Circular dichroic study of conformational changes in ovalbumin

By simulation of the circular dichroic spectra (Greenfield and Fasman (1969)) and using reference spectra of Chen et al. (1974), native ovalbumin was estimated to contain 33% -helix, 5% -structure, and 62% random coil. Ovalbumin resisted conformational changes in solutions of urea and of SDS. However, guanidine induced transition, starting at about 2 M and completing at about 4.5 M. At concentrations exceeding 4.5 M guanidine, ovalbumin existed as 6–7% -helical, 12–13% -structure, and 80–81% random coil. Ovalbumin after denaturation in 6 M guanidine or in 8 M urea (incubated at 4°C for 24 hr) did not recover the native conformation but acquired a new conformation in each case, with a somewhat destabilized helical structure.Abbreviation used CD circular dichroism - SDS sodium dodecyl sulfate     

Helix formation in reduced, S-carboxymethylated human choriogonadotropin beta subunit and tryptic peptides

The subunit of human choriogonadotropin (hCG) and its asialoderivative were digested with trypsin and then reduced and S-carboxymethylated. A series of peptides were purified which corresponded to residues 1–43, 44–95, 96–114, and 123–145 of the 145 amino acid residue glycoprotein. The two N-linked oligosaccharides were present on the amino terminal peptide, and three of the four O-linked oligosaccharides were present on the carboxy terminal peptide. Circular dichroic spectra between 190–240 nm were obtained on reduced, S-carboxymethylated (RCM) hCG and the above peptides, both in aqueous solution and in the helicogenic solvent 80% (vol/vol) trifluoroethanol (TFE). In aqueous solution there was evidence of only limited helicity in the peptides and RCM-hCG however, in the presence of TFE, peptides 1–43 and 44–95 exhibited significant helicity, as did the full-length linear chain. The helicity developed in TFE by RCM-hCG appears much greater than that which occurs in the native, disulfide-intact form, thus suggesting that the disulfides prevent expression of helicity in regions with -helix potential. Application of the Chou-Fasman secondary structure predictive algorithm to hCG suggested that several regions of helix potential, in particular regions 14–21, 59–69, and perhaps 80–88, may account for much of the helicity observed in peptides 1–43 and 44–95, respectively, in TFE. The region from 96–145 has no significant potential for helicity, consistent with the measured circular dichroic spectra of peptides 96–114 and 123–145. These results demonstrate that helicity can occur in the linear form of hCG, and this secondary structure can best be attributed to the amino terminal and the middle portion of the molecule. Several potential regions of -structure and -turns were also suggested.     

Enzymatic fragmentation of tetanus toxin. Identification and characterization of an atoxic,immunogenic fragment

Summary Purified filtrate tetanus toxin was subjected to limited digestion with papain and the resulting fragments were separated by gel exclusion chromatography and characterized. One atoxic fragment was shown to react with antiserum against tetanus toxoid and was capable of inducing antibodies in rabbits that neutralized native tetanus toxin. The fragment had an estimated molecular weight of 56,000 by SDS polyacrylamide gel electrophoresis and 62,000 by sedimentation equilibrium. In the presence of a reducing agent, the fragment yielded two components with approximatec molecular weights of 23,000 and 32,000. Thus, it appears that the atoxic, immunogenic fragment is composed of two peptides joined by at least one disulfide bond. The fragment was examined by circular dichroism and data analysis indicated the presence of considerable -structure, but little, if any, -helicity. This is significantly different from the estimates for filtrate toxin, 29% -helicity and 23% -structure. Above 250 nm, the circular dichroic spectrum of the fragment was also distinct from that of intact toxin.Portions of this workwere presented at the 1977 Federation Meeting, Chicago, April 4, 1977 (Fed. Proc. 36, 2099, 1977).Recipient of a Research Career Development Award AM-00055.     

