The effects of environmental factors on growth and the chemical and biochemical composition of marine diatoms. I. Light and temperature effects

Temperature and light interact to modify the chemical and biochemical composition of a nitrogen-limited marine diatom, Thalassiosira allenii Takano, grown at a constant dilution rate in continuous culture and under a light:dark cycle.The percent of the total ¹⁴C incorporated into protein, polysaccharide and lipid, the N/C ratio and the C/cell varied with temperature in a markedly non-linear manner. The N/cell was negatively correlated to temperature. The Chl $\text{a}\text{C}$ ratio was positively correlated with temperature under saturating light and non-saturating light for temperatures > 25 °C, but was constant under non-saturating light conditions for temperatures < 25 °C.Productivity index (PI) was negatively correlated to temperature under saturating light conditions, but did not vary under low light. In each case, the variation in PI with temperature was governed by the variation in Chl $\text{a}\text{C}$.The dark carbon loss rate was exponentially related to temperature and independent of light. Variation in the percent of the total ¹⁴C incorporated into protein and polysaccharide, the $\text{N}\text{C}$ ratio and C/cell was primarily due to the effects of N-limitation < 20 °C and primarily due to the effects of temperature > 20 °C. Variation in N/cell was primarily due to the effects of temperature over the entire range of temperature studied. Variation in Chl $\text{a}\text{C}$ was caused by the interaction of temperature and light effects.In most cases, temperature and nutrient effects interacted to govern how a particular parameter varied with temperature while light affected the range of values over which the parameter varied.The percent of the total ¹⁴C incorporated into protein exhibited a significant linear relationship with $\text{N}\text{C}$.The dark carbon loss rate, $\text{N}\text{C}$ ratio and Chl $\text{a}\text{C}$ ratio data were used to test the applicability of a model for the physiological adaptation of unicellular algae. The model, with parameters derived from a non-linear least-squares fit of the dark carbon loss rate data, adequately described the $\text{N}\text{C}$ ratio between 15 and 25 °C at 290 and 137 μE · m^?2 · s^?1, but failed to describe the data at 28 °C and at 48 μE · m^?2 · s^?1. The Chl $\text{a}\text{C}$ ratio was adequately described by the model under all light and temperature conditions.     

Production of carbohydrates by the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt. I. Effect of the concentration of nutrients in the culture medium

The chemical composition of cells of Chaetoceros affinis var. willei (Gran) Hustedt grown in batch culture was markedly influenced by the concentration of nutrients in the medium. In the logarithmic growth phase the content of cellular glucan was relatively low, but in the stationary phase the glucan content showed a rapid increase which seemed to coincide with the depletion of nitrate from the medium. This led to very pronounced variations in the ratio of protein to carbohydrate. This ratio can easily be determined and seems to be a sensitive and convenient parameter for characterizing the physiological state of the diatom cells.     

Studies on the biochemistry and fine structure of silica-shell formation in diatoms

Summary The distribution of radioactivity in ethanol-water-soluble compounds after short periods of photosynthetic incorporation of ¹⁴CO₂ is consistent with the operation of the photosynthetic carbon reduction (PCR) cycle in the fresh water diatom Navicula pelliculosa. Incorporation of ¹⁴CO₂ for extended time periods established the presence of the intermediates of the PCR and tricarboxylic acid (TCA) cycles, amino acids, and organic acids; free sugars were not observed. The main labelled soluble carbohydrate was a glucan. Hydrolysis of the radioactive insoluble material indicated the presence of carbohydrates containing several distinct sugars, and proteins with the usual amino-acid composition. During silicon starvation of exponentially growing cultures, rates of incorporation of both ³²P _i and ¹⁴CO₂ decreased. Incorporation into the lipid increased, with a corresponding decrease into protein and carbohydrate. Reintroduction of Si to staryed cells led to an increased rate of incorporation of both isotopes, and transient changes in the radioactivity in most metabolic intermediates investigated. After 30 min the radioactivity in all PCR cycle intermediates, except phosphoglyceric acid, had increased by about 300%. The radioactivity of citrate and -keto-glutarate increased, whereas that of other TCA-cycle intermediates decreased. An initial decrease in the levels of glutamate, aspartate and glutamine was apparently reversed by cleavage of glutamate-aspartate peptides, as radioactivity of other amino acids increased. Incorporation into the soluble glucan and into protein increased markedly although the rate of incorporation into insoluble carbohydrates remained constant.     

