Reflections on Benner: a critical perspective.

This paper examines the theoretical perspective of Patricia Benner (1984; 1989) as outlined in two of her recent works. The evaluative criteria used are the critical perspective developed by Carr and Kemmis (1986). The interpretive strengths of her work are identified and extended by comparison with the work of Lawler (1991). Problems in Benner's analyses are found to centre around the weaknesses of the interpretive perspective. An analysis of the structural constraints that act to distort the interpretation of nurses' work is offered as an alternative. An evaluation is given of the relevance to practice of Benner's work.


Physical assessment by nurses: a study of nurses' use of chest auscultation as an indicator of their assessment practices.

A non-experimental survey was conducted to determine if the factors such as years of practice, how physical assessment skills were learnt and how often these skills were utilised, influenced nurses' reluctance to implement physical assessment. A sample of 150 registered nurses was surveyed. Analysis indicated that the majority of respondents has been taught to listen to chest sounds (auscultation) but did not implement this skill on a daily basis. Chi square analysis indicated that younger nurses with less years of experience were more keen to learn chest auscultation skills than nurses who were older and had more years of nursing experience.


Metabolic activation of phenols by stimulated neutrophils: a concept for a selective type of anti-inflammatory drug.

Apocynin (4-hydroxy-3-methoxy-acetophenone) is a potent and selective inhibitor of neutrophil oxyradical production. The mechanism of action involves metabolic activation in a (myelo)-peroxidase-dependent reaction. The reaction product(s) prevent(s) the assembly of the superoxide anion-generating NADPH:O2 oxidoreductase by conjugation to essential thiol groups. Different neutrophil functions that are essential to their bactericidal activity, however, remain intact. When administered orally a potent anti-inflammatory activity was found in rats with experimentally induced local or systemic inflammation.


Human B-cell growth-inhibitory activity of eosinophil cationic protein.

The effect of eosinophil cationic protein (ECP) upon proliferation of human B cell lines or purified B cells was studied. ECP inhibited proliferation of the human lymphoblastoid cell lines CBL and GM-1056 at doses of 0.1-5 ng/mL during 2-4 days of culture. The inhibitory effect of ECP was reversible and not due to toxic damage. Moreover, inhibition could be blocked by anti-ECP serum while the control serum failed to do so. Of various cytokines tested--including interleukin (IL)-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6; interferon (IFN)-alpha or IFN-gamma--IL-4 reduced the inhibition, while other cytokines failed to do so. The reduction of inhibition was specific to IL-4 since reduction by IL-4 was blocked by anti-IL-4 antibody but not by the control antibody. ECP also inhibited proliferation of tonsillar small resting B cells stimulated with anti-mu antibody plus low molecular weight B-cell growth factor (BCGF) or of large activated B cells. In contrast, ECP had no effect on proliferation of unstimulated small resting B cells. This inhibition was also reduced by IL-4 specifically. These results indicate that ECP may also act as a B-cell regulating factor.


An immunodominant antigen of Giardia lamblia is a heat shock protein.

In this study we demonstrate that an immunodominant antigen of Giardia lamblia is a heat shock protein. The expression of the antigen was induced not only by heat shock but also when Giardia trophozoites were placed in media more closely resembling the environment in the host, indicating that this antigen may play an important role in host-parasite interactions.


Solid-phase syntheses of two deacetyl-thymosin alpha 1 analogues with substitution at position 21 and their effects on low E-rosette-forming lymphocytes of uremic patients.

Two deacetyl-thymosin alpha 1 analogues containing Phe or Phe(4F) at position 21 were synthesized by the manual solid-phase method and their immunological effects on the low E-rosette-forming lymphocytes of uremic patients were studied. Fluorination of the p-position of Phe21 resulted in a marked restorative effect on the low E-rosette-forming lymphocytes of uremic patients compared with that of [Phe21]deacetyl-thymosin alpha 1. The synthetic [Phe21]deacetyl-thymosin alpha 1 was approximately equal in potency to our synthetic deacetyl-thymosin alpha 1 in uremic patients.


Transforming growth factor-beta 1 inhibits postischemic increases in splanchnic vascular resistance.

Anesthesized male rabbits having a resting mean arterial pressure of 81 +/- 4 mm Hg and superior mesenteric artery blood flow of 91 +/- 7 mL min-1 were subjected to 60 min of splanchnic ischemia followed by 60 min of reperfusion. Upon reperfusion, mean arterial pressure fell. Splanchnic blood flow also decreased but not in parallel with blood pressure; consequently, vascular resistance was increased over the reperfusion period. This increase in splanchnic vascular resistance was not affected by intravenous t-PA (0.5 mg kg-1 + 5 mg kg-1 hr-1) for 30 min prior to and throughout the reperfusion period or by intravenous L-NAME (1 mg kg-1 x 2). However, intravenous infusions of TGF-beta (18 or 54 micrograms kg-1) at the time of reperfusion dose dependently attenuated the increases in vascular resistance (p < 0.05). This effect of TGF-beta was enhanced by coadministration of t-PA and inhibited by the coadministration of L-NAME. We propose that the effects of TGF-beta are ultimately mediated via nitric oxide release, and conclude that this may be useful therapy for the prevention of reperfusion-associated injury following surgery or as an adjunct to thrombolytic therapy.


Localization of subspecies of protein kinase C in the mammalian central nervous system.

Activation of protein kinase C (PKC) is regulated by dual second messengers; diacylglycerol (DG) produced by receptor mediated hydrolysis of phosphatidylinositol and Ca2+ which is released by inositol 1,4,5-triphosphate (IP3) from intracellular stores in the endoplasmic reticulum. In the mammalian central nervous system, available evidence suggests that PKC plays a prominent role in the processing of neuronal signals and in the short-term or long-term modulation of synaptic transmission. This enzyme is a member of a family consisting of at least eight subspecies, alpha, beta I, beta II, gamma, delta, epsilon, zeta and eta. The homologous structure of each subspecies makes difficult resolution of the enzymological properties of the enzyme. The distinct functional roles of PKC subspecies in mammalian tissues have been elucidated by defining the localization of each subspecies. We identified alpha-, beta I-, beta II- and gamma-PKC subspecies in the rat brain by in situ hybridization and by light and electron microscopic immunohistochemistry, using antibodies specific for each subspecies. Most immunoreactions of the alpha, beta I, beta II and gamma subspecies were evident in neurons and there were few, if any, in glial cells. In this article, we summarize known cellular and subcellular localizations of PKC subspecies in mammalian CNS and some aspects of current studies in neuronal functions regulated by this enzyme are discussed.


Blood-brain barrier transfer of L-Trp and alpha-MTrp in Li-treated rats.

Blood-brain barrier (BBB) transport for L-Trp and alpha-methyl-L-tryptophan was evaluated in Li-treated rats. Five different brain areas as well as left to right differences were examined. No left to right difference in the PS product was observed. Lithium treatment had a significant effect on the plasma concentration of Val, Leu and Ile but no effect on plasma total or free Trp. The ratio of plasma Trp to the sum of Leu, Val, Ile, Phe, Met and Tyr is increased in the Li-treated rats but not significantly. However, the ratio of Trp/(Val+Leu+Ile) is significantly increased in the Li-treated rats. The Km apparent (Kmapp) for the BBB Trp transport is significantly decreased (affinity of the carrier for Trp is increased) in the Li-treated rats. A decrease in the Kmapp is one of the possible factors responsible for an increase in the brain Trp concentration and subsequent increase in the brain serotonin synthesis in Li-treated rats.


Characterization of cocaine-sensitive dopamine uptake in PC12 cells.

Dopamine uptake in rat pheochromocytoma (PC12) cells is a carrier-mediated process which follows Michaelis-Menten kinetics. Uptake was saturable with an apparent Km of 0.71 microM for dopamine and a Vmax of 3.2 pmol/2 x 10(5) cells/min. The rank order of potency for various amines was norepinephrine > or = dopamine > epinephrine. Uptake increased with increasing temperature and showed a sharp break in the Arrhenius plot at 27.5 degrees C. The Q10 was 1.39 above and 2.95 below 27.5 degrees C. Cocaine inhibited uptake in a dose-dependent manner with a Ki of 0.97 microM. The presence of cocaine lowered the apparent Km but did not affect the Vmax, indicating competitive inhibition. Tunicamycin inhibited [3H]dopamine accumulation in a dose- and time-dependent fashion suggesting the dopamine uptake site in PC12 cells is an asparagine-linked glycoprotein. Kinetic analysis showed a decrease in Vmax but not in the apparent Km after tunicamycin treatment, consistent with the notion that tunicamycin treatment results in the loss of a substantial amount of active carrier molecules.


Protease activities in carp retina.

Protease of carp retina were examined by electrophoresis and fluorogenic assays. A 70 kD serine protease with an alkaline pH optimum was detected in gelatin-containing polyacrylamide gels. A similar enzyme was found in carp brain and muscle, but not in lens. Using aminomethylcoumarin (MCA) substrates, activities that hydrolysed Z-Phe-Arg-MCA, Boc-Ala-Gly-Pro-Arg-MCA and various aminoacyl-MCAs were detected. The Z-Phe-Arg-MCA hydrolase was an acidic cysteine protease, whereas the Boc-Ala-Gly-Pro-Arg-MCA hydrolase was an alkaline cysteine protease. All aminoacyl hydrolase activities tested were inhibited by bestatin and o-phenanthroline, but not by inhibitors of serine, cysteine and aspartic proteases, suggesting they are metalloaminopeptidases. Of the substrates tested, Tyr-MCA was the most readily hydrolysed aminoacyl substrate. Preliminary evidence was obtained suggesting that levels of these activities do not differ between light- and dark-adapted retinae. The proteases have a potential involvement in retinal functioning and show similarities to other proteases known to act in the central nervous system. In particular, the Tyr-MCA hydrolase may be related to an enzyme known to remove the N-terminal tyrosine residue from enkephalin.


Regulation of tubulin, Tau and microtubule associated protein 2 expression during mouse brain development.

