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Biomedical subjects

H Guo

Publications and source records attributed to H Guo.

At least 19 recordsLinked to original sources
PubMed2000

Group II introns designed to insert into therapeutically relevant DNA target sites in human cells.

Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a >14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron Ll.LtrB and to select introns that insert into desired target sites. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy.

Base Pairing↗
PubMed2000

Specificity and efficiency of Cre-mediated recombination in Emx1-Cre knock-in mice.

Emx1 is a mouse homologue of the Drosophila homeobox gene empty spiracles and its expression is restricted to the neurons in the developing and adult cerebral cortex and hippocampus. We reported previously the creation of a line of transgenic mice in which the cre gene was placed directly downstream of the putative Emx1 promoter using ES cell technology. We showed that Cre protein was present in the cerebral cortex of the transgenic mice and was able to mediate loxP-specific recombination in vitro. In the present study, the specificity and efficiency of the cre-mediated recombination were determined using three independent lines of reporter mice and a combination of histochemical staining, neuronal culture, and Southern detection of the genomic DNA. Our results showed that the recombination was highly efficient in all three lines of reporter mice tested and confirmed that the deletion was restricted to the neurons in the cerebral cortex and hippocampus. Furthermore, we have determined that the recombination efficiency in the cerebral cortex was 91%. Our results suggest that Emx1 is not expressed in every neuron in the developing and adult cerebral cortex. This line of cre mice should contribute to the studies of cortical development and plasticity.

Animals↗
PubMed2000

Emx1-specific expression of foreign genes using "knock-in" approach.

Emx1 is a mouse homologue of the Drosophila homeobox gene empty spiracles. Its expression is limited to the neurons in developing and adult cerebral cortex and hippocampus. Because of the highly restricted expression pattern of the Emx1 gene, it would be quite desirable to characterize the promoter of the Emx1 for directing foreign gene expression in the transgenic mouse. We report here that we have achieved the Emx1-specific expression in transgenic mice by inserting the lacZ reporter and cre genes directly into the exon 1 of the Emx1 gene using embryonic stem (ES) cell technology. The distribution of the beta-galactosidase activity in the transgenic mice was consistent with the published results obtained using in situ hybridization and immunohistochemistry. Furthermore, we have demonstrated that Cre protein was present in the cerebral cortex of the transgenic mice and was able to mediate loxP-specific recombination in vitro. The creation of this line of cre transgenic mice, and the demonstration that the insertion site located in the exon 1 of the Emx1 gene could render foreign genes a specific expression pattern restricted to the developing and adult cerebral cortex and hippocampus, should be conducive to further studies of the effect of a gene mutation or overexpression upon the development and plasticity of cerebral cortex and hippocampus.

Animals↗
PubMed2000

The major myosin-binding site of caldesmon resides near its N-terminal extreme.

The primary myosin-binding site of caldesmon was thought to be in the N-terminal region of the molecule, but the exact nature of the caldesmon-myosin interaction has not been well characterized. A caldesmon fragment that encompasses residues 1-240 (N240) was found to bind full-length smooth muscle myosin on the basis of co-sedimentation experiments. The interaction between myosin and N240 was not affected by phosphorylation of myosin, but it was weakened by the presence of Ca(2+)/calmodulin. To locate the myosin-binding site, we have designed several synthetic peptides based on the N-terminal caldesmon sequence. We found that a peptide stretch corresponding to the first 27 residues (Met-1 to Tyr-27), but not that of the first 22 residues (Met-1 to Ala-22), exhibited a moderate affinity toward myosin. We also found that a peptide containing the segment from Ile/Leu-25 to Lys-53 bound both myosin and heavy meromyosin more strongly and was capable of displacing caldesmon from myosin. Our results demonstrate that the sequence near the N-terminal extreme of caldesmon harbors a major myosin-binding site of caldesmon, in which both the nonpolar residues and clusters of positively and negatively charged residues confer the specificity and affinity of the caldesmon-myosin interaction.

