[Heat shock modulates the ability of A-549 cell line from human lung adenocarcinoma to regulate the intensity of DNA and protein biosynthesis by an autocrine mechanism].

A-549 cells of human lung adenocarcinoma were subjected to heat shock (30 min, 44 degrees C) which caused substantial decreases in the rates of biosynthesis of the great bulk of cellular proteins with simultaneous increases in the synthesis rates of the 70 kDa protein predominantly localized in cell cytosol. By the 6th hour after the heat shock cessation this protein synthesis reached its maximum; by the 18th hour it was no longer detectable, while the protein itself was not denatured. During the recovery after the heat shock the ability of the serum-free culture medium conditioned by A-549 cells in autocrine regulation of [3H]thymidine incorporation into DNA and [3H]leucine incorporation into proteins changed also. The conditioned medium obtained within 1-3 hours after the heat shock did not influence the intensity of DNA synthesis, while the medium obtained 4-48 hours after the heat shock stimulated this process, the maximal effect (3.3-fold stimulation) being observed in the case of the 48-hour conditioned medium. Temporary (1 hour) acidification of the conditioned media down to pH 2.0 resulted in complete inhibition of the stimulating activity. Besides, these media acquired an ability to inhibit [3H]thymidine incorporation into the DNA of tracer cells. Study of effects of conditioned media on the rate of [3H]leucine incorporation into A-549 cell proteins revealed that the media obtained 1-4 hours after the heat shock inhibited this process, while the media obtained 6-18 hours thereafter stimulated it 1.2-2.1-fold. In the test systems under study temporary acidification of the media increased their stimulating influence on [3H]leucine incorporation into cellular proteins.


[Blood cobalamins and xenobiotic-metabolizing enzymes in rat liver in adjuvant arthritis].

Changes in the total cobalamin content and spectrum of individual forms of these vitamins in blood cells and plasma as well as the activities of enzymatic systems of xenobiotic metabolism in liver microsomes of rats with experimental adjuvant arthritis (AA) have been studied. The total cobalamin content in the blood plasma of rats with AA was increased in comparison with intact animals; however, leucocytes from AA rats were deficient in methylcobalamin (MeCbl). A correlation was found between the ratios of individual cobalamin forms and their total content which was differently expressed in experimental and control animals. The development of AA was associated with marked inhibition of the cytochrome P-450-dependent monooxygenase system of the liver and glutathione transferase. The possibility of correction of these disturbances by MeCbl is discussed.


[The effect of substrates on stability of firefly luciferase, embedded in phosphatidylcholine liposomes].

The effects of substrates (ATP and luciferin) on stability of firefly luciferase embedded into phosphatidylcholine liposomes have been studied. Luciferin did not exert any appreciable influence on enzyme inactivation. Minor concentrations of adenosine 5'-triphosphate destabilized the enzyme; however, the increase in ATP concentration markedly stabilized the enzyme entrapped into liposomes. A kinetic scheme of ATP action on enzyme inactivation is proposed. According to this scheme, the enzyme has two ATP-binding sites.


[Purification and physico-chemical properties of soluble carboxypeptidase N from cat brain gray matter].

Using affinity chromatography on diasorb-L-arginine and polyacrylamide gel electrophoresis, soluble carboxypeptidase H (E. C. 3.4.17.10) has been isolated from cat brain cortex and purified 598-fold with a 16% yield. The enzyme has a molecular mass of 50 kDa, consists of one polypeptide chain, and displays the maximum activity at pH 5.6. Carboxypeptidase H is a thiol-dependent metalloenzyme and contains a Zn2+ ion in its active center. The Km and V values for dansyl-Phe-Leu-Arg are 100 +/- 5 microM and 12.5 +/- 1.4 microM/min/mg of protein, respectively. The existence of two forms of soluble carboxypeptidase differing in isoelectric points and pH optima has been demonstrated. The enzyme with a pI of 4.8 has a pH optimum at 5.5-5.6, while that with a pI of 5.25-at 6.0.


[Rabbit annexins: comparison of a series of proteins in cell-free homogenates and membranes from various tissues and a method of rapid preliminary identification of individual components after two-dimensional electrophoresis].

The annexin sets in cell-free homogenates and membranes of rabbit skeletal and heart muscles, liver, kidney, lung, and brain, have been compared by one- and two-dimensional electrophoresis. The pIs and M(r)s of the proteins identified have been determined. The data on two-dimensional electrophoresis of annexins from different animals have been systematized. Simple graphs are proposed which allow to identify annexins on electrophoregrams. The technique has definite potentialities in recognition of some unidentified Ca(2+)-dependent membrane-binding proteins and may be used to predict the trend of search for novel members of the annexin family.


[Utilization of oxygen radicals by synthetic proline-rich oligopeptides].

Inhibition of superoxide by two synthetic proline-rich hexapeptides simulating the hinge region of the IgG molecule has been studied. The CPPPEL (P-Cys) peptide was active in utilizing superoxide (O2.-), while the APPPEL (P-Ala) peptide had no such activity. Spontaneous formation of clusters with six and/or eight monomers was shown for P-Cys, but not P-Ala. Preincubation of mixed P-Cys and P-Ala resulted in the appearance of a product with a high affinity for O2.-. Incubation of the mixture at 56 degrees C led to inactivation. Preincubation of P-Cys with ZnCl2 reversed the main pathway of O2.- utilization by P-Cys. The relationship of the P-Cys activity to its clusterization is discussed.


[Inhibiting effect of ADP-ribosylation by pertussis toxin on passive transport of Ca2+ into the cell membrane of porcine myometrium].

ADP-ribosylation by whooping cough toxin of protein components of inside-out oriented vesicles of pig myometrium plasma membranes under conditions of their depolarization results in significant inhibition of passive transport of Ca2+ ions. The inhibiting effect is dose- and time-dependent. rho-Chloromercuribenzoate (0.5 mM) blocks the effect of whooping cough toxin, no such effect on Ca2+ transport being observed in control preparations.


Applications of DAPI cytochemistry to neurobiology.

4',6-Diamidino-2-phenylindole hydrochloride (DAPI) is a fluorescent dye with high affinity for DNA. We have employed it as a fluorescent chromatin counterstain on sections immunofluorescent-stained using rhodamine and on tissues enzymatically stained using beta-galactosidase. DAPI also allows easy identification of mitotic figures and can be used to supplement cytochemical studies involving cell division in the nervous system.


Activation of mouse oocytes, fertilization and development of mouse embryos in vitro after staining with trypan blue or fluorescein diacetate.

The influence of vital staining with trypan blue or fluorescein diacetate on the fertilization of mouse oocytes and the developmental potential of mouse embryos was assessed. Neither stain induced spontaneous activation in mouse oocytes, nor did they impair the in vitro development and implantation of mouse zygotes, two-cell embryos, stressed morulae or blastocysts. However, fertilization and subsequent development of mouse oocytes have been shown to be reduced by vital staining.


Rapid assessment of maturation stage and reproductive mode in centrolecytic eggs of stick insects (Phasmatodea) using DAPI stain.

A rapid three-step DAPI technique is proposed for detecting meiotic stages and sperm head evolution in yolky, fertilized stick insect eggs, which were difficult to analyze with other methods. Fixed eggs were freed from chorionic envelopes and stained directly in DAPI/PBS solution. After rinsing, eggs were singly squashed in a drop of mounting buffer and examined under a microscope with incident fluorescent illumination. The method was almost uniformly successful, and direct observation of nuclear structures, coupled with fluorometry, allowed easy recognition of bivalents, diads, pronuclei and their DNA content. The DAPI method proposed here appears particularly helpful for investigating unusual reproductive modes in eggs with large amounts of yolk.


Light microscopic lectin histochemistry on celloidin stabilized cryostat sections of rat colon.

Celloidin stabilized pre-epithelial mucus gel (PMG) is stained in cryostat sections of the rat colon for light microscopic lectin histochemistry. Specific sugar residues of the mucins are demonstrated both in the PMG and in the mucin-containing cells of the mucosa.


A double-embedding technique for thin tissue membranes.

A new double-embedding technique for thin tissue membranes is presented. This technique is useful for thin membranes such as mesenteric membranes from rodents, which usually measure only 10 microns in thickness. Several membranes are fixed and mounted on four needles located at the bottom of a plastic box. The box is filled with agarose at 50 C and then allowed to solidify. The agarose block is then removed, dehydrated in alcohol, cleared with HistoPetrol (isoparaffin hydrocarbons), permeated with paraffin and sectioned. The morphology is comparable to that obtained with methacrylate plastic embedding but is less time-consuming, less hazardous since no plastic hardener and activator are used and makes immunohistochemical studies easier.


A comparative study of avidin-biotin-peroxidase complexes for the immunohistochemical detection of antigens in neural tissue.

It has been suggested that the use of avidin-biotin immunohistochemical techniques for antigen detection in neural tissue produces nonspecific background staining. For this reason neural tissue was used to test the quality, sensitivity and specificity of four commercially available antibody detection kits which use avidin or streptavidin binding to biotin. Free-floating, thick-section immunohistochemistry on perfusion fixed rat central nervous system revealed variability among staining kits for all parameters analyzed under the same experimental conditions. The reagents from the Vector 'Elite' kit were the most sensitive and specific, and received the highest overall rating for quality. Most commercial products tested could be used at greater dilutions than those recommended by the manufacturers without compromising specific staining. No staining was evident when the primary and secondary antibodies were omitted. This suggests that nonspecific binding is unlikely to be due to endogenous ligands, charge or hydrophilic reactions between these tertiary complexes and the tissue sections.


Lead tetraacetate-thiocarbohydrazide-silver proteinate method for light microscopy of polysaccharides.

Lead tetraacetate-thiocarbohydrazide-silver proteinate reaction sequence for light microscopy of polysaccharides was evaluated on Carnoy's fixed rat liver sections. The results of this evaluation suggest that, on the light microscopic level, the lead tetraacetate-thiocarbohydrazide-silver proteinate method may serve as a practical and histochemically specific alternative to the lead tetraacetate-Schiff reaction for the localization of tissue carbohydrates.


A method for embedding thin membranes in historesin.

A method is described for flat-embedding thin membranous tissues in Historesin. It allows easy orientation for sectioning large areas parallel to the surface. Selected fields can be monitored from the unfixed specimen, throughout preparation, to mounting on the microscope slide. For cross-sectioning, the flat-embedded tissue can be stacked and re-embedded to increase the amount of material examined per section.


Familial defective apolipoprotein B-100 in 12 subjects and their kindred.

