Biomedical subjects

Cheng Zhu

Publications and source records attributed to Cheng Zhu.

At least 37 records · Page 2Linked to original sources

PubMed2006

Marked protection by selective cerebral profound hypothermia after complete cerebral ischemia in primates.

Hypothermia has been demonstrated to protect the brain from ischemia or traumatic brain injury. Achieving profound hypothermia has relied on techniques requiring total body cooling, which may result in serious cardiovascular and pulmonary complications. A technique to selectively cool the brain could conceivably exert a marked protection on cerebral structures and provide a relatively bloodless operative surgical field without systemic complications. Accordingly, this approach was tried in 7 rhesus monkeys after induction of general anesthesia. The right internal carotid artery and both internal jugular veins were each occlusively cannulated and connected to a circulation pump. The left internal carotid artery, both external carotid arteries, and both external jugular veins were temporarily clamped to establish severe cerebral ischemia. Using a closed-circuit system, cooled Ringer's lactate liquid (4 degrees C) was infused through right internal carotid artery with outflow draining though both internal jugular veins. Cooled perfusate decreased cerebral temperature to the target temperature of 15 degrees C. Thereafter, pump flow was discontinued, and brains were rewarmed spontaneously, while the temporarily clamped carotid arteries and jugular veins were opened to resume normal cerebral blood circulation. Neurological functions were recorded daily and cerebral histology was examined at the conclusion of the experiment. Magnetic resonance (MR) scans were routinely taken before and 3 weeks after ischemia. In the normothermia control group of five rhesus monkeys, Ringer's solution at 37 degrees C was infused in the same manner as the cold solution with cerebral temperature maintained at 36.7 +/- 0.32 degrees C. Right cerebral temperature decreased from 36.5 +/- 0.49 to 15.5 +/- 2.29 degrees C, and simultaneously the left cerebral temperature decreased from 36.4 +/- 0.38 to 16.3 +/- 2.4 degrees C for 62.8 +/- 9.76 min during selective cerebral cooled Ringer's liquid perfusion. In contrast, rectal temperature was only reduced to 32.4 +/- 0.96 degrees C from a baseline of 37.2 +/- 0.76 degrees C. Internal jugular vein hematocrit was 38.2 +/- 0.31% before perfusion and 2.82 +/- 0.46% at the end of perfusion in profound hypothermia group; hematocrit was 39.7 +/- 0.62% before perfusion and 3.42 +/- 0.38% at the end of perfusion in the normothermia group. In the hypothermic group, neurological functions were normal during 6 months of follow-up, and microscopic examination of brain tissue did not show evidence of pathological changes in hippocampus or medulla. MR scans did not show any cerebral infarction. In contrast, none of the monkeys in normothermia group survived for more than several hours, and microscopic examination of the brain revealed extensive neuronal necrosis within the medulla. Selective cerebral profound hypothermia provides significant histologic and neurologic protection after severe cerebral ischemia. In addition, there were no major complications, and the operative field remained relatively bloodless in the profound hypothermic group.

Anesthesia, General↗

PubMed2006

p27kip1 siRNA induces proliferation in corneal endothelial cells from young but not older donors.

