Relationships between resilience and quality of life in parents of children with cancer.

CLINICALTRIALS.GOV ID: NCT03631485.

Adventure-based training to promote physical activity and reduce fatigue among childhood cancer survivors: A randomized controlled trial.

BACKGROUND: Cancer-related fatigue is one of the most distressing symptoms reported by childhood cancer survivors. Despite the body of evidence that regular physical activity helps alleviate cancer-related fatigue, insufficient participation in physical activity is frequently observed among childhood cancer survivors. OBJECTIVES: This study examined the effectiveness of an adventure-based training programme in promoting physical activity, reducing fatigue, and enhancing self-efficacy and quality of life among Hong Kong Chinese childhood cancer survivors. DESIGN: A prospective randomised controlled trial. SETTINGS: A paediatric oncology outpatient clinic, a non-governmental organisation, and a non-profit voluntary organisation. PARTICIPANTS: Hong Kong Chinese childhood cancer survivors aged 9-16 years who reported symptoms of fatigue and had not engaged in regular physical exercise in the past 6 months. METHODS: The experimental group underwent a 4-day adventure-based training programme. The control group received a placebo intervention. The primary outcome was fatigue at 12 months. Secondary outcomes were physical activity levels, self-efficacy and quality of life at 12 months. Data collection was conducted at baseline, and 6 and 12 months after the intervention began. We performed intention-to-treat analyses. RESULTS: From 6 January, 2014 to 8 June, 2015, we randomly assigned 222 eligible childhood cancer survivors to either an experimental (n = 117) or a control group (n = 105). The experimental group showed statistically significantly lower levels of cancer-related fatigue (P < 0.001), higher levels of self-efficacy (P < 0.001) and physical activity (P < 0.001), and better quality of life (P < 0.01) than the control group at 12 months. CONCLUSIONS: This study provides evidence that adventure-based training is effective in promoting physical activity, reducing cancer-related fatigue, and enhancing self-efficacy and quality of life among Hong Kong Chinese childhood cancer survivors. These results may help inform parents and healthcare professionals that regular physical activity is crucial for the physical and psychological wellbeing and quality of life of childhood cancer survivors.

The in vitro protection of human decay accelerating factor and hDAF/heme oxygenase-1 transgenes in porcine aortic endothelial cells against sera of Formosan macaques.

To mitigate hyperacute rejection, pigs have been generated with alpha-Gal transferase gene knockout and transgenic expression of human decay accelerating factor (hDAF), MCP, and CD59. Additionally, heme-oxygenase-1 (HO-1) has been suggested to defend endothelial cells. Sera (MS) (0%, 1%, 5%, 10%, and 15%) from Formosan macaques (Macaca cyclopis, MC), an Old World monkey wildly populated in Taiwan, was used to test the protective in vitro, effects of hDAF or hDAF/hHO-1 on porcine aortic endothelial cells (pAEC) derived from hDAF(+), hDAF(+)/hHO-1(+), and hDAF(+)/hHO-1(-) and 1 nontransgenic pAEC. Ten percent human serum (HS) served as a positive control. When MS addition increased to 10% or 15%, all transgenic pAEC exhibited a greater survival than nontransgenic pAEC. Noticeably, 15% MS reduced survived to <10% versus >40% in nontransgenic and transgenic pAEC, respectively. These results revealed that hDAF exerted protective effects against MC complement activation. However, comparing with 10% MS and HS in pAEC of nontransgenic pigs, the survivability was higher in HS, suggesting that complement activation by MS was more toxic than that by HS. Furthermore, hDAF(+)/hHO-1(+) showed no further protection against effects of MS on transgenic pAEC.

Long-term survival of HLA-DR15+ pig skin in SCID mice after reconstitution with human peripheral blood mononuclear cells and under short-term immunosuppression.