Twelve unrelated subjects with heterozygous familial defective apolipoprotein B-100 were identified in a group of 252 patients with type IIa hypercholesterolaemia. Approximately 5% of hypercholesterolaemia can be explained by this mutation in the collective studied. Familial defective apolipoprotein B-100 is therefore the most common known mutation causing primary hypercholesterolaemia. Family studies revealed an additional 14 affected subjects. All family members with the mutation had elevated cholesterol concentrations. In a normolipidaemic control group of 146 subjects the mutation was not present. In the affected individuals a variable expression of total cholesterol concentrations and atherosclerosis was observed. Plasma cholesterol ranged from 6.60 to 14.89 mmol/l with a mean of 9.43 mmol/l. Premature atherosclerosis was present in 4 patients, while one affected woman is now 92 years old and has no symptoms of coronary heart disease or peripheral atherosclerosis. Analysis of the haplotypes and genotypes by 3 biallelic and 1 multi-allelic DNA marker suggests that the disorder is caused in all affected patients by the same rare allele. The fact that the same mutant allele was also identified in other European populations and in a North American population of Caucasian origin argues for a common European origin of this mutation.


Alteration of the permeability of the human erythrocyte membrane to cations by liposome-incorporated amphotericin B.

Some effects of liposome-incorporated amphotericin B on the permeability of the human erythrocyte membrane to potassium and sodium ions is reported. The influence of cholesterol and amphotericin B in causing a shift towards smaller and larger liposomes, respectively, is also described. Phosphatidylcholine liposomes containing amphotericin B in a molar ratio of 7.4 +/- 0.1 (mean +/- SD) antibiotic to 1000 phospholipid reduced the initial rates of K+ and Na+ transport across the erythrocyte membrane to 40 +/- 2.6% and 0%, respectively, of their rates in the presence of comparable concentrations of free amphotericin B. Amphotericin B incorporated into liposomes (8.2 +/- 0.15 mumol antibiotic per 1000 mumol total lipid) composed of cholesterol and phosphatidylcholine (in a molar ratio of 3:7) reduced the initial rate of K+ transport to 19 +/- 0.8% of its value measured in the presence of a comparable concentration of free antibiotic. These results suggest that liposomes containing specified amounts of amphotericin B, especially liposomes also containing cholesterol in addition to phosphatidylcholine, could be used as a method of controlling K+ transport across the erythrocyte and possibly other types of cellular membranes, thereby limiting antibiotic toxicity to some mammalian tissues.


Reference limits of plasma fibrinogen.

Fibrinogen is considered to be a strong predictor and independent factor of cardiovascular diseases. The data presented here describe the baseline measurements of fibrinogen in 1008 apparently healthy subjects, aged 4-60 years and their relationship to age, sex, body weight, smoking, alcohol, and use of oral contraceptives. Pearson's correlations and a linear multiple regression model were used. Plasma fibrinogen was measured kinetically in a photometer, the Behring Chromotimer, using the CTS-fibrinogen method. There were neither statistical difference between girls and boys aged 4-20 years nor correlation with variables related to cardiovascular diseases. In adults, we found an increase of plasma fibrinogen concentration with age and no statistical difference between men and women, except in subjects aged 40-50 years. There was a positive correlation between fibrinogen and ponderal index. In women aged 20-30, 30-40, 40-50 and 50-60 years, the mean fibrinogen concentrations increased of 0.009, 0.021, 0.010 and 0.015 g/l for one percent of overweight, in each subgroup respectively. In women aged 20-30 years using oral contraceptives, the mean fibrinogen concentration was 0.19 g/l higher than in women not using oral contraceptives. The smoking effect was observed only in 30-40 year-old men. Each cigarette smoked per day increases of the mean fibrinogen by 0.35 g/l after standardization for ponderal index and alcohol consumption. Alcohol consumption was negatively correlated to plasma fibrinogen in subjects 30-40 years old. In women, 1 g of alcohol per day induces a 0.008 g/l decrease in the mean fibrinogen while in men the decrease is 0.004 g/l.(ABSTRACT TRUNCATED AT 250 WORDS)


Mucin-like high molecular mass protein fractions from total pig gallbladder bile mucus, pig gallbladder wall mucus, and total human gallbladder bile mucus.

Native mucin-like complexes were obtained from both pig gallbladder bile and pig gallbladder wall mucus by precipitation, centrifugation, and gel permeation chromatography. Crude preparations by either dialysis (native mucus from bile, and native gallbladder wall mucus) or by precipitation (crude total bile mucus, and lipomucoid) were purified by gel permeation chromatography on Sephacryl S-300HR and Sephacryl S-500HR (Pharmacia). The elution profiles obtained with a reversibly denaturing and detergent-containing eluent showed the same pattern for all samples, although the amounts of the four main fractions differed somewhat. The excluded fraction with the highest carbohydrate portion had an apparent M(r) > 10(7). This fraction and the following included lipomucoid (in physiological solution tightly bound to fraction I), and an eluent-insoluble mucus portion from all samples were characterized by determination of the protein concentration, carbohydrates, sialic acids, and lipids, using standard methods. Sugar analysis was performed by gas-liquid chromatography. Human gallbladder bile was subjected to the same procedures of mucus precipitation and separation. Human gallbladder bile mucus showed identical behaviour to that of pig gallbladder bile mucus, and showed a very similar elution pattern in gel chromatography.


Concentrations of purine nucleotides and purine and pyrimidine bases in cerebrospinal fluid of neurologically healthy children.

The concentrations of the nucleotides AMP and IMP, the nucleosides adenosine, guanosine and inosine, the purine bases adenine, guanine, hypoxanthine and xanthine, urate, and the pyrimidine bases cytosine, thymine and uracil were determined by high performance liquid chromatography in the cerebrospinal fluid of 63 children aged between 1 month and 13 years who showed no sign of neurological disease. The results are compared with those of other authors, and used to establish reference ranges for the above metabolites in the cerebrospinal fluid of children.


Stability of pO2, pCO2 and pH in heparinized whole blood samples: influence of storage temperature with regard to leukocyte count and syringe material.

Influences of storage temperature and blood cell metabolism in different types of syringes were investigated. Experiments were performed on blood samples with normal and elevated leukocyte counts. After equilibration with gas mixtures at normal pO2 (86 mm Hg/11.5 kPa) and elevated pO2 (140 mm Hg/18.7 kPa), sequential blood gas analyses were done within one hour. Storage temperatures were 4 degrees C or 22 degrees C. In the first group of experiments we compared glass samplers with plastic syringes at different storage temperatures with regard to deviations of blood gas concentrations. The analysed samples had a normal cell count. Blood stored in glass syringes in ice water served as the reference, and it displayed virtually no changes. The deviations of pCO2 and pH were relatively small. In plastic syringes the greatest increases for pO2 occurred after storage at 4 degrees C, which can be explained by the increased solubility of oxygen and the higher O2 affinity of haemoglobin at 4 degrees C. When stored at room temperature, the deviations in plastic syringes were smaller. In a second group of experiments, the influence of cell metabolism was studied. Blood gases were analysed in samples with elevated leukocyte counts (20 x 10(9)/l, 40 x 10(9)/l, 60 x 10(9)/l), and only glass syringes were used. It was demonstrated that after storage at 22 degrees C considerable losses in pO2 occurred, while at 4 degrees C there was virtually no change. Deviations of pO2, pCO2 and pH are described in detail.(ABSTRACT TRUNCATED AT 250 WORDS)


Concentrations in serum of IgG, IgM and IgA and their age-dependence in beagle dogs as determined by a newly developed enzyme-linked-immuno-sorbent-assay (ELISA).

The concentrations of immunoglobulins IgG, IgM and IgA and their age dependence were determined in the serum of normal, untreated beagle dogs by a newly developed sandwich enzyme-linked-immuno-sorbent-assay (ELISA). A clear age-dependent increase between approximately 0.8 and 1.6 years of age was observed for the immunoglobulin IgA, whereas IgG and IgM showed only a slight tendency to an age-dependent increase. For immunotoxicological characterization of various compounds especially in long term studies, this IgA age-dependence has to be considered in the planning and interpretation of studies with beagle dogs.


Creatinine and automatic analysers in relation to icteric specimens.

A study is described on the interference of bilirubin with the determination of creatine in serum using three automatic analysers i.e. the Technicon Chem-1, the DuPont Dimension and the Baxter Paramax. In all instruments a kinetic Jaffé reaction is applied. As comparison method we used a HPLC based selected method. All methods proved to be sensitive to interference, the Paramax showing the best performance with our set of specimens. Results regarding modifications leading to improvement and possible future experiments are discussed.


Determination of human neutrophil elastase in bronchoalveolar lavage fluid: homogeneous immunoactivation versus heterogeneous enzyme immunoassay.

Elastase mass concentrations of bronchoalveolar lavage fluid were determined by a homogeneous immunoactivation and a heterogeneous enzyme immunoassay. There was an excellent correlation between both assay systems (y = 1.0376 . x + 1.311; r = 0.9901; n = 43) indicating the suitability of the immunoactivation method for the determination of elastase concentrations in bronchoalveolar lavage fluid as a matrix. Furthermore, dilution of bronchoalveolar lavage fluid samples did not influence the elastase recovery of either assay system.


The C1 inhibitor deficiency. A review.

C1 inhibitor (C1I), a member of the serine protease inhibitor superfamily, is the principal regulator of the activation classical pathway of complement by reducing the proteolytic activity of activated C1r and C1s. A deficiency of active C1 inhibitor is the most commonly identified genetic defect of the complement system. It is associated with a pathology called angioedema. There are three forms of hereditary angioedema. The first type is characterized by an insufficient production of a normal protein. The two other forms are characterized by the presence of an abnormal C1 inhibitor protein. Moreover a reduction of functional C1 inhibitor may also be acquired. There are two types of acquired angioedema, a form associated with malignancy (B cell lineage, breast cancer, ...) and an autoimmune form. Angioedema manifests itself by attacks of swelling of the extremities, face, trunk, airways, or abdominal viscera, occurring spontaneously or as a result of trauma. Three main categories of substances have been proposed for the treatment of C1 deficiencies: the androgens, the antifibrinolytics and fresh plasma or purified C1 inhibitor. To distinguish between the different forms of C1 inhibitor deficiencies, it is necessary to determine the amount of C1 inhibitor protein and the level of its functional activity. Several methods for the determination of C1 inhibitor have been proposed: titrimetric and spectrophotometric assays, inhibition of complement haemolytic activity, radioimmunoassay, enzyme-linked immunosorbent assay, ...), in order to improve the diagnosis and the treatment of angioedema.


