Napier grass (Pennisetum purpureum Schum) management strategies for dairy and meat production in the tropics and subtropics: yield and nutritive value.

Napier grass (Pennisetum purpureum Schumach) comprises up to 80% of the cattle diet in many tropical and subtropical regions and is used primarily by smallholder farmers. Despite the grass's high yield, resulting animal productivity from this grass is low. One of the key reasons for the low animal productivity of Napier grass is its low nutritive value under current management. Taken together, previous work has shown the current yield, crude protein (CP), and metabolisable energy (ME) of Napier grass to be 26 t dry matter (DM)/ha/year, 96 g/kg DM, and 8.7 MJ/kg DM, respectively, ranging from 2 to 86 t DM/ha/year, 9 to 257 g CP/kg DM, and 5.9 to 10.8 MJ ME/kg DM, respectively, suggesting an opportunity for significant improvement on both yield and nutritive value of this grass. The DM yield and nutritive value of this grass are inversely related, indicating a trade-off between yield and quality; however, this trade-off could be minimised by increasing sowing density and harvesting frequency. Available literature shows that this simple management strategy of increasing sowing density (50 cm × 40 cm) and harvesting frequency (11-12 harvests/year) provides 71 t DM/ha with 135 g/kg DM CP and 10.8 MJ ME/kg DM. This quality of Napier grass has the potential to increase both milk and meat production substantially in the tropics and subtropics, and the farmers will likely find this simple management acceptable due to the high yield obtained through this management. However, there is a paucity of work in this field. Therefore, management strategies to improve the nutritive value of Napier grass are required to increase milk and meat production in the tropics and subtropics and in doing so improve the food security of more than half of the global population living in these regions.

Field and in-silico analysis of harvest index variability in maize silage.

Maize silage is a key component of feed rations in dairy systems due to its high forage and grain yield, water use efficiency, and energy content. However, maize silage nutritive value can be compromised by in-season changes during crop development due to changes in plant partitioning between grain and other biomass fractions. The partitioning to grain (harvest index, HI) is affected by the interactions between genotype (G) × environment (E) × management (M). Thus, modelling tools could assist in accurately predicting changes during the in-season crop partitioning and composition and, from these, the HI of maize silage. Our objectives were to (i) identify the main drivers of grain yield and HI variability, (ii) calibrate the Agricultural Production Systems Simulator (APSIM) to estimate crop growth, development, and plant partitioning using detailed experimental field data, and (iii) explore the main sources of HI variance in a wide range of G × E × M combinations. Nitrogen (N) rates, sowing date, harvest date, plant density, irrigation rates, and genotype data were used from four field experiments to assess the main drivers of HI variability and to calibrate the maize crop module in APSIM. Then, the model was run for a complete range of G × E × M combinations across 50 years. Experimental data demonstrated that the main drivers of observed HI variability were genotype and water status. The model accurately simulated phenology [leaf number and canopy green cover; Concordance Correlation Coefficient (CCC)=0.79-0.97, and Root Mean Square Percentage Error (RMSPE)=13%] and crop growth (total aboveground biomass, grain + cob, leaf, and stover weight; CCC=0.86-0.94 and RMSPE=23-39%). In addition, for HI, CCC was high (0.78) with an RMSPE of 12%. The long-term scenario analysis exercise showed that genotype and N rate contributed to 44% and 36% of the HI variance. Our study demonstrated that APSIM is a suitable tool to estimate maize HI as one potential proxy of silage quality. The calibrated APSIM model can now be used to compare the inter-annual variability of HI for maize forage crops based on G × E × M interactions. Therefore, the model provides new knowledge to (potentially) improve maize silage nutritive value and aid genotype selection and harvest timing decision-making.

Genetic loci regulating cadmium content in rice grains.

