Further characterization of the acidic GPI-hydrolyzing phospholipase present in human sera

A phospholipase from human serum capable of hydrolyzing glycosylphosphatidylinositol membrane anchors was described and partially characterized by our group some years ago. This activity presented a pH optimum between 5.0 and 6.0 and was inhibited by EDTA, EGTA and 1,10-phenanthroline. Partial purification showed that the enzyme was a glycoprotein with an apparent molecular weight of 140 kDa as judged by gel filtration. Other investigators characterized at the same time a phospholipase D with similar properties but with a pH optimum near 7.5. We now confirm that the human serum enzyme is indeed a phospholipase D capable of hydrolyzing mfVSG and glycolipids A and C from T. brucei. Isoelectric focusing of whole sera suggests the presence of two isoforms, one with a pI of 4.7 which was the form previously purified by our group, and others with pI from 6.2 to 7.4.     

Alpha-adrenergic receptors in human myometrium: changes during pregnancy

In order to investigate the effect of fibrin glueing on the treatment or prevention of air leakages, 114 patients undergoing pulmonary resections and pneumonectomies were studied in two treatment groups: surgery alone (59 patients) or analogous surgical treatment followed by the application of fibrin glue (55 patients). The patients were randomly assigned to treatment groups within two strata: pulmonary resections (63 patients) and pneumonectomies (51 patients). Intraoperatively, 81% of the patients undergoing pulmonary resection who suffered from air leakages after conventional suturing showed improved results of the airway-tolerance-pressure test after the application of fibrin glue (one-sided P value < 0.01; 95% confidence interval: 58-95%). Treatment with fibrin glue reduced the incidence of postoperative leakages significantly from 66% in the control group to 39% in the treatment group (one-sided P-value < 0.02; estimated risk reduction 41%; 95% confidence interval 2-65%). An additional reduction of the duration of post-operative air leakages by the treatment with fibrin glue could not be shown. In terms of minor response criteria, slight trends for an advantage of treatment with fibrin glue could be observed for the duration of stay in hospital and the number of patients with complications. There were no obvious trends concerning fever, intraoperative and postoperative intubation times, the amount of secretion from thoracic tubes and the general condition of the patients. No adverse drug event related to fibrin glueing was observed.     

Dietary oxysterols are incorporated in plasma triglyceride-rich lipoproteins, increase their susceptibility to oxidation and increase aortic cholesterol concentration of rabbits

Early fatty streaks and advanced lesions are characterized by the deposition of cholesterol and cholesterol oxidation products (oxysterols). Oxysterols have been shown to be cytotoxic and pro-atherogenic compared to cholesterol and are found in cholesterol-rich processed foods. The consumption of dietary oxysterols may be significant in the onset and development of vascular disease. In order to study the short term effects of low levels of ingested dietary oxysterols on lipoprotein and aortic cholesterol and oxysterol levels, rabbits were fed either standard chow, chow supplemented with 1.0% oxidized cholesterol (containing 6% oxysterols), or 1.0% purified cholesterol (control). To determine the distribution and uptake of oxysterols after a 2-week dietary period, triglyceride-rich plasma lipoproteins, low density lipoproteins and aorta were analyzed by GC-MS. The concentration of 7beta-hydroxycholesterol was similar in all groups but the oxidized cholesterol-fed animals showed five times the concentration of 5alpha,6alpha-epoxycholesterol and double the level of 7-ketocholesterol in triglyceride-rich lipoproteins compared to the purified cholesterol-fed animals. The presence of 7-ketocholesterol in LDL was exclusive to animals fed the oxidized cholesterol diet. In addition, oxidation of triglyceride-rich lipoproteins was significantly greater in rabbits fed oxidized cholesterol compared to the pure cholesterol-fed animals. The oxidized cholesterol-fed animals also had a 64% increase in total aortic cholesterol, despite lower plasma cholesterol levels compared to the pure cholesterol control animals. Taken together these results suggest that dietary oxysterols may substantially increase the atherogenicity of lipoproteins.     

