Regulation of vitamin D receptor and Genistein on bone metabolism in mouse osteoblasts and the molecular mechanism of osteoporosis.

The aim of this work was to study the mechanisms of vitamin D receptor (VDR) and Genistein (Gen) on the regulation of bone metabolism of phytoestrogens from cellular and epidemiological perspectives. MC3T3-E1 cells were treated with different concentrations of Gen, and the cell-proliferation rate was detected by an MTT colorimetric assay. The effect of the VDR receptor blocker ZK159222 on the Gen effect was then observed; after adding Gen to MC3T3-E1 cells, we detected the expression of VDR protein via Western blotting. After adding estrogen receptor α-blocker MPP and estrogen receptor ß-blocker PHTPP, we observed the effect of Gen on the regulation of the VDR protein. DNA was extracted from the blood samples of 200 postmenopausal women in the early epidemiological survey, and the restriction fragment length polymorphism of VDR gene Apa I and Bsm I in each sample was observed. The results were analyzed using dietary survey and bone mineral density examination. The results show that 10-8mol/L Gen can promote the proliferation of MC3T3-E1 cells (P less than 0.05). This effect can be canceled by the VDR blocker ZK159222. By analyzing the Apa I and Bsm I genotypes of VDR restriction sites, we discovered no significant difference in bone mineral density (BMD) between different genotypes (P>0.05). In addition, there was no significant correlation between dietary phytoestrogen intake and BMD in different genotypes (P>0.05). In conclusion, VDR can mediate the effect of Gen on the proliferation of MC3T3-E1 cells. The up-regulated expression of VDR protein in Gen is not mediated by the estrogen receptor. Moreover, the VDR gene polymorphism is not related to the BMD in various parts and is not related to the bone metabolism effect of the dietary plant estrogen intake.

Establishment of a Quantitative Polymerase Chain Reaction Assay for Monitoring Chimeric Antigen Receptor T Cells in Peripheral Blood.

BACKGROUND: The chimeric antigen receptor (CAR) consists of an antigen recognition moiety from a monoclonal antibody fused to an intracellular signalling domain capable of activating T cells. The specific structure of the CAR molecule has been used in various basic research and clinical settings to detect CAR expression, but it is necessary to develop more specific and simpler monitoring methods to observe real-time changes. MATERIALS AND METHODS: To develop a quantitative assay for the universal detection of DNA from anti-CD19 CAR-T cells, a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay was developed using primers based on FMC63-28Z gene sequences. We identified the numbers of copies of CAR gene on T cells transduced with the CAR gene that were obtained from peripheral blood. RESULTS: The assay had a minimum detection limit of 10 copies/µL and a strong linear standard curve (y = -3.3682x + 38.594; R2 = 0.999) within the range of the input CAR gene (10-107 copies/µL). The reproducibility test showed a coefficient of variation ranging from 0.63%-1.65%. Real-time qPCR is a highly sensitive, specific, reproducible, and universal method that can be used to detect anti-CD19 CAR-T cells in peripheral blood.

[Analysis of prognostic factors of portal hypertension treated with devascularization].

