In vitro effect of various mitogens on starfish (Asterias rubens) axial organ cells

The effects of various mitogens on axial organ (AO) cells of the sea star have been studied. Pokeweed mitogen (PWM) stimulates [³H]thymidine incorporation by the whole population of axial organ cells of the sea star. This effect occurs 24 hr after the addition of PWM and is maximal at 40 μg/ml. In contrast, no stimulation is observed when coelomocytes are treated with PWM under the same conditions. No stimulation of the whole AO cell population is observed in the presence of Con A or LPS. However, the AO cell population can be divided, on the basis of surface adherence properties, into two subpopulations, adherent and nonadherent. Con A stimulates the nonadherent cells, but not the adherent cells: The stimulating effect is maximal 24 hr after addition of Con A and at 0.2–0.5 μg/ml. In contrast, LPS stimulates the adherent but not the nonadherent cells and the stimulating effect is maximal at 24 hr and at 45 μg/ml.     

The production of T-cell growth factor (TCGF) in vivo in sheep

Lymph and supernatants derived from efferent lymphocytes leaving the popliteal lymph nodes of sheep responding to human red cells or dinitrophenylated bovine serum albumin were examined for the presence of T-cell growth factor (TCGF). Efferent cells from normal sheep, but not from antigen-stimulated sheep, were found to release low levels of TCGF when incubated in medium for 12 hr in the absence of any exogenous stimulus. High levels of TCGF were found in normal lymph and also in immune lymph collected from sheep during the first 6 hr of immune responses. There were no detectable levels of TCGF in lymph collected later in the response. The lymphokine appeared to be a single molecular species of 10,000–20,000 molecular weight as assessed by exclusion chromatography. Efferent cells expressing receptors for TCGF were found in efferent lymph during the first 12 hr of the response. The results demonstrate for the first time that TCGF is produced in vivo and that asynchrony exists between TCGF production and expression of receptors for TCGF on efferent cells released by the stimulated node. Based on the known kinetics of previously reported synergistic factors, mitogenic factors, and T-cell-replacing factors in sheep efferent lymph and their physical characteristics it was concluded that the TCGF detected in lymph is distinct from these factors.     

Mutagenicity and cytotoxicity of the carcinogen-mutagen aflatoxin B1 in Streptococcus pneumoniae (Pneumococcus) and Salmonella typhimurium: dependence on DNA repair functions

Strains R6, R6x and R6uvr-1 of Streptococcus pneumoniae (Pneumococcus) are sensitive to the cytotoxic effects of the mutagen/carcinogen aflatoxin B₁ (AFB₁). R6uvr-1 is more prone to the cytotoxic effects of AFB₁ than the repair-proficient parental strain, R6. The same differential susceptibility of strains R6, R6x and R6uvr-1 was observed when UV light replaced metabolically activated AFB₁. All pneumococcal strains were immutable by AFB₁. AFB₁ mutagenesis in Salmonella typhimurium strains was dependent on a functional RecA gene product. The enhancing effects of ΔuvrB and plasmid pKM101 were found to be additive. Data presented are consistent with the following: (i) AFB₁ toxic effects are due mainly to DNA binding of AFB₁; (ii) AFB₁ mutagenesis is dependent on error-prone DNA repair; (iii) Pneumococcus lacks an active error-prone (SOS) DNA-repair system.     

Chemical investigations on the gum exudate from sajna (Moringa oleifera)

The purified, whole-gum exudate from the drum-stick plant (Moringa oleifera) was found to contain l-arabinose, d-galactose, d-glucuronic acid, l-rhamnose, d-mannose, and d-xylose in the molar ratios of ～ 14.5:11.3:3:2:1:1. A homogeneous, degraded-gum polysaccharide consisting of d-galactose, d-glucuronic acid, and d-mannose in the molar ratios of ～ 11.7:3.9:1, was obtained on mild hydrolysis of the whole gum with acid. Permethylation studies were conducted on the whole gum, the degraded gum, and their carboxyl-reduced products, and the results were in good agreement with those obtained from periodate oxidation followed by Smith degradation. Also, isolation and characterization of the oligosaccharides obtained from the mother liquor during preparation of the degraded gum, and by graded hydrolysis of the degraded gum, were achieved. On the basis of the results obtained from these studies, a tentative structure was assigned to the average repeating-unit of the gum.     