Structural analysis of botulinum neurotoxin types A and E in aqueous and nonpolar solvents by Fourier transform infrared, second derivative UV absorption, and circular dichroic spectroscopies

Two pharmacologically similar but antigenically distinct botulinum neurotoxins, types A and E with a 1000-fold difference in their toxicity, were examined for nonpolar solvent-induced changes in secondary structures and polypeptide foldings to understand their structural differences and their comparative responsiveness/susceptibility to solvent perturbation. Analysis of far UV circular dichroic spectra in aqueous buffer for types A and E neurotoxins yielded the following: the -helix contents were 27 and 20%; the -sheets were 36 and 44%, the -turns were 6.0 and 0%, and the random coils were 31 and 36%, respectively. Fourier transform infrared spectra, obtained by using attenuated total reflection technique, indicated high content of -helix and -pleated sheet structures for both neurotoxins as judged by strong bands at 1651 and 1633 cm^–1 in the amide I frequency region and bands at 1314 and 1245 cm^–1 in the amide III frequency region. The peak height ratio of 1314 and 1245 cm^–1 bands, suggests that the type A neurotoxin has slightly higher -helical content than the type E neurotoxin. These observations are consistent with the secondary structures estimated from far UV circular dichroic spectra. Fourier transform infrared spectra of the neurotoxins, exposed to methanol, showed sharp increases of the 1651 cm^–1 band and a significant increase in the height of the 1314 cm^–1 band, suggesting increases in the -helical contents of the proteins. The changes were more in the type A than in the type E neurotoxin. The changes were reversible upon reexposure of the proteins to the aqueous buffer. Second derivative absorption spectroscopy demonstrated that methanol also induced changes in the degree of Tyr exposure to solvent. The results are discussed in terms of structural differences between the single and dichain neurotoxins and in terms of their mode of action.     

Contributions of tryptophan side chains to the far-ultraviolet circular dichroism of proteins

It has often been assumed that the role of aromatic side chains in the far-ultraviolet region of protein circular dichroism (CD) is negligible. However, some proteins have positive CD bands in the 220–230 nm region which are almost certainly due to aromatic side chains. The contributions to the CD of interactions between tryptophan side chains and the nearest neighbor peptide groups have been studied, focusing on the indole B_b transition which occurs near 220 nm. Calculations on idealized peptide conformations show that the CD depends strongly on both backbone and side-chain conformation. Because of the low symmetry of indole, rotation about the CC bond (dihedral angle ₂) by 180° generally leads to large changes in the CD, often causing the B_b band to reverse sign. When side-chain conformational preferences are taken into account, there is no strong bias for either positive or negative B_b rotational strengths. The observation that simple tryptophan derivatives such as N-acetyl-L-tryptophan methylamide have positive CD near 220 nm implies either that these derivatives prefer the _R region over the region, or that there is little preference for ₂ < 180° over ₂ > 180°. Nearest-neighbor-only calculations on individual tryptophans in 15 globular proteins also reveal a small bias toward positive B_b bands. Rotational strengths of the B_b transition for some conformations can be as large as 1.0 Debye-Bohr magnetons in magnitude, corresponding to maximum molar ellipticities greater than 10⁵ degcm²/dmol. Although a substantial amount of cancellation occurs in most of the examples considered here, such CD contributions could be significant, especially in proteins of low helix content.     

Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

Spectroscopic and binding studies of azide to type-2-copper-depleted ascorbate oxidase from zucchini

Summary Binding of azide to type-2-copper-depleted (T2D) zucchini ascorbate oxidase, containing reduced type-3 Cu centers, and met-T2D ascorbate oxidase, containing oxidized type-3 Cu centers, has been studied spectroscopically. In both cases titration with azide in 0.1 M phosphate pH 6.8 produces a broad near-ultraviolet band with maximum at 455 nm (e 2500 M^–1 cm^–1, with respect to the met-T2D enzyme) and shoulder at 390 nm (e 1700 M^–1 cm^–1), that are assigned to(azide)Cu(II) ligand-to-metal charge transfer (LMCT) transitions. This is accompanied by a reduction of absorbance at 330 nm in the met-T2D) enzyme adduct (e –1400 M^–1 cm^–1). A broad circular dichroic band of negative sign between 370–480 nm corresponds to the LMCT absorption band. Analysis of the titration data indicates that one azide ion binds independently to each of the binuclear T3 Cu couples with low affinity (K = 50 M^–1). The ESR signal of the T1 Cu observed in frozen solutions of the T2D enzyme is also perturbed by the addition of azide. The analogies in the azide-binding characteristics between ascorbate oxidase and laccase are discussed.     

