[Efficacy and safety of multiple-dose intravenous tranexamic acid for reducing blood loss in complex tibial plateau fractures: A prospective randomized controlled trial].

Objective: To investigate the efficacy and safety of multiple-dose intravenous tranexamic acid (TXA) for reducing blood loss in complex tibial plateau fractures with open reduction internal fixation by a prospective randomized controlled trial. Methods: A study was conducted on patients with Schatzker type Ⅳ-Ⅵ tibial plateau fractures admitted between August 2020 and December 2022. Among them, 88 patients met the selection criteria and were included in the study. They were randomly allocated into 3 groups, the control group (28 cases), single-dose TXA group (31 cases), and multiple-dose TXA group (29 cases), using a random number table method. There was no significant difference ( P>0.05) in terms of age, gender, body mass index, the Schatzker type and side of fracture, laboratory examinations [hemoglobin (Hb), activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen (Fib), international normalized ratio (INR), D-dimer, and interleukin 6 (IL-6)], and preoperative blood volume. The control group received intravenous infusion of 100 mL saline at 15 minutes before operation and 3, 6, and 24 hours after the first administration. The single-dose TXA group received intravenous infusion of 1 g TXA (dissolved in 100 mL saline) at 15 minutes before operation, followed by an equal amount of saline at each time point after the first administration. The multiple-dose TXA group received intravenous infusion of 1 g TXA (dissolved in 100 mL saline) at each time point. The relevant indicators were recorded and compared between groups to evaluate the effectiveness and safety of TXA, including hospital stays, operation time, occurrence of infection; the occurrence of lower extremity deep vein thrombosis, intermuscular vein thrombosis, and pulmonary embolism at 1 week after operation; the lowest postoperative Hb value and Hb reduction rate, the difference (change value) between pre- and post-operative APTT, PT, Fib, and INR; D-dimer and IL-6 at 24 and 72 hours after operation; total blood loss, intraoperative blood loss, hidden blood loss, drainage flow during 48 hours after operation, and postoperative blood transfusion. Results: ① TXA efficacy evaluation: the lowest Hb value in the control group was significantly lower than that in the other two groups ( P<0.05), and there was no significant difference between the single- and multiple-dose TXA groups ( P>0.05). The Hb reduction rate, total blood loss, intraoperative blood loss, drainage flow during 48 hours after operation, and hidden blood loss showed a gradual decrease trend in the control group, single-dose TXA group, and multiple-dose TXA group. And differences were significant ( P<0.05) in the Hb reduction rate and drainage flow during 48 hours after operation between groups, and the total blood loss and hidden blood loss between control group and other two groups. ② TXA safety evaluation: no lower extremity deep vein thrombosis or pulmonary embolism occurred in the three groups after operation, but 3, 4, and 2 cases of intermuscular vein thrombosis occurred in the control group, single-dose TXA group, and multiple-dose TXA group, respectively, and the differences in the incidences between groups were not significant ( P>0.05). There was no significant difference in the operation time between groups ( P>0.05). But the length of hospital stay was significantly longer in the control group than in the other groups ( P<0.05); there was no significant difference between the single- and multiple-dose TXA groups ( P>0.05). ③ Effect of TXA on blood coagulation and inflammatory response: the incisions of the 3 groups healed by first intention, and no infections occurred. The differences in the changes of APTT, PT, Fib, and INR between groups were not significant ( P>0.05). The D-dimer and IL-6 in the three groups showed a trend of first increasing and then decreasing over time, and there was a significant difference between different time points in the three groups ( P<0.05). At 24 and 72 hours after operation, there was no significant difference in D-dimer between groups ( P>0.05), while there was a significant difference in IL-6 between groups ( P<0.05). Conclusion: Multiple intravenous applications of TXA can reduce perioperative blood loss and shorten hospital stays in patients undergoing open reduction and internal fixation of complex tibial plateau fractures, provide additional fibrinolysis control and ameliorate postoperative inflammatory response.

