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K K Wang

Publications and source records attributed to K K Wang.

At least 73 records · Page 4Linked to original sources
PubMed1998

Regional calpain and caspase-3 proteolysis of alpha-spectrin after traumatic brain injury.

Activity of calpains and caspase-3 inferred from proteolysis of the cytoskeletal protein alpha-spectrin into signature spectrin breakdown products (SBDPs) was used to provide the first systematic and simultaneous comparison of changes in activity of these two families of cysteine proteases after traumatic brain injury (TBI) in rats. Distinct regional and temporal patterns of calpain/caspase-3 processing of alpha-spectrin were observed in brain regions ipsilateral to the site of injury after TBI, including large increases of 145 kDa calpain-mediated SBDP in cortex (up to 30-fold), and enduring increases (up to 2 weeks) of 145 kDa SBDP in hippocampus and thalamus. By contrast, 120 kDa caspase-3-mediated SBDP was absent in cortex and showed up to a 2-fold increase in hippocampus and striatum at early (hours) after TBI. Future studies will clarify the pathological significance of large regional differences in activation of calpain and caspase-3 proteases after TBI.

Animals↗
PubMed1998

Temporal relationships between de novo protein synthesis, calpain and caspase 3-like protease activation, and DNA fragmentation during apoptosis in septo-hippocampal cultures.

Caspase 3-like proteases are key executioners in mammalian apoptosis, and the calpain family of cysteine proteases has also been implicated as an effector of the apoptotic cascade. However, the influence of upstream events on calpain/caspase activation and the role of calpain/caspase activation on subsequent downstream events are poorly understood. This investigation examined the temporal profile of apoptosis-related events after staurosporine-induced apoptosis in mixed glial-neuronal septo-hippocampal cell cultures. Following 3 hr exposure to staurosporine (0.5 microM), calpain and caspase 3-like proteases processed alpha-spectrin to their signature proteolytic fragments prior to endonuclease-mediated DNA fragmentation (not evident until 6 hr), indicating that endonuclease activation is downstream from calpain/caspase activation. Cycloheximide, a general protein synthesis inhibitor, completely prevented processing of alpha-spectrin by calpains and caspase 3-like proteases, DNA fragmentation and cell death, indicating that de novo protein synthesis is an upstream event necessary for activation of calpains and caspase 3-like proteases. Calpain inhibitor II and the pan-caspase inhibitor Z-D-DCB each inhibited their respective protease-specific processing of alpha-spectrin and attenuated endonuclease DNA fragmentation and cell death. Thus, activation of calpains and caspase 3-like proteases is an early event in staurosporine-induced apoptosis, and synthesis of, as yet, unknown protein(s) is necessary for their activation.

Animals↗
PubMed1998

pH dependency of mu-calpain and m-calpain activity assayed by casein zymography following traumatic brain injury in the rat.

Studies employing casein zymographic assays analyzed the effects of varying pH (from pH 6.8 to pH 8.0) on changes in mu-calpain and m-calpain activity in naive, sham-injured and injured rat cortex 3 h following unilateral cortical impact injury. Mu-calpain activity following cortical impact injury was enhanced between pH values of 7.2 and 7.8, with pH 7.5 being optimal. m-Calpain activity was readily detected only between pH values of 7.2 and 7.4, with pH 7.3 producing the most prominent proteolytic activity. These observations suggest that strict control of pH is an important consideration in assessments of brain pH activation by casein zymography. Moreover, activation of different calpain isoforms, especially after traumatic brain injury, may be differentially influenced by smaller changes in physiological pH than previously recognized.

Animals↗
PubMed1998

Mono-L-aspartyl chlorin e6 (NPe6) and hematoporphyrin derivative (HpD) in photodynamic therapy administered to a human cholangiocarcinoma model.

