[Effect of hydrochlorothiazide on various components of the kinin system and plasma renin activity in patients with arterial hypertension treated with captopril]. Combined hypertension treatment with inhibitors of the converting enzyme (ICE) and diuretocs gives manifold advantages, the most important of them is a synergistic action of both drugs resulting in blood pressure decrease and prevention of hypokaliaemia. The purpose of the present study was assessment of the effect of hydrochlorothiazide in 50 mg daily doses on blood levels of kininogen and prekallikrein and on plasma renin activity (PRA) in hypertensive patients treated during captopril 150 mg daily alone during 3 weeks, without success. The study was carried out on 15 patients with essential hypertension stage III after WHO. A control group comprised 18 healthy subjects. The determinations were done three time: before treatment, after 3 weeks on captopril, and after 2 weeks of combined treatment with captopril and hydrochlorothiazide. It was found combined treatment with captopril and hydrochlorothiazide produced blood pressure normalization in patients insufficiently responding to captopril alone. During the combined treatment normalization of blood pressure was associated with a higher fall of kininogen concentration, higher rise of prekallikrein and PRA in relation to the values obtained with captopril alone. This evidenced a more pronounced reaction of the kinin system and the renin-angiotensin-aldosterone system to the combined treatment as compared with the response of both systems to captopril alone.


[Maternal viral infections as a fetal risk factor]. After a review of the literature reports from recent years viral infections in pregnant women are presented as a fetal and neonatal risk factor. Maternal infections with rubella virus, cytomegalovirus++, hepatitis, Coxsackie B, varicella, herpes, poliomyelitis, parvovirus B19 and HIV are discussed stressing their unfavourable effect on the developing embryo, fetus or newborn.


[Experience with surgical treatment of liver neoplasms]. Six cases of primary and metastatic hepatic tumors are reported. The diagnoses, surgical treatment and the possibilities of complications after rarely performed liver resection are presented.


[The EBNA2 coding region in the viral genome of Epstein-Barr virus (EBV)-positive cases of Hodgkin's disease]. Epstein-Barr virus (EBV)-genome containing cases of Hodgkin's disease (HD) are known to express latent membrane protein (LMP) of EBV but no EBV nuclear antigen 2 (EBNA2). We investigated 35 cases of HD for the presence of EBV genome sequences to know whether EBNA2 coding region is deleted in HD. 27 cases had general EBV sequences, none had evidence of a deletion of EBNA2. 26 cases turned out to harbour EBV type 1, one case had EBV type 2. The absence of EBNA2 protein expression in HD can not be explained by a deletion of EBNA2 coding region. EBV type 1 is the prevalent subtype of EBV in our series of HD.


Smoking and reproduction. Short and long term effects and benefits of smoking cessation. There have been many attempts during the last years to restrict smoking because of the significant health hazards. In particular the high prevalence of smoking among women in their reproductive years has been a matter of concern. This review was prompted in order to make the medical profession, and gynecologists in particular, confident with the underlying data. This will hopefully result in enabling them to provide balanced information to their patients, not overemphasizing the dangers of smoking, but rather presenting ways to properly recognize and treat tobacco dependence. It is a widely held view that the various risks imposed by smoking are very modest. A doubling of the risk for a rare problem may not prevent many women from continuing to smoke. However, a doubling of the risk for early miscarriage or a significant reduction of success rate in the treatment of infertility may be much more discouraging. The purpose of this review is to list the various effects smoking may have on the different phases of reproduction and to present an overview of the explanation models which have been suggested. Late effects on the child and its development will also be discussed. Hopefully, this will lead to proper concern about the problems and thus increase the motivation to quit smoking even before attempting a pregnancy. In addition, we give some hints on smoking cessation programs.


Long-term treatment with transcutaneous estradiol and oral medroxyprogesterone acetate. In an open prospective study with therapeutic monitoring, 34 women with climacteric symptoms, FSH > 40 IU/L and LH > 25 IU/L were treated for 12 months with an estradiol-depot-patch (Estraderm TTS) 50 micrograms twice a week and medroxyprogesterone acetate 10 mg tablets from 12th to 25th day of cycle. During the first months a significant improvement was seen in hot flushes and other climacteric inconveniences in terms of Kupperman's menopause index. During the study period FSH and LH were suppressed and the estrogen values were normalized. The fraction of free estradiol compared to protein bound estradiol remained unchanged during the whole treatment. The serum-lipids and serum-SHBG at inclusion were within normal limits and did not change during 12 months of treatment. Thus from these parameters no sign of any liver induction was seen. Ten patients had short term skin symptoms while four withdrew from the study because of persistent skin symptoms. Nine patients withdrew from the study, in five cases this was related to the therapy while in the other four it was due to other causes. Twenty-five (74%) women wished to continue the treatment after 12 months.


Polycystic ovary syndrome and risk for myocardial infarction. Evaluated from a risk factor model based on a prospective population study of women. In order to estimate whether women with polycystic ovary syndrome (PCOS) have an increased risk of developing myocardial infarction, a risk factor model was applied on 33 women with PCOS and 132 age matched referents. The risk factor model was established from independent risk factors for myocardial infarction in a prospective population study of 1462 women in Göteborg, Sweden. The independent risk factors were age, manifest hypertension, manifest diabetes mellitus, central obesity measured as increased waist to hip circumference ratio and serum triglyceride concentration. A considerably increased risk (relative risk of 7.4) of developing myocardial infarction was observed for women with PCOS compared to age-matched referents. Since the risk factors include variables correlated to obesity, the results indicate that advice on dietary restriction is an important part of the treatment once the diagnosis is established.


Lupus anticoagulant and anticardiolipin antibodies in an obstetric population. Lupus anticoagulant (LA) and anticardiolipin antibodies (ACA) have been reported to be associated with fetal loss. OBJECTIVE. Our aim was to estimate the incidence of LA and to examine the correlation between LA and ACA in pregnant women. To investigate the clinical significance of LA and ACA in an obstetric population. STUDY DESIGN. A prospective, cross sectional study of 2856 consecutive women admitted to a department of obstetrics and gynecology for delivery or due to pregnancy complications during an 11 month period. METHODS. Activated partial thromboplastin time (APTT) was determined in all patients. LA and ACA were determined if APTT > or = 35 sec. For reference ACA was determined in a group of randomly selected patients with APTT < 35 sec. The results were analyzed in relation to the obstetrical records. RESULTS. Overall incidence of APTT > or = 35 sec.: 7.0%, significantly more frequent in patients with early spontaneous abortion (18.6%) and intrauterine growth retardation (17.5%). Incidence of LA 0.07%. The patients had undetectable ACA and no clinical condition related to LA. Incidence of ACA class IgM (IgM-ACA) in patients with APTT > or = 35: 20.4%, significantly higher than in the reference group (9.6%). Uncomplicated pregnancy in 84% of patients with IgM-ACA. No cases of ACA class IgG (IgG-ACA) in patients with APTT > or = 35 but two cases in the reference group (one normal pregnancy, one spontaneous abortion). CONCLUSION. LA is a rare manifestation with uncertain significance in otherwise healthy pregnant women. IgM-ACA in low titer occurs relatively frequently during normal pregnancy.


Fetal heart rate response to acoustic stimulation in relation to fetal development and hearing impairment. In twelve healthy pregnant women the fetal heart rate response to sound stimulation was tested every second week between the 22nd and the 34th week of the pregnancy. Habituation to the acoustic stimuli used was also investigated at the last examination. A high risk material for hearing impairment comprised 31 pregnant women. The stimuli were presented by a vibrator placed on the maternal abdomen over the fetal head. As stimuli pure tones were used in the form of pulses of one second at a frequency of 3000 Hz and with an onset and a decay time chosen not to cause audible clicks to a normal ear. The intensity of the tone pulse was set at a level calculated to correspond at the position of the fetal head to 110 dB sound pressure level in free field. Roughly 50% of the fetuses tested started to react with an increased heart rate in the 24th week of the development. Four weeks later all tested subjects responded to the test stimuli. Habituation of the motor 'startle' response was shown but the fetus heart rate change was consistent over 20 consecutive stimulations one min in between each. In 31 high risk subjects the responses from three fetuses must be considered as pathologic. These three children exhibited impaired auditory tests after birth and severe hearing impairment at the age of three years. All the others disclosed normal hearing at birth as well as at three years of age. Repeated significant increases in fetal heart rate following acoustic stimulation may appear as an indication of normal function of the hearing organ.(ABSTRACT TRUNCATED AT 250 WORDS)


Women's evaluation of three early abortion methods. Today several very early abortion methods are available; effective and safe, medical as well as surgical. The aim of the present study is to estimate women's attitudes towards three early abortion methods, to see if one or several of these methods are preferable--or best avoided--for psychological reasons, and to assess if women of a certain social or psychological background would benefit from a specific abortion method. Three groups of women were interviewed two weeks after a first trimester abortion; 1/43 women having an 'ordinary abortion' with preoperative ripening of the cervix followed by a vacuum aspiration under heavy sedation, 2/40 women having a modified Karman exeresis with a paracervical block, 3/45 women having a medical abortion with RU 486 and prostaglandin. There was a noticeable difference in the effect the three abortion methods had on the women taking part in the study. Women with a medical abortion reported more bleeding and somewhat more pain and moral considerations than the other women, but also a relief to be spared a surgical procedure. Most of the women said they preferred the abortion method they had just experienced. A short waiting time appeared to be more important than whether a surgical or a medical method was used. Which abortion method is the best in each case is dependent on a variety of personal reasons, and can only be decided by each woman, in consultation with her doctor.


Sonographic evaluation of the endometrium in in vitro fertilization IVF cycles. A way to predict pregnancy? The endometrial thickness and echogenic pattern were prospectively evaluated in 100 patients undergoing IVF-treatment. On the day before oocyte aspiration (day-1) the endometrium was significantly thicker in the group of patients achieving an ongoing pregnancy than in the group that failed to conceive. The minimal endometrial thickness on day-1 compatible with an ongoing pregnancy was 9 mm. Before oocyte aspiration two different endometrial patterns could be distinguished, a multilayered, 'triple line', hypoechogenic endometrium (A) and an isoechogenic pattern (B). Other studies have, in addition, described a hyperechogenic endometrium with a poor pregnancy rate but that pattern could in the present study only be demonstrated after follicle aspiration, 32 clinical pregnancies occurred. Among patients with endometrial pattern A, 36% conceived and among patients with pattern B 23% conceived which was not significantly different. It is concluded that the day before aspiration an endometrial thickness of at least 9 mm appears to be required for an ongoing pregnancy. In all cases a triple line (hypo-or isoechogenic) pattern was evident before aspiration.


