The PTPROt tyrosine phosphatase functions as an obligate haploinsufficient tumor suppressor in vivo in B-cell chronic lymphocytic leukemia.

The tyrosine phosphatase PTPROt is a suggested tumor suppressor (TS) in B-cell chronic lymphocytic leukemia (CLL), and its expression is reduced in this disease. In order to examine how reduced PTPROt expression affects CLL in vivo we induced CLL in PTPROt-targeted mice. Unexpectedly, loss of both Ptprot alleles delayed disease detection and progression and lengthened survival relative to mice carrying two intact alleles, indicating that PTPROt fulfills a novel tumor-promoting role in CLL. Tumor cells from mice lacking PTPROt exhibited reduced B-cell receptor (BCR)-induced signaling, as well as increased apoptosis and autophagy. Inhibition of BCR/Src signaling in CLL cells induced their apoptosis, indicating that these findings are linked causally. These results suggest a cell-autonomous mechanism for the weakened CLL phenotype of PTPROt-deficient mice and uncover non-redundant roles for PTPROt in support of BCR signaling and survival of CLL cells. In contrast, loss of only one Ptprot allele induced earlier detection and progression of CLL and reduced survival, consistent with a tumor-suppressing role for PTPROt. Tumor cells from mice lacking one or both Ptprot allele exhibited increased interleukin-10 (IL-10) expression and signaling, factors known to support CLL; cells lacking one Ptprot alleles exhibited normal BCR signaling and cell death rates. We conclude that loss of one Ptprot allele promotes CLL, most likely by activating IL-10 signaling. Loss of both Ptprot alleles also reduces BCR signaling and increases cell death rates, offsetting the IL-10 effects and reducing the severity of the disease. PTPROt thus functions as an obligate haploinsufficient TS in CLL, where its expression levels determine its role as a promoter or inhibitor of the tumorigenic process in mice. Partial loss of PTPROt generates the strongest disease phenotype, suggesting that its intermediate expression levels in CLL are selected for.

Conversions in laparoscopic cholecystectomy.

Authors in this article emphasize the wide use of laparoscopic cholecystectomy in gall bladder surgery. Indications for laparoscopic operations are increasing. In spite of increasing practical experience of surgeons, availability of new instruments, advances of techniques, this problem of conversion is always actual. The question of conversion may depend on subjective and objective causes. It's the decision of the surgeon whether a conversion is necessary taking into account uncomplicated operation and postoperative state of the patient. In the Surgery clinic of FN Nitra 2078 cholecystectomies were performed in the period from 1.1.2002 to 31.1. 2007. Out of this number, there were 1535 (74%) laparoscopic operations and 543 (26%) classic operations. From the group of 1535 laparoscopic operations conversion was necessary in 89 patients (5.7%) (Tab. 4, Ref. 9).

Enterovesical fistulas in Crohn's disease.

BACKGROUND: Crohn's disease is a chronic inflammatory disease of the bowel, that may affects the urinary system. Although fistula formation has been reported in up to 35% patients suffering from Crohn's disease, urinary fistulas affect only 2 to 8% patients. PATIENTS AND METHODS: Authors have done a retrospective study with the aim to investigate the incidence of enterovesical fistulas in patients admitted due to Crohn's disease to the IInd Department of Surgery of the Comenius University Medical School and Department of Surgery of University Hospital Nitra during 10 years long period. RESULTS: The overall incidence of enterovesical fistulas in our clinical material was 6.83%. All patiens underwent elective surgery. There were no serious postoperative complication. A two stage approach was necessary due to severe inflammation in one patient. Severity of inflammation decreased later on, after treatment with anti TNF *, which allowed subsequent elective surgery. CONCLUSION: Authors consider elective surgery as a treatment of choice in the managenet of enterovesical fistulas in Crohn's disease. Surgery is effective and safe (Fig. 2, Ref. 3). Full Text (Free, PDF) www.bmj.sk.