In allotransplantation, donor-recipient human leukocyte antigen (HLA) matches improve graft survival. For studies of the role of donor-recipient HLA II matching on xenotransplantation, we successfully generated HLA-DR15+ transgenic pigs the the skins of which were transplanted to SCID mice, which were thereafter reconstituted with HLA-DR15+ or -DR15(-) hPBMC. Cyclosporine was given intraperitoneally to SCID mice for 12 days. Human T cells were observed in SCID mice after reconstitution. Mixed lymphocytes responses showed greater responses by HLA-DR15(-) human peripheral blood mononuclear cells (hPBMC) against HLA-DR15+ porcine PBMC. HLA-DR15+ porcine skins survived more than 100 days in all SCID mice. HLA-DR15+ porcine skins were rejected in all non-SCID (Balb/c) mice. The histologic pictures of transplanted HLA-DR15+ porcine skins showed surviving porcine epithelium in remodeling murine dermis and little lymphocyte infiltration into the murine dermis. The long-term survival of HLA-DR15+ pig skin in all hPBMC-SCID mice might be due to poor engraftment or function of reconstituted T cells. Further studies are needed to clarify the role of donor-recipient matching of HLA-DR15.

The pathology of systemic iridoviral disease in fish.

Iridoviruses have been associated with severe disease and economic loss in farmed food fish and ornamental fish, with mortality often reported to reach 50% or more. In the present study, three tropical marine food fish species and four tropical freshwater ornamental fish species with systemic iridovirus infections were examined histopathologically and ultrastructurally. Light microscopy consistently revealed pale to intensely basophilic hypertrophied virus-infected cells in spleen, kidney and intestine from all seven species. Ultrastructural examination showed changes in the vascular endothelium overlying hypertrophied virus-infected cells suggestive of pressure necrosis. Viral isolation was improved by the use of fibroblastic cell lines. This, together with the sub-endothelial location of infected cells in all infected species examined, suggests that systemic iridoviruses are mesotheliotropic.

Missense C168T in the Wiskott--Aldrich Syndrome protein gene is a common mutation in X-linked thrombocytopenia.

We describe a large Syrian--Lebanese family who clinically manifest X-linked thrombocytopenia (XLT). To date, five family members have undergone splenectomy with rapid and sustained normalization of their platelet numbers. Genomic analysis demonstrated that affected men in this cohort had the missense C168T (Thr45Met) mutation in exon 2 of the Wiskott-Aldrich Syndrome protein (WASp) gene. Exon 2 is the commonest site for mutations associated with XLT and mild forms of WAS, and the C168T missense mutation is the most frequent. Detection of this mutation by restriction enzyme digestion provides an efficient screening test for prompt identification and for assessment of female carrier status.

Current trends and new approaches in the management of diabetes mellitus.

Current trends in the management of type 2 diabetes mellitus, based on the 20-year United Kingdom Prospective Diabetic Study, include intensive treatment to control the blood glucose level and blood pressure in order to prevent or delay microvascular and cardiovascular complications. In the new millennium, type 2 diabetes will become epidemic in developing countries. If diabetes were to develop in 10% of the 1.2 billion population of China, the expense of intensive treatment would be immense. Laboratory tests are useful for detecting risk factors before the onset of the disease and convincing the general public to take preventive measures. Glucose tolerance testing is one of these tests. When glucose tolerance is impaired, 25% of beta-cell function is lost. Determining the plasma proinsulin level is another useful evaluation; impaired glucose tolerance accompanied by increased plasma proinsulin level is indicative of an enhanced risk that type 2 diabetes will develop within 5 years. Educating the public about eating a healthy diet and exercising may prevent the development of diabetes and thereby reduce the global prevalence of type 2 diabetes.

Regulation of Ca(2+)-activated K+ currents by ciglitazone in rat pituitary GH3 cells.

