Maintaining Protein Levels Research Articles

Soybean seed yield, protein, and oil concentration for a modern and old genotype under varying row spacings

Progress in soybean (Glycine max L.) breeding has led to a reduction in optimal seeding rates due to enhanced branching capacity over time. However, less is known about the changes in canopy architecture between old and modern soybean genotypes at varying row spacing and their impact on yield and seed quality through the main stem and branches. Therefore, this study aimed to i) evaluate yield and seed quality responses of an old and modern soybean genotype at different row spacings and ii) examine the yield and seed quality of branches and the main stem. Trials were conducted in Kansas (United States, US) during 2020 and 2021, comparing two genotypes (old, released in 1980, and modern, released in 2013) at four row spacings (0.19, 0.38, 0.76, and 1.52 m) under rainfed conditions. Seed yield and quality (protein and oil concentrations, %) were assessed at the end of each growing season. In 2021, both genotypes had low and similar yields at all row spacings (averaging 2481 kg ha−1) with 2.5 % less protein on branches compared to the main stem. However, 2022 resulted in a high-yielding environment, with the modern yielding 50 % more (3584 kg ha−1) than the old (2315 kg ha−1) genotype in narrow row spacings (<0.38 m). Additionally, the modern genotype showed a three-fold greater contribution to yield from branches (1113 kg ha−1) relative to the old genotype (379 kg ha−1). Despite the high yields observed in narrow rows, the modern genotype maintained protein levels. These results highlight the importance of row spacing as a key management practice for improving yield while maintaining protein levels in high yield conditions.

Arabidopsis PROTODERMAL FACTOR2 binds lysophosphatidylcholines and transcriptionally regulates phospholipid metabolism.

Plant homeodomain leucine zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. While the START domain is required for TF activity, its presumed role as a lipid sensor is not clear. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative member that controls epidermal differentiation, recruits lysophosphatidylcholines (LysoPCs) in a START-dependent manner. Microscale thermophoresis assays confirmed that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally found that PDF2 acts as a transcriptional regulator of phospholipid- and phosphate (Pi) starvation-related genes and binds to a palindromic octamer with consensus to a Pi response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments revealed a role for START in maintaining protein levels, and Pi starvation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Our data provide insights toward understanding how the lipid metabolome integrates Pi availability with gene expression.

Magnetic Nanoparticles for Protein Separation and Purification.

Proteins are essential for various functions such as brain activity and muscle contraction in humans. Even though food is a source of proteins, the bioavailability of proteins in most foods is usually limited due to matrix interaction with other biomolecules. Thus, it is essential to extract these proteins and provide them as a nutraceutical supplement to maintain protein levels and avoid protein deficiency. Hence, protein purification and extraction from natural sources are highly significant in biomedical applications. Chromatography, crude mechanical disruption, use of extractive chemicals, and electrophoresis are some of the methods applied to isolate specific proteins. Even though these methods possess several advantages, they are unable to extract specific proteins with high purity. A suitable alternative is the use of nanoparticles, which can be beneficial in protein purification and extraction. Notably, magnetic iron and iron-based nanoparticles have been employed in protein extraction processes and can be reused via demagnetization due to their magnetic property, smaller size, morphology, high surface-to-volume ratio, and surface charge-mediated property. This chapter is a summary of various magnetic nanoparticles (MNPs) that can be used for the biomolecular separation of proteins.

Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism

Proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity clamp for eukaryotic DNA polymerases and a binding platform for many DNA replication and repair proteins. The enzymatic activities of PCNA loading and unloading have been studied extensively in vitro. However, the subcellular locations of PCNA loaders, replication complex C (RFC) and CTF18-RFC-like-complex (RLC), and PCNA unloader ATAD5-RLC remain elusive, and the role of their subunits RFC2-5 is unknown. Here we used protein fractionation to determine the subcellular localization of RFC and RLCs and affinity purification to find molecular requirements for the newly defined location. All RFC/RLC proteins were detected in the nuclease-resistant pellet fraction. RFC1 and ATAD5 were not detected in the non-ionic detergent-soluble and nuclease-susceptible chromatin fractions, independent of cell cycle or exogenous DNA damage. We found that small RFC proteins contribute to maintaining protein levels of the RFC/RLCs. RFC1, ATAD5, and RFC4 co-immunoprecipitated with lamina-associated polypeptide 2 (LAP2) α which regulates intranuclear lamin A/C. LAP2α knockout consistently reduced detection of RFC/RLCs in the pellet fraction, while marginally affecting total protein levels. Our findings strongly suggest that PCNA-mediated DNA transaction occurs through regulatory machinery associated with nuclear structures, such as the nuclear matrix.

Quercetin Antagonizes Glucose Fluctuation Induced Renal Injury by Inhibiting Aerobic Glycolysis via HIF-1α/miR-210/ISCU/FeS Pathway.

Background and Objective: Glucose fluctuation (GF) has been reported to induce renal injury and diabetic nephropathy (DN). However, the mechanism still remains ambiguous. Mitochondrial energy metabolism, especially aerobic glycolysis, has been a hotspot of DN research for decades. The activation of HIF-1α/miR210/ISCU/FeS axis has provided a new explanation for aerobic glycolysis. Our previous studies indicated quercetin as a potential therapeutic drug for DN. This study aims to evaluate levels of aerobic glycolysis and repressive effect of quercetin via HIF-1α/miR210/ISCU/FeS axis in a cell model of GF.Methods: The mouse glomerular mesangial cells (MCs) were exposed in high or oscillating glucose with or without quercetin treatment. Cell viability was measured by CCK8 assay. Aerobic glycolysis flux was evaluated by lactate acid, pH activity of PFK. Apoptosis level was confirmed by Annexin V-APC/7-AAD double staining and activity of caspase-3. TNF-α and IL-1β were used to evaluate inflammation levels.Results: GF deteriorated inflammation damage and apoptosis injury in MCs, while quercetin could alleviate this GF-triggered cytotoxicity. GF intensified aerobic glycolysis in MCs and quercetin could inhibit this intensification in a dose-dependent manner. Quercetin prevented activities of two FeS-dependent metabolic enzymes, aconitase, and complex I, under GF injury in MCs. The mRNA expression and protein contents of HIF-1α were increased after GF exposure, and these could be alleviated by quercetin treatment. Knockdown of ISCU by siRNA and Up-regulating of miR-210 by mimic could weaken the effects of quercetin that maintained protein levels of ISCU1/2, improved cell viability, relieved inflammation injury, decreased apoptosis, and reduced aerobic glycolysis switch in MCs.Conclusion: Quercetin antagonizes GF-induced renal injury by suppressing aerobic glycolysis via HIF-1α/miR-210/ISCU/FeS pathway in MCs cell model. Our findings contribute to a new insight into understanding the mechanism of GF-induced renal injury and protective effects of quercetin.

Changes of the Proteome and Acetylome during Transition into the Stationary Phase in the Organohalide-Respiring Dehalococcoides mccartyi Strain CBDB1.

The strictly anaerobic bactGIerium Dehalococcoides mccartyi obligatorily depends on organohalide respiration for energy conservation and growth. The bacterium also plays an important role in bioremediation. Since there is no guarantee of a continuous supply of halogenated substrates in its natural environment, the question arises of how D. mccartyi maintains the synthesis and activity of dehalogenating enzymes under these conditions. Acetylation is a means by which energy-restricted microorganisms can modulate and maintain protein levels and their functionality. Here, we analyzed the proteome and Nε-lysine acetylome of D. mccartyi strain CBDB1 during growth with 1,2,3-trichlorobenzene as an electron acceptor. The high abundance of the membrane-localized organohalide respiration complex, consisting of the reductive dehalogenases CbrA and CbdbA80, the uptake hydrogenase HupLS, and the organohalide respiration-associated molybdoenzyme OmeA, was shown throughout growth. In addition, the number of acetylated proteins increased from 5% to 11% during the transition from the exponential to the stationary phase. Acetylation of the key proteins of central acetate metabolism and of CbrA, CbdbA80, and TatA, a component of the twin-arginine translocation machinery, suggests that acetylation might contribute to maintenance of the organohalide-respiring capacity of the bacterium during the stationary phase, thus providing a means of ensuring membrane protein integrity and a proton gradient.

