[Acquisition of developmental competence by nuclear transfer of immature oocytes].
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Animals↗
Biomedical subjects
S Bao
Publications and source records attributed to S Bao.
[Construction of cDNA library of Mortierella and screening of delta 9 fatty acid desaturase cDNA sequence].
Mortierella species have potential for fermentative production of polyunsaturated fatty acids including gamma-linolenic acid, Arachidonic acid and EPA, etc. In order to clone genes encoding enzymes in the unsaturated fatty acid biosynthetic pathway, cDNA library of Mortierella was constructed using lambda gt 10 vector. Using cDNA encoding conserved region of delta 9 fatty acid desaturase gene as probe, Mortierella cDNA library was screened. After two rounds of screening one positive clone was identified which has insert length of larger than 1.6 kb.
Base Sequence↗
B1 B cell numbers and antibodies against phosphorylcholine and LPS are increased in IL-6 gene knockout mice.
Peritoneal cavity cells were isolated from IL6-gene knockout (IL6(-/-)) and wild-type mice and stained for expression of IgM, CD5, and CD23. B1 cell (IgM(+)/CD23(-), CD5(+)/IgM(+)) numbers were increased twofold in IL6(-/-) mice compared to normals while IgM(+)/CD23(+) (B2) cell numbers were reduced threefold. Intestinal antibody levels were also determined for both total immunoglobulin and phosphorylcholine (PC)-specific and LPS-specific antibody following oral challenge with attenuated Salmonella typhimurium. Total immunoglobulin levels (IgM, IgG, and IgA) were reduced 60-80% in intestinal secretions of IL6(-/-) mice compared to wild-type controls; however, PC-specific antibody was significantly higher in IL6(-/-) mice. Anti-LPS antibodies were also three- to sevenfold higher in IL6(-/-) mice compared to controls following Salmonella challenge. These data suggest that in IL6(-/-) mice the development of mucosal B2 cells is impaired but that intestinal B1 cells responding to microbial antigens such as PC and LPS develop normally and are fully functional.
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Analysis of the mucosal microenvironment: factors determining successful responses to mucosal vaccines.
The predominance of IgA antibodies in mucosal sites reflects a combination of high rate IgA isotype switching among precursor cells in induction sites, their selective localisation in mucosal effector tissues and vigorous proliferation of these cells after extravasation. Each of these steps leading to IgA expression at the mucosa is under cytokine control. This paper will address the role of cytokines in induction and expression of IgA responses, the contribution of various precursor cell subsets and their differential responses to cytokine signals and strategies for manipulating cytokine expression. With respect to IgA antibody production in the gut whereas IL-4 and TGF-beta have been implicated in isotype switching of precursor cells to IgA commitment, their subsequent localisation, proliferation and effector activity expression is dependent on IL-5 and IL-6 expression locally. Most IgA plasma cells in the intestine derive from cells of the B2 lineage in the Peyer's patch, but a subpopulation of cells derived from the peritoneal cavity (B1 cells) also contribute to the IgA plasma cell population in the intestinal lamina propria. Whereas IgA+ cells of the B2 lineage are IL-6 dependent but IL-5 independent, B1-derived IgA+ cells are IL-5 dependent and IL-6 independent. On the other hand, cell mediated immune responses in the gut are highly dependent on IFN-gamma production by both Th1 CD4 cells and CD8 cells and in enteric Salmonella infection IFN-gamma production is essential but antibody has little effect on this process.Therapeutic interventions based on the information emerging from these studies will lead to improved vaccination responses and correction of immunodeficiencies especially in young animals.
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Filtroporation: A simple, reliable technique for transfection and macromolecular loading of cells in suspension.
