A tobacco DNA binding protein that interacts with a light-responsive box II element. Ribulose-1,5-bisphosphate carboxylase/oxygenase plays a key role in photosynthetic carbon fixation in higher plants. The small subunit of this chloroplast enzyme (rbcS), encoded by a family of nuclear genes, is regulated at the transcriptional level by light. Promoter analyses have previously identified the box II sequence as a cis element critical for the light-regulated expression of rbcS genes. Nuclear factor GT-1 binds specifically to this element and is one of the plant nuclear factors that has been detected and studied in great detail. Here we describe the cloning and characterization of a tobacco cDNA encoding a protein, designated B2F (Box II Factor), with similar binding specificity and mobility in gel retardation assays as nuclear GT-1. Steady state levels of mRNA encoding B2F do not appear to be regulated by light; this is consistent with the previous observation that nuclear GT-1 activity is present in extracts from both light-grown and dark-adapted plants. Sequence comparison with another plant trans-acting factor, GT-2, which binds to a GT-like element in the rice phytochrome promoter, shows striking homology in three putative alpha-helices that may be involved in DNA binding.


Characterization of a gene encoding a DNA binding protein with specificity for a light-responsive element. The sequence element of box II (GTGTGGTTAATATG) is a regulatory component of a light-responsive element present within the upstream region of pea rbcS-3A. The nuclear protein GT-1 was defined previously as a DNA binding activity that interacts with box II. Here, we describe the isolation and characterization of cDNA sequences that encode a DNA binding protein with specificity for this element. The recombinant protein, tobacco GT-1a, shows similar sequence requirements for DNA binding to nuclear GT-1, as assayed by its ability to interact with previously defined 2-bp scanning mutations of box II, and is shown to be immunologically related to nuclear GT-1. The predicted structure of the 43-kD protein derived from the cDNA sequence suggests the presence of a novel helix-helix-turn-helix (HHTH) motif. Comparison between the predicted protein sequence encoded by the tobacco GT-1a cDNA and that of another GT binding protein, rice GT-2, reveals strong amino acid conservation over the HHTH region; this motif appears to be involved in the interaction between the recombinant protein and box II. Genomic DNA gel blot analysis indicated the presence of a small gene family of related sequences within the tobacco nuclear genome. RNA gel blot analysis of tobacco mRNA using the isolated cDNA as a probe showed that transcripts are present in several tissues, including both light-grown and dark-adapted leaves.


The use of antisense mRNA to inhibit the tonoplast H+ ATPase in carrot. Carrot root cells were transformed with the coding or 5' noncoding regions of the carrot vacuolar H+ ATPase A subunit cDNA cloned in the antisense orientation behind the cauliflower mosaic virus 35S promoter. Bafilomycin-sensitive ATPase, H(+)-pumping, and 14C-O-methyl-glucose uptake activities were specifically inhibited in the tonoplast fractions of mutant cell lines. Protein gel blotting confirmed that the expression of the A subunit was inhibited in the tonoplast fraction, but not in the Golgi fraction. Two-dimensional protein gel blots of total microsomes of wild-type and control transformant cell lines revealed two major immunoreactive polypeptides in the acidic pI range. In contrast, highly purified tonoplast membranes contained only the less acidic polypeptide. Because the less acidic polypeptide was preferentially diminished in the two antisense cell lines, we infer that the antisense constructs specifically blocked expression of a tonoplast-specific isoform of the V-ATPase A subunit in carrot. Regenerated plants containing the antisense constructs exhibited altered leaf morphologies and reduced cell expansion. The altered phenotype was correlated with the presence of the antisense construct.


Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants. Expression of the tobacco mosaic virus 30-kD movement protein (TMV MP) gene in tobacco plants increases the plasmodesmatal size exclusion limit (SEL) 10-fold between mesophyll cells in mature leaves. In the present study, we examined the structure of plasmodesmata as a function of leaf development. In young leaves of 30-kD TMV MP transgenic (line 274) and vector control (line 306) plants, almost all plasmodesmata were primary in nature. In both plant lines, secondary plasmodesmata were formed, in a basipetal pattern, as the leaves underwent expansion growth. Ultrastructural and immunolabeling studies demonstrated that in line 274 the TMV MP accumulated predominantly in secondary plasmodesmata of nonvascular tissues and was associated with a filamentous material. A developmental progression was detected in terms of the presence of TMV MP; all secondary plasmodesmata in the tip of the fourth leaf contained TMV MP in association with the filamentous material. Dye-coupling experiments demonstrated that the TMV MP-induced increase in plasmodesmatal SEL could be routinely detected in the tip of the fourth leaf, but was restricted to mesophyll and bundle sheath cells. These findings are discussed with respect to the structure and function of plasmodesmata, particularly those aspects related to virus movement.


Light-independent chlorophyll biosynthesis: involvement of the chloroplast gene chlL (frxC). The Chlamydomonas reinhardtii chloroplast gene chlL (frxC) is shown to be involved in the light-independent conversion of protochlorophyllide to chlorophyllide. The polypeptide encoded by chlL contains a striking 53% amino acid sequence identity with the bacteriochlorophyll (bch) biosynthesis bchL gene product in the photosynthetic bacterium Rhodobacter capsulatus. In a previous analysis, we demonstrated that bchL was involved in light-independent protochlorophyllide reduction, thereby implicating chlL in light-independent protochlorophyllide reduction in photosynthetic eukaryotes. To perform a functional/mutational analysis of chlL, we utilized particle gun-mediated transformation to disrupt the structural sequence of chlL at its endogenous locus in the chloroplast genome of Chlamydomonas. Transformants for which the multicopy chloroplast genome was homoplasmic for the disrupted chlL allele exhibit a "yellow-in-the-dark" phenotype that we demonstrated to be a result of the dark accumulation of protochlorophyllide. The presence of a chlL homolog in distantly related bacteria and nonflowering land plants, which are thought to be capable of synthesizing chlorophyll in the dark, was also demonstrated by cross-hybridization analysis. In contrast, we observed no cross-hybridization of a probe of chlL to DNA samples from representative angiosperms that require light for chlorophyll synthesis, in support of our conclusion that chlL is involved in light-independent chlorophyll biosynthesis. The role of chlL in protochlorophyllide reduction as well as recent evidence that both light-independent and light-dependent protochlorophyllide reductases may be of bacterial origin are discussed.


Expression of an outward-rectifying potassium channel from maize mRNA and complementary RNA in Xenopus oocytes. Injection of Xenopus oocytes with poly(A)+ mRNA isolated from different plants (maize, cucumber, and squash) results in the appearance of a voltage- and time-dependent, potassium-selective, outward current that is similar to the outward-rectifying potassium current recorded in many higher plant cells. Maize shoots were found to be especially enriched in mRNA encoding such activity. A cDNA library of maize shoot mRNA was constructed in the vector lambda ZAPII and was used to synthesize RNA complementary to the cDNA (cRNA). Injection of the cRNA gave rise to an outward-rectifying potassium current with properties similar to the currents obtained by poly(A)+ mRNA injection. These results demonstrate that higher plant mRNA can be properly translated into a product that produces a voltage-regulated potassium channel in the plasma membrane of Xenopus oocytes. Thus, Xenopus oocytes can be used as a heterologous expression system for the functional identification and isolation of plant ion channel genes as well as for the study of structure-function relationship of plant ion channels.


A rice cab gene promoter contains separate cis-acting elements that regulate expression in dicot and monocot plants. The major light-harvesting chlorophyll a/b binding proteins of the photosynthetic apparatus are encoded by families of nuclear cab genes. The expression of most cab genes is tissue specific and photoregulated in angiosperms. In transgenic tobacco plants, expression of the reporter gene beta-glucuronidase (GUS) is photoregulated and tissue specific from 5' upstream sequences of the rice cab1R gene; deletion of sequences upstream from position -170 with respect to the transcription start site eliminates the enhanced and photoregulated expression in the transgenic plants. Using an in situ transient expression assay, we have determined that the sequence OCT-R, an octamer repeat that lies within the -269 to -170 region of cab1R, is essential for photoregulated expression of the chimeric GUS gene in leaf cells of maize and rice but is not required for expression in illuminated tobacco leaves. Conversely, box III*- and G-box-like sequences found near OCT-R in cab1R are necessary for high-level transient expression of the reporter gene in tobacco leaf tissue but are not required for transient expression in maize or rice leaves.


Protein sorting to the vacuolar membrane. The vacuolar membrane (tonoplast) of plant cells contains a polytopic integral membrane protein with six membrane-spanning domains and cytoplasmically oriented amino-terminal and carboxy-terminal domains. This protein, tonoplast intrinsic protein (TIP), is a member of the membrane intrinsic protein (MIP) family of proteins, a family of channel proteins found in a variety of organisms. In bean seeds, alpha-TIP is synthesized on the rough endoplasmic reticulum and its transport to the tonoplast is mediated by the secretory system. In this study, we report that a polypeptide segment that includes the sixth membrane domain and the cytoplasmic tail of 18 amino acids of alpha-TIP is sufficient to target the reporter protein phosphinotricine acetyltransferase to the tonoplast of stably transformed tobacco cells. To determine if the carboxy-terminal cytoplasmic tail of alpha-TIP contains important tonoplast targeting information, a deletion construct lacking the 15 carboxy-terminal amino acids was introduced for transient expression in tobacco cells; we found that the slightly truncated protein still accumulated in the tonoplast. From these results, we concluded that a transmembrane domain of a tonoplast protein probably contains sufficient information for transport to the tonoplast. Whether such transport occurs by bulk flow or involves specific cellular machinery remains to be determined.


Developmental expression of tobacco pistil-specific genes encoding novel extensin-like proteins. We have sought to identify pistil-specific genes that can be used as molecular markers to study pistil development. For this purpose, a cDNA library was constructed from poly(A)+ RNA extracted from tobacco stigmas and styles at different developmental stages. Differential screening of this library led to the isolation of cDNA clones that correspond to genes preferentially or specifically expressed in the pistil. Seven of these cDNA clones encode proteins containing repetitions of the pentapeptide Ser-Pro4, which is a typical motif found in extensins. Unlike extensin genes, the extensin-like genes described here are not induced under stress conditions. RNA gel blot hybridizations demonstrated the organ-specific expression of the extensin-like genes and their temporal regulation during pistil development. After pollination, the transcript levels of the pistil-specific extensin-like genes change relative to levels in unpollinated pistils. In situ hybridization experiments showed that at least one of these pistil-specific genes is specifically expressed in cells of the transmitting tissue. The possible roles of the extensin-like proteins in pistils are discussed.