Change in the concentration of total lipids, phospholipids and neutral lipids in rat liver mitochondria and microsomes during chemical carcinogenesis

Sharp increase in the content of total lipids and, especially, neutral lipids, and considerable decrease in the content of phospholipids were observed in liver microsomes and mitochondrias from liver of tumour-bearing rats in the process of the growth of sarcoma, induced by a single injection of 3,4-benzpyrene. The injection of anthracene, a non-cancerogenous hydrocarbon, practically did not affect the lipid composition of rat liver subcellular particles. Thus, the disturbances in normal functioning of subcellular particles under cancerogenesis are due to considerable change in the chemical composition of biomembranes. The data obtained confirm our hypothesis on lipid mobilization under tumour growth.     

Chorionic gonadotropin concentration in early human pregnancy: comparison of specific and nonspecific assays

Daily blood samples were collected during 10 menstrual cycles in which conception had occurred, and changes in circulating concentrations of human chorionic gonadotropin (hCG) were measured simultaneously in specific and nonspecific radioimmunoassays. The hCG concentration profiles were not identical, and the fundamental differences could not be related to differences in technique. Both specific and nonspecific parameters of hCG concentration rose exponentially for the first 2 to 3 weeks following first detection and were thus amenable to linear regression analysis. Doubling times were significantly different at 2.3 and 1.8 days, respectively. Extrapolation of the regression data gave a (theoretical) plasma concentration of 3 IU/liter at 9.5 days after the luteinizing hormone peak using the specific assay but at only 5.5 days using the nonspecific assay. This difference is sufficiently large to suggest the presence of other forms of hCG or its subunits about the time of implantation and during early pregnancy.     

Changes in the expression of adenylyl cyclases in the rat uterus during the course of pregnancy

The changes in the expression of the various adenylyl cyclases (ACs) in the rat uterus during the course of pregnancy and after delivery were examined by Northern blot analysis and AC assay. Northern blot analysis revealed that five isoforms of ACs (AC2, AC4, AC6, AC7, and AC9) were expressed in the rat uterus, AC6 being the most abundant. The level of expression of these ACs increased 1.7- to 3.4-fold during the course of pregnancy and diminished near term and after delivery. The highest level of expression in each type of AC was consistently seen on Day 17 of pregnancy, and the relative increase of expression, as compared to that in nonpregnant rats, was as follows: AC2 > AC4 > AC7 > AC9 > AC6. In agreement with these findings, both basal and forskolin-stimulated AC activities exhibited a 2- to 3-fold increase during the course of pregnancy, followed by a decrease near term. Our data indicate that post-receptor events, namely marked changes in the level of AC mRNA (and presumably proteins) occur during pregnancy and after delivery and that they contribute to the essential role of cAMP in maintaining uterus quiescence.     

Fear of childbirth during pregnancy may increase the risk of emergency cesarean section

The clinical features of progression sensorineural hearing loss and vertigo in combination with the radiologic finding of a contrast-enhancing mass within the inner auditory canal are suggestive of an acoustic neuroma. We report our findings in a 57-year-old woman with known mixed connective tissue disease who was presumed to have a neuroma. A large malignant lymphoma of the cerebellopontine angle presented clinically with a primary acoustic none palsy and no other central neurological deficits. Both the primary radiological examinations and the exclusively peripheral nerve palsy failed to indicate manifestations of a lymphoma. The development of a progressive facial palsy within 8 weeks of presentation and an atypical occipital headache were uncommon findings for an acoustic neuroma. Such changes in symptoms despite the occurrence of cardinal symptoms require further diagnostic measures. Manifestations of a malignant lymphoma in the cerebellopontine angle are extremely rare. To our knowledge a case of an intracerebral lymphoma in a patient with Sj?gren's syndrome has never been reported before.     

