Protective effect of recombinant neutrophil elastase inhibitor (R-020) on sepsis-induced organ injury in rat

Severe inflammatory responses after major surgeries, trauma, and infection develop multiple organ dysfunction. In the mechanisms of the pathogenesis of these responses, activated neutrophils are thought to be important in terms of their ability to produce various kinds of proteinases, which can degrade various proteins constructing human tissues. Among their proteinases, neutrophil elastase is the strongest serine proteinase secreted from activated neutrophils. Thus, we examined in this study the inhibitory effect and therapeutic efficacy of newly produced recombinant human Kunitz-type proteinase inhibitor (R-020), which coded the second domain of human urinary trypsin inhibitor. R-020 was effective in significantly improving the survival rate after induction of the rat lethal peritonitis model (cecal ligation and punctureinduced septic shock model). We suggest that various serine proteinases are implicated in the pathogenesis of neutrophil-related multiple organ failure and that recombinant human Kunitz-type proteinase inhibitor might be effective in the treatment of these kinds of organ dysfunction.     

Distinct progression pathways involving the dysfunction of DUSP6/MKP-3 in pancreatic intraepithelial neoplasia and intraductal papillary-mucinous neoplasms of the pancreas.

DUSP6/MKP-3 is identified as a candidate tumor suppressor gene for pancreatic cancer. The aim of this study was to elucidate the roles of DUSP6 in the pancreatic carcinogenesis through the pancreatic intraepithelial neoplasia and/or intraductal papillary-mucinous neoplasms, both of which are considered to be precursor lesions of invasive carcinoma of the pancreas, by comparing with involvements of other major tumor suppressive pathways. Expressions of DUSP6, CDKN2A, TP53, and SMAD4 were investigated by immunohistochemistry in a total of 206 lesions of dysplastic ductal precursors and carcinomas retrieved from 52 pancreata with invasive ductal carcinomas and 51 of those with intraductal papillary-mucinous neoplasms. The intensity of staining was evaluated in lesions at different atypical grades and statistically compared among them. Mutations of KRAS2 were analyzed by methods of the allele-specific oligonucleotide hybridization and nucleotide sequencing. In pancreata with invasive ductal carcinomas, expressions of DUSP6 were abrogated exclusively in the invasive carcinoma cells in contrast to its fairly preserved expressions in pancreatic intraepithelial neoplasia. In pancreata with intraductal papillary-mucinous neoplasms, abrogated expressions of DUSP6 were observed in a relatively small fraction of intraductal adenoma/borderlines and intraductal carcinomas. Most of the intraductal adenoma/borderline lesions with abrogation of DUSP6 harbored mutations of KRAS2. None of the molecules was associated with each other in any grade of lesions. Morphological variations of papillae of the intraductal papillary-mucinous neoplasms were evaluated and analyzed for their associations with abrogations of the molecules, which resulted in finding of no significant associations. Our results suggest that the abrogation of DUSP6 is associated exclusively with progression from pancreatic intraepithelial neoplasia to the invasive ductal carcinoma while it is potentially associated with initiation of intraductal papillary-mucinous neoplasms with mutated KRAS2, which is independent of other major tumor suppressive pathways in both types of neoplasms.     

Expression of serotonin2A receptors in Purkinje cells of the developing rat cerebellum

Previous physiological and pharmacological studies have shown that the serotonin_2A (5-HT_2A) receptor is involved in cerebellar functions. However, the expression of 5-HT_2A receptors in the developing cerebellum has not been elucidated to date. In the present immunohistochemical study, we examined developmental changes of the distribution of 5-HT_2A receptors in Purkinje cells of the rat cerebellum from embryonic day 18 (E18) to postnatal day 21 (P21). The weak immunoreaction to 5-HT_2A receptors was found in the deep cerebellar nuclei on E19. In the cerebellar cortex of the hemisphere and the posterior vermis, somata of Purkinje cells became weakly immunoreactive on P0. With the dendritic elongation and arborization, the immunoreaction appeared in the proximal parts of Purkinje cell dendrites. Distal parts of the dendrites became immunoreactive after P12, and were strongly immunolabeled by P21. The present study may provide a structural basis to investigate the roles of 5-HT_2A receptors during the cerebellar development.     