Specific expression of an extensin-like gene in the style of Nicotiana alata. cDNAs and corresponding genomic clones encoding a putative proline-rich protein (NaPRP3) were isolated from libraries prepared from Nicotiana alata style mRNA and genomic DNA. The predicted NaPRP3 protein is structurally similar to extensin in containing six copies of the characteristic extensin sequence Ser-Pro4, but differs in being smaller (151 residues compared with greater than 300 residues) and lacking Tyr residues. In contrast to most extensin genes, the NaPRP3 gene is not induced by mechanical wounding, and its expression is restricted to cells of the transmitting tract of the style.


A novel circadian phenotype based on firefly luciferase expression in transgenic plants. A 320-bp fragment of the Arabidopsis cab2 promoter is sufficient to mediate transcriptional regulation by both phytochrome and the circadian clock. We fused this promoter fragment to the firefly luciferase (Luc) gene to create a real-time reporter for regulated gene expression in intact plants. Cab2::Luc transcript accumulated in the expected patterns and luciferase activity was closely correlated to cab2::Luc mRNA abundance in both etiolated and green seedlings. The concentration of the bulk of luciferase protein did not reflect these patterns but maintained a relatively constant level, implying that a post-translational mechanism(s) leads to the high-amplitude regulation of luciferase activity. We used a low-light video imaging system to establish that luciferase bioluminescence in vivo accurately reports the temporal and spatial regulation of cab2 transcription in single seedlings. The unique qualities of the firefly luciferase system allowed us to monitor regulated gene expression in real time in individual multicellular organisms. This noninvasive marker for temporal regulation at the molecular level constitutes a circadian phenotype, which may be used to isolate mutants in the circadian clock.


The internal meristem layer (L3) determines floral meristem size and carpel number in tomato periclinal chimeras. Cell-cell interactions are important during plant development. We have generated periclinal chimeras between plants that differ in the number of carpels per flower to determine the roles of cells occupying specific positions in the floral meristem in determining the number of carpels initiated. Intraspecific chimeras were generated between tomato (Lycopersicon esculentum) expressing the mutation fasciated, which causes an increased number of floral organs per whorl, and tomato wild type for fasciated. Interspecific chimeras were generated between tomato and L. peruvianum, which differ in number of carpels per flower. In both sets of chimeras, carpel number as well as the size of the floral meristem during carpel initiation were not determined by the genotype of cells in the outer two layers of the meristem (L1 and L2) but were determined by the genotype of cells occupying the inner layer (L3) of the meristem. We concluded from these experiments that during floral organ initiation, cells in certain layers of the meristem respond to information supplied to them from other cells in the meristem.


The beta subunit of tomato fruit polygalacturonase isoenzyme 1: isolation, characterization, and identification of unique structural features. We have purified and isolated cDNAs encoding the beta subunit of tomato fruit polygalacturonase isoenzyme 1 (PG1), a cell wall protein that associates with, and apparently regulates, the catalytic PG2 polypeptides. Expression of the beta subunit is fruit specific and temporally separated from the expression of PG2 during fruit development. The 37- to 39-kD beta subunit is encoded as a 69-kD precursor protein containing a signal sequence and two propeptide domains. The mature protein is composed almost entirely of the novel 14-amino acid motif FTNYGxxGNGGxxx in which many of the phenylalanine residues are post-translationally modified. The unique structural features of the motif suggest an important role in the function of the protein and hence in the activity of PG1. The beta subunit may represent a class of bifunctional plant proteins that interact both with structural components of the cell wall and catalytic proteins to localize and/or regulate metabolic activities within the cell wall.


General roles of abscisic and jasmonic acids in gene activation as a result of mechanical wounding. Exogenous application of abscisic acid (ABA) has been shown to induce a systemic pattern of proteinase inhibitor II (pin2) mRNA accumulation identical to that induced by mechanical wounding. Evidence is presented that the ABA-specific response is not restricted to pin2 genes but appears to be part of a general reaction to wound stress. Four other wound-induced, ABA-responsive genes that encode two additional proteinase inhibitors, the proteolytic enzyme leucine aminopeptidase, and the biosynthetic enzyme threonine deaminase were isolated from potato plants. Wounding or treatment with ABA resulted in a pattern of accumulation of these mRNAs very similar to that of pin2. ABA-deficient plants did not accumulate any of the mRNAs upon wounding, although they showed normal levels of expression upon ABA treatment. Also, application of methyl jasmonate (MeJA) induced a strong accumulation of these transcripts, both in wild-type and in ABA-deficient plants, thus supporting a role for jasmonic acid as an intermediate in the signaling pathway that leads from ABA accumulation in response to wounding to the transcriptional activation of the genes.


Protein expression from an Escherichia coli/Bacillus subtilis multifunctional shuttle plasmid with synthetic promoter sequences. A plasmid shuttle vector (pSP10) was designed and constructed to simplify screening of cloned DNA and to facilitate expression of the protein products. The plasmid contained the following features: (i) a selection gene, chloramphenicol acetyltransferase; (ii) an indicator gene encoding beta-galactosidase for visual identification of colonies containing DNA inserts; (iii) a cloning region immediately upstream from the indicator gene; (iv) origins of replication recognized by both Escherichia coli and Bacillus subtilis; and (v) a synthetic DNA expression control sequence, including -35 and -10 regions, ribosomal binding site, and transcriptional and translational start sites. The promoter region is a synthetic consensus sequence derived from published B. subtilis promoters. The plasmid has been shown to replicate actively in E. coli and B. subtilis and to confer chloramphenicol resistance to both hosts. DNA inserted at the cloning region inactivates the indicator gene, resulting in white colonies on 5'-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside plates. beta-Galactosidase has been expressed from pSP10 in both E. coli and B. subtilis. A comparison was made of the expression levels of beta-galactosidase from the same plasmid which had been modified to contain: (i) the synthetic control region, (ii) no promoter region, (iii) the synthetic control region cloned in the opposite orientation, or (iv) the tac promoter.


An import-competent precursor of small subunit of ribulose-1,5-bisphosphate carboxylase generated by factor Xa cleavage from a beta-galactosidase fusion expressed in Escherichia coli. A plasmid-encoding fusion protein interlinked by factor Xa recognition sequence between beta-galactosidase and a precursor of the small subunit of wheat ribulose-1,5-bisphosphate carboxylase has been constructed. The plasmid directed abundant synthesis of the fusion protein in Escherichia coli. The recombinant protein was accumulated in an aggregated form that was associated with the bacterial membranes. A procedure was developed to isolate the fusion protein in a relatively pure and soluble form. Bovine factor Xa cleaved the isolated chimera to generate the complete chloroplast precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase from the fused beta-galactosidase. The cleaved precursor protein was imported into the isolated chloroplasts and processed to yield its mature counterpart.


Characterization of recombinant antistasin secreted by Saccharomyces cerevisiae. Secretion from recombinant yeast was used as a potential source of large quantities of the leech protein antistasin (ATS), a potent and highly specific inhibitor of the serine protease coagulation factor Xa. Mature recombinant ATS (r-ATS) is obtained after intracellular cleavage by the yscF protease of the mating factor alpha-1 pre-proleader from the fusion protein at the Lys-Arg sequence junction. Production levels are relatively low (ca. 1 mg/liter). Purification of the secreted product from a complex growth medium involved cell removal by microfiltration and diafiltration, cation-exchange capture and concentration on S-Sepharose Fast Flow, C-4 reverse-phase high-performance liquid chromatography (RP-HPLC), and HPLC cation-exchange chromatography step, and RP-HPLC concentration and desalting. The process was scaled up from the 16- to the 250-liter level with a corresponding increase in amount of r-ATS. From the 250-liter fermentation two major forms, r-ATS-I and r-ATS-II, distributed approximately 60:40, and a minor form, r-ATS-minor (ca. 1% of the purified r-ATS), were characterized. Limited N-terminal sequence analysis by Edman degradation indicated that r-ATS-I has the predicted mature N-terminus starting with Gln, that r-ATS-II is N-terminally blocked with pyroglutamate, and that r-ATS-minor is an incompletely processed form. RP-HPLC, hydrophilic-interaction HPLC, cation-exchange HPLC analysis, and electrophoresis results are consistent with the differences observed by sequencing. Preliminary in vitro characterization by intrinsic Ki determination for factor Xa inhibition indicated that the yeast r-ATS forms are indistinguishable from each other as well as from r-ATS expressed by the insect baculovirus host-vector system. Nevertheless, r-ATS-I and r-ATS-II appear less potent than insect-derived r-ATS in the activated partial thromboplastin time clotting assay. Further characterization indicated that C-terminal cleavage at Pro-116 had occurred in r-ATS-I and r-ATS-II as well as oxidation of methionine residues to methionine sulfoxide. The possible role of the C-terminus in inhibition of the prothrombinase complex is discussed.


Purification and characterization of a 65-kDa tumor-associated phosphoprotein from rat transplantable hepatocellular carcinoma 1682C cell line. We have isolated a homogeneous tumor-associated phosphoglycoprotein of about 65 kDa (p65) by ammonium sulfate precipitation of proteins from conditioned medium containing the rat transplantable hepatocellular carcinoma 1682C cell line, followed by high-performance liquid chromatography on molecular-sieving and phenyl hydrophobic interaction columns. The protein was concentrated in a Rotofor isoelectric focusing cell and finally separated by isoelectrofocusing followed by SDS--polyacrylamide gel electrophoresis. We achieved a purification of approximately 11,000-fold after the Rotofor concentration step. This protein migrated as a single band upon electrophoresis in SDS-PAGE and had a pI of 5.8 in isoelectrofocusing gels. The carbohydrate content of the blotted phosphoglycoprotein was analyzed by probing the blots with biotinylated lectins; a positive reaction was detected with concanavalin A, wheat-germ agglutinine, and Ricinus communis agglutinine. To confirm the tumor origin of this molecule, hepatocellular carcinoma cells were labeled in vivo using [32P]orthophosphate as well as [35S]methionine and cell culture medium was analyzed for the presence of radioactive band that corresponds with our protein. Phosphoamine acid analysis by thin-layer chromatography showed the presence of phosphotyrosine, phosphothreonine, and phosphoserine, which was later confirmed by analysis of the amino acid composition. Using the method described by Marchalonis and Weltman for comparative analysis of protein structure and evolution, we compared the protein isolated by us with other tumor markers and proteins showing similar properties and found no significant similarities.


