Impaired function of skeletal stem cells derived from growth plates in ovariectomized mice.

INTRODUCTION: Mouse skeletal stem cells (mSSCs, CD45-Ter119-Tie2-CD51+Thy-6C3-CD105-CD200+population) are identified in growth plates (GP) and play important roles in bone regeneration. However, the role of mSSCs in osteoporosis remains unclear. MATERIALS AND METHODS: The GP were stained by HE staining, and the mSSC lineage was analyzed by flow cytometry at postnatal of 14 days and 30 days in wild-type mice. The mice (8 weeks) were either sham operated or ovariectomy (OVX) and then sacrificed at 2, 4 and 8 w. The GP were stained by Movat staining, and mSSC lineage was analyzed. Then, mSSCs were sorted by fluorescence-activated cell sorting (FACS); the clonal ability, chondrogenic differentiation and osteogenic differentiation were evaluated, and the changed genes were analyzed by RNA-seq. RESULTS: The percentage of mSSCs were decreased with the narrow GP. Heights of GP were decreased significantly in 8w-ovx mice compared with 8w-sham mice. We found the percentage of mSSCs were decreased in mice at 2w after ovx, but the cell numbers were not changed. Further, the percentage and cell numbers of mSSCs were not changed at 4w and 8w after ovx. Importantly, the clonal ability, chondrogenic differentiation and osteogenic differentiation of mSSCs were impaired at 8w after ovx. We found 114 genes were down-regulated in mSSCs, including skeletal developmental genes such as Col10a1, Col2a1, Mef2c, Sparc, Matn1, Scube2 and Dlx5. On the contrary, 526 genes were up-regulated, including pro-inflammatory genes such as Csf1, Nfkbla, Nfatc2, Nfkb1 and Nfkb2. CONCLUSION: Function of mSSCs was impaired by up-regulating pro-inflammatory genes in ovx-induced osteoporosis.

Early Signal of Emerging Nuclear Collectivity in Neutron-Rich

Radioactive ^{129}Sb, which can be treated as a proton plus semimagic ^{128}Sn core within the particle-core coupling scheme, was studied by Coulomb excitation. Reduced electric quadrupole transition probabilities, B(E2), for the 2^{+}⊗πg_{7/2} multiplet members and candidate πd_{5/2} state were measured. The results indicate that the total electric quadrupole strength of ^{129}Sb is a factor of 1.39(11) larger than the ^{128}Sn core, which is in stark contrast to the expectations of the empirically successful particle-core coupling scheme. Shell-model calculations performed with two different sets of nucleon-nucleon interactions suggest that this enhanced collectivity is due to constructive quadrupole coherence in the wave functions stemming from the proton-neutron residual interactions, where adding one nucleon to a core near a double-shell closure can have a pronounced effect. The enhanced electric quadrupole strength is an early signal of the emerging nuclear collectivity that becomes dominant away from the shell closure.

The magic nature of (132)Sn explored through the single-particle states of (133)Sn.

Atomic nuclei have a shell structure in which nuclei with 'magic numbers' of neutrons and protons are analogous to the noble gases in atomic physics. Only ten nuclei with the standard magic numbers of both neutrons and protons have so far been observed. The nuclear shell model is founded on the precept that neutrons and protons can move as independent particles in orbitals with discrete quantum numbers, subject to a mean field generated by all the other nucleons. Knowledge of the properties of single-particle states outside nuclear shell closures in exotic nuclei is important for a fundamental understanding of nuclear structure and nucleosynthesis (for example the r-process, which is responsible for the production of about half of the heavy elements). However, as a result of their short lifetimes, there is a paucity of knowledge about the nature of single-particle states outside exotic doubly magic nuclei. Here we measure the single-particle character of the levels in (133)Sn that lie outside the double shell closure present at the short-lived nucleus (132)Sn. We use an inverse kinematics technique that involves the transfer of a single nucleon to the nucleus. The purity of the measured single-particle states clearly illustrates the magic nature of (132)Sn.