Objective: To explore the prognostic factors of portal hypertension treated with devascularization. Methods: A total of 397 patients with portal hypertension underwent devascularization in Nanjing Drum Tower Hospital from February 1993 to April 2014, among which there were 242 male and 155 female patients with median age of 48 years. The perioperative data were retrospectively collected. Logistic regression was used to find the risk factors which affect the operative complications. Follow-up evaluation was in progress regularly. Kaplan-Meier survival curve, Log-rank test and Cox regression model were used to find out factors which affect the long-term results. Results: All together 397 patients underwent devascularization, in whom 8 patients died perioperative, 389 patients discharged successfully. Logistic regression showed that age (≥48 years) (χ2=4.559, OR=2.048, P=0.033), red color sign before surgery (χ2=4.959, OR=2.129, P=0.026) and without portosystemic collateral vessels reserved (χ2=13.348, OR=5.122, P=0.000) were risk factors of perioperative complications. The follow-up time was (5.7±4.6) years. Totally 27 patients were lost from follow-up, 103 patients died for the disease during follow-up. The survival rate at 1-, 3-, 5-, 10-, 15- and 20-years was 93.6%, 86.9%, 80.1%, 59.3%, 54.1% and 38.5% respectively.Univariate analysis showed that gender (male), age (≥48 years), hemorrhage before surgery (≥500 ml per time), hepatitis virus and without portosystemic collateral vessels reserved were risk factors of the long-term survival (P<0.05). Cox regression analysis showed that age (≥48 years) (χ2=9.850, RR=1.904, P=0.002), hemorrhage before surgery (≥500 ml per time) (χ2=34.402, RR=3.273, P=0.000), hepatitis virus (χ2=7.573, RR=2.525, P=0.006) and without portosystemic collateral vessels reserved (χ2=5.905, RR=1.889, P=0.015) were independent risk factors that affect the long-term survival. Conclusion: Devascularization with portosystemic collateral vessels reserved has favorable perioperative and long-term outcome, and it definitely is a very safe and effective technique for portal hypertension.

Genetic diversity and pathogenicity differentiation of Sclerotinia sclerotiorum on rapeseed (Brassica napus L.) in Anhui Province, China.

The pathogenicity of 47 isolates of Sclerotinia sclerotiorum from oilseed rape (Brassica napus L.) in Anhui, China, was tested by detached leaf inoculation using the susceptible rape cultivar Wanyou-14. All isolates were pathogenic to the cultivar and could be grouped into 3 categories based on the lesion length on the leaves tested: weak pathogenicity type, intermediate pathogenicity type, and strong pathogenicity type. This suggested that there was differentiation in the pathogenicity among the strains tested of S. sclerotiorum. Additionally, the intraspecific DNA polymorphisms among 47 strains of S. sclerotiorum were investigated by screening 40 pairs of inter-simple sequence repeat (ISSR) primers. Unweighted pair-group method with arithmetic average cluster analysis of these ISSR data distinguished all strains from each other and revealed considerable genetic variability among them. These strains were classified into 7 clusters according to their branching in the dendrogram, and partial correlation was observed between the genetic polymorphisms and the pathogenicity of S. sclerotiorum strains.

L-3-n-Butylphthalide protects rats' cardiomyocytes from ischaemia/reperfusion-induced apoptosis by affecting the mitochondrial apoptosis pathway.

AIMS: This study investigated the role of L-3-n-Butylphthalide (NBP) in cardiac protection. METHODS: The left anterior descending coronary arteries (LAD) of the rats were occluded for 30 min following by 2-h reperfusion to make the ischaemia/reperfusion models. Neonatal cardiomyocytes were cultured and subjected to hypoxia. L-3-n-Butylphthalide was administered intraperitoneally 2 h before the surgery and right after the reperfusion in the in vivo experiments or added to the culture medium in vitro. Haemodynamic parameters were recorded to evaluate the cardiac functions, triphenyltetrazolium chloride (TTC) and Evens blue staining were used to determine the area of risk and infarct area, apoptotic cell numbers were counted with terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. Western blotting was used to determine the apoptotic protein levels and immune staining to determine the translocation of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. RESULTS: Our research showed for the first time that L-3-n-Butylphthalide had great effects in improving cardiac hemodynamic function and decreasing cardiac infarct areas and apoptotic cell numbers in the peri-infarct areas. The apoptotic signals investigation showed that L-3-n-Butylphthalide affected the mitochondrial pathway including Bcl-2 protein expression, inhibition of caspase 3 activation and cytochrome C releasing. Besides, Glyceraldehyde-3-phosphate dehydrogenase protein translocation was inhibited by L-3-n-Butylphthalide treatment, and this effect was mediated by endogenous reactive oxygen species (ROS). CONCLUSION: L-3-n-Butylphthalide protects cardiomyocytes from ischaemia/reperfusion-induced apoptosis by antioxidant effect and affecting mitochondrial apoptotic pathway.

Confocal laser endomicroscopy for detection of neoplasia in Barrett's esophagus: a meta-analysis.

