Urinary LAM grade, culture positivity, and mortality among HIV-infected South African out-patients.

SETTING: Four ambulatory clinics in Durban, South Africa. OBJECTIVE: To test the relationships of patient characteristics, time to mycobacterial culture positivity, and mortality with urinary lipoarabinomannan (LAM) grade category. DESIGN: Newly diagnosed human immunodeficiency virus (HIV) infected adults were screened for tuberculosis (TB) using sputum culture, tested for urinary LAM, and followed for up to 12 months. We performed multivariable ordinal logistic regression of risk factors for low (1 or 2) or high (3, 4, or 5) LAM grade. We used adjusted Cox regression models to determine the hazard ratios of time to culture positivity and death. RESULTS: Among 683 HIV-infected adults, median CD4 count was 215 cells/mm³ (interquartile range 86-361 cells/mm³), 17% had culture-confirmed TB, and 11% died during follow-up. Smoking, tachycardia (pulse > 100 beats/minute), CD4 count < 100 cells/mm³, and TB culture positivity were each associated with higher LAM grade. In multivariate models, a high urine LAM grade was associated with four-fold increased hazard of culture positivity (P = 0.001) and two-fold increased hazard of mortality (P = 0.02). Among patients treated for TB, these associations were no longer statistically significant. CONCLUSION: In this population, a higher urine LAM grade was associated with shorter time to culture positivity and mortality; however, these associations were not present for those starting anti-tuberculosis treatment.

Experience with fully covered self-expandable metal stents for anastomotic stricture following esophageal atresia repair.

There is a lack of experience with fully covered self-expandable metal stents (SEMSs) for the treatment of benign esophageal conditions in the pediatric population. This is the evaluation of our institutional experience of placing SEMSs for anastomotic stricture (AS) formation following esophageal atresia (EA) repair. Patients were jointly managed from the Department of Pediatric Surgery and Central Interdisciplinary Endoscopy at our institution. Thirteen children (8 male, 5 female) with a median age of 4 months (range: 1-32 months) who underwent treatment with SEMSs for a postoperative AS following EA repair between February 2006 and April 2016 were recruited into this retrospective study. SEMSs that are originally designed for other organs such as trachea, bronchus, biliary tract, or colon were inserted under general anesthesia via endoscopic guidance. Simultaneous fluoroscopy was not required in any case. In five infants, the stents were inserted primarily without previous therapy. Seven patients underwent stenting following dilatation with or without adjuncts (e.g. Mitomycin C, Triamcinolone). In one case with an AS and a simultaneous persistent tracheoesophageal fistula (TEF), multiple SEMSs were applied after failure to close the fistula with fibrin glue.The median duration of individual stent placement was 30 days (range: 5-91 days). In five children up to four different biliary, bronchial or colonic SEMSs were placed successively over time. There were no problems noted at stent insertion or removal. Eight children (62%) developed complications associated with stenting. At follow-up, in eight patients (62%) AS was resolved, including all of those five cases, who had their stents inserted without previous therapy. Five children (38%), who underwent dilatation prior to stenting did not improve their AS and required further intervention. Overall, the cohort exhibited a slight, but not significant weight gain between stent insertion and (final) stent removal.Insertion of SEMSs for AS following EA repair is safe and often successful with only one single application. It can be used as a primary procedure (without previous therapy) or after failed dilatations.There was one death in this study that was unrelated to stenting and occurred 12 months after stent removal. Because of the absence of manufactured, age-related devices, SEMSs that are originally designed for other organs can be applied. Establishment of a standardized management including stent placement for the treatment of AS following EA repair in the pediatric population is required.

Alternative ileal flap for bladder augmentation if mesentery is short.

