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Objective: The present study aims to explore the effects of enteral nutrition (EN) semi-curing feeding on nutritional diarrhoea in the patients with stroke., Methods: The patients admitted to the neurological intensive care unit of a tertiary care hospital with stroke- and EN-related diarrhoea between May 2019 and October 2020 were included in the study. The 60 patients, who met the inclusion criteria were divided into the two groups (30 patients each), the experimental group (EN solution+probiotics+90 ml of pectin) and the control group (EN solution+probiotics), in accordance with the random number table method. The stool number, total stool amount, perineal skin score, and time required to achieve the nutritional target of each patient were recorded at admission and on days 1, 3, and 7 of pectin intervention. The lymphocyte count and the haemoglobin, serum pre-albumin (PA), and total cholesterol (TC) levels were measured in order to assess the patients' nutritional statuses., Results: The stool number and total stool amount on days 1 and 3 of pectin intervention in the experimental group were better than in the control group; the differences were statistically significant (p <0.05). On day 7 of the intervention, the stool number, total stool amount, and perineal skin score had decreased in the experimental group; the differences were statistically significant, when compared to the control group (p <0.05). Furthermore, the PA levels increased, and TC levels decreased in the experimental group; the differences were statistically significant, when compared to the control group (p <0.05). On day 3 of the intervention, the PA levels in the experimental group were increased compared to the control group; the difference was statistically significant (p <0.05)., Conclusion: EN semi-curing feeding could improve EN-related diarrhoea and nutritional status levels in patients with stroke (Tab. 3, Ref. 9).

We have developed a rapid Raman spectroscopy-based method for the detection and quantification of early innate immunity responses in Arabidopsis and Choy Sum plants. Arabidopsis plants challenged with flg22 and elf18 elicitors could be differentiated from mock-treated plants by their Raman spectral fingerprints. From the difference Raman spectrum and the value of p at each Raman shift, we derived the Elicitor Response Index (ERI) as a quantitative measure of the response whereby a higher ERI value indicates a more significant elicitor-induced immune response. Among various Raman spectral bands contributing toward the ERI value, the most significant changes were observed in those associated with carotenoids and proteins. To validate these results, we investigated several characterized Arabidopsis pattern-triggered immunity (PTI) mutants. Compared to wild type (WT), positive regulatory mutants had ERI values close to zero, whereas negative regulatory mutants at early time points had higher ERI values. Similar to elicitor treatments, we derived an analogous Infection Response Index (IRI) as a quantitative measure to detect the early PTI response in Arabidopsis and Choy Sum plants infected with bacterial pathogens. The Raman spectral bands contributing toward a high IRI value were largely identical to the ERI Raman spectral bands. Raman spectroscopy is a convenient tool for rapid screening for Arabidopsis PTI mutants and may be suitable for the noninvasive and early diagnosis of pathogen-infected crop plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chung, Singh, Huang, Koyyappurath, Seo, Mao, Diloknawarit, Ram, Sarojam and Chua.)

Several SQUAMASA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors are involved in plant developmental transition from vegetative to reproductive growth. However, the function of SPL10 in regulating floral transition is largely unknown. It is also not known which Mediator subunit mediates SPL10 transcriptional activity. Here, we used overexpression lines and knockout mutants to examine the role of SPL10 in flowering-time regulation and we investigated possible interactions of SPL10 with several mediator subunits in vitro and in vivo. Plants overexpressing SPL10 showed precocious flowering, whereas the triple loss-of-function mutants of SPL10 and its two homologous genes, SPL2 and SPL11, flowered late compared with wild-type plants. We found that SPL10 interacts with MED25, a subunit of the Mediator complex, which bridges transcription factors and RNA polymerase II to facilitate transcription initiation. Genetic analysis showed that MED25 acts downstream of SPL10 to execute SPL10-regulated floral transition. Furthermore, SPL10 was required for MED25 association with the promoters of two target genes, FUL and LFY. We provide evidence that SPL10 recruits MED25 to the promoters of target genes to regulate flowering time. Our results on the SPL10/MED25 module are relevant to the molecular mechanism of other SPL family members., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Background: Stevia rebaudiana produces sweet-tasting steviol glycosides (SGs) in its leaves which can be used as natural sweeteners. Metabolic engineering of Stevia would offer an alternative approach to conventional breeding for enhanced production of SGs. However, an effective protocol for Stevia transformation is lacking., Results: Here, we present an efficient and reproducible method for Agrobacterium-mediated transformation of Stevia. In our attempts to produce transgenic Stevia plants, we found that prolonged dark incubation is critical for increasing shoot regeneration. Etiolated shoots regenerated in the dark also facilitated subsequent visual selection of transformants by green fluorescent protein during Stevia transformation. Using this newly established transformation method, we overexpressed the Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1 (SrDXS1) and kaurenoic acid hydroxylase (SrKAH), both of which are required for SGs biosynthesis. Compared to control plants, the total SGs content in SrDXS1- and SrKAH-overexpressing transgenic lines were enhanced by up to 42-54% and 67-88%, respectively, showing a positive correlation with the expression levels of SrDXS1 and SrKAH. Furthermore, their overexpression did not stunt the growth and development of the transgenic Stevia plants., Conclusion: This study represents a successful case of genetic manipulation of SGs biosynthetic pathway in Stevia and also demonstrates the potential of metabolic engineering towards producing Stevia with improved SGs yield.

