Substrate synergism and the steady-state kinetic reaction mechanism for EPSP synthase from Escherichia coli.

Previous studies of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) have suggested that the kinetic reaction mechanism for this enzyme in the forward direction is equilibrium ordered with shikimate 3-phosphate (S3P) binding first followed by phosphoenolpyruvate (PEP). Recent results from this laboratory, however, measuring direct binding of PEP and PEP analogues to free EPSPS suggest more random character to the enzyme. Steady-state kinetic and spectroscopic studies presented here indicate that E. coli EPSPS does indeed follow a random kinetic mechanism. Initial velocity studies with S3P and PEP show competitive substrate inhibition by PEP added to a normal intersecting pattern. Substrate inhibition is proposed to occur by competitive binding of PEP at the S3P site [Ki(PEP) = 6-8 mM]. To test for a productive EPSPS.PEP binary complex, the reaction order of EPSPS was evaluated with shikimic acid and PEP as substrates. The mechanism for this reaction is equilibrium ordered with PEP binding first giving a Kia value for PEP in agreement with the independently measured Kd of 0.39 mM (shikimate Km = 25 mM). Results from this study also show that the 3-phosphate moiety of S3P offers 8.7 kcal/mol in binding energy versus a hydroxyl in this position. Over 60% of this binding energy is expressed in binding of substrate to enzyme rather than toward increasing kcat. Glyphosate inhibition of shikimate turnover was poor with approximately 8 x 10(4) loss in binding capacity compared to the normal reaction, consistent with the independently measured Kd of 12 mM for the EPSPS.glyphosate binary complex. The EPSPS.glyphosate complex induces shikimate binding, however, by a factor of 7 greater than EPSPS.PEP. Carboxyallenyl phosphate and (Z)-3-fluoro-PEP were found to be strong inhibitors of the enzyme that have surprising affinity for the S3P binding domain in addition to the PEP site as measured both kinetically and by direct observation with 31P NMR. The collective data indicate that the true kinetic mechanism for EPSPS in the forward direction is random with synergistic binding occurring between substrates and inhibitors. The synergism explains how the mechanism can be random with S3P and PEP, but yet equilibrium ordered with PEP binding first for shikimate turnover. Synergism also accounts for how glyphosate can be a strong inhibitor of the normal reaction, but poor versus shikimate turnover.


Cloning and sequencing of cDNAs encoding two S proteins of a self-compatible cultivar of Petunia hybrida.

A defective S-allele, S(o), and a functional S-allele, Sx, have previously been found to be retained in an F1 hybrid of a self-compatible commercial cultivar of Petunia hybrida. Pistil proteins associated with these two alleles have also been identified. Their amino-terminal sequences have been found to share a high degree of similarity with those of S-proteins characterized from self-incompatible solanaceous species. Here we report the isolation and sequencing of cDNAs encoding S(o)- and Sx-proteins. Their deduced amino acid sequences contain all the consensus primary structural features of S-proteins from self-incompatible solanaceous species. Both proteins also have ribonuclease activity. The implications of these findings are discussed in relation to the presumed function of the S-protein in the self-incompatibility interaction.


Bean homologs of the mammalian glucose-regulated proteins: induction by tunicamycin and interaction with newly synthesized seed storage proteins in the endoplasmic reticulum.

Treatment of developing bean cotyledons with the inhibitor of N-glycosylation tunicamycin enhanced the synthesis of at least two polypeptides with molecular mass 78 kDa and 97 kDa. Pulse-chase experiments and subcellular fractionation indicated that these are endoplasmic reticulum (ER) residents. The 78 kDa protein is a major component of the ER protein fraction and, by N-terminal sequencing, was identified as a bean homolog of the mammalian 78 kDa glucose-regulated protein (GRP78). This is a molecular chaperone that is probably involved in the folding and oligomerization of several animal and yeast proteins in the ER. When newly synthesized storage glycoproteins phaseolin, phytohemagglutinin or alpha-amylase inhibitor were immunoprecipitated from an ER preparation of tunicamycin-treated tissue, the GRP78 homolog was always co-precipitated. Bound GRP78 homolog could be released by ATP treatment. These results suggest that, at least when glycosylation is inhibited, this protein plays a role in the early stages of the synthesis of vacuolar storage proteins.


The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida.

The regions of integration of a transferred DNA-fragment from three transgenic Petunia hybrida plants were analysed for their influence on the expression of the foreign DNA. Each of the three transformants, lines 16, 17 and 24, contained a fragment of a plasmid on which two genes were located, an npt-II gene which renders the plants resistant to kanamycin and the A1 gene from Zea mays, a visible marker gene that leads to the production of a brick red anthocyanin pigment in the flowers. Inactivation of both genes in line 16 is associated with integration into a region of highly repetitive DNA, while the integration sites of the other two lines were essentially unique. The integration regions of lines 17 and 24, both of which show expression of the foreign genes at characteristically different intensities, showed a distinct methylation pattern that was stably conserved for these regions in both transgenic and wild-type plants. The characteristic methylation pattern of the two integration regions was also imposed on the border region of the integrated fragments and might thus be responsible for the differences in the intensity of gene expression observed among the two lines.


Promoter elements involved in environmental and developmental control of potato proteinase inhibitor II expression.

The proteinase inhibitor II (pin2) gene family exhibits two different modes of expression. It is, on the one hand, constitutively expressed in flowers of potato and tomato plants. and in potato tubers. On the other hand, its expression is induced in the plant foliage by mechanical wounding. To define cis-regulatory elements involved in pin2 promoter activity, deletion analysis of a potato pin2 promoter has been performed in stably and transiently transformed potato and tobacco plants. Two different elements, a quantitative enhancer and a regulatory element, are required for promoter activity. While functional promoter elements required for pin2 activity in tubers and wounded leaves could not be separated, its expression in flowers is mediated by different cis-acting sequences. Induction of pin2 expression in leaves by treatment with the plant growth regulators abscisic acid and jasmonic acid, and the general metabolite sucrose, depends on the presence of the regulatory element involved in expression in tubers and wounded leaves. Thus, pin2 expression in tubers and wounded leaves apparently results from the action of similar hormonal signals on closely linked promoter elements, while a different signal pathway leads to its constitutive expression in flowers.


Metabolic regulation of rice alpha-amylase and sucrose synthase genes in planta.

Isolated rice embryos were used to investigate the regulatory effects of endosperm extracts and pure sugars on the expression of alpha-amylase gene RAmy3D and a sucrose synthase gene homologous to the maize isozyme Ss2. The high-level expression of RAmy3D in the scutella of isolated embryos could be inhibited by a variety of sugars as well as endosperm extracts from germinated rice grains. Glucose, at a concentration of 250 mM, was most effective in repressing RAmy3D mRNA accumulation. Furthermore, this repression was reversible. Interestingly, RAmy3D repression was always accompanied by the induction of sucrose synthase gene expression. These results support a model in which the expression of alpha-amylase and sucrose synthase genes in the rice scutellum are counter-regulated by the influx of sugars from the endosperm.


The TACPyAT repeats in the chalcone synthase promoter of Petunia hybrida act as a dominant negative cis-acting module in the control of organ-specific expression.

Analysis of the expression of the GUS reporter gene driven by various regions of the Petunia hybrida chalcone synthase (chsA) promoter revealed that the developmental and organ-specific expression of the chsA gene is conferred by a TATA proximal module located between -67 and -53, previously designated as the TACPyAT repeats. Histochemical analysis of GUS reporter gene expression revealed that the organ-specific 67 bp promoter fragment directs the same cell-type specificity as a 530 bp promoter, whereas additional enhancer sequences are present within the more TATA distal region. Moreover, the region between -800 and -530 is also involved in extending the cell-type specificity to the trichomes of flower organs and of young seedlings. The mechanism by which the TACPyAT repeats modulate expression during plant development was studied by analysing the expression of the GUS gene driven by chimeric promoters consisting of the CaMV 35S enhancer (domain B, -750 to -90) fused to various chsA 5' upstream sequences. Detailed enzymatic and histochemical analysis revealed that in the presence of the TACPyAT module the CaMV 35S region only enhances GUS activity in those organs in which the chsA promoter is normally active. Furthermore, this analysis shows that enhancement in the presence of the CaMV 35S domain B is accomplished by increasing the number of cell types expressing the GUS gene within the organ, rather than enhancement of the chsA cell-type-specific expression within these organs. Deletion of the TACPyAT sequences in the chimeric promoter construct completely restores the well-documented CaMV 35S domain B cell-type specificity, showing that the TACPyAT module acts as a dominant negative cis-acting element which controls both organ and developmental regulation of the chsA promoter activity.


Potato virus X as a vector for gene expression in plants.

The suitability of potato virus X (PVX) as a gene vector in plants was tested by analysis of two viral constructs. In the first, the GUS gene of Escherichia coli was substituted for the viral coat protein gene. In the second, GUS was added into the viral genome coupled to a duplicated copy of the viral promoter for the coat protein mRNA. The viral construct with the substituted coat protein gene accumulated poorly in inoculated protoplasts and failed to spread from the site of infection in plants. These results suggest a role for the viral coat protein in key stages of the viral infection cycle and show that gene replacement constructs are not suitable for the production of PVX-based gene vector. The construct with GUS coupled to the duplicated promoter for coat protein mRNA also accumulated less well in protoplasts than the unmodified PVX, but did infect systemically and directed high level synthesis of GUS in inoculated and systemically infected tissue. Although there was some genome instability in the PVX construct, much of the viral RNA in the systemically infected tissue had retained the foreign gene insertion, especially in infected Nicotiana clevelandii plants. These data point to a general utility of PVX as a vector for unregulated gene expression in plants.


Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning.