[Relationship between microsatellite instability and hepatocyte growth factor expression and their prognostic significance in colorectal cancer].

OBJECTIVE: To investigate the expression of hepatocyte growth factor (HGF) and its relationship with microsatellite instability (MSI) and their influence on survival in patients with colorectal cancer. METHODS: Immunohistochemistry (IHC) was used to detect the expression of HGF and MSI in 98 specimens of colorectal cancer. Tumors lacking protein expression of any of the four mismatch repair genes (MLH1, PMS2, MSH2 or MSH6) were labelled as MSI, and the rest were considered as microsatellite stable (MSS). The associations between expression and clinicopathological factors were assessed using Chi-square tests. Kaplan-Meier curves, log-rank test, and Cox regression were used to analyze the association between biomarker expressions and overall survival. RESULTS: The incidence rate of MSI in 98 colorectal specimens was 32.7%, and was statistically significantly correlated with the location of tumor and differentiation degree (P<0.05). The HGF-expression rate was 71.4%. The patients with an MSI tumor had a significantly higher HGF expression, compared with the patients with an MSS tumor (P=0.048). The 5-year survival rate of MSI group and MSS group were 39.8% and 58.7%, respectively (P=0.009). The 5-year survival rate of HGF-positive group and HGF-negative group were 46.2% and 67.9% (P=0.035). The multivariate analysis showed that lymphocytic infiltration, TMN stage, MSI and HGF are independent prognostic factors in colorectal cancer (P<0.05 for all). CONCLUSIONS: HGF is highly expressed in colorectal cancer patients with microsatellite instability. Both microsatellite instability and HGF are independent factors affecting the prognosis in patient with colorectal cancer.

Constraint of the astrophysical

The Galactic 1.809-MeV γ-ray signature from the ß decay of ^{26g}Al is a dominant target of γ-ray astronomy, of which a significant component is understood to originate from massive stars. The ^{26g}Al(p,γ)^{27}Si reaction is a major destruction pathway for ^{26g}Al at stellar temperatures, but the reaction rate is poorly constrained due to uncertainties in the strengths of low-lying resonances in ^{27}Si. The ^{26g}Al(d,p)^{27}Al reaction has been employed in inverse kinematics to determine the spectroscopic factors, and hence resonance strengths, of proton resonances in ^{27}Si via mirror symmetry. The strength of the 127-keV resonance is found to be a factor of 4 higher than the previously adopted upper limit, and the upper limit for the 68-keV resonance has been reduced by an order of magnitude, considerably constraining the ^{26g}Al destruction rate at stellar temperatures.

Seasonal variation and source apportionment of organic tracers in PM10 in Chengdu, China.

Organic compound tracers including n-alkanes, triterpane, sterane, polycyclic aromatic hydrocarbons (PAHs) and dicarboxylic acids of airborne particulate matter (PM10) were characterized for samples collected at five sites from July 2010 to March 2011 using GC/MS. Spatial and temporal variations of these organic tracers in PM10 were studied, and their sources were then identified respectively. Average daily concentrations of PM10 varied in different seasons with the trend of PM10 in winter (0.133 mg/m(3)) > autumn (0.120 mg/m(3)) > spring (0.103 mg/m(3)) > summer (0.098 mg/m(3)). Daily concentrations of n-alkanes (C11-C36) ranged from 12.11 to 163.58 ng/m(3) with a mean of 61.99 ng/m(3). The C max and CPI index of n-alkanes indicated that vehicle emissions were the major source in winter, while the contributions of high plant wax emissions became significant in other seasons. It was discovered that the main sources of triterpenoid and steranes were gasoline and diesel engine emissions. Concentrations of ∑15PAHs in PM10 also varied (12.25-58.56 ng/m(3)) in different seasons, and chrysene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(ghi) perylene and fluoranthene were the dominant components. In the four seasons, the concentration of ∑15PAHs was relatively higher at the northern site because of traffic congestion. The main source of airborne PAHs was traffic emissions and coal combustion. Average daily concentrations of dicarboxylic acids (C4-C10) in PM10 ranged from 12.11 to 163.58 ng/m(3), of which azeleic acid was the major compound (0.49-52.04 ng/m(3), average 14.93 ng/m(3)), followed by succinic acid (0.56-19.08 ng/m(3), average 6.84 ng/m(3)). The ratio of C6/C9 showed that the major source in winter was biological, while the contributions of emissions from anthropogenic activities were much higher in summer.