Effects of short-term and continuous exposure to reduced salinities on the biochemical composition of larval lobster, Homarus gammarus

In coastal areas with estuarine influence, exposure to hypo-osmotic conditions may affect larval survival, development and growth. Most knowledge about effects of reduced salinity on coastal organisms is based on keeping individuals under constant conditions in the laboratory. By contrast, little is known about the effects of more realistic situations where organisms are exposed to low salinity over short time scales. Such environmental short-term fluctuations are expected to increase due to climate change. Here, we experimentally evaluated the sublethal effects of both short-term and continuous exposure to moderately reduced salinities (salinity 20 and 25; compared to seawater, salinity 32) in larvae of European lobster Homarus gammarus. Total body dry mass and biochemical composition (measured as: protein and lipid contents) were measured as response variables in Mysis stages I to III. Short-term effects of low salinity were quantified in a group of larvae kept in seawater from hatching until the time of transfer to the test salinities. After ca. 40 % of each moult cycle in seawater (determined in preliminary experiments for Mysis I, II and III), larvae were assigned to a seawater control or reduced salinities lasting for 16 h (i.e. until ca. 50 % of the time spent within the moulting cycle). Effects of continuous exposure to low salinity were quantified when larvae were exposed to the different salinities from hatching, until they reached ca. 50 % of the successive moulting stage. Surprisingly, in the Mysis II and III stages, short-term exposure to low salinity had much stronger effects on accumulation of reserves than the continuous exposure. Such effects were manifested mostly as limited accumulation, or even losses, in the lipid content as compared to reductions in the amount of protein accumulated. The most sensitive stage to exposure to low salinity was the Mysis III; by contrast in Mysis I such effects were relative weak (not always significant). Chronic exposure to low salinity also led to an increase in developmental time especially at the advanced stages. Our results highlight the importance of quantifying effects of environmental fluctuations at different time scales in order to better understand how organisms cope with realistic environmental change in the coastal zones. For H. gammarus, our results suggest that larvae respond adaptively to low salinity by maintaining protein levels at expenses of reductions in lipid accumulation and by extending the developmental time, but the capacity to elicit a fully compensatory response varies ontogenetically.

Terminal Regions Confer Plasticity to the Tetrameric Assembly of Human HspB2 and HspB3

Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes—from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterization. Atomic coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallized as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four α-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible “nuts and bolts” involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps.

A Proposed Chaperone of the Bacterial Type VI Secretion System Functions To Constrain a Self-Identity Protein.

The bacterium Proteus mirabilis can communicate identity through the secretion of the self-identity protein IdsD via the type VI secretion (T6S) system. IdsD secretion is essential for self-versus-nonself recognition behaviors in these populations. Here we provide an answer to the unresolved question of how the activity of a T6S substrate, such as IdsD, is regulated before secretion. We demonstrate that IdsD is found in clusters that form independently of the T6S machinery and activity. We show that the IdsC protein, which is a member of the proposed DUF4123 chaperone family, is essential for the maintenance of these clusters and of the IdsD protein itself. We provide evidence that amino acid disruptions in IdsC are sufficient to disrupt IdsD secretion but not IdsD localization into subcellular clusters, strongly supporting the notion that IdsC functions in at least two different ways: maintaining IdsD levels and secreting IdsD. We propose that IdsC, and likely other DUF4123-containing proteins, functions to regulate T6S substrates in the donor cell both by maintaining protein levels and by mediating secretion at the T6S machinery.IMPORTANCE Understanding the subcellular dynamics of self-identity proteins is crucial for developing models of self-versus-nonself recognition. We directly addressed how a bacterium restricts self-identity information before cell-cell exchange. We resolved two conflicting models for type VI secretion (T6S) substrate regulation by focusing on the self-identity protein IdsD. One model is that a cognate immunity protein binds the substrate, inhibiting activity before transport. Another model proposes that DUF4123 proteins act as chaperones in the donor cell, but no detailed molecular mechanism was previously known. We resolve this discrepancy and propose a model wherein a chaperone couples IdsD sequestration with its localization. Such a molecular mechanism restricts the communication of identity, and possibly other T6S substrates, in producing cells.