Significance of bone alkaline phosphatase, CA 15-3 and CEA in the detection of bone metastases during the follow-up of patients suffering from breast carcinoma.

After the introduction (1, 2) and methodical evaluation (3, 4) of a new method for the quantitative measurement of the bone isoenzyme of alkaline phosphatase (test-combination bone alkaline phosphatase, Boehringer Mannheim), we started a retrospective clinical study for the follow-up investigations of breast cancer patients. Our aim was to establish the significance of the routinely used tumour markers, CEA and CA 15-3, in combination with bone alkaline phosphatase for the early detection of metastatic spread to the bone. We investigated 492 sera from 92 patients suffering from breast carcinoma, and we compared each date of investigation with the results of the clinical examination and with the results of medical imaging, if that had been performed. From a previous study involving skeleton scintigraphy (5) we knew that single examinations do not allow a differential diagnosis between benign and malignant disorders of the bone, so we based our calculations on differences between sequential investigations. We found that in follow-up investigations of patients with breast carcinoma the combined determination of CEA, CA 15-3 and bone alkaline phosphatase may be indicative for the localisation of metastatic disease. The determination of the bone alkaline phosphatase is easy to handle with a short assay time and good reproducibility; it can therefore be recommended.


Calcium regulating hormones after oral and intravenous calcium administration.

The aim of this study was to determine the changes in serum calcium concentration and in the concentrations of calcium regulating hormones after a single oral or intravenous calcium administration. Standard dosages of calcium, as used in routine patient care, were employed. Intact parathyrin, calcitonin, calcitriol, calcidiol, total calcium, ionized calcium, total protein and phosphate were determined in 12 healthy young men before and up to 8 h after oral and intravenous administration of calcium. During a fortnight there were four study days with 1000 mg calcium orally (p.o.), 2000 mg orally, 180 mg calcium intravenous (i.v.) and a control day without calcium. During the study the men were on a low calcium diet. We observed a sharp increase in the calcium concentration after i.v. administration (15 min: total Ca: + 0.48 +/- 0.32 mmol/l; ionized Ca: + 0.25 +/- 0.15 mmol/l; p < 0.01). The concentration increase after the two oral loads was nearly identical. The maximal concentration of total calcium was reached after 120 min (1000 mg: + 0.1 +/- 0.04 mmol/l; p < 0.001; 2000 mg: + 0.12 +/- 0.04 mmol/l; p < 0.001). There was a significant increase in urinary calcium after all modes of calcium administration. Calcitonin increased significantly only after i.v. injection of calcium (+ 9.2 +/- 3.4 pmol/l; p < 0.001) while parathyrin decreased significantly after all modes of calcium administration (i.v.: 15 min: -1.9 +/- 0.88 pmol/l; p < 0.01; 1000 mg: 90 min: -0.78 +/- 0.75 pmol/l; p < 0.001; 2000 mg: 90 min: -1.02 +/- 0.57 pmol/l; p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Acute myocardial infarction size and myoglobin release into serum.

The kinetics of myoglobin release after acute myocardial infarction were studied. Various algorithms for calculation of infarct size, based on immunonephelometric determination of myoglobin and cumulative myoglobin release into the circulation were compared. The cumulative myoglobin release and maximal serum myoglobin concentration were compared with various measures of infarct size: cumulative release of creatine kinase, electrocardiographic changes, and left ventricular ejection fraction. After acute myocardial infarction, time to peak for myoglobin in serum was correlated with time to peak for creatine kinase (r = 0.645). On average, the myoglobin concentration peaked 8.8 h earlier than creatine kinase activity. The rate of elimination of myoglobin showed a large variation (0.041-0.628 h-1) and was not correlated with the elimination rate of creatine kinase. The elimination rate of myoglobin after acute myocardial infarction was shown to depend on the patient's age and infarct size. The elimination constant of myoglobin is preferably estimated on an individual basis in large and complicated infarctions. Cumulative myoglobin release correlated with algorithms based on the cumulative release of creatine kinase (r = 0.622) and its isoenzyme MB (r = 0.660), and to a lesser extent with the residual left ventricular ejection fraction (r = 0.513) and the sum of ST-segment deviations on electrocardiography (r = 0.469). Maximal myoglobin values in serum correlated moderately with the calculated infarct size (r = 0.488; based on creatine kinase-MB) and electrocardiographic changes (r = 0.554). In combination with fast immunological methods for myoglobin determination, myoglobin peak height offers the advantage of providing reliable results within 12 h after onset of symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)


Determination of serum calcitonin by immunometric two-site assays in normal subjects and patients with medullary thyroid carcinoma.

We evaluated the clinical usefulness of a commercial two-site immunoassay for calcitonin, which is based on two monoclonal antibodies against distinct epitopes of calcitonin. The potential lower detection limit of the assay was 2 ng/l. Inter-assay and intra-assay variation were both < 11%. No cross reaction with calcitonins from different species (salmon and eel calcitonin) was found. Of the various fragments of human calcitonin, only the 11-32 fragment could be detected. Basal calcitonin concentrations in normals (n = 69) ranged from < 2 ng/l to 50 ng/l. Basal calcitonin concentrations in 30% of the men (< 2-48 ng/l) and 51% of the women (< 2-10 ng/l) were undetectable. Stimulation of calcitonin with pentagastrin (0.5 microgram/kg body weight) in normal subjects was followed by an increase in calcitonin in all men (n = 17) and in 60% of the women (n = 12). The upper reference value for pentagastrin-stimulated calcitonin concentrations for all 29 normals was 78 ng/l (defined as the 95th percentile): 79 ng/l for men, and 50 ng/l for women. Using a different immunoradiometric two-site assay, a similar percentage of "positive" responses to pentagastrin in normals was obtained (20 out of 29). Four previously thyroidectomized patients with medullary thyroid carcinoma had normal basal, but pathological stimulated calcitonin values at follow-up. At reoperation, cervical lymph nodes with tumour tissue were removed. Postoperatively, the patients had basal and stimulated calcitonin concentrations within the limits of the established reference range. In conclusion, reference ranges for basal and pentagastrin-stimulated calcitonin concentrations have been established for an immunometric two-site assay.(ABSTRACT TRUNCATED AT 250 WORDS)


Serum lipids and apolipoprotein concentrations and plasma fibronectin concentrations in renal transplant patients.

In those cases where hypertriglyceridaemia was present before renal transplantation, it persisted after transplantation, and hypercholesterolaemia also developed. We studied serum lipid, lipoprotein, and apolipoprotein concentrations and plasma fibronectin concentrations in 57 renal transplantation patients and 29 healthy controls. We concluded that atherosclerosis in renal transplantation patients might be related to alterations in the constitutions of lipoproteins and apolipoproteins, but fibronectin synthesized by vascular endothelial cells seemed not to be associated with the atherosclerotic process.


Perspective: measurement of circulating glycated proteins to monitor intermediate-term changes in glycaemic control.

The effective clinical management of diabetes requires accurate means to measure and monitor prevailing blood glucose concentrations. This can be accomplished with regular home glucose monitoring and periodic measurement of indicators that reflect ambient glycaemia during defined temporal intervals. The amount of glycated haemoglobin provides an index of integrated long-term glycaemia, whereas the amount of glycated albumin provides an index of short to intermediate term integrated glycaemic control. However, there is confusion regarding the various methods for estimation of glycated serum or plasma proteins, in part because the different methods use different reaction principles, measure different substances, and express results in different units. This article reviews the principles underlying different assays for measurement of glycated albumin and glycated serum proteins, compares their ranges of values, and discusses their clinical relevance assessed by studies published in the recent medical literature. The methods evaluated include commercially available assays for measuring glycated albumin with monoclonal antibodies and with affinity chromatography (including HPLC), and the fructosamine assay for glycated serum proteins.


Validation of the ultrafiltration technique for creatinine analysis by HPLC: a comparison with direct serum injection.

The determination of creatinine by HPLC was performed by direct injection of serum onto the column, and after ultrafiltration of the sample, and the results were compared. A modified weak cation exchange column was used for HPLC. This eliminates the protein displacement effect and the Gibbs-Donnan effect, thus permitting a rapid direct analysis. Three of the four ultrafiltration methods gave almost identical analytical results. Regression analyses of the results from direct serum injection versus ultrafiltration showed a bias of 5% on the slope y = 1.050 . xpure serum + 1.856, which was in our view solely due to the volume displacement effect of the proteins; correction of each data point by a protein-dependent correction factor resulted in an almost perfect regression line.


Determination of human mast cell tryptase by bioluminescence-enhanced two-site immunometric assay.

A highly sensitive bioluminescence-enhanced two-site immunometric assay for human mast cell tryptase was developed. The usable range of the standard curve was from 10 ng to 10 micrograms per litre. The intra-assay coefficient of variation of the test was between 8.0 and 12.3%, and the inter-assay coefficient of variation between 8.3 and 13.5%. Recovery of human mast cell tryptase in human serum was 100% (fraction of serum in the test sample: 0.30).


The influence of pCO2 on the rate of ammonia formation in blood.

The influence of the sample pCO2 on the rate of ammonia formation was studied with gas equilibrated blood samples, using different gas mixtures for the equilibration. The rate of increase in plasma ammonia concentration at a mean pCO2 of 62 mm Hg = 8.2 kPa (mean pH = 7.282) was significantly lower than at 36 mm Hg = 4.8 kPa (pH = 7.438). In CO2-depleted blood (pH > 8) ammonia formation was strongly accelerated. This was reversible by readjusting the pH to 7.4 by addition of Tris-HCl solution. In stoppered containers with or without enclosed atmospheric air, a decrease of blood pCO2 or an increase of pH values was not observed during storage over 15 minutes at 0 or 20 degrees C. Although this study confirms that the pCO2 (or rather the pH) is an important analytical influence quantity in the determination of plasma ammonia, strictly anaerobic processing of the blood samples is not necessary; the usual technique of transporting and preprocessing blood samples in partially filled and stoppered containers appears to be adequate. Mainly due to the deamination of intracellular AMP (1), the ammonia1) concentration in blood increases continuously after sampling. Rates of increase in plasma ammonia concentration have recently been investigated thoroughly with blood samples from healthy probands to define the maximum delay between sampling and separation of the blood cells that can be tolerated if the in vivo existing plasma ammonia concentration has to be measured (2). Strictly speaking, the guidelines for handling the blood samples (2) apply to blood stored under anaerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunoassay of CEA, CA 19-9, CA 125, and CA 15-3 on the automated systems ES 300 and ES 600: methodological evaluation from a multicentre collaborative study.