Isolation and purification of a biologically active human platelet-derived growth factor BB expressed in Escherichia coli. Human platelet-derived growth factor (PDGF) was expressed in Escherichia coli from a high-level cytoplasmic expression vector. A cDNA fragment encoding the mature form of the human PDGF B chain (hPDGF-B) was cloned into a plasmid under transcriptional control of the inducible E. coli Tac promoter. Expression of hPDGF-B from the final construct, pTacBIq, is regulated by the lactose repressor (LacIq). Upon induction, a polypeptide of approximately 14 kDa that had the same molecular mass and immunoreactivity as authentic hPDGF-B was produced. The production of recombinant hPDGF-B was significantly increased in an E. coli strain (CAG629) defective in expression of the lon protease. Expression of hPDGF-B in the CAG629 strain accounted for approximately 1% of total cell protein. In this system, hPDGF-B is expressed as an insoluble, intracellular protein and can readily be obtained in a partially purified form after differential centrifugation. Amino acid sequence determination of the purified protein has verified that the amino-terminal portion of the recombinant PDGF is correct. After renaturation into dimers, the purified recombinant hPDGF is fully functional in assays for receptor binding and mitogenesis.


One-step immunoaffinity purification of complex I subunits from beef heart mitochondria. Polypeptides of beef heart mitochondrial complex I were isolated from 15 mg of solubilized beef heart mitochondria using antibodies immobilized on an agarose chromatography column. The preparation was examined by SDS electrophoresis and Western blotting using affinity-purified antibodies to complex I and compared to beef heart complex I purified according to the conventional method of Hatefi and Rieske. There was a high degree of homology between the two preparations as judged by SDS-polyacrylamide electrophoresis and by immunoblotting with seven affinity-purified antibodies to various complex I subunits. This method could be applied to the preparation of complex I subunits from small samples such as human muscle biopsy specimens.


Ferredoxin:NADP oxidoreductase of Cyanophora paradoxa: purification, partial characterization, and N-terminal amino acid sequence. The ferredoxin:NADP+ oxidoreductase of the protist Cyanophora paradoxa, as a descendant of a former symbiotic consortium, an important model organism in view of the Endosymbiosis Theory, is the first enzyme purified from a formerly original endocytobiont (cyanelle) that is found to be encoded in the nucleus of the host. This cyanoplast enzyme was isolated by FPLC (19% yield) and characterized with respect to the uv-vis spectrum, pH optimum (pH 9), molecular mass of 34 kDa, and an N-terminal amino acid sequence (24 residues). The enzyme shows, as known from other organisms, molecular heterogeneity. The N-terminus of a further ferredoxin:NADP+ oxidoreductase polypeptide represents a shorter sequence missing the first four amino acids of the mature enzyme.


Human müllerian inhibiting substance: enhanced purification imparts biochemical stability and restores antiproliferative effects. Separation of copurifying protease activity from recombinant human Müllerian inhibiting substance (rhMIS) bound to a monoclonal antibody immunoaffinity column by a high-salt wash results in cleaner preparations of rhMIS resistant to cleavage upon storage. In addition, an inhibitor of rhMIS antiproliferative activity is removed. Proteolytic cleavages produced by either a copurifying protease or exogenous plasmin occur at residues 229 and 427 but do not abolish rhMIS biological activity. This report details the modified immunoaffinity column isolation protocol suitable for proteins such as rhMIS and describes the biochemical and antiproliferative properties of this protein.


Expression of the adenovirus E1B 175R protein and its association with membranes of Escherichia coli. The E1B 175-amino-acid (175R) protein of adenovirus 2 is required for cellular transformation of primary cells and establishing cell morphology in lytically infected cells. To investigate the biochemical function of this protein, we constructed a bacterial expression vector (pKHB1-T) to produce the 175R protein in sufficient amounts for purification and biochemical analysis. On the basis of DNA sequencing, gel electrophoresis, and immunoblot analysis, the pKHB1-T-encoded 175R protein appears to be identical to that expressed transiently in mammalian or adenovirus-transformed cells. The bacterially produced viral protein was also found to be quite stable and without any modifications. Partial purification of the pKHB1-T-encoded protein revealed that the majority of its associates with the inner membrane of the bacterial cell. This, together with the possibility of the 175R protein containing an N-terminal amphipathic alpha-helix as a potential translocation signal, suggests that there may be a common mechanism of protein transport operating in both eucaryotic and procaryotic systems.


Expression of human NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in Escherichia coli: purification and partial characterization. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is a bifunctional enzyme synthesized as a 37-kDa precursor that is imported into the mitochondria of embryonic and transformed mammalian cells. The cDNA encoding the human bifunctional enzyme was modified to remove nucleotides corresponding to the mitochondrial targeting sequence and was subcloned into a procaryotic expression vector under the control of the T7 RNA polymerase promoter. The soluble dehydrogenase-cyclohydrolase was expressed in Escherichia coli at levels up to 150-fold higher than those found in transformed mammalian cells. Forms of the recombinant enzyme with one, three, or seven additional amino-terminal residues were purified to homogeneity and shown to have similar kinetic properties. Investigation of the absolute requirement of the enzyme for Mg2+ using fluorescence quenching indicates that this ion binds in the absence of substrates.


Chemical and physiological properties of polysucrose, a new marker of intestinal permeability to macromolecules. To measure intestinal absorption of macromolecules we have developed a new technique employing synthetic polysucrose polymers as probe molecules. Polysucrose (PS) is water-soluble, nontoxic, resistant to intestinal enzymes, spherical and can be produced with a molecular weight distribution that relates to the size of many normal food proteins. Normally, a very small fraction of large molecules passes the exclusion barrier of the healthy intestine. Thus, quantification of resorbed macromolecules requires assays of high sensitivity. For detection of PS in various biological fluids, micro-ELISAs have been established. PS with a mean molecular weight of 14,700 daltons (PS 15,000) is rapidly excreted into the urine. Twenty-one healthy volunteers who orally ingested 1 g of this preparation showed a 12-hour urine excretion of 0.018% (interquartile range 0.014-0.022).


Detection of IgG subclasses with anti-IgE activity in patients with atopic diseases. Significantly increased levels of IgG anti-IgE were seen in atopic patients as compared with controls. A correlation was observed between the levels of anti-IgE autoantibodies and serum IgE in the groups of atopic patients studied. No significant correlation was found between IgG anti-IgE levels and severity of the disease or between IgG subclasses with anti-IgE activity and clinical status. Analysis of IgG subclasses with anti-IgE activity showed that IgG1 and IgG4 were clearly factors in differentiating the atopics from the controls. IgG2 and IgG3 anti-IgE levels were not statistically significantly elevated. The lack of these autoantibodies may be explained by the presence of immune complexes or the lack of specificity of the monoclonal antibodies used in this study. These observations have not yet determined whether these autoantibodies and the isotypic selection and restriction observed play a role in the dysregulation of the immune response and in the evolution towards atopy.


Increased eosinophil chemotactic activity in bronchial washes obtained from patients with asymptomatic allergic alveolitis. The chemotactic activities of eosinophils and neutrophils were measured in bronchial washes obtained from 6 symptom-free patients with previous extrinsic allergic alveolitis and 9 healthy volunteers. An increased chemotactic activity was found in the washes obtained from the patients, and the recovery of eosinophils was correlated with the eosinophil chemotactic activity, suggesting a causal relationship. Chromatography of the fluids obtained suggested that low molecular weight compounds were principally responsible for the increased chemotactic activity. A mild disturbance of the integrity of the alveolocapillary barrier could to some extent add to the explanation of the increased chemotactic activity in the washes obtained from the patients.


Antiasthmatic activity of a novel thromboxane A2 antagonist, S-1452, in guinea pigs. We examined the effect of a potent thromboxane (Tx) A2 receptor antagonist, calcium (1R, 2S, 3S, 4S)-(5Z)-7-(((phenylsulfonyl)amino)bicyclo[2.2.1] hept-2-yl)-5-heptenoate dihydrate (S-1452), on antigen- and various allergic-spasmogen-induced contractions of guinea pig lung parenchymal strips and on the increase in insufflation pressure, an index of bronchoconstriction, in anesthetized guinea pigs. In isolated guinea pig lung parenchymal strips, S-1452 showed competitive antagonism of the contractile activity of U-46619, a TxA2 mimetic, with a pA2 value of 8.9. The compound also inhibited the contraction induced by prostaglandin (PG) D2 and PGF2 alpha, but a TxA2 synthetase inhibitor, OKY-046, did not. In contrast, both drugs inhibited not only leukotriene (LT) D4-induced contraction but also antigen-induced contraction in the presence of a histamine antagonist. In anesthetized guinea pigs, oral administration of S-1452 markedly inhibited the bronchoconstrictions induced by intravenous injection of U-46619, PGD2, PGF2 alpha, LTD4 and platelet-activating factor (PAF) with ED50 values of 0.006, 0.031, 0.112, 0.033 and 0.115 mg/kg, respectively, but OKY-046 inhibited only that by LTD4 and PAF. Additionally, bronchoconstriction following intravenous injection of antigen was almost completely suppressed by S-1452 (0.1 mg/kg) and partially by OKY-046 (300 mg/kg) in passively sensitized guinea pigs which were treated with diphenydramine and propranolol. The inhibitory effect of S-1452 against U-46619-induced broncho-constriction persisted up to 7 h after oral administration.(ABSTRACT TRUNCATED AT 250 WORDS)


Long- and short-term variability of airborne bovine epithelial antigen concentrations in cowsheds. We have previously demonstrated an association between the level of specific airborne animal-derived dusts in animal houses and farmers' humoral responses. These findings suggested that allergenic dusts may be able to cause a response in a concentration-dependent manner. In order to determine the reliability of a single measurement for the evaluation of general levels of dust, we studied the long- and short-term variability of airborne bovine epithelial antigen (BEA) concentrations in cowsheds. It was found that individual cowsheds seem to have characteristic levels of BEA in the air; some cowsheds tend to have consistently high concentrations of BEA, whereas in other cowsheds BEA concentrations tend to be low. Thus, single determinations do appear to reflect the general level of BEA in cowsheds. These findings corroborate the previously observed association between the levels of airborne-specific dusts based on single determinations and farmers' humoral responses.


