Unit Protein Basis Research Articles

Comparisons of Modern United States and Canadian Malting Barley Cultivars with Those from Pre-Prohibition: V. Bmy1 Intron III Alleles and Grain β-Amylase Activity and Thermostability

This study was conducted to determine the variation in Pre-Prohibition and modern barley malting genotype β-amylase activities and thermostabilities with differing Bmy1 intron III alleles. Sequencing of the endosperm specific β-amylase gene Bmy1 intron III of two- and six-row of Pre-Prohibition barley varieties revealed that both two- and six-row varieties had the Bmy1.a intron III allele. This is unusual in that previously there has been only one documented case of a two-row cultivar having the Bmy1.a intron III allele. In this study, all modern cultivars were found to have the expected Bmy1.b intron III allele and Bmy1.a intron III allele for two- and six-row cultivars, respectively. On a fresh weight or unit protein basis, the mean grain β-amylase activities of modern malting cultivars were significantly higher (P < 0.0001) than the mean for Pre-Prohibition malting varieties. This indicates that the many years of selection for diastatic power (DP) since the end of Prohibition in North America have increased the β-amylase activity significantly. Many studies have shown that DP strongly correlates with β-amylase activity. The β-amylase activities of modern cultivars with the highest β-amylase activity were significantly higher (P < 0.0001) than all Pre-Prohibition varieties. The β-amylase activities of Pre-Prohibition varieties with the lowest β-amylase activity were significantly lower (P < 0.0001) than all modern cultivars. Modern cultivars were significantly higher (P = 0.022) than for Pre-Prohibition varieties; although, the differences for most genotypes, be they Pre-Prohibition or modern, were not large. The two-row Pre-Prohibition varieties Hanna and Hannchen were the exceptions, being highly significantly lower (P < 0.0001) in thermostability than all other Pre-Prohibition and modern genotypes.

Distiller’s dried grains with solubles as an alternative protein source in diets of tilapia

AbstractDistiller’s dried grains with solubles (DDGS), a dried residue that remains after the fermentation of grain mash by selected yeasts and enzymes to produce ethanol and carbon dioxide, is currently readily available and less expensive than other conventional protein sources on a per unit protein basis. The nutrient content and nutritional value of DDGS varies with the source and quality of the grains, fermentation time and efficiency, the drying process and the quantity of distiller’s solubles added. In general, corn and wheat DDGS are deficient in lysine and methionine for tilapia, with lysine being the most limiting. Research evaluating the nutritional value of DDGS showed that 20–30% DDGS derived from corn or wheat can be included in tilapia diets without requiring lysine supplementation. With supplementation of lysine, DDGS at levels of 40% or higher can be used without affecting growth performance and feed utilization efficiency. Distiller’s dried grains with solubles also contain yeast, a rich source of beta glucan and nucleotides that have been reported to enhance immunity and disease resistance in fish. Corn DDGS, owing to its high oil content, is rich in linoleic acid and is an excellent source of energy and essential fatty acids for tilapia. High concentrations of xanthophylls present in corn DDGS may impart yellow pigment in fish flesh if included at high levels. Taking into consideration various factors affecting the nutritional value of diets and the quality of pellet and fish products, 15–20% DDGS appears to be optimum in the diets of tilapia.

Partition of minerals in body components from a high- and low-lean genetic line of barrows and gilts from 20 to 125 kilograms of body weight12

