Determination of total protein in highly purified factor IX concentrates. Protein determination by the methods of Kjeldahl, Biuret, Bradford and UV absorbance at 280 nm have been studied in regard to accuracy, precision and simplicity. A reference preparation of a highly purified factor IX concentrate, Nanotiv, reconstituted to 1/5 of ordinary volume was used in the study in order to make a comparison between the different procedures. The Kjeldahl method resulted in a protein concentration of 3.7 mg/ml, whereas the Biuret, Bradford (BSA) and UV absorbance at 280 nm resulted in protein concentrations of 3.6, 2.5 and 2.8 mg/ml, respectively. The corresponding values for specific activity were 136, 140, 200 and 179 IU/mg, respectively. These results demonstrate a great variation in the response obtained by different methods for determination of total protein.


Manufacture and in vitro characterization of a solvent/detergent-treated human plasma. We have developed a modified solvent/detergent (S/D) treatment to inactivate viruses in human plasma using 1% w/w final concentration of tri(n-butyl) phosphate (TNBP) and Triton X-100 and an incubation period of 4 h at 30 degrees C. The procedure inactivates > or = 10(6) chimpanzee-infectious doses (CID50) of HBV, > or = 10(5) CID50 of HCV, and > or = 10(6.2) tissue culture infectious doses (TCID50) of HIV. After virus inactivation, eleven plasma batches were lyophilized and 12 batches were deep-frozen until further use. The batches were characterized by extensive laboratory tests including measurement of clotting factors I-XIII, von Willebrand factor, plasminogen, inhibitors of blood coagulation and fibrinolysis, and other clinically important plasma proteins. All parameters were determined before and after S/D treatment. Twelve conventional single donor plasma units served as control. There were no marked losses of activities of clotting factors, antithrombin III, protein C, plasminogen, and C1-esterase inhibitor due to treatment. After the S/D step, the levels of these parameters were within the normal range in all batches. The same holds true for total protein, immunoglobulins, albumin, complement factors C3 and C4, haptoglobin, hemopexin, caeruloplasmin, alpha 1-antitrypsin, and pH. Protein S and alpha 2-antiplasmin activities decreased by about 50% and were frequently found to be slightly below the lower limit of the respective normal range after treatment. The interindividual variations of all proteins analysed were significantly lower than in the single donor plasma units. The S/D procedure did not lead to increases of markers indicating activation of hemostasis.(ABSTRACT TRUNCATED AT 250 WORDS)


A decade of rare donor services in the United States. Report of the American Red Cross Rare Donor Registry (1981-1990). Between 1981 and 1990, the American Red Cross Rare Donor Registry supplied 9,872 units of red cell components with rare phenotypes to blood centers in the United States and abroad. Approximately 51% were from donors with high-frequency antigen-negative phenotypes and 49% were from donors with multiple antigen-negative phenotypes. Since 1989, the disease category requiring the largest number of units has been sickle cell disease. Strategies to ensure that the Registry will have adequate resources to meet future requirements include testing selected donors for rare phenotypes and blood conservation programs, such as intraoperative salvage and the treatment of anemia of chronic renal failure with recombinant erythropoietin.


Elevated platelet-associated IgG in PlA1-negative mothers following sensitization to the PlA1 antigen during pregnancy. Between 1984 and 1990, we studied 25 infants with neonatal alloimmune thrombocytopenia caused by alloimmunization to the PlA1 alloantigen. We investigated whether mothers of these infants developed elevated platelet-associated immunoglobulin (PAIgG) in addition to anti-PlA1. Eight of the women were found to have PAIgG which persistent at least 7-10 days postdelivery. Eluates prepared from six of the women's platelets reacted with PlA1-positive and PlA1-negative donor platelets, and platelets from donors with Glanzmann's thrombasthenia. None of the women with elevated PAIgG had thrombocytopenia, eclampsia, or infections. Two women were found to have autoreactive antibodies present in plasma. These results indicate that elevated PAIgG may be found in women immunized to the PlA1 antigen. Some women may also have autoreactive antiplatelet antibodies in their plasma. These findings may lead to confusing serologic findings in evaluating the cause of neonatal alloimmune thrombocytopenia.


A semiautomated method for erythrocyte antigen typing on microtitration plates. We describe a method for erythrocyte antigen typing on U-bottom microtitration plates. This method permitted phenotyping of erythrocytes by an enzyme-enhanced hemagglutination assay simultaneously with an indirect antihuman globulin assay within the same microtitration plate. A robotic sample processor identified the samples, prepared the red cell suspensions and distributed the suspension along with the diluted antisera onto the microtitration plates. The assay results were read and interpreted using a microtitration plate photometer controlled by software specially designed to interpret agglutination reactions. Sample identities and assay results were automatically connected by the computer system and transmitted to a minicomputer system.


Complement activation by cold agglutinins. Purified monoclonal human IgM cold agglutinins (CA) of different specificities (anti-I, anti-i, anti-Pr) were investigated for their complement-activating capacity in a homologous system. Incubation of human RBC with excess of IgM CA in the cold, and subsequently with human serum at 37 degrees C, resulted in striking differences in hemolysis. Hemolysis did not correlate to the amount of antibodies bound to RBC at 4 or 20 degrees C. Despite the hemolytic inefficiency of anti-i and anti-Pr CA tested, C1 fixation and subsequent activation of the classical pathway of complement could be assessed in all cases. Absolute numbers of C3 molecules bound to RBC, exceeding the critical level to initiate the terminal sequence of the complement cascade, could not fully explain the differences in the hemolytic activity of the CA. Since C8 binding protein (C8bp) carries I determinants it is hypothesized that anti-I-induced complement-mediated hemolysis might also be favored by the binding of the autoantibody to and probably steric hindrance of this major regulatory protein of the terminal complement sequence. The prominent role of homologous restriction of complement-mediated lysis as a protective mechanism can also be deduced from the fact that rabbit as well as rat serum as a source of heterologous complement lysed cold agglutinin-sensitized red blood cells more efficiently than human serum.


11-Oxycorticosteroid-dependent antibody in the serum of a patient with bladder neoplasm. An IgM 11-oxycorticosteroid-dependent antibody was identified in the serum of a patient with bladder malignancy, which at 4-37 degrees C reacted with all the 11-oxycorticosteroids tested but not with desoxycorticosterone, testosterone, nortestosterone and progesterone. The resulting drug-antidrug antibody complex combines nonspecifically with red blood cells causing agglutination. This reactivity is enhanced both by acid pH and by high drug solution concentrations as well as by ficin test. The antibody is complement-independent and has no blood group specificity. No in vivo or in vitro hemolysis was observed.


Immediate haemolytic transfusion reaction due to anti-Inb. An immediate haemolytic transfusion reaction was investigated in a patient with intestinal cancer. The causative antibody was directed against a high-frequency antigen Inb that was presumably produced as a result of pregnancies. This is the first case of a severe transfusion reaction due to this alloantibody.


[Medical workers and AIDS: the attitude to the problem]. A questionnaire was spread among Kiev physicians with the purpose of evaluating their level of information concerning the AIDS problem. An interrelation of level of knowledge and character of relation to the HIV infection was found. It was established that the idea of air-drop ways of HIV transmission is widely spread among the medical personnel with a tendency to stigmatization of the infected, underestimation of the AIDS problem significance. The authors emphasize the requirement of state-backed program on system of professional training of medical personnel in the AIDS problem.


[Problems in improving the public health of a rural area based on a study of statistical data on population health]. The dynamics of health indices of the Khust District (Transcarpathian Province [correction of Zacarpathye region]) population was studied from 1970 through 1990. It is concluded that statistical data on the increase of morbidity are related to improvement of diagnostics of nosological entities related to increase of the availability of doctors, nursing personnel, number of beds in the district. Reserves of further medical services to the population are discussed.


[The characteristics of the course of acute pneumonia in patients subjected to prolonged exposure to low doses of ionizing radiation as a result of the accident at the Chernobyl Atomic Electric Power Station]. Distinct changes in the clinical picture of acute pneumonia were noted in patients subjected to constant prolonged (1986-1990) effect of small doses of ionizing radiation as a result of residing in the contaminated territory after the Chernobyl atomic station disaster. These changes included increased duration of the disease, frequency of protracted forms, suppression of the immune system. At the same time a general decrease of the incidence of acute pneumonia, increase of the frequency of severe cases of concomitant pathology indicate some stimulating effect of small doses of ionizing radiation on the body with a healthy immune system and suppressing effect on inadequately functioning immune system. Changes of the clinico-laboratory indices of the course of acute inflammatory process depended on the duration of residing in the contaminated locality and to a lesser degree of the dose.


[A scientific knowledge analysis of the problem of the biomedical sequelae of the accident at the Chernobyl Atomic Electric Power Station]. The authors carried out a scientific-research analysis of publication on the problem of medico-biological sequels of the Chernobyl Atomic Station. Systematization of 282 native and 45 foreign publications are presented as information models emphasizing the main trends of research, especially changes in the nervous system as well as in related systems of the body. Further detailed studies of the Chernobyl disaster sequels are important.


[The effect of quantum hemotherapy on cellular and humoral immunity and on erythrocyte morphology in patients with ischemic heart disease]. A study is presented of the effect of reinfusions of ultraviolet-irradiated autologous blood on the number of lymphocytes, titer of circulating immune complexes and theophylline-sensitive cells, number of immunoglobulins A, G and M, number and form of erythrocytes in 30 patients with stable and progressive exertion stenocardia. Thirty control patients were treated with nitrates, calcium antagonists and acetylsalicylic acid. It was established that in patients with stenocardia quantum hemotherapy produces an immunocorrective action reduces the titer of circulating immune complexes, the content of T-lymphocytes, the number of immunoglobulins A and G, normalizes the morphological and functional activity of erythrocytes. Use of this method in the treatment of ischemic heart disease is pathogenetically justified and prevents development of myocardial infarction.


[Immunity and the prostaglandin level in gastritis B]. The indices of systemic and local immunity, level of prostaglandins, presence of Helicobacter pylori in the gastric mucosa was studied in 53 patients with gastritis B. Patients with superficial antral gastritis showed independent of the presence of Helicobacter pylori in the gastric mucosa changes of the systemic immunity and level of PGE2 and PGF1 that were abnormal. In atrophy of the antral gastric mucosa and presence there of H. pylori one observed a reduction of the number of T-lymphocytes and proliferative activity. Suppression of the system of prostaglandins in the blood and gastric mucosa, the toxic effect of H. pylori results in autosensibilization, increase of IgA and IgG finally leading to development of erosions in the gastric antrum.


