The cytoplasmic membrane is a primary target for the staphylocidal action of thrombin-induced platelet microbicidal protein

Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, cationic peptide released from rabbit platelets exposed to thrombin in vitro. tPMP-1 is microbicidal against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. Preliminary evidence suggests that tPMP-1 targets and disrupts the staphylococcal cytoplasmic membrane. However, it is not clear if the cytoplasmic membrane is a direct or indirect target of tPMP-1. Therefore, we assessed the in vitro activity of tPMP-1 versus protoplasts prepared from logarithmic-phase (LOG) or stationary-phase (STAT) cells of the genetically related S. aureus strains 19S and 19R (tPMP-1 susceptible and resistant, respectively). Protoplasts exposed to tPMP-1 (2 microg/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and ultrastructural alterations (by transmission electron microscopy [TEM]). Exposure to tPMP-1 resulted in substantial lysis of LOG but not STAT protoplasts of 19S, coinciding with protoplast membrane disruption observed by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the staphylococcal cell wall. In contrast to 19S, neither LOG nor STAT protoplasts of 19R were lysed by tPMP-1. tPMP-1-induced membrane damage was further characterized with anionic planar lipid bilayers subjected to various trans-negative voltages. tPMP-1 increased conductance across bilayers at -90 mV but not at -30 mV. Once initiated, a reduction in voltage from -90 to -30 mV diminished conductance magnitude but did not eliminate tPMP-1-mediated membrane permeabilization. Therefore, tPMP-1 appears to directly target the staphylococcal cytoplasmic membrane as a primary event in its mechanism of action. Specifically, tPMP-1 likely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner.     

Activation of the mitogen-activated protein kinase pathway is involved in and sufficient for megakaryocytic differentiation of CMK cells

Activation of the mitogen-activated protein (MAP) kinase pathway has been associated with both cell proliferation and differentiation. Constitutively activated forms of Mek (MAP kinase/Erk kinase) and Erk (MAP kinase) have been previously shown capable of inducing differentiation or proliferation in nonhematopoietic cells. To specifically examine the role of Erk activation in megakaryocytic growth and development, we activated the MAP kinase pathway by the transfection of constitutively activated Mek or Erk cDNA into a human megakaryoblastic cell line, CMK, by electroporation. The CMK transfectant clones that expressed constitutively activated Mek or Erk showed morphologic changes of differentiation. Transfected cells also showed expression of mature megakaryocytic cell surface markers. The MAP kinase pathway was also activated by treatment of the hematopoietic cells with a cytokine that activates Erk. The treatment of CMK cells with stem cell factor (SCF ) caused MAP kinase activation and induced differentiation by the expression of mature megakaryocytic cell surface markers. The effects of the SCF treatment were inhibited by pretreatment with a specific inhibitor of the MAP kinase pathway, PD98059. In this report, we conclude that activation of the MAP kinase pathway was both necessary and sufficient to induce differentiation in this megakaryoblastic cell line.     

Polyclonal antibodies from blood and cerebrospinal fluid of patients with multiple sclerosis effectively hydrolyze DNA and RNA

It is known that in the blood of patients with some autoimmune diseases catalytically active antibodies hydrolyzing proteins, DNA, and RNA may be detected. In the present work homogeneous preparations of IgG antibodies (Ab) possessing high affinity for nucleic acids (NA) were obtained for the first time from blood and cerebrospinal fluid of patients with multiple sclerosis (MS). The fraction of IgG Ab as well as its Fab fragments and isolated light chains of both kappa- and lambda-types were shown to catalyze effectively the hydrolysis of DNA and RNA. It is shown by different methods that the capability for nucleic acid hydrolysis is an intrinsic property of the polyclonal Ab. NA-hydrolyzing Ab were detected in the blood of 69 of 72 and in the cerebrospinal fluid of 5 of 5 examined MS patients, while they were not detected in the blood of any of 50 healthy donors examined. Comparison of relative rates of RNA hydrolysis and of the substrate specificity in hydrolysis of various model RNAs--cCMP, poly(U), poly(A), and poly(C)--revealed pronounced differences of MS antibodies from ribonucleases of human blood, ribonuclease A, and all earlier described abzymes. The abzymes are usually characterized by relatively low specific activities in comparison with that of normal enzymes catalyzing analogous reactions. Ab from the blood of MS patients are the first example of autoabzymes whose specific activity in RNA hydrolysis is comparable or even higher than that of pancreatic ribonuclease A--one of the most active RNA-hydrolyzing enzymes.     

