Improved method for detecting poliovirus negative strands used to demonstrate specificity of positive-strand encapsidation and the ratio of positive to negative strands in infected cells.

We have developed a ribonuclease protection method suitable for sensitive detection of an RNA species in the presence of a large excess of its complementary strand, as for the detection of negative strands of positive-strand RNA viruses. By using this method to probe for poliovirus negative strands in virions, we found that positive strands are present in at least 40,000-fold excess over negative strands. Thus, we have confirmed that poliovirus encapsidation is highly specific for positive strands and have demonstrated that the genome-linked protein VPg, which is covalently attached to the 5' ends of both positive and negative strands, cannot be the sole determinant of RNA packaging. We tested the ratios of viral positive strands to negative strands in cells at different times during infection; this value ranged from approximately 40/1 to 70/1, being highest at 4 h and lower at 2 and 6 h postinfection.     

The influence of topography on the nature of humic substances in soil organic matter at a site in the Atlantic Coastal Plain of South Carolina

The effect of topography on the nature of humic substances, isolated as water soluble organic carbon (WSOC), fulvic acid (FA), and humic acid (HA) was evaluated by comparing relative proportion and chemical characteristics of these fractions in upland and bottomland Coastal Plain soils in South Carolina. The fractions were characterized by elemental analysis and¹³C cross-polarization magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectroscopy. The majority of humic substances occur as humic acids, with bottomland soils having higher HA/FA ratios when compared to upland soils. We found no significant differences between upland and bottomland humic substances with respect to yields of WSOC and fulvic acids, and in the C and N content of humic and fulvic acids. Carbon-13 CPMAS NMR spectroscopy revealed that the WSOC and fulvic acid fractions were composed largely of O-alkyl-C structures with bottomland soils having higher amounts of these groups. Humic acid C distribution was similar between upland and bottomland soils and was largely composed of aromatic groups. Our results demonstrate that topography influences the formation of humic acid and the structural and chemical properties of the various humic fractions.requests for offprints     

Characterization of a heat-stable NADP-dependent isocitrate dehydrogenase from the obligate thermophile Thermoleophilum minutum YS-4

Summary A thermostable NADP-dependent isocitrite dehydrogenase (IDH; EC. 1.1.1.42) was purified from the obligately thermophilic hydrocarbonoclastic bacterium Thermoleophilum minutum YS-4 (ATCC 35265). This was accomplished by affinity chromatography and electroelution from a nondenaturing polyacrylamide gel. The enzyme has an M _r of 60 000 and is composed of two identical subunits of M _r 30 500. The amino acid composition has an Arg/Lys ratio of 4:1 and very high levels of glycine. Under nondenaturing conditions, the enzyme has a distinct difference in electrophoretic mobility relative to IDHs obtained from other genera including the genus Thermus. The secondary strcuture consists of 16% -helix, 20% -sheet, 25% -turn and 37% random coil as determined by circular dichroism spectroscopy. The optimum pH and temperature for activity were 7.2 and 75° C respectively and the apparent K _mvalues for DL-isocitrate adn NADP⁺ were 33 M, and 48 M, respectively. The enzyme requires divalent cations, such as Mn²⁺ or Mg²⁺ for activity. NAD⁺ cannot substitute for NADP⁺. Oxaloacetate plus glyoxylate exert considerable inhibition on IDH activity while other glycolytic and tricarboxylic acid cycle intermediates have a lesser effect. p-Chloromercuribenzoic acid was inhibitory to the IDH although isocitrate and Mn²⁺ offered some protection from this inactivation. The enzyme is thermostable, retaining 84% and 57% of initial activity after incubation for 1 h at 60° and 70° C, respectively. Isocitrate provided protection from thermal inactivation allowing the IDH to maintain 21% activity after 1 h at 80° C. Offprint requests to: J. J. Perry     

The effects of endogenous or exogenous catecholamines on blood respiratory status during acute hypoxia in rainbow trout (Oncorhynchus mykiss)