[The efficacy of antibacterial treatment in chronic gastritis of Helicobacter etiology]. A study of 140 patients with chronic gastritis revealed Helicobacter pylori in the gastric antral mucosa in 100% and in the duodenum in 50%. There is a direct correlation between the presence and amount of Helicobacter pylori in the mucosa, lymphoplasmocytic infiltration, dystrophic changes of epithelium. Ampiox treatment proved beneficial both clinically and morphologically. After treatment Helicobacter pylori was absent in 65.7%, in the remaining cases it reduced by 5-10 times.


[The level and cryoprecipitating activity of fibronectin in bronchial asthma patients]. 125 patients with bronchial asthma were under observation. It has been established that the level of fibronectin decreases as of severity of clinical symptoms of the disease growth. Parallelly cryoprecipitating activity of fibronectin decreases, testifying functional inferiority of the pointed glycoprotein in the patients with bronchial asthma. Under the influence of undergoing therapy restoration of fibronectin concentration and its complex-forming ability takes place except of the hormone-dependent cases of asthma.


[The erythrocyte composition of the peripheral blood in tobacco smokers]. Data of cytometry and acid erythrography were evaluated in 60 tobacco smokers and 30 non-smoking healthy persons depending on length of smoking. Tobacco smoking was found to effect blood erythrocytes. Five-year smoking resulted in an increase of macrocytes, young resistant cells. Toxic signs were less pronounced concerning the resistance of circulating red cells. 6-10 years duration of smoking resulted in toxic lesions of the circulating erythrocytes confirmed by an increase of the number of spherulation-altered and low-resistant cells, early onset and late termination of hemolysis, increase of the percent of perished cells.


[The dynamics of the urinary glycosaminoglycan indices in the pathogenetic therapy of glomerulonephritis]. A positive dynamics of the course of glomerulonephritis under the effect of pathogenetic therapy evaluated in 140 patients was accompanied by a statistically valid reduction of glycoseaminoglycanes in the urine. This allows to use these indices as adjunctive criteria of treatment efficiency, especially in patients with chronic forms of the disease.


[The use of ethonium electrophoresis in treating patients with atherosclerotic encephalopathy]. The new property of ethonium as an angioprotector makes it possible to use ethonium electrophoresis in the treatment of patients with dyscirculatory encephalopathy of atherosclerotic genesis. Treatment results in a hypocholesterolemic effect, improvement of rheoencephalography data and lipid peroxidation.


[The hemostatic system in traumatic subarachnoid hemorrhage in the Far North]. It was found that in conditions of the extreme North sanation of the cerebrospinal fluid occurs later in patients with traumatic subarachnoidal hemorrhage than in the central regions of the country. In connection with this the hemostasis system was studied in 20 patients with traumatic subarachnoidal hemorrhage and 10 patients with brain tumours (control group). Patients with traumatic subarachnoidal hemorrhage showed significant hypocoagulation changes.


[The clinico-physiological validation of the use of the endolymphatic route for antibiotic administration for preventing the translocation of intestinal microflora during the treatment of sepsis patients]. One of the main mechanisms of generalization of infection in purulent-inflammatory processes of the abdominal cavity, retroperitoneal space and pelvis minor is translocation of the pathogenic microflora of the intestine in the lymphatic system via regional lymphatic nodes with subsequent rupture into the systemic circulation. Endolymphatic administration of antibacterial agents into the peripheral lymphatic vessel favours saturation of the reticuloendothelial structures of the lymph nodes by the antibiotic agent that increases their barrier function and, thus, prevents generalization of the infection and development of polyorganic insufficiency.


[An electron microscopic study and the morphofunctional status of the lungs in an experimental reproduction of acute pneumonia]. An analysis of the ultrastructural organization of alveocytes (types I and II), endotheliocytes, interstitium alveolar, macrophages and aerohematic barrier in rabbits with experimental acute pneumonia revealed disorders of synthesis and release of surfactant on the alveolar surface. The observed in pneumonia edema and swelling of the aerohematic barrier components as well as the interstitium reduces the protective mechanism of alveols favouring transition of the disease to a protracted form.


[The effect of elevated environmental temperature on the development of a potassium deficiency in the body]. Electron microscopy and roentgen microanalysis were used to study the ultrastructure and electrolytic balance of K+, Na+ and Cl- of the animal myocardial tissue during general body hyperthermia. It was established that long-term effect of heating microclimate causes a distinct reduction of K+ in the cardiomyocytes accompanied by essential changes of the ultrastructure of ischemic character.


[The toxicological-hygienic characteristics of surface-active substances used in the technological processes of the oil-producing industry]. The authors suggest two stages of ecological and hygienic evaluation of surface active substances widely used in oil extraction. Stage 1. Toxicological-hygienic evaluation of these substances. Stage 2. Ecological-hygienic aspects of surface active substances (migration into soil, phytotoxicity, effect on the water quality and oth.). It was established at stage 1 that proxanol, proxamin, diproxamin are compounds of minor hazard (class 4 hazard). These substances belong to local skin-irritating substances. Proxanol produces skin-resorptive effects which is confirmed by clinical signs of intoxication, reduction of weight of the experimental animals.


[The peripheral hemodynamics of pregnant women with diabetes mellitus]. The state of the peripheral hemodynamics was studied rheovasographically in 26 pregnant patients suffering of diabetes mellitus and 30 healthy pregnant (pregnancy terms: 26, 27 and 34-40 weeks). Results of the study revealed changes of the peripheral hemodynamics in the pregnant diabetic patients as compared with pregnant women non-diabetics. These changes consisted in non-adequate response of the vascular bed to increased hemodynamic loads at the 27-33 pregnancy week as well as in more significant increase of the tone in the diabetic patients during the first half of pregnancy as compared with the healthy women. These changes may become supplementary criteria for revealing preclinical manifestations of diabetic angiopathies in pregnant diabetic women.


[Natural killer activity--an index of treatment efficacy in patients with acute leukemia]. Treatment results were analyzed in 60 patients with acute non-lymphoblastic leucosis. In 30 patients treatment was carried out with consideration of sensitivity of blast cells to chemotherapeutic agents, in 30 polychemotherapy programs were used. Results indicate that individual choice of agents produced a large number of complete clinico-hematological remissions. In 50% of patients with clinico-hematological improvement natural killers were found in the blood and they showed activity after a course of selected treatment.


[The comprehensive diagnosis and treatment of lymphosarcoma of the pharynx]. Modern methods (intrascopic and other instrumental) were used to increase the diagnostic efficiency and extension of the tumour process in 22 patients with lymphosarcoma of the pharynx. Optimal treatment efficacy is achieved in lymphosarcoma of the pharynx by combined use of surgical, radiation (non-traditional course) treatment as well as chemotherapy which is used at early stages as an adjunctive method.


[The rehabilitative treatment of patients after a myocardial infarct who reside in an area close to the Chernobyl Atomic Electric Power Station]. Rehabilitation treatment is evaluated in 55 patients (long-term results) with a history of myocardial infarction residing in the Chernobyl atomic station zone. It was established that the course of the disease in persons who participated in liquidation of the Chernobyl disaster was characterized by a more frequent development of reinfarctions as compared with the Kiev population. Patients residing in the zone neighbouring the Chernobyl station developed more frequently cardiac failure. This requires detailed examination of these patients during their return to work.


[The significance of the lymphocyte adherence inhibition test in patients with diffuse toxic goiter]. Patients with diffuse toxic goiter reveal an increased adhesive capacity of lymphocytes evidencing a reduction of lymphokine production by lymphocytes which would suppress adhesiveness. The degree of increase of adhesive properties of lymphocytes depended on the severity of the clinical picture of diffuse toxic goiter, being higher in those with a severe course. Results of the test of lymphocyte adhesion suppression reflect the state of cellular immunity, severity of the clinical course of diffuse toxic goiter and efficacy of treatment.


[Occupational morbidity among the workers in enterprises for secondary ferrous metal processing and in refractory materials plants]. The authors discuss the level of professional morbidity among workers of ferrous metal processing enterprises and of refractory factories depending on the profession, age, length of work and sex. Recommendations on the reduction of professional diseases in this branch of industry are given.


Prognostic indicators in fulminant hepatic failure. Because of the inhomogeneous prognosis in fulminant hepatic failure, prognostic criteria are required which help to establish the indication for liver transplantation as a successful therapeutic procedure. In our study of 33 patients with fulminant hepatic failure (94% viral hepatitis, 67% hepatitis B), serum bilirubin > 320 or < 160 mumol/L, serum creatinine > 110 mumol/L, prothrombin time < 15% of the normal value and duration of jaundice until onset of encephalopathy > 7 days indicated a fatal outcome. When criteria described by O'Grady et al. were used, only limited predictability was achieved. This, as well as the frequently contradictory results of the few prognostic studies published so far, is probably due to the regional differences in the etiology and the different clinical courses of fulminant hepatic failure.


[Orthotopic liver transplantation in hepatic cirrhosis: on the problem of infection of the transplant with persistent hepatitis viruses]. 269 orthotopic liver transplantations (OLT) were performed in 253 patients at our institution from September 1988 to May 1992. 121 patients had end-stage cirrhosis secondary to viral hepatitis type B, delta, or type non-A non-B and C respectively. Reinfection of the graft by persistent viruses is a potential complication in these cases. Passive immunization with anti-HBs hyperimmunoglobulin (HIg) can prevent clinically relevant reinfection of the graft in patients with hepatitis B virus (HBV) infection and low replication rates. Patients with high replication rates will rarely benefit from OLT. Patients with hepatitis delta virus (HDV) infection usually experience HDV reinfection of the graft with subsequent chronic hepatitis although prophylaxis with anti-HBs-HIg was performed. Treatment with interferon alpha had no apparent effect on the incidence of graft reinfection with HBV in this series, but the replication rate of HDV was reduced. Persistent hepatitis C viruses (HCV) usually infect the graft; this was demonstrated in 17 patients by means of the polymerase chain reaction. HCV infection usually causes a mild form of acute hepatitis with transition to a chronic course. Therefore the significance of persistent viral infection lies in the potential for chronic hepatitis in the transplanted organ rather than in the danger of acute injury of the allograft.


[Extraction of an artery in rectal biopsy]. A biopsy of a rectal carcinoma has led to the extraction of a 2.5 cm arterial specimen, followed by an arterial bleeding. The cause of this exceptional complication was the presence of atypical vessels reaching the rectal lumen due to tumor ulceration.