It has been estimated that up to 90% of human exposure to cadmium is through food, and that cadmium within rice grains can be a major contributor to that dietary source. In this study genome wide association mapping was conducted on the Bengal and Assam Aus Panel (BAAP) of rice to identify quantitative trait loci and candidate genes for lowering grain cadmium. Field experiments were conducted over two years under two different irrigation systems: continually flooded and alternate wetting and drying (AWD). There was significant effects of water treatment, genotype, and genotype by water treatment interaction. Importantly, AWD increased grain cadmium, on average, by 49.6% and 108.8% in year 1 and 2 respectively. There was between 4.6 and 28 fold variation in cadmium concentration. A total of 58 QTLs were detected but no loci are clearly specific to one water regime despite approximately 20% of variation attributable to genotype by water regime interaction. A number of QTLs were consistent across most water treatments and years. These included QTLs on chromosome 7 (7.23-7.61, 8.93-9.04, and 29.12-29.14 Mbp), chromosome 5 (8.66-8.72 Mbp), and chromosome 9 (11.46-11.64 Mbp). Further analysis of the loci on chromosome 7 (8.93-9.04 Mbp), identified the candidate gene OsNRAMP1, where cultivars with a deletion upstream of the gene had higher concentrations of cadmium compared to the cultivars that did not have the deletion. The distribution of alleles within the BAAP suggest this QTL is easily detected in this population because it is composed of aus cultivars. Local genome cluster analysis suggest high Cd alleles are uncommon, but should be avoided in breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s10681-020-02752-1).

Plasmid DNA nicking- a Novel Activity of Soybean Trypsin Inhibitor and Bovine Aprotinin.

Protease inhibitors, such as trypsin inhibitor, serum alpha-1 antitrypsin, or liver aprotinin, are a class of proteins that competitively bind and block the catalytic activity of proteolytic enzymes with wide ranging biological functions. A significant number of protease inhibitors have also been shown to possess antimicrobial activity, presumed to contribute in defense against pathogenic microorganisms as plants with higher levels of protease inhibitors tend to exhibit increased resistance towards pathogens. Two proposed mechanisms for the antimicrobial activity are combating microbial proteases that play roles in disease development and disruption of microbial cell wall & membrane necessary for survival. Here we show for the first time a novel activity of soybean trypsin inhibitor and bovine aprotinin that they nick supercoiled, circular plasmid DNA. A number of experiments conducted to demonstrate the observed DNA nicking activity is inherent, rather than a co-purified, contaminating nuclease. The nicking of the plasmid results in markedly reduced efficiencies in transformation of E. coli and transfection of HEK293T cells. Thus, this work reveals yet a new mechanism for the antimicrobial activity by protease inhibitors.

Nuclear Localization Signal and p53 Binding Site in MAP/ERK Kinase Kinase 1 (MEKK1).

Previously, we showed that Mekk1 translocates to the nucleus, interacts with tumor suppressor protein p53, and co-represses PKD1 transcription via an atypical p53 binding site on the minimal PKD1 promoter (JBC 285:38,818-38,831, 2010). In this study, we report the mechanisms of Mekk1 nuclear transport and p53 binding. Using GFP-linked constitutively active-Mekk1 (CA-Mekk1) and a deletion strategy, we identified a nuclear localization signal (HRDVK) located at amino acid (aa) residues 1,349-1,353 in the C-terminal Mekk1 catalytic domain. Deletion of this sequence in CA-Mekk1 and full-length Mekk1 significantly reduced their nuclear translocation in both HEK293T and COS-1 cells. Using co-immunoprecipitation, we identified an adjacent sequence (GANLID, aa 1,354-1,360) in Mekk1 responsible for p53 binding. Deletion of this sequence markedly reduced the interaction of Mekk1 with p53. Mekk1 does not appear to affect phosphorylation of Ser15, located in the Mdm2 interaction site, or other Ser residues in p53. However, Mekk1 mediates p53 protein stability in the presence of Mdm2 and reduces p53 ubiquitination, suggesting an interference with Mdm2-mediated degradation of p53 by the ubiquitin-proteasome pathway.

Regulation of HFE expression by poly(ADP-ribose) polymerase-1 (PARP1) through an inverted repeat DNA sequence in the distal promoter.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron overload among Caucasians of northern European descent. Over 85% of all cases with HH are due to mutations in the hemochromatosis protein (HFE) involved in iron metabolism. Although the importance in iron homeostasis is well recognized, the mechanism of sensing and regulating iron absorption by HFE, especially in the absence of iron response element in its gene, is not fully understood. In this report, we have identified an inverted repeat sequence (ATGGTcttACCTA) within 1700bp (-1675/+35) of the HFE promoter capable to form cruciform structure that binds PARP1 and strongly represses HFE promoter. Knockdown of PARP1 increases HFE mRNA and protein. Similarly, hemin or FeCl3 treatments resulted in increase in HFE expression by reducing nuclear PARP1 pool via its apoptosis induced cleavage, leading to upregulation of the iron regulatory hormone hepcidin mRNA. Thus, PARP1 binding to the inverted repeat sequence on the HFE promoter may serve as a novel iron sensing mechanism as increased iron level can trigger PARP1 cleavage and relief of HFE transcriptional repression.