Rapid DNA interstrand cross-linking of Pt(IV) compound.

Pt(IV) anticancer compounds have been developed for several decades to overcome the drawbacks of their Pt(II) congeners, and the reduction of Pt(IV) to Pt(II) has been commonly regarded as a necessary step in the activation of Pt(IV) compounds prior to targeting DNA. However, blockage of glutathione (GSH) biosynthesis resulted in a slight effect on the cytotoxicity of oxoplatin in yeast Saccharomyces cerevisiae strains, urging us to reconsider the mechanism of actions for the "inert" Pt(IV) complexes. Using X-ray absorption near-edge spectroscopy (XANES), our data demonstrated that Pt(IV) complex oxoplatin could bind to DNA in a tetravalent state. Both alkaline denaturing agarose electrophoresis and thermal denaturation-renaturation assay revealed that oxoplatin could rapidly produce stable interstrand crosslinks (ICLs), which can further translate into a fast cell-killing process in cancer cells. Using quantitative real-time PCR and immunofluorescence analysis, we also proved that Pt(IV) complex oxoplatin could induce a quick intracellular response of the FA/BRCA pathway in cancer cells that involves the DNA interstrand crosslinking repair system, and this quick response to ICLs was independent with the intracellular GSH levels. Cell cycle analysis showed that short incubation with oxoplatin can induce a strong S phase arrest in HeLa cells, indicating that the rapid interstrand crosslinks produced by oxoplatin might stall the replication fork, result in the double-strand breaks, and eventually induce cell death. Our results implied that, besides the reduction mechanism to release the Pt(II) congeners, direct and rapid interstrand cross-linking with DNA by Pt(IV) compounds might be a unique mechanism for Pt(IV) compounds, which may provide new insight for the development of next-generation platinum-based drugs.

MIDCAB versus off-pump CABG: Comparative study.

OBJECTIVE: To investigate the clinical significance of minimally invasive direct coronary artery bypass (MIDCAB). METHODS: Three hundred patients admitted to the Second Hospital of Shandong University from March 2006 to February 2010 were retrospectively analyzed and received off-pump operation under general anesthesia. Depending on surgical methods, patients were divided into the MIDCAB group (n = 90) and the median sternotomy off-pump coronary artery bypass (OPCAB) group, named as the OPCAB group (n = 210). Preoperative clinical data, perioperative clinical data, and mid-term follow-up at 7 years after operation were analyzed and compared between the two groups. RESULTS: Age and the number of vascular lesions in the OPCAB group were higher than those in the MIDCAB group, whereas left ventricular ejection fraction (LVEF) in the former was lower than that in the latter (p<0.05). The number of distal anastomotic stomas, operation time, blood transfusion volume, postoperative cardiac troponin I (cTnI) peak value, ventilator use time, intensive care unit (ICU) stay, and hospital stay in the MIDCAB group were significantly lower than those in the traditional OPCAB group (p<0.05). Among the 294 recovered cases, 201 cases completed 7 years of continuous follow-up, with a follow-up rate of 67.91%. The LVEF and the patency rate of vessels in the MIDCAB group were higher than those in the OPCAB group (p<0.05). CONCLUSIONS: Compared with the median sternotomy OPCAB, MIDCAB is characterized by small incision, small trauma, positive efficacy, and safety; therefore, it is worthy of being promoted.

Rapid induction of apoptosis in tumor cells treated with a new platinum(II) complex based on amino-thiazolidinone.

