Effects of feed supplementation on growth, blood parameters and reproductive performance in Sanga and Friesian-Sanga cows grazing natural pasture.

This study determined the effects of feed supplementation during the postpartum period on the weight gain, milk yield, blood profiles and reproductive performance of Sanga and Friesian-Sanga cows grazing on natural pasture. 20 Sanga and 20 Friesian-Sanga cows were randomly allocated either to serve as a control on grazing only or to be supplemented with 2.5 kg of concentrate a day for 10 weeks during the dry season. Each week, all cows were weighed and scored for body condition. Partial milk yield of cows was determined daily. Plasma concentrations of blood metabolites were assessed fortnightly from weeks 1 to 10 postpartum. Resumption of postpartum ovarian activity was determined by measuring progesterone concentration in the plasma from weeks 1 to 10. Supplemented cows had a better body condition score (6.2 versus 5.8; P < 0.05) and higher partial milk yield (1.94 versus 1.55 L/day; P < 0.01) than non-supplemented cows. Sanga cows had a better body condition score (6.2 versus 5.8; P < 0.05) but lower milk yield (1.58 versus 1.92 L/day; P < 0.01) than the Friesian-Sanga crossbreds. Total protein (P < 0.05) and albumin (P < 0.01) concentrations were higher in the supplemented than in the non-supplemented cows. Sanga cows recorded higher globulin (P < 0.05) and total cholesterol (P < 0.01) but lower albumin (P < 0.01) concentrations than Friesian-Sanga crossbred cows. Feed supplementation did not affect (P < 0.05) the interval from calving to resumption of ovarian activity, and the days to resumption of ovarian activity in the Sanga and Friesian-Sanga cows were also similar (P > 0.05). The results demonstrate the beneficial effects of feed supplementation in terms of improved body condition and metabolic status and increased milk yield.

Impairment of liver synthetic function and the production of plasma proteins in primary breast cancer patients on doxorubicincyclophosphamide (AC) protocol.

Doxorubicin and Cyclophosphamide (AC protocol) combination is usually considered as a first line therapy in newly diagnosed breast cancer patients. Thus, a retrospective observational study was conducted to monitor the effect of AC protocol on liver synthetic functions and production of plasma proteins in breast cancer patients, reporting to specialized cancer care hospital of Lahore, Pakistan. A total of 75 patients (n=75) on AC protocol with breast cancer were observed in this study. The patient data including age, gender, body surface area, dosage, disease status and laboratory biochemical values were recorded by reviewing historical treatment records. Pre-treatment values were taken as baseline values for albumin, globulin, blood urea nitrogen (BUN), albumin/globulin (A/G) ratio and total proteins. The baseline values were compared after each cycle of by applying ANOVA using statistical tool SPSS® version 21. The plasma levels of blood urea nitrogen (BUN), total protein and globulin dropped significantly (p<0.05) in patients of all age groups. However, the albumin levels were not significantly changed (p>0.05). The A/G ratio level increased (p<0.05) as a result of reduction in globulin levels. Significant changes in plasma protein levels were observed in the elderly patients (50 to 65 years) than patients between 20 to 50 years of age. AC protocol impairs liver synthetic functions as observed by decreased blood urea nitrogen (BUN) and plasma protein levels.

Lack of Globulin Synthesis during Seed Development Alters Accumulation of Seed Storage Proteins in Rice.

The major seed storage proteins (SSPs) in rice seeds have been classified into three types, glutelins, prolamins, and globulin, and the proportion of each SSP varies. It has been shown in rice mutants that when either glutelins or prolamins are defective, the expression of another type of SSP is promoted to counterbalance the deficit. However, we observed reduced abundances of glutelins and prolamins in dry seeds of a globulin-deficient rice mutant (Glb-RNAi), which was generated with RNA interference (RNAi)-induced suppression of globulin expression. The expression of the prolamin and glutelin subfamily genes was reduced in the immature seeds of Glb-RNAi lines compared with those in wild type. A proteomic analysis of Glb-RNAi seeds showed that the reductions in glutelin and prolamin were conserved at the protein level. The decreased pattern in glutelin was also significant in the presence of a reductant, suggesting that the polymerization of the glutelin proteins via intramolecular disulfide bonds could be interrupted in Glb-RNAi seeds. We also observed aberrant and loosely packed structures in the storage organelles of Glb-RNAi seeds, which may be attributable to the reductions in SSPs. In this study, we evaluated the role of rice globulin in seed development, showing that a deficiency in globulin could comprehensively reduce the expression of other SSPs.

Generation of recombinant hyperimmune globulins from diverse B-cell repertoires.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

Co-expression of alpha' and beta subunits of beta-conglycinin in rice seeds and its effect on the accumulation behavior of the expressed proteins.