The organic and inorganic composition of some cirripede shells

The protein, chitin, calcium, and magnesium present in the shells of several species of cirripedes have been determined; the powdered shell material was also examined by differential thermal analysis (DTA). Chthamalus sp., in which lamina of epicuticular matter are present within the shell, show the greatest organic content. When the organic material is separated into its protein and chitin components the latter is present in similar quantities in all species; this reflects a similar quantity of the chitin-protein matrix. Reduction in the protein content of heavily eroded C. depressus seems to be due to the removal of protein by endolithic algae. The $\text{Ca}\text{Mg}$ ratio increases from extreme hypobiotic to exposed C. depressus. There is a marked correlation between the $\text{Ca}\text{Mg}$ ratio and the organic content and it is suggested that the magnesium is largely associated with protein rather than with the calcitic lattice. This receives support from the thermal analyses. Small quantities of quartz were detected by X-ray analysis.     

The characterization of a chitin-associated D-glucan from the cell walls of Aspergillus niger

Exhaustive extraction of the cell walls of Aspergillus niger with 10% NaOH solution leaves an alkali-resistant residue containing chitin and glucan as the major components. The glucan in this residue comprises 58.7% of the total cell wall glucan and was characterized by permethylation, and identification of the resulting $\text{O-}\text{methyl-}\text{D}\text{-glucoses}$ obtained after hydrolysis by gas-liquid chromagtography and mass spectrometry of the derived partially acetylated, partially methylated, [1-²H]alditols. The glucan was separated from the chitin by acetylation of the alkali-resistance material, a procedure which separates a large portion of the total glucan as a chloroformsoluble acetate, abd by treatment of the alkali-insoluble residue with nitrous acid, a procedure which was found to render the complex soluble in dimethylsulfoxide and amenable, therefore, to permethylation. The data collected suggests that the preparation is an essentially linear glucan containing 85–95% 1 → 3 linkages and 10–15% 1 → 4 linkages. An analysis of the glycosidic linkages using NMR spectroscopy indicate that both α and β linkages are present in the ratio of 4:1. An identical glucan appears to be present in the cell walls of Penicillium chrysogenum as well as the spore cell walls of both organisms, as evidenced by methylation studies.     

Studies on the phytoplankton ecology of the trondheemsfjord. II. Chloroplast pigments in relation to abundance and physiological state of the phytoplankton

The quantitative composition of the chloroplast pigments of phytoplankton sampled weekly at one station in the Trondheimsfjord was studied by circular paper chromatography throughout 18 months. The concentrations of total chlorophyll a (T-chl a obtained by the trichromatic method) as well as of chromatographically purified chlorophyll a (chl a) followed the variations in phytoplankton concentration. Two spring blooms and a weak autumn flowering of phytoplankton were clearly reflected in the pigment contents found, namely 14–16 mg T-chl a/m³ for the spring maxima, corresponding to nearly 300 mg T-chl a/m² for the euphotic zone; and 3–4 mg/m³ or 32 mg/m² for the autumn peak. The concentrations between blooms amounted to ≈ 1 mg T-chl a/m³, while concentrations down to 0.03 mg/m³ were found for winter samples.The content of T-chl a was high in diatom cells prior to a bloom (20–40 × 10^?9 mg/cell). During rapid growth (a more or less exponential phase) the cell content of chloroplast pigments decreased (to 5–10 × 10^?9 mg). No degradation product of chlorophylls could be detected during this phase and the percentage of chl a (of T-chl a) was high (70–80 %). At the peak of the bloom, and especially when the nitrate content in the surrounding water had been exhausted, low values for T-chl a were found ($\text{0.3–0.5 × 10}{}^{\mathrm{<mtext>9</mtext><mtext>?</mtext>}}\text{mg/cell}$). As soon as the cell counts started to fall, or even before the decline could be clearly detected, the percentage of chl a dropped (to 40-20 %) and derived chlorophylls (not phaeophytin a) were present in the samples. Model studies with cultured algae showed a similar behaviour.It is concluded that the proportion of chl a to T-chl a and the occurrence of chlorophyll derivatives in phytoplankton samples can give valuable information on the stage of development of the algal populations involved.     