Thiazolidinone derivatives have been previously shown significant anti-cancer activities. Two amino-thiazolidinone complexes, [Pt(HTone)Cl] (1) and [Cu(HTone)Cl] (3) (HTone = (Z)-2-((E)-(1-(pyridin-2-yl)ethylidene)hydrazono)thiazolidin-4-one) and one ethyl-modified [Pt(ETone)Cl2] (2) (ETone = (Z)-3-ethyl-2-((E)-(1-(pyridin-2-yl)ethylidene) hydrazono)thiazolidin-4-one)], were designed and synthesized in order to explore novel metal-based antitumor agents. MTT assay indicated that 1 and 3 were markedly cytotoxic to MCF-7, HepG-2 and NCI-H460 tumor cells, superior to both cisplatin and the HTone ligand. Massive dead cells were observed as early as 6 h when treated with 1, indicating rapid action of 1 as compared to that of other compounds. More interestingly, Hoechst 33342 staining and flow cytometry analysis illustrated that only complex 1 could induce obvious cell apoptosis within 12 h, which was associated with the high-expression of Bax and cleavage of caspase-3 from 35 kDa to 17 kDa. By means of ICP-MS assay, we found complex 1 could largely accumulate in tumor cells in a short time. Additionally, complex 1 showed no cross resistance against the cisplatin-resistant cells.

Comparison of the clinical effects of zero-profile anchored spacer (ROI-C) and conventional cage-plate construct for the treatment of noncontiguous bilevel of cervical degenerative disc disease (CDDD): A minimum 2-year follow-up.

Comparing the clinical and radiographic outcomes in anterior cervical discectomy and fusion (ACDF) using a zero-profile anchored spacer (ROI-C) or a conventional cage-plate construct (CPC) for treating noncontiguous bilevel of cervical degenerative disc disease (CDDD).Overall, 46 patients with 2 noncontiguous segments of CDDD, treated with ACDF from January 2011 to October 2015, were included in this study. ROI-C was used in 22 patients (group A) and CPC in 24 patients (group B). The clinical and radiographic outcomes and complications were compared pre- and postoperatively. All patients were followed up for at least 24 months after surgery.No significant difference was found in fusion rate, cervical curvature, height of fused segment (FSDH), intraoperative blood loss, and Japanese Orthopaedic Association (JOA), and Neck Disability Index (NDI) scores between the 2 groups. Group A had a shorter operation time and significantly lower incidence of dysphagia (3 and 24 months postoperatively) than group B (P < .001 and P < .05, respectively). Moreover, group A had a higher loss of FSDH than group B, but with no difference between the 2 groups (P > .05). Two cages developed subsidence in group A (4.5%) and 2 adjacent levels developed degeneration in group B (2,8%).ACDF with ROI-C device was superior to CPC for noncontiguous bilevel of CDDD because it avoided postoperative dysphagia and required a shorter operation time. Moreover, the clinical outcomes were comparable. Prospective trials with larger samples and longer follow-up are required to confirm the results.

Protective effects of recombinant human brain natriuretic peptide in perioperative period during open heart surgery.

The aim of the present study was to evaluate the protective effects and safety aspects of recombinant human brain natriuretic peptide (rhBNP) on cardiac functions of patients undergoing open-heart surgery during perioperative period. In total, 150 patients undergoing open heart surgery in the Second Hospital of Shandong Universty from August 2015 to July 2016 were randomly divided into control group and observation group each with 75 cases. Patients in control group were treated by routine rehabilitation while patients in the observation group were treated by both the routine rehabilitation and rhBNP. All the observations were made before operation, after operation and 7 days after operation. The changes of N-terminal pro-brain natriuretic peptide (NT-proBNP) of patients, the left ventricular ejection fraction (LVEF), cardiac function [Cardiac output (CO), pulmonary capillary wedge pressure (PAWP) and central venous pressure (CVP)] of patients were measured. Further, respirator support time, ICU stay time, incidence of complications and vital signs (BP, HR, SaO2) of patients in the two groups were also compared. NT-proBNP levels of all patients improved after operation but it decreased in both groups after 7 days of operation. The decrease of NT-proBNP levels in observation group was significantly higher than that of control group. Whereas, LVEF, CO, PAWP and CVP of patients in both the groups increased after operation but effects were significantly higher in the observation group after 7 days of medication. Respirator support time and ICU stay time of patients in observation group were significantly shorter than those in control group, and the incidence of postoperative complications of patients in the observation group were significantly lower than the control group. Moreover, BP, HR and SaO2 of patients in observation group were significantly elevated in comparison to control group (P<0.05). Recombinant human brain natriuretic peptide (rhBNP) could significantly improve the cardiac functions of patients after open heart surgery, and is safe as well as reliable.

