Prophenol oxidase A3 in Drosophila melanogaster: activation and the PCR-based cDNA sequence

Phenol oxidase exists in Drosophila hemolymph as a prophenol oxidase, A₁ and A₃, that is activated in vivo with a native activating system, AMM-1, by limited proteolysis with time. The polypeptide in purified prophenol oxidase A₃ has a molecular weight of approximately 77,000 Da. A PCR-based cDNA sequence coding A₃ has 2501 bp encoding an open reading frame of 682 amino acid residues. The potential copper-binding sites, from Trp-196 to Tyr-245, and from Asn-366 to Phe-421, are highly homologous to the corresponding sites in other invertebrates. The availability of prophenol oxidase cDNA should be useful in revealing the biochemical differences between A₁ and A₃ isoforms in Drosophila melanogaster that are refractory or unable to activate prophenol oxidase.     

4-hydroxynonenal triggers multistep signal transduction cascades for suppression of cellular functions

4-hydroxynonenal (HNE), an aldehyde product of membrane lipid peroxidation, has been suggested to mediate a number of oxidative stress-linked pathological events in humans, including cellular growth inhibition and apoptosis induction. Because HNE is potentially reactive to a number of both cell surface and intracellular proteins bearing sulfhydryl, amino and imidazole groups, it seems that there are multiple signal transduction cascades. Here we briefly review the HNE-triggered signal transduction cascades that lead to suppression of cellular functions and to cell death, based mainly on our own recent study results.We first showed that formation of HNE-cell surface protein adducts, which mimicked ligand-cell surface receptor binding, induced activation of receptor-type protein tyrosine kinases such as epithelial growth factor receptor (EGFR) and that this caused growth inhibition through a cascade of activation of EGFR, Shc and ERK. Next, we showed that HNE-mediated scavenging of cellular glutathione led to activation of caspases and to DNA fragmentation through a Fas-independent and mitochondria-linked pro-apoptotic signal pathway. More recently, we have obtained evidence that the HNE-triggered signal cascade for caspase activation encounters complex positive feedback regulatory mechanisms that are linked to the inhibition of anti-apoptotic signals and are dependent on caspase activity. Underlying multiple regulatory mechanisms, including mechanisms of activation of Akt-dephosphorylating PP2A activity, activities of protein tyrosine kinases have been shown to be biphasically controlled by HNE. In addition, we have obtained results suggesting that HNE inhibits phosphorylation of IkappaB, possibly by targeting some elements upstream of IkappaB, which might downregulate the NF-kappaB-mediated cellular responses, including serum deprivation-induced iNOS expression and generation of anti-apoptotic signals.These results suggest that HNE reacts with multiple cell surface and intracellular sites for triggering a network of signal transduction that is ultimately focused on suppression of cellular functions.     

A secreted type of beta 1,6-N-acetylglucosaminyltransferase V (GnT-V) induces tumor angiogenesis without mediation of glycosylation: a novel function of GnT-V distinct from the original glycosyltransferase activity

Angiogenesis is the first regulatory step of tumor progression. Herein, we report on some findings that show that beta1,6-N-acetylglucosaminyltransferase V (GnT-V) functions as an inducer of angiogenesis that has a novel and completely different function from the original function of glycosyltransferase. A secreted type of GnT-V protein itself promoted angiogenesis in vitro and in vivo at physiological concentrations. The highly basic domain of GnT-V induced the release of fibroblast growth factor-2 from heparan sulfate proteoglycan on the cell surface and/or extracellular matrix, leading to angiogenesis. These findings provide some novel information on the relationship between GnT-V and tumor metastasis. The inhibition of GnT-V secretion or its expression represents a novel potential strategy for the inhibition of tumor angiogenesis.     

Purification and characterization of a novel fungal alpha-glucosidase from Mortierella alliacea with high starch-hydrolytic activity

The fungal strain Mortierella alliacea YN-15 is an arachidonic acid producer that assimilates soluble starch despite having undetectable alpha-amylase activity. Here, a alpha-glucosidase responsible for the starch hydrolysis was purified from the culture broth through four-step column chromatography. Maltose and other oligosaccharides were less preferentially hydrolyzed and were used as a glucosyl donor for transglucosylation by the enzyme, demonstrating distinct substrate specificity as a fungal alpha-glucosidase. The purified enzyme consisted of two heterosubunits of 61 and 31 kDa that were not linked by a covalent bond but stably aggregated to each other even at a high salt concentration (0.5 M), and behaved like a single 92-kDa component in gel-filtration chromatography. The hydrolytic activity on maltose reached a maximum at 55 degrees C and in a pH range of 5.0-6.0, and in the presence of ethanol, the transglucosylation reaction to form ethyl-alpha-D-glucoside was optimal at pH 5.0 and a temperature range of 45-50 degrees C.     