[Doppler ultrasound images of the uterine artery and uterine involution in normal puerperium]. Serial sonographic studies of the involution of the uterus and Doppler ultrasound studies of the uterine artery blood flow were done with a pulsed Doppler apparatus on 102 patients with uncomplicated pregnancy, normal vaginal develivery, and normal puerperal course on the 2nd and 4th post-partum days and 1 and 2 months post partum. Length of the uterus, anteroposterior and transversal uterine diameter, and diameter of the myometrium on the anterior and posterior uterine wall underwent main involution during the 1st month post partum and showed only a slight decrease during the 2nd month post partum. Systolic/diastolic ratio and resistance index of the uterine artery increased slightly, but not significantly, between the 2nd and 4th post-partum days, but significantly until the end of the 1st month post partum. Until the end of the 2nd month post partum systolic/diastolic ratio and resistance index of the uterine artery showed, although the involution of the uterus was largely completed, a further significant increase.


[Regulation of sterol carrier protein-2 in human luteal cells by LH and LH-RH]. Conversion of cholesterol to pregnenolone is the most important and, at the same time, the rate-limiting step of steroidogenesis. Sterol carrier protein-2 (SCP2) or nonspecific lipid transfer protein (nsL-TP) is an intracellular protein, which plays an important role in the pre- and transmitochondrial transport of cholesterol and for the mitochondrial synthesis of pregnenolone. Synthesis of pregnenolone in rat Leydig cells can be increased by LH and LH-RH; however, only LH leads to characteristic changes in intracellular concentrations of SCP2. This means that synthesis of pregnenolone is regulated in two different ways. In this study we aimed to find out whether such a 'second way' of steroidogenesis is also demonstrable for the human corpus luteum (i.e. human luteal cells). Human luteal cells were collected during follicle punctures and were cultured as described previously. We demonstrate that (human) LH/hCG are able to enhance pregnenolone synthesis; this process is accompagnied by typical changes of SCP2 and an increase in activity of 7-dehydrocholesterol reductase, which is a marker enzyme for SCP2. LH-RH was shown to exert no effect. Thus, we conclude that a second way of steroidogenesis (i.e. synthesis of pregnenolone) cannot be proved for the human corpus luteum.


[Prognostic factors in endometrial cancer. A retrospective study based on 564 patients]. 564 patients with cancer of the endometrium were treated at the Department of Obstetrics and Gynaecology of the University Hospital in Zurich during the years 1970-1989. The most important clinical and pathologic-anatomic prognostic factors were evaluated and compared with the literature in a retrospective study. Myometrial depth of infiltration and histological tumour grading could be confirmed as reliable prognostic factors. Their influence on survival curves was highly significant (log rank test: p = 0.0001). In agreement with other authors the prognostic influence of the uterine length is minimal and not significant. Therefore the replacement of the criterion uterine-sond length by depth of myometrial infiltration in the new FIGO classification of 1989 is justified also in view of our data.


[Effectiveness of estriol depot vaginal suppositories in postmenopausal women with urogenital climacteric manifestations]. In this study, 43 postmenopausal patients with urogenital complaints such as vaginal dryness, incontinence, or bleeding with intercourse were treated with weekly applications of Orthogynest vaginal estriol suppositories in a new depot form. A marked increase in the vaginal epithelium (A. Schmitt score) and a significant decrease in symptoms due to vaginal atrophy were noted. The depot form allows for decreased dosing frequency, leading to improved compliance. Systemic symptoms, such as hot flashes and emotional lability, were also noted to respond to the Orthogynest suppositories.


[Intra-epithelial neoplasia of the vulva and smoking]. VIN III etiology is multifactorial with a predominant role held by human papillomavirus infections, especially infections with HPV type 16. Other cofactors are also involved. We reviewed our patients presenting with VIN III, focusing our attention on smoking. Out of 37 patients 29 (78%) were smokers and among those who presented with a relapse after treatment (11 patients) all were smokers. We discuss mechanisms by which tobacco could act as a cofactor in VIN III.


Biological gestational age and its calendar assessment with ultrasound. Part 1: Physiological considerations. Biological gestational age differs from calendar age, which is only its measurement. In spite of that in many countries all over the world, the rule for termination of pregnancy exclusively because its calendar duration is longer than 293 days (so-called postterm pregnancy) is widely applied. Constant increase in the blood oxytocinase level of pregnant women, just like the sonographic measurements of fetal bodies, proves the appropriate development of pregnancy with all the more accuracy, the more similar the subsequent values of the enzymatic and ultrasonographic determinations. From medical and ethical points of view the 'obstetrical' scale comprising sequential periods' pre-, at- and post-term' is an incorrect way of calculating the duration of pregnancy. Gynecologists have to use the word 'term' only to describe an individual's date of fetal maturity (birth-date) and the possible true pre- and post-term of an individual should be placed within as well as outside the normal range of deliveries.


The past and present of the Polish National Health Services. Reform project. This paper presents a short historical outline of the national health service (NHS) system in Poland. Consecutive stages of the NHS system reform are described (up to October 1991), including the period of early 80's and the Round Table Conference. The general principles of the project of the Polish NHS system reform, which is intended to be implemented with support from the World Bank, are presented. These principles are related particularly to the scope of the questions assigned to the task forces established to solve the basic problems of the present system. Those include: 1. Health Promotion Task Force, 2. Primary Health Care Task Force, 3. Occupational Health Task Force, 4. Health Information System Task Force, 5. Cost Accounting Task Force, 6. Resource Allocation Task Force, 7. Pharmaceutical Monitoring and Drug Control Task Force, 8. Management Development Task Force, 9. Regional Health Services (Consortia) Task Force.


The impact of the chemical industry on the human environment. The magnitude of the emitted to the air dusts and gases, liquid and solid wastes disposal produced by chemical industry is presented. Impact of chemical industry on the environment is discussed. Some hazardous agents, occurring in the work environment, morbidity and sickness absenteeism rates, noted among employees in chemical industry, are considered.


Toxic effects of combined exposure to toluene and m-xylene in animals. III. Subchronic inhalation study. Effects of combined exposure to toluene and m-xylene in the conditions of subchronic inhalation experiments in rats were examined. Rats were exposed to vapours of individual solvents and their 1:1 mixture at concentrations of 1000 ppm or 100 ppm, 6 h/day, 5 days/week, for 3 or 6 months, respectively. In rats exposed for 3 or 6 months to toluene, m-xylene and their mixtures (1:1) at concentrations of 1000 ppm and 100 ppm, respectively, the observed disturbances in rotarod performance test and decrease in spontaneous motor activity were statistically significant in comparison to control. In animals exposed to mixtures (1:1) of toluene and m-xylene, changes were not significantly different but more pronounced when compared to single solvent groups. The decrease of red blood cells count and increase of rod neutrophil cell counts were observed only in rats exposed for 3 months to mixture of solvents. Results obtained in condition of acute and subchronic inhalation exposure and toxicokinetics data interpreted jointly indicate the more than additive toxic effects of combined exposure to toluene and m-xylene.


The toxic effects of combined exposure to toluene and m-xylene in animals. IV. Liver ultrastructure after subchronic inhalatory exposure. The effects of combined inhalatory exposure to m-xylene and toluene on the rat hepatocytes were investigated. Limited proliferation of smooth endoplasmic reticulum and increase of the number of lysosomes were observed in hepatocytes after 3-months of single exposure to 1000 ppm of m-xylene and toluene. Moreover, mitochondria increased in most of hepatocytes. After combined exposure to mixture of m-xylene and toluene at concentrations of 500 + 500 ppm, respectively, the types of ultrastructural changes in hepatocytes followed the pattern observed after exposure of each of the single organic solvent. However, proliferation of the smooth endoplasmic reticulum was more eminent. The hepatocytes ultrastructure of rats exposed to 100 ppm of m-xylene and toluene for 6 months were similar to that observed in rats exposed to 1000 ppm of m-xylene for 3 months. In rats exposed to a mixture of m-xylene and toluene for 6 months, the ultrastructural changes in hepatocytes were a combination of those observed after a single exposure to each of the organic solvent. It may be concluded that changes in the hepatocytes ultrastructure were an adaptive rather than a toxic effect. Because in the case of combined exposures the doses were half of those used in single exposures, the additive effect on the proliferation of smooth endoplasmic reticulum was observed in the rat liver after combined exposure to toluene and m-xylene using electron microscopy.


Tolerance to chlorphenvinphos in rats assessed on the basis of changes in locomotor behavior in rotating wheels. Spontaneous locomotor activity in rotating wheels was investigated in rats exposed repeatedly (i.p. daily injections, five days a week for two weeks) to an agricultural organophosphorus pesticide, chlorphenvinphos (CVP) at doses of 1.0 and 3.0 mg/kg. After a seven day interval each rat was injected with a single 3.0 mg/kg test dose of CVP in order to assess the stability of tolerance. Concomitant changes in blood and the brain ChE activity were also investigated. It was found that exposure to CVP at a low dose (1.0 mg/kg), resulting in less than 50% reduction of ChE activity in blood and in the brain, did not produce changes in spontaneous locomotion in rotating wheels in the rat. Higher doses (3.0 mg/kg) inhibited blood and the brain ChE by more than 50% and reduced locomotion. Under conditions of repeated exposure to CVP at the symptomatic (3.0 mg/kg) dose ChE activity remained low throughout the exposure period, however, locomotor activity returned to a normal level, i.e. tolerance developed, within less than five days. Seven days after termination of the repeated exposure, the behavioral subsensitivity to CVP still remained. The biochemical data suggest that it may be related, at least partially, to a diminished vulnerability of ChE in some parts of the brain to CVP induced inhibition.


A comparison of changes in spontaneous (EEG) and evoked brain activity induced by chlorphenvinphos and physostigmine in rats and rabbits. The effects of single i.p. injections of two cholinesterase inhibitors, chlorphenvinphos (CVP) and physostigmine, on hippocampal and cortical EEG and flash evoked potentials in occipital cortex were compared in rabbits and rats. A comprised method of spectral analysis was employed for evaluation of changes in EEG. The obtained results showed that in both species the changes in hippocampal and cortical EEG after administration of CVP were relatively small or negligible in comparison with those after physostigmine administered in doses resulting in comparable (or even lesser) inhibition of blood cholinesterase (ChE). Neither CVP nor physostigmine resulted in significant changes in the morphology of the flash evoked potentials. The data do not confirm the suggestion that brain electrical activity is the most sensitive index of neurotoxicity resulting from exposure to organophosphate ChE inhibitors.