The level of three microtubule proteins, tubulin, Tau and MAP2 and of their encoding mRNA was studied in the mouse brain at an early developmental stage (3 days postnatal) and in adulthood. The level of the mRNA encoding both tubulin and Tau decreased by 85% between these two stages whereas the encoded proteins decreased only by 50% during the same period. Thus, the level of these proteins seems to be regulated both negatively and positively by transcriptional and post translational mechanisms. In vitro transcription assays, performed with nuclei isolated at different postnatal stages, showed that the tubulin and Tau transcripts are produced with some variations during mouse brain development. However these fluctuations are much less important than the drops of the steady state levels of tubulin and Tau mRNA seen in vivo. Thus, the decrease in transcripts levels does not seem to result from reduced transcriptional activities, and can be ascribed to changes in mRNA stability occurring during brain development, i.e. to a post transcriptional mechanism. The situation is even more complex for MAP2: its encoding mRNA level remains constant during development whereas the in vitro transcription activity decreases markedly during the same period. Finally, MAP2 protein level increases during development although its encoding mRNA level remains constant suggesting that this protein is stabilized by a post translational mechanism.


Grafting of genetically modified cells: effects of acetylcholine release in vivo.

In this study, microdialysis was used to investigate functional recovery of central cholinergic neurons in the forebrain of rats with cortical devascularizing lesions. Mature male rats were unilaterally lesioned by disruption of the pia arachnoid vessels and genetically modified fibroblasts secreting nerve growth factor (NGF) were placed at the site of the lesion. One month following surgery, microdialysis probes were installed in the remaining cortex and were perfused with artificial cerebrospinal fluid (csf) containing neostigmine (5 nM) and/or KCl (100 mM). The basal (non-stimulated) release of acetylcholine (ACh) in the cortex was similar in all experimental groups, whereas KCl stimulated release of ACh was significantly augmented (P < 0.05) in the ipsilateral remaining cortex in lesioned animals that have been implanted with fibroblasts secreting NGF. These results suggest that NGF secreted by genetically engineered fibroblasts modulates neuroplasticity in the adult mammalian CNS and may favour recovery of cortical function following injury.


A novel glycosignaling system: GQ1b-dependent neuritogenesis of human neuroblastoma cell line, GOTO, is closely associated with GQ1b-dependent ecto-type protein phosphorylation.

Previously, we reported that ganglioside GQ1b specifically promoted neuritogenesis of human neuroblastoma cells (GOTO), and also that is specifically stimulated the phosphorylation of several cell surface proteins on the same cells. To disclose the relationship between the two events, we examined them using a novel protein kinase inhibitor, K-252b, which is a derivative of K-252a and cannot pass through cell membrane. K-252b inhibited the GQ1b-dependent neuritogenesis as well as the GQ1b-stimulated phosphorylation. This suggests the direct coupling between the two cell events and the occurrence of a new biosignal transduction system.


Characterization of phospholipase A2 and acyltransferase activities in squid (Loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions.

Phospholipase A2 and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A2 activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A2 was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating. The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. Km (apparent) and Vmax (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide. Phospholipase A2 activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A2 activity was stimulated by Ca++ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca(++)-independent phospholipase A2, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.


Differential stimulation of hepatic and brain metallothioneins by ethanol.

Administration of ethanol induces the synthesis of hepatic metallothionein and metallothionein mRNA in the liver but not in the brain. Furthermore, ethyl alcohol, methyl alcohol and isopropyl alcohol enhance the synthesis of metallothionein in Chang cells but not in neuroblastoma IMR-32 cells in culture. The results of this study are interpreted to suggest that the mechanisms of synthesis of metallothionein and the utilization of essential metal nutrients in the brain and peripheral tissues are not identical.


Striatal quinolinic acid lesions increase [3H]WIN 35,428 binding to the dopamine transporter.

Striatal quinolinic acid lesions have been used as a model for the pathology seen in Huntington's disease. Seven days following a unilateral injection of 100 nmol of quinolinic acid into the rat caudate-putamen (striatum), a large increase in the binding of [3H]WIN 35,428 to the lesioned caudate-putamen was seen when compared to the unlesioned side. Binding in other brain regions was unchanged. These data indicate that there is an increase in dopaminergic uptake sites in the lesioned caudate-putamen and suggest a target for further study in Huntington's disease.


Differential production of prostaglandins D2 and E2 and of an unusual prostanoid by chick spinal cord and meninges in vitro.

In order to specify the source of locally synthesized prostaglandin (PG) E2 which is able to saturate the large class of low affinity PGE2 receptors in chick spinal cord, bioconversion of [1-14C]arachidonic acid into prostanoids was studied in homogenates of chick spinal cord and meninges first without addition of exogenous glutathione (GSH). Homogenates of spinal cord produced 14C-labeled PGE2, PGD2 and PGF2 alpha. Homogenates of meninges accumulated much larger amounts of [14C]PGE2 than spinal cord and surprisingly a 14C-labeled arachidonate metabolite referred to as compound Y. Compound Y generation, which was inhibited by indomethacin and enhanced by esculetin, was therefore mediated through the cyclooxygenase pathway. The fact that no labeled compound Y was detected in homogenates incubated with [3H]PGD2 or [3H]PGE2 indicated that compound Y was not a degradation product of PGs. Secondly, after addition of exogenous GSH, 14C-labeled compound Y was totally converted into [14C]PGE2. The compound Y which is converted into PGFs after a strong reduction with NaBH4 and into PGE2 after a mild reduction with GSH-hemin system or SnCl2 was therefore assumed to be a 15 hydroperoxy-PGE2 (15 HP-PGE2). These results suggest that PGE2 can be synthesized in meninges either by the classical isomerization of PGH2 or by isomerization of PGG2 followed by a GSH-sensitive reaction.


Malnutrition increases insoluble-to-soluble tubulin ratio and in vitro incorporation of 32ATP in rat cerebral cortex.

Wistar rats were fed a normal protein (25% casein) or an isoenergetic low protein (8% casein) diet from the day of birth to weaning on day 21. Litters were killed at weaning and cerebral cortex was removed. Tubulin was prepared by centrifugation at 100,000 g, 4 degrees C, as described by Shelansky et al. [Proc. Natn. Acad. Sci. U.S.A. 70, 765-768 (1973)]. Cold-insoluble tubulin was recovered in the pellet (P1) fraction and cold-soluble tubulin in the supernatant (S1) fraction. Alpha and beta tubulin were quantified by electrophoretic and immunological methods in both fractions. Our results indicated that malnutrition enhanced the ratio of cold-insoluble-tubulin-to-cold-soluble-tubulin. Furthermore malnutrition induced an increased in vitro incorporation of 32P into both soluble and insoluble tubulins. Although tubulin phosphorylation has been related to tubulin stability properties, we cannot unequivocally ascribe the increased insoluble/soluble tubulin ratio with malnutrition to increased in vitro incorporation of 32P.


Time-resolved protein crystallography.

Advances in synchrotron radiation technology have allowed exposure times from protein crystals of the order of milliseconds to be used routinely, and in exceptional circumstances exposure times of 100 ps have been obtained. However, many data sets take seconds to record because of the slow time scale of film change or crystal reorientation or translation when more than one exposure is required. This problem has been addressed by Amemiya et al. (1989). There has been considerable progress in methods to initiate reactions in protein crystals, especially the development of photolabile caged compounds but also temperature jump, pH jump, and diffusion. Although flash lamps deliver pulses of 100 mJ/ms, often several pulses are required to release sufficient product, and reaction initiation can take several seconds. Laser illumination can provide more powerful input, but the laser must be accommodated within the restricted space at the synchrotron station. The requirement to maintain synchrony among the molecules in the crystal lattice as the reaction proceeds and to ensure that the lifetime of intermediates is longer than data collection rates emphasizes the need for chemical characterization of the reaction under study. As Ringe advocated in the studies with chymotrypsin, it may be more profitable to devise conditions under which certain intermediates along the reaction pathway accumulate in the crystal and to record these in a series of discrete steps rather than continuous monitoring of the reaction. The Laue method is limited to those proteins that give well-ordered crystals and problems of transient disorder on initiation of reaction and problems of radiation damage need to be overcome or avoided by suitable experimental protocols.(ABSTRACT TRUNCATED AT 250 WORDS)


Macromolecular crystal growth experiments on International Microgravity Laboratory--1.

Macromolecular crystal growth experiments, using satellite tobacco mosaic virus (STMV) and canavalin from jack beans as samples, were conducted on a US Space Shuttle mission designated International Microgravity Laboratory--1 (IML-1), flown January 22-29, 1992. Parallel experiments using identical samples were carried out in both a vapor diffusion-based device (PCG) and a liquid-liquid diffusion-based instrument (CRYOSTAT). The experiments in each device were run at 20-22 degrees C and at colder temperatures. Crystals were grown in virtually every trial, but the characteristics of the crystals were highly dependent on the crystallization technique employed and the temperature experience of the sample. In general, very good results, based on visual inspection of the crystals, were obtained in both PCG and CRYOSTAT. Unusually impressive results were, however, achieved for STMV in the CRYOSTAT instrument. STMV crystals grown in microgravity by liquid-liquid diffusion were more than 10-fold greater in total volume than any STMV crystals previously grown in the laboratory. X-ray diffraction data collected from eight STMV crystals grown in CRYOSTAT demonstrated a substantial improvement in diffraction quality over the entire resolution range when compared to data from crystals grown on Earth. In addition, the extent of the diffraction pattern for the STMV crystals grown in space extended to 1.8 A resolution, whereas the best crystals that were ever grown under conditions of Earth's gravity produced data limited to 2.3 A resolution. Other observations indicate that the growth of macromolecular crystals is indeed influenced by the presence or absence of gravity. These observations further suggest, consistent with earlier results, that the elimination of gravity provides a more favorable environment for such processes.


Correlation functions as a tool for protein modeling and structure analysis.

