C-myb Regulates Autophagy for Pulp Vitality in Glucose Oxidative Stress.

Diabetes mellitus is closely related to oral-complicated diseases by oxidative stress. This study investigates whether cellular myeloblastosis (c-myb) could protect human dental pulp cells against glucose oxidative stress and regulate autophagy activity for pulp vitality. Diabetes mellitus was induced by streptozotocin in Sprague-Dawley rats, and their pulp tissue in teeth was analyzed in terms of pulp cavity and molecules by hematoxylin and eosin and immunohistochemistry staining. Human dental pulp cells were serially subcultured and treated with glucose oxidase in the presence of elevated glucose to generate glucose oxidative stress. The replication-deficient adenovirus c-myb and small interfering RNA c-myb were introduced for c-myb expression. The pulp tissue from the diabetic rats was structurally different from normal tissue in terms of narrow pulp capacity, reduced c-myb, and dentinogenesis molecules. Glucose oxidase treatment decreased c-myb and dentinogenesis molecules (bone morphogenetic protein 2 and 7, dentin matrix protein 1, and dentin sialophosphoprotein) in human dental pulp cells. However, overexpression of c-myb by adenovirus c-myb increased dentinogenesis, autophagy molecules (autophagy protein 5, microtubule-associated protein 1A/1B-light chain 3, and Beclin-1), and cell survival via p-AMPK/AKT signaling even with glucose oxidative stress. In contrast, the lack of c-myb decreased the above molecules and cell survival by downregulating p-AMPK/AKT signaling. The results indicate that diabetes leads to irreversible damage to dental pulp, which is related to downexpression of autophagy via the p-AMPK/AKT pathway by decline of c-myb. The findings of this study provide a new insight that c-myb could ameliorate autophagy activity and that it is applicable for monitoring complicated diseases of dental pulp. The involvement of c-myb in pulp pathology could serve a therapeutic target in oral-complicated diseases.

Direct effect of streptozotocin on periodontal ligament cells through myeloid cell leukemia-1.

OBJECTIVE: We hypothesized that streptozotocin (STZ) has a direct impact on periodontal ligament cell (PDL) damage as a potential direct inducer of periodontitis. BACKGROUND: Since diabetes was accepted as one of the risk factors for the development of periodontal disease, various scientific studies have been undertaken in the STZ-induced periodontal disease models. STZ induces ß-cell damage and subsequent diabetes development in vivo. Until now, assessment of the impacts of STZ-induced experimental diabetes on periodontitis has generally been conducted on the fundamental assumption that STZ have no direct action on PDL and its function. However, several recent studies suggest that STZ also directly affect many different biological functions in various tissues or organs. MATERIAL AND METHODS: To assess the apoptotic effects of STZ on PDLs, they were treated with or without STZ at different concentrations. Qualitative estimation of apoptotic cell death was obtained by live/dead assay. The expression levels of apoptosis-related proteins were evaluated by western blot analysis. RESULTS: STZ inhibits growth and induces apoptosis in PDLs in a dose-dependent manner. Furthermore, STZ dramatically induced Mcl-1 downregulation in a proteasome-dependent manner and thereby induced apoptosis of PDLs through the Bak/Bax apoptotic signaling pathway. CONCLUSION: Our results support the hypothesis that suppression of the cellular Mcl-1 levels by STZ may be at least partly attributed to the development of periodontitis in STZ-induced diabetic animal models.

The expression of vascular endothelial growth factor and Syndecan-4 in cartilage from osteoarthritic knees.