The analytical performance of the automated Enzymun Test System ES 300 and ES 600 (developed by Boehringer Mannheim) for the assay of the tumour markers CEA, CA 19-9, CA 125, and CA 15-3, was assessed from data collected in a multicentre collaborative study in which eleven laboratories were involved. Results of the 1990 cycle of the external quality assessment (EQA) scheme for tumour markers, supported by the Italian National Research Council (CNR), were also used in this evaluation. The within-assay and between-assay precision was found to be 2.0 and 4.3 CV% for CEA, 2.9 and 6.8 CV% for CA 19-9, 3.6 and 9.4 CV% for CA 125, 2.9 and 6.0 CV% for CA 15-3. The between-lab variability of the four tumour markers on ES 300 and ES 600 systems was 9.4, 10.6, 11.9, 9.2 CV% for CEA, CA 19-9, CA 125 and CA 15-3 respectively. These values were comparable to or better than those obtained with the most precise manual kits used by laboratories participating in the 1990 EQA cycle. The agreement between the results from the Enzymun Test and those obtained using other method/kits was evaluated by assaying control samples previously circulated either in the CNR EQA or in the German EQA. The regression analysis indicates that for CEA, CA 125 and CA 15-3 assays the results produced by ES 300 and ES 600 are in good agreement with the consensus means of the EQAs; CA 19-9 results exhibit a worse correlation and are generally lower than the consensus mean. The linearity of the assays for the four tumour markers was checked by dilution tests performed by participants in the collaborative study; in all cases the dilution of the sample did not affect the values obtained.


Multicentre evaluation of the Boehringer Mannheim/Hitachi 747 analysis system.

Analytical performance and practicability of the new Boehringer Mannheim/Hitachi 747 analysis system were assessed in a multicentre evaluation involving four laboratories. The analytical performance was evaluated according to a protocol similar to the ECCLS guidelines and comprised 13 analytes including enzymes, substrates and electrolytes. About 65,000 results were obtained within three months. The evaluation was planned and supported by a program system called "Computer Aided Evaluation". Acceptance criteria have been established for judging the results. The median of the within-run coefficients of variation (CVs) in control sera of all methods was below 1%, being far below the acceptance limit of 2%. The median of CVs of between-days imprecision was below 2% (acceptance criterion 3%). The high degree of precision prompted us to set up a biometrical model suitable for the differentiation between deviant points, outliers and measurements that can still be explained by the system performance. No relevant drift effects were observed during eight hours. The methods were linear over a wide range, avoiding rerun analysis in most cases. No sample-related carry-over was found. Reagent-dependent carry-over outside the acceptance limits was measured from uric acid to phosphorus to a slight extent, and from triacylglycerols to lipase, as well as from total protein to bilirubin to a perceptible degree. It can be avoided by separating these reagent combinations in the channel arrangement. Taking a systematic deviation of more than 10% as unacceptable, four of the 13 analytes suffered from interference by haemoglobin, one by bilirubin and one by turbidity. The Boehringer Mannheim/Hitachi 747 analysis system is capable of determining serum indices which in combination with the interferogram allow an assessment of the interference. With the exception of chloride the recovery of the assigned values for all control sera showed values between 95 and 105%. Out of 40 method comparison studies for enzymes and substrates, 31 yielded regression equations with less than 5% proportional errors and less than 5% constant errors. Deviations exceeding these acceptance criteria can be explained by differences in the reagent formulation, in the method employed or in calibration. The agreement of the ISE method comparisons was within a +/- 5% deviation over a wide analytical range. Practicability of the Boehringer Mannheim/Hitachi 747 analysis system was assessed with the help of a questionnaire, in which properties of the instrument were quantified, thus permitting a relatively objective rating. The 190 questions were placed in 14 groups, each dealing with an attribute of the instrument.(ABSTRACT TRUNCATED AT 400 WORDS)


Fine structure of cells in the young regenerating spinal cord of the lizard Anolis carolinensis after H3-thymidine administration.

H3-thymidine was injected in a single dose into adult lizards Anolis carolinensis during early stages of tail regeneration when the new tail measured in length 2-5 mm (about 3 weeks). Electron microscopic autoradiography revealed that in the regenerating spinal cord three different cell types were recognizable four hours post-injection. By far the most common type was the ependymal cell. Ependymal or roundish cells with clear electron-density or storing bundles of intermediate filaments were occasionally found. A second rarer, cell type devoid of intermediate filaments was a pale round cell, often highly labelled. This cell showed a very poor ultrastructural differentiation and probably represents the precursor of the few neurons and glial cells observed in older stages of regeneration. The third type was a neuroblast at different degrees of differentiation and did not uptake H3-thymidine revealing it was a post-mitotic cell. Despite the relatively advanced ultrastructural differentiation of these neurons, synapses were rarely seen after three weeks of SC regeneration in Anolis.


[Erythropoietin--physiology and therapeutic potentialities].

Erythropoietin (EPO) is the main regulatory hormone for the control of erythropoiesis. EPO leads to enhanced mitosis and differentiation of erythroid precursors in the bone marrow. The major stimulus for EPO-formation is anaemia of various origin, resulting in an exponential relation between EPO levels and a decrease in haematocrit. Another important stimulus for increased EPO production is a fall of the arterial oxygen tension caused by either cardiopulmonary disorders or by a decrease of the oxygen tension in the inspiratory gas. Human erythropoietin was first isolated and purified from a large amount of urine of patients with aplastic anaemia. After the EPO gene had been cloned and expressed, biotechnically produced recombinant human erythropoietin (rHu-EPO) became available for clinical trials. EPO deficiency appears to be the major cause of renal anaemia, and hence the treatment of these patients is the most important indication for clinical use. Encouraging results in patients whose anaemia is not of renal origin have also been reported, using treatment with rHu-EPO. In preoperative autologous blood donation programmes prior to elective surgery, rHu-EPO therapy improved the amount of donated blood and ameliorated the decrease of haematocrit values. Side effects such as hypertension, thrombosis, hypercalcaemia, elevated liver enzymes were rare and were mostly related to the underlying disease.


[Muscle relaxation with no effect on oxygen uptake during isoflurane anesthesia?].

To determine the effects of vecuronium neuromuscular blockade on O2 consumption (VO2) during isoflurane anaesthesia 12 patients were studied. 12 patients (ASA-PS I-II, 37.1 +/- 12.1 yr, 173 +/- 8 cm, 70.1 +/- 8.6 kg), scheduled for urological lower abdominal surgery, received isoflurane-N2O-O2-anaesthesia under steady-state conditions (1.3 MAC). Duration of anaesthesia was 169 +/- 32 min and 0.057 +/- 0.016 mg/kg/h vecuronium were needed. The desired level of neuromuscular transmission was set to 10% of control. This level of neuromuscular blockade was kept constant for 60 min by a negative feedback controlled infusion of vecuronium. VO2 was measured by an indirect calorimetry device (MMC Horizon, STPD). During and after recovery of neuromuscular function anaesthesia was maintained and oxygen measurements were continued. Preanaesthetic values of VO2 were in the predicted range for basal metabolism. Steady-state general anaesthesia lead to an 26-28% reduction of VO2 (Range: 144-232 ml/min) compared to the preanaesthetic values (202-288 ml/min, p < 0.01). Neuromuscular blockade showed no significant effect on O2 uptake. We conclude that in patients with adequate depth of anaesthesia vecuronium-induced neuromuscular blockade does not lead to a further reduction of oxygen consumption, since muscular tone is already reduced by general anaesthesia.


[The determination of end-expiratory CO2 during resuscitation. Experience and results with the Normocap 200 (Fa. Datex) in preclinical resuscitation conditions].

Capnometers measure carbon dioxide (CO2) in inspired and expired air. Under physiological conditions end-tidal CO2 (peCO2) measurements closely reflect arterial pCO2 (paCO2). End-tidal CO2 concentration has been found to correlate with cardiac output in animal models and in clinical studies with cardiac arrest during cardiopulmonary resuscitation (CPR). In the present study in 23 cases of CPR end-tidal CO2 concentrations were registered during precordial compression with a transportable, battery-carried capnometer. In 7 cases of successful CPR mean concentrations of end-tidal CO2 were higher than in unsuccessful CPR (13 +/- 7 mmHg versus 8 +/- 6 mmHg). No statistical significance was found. In case of successful CPR there was a sudden rise in end-tidal CO2 up to 54 +/- 6 mmHg, indicating satisfactory spontaneous circulation. With capnometry it is possible to assess the efficacy of precordial compression during resuscitation and the return of spontaneous circulation in cardiopulmonary arrest.


[Pulmonary function one year after extracorporeal lung assist. A long-term follow-up of patients with acute adult respiratory distress syndrome].

This retrospective follow-up studied lung function and reconvalescence in 38 young patients without primary lung disease, who suffered from severe ARDS and survived by means of extracorporeal lung assist (ELA) treatment. Over a period of 3, 6, 9 and 12-20 months dynamic and static lung volumes and the results of X-ray and CT scan of the thorax were studied. Within the third and sixth month the forced vital capacity, FEV1, and arterial blood gases reached the lower range of normal values. Obvious relative emphysema RV/TLC was observed at the time of discharge from hospital and during the first four months. This was found to reverse during the following months. After a period of 12-20 months all patients had an abnormal diffusion capacity (TLCO) but with normal transfer coefficients (TLCO/VA). Even though ARDS did not induce obstructive changes, the diminished diffusion capacity and the slightly reduced expiratory peak flow in 25-75% of FVC (FEF 25-75) indicates slight changes in the small airways. Following up the X-ray and CT results we found extraordinary morphological restitution. Spiroergometry results showed a normal cardiopulmonary pattern for untrained individuals. 36 of the 38 patients were integrated in normal working and social life within 12-20 months.


[Artificial intravascular oxygenation (IVOX). Application to the treatment of postoperative respiratory failure].

Very recently, the concept of artificial intracorporeal oxygenation of blood for patients suffering from respiratory failure has been introduced into clinical practice through development of a totally implantable intravascular oxygenator (IVOX). We report on the use of such a device in a patient who developed severe respiratory insufficiency secondary to prolonged hypovolaemic shock and pneumonia following successful repair of a ruptured abdominal aortic aneurysm in September, 1990. Postoperatively, severe hypoxaemia occurred (AaDO2 548-602 torr) despite extensive mechanical ventilatory support. There was no obvious chance to overcome this situation by conventional therapeutic measures and the decision was made to institute IVOX therapy. Hypoxaemia was resolved immediately and both FiO2 and tidal volume could be reduced within hours. The patient's respiratory condition continued to improve over the next days leading to termination of IVOX therapy after 71 hours. However, the necessity of long-term ventilatory support secondary to recurrent pneumonia and sepsis, multiple abdominal reoperations for ischemic colitis and retroperitoneal abscess prolonged his recovery. He was discharged from the hospital after four months and is alive and well now 14 months after his operation. He is the first long-term survivor after IVOX therapy in Europe. IVOX may be successfully used in selected patients while the indications and it's potential role in the therapy of severe respiratory failure still need to be defined.