Proteins present unique folding structures whose conformations are determined primarily by their amino acid sequences. At present, there is no algorithm that would correlate the sequences with the structures determined by X-ray analysis or NMR. Comparative modeling of a new protein sequence based on the known structure of a functionally related protein promises to yield model structures that may provide relevant properties of the protein. To analyze the quality of a model structure, a set of correlation functions was derived from calculations on a subset of proteins from the structure database. Twenty-three highly resolved protein structures with resolutions of at least 1.7 A from various protein families were used as the primary database. The purpose of this initial work was to find highly sensitive functions (including statistical error limits for the parameters) that describe properties of "real" proteins. Each correlation described is characterized by the correlation coefficient, the parameters for linear or nonlinear regression (coefficients of the equation), standard deviation and variance, and the confidence limits describing the statistical probability for values to occur within these limits, e.g., the natural variability of the property under examination. In addition, a method was developed for creating reasonably misfolded proteins. The ability of a correlation function to discriminate between the native structure and the misfolded conformations is expressed by the reliability index, which indicates the sensitivity of a correlation function. The term correlation functions thus summarizes a variety of efforts to find a mathematical description for the properties of protein structures, for their correlation, and for their significance.


On the multiple simultaneous superposition of molecular structures by rigid body transformations.

A method of optimally superimposing n coordinate sets on each other by rigid body transformations, which minimizes the sum of all n (n - 1)/2 pairwise residuals, is presented. In the solution phase the work load is approximately linear on n, is independent of the size of the structures, is independent of their initial orientations, and terminates in one cycle if n = 2 of if the coordinate sets are exactly superposable, and otherwise takes a number of cycles dependent only on genuine shape differences. Enantiomorphism, if present, is detected, in which case the option exists to reverse or not to reverse the chirality of relevant coordinate sets. The method also offers a rational approach to the problem of multiple minima and has successfully identified four distinct minima in such a case. Source code, which is arranged to enable the study of the disposition of domains in multidomain structures, is available from the author.


Disulfide bridges in tomato pectinesterase: variations from pectinesterases of other species; conservation of possible active site segments.

Analysis of tomato pectinesterase by carboxymethylation, with and without reduction, shows that the enzyme has two intrachain disulfide bridges. Analysis of fragments obtained from the native enzyme after digestion with pepsin identified bridges connecting Cys-98 with Cys-125, and Cys-166 with Cys-200. The locations of disulfide bridges in tomato pectinesterase are not identical to those in three distantly related pectinesterases (18-33% residue identities) from microorganisms. However, one half-Cys (i.e., Cys-166) position is conserved in all four enzymes. Sequence comparisons of the overall structures suggest a special importance for three short segments of the entire protein. One segment is at the N-terminal part of the tomato pectinesterase, another in the C-terminal portion near the distal end of the second disulfide loop, and the third segment is located in the central part between the two disulfide bridges. The latter segment, encompassing only 40 residues of the entire protein, appears to high-light a functional site in a midchain segment.


Specificity mapping of cellulolytic enzymes: classification into families of structurally related proteins confirmed by biochemical analysis.

The specificities of 15 cellulolytic enzymes have been examined using chromophoric glycosides derived from D-glucose, cellobiose, higher cellooligosaccharides, lactose, D-xylose, and beta-(1,4)-xylobiose. Coinciding with a classification based on hydrophobic cluster analysis of amino acid sequences, six families each showing a characteristic specificity pattern were observed. Furthermore, in these cases where the anomeric forms of reaction products were determined, results seem to indicate conservation of intrinsic reaction mechanism (single or double displacement) within each family. On the other hand, the low molecular weight substrates do not discriminate exo- from endocellulases. This functional differentiation is speculated to originate from the presence, in exoenzymes, of a tunnel-shaped active site formed by extra loops in their structure.


Inactivation and covalent modification of CTP synthetase by thiourea dioxide.

Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.


Unfolding domains of recombinant fusion alpha alpha-tropomyosin.

The thermal unfolding of the coiled-coil alpha-helix of recombinant alpha alpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 degrees C involving the middle of the molecule; (2) a major transition at 46 degrees C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56 degrees C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70 degrees C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 degrees C or 70 degrees C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a cross-link to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46 degrees C from the stabilized N-terminal unfolding domain at 56 degrees C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.


A new subfamily of bacterial ABC-type transport systems catalyzing export of drugs and carbohydrates.

Sequence comparison studies revealed that the drug resistance transporter of Streptomyces peucetius (DrrAB) and two nodulation gene products (NodIJ) of Rhizobium leguminosarum are homologous to proteins encoded by three sets of genes that comprise capsular polysaccharide export systems in gram-negative bacteria: KpsTM of Escherichia coli, BexABC of Haemophilus influenzae, and CtrDCB of Neisseria meningitidis. These five systems comprise a new subfamily within the family of ATP binding cassette (ABC)-type transporters. We have termed this subfamily the ABC-2 subfamily. For three of the systems comprising this subfamily (Drr, Nod, and Kps) only one integral membrane constituent has been identified, whereas for the other two systems (Bex and Ctr) two dis-similar integral membrane constituents have been found. This observation suggests that the transmembrane channels of ABC-2-type transporters can be formed of homo- or heterooligomers as is true of several other classes of transport systems.


Differences in the amino acid distributions of 3(10)-helices and alpha-helices.

Local determinants of 3(10)-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3(10)-helices and multiple-turn conformations, had strong amino acid sequence patterns not evident among alpha-helices. Aspartate occurred over twice as frequently in the N-cap position of 3(10)-helices as in the N-cap position of alpha-helices. Unlike alpha-helices, 3(10)-helices had few C-termini ending in a left-handed alpha conformation; most 3(10) C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3(10)- and alpha-helices were also apparent, producing amphipathic 3(10)-helices. Local interactions that stabilize 3(10)-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of alpha-helices have been postulated to proceed through reverse-turn and 3(10)-helix intermediates, sequence differences between 3(10)- and alpha-helices can also lend insight into factors influencing alpha-helix initiation and propagation.


NMR analysis of regioselectivity in dephosphorylation of a triphosphotyrosyl dodecapeptide autophosphorylation site of the insulin receptor by a catalytic fragment of LAR phosphotyrosine phosphatase.

An autophosphorylation site in the activated insulin receptor tyrosine kinase domain has three tyrosines phosphorylated when fully activated. To begin to examine recognition of triphosphotyrosyl sites by protein tyrosine phosphatases in possible control of signal transduction a triphosphotyrosyl dodecapeptide TRDIpYETDpYpYRK corresponding to residues 1,142-1,153 of the insulin receptor was prepared and incubated with the 40-kDa catalytic domain of the human PTPase LAR. To assess regioselectivity of recognition, the three diphosphotyrosyl regioisomers, and the three monophosphotyrosyl regioisomers were prepared and assayed. All seven peptides were PTPase substrates. To identify any preferences in dephosphorylation at pY5, pY9, or pY10, 1H-NMR analyses were conducted during enzyme incubations and distinguishing fingerprint regions determined for each of the seven phosphotyrosyl peptides. LAR PTPase shows strong preference for dephosphorylation first at pY5 (at tri-, di-, and monophosphotyrosyl levels). Initially this regioselectivity gives the Y5(pY9)(pY10) diphospho regioisomer, followed by equal dephosphorylation at pY9 or pY10 to give the corresponding monophosphoryl species on the way to fully dephosphorylated product. The NMR methodology is applicable to other peptides with multiple sites of phosphorylation that undergo attack by any phosphatase.


Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two-dimensional NMR spectroscopy.

The solution structure of the phosphocarrier protein, HPr, from Bacillus subtilis has been determined by analysis of two-dimensional (2D) NMR spectra acquired for the unphosphorylated form of the protein. Inverse-detected 2D (1H-15N) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann-Hahn (HOHAHA) spectra utilizing 15N assignments (reported here) as well as previously published 1H assignments were used to identify cross-peaks that are not resolved in 2D homonuclear 1H spectra. Distance constraints derived from NOESY cross-peaks, hydrogen-bonding patterns derived from 1H-2H exchange experiments, and dihedral angle constraints derived from analysis of coupling constants were used for structure calculations using the variable target function algorithm, DIANA. The calculated models were refined by dynamical simulated annealing using the program X-PLOR. The resulting family of structures has a mean backbone rmsd of 0.63 A (N, C alpha, C', O atoms), excluding the segments containing residues 45-59 and 84-88. The structure is comprised of a four-stranded antiparallel beta-sheet with two antiparallel alpha-helices on one side of the sheet. The active-site His 15 residue serves as the N-cap of alpha-helix A, with its N delta 1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme I. The existence of a hydrogen bond between the side-chain oxygen atom of Tyr 37 and the amide proton of Ala 56 is suggested, which may account for the observed stabilization of the region that includes the beta-turn comprised of residues 37-40. If the beta alpha beta beta alpha beta (alpha) folding topology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of beta alpha beta beta alpha beta proteins that include acylphosphatase and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins.


Total chemical synthesis, characterization, and immunological properties of an MHC class I model using the TASP concept for protein de novo design.

The design, total chemical synthesis, and immunological properties of a four-alpha-helix bundle template-assembled synthetic protein (TASP) mimicking some of the structural features of the major histocompatibility complex (MHC) class I is described. In a first approach, the native sequence 58-74 of the alpha 1 heavy chain domain of HLA-A2 was modeled in order to increase helix stability and amphiphilicity of the 17-mer peptide, preserving the residues for potential T-cell receptor (TcR) binding properties. According to the TASP concept, these helical segments were covalently attached to a cyclic template molecule designed for the induction of a four-helix-bundle topology of the assembled peptide blocks. After extensive HPLC purification, stepwise solid-phase synthesis resulted in a TASP molecule of high chemical purity as demonstrated by analytical HPLC, mass spectrometry, and amino acid analysis. CD spectroscopic investigations are consistent with the onset of a partial alpha-helical conformation in aqueous buffer as well as in TFE. Antibodies raised directly against this four-alpha-helix bundle TASP molecule (without prior conjugation to a carrier molecule) were detected by ELISA. Flow cytometry studies showed that these antibodies recognize the native MHC class I molecule on the surface of HLA-A2-positive cells. The results indicate that the TASP approach represents a versatile tool for mimicking conformational epitopes.


Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA-binding domain as studied by amide proton exchange.