PURPOSE: To date the clam ileocystoplasty is the preferred method of bladder augmentation in children when the urodynamic problem is non-compliance and/or detrusor overactivity. The key to this technique is the incision of the bladder wall deep into the pelvis down to the trigone in order to avoid a diverticulum like neobladder and to provide adequate margin for augmentation. The detubularised ileum flap therefore has to reach to the bottom of the divided bladder on a reliable vascular pedicle without significant tension. A short ileal mesentery caused by previous surgery, peritonitis, peritoneal dialysis or ventriculo-peritoneal shunt may complicate surgery and compromise outcome. We hypothesized we can rely on the communication of the intramural vessels within the intestine and can detubularise the ileum adjacent to the mesentery rather than along the antimesenteric line and this could be combined with ligation of some vasa recta (VR) in order to create alternative ileum flaps, which reach further into the pelvis. Our aim was to assess the viability of the alternative flaps detubularised along the paramesenteric line and measure how many VR could be sacrificed beyond the tertiary arcades. MATERIALS AND METHODS: After ethical approval adjacent ileal segments were detubulirased along the antimesenteric line (Group 1) and along the paramesenteric line (Group 2) in 5 minipigs in general anaesthesia. Ligation of 0,1,2,3 and 4 VR has been performed starting from the free end of the segments. The length of the ileal flaps was recorded. The microcirculation of flap edges was detected by in vivo microscopy using orthogonal polarising spectral imaging (Cytoscan A/R Cytometrics, PA, USA). Clam ileocystoplasty was performed with the ileum detubularised along the paramesenteric line without ligation of VR. Specimens of the augmented bladder were obtained after 4 weeks and stained with Hematoxilin + Eosin. RESULTS: No alteration in capillary red blood cell velocity (RBCV) and perfusion rate (PR) was observed after paramesenteric detubularisation. The flaps in Group 2 reached 20.25 ± 0.5 mm longer vs. CONTROL: This is 98% of the mean bowel width (20.5 ± 0.57 mm) measured in the animals. Ligation of each VR further increased the length of both flaps (mean: 10.59 ± 3.18 mm) however ligation of more than 2 VR gradually decreased the microcirculation in both groups. All animals augmented with alternative flap survived, there was no urine leak or suture break down. Histology confirmed viable bowel flaps. CONCLUSION: Paramesenteric detubularisation of the ileum is fully tolerated and results in longer reaching ileal flap vs. CONTROL: Only limited ligation of VR is tolerated. DISCUSSION: This study showed the first time that clam ileocystoplasty is feasible with ileal flap detubularised along the paramesenteric line. The use of the animal model and the relative short postoperative observation are the main limitations of this study.

Membrane expression of trail receptors DcR1 and DcR2 in the normal endometrium, endometrial atypical hyperplasia and endometrioid endometrial cancer.

We aimed to evaluate the membrane expression of DcR1 and DcR2 in the normal endometrium (NE), endometrial atypical hyperplasia (EAH) and endometrioid endometrial cancer (EEC). The study comprised 101 patients: 20 NE, 14 EAH and 67 EEC. Membrane expression of DcR1 and DcR2 was examined and presented as total score (TS). The membrane expression of both DcR1 and DcR2 was more common in EEC than in NE (p < 0.001; p < 0.001). A strong correlation was found between type of endometrial tissue (NE/EAH/EEC) and the TS of DcR1 (p = 0.001) and DcR2 (p < 0.001). In EEC, the TS of DcR1 and DcR2 was not related to grading and survival. The TS of DcR1 negatively correlated with staging (p = 0.018), but DcR2 did not. The membrane expression of decoy receptors for TRAIL DcR1 and DcR2 is greater in NE than EEC. In EEC patients, membrane expression of DcR1 and DcR2 are not independent predictors of survival.

Membrane expression of the death ligand trail receptors DR4 and DR5 in the normal endometrium, endometrial atypical hyperplasia and endometrioid endometrial cancer.

To assess membrane expression of DR4 and DR5 in the normal endometrium (NE), endometrial atypical hyperplasia (EAH) and endometrioid endometrial cancer (EEC), the study examined 101 patients: 20 NE, 14 EAH and 67 EEC. The expression of DR4 and DR5 was examined and presented as the total score (TS). DR4 expression was seen in 18 NE, 11 EAH and 10 EEC. DR5 expression was seen in 20 NE, 13 EAH and 21 EEC. A strong correlation between type of endometrial tissue and TS of both receptors was identified. In EEC TS of DR4 and DR5 was not related to grading, staging or survival. Malignant transformation in the endometrium is related to reduction of membrane DR4 and DR5 expression. The level of membrane staining of the receptors in EEC is not dependent on grading and staging, and is not sufficient to predict survival in EEC patients.