CMV promoter is repressed by p53 and activated by JNK pathway.

Viral promoters are widely utilized in commercial and customized vectors to drive expression of genes of interest including reporter, effector and transfection control, because of their high transcription efficiency in a variety of primary and transformed cell lines. However, we observed altered rate of transcription for these promoters under conditions such as presence of an effector protein. These variations in viral promoter driven expressions can potentially lead to incorrect conclusion, especially in comparative and quantitative experiments. We found significantly reduced viral promoter activity in cells overexpressing tumor suppressor protein p53, whereas markedly induced transcription in cells overexpressing MAP/ERK kinase kinase 1 (Mekk 1). Using deletion constructs generated from the CMV promoter, we found the transcription reduction by p53 is possibly mediated through the TATA motif present in proximal CMV promoter. The activation of the CMV promoter by Mekk 1, on the other hand, is attributed to the proximal CRE binding site in the promoter. These findings may be of interest to investigators who use CMV (or other viral) promoter driven vectors for either comparative or quantitative gene expression, or effect on promoter activity.

Cytotoxicity analysis of active components in bitter melon (Momordica charantia) seed extracts using human embryonic kidney and colon tumor cells.

Bitter melon (Momordica charantia) seed extracts (BMSE) have been used as traditional medicine for treating various ailments, although in many cases, the active component(s) are unidentified. In this study, bitter melon seeds were extracted in water, ethanol, or ethanol: water (1:1). The aqueous seed extracts (BMSE-W) exhibited marked cytotoxicity towards human embryonic kidney 293T (HEK293T) and human colon tumor 116 (HCT1116) cells. The activity in BMSE-W was unaffected by heat and proteinases treatments, and eluted in the total volume of size-exclusion HPLC, suggesting the small, organic nature of the active component(s). Gas chromatographic-mass spectrometic (GC-MS) analysis of the HPLC fractions identified methoxy-phenyl oxime (MPO) as a major active component. Acetophenone oxime, a commercially available structural homolog of MPO, demonstrated cytotoxicity comparable with that of the BMSE-W. The oxime functional group was found to be critical for activity. Increased poly-(ADP-ribose)-polymerase and beta-actin cleavage, and chromatin condensation observed in treated cells suggested apoptosis as a plausible cause for the cytotoxicity. This study, for the first time, identified a cytotoxic oxime in BMSE-W.

Identification of bound nitro musk-protein adducts in fish liver by gas chromatography-mass spectrometry: biotransformation, dose-response and toxicokinetics of nitro musk metabolites protein adducts in trout liver as biomarkers of exposure.

Ubiquitous occurrences of synthetic nitro musks are evident in the literature. The in vivo analysis of musk xylene (MX) and musk ketone (MK)-protein adducts in trout liver has been performed by gas chromatography-mass spectrometry using selected ion monitoring (GC-SIM-MS). Biotransformation, dose-response and toxicokinetics studies of 2-amino-MX (2-AMX), 2-amino-MK (2-AMK) and 4-amino-MX (4-AMX) metabolites, covalently bound to cysteine amino acids in proteins in fish liver, formed by enzymatic nitro-reduction of MX and MK, have been described. Trouts were exposed to single exposures of 0.010, 0.030, 0.10, and 0.30 mg/g MX and/or MK. Forty-two fish liver samples were collected from exposed- and control-fish subsequent to exposure intervals of 1 day, 3 days, and 7 days and were composited as per exposure schedules and times. Alkaline hydrolysis released bound metabolites from exposed liver composites that were extracted into n-hexane and then concentrated and analyzed by GC-SIM-MS. The presence of the metabolites in liver extracts was confirmed based on agreement of similar mass spectral properties and retention times with standards. In the dose-response study, the maximum adduct formation was 492.0 ng/g for 2-AMX, 505.5 ng/g for 2-AMK and 12588.5 ng/g for 4-AMX in liver at 0.03 mg/g MX and MK fish in 1 day after exposure. For toxicokinetics investigation, the highest amount of the target metabolites was found to be the same concentration as observed in the dose-response study for 1 day after exposure with 0.03 mg/g MX and MK fish and the half-lives of the metabolites were estimated to be 2-9 days based on assumption of first-order kinetics. Average recoveries exceeded 95% with a relative standard deviation (RSD) around 9%, and the limit of detection (LOD) ranged from 0.91 to 3.8 ng/g based on a signal to noise ratio of 10 (S/N=10) could be achieved for the metabolites. No metabolites were detected in the controls and exposed non-hydrolyzed liver extracts. This is the first report on dose-response and toxicokinetics of nitro musk-cysteine-protein adducts in fish liver.