Alkaline and acid phosphatases of the marine diatoms Chaetoceros affinis var. willei (Gran) Hustedt and Skeletonema costatum (Grev.) Cleve

Alkaline phosphatase activity in cultures of the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt was higher than in Skeletonema costatum (Grev.) Cleve. The enzyme activity was localized in coarse cell particles. Acid phosphatase activity was found in the cytoplasmic fraction. Induction of alkaline phosphatase depended on the $\text{N}\text{P}$ ratio in the culture medium. A $\text{N}\text{P}$ ratio > 40 in dilution/batch culture and > 30 in large scale batch culture, respectively, induced alkaline phosphatase.Cell phosphorus showed a critical value below which alkaline phosphatase was induced. Alkaline phosphatase in natural phytoplankton from the Trondheimsfjord is unlikely to occur except possibly in special situations.     

On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

Identification of the outer membrane protein of E.coli that produces transmembrane channels in reconstituted vesicle membranes

Outer membrane of $\text{Escherichia}$$\text{coli}$ allows a rapid diffusion of saccharides of molecular weights less than 550. This permeability property could be restored in vesicle membranes reconstituted from isolated phospholipids, lipopolysaccharide, and an outer membrane protein. The active protein aggregates were isolated from the insoluble material left after solubilization of cell envelope of $\text{Escherichia}$$\text{coli}$ B with sodium dodecyl sulfate at 35°. Analysis by acrylamide gel electrophoresis, isoelectric focusing and amino terminal amino acid determination revealed that only a single species of protein, with a molecular weight of 36,500 forms the oligoprotein aggregates which produces diffusion channels.     

Improving utilization of crop residues. Evaluation of two chemicals used to improve digestibility of crop residues

Two chemical treatments involving chelating metal caustic swelling (CMCS) and sodium hydroxide (NaOH) were evaluated for their ability to affect in vitro dry matter and in vitro cellulose disappearance (IVDMD and IVCD, respectively), dry matter disappearance in sacco and the chemical composition of two low-quality crop residues, cornstalks (CS) and soya bean residue (SBR). At $\text{chemical}\text{substrate}$ ratios ranging from 0.25:1 to 5:1, linear increases in IVCD of both roughages were noted. Improvements in IVCD of CS and SBR were noted at 1:1, 3:1 and 5:1$\text{chemical}\text{substrate}$ ratios, regardless of whether the treated roughages were used immediately after treatment (fresh basis) or preserved by drying or freezing. Decreased concentrations of neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL) and crude protein (CP) resulted from chemical treatment. No effects of the water component of the solvent solutions were observed. Dry matter disappearance in sacco of CMCS- and NaOH-treated roughages was increased at all $\text{chemical}\text{substrate}$ ratios tested. Treatment of either roughage with CMCS resulted in greater digestibility in sacco than did treatment with NaOH. Treatment of CS with CMCS resulted in higher rates of digestion than did treatment with NaOH. No differences in rates of digestion of SBR were noted between chemicals. Both chemicals were more effective in improving digestibility of CS than of SBR.     

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD⁺ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio brought about by glucagon may be secondary to the increase in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ and $\text{[ATP]}\text{[ADP]}$ ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD⁺ system.     

The dependence of the conformation of a (1→3)-β-D-glucan on chain-length in alkaline solution

(1→3)-β-D-Glucans of various degrees of polymerization were prepared by degradation of a gel-forming D-glucan with formic acid. The degraded D-glucans were separated into a water-soluble fraction (soluble D-glucan) and an insoluble fraction (insoluble D-glucan). Both D-glucans were further fractionated. The optical rotation including determination of the o.r.d. curves of the fractions and of the original gel-forming D-glucans was measured at various sodium hydroxide concentrations (0–5M). The results indicate that (1→3)-β-D-glucans of $\text{DP}$n below ca. 25 (the soluble D-glucan) took a disordered form in both neutral and alkaline solutions, whereas the D-glucans of higher $\text{DP}$n (the insoluble and the original D-glucans) took an ordered structure in dilute alkaline solution (0.1M). The proportion of ordered structure in the insoluble D-glucan increases with $\text{DP}$n to attain a maximum value at a $\text{DP}$n of around 200; this may be the lower limit of $\text{DP}$n to permit gel formation in neutral media. The formation of complexes with Congo Red in alkaline solutions by the soluble and the insoluble D-glucans supports the same conclusions.     