Backbone amide proton exchange rates in the DNA-binding domain of GAL4 have been determined using 1H-15N heteronuclear correlation NMR spectroscopy. Three forms of the protein were studied-the native Zn-containing protein, the Cd-substituted protein, and a Zn-GAL4/DNA complex. Exchange rates in the Zn-containing protein are significantly slower than in the Cd-substituted protein. This shows that Cd-substituted GAL4 is destabilized relative to the native Zn-containing protein. Upon DNA binding, global retardation of amide proton exchange with solvent was observed, indicating that internal fluctuations of the DNA-recognition module are significantly reduced by the presence of DNA. In all forms of the protein, the internal dyad symmetry of the DNA-recognition module of GAL4 is reflected by the backbone amide proton exchange rates.


Structure of the oxidized long-chain flavodoxin from Anabaena 7120 at 2 A resolution.

The structure of the long-chain flavodoxin from the photosynthetic cyanobacterium Anabaena 7120 has been determined at 2 A resolution by the molecular replacement method using the atomic coordinates of the long-chain flavodoxin from Anacystis nidulans. The structure of a third long-chain flavodoxin from Chondrus crispus has recently been reported. Crystals of oxidized A. 7120 flavodoxin belong to the monoclinic space group P2(1) with a = 48.0, b = 32.0, c = 51.6 A, and beta = 92 degrees, and one molecule in the asymmetric unit. The 2 A intensity data were collected with oscillation films at the CHESS synchrotron source and processed to yield 9,795 independent intensities with Rmerg of 0.07. Of these, 8,493 reflections had I > 2 sigma and were used in the analysis. The model obtained by molecular replacement was initially refined by simulated annealing using the XPLOR program. Repeated refitting into omit maps and several rounds of conjugate gradient refinement led to an R-value of 0.185 for a model containing atoms for protein residues 2-169, flavin mononucleotide (FMN), and 104 solvent molecules. The FMN shows many interactions with the protein with the isoalloxazine ring, ribityl sugar, and the 5'-phosphate. The flavin ring has its pyrimidine end buried into the protein, and the functional dimethyl benzene edge is accessible to solvent. The FMN interactions in all three long-chain structures are similar except for the O4' of the ribityl chain, which interacts with the hydroxyl group of Thr 88 side chain in A. 7120, while with a water molecule in the other two. The phosphate group interacts with the atoms of the 9-15 loop as well as with NE1 of Trp 57. The N5 atom of flavin interacts with the amide NH of Ile 59 in A. 7120, whereas in A. nidulans it interacts with the amide NH of Val 59 in a similar manner. In C. crispus flavodoxin, N5 forms a hydrogen bond with the side chain hydroxyl group of the equivalent Thr 58. The hydrogen bond distances to the backbone NH groups in the first two flavodoxins are 3.6 A and 3.5 A, respectively, whereas in the third flavodoxin the distance is 3.1 A, close to the normal value. Even though the hydrogen bond distances are long in the first two cases, still they might have significant energy because their microenvironment in the protein is not accessible to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)


Arginine 54 in the active site of Escherichia coli aspartate transcarbamoylase is critical for catalysis: a site-specific mutagenesis, NMR, and X-ray crystallographic study.

The replacement of Arg-54 by Ala in the active site of Escherichia coli aspartate transcarbamoylase causes a 17,000-fold loss of activity but does not significantly influence the binding of substrates or substrate analogs (Stebbins, J.W., Xu, W., & Kantrowitz, E.R., 1989, Biochemistry 28, 2592-2600). In the X-ray structure of the wild-type enzyme, Arg-54 interacts with both the anhydride oxygen and a phosphate oxygen of carbamoyl phosphate (CP) (Gouaux, J.E. & Lipscomb, W.N., 1988, Proc. Natl. Acad. Sci. USA 85, 4205-4208). The Arg-54-->Ala enzyme was crystallized in the presence of the transition state analog N-phosphonacetyl-L-aspartate (PALA), data were collected to a resolution limit of 2.8 A, and the structure was solved by molecular replacement. The analysis of the refined structure (R factor = 0.18) indicates that the substitution did not cause any significant alterations to the active site, except that the side chain of the arginine was replaced by two water molecules. 31P-NMR studies indicate that the binding of CP to the wild-type catalytic subunit produces an upfield chemical shift that cannot reflect a significant change in the ionization state of the CP but rather indicates that there are perturbations in the electronic environment around the phosphate moiety when CP binds to the enzyme. The pH dependence of this upfield shift for bound CP indicates that the catalytic subunit undergoes a conformational change with a pKa approximately 7.7 upon CP binding. Furthermore, the linewidth of the 31P signal of CP bound to the Arg-54-->Ala enzyme is significantly narrower than that of CP bound to the wild-type catalytic subunit at any pH, although the change in chemical shift for the CP bound to the mutant enzyme is unaltered. 31P-NMR studies of PALA complexed to the wild-type catalytic subunit indicate that the phosphonate group of the bound PALA exists as the dianion at pH 7.0 and 8.8, whereas in the Arg-54-->Ala catalytic subunit the phosphonate group of the bound PALA exists as the monoanion at pH 7.0 and 8.8. Thus, the side chain of Arg-54 is essential for the proper ionization of the phosphonate group of PALA and by analogy the phosphate group in the transition state. These data support the previously proposed proton transfer mechanism, in which a fully ionized phosphate group in the transition state accepts a proton during catalysis.


A molecular model for membrane fusion based on solution studies of an amphiphilic peptide from HIV gp41.

The mechanism of protein-mediated membrane fusion and lysis has been investigated by solution-state studies of the effects of peptides on liposomes. A peptide (SI) corresponding to a highly amphiphilic C-terminal segment from the envelope protein (gp41) of the human immunodeficiency virus (HIV) was synthesized and tested for its ability to cause lipid membranes to fuse together (fusion) or to break open (lysis). These effects were compared to those produced by the lytic and fusogenic peptide from bee venom, melittin. Other properties studied included the changes in visible absorbance and mean particle size, and the secondary structure of peptides as judged by CD spectroscopy. Taken together, the observations suggest that protein-mediated membrane fusion is dependent not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form an amphiphilic structure that promotes the mixing of the lipids between membranes. A speculative molecular model is proposed for membrane fusion by alpha-helical peptides, and its relationship to the forces involved in protein-membrane interactions is discussed.


Modeling the antigen combining site of an anti-dinitrophenyl antibody, ANO2.

A model structure has been constructed for a monoclonal anti-dinitrophenyl antibody. The antibody, ANO2, has been sequenced and cloned (Anglister, J., Frey, T., & McConnell, H.M., 1984, Biochemistry 23, 1138-1142). Its amino acid sequence shows striking homology with the anti-lysozyme Fab fragments HyHel5 (83%) and HyHel10 (73%). Based on this homology, a model for the ANO2 variable heavy and variable light chain framework was constructed using a hybrid of the HyHel5 light chain and the HyHel10 heavy chain backbone, omitting the hypervariable loops. These coordinates were used as scaffolds for the model building of ANO2. The CONGEN conformational sampling algorithm (Bruccoleri, R.E. & Karplus, M., 1987, Biopolymers 26, 127-196) was used to model the six hypervariable loops that contain the antigen-combining site. All the possible conformations of the loop backbones were constructed and the best loop structures were selected using a combination of the CHARMM potential energy function and evaluation of the solvent-accessible surface area of the conformers. The order in which the loops were searched was carried out based on the relative locations of the loops with reference to the framework of the beta-barrel, namely, L2-H1-L3-H2-H3-L1. The model structures thus obtained were compared to the high resolution X-ray structure (Brünger, A.T., Leahy, D.J., Hynes, T.R., & Fox, R.O., 1991, J. Mol. Biol. 221, 239-256).


Conformational stability of porcine serum transferrin.

The conformation of porcine serum ferric transferrin (Tf) and its stability against denaturation were studied by circular dichroism. Tf was estimated to have 19-24% alpha-helix and 50-55% beta-sheet based on the methods of Chang et al. (Chang, C.T., Wu, C.-S.C., & Yang, J.T., 1978, Anal. Biochem. 91, 13-31) and Provencher and Glöckner (Provencher, S.W. & Glöckner, J., 1981, Biochemistry 20, 33-37). Removal of the bound ferric ions (apo-Tf) did not alter the overall conformation, but there were subtle changes in local conformation based on its near-UV CD spectrum. The Tfs were stable between pH 3.5 and 11. Denaturation by guanidine hydrochloride (Gu-HCl) showed two transitions at 1.6 and 3.4 M denaturant. The process of denaturation by acid and base was reversible, whereas that by Gu-HCl was partially reversible. The irreversible thermal unfolding of Tfs began at temperatures above 60 degrees C and was not complete even at 80 degrees C. The bound irons (based on absorbance at 460 nm) were completely released at pH < 4 or in Gu-HCl solution above 1.7 M, when the protein began to unfold, but they remained intact in neutral solution even at 85 degrees C. The NH2- and COOH-terminal halves of the Tf molecule obtained by limited trypsin digestion had CD spectra similar to the spectrum of native Tf, and the COOH-terminal fragment had more stable secondary structure than the NH2-terminal fragment.


Kinetic studies of the refolding of yeast phosphoglycerate kinase: comparison with the isolated engineered domains.

Unfolding and refolding kinetics of yeast phosphoglycerate kinase were studied by following the time-dependent changes of two signals: the ellipticity at 218 nm and 222 nm, and the fluorescence emission at 330 nm (following excitation at 295 nm). The protein is composed of two similar-sized structural domains. Each domain has been produced by recombinant DNA techniques. It has been previously demonstrated that the engineered isolated domains are able to fold into a quasinative structure (Minard, P., et al., 1989b, Protein Eng. 3, 55-60; Missiakas, D., Betton, J.M., Minard, P., & Yon, J.M., 1990, Biochemistry 29, 8683-8689). The behavior of the isolated domains was studied using the same two conformational probes as for the whole enzyme. We found that the refolding kinetics of each domain are multiphasic. In the whole protein, domain folding and pairing appeared to be simultaneous events. However, it was found that some refolding steps occurring during the refolding of the isolated C-domain are masked during the refolding of yeast phosphoglycerate kinase. The N-domain was also found to refold faster when it was isolated than when integrated.


X-ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium Pyrococcus furiosus.