Miniinvasive treatment of pericardial effusions.

The aim of our study is to show the clinical potential of laparoscopic treatment of pericardial effusions. In spite of the small number of the patients we want to bring to attention the benefit of this mininvasive procedure. The laparoscopic fenestration is indicated when the pericardial effusion persists after unsuccessful medical treatment and when clinical and echocardiografic signs of tamponade develop (Ref 13). Full Text (Free, PDF) www.bmj.sk.

[Quadruparesis as a complication of acute pancreatitis--case report]. / Kvadruparéza ako komplikácia akútnej pankreatitídy--kazuistika.

Pancreatic encephalopathy is a rare complication of acute pancreatitis. Clinical features include focal neurological signs and acute oncet of dementia. Clinical picture can fiuctuate over the time and depends on phase of this disease. The progression and the regression of encephalopathy with relapse and remission of acute pancreatitis has been often described.

[Choledochal cyst complication of pregnancy]. / Cysta choledochu ako komplikácia gravidity.

Authors in this case report describe rare complication of pregnancy. Ultrasound examination showed cystic lesion in abdominal cavity. It was indicated caesarean section because of deterioration of patient's condition. After the removal of foetus it was peroperative found cystic lesion in subhepatal area in right mesogastrium. Cystic lesion compressed stomach, colon transverse and loops of small intestine. After the revision of abdominal cavity and local finding surgeon detected choledochal cyst. It was resected and sutured hepaticojejunoanastomosis.

[Acute intestinal ischaemia]. / Akútna intestinálna ischémia.

In spite of introduction modern diagnostics and therapeutics modalities in clinical practice diagnostics of acute intestinal ischaemia is very difficult. Acute intestinal ischaemia is rare cause of acute abdominal dissease but results of surgical treatment and prognosis of the patients with acute intestinal ischaemia is very poor. The aim of study is occurence, diagnostics and therapeutics possibilities of acute intestinal ischaemia, because tretament of acute intestinal ischaemia have high rate of mortality. Authors claiming on own small set of patients that in diagnostics of acute abdominal pain everything in once mind on acute intestinal ischaemia.

Atypical cyst of the right ductus hepaticus.

Cystic dilatation is a rare disease of extra- or intra-hepatic bile ducts. According to the literature it is an disorder accompanying other diseases rather than an independent diagnosis. In the case of smaller dimensions it is often found by coincidence because of its asymptomatic course of development. Mostly it manifests itself through abdominal pain or icterus. The authors show a case report of 14-months old patient with the finding of a cyst in the right ductus hepaticus with extraordinary dimensions (Fig. 2, Ref. 11). Full Text (Free, PDF) www.bmj.sk

Combined parasacral, vaginal and endorectal approach in surgical treatment of giant rectocele.

Authors operated on their Surgical departement 67 years old women with incomplete evacuation, and digital support during defecation, giant rectocele and massive vaginal vault prolaps. Authors realized cinedefecography and detected giant rectocele, depth was 8 cm, anorectal angle was 120 degrees. They stated Resting pressure 40 cm H2O, and Maximum squeeze pressure 50cm H2O by anorectal manometry. Authors verified external anal sphincters defect by endoanal ultrasound and determined Pudendal nerve terminal motor latency (PN TML) and recorded pathologic values of n.pudendal latency ( left branch 2,7 msec., right branch 4,3 msec). In concerning massive vaginal vault prolaps, huge rerectocele and clinical incompletely evacuation with self digital support during defecation with present defect od external anal sphincters and pathologic values of PN TML, authors indicated and made combined transvaginal, endorectal and perineal reconstructive operative performance. In the present time two years after the surgery radiologic mean depth of the rectocele was significantly reduced (preoperatively 8cm; postoperatively 1 cm). Anorectal angle is 100 degrees. Values of the PN TML is normaly (left branch of n. pudendalis 1,7 msec and right branch of n. pudendalis 1,9 msec). Authors recorded Resting pressure 60 cm H2O and Maximum squeeze pressure 110 cm H2O by anorectal manometry. They didnt visualized any external anal sphincters defect by anal ultrasound. Postoperatively difficult evacuation completely disappeared and digital support was no longer necessary during evacuation.