BACKGROUND: Ciglitazone, an antidiabetic agent of the thiazolidinedione family, is known to be an activator of the peroxisome-proliferator activator receptor (PPAR)-gamma. The underlying mechanism of ciglitazone actions on ionic currents in neuroendocrine cells remains unclear. METHODS: The effects of ciglitazone on ionic currents were investigated in rat pituitary GH3 cells using the whole-cell and inside-out configurations of the patch-clamp technique. RESULTS: In GH3 cells, ciglitazone at 3-300 mumol/L caused a reversible increase in the amplitude of the Ca(2+)-activated K+ current (IK(Ca)) with a half-maximal concentration of 16 mumol/L. Under the inside-out patch recording mode, ciglitazone applied intracellularly increased the activity of the large-conductance Ca(2+)-activated K+ (BKCa) channels, but did not affect their single-channel conductance. However, troglitazone (30 mumol/L) caused a reduction in the channel activity. The ciglitazone-induced change in the kinetic behavior of BKCa channels is due to an increase in mean open time and a decrease in mean closed time, whereas the troglitazone-induced decrease in the channel activity is related to a decrease in mean open time and an increase in mean closed time. Ciglitazone caused a left shift in the midpoint for voltage-dependent opening. The ciglitazone-stimulated activity of BKCa channels is independent of internal Ca2+. Under the current clamp mode, ciglitazone (30 mumol/L) hyperpolarized the membrane potential. CONCLUSIONS: This study shows that in addition to its activation of PPAR-gamma, ciglitazone can stimulate the activity of BKCa channels expressed in GH3 cells. These effects may affect membrane potentials and contribute to the ciglitazone-induced change in the functional activity of neurons or neuroendocrine cells.

An Asian multicenter clinical trial to assess the efficacy and tolerability of acarbose compared with placebo in type 2 diabetic patients previously treated with diet. Asian Acarbose Study Group.

OBJECTIVE: To assess the efficacy, safety, and tolerability of acarbose versus placebo during a 24-week treatment period in Asian type 2 diabetic patients with dietary failure. RESEARCH DESIGN AND METHODS: After a 6-week screening period, 126 multiethnic Asian type 2 diabetic patients (64 men, 62 women; mean age +/- SD, 53.4 +/- 10 years) were randomized to receive acarbose (n = 63) or placebo (n = 63). The dosage was increased from 50 mg t.i.d. at week 0 to 100 mg t.i.d. at week 4. Patients were then followed up at weeks 10, 16, and 24. At each visit, body weight, blood pressure, and metabolic indexes were measured. At weeks 0 and 24, fasting plasma glucose and insulin were measured before and 1 h after the administration of an individually tailored breakfast. RESULTS: Using the intention-to-treat analysis, there were greater reductions in (mean [95% CI]) HbA1c (-0.70 [-1.00 to -0.39] vs. -0.27% [-0.54 to 0]; P = 0.04), fasting plasma glucose (-0.37 [-0.75 to 0.02] vs. 0.41 mmol/l [-0.08 to 0.90]; P = 0.017) and 1-h plasma glucose (-0.77 [-1.44 to -0.10] vs. 0.65 mmol/l [-0.07 to 1.36]; P = 0.05) in the acarbose group compared with the placebo group. With acarbose treatment, 78% of patients achieved an HbAlc < 8% compared with 56% in the placebo group (P = 0.003). There was a greater reduction in body weight (-1.31 [-2.46 to -0.15] vs. 0.16 kg [-3.36 to 0.10]; P = 0.02) and higher incidence of flatulence (56 vs. 37%; P = 0.032) in the acarbose than in the placebo group. Using baseline HbA1c and race as covariates, there were no significant interethnic differences in treatment responses (P = 0.232 for treatment-race interaction; P < 0.001 for treatment effect). The dropout rates were similar between the two groups (acarbose, 11 of 63; placebo, 6 of 63). There were no significant laboratory adverse events in either group. CONCLUSIONS: In this multicenter study involving six ethnic groups, acarbose 100 mg t.i.d. was an effective, safe, and generally well-tolerated therapy in Asian type 2 diabetic patients with dietary failure. In some patients with troublesome gastrointestinal symptoms, a lower dosage may be necessary.

Simulation of the enzyme reaction mechanism of malate dehydrogenase.

