Serum lipoprotein profile in the Mediterranean variant of glucose-6-phosphate dehydrogenase deficiency.

Sardinian males with erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) deficiency have lower serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (ApoB), compared to normals. Since the enzyme deficiency is expressed also in nucleated cells, we studied cholesterol (C) and DNA synthesis and LDL-receptor expression in freshly isolated circulating mononuclear cells from normal and G-6-PD-deficient Sardinians. Synthesis of C (as 14C-acetate incorporation) and of DNA (as 3H-thymidine incorporation) was clearly reduced, both in basal state and after PHA stimulation, in G-6-PD-deficient cells compared to normal cells. On the other hand, no clear influence of G-6-PD deficiency on LDL-receptor expression could be demonstrated. The Mediterranean variant of G-6-PD deficiency is characterized, whatever the metabolic mechanism may be, by a serum lipoprotein pattern of reduced atherogenicity.


Association of a polymorphism in the Apo AI gene promoter with hyperalphalipoproteinemia.

The distribution of a polymorphism due to an Adenine to Guanine transition in the ApoAI gene has been studied in 136 women and 108 men, through amplification of the promoter region of the gene and allele-specific oligonucleotide hybridization. The allelic frequencies for the A allele were 0.10, 0.14 and 0.27 in women and 0.08, 0.17 and 0.14 in men for the lowest decile, intermediate group and the highest decile of HDL-cholesterol levels, respectively. Statistical analysis showed that the A allele was associated with high HDL-cholesterol levels in women, but not in men.


The molecular defects in lipoprotein lipase deficient patients.

The underlying molecular defects that lead to a deficiency of lipoprotein lipase in two patients from different kindreds presenting with the familial hyperchylomicronemia syndrome have been identified. Sequence analysis of amplified LPL cDNA of the patient from the Bethesda kindred revealed a single point mutation (G to A) at position 781 of the normal gene that resulted in the substitution of an alanine for a threonine at residue 176 and the loss of an SfaN1 site present in the normal LPL gene. Amplification of patient cDNA by the PCR followed by restriction enzyme digestion with SfaN1 established that the patient is a true homozygote for the defect. The proband from the second kindred was found to be a compound heterozygote for two separate allelic mutations, including a T to C transition at nucleotide 836 and a G to A mutation at base 983 that led to the substitution of Ile194 by Thr and Arg243 by His, respectively. Transient expression of the mutant LPL cDNAs from both kindreds in human embryonal kidney-293 cells resulted in the synthesis of enzymatically inactive proteins, establishing the functional significance of the mutations.


Factor VIII and coronary heart disease.

In plasma, Factor VIII is bound to its carrier protein, von Willebrand Factor (vWF). Therefore, the levels of these two factors vary together. From studies in the general population, in patients with coronary heart disease and in hemophilia patients, an association between Factor VIII levels and coronary heart disease clearly emerges.


Plasma fibrinogen and factor VII as risk factors for cardiovascular disease.

The importance of the thrombotic component of coronary heart disease is increasingly recognised, and in particular the role of the coagulation system in this process. The Northwick Park Heart study was the first major prospective study to identify both fibrinogen and factor VIIc as risk factors, as powerful as total cholesterol in predicting ischaemic events. Since then, a number of epidemiological studies have confirmed the importance of fibrinogen, not just in CHD but in stroke as well. A variety of environmental factors are known to influence levels of factor VII and fibrinogen and therefore support their role in the development of coronary thrombosis. Both are known to increase with age and body weight and are relatively elevated in diabetes. Fibrinogen is strongly related to smoking habit and a substantial proportion of the IHD risk associated with smoking is mediated through this relationship. There is a dose response effect between number of cigarettes smoked and level of fibrinogen and an inverse relationship with time since cessation of the habit. Factor VII is known to correlate with total cholesterol level, and there is a relationship between dietary variability of fat intake and factor VII, which is likely to play an important role in the risk of CHD. The case for using either anticoagulation or anti platelet agents in secondary prevention of myocardial infarction is now clear, but there are still uncertainties in primary prevention which relate to the ideal dose intensity of either aspirin or anti-coagulation and the type of patient most likely to benefit. The ongoing Thrombosis Prevention Trial identifies middle-aged males at high risk of a myocardial infarction.(ABSTRACT TRUNCATED AT 250 WORDS)


Fibrinogen and mechanisms of thrombosis. A difficult link.

Epidemiological observations indicate that high plasma fibrinogen levels are strongly correlated to the frequency of two major thrombotic complications of atherosclerosis: stroke and myocardial infarction. Thrombosis is increasingly recognized as a central mechanism in stroke and myocardial infarction, and fibrinogen is involved in events thought to play a major role in thrombosis. Therefore, elucidation of the relationship between fibrinogen and thrombosis may strengthen the predictive value of this protein and define new interventions against stroke and myocardial infarction. In addition, advances in the understanding of the atherogenic potential of several risk factors of coronary heart disease took advantage of information emerging from the measurement of the factor in population-based studies. Thus, it is conceivable that measuring plasma fibrinogen to predict stroke and myocardial infarction is a major direction to be followed to gain insight into the thrombogenic potential on this protein and inspire new strategies against thrombotic complications of atherosclerosis.


IDL, VLDL, chylomicrons and atherosclerosis.

In humans with the lipoprotein lipase deficiency disorder large amounts of chylomicrons and large very low-density lipoprotein (VLDL) accumulate in plasma. In spite of this, atherosclerosis does not seem to develop at an accelerated rate, suggesting that these lipoproteins do not promote atherogenesis. In humans with dysbetalipoproteinemia remnant lipoproteins (intermediate density lipoprotein (IDL) plus beta-VLDL) accumulate in plasma and these particles may therefore be the factor causing accelerated atherosclerosis in this disorder. Epidemiological studies in humans suggest that IDL or remnant lipoproteins are predictors of the severity or progression of atherosclerosis. Similar studies in the St. Thomas' Hospital rabbit strain, an animal model with genetically elevated plasma levels of VLDL, IDL and low-density lipoprotein (LDL), showed that IDL or remnant lipoproteins were better predictors of the extent of atherosclerosis than were LDL or VLDL. Studies of lipoprotein/arterial wall interactions have demonstrated that the larger the lipoprotein particle, the lower the influx into intima. Very large VLDL and chylomicrons do not seem to enter intima. Although high-density lipoprotein (HDL) enters intima faster than other lipoproteins, the small HDL particles seem to penetrate the entire arterial wall and leave via lymphatics and vasa vasorum in the outer media and adventitia. In contrast, LDL, and possibly also IDL and smaller VLDL, may only leave the intima via the lumen of the artery. In conclusion, a substantial body of evidence suggests that remnant lipoproteins (IDL and smaller VLDL) share with LDL the potential for promoting atherosclerosis, whereas very large VLDL and chylomicrons do not seem to have this effect.


The importance of triglycerides: results from the Prospective Cardiovascular Münster (PROCAM) Study.

In the PROCAM study hypertriglyceridaemia was much more common among men (18.6%) than women (4.2%). Prevalence increased with age in women, but remained nearly constant at about 20% in men aged 35 years or more. In a multiple regression analysis a strong negative correlation between triglycerides and HDL cholesterol was found. A positive relationship was observed with cholesterol, blood glucose, factor VIIc, and PAI-1 in both sexes. Only in men, triglycerides are positively correlated with Apo A-I, body mass index, cigarette smoking, and alcohol consumption. Postmenopausal women showed higher triglyceride levels than premenopausal women. No independent relationships were observed to age, blood pressure, Lp(a), Apo A-II, Apo B, AT-III, protein C, fibrinogen, and oral contraceptives. A longitudinal analysis of data from 4474 male PROCAM participants aged 40-64 years with a follow-up of 4 years did not identify triglycerides as an independent risk factor, but the data suggest hypertriglyceridaemia is an additional risk factor for CHD, when excessive triglycerides coincide with a high ratio of plasma cholesterol to HDL-cholesterol and with low HDL-cholesterol values.


Protein engineering of Drosophila alcohol dehydrogenase. The hydroxyl group of Tyr152 is involved in the active site of the enzyme.

Drosophila alcohol dehydrogenase is the most studied member of the family of short-chain alcohol dehydrogenases, although its tridimensional structure still remains unknown. We have engineered a Drosophila alcohol dehydrogenase in which tyrosine-152, an invariant residue in all members of the family, has been substituted by phenylalanine. The mutated gene has been expressed in yeast and pure mutant enzyme has been prepared by a one-step FPLC chromatographic procedure. Drosophila alcohol dehydrogenase-phenylalanine-152 shows no enzymatic activity. This result suggests not only that tyrosine-152 could constitute an essential building block of the active site but also that its hydroxyl group is directly involved in the redox reaction catalyzed by the enzyme.


Tobacco chloroplast ribosomes contain a homologue of E. coli ribosomal protein L28.

The genes for ribosomal proteins L28 and L33 constitute an operon (rpmBG) in E. coli, but in plant chloroplasts L33 is encoded by the chloroplast DNA and L28 seems to be encoded by the nuclear genome. A 15 kDa protein was isolated from the 50 S subunit of tobacco chloroplast ribosomes and its N-terminal amino acid sequence was determined. A cDNA for this protein was cloned and analyzed. The cDNA encodes a 151 amino acid protein consisting of a predicted transit-peptide of 74 amino acids and a mature protein of 77 amino acids. The mature protein is homologous to E. coli L28, hence we named it chloroplast L28 (CL28). This is the first report on the presence of an E. coli L28-like protein in another organism.


Regions of beta 4.beta 2 subunit chimeras that contribute to the agonist selectivity of neuronal nicotinic receptors.

Fifteen chimeric nicotinic receptor beta subunits were constructed consisting of N-terminal neuronal beta 4 sequences and C-terminal beta 2 sequences. Responses to cytisine, nicotine, or tetramethylammonium were compared to acetylcholine responses for these subunits expressed in Xenopus oocytes with alpha 3 subunits. The results show that (i) two residues in the extracellular domain of chimeric beta 4.beta 2 subunits (108 beta 2F/beta 4V, 110 beta 2S/beta 4T) account for much of the relative cytisine sensitivity; and (ii) four extracellular residues of chimeric beta 4.beta 2 subunits (112 beta 2A/beta 4V, 113 beta 2V/beta 4I and 115 beta 2S/beta 4R, 116 beta 2Y/beta 4S) account for most of the relative tetramethylammonium sensitivity. The data did not permit localization of nicotine sensitivity to any particular region.


