Mature wheat (Triticum aestivum L.) endosperm contains two types of starch granules: large A‐type and small B‐type. Two methods, microsieving or centrifugal sedimentation through aqueous solutions of sucrose, maltose, or Percoll were used to separate A‐ and B‐type starch granules. Microsieving could not completely separate the two types of starch granules, while centrifuging through maltose and sucrose solutions gave a homogenous population for B‐type starch granules only. Centrifuging through two Percoll solutions (70 and 100%, v/v) produced purified populations of both the A‐ and B‐type starch granules. Analysis of starch granule size distribution in the purified A‐ and B‐type granule populations and in the whole‐starch granule population obtained directly from wheat endosperm confirmed that the purified A‐ and B‐type starch granule populations represented their counterparts in mature wheat endosperm. Centrifugations through two Percoll solutions were used to purify A‐ and B‐type starch granule populations from six wheat cultivars. The amylose concentrations and gelatinization properties of these populations were analyzed. All of the A‐type starch granules contained higher amylose concentrations and had higher gelatinization enthalpies than did B‐type starch granules. Although A‐ and B‐type starch granules started to gelatinize at a similar temperature, B‐type starch granules had higher gelatinization peak and completion temperatures than did A‐type starch granules

Cardiac dysfunction is a major consequence of sepsis/septic shock and contributes to the high mortality of sepsis. Innate and inflammatory responses mediated by Toll-like receptors (TLRs) play a critical role in sepsis-induced cardiac dysfunction. MicroRNA-146 was first identified as a negative regulator in innate immune and inflammatory responses induced by LPS. This study examined whether miR-146a will have a protective effect on sepsis-induced cardiac dysfunction. Lentivirus expressing miR-146a (LmiR-146a) or lentivirus expressing scrambled miR (LmiR-control) were delivered into the myocardium via the right carotid artery. Seven days after transfection, mice were subjected to CLP. Untransfected mice were also subjected to CLP-induced sepsis. Cardiac function was examined by echocardiography before and 6 h after CLP. In vitro studies showed that increased miR-146a levels suppresses LPS-induced IκBα phosphorylation and inflammatory cytokine production in both H9C2 cardiomyocytes and J774 macrophages. In vivo transfection of LmiR-146a attenuated sepsis-induced cardiac dysfunction. The values for EF% and FS% in LmiR-146a transfected CLP mice were significantly greater than in untransfected CLP control. LmiR-146a transfection prevented sepsis-induced NF-κB activity, suppressed IRAK and TRAF6 expression in the myocardium, and attenuated sepsis-induced inflammatory cytokine production in both plasma and peritoneal fluid. In addition, LmiR-146a transfection decreased sepsis-induced infiltration of neutrophils and macrophages into the myocardium. LmiR-146a can also transfect macrophages in the periphery. We conclude that miR-146a attenuates sepsis-induced cardiac dysfunction by preventing NF-κB activation, inflammatory cell infiltration, and inflammatory cytokine production via targeting of IRAK and TRAF6 in both cardiomyocytes and inflammatory monocytic cells.

Objective To determine the role of Toll-like receptor 3 in cardiac dysfunction during polymicrobial sepsis. Design controlled animal study Setting University Research Laboratory Subjects Male C57BL/6, Wild type, Toll-like receptor 3−/− Intervention Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. Toll-like receptors (TLRs) play a critical role in the pathophysiology of sepsis/septic shock. TLR3 is located in intracellular endosomes and recognizes double stranded RNA. This study examined the role of TLR3 in cardiac dysfunction following cecal ligation and puncture (CLP)-induced sepsis. TLR3 knockout (TLR3−/−, n=12) and age-matched wild type (WT, n=12) mice were subjected to CLP. Cardiac function was measured by echocardiography before and 6 hrs after CLP. Measurements and results CLP resulted in significant cardiac dysfunction as evidenced by decreased ejection fraction by 25.7% and fractional shortening by 29.8%, respectively. However, TLR3−/− mice showed a maintenance of cardiac function at pre-CLP levels. Wild type mice showed 50% mortality at 58 hrs and 100% mortality at 154 hrs after CLP. In striking contrast, 70% of TLR3−/− mice survive indefinitely, i.e. >200 hrs. TLR3 deficiency significantly decreased CLP-induced cardiac myocyte apoptosis and attenuated CLP-induced Fas and FasL expression in the myocardium. CLP-activation of TLR4-meidated NF-κB and TRIF-dependent IFN signaling pathways was prevented by TLR3 deficiency. In addition, CLP-increased VCAM-1 and ICAM-1 expression and neutrophil and macrophage sequestration in the myocardium were also attenuated in septic TLR3−/− mice. More significantly, adoptive transfer of WT bone marrow stromal cells to TLR3−/− mice abolished the cardioprotective effect in sepsis. Conclusions These data indicate that TLR3 plays a deleterious role in mediating cardiac dysfunction in sepsis. Thus, modulation of TLR3 activity may be useful in preventing cardiac dysfunction in sepsis.