Amino Acid Sequence↗
PubMed2000

Cre-mediated cerebellum- and hippocampus-restricted gene mutation in mouse brain.

Using the phage P1-derived Cre/loxP recombination system, we have created a line of cre-transgenic mice in which the Cre-mediated gene deletion is restricted to granule cells of cerebellum and dentate gyrus of hippocampus. Low levels of deletion were also present in pyramidal cells of hippocampal CA1 and CA3 fields. The Cre/loxP recombination occurred prenatally. The recombination efficiencies in the granular layer of the cerebellum, the granular layer of the dentate gyrus, and the CA1 and CA3 pyramidal cells of the hippocampus were 34.0%, 23.1%, 3.0%, and 9.8%, respectively. This line of cre-transgenic mice should be conducive to studies of the effect of a gene mutation upon brain development and plasticity.

Animals↗
PubMed2000

EMMPRIN (CD147), an inducer of matrix metalloproteinase synthesis, also binds interstitial collagenase to the tumor cell surface.

Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as basigin or CD147, is a glycoprotein that is enriched on the surface of tumor cells and stimulates production of several matrix metalloproteinases by adjacent stromal cells. In this study, we have found that EMMPRIN not only stimulates the production of interstitial collagenase (MMP-1) but also forms a complex with MMP-1 at the tumor cell surface. Complex formation was demonstrated by phage display, affinity chromatography, and immunocytochemistry. Presentation of MMP-1 complexed to EMMPRIN at the tumor cell surface may be important in modifying the tumor cell pericellular matrix to promote invasion.

Antigens, CD↗
PubMed2000

Regulation of vascular smooth muscle tone by N-terminal region of caldesmon. Possible role of tethering actin to myosin.

To assess the functional significance of tethering actin to myosin by caldesmon in the regulation of smooth muscle contraction, we investigated the effects of synthetic peptides, containing the myosin-binding sequences in the N-terminal region of caldesmon, on force directly recorded from single permeabilized smooth muscle cells of ferret portal vein. Two peptides were used, IK29C and MY27C, containing residues from Ile(25) to Lys(53) and from Met(1) to Tyr(27) of the human and chicken caldesmon sequence, respectively, plus an added cysteine at the C terminus. In cells clamped at pCa 6. 7, both peptides increased basal tone. Pretreatment of cells at pCa 6.7 with IK29C or MY27C decreased the amplitude of subsequent phenylephrine-induced contractions but not microcystin-racemic mixture-induced contractions. In all cases the effects of the peptides were concentration-dependent, and IK29C was more potent than MY27C, in agreement with their relative affinity toward myosin. The peptides were ineffective after the phenylephrine contraction was established. MY27C did not further increase the magnitude of contraction caused by a maximally effective concentration of IK29C, consistent with the two peptides having the same mechanism of action. Neither polylysine nor two control peptides containing scrambled sequences of IK29C, which do not bind myosin, had any effect on basal or phenylephrine-induced force. Our results suggest that IK29C and MY27C induce contraction by competing with the myosin-binding domain of endogenous caldesmon. Digital imaging of fluoroisothiocyanate-tagged IK29C confirmed the association of the peptide with intracellular filamentous structures. The results are consistent with a model whereby tethering of actin to myosin by caldesmon may play a role in regulating vascular tone by positioning the C-terminal domain of caldesmon so that it is capable of blocking the actomyosin interaction.

Amino Acid Sequence↗
PubMed2000

Neuronal fractalkine expression in HIV-1 encephalitis: roles for macrophage recruitment and neuroprotection in the central nervous system.

