Cellular compartmentation of ureide biogenesis in root nodules of cowpea (Vigna unguiculata (L.) Walp.)

Cowpea (Vigna unguiculata (L.) Walp.) nodules have been investigated by means of cytochemical and immunocytochemical procedures at the ultrastructural level in order to assess the role of the uninfected cells in ureide biogenesis. Uricase activity in the nodules was shown by cytochemical methods to be localized exclusively in the numberous large peroxisomes confined to the uninfected cells; the small peroxisomes in the infected cells did not stain for uricase. Uricase was also localized in the peroxisomes of uninfected cells by immunogold techniques employing polyclonal antibodies against nodule-specific uricase of soybean. There was no labeling above background of any structures in the infected cells. The results indicate that the uninfected cells are essential for ureide biogenesis in cowpea. Although tubular endoplasmic reticulum, the presumptive site of allantoinase, increases greatly in the uninfected cells during nodule development, it virtually disappears as the nodules mature. The inconsistency between the disappearance of the tubular endoplasmic reticulum from older nodules and the high allantoinase activity reported for older plants remains to be explained.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum - GARG goat anti-rabbit immunoglobulin G - IgG immunoglobulin G - kDa knodalton - Mr apparent molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Steroids and related products. L. The synthesis of 17-methoxymethylprogesterone

A new progesterone analogue, 17-methoxymethylprogesterone, was synthesized from pregnenolone by two pathways, one involving as intermediate a 17-hydroxymethylated adduct, the other one by methoxymethylation of a 17,20-lithium enolate with bromomethoxymethane. The product shows significant progestational activity.     

Detection of oxovanadium(IV) and characterization of its ligand environment in subcellular fractions of the liver of rats treated with pentavalent vanadium(V)

Tetravalent oxovanadium(IV) was detected in subcellular fractions of liver by ESR spectroscopy after i.p. injection of pentavalent vanadium(V) as sodium vanadate into rats for three days. This indicates that the metal ion was reduced from the pentavalent state to oxovanadium(IV). The ligand environment around this oxovanadium center was characterized using ESR parameters (g_o and A_o) and the empirical bonding coefficients calculated from the ESR parameters. These values indicate that most of the ligand atoms around the oxovanadium(IV) are oxygens and that the metal may exist in a protein-bound form.     

Cell-free synthesis of Chironomus thummi (Diptera) globins

RNA isolated from Chironomus thummi (Diptera) larvae directs the incorporation of amino acid into newly synthesized products in a cell-free translation system prepared from wheat germ. A fraction of the total cell-free product was specifically immunoprecipitable with antibody against total C. thummi hemoglobin. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the immunoreactive material revealed the cell-free product to have an apparent molecular mass approximately 3000 daltons greater than secreted C. thummi globin purified from hemolymph. In contrast, analysis of the immunoreactive material by polyacrylamide gel electrophoresis under nondenaturing conditions indicated several chemically distinct globins to be present in the cell-free immunoreactive products. These results provide evidence suggesting the possible existence of a preglobin and the data further provide the initial foundation required for elucidating the regulatory mechanisms that control the developmental stage-specific expression of the globin genes in C. thummi.     

An improved synthesis of thyrotropin releasing hormone (TRH) and crystallization of the tartrate

An improved synthesis of thyrotropin releasing hormone (TRH), pGlu-His-Pro-NH₂, is reported. Z-pGlu-ONB (N-hydroxy-5-norbornene-2,3-dicarboximide ester) was reacted with H-His-OH to yield a crystalline Z-pGlu-His-OH which was coupled with H-Pro-NH₂ by the $\text{HONB}\text{DCC}$ method to give Z-pGlu-His-Pro-NH₂ as a fine crystal. Hydrogenation of this protected tripeptide yielded pure TRH nearly quantitatively. The optical purity of TRH thus obtained was confirmed by the method L- and D- amino acid oxidase digestion. The crystallization of TRH was achieved as a tartrate, and the properties of the crystalline TRH-tartrate are described.     