Synthesis of the proline analogue [2,3-3H]azetidine-2-carboxylic acid. Uptake and incorporation in Arabidopsis thaliana and Escherichia coli.

Azetidine-2-carboxylic acid, the 4-membered ring noranalogue of proline, is regularly used in the study of proline metabolism as well as the study of protein conformation. We prepared D,L-[2,3-3H]azetidine-2-carboxylic acid with an optimized 10% yield from commercially available 4-amino-[2,3-3H]butyric acid. Purification was performed by fast-protein liquid chromatography. The biological activity was checked in both Arabidopsis thaliana and Escherichia coli. The obtained specific activity of 10 mCi/mmol was sufficient for most uptake and incorporation studies.


Vitamin E dietary supplementation inhibits transforming growth factor beta 1 gene expression in the rat liver.

Overexpression of transforming growth factor beta 1 (TGF beta 1) and increased transcription of pro-collagen type I, are known to represent major events implicated in the development of liver fibrosis under either experimental or clinical conditions. Here we report that long-term dietary vitamin E supplementation in animals undergoing an experimental model of liver fibrosis (induced by chronic treatment of rats with carbon tetrachloride) results in a net inhibition of both hepatic TGF beta 1 and alpha 2 (I) procollagen mRNA levels. Moreover, of striking interest is the observation that vitamin E supplementation per so down-modulates basal levels of TGF beta 1 mRNA in the liver of untreated animals, suggesting that a dietary regimen rich in vitamin E may potentially interfere with both the initiation and progression of the fibrosclerotic processes.


Trimethylamine dehydrogenase of bacterium W3A1. Molecular cloning, sequence determination and over-expression of the gene.

The gene encoding trimethylamine dehydrogenase (EC 1.5.99.7) from bacterium W3A1 has been cloned. Using the polymerase chain reaction a 530 bp DNA fragment encoding a distal part of the gene was amplified. Using this fragment of DNA as a probe, a clone was then isolated as a 4.5 kb BamHI fragment and shown to encode residues 34 to 729 of trimethylamine dehydrogenase. The polymerase chain reaction was used also to isolate the DNA encoding the missing N-terminal part of the gene. The complete open reading frame contained 2,190 base pairs coding for the processed protein of 729 amino acids which lacks the N-terminal methionine residue. The high-level expression of the gene in Escherichia coli was achieved by the construction of an expression vector derived from the plasmid pKK223-3. The cloning and sequence analysis described here complete the partial assignment of the amino acid sequence derived from chemical sequence [1] and will now permit the refinement of the crystallographic structure of trimethylamine dehydrogenase and also a detailed investigation of the mechanism and properties of the enzyme by protein engineering.


Flow-induced prostacyclin production is mediated by a pertussis toxin-sensitive G protein.

Fluid flow and several other agonists induce prostacyclin (PGI2) production in endothelial cells. G proteins mediate the response of a large number of hormones such as histamine, but the transduction pathway of the flow signal is unclear. We found that GDP beta S and pertussis toxin inhibited flow-induced prostacyclin production in human umbilical vein endothelial cells. In addition, flow potentiated the histamine-induced production of PGI2. This suggests that flow stimulates prostacyclin production via a pertussis toxin-sensitive G protein and modulates the stimulus-response coupling of other agonists.


Molecular cloning and permanent expression in a neuroblastoma cell line of a fast inactivating potassium channel from bovine adrenal medulla.

Using a cDNA library from bovine adrenal medulla, we have isolated cDNAs coding for a potassium channel. These cDNAs encode a 660-amino acid protein that has a molecular weight of 73,288 kDa and no amino-terminal signal peptide. We have called it BAK4. Analysis of its sequence reveals close similarity (94% homology) with a recently described potassium channel from rat brain (RCK4) and heart (RHK1). Neuroblastoma cells (Neuro-2a cell line) were stably transfected with BAK4 DNA. Expression of the DNA was under the control of a heat-shock promoter. Several clones, that were isolated by neomycin resistance selection, had integrated the plasmid DNA in a stable form. Upon heat induction, these cells produced BAK4 RNA and a potassium outward current, not present in control non-transfected cells. The current, which was transient and decayed markedly during the duration of 200 ms-pulses, can be described as a Ik(A) potassium current. The expression of these types of channels in brain (RCK4,RHK1), heart (RHK1) and adrenal medulla (BAK4) suggest their possible implication in important functions for the cell.


Specific release of a 9-kDa extrinsic polypeptide of photosystem I from spinach chloroplasts by salt washing.

The newly reported 9-kDa polypeptide in photosystem I [(1991) FEBS Lett. 280, 332-334] is an extrinsic component located on the lumenal side of the thylakoid membrane. This subunit can be solubilized with high salt buffer and does not bind any cofactors. The photosystem I electron transfer chain remains intact and functional in the absence of this component as characterized by the photoreduction of NADP+.


Quantitative study of the binding of cysteine proteinases to basement membranes.

Binding of cysteine proteinases of the papain superfamily (papain and cathepsins B, B-like and L) to basement membranes was studied by using the enzymatic activity of these proteinases against their specific fluorogenic substrates. Papain inactivated by E64 was used for Kd determination by competition experiments. The binding was characterized using the following parameters, the equilibrium constant, Kd, and the number of substrate sites, n, values of which were in the range of 10(-7) M and 10(12), respectively. Such results would be of significant interest for the understanding of the biological role of cysteine proteinases in tumour invasion and other types of tissue remodeling.


Characterization of myocardial extracellular ATP receptors by photoaffinity labelling and functional assays.

Extracellular ATP receptors in rat ventricular myocytes were investigated through intact cell photolabelling followed by protein isolation. 8-Azido-ATP (8Az-ATP) was used for labelling under specific conditions determined by parallel functional studies. In those studies ATP-induced cytosolic Ca2+ transients were irreversibly and specifically inhibited by UV-photolyzed 8Az-ATP, but not by 2-azido-ATP (2Az-ATP), even in the presence of high concentrations of phosphonucleotides not affecting myocardial ATP receptors. Under those conditions background labelling is minimized and radioactive 8Az-ATP specifically labels a band of 45-48 kDa on a SDS gel. Labelling under the above conditions in the presence of ATP gamma S or 2-methylthio-ATP (2-meSATP), which are distinct for two functionally different cardiac ATP receptors, shows two different proteins within the same band consistent with the possible labelling of these two receptors.


Lipid interaction of tetanus neurotoxin. A calorimetric and fluorescence spectroscopy study.

The interaction of Tetanus toxin with phospholipid vesicles containing gangliosides (GD1a, GD1b or GT1b) or phosphatidic acid has been investigated at neutral or acidic pH. Change in the thermotropic properties of the vesicles occurred only after addition of the toxin at acidic pH, and led to surface binding or membrane insertion of the protein, dependent on the physical state of the membrane. Most remarkably, toxin addition at acidic pH to dipalmitoyl-phosphatidylcholine vesicles containing GT1b ganglioside, caused formation of ganglioside microdomains on the vesicle surface.


Relocalization of alpha B-crystallin by heat shock in ovarian carcinoma cells.

alpha B-Crystallin, a major lens protein, is present in clearly detectable amounts in cultured ovarian carcinoma cells. After heat-shock treatment of these cells at 45 degrees C alpha B-crystallin relocalizes from the detergent-soluble, cytosolic fraction to the non-ionic detergent-insoluble nuclear/cytoskeletal fraction. Colchicine treatment of the cells, although giving rise to a vimentin collapse on the nucleus, does not result in redistribution of alpha B-cyrstallin. When this colchicine treatment is followed by heat shock, alpha B-crystallin relocalizes again to the insoluble fraction, indicating that this relocalization is independent of the collapse of the vimentin network.


The complete primary structure of abrin-a B chain.

The complete 267 amino acid sequence of abrin-a B chain was determined by analysis of peptides obtained by digestion with trypsin, chymotrypsin, lysyl endopeptidase, Staphylococcus aureus V8 protease and thermolysin. The sequence is not identical with that predicted previously by nucleotide sequencing, indicating the presence of isoforms of abrin. Comparison of the amino acid sequence of abrin-a B chain with that of ricin-D B chain reveals a high degree of sequence identity (59%). Abrin-a B chain appears to consist of two domains, each domain with subdomains (alpha, beta, gamma) of about 40 amino acid residues.


Structure of the small ribosomal subunit RNA of the pulmonate snail, Limicolaria kambeul, and phylogenetic analysis of the Metazoa.

The complete nucleotide sequence of the small ribosomal subunit RNA of the gastropod, Limicolaria kambeul, was determined and used to infer a secondary structure model. In order to clarify the phylogenetic position of the Mollusca among the Metazoa, an evolutionary tree was constructed by neighbor-joining, starting from an alignment of small ribosomal subunit RNA sequences. The Mollusca appear to be a monophyletic group, related to Arthropoda and Chordata in an unresolved trichotomy.


TATA box-independent transcription of the human tissue plasminogen activator gene initiates within a sequence conserved in related genes.

Transcription of the human tissue-type plasminogen activator (tPA) gene has been reported to initiate from a single site proximal to a TATA box motif [1985, J. Biol. Chem. 260, 11223-11230]. In this study, we utilized primer extension analysis to evaluate the tPA mRNA start site in phorbol-12-myristate 13-acetate (PMA) induced WI-38 human lung fibroblast cells. Whilst some tPA mRNA initiated from the predicted TATA-proximal location (+1), a 10-fold greater proportion of tPA mRNA transcripts initiated 110 bases downstream from a sequence conserved and utilized as the TATA-independent transcription start site in the rodent tPA genes. Moreover, the transfection and expression in different cell types of a cosmid containing the entire human tPA gene resulted in utilization of the same downstream (+110) start site. We propose that this, rather than the previously published position, is the major transcriptional initiation point for the human tPA gene. A core sequence (5'-CAGAGCTG-3') was identified which is common to the TATA-independent mRNA start sites of the human, mouse and rat tPA genes, and which demonstrates only partial similarity to sequences found at the initiation point of other TATA-independent genes.


A protective role for nitric oxide in the oxidative modification of low density lipoproteins by mouse macrophages.