Barrett's esophagus (BE) is associated with an increased risk of esophageal adenocarcinoma, and the recommended guideline for detection of neoplasia is surveillance endoscopy with random four-quadrant biopsies. Recently, a novel technique, confocal laser endomicroscopy (CLE), has emerged and enabled the endoscopist to perform a real-time histologic assessment of the gastrointestinal tract. We aimed to assess the accuracy of CLE in diagnosing BE-associated neoplasia by pooling data of existing trials. Databases including PubMed, EMBASE, the Cochrane Library, the Science Citation Index and momentous meeting abstracts were searched and evaluated by two reviewers independently. Meta-analysis was performed. Pooling data were conducted in a fixed effect model or a random effects model. Eight studies involving 709 patients and 4008 specimens were analyzed. In a per-patient analysis, the pooled sensitivity of CLE for detection of neoplasia was 89% (95% confidence interval [CI], 0.80-0.95), and the specificity was 75% (95% CI, 0.69-0.81). The area under the curve under the summary receiver operating characteristic was 0.9472. In a per-location analysis, the pooled sensitivity of CLE for detection of neoplasia was 70% (95% CI, 0.65-0.74), and the specificity was 91% (95% CI, 0.90-0.92). The area under the curve under the summary receiver operating characteristic was 0.9509. CLE is a reasonable, promising modality for management of patients with BE; more prospective trials need doing to determine whether it is superior to traditional method in diagnosing BE-associated neoplasia.

First Report of Brown Blight Disease Caused by Colletotrichum gloeosporioides on Camellia sinensis in Anhui Province, China.

Yellow Mountain fuzz tip, a cultivar of Camellia sinensis (L.) Kuntze, is commonly grown in the Yellow Mountain region in Anhui Province of China. During 2011 to 2012, leaf and twig blight on tea plants occurred from July to September in growing regions. Symptoms of blight on leaves of infected plants were detected in 30 to 60% of the fields visited and up to 500 ha were affected each year. Symptoms began as small, water-soaked lesions on young leaves and twigs and later became larger, dark brown, necrotic lesions, 1 to 3 mm in diameter on leaves and 2 to 5 mm long on twigs. To determine the causal agent, symptomatic leaf tissue was collected from plants in Gantang and Tangkou townships in September 2012. Small pieces of diseased tea leaves and twigs were surface-disinfested in 2% NaClO for 3 min, rinsed twice in distilled water, plated on potato dextrose agar, and incubated at 28°C for 5 days. Eleven isolates were recovered and all cultures produced white-to-gray fluffy aerial hyphae and were dark on the reverse of the plate. The hyphae were hyaline, branching, and septate. Setae were 2- to 3-septate, dark brown, acicular, and 78.0 to 115.0 µm. Conidiogenous cells were hyaline, short, branchless, cylindrical, and 11.3 to 21.5 × 4.2 to 5.3 µm. Conidia were hyaline, aseptate, guttulate, cylindrical, and 12.5 to 17.3 × 3.9 to 5.8 µm. Appresoria were ovate to obovate, dark brown, and 8.4 to 15.2 × 7.8 to 12.9 µm. DNA was amplified using the rDNA-ITS primer pair ITS4/ITS5 (3), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) primer pair GDF/GDR (2) and beta-tubulin 2 gene (Tub2) primer pair Btub2Fd/Btub4Rd (4). Sequences (GenBank Accession Nos. KC913203, KC913204, and KC913205) of the 11 isolates were identical and revealed 100% similarity to the ITS sequence of strain P042 of Colletotrichum gloeosporioides (EF423527), 100% identity to the GAPDH of isolate C07009 of C. gloeosporioides (GU935860), and 99% similarity to Tub2 of isolate 85 of C. gloeosporioides (AJ409292), respectively. Based on the above data, the 11 isolates were identified as C. gloeosporioides (Penz.) Penz. & Sacc. To confirm pathogenicity, Koch's postulate was performed and 4 ml of conidial suspension (1 × 105 conidia/ml) of each of the 11 isolates was sprayed on five leaves and five twigs per plant on four 12-month-old Yellow Mountain fuzz tip plants. Control plants were sprayed with distilled water. The inoculated plants were maintained at 28°C in a greenhouse with constant relative humidity of 90% and a 12-h photoperiod of fluorescent light. Brown necrotic lesions appeared on leaves and twigs after 7 days, while the control plants remained healthy. The experiments were conducted three times and the fungus was recovered and identified as C. gloeosporioides by both morphology and molecular characteristics. Tea plant blight caused by C. gloeosporioides was identified in Brazil (1), but to our knowledge, this is the first report of C. gloeosporioides causing tea leaf and twig blight on Yellow Mountain fuzz tip plants in Anhui Province of China. References: (1) M. A. S. Mendes et al. Page 555 in: Embrapa-SPI/Embrapa-Cenargen, Brasilia, 1998. (2) M. D. Templeton et al. Gene 122:225, 1992. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (4) J. H. C. Woudenberg et al. Persoonia 22:56, 2009.