Dispersed H3K27 trimethylation (H3K27me3) of the AGAMOUS (AG) genomic locus is mediated by CURLY LEAF (CLF), a component of the Polycomb Repressive Complex (PRC) 2. Previous reports have shown that the AG second intron, which confers AG tissue-specific expression, harbors sequences targeted by several positive and negative regulators. Using RACE reverse transcription polymerase chain reaction, we found that the AG intron 2 encodes several noncoding RNAs. RNAi experiment showed that incRNA4 is needed for CLF repressive activity. AG-incRNA4RNAi lines showed increased leaf AG mRNA levels associated with a decrease of H3K27me3 levels; these plants displayed AG overexpression phenotypes. Genetic and biochemical analyses demonstrated that the AG-incRNA4 can associate with CLF to repress AG expression in leaf tissues through H3K27me3-mediated repression and to autoregulate its own expression level. The mechanism of AG-incRNA4-mediated repression may be relevant to investigations on tissue-specific expression of Arabidopsis MADS-box genes., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7., (© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The human transmembrane 6 superfamily member 2 (TM6SF2) gene has been implicated in plasma lipoprotein metabolism, alcoholic and non-alcoholic fatty liver disease and myocardial infarction in multiple genome-wide association studies. To investigate the role of Tm6sf2 in metabolic homeostasis, we generated mice with elevated expression using adeno-associated virus (AAV)-mediated gene delivery. Hepatic overexpression of mouse Tm6sf2 resulted in phenotypes previously observed in Tm6sf2-deficient mice including reduced plasma lipid levels, diminished hepatic triglycerides secretion and increased hepatosteatosis. Furthermore, increased hepatic Tm6sf2 expression protected against the development of atherosclerosis in LDL-receptor/ApoB48-deficient mice. In cultured human hepatocytes, Tm6sf2 overexpression reduced apolipoprotein B secretion and resulted in its accumulation within the endoplasmic reticulum (ER) suggesting impaired ER-to-Golgi trafficking of pre-very low-density lipoprotein (VLDL) particles. Analysis of two metabolic trait-associated coding polymorphisms in the human TM6SF2 gene (rs58542926 and rs187429064) revealed that both variants impact TM6SF2 expression by affecting the rate of protein turnover. These data demonstrate that rs58542926 (E167K) and rs187429064 (L156P) are functional variants and suggest that they influence metabolic traits through altered TM6SF2 protein stability. Taken together, our results indicate that cellular Tm6sf2 level is an important determinant of VLDL metabolism and further implicate TM6SF2 as a causative gene underlying metabolic disease and trait associations at the 19p13.11 locus., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In many aromatic plants including spearmint (Mentha spicata), the sites of secondary metabolite production are tiny specialized structures called peltate glandular trichomes (PGT). Having high commercial values, these secondary metabolites are exploited largely as flavours, fragrances and pharmaceuticals. But, knowledge about transcription factors (TFs) that regulate secondary metabolism in PGT remains elusive. Understanding the role of TFs in secondary metabolism pathway will aid in metabolic engineering for increased yield of secondary metabolites and also the development of new production techniques for valuable metabolites. Here, we isolated and functionally characterized a novel MsYABBY5 gene that is preferentially expressed in PGT of spearmint. We generated transgenic plants in which MsYABBY5 was either overexpressed or silenced using RNA interference (RNAi). Analysis of the transgenic lines showed that the reduced expression of MsYABBY5 led to increased levels of terpenes and that overexpression decreased terpene levels. Additionally, ectopic expression of MsYABBY5 in Ocimum basilicum and Nicotiana sylvestris decreased secondary metabolite production in them, suggesting that the encoded transcription factor is probably a repressor of secondary metabolism., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The central nervous system is known to play important roles in the regulation of renal sodium excretion. The present study was designed to reveal the interrelationship between cholinergic pathway in the magnocellular paraventricular nucleus (PVN) and the natriuresis induced by brain cholinergic stimuli. The results indicated that urinary sodium excretion was significantly increased at 40 min after intracerebroventricular (ICV) injection of carbachol (CBC). Immunohistochemical studies showed that CBC increased choline acetyltransferase-immunoreactivity (ChAT-IR) in the magnocellular PVN and renal proximal convoluted tubule (PCT), respectively. After pretreatment with atropine, urinary sodium excretion was significantly reduced, and carbachol-increased ChAT-IR in the magnocellular PVN and PCT was also significantly decreased. These results suggested that brain cholinergic stimuli induced the natriuresis and increased the activity of cholinergic neurons in the magnocellular PVN and cholinergic system in the PCT. The blockade of muscarinic receptor completely abolished the natriuresis and partially inhibited carbachol-exerted stimulatory effects in the magnocellular PVN and PCT. To summarize, brain cholinergic pathway and peripheral cholinergic system in kidney were found to contribute to the natriuresis following brain cholinergic stimulation. Our findings revealed novel evidence that PVN was involved in the natriuresis via humoral mechanisms.

Background: Jatropha curcus is a good candidate plant for biodiesel production in tropical and subtropical regions. However, J. curcus is susceptible to the geminivirus Indian cassava mosaic virus (ICMV), and frequent viral disease outbreaks severely limit productivity. Therefore the development of J. curcus to carry on durable virus resistance remains crucial and poses a major biotechnological challenge., Results: We generated transgenic J. curcus plants expressing a hairpin, double-stranded (ds) RNA with sequences homologous to five key genes of ICMV-Dha strain DNA-A, which silences sequence-related viral genes thereby conferring ICMV resistance. Two rounds of virus inoculation were conducted via vacuum infiltration of ICMV-Dha. The durability and heritability of resistance conferred by the dsRNA was further tested to ascertain that T1 progeny transgenic plants were resistant to the ICMV-SG strain, which shared 94.5% nucleotides identity with the ICMV-Dha strain. Quantitative PCR analysis showed that resistant transgenic lines had no detectable virus., Conclusions: In this study we developed transgenic J. curcus plants to include a resistance to prevailing geminiviruses in Asia. These virus-resistant transgenic J. curcus plants can be used in various Jatropha breeding programs.