Properties and subcellular localization of adenosine diphosphatase in rat heart

Some properties and subcellular localization of adenosine diphosphatase (ADPase) activity from rat heart have been investigated. The pH optimum was 7.4, maximal activity was found with 5 mM MgCl2, and the apparent Km was 20 microM. ADPase activity was strongly inhibited by NaF and AppNHp, and to a lesser extent by AMP and GppNHp. The enzyme was not inhibited by p-nitrophenylphosphate, beta-glycerophosphate, or pyridoxal phosphate. The distribution of ADPase activity in subcellular fractions obtained by differential centrifugation parallel ouabain-sensitive (Na+-K+)ATPase and 5'-nucleotidase activities, suggesting a plasma membrane-bound localization. The functional significance of ADPase in adenosine production and hemostasis is discussed.     

Quantitative studies on brown algal phenols. II. Seasonal variation in polyphenol content of Ascophyllum nodosum (L.) Le Jol. and Fucus vesiculosus (L.)

The content of extractable polyphenols in the brown algae Ascophyllum nodosum (L.) Le Jol. and Fucus vesiculosus (L.) was measured at ≈28-day intervals for one year. Colorimetric methods based on the Folin-Denis, Brentamine, and vanillin-H₂SO₄ reactions were used to estimate relative contents of polyphenols, and these values were converted to absolute contents using the gravimetric method introduced earlier. Relatively little error was introduced by variations in the qualitative composition of the extracted polyphloroglucinols.There appeared to be a significant temporal correlation between polyphenol content and the reproductive state of the algae. The content of polyphenols in A. nodosum was at a minimum (≈9–10% of dry matter) during the period of maximum fruit body shedding (late May), and reached a maximum (≈12–14% of dry matter) during the ‘winter season’. In F. vesiculosus, the minimum (≈8–10% of dry matter) was one to two months later, just before the period of maximum fertility, and thereafter rose to a maximum (≈11–13% of dry matter) during the period of sterility. These results furthermore suggest that the bulk of the polyphenols are not readily accessible as reserve components, and indicate that modifications may be needed in the ‘chemical defense’ and ‘waste product’ hypotheses concerning the significance of brown algal polyphenols.     

Thermodynamics of base interaction in (A)n and (A.U)n

Using precision scanning microcalorimetry we studied (A)_n and (A·U)_n melting in highly diluted solutions (0.3 to 5.0 mm) with different Na⁺ activity. This permitted us to determine directly the thermodynamic functions of stacking interaction in (A)_n and base-pairing in (A·U)_n. For (A-A) stacking at (A)_n melting temperature we obtained ΔH^(A)n_m = 12.6 kJ mol^?1; ΔS^(A)n_m = 41 J K^?1 mol^?1. For A·U base-pairing at a standard temperature of 298 K and 0.1 m-Na⁺ we have: ΔH^(A·U) = 34 kJ mol^?1; ΔS^(A·U) = 102 J K^?1 mol^?1ΔG^(A·U) = ?3.5 kJ mol^?1.     

Modulation of platelet-activating factor (PAF) synthesis and release from human polymorphonuclear leukocytes (PMN): role of extracellular Ca2+

Extracellular Ca2+ regulated the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear leukocytes (PMN) stimulated with N'-formyl-methionyl-leucyl-phenylalanine (FMLP) in the presence of cytochalasin B. Maximum PAF synthesis and release required the presence of 0.14 mM Ca2+ whereas 1.4 mM Ca2+ was necessary for maximum lysosomal enzyme secretion. The synthesis of PAF occurred within 2.5 min after PMN stimulation in the presence of 1.4 mM Ca2+; however, PAF release did not occur until 5 min after stimulation. Peak PAF release occurred by 7.5 min but accounted for only 30-40% of the total amount of PAF synthesized, the remainder being retained on or within the PMN. Stimulation of PMN in the presence of 0.01 M EDTA or EGTA decreased PAF synthesis and release by greater than 95%. In the absence of extracellular Ca2+, stimulated PMN synthesized PAF in amounts that were 10-30% of maximum, but there was no release of the newly synthesized PAF. At Ca2+ concentrations greater than 0.01 mM, there was a dose-dependent (up to 0.14 mM) increase in PAF synthesis that was associated with the initiation and concomitant increase in the amount of PAF released. These data suggest the presence of a PAF synthesis-release coupling mechanism in which the extracellular Ca2+-dependent release of PAF stimulates additional PAF synthesis.     

