Equivalent Protein Levels Research Articles

Anti-tumor effect of antibody drug conjugate ASP1235 targeting Fms-like tyrosine kinase 3 with venetoclax plus azacitidine in an acute myeloid leukemia xenograft mouse model.

Antibody drug conjugates (ADC) are one of the attractive modalities for the treatment of acute myeloid leukemia (AML). Previously, we have developed ASP1235, a novel ADC targeting Fms-like tyrosine kinase 3 (FLT3) which is widely expressed on the leukemic blasts of AML patients. In this study, we sought to evaluate the therapeutic effect of ASP1235 in combination with venetoclax plus azacitidine, a novel standard-of-care treatment for elderly AML patients, in ASP1235 poor sensitive AML cells. To identify the suitable preclinical model, we first evaluated the growth inhibitory effect of ASP1235 on several leukemia cell lines expressing FLT3 and found that THP-1 cells were partially sensitive to ASP1235 in vitro. Furthermore, ASP1235 showed marginal anti-tumor activity in a THP-1 xenograft model. Compared to the leukemic blasts in most of the relapsed or refractory (R/R) AML patients tested, THP-1 cells expressed equivalent protein levels of Bcl-2, suggesting that ASP1235 in combination with venetoclax plus azacitidine is a rational treatment in the THP-1 model. In vitro, ASP1235 showed a cytotoxic effect on THP-1 cells in combination with venetoclax, and the combination effect was greater than the additive effect. Furthermore, ASP1235 also showed a combination effect with venetoclax plus azacitidine treatment. Similarly, the combination of ASP1235, venetoclax and azacitidine showed a superior anti-tumor effect in a THP-1 xenograft model without obvious body weight loss. These findings provide supportive evidence that the triple combination of ASP1235, venetoclax and azacitidine would improve the clinical outcome of ASP1235 monotherapy and venetoclax plus azacitidine regimen in AML patients.

The HCM-linked W792R mutation in cardiac myosin-binding protein C reduces C6 FnIII domain stability.

Cardiac myosin-binding protein C (cMyBP-C) is a functional sarcomeric protein that regulates contractility in response to contractile demand, and many mutations in cMyBP-C lead to hypertrophic cardiomyopathy (HCM). To gain insight into the effects of disease-causing cMyBP-C missense mutations on contractile function, we expressed the pathogenic W792R mutation (substitution of a highly conserved tryptophan residue by an arginine residue at position 792) in mouse cardiomyocytes lacking endogenous cMyBP-C and studied the functional effects using three-dimensional engineered cardiac tissue constructs (mECTs). Based on complete conservation of tryptophan at this location in fibronectin type II (FnIII) domains, we hypothesized that the W792R mutation affects folding of the C6 FnIII domain, destabilizing the mutant protein. Adenoviral transduction of wild-type (WT) and W792R cDNA achieved equivalent mRNA transcript abundance, but not equivalent protein levels, with W792R compared with WT controls. mECTs expressing W792R demonstrated abnormal contractile kinetics compared with WT mECTs that were nearly identical to cMyBP-C-deficient mECTs. We studied whether common pathways of protein degradation were responsible for the rapid degradation of W792R cMyBP-C. Inhibition of both ubiquitin-proteasome and lysosomal degradation pathways failed to increase full-length mutant protein abundance to WT equivalence, suggesting rapid cytosolic degradation. Bacterial expression of WT and W792R protein fragments demonstrated decreased mutant stability with altered thermal denaturation and increased susceptibility to trypsin digestion. These data suggest that the W792R mutation destabilizes the C6 FnIII domain of cMyBP-C, resulting in decreased full-length protein expression. This study highlights the vulnerability of FnIII-like domains to mutations that alter domain stability and further indicates that missense mutations in cMyBP-C can cause disease through a mechanism of haploinsufficiency. NEW & NOTEWORTHY This study is one of the first to describe a disease mechanism for a missense mutation in cardiac myosin-binding protein C linked to hypertrophic cardiomyopathy. The mutation decreases stability of the fibronectin type III domain and results in substantially reduced mutant protein expression dissonant to transcript abundance.