[Risk of colorectal cancer in ulcerative colitis--monitoring strategies and identification of risk patients]. Patients with ulcerative colitis carry an increased colorectal cancer risk. The cumulative cancer risk for patients with pancolitis is 0.5-0.7% per patient year. Dysplasia as a histologic marker of a neoplastic transformation is used to identify patients with an increased cancer risk during colonoscopic surveillance. Because the classification of dysplasia is subject to inter- and intraobserver variation new methods for the detection of risk patients have been investigated. DNA analysis by flow cytometry seems to be of value for the detection of DNA aneuploidy and the identification of patients who are at risk for neoplastic progression. The significance of DNA aneuploidy is being evaluated in prospective studies. Surveillance guidelines depend on duration and anatomical extent of the colitis as well as on the detection of dysplasia.


[Ultrasound morphology of gallstones].

[Ultrasound morphology of gallstones]. Using a subtile examination technique genuine information can be obtained from the interior of a gallstone--at least to a depth of 15 mm. This information should provide an indication of the homogeneous (crystalline) or inhomogeneous (non-cholesterol) structure of the stone. Further systematic use of differentiating gallstone ultrasonography could become a useful additional decision making criterion for selection of gallstones suitable for conservative treatment.


[Pathogenesis of liver fibrosis. Synthesis of collagen and non-collagen proteins in cell culture and in vivo]. In vitro and in vivo studies have shown that Ito-cells are able to express the "smooth-muscle"-isoform of alpha-actin which seems to characterize the activated stage of the cells. "Activated" Ito-cells synthesize and secrete collagen- and non-collagen components of the extracellular matrix like fibronectin, laminin, tenascin and entactin. "Quiscent" Ito-cells synthesize mostly collagen type IV and III while "activated" Ito-cells also synthesize alpha-2-macroglobulin, a potent protease inhibitor. The in vitro obtained results were confirmed in vivo as demonstrated on liver sections from chronically damaged liver (carbon tetrachloride administration) by immunohistochemistry and by in situ hybridisation technique. Sinusoidal endothelial cells were shown to synthesize collagen type IV and fibronectin during the time in culture.


[Pathomorphology of acute and chronic stages of CCl4-induced liver fibrosis: immunohistochemical and in situ hybridization studies]. The cellular localization of expression of various genes activated during the course of liver fibrosis and regeneration was studied by immunohistology and in situ hybridization in rat and human liver tissues. Mesenchymal cells proved to be the principal sources of extracellular matrix proteins and of fibrogenic growth factors, whereas the collagenase-activating protease transin/stromelysin gene was transcribed in parenchymal cells as well. Fibrogenesis by the mesenchymal compartment appears to be balanced by fibrolysis controlled by parenchymal cell functions. Continuous parenchymal damage may thus disrupt this balance between fibrogenesis and fibrolysis, resulting in fibrosis.


[Connective tissue polypeptides in serum: new parameters of connective tissue synthesis and degradation in liver fibrosis]. With the invention of drugs that effectively and specifically inhibit excessive collagen synthesis in the liver, a growing interest in serum assays that assess fibrogenesis, i.e. the de novo formation and fibrolysis, i.e. the removal of connective tissue in the liver, may be anticipated. Several serum assays for connective tissue polypeptides fulfil the criteria of sensitivity and specificity for chronic liver diseases. The aminoterminal procollagen type III peptide (PIIINP) correlates with hepatic fibrogenesis, whereas the propeptides of basement membrane collagen (PIVNP and PIVCP) and the basement membrane glycoprotein laminin reflect the enhanced turnover of basement membranes of the sinusoids as well as of proliferating bile ducts and blood vessels in active fibrosis. Circulating collagen type VI results from degradation of interstitial microfilaments and undulin appears to be released upon remodeling of the hepatic architecture. Combined measurement of selected parameters may allow a non-invasive assessment of the balance between fibrogenesis and fibrolysis on a day-to-day basis, especially in the light of potential antifibrotic therapy.


Pharmacokinetics of colchicine: a review of experimental and clinical data. Thanks to the development of a sensitive and specific radioimmunoassay for colchicine, the pharmacokinetics of colchicine is now well-established after single oral doses. Absorption is characterized by a zero-order rate constant while disposition appears biexponential with a rapid distribution phase (t1/2 = 1.8 h) and a long elimination phase (t1/2 = 20 h). All studies confirm the large total body clearance (35 l/h) predominantly by the extrarenal route and the large distribution volume (700 l). Further studies need to be performed to investigate colchicine absorption and to describe the metabolic pathway of the drug. To date, relationships between colchicine plasma levels and pharmacological effects have not been defined. Monitoring of plasma levels in patients with familial Mediterranean fever should improve treatment with colchicine. However, the therapeutic range has not been precisely determined. The use of colchicine in the treatment of liver cirrhosis and primary biliary cirrhosis is a recent development; so, assuming that a large part of total body clearance depends on hepatic function, the influence of hepatic diseases on colchicine disposition needs to be investigated in order to define the most appropriate therapeutic dosing.


[Colchicine in toxic liver cirrhosis and primary biliary cirrhosis]. In animals, colchicine has been shown hepatoprotective, which is related to its anti-fibrotic and anti-inflammatory effect. In man, colchicine has been used for the treatment of alcoholic liver disease and primary biliary cirrhosis. Although controlled trials observed an improvement of liver function tests under colchicine, the survival rate in most studies was only slightly improved without reaching statistical significance. Therefore, at present, no final evaluation of colchicine for the treatment of alcoholic liver diseases and primary biliary cirrhosis can be given.


[Pathophysiology of cholestasis: correlation between bile acid metabolism and liver damage]. In patients with cholestasis the biliary excretion of bile acids is diminished. Hepatic sulfation, glucuronidation and hydroxylation of bile acids are increased and enhance their urinary excretion. These metabolic pathways however, are not sufficient to prevent hepatic damage by bile salts. Ursodeoxycholic acid, a very hydrophilic and therefore untoxic bile acid, in part prevents the hepatotoxicity of more hydrophobic bile acids.


Treatment of chronic active hepatitis and primary biliary cirrhosis with ursodeoxycholic acid. Ursodeoxycholic acid (UDCA) improves liver function tests in patients with chronic active hepatitis (CAH) and primary biliary cirrhosis (PBC). UDCA will reduce biochemical parameters of both cholestasis and hepatocellular damage. The effects may be less beneficial in patients with advanced stages of chronic liver disease: in PBC we found the improvement of laboratory parameters in stage I and II very impressive, in stage III and IV it was less marked. Data of two controlled trials in PBC showed an improvement in liver histology, in one study the improvement was statistically significant. UDCA can be administered for at least 10 years without any adverse effects, the treatment is safe and improves life quality. The mode of action of UDCA seems to be in its displacement of toxic hydrophobic bile salts from the bile acid pool and the hepatocellular membrane. In in-vitro investigations a direct protective effect of UDCA on isolated sinusoidal hepatocyte membranes against toxic bile salts has been shown. This protective effect of a more general nature may explain the efficacy of UDCA in different chronic, especially cholestatic liver diseases.


Hepatic fibrogenesis: the puzzle of interacting cells, fibrogenic cytokines, regulatory loops, and extracellular matrix molecules. Fibrotic lesions affect a wide variety of organs but liver fibrosis is probably the most abundant form of organ fibrosis. Disturbances of the equilibrium between parenchyma and extracellular matrix (ECM) leading to a disproportionate increase and irregular deposition of newly formed connective tissue components (fibrosis) is a common and clinically most important sequelae of chronic active liver diseases. Significant progress has been made during the last years in the analysis of the structural composition of ECM in normal and fibrotic liver and in the dissection of the molecular and cellular mechanisms of exaggerated ECM-deposition in necroinflammatory areas using experimental models of hepatic fibrosis, isolated cells in mono- and coculture systems and the growing repertoire of molecular biological tools. Perisinusoidal, retinoid-storing cells (Ito cells, parasinusoidal lipocytes) transform under inflammatory stimuli to myofibroblast-like cells being qualitatively and quantitatively the most important connective tissue producing cell type in human and animal liver. Its activation and transformation is mediated by paracrine and autocrine loops involving transforming growth factor (TGF) beta as the main fibrogenic mediator secreted by activated liver macrophages, possibly also by endothelial cells and liberated by disintegrated thrombocytes. In advanced stages of retinoid-storing cell transformation additional growth factors like platelet derived growth factor (PDGF) become important indicating a sequential action of cytokines and fibrogenic mediators during the fibrogenic process. The molecular and cellular interactions in the course of liver fibrogenesis have gained model character for a number of other organ fibrotic processes, wound repair, and atherogenesis included.(ABSTRACT TRUNCATED AT 250 WORDS)


[Combined ursodeoxycholic acid plus colchicine--treatment of primary biliary cirrhosis: results of a placebo-controlled double-blind study]. In the present pilot study we investigated the effects of urso treatment alone in comparison to a combined treatment with urso plus colchicine in PBC. 22 patients with PBC in the histological stages 1-3 entered the study. All patients were pretreated with urso alone (10-12 mg/kg) for 12 months. Thereafter treatment was continued in a double-blind randomized fashion with urso plus placebo or urso plus colchicine (1 mg/day) for another 12 months. During the initial 12 months urso treatment liver function tests improved significantly in all patients, pruritus improved in 60% of patients. After randomization to the different treatment groups the biochemical parameters stabilized at the lower level and no significant differences could be found between urso plus placebo and urso plus colchicine treatment concerning aminotransferases, alkaline phosphatase, bilirubin, cholinesterase, albumin or cholesterol. The results of this pilot study suggest that the addition of colchicine to an initial urso treatment does not lead to further improvement of aminotransferases, alkaline phosphatase, bilirubin or clinical symptoms like pruritus.


[The dependence of respiratory intensity on body mass in representative Caudata and Salientia]. Experimental data indicate dependence of the respiration rate on the body mass in both caudate and ecaudate amphibians. The regression line slopes are similar in the both groups, while the a factor value is 3 times lower in the caudates than it is in ecaudates. There certain sex differences in the respiration rate which have no clear biological interpretation.


[The motor activity of the chick embryo amnion in the late stages of development. The role of serotonin and noradrenaline]. Experiments with 13- to 19-day-old chicken embryos maintained at 37 degrees C were conducted using direct registration of embryonic movements and amnion pulsation. It is shown that in the non-innervated and lacking blood vessels non-striated-muscled amnion, motoric activity could be observed nearly up to the end of the embryonic development, not only during 5 to 14 days old interval as it was supposed earlier. In accordance with our previous results indicating important role of biogenic amines (serotonin and noradrenaline) in the regulation of motoric activity of the chicken embryo amnion at earlier and middle ages, this study provides some evidences of the humoral regulatory mechanism even at later embryonic stages. After being injected into amniotic fluid of older embryos, the serotonin stimulates and noradrenaline inhibits amnion motoric activity (the both are taken at final concentration of 10(-7) to 10(-6) M). Serotonin's receptors blocator, the cyprogeptadin, suppress while beta-adrenoreceptors' blocator, the propranolol, activates intact amnion motorics ( both are taken at final concentration of 10(-7) to 10(-5) M).