[CT findings of a cerebral air embolism as a consequence of an accidental subclavian catheter disconnection].

This report describes the definitive diagnosis of cerebral air embolism following accidental disconnection of a subclavian catheter by documentation of cranial CT's. We also discuss critically the pathophysiology of cerebral air embolism and its differentiation from "the bends". The necessity of recognising this entity is important in differential diagnosis. The pathophysiology of air embolism is discussed.


[Acute angioneurotic edema due to ACE inhibitors].

Acute angioneurotic edema due to angiotensin converting enzyme (ACE) inhibitors usually develops shortly after therapy has been started. In this case, hypopharyngeal edema occurred with a delay of nine days. It required endotracheal intubation and could only be differentiated from an inflammatory process by examination under general anesthesia. The mechanism of action of ACE inhibitors, the pathogenesis of angioneurotic edema, its therapy outside as well as in the hospital, and the case described are being discussed. It is concluded that severe reactions induced by ACE inhibitors must be expected even after considerable time of therapy.


An appraisal of spontaneous adverse event monitoring.

Awareness of the need to continue the safety assessment of new drugs into the post-marketing period has continued to increase over the past few years. The stage has been reached where there is, for all practical purposes, unanimous agreement that all new drugs, particularly those intended for widespread use and/or long-term administration, be subjected to some form of post-marketing monitoring. In many cases nationally sponsored spontaneous reporting systems are claimed to be sufficient to meet the needs without any critical appraisal being made of the suitability or reliability of this method. The adoption of 'pharmacovigilance' (spontaneous reporting) as the system for the whole European Community (EC) emphasizes the faith that has been placed in the system. At a recent symposium (Management Forum, 9-10 March, 1992) on post-marketing safety evaluation, more than half of the 16 papers presented were entirely or predominantly concerned with spontaneous reporting systems, which is a pattern common to other meetings on this topic. The Committee on Safety of Medicines (CSM) has referred to spontaneous reporting as the cornerstone of post-marketing surveillance which has further endorsed a general feeling of confidence that the system will meet our societal needs even though its scientific capability may be deficient. It is the purpose of this paper to challenge these assumptions and to make an objective appraisal of the various methods available.


Reduction of vein graft intimal hyperplasia by ex vivo treatment with desferrioxamine manganese.

Reversed vein grafting exposes the venous tissue to a period of ischemia, reperfusion and subsequent free radical generation which may contribute to endothelial injury and/or damage, smooth muscle cell proliferation and the later development of intimal hyperplasia. The effects of ex vivo treatment with desferrioxamine Mn+3 (DFMn), a cell-permeable free radical scavenger, on the development of intimal hyperplasia in experimental vein grafts was examined. Twenty New Zealand white rabbits received a reversed vein interposition bypass graft into the ipsilateral common carotid artery. Ten explanted veins were immersed in a heparinized (5 IU/ml) saline solution, and 10 others were immersed in a similar solution containing DFMn (1 mM) for 45 min prior to reimplantation. There were no short-term functional or morphologic toxic side effects associated with DFMn treatment on either the endothelial or smooth muscle cells of the veins. At 28 days, grafts (n = 20) were perfusion-fixed in vivo for histological and morphometric studies. There was a significant reduction in intimal thickening in the DFMn-treated group compared to the untreated group. The thicknesses of the intimal hyperplasia in the proximal segments were 50.6 +/- 6.3 vs. 76.9 +/- 3.2 microns (p < 0.05), in the middle segments 42.0 +/- 5.0 vs. 84.3 +/- 5.4 microns (p < 0.05) and in the distal segments 55.7 +/- 5.0 vs. 88.3 +/- 6.2 microns (p < 0.05) for treated and untreated animals, respectively. No evidence of long-term toxicity was found.(ABSTRACT TRUNCATED AT 250 WORDS)


Increased shear stress leads to differential release of endothelin and ATP from isolated endothelial cells from 4- and 12-month-old male rabbit aorta.

Freshly isolated thoracic aortic endothelial cells from 4- and 12-month-old male rabbits were exposed to two periods of increased flow rate and the levels of ATP, endothelin (ET) and arginine vasopressin (AVP) released into the perfusate were measured. The rapid, significant release of ATP and ET with increased flow from cells isolated from 12-month-old rabbits was not seen when cells isolated from 4-month-old rabbits were exposed to the same conditions. Increased flow did not stimulate AVP release from cells from either age group. Basal release with low flow also differed in endothelial cells from 4-month-old compared to 12-month-old rabbits; thus in 4-month preparations basal release of AVP was greater, that of ET was smaller, while that of ATP was unchanged relative to 12-month preparations. Immunohistochemical studies showed that almost half as many endothelial cells isolated from 4-month-old rabbits were immunoreactive to ET and AVP compared to cells isolated from 12-month-old animals. It is suggested that the endothelial contribution to the control of vascular tone in rabbit thoracic aorta changes during ageing.


Evidence that an Arg79-->Gln substitution in human factor VII is not associated with a reduction in coagulant activity.

A recent report hypothesized that an Arg79-->Gln mutation in the first epidermal growth factor-like domain of human factor VII is the molecular basis for a severe (< 1%) factor VII functional deficiency. In the present study, a site-specific mutant human factor VII cDNA (Arg79-->Gln) was constructed, subcloned and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity and characterized with respect to gamma-carboxyglutamic acid content, ability to activate, tissue factor-dependent amidolytic activity and expression of factor VIIa proteolytic activity on tissue factor-bearing cells. Mutant factor VII was fully carboxylated and exhibited the same molecular weight and coagulant activity as plasma factor VII. Mutant factor VII was activated by factor Xa at the same rate, and to the same extent, as plasma factor VII. In the presence of tissue factor, mutant factor VII was converted to factor VIIa in an autocatalytic manner at a rate indistinguishable from that observed with plasma factor VII. In addition, the amidolytic activities of mutant factor VIIa and plasma factor VIIa towards S-2288 in the presence of relipidated tissue factor were identical. Finally, following complex formation with cell surface tissue factor, mutant factor VIIa activated factor X at essentially the same rate as plasma factor VIIa under comparable conditions. These results are not consistent with the notion that the arginine-79 residue in the first epidermal growth factor-like domain of human factor VII is essential for the expression of tissue factor-dependent factor VIIa proteolytic activity.


Thrombogenicity and intimal hyperplasia after conventional and thermal balloon dilation in normal rabbit iliac arteries.

Acute occlusion and restenosis are the major complications of percutaneous transluminal coronary balloon angioplasty. Application of heat during balloon dilation was postulated to reduce these complications. We evaluated thrombogenicity and intimal hyperplasia of normal rabbit iliac arteries after conventional (37 degrees C) and thermal balloon dilation. Thermal dilation was performed with a radio-frequency-heated balloon, provided with three thermocouples attached to the inside of the balloon skin. In a previous in vitro study, thrombogenicity of human subendothelium was increased at 55 degrees C and greatly decreased at temperatures over 70 degrees C. Thermal balloon dilation was therefore performed at 55 and 90 degrees C in vivo. Rabbits survived 2 h for evaluation of platelet adhesion or either 3 or 8 weeks for intimal hyperplasia. Angiograms revealed no occlusions or thrombi after any procedure. Platelet adhesion was quantified on 20 scanning electron microscopic pictures per balloon dilation site and was expressed as the percentage of the luminal surface covered by platelets. Platelet adhesion was similar in all groups, although large thrombi were present in the 90 degrees C group. Intimal hyperplasia was measured morphometrically at regular intervals over the balloon site. After 3 weeks, the average intimal hyperplasia was significantly reduced in the 90 degrees C balloon dilation group, which was mainly due to the absence of intimal hyperplasia in the midpart of these segments. After 8 weeks, intimal hyperplasia was equal in all groups. Thus, in the applied model, platelet coverage was equal after conventional balloon angioplasty and after 55 and 90 degrees C balloon angioplasty.(ABSTRACT TRUNCATED AT 250 WORDS)


Mechanisms of flow-mediated signal transduction in endothelial cells: kinetics of ATP surface concentrations.

Intracellular free calcium ([Ca2+]i) was measured in single cells of a confluent endothelial monolayer subjected to defined flow. Flow medium containing adenosine triphosphate (ATP) was used to study the influence of flow forces upon agonist-response coupling as mediated via the P2y-purinoceptor. [Ca2+]i responses were highly sensitive to the fluid motion at the cell surface; consecutive small increases of flow stimulated large [Ca2+]i transients with the levels returning to baseline at the new flow rate within 250 s. The characteristics of [Ca2+]i transients were also influenced by decreasing flow. Since potent ectonucleotidases at the endothelial cell surface rapidly degrade ATP, we postulated that a combination of flow and degradative enzymes regulates the mass transport of ATP in the boundary layer. The hypothesis predicts that step increases of flow exceed the capacity of the ectonucleotidases and allow ATP to reach the receptor. Experiments were conducted to compare ATP and ADP beta S, a nonhydrolyzable ATP analog that resists degradation by surface ectonucleotidases, and calculations of ATP mass transport to the cell surface were compared to estimates of surface clearance rates. Calculations of mass transport coefficients for ATP in the boundary layer demonstrated that changes of flow which elicited a prominent [Ca2+]i response represented 26-73% changes in the mass transport of ATP from the bulk fluid. When steadystate mass transport coefficients for ATP under various flow conditions were compared with the estimated rate constant for surface degradation of ATP, ratios close to unity were obtained. These results suggest that both boundary layer mass transport and ATP clearance rates can be rate-limiting for flow-mediated activation of the P2y-receptor. The experiments provide evidence for differential signal transduction responses in the endothelium driven by diffusion gradients (derived from both the blood and the vessel wall), which are likely to vary widely in the complex flow fields encountered in vivo.


Composition and susceptibility to thrombolysis of human arterial thrombi and the influence of their age.