PURPOSE: To determine whether small interfering (si)RNA downregulation of the cyclin-dependent kinase inhibitor p27kip1 overcomes G(1)-phase arrest and promotes cell-cycle progression in human corneal endothelial cells (HCECs) from young (<30 years old) and older (>60 years old) donors. METHODS: Transfection of siRNA was confirmed by incubating confluent cultures of HCECs with FITC-labeled nonsilencing siRNA. Confluent cultures were transfected for 48 hours with p27kip1 siRNA (2.5, 5, 25, or 100 nM) or nonsilencing siRNA, with a lipid transfection reagent. As a comparison, cultures were also transfected for 48 hours with p27kip1 antisense (AS) or missense (MS) oligonucleotides (oligo). At various times after transfection, cells were fixed for immunocytochemical localization of p27kip1 or extracted for Western blot analysis to assess relative p27kip1 protein levels. Cultures were also prepared for ZO-1 immunolocalization, to assess the effect of transfection on the morphology of the monolayer. The number of cells was counted at 0, 48, 96, 144, and 192 hours after incubation, and a cell-viability assay was performed. RESULTS: A dose-dependent decrease in p27kip1 protein level was observed in Western blot analysis, and nuclear staining for p27kip1 was greatly reduced in HCECs incubated with p27kip1 siRNA. No change in p27kip1 levels or in nuclear staining was observed in the nonsilencing control. p27kip1 siRNA (25 nM) appeared to be quantitatively more efficient than antisense oligonucleotide (500 nM) in reducing p27kip1 protein levels. Viability was less affected by siRNA treatment than by AS oligo transfection. ZO-1 staining showed no effect on morphology of the monolayer. The number of HCECs from young donors (<30 years old) transfected with p27kip1 siRNA increased up to 144 hours after incubation, whereas no change in the number of cells was observed in HCECs transfected with nonsilencing siRNA. In contrast to the results from young donors, no change in the number of cells was observed at any time point tested in HCECs from older donors (>60 years old) after p27kip1 siRNA transfection. CONCLUSIONS: Transfection of p27kip1 siRNA was sufficient to promote proliferation in confluent cultures of HCECs from younger, but not older donors. These results suggest that inhibition of proliferation in older donors is regulated by other mechanisms in addition to p27kip1.

Adult↗

PubMed2005

Expression of tissue inhibitor of metalloproteinase-4 (TIMP-4) in endometrium and placenta of rhesus monkey (Macaca mulatta) during early pregnancy.

The tissue inhibitors of metalloproteinases (TIMPs) are secreted as important regulators of the matrix metalloproteinases (MMPs). TIMP-4 is the most recently characterized member of the TIMPs family. In the present study, we examined the expression and localization of the TIMP-4 transcript and protein in endometrium and placenta of rhesus monkey (Macaca mulatta) on days 12, 18 and 26 of pregnancy using RT-PCR, in situ hybridization, and immunohistochemistry. The fragment of TIMP-4 gene from rhesus monkey uterine samples shared 95% identity with the corresponding region of human homologue. On day 12 of pregnancy, TIMP-4 mRNA was mainly expressed in the glandular and luminal epithelium. On days 18 and 26 of pregnancy, the expression of TIMP-4 mRNA tended to decline in glandular epithelium and there were strong staining in the placental villi. Furthermore, TIMP-4 mRNA was very faint or undetectable in the stromal cells, endothelial cells of arterioles and myometrium at any stages of pregnancy. The results of immunohistochemical analysis were similar to that of its mRNA. These findings indicate that TIMP-4 might play an important role in glandular secretion, endometrial tissue remodeling and invasion of the trophoblast cells by regulating MMPs in a localized manner in the uteri of rhesus monkey during early pregnancy.

Animals↗

PubMed2005

Measuring molecular elasticity by atomic force microscope cantilever fluctuations.

In single-molecule mechanics experiments the molecular elasticity is usually measured from the deformation in response to a controlled applied force, e.g., via an atomic force microscope cantilever. We have tested the validity of an alternative method based on a recently developed theory. The concept is to measure the change in thermal fluctuations of the cantilever tip with and without its coupling to a rigid surface via the molecule. The new method was demonstrated by its application to the elasticity measurements of L- and P-selectin complexed with P-selectin glycoprotein ligand-1 or their respective antibodies, which showed values comparable to those measured from the slope of the force-extension curve. L- and P-selectin were found to behave as nearly linear springs capable of sustaining large forces and strains without sudden unfolding. The measured spring constants of approximately 4 and approximately 1 pN/nm for L- and P-selectin, respectively, suggest that a physiological force of approximately 100 pN would result in an approximately 200% strain for the respective selectins.

Calibration↗

PubMed2005

Two-dimensional kinetics regulation of alphaLbeta2-ICAM-1 interaction by conformational changes of the alphaL-inserted domain.