Experimental assessment of the effect of coal dust with polymetallic salts on the respiratory tract epithelium. Experimental assessment of the effect of coal dust from the Debiensko coal mine on the rat respiratory tract epithelium was carried out. The coal mine there contains up to 0.6 per cent of polymetallic salts including such heavy metals as Co, Cu, Mn, Ni, Pb, Sr, Zn. Inflammation of the upper respiratory tract caused by the coal dust was proved as well as mucous membrane ulceration and epithelial dysplasia.


Transformation of field monitoring results into miners' annual exposures to radon progeny. This paper presents the theory of estimating miners' radiation hazard, using the values of radon daughters' concentration in the work-environment. All measurement results can be obtained with any technique beginning with instant measurement and ending with long-term dosimetry. The measurements carried out in metal ore mines show that the seasonal changes in radon daughters' concentration have their main influence in the estimation of the miners' hazard. The estimation of the hazard which was obtained from equations, was compared with the results given from the computer simulation.


The role of biological markers in toxicology. Biological markers are tools that can be used to clarify the relationship, between exposure to a xenobiotic compound and health impairment and to indicate individual or population differences that affect the biologically effective dose. Biological markers, broadly defined, are indicators of variation in cellular or biochemical components or processes, structure, or function that are measurable in biological systems or samples. There is growing interest in the use of biological markers to study the health effects of exposure to environmental toxicants in occupational medicine, epidemiology, toxicology, and related biomedical fields.


Comparison of results derived from follow-up examination of respiratory systems in chosen groups of metallurgists. In a 16.5-year follow-up study of the steel industry we investigated the relation of chronic occupational exposure to the changes of ventilatory efficiency and to the frequency of chronic bronchitis (Chronic Obstructive Pulmonary Disease--COPD) in a group of 65 men working in the harmful environment of a Coking Plant (CP). The reference group comprised 34 employees of Cold Rolling Mill (CRM) working in favorable hygienic conditions. The faster decline of VC and FEV1 were noted in the group of CP in comparison to the control group. Also the frequency of pathologic values of RT was significantly higher (p < or = 0.001) in the exposed group. The incidence of COPD increased more in the group of CP than in the group of rollers. No differences in the annual decline of FEV1 and VC between smokers and nonsmokers from CP were noted, while in the group of men working in favorable environmental conditions the differences between smoking categories were significant. It suggests that the impact of occupational exposure is so powerful that it can mask the unfavorable influence of cigarette smoking on the ventilatory function of men working in a Coking Plant.


Coughing as the sole symptom of occupational bronchial allergy. In all of 11 observed patients with bakers' asthma, coughing preceded the development of dyspnea. Seventeen coughing atopic patients working in a bakery were observed for 2 years and treated with ketotifen and disodium cromoglycate (DSCG). Four of the patients developed asthma. Ketotifen prevented coughing attacks but did not prevent asthma development. Bronchodilators interrupted cough attacks. DSCG was ineffective.


Biological monitoring of risk of bladder cancer in persons occupationally exposed to aromatic amines. Recent advances in molecular biology and toxicology have greatly contributed to the early diagnosis of biological changes which may evoke neoplasms. This paper reviews the issues regarding the screening of persons occupationally exposed to carcinogenic aromatic amines. The screening was designed for an early detection of bladder cancer by means of biochemical tests. The applied tests facilitated the estimation of the level of aromatic amines which penetrate an organism (biomarkers of exposure), early diagnosis of the biochemical disorders which may influence cancer development (biomarkers of early effects) and the detection of genetic predispositions which enhance risk of such disorders (biomarkers of susceptibility).


Effects of underwater noise on human hearing. Hearing conservation for divers and swimmers has been overlooked nearly everywhere in the world. Because submerging a listener changes his or her auditory physiology dramatically, the research upon which we base exposure limits for airborne noise is not pertinent under water. Of the research that is necessary for developing a damage-risk standard for underwater noise exposure, only a negligible amount has been done. The value of Poland's marine industries and the motivation of its audiologists make Poland and ideal country in which to accomplish this research and to develop the foundation for national and international standards.


The effect of maternal exposure to dioxolane on prenatal and postnatal development in rats. Female rats were given by gavage every other day from days 8-20 of gestation an aqueous solution of dioxolane at daily doses equal to 0.025, 0.1 and 0.2 LD50 (first series--prenatal development) or from days 2-20 of gestation at daily doses equal to 0.025, 0.075 and 0.15 LD50 (second series--postnatal development). At doses toxic or subtoxic to maternal rats (0.1 and 0.2 LD50) dioxolane did not cause increased embryo or fetus intrauterine death rates or congenital defects, it did cause, however, dose-related delays in fetal development. Dioxolane does not cause impairment of physical development or behavioral disturbances. Exposure to higher doses of the compound (0.2 LD50) leads to increased perinatal death rates in the offspring, without causing, however, disturbances in the maternal instinct. The exposure of pregnant rats to dioxolane decreased haemoglobin levels in 5-week-old offspring. At a dose 1.15 g/kg (0.2 LD50) the chemical significantly increased exploratory motor activity of female offspring at the age of 8 weeks, but did not affect significantly locomotor activity of males and the active avoidance acquisition of adult offspring.


Genotoxicity assessment of sulfosuccinate IO-5, Rokamid MRZ 17, Rokacet RZG7P2 and Roksol TL-7 using bacteria and bone marrow rodent cells. Three short-term tests were used for evaluation of the genotoxic activity of four surface active agents. These were: Ames, Salmonella reversion assay using 4 tester strains (TA97a, TA98, TA100 and TA102), the micronucleus test int the bone marrow of Balb C mice and in vivo sister chromatid exchange (SCE) analysis in SFIS or Balb C mice. The frequency of micronucleated PCEs did not exceed the control values in mice of both sexes after intraperitoneal administration of all four compounds. Three preparations--Sulfosuccinate IO-5, Rokamid MRZ 17 and Rokacet RZG7P2 produced a negative response in Salmonella strains gene mutation assay and SCE induction test in mouse bone marrow cells. The Roksol TL-7 induced frameshift and base-pair substitution mutations in Salmonella tester strains both with and without S9 (prepared from the liver of rats which had been pretreated with Aroclor 1254). Evidently positive results (more than a twofold increase in the number of revertants per plate) were observed in tester strain S. typhimurium TA97a (with and without S9 metabolic activation) and S. typhimurium TA100 (with S9 metabolic activation) at a dose of 0.2 microliter Roksol TL-7 per plate. Roksol TL-7 caused slight increase in the SCE level in mouse bone marrow cells. A significant increase in SCE frequency was observed at doses of 50, 75 and 100 mg/kg.


Oral anticoagulant therapy: practical considerations. The key to safe and effective oral anticoagulation is to have an understanding of the rationale for dosing guidelines and therapeutic ranges; an appreciation of the imprecision of prothrombin time testing and its standardization; knowledge of the factors influencing prothrombin time response; and awareness of the importance of patient empowerment via ongoing patient education. This review focuses on the routine management of oral anticoagulant therapy to provide these practical insights and to promote safe and effective therapy.


Anticoagulation during pregnancy.

Anticoagulation during pregnancy. When anticoagulants are indicated during pregnancy, the needs of the mother as well as those of the fetus must be considered. Although anticoagulants may be indicated to prevent life threatening maternal thrombosis, oral anticoagulants such as warfarin have been associated with fetal anomalies. Patient education and early intervention with carefully monitored subcutaneous heparin can reduce both fetal and maternal risks.


Components and defects of the coagulation system. The purpose of this review article is to present a simplified view of coagulation in five parts (vessels, platelets, cascade, natural inhibitors, and fibrinolysis), update the NP with current concepts regarding coagulation, discuss cellular and protein functions, and provide an overall understanding of how the five components work together. Discussion of disease states, genetic deficiencies, and acquired inhibitors of the coagulation system is included in this article.


Acquired hypercoagulable states.

Acquired hypercoagulable states. Acquired hypercoagulable states comprise a diverse group of clinical conditions that are associated with an increased risk of thrombosis. These clinical conditions include malignancy, diabetes mellitus, venous stasis, pregnancy, oral contraceptive use, lupus anticoagulant, postoperative state, immobilization, myeloproliferative disorders, and nephrotic syndrome. Recognition of these associations, possible underlying mechanisms, identification of high risk individuals, thromboembolic prophylaxis, and other clinical implications are discussed.


Inherited hypercoagulable states: questions and controversies. Inherited hypercoagulable states such as protein C, protein S, and antithrombin III deficiencies account for 15% to 20% of recurrent thromboembolic episodes. A common risk profile is associated with these disorders and guides the use of laboratory screening. The use of anticoagulants in inherited hypercoagulable states varies with a patient's personal and familial history of thrombosis. Special consideration is given to the need for anticoagulants perioperatively and during pregnancy.


Oral anticoagulation therapy: indications, contraindications, and complications. The goal of oral anticoagulation therapy is to prevent thromboembolic events while minimizing the possibility of hemorrhage. This article discusses indications for, contraindications to, and possible complications of anticoagulant therapy. Guidelines for the use of low intensity and high intensity anticoagulation and duration of anticoagulant therapy are also included.


Factors that influence therapeutic anticoagulation control. A wide range of variables can influence warfarin anticoagulation control. This report summarizes the effects of medication, alcohol, diet, and other factors on prothrombin times. Useful summary tables are included.


Local anesthesia: a review.

Local anesthesia: a review. Local anesthetics are the most widely administered drugs in dentistry. Significant advances have been made in past decades that have greatly increased both the safety and the efficacy of these important drugs. This paper reviews the history of local anesthesia, pharmacokinetics and clinical implications, techniques, complications, and future directions in the quest for more effective pain control in dentistry.