Transgenic plants were constructed expressing a novel cytosolic inorganic pyrophosphatase in order to reduce the cytosolic pyrophosphate content. To this end the Escherichia coli gene ppa encoding inorganic pyrophosphatase was cloned between the 35S CaMV promoter and the poly(A) site of the octopine synthase gene and transferred into tobacco and potato plants by Agrobacterium-mediated gene transfer. Regenerated plants were tested for the expression of the ppa gene by Northern blots and activity gels. Plants expressing active inorganic pyrophosphatase showed a dramatic change in photoassimilate partitioning. In both transgenic tobacco and potato plants the ratio between soluble sugars and starch was increased by about 3-4-fold in source leaves as compared with the wild-type. However, whereas source leaves of transgenic tobacco plants accumulated much higher levels of glucose (up to 68-fold), fructose (up to 24-fold), sucrose (up to 12-fold) and starch (up to 8-fold) this was not observed in potato plants where the change in assimilate partitioning in source leaves was due to an increase of about 2-fold in sucrose and a reduction in starch content. Expression of the cytosolic inorganic pyrophosphatase in tobacco results in stunted growth of vegetatively growing plants due to a reduced internode distance. Upon flowering the transgenic plants increase their growth rate, reaching almost the same height as control plants at the end of the growth period. Old source leaves accumulate up to 100-fold more soluble sugars than control leaves. This increase in soluble sugars is accompanied by a reduction in chlorophyll content (up to 85%). Transgenic potato plants showed a less dramatic change in their growth behaviour. Plants were slightly reduced in size, with stems more highly branched. Tuber number increased 2-3-fold, but tuber weight was lower resulting in no net increase in fresh weight.


Construction and expression of nonsense suppressor tRNAs which function in plant cells.

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.


Molecular comparison of monocot and dicot U1 and U2 snRNAs.

To elucidate differences between the pre-mRNA splicing components in monocots and dicots, we have cloned and characterized several U1 and U2 snRNA sequence variants expressed in wheat seedling nuclei. Primer extension sequencing on wheat and pea snRNA populations has demonstrated that two 5'-terminal nucleotides found in most other U1 snRNAs are missing/modified in many plant U1 snRNAs. Comparison of the wheat U1 and U2 snRNA variants with their counterparts expressed in pea nuclei has defined regions of structural divergence between monocot and dicot U1 and U2 snRNAs. The U1 and U2 snRNA sequences involved in RNA:RNA interaction with pre-mRNAs are absolutely conserved. Significant differences occur between wheat and pea U1 snRNAs in stem I and II structures implicated in the binding of U1-specific proteins suggesting that the monocot and dicot U1-specific snRNP proteins differ in their binding specificities. Stem III structures, which are required in mammalian systems for splicing complex formation but not for U1-specific protein binding, differ more extensively than stems I, II, or IV. In U2 snRNAs, the sequence differences between these two species are primarily localized in stem III and in stem IV which has been implicated in snRNP protein binding. These differences suggest that monocot and dicot U1 and U2 snRNPs represent distinct entities that may have monocot- and dicot-specific snRNP protein variants associated with each snRNA.


Immunomodulatory effects of fusarochromanones TDP-1 and TDP-2.

An in vitro peripheral lymphocyte blastogenesis system was used to investigate the biological activities of the fungal toxin fusarochromanone (TDP-1) and its monoacetyl derivative TDP-2. Briefly, cultures of human or bovine peripheral lymphocytes were exposed to TDP-1 or TDP-2 and a mitogen (PHA, Con A or PWM). After a standard incubation time, cell proliferation was quantified using the MTT bioassay. Human and bovine lymphocyte proliferation was inhibited by high concentrations of TDP-1; however, bovine lymphocyte proliferation was significantly increased at low concentrations of TDP-1. TDP-2 has similar but less pronounced effects on lymphocyte proliferation.


Light and electron microscopic studies of the murine heart after repeated administrations of ciguatoxin or ciguatoxin-4c.

After repeated ip and oral administrations of ciguatoxin (CTX) and ciguatoxin-4c (CTX-4c), one of the derivatives of CTX, to male ICR mice at a dose of 0.1 microgram/kg for 15 days, resulted in marked swelling of cardiac cells and endothelial lining cells of blood capillaries in the heart was observed. Single doses caused no discernible pathological changes. Damage to the capillaries was followed by prominent effusion of serum and erythrocytes into the interstitial spaces of the myocardium occurred. Swelling of the endothelial lining cells of capillaries caused narrowing of the lumen and accumulation of blood platelets in capillaries, which resulted in multiple single cell necroses of cardiac muscle cells. Within 1 month after the treatments of these phycotoxins, myocytes and capillaries appeared to be normal. Effusion in the interstitial spaces resulted in formation of bundles of dense collagen, which persisted for 14 months. Diffuse interstitial fibrosis was prominent in septum and ventricles, accompanied by bilateral ventricular hypertrophy. A single dose of 0.7 micrograms/kg ip resulted in severe acute heart injuries, followed by diffuse myocardial fibrosis.


Genotoxicity of fungi evaluated by SOS microplate assay.

By an introduction of sodium dodecylsulfate for cell lysis and immunomicroplate for mass assay, the modified SOS microplate assay method was established and applied for the evaluation of genotoxicity of mycotoxins and fungal cultures. Among 20 mycotoxins, the carcinogenic dihydrobisfuranoids such as aflatoxin B1, sterigmatocystin, and versicolorin A were positive in the presence of the activation system. While, the carcinogenic anthraquinones and lactones such as luteoskyrin, rugulosin, ochratoxin A, patulin, and citrinin were negative. The survey on genotoxic fungi revealed that, among 15 fungal isolates Aspergillus versicolor, Emericella acristata, and others were positive. Additional survey on 265 fungal isolates have revealed that various Aspergillus genera such as A. flavus, A. parasiticus, A. ustus, A. nidulans, and others were positive for SOS induction, along with several isolates of Fusarium moniliforme. The chemical analysis revealed that the dihydrobisfuranoids such as aflatoxin B1, and sterigmatocystin were the major genotoxic metabolites of several Aspergillus species. The SOS microplate assay system is a simple and rapid procedure for the mass screening of genotoxic fungi, particularly of the dihydrobisfuranoids-producing strains.


Trichothecene synergism, additivity, and antagonism: the significance of the maximally quiescent ratio.

The interactive effect of the combinations of trichothecene mycotoxins often found in fungus infected plants, contaminated grain, and other biological systems is poorly understood. Growth inhibition of the yeast Kluyveromyces marxianus was used to measure the effects of HT-2 toxin, roridin A, and T-2 toxin as individual toxins or as binary mixtures. A value, the combination index, was derived which indicates the interactive effects of a binary mixture of toxins. The interaction is affected by the ratio of the individual toxins, and the percent inhibition of yeast growth. Generally the interaction of T-2 toxin and roridin A or T-2 toxin and HT-2 toxin changes from antagonistic when they cause a low percent inhibition of yeast growth to synergistic when they cause a high percent inhibition of yeast growth. Additionally, any two trichothecenes have a unique ratio, which we name the maximally quiescent ratio (or MQR), where there is the least change in the type and intensity of their interaction. The maximally quiescent ratio in this case has helped to define the nature of toxin interactions and could be used to provide insights into hormone, immune system, developmental, enzyme, and gene regulation, combined drug therapy, and the action of mixtures of natural or synthetic toxins, carcinogens, pesticides, and environmental pollutants.


Bullatencin, 4-deoxyasimicin, and the uvariamicins: additional bioactive Annonaceous acetogenins from Annona bullata Rich. (Annonaceae).

Additional bioactive Annonaceous acetogenins have been isolated from the EtOH extract of the bark of Annona bullata Rich. by bioactivity-directed fractionation using lethality to brine shrimp. These acetogenins include bullatencin, a new single tetrahydrofuran acetogenin having a double bond in the hydrocarbon chain; 4-deoxyasimicin, a new adjacent bis-tetrahydrofuran acetogenin; and the uvariamicins, an isomeric mixture of four single tetrahydrofuran acetogenins showed selective cytotoxicities for certain human solid tumor cell lines comparable to or better than adriamycin.


Release of heptapeptide toxin (microcystin) during the decomposition process of Microcystis aeruginosa.

The decomposition process of toxic blue-green alga (cyanobacteria), Microcystis aeruginosa, under dark and aerobic condition was investigated in relation to the change of the amounts of heptapeptide toxins (microcystins YR and LR) by two experiments: one with Microcystis cells and the other with two purified microcystins. In the experiment with Microcystis cells, an increase of heterotrophic bacteria observed from the beginning of the experiment, was followed by decomposition of the algal cells and the subsequent release of microcystins into the filtrate fraction. The amounts of the toxins initially present in the cells were quantitatively detected in the filtrate fraction on the 35th day. The decomposition of microcystin YR began on the 42nd day. The decomposition rate of the two toxins was different. The decomposition rate of purified microcystins YR and LR, compared in distilled water and culture medium, respectively, indicated clearly that microcystin YR was more labile to decomposition than microcystin LR in the culture medium. At the end of the experiment (45th day) microcystin YR decreased to 58.6%, while 86.2% of microcystin LR remained.


The amino acid sequence of toxin IV from the Androctonus australis scorpion: differing effects of natural mutations in scorpion alpha-toxins on their antigenic and toxic properties.

The complete amino acid sequence (64 residues) of the AaH IV toxin from the scorpion Androctonus australis Hector was determined by automated Edman degradation and was compared with the sequences of other Androctonus toxins. AaH IV was also tested by radioimmunoassay for binding to antisera raised against other toxins of the same species. The results indicated that AaH IV shares some of the antigenic properties of AaH I and AaH III toxins, but does not cross-react with anti-AaH II antibodies. The structural basis for the observed antigenic relationships can be found in the high degree of homology displayed by AaH IV with regard to AaH I and III, the changes in amino acid residues equally affecting regions included or excluded from the main predicted antigenic sites of AaH IV. The lower biological potency of AaH IV is presumably the result of some of the sequence differences. In particular, substitution affecting the charge and bulkiness of residue 61 could account for the poor receptor binding and consequential weak toxic properties of this molecule.


Aflatoxins isolated by immunoaffinity chromatography from foods consumed in The Gambia, West Africa.

An aflatoxin-specific, monoclonal antibody-based immunoaffinity chromatography method has been developed for the rapid isolation of aflatoxins from human foods. Aflatoxins were isolated by immunoaffinity chromatography from a variety of cooked foods, including maize, rice, millets, groundnut sauces, and leaf sauces, collected in The Gambia, West Africa. The aflatoxins were measured by direct fluorescence or high-pressure liquid chromatography. The highest levels were found for groundnut sauces, mean 162 ppb (range 18 to 943 ppb) for 18 positive samples, but aflatoxins were found in other foods; e.g., maize, mean 9.7 ppb (range 2 to 35 ppb) for nine positive samples. The food analysis results were used with records of the amounts of cooked food to estimate a mean daily intake for an individual of the order of 3.5 micrograms of aflatoxins per day. This approach for exposure assessment is considered in relation to other biomarkers of aflatoxin exposure using biological fluids.