The structures of the oxidized and reduced forms of the rubredoxin from the archaebacterium, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C, have been determined by X-ray crystallography to a resolution of 1.8 A. Crystals of this rubredoxin grow in space group P2(1)2(1)2(1) with room temperature cell dimensions a = 34.6 A, b = 35.5 A, and c = 44.4 A. Initial phases were determined by the method of molecular replacement using the oxidized form of the rubredoxin from the mesophilic eubacterium, Clostridium pasteurianum, as a starting model. The oxidized and reduced models of P. furiosus rubredoxin each contain 414 nonhydrogen protein atoms comprising 53 residues. The model of the oxidized form contains 61 solvent H2O oxygen atoms and has been refined with X-PLOR and TNT to a final R = 0.178 with root mean square (rms) deviations from ideality in bond distances and bond angles of 0.014 A and 2.06 degrees, respectively. The model of the reduced form contains 37 solvent H2O oxygen atoms and has been refined to R = 0.193 with rms deviations from ideality in bond lengths of 0.012 A and in bond angles of 1.95 degrees. The overall structure of P. furiosus rubredoxin is similar to the structures of mesophilic rubredoxins, with the exception of a more extensive hydrogen-bonding network in the beta-sheet region and multiple electrostatic interactions (salt bridge, hydrogen bonds) of the Glu 14 side chain with groups on three other residues (the amino-terminal nitrogen of Ala 1; the indole nitrogen of Trp 3; and the amide nitrogen group of Phe 29). The influence of these and other features upon the thermostability of the P. furiosus protein is discussed.


Solution-state structure by NMR of zinc-substituted rubredoxin from the marine hyperthermophilic archaebacterium Pyrococcus furiosus.

The three-dimensional solution-state structure is reported for the zinc-substituted form of rubredoxin (Rd) from the marine hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. Structures were generated with DSPACE by a hybrid distance geometry (DG)-based simulated annealing (SA) approach that employed 403 nuclear Overhauser effect (NOE)-derived interproton distance restraints, including 67 interresidue, 124 sequential (i-j = 1), 75 medium-range (i-j = 2-5), and 137 long-range (i-j > 5) restraints. All lower interproton distance bounds were set at the sum of the van Der Waals radii (1.8 A), and upper bounds of 2.7 A, 3.3 A, and 5.0 A were employed to represent qualitatively observed strong, medium, and weak NOE cross peak intensities, respectively. Twenty-three backbone-backbone, six backbone-sulfur (Cys), two backbone-side chain, and two side chain-side chain hydrogen bond restraints were include for structure refinement, yielding a total of 436 nonbonded restraints, which averages to > 16 restraints per residue. A total of 10 structures generated from random atom positions and 30 structures generated by molecular replacement using the backbone coordinates of Clostridium pasteurianum Rd converged to a common conformation, with the average penalty (= sum of the square of the distance bounds violations; +/- standard deviation) of 0.024 +/- 0.003 A2 and a maximum total penalty of 0.035 A2. Superposition of the backbone atoms (C, C alpha, N) of residues A1-L51 for all 40 structures afforded an average pairwise root mean square (rms) deviation value (+/- SD) of 0.42 +/- 0.07 A. Superposition of all heavy atoms for residues A1-L51, including those of structurally undefined external side chains, afforded an average pairwise rms deviation of 0.72 +/- 0.08 A. Qualitative comparison of back-calculated and experimental two-dimensional NOESY spectra indicate that the DG/SA structures are consistent with the experimental spectra. The global folding of P. furiosus Zn(Rd) is remarkably similar to the folding observed by X-ray crystallography for native Rd from the mesophilic organism C. pasteurianum, with the average rms deviation value for backbone atoms of residues A1-L51 of P. furiosus Zn(Rd) superposed with respect to residues K2-V52 of C. pasteurianum Rd of 0.77 +/- 0.06 A. The conformations of aromatic residues that compose the hydrophobic cores of the two proteins are also similar. However, P. furiosus Rd contains several unique structural elements, including at least four additional hydrogen bonds and three potential electrostatic interactions.(ABSTRACT TRUNCATED AT 400 WORDS)


Comparison of the X-ray structure of native rubredoxin from Pyrococcus furiosus with the NMR structure of the zinc-substituted protein.

The three-dimensional X-ray structures of the oxidized and reduced forms of rubredoxin from Pyrococcus furiosus, determined at -161 degrees C, and the NMR structure of the zinc-substituted protein, determined in solution at 45 degrees C, are compared. The NMR and X-ray structures, which were determined independently, are very similar and lead to similar conclusions regarding the interactions that confer hyperthermostability.


[Effect of loratadine--selective antagonist of histamine (H1) receptor--on allergen-induced bronchoconstriction in atopic asthmatics].

The aim of this study was to evaluate the effect of loratadine on allergen-induced bronchoconstriction. The study was performed in 7 asthmatic patients with sensitivity to Dermatophagoides pteronyssinus, 4 women and 3 men. aged from 19 to 37 years. The allergen inhalatory--challenge test was performed according to Chai et al. Early phase of Dermatophagoides pteronyssinus--induced bronchoconstriction appeared in all examined patients, but late phase of bronchial obstruction was observed only in 3 persons. 30 mg orally administered of loratadine have no protect effect on early phase of allergen--induced bronchoconstriction. After loratadine ingestion the late phase of this bronchial obstruction disappeared.


[Effect of loratadine (LO), a selective H1 antagonist, on histamine-induced bronchoconstriction].

The influence of single and repeated LO (10 mg/day) doses on histamine-induced bronchoconstriction was studied in 11 patients with stabile bronchial asthma. Bronchial reactivity was estimated as histamine content potent to elicit a 20% FEV, fall, i.e. PC20H. A single dose gave a significant (5.5 fold) reduction in bronchial reactivity to inhaled histamine. Application of LO during 6 days gave a similar effect. Withdrawal of LO for 6 days yielded an increase in bronchial hyperreactivity.


[Effect of cetirizine, selective H1 antagonist of histamine on skin and bronchial reactivity and cellular histamine release in allergic diseases].

The study aimed at evaluating an effect of cetirizine--selective antagonist of histamine H1 receptor--on skin and bronchial reaction to histamine, and histamine release from the cells by pollen antigen and anti-IgE. Skin reactivity was tested thrice in 33 patients treated with Zyrtec (Polfa) for the chronic urticaria or allergic rhinitis. Reaction of the bronchi to histamine was tested with a technique described by Ryan et al in 10 asthmatic patients. As effect of cetirizine on histamine release from neutrophils was evaluated in patients with pollinosis before and on the fourth day of therapy. It was shown that the treatment with Zyrtec markedly decreases skin reaction to histamine. Single dose of drug administered 4 hours before the tests markedly restricts reactivity of the bronchi. Daily dose of 10 mg administered to patients with pollinosis stabilizes membranes of neutrophils.


[Cetirizine for treating allergic seasonal rhinitis].

In 14 persons suffering from allergic, seasonal rhinitis the efficacy of 14-days treatment of cetirizine (Zyrtec 10 mg/day) was evaluated. The study was performed in double blind randomised method. Cetirizine was significantly more effective than placebo in reducing the clinical symptoms of rhinitis near in 60% patients. The improvement of nasal mucosa state in laryngological investigation was observed in patients who were better clinically. The inhalatory challenge test with histamine in 9 patients was positive and after cetirizine treatment the bronchial hyperreactivity disappeared.


[The effect of muscarinic receptor blockers on non-specific bronchial reactivity in patients with bronchial asthma].

The effect of two anticholinergic drugs, Ipratropium bromide, nonselective muscarinic receptor antagonist and Pirenzepine, a selective M1 receptor blocker on bronchospasm induced by metacholine and histamine was investigated. The studies were carried out in a group of 52 asthmatic patients. The provocation tests were made by the Ryan method using a De Vilbiss nebuliser connected to a Rosenthal-French dosimeter. It was shown that both drugs protect significantly metacholine induced bronchospasm, but Ipratropium was more effective than Pirenzepine, which suggest that blockade of the M1 receptors only is not sufficient for a complete limitation of bronchi response to metacholine challenge. After histamine provocation some limitation of bronchial reactivity by two investigated drugs was also observed, but not so marked as after metacholine provocation. No significant differences between the effects of Pirenzepine and Ipratropium on histamine induced bronchospasm were observed.


[Late effort induced asthmatic response in patients with atopic bronchial asthma after administration of cromoglycate disodium with concurrent evaluation of neutrophil chemotactic factor (NCG) in serum].

To evaluate an effect of Intal on the late post-provocation bronchospasm in 30 patients with atopic asthma (with early bronchospasm) the treatment was accompanied by the assessment of NCF activity (as a marker of mastocytes degranulation. It was shown that after therapy with Intal exerted an effect on the NCF activity not only in the early but also late bronchospasm in comparison with its activity in patients after exercise test but prior to therapy--9/22 and 6/29. The authors share an opinion tha the presence of NCF in part of patients with the late postexercise bronchospasm (treated with Intal) may indicate that its origin is different (not from mastocytes).


[Clinical evaluation of procaterol and salbutamol in patients with bronchial asthma].

When the beta-2-selectivity of beta-agonists is assessed in man it is always considerably less than would be expected from studies on isolated tissues. That was a reason why this study was taken. As we know procaterol has been included into group of beta-2-selective non-catecholamines with a reasonably long duration of action. We performed clinical evaluation of procaterol and salbutamol on 24 patients with bronchial asthma. Procaterol was administered per os (tabl. a 50 micrograms twice daily) during 4 weeks, and next salbutamol was replaced above mentioned (tabl. a 8 mg twice daily) also during 4 weeks. Clinical symptoms and spirometric values were analyzed. Obtained results indicated that procaterol and salbutamol showed a similar potency in spirometric examination in patients with bronchial asthma. Observed side effects after procaterol were less often than after salbutamol.


[On the late post-provocation spasm of the bronchi].

To assess the incidence of the late reaction of the bronchi in patients with atopic asthma the following methods of the provocation with atopic asthma the following methods of the provocation were applied: a) exercise test, b) a 3-minute ventilation with cold air and c) inhalation of the distilled water Disturbances of ventilation were assessed spirographically. An emphasis was also on the activity of NCF in the blood serum following the applied provocation tests. It was found that the late bronchospasm followed the exercise test in about half of the examined patients (48%). It was less frequent after hyperventilation with cold air (33%) and inhalation of the distilled water (38%). Bronchospasm was accompanied by the increase in NCF activity in the blood serum. The late bronchospasm after provocation tests in asthmatics is a real fact, not depending on the accidental ventilation disorders.