First Report of Leaf Spot Caused by Colletotrichum spaethianum on Peucedanum praeruptorum in China.

Peucedanum praeruptorum Dunn, a traditional Chinese medicinal herb, is an important crop in Ningguo, China. Since 2010, leaf spot symptoms were observed yearly starting in June. Blighted leaf areas on individual plants ranged from 10 to 25% in many fields, and up to 200 ha were affected each year. Symptoms consisted of small, brown, necrotic spots uniformly distributed on the 1- to 2-week-old leaves. Small tissue pieces from the edges of lesions were disinfected in 2% NaClO for 3 min, rinsed twice in distilled water, plated on potato dextrose agar (PDA), and incubated at 25°C in darkness for 4 days. Single spore isolations were obtained for six strains. When inoculated on SNA media, the six strains produced typical septate mycelium, with the young hyphae hyaline and aged ones white greyish. Setae of the strains on SNA were brown, tip acute, 2- to 3-septate, and 32.5 to 85.6 µm long. Conidiogenous cells were hyaline, cylindrical, 2- to 3-septate, 6.2 to 16.5 µm in length, and 2.8 to 4.3 µm in width. The mature conidia were slightly curved, with round apex and truncate base, 1 to 5 oil globules, and were 13.3 to 23.8 µm in length and 3.0 to 3.9 µm in width, respectively. Appressoria were solitary or in loose groups, dark brown, irregular shapes, and were 6.8 to 9.2 µm in length and 4.3 to 7.1 µm in width. PCR amplification was carried out by utilizing the universal rDNA-ITS primer pair ITS4/ITS5 (1) and the actin gene primer pair ACT-512F and ACT-783R (2). The PCR products of ITS (GenBank Accession No. KC913201) and actin gene (KC913202) from six isolates were identical, respectively, and shared 100% identity to the ITS sequence of strain CBS 167.49 of Colletotrichum spaethianum (GU227807.1) and 99% similarity to the actin gene of strain CBS 167.49 of C. spaethianum (GU227905.1), which was isolated from Hosta sieboldiana in Germany (3). Based on the above, the isolates were identified as C. spaethianum. To confirm pathogenicity, conidial suspensions (105 conidia ml-1) of each of the six isolates were sprayed on four leaves per plant on five 6-month-old P. praeruptorum plants. Control plants were sprayed with water. Plants were maintained at 28°C in a greenhouse with constant humidity (RH 90%) and a 12-h photoperiod of fluorescent light. Symptoms similar to the original ones started to appear after 10 days, while the control plants remained healthy. The tests were repeated three times and the fungus was recovered and identified as C. spaethianum by both morphology and molecular characterization. To our knowledge, this is the first report of C. spaethianum causing leaf spot on P. praeruptorum in China. Since the C. spaethianum infections pose a serious threat to P. praeruptorum production, this disease needs to be considered for developing effective control strategies. References: (1) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (2) U. Damm et al. Fung. Divers. 39:45, 2009. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Genetic diversity of Phytophthora capsici (Pythiaceae) isolates in Anhui Province of China based on ISSR-PCR markers.

