially irreversible degenerative neuronal changes in the spinal cord in association with the development of opioid tolerance. Interestingly, similar cellular and intracellular changes occur in the spinal cord following peripheral nerve injury. These findings indicate that interactions exist in the spinal cord neural structures between two seemingly unrelated conditions-chronic opioid exposure and a pathological pain state. These observations may help understand mechanisms of chemical intolerance and multiple chemical sensitivity as well as have significant clinical implications in pain management with opioid analgesics.

Analgesics↗
PubMed2001

Oligonucleotide-mediated, PCR-independent cloning by homologous recombination.

We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiae that allows precise DNA sequences to be transferred independent of restriction enzymes and PCR. In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides. The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert. The linkers can also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, preferred codons, etc.), thus allowing modification of the transferred DNA. Linkers can be designed such that DNA sequences can be transferred with just two reusable universal oligonucleotides and two gene-specific oligonucleotides. This cloning method, which is performed by co-transforming yeast with linear vector, substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based, oligonucleotide-mediated gap repair technique (YOGRT).

Cloning, Molecular↗
PubMed2001

Effects of different patterns of feed restriction and insulin treatment during the luteal phase on reproductive, metabolic, and endocrine parameters in cyclic gilts.

The objectives of the present study were 1) to study potential effects of previous nutritional treatment on developmental competence of early fertilized oocytes in vitro; 2) to study responses to insulin treatment during the period of feed restriction in the late luteal phase which has deleterious effects on subsequent fertility; and 3) to establish the metabolic and endocrine status of gilts during treatment and the subsequent periestrous period. Nineteen trios of littermate gilts were subjected to feed restriction during the first (RH) or second (HR) week of the estrous cycle. A second group of HR gilts received injections of long-acting insulin during their period of feed restriction (HR+I). Intensive sampling was performed in a subgroup of 23 animals on d 15 and 16 of the cycle for analyses of endocrine (gonadotropins and steroid hormones) and metabolic (insulin, IGF-I, leptin, total triiodothyronine [T3], and free T3) variables. Gilts were checked for estrus every 6 h, and time of ovulation was monitored by transcutaneous ultrasonography. Surgeries were performed 12 to 20 h after ovulation, and the early-fertilized oocytes recovered were cultured in vitro under standardized conditions. There was no treatment effect on the developmental competence of fertilized oocytes in vitro; however, ovulation rate was increased in HR+I gilts. No effect of treatment was observed on plasma leptin and IGF-I concentrations on d 15 and 16. However, HR+I gilts had higher (P < 0.05) postprandial insulin and lower (P < 0.05) postprandial total and free T3 on d 15. Plasma concentrations of LH, FSH, and progesterone on d 15 and 16 and plasma estradiol concentrations on d 16 were not affected by previous nutritional or insulin treatment. In the periestrous period, plasma concentrations of LH, FSH, and estradiol were higher (P < 0.05) in RH and HR+I, and the rise in plasma progesterone after the LH surge was lower (P < 0.05), than in HR gilts. No effect of treatment was observed on plasma concentrations of metabolic hormones, except on plasma leptin concentrations, which were higher (P < 0.05) at the time of the LH surge in RH gilts. These results suggest that feed restriction during the late luteal phase may have deleterious effects on ovarian function in the periestrous period, which may be counteracted by insulin.

Animals↗
PubMed2001

Histochemical study of transmyocardial laser channels in dogs.

OBJECTIVE: Laser-induced myocardial injury was investigated in a canine transmyocardial laser revascularization model. METHODS: A total of 23 channels were created in the left ventricles of 5 animals with a CO2 laser. Four animals were sacrificed immediately and one at week 2. Lactate dehydrogenase and succinate dehydrogenase in channel regions were examined with a Nitro-BT method staining. RESULTS: Immediately after the procedure, laser channels and surrounding injured myocardium could be stratified from the center to the border into 5 concentric zones, i.e., vaporized zone, carbonized zone, enzyme-disappeared zone, enzyme-decreased zone and enzyme-concentrated zone. Two weeks later, the phenomenon disappeared. CONCLUSIONS: Immediately after transmyocardial revascularization, there is a ring of enzyme-concentrated zone at the outermost region of injured myocardium. Two weeks later, enzyme activities of reversibly injured myocytes appear to have recovered.