Cultured Chinese hamster ovary (CHO) cells suspended in their growth medium were forced by gas pressure through the uniformly sized micropores of filter membranes. This procedure caused transient damage to the plasma membrane, which increased the permeability of the cells to exogenous molecules. This "filtroporation" was indicated by uptake of fluorescent dextran molecules up to 500,000 MW in cells deemed viable by trypan blue dye exclusion. The macromolecular uptake was increased if the driving pressure was increased at constant micropore size, or if the micropore size was decreased at constant driving pressure. Larger membrane perturbations permitted uptake of a luciferase reporter plasmid, which resulted in transfection of the CHO cells with the surviving cells expressing luciferase activity after 2 days in culture. This simple and general new method of porating cells in suspension may be optimized to incorporate the desired macromolecules while retaining the maximum viability.
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Impaired cerebellar synapse maturation in waggler, a mutant mouse with a disrupted neuronal calcium channel gamma subunit.
The waggler, a neurological mutant mouse with a disrupted putative neuronal Ca(2+) channel gamma subunit, exhibits a cerebellar granule cell-specific brain-derived neurotrophic factor deficit, severe ataxia, and impaired eyeblink conditioning. Here, we show that multiple synapses of waggler cerebellar granule cells are arrested at an immature stage during development. Synaptic transmission is reduced at parallel fiber-Purkinje cell synapses. The Golgi cell-granule cell synaptic currents show immature kinetics associated with reduced gamma-aminobutyric acid type A receptor alpha6 subunit expression in granule cells. In addition, the mossy fiber-granule cell synapses exhibit N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs), but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated EPSCs. Our results suggest that voltage-dependent Ca(2+) channels are involved in synapse maturation. This deficient synaptic transmission in the waggler cerebellum may account for their behavioral deficits.
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HRad17, a human homologue of the Schizosaccharomyces pombe checkpoint gene rad17, is overexpressed in colon carcinoma.
Using the palindromic PCR-cDNA display method, we have cloned a novel gene overexpressed by human colon carcinoma relative to normal colon. Among normal tissues examined, only testis expresses it at a high level. Sequence analysis revealed its extensive homology with checkpoint genes rad17 of Schizosaccharomyces pombe and RAD24 of Saccharomyces cerevisiae. This novel gene designated as hRad17 is localized to chromosome 5q12,13.1, a region known to be deleted in a variety of human cancers. Promoter region and one pseudogene of hRad17 have been identified. Whereas the increased expression of hRad17 by human colon carcinomas may be related to the known resistance of these cells to DNA-damaging agents during therapy, the deletion of hRad17 in a variety of cancers may predispose them to increased rate of mutation and heightened sensitivity to DNA-damaging agents, including radiation and anticancer drugs.
Animals↗
The inhibitory effect of hCG on counter current transfer of GnRH and the presence of LH/hCG receptors in the perihypophyseal cavernous sinus--carotid rete vascular complex of ewes.
The existence of the hormone passage from venous blood into arterial blood in the area of the perihypophyseal vascular complex has been demonstrated in some mammals, but its mechanism has not been defined. To study the regulatory mechanism we infused hCG into perihypophyseal cavernous sinus of ovariectomized, conscious ewes to test if the hCG would affect putative LH/hCG receptors and inhibit counter-current transfer of GnRH from the venous cavernous sinus to the arterial carotid rete. The latter study was done on an isolated head model. Ewes were ovariectomized in mid-anestrus and, after 4 to 5 wk were used in the experiments. On the day of experiment ewes were treated intramuscularly with estradiol benzoate or oil vehicle, and 18 to 20 h later were infused either with a multielectrolyte solution or hCG for 2 h via the venae angularis oculi. Immediately thereafter the ewes were anesthetized and exanguinated, and subsequently decapitated. The isolated head was perfused with Dextran in multielectrolyte. The 125I-GnRH was infused into the cavernous sinus via the venae angularis oculi for 5 min; contemporaneous samples were taken from the carotid rete and both jugular veins at 1-min intervals. Transfer of 125I-GnRH from the cavernous sinus to the carotid rete was inhibited by hCG in ewes pretreated with estradiol benzoate but not with oil (P<0.005). We collected tissue samples from the vascular complex of the cavernous sinus and carotid rete of cyclic ewes to determine the presence of LH/hCG receptors. In situ hybridization showed the presence of LH/hCG receptor mRNA transcripts in the walls of both arterial and venous compartments of the cavernous sinus-carotid rete complex, and immunohistochemistry revealed the presence of receptor proteins. These novel findings confirm previously obtained data suggesting that LH is a modulatory factor for the counter-current transfer of neuropeptides from the venous blood of the cavernous sinus to the arterial blood supplying the brain and hypophysis. The LH could modulate 125I-GnRH transfer acting directly on the vascular smooth muscle.
