Favourable associations between the myosin heavy-chain and light-chain isoforms in human skeletal muscle

Histochemical and biochemical analyses were performed in order to examine the relationship between myosin light-chain (LC) isoforms and fibre-type distributions in whole human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis muscle in six healthy men, and analysed for the relative area occupied by each fibre type (percentage of fibre type area) and the molar ratio of each LC isoform. The percentage of type I fibre area was positively correlated with the molar ratio of slow LC (LC1s and LC2s) to total LC. The regression line was located below the line of unity. Also, the ratio of percentage of type II fibre area to that of type II fibre area was positively correlated with the molar ratio of the fast alkali LC LC1f to fast alkali LCs LC1f and LC3f. These results support previous study, having shown that in human skeletal muscle some type I fibres express various amounts of fast LC in addition to slow LC and suggest that fast myosin heavy-chain HCII a is favourably associated with LC1f, whereas HCIIb is favourably associated with LC3f.     

ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium

Ciliary beat frequency (CBF) was measured by video-optical microscopy in rat tracheal and distal airway ciliary cells using a slice preparation. In tracheal ciliary cells (tracheal slice), ATP or 2-methylthio ATP (MeSATP) increased CBF, which was inhibited by suramin (100 microm, an inhibitor of purinergic receptor). Ionomycin (5 microm) or thapsigargin (2 microm) increased CBF similarly. Ca2+-free solution or addition of Ni2+ (1 mm) decreased CBF gradually by approximately 25% and subsequent stimulation with ATP (10 microm) increased CBF transiently. The purinergic agonist experiments demonstrated that ATP increases CBF in tracheal ciliary cells via both P2X and P2Y receptors. ATP increased the intracellular calcium concentration ([Ca2+]i) in tracheal ciliary cells. However, in distal airway ciliary cells (lung slice), ATP did not increase CBF and [Ca2+]i, although a Ca2+-free solution decreased CBF, and ionomycin (5 microm) or thapsigargin (2 microm) increased it. Moreover, acetylcholine (100 microm) did not increase CBF in distal airway ciliary cells, although it increased CBF in tracheal ciliary cells. Terbutaline (10 microm), a selective beta2-adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca2+-mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. In conclusion, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.     

Effect of Bcl-2 antisense oligonucleotide on drug-sensitivity in association with apoptosis in undifferentiated thyroid carcinoma

Although attempts have been made to treat undifferentiated thyroid carcinoma using multidisciplinary therapeutic procedures including surgery, radiotherapy, and chemotherapy, the prognosis of undifferentiated thyroid carcinoma remains quite poor. New approaches to increase the sensitivity of patients to anticancer drugs and radiation will be needed to improve the survival rate for undifferentiated thyroid carcinoma. We examined the effect of Bcl-2 antisense oligonucleotide on drug-sensitivity in association with apoptosis in the 8305C undifferentiated thyroid carcinoma cell line. The drug sensitivity was evaluated by MTT assay for 48 h, while apoptosis was assessed according to the formation of internucleosomal DNA ladders. The Bcl-2 antisense was introduced into 8305C cells by using a 18-mer phosphorothioate oligonucleotide by lipopolyamine-mediated transfection twice for 12 h. The expression of apoptosis genes was assessed by Western blotting. The 8305C cells were sensitive to adriamycin (ADM), mitomycin (MMC), docetaxel (TXT), and paclitaxel (TXL), showing mean IC50 values of 0.72, 1.1, 1.3, and 4.1 microM, respectively. In contrast, the 8305C cells were resistant to cisplatin (CDDP) and 5-fluorouracil (5-FU), with mean IC50 values of 42.0 and 48.0 microM, respectively. Treatment with Bcl-2 antisense suppressed the protein level of Bcl-2 in 8305C cells in a dose-dependent manner up to 1.0 microM. Drug-sensitivity was increased by pretreatment with Bcl-2 antisense as assessed by the IC50 (x-fold): 0.48 (1.5-fold) in ADM; 0.42 (2.6-fold) in MMC, 0.56 (2.3-fold) in TXT, 1.5 (2.7-fold) in TXL, 8.6 (4.9-fold) in CDDP, and 25.0 (1.9-fold) in 5-FU, respectively. The increased drug-sensitivity was associated with the induction of apoptosis-related proteins, Fas, caspase 8, cytochrome c, caspase 3, and to subsequent apoptosis, as determined by the formation of internucleosomal DNA ladders and PARP in the treated cells. Susceptibility in apoptotic cell death following treatment with anticancer drugs was associated with induction of apoptosis-related genes in undifferentiated thyroid carcinoma cells, and induction of apoptosis was enhanced by pretreatment with Bcl-2 antisense oligonucleotide. These results imply a potential new strategy targeting an antiapoptotic protein, Bcl-2, by its antisense oligonucleotide for enhancement of chemotherapeutic efficacy in undifferentiated thyroid carcinomas.     