Low density lipoproteins (LDL) oxidatively modified by macrophages have been shown to be atherogenic in ex vivo studies. We studied the potential role of nitric oxide (NO), a free radical produced by macrophages, in LDL modification. Human LDL (1 mg/ml) were incubated with mouse peritoneal macrophages in Ham's F-10 medium. The cells were then stimulated by interferon-gamma and tumor necrosis factor-alpha to increase their production of NO from 1.3 to 12.2 microM in 24 h, as measured by nitrite. Lipid peroxidation of LDL, as measured by thiobarbituric acid-reactive materials (TBARS), was reduced in stimulated cells in a time-dependent manner. At 24 h, the decrease was about 27%. In the presence of an NO synthase inhibitor (NG-aminophomoarginine), the generation of NO was diminished and the protection against LDL lipid peroxidation was reversed. The extent of LDL protein modification was also assessed by examining its electrophoretic mobility. It was found that macrophage NO reduced the change in LDL electromobility. These data indicate that the production of NO may inhibit the oxidative modification of LDL with cytokine-stimulated macrophages. We suggest that NO plays a protective role in limiting macrophage-induced LDL modification.


X-ray crystal structure of the serine proteinase inhibitor eglin c at 1.95 A resolution.

The crystal structure of eglin c, naturally occurring in the leech Hirudo medicinalis, is known from its complexes with various serine proteinases, but the crystallization of free eglin c has not yet been reported. A method is described for growing well-diffracting crystals of free eglin c from highly concentrated protein solutions (approximately 200 mg/ml). The space group of the orthorhombic crystals was determined to be P2(1)2(1)2(1) with unit cell parameters a = 32.6, b = 42.0, c = 44.1 A. The structure of free eglin c was resolved at 1.95 A resolution by Patterson search methods. The final model contains all 70 amino acids of eglin c and 125 water molecules. In comparison to the eglin structure known from its complexes with proteinases, only small differences have been observed in free eglin c. However, the reactive site-binding loop and a few residues on the surface of eglin have been found in different conformations due to crystal contacts. In contrast to the complex structures, the first seven amino acids of the highly flexible amino terminus can be located. Crystallographic refinement comprised molecular dynamics refinement, classical restrained least-squares refinement and individual isotropic atomic temperature refinement. The final R-factor is 15.8%.


Insulin receptor and glucose transporter mRNA expression in skeletal muscle of genetically obese Zucker rats.

Insulin resistance and defective glucose transport are associated with muscle tissue in the genetically obese Zucker rat and are accompanied by changes in the number of insulin receptors and the availability of glucose transporters. The present study was carried out to assess whether, in male Zucker rats at 10 weeks of age, these defects were reflected by changes in the levels of mRNAs for the insulin receptor and for the insulin responsive glucose transporter (GLUT-4). Total RNA was extracted from plantaris and soleus muscles and the levels of insulin receptor and GLUT-4 mRNAs and 18 S rRNA were determined by Northern hybridization and quantified by image analysis of the autoradiographs. A 50% increase in the level of insulin receptor mRNA was detected in both the plantaris and the soleus muscle from the obese rats compared to the lean rats. No change in the level of GLUT-4 mRNA was detected in the plantaris muscle although increases were observed in the soleus muscle from the obese rats.


Characterization of a 41 kDa photoinhibition adduct in isolated photosystem II reaction centres.

When isolated reaction centres of photosystem II are subjected to photoinhibitory illumination, a 41 kDa SDS-PAGE band is observed under all experimental conditions. The same band is also found, together with lower molecular weight fragments of the D1 protein, in whole thylakoids and in all PSII sub-particles investigated up to now. In the case of isolated reaction centres the 41 kDa band is represented by a heterodimer of the D1 polypeptide and the alpha-subunit of cytochrome b559. The cross-linkage between D1 and alpha-cyt b559 involves a region on D1 between the N-terminal residue and Arg-225, and is an early event in photo-induced damage to the D1 protein.


Occupancy of a C2-C2 type 'zinc-finger' protein domain by copper. Direct observation by electrospray ionization mass spectrometry.

The metal ion specificity of most 'zinc-finger' metal binding domains is unknown. The human estrogen receptor protein contains two different C2-C2 type 'zinc-finger' sequences within its DNA-binding domain (ERDBD). Copper inhibits the function of this protein by mechanisms which remain unclear. We have used electrospray ionization mass spectrometry to evaluate directly the 71-residue ERDBD (K180-M250) in the absence and presence of Cu(II) ions. The ERDBD showed a high affinity for Cu and was completely occupied with 4 Cu bound; each Cu ion was evidently bound to only two ligand residues (net loss of only 2 Da per bound Cu). The Cu binding stoichiometry was confirmed by atomic absorption. These results (i) provide the first direct physical evidence for the ability of the estrogen receptor DNA-binding domain to bind Cu and (ii) document a twofold difference in the Zn- and Cu-binding capacity. Differences in the ERDBD domain structure with bound Zn and Cu are predicted. Given the relative intracellular contents of Zn and Cu, our findings demonstrate the need to investigate further the Cu occupancy of this and other zinc-finger domains both in vitro and in vivo.


Reactive oxygen species are involved in the activation of cellular phospholipase A2.

Vanadate (V) potentiated (4- to 10-fold) the activation of cellular phospholipase A2 (PLA2) induced by H2O2 (H), a phorbol ester (T), a Ca(2+)-ionophore (A) and opsonized zymosan in macrophages. V+H induced in intact cells the activation and translocation of PLA2 and protein kinase C (PKC) to the plasma membrane. V+H and V+T+A induced strong chemiluminescence (CL) which was abrogated by a specific NADPH oxidase inhibitor diphenylene iodonium (DPI). DPI markedly suppressed the stimulation of PLA2 by V+T+A and V+OZ. The results suggest that the formation of endogenous reactive oxygen species (ROS) is important for PLA2 activation.


The microtubule binding repeats of tau protein assemble into filaments like those found in Alzheimer's disease.

The paired helical filament, which comprises the major fibrous element of the neurofibrillary tangle in Alzheimer's disease, contains abnormally phosphorylated microtubule-associated protein tau as its principal constituent. The repeat region of tau protein, which represents the microtubule binding domain, forms the core of the filament. Here we show that an expressed fragment of tau protein spanning the repeat region can assemble in vitro into filaments like those found in Alzheimer's disease.


Molecular cloning of the alpha-subunit of rat endopeptidase-24.18 (endopeptidase-2) and co-localization with endopeptidase-24.11 in rat kidney by in situ hybridization.

Endopeptidase-24.18 (endopeptidase-2, EC 3.4.24.18, E-24.18) is a Zn-ectoenzyme of rat renal and intestinal microvillar membranes exhibiting an oligomeric structure, alpha 2-beta 2. The primary structure of the alpha-subunit of E-24.18 has been defined by molecular cloning and its expression mapped in rat kidney by in situ hybridization. A 2.9-kb cDNA coding for the alpha-subunit was isolated and sequenced. It had an open reading frame of 2,244 base pairs coding for a type I membrane protein of 748 amino acids. The deduced amino acid sequence showed 87% identity with that of meprin A, a mouse metallo-endopeptidase, sharing common properties with the rat enzyme, and 85% identity with the human intestinal enzyme, 'PABA-peptide hydrolase'. Northern blot analysis revealed the alpha-subunit to be encoded by a single mRNA species of 3.2-kb. In situ hybridization performed on rat kidney showed a co-localization of E-24.18 with endopeptidase-24.11 in proximal tubules of juxtamedullary nephrons, suggesting that the two enzymes have similar or complementary physiological functions in kidney.


Vanadate potentiates the glycogenic action of insulin-like growth factors on isolated diaphragm.

Na3VO4 (6.5 mumol/100 g rat weight), co-injected with a trace amount of [14C]glucose, increased within 15 min the incorporation of radiolabel in diaphragmal glycogen. After 2 h the vanadate-induced increases were 12-fold in the diaphragm and 7-8-fold in heart and liver. In contrast, when added to isolated diaphragms for up to 1 h, vanadate (0.1-5 mM) had no effect on the synthesis of glycogen from 5 mM glucose. In search of a putative mediator of vanadate's action in vivo, insulin and the insulin-like growth factors (IGFs) were considered. Their plasma concentration was not affected by vanadate treatment. In isolated diaphragms, 1 mM vanadate did not potentiate insulin-induced glycogen synthesis, but it caused a several-fold increase in glycogen synthesis in the presence of concentrations of IGF-I which, alone, had no effect. A similar synergism occurred between vanadate and IGF-II. We propose that the glycogenic action of vanadate in vivo, at least in some tissues, involves a potentiation of the action of IGF-I.


Fourier transform infrared (FTIR) spectroscopic investigation of the nicotinic acetylcholine receptor (nAChR). Investigation of agonist binding and receptor conformational changes by flash-induced release of 'caged' carbamoylcholine.

The binding and interaction of carbamoylcholine with the nicotinic acetylcholine receptor was investigated using photolytically released carbamoylcholine ('caged' carbamoylcholine). Upon UV flash activation of this photolabile substrate analog, characteristic changes in the IR absorbance spectrum were detected. Apart from difference bands arising from the changes of molecular structure upon photolytical release, spectral features can be attributed to the agonist upon binding to the receptor as well as to conformational changes of the receptor itself. The two photo-labile agonist analogs N-[1-(2-nitrophenyl)ethyl] carbamoylcholine iodide (cage I) and N-(alpha-carboxy-2-nitrobenzyl) carbamoylcholine trifluoroacetate (cage II), with different structures for comparison of the 1680-1540 cm-1 region sensitive for protein conformation, yielded consistent results. A preliminary interpretation in terms of substrate binding and local conformational changes of the receptor upon carbamoylcholine binding is provided, in analogy to the binding of acetylcholine, activation, and subsequent deactivation taking place during signal transduction.


Adaptability of the coccidian Coelotropha to parasitism.

The coccidian, Coelotropha durchoni, manages to develop in its host, the polycheate annelid, Nereis diversicolor, because of its ability to circumvent the host's internal defence system. First, it avoids phagocytes by penetrating other cells, principally eleocytes and muscular cells, where it undergoes a phase of intracellular development. After becoming extracellular, a thick coat protects it from being attacked by granulocytes. This coat then breaks and is shed from its surface to permit fertilization. Parasites that have lost their coats and remain unfertilized are surrounded with granulocytes and destroyed by encapsulation. A strict hormonal correlation exists between the biological cycles of the parasite and its host. Thus, the mature spores of the coccidian parasite are disseminated when the worm lays its gametes by rupture of teguments. C. durchoni and N. diversicolor have established a biological equilibrium that permits mutual survival of both partners and constitutes a simple model for the study of the host-parasite relationship.