Purification and characterization of mojave (Crotalus scutulatus scutulatus) toxin and its subunits

An acidic lethal protein, Mojave toxin, has been isolated from the venom of Crotalus scutulatus scutulatus. The purified toxin had an i.v. LD₅₀ of 0.056 μg/g in white mice. Disc polycrylamide gel electrophoresis at pH values of 9.6 and 3.8 and isoelectric focusing in polyacrylamide gels with a pH 3.5–10 Ampholyte gradient were used to establish the presence of one major protein band. The pI of the most abundant form of the toxin was determined to be 5.5 by polyacrylamide gel isoelectric focusing experiments. The molecular weight was established to be 24,310 daltons from amino acid composition data. Mojave toxin was shown to consist of two subunits, one acidic and one basic with isoelectric point (pI) values of 3.6 and 9.6, respectively. Amino acid analyses established molecular weights of 9593 for the acidic component and 14,673 for the basic component. The acidic subunit consisted of three peptide chains intermolecularly linked by cystine residues. The basic subunit was a single polypeptide chain with six intramolecular disulfide bonds. The basic subunit was lethal to test animals with an intravenous LD₅₀ of 0.58 μg/g. Following recombination of the subunits a recombinant toxin was isolated which was identical to the native toxin by comparisons of electrophoretic mobility and toxicities. Comparisons of circular dichroism spectra also indicated reassociation to the native toxin structure. Phospholytic activity was associated with Mojave toxin and the basic subunit was responsible for this enzymic activity. Phospholipase activity of the basic subunit was inhibited by addition of the acidic subunit.     

Fatty acid composition of the lipids of Puffinus tenuirostris (Temminck) in relation to its diet

The fatty acid composition of lipids isolated from the depot fat, stomach contents, and proventricular oil of adult and chick Puffinus tenuirostris (Temminck) has been analysed. The diet of both adults and chicks is almost exclusively derived from the euphausiid Nyctiphanes australis Sars, and an attempt was made to determine whether dietary lipid affects the composition of depot fat, and whether individual fatty acids in the stomachs and proventricular oil can be used as markers for the origin of the diet. An apparent selectivity in the deposition of fatty acids in the fat depots can be explained by the conversion of fatty alcohols, derived from the euphausiid wax ester, into fatty acids of equivalent chain length and unsaturation. Hexadecadienoic acid appeared to be the only possible marker fatty acid from the euphausiid, but wide variations in its level limits its usefulness as a reliable index of the diet of Puffinus tenuirostris.     

Structural investigations on two hemicellulosic polysaccharides from groundnut (Arachis hypogea) seed endosperm

A highly branched xylan and a linear, β-d-(1→4)-linked glucomannan are the two hemicellulosic components isolated from the endosperms of groundnut (Arachis hypogea). Electrophoretic, sedimentation, and sugar analysis indicate the polysaccharides to be fairly homogeneous. The O-methyl derivatives of the polysaccharides were analysed, after reduction and O-acetylation, by gas-liquid chromatography and g.l.c.-mass spectrometry. 2,3,4-Tri-O-methyl-d-xylose (3.6 mol), 2,3-di-O-methyl-d-xylose (21.0 mol), 3-O-methyl-d-xylose (2.8 mol), and d-xylose (4.2 mol) were detected in the xylan, whereas 2,3,4,6-tetra-O-methyl-d-glucose and/or mannose (1.6 mol), 2,3,6-tri-O-methyl-d-mannose (5.6 mol), and 2,3,6-tri O-methyl-d-glucose (21.2 mol) were found in the glucomannan. Periodate and Smith-degradation studies substantiate the results of methylation analysis on the xylan. A glucose: mannose ratio of 3:1 for the glucomannan, however, suggests that this fraction may be an aggregate of true glucomannan and glucan or degraded cellulose.     