Phytophthora capsici is a plant pathogenic oomycete that damages numerous crops worldwide. Consequently, interest in research on the genetic structure of this species has grown in recent decades. However, there is little information about P. capsici in eastern China. We investigated the genetic diversity of P. capsici isolates from three large regions of Anhui Province in eastern China based on ISSR-PCR technology. Thirteen random primers were screened and used to amplify DNA from 51 samples. We obtained 158 reproducible ISSR fragments, of which 90% were polymorphic, revealing a high degree of polymorphism among the isolates. Genetic similarity coefficients among all the isolates ranged from 0.56 to 0.94, with a mean of 0.84 based on the ISSR data, indicating a high level of genetic variation in these P. capsici isolates. Cluster analysis using UPGMA indicated that the Anhui isolates were divided into seven groups according to the DNA fingerprints, although there was no correlation between the ISSR group and geographic origin. Isolates from the same location showed no clustering based on the year of sampling. AMOVA partitioned variability among (13.6%) and within populations (86.4%). The gene flow among populations ranged from 2.804 to 4.937, with a mean of 3.545, indicating highly frequent gene exchange. Genetic distances and genetic differentiation were negatively correlated with geographic distances. These results lead us to suggest that this pathogen has considerable evolutionary potential, which will enable it to adapt to and overcome management strategies over time.

First Report of Medical Tree Peony Root Rot Caused by Fusarium solani in Tongling, China.

Tree peony bark, a main component of Chinese traditional medicine used for alleviating fever and dissipating blood stasis, is mainly produced in Tongling, China. Recently, tree peony cultivation in this area was seriously affected by root rot, with approximately 20 to 30% disease incidence each year. The disease severely affects yield and quality of tree peony bark. During the past 2 years, we collected 56 diseased tree peony plants from Mudan and Fenghuang townships in Tongling. We found reddish brown to dark brown root rot in mature roots, especially on those with injuries. Plant samples collected were disinfected with 2% sodium hypochlorite and isolations were conducted on potato sucrose agar (PSA). Eleven isolates were obtained and all had white fluffy aerial hypha on PSA. Two types of conidia were produced; the larger, reaphook-shaped ones had three to five septa and the smaller, ellipse-shaped ones had one or no septum. The reaphook-shaped conidia were 20.15 to 37.21 × 3.98 to 5.27 µm and the ellipse-shaped conidia were 6.02 to 15.52 × 2.21 to 5.33 µm in size. Chlamydospores were produced, with two to five arranged together. Biological characteristics of the fungi indicated that the optimum temperature for the mycelial growth on PSA was 25 to 30°C and the optimum pH range was 5.5 to 7.0. The above morphological characteristics point the fungal isolates to be Fusarium solani. To confirm pathogenicity, 30 healthy 1-year-old tree peony seedling plants were grown in pots (25 cm in diameter) with sterilized soil and a conidial suspension from one isolate (FH-1, 5 × 105 conidia/ml) was used for soil inoculation. Inoculated seedlings were maintained at 28°C in a greenhouse with a 12-h photoperiod of fluorescent light. Seedlings inoculated with distilled water were used as controls. After 3 weeks, the roots were collected and rinsed with tap water. Dark brown lesions were observed in the inoculated mature roots but not in the control roots. To confirm the identity of the pathogen, F. solani strains were reisolated from the lesions and total genomic DNA was extracted with the cetyltriethylammnonium bromide method from the mycelia of the reisolated strains (1). PCR was performed using the fungal universal primers ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') to amplify a DNA fragment of approximately 590 bp. The purified PCR products were sequenced (Invitrogen Co., Shanghai, China) and shared 100% sequence identity with each other. A comparison of the sequence (JQ658429.1) by the Clustal_W program (2) with those uploaded in GenBank confirmed with the fungus F. solani (100% sequence similarity to isolate S-0900 from the Great Plains of the United States; EU029589.1). To our knowledge, this is the first report of F. solani causing medical tree peony root rot in China. The existence of this pathogen in China may need to be considered for developing effective control strategies. References: (1). C. N. Stewart et al. Biotechniques 14:748, 1993. (2). J. D. Thompson et al. Nucleic Acids Res. 22:4673, 1994.