[Postoperative adhesions, the everlasting topical subject]. / Pooperacné adhézie, stále aktuálny problém.

Creation of postoperative adhesions is a part of every abdominal operation. The authors analyse 320 patients operated for ileus in last 7 years. 118 patients were operated for adhesive ileus. Most common reoperations for ileus are after radical gynecological operations and inflammatory intraabdominal diseases. The creation of adhesiones depends on preoperative mechanical or chemical damage of tissues and peritoneum, bacterial infection and irradiation. The major complication of intraabdominal adhesions are disturbances of bowel function what leads to subileus or ileus. Authors present therapeutical possibilities and prefer laparoscopic operations.

[Primary tumors of the duodenum and the small intestine in our clinical study subjects over a ten-year period]. / Primárne tumory dvanástorníka a tenkého creva v nasom klinickom materiáli za desat'rocné obdobie.

The authors are evaluating occurrence of primary tumors of duodenum and small intestine during last 10 years (1995-2004) as a retrospective study. 13 patients were operated in this period, three of them are presented as case reports. The main clinical symptoms were bleeding and obstruction, two patients had no symptoms. To define the preoperative diagnosis of this kind of tumors is difficult and needs an essential combination of exams.

[Internal hernias--uncommon causes of the status ileosus]. / Vnútorne hernie--menej castá prícina ileóznych stavov.

Internal hernia is an uncommon cause of acute intestinal obstruction with. Preoperative diagnosis of an internal hernia is difficult because of the lack of specific signs. Only a few cases of internal hernia have been successfully diagnosed, however, most cases (74%) were initially diagnosed as intestinal obstructions. Authors present the case of the patient with the history of resection of gaster sec. Billroth II with gastroenteroanastomosis. The symptoms of the internal hernia were vomiting, abdominal pain and leaded to an emergency operation. An incarceration of the bowel retroanastomotic under the Braun anastomosis. The obstruction was released by the traction of the bowel and the abnormal opening was sutured to prevent recurrence.

Functional studies on the MRP1 multidrug transporter: characterization of ABC-signature mutant variants.

BACKGROUND: MRP1 is a key multidrug resistance ATP-binding Cassette (ABC) transporter in tumor cells. A functionally important signature motif is conserved within all ABC domains. Our current studies aimed to elucidate the role of these motifs in the cooperation of MRP1 ABC domains. MATERIALS AND METHODS: We designed human MRP1 mutants based on a bacterial ABC structure. Conserved leucines (Leu) were replaced by arginines (Arg), while glycines (Gly) were substituted for aspartic acids (Asp). The activity of these mutants was assayed by measuring ATPase activity and vesicular transport. ATP-binding and transition-state formation were studied by a photoreactive ATP analog. RESULTS: The Leu to Arg mutants retained both ATPase and transport activity, while the Gly to Asp mutants were inactive in all functional assays, while showing normal ATP-binding. CONCLUSION: Our results reinforce the notion that a single mutation in one of the ABC-signature regions affects the function of the whole protein. The relative role of the conservative leucines and glycines in MRP1 indicates a similar three-dimensional structure within the catalytic center of various ABC proteins.

Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1).