HIV-1 infection of the brain results in chronic inflammation, contributing to the neuropathogenesis of HIV-1 associated neurologic disease. HIV-1-infected mononuclear phagocytes (MP) present in inflammatory infiltrates produce neurotoxins that mediate inflammation, dysfunction, and neuronal apoptosis. Neurologic disease is correlated with the relative number of MP in and around inflammatory infiltrates and not viral burden. It is unclear whether these cells also play a neuroprotective role. We show that the chemokine, fractalkine (FKN), is markedly up-regulated in neurons and neuropil in brain tissue from pediatric patients with HIV-1 encephalitis (HIVE) compared with those without HIVE, or that were HIV-1 seronegative. FKN receptors are expressed on both neurons and microglia in patients with HIVE. These receptors are localized to cytoplasmic structures which are characterized by a vesicular appearance in neurons which may be in cell-to-cell contact with MPs. FKN colocalizes with glutamate in these neurons. Similar findings are observed in brain tissue from an adult patient with HIVE. FKN is able to potently induce the migration of primary human monocytes across an endothelial cell/primary human fetal astrocyte trans-well bilayer, and is neuroprotective to cultured neurons when coadministered with either the HIV-1 neurotoxin platelet activating factor (PAF) or the regulatory HIV-1 gene product Tat. Thus focal inflammation in brain tissue with HIVE may up-regulate neuronal FKN levels, which in turn may be a neuroimmune modulator recruiting peripheral macrophages into the brain, and in a paracrine fashion protecting glutamatergic neurons.

Adult↗
PubMed2000

TGF-beta1 inhibits protracted-relapsing experimental autoimmune encephalomyelitis by activating dendritic cells.

Protracted-relapsing experimental autoimmune encephalomyelitis (PR-EAE) in DA rats is an animal model closely related to multiple sclerosis (MS). Previous studies showed that nasal administration of TGF-beta1 suppressed the development and relapse of PR-EAE clinically and pathologically. Here we demonstrate that this suppressive effect was associated with activation of dendritic cells (DC), showing elevated proliferative response and IFN-gamma and nitric oxide (NO) production by DC. DC derived from TGF-beta1-treated rats with PR-EAE also showed high expression of nitric oxide synthase 2 (NOS2) at both mRNA and protein levels. Apoptotic cells were increased in spleen sections of TGF-beta1-treated rats compared to control rats. In studying mechanisms of apoptosis in TGF-beta1-treated rats, in vitro experiments demonstrated that TGF-beta1-treated DC induced apoptosis of CD4(+)T cells by a NO pathway after co-culture with T cells. These results support the hypothesis that TGF-beta1-induced suppression of PR-EAE is associated with apoptosis of CD4(+)T cells induced by DC-derived NO.

Animals↗
PubMed2000

Radiofrequency application to the posteroseptal region alters retrograde accessory pathway activation.

INTRODUCTION: Radiofrequency applications to the posteroseptal region can ablate the atrioventricular accessory pathway residing in this area. In conjunction with the adjacent anatomic structures, however, ablative lesions which do not effectively ablate the accessory pathway could markedly alter retrograde atrial activation sequence and confound interpretation of further mapping of an accessory pathway. METHODS AND RESULTS: Electrophysiologic studies, endocardial activation mapping and radiofrequency catheter ablation were undertaken in three patients with recurrent supraventricular tachycardia. Patients were initially thought to have a single posteroseptal accessory pathway; earliest ventrioatrial activation during tachycardias and during ventricular pacing was at the coronary sinus ostium, but initial radiofrequency applications were unsuccessful to ablate the pathway. After initial radiofrequency applications to the posteroseptal region, the earliest retrograde atrial activation changed to the right atrial free wall in two patients. Additional radiofrequency application to the posteroseptal area was able to ablate the single posteroseptal accessory pathway in one patient. Radiofrequency application to the right atrial free wall was required to stop tachycardia initiation in other patient. The third patient was suspected of having a slow-slow atrioventricular nodal reentry tachycardia. Radiofrequency application to the posteroseptal area changed the earliest retrograde atrial activation to the distal coronary sinus recording site, mimicking an accessory pathway at the left atrial free wall. Radiofrequency application to the anteroseptum was able to ablate the concealed accessory pathway. CONCLUSION: Radiofrequency applications to the posteroseptal region can markedly alter retrograde atrial activation, thereby confounding further mapping of the accessory pathway.