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Sonoporation of cultured cells in the rotating tube exposure system.
Suspensions of Chinese hamster ovary cells were exposed to ultrasound in the presence of fluorescent dextran to determine the conditions needed for sonoporation with uptake of the large molecules. Albunex, a gas-body- based ultrasound contrast agent, was added to enhance cavitation. Ultrasound was continuous wave at frequencies of 1.0, 1.68, 2.25, 3.3, 5.3, and 7.15 MHz. Sterile 4.5-mL exposure chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. After exposure, cells were tested for sonoporation by counting fluorescent cells and for cell lysis by counting cells stained by trypan blue. Sonoporation was a sensitive bioeffects indicator that was detected at pressure amplitudes lower than were needed for transient cavitation or cavitation-induced cell lysis. For 10% Albunex, apparent thresholds for sonoporation, which were comparable to the levels required to perturb the gas bodies, were 0.084 MPa (spatial peak negative pressure amplitude) from 1.0-3.3 MHz and 0.27 MPa at 5.3 and 7.15 MHz. Sonoporation decreased slightly if the tube was not rotated. The effects increased for increasing Albunex concentration (with rotation). The plating efficiency of cells exposed to 0.2 MPa at 2.25 MHz and sorted by a flow cytometer was 19% (3.6% standard deviation [SD]) for fluorescent cells, compared to 67% (1% SD) for nonfluorescent exposed cells and 62% (6% SD) for sham-exposed cells. The reduced viability represents an important consideration for possible applications of sonoporation.
Albumins↗
Ultrasonic enhancement of gene transfection in murine melanoma tumors.
The enhancement of gene transfection by ultrasound (US) was evaluated in vitro and in vivo using the B16 mouse melanoma model. Cultured cells were either exposed in suspensions in vitro or implanted subcutaneously in female C57BL/6 mice for 10-14 days and, subsequently exposed, in vivo. For comparison to results with a luciferase plasmid, a reporter plasmid for green fluorescent protein (GFP) was used to evaluate transfection efficiency. US was supplied by a system, similar to a Dornier HM-3 lithotripter, that produced shock waves (SW) of 24.4 MPa peak positive and 5.2 MPa peak negative pressure amplitudes at the focus. The plasmids were mixed with the suspensions to achieve 20 ,microL mL(-1), or were injected intratumorally to provide 0.2 mg DNA per mL of tumor. Acoustic cavitation was promoted by retaining 0.2 mL of air in the 1.2-mL exposure chambers in vitro and by injecting air at 10% of tumor volume in vivo. In vitro, cell counts declined to 5.3% of shams after 800 SW exposure, with 1.4% of the cells expressing GFP after 2 days of culture. In vivo, 2 days after 400 SW exposure, viable-cell recovery from excised tumors was reduced to 4.2% of shams and cell transfection was enhanced by a factor of about 8, reaching 2.5% of cell counts (p < 0.005 in t-test). These results show that strong tumor ablation induced by US shock wave treatment can be coupled with simultaneous enhancement of gene transfection.
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Spin-Echo planar spectroscopic imaging for fast lipid characterization in bone marrow.