Construction of a tissue microarray with two millimeters cores of endometrioid endometrial cancer: factors affecting the quality of the recipient block.

The tissue microarray (TMA) method currently is not used to render a primary diagnosis of cancer, but its scientific value has been proved in studies of various cancer types. TMA technology still is not used often for uterine tumors, however. We investigated the repeatability of histological diagnosis of endometrioid endometrial cancer (EEC) using conventional histology and TMA using 2 mm cores. We examined EEC tissues from 171 patients. Formalin fixed, paraffin embedded tissue donor blocks from EEC specimens were selected and examined histologically. Duplicate 2 mm tissue cores were inserted into a TMA recipient block. EEC tissues were examined as hematoxylin-eosin stained sections from the TMAs. EEC tissue was identified in the TMAs in 158 cases (92.4%) and not found in 13 cases (7.6%). On the TMA slides, both EEC positive cores were identified in 129 cases (75.4%), but only one core in 29 cases (17.0%). Among 342 biopsies of the donor blocks (each case in duplicate), EEC was found in 287 cases (83.9%) using the TMA: 124/146 (84.9%) with superficial infiltration, 153/178 (86.0%) with deep myometrial infiltration, and 10/18 (55.6%) without myometrial infiltration. We concluded that two 2 mm tissue cores from a biopsy of a donor block inserted into a TMA recipient block were sufficient to diagnose EEC in more than 90% of cases. EEC was identified in the TMAs with similar frequency with respect to superficial and deep myometrial infiltration. Cases without myometrial infiltration were identified less often.

Apoptosis-, proliferation, immune function-, and drug resistance- related genes in ER positive, HER2 positive and triple negative breast cancer.

The aim of our study was to examine an association between gene expression assessed using a 23-gene microarray and receptor status of breast cancer samples categorized as ER positive, HER2 positive and triple negative subtypes. The ER positive cohort was subsequently divided into Luminal A, Luminal B HER2 negative and Luminal B HER2 positive subtypes. Core- needle biopsies were collected from 78 female patients with inoperable locally advanced breast cancer or resectable tumors suitable for downstaging, before any treatment. Expressions of 23 genes were determined by means of TagMan Low Density Arrays. Analysis of variance was used to select genes with discriminatory potential between receptor subtypes. We introduced a correction for false discovery rates (presented as q values) due to testing multiple hypothesis. Pairwise post-hoc comparisons of receptor subtypes were performed using Tukey 's HSD test. Five genes out of a 23-gene microarray differed significantly in relation to breast cancer receptor-based subtypes. Among these five genes, we identified: BCL2 (p=0.0002, q=0.0009), MKI67 (p=0.0037, q=0.0064), IGF1R (p=0.0040, q=0.0064), FOXC1 (p=0.0113, q=0.0135) and IRF1 (p=0.0435, q=0.0416) as ones showing ER positive, HER2 positive and triple negative -subtype specific expression profiles. When incorporating Luminal A, Luminal B HER2 negative, Luminal B HER2 positive subtypes into analysis, four genes: BCL2 (p=0.0006, q=0.0034), MKI67 (p=0.0078, q=0.0198), FOXC1 (p=0.0102, q=0.0198) and IGF1R (p=0.0174, q=0.0254) were selected. Elevated levels of IGF1R and BCL2 were significantly linked with Luminal A subtype. Triple negative breast cancer subtype was associated with higher expression of IRF1, FOXC1 and MKI67. In HER2 positive cohort lower expression of all five analyzed genes was noted.

Evaluation of oestrogen receptor expression in breast cancer by quantification of mRNA.