Studies on the characterization of human erythrocyte acetylcholinesterase and its interaction with antibodies

Human erythrocyte membranes were solubilized in 5% Triton X-100 and the acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was isolated by affinity chromatography utilizing a specific inhibitor, $\text{trimethyl}\text{-p-}\text{aminophenyl}$ ammonium chloride, bound to Sepharose 4B. After a repeated chromatography acetylcholinesterase was found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunization of rabbits with acetylcholinesterase elicited the formation of an antiserum which gave single precipitin lines with the enzyme on immunodiffusion and rocket, crossed and immuno-electrophoreses. The purified enzyme had a specific activity of 418 units/mg protein. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of acetylcholinesterase with acetylthiocholine as substrate was $\text{1.5 × 10}{}^{\mathrm{?4}}\text{M}$. Isoelectric focusing of acetylcholinesterase in the presence of Triton X-100 and within the pH ranges of 3–10 and 3–6 exhibited a single peak of enzyme activity with a $\text{P}\text{I}$ of 4.8. The results of amino acid and carbohydrate analyses showed that acetylcholinesterase is a glycoprotein with a carbohydrate/protein weight ratio of 0.16 and glucose, galactose, mannose, glucosamine, galactosamine and sialic acid as the sugar components. The N-terminal amino acid was blocked. Lipid, phosphorus and fatty acid analyses indicated phosphatidylserine and cholesterol as the major lipid components of acetylcholinesterase. The apparent subunit molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol was 160 000 and in its presence, 80 000. The kinetic studies showed a competitive inhibition of acetylcholinesterase by its antibodies. Agglutination of human red cells by monospecific antiserum to acetylcholinesterase confirmed that the antigenic site(s) of the enzyme is localized on the outer surface of the erythrocyte membrane.     

The composition and nutritional status of the diet of Oreochromis niloticus in Lake Chamo, Ethiopia

The stomach contents of Oreochromis niloticus from L. Chamo were microscopically examined and chemically analysed in order to study the relationship between food quality, growth and condition. More than 20 genera of phytoplankton plus Copepoda, Rotifera and nauplii were present in the stomach contents. The diatom, Melosira , was the most dominant alga in the diet, contributing about 30% of the organic matter. The composition of the food in terms of organic nutrients was dominated by carbohydrate (20.3% dry weight). Both protein and fat made low contributions to the diet; 10% and 5.8% dry weight, respectively. The overall performance of the fish in assimilating food was high: 43.3% for ash free dry weight, 61.2% for protein, 57.2% for lipid, 29% for carbohydrate and 55.7% for energy. The digestible protein-energy ratio was 15.7 mg kJ⁻¹, suggesting that the food was qualitatively good both for fast growth and for maintaining good condition. It was concluded that the small amount of good quality food ingested, the low fish population density in the lake and the high temperature of the water are the major factors responsible for making O. niloticus a fast-growing fish in Lake Chamo.     

Feeding value of alkali-treated whole rye (secale cereale L.) grain for lactating cows and its digestibility for sheep

Alkali-treated whole rye grain (35 g NaOH/kg grain) and rolled rye grain were given to cows in diets with $\text{concentrate}\text{roughage}$ ($\text{C}\text{R}$) ratios of 65:35 and 75:25. Alkali treatment tended to increase intake (by 13% for the 65:35 diet), but non-significantly. Milk yield and composition did not differ significantly among the four treatments.Alkali treatment of rye tended to increase the molar proportion of acetic acid and decrease that of propionic acid, although non-significantly.Apparent digestibility of dry matter, crude protein and energy, determined by the acid-insoluble ash (AIA) technique, did not differ significantly among treatments.In another trial, alkali treatment of rye grain for sheep resulted in no significant improvement in dry matter, energy or cellulose digestion, but alkali treatment of barley increased the digestibility of acid detergent fibre and cellulose. The present studies show a slight tendency for improvement with alkali treatment of whole rye grain, but further investigations are needed.     