Effect of heparin on lecithin: cholesterol acyltransferase and lipids in cholestasis

Injury to the hippocamp disturbs learning processes and short-term memory in 20-, 50- and 110-day rats. In 50-day rats, hippocampectomy results in lesser changes in learning and memory than in 20- and 110-day animals. Anatomo-physiological characteristics of the hippocamp in 20-day rats presumably indicate a particular importance of this structure at early stages of ontogenesis, when the brain cortex is not yet sufficiently mature and its connections with other structures are not completely formed. Non-linear dependence of learning disturbance in rats of varying age after hippocampectomy suggests that in rat hippocampal function undergoes changes during individual development of animals.     

Accumulation of co-localised unesterified cholesterol and neutral lipids within vacuolised elastin fibres in athero-prone areas of the human aorta

To investigate whether there are alterations of elastin fibres in the arterial intima at the pre-atherosclerotic stage, grossly normal areas of human thoracic aorta were taken soon after death from 13 healthy trauma victims whose ages ranged from 16 to 40 years. Two areas were compared: atherosclerosis-prone (AP) areas localised to the dorsal aspect of the aorta along the rows of intercostal branch origins, and atherosclerosis-resistant (AR) areas from the ventral aorta. Electron microscopic analysis combined with cytochemical staining was applied. Unesterified cholesterol was identified using the filipin-staining technique while neutral lipids were visualised by the OTO-technique. Intimal features were studied by combining the filipin-staining and the OTO-technique. Electron microscopical examination showed that in both AR and AP areas, some elastin fibres in the intima were vacuolised. Unesterified cholesterol was found to be predominantly localised in the musculoelastic layer, in particular, inside the vacuolised elastin fibres. This localisation was seen in all 13 AP areas studied in contrast to the AR areas where it was observed in only four of 13 aortas studied (P < 0.0005, chi2-test). Accumulation of neutral lipids inside vacuolised elastin fibres was found in five out of 13 AP areas but was not observed in any of the AR areas (P=0.01, chi2). A combination of the filipin-staining and OTO-techniques showed that some deposits of neutral lipids and unesterified cholesterol within vacuolised elastin fibres were independently located from each other, but more frequently, neutral lipids were co-located with unesterified cholesterol. The present observations indicate a difference between AP and AR intimal areas which, in particular, relates to the structure of elastin fibres in the musculoelastic layer. The observations suggest that alterations of the extracellular matrix are involved in the trapping and retention of cholesterol and neutral lipids within the intima at an early stage in the development of atherosclerotic lesions.     

Methylation status of DNA cytosine during the course of induction of liver cancer in hamsters by hydrazine sulfate

Hydrazine, which is toxic and carcinogenic to rodent liver, has been shown to react with endogenous formaldehyde in the liver to form formaldehyde hydrazone (CH2 = N-NH2), an alkylating intermediate that methylates DNA guanine at the N7- and O6-positions. Studies were conducted to investigate the role of chronic hydrazine-induced hepatotoxicity on DNA maintenance methylation (formation of 5-methyldeoxycytosine) and the development of liver cancer. Male Syrian golden hamsters were given hydrazine sulfate (0, 170, 340 and 510 mg/l) in drinking water for 21 months (average dose 0, 4.2, 6.7 and 9.8 mg/kg body wt hydrazine as the free base). Hepatotoxicity was evaluated histologically, and regenerative DNA synthesis and maintenance methylation were measured as the incorporation of [methyl-14C]thymidine into DNA and the methyl moiety of [methyl-3H]methionine into 5-methyldeoxycytosine in DNA, respectively. Methylguanines were detected in liver DNA at the first observation time of 6 months of treatment; levels of these aberrant bases decreased or became undetectable at 14 months, and increased in a dose-related manner for the remainder of the study. DNA adducts persisted in the highest dose group throughout the study, repeating the results of a similar study previously reported by this laboratory (Bosan et al., Carcinogenesis, 8, 439-444, 1987). Linear regression analysis of thymidine and methionine methyl moiety incorporation into liver DNA suggested impairment of maintenance methylation of DNA (5-methyldeoxycytosine) in the middle and high exposure animals. Hepatic adenomas and hepatocellular carcinomas developed in a dose-related manner and were highly correlated to decreased uptake of radiolabel from methionine into DNA 5-methylcytosine. These results are part of a continuing study on alteration of maintenance methylation during hydrazine induction of liver cancer.     