Age and Growth of the Whiskery Shark, Furgaleus macki, from Southwestern Australia

Age and growth of the whiskery shark, Furgaleus macki, from southwestern Australia were examined using vertebral ageing and tag-recapture data. The readability of bands on the vertebral centra varied markedly between individuals. Four readers were used to make band counts, with the most experienced reader having the lowest index of average percent error and the highest level of agreement with final counts. Marginal increment analysis indicated that opaque bands form in January. With parturition occurring from August to October, size data suggests that the first band is probably formed 15–17 months after birth. The age at maturity was estimated to be 4.5 years for males, and 6.5 years for females. The oldest male was 10.5 years, and oldest female was 11.5 years. Von Bertalanffy growth parameters for males were L =121.5cm fork length, K=0.423 year^–1, t ₀=–0.472 years, were L =120.7cm fork length, K=0.369 year^–1, t ₀=–0.544 years for females, and were L =118.1cm fork length, K=0.420 year^–1, t ₀=–0.491 years for combined sexes. Data from a tag recapture study were analysed using a maximum likelihood method to verify the estimates of growth parameters from vertebral ageing. Von Bertalanffy growth parameters from the tag recapture study were L =128.2cm fork length, K=0.288 year^–1, t ₀=–0.654 years. The two methods of estimating growth parameters produced similar results, with rapid growth until approximately 5 years of age, after which there was little increase in length.     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

Spectral sensitivity of visual cells of the compound eye ofBlatta orientalis

Responses of single visual cells of the anterior part of the compound eye of the oriental cockroachBlatta orientalis were recorded intracellularly. Two spectral types of cells were discovered: ultraviolet receptors with _max 361 nm and green-sensitive receptors with _max 503 nm. The spectral curve of the whole eye, measured by the electroretinogram, included two peaks (=350–370 and 500 nm) and a minimum between 400 and 430 nm. This last fact is interpreted as additional evidence of the dichromatic vision of the cockroach.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 17, No. 1, pp. 57–61, January–February, 1985.     

Extracellular Poly(α-L-guluronate)lyase from Corynebacterium sp.: Purification, Characteristics, and Conformational Properties

Extracellular alginate lyase was purified from the culture supernatant of Corynebacterium sp. isolated from the sewage of a sea tangle processing factory in order to elucidate the structure—function relationship of alginate lyase. The electrophoretically homogeneous enzyme was shown to have a molecular mass of 27 kDa by sodium dodecyl sulfate (SDS)—polyacrylamide gel electrophoresis (PAGE) and by gel filtration, with an isoelectric point of 7.3. The molecular mass from amino acid analysis was 28.644 kDa. The optimal pH and temperature for the enzyme reaction were around 7.0 and 55°C, respectively. Metal compounds such as MnCl₂ and NiCl₂ increased the enzyme activity. The enzyme was identified as the endolytic poly(-L-guluronate)lyase, which was active on poly(-L-1,4-guluronate) and caused a rapid decrease in the viscosity of alginate solution. Measurement of the far-UV circular dichroic spectrum of the enzyme molecule gave a spectrum with a deep trough at 215nm accompanied by a shallow one at around 237 nm, and with a high peak at 197 nm and a much lower one at 230 nm. This spectrum was most likely to be that of the -form of the enzyme molecule and resembled poly(-D-mannuronate)lyase from Turbo cornutus (wreath shell) and poly(-L-guluronate)lyase from Vibrio sp. (marine bacterium). The near-UV circular dichroic spectrum was characteristic for aromatic amino acid residues. In the presence of 6 M urea, these spectra changed drastically in the near-UV and a little in the far-UV with the disappearance of the enzyme activity. Removal of the denaturant in the enzyme solution by dialysis restored both the activity and inherent circular dichroic spectra. The -sheets observed in alginate lyases as the major ordered structure seem to be a common conformation for the lyases.     