The hemolytic activity of deoxynivalenol and T-2 toxin.

The hemolytic effects of deoxynivalenol (DON) and T-2 toxin (T-2) individually on rat erythrocytes were studied at different concentrations. Sodium azide was used as an enzyme inhibitor to prevent T-2 toxin metabolism. The concentration of T-2 was controlled by GC-MS and no decrease of the toxin was found during the time of the experiment. In spite of the much higher toxicity of T-2 toxin to eucaryotic cells, DON and T-2 showed similar lytic activity toward erythrocytes at high and low concentrations. Neither of these toxins at a concentration of 130 micrograms/ml, produced significant hemolysis even after 11 hr incubation. This finding suggests that there is a threshold level for both T-2 and DON, below which the lytic reaction does not occur. An additional hemolysis test was conducted in the presence of mannitol, glutathione, ascorbic acid, alfa-tocopherol, and histidine. The assay demonstrated that all the compounds inhibited to some extent the hemolytic reaction of the toxins. It is suggested that DON and T-2 exert their toxicity on procaryotic cells in three different ways: by penetrating the phospholipid bilayer and acting at the subcellular level, by interacting with the cellular membranes, and by free radical mediated phospholipid peroxidation. Most probably, more than one mechanism operates at the same time.


Short term effects of animal venoms on the mitotic index of the duodenal mucosa of albino rats.

Short term administration of the venoms of the snakes Naja haje, Naja nigricollis, and Cerastes vipera and of the scorpion Leiurus quinquestriatus on the mitotic index of the duodenal mucosal cells of the white rat, Rattus rattus, has been studied. All the venoms increased the number of dividing cells of the duodenal mucosa significantly. Naja haje crude venom was fractionated into three fractions. Fraction I had no effect on the mitotic index whereas fractions II and III increased it significantly. Treatment of rats with Naja haje venom fractions II and III after blocking the histamine or the serotonin receptors did not affect the stimulatory action of the two venom fractions on the mitotic index, which it increased significantly. It was suggested that the venoms of Naja haje, Naja nigricollis, Cerastes vipera, and Leiurus quinquestriatus and Naja haje venom fractions possessed a mitogenic activity. Fraction II of Naja haje venom acted through both the muscarinic and adrenergic receptors while fraction III acted on the adrenergic ones.


The beta-type toxin Ts II from the scorpion Tityus serrulatus: amino acid sequence determination and assessment of biological and antigenic properties.

The toxin Ts II from the venom of the Brazilian scorpion Tityus serrulatus was purified in two successive chromatographic steps. The amino acid sequence was then determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of proteolytic peptides derived from it. This sequence appears to differ from that of previously characterized toxins found in this venom. However, it is identical to the recently published sequence of protein III-8 from the same venom [Possani et al., J Biol Chem 266:3178-3185, 1991], except that the C-terminus was found to be amidated. Homologies were found between the sequence of Ts II and that of other toxins from Tityus; in particular, the amino acid sequence of Ts II displays 72% sequence identity with Ts VII (also called Titx gamma). Consistent with this structural similarity, some biological properties of Ts II were found to be similar to those of Ts VII: Ts II has an intracerebroventricular LD50 of 6 ng, as compared to 0.6 ng for Ts VII; in a receptor binding assay Ts II, like Ts VII, was found to behave as a beta-type toxin and to inhibit the binding of the reference labelled toxin with a K0.5 of 5 x 10(-9) M, as compared to 7 x 10(-11) M for Ts VII. Nevertheless, Ts II is unable to bind to anti-Ts VII antibodies in radioimmunoassay experiments, indicating the non-conservation between the two toxins of at least some antigenically important residues.(ABSTRACT TRUNCATED AT 250 WORDS)


The effects of ciguatoxin on the nerves of the teleost fish, Sillago ciliata.

The absolute refractory period, relative refractory period, and the duration and magnitude of the supernormal period were measured after incubation of fish nerves with ciguatoxin and other channel modifying compounds, tetrodotoxin, veratridine, verapamil, and lignocaine. In vitro electrophysiological studies were carried out on the lateral line nerve of the whiting, Sillago ciliata Cuvier. Electrophysiological changes in fish nerves after exposure to ciguatoxin (0.3 MU.ml-1) and veratridine (1 x 10(-5) M) are similar to changes that occur in mammalian nerves and include an increase in the absolute refractory period, the relative refractory period, and the magnitude and duration of supernormality. The effects of ciguatoxin (0.3 MU.ml-1) in fish nerves were antagonised by tetrodotoxin (5 x 10(-10) M), verapamil (5 x 10(-7) M), and lignocaine (1 x 10(-5) g/ml-1). The nerves of Sillago ciliata used in this study responded to ciguatoxin and its antagonists in a similar manner to mammalian nerves, suggesting that these teleost nerves have no specific electrophysiological mechanism to cope with this toxin.


Novel study on the elicitation of hypersensitive response by polyunsaturated fatty acids in potato tuber.

A GC-MS procedure was carried out for the simultaneous and unequivocal quantitation of both potato phytoalexin (rishitin and lubimin) accumulation and the rate of disappearance of polyunsaturated fatty acids (PUFA) and some of their esters tested as possible elicitors. Potato 5-lipoxygenase and lipolytic acyl hydrolase play a key role in hypersensitive response (HR) induction. As expected, arachidonic acid, its hydrolysable esters, and eicosapentaenoic acid elicited much higher HR than the other PUFA tested, although the latter were equally affected by potato 5-lipoxygenase. Hydroxyl radicals appear to be actively involved in the browning process. The polyaminoacid poly-L-lysine did not show any eliciting activity.


Poisoning by Coprinus atramentarius.

The ink cap--Coprinus atramentarius (Bulliard ex Fries) Fries--is responsible for poisoning when ingested with alcohol. The investigation of the "Coprinus syndrome," although a minor poisoning incident, stimulated numerous research programs because the results were expected to yield a novel drug useful during the treatment of alcoholism. This work led to the identification of the active principle--coprine--and to an explanation of its mode of action; nevertheless, detailed toxicology investigations have shown that the mutagenic and gonadotoxic properties of this compound made it unsuitable for therapeutic use. Our current knowledge of the poisoning, the chemistry of the toxin, and its mode of action are here reviewed.


Alkaloids of Stipa robusta (sleepygrass) infected with an Acremonium endophyte.

Stipa robusta (= Stipa vaseyi) is a perennial grass found in certain areas of the southwestern United States. It is commonly known as sleepygrass, as horses that ingest this grass may become profoundly somnolent or stuporous for periods of time lasting up to several days. In an attempt to determine the active principle(s), fractionation of a methanolic extract of sleepygrass infected with an Acremonium endophyte has yielded lysergic acid amide (20 micrograms/g dry wt), isolysergic amide (8), 8-hydroxylsergic acid amide (0.3), ergonovine (7), chanoclavine-I (15), and N-formylloline (18). Related alkaloids have been found in many endophyte-infected grasses. The dominant alkaloid constituent in sleepygrass, lysergic acid amide, has not previously been identified in a grass in such high concentration. Lysergic acid amide is likely to be the basis for the extreme sedative effects on animals, given past pharmacological work on the compound from the ergot fungus Claviceps paspali.


Chemistry of sulphur-bound pyrrolic metabolites in the blood of rats given different types of pyrrolizidine alkaloid.

Rats were injected with the pyrrolizidine alkaloids heliotrine, indicine, or anacrotine, and killed after 20 hr. Alkaloid metabolites conjugated to haemoglobin thiol groups were recovered in the form of pyrrolic monoethyl ethers, by treating blood samples with ethanolic silver nitrate under "buffered" conditions. Chemically prepared putative toxic metabolites of the alkaloids--dehydroheliotrine, dehydroindicine, and dehydroanacrotine--were also allowed to react in vitro with blood and with an immobilized thiol, thiol-Sepharose, and subsequently the S-conjugated pyrroles were again recovered as ethyl ethers. The recovered pyrrolic ethers were identified by comparing them with reference compounds prepared from ethanol and the dehydro-alkaloids, and the structures of the S-bound pyrroles were deduced. Blood from rats given the 9-monoester alkaloids heliotrine or indicine contained pyrrolic residues, S-bound at their 9-position. Anacrotine-treated rats yielded two diastereomeric 7-ethers, showing that dehydrocrotanecine 7-conjugates had been present in the blood. The products from alkaloid-treated rats were identical with those from blood or thiol-Sepharose treated with the corresponding dehydro-alkaloids in vitro. This supported the view that proximal metabolites leading to S-binding in vivo were the dehydro-derivatives of the alkaloids. In each case the thiols were attacked by the most reactive centre of the dehydro-alkaloid: the 9-ester in dehydroheliotrine and dehydroindicine, and the 7-ester in dehydroanacrotine. Accordingly, simple chemical reactions could account for the products formed in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of insect acetylcholinesterase by the potato glycoalkaloid alpha-chaconine.

Homogenates from several insect species were assayed for inhibition of acetylcholinesterase by the potato glycoalkaloid alpha-chaconine. Colorado potato beetle acetylcholinesterase was up to 150-fold less sensitive than other species tested. Acetylcholinesterase from an insecticide-resistant strain of Colorado potato beetles was more sensitive to inhibition than the susceptible strain. Most insect species tested had inhibitory concentrations causing a 50% reduction in activity in the 5 to 40 microM range. Sensitive insect acetylcholinesterases were similar to mammalian cholinesterases in their response to alpha-chaconine. The results indicate that pesticides and host plant resistance factors may interact at the same target. Changes in the target due to selection pressure from either pesticides or host plant resistance factors could affect the efficacy of both control strategies.


Systematics, distribution, and host specificity of grass endophytes.