Double-magic nature of 132Sn and 208Pb through lifetime and cross-section measurements.

Single-neutron states in (133)Sn and (209)Pb, which are analogous to single-electron states outside of closed atomic shells in alkali metals, were populated by the ((9)Be, (8)Be) one-neutron transfer reaction in inverse kinematics using particle-γ coincidence spectroscopy. In addition, the s(1/2) single-neutron hole-state candidate in (131)Sn was populated by ((9)Be, (10)Be). Doubly closed-shell (132)Sn (radioactive) and (208)Pb (stable) beams were used at sub-Coulomb barrier energies of 3 MeV per nucleon. Level energies, γ-ray transitions, absolute cross sections, spectroscopic factors, asymptotic normalization coefficients, and excited-state lifetimes are reported and compared with shell-model expectations. The results include a new transition and precise level energy for the 3p(1/2) candidate in (133)Sn, new absolute cross sections for the 1h(9/2) candidate in (133)Sn and 3s(1/2) candidate in (131)Sn, and new lifetimes for excited states in (133)Sn and (209)Pb. This is the first report on excited-state lifetimes of (133)Sn, which allow for a unique test of the nuclear shell model and (132)Sn double-shell closure.

Neutron single particle structure in 131Sn and direct neutron capture cross sections.

Recent calculations suggest that the rate of neutron capture by (130)Sn has a significant impact on late-time nucleosynthesis in the r process. Direct capture into low-lying bound states is expected to be significant in neutron capture near the N=82 closed shell, so r-process reaction rates may be strongly impacted by the properties of neutron single particle states in this region. In order to investigate these properties, the (d,p) reaction has been studied in inverse kinematics using a 630 MeV beam of (130)Sn (4.8 MeV/u) and a (CD(2))(n) target. An array of Si strip detectors, including the Silicon Detector Array and an early implementation of the Oak Ridge Rutgers University Barrel Array, was used to detect reaction products. Results for the (130)Sn(d, p)(131)Sn reaction are found to be very similar to those from the previously reported (132)Sn(d, p)(133)Sn reaction. Direct-semidirect (n,γ) cross section calculations, based for the first time on experimental data, are presented. The uncertainties in these cross sections are thus reduced by orders of magnitude from previous estimates.

Halo nucleus 11Be: a spectroscopic study via neutron transfer.

The best examples of halo nuclei, exotic systems with a diffuse nuclear cloud surrounding a tightly bound core, are found in the light, neutron-rich region, where the halo neutrons experience only weak binding and a weak, or no, potential barrier. Modern direct-reaction measurement techniques provide powerful probes of the structure of exotic nuclei. Despite more than four decades of these studies on the benchmark one-neutron halo nucleus 11Be, the spectroscopic factors for the two bound states remain poorly constrained. In the present work, the 10Be(d,​p) reaction has been used in inverse kinematics at four beam energies to study the structure of 11Be. The spectroscopic factors extracted using the adiabatic model were found to be consistent across the four measurements and were largely insensitive to the optical potential used. The extracted spectroscopic factor for a neutron in an nℓj=2s(1/2) state coupled to the ground state of 10Be is 0.71(5). For the first excited state at 0.32 MeV, a spectroscopic factor of 0.62(4) is found for the halo neutron in a 1p(1/2) state.