Differences in human phospholipid transfer protein activity following incubation of Fungizone® compared to lipid-based Amphotericin-B formulations in normolipidemic and hyperlipidemic plasma

Aim: To investigate how different formulations of Amphotericin-B (Amp-B) affect the activity of phospholipid transfer protein (PLTP) when incubated with hyperlipidemic and normolipidemic plasma at physiological temperature (37°C). Methods: Six hyperlipidemic and six normolipidemic plasma samples were collected and tested for protein concentration. Equivalent protein levels (25 μg) were then tested for PLTP activity using an in vitro established kit at physiological temperature (37°C). Increasing concentrations of different Amp-B formulations (1, 2, and 5 μg/mL) in the pharmacological range were then added to the plasma and tested for activity from 5 to 90 minutes. The Amp-B formulations used in the study were Fungizone®, Abelcet®, and AmBisome®. Results: In normolipidemic plasma, PLTP activity was found to be increased by Abelcet® and AmBisome® but inhibited by Fungizone®. In hyperlipidemic plasma, PLTP activity was found to be increased by Abelcet® and AmBisome® but not changed by Fungizone®. The Vm value for Abelcet® and AmBisome® was higher than Fungizone®; although, no difference was observed in the Km values between formulations. Conclusions: Findings suggest that lipid-based formulations of Amp-B promote the transfer of Amp-B into high-density lipoprotein fractions at a degree of increase inversely proportional to the lipid levels in the plasma.

Tissue Expression and Genomic Sequences of Rat N-acetyltransferases rNat1, rNat2, rNat3, and Functional Characterization of a Novel rNat3*2 Genetic Variant

Human arylamine N-acetyltransferases NAT1 and NAT2 are highly polymorphic genes that modify individual susceptibility to cancers caused by exposure to arylamine procarcinogens. Strong similarities exist between rat Nats and human NATs, and rat Nat2 polymorphisms result in slow acetylator phenotype. Recently, a third rat Nat, rNat3*1, was reported. Although in vivo toxicological and carcinogenic studies are often conducted in rats, relatively little is known about Nat sequences among available inbred rat strains. We report here that rNat1 and rNat2 open reading frames (ORFs) in 12 inbred rat strains (ACI, BN, BUF, CDF, COP, DA, LEW, LOU/M, MW, PVG, SHR, WF) corresponded to reference rNat1*13 and rNat2*20. While 10 of the 12 strains had reference rNat3*1 ORFs, strains ACI and COP had a variant rNat3*2 ORF characterized by a G619>T transversion (A207S). The rNat3*2 single nucleotide polymorphism reduced Nat3 protein levels and N- and O-acetyltransferase activity when recombinantly expressed in bacteria. Recombinant expression of rNat3 1 and rNat3 2 in COS-1 cells yielded equivalent protein levels but undetectable catalytic activities. Relative tissue expressions of rNat1, rNat2, and rNat3 mRNAs were assessed in liver and 12 extrahepatic tissues (lung, spleen, kidney, heart, esophagus, stomach, urinary bladder, prostate, colon, duodenum, jejunum, ileum) from male F344 rats exsanguinated prior to sacrifice. Semiquantitative RT-PCR experiments demonstrated that the relative expression of the rNat transcripts in liver and 12 extrahepatic tissues was rNat1 > rNat2, while rNat3 transcripts were not detected. This study concludes that rNat1 and rNat2 are primarily responsible for acetylation phenotype in rats.

Species-Specific Differences in Intracellular Transport Reveal a Golgi-Localization Requirement for D816V Mutant Kit Receptor To Induce Leukemia.