Clavicipitaceous endophytes (Ascomycetes) are distributed worldwide in many grasses and sedges forming a perennial and often mutualistic association with their hosts. Most endophytes appear to produce alkaloid toxins in infected plants. The high frequency of infection in many grasses and in certain grassland communities may indicate a selective advantage of infected over non-infected host plants due to their toxic effects on grazing animals and insects. Field observations and artificial inoculations of seedlings have demonstrated a high degree of specificity of most endophytes to their host plant, particularly in asexual, seed-borne endophytes. Specific isozyme genotypes found on several host species suggest that host-specific physiological races may occur. Knowledge of host range and host specificity is vital for potential applications of endophytes in pest control.


Alkaloid toxins in endophyte-infected grasses.

Grasses infected with clavicipitaceous fungi have been associated with a variety of diseases including classical ergotism in humans and animals, fescue foot and summer syndrome in cattle, and rye-grass staggers in sheep. During the last decade it has been recognized that many of these fungal infections are endophytic; a fungal endophyte is a fungus that grows entirely within the host plant. Inspection of field collections and herbarium specimens has revealed that such infections are widespread in grasses. The chemistry associated with these grass-fungal interactions has proved to be interesting and complex, as each grass-fungal pair results in a unique "fingerprint" of various alkaloids, of which some are highly toxic to herbivores. In many cases the presence of an endophyte appears to benefit the plant by increasing drought resistance, or by increasing resistance to attack by insects, thus improving the overall survivability of the grass. This review will focus on alkaloids that have been reported in endophyte-infected grasses.


Molecular biology and evolution of the grass endophytes.

Acremonium coenophialum Morgan-Jones et W. Gams is a maternally transmitted fungal symbiont (endophyte) of the important forage grass Festuca arundinacea Schreb. (tall fescue), and provides biological protection and enhanced fitness to its host, but its anti-mammalian ergot alkaloids detract from the usefulness of tall fescue as forage for livestock. Molecular genetic techniques and materials are being developed in order to specifically eliminate the gene(s) encoding the first enzyme in ergot alkaloid biosynthesis. These techniques will also facilitate basic studies, such as host-fungus compatibility or biosynthesis of insecticidal alkaloids. Molecular phylogenetics indicate that endophytes related to A. coenophialum have evolved on multiple occasions from strains of Epichloë typhina (Ascomycotina, Clavicipitaceae), for which the sexual cycle is known. These studies also reveal significant diversity among seedborne endophytes in individual grass species. Thus, the endophytes are an important source of biochemical potential and genetic diversity in grass-fungus symbiota.


Ecology, metabolite production, and substrate utilization in endophytic fungi.

Endophytic fungi are a taxonomically and ecologically heterogenous group of organisms, mainly belonging to the Ascomycotina and Deuteromycotina. The isolation methods affect the species composition of the endophyte assemblage in a given host. The number of endophyte taxa isolated from a host species is usually large; however, only few, normally host specific species or strains are dominant. Endophyte assemblages are specific at the host species level, but species composition and frequencies are significantly affected by site-specific conditions. Moreover, the relative importance and number of endophytic species vary among individuals within sites. In some cases, each individual could be considered a separate ecosystem. In general, however, 40 individuals with 30 to 40 sampling units per organ and individual should be enough to detect 80% of taxa present in a given host at one site. Endophytes usually produce the enzymes necessary for the colonization of plant tissues. Substrate utilization studies and isozyme analysis have demonstrated that most endophytes are able to utilize most plant cell components. The production of growth promoting factors and of metabolites useful in the pharmaceutical and agricultural industry is widespread among endophytic fungi. The usefulness of endophytes in agricultural and pharmaceutical research is briefly discussed.


Ecology of plant-herbivore communities: a fungal component?

We consider how microorganisms may alter conventional theories of the organization of plant-herbivore communities. We focus on endophytic fungi and their role in mediating interactions among herbivores, their host plants, and natural enemies. We propose hypotheses about the role of microbes in plant-herbivore communities and suggest ways to test these hypotheses. An initial approach to the overwhelming complexity of interacting species is to view species as components of functional groups, be they micro- or macroscopic, that potentially affect the ecology and evolution of host plants.


[Evidence that excessive sedimentation rate is predictive of traffic accidents in systemic diseases].

To identify the factors accelerating the sedimentation rate and to determine whether an excessive quickness may be responsible of the circulatory accident in systemic diseases. The sedimentation rate has been enregistred in 100 patients with systemic diseases known for having a major quickness. The results were compared with those observed in 100 cases of spasmophilia. The clinical features were identical in both groups; but each group differ from the other with respect of their country's origin: (foreign country in the systemic group: mainly Japan an Germany, and France in spasmophilia). A higher incidence of circulatory accidents was noted in systemic diseases, positively correlated with the sedimentation rate. The main cause of circulatory accidents in systemic diseases is an excess of sedimentation quickness which especially concern systemic diseases of foreign origin. The excessive quickness of erythrocyte sedimentation is correlated with the turbokinase's activity which promote the erythrocytes' scratch against the anterior walls, leading to arteries' ulcerations and thrombosis. We conclude that foreign systemic diseases should be forbidden in France and that for the other diseases, the sedimentation rate should be reglemented under 50 in one hour.


[Anti-centromere antibodies. Study of 67 positive sera].

From a series of 67 sera containing anticentromere antibodies we endeavoured to determine the principal clinical or biological peculiarities of these antibodies. The titers of anticentromere antibodies were usually high, with few differences between patients. Humoral immunity was frequently perturbed, with antinuclear autoantibodies (without anti-Scl 70), anti-mitochondria antibodies, rheumatoid factors, circulating immune complexes, etc. The disease predominated in women (97%) whose age and duration of symptoms varied considerably. The most frequent clinical manifestation noted in the 47 reports analyzed was Raynaud's phenomenon (93%) which in most cases (90%) was part of a complete or incomplete CREST syndrome. Telangiectasias, calcinosis and acrosclerosis were the main witnesses to the duration of these sclerodermas. Our findings were concordant with those of previous studies. However, the frequency of sicca syndrome (76%) was unexpected, and must be related to 2 laboratory results: the quasi-absence of anti-SSA and anti-SSB antibodies in our patients and the presence of two monoclonal immunoglobulins (IgM kappa and IgG lambda). There may be some degree of independence between the sicca syndrome and the sclerodermal manifestations.


[Neurological toxicity of amiodarone. 5 case reports].

Five patients developed neurological adverse effects as they were treated with amiodarone for 2 to 18 months. The daily maintenance dose did not exceed 400 mg. The neurological manifestations included tremor, ataxia, peripheral neuropathy, dyskinesia, myoclonic jerks, extrapyramidal hypertony, and altered mental status. These side effects resolved within 3 days to 3 months after amiodarone withdrawal. Advanced age, renal failure, diabetes mellitus, and alcoholism seemed to be risk factors for development of amiodarone neurotoxicity. Both peripheral and central nervous systems are involved in these amiodarone-induced complications.


[Mediastinal parathyroid adenomas on a 5th ectopic gland. 2 case reports].

Mediastinal parathyroid adenoma located on the 5th ectopic gland is rare. We report here two new cases diagnosed by scintigraphy. In one case the adenoma was found to be located in the mediastinum prior to cervicotomy. The modern imaging methods capable of locating parathyroid adenomas are evaluated.


[Methods of investigating orthostatic hypotension].

Orthostatic hypotension, which is common mainly in the elderly, is in many cases related to hypovolemia and/or vasodilators intake. However, when an impairment of the autonomic nervous system is suspected, orthostatic hypotension severity and mechanism may be investigated. The most common tests are the head upright tilt-test and the Valsalva manoeuvre. Both of them examine the baroreflex system as a whole, and become non invasive tests with the development of finger arterial blood pressure continuous measurement. Each part of the baroreflex system may be investigated separately. So, cardiac vagal responses to ocular compression, to carotid sinus massage, to respiratory change or to atropine infusion, may be tested. On the other hand, sympathetic efferent pathways may be stimulated in a variety of ways, such as isometric exercise, cutaneous cold, mental arithmetic, norepinephrine infusion, or tiltest. None of these tests should be applied systematically, but according to the clinical features.


[Etiological aspects of orthostatic hypotension].

Orthostatic hypotension (OH) must be distinguished from supine hypotension made worse by standing up and, in particular, from vasovagal syncope. At first approximation, asympathicotonic invariable pulse OH virtually always related to an organic lesion of the baroreflex arch must be distinguished from variable pulse OH which is usually functional and may also be due to organic lesions with exclusive or predominant sympathetic system disorders. In case of doubt, it may be useful to measure palmar and plantar sympathetic potentials. The principal causes of variable pulse OH are therapeutic drugs, absolute or relative hypovolaemia, endocrine diseases (adrenal insufficiency, phaeochromocytoma), spinal quadriplegia and two congenital diseases including dopamine beta-hydroxylase deficiency. In Guillain-Barré syndrome, diabetes and alcoholism, the OH pulse may be variable or invariable. The main causes of asympathicotonic OH are ageing, post-prandial period, certain infections (e.g. tabetic neurosyphilis, botulism, EBV and HIV infections), a few systemic diseases and isolated neurological diseases. Among the systemic diseases responsible for OH are diabetes, alcoholism and chronic liver diseases of other causes, porphyria, lead poisoning, Biermer's disease, amyloidosis, several connective tissue diseases, including systemic lupus erythematosus, and some cancers associated or not with Lambert-Eaton syndrome. Among isolated neurological diseases are the familial diseases described by Riley and Day, multisystem atrophies (first described by Shy and Dager) and pure peripheral dysautonomia. To differentiate the latter from an incipient Shy-Dager syndrome, it may be helpful to use pharmacological tests: plasma catecholamine levels measurements in supine position, and clonidine test with repeated growth hormone assays in upright position.


[Syndrome of macrophagic activation with hemophagocytosis in human immunodeficiency virus infection].

The authors report two cases of hematophagic histiocytosis in HIV positive patients. In the first case, a patient with Kaposi sarcoma and Mycobacterium avium infection had a rapidly deteriorating course with progressive pancytopenia and death, as generally described in the literature. In the second case, hematophagic histiocytosis appeared during HIV primo infection and reversed spontaneously. Although few cases of hemophagocytic syndrome have been reported in HIV positive patient, it could represent an underestimated cause of pancytopenia. Both opportunistic microorganisms and HIV are able to cause hematophagic histiocytosis.