Implantation of a modified stented bovine pulmonary valve in a beating heart sheep model.

PURPOSE: The aim of this research was to assess the performance of a modified bovine stent valve implanted transventricularly in the pulmonary position in sheep with a 3-month follow-up period. MATERIALS AND METHODS: Seven modified pulmonary bovine stent valves were transventricularly implanted in the pulmonary position into seven sheep using a delivery system. Stent valve performance was investigated and evaluated hemodynamically, angiographically, and with echocardiograms before, immediately after, and 3 months following implantation. Macroscopic, histologic, and radiographic examinations were performed on the explanted graft at 3 months. RESULTS: The modified stent valves were all deployed and implanted successfully in the pulmonary position in seven sheep. Angiographic, echocardiographic, hemodynamic, and macroscopic analyses confirmed firm anchoring of the stents in the target position in the early and 3-month follow-up period. All modified stent valves showed satisfactory function, except one moderate stenosis (32 mmHg gradient) with mild regurgitation that was discovered at 3 months. All seven valves were free of any calcification and thrombus formation at postmortem macroscopic examination, which was confirmed by histologic and radiographic examination. All stents were intact without any fracture at microscopic or radiographic examination. CONCLUSIONS: Transventricular implantation of a modified nitinol pulmonary valve stent showed good structural and functional outcomes without stent fracture or migration.

Four Cu(ii) complexes based on antitumor chelators: synthesis, structure, DNA binding/damage, HSA interaction and enhanced cytotoxicity.

Four novel copper(ii) complexes [Cu(II)(Bp4mT)(µ-Cl)]2 (), [Cu(II)(µ-Bp4mT)Br]2 (), [Cu(II)(HBpT)Cl] (), and [Cu(II)(HBpT)Br] () (Bp4mT = 2-benzoylpyridine-4-methylthiosemicarbazone, HBpT = 2-benzoylpyridine thiosemicarbazone), were synthesized and characterized using single-crystal X-ray diffraction, elemental analysis, infrared, and ultraviolet-visible spectroscopy. X-ray analysis revealed that complexes and based on the Bp4mT ligand presented dimeric structures in which the Cu(ii) ions were located in a five-coordinated distorted square-pyramidal geometry, whereas both and complexes were mononuclear with the Cu(ii) ions exhibiting a tetracoordinated square planar configuration. Their interactions with calf thymus DNA (CT-DNA) were investigated using viscosity measurements and fluorescence spectroscopy. Multispectroscopic evidence has shown interactions between these complexes and human serum albumin (HSA). All these complexes have exhibited efficient oxidative cleavage of supercoiled DNA in the presence of hydrogen peroxide, presumably via an oxidative mechanism. Furthermore, in vitro cytotoxicity studies of against human liver hepatocellular carcinoma cells (HepG-2), human large cell lung carcinoma cells (NCI-H460), and human cervical carcinoma cells (HeLa) indicated their promising antitumor activity with quite low IC50 values in the range of 0.08-1.98 µM, which are 83 times lower than those of cisplatin. The mechanistic studies revealed that four complexes, which induced early apoptosis, were involved in reactive oxygen species generation and DNA cleavage for their antitumor activities.

Association of structural modifications with bioactivity in three new copper(II) complexes of Schiff base ligands derived from 5-chlorosalicylaldehyde and amino acids.