Differential effect of walnut oil and safflower oil on the serum cholesterol level and lesion area in the aortic root of apolipoprotein E-deficient mice

Walnut oil (WO) is a good source of alpha-linolenic acid. We compared the effects of WO and high-linoleic safflower oil (HLSO) on the serum lipid level and atherosclerosis development in male and female apolipoprotein (apo) E-deficient mice. The WO diet resulted in a higher level of serum cholesterol than with HLSO. Female mice fed on the WO diet had a greater lesion area in the aortic root than did those on the HLSO diet. There was no diet-dependent difference in the level of cholesterol and its oxidation products in the abdominal and thoracic aorta. These results suggest that the unpleasant effects of the WO diet on apo E-deficient mice may be attributable to alpha-linolenic acid.     

Production of cycloisomaltooligosaccharides from dextran using enzyme immobilized in multilayers onto porous membranes

Anion-exchange porous hollow-fiber membranes with a thickness of about 1.2 mm and a pore size of about 0.30 microm were used as a supporting matrix to immobilize cycloisomaltooligosaccharide glucanotransferase (CITase). CITase was immobilized to the membrane via anion-exchange adsorption and by subsequent enzymatic cross-linking with transglutaminase, the amount of which ranged from 3 to 110 mg per gram of the membrane. The degree of enzyme multilayer binding was equivalent to 0.3-9.8. Dextran, as the substrate, was converted into seven- to nine-glucose-membered cycloisomaltooligosaccharides (CI-7, -8, and -9) at a maximum yield of 28% in weight at a space velocity of 10 per hour during the permeation of 2.0% (w/w) dextran solution across the CITase-immobilized porous hollow-fiber membrane. The yield of CIs increased with increasing degree of CITase multilayering.     

The tear "TROUF" procedure: transconjunctival repositioning of orbital unipedicled fat

Aging of the lower lid and cheek results in bulging of orbital fat and deepening of the orbital rim groove, known as the "tear trough." As an alternative to orbital fat excision, pedicles of fat can be spread in a submuscular plane to obliterate the groove of the tear trough. In a 1-year period, 25 patients had correction of their tear trough deformities through a transconjunctival approach. This technique included incision of the septum near the arcus marginalis, dissection of a suborbicularis pocket, and placement of medial and central fat pedicles over the orbital rim with buried 5-0 plain gut sutures. Clinically, after a follow-up of 17 to 31 months, there was leveling of the tear trough, smooth contour of the lower lid and cheek, minimal complications, and high patient satisfaction.     

Variation in the distribution of trace elements in hepatoma

There are many reports of reduction of zinc level and rise of copper level in serum of patients with liver disease. However, there are a few reports that compare the trace elements in tumor tissues and nontumor tissues of the liver with hepatoma. We studied trace element distribution in tumor tissues and nontumor tissues of liver with hepatoma and compared them with data from normal liver tissues. Zinc (Zn), copper (Cu), selenium (Se), cadmium (Cd), mercury (Hg), and iron (Fe) were chosen as the trace elements to be observed. We observed falls of Zn, Cd, and Hg levels in tumor tissues and the rise of Cu level as a result of this investigation. Zn, Cd, and Hg levels in tumor tissues were significantly lower than those in nontumor tissues and Zn, Cd, and Hg levels in nontumor tissues were significantly lower than in normal liver tissues. This tendency was clearer for Cd and Hg than for Zn. Although the distribution of Cu was not significant, a distribution contrary to that of Zn was shown. These findings indicate that the distribution of Zn, Cd, and Hg can serve as supportive evidence that could be useful as a tumor marker. Selenium showed almost the same accumulation tendency among tumor tissues, nontumor tissues, and normal livers. Although correlation was observed among most metals in the normal liver, there was almost no correlation in tumor tissues.     

Evolution of the cystatin B gene: implications for the origin of its variable dodecamer tandem repeat in humans

The human cystatin B gene contains a variable number of 12-bp tandem repeats in its promoter region, of which the common alleles contain two or three copies and unusual expansion causes progressive myoclonus epilepsy of the Unverricht-Lundborg type. We undertook a comprehensive analysis of the genomic sequence to address the evolutionary events of this variable repeat. By examination of a contiguous genome sequence spanning 5.0 kb and linkage analysis of detected polymorphic changes, we identified six major intragenic haplotypes in unrelated Japanese subjects. The number of normal repeats was closely correlated with these alleles, indicating that changes in the array should be comparatively rare events during human evolution. To examine the origin of the repeat array further, we also analyzed five primate genomes. Repetitive polymorphism was unlikely in hominoids, and the array originated with the dodecamer itself in the course of primate evolution. The variability conceivably developed after the separation to humans.