Previous studies support the important role of vascular endothelial growth factor (VEGF) and syndecan-4 in the pathogenesis of osteoarthritis (OA). Both VEGF and syndecan-4 are expressed by chondrocytes and both are involved in the regulation of matrix metalloproteinase-3, resulting in the activation of aggrecanase II (ADAMTS-5), which is essential in the pathogenesis of OA. However, the relationship between VEGF and syndecan-4 has not been established. As a pilot study, we assayed the expression of VEGF and syndecan-4 in cartilage samples and cultured chondrocytes from osteoarthritic knee joints and analysed the relationship between these two factors. Specimens were collected from 21 female patients (29 knees) who underwent total knee replacement due to severe medial OA of the knee (Kellgren-Lawrence grade 4). Articular cartilage samples, obtained from bone and cartilage excised during surgery, were analysed and used for chondrocyte culture. We found that the levels of expression of VEGF and syndecan-4 mRNA did not differ significantly between medial femoral cartilage with severe degenerative changes and lateral femoral cartilage that appeared grossly normal (p = 0.443 and 0.622, respectively). Likewise, the levels of expression of VEGF and syndecan-4 mRNA were similar in cultured chondrocytes from medial and lateral femoral cartilage. The levels of expression of VEGF and syndecan-4 mRNAs were significantly and positively correlated in cartilage explant (r = 0.601, p = 0.003) but not in cultured chondrocytes. These results suggest that there is a close relationship between VEGF and syndecan-4 in the cartilage of patients with OA. Further studies are needed to determine the exact pathway by which these two factors interact in the pathogenesis of OA.

The in vitro apoptotic effects of A248 and A1659, newly synthetic histone deacetylase inhibitors in oral cancer cells.

OBJECTIVES: Histone deacetylase (HDAC) inhibitors represent potential therapeutic agents against various cancers. In this study, we attempt to identify whether newly synthesized HDAC inhibitors, A248 and A1659, can be effective anti-cancer drug candidates for oral cancer. MATERIALS AND METHODS: The anti-cancer activities of A248 and A1659 in MC-3 and HN22 human oral cancer cells were evaluated by 3-(4,5-dimethylthiazol-20yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assay, 4'-6-diamidino-2-phenylindole (DAPI) staining, Western blot analysis, immunocytochemistry, and small interference RNA (siRNA) technology. RESULTS: A248 and A1659 enhanced histone acetylation and decreased the viability of MC-3 and HN22 cells. A248 and A1659 also induced apoptosis, as evidenced by altered nuclear features and poly(ADP-ribose)polymerase (PARP) cleavage. A248 and A1659 markedly decreased Sp1 expression in a concentration- or time-dependent manner and blocked nuclear translocation of Sp1 protein from the cytosol, which contributed to an increase in p27 expression and a decrease in cyclin D1 expression. Furthermore, the knockdown of Sp1 protein with siRNA caused marked alteration of p27 and cyclin D1 expression to induce apoptosis. The most popular HDAC inhibitor, trichostatin A (TSA) also induced apoptosis and reduced the expression level of Sp1 protein. CONCLUSION: These results suggest that A248 and A1659, two new HDAC inhibitors, may be attractive therapeutic drug candidates for targeting Sp1 in human oral cancer cells.

Apoptotic effect of dibenzylideneacetone on oral cancer cells via modulation of specificity protein 1 and Bax.

OBJECTIVE: Dibenzylideneacetone (DBA), an analogue of curcumin, has been shown to have potential anticancer effects against several cancers. However, the molecular mechanism underlying anticancer activity of DBA has not been well established yet. In this study, we investigated the function and molecular mechanism of DBA in human oral cancer cells. MATERIALS AND METHODS: The growth-inhibitory and apoptotic effects and related signaling pathways of DBA were evaluated using trypan blue exclusion assay, 4'-6-diamidino-2-phenylindole staining, Western blot analysis, siRNA, and reverse transcription-polymerase chain reaction. RESULTS: DBA inhibited cell growth and induced apoptosis, as evidenced by PARP cleavage, activation of caspase-3, and nuclear condensation. DBA also decreased specificity protein 1 (Sp1) expression through facilitating protein degradation. In addition, DBA enhanced the induction of pro-apoptotic protein Bax, resulting in their conformational change, translocation into mitochondrial outer membrane, and its oligomerization. The down-regulation of Sp1 by siRNA targeting Sp1 and mithramycin A increasingly activated Bax to trigger apoptosis. Moreover, DBA-induced growth inhibition and apoptosis in various human oral cancer cell lines were associated with Sp1 down-regulation and induction of Bax. CONCLUSION: These findings suggest that DBA may be a potential anticancer drug candidate to induce apoptosis through down-regulation of Sp1 in human oral cancer.

Effect of surface treatments on microtensile bond strength of repaired aged silorane resin composite.