[Effect of cyclosporine A on the clinical state and spirometric parameters in patients with steroid-dependent bronchial asthma].

The study aimed at assessing an effect of Cs A on spirometric indices in 30 patients with steroid-dependent bronchial asthma. All patients were treated with triamcinolone in a mean daily dose of 6-8 mg for at least 5 years. Half of patients were given in a double blind trial cyclosporin in a daily dose of 100 mg for 2 years. The remaining 15 patients were not given cyclosporin. All patients were also given corticosteroids, methylxanthines, and mucolytics. Statistically significant increase in FVC, FEV1, and PEFR was noted in patients treated with cyclosporin after 2 years while no change was seen in patients not given Cs. At the same time, a reduction of corticoids doses and diminished of post-steroid symptoms were noted in patients treated with cyclosporin.


[The effect of peat preparation on the release of histamine from basophils of atopic bronchial asthma].

Antigen-induced basophil histamine releasing (BHR) is an in vitro model of the immediate-type allergic reaction. We studied the influence of immunologically active peat preparation (PTT). On BHR induced with specific antigen or Con A. The highest concentration of PTT enhanced, but lower concentrations decreased antigen-induced BHR. Surprisingly, similar effect was observed with placebo, suggestions that immunologically inactive components of both preparations may be responsible for the modulatory activity.


[Effect of long term systemic steroid therapy on the cardiac muscle].

In asthmatic patients treated on a long term basis with steroids echocardiographic examination of the heart disclosed morphological and functional changes in the heart muscle. Such changes were not found in asthmatic treated occasionally with steroids. Differences between both groups depended on decreased contractibility of the heart muscle and concentric hypertrophy of the left ventricle present in asthmatic treated on a long term basis with systemic steroids.


[Paradoxic bronchial constriction after inhalation of fenoterol and ipratropium bromide].

Two cases of paradoxic bronchial constriction after inhalation of fenoterol and ipratropium bromide are presented. Both patients had bronchial asthma. In the first case the inhalation of fenoterol produced dyspnea and a decrease of FEV1 and FEF25-75. In the second similar observations were made after inhalation of ipratropium bromide. The above mentioned observations illustrate the risk of drug related paradoxic reactions, especially in atopic patients.


[Clinical and biochemical correlations in certain metabolic myopathies].

Muscular glycogenosis is a disease resulting from genetical abnormalities altering an enzyme which is involved in glycogen metabolism. In addition to disorders of glycogenosis and glycolysis, there are other pathological processes such as alpha-glycosidase deficiency and diseases associated with abnormal polysaccharide structure. A short review of the various diseases with their particular features is reported.


[Regulation of kidney hemodynamics. Role of endothelial factors].

The vascular endothelium produces relaxing (PGI2 and EDRF) and contracting (endothelin) factors of the vascular smooth muscle cells. Among these, EDRF, recently identified as NO, is most important. Indeed, NO is continuously formed in the endothelial cells and can thus maintain a state of active and permanent vasodilatation. Consequently, NO may also set the systemic arterial pressure. Our experimental observations suggest that NO sets approximately 50% of the vasomotor tone of the arterial circulation including the renal circulation. In this regard, the activity of NO is not indispensable to renal blood flow autoregulation. Alterations of NO's and/or other endothelial factors' synthesis occur in a variety of cardio-vascular and renal diseases. A better understanding of the physiology and pathophysiology of the vascular endothelium will certainly allow to consider new and exciting therapeutic perspectives in these areas.


The early nodulin gene SrEnod2 from Sesbania rostrata is inducible by cytokinin.

The structure and expression of the early nodulin gene Enod2 from the stem-nodulated tropical legume Sesbania rostrata (SrEnod2) was examined. Genomic clones carrying the single SrEnod2locus were isolated and the DNA sequence of the gene was determined. The SrEnod2 gene was found to lack introns and to encode a protein consisting primarily of a 55-fold repeat of short proline-rich oligopeptides. A putative signal sequence, which may be responsible for targeting of the Enod2 protein to the cell wall, was found to precede this repeat. The temporal expression of the SrEnod2 gene was found to be different in S. rostrata stem versus root nodules induced by Azorhizobium caulinodans ORS571. SrEnod2 gene expression was shown to be induced specifically and rapidly by physiologically significant concentrations of exogenously supplied cytokinins. The SrEnod2 gene was also found to be highly expressed in S. rostrata crown gall tumors induced by wild-type Agrobacterium tumefaciens strains, but not in tumors induced by an A. tumefaciens strain carrying a mutation in the cytokinin biosynthesis gene 4. Implications of these observations with regard to cytokinin-induced plant gene expression and a possible role for cytokinin as a symbiotic signal are discussed.


Molecular characterization of glutathione reductase cDNAs from pea (Pisum sativum L.).

A cDNA for pea glutathione reductase has been cloned and sequenced. The derived amino acid sequence of 562 residues shows a high degree of homology to the previously published GR sequences from human erythrocytes and from two prokaryotes: Escherichia coli and Pseudomonas aeruginosa. The pea enzyme differs from other GRs in having an N-terminal leader sequence of about 60-70 residues which may be a chloroplast transit peptide and a 20 amino acid C-terminal extension of unknown function.


Analysis of large DNA from soybean (Glycine max L. Merr.) by pulsed-field gel electrophoresis.

The technique of pulsed-field gel electrophoresis (PFE) has been used to study chromosomal regions and entire genomes of several organisms. Techniques are presented for the isolation of high molecular weight DNA from embedded soybean protoplasts and the conditions for separating large DNA fragments using PFE. Digestion was detected by Southern hybridization using single copy nodulin clones. These data are being used to generate a physical map of the nodulin region(s) of the soybean genome.


Identification of a cDNA for the plastid-located geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening.

Geranylgeranyl pyrophosphate synthase is a key enzyme in plant terpenoid biosynthesis. Using specific antibodies, a cDNA encoding geranylgeranyl pyrophosphate synthase has been isolated from bell pepper (Capsicum annuum) ripening fruit. The cloned cDNA codes for a high molecular weight precursor of 369 amino acids which contains a transit peptide of approximately 60 amino acids. In-situ immunolocalization experiments have demonstrated that geranylgeranyl pyrophosphate synthase is located exclusively in the plastids. Expression of the cloned cDNA in E. coli has unambiguously demonstrated that the encoded polypeptide catalyzes the synthesis of geranylgeranyl pyrophosphate by the addition of isopentenyl pyrophosphate to an allylic pyrophosphate. Peptide sequence comparisons revealed significant similarity between the sequences of the C. annuum geranylgeranyl pyrophosphate synthase and those deduced from carotenoid biosynthesis (crtE) genes from photosynthetic and non-photosynthetic bacteria. In addition, four highly conserved regions, which are found in various prenyltransferases, were identified. Furthermore, evidence is provided suggesting that conserved and exposed carboxylates are directly involved in the catalytic mechanism. Finally, the expression of the geranylgeranyl pyrophosphate synthase gene is demonstrated to be strongly induced during the chloroplast to chromoplast transition which occurs in ripening fruits, and is correlated with an increase in enzyme activity.


Molecular characterization and expression of an alfalfa protein with sequence similarity to mammalian ERp72, a glucose-regulated endoplasmic reticulum protein containing active site sequences of protein disulphide isomerase.

A complementary DNA clone (G1) containing sequence similarity to the mammalian lumenal endoplasmic reticulum protein ERp72 was isolated from an alfalfa (Medicago sativa L.) cDNA library by screening with a cDNA encoding human protein disulphide isomerase (PDI), which contains two thioredoxin-like active site regions which are highly conserved in ERp72. The polypeptide encoded by G1 consists of 364 amino acids, possesses a putative N-terminal secretory signal sequence and two regions, 113 amino acids apart, identical to the active sites of PDI and ERp72. G1 appears to be encoded by a small gene family in alfalfa, whose transcripts are constitutively expressed in all major organs of the plant. In alfalfa cell suspension cultures, G1 transcripts were markedly induced by treatment with tunicamycin, but not in response to calcium ionophore, heat shock or fungal elicitor. A similar expression pattern was observed for transcripts encoded by B2, a recently cloned alfalfa cDNA with strong sequence similarity to PDI. We discuss potential roles of plant proteins resembling vertebrate PDI and ERp72.


A 126 bp fragment of a plant histone gene promoter confers preferential expression in meristems of transgenic Arabidopsis.

The tissue-specific pattern of expression directed by the H4A748 Arabidopsis histone promoter was investigated by analysis of beta-glucuronidase (GUS) activity in transgenic Arabidopsis containing H4A748-GUS gene fusions. As determined by fluorimetric and histochemical tests, the H4A748 promoter directs preferential expression in meristems of young seedlings and adult plants. The low activity found in nonproliferating tissues may relate to basal constitutive expression of the histone promoter and/or to endoreduplication occurring in some tissues. The endogenous histone mRNA levels parallel the GUS activity found in different tissues. Analysis of the regulatory properties of 5' deleted promoters showed that multiple positive elements exist between -900 and -219 and that the proximal region of the promoter to -219 is sufficient to establish the full tissue-specific pattern of expression. Further deletion to -93 nearly abolished the promoter activity thus suggesting that the 126 bp fragment located between -219 and -93 contains the elements responsible for the specific expression pattern. The presence of several remarkable sequences within this fragment is discussed.


Characterization of DNA sequences that mediate nuclear protein binding to the regulatory region of the Pisum sativum (pea) chlorophyl a/b binding protein gene AB80: identification of a repeated heptamer motif.

Two protein factors binding to the regulatory region of the pea chlorophyl a/b binding protein gene AB80 have been identified. One of these factors is found only in green tissue but not in etiolated or root tissue. The second factor (denominated ABF-2) binds to a DNA sequence element that contains a direct heptamer repeat TCTCAAA. It was found that presence of both of the repeats is essential for binding. ABF-2 is present in both green and etiolated tissue and in roots and factors analogous to ABF-2 are present in several plant species. Computer analysis showed that the TCTCAAA motif is present in the regulatory region of several plant genes.