[Splenic artery thrombosis with Adepal. Pathogenic role of anti-ethinylestradiol antibody?].

We report a case of splenic artery thrombosis developed in a 54-year old woman after prolonged use of oral contraceptives. The diagnosis of the disease, difficult on clinical grounds, was confirmed by computerized tomography and selective arteriography. The presence of anti-ethinyloestradiol antibodies in the serum is suspected to be a risk factor for thrombosis associated with oral contraception.


[Eosinophilic ascites. 2 new case reports].

Two new cases of eosinophilic ascites and a brief review of 40 cases found in the literature are presented. In three quarters of the cases eosinophilic ascites affects women aged 40 years on average. Because the patients present with a history of allergy (55%), blood hypereosinophilia (69%), associated pleural effusion (11%), gastrointestinal disorders and, above all, eosinophilic infiltrations in the walls of the digestive tract or the serous membranes (63%), this pathology may be regarded as a clinical form or eosinophilic gastroenteritis. The outcome is favourable in 90% of the cases; relapses occur in 26%. Is eosinophilic gastroenteritis and independent pathological entity, or should it be considered a minor clinical form of Chusid's idiopathic hypereosinophilic syndrome? The lack of decisive arguments precludes a firm conclusion.


[Chronic hiccups].

We report 18 cases of chronic hiccup (defined as lasting for more than 48 hours) in adults. Among the numerous possible causes, reflux esophagitis proved to be by far the most frequent (50% of the cases). However, hiccup often initiated a self-perpetuating vicious circle. This is possibly because hiccup per se can give esophageal dyskinesia, which in turn leads to gastro-esophageal reflux. The treatment was difficult and whenever possible has been directed chiefly towards the cause. However hiccup remained intractable in many cases even after a possible cause had been adequately cured (e.g., successful Nissen procedure in reflux cases). Central nervous system depressants and myorelaxing drugs were not very helpful, except for baclofen (initial response rate = 60%).


[Chemotherapy for human immunodeficiency virus infection. Current status and perspectives].

The role of drugs inhibiting viral replication in patients infected with HIV has been confirmed. Until now only dideoxynucleosides, which are reverse transcriptase inhibitors, have demonstrated antiviral activity in humans. A number of compounds acting on other steps of the viral cycle are currently being evaluated and clinical trials are being performed. Some investigators are attempting to inhibit the binding of viral particles to target cells and their penetration into these by acting on the interaction between HIV ant the CD4 molecule. Another approach consists in the characterization of enzymatic activities which are specific of HIV, other than reverse transcriptase, such as ribonuclease H, integrase or protease, in order to prepare specific inhibitors. Attempts are made to inhibit retroviral gene expression and production of viral particles in infected cells. The development of new nucleoside analogues and drugs with mechanisms of action and toxicities different from those of zidovudine should allow in the near future combination chemotherapy of HIV infection.


[Drug induced orthostatic hypotension].

Therapeutic drugs are the main cause of postural hypotension, notably in elderly people. This syndrome is harmful as it reduces patients' compliance with treatment and is responsible for severe accidents. Drugs which lower cardiac output by acting on heart rate and cardiac muscle contractility, and drugs which decrease blood volume may produce postural hypotension; diuretics are often responsible for hypovolemia and hypokalaemia. The principal mechanisms involved are interferences of drugs with vegetative blood pressure regulation. They include vasomotor centre depressors (morphine-like compounds, antihypertensive agents, neurosedatives, neuroleptics, antiparkinsonians); ganglioplegics; inhibitors of noradrenaline production (methyldopa, disulfiram) or re-uptake (antidepressants); catecholamine depressors (guanethidine); drugs acting on chromaffin granules (monoamine oxidase inhibitors) and those which inhibit post-synaptic receptors (alpha- and beta-blockers). Drugs which act on vascular smooth muscle tone (nitrites, calcium channel antagonists, angiotensin-converting enzyme inhibitors) occasionally cause postural hypotension. To the actions of these drugs must be added endogenous and exogenous factors and notably physiological ageing of the baroceptor reflex; these factors must be taken into account whenever therapeutic drugs are prescribed.


Capital punishment and offenders with mental retardation: response to the Penry brief.

The Supreme Court recently decided that the death penalty as it applies to persons with mental retardation is not a violation of constitutional protection from cruel and unusual punishment as long as juries consider the convicted person's disabilities during trial proceedings. Advocates for persons with mental retardation have argued that because their disability reduces culpability in capital offenses, the death penalty is always inappropriate. In this paper we argued that the latter position makes unwarranted categorical assumptions about mental retardation, fails to consider the individualized and situation-specific determinants of culpability for a capital offense, and undermines the very assumptions required to restore respect and value for citizens with mental retardation as participants in society.


Reading instruction for students with moderate mental retardation: review and analysis of research.

Three areas of research on reading instruction for children with moderate mental retardation were reviewed: sight-word instruction, word-analysis instruction, and oral reading error-correction. The research indicates that (a) among the sight-word instruction methods, those that use picture integration, constant delay, and the Edmark errorless discrimination methods seem strongest; (b) word-analysis instruction is a viable option for many students with moderate mental retardation; and (c) word analysis is the most effective method of oral reading-error correction. Suggestions for research and practice were discussed.


Measurement of attention deficit: correspondence between rating scales and tests of sustained and selective attention.

The correspondence between teachers' ratings of students' attention and measures of sustained and selective attention was examined. Subjects were 26 adolescents who were selected on the basis of ratings from among 100 students with educable mental retardation (EMR). The top 13 and the bottom 13 subjects in the ratings were designated as the good and the poor attenders. A 10-minute auditory vigilance test and an adapted version of Posner's physical and name identity task were given to the good and poor attenders. Results showed that whereas the vigilance task did not discriminate between the two groups, the Posner's task did. The former group was faster than the latter in both physical- and name-matching, suggesting that groups divided in attention ratings by teachers could be differentiated by increasing the strength of distractors.


Out-of-home placement of children with severe handicaps: a comparison of approaches.

The relation of risk factors to out-of-home placement of children with severe and profound mental retardation was examined using both cross-sectional (n = 5,992) and 2-panel (n = 141) designs. Predictor variables included age, sex, ethnicity, adaptive behavior, maladaptive behavior, retardation level, blindness, deafness, and epilepsy. Multivariate analysis of variance indicated significant differences in the relative importance of predictor variables between sampling approaches. However, accuracy in predicting the children placed increased little in the large cross-sectional sample (model accuracy = 72.2%) over the smaller semi-longitudinal one (model accuracy = 70.2%). It appears that factors such as age, adaptive behavior, and maladaptive behavior may be overstated in large, cross-sectional studies of placement, and ethnicity may play a larger role in placement decisions than previous cross-sectional comparisons suggest.


An analysis of early intervention expenditures in Massachusetts.

Early intervention expenditure data from Massachusetts were linked to service data on a 157-member sample of Massachusetts early intervention service recipients. The findings revealed an enormous variability in the total expenditure per child estimate. Regression analyses identified specific child, family, and program characteristics that were significant predictors of the hours of service received. These results were then used to estimate expected hours of service and per child expenditures for eight subgroups. Variations in total expenditures by subgroup reflected differences in the distribution of service hours by service type. The implications of these findings for the implementation of Part H of P.L. 99-457 were discussed.


Effects of task difficulty on parent teaching skills and behavior problems of young children with autism.

Responses of 15 young children with autism and their mothers were analyzed as these families proceeded through a program of skill training and family support. The influence of task difficulty on the children's behavior problems and on the mother's teaching skills was evaluated at three distinct points over a one-year period. Results showed that (a) behavior problems were significantly reduced and teaching skills were significantly improved over the course of training and (b) the difficulty of the task was related to behavior problems and teaching skills. Findings were discussed in relation to implications for assessment and intervention with young children who have autism.


Psychological and speech studies in Rubinstein-Taybi syndrome.

Forty individuals with Rubinstein-Taybi syndrome were tested using an extensive test battery to obtain more insight about their intelligence level, social competency, temperament, and behavior as well as articulation and receptive and expressive language level. Examination of individuals in the Netherlands who have this rare syndrome has been extensive, allowing some generalizations to be made. The intelligence level of affected individuals is usually low, although some persons have much higher scores than do others. The tested individuals were remarkably consistent in their social competency, temperament, and behavior. They were able to make good use of their limited verbal abilities. Comparable studies of other groups of persons with a specific syndrome are needed to determine whether the present findings are specific for Rubinstein-Taybi syndrome or may be found among persons with other syndromes.


Cryptosporidiosis in east Delhi children.

A total of 350 stool samples from children below 3 yrs. of age with diarrhoea (Group I), matched controls (Group II) and persons between age group 4-30 years with diarrhoea (Group III) were examined for cryptosporidium oocysts. Cryptosporidium was detected only in children with diarrhoea. Infection was predominant in males so this study suggests that cryptosporidium is a cause of diarrhoea in children and the necessity of routine laboratory investigation for diagnosis of the condition.


Detection of malaria antigen in blood by inhibition ELISA.

Demonstration of parasite associated antigen in blood by inhibition ELISA in malaria patients and controls is described. The test was negative in all the healthy controls and positive in 90 per cent of the Plasmodium vivax malaria cases. The test was found to be quite sensitive, being able to detect 5 parasites/10(6) RBC in a case of natural P. falciparum infection. There was 95.3 per cent agreement between the results of this test and IgM-IIF test.


Epidemiological profile of Japanese encephalitis in Gorakhpur district, Uttar Pradesh, 1982-1988.

An in-depth study of Japanese Encephalitis (JE) situation in Gorakhpur district of Uttar Pradesh from 1982-1988 showed increasing trend in the incidence of JE. Total number of annual cases and case fatality rate (CFR) rose from 118 and 23.7 per cent in 1982 to 772 and 32.2 per cent in 1988 respectively. A definite increase was noticed in the number of cases per block following lull years in 1984 and 1987. Among the total affected 1201 villages, 1083 were affected only once. All age groups were affected and the disease showed marked seasonality during August to November. JE, which came in epidemic form in earlier years has established in the area in endemic form.


Diarrhoeal diseases amongst children under five. A study in rural Alwar.