Hemoglobin Constant Spring: hemoglobin synthesis in heterozygous and homozygous states.

Thirteen adult and one newborn heterozygotes, and three homozygotes for hemoglobin Constant Spring were examined for globin chain synthesis. Reticulocytes from venous blood were incorporated with [³H]-leucine in an incubation mixture for 3 hours. Globin prepared from the radioactive, washed red cells was fractionated by CM-cellulose chromatography in 8 M urea and the total radioactivity of each globin chain was determined. The mean of $\text{α}\text{β}$ ratio in the heterozygotes was 1.34 ± SD 0.08, which is significantly different from that of 1.07 ± SD 0.03 in eleven normal controls. The $\text{α}\text{β+γ}$ ratio in the heterozygous neonate was also 1.39. The $\text{α}\text{β}$ ratios in the three homozygotes were around 1.6. The α-Constant Spring chain appears to be over produced, but it may be unstable or labile, not fully available for conjugation with the non alpha chains.     

The subcellular localization of calmodulin, cyclic AMP phosphodiesterase, and adenylate cyclase in bovine adrenal medulla

The subcellular localization of calmodulin, cyclic nucleotide phosphodiesterase, and adenylate cyclase was studied in bovine adrenal medulla. Approximately 70% of the calmodulin and 90% of the cAMP phosphodiesterase activities were found colocalized in the cytoplasm. The subcellular distribution of adenylate cyclase closely paralleled the distribution of acetylcholinesterase, a marker for plasma membranes. The fraction of calmodulin which is particulate in nature has a distribution profile very similar to that of adenylate cyclase. The chromaffin granule fraction contained only 0.86% of the total cAMP phosphodiesterase, 0.41% of the total adenylate cyclase, and 1.4% of the total calmodulin.     

Defective interfering particles of poliovirus: mapping of the deletion and evidence that the deletions in the genomes of DI(1), (2) and (3) are located in the same region.

The deletions in RNAs of three defective interfering (DI) particles of poliovirus type 1 have been located and their approximate extent determined by three methods. (1) Digestion with RNase III of DI RNAs yields the same 3′-terminal fragments as digestion with RNase III of standard virus RNA. The longest 3′-terminal fragment has a molecular weight of 1.55 × 10⁶. This suggests that the deletions are located in the 5′-terminal half of the polio genome. (2) Fingerprints of RNase T₁-resistant oligonucleotides of all three DI RNAs are identical and lack four large oligonucleotides as compared to the fingerprints of standard virus, an observation suggesting that the deletions in all three DI RNAs are located in the same region of the viral genome. The deletion-specific oligonucleotides have also been shown to be within the 5′-terminal half of the viral genome by alkali fragmentation of the RNA and fingerprinting poly (A)-linked (3′-terminal) fragments of decreasing size. (3) Virion RNA of DI(2) particles was annealed with denatured double-stranded RNA (RF) of standard virus and the hybrid heteroduplex molecules examined in the electron microscope. A single loop, approximately 900 nucleotides long and 20% from one end of the molecules, was observed. Both the size and extent of individual deletions is somewhat variable in different heteroduplex molecules, an observation suggesting heterogeneity in the size of the deletion in RNA of the DI(2) population. Our data show that the DI RNAs of poliovirus contain an internal deletion in that region of the viral genome known to specify the capsid polypeptides. This result provides an explanation as to why poliovirus DI particles are unable to synthesize viral coat proteins.     

Evidence for peptidoglycan-associated protein(s) in Neisseria gonorrhoeae.

Growth pH markedly influenced the composition of the cell envelope of $\text{Neisseria gonorrhoeae}$. The composition of the peptidoglycan from cells grown at pH 7.2 and 8.0 consisted primarily (91%) of muramic acid, glutamic acid, alanine, $\text{meso}$-diaminopimelic acid, and glucosamine in approximate molar ratios of 1:1:2:1:1. The peptidoglycan from cells grown at pH 6.0 contained an accessory protein(s) which accounted for 42% of the weight of the isolated complex.     