Clinical investigation of potency and onset of different lidocaine sprays for topical anesthesia in dentistry. The clinical effects of three lidocaine-containing solutions with and without frigen (freon-113) as a propellant, after different waiting periods, and with different dosages applied were investigated in 130 outpatients who were undergoing dental treatment in the maxilla under local anesthesia. They were divided randomly into five groups (A through E): (A) Xylocaine spray with frigen, two applications (20 mg lidocaine); (B) Xylestesin spray with frigen, two applications (14 mg lidocaine); (C) Xylestesin spray with frigen, three applications (21 mg lidocaine); (D) Xylestesin pump spray without frigen, two applications (14 mg lidocaine); and (E) no topical anesthesia. They were further divided into 12 subgroups to evaluate waiting periods between the application of the topical anesthesia and the injection (1, 2, or 3 minutes). Patients assessed the pain of the injection, intensity of numbness, and intensity of the taste on a visual analog scale; they also assessed the pain of the injection compared to former injections. Pain during injection was reduced by topical application of lidocaine. A waiting period of 2 minutes proved to be sufficient and can be justified to avoid impatience and increased numbness in patients. However, a 3-minute waiting period may be appropriate for sensitive patients. An increase in the dosage failed to show better analgesia. The pump spray without frigen proved to be effective.


Displacement of the endotracheal tube caused by postural change: evaluation by fiberoptic observation. Unexpected displacement of the endotracheal tube during anesthesia caused by postural change of the neck or passive compression by the mouth gag was investigated under transluminal fiberoptic observation. Twenty-two patients were divided into orotracheal and nasotracheal intubation groups according to the technical requirements of the planned oral and maxillofacial surgery. Under nasotracheal intubation, the mean length of displacement from the carina was 21 mm by extension of the neck, and 8 and 7 mm by lateral rotation of the neck to the right and left sides, respectively. Under orotracheal intubation, the mean length of displacement from the carina was 12 mm by extension of the neck and almost 28 mm with application of the mouth gag. To avoid accidental extubation or one-sided bronchial intubation during anesthesia, the tip (distal end) of the endotracheal tube should be located less than 32 mm from the carina before extension of the neck and more than 41 mm from the carina before application of the mouth gag.


Ingestion and aspiration of foreign bodies. Foreign bodies may be swallowed as well as aspirated, but patients and their relatives may not understand the distinction. Fortunately, swallowing occurs more often than aspiration. Dental prostheses present specific problems in this regard. Their configuration impedes easy gliding through the esophagus and makes their extraction difficult. Swallowing of foreign bodies in elder patients is often explained by a decrease in psychological or neurological function, which undoubtedly may occur. The loss of tactile sense of the hard and soft palate as a result of complete denture use is another reason for frequent occurrence of this problem in elder patients.


How safe is dental anesthesia?

How safe is dental anesthesia? In the United Kingdom, the number of general anesthetics given under the National Health Service in general dental practice (excluding those given in hospitals) has declined steadily during the past 10 years. The mortality rate has also declined from approximately 1 in 200,000 (1952) to 1 in 2,000,000 (1990). These figures include deaths that occurred following treatment in hospitals and are divided almost equally between anesthetics given in hospitals and those given in dental practices. This paper examines the causes of deaths under dental general anesthesia and evaluates the proposals that have been made by the various official bodies appointed to consider the subject.


Victory over pain: an historical perspective. Horace Wells, a dentist, is credited with the discovery of anesthesia. However, there are others who experimented with inhalation agents long before Wells' time. This paper reviews the history of anesthesia and recounts its discovery by Wells in 1844 as we approach the 150th anniversary of the event.


Practical application of meridian acupuncture treatment for trigeminal neuralgia. This report evaluates the effect of meridian acupuncture treatment on trigeminal neuralgia. Ten patients aged 26 to 67 years (mean 55.4 years) who visited the outpatient Dental Anesthesiology Clinic at Tsurumi University Dental Hospital from 1985 to 1990 were studied. Five of the patients suffered from idiopathic and five from symptomatic trigeminal neuralgia. The patients underwent meridian treatment by acupuncture alone or acupuncture combined with moxibustion. The acupuncture method used was primarily basic treatment employing only needles without electrical stimulation. Meridian acupuncture treatments were repeated from two to four times a month. Five patients were restored to a pain-free state. The other five patients noted a decrease in pain, but with some level of pain remaining (significant pain in one patient). It is concluded that meridian acupuncture treatment is useful and can be one therapeutic approach in the management of trigeminal neuralgia.


The baboon: an ideal model in biomedical research. The baboon is a good animal model for research and investigations in physiology and pathophysiology, also using radiopharmaceutical techniques. It has several similarities to the human being, and all parameters in human physiology can be measured in the baboon model with the same or equal technical equipment. Fourier phase analysis in radionuclide ventriculography, hemodynamic reactions in a septic shock model, and investigations in conjunction with local anesthetics (eg, effects on cerebral blood flow) are typical types of research that have been or are currently being performed on the baboon. Institutes using the baboon model must be equipped with all instruments and operating facilities as would be needed for investigations in human beings. Ethical considerations must be regarded strictly and supervised by an ethics committee. Protocols must determine exactly why in vivo experimentation is preferred to in vitro tests. Anesthesia techniques in a baboon model allow study on the animal itself, eliminate pain (and stress) to the animal, and should not interfere with the aims of the investigation being performed.


Incidence of positive aspiration in the Gow-Gates mandibular block. Evidence strongly suggests that rigorous precautions should be taken to avoid accidental intravascular injection of local anesthetic solutions. In this study, 3,000 patients each received a Gow-Gates mandibular block, resulting in a positive aspiration frequency of 1.6%. In the conventional inferior alveolar block technique, a range of positive aspirations from 3.6% to 22% has been noted. The Gow-Gates block has other important advantages over the conventional block. By observing the landmarks and the depth of penetration, it is possible to reinsert the needle with a reasonable assurance of a negative aspiration at the second attempt. The conventional technique suffers the major disadvantage that needle insertion techniques which produce the highest rates of clinically successful pain control run the greatest risk of vascular penetration. Operators skilled in the Gow-Gates technique should be able to achieve a positive aspiration rate of less than 2%. The technique is unique among oral nerve block procedures in that the preferred injection site for maximum efficacy is also the site that is least likely to result in vascular penetration.


Intraoral conduction anesthesia with epinephrine-containing local anesthetics and arterial epinephrine plasma concentration. Following conduction anesthesia using either lidocaine 2% with epinephrine 1:80,000, articaine 4% with epinephrine 1:100,000, or articaine 4% with epinephrine 1:200,000, the arterial plasma epinephrine concentration was measured. Eighteen healthy young patients scheduled for osteotomy of a mandibular third molar were studied. Each local anesthetic-epinephrine combination was tested in six patients. There was no significant difference in the arterial plasma epinephrine concentration after injection of 2 mL of the studied anesthetic-epinephrine combinations. The result was explained by the concentration difference in the local anesthetics. Although the vasodilating action of lidocaine and articaine is almost identical, there will be enhanced vasodilation by the doubled concentration in the case of articaine (4%) and the local resorption of epinephrine may be facilitated. There were no significant changes in the measured cardiovascular parameters.


Local anesthesia of the head and neck.

Local anesthesia of the head and neck. In theory, most ear, nose, and throat surgery involving the soft tissues of the head and neck may be performed under local anesthesia. This includes surgery on the middle ear, even mastoidectomy; partial or total laryngectomies; surgery on the nose and paranasal sinuses; and surgery on the major salivary glands. For this purpose, local anesthetics, with or without epinephrine, are administered either by infiltration injection or by topical application. The selection of either local or general anesthesia for a surgical procedure will depend on many important factors, not the least of which is the preference of the patient. This paper presents factors to be considered in making this choice, as well as surgical indications and contraindications to the use of local anesthesia.


The management of craniofacial pain in a pain relief unit. This paper reports the results of 34 craniofacial pain sufferers who were treated at the Dudley Pain Relief Unit over a 1-year period. Most of the patients were referred by their general medical practitioners. They were adults representing all age groups, with a female-male ratio of 4:1. The average history of pain was 5.5 years. Neuralgic pain (as distinct from temporomandibular joint dysfunction syndrome, migrainous disorders, and pain of iatrogenic origin) was most frequently seen. Oral drug therapy, local injection of corticosteroids and analgesics, peripheral neurolysis, magnetotherapy, hypnotherapy, and acupuncture were the lines of management available. By the end of this study period, pain had been relieved or eliminated in 30 of the patients (88%).


Management of an emergency: to be prepared for the unwanted event. Every dental office needs an adequate emergency treatment structure. While theoretical and practical pregraduate education in emergency medicine is included (in differing degrees) in the curricula of dental schools throughout the world, postgraduate training is generally undertaken on a voluntary basis. Repeated training in emergency techniques is encouraged for the dentist and dental office staff, as the trained and coordinated action of the whole team in the dental office is necessary when an emergency occurs. Whenever possible, a recognized risk factor should be eliminated or its possible influence minimized prior to dental treatment. Patients requiring immediate dental therapy will need appropriate monitoring, adequate application of systemically acting drugs, or even treatment under standby conditions. Diagnostic equipment as well as equipment for monitoring the respiratory and cardiovascular systems should be available. Several monitors (blood pressure, ECG, and pulsoximetry) have been introduced to dentistry and have their indications in special patient groups. Clear therapeutic concepts are the endpoint of proper emergency management. The restoration of an undisturbed vital function has to be achieved, followed by detailed therapy. Dentists should refresh their knowledge of emergency treatment techniques and prepare themselves with therapeutic guidelines.


Insulin-like growth factor-II increases plasma osteocalcin concentration in newborn lambs. The influence of GH, IGF-I and IGF-II on plasma 1,25-(OH)2D and osteocalcin (OC) concentrations was studied in four groups of five newborn lambs each. They received a single i.v. injection of either purified bovine GH (1 IU/mg 600 micrograms/kg body wt), synthetic human IGF-I (10 or 200 micrograms/kg body wt) or synthetic human IGF-II (10 micrograms/kg body wt). Five controls were injected the same volume (1 ml) of solvent. IGF-II, but not GH or IGF-I, induced a rapid (1.5 h) and sustained (up to 18 h) increase of serum osteocalcin. This effect of IGF-II on plasma OC concentration was not mediated by 1,25-(OH)2D, which increased in plasma only 18 h after IGF-II injection. These data suggest that IGF-II stimulates, the in vivo secretion of OC by osteoblasts through a mechanism independent of 1,25-(OH)2D. Further studies are necessary to elucidate the mechanism of IGF-II action on osteocalcin synthesis and to explain the late increase of plasma 1,25-(OH)2D after IGF-II injection.