The spliceosome U2 snRNP factors promote genome stability through distinct mechanisms; transcription of repair factors and R-loop processing.

Recent whole-exome sequencing of malignancies have detected recurrent somatic mutations in U2 small nuclear ribonucleoprotein complex (snRNP) components of the spliceosome. These factors have also been identified as novel players in the DNA-damage response (DDR) in several genome-wide screens and proteomic analysis. Although accumulating evidence implies that the spliceosome has an important role in genome stability and is an emerging hallmark of cancer, its precise role in DNA repair still remains elusive. Here we identify two distinct mechanisms of how spliceosome U2 snRNP factors contribute to genome stability. We show that the spliceosome maintains protein levels of essential repair factors, thus contributing to homologous recombination repair. In addition, real-time laser microirradiation analysis identified rapid recruitment of the U2 snRNP factor SNRPA1 to DNA-damage sites. Functional analysis of SNRPA1 revealed a more immediate and direct role in preventing R-loop-induced DNA damage. Our present study implies a complex interrelation between transcription, mRNA splicing and the DDR. Cells require rapid spatio-temporal coordination of these chromatin transactions to cope with various forms of genotoxic stress.

SGT1 contributes to maintaining protein levels of MEK2DD to facilitate hypersensitive response-like cell death in Nicotiana benthamiana

Gene silencing revealed that the mitogen-activated protein kinase (MAPK) cascade in Solanaceae consisted with MEK2-WIPK/SIPK, is required for R protein-induced hypersensitive response (HR) cell death and/or resistance. Overexpression of MEK2DD results in HR-like cell death. MEK2DD is a phospho-mimic and constitutive active form harboring mutations at putative phosphorylation sites of upstream MAPKKK. The molecular mechanism that induces HR-like cell death is unknown. Here we report SGT1 is required for the accumulation of MEK2DD protein, not MEK2WT. Virus-induced gene silencing of SGT1 resulted in low protein accumulation of MEK2DD. This result suggests that SGT1 has a positive role in the accumulation of the MEK2 active form protein to facilitate signal transduction.

Temporal profiles of high-mobility group box 1 expression levels after cortical spreading depression in mice.

Cortical spreading depression (CSD) has recently been shown to induce the release of the nuclear protein termed high-mobility group box 1 from neurons, causing activation of the trigeminovascular system. Here, we explored the effects of single and multiple cortical spreading depression inductions on high-mobility group box 1 (HMGB1) transcriptional activity relative to high-mobility group box 1 protein expression levels and intracellular localization in cortical neurons and astrocytes. Single or multiple cortical spreading depression inductions were achieved by KCl application to the mouse cerebral cortex. The animals were sacrificed at 30 minutes, 3 hours and 24 hours after cortical spreading depression induction. High-mobility group box 1 expression levels were explored with in situ hybridization, Western blotting and immunostaining. Cortical spreading depression up-regulated high-mobility group box 1 transcriptional activity in neurons at 3 hours in a manner that was dependent on the number of cortical spreading depression inductions. At 24 hours, the high-mobility group box 1 transcriptional activity had returned to basal levels. Cortical spreading depression induced a reduction in high-mobility group box 1 protein expression at 3 hours, which was also dependent on the number of cortical spreading depression inductions. Following cortical spreading depression, the release of high-mobility group box 1 from the nucleus was observed in a small proportion of neurons, but not in astrocytes. Cortical spreading depression induced translocation of high-mobility group box 1 from neuronal nuclei, driving transcriptional up-regulation of high-mobility group box 1 to maintain protein levels.