The maize dull1 ( du1 ) gene is a determinant of the structure of endosperm starch, and du1 -mutations affect the activity of two enzymes involved in starch biosynthesis, starch synthase II (SSII) and starch branching enzyme IIa (SBEIIa). Six novel du1 -mutations generated in Mutator -active plants were identified. A portion of the du1 locus was cloned by transposon tagging, and a nearly full-length Du1 cDNA sequence was determined. Du1 codes for a predicted 1674-residue protein, comprising one portion that is similar to SSIII of potato, as well as a large unique region. Du1 transcripts are present in the endosperm during the time of starch biosynthesis, but the mRNA was undetectable in leaf or root tissue. The predicted size of the Du1 gene product and its expression pattern are consistent with those of maize SSII. The Du1 gene product contains two repeated regions in its unique N terminus. One of these contains a sequence identical to a conserved segment of SBEs. We conclude that Du1 codes for a starch synthase, most likely SSII, and that secondary effects of du1 -mutations, such as reduction of SBEIIa, result from the primary deficiency in this starch synthase. INTRODUCTIONStarch is the most significant carbohydrate reserve in plant storage tissues and comprises the glucose polymers amylose and amylopectin. Amylose is predominantly linear chains of ␣ (1 → 4)-linked glucose residues, whereas amylopectin is a highly branched glucan with a specific "clustered" distribution of ␣ (1 → 6) glycosidic bonds (i.e., branch linkages) connecting linear chains (reviewed in French, 1984;Manners, 1989). Despite the relatively simple chemical structure of amylopectin, the enzymatic processes responsible for the formation of the highly specific and complex branching patterns in this polysaccharide are not clearly understood. Biosynthesis of amylose and amylopectin involves the activities of four groups of enzymes, of which each comprises multiple isozymes. These enzymes are ADPglucose pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDBE) (reviewed in Preiss, 1991;Hannah et al., 1993;Martin and Smith, 1995;Nelson and Pan, 1995;Ball et al., 1996;Preiss and Sivak, 1996;Smith et al., 1997). These enzymatic steps can account for all chemical linkages in starch; however, the specific roles of individual isozymes in the formation of specific branching patterns in amylopectin and determination of starch granule structure and properties remain unknown.Analysis of maize mutants with abnormal endosperm phenotypes has contributed greatly to the understanding of starch synthesis (reviewed in Shannon and Garwood, 1984;Nelson and Pan, 1995) and facilitated the identification of many genes coding for starch biosynthetic enzymes. Cloned maize genes involved in starch biosynthesis are waxy ( wx ), coding for granule-bound starch synthase I (GBSSI) (Shure et al., 1983;Klösgen et al., 1986), amylose extender ( ae ), coding for SBEIIb (Fisher et al., 1993;Stinard et al., 1993), s...

Centella asiatica (also known as Centella asiatica (L.) Urb. or Gotu kola) is a traditional Chinese medicine with extensive medicinal value, which is commonly used in Southeast Asian countries. This study aimed to summarize the effects of C. asiatica and its main components on neurological diseases, endocrine diseases, skin diseases, cardiovascular diseases, gastrointestinal diseases, immune diseases, and gynecological diseases, as well as potential molecular mechanisms, to study the pathological mechanism of these diseases based on the changes at the molecular level. The results showed that C. asiatica and its triterpenoids had extensive beneficial effects on neurological and skin diseases, which were confirmed through clinical studies. They exhibited anti-inflammatory, antioxidative stress, anti-apoptotic effects, and improvement in mitochondrial function. However, further clinical studies are urgently required due to the low level of evidence and lack of patients.