Isolation and partial characterization of a growth factor from human cementum. Cementum is the mineralized interface through which collagen fibers of periodontal connective tissues are anchored onto the tooth surface. We have isolated and partially characterized a mitogenic factor from human cementum which has properties different from other growth factors. Cementum was harvested from healthy human teeth, extracted in 1.0 M CH3COOH and mitogenic activities were fractionated by heparin-affinity chromatography. Proteins eluted by 0.4-0.6 M NaCl, which contained most of the cementum mitogenic activity, were precipitated by trichloroacetic acid and resolved by HPLC through ion-exchange and reverse-phase columns. NaDodSO4-polyacrylamide gel electrophoresis revealed that the purified preparation contained a M(r) 23,000 protein and this protein was associated with mitogenic activity. The purified cementum-derived growth factor (CGF) was active alone, but at suboptimal concentrations its activity was potentiated by small quantities of plasma-derived serum and epidermal growth factor (EGF). The activity was resistant to heat, but it was destroyed by trypsin digestion. Reduction and alkylation destroyed the mitogenic activity, however electrophoretic mobility was not affected. Binding of EGF to fibroblast membranes was not affected by the CGF and assays to detect platelet-derived growth factor were negative. These characteristics indicated that CGF is a distinct molecular species. Our data show that cementum contains several mitogenic factors and that CGF is the major cementum mitogen.


Bone marrow stromal colony formation requires stimulation by haemopoietic cells. In mouse bone marrow cultures plated at low cell density, stromal colonies formed from colony-forming unit fibroblastic (CFUf) failed to develop unless the cultures were supplemented with irradiated feeder cells. Colony-stimulating activity was produced by irradiated bone marrow and spleen cells and by platelets, was dose dependent, not species specific and was maximal at high serum concentration. The efficiency of CFUf colony formation was 1.7 x 10(-4) for mechanically disaggregated and 14.6 x 10(-4) for trypsinised bone marrow cells. The colonies formed in the presence of feeder cells comprised hundreds of fibroblasts. In the absence of feeder cells, small fibroblast foci and single fibroblasts only were present in cultures. PDGF, IL-3 and EGF did not substitute for the colony-stimulating activity of feeder cells. These results suggest that CFUf colony formation requires growth factor(s) released by platelets and megakaryocytes which remain to be identified.


Mechanisms of fibronectin-mediated attachment of osteoblasts to substrates in vitro. Adhesive proteins of plasma and the extracellular matrix, such as fibronectin, adsorbed onto surfaces mediate cell/substrate adhesion. In a series of experiments, the roles of the type III connecting segment (IIICS) adhesion sites (specifically, CS1 and CS5 peptides) of fibronectin, heparan sulfate proteoglycan, endogenous proteins, and passive attachment in fibronectin-mediated osteoblast attachment were examined in vitro. The CS1 and CS5 peptides of the IIICS of fibronectin had no effect on osteoblast attachment. Blocking the heparin-binding domains of fibronectin inhibited osteoblast attachment by 40-45%, which is complementary to inhibition results previously obtained with the RGDS tetrapeptide. Endogenously synthesized and secreted proteins played a role in maintaining and repairing the osteoblast surface. Osteoblast attachment to fibronectin, but not to the nonadhesive protein albumin, occurred via active mechanisms in that the process was dependent on free sulfhydryl groups, divalent cations and temperature.


On the rat model of human osteopenias and osteoporoses. The idea that rats cannot model human osteopenias errs. The same mechanisms control gains in bone mass (longitudinal bone growth and modeling drifts) and losses (BMU-based remodeling), in young and aged rats and humans. Furthermore, they respond similarly in rats and man to mechanical influences, hormones, drugs and other agents.


Comparison of a rapid (2-h) versus a slow (24-h) infusion of alendronate in the treatment of hypercalcemia of malignancy. Alendronate (aminohydroxybutylidene bisphosphonate) is a potent inhibitor of bone resorption but the role of the duration of intravenous infusion in its efficacy profile is unclear. In a two-centre, parallel, randomized, double-blind study, 20 patients with tumoral hypercalcemia received a single 10-mg i.v. infusion over either 2 h (group A, n = 10) or 24 h (group B, n = 10). Recurrences (n = 6) were retreated using the same regimen. Pretreatment plasma calcium (Ca) was 3.32 +/- 0.08 mM (mean +/- SEM) for all patients. Treatment A and B were associated with similar temporal profiles for onset, time to reach normocalcemia, (6 vs 5 days), nadir (day 6: 2.45 +/- 0.06 vs 2.43 +/- 0.08 mM) and time to relapse (day 21). Normocalcemia (2.15-2.55 mM) was achieved in seven (A) and nine (B) patients with other cases being partial responders (Ca: 2.65-2.76 mM). A significant decrease of urinary calcium and hydroxyproline excretion and a significant increase of PTH accompanied Ca normalization in both groups. Ca response was 50% lower on 2nd treatment with alendronate. Both treatments were well tolerated with transient mild fever being the most common adverse experience. In conclusion, whether infused over 2 or over 24 h, a single dose of 10 mg alendronate led to normalization of tumoral hypercalcemia in a large majority of cases.


Can nandrolone add to the effect of hormonal replacement therapy in postmenopausal osteoporosis? Thirty-six women with postmenopausal osteoporosis (31 of them with at least one non-traumatic vertebral compression fracture) were matched pair-wise as to age, years since menopause and body mass index and randomized to receive either cyclical estrogen-progestagen replacement treatment (group 1) or the same treatment plus nandrolone decanoate (group 2). During the first year of treatment in both groups the forearm BMC (SPA) rose proximally and distally 2-3%. Over 2 years the increments of forearm BMC in both groups were up to 4.5%. Lumbar BMC (DPA) rose in both groups nearly 10% over the first year and 12-12.5% over 2 years. The cancellous bone density of L3 (QCT) showed in 6 months an increase of 21% in group 1 and 29% in group 2 to subsequently stay at that level. All these changes from the basal levels were highly significant but there were no significant differences between the two groups. These two conclusions were also drawn with regard to the induced fall of serum alkaline phosphatase (-23%), osteocalcin (-35% to -44%) and procollagen I (-15% to -22%) and of the fasting urinary hydroxyproline (-33% to -36%). No significant increase in the number of newly deformed vertebrae occurred in 2 years.


The effect of vertebral collapse on spinal bone mineral density measurements in osteoporosis. Bone mineral density (BMD) of the lumbar spine (L1-L4) was measured using dual-energy x-ray absorptiometry (DXA) in 57 postmenopausal women with spinal osteoporosis aged 50-82 years (average age 64). For each vertebra between L1 and L4, the BMD was compared to age-matched normal population values and a Z score obtained. Twenty three patients had between one and three collapsed vertebrae from L1 to L4. The average Z score for fractured vertebrae was -1.62 but for uncollapsed vertebrae was -2.26 (P less than 0.001). The difference in Z scores obtained for collapsed and uncollapsed vertebrae was greater where only one fracture was present (0.803) compared to two (0.577) or three fractures (0.245). The average increase in density for a fractures vertebra was 0.070 g/cm2. For L1 vertebral fractures alone the average increase in BMD was 0.096 g/cm2, L2 vertebral fractures 0.041 g/cm2, L3 fractures 0.029 g/cm2 and L4 fractures 0.062 g/cm2. It is concluded that as vertebral collapse is not alway detected using DXA and usually causes a rise in BMD, spinal x-rays are necessary to avoid misinterpretation of a falsely elevated BMD in osteoporotic patients, particularly in longitudinal studies or when monitoring therapy, where small changes in BMD are important.


Relation of infant feeding to adult serum cholesterol concentration and death from ischaemic heart disease. To examine whether method of infant feeding is associated with adult serum lipid concentrations and mortality from ischaemic heart disease. Follow up study of men born during 1911-30. Hertfordshire, England. 5718 men, for 5471 of whom information on infant feeding had been recorded by health visitors and 1314 of whom had died. 485 of the men born during 1920-30 and still living in Hertfordshire who had blood lipid measurements. Death from ischaemic heart disease; serum cholesterol and apolipoprotein concentrations. 474 men had died from ischaemic heart disease. Standardised mortality ratios were 97 (95% confidence interval 81 to 115) in men who had been breast fed and had not been weaned at 1 year, 79 (69 to 90) in breast fed men who had been weaned at 1 year, and 73 (59 to 89) in men who had been breast and bottle fed. Compared with men weaned before one year men not weaned had higher mean serum concentrations of total cholesterol (6.9 (not weaned) v 6.6 (weaned) mmol/l), low density lipoprotein cholesterol (5.0 v 4.6 mmol/l) and apolipoprotein B (1.14 v 1.08 g/l). Men who had been bottle fed also had a high standardised mortality ratio for ischaemic heart disease (95; 68 to 130) and high mean serum concentrations of total cholesterol (7.0 mmol/l), low density lipoprotein cholesterol (5.1 mmol/l), and apolipoprotein B (1.14 g/l). In all feeding groups serum apolipoprotein B concentrations were lower in men with higher birth weight and weight at 1 year. Age of weaning and method of infant feeding may influence adult serum low density lipoprotein cholesterol concentrations and mortality from ischaemic heart disease. Adult serum apolipoprotein B concentrations are related to growth in fetal life and infancy.


Reliability and effectiveness of screening for hearing loss in high risk neonates. To establish the reliability and effectiveness of screening for hearing loss by brainstem auditory evoked potential testing in high risk neonates. Seven year investigation of newborn babies admitted to a special care baby unit and monitored through a regional children's audiology unit. Special care baby unit and children's audiology department, Belfast. 405 neonates admitted to the baby unit, during 1 October 1982 to 31 March 1987. Presence of hearing impairment, type and severity of hearing impairment, mortality. 85 children failed the screening test, 62 of whom were followed up. Five children had severe bilateral sensorineural impairment and 12 had conductive impairment requiring surgical intervention. A further 18 had severe neurological disorder detected. The sensitivity of screening was 100% and specificity was 88%. If the procedure was introduced into routine clinical practice the mean age at diagnosis for all children with severe perinatal hearing impairment would be 11 (median 1) months. The mean age at diagnosis with the health visitor screening service was 23 (19) months (difference 10 months, 95% confidence interval 6 to 16 months; p < 0.0001). Screening for hearing loss in high risk neonates is highly reliable and cost effective. It also provides valuable neurophysiological information. Routine testing of these infants would result in over half of all children with severe bilateral perinatal sensorineural hearing impairment being identified by 2 months of age. This would make an important contribution to the habilitation of this socially, emotionally, and educationally vulnerable group.