Members of the acetohydroxyacid synthase multigene family of Brassica napus have divergent patterns of expression.

Brassica species possess the most complex acetohydroxyacid synthase (AHAS) multigene family reported for plants. The AHAS genes code for an essential enzyme in branched-chain amino acid biosynthesis. In the allotetraploid species, B. napus, four (AHAS1-4) of the five AHAS genes have been cloned and sequenced. The transcripts were examined by RNase protection assays using gene-specific, antisense RNA probes. Only AHAS1, AHAS2 and AHAS3 were shown to be expressed in B. napus and one of the diploid progenitor species B. campestris or B. oleracea. AHAS1 and AHAS3 are highly conserved genes that presumably code for the essential AHAS housekeeping functions. They were expressed as low abundance mRNA in all somatic and reproductive tissues examined. AHAS2, which is structurally distinct from all other plant AHAS genes, was only expressed in mature ovules and extraembryonic tissues of immature seeds. This study provides direct evidence for multiple AHAS isoforms in plants and for an AHAS gene which is developmentally regulated in a tissue-specific manner. The discovery raises questions concerning the functional significance of AHAS in seed development.


A Brassica napus gene family which shows sequence similarity to ascorbate oxidase is expressed in developing pollen. Molecular characterization and analysis of promoter activity in transgenic tobacco plants.

The genomic clone named Bp10 contains a member of a small pollen-specific gene family of B. napus. The expression of the Bp10 gene family is maximal in early binucleate microspores and declines considerably in mature trinucleate pollen. Homologues of the Bp10 genes are expressed in the pollen of other plant species. The pollen-specific expression of the gene contained in the genomic clone was confirmed in tobacco plants transformed with a chimeric Bp10 promoter/GUS construct. A promoter fragment of 396 bp is sufficient to direct a strong and correct spatial and temporal expression in transgenic plants. The Bp10 gene family codes for proteins of 62 kDa showing approximately 30% sequence identify to cucumber and pumpkin ascorbate oxidases (AAOs). However, the AAO active centres are not conserved in the Bp10 products, suggesting an evolutionary relationship but a different enzymatic activity for these proteins. Expression of a recombinant Bp10 protein in E. coli inhibits bacterial growth on minimal medium, suggesting the production of an enzymatically active polypeptide in bacteria. No AAO activity could be correlated with the expression of the recombinant protein. Moreover, substances affecting AAO activity do not appear to influence the inhibitory activity of the protein produced in bacteria. However, as indicated by the rescue of bacterial growth in the presence of sodium bicarbonate or gaseous CO2, the Bp10 protein activity could be modulated by CO2 levels.


Molecular analysis of the avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum fully supports the gene-for-gene hypothesis.

The interaction between the fungal pathogen Cladosporium fulvum and tomato is supposed to have a gene-for-gene basis. Races of C. fulvum which have 'overcome' the resistance gene Cf9 of tomato, lack the avirulence gene avr9 which encodes a race-specific peptide elicitor. Races avirulent on tomato genotypes carrying the resistance gene Cf9 produce the race-specific peptide elicitor, which induces the hypersensitive response (HR) on those genotypes. The causal relationship between the presence of a functional avr9 gene and avirulence on tomato genotype Cf9 was demonstrated by cloning of the avr9 gene and subsequent transformation of C. fulvum. A race virulent on tomato genotype Cf9 was shown to become avirulent by transformation with the cloned avr9 gene. These results clearly demonstrate that the avr9 gene is responsible for cultivar specificity on tomato genotype Cf9 and fully support the gene-for-gene hypothesis. The avr9 gene is the first fungal avirulence gene to be cloned.


Cloning and characterization of the gene encoding the endopolygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L.

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein purified from hypocotyls of true bean (Phaseolus vulgaris L.). PGIP inhibits fungal endopolygalacturonases and is considered to be an important factor for plant resistance to phytopathogenic fungi (Albersheim and Anderson, 1971; Cervone et al., 1987). The amino acid sequences of the N-terminus and one internal tryptic peptide of the PGIP purified from P. vulgaris cv. Pinto were used to design redundant oligonucleotides that were successfully utilized as primers in a polymerase chain reaction (PCR) with total DNA of P. vulgaris as a template. A DNA band of 758 bp (a specific PCR amplification product of part of the gene coding for PGIP) was isolated and cloned. By using the 758-bp DNA as a hybridization probe, a lambda clone containing the PGIP gene was isolated from a genomic library of P. vulgaris cv. Saxa. The coding and immediate flanking regions of the PGIP gene, contained on a subcloned 3.3 kb SalI-SalI DNA fragment, were sequenced. A single, continuous ORF of 1026 nt (342 amino acids) was present in the genomic clone. The nucleotide and deduced amino acid sequences of the PGIP gene showed no significant similarity with any known databank sequence. Northern blotting analysis of poly(A)+ RNAs, isolated from various tissues of bean seedlings or from suspension-cultured bean cells, were also performed using the cloned PCR-generated DNA as a probe. A 1.2 kb transcript was detected in suspension-cultured cells and, to a lesser extent, in leaves, hypocotyls, and flowers.(ABSTRACT TRUNCATED AT 250 WORDS)


Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants.

A cauliflower mosaic virus (CaMV) 35S promoter derivative, which is tightly repressed by the Tn 10 encoded Tet repressor in a transient expression system as well as in transgenic plants has been constructed. After treatment of transgenic plants with tetracycline (Tc) the activity of the reporter enzyme beta-glucuronidase (GUS) increased up to 500-fold in tissue culture as well as under greenhouse conditions. Efficient de-repression was achieved by Tc uptake through the roots as well as by Tc treatment of leaves of intact plants. As Tc is not very stable in the plants, this system can also be used for a transient expression of a transgene. This system provides a unique tool for regenerating transgenic plants carrying a repressed transgene and for efficiently de-repressing its activity by a specific inducer at any time point of further development.


Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs.

An Arabidopsis thaliana cDNA bank has been constituted in a Saccharomyces cerevisiae expression vector based on the phosphoglycerate kinase (PGK) promoter and terminator. This bank was used to complement eight S. cerevisiae auxotrophic markers. All of them were corrected. These results confirm the quality of the bank and the feasibility of cloning plant genes by yeast mutant complementation. The cDNA complementing the ura1 yeast mutant was sequenced, analysed and shown to encode a dihydroorotic (DHO) dehydrogenase sequence.


[A physiological investigation of chronic electrical stimulation with scala tympani electrodes in kittens].

A physiological investigation of cochlear electrical stimulation was undertaken in six two-month-old kittens. The scala tympani electrodes were implanted and electrically stimulated using biphasic balanced electrical pulses for periods of 1000-1500h in four ears. Four ears received implants for same period but without electrical stimulation. The other two ears served as normal control. The results indicated: 1) Chronic electrical stimulation of the cochlea within electrochemically safe limits did not influence the hearing of kittens and the normal delivery of impulses evoked by acoustic and electrical signals on the auditory brainstem pathway. 2) The wave shapes of EABRs were similar to those of ABRs. The amplitudes of EABRs showed a significant increase following chronic electrical stimulation, resulting in a leftward shift in the input/output function. The absolute latencies and interwave latencies of waves II-III, III-IV and II-IV were significantly shorter than those of ABRs. These results imply that there was no adverse effect of chronic electrical stimulation on the maturing auditory systems of kittens using these electrical parameters and the mechanism of electrical hearing should be further studied.


[Effect of flavone glycosides of Epimedium koreanum on murine fibrinolytic system and apoplectic mortality].

The enhancement of fibrinolytic ability of stimulated murine macrophages by flavone glycosides of Epimedium koreanum (TFG) was measured by the [125I]-fibrin-coated plate method. The activity of the plasminogen activator (PA) induced by TFG was determined by a spectrophotometric assay. The activity of PA produced by TFG-stimulated macrophages was 0.731 IU/ML. (P < 0.01). TFG-stimulated macrophages showed rapid fibrinolysis. The activity of stimulated macrophages was approximately 2.8 fold that of the control. In in vivo experiments, the effect of TFG on spontaneously hypertensive and apoplexy rats (SHRsp) was very evident. The abiotic rate of TFG-stimulated rats was 5%, while that of the control group was 90%. TFG showed obvious hypotensive activity as well.


[The kinetic observation of serum ketone body levels in surgical patients receiving MCT or LCT emulsions].

The changes of serum beta-hydroxybutyrate concentrations in patients after receiving medium chain triglycerides (MCT) or long chain triglycerides (LCT) were studied. The patients were divided into two groups: in Group 1, the dose of fat was 0.14 g/kg.h for fat emulsion clearance test: and in Group 2, the dose was 0.06 g/kg.h for total parenteral nutrition (TPN) support. The beta-hydroxybutyrate concentrations were estimated by an enzymatic method using Sigma's beta-hydroxybutyrate kit (Sigma Diagnostics, MO 63178, USA). The normal range of beta-hydroxybutyrate concentration in 30 healthy Chinese subjects was 1.04 +/- 1.42 mg/dl (range 0-3.88 mg/dl). In Group 1, we found that the concentrations of beta-hydroxybutyrate rose steadily after fat emulsion infusion. The concentrations in the medium chain triglyceride group were higher than those in long chain triglyceride group. The elevated concentrations of beta-hydroxybutyrate gradually returned to normal levels when the emulsion infusion was stopped. In Group 2, the concentrations of beta-hydroxybutyrate in the patients were within the normal range.


[Effect of lipopolysaccharide on protein kinase C activity in human erythrocytes].

Membrane-associated protein kinase C activity was significantly increased after normal human erythrocytes were incubated with lipopolysaccharide. The effect was dose and time dependent. The results indicate that protein kinase C is activated by lipopolysaccharide in intact human erythrocytes.


[Investigation of maternal peripheral plasma concentration of PG in induction of labor by nutrition food inductive drug].