MAP/ERK kinase kinase 1 (MEKK1) mediates transcriptional repression by interacting with polycystic kidney disease-1 (PKD1) promoter-bound p53 tumor suppressor protein.

Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNFα, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation.

Transfer buffer containing methanol can be reused multiple times in protein electrotransfer.

We investigated the feasibility of repeated use of transfer buffer containing methanol in electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to polyvinylidene difluoride (PVDF) membrane using a prestained protein marker of broad molecular sizes. Transfer of the antitumor protein p53 in HEK293T cell extracts, using fresh and used transfer buffer, followed by detection with anti-p53 antibody was also performed to test detectability in immunoblot. Results from these experiments indicate that the transfer buffer can be reused at least five times and maintain a similar extent of protein transfer to PVDF membrane. Repeated use of the transfer buffer containing methanol will significantly reduce the volume of hazardous waste generated and its disposal cost as well as its adverse effect on environment.

Retinoic acid-dependent activation of the polycystic kidney disease-1 (PKD1) promoter.

The retinoic acids all-trans retinoic acid (AT-RA) and 9-cis retinoic acid (9C-RA) and the retinoic acid receptors RAR and RXR significantly induce transcriptional activity from a 200-bp PKD1 proximal promoter in transfected mammalian cells. This PKD1 promoter region contains Ets, p53, and GC box motifs, but lacks a canonical RAR/RXR motif. Mutagenesis of the Ets sites did not affect RA induction. In contrast, GC box mutations completely blocked stimulation by AT-RA and by RXRbeta or RARbeta. Mithramycin A, which prevents Sp1 binding, significantly reduced basal promoter activity and suppressed upregulation by AT-RA and RXR. The 200-bp proximal promoter could not be induced by AT-RA in Drosophila SL2 cells, which lack Sp1, but could be activated in these cells transfected with exogenous Sp1. Small interfering RNA knockdown of Sp1 in mammalian cells completely blocked RXRbeta upregulation of the promoter. These data indicate that induction of the PKD1 promoter by retinoic acid is mediated through Sp1 elements. RT-PCR showed that AT-RA treatment of HEK293T cells increased the levels of endogenous PKD1 RNA, and chromatin immunoprecipitation showed the presence of both RXR and Sp1 at the PKD1 proximal promoter. These results suggest that retinoids and their receptors may play a role in PKD1 gene regulation.

PCR-based restriction fragment length polymorphism and haplotype of the most common mutation L176F in the beta-glucuronidase gene.

Mucopolysaccharidosis type VII or Sly syndrome is an autosomal recessive disorder of glycosaminoglycan storage leading to variable clinical symptoms, such as hepatosplenomegaly, bone deformities, hearing loss, corneal opacities, mental retardation, and hydrops fetalis in affected individuals. The disease is caused by approximately 40 different mutations in the beta-glucuronidase gene. Detection of the most common mutation L176F by single-strand conformation polymorphism (SSCP) was not always successful. Although DNA sequencing followed by PCR amplification can easily detect this mutation, accessibility to a DNA sequencer or useful reagents in the sequencing procedure is not readily available in many countries. A PCR-based restriction fragment length polymorphism (RFLP) developed in this report would allow rapid and easier detection of this mutation for screening new patients or neonates of heterozygous parents. Analysis of intragenic polymorphic sites in the L176F patients identified two distinct alleles; the predominant one probably originated in Spain.

Ets factors regulate the polycystic kidney disease-1 promoter.