Three novel mutations of the fibrillin-1 gene and ten single nucleotide polymorphisms of the fibrillin-3 gene in Marfan syndrome patients

Marfan syndrome (MFS) is an autosomal dominant disorder of the extracellular matrix. Allelic variations in the gene for fibrillin-1 (FBN1) have been shown to cause MFS. To date, over 550 mutations have been identified in patients with MFS and related connective tissue diseases. However, about a half of MFS cases do not possess mutations in the FBN1 gene. These findings raise the possibility that variants located in other genes cause or modify MFS. To explore this possibility, firstly we analyzed FBN1 allelic variants in 12 Japanese patients with MFS, and secondly we analyzed fibrillin-3 gene (FBN3) in patients without FBN1 mutations using conformation sensitive gel electrophoresis (CSGE) and direct sequencing analysis. We identified three novel FBN1 mutations and ten FBN3 single nucleotide polymorphisms (SNPs). In this report, we could not detect a responsible mutation of the FBN3 gene for MFS. Although the number of the cases in this report is small, at least these results suggest that disease-causing mutations in exon regions of the FBN3 gene are very rare in MFS.Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers: AB177797, AB177798, AB177799, AB177800, AB177801, AB177802, AB177803     

Neuronal protein NP25 interacts with F-actin

Neuronal protein NP25 is a neuron-specific protein present in highly differentiated neural cells, but its functional properties have not been well characterized. NP25 shows high amino acid sequence homology with the smooth muscle cell cytoskeleton-associated proteins, SM22, mp20, and calponin. To gain an insight into the biological functions of NP25, we first examined its subcellular localization in the human neuroblastoma cell line, SK-N-SH. NP25 diffusely distributed in the cytoplasm and fiber-like staining was also observed. It showed that NP25 co-localized with F-actin on stress fibers. A co-sedimentation assay demonstrated that NP25 bound to filamentous actin. Further investigations using fluorescence resonance energy transfer (FRET) technique revealed intracellular binding of NP25 and actin. The significance of the interaction between NP25 and F-actin is discussed.     

Adaptive Predictive Control of Arterial Blood Pressure Based on a Neural Network During Acute Hypotension

In acute hypotension, an automated drug infusion system to control mean arterial blood pressure (MAP) has not been previously studied, though many investigations have examined the use of vasodilating drugs to control MAP in postoperative hypertension. Therefore, we examined an automated control of MAP during acute hypotension using a neural network (NN) approach. A proportional-integral-derivative (PID) control, an adaptive predictive control using a NN (APC(NN)), a combined control of APC(NN) and PID (APC(NN-PID)), a fuzzy control, and a model predictive control were tested in computer simulation based on the MAP response to norepinephrine (NE) of 25 microg ml(-1). In six anesthetized rabbits, using the NE of 25 microg ml(-1), the PID control, APC(NN), and APC(NN-PID) prevented severe hypotension compared to an uncontrolled condition. Under PID control, four of the six animals showed MAP oscillation. Using NE of 50 microg ml(-1), the rabbits recovered from acute hypotension for all systems tested but showed sustained MAP oscillation during PID control. In conclusion, utilization of a NN for adaptive predictive control systems could facilitate the development of an automated drug infusion apparatus because it provides robust control even when acute or large perturbations and inter-individual differences in the sensitivity to therapeutic agents occur.     