Mutations in the c-Kit gene (KIT) that result in constitutive tyrosine kinase activity are associated with mastocytosis/mast cell leukemia, gastrointestinal stromal tumors (GIST), and acute myeloid leukemia (AML). Expression of a murine Kit allele with a mutation in the phosphotransferase domain (Kit D814V) renders murine Ba/F3 cells factor-independent and causes lymphocytic leukemia in mice. An animal model using the homologous human allele (KIT D816V) has not been described. We generated retroviral expression constructs using wild-type and mutant human KIT and murine Kit that achieved equivalent protein levels in human-derived 293T cells. When expressed in murine cells however, despite equivalent transcript levels, the KITD816V protein specifically was present in lower abundance, and unlike the murine mutant, the human D816V was unable to transform Ba/F3 cells without prior selection. In contrast, human KITD816V rapidly transformed human TF-1 cells to factor independence. Fusion of the extracellular and transmembrane domains of murine Kit to the intracellular domain of human KIT (Hybrid KIT WT and Hybrid KITD816V) facilitated higher protein expression in murine cells, and rapid transformation of both Ba/F3 and TF-1 cells. Endoglycosidase digestion revealed that both human and murine mutant Kit receptors were incompletely glycosylated, and by immunohistochemistry we found that the murine and hybrid mutant KIT receptors were predominantly localized to the golgi. However, the human KIT D816V protein, in murine cells, failed to traffic past the endoplasmic reticulum (ER), while in human cells, KITD816V was able to traffic through the ER to the golgi. In murine cells expressing human KITD816V, downstream signaling pathways, including Stat3, Stat5 and Akt were activated to a lesser extent compared to cells expressing the murine KitD814V or hybrid KITD816V. Treatment of cells with small molecule inhibitors of protein trafficking demonstrated that activation of downstream signaling pathways activated by Hybrid KITD816V required golgi but not cell membrane localization. Lastly, mice transplanted with bone marrow transduced with the human KITD816V remained free of disease while mice transplanted with the golgi-localized Hybrid KITD816V succumbed to fatal myeloproliferative disease (MPD) with 100% penetrance. Taken together, our data demonstrate that murine and human cells traffic the KITD816V protein differently and that leukemogenic signals require localization of the mutant receptor to the golgi apparatus. This is the first report of an animal model of myeloid leukemia/MPD using the KIT oncogene and will be useful for elucidating the mechanisms by which KITD816V contributes to leukemogenesis and for testing novel therapeutic strategies.

Nutritive value of rubber seed (Hevea brasiliensis) meal: utilization by growing pigs of semipurified diets in which rubber seed meal partially replaced soybean meal.

Thirty-six four-way cross (Chester White X Landrace X Large White X Yorkshire) growing barrows and gilts were used to investigate the effects of replacing graded levels of protein from soybean meal with equivalent levels of protein from rubber seed meal in 16% CP semipurified diets on the performance characteristics, hematocrit, plasma metabolites and N utilization of pigs. The first diet (control) was largely cornstarch-soybean meal in which the soybean meal supplied all of the CP. In other diets, rubber seed meal replaced 10%, 20% and 30%, respectively, of the protein of soybean meal in the control diet. Twenty gilts were used in a 28-d growth trial and 16 barrows were used in an 8-d digestion trial (4-d collection). There were no differences (P greater than .05) in ADG, ADF intake and in the feed:gain ratios, even though there was a trend for lower ADG and gain:feed ratio as the level of rubber seed meal increased in the diet. There were no differences in hematocrit, but plasma protein and albumin tended to be depressed when rubber seed meal provided more than 10% of the dietary protein. Apparent digestibilities of GE, DM and N were lower with rubber seed meal at 20% of the protein than with any other diet, but apparent N retained and the percent of digested N retained were not depressed significantly (P greater than .05). Although rubber seed meal protein is of poorer quality than soybean meal protein for growing pigs, at least 10% of dietary protein can be provided by rubber seed meal without adversely affecting growth and N utilization.

Activation and detoxification of 1, 8-dinitropyrene by mammalian hepatic fractions in the Salmonella mutagenicity assay

1,8-Dinitropyrene (DNP) is a potent, direct-acting mutagen to Salmonella typhimurium TA98 due to activation by bacterial metabolism. We have studied the effects of uninduced and Aroclor-induced rodent liver post-mitochondrial supernatant (S9), microsomes or cytosolic preparations on its activity. With the plate incorporation protocol, the reported S9-mediated inhibition of mutagenicity was confirmed and shown to be greater after induction when equivalent levels of protein were plated. Cytosol increased mutagenicity considerably whereas the inhibitory effect of S9 could be attributed to the microsomal fraction. The activating and detoxifying capacities of the various hepatic fractions derived from male Sprague-Dawley rats and female BALB/c mice were similar. In the absence of hepatic preparations, DNP mutagenicity was much lower in TA98/1,8DNP6 (deficient in transacetylase) than in TA98, although mutagenic activity could be still increased in the former strain by addition of uninduced cytosol. When DNP was pre-incubated in a liquid assay with S9 before addition of TA98, inhibition occurred and was time dependent. Thus the overall detoxifying effect of S9 is independent of bacterial metabolism. Furthermore, inhibition occurred under conditions where cytosol and microsomal components of S9 were kept together, indicating that the detoxifying effect of the microsomes predominates. The data therefore suggest that the microsomal-mediated detoxification by S9 may be due to the presence of an inhibitory component, enzymatic or otherwise, whose concentration is increased after Aroclor-induction. Nitro-reductase activities detected in TA98 and TA98/1,8-DNP6 were 10-fold higher than those in the mammalian fractions, although the activities in the latter increased to the bacterial enzyme levels after incubation of induced S9 or microsomes, but not cytosol, with an NADPH-generating system.(ABSTRACT TRUNCATED AT 250 WORDS)