BACKGROUND: Despite their relatively localized nature, the therapy for surgically unresectable cholangiocarcinomas has been largely unsuccessful. Photodynamic therapy is a promising technique for both curative and palliative treatment of this malignancy. The effectiveness of a potential new photosensitizer, mono-l-aspartyl chlorin e6 (NPe6), was compared with that of a traditional drug, hematoporphyrin derivative (HpD), in photodynamic therapy administered to a human cholangiocarcinoma model. METHODS: An established cholangiocarcinoma cell line was inoculated subcutaneously in the left back of male nude mice age 8 weeks. After a predetermined tumor size was reached, the mice were randomly assigned to either a control group or an experimental group. Experimental tumor-bearing mice received either HpD (5 mg/kg or 10 mg/kg) or NPe6 (2 mg/kg, 5 mg/kg, or 8 mg/kg) followed by photoradiation. HpD and NPe6 were administered intraperitoneally at 24 and 2 hours, respectively, prior to light exposure. Photoradiation was conducted using a xenon-mercury arc lamp with a 405-650 nm filter at a light flux of 80 J/cm2. Tumor response was assessed by serial tumor volume measurements. RESULTS: Control mice showed an estimated mean tumor volume doubling rate of 9.0 days. Triaxial tumor measurements correlated well with autopsy measurements (correlation coefficient = 0.9). Overall differences in tumor volume reduction were detected (P < 0.001) among the three groups: HpD, NPe6, and controls (photoradiation only, HpD only, or NPe6 only). The degree of tumor volume reduction was superior for dosages of NPe6 compared with all dosages of HpD (P < 0.05). Although a dose effect was detected (P < 0.05) for HpD and separately for NPe6, a consistent dose-response relationship was not observed for either. Inhibition of tumor regrowth was better for NPe6 compared with HpD. The depth of tissue injury was significantly increased (P < 0.05), by 67%, for 5-8 mg/kg of NPe6 compared with 5-10 mg/kg of HpD. The duration of cutaneous photosensitization was also decreased for NPe6 compared with HpD. CONCLUSION: Photodynamic therapy with HpD or NPe6 was effective inducing tumor regression in the cholangiocarcinoma model in this study. At the dosages studied, NPe6 appeared to induce greater tumor regression than HpD, with decreased tumor regrowth and duration of cutaneous photosensitization.

Animals↗
PubMed1998

Transcutaneous access to retrograde axonal flow.

Transcutaneous entry of fluorescent tracer and subsequent retrograde neuronal transport achieved by the use of dimethyl sulfoxide (DMSO) as a vehicle fluorescent dye dissolved in DMSO and applied topically to the hind limb of rats was found in corresponding dorsal root ganglia; aqueous absorption of tracer dye by neuronal tissue was not demonstrated. This example of transcutaneous access and retrograde transport may have implications as to the entry of various toxins, viruses, chemicals, and therapeutic agents to the nervous system.

Administration, Cutaneous↗
PubMed1998

Hyaluronic acid enhances peripheral nerve regeneration in vivo.

Hyaluronic acid has been shown to enhance peripheral nerve regeneration in vitro. It has been proposed that, during the fibrin matrix phase of regeneration, hyaluronic acid organizes the extracellular matrix into a hydrated open lattice, thereby facilitating migration of the regenerating axons. Hyaluronic acid solutions and saline control solutions were injected into a nerve guide spanning a transected gap in the sciatic nerve of Sprague-Dawley rats (five in each group). Nerve conduction velocities were measured at 4 weeks by electromyography (EMG) before sacrifice of the animals. These studies demonstrated increased conduction velocities in the hyaluronic acid group compared with control animals (P = 0.006). After the animals were sacrificed, regenerated axon cables were quantified histologically, and axon branching was delineated by retrograde tracer analysis. In addition, the hyaluronic acid group showed an increase in myelinated axon counts at 4 weeks (P= 0.03). An increase in retrograde flow was demonstrated in the hyaluronic acid groups compared with animals receiving saline solution.

Animals↗
PubMed1998

Evidence for activation of caspase-3-like protease in excitotoxin- and hypoxia/hypoglycemia-injured neurons.