Increased expression of sonic hedgehog in temporal lobe epileptic foci in humans and experimental rats.

Sonic hedgehog (Shh) is a soluble signaling protein that is crucial in regulating cell proliferation, migration and differentiation, axonal guidance and neural progenitor cell survival during nervous system development. Recent evidence suggests that the Shh plays an important role in adulthood in regulating structural plasticity. Here we investigated the expression of Shh in temporal lobe epileptic foci in patients and experimental animals in order to explore the probable relationship between Shh expression and temporal lobe epilepsy (TLE). The Shh expression was assessed in 30 human brain tissues derived from patients undergoing operation for intractable TLE and was also detected in 12 histological normal temporal lobes from controls. Meanwhile, we investigated the Shh expression in the temporal neocortex and hippocampus from lithium-pilocarpine-treated rats on days 1, 3, 7, 14, 30, and 60 post-seizure, and from control rats. Expression of Shh was assessed by immunohistochemistry, immunofluorescence, and Western blot analysis. Shh was mainly expressed in neurons in temporal lobe epileptic foci in humans and experimental rats. Compared to the control group, Shh expression was enhanced in the temporal neocortex of patients with intractable TLE. In experimental rats, Shh expression gradually increased from 7 to 60 days post-seizure in temporal neocortex and elevated from 3 to 60 days in hippocampus with the peak levels at 30 days as compared with the control group. These results suggest that Shh may play an important role in the development of TLE.

[Clinical study of 1949 cases of port wine stains treated with vascular photodynamic therapy (Gu's PDT)]. / Analyse clinique de 1 949 angiomes plans traités par thérapie photodynamique vasculaire (Gu's PDT).

BACKGROUND: Complete fading of port wine stains (PWS) is difficult to achieve with current laser treatments. Photodynamic therapy (Gu's PDT) could offer a very efficient alternative for PWS therapy. PATIENTS AND METHOD: 1949 lesions in 1385 patients were treated by PDT. Each patient received an intravenous injection of hematoporphyrin derivative (HpD) or hematoporphyrin monomethyl ether (HMME) at 3-7 mg/kg. Laser irradiation was performed on a 2 to 8 cm spot size. Different wavelengths (488.0 nm to 578.2 nm) were evaluated with a power density of 50-100 mW/cm2. Fluences ranged from 90 to 540 J/cm2. RESULTS: Among the 1942 lesions, PWS clearance was observed in 99.7% of cases. Excellent results were achieved in 128 lesions (6.6%) (100% clearance), 746 lesions (38.3%) yielded to good results (clearance > 75%), 923 lesions (47.4%) showed moderate results (clearance 50-75%), 145 lesions (7.4%) showed poor results (clearance<50%) and in 7 lesions (0.3%) no visible change was observed. The pink port wine stains revealed better response to Gu's PDT with only one session. Conversely, purple stains in adult patients required 2 sessions or more. CONCLUSION: This new PDT technique is effective and highly selective, with almost no risk of scarring.

Improved procedure for the analysis of gamma-hydroxybutyrate and ethylene glycol in whole blood.

The modification of a procedure originally developed for the analysis of ethylene glycol (EG) in serum was also found to permit the simultaneous analysis of gamma-hydroxybutyrate (GHB) in whole blood. The primary feature of the EG procedure was that it employed a water scavenger, 2,2-dimethoxypropane, which reacted with water to produce volatile methanol. Water scavenging is a technique that could be adapted for the analysis of drugs such as GHB as their respective di-t-butyldimethylsilyl derivatives. A close structural analogue of GHB, 2-hydroxy-3-methylbutyric acid, was successfully employed as the internal standard for both EG and GHB. The advantages of the modified procedure are that it is very quick and easy to perform and produces remarkably clean extracts for GHB, especially when compared to other liquid-liquid techniques. We have successfully applied this technique for the analysis of GHB and EG in several postmortem and driving-under-the-influence cases. There is an apparently wide variability between levels of GHB that can be associated with impairment versus those levels that can be associated with death.