Prion domain interaction responsible for species discrimination in yeast [PSI+] transmission

BACKGROUND: The yeast [PSI+] factor is transmitted by a prion mechanism involving self-propagating Sup35 aggregates. As with mammalian prions, a species barrier prevents prion transmission between yeast species. The N-terminal of Sup35 of Saccharomyces cerevisiae, necessary for [PSI+], contains two species-signature elements-a Gln/Asn-rich region (residues 1-41; designated NQ) that is followed by oligopeptide repeats (designated NR). RESULTS: In this study, we show that S. cerevisiae[PSI+] is transmissible through plasmid shuffling and cytoplasmic transfer to heterotypic Sup35s whose NQ is replaced with the S. cerevisiae NQ. In addition to homology, the N-terminal location is essential for NQ mediated susceptibility to [PSI+] transmission amongst heterotypic Sup35s. In vitro, a swap of NQ of S. cerevisiae Sup35 led to cross seeding of amyloid formation. CONCLUSIONS: These findings suggest that NQ discriminates self from non-self, and is sufficient to initiate [PSI+] transmission irrespective of whether NR is heterotypic. NR as well as NQ alone coalesces into existing [PSI+] aggregates, showing their independent potentials to interact with the identical sequence in the [PSI+] conformer. The role of NQ and NR in [PSI+] prion formation is discussed.     

Existence of a small population of IL-2Rβhi TCRint cells in SCG and MRL-lpr/lpr mice which produce normal Fas mRNA and Fas molecules from the lpr gene

Mice carrying the lpr gene, SCG and MRL-lpr/lpr mice, were used to characterize the phenotype and lpr gene of abnormally proliferating T cells in these mice. A major population which expanded in these mice were T cells expressing intermediate (int) levels of T cell receptor (TCR) (and CD3) and the phenotype of interleukin-2 receptor (IL-2R)β^lo α^? (possibly abnormal TCR^int cells). The levels of TCR^hi cells of thymic origin (generated through the mainstream of T cell differentiation in the thymus) profoundly decreased after the onset of disease. However, a small population of normal TCR^int cells (i.e. IL-2Rβ^hi α^?) were also found to exist in all tested organs. For example, the majority of abnormal IL-2Rβ^lo TCR^int cells were CD4^?8^? CD2^?, while normal IL-2Rβ^hi TCR^int cells were a mixture of single-positive cells (mainly CD8⁺), CD4^?8^? cells and CD2⁺ cells. Moreover, normal TCR^int cells preferentially produced normal Fas mRNA and Fas molecules from the lpr gene. This phenomenon explains the leaky appearance of normal Fas mRNA and Fas molecules in mice carrying the lpr gene. It is suggested that a small population of IL-2Rβ^hiTCR^int cells are resistant to the lpr genetic abnormality.     

Quantitative evaluation of hand cranking a roller pump in a crisis management drill

The heart-lung machines for open-heart surgery have improved over the past 50 years; they rarely break down and are almost always equipped with backup batteries. The hand-cranking procedure only becomes necessary when a pump breaks down during perfusion or after the batteries have run out. In this study, the performance of hand cranking a roller pump was quantitatively assessed by an objective method using the ECCSIM-Lite educational simulator system. A roller pump connected to an extracorporeal circuit with an oxygenator and with gravity venous drainage was used. A flow sensor unit consisting of electromagnetic sensors was used to measure arterial and venous flow rates, and a built-in pressure sensor was used to measure the water level in the reservoir. A preliminary study of continuous cranking by a team of six people was conducted as a surprise drill. This system was then used at a perfusion seminar. At the seminar, 1-min hand-cranking drills were conducted by volunteers according to a prepared scenario. The data were calculated on site and trend graphs of individual performances were given to the participants as a handout. Preliminary studies showed that each person's performance was different. Results from 1-min drills showed that good performance was not related to the number of clinical cases experienced, years of practice, or experience in hand cranking. Hand cranking to maintain the target flow rate could be achieved without practice; however, manipulating the venous return clamp requires practice. While the necessity of performing hand cranking during perfusion due to pump failure is rare, we believe that it is beneficial for perfusionists and patients to include hand-cranking practice in periodic extracorporeal circulation crisis management drills because a drill allows perfusionists to mentally rehearse the procedures should such a crisis occur.