Biomedical subjects
Publications and source records attributed to J Gao.
This paper reports the new prepared method of Prepared Soypean by purebred culture of Aspergillus niger. The technology condition is that temperture is 28 +/- 2 degrees C and comparative humidity is 95%, time of fermented culture is 15-20 days. After improvement, polluttion of other bacteria is easily avoided.
The family size biogas tanks were built in the study site. Their effects were monitored. Faecal coliform and parasite eggs were examined before and after biogas digestion. The results of whole-year operation indicate that faecal coliform and parasite eggs can be reduced by 99.7% and 99.8%, respectively. The schistosome eggs survival experiment showed that the eggs all died off in 100 days of retention in biogas tanks. Therefore, the appropriate technology of nightsoil treatment can effectively interrupt the life cycle of schistosome.
PURPOSE: It is well-known that after trauma to the ocular surface such as that seen in refractive surgeries like photorefractive keratectomy (PRK; laser corneal surface ablation), there is a "dropout" of underlying stromal keratocytes. Recently it was proposed by Wilson et al. that this loss of keratocytes may be the result of apoptosis. This process can be initiated by several cellular factors that result in release of endonucleases, DNA segmentation, and eventual cellular destruction. One protein, bcl-2, has been shown to have a profound role in its inhibition. The goal of this study was to evaluate the mechanisms and level of apoptosis in the cornea after PRK. METHODS: Rabbits were anesthetized systemically with ketamine/xylazine. The corneal epithelium was removed by using phototherapeutic keratectomy (PTK, 100 pulses). PRK was then performed (-9.0 D, 5.0 mm optical zone). Rabbits were killed at 4 days and 4 weeks, and the corneas were prepared for light and electron microscopy, as well as for apoptosis evaluation. RESULTS: The apoptosis assay demonstrated that (a) the normal cornea exhibited a limited level of apoptosis, primarily in the superficial epithelium, very little in the basal epithelium, with none in the keratocytes and endothelium; (b) the entire epithelial layer was found to be apoptotic 4 days and 4 weeks after PRK; (c) an elevated level of apoptosis was detected in both keratocytes and endothelial cells after PRK at the same time points. The immunohistochemical staining showed that (a) the normal cornea had a low level of bcl-2 protein, exclusively in the superficial epithelium; (b) bcl-2 was induced in the basal epithelial cells and anterior keratocytes under the wound bed 4 days after PRK and increased 4 weeks after PRK. CONCLUSIONS: These findings suggest that apoptosis plays an important role in the corneal wound-healing process, which appears to be mediated in part by bcl-2. Bcl-2 induction may protect corneal epithelial cells from apoptosis after trauma such as PRK.
Integrin alpha IIb beta 3 is necessary for platelet thrombus formation by virtue of its ability to bind fibrinogen and von Willebrand factor in a regulated fashion and to mediate platelet aggregation. Moreover, alpha IIb beta 3 transduces inward signals in response to ligand binding which promote cytoskeletal reorganization and other post-ligand binding events. Anchorage and signaling through alpha IIb beta 3 appear to be controlled by interactions of intracellular proteins with the cytoplasmic tails of alpha IIb and/or beta 3. The cytoplasmic tails and associated cytoskeletal components may function as a scaffold or nucleation site for the assembly of enzymes, substrates and adaptor molecules into a signaling complex. Identification of the components of this signaling complex and characterization of their dynamic interactions should lead to insights into the molecular basis of platelet activation defects and may provide a rationale for the development of new anti-thrombotic drugs.
Research over the past few years on the function of intermediate filaments in cells in culture has not produced convincing results, because the key role of intermediate filaments is within tissues and at certain periods of development. Only recently the technique of gene knockout has been used to examine intermediate filaments in mice and has provided the first evidence that intermediate filaments are directly involved in cell resilience and the maintenance of tissue integrity. Knockout of the gene encoding keratin K8 is lethal in the embryo, and results in hepatic or intestinal lesions, while knockout of the K14 or K10 genes leads to rupture of stratified epithelia. Knockout of the gene encoding desmin causes the rupture of skeletal and cardiac muscle, and collapse of blood vessel walls. Knockout of the gene coding for GFAP leads to a loss of cerebral white matter, and knockout of the gene coding for vimentin causes degeneration of the cerebellar Purkinje cells. The results reveal the lack of compensation by another intermediate filament. Tissues without intermediate filaments fall apart; they are mechanically unstable, unable to resist physical stress, and this leads to cell degeneration. By maintaining the shape and plasticity of the cell, the intermediate filament network acts as an integrator within the cell space. The state of mechanical force imposed on a tissue or a cell can alter the shape of certain elements of the cytoskeleton and thus participate to the control of cell functions.