The human multidrug resistance protein (MRP1) contributes to drug resistance in cancer cells. In addition to an MDR1-like core, MRP1 contains an N-terminal membrane-bound (TMD(0)) region and a cytoplasmic linker (L(0)), both characteristic of several members of the MRP family. In order to study the role of the TMD(0) and L(0) regions, we constructed various truncated and mutated MRP1, and chimeric MRP1-MDR1 molecules, which were expressed in insect (Sf9) and polarized mammalian (MDCKII) cells. The function of the various proteins was examined in isolated membrane vesicles by measuring the transport of leukotriene C(4) and other glutathione conjugates, and by vanadate-dependent nucleotide occlusion. Cellular localization, and glutathione-conjugate and drug transport, were also studied in MDCKII cells. We found that chimeric proteins consisting of N-terminal fragments of MRP1 fused to the N terminus of MDR1 preserved the transport, nucleotide occlusion and apical membrane routing of wild-type MDR1. As shown before, MRP1 without TMD(0)L(0) (Delta MRP1), was non-functional and localized intracellularly, so we investigated the coexpression of Delta MRP1 with the isolated L(0) region. Coexpression yielded a functional MRP1 molecule in Sf9 cells and routing to the lateral membrane in MDCKII cells. Interestingly, the L(0) peptide was found to be associated with membranes in Sf9 cells and could only be solubilized by urea or detergent. A 10-amino-acid deletion in a predicted amphipathic region of L(0) abolished its attachment to the membrane and eliminated MRP1 transport function, but did not affect membrane routing. Taken together, these experiments suggest that the L(0) region forms a distinct domain within MRP1, which interacts with hydrophobic membrane regions and with the core region of MRP1.

Transition-state formation in ATPase-negative mutants of human MDR1 protein.

In this work we have studied the partial catalytic reactions in MDR1 variants carrying mutations in the conserved Walker A region (K433M and K1076M) of either the N-terminal or C-terminal ABC domain. Both mutations have been demonstrated to cause a loss of drug transport, drug-stimulated ATPase, and vanadate-dependent nucleotide trapping activity. Here we show that these mutants still allow transition state formation (nucleotide trapping) when fluoro-aluminate or beryllium fluoride is used as a complex-stabilizing anion. Drug stimulation of nucleotide trapping was found to be preserved in both mutants. Limited trypsin digestion revealed that whenever MDR1-nucleotide trapping occurred, both ABC domains were involved in the formation of the catalytic intermediates. Our results show that details of the MDR1-ATPase cycle can be studied even in ATPase-negative mutants. These data also demonstrate that the conformational alteration caused by a mutation in one of the ABC domains is propagated to the other, nonmutated domain, indicating a tight coupling between the functioning of the two ABC domains.

Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions.

The human multidrug resistance protein MRP1 and its homolog, MRP2, are both suggested as being involved in cancer drug resistance and the transport of organic anions. We expressed MRP1 and MRP2 in Spodoptera frugiperda ovarian cells and compared their ATP-dependent transport properties and vanadate-sensitive ATPase activities in isolated membrane vesicles. Both MRP1 and MRP2 actively transported leukotriene C(4) and N-ethylmaleimide glutathione (NEM-GS), although the relative affinity of MRP2 for these substrates was found to be significantly lower than that of MRP1. Methotrexate was actively transported by both proteins, although more efficiently by MRP2. ATP-dependent NEM-GS transport by MRP1 and MRP2 was variably modulated by organic anions. Probenecid and furosemide inhibited, whereas under certain conditions sulfinpyrazone, penicillin G, and indomethacin greatly stimulated, MRP2-mediated NEM-GS uptake. Vanadate-sensitive ATPase activity in isolated membranes containing MRP1 or MRP2 was significantly stimulated by NEM-GS and reduced GS, although these compounds acted only at higher concentrations in MRP2. ATP hydrolysis by MRP2 was also effectively stimulated by methotrexate. Probenecid, sulfinpyrazone, indomethacin, furosemide, and penicillin G all significantly increased MRP2-ATPase activity, whereas these compounds acted more as ATPase inhibitors on MRP1. These results indicate that MRP1 is a more efficient transporter of glutathione conjugates and free glutathione than MRP2, whereas several anions are preferred substrates for MRP2. Our data suggest that MRP2 may be responsible for the active secretion of pharmacologically relevant organic anions, such as diuretics and antibiotics, and indicate different modulation possibilities for MRP1 or MRP2 in drug-resistant tumor cells.