Monocyte procoagulant activity induced by adherence to an artificial surface is reduced by end-point immobilized heparin-coating of the surface

Heparin-coating improves the biocompatibility of blood contacting artificial surfaces. This led us to investigate the impact of heparin-coating (Carmeda AB, Stockholm) of polymetylmetacrylate on the expression of monocyte tissue factor procoagulant activity (TF-PCA) by surface adhesion. Also, the anticoagulant effect of heparin-coating in the presence or absence of adherent procoagulant monocytes was assessed. This is of particular interest, since activation of extrinsic coagulation by adherent monocyte TF-PCA may play a significant role in thrombin generation during extracorporeal circulation. Monocytes exposed to heparin-coated or non-coated polymetylmetacrylate expressed TF-PCA. The heparin coat did not affect the rate of monocyte adhesion. However, heparin-coating reduced the induction of TF-PCA of non-adherent and adherent monocytes by 17 and 33% (p <0.001 and p <0.0003), respectively. Heparin-coating in the absence of monocytes, totally inhibited the clotting of recalcified plasma (p <0.003). In contrast, in the presence of adherent monocytes expressing TF-PCA, surface-bound heparin did not inhibit clotting. However, inclusion of heparin in a plasma concentration of 8.9 IU/ml totally inhibited the activation of coagulation. It is apparent that heparin-coating of an artificial surface is an efficient means to inhibit coagulation of recalcified plasma, but much less so when procoagulant monocytes are adherent to the coated surface. The present findings are of clinical relevance, since monocytes will adhere to blood contacting surfaces of extracorporeal circuits or to implanted vascular prostheses and subsequently express TF-PCA, and this may promote thromboembolism.     

Prognostic features of teratomas with malignant transformation: a clinicopathological study of 21 cases

PURPOSE: Teratomas with malignant transformation comprise up to 6% of metastatic teratomas. The prognosis of patients with these tumors can vary considerably. We delineate factors that may be related to prognosis in a cohort of men with teratoma with malignant transformation. MATERIALS AND METHODS: We analyzed pathological features, treatment, response, recurrence, time to recurrence, subsequent followup and survival for 21 patients (median age 28 years) diagnosed with teratoma with malignant transformation during a 7-year period at our institution. RESULTS: Malignant nongerm cell elements were present in the primary tumor in 11 cases (52%). Of 18 patients with testicular primaries 17 (94%) presented with metastatic disease. Despite aggressive treatment with surgery and chemotherapy 17 of 21 cases (81%) recurred (median time 6 months). Overall, 5 patients (24%) died of disease (median survival 23 months), 5 (24%) are alive with metastases (median followup 41 months) and 11 (52%) have no evidence of disease (median followup 50 months). Progression/recurrence was substantially greater for 2 of 2 cases with a mediastinal origin, 3 of 4 with rhabdomyosarcomatous differentiation and 5 of 6 with neural differentiation compared with the remainder of the cohort (p < 0.05). CONCLUSIONS: Teratomas with malignant transformation are usually metastatic at presentation, have a high recurrence rate and are more aggressive than teratomas without malignant transformation. Prognosis is especially poor for mediastinal teratomas with malignant transformation and for those with neural or rhabdomyosarcomatous differentiation. Complete surgical resection of residual or recurrent disease appears to offer the best chance for prolonged survival.     