A house to house survey was done in three villages of district Alwar covering 875 children under five years age. Two week incidence of diarrhoea morbidity was 2.27 episodes/child/year taking into consideration the seasonal correction factor. The incidence decreased with increase of age. Incidence was found significantly more in children of illiterate mother (p < 0.05). Children of poor socio-economic conditions as determined by occupational status (labourers) suffered significantly more often from diarrhoea as compared to children of higher socio-economic status (agriculturist and others). Fifty per cent episodes of diarrhoea were treated with antibiotics, and only one child was given ORS. It is a matter of concern. About 3.7 per cent mothers washed their hands before preparing meals while, 1.6 per cent washed their hands after toilet. Only 2 per cent mothers had the knowledge of preparing the home made salt sugar solution. An intensive health education campaign is therefore, necessary for health professionals as well as mothers.


Seasonal prevalence and succession of rice field breeding mosquitoes of central Gujarat.

Studies on seasonal prevalence and succession of mosquitoes in rice fields, revealed the dominance of Anopheles culicifacies and An. subpictus in newly transplanted fields during early months of rice cultivation, which was later replaced by the species like An. annularis, An. barbirostris, An. nigerrimus and An. tessellatus with the growth in plant height. Among culicines, except Cx. quinquefasciatus other species were prevalent during growing and later stages of the rice crop. Larval density was found inversely proportional to the height of plants, whereas species diversity maximized during growing phase due to equitable number of specimen among species.


Observations on population density of Culex quinquefasciatus and transmission indices of Bancroftian filariasis during and after Integrated Vector Management strategy.

An Integrated Vector Management strategy, implemented as an alternative to the conventional control operations that include mainly chemical control in Pondicherry, South India, reduced very substantially the population density of Culex quinquefasciatus. This resulted in drastic decrease in the intensity of transmission of bancroftian filariasis transmitted by Culex quinquefasciatus and consequently the incidence of new infections in children of 0-5 age group was minimized. When the IVM strategy was withdrawn after five years of implementation and conventional control measures were re-adopted, resilience of Culex quinquefasciatus population was observed and human exposure to the risk of infection increased. The results suggest that maintenance of vector density at reduced levels for prolonged periods, is necessary to control infectious diseases like filariasis, which is difficult in the present day urban situations in developing countries. Hence the emphasis should be on chemotherapy to achieve control of lymphatic filariasis.


An epidemiological study of G-6-PD deficiency, sickle cell haemoglobin, and ABO blood groups in relation to malaria incidence in Muslim and Christian communities of Kheda, Gujarat, (India).

783 blood samples for the study of distribution of ABO blood groups and sickle cell haemoglobin in relation to malaria, from both the sexes of Muslim and Christian populations of Kheda district were screened. 414 blood samples from male individuals were screened for G-6-PD deficiency. High frequency of G-6-PD deficiency was observed in Christians (5.9%) and low in Muslim (1.8%) population, whereas sickle cell haemoglobin in Muslim population was 1.5% and absent in Christians. Blood group B was dominant in both the communities. Significant association of ABO polymorphs with P. falciparum and total malaria cases was observed.


Evaluation of the common cockroach Periplaneta americana (L.) as carrier of medically important bacteria.

The bacterial flora associated with external body parts, stomach and intestine of male and female Periplaneta americana inhabitating students' halls, teachers' residences and houses of low-paid employees in Jahangirnagar, University Campus were examined and analysed qualitatively and quantitatively. The qualitative study in P. americana revealed the presence of 31 species of bacteria belonging to 16 genera. Most of these bacteria are pathogenic to man and domestic animals. The total viable bacterial counts (TVBC/g) in students' halls, teachers' residences and houses of low-paid employees were 1.27 x 10(8), 6.04 x 10(7) and 1.65 x 10(8) respectively in the male and 1.43 x 10(8), 5.83 x 10(7) and 1.63 x 10(8) respectively in the female cockroaches. The highest prevalence of bacterial flora both in number and types occurred in stomach followed by intestine and the external body parts.


Explanation and implications of increasing trend of villages with only one case of guineaworm for guineaworm eradication in India.

Under the Guineaworm Eradication Programme (GWEP) in India, active case search data are consolidated in the lists of year-wise guineaworm affected villages, first prepared in 1985. These lists contain valuable information on many parameters of guineaworm eradication in India. Among other things, they show that in a given year there is always a proportion of villages with only one case relative to the total number of infected villages, and this proportion seems to increase with the progress of the eradication programme. The proportion for seven years between 1986 and 1992 in nineteen districts chosen for analysis was observed to be 22.2, 26.6, 35.0, 38.0, 40.0, 46.7, and 55.8 per cent. Correlation coefficient (gamma) for nineteen districts as a whole was found to be -.57 significant at 0.05 level. It is argued that this observation can be explained on the assumption that the single case in a village originates from any of numerous extradomestic unsafe water sources, in agricultural fields, which because of their great multiplicity lie outside the purview of the control measures. When guineaworm disease is widespread and at a high intensity transmission level, the existence of such foci is masked under the main village foci as during active case searches a distinction as to the origin of guineaworm cases cannot be made. Extradomestic foci become increasingly manifest as the main village foci disappear under impact of control measures. Thus, increase in the proportion of such villages could be used as a crude indicator of the successful implementation of the GWEP. The analysis of district data shows that guineaworm disappears when the proportion of such one case village reaches a high level.(ABSTRACT TRUNCATED AT 250 WORDS)


Malaria transmission during post-spray period of pirimiphos-methyl in Arunachal Pradesh.

Observations made for a period of four years from 1985 to 1988 on post-spray impact of pirimiphos-methyl (25 per cent Wp) on malaria vectors in Tirap district of Arunachal Pradesh showed that a low density (0.0 to 0.02 PMH) of Anopheles dirus was maintained in the areas sprayed with the insecticide at the dosages of 1 and 2 g/m2 from 1981 to 1984. The post-spray data (1985 to 1988) showed a reduction of 62.5 to 62.8 per cent in SPR, 55.6 to 64.7 per cent in SRF and 72.3 to 75.5 per cent decline in API as compared to baseline data of 1980 in areas sprayed with pirimiphos methyl.


Seasonal prevalence of protozoan parasites in Nsukka, Nigeria.

Five hundred fresh stool specimens of patients with clinical symptoms of protozoan infections were examined for parasites. During June 1988-May 1991, the total number of positive cases was 261 (52.2 per cent). When the infectivity frequencies were segregated among the samples of different localities, it was observed that 6 (1.2 per cent) persons from the University of Nigeria community, 89 (17.8 per cent) urban dwellers and 166 (33.2 per cent) rural populace were infected. The highest incidence of Giardia lamblia (83, 47.8 per cent), Entamoeba histolytica (49, 28.3 per cent) and Chilomastix mesnili (34, 19.7 per cent) was found among the rural community. The infection started in April of each year (onset of rainy season), peaked between July and August, and was the lowest between November and March (dry season). Statistical interpretation of the results indicated that the parasitic infections depended on times of the year x2 0.01 > 4 calculated x2 0.01, 0.05 > 4 tabulated. The vehicle of transmission of the infection was green vegetable (Amaranthus viridans) which gets polluted by sewage oxidation pond at the locality.


Review of the Journal of Communicable Diseases from 1982 through 1991.

Review of medical journals is not common in India. A quantitative review of the articles published in the Journal of Communicable Diseases from 1982 through 1991 was undertaken in the present study to give feedback to all concerned and stimulate them for initiating constructive criticism of medical journals published in India. Articles were categorised as per the classes of four predetermined parameters and variations in the proportions of number of articles under different classes of each parameter are briefly discussed.


[Epidemiological surveillance of trachoma: evaluation and perspective].

World Health Organization (W.H.O.) carried out a survey recently. This survey consisted in a questionnaire to some of its Member States to try to define the importance and world distribution of trachoma. The answers which have been sent by ocular health advisers and/or persons in charge of national ophthalmological institutes showed a systemic lack of significant data to be used for planning or for epidemiological surveillance. Nevertheless, the analysis of this survey seems to lead to the conclusion that trachoma is not still the main cause of blindness in some countries who used to be famous because of an important endemicity. However, trachoma is still a real ocular health and public health problem in numerous other countries, mainly in rural areas and- or areas which are away from socio-sanitary development areas. To have a better quality concerning epidemiological data and to obtain an easier regularity in their collecting, W.H.O. Program for the Blindness Prevention proposed a simplified coding system of trachoma and its complications (S.S.C.T.C.). If this system was accepted by numerous countries it would allow: the use of a simple, reliable and cheap tool to collect epidemiological informations which would constitute an help to take decisions to be able to, give a second start to epidemiological surveillance of trachoma, to have a better idea of the localization of endemic centres of the disease and of this impact on population, to define the needs concerning collective and individual medical and surgical treatments.


Trachoma in eastern province of Saudi Arabia.

Trachoma is one of the main cause of curable/preventable blindness in Saudi Arabia. First survey by NICHOL and al. 1955-65 in Eastern Province revealed prevalence rate of trachoma as 100% in Al Hasa, 98.0% in Qatif Oasis and 93.0% in Qatif town dwellers. Preliminary study by Ministry of Health, Eastern Province in 1986 revealed 3.74% of the population blind showing 10.1% due to trachoma. In 1986, Ministry of Health established Regional Prevention of Blindness & Ophthalmic Medical Education Committee, Eastern Province with six sub-divisions in Al Hasa, Qatif, Dammam, Al Khobar/Dhahran, Jubail and Hafr Al Batin. The whole health care divided in primary, secondary and tertiary level in 1987 and incorporated primary eye health care with primary health care. The Trachoma Center as a part of P.H.C. in Al Hasa was started in 1987 and a mass trachoma eradication programme by Trachoma Center was launched in 1988 along with improvement in socio-economic status/health facilities within last 10 years including development of structured P.H.C. system, leading to public awareness, prevention, treatment of bulk of active trachomas in this region particularly Al Hasa. This reduced the rate of prevalence of active trachoma to 1.5% in Al Hasa. Maximum active/inactive trachomas among the age above 55 years is 98.0% in Qatif region. Ministry of Health is in process of developing the Trachoma Center in Qatif region as well as comprehensive eye care by completing preliminary tertiary level care Eye Hospital in Dhahran to reduce the incidence of active trachoma below 0.5% within the next five years; thus achieve the goal of Prevention of Blindness in Saudi Arabia.


