Amino acid sequence of heat-labile enterotoxin from chicken enterotoxigenic Escherichia coli is identical to that of human strain H 10407

Abstract The DNA sequence of heat-labile enterotoxin from the chicken enterotoxigenic Escherichia coli 21d strain was determined by direct dideoxy sequencing of polymerase chain reaction (PCR)-amplified DNA and was compared with those of heat-labile enterotoxins from porcine and human enterotoxigenic E. coli strains EWD 299 and H 10407. The structural genes of the A and B subunits of chicken heat-labile enterotoxin were identical to those of human heat-labile enterotoxin from the human H 10407 strain. Moreover, 67 base pairs of the upstream and 60 base pairs of the downstream region of the chicken heat-labile enterotoxin gene were also identical to those of the human heat-labile enterotoxin from strain H 10407. However, the patterns of plasmids from the 21d and H 10407 strains were different. The 21d strain had no band corresponding to the 42-MDa plasmid of the H10407 strain encoding the heat-labile enterotoxin gene but it had a smaller plasmid. These data suggest that although the DNA sequence of chicken heat-labile enterotoxin is identical to that of human heat-labile enterotoxin, the plasmid encoding the chicken heat-labile enterotoxin gene in the chicken might be different from that encoding the human heat-labile enterotoxin gene in the H10407 strain.     

Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the expressed-protein stability

Human pro-urokinase (pro-UK) gene was engineered for expression in mammalian cells. The stability of recombinant pro-UKs produced by two kinds of cells, Chinese hamster ovary (CHO) and human lymphoblastoid Namalwa KJM-1 cells, were compared. The pro-UK expressed in CHO cells in serum-free medium was degraded by cysteine endopeptidase secreted by CHO cells. This endopeptidase was inhibited by pchloromercuribenzonate (PCMB) and leupeptin more efficiently than by aprotinin. On the other hand, the pro-UK expressed in Namalwa KJM-1 cells was not degraded, resulting in the stable production of pro-UK at a rate of 2–3 g/10⁶ cells/day by use of a gene amplification method with dihydrofolate reductase (DHFR) in serum-free medium. Thus, Namalwa KJM-1 cells showed the desired characteristics as a host cell for the production of recombinant proteins. The stability of recombinant proteins produced in heterologous systems may vary depending on the host cells.Abbreviations ABTS 2,2-azino-di-(3-ethylbenzothiazoline) sulfonic acid diammonium salt - AMC 7-amino-4-methylcoumarin - CHO Chinese hamster ovary - DFP diisopropylfluorophosphate - DHFR dihydrofolate reductase - ELISA enzyme-linked immunosorbent assay - MCA 4-methylcoumaryl-7-amide - MTX methotrexate - NEAA non essential amino acid - NEM N-ethylmaleimide - PCMB p-chloromercuribenzonate - PMSF phenylmethanesulfonyl fluoride - pro-UK pro-urokinase     

Orcadian Variation in Amikacin Clearance and Its Effects on Efficacy and Toxicity in Mice with and Without Immunosuppression

A once-daily dosage regimen has been recently recommended in the use of aminoglycoside antibiotics since they induce a postantibiotic effect. In choosing this regimen, one must determine the most appropriate time of day for administration of the drug. We investigated the effects of the timing of amikacin (AMK) administration on the kinetics, the efficacy against intraperitoneal infection with Pseudomonas aeruginosa, and the toxicity of AMK in mice with and without immijnosuppression. We found circadian variations in the kinetics, efficacy, and toxicity of the drug in mice. Male and female ICR mice, which were housed under a light-dark (12:12 h) cycle with free food and water intake, were injected subcutaneously with AMK sulfate 50 mg/kg body wt. There was a circadian variation in AMK clearance for both sexes with the maximum value in the dark phase and the minimum in the light phase after a single administration. When AMK 500 mg/kg/day was repeatedly administered once daily for 30 days, higher toxicity was demonstrated in mice injected with the drug at the time of day with lower AMK clearance, although no difference was demonstrated in the toxicity between the two time points with different AMK clearance when AMK 1,500 mg/kg was administered in a single dose. The ED₅₀ of AMK to cure the infected mice in the midlight phase (13:00 h) with lower clearance was significantly lower than that in the middark phase (01:00 h) with higher clearance. In contrast, the ED₅₀ in the early light phase (09:00 h) was significantly lower than that in the early dark phase (21:00 h), although AMK clearance was not different between these two different time points. In mice premedicated with cyclophosphamide to suppress immune functions, the difference in the ED₅₀ of AMK was still demonstrated between 13:00 and 01:00 h, but not between 09:00 and 21:00 h. The present study shows not only that there were circadian variations in both AMK clearance and toxicity after repeated administration, but also that there was a circadian variation in the efficacy of AMK in mice infected with P. aeruginosa. These results suggest that the timing of drug administration should be considered in pharmacotherapy with AMK and that the most appropriate time of administration in mice and nocturnal animals may be in the midlight (resting) phase. They also suggest that the ED₅₀ of AMK. against P. aeniginosa infection may be influenced not only by the circadian variation in pharmacokinetics but also by the variations in immune systems suppressed by cyclophosphamide.     