Lipase maturation factor 1 (Lmf1) is a critical determinant of plasma lipid metabolism, as demonstrated by severe hypertriglyceridemia associated with its mutations in mice and human subjects. Lmf1 is a chaperone localized to the endoplasmic reticulum (ER) and required for the post-translational maturation and activation of several vascular lipases. Despite its importance in plasma lipid homeostasis, the regulation of Lmf1 remains unexplored. We report here that Lmf1 expression is induced by ER stress in various cell lines and in tunicamycin (TM)-injected mice. Using genetic deficiencies in mouse embryonic fibroblasts and mouse liver, we identified the Atf6α arm of the unfolded protein response as being responsible for the up-regulation of Lmf1 in ER stress. Experiments with luciferase reporter constructs indicated that ER stress activates the Lmf1 promoter through a GC-rich DNA sequence 264 bp upstream of the transcriptional start site. We demonstrated that Atf6α is sufficient to induce the Lmf1 promoter in the absence of ER stress, and this effect is mediated by the TM-responsive cis-regulatory element. Conversely, Atf6α deficiency induced by genetic ablation or a dominant-negative form of Atf6α abolished TM stimulation of the Lmf1 promoter. In conclusion, our results indicate that Lmf1 is an unfolded protein response target gene, and Atf6α signaling is sufficient and necessary for activation of the Lmf1 promoter. Importantly, the induction of Lmf1 by ER stress appears to be a general phenomenon not restricted to lipase-expressing cells, which suggests a lipase-independent cellular role for this protein in ER homeostasis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Triacylglycerols (TAGs) are the most abundant form of storage oil in plants. They consist of three fatty acid chains (usually C16 or C18) covalently linked to glycerol. SDP1 is a specific lipase for the first step of TAG catabolism in Arabidopsis seeds. Arabidopsis mutants deficient in SDP1 accumulate high levels of oils, probably due to blockage in TAG degradation. We applied this knowledge from the model plant, Arabidopsis thaliana, to engineer increased seed oil content in the biodiesel plant Jatropha curcas using RNA interference (RNAi) technology., Results: As Jatropha is a biodiesel crop, any significant increase in its seed oil content would be an important agronomic trait. Using A. thaliana as a model plant, we found that a deficiency of SDP1 led to higher TAG accumulation and a larger number of oil bodies in seeds compared with wild type (Columbia-0; Col-0). We cloned Jatropha JcSDP1, and verified its function by complementation of the Arabidopsis sdp1-5 mutant. Taking advantage of the observation with Arabidopsis, we used RNAi technology to generate JcSDP1 deficiency in transgenic Jatropha. We found that Jatropha JcSDP1-RNAi plants accumulated 13 to 30% higher total seed storage lipid, along with a 7% compensatory decrease in protein content, compared with control (CK; 35S:GFP) plants. Free fatty acid (FFA) content in seeds was reduced from 27% in control plants to 8.5% in JcSDP1-RNAi plants., Conclusion: Here, we showed that SDP1 deficiency enhances seed oil accumulation in Arabidopsis. Based on this result, we generated SDP1-deficient transgenic Jatropha plants using by RNAi technology with a native JcSDP1 promoter to silence endogenous JcSDP1 expression. Seeds of Jatropha JcSDP1-RNAi plants accumulated up to 30% higher total lipid and had reduced FFA content compared with control (CK; 35S:GFP) plants. Our strategy of improving an important agronomic trait of Jatropha can be extended to other oil crops to yield higher seed oil.

Objective: Lipoprotein lipase (LPL) is a principal enzyme in lipoprotein metabolism, tissue lipid utilization, and energy metabolism. LPL is synthesized by parenchymal cells in adipose, heart, and muscle tissues followed by secretion to extracellular sites, where lipolyic function is exerted. The catalytic activity of LPL is attained during posttranslational maturation, which involves glycosylation, folding, and subunit assembly within the endoplasmic reticulum. A lipase-chaperone, lipase maturation factor 1 (Lmf1), has recently emerged as a critical factor in this process. Previous studies demonstrated that loss-of-function mutations of Lmf1 result in diminished lipase activity and severe hypertriglyceridemia in mice and human subjects. The objective of this study is to investigate whether, beyond its role as a required factor in lipase maturation, variation in Lmf1 expression is sufficient to modulate LPL activity in vivo., Methods and Results: To assess the effects of Lmf1 overexpression in adipose and muscle tissues, we generated aP2-Lmf1 and Mck-Lmf1 transgenic mice. Characterization of relevant tissues revealed increased LPL activity in both mouse strains. In the omental and subcutaneous adipose depots, Lmf1 overexpression was associated with increased LPL specific activity without changes in LPL mass. In contrast, increased LPL activity was due to elevated LPL protein level in heart and gonadal adipose tissue. To extend these studies to humans, we detected association between LMF1 gene variants and postheparin LPL activity in a dyslipidemic cohort., Conclusions: Our results suggest that variation in Lmf1 expression is a posttranslational determinant of LPL activity.