Adolescent↗
PubMed2000

Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability?

Azurin has a beta-barrel fold comprising eight beta-strands and one alpha helix. A disulfide bond between residues 3 and 26 connects the N-termini of beta strands beta1 and beta3. Three mutant proteins lacking the disulfide bond were constructed, C3A/C26A, C3A/C26I and a putative salt bridge (SB) in the C3A/S25R/C26A/K27R mutant. All three mutants exhibit spectroscopic properties similar to the wild-type protein. Furthermore, the crystal structure of the C3A/C26A mutant was determined at 2.0 A resolution and, in comparison to the wild-type protein, the only differences are found in the immediate proximity of the mutation. The mutants lose the 628 nm charge-transfer band at a temperature 10-22 degrees C lower than the wild-type protein. The folding of the zinc loaded C3A/C26A mutant was studied by guanidine hydrochloride (GdnHCl) induced denaturation monitored both by fluorescence and CD spectroscopy. The midpoint in the folding equilibrium, at 1.3 M GdnHCl, was observed using both CD and fluorescence spectroscopy. The free energy of folding determined from CD is -24.9 kJ.mol-1, a destabilization of approximately 20 kJ.mol-1 compared to the wild-type Zn2+-protein carrying an intact disulfide bond, indicating that the disulfide bond is important for giving azurin its stable structure. The C3A/C26I mutant is more stable and the SB mutant is less stable than C3A/C26A, both in terms of folding energy and thermal denaturation. The folding intermediate of the wild-type Zn2+-azurin is not observed for the disulfide-deficient C3A/C26A mutant. The rate of unfolding for the C3A/C26A mutant is similar to that of the wild-type protein, suggesting that the site of the mutation is not involved in an early unfolding reaction.

Azurin↗
PubMed2000

Evaluation of total volatile organic compound emissions from adhesives based on chamber tests.

In 1997, Homeswest in western Australia and Murdoch University developed a project to construct low-allergen houses (LAHs) in a newly developed suburb. Before the construction of LAHs, all potential volatile organic compound (VOC) emission materials used in LAHs are required to be measured to ensure that they are low total VOC (TVOC) emission materials. This program was developed based on this purpose. In recent times, the number of complaints about indoor air pollution caused by VOCs has increased. A number of surveys of indoor VOCs have indicated that many indoor materials contribute to indoor air pollution. Although some studies have been conducted on the characteristics of VOC emissions from adhesives, most of them were focused on VOC emissions from floor adhesives. Few measurements of VOC emissions from adhesives used for wood, fabrics, and leather are available. Furthermore, most research on VOC emissions from adhesives has been done in countries with cool climates, where ventilation rates in the indoor environment are lower than those in Mediterranean climates, due to energy conservation. VOCs emitted from adhesives have not been sufficiently researched to prepare an emission inventory to predict indoor air quality and to determine both exposure levels for the Australian population and the most appropriate strategies to reduce exposure. An environmental test chamber with controlled temperature, relative humidity, and airflow rate was used to evaluate emissions of TVOCs from three adhesives used frequently in Australia. The quantity of TVOC emissions was measured by a gas chromatography/flame ionization detector. The primary VOCs emitted from each adhesive were detected by gas chromatography/mass spectrometry. The temporal change of TVOC concentrations emitted from each adhesive was tested. A double-exponential equation was then developed to evaluate the characteristics of TVOC emissions from these three adhesives. With this double-exponential model, the physical processes of TVOC emissions can be explained, and a variety of emission parameters can be calculated. These emission parameters could be used to estimate real indoor TVOC concentrations in Mediterranean climates.