Long-term intraocular pressure control after cataract extraction with trabeculectomy: phacoemulsification versus extracapsular technique

Mutations of the p53 gene are associated with a number of non-lymphoid cancers of the dog. The present study investigates the p53 gene status within canine patients treated for primary and secondary lymphoma. Three out of eight patients exhibited p53 gene mutations. These included one patient with a germ line mutation and two patients with de novo p53 mutations associated with the secondary lymphoma. Allelic loss of the p53 gene was also observed within primary and secondary tumours of the three canine patients. The results indicate that germ line p53 mutations exist in dogs and may be involved in the known predisposition of some breeds to cancer. The presence of therapy-related p53 point mutations was found to be associated with chemoresistant secondary lymphomas. A causative role for DNA-damaging chemotherapy in de novo mutation of the p53 gene is discussed. Characterization of p53 inactivation in canine tumorigenesis may provide a valuable clinical model for assessing the efficacy and optimal therapeutic regimens of anti-cancer agents.     

Isolation and characterization of the Saccharomyces cerevisiae DPP1 gene encoding diacylglycerol pyrophosphate phosphatase

Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway. DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme from Saccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol. Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 (diacylglycerol pyrophosphate phosphatase) gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S. cerevisiae. The heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S. cerevisiae. DGPP phosphatase possesses a Mg2+-independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S. cerevisiae and in insect cells. DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains. The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases. A dpp1Delta mutant was constructed by deletion of the chromosomal copy of the DPP1 gene. The dpp1Delta mutant was viable and did not exhibit any obvious growth defects. The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP. Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg2+-independent PA phosphatase activity in S. cerevisiae.     

Treatment of early T1 small T2N breast cancers. Our experience at the Yaounde General Hospital. 21 cases]

OBJECTIVE: To modify the classic fetal biophysical profile (FBP) with the aim of obtaining rapid and accurate information about actual fetal condition in non-compromised fetuses with a subsequent favorable outcome and to be suitable for a number of outclinic patients. METHODS: Four-hundred and ninety-four fetuses from singleton pregnancies in two randomized groups were monitored by the modified FBP (mFBP) and 168 of them after the external vibratory acoustic stimulation (VAS/mFBP). The mFBP was characterized by two main characteristics: non-stress test was excluded and the testing was finished at the moment when all of the three fetal biophysical activities became normal. The external VAS was applied only in cases with no evidence of fetal activity at the start of the FBP. RESULTS: Of the examined fetuses, 326 fetuses in the control group were monitored by the mFBP and there were 316 (96.9%) favorable outcomes and 10 (3.1%) adverse perinatal outcomes. The sensitivity, specificity and positive and negative predictive values of the mFBP score in predicting adverse perinatal outcome were 60, 99, 66.7 and 98.7%, respectively. In the study group of 168 fetuses there were 165 (98.2%) favorable outcomes and three (1.8%) adverse perinatal outcomes. The sensitivity, specificity and positive and negative predictive values of the VAS/mFBP were 66.7, 100, 100 and 99.4%, respectively. The efficiency of the VAS/mFBP in predicting perinatal mortality alone was even higher. After the external VAS and the first 5 min of the modified testing approximately two-fifths (41.8%) of healthy fetuses with a subsequent good outcome exhibited normal in all of the three biophysical activities and approximately two-thirds (65.5%) of them after 10 min. In the VAS/mFBP group of healthy fetuses, during the same time periods, normal breathing movements were observed in 72% and 87% of fetuses, respectively. CONCLUSIONS: According to our results the mFBP and particularly the VAS/mFBP antenatal protocol as a new and rational variant of the FBP could improve fetal assessment allowing in cases of non-compromised fetuses rapid and accurate information about actual fetal well-being. Because of its high accuracy and a reduced testing time the antepartal method with observation of fetal breathing movements after VAS is becoming acceptable as a screening of fetal well-being evaluation in outclinic conditions.     

Plasma insulin and growth hormone during antarctic residence

A 50-year-old female developed a swelling in the epigastrium which later ruptured to form a sinus. A diagnosis of primary cutaneous cryptococcosis was made with the help of histopathology and microbiological studies. Treatment with amphotericin-B and 5-flucytosine gave a good therapeutic response and the sinus healed within 2 months.