The leukocyte integrin alphaLbeta2 mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of alphaLbeta2 is regulated by or induces conformational changes in the inserted (I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional (2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with alphaLbeta2 or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by approximately 8000- and approximately 30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type alphaLbeta2, but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the alphaLbeta2, is required for affinity and on-rate up-regulation.

Animals↗

PubMed2005

Expressions and regulation of endothelial and inducible nitric oxide synthases in mouse uterus during the estrous cycle and early pregnancy.

Nitric oxide (NO) has been implicated in many cellular processes. We examined the temporal and spatial expressions of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in mouse uteri during the estrous cycle and early pregnancy, as well as the regulation of eNOS and iNOS by estradiol (E2) and progesterone (P4) in ovariectomized mouse uteri using in situ hybridization and immunohistochemistry. Our results showed that positive eNOS and iNOS signals were localized in the uterine luminal epithelium and glandular epithelium during the estrous cycle. In ovariectomized mice, both E2 and P4 regulated the expression of eNOS and iNOS. During early pregnancy, eNOS and iNOS were detected not only in epithelium, but also in the primary decidual zone surrounding implanting embryos on day 6 of pregnancy, and in the whole decidualized stroma on day 7 of pregnancy. In conclusion, the results demonstrated that two NOS isoforms were localized in mouse uteri in specific temporal and spatial patterns during the estrous cycle and early pregnancy, and ovarian hormones can regulate their expression. Furthermore, the data suggest that the expression of NOS during the peri-implantation period might lead to enhance NO production, which could promote embryo implantation.

Animals↗

PubMed2005

Expression of matrix metalloproteinase-26 (MMP-26) mRNA in mouse uterus during the estrous cycle and early pregnancy.

Matrix metalloproteinases (MMPs) and their tissue inhibitors play important roles in the remodeling of extracellular matrix (ECM). MMP-26, also called endometase or matrilysin-2, is a novel member of the MMP family. The present study was to investigate the temporal and spatial expression of MMP-26 mRNA in mouse uterus during the estrous cycle and early pregnancy by using in situ hybridization and semi-quantitative RT-PCR. In this study, MMP-26 mRNA was found to be localized to the luminal and glandular epithelium at proestrus and estrus, and the expression level was decreased significantly from metestrus to dioestrus. During pre-implantation period, MMP-26 mRNA was predominantly expressed in luminal and glandular epithelium at much higher level; whereas it switched to stroma during peri-implantation period, and also appeared in the blastocysts and the implantation sites. The results suggested that MMP-26 might play a role in the cycling changes of mouse uterus during the estrous cycle and embryo implantation.

Animals↗

PubMed2005

Expression of adamalysin 19/ADAM19 in the endometrium and placenta of rhesus monkey (Macaca mulatta) during early pregnancy.

A disintegrin and metalloproteinase (ADAM) 19 may contribute to multiple processes including proteolysis, adhesion and intracellular signalling. These processes are also critical for embryo implantation. The aim of this study was to investigate the spatio-temporal expression of the ADAM19 in rhesus monkey uteri on days 12, 18 and 26 of pregnancy. The results showed that in the cloned monkey 346 bp ADAM19 gene fragment and 114 amino acid residues were 98 and 100% identical to those of human homologues, respectively. In-situ hybridization confirmed that the ADAM19 mRNA was located in the luminal and glandular epithelium on day 12 of pregnancy. On day 18 of pregnancy, strong signals of the ADAM19 mRNA were detected in the placental villi, trophoblastic column and glandular epithelium near the myometrium. Moderate expression of the ADAM19 mRNA was seen in the trophoblastic shell and stromal cells. The placental villi and trophoblastic column expressed abundant ADAM19 mRNA, and ADAM19 transcripts were also detected in the trophoblastic shell and fetal-maternal border on day 26 of pregnancy. The expression pattern of the ADAM19 protein was similar to its transcript, but signals for the ADAM19 protein in the stromal cells and trophoblastic shell increased more than its mRNA on day 18 of pregnancy. Statistical analysis demonstrated that the expression level of ADAM19 significantly increased on day 18 of pregnancy. These data suggest that the ADAM19 may be involved in the key processes of glandular secretion, trophoblast invasion and degradation of extracellular matrix during early pregnancy.