In vitro controllability of the MagScrew total artificial heart system

The purpose of this study was to evaluate the in vitro responses to preload and afterload of our total artificial heart (TAH), the MagScrew TAH. The TAH consists of two blood pumps and a control logic, developed at the Cleveland Clinic, OH, and the MagScrew actuator and its electronic control system, developed by Foster-Miller Technologies, Inc., Albany, NY. Tests were performed on a mock circulatory loop, using water as a test fluid. Preload sensitivity of the Mag-Screw TAH demonstrated a Frank-Starling response to preload in automatic mode. A peak flow of 10 L/min was obtained, with a left atrial pressure of 13 mm Hg. The relationship between right atrial pressure and left atrial pressure was well balanced when tested with a left bronchial shunt flow of 5% and a range of pulmonary artery and aortic pressures. With respect to afterload response, the left pump showed a relatively low sensitivity, which allowed the pump to maintain perfusion over a wide range of aortic pressures. The right pump, on the other hand, was much more sensitive to pulmonary artery pressure, which provided a measure of protection against pulmonary congestion. The very effective physiologic response of the MagScrew TAH is believed to result from employment of a left master, alternating ejection control logic, high inherent sensitivity of the blood pumps to atrial pressure, a lower effective stroke volume for the right pump, and a scaling of right side motor ejection voltage to 80% of that used for the left side ejection.     

Development of a new calcium phosphate cement that contains sodium calcium phosphate

A cement powder consisting of sodium calcium phosphate, Na3Ca6(PO4)5, in addition to tetracalcium phosphate and beta-tricalcium phosphate was prepared by pulverizing blocks of 4 wt% sodium-, 11 wt% carbonate-containing apatite samples that were heated at 1700 degrees C for 5 h. When mixed with 30 wt% malic acid or citric acid at a powder liquid ratio of 3:1, the cement set in 3 or 7 min at room temperature with compressive strength being around 52 or 27 MPa. In HeLa-cell cultures, the cement mixed with malic acid was less cytotoxic than the cement mixed with citric acid, which was far less cytotoxic than a commercial carboxylate cement used as a negative control, suggesting malic acid to be superior to citric acid as a liquid in this regard. Similar findings were also obtained with osteoclasts, of which culture experiments clearly suggested that the number of osteoclasts on the cement mixed with malic acid was significantly greater than that on the cement mixed with citric acid. Since osteoclastic response to substrates could be used as a maker in evaluating their bioresorbability associated with osteoclasts, the above finding may suggest that the cement that is to be mixed with malic acid would be more useful as bone substitutes.     

Influence of internal and external donors on activity and stereospecificity of ziegler-natta catalysts

Three kinds of MgCl₂-supported TiCl₄ catalysts (ester-free, ethyl benzoate (EB, monoester) and dibutyl phthalate (DNBP, diester) as internal donors) were prepared. Polymerization of propylene was carried out using these catalysts with and without phenyltriethoxysilane (PTES) as an external donor. Significant differences were observed in the kinetic behavior of polymerization among ester-free, monoester, and diester systems. Addition of PTES, however, did not change the kinetic behavior. The polymers produced were separated into three fractions by extraction with boiling heptane and octane, and the molar mass distribution (MMD) curves were measured for these three fractions. The heptane-insoluble, isotactic polymer obtained in the absence of PTES was found to consist of two fractions, octane-soluble and -insoluble. The octane-soluble polymer having lower molar mass contained appreciable amounts of syndiotactic stereoblocks in the chains, whereas the octane-insoluble polymer did not contain such stereoblocks. Addition of PTES drastically increased the octane-insoluble fraction without changing the average molar mass. On the other hand, the MMD curve for the heptane-soluble, atactic polymer gradually shifted to the lower molar mass range with an increase in the concentration of PTES. From these results, the effects of internal and external donors on catalyst activity and stereospecificity were discussed putting emphasis on the differences between monoesters and diesters.     