Maximal electrical stimulation of patients with frequency, urgency and urge incontinence. Report of 38 cases. Thirty-eight consecutive female patients with frequency, urgency or urge incontinence were treated with maximal electrical pelvic floor stimulation. Diagnostic cystometry was performed in 34 cases. Detrusor instability was found in 13 patients, sensory urgency in 13 and hyperreflexia in eight cases. The effect of the treatment was evaluated by subjective assessment and micturition charts filled in for 48 hours. 63% were cured or improved. The success rate was the same among elderly and younger patients. Detrusor instability and hyperreflexia improved in about 75% of the cases. A good correlation was found between the subjective assessment and the micturition chart recordings. Maximal electrical stimulation has a good effect on certain types of lower urinary tract dysfunction with few side-effects. It is well tolerated even by elderly patients.


Uterine remodelling following conservative myomectomy. Ultrasonographic evaluation. Uterine leiomyomata develop during the reproductive years and may interfere with fertility through mechanisms as yet not fully understood. A developing intramural or large submucosal leiomyoma causes hypertrophy of the myometrium. Conservative myomectomy, as opposed to hysterectomy, is the preferred surgical procedure for patients who wish to preserve their fertility. Ten patients were ultrasonographically evaluated at monthly intervals following myomectomy and the changes in uterine (myometrial) volume were assessed. There was a gradual decrease in uterine volume in all patients during the six months following removal of intramural and submucosal leiomyomata. The most remarkable decrease in size occurred during the initial two to three months for the majority of the patients. The impact of this process on fertility should be further evaluated. One of the intriguing questions is whether the period of uterine shrinkage represents the time of the healing process during which conception should be prevented.


Choriocarcinoma associated with ectopic pregnancy after tubal sterilisation. A 33-year-old highly parous woman developed severe abdominal pain and signs of circulatory collapse 10 months after tubal sterilisation in the absence of symptoms of pregnancy. A ruptured ectopic pregnancy sited interstitially in the right tube and extending into the myometrium and parametrium was found at laparotomy. Histopathologic examination revealed an ectopic pregnancy consisting of choriocarcinoma--a rare but life-threatening combination in a sterilised woman.


Lipid-independent therapeutic properties of transdermal 17 B-estradiol on cardiovascular diseases. A low dose of transdermally administered 17 B-estradiol promptly improved a severe, treatment-unresponsive, cardiac arrhythmia initiated after the menopause in a woman with type-II familial hyperlipoproteinemia. The same treatment normalized the hypertension initiated after ovariectomy, in a woman who was only poorly controlled by anti-hypertensive drugs. These two cases are the first clear report of cardiovascular therapeutic properties of transdermal estradiol via mechanisms independent of lipoprotein modifications.


Multicystic mesothelioma with endometriosis. A multicystic mesothelioma of the omentum in a 36 year old woman consisted of a multicystic mass with foci of typical endometriosis and 'necrotic pseudoxanthomatous nodules'. The presence of endometriosis within multicystic mesothelioma has never been reported. Our findings support the hypothesis that endometriosis plays a rôle in the pathogenesis of multicystic mesothelioma and that this is a reactive rather than a neoplastic lesion.


Megacystis-microcolon-intestinal hypoperistalsis syndrome. Antenatal appearance in two cases. Two cases with severe congenital megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) are presented. This is a rarely encountered syndrome in neonates and 45 cases have earlier been reported. The disease is usually lethal and it now seems clear that MMIHS is an autosomal recessive disorder. The enlarged bladder, typical of this syndrome, is however easy to define by ultrasound, sometimes even in early pregnancy. The concomitant finding of a dilatation of the urinary tract and the absence of oligohydramnios may lead the physician to suspect the diagnosis. Because of the information available from sonography, appropriate investigations can be undertaken immediately after delivery. Prenatal ultrasound examination in subsequent pregnancies is recommended.


Intracellular pH regulation by a Na+/H+ exchanger in cultured bovine trabecular cells. Intracellular pH (pHi) of cultured bovine trabecular cells was measured using video-imaging techniques with a pH-sensitive intracellular fluorescent dye, BCECF. In bicarbonate-rich Ringer at pH 7.4, pHi was 7.29 +/- 0.03 (+/- SEM, n = 12 monolayers, 120 cells sampled). Exposure to 20 mM NH4Cl immediately alkalinized pHi: replacement with a Na(+)-rich solution acidified pHi before recovery to resting levels. When NH4Cl was replaced by a low Na+ solution, acidification was sustained but pHi recovery occurred after Na(+)-rich solution. A pHi of 7.11 +/- 0.02 (n = 2 monolayers, 20 cells) occurred in pH 6.8 and pHi was 7.72 +/- 0.03 (n = 2 monolayers, 20 cells) in pH 8.0. Amiloride (1 mM) acidified pHi but DIDS (1 mM) treatment, HCO3(-)-free condition, 1 mM ouabain, 50 mM K+, and 2 mM BaCl2 failed to change pHi. Hydrogen peroxide (1 mM) acidified pHi but no change occurred with 50 microM. Trabecular cells possess an Na+/H+ exchanger similar to that in other cell types.


Association between feline immunodeficiency virus antibodies and host characteristics in Finnish cats. Toward the end of 1989 the largest private veterinary laboratory in Finland (Vet/lab) began using a commercial combined ELISA test for Feline Immunodeficiency Virus (FIV) antibodies and Feline Leukemia Virus (FeLV) antigens (Cite Combo). The overall proportion of FIV seropositive feline samples was 5% during the 22 month study period. The number of tests performed increased slowly while the positive test results decreased with time (7% in 1990 and 4% in 1991). The decrease in prevalence was assumed to reflect a change in the sample population rather than an actual change in the general cat population. There were more symptomatic and domestic cats tested in 1990 than 1991. The lower-risk groups in the second year of the study may simply be an indication that the cat owners became more aware of FIV and the motivation to send samples switched from the veterinarian's interest to diagnose the disease in a symptomatic cat to the owner's interest to survey their cats for possible FIV infection. In a multivariable analysis, breed, symptoms, age and sex were associated with the risk of FIV seropositivity. The risk increased faster with age in males than in females (i.e., the age effect was not constant between sexes). The cats with symptoms had a higher risk than those without symptoms and non-purebred cats were at a higher risk than purebred cats. FeLV infection was not associated with FIV.


Diet in the epidemiology of postmenopausal breast cancer in the New York State Cohort. A number of authors have presented evidence that high dietary fat increases the risk of breast cancer, and a number have presented evidence to the contrary. In this study, dietary histories were obtained in 1980 from 18,586 postmenopausal women in New York State. These women were followed through 1987 to ascertain their incidence of breast cancer and other cancers and deaths from all causes, as registered in the New York State Tumor Registry and Office of Vital Statistics. Survival analysis revealed that the incidence of breast cancer increased with age, was higher among the nulliparous, was higher for those with a late (> 26 years) age at first pregnancy, and increased with increasing socioeconomic status--all risk factors discovered before for breast cancer. No increase in risk was related to the ingested amount of calories, vitamins A, C, or E, dietary fiber, or fat. Although dietary fat has been found to be associated with higher risk of cancer at a number of other sites, e.g., the lung, colon, and rectum, and although some previous writers have suggested an association with risk of breast cancer, the findings in three cohort studies as well as in eight substantial case-control studies are negative and suggest that a relation is far from established.


Bannayan-Riley-Ruvalcaba syndrome.

Bannayan-Riley-Ruvalcaba syndrome. Here we report on 12 affected members of a family with Bannayan-Riley-Ruvalcaba syndrome. We present clinical evidence of overlap between Bannayan-Zonana syndrome. Riley-Smith syndrome, and Ruvalcaba-Myhre syndrome in this autosomal dominantly inherited condition. We expand the phenotypic spectrum to include Hashimoto thyroiditis, which occurred in 7 of our cases. Finally, we discuss the relationship between the syndrome and juvenile polyposis of infancy.


Hepatitis C in chronic renal failure patients. The occurrence of hepatitis C virus (HCV) infection amongst chronic renal failure (CRF) patients in our Nephrology Unit was investigated over a period of 1 year. A total of 71 patients was studied comprising 26 chronic haemodialysis (CHD) patients, 6 acute haemodialysis patients, 4 peritoneal dialysis patients and 35 CRF patients not on dialysis. Patients were screened before and after haemodialysis, and their baseline and postdialysis values of liver enzymes were determined. Eleven (15.5%) of the total 71 patients were HCV antibody positive. Analysis of the individual patient groups showed that 8 (30.7%) of the 26 CHD patients were positive for HCV. Our data showed a statistically significant relationship between seroconversion and duration of dialysis (p < 0.05). A high statistically significant (p < 0.0001) correlation was observed between the HCV antibodies and CRF. The relative risk of hepatitis C was about 22 times greater for those with CRF compared with the normal controls, which makes CRF an important risk factor. A high proportion of the HCV seroconverters had elevated liver enzyme (serum glutamic pyruvic transaminase). The data presented show a positive correlation between HCV seroconversion, CRF, duration on dialysis and elevated serum liver enzymes.


[Therapy of post-herpetic neuralgia. Pain therapy using an anti-varicella zoster immunoglobulin]. After remission of the dermatological symptoms of herpes zoster infection, post-zoster neuralgia (PZN) can persist or recur for months and years. Most frequently, satisfactory therapy of PZN is not possible. During recent years the persistence of viruses on the surface of neuronal cells has been discussed as the possible reason for chronic pain. Virostatic therapy as well as polyvalent 7s-IgG-immunoglobulins exerted a minimal effect on the pain level. In an open trial we therefore used a specific anti-varicella zoster immunoglobulin (VZI) for treatment of PZN. METHODS. In our study ten patients (six female, four male; age 55-87 years) with latencies between the acute infection and onset of immunoglobulin therapy between 10 months and 3 years were included. PZN was located according to the dermatomes involved: one trigeminal nerve, one cervical, four thoracic and four lumbal segments. All patients had received more than two different drugs or had had therapeutic procedures without success. On the visual analogue scale (VAS: 0-100%) all patients rated their subjective pain with at least 49%. Before starting the study the preexisting specific drug therapy was discontinued in all patients. VZI infusion was given i.v. at a dose of 2 ml/kg body weight within 120 min. All patients had to rate their pain at the VAS every 10 min during the 120 min infusion time, at day 1, 2, 7, 14, 30 and than every month after therapy with VZI. RESULTS. Even during the infusion all ten patients reported a sharp decrease in pain of at least 47% (average 87%) on the VAS. Fourteen days later the remaining pain level averaged 6% VAS. Only the first of the 10 patients reported twice a recurring increase of pain. He therefore received the standard dosage of VZI again at day 2 and day 214, respectively. Thereafter until day 566 the pain level of this patient was lower than 10% VAS. The maximum surveillance interval has so far been 600 days. No side-effects due to the infusion of VZI have been encountered. CONCLUSIONS. Treating pain in persistent PZN is extremely difficult and mostly results in a small diminution of the pain level. Persistence of viruses on the neuronal cell surface and resulting reduction of "luxury functions" of those cells may explain algogenesis by PZN and resistance to therapeutic efforts. We used VZI for the first time for therapy of PZN and observed a striking analgesic effect in all patients for the entire surveillance time.