The used nutrition food inductive drug to induce labor in 33 pregnant women, 22 of 33 succeeded and 11 failed. The PGs level in maternal peripheral blood was measured twice. One before taking and the second sample was drawn blood during second stage of labor. Another 13 pregnant women of spontaneous labor were measured as control group. The 6-keto-PGF1 alpha level was significantly increased in the succeeding group than in the tailed induced group, the PGE concentration seems no significant change in these three groups. The data suggested that the nutritional food inductive drug may accelerate the synthesis of prostaglandin in the succeeding group.


[Clinical and histopathological observation and determination of serum IgE levels in patients with dermographism].

In 50 cases of dermographism, clinical and histopathological observations were made and serum IgE levels were measured. The results showed that serum IgE levels in patients with dermographism were increased significantly (P < 0.01) as compared with the control group. The role of increased IgE in the pathogenesis of the disease is discussed.


[The value of serum tumor-associated glycoprotein 72 (TAG 72) in the diagnosis of malignancies].

Serum TAG 72 concentrations of 110 normal subjects, 110 patients with benign diseases and 109 with malignant diseases were determined by a solid phase radioimmunoassay. Its levels in normal subjects were 1.6 +/- 1.5 u/ml (mean +/- SD, range 0-8.2 u/ml) and the cut-off limit was set at 4.6 u/ml. The positive rates in benign and malignant patients were 5.5% and 48.6%, respectively. Among the malignancies, 80.0% of ovarian and 63.6% of colon carcinomas had positive test results. The specificity for differentiating malignancies from benign diseases was 94.5% and the accuracy of diagnosis was 71.7%. The present study indicates that TAG 72 as a tumor marker is helpful in the diagnosis of malignancies.


[DNA probe labeling with digoxigenin-dUTP and its application in gene diagnosis].

In this paper, DNA probe labeling by the randomly primed incorporation of digoxigenin-dUTP is reported. The sensitivity of color reaction and hybridization were 32 fg and 200 fg, respectively, and both were specific for the target. Single-copy and multi-copy gene fragments among 2 micrograms human genomic DNA were detected by beta IVS II, Fr 3-42 and 3'HVR labeled with digoxigenin-dUTP. The results were consistent with a radioactive control assay. This method has been successfully used in the gene diagnosis of adult polycystic kidney disease.


[Effect of the tumor promoter phorbol myristate acetate (PMA) on cell-mediated immunity. IV. PMA-induced modulation of mouse dendritic cell function].

In the present study we investigated the in vitro effects of the tumor promoter phorbol myristate acetate (PMA) on the accessory function of mouse spleen dendritic cells (DC) in mitogenesis. The effects of the PMA, a protein kinase C (PKC) activator, depended on dose. If pretreated with < 50 ng/ml of PMA for 3 h, DC activity was enhanced by about two-fold; whereas > or = 200 ng/ml of PMA decreased DC activity with an inhibition rate about 50% (in early response DC activity showed a moderate increase). Our results indicate that the function of DC, a potent accessory population, can be further enhanced by a low dose of PMA. This is circumstantial evidence that DC activity can be up-regulatal via PKC activation. That a high dose of PMA inhibits DC activity might be a mechanism by which PMA promotes carcinogenesis.


[Studies on human cervical carcinoma cell line. II. Establishment of HGPRT- cell line CC-80IAR2 and its biological characteristics].

An HGPRT- cell line, CC-801AR2, was established from cloned human cervical carcinoma cell line CC-801 by using MNNG to induce cell mutation and 8-azaguanine (8-AG) to select the 8-AG resistant cells. The deficiency of HG-PRT in CC-801AR2 cells was proved by enzyme activity assay. This cell line was very sensitive to HAT medium. It was very similar in biological characteristics to CC-801, especially in that it retained its tumorigenicity when transplanted into nude mice. CC-801AR2 can therefore be used in somatic cell hybridization and gene transfer experiments.


[Flow cytometric analysis of surface antigens on liquid nitrogen-stored cells].

A high non-specific fluorescence background imposes serious interference on a flow cytometer in surface antigen assay for those cells stored in liquid nitrogen. The non-specific fluorescence comes from dead and dying cells, the permeability of whose membrane has been increased so that the fluorescence reagent can enter into the cells. To solve this problem, liquid nitrogen-stored PBMNC and Raji cells were screened on different gatings for surface antigen stained with fluorescence. It was observed that the high fluorescence background as well as debris can be gated out by means of gating on FALS, angle 90 degrees angle and PRFL. As a result, the percentage of surface antigen positive cells determined was close to that assayed in fresh samples.


[The preparation and clinical use of a radioimmunoassay CA125 kit for the diagnosis of epithelial ovarian cancer].

A self-made radioimmunoassay CA125 kit (using OC125 monoclonal antibody ascites offered by Dr. Bast Laboratory which was purified, solidified and labelled with 125I) was used for serum determination in 80 patients with epithelial ovarian cancer and in 40 standard antigen samples. The results demonstrated a statistically significant correlation between our self-made CA125 kit and an imported CENTOCOR CA125 kit (P < 0.001). Clinical experience before operation in 40 patients with epithelial ovarian cancer, 34 with endometriosis and 39 with other malignant ovarian tumors suggested that the self-made CA125 kit was more sensitive in diagnosing epithelial ovarian cancer than the other two diseases. Post-operative follow-up in 86 non-recurrent cases and 101 recurrent cases showed a 96% positive predictive rate and 91% negative predictive rate. It is concluded that self-made CA125 kit is very useful in monitoring epithelial ovarian cancer.


[A case of acute lymphocytic leukemia transformed from malignant lymphoma].

We report a case of malignant lymphoma in a 15-year-old male. He had erythema and nodular lesions, hepatomegaly, and splenomegaly with high fever for one month. Laboratory examination revealed a normal blood routine test, a few lymphomatic cells in the bone marrow, and histopathologic changes characteristic of malignant lymphoma in a skin biopsy. Two months later, a second bone marrow examination showed 90% prolymphoblasts. The peripheral blood count revealed WBC 108 x 10(9)/L; among these, prolymphoblasts accounted for 92%. The patient was diagnosed as acute lymphocytic leukemia, and died 2 days later. The diagnosis and transformation from malignant lymphoma to acute lymphocytic leukemia are discussed.


[Application of 3 dimensional computer tomography to maxillofacial surgery].

Three dimensional CT examination is a recently developed technique. Nine patients suffering from traumatic and developmental abnormalities were examined with this new method and the results were satisfying. Applying this technique, we routinely obtain a set of equal spaced, nonoverlapping, high resolution CT scans of the maxillofacial deformities. It provided a condition for facilitating surgical planning and improving quantitative of postoperation evaluation.


[The nerve fiber projection of trigeminal ganglion cell to dental pulp, dentine and periodontal membrane in the rat].

The nerve fiber projection of the trigeminal ganglion cell to dental pulp, dentine and periodontal membrane was investigated with the technique of anterograde radioautographic neuroanatomical tracing (ARNT) in the rat. Results showed that the innervation in dentine and pulp was not uniform. The nerve endings were most numerous in the pulp horn, and in the tubular dentine of the cusp and cervical regions. In the periodontal membrane, both large- and small-diameter nerve fibers were found. And at least part of the innervation in the periodontal membrane was of trigeminal ganglion origin.


[Chronic obstructive parotitis: a report of 92 cases].

This article presents 92 cases (117 diseased parotid glands) of chronic obstructive parotitis with studies by clinical, sialographical, sequential quantitative scintigraphy methods and with investigation of the treatment. The nature of this disease is that various local factors cause obstruction of saliva flow resulting in recurrent swelling of parotid, in which the main feature in sialography is irregular dilatation of the main duct and branch duct. Sialographical manifestations can be divided into four types. Conservative, operative therapy and injection into diseased gland with 1% methyl violet are introduced to treat this disease. Long follow-up findings show that type I in sialography can be cured by conservative method, but other types to not respond to this method and should be treated by other methods, such as parotidectomy. We have used 1% methyl violet to treat cases who have swelling of parotid after conservative therapy. It is considered to be a simple, practical and good method and should only be used in this disease. There are no other side effects except swelling of parotid for a few weeks. Especially it can be used to treat the anterior part of main duct left over by parotidectomy, which still gives off purulent discharge.


[Study on the recovery of 4 types of cultured cell after contacting varieties of dental casting alloys].

Four types of cultured cell (Gin-1, Chang Liver, HEP-2 and L-929) were used in vitro to determine the cytotoxicity of 12 Chinese-Japanese Dental Casting Alloys from cell recovery ability. A new cytocompatibility detecting method--cell recovery test was established and recommended. This method can be able to observe the state of cell recovery through self growth cycle, diminish the side-effect of dead cell and its products on the living cells, nearly mimic the environment in vivo and study the inherited toxicology of cells. The application of computer photo-pattern analysis technique can acquire the objective and accurate data directly.


[Clinical observation of the superoxide dismutase (SOD) and lipid peroxide (LPO) changes in oral-maxillofacial surgical patients].

This article reports examination of superoxide dismutase (SOD) activity in RBC and lipid peroxide (LPO) content in the plasma of the oral-maxillofacial operation patients. The results showed that the LPO content in postoperation ascended remarkably compared with pre-operation (P < 0.001) and that the SOD activity rose also (P < 0.05). It was discovered that the difference of LPO content between the pre-operation and post-operation was closely relative with the difference of SOD activity, the operative time and the operation method (intra- or extra-oral approach). The results demonstrated that the operative damage resulted in the increasing of the reactivity of superoxide radical and LPO and that the rising of SOD activity was probably the compensation of the body.


[Binding of Actinomyces viscosus to collagen].

The ability of collagen adsorbed on apatitic surfaces to promote adhesion of A. viscosus was studied. Treatment of hydroxyapatite (HA) with human type I or type III collagen strongly promoted adhesion of A. viscosus LY7. TEM observations revealed that A. viscosus also attached to fibrils prepared from human type I collagen. The alpha(2) polypeptide derived from type I collagen exhibited moderate activity in promoting binding. Mutans of A. viscosus which possess type I fimbriae, but not type 2 fimbriae or no fimbriae, also bound to collagen-treated HA. This suggests that the adhesin responsible was associated with type I fimbriae, strains of A. israelii and A. odontolyticus also exhibited strong binding to collagen-treated HA. The avidity of Actinomyces species for collagen would seem to be partially responsible for the high proportions of these organisms found on root surfaces.