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OBJECTIVE: To examine the IP3 amounts, invasive potentials, and response to LN and EGF of the substrains with high or low metastatic ability from two kinds of carcinoma cell lines (MFC and CNZ-2Z). METHOD: Radio-ligand binding assay and matrigel invasive assay. RESULTS: The low metastatic substrains from the two lines had higher amounts of IP3 and stronger response to EGF than their responsive high ones, but high metastatic substrains from CNZ-2Z had stronger response to LN than the low ones while MFC substrains had not significant difference in response to LN. CONCLUSION: There is internal difference in signal transduction between the high and low metastatic cancer cells, and it is significant to study the difference in detail.
Endothelins are potent vasoconstrictors released by endothelium, and their concentrations, which may be involved in the pathophysiology of burn injury, are increased in burn patients. The purpose of this study was to investigate the role of endogenous endothleins in postburn renal injury. 30% TBSA III degree burnt rat model was used, and 72 male Wistar rats were studied: 32 burnt rats received intravenous infusion of the non-selective ETA-ETB receptor antagonist PD145065 (1 mumol/Kg body weight) for 3 hours (group 1); other 32 burnt rats received only the vehicle (0.5 ml/250 g/h) (group 2). The rats both in group 1 and group 2 were given adequate resuscitation via different ways. 8 healthy rats were normal controls. In group 2, the blood flow of kidney decreased sharply, while marked lipoperoxidation damage and ATP depletion in kidney occurred although the arterial blood pressure remained unchanged postburn after sufficient fluid was given. Administration of endothelin receptor antagonist PD145065 has been demonstrated to be able to prevent kidney from damage to some extents both morphologically and biochemically by means of increment of blood flow, stimulation of the activity of SOD, enhancement of the contents of ATP and ADP, and reduction of the severity of edema in kidney. These results suggest that endogenous endothelins contribute to renal injury in the early postburn stage in rats through vascular constriction and tissue hypoxia which in turn induced ATP depletion and lipoperoxidation damage.
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We have used frequency-domain fluorescence resonance energy transfer (FRET) and anisotropy measurements to identify the structural properties of wheat germ calmodulin (CaM) bound to either the plasma membrane Ca-ATPase (PM-Ca-ATPase) in native erythrocyte ghost membranes or a peptide (C25W) that has an identical sequence to the CaM-binding domain on the PM-Ca-ATPase. Cross-linking experiments using benzophenone labeled CaM in conjunction with immunoblots using antibodies specific for either CaM or the PM-Ca-ATPase indicate that one molecule of CaM selectively binds one PM-Ca-ATPase polypeptide chain in native erythrocyte ghost membranes. There are no other proteins in the erythrocyte membrane that bind CaM with high affinity, permitting the measurement of the structural properties of CaM bound to the PM-Ca-ATPase in native erythrocyte ghost membranes. FRET measurements between the fluorophore pyrene maleimide (PMal) located at Cys27 in calcium binding loop I and nitrotyrosine139 in calcium binding loop IV on wheat germ CaM indicate that the average spatial separation and conformational heterogeneity associated with the two opposing globular domains of CaM are virtually identical upon CaM binding to either the PM-Ca-ATPase or C25W. Measurements of the solvent accessibility and segmental rotational dynamics of PMal-CaM bound to either the PM-Ca-ATPase or C25W further indicate that the local environment around the pyrene label located at Cys27 is very similar. However, the overall rotational dynamics of CaM bound to the PM-Ca-ATPase is much slower (phi 2 = 83 +/- 14 ns) than observed when CaM binds C25W (phi 2 = 10.3 +/- 0.5 ns). This implies that CaM is tightly associated with the CaM-binding domain of the PM-Ca-ATPase and that the observed rotational motion of pyrenylmaleimide labeled CaM is characteristic of the global motion of the CaM-binding domain on the PM-Ca-ATPase. The similar conformational heterogeneity and local environment of CaM bound to either the PM-Ca-ATPase or C25W indicates that CaM binds to a contiguous sequence of amino acids on the Ca-ATPase that are analogous to C25W and that there are no significant interactions with other structural elements within the PM-Ca-ATPase. The rate of rotational motion associated with CaM bound to the PM-Ca-ATPase is consistent with hydrodynamic calculation in which the calmodulin-binding domain located at the carboxyl-terminus of the PM-Ca-ATPase has a stable and defined tertiary structure that is independent of the other cytoplasmic domains of the PM-Ca-ATPase.