Ultrastructural localization of immunoreactive corticotropin, β-lipotropin, α- and β-endorphin in Cells of the human fetal anterior pituitary

Summary Cells immunoreactive with anti--(17–39) ACTH, -(1–24) corticotropin, -LPH, - and -EP were identified in the human fetal anterior pituitary at the ultrastructural level using the peroxidase-antiperoxidase complex method on ultrathin sections.Only one definite cell type was revealed by all these antisera. All granules of each individual immunostained cell reacted regardless of the antiserum used. The immunostained cells occurred in groups and were sometimes located in the wall of the follicle-like structures commonly observed in the fetal anterior pituitary. The cells revealed two main aspects: 1) The largest elements were rich in organelles, and their numerous secretory granules showed significant variations in size (250–500 nm in diameter), electron density of their content and stain-deposit intensity. The ergastoplasm, consisting of irregular tubules, was poorly developed. In the vicinity of the conspicuous Golgi apparatus, organelles related to the GERL complex were commonly observed. Multivesicular bodies were frequent. Some of these cells showed bundles of microfilaments (60 nm in thickness). 2) The smaller cells had an electron-lucent hyaloplasm with sparse organelles; they contained fewer granules and never showed microfilaments.The immunocytological results are consistent with the synthesis of a molecule similar to pro-opiocortin by this type of endocrine cell in human fetuses. Morphological evidence for the maturation process of this precursor and for the secretory activity of these cells and its possible regulation is presented and discussed.Abbreviations used ACTH corticotropin (39 amino acid polypeptide) - -MSH -melanotropin (ACTH [1–13]) - CLIP corticotropin-like intermediate lobe peptide (ACTH [18–39]) - -LPH -lipotropin (91 amino acid polypeptide) - -MSH -melanotropin (-LPH [41–58]) - -EP -endorphin (-LPH [61–91]) - -EP -endorphin (-LPH [61–76]) - PTA phosphotungstic acid Acknowledgements: The authors would like to thank Professors P. Magnin and J. Liaras, Hôpital Edouard Herriot; M. Dumont, Hôpital de la Croix-Rousse; A. Notter and R. Garmier, Hôtel Dieu; M. Bethenod, Hôpital Debrousse, Lyon, and the entire staff whose cooperation enabled samples to be taken under optimal conditions. The authors also thank Professor L. Graf, Research Institute for Pharmaceutical Chemistry (Budapest), and Professor R. Guillemin (Salk Institute, La Jolla) for their generous gift of antigensThis work was supported by a grant from I.N.S.E.R.M., ATP 46.77.78 (P.M. Dubois)     

Intraspecific variation of spectral sensitivity in threespine stickleback (Gasterosteus aculeatus) from different photic regimes

To examine the influence of the spectral characteristics of underwater light on spectral sensitivity of the ON and OFF visual pathways, compound action potential recordings were made from retinal ganglion cells of threespine stickleback from different photic regimes. In fish from a red-shifted photic regime (P₅₀ 680 nm for downwelling light at 1m), peak sensitivity of both the ON and OFF pathways was limited to long wavelength light (_max 600–620). In contrast, the ON pathway of fish from a comparatively blue-shifted (P₅₀ 566 nm) photic regime exhibited sensitivity to medium (_max 540–560) and long (_max 600 nm) wavelengths, while the OFF pathway exhibited peak sensitivity to only medium (_max 540 nm) wavelength light. In a third population, where the the ambient light is moderately red-shifted (P₅₀ 629 nm), the ON pathway once again exhibited only a long wavelength sensitivity peak at 620 nm, while the OFF pathway exhibited sensitivity to both medium (_max 560 nm) and long (_max 600–620 nm) wavelength light. These findings suggest that the photic environment plays an integral role in shaping spectral sensitivity of the ON and OFF pathways.     