AIMS: cDNA microarrays have subclassified breast carcinomas into molecular subtypes with oestrogen receptor-alpha (ER) gene expression as a main marker. The aim was to compare ER expression in 97 patients with operable breast cancer estimated by real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) and by routine immunohistochemistry, and to determine which method was reliable for molecular subtyping in relation to basal-type keratins and HER2 gene expression. METHODS AND RESULTS: Frozen tumour samples were analysed by real-time RT-PCR for the expression of ER, HER2, keratin 5 and keratin 17 genes. In a group of 27 tumours with a low level of ER mRNA (<1.00), there were eight ER+ cases as assessed by immunohistochemistry, and of 70 cases with a high level of ER mRNA (>or=1.00), 26 were ER- by immunohistochemistry (P = 0.003). Lack of prognostic relevance of ER mRNA level was demonstrated, whereas assessment by immunohistochemistry was related to clinical outcome. Expression of basal keratins and HER2 genes differed significantly between ER+ and ER- tumours based on immunohistochemistry, but not on mRNA level. CONCLUSIONS: These results throw doubt on the assessment of ER mRNA as a key factor in the molecular distinction between breast tumours.

Expression of tumour necrosis factor-related apoptosis-inducing ligand and caspase-3 in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C.

AIM: To assess whether the distribution of the recently described proapoptotic ligand, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and the apoptosis effector, caspase-3 alters with the degree of inflammation and fibrosis present in liver biopsy specimens from patients with chronic hepatitis C virus infection. METHODS AND RESULTS: Expression of TRAIL and caspase-3 was assessed immunohistochemically in liver biopsy specimens obtained from 89 adults with chronic hepatitis C. Expression of TRAIL in hepatocytes correlated inversely with stage of fibrosis (P = 0.001), classified according to the Scheuer score; expression of caspase-3 in hepatocytes correlated with grade of inflammation (P = 0.012). Expression of TRAIL in hepatocytes was not correlated with grade of inflammation (P > 0.05); expression of caspase-3 was not correlated with stage of fibrosis (P > 0.05). Maximum expression of proapoptotic TRAIL protein was observed in cases with low grade inflammation (G0) and low stage fibrosis (S1). Maximum expression of caspase-3 in hepatocytes was observed in cases with high grade inflammation (G3-4) and high stage fibrosis (S3), but not with liver cirrhosis (S4). CONCLUSIONS: There is a significant decrease in TRAIL expression with increasing grade of inflammation, whereas caspase-3 expression is significantly increased with advanced fibrosis, short of cirrhosis.

Pulmonary function following completion of Minimally Invasive Repair for Pectus Excavatum (MIRPE).

UNLABELLED: Pulmonary function testing remains part of the routine preoperative investigations in patients with pectus excavatum, although there is evidence that reduced exercise capacity is predominantly due to impaired cardiovascular performance rather than ventilatory limitation. AIM OF THE STUDY: To evaluate the change of pulmonary function in patients after completion (metal bar removal) of minimally invasive repair for pectus excavatum compared to the preoperative functional results. PATIENTS AND METHODS: All patients who underwent minimally invasive repair for pectus excavatum (MIRPE) between February 2000 and June 2006 and subsequently had their metal bars removed were examined. Pulmonary function tests were performed as part of the routine pre- and postoperative protocol. Vital capacity (VC), forced expiratory volume in one second (FEV1), total lung capacity (TLC), residual volume (RV) and maximal expiratory flow rate at 25 % (MEF25) were evaluated. In addition, comparisons were made between patients with a low ( 6) pectus severity index (PSI) to establish the relative pre- to postoperative change of the above-mentioned lung function parameters. RESULTS: Fifteen patients were included in the study (14 males, 1 female). The median age at surgery was 15.9 years. The metal bar(s) were removed after a median of 37 months following the initial operation. Lung function tests were performed at a median of 32 days preoperatively and 129 days after completion of the procedure. Preoperative lung function values lay below population averages. The majority of patients had either a restrictive, obstructive or combined pulmonary disorder. Improvement was observed in all lung function parameters after metal bar removal compared to preoperative values and was significant for VC, FEV1 and the RV/TLC ratios. With regard to the severity of the deformity, although not statistically significant, patients with a higher PSI (> 6) appeared to benefit particularly from the operation. CONCLUSION: Our findings indicate that pulmonary function improves in patients after completion of minimally invasive repair of pectus excavatum. However, a review of the literature suggests that, in all probability, following surgery, patients benefit more with regard to enhanced cardiovascular performance than from improved pulmonary limitations. The value of routine testing of pre- and postoperative lung function in patients with pectus excavatum is questionable.