Three novel structurally associated copper(II) complexes [Cu(II)(SalCl-Gly)(H2O)2] (1), [Cu(II)(SalCl-Ala)(H2O)] (2) and [Cu(II)(SalCl-Gly)(bipy)]·0.5H2O (3) (SalCl-Gly=5-chloro-2-hydroxybenzylidene-glycine, SalCl-Ala=5-chloro-2-hydroxybenzylidene-alanine, bipy=2,2'-bipyridine) have been synthesized and characterized by X-ray crystallography, elemental analysis, IR and fluorescence spectroscopy. Single-crystal diffraction reveals that complex 1 is an infinite 1D zigzag chain in which SalCl-Gly serves as both a chelating and a bridging ligand, while complexes 2 and 3 are mononuclear. Cu(II) ions in complexes 1-3 exhibit distorted quasi-hexacoordinated octahedral, tetracoordinated square planar, and pentacoordinated square pyramid geometry, respectively. Their interactions with calf thymus DNA (CT-DNA) have been investigated by viscosity measurements and fluorescence spectroscopy. The apparent binding constant (Kapp) values for 1-3 are 1.02×10(5), 0.98×10(5) and 1.57×10(5)M(-1), respectively. All complexes displayed efficient oxidative cleavage of supercoiled DNA in the presence of H2O2. Complex 2, whose ligand can be regarded as a methyl-modification of SalCl-Gly of 1, showed a reduced DNA cleavage activity and a little-changed DNA-binding ability compared with 1. While attaching a 2,2'-bipyridine group to 1, the resulting complex 3 was conferred an enhanced intercalation into DNA. Moreover, cytotoxicity studies of three complexes against HepG-2 (human liver hepatocellular carcinoma) and NCI-H460 (human large-cell lung carcinoma) cells indicated that, thereto, complex 3 possessed the highest inhibition on viability of tested cells.

Functional study of the Hap4-like genes suggests that the key regulators of carbon metabolism HAP4 and oxidative stress response YAP1 in yeast diverged from a common ancestor.

The transcriptional regulator HAP4, induced by respiratory substrates, is involved in the balance between fermentation and respiration in S. cerevisiae. We identified putative orthologues of the Hap4 protein in all ascomycetes, based only on a conserved sixteen amino acid-long motif. In addition to this motif, some of these proteins contain a DNA-binding motif of the bZIP type, while being nonetheless globally highly divergent. The genome of the yeast Hansenula polymorpha contains two HAP4-like genes encoding the protein HpHap4-A which, like ScHap4, is devoid of a bZIP motif, and HpHap4-B which contains it. This species has been chosen for a detailed examination of their respective properties. Based mostly on global gene expression studies performed in the S. cerevisiae HAP4 disruption mutant (ScΔhap4), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not. Moreover HpHAP4-B is able to complement the H2O2 hypersensitivity of the ScYap1 deletant, YAP1 being, in S. cerevisiae, the main regulator of oxidative stress. Finally, a transcriptomic analysis performed in the ScΔyap1 strain overexpressing HpHAP4-B shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4. Deletion of these two genes in their natural host, H. polymorpha, confirms that HpHAP4-A participates in the control of the fermentation/respiration balance, while HpHAP4-B is involved in oxidative stress since its deletion leads to hypersensitivity to H2O2. These data, placed in an evolutionary context, raise new questions concerning the evolution of the HAP4 transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.

The PI3K/AKT Pathway and FOXO3a Transcription Factor Mediate High Glucose-Induced Apoptosis in Neonatal Rat Ventricular Myocytes.

