[Laparoscopic "single-port" appendectomy in children]. / Laparoskopische "Single Port"-Appendektomie im Kindesalter.

BACKGROUND: In contrast to the laparoscopic three trocar-technique and to the single incision laparoscopic surgery (SILS), the "single-port" appendectomy (SPA) requires only one port with one integrated instrument channel. We report on our experience with this half-open surgical technique. PATIENTS / MATERIALS AND METHODS: Between September 2006 and August 2008 a total of 285 children underwent an appendectomy, 265 in SPA technique. Through a 10 mm subumbilical inserted ballon trocar, diagnostic laparoscopy was routinely performed and, afterwards, the appendix was grasped with a 450 mm forceps, exteriorised and dissected outside the abdomen as in open surgery. Patients with perforated appendicitis detected by preoperative ultrasonography were operated by open appendectomy. RESULTS: 94 % of the SPA were performed successfully with no conversion. In six patients (2.3 %), a second trocar was inserted, in seven children (2.6 %), an extension of the incision became necessary. There were three conversions (1.1 %) to open surgery. The median operating time was 50 min and the median length of hospital stay 4 days. Three children had postoperative wound infections (1.1 %). CONCLUSIONS: SPA is a safe alternative to conventional appendectomy techniques, in part also in cases of perforated appendicitis. The minimal scarring guarantees an attractive cosmetic result. The diagnostic laparoscopy enables one to obtain additional information. In the case of extended adhesions, an extension of the incision and / or the use of a second trocar may be helpful.

Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product.

Krev-1 is known to suppress transformation by ras. However, the mechanism of the suppression is unclear. The protein product of Krev-1, Rap1A-p21, is identical to Ras-p21 proteins in the region where interaction with guanosine triphosphatase (GTPase) activating protein (GAP) is believed to occur. Therefore, the ability of GAP to interact with Rap1A-p21 was tested. Rap1A-p21 was not activated by GAP but bound tightly to GAP and was an effective competitive inhibitor of GAP-mediated Ras-GTPase activity. Binding of GAP to Rap1A-p21 was strictly guanosine triphosphate (GTP)-dependent. The ability of Rap1A-p21 to bind tightly to GAP may account for Krev-1 suppression of transformation by ras. This may occur by preventing interaction of GAP with Ras-p21 or with other cellular proteins necessary for GAP-mediated Ras GTPase activity.

Rapamycin-sensitive phosphorylation of PKC on a carboxy-terminal site by an atypical PKC complex.

BACKGROUND: The protein kinase C (PKC) family has been implicated in the control of many cellular functions. Although PKC isotypes are characterized by their allosteric activation, phosphorylation also plays a key role in controlling activity. In classical PKC isotypes, one of the three critical sites is a carboxy-terminal hydrophobic site also conserved in other AGC kinase subfamily members. Although this site is crucial to the control of this class of enzymes, the upstream kinase(s) has not been identified. RESULTS: A membrane-associated kinase activity that phosphorylates the hydrophobic site in PKCalpha was detected. This activity was suppressed when cells were pretreated with the immunosuppresant drug rapamycin or the phosphoinositide (Pl) 3-kinase inhibitor LY294002. These pretreatments also blocked specifically the serum-induced phosphorylation of the hydrophobic site in PKCdelta in vivo. The most highly purified hydrophobic site kinase preparations ( approximately 10,000-fold) reacted with antibodies to PKCzeta/iota. Consistent with this, rapamycin and LY294002 reduced the recovery of PKCzeta from the membrane fraction of transfected cells. An activated mutant of PKCzeta, but not wild-type PKCzeta, induced phosphorylation of the PKCdelta hydrophobic site in a rapamycin-independent manner, whereas a kinase-dead PKCzeta mutant suppressed this serum-induced phosphorylation. The immunopurified, activated mutant of PKCzeta could phosphorylate the PKCdelta hydrophobic site in vitro, whereas wild-type PKCzeta could not. CONCLUSIONS: PKCzeta is identified as a component of the upstream kinase responsible for the phosphorylation of the PKCdelta hydrophobic site in vitro and in vivo. PKCzeta can therefore control the phosphorylation of this PKCdelta site, antagonizing a rapamycin-sensitive pathway.