An experiment was conducted to determine if the macro- and micromineral contents of the ham and loin or the remaining body component differed by genetic line, sex, or BW. The experiment was a completely randomized design with a factorial arrangement (2 × 2 × 5) using barrows and gilts of 2 genetic lines at 5 BW intervals in 2 groups with 6 replicates (n = 120 pigs). Pigs were housed in groups of 5 per pen and removed when individual pigs reached their targeted BW. Twelve pigs (3 from each genetic line and sex) were killed at 23 kg of BW and at 25-kg intervals up to 125 kg of BW. After slaughter, loin and ham muscles were dissected and trimmed of fat, with the ham deboned. This muscle mass constituted the first body compartment. The trimming from these muscles, ham bones, the remaining body, internal tissues, skin, and head were combined and constituted the second body component. The data were analyzed by PROC MIXED using the animal as the experimental unit. Muscle weights and their protein contents differed (P < 0.01) between the high- and the low-lean pigs and barrows and gilts and also among 5 BW groups/intervals. Total macro- and micromineral contents in the loin and ham were greater (P < 0.01) in the high-lean genetic line and gilts and increased (P < 0.01) as BW increased. Genetic line × BW and sex × BW interactions (P < 0.01) occurred for the macrominerals and for Fe, Se, and Zn, with contents diverging, and were greater as BW increased in high-lean pigs and gilts. The weight and protein content of the remaining body component was greater (P < 0.01) in the high-lean genetic line but not for the 2 sexes. In this body component, macromineral contents were greater as BW increased (P < 0.01), as were the microminerals Fe, Se, and Zn (P < 0.01). When the minerals were expressed on a per kilogram of body component basis, the ham and loin mineral compositions were similar for both genetic lines and sexes, but Na and Cl declined (P < 0.01) as BW increased. Most microminerals showed a small increase with BW. In the remaining body component, Ca increased (P < 0.03) in the low-lean line, whereas K was greater (P < 0.01) in the high-lean genetic line. When expressed on a unit protein basis, the low-lean genetic line had more macrominerals in the loin and ham than the high-lean genetic line. These results indicate that high-lean genetic line pigs and gilts have greater total macro- and micromineral contents in the ham and loin than the low-lean pigs, thus indicating that their dietary mineral needs are greater during the latter part of the finisher period in heavier muscled pigs.

Ethylene‐binding activity, gene expression levels, and receptor system output for ethylene receptor family members from Arabidopsis and tomato†

Ethylene signaling in plants is mediated by a family of ethylene receptors related to bacterial two-component regulators. Expression in yeast of ethylene-binding domains from the five receptor isoforms from Arabidopsis thaliana and five-receptor isoforms from tomato confirmed that all members of the family are capable of high-affinity ethylene-binding activity. All receptor isoforms displayed a similar level of ethylene binding on a per unit protein basis, while members of both subfamily I and subfamily II from Arabidopsis showed similar slow-release kinetics for ethylene. Quantification of receptor-isoform mRNA levels in receptor-deficient Arabidopsis lines indicated a direct correlation between total message level and total ethylene-binding activity in planta. Increased expression of remaining receptor isoforms in receptor-deficient lines tended to compensate for missing receptors at the level of mRNA expression and ethylene-binding activity, but not at the level of receptor signaling, consistent with specialized roles for family members in receptor signal output.

Patterns of Sorbitol Metabolism and Availability during Apple Fruit Set

Acquiring sufficient carbohydrate is essential for successful apple fruit set. Sorbitol may be the dominant carbohydrate imported by growing fruit, and the rate of sorbitol accumulation may be a function of NAD-dependent sorbitol dehydrogenase (SDH; EC 1.1.1.14) activity. Prior work indicated that SDH activity from whole fruit (seeds plus cortex) increased for 2 or 3 weeks after initiation of fruit growth and then declined through 5 weeks. Using SDH activity assays, an SDH-specific antibody, and SDH-specific probes in Northern analyses, it is evident that SDH is expressed and is active in both apple seed and cortex tissue during the first few weeks of fruit growth. On a per unit protein basis, SDH activity in seeds increased by the pattern described above while that in fruit was generally lower and constant. During this same period of time, the sorbitol content of the expressed sap of apple shoots was analyzed. The sorbitol concentration was 50- to 100-fold higher than the sucrose concentration. The concentrations of both carbohydrates changed in parallel to the change in SDH activity of whole fruit and seeds. The lowest SDH activity and sap sorbitol levels preceded and/or coincided with the beginning of the natural fruit drop (or June drop) period.