Amino Acid Sequence↗

PubMed2005

Thermo-mechanical responses of a surface-coupled AFM cantilever.

Atomic force microscopy (AFM) has been widely used for measuring mechanical properties of biological specimens such as cells, DNA, and proteins. This is usually done by monitoring deformations in response to controlled applied forces, which have to be at ultralow levels due to the extreme softness of the specimens. Consequently, such experiments may be susceptible to thermal excitations, manifested as force and displacement fluctuations that could reduce the measurement accuracy. To take advantage of, rather than to be limited by, such fluctuations, we have characterized the thermomechanical responses of an arbitrarily shaped AFM cantilever with the tip coupled to an elastic spring. Our analysis shows that the cantilever and the specimen behave as springs in parallel. This provides a method for determining the elasticity of the specimen by measuring the change in the tip fluctuations in the presence and absence of coupling. For rectangular and V-shaped cantilevers, we have derived a relationship between the mean-square deflection and the mean-square inclination and an approximate expression for the specimen spring constant in terms of contributions to the mean-square inclination from the first few vibration modes.

Computer Simulation↗

PubMed2005

Comparison of the proliferative capacity of human corneal endothelial cells from the central and peripheral areas.

PURPOSE: To compare the relative proliferative capacity between human corneal endothelial cells (HCECs) cultured from the central and peripheral areas of the cornea. METHODS: Human corneas were divided into two groups based on donor age (younger group, < or =30 years of age; older group, > or =50 years of age). Corneas were trephined, and Descemet's membrane with HCECs was stripped from the central (0-6.75 mm) and peripheral (6.75-9.5 mm) areas. HCECs were then isolated from Descemet's membrane and cultivated. An equal number of passage-1 endothelial cells from each area were seeded, and the number of cells was determined at various times after seeding. Doubling times of cells from each area were compared. The antibody against minichromosome maintenance-2 (MCM2) protein was tested for replication competence. RESULTS: Morphologically, HCECs from the central area were similar to cells from the peripheral area. The doubling time (in hours) of HCECs from the central area was 35.20 in the younger group (n = 4) and 54.54 in the older group (n = 4) and from the peripheral area, 29.37 in the younger group and 46.23 in the older group. There was no significant difference (younger: P = 0.515; older: P = 0.222) between the central and peripheral area in each age group. MCM2-positive cells were consistently observed in cultures from the central, as well as peripheral, area. There was no significant difference (younger: P = 0.929; older: P = 0.613) in the percentage of MCM2-positive cells between these two areas in either age group. Even though there was no significant difference, there was a tendency toward increased doubling time and decreased percentage of MCM2 in the central area of the older group. CONCLUSIONS: These results indicate that corneal endothelial cells from both the central and peripheral areas retain potential proliferative capacity.

Adolescent↗

PubMed2005

Catch bonds: physical models, structural bases, biological function and rheological relevance.

Force can shorten the lifetimes of macromolecular complexes (e.g., receptor-ligand bonds) by accelerating their dissociation. Perhaps paradoxical at first glance, bond lifetimes can also be prolonged by force. This counterintuitive behavior was named catch bonds, which is in contrast to the ordinary slip bonds that describe the intuitive behavior of lifetimes being shortened by force. Fifteen years after their theoretical proposal, catch bonds have finally been observed. In this article we review recently published data that have demonstrated catch bonds in the selectin system and suggested catch bonds in other systems, the theoretical models for their explanations, possible structural bases, their relation to flow-enhanced adhesion, and the potential biorheological relevance.

Cell Adhesion↗

PubMed2005

Catch bonds: physical models and biological functions.

