Construct and concurrent validity of a Nintendo Wii video game made for training basic laparoscopic skills.

BACKGROUND: Virtual reality (VR) laparoscopic simulators have been around for more than 10 years and have proven to be cost- and time-effective in laparoscopic skills training. However, most simulators are, in our experience, considered less interesting by residents and are often poorly accessible. Consequently, these devices are rarely used in actual training. In an effort to make a low-cost and more attractive simulator, a custom-made Nintendo Wii game was developed. This game could ultimately be used to train the same basic skills as VR laparoscopic simulators ought to. Before such a video game can be implemented into a surgical training program, it has to be validated according to international standards. METHODS: The main goal of this study was to test construct and concurrent validity of the controls of a prototype of the game. In this study, the basic laparoscopic skills of experts (surgeons, urologists, and gynecologists, n = 15) were compared to those of complete novices (internists, n = 15) using the Wii Laparoscopy (construct validity). Scores were also compared to the Fundamentals of Laparoscopy (FLS) Peg Transfer test, an already established assessment method for measuring basic laparoscopic skills (concurrent validity). RESULTS: Results showed that experts were 111 % faster (P = 0.001) on the Wii Laparoscopy task than novices. Also, scores of the FLS Peg Transfer test and the Wii Laparoscopy showed a significant, high correlation (r = 0.812, P < 0.001). CONCLUSIONS: The prototype setup of the Wii Laparoscopy possesses solid construct and concurrent validity.

Different oligomeric forms of protein phosphatase 2A activate and inhibit simian virus 40 DNA replication.

The ability of simian virus 40 (SV40) large T antigen to catalyze the initiation of viral DNA replication is regulated by its phosphorylation state. Previous studies have identified the free catalytic subunit of protein phosphatase 2A (PP2Ac) as the cellular phosphatase which can remove inhibitory phosphoryl groups from serines 120 and 123. The catalytic C subunit exists in the cell complexed with a 65-kDa A subunit and one of several B subunits. To determine if any of the holoenzymes could activate T antigen, we tested the ability of the heterodimeric AC and two heterotrimeric ABC forms to stimulate T-antigen function in unwinding the origin of SV40 DNA replication. Only free catalytic subunit C and the heterotrimeric form with a 72-kDa B subunit (PP2A-T72) could stimulate T-antigen-dependent origin unwinding. Both the dimeric form (PP2A-D) and the heterotrimer with a 55-kDa B subunit (PP2A-T55) actively inhibited T-antigen function. We found that PP2A-T72 activated T antigen by dephosphorylating serines 120 and 123, while PP2A-D and PP2A-T55 inactivated T antigen by dephosphorylating the p34cdc2 target site, threonine 124. Thus, alterations in the subunit composition of PP2A holoenzymes have significant functional consequences for the initiation of in vitro SV40 DNA replication. The regulatory B subunits of PP2A may play a role in regulating SV40 DNA replication in infected cells as well.

Characterization of glycogen-synthase phosphatase and phosphorylase phosphatase in subcellular liver fractions.

Upon fractionation of a postmitochondrial supernatant from rat liver, the synthase phosphatase (EC 3.1.3.42) activity (assayed at high tissue concentrations) was largely recovered in the glycogen fraction and to a minor extent in the cytosol. In contrast, the phosphorylase phosphatase (EC 3.1.3.17) activity was approximately equally distributed between these two fractions, a lesser amount being recovered in the microsomal fraction. The phosphatase activities in the microsomal and glycogen fractions were almost completely inhibited by a preincubation with the modulator protein, a specific inhibitor of type-1 (ATP,Mg-dependent) protein phosphatases. In the cytosolic fraction, however, type-2A (polycation-stimulated) phosphatase(s) contributed significantly to the dephosphorylation of phosphorylase and of in vitro phosphorylated muscular synthase. Liver synthase b, used as substrate for the measurement of synthase phosphatase throughout this work, was only activated by modulator-sensitive phosphatases. Trypsin treatment of the subcellular fractions resulted in a dramatically increased (up to 1000-fold) sensitivity to modulator, a several-fold increase in phosphorylase phosphatase activity and a complete loss of synthase phosphatase activity. Similar changes occurred during dilution of the glycogen-bound enzyme. A preincubation with the deinhibitor protein, which is known to counteract the effects of inhibitor-1 and modulator, increased several-fold the phosphorylase phosphatase activity, but exclusively in the cytosolic and microsomal fractions. It did not affect the synthase phosphatase activity. Taken together, the results indicate the existence of distinct, multi-subunit type-1 phosphatases in the cytosolic, microsomal and glycogen fractions.