Mutational and kinetic analyses of the GTPase-activating protein (GAP)-p21 interaction: the C-terminal domain of GAP is not sufficient for full activity.

The GTPase-activating protein (GAP) stimulates the GTPase reaction of p21 by 5 orders of magnitude such that the kcat of the reaction is increased to 19 s-1. Mutations of residues in loop L1 (Gly-12 and Gly-13), in loop L2 (Thr-35 and Asp-38), and in loop L4 (Gln-61 and Glu-63) influence the reaction in different ways, but all of these mutant p21 proteins still form complexes with GAP. The C-terminal domain of the human GAP gene product, GAP334, which comprises residues 714 to 1047, is 20 times less active than full-length GAP on a molar basis and has a fourfold lower affinity. This finding indicates that the N terminus of GAP containing the SH2 domains modifies the interaction between the catalytic domain and p21.

Deregulated estrogen receptor alpha expression in mammary epithelial cells of transgenic mice results in the development of ductal carcinoma in situ.

A conditional tetracycline-responsive transgenic mouse model with deregulated estrogen receptor alpha expression in mammary epithelial cells developed ductal hyperplasia (DH), lobular hyperplasia, and ductal carcinoma in situ (DCIS) by 4 months of age. Higher proliferative rates were found in both normal and abnormal ductal and lobular structures. DH and DCIS but not normal ductal structures showed an increased percentage of cells with nuclear-localized cyclin D1. No differences in either the prevalence or extent of these phenotypes following exogenous 17beta-estradiol treatment were found suggesting that alteration of ERalpha expression was the rate-limiting factor in initiation of DH, lobular hyperplasia, and DCIS.

Protein conformational flexibility modulates kinetics and thermodynamics of drug binding.

Structure-based drug design has often been restricted by the rather static picture of protein-ligand complexes presented by crystal structures, despite the widely accepted importance of protein flexibility in biomolecular recognition. Here we report a detailed experimental and computational study of the drug target, human heat shock protein 90, to explore the contribution of protein dynamics to the binding thermodynamics and kinetics of drug-like compounds. We observe that their binding properties depend on whether the protein has a loop or a helical conformation in the binding site of the ligand-bound state. Compounds bound to the helical conformation display slow association and dissociation rates, high-affinity and high cellular efficacy, and predominantly entropically driven binding. An important entropic contribution comes from the greater flexibility of the helical relative to the loop conformation in the ligand-bound state. This unusual mechanism suggests increasing target flexibility in the bound state by ligand design as a new strategy for drug discovery.

Loss of protein phosphatase 2A expression correlates with phosphorylation of DP-1 and reversal of dysplasia through differentiation in a conditional mouse model of cancer progression.

A conditional mouse model of time-dependent dysplasia reversal demonstrated that reversal and differentiation of dysplastic salivary gland tissue at the 4-month reversible stage was characterized by the appearance of a phosphorylated slower mobility form of Differentiation Related Transcription Factor 1-polypeptide-1 that was correlated with cellular differentiation. The phosphorylated form of DP-1 was not found at the 7-month irreversible stage or in adenocarcinomas. At the 4-month reversible stage, protein phosphatase 2A expression was down-regulated coincident with loss of oncogene expression, whereas PP2A expression persisted at the 7-month irreversible stage. Results are consistent with the hypothesis that persistent PP2A expression prevented the appearance of the phosphorylated form of DP-1 required for cellular differentiation and reversal of dysplasia after loss of oncogene expression.

Differences in GTPase-activating protein activity between liver tumors and normal liver tissue in mice.