A preliminary study of the suitability of dehydrated poultry slaughterhouse wastewater as a constituent of broiler feeds

In a preliminary experiment the effects of six levels of effluent mixture in the diet were compared with the effect of a control diet (2, 3, 4, 5, 6, 7% pure effluent after correction balance) and the effects of 4 and 7% of effluent were compared with those of equivalent protein levels supplied by meat, blood and herring meal. All diets were calculated on a linear programming basis isonitrogenously and isoenergetically and contained the same calcium and phosphorus levels. Methionine and lysine were added when needed. A number of 910 male white-shanked (Hubbard) broiler chicks were kept in cages, 13 groups, seven times replicated. 1. Effluent (sludge) was found to be a reasonable animal protein feedstuff as far as methionine, cystine and energy (ME) are concerned. Its disadvantages are the high cost of drying and its high iron content. 2. Increasing the proportion of effluent in the feed from 2 to 7% leads to a clear growth depression at 4 and 7 weeks of age because of the high iron concentration. 3. Herring meal was significantly superior to that of dried effluent protein, fed on equivalent protein levels. Blood and meat meal were comparable to effluent protein.

The influence of cooked kidney beans (Phaseolus vulgaris) on intestinal cell turnover and faecal nitrogen excretion in the rat.

Male Wistar rats were fed on semi-synthetic diets containing cooked white kidney beans (Phaseolus vulgaris) or equivalent levels of protein and carbohydrate. No change was observed in over-all nitrogen balance in animals fed on the bean diet, but there was a two- to three-fold increase in their faecal excretion, compared with control rats. This was compensated by a decrease in urinary-N excretion. Homogenized small intestinal mucosa, prepared from bean-fed animals, showed a 28% increase in protein content compared with control material. Measurements of 3H-labelled thymidine turnover indicated that mucosal cell exfoliation was increased by approximately 35% in the small intestines of bean-fed rats compared with controls. It is concluded that though a diet rich in cooked P. vulgaris leads to some increase in mucosal cell turnover in the small intestine of rats, the consequent increase in mucosal protein loss could not account for the increased faecal-N excretion seen in these animals.

The baking quality and protein characteristics of a winter wheat grown at different levels of nitrogen fertilisation

AbstractThree samples of field‐grown winter wheat (Triticum aestivum cv. Atou) with different protein contents were produced by late application of urea as a nitrogenous fertiliser. Baking tests (a conventional fermentation procedure) indicated that the breadmaking quality of the flours increased as protein content was raised from the lowest to the intermediate level, but that the flours of intermediate and highest protein content were of equivalent breadmaking quality. To compare gluten baking quality independently of protein quantity, loaves were also baked from ‘flours’ reconstituted to equivalent protein levels using the isolated glutens. The flours of low and intermediate protein content yielded glutens of similar baking quality. The gluten derived from the flour of highest protein content gave a lower loaf volume and texture score: subsequent biochemical investigations suggested that this was due to an effect of the relative levels of nitrogen and sulphur available to the plants grown on this particular soil. Analysis of the flours and glutens indicated that the ratio of sulphur:nitrogen fell as grain protein content increased and this correlated with a lower proportion of the sulphur amino acids cyst(e)ine and methionine. Gel electrophoresis studies revealed, in particular, an increase in the proportion of the sulphur‐deficient, ω‐gliadin species as grain protein content increased. Agarose gel filtration chromatography of the flour and gluten proteins also suggested a correlation between the extent of aggregation of their glutenin components (mediated by disulphide bonds involving cystine residues) and their functional properties. The results of this study suggest that for wheat grown on this particular soil late application of high levels of a nitrogenous fertiliser in the absence of sulphur fertilisation led to a change in the balance between available nitrogen and available sulphur, such that the available sulphur levels became insufficient for ‘normal’ grain development. Nevertheless, the results indicated that considerable alteration in the biochemical characteristics of the flour proteins occurred before gluten baking quality was noticeably affected.