Identification of Dental Stem Cells Similar to Skeletal Stem Cells.

Stem and progenitor cells play important roles in the development and maintenance of teeth and bone. Surface markers expressed in bone marrow-derived mesenchymal stem cells are also expressed in dental tissue-derived stem cells. Mouse skeletal stem cells (mSSCs, CD45-Ter119-Tie2-CD51+Thy-6C3-CD105-CD200+) and human skeletal stem cells (hSSCs, CD45-CD235a-TIE2-CD31-CD146-PDPN+CD73+CD164+) have been identified in bone and shown to play important roles in skeletal development and regeneration. However, it is unclear whether dental tissues also harbor mSSC or hSSC populations. Here, we employed rainbow tracers and found that clonal expansion occurred in mouse dental tissues similar to that in bone. We sorted the mSSC population from mouse periodontal ligament (mPDL) tissue and mouse dental pulp (mDP) tissue in the lower incisors by fluorescence-activated cell sorting (FACS). In addition, we demonstrated that mPDL-derived skeletal stem cells (mPDL-SSCs) and mDP-derived skeletal stem cells (mDP-SSCs) have similar clonogenic capacity, as well as cementogenic and odontogenic potential, but not adipogenic potential, similar to the characteristics of mSSCs. Moreover, we found that the dental tissue-derived mSSC population plays an important role in repairing clipped incisors. Importantly, we sorted the hSSC population from human periodontal ligament (hPDL) and human dental pulp (hDP) tissue in molars and identified its stem cell characteristics. Finally, hPDL-like and hDP-like structures were generated after transplanting hPDL-SSCs and hDP-SSCs beneath the renal capsules. In conclusion, we demonstrated that mouse and human PDL and DP tissues harbor dental stem cells similar to mSSCs and hSSCs, respectively, providing a precise stem cell population for the exploration of dental diseases.

Near-barrier fusion of Sn + Ni and Te + Ni systems: examining the correlation between nucleon transfer and fusion enhancement.

The fusion excitation functions for radioactive (132)Sn + (58)Ni and stable (130)Te + (58,64)Ni were measured at energies near the Coulomb barrier. The coupling of transfer channels in heavy-ion fusion was examined through a comparison of Sn + Ni and Te + Ni systems, which have large variations in the number of positive Q-value nucleon transfer channels. In contrast with previous experimental comparisons, where increased sub-barrier fusion cross sections were observed in systems with positive Q-value neutron transfer channels, the reduced excitation functions were equivalent for the different Sn + Ni and Te + Ni systems. The present results suggest a dramatically different influence of positive Q-value transfer channels on the fusion process for the Sn + Ni and Te + Ni systems.

[Management of major complications in surgical treatment of mandibular osteoradionecrosis by using vascularized free flaps].

Objective: To retrospectively analyze of the prevention and management of major complications in surgical treatment of osteoradionecrosis (ORN) of the mandible by using vascularized free flaps and to provide a reference for improving clinical treatments. Methods: All cases diagnosed as mandibular ORN and received surgical treatment in the Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University from August 2014 to March 2019 were included. The patients' clinical data, mainly including general information, primary tumor, interval time between radiotherapy and osteoradionecrosis, surgical methods and postoperative complications, were collected. The data of surgical methods and postoperative complications of these patients were compared with the similar data of patients with malignant tumor surgically treated by using vascularized free flaps during the same period. Results: The postoperative follow-up data of 104 patients with mandibular ORN, who underwent surgery in our hospital, were collected. In the control group, surgeries of vascularized free flap transfer were performed in 430 patients with malignant tumor. Among the 34 ORN cases (34/104, 32.7%) of segmental resection with vascularized free flap transfer, there were 13 cases (13/34, 38.2%) of postoperative local infection in maxillofacial area, 6 cases (17.6%) of pulmonary infection, 2 cases (5.9%) of venous thrombosis, 3 cases (8.8%) of anastomotic artery rupture and 5 cases (14.7%) of vascular crisis. One case died of depression and misanthropy. Meanwhile, among 430 patients with malignant tumor who underwent surgeries of vascular free flap reconstruction, 25 cases (5.8%) had postoperative local infection and 29 cases (6.7%) had pulmonary infection. Vascular crisis occurred in 12 cases (2.8%) and anastomotic artery rupture in 1 case (0.2%). No venous thrombosis and misanthropy occurred. The incidence of postoperative complications in mandibular ORN was much higher than that of vascularized free flap transfer surgeries in malignant tumor cases (P<0.05). Conclusions: Compared with patients with malignant tumors undergoing vascularized free flap transfer surgeries, patients with mandibular ORN undergoing same surgeries are more likely to have postoperative complications. This study may help clinicians to fully understand the local, general and psychological conditions during the perioperative period of ORN patients.