2-(2'-phosphoryloxyphenyl)-4(3H)-quinazolinone derivatives as fluorogenic precipitating substrates of phosphatases. Characterization of 2-(2'-phosphoryloxyphenyl)-4(3H)-quinazolinone (PPQ) derivatives as fluorogenic precipitating substrates of phosphatases is reported in this work. Soluble and colorless PPQ derivatives can be specifically hydrolyzed by acid and alkaline phosphatases into insoluble products, 2-(2'-hydroxyphenyl)-4(3H)-quinazolinone (HPQ) derivatives which appear as fluorescent precipitates in water. The fluorescence and precipitation of HPQ depend on the concentration of its neutral phenolic form and therefore are related to the aqueous pH and PPQ concentration converted. Since HPQ formed from corresponding PPQ hydrolysis by phosphatases instantly precipitates and simultaneously fluoresces with a high photostability and large Stokes shift in water, PPQ can serve as a novel class of substrate dyes for detecting any immobilized phosphatase activities in situ, especially for applications of sensitive fluorescence histochemistry and cytochemistry. This is demonstrated by the alkaline phosphatase-aided visualization of static concanavalin A (Con A) receptors. By a linkage-amplification technique involving biotinylated Con A, streptavidin-alkaline phosphatase conjugate, and a PPQ substrate, the Con A receptors on the membrane of fixed NIH 3T3 cell were specifically viewed as dense, contrasting, durable, and cytologically resolved fluorescent stains under a conventional fluorescent microscope.


An easy cAMP extraction method facilitating adenylyl cyclase assays. We present a facile procedure for measuring adenylyl cyclase activity which circumvents the two-step chromatographic purification of 32P-labeled cAMP. cAMP produced by stimulated cell membrane preparations is easily purified by organic extraction and thus available for quantification using tritium-labeled tracer cAMP and commercially available cAMP-binding protein. The quantification of cAMP is unaffected by the extraction procedure and sample handling. Data obtained by this method were identical to those obtained by the chromatographic method. This procedure could be shown to be suitable for measuring receptor-mediated adenylyl cyclase modulation.


Correction for creatine interference with the direct indophenol measurement of NH3 in steady-state nitrogenase assays. Creatine was identified as a major source of interference with the direct phenol/hypochlorite colorimetric determination of ammonia in nitrogenase reaction mixtures. A method is described for removing other compounds which inhibit color development and for compensating for the interference produced by creatine. This method avoids time-consuming microdiffusion and also routinely makes available the efficiency of ATP hydrolysis coupled to substrate reduction (ATP/2e ratio) with N2 as a reducible substrate. Using this method we determined values for this ratio at 30 degrees C of 4.87 +/- 0.03 during the reduction of protons to H2 and 7.16 +/- 0.14 during the reduction of N2 by the vanadium-containing nitrogenase of Azotobacter chroococcum.


Spectrophotometric pyrophosphate assay of 2',5'-oligoadenylate synthetase. A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2',5'-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC1.1.1.49). The assay is at least as sensitive for measurements of 2',5'-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH.


Platelet-activating factor in bronchoalveolar lavage from patients with sarcoidosis. Platelet-activating factor (PAF), a lipid mediator of inflammation and anaphylaxis, may play a role in several physiopathologic alterations of the lung. A lipid compound with physicochemical and biologic characteristics similar to synthetic PAF was extracted and purified from bronchoalveolar lavage (BAL) fluid of 15 of 34 patients with sarcoidosis. PAF was quantitated by a bioassay on washed rabbit platelets. The specificity of platelet aggregation was assessed by using two different PAF receptor antagonists. The incidence of detectable amounts of PAF in BAL fluid of sarcoid patients was statistically significant (chi 2 = 4.064, p = 0.044) when compared with the 14 normal control subjects. The results demonstrated an increased production of PAF in the lower respiratory tract of patients with sarcoidosis. The presence of PAF in BAL fluid, however, did not correlate with radiologic stage, intensity of alveolitis, gallium scanning positivity, angiotensin-converting enzyme serum level, or lung function tests. Therefore, a direct relationship between presence of PAF in BAL fluid and activity of lung disease in patients with sarcoidosis was not directly established.


Radioimmunoassays of arginine vasopressin and atrial natriuretic peptide: application of a common protocol for plasma extraction using Sep-Pak C18 cartridges. A rapid vacuum-driven procedure, using pre-treated Sep-Pak C18 cartridges, has been developed for the simultaneous extraction of arginine vasopressin (AVP) and atrial natriuretic peptide (ANP) from plasma. Non-specific interference was removed by fractional elution with an aqueous methanol/trifluoroacetic acid (TFA) mixture. AVP and ANP were coeluted under positive pressure with a methanol/TFA mixture and the eluates air-dried before measurement using separate radioimmunoassays. Assay ranges for AVP and ANP were 0.12-29.5 pmol/L and 0.65-162 pmol/L, respectively, with mean recoveries (standard deviation in parentheses) for AVP of 96.4% (5.5%) at a level of 11.8 pmol/L and for ANP of 94.8% (5.9%) at a level of 32.4 pmol/L. The extraction and assay procedures were validated by observing the changes in plasma AVP and ANP concentrations in normal subjects at different stages of hydration and in elderly patients during treatment for congestive cardiac failure.


[Role of in situ alkalinization in the treatment of obstructive uric acid lithiasis]. The authors report a series of nine reno-ureteral obstructive uric acid calculi initially treated by percutaneous nephrostomy. After attempting stone dissolution by in situ alkalinization, they discuss the place of such a method among the various treatments for uric acid stones.


Fluoroquinolone supersusceptibility mediated by outer membrane protein OprH overexpression in Pseudomonas aeruginosa: evidence for involvement of a nonporin pathway. Overexpression of Pseudomonas aeruginosa outer membrane protein OprH led to an 8- to 32-fold increase in susceptibility to chloramphenicol and the quinolones nalidixic acid, norfloxacin, ciprofloxacin, and fleroxacin in comparison with the susceptibility of the wild-type strain H103 grown on Mg(2+)-sufficient medium. This was true regardless of whether OprH overexpression was induced by growth of strain H103 on Mg(2+)-deficient medium, the addition of 5 mM m-toluate to cells containing the cloned oprH gene behind the inducible tol promoter in plasmid pGB25, or mutation in the polymyxin-resistant derivative strain H181. In contrast, OprH overexpression failed to reverse the quinolone resistance phenotype of a nalB mutant. OprH was purified to homogeneity by selective detergent solubilization and fast protein liquid chromatography. The addition of OprH to the solution bathing a black lipid bilayer membrane failed to give rise to an increase in membrane conductance. This suggests that OprH is not a porin but, instead, may cause increased uptake of quinolones and chloramphenicol via a non-porin pathway.


Sparfloxacin, ethambutol, and cortisol receptor inhibitor RU-40 555 treatment for disseminated Mycobacterium avium complex infection of normal C57BL/6 mice. Sparfloxacin (50 mg/kg of body weight given subcutaneously each day), alone or in combination with ethambutol (50 mg/kg given subcutaneously each day), was examined for its therapeutic efficacy against experimental infection induced with the Mycobacterium avium complex in normal C57BL/6 mice. In addition, the potential anti-infective role of RU-40 555 (100 mg/kg given intraperitoneally each day), a drug that inhibits the cortisol receptors, was examined in the same model. Treatments were started 24 h after intravenous bacterial challenge and were continued for 21 days. Compared with controls, sparfloxacin or ethambutol decreased the CFU counts in spleens and lungs (P < 0.001). The sparfloxacin plus ethambutol combination was more effective than sparfloxacin alone in spleens (P < 0.001) but not in lungs. The sparfloxacin plus ethambutol plus RU-40 555 combination was more effective than the sparfloxacin plus ethambutol combination in spleens and lungs (P < 0.001). Thus, in this model, RU-40 555 enhanced the antibacterial activities of the antibiotics tested. Results of the study showed that normal C57BL/6 mice infected with the M. avium complex can be used for the evaluation of antimicrobial agents.


Activity of clarithromycin against Mycobacterium avium complex infection in beige mice. The activity of clarithromycin alone and in combination with other antimycobacterial agents was evaluated in the beige (C57BL/6J bgj/bgj) mouse model of disseminated Mycobacterium avium complex (MAC) infection. A dose-response experiment was performed with clarithromycin at 50, 100, 200, or 300 mg/kg of body weight administered daily by gavage to mice infected with approximately 10(7) viable MAC. A dose-related reduction in spleen and liver cell counts was noted with treatment at 50, 100, and 200 mg/kg. The difference in cell counts between treatment at 200 and 300 mg/kg was not significant. Clarithromycin at 200 mg/kg of body weight was found to have activity against three additional MAC isolates (MICs for the isolates ranged from 1 to 4 micrograms/ml by broth dilution). Clarithromycin at 200 mg/kg in combination with amikacin, ethambutol, temafloxacin, or rifampin did not result in increased activity beyond that seen with clarithromycin alone. Clarithromycin in combination with clofazimine or rifabutin resulted in an increase in activity beyond that seen with clarithromycin alone. The combination of clarithromycin with clofazimine or rifabutin should be considered for evaluation in the treatment of human MAC infections.


Pharmacokinetics and metabolism of racemic 2',3'-dideoxy-5-fluoro-3'-thiacytidine in rhesus monkeys. 2',3'-Dideoxy-5-fluoro-3'-thiacytidine (FTC) is a nucleoside analog that selectively inhibits human immunodeficiency and hepatitis B viruses in vitro. In this study, the preclinical pharmacokinetics of racemic FTC in rhesus monkeys following intravenous and oral administration were characterized. The terminal half-life of FTC was independent of the route of administration and averaged 1.34 +/- 0.18 h (mean +/- standard deviation). Total clearance of FTC was moderate to high, averaging 1.49 +/- 0.24 liters/h/kg. Qualitative assessment of urine samples suggests that renal excretion of unchanged FTC was the major route of elimination of the nucleoside. The compound was also eliminated by metabolism and the deaminated biotransformation product 2,3'-dideoxy-5-fluoro-3'-thiauridine (FTU) was detected in serum and urine. This metabolite has no antiviral activity in human lymphocytes and liver cells. FTC and the metabolite FTU were conjugated, to a minor extent yielding the corresponding glucuronides. No 5-fluorouracil was detected in serum or urine. This is consistent with chromatographic studies using a chiral column that indicated that when racemic FTC is treated with cellular cytidine-deoxycytidine deaminase, the D-(+)-enantiomer of FTC is slowly deaminated to D-(+)-FTU, whereas the L-(-)-enantiomer is essentially resistant to this enzyme. The steady-state volume of distribution of FTC in serum averaged 2.23 +/- 0.42 liters/kg, and the nucleoside analog was distributed into the cerebrospinal fluid, which suggests that this drug penetrated the blood-brain barrier. Absorption of FTC after oral administration was rapid, with bioavailability averaging 73 +/- 6%. Taken together, the results indicate that the unusual L-(-)-enantiomer of FTC should be evaluated further in rhesus monkeys prior to determination of whether this compound is useful for treatment of human immunodeficiency and hepatitis B virus infections.