The Saccharomyces cerevisiae homologue YPA1 of the mammalian phosphotyrosyl phosphatase activator of protein phosphatase 2A controls progression through the G1 phase of the yeast cell cycle.

The Saccharomyces cerevisiae gene YPA1 encodes a protein homologous to the phosphotyrosyl phosphatase activator, PTPA, of the mammalian protein phosphatase type 2A (PP2A). In order to examine the biological role of PTPA, we disrupted YPA1 and characterised the phenotype of the ypa1Delta mutant. Comparison of the growth rate of the wild-type strain and the ypa1Delta mutant on glucose-rich medium after nutrient depletion showed that the ypa1Delta mutant traversed the lag period more rapidly. This accelerated progression through "Start" was also observed after release from alpha-factor-induced G1 arrest as evidenced by a higher number of budding cells, a faster increase in CLN2 mRNA expression and a more rapid reactivation of Cdc28 kinase activity. This phenotype was specific for deletion of YPA1 since it was not observed when YPA2, the second PTPA gene in budding yeast was deleted. Reintroduction of YPA1 or the human PTPA cDNA in the ypa1Delta mutant suppressed this phenotype as opposed to overexpression of YPA2. Disruption of both YPA genes is lethal, since sporulation of heterozygous diploids resulted in at most three viable spores, none of them with a ypa1Delta ypa2Delta genotype. This observation indicates that YPA1 and YPA2 share some essential functions. We compared the ypa1Delta mutant phenotype with a PP2A double deletion mutant and a PP2A temperature-sensitive mutant. The PP2A-deficient yeast strain also showed accelerated progression through the G1 phase. In addition, both PP2A and ypa1Delta mutants show similar aberrant bud morphology. This would support the notion that YPA1 may act as a positive regulator of PP2A in vivo.

Phosphorylation and negative regulation of the transcriptional activator CREM by p34cdc2.

Transcription factors that bind to cAMP-responsive elements (CREs) regulate the expression of target genes in response to activation of the adenylyl cyclase pathway. It is generally thought that activation is obtained through direct phosphorylation by the cAMP-dependent protein kinase-A. We have isolated the gene CRE modulator (CREM), which encodes multiple members of the CRE-binding protein family, by cell-specific alternative splicing. Various isoforms have been characterized, encoding both repressors (CREM alpha, -beta, and -gamma) as well as activators (CREM tau). Here we show that the function of the activator CREM tau is regulated by the p34cdc2 kinase. Multiple serine and threonine residues are phosphorylated in vivo as well as in vitro by p34cdc2. Although there is no effect of p34cdc2-mediated phosphorylation on CREM tau DNA binding, we observed a dramatic effect on the trans-regulatory function. Coexpression of a constitutively active p34cdc2 mutant shows that the trans-activation potential of CREM tau is strongly reduced by p34cdc2. This represents the first example of negative regulation of a transcription factor of this class by p34cdc2.

Dephosphorylation of the deinhibitor protein by the PCSH protein phosphatase.

The deinhibitor protein, responsible for the decreased sensitivity of the ATP,Mg-dependent protein phosphatase to inhibitor-1 and the modulator protein, is inactivated by cyclic AMP-dependent protein kinase and reactivated by dephosphorylation. The specificity of this reaction was tested with the ATP,Mg-dependent phosphatase in its activated or spontaneously active form, four different forms of polycation-stimulated phosphatases (PCSH, PCSM, PCSL and PCSC) and calcineurin. Only the high -Mr polycation-stimulated protein phosphatase (PCSH), but not its catalytic subunit (PCSC), shows a high degree of specificity for the deinhibitor protein. Deinhibitor phosphatase activity of PCSH is affected neither by polycations nor by Mn ions.

Activation of the PCSM-protein phosphatase by a Ca2+-dependent protease.

