Incidence of elevated procalcitonin and presepsin levels after severe trauma: a pilot cohort study.

Procalcitonin (PCT) and presepsin (PSEP) are useful biomarkers for diagnosing sepsis; however, elevated PCT and PSEP levels may be observed in conditions other than sepsis. We hypothesised that PCT and PSEP levels could increase after severe traumatic injuries. Trauma patients with an Injury Severity Score of ≥16 from October 2013 to September 2015 were enrolled in our study. We examined PCT and PSEP levels and their positive rates on days 0 and 1. PCT and PSEP levels on days 0 and 1 were compared. Risk factors for increasing sepsis biomarker levels were identified by multivariate logistic regression analyses. In this study, 75 patients were included. PCT levels on days 0 and 1 were 0.1±0.4 and 1.8±6.3 ng/ml, respectively (P=0.02). PSEP levels on days 0 and 1 were 221±261 and 222±207 pg/ml, respectively (P=0.98). As per multivariate logistic regression analyses, packed red blood cell (PRBC) transfusion was the only independent risk factor for higher PCT levels on day 1 (P=0.04). Using PCT to diagnose sepsis in trauma patients on day 1 requires caution. PRBC transfusion was found to be a risk factor for increasing PCT levels. On the other hand, PSEP levels were not affected by trauma during the early phases.

Phospholipase D activity of human amnion cells stimulated with phorbol ester and bradykinin.

We investigated the activity of phospholipase D (PLD) in human amnion cells labeled with [3H]oleate. The PLD activity was detected as signal-induced synthesis of phosphatidic acid (PA) and in the presence of ethanol, phosphatidylethanol (PEt). The PLD was shown to be activated by phorbol, 12-myristate, 13-acetate (PMA), calcium ionophore A23187, oxytocin, bombesin and bradykinin, but not by platelet-activating factor (PAF) and epidermal growth factor (EGF). The amniotic PLD thus appeared to be activated by a variety of agonists but with a certain specificity to stimulators. We examined the mode of the PLD activation using PMA (20 nM) and bradykinin (1 microM) as model stimulators. PMA and bradykinin elicited a rapid and sustained response with the peaks of PA-labeling attained at 5 and < 1 min after stimulation, respectively. In both cases, there was a concomitant rise of diacylglycerol (DG), and the PA accumulation was suppressed by ethanol at the expense of labeling of PEt. The PA synthesis caused by the two stimulators was similarly inhibited by staurosporine and by a chronic treatment with PMA (100 nM for 24 h), suggesting that the activation of PLD is linked to the action of protein kinase C. With the cells labeled with radioactive choline and ethanolamine, we found that the amniotic PLD hydrolyzed almost equally phosphatidylcholine and phosphatidylethanolamine. Although bradykinin and PMA stimulated cellular PLD to a comparable extent, prostaglandin (PG)E2 release was not stimulated by bradykinin in contrast to the marked effect by PMA. Further work is thus needed to clarify the significance of the novel PLD signaling pathway in the function of amnion cells.

A recurrent large Alu-mediated deletion in the hypoxanthine phosphoribosyltransferase (HPRT1) gene associated with Lesch-Nyhan syndrome.

We identified the identical large genomic deletion in the hypoxanthine phosphoribosyltransferase (HPRT1) gene in two Japanese patients with Lesch-Nyhan (LN) syndrome. This deletion spanned from an Alu sequence in the promoter region to another Alu-sequence in intron 1, a length of 2,969 base pairs including exon 1. In order to ask whether this deletion was a recurrent mutation, we developed a simple alternative method to determine the separate origin of the HPRT1 mutation of the patients as assessed with an apparent mtDNA polymorphism. Considering that an LN syndrome-causing mutation is not transmitted from patient to offspring as LN syndrome is a fatal disease in childhood and that mtDNA is maternally inherited, HPRT1 mutations and mtDNA would be co-transmitted from carrier mother to offspring since both appeared in females. Two bases were different in the hypervariable region I of the mtDNA between the two patients, indicating the separate origin of their mtDNA over at least several thousand years as calculated based on the molecular evolution rate in this region. We thus conclude that the identical deletion found in HPRT1 of the two patients was derived from recurrent events of genomic recombination. Given that the same Alu-mediated deletion of HPRT1 has not been reported among somatic mutations at the same locus, this region of the HPRT1 gene flanked by Alu-sequences is likely a mutational hot spot in the germline but not in somatic cells. In addition, we also report novel LN-syndrome-conferring mutations in intron 6 (IVS6+1G --> C) and intron 8 (IVS7-9T --> G) that resulted in exclusions of exon 6 and exon 8, respectively.