Pharmacokinetics and inflammatory fluid penetration of sparfloxacin. A single 400-mg oral dose of sparfloxacin was given to each of six healthy male volunteers, and the concentrations of the drug were measured in plasma, cantharides-induced inflammatory fluid, and urine over the subsequent 52 h. The mean peak concentration in plasma of 1.6 micrograms/ml was attained at a mean time of 2.7 h postdose. The mean peak concentration in inflammatory fluid of 1.3 micrograms/ml was attained at a mean time of 5 h postdose. The mean elimination half-life in plasma was 17.6 h, and that in inflammatory fluid was 19.7 h. The overall penetration into inflammatory fluid was 117%. Urinary recovery within the first 52 h postdose was 8.8% of the administered dose. Our results indicate that a once-daily dosage of sparfloxacin should be adequate to treat systemic infections caused by most common bacterial pathogens.


Steady-state pharmacokinetics and sputum penetration of lomefloxacin in patients with chronic obstructive pulmonary disease and acute respiratory tract infections. Oral doses of 400 mg of lomefloxacin were administered once daily prior to breakfast to 10 middle-aged to elderly hospitalized patients with chronic obstructive pulmonary disease during treatment for bronchopulmonary infections. Serial plasma and sputum samples and fractional urine samples were obtained over a steady-state dosing interval. Lomefloxacin concentrations were determined in duplicate by a validated agar well diffusion microbiological assay. The maximum plasma lomefloxacin concentration (4.5 +/- 1.8 mg/liter), the time of occurrence of the maximum concentration (1.7 +/- 1.6 h), and the apparent volume of distribution associated with the terminal phase (2.19 +/- 1.05 liter/kg) were comparable to the values reported for healthy, young volunteers. Compared with the data reported for young adults, the elimination half-life (12.7 +/- 4.67 h) was longer and the apparent total body clearance (132 +/- 36.6 ml/min/1.73 m2) was lower in middle-aged to elderly patients. These differences were most likely attributable to age-related decreases in renal function, as evidenced by the lower lomefloxacin renal clearance (70.3 +/- 33.5 ml/min) in patients. The presence of acute respiratory infection per se did not appear to alter lomefloxacin pharmacokinetics. The peak lomefloxacin concentration in purulent, expectorated sputum samples of 4.3 +/- 1.2 mg/liter occurred 3.1 +/- 1.7 h after dose administration and subsequently declined to 1.7 +/- 0.5 mg/liter at the end of the 24-h dosing interval. The percent penetration into sputum, as assessed by comparing the area under the curve for sputum and plasma samples, was 120 +/- 39.8 (range, 70 to 185). The steady-state lomefloxacin concentrations in plasma and sputum samples from ill, older patients were in excess of the MICs for 90% of the strains tested of common, susceptible respiratory pathogens over most of the dosing interval.


Synergy of itraconazole with macrophages in killing Blastomyces dermatitidis. We examined in vitro interaction between the azole antifungal agents itraconazole and ketoconazole and macrophages and their activities against Blastomyces dermatitidis. Fungistatic and fungicidal concentrations for B. dermatitidis in vitro were assessed in a microculture system in which fungistasis was measured as inhibition of multiplication and fungicidal activity was measured as reduction of inoculum CFU. Resident peritoneal murine macrophages, which surround but do not phagocytize the fungus, were not fungicidal for B. dermatitidis isolates but were fungistatic for some isolates studied. Synergy was demonstrated when fungistatic concentrations (e.g., 0.01 micrograms/ml) of itraconazole, which limited growth 55% compared with that of controls, were cocultured with macrophages; this resulted in fungicidal activity (85% killing) against B. dermatitidis (ATCC 26199) in 72-h assays. This synergy could occur even if itraconazole was added after the macrophages had surrounded the fungus. Ketoconazole at fungistatic concentrations did not act synergistically with macrophages to kill B. dermatitidis. Lymph node lymphocytes could not substitute for macrophages in synergy with itraconazole to kill B. dermatitidis. When B. dermatitidis was separated by a filter from macrophages in Transwell cultures, fungicidal synergy with itraconazole was less efficient. Pretreatment of B. dermatitidis with itraconazole for 24 h did not render the fungus susceptible to killing by macrophages in the absence of itraconazole, whereas pretreatment of nonfungistatic macrophages with itraconazole rendered them fungistatic in a dose-dependent manner. Three other isolates were killed by otherwise fungistatic concentrations of itraconazole when the isolates were cocultured with macrophages. These findings indicate that one basis for the efficacy of itraconazole versus ketoconazole in treating blastomycosis could be synergy of a fungistatic concentration of itraconazole with macrophages in killing of B. dermatitidis.


Susceptibility of Rickettsia conorii, R. rickettsii, and Coxiella burnetii to PD 127,391, PD 131,628, pefloxacin, ofloxacin, and ciprofloxacin. Plaque formation and dye uptake assays were used to measure the MICs of PD 127,391 and PD 131,628 against Rickettsia species. The MICs of PD 127,391 were 0.25 microgram/ml for Rickettsia rickettsii and 0.125 to 0.25 microgram/ml for Rickettsia conorii. The MICs of PD 131,628 were 0.25 to 0.5 microgram/ml for R. rickettsii and 0.5 microgram/ml for R. conorii. As determined by the shell vial technique, 15 strains of Coxiella burnetii were susceptible to PD 127,391 and PD 131,628 (MIC, < or = 1 microgram/ml), while one strain of C. burnetii (MP10) was of intermediate susceptibility.


Activities of various quinolone antibiotics against Mycobacterium leprae in infected mice. Previously, pefloxacin and ofloxacin were found to be active against Mycobacterium leprae in vitro, in experimental animals, and in clinical trials of lepromatous leprosy patients. In this study, we compared certain more recently developed fluoroquinolones (lomefloxacin, PD 124816, WIN 57273, temafloxacin, and sparfloxacin) with pefloxacin and ofloxacin in M. leprae-infected mice at doses of 50, 150, and 300 mg/kg given five times weekly. All seven of the fluoroquinolones studies were active against M. leprae; temafloxacin and sparfloxacin were the most active, being fully bactericidal at all three dosage schedules. Additionally, sparfloxacin was found to be fully bactericidal at 15 and 30 mg/kg given five times weekly.


Role of Pseudomonas aeruginosa outer membrane protein OprH in polymyxin and gentamicin resistance: isolation of an OprH-deficient mutant by gene replacement techniques. The cloned oprH gene of Pseudomonas aeruginosa was mutated by inserting a 1.4-kbp fragment that encodes tetracycline resistance. This mutated gene was then incorporated into the P. aeruginosa chromosome by homologous recombination. Growth of the resultant oprH::tet mutant in Mg(2+)-deficient medium had little effect on susceptibility to polymyxin B, gentamicin, or EDTA unless the mutant was complemented by the cloned oprH gene in plasmid pGB25. In contrast, growth of the parent strain on Mg(2+)-deficient medium resulted in resistance to all three agents. These data support the hypothesis that overexpression of OprH under Mg(2+)-deficient growth conditions is conditionally required for resistance to these three agents.


Urinary protein/creatinine index in follow up of patients with Wilms' tumour after nephrectomy. The protein/creatinine index (p/c) was determined in early morning urine (EMU) samples from available patients with Wilms' tumour who had had a nephrectomy and whose diagnosis had been made between January 1970 and December 1989. Clinical details were obtained by case note review. Results were obtained from 36 boys and 40 girls. The mean interval between nephrectomy and measurement of the EMUp/c was 9.0 years (2-23). Eleven patients had a EMUp/c greater than 20 mg/mmol (normal range less than 20). Of the 11 patients with proteinuria, there were in addition to nephrectomy other adverse features including bilateral tumours, treatment with nephrotoxic drugs, and dysplastic kidneys. Renal dysfunction seems most likely to occur where there are adverse factors in addition to unilateral nephrectomy. There was a significant correlation between the glomerular filtration rate and the EMUp/c, and it is thought that this is a simple tool which can be used for the regular monitoring of renal function in these patients.


Fatal encephalitis/encephalopathy in primary human herpesvirus-6 infection. An encephalitic illness with a fatal outcome occurred in a 9 month old girl with virologically confirmed exanthem subitum. Human herpes-virus-6 (HHV-6) DNA was found in the cerebrospinal fluid at the acute stage of the disease by the polymerase chain reaction, but the virus antigen was not detected in her brain tissue. This suggests that HHV-6-induced encephalitis/encephalopathy may be due to a non-infectious process.


[Aging processes in the brain].

[Aging processes in the brain]. In 1991 the brains of 15 persons with an age of at least 60 years and without neurologic or psychiatric disorders were investigated prospectively. Different brain-areas (topographically exactly defined) were investigated for so-called brain-aging. Only the frequency of corpora amylacea and primitive senile plaques showed a tendency to increase in the elderly. A defined correlation to the calendaric age could not be stated.


Physiological and clinical aspects of vitamin A and its metabolites. Retinoids, including retinol and retinoic acid (RA), are a group of naturally occurring and synthetic compounds that exhibit vitamin A-like biological activity. They achieve their effects by binding to intracellular proteins. Important sites of action are the nuclear retinoic acid receptors (RAR). These receptors, namely, RAR alpha, RAR beta, and RAR gamma, function as transcription factors by binding to RA-responsive elements (RARE) of multiple genes. Retinoids play a role in vision, embryogenesis, immune modulation, growth and differentiation of normal, premalignant and malignant tissues, the suppression of carcinogenesis, and the inhibition of tumor growth in experimental systems and humans. Reports of the significant antitumor effect of all-trans-RA in acute promyelocytic leukemia and the synthesis of new, less toxic, and more potent retinoids has generated renewed interest in these compounds. Retinoids may have an important role to play in the chemoprevention and therapy of cancer.