Comparison of female to male and male to female transmission of HIV in 563 stable couples. European Study Group on Heterosexual Transmission of HIV. To identify risk factors for heterosexual transmission of HIV and to compare the efficiency of male to female and female to male transmission. Cohort study of heterosexual couples. Regular partners of HIV infected subjects were tested and both members of the couples interviewed every six months. HIV prevalence in partners was analysed according to the characteristics of the couples. Nine European countries. 563 couples comprising 156 female index patients with their 159 male partners and 400 male index patients with their 404 female partners. Partners reporting risk factors other than sexual contacts with the index patient were excluded. HIV infection in partners and high risk sexual behaviour. Overall, 19 (12%) male partners and 82 (20%) female partners were infected with HIV, suggesting that male to female transmission is 1.9 (95% confidence interval 1.1 to 3.3) times more effective than female to male transmission. An advanced stage of HIV infection in the index patient (odds ratio 17.6; 4.9 to 62.7) and sexual contacts during menses (3.4; 1.0 to 11.1) increased the risk of female to male transmission and stage of infection (2.7; 1.5 to 4.9), anal sex (5.1; 2.9 to 8.9), and age of the female partner (3.9; 1.2 to 13.0 for age > 45 years) increased the risk of male to female transmission. None of the 24 partners who had used condoms systematically since the first sexual contact was infected. Several factors which potentiate the risk of transmission through unprotected vaginal intercourse have been identified. Knowledge of these factors could be helpful for counselling patients infected with HIV and their sexual partners.


Is Bordetella pertussis clonal?

Is Bordetella pertussis clonal? To establish whether Bordetella pertussis is essentially clonal. Analysis of restriction fragments of XbaI digests of DNA from clinical and control isolates of B pertussis by pulse field gel electrophoresis. 105 isolates of B pertussis: 67 clinical isolates from throughout the United Kingdom and 23 from Germany (collected during the previous 18 months); vaccine strains 2991 and 3700; and 13 control isolates from Manchester University's culture collection. Frequency of DNA types according to country of origin and classical serotyping. 17 DNA types were identified on the basis of the variation in 11 fragments, banding at 200-412 kilobases; 15 types were found in the clinical and control isolates from the United Kingdom and seven in those from Germany. There was no correlation with serotype. DNA type 1 was the commonest overall (22/105 strains, 22%), predominating in serotypes 1,2 and 1,2,3 and including the vaccine strains but not the isolates from Germany. Current infections due to B pertussis are not caused by a clonal pathogen as multiple strains are circulating in a given population at one time. There is also considerable epidemiological variation in the pathogen population between countries. These findings may have implications for the design of acellular vaccines.


Primary and preschool immunisation in Grampian: progress and the 1990 contract. To examine changes in immunisation performance in Grampian region after the introduction of the 1990 contract for general practitioners. Retrospective descriptive study using data held on the Grampian immunisation record system's computer. All 95 general practices in Grampian region (313 general practitioners). All children in the primary immunisation and preschool booster age groups. This formed two groups of children for each of the four calendar quarters of 1990 and first three quarters of 1991 analysed as (a) those aged 2 years on the first day of the relevant quarter and (b) those aged 5 years on the first day of the relevant quarter, with an average population of 6600 and 6400 respectively. Percentage immunised by practice. For primary immunisation the number of practices achieving immunisation rates of at least 95% increased from 29 (31%) to 76 (81%), and practices achieving 90% rates rose from 69 (73%) to 87 (93%). For preschool boosters, the number of practices achieving at least 95% immunisation rates increased from 22 (23%) to 61 (64%). By the end of September 1991, 76 (80%) practices were achieving at least 90% levels compared with 36 (39%) at the beginning of 1990. Since the beginning of 1989 the proportion of immunisations not given by general practitioners declined from 14% to 2%. Primary and preschool immunisation rates for preschool children in Grampian showed a sustained improvement during 1990 and consolidation in 1991. Although overall trends were unchanged, 18 months after the introduction of the 1990 contract only one practice failed to meet lower target levels of 70% for both primary and preschool immunisation. By September 1991 more than three out of four practices had reached levels of at least 95% for primary immunisation.


Communication between general practitioners and consultants: what should their letters contain? To canvass the views of all general practitioners and consultants working in Newcastle upon Tyne on the content of referral letters and replies, the feasibility of standardising certain aspects of referral letters, and the use of communications data for audit purposes. A postal questionnaire was sent to all general practitioners and consultants in Newcastle upon Tyne in May 1991. Questions were asked about the clinical and administrative content of letters, the utility of standard categories to state the reason for referral, the idea of using letters for feedback purposes, and communications as a potential topic for professionally led audit. Area served by Newcastle upon Tyne Family Health Services Authority and District Health Authority. Replies were received from 274 (77%) doctors (115 general practitioners and 159 consultants). A majority (225; 82%) were in favour of items defined as "always important" forming a minimum requirement for referral letters and for consultants' replies. Using standardised categories to state the reason for referral was not endorsed: 102 (89%) general practitioners and 132 (83%) consultants preferred referrers to use their own words. Using referral communications to provide feedback was less popular with consultants (54; 34%) than general practitioners (72; 63%). Finally, a majority of doctors (179; 65%) were in favour of using written communications as a topic for professionally led audit. A high degree of consensus exists among clinicians about the content of referral communications. Although doctors may still reject the concept of standardised communications, they have unambiguously endorsed a standard for communication that they can aspire to, and they are prepared to use it as a yardstick for their actual performance.


Fetal nuchal translucency: ultrasound screening for chromosomal defects in first trimester of pregnancy. To examine the significance of fetal nuchal translucency at 10-14 weeks' gestation in the prediction of abnormal fetal karyotype. Prospective screening study. The Harris Birthright Research Centre for Fetal Medicine, King's College Hospital, London. 827 fetuses undergoing first trimester karyotyping by amniocentesis or chorionic villus sampling. Incidence of chromosomal defects. The incidence of chromosomal defects was 3% (28 of 827 cases). In the 51 (6%) fetuses with nuchal translucency 3-8 mm thick the incidence of chromosomal defects was 35% (18 cases). In contrast, only 10 of the remaining 776 (1%) fetuses were chromosomally abnormal. Fetal nuchal translucency > or = 3 mm is a useful first trimester marker for fetal chromosomal abnormalities.


Respiratory symptoms and atopy in Aberdeen schoolchildren: evidence from two surveys 25 years apart. To estimate changes in the prevalence of respiratory symptoms and the reported diagnoses of asthma, eczema, and hay fever in primary school children in Aberdeen between 1964 and 1989. Determination of incidence prevalence and prevalence from survey data. Aberdeen, Scotland. 2743 primary school children (aged 8-13) from 1964 and 4003 [corrected] from 1989. Survey data on whether, according to the parent or guardian, the child wheezed or was troubled with shortness of breath; the number of episodes of breathlessness in the past year; and whether asthma, eczema, or hay fever had ever been diagnosed. Questionnaires were completed by the parents of 2510 children in 1964 and 3403 children in 1989. The prevalence of wheeze rose from 10.4% in 1964 to 19.8% in 1989, and the prevalence of episodes of shortness of breath increased from 5.4% to 10.0%. In both surveys wheeze and shortness of breath were more prevalent in boys than in girls. The reported diagnosis of asthma rose from 4.1% to 10.2%, hay fever from 3.2% to 11.9%, and eczema from 5.3% to 12%. The proportion of boys suffering from eczema rose from 47.7% to 60.0%. Hay fever showed a similar increase, from 49.4% to 60.1%, in boys over the 25 year period. Though the parents of a higher proportion of children with wheeze were aware of the diagnosis of asthma in 1989, because of the increased prevalence of wheeze the absolute number of parents of wheezy children who were not aware of a diagnosis of asthma increased from 7.4% to 9.6% of the population studied. The higher diagnosis rate for asthma is due not simply to changes in diagnostic fashion but reflects an increase over the past 25 years in the prevalence of respiratory symptoms, which in turn may reflect a more general change in the prevalence of atopy, the increase in which was particularly noticeable in boys. This increase explains some of the increase in hospital admission rates for children with asthma.


A survey of hospital toilet facilities.

A survey of hospital toilet facilities. To assess the quality of toilet facilities available for disabled people in a large provincial teaching hospital. Survey of toilet facilities for patients on the wards and in the outpatient department. Teaching hospital in Leeds. Although the quality of toilet facilities varied, none met the standards recommended by the British Standards Institution. The worst facilities were found on a ward accommodating elderly patients, where the toilets were unsuitable for use by disabled people and bedside commodes had to be used instead. Toilet provision within a major hospital failed to meet standards required for disabled people. Admission to hospital may therefore result in loss of independence and dignity. If hospitals are to be centres of excellence, greater consideration must be given to the requirements of disabled people in the design of new wards, and current inadequate facilities should be upgraded.


Edinburgh primary care depression study: treatment outcome, patient satisfaction, and cost after 16 weeks. To compare the clinical efficacy, patient satisfaction, and cost of three specialist treatments for depressive illness with routine care by general practitioners in primary care. Prospective, randomised allocation to amitriptyline prescribed by a psychiatrist, cognitive behaviour therapy from a clinical psychologist, counselling and case work by a social worker, or routine care by a general practitioner. 121 patients aged between 18 and 65 years suffering depressive illness (without psychotic features) meeting the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Third Edition for major depressive episode in 14 primary care practices in southern Edinburgh. Standard observer rating of depression at outset and after four and 16 weeks. Numbers of patients recovered at four and 16 weeks. Total length and cost of therapist contact. Structured evaluation of treatment by patients at 16 weeks. Marked improvement in depressive symptoms occurred in all treatment groups over 16 weeks. Any clinical advantages of specialist treatments over routine general practitioner care were small, but specialist treatment involved at least four times as much therapist contact and cost at least twice as much as routine general practitioner care. Psychological treatments, especially social work counselling, were most positively evaluated by patients. The additional costs associated with specialist treatments of new episodes of mild to moderate depressive illness presenting in primary care were not commensurate with their clinical superiority over routine general practitioner care. A proper cost-benefit analysis requires information about the ability of specialist treatment to prevent future episodes of depression.