Allogeneic resorption in colonial protochordates: consequences of nonself recognition.

Colonial tunicates (protochordates of the subfamily Botryllinae) have the property of forming natural chimeras in the wild or in the laboratory with other members of the same species if both individuals share at least one allele at a single highly polymorphic locus, termed the fusibility/histocompatibility (Fu/HC) locus. Laboratory studies revealed that after the establishment of a common blood circulatory system between a fusible pair one of the colonies in the chimera is usually resorbed, a phenomenon that occurs after an interval of 1 week or extends to up to 8 months. In the present article, we review this allogeneic resorption in Botryllus schlosseri, a cosmopolitan protochordate. The studies on allogeneic resorption in B. schlosseri revealed 13 typical characteristics. However, several processes interrupt successful resorption, such as the retreat growth phenomenon, chimeric death, and others. It was also found that allogeneic resorption is not only genetically controlled but is also controlled by a polymorphic hierarchial phenomenon, including the Fu/HC locus and additional unrelated loci. One basic rule for this genetic system is that colonies heterozygotic on the resorption elements will resorb more homozygotic partners. This colony resorption, expressed by a morphological elimination of one partner, is probably manifested by cellular elements circulating in the tunicate blood system. However, it was also shown that some blood-borne cells of the inferior partner, such as the stem cells, may escape resorption, a phenomenon that raises the threat of germ/somatic cell parasitism.


Phagocytosis and hydrogen peroxide production by phagocytes of the sea urchin Strongylocentrotus nudus.

Phagocytosis of erythrocytes by phagocytes from the sea urchin Strongylocentrotus nudus can occur in vitro, and is enhanced by opsonization with the coelomic fluid. This opsonic activity of coelomic fluid can be elevated over a 5-day period by injecting erythrocytes into the coelom. Phagocytes produce hydrogen peroxide during both resting and stimulated states. This result on hydrogen peroxide production is the first to be observed in echinoderms. During the stimulated state, phagocytes produce more hydrogen peroxide than resting phagocytes. However, hydrogen peroxide production by phagocytes is not affected by opsonic activity of the coelomic fluid. Phagocytes share similar functional properties with vertebrate macrophages and granulocytes.


Identification of a second immunoglobulin in the most primitive shark, the frill shark, Chlamydoselachus anguineus.

Three molecular forms of immunoglobulins: pentamer, dimer and monomer, were isolated from serum of the frill shark, Chlamydoselachus anguineus, the most primitive extant shark. A pentamer having an apparent mass of 900 kDa, consisting of 68 kDa heavy (H) chains and 22-24 kDa light (L) chains, was considered to be IgM because of its similarity to mammalian IgM in both molecular form and H chain molecular weight. The dimer and monomer with apparent masses of 300 kDa and 150 kDa, respectively, were composed of the same H chains of 45-50 kDa, a value smaller than that of the H chains from the pentamer, and light chains identical to those of the pentamer. The H chains of 68 kDa and 45-50 kDa represented individual epitopes and were synthesized by different plasma cells. We thus concluded that the frill shark has two distinct classes of immunoglobulins: one a pentameric IgM and the other a second class of immunoglobulin with dimeric and monomeric structure. The presence of the second Ig is of considerable interest with respect to antigen recognition and exclusion.


The cellular proliferative response, humoral antibody response, and cross reactivity studies of Tetrahymena pyriformis with Ichthyophthirius multifiliis in juvenile carp (Cyprinus carpio L.).

The immune response of 10-12-week-old juvenile carp to T. pyriformis [CCAP 1630/w, 1939 (w)] was investigated. The humoral antibody response following one and two intraperitoneal injections of whole live T. pyriformis (protein concentration of 25 micrograms/g body weight), separated by an interval of 4 weeks, was monitored over 12-16 weeks. Peak antibody titres were detected 6 weeks following antigen administration. Antibody titres were elevated following the second injection, and relatively high levels were still maintained by week 16. Proliferative responses measured by autoradiography following intraperitioneal injection of methyl 3H thymidine (1-2 microCi/g body weight) were recorded in the pronephros, opisthonephros, and spleen at weekly intervals for 4 weeks following each injection of T. pyriformis. Immunised fish had higher counts than nonimmunised controls, with the greatest counts occurring at 2 and/or 3 weeks following one injection of antigen and 2 weeks following two injections. Activity was recorded prior to peak antibody production with the pronephros and opisthonephros having greater proliferative responses than the spleen. Cross antigenicity studies to examine protection of carp against I. multifiliis following administration of T. pyriformis, showed little evidence in support of protection using this strain of T. pyriformis. Further, antisera raised in either carp, rabbits, or rats failed to show any cross antigenicity between these two species of protozoa.


The effects of MHC chromosome dosage on bursal B-cell subpopulations in embryonic and neonatal chickens.

The effect of MHC dosage on the population structure of developing bursal lymphocytes was studied in the Trisomic strain of chickens. These chickens contain either 2 (disomic), 3 (trisomic), or 4 (tetrasomic) copies of the MHC-encoding chromosome. To assess the developmental profile of bursal cell populations, bursal cells were characterized in the older embryo (18 days of incubation) and at two neonatal ages (1 day and 1 week post hatch) by a number of cellular parameters. Both transient and persistent alterations in cellular composition were revealed in the bursal subpopulations of trisomics and tetrasomics (aneuploids). Aneuploid bursal cells showed increased cell surface expression of MHC class II Ia antigen, at each age, compared to normal disomic cells. Aneuploid cell populations showed an increased incidence of small cells, at each age, a decreased incidence of Ia+ and IgM+ cells during late embryogenesis, and an increased incidence of IgG+ cells in the late embryo and 1-day-old chick. Significantly fewer proliferating cells were observed in the aneuploids, as compared to disomics, in the late embryo and 1-week-old chick. Three subpopulations were observed to be persistently altered in the MHC-aneuploids: an increased pool of Ia , small, IgM+ cells; a decreased pool of Ia+, large, IgM+ cells; and a decreased pool of Ia+, large, IgM- cells. Thus, increasing MHC-chromosome dosage is accompanied by specific alterations in B-cell differentiation. The elevated surface Ia antigen expression found on developing aneuploid B cells may play a role in these alterations.


Characterization of a cDNA clone encoding for a porcine immunoglobulin mu chain.

We present the complete sequence of cDNA clone BLM6, isolated from a porcine spleen cDNA library. BLM6 encodes for the constant region of a secreted mu heavy chain. The cDNA is 1342 bp in length, begins within C mu 1, and terminates in the 3' translated region. The deduced amino acid sequence indicates there are 111 amino acids in C mu 2, 106 in C mu 3, and 113 in C mu 4, along with 19 carboxy-terminal residues that comprise the secreted terminus. The full length transcript is approximately 2.4 kb as determined by Northern blot analysis. Southern blot analysis suggests there is only one mu chain gene within the porcine genome. Comparison of the deduced amino acid sequence of porcine C mu with other mammalian C mu sequences indicates that the overall identity is approximately 60%, with the greatest identity seen in the secreted terminus (approximately 85%) and C mu 4 (approximately 70%); and less identity in C mu 3 (60%) and C mu 2 (50%).


Euthanasia and assisted suicide. I. How often is it practised by family doctors in The Netherlands?

A survey was conducted among family doctors to determine the frequency with which they were requested to administer euthanasia or assist in suicide, and how often they actually applied these procedures. Two random samples (in each n = 521) were taken from the population of Dutch family doctors (n = 6300) and requested to complete an anonymous questionnaire. The response was 67%. The entire body of Dutch family doctors practices euthanasia or assisted suicide about 2000 times per annum; 48% have never engaged in these practices. An average of 40% of all requests are complied with. We conclude that far fewer family doctors are involved in euthanasia and assisted suicide than was previously supposed. Euthanasia or assisted suicide was administered to 1 in 25 persons who died in their own homes.


Euthanasia and assisted suicide. II. Do Dutch family doctors act prudently?

We conducted a survey among two random samples of Dutch doctors in order to determine whether they acted prudently with regard to euthanasia and assisted suicide. The doctors completed an anonymous questionnaire and those who at one time or another had applied euthanasia or assisted suicide (52%) were asked about several aspects of the requirements for prudent practice. 'Pointless suffering' was the most important and most common reason for requesting euthanasia or assisted suicide; 'pain' was rarely the most important reason. In 7% of the cases alternative forms of treatment were still available; these were hardly ever therapeutic. A total of 12% of the doctors had applied euthanasia or assisted suicide without having had any kind of consultation or discussion with a colleague, a nurse or any other health care professional; 26% had not issued a certificate testifying to death from natural causes. We conclude that some of the family doctors do not observe the procedural requirements, but that the majority satisfies the material requirements for prudent practice.


Experience of bereaved relatives who granted or refused permission for organ donation.

A group of individuals who had granted or refused permission to have organs removed from a decreased relative for transplantation were interviewed. Organ donation was found to be a very delicate subject: the request for organ donation often comes unexpectedly and most relatives are not aware of the wishes of the decreased (donor codicil), which complicates the decision process. Some of the relatives who refused regretted their decision later. Remorse was also encountered in the group who gave their permission, although positive feelings were more common. The position of the member of staff who asks for permission is complicated. Suggestions are presented to improve the procedure.


Overall use of proctoscopy in general practice and possible relation to the stage of rectal cancer.

Data from the Danish National Health Service records on activities of each of 146 general practices in the county of North Jutland, Denmark, were studied to determine whether the use of proctoscopy influenced the stage at which cancer of the rectum was recognized. Information for all patients in the county who received the diagnosis of cancer of the rectum was obtained from the Danish Cancer Registry. Proctoscopy was performed significantly more often in partnership practices (96%) than in single practices (81%). The frequency with which the test was used varied from 1 to 107 proctoscopies per general practitioner per year. In the 95 patients with cancer of the rectum, no relationship was found between the stage (Dukes') at the time of diagnosis and work-load, size and activity of practice, or use of proctoscopy.


Evaluating patients with chest pain using classification and regression trees.