Effects of quorum-sensing deficiency on Pseudomonas aeruginosa biofilm formation and antibiotic resistance

Variations in biofilm formation by, and antibiotic resistance of, Pseudomonas aeruginosa PAO1 (wild type) and the quorum-sensing-deficient mutants PDO100 (Delta rhlI), JP1 (Delta lasI) and JP2 (Delta lasI Delta rhlI) were studied. For PAO1, the maximum-accumulation phase of biofilm formation began immediately and a plateau phase was reached after 24 h, whereas the quorum-sensing mutants showed 36-48 h lags before entering the maximum-accumulation phase. After 72 h, the cell density of the PAO1 biofilms was c. 0.8-1.2 log greater than for the mutants. On a unit protein basis, total polysaccharide production was similar for PAO1 and PDO100, whereas JP1 and JP2 biofilms accumulated only c. 36% of the PAO1 level after 72 h. Fluorescent micrographs revealed that the PAO1 biofilms were much thicker than those of the quorum-sensing-deficient mutants. In the case of the PAO1 and PDO100 biofilms, most cells were attached to the top of the biofilm layer, whereas the bottom layer consisted predominantly of polysaccharides. The JP1 and JP2 biofilms were closely packed with cells, and little polysaccharide was visible. Cells in PAO1 biofilms were little affected by kanamycin, even at 100 mg/L, whereas those in PDO100 biofilms were susceptible to the highest concentration of kanamycin (100 mg/L) but not to lower concentrations (10 and 50 mg/L). In contrast, cells in JP1 and JP2 biofilms were susceptible to kanamycin at all three concentrations.

Seasonal protease activity in storage tissue of the deciduous grass Molinia caerulea

Plants of Molinia caerulea were grown in sand culture for two seasons. All nitrogen (N) supplied to the plants was enriched with 15N throughout the first season, and at natural isotopic abundance throughout the second season. A series of destructive harvests was taken during the second season. At each harvest, the N mobilized from roots and swollen basal internodes and the protease activity (ability to degrade azocasein) of these storage tissues were measured. The pH response curves of protease activity of both basal internodes and roots exhibited optima at pH 5 throughout the season. The protease activity of roots and basal internodes increased in spring, both on a unit fresh weight and unit protein basis, concomitant with mobilization of N from both these tissues to new shoot growth. In absolute terms more N was mobilized from roots than from basal internodes. However, basal internodes which, compared with roots, showed the greatest protease activity (on a f. wt or unit protein basis) were also the tissues that mobilized a greater proportion of N present in the tissue over winter to new shoot material. Individual clones of M. caerulea varied both in the amount of N mobilized from roots and in root protease activity (per plant). Individuals with a higher protease activity mobilized more N compared with individuals of lower protease activity. Therefore in M. caerulea, relationships between N mobilization and protease activity exist at several levels: (1) between different tissues; (2) temporally throughout the season; and (3) between individual clones.

Oxidative damage and motor neurone disease difficulties in the measurement of protein carbonyls in human brain tissue.

It has been suggested in the literature that elevated oxidative protein damage, measured as protein carbonyls, is present in the nervous system of patients with sporadic motor neurone disease (MND). However, the actual reported levels of brain protein carbonyls vary over a wide range. We show here that this is probably due to the use of the different protocols for the carbonyl assay; results differ depending on when the dinitrophenylhydrazine reagent is added and at what stage in the procedure protein is assayed for the calculation of carbonyls on a unit protein basis. Using a range of different procedures, we were unable to confirm reports of elevated protein carbonyls in motor cortex from brains of patients with MND. We also measured thiobarbituric acid-reactive material in the brain samples using an HPLC-based TBA test in the presence of butylated hydroxytoluene. In general, there was no significant elevation of TBARS in MND motor cortex. However, four patients showed values higher than any of the control patients (both 'normal' control and 'disease control'). There was no correlation of TBARS with protein carbonyl values. We suggest that oxidative damage in motor cortex in sporadic MND, if it occurs, may be confined to a small group of patients and may affect different molecular targets in each patient.