A transgenic rice that produces both the alpha' and beta subunits of beta-conglycinin has been developed through the crossing of two types of transgenic rice. Although the accumulation level of the alpha' subunit in the alpha'beta-transgenic rice was slightly lower than that in the transgenic rice producing only the alpha' subunit, the accumulation level of the beta subunit in the alpha'beta-transgenic rice was about 60% higher than that in the transgenic rice producing only the beta subunit. Results from sequential extraction and gel-filtration experiments indicated that part of the beta subunit formed heterotrimers with the alpha' subunit in a similar manner as in soybean seeds and that the heterotrimers interacted with glutelin via cysteine residues. These results imply that the accumulation level of the beta subunit in the alpha'beta-transgenic rice increases by an indirect interaction with glutelin. Immunoelectron microscopy revealed that the alpha' and beta subunits are localized in a low electron-dense region of protein body-II (PB-II) and that alpha' homotrimers in the alpha'beta-transgenic rice seeds seem to accumulate outside of this low electron-dense region.

Initiation of protein synthesis at an unusual position in an immunoglobulin gene?

The amino acid sequence of urinary beta(2)-microglobulin has been partially determined and found to be related to the constant region of IgG immunoglobulin heavy chain. beta(2)-Microglobulin is present in normal individuals. Its gene may have evolved from an immunoglobulin gene by the use of an unusually located start signal for initiating synthesis of the polypeptide.

Recombinant soybean protein beta-conglycinin alpha'-subunit expression and induced hypersensitivity reaction in rats.

BACKGROUND: The major storage protein in soybean seed is beta-conglycinin and this protein has been identified as being responsible for food-allergic reactions in several species. However, the mechanism through which beta-conglycinin induces an allergic reaction has not yet been elucidated. In addition, assessing the antigenic activity of beta-conglycinin by studying the activity of a subunit has rarely been conducted. Therefore, the objective of the present study was to characterize the antigenic specificity of the beta-conglycinin alpha'-subunit. METHODS: We established an Escherichia coli expression system to obtain beta-conglycinin alpha'-subunit. The fusion proteins were then used in a rat model to induce a hypersensitive reaction. Immunoblotting, IgE and IgG1 level, histamine release, and passive cutaneous anaphylaxis reactions and intestinal histology were tested to assess the allergenic activity of the beta-conglycinin alpha'-subunit. RESULTS: Pure beta-conglycinin alpha'-subunit was obtained by expression in E. coli. The recombinant proteins were shown to have the same biological activity as the natural beta-conglycinin alpha'-subunit using immunoblotting analysis. Both the IgE and IgG1 level in serum and the histamine concentration in the intestine were increased while passive cutaneous anaphylactic reactions were induced in Brown Norway rats by intragastric gavage with the alpha'-subunit. Histamine release of mast cells was also elevated in vitro. CONCLUSIONS: Our results indicate that the beta-conglycinin alpha'-subunit possesses an intrinsic immune-stimulating capacity and that it can induce an allergic reaction. Moreover, this study showed that beta-conglycinin alpha'-subunit-induced anaphylaxis is IgE mediated, and mast cell degranulation and histamine release are associated with anaphylactic symptoms.

Creation of soybean beta-conglycinin beta with strong phagocytosis-stimulating activity.

beta-Conglycinin is composed of three kinds of subunit: alpha, alpha' and beta. A phagocytosis-stimulating peptide sequence (MITLAIPVNKPGR), soymetide, exists in the alpha' subunit of beta-conglycinin. Met at N terminus of the soymetide is essential for the activity. When Thr at the third residue from N terminus of the soymetide is replaced by Phe or Trp, the phagocytosis-stimulating activity greatly increases (ThrMet, Lys-->Thr, Phe, or Trp) into the beta subunit after confirmation of the effects of residue replacements by molecular modeling, suggesting that the introduced mutations might not prevent the correct folding. The studies of circular dichroism (CD), gel filtration and differential scanning calorimetry (DSC) of the mutants (I122M/K124T, I122M/K124F, I122M/K124W) expressed in E. coli demonstrated that they folded and self-assembled similarly to the wild type. This was confirmed by X-ray analysis of I122M/K124W crystal where the biggest residue tryptophane was introduced. The three mutants exhibited phagocytosis activities after digestion by trypsin, and the order was the wild type

Synthesis of alpha- and beta-globin directed by messenger ribonucleoprotein from rabbit reticulocytes.