Repetitive transients in intracellular Ca2+ in cultured human vascular smooth muscle cells. Human uterine vascular smooth muscle cells have been isolated and maintained in culture. When these cells are exposed to bathing solutions with nominally zero sodium, using potassium, N-methyl-D-glucamine or Tris as substitutes, repetitive transient increases in intracellular calcium are observed. These transients are abolished when the calcium concentration of the bathing solution is reduced to nominally zero suggesting a role for extracellular calcium in the activation or maintenance of the transients. The hypothesis is proposed that the underlying mechanism involves a calcium influx through the reversed operation of a sodium-calcium exchange mechanism and the cyclical activation of calcium-induced calcium release from the sarcoplasmic reticulum. Noradrenaline (10(-6) M) and caffeine (20-30 mM) reversibly inhibited the transients. The inhibitory action of these agents could not be mimicked by dibutyryl cAMP suggesting that cAMP does not mediate the inhibition. Caffeine alone had no effect on resting calcium. Thimerosal (1-100 microM), an agent thought to activate a second type of calcium-induced calcium release mechanism activated repetitive transient increases in intracellular calcium which behave in a similar manner to those activated by sodium removal. These data are consistent with the presence of a thimerosal-activated calcium-induced calcium release mechanism in these cultured human cells. It is proposed that this mechanism is different from the calcium-induced calcium release mechanism, described in other cell types, which is activated by caffeine.


Modulation of voltage-dependent calcium current in Helix aspersa buccal neurones by serotonin and protein kinase C activators. In Helix aspersa, activation of the cerebral giant serotonin neurones (GSNs) evokes a biphasic, excitatory synaptic response in the M neurones of the buccal ganglia. Local application of serotonin to the current-clamped M neurones also evokes fast and slow depolarizing responses. The slow response is thought to be dependent on calcium ions, whereas sodium ions have been implicated in the fast response. Here we provide further evidence that the slow response results from an increase in conductance to calcium ions, and show that okadaic acid, an antagonist of protein phosphatases 1 and 2A, potentiates the effect of serotonin, suggesting that the response is phosphorylation dependent. Further, agents known to activate protein kinase C, such as 1-oleoyl-2-acetyl-rac-glycerol and active phorbol esters (but not an inactive one) were found to increase the calcium current (actually carried by barium ions) of the M neurones. Such data suggest that the slow synaptic response mediated by serotonin can occur by activation of protein kinase C and phosphorylation of the affected voltage-sensitive calcium channels, or some closely associated protein(s).


Exercise-induced cholesterol depletion and Na+,K(+)-ATPase activities in human red cell membrane. Red cell membranes were isolated from blood samples obtained from athletes during exhaustive exercise on a bicycle ergometer and during the subsequent recovery period of 60 min. Plasma lactate levels were also determined. During exercise, cell membranes were progressively depleted of cholesterol and, at exhaustion, membrane cholesterol was less than 80% of the initial level. A parallel decline in Na+,K(+)-ATPase was also noted, while phospholipid reduction was around 5%. During recovery, the erythrocyte membrane cholesterol and Na+,K(+)-ATPase increased, but at a slow rate and were inversely proportional to plasma lactate content.


Expression of B-type Epstein-Barr virus in HIV-infected patients and cardiac transplant recipients. In the present study, peripheral blood mononuclear cells (PBMCs) were obtained from HIV+ subjects as well as cardiac transplant recipients, and the presence of A- and B-type Epstein-Barr virus (EBV) was determined using the polymerase chain reaction (PCR). Of the HIV+ patients studied, 24% were found to be infected with A-type EBV, 27% with B-type EBV, and 39% with both A and B virus types. Analysis of PBMCs from cardiac transplant recipients revealed that 39% were infected with A-type EBV, 33% with B-type EBV, and 28% with both EBV types. These results demonstrate a higher prevalence of infection with B-type EBV in HIV+ patients, than had been found previously by an analysis of spontaneously generated lymphoblastoid cell lines. The data indicated that it is not HIV per se which is responsible for the high incidence of B-type EBV in HIV+ individuals.


Accumulation of 99mTc-HM-PAO in photon deficient areas in bone scan of bone metastasis from hepatocellular carcinoma. To evaluate bone metastasis from hepatocellular carcinoma (HCC), both bone and 99mTc-HM-PAO scintigraphies were performed in six patients with clinically and pathologically confirmed HCC. Two patients had a bone scintigram which revealed abnormal accumulation in the skull base, pelvic bone and thoracic spine. The 99mTc-HM-PAO scans of both these patients also showed abnormal accumulation in the same sites. The bone scintigrams in one patient revealed not only abnormal accumulation in the ribs but also photon deficient areas in the sternum, thoracic spine and femur, while 99mTc-HM-PAO scans showed abnormal accumulation in all these sites. In three patients, bone scintigraphy revealed photon deficient areas in the ribs, pelvic bone and femur, and their 99mTc-HM-PAO scintigrams showed abnormal accumulation in the same sites. Thus, it was shown that, in the detection of bone metastasis from HCC by means of bone scintigraphy, it was necessary to pay attention to hot and cold lesions, and that a combination study with 99mTc-phosphorous compounds and 99mTc-HM-PAO was useful in evaluating these lesions.


Comparison of Indium-111-labeled leukocyte scintigraphy and Technetium-99m joint scintigraphy in rheumatoid arthritis and osteoarthritis. This study was undertaken to evaluate the use of Indium-111-labeled leukocyte (111In-WBC) imaging compared with Technetium-99m pertechnetate (99mTcO4-) imaging in 19 patients with rheumatoid arthritis (RA) and 8 with osteoarthritis. Knee and wrist joints were evaluated for both radionuclides. The results indicated a good correlation of the clinical assessment of pain and swelling with joint uptake ratio (JUR) between 111In-WBC and 99mTcO4- in RA and osteoarthritis patients. We observed a discrepancy in both imagings in "burned out" cases. It was concluded that a JUR of 111In-WBC could distinguish active RA from inactive RA or osteoarthritis at a value of 1.15 and that the use of 111In-WBC was a more reliable procedure than 99mTcO4-.


Positive imaging of cardiomyopathy with 99mTc(V)-DMSA. A case of histologically proven dilated cardiomyopathy and a case of clinically diagnosed cardiomyopathy (cardiac amyloidosis was strongly suspected but was not confirmed) were examined with 99mTc(V)-dimercaptosuccinic acid (DMSA). 99mTc(V)-DMSA accumulation in the damaged myocardium was clearly demonstrated. These results suggested the possibility that 99mTc(V)-DMSA could be used as a positive agent for cardiomyopathy.


1H-NMR imaging of fimbria fornix lesions in the rat brain. Mechanical lesions of the fimbria fornix (FF) have been widely used as a model to investigate the recovery of damaged brain tissue. 1H-NMR imaging was employed to non-invasively measure changes in the brain after unilateral FF transection. Rats were subjected to NMR imaging at various times after the lesions were made. The experimental protocol included (multislice) T2-weighted and diffusion-weighted imaging thereby allowing the construction of two-dimensional maps of the relaxation time T2 (transverse or spine-spin relaxation time) and the apparent diffusion coefficient (ADC) of water. FF transection induced considerable changes in the status of the brain tissue at a number of different locations which were exclusively present in the affected hemisphere. At 1 day post-lesion the region of the lateral ventricle and hippocampus started to display pronounced changes in that T2- and diffusion-weighted images showed a hyperintensity and a hypointensity, respectively. These effects were maximal around day 2 to 4 whereafter a slow recovery towards the control situation was observed. Immediately after transection the FF lesion itself could be visualized. These early images pointed to an aspecific disruption of the tissue due to the mechanical intervention. Interestingly, however, from day 2 post-lesion a number of changes became evident in this region which seemed to be localized to specific structures, including the ventricle and hippocampus. After one month the presumably ventricle effect dominated and was predominantly localized to the anterior side of the FF lesion.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of oxytocin-stimulated phosphoinositide turnover in rat myometrium by pertussis and cholera toxins may involve protein kinase A activation. Both pertussis and cholera toxins inhibit oxytocin-stimulated phosphoinositide turnover in rat myometrium. The actions of pertussis and cholera toxins as well as those of CPTcAMP are reversed by H-8, an inhibitor of protein kinase A. H-8 does not have a major effect on cAMP elevation by the toxins in the presence of oxytocin. The results suggest that the stimulation by oxytocin of phosphoinositide turnover does not involve direct obligatory coupling to a pertussis toxin-sensitive GTP-binding protein. Rather, indirect effects on protein kinase A activation may contribute to the inhibitory effects of both cholera and pertussis toxins. This study suggests that caution must be exercised in interpreting inhibition of phosphoinositide turnover by pertussis toxin in whole cell experiments as indicative of direct involvement of a toxin-sensitive GTP-binding protein.


Mechanism of inhibition by cyclic AMP of protein kinase C-triggered respiratory burst in Ehrlich ascites tumour cells. The superoxide anion generation in Ehrlich ascites tumour (EAT) cells increased more than two-fold in the presence of the tumour promoter, tetradecanoyl phorbol myristate acetate (TPA). Epinephrine and dibutryl cAMP (Bt2 cAMP) inhibited in a dose-dependent manner, both basal and TPA-triggered superoxide generation in EAT cells. The kinetics of inhibition of superoxide generation showed a maximum inhibition between 30 and 40 min of preincubation with epinephrine or Bt2 cAMP of EAT cells and coincided with an increase in activity of a phosphoprotein phosphatase. In TPA-treated EAT cells, epinephrine or Bt2 cAMP increased the phosphatase activity in a dose-dependent manner. In vitro EGTA, EDTA and sodium fluoride inhibited phosphatase activity. Superoxide generation in response to TPA in Triton-permeabilized EAT cells was inhibited by inclusion of the phosphatase in the assay. Taken together, these results clearly suggest that the phosphatase activity in EAT cells develops as a result of protein kinase A (PKA) and protein kinase C (PKC)-mediated phosphorylation of the phosphatase which then mediates dephosphorylation of the PKC-triggered phosphorylation of proteins to inhibit respiratory burst. A cross-talk between PKA and PKC pathways negatively modulates superoxide generation in EAT cells.


Phorbol ester effects on hormonal responses in freshly isolated short-term incubated and cultured hepatocytes. Beta-adrenergic, alpha-1-adrenergic and glucagon stimulation of glucose release were compared between hepatocytes which were freshly isolated, incubated for 3 h in suspension or cultivated for 4 or 24 h in plastic culture flasks in the presence and absence of the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast to the absence of an isoproterenol effect in freshly isolated hepatocytes, an increased sensitivity of glucose liberation towards isoproterenol could be observed 4 h after the start of culture, whereas the beta-receptor number was not found to be increased before 24 h. TPA has no effect on isoproterenol-stimulated glucose release at all investigated conditions. The alpha-1-adrenergic responses tested by using the alpha-1-adrenergic agonist phenylephrine is blocked completely in freshly isolated hepatocytes preincubated with 10(-6) M TPA. However, after 3 h incubation of hepatocytes in suspension or in primary culture, TPA had no effect on phenylephrine-stimulated glucose release. The effect of 10(-9) M glucagon on glucose release from freshly isolated hepatocytes was not influenced by TPA, whereas after 90 and 180 min incubation a significant decrease could be observed. On the other hand, TPA inhibited stimulation of adenylate cyclase activity by glucagon concentrations of 10(-5) M in freshly isolated hepatocytes, but no effect was found in hepatocytes incubated for 3 h in suspension or maintained for 24 h in primary culture. The different TPA effects may be an expression of changes of the accessibility of protein kinase C to TPA caused by translocation and/or intracellular activation of this enzyme at the tested experimental conditions.