Somatotropin Enhances the Rate of Amino Acid Deposition but Has Minimal Impact on Amino Acid Balance in Growing Pigs1

This experiment was conducted to establish the influence of porcine somatotropin on tissue distribution and deposition rates of amino acids in growing pigs. Barrows were treated daily with buffer or porcine somatotropin (100 µmg/kg body wt) when they weighed between 30 and 64 kg (eight pigs/group). Pigs were restrictively fed so that the average food intake was 1.86 kg/d. The corn-soybean meal-skim milk-based diet contained 18% crude protein and 1.2% lysine, and was designed to meet muscle amino acid ratio profile with respect to lysine as the first limiting amino acid for growth. Tissue levels of eighteen amino acids were determined on lyophilized samples that were appropriately hydrolyzed and analyzed by HPLC. The concentrations (mg/g dry wt) of all amino acids were greater in carcass, skin, head and empty body of porcine somatotropin-treated pigs; amino acid concentration in viscera was not influenced by porcine somatotropin. However, when the concentration of each amino acid was expressed on a per unit protein basis, the amino acid profiles of control and porcine somatotropin-treated pigs were quite similar. The average deposition rate of each amino acid was increased ∼67% by porcine somatotropin. When the deposition of each amino acid was calculated in relation to lysine, however, the pattern of amino acid utilization for growth was similar for control and porcine somatotropin-treated pigs; exceptions were arginine, glycine and tryptophan. The ratio of indispensable to dispensable amino acids that were deposited was also similar for control and porcine somatotropin-treated pigs. These data indicate that the synthesis rate of individual proteins can be influenced by porcine somatotropin, but the balance of amino acids remains largely unaffected, suggesting that the changes in protein and amino acid metabolism elicited by porcine somatotropin are consistent with normal growth processes.

Temporal nature of the promoter and not relative strength determines the expression of an extensively processed protein in a baculovirus system

We demonstrate that the expression of extensively modified and secreted heterologous proteins synthesized in the baculovirus expression vector system (BEVS) depends on the temporal nature of the promoter transcribing the foreign gene. The β subunit of the human chorionic gonadotropin, an extensively modified secretory glycoprotein hormone was expressed under the transcriptional control of the AcNPV basic protein gene promoter (MP) and the polyhedrin gene promoter (POL), respectively. MP, activated late in the infection cycle, is a weaker promoter when compared to the stronger very late POL promoter. Levels of secretion, immunoreactivity and bioactivity of recombinant proteins, βhCG MP and βCG POL. synthesized under control of the MP and POL promoter were compared. Secretion of βhCG MP was relatively higher. Enzymatic analysis revealed that the synthesized protein was sialylated. Receptor binding assays and testosterone stimulation assays in a mouse Leydig cell system demonstrated that on a unit protein basis, βhCG MP was biologically more active than βhCG POL.

Intraplastidic Localization of the Enzymes That Convert δ-Aminolevulinic Acid to Protoporphyrin IX in Etiolated Cucumber Cotyledons

The intraplastidic localization of the enzymes that catalyze the conversion of delta-aminolevulinic acid (ALA) to protoporphyrin IX (Proto) is a controversial issue. While some researchers assign a stromal location for these enzymes, others favor a membranebound one. Etiochloroplasts were isolated from etiolated cucumber cotyledons (Cucumis sativus, L.) by differential centrifugation and were purified further by Percoll density gradient centrifugation. Purified plastids were highly intact, and contamination by other subcellular organelles was reduced five- to ninefold in comparison to crude plastid preparations. Most of the ALA to Proto conversion activity was found in the plastids. On a unit protein basis, the ALA to Proto conversion activity of isolated mitochondria was about 2% that of the purified plastids, and could be accounted for by contamination of the mitochondrial preparation by plastids. Lysis of the purified plastids by osmotic shock followed by high speed centrifugation, yielded two subplastidic fractions: a soluble clear stromal fraction and a pelleted yellowish one. The stromal fraction contained about 11% of the plastidic ALA to Proto conversion activity while the membrane fraction contained the remaining 89%. The stromal ALA to Proto conversion activity was in the range of stroma contamination by subplastidic membrane material. Complete solubilization of the ALA to Proto activity was achieved by high speed shearing and cavitation, in the absence of detergents. Solubilization of the ALA to Proto conversion activity was accompanied by release of about 30% of the membrane-bound protochlorophyllide. It is proposed that the enzymes that convert ALA to Proto are loosely associated with the plastid membranes and may be solubilized without the use of detergents. It is not clear at this stage whether the enzymes are associated with the outer or inner plastid membranes and whether they form a multienzyme complex or not.