The effect of high levels of molasses in combinations with hay on digestibility of organic matter,microbial protein synthesis and volatile fatty acid production in vitro

Fermentation in vitro of $\text{0}\text{100}$, $\text{10}\text{90}$, $\text{40}\text{60}$ and $\text{70}\text{30}$ cane molasses/hay mixtures in media containing urea, trace minerals and vitamins showed a progressive improvement of digestible organic matter in vitro (INVDOM) of hay as the level of molasses increased. Replacement of urea by soya bean meal induced a lower INVDOM of hay in the mixture with 70% molasses.Structural carbohydrates (hay) induced higher microbial synthesis per unit digestible organic matter (DOM) than simple sugars (molasses).The results observed emphasized the importance of adding urea to molasses for improved digestion of hay and a higher percentagge of propionic acid in the rumen. The effect of formulation of molasses/hay rations with soya bean meal or urea on the pattern of fermentation is discussed.     

Phosphate transport and the stoicheiometry of respiratory driven proton translocation in Escherichia coli.

The stoicheiometry of respiratory driven proton translocation associated with the oxidation of endogenous substrates has been measured in an organic phosphate auxotrophic mutant of $\text{Escherichia coli}$. The results obtained indicate that movements of inorganic phosphate do not result in an experimental underestimation of the observed H⁺/site ratio in $\text{E. coli}$, as has previously been suggested for mitochondria.     

Electron paramagnetic resonance studies of membrane proteins in hepatic microsomes

Hepatic microsomal membranes, prepared under various conditions that yield either ‘intact’ or ‘disrupted’ microsomal vesicles, have been labeled via the sulfhydryl groups of intrinsic membrane proteins using nitroxide analogs of $\text{N-}\text{ethylmaleimide}$. Electron paramagnetic resonance spectra revealed the presence of two dominant classes of bound label corresponding to differing degrees of immobilization, the ratio of which were quantitated using a parameter designated the ‘$\text{W/S}$’ ratio. For latent microsomes, the value of this parameter was determined to be $\text{0.65 ± 0.02}$ and was influenced by factors such as label/protein ratio, incubation period, nitroxide structure, temperature and pH. The $\text{W/S}$ ratio was also sensitive to the degree of membrane integrity as revealed by the latency of mannose 6-phosphate activity of glucose-6-phosphohydrolase. In addition, membrane disruption resulted in a corresponding decrease in the order parameter for nitroxide-labeled fatty acids intercalated within the lipid bilayer. The $\text{W/S}$ ratio was observed to be dependent upon the method of microsome preparation yielding values of $\text{1.02 ± 0.02}$ for ‘hypertonically disrupted’ vesicles and $\text{1.28 ± 0.02}$ for ‘mechanically disrupted’ vesicles. Microsomal marker enzymes such as cytochrome $\text{P-450}$ and FAD-containing monooxygenase retained significant levels of functionally following nitroxide incorporation.     

Association of myelin basic protein with detergent micelles

Equilibrium measurements of the binding of central nervous system myelin basic protein to sodium dodecyl sulphate, sodium deoxycholate and lysophosphatidylcholine have been obtained by gel permeation chromatography and dialysis. This protein associates with large amounts of each of these surfactants: the apparent saturation weight ratios (surfactant/protein) being $\text{3.58 ± 0.12}\text{and}\text{2.30 ± 0.15}$ for dodecyl sulphate at ionic strengths 0.30 and 0.10, respectively, $\text{1.34 ± 0.10}$ for deoxycholate (at 0.12 ionic strength) and $\text{4.0 ± 0.5}$ for lysophosphatidylcholine. Binding to the ionic surfactants increases markedly close to their critical micelle concentrations. Sedimentation analysis shows that at 0.30 ionic strength in excess dodecyl sulphate the protein is monomeric. It becomes dimeric when the binding ratio falls below 1 at a free detergent concentration of approximately 0.25 mM: below this concentration much of the protein and detergent forms an insoluble complex. The amount of dodecyl sulphate bound at high concentrations and at both above-mentioned ionic strengths corresponds closely to that expected for interaction of a single polypeptide with two micelles. Variability of deoxycholate micelle size on interaction with other molecules precludes a similar analysis for this surfactant. Association was observed only with single micelles of lysophosphatidylcholine. The results provide strong evidence for dual lipid-binding sites on basic protein and indicate that lipid bilayer cross-linking by this protein may be effected by single molecules.