Estrogen receptors in uterine plasma membrane

In order to assess the subcellular distribution of estrogen-binding components in their native state, plasma membrane and other cell fractions were prepared by quantitative methods from uterine cells in the absence of [ 3H]-estradiol-17β (E 2β). Cells isolated from uteri of ovariectomized rats were disrupted with minimum homogenization in buffered isotonic sucrose with CaCl 2 and fractionated using isotonic media throughout. Activities of succinate dehydrogenase and acid phosphatase were concentrated in the mitochondria:lysosome(M + L) fraction. Glucose-6-phosphatase occurred predominantly in the microsome-rich (P) fraction. Alkaline phosphatase and 5'-nucleotidase were found principally in P and crude nuclear (N) fractions, with N also retaining 96% of DNA. Determinations of specific [ 3H]-E 2β binding to cell fractions at equivalent protein levels were conducted by equilibration for 2 h at 4°C. Binding-sites for E 2β were present in N > P > M + L > 105,000 g supernates (S). However, by using alternative homogenization procedures known to elicit lysis, fragmentation, and stripping of cell structures, E 2β binding-sites as well as 5'-nucleotidase, a plasma membrane marker enzyme, both occurred predominantly in S. Using the more conservative cell disruption and fractionation procedures, plasma membrane subfractions (F2) with densities of 1.13–1.16 were partially purified, principally as smooth membrane vesicles and large “ghosts”, by further centrifugation of N in a discontinuous sucrose density gradient. Activity of 5'-nucleotidase in F2 was enriched to 12 times that of the homogenate. Specific binding-sites for E 2β were concentrated in F2 to 23 times homogenate levels. Such binding of E 2β in F2 was saturable, with an association constant of 4.3 × 10 10 M −1. At saturation, E 2β receptors in F2 correspond to 2.0 pmol/mg membrane protein. Ligand specificity of [ 3H]-E 2β binding to F2 was established by negligible competition by 200-fold molar excess of unlabelled estradiol-17α, cortisol, testosterone, or progesterone, whereas E 2β and diethylstilbestrol were effective inhibitors. Specific binding of E 2β to F2 at 4°C was blocked by prior exposure of membranes to trypsin or to 60°C, but remained essentially undiminished by extraction of membranes with either severely hypotonic or high-salt buffers. In controlled experiments, it was found that only 6–9% of [ 3H]-E 2β-labelled cytosol components became associated with F2 during 2 h incubation at 4°C; the bulk of such adsorbed material was readily extracted by high-salt buffers. Thus, enrichment of F2 in E 2β-binding activity cannot be attributed to gross entrapment or adsorption of cytosolic components. These data indicate that high-affinity membrane binding-sites with specificity for E 2β must be further considered in investigations of the uterine cell recognition of and response to the hormone.

Feed Texture and Light Treatment Effects on the Performance of Chicken Broilers

A total of 4800 broiler chicks from a commercial parent genotype was used in two experiments to evaluate effects of feeding finisher diets containing different levels of “fines”. These experiments also included a comparison of incandescent continuous green lighting and white lighting with continuous and intermittent exposure at different intensities, including one treatment which was phased down to darkness at 21 days of age and continued to 49 days of age. Fines used in these diets were ground pellets having equivalent levels of protein, fat and fiber. Performance of birds fed crumbled starters (1–28d) and pelleted finishers (29–49d) was superior to birds fed all-mash starters and finishers. As the proportion of fines in finisher diets increased, there was a significant decrease in body weights and monetary returns.Performance under both intermittent white lighting vs. continuous white or green lighting had no differential impact with light intensity of .5 lux or 7 lux from 21 to 49 days of age. Broilers grown in darkness from 21 to 49 days performed well although body weights were reduced compared with birds grown in light. There was no evidence of an interaction between light and feed texture treatments.