Each of three distinct, concentric layers of human arterial thrombi, was analysed immunochemically for the plasminogen and fibrin content, and for the ex vivo susceptibility to thrombolysis by various thrombolytic agents in a saline or plasma milieu. The age of the thrombus layer determined: (a) the plasminogen content; (b) the fibrin content, inferred from the recovery of fibrin degradation products after complete lysis and from the binding of a monoclonal anti-fibrin antibody in a perfusion system, and (c) the lysibility of the thrombus. Plotting concentration of the various thrombolytic agents against percentage of lysis at several time points allows for reading of equivalent potencies of the respective units. Undiluted solutions of APSAC and rt-PA, prepared according to the manufacturer's directions, were less effective than diluted solutions, which has consequences for local therapy. All agents were at least as effective in saline as in a plasma milieu. We conclude that the plasminogen content of aged arterial thrombi is sufficient for complete and rapid thrombolysis. Only after several months do fibrin and plasminogen become so far degraded or replaced that the thrombi become resistant to thrombolysis.


Wall thickness to lumen diameter ratios of arteries from SHR and WKY: comparison of pressurised and wire-mounted preparations.

Passive properties (diameter, wall-to-lumen ratio and axial length) of small mesenteric arteries from SHR and WKY rats were measured with the artery segments cannulated and pressurised, or mounted on wires in a myograph. The measurements were made with a range of distending pressures (or calculated equivalent distending pressures when wire-mounted) from 0 to 180 mm Hg. The axial length of artery segments increased with increasing distending pressure when cannulated, but not when wire-mounted. The axial extension was greater for arteries from WKY (up to 105%) than for arteries from SHR (up to 65%). The arteries from SHR had significantly smaller diameters and greater wall-to-lumen ratios than the arteries from WKY. However, the diameters calculated for the arteries when wire-mounted were less than the measured diameters, and the wall-to-lumen ratio was always greater when wire-mounted than when cannulated because of the underestimated diameter and the absence of axial extension. Wall-to-lumen ratios decreased with increased distending pressure; values at 180 mm Hg were only 18 and 25% of those at 0 mm Hg for WKY and SHR arteries, respectively. The large degree of variability of wall-to-lumen ratios obtained from the two different preparations and the large range of values that are obtained from a single artery at different distending pressures must call into question the validity of characterising vascular hypertrophy by any single estimation of this parameter.


Non-vitamin K-dependent clotting factors during oral anticoagulant treatment.

The non-vitamin K-dependent coagulation factors fibrinogen, factor V, von Willebrand factor and factor XIII were measured in plasma of patients receiving oral anticoagulant treatment. Levels of fibrinogen, von Willebrand factor and factor XIII were above the upper normal limit in 60.0%, 58.6% and 53.4% of patients respectively. Factor V levels were below the lower normal limit in 56.3% of patients. Compared with a control group we found significant differences in the anticoagulated group for fibrinogen (P < 0.0001), factor V (P < 0.0001), von Willebrand factor (P < 0.0001) and factor XIII (P < 0.0001). It appears that the down-regulation of factors II, VII, IX and X during oral anticoagulation is accompanied by a subsequent up-regulation of fibrinogen, von Willebrand factor and factor XIII. This up-regulation or its absence could explain the thrombotic and bleeding complications seen in some patients on anticoagulant therapy and might also reduce the beneficial effect of oral anticoagulation in cardiovascular disease.


Early cross-linked fibrin in human plasma contains alpha-polymers with intact fibrinopeptide A.

alpha-polymer formation, as opposed to gamma-chain dimerization has been considered a relatively late event in factor XIII-induced fibrin stabilization. Recently it has been shown, however, that plasma from healthy individuals and from patients with fibrinaemia contains small amounts of soluble fibrin/fibrinogen oligomers interlinked through dimerized gamma-chains as well as cross-linked alpha-chains. The present work was carried out to see if these early alpha-chain polymers also arise during coagulation of plasma in vitro. Plasma samples from healthy individuals, prepared by immediate centrifugation of blood collected without anticoagulant, were allowed to clot spontaneously for varying periods. The plasma clots were solubilized in SDS-urea-mercaptoethanol and samples were subjected to SDS-PAGE and Western blotting using polyclonal antibodies to human fibrinogen, or monoclonal antibodies specific either for A alpha/alpha-chains, for fibrinopeptide A-containing chains, for the N-terminus of the fibrin beta-chain or for the gamma-chains. Fibrin/fibrinogen oligomers were seen to form long before visible gelation of plasma. These oligomers were cross-linked through gamma-chain dimerization, but also through A alpha- or alpha-chain polymerization. The number and amount of alpha-polymers containing A alpha-chains increased immediately after clot formation, but these disappeared about 20 min later, due to complete removal of fibrinopeptide A (FPA) by thrombin. It is concluded that alpha-polymer formation is a very early event during plasma coagulation in vitro, and that both A alpha- and alpha-chains are involved.


Fluid shear stress stimulates membrane phospholipid metabolism in cultured human endothelial cells.

There is evidence suggesting that fluid shear stress activates phospholipid turnover in endothelial cells, but it is not clear which phospholipids are involved in the transduction of the flow signal. Cultured human umbilical-vein endothelial cells were prelabeled with [14C]-arachidonic acid and subjected to laminar shear stresses of 0.4, 1.4 and 22 dyn/cm2 for times up to 30 min, after which the distribution of the radioactivity in the phospholipids was determined. We observed decreases in labeled phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid at 10-30 s, and increases in labeled diacylglycerol (DG) and free arachidonate, as well as a simultaneous elevation in inositol 1,4,5-triphosphate (IP3) levels. A second peak in IP3 levels was observed 10 min after the onset of shear. This is in contrast with agonist-stimulated endothelial cells, where IP3 levels go back to initial values within a few minutes after stimulation. The flow-induced IP3 response was the same in the presence or absence of ATP and serum in the perfusing medium. These results are consistent with the activation of phospholipase C, phospholipase A2 and DG lipase by shear stress. This suggests that several phospholipids are involved in the production of free arachidonic acid and DG, which are likely to be important mediators of the shear stress signal. In addition, flow may lead to a chronic stimulation of endothelial-cell metabolism.


Congenital thrombophilia among patients with venous thromboembolism.

During a 3-year period we studied 393 adult patients (382 of whom were unrelated) with a history of acute venous thromboembolism. A congenital deficiency state known to predispose to thrombosis was found in 27.2%. Of these, most were due to deficiencies of protein C (9.2%), protein S (7.6%), antithrombin III (5%) or to increased plasma PAI-1 concentration (3.1%) which, in the absence of any known factor that predisposes towards thrombosis, results in a diminished fibrinolytic activity. There was a characteristic pattern between the age of onset (mean 34 years) of thrombosis and individual protein deficiency. Thrombosis appeared spontaneously in 73% of cases with recurrence in 80%. In contrast, in the remaining unrelated patients, 138 (35.1%) in whom venous thromboembolism was secondary and occurred at a mean age of 43 years, and in the other 140 (35.6%) who suffered thromboembolism spontaneously at a later age (mean age 55), there was no permanent protein deficiency state or alteration in fibrinolytic activity and thrombosis recurrence was lower (53.6% and 20.7% respectively). Of the 393 patients, deep vein thrombosis was the most common manifestation; however, in congenital thrombophilia, thrombosis of visceral vessels and Raynaud's syndrome (6%) were also detected.


Natural inhibitors of blood coagulation and fibrinolysis in patients with lupus anticoagulant.

We studied the natural inhibitors (NI) of blood coagulation and fibrinolysis in 50 patients with lupus anticoagulant (LA), in order to identify possible alterations of these NI, that could favour thrombotic manifestations. We found no statistically significant difference in antithrombin III, protein C and alpha 2-antiplasmin between controls and patients with LA, irrespective of their clinical manifestations. We found an increase of plasminogen activator inhibitor (PAI, P < 0.001) and a decrease of free protein S (PSf, P < 0.001) and total protein S (PSt, 0.01 < P < 0.05) in the patients with LA when compared with the control group. We found no difference in the levels of NI between patients with thrombosis (n = 19) and without thrombosis (n = 31) nor between patients with (n = 25) or without thrombosis and/or foetal loss (n = 25). In contrast, we observed a decrease of PSf in women with foetal loss (n = 10) as compared with women without foetal loss (n = 22, 0.01 < P < 0.05) and a decrease of PSf when comparing 19 patients with systemic lupus erythematosus (SLE) with 31 patients without SLE (0.01 < P < 0.05). These findings show that the patients with LA had several abnormalities in the NI system, but there was no significant association between levels of PAI, PSf, PSt and a history of thrombosis.


Evidence for activation of coagulation in Crohn's disease.

Haemostatic changes in 16 patients with Crohn's disease were studied from active disease into clinical remission and beyond. Elevated concentrations of fibrinopeptide A (FpA) and prothrombin fragments F1 + 2 (F1 + 2) were found at times of both active (FpA median 3.2, range [0.3-40] ng/ml and F1 + 2 median 2.3, range [0.3-18] nm/l) and inactive disease (FpA median 2, range [0.4-40] ng/ml and F1 + 2 median 1.3, range [0.2-20) nm/l]. We also measured the physiological inhibitors of coagulation and fibrinolysis; there was no significant difference in the levels of antithrombin III, protein C or the Exner ratio between active and inactive disease. Free protein S levels were significantly lower in active disease (median 34, range 9-54 U/dl) than in remission (median 40, range 12-65 U/dl). Plasminogen activator inhibitor type 1 (PAI-1) was significantly raised in remission (median 11, range 3-32 ng/ml) when compared to active disease (median 7, range 3-42 ng/ml). The D-dimer correlated significantly with fibrinopeptide A (P < 0.001), suggesting reactive fibrinolysis in some patients. Most (35/52, 67%) samples showed evidence of persistent haemostatic activation (elevated FpA and/or F1 + 2) during phases of apparent clinical remission in Crohn's disease, a factor that is not reflected by clinical activity scores. This study supports the hypothesis that coagulation is activated in the mesenteric vasculature of patients with Crohn's disease.


Monoclonal antibodies against the C-terminal peptide of human tissue factor for studies of the cytoplasmic domain.

The carboxyl-terminal cytoplasmic domain of human tissue factor (the essential cofactor for coagulation factor VII) is a prospective regulatory domain through which viable cells may control the expression of tissue factor activity. Furthermore, this domain is subject to post-translational modifications of as yet unknown functional significance. Hybridomas producing antibodies against the C-terminal domain of tissue factor were obtained using splenocytes harvested from mice immunized with a synthetic peptide corresponding to the nine terminal residues of the protein sequence. These antibodies, C28 1.1 and C28 2.1, react with purified placental tissue factor, but not with a recombinant soluble tissue factor lacking the cytoplasmic and membrane-spanning domains. This confirms that tissue factor from biological membranes contains the entire cytoplasmic domain predicted from the cDNA sequence, and provides a mechanism to determine whether tissue factor used experimentally retains this proteolytically sensitive epitope. The antibodies have been employed to demonstrate selective proteolytic removal of the carboxyl-terminal peptide from tissue factor. Limited experiments indicate that they will be useful for characterizing the distribution of vesicles with regard to the possible orientations of tissue factor on their surface.