We collected data on 320 patients complaining to their general practitioner of a new episode of chest pain, discomfort or oppression. Relationships were examined between initial signs and symptoms and a follow-up diagnosis after a period of 2 weeks to 2 months. The data were analysed with CART, a statistical decision theory software package. In our first run, the number of misclassifications by CART was 56%. After regrouping of the data and diagnostic categories, there were 37% misclassifications. The most discriminating variable turned out to be pain on palpation. When comparing each of five diagnostic groups to all others, we found a positive predictive value of 27% for gastrointestinal diseases, 72% for cardiovascular disorders, 69% for respiratory diseases, 58% for psychopathology and 73% for chest wall pathology. The CART methodology needs further investigation and testing before any clinical application will be possible in general practice.


An evaluation of a nurse-run asthma clinic in general practice using an attitudes and morbidity questionnaire.

A before and after longitudinal study was conducted to evaluate a nurse-run asthma clinic in general practice. Sibbalds morbidity and attitudes questionnaire was used to monitor the changes in association with attendance at the clinic. One hundred and five patients aged 3-83 years attended the clinic. Over the 12 months of the study patients reported significant reductions in attacks of wheeze, nocturnal attacks, overall trouble (P eta 0.005) and GP home visits (P = 0.02). Patients also reported a significant improvement in feelings of stigma (P less than 0.001), and confidence in self-care (P less than 0.001). There was a significant association between morbidity criteria such as 'overall interference' and stigma at the outset of the clinic (R = -0.44; P less than 0.001; 95% confidence interval -0.58 to -0.26). A patient's feelings of stigma towards their asthma at the outset did not predict the eventual improvement in morbidity. As the patient's asthma improved the feelings of stigma became less apparent. This would suggest that improving a patient's morbidity should be one of the first priorities in asthma care. Confidence in the doctor was high at the outset and did not change throughout the study, despite the fact that a large proportion of patients wanted more information about their asthma but found it difficult to talk to their doctor. General practitioners are often restrained by how much time and resources they can devote to each patient. The nurse-run asthma clinic with its programme of structured care represents a possible solution to these problems.


Treatment with inhaled steroids in asthma and chronic bronchitis: long-term compliance and inhaler technique.

We investigated compliance and inhaler technique in 50 patients with airway obstruction (26 asthma, 24 chronic bronchitis) being treated with inhaled steroid (beclomethasone dipropionate, BDP) via a dry powder inhaler (Rotahaler omega). Patients had already participated for one year in a 2-year trial of BDP in general practice. They were treated daily with two dry powder inhalations of 400 micrograms BDP in combination with a bronchodilator. Compliance with BDP was measured by counting capsules (single-blind) at the end of a 4-month period and through a questionnaire. Counting capsules revealed non-compliance in 46% of the patients. Compliance was not related to age, sex, diagnosis or side-effects of BDP. In chronic bronchitis, but not in asthma, compliance was related to the outcome parameters of steroid treatment (pulmonary symptoms, change in lung function and non-specific bronchial responsiveness). The inhaler technique was judged insufficient in 27% of the patients. This study stresses the importance of regular instruction in inhaler technique and proper information about prophylactic steroid treatment by the general practitioner during the treatment of asthma and chronic bronchitis.


A practical intervention programme aimed at decreasing high serum cholesterol levels in primary care.

A practical, primary care based intervention programme which aimed to lower serum cholesterol in a large percentage of hypercholesterolaemic subjects in general practice is described. Intervention consisted of a 8-10 minute interview supplying oral and written diet counselling. The programme was tried in a kibbutz (agricultural settlement) with 93 adult members: 89 had their serum cholesterol determined, 35 of whom (39%) were hypercholesterolaemic--19 had borderline high cholesterol (5.2-6.2 mmol/l), 16 had definitely high cholesterol (greater than or equal to 6.2 mmol/l). Repeat blood samples were taken from 33 of the 35 hypercholesterolaemic patients 6-9 months after exposure to the intervention programme. The initial cholesterol level of the hypercholesterolaemic group was 6.31 +/- 0.2 mmol/l (mean +/- SE): at follow up it was 5.3 +/- 0.2 mmol/l (P less than 0.001), a decrease in serum cholesterol (mean 22.2 +/- 2.1%) being seen in 28 of 33 patients (84%). In 19 patients (58%) cholesterol dropped to normal values, and in six additional cases (18%) it fell to borderline values. These data suggest that high cholesterol levels in otherwise normal adults may be reduced with little effort by the primary care physician and nurse.


Controlled evaluation of management of schizophrenia in one general practice: a pilot study.

The study reports on a controlled evaluation of services offered to patients with a diagnosis of schizophrenia in comparison to diabetic patients and matched attenders with no chronic illness. Data were collected from one inner city London group practice, by a retrospective analysis of patients' notes over a period of 6 years. Although patients with schizophrenia attended significantly more often than other patients, clinical assessments in the surgery were rarely undertaken. Communications with hospital consultant services were rare for both chronic groups of patients but in the case of those with schizophrenia the doctor received on average only one letter every 2 years. The findings support other recommendations for a more structured approach to the care of schizophrenia in general practice with regular attention to mental state assessment and monitoring of drug treatment.


Perceptions of psychological problems in general practice: a comparison of general practitioners and psychiatrists.

All general practitioners and psychiatrists working in a single health district were sent a questionnaire on their perceptions of the prevalence of psychological problems among patients consulting in general practice. One hundred and twenty-one GPs (75%) and 10 (83%) psychiatrists responded. GPs and psychiatrists agreed that up to 20% of consulting patients were likely to be clinically depressed; they also agreed on the most appropriate management, although GPs tended to be more conservative than psychiatrists would advise on making use of hospital services. GPs, however, believed non-specific psychological problems to be significantly less common than did psychiatrists. This expectation may help explain the reported failure of GPs to diagnose all psychological problems identified by formal psychiatric instruments.


Attaching prices to decision making in general practice.

This paper is an attempt to evaluate the cost implications of decisions made by general practitioners during consultations. Prices were attached to the cost generating decisions of 85 general practitioners in South East Scotland who participated in a 12-month study of their workload. When the price attached to each referral decision was one out-patient attendance, 56 of the 85 general practitioners' costs per consultation were within 15% of the average cost, and 76 were within 25% of the average. However, when the price attached to referral activity was adjusted upwards (to allow for the inclusion of further hospital care), variations between doctors became more pronounced and the decision to refer replaced prescribing as the most important element of variable cost. No important relationships were discovered which linked cost generating activities with characteristics of the doctor or the practice. Implications for fund-holding are discussed.


Principle agendas of doctors and patients in general practice consultations.

Seventy-three general practice interviews were analysed according to the method described by Butler et al. Results for the three principal agenda types (physical, emotional and social) are presented in terms of the control exerted by either doctor or patient in determining the content of the interview. Both doctor and patient address physical agendas to a similar high degree. In contrast, patients present emotional agendas to a far greater extent than doctors address these concerns. The findings for social agendas are intermediate between those for physical and those for emotional agendas. It is argued that doctors can facilitate the expression of emotional and social agendas by giving explicit or implicit permission for their presence in the interview.


Reasons for encounter and diagnosed health problems: convergence between doctors and patients.

This study of 521 encounters in 25 urban general practices in Australia, compares both patient and doctor reported reasons for encounter (RFE) and diagnoses. Although doctors and their patients generally agreed on the overall distribution of RFE and diagnoses that arose, there was disagreement in at least 30% of paired comparisons within individual encounters. There was better agreement for RFE than for diagnoses. This may have been partly due to differences in the classification systems used. However, it suggests that diagnoses recalled by patients at later household interview are at best only a rough approximation of the diagnoses recorded by the doctor. These findings are important both for patient care and for the conduct of general practice morbidity research.


Examination and communication: a study of first encounters between patients and physiotherapists.

What happens in first encounters between patients and physiotherapists? Videotaped encounters between two patients and their physiotherapists were analysed in depth to try to answer this question. It is advocated that attention must not be restricted to either clinical decision making or communication, but needs to be directed towards the relationship between communication and diagnostic approaches. In other words, in order to understand how a health problem is defined, the complexity and social organization of the process of interaction cannot be ignored. Therapies are chosen and cooperation is built on such socially and professionally constructed basis, and it is suggested that the nature of this construction needs to be grasped. To this end, the author analyses both the therapists' examination and the communication between patient and therapist. Focus is thereby shifted from the 'pure' diagnostic activity to the frame of reference underlying it. The analysis demonstrates differences in the two therapists' practice from the very moment patient and therapist greet one another till they say farewell. The therapists' actions mirror the dissimilar conceptual frameworks underlying their practice, and the therapists' conceptual lenses therefore need to be scrutinized further.


Do physicians care about patients with dysphagia? A study on confirming communication.

Thirty-nine patients with longstanding oesophageal dysphagia took part in an interview and a questionnaire study to investigate whether they considered themselves met with interest, respect, understanding and knowledge (felt confirmed) when consulting their physician for the first time (mostly general practitioner), whether non-confirmed patients have any distinguishing attributes, and whether confirmation affects the patient's impression of being helped. Twenty-five of these considered themselves as non-confirmed. There was no difference between the groups as regards sex, age, education, degree of swallowing difficulties and incidence of chest symptoms other than dysphagia. There was a greater fear of cancer (P less than 0.05) and a stronger indirect aggression (P less than 0.05) among the non-confirmed patients. Patients who felt confirmed reported more often that a correct oesophageal diagnosis had been given (P less than 0.05), felt they had received adequate help (P less than 0.001) and experienced improvement in swallowing difficulties (P less than 0.05). It is concluded that patient attributes seem to slightly influence the patients' possibilities of being confirmed, and that confirmation is of great importance for the patient's feeling of being helped and improved.


A negotiation model for the doctor-patient relationship.

A model has been developed to help physicians negotiate with patients in more explicit and effective ways. This model provides physician teachers and learners with a framework and a common language to describe the dynamic nature of the doctor-patient negotiation. This framework consists of three dimensions: content, relationship levels, and the problem-solving phases. The constructs of disease, illness, sickness and the patient's context are used to describe the content of negotiation: this is what the doctor and patient are talking about. Autonomy, power, control and responsibility are the constructs that define the relationship levels: autonomism, egalitarianism, parentalism, and autocracy. These levels describe how the doctor and patient relate to one another during their negotiation. The problem-solving phases are relationship building, agenda setting, assessment, problem clarification, management and closure. Teachers and learners can use this model to describe how the doctor and the patient affect the negotiation process, and how the process in turn affects the doctor-patient relationship and medical care. With practice using this model, physicians can increase their repertoire of negotiating strategies that will efficiently enhance doctor-patient collaboration, the problem-solving process and the health of the patient and family.