Control of B lymphoma through regulation of TRAF protein degradation during nutrient stress (TUM7P.932)

Abstract The TNFR family of receptors includes members, such as CD40, that mediate important immune functions. TNFRs play critical roles in regulating cellular survival, where they utilize TNFR associated factors (TRAFs), including TRAF1 and TRAF2, as adaptors to signal through NF-κB and MAPK pathways. TRAF1 is linked with increased lymphoma cell survival and is overexpressed in over 50% of human lymphomas, with highest expression in most refractory B-CLL. Thus there is interest in blocking TRAF1 induced survival signaling in lymphoma. Here we show that during nutrient deprivation, induced by low glutamine in the media or by treatment with the licensed drug Erwinaze, TRAF1 is essential for maintaining protein levels of another pro-survival signaling adaptor, TRAF2, in RAJI cells (Burkitt's lymphoma). The PKC, PKN1, has been shown to phosphorylate TRAF1. Utilizing shRNA knockdown, we show that loss of PKN1 reduces TRAF1 expression, suggesting that PKN1 phosphorylation stabilizes TRAF1. Importantly, we demonstrated that Erwinaze treatment causes cell death in TRAF1 deficient RAJI cells but not in wild type cells. Moreover, PKN1 deficient RAJI cells, which exhibit lower TRAF1 expression, were also susceptible to treatment with Erwinaze. Together, these findings suggest that a combination therapy that targets TRAF1 for degradation (i.e. PKN1 inhibitor) along with Erwinaze to cause nutrient stress mediated loss of TRAF2 constitutes an effective and aggressive therapy for B cell lymphoma

Uncovering New Functions for MicroRNAs in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, microRNA (miRNA) regulation of development was first observed in the striking abnormalities of lin-4 and let-7 loss of function mutants. However, after these first two miRNA mutant phenotypes were described, progress on the identification of miRNA functions in worms slowed considerably. Recent advances reveal new functions for miRNAs in embryonic and larval development as well as in the regulation of lifespan and stress response. Results from a combination ofcomputational, biochemical, and genetic approaches have deepened our understanding of miRNA regulation of target mRNAs and support the hypothesis that miRNAs have an important role in ensuring the robustness of developmental and physiological pathways.

NBA1/MERIT40 and BRE Interaction Is Required for the Integrity of Two Distinct Deubiquitinating Enzyme BRCC36-containing Complexes

BRCC36-deubiquitinating enzyme (DUB) forms two different complexes through interactions with two different adaptor proteins Abraxas and ABRO1 in cells. Abraxas mainly localizes in the nucleus, mediating the interaction of BRCC36 with BRCA1. ABRO1 is mainly localized in the cytoplasm. Because it lacks the BRCA1-interacting motif, the ABRO1 complex does not interact with BRCA1. Both BRCC36-containing complexes contain common components including BRE and NBA1/MERIT40. Here, we found that the two complexes are assembled in a similar manner and NBA1 and BRE interaction is critical for maintaining the integrity of both of the complexes. Knockdown of NBA1 or BRE leads to decreased levels of components of the two BRCC36-containing complexes. We provided evidence that NBA1 interacts with BRE through a C-terminal conserved motif of the NBA1 protein and a C-terminal UEV domain of the BRE protein. Furthermore, the NBA1-BRE interaction is required for cellular resistance to ionizing irradiation and NBA1's role in recruiting BRCA1 to DNA damage sites. Together, these studies reveal critical interactions required for the formation and function of BRCC36-containing DUB complexes.