The involvement of the Saccharomyces cerevisiae multidrug resistance transporters Pdr5p and Snq2p in cation resistance.

The ATP-binding cassette superfamily proteins Pdr5p and Snq2p of Saccharomyces cerevisiae are implicated in multidrug resistance. Here, we show that these transporters are also involved in cation resistance. Null mutants of PDR5 and SNQ2 genes exhibit increased sensitivity to NaCl, LiCl and MnCl2. The mutant cells grown in the presence of high concentrations of these metal salts contain higher levels of the metals than wild-type cells. The expression of PDR5 and SNQ2 is induced by the metal salts. These results provide evidence that the yeast drug transporters contribute to cation resistance by regulating cellular cation homeostasis under ionic stress conditions.

Bombesin and bradykinin increase inositol phosphates and cytosolic free Ca2+, and stimulate DNA synthesis in human endometrial stromal cells.

The present studies were carried out to investigate the effect of several growth factors on human endometrial stromal cells. In human endometrial stromal cells, bombesin and bradykinin provoked an increase in intracellular free Ca2+ and in labelled inositol phosphates when pre-incubated with [3H]myoinositol. Some or possibly all of the initial increase in intracellular free Ca2+ represented a mobilization of Ca2+ from intracellular stores and the second phase of the response depended on Ca2+ influx from the extracellular medium. [3H]Thymidine was added to human cultured endometrial stromal cells with bombesin, bradykinin, epidermal growth factor (EGF), prostaglandin F2 alpha, vasopressin and platelet-derived growth factor. Bombesin, bradykinin and EGF stimulated the incorporation of [3H]thymidine into DNA in quiescent cells. In conclusion, bombesin and bradykinin are growth factors which activate phospholipase C in human endometrial stromal cells, while EGF stimulates DNA synthesis without the activation of phospholipase C.

Can dog-ear formation be decreased when an S-shaped skin resection is used instead of a spindle skin resection? A three-dimensional analysis of skin surgery techniques using the finite element method.

Dog-ear formation is often unavoidable with resection and suturing of the skin, including spindle excision. Regarding dog-ear formation after basic spindle skin resection during removal of a round tumor of the skin, we quantitatively analyzed the frequency of dog-ear formation with respect to the following three techniques: previous spindle skin resection, S-shaped skin resection, which has been experientially considered to induce limited deformity, and mosque-shaped skin resection for control. To date, by using paper models or sponges, various techniques of skin resection have been simulated in the field of plastic surgery. In the present study, we performed three-dimensional simulation and analyzed three different techniques of skin resection by using the finite element method. As a result, image simulation demonstrated that the frequency of dog-ear formation was limited by S-shaped, spindle, and mosque-shaped skin resection, in descending order.

Three-dimensional finite element analysis of skin suture. Part 1: spindle model and S-shaped modified model.

Suturing of postoperative wounds in skin unfortunately leads to extrusion of the skin, resulting in so-called "dog ear". We performed three-dimensional finite element method (FEM) analyses to investigate how suture methods affect the height of the extrusion. Three models were prepared: (1) conventional suture method Sp; (2) S-shaped modified suture model Si-1, in which one side of the curves is introverted; and (3) another S-shaped suture model Si-2, in which both sides of the curves are introverted. The results of FEM analysis agreed well with the figure and location of the extrusions in clinical suture surgery and the height of the extrusion was mimicked visually in three dimensions. The height of the extrusion peak of the S-shaped modified suture method Si-1 was decreased by 40% in comparison with the conventional suture method Sp.

A surgical simulation system of skin sutures using a three-dimensional finite element method.

OBJECTIVE: To establish a surgical simulation system of skin sutures using a three-dimensional finite element method. DESIGN: Three-dimensional finite element models were developed from point data obtained with a rapid three-dimensional surface-measuring device and postoperative profiles were evaluated using these models. BACKGROUND: Since suturing a wound may result in undesirable skin extrusion, it is important to make the extrusion as inconspicuous as possible. We have investigated a means of determining appropriate suture methods to decrease the extrusion. METHODS: Affected body parts were measured non-invasively with a rapid three-dimensional surface-measuring device. Finite element models were prepared, and an appropriate method for reducing skin extrusion was evaluated by attempting various suturing methods. RESULTS: Two kinds of finite element models were prepared: a conventional spindle model and a modified S-shape model. The height of the extrusion of the modified S-shape model was decreased by 40% in comparison with that of the spindle model. These results agreed with clinical findings. CONCLUSIONS: Due to this surgical simulation system of skin sutures, with a rapid three-dimensional surface-measuring device and three-dimensional finite element analysis, it was possible to design an appropriate suturing method and to evaluate the postoperative skin profiles. The modified S-shape suture method would be a recommendable method. RELEVANCE: Using this surgical simulation system of skin sutures, a surgeon can evaluate an appropriate suturing method before operation. It is expected that this system will reduce a surgeon's labor.