The interrupted consultation.

This paper focuses attention, in a modest empirical way, on a hitherto unexplored phenomenon. Widespread in Israel, the interrupted patient-physician consultation may or may not be found extensively elsewhere. It is assumed that interruptions coming from the social environment affect communication and hence diagnosis and therapy, as well as satisfaction of both doctor and patient. Observations were conducted in a random sample of 100 consultations, averaging 9.4 minutes, of four physicians in a neighbourhood health centre in Israel. Interruptions were recorded in 94 cases, with an average of 1.36 interruptions per consultation. Contrary to Israeli myth, most interruptions were not by other patients but by clinic staff. The phenomenon is considered in terms of cultural practice and the consequences of group practice, nurse autonomy and medical student teaching. Further research is proposed to study the extent, sources and consequences of interruptions in family practice and, if warranted, methods of coping.


Problems and issues in family medicine psychosocial research.

The authors believe that, despite their complexity, psychosocial factors should be included with biological variables in family medicine research. To aid this process, this article reviews some problems and issues in selected areas of psychosocial research relevant to family medicine. Basic background material, dimensions, models, intra-individual, intra-familial physiological mechanisms of transmission, and factors related to stress, support and coping, are discussed. This is followed by methodological issues that include research design, psychometric properties of measures, data collection, quantitative and qualitative methods, family assessment problems and techniques, and analytic procedures. The latter includes scoring problems, global versus specific questions, and statistical analyses issues. Finally, a discussion of biological relationships and potential biological markers for psychosocial processes or variables concludes this review.


In vitro photosensitivity of ecdysteroid synthesis by prothoracic glands of Rhodnius prolixus (hemiptera).

The effects of light:dark transitions on in vitro ecdysteroid synthesis by prothoracic glands (PGs) of male fifth instar Rhodnius have been studied. PGs were rendered arrhythmic by prolonged maintenance of the animals in continuous light (LL). PGs were then explanted and ecdysteroid synthesis was measured hourly by radioimmunoassay. PGs were transferred from LL to darkness at various times after explanation and hourly synthesis was measured for several more hours. Transfer of PGs to darkness in vitro invariably elicited a prompt and substantial (up to fivefold) increase in ecdysteroid synthesis. This response was unaffected by the presence of tetrodotoxin in the medium at a concentration that blocks peripheral nervous activity. It is concluded that the PGs are themselves directly photosensitive. In contrast, PGs from animals raised in continuous darkness and incubated in vitro in darkness showed no response when exposed to light. It is inferred that PGs do not respond to maintained states of either light or dark, or to "lights on." Rather, it is argued that transfer from LL to darkness in vitro constitutes a "lights-off" Zeitgeber which is detected by a photosensitive circadian clock in the PGs. This is the first report of photosensitivity in a steroidogenic tissue and provides primary evidence of a photosensitive pacemaker in a nonneural endocrine tissue.


An enzyme-linked immunosorbent assay for stanniocalcin, a major hypocalcemic hormone in teleost.

An enzyme-linked immunosorbent assay (ELISA) was developed to quantify stanniocalcin (STC) levels in tissue extracts and plasma samples. The detection limit of the competitive ELISA described is 0.2 ng STC per well, allowing detection of 3.7 pmol.liter-1 (assuming a molecular mass of 54,000 Da for native STC). The particular antiserum detects STC in plasma obtained from a variety of freshwater and seawater species. In freshwater post-smolt Salmo salar plasma, STC levels were significantly lower (around 0.74 nmol.liter-1) than those of seawater smolts (around 2.78 nmol.liter-1). Seven days after removal of Stannius corpuscles from freshwater eels a significant hypercalcemia was observed as well as a drop in plasma STC levels (from 2.33 to 0.67 nmol.liter-1).


Sexual development and free-running period in quail kept in constant darkness.

In Japanese quail (Coturnix coturnix japonica) sexual development may occur in total darkness (DD). Linked to sexual maturation, variations of the rhythm of feeding activity were observed: in DD, the amount of activity increased and the period of the free-running rhythm lengthened. During the first weeks in DD all the quails presented a free-running period less than 24 hr (22.3 +/- 0.5 hr; N = 50). At the end of the experiment, the more a bird was mature, the greater the lengthening of its period. In DD, testosterone implants in castrated male quails can mimic these modifications but we failed to find any correlation between the number of implants (i.e., the level of testosterone) and the extent of the lengthening.


Growth hormone binding sites in tilapia (Oreochromis mossambicus) liver.

125I-labeled bovine and tilapia growth hormones were used to assess the presence of growth hormone receptors in membranes prepared from tissues of the tilapia Oreochromis mossambicus. The highest level of specific binding was detected in liver membranes from animals of both sexes and the binding was protein-dependent. Tilapia growth hormone, bovine growth hormone, and ovine prolactin, but not tilapia prolactin, potently inhibited the hepatic binding of 125I-labeled bovine growth hormone. Scatchard analysis of the 125I-labeled bovine growth hormone binding data revealed a Bmax (maximum binding) value of 180 fmol/mg protein and a Kd (dissociation constant) value of 13 nM. Tilapia growth hormone potently inhibited hepatic binding of 125I-labeled tilapia growth hormone. Scatchard analysis revealed a single class of binding sites with Bmax and Kd values of 390 fmol/mg protein and 2.5 nM, respectively. Bovine growth hormone and ovine prolactin were less potent while tilapia prolactin was inactive in inhibiting hepatic 125I-labeled tilapia growth hormone binding.


Primary structures of the hypertrehalosemic peptides from corpora cardiaca of the primitive cockroach Polyphaga aegyptiaca.

Two hypertrehalosemic neuropeptides from the corpus cardiacum of the cockroach Polyphaga aegyptiaca were isolated by reversed-phase high-performance liquid chromatography, and their primary structures were determined by pulsed-liquid phase sequencing employing Edman chemistry, after enzymically deblocking the N-terminal pyroglutamate residue. As neither peptide was cleaved by carboxypeptidase, the C-terminus of each peptide was also blocked. Both peptides were found to be uncharged octapeptides with the sequences: Peptide 1: pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-NH2; and peptide 2: pGlu-Ile-Thr-Phe-Thr-Pro-Asn-Trp-NH2. Both peptides are clearly defined as members of the adipokinetic hormone/red pigment-concentrating hormone family of peptides. Whereas peptide 1 is identical in structure to the previously sequenced hypertrehalosemic neuropeptide from tenebrionid beetles (and is therefore designated the acronym Tem-HrTH), peptide 2 is a novel peptide and is designated the acronym Poa-HrTH. Both synthetic peptides caused an increase in the hemolymph carbohydrate concentration in P. aegyptiaca, specifically changing the trehalose concentration. The novel peptide Poa-HrTH was not very potent in elevating blood carbohydrates in the American cockroach.


Annual variations in the binding of insulin to hepatic membranes of the frog Rana esculenta.

Amphibia undergo regular annual cycles of metabolic activity that are influenced by both exogenous factors and hormones. Insulin binding to crude frog hepatic membranes was studied throughout the year. The general character of insulin binding was similar to that in other vertebrates; the maximum specific binding was achieved after 4 hr at 4 degrees, the optimum pH was 7.8, half-maximal displacement of bound insulin was from 9 x 10(-10) to 1 x 10(-9) M, and insulin analogs competed for the insulin receptor in line with their relative biological potencies. A biphasic Scatchard plot and negative cooperativity of the receptor were also observed in frog liver membranes. Affinity constants from Scatchard plots revealed high and low affinity binding sites which were unchanged during the year. The seasonal cycle, however, markedly affected the binding capacity for both sites. Maximum binding occurred in May-June and the minimum in November-December for both classes of receptors. Binding capacities ranged from 1.71 to 11.33 fmol/mg protein for the high affinity sites and from 432 to 3171 fmol/mg protein for the low affinity sites. It is concluded that annual cycles of insulin binding reflect modulation of receptor number rather than receptor affinity.


Thyroid and adrenal function in the ring dove (Streptopelia risoria) during food deprivation and a breeding cycle.

The concentration of plasma triiodothyronine (T3), thyroxine (T4), corticosterone, and glucose as well as body weight and food intake were measured in pairs of ring doves throughout a breeding cycle and during food deprivation. During courtship and incubation the levels of plasma T4 were significantly higher in males than in females. A transitory decrease in food intake in both sexes was measured at the onset of incubation but was not associated with a change in body weight or in levels of plasma thyroid hormones. After the eggs had hatched, food intake increased but was associated with a reduction in body weight and concentrations of plasma T3 and T4, although plasma corticosterone and glucose both increased. Food deprivation for 48 hr resulted in a significant fall in the concentration of plasma T3 and an increase in plasma corticosterone. These changes suggest that all the food was not being digested by the adult birds during brooding but was almost exclusively regurgitated to feed the squabs.


Influence of dihydrotestosterone on sex determination in channel catfish and blue catfish: period of developmental sensitivity.

Treatment of channel catfish with 0.2, 20, or 200 mg/liter of dihydrotestosterone (DHT) in the water during the egg stage or during egg and sac-fry stages did not alter the expected 1:1 sex ratio of the progeny. Feeding DHT at 200 mg/kg of feed for the first 21 days after yolk sac absorption resulted in 80% females; this proportion was increased by combining feeding with treatment of 200 mg DHT/liter in the sac-fry stage (90%) or in the egg and sac-fry stage (97%). In contrast, treatment of blue catfish sac-fry with 200 mg DHT/liter, with or without the combination of feeding DHT at 200 mg/kg food, resulted in 100% female populations. Neither clomiphene citrate, an estrogen-receptor blocking agent, nor clofibrate, an inhibitor of hepatic synthesis of cholesterol, affected the sex ratio of channel catfish, and neither of these compounds altered the feminizing effect of 200 mg DHT/kg when fed in combination with DHT. The nonaromatizable androgen DHT is not as effective as many other androgens in producing paradoxical female populations of channel catfish. However, feminization of blue catfish by treatment of sac-fry indicates that this species is more susceptible to hormonal manipulation and that the period of sex determination may occur earlier in development than in channel catfish.


Acid-base-electrolyte balance responses of Bufo marinus to aminoglutethimide, corticosterone, and aldosterone during hypercapnia.