Coexpression of cholesterol sulfate and cytokeratin as tumor markers in well-differentiated squamous cell carcinoma of the human uterine cervix

The expression of cholesterol sulfate (CS) is known to increase during squamous differentiation of keratinocytes and to activate the epsilon, eta, and zeta forms of protein kinase C as a signal transduction molecule for the subsequent expression of transglutaminase-1 (TG-1) and cytokeratins. To gain further insight into the regulation of cellular differentiation and tumorigenesis by CS, we examined the concentration and the potential for synthesis of CS in seven and four surgical specimens from human ovarian and uterine cervical cancer patients, respectively, and eight cell lines established from human uterine cervical cancer patients and compared them for the rate of expression of cytokeratin. CS was present in all of the uterine cervical cancer tissue specimens but only in the mucinous type of cystadenocarcinoma among ovarian cancer tissue specimens, and cytokeratin was highly expressed in the tissues with a high concentration of CS, which were classified as well-differentiated on the basis of morphological examination. Similarly, cells derived from a keratinizing type of well-differentiated cervical carcinoma demonstrated strong potential for synthesis of CS, stained positive with anti-cytokeratin antibody, and exhibited a higher specific activity of TG-1, whereas the cells without CS did not stain positive with anti-cytokeratin antibody and exhibited a lower specific activity of TG-1. These findings indicate that CS is coexpressed with TG-1 and cytokeratin in the well-differentiated types of squamous cell cancers as a tumor marker.     

Cytophotometric determination of DNA concentration in the nuclei of the human placental trophoblast in late toxemias of pregnancy

The distribution of trophoblast nuclei of the human placenta by their ploidy was studied in normal pregnancy and in late pregnancy toxemias by the monowave emthod of cytophotometry in visible light. In the plasmodiotrophoblast a pronounced increase in the amount of nuclei in the S-phase (up to 40%) was noted in late toxemias which suggests a considerable activation of the DNA synthesis. The placenta cytotrophoblast in late toxemias is substantially different from that in normality. The Langhans cells disappear almost completely while in normality they are present in young terminal villi till the very end of pregnancy. The amount of the island cytotrophoblasts is much greater, the distribution of nuclei according t the DNA content being of absolutely different character than in normality. A great amount of hypodiploid nuclei (up to 36%) was observed which are absent in normal conditions as well as much greater fluctuations in the amount of diploid nuclei (from 4% to 37%) as compared with the normal amount (25.9%). These data show a high amitotic activity in the island cytotrophoblast in late toxemias. The above changes in the plasmodiotrophoblast and cytotrophoblast might be considered as a compensatory reaction of the placenta to a considerably increased vacuolization and deep atrophic processes in degenerating villi.     

Higher cholesterol in human LDL is associated with the increase of oxidation susceptibility and the decrease of antioxidant defence: experimental and simulation data

BACKGROUND: Kaposi's sarcoma (KS) is an human herpesvirus 8-associated tumor, occurring in immunocompromised patients. We report here an increased incidence of KS among kidney graft recipients (KGRs) during the last 2 years, concomitant to the introduction of the immunosuppressant mycophenolate mofetil (MMF). METHODS: A total of 1835 KGRs, receiving organs between 1987 and 1997, were surveyed for the development of KS. A total of 371 patients received therapy including MMF (group A), whereas 1464 patients were treated with an MMF-free protocol (group B). RESULTS: 3/371 patients (0.8%) of group A versus 2/1464 patients (0.1%) of group B developed KS. In group A, KS became evident 7+/-2 months after initiation of MMF therapy. CONCLUSIONS: At our center, during the last 2 years, the incidence of KS has increased in KGRs, and it is not clear whether the introduction of MMF contributes to the phenomenon.     

