Chlorophyll-deficient gene and morphological variations in Korean populations of maize (Zea mays)

We studied cultivated and naturalized Korean maize populations to determine the extent to which the chlorophylldeficient mutation and the phenotypic variations of two morphological characters (i.e., red coleoptiles and epicotyls, and the number of the first root hairs) are maintained. The frequency of the chlorophyll-deficient mutant gene (2.73% on average) was highly variable. Frequencies of red coleoptiles and epicotyls also were higher than expected from a mutation-selection balance. The average number of hairy phenotypes within populations was 1.8, ranging from 0.0 to 4.0. Naturalized populations were closely related to with cultivated communities. Most striking, however, was the more significant difference among populations than within populations with regard to both the frequency of chlorophyll-deficient mutant genes and the phenotypic variations of our two morphological characters. On a per-gene basis, the majority of the phenotypic variation (mean of 73.3%) resided among populations.     

Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy

Macroautophagy allows for bulk degradation of cytosolic components in lysosomes. Overexpression of GFP/RFP-LC3/GABARAP is commonly used to monitor autophagosomes, a hallmark of autophagy, despite artifacts related to their overexpression. Here, we developed new sensors that detect endogenous LC3/GABARAP proteins at the autophagosome using an LC3-interacting region (LIR) and a short hydrophobic domain (HyD). Among HyD-LIR-GFP sensors harboring LIR motifs of 34 known LC3-binding proteins, HyD-LIR(TP)-GFP using the LIR motif from TP53INP2 allowed detection of all LC3/GABARAPs-positive autophagosomes. However, HyD-LIR(TP)-GFP preferentially localized to GABARAP/GABARAPL1-positive autophagosomes in a LIR-dependent manner. In contrast, HyD-LIR(Fy)-GFP using the LIR motif from FYCO1 specifically detected LC3A/B-positive autophagosomes. HyD-LIR(TP)-GFP and HyD-LIR(Fy)-GFP efficiently localized to autophagosomes in the presence of endogenous LC3/GABARAP levels and without affecting autophagic flux. Both sensors also efficiently localized to MitoTracker-positive damaged mitochondria upon mitophagy induction. HyD-LIR(TP)-GFP allowed live-imaging of dynamic autophagosomes upon autophagy induction. These novel autophagosome sensors can thus be widely used in autophagy research.     

Cloning and functional expression of the gene encoding an inhibitor against <Emphasis Type="Italic">Aspergillus flavus</Emphasis> α-amylase,a novel seed lectin from <Emphasis Type="Italic">Lablab purpureus</Emphasis> (<Emphasis Type="Italic">Dolichos lablab</Emphasis>)

Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection.     

Hydrotropic polymeric micelles for enhanced paclitaxel solubility: in vitro and in vivo characterization

The purpose of this investigation was to characterize the in vitro stability and in vivo disposition of paclitaxel in rats after solubilization of paclitaxel into hydrotropic polymeric micelles. The amphiphilic block copolymers consisted of a micellar shell-forming poly(ethylene glycol) (PEG) block and a core-forming poly(2-(4-vinylbenzyloxy)-N,N-diethylnicotinamide) (P(VBODENA)) block. N,N-Diethylnicotinamide (DENA) in the micellar inner core resulted in effective paclitaxel solubilization and stabilization. Solubilization of paclitaxel using polymeric micelles of poly(ethylene glycol)-b-P(D,L-lactide) (PEG-b-PLA) served as a control for the stability study. Up to 37.4 wt % paclitaxel could be loaded in PEG-b-P(VBODENA) micelles, whereas the maximum loading amount for PEG-b-PLA micelles was 27.6 wt %. Thermal analysis showed that paclitaxel in the polymeric micelles existed in the molecularly dispersed amorphous state even at loadings over 30 wt %. Paclitaxel-loaded hydrotropic polymeric micelles retained their stability in water for weeks, whereas paclitaxel-loaded PEG-b-PLA micelles precipitated in a few days. Hydrotropic polymer micelles were more effective than PEG-PLA micelle formulations in inhibiting the proliferation of human cancer cells. Paclitaxel in hydrotropic polymer micelles was administered orally (3.8 mg/kg), intravenously (2.5 mg/kg), or via the portal vein (2.5 mg/kg) to rats. The oral bioavailability was 12.4% of the intravenous administration. Our data suggest that polymeric micelles with a hydrotropic structure are superior as a carrier of paclitaxel due to a high solubilizing capacity combined with long-term stability, which has not been accomplished by other existing polymeric micelle systems.     

Lipid-Overloaded Enlarged Adipocytes Provoke Insulin Resistance Independent of Inflammation

In obesity, adipocyte hypertrophy and proinflammatory responses are closely associated with the development of insulin resistance in adipose tissue. However, it is largely unknown whether adipocyte hypertrophy per se might be sufficient to provoke insulin resistance in obese adipose tissue. Here, we demonstrate that lipid-overloaded hypertrophic adipocytes are insulin resistant independent of adipocyte inflammation. Treatment with saturated or monounsaturated fatty acids resulted in adipocyte hypertrophy, but proinflammatory responses were observed only in adipocytes treated with saturated fatty acids. Regardless of adipocyte inflammation, hypertrophic adipocytes with large and unilocular lipid droplets exhibited impaired insulin-dependent glucose uptake, associated with defects in GLUT4 trafficking to the plasma membrane. Moreover, Toll-like receptor 4 mutant mice (C3H/HeJ) with high-fat-diet-induced obesity were not protected against insulin resistance, although they were resistant to adipose tissue inflammation. Together, our in vitro and in vivo data suggest that adipocyte hypertrophy alone may be crucial in causing insulin resistance in obesity.     

Structural basis of cooperativity in human UDP-glucose dehydrogenase

Background

UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics.

Methodology

Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle.

Conclusion

In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD⁺ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD⁺ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.     

The effect of rebamipide on gastric xanthine oxidase activity and type conversion in ethanol-treated rats

Rebamipide, a novel antipeptic ulcer drug, 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinone-4-yl]-propionic acid, was studied for its inhibitory effect on gastric xanthine oxidase activity and type conversion of the enzyme that has a profound role in free radical generation. Intraperitoneal administration of rebamipide at 60 mg/kg body weight reduced gastric mucosal hemorrhagic lesions and lipid peroxidation, which was proportional to the inhibitory effect of rebamipide on alcohol-induced xanthine oxidase-type conversion and enzyme activity. It was also observed that the activity of xanthine oxidase was significantly inhibited by administration of rebamipide at 60 mg/kg body weight, leading to a significant reduction of lipid peroxide content in alcohol-treated rats. The results suggest that alcohol-induced gastric mucosal lesions might be, in part, due to the increased activity of xanthine oxidase and type conversion rate of the enzyme and the protective effect of rebamipide on gastric mucosal lesions would result from its ability to protect against oxidative stress on gastric mucosal lesions of alcohol-treated rats.