OBJECTIVE: This laboratory study compared the repaired microtensile bond strengths of aged silorane resin composite using different surface treatments and either silorane or methacrylate resin composite. METHODS: One hundred eight silorane resin composite blocks (Filtek LS) were fabricated and aged by thermocycling between 8°C and 48°C (5000 cycles). A control (solid resin composite) and four surface treatment groups (no treatment, acid treatment, aluminum oxide sandblasting, and diamond bur abrasion) were tested (N=12 blocks, 108 beams/group). Each treatment group was randomly divided in half and repaired with either silorane resin composite (LS adhesive) or methacrylate resin composite (Filtek Z250/Single Bond Plus). After 24 hours in 37°C distilled water, microtensile bond strength testing was performed using a non-trimming technique. Surface topography after surface treatment was analyzed using scanning electron microscopy (SEM). Failure mode was examined using optical microscopy (50×). RESULTS: Weibull-distribution survival analysis revealed that aluminum oxide sandblasting followed by silorane or methacrylate resin composite and acid treatment with methacrylate resin composite provided insignificant differences from the control (p>0.05). All other groups were significantly lower than the control. Failure was primarily adhesive in all groups. CONCLUSION: Aluminum oxide sandblasting produced microtensile bond strength not different from the cohesive strength of silorane resin composite. After aluminum oxide sandblasting, aged silorane resin composite can be repaired with either silorane resin composite with LS system adhesive or methacrylate resin composite with methacrylate dental adhesive.

Effect of Er,Cr:YSGG laser, air abrasion, and silane application on repaired shear bond strength of composites.

UNLABELLED: Aged resin composites have a limited number of carbon-carbon double bonds to adhere to a new layer of resin. Study objectives were to 1) evaluate various surface treatments on repaired shear bond strength between aged and new resin composites and 2) to assess the influence of a silane coupling agent after surface treatments. METHODS: Eighty disk-shape resin composite specimens were fabricated and thermocycled 5000 times prior to surface treatment. Specimens were randomly assigned to one of the three surface treatment groups (n=20): 1) air abrasion with 50-µm aluminum oxide, 2) tribochemical silica coating (CoJet), or 3) Er,Cr:YSGG (erbium, chromium: yttrium-scandium-gallium-garnet) laser or to a no-treatment control group (n=20). Specimens were etched with 35% phosphoric acid, rinsed, and dried. Each group was divided into two subgroups (n=10): A) no silanization and B) with silanization. The adhesive agent was applied and new resin composite was bonded to each conditioned surface. Shear bond strength was evaluated and data analyzed using two-way analysis of variance (ANOVA). RESULTS: Air abrasion with 50-µm aluminum oxide showed significantly higher repair bond strength than the Er,Cr:YSGG laser and control groups. Air abrasion with 50-µm aluminum oxide was not significantly different from tribochemical silica coating. Tribochemical silica coating had significantly higher repair bond strength than Er,Cr:YSGG laser and the control. Er,Cr:YSGG laser and the control did not have significantly different repair bond strengths. Silanization had no influence on repair bond strength for any of the surface treatment methods. CONCLUSION: Air abrasion with 50-µm aluminum oxide and tribochemical silica followed by the application of bonding agent provided the highest repair shear bond strength values, suggesting that they might be adequate methods to improve the quality of repairs of resin composites.

ß-Phenethyl isothiocyanate induces death receptor 5 to induce apoptosis in human oral cancer cells via p38.

OBJECTIVES: ß-Phenylethyl isothiocyanate (PEITC) has been demonstrated to fight many types of cancers through various molecular pathways. In this study, we focused on its effect on the induction of apoptosis to inhibit cell growth and molecular mechanism in oral cancer. MATERIALS AND METHODS: 3-(4,5-dimethylthiazol-2-yl)-5-(2,4-disulfophenyl)-2-(4 sulfophenyl)-2H-tetrazolium (MTS) assay was used to examine cell viability. The apoptotic effect was investigated using 4'-6-Diamidino-2-phenylindole (DAPI) staining or Western blotting. Inhibitors were used to determine the molecular target and mechanism of PEITC-mediated apoptosis. RESULTS: ß-Phenylethyl isothiocyanate inhibited the growth of HN22 human oral cancer cells and induced caspase-dependent apoptosis in HN22 cells as evidenced by nuclear fragmentation and the activation of caspase 3. It increased cleaved caspase 8, truncated BID, and death receptor 5 (DR5) through the activation of p38 MAPK. This result was confirmed by blockage of PEITC-induced cleavages of Poly(ADP-ribose) Polymerase, caspase-3, caspase-8, and DR5 by p38 MAPK inhibitor, SB203580. We also found that PEITC activated p38 and augmented DR5 to induce apoptosis in other human oral cancer cells. CONCLUSIONS: These results suggest that DR5 is a potential molecular target for PEITC-induced apoptosis in oral cancer via p38 MAPK.