Background: Jatropha curcas is recognized as a new energy crop due to the presence of the high amount of oil in its seeds that can be converted into biodiesel. The quality and performance of the biodiesel depends on the chemical composition of the fatty acids present in the oil. The fatty acids profile of the oil has a direct impact on ignition quality, heat of combustion and oxidative stability. An ideal biodiesel composition should have more monounsaturated fatty acids and less polyunsaturated acids. Jatropha seed oil contains 30% to 50% polyunsaturated fatty acids (mainly linoleic acid) which negatively impacts the oxidative stability and causes high rate of nitrogen oxides emission., Results: The enzyme 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in plants. We identified three putative delta 12 fatty acid desaturase genes in Jatropha (JcFAD2s) through genome-wide analysis and downregulated the expression of one of these genes, JcFAD2-1, in a seed-specific manner by RNA interference technology. The resulting JcFAD2-1 RNA interference transgenic plants showed a dramatic increase of oleic acid (> 78%) and a corresponding reduction in polyunsaturated fatty acids (< 3%) in its seed oil. The control Jatropha had around 37% oleic acid and 41% polyunsaturated fatty acids. This indicates that FAD2-1 is the major enzyme responsible for converting oleic acid to linoleic acid in Jatropha. Due to the changes in the fatty acids profile, the oil of the JcFAD2-1 RNA interference seed was estimated to yield a cetane number as high as 60.2, which is similar to the required cetane number for conventional premium diesel fuels (60) in Europe. The presence of high seed oleic acid did not have a negative impact on other Jatropha agronomic traits based on our preliminary data of the original plants under greenhouse conditions. Further, we developed a marker-free system to generate the transgenic Jatropha that will help reduce public concerns for environmental issues surrounding genetically modified plants., Conclusion: In this study we produced seed-specific JcFAD2-1 RNA interference transgenic Jatropha without a selectable marker. We successfully increased the proportion of oleic acid versus linoleic in Jatropha through genetic engineering, enhancing the quality of its oil.

Objective: To identify metabolic derangements contributing to diabetes susceptibility in the leptin receptor-deficient obese C57BLKS/J-db/db (BKS-db) mouse strain., Research Design and Methods: Young BKS-db mice were used to identify metabolic pathways contributing to the development of diabetes. Using the diabetes-resistant B6-db strain as a comparison, in vivo and in vitro approaches were applied to identify metabolic and molecular differences between the two strains., Results: Despite higher plasma insulin levels, BKS-db mice exhibit lower lipogenic gene expression, rate of lipogenesis, hepatic triglyceride and glycogen content, and impaired insulin suppression of gluconeogenic genes. Hepatic insulin receptor substrate (IRS)-1 and IRS-2 expression and insulin-stimulated Akt-phosphorylation are decreased in BKS-db primary hepatocytes. Hyperinsulinemic-euglycemic clamp studies indicate that in contrast to hepatic insulin resistance, skeletal muscle is more insulin sensitive in BKS-db than in B6-db mice. We also demonstrate that elevated plasma triglyceride levels in BKS-db mice are associated with reduced triglyceride clearance due to lower lipase activities., Conclusions: Our study demonstrates the presence of metabolic derangements in BKS-db before the onset of beta-cell failure and identifies early hepatic insulin resistance as a component of the BKS-db phenotype. We propose that defects in hepatic insulin signaling contribute to the development of diabetes in the BKS-db mouse strain.

ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. The F1 subcomplex has three catalytic nucleotide binding sites, one on each beta subunit, at the interface to the adjacent alpha subunit. In the x-ray structure of F1 (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the three catalytic beta/alpha interfaces differ in the extent of inter-subunit interactions between the C termini of the beta and alpha subunits. At the closed betaDP/alphaDP interface, a hydrogen-bonding network is formed between both subunits, which is absent at the more open betaTP/alphaTP interface and at the wide open betaE/alphaE interface. The hydrogen-bonding network reaches from betaL328 (Escherichia coli numbering) and betaQ441 via alphaQ399, betaR398, and alphaE402 to betaR394, and ends in a cation/pi interaction between betaR394 and alphaF406. Using mutational analysis in E. coli ATP synthase, the functional importance of the betaDP/alphaDP hydrogen-bonding network is demonstrated. Its elimination results in a severely impaired enzyme but has no pronounced effect on the binding affinities of the catalytic sites. A possible role for the hydrogen-bonding network in coupling of ATP synthesis/hydrolysis and rotation will be discussed.

Hypertriglyceridemia is a hallmark of many disorders, including metabolic syndrome, diabetes, atherosclerosis and obesity. A well-known cause is the deficiency of lipoprotein lipase (LPL), a key enzyme in plasma triglyceride hydrolysis. Mice carrying the combined lipase deficiency (cld) mutation show severe hypertriglyceridemia owing to a decrease in the activity of LPL and a related enzyme, hepatic lipase (HL), caused by impaired maturation of nascent LPL and hepatic lipase polypeptides in the endoplasmic reticulum (ER). Here we identify the gene containing the cld mutation as Tmem112 and rename it Lmf1 (Lipase maturation factor 1). Lmf1 encodes a transmembrane protein with an evolutionarily conserved domain of unknown function that localizes to the ER. A human subject homozygous for a deleterious mutation in LMF1 also shows combined lipase deficiency with concomitant hypertriglyceridemia and associated disorders. Thus, through its profound effect on lipase activity, LMF1 emerges as an important candidate gene in hypertriglyceridemia.

ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. The F(1) subcomplex has three catalytic nucleotide binding sites, one on each beta subunit, with widely differing affinities for MgATP or MgADP. During rotational catalysis, the sites switch their affinities. The affinity of each site is determined by the position of the central gamma subunit. The site with the highest nucleotide binding affinity is catalytically active. From the available x-ray structures, it is not possible to discern the high-affinity site. Using fluorescence resonance energy transfer between tryptophan residues engineered into gamma and trinitrophenyl nucleotide analogs on the catalytic sites, we were able to determine that the high-affinity site is close to the C-terminal helix of gamma, but at considerable distance from its N terminus. Thus, the beta(TP) site in the x-ray structure [Abrahams JP, Leslie AGW, Lutter R, Walker JE (1994) Nature 370:621-628] is the high-affinity site, in agreement with the prediction of Yang et al. [Yang W, Gao YQ, Cui Q, Ma J, Karplus M (2003) Proc Natl Acad Sci USA 100:874-879]. Taking into account the known direction of rotation, the findings establish the sequence of affinities through which each catalytic site cycles during MgATP hydrolysis as low --> high --> medium --> low.