Caspase activation has been shown to be a critical step in several models of neuronal apoptosis such as staurosporine treatment of human neuroblastoma SH-SY5Y cells and potassium deprivation of rat cerebellar granule neurons. One common event is the appearance of caspase-mediated 120-kDa nonerythroid alpha-spectrin breakdown product (SBDP120). Second, inhibitors of the caspase family are effective blockers of such neuronal death. In this study, we report the appearance of caspase-mediated SBDP120 in excitotoxin-challenged fetal rat cerebrocortical neurons [N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate] and rat cerebellar granule neurons (NMDA and kainate). A general caspase inhibitor, carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB), blocked the formation of SBDP120 under these conditions and attenuated the observed NMDA-induced lactate dehydrogenase (LDH) release in both cell types. Furthermore, hydrolytic activity toward a caspase-3-preferred synthetic peptide substrate, acetyl-DEVD-7-amido-4-methylcoumarin, was significantly elevated in NMDA-treated granule neurons. Lastly, oxygen-glucose deprivation (OGD)-challenged cerebrocortical cultures also showed the appearance of SBDP120. Again, Z-D-DCB blocked the SBDP120 formation as well as attenuated the LDH release from the OGD-challenged neurons. Taken together, the presence of caspase-specific SBDP120 and the neuroprotective effects of Z-D-DCB strongly suggest that caspase activation contributes at least in part to excitotoxin- and OGD-induced neuronal death.

Animals↗
PubMed1998

Subcellular localization and duration of mu-calpain and m-calpain activity after traumatic brain injury in the rat: a casein zymography study.

Casein zymographic assays were performed to identify changes in mu-calpain and m-calpain activity in naive, sham-injured, and injured rat cortex at 15 minutes, 3 hours, 6 hours, and 24 hours after unilateral cortical impact brain injury. Cortical samples ipsilateral and contralateral to the site of injury were separated into cytosolic and total membrane fractions. Marked increases in mu-calpain activity in cytosolic fractions in the ipsilateral cortex occurred as early as 15 minutes, became maximal at 6 hours, and decreased at 24 hours to levels observed at 15 minutes after injury. A similar temporal profile of cytosolic mu-calpain activity in the contralateral cortex was observed, although the increases in the contralateral cortex were substantially lower than those in the ipsilateral cortex. Differences were also noted between cytosolic and total membrane fractions. The detection of a shift in mu-calpain activity to the total membrane fraction first occurred at 3 hours after traumatic brain injury and became maximal at 24 hours after traumatic brain injury. This shift in mu-calpain activity between the two fractions could be due to the redistribution of mu-calpain from the cytosol to the membrane. m-Calpain activity was detected only in cytosolic fractions. m-Calpain activity in cytosolic fractions did not differ significantly between ipsilateral and contralateral cortices, and increased in both cortices from 15 minutes to 6 hours after injury. Relative magnitudes of m-calpain versus mu-calpain activity in cytosolic fractions differed at different time points after injury. These studies suggest that traumatic brain injury can activate both calpain isoforms and that calpain activity is not restricted to sites of focal contusion and cell death at the site of impact injury but may represent a more global response to injury.

Animals↗
PubMed1998

Skin tightening effects of the ultrapulse CO2 laser.

This study analyzed the skin tightening or contracture effect of the Ultrapulse carbon dioxide (CO2) laser on the skin of hairless guinea pigs by light and electron microscopic, histologic, and tensiometric evaluations. Two 2 X 2 cm squares of back skin were precision tattooed on each of the animals in the study (n = 12). One square served as the control, and the other square was used as experimental skin. The experimental skin was treated with three passes of the CO2 laser at 500 mJ and 5 W using a 3-mm collimated hand-piece. Skin specimens from three animals were analyzed at 1, 4, 8, and 12 weeks. After three passes, the length of the square was reduced by 27 percent, and the width was reduced by 40 percent. Over the next 12 weeks, as the animals grew, the dimensions of the control areas also increased. The laser-treated areas continued to maintain their contracted dimensions, however. By the 12th week, the laser-treated areas were 28.35 percent shorter in length and 15.5 percent shorter in width than the control areas. Histologic examination demonstrated a significantly higher content of collagen in the reticular layer, which was more compact than that of the normal skin. Electron microscopy revealed that the laser had induced shortening of the collagen fibers (7.45 percent; p = 0.026), which persisted beyond the 12th week. Laser treatment did not significantly alter the tensile strength of the skin, although, at the 8th week, the treated areas had a slightly higher tensile strength.

Animals↗
PubMed1998

Potential contribution of proteases to neuronal damage.