In maize (Zea mays L.) three isoforms of starch-branching enzyme (SBEI, SBElla, and SBEllb) are involved in the synthesis of amylopectin, the branched component of starch. To isolate a cDNA encoding SBElla, degenerate oligonucleotides based on domains highly conserved in Sbe2 family members were used to amplify SbeZ-family cDNA from tissues lacking SBEllb activity. l h e predicted amino acid sequence of a SbeZa cDNA matches the N-terminal sequence of SBElla protein purified from maize endosperm. l h e size of the mature protein deduced from the cDNA also matches that of SBElla. Features of the predicted protein are most similar to members of the SBEll family; however, it differs from maize SBEllb in having a 49-amino acid N-terminal extension and a region of substantial sequence divergence. SbePa mRNA levels are 1 O-fold higher in embryonic than in endosperm tissue, and are much lower than SbeZb in both tissues. Unlike SbeZb, SbeZa-hybridizing mRNA accumulates in leaf and other vegetative tissues, consistent with the known distribution of SBElla and SBEllb activities.SBE catalyzes the formation of a(1-6) branches in amylopectin, the highly branched component of starch. It cleaves a ( l 4 ) bonds on linear glucosyl chains and reattaches the released glucan segments to the same or another glucosyl chain by a(1-6) linkages. The reaction creates not only branches, but also new, nonreducing ends for further a(14) glucan elongation. Multiple forms of SBE have been characterized in many species, including rice (Mizuno et al

BackgroundThis study aims to clarify the underlying mechanism for the tumor suppressive function of lnc TUSC7 in chemotherapy resistance of esophageal squamous cell carcinoma (ESCC).MethodsTUSC7, miR-224 and DESC1 expressions in ESCC tissues and cells were detected by qRT-PCR. Protein level of DESC1, EGFR and p-AKT were observed by Western blot. Overall survival was calculated using the Kaplan-Meier method. Dual-luciferase reporter gene assay and RIP assay were used to comfirm TUSC7 binding to miR-224, and miR-224 binding to DESC1. Cell proliferation, apoptosis, and colony formation was detected by MTT, Flow Cytometry and Colony formation assays.ResultsTUSC7 was downregulated in ESCC tissues and cells, and low TUSC7 indicated worse overall survival. The analysis of bioinformatics softwares showed that TUSC7 specifically bound to miR-224, and we proved miR-224 was upregulated in ESCC and negatively correlated with TUSC7 expression. Overexpression of TUSC7/inhibition of miR-224 suppressed cell proliferation, colony formation and chemotherapy resistance of ESCC cells, and promoted cell apoptosis. In addition, we confirmed that miR-224 specifically bound to DESC1, and negatively correlated with DESC1. TUSC7 suppressed the proliferation and chemotherapy resistance of ESCC cells by increasing DESC1 expression via inhibiting miR-224. We also confirmed DESC1 inhibited chemotherapy resistance of ESCC cells via EGFR/AKT. Finally, in vivo experiments demonstrated that overexpression of TUSC7 decreased tumor growth and chemotherapy resistance.ConclusionThese findings suggested TUSC7 suppressed chemotherapy resistance of ESCC by downregulating miR-224 to modulate DESC1/EGFR/AKT pathway.

Recent evidence suggests that the macrophage scavenger receptor class A (SR-A, aka, CD204) plays a role in the induction of innate immune and inflammatory responses. We investigated whether SR-A will cooperate with Toll-like receptors (TLRs) in response to TLR ligand stimulation. Macrophages (J774/a) were treated with Pam2CSK4, (TLR2 ligand), Poly I:C (TLR3 ligand), and LPS (TLR4 ligand) for 15 min in the presence or absence of fucoidan (the SR-A ligand). The levels of phosphorylated IκBα (p-IκBα) were examined by Western blot. We observed that Poly I:C and LPS alone, but not Pam2CSK4 or fucoidan increased the levels of p-IκBα. However, LPS-induced increases in p-IκBα levels were further enhanced when presence of the fucoidan. Immunoprecipitation and double fluorescent staining showed that LPS stimulation promotes SR-A association with TLR4 in the presence of fucoidan. To further confirm our observation, we isolated peritoneal macrophages from SR-A deficient (SR-A−/−), TLR4−/− and wild type (WT) mice, respectively. The peritoneal macrophages were treated with LPS for 15 min in the presence and absence of fucoidan. We observed that LPS-stimulated TNFα and IL-1β production was further enhanced in the WT macrophages, but did not in either TLR4−/− or SR-A−/− macrophages, when fucoidan was present. Similarly, in the presence of fucoidan, LPS-induced IκBα phosphorylation, NF-κB binding activity, and association between TLR4 and SR-A were significantly enhanced in WT macrophages compared with LPS stimulation alone. The data suggests that SR-A is needed for LPS-induced inflammatory responses in macrophages.