The translation of globin messenger ribonucleoprotein (mRNP) obtained from high salt-washed rabbit reticulocyte ribosomes by treatment with EDTA was investigated using a cell-free system from mouse Krebs II ascites tumour cells. The messenger activity of the mRNP and the mRNA derived from it by mild deproteinization was compared in the presence and absence of reticulocyte initiation factors. Both forms gave identical products over a wide range of messenger concentration and there was no qualitative or quantitative difference in their efficiency as messengers. It is concluded that the proteins associated with polysomal mRNA do not alter the specificity of translation of alpha- and beta-globin messengers or the requirement for initiation factor.

In vitro and in vivo alterations of long-term murine plasmacytoma cultures.

Mouse myeloma cell line (SLU-5) that has undergone lengthy propagation in vitro and become nononcogenic was compared with an earlier oncogenic passage of cells. Immunogenicity of cells cultured for various periods of time was compared with that of the original tumor (MOPC-21). The nononcogenic cells were most immunogenic. Cells that were nononcogenic in normal mice produced tumors in nonlethally irradiated mice. Clones isolated from oncogenic and nononcogenic populations varied widely with respect to ability to produce tumors in mice and to specific globulin production.

Expression of soybean glycinin subunit precursor cDNAs in Escherichia coli.

As the cDNAs encoding A1aB1b and A2B1a subunit precursors of the glycinin A2 subfamily contain a unique NcoI site sequence, (A)CCATGG, occurring at their translation initiation sites, plasmids were constructed to direct the synthesis of those precursor proteins by inserting NcoI/PstI fragments derived from those cDNA clones into the NcoI/PstI-pKK233-2 expression vector in Escherichia coli MV1190, respectively. The resultant plasmids directed the expression of 57-kDa protein components that have molecular masses in agreement with those of the in vitro translation products directed by glycinin A2 subfamily mRNAs, by the addition of isopropyl beta-D-thiogalactoside. These proteins, which comprised as much as 1% of the total bacterial protein, are immunoprecipitable with rabbit antibodies specific for glycinin subunits. This procedure makes glycinin subunits available as a model for studying structure-function relationships in seed proteins using site-directed mutagenesis. This is the first expression of glycinin-like storage protein in E. coli.

Preparation, characterization, and primate testing of monoclonal antithymocyte globulin.

Monoclonal antibodies specific for T cells from both the human and rhesus primate species were detected by their ability to inhibit T cell rosette formation with sheep erythrocytes. The antibodies were shown by fluorescence techniques to react with all thymocytes and peripheral blood T cells but not to B cells, monocytes, platelets, or erythrocytes. Rosette inhibition titers of these antibodies were 30-fold lower when rhesus, rather than human T, cells were used as the rosette-forming cell in assay. Nonetheless, two monoclonal antibodies, of the IgG3 isotype, termed antithymocyte monoclonal (ATM) e.1 and 2.2, were shown to depress selectively circulating T cells to nondetectable levels following single dose administration to rhesus primates and to prolong skin allograft survival in a rhesus primate given a 6-dose course of treatment. The rhesus primates suffered no ill effects and no peripheral blood cellular component other than T cells was depressed. Monoclonal antibody secreting hybridoma cells are capable of producing ATM 3.1 and 3.2 in quantity when grown as peritoneal tumors in selected mouse hybrids. Purification of ATM 3.1 or 3.2 is easily accomplished by affinity chromatography on protein A. These properties suggest that ATM antibodies may become useful immunosuppressive agents in clinical transplantation.

Effects of short-term exposure to pulsed electromagnetic field on some biochemical parameters in mice.

Five-months-old male albino mice were subjected to an electromagnetic field (EMF) of 5 mT of magnitude with a frequency of 60 Hz for 8hr of single application. Analysis of blood sampled on hourly basis (up to 8 hr) for levels/activities of total protein, albumin, globulin, uric acid, creatinine, cholesterol, and alkaline phosphatase indicated no significant differences (p > 0.05) from that of the control group.

OsAAP6 functions as an important regulator of grain protein content and nutritional quality in rice.

Grains from cereals contribute an important source of protein to human food, and grain protein content (GPC) is an important determinant of nutritional quality in cereals. Here we show that the quantitative trait locus (QTL) qPC1 in rice controls GPC by regulating the synthesis and accumulation of glutelins, prolamins, globulins, albumins and starch. qPC1 encodes a putative amino acid transporter OsAAP6, which functions as a positive regulator of GPC in rice, such that higher expression of OsAAP6 is correlated with higher GPC. OsAAP6 greatly enhances root absorption of a range of amino acids and has effects on the distribution of various amino acids. Two common variations in the potential cis-regulatory elements of the OsAAP6 5'-untranslated region seem to be associated with GPC diversity mainly in indica cultivars. Our results represent the first step toward unravelling the mechanism of regulation underlying natural variation of GPC in rice.

Stable accumulation of seed storage proteins containing vaccine peptides in transgenic soybean seeds.