Comparative Study of Culture Conditions for Maintaining CYP3A4 and ATP-Binding Cassette Transporters Activity in Primary Cultured Human Hepatocytes

The aim of this study was to determine suitable culture conditions for maintaining the activity of cytochrome p450 (CYP) 3A4 and drug transporters in primary cultured human hepatocytes. Human hepatocytes were isolated using the two-step collagenase perfusion technique and were cultured with four different media, serum-free William’s E medium (serum-free WEM), WEM containing fetal calf serum (FCS-WEM), WEM with human serum (HS-WEM), and Lanford’s medium. The albumin levels were maintained for 7 days in hepatocytes. Although CYP3A4 mRNA levels gradually decreased from 3 days, CYP3A4 and hepatocyte nuclear factor-4α alpha protein levels and activities were maintained for 7 days in hepatocytes cultured with serum-free WEM and Lanford’s but not in those with FCS-WEM and HS-WEM. Furthermore, CYP3A4 protein levels were significantly increased by the addition of rifampicin and dexamethasone to the culture media, indicating that the induction potential was maintained. The protein levels of P-glycoprotein, multi-drug-resistance-2, and breast cancer-resistance protein were maintained for 7 days in all media. Serum-free WEM and Lanford’s also maintained protein levels of CYP2C19, CYP2D6, and organic anion transporter polypeptide in the hepatocytes. Serum-free WEM and Lanford’s may be appropriate culture media for maintaining CYP3A4 and drug transporter protein levels in primary cultured hepatocytes.

G protein-coupled estrogen receptor 1 (GPER, GPR 30) in normal human endometrium and early pregnancy decidua

The recently identified trans-membrane G protein-coupled estrogen receptor 1 (GPER, GPR30) has been implicated in rapid non-genomic effects of estrogens. This focuses on expression and localization of GPER mRNA and protein in normal cyclic endometrium and early pregnancy decidua. Real-time PCR, western blotting, in situ hybridization and immuno-histochemistry were used. Endometrial expression of GPER mRNA was lower in the secretory phase than in the proliferative phase, and even lower in the decidua. The expression pattern was similar to that of ERα mRNA, but different from that of ERβ mRNA. Western blot detected GPER protein as a 54 kDa band in all endometrial and decidual samples. In contrast to the mRNA, GPER protein did not show cyclic variations. Apparently, a lower amount of mRNA is sufficient to maintain protein levels in the secretory phase. GPER mRNA was predominantly localized in the epithelium of mid- and late-proliferative phase endometrium, whereas expression in early proliferative and secretory glands could not be distinguished from the diffuse stromal signal, which was present throughout the cycle. Immuno-staining for GPER was stronger in glandular and luminal epithelium than in the stroma throughout the cycle. The cyclic variations of GPER mRNA obviously relate to strong epithelial expression in the proliferative phase, and the expression pattern suggests regulation by ovarian steroids. GPER protein is present in endometrial tissue throughout the cycle, and the epithelial localization suggests potential functions during sperm migration at mid-cycle, as well as decidualization and blastocyst implantation in the mid-secretory phase.

Hepatoprotective effects of sea buckthorn (Hippophae rhamnoides L.) against carbon tetrachloride induced liver injury in rats

AbstractBACKGROUND: Liver injuries induced by carbon tetrachloride are the best‐characterized system of xenobiotic‐induced hepatotoxicity and commonly used model for the screening of hepatoprotective activities of drugs. The present study evaluates the hepatoprotective activity of sea buckthorn (Hippophae rhamnoides L.), family Elaeagnaceae, on carbon tetrachloride (CCl4)‐induced liver injury in male albino rats. The study was performed on Sprague–Dawley male albino rats weighing about 180–200 g. The animals were pretreated with three different doses of leaf extract (50, 100 and 200 mg kg−1 body weight) for 5 days. Hepatotoxicity was induced by single oral administration of 1.5 mL CCl4 kg−1 body weight on the fifth day. The animals were then sacrificed and assessed for various biochemical parameters.RESULTS: Administration of CCl4 significantly enhanced glutamate oxaloacetate transferase (GOT), glutamate pyruvate transferase (GPT), alkaline phosphatase (ALP) and bilirubin, and decreased total protein levels in the serum. Treatment with CCl4 also significantly decreased reduced glutathione (GSH), and decreased glutathione peroxidase and superoxide dismutase activity. CCl4 treatment also caused a significant increase in hepatic lipid peroxidation as assessed by malondialdehyde (MDA) levels in the tissue. Pretreatment of leaf extract at a concentration of 100 and 200 mg kg−1 body weight significantly (P &lt; 0.05) protected the animals from CCl4‐induced liver injury. The extract significantly restricted the CCl4‐induced increase of GOT, GPT, ALP and bilirubin and better maintained protein levels in the serum. Further, it also enhanced GSH and decreased MDA levels.CONCLUSION: The results show that sea buckthorn leaf extract has significant hepatoprotective effects which might be due to its antioxidant activity and can be developed as a nutraceutical or food supplement against liver diseases. Copyright © 2008 Society of Chemical Industry