BACKGROUND: PI3K/AKT pathway plays major roles in regulating cardiomyocyte metabolism. The roles of PI3K/AKT pathway and FOXO3a in mediating high glucose-induced apoptosis in cardiomyocytes remain unclear. OBJECTIVES: In this experimental study, we investigated the mechanisms of the PI3K/AKT pathway and FOXO3a in mediating hyperglycemia-induced apoptosis in neonatal rat ventricular myocytes (NRVMs). MATERIALS AND METHODS: NRVMs were adopted as the cell model to investigate the roles of PI3K/AKT and FOXO3a in mediating hyperglycemia-induced apoptosis in cardiomyocytes. Annexin-V-FITC staining and PI staining were used to evaluate the apoptosis in NRVMs under indicated conditions of serum starvation, high glucose exposure, and pharmacological or genetic manipulations on the expressions of PI3K/AKT and FOXO3a. Western blotting was conducted to evaluate the cytoplasmic/nuclear localization of FOXO3a in NRVMs exposed to high glucose. FOXO3a transcriptional activity was measured by luciferase reporter assay. RESULTS: High glucose (30 mM) induced significant apoptosis in serum-starved NRVMs as compared with normal glucose (5 mM) control (12.01 ± 0.76% vs. 2.86 ± 0.55%; P < 0.001). Treatment with IGF1 attenuated hyperglycemia-induced apoptosis by 68% (3.23 ± 0.76% vs. 9.97 ± 1.29%; P < 0.001; n = 3) in comparison with the non-treated control. Treatment with PI3K inhibitor LY294002 enhanced hyperglycemia-induced apoptosis by 109% (20.83 ± 1.87% vs. 9.97 ± 1.29%; P < 0.001; n = 3) in comparison with the non-treated control. Over-expression of AKT by transduction with CA-AKT attenuated hyperglycemia-induced apoptosis by 47% (5.48 ± 0.35% vs.10.31 ± 0.94%; P < 0.001; n = 3) in comparison with the empty-vector control. Transduction with DN-AKT enhanced high glucose-induced apoptosis by 105% (21.13 ± 1.11% vs. 10.31 ± 0.94%; P < 0.001; n = 3) in comparison with the empty-vector control. Western blotting showed that high glucose induced a significant increase in FOXO3a nuclear localization. Luciferase reporter assay showed that high glucose induced a significant increase of 310% (P < 0.001; n = 3) in FOXO3a transcriptional activity against Fas ligand when NRVMs were transducted with TM-FOXO3a in comparison with the empty-vector control. CONCLUSIONS: The PI3K/AKT pathway mediated hyperglycemia-induced apoptosis of NRVMs through the translocation of FOXO3a to nuclei and the resultant enhanced transcriptional activity of FOXO3.

Perventricular closure of a post-traumatic muscular ventricular septal defect guided by transesophageal echocardiogram.

Minimally invasive perventricular closure is emerging as an alternative to conventional open surgery in treating traumatic ventricular septal defects (VSDs). We report a case of successful perventricular closure of a post-traumatic muscular VSD in a patient who sustained blunt chest trauma in a motor vehicle accident. A larger Amplatzer muscular VSD occluder (16 mm in diameter) was used to close the VSD near the apex.

Nuclease activity and protein-binding properties of a novel tetranuclear thiosemicarbazide Pt(II) complex.

A novel tetranuclear [Pt4(Am4M)4]·16.25H2O (1), where H2Am4M is the thiosemicarbazone ligand (Z)-2-(amino(pyridin-2-yl)methylene)-N-methylhydrazinecarbothioamide, has been synthesized and characterized by using various physico-chemical techniques. X-ray analysis revealed that the Pt(II) complex consists of a neutral tetranuclear [Pt4(Am4M)4] unit and abundant water molecules. The tetranuclear unit is stabilized by strong intramolecular π-π stacking and thus presents a ship-like eight-membered ring [Pt4S4] with four tridentate ligands peripherally coordinated to four Pt(II) ions, constituting a novel type of "well" structure. DNA and protein binding properties of complex 1 have been studied by UV and fluorescence spectroscopic methods. The binding constant (Kb = 3.05 × 10(6) M(-1)) and the apparent binding constant (Kapp = 7.732 × 10(6) M(-1)) to calf thymus DNA (CT-DNA) were accurately fitted by two respective non-linear equations, suggesting an efficient DNA intercalative binding mode which was confirmed by viscometric experiments. Furthermore, following agarose gel electrophoresis experiments, we observed that the plasmid DNA pUC19 could be completely digested by complex 1, depending on incubation time and concentration, indicating that complex 1 may possess an effective nuclease activity on DNA. Singlet oxygen ((1)O2) inhibitors showed an inhibitory effect on the cleavage. Fluorescence spectrometry also detected a medium affinity of complex 1 to bovine serum albumin (BSA) at different temperatures, which was tentatively assigned to a static binding mode. All these results suggested that this poly-Pt(II) complex might exhibit biological action as a potential chemotherapy agent.