Flame atomic absorption spectrometry with air-acetylene flame was used for the determination of inorganic metal elements in different parts ( flower, leaf, stem and root) of Sonchus oleraceus L. The contents of Ca, Mg, K, Na, Fe, Mn, Cu, Zn, Cr, Co, Ni, Pb and Cd in the flower, leaf, stem and root of Sonchus oleraceus L were compared. The order from high to low of the additive weight (microg x g(-1)) for the 13 kinds of metal elements is as follows: leaf (77 213.72) > flower (47 927.15) > stem(42 280.99) > root (28 131.18). From the experimental results it was found that there were considerable differences in the contents of the metal elements in different parts, and there were richer contents of Fe, Zn, Mn and Cu in root and flower, which are necessary to human health, than in other parts.

Combined lipase deficiency (cld) is a recessive, lethal mutation specific to the tw73 haplotype on mouse Chromosome 17. While the cld mutation results in lipase proteins that are inactive, aggregated, and retained in the endoplasmic reticulum (ER), it maps separately from the lipase structural genes. We have narrowed the gene critical region by about 50% using the tw18 haplotype for deletion mapping and a recombinant chromosome used originally to map cld with respect to the phenotypic marker tf. The region now extends from 22 to 25.6 Mbp on the wild-type chromosome, currently containing 149 genes and 50 expressed sequence tags (ESTs). To identify the affected gene, we have selected candidates based on their known role in associated biological processes, cellular components, and molecular functions that best fit with the predicted function of the cld gene. A secondary approach was based on differences in mRNA levels between mutant (cld/cld) and unaffected (+/cld) cells. Using both approaches, we have identified seven functional candidates with an ER localization and/or an involvement in protein maturation and folding that could explain the lipase deficiency, and six expression candidates that exhibit large differences in mRNA levels between mutant and unaffected cells. Significantly, two genes were found to be candidates with regard to both function and expression, thus emerging as the strongest candidates for cld. We discuss the implications of our mapping results and our selection of candidates with respect to other genes, deletions, and mutations occurring in the cld critical region.

During ATP synthesis, ATP synthase has to bind MgADP in the presence of an excess of MgATP. Thus, for efficient ATP synthesis it would be desirable if incoming substrate could be bound to a catalytic site with a preference for MgADP over MgATP. We tested three hypotheses predicting the existence of such a site. However, our results showed that, at least in absence of an electrochemical proton gradient, none of the three catalytic sites has a higher affinity for MgADP than for MgATP.

The C57BLKS/J (BKS) inbred mouse strain is a widely used animal model of type 2 diabetes. In the presence of the diabetes (db) mutation, obese BKS-db mice develop severe diabetes. Genetic studies of diabetes-susceptibility in this strain are facilitated by the fact that BKS is a genetic composite between the diabetes-resistant C57BL/6J (B6) and susceptible DBA/2J (DBA) strains. On this basis, it has been hypothesized that diabetes-susceptibility in BKS is conferred by DBA-derived alleles. However, recent studies revealed non-B6/non-DBA genetic material in BKS. To identify the origin of this genetic component, we generated a genomic map of BKS using 537 microsatellite markers. Our results demonstrate that, in addition to B6 and DBA, BKS contains alleles from at least three other strains, including 129, C57BL/10 and an unidentified mouse strain. We also analyzed two congenic strains, B6-db and BKS-db, which are widely used for the genetic mapping of diabetes-susceptibility loci. We identified several donor-derived genomic regions introduced during the generation of these congenic strains. In summary, our study reveals novel aspects of the genetic fine-structure of BKS and related strains and facilitates the identification of diabetes-susceptibility loci in this mouse model.

A protocol was developed for Agrobacterium-mediated genetic transformation of monocotyledon foliage plant Caladium bicolor cv. Jackie Suthers using leaf disc and petiole as the explants. The explants were inoculated with Agrobacterium strain LBA4404 harboring a binary vector with the maize anthocyanin regulatory gene Lc under the control of the cauliflower mosaic virus promoter. Callus formation was induced in MS medium supplemented with 0.5 mg/l 6-benzylaminopurine (6-BA), 0.1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g/l sucrose and kanamycin 50 mg/l for selection. Resistant calli were induced for shoot generation in MS medium with 2 mg/l 6-BA and 0.2 mg/l alpha-naphthaleneacetic acid. As much as 10% of the explants gave rise to kanamycin-resistant shoots with our procedure. Transformed plants had enhanced anthocyanin accumulation in the roots, leaves and stems (epidermis and vascular bundles). Integration of the transgene into the host genome was confirmed by genomic Southern blot hybridization, and RNA blot hybridization analysis indicated that the expression of the transgene correlated with anthocyanin accumulation. This investigation illustrates the utility of anthocyanin regulatory genes in the genetic manipulation of the color of foliage plants. It also supports the premise that the Lc gene can be used as a powerful non-destructive cell autonomous visual marker in a wide variety of plants, as exemplified by the perfect symmetrical half-green/half-red plant presumably derived from the symmetrical division of one transgenic and one non-transgenic precursor meristematic cell.