Morphological and functional characterization of vascular muscle cells enzymatically dispersed from dog mesenteric arteries.

The objective of this study was to compare the properties of single smooth muscle cells enzymatically dispersed from the dog mesenteric arteries to the properties of similar cells functioning in tissue strips. The isolated cells remained relaxed in nominally Ca(2+)-free medium for about 1-2 h after exposure to 1 mM Ca2+ and like intact mesenteric artery rings did not contract spontaneously. Enzymatically dispersed cells maintained all the characteristic morphological features observed in strips of muscle prior to isolation except that the amorphous materials covering the smooth muscle cell surfaces (basal lamina) were absent after enzymatic dispersion. Addition of 100 mM KCl to these vascular muscle cells elicited maximal shortening in the presence but not in the absence of extracellular Ca2+ and KCl-induced cell shortening was prevented by 10(-7) M nifedipine indicating the presence of functional voltage-operated Ca2+ channels. However, in contrast to the vascular muscle strips, in which graded contractile responses were observed with increasing KCl concentrations, isolated vascular muscle cells underwent nearly maximal contraction at concentrations as low as 15 mM KCl. Both intact tissue and isolated cell preparations responded similarly to phenylephrine in a concentration-dependent manner and the responses were blocked by prazosin. In contrast to muscle strips, the isolated cells did not shorten in response to phenylephrine in Ca(2+)-free medium. Isolated muscle shortened in the presence of sarcoplasmic reticulum selective Ca2+ transport ATPase inhibitors, cyclopiazonic acid or thapsigargin. Ryanodine also caused contraction. We conclude that enzymatically dispersed smooth muscle cells from dog mesenteric arteries are potentially useful for studies of the regulation of smooth muscle contractility, but have significantly increased sensitivity to external K+, implying an altered membrane potential or voltage dependence of ion channels. Their impaired ability to contract to phenylephrine in Ca(2+)-free medium implies some alteration in intracellular Ca2+ stores of their coupling to cellular activation. These differences will affect how the data obtained from freshly isolated enzymatically dispersed vascular muscle cells may be extrapolated to cell studies in intact tissues.


The fibrinolytic potential in patients with Cushing's disease: a clue to their hypercoagulable state.

The aim of our study was to determine the fibrinolytic potential in a large group of patients with Cushing's disease. These patients had a significant shortening of the activated partial thromboplastin time and increase in factor VIII/von Willebrand factor complex compared to normal controls. The mean levels of plasminogen, tissue plasminogen activator (t-PA) antigen and plasminogen activator inhibitor (PAI) activity were significantly higher than in normal subjects, whereas the basal fibrinolytic activity was similar to that seen in the control group. In 17 out of 30 Cushing patients and in 17 normal subjects the fibrinolytic potential was determined with the venous occlusion test. In the Cushing group, the release of t-PA antigen after 20 min of venous occlusion was comparable to that observed in the control group. However, Cushing patients showed a lower fibrinolytic activity than normal subjects, since a lesser shortening of the euglobulin lysis time and a non-significant rise of plasminogen activator activity levels were found. Moreover, in these patients the PAI activity values remained unchanged and significantly increased after venous occlusion test also. In conclusion, the impaired fibrinolytic activation seen in Cushing patients after venous occlusion can be explained by the inhibitory effect of the high PAI levels on plasminogen activators. The defective fibrinolytic potential could further contribute to the hypercoagulable state in Cushing's disease. High PAI levels before surgery may represent an additional risk factor for post-surgical thromboembolic complications in Cushing patients.


A microtitre plate ELISA to measure thrombin-antithrombin complex using pan-specific antibodies.

A sandwich enzyme-linked immunosorbent assay (ELISA) has been developed to measure plasma levels of thrombin-antithrombin complex (TAT). The assay is performed in a microtitre plate using polyclonal antibodies specific for antigenic determinants on prothrombin and antithrombin. Antibody to prothrombin was immobilized on a solid phase, using a titre predetermined to optimize capture of TAT. The performance of the microtitre plate ELISA for TAT has been extensively investigated and compared with the performance characteristics of a tube-based ELISA for TAT which is available commercially (Enzygnost-TAT, from Behringwerke, Marburg, Germany). Studies with plasma containing various levels of prothrombin showed that the zymogen competed with TAT for capture antibody in both assays. Variations in prothrombin levels between plasma samples present a potential source of artifact, but one which does not critically affect the performance of either assay in detecting large elevations in TAT. A high correlation (r = 0.88) was established between the results of plasma samples assayed by both assays, whether citrate or EDTA anticoagulant was used to prepare plasma. High correlations (r > 0.90) were also established for each assay between the results of plasma prepared with EDTA as compared to citrate anticoagulant. Both assays were able to discriminate completely between a group of 16 normal controls and a group of 31 patients with disseminated intravascular coagulation (DIC).


The pathogenesis of accelerated fibrinolysis in hepatosplenic schistosomiasis.

Seventy patients with different stages of hepatosplenic schistosomiasis and 18 non-bilharzial normal controls were studied. Plasminogen, plasminogen activators (PA), tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), alpha 2-antiplasmin (alpha 2-AP), plasminogen activator inhibitor (PAI), fibrinogen/fibrin degradation products (FDP) and D-dimer were determined to elucidate the role of plasminogen activators and inhibitors in the pathogenesis of accelerated fibrinolysis in schistosomiasis. There was a progressive increase in the levels of PA, t-PA, u-PA, FDP and D-dimer indicating enhanced fibrinolytic activity with advancing disease. In addition, there was progressive decrease of plasminogen, alpha 2-AP and PAI levels which might be due to decreased hepatic synthesis and/or increased peripheral consumption. These findings suggest that the pathogenesis of accelerated fibrinolysis in schistosomiasis is multifactorial, but may be due to the progressive increase in the levels of plasminogen activators. In addition, the increase of FDP and D-dimer levels are evidence of secondary fibrinolysis following thrombin generation.


The fibrinolytic system and proteins C and S in treated polycythaemia rubra vera.

This study was designed to assess whether factors other than high haemoglobin, thrombocytosis and abnormal platelet function predispose to thrombosis in polycythaemia rubra vera (PRV). Components of the fibrinolytic system and concentrations of the naturally occurring anticoagulants were measured in patients and controls in the resting state; the fibrinolytic capacity was reassessed after venous occlusion. The results were related to presence or absence of a history of thromboembolism. Under resting conditions, patients with PRV had reduced plasminogen activator inhibitor antigen levels and higher fibrin plate lysis area and tissue plasminogen activator activity. Protein C, protein S and factor V levels were reduced. Those patients with a history of thromboembolism had decreased tissue plasminogen activator activity after venous occlusion compared to those who had not experienced a thrombosis. We conclude that reduced fibrinolytic capacity may predispose to thrombosis in PRV. Despite treatment to normalize haemoglobin levels, the patients have persistent activation of their fibrinolytic systems. This, and reduced levels of proteins C and S, may be secondary to a chronic, clinically occult, disseminated intravascular coagulation.


Rod-like cholesterol micelles in aqueous solution studied using polarized and depolarized dynamic light scattering.

Micelles of cholesterol in aqueous solution have been investigated using polarized and depolarized dynamic light scattering. They are shown to be highly extended and characterized by a narrow size distribution. It is shown that a rod-like model is applicable with length, L = 580 nm. Determination of the rotational diffusion coefficient by analysis of the autocorrelation function gave a value of theta = 150 s-1, which is close to the calculated value for the rod with this dimension. Depolarized dynamic light scattering measurements as a function of angle gave a value of 110 s-1.


Structure of the myosin head in solution and the effect of light chain 2 removal.

Structural properties of rabbit skeletal myosin head (S1) and the influence of the DTNB light chain (LC2) on the size and shape of myosin heads in solution were investigated by small angle x-ray scattering. The LC2 deficient myosin head, S1 (-LC2), and the S1 containing LC2 light chain, S1 (+LC2) were studied in parallel. The respective values of the radius of gyration were found to be (40.2 +/- 0.5) A and (46.7 +/- 1) A, while the maximum dimension was (190 +/- 15) A for both species. The large difference between the two Rg values suggest that LC2 is located close to one extremity of the myosin head, in agreement with most electron microscopy observations. All models derived from the x-ray scattering pattern of the native myosin head share a common overall morphology, showing two main regions, an asymmetric globular portion which tapers smoothly into a thinner domain of roughly equivalent length making an angle of approximately 60 degrees, with a contour length of approximately 210 A.


Motion of actin filaments in the presence of myosin heads and ATP.

We measured, by fluorescence correlation spectroscopy, the motion of actin filaments in solution during hydrolysis of ATP by acto-heavy meromyosin (acto-HMM). The method relies on the fact that the intensity of fluorescence fluctuates as fluorescently labeled actin filaments enter and leave a small sample volume. The rapidity of these number fluctuations is characterized by the autocorrelation function, which decays to 0 in time that is related to the average velocity of translation of filaments. The time of decay of the autocorrelation function of bare actin filaments in solution was 10.59 +/- 0.85 s. Strongly bound (rigor) heads slowed down the diffusion. Direct observation of filaments under an optical microscope showed that addition of HMM did not change the average length or flexibility of actin filaments, suggesting that the decrease in diffusion was not due to a HMM-induced change in the shape of filaments. Rather, slowing down of translational motion was caused by an increase in the volume of the diffusing complex. Surprisingly, the addition of ATP to acto-HMM accelerated the motion of actin filaments. The acceleration was the greatest at the low molar ratios of HMM:actin. Direct observation of filaments under an optical microscope showed that in the presence of ATP the average length of filaments did not change and that the filaments became stiffer, suggesting that acceleration of diffusion was not due to an ATP-induced increase in flexibility of filaments. These results show that some of the energy of splitting of ATP is impaired to actin filaments and suggest that 0.06 +/- 0.02 of HMM interferes with the diffusion of actin filaments during hydrolysis of ATP.


Residue solvent accessibilities in the unfolded polypeptide chain.

The difference of solvent accessibilities in the native and unfolded states of the protein is used as a measure of the hydrophobic contribution to the free energy of folding. We present a new approximation of amino acids solvent accessibilities in the unfolded state based on the 1-ns molecular dynamics simulation of Ala-X-Ala tripeptides at a temperature of 368 K. The standard accessibility values averaged from the molecular dynamics study are significantly lower from those previously obtained by considering only selected conformations of Ala-X-Ala tripeptides.