Biological evaluation of a cytotoxic 2-substituted benzimidazole copper(II) complex: DNA damage, antiproliferation and apoptotic induction activity in human cervical cancer cells.

Exploring novel chemotherapeutic agents is a great challenge in cancer medicine. To that end, 2-substituted benzimidazole copper(II) complex, [Cu(BMA)Cl2]·(CH3OH) (1) [BMA = N,N'-bis(benzimidazol-2-yl-methyl)amine], was synthesized and its cytotoxicity was characterized. The interaction between complex 1 and calf thymus DNA was detected by spectroscopy methods. The binding constant (K b = 1.24 × 10(4 )M(-1)) and the apparent binding constant (K app = 6.67 × 10(6 )M(-1)) of 1 indicated its moderate DNA affinity. Complex 1 induced single strand breaks of pUC19 plasmid DNA in the presence of H2O2 through an oxidative pathway. Cytotoxicity studies proved that complex 1 could inhibit the proliferation of human cervical carcinoma cell line HeLa in both time- and dose-dependent manners. The results of nuclei staining by Hoechst 33342 and alkaline single-cell gel electrophoresis proved that complex 1 caused cellular DNA damage in HeLa cells. Furthermore, treatment of HeLa cells with 1 resulted in S-phase arrest, loss of mitochondrial potential, and up-regulation of caspase-3 and -9 in HeLa cells, suggesting that complex 1 was capable of inducing apoptosis in cancer cells through the intrinsic mitochondrial pathway.

Gene responses to oxygen availability in Kluyveromyces lactis: an insight on the evolution of the oxygen-responding system in yeast.

The whole-genome duplication (WGD) may provide a basis for the emergence of the very characteristic life style of Saccharomyces cerevisiae-its fermentation-oriented physiology and its capacity of growing in anaerobiosis. Indeed, we found an over-representation of oxygen-responding genes in the ohnologs of S. cerevisiae. Many of these duplicated genes are present as aerobic/hypoxic(anaerobic) pairs and form a specialized system responding to changing oxygen availability. HYP2/ANB1 and COX5A/COX5B are such gene pairs, and their unique orthologs in the 'non-WGD' Kluyveromyces lactis genome behaved like the aerobic versions of S. cerevisiae. ROX1 encodes a major oxygen-responding regulator in S. cerevisiae. The synteny, structural features and molecular function of putative KlROX1 were shown to be different from that of ROX1. The transition from the K. lactis-type ROX1 to the S. cerevisiae-type ROX1 could link up with the development of anaerobes in the yeast evolution. Bioinformatics and stochastic analyses of the Rox1p-binding site (YYYATTGTTCTC) in the upstream sequences of the S. cerevisiae Rox1p-mediated genes and of the K. lactis orthologs also indicated that K. lactis lacks the specific gene system responding to oxygen limiting environment, which is present in the 'post-WGD' genome of S. cerevisiae. These data suggested that the oxygen-responding system was born for the specialized physiology of S. cerevisiae.

Oxygen-dependent transcriptional regulator Hap1p limits glucose uptake by repressing the expression of the major glucose transporter gene RAG1 in Kluyveromyces lactis.