Lipid characterization of bone marrow in vivo with proton magnetic resonance spectroscopy was performed using Spin-Echo Planar Spectroscopic Imaging sequences. The methods are shown capable of rapidly generating two-dimensional chemical shift imaging data sets suitable for measuring lipid indices that reflect unsaturation levels among triglycerides, as demonstrated in oil phantoms and bone marrow from a healthy volunteer. The volume coverage, spatial resolution, acquisition speed, and spectral characteristics of Spin-Echo Planar Spectroscopic Imaging should make it attractive for clinical studies of diseases affecting normal lipid chemical composition.
Adult↗
Bilateral lesions of the interpositus nucleus completely prevent eyeblink conditioning in Purkinje cell-degeneration mutant mice.
The authors have previously demonstrated that Purkinje cell-degeneration (pcd) mutant mice are impaired in eyeblink conditioning (L. Chen et al., 1996a). The present study addresses the following 3 questions: (a) whether pcd mice perceive the conditioned and unconditioned stimuli as well as the wild-type mice, (b) whether pcd mice have a normal sensitization level, and (c) whether the residual learning in pcd mice is cerebellum-dependent. Results indicated that the pcd mice exhibited normal tone-induced responses in the cochlear nucleus and normal sensitivity to heat-induced pain. They showed a similar level of sensitization as the wild-type mice and were completely unable to learn conditioned eyeblinks after bilateral lesions aimed at the anterior interpositus nucleus. Thus, pcd mice are partially impaired in eyeblink conditioning because of a deficiency in learning mechanisms, and the residual learning in the pcd mice is mediated by the cerebellar nuclei.
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TNF-alpha production in the cornea in response to Pseudomonas aeruginosa challenge.
Pseudomonas aeruginosa can cause ulcerative bacterial keratitis or contact lens-induced acute red eye (CLARE) in humans. The present study used a mouse model of ocular infection and inflammation to examine the relationship between TNF-alpha and inflammation in the cornea in response to challenge with either a strain of P. aeruginosa causing keratitis or a CLARE strain. Constitutive TNF-alpha mRNA was detected in the epithelium, mainly towards the periphery. After infection with the keratitis-inducing strain (6294), TNF-alpha expression was elevated four-fold by 24 h post-challenge. No detectable induction of TNF-alpha mRNA was seen with CLARE strain (Paer1) challenge at any time point. The TNF-alpha protein production detected by ELISA showed a corresponding pattern to the mRNA expression, which also correlated with pathological changes. These results suggest that invasive strains of P. aeruginosa create greater pathological changes as a result of elevated TNF-alpha production, which contributes to inflammation during keratitis in vivo.
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Modulation of IL-10, IL-12, and IFN-gamma in the epidermis of hairless mice by UVA (320-400 nm) and UVB (280-320 nm) radiation.
We have observed recently that the suppression of contact hypersensitivity (CHS) induced in mice by UVB irradiation may be prevented by suberythemal exposure to UVA radiation. Because the UVB-immunosuppressed state is associated with an upregulation of the Th2-associated cytokines IL-10 and IL-4, and a deficiency in Th1-associated IL-2, IL-12, and IFN-gamma, and because UVA photoimmunoprotection appeared to be IFN-gamma- dependent, we tested the hypothesis that UVA immunoprotection results from an ability to prevent the UVB-induced cytokine disarray. This study describes changes in epidermal IL-10, IL-12 and IFN-gamma for 5 d following irradiation of hairless mice with the CHS-modulating doses of UVB, UVA, or UVA + UVB, using immuno-histochemical detection in paraffin embedded skin sections, followed by image analysis quantitation. We found that UVB, but not UVA exposure, caused an increase in epidermal IL-10 expression, peaking at 3 d. UVA irradiation, but not UVB, resulted in increased epidermal IL-12 expression, peaking at 3 d, and increased epidermal IFN-gamma expression peaking earlier at 1 d. Irradiation with UVA + UVB abrogated the UVB-enhanced expression of IL-10, and caused small but significant increases in IL-12 and IFN-gamma at 3 d and 1 d, respectively. These findings suggest that UVA photoimmunoprotection is mediated via prevention of IL-10 release, and thus the maintenance of the Th1/Th2 balance, probably by upregulation of IL-12 and IFN-gamma, which are known to antagonize IL-10 in numerous models. The time course suggests that IFN-gamma responds initially to UVA radiation, and may stimulate the increased expression of IL-12.