Effects of domain connection and disconnection on the yields of in-plane bimolecular reactions in membranes.

It has recently been shown (Vaz, W.L.C., E.C.C. Melo, and T.E. Thompson. 1989. Biophys. J. 56:869-875; 1990. Biophys. J. 58:273-275) that in lipid bilayer membranes in which ordered and disordered phases coexist, the ordered phase can form a two-dimensional reticular structure that subdivides the coexisting disordered phase into a disconnected domain structure. Here we consider theoretically the yields of bimolecular reactions between membrane-localized reactants, when both the reactants and products are confined to the disordered phase. It is shown that compartmentalization of reactants in disconnected domains can lead to significant reductions in reaction yields. The reduction in yield was calculated for classical bimolecular processes and for enzyme-catalyzed reactions. These ideas can be used to explain certain experimental observations.


Computer modelling of glycolipids at membrane surfaces.

Interactions of membrane anchored molecules such as glycolipids with a membrane surface are important in determining headgroup conformation. It is therefore essential to represent these membrane surface interactions in molecular modeling studies of glycolipids and other membrane bound molecules. We introduce here an energy term that represents the interaction of molecules with a membrane bilayer. This membrane interaction energy term has been added to the potential energy function of a molecular dynamics and mechanics program and has been parameterized using partition coefficients between an aqueous solution and a vesicular membrane for two model glycolipids.


A synthetic analogue of melittin aggregates in large oligomers.

An analogue of melittin synthesized in the group of E. T. Kaiser (DeGrado, W. F., F. J. Keźdy, and E. T. Kaiser. 1981. J. Am. Chem. Soc. 103:679-681) was investigated by Raman spectroscopy and fluorescence anisotropy decay. In water, the analogue is completely alpha-helical and aggregates in large oligomers of about 50 monomers. In vesicle membranes, it undergoes orientational fluctuations similar to melittin. The most significant difference from melittin, therefore, is the formation of straight helixes and their aggregation in large oligomers in water. We interpret this as a consequence of the lacking proline residue in the analogue. We, furthermore, hypothesize that the increased tendency for aggregation causes the increased hemolytic activity of the analogue.


Photogating of ionic currents across lipid bilayers. Electrostatics of ions and dipoles inside the membrane.

The conductances of the lipophilic ions tetraphenylboride and tetraphenylphosphonium across a lipid bilayer can be increased or decreased, i.e., gated, by the photoformation of closed-shell metalloporphyrin cations within the bilayer. The gating can be effected by pulsed or continuous light or by chemical oxidants. At high concentrations of lipophilic anions where the dark conductance is saturated due to space charge in the bilayer, the photogated conductance can increase 15-fold. The formation of porphyrin cations allows the conductance to increase to its nonspace charge limited value. Conversely, the decrease of conductance in the light of phosphonium cations diminishes toward zero as the dark conductance becomes space charge limited. We present electrostatic models of the space charge limited conductance that accurately fit the data. One model includes an exponentially varying dielectric constant for the polar regions of the bilayer that allows an analytical solution to the electrostatic problem. The exponential variation of the dielectric constant effectively screens the potential and implies that the inside and outside of real dielectric interfaces can be electrically isolated from one another. The charge density, the distance into the membrane of the ions, about one-quarter of its thickness, and the dielectric constant at that position are determined by these models. These calculations indicate that there is insufficient porphyrin charge density to cancel the boride ion space charge and the following article proposes a novel ion chain mechanism to explain these effects. These models indicate that the positive potential arising from oriented carbonyl ester groups, previously used to explain the 10(3)-fold larger conductance of hydrophobic anions over cations, is smaller than previously estimated. However, the synergistic movement of the positive choline group into the membrane can account for the large positive potential.


Photogating of ionic currents across lipid bilayers. Hydrophobic ion conductance by an ion chain mechanism.

The photogating of hydrophobic ion currents across the lipid bilayer membrane allows the direct study of their kinetics by symmetrically forming charge within the membrane and across each interface, rather than across the membrane. We find that the photoinduced conductance continues to increase beyond the region where the tetraphenylboride charge density in the membrane exceeds the estimated porphyrin cation density. This photoconductance is proportional to the tetraphenylboride charge density raised to the second to third power. The risetime of the photogating effect increases with increasing concentration of tetraphenyl boride. The porphyrin cation mobility is increased when the tetraphenylboride anion is present, and low concentrations of tetraphenylphosphonium cation increase the dark conductivity while inhibiting the photoconductivity. The activation energy for both the porphyrin and phosphonium cation induced conductance is more positive than that of the tetraphenylboride conductance. From these results we conclude that in addition to some cancellation of space charge within the membrane, the mechanism of increased conductance involves the transport of these hydrophobic anions via an alternating anion-cation chain, analogous to the Grotthuss mechanism for excess proton conduction in water. This ion chain conductance can be viewed as an evolutionary prototype of an ion channel across the membrane. It also underscores the importance of the counter ion in the transport of large ions such as peptides across the lipid bilayer.


Low temperature structures of dCpG-proflavine. Conformational and hydration effects.

The structure of the complex of dCpG with proflavine was determined using x-ray data taken at -130 degrees C (low temperature) and at -2 degrees C (cold temperature) and compared with the structure of the complex determined previously at room temperature (Shieh, H. S., H. M. Berman, M. Dabrow, and S. Neidle. 1980. Nucleic Acids Res. 8:85-97). Low temperature was refined with 5,125 reflections between 8.0 and 0.93 A, Anisotropically modeled temperature factors were used for DNA/drug atoms and isotropic ones for water oxygens to R factor of 12.2% in P2(1)2(1)2; a = 32.853, b = 21.760, c = 13.296 A. Cold temperature was refined isotropically with 2,846 reflections 8.0-0.89 A to R = 15.1% in P2(1)2(1)2; a = 32.867, b = 22.356, c = 13.461 A. Both structures are very similar to the room temperature one, though some important differences were observed: one guanine sugar moiety is disordered and additional water molecules have been located that give rise to infinite polyhedral hydration networks.


Pressure and low temperature effects on the fluorescence emission spectra and lifetimes of the photosynthetic components of cyanobacteria.

The effects of hydrostatic pressure on the excited state reactions of the photosynthetic system of cyanobacteria were studied with the use of stationary and dynamic fluorescence spectroscopy. When the cells were excited with blue light (442 nm), hydrostatic pressure promoted a large increase in the fluorescence emission of the phycobilisomes (PBS). When PBS were excited at 565 nm, the shoulder originating from photosystem II (PSII) emission (F685) disappeared under 2.4 kbar compression, suggesting suppression of the energy transfer from PBS to PSII. At atmospheric pressure, the excited state decay was complex due to energy transfer processes, and the best fit to the data consisted of a broad Lorentzian distribution of short lifetimes. At 2.4 kbar, the decay data changed to a narrower distribution of longer lifetimes, confirming the pressure-induced suppression of the energy transfer between the PBS and PSII. When the cells were excited with blue light, the decay at atmospheric pressure was even more complex and the best fit to the data consisted of a two-component Lorentzian distribution of short lifetimes. Under compression, the broad distribution of lifetimes spanning the region 100-1,000 ps disappeared and gave rise to the appearance of a narrow distribution characteristic of the PBS centered at 1.2 ns. The emission of photosystem I underwent 2.2-fold increase at 2.4 kbar and room temperature. A decrease in temperature from 20 to -10 degrees C at 2.4 kbar promoted a further increase in the fluorescence emission from photosystem I to a level comparable with that obtained at temperatures below 120 degrees K and atmospheric pressure. On the other hand, when the temperature was decreased under pressure, the PBS emission diminished to very low value at blue or green excitation, suggesting the disassembly into the phycobiliprotein subunits.


A model for binding of an antifreeze polypeptide to ice.

A model is proposed, based on recent peptide analog and ice crystal etching studies, whereby an alanine-rich, alpha-helical antifreeze polypeptide (AFP) from the winter flounder inhibits the growth of ice crystals by hydrogen bonding of Thr, Asn, and Asp side chains in a specific pattern to the [2021] hexagonal bipyramidal planes of ice. It is further suggested that this mode of binding is unidirectional, maximizing opportunities for packing of AFPs on the ice surface, and that ice crystal growth inhibition occurs by a two-step mechanism involving hydrogen bonding and hydrophobic interpeptide interactions.


Locomotion forces generated by a polymorphonuclear leukocyte.

There have been very few studies which have measured the physical forces generated by cells during active movements. A special micropipette system has been designed to make it possible to observe cell motion within the pipette and to apply a pressure to counter the chemotactic migration of the cell. This provides a direct measure of the locomotion force generated by the cell. The average velocity of forward motion is 0.33 microns/s in the absence of counter-pressure. The application of a positive counter-pressure (C-P) causes a decrease in the velocity of the forward motion of the cell. At 17 cm H2O of C-P, the cell velocity drops to zero and even moves backward with a higher C-P. The results show that the decrement of velocity is linearly related to the magnitude of the C-P with a complete stoppage at a pressure of 17 cm H2O which corresponds to a force of 0.003 dyn. The maximum work rate of the cell is approximately 2.5 x 10(-8) erg/s.


Conformational adaptability of the terminal regions of flagellin.

Secondary structure formation in the disordered terminal regions of flagellin were studied by circular dichroic (CD) spectroscopy, Fourier transform infrared spectroscopy, and x-ray diffraction. The terminal regions of flagellin are known to form alpha-helical bundles upon polymerization into flagellar filaments. We found from comparative CD studies of flagellin and its F40 tryptic fragment that a highly alpha-helical conformation can be induced and stabilized in the terminal regions in 2,2,2-trifluoroethanol (TFE) containing solutions, which is known to promote intra-molecular hydrogen bonding. Two oligopeptides, N(37-61) and C(470-494), each corresponding to a portion of terminal regions and predicted to have a high alpha-helix forming potential, were synthesized and studied. Both peptides were disordered in an aqueous environment, but they showed a strong tendency to assume alpha-helical structure in solutions containing TFE. On the other hand, peptides were found to form transparent gels at high concentrations (> 15 mg/ml) and all three methods confirmed that the peptides become ordered into a predominantly beta structure upon gel formation. Our results show that large segments of the disordered terminal regions of flagellin can adopt alpha-helical as well as beta structure depending on the environmental conditions. This high degree of conformational adaptability may be reflecting some unique characteristics of the flagellin termini, which are involved in self-assembly and polymorphism of flagellar filament.