The inhibitor-1 phosphatase but not the phosphorylase phosphatase activity of a newly discovered 250 kDa polycation-stimulated (PCSM) protein phosphatase in rabbit skeletal muscle is increased up to 10-fold by a Ca2+-dependent protease. The enzyme-directed protease effect to which the PCSH and PCSL phosphatases are insensitive was progressively lost during purification of the enzyme. This could be explained by either a slow conversion of the enzyme to an active form of the enzyme with a change in specificity, or the loss of a protease-sensitive inhibitor of the inhibitor-1 phosphatase activity, resulting in a PCS phosphatase characterized by its high inhibitor-1/phosphorylase alpha activity ratio. The Ca2+-dependent protease is completely inhibited by EGTA or leupeptin.

Okadaic acid, a specific protein phosphatase inhibitor, induces maturation and MPF formation in Xenopus laevis oocytes.

Micro-injection of, or incubation with okadaic acid (OA), a specific phosphatase inhibitor, can induce formation of maturation-promoting factor (MPF) and germinal vesicle breakdown (GVBD) in Xenopus laevis oocytes. Comparison of the dose-response curves of OA on maturation, isolated enzymes and phosphatase activities in crude oocyte preparations suggests that inhibition of both polycation-stimulated (PCS) and ATP,Mg-dependent (AMD) phosphatases is sufficient but requires that a critical phosphorylation level is attained of one or several of their substrates, resulting in the formation of active MPF and meiotic maturation.

Dephosphorylation of tau protein and Alzheimer paired helical filaments by calcineurin and phosphatase-2A.

We have shown previously that brain tissue contains protein kinases which can phosphorylate tau protein to a state reminiscent of the pathological state of Alzheimer paired helical filaments (PHFs); these include proline-directed kinases which phosphorylate SP or TP motifs (such as MAP kinase and GSK-3) [Drewes et al. (1992); Mandelkow et al. (1992)], as well as a novel kinase which phosphorylates S262 of tau protein and thereby strongly reduces the binding of tau to microtubules [Biernat et al. (1993)]. Here we report on the corresponding phosphatases in brain which normally keep the 'pathological' sites free of phosphate. The major phosphatases acting on tau are calcineurin and PP-2A, but not PP-1. Both are present and active in brain extracts, they can dephosphorylate recombinant tau after prior phosphorylation with either MAP kinase, GSK-3, or brain extract, and the course of dephosphorylation can be monitored with antibodies diagnostic of the pathological state of tau. Both phosphatases also act directly on PHF tau isolated from Alzheimer brains.

The ATP,Mg-dependent protein phosphatase: regulation by casein kinase-1.

The free modulator subunit of the ATP,Mg-dependent phosphatase is phosphorylated up to 1 mol per mol by casein kinase-1, up to 1.85 mol per mol after dephosphorylation by the PCSH1 phosphatase, but 10-fold less when purified in the presence of NaF, suggesting an in vivo phosphorylation of the casein kinase-1 sites. Peptide mapping of 32P-modulator labeled by casein kinase-1 or -2 shows a different phosphorylation pattern. Phosphorylation of the inactive phosphatase by casein kinase-1 prevents the subsequent kinase FA-mediated activation, while it does not impair the activated phosphatase.

A synthetic peptide substrate specific for casein kinase I.

The synthetic peptide, Asp-Asp-Asp-Glu-Glu-Ser-Ile-Thr-Arg-Arg, derived from the phosphorylation site of casein kinase-1 (CK-1) in beta-casein A(2), is readily phosphorylated by CK-1, but not by casein kinase-2(CK-2), cyclic AMP-dependent protein kinase, protein kinase C, phosphorylase kinase and protein kinase FA. Phosphorylation by CK-1 occurs only at Ser-6, Thr-8 being unaffected. The Km for the peptide is higher (1 mM) than for beta-casein A(2) (40 microM), while the Vmax is quite comparable. This is the first synthetic peptide substrate for CK-1 described so far, and can be used for the rapid and specific estimation of CK-1 activity in crude extracts.

Phosphorylation of the modulator protein of the ATP, Mg-dependent protein phosphatase by casein kinase TS. Reversal by PCS phosphatases and control by distinct phosphorylation site(s).