Adhesives↗
PubMed1999

Neuronally modulated transcription of a glycine transporter in rat dorsal cochlear nucleus and nucleus of the medial trapezoid body.

Neurotransmitter transporters limit transmitter concentration at the postsynaptic membrane by removing neurotransmitters from the synaptic cleft. Not only do neurotransmitter transporters contribute to the regulation of synaptic transmission, but they themselves might be dynamically regulated by neuronal activity of the neurons in which they are expressed. In this experiment, we investigated the question of whether the transcription of two different glycine transporters, Glyt1 and Glyt2, is influenced by neuronal activity. These transporters are found in the dorsal cochlear nucleus (DCN) and medial nucleus of the trapezoid body. Glyt1 and Glyt2 mRNA were measured by using hybridization histochemistry and a semiquantitative reverse transcription polymerase chain reaction. Decreases in auditory primary afferent activity, caused by either unilateral labyrinthectomy or disruption of the middle ear ossicles, caused a reduction in Glyt2, but not Glyt1 mRNA in the ipsilateral DCN and in the contralateral medial nucleus of the trapezoid body. Acoustic stimulation at either 10 kHz or 40 kHz was used to provide controlled increases in primary afferent activity, evoking localized increases in Glyt2 mRNA in clusters of neurons in the DCN. The location of these clusters corresponded to the regions of the auditory tonotopic map devoted to these frequencies. The duration of changes in Glyt2 mRNA evoked by unilateral labyrinthectomy, measured with the reverse transcription polymerase chain reaction, was 5-10 days. These data provide the first example of in vivo regulation of transporter transcription by neuronal activity.

Amino Acid Transport Systems, Neutral↗
PubMed1999

Microfabrication techniques using focused ion beams and emergent applications.

The application of focused ion beam (FIB) machining in several technologies aimed at microstructure fabrication is presented. These emergent applications include the production of micromilling tools for machining of metals and the production of microsurgical tools. An example of the use of microsurgical manipulators in a circulatory system measurement is presented. The steps needed to transform the laboratory fabrication of these tools and manipulators into a routine FIB production process are discussed. The ion milling of three-dimensional cavities by the exact solution of a mathematical model of the FIB deflection is demonstrated. A good agreement between the model calculation and the ion beam control has been obtained for parabolic and cosine cross-section features with planes of symmetry.

Animals↗
PubMed1999

GRAB proteins, novel members of the NAC domain family, isolated by their interaction with a geminivirus protein.

Geminiviruses encode a few proteins and depend on cellular factors to complete their replicative cycle. As a way to understand geminivirus-host interactions, we have searched for cellular proteins which interact with viral proteins. By using the yeast two-hybrid technology and the wheat dwarf geminivirus (WDV) RepA protein as a bait, we have isolated a family of proteins which we termed GRAB (for Geminivirus Rep A-binding). We report here the molecular characterization of two members, GRAB1 and GRAB2. We have found that the 37 C-terminal amino acids of RepA are required for interaction with GRAB proteins. This region contains residues conserved in an equivalent region of the RepA proteins encoded by other viruses of the WDV subgroup. The N-terminal domain of GRAB proteins is necessary and sufficient to interact with WDV RepA. GRAB proteins contain an unique acidic C-terminal domain while their N-terminal domain, of ca. 170 amino acids, are highly conserved in all of them. Interestingly, this conserved N-terminal domain of GRAB proteins exhibits a significant amino acid homology to the NAC domain present in proteins involved in plant development and senescence. GRAB1 and GRAB2 mRNAs are present in cultured cells and roots but are barely detectable in leaves. GRAB expression inhibits WDV DNA replication in cultured wheat cells. Our studies highlight the importance that the pathway(s) mediated by GRAB proteins, as well as by other NAC domain-containing proteins, might have on geminivirus DNA replication in connection to plant growth, development and senescence pathways.

Amino Acid Sequence↗