Comparison of cellular phenotypes in tumor cyst and ascitic fluid from ovarian serous carcinoma. Density gradient centrifugation was applied to isolate cell subsets from tumor cyst and ascitic fluid in eight patients with ovarian serous carcinoma. A comparison of cellular composition and immunologic reactivity of cells from the cysts and from ascitic fluid in each patient was performed. Some differences in density profiles were found, but in each case the consistency of morphologic cell forms in the primary tumor and ascites was documented. Immunophenotypic analyses of isolated cellular fractions using polyclonal and monoclonal antibodies against ovarian carcinoma-associated antigens showed significant immunologic intratumoral heterogeneity. However, there was a similarity of antigen expression in cells from the primary tumors and ascitic fluids. Our study indicated that morphologic and antigenic characterization of a given tumor could be determined in a single representative sample of ascitic fluid.


Diagnosis of follicular lymphoma by fine needle aspiration biopsy. This study evaluated the diagnostic accuracy of fine needle aspiration biopsy (FNAB) of follicular lymphoma (FL). Fourteen aspirates of lymph nodes in which follow-up surgical biopsy revealed FL were studied. Two aspirates were deemed unsatisfactory because of a paucity of cells. The remaining 12 cases received the following diagnoses: 4 positive for malignant lymphoma, 4 highly suspicious for malignant lymphoma and 4 false negatives. FNAB of FL can show a monomorphic or polymorphic cell population. The aspirates with a positive or suspicious diagnosis showed monomorphic cell populations. False-negative diagnoses were attributable to misleading sampling or preparation methods in most cases. We conclude that FNAB of FL is less accurate than FNAB of non-Hodgkin's malignant lymphoma (NHL) in general, but the accuracy rate is similar to that of FNAB of all low-grade NHL. The value of current approaches to the diagnosis of suspected lymphoma by FNAB is emphasized.


Influence of endocervical status on the cytologic prediction of cervical intraepithelial neoplasia. A case-control analysis of the endocervical status of smears that preceded a histologic diagnosis of cervical intraepithelial neoplasia (CIN) showed that smears which correctly predicted CIN were significantly more likely to include metaplastic cells than were smears reported to be negative. There was no significant difference between the smears with respect to the presence of columnar cells. A high level of agreement was apparent between scientists in determining both columnar and metaplastic cell status. A discussion of the definition, role and potential impact of endocervical status in the prevention of cervical cancer is presented.


Fine needle aspiration of pancreatic cystic epithelial neoplasms. Pancreatic cystic epithelial neoplasms present diagnostic challenges in cytology. An accurate diagnosis is important since the prognosis and treatment may vary. We report the cytologic features in fine needle aspirates of four cases of cystic neoplasms of the pancreas (two micro-cystic adenomas, one mucinous cystic neoplasm and one mucinous cystadenocarcinoma). Smears were evaluated as to their cellularity, content and predominant cell type. Aspirates from the microcystic adenomas yielded hypocellular material with rare strips of cuboidal cells having bland nuclei and pale cytoplasm. No mucinous material was identified in the background, but the cells stained positively with periodic acid-Schiff stain. Smears from the mucinous cystic neoplasm were moderately cellular and contained abundant mucinous material. The columnar epithelial cells were arranged in tight sheets, clusters and strips. Most cells had benign nuclear features with focal mild nuclear atypia. Key cytologic findings noted in the mucinous cystadenocarcinoma were moderate cellularity, loose clusters of cells, single cells, overt malignant nuclear features and occasional signet ring cells. Pancreatic pseudocysts can be distinguished from pancreatic cystic epithelial neoplasms by the predominance of histiocytes and inflammatory cells and absence or paucity of epithelial cells. To differentiate microcystic adenomas from mucinous cystic neoplasms, the above criteria coupled with periodic acid-Schiff and mucin staining should effectively differentiate these diagnostic entities.


Fine needle aspiration of lymphoblastic lymphoma. A multiparameter diagnostic approach. Sixteen fine needle aspiration (FNA) biopsies of lymphoblastic lymphoma (LBL) that were used to either initially diagnose disease (12) or document relapse (4) were reviewed. Cellular aspirates (2 x 10(7) cells) were readily obtained for immunologic, DNA/RNA flow cytometric and immunoglobulin and/or T-cell receptor gene rearrangement studies. Cytologically, aspirates were characterized by intermediate-sized cells (9.5-18.5 microns) with fine nuclear chromatin, small, inconspicuous nucleoli, irregular nuclear contours and scant basophilic cytoplasm. Frequent mitotic figures were seen (1-14 figures per 1,000 cells). Fourteen cases demonstrated a T-cell phenotype with considerable phenotypic variability. One case demonstrated a precursor B-cell phenotype, and another demonstrated biphenotypic expression with both T-cell and myeloid differentiation. Eleven of 14 cases (79%) were positive for terminal deoxynucleotidyl transferase. Thirteen of 15 cases (87%) manifested diploid DNA content by flow cytometric analysis and were characterized by intermediate proliferative activity (S+G2M 12.7 +/- 8.7% SD) and intermediate mean RNA index (1.3 +/- .5 SD). T beta gene rearrangements were demonstrated in four of five phenotypic T-cell LBL cases analyzed, with concomitant JH gene rearrangements observed in three cases, confirming that bigenotypic rearrangements characterize some T-cell LBLs. We conclude that FNA samples are adequate for accurate characterization of LBL and may obviate the need for surgical biopsy.


Cytopathology of the tall cell variant of thyroid papillary carcinoma. The tall cell variant of thyroid papillary carcinoma differs from classic papillary carcinoma in its more aggressive clinical behavior, cell type (columnar amphophilic to oxyphilic) and higher frequency of stromal lymphoid infiltrate. A retrospective study of three such cases was made, with an emphasis given to the utility of fine needle aspiration cytology in their identification. Aspirates revealed papillary fronds and cyanophilic and oxyphilic neoplastic cells with a high proportion of nuclear grooves and cytoplasmic inclusions. These nuclear details allowed a specific diagnosis of papillary carcinoma with oxyphil cells as compared to oxyphilic cell follicular tumors. Smears from two cases showed, in addition, lymphoid cells and multinucleate giant cells. In them a diagnosis of coexisting Hashimoto's disease, granulomatous thyroiditis or inflammatory tumor stroma could not be excluded cytologically.


Fine needle aspiration cytology of primary soft tissue tumors. Morphologic analysis of the most frequent types. We reviewed 98 cases of fine needle aspiration of soft tissue tumors with histologic confirmation performed during an eight-year period and propose a working morphologic classification based on the most prominent cytologic features. Six main tumor groups are recognized: myxoid, round cells, spindle cells, pleomorphic cells, polygonal cells and well differentiated. We believe that this classification allows recognition of the most common soft tissue tumors while helping with the differential diagnosis of other neoplasia, primary or secondary, with similar morphology and a similar presentation.


Cerebrospinal fluid cell preparation methods. An evaluation. Although the cells from cerebrospinal fluid are widely used for diagnostic purposes, the cell yield on the microscopic slide is only rarely considered and mostly unsatisfactory. A reevaluation of several preparatory methods showed a very low yield with the Sayk-type chamber, a modest yield with the cytocentrifuge and the best yield with a simple, self-adhering chamber on slides coated with a polycation.


Presacral myelolipoma. Report of a case with fine needle aspiration cytology and immunohistochemical and histochemical studies. The cytomorphologic features of a case of the rare presacral myelolipoma, diagnosed by computed tomography (CT)-guided percutaneous fine needle aspiration (FNA) biopsy, are presented. It occurred in a 40-year-old man on prolonged steroid therapy for bronchial asthma. On the air-dried, Diff-Quik-stained smear, hematopoietic cells of all lineages and fragments of collapsed stroma were seen in a background of fat droplets. Megakaryocytic, myelocytic and eosinophilic lineages were further demonstrated by Factor VIII immunostain, chloroacetate esterase and LUNA-E histochemical stain, respectively. The reticulum stain of the resected tumor demonstrated that the hematopoietic cells and fat were supported by numerous reticulum fibers of varying thickness; that accounted for the collapsed stroma in the aspirated material. Presacral myelolipoma, occurring predominantly in female adults and presenting secondarily as a space-occupying lesion, is best regarded as encapsulated, heterotopic bone marrow supported by a network of reticulum fibers; it has a characteristic image on CT scan and can be diagnosed by FNA.


Fine needle aspiration cytology of clear cell sarcoma. Report of a case with immunocytochemical, immunohistochemical and ultrastructural studies. Cytologic findings of clear cell sarcoma obtained by fine needle aspiration (FNA) of a tumor are described. The tumor probably originated in the retroperitoneal tissue, and the diagnosis was confirmed histologically by open biopsy. Percutaneous needle aspirates of the intraabdominal tumor and touch preparations obtained from the open biopsy specimen revealed numerous atypical cells with an extremely hyperchromatic nucleus, prominent nucleoli and clear cytoplasm. The cytoplasm was rich in glycogen. The immunocytochemical technique demonstrated S-100 protein and neuron-specific enolase in the cytoplasm, both of which were exhibited also in the histologic specimen. Clear cell sarcoma is a rare tumor of soft tissue, and to our knowledge, detailed cytologic appearances of this tumor obtained by FNA have not been reported. In addition, the present tumor was unique in location. It is possible to diagnose clear cell sarcoma accurately on an FNA cytologic specimen if the periodic acid-Schiff stain and immunocytochemical technique are utilized in addition to the routine Papanicolaou method.


Cytology of pyelitis glandularis cystica. A case report. Cytologic findings that occur in association with proliferative lesions involving von Brunn's nests in the urinary tract are rare. Such lesions are probably far more common than recognized and are probably considered in the clinicoradiologic differential diagnosis when space-occupying lesions are found, particularly in the upper urinary tract. Reported here are the cytologic findings in a case of pyelitis glandularis cystica that presented as a renal pelvic lesion.