Expression of Bax, Bcl-xL and Bcl-2 proteins in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C.

AIMS: To determine the expression of regulators of apoptosis in chronic hepatitis C. METHODS AND RESULTS: Expression of Bax, Bcl-xL and Bcl-2 proteins was assessed immunohistochemically in liver biopsy specimens obtained from 89 adults with chronic hepatitis C. Expression of Bax in hepatocytes correlated inversely with grade of inflammation (P < 0.001) and stage of fibrosis (P = 0.011), classified according to the Scheuer score; expression of Bcl-xL in hepatocytes did not correlate with grade of inflammation (P = 0.106) or stage of fibrosis (P = 0.078); maximum Bcl-xL expression was observed in grade 3 inflammation and stage 4 fibrosis. Expression of Bcl-2 protein in hepatocytes was present in only two cases (both with advanced disease); the expression of Bcl-2 protein in interlobular bile duct epithelial cells correlated with the grade of inflammation (P = 0.018), but not with stage of fibrosis (P = 0.154). The expression of Bcl-2 protein in lymphoid cells infiltrating portal zones and lobules did not correlate with grade of inflammation (P = 0.113) or stage of fibrosis (P = 0.815). CONCLUSION: Major differences in expression of studied proteins were observed in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C.

WWOX--the FRA16D cancer gene: expression correlation with breast cancer progression and prognosis.

AIMS: WWOX is a tumour suppressor gene involved in various tumours including breast cancer. High chromosomal abnormalities in a genomic region spanned by WWOX are associated with the fact that this gene covers approximately 1 million base pairs of the second most affected among common chromosomal fragile sites FRA16D. We evaluated WWOX expression levels in breast cancer samples in association with diagnostics-prognostics markers. METHODS: We performed quantitative real-time RT-PCR to analyse levels of expression of WWOX in 132 cases of breast cancer. We evaluated the relationship between WWOX mRNA levels, clinico-pathological factors, expression of aberrant WWOXDelta6-8 mRNA and other cancer related genes. RESULTS: Expression of WWOX was higher in patients younger than 50 years old, in ER and PR positive tumours vs negative for those receptors and tumours without lymph node metastasis vs LN+. WWOX mRNA levels were also higher in tumours with higher apoptotic index (Bcl2/Bax ratio). Negative associations were found between WWOX expression and cytokeratins 5/6 and 17 (P<0.05). High level expression of WWOX was also associated with better disease free survival. Presence of WWOXDelta6-8 transcripts were accompanied with lower WWOX wild type mRNA level. CONCLUSIONS: Reduced WWOX expression commonly observed in various neoplasias in cases of breast cancer is associated with markers of bad prognosis. Our findings reveal additional evidence that WWOX may be involved in steroid (estrogens) metabolism and signaling pathways. WWOX can be considered as a new target for gene therapy development due to the association of high WWOX expression with improved disease free survival.

Usefulness of polyclonal antibodies raised against P65 oncofetal protein in immunohistochemical diagnosis of ductal breast cancer.

Paraffin-embedded infiltrating ductal breast cancer tissue slides (135) were analyzed by immunohistochemistry with the use of rabbit polyclonal anti-P65 oncofetal protein and mouse monoclonal anti-estrogen/progesterone receptor (ER, PR) antibodies. Analysis with anti-P65 antibody revealed the positive cytoplasmic reaction in 83 cases, 98 showed the nucleic reaction and 3 were immunologically negative. Among the analyzed cases 49 revealed both cytoplasmic and nucleic reactions. For the whole group of cancers the correlation was found between ER or PR level and P65 cytoplasmic reaction (r = 0.77 and 0.66, respectively) and low inverse correlation with nucleic localization of P65 protein. The percentage of positive cells with cytoplasmic expression of P65 was significantly higher in more histologically differentiated cancers (grade I and II according to Bloom and Richardson) than in grade III. Opposite tendency was observed for the nucleic expression of P65 protein. The percentage of immunopositive nuclei grew with the advance of the disease and was the highest in poorly-differentiated (grade III) tumors. The tumors with P65 cytoplasmic reaction were mainly small (T1, T2), without metastases to lymph nodes (N0) and distant metastases (M0). The dependence between P65 protein localization and clinical stage of disease (TNM classification) was evaluated statistically. The straight dependence existed between P65 nucleic reaction and tumor size (p = 0.0002), metastases to lymph nodes (p = 0.0032) and distant metastases (p = 0.0006). The obtained results suggest that the transfer of P65 protein from cytoplasm to nuclei of the breast cancer cells is connected with more clinically advanced stages and worse prognosis for the patients.