Force can shorten the lifetimes of receptor-ligand bonds by accelerating their dissociation. Perhaps paradoxical at first glance, bond lifetimes can also be prolonged by force. This counterintuitive behavior was named catch bonds, which is in contrast to the ordinary slip bonds that describe the intuitive behavior of lifetimes being shortened by force. Fifteen years after their theoretical proposal, catch bonds have finally been observed. In this article we review recently published data that have demonstrated catch bonds in the selectin system and suggested catch bonds in other systems, the theoretical models for their explanations, and their function as a mechanism for flow-enhanced adhesion.

Animals↗

PubMed2004

Force history dependence of receptor-ligand dissociation.

Receptor-ligand bonds that mediate cell adhesion are often subjected to forces that regulate their dissociation via modulating off-rates. Off-rates control how long receptor-ligand bonds last and how much force they withstand. One should therefore be able to determine off-rates from either bond lifetime or unbinding force measurements. However, substantial discrepancies exist between the force dependence of off-rates derived from the two types of measurements even for the same interactions, e.g., selectins dissociating from their ligands, which mediate the tethering and rolling of leukocytes on vascular surfaces during inflammation and immune surveillance. We used atomic force microscopy to measure survival times of P-selectin dissociating from P-selectin glycoprotein ligand 1 or from an antibody in both bond lifetime and unbinding force experiments. By a new method of data analysis, we showed that the discrepancies resulted from the assumption that off-rates were functions of force only. The off-rates derived from forced dissociation data depended not only on force but also on the history of force application. This finding provides a new paradigm for understanding how force regulates receptor-ligand interactions.

Algorithms↗

PubMed2004

Catch bonds govern adhesion through L-selectin at threshold shear.

Flow-enhanced cell adhesion is an unexplained phenomenon that might result from a transport-dependent increase in on-rates or a force-dependent decrease in off-rates of adhesive bonds. L-selectin requires a threshold shear to support leukocyte rolling on P-selectin glycoprotein ligand-1 (PSGL-1) and other vascular ligands. Low forces decrease L-selectin-PSGL-1 off-rates (catch bonds), whereas higher forces increase off-rates (slip bonds). We determined that a force-dependent decrease in off-rates dictated flow-enhanced rolling of L-selectin-bearing microspheres or neutrophils on PSGL-1. Catch bonds enabled increasing force to convert short-lived tethers into longer-lived tethers, which decreased rolling velocities and increased the regularity of rolling steps as shear rose from the threshold to an optimal value. As shear increased above the optimum, transitions to slip bonds shortened tether lifetimes, which increased rolling velocities and decreased rolling regularity. Thus, force-dependent alterations of bond lifetimes govern L-selectin-dependent cell adhesion below and above the shear optimum. These findings establish the first biological function for catch bonds as a mechanism for flow-enhanced cell adhesion.

Antibodies, Monoclonal↗

PubMed2004

Quantifying the effects of molecular orientation and length on two-dimensional receptor-ligand binding kinetics.

Surface presentation of adhesion receptors influences cell adhesion, although the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the molecular orientation and length of adhesion receptors on the cell membrane affected two-dimensional kinetic rates of interactions with surface ligands. Interactions of P-selectin, E-selectin, and CD16A with their respective ligands or antibody were used to demonstrate such effects. Randomizing the orientation of the adhesion receptor or lowering its ligand- and antibody-binding domain above the cell membrane lowered two-dimensional affinities of the molecular interactions by reducing the forward rates but not the reverse rates. In contrast, the soluble antibody bound with similar three-dimensional affinities to cell-bound P-selectin constructs regardless of their orientation and length. These results demonstrate that the orientation and length of an adhesion receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding.

Animals↗

PubMed2004

Mechanical switching and coupling between two dissociation pathways in a P-selectin adhesion bond.