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Expression of interleukin-6 in the cornea in response to infection with different strains of Pseudomonas aeruginosa.
Strains of Pseudomonas aeruginosa causing keratitis can be either cytotoxic (6206) or invasive (6294), while a strain (Paer1) causing contact lens-induced acute red eye has been shown to be neither. In situ hybridization was used to examine the location and identity of cells expressing interleukin-6 (IL-6) mRNA in the murine cornea and changes in expression in response to infection with different strains of P. aeruginosa. The number of IL-6-positive cells was determined by image analysis. IL-6 protein levels were measured by an enzyme-linked immunosorbent assay. BALB/c mice were challenged by use of the wounded-cornea model with P. aeruginosa 6294, 6206, or Paer1 (2 x 10(6) CFU). At time intervals up to 24 h, postchallenge corneal tissue was probed for IL-6 mRNA. IL-6 mRNA expression was rapidly elevated in the epithelium in response to strains 6294 and 6206. At the conclusion of the experiments, infiltrating inflammatory cells also stained positively for IL-6 mRNA. In contrast, corneas challenged with strain Paer1 showed significant upregulation of IL-6 mRNA only at 4 h postchallenge. Three distinct patterns of IL-6 mRNA expression in the mouse cornea occur in response to these three ocular isolates of P. aeruginosa. The data obtained for mRNA expression in the cornea for all three strains of P. aeruginosa correlated well with IL-6 protein analysis of whole-eye homogenates. Differences in the cytokine responses to these strains correlate with differences in the pathology associated with each strain and may offer an opportunity to develop strategies for the improved management of ocular inflammation.
Animals↗
[Principle and application of scattering power function method in X-ray fluorescence analysis. II. Incoherent scattering radiation].
It was discovered that the relationship between mass attenuation coefficient and incoherent scattering is not an inverse proportion but a power function in a wide range of matrix compositon. Experimental results are in good agreement with this theoretical predition. A new method is recommended for matrix absorption correction according to the discovery and used for the determination of microelement Sr in geological samples as an application example. The accuracy of the results of heavy absorption samples obtained by this method is much better than those obtained by the traditional scattering internal method.
English Abstract↗
[Principle and application of scattering power function method in X-ray fluorescence analysis. III. Continuous scattering radiation].
The study in this paper shows that the relationship between mass attenuation coefficient and continuous scattering radiation at wavelength 0.081 nm is not an inverse proportion supposed as the scattering internal standard method but a power function in a wide range of matix composition. Experimental results are in good agreement with that of theoretical prediction. A new method is recommended for matrix absorption correction using the 1.22 power function of continuous scattering radiation at wavelength 0.081 nm and used for microelement Sr determination in geological samples as an application example. The accuracy of the results by this method is nearly 4 times better than that from the traditional scattering internal method in a wide range of matrix composition.
English Abstract↗
Elevation of immunoglobulin deposition in the synovial membrane of dogs with cranial cruciate ligament rupture.
Synovial membrane biopsies were collected from 32 dogs with spontaneous cranial cruciate ligament rupture and seven control dogs. The subclasses of immunoglobulins in the synovial membrane of both the diseased and control dogs were examined with immunohistochemistry in combination with image analysis. IgG deposition was four-fold higher and IgM was eight-fold higher in dogs with spontaneous cranial crucial ligament (CrCL) rupture. These findings support the theory that there is an immune component to the inflammation associated with canine CrCL rupture. Further investigation is required to determine if these immune or inflammatory responses in CrCL rupture joints are the primary factor in ligament rupture or if they develop secondary to ligament rupture and instability.
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