Prominent increase of macrophage migration inhibitory factor in the sera of patients with uveitis

Ciguatera is a significant food-borne disease caused by potent polyether toxins (ciguatoxins) which accumulate in the flesh of ciguateric reef fish at risk levels > 0.1 ppb for Pacific ciguatoxins. Research on ciguatera has been severely hindered by the lack of analytical methods that detect and characterize low levels of ciguatoxin in crude extracts of fish. Here we report a new procedure for ciguatoxin analysis based on gradient reversed-phase HPLC/tandem mass spectrometry (HPLC/MS/MS). The method gave a linear response to pure Pacific and Caribbean ciguatoxins (P-CTX-1 and C-CTX-1) and the structurally related brevetoxin (PbTx-2) spiked into crude extracts of fish. Levels equivalent to 40 ppt P-CTX-1, 100 ppt C-CTX-1, and 200 ppt PbTx-2 in fish flesh could be detected by HPLC/MS/MS. Using P-CTX-1 as an internal standard, the analysis of extracts of 30 ciguateric fish from the Caribbean Sea (8 toxic, 12 borderline, and 10 nontoxic by mouse bioassay) confirmed the reliability of the method and allowed an estimated risk level of > 0.25 ppb C-CTX-1 to be established. HPLC/MS/MS provides a sensitive analytical approach, not previously available, for the determination of Pacific and Caribbean ciguatoxins at sub-ppb levels in fish flesh.     

The effect of oxytocics on the human cervix during midtrimester pregnancy

Observations of the effects of oxytocics on the human pregnant cervix have been made in vivo using a double open ended catheter technique. Prostaglandin E, prostaglandin F2alpha and oxytocin had similar but no specific effects upon the intracervical canal pressure; ergometrine caused contractions of the cervix. The significance of these findings is discussed in relation to cervical rupture and cervico-vaginal fistulae that have been reported following second trimester abortion induced with prostaglandins.     

C19 steroids estrogenic activity in human breast cancer cell lines: importance of dehydroepiandrosterone sulfate at physiological plasma concentration

The estrogenic action of C19 steroids on breast cancer cells was measured by bioluminescence in stably transfected human breast cancer MCF-7 and T47D cell lines with a reporter gene that allows expression of the firefly luciferase enzyme under control of an estrogen regulatory element. The "estrogenic activity" of C19 steroids, such as dehydroepiandrosterone (DHEA) and its sulfate (DHEAS), androst-5-en-3 beta,17 beta-diol, androst-4-en-3,17-dione, dihydrotestosterone, testosterone, and 5 alpha-androstan-3 beta,17 beta-diol was studied. This showed that DHEAS, at concentration observed in physiological conditions (10(-6) M), had a high "estrogen-like effect" in MCF-7 and T47D cell lines. Other C19 steroids, at physiological plasma concentration, alone or together did not have any significant effect on the luciferase activity. Moreover aminoglutethimide, an inhibitor of the aromatase enzyme, in the presence of C19 steroids, partially decreased the luciferase activity. These results suggest that MCF-7 and T47D cell lines could convert DHEAS to estrogen-like compounds by different enzymatic systems.     

Increased concentration of beta-endorphin in the sera of patients with severe atopic dermatitis

OBJECTIVES: To assess the use and content of written information relating to transurethral resection of the prostate (TURP). MATERIALS AND METHODS: All 328 full members of the British Association of Urological Surgeons (BAUS) were requested to submit any information sheets or specialized consent forms given to patients about to undergo TURP. They were asked to record when the information was given and whether this was recorded in the notes. The sheets were analysed with reference to a nominally comprehensive list of topics suitable for inclusion in such sheets. RESULTS: One-hundred and eleven replies were received (34%) which represents 44% of urological departments. Of those replying, only 56 used information sheets for TURP. The overall comprehensiveness of the information was surprisingly poor, with 12% omitting to mention retrograde ejaculation and 19% failing to discuss the need for a catheter. CONCLUSION: The written information given to patients about to undergo TURP appears inadequate. There is a need for a nationally accepted information sheet that can be edited locally.