The relationship between first-phase insulin secretion and glucose metabolism. A possible pathogenetic link between absence of first-phase insulin secretion and development of impaired glucose metabolism has been suggested by the results of several cross-sectional studies. First-phase insulin secretion measured during a +7 mmol/l hyperglycemic glucose clamp correlated with total glucose disposal during the clamp (r = 0.65, p < 0.001, N = 59). To examine whether restoration of first-phase insulin secretion improves peripheral glucose uptake in subjects with impaired glucose utilization, seven insulin-resistant subjects (age 54 (38-62) years: BMI 29.3 (21.7-35.8); fasting plasma glucose 5.5 (4.8-7.2) mmol/l; fasting insulin 57 (37-105) pmol/l with impaired first-phase (148 (29-587) vs controls 485 (326-1086) pmol/l x 10 min; p < 0.05) and normal second-phase (1604 (777-4480) vs controls (1799 (763-2771) pmol/l x 110 min) insulin secretion were restudied. The impaired first-phase insulin secretion was restored by an iv insulin bolus at the start of the hyperglycemic clamp. Substrate oxidation rates and hepatic glucose production were determined by indirect calorimetry and [3-3H]glucose infusion. Total glucose uptake was impaired in the insulin-resistant subjects with impaired first-phase insulin secretion compared to controls (18.8 (13.2-22.2) vs 34.8 (24.3-62.1) mumol.kg-1 x min-1; p < 0.01). Restoration of first-phase insulin secretion (1467 (746-2440) pmol/l x 10 min) did not affect glucose uptake (20.2 (9.9-23.8) mumol.kg-1.min-1), with no difference in oxidative and non-oxidative glucose metabolism between the experiments. Second-phase insulin secretion was similar during both experiments.(ABSTRACT TRUNCATED AT 250 WORDS)


Adamantinoma. A case report with aspiration cytology and differential diagnostic and immunohistochemical considerations. Fine needle aspiration biopsy of bone lesions is routinely used in the metastatic workup of patients with radiographically suspicious areas. However, caution must be used when interpreting smears from aspirates performed on primary bone neoplasms. These tumors are often heterogeneous, and problems with sampling may be encountered. We report a case of a 25-year-old male who presented with a 3-cm lytic lesion in the tibia. A diagnosis of benign fibroosseous lesion was based on the clinical presentation, radiographic appearance and presence of numerous sheets and single cytologically bland spindle cells. Subsequent curettage of the specimen revealed an adamantinoma with a prominent fibrous component. Most of these rare, locally aggressive neoplasms are located in the tibia. They are characterized histologically as having a fibrous background with islands of basaloid, spindle or squamoid cells. Furthermore, a differentiated, regressing variant with an osteofibrous dysplasia-like appearance also exists. Smears consisting primarily of spindle cells or fibrous tissue may lead to an erroneous diagnosis of a fibrohistiocytic neoplasm, fibrous dysplasia, fibrous cortical defect or ossifying fibroma. Pertinent cytomorphologic features should aid in establishing the correct diagnosis of adamantinoma.


Parathyroid hormone-related peptide, measured by a midmolecule radioimmunoassay, in various hypercalcaemic and normocalcaemic conditions. The diagnosis of humoral hypercalcaemia of malignancy often presents considerable clinical problems. We have studied parathyroid hormone-related peptide (PTHrP) in serum from patients with humoral hypercalcaemia of malignancy (N = 22), hypercalcaemia of malignancy with skeletal metastases (17), histologically confirmed primary hyperparathyroidism (21) and hypercalcaemic patients with various benign diseases (9). PTHrP measurements were also made in normocalcaemic patients with various malignancies (23), endocrine diseases (13), sarcoidosis (22) and chronic renal failure (17). PTHrP was measured by a novel radioimmunoassay using rabbit antibodies directed towards the midregion of the molecule. Immuno- or silica cartridge extraction of serum before radioimmunoassay enabled us to measure PTHrP in all samples, which may add further information about circulating forms of PTHrP. PTHrP was clearly elevated in patients with humoral hypercalcaemia of malignancy (5.0 +/- 4.7 pmol/l) (mean +/- SD, N = 12) and when the kidney function was impaired (4.0 +/- 0.9 pmol/l) (N = 15) (silica cartridge extraction), whether the subject was hypercalcaemic or not. Some patients with endocrine diseases, including two with primary hyperparathyroidism, had slightly elevated serum PTHrP concentrations, while they were normal in sarcoidosis. In healthy subjects the levels were 1.1 +/- 0.5 pmol/l (N = 15) after immunoextraction and 0.8 +/- 0.2 pmol/l (N = 33) after silica cartridge extraction.


Treatment of juvenile goitre with levothyroxine, iodide or a combination of both: the value of ultrasound grey-scale analysis. The effects of oral iodide, levothyroxine and of iodide and levothyroxine in combination were studied in three groups of 30 children, age 13-15 years, with euthyroid goitre. As endpoints of this study, we used thyroid volume reduction, thyroid hormones, thyrotropin and thyroid grey-scale histograms by computerized analysis. The three groups were well matched with respect to mean age, body weight and pretreatment thyroid volumes and thyroid hormones. Mean urinary iodide excretion before treatment was in the range of 30 micrograms/g creatinine, since the study was conducted in an iodine-deficient area. All three treatment regimens led to significant reductions in thyroid volume within one month. After six months on 100 micrograms of levothyroxine, thyroid volume had decreased from 14.1 +/- 4.2 ml to 8.3 +/- 2.6 ml (mean +/- SD); on 150 micrograms of iodide, from 18.5 +/- 6.2 ml to 8.8 +/- 2.7 ml; and on 100 micrograms of iodide plus 50 micrograms of levothyroxine, from 17.2 +/- 3.1 ml to 8.3 +/- 2.0 ml. When treatment was discontinued for three months, or the dosage reduced, thyroid volume increased again in the levothyroxine (to 11.3 +/- 2.5 ml) but not in the iodide group. Grey-scale values (by ultrasound, computer-aided estimation) after nine months were significantly different between the three treatment groups; no change was observed with levothyroxine, but after 150 micrograms of iodide as well as after combined treatment with levothyroxine and iodide there were marked decreases of grey-scale values; this is interpreted as reflecting a decrease in follicle size and colloid content of the thyroid which takes place after iodide supplementation.(ABSTRACT TRUNCATED AT 250 WORDS)


Transitional cell carcinoma of the renal pelvis metastatic to the metacarpal. A case report correlating cytologic and histologic findings. This report describes the cytologic, radiologic and histologic findings in a 76-year-old male who presented with a pathologic fracture of the first metacarpal bone as the result of metastatic transitional cell carcinoma. The primary neoplasm was sited in the right renal pelvis. Metastases were also detected in the liver and confirmed cytologically. Problems encountered with the cytologic diagnosis are explained by correlation with the histologic findings. The case also illustrates the importance of clinicopathologic correlation when interpreting fine needle aspiration biopsies.


Combined treatment with growth hormone and gonadotropin-releasing hormone analogues in children with isolated growth hormone deficiency. In subjects with an isolated GH deficiency the inhibition of puberty by GnRH-analogue administration may be attempted to delay the onset, or to prolong the duration, of pubertal maturation in order to improve final height. We report our experience on the matter in 10 subjects (6M, 4F) suffering from isolated GH deficiency with a chronological age ranging from 6.5 to 10.6 years at diagnosis. After a period of 1-5.1 years of GH treatment, GnRH-analogues (long-acting D-Trp-6-GnRH) were added to GH for 12 months, when six subjects were still prepubertal and four in early puberty. During combined therapy, a regression in pubertal development was shown in three out of four children in early puberty, while serum testosterone or estradiol decreased. Height velocity decreased (from 5.23 +/- 1.49 (mean +/- SD) to 4.12 +/- 0.67 cm/year; p < 0.02), whereas height SD scores for bone age increased (from -0.75 +/- 0.42 to -0.47 +/- 0.55; p < 0.02). During the year of combined therapy, bone age increased only 0.57 +/- 0.27 years. The values for predicted height (TW2 and Bayley-Pinneau method) after combined treatment were also higher than those after treatment with GH alone (p < 0.02 and p < 0.001, respectively). Our preliminary data showed that the addition of GnRH-analogues to GH in subjects with isolated GH deficiency reduces the effect of GH on height velocity, but determines an improvement in statural prognosis, although a proper answer will not be obtained until final height has been achieved.


Cytologic and immunocytochemical studies of cerebrospinal fluid in meningeal sarcoidosis. A case report. Serial studies were done on cerebrospinal fluid (CSF) from a patient with sarcoidosis involving the meninges. Initially when the disease was active, the CSF protein was increased and glucose decreased. The number of cells in the CSF was moderately increased, and many mononuclear cells were present. Cytologic studies of the CSF showed many normal and some atypical lymphocytes. Immunochemical studies showed that most of these lymphocytes were T cells, with T-helper cells predominating over T-suppressor cells by a ratio of 3.92; B-lymphocytes were polyclonal. Subsequent studies of the CSF over the following three and one-half years showed that the protein and glucose content and the cell counts in the fluid did not correlate well with the activity of the disease. The number of atypical lymphocytes seemed to be a more useful marker of disease activity in the patient. Cytologic studies, when interpreted within the context of other CSF and clinical findings, are useful for the assessment of patients with sarcoidosis involving the meninges.


Serum levels of intact parathyroid hormone and alkaline phosphatase correlate with cortical and trabecular bone loss in primary hyperparathyroidism. We examined the relationship between bone loss and several biochemical indices in 38 patients with primary hyperparathyroidism. Bone mineral density was reduced by 12 +/- 4.0% in the lumbar spine, 18 +/- 4.2% at the distal radius and 21 +/- 2.8% at the proximal radius (mean +/- SEM). There were significant negative correlations between the serum concentrations of intact parathyroid hormone (PTH) and the Z-scores of the bone mineral content at the proximal and distal radius. In the lumbar spine, bone mineral density was greater in patients with mildly elevated PTH and less in patients whose PTH levels exceeded 8.6 pmol/l. We also observed a strong association between increased levels of serum alkaline phosphatase and low bone mineral Z-scores. Our data thus indicate that cortical and, with the exception of mild primary hyperparathyroidism, trabecular bone loss is proportional to the concentration of circulating PTH and the severity of PTH-induced bone turnover. For the individual patient, however, the usefulness of intact PTH and alkaline phosphatase measurements for assessing bone loss associated with primary hyperparathyroidism seems to be only limited.