Inhibition of myeloid cell leukemia-1 by tolfenamic acid induces apoptosis in mucoepidermoid carcinoma.

OBJECTIVES: The aim of this study was to evaluate the role of tolfenamic acid (Tol) and ampiroxicam (Amp) in the apoptotic regulation of YD-15 salivary mucoepidermoid carcinoma (MEC). MATERIALS AND METHODS: The effect of Tol on apoptosis and its mechanism were examined using a 3-(4,5-dimethylthiazol-2-yl)-5-(2,4-disulfophenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, Sub-G(1) population, Western blot analysis, 4'-6-Diamidino-2-phenylindole staining, reverse transcriptase polymerase chain reaction, immunostaining and small interfering RNA transfection. RESULTS: Tol inhibited cell growth of YD-15 cells but Amp did not. Tol induces apoptosis in YD-15 cells as evidenced by nuclear fragmentation, accumulation of the sub-G1 phase and the activation of caspase 3. Tol inhibited myeloid cell leukemia-1 (MCL-1) at the protein and mRNA levels. The treatment of MCL-1 siRNA to YD-15 cells resulted in the activation of caspase 3 and the inhibition of cell growth. Moreover, MCL-1 was regulated by specificity protein 1, but not by mitogen-activated protein kinases. CONCLUSION: These results suggest that Tol could be a potent anti-cancer drug for YD-15 MEC cells that acts by regulating the MCL-1 protein.

Management and outcomes of traumatic colon injury in civilian and military patients.

Divergent injury patterns may indicate the need for differing strategies in combat and civilian trauma patients. This study aims to compare outcomes of colon injury management in these two populations. Parallel retrospective reviews were conducted comparing warfighters (n = 59) injured downrange and subsequently transferred to the United States with civilians (n = 30) treated at a United States Level I trauma center. Patient characteristics, mechanisms of injury, treatment course, and complications were compared. The civilian (CP) and military (MP) populations did not differ in Injury Severity Score (MP 20 vs CP 26; P = 0.41). The MP experienced primarily blast injuries (51%) as opposed to blunt trauma (70%; P < 0.01) in the CP. The site of colon injury did not differ between groups (P = 0.15). Initial management was via primary repair (53%) and resection and anastomosis (27%) in the CP versus colostomy creation (47%) and stapled ends (32%) in the MP (P < 0.001). Ultimately, the CP and MP experienced equivalent continuity rates (90%). Overall complications (MP 68% vs CP 53%; P = 0.18) and mortality (MP 3% vs CP 3%; P = 0.99) did not differ between the two groups. The CP and MP experience different mechanisms and initial management of colon injury. Ultimately, continuity is restored in the majority of both populations.

Apoptotic effect of Polygonum Cuspidatum in oral cancer cells through the regulation of specificity protein 1.

OBJECTIVES: The aim of this study was to evaluate the growth inhibitory and apoptosis-inducing effects and mechanisms of Polygonum cuspidatum root in oral cancer cells. MATERIALS AND METHODS: The testing materials were separated by normal-phase silica gel liquid chromatography. The effect of P. cuspidatum root on apoptotsis and its mechanism were performed using 3-(4,5-dimethylthiazol-20yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) (MTS) assay, western blot analysis, RT-PCR, promoter assay, and (4'-6-Diamidino-2-phenylindole) (DAPI) staining. RESULTS: The methanol extract of P. cuspidatum (MEPC) inhibited the proliferation of oral cancer cells by inducing caspase-dependent apoptosis. Protein and mRNA expression levels and the transactivation of Specificity protein 1 (Sp1) were markedly decreased in KB cells treated with MEPC. Ethyl acetate fraction (EA) from MEPC was more potent than aqueous fraction (AQ) from MEPC to induce apoptosis. F2, F3, and F4 from EA differentially inhibited the growth of KB cells, and it depends on the amount of Emodin in F2, F3, and F4. Moreover, Emodin inhibited oral cancer cell growth and induced caspase-dependent apoptosis by decreasing Sp1. MEPC also decreased an apoptosis-related downstream target of Sp1 protein, survivin. CONCLUSION: The results from this study strongly suggest that MEPC, its fraction, and Emodin may be potential bioactive materials to cause apoptosis mechanism via the down-regulation of Sp1 in oral cancer cells.