Time-dependent inhibition of inositol-1,4,5-trisphosphate-5-phosphatase by calmidazolium chloride in rat GH3 cells. The calmodulin inhibitor calmidazolium chloride inhibited the activity of soluble and particulate Ins(1,4,5)P3-5-phosphatase from GH3 cells, with an IC50 value of approximately 100 microM following a 10-min preincubation with the enzyme. The inhibition was time-dependent and could not be reversed by washing of the particulate fraction. It is concluded that although the inhibitory effect of calmidazolium chloride cannot be related per se to inhibition of calmodulin function, effects of this compound unrelated to actions upon calmodulin function may be found when concentrations that are only moderately supramaximal are used.


The alpha 1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca(2+)-ATPase. Membrane Ca(2+)-ATPase activity was stimulated in vitro separately by T4 (10(-10) M) and by epinephrine (10(-6) M). In the presence of a fixed concentration of T4, additions of 10(-8) and 10(-6) M epinephrine reduced the T4 effect on the enzyme. beta-Adrenergic blockade with propranolol (10(-6) M) prevented stimulation by epinephrine of Ca(2+)-ATPase activity, but did not prevent the suppressive action of epinephrine on T4-stimulable Ca(2+)-ATPase. In contrast, alpha 1-adrenergic blockade with unlabelled prazosin restored the effect of T4 on Ca(2+)-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulation by T4 of red cell Ca(2+)-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (Kd, 1.2 x 10(-8) M; Bmax, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains alpha 1-adrenergic receptor sites that are capable of regulating Ca(2+)-ATPase activity.


The Drosophila segmentation gene runt has an extended cis-regulatory region that is required for vital expression at other stages of development. The Drosophila runt gene functions in several developmental pathways during embryogenesis. This gene was initially characterized due to the pivotal role that it plays in the genetic regulatory network that establishes the segmented body pattern. Recently it was found that this X-chromosome-linked gene is one of several dosage-sensitive, X-linked components that is involved in activating the Sex-lethal gene in blastoderm stage female embryos. Finally, this gene is also extensively re-expressed in later stages of embryogenesis in the developing nervous system where it plays an important role in the development of specific neural lineages. We have initiated an analysis of the runt cis-regulatory region in order to investigate runt's roles in these (and other) developmental pathways. Analysis of both the function and the expression patterns of runt genes with truncated cis-regulatory regions indicates that there are multiple elements that make quantitative contributions to runt regulation during segmentation. We find that sequences that are more than 8.5 kb upstream of the runt promoter are necessary for normal expression during the post-blastoderm stages of embryogenesis. Genetic experiments indicate that the post-blastoderm expression of runt is vital to the organism.


Two isoforms of retinoic acid receptor alpha expressed during Xenopus development respond to retinoic acid. Retinoic acid regulates gene expression by binding to receptors that are ligand-dependent transcription factors. The different types of RARs may mediate the diverse effects of RA on different tissues at different stages of development. This implies RAR expression will be spatially and temporally regulated during normal development. To examine this point, cDNA clones encoding RARs have been isolated from Xenopus embryos. Two different classes of RAR alpha have been characterised, they both show most similarity to the RAR alpha 2 isoform. In response to exogenous RA, XRAR alpha 2 transcripts accumulate whilst the level of XRAR gamma decreases. In addition, transcripts from the two XRAR alpha 2 genes are differentially distributed in the neurectoderm of RA treated embryos. Modulation of RAR transcript levels by RA may provide one level of control of RAR gene expression during development.


High efficiency of endogenous antigen presentation by MHC class II molecules. MHC class II molecules are involved in the presentation of both exogenous and endogenous antigens to CD4 T cells. Using the trans-membrane hemagglutinin (HA) from measles virus and the secreted hen egg lysozyme (HEL) as antigen models, we have compared the efficiency of MHC class II presentation by naive antigen presenting cells (APCs) pulsed with exogenous antigen with that of their transfected counterparts synthesizing endogenous antigen. B cells expressing even a very low amount of trans-membrane HA were found to present endogenous HA to I-Ed restricted T cell hybridomas with a high efficiency whereas their naive counterparts required to be pulsed with a comparatively high amount of exogenous HA. Similarly, MHC class II presentation of endogenous secreted HEL was found to be much more efficient when compared with that of exogenous HEL. Biochemical studies did not reveal any enhanced intracellular degradation of endogenous HEL. As expected, HEL was released in the surrounding medium within < 1 h. MHC class II presentation of endogenous HEL could not be explained by re-uptake by bystander APCs of HEL secreted in the surrounding medium. No sensitization of naive APCs could be observed either when co-cultured with HEL secreting cells or when cultured for 10 days with a sub-threshold amount of exogenous HEL. At the cell surface, I-Ed molecules immunoprecipitated from HEL secreting cells were found to be slightly enriched in SDS-resistant forms. These data raised the question of how peptides derived from endogenous transmembrane and secreted antigens can so efficiently reach an MHC class II loading compartment.


[Annual cost of microbial immunological tests for HIV-positive individuals in a hospital center]. The goal of this study is to increase the knowledge of the AIDS cost in a hospital. We have studied the HIV patients diagnosed during a year in our hospital and the charges of the serological tests performed to them. 20.8% of the tests were to study antibodies against HIV (only 20% were positive). After patient distribution between extra or intrahospital origin, we highlight that 61% of the charges are attributed to patients who do not belong to the hospital but to its catchment area. We emphasize the problem that AIDS patients are creating to hospital, being necessary that personal select only useful tests honestly.


Localized proton NMR spectroscopy of experimental gliomas in rat brain in vivo. Experimental brain tumors produced in rats (n = 10) by stereotactic implantation of cells from the F98 anaplastic glioma clone into the right caudate nucleus were studied in vivo using localized proton NMR and in vitro using high-resolution proton NMR, bioluminescent imaging of lactate, ATP and glucose distributions, and fluorescent imaging of regional pH. In vivo spectra from normal brain contralateral to the tumor regions showed resonances assignable to N-acetyl aspartate (NAA), creatines, choline-containing compounds, myo-inositol, glutamate and glucose in a pattern similar to those obtained from normal anaesthetized rats. In vivo tumor spectra were characterized by the almost complete absence of NAA, a substantial reduction of total creatine and glucose, and an increase of cholines. Based on the in vitro spectra the increase of the myo-inositol signal observed in vivo was mainly attributed to glycine. Histological examination as well as bioluminescent and fluorescent imaging indicated two stages of tumor development, i.e., solid vital tumors and tumors with necrosis. However, there was no consistent relationship between proton NMR observations and tumor development.


[Gastroenteritis caused by adenoviruses 40/41: epidemiological and clinical aspects]. The 40/41 adenovirus plays an important role as the productive agent of gastroenteritis at a pediatric age according to recent studies. With the aim of evaluating the clinical-epidemiological importance of this agent in our medium a prospective study of the 40/41 adenovirus was carried out in 1,697 stools of children of under 14 years of age over a period of 2 years. An enzyme immunoassay technique with specific monoclonal antibodies was used for detection. The 40/41 adenovirus was the third cause of gastroenteritis following Salmonella sp. and rotavirus with a percentage of infection of 2.1%. In 87.1% of the patients in which it was detected the patients had an age of less than 24 months. A clear peak of infection was found in the months of September of the two years studied. The infection was of intrahospital origin in 46.2% of the patients. Most of the children presented a self-limited diarrheic picture. The evolution of the patients was good with adequate rehydration measures. The study of the 40/41 adenovirus is of indubitable interest with the aim of obtaining greater knowledge of its importance in the epidemiology and clinical manifestations of gastroenteritis.


[Receptor binding oscillations].

[Receptor binding oscillations]. A kinetic analysis of the delta-opiate receptor agonist, [3H] DADLE, binding to cell (NG108-15) suspensions has led to the discovery of a new periodic biological phenomenon, namely, receptor activity oscillations. The absence of oscillations for the antagonist binding to the receptors suggests that oscillations are generated only as a result of receptor signal transformation. The correlation between the quantitative characteristics of the kinetic curves and the experimental conditions points to the fact that receptor binding oscillations may be due either to the receptor binding to the G-protein or the interaction of the receptor (or G-protein) with microtubules.


[Interaction of (3H)naloxone with immunocompetent murine cells: the effect of mitogenic and antigenic activation]. The binding of the opioid antagonist [3H]-naloxone to immunocompetent cells of the mouse, F1(CBA x C57B1/6), in medium 199 has been studied. The binding was reversible and reached a maximum during 15-20 min at 37 degrees C. The stereospecificity profile was proven to correspond to mu-type receptors. The binding curve was characterized by high positive cooperativity (nH = 2.3, IC50 = 5 nM). Mitogenic stimulation by Con A, SEA, and ML caused an increase in the number of receptors. Besides, stimulation by an antigen (ovalbumin) changed the binding parameters. The distribution of binding sites for naloxone on various immunocompetent cells was investigated. The maximal number of sites was found on lymphocytes of lymph nodes and bone marrow cells. A conclusion is drawn that both T- and non-T-cells play a role in naloxone binding.


An attempt to standardize APTT reagents used to monitor heparin therapy. Citrated samples from 100 patients on i.v. heparin and 20 normal patients were tested with three batches each of three activated partial thromboplastin time (APTT) reagents: Thrombosil I (Ortho); Automated APTT (Organon Teknika) and Actin FSL (Baxter). The ratio of APTT over the geometric mean normal APTT for each heparinized sample was calculated. One batch of reagent arbitrarily chosen as a reference gave the ratios APTRREF (y). The remaining reagents to be standardized against the reference system gave the ratios APTRTEST (x). The best correlation between systems was given by log vs log x. Standard curves were prepared from the APTT ratios of the 20 normal patients and 65 of the heparinized samples. On plotting log APTRTEST vs log APTRREF the y intercept was close to zero so x was expressed in terms of y using; log x = HSI. log y, where HSI (Heparin Sensitivity Index) = slope. The APTRTEST results of the remaining 35 heparinized samples were transformed using; APTRTRANS = (APTRTEST)HSI.APTRTRANS was then compared to APTRREF to determine whether the transformation brought the results closer to the reference. We conclude that although some improvement was found by using the transform, it was not possible to mathematically relate APTT results due to a high degree of variation between results using different reagents. A standard APTT reagent for the monitoring of heparin therapy is recommended. A separate APTT reagent may be required for the screening of factor deficiencies and lupus anticoagulants.