Calpain was first discovered 30 years ago. Two major isoforms were subsequently isolated and purified. The presence of an endogenous protein inhibitor, calpastatin, was later discovered. Calpain activity is tightly regulated by Ca(2+). At physiological levels of Ca(2+), the role of calpain remains poorly understood, but is believed to be involved in mitosis and muscle cell differentiation. Calpain has also been implicated in various membrane fusion events through remodeling of the cytoskeletal network. Calpain activation has been shown to be increased during normal aging and in muscular dystrophy, cataract, arthritis and Alzheimer's disease, and in acute traumas such as traumatic brain injury (TBI), spinal cord injury and cerebral and cardiac ischemia. Early work on calpain inhibitors was limited to protein inhibitors and other nonselective enzyme inhibitors. Peptidyl aldehydes such as leupeptin and antipain are also among the earliest reported calpain inactivators. Irreversible inhibitors such as the E64 family have also been studied, and peptidyl halomethanes and diazomethanes have long been used as protease inhibitors. A variety of calpain inhibitors are under development. From a therapeutic perspective, calpain inhibitors may have several advantages over other more conventional targets such as ion channel blockers and glumate antagonists, since calpain proteolysis represents a later component of a pathway mediating cell death initiated by excitotoxicity and elevated Ca(2+) levels. Although the potential clinical utility of calpain inhibitors seems well established, a number of important considerations remain to be addressed. The role of other proteolytic cascades contributing to neuronal cell damage following TBI must also be considered.

Journal Article↗
PubMed1997

A purine nucleoside phosphorylase (PNP) inhibitor induces apoptosis via caspase-3-like protease activity in MOLT-4 T cells.

Children with congenital homozygous deficiency of purine nucleoside phosphorylase (PNP) have abnormalities in purine metabolism that result in T-cell selective immune deficiency. The mechanism of action for cell death has been attributed to intracellular accumulation of dGTP, a potent inhibitor of ribonucleotide reductase and subsequently DNA synthesis, in thymocytes and T-cells but not B-cells. However, the mode of cell death has not been determined to be either necrosis or apoptosis. To examine the involvement of apoptosis in T-cells following PNP inhibition, MOLT-4 cells, a human T cell leukemia cell line, were co-treated with the PNP inhibitor, CI-1000 (2-amino 3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]-pyrimidin-4-one HCl), and 2'-deoxyguanosine (dGuo) which resulted in a concentration-dependent loss of cell viability (trypan blue) and inhibition of tritiated thymidine ([3H]-TdR) uptake. Staining of cells with the DNA dye Hoechst 33,258 showed nuclear morphology characteristic of apoptosis. Western blots (24 h lysates) were probed with antibodies against several proteins implicated in apoptosis. Anti-PARP revealed the presence of an 85 kD PARP breakdown product while, anti-alpha-spectrin revealed the accumulation a 120 kD breakdown product, both suggestive of CPP32 cleavage (caspase-3; an ICE-like cysteine protease). Western blots also detected the loss of the intact 32 kD caspase-3 isoform, a biochemical event associated with caspase-3 activation. Corresponding fluorometric activity assays detected a marked increase in caspase-3-like activity using the substrate Ac-DEVD-MCA. Lastly, a pan caspase inhibitor (Z-D-DCB) and 2'-deoxycytidine (dCyd), which is known to prevent dGTP accumulation following PNP inhibition, were able to prevent cell death and all indicators of caspase-3-like activity in MOLT-4 cells co-treated with dGuo and CI-1000. In summary, we provided several lines of evidence for the role of apoptosis and the contribution of caspase-3-like proteases in T-cell death following PNP inhibition.

Apoptosis↗
PubMed1997

Differential expression of a cDNA clone in human liver versus hepatic cancer--highly homologous to aryl-dialkyl-phosphatase.

We applied the technique of mRNA differential display to normal liver tissue and hepatoma cell line Hep3B. One of the isolated cDNA clones was expressed in human normal liver tissue but not in the human hepatocarcinoma cell line. Northern Blot analysis confirmed that high level of mRNA was expressed in human normal liver tissue but the level was decreased in non-cancerous liver tissue from hepatoma patients. Low level or no expression was observed in human hepatoma tissue. One of these transcripts was about 1.8 kb in length. Southern Blot analysis showed that it was a single copy gene. We obtained a full length cDNA clone of 2,395 bp by screening human liver 5'-stretch plus cDNA library. Nucleotide sequence indicated that this clone was highly homologous to aryl-dialkyl-phosphatase and possessed two polymorphic sites. Aryl-dialkyl-phosphatase which has a prominent role in the metabolism of several toxic, synthetic compounds, may be potentially related to human hepatocarcinoma susceptibility. The biological significance of its differential expression in normal versus malignant tissue is discussed.