Preemptive analgesia by zaltoprofen that inhibits bradykinin action and cyclooxygenase in a post-operative pain model

The post-operative pain state results from a barrage of primary afferent inputs exposed to products of tissue damage such as bradykinin and prostaglandins and the central sensitization by the continuing inputs. This provides the rationale for preemptive analgesia, whereby the blockade of primary afferent inputs prior to injury may result in a reduction of post-operative pain. 2-(10,11-dihydro-10-oxo-dibenzo[b,f]thiepin-2-yl) propionic acid (zaltoprofen) is a unique compound that inhibits cyclooxygenase (COX) and exhibits anti-bradykinin activity. The present study evaluated the preemptive analgesic effect of zaltoprofen in a post-operative pain model produced by plantar incision. When orally, but no intrathecally, administered 30 min prior to incision, zaltoprofen significantly increased the withdrawal threshold 2 h and 1-3 days after incision at 10 mg/kg. While the bradykinin B1 antagonist des-Arg10-HOE-140, the selective COX-1 inhibitor SC-560, and the selective COX-2 inhibitor celecoxib did not affect post-operative pain, the B2 antagonist HOE-140 dose-dependently relieved the post-operative pain at 2-200 microg/kg with a time course similar to that of zaltoprofen. The B2 receptor mRNA was expressed in the hindpaw and the expression did not change before and 24 h after surgery. These results suggest that zaltoprofen produces the preemptive analgesic effect peripherally by blocking the B2 pathway.     

Ultrastructural localization of laminin-5 (gamma2 chain) in the rat peri-implant oral mucosa around a titanium-dental implant by immuno-electron microscopy

Laminin-5 (Ln-5) is an important molecule associated with epithelial cell adhesion and migration. In the gingiva around the tooth, Ln-5 localizes within basement membranes between the junctional epithelium (JE) and the tooth or connective tissue. Recently, we reported that in the oral mucosa around a dental implant, Ln-5 is expressed within the basement membranes at the implant-peri-implant epithelium (PIE) interface, and at the PIE-connective tissue interface. However, the ultrastructural localization of Ln-5 within or along the PIE has not yet been reported. Therefore, peri-implant oral mucosa was treated with anti-Ln-5 (gamma2 chain) antibody and examined using immuno-electron microscopy. Ln-5 was localized in the cells of the innermost-third layer and basal layer of the PIE. A 100-nm-wide Ln-5-positive internal basal lamina (basement membrane) and hemidesmosomes as adhesion structures were formed at the apical portion of the implant-PIE interface. However, at the upper-middle portion of the interface, these adhesion structures were not observed. Furthermore, at the PIE-connective tissue interface, the Ln-5-positive external basal lamina (basement membrane) and hemidesmosomes were partially deficient. Judging from these findings, we concluded that Ln-5 contributes to the attachment of the PIE to the titanium surface, and that PIE attached to titanium at the apical portion of the dental implant-PIE interface.     

Pneumocystis carinii carriage in immunocompromised patients with and without human immunodeficiency virus infection

Eighty-one bronchoalveolar lavage (BAL) specimens obtained from 26 HIV-infected, 45 non-HIV immunosuppressed and 10 immunocompetent patients with primary pulmonary diseases were analysed for the presence of Pneumocystis carinii by staining and by P. carinii 5S rDNA determined by PCR. P. carinii was observed by staining of BAL specimens from HIV-infected patients significantly more frequently than those from immunocompromised hosts without HIV infection (57.7% versus 20.0%, respectively). P. carinii 5S rDNA was detected by PCR assay in seven (26.9%) HIV-infected individuals, which was significantly more frequent than for four (8.9%) immunosuppressed patients without HIV infection, for whom staining was negative. None of these patients developed P. carinii pneumonia (PCP) within the follow-up period. BAL specimens from 10 immunocompetent patients with pulmonary disorders were negative for PCP by both staining and PCR assay.