Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

Promotion of plantlet formation from somatic embryos of carrot treated with a high molecular weight extract from a marine cyanobacterium

Summary Somatic embryos of Daucus carota L. developed into plantlets at high frequency after addition of an extract from a marine cyanobacterium, Synechococcus sp. NKBG 042902. High molecular weight, nondialyzing fraction, separated from the extract, possessed enhanced plantlet formation promoting activity. Plantlet formation frequency was 60 % after addition of nondialysate (100 mg/l) compared to 28 % without addition. Embryos treated with the nondialysate contained five times more chlorophyll than nontreated embryos after 6 days of culture. The chlorophyll a/b ratio of 4-day old treated somatic embryos was found to be similar to that of zygotic embryos. However, the chlorophyll a/b ratio of plantlets induced from nontreated somatic embryos was variable. Nondialysate was fractionated by ultracentrifugation and an active component obtained, which gave a maximum plantlet formation frequency of 71 %, and induced rapid greening of shoots.Abbreviations MS Murashige and Skoog medium - 2,4D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - E Einstein - Chl Chlorophyll - vvm volume of air, volume of medium per minute     

Effects of Maillard reaction products on the oxidative cleavage and polymerization of protein under ascorbic acid-transition metal system.

This study investigated the effects of Maillard reaction products (MRPs) on the oxidative cleavage and polymerization of BSA (bovine serum albumin) in an aqueous system. In L-ascorbic acid (AsA) and Cu(II) or Fe(III) reaction system, 50-60% of BSA was cleaved under physiological conditions (37 degrees C, pH 7.2). The oxidative cleavage induced by AsA-Cu(II) system was suppressed to the extent of 32-86% by model melanoidins or brown pigments from amino acids and foodstuffs. In the AsA-Fe(III) system, the oxidative cleavage was inhibited to the extent of 45-93% by melanoidins and brown pigments. However, this cleavage was promoted by amino acid Amadori rearrangement products and brown pigment from soy paste. Therefore, MRPs show both suppression and promotion activity on oxidative cleavage of BSA in the system of AsA and a transition metal. The quantity of Amadori rearrangement moiety (ARM) in melanoidins from Lysine and brown pigments molecules from foods was also measured. From these data, it was estimated that the suppression and/or promotion of oxidative cleavage of BSA did not only depend on the quantity of ARM, but also depended on the chemical structure of ARM in melanoidins or brown pigments.     

Improved haplotype analysis of human myelin basic protein short tandem repeat loci

We report an improved haplotype analysis of the human myelin basic protein gene (MBP) short tandem repeat (STR) polymorphism. The polymorphic G-->A transition and 2 conventional STR polymorphisms, MBPA and MBPB, were simultaneously determined by an amplified product length polymorphism technique. After the MBPC fragments containing MBPA and MBPB were amplified, the linkage of these 2 STR loci was determined by a second amplification, using polymerase chain reaction (PCR) technique, of the isolated MBPC fragments. The present haplotype analysis dispensed with family studies for the haplotyping of MBPA and MBPB. Polymorphisms of the MBP loci studied in German and Japanese populations showed a high genomic variation. Haplotype analysis of the MBP loci showed distinct differences between the German and the Japanese populations. Consequently, haplotype analysis of the MBP loci promises to be useful in forensic identification and paternity testing.     

Respiratory infection with influenza A virus interferes with the induction of tolerance to aeroallergens

Viral respiratory infections have been implicated in influencing allergen sensitization and the development of asthma, but their exact role remains controversial. Because respiratory exposure to Ag normally engenders T cell tolerance and prevents the development of airway hyperreactivity (AHR) and inflammation, we examined the effects of influenza A virus infection on tolerance induced by exposure to intranasal (i.n.) OVA and the subsequent development of AHR. We found that concurrent infection with influenza A abrogated tolerance induced by exposure to i.n. OVA, and instead led to the development of AHR accompanied by the production of OVA-specific IgE, IL-4, IL-5, IL-13, and IFN-gamma. When both IL-4 and IL-5 were neutralized in this system, AHR was still induced, suggesting that influenza-induced cytokines such as IL-13, or mechanisms unrelated to cytokines, might be responsible for the development of AHR. The length of time between influenza A infection and i.n. exposure to OVA was crucial, because mice exposed to i.n. OVA 15-30 days after viral inoculation developed neither AHR nor OVA-specific tolerance. These mice instead acquired Th1-biased OVA-specific immune responses associated with vigorous OVA-induced T cell proliferation, and reduced production of OVA-specific IgE. The protective effect of influenza A on AHR was dependent on IFN-gamma, because protection was abrogated with a neutralizing anti-IFN-gamma mAb. These results suggest that viral respiratory infection interferes with the development of respiratory allergen-induced tolerance, and that the time interval between viral infection and allergen exposure is critical in determining whether viral infection will enhance, or protect against, the development of respiratory allergen sensitization and AHR.     