1. The whole cell patch clamp technique was used to study effects of the beta agonist isoprenaline (Iso) on the current-voltage (I-V) relationship of the Na(+)-K+ pump current (Ip) in acutely isolated guinea-pig ventricular myocytes. 2. The effect of Iso on Ip at high [Ca2+]i (1.4 microM) was voltage dependent. The I-V relationship of Ip in Iso shifted by approximately 30 mV in the negative direction on the voltage axis, increasing Ip at negative voltages but leaving Ip unchanged at positive voltages. 3. Intracellular application of the calmodulin antagonist, calmodulin-dependent protein kinase II fragment 290-309, did not eliminate or reduce the Iso-induced voltage shift, suggesting calmodulin-dependent protein kinase II was not involved. 4. The Iso inhibition of Ip at low [Ca2+]i (15 nM) was not voltage dependent. Ip was reduced by 20 to 30% in the presence of Iso at each holding potential. 5. When the voltage dependence of Ip was largely reduced by substitution of N-methyl-D-glucamine+ for external Na+, the magnitude of the low [Ca2+]i, Iso-induced inhibition of Ip was progressively eliminated by increasing the [Ca2+]i. At a [Ca2+]i of 1.4 microM, this inhibition disappeared. 6. At intermediate values of [Ca2+]i, the I-V curves in Na(+)-containing solution in the presence and the absence of Iso crossed over. The higher the [Ca2+]i, the more positive the voltage at which the two I-V curves intersected. 7. During beta-adrenergic activation our results suggest intracellular Ca2+ has two effects: (a) It prevents protein kinase A (PKA) phosphorylation-induced inhibition of Ip. (b) It causes a PKA phosphorylation-induced shift of the pump I-V relationship in the negative direction on the voltage axis. These effects may have important physiological significance in the regulation of heart rate and cardiac contractility.
This paper describes the use of electrospray ionization-mass spectrometry (ESI-MS) to screen two libraries of soluble compounds to search for tight binding inhibitors for carbonic anhydrase II (EC 4.2.1.1). The two libraries, H2NO2SC6H4C(O)NH-AA1-AA2-C(O)NHCH2CH2CO2H where AA1 and AA2 are L-amino acids (library size: 289 compounds) or D-amino acids (256 compounds), were constructed by attaching tripeptides to the carboxyl group of 4-carboxybenzenesulfonamide. Screening of both libraries yielded, as the tightest binding inhibitor, compound 1 (AA1 = AA2 = L-Leu; binding constant Kb = 1.4 x 10(8) M-1). The ability of ESI-MS to estimate simultaneously the relative binding affinities of a protein to soluble ligands in a library, if general, should be useful in drug development.
Electrostatic interactions between charges on ligands and charges on proteins that are remote from the binding interface can influence the free energy of binding (delta Gb). The binding affinities between charged ligands and the members of a charge ladder of bovine carbonic anhydrase (CAII) constructed by random acetylation of the amino groups on its surface were measured by affinity capillary electrophoresis (ACE). The values of delta Gb derived from this analysis correlated approximately linearly with the charge. Opposite charges on the ligand and the members of the charge ladder of CAII were stabilizing; like charges were destabilizing. The combination of ACE and protein charge ladders provides a tool for quantitatively examining the contributions of electrostatics to free energies of molecular recognition in biology.
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The scavenging activities of six phenylpropanoid glycosides, i.e. leucosceptoside A and martynoside, isolated from Pedicularis alashanica Maxim, verbascoside, and pediculariosides A, M and N isolated from Pedicularis striata Pall ssp. arachnoidea Franch Tsoong, on superoxide anion and hydroxyl radicals have been studied by the spin trapping method. The results demonstrated that the number of phenolic hydroxyl groups in the structures of the phenylpropanoid glycosides is related to their scavenging activities. The scavenging effects of the phenylpropanoid glycosides possessing two o-dihydroxyl groups were stronger than the effects of the compounds possessing two o-hydroxy-methoxy groups.