In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. The microsporocytes generate microspores, whereas the tapetal cells support the development of microspores into mature pollen grains. Despite their importance to plant reproduction, little is known about the underlying genetic mechanisms that regulate the differentiation and interaction of these highly specialized cells in the anther. Here, we report the identification and characterization of a novel tapetum determinant1 (TPD1) gene that is required for the specialization of tapetal cells in the Arabidopsis anther. Analysis of the male-sterile mutant, tpd1, showed that functional interruption of TPD1 caused the precursors of tapetal cells to differentiate and develop into microsporocytes instead of tapetum. As a results, extra microsporocytes were formed and tapetum was absent in developing tpd1 anthers. Molecular cloning of TPD1 revealed that it encodes a small protein of 176 amino acids. In addition, tpd1 was phenotypically similar to excess microsporocytes1/extra sporogenous cells (ems1/exs) single and tpd1 ems1/exs double mutants. These data suggest that the TPD1 product plays an important role in the differentiation of tapetal cells, possibly in coordination with the EMS1/EXS gene product, a Leu-rich repeat receptor protein kinase.

Administration of nitric oxide (NO) or NO donors to isolated carotid sinus and carotid bodies inhibits the activity of baroreceptor and chemoreceptor afferent nerves. Furthermore, NO synthase (NOS) is present in endothelial cells and in sensory nerves innervating the carotid sinus region. The major goal of this study was to determine whether overexpression of NOS in carotid sinus modulates baroreceptor activity. Rabbits were anesthetized, and adenoviral vectors (5 x 10(8) plaque-forming units) encoding genes for either beta-galactosidase (beta-Gal) or endothelial type III NOS (eNOS) were applied topically to the adventitial surface of one carotid sinus. In some experiments, the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) was applied to the carotid sinus immediately after the vector. Four to five days later, baroreceptor activity and carotid sinus diameter were measured from the vascularly isolated carotid sinus of the anesthetized rabbits. Transgene expression was confirmed by X-Gal staining of beta-Gal and measurement of NOS activity by citrulline assay. The expression was restricted to the carotid sinus adventitia. Baroreceptor activity was decreased significantly, and the pressure-activity curve was shifted to higher pressures in eNOS-transduced (n = 5) compared with beta-Gal-transduced (n = 5) carotid sinuses. The pressure corresponding to 50% of maximum activity averaged 55 +/- 6 and 76 +/- 7 mmHg in beta-Gal- and eNOS-transduced carotid sinuses, respectively (P < 0.05). Decreased baroreceptor activity was accompanied by a significant increase in carotid diameter in the eNOS-transduced carotid sinuses (n = 5). l-NAME prevented the inhibition of baroreceptor activity and the increase in carotid diameter in eNOS-transduced carotid sinuses (n = 5). We conclude that adenoviral-mediated gene transfer of eNOS to carotid sinus adventitia causes sustained, NO-dependent inhibition of baroreceptor activity and resetting of the baroreceptor function curve to higher pressures.

The maturation of lipoprotein lipase (LPL) into a catalytically active enzyme was believed to occur only after its transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To test this hypothesis, LPL located in these two subcellular compartments was separated and compared. Heparin affinity chromatography resolved low affinity, inactive LPL displaying ER characteristics from a high affinity, active fraction exhibiting both ER and Golgi forms. The latter forms were further separated by beta-ricin chromatography and were found to have comparable activities per unit of LPL mass. Thus, LPL must reach a functional conformation in the ER. Active LPL, regardless of its cellular location, exhibited the expected dimer conformation. However, inactive LPL, found only in the ER, was highly aggregated. Kinetic analysis indicated a concurrent formation of LPL dimer and aggregate and indicated that the two forms have dissimilar fates. Whereas the dimer remained stable even when confined to the ER, the aggregate was degraded. Degradation rates were not affected by proteasomal or lysosomal inhibitors but were markedly reduced by ATP depletion. Lowering the redox potential in the ER by dithiothreitol caused the dimer to associate with calnexin, BiP, and protein-disulfide isomerase to form large, inactive complexes; dithiothreitol removal induced complex dissociation with restoration of the functional LPL dimer. In contrast, the LPL aggregate was only poorly associated with ER chaperones, appearing to be trapped in an irreversible, inactive conformation destined for ER degradation.

Cotyledons, each with a 1-2 mm petiole at its base, were cut from axenic seedlings and infected with Agrobacterium tumefaciens. After 2-3 days of cocultivation, the cotyledon explants were transferred to MS selection medium containing 15 mg/L kanamycin and 4.5 mg/L 6-BA to induce shoot differentiation. Kanamycin-resistant shoots were subcultured on selection medium with 20-50 mg/L kanamycin for 3-6 months for eliminating escaped non-transformants, and then rooted on MS medium containing 25 mg/L kanamycin and 0.1 mg/L NAA. Whole plants were transplanted into soil and grew in the field. DNA Southern blot hybridization and polymerase chain reaction showed that some of the plants were positive when probed with the insecticidal crystal protein gene. The transgenic plants exhibited tolerant to pest insects such as Laphygma exigua and Pieris rapae in leaf feeding experiments Kanamycin-resistance and insect-resistance were maintained in the progeny. The foreign genes were delivered to the progeny according to Mendelian Law of single gene segregation.

The role that the central sympathetic nervous system plays in the development of obesity hypertension and insulin was evaluated by feeding dogs a high fat diet with or without clonidine treatment. Thirteen adult mongrel dogs were chronically instrumented and randomly assigned to receive either a high fat diet and no clonidine (n=6) or a high fat diet plus clonidine (n=7), 0.3 mg BID. Blood pressure, heart rate, plasma insulin, and electrolytes were measured daily. Insulin resistance was assessed with a multiple-dose euglycemic clamp (1, 2, and 30 mU. kg-1. min-1) before and after 1, 3, and 6 weeks of the high fat diet. Clonidine prevented the hypertension, tachycardia, and insulin resistance associated with feeding dogs the high fat diet but did not affect weight gain. The present study suggests that the central sympathetic nervous system plays a critical role in the development of both insulin resistance and hypertension associated with feeding dogs a high fat diet.