MDM2 Binding Induces a Conformational Change in p53 That Is Opposed by Heat-shock Protein 90 and Precedes p53 Proteasomal Degradation

p53 protein conformation is an important determinant of its localization and activity. Changes in p53 conformation can be monitored by reactivity with wild-type conformation-specific (pAb-1620) or mutant conformation-specific (pAb-240) p53 antibodies. Wild-type p53 accumulated in a mutant (pAb-240 reactive) form when its proteasome-dependent degradation was blocked during recovery from stress treatment and in cells co-expressing p53 and MDM2. This suggests that conformational change precedes wild-type p53 degradation by the proteasome. MDM2 binding to the p53 N terminus could induce a conformational change in wild-type p53. Interestingly, this conformational change was opposed by heat-shock protein 90 and did not require the MDM2 RING-finger domain and p53 ubiquitination. Finally, ubiquitinated p53 accumulated in a pAb-240 reactive form when p53 degradation was blocked by proteasome inhibition, and a p53-ubiquitin fusion protein displayed a mutant-only conformation in MDM2-null cells. These results support a model in which MDM2 binding induces a conformational change that is opposed by heat-shock protein 90 and precedes p53 ubiquitination. The covalent attachment of ubiquitin may "lock" p53 in a mutant conformation in the absence of MDM2-binding and prior to its degradation by the proteasome.

Improvements in malting barley grain yield by manipulation of genes influencing grain protein content

Malting barley (Hordeumvulgare L.) is traditionally grown under low soil fertility as a conservative approach to achieve the malting requirement of grain below a maximum prescribed limit for protein concentration, typically 12.0%. Such strategies severely limit grain yield. Traditionally, barley breeders have sought to improve yield in malting barley through selection for higher yield per se, without considering an alternative approach in which genetic manipulation of inherent grain protein concentration would allow higher yields through the adoption of an alternative farming system for malting barley production. The present study examined whether greater yield improvements could be achieved by manipulating genes influencing grain protein content, and allowing malting barley to be grown on crop rotations with higher fertility. Doubled haploid lines, selected from a mapping population based on allelic differences at two genes (quantitative trait loci; QTL) influencing grain protein content, were evaluated for productivity over a range of agronomic practices and environments (sowing date, nitrogen application, and a range of field histories). QTL effects were highly repeatable over the range of practices and environments, with individuals carrying two low-protein alleles (q1q1q2q2) producing grains with comparatively lower grain protein than individuals carrying the alternate allelic configuration (Q1Q1Q2Q2). Based on two years of data, the yield advantage of growing barley on legume stubble as opposed to wheat stubble was approximately 1.0 t ha−1, or a yield increase of 53.3%. When grown after a pulse crop in 2005, average yields of those mapping population genotypes carrying low-protein alleles was 4.28 t ha−1, with average grain protein of 12.0% whilst the average yield of their high protein counterparts was 3.76 t ha−1 with an average protein level of 12.7%. We conclude that, as an alternative means of malting barley yield improvement, varieties can be developed that allow barley to be grown in highly fertile conditions resulting in yields up to 53% over current farming practices, while maintaining protein levels within malting specifications.