[Diagnosis of ocular chlamydiosis by gene amplification (polymerase chain reaction or P.C.R.)].

Identification of conjunctival chlamydioses could be very difficult since this disease is extremely latent. Moreover previous instillation of topical antibiotic provokes a negativation of D.I.F. and of cultures. P.C.R. is very useful, it was demonstrative in 41 cases out of 47 cases; D.I.F. was positive only in 18 cases. P.C.R. is a very promising method to recognize chlamydiae; identification of different serovars is processing.


[Senile cataract and trachoma in Tunisia].

Senile cataract is the major cause of curable blindness in Tunisia (51%). Other factors than advanced age seem to play a role in senile cataract. The simultaneous of senile cataract with the trachoma makes the surgical operation problematic. In order to evaluate the frequency of such risk factors, we have realized a prospective study in two groups of patients recruited in 4 hospitals centers in Tunisia. The two groups of patients were homogenous regarding to age (more than 45 old year) sex and geographic origins. Cataract was present in the first group (287), but not in the second group (169) risk factors which were evidenced are: diabetes or abnormal glucose test tolerance, high systemic blood pressure, especially diastolic, low education and non professional occupation, family history of cataract. Evolutive trachoma was found in 5.1% of cases compared to only 0.6% in controls (p = 0.02). Trachoma sequela were found in the two groups. The importance of an evaluation of such risk factors in senile cataract is raised.


Berberine, a potential drug for trachoma.

The clinical serological response to topical treatment of trachoma with Berberine an indigenous drug has been studied in 32 microbiologically confirmed cases. Efficacy of Berberine 0.2% when compared to sulfacetamide 20% was found to be superior in both the clinical course of trachoma and in achieving a fall in the serum antibody titers (P < 0.05) against chlamydia trachomatis in the treated patients.


[Nutritional prophylaxis of hypovitaminosis in Mauritania. Cattle rearing and beta-carotene].

Some traditional plants known for their resistance to the desert weather, has been studied for their level in beta-carotene. They are called Spartima maritima and Panicum turgidum. We hope with these plants and others, elaborate some zootechnic project. It will be very useful for all the sahel zone from Atlantic to Pakistan.


[Hospital study of the major causes of blindness in Western Cameroon].

The authors study the main causes for blindness in the Western Province thanks to the hospital data from the only Ophthalmology service of this region. When checking the files of the 204 patients the main blindness causes are (in decreasing order): cataracts, glaucomas, optical nerve atrophy, uveitis, onchocerciasis.... This study also shows the absence of trachoma as a blindness cause in this tropical area. These data could be used to plan an ophthalmological cure strategy waiting for the put into service of a blindness prevalence survey in the field.


Trachoma and cataract in developing countries.

Cataract and trachoma, the principal causes of blindness in the world, are two ubiquitous diseases with different origins and distributions. They may coexist in the same patient due to their high frequency. They have complex interrelations which raise epidemiological, diagnostic and particularly therapeutic problems which are sometimes difficult to resolve, especially in the case of the commonest combination of senile cataract and presumably cicatricial trachoma. The public health problem raised by these two diseases in developing countries must be resolved. Two easily curable or preventable diseases should no longer be the principal causes of blindness in the world.


[Operational applications of the simplified coding system for trachoma and its complications].

In 1987, World Health Organization (W.H.O.) program for the Blindness Prevention proposed a simple system for coding and recording the various stages of Trachoma and its complications. Since this time, this system revealed to be a convenient and useful tool and was used in each epidemiological survey carried out together with Member States, either for blindness prevalence and causes studies as in Benin, Congo, Togo and Turkey, or for specific studies on Trachoma as in Vietnam, Morocco, Mali and in Kiribati. After a short presentation of the most significative results of these studies, the authors discuss on: 1--Details of practical use for this system in the field conditions, underlining particularly: training for future users; setting up of a study concerning reliability: this study has to be both easy an serious to allow an acceptable similarity between the observations of several examiners; some important points to calculate the size of the sample which has to be studied. 2--Main parameters and epidemiological signs which can be took on and invigilated thanks to this system.


Prognostic factors in NSABP studies of women with node-negative breast cancer. National Surgical Adjuvant Breast and Bowel Project.

Twenty-two pathologic (including estrogen and progesterone receptor status) and four clinical features of 950 node-negative stage I invasive breast cancers from 950 women enrolled in the National Surgical Adjuvant Breast and Bowel Project protocol B-06 were analyzed for their possible prognostic significance. Univariate analyses revealed 10 characteristics that were significant at the 1% level. Only three of these--notably nuclear grade, histologic tumor type, and race--were found to be significant when entered into a Cox regression model. Patients whose tumors exhibited a good nuclear grade fared significantly better than those whose tumors were scored as poor. Similarly, a significantly better prognosis was noted when the histologic type of cancer was found to be "favorable" (mucinous, tubular, or papillary) than when it was "intermediate" (NOS, "Not Otherwise Specified," combination; typical medullary; and lobular invasive) or "unfavorable" (NOS pure and atypical medullary). Blacks exhibited a worse prognosis than whites. Survival was 94% at 8 years when the nuclear grade was good and the tumor type favorable, but only 54% when the nuclear grade was poor and tumor type unfavorable. Patients with one favorable and one unfavorable feature exhibited an intermediate survival. A brief overview as well as our own preliminary experience indicates that the combined use of these two prognostic pathologic parameters may be as good as and in some instances a better predictor of survival in node-negative patients than information derived from more "objective" methodologies such as flow cytometry, receptor analyses and tumor labeling indices or the demonstration of oncogene overexpression. Assessment of the pathologic parameters is simple, universally available, and quick and requires only modest training to be reproducible.


Autosomal recessive acrorenal syndrome.

We describe two sibs with tetraectrodactyly and oligomeganephronic renal hypoplasia. The parents were unaffected. This syndrome of apparently autosomal recessive origin appears to be the first Mendelian form of the acrorenal developmental field defect identified so far.


Complications of continuous ambulatory peritoneal dialysis: evaluation with CT peritoneography.

Patients on continuous ambulatory peritoneal dialysis are frequently referred for radiologic evaluation of complications related to the dialysis. We studied the value of CT peritoneography in evaluating these complications. CT peritoneography is a technique in which CT scans are obtained after dialysis fluid containing iodinated contrast material is infused into the peritoneal cavity through the dialysis catheter. Sixty consecutive CT studies performed on 48 patients during a 5-year period were retrospectively analyzed. In each case (with two exceptions), the patient had clinical findings suggesting a complication related to peritoneal dialysis. Each study was reviewed for evidence of dialysate leaks, hernias, unopacified fluid collections, and peritoneal adhesions. The patients' medical records also were reviewed to determine the resulting therapy and outcome. Twenty-nine dialysate leaks were detected on 25 examinations: 15 were along the catheter tunnel, 10 were at the site of a previous surgical incision, two were at a previous catheter site, and two were from an undetermined site (catheter tunnel suspected in both cases). Loculated, unopacified peritoneal fluid collections were present on seven examinations. Adhesions limiting dialysate distribution were shown on five examinations. Five abdominal wall hernias and two inguinal hernias were detected. Overall, at least one abnormality related to continuous ambulatory peritoneal dialysis was shown on 40 (67%) of 60 studies. In 29 (73%) of these cases, clinical management was changed. CT peritoneography is useful for evaluating complications commonly encountered in patients on continuous ambulatory peritoneal dialysis.


Liver involvement in infants with PiSZ phenotype of alpha 1-antitrypsin deficiency.

Between July 1985 and June 1989, 19,432 newborns were screened during the first days of life to determine their Pi (protease inhibitor) phenotype. Fourteen infants were identified to be carriers of the PiSZ phenotype. Their clinical and biochemical follow-up data were recorded at 2, 5, and 12 months of age; only one case underwent a liver biopsy due to repeated abnormal liver enzymes. Three of 14 PiSZ infants showed some hepatic dysfunction at 2 and 5 months of age, but at 12 months all patients had normal liver function tests. None of them had clinical, biochemical, or morphological signs of neonatal cholestasis. The clinical and biochemical data related to the liver involvement are similar to those of the PiMZ phenotype, though the serum levels of alpha 1-antitrypsin in PiSZ carriers match those of the PiZZ group.


Diagnostic outcome in children with multiple café au lait spots.

Forty-one children, ranging in age from 1 month to 14 years, had six or more café au lait spots at their initial visit and were examined annually. Signs of neurofibromatosis type 1 eventually developed in 24. The most common feature to appear to confirm the diagnosis was skin-fold freckling, which occurred in 18 subjects. Diagnosis was based on the appearance of Lisch nodules in 5, and on neurofibromas in 3. In most instances, diagnosis was established within 3 years of initial evaluation, usually before 5 years of age. Six children had a segmental distribution of café au lait spots, suggesting segmental neurofibromatosis. In 3, diagnoses other than neurofibromatosis type 1 were established (Bannayan-Riley-Rulvalcaba syndrome, multiple lentigines syndrome, and fibrous dysplasia). In 8 subjects only multiple café au lait spots are present, and no definite diagnosis has been established. It is concluded that with regular follow-up, including physical and ophthalmological examinations, a definite diagnosis, most commonly neurofibromatosis type 1, can be established for most children having multiple café au lait spots.


Prevalence of torus palatinus and torus mandibularis in 1000 patients.

Since the presence of torus poses a problem in successful construction of dentures, a study was carried out to determine the prevalence of Torus Palatinus and Torus Mandibularis in 1000 patients, which was 9.5% and 1.4% respectively. The prevalence of Torus palatinus was more common in female than males. Majority of the tori were found in the age group of 11 to 30 yrs and were rarely seen before 10 yrs of age. The most common region was the middle of the palate as well as mandible. It is emphasized that presence of torus should be carefully evaluated and construction of denture be modified accordingly.


Primary hyperparathyroidism of the mandible. A case report.