Evaluation of the CEDIA DAU assays and the AxSym system for the analysis of cannabinoids in whole blood.

The Microgenics CEDIA DAU (EIA) and the Abbott AxSym system (FPIA) cannabinoids assays were evaluated for their combined effectiveness in the analysis of cannabinoids in whole blood. Blood samples were treated with acetone, evaporated, and reconstituted, and the supernatant was analyzed by the EIA cannabinoids assay. Blood samples determined positive by EIA were then treated with acetonitrile and sodium sulfate, and the resultant protein-free supernatant was analyzed using the FPIA cannabinoids assay. A total of 98 blood samples determined to be presumptively positive by both EIA and FPIA were further analyzed for the presence of 11-nor-carboxy-delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH). All 98 blood samples could be confirmed for the presence of THCCOOH by gas chromatography-mass spectrometry (GC-MS) at concentrations greater than the 10 ng/mL cutoff. The GC-MS results were found to correlate significantly better with those of the FPIA cannabinoids assay (r = 0.75) than with EIA (r = 0.22). Procedures for the rapid analysis of whole blood for cannabinoids using CEDIA DAU reagents and the AxSym system are presented.

Characterization and partial purification of microsomal casein kinase II from osteoblast-like cells: an enzyme that phosphorylates osteopontin and phosphophoryn.

Microsomal casein kinase II (mCKII) is a membrane-bound enzyme present in the microsomal fractions of ROS 17/2.8 osteoblast-like cells. It phosphorylates acidic matrix phosphoproteins such as phosphophoryn and osteopontin. Addition of 1.0% Nonidet P-40 facilitates extraction of the optimum amount of detergent-solubilized and -activated enzyme from microsomal fractions. mCKII was partially purified over 3000-fold by sequential chromatography over DEAE-cellulose and heparin-agarose. SDS-polyacrylamide gels, showed that mCKII contained 43 kDa and 31 kDa polypeptides, corresponding to the alpha- and beta-subunits of the enzyme, respectively. The alpha subunit was identified by anti-CKII antiserum and the beta subunit, by its ability to undergo autophosphorylation. The enzyme was inhibited by 50% with 0.4 micrograms/ml heparin and stimulated by 100% with 1.0 mM spermine when casein was used as a substrate. The phosphorylation of phosphophoryn was reduced to 50% by 0.8 micrograms/ml heparin, but was increased to 2-2.5 fold by 5 to 15 mM spermine, which may be due to substrate-directed effects. Kinetic analysis showed that the apparent Km values for phosphophoryn (0.39 microM) and for osteopontin (2.1 microM) were lower than that for casein (21.3 microM). Vmax values of phosphophoryn and osteopontin were 2.2-fold and 4.6-fold higher than that of casein. Using the ratio Vmax/Km as a measure of kinetic specificity, osteopontin and phosphophoryn appear to be the more specific substrates than casein for mCKII. Thus, both proteins can be considered as physiological substrates for mCKII.

Microsomal casein kinase II in endoplasmic reticulum- and Golgi apparatus-rich fractions of ROS 17/2.8 osteoblast-like cells: an enzyme that modifies osteopontin.

Osteopontin is an acidic phosphoprotein containing casein kinase II (CKII) phosphorylatable sites and an acidic amino acid cluster. The metabolically 32P-labelings of both serines and threonines in vitro in osteopontin immunoprecipitated from rat osteoblast-like ROS 17/2.8 cells may suggest that casein kinase II catalyzes this modification. The enzyme occurs in microsomal fractions of rat osteoblast-like ROS 17/2.8 cells. Subcellular fractions containing endoplasmic reticulum and Golgi apparatus were isolated by differential centrifugation and were identified according to their ultrastructures and the presence of marker enzymes such as glucose-6-phosphatase and thiamine pyrophosphatase, respectively. both fractions phosphorylated the partially dephosphorylated osteopontin and the specific substrate peptide RRREEETEEE. Endoplasmic reticulum-catalyzed peptide phosphorylation was 2.7 times lower than that of Golgi although both endoplasmic reticulum- and Golgi-catalyzed peptide reactions were 50% inhibited by 20 and 100 ng/ml heparin, respectively. Western blot analysis revealed that both fractions contained osteopontin and microsomal CKII. Furthermore, microsomal CKII was immunogold-labeled in endoplasmic reticulum and Golgi apparatus. Heparin inhibition and utilization of [gamma-32P]GTP as a phosphate donor by both fractions confirmed their capacity to phosphorylate osteopontin. The results suggest that microsomal CKII modifies the acidic matrix proteins during transportation. These matrix phosphoproteins may participate in the mineralization process of hard tissues.