Comparison of abdominal damage control surgery in combat versus civilian trauma.

BACKGROUND: The majority of individuals who perform damage control surgery in the military arena are trained in civilian venues. Therefore, it is important to compare and contrast damage control performed in civilian and military settings. In contrast to civilian trauma, which is primarily caused by blunt injury and addressed at one or two surgical facilities, combat casualties primarily sustain explosion-related injuries and undergo treatment at multiple levels of care across continents. We aimed to compare patients undergoing abdominal damage control surgery across these two very different settings. METHODS: Parallel retrospective reviews were conducted over 2 years (2005-2006) in a combat setting and at a US Level I trauma center. Patients were examined during the first 7 days after injury. RESULTS: The civilian population (CP) was older (40 vs. 23; p < 0.01) with a higher injury severity score (35 vs. 27; p < 0.02). The CP experienced greater blunt injury than the military population (MP) (83 vs. 4%; p < 0.01). Explosion-related injury was only present in the MP (64%). At baseline, the CP presented with lower systolic blood pressure (108 vs. 126) and larger base deficit (9.8 vs. 6.5; p < 0.05). The MP underwent more surgeries (3.5 vs. 2.9; p = 0.02) with similar rates of fascial closure (48.7% vs. 70.0%; p = 0.11). Complication rates were similar between the CP and the MP (43% vs. 58%, respectively; p = 0.14). CONCLUSIONS: Military and civilian trauma patients who undergo damage control surgery experience similar fascial closure rates despite differing demographics and widely disparate mechanisms of injury. The MP undergoes a greater number of procedures than the CP, but complication rates do not differ between the groups.

Toluene-induced accumulation of trehalose by Pseudomonas sp. BCNU 106 through the expression of otsA and otsB homologues.

AIM: The objective of this study was to investigate toluene-induced accumulation mechanism of trehalose in a toluene-tolerant bacterium Pseudomonas sp. BCNU 106. METHODS AND RESULTS: The accumulation of trehalose by a toluene-tolerant bacterium Pseudomonas sp. BCNU 106 was examined at various cultivation time by measuring the total intracellular trehalose content, trehalase activity and mRNA levels of the trehalose-biosynthetic genes. The pattern of trehalose accumulation corresponded to the mRNA expression pattern of the trehalose-biosynthetic genes with the maximum level at 12 h or 4 h of cultivation with 10% (v/v) toluene, respectively. The trehalose-biosynthetic genes were also cloned and sequenced. Furthermore, the effects of toluene addition on the intracellular osmotic pressure and pH were investigated. It was shown that homeostasis was maintained in the bacterial cells. CONCLUSIONS: In a toluene-tolerant bacterium Pseudomonas sp. BCNU 106, a significant amount of trehalose was accumulated through the toluene-induced expression of the trehalose-biosynthetic genes after the exposure to toluene. SIGNIFICANCE AND IMPACT OF THE STUDY: The accumulation of the high level of intracellular trehalose was preceded by the expression of otsA/B genes in toluene-tolerant bacteria, contributing to the elucidation of the tolerance mechanism.

Induction of MMP-13 expression by soluble human glucocorticoid-induced tumor necrosis factor receptor in fibroblast-like synovial cells.