Clinically silent Paget disease of male nipple.

Male breast cancer constitutes less than 1% of all breast cancer cases. Special types, such as Paget disease of the breast, are extremely rare. To our knowledge there are less than 50 cases of Paget disease of male breast described in world medical literature. Authors present pathologically proven but clinically silent case of Paget disease of the nipple in an 86-year old male with breast cancer. Literature data suggest that the prognosis of male Paget cancer is worse than in "ordinary" male breast cancer. Therefore, the histologic diagnosis of Paget disease, even in an asymptomatic male patient, may have negative prognostic value.

p65 and c-erbB2 genes expression in breast tumors: comparison with some histological typing, grading and clinical staging.

Using PCR technique we have analyzed p65 and c-erbB2 genes expression in 47 frozen tissue slides taken from patients diagnosed as ductal and lobular breast cancer, classified as G3, and in a limited panel of proliferative breast disease cases. Expression of p65 was generally connected with small tumor size and with absence of metastases in regional lymph nodes. We have found interdependence between p65 gene expression and negative states of lymph nodes. On the contrary, c-erbB2 expression was observed in patients with large tumors and with metastases to the regional lymph nodes. Between both genes (p65 and c-erbB2) opposite interdependence was found. No statistical dependence between estrogen/progesterone receptor levels and p65 or c-erbB2 expression were noticed. The presence of p65 expression appeared in the group of proliferating breast disease cases which were connected with higher risk of breast cancer. Lack of p65 expression accompanied cases which were classified as fibroadenoma.

Quadruplication of the ureter in combination with a large ureteral cyst.

A newborn female was found to have a quadruplication of the ureter: four proximal, slim ureters opened into a large ureteral cyst and a distal monoureter opened into the bladder. Only five cases of ureteral quadruplication have been reported in the literature, one of which presented with identical pathology. These two cases may have been caused by similar, unidentified abnormal embryonic development.

Expression of CD34 in gastric cancer and its correlation with histology, stage, proliferation activity, p53 expression and apoptotic index.

The formation of new blood vessels is essential for tumor growth and progression. Until today there are only few studies of the immunohistochemical assessment of angiogenesis in gastric cancer by the evaluation of the expression of CD34 antigen. The aim of this study was to analyze the relationship between microvessel density (MVD) expressed as the mean count of CD34 immunostained vessels and clinicopathologic features of gastric tumors (the histological type according to the Lauren classification, tumor grade G; presence of lymph node metastases N; depth of tumor invasion; stage of disease (UICC-AJCC 1988 1992), p53 expression, tumor cell proliferative activity described as the Ki67 labelling index and apoptotic index of tumor cells TUNEL method). We assessed formalin-fixed, paraffin-embedded tissue samples obtained during potentially radical gastrectomy from 58 patients with primary gastric adenocarcinoma. The representative tissue blocks from each tumor were used for the immunohistochemical assay and examined by two pathologists independently. MVD was counted in five tumor areas of the most intensive neovascularization (x 200 field by light microscopy) and the mean counts were recorded. The mean MVD (CD34 expression value+/-SD) in this study was 43,15+/-19,8 per x 200 field. The study demonstrated the statistically significant correlation between MVD and two main histological parameters: tumor grading (p < 0.001) and tumor histological type according to Lauren s classification (p<0.05). In well and moderately differentiated tumors (G1/2) MVD was significantly lower in comparison to the group of poorly differentiated cancer G3 (mean value: 31,62 vs. 49,89). MVD was higher in diffuse type of gastric cancer comparing to intestinal type (50.05+/-19,03 vs. 39.17+/-20,09). However, the authors failed to find a significant correlation between MVD and other investigated histopathological features in malignant gastric tumors. The close relationship between CD34 immunostaining, gastric cancer tumor vascularity and main histological parameters was shown in this study. It can be stated that analysis of expression of angiogenesis in gastric cancer may be helpful for better estimation of hematogenous recurrence and the selection of the group of patients for adjuvant antiangiogenic treatment.