The phosphorylation by casein kinase TS (II) of the modulator protein of the ATP, Mg-dependent phosphatase increases after preincubation with the PCSH1 phosphatase or with the catalytic subunit of the ATP, Mg-dependent phosphatase. Dephosphorylation by the two phosphatases combined leads to the incorporation of 2 mol phosphate per mol modulator (at Ser residues). Occupancy of the ATP, Mg-dependent phosphatase phosphorylation site(s) is a negative determinant in the phosphorylation of the modulator by kinase TS. Among the PCS phosphatases PCSH1 shows the highest activity toward the 32P-Ser residues labeled by kinase TS in untreated or previously dephosphorylated modulator, while the ATP, Mg-dependent phosphatase is totally ineffective. Protamine stimulates all phosphatase activities, so that the catalytic subunit of the ATP, Mg-dependent phosphatase becomes almost as effective as the PCSC phosphatase in dephosphorylating the kinase TS sites.

Multiple molecular forms of phosphorylase phosphatase associated with particulate glycogen and extracted from the cytosol of dog liver.

The glycogen pellet of dog liver extracts contains a phosphorylase phosphatase which has characteristics different from those of the phosphatases extracted from the cytosol. The phosphatase associated with glycogen is characterized by a M, of 51,000, a half maximal inhibition at 0.3 mM ATP (Hill coefficient : 2) and a Ki for Mg2+ of 1 mM. Treatment with urea or mercaptoethanol of the phosphatase associated with glycogen does not influence the activity, the Mr or the half maximal inhibition by ATP, but a decrease of the Hill coefficient for ATP is observed. A similar treatment of the phosphatases extracted from the high speed supernatant results in a decrease of the Mr of the spontaneously active form from 215,000 to 43,000, without an effect on the Ki for ATP (7 micronM), but accompanied by an increase in activity. The ATP-Mg dependent form of the phosphatase from the high speed supernatant (Mr : 138,000 ; Ka for ATP in the presence of 0.1 mM Mg2+ : 0.3 micronM), is denatured by urea or mercaptoethanol. The phosphatase associated with particulate glycogen cannot be found in the supernatant, nor the phosphorylase phosphatases present in the supernatant in the glycogen pellet. When all the glycogen is mobilized (starvation, glucagon) the phosphatase specifically associated with glycogen cannot be found as such in the cytosol. No activation of synthase beta can be detected neither with the phosphatases extracted from the cytosol nor with the enzyme released from the glycogen pellet.

Photosensitized inhibition of growth factor-regulated protein kinases by hypericin.

The naphthodianthrone hypericin causes a photosensitized inhibition of protein kinases involved in growth factor signalling pathways. Nanomolar concentrations of hypericin inhibit the protein tyrosine kinase activities (PTK) of the epidermal growth factor receptor and the insulin receptor, while being ineffective towards the cytosolic protein tyrosine kinases Lyn, Fgr, TPK-IIB and CSK. Photosensitized inhibition by hypericin is not restricted to receptor-PTKs since the Ser/Thr protein kinases (protein kinase CK-2, protein kinase C and mitogen-activated kinase) are also extremely sensitive to inhibition (IC50 value for protein kinase CK-2 = 6 nM). A comparison of the hypericin-mediated inhibition of the epidermal growth factor-receptor PTK and protein kinase CK-2 revealed that the inhibition is irreversible, strictly dependent upon irradiation of the enzyme-inhibitor complex with fluorescent light and likely mediated by the formation of radical intermediates (type I mechanism). Although the exact molecular basis for the selectivity of enzyme inhibition by hypericin remains unknown, our results suggest that distantly related protein kinases could still share common reactive domains for the interaction with hypericin.

Posttraumatic multiple organ failure--a report on clinical and autopsy findings.

In a retrospective analysis, clinical data and histological specimens were obtained from patients (n = 59) who died of severe injury. Three groups with comparable injury severity were differentiated according to the time of death. In group A (death, within 24 h) (n = 15) despite multiple injuries, patients almost always died from brain injury. Pulmonary failure was the leading cause of death in group B (death, days 2-7) (n = 16). The majority of group C patients (death, > 7 days) (n = 28) died of multiple organ failure. Organ weights at autopsy were all pathologically high but did not show an association with the amount of intravenous volume infused during intensive care. Organ histology revealed signs of interstitial edema and infiltration of polymorphonuclear leukocytes in group B patients especially in the lung, and in all groups to a lower degree in liver and kidney. The distribution of interstitial edema and cell necrosis appeared to be organ-specific. Our data confirm the presence of a generalized inflammatory reaction in patients with severe trauma. The pattern of organ failure, in addition to known pathogenetic changes (mediators, endotoxemia, etc.), appears to be influenced by organ structure and perfusion.