OBJECTIVE: We tested the hypothesis that human glucocorticoid-induced tumor necrosis factor receptor (hGITR/TR11) expressed on the surface of activated CD4(+) T cells is responsible for up-regulating the production of matrix metalloproteinase (MMP)-13 by fibroblast-like synoviocytes (FLSs). METHODS: The level of MMP-13 was measured by Western blot and reverse transcriptase polymerase chain reaction (RT-PCR). Expressions of hGITR ligand (hGITRL) on the surface of FLSs and hGITR on the surface of human CD4(+) T cells were analyzed by flow cytometry and RT-PCR. Neutralizing antibodies (Abs) were used to block hGITRL and hGITR on the surface of FLSs and human CD4(+) T cells, respectively. Human CD4(+) T cells were cocultured with FLSs to facilitate interaction between hGITR on CD4(+) T cells and hGITRL on FLSs. RESULTS: Soluble hGITR (shGITR) stimulated FLSs to produce MMP-13, and blockade of hGITRL reduced this effect. Direct contact between activated CD4(+) T and FLSs also induced the production of MMP-13, and neutralization of hGITR on activated CD4(+) T cells during coculture decreased the amount of MMP-13 produced by FLSs. CONCLUSION: shGITR stimulated FLSs to produce MMP-13 via a signal through hGITRL. Direct contact between activated CD4(+) T cells and FLSs facilitated hGITR-hGITRL interaction, and resulted in inducing MMP-13. This effect may increase tissue destruction in chronic inflammation such as rheumatoid arthritis (RA).

Requirement of metabolic activation for estrogenic activity of Pueraria mirifica.

A wide range of chemicals derived from plant and human-made xenobiotics are reported to have hormonal activities. The present study was performed to examine the estrogenic effect of Kwao Keur, Pueraria mirifica (PM), that has been used as a rejuvenating folk medicine in Thailand, using recombinant yeast, MCF-7 cell proliferation and HepG2 cell transient transfection assay. In recombinant yeast assay, 0.025, 0.25, 2.5, 25, 2.5 x 10(2), 2.5 x 10(3), 2.5 x 10(4) ng/ml concentrations of PM did not show any estrogenic activities, while 10(-9) of 17 beta-estradiol (positive control) showed high estrogenic activity. Estrogenic activities were induced at 2.5 ng/ml to 25 microg/ml concentrations of PM in a dose-dependent manner on MCF-7 cells and the estrogenic effect of PM was blocked by tamoxifen treatment, a well-known anti-estrogen. PM also showed estrogenic effect on human hepatoma cell line, HepG2 cells, containing estrogen receptor and luciferase reporter gene. Taken together, PM in itself may have no estrogenicity in yeast system, but it has estrogenicity in MCF-7 & HepG2 cells that have human metabolic enzymes. The results indicated that PM may require metabolic activation for estrogenic activity.

Strategies for combining and scaling sexual maturity indicators: the Heartfelt Study.

PRIMARY OBJECTIVE: To identify the best variable or combination of sexual maturity variables to use in investigations of adolescent health, and explore possible re-scaling of five-stage sexual maturity stages into dummy variables' ('advanced'/'delayed'). RESEARCH DESIGN: Cross-sectional. SUBJECTS: Tri-ethnic sample of 384 US adolescents 11-16 years of age (Heartfelt Study). METHODS: Sexual maturity variables are genital. breast and pubic hair stages obtained by clinical examination by nurse practitioners, testes size, menarche and age at menarche. RESULTS: Principal factor analysis of sexual maturity variables, height and age, revealed high commonality among the variables, as no more than a single factor appeared in 27 of 28 factor analyses (by sex and age groups). Genital stage (boys) and breast stage (girls) were most highly related to the first principal factor independent of age and ethnicity. ANOVA of 'key variables' indirectly reflecting endocrine function suggested that some stages could be combined. CONCLUSIONS: A single variable should suffice to describe sexual maturity at the studied ages, or variables could be combined in a (unstandardized) sexual maturity index (SMI): SMI (Girls)=0.998 x BREAST+0.900 x PUBIC HAIR + 1.815 x YEARS SINCE MENARCHE. SMI (Boys)= 1.104 x GENITAL + 1.328 x PUBIC HAIR + 5.997 x TESTES SIZE (mL).

Blood pressure and sexual maturity in adolescents: the Heartfelt Study.