[A survey of knowledge regarding pediatric clinical trial among children at 8-18 years of age].

Objective: To investigate the knowledge regarding clinical research among children at 8-18 years of age. The survey results will form the basis for developing public education program for this population. Methods: The survey was conducted among children at 8-18 years of age using WeChat and spot investigation between January 2016 and January 2017. According to different developmental stages, the survey population was divided into four groups: age 8-10, 11-13, 14-15 and 16-18 years. The level of knowledge regarding clinical research was analyzed. Results: Totally 1 329 questionnaires were issued and 1 233 effective questionnaires were returned with a recovery rate of 92.8%. The overall awareness rate regarding clinical research was 32.8% (405/1 233) . It revealed that 282 (22.9%) individuals thought that clinical research was to treat people like experimental rats. When asked "who have the final decision on research participation", the percentages of those who chose oneself, parents or guardian and doctor were 44.6% (550/1 233), 74.2% (915/1 233) and 36.8% (454/1 233) respectively. When asked "If you want to participate a study, but your parents or guardian do not agree, what would you do?", 33.9% (418/1 233) of individuals will "give up". As to "If you do not want to participate a study, but your parents or guardian think you should, what would you do?", 51.3% (632/1 233) chose "listen to parents" and 28.8% (355/1 233) chose "refuse the suggestions of parents or guardian". As to "what are your greatest concerns of participating an investigation?" , 68.1% (840/1 233) chose "worry about added pain or discomfort". but 58.0% (715/1 233) thought if "doctors and nurses take good care of me" their "concerns will reduce" or "feel better to participate in the research?". 55.6% (686/1 233) and 49.3% (608/1 233) individuals responded that they will "participate in an research?" when they "know that other people also participate the research" and when they "know the details regarding what will happen after the enrollment". Conclusions: The knowledge level of clinical research among children aged 8-18 years were not high. It is very necessary to promote the public education of clinical research for this population and also very necessary to address their concern regarding the research.

Clinical features, polysomnography and outcome in patients with hypnic headache.

Hypnic headache is rarely reported in Asians, and the proposed International Classification of Headache Disorders (ICHD)-2 criteria have never been field-tested. We studied 17 consecutive Taiwanese patients (M/F: 9/8, mean age at onset 69.6 years) with hypnic headache from a headache clinic. Fifteen patients (88%) reported >15 headache attack days per month. Polysomnography studies done on 11 patients recorded 12 attacks in seven patients: two during rapid eye movement (REM) sleep, three during non-REM sleep and two having both. Five of the seven patients reported their headache profile during polysomnography studies. The clinical course was mostly episodic without recurrence (n = 9, 53%), followed by relapsing/remitting (n = 5) and chronic (n = 3). The ICHD-2 criteria were not fulfilled in 35% (6/17) patients based on patient recall or 60% (3/5) patients based on direct questioning during polysomnography studies. The major reason was the presence of pulsatile rather than dull headache in our patients. Unlike previous studies, our study showed hypnic headache occurred equally in both REM and non-REM sleep, and most patients ran an episodic course.