A hybrid numerical method, which employs molecular mechanics to describe the bulk of the solvent-protein matrix and a semiempirical quantum-mechanical treatment for atoms near the reactive site, was utilized to simulate the minimum energy surface and reaction pathway for the interconversion of malate and oxaloacetate catalyzed by the enzyme malate dehydrogenase (MDH). A reaction mechanism for proton and hydride transfers associated with MDH and cofactor nicotinamide adenine dinucleotide (NAD) is deduced from the topology of the calculated energy surface. The proposed mechanism consists of (1) a sequential reaction with proton transfer preceding hydride transfer (malate to oxaloacetate direction), (2) the existence of two transition states with energy barriers of approximately 7 and 15 kcal/mol for the proton and hydride transfers, respectively, and (3) reactant (malate) and product (oxaloacetate) states that are nearly isoenergetic. Simulation analysis of the calculated energy profile shows that solvent effects due to the protein matrix dramatically alter the intrinsic reactivity of the functional groups involved in the MDH reaction, resulting in energetics similar to that found in aqueous solution. An energy decomposition analysis indicates that specific MDH residues (Arg-81, Arg-87, Asn-119, Asp-150, and Arg-153) in the vicinity of the substrate make significant energetic contributions to the stabilization of proton transfer and destabilization of hydride transfer. This suggests that these amino acids play an important role in the catalytic properties of MDH.

Regulation of thyroid hormones in the secretion of insulin and gastric inhibitory polypeptide in male rats.

The effect of thyroid hormones on glucose-induced secretion of gastric inhibitory polypeptide (GIP) and insulin was studied. Male rats were thyroidectomized (Tx) or sham Tx. Sham Tx rats were injected with either propylthiouracil ([PTU] 20 mg/kg intraperitoneally) or saline for 2 weeks. In addition, thyroid-intact rats were injected intravenously with triiodothyronine ([T3]5 microg/kg) or saline 10 minutes before an oral glucose load (3.2 g/kg). Blood samples were collected from each animal via a jugular catheter at 0, 10, 20, 30,45, 60, and 90 minutes following glucose ingestion. Plasma levels of GIP and insulin were measured by specific radioimmunoassays (RIAs). Thyroidectomy-induced hypothyroidism increased the basal level of plasma GIP, but decreased that of insulin. Insulin levels at 10, 20, and 30 minutes following oral glucose were lower in hypothyroid rats than in euthyroid rats. Conversely, GIP levels at 60 and 90 minutes following glucose ingestion in PTU-induced hypothyroid rats were higher than those in euthyroid rats. Furthermore, glucose-stimulated insulin secretion was unaltered by pretreatment with T3, whereas the glucose-induced increase in plasma GIP was completely abolished by preinjection of T3 in thyroid-intact rats. These results suggest that thyroid functions are involved in the regulation of insulin and GIP secretion in rats.

Progress toward chemical accuracy in the computer simulation of condensed phase reactions.

We describe a procedure for the generation of chemically accurate computer-simulation models to study chemical reactions in the condensed phase. The process involves (i) the use of a coupled semiempirical quantum and classical molecular mechanics method to represent solutes and solvent, respectively; (ii) the optimization of semiempirical quantum mechanics (QM) parameters to produce a computationally efficient and chemically accurate QM model; (iii) the calibration of a quantum/classical microsolvation model using ab initio quantum theory; and (iv) the use of statistical mechanical principles and methods to simulate, on massively parallel computers, the thermodynamic properties of chemical reactions in aqueous solution. The utility of this process is demonstrated by the calculation of the enthalpy of reaction in vacuum and free energy change in aqueous solution for a proton transfer involving methanol, methoxide, imidazole, and imidazolium, which are functional groups involved with proton transfers in many biochemical systems. An optimized semiempirical QM model is produced, which results in the calculation of heats of formation of the above chemical species to within 1.0 kcal/mol (1 kcal = 4.18 kJ) of experimental values. The use of the calibrated QM and microsolvation QM/MM (molecular mechanics) models for the simulation of a proton transfer in aqueous solution gives a calculated free energy that is within 1.0 kcal/mol (12.2 calculated vs. 12.8 experimental) of a value estimated from experimental pKa values of the reacting species.

Aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation with isolated hepatocytes from rats and hamsters.

Binding of aflatoxin B1 (AFB1) to DNA and AFB1-glutathione conjugation during the metabolism of AFB1 have been examined with freshly isolated hepatocytes from male Fischer rats and Syrian hamsters. Even though there was no significant difference in cytochrome P450 and glutathione contents, there were marked differences in the metabolism of AFB1 (33 nM) in hepatocytes from these two species. Thus, AFB1-DNA binding was six-fold higher in the rat than in hamster hepatocytes, whereas AFB1-glutathione conjugation was 12-fold higher in hamster than in rat hepatocytes. The addition of 0.5 mM diethylmaleate had no significant effect in rats, whereas its presence produced a nine-fold increase in AFB1-DNA binding with 85% inhibition of thiol conjugation in hamster hepatocytes. Styrene oxide (1 mM) produced 50% and 25-fold increases in AFB1-DNA binding in rat and hamster hepatocytes, respectively, with corresponding decreases in thiol conjugation. Triethyltin bromide (50 microM) inhibited both processes by 50% in rat hepatocytes, whereas it produced a nine-fold increase in AFB1-DNA binding with a concomitant decrease in thiol conjugation in hamster hepatocytes. These results suggest that glutathione S-transferases play a more significant role in modulating AFB1-DNA binding in hamster than in rat hepatocytes.

Effect of butylated hydroxyanisole pretreatment on aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation in isolated hepatocytes from rats.

The effect of 2(3)-tert-butyl-4-hydroxyanisole (BHA) pretreatment of rats on both aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione has been examined with isolated hepatocytes and in intact rats. Young male F344 rats were fed AIN-76A diet with or without 0.75% BHA for 2 weeks. Even though there were no significant differences in either cytochrome P-450 or reduced glutathione contents, there were marked differences in AFB1 metabolism in isolated hepatocytes from these two groups. Thus, at the 33 nM AFB1 level, AFB1-DNA binding was 3-fold higher in control compared to BHA-treated hepatocytes whereas AFB1-glutathione conjugation was 5-fold higher in treated compared to controls. Even at higher AFB1 concentrations (2 and 10 microM), DNA binding was 4-6-fold higher in controls whereas thiol conjugation was 5-9-fold higher in treated compared to control hepatocytes. Addition of 0.5-1.0 mM diethylmaleate did not have any significant effect in control hepatocytes whereas its presence produced about 70-100% increase in DNA binding with 65-80% inhibition of thiol conjugation in treated hepatocytes. Addition of 1 mM styrene oxide caused 75-100% and 4-8-fold increase in AFB1-DNA binding in control and treated hepatocytes, respectively, with corresponding decreases in thiol conjugation. In intact rats, BHA treatment reduced hepatic AFB1-DNA binding to 15% of controls with concomitant increase in biliary excretion of AFB1-reduced glutathione conjugate. It appears that the induced cytosolic GSH S-transferases after BHA treatment of rats play a significant role in inhibiting hepatic AFB1-DNA binding and AFB1 hepatocarcinogenesis presumably by inactivation of the reactive AFB1-epoxide.

A mechanism of inhibition of aflatoxin B1-DNA binding in the liver by phenobarbital pretreatment of rats.