This study investigates sexual maturity as a predictor of resting blood pressures independent of other known predictors, in 179 boys and 204 girls 11-16 years of age from the Heartfelt Study. The sample included youth of African (n = 140), Mexican (n = 117), and European and "other" (n = 126) backgrounds. Sexual maturity was assessed during clinical examination of three standard indicators for each sex. Systolic and diastolic blood pressures were higher in children of maturity stages IV and V, compared to stages I-III, in each gender/ethnic group (P < 0.01 in almost all groups). Boys and girls advanced in sexual maturity for their age group, had significantly higher systolic blood pressures (but not diastolic) than the less advanced in linear models that included height, body mass index (BMI), ethnicity, and age as co-predictors. Diastolic blood pressures were predicted by height in boys and by age and the BMI in girls. This analysis, using a very conservative approach, suggests that sexual maturity provides important and independent information on systolic blood pressure in adolescents. Further investigation of its role in 24-hr blood pressures and in blood pressures taken during physical and emotional stress, is recommended.

Thrombopoietin is synergistic with other cytokines for expansion of cord blood progenitor cells.

We investigated the effects of recombinant human thrombopoietin (TPO) in combination with various cytokines including erythropoietin (EPO), interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor (SCF) on megakaryopoiesis, and the expansion of CD34+CD41a+ cells from human cord blood CD34+ cells with these cytokines under serum-free conditions. Human cord blood CD34+ cells were cultured in Megacult (Stem Cell Technologies Inc. Vancouver, Canada) in the presence of recombinant growth factors. Colony-forming unit-megakaryocyte (CFU-M) colonies were counted on day 14. CD34+CD41a+ and CD34-CD41a+ cell expansion was analyzed using a serum-free liquid culture system for 7 days with recombinant growth factors. TPO alone had a concentration-dependent effect on megakaryocyte colony growth. At concentrations above 1 ng/ml, TPO supported significant CFU-Meg colony formation in a concentration-dependent manner. The combination of TPO plus other cytokines, including EPO, IL-3, and SCF, resulted in a synergistic enhancement of the number of CFU-Meg colonies, but IL-6 failed to enhance the effect of TPO. The number of CD41a+ cells increased after 7 days in liquid culture of human cord blood CD34+ cells with various cytokines (EPO, IL-3, IL-6, SCF) combined with TPO, but SCF plus TPO only resulted in a significant synergistic increment of CD34+CD41a+ cells compared with TPO alone. The results of the present study indicate that EPO, IL-3, and SCF can be synergistic with TPO to stimulate proliferation of CFU-Meg and suggest that SCF plus TPO can expand CD34+CD41a+ cells to effect the rapid recovery of platelets in patients following stem cell transplantation.

Pharmacological characterization of effects of verapamil and GS 283 on isolated guinea pig and rat trachealis.

The pharmacological effects of verapamil and GS 283, 1-(4'-methoxybenzyl)-6,7-dihydroxy-3,4-dihydroxyisoquinoline, were investigated using isolated rat and guinea pig trachealis. Both verapamil and GS 283 inhibited carbachol-, histamine (only guinea pig)-, and high-K(+)-induced contraction in a dose-dependent manner. GS 283 acted as a weak histamine H1 and muscarinic receptor antagonist in guinea pig and rat trachealis with respective pKB values in the range of 5.60 approximately 6.12 and 5.17 approximately 5.83. On the other hand, pyrilamine and atropine showed a typical competitive antagonism on histamine (guinea pig) and on muscarinic receptors (rat trachea) with pKB values of 9.25 +/- 0.21 and 9.37 +/- 0.32, respectively. GS 283 inhibited Ca(2+)-induced contraction on guinea pig trachealis in Ca(2+)-free media. Furthermore, very high concentrations of GS 283 and verapamil completely abolished a phasic contraction induced by carbachol in Ca(2+)-free media, suggesting that verapamil and GS 283 can enter into the cytoplasm, where they may exert secondary actions on internal sites of the muscle, such as the sarcoplasmic reticulum. It is concluded that GS 283 has a Ca2+ antagonistic action along with weak histamine and muscarinic receptor blocking activity in isolated rat and guinea pig tracheal smooth muscle and its mode of action is likely inhibition of Ca2+ influx from plasma membrane and also release from the sarcoplasmic reticulum.