Cytodiagnosis and comparison of nondecidualized and decidualized endometriosis of the abdominal wall. A report of two cases. We describe two cases of endometriosis of the abdominal wall occurring in young, multiparous women in which the diagnosis was made by fine needle aspiration biopsy. One case illustrates the cytologic features of non-decidualized endometriosis: a biphasic population of stromal and glandular cells. In contrast, the other case showed large, plump stromal cells in a distinctive myxoid background, creating a picture of decidualized endometriosis. The differential diagnoses of palpable masses in the abdominal wall and the importance of clinicopathologic correlation are discussed.


Effects of short-term growth hormone treatment on PTH, calcitriol, thyroid hormones, insulin and glucagon. We measured changes in serum insulin-like growth factor-1 (IGF-1), calcitriol, parathyroid hormone (PTH), thyroid hormones, insulin, and plasma glucagon in response to seven days of treatment with a pharmacological dosage of recombinant human growth hormone (r-hGH) (0.1 IU/kg sc twice daily) or placebo in 20 normal male volunteers to evaluate whether the effect of r-hGH on biochemical bone markers could be attributed to changes in these hormones. Serum IGF-1 (p < 0.001) and vitamin D-binding protein (p < 0.001) increased steadily during treatment returning to baseline at day 14. Total calcitriol (p < 0.01) and free calcitriol index (p < 0.001) increased transiently at day 4. Furthermore, serum insulin (p < 0.001) and both total (p < 0.001) and free triiodothyronine (p < 0.02) increased during treatment, while serum PTH and plasma glucagon remained unchanged. In conclusion, pharmacological doses of r-hGH increased not only IGF-1 but also free-calcitriol index, insulin, and free T3. The increase in these hormones may be co-responsible for some of the observed effects of r-hGH on bone turnover and calcium homeostasis.


Growth hormone stimulates galactopoiesis in healthy lactating women. Sixteen normally lactating women underwent a double-blind randomized placebo-controlled trial of recombinant human growth hormone (hGH) to assess the effect of hGH on milk production in early lactation. Milk volumes were measured by test weighing procedures of the infants and removal of residual milk on a control day and after 7 days of treatment with recombinant hGH (0.1 IU.kg-1 body weight.d-1) or placebo treatment. Although all women were lactating normally before the study commenced, milk volume in 8 hGH treated mothers was increased (p < 0.02) by 18.5 +/- 1.4% (mean +/- SEM) compared to 11.6 +/- 2.0% in the placebo-treated group (N = 8). No adverse effects were seen with hGH treatment and no major changes noted in milk constituents. The hGH concentrations in milk were low and did not change with therapy. Plasma concentrations of IGF-1 increased significantly within 24 h of hGH treatment and increased further towards the end of the trial to values of 2.6-fold above the pretreatment values. The concentration of IGF-1 in milk was approximately 100-fold lower than those observed in plasma and could only be reliably measured after size exclusion chromatography to remove the interfering influence of IGF binding proteins in the radioimmunoassay. All women treated with hGH showed a small increase in milk IGF-1 concentrations but the values remained within the range of values observed in women receiving the placebo treatment (1.2-4.4 micrograms/l). Growth hormone treatment increased milk volume in normal lactating women during early lactation.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of prolonged Acipimox treatment on glucose and lipid metabolism and on in vivo insulin sensitivity in patients with non-insulin dependent diabetes mellitus. The effect of prolonged treatment with Acipimox on in vivo peripheral insulin sensitivity, and on glucose and lipid metabolism, was investigated in patients with NIDDM in a double-blind study. Twelve NIDDM patients were randomized to treatment with either placebo or Acipimox in pharmacological doses (250 mg x 3) for three months. Fasting plasma glucose, insulin, C-peptide and HbA1c concentrations were unaffected after three months of acipimox treatment. However, fasting plasma non-esterified fatty acid (NEFA) concentrations were twofold elevated after Acipimox treatment (1.34 +/- 0.09 vs 0.66 +/- 0.09 mmol/l; p < 0.05). Despite this, repeated acute Acipimox administration after the three months' treatment period enhanced total insulin-stimulated glucose disposal to the same extent as acute Acipimox administration before the treatment period (367 +/- 59 vs 392 +/- 66 mg.m-2.min-1, NS; both p < 0.05 vs placebo glucose disposal) (267 +/- 44 mg.m-2.min-1). In conclusion, insulin resistance or tachyphylaxis towards the effects of Acipimox on insulin stimulated glucose disposal was not induced during prolonged Acipimox treatment. The lack of improvement of blood glucose control in the patients with NIDDM may be due to the demonstrated rebound effect of lipolysis.


Effect of melatonin on the in vitro secretion of progesterone and estradiol 17 beta by ovine granulosa cells. This study was undertaken to determine the effect of melatonin on steroid hormone production by ovine granulosa and luteal cells in vitro. Granulosa and luteal cells from ovine ovaries were cultured for nine days either in D-MEM only or in the presence of melatonin (0.86, 8.6, 86 nmol/l), ovine luteinizing hormone (oLH, 2 micrograms/l) or a combination of both these hormones. Progesterone (P4) and estradiol 17 beta (E2) were determined by validated RIAs. Melatonin stimulation began at either day 1 or day 5 of culture. Melatonin (0.86 nmol/l) significantly increased (p < 0.001) progesterone secretion by granulosa cells both when administered alone and when administered in combination with oLH; the more marked response was observed in the latter case. When the stimulation began at day 5, at a more advanced degree of differentiation of the cells, higher levels of P4 were observed. Higher concentrations of melatonin did not further increase progesterone production. Melatonin alone did not have a significant effect on the production of estradiol 17 beta; neither did melatonin stimulate progesterone production in either long-term cultured luteal cells or in short-term (1-2 h) cultured luteal and granulosa cells. The results of this study document a direct effect of melatonin in stimulating granulosa cells to produce progesterone, a synergistic activity between melatonin and luteinizing hormone and a different ability of granulosa cells to secrete P4 depending on the degree of differentiation.


Diaphragmatic movement using ultrasound during spontaneous and mechanical ventilation: effect of tidal volume. Using ultrasound (US) the effect of various tidal volumes on the movement of ventral, dome and dorsal parts of the right hemidiaphragm was studied, both during spontaneous and mechanical ventilation. Six healthy non-medicated volunteers who were in the supine position breathed spontaneously shallowly (tidal volume (VT) being 400 ml) (SB), and deeply (VT 1000 ml) (SB-deep). In addition, they were mechanically ventilated with intermittent positive pressure ventilation at three different VT's: 500 ml (IPPV-500), 1000 ml (IPPV-1000) and 1700 ml (IPPV-1700). The maximal movement was recorded in the ventral part in 2 volunteers during SB, in 3 during SB-deep, and in 3 and 5 subjects during IPPV-500 and IPPV-1700, respectively. The movement in dome was 100% during SB (all others standardized to this), 303 +/- 107% during SB-deep, 82 +/- 30% during IPPV-500, 165 +/- 70% during IPPV-1000 and 266 +/- 153% during IPPV-1700. An increased tidal volume is associated with an increase in the diaphragmatic movement studied by US. However, a larger VT is needed during mechanical ventilation to achieve the same amount of change as occurred with deep spontaneous breathing.


Behavioural changes in children following minor surgery--is premedication beneficial? One hundred and twenty-three male children, aged one to ten years, were studied to determine the influence of premedication on changes in patterns of behaviour following hospitalization for repair of inguinal hernias. Four comparable groups were selected for premedication regimen: (1) A control group without premedication; (2) oral trimeprazine tartrate 2 mg/kg, methadone 0.1 mg/kg and droperidol 0.15 mg/kg; (3) oral midazolam 0.45 mg/kg; (4) intramuscular midazolam 0.15 mg/kg. Standard inhalational anesthesia was used and caudal blocks employed for analgesia. The parents returned a questionnaire at two weeks. Changes in behaviour were reported in 78% of the children and overall, premedication showed little benefit. However, midazolam premedication was associated with a significantly lower incidence of night-time crying and awakening, compared with no premedication. Only for night-time crying and day-time toilet training did age below five years prove to be a significant contributing factor.


Aging and immunity.

Aging and immunity. The function of the immune system peaks at around puberty and gradually declines thereafter with advance in age. The age-related decline of immunological function primarily occurs in the T cell-dependent immune system and is generally associated with increase in susceptibility to infections as well as in incidence of autoimmune phenomena in the elderly. The age-related change in T cell-dependent immune functions can be ascribed to the physiological thymic atrophy which starts in an early stage of life. Emigration of T cells from the thymus to the periphery mainly takes place in the late fetal and newborn stage, and dramatically declines after puberty. In other words, the thymic capacity to promote T cell differentiation starts to change in the early stage of life in terms of quantity and quality of T cells. Thus, the composition of T cell-subsets in the periphery gradually changes with age, resulting in the alteration of T cell functions in the elderly. The restoration of immunological functions of the aged individuals is possible and might be beneficial for them to cope with various diseases associated with aging. Physiological thymic atrophy is controlled by both extrathymic and intrathymic factors, and is not a totally irreversible process. The process of thymic atrophy might be explained by further understanding of the relationship between the neuroendocrine and the immune systems.


Effects of alkyl alcohols and related chemicals on rat liver structure and function. III. Physiochemical properties of ethanol-, propanol- and butanol- treated rat liver mitochondrial membranes. The physicochemical properties of mitochondria in liver tissue obtained from rats given 32% ethanol, 32% propanol or 6.9% butanol in drinking water for up to 3 months were investigated using differential scanning calorimetry and fluorescence polarization measurements. The results obtained were as follows: 1) Phospholipids extracted from mitochondria showed increases in the relative amounts of phosphatidylcholine, phosphatidylinositol and phosphatidylserine, and a decrease in the relative amount of phosphatidylethanolamine. An increase in the unsaturated/saturated fatty acid ratio of phospholipids was also observed. 2) Elevation of the thermotropic lipid phase transition temperature with a decrease in the enthalpy value (delta H) was revealed by differential scanning calorimetry. 3) The elevation of the lipid phase transition temperature was detected also by fluorescence polarization measurements using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Elevation of mitochondrial membrane fluidity was found in some of the experimental animals, but most showed no changes in comparison with the control. A possible role of membrane fusion in the mechanism of formation of ethanol-, propanol- and butanol-induced hepatic megamitochondria is discussed on the basis of these results.