The effect of phenobarbital (PB) pretreatment of rats on both hepatic aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione (AFB1-SG) conjugation have been examined in studies in vivo and in vitro. Male Sprague-Dawley rats fed a commercial diet with 0.1% PB in their drinking water for 1 week had total wet liver weight and microsomal protein content about 27% and 38% higher, respectively, than controls. Hepatic cytochrome P-450 content, microsomal cytochrome P-450 mediated AFB1 binding to exogenous DNA and formation of hydroxy metabolites of AFB1 were also about threefold higher in PB-treated rats and cytosolic reduced glutathione S-transferase activities were about doubled. Microsome-mediated AFB1-DNA binding, when examined at 2 microM and 10 microM levels of AFB1, was inhibited two-to threefold more by cytosols of treated rats whereas AFB1-SG conjugation was two- to threefold higher by cytosols of treated rats. In reconstitution experiments with 2 microM AFB1, with intact nuclei serving as a source of endogenous DNA, addition of microsomes from either group generated a large amount of AFB1-DNA binding (68-105 pmol) and a smaller amount of AFB1-SG conjugate (12-21 pmol). The presence of cytosol from the controls reduced AFB1-DNA binding to a much lesser extent than the cytosol from the treated group whereas AFB1-SG conjugation was much higher with the cytosol from the treated group. These results are in agreement with the studies in vivo. In isolated hepatocytes at 33 nM, 2 microM and 10 microM AFB1 levels, AFB1-DNA binding was decreased 50 to 70% by prior PB-treatment whereas AFB1-SG conjugation was two- to threefold higher in treated compared to control hepatocytes. In hepatocytes, addition of 1 mM diethylmaleate increased DNA binding two- to threefold with a corresponding decrease in AFB1-SG conjugation. Addition of 1 mM styrene oxide caused 5- to 10-fold increases in AFB1-DNA binding at levels of AFB1 of 33 nM and 2 microM; but at 10 microM AFB1, increases in AFB1-DNA binding were two- to threefold. In intact rats, PB treatment reduced hepatic AFB1-DNA binding to 30% of controls with concomitant increase in biliary excretion of AFB1-SG conjugate. It appears that the induced cytosolic GSH S-transferases after PB treatment of rats plays a significant role in inhibiting hepatic AFB1-DNA binding and hepatocarcinogenesis presumably by inactivation of the reactive AFB1-epoxide.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of butylated hydroxyanisole pretreatment on in vitro hepatic aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation in rats.

The effect of 3(2)-tert-butyl-4-hydroxyanisole (BHA) pretreatment of rats on both in vitro hepatic aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione (AFB1-SG) conjugation has been examined. For these studies, young male F344 rats were fed AIN-76 A diet with or without 0.75% BHA for 2 weeks. There were no significant differences either in microsomal cytochrome P-450 content or microsome-mediated exogenous DNA binding to AFB1 with cytochrome P-450 from control or BHA-treated animals. There were large differences in reduced glutathione S-transferase activity with treated cytosols showing 2.5-fold higher activity than the controls. Hepatic reduced glutathione levels were 25% higher in treated than in controls. Kinetics of cytosolic inhibition of microsome-mediated AFB1-DNA binding and formation of AFB1-SG conjugate when examined at two levels of AFB1 (2 and 10 microM) and a 4-fold range of cytosolic concentrations showed that inhibition of AFB1-DNA binding was greater with cytosol from the treated compared to the controls. However, AFB1-SG conjugation was 3- to 4-fold greater in treated than in controls. Inhibition of AFB1-DNA binding by cytosol was reversed in the presence of 1 mM level of various epoxides with concomitant inhibition of AFB1-SG conjugation. In reconstitution studies with 2 microM AFB1, intact nuclei alone from either group did not yield significant amounts of either DNA binding or AFB1-SG conjugation. However, addition of microsomes from either group to these nuclei generated a large amount of AFB1-DNA binding (82-111 pmol) and a smaller amount of AFB1-SG conjugate (9-28 pmol). The presence of cytosols from the control group reduced AFB1-DNA binding to a much lesser extent than the cytosols from the treated group. However, AFB1-SG conjugation was much higher with the cytosol from treated than with the controls. These reconstitution studies with endogenous DNA show more AFB1-DNA binding with the control than with BHA-treated animals and are in agreement with the studies in vivo. It appears that induced levels of cytosolic reduced glutathione S-transferase modulate AFB1-DNA binding and AFB1 hepatocarcinogenesis.

Effect of thyroidectomy on the release of prolactin in vitro response to calcitonin.