Structural features of two amyloids from the hemi-cellulosic fraction of field-bean (Dolichos lablab) hulls

Two amyloid-type fractions were isolated from field-bean (Dolichos lablab) hulls by 10% alkali extraction followed by acetylation and solvent fractionation. The major, chloroform-insoluble fraction and a minor, chloroform-soluble fraction were found to be homogeneous in sedimentation analysis and molecular-sieve chromatography. The polysaccharides contained xylose and glucose in various proportions. Methylation analysis, periodate oxidation, Smith degradation, oxidation by chromium trioxide, and oligosaccharide studies indicated a new type of structure for the major fraction (glucose:xylose ratio of 1.9:1) in that it had a backbone of (1→4)-linked β-d-glucose residues interspersed with single or multiple residues of (1→4)-linked β-d-xylose, and to which some single d-xylosyl groups are attached through O-6 of d-glucose. In contrast, the minor fraction (glucose:xylose ratio of 1:3.7) had a backbone of (1→4)-linked β-d-xylose interspersed with (1→4)-β-d-glucose and having a side chain of d-xylose, attached through O-6 of d-glucose. The third fraction was found to be a mixture of linear (1→4)-d-glucan and (1→4)-d-xylan.     

Environmental regulation of female calling and male pheromone response periodicities in the codling moth (Laspeyresia pomonella)

Codling moth female calling and male pheromone responsiveness under the defined conditions of 23°C and light:dark (LD) 16:8 occurred primarily during scotophase. Under either continuous photophase or scotophase females called with periodicities very similar to their periodicity under the LD cycle, indicating that the rhythmicity is circadian. Male response rhythmicity was maintained under continuous photophase. A decrease in the temperature from 23° to 16°C resulted in a reduction in the proportion calling when the decrease in temperature occurred during scotophase and a shift of maximal calling into photophase when the decrease in temperature occurred 3 hr prior to the initiation of scotophase. Decreases of temperature from 23° to 16°C and of light intensity did not produce similar shifts in the periodicity of male upwind orientation. Of 6 pheromone dosages from 10^?5 to 10² μg, 10^?1 and 10⁰ μg elicited the most male upwind orientation.     

Complete turnover of poly(A) on maternal mRNA of sea urchin embryos

Measurement of the incorporation of radioactive adenosine into precursor pools and into poly(A) of fertilized sea urchin eggs showed that the amount of adenosine incoporated into poly(A) after a 2 hr incubation approximated the total poly(A) content of the embryos. This was observed whether the incubation was begun at fertilization when the poly(A) content is tripling or at 2.5 hr after fertilization when the poly(A) levels are not changing, and thus indicates that poly(A) turns over continually and completely. The turnover appears to take place on polysomal mRNA, since after either 10 or 120 min of incubation, 75% of the ³H-adenosine incorporated into poly(A) is on polysomes. Poly(A) lengths before and after fertilization are not significantly different, indicating that the increase in poly(A) content reflects the addition of poly(A) sequences onto mRNA molecules which previously contained no poly(A) sequences or only short poly(A) sequences. Both the new as well as the preexisting poly(A) tracts must turn over to produce the incorporation we observe. The radioactive poly(A) tracts measured by alkaline release of adenosine begin as short sequences and gradually extend their lengths until they have reached a size consistent with the idea that the poly(A) sequences have become fully radioactive. This labeling pattern shows that the poly(A) is turning over from the 3′ end terminal probably by a shortening and lengthening mechanism.     

Semiquantitative paper chromatographic separation of chlorophylls a and b

[¹⁴C]Chlorophyll (chl) a has been utilized to demonstrate the contamination of chl b by (probably) oxidation products of chl a in thin-layer or paper chromatography. By circular chromatography of both chlorophylls as their pheophytins, the contamination of chl a (as pheophytin a) in chl b (as pheophytin b) may be reduced to 0.15–0.35.     