The Ets family of transcription factors consists of a group of highly conserved sequence-specific DNA binding proteins that functionally cooperate with other transcription factors to regulate a number of diverse cellular processes including proliferation, differentiation, and apoptosis. We have analyzed a 3.3kb 5'-upstream region of the human PKD1 promoter, using transient transfection in HEK293T cells and Drosophila SL2 cells, to demonstrate that the PKD1 promoter is a target of Ets family transcription factors. Our studies showed that PKD1 promoter-luciferase reporter gene expression is downregulated by cotransfected Fli-1 and is upregulated by cotransfected Ets-1. Using deletion constructs, we demonstrated that the sequences responding to Fli-1 and Ets-1 lie within the -200 to +33bp proximal promoter. This region was found to contain two putative Ets response elements (EREs): an upstream (Ets-A) sequence 5'-CGGAA-3' (-181 to -185) and a downstream (Ets-B) sequence 5'-CGGAT-3' (-129 to -133). Site-directed mutagenesis indicated that both EREs are functional. A Fli-1 DNA binding domain mutant construct (W321R), which is incapable of binding DNA, was unable to inhibit basal promoter activity. In contrast, a Fli-1 DNA binding domain truncation mutant construct, which only contains the DNA binding domain and lacks the transactivation domain, was able to inhibit. These results suggest that the effect of Fli-1 is through direct binding to these EREs. Direct binding of Fli-1 and Ets-1 to the Ets-A and Ets-B sites was supported by electrophoretic mobility shift assays. Lastly, competition between Fli-1 and Ets-1 for the two EREs was demonstrated by showing that increasing amounts of Ets-1 could overcome Fli-1 repression of promoter activity. Taken together, these experiments define the proximal PKD1 promoter region as a potential target of Ets family transcription factors.

Remarkable sequence conservation of the last intron in the PKD1 gene.

The last intron of the PKD1 gene (intron 45) was found to have exceptionally high sequence conservation across four mammalian species: human, mouse, rat, and dog. This conservation did not extend to the comparable intron in pufferfish. Pairwise comparisons for intron 45 showed 91% identity (human vs. dog) to 100% identity (mouse vs. rat) for an average for all four species of 94% identity. In contrast, introns 43 and 44 of the PKD1 gene had average pairwise identities of 57% and 54%, and exons 43, 44, and 45 and the coding region of exon 46 had average pairwise identities of 80%, 84%, 82%, and 80%. Intron 45 is 90 to 95 bp in length, with the major region of sequence divergence being in a central 4-bp to 9-bp variable region. RNA secondary structure analysis of intron 45 predicts a branching stem-loop structure in which the central variable region lies in one loop and the putative branch point sequence lies in another loop, suggesting that the intron adopts a specific stem-loop structure that may be important for its removal. Although intron 45 appears to conform to the class of small, G-triplet-containing introns that are spliced by a mechanism utilizing intron definition, its high sequence conservation may be a reflection of constraints imposed by a unique mechanism that coordinates splicing of this last PKD1 intron with polyadenylation.

The polycystic kidney disease-1 promoter is a target of the beta-catenin/T-cell factor pathway.

Polycystic kidney disease (PKD) results from loss-of-function mutations in the PKD1 gene. There are also reports showing abnormally high levels of PKD1 expression in cystic epithelial cells. At present, nothing is known about the molecular mechanisms regulating the normal expression of the PKD1 gene or whether transcriptional disregulation of the PKD1 gene has a role in cyst formation. We have analyzed a 3.3-kb 5'-proximal portion of the human PKD1 gene. Sequence analysis revealed the presence of consensus sequences for numerous transactivating factors, including four T-cell factor (TCF) binding elements (TBEs). Transcriptional activity of the 3.3-kb fragment and a series of deletion constructs was assayed in HEK293T cells. A 2.0-kb proximal promoter region containing one of the four TBEs (TBE1) was inducible up to 6-fold by cotransfection with beta-catenin. beta-catenin-mediated induction was inhibited by dominant-negative TCF and by deletion of the TBE1 sequence. 15- or 109-bp sequences containing the TBE1 site, when cloned upstream of a minimal promoter, were shown to respond to beta-catenin induction. Gel shift assays confirmed that the TBE1 site is capable of forming complexes with TCF and beta-catenin. To determine whether expression of the endogenous PKD1 gene responds to beta-catenin, HT1080 cells were treated with LiCl, and HeLa cells were stably transfected with beta-catenin. In both cases, endogenous PKD1 mRNA levels were elevated in response to these treatments. Taken together, these studies define an active PKD1 promoter region and suggest that the PKD1 gene is a target of the beta-catenin/TCF pathway.