Chinese J. Physiol. 31(2):105-112, 1988. The effect of calcitonin (CT) on the release of prolactin (PRL) and thyrotropin (TSH) in vitro from anterior pituitary glands (APs) of thuroidectomized (Tx) and intact male rats was investigated. Male rats were Tx or sham Tx, then sacrificed 47 days later. APs were removed, quartered and incubated with CT (0 or 32 microIU/ml) and/or thyroxine (0 or 0.1 microgram/ml) at 37 degrees C for 4 h. Thyroidectomy significantly decreased PRL release in vitro. CT inhibited the release of PRL in vitro from APs of intact rats but increased PRL release from APs of Tx animals. Neither CT nor T4 affected the release of TSH in vitro. These results suggest that the modulation of CT on the release of PRL from rat anterior pituitary glands is thyroxine-dependent.

Assessment of the relative locations of the greater palatine foramen in adult Chinese skulls.

One hundred dry skulls of adult Chinese of both sexes were studied. They were homogeneous in the form of maxillary arch and having full eruption of the upper third molar, without missing teeth and malposition of teeth. Our findings revealed that the mean distance from the center of the greater palatine foramen (GPF) to the midsagittal plane of the hard palate was 16.00 mm, and to the posterior border of the hard palate, 4.11 mm. The location of the GPF related to the maxillary molars was expressed as percentage in 5 relations. We found that the most common location of the GPF was between the maxillary second and third molars (relation III: 48%), and less common was lingual to the maxillary third molar (relation IV: 33.5%). The usually accepted description of the GPF location was lingual to the second molar (relation II), but in our study this relative position occurred in only 17% of the skulls. The long axis of the greater palatine canal directing to the GPF in the oral cavity was found to be directed anteriorly in 181 openings (90.5%) of the 200 GPF, and only 19 openings (9.5%) directed vertically. The bilateral symmetry of GPF on both sides of each skull was remarkable. The discrepancy of our observations on the Chinese skulls from those on other ethnic groups was discussed. Our findings suggest, therefore, the existence of an ethnic variation and the necessity of a more accurate method of locating the GPF in clinical practice.

Enzymatic methylation of in vitro synthesized apocytochrome c enhances its transport into mitochondria.

The gene for iso-1-cytochrome c from Saccharomyces cerevisiae was recloned into a pSP65 vector containing an active bacteriophage SP promoter. The iso-1-cytochrome c gene was cloned as an 856-base pair XhoI-HindIII fragment. When the resulting plasmid was digested at the HindIII site 279 bases downstream from the termination codon of the gene and transcribed in vitro using SP6 RNA polymerase, full length transcripts were produced. The SP6 iso-1-cytochrome c mRNA was translated using a rabbit reticulocyte lysate system, and the protein products were analyzed on sodium dodecyl sulfate-polyacrylamide gels. One major band with a molecular weight of 12,000 was detected by autofluorography and coincided with the Coomassie staining band of apocytochrome c from S. cerevisiae. The product was also shown to be identical with that of standard yeast apocytochrome c on an isoelectrofocusing gel. The in vitro synthesized iso-1-apocytochrome c was enzymatically methylated by adding partially purified S-adenosyl-L-methionine:cytochrome c-lysine N-methyltransferase (protein methylase III, EC 2.1.1.59) from S. cerevisiae along with S-adenosyl-L-methionine to the in vitro translation mixtures. The methylation was shown to be inhibited by the addition of the methylase inhibitor S-adenosyl-L-homocysteine or the protein synthesis inhibitor puromycin. The principal type of methylated amino acid in the protein was found to be epsilon-N-trimethyllysine which accounted for 77% of the total. Finally, the methylation of in vitro synthesized iso-1-apocytochrome c was found to increase its import into mitochondria isolated from S. cerevisiae 2-4-fold over unmethylated protein, but not into rat liver mitochondria. This suggests that methylation facilitates the import of apocytochrome c into mitochondria by a specific receptor mechanism.