Experiments were conducted to test the hypothesis that one or more interrenal steroids are active in regulatory responses to respiratory acidosis in the toad, Bufo marinus. Toads were divided into four experimental groups. The first group received sham injections. The second group received 1-3 mg of aminoglutethimide (AG) every 8 hr. AG inhibits the conversion of cholesterol to pregnenolone, thus inhibiting all steroid hormone synthesis. The third group received AG + 5 micrograms of aldosterone on the same schedule. The fourth group received AG + 25 micrograms of corticosterone on the same schedule as the other groups. All four groups were subjected to hypercapnia using 5% CO2 to induce a respiratory acidosis. The sham-operated animals displayed the normal compensatory pattern of producing a metabolic alkalosis (elevated plasma HCO3-) after 24 hr. AG-treated toads failed to elevate plasma HCO3-. Administration of interrenal steroids produced compensation in varying degrees. Aldosterone produced a small compensation while corticosterone produced a compensation similar to that seen in sham-operated animals. Analysis of steroid titers in toad plasma during hypercapnia showed that Bufo marinus does not elevate aldosterone during respiratory acidosis, but that corticosterone is elevated. AG blocked the corticosterone elevation, however. AG also produced a hyponatremia that was corrected with aldosterone or corticosterone. Normocapnic controls showed that AG does not produce deleterious effects on pH or blood gases in toads in the absence of a respiratory acidosis. We conclude that corticosterone is important in acid-base regulatory responses to respiratory acidosis in this amphibian.


Duodenal acidification and intra-arterial injection of CCK8 increase gallbladder motility in the rainbow trout, Oncorhynchus mykiss.

A sensitive method for in vivo measurement of intra-gallbladder pressure in free swimming rainbow trout (Oncorhynchus mykiss) is described. The method gives information on changes in gallbladder pressure after either instillation of liquids into the duodenum or infusion/injection of agents into the circulatory system. Blood samples can also be taken. Duodenal acidification is a potent initiating factor for gallbladder motility in the rainbow trout. Intra-arterially injected sulfated octapeptide of cholecystokinin is also a potent excitatory agent for activating the gallbladder. Acidification of the upper intestine causes release of cholecystokinin, which affects gallbladder motility in trout as in mammals.


Effects of photoperiod and temperature on rhythmic melatonin secretion from the pineal organ of the white sucker (Catostomus commersoni) in vitro.

The secretion rate of melatonin from cultured pineal organs of the white sucker was examined for several days under either a 12:12-hr light:dark (LD) cycle or continuous darkness (DD) at either 10 degrees or 20 degrees. The incubation medium was changed at 3-hr intervals and secreted melatonin was measured by RIA. Under a 12:12-hr LD cycle (0800 light on, 2000 light off) melatonin secretion was suppressed during the day and highly active at night, with larger amplitudes at 20 than at 10 degrees. In DD at 10 degrees no circadian rhythmicity in secretion was found in October or January, whereas at 20 degrees a circadian-like pattern was detected in pineals which were derived from animals reared at either 10 degrees or 20 degrees for 1 week prior to the experiment in October or January. The pineals in the DD experiment still responded to an additional 24-hr LD cycle at both temperatures even after 6 or more days. These results clearly reveal the influence of photoperiod and temperature on melatonin secretion of organ-cultured pineal glands. The existence of a circadian oscillator for melatonin secretion in the pineal gland of the white sucker is suggested.


Amplexus induces surge of luteinizing hormone in male toads, Bufo japonicus.

At mating time in the wild, male toads (Bufo japonicus) exhibit a surge in circulating luteinizing hormone (LH) that is thought to result in spermiation. Experiments were performed to identify proximate control of this gonadotropin surge. Male toads migrating to a breeding pond were captured and put in plastic containers either with equal numbers of females or without females. All the males placed with the females clasped them in amplexus that lasted for about 12 hr. During this period, plasma LH and follicle-stimulating hormone levels increased progressively to about 25 and 2 times the initial levels, respectively, and then returned to the initial levels. No significant increases in plasma gonadotropin levels were observed in the males kept without females. These results suggest that the amplexus directly induced the LH surge, but do not preclude control by pheromonal or visual stimuli. To isolate effects of these factors, solitary male toads were kept with dummies of the female. The dummy was a block of "konnyaku," Japanese food whose component is mannan extracted from the root of a plant. Konnyaku is highly elastic, white in color, and virtually odorless. Thus, these dummies felt, but did not look or smell, realistic. All the males clasped their dummy and formed amplexus for 12 hr or more; an LH surge indistinguishable from that with the females was observed. It is concluded that the amplexus itself is the stimulus that induces the LH surge, and subsequent spermiation, in male toads. A strongly male-biased sex ratio, combined with a need to time spermiation precisely with actual mating, may have resulted in the evolution of this neuroendocrine reflex in male Bufo.


Thyroid hormone receptor induction by triiodothyronine in tadpole erythrocytes in vivo and in vitro and the effect of cycloheximide and actinomycin-D.

Tadpole erythrocyte nuclei contain specific T3 binding sites which increase in number during spontaneous or T3-induced metamorphosis. In the present studies the increase in the number of T3 binding sites after a T3 injections appeared to be completely prevented by cycloheximide and actinomycin D, inhibitors of protein synthesis and RNA synthesis, respectively. However, in some experiments the effect was not statistically significant. The increase in sites was prevented only if the inhibitors were administered at 0 or 24 hr after T3 injection, but not at 48 or 72 hr after T3. When tadpole erythrocytes were incubated with T3 in vitro in M199 culture medium, the number of nuclear T3 binding sites increased within 48 hr. This increase was highly sensitive to inhibition by cycloheximide (maximal at 1 x 10(-6) M) or actinomycin D (maximal at 0.02 micrograms/ml). These inhibitor concentrations only slightly reduced the incorporation of labeled precursors. The T3 concentration required to induce a half-maximal increase in binding sites in vitro was about the same as the T3 concentration at which half the binding sites were occupied. The T3 binding sites had a high affinity for thyroid hormone analogs which stimulate metamorphosis. These results support the designation of the T3 binding sites as T3 receptors. They show that the tadpole erythrocytes respond to T3 with an increase in the number of T3 binding sites without the involvement of other tissues. It is proposed that this is a receptor induction involving synthesis of RNA and protein.


Interactions between progesterone and androgens in the stimulation of sex behaviors in male little striped whiptail lizards, Cnemidophorus inornatus.

Progesterone is believed to have a suppressive or inhibitory role in regulating androgen-dependent sex behaviors in male mammals and birds. Previous studies in this laboratory have revealed that in the little striped whiptail lizard (Cnemidophorus inornatus), progesterone (P) can stimulate sex behavior in a proportion of the males. The present study sought to determine (i) interactions between androgens and P in activating sex behaviors, and (ii) the overlap in behavioral sensitivities to androgens and P in male C. inornatus. With an increasing length of castration the behavioral sensitivity of males to exogenous P was reduced. However, priming of castrated males with subthreshold doses of exogenous dihydrotestosterone (DHT) greatly facilitated subsequent behavioral responses to exogenous P. Progesterone treatments of castrated males were more effective at reinstating sex behaviors in males that exhibited high-intensity sex behaviors prior to castration compared to males that exhibited low-intensity sex behaviors. Finally, exogenous DHT is more effective at reinstating sex behaviors in P-sensitive males than in P-insensitive males. These data are discussed in light of possible mechanisms underlying the unusual behavioral effects of P in the Cnemidophorus model system.


Ultrastructural investigation of the corpora atretica of the electric ray, Torpedo marmorata.

Follicular atresia was studied in the ovary of the electric ray, Torpedo marmorata, by light and electron microscopy. The course of atresia may be divided into four stages. The first two comprise the dissolution of the oocyte and its phagocytosis by the small cells of the granulosa epithelium. The third stage consists of the transformation of the granulosa epithelium into an active glandular structure and is accompanied by the development of a smooth endoplasmic reticulum. The fourth stage is marked by sclerosis and pigmentary degeneration of the atretic follicle. Together these observations suggest an endocrine steroidogenic role for the corpora atretica (preovulatory corpora lutea) in T. marmorata.


Immunohistochemical demonstration of the presence and localization of diverse molecular forms of gonadotropin-releasing hormone in the lizard (Podarcis s. sicula) brain.

The immunohistochemical presence and the distribution pattern of four different molecular forms of gonadotropin-releasing hormone (GnRH) were investigated in the brain of both sexes of the lizard, Podarcis s. sicula. Animals used in this study were collected in November and April, representing two different periods of the reproductive cycle. The antisera used were those raised against synthetic mammalian GnRH, chicken GnRH-I and II, and salmon GnRH. Strong immunoreaction was obtained for salmon, chicken-I, and chicken-II GnRHs, whereas a very weak reaction was seen for the mammalian form of GnRH. The distribution of immunoreactive-GnRH perikarya and fibers did not vary with the sex, the reproductive condition of the animals, or the antiserum used. Also, the intensity of immunoreaction with any one antiserum was quite similar in both periods of the year and in all brains examined. The immunoreactive perikarya was seen as two distinct groups, one in the mesencephalon and the other in the infundibulum. Immunoreactive fiber endings were seen in the telencephalon, the optic tectum, the anterior preoptic area, the median eminence, the central grey matter, the rhombencephalon, and the cerebellum. No immunoreactive perikarya were seen in the telencephalon or the anterior preoptic area.


Effects of growth hormones on bone formation and resorption in rainbow trout (Oncorhynchus mykiss), as examined by histomorphometry of the pharyngeal bone.

Effects of salmon and eel growth hormones (GHs) on bone metabolism in rainbow trout (Oncorhynchus mykiss) were studied using histomorphometry of their pharyngeal bones. When salmon GH (sGH) was injected intraperitoneally at a dose of 0.01 or 0.1 microgram/g/week for 10 times into fed trout, body growth in both length and weight was accelerated. Formation and resorption of bone were enhanced only by the higher dose of sGH. When a cholesterol pellet containing recombinant eel GH (reGH-pellet) was implanted subcutaneously into starved trout for 1 week (37 micrograms reGH/18 g fish), formation and resorption of bone increased, although activity of bone formation was low. The results indicate that the GHs increased both formation and resorption of bone, regardless of the nutritional status of the fish.


Identification of putative egg-laying hormone containing neuronal systems in gastropod molluscs.