Ferric leghemoglobin reductase from soybean root nodules

An NADH: (acceptor) oxidoreductase from the cytosol of soybean root nodules was purified by ammonium sulfate fractionation, hydroxylapatite adsorption, and Sephacryl S-200 Superfine chromatography. The native molecular weight of the reductase was found to be 100,000 by analytical gel filtration and 83,000 by equilibrium ultracentrifugation. The subunit molecular weight was 54,000 as determined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The pI of the enzyme was 5.5. With ferric leghemoglobin (Lb) as the substrate, nearly identical initial velocities were obtained using either CO or O2 to ligate the enzymatically produced ferrous leghemoglobin. With CO as the ligand in the reaction, the product of the enzyme-catalyzed, NADH-dependent reduction of ferric Lb was spectrally identified as LbCO. Initial velocity was a linear function of increasing enzyme concentration. NADPH was only 31% as effective an electron donor as NADH as determined by initial velocity. The Michaelis constants (Km) for ferric Lba and NADH were 9.5 and 18.8 microM, respectively. Myoglobin, Lba, Lbc1, Lbc2, Lbc3, and Lbd were reduced at similar rates by the reductase. At pH 5.2, acetate-bound ferric Lb and nicotinate-bound ferric Lb were reduced by the enzyme at 83 and 5%, respectively, of rates observed in the absence of these ligands. The rate of enzymatic reduction of ferric Lb was constant between pH 6.5 and 7.6 but increased approximately threefold at pH 5.2. The results indicate that the NADH: (acceptor) oxidoreductase could be identified as a ferric Lb reductase.     

Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

Inducibility of 6-thioguanine-resistant (6TGr) mutants and single-strand scission of DNA by cadmium chloride (CdCl2) was investigated in cultured Chinese hamster V79 cells. Frequency of 6TGr mutants increased concentration dependently by 24-h treatment with CdCl2 up to 3 X 10(-6) M but decreased beyond 3 X 10(-6) M. Mutagenic potency of cadmium in the absence of S9 was about half that of benzo[a]pyrene in the presence of S9 at equitoxic concentrations. Treatment of the cultured cells with cadmium after benzo[a]pyrene treatment was not synergistic but additive to the mutagenicity of benzo[a]pyrene. Single-strand scission of DNA by alkaline elution techniques was observed in the cells treated with CdCl2 for 2 h in a concentration-dependent manner. The single-strand scission by cadmium was detected only in combination with proteinase K digestion of the cell lysates, indicating formation of DNA--protein cross-linking by the metal. These biological and biochemical findings indicate that cadmium is mutagenic in mammalian cells, and its mutagenic effect seems to be accompanied by single-strand scission of DNA.     

The conversion of benzo(alpha)pyrene 4,5-oxide into 4-hydroxybenzo(alpha)pyrene in the presence of polyriboguanylic acid.

Incubation of benzo[alpha] pyrene 4,5-oxide with poly(G) in neutral aqueous ethanol resulted in the formation of covalent adducts and in the production of free 4-hydroxybenzo[alpha]pyrene. This phenol, which was identified by its UV spectral properties and by its chromatographic characteristics, was also formed but at a much slower rate when the epoxide was incubated with DNA or with GMP. Phenol formation was not detected when benzo[alpha]-pyrene 4,5-oxide was incubated for prolonged periods in the presence of poly(A), poly(C) or poly(U) or in the absence of nucleic acid. Formation of 4-hydroxybenzo[alpha] pyrene from the epoxide in the presence of poly(G) was not accompanied by detectable base modifications or by breakage of phosphodiester linkages.     

Chemical composition and ultrastructure of the epicuticular wax in four mutants of Pisum sativum (L)

The action of mutations affecting the epicuticular wax of Pisum sativum has been investigated at the chemical and ultrastructural level. Upper and lower surfaces of the leaves were found to differ markedly in both ultrastructure and chemistry. Mutations affected primarily either the lower (wa, wb and wsp) or the upper surface (wlo), but some effects of all 4 genes could be seen on both surfaces. Specific biochemical lesions could be implied for wsp and wa but the chemical effects of wb and wlo were more diffuse. Generally a close relation between chemical composition and crystallite form of the wax was evident throughout the work.