Absence of a 45 or 60 Hz Electric Field-Induced Respiratory Effect in Physarum polycephalum

Microplasmodia of Physarum polycephalum strain ${\rm M}_{3}{\rm C}$ were exposed to 60 Hz continuous wave electric fields of 0.7 and 70 V/m for more than 13 weeks and to 45 Hz CW fields of the same strengths for nearly 7 weeks. At a particular field strength, duplicate subcultures of sham-exposed and field-exposed microplasmodia were independently maintained and measured to allow comparison of variation between subcultures within an exposure treatment group. Microplasmodial oxygen uptake rates were measured using a Clark-type oxygen electrode system, and respiratory rates were expressed on a unit protein basis. Few consistent respiratory rate differences between sham- and field-exposed subcultures were detected at either 0.7 or 70 V/m. These findings are in contrast to earlier reports of significant microplasmodial respiratory rate depression in response to 45 or 75 Hz, combined 0.7 V/m, 2.0 G electric and magnetic fields and in response to 75 Hz, 0.7 V/m electric field exposure [Greenebaum et al., DHEW R...

On the disposition of a phosphorylated protein ("synapsin I") and its associated kinases in synaptosomes from rat brain.

Endogenous phosphorylation of synapsin I (protein I), a phosphoprotein located on the surface of synaptic vesicles, was studied in vesicles prepared from synaptosomes lysed in the absence (control) or presence of 50 microM-cyclic AMP ("cAMP-treated"). Compared to synaptic plasma membrane (SPM) fractions prepared in parallel, and confirming previous work, the vesicle fractions were highly enriched on a unit protein basis in Ca2+-calmodulin-dependent kinase activity towards synapsin I. In contrast, with control vesicles the magnitude of the total phosphorylation of synapsin I in the presence of cyclic AMP was similar to that observed in SPM, but regulation by cyclic AMP was only partial. In "cAMP-treated" vesicles, however, synapsin I phosphorylation was highly enriched compared to SPM and the activity was virtually independent of cyclic AMP. The results show that while the free catalytic subunit of the cyclic AMP-dependent kinase remains associated with synapsin I during vesicle isolation the holoenzyme remains bound to membrane fragments, probably through its regulatory subunit.

The nucleoids of rat liver cell microbodies. Fine structure and enzymes.

The nucleoids of microbodies of rat liver cells were isolated in a highly homogeneous and pure state, by treating the microbody-rich fraction, prepared from 10% polyvinylpyrrolidone-0.25 M sucrose homogenate, with Triton X-100. Three treatments with 0.1% detergent were enough to render the nucleoids free from contamination with mitochondria, microsomes, lysosomes, and intact microbodies. Electron microscopically, the nucleoids were found to consist of parallel bundles of highly dense hollow tubules, the outer and inner diameters of which are approximately 150 and 50 A, respectively. Ten tubules are arranged around a longitudinal space 190 x 200 A in width. The nucleoids thus show a honeycomb appearance in the cross-plane and a parallel-packed structure in the longitudinal plane. Biochemically, the nucleoids were found to bear only urate oxidase among probably microbody-enzymes, and they might be the only cytoplasmic particles of rat liver cells in which the enzyme locates. Urate oxidase activity, on a unit protein basis, of the nucleoid preparation is approximately 380 times as high as that of the whole homogenate, and is almost comparable with that of a commercial type I enzyme preparation. No enzymes of mitochondrial, microsomal, and lysosomal origins were detected in the nucleoids. The fine structure of the nucleoids is described in detail, and a probable schematic diagram is presented.