Animals↗
PubMed2001

Genetic analysis of wild-type hepatitis A virus strains.

OBJECTIVE: To clarify the distribution of hepatitis A virus (HAV) genotype in geographical regions of China. METHODS: Seventeen representative HAV strains were isolated from the stool or serum of hepatitis A patients in different geographical regions. Viral RNA was recovered from stool or serum by proteinase K digestion and phenol-chloroform extraction, followed by ethanol precipitation prior to reverse transcription and polymerase chain reaction (RT-PCR) amplification. The nucleotide sequences of VP1/2A junction region were tested by using a direct sequencing technique. RESULTS: A pairwise comparison of sequences within 168 bases at the VP1/2A junction revealed that all the sequences clustered within genotype I. About 53% of strains clustered in genotype I B, with less than 6% variability; while the others clustered in genotype I A, with less than 5.3% variability. Sequence homology between genotype I A and I B varied from 88.7% to 92.3%. CONCLUSION: Epidemic or sporadic HAV strains in China may belong to HAV genotype I A or I B. Epidemiologically related strains may be identical or closely related in sequence.

Genotype↗
PubMed2001

[Influence of calcium carbonate on the microstructure of bovine serum albumin].

The influence of calcium carbonate on the microstructure of bovine serum albumin (BSA) in aqueous solution was studied by ultraviolet spectrum and ultraviolet second order derivative spectrum and Fourier transform infrared spectrum. The results showed the changes of the microenvironment of the aromatic amino acid residues and secondary structure of BSA. It was the changes that provided a favorable condition for biomineral formation.

Amino Acids↗
PubMed2000

PTRF (polymerase I and transcript-release factor) is tissue-specific and interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor which binds to the two mouse type-I collagen gene promoters.

We have used the yeast two-hybrid system to clone the protein that interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor that was cloned previously as the factor that binds to the two mouse proximal promoters of the type-I collagen genes. We utilized as bait the N-terminal domain of BFCOL1 that includes the zinc-finger DNA-binding domain. One cDNA contained a potential open reading frame for a polypeptide of 392 amino acids and was identical to PTRF (polymerase I and transcript-release factor), which is involved in transcription termination of the RNA polymerase I reaction. Northern-blot analysis revealed that the pattern of mRNA expression was similar to that of the type-I collagen gene. In addition, we detected the mRNA expression only in a fibroblast cell line and two bone cell lines, but not in other blood and neuronal cell lines. Recombinant protein was shown to enhance the binding of BFCOL1 to its binding site in the mouse proalpha2(I) collagen proximal promoter in vitro. The transient-transfection experiment showed that PTRF had a suppressive effect on the mouse proalpha2(I) collagen proximal promoter activity. We speculate that PTRF might play a role in the RNA polymerase II reaction as well as that of RNA polymerase I.

Animals↗
PubMed2000

Two distinctive antinociceptive systems in rats with pathological pain.

A common obstacle in clinical management of pathological pain is the poor response to opioid analgesics. We now report that delta9-tetrahydrocannabinol (delta9-THC)-induced antinociception remained effective in rats with pathological pain. The selective central cannabinoid receptor antagonist SR141716A, but not the generic opioid receptor antagonist naloxone, blocked the delta9-THC antinociception. Moreover, there is no cross-tolerance between the antinociceptive effects of morphine and delta9-THC in pathological pain states. The results indicate that delta9-THC antinociception is both effective and independent of opioid receptors in rats with pathological pain. Thus, the cannabinoid analgesic system may be superior to opioids in alleviating intractable pathological pain syndromes.

Analgesics, Non-Narcotic↗
PubMed2000

Isolation and identification of metabolites of porfiromycin formed in the presence of a rat liver preparation.