Mitochondrial GTPase mitofusin 2 mutation in Charcot–Marie–Tooth neuropathy type 2A

Charcot–Marie–Tooth disease (CMT) has been classified into two types, CMT1 and CMT2, demyelinating and axonal forms, respectively. CMT2 has been further subdivided into eight groups by linkage studies. CMT2A is linked to chromosome 1p35–p36 and mutation in the kinesin family member 1B-ß (KIF1B) gene had been reported in one pedigree. However, no mutation in KIF1B was detected in other pedigrees with CMT2A and the mutations in the mitochondrial fusion protein mitofusin 2 (MFN2) gene were recently detected in those pedigrees. MFN2, a mitochondrial transmembrane GTPase, regulates the mitochondrial network architecture by fusion of mitochondria. We studied MFN2 in 81 Japanese patients with axonal or unclassified CMT and detected seven mutations in seven unrelated patients. Six of them were novel and one of them was a de novo mutation. Most mutations locate within or immediately upstream of the GTPase domain or within two coiled-coil domains, which are critical for the functioning or mitochondrial targeting of MFN2. Formation of a mitochondrial network would be required to maintain the functional peripheral nerve axon.     

Batch anaerobic co-digestion of cow manure and waste milk in two-stage process for hydrogen and methane productions

Anaerobic co-digestion of cow manure (CM) and waste milk (WM), produced by sick cows during treatment with antibiotics, was evaluated in two-stage process under thermophilic condition (55 °C) to determine the effect of WM addition on hydrogen (H₂) and methane (CH₄) production potentials, volatile solids (VS) removal, and energy recovery. Six CM to WM VS ratios of 100:0, 90:10, 70:30, 50:50, 30:70, and 10:90 were examined using 1-L batch digesters. The WM VS ratio of 30 % was found to be the minimum limit for significant increases in specific H₂ and CH₄ yields, and VS removal as compared to digestion of manure alone (P < 0.05). The highest specific H₂ and CH₄ yields, VS removal and energy yield were 38.2 mL/g VS, 627.6 mL/g VS, 78.4 % and 25,459.8 kJ/kg VS, respectively, in CM:WM 30:70. Lag phases to H₂ and CH₄ productions were observed in CM–WM mixtures, increased with increasing the amount of WM in the feedstock and were greater than 72 h in CM:WM 50:50 and 30:70. The digestion system failed in CM:WM 10:90. The results suggest that CM:WM 30:70 was optimum, however, due to limited amount of WM usually generated and long lag phase at this ratio which may make the process uneconomical, CM:WM 70:30 is recommended in practice.     

1H, 13C, and 15N resonance assignment of the first PDZ domain of mouse ZO-1

Zonula occludens-1 (ZO-1) is a scaffolding molecule critical to the formation of intercellular adhesion structures, such as tight junctions (TJs) and adherens junctions (AJs). ZO-1 contains three PDZ domains followed by a GUK domain and a ZU5 domain. The first PDZ of ZO-1 (ZO-1(PDZ1)) serves as a protein–protein interaction module and interacts with the C-termini of almost all claudins to initiate the formation of a belt-like structure on the lateral membranes, thereby promoting TJ formation. It has been recently reported that approximately 15% of all PDZ domains bind phosphoinositides, and ZO-1(PDZ1) is the one of these. Here we report the ¹⁵N, ¹³C, and ¹H chemical shift assignments of the first PDZ domain of mouse ZO-1. The resonance assignments obtained in this work may contribute in clarifying the interplay between the two binary interactions, ZO-1(PDZ1)–claudins and ZO-1(PDZ1)–phospholipids, and suggesting a novel regulation mechanism underlying the formation and maintenance of cell–cell adhesion machinery downstream of the phospholipid signaling pathways.