Involvement of histidine permease (Hip1p) in manganese transport in Saccharomyces cerevisiae.

In a search for components involved in Mn2+ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn2+ transport. The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine. The mutation, designated hip1-272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF. This mutation led to Mn2+ resistance, whereas the corresponding null mutation did not. Both hip1-272 cells and the null mutant exhibited low tolerance to divalent cations such as Co2+, Ni2+, Zn2+, and Cu2+. The Mn2+ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine. The cellular Mn2+ content of the hip1-272 mutant was lower than that of wild type or null mutant, due to increased rates of Mn2+ efflux. We propose that Hiplp is involved in Mn2+ transport, carrying out a function related to Mn2+ export.

GSK-3 kinase Mck1 and calcineurin coordinately mediate Hsl1 down-regulation by Ca2+ in budding yeast.

The Ca2+-activated pathways of Saccharomyces cerevisiae induce a delay in the onset of mitosis through the activation of Swe1, a negative regulatory kinase that inhibits the Cdc28-Clb complex. Calcineurin and Mpk1 activate Swe1 at the transcriptional and post-translational level, respectively, and both pathways are essential for the cell cycle delay. Our genetic screening identified the MCK1 gene, which encodes a glycogen synthetase kinase-3 family protein kinase, as a component of the Ca2+ signaling pathway. Genetic analyses indicated that Mck1 functions downstream of the Mpk1 pathway and down-regulates Hsl1, an inhibitory kinase of Swe1. In medium with a high concentration of Ca2+, Hsl1 was delocalized from the bud neck and destabilized in a manner dependent on both calcineurin and Mck1. Calcineurin was required for the dephosphorylation of autophosphorylated Hsl1. The E3 ubiquitin ligase complex SCF(Cdc4), but not the anaphase-promoting complex (APC), was essential for Hsl1 destabilization. The Ca2+-activated pathway may play a role in the rapid inactivation of Hsl1 at the cell cycle stage(s) when APC activity is low.

Loss of sperm in juvenile spermatogonial depletion (jsd) mutant mice is ascribed to a defect of intratubular environment to support germ cell differentiation.

C57BL/6(B6)-jsd/jsd mice are sterile due to the defective spermatogenesis in the testes. To know the cause of the deficient spermatogenesis in B6-jsd/jsd mice, we examined whether the problem is within or outside the seminiferous tubules by transplanting tubules from cryptorchid testes of B6- +/+ mice into B6-jsd/jsd testes or tubules from B6-jsd/jsd mice into testes of (WB x C57BL/6)F1-W/Wv (hereafter, WBB6F1-W/Wv) mice. Type A spermatogonia differentiated into spermatids in seminiferous tubules from cryptorchid testes transplanted into B6-jsd/jsd testes. In contrast, in B6-jsd/jsd tubules transplanted into WBB6F1-W/Wv testes, type A spermatogonia were stimulated to mitotic proliferation, but didn't proceed to any differentiated germ cells. The present results suggest that the cause of the deficient spermatogenesis in B6-jsd/jsd mice is a defect of intratubular environment to support germ cell differentiation.

A positive screening for drugs that specifically inhibit the Ca2+-signaling activity on the basis of the growth promoting effect on a yeast mutant with a peculiar phenotype.

An inappropriate activation of a signaling pathway in yeast often has a deleterious physiological effect and causes various defects, including growth defects. In a certain genetic background (deltazds1) of Saccharomyces cerevisiae, the cell-cycle progression in G2 is specifically blocked in the medium with CaCl2 by the hyperactivation of the Ca2+-signaling pathways. Here, we developed a novel drug screening procedure designed to detect the active compounds that specifically attenuate the Ca2+-signaling activity on the basis of the ability to abrogate the growth defect of the cells suffering from the hyperactivated Ca2+ signal. Using known calcineurin inhibitors as model compounds, we have established the screening conditions for the drugs that suppress the Ca2+-induced growth inhibition. An indicator strain with an increased drug sensitivity was constructed with a syr1/erg3 null mutation.