Amino Acid Sequence↗
PubMed1997

Crystal structure of calcium bound domain VI of calpain at 1.9 A resolution and its role in enzyme assembly, regulation, and inhibitor binding.

The three dimensional structure of calcium-bound domain VI of porcine calpain has been determined to 1.9 A resolution. The crystal structure reveals five EF-hands, one more than previously suggested. There are two EF-hand pairs, one pair (EF1-EF2) displays an 'open' conformation and the other (EF3-EF4) a 'closed' conformation. Unusually, a calcium atom is found at the C-terminal end of the calcium binding loop of EF4. With two additional residues in the calcium binding loop, the fifth EF-hand (EF5) is in a 'closed' conformation. EF5 pairs up with the corresponding fifth EF-hand of a non-crystallographically related molecule. Considering the EF5's role in a homodimer formation of domain VI, we suggest a model for the assembly of heterodimeric calpain. The crystal structure of a Ca2+ bound domain VI-inhibitor (PD150606) complex has been refined to 2.1 A resolution. A possible mode for calpain inhibition is discussed.

Acrylates↗
PubMed1997

Characterization of CPP32-like protease activity following apoptotic challenge in SH-SY5Y neuroblastoma cells.

We characterized the activation of interleukin-1beta-converting enzyme (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti-poly(ADP-ribose) polymerase (anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32-like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH-SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH-SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.

Antibody Specificity↗
PubMed1997

Neuronal nitric oxide synthase and calmodulin-dependent protein kinase IIalpha undergo neurotoxin-induced proteolysis.

Calpain (calcium-activated neutral protease) has been implicated as playing a role of neuronal injury in cerebral ischemia and excitotoxicity. Here we report that, in addition to extreme excitotoxic conditions [N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate challenges], other neurotoxins such as maitotoxin, A23187, and okadaic acid also induce calpain activation, as detected by m-calpain autolytic fragmentation and nonerythroid alpha-spectrin breakdown. Under the same conditions, calmodulin-dependent protein kinase II-alpha (CaMPK-IIalpha) and neuronal nitric oxide synthase (nNOS) are both proteolytically cleaved by calpain. Such fragmentation can be reduced by calpain inhibitors (acetyl-Leu-Leu-Nle-CHO and PD151746). In vitro digestion of protein extract from cortical cultures with purified mu- and m-calpain produced fragmentation patterns for CaMPK-IIalpha and nNOS similar to those produced in situ. Also, several other calpain-sensitive calmodulin-binding proteins (plasma membrane calcium pump, microtubule-associated protein 2, and calcineurin A) and protein kinase C-alpha are also degraded in neurotoxin-treated cultures. Lastly, in a rat pup model of acute excitotoxicity, intrastriatal injection of NMDA resulted in breakdown of CaMPK-IIalpha and nNOS. The degradation of CaMPK-IIalpha, nNOS, and other endogenous calpain substrates may contribute to the neuronal injury associated with various neurotoxins.

Animals↗
PubMed1997

Thapsigargin induces apoptosis in SH-SY5Y neuroblastoma cells and cerebrocortical cultures.

Thapsigargin, a specific inhibitor of the endoplasmic reticular Ca(2+)-ATPase, has been used previously to mobilize calcium release from intracellular calcium stores. We now show that thapsigargin (1-10 microM) induces apoptosis in a neuroblastoma cell line (SH-SY5Y) and in fetal rat cerebrocortical cultures. Cell death measured by lactate dehydrogenase release was observed 24-48 hours after treatment with thapsigargin. In both cases, DNA extracts from thapsigargin treated cells showed laddering, typical of endonuclease-mediated internucleosomal cleavages. The presence of DNA fragments was also confirmed by an ELISA designed for detecting nucleosomes in apoptotic cells. Cycloheximide reduced the extent of DNA fragmentation and injury in thapsigargin-treated cells. Dantrolene, an inhibitor of calcium release from intracellular stores partially abolished the effect of thapsigargin, suggesting that the initial Ca2+ rise may be the signalling event in this apoptotic cell death pathway. We propose that thapsigargin-induced cell death in cultured neuronal cells may be a useful system to study the molecular and genetic events involved in apoptosis.

Animals↗