There has been a significant increase in the use of transgenic plants for the large-scale production of pharmaceuticals and industrial proteins. Here, we report the stable accumulation of seed storage proteins containing disease vaccine peptides in transgenic soybean seeds. To synthesize vaccine peptides in soybean seeds, we used seed storage proteins as a carrier and a soybean breeding line lacking major seed storage proteins as a host. Vaccine peptides were inserted into the flexible disordered regions in the A1aB1b subunit three-dimensional structure. The A1aB1b subunit containing vaccine peptides in the disordered regions were sorted to the protein storage vacuoles where vaccine peptides are partially cleaved by proteases. In contrast, the endoplasmic reticulum (ER)-retention type of the A1aB1b subunit containing vaccine peptides accumulated in compartments that originated from the ER as an intact pro-form. These results indicate that the ER may be an organelle suitable for the stable accumulation of bioactive peptides using seed storage proteins as carriers.

Differential expression and elution behavior of basic 7S globulin among cultivars under hot water treatment of soybean seeds.

Basic 7S globulin (Bg7S), which accumulates in mature soybean (Glycine max) seeds, is an extracellular matrix protein. A large amount of Bg7S is synthesized de novo and is eluted from soybean seeds when immersed in 50-60°C water (hot water treatment, HWT). However, the Bg7S elution mechanism remains unclear. Under HWT, the seeds probably undergo heat stress and flooding stress. To obtain fundamental knowledge related to how Bg7S is eluted from hot-water-treated seeds, this study compared Bg7S elution among soybean cultivars having different flooding tolerance during pre-germination. The amounts of Bg7S eluted from seeds varied significantly among cultivars. Elution was suppressed by seed coats regarded as preventing the leakage of seed contents by rapid water imbibition. Furthermore, Bg7S expression levels differed among cultivars, although the difference did not result from any variation in Bg7S promoter sequences. However, the expression levels of Bg7S under HWT were not associated with the flooding tolerance level. Immunoelectron microscopy revealed that the Bg7S accumulated in the intercellular space of hot-water-treated seeds. Plasma membrane shrinkage was observed. The main proteins eluted from seeds under HWT were located in the extracellular space. This study clarified the mechanism of Bg7S elution from seeds under HWT.

Overexpression of a modified amaranth protein in Escherichia coli with minimal media and lactose as inducer.

In this research it was attempted to overexpress the acidic subunit, from the 11S amaranth seed globulin termed amarantin, modified with antihypertensive peptides in Escherichia coli Rosetta (DE3) by manipulating some factors in batch fermenter such as growth medium composition, inducer (isopropyl ß-D-thiogalactopyranoside [IPTG] or lactose), air flow, cultivation temperature, agitation speed and induction time. The possibility of using several minimal media and lactose as inducer to increase yields of the recombinant protein was investigated. Previous fermentations at flask level showed that two minimal culture media (A6 and A7) and 0.5% (w/v) lactose presented high yields of the engineered protein expression. Thus, the latter two media were tested at fermenter level, the lactose inducer, and different environmental conditions. Factors with significant effects were identified by Plackett-Burman design with center points and were adjusted at the level suggested and the yields of the recombinant protein were increased from 303.2 to 1,531 mg L(-1) in A6 and from 363.4 to 1,681 mg L(-1) in A7. Unlike some patents where the highest productivity was achieved at 24 h or afterwards, in this research the best productivity of the recombinant acidic subunit was attained at 4 and 6 h of induction using both media, respectively.

Effect of environmental conditions on the expression levels of a recombinant 11S amaranth globulin in Escherichia coli.

The expression in Escherichia coli strain Rosetta of the recombinant acidic subunit from the 11S amaranth seed storage protein (protein ACM3) was studied at flask and at bioreactor levels. This subunit was modified by inserting four Val-Tyr antihypertensive peptides in tandem into its third variable region and also with the tripeptide Ile-Pro-Pro in the Cterminal region. Flasks experiments allowed us to define the best conditions for the preparation and expression and accumulation of the protein ACM3, including the certainty of its presence within the cells especially as an insoluble fraction. The effects of cultivation temperature, aeration rate and agitation speed on the production of the protein ACM3 was tested in a 5-L batch bioreactor. Applying response surface methodology (RSM) we found that the aeration rate was the most significant factor affecting in a positively way the production yields and productivity of the recombinant protein. Temperature had effect only in conjunction to aeration. The highest recombinant acidic subunit concentration (747 mg L-1) and the highest productivity (186 mg L-1 h-1) were attained in 4 h of cultivation when the factors evaluated were controlled at its central values: 0.1 vvm, 300 rpm, and 30.5° C. Results from this study indicate that RSM is an effective technique to maximize the production of this recombinant protein.