Isolation and characterization of cathepsin B from bovine brain

Cathepsin B (EC 3.4.22.1) was purified 746-fold with a 21% recovery from bovine brain by autolysis, fractional precipitation with acetone, carboxy-methyl-Sephadex chromatography, affinity chromatography on a cystamine containing column and gel filtration chromatography. The purified cathepsin B eluted on gel filtration with an apparent molecular weight of 27,000 but was resolved into three bands of 30,000, 25,000 and 5,000 molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Antibodies to cathepsin B, raised against the 30,000 dalton band, were shown by immunoblots to react with both the 30,000 and 25,000 dalton proteins with results suggesting that the former predominated as the immunoreactive form in bovine brain homogenates. Isoelectric focusing demonstrated multiple bands, ranging from pH 4.75–5.2 with the major band at pH 5.1–5.2, all of which were capable of degrading N-carbobenzoxy-l-arginyl-l-arginine 4-methoxy--naphthylamide. The cathepsin B activity against N-benzoyl-dl-arginine -naphthylamide (BANA) and bovine myelin basic protein (MBP) had a pH optimum of pH 6.0. The Km for the degradation of BANA was 1.0 mM and 5.1 mM when assayed in the presence of 1% and 2.5% dimethylsulfoxide, respectively. Cathepsin B from bovine brain has many properties similar to cathepsin B isolated from other organs. The degradative effect of cathepsin B on MBP suggests a role for this proteinase in inflammatory demyelination.     

Water chemistry changes in the gill micro-environment of rainbow trout: experimental observations and theory

Summary Soft water of low buffer capacity was drawn from near the branchial surface of rainbow trout (Salmo gairdneri) at 15°C, using opercular catheters, to determine pH changes in water passing over the gills. Latex masks allowed measurement of ventilation volume, and concentrations of carbon dioxide, oxygen, ammonia, and titratable base in expired water were compared to concentrations in inspired water. Water passing over the gills was more basic than inspired water if the inspired water was pH 4–6 (maximum increase: +0.7 pH units near pH 5). Expired water was more acidic than inspired water if the inspired water was pH 6–10 (maximum decrease: –1.7 pH units near pH 9). Ventilation volume (0.37 l·kg^–1·min^–1) and oxygen consumption (1.7 mmol·kg^–1·h^–1) were constant in the pH range 4.6–10.1, but both increased by 1.6–2.4× near pH 4. Carbon dioxide transfer near the gills was about 100 M, ammonia transfer about 15 M, and titratable base added at the gills was about 30 M. A theoretical model using CO₂, titratable base, and ammonia added at the gills, the titration characteristics of the defined soft water medium, and aquatic equilibria for CO₂ and ammonia, adequately explained the experimentally observed changes in pH near trout gills. Our observations and predictive model indicate that any gill contaminant whose toxicity varies with pH may be more or less toxic at the gills than predicted from bulk water chemistry alone.Abbreviations pH _ex expired pH - pH _in inspired pH     

Accumulation of fumonisins B1 and B2 in freshly harvested Brazilian commercial maize at three locations during two nonconsecutive seasons

Fifty-six Brazilian commercial maize cultivars were examined for FB₁ and FB₂ accumulation after two non-consecutive growing seasons. During the 94/95 growing season 35 cultivars were planted at three locations in the state of São Paulo, Brazil. All samples (total of 105) were contaminated (0.10 g/g–6.58 g/g FB₁ and 0.04 g/g–2.15 g/g FB₂). During the 97/98 growing season, 8 of the cultivars used during 94/95 and 21 others were replanted at the same locations. All 87 samples were contaminated (1.15 g/g–43.80 g/g FB₁ and 0.08 g/g–11.65 g/g FB₂). One cultivar accumulated significantly less fumonisins in all locations during both growing seasons, indicating that some degree of selection may be possible even in climates that favor F. moniliforme (verticillioides) infection of maize. The presence of water surplus in soil from kernel maturity to harvest correlated with concentrations of FB₁ in the grain for the 8 cultivars planted during both seasons at three locations. Observed trends indicated that water excesses and deficits from silking to harvest increased fumonisin levels. The difference in the incidence of FB₁, FB₂, and FB₁ + FB₂ was significant between growing seasons, planting locations and between cultivars. Neither the level of hybridization, nor the type of endosperm, nor the length of the vegetative cycle showed any effect on the FB₁ contamination.This revised version was published online in October 2005 with corrections to the Cover Date.     