Involvement of the Arg-Asp-His catalytic triad in enzymatic cleavage of the phosphodiester bond.

Phosphatidylinositol-specific phospholipase C (PI-PLC) catalyzes the cleavage of the P-O bond in phosphatidylinositol via intramolecular nucleophilic attack of the 2-hydroxyl group of inositol on the phosphorus atom. Our earlier stereochemical and site-directed mutagenesis studies indicated that this reaction proceeds by a mechanism similar to that of RNase A, and that the catalytic site of PI-PLC consists of three major components analogous to those observed in RNase A, the His32 general base, the His82 general acid, and Arg69 acting as a phosphate-activating residue. In addition, His32 is associated with Asp274 in forming a catalytic triad with inositol 2-hydroxyl, and His82 is associated with Asp33 in forming a catalytic diad. The focus of this work is to provide a global view of the mechanism, assess cooperation between various catalytic residues, and determine the origin of enzyme activation by the hydrophobic leaving group. To this end, we have investigated kinetic properties of Arg69, Asp33, and His82 mutants with phosphorothioate substrate analogues which feature leaving groups of varying hydrophobicity and pK(a). Our results indicate that interaction of the nonbridging pro-S oxygen atom of the phosphate group with Arg69 is strongly affected by Asp33, and to a smaller extent by His82. This result in conjunction with those obtained earlier can be rationalized in terms of a novel, dual-function triad comprised of Arg69, Asp33, and His82 residues. The function of this triad is to both activate the phosphate group toward the nucleophilic attack and to protonate the leaving group. In addition, Asp33 and His82 mutants displayed much smaller degrees of activation by the fatty acid-containing leaving group as compared to the wild-type (WT) enzyme, and the level of activation was significantly reduced for substrates featuring the leaving group with low pK(a) values. These results strongly suggest that the assembly of the above three residues into the fully catalytically competent triad is controlled by the hydrophobic interactions of the enzyme with the substrate leaving group.

Mechanism of phosphatidylinositol-specific phospholipase C: origin of unusually high nonbridging thio effects.

Phosphatidylinositol-specific phospholipase C (PI-PLC) has been proposed previously to employ a catalytic mechanism highly reminiscent of that of ribonuclease A (RNase A). Both catalytic sites are comprised of two histidine side chains acting as a general base-general acid pair and a phosphate-activating residue: an arginine in the case of PI-PLC and a lysine in RNase A. Despite these structural similarities, the PI-PLC reaction is slowed 10(5)-fold upon substitution of one of the phosphate nonbridging oxygen atoms with sulfur, whereas a much smaller effect is observed in the analogous RNase A reaction. Here, we report a systematic study of this property in PI-PLC, conducted by means of site-directed chemical modification of a cysteine residue replacing the arginine at position 69. The results show that mutant enzymes featuring bidentate side chains at this position display significantly higher activity, higher thio effects, and greater stereoselectivity than do those with monodentate side chains. The results suggest that the bidentate nature of Arg69 is the origin of the large thio effects and stereoselectivity in PI-PLC. We propose that in addition to binding the phosphate, the function of arginine 69 is to bring the phosphate group and the 2-OH group of inositol into proximity and to induce proper alignment for nucleophilic attack, and possibly to lower the pK(a) of the 2-OH. The results presented here could be important to mechanisms of phosphoryl transfer enzymes in general, suggesting that a major part of thio effects observed in enzymatic phosphoryl transfer reactions can originate from factors other than direct interaction between a side chain and a phosphate group, and caution the use of the absolute magnitude of the thio effect as an indicator of the strength of such interactions.