Improved gene expression using low molecular weight peptides produced from protamine sulfate.

DNA condensation plays a key role in non-viral gene delivery by affecting gene transfection, nuclear targeting, and eventual gene expression efficiency. Theoretically, a DNA condenser with the appropriate DNA condensation ability but without affecting DNA dissociation from DNA condensates inside the cytoplasm should be a perfect carrier for gene delivery. Protamine is a natural DNA condensation agent and has been widely used in gene delivery. In this work, protamine was selectively digested enzymatically to produce low molecular weight protamine fragments (LMWPs) of various lengths and amino acid compositions. The DNA condensation ability and gene transfection efficiency of these LMWP peptides were tested. Compared to protamine, all the LMWP peptides showed lower DNA binding strength. However, some LMWP peptides demonstrated excellent DNA condensation ability and could form very compact DNA condensates with small particle size (approximately 100 nm). More interestingly, LMWP peptide-mediated in vitro gene delivery showed prolonged (up to 12 days) gene expression. Results from this study suggest that designing DNA condensers with appropriate and tunable DNA binding strengths and condensation abilities would be an effective means to improve gene expression and thus gene therapy efficiency. Since LMWP peptides have low immunogenicity, they would be safer than protamine for use in gene therapies.

Protective effect of linoleic acid on IFN gamma-induced cellular injury in primary culture hepatocytes.

We have previously demonstrated that treatment of hepatocytes with IFN gamma results a series of cellular injury processes, including DNA synthesis arrest, membrane breakage and apoptosis. In the present work, we show that IFN gamma suppresses cellular respiration and protein synthesis in hepatocytes, and that cellular respiration suppression is an early event in the IFN gamma-induced cellular injuries. Polyunsaturated fatty acids (PUFAs) increased cellular respiration of hepatocytes, but only linoleic acid showed some protective effect against IFN gamma-induced cellular respiration suppression. Linoleic acid also reduced other IFN gamma-mediated cellular injuries, including membrane breakage and protein synthesis inhibition. Like linoleic acid, fetal bovine serum also inhibited IFN gamma-induced cellular damage. Increased NAD levels were found in both IFN gamma-treated and non-treated hepatocytes following the addition of PUFAs, but clofibrate, a peroxisome proliferator, bromophenacyl bromide (BPB), an inhibitor of phospholipase, nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, and arachidonic acid, a metabolite of linoleic acid, did not inhibit IFN gamma-induced cellular injury. In addition, the combination of linoleic acid and IFN gamma induced nitric oxide (NO) synthesis in hepatocytes. These results suggest that fatty acid may play an important role in liver homeostasis during chronic inflammatory states and sepsis.

ATTEMPTS: a heparin/protamine-based prodrug approach for delivery of thrombolytic drugs.

The aim of this study is to develop a heparin/protamine-based prodrug system for the controlled delivery of enzyme such as tissue-type plasminogen activator (tPA). This approach, termed antibody targeted, triggered, electrically modified prodrug-type strategy (ATTEMPTS), would permit antibody-directed administration of inactive tPA, and allow a subsequent triggered release of the active tPA at the target site. Cation-modified tPA (mtPA) was attached to a heparin--antifibrin complex via ionic interaction. The active tPA can be subsequently released by the addition of protamine, a competitive heparin inhibitor. Anti-fibrin IgG was conjugated to heparin via an end-point attachment to form the heparin--antifibrin--complex which provides the targeting efficiency of the final heparin--mtPA complex. Cation-modification was performed either by chemical conjugation by linking (Arg)(7)Cys to tPA with N-succinimidy-3-(2-pyridyldithio) propionate or by recombinant DNA method. Results show that the chemical modification process did not significantly alter specific activity of tPA with regard to plasminogen activation, fibrin-binding ability, and response toward fibrinogen. Expressed modified tPA (EmtPA) produced by recombinant DNA methods retained the same catalytic activity of the parent tPA, as well as a dynamic catalytic behavior depending upon the presence of heparin and protamine. Both types of modified tPA, especially the mtPA demonstrated a significantly higher affinity toward heparin or heparin--antifibrin complex than native tPA. In addition, the complexes of mtPA--heparin did not yield any intrinsic clot lysis activity owing to the blockage of the active site of tPA by attached heparin. On the other hand, heparin-induced inhibition of both mtPA and EmtPA activity was reversed by adding protamine, as confirmed by chromogenic and in vitro clot lysis assays. These results suggested that a heparin/protamine-based tPA delivery system may be a useful tool to improve current thrombolytic therapeutic status, by both precisely regulating the release of active tPA and aborting the associated bleeding risk. Alternatively, this ATTEMPTS approach could also be used to deliver enzyme drugs while diminishing their associated toxic effects.