Synthesis of affinity chromatography resins for the purification of brain glutamate decarboxylase

In the present paper we describe the synthesis of Sepharose-boundN-(5-phosphopyridoxyl)-amino-oxyacetic acid,N-(5-phosphopyridoxyl)-canaline, andN-(5-phosphopyridoxyl)-,-diaminobutyric acid (Seph-DAB-PLP), designed for binding specifically brain glutamate decarboxylase (GAD). The three Sepharose-N-(phosphopyridoxyl)-amino acids were capable of binding GAD from a mouse brain soluble preparation. The enzyme can be purified up to 25-fold in one step by affinity chromatography on Seph-DAB-PLP.Part of this work was presented at the VI Meeting of the American Society for Neurochemistry, Mexico City, March 10–14, 1975.     

The occurrence and identification of intracellular polyglucose storage granules inMethylococcus NCIB 11083 grown in chemostat culture on methane

The accumulation of intracellular storage granules (0.03–0.5 m) byMethylococcus NCIB 11083 when grown under conditions of ammonia limitation with methane as the sole source of carbon and energy was inversely proportional to the dilution rate. The isolated material was composed entirely of glucose residues and the infra-red spectrum exhibited characteristic absorption bands at 925 cm^-1, 845 cm^-1 and 745±4 cm^-1, indicating the presence of (14) glycosidic linkages. The polymer dissolved in hot water to give an opalescent solution that formed a violet iodine complex with an absorption maximum at 550 nm, identical to that observed with reference amylopectin. The percentage of the polysaccharide released as maltose by the action of - and -amylases was 55–64% and 80–90% respectively, values very similar to those obtained by the action of these enzymes on reference amylopectin and glycogen. Methylation analysis indicated that the average interior and exterior chain lengths of the polymer were 2.7 and 10.0 glucose units respectively and confirmed that theMethylococcus polyglucose is a branched polymer composed of units joined by 14 and 16 linkages. The number average molecular weight of the polymer is 2–4.5×10⁵. The stored polymer was metabolised by the organism and its metabolism resulted in the synthesis of protein.Abbreviations GLC gas liquid chromatography - MS mass spectroscopy - PAAN peracetylated aldononitriles     

Production of trichothecene and non-trichothecene mycotoxins by Fusarium species isolated from maize in Minnesota

Eighty-two cultures of Fusarium species isolated in 1986 from moldy maize in Minnesota were each cultured on rice for 4 weeks and found to produce the following mycotoxins: F. graminearum isolates, deoxynivalenol (DON, 4–225 g/g), 3-acetyldeoxynivalenol (3-ADON, 2–4g/g), 15-acetyldeoxynivalenol (15-ADON, 1–35 g/g) and zearalenone (ZEA, 5–4350 g/g); F. moniliforme, fusarin C (detectable amounts to 1000 g/g); F. mòniliforme, F. oxysporum, F. proliferatum and F. subglutinans isolates, moniliformin (15–6775 g/g); F. moniliforme, F. proliferatum, and F. subglutinans isolates, fusaric acid (detectable amounts). Other mycotoxins screened for in each rice sample and not detected were T-2 toxin, HT-2 toxin, neosolaniol, T-2 tetraol, nivalenol, fusarenon-X, scirpenols, alpha and beta trans-zearalenols, wortmannin, and fusarochromanone. The rat feeding bioassay indicated that other, unidentified toxins may be present.     

Temporal shifts in visual pigment absorbance in the retina of Pacific salmon

The visual pigments and photoreceptor types in the retinas of three species of Pacific salmon (coho, chum, and chinook) were examined using microspectrophotometry and histological sections for light microscopy. All three species had four cone visual pigments with maximum absorbance in the UV (_max: 357–382 nm), blue (_max: 431–446 nm), green (_max: 490–553 nm) and red (_max: 548–607 nm) parts of the spectrum, and a rod visual pigment with _max: 504–531 nm. The youngest fish (yolk-sac alevins) did not have blue visual pigment, but only UV pigment in the single cones. Older juveniles (smolts) had predominantly single cones with blue visual pigment. Coho and chinook smolts (>1 year old) switched from a vitamin A₁- to a vitamin A₂-dominated retina during the spring, while the retina of chum smolts and that of the younger alevin-to-parr coho did not. Adult spawners caught during the Fall had vitamin A₂-dominated retinas. The central retina of all species had three types of double cones (large, medium and small). The small double cones were situated toward the ventral retina and had lower red visual pigment _max than that of medium and large double cones, which were found more dorsally. Temperature affected visual pigment _max during smoltification.