Baroreceptor nerve endings are located in the adventitia of the carotid sinuses and aortic arch. The goal of the present study was to develop a method for gene transfer to the carotid sinus adventitia. Replication-deficient adenovirus containing the gene for Escherichia coli beta-galactosidase (beta-Gal) was applied topically to the carotid sinuses of anesthetized rabbits. Transgene expression was localized by histochemical staining and quantified by chemiluminescence assay (Galacto-Light). Possible effects of adenovirus on baroreceptor sensitivity were investigated by recording baroreceptor activity from the vascularly isolated carotid sinus over a pressure range of 0 to 160 mm Hg. Beta-Gal expression in carotid sinus was evident 1 day after virus application, was dose dependent, and was markedly enhanced after 4 days. Expression was restricted to the adventitia of the vessel wall and was not present in vehicle-treated carotid sinuses. Baroreceptor sensitivity measured from carotid sinuses exposed to adenovirus 4 to 5 days beforehand was not altered compared with that measured from control carotid sinuses. In summary, topical application of adenoviral vectors to the carotid sinus provides transgene expression restricted to the region of baroreceptor innervation. The technique provides a novel approach to delineate mechanisms involved in baroreceptor activation and to deliver neuroactive gene products to the baroreceptors.

The goal of the present study was to determine whether oxygen-derived free radicals contribute to baroreceptor dysfunction in atherosclerosis. Baroreceptor activity was measured from the carotid sinus nerve during pressure ramps in isolated carotid sinuses of anesthetized rabbits. Rabbits fed a 0.5% to 1.0% cholesterol diet for 7.9 +/- 0.4 months (mean +/- SE; range, 5.5 to 10) developed atherosclerotic lesions in the carotid sinuses. Maximum baroreceptor activity measured at 140 mm Hg and the slope of the pressure-activity curve were reduced in atherosclerotic (n = 15) compared with normal (n = 13) rabbits (425 +/- 34 versus 721 +/- 30 spikes per second and 6.2 +/- 0.6 versus 10.8 +/- 0.8 spikes per second per mm Hg, respectively, P < .05). The level of activity was inversely related to plasma cholesterol concentration (r = .86, P < .001) and total cholesterol load (plasma concentration x duration of diet, r = .92). Mean arterial pressure was normal in both groups. Exposure of the carotid sinus to the free-radical scavengers superoxide dismutase (SOD) and catalase significantly increased maximum baroreceptor activity by 25 +/- 4% in atherosclerotic rabbits (n = 6) but caused only small and irreversible changes in activity in normal rabbits (n = 8). Catalase alone but not SOD also increased baroreceptor activity in atherosclerotic rabbits (n = 7). Exposure of the carotid sinus of normal rabbits to exogenous free radicals generated from the reaction between xanthine and xanthine oxidase inhibited baroreceptor activity in a dose-dependent and reversible manner (n = 8, P < .05). The inhibition of activity was attenuated by SOD and catalase but was not attenuated by the inhibitor of hydroxyl radical formation, deferoxamine. Neither restoration of baroreceptor activity in atherosclerotic rabbits by catalase nor inhibition of activity by xanthine/xanthine oxidase could be explained by changes in the carotid pressure-diameter relation or prostacyclin formation. These results indicate that oxidant stress inhibits baroreceptor activity and that endogenous oxyradicals produced in atherosclerotic carotid sinuses contribute to baroreceptor dysfunction.

The carotid sinuses, one of the major sites of baroreceptor innervation, are also a common site of atherosclerotic lesions and platelet aggregation. The goal of the present study was to determine whether platelet activation in carotid sinuses causes reflex-mediated changes in renal sympathetic nerve activity and arterial pressure. Rabbit platelets were isolated, resuspended in Krebs' buffer, and activated by thrombin. Injection of activated platelets (3 x 10(8) platelets/mL) into the vascularly isolated carotid sinuses of anesthetized rabbits essentially eliminated sympathetic nerve activity and acutely decreased mean arterial pressure from 126 +/- 5 to 53 +/- 4 mm Hg (n=16; P < .05). Sympathetic activity and arterial pressure returned to control levels over a period of minutes despite sustained exposure to activated platelets. Injection of U-46619, a thromboxane analogue and vasoconstrictor, into carotid sinuses did not alter sympathetic activity or arterial pressure. However, serotonin (5-hydroxytryptamine [5-HT]), which is known to be released from activated platelets, and the 5-HT3 receptor agonist phenylbiguanide mimicked the effect of platelets. Furthermore, the platelet-induced reflex inhibition of sympathetic activity and hypotension were not altered by the cyclooxygenase inhibitor indomethacin but were attenuated significantly by 5-HT receptor antagonists. Platelet activation inhibited sympathetic activity to 5 +/- 2% of control in the absence of antagonists but to only 35 +/- 11 and 76 +/- 4% of control after selective blockade of 5-HT2 and 5-HT3 receptors with ketanserin and MDL-72222, respectively. The results indicate that (1) platelet activation in carotid sinuses triggers reflex inhibition of sympathetic nerve activity and hypotension; (2) the reflex is not caused by carotid vasoconstriction and is not mediated by prostanoids; and (3) the reflex is mediated by 5-HT acting primarily on 5-HT3 and to a lesser extent on 5-HT2 receptors. We speculate that this reflex may contribute to arterial pressure lability and susceptibility to stroke in patients with carotid atherosclerotic disease.