3D-CT stereoscopic imaging in maxillofacial surgery.

We obtained stereoscopic 3D-CT images in maxillofacial bone fracture patients. These images are made at two different angles. One is equivalent to the view obtained by a subciliary incision during surgery. Another is equivalent to the view obtained by oral incision during surgery. A stereoscopic image is created with a pair of images that differ from each other by a 6 degree shift of the z axis.

Role of calcineurin and Mpk1 in regulating the onset of mitosis in budding yeast.

Signalling via calcium is probably involved in regulating eukaryotic cell proliferation, but details of its mechanism of action are unknown. In Schizosaccharomyces pombe, the onset of mitosis is determined by activation of a complex of the p34cdc2 protein kinase and a cyclin protein that is specific to the G2 phase of the cell cycle. This activation requires dephosphorylation of p34cdc2. Weel, a tyrosine kinase that inhibits p34cdc2 by phosphorylating it, is needed to determine the length of G2 phase. Here we show that calcium-activated pathways in Saccharomyces cerevisiae control the onset of mitosis by regulating Swel, a Weel homologue. Zds1 (also known as Oss1 and Hst1) is important in repressing the transcription of SWE1 in G2 phase. In the presence of high calcium levels, cells lacking Zds1 are delayed in entering mitosis. Calcineurin and Mpk1 regulate Swel activation at the transcriptional and posttranslational levels, respectively, and both are required for the calcium-induced delay in G2 phase. These cellular pathways also induce a G2-phase delay in response to hypotonic shock.

Inhibition of human telomerase by rubromycins: implication of spiroketal system of the compounds as an active moiety.

We found that a group of rubromycins and their analogues, a class of quinone antibiotics that possesses benzofuran and benzodipyran rings to form a spiroketal system, strongly inhibited human telomerase as assessed with a modified telomeric repeat amplification protocol. beta- and gamma-Rubromycins and purpuromycin appeared to be the most potent telomerase inhibitors, with 50% inhibitory concentrations (IC(50)) of about 3 microM, and griseorhodins A and C also showed comparable potencies for the inhibition (IC(50) = 6-12 microM). In contrast, opening of the spiroketal system of beta-rubromycin, giving rise to alpha-rubromycin, substantially decreased its inhibitory potency toward telomerase (IC(50) > 200 microM), indicating the essential role of the spiroketal system in telomerase inhibition. A kinetic study of the inhibition by beta-rubromycin revealed a competitive interaction with respect to the telomerase substrate primer, with a K(i) of 0.74 microM, whereas a mixed type inhibition was observed with respect to the nucleotide substrate. beta-Rubromycin was also potent in inhibiting retroviral reverse transcriptases but had virtually no effect on other DNA/RNA-modifying enzymes including DNA and RNA polymerases, deoxyribonuclease, and topoisomerase. Although beta-rubromycin showed nonspecific cytotoxicities, reducing proliferation of cancer cells (IC(50) approximately 20 microM), we conclude that beta-rubromycin appears to be a lead structure for the development of more potent and selective inhibitors of human telomerase.

Isolation and characterization of monoclonal antibodies that inhibit hepatitis C virus NS3 protease.

A series of mouse monoclonal antibodies (MAbs) to the nonstructural protein 3 (NS3) of hepatitis C virus was prepared. One of these MAbs, designated 8D4, was found to inhibit NS3 protease activity. This inhibition was competitive with respect to the substrate peptide (K(i) = 39 nM) but was significantly decreased by the addition of the NS4A peptide, a coactivator of the NS3 protease. 8D4 also showed marked inhibition of the NS3-dependent cis processing of the NS3/4A polyprotein but had virtually no effect on the succeeding NS3/4A-dependent trans processing of the NS5A/5B polyprotein in vitro. Epitope mapping of 8D4 with a random peptide library revealed a consensus sequence, DxDLV, that matched residues 79 to 83 (DQDLV) of NS3, a region containing the catalytic residue Asp-81. Furthermore, synthetic peptides including this sequence were shown to block the ability of 8D4 to bind to NS3, indicating that 8D4 interacts with the catalytic region of NS3. The data showing decreased inhibition potency of 8D4 against the NS3/4A complex suggest that 8D4 recognizes the conformational state of the protease active site caused by the association of NS4A with the protease.