Cytopathic effects of Treponema denticola chymotrypsin-like proteinase on migrating and stratified epithelial cells.

The effects of Treponema denticola and its outer membrane-bound chymotrypsin-like proteinase on periodontal ligament epithelial cell cultures at different stages of maturity were studied. In sparse cultures with migrating epithelial cells, large intracellular vacuoles were formed rapidly following exposure to live T. denticola. Treponemes showing structural damage were seen occasionally inside membrane-bound vesicles. Intensive membrane blebbing occurred in infected cells and continued for up to 48 h before the cell died. Blebbing could also be induced by a purified chymotrypsin-like proteinase of T. denticola. Cortical actin and alpha-actinin of the bacterium-treated cells showed disorganization, and pericellular fibronectin was degraded by both whole T. denticola and the isolated proteinase. Epithelial cells with well-formed lateral cell contacts appeared to be more resistant to the effects of T. denticola than migrating isolated cells. In multilayer epithelial cultures, adhesion of T. denticola and membrane blebbing were observed infrequently. There was no evidence of invasion of T. denticola into epithelial multilayers. However, immunogold electron microscopy showed rapid transport of T. denticola chymotrypsin-like proteinase into newly formed large intracellular vacuoles within the epithelial layers. These vacuoles were lined by membranes studded with ribosomes. T. denticola-treated epithelial multilayers had loose cell contacts, collapsed intercellular spaces, and increased permeability. Through its capacity to cause these unique cytopathic effects, the chymotrypsin-like proteinase of T. denticola has the potential to contribute to the initiation of periodontal disease.

Multilayer culture of periodontal ligament epithelial cells: a model for junctional epithelium.

The unique features of junctional epithelium involve lack of keratinization, limited differentiation and a relatively permeable structure. In order to study the relationship between differentiation and permeability of stratified epithelium a model system was developed. Porcine periodontal ligament epithelial cells were cultured on the polycarbonate nucleopore membrane of the Transwell two-compartment culture system. Within 5 days of culture the cells formed a confluent multilayered structure. Subsequently, maturation of the structure and differentiation of surface cells took place. Transmission electron microscopy showed that the cells were arranged into basal and suprabasal layers with sparse desmosomal attachments and wide intercellular spaces resembling the organization of junctional epithelium. The basal cells attached to a subepithelial basal lamina through numerous hemidesmosomes. The cytokeratin profile of the cultured epithelium (K5, 6, 14, 16, 19) resembled that of the cells of junctional epithelium attached to the tooth surface. The older cultures expressed differentiation markers, K4, K13 and involucrin, thereby resembling sulcular epithelium. The epithelial permeability, measured by diffusion of phenol red, radioactive dextran or methionine tracers, and as transepithelial electrical resistance, decreased with the increased cell number and maturation of the cultures. The new model provides an organotypic culture system which allows to control differentiation of a multilayered periodontal epithelium. It thus may serve as a valuable new tool for studies on the permeability and behaviour of periodontal epithelium under the influence of exogenous and endogenous factors.

An experimental study of trachea anastomosis in rabbits using carbon dioxide laser.

A technique of sutureless trachea anastomosis in rabbits using carbon dioxide laser was reported herein. In 6 rabbits with laser-assisted trachea anastomoses, only one was found to have slight anastomotic stenosis after operation, whereas 6 rabbits with conventional sutures all were found to have anastomotic stenosis. Results show that laser-assisted trachea anastomosis has certain advantages over the conventional suturing technique, thereby finding wide application in clinical tracheal reconstruction and lung transplantation.