Cotyledons cut from axenic seedlings were immersed inAgrobacterium tumefaciens suspension which was treated with acetosyringone and nopaline at low pH overnight. The infected cotyledon explants were cultured on MSB medium (MS salts + B5 Vitamins) containing 6-BA 3mg/1 for 2-3 days, and transferred onto selective medium (MSB with kanamycin 50-100 mg/l). Kanamycin-resistant shoots were selected. More than 60 regenerated plants were obtained. About 60% of the plants showed high NPT II activity. Southern blot hybridization showed that some of the plants gave a positive signal with the insecticidal crystal protein gene (cry IA gene) probe, and exhibited tolerant to insects such asPieris rapae (cabbage caterpillar) in leaf feeding experiments. Kanamycin-resistance and insect-resistance were maintained in the progeny.

Gadolinium (Gd3+) has been shown to prevent mechanoelectrical transduction believed to be mediated through stretch-activated channels. We investigated the possible role of Gd(3+)-sensitive channels in mediating baroreceptor activity in the carotid sinus of rabbits. Baroreceptor activity induced by a ramp increase of carotid sinus pressure was reduced significantly during exposure to Gd3+. The inhibition was dose-related and reversible, and was not associated with alteration of carotid sinus wall mechanics as the pressure-strain relationship was unaffected. Veratrine triggered action potentials from single- and multiple-baroreceptor fibers when their response to pressure was inhibited by Gd3+. This suggests that the effect of Gd3+ on baroreceptors in the isolated carotid sinus was specific to their mechanical activation. The results suggest that stretch-activated ion channels sensitive to Gd3+ may be the mechanoelectrical transducers of rabbit carotid sinus baroreceptors.

The selectins are a three-member family of leukocyte, platelet, and endothelial cell adhesion proteins that mediate leukocyte traffic into normal and inflamed tissues. P-selectin is expressed by endothelial cells and platelets, E-selectin by endothelial cells, and L-selectin by circulating leukocytes. To determine if selectin-mediated leukocyte adhesion influences the development of lung reperfusion injury, we studied hemodynamics and respiratory and inert gas exchange in sheep subjected to 3-hour in situ left lung ischemia followed by 6-hour left lung reperfusion with the right lung excluded. Ten minutes before reperfusion, eight animals received EL-246 (1 mg/kg intravenously), a novel antihuman selectin antibody that recognizes and blocks both L- and E-selectin and cross-reacts in sheep. Eight control animals with ischemia received no treatment, whereas three received an isotype-matched antihuman L-selectin antibody that does not cross-react in sheep (DREG-56, 1 mg/kg intravenously). Eight sham control sheep underwent an identical operative procedure but were never subjected to ischemia. Volume-cycled, pressure-limited (20 cm H2O) mechanical ventilation was consistent in all animals throughout the experiment. Six-hour survival in EL-246 recipients (100%) was significantly higher than in either ischemic control sheep (37.5%) or DREG-56 recipients (33.3%), but gravimetric lung water was equivalent in EL-246 recipients (5.9 +/- 1.7 ml/kg), ischemic control sheep (8.3 +/- 3.0 ml/kg), and DREG-56 recipients (9.1 +/- 2.6 ml/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

CD18 designates a component of a leukocyte surface glycoprotein complex that mediates endothelial adherence. To determine whether interference with CD18-dependent leukocyte adhesion modifies reperfusion injury, we transplanted 16 canine left lungs after 4-hour preservation with modified Euro-Collins solution. Anti-canine CD18 monoclonal antibody (R15.7, 1 mg/kg, intravenously) was administered to eight lung recipients 5 minutes before reperfusion; eight control recipients were not treated. Ventilation was identical in donor-recipient pairs (tidal volume, 600 ml; fraction of inspired oxygen, 0.53; positive end-expiratory pressure, 5 cm H2O). Respiratory and inert gas exchange and hemodynamics were assessed in left lung donors one-half hour after right lung exclusion and in allograft recipients at 0.5, 1.5, 2.5, 3.5, and 6.0 hours after transplantation and right lung exclusion. Reperfusion injury was evident in both recipient groups at 6 hours after transplantation, but inert gas shunt was lower in monoclonal antibody-treated dogs (13% +/- 6%) than in controls (30% +/- 17%, p < 0.05); comparisons of arterial blood gases in monoclonal antibody recipients (PaO2, 209 +/- 83 mm Hg; PaCO2, 45 +/- 7 mm Hg) and controls (PaO2, 108 +/- 54, p < 0.05; PaCO2, 64 +/- 25, p < 0.05) at 6 hours indicated that monoclonal antibody administration distinctly improved respiratory gas transfer. Gravimetric lung water was less in monoclonal antibody recipients (5.78 +/- 1.01 ml/kg) than in controls (8.02 +/- 1.90 ml/kg, p < 0.05), but lung compliance at 6 hours was equally reduced in monoclonal antibody recipients (40 +/- 9 ml/cm H2O) and in controls (39 +/- 7 ml/cm H2O, p = not significant). Pulmonary vascular resistance doubled immediately after transplantation but was identical in monoclonal antibody-treated dogs (890 +/- 168 dynes.sec.cm-5) and in controls (874 +/- 162 dynes.sec.cm-5, p = not significant) at 6 hours. We conclude that inhibition of CD18-dependent leukocyte function attenuates the development of both shunt and abnormal respiratory gas exchange in lung reperfusion injury. Significant physiologic abnormalities occurred despite R15.7 treatment and may represent inadequate preservation or the effect of CD18-independent adhesion mechanisms.