The HAP1 (CYP1) gene product of Saccharomyces cerevisiae is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative S. cerevisiae, Kluyveromyces lactis is an aerobic yeast species which shows different oxygen responses. We examined the role of the HAP1-equivalent gene (KlHAP1) in K. lactis. KlHap1p showed a number of sequence features and some gene targets (such as KlCYC1) in common with its S. cerevisiae counterpart, and KlHAP1 was capable of complementing the hap1 mutation. However, the KlHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28 degrees C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The DeltaKlhap1 mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis.

Effects of extended-release felodipine on endothelial vasoactive substances in patients with essential hypertension.

BACKGROUND: Endothelial dysfunction plays a significant role in the pathogenesis of essential hypertension (EH). This trial was undertaken to reveal the effects of extended-release felodipine on endothelial vasoactive substances in EH patients. METHODS: A colorimetric chemical method was employed to measure the level of nitric oxide (NO) and nitric oxide synthase (NOS), while radioimmunoassay was employed to measure endothelin (ET), angiotensin-II (Ang-II), thromboxane A2 (TXA(2)) and prostacyclin I(2) (PGI(2)) in plasma of the subjects. Group 1 consisted of 120 patients who were treated with a 4-week mono-therapy of felodipine. Group 2 consisted of 70 patients who were participating in fitness programs during this period. Another group comprising 80 individuals was selected as controls. Data from both the starting point and the ending point were collected and analyzed. RESULTS: After a 4-week mono-therapy of extended-release felodipine in Group 1, the levels of plasma ET, Ang-II and TXA(2) decreased significantly, while levels of NO, NOS and PGI(2) did not noticeably change. In Group 2, there were almost no changes in levels of ET, Ang-II, TXA(2), NO, NOS and PGI(2). CONCLUSIONS: From these results, we conclude that felodipine reduced blood pressure by decreasing the secretion of ET, Ang-II and TXA(2). Consequently, felodipine can revitalize the endothelial function in EH patients.

Why do some yeast species require niacin for growth? Different modes of NAD synthesis.

NAD holds a key position in metabolism and cellular regulatory events as a major redox carrier and a signalling molecule. NAD biosynthesis pathways have been reconstructed and compared in seven yeast species with completely sequenced genomes, including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica and Schizosaccharomyces pombe. Both amino acid and nucleotide sequence similarity analysis in silico indicated that de novo NAD biosynthesis might not exist in K. lactis, C. glabrata and Schiz. pombe, while other species have the kynurenine pathway. It also showed that the NAD salvage pathway via nicotinic acid and nicotinic acid mononucleotide is conserved in all of these yeasts. Deletion of KlNPT1 (the gene for nicotinate phosphoribosyl-transferase) is lethal, which demonstrates that this salvage pathway, utilizing exogenous nicotinic acid, is the unique route to synthesize NAD in K. lactis. The results suggested that the basis of the variation of niacin requirements in yeasts lies in their different combinations of NAD biosynthesis pathways. The de novo pathway is absent but the salvage pathway is conserved in niacin-negative yeasts, while both pathways coexist in niacin-positive yeasts.

Cloning of the LAC4 Promoter from K. lactis and Studies on the Function of its UASII.

The -661 - +21 bp region of the LAC4 gene form K. lactis CBS141 was obtained by PCR technique and fused with the lacZ gene from E. coli to construct and expression vector YFD 114. It was then transformed into K. lactis Y167. The function of the cloned LAC4 promoter was tested by induction with galactose, lactose, sorbitol or IPTG. The results revealed that galactose had higher inducing effect than lactose while sorbitol and IPTG had no inducing effect. The insertion of chemically synthesized LAC4 UASII at just the upstream of the cloned LAC4 promoter could increase its basic and induced expression level. The extents of increase were different with the orientation and the copy number of the inserted UASII.