Immunohistochemical localization of gangliosides in ENU-induced rat glioma. Biochemical studies have indicated that the structurally simple gangliosides, including GD3 and GM3, are major glycolipid components of glioma tissues. In order to clarify the localization of the gangliosides in ethylnitrosourea-induced rat glioma, an immunohistochemical study was performed using antibodies against GM1, GM3, and GD3. The results obtained in normal fetus, newborn, and adult rat brain, and also in human glioma, were compared. In fetal and newborn rat brain, GD3 was present mainly in the neuroepithelial cell surface of the matrix and subependymal layers of the ventricular wall, but GM3 and GM1 were not detected. In adult rat brain, GD3-positive cells were absent, or present in diminished number, and GM1 was found chiefly in the neuropil of the cerebral cortex. Most of the rat glioma cells were positive for GD3, but not for GM1. It was demonstrated that the ganglioside composition of glioma cells was similar to that of immature neuroectodermal cells in fetal and newborn rat brain. Furthermore, the number of GD3-positive oligodendroglioma cells increased with tumor growth. In anaplastic gliomas and gross oligodendrogliomas, most tumor cells expressed not only GD3 but also GM3. These results suggest that GD3 is a marker of proliferating neuroectodermal cells, and that activity of the key enzymes in ganglioside synthesis alters with tumor growth and anaplastic change.


Incidence of Paneth cells in minute tubular adenomas and adenocarcinomas of the large bowel. This study attempted to demonstrate the incidence of Paneth cells within large bowel tubular adenoma and adenocarcinoma according to location and macroscopic appearance using minute tumors (up to 5 mm in size). We have shown that Paneth cells were sometimes seen in the early stage of the development of large bowel epithelial neoplasia. According to the macroscopic appearance (elevated or depressed type), in large bowel epithelial neoplasia, there was a statistical difference between the depressed type (32.5%, 14 of 40 cases) and the elevated type (16.6%, 24 of 145 cases) (Chi square analysis, p < 0.05) in the incidence of Paneth cells. Paneth cells were seen more frequently in adenocarcinoma (45.8%, 11 of 24 cases) than in tubular adenoma (16.8%, 27 of 161 cases), with a significant statistical difference (Chi square analysis, p < 0.01). According to location, in both tubular adenoma and adenocarcinoma, Paneth cells were more frequently observed in the proximal colon (tubular adenoma: p < 0.01, adenocarcinoma: p < 0.05, Chi square analysis).


Three human osteosarcoma cell lines exhibiting different phenotypic expressions. to elucidate the differences in histological features and biological behavior of osteosarcomas, three human osteoblastic-type osteosarcomas were studied in vitro and in nude mice. The secretory processing and extracellular fiber formation of type I collagen proved to be the most important factor in bone formation in the osteosarcomas. Alkaline phosphatase also seemed to be important. However, we were unable to find any particular protooncogene abnormalities which could have been implicated in the occurrence or biological behavior of these osteosarcomas.


An autopsy case of Kawasaki disease with reference to occurrence of acute coronary thrombosis in the convalescent stage. A one-year, four month-old boy who had suffered from Kawasaki disease died suddenly during convalescence despite intensive gamma-globulin treatment. Autopsy revealed a) sausage-like aneurysms of the left and right coronary arteries and fresh thrombosis in the right coronary aneurysm, b) fresh transmural myocardial necrosis in the whole wall of the left ventricle and the anterior part of the wall of the right ventricle, and c) swelling of the cervical lymph nodes and thymus (60 g). Histologically, fibrocellular thickening of the intima and destruction of the media and internal elastic lamina were conspicuous in the area of the aneurysm, but those of the intima and media in the areas adjacent to the aneurysm were mild. Abrupt narrowing of the lumen at the border between the aneurysm and periphery of the right coronary artery was detected, and this may have been responsible for formation of the thrombus in the right coronary aneurysm. In the systemic arteries, perivascular fibrosis was very noticeable despite less severe injury to the intima and media. These findings suggest that severe inflammation of the periarterial regions was present in the acute phase. The lymph system still showed inflammation, supporting the infectious or toxic nature of Kawasaki disease.


Carcinoid tumor of the middle ear containing serotonin and multiple peptide hormones. A case report and review of the pathology literature. A 69-year-old man complaining of longstanding hearing loss and mild otorrhea was found to have a mass obliterating the external auditory canal and polypous tympanic mucosa with accompanying absence of the tympanic membrane and ossicular chain. Tumors excised from the external auditory canal and tympanum showed histologic features essentially characteristic of a carcinoid tumor: a ribbon or festoon arrangement of tumor cells, formation of anastomosing cords and glandular spaces, presence of numerous argyrophilic as well as argentaffin secretory granules within many of the tumor cells, and ultrastructural evidence of neurosecretory granules in the tumor cell cytoplasm. Immunohistochemically, the tumor was found to contain not only neuronal marker substances such as neuron-specific enolase, S-100 protein and chromogranin A, but also serotonin and multiple peptide hormones such as pancreatic polypeptide, glucagon, cholecystokinin and leucine-enkephalin. A review of the pathology of 17 previous cases of carcinoid of the middle ear suggested that this type of carcinoid may have a variegated hormone profile among carcinoids of foregut origin, and hormonally may resemble ileal carcinoid arising from the midgut, although their histogenetic origins may differ, because of frequent production of serotonin.


The regulatory domain of fungal and plant plasma membrane H(+)-ATPase. The activity of fungal and plant plasma membrane H(+)-ATPases seems to be regulated by modulation of the interaction of an inhibitory domain at the C-terminus with the active site. In the yeast ATPase, a mutation at the active site (Ala547- > Val) and a deletion of the C-terminus result in constitutive activation. A double Ser911- > Ala, Thr912- > Ala mutation at the C-terminus (defining putative phosphorylation sites) locks the enzyme in the inhibited state and can be suppressed by the Ala547- > Val mutation at the active site. This provides genetic evidence for domain interaction. In plant ATPase, proteolytic removal of the C-terminus also results in constitutive activation. A peptide covering a region of the plant C-terminus with homology to the yeast C-terminus inhibits the truncated plant ATPase. This suggests similar regulatory mechanisms in fungal and plant ATPases.


Site-directed mutagenesis of the Ca2+ ATPase of sarcoplasmic reticulum. We have cloned cDNA encoding the Ca2+ ATPase from fast-twitch skeletal muscle and, on the basis of its amino acid sequence and immunological studies of its topology, have made deductions concerning its secondary structure and active sites. These deductions have led us to test models for Ca2+ transport through expression of the protein in functional form in COS-1 cells, mutagenesis, and measurement of altered function. Mutation of about 250 of the 1000 amino acids making up the Ca2+ pump has indicated that the sites of high affinity Ca2+ binding are located in the center of the transmembrane domain and are made up from residues located in transmembrane sequences M4, M5, M6 and M8. The ATP binding site appears to be located in the headpiece and is made up from a series of loop sequences connecting alternating alpha helices and beta strands. Sites of conformational interaction appear in all domains throughout the Ca2+ pump. In our present model, Ca2+ transport occurs through binding to high affinity sites accessible to the cytoplasm in the E1 conformation, followed by release to the lumen from low affinity sites which form during the ATP-induced transition of the protein from the E1 to the E2 conformation.


Structural basis for the E1/E1P-E2/E2P conformation changes in the sarcoplasmic reticulum Ca(2+)-ATPase studied by site-specific mutagenesis. Site-specific mutagenesis and functional expression of cloned cDNA of the sarcoplasmic reticulum Ca(2+)-ATPase have been used to demonstrate that exchange of single amino acid residues in the Ca(2+)-ATPase can lead to forms with either "E1/E1P" or "E2/E2P" characteristics. The localization of the mutated residues identifies the M4S4 segment of the ATPase molecule as central to the energy-tranducing conformation change, and on the basis of this information a model for the pump mechanism is discussed.


Genetic probing of the yeast plasma membrane H(+)-ATPase. The H(+)-ATPase from Saccharomyces cerevisiae has been probed by a random genetic approach that has led to the isolation of primary and secondary site mutations. These H(+)-ATPase (PMA1) mutants help define specific functional, as well as interacting, regions of the H(+)-ATPase. Cellular resistance to hygromycin B has been an important selection tool for the isolation of pmal mutants. One prominent hygromycin B-resistant mutant, pmal-105, was found to have a S368F mutation near the site of phosphorylation (D378) in the catalytic core. This mutation prevents growth in low pH or NH(4+)-containing medium and induces an acid-sensitive Vmax for ATP hydrolysis, as well as a pronounced insensitivity to vanadate. The prominent cellular and biochemical phenotypes of this strain facilitated a detailed revertant analysis to identify protein structure domains that interact directly or indirectly with the localized region defined by the F368 mutation. Partial revertants were isolated which were resistant to low pH or NH4+ but retained hygromycin resistance. Second site mutations were found within the first and second cytoplasmic loop domains, as well as in transmembrane segments 1-3 & 7. All of the revertant enzymes have a stable Vmax but some show changes in the pH optimum for ATP hydrolysis; all display vanadate sensitivities ranging between the insensitive F368 mutant and the fully-sensitive wild type enzyme. Revertant analyses have also been performed on two other pma1 mutants which carry the mutations A135V and G158D in transmembrane segments 1 and 2, respectively. Compensating second site mutations to these mutations were identified in transmembrane segments 1, 2, 4 & 7, as well as within the central catalytic domain. These analyses have helped identify interacting protein structure domains that may participate in coupling ATP hydrolysis to proton transport. Furthermore, they facilitate the construction of structural models to account for these interactions.


Kdp, a bacterial P-type ATPase whose expression and activity are regulated by turgor pressure. The Kdp ATPase is a P-type ATPase consisting of three large protein subunits in a complex that probably contains 2 copies of each subunit. A small hydrophobic peptide, encoded in the same operon as the large subunits, may also participate. Kdp has very high affinity for K+ and serves to scavenge this ion when its concentration is low. Kdp responds to turgor pressure at two levels, at the level of activity and of expression. Kdp mediates net uptake when turgor is low, but mediates exchange without net change when turgor is normal. Kdp is expressed only when turgor is low. This control is mediated by a pair of regulatory proteins, members of the class of 'sensor-effector' regulators widely distributed in bacteria. It is suggested that low turgor changes the conformation of the KdpD 'sensor' protein, activating its presumed kinase activity to phosphorylate KdpE, the 'effector' protein, and phospho-KdpE in turn turns on expression of the operon that encodes the Kdp complex.


The Na+/glucose cotransporter (SGLT1).