Cloning and differential expression of new calcium, calmodulin-dependent protein kinase II isoforms in Xenopus laevis oocytes and several adult tissues.

The different oligomers composing the high molecular weight calcium/calmodulin-dependent protein kinase II (CaMKII) holoenzyme, previously shown to be transiently activated during Xenopus oocyte maturation, migrate on SDS-PAGE as proteins of 83, 72, 62, 56, and 52 kDa and have all been cloned. The holoenzyme consists of the CaMKII isoforms gammaB, gammaC, and delta12, already described in other species, while gammaJ, gammaK, gammaL, gammaM, and gammaN are now described for the first time. The gamma-isoforms are splice variants of the gamma-gene, containing in their variable region different combinations of known exons and one, two or three novel exons. Semi-quantitative RT-PCR revealed that all isoforms identified in prophase oocytes are also expressed in adult tissues with a tissue-specific expression pattern. At least thirty different CaMKII isoforms could be identified in different Xenopus adult tissues, most of which are described here for the first time.

Ogilvie's method applied to infected wound disruption.

Nylon mesh was used without closing the skin in 26 patients in whom it was not possible to close the infected abdominal wall without undue tension. A total of 73 meshes were implanted. Major complications consisted of three wound disruptions in two patients and formation of a sinus tract necessitating excision of the mesh in one patient. Definitive closure generally consisted of skin grafting on the granulation tissue growing through the mesh. Mortality was high, due to underlying diseases. In three of 13 survivors, an incisional hernia developed.

Intestinal permeability after severe trauma and hemorrhagic shock is increased without relation to septic complications.

After thermal injury, alterations in intestinal permeability have been demonstrated and have correlated with subsequent infections. We measured intestinal permeability on the second day after severe trauma and hemorrhagic shock (ruptured abdominal aneurysm). The mean (+/- SD) lactulose-mannitol (L/M) excretion ratio was 0.012 +/- 0.005 in seven healthy control subjects, 0.069 +/- 0.034 in 11 severely traumatized patients, and 0.098 +/- 0.093 in eight patients with aneurysm, indicating a significant increase of intestinal permeability in both patient groups. No significant correlation was found between L/M ratios and age, severity of injury or shock, lactate levels on admission, APACHE (acute physiology and chronic health evaluation) II score, daily pulmonary gas exchange parameters, or mean multiple organ failure scores. No difference in intestinal permeability between patients with and without subsequent infections could be demonstrated. In 11 patients we looked for endotoxin in the systemic circulation. In six patients endotoxemia was present immediately after admission and before the L/M test. However, during the L/M test and 1 day afterward no circulating endotoxin was observed. The present data provide evidence for the hypothesis that increased intestinal permeability and subsequent infectious complications are independent phenomena, frequently seen in patients after severe trauma or hemorrhagic shock.

Whole-body inflammation in trauma patients. An autopsy study.

In a review of autopsy specimens and reports in 35 trauma cases, we found signs of generalized inflammation and tissue damage with increases in organ weights in organs not primarily injured. These abnormalities occurred independent of the time of death and were also found in patients who died of brain injury alone. The most pronounced signs of inflammation and increases in organ weights were found when the adult respiratory distress syndrome, hypovolemic shock, or multiple organ failure were the causes of death. These findings are similar to those found in several organs of rabbits after four hours of complement activation in combination with 20 minutes of hypoxia. Therefore, the autopsy findings in this series of trauma patients might represent the morphologic features of adult respiratory distress syndrome and multiple organ failure in an early, preclinical stage.

Posttraumatic complications and inflammatory mediators.

In a series of 71 patients with trauma, we measured weekly the blood levels of a number of complement proteins and activation products. We also measured the following: leukocytes, platelets, granulocyte enzyme elastase, alpha 1-antitrypsin, total protein, albumin, haptoglobin, and fibronectin. The intensity of complement activation and the blood levels of elastase correlated with the following factors: injury severity (especially the severity of limb injury), development of adult respiratory distress syndrome, development and severity of multiple organ failure, and probability of a fatal outcome. The plasma elastase level seemed to be the best predictor of adult respiratory distress syndrome and the best correlate of injury severity and multiple organ failure severity. Our findings support the hypothesis that posttraumatic activation of the complement system leads to activation of granulocytes, followed by microvascular injury and finally by organ failure.