The isolation and identification of the major metabolites of porfiromycin formed in the presence of a rat liver preparation under aerobic conditions were performed with high-performance liquid chromatography and electrospray ionization mass spectrometry. Porfiromycin was extensively metabolized by the rat liver preparation in an aqueous 0.1 M potassium phosphate buffer (pH 7.4) containing an NADPH generating system at 37 degrees C. A total of eight metabolites was identified as mitosene analogs. Of these, three primary metabolites are 2-methylamino-7-aminomitosene, 1,2-cis and 1,2-trans-1-hydroxy-2-methylamino-7-aminomitosene, which are consistent with those previously observed in hypoxia using purified rat liver NADPH-cytochrome c reductase. Interestingly, 2-methylamino-7-aminomitosene is a reactive metabolite, which undergoes further activation at the C-10 position by the loss of carbamic acid and then links with the 7-amino group of the primary metabolites to yield two dimeric adducts. In addition, three phosphate adducts, 10-decarbamoyl-2-methylamino-7-aminomitosene-10-phosphate, 1,2-cis and 1,2-trans-2-methylamino-7-aminomitosene-1-phosphate, were also identified in the incubation system. The configurations of the diastereoisomeric metabolites were determined with (1)HNMR and phosphatase digestion. On the basis of the metabolite profile, we propose in vitro metabolic pathways for porfiromycin. The findings provide direct evidence for understanding the reactive nature and hepatic metabolism of the drug currently in phase III clinical trials.

Acetylcysteine↗
PubMed2000

Filopodial behavior is dependent on the phosphorylation state of neuronal growth cones.

Several lines of evidence suggest that phosphorylation events play an important role in transducing neurite outgrowth signals. Here we tested if such phosphorylation events altered filopodial dynamics on neuronal growth cones and thereby might affect pathfinding decisions. The general protein kinase inhibitor K252a caused an increase in the overall length of filopodia, thereby increasing the action radius of a growth cone. Application of specific kinase inhibitors demonstrated that myosin light chain kinase, Ca/calmodulin-dependent kinase II, and protein kinase A were likely not involved in this filopodial response. Inhibition of protein kinase C (PKC) with calphostin C or cerebroside, however, induced filopodial elongation similar to that seen with K252a. Activation of PKC with the phorbol ester PMA produced the opposite effect, namely filopodial shortening. Consistent with this finding, the protein phosphatase activator C(2)-ceramide resulted in a significant increase in filopodial length, whereas application of the protein phosphatase inhibitor okadaic acid caused the opposite effect, filopodial shortening. Lastly, the tyrosine kinase inhibitor genistein also caused filopodial elongation, and this effect could be negated by the tyrosine phosphatase inhibitor sodium ortho-vanadate. Using the calcium indicator fura-2, we further showed that these drugs did not cause a measurable change in the free intracellular calcium concentration ([Ca(2+)](i)) in growth cones. Taken together, these results suggest that the action radius of a growth cone and its resulting pathfinding abilities could be rapidly altered by contact with extracellular cues, leading to changes in the activity of protein kinases and phosphatases.

Animals↗
PubMed2000

NMDA-receptor antagonists and opioid receptor interactions as related to analgesia and tolerance.

A model proposing that N-methyl-D-aspartate (NMDA) receptor and opioid receptor mechanisms overlap and interact within the same dorsal horn nociceptive neurons makes several predictions. First, hyperalgesia should be associated with opioid tolerance. Second, both hyperalgesia and tolerance to opioid-analgesia should be blocked by an NMDA-receptor antagonist. Results from our laboratory and others support these predictions and point to several clinical implications. One is that, in addition to preventing tolerance and dependence, combining NMDA-receptor antagonists with both opioid and nonopioid analgesics may increase their analgesic potency. Preclinical animal studies demonstrate these advantages and underscore the practicality of the combined administration of nontoxic NMDA-receptor antagonists with various types of analgesic drugs.

Analgesia↗
PubMed2000

Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays.

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

Cell Line↗
PubMed2000

Structural basis of the recognition of the dishevelled DEP domain in the Wnt signaling pathway.

The DEP domain of Dishevelled (Dvl) proteins transduces signals to effector proteins downstream of Dvl in the Wnt pathway. Here we report that DEP-containing mutants inhibit Wnt-induced, but not Dvl-induced, activation of the transcription factor Lef-1. This inhibitory effect is weakened by a K434M mutation. Nuclear magnetic resonance spectroscopy revealed that the DEP domain of mouse Dvl1 comprises a three-helix bundle, a beta-hairpin 'arm' and two short beta-strands at the C-terminal region. Lys 434 is located at the tip of the beta-hairpin 'arm'. Based on our findings, we conclude that DEP interacts with regulators upstream of Dvl via a strong electric dipole on the molecule's surface created by Lys 434, Asp 445 and Asp 448; the electric dipole and the putative membrane binding site are at two different locations.

3T3 Cells↗