Molecular cloning of the 31 kDa cytosolic phospholipase A2, as an antigen recognized by the lung cancer-specific human monoclonal antibody, AE6F4

The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A₂ (cPLA₂), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA₂ expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA₂ was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA₂, but they also play a crucial role in antigen recognition by the monoclonal antibody.     

Plant Microtubules can be Translocated by a Dynein ATPase from Sea Urchin in Vitro

The latent ability of plant microtubules to translocate or slidein the presence of motor proteinwas examined in a motility assayin vitro. Plant microtubules isolated from tobacco BY-2 cellsmoved over a glass surface covered with outer arm 21S dyneinfrom flagella of sea urchinsperm with an average velocity of3.7 µm s^-1. This velocity was similar to that of microtubulesisolated from bovine brain under the same conditions (averagevelocity, about 4.1 µm s^-1). These results suggest thatplant microtubules have an intrinsic ability to interact withand to be translocated by dynein. It is postulated that microtubule-basedmotor proteins, including dynein ATP-ase,are involved in thefunctioning of microtubules in plant cells. (Received May 20, 1995; Accepted September 18, 1995)     

Mval polymorphism in the proteolipid protein (PLP) gene

We report a rare polymorphism in the human proteolipid protein (PLP) gene. A synonymous mutation, 168 AG, was detected in exon 2 of the PLP gene. Mutations in this gene have been reported in some cases of Pelizaeus-Merzbacher disease.     

Polymerase chain reaction detection of two novel human N-acetylgalactosamine-6-sulfate sulfatase gene polymorphisms by single-strand conformation polymorphism analysis or by StyI and StuI cleavages

A novel polymorphism (6376 G/T) in intron 7 (I7) of the human PROC gene has been identified by direct DNA sequencing. Restriction analysis with the use of mutagenic primers indicate that the allele frequencies are 0.17 (allele T) and 0.83 (allele G), with a calculated heterozygosity of 28%.     

Application of N-terminally Truncated DNA Polymerase from Thermus thermophilus ({Delta}Tth Polymerase) to DNA Sequencing and Polymerase Chain Reactions: Comparative Study of {Delta}Tth and Wild-Type Tth Polymerases

N-Terminally truncated DNA polymerase from Thermus thermophilus(Tth polymerase) lacking 5'-3' exonuclease activity was usedfor DNA sequencing and polymerase chain reaction (PCR). In contrastto the high background of the sequencing ladder observed withthe wild-type Tth polymerase, Tth polymerase gave readable sequencingpatterns which extend up to more than 500 bases from the primersite on cycle sequencing and automated sequencing. The Tth polymerasewas used for the standard and mutagenic PCR, and net amplificationof the DNA and the mutations accumulated during PCR were analyzed.Under mutagenic PCR, the mutation rates were 7.0 x 10^–4(Tth) and 8.3 x 10^–4 (Tth) per nucleotide per cycle ofamplification, which were 4–9 times higher than the ratesunder standard PCR.     

Mycofloral succession onPinus densiflora needles on a moder site

Mycofloral succession on decaying pine needles in aPinus densiflora forest on a moder site was investigated in Sugadaira, Nagano Pref., central Japan. Dead needles on the tree, fallen needles obtained from two recognizable sublayers of the L layer and the upper sub-layer of the F₁ layer in the organic horizon were examined for their fungal flora using both washing and surface sterilization techniques. The major interior colonizer in freshly fallen needles varied with the season:Chaetopsina fulva in summer andSelenosporella curvispora in the other seasons.Thysanophora penicillioides was a remarkable external colonizer of freshly fallen needles in summer, while soil fungi were external colonizers of such needles in the other seasons. A possible successional change of major fungi with the needle decay was suggested. The observed seasonal alternation of the species colonizing freshly fallen needles was discussed in relation to climatic conditions. Contributions from Sugadaira Montane Research Center, No. 152.