Many biomolecular bonds exhibit a mechanical strength that increases in proportion to the logarithm of the rate of force application. Consistent with exponential decrease in bond lifetime under rising force, this kinetically limited failure reflects dissociation along a single thermodynamic pathway impeded by a sharp free energy barrier. Using a sensitive force probe to test the leukocyte adhesion bond P-selectin glycoprotein ligand 1 (PSGL-1)-P-selectin, we observed a linear increase of bond strength with each 10-fold increase in the rate of force application from 300 to 30,000 pN/sec, implying a single pathway for failure. However, the strength and lifetime of PSGL-1-P-selectin bonds dropped anomalously when loaded below 300 pN/sec, demonstrating unexpectedly faster dissociation and a possible second pathway for failure. Remarkably, if first loaded by a "jump" in force to 20-30 pN, the bonds became strong when subjected to a force ramp as slow as 30 pN/sec and exhibited the same single-pathway kinetics under all force rates. Applied in this way, a new "jump/ramp" mode of force spectroscopy was used to show that the PSGL-1-P-selectin bond behaves as a mechanochemical switch where force history selects between two dissociation pathways with markedly different properties. Furthermore, replacing PSGL-1 by variants of its 19-aa N terminus and by the crucial tetrasaccharide sialyl LewisX produces dramatic changes in the failure kinetics, suggesting a structural basis for the two pathways. The two-pathway switch seems to provide a mechanism for the "catch bond" response observed recently with PSGL-1-P-selectin bonds subjected to small-constant forces.

Gangliosides↗

PubMed2004

Expression of proteasome subunits low molecular mass polypeptide (LMP) 2 and LMP7 in the endometrium and placenta of rhesus monkey (Macaca mulatta) during early pregnancy.

Previous studies have demonstrated that the ubiquitin-proteasome pathway plays an important role in embryo implantation. Low molecular mass polypeptide (LMP) 2 and LMP7 are the two subunits of 20S proteasome, which are critical for proteasome activity. To further elucidate the roles of LMP2 and LMP7 in embryo implantation during early pregnancy, we cloned partial sequences of the LMP2 and LMP7 genes and studied the spatiotemporal expression of LMP2 and LMP7 in rhesus monkey (Macaca mulatta) uteri on Days 12, 18, and 26 of pregnancy. The results showed that the 349-base pair (bp) LMP2 fragment and the 340-bp LMP7 fragment were 97% and 99% identical, respectively, to those of human homologues. From the statistical results of reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry, we found that the expression levels of LMP2 and LMP7 significantly increased with the elongation of pregnancy. The LMP2 and LMP7 mRNAs were mainly expressed in the luminal and glandular epithelium on Day 12 of pregnancy. On Days 18 and 26 of pregnancy, strong signals of LMP2 and LMP7 mRNAs were detected in the placental villi, trophoblastic column, and arterial endothelial cells close to the implantation site, and moderate expressions were found in the trophoblastic shell and glandular epithelium. The LMP2 and LMP7 mRNAs were extensively distributed in the stroma on Day 26 of pregnancy. The expression patterns of LMP2 and LMP7 proteins were similar to those of their transcripts, but weak immunostaining of LMP2 and LMP7 proteins was detected in stroma at all stages of pregnancy. These results suggest that LMP2 and LMP7 may be involved in some key processes of trophoblastic invasion, angiogenesis, degradation of the extracellular matrix, immune tolerance, and glandular secretion.

Animals↗

PubMed2004

Cinchona alkaloid-lewis acid catalyst systems for enantioselective ketene-aldehyde cycloadditions.

Asymmetric cinchona alkaloid-catalyzed acid chloride-aldehyde cyclocondensation (AAC) reactions afford enantioenriched 4-substituted and 3,4-disubstituted beta-lactones with near perfect absolute and relative stereocontrol. These reactions are characterized by the operational simplicity derived from using commercially available or easily obtained (one-step) reaction catalysts and in situ ketene generation from acid chlorides. The range of aldehyde substrates that serve as effective AAC substrates include sterically hindered aldehydes such as cyclohexanecarboxaldehyde and pivaldehyde.

Acids↗