The Na+/glucose cotransporter (SGLT1). An important class of Na+ transport proteins is the cotransporters. They exist in bacteria and animal cells and are responsible for the "active" accumulation of sugars, amino acids, carboxylic acids and some ions, e.g., I-, Cl-, and PO-4, in cells. In the small intestine and renal proximal tubule the cotransporters play an important role in the transport of salt and water across the epithelia. The most well known and best characterized Na+ cotransporter is the intestinal brush border Na+/glucose cotransporter. We have cloned, sequenced, and expressed both the rabbit and human Na+/glucose cotransporters. The cDNAs code for 73kDa proteins with 662-664 residues (86% identity). Secondary structure analysis suggests a 12 membrane-spanning helical model with the N- and C-termini in the cytoplasm. A single N-linked glycosylation site is utilized at Asn248. These sugars are not required for function. Two essential residues for functional expression in oocytes have been identified, Asp28 and Arg300. In two sisters with glucose-galactose malabsorption the transport defect is caused by a missense mutation changing Asp28 to Asn28, and we have found that changing Arg300 to Cys300 eliminated transport. Current research is directed to finding residues and domains essential for ligand binding and transport, and we are using electrophysiological techniques to correlate structure and function.


Regulation of the (Ca(2+)+Mg2+)-ATPase of basolateral membranes from kidney proximal tubules by kinase-mediated phosphorylation and by insulin. Membrane vesicles derived from the basolateral aspect of kidney proximal tubule cells are phosphorylated by ATP in the absence of Ca2+. This Mg(2+)-dependent, hydroxylamine-resistant phosphorylation was associated with a 50% inhibition of the (Ca(2+)+Mg2+)-ATPase activity measured upon addition of micromolar Ca2+ concentrations, enough to saturate the high-affinity sites of the Ca2+ pump. The presence of either the protein kinase inhibitor H7 or insulin during phosphorylation virtually eliminated the inhibitory effect associated with phosphorylation. However, insulin itself inhibited ATP hydrolysis by the (Ca(2+)+Mg2+)-ATPase when it was present in the assay medium containing buffer, ATP, Mg2+ and Ca2+, the hydrolytic activity being initiated by addition of the membranes without prior phosphorylation. These results suggest that insulin may play a role in regulating transepithelial Ca2+ transport in renal proximal tubules, and that its effects may be linked with a kinase-mediated process that depends on the functional state of the (Ca(2+)+Mg2+)-ATPase.


Numerical simulation of uni-site and bi-site ATP-hydrolysis catalyzed by the membrane-bound H(+)-ATPase from chloroplasts. The kinetics of ATP-hydrolysis catalyzed by the H(+)-ATPase from chloroplasts was described by an enzyme kinetic model where either one (uni-site catalysis) or two (bi-site catalysis) sites are operating. Numerical simulations are carried out with the software package LARKIN using measured rate constants as a basis. When the ATP-concentration is increased up to 20 nM (at 20 nM enzyme concentration) a change from uni-site to bi-site catalysis occurs at about 7 nM. The measured effective rate constants change by a factor two. However, the calculated rate constants for the second site are drastically increased.


Reaction cycle and thermodynamics in bacteriorhodopsin. Light causes the all-trans to 13-cis isomerization of the retinal in bacteriorhodopsin; the thermal relaxation leading back to the initial state drives proton transport first via proton transfer between the retinal Schiff base and D85 and then between the Schiff base and D96. The reaction sequence and thermodynamics of this photocycle are described by measuring time-resolved absorption changes with a gated multichannel analyzer between 100 ns and 100 ms, at six temperatures between 5 degrees C and 30 degrees C. Analysis of the energetics of the chromophore reaction sequence is on the basis of a recently proposed model (Váró & Lanyi, Biochemistry 30, 5016-5022, 1991) which consists of a single cycle and many reversible reactions: BR -hv-->K<==>L<==>M1-->M2<==>N<==>O-->BR. The existence of the M1-->M2 reaction, which functions as the switch in the proton transfer, is confirmed by spectroscopic evidence. The calculated thermodynamic parameters indicate that the exchange of free energy between the protein and the protons is at the switch step. Further, a large entropy decrease at this reaction suggests a protein conformation change which will conserve delta G for driving the completion of the reaction cycle. The results provide insights to mechanism and energy coupling in this system, with possible relevance to the general question of how ion pumps function.


Characterization of proton transport in bone-derived membrane vesicles. ATP-dependent proton transport was characterized in membrane vesicles, prepared by differential centrifugation from medullary bone of egg laying hens, a source rich in osteoclasts. The H(+)-ATPase present in this preparation showed the characteristics of a vacuolar H(+)-ATPase in its sensitivity to inhibitors, including bafilomycin A1, its sensitivity to cold treatment and its electrogenic mechanism. There was no evidence for a direct activation of the H(+)-ATPase by anions, including Cl-. These results are consistent with the view that the osteoclast, the cell responsible for bone resorption, secrets acid by means of a vacuolar H(+)-ATPase.


A novel type haem group of cytochrome o from Escherichia coli. The b-type haem groups of cytochrome o have generally been thought to be protohaems. However, we have recently shown that they are of a novel kind, for which we propose the name haem O. On the basis of its pyridine haemochrome spectrum, chromatographic behaviour and molecular mass of 839 Da, we suggest that haem O is a haem A-like molecule with the formyl group in position 8 of the latter replaced by methyl, but with retention of the 17-carbon hydroxyethyl-farnesyl side chain in position 2, characteristic of haem A. This structure has now been unambiguously verified by resonance Raman, 1H-NMR, and infrared spectroscopy, as well as by mass spectroscopy of molecular fragments, in collaboration with C. K. Chang, G. T. Babcock and their co-workers at Michigan State University. Properties of haem O are compared with haem A and protohaem, and possible contributions of haem O structure to the cytochrome o oxidase function are discussed.


Expression of subunit II of chloroplast H(+)-ATPase in an Escherichia coli mutant lacking subunit b of its H(+)-ATPase. The DNA of subunit II of the H(+)-ATPase from spinach chloroplast was expressed in Escherichia coli. It was found that a high gene dose is lethal to E. coli. With a lower gene dose subunit II was not able to substitute for the homologous subunit b in the E. coli ATP synthase.


Mutational analysis of the role of Lys684 in the Ca(2+)-ATPase of sarcoplasmic reticulum. Site-specific mutagenesis of the sarcoplasmic reticulum Ca(2+)-ATPase was used to investigate the functional role of Lys684 located in the "hinge-domain", a highly conserved region of the cation-transporting ATPases. Mutation of Lys684 to Arg, Ala, His and Gln resulted in complete loss of calcium transport function and ATPase activity. For the Lys684- > Ala, His, Gln mutants, this coincided with a loss of the ability to form a phosphorylated intermediate from ATP or Pi, whereas the Lys684- > Arg mutant retained the ability to phosphorylate from ATP with normal apparent affinity, demonstrating the importance of the positive charge. On the other hand, no phosphorylation was observed with Pi as substrate in this mutant. Examination of the partial reactions following phosphorylation from ATP in the Lys684- > Arg mutant demonstrated a reduction of the rate of transformation of the ADP-sensitive phosphoenzyme intermediate (E1P) to the ADP-insensitive phosphoenzyme intermediate (E2P), which could account for the loss of transport function. Once accumulated, the E2P intermediate was able to decompose rapidly in the presence of K+ at neutral pH. In the Lys684- > Ala mutant, nucleotides were found to protect with normal affinity against intramolecular cross-linking induced with glutaraldehyde, indicating that the nucleotide binding site was intact. These data point to a role of Lys684 in the binding and transfer of phosphate to the protein, and in the transport-associated conformational changes of the phosphorylation site.


An atomic model for the structure of bacteriorhodopsin, a seven-helix membrane protein. A three-dimensional map of bacteriorhodopsin has been obtained, at near-atomic resolution, by collecting and analysing electron diffraction patterns and electron micrographs from crystals of bacteriorhodopsin preserved at very low temperatures. The map shows a resolution of 3.5 degrees in a direction parallel to the plane of the membrane, but poorer resolution perpendicular. It shows many features well resolved from the main density of the seven alpha-helices, which we interpret as the bulky sidechains of tyrosine, phenylalanine and tryptophan, as well as a very dense feature, which is the beta-ionone ring of the retinal chromophore. Using these bulky side chains as starting points and taking account of bulges of density for the smaller side chains such as leucine, we built an atomic model for the residues between 8 and 225. There are 21 amino acids from all 7 helices surrounding the retinal and 26 amino acids contributed by 5 helices that form the proton channel. Ten of the amino acids in the middle of the proton channel are also part of the retinal-binding site. The model provides a useful basis for considering the mechanism of proton pumping and in the interpretation of other experimental data. In particular, the model suggests that the pK changes in the Schiff base must act as the means by which light energy is converted to proton pumping through the channel. Asp-96 is on the pathway from the cytoplasm to the Schiff base and asp-85 on the pathway from the Schiff base to the extracellular surface. The experimental map and the building of the model of the structure will be described, as well as our interpretation of the structural basis of the mechanism.


Purification of the H(+)-ATPase from chloroplasts. Triton X-100* was replaced in the purification of the H(+)-ATPase from chloroplasts by either octyl glucoside, octyl thio glucoside or the zwitterionic detergent CHAPS. The results indicate that the enzyme can be purified without Triton X-100 and retain full activity.


Structure and function of the Na(+)-translocating ATPase of Propionigenium modestum. The strict anaerobic bacterium Propionigenium modestum performs a Na+ cycle over the membrane to couple ATP synthesis to the decarboxylation of methyl-malonyl-CoA. The responsible ATPase has a typical F1F0 structure, the water-soluble F1 moiety being composed of five different subunits and the more firmly membrane-bound F0 part consisting of three different subunits. The F1F0 ATPase but not F1 alone was specifically activated by Na+ ions, suggesting that the Na+ binding site is located on the F0 moiety. The ATPase reconstituted into proteoliposomes catalyzed an ATP-dependent Na+ accumulation that was stimulated to the same extent by dissipating the membrane potential with valinomycin or with the uncoupler carbonylcyanide-m-chloro phenylhydrazone. The transport of Na+ is therefore a primary event, not a secondary event involving the intermediate formation of a proton gradient. The ATPase also catalyzed H+ translocation at Na+ concentrations below 1 mM. Our results indicate a common mechanism of the ATPase for Na+ and H+ (H3O+) translocation and a switch from H+ to Na+ translocation by increasing the Na+ concentration. A hybrid ATPase consisting of F1 from E. coli and F0 from P. modestum had the same specificity with respect to the translocated cations as the homologous F1F0 ATPase of P. modestum, indicating again that the Na+ (H+) binding site is located on the F0 part. Also in accord with this supposition is a diffusion potential-induced translocation of Na+ or H+ through the F0 part of the enzyme complex. The phylogenetic relationship between the Na(+)-translocating ATPase of P. modestum and other F1F0 ATPases has been clearly demonstrated by sequencing studies.