Of gastropod molluscs, only in the Aplysiidae and the Lymnaeidae have the genes encoding the respective egg-laying hormones been cloned and the neurons controlling egg laying and egg-laying behavior been identified. Immunocytochemistry, using antibodies raised against alpha-CDCP (one of the neuropeptides encoded on the egg-laying hormone gene of Lymnaea stagnalis), identified neurons in various species of gastropods. In the basommatophoran snail, Biomphalaria glabrata, large and small neurons were observed in areas of the central nervous system similar to where immunoreactive cells exist in L. stagnalis, i.e., in the cerebral and pleural ganglia. In the stylommatophoran snail (Helix aspersa) and the slug (Limax maximus), large immunopositive neurons occur in the visceral and right parietal ganglia. In L. maximus, small immunoreactive neurons were found in the cerebral ganglia while in H. aspersa similar cells were observed intermingled with the large cells in the visceral and right parietal ganglia. Similar to the situation in L. stagnalis, in the female part of the reproductive tract of B. glabrata, L. maximus, and A. californica, but not in H. aspersa, neurons and/or fiber tracts are present. The results indicate that egg-laying hormone precursor molecules of gastropod molluscs are phylogenetically closely related. The alpha-CDCP antiserum may allow the identification of hitherto unknown egg-laying regulating systems of gastropod molluscs.


High-level resistance to ethidium bromide and antiseptics in Staphylococcus aureus.

A gene encoding high-level resistance to ethidium bromide (EB) and antiseptics was isolated from a transferable plasmid, pTZ22, in Staphylococcus aureus. DNA sequencing revealed that the plasmid has two copies of the ebr gene that normally mediates low-level resistance to EB and antiseptics. The efflux rate for EB of strains with duplicated ebr genes was twice the rate of strains with a single ebr gene. It was concluded that the duplication of ebr is responsible for the high-level resistance to EB and antiseptics.


A method of enhancing verocytotoxin production by Escherichia coli.

The number of verocytotoxin producing Escherichia coli (VTEC) present in the faeces during an infection may be very low, making their detection difficult. We report a method for enhancing toxin production by VTEC using mitomycin C as an inducing agent with the aim of improving the detection of VTEC. In pure culture, mitomycin C enhanced toxin production up to 100-fold. When applied to mixed faecal culture, toxin could be detected in mitomycin C treated samples when standard cultures were negative and when substantially fewer verocytotoxin-producing bacteria were present. Use of this method may aid in the detection of VTEC and is appropriate for use in the routine diagnostic laboratory.


Bordetella heat-labile toxin causes release of radioactivity from smooth muscle cells labeled with [14C]arachidonic acid.

The effect of Bordetella heat-labile toxin (HLT) on cultured vascular or tracheal smooth cells labeled with [14C]arachidonic acid for 3 h was examined. At 37 degrees C, in the presence of Ca2+, HLT induced the release of radioactive substances from the cells in a dose-dependent manner but HLT had no effect on release of radioactivity from cells at 0 degrees C or in the absence of Ca2+. After cells were exposed to HLT, a 2-h lag period occurred before release of radioactivity was detected. The substances released from cells by HLT were identified as arachidonic acid and phosphatidylcholine.


Incorporation of S-[3H]methyl-D-cysteine into the peptidoglycan of ether-treated cells of Escherichia coli.

Certain D-amino acids can be incorporated into the murein sacculus of Escherichia coli apparently through a direct transpeptidation reaction independent of the normal biosynthetic pathway. Investigation of this process is important because it could lead to the identification of hitherto unknown enzymes involved in murein metabolism. However, a serious drawback is the lack of an appropriate in vitro assay. We have analysed the suitability of a system based on the incorporation of a radioactive substrate (S-[3H]methyl-D-cysteine) by ether-treated cells, a method successfully applied before to the study of murein biosynthesis. The results reported here indicate that ether-treated cells are indeed proficient in the incorporation of D-amino acids, matching closely the properties of the reaction in growing cells.


Two variants of transferrable extended-spectrum TEM-beta-lactamase successively isolated from a clinical Escherichia coli isolate.

In a leukaemic patient presenting a septicaemia treated with ceftazidime and amikacin, two clinical Escherichia coli isolates distinguished by their level of resistance to oxyimino-beta-lactams were isolated at an interval of 24 h. The isolates were identified by biotyping and esterase electrophoretic typing and the two host strains were shown to be identical. However, each of these strains exhibited a different transferrable extended-spectrum beta-lactamase. These enzymes had different pI values (5.25 and 5.58), but were both blaTEM-1 mutants. The enzyme with pI 5.25 was identical to TEM-101 (TEM-12) (serine 162 substitution). The enzyme with pI 5.58 showed an additional amino acid substitution (lysine residue instead of an arginine at position 237) and was denominated TEM-23. These data indicate that point-mutations can be successively cumulated in vivo by blaTEM mutants, leading to expression of beta-lactamases with increased hydrolysis rates.


Phylogenetic analysis of the genera Planococcus, Marinococcus and Sporosarcina and their relationships to members of the genus Bacillus.

A phylogenetic analysis based on 16S rRNA was performed on the genera Planococcus, Marinococcus, Sporosarcina and endospore-forming rods. In agreement with earlier 16S rRNA cataloguing data, Planococcus citreus and Sporosarcina ureae clustered with Bacillus pasteurii and other bacilli containing lysine in their cell walls. Sporosarcina halophila was shown to be genetically distinct from S. ureae and formed a loose association with the main Bacillus subtilis grouping. Marinococcus halophilus (formerly Planococcus halophilus) exhibited low levels of relatedness to all reference species examined and formed a distinct line of descent.


Polyclonal antibodies against Staphylococcus aureus ATCC 12600 catalase do not recognize any protein in cellular extracts from S. aureus subsp. anaerobius.

Rabbits were immunized with electrophoretically pure catalase from Staphylococcus aureus ATCC 12 600. The antiserum was used to study whether S. aureus subsp. anaerobius was able to synthesize the apoprotein of catalase. Proteins were separated on polyacrylamide gels (SDS-PAGE) and transferred to nitrocellulose membranes and were detected by immunoblotting. No protein reacting with the purified immunoglobulins against S. aureus ATCC 12,600 catalase could be detected in crude and partially purified cellular extracts from S. aureus subsp. anaerobius or its aerotolerant mutants.


Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus.

Conjugative transfer, in the apparent absence of plasmid DNA, of high-level vancomycin resistance from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus B111 has been demonstrated in vivo and in vitro. Selection of transconjugants on media containing erythromycin or chloramphenicol may result in the transfer of resistance to erythromycin, chloramphenicol, gentamicin, streptomycin and vancomycin though these are capable of separate transfer. Vancomycin resistance has not been transmitted from staphylococcus to staphylococcus though transfer of erythromycin and of chloramphenicol resistance has been achieved.


Serious physical illness as a stressor: effects on family use of medical services.

The purpose of this study was to investigate the effect of stress on the family due to the hospitalization of a family member for a serious chronic illness. The data were the health insurance claims of 3,591 families obtained from the largest U.S.A. insurer of federal employees. It was hypothesized that the nonhospitalized family members would have a stress-related increase in medical expenses for some period of time after the chronically ill person was hospitalized. Multiple regression analysis with adjustments for a number of covariates found increases in mean medical charges of $326 per person (p less than 0.01) (excluding the hospitalized person) in the 3 years following the hospitalization. This finding lends support to the theory that stress--in this case, hospitalization of one family member--affects the entire family system. No significant differences were found in medical charges between families who did and did not receive mental health treatment following the hospitalization.


Epidemiology of depression in primary care.

Major depressive disorder has been recently found to be associated with high medical utilization and more functional impairment than most chronic medical illnesses. Major depression is a common illness among persons in the community, in ambulatory medical clinics, and in inpatient medical care. Studies have estimated that major depression occurs in 2%-4% of persons in the community, in 5%-10% of primary care patients, and 10%-14% of medical inpatients. In each setting there are two to three times as many persons with depressive symptoms that fall short of major depression criteria. Recent studies have found that in one-third to one-half of patients with major depression, the symptoms persist over a 6-month to one-year period. The majority of longitudinal studies have determined that severity of initial depressive symptoms and the presence of a comorbid medical illness were predictors of persistence of depression.


Screening laboratory evaluation in psychiatric patients: a review.

Routine laboratory screening of psychiatric patients is a common clinical practice. Several studies have demonstrated the limited utility and higher cost of ambulatory and preadmission screening in the evaluation of general medical patients. The data concerning the use of such screening profiles in psychiatric patients are reviewed. Widespread use of extensive screening batteries, consisting of complete blood cell count (CBC), complete blood chemistry analysis, erythrocyte sedimentation rate (ESR), urinalysis, B12, folate, electroencephalogram (EEG), electrocardiogram (EKG), and chest x-ray film, is not indicated in the majority of psychiatric patients. Such investigations result in many abnormal findings, most of which are clinically insignificant and do not affect patient management and outcome. Most abnormal results can be predicted by information obtained from a careful history, review of systems, and physical examination. Certain populations appear to benefit from more extensive evaluation, including those older than 65 years of age or of low socioeconomic status, state hospital patients, patients with drug and alcohol histories, and patients with evidence of disorientation, self neglect, or organic mental disorders. The few tests that have merit as broader screening tests in asymptomatic patients include serum glucose, blood urea nitrogen (BUN), creatinine, and urinalysis. Patients on psychotropic medications should be monitored for side effects of that particular therapy. Further prospective data are needed to develop cost-efficient, population-specific diagnostic strategies.


The depressed alcoholic. Clinical features and medical management.

A relationship between depression and alcoholism has long been postulated. A review of prior research studies reveals that though patients with depression do not appear to develop alcoholism to any great extent, recently detoxified alcoholics have a depressive syndrome about 20% of the time. This cannot be accounted for readily from data on family studies or genetic studies, which generally suggest that alcoholism and depression are two independent illnesses, albeit both quite common. Clinically, depressed alcoholics resemble alcoholics more than they resemble depressives. The clinical course of depression when it coexists with alcoholism is generally benign and self-limited, with most patients becoming euthymic over the course of 2-4 weeks without specific antidepressant treatment. In some depressed alcoholics, however, a more chronic depression persists, and may predict a worse outcome for the alcoholism. Treatment of depression in alcoholics should be initially conservative. Tricyclic and other antidepressants should be used with extreme care as they may potentiate toxic effects of alcohol.