The intrinsic GTPase activity of the cellular protein p21ras is strongly increased by two cytosolic proteins, the GTPase-activating protein (GAP) produced by the neurofibromatosis type 1 gene (NF1-GAP) and a GAP of 120 kDa molecular mass (p120-GAP). The GAP-mediated stimulation of p21ras GTPase activity was measured in cytosol obtained from carcinogen-induced liver tumors and normal liver tissues of mice of two strains, namely C3H/He and C57BL/6J. For this purpose, cytosolic extracts were incubated with recombinant human p21ras complexed to [gamma-32P]GTP and the time-dependent decrease in p21ras bound radioactivity was measured. Liver cytosolic extracts mediated an increase in the GTPase activity of wild-type p21ras. There were great differences between tumor and normal tissues in the maximal velocity (Vmax) and in the apparent Michaelis constant (KM) of the p21ras GTPase reaction. Both Vmax and apparent KM were decreased in the liver tumors. Cytosolic extracts isolated from liver tumors that harbored point mutations in codon 61 of the c-H-ras gene did not differ in their activity from extracts obtained from non-mutated liver tumors. Since both GAP proteins are important cellular regulators of the ras signaling pathway and probably also effectors of p21ras, the observed differences in GAP activity may be of relevance for the tumorigenic process in mouse liver.

Effect of alterations in extracellular fluid volume on urinary kallikrein in the conscious rat.

The effect of alterations in extracellular fluid volume (ECV) and solute concentration on excretion of urinary kallikrein was examined in conscious Sprague-Dawley rats. Animals were given infusions of either dextrose and water, saline, or albumin according to a variety of protocols. These were designed to evaluate possible relationships between excretion of kallikrein, volume, sodium, and potassium. A reproducible pattern of kallikrein excretion was noted in all volume expanded groups. This consisted of a short lived increase during the initial hour of expansion with a subsequent fall to lower levels than baseline and a gradual recovery. To define the role of aldosterone in these studies, an adrenalectomized group and a group of appropriately prepared sham controls were expanded with saline. Adrenalectomy did not effect this pattern. We postulate a tubular "washout" phenomenon as the etiology of these observations. Results of these studies fail to demonstrate a consistent relationship between urinary volume, sodium, or potassium and the simultaneous amount of kallikrein found in the urine.

Naloxone attenuates the hypotension induced by Hageman factor.

The hypotension induced in the pentobarbital anesthesized rat by the i.v. administration of an active Hageman factor fragment (Hff) is significantly attenuated by naloxone. This effect is specific because the opiate antagonist does not modify the hypotension elicited by rat urinary kallikrein, bradykinin or nitroglycerin. These results suggest that the contact activation of endogenous Hageman factor could result in the generation of vasoactive opioid peptides derived from circulating large molecular weight precursors.

[Inversion of the right hemi-diaphragm associated with abundant pleural effusion (author's transl)]. / Inversion diaphragmatique droite lors des épanchements pleuraux abondants. Etude échotomographique.

The radiological appearance and the physiopathological consequences of the phenomenon of the inversion of the diaphragm on the left side associated with abundant pleural effusion, were studied by Felson and his school as early as 1965. Echotomography reveals that the phenomenon occurs on the right side in exactly the same way.

Combined therapy with cyclodextrin/allopregnanolone and miglustat improves motor but not cognitive functions in Niemann-Pick Type C1 mice.

Niemann-Pick Type C1 (NPC1) is an autosomal recessive disorder characterized by the accumulation of cholesterol and glycosphingolipids. Combination-treatment utilizing cyclodextrin, allopregnanolone and miglustat (CYCLO/ALLO/miglustat) can ameliorate NPC1 disease in a mutant mouse model. The present study was designed to add behavioral analysis in NPC1 mutant mice upon CYCLO/ALLO/miglustat therapy. NPC1 mutant (BALB/cJ NPC1NIH) and control mice were used. For the combination treatment mice were injected with CYCLO/ALLO weekly, starting at P7. The miglustat injection was performed daily from P10 till P23. Starting at P23, miglustat was added to the powdered chow. For the sham treatment of control and mutant mice the same schedule was used with 0.9% NaCl injection. Locomotor activity was assessed in open field, elevated plus maze and accelerod tests. For assessment of spatial learning and memory the Morris water maze test was conducted. Electron microscopy has been performed to support the behavioral data. The sham-treated mutant mice exhibited motor impairments in all performed tests. In the water maze the sham-treated mutants exhibited impairment in remembering the location of the hidden platform. CYCLO/ALLO/miglustat treatment positively influenced motor dysfunction: total distance and number of visits significantly increased, and accelerod performance improved. The spatial learning, however, did not benefit from therapy. At the morphological level, an excessive accumulation of electron-dense material was seen in the cerebellar Purkinje cells of mutant mice. A regression of these autophagosomal inclusions was seen upon therapy. CYCLO/ALLO/miglustat therapy ameliorates motor but not cognitive deficits in NPC1 mutant mice, suggesting unequal vulnerability of different brain areas to the treatment.