From a CC1.2 embryonic stem cell genomic library, we isolated and sequenced a 10.4-kb DNA segment (GenBank/EMBL Data Bank accession number L49022) containing the entire gene encoding mouse h1-calponin, an actin-associated smooth muscle-specific protein and a potential modulator of contraction. Sequence data revealed that there are seven exons and six introns in the h1-calponin gene. Determined by primer extension mapping of the RNA transcripts, the transcription of h1-calponin gene initiates at the same site in stomach, urinary bladder and pregnant uterus smooth muscles. The genomic organization suggests that the previously identified alpha- and beta-calponin isoforms are produced by splicing of exon 7 at two alternative acceptor sites. Isolation and structural characterization of the h1-calponin gene provides information to further investigate the expression regulation of this smooth muscle-specific gene.
The rpL34 gene, which encodes a cytoplasmic ribosomal protein with a high homology to the rat 60S r-protein L34, was isolated from a genomic library of tobacco (Nicotiana tabacum L. cv. Xanthi-nc). A 1500 bp upstream promoter fragment was fused to the chloramphenicol acetyltransferase (CAT) reporter gene or beta-glucuronidase (GUS) reporter gene and transferred into tobacco plants by the Agrobhacterium-mediated leaf disk transformation method. Analysis of CAT activity in leaf tissues showed that mechanical wounding increased the rpL34 promoter activity about 5 times as compared to untreated controls and that the promoter activity was further enhanced by plant growth regulators, 2,4-dichlorophenoxyacetic acid and benzyladenine. Histochemical GUS staining patterns of the transgenic plants showed that the rpL34 promoter activity is high in actively growing tissues, including various meristems, floral organs, and developing fruits. A series of 5' deletion analyses of the rpL34 promoter indicated that a 50 bp region located between -179 and -129 is essential for wound, auxin and cytokinin responses. Deletion of this region reduced the promoter activity to an undetectable level. Insertion of the 50 nucleotide sequence into a minimal promoter restored the promoter activity and the promoter strength was proportional to the copy number of the upstream sequence. The role of TATA and CAAT box regions was studied by a series of 3' deletion analyses. A 3' deletion up to -28 did not significantly affect the promoter strength. However deletion of the promoter up to 70 bp, which deleted the TATA box region, significantly reduced promoter activity. Further deletion of the promoter up to - 104. eliminating the CAAT box region, abolished the promoter activity. These results suggest that the TATA box and CAAT box regions are also important for the rpL34 promoter activity in addition to the 50 bp upstream region.
The changing distributions of collagens and glycosaminoglycans have been studied at the attachments of the medial collateral ligament during postnatal development. The ligament is of particular interest because it has a fibrocartilaginous attachment to the femoral epiphysis, but a fibrous one to the tibial metaphysis. Ligaments were examined in rats killed at birth and at 2, 4, 6, 8, 10, 20, 30, 45, 60, 90 and 120 days after birth. Cryosections were immunolabelled with monoclonal and polyclonal antibodies against types I and II collagen, chondroitin 4 and 6 sulfate, dermatan and keratan sulfate. Although the ligament is attached at both ends to bones that develop from cartilage, there was a striking difference in collagen labelling. Type II collagen was only found in spicules of calcified cartilage in bone beneath the tibial enthesis after ossification had commenced, but there was a continuous band of labelling at all stages of development at the femoral enthesis. Initially, the cartilage at the femoral attachment lacked type I collagen, but by 45 days labelling was continuous from ligament to bone. Continuity of labelling was seen much earlier at the tibial enthesis, as soon as bone had formed. There were also marked changes in glycosaminoglycan distribution. Keratan sulfate was present at both entheses up to 45 days, but only at the femoral enthesis thereafter. Both attachments labelled throughout life for dermatan sulfate, but chondroitin 4 and 6 sulfate were only found at the femoral end. The results suggest that enthesial cartilage at the femoral attachment was initially derived from the cartilaginous bone rudiment but was quickly eroded on its deep surface by endochondral ossification as bone formed at the attachment site. It was replaced by fibrocartilage developing in the ligament. This mechanism allows enthesis cartilage/fibrocartilage to contribute to the growth of a bone at a secondary centre of ossification in addition to dissipating stress at the ligament-bone junction.