Purification and physicochemical properties of an extra-cellular cycloamylose (cyclodextrin) glucanotransferase from Bacillus macerans

An extracellular cycloamylose (cyclodextrin) glucanotransferase (EC 2.4.1.19) from Bacillus macerans was purified to homogeneity by adsorption on starch, ammonium sulfate fractionation, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of 67,000 and consisted of one polypeptide chain. The isoelectric point was pH 5.4. Temperature and pH optima were 60° and 5.45.8, respectively. The purified enzyme was quite stable at 50° (pH 6.0), but lost ≈80% of its activity at 60° for 30 min (pH 6.0). Prolonged digestion by trypsin did not affect the catalytic properties of the enzyme. The K_m for starch was 5.7 mg/ml.     

Regulation of C4 photosynthesis: Physical and kinetic properties of active (dithiol) and inactive (disulfide) NADP-malate dehydrogenase from Zea mays

NADP-malate dehydrogenase was purified from leaves of Zea mays in the absence of thiol-reducing agents by (NH₄)₂SO₄, polyethylene glycol, and pH fractionation followed by dye-ligand affinity chromatography and gel filtration. The purified enzyme is completely inactive (no activity detected between pH 6 and 9) but can be reactivated by thiol-reducing agents including dithiothreitol and thioredoxin. The active enzyme shows distinctly alkaline pH optima when assayed in either direction; K_m values at pH 8.5 are oxaloacetate, 18 μm; malate, 24 mm; NADPH, 50 μm; and NADP, 45 μm. The reduction of oxaloacetate is inhibited by NADP (competitive with respect to NADPH, K_i = 50 μm). The molecular weight of the native inactive or active enzyme is 150,000 with subunits of M_r 38,000. Active enzyme is much more sensitive (>50-fold) to heat denaturation than is the inactive enzyme and is irreversibly inactivated by N-ethylmaleimide whereas the inactive enzyme is insensitive to this reagent. The active and inactive forms of NADP-malate dehydrogenase are assumed to correspond to dithiol and disulfide forms of the enzyme, respectively. The relative coenzyme-binding affinities of inactive NADP-malate dehydrogenase differ by a factor of 10² from the binding affinities for active NADP-malate dehydrogenase and 10⁴ for non-thiol-regulated NAD-specific malate dehydrogenase. It is proposed that the 100-fold change in differential binding of NADP and NADPH upon conversion of NADP-malate dehydrogenase to the disulfide form may sufficiently alter the equilibrium of the central enzyme-substrate complexes, and hence the catalytic efficiency of the enzyme, to explain the associated loss of activity.     

Conformation of the antifreeze glycoprotein of polar fish

High-field proton and 13C NMR spectroscopy has been used to test and refine the recent proposal, based on vacuum uv circular dichroism results, of a threefold left-handed helical conformation for antifreeze glycoprotein (AFGP). Partial assignment of the protons of the glycotripeptide repeating unit has been made by comparison with spectra of model compounds, by selective decoupling, and by measurements of nuclear Overhauser effect (nOe). At 40 degrees C, AFGP fraction 8 (Mr 2600) shows 2-Hz linewidths which broaden at lower temperature. Neither 1H nor 13C chemical shifts depend strongly on temperature, suggesting no abrupt conformational transition. The nOe between alanine alpha and beta protons vary with temperature and with field strength, from small positive enhancements at 50 degrees C and 80 MHz to large negative effects at 3 degrees C and 300 MHz, indicating a substantial change of rotational correlation time with temperature. The higher-molecular-weight fraction 1-4 shows negative nOe at all temperatures. The CD spectra of fraction 1-4 show bands characteristic of the polyproline II structure at both 3 and 50 degrees C, while those bands in fraction 8 are weaker at 50 than 3 degrees C. The 1H nOe, the 13C T1, and CD data are interpreted as indicating that AFGP fraction 8 is an extended "rod-like" conformation at low temperature which becomes a flexible coil at high temperature, while fraction 1-4 is a flexible rod with sufficient segmental mobility to eliminate any long-range order.     