Inhibition of PAF-induced activation of human platelets by verapamil. Verapamil, an inhibitor of calcium channels, was shown to inhibit PAF-induced platelet activation. In the presence of 50 microM Verapamil both thromboxane (Tx) formation and release of ATP from dense granules induced by 100 nM PAF was completely inhibited. This concentration of Verapamil only produced partial inhibition of PAF-induced aggregation. It also reduced the size of the PAF-induced calcium (Ca2+) transient demonstrated in Fura-2 loaded platelets. In the absence of extracellular Ca2+, following chelation by EGTA, PAF was still able to induce a Ca2+ transient confirming the requirement of both intra and extracellular Ca2+ for PAF-induced platelet activation. 100 microM Verapamil was able to completely abolish the calcium transient induced by low doses of PAF. These results further suggest that Verapamil is able to inhibit PAF-induced platelet activation by mechanisms apart from blocking Ca2+ channels.


Comparison of prothrombin fragment 1 + 2 with thrombin-antithrombin III complex in plasma of patients with disseminated intravascular coagulation. Plasma levels of prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin III complex (TAT) are thought to be specific indicators of thrombin generation. To assess whether these two parameters behave similarly in vivo, we compared the plasma levels of F1 + 2 with TAT in 41 patients with disseminated intravascular coagulation (DIC). Both F1 + 2 and TAT were markedly elevated in patients with DIC compared to healthy subjects. Although a positive correlation was found between F1 + 2 and TAT (r = 0.585, P < 0.001) there was a large scatter among individuals. In addition, plasma concentrations of TAT were much lower than F1 + 2. The correlation between the TAT/F1 + 2 ratio and antithrombin III was weak (r = -0.268, P = 0.09). The TAT/F1 + 2 ratio was positively correlated with TAT (r = 0.481, P = 0.002), indicating that the difference in molar concentrations between F1 + 2 and TAT decreased as the TAT value increased. Serial determinations of these parameters showed that plasma TAT values changed roughly in parallel with F1 + 2 in the majority of patients. Although further studies are required to further clarify the observed differences between F1 + 2 and TAT, both parameters would be equally useful for the precise detection of haemostatic activation in patients with DIC.


Effects of melittin on lipid-protein interactions in sarcoplasmic reticulum membranes. To investigate the physical mechanism by which melittin inhibits Ca-adenosine triphosphatase (ATPase) activity in sarcoplasmic reticulum (SR) membranes, we have used electron paramagnetic resonance spectroscopy to probe the effect of melittin on lipid-protein interactions in SR. Previous studies have shown that melittin substantially restricts the rotational mobility of the Ca-ATPase but only slightly decreases the average lipid hydrocarbon chain fluidity in SR. Therefore, in the present study, we ask whether melittin has a preferential effect on Ca-ATPase boundary lipids, i.e., the annular shell of motionally restricted lipid that surrounds the protein. Paramagnetic derivatives of stearic acid and phosphatidylcholine, spin-labeled at C-14, were incorporated into SR membranes. The electronic paramagnetic resonance spectra of these probes contained two components, corresponding to motionally restricted and motionally fluid lipids, that were analyzed by spectral subtraction. The addition of increasing amounts of melittin, to the level of 10 mol melittin/mol Ca-ATPase, progressively increased the fraction of restricted lipids and increased the hyperfine splitting of both components in the composite spectra, indicating that melittin decreases the hydrocarbon chain rotational mobility for both the fluid and restricted populations of lipids. No further effects were observed above a level of 10 mol melittin/mol Ca-ATPase. In the spectra from control and melittin-containing samples, the fraction of restricted lipids decreased significantly with increasing temperature. The effect of melittin was similar to that of decreased temperature, i.e., each spectrum obtained in the presence of melittin (10:1) was nearly identical to the spectrum obtained without melittin at a temperature approximately 5 degrees C lower. The results suggest that the principal effect of melittin on SR membranes is to induce protein aggregation and this in turn, augmented by direct binding of melittin to the lipid, is responsible for the observed decreases in lipid mobility. Protein aggregation is concluded to be the main cause of inactivation of the Ca-ATPase by melittin, with possible modulation also by the decrease in mobility of the boundary layer lipids.


Effects of crystallization on the heme-carbon monoxide moiety of bovine heart cytochrome c oxidase carbonyl. Cytochrome c oxidase isolated from bovine heart was crystallized in the fully reduced carbon monoxide (CO)-bound form. To evaluate the structure of the O2 reaction site in crystals and in solution, the bound C-O stretch infrared band in protein crystals was compared with the band for protein solution. In solution, the C-O stretch band could be deconvoluted into two extremely narrow bands, one at 1963.6 cm-1 with delta v1/2 = 3.4 cm-1 of 60% Gaussian/40% Lorentzian character represented 86% of the total band area and the other at 1960.3 cm-1 with delta v1/2 = 3.0 cm-1 of 47% Gaussian/53% Lorentzian character represented 14% of the total band area. The crystals exhibited two deconvoluted C-O infrared bands having very similar band parameters with those in solution. These findings support the presence of two structurally similar conformers in both crystals and solution. Thus crystallization of this enzyme does not affect the structure at the CO-binding site to as great extent as has been noted for myoglobin and hemoglobin carbonyls, indicating that the active (CO- or O2-binding) site of cytochrome c oxidase must be conformationally very stable and highly ordered compared to other hemoproteins such as hemoglobin.


Redox interactions in cytochrome c oxidase: from the "neoclassical" toward "modern" models. Because of recent experimental data on the redox characteristics of cytochrome c oxidase and renewed interest in the role of cooperativity in energy coupling, the question of redox cooperativity in cytochrome c oxidase is reexamined. Extensive redox cooperativity between more than two redox centers, some of which are spectrally invisible, may be expected for this electron transfer coupled proton pump. Such cooperativity, however, cannot be revealed by the traditional potentiometric experiments based on a difference in absorbance between two wavelengths. Multiwavelength analyses utilizing singular value decomposition and second derivatives of absorbance vs. wavelength have revealed a stronger cooperativity than consistent with the "neoclassical" model, which allowed only for weak negative cooperativity between two equipotential one-electron centers. A thermodynamic analysis of redox cooperativity is developed, which includes the possibilities of strong cooperative redox interactions, the involvement of invisible redox centers, conformational changes, and monomer/dimer equilibrations. The experimental observation of an oxidation of one of the cytochromes (a3) with a decrease in applied redox potential is shown to require both strong negative cooperativity and the participation of more than two one-electron centers. A number of "modern" models are developed using the analytical approaches described in this paper. By testing with experimental data, some of these models are falsified, whereas some are retained with suggestions for further testing.


Recombinant human growth hormone promotes human lymphocyte engraftment in immunodeficient mice and results in an increased incidence of human Epstein Barr virus-induced B-cell lymphoma. Recombinant human growth hormone (rhGH) previously has been demonstrated to promote human or mouse T-cell engraftment in immunodeficient mice. We then wanted to examine long-term effects of rhGH on human cell engraftment in these mice. Mice with severe combined immune deficiency (SCID) were given human peripheral blood lymphocytes or human bone marrow cells and daily injections of rhGH (20 micrograms ip every other day). Upon later assessment for engraftment by flow cytometric analysis, it was determined that rhGH strongly promoted human T-cell engraftment in the thymus and spleens of these mice. However, there was considerable variability in both the incidence and extent of engraftment which appears to be due to donor-to-donor variation. Additionally, rhGH promoted B lymphomagenesis in these mice since long-term treatment of these xenogeneic chimeras with rhGH resulted in the increased incidence of human Epstein-Barr virus (EBV)-infected B-cell lymphoma. Thus, while rhGH can be used to optimize human T-cell engraftment in SCID mice, it also increases the likelihood of B-cell lymphoma generation when the donor is EBV infected. The results suggest that the activation of human T cells by rhGH results in an increased ability of these cells to traffic to the peripheral lymphoid organs of the SCID mice and results in a lymphoid microenvironment conducive to the outgrowth of EBV-transformed B lymphocytes.


Prolactin and prolactin secretagogues reverse immunosuppression in mice treated with cysteamine, glucocorticoids, or cyclosporin-A. Suppression of prolactin (PRL) secretion with the dopamine agonist, bromocriptine, has been shown in rodents to diminish a variety of immunologic responses, including delayed type hypersensitivity, primary antibody response, T-cell dependent macrophage activation, and ex vivo T- and B-lymphocyte proliferation in response to mitogens. These same responses can be suppressed by endogenous or exogenous glucocorticosteroids and, in large measure, the immunosuppressant peptide cyclosporin A. The sulfhydryl reducing agent cysteamine (2-aminoethanethiol) is known to reduce pituitary and plasma prolactin levels. Treatment of mice with cysteamine at doses which suppressed circulating PRL levels resulted in suppression of ex vivo blastogenic responses of lymphocytes from treated mice. The T-cell-dependent primary IgM response to immunization with sheep red blood cells was also suppressed by cysteamine treatment. Treatment of mice with drugs stimulating the release of endogenous PRL, or with exogenous ovine PRL, was found to antagonize the suppression of lymphocyte proliferative responses to mitogens induced in mice by glucocorticoid or cyclosporin treatment. These data suggest that many drugs in common clinical use could have potential immunomodulatory actions due to suppression or stimulation of pituitary PRL secretion. Furthermore, lactogenic hormones appear to exert counterregulatory actions which may modify glucocorticosteroid actions on immune and other target issues.


Synthesis and characterization of estrogen 2,3- and 3,4-quinones. Comparison of DNA adducts formed by the quinones versus horseradish peroxidase-activated catechol estrogens. Catechol estrogens (CE) are among the major metabolites of estrone (E1) and 17 beta-estradiol (E2). Oxidation of these metabolites to semiquinones and quinones could generate ultimate carcinogenic forms of E1 and E2. The 2,3- and 3,4-quinones of E1 and E2 were synthesized by MnO2 oxidation of the corresponding CE, following the method for synthesizing E1-3,4-quinone [Abul-Hajj (1984) J. Steroid Biochem. 21, 621-622]. Characterization of these compounds was accomplished by UV, nuclear magnetic resonance, and mass spectrometry. The relative stability of these compounds was determined in DMSO/H2O (2:1) at room temperature, and the 3,4-quinones were more stable than the 2,3-quinones. The four quinones directly reacted with calf thymus DNA to form DNA adducts analyzed by the 32P-postlabeling method. The adducts were compared to those formed when the corresponding CE were activated by horseradish peroxidase (HRP) to bind to DNA. The E1- and E2-2,3-quinones formed much higher levels of DNA adducts than the corresponding 3,4-quinones. In addition, many of the adducts (70-90%) formed by the E1- and E2-2,3-quinones appeared to be the same as those formed by activation of 2-OHE1 or 2-OHE2 by HRP to bind to DNA. Little overlap was observed between the adducts formed by E1- and E2-3,4-quinones and HRP-activated 4-OHE1 and 4-OHE2. These results suggest that semiquinones and/or quinones are ultimate reactive intermediates in the peroxidatic activation of catechol estrogens.


Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Peroxynitrite [oxoperoxonitrate(1-), ONOO-] may be formed in vivo from superoxide and nitric oxide. The anion is stable, but the acid (pKa = 6.8) decays to nitrate with a rate of 1.3 s-1 at 25 degrees C. The experimental activation parameters of this process are delta H++ = +18 +/- 1 kcal/mol, delta S++ = +3 +/- 2 cal/(mol.K), and delta G++ = +17 +/- 1 kcal/mol. Peroxynitrite (or its protonated form) oxidizes some compounds such as thiols and thioethers in a biomolecular reaction. The reactions with glutathione and cysteine have activation enthalpies of 10.9 and 9.7 kcal/mol, respectively, which are lower than that of the isomerization reaction. Peroxynitrite reacts with other compounds such as dimethyl sulfoxide and deoxyribose in a unimolecular reaction for which the activation of peroxynitrite is rate-limiting. In theory, activation could involve (1) heterolysis to OH- and NO2+ (delta rxn Gzero' = 13 kcal/mol at pH 7) or (2) homolysis to .OH and .NO2 (delta rxn Gzero = 21 kcal/mol), and these processes also could be involved in the isomerization to nitrate. However, thermodynamic and kinetic considerations indicate that neither process is feasible, although binding to metal ions may reduce the large activation energy associated with heterolysis. An intermediate closely related to the transition state for isomerization of ONOOH to HONO2 may be the strongly oxidizing intermediate responsible for hydroxyl radical-like oxidations mediated by ONOOH. Thus, peroxynitrite reacts with different compounds by at least two distinct mechanisms, and the hydroxyl radical is not involved in either.


Endocrine control of the immunosuppressive activity of the submandibular gland. Extracts of the submandibular gland (SMG) of rats contain fractions that stimulate the in vitro proliferation of Con A-treated lymphocytes. One of the stimulatory fractions was also shown to induce in vivo immunosuppression in rats and mice in several experimental models. Since many other biologically active factors of the SMG had been found to be hormone dependent, we investigated the effects on the immunosuppressive factor of hypophysectomy (Hx) and of hormonal reconstitution in male Fischer rats. Hx induced a marked atrophy of the SMG together with an almost complete disappearance of both the in vitro lymphocyte-stimulating activity and the in vivo immunosuppressive activity, the latter assayed with the contact sensitivity reaction in mice. The treatment of the Hx rats with pituitary hormones demonstrated that prolactin (PRL), thyroid stimulating hormone (TSH), and luteinizing hormone (LH) induced a significant reconstitution of these biological activities, growth hormone led to the recovery of the lymphocyte-stimulating activity but not of the immunosuppressive activity, while follicle-stimulating hormone, and adrenocorticotropic hormone did not induce any recovery of these biological activities. In view of the positive results obtained with TSH and LH further experiments were done to compare the effects of thyroid and sex hormones with those of PRL. The results demonstrated that testosterone and thyroid hormones induced significant recovery of the lymphocyte-stimulating and the immunosuppressive activity. The combination of these two hormones with PRL produced the most effective results. On the other hand, estrogens and progesterone had no significant effects. These results confirm the effectiveness of androgens and thyroid hormones in stimulating the production of biologically active factors by the SMG. Moreover, they demonstrate that PRL, a hormone not previously considered to increase the activity of the SMG, stimulates the production of immunoregulatory factors in Hx animals.


Stimulation of calcium influx in HL60 cells by cholesteryl-modified homopolymer oligodeoxynucleotides. Cholesteryl-modified 15-mer homopolymers of cytidine and thymidine phosphodiester oligodeoxynucleotides (chol-OdC15 and chol-OdT15), but not chol-modified heteropolymeric oligos or chol-modified phosphorothioate oligos, were found to increase cytosolic free Ca2+ in HL60 cells. A flow cytometer and the calcium-sensitive dye indo-1 were used to make multiparameter measurements on the HL60 cells. Chol-OdC15 (5-10 microM) triggered a rapid increase (within 1 min) in [Ca2+]i, with a subsequent slow decline to baseline over 15 min in the continuous presence of agonist. The effect was preserved after unloading the intracellular Ca2+ stores with caffeine and ryanodine. The effect was not sensitive to membrane depolarization by KCl (60 mM) or nimodipine, a dihydropyridine calcium channel antagonist. An increase in [Ca2+]i was absent in a Ca(2+)-free solution and was inhibited by the inorganic Ca2+ channel blocker Cd2+. The results suggest that Ca2+ influx activated by the chol-oligomer is probably mediated by receptor-operated Ca2+ channels. This effect may be due to direct binding of the chol-oligo to the channel or to induced conformational changes due to modification of the local microenvironment.


A new structure-activity model for Ah receptor binding. Polychlorinated dibenzo-p-dioxins and dibenzofurans. A new structure-affinity model for the aromatic hydrocarbon (Ah) receptor is reported. The proposed mathematical model completely eliminates multiple regression analysis in its formulation and overcomes the cross-class comparison inherent to classical quantitative structure-activity relationships. Taking the polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) as model xenobiotics, the binding affinity of a PCDD relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is shown to be analytically related to the electron affinities, entropies, and lipophilicities of PCDD and TCDD. From the calculated dissociation constants of PCDD-Ah receptor complexes, the corresponding equilibrium constants of PCDF-Ah complexes could be computed, in agreement with the experimental observation that the trend in the binding affinities of PCDDs and PCDFs to the Ah receptor are similar. The reported model is capable of quantitatively explaining the quantitatively estimating the in vitro binding affinities of PCDDs, PCDFs, and related xenobiotics to the Ah receptor. Therefore, a halogenated aromatic compound is expected to have a higher affinity for the cytosolic protein than TCDD if it is less lipophilic and has a higher electron affinity and lower entropy. Furthermore, the affinities of structurally related polychlorinated aromatic xenobiotics for the Ah receptor could be computed from their entropies and electron affinities.


Reliability of the herpes simplex virus immunofluorescent test in corneal disease. Herpetic ocular disease never produces well-identifiable morphological aspects. It is expensive, complicated and slow to culture Herpes simplex virus. The validity of the fluorescent monoclonal antibodies test was investigated using corneal cells with or without a sure herpetic lesion. The tests, on 36 cases without sure herpetic lesions, were always negative, while of the 42 patients with a clinically ascertained herpetic lesion, 38 were positive (90.5%) and 4 negative (9.5%), two of these on account of inadequate tissue preparations. Thus, this method can prove useful and reliable for diagnostic purposes.


Novel N-linked oligo-mannose type oligosaccharides containing an alpha-D-galactofuranosyl linkage found in alpha-D-galactosidase from Aspergillus niger. Structures of oligosaccharides from Aspergillus niger alpha-D-galactosidase [EC 3.2.1.22] were studied. Purified alpha-D-galactosidase was treated with N-glycosidase F, and six kinds of oligosaccharides were isolated by gel chromatography and anion-exchange chromatography. The structures of the oligosaccharides were determined by 1H-NMR and compositional analysis to be Man5GlcNAc2, Man6GlcNAc2, Man9GlcNAc2, GlcMan9GlcNAc2, GalMan4GlcNAc2 and GalMan5GlcNAc2. From mild acid hydrolysis, methylation analysis and ROESY spectral analysis, it was ascertained that the galactosyl residue in two oligosaccharides was in the furanose form and was bound to mannose at the nonreducing end with an alpha 1-2 linkage (GalfMan4GlcNAc2 and GalfMan5GlcNAc2).


Proton, carbon, and nitrogen chemical shifts accurately delineate differences and similarities in secondary structure between the homologous proteins IRAP and IL-1 beta. 1H alpha, 13C alpha, and 15N alpha secondary shifts, defined as the difference between the observed value and the random coil value, have been calculated for interleukin-1 receptor antagonist protein and interleukin-1 beta. Averaging of the secondary chemical shifts with those of adjacent residues was used to smooth out local effects and to obtain a correlation dependent on secondary structure. Differences and similarities in the placement of secondary structure elements in the primary sequences of these structurally homologous proteins are manifested in the smoothed secondary chemical shifts of all three types of nuclei. The close correlation observed between the secondary chemical shifts and the previously defined locations of secondary structure, as defined by traditional methods, exemplifies the advantage of chemical shifts to delineate regions of secondary structure.


Stimulation of the pituitary-adrenal axis with a low dose [Arg8]-vasopressin in depressed patients and healthy subjects. Graded doses arginine-vasopressin (AVP) were administered to depressed patients and control subjects to compare the sensitivity of the pituitary-adrenal system of these subjects for this compound. The plasma levels of cortisol, adrenocorticotropic hormone (ACTH) and beta-endorphin were measured before and after intravenous AVP injection. The hormonal output was taken as a measure of pituitary-adrenal function. In control subjects 3 doses AVP and placebo were used, whereas in patients two doses AVP, a low and a high dose, and placebo were tested. All tests were carried out in the afternoon when the pituitary-adrenal system is stable and more susceptible for stimulation. Patients were subdivided into dexamethasone suppressors and nonsuppressors based on their DST status before testing to look for differences among these groups. Control subjects showed no response of the hormones to the lowest dose AVP and a moderate response to the higher doses. Interestingly, depressed patients as compared to controls responded more to the lowest dose AVP in particular with respect to ACTH. DST status did not influence the results. These findings suggest an enhanced sensitivity of the pituitary to low doses AVP in depressed patients. Thus, AVP might play a role in HPA dysfunction in depression.


Sequential assignment of the backbone nuclei (1H, 15N and 13C) of c-H-ras p21 (1-166).GDP using a novel 4D NMR strategy. The c-H-ras p21 protein is the product of the human ras proto-oncogene, a member of a ubiquitous eukaryotic gene family which is highly conserved in evolution. These proteins play an important role in the control of cellular growth. We report here the sequential assignment of the backbone nuclei in a truncated form of the 21-kD gene product, using our recently proposed 4D NMR strategy (Boucher et al., 1992). These assignments are the first step towards a full investigation of the structure, dynamics and interactions of wild-type and oncogenic ras p21 using NMR spectroscopy. Some of the data were presented at the 33rd ENC held at Asilomar, California, U.S.A., in April 1992.