Overriding of cyclin-dependent kinase inhibitors by high and low risk human papillomavirus types: evidence for an in vivo role in cervical lesions.

High risk types of human papillomavirus (HPV) are agents in the aetiology of cervical carcinoma. The products of two early genes, E6 and E7, appear to be the principal transforming proteins. Studies of various monolayer cell culture systems have shown that the E7 oncoprotein of human papillomavirus type 16 is able to neutralize or bypass the inhibitory effect of the cell cycle-dependent kinase (CDK) inhibitors (CKIs) p21WAF1/CIP1 and p27KIP1. To understand whether the p21WAF1/CIP1 or p27KIP1 neutralization also plays a role in vivo, we performed studies on clinical specimens. Forty-five cervical biopsies, including HPV-negative mucosa, HPV 16-positive preinvasive (low and high grade lesions) and invasive neoplasia as well as HPV 6-positive condyloma acuminatum were analysed by single and double immunohistology. We examined the positive cell cycle regulator cyclin A and the universal cell cycle marker Ki67 as well as the negative cell cycle regulators p21WAF1/CIP1 and p27KIP1. Here, we show that in a significant fraction of cells the G1 block can be overcome despite high levels of CKIs in HPV lesions. This phenomenon, which was more evident for p21WAF1/CIP1 than for p27KIP1 was most marked in low grade lesions and in condylomata acuminata, in which a high viral productivity is expected. These results indicate that the overriding of CKI inactivation by viral oncoproteins appears to be a conserved property between low and high risk HPV types. We conclude that the CKI neutralization by HPVs is likely to be required for viral DNA replication rather than for malignant transformation of the host cell.

Functional multidrug resistance protein (MRP1) lacking the N-terminal transmembrane domain.

The human multidrug resistance protein (MRP1) causes drug resistance by extruding drugs from tumor cells. In addition to an MDR-like core, MRP1 contains an N-terminal membrane-bound region (TMD0) connected to the core by a cytoplasmic linker (L0). We have studied truncated MRP1 versions containing either the MDR-like core alone or the core plus linker L0, produced in the baculovirus-insect (Sf9) cell system. Their function was examined in isolated membrane vesicles. Full-length MRP1 showed ATP-dependent, vanadate-sensitive accumulation of leukotriene C4 and N-ethylmaleimide glutathione. In addition, leukotriene C4-stimulated, vanadate-dependent nucleotide occlusion was detected. The MDR-like core was virtually inactive. Co-expression of the core with the N-terminal region including L0 fully restored MRP1 function. Unexpectedly, a truncated MRP1 mutant lacking the entire TMD0 region but still containing L0 behaved like wild-type MRP1 in vesicle uptake and nucleotide trapping experiments. We also expressed the MRP1 constructs in polarized canine kidney derived MDCKII cells. Like wild-type MRP1, the MRP1 protein without the TMD0 region was routed to the lateral plasma membrane and transported dinitrophenyl glutathione and daunorubicin. The TMD0L0 and the MRP1 minus TMD0L0 remained in an intracellular compartment. Taken together, these experiments strongly suggest that the TMD0 region is neither required for the transport function of MRP1 nor for its proper routing to the plasma membrane.

Babesia equi field isolates cultured from horse blood using a microcentrifuge method.

Babesia equi, a causative agent of equine piroplasmosis, was isolated from horses in the Chaco Province of Argentina, a known piroplasmosis endemic region. Fifteen B. equi field isolates were acquired by culture from 23 actively working horses from 2 ranches. The horses appeared healthy with no clinical signs or histories indicative of equine piroplasmosis. All 23 horses had B. equi-specific antibody activity by the indirect fluorescent antibody test and 18 were also complement fixation test positive for B. equi. Equine erythrocytes were prepared for parasite culture using a microcentrifuge tube method. This method greatly reduces the time involved in cell handling and parasite exposure to ambient conditions. By this method, B. equi cultures can be initiated from very small quantities of blood.