Synthesis and characterization of lipid-linked mannosyl oligosaccharides in Volvox carteri f. nagariensis

Particulate membrane fractions from Volvox carteri catalyze the transfer of mannose from GDP-mannose to dolichyl diphosphate-[¹⁴C]chitobiose to form lipid-linked oligosaccharides up to a dolichyl diphospnate-chitobiose-(mannose)₅ structure. Mannosylation of the chitobiosyl lipid requires divalent cations and detergents as solubilizing agents. Depending on the nature of the detergent, the oligosaccharide pattern differs markedly: With deoxycholate or the zwitterionic detergent 314 a lipid-linked trisaccharide accumulates. The nonionic Triton X-100, however, gives rise to a spectrum of compounds up to a heptasaccharide. Enzyme digestion of the tri- and pentasaccharide structure, obtained after mild acid hydrolysis of the corresponding [¹⁴C]glycolipids, revealed that the first mannose is bound via a β-glycosidic linkage to the chitobiosyl core, whereas the outer mannose residues are linked as α-mannosides. Our studies indicate that, in agreement with recent findings in other organisms, the innermost α-mannosidic residues are donated directly from GDP-mannose. The structure of oligosaccharides synthesized by Volvox membranes is thus consistent with results from other eucaryotic species, suggesting a common pathway of N-glycosylation of glycoproteins.     

In Vivo and in Vitro binding of platinum to metallothionein

The in vivo binding of platinum to metallothionein (MT) has been observed in rat tissues following injections of the cis and trans isomers of DDP (dichlorodiammine-platinum(II)). Platinum in either cis-DDP or trans-DDP does not directly induce MT; platinum-MT is produced by the replacement of previously bound zinc in the protein. The binding of Pt(II) to MT depends on the availability of SH groups in MT. Preinjection with CdCl₂ significantly enhances the association of Pt(II) with MT fractions compared to the degree of association resulting from injections with either cis-DDP or trans-DDP without CdCl₂ pretreatment. In vitro experiments in which tissue extracts including a known (Cd,Zn)-MT were incubated with either cis-DDP or trans-DDP show that these isomers differ with respect to the transfer of Pt to MT; the equilibrium in both cases was reached when approximately 40% of the available Pt is bound to MT but with this equilibrium value attained in 2 h in the case of trans-DDP and only after 72 h in the case of cis-DDP. Pt-MTs were also formed by a series of incubation steps in which a native MT was used to prepare the apoprotein which was subsequently incubated with either cis-DDP or trans-DDP. Spectrophotometry established that a shoulder occurs at 285 nm for the Pt-MTs resulting from the incubation with either isomer. A competitive double-antibody radioimmunoassay for MT demonstrated that these Pt-MTs had complete cross-reactivity with a native (Cd,Zn)-MT. Gel filtration of tissue extracts after either in vivo or in vitro treatment with DDP showed that Pt was bound to a molecular species with properties characteristic of MT. These results were verified by atomic absorption spectrophotometry and polyacrylamide gel electrophoresis assays.     

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.     

Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and Black mutant Manduca sexta larvae

The absence of juvenile hormone at the time of head cap slippage during the last-larval moult of the tabacco hornworm, Manduca sexta, causes deposition of premelanin granules into the outer regions of the newly forming endocuticle beginning 13 h later. These granules were found to contain an inactive phenoloxidase which becomes activated about 9 h later, 4 h before body melanization begins. The onset of melanization was not accelerated by melanization and reddish colouration hormone from Bombyx heads, extracts of pharate-adult corpora cardiaca or pharate-larval ventral nerve cords (sources of eclosion hormone), or extracts of pharate-larval suboesophageal ganglia or corpora cardiaca-corpora allata complexes. Instead the fall of the ecdysteroid titre to below 250 ng/ml 20-hydroxyecdysone equivalents appeared to be the cue that allowed melanization about 4.5 h later. Up to, but not after, this time both melanization and ecdysis could be delayed by exogenous 20-hydroxyecdysone in a dose-dependent fashion above 0.1 μg per larva. In vitro studies published elsewhere indicate that 20-hydroxyecdysone prevents the activation of the premelanin granules. Thus the granules can be deposited at the proper time in the newly forming endocuticle but their melanization is regulated by the declining ecdysteroid titre and it thus synchronized with other events occurring just before ecdysis.