ATTEMPTS: a heparin/protamine-based delivery system for enzyme drugs.

A prodrug delivery system termed "Antibody Targeted, Triggered, Electrically Modified Prodrug-Type Strategy (ATTEMPTS)" has been developed to permit the antibody-directed administration of inactive enzyme drug including tissue-type plasminogen activator (tPA), and allow a subsequent triggered release of the active tPA at the target site. Cation-modified tPA (mtPA) was attached to a heparin-antifibrin complex via ionic interaction, and the active tPA can subsequently be released by the addition of protamine, a competitive heparin inhibitor. Anti-fibrin IgG was conjugated to heparin via an end-point attachment to form the heparin-antifibrin complex which provides the targeting efficiency of the final heparin/mtPA complex. Cation modification was performed by either chemical conjugation by linking (Arg)7Cys to tPA with N-succinimidy-3-(2-pyridyldithio) propionate or by recombinant DNA methods. Results show that the modification process did not significantly alter the specific activity of tPA with regard to plasminogen activation, fibrin-binding ability, and response toward fibrinogen. The complexes of both modified tPA-heparin did not yield any intrinsic catalytic activity owing to the blockage of the active site of tPA by the attached heparin. On the other hand, heparin-induced inhibition of modified tPA activity was reversed by adding protamine, which is similar to that of a prodrug delivery system. These results suggest that heparin/protamine-based enzyme delivery systems may be a useful tool to improve current enzyme therapeutic status, as well as thrombolytic therapy, by both regulating the release of active enzyme and aborting the associated systemic toxic effect. Currently, modification of enzyme drugs has been optimized by recombinant DNA technology assisted by computer simulation. In addition, the original strategy has been revised to obtain enhanced therapeutic efficacy.

The potential mechanism for the effect of heparin on tissue plasminogen activator-mediated plasminogen activation.

The effects and possible role of heparin on tissue plasminogen activator-mediated plasminogen activation was thoroughly investigated. Direct analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that heparin increased the conversion of plasminogen to plasmin. Experiments by fluorescence quenching suggested that the stimulation of tissue plasminogen activator activity probably was due to a direct binding of heparin to tissue plasminogen activator, causing a conformational change of tissue plasminogen activator and rendering it more accessible to plasminogen interaction. The absence of additive stimulation effects on tissue plasminogen activator-mediated plasminogen activation when both heparin and fibrinogen were present also implied that both compounds interacted with tissue plasminogen activator via the same domain; it appeared to be most likely via the kringle-2 domain in tissue plasminogen activator based on studies using epsilon-aminocaproic acid as an inhibitor. Unlike heparin-induced stimulation of antithrombin-thrombin interaction, the heparin-induced stimulation of tissue plasminogen activator did not seem to follow a template model. Only in the presence of a high plasminogen or a low tissue plasminogen activator concentration, massive stimulation of tissue plasminogen activator activity was observed via a pseudotemplate model. The results suggest that precautions concerning high heparin dose should be given during its conjunctive clinical use with tissue plasminogen activator in thrombolytic therapy to reduce the risk of hemorrhage.