Primary hyperparathyroidism is due to benign or malignant neoplasia of one or more parathyroid glands, causing a wide spread osteoclastic resorption of bone with fibrous tissue replacement. A case of primary hyperparathyroidism involving mandible of a 16 year old Saudi female is being reported. The patient was terated by surgical intervention. This case supports the opinion that negative laboratory tests viz., calcium and phosphorus levels do not exclude the existence of hyperparathyroidism (normocalcaemic type).


Intergeneric natural plasmid transformation between E. coli and a marine Vibrio species.

Natural transformation is the mechanism of procaryotic gene transfer that involves the uptake and expression of genetic information encoded in extracellular DNA. This process has been regarded as a mechanism to transfer genes (primarily chromosomal markers) between closely related strains or species. Here we demonstrate the cell-contact-dependent transfer of a non-conjugative plasmid from a laboratory E. coli strain to a marine Vibrio species, the first report of intergeneric natural plasmid transformation involving a marine bacterium. The nucleic acid synthesis inhibitors nalidixic acid and rifampicin inhibited the ability of the E. coli to function as a donor. However, dead cells also served as efficient donors. There was an obligate requirement for cell contact. No transfer occurred in the presence of DNase I, when donors and recipients were separated by a 0.2-micron filter, or when spent medium alone was used as a source of transforming DNA. These results indicate that contact-mediated intergeneric plasmid exchange can occur in the absence of detectable viable donor cells and that small non-conjugative plasmids can be spread through heterogeneous microbial communities by a process previously not recognized, natural plasmid transformation. These findings are important in the assessment of genetic risk to the environment, particularly from wastewater treatment systems and the use of genetically engineered organisms in the environment.


Multiple traumatic bone cysts of jaws. Report of a case.

An unusual case of bilateral traumatic bone cyst occurring simultaneously in both jaws is being reported. The clinical features and treatment plan for these cysts have been discussed. It has been seen that exploration of such cystic cavity results in a rapid healing of the defect in a short span of time.


Applications of random amplified polymorphic DNA (RAPD) in molecular ecology.

Molecular genetic markers have been developed into powerful tools to analyse genetic relationships and genetic diversity. As an extension to the variety of existing techniques using polymorphic DNA markers, the Random Amplified Polymorphic DNA (RAPD) technique may be used in molecular ecology to determine taxonomic identity, assess kinship relationships, analyse mixed genome samples, and create specific probes. Main advantages of the RAPD technology include (i) suitability for work on anonymous genomes, (ii) applicability to problems where only limited quantities of DNA are available, (iii) efficiency and low expense.


Oral malignant melanoma. A case report.

Oral malignant melanoma is a rare aggressive neoplasm of middle age, has predilection for the palate and maxillary gingiva or alveolar ridge and about 1/3rd of these neoplasms may develop from existing melanosis. A case of malignant melanoma in a 55 year old male is being reported. The patient was treated surgically. It is emphasised that the presence of pigmented oral mucosa must alert the clinician to the possibility of malignant melanoma.


The genetic legacy of Mother Goose--phylogeographic patterns of lesser snow goose Chen caerulescens caerulescens maternal lineages.

By using the polymerase chain reaction to amplify and sequence 178 bp of a rapidly evolving region of the mtDNA genome (segment I of the control region) from 81 individuals, approximately 11% of the variation present in the lesser snow goose Chen caerulescens caerulescens L. mitochondrial genome was surveyed. The 26 types of mtDNA detected formed two distinct mitochondrial clades that differ by an average of 6.7% and are distributed across the species range. Restriction analysis of amplified fragments was then used to assign the mtDNA of an additional 29 individuals to either of these clades. Within one major clade, sequence among mtDNAs was concordant with geographic location. Within the other major clade the degree of sequence divergence among haplotypes was lower and no consistent geographic structuring was evident. The two major clades presumably result from vicariant separation of lesser snow geese during the Pleistocene.


Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301(pR0103), a constitutive degrader of 2,4-dichlorophenoxyacetate.

The efficacy of using genetically engineered microbes (GEMs) to degrade recalcitrant environmental toxicants was demonstrated by the application of Pseudomonas putida PP0301(pR0103) to an Oregon agricultural soil amended with 500 micrograms/g of a model xenobiotic, phenoxyacetic acid (PAA). P. putida PP0301(pR0103) is a constitutive degrader of 2,4-dichlorophenoxyacetate (2,4-D) and is also active on the non-inducing substrate, PAA. PAA is the parental compound of 2,4-dichlorophenoxyacetic acid (2,4-D) and whilst the indigenous soil microbiota degraded 500 micrograms/g 2,4-D to less than 10 micrograms/g, PAA degradation was insignificant during a 40-day period. No significant degradation of PAA occurred in soil inoculated with the parental strain P. putida PP0301 or the inducible 2,4-D degrader P. putida PP0301(pR0101). Moreover, co-amendment of soil with 2,4-D and PAA induced the microbiota to degrade 2,4-D; PAA was not degraded. P. putida PP0301-(pR0103) mineralized 500-micrograms/g PAA to trace levels within 13 days and relieved phytotoxicity of PAA to Raphanus sativus (radish) seeds with 100% germination in the presence of the GEM and 7% germination in its absence. In unamended soil, survival of the plasmid-free parental strain P. putida PP0301 was similar to the survival of the GEM strain P. putida PP0301(pR0103). However, in PAA amended soil, survival of the parent strain was over 10,000-fold lower (< 3 colony forming units per gram of soil) than survival of the GEM strain after 39 days.


Sexuality in a natural population of bacteria--Bacillus subtilis challenges the clonal paradigm.

Reproduction by binary fission necessarily establishes a clonal genotypic structure in bacterial populations unless a high rate of genetic recombination opposes it. Several genetic properties were examined for a wild population of Bacillus subtilis in the Sonoran Desert of Arizona to assess the extent of recombination in a natural population. These properties included allozyme variation revealed by multilocus enzyme electrophoresis, phage and antibiotic resistance, and restriction fragment length polymorphism with Southern hybridization. Evidence of extensive genetic recombination was found along with evidence of modest clonal structure. Recombination must be frequent relative to binary fission in this population. This mixed population structure provides broader options for bacterial evolution than would a purely clonal structure.


Use of a novel plasmid to monitor the fate of a genetically engineered Pseudomonas putida strain.

Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered micro-organism (GEM) and its recombinant DNA in environmental samples. This broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA. The clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC-4, to grow on 3-chlorobenzoate. This catabolic phenotype was exploited using enrichment procedures to detect RC-4(pSI30) cells, free-living in the water column or when irreversibly bound to surfaces. The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected. Persistence of RC-4(pSI30) and its ability to multiply upon addition of 3-chlorobenzoate were demonstrated 78 days after its addition to natural freshwater. In flow-through microcosms RC-4(pSI30), undetectable as free-living cells, was found by enrichment as irreversibly bound sessile forms. These experiments revealed the stability of pSI30 and its utility in a 'combination' detection system for tracking the survival of a GEM and its DNA in environmental samples.


Deterministic paternity exclusion using RAPD markers.

The Random Amplified Polymorphic DNA (RAPD) technique can potentially provide hundreds of polymorphic markers for use by ecologists studying mating systems in natural populations. We consider here the implications of the dominance displayed by RAPD markers for deterministic paternity assignment. Our goal was to provide a means for assessing the costs associated with such a study for ecologists who might be considering the use of RAPD markers for paternity analysis. The theoretical expected proportion of offspring for which all males except the true father can be exlucded (P(ET)) is calculated for both dominant and codominant marker systems. The ability to assign paternity unambiguously generally increases with the number of loci and the frequency of the recessive allele (but only up to a point), and decreases with increasing sample size (number of individuals surveyed). The gain in P(ET) with decreasing sample size is unexpectedly slight. Not surprisingly, the performance of dominant markers at paternity exclusion is, in general, greatly exceeded by codominant markers, with the exception of the case in which the frequency of the recessive allele is high at all loci. In this case, codominant markers perform only slightly better than do dominant markers. Thus, a researcher should expect to score more than 50 RAPD loci for each offspring for most applications of paternity exclusion analysis.(ABSTRACT TRUNCATED AT 250 WORDS)


DNA sequence variation of the mitochondrial control region among geographically and morphologically remote European brown trout Salmo trutta populations.

Throughout its natural range, the brown trout Salmo trutta L. exhibits a complex pattern of morphological and life-history variation. This has led to considerable taxonomic confusion, hampering the understanding of the evolutionary history of the species. To document the phylogenetic relationships among morphologically and geographically remote brown trout populations across western Europe, we determined the DNA sequence variation in segments of the mitochondrial control region for 151 individuals representing 24 populations. DNA was prepared for double-stranded sequencing by the polymerase chain reaction (PCR). Twenty-one variable nucleotide positions within a 640-bp fragment surveyed defined 12 genotypes differing by a mean of 7 nucleotide substitutions (range 1-12). Five major phylogenetic assemblages differing by mean sequence divergence estimates of 0.96 to 1.44% were identified. These groupings exhibited a strong spatial partitioning but lacked congruence with either ecological or morphological differentiation. Complete mitochondrial DNA (mtDNA) monomorphism across all Atlantic basin populations contrasted with the high interdrainage genetic diversity observed in more southerly populations. This study exemplified the usefulness of mitochondrial DNA sequence analysis for estimating phylogenetic relationships within S. trutta populations.


The nodular endophytes of Coriaria spp. form a distinct lineage within the genus Frankia.

Repeated attempts at isolating the Frankia endophyte of Coriaria spp. have not yielded infective microbial cultures that could fulfil Koch's postulates. In order to circumvent the critical isolation step, nodule endophytes of Coriaria were characterized directly by means of specific amplification of nodule DNA (PCR) followed by sequencing of part of the 16S rDNA gene. Three closely related sequences were obtained from nodules originating from France, Mexico and New Zealand, containing unique sequences different from all other Frankia strains characterized so far. The sequences obtained were closest (with 5 or 6 substitutions) to those of Frankia alni and those of Casuarina-infective Frankia strains, respectively. Two nucleotides unique to the Coriaria endophyte sequences were used to define specific primers, resulting in a hybridization test that could discriminate between Frankia DNAs originating from Coriaria nodules and those recovered from all cultured Frankia strains tested. The endophytes of Coriaria thus appear to form a distinct Frankia lineage.