Direct interaction of PU.1 with oncogenic transcription factors reduces its serine phosphorylation and promoter binding.

The homeostasis of hematopoiesis in the bone marrow is governed by a small number of key transcription factors, including PU.1, GATA-1 and c/EBPα. PU.1, a member of the E-twenty-six family of transcription factors, is indispensable for normal hematopoiesis. Inactivation of PU.1 induces acute leukemia in mice. Recent data suggest that the leukemia-associated fusion protein pro-myelocytic leukemia/retinoic acid receptor alpha (PML/RARα) inhibits PU.1, but the mechanism mediating this inhibition is unclear. Here, we investigated the mechanisms by which the fusion proteins PML/RARα and pro-myelocytic leukemia zinc finger/RARα (PLZF/RARα) (X-RARα) interfere with the function of PU.1. We found that X-RARα proteins functionally inactivate PU.1 by reducing its promoter-binding capacity, resulting in a reduction in PU.1-dependent transcriptional transactivation. In fact, X-RARα proteins directly interact with PU.1, leading to both the sequestration of PU.1 from its target promoters and a reduction in its serine phosphorylation, which is crucial for its promoter binding and transcriptional activity. We found that the functional inactivation of PU.1 could be overcome by the forced overexpression of PU.1 in PML/RARα- or PLZF/RARα-positive murine hematopoietic progenitor cells; evidently, this overexpression rescued the leukemic differentiation block induced by X-RARα proteins. Our data thus provide strong evidence that X-RARα proteins functionally inhibit PU.1, shedding light on the mechanism by which X-RARα proteins induce leukemogenesis.

Loss of cyclin D1 in concert with deregulated estrogen receptor alpha expression induces DNA damage response activation and interrupts mammary gland morphogenesis.

We have previously shown that increased and deregulated estrogen receptor alpha expression in the mammary gland leads to the development of proliferative disease and cancer. To address the importance of cyclin D1 in ERalpha-mediated mammary tumorigenesis, we crossed ERalpha-overexpressing mice with cyclin D1 knockout mice. Mammary gland morphogenesis was completely interrupted in the ERalpha-overexpressing cyclin D1-deficient triple transgenic mice. In addition to a highly significant reduction in mammary epithelial cell proliferation, cyclin E was upregulated resulting in DNA damage checkpoint activation and apoptosis. This imbalance between proliferative and apoptotic rates in conjunction with remarkable structural defects and cellular disorganization in the terminal end buds interrupted ductal morphogenesis. Interestingly, the structure of the mammary fat pad was fundamentally altered by the consequences of overexpressing ERalpha in the epithelial cells in the absence of cyclin D1 illustrating how alterations in the epithelial compartment can impact surrounding stromal composition. Transplantation of embryonic ERalpha-overexpressing and cyclin D1-deficient mammary epithelium into the cleared fat pad of wild-type mice did not rescue the aberrant mammary gland phenotype indicating that it was intrinsic to the mammary epithelial cells. In conclusion, although cyclin D1 is not essential for proliferation of normal mammary epithelial cells, ERalpha-overexpressing cells are absolutely dependent on cyclin D1 for proliferation. This differential requirement for cyclin D1 in normal vs abnormal mammary epithelial cells supports the application of cyclin D1 inhibitors as therapeutic interventions in ERalpha-overexpressing breast cancers.