Summary An extracorporeal circulation of rainbow trout (Oncorhynchus mykiss) was utilized to continuously monitor the rapid and progressive effects of endogenous or exogenous catecholamines on blood respiratory/acid-base status, and to provide in vivo evidence for adrenergic retention of carbon dioxide (CO₂) in fish blood (cf. Wood and Perry 1985). Exposure of fish to severe aquatic hypoxia (final P _wO₂=40–60 torr; reached within 10–20 min) elicited an initial respiratory alkalosis resulting from hypoxia-induced hyperventilation. However, at a critical arterial oxygen tension (P _aO₂) between 15 and 25 torr, fish became agitated for approximately 5 s and a marked (0.2–0.4 pH unit) but transient arterial blood acidosis ensued. This response is characteristic of abrupt catecholamine mobilization into the circulation and subsequent adrenergic activation of red blood cell (RBC) Na⁺/H⁺ exchange (Fievet et al. 1987). Within approximately 1–2 min after the activation of RBC Na⁺/H⁺ exchange by endogenous catecholamines, there was a significant rise in arterial PCO₂ (P _aCO₂) whereas arterial PO₂ was unaltered; the elevation of P _aCO₂ could not be explained by changes in gill ventilation. Pre-treatment of fish with the -adrenoceptor antagonist phentolamine did not prevent the apparent catecholamine-mediated increase of P _aCO₂. Conversely, pre-treatment with the -adrenoceptor antagonist sotalol abolished both the activation of the RBC Na⁺/H⁺ antiporter and the associated rise in P _aCO₂, suggesting a causal relationship between the stimulation of RBC Na⁺/H⁺ exchange and the elevation of P _aCO₂. To more clearly establish that elevation of plasma catecholamine levels during severe hypoxia was indeed responsible for causing the elevation of P _aCO₂, fish were exposed to moderate hypoxia (final P _wO₂=60–80 torr) and then injected intraarterially with a bolus of adrenaline to elicit an estimated circulating level of 400 nmol·l^-1 immediately after the injection. This protocol activated RBC Na⁺/H⁺ exchange as indicated by abrupt changes in arterial pH (pHa). In all fish examined, P _aCO₂ increased after injection of exogenous adrenaline. The effects on P _aO₂ were inconsistent, although a reduction in this variable was the most frequent response. Gill ventilation frequency and amplitude were unaffected by exogenous adrenaline. Therefore, it is unlikely that ventilatory changes contributed to the consistently observed rise in P _aCO₂. Pretreatment of fish with sotalol did not alter the ventilatory response to adrenaline injection but did prevent the stimulation of RBC Na⁺/H⁺ exchange and the accompanying increases and decreases in P _aCO₂ and P _aO₂, respectively. These results suggest that adrenergic elevation of P _aCO₂, in addition to the frequently observed reduction of P _aO₂ are linked to activation of RBC Na⁺/H⁺ exchange. The physiological significance and the potential mechanisms underlying the changes in blood respiratory status after addition of endogenous or exogenous catecholamines to the circulation of hypoxic rainbow trout are discussed.Abbreviations P _aCO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen tension - P _da dorsal aortic pressure - pHa arterial pH - P _wO₂ water oxygen tension - RBC red blood cell - V _f breathing frequency     

Cloning the cDNA encoding the AmbtV allergen from giant ragweed (Ambrosia trifida) pollen.

Ragweed (Ambrosia) pollens contain a number of proteins that cause allergic disease in ragweed-sensitive people. The cloning of the AmbtV cDNA is important, since the 4.4-kDa AmbtV, one of the allergens in giant ragweed (Ambrosia trifida) pollen, serves as a simple model system to study the basic structural requirements for immune recognition of foreign protein allergens. We report the cloning of the AmbtV cDNA by means of the polymerase chain reaction (PCR) using degenerate primers. We generated three sets of overlapping cDNA clones by a combination of PCR and anchored-PCR, and determined the complete nucleotide (nt) sequence. From the nt sequence, the amino acid (aa) sequence of the protein was confirmed and the leader sequence was deduced. This general approach can be used to clone allergen and other cDNAs from complex biological sources provided partial aa sequence information is available. It may be the best available approach in cases where the isolation of clones from a cDNA library is difficult, which proved to be the case for AmbtV.     

The amyloid percursor protein of Alzheimer disease is expressed as a 130 kDa polypeptide in various cultured cell types

The vascular and parenchymal amyloid deposits in Alzheimer disease (AD), normal aging and Down syndrome are mainly composed of a 4 kDa polypeptide (A4), which derives from a larger precursor protein (APP). There is evidence that APP is a transmembrane glycoprotein present in most tissues, but the characteristics of APP in intact cells are not yet known. In order to investigate this issue, we examined the immunoreactivity of fibroblasts of human and nonhuman cell lines with antisera raised to synthetic peptides corresponding to A4 and to two other domains of the APP. All three antisera recognized a 130 kDa polypeptide (APP-130) in immunoblots from all cell lines. In fibroblasts, an additional polypeptide of 228 kDa (APP-228) was recognized by the antiserum to A4. In immunoblots of two dimensional gels, APP-130 showed a pI of 6.2, while APP-228 failed to focus in the pH range of 4.7-7.0. Sequential extractions of cells with buffer and with Triton X-100 indicate that APP-130 is extractable with nonionic detergents at high ionic strength, whereas 228 kDa APP is a cystolic component. Immunofluorescence staining is consistent with an intracellular perinuclear and plasma membrane localization. It is concluded that APP-130 and APP-228 are two forms of the APP which result from extensive posttranslational modifications of a smaller original gene product. It is likely that APP undergoes similar posttranslational modifications in different cell types.     

The interaction of troponin-I with the N-terminal region of actin

The interaction between troponin-I and actin that underlies thin-filament regulation in striated muscle has been studied using proton magnetic resonance spectroscopy. A restricted portion of skeletal muscle troponin-I (residues 96-116) has previously been shown to be capable of inhibiting the MgATPase activity of actomyosin in a manner enhanced by tropomyosin [Syska et al. (1976) Biochem. J. 153, 375-387]. On the basis of homologous spectral effects for signals of specific groups observed in different complexes formed using the native proteins and a variety of defined peptides, it is concluded that the segment of troponin-I which has inhibitory activity interacts with the N-terminal region of actin. The surface of contact of the inhibitory segment of troponin-I with actin involves two regions of the N-terminal of actin. These are located between residues 1-7 and 19-44. The data are discussed in the context of a structural mechanism for the inhibition of myosin ATPase activation.     

Structural analysis of the specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72)

The specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72) was found to be a high molecular weight polymer composed of D-galactose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-L-fucose, L-rhamnose, glycerol, and phosphate (2:1:1:1:1:1:1). Partial hydrolysis, dephosphorylation, methylation analysis, periodate oxidation studies, and one- and two-dimensional 1H and 13C high-field nuclear magnetic resonance experiments showed the polysaccharide to be a branched polymer of a 1-phosphoglycerol-substituted hexasaccharide repeating unit having the structure: (formula; see text).