Transient Expression of CD20 Antigen (Pan B Cell Marker) in Activated Lymph Node T Cells

In contrast to the case of peripheral T cells, the surface expression of CD20 antigen and the expression of CD20 mRNA in monkey lymph node (LN) T cells underwent a noticeable increase when they were cultured with mitogen and interleukin-2 (IL-2). To confirm in vivo regulation of CD20 expression during the activation of LN T cells, we examined LNs derived from monkeys experimentally inoculated with simian immunodeficiency virus (SIV). Significant expression of CD20 antigen was detected in the T cells of the LNs at the stage of lymphadenopathy. These findings suggest that lymphocyte activation in the LNs induced expression of the CD20 molecule in some T cells.     

Association of neutral α-glucosidase with cytoplasmic granules of peritoneal macrophages

A neutral α-glucosidase (EC 3.2.1.20) activity was shown to be associated with granules which are sedimentable at 10 000 g after differential centrifugation of mouse peritoneal macrophage homogenates. When the post-nuclear supernatant was centrifuged in a sucrose density gradient, high activities for neutral α-glucosidase and β-glucuronidase (EC 3.2.1.31) were detected in the bottom fractions because of aggregation of the granules. Neutral α-glucosidase-containing granules were completely disaggregated by the addition of 20 units/ml of heparin and 10 mM Tris-HCl (pH 7.2), which caused only a partial disaggregation of β-glucuronidase-containing granules. The addition of a high concentration of heparin, Tris buffer, or KCl to the gradient gave the same patterns of disaggregation of the granules. Under the condition in which about 50% of the total β-glucuronidase activity was released into the medium, depending on phagocytosis, very little α-glucosidase was released. These observations suggested that neutral α-glucosidase may localize in non-lysosomal granules.     

Cloning and Expression of the Pseudomonas Gene for Utilization of d-Leucine in Escherichia coli

Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in l-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80 ~ 90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8 kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no d-leucine transaminase activity, but five-times higher d-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for d-amino acid dehydrogenase, a key enzyme in d-amino acid metabolism.     

Egg-Cracking Vibration as a Cue for Stink Bug Siblings to Synchronize Hatching

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Dose profiles for lung and breast regions at prospective and retrospective CT coronary angiography using optically stimulated luminescence dosimeters on a 64-detector CT scanner

PurposeThe purpose of our study was to acquire dose profiles at critical organs of lung and breast regions using optically stimulated luminescence (OSL) dosimeters; assess the actual radiation dose delivered at retrospective and prospective computed tomography coronary angiography (CTCA).Materials and methodsUsing a chest CT phantom we applied a prospectively-gated step-and-shoot- and a retrospectively-gated helical mode on a 64-detector row CT scanner. Retrospective scan mode was used with and without electrocardiogram (ECG) based tube current modulation. OSL dosimeters were used to measure dose profiles. In the both scan modes we acquired dose profiles and determined the mean and maximum dose in left lung and in left breast regions.ResultsIn prospective mode, the mean dose was 21.53 mGy in left lung- and 23.59 mGy in left breast region. With respect to the retrospective mode, the mean dose with tube current modulation was 38.63 mGy for left lung- and 46.02 mGy for left breast region, i.e. 0.56 and 0.55 times lower than the mean dose without modulation.ConclusionThe OSL dosimeter is useful for measurement of the actual radiation dose along z-axis at lung and breast regions in the prospective and the retrospective CTCA.     

Oxidative phosphorylation in Micrococcus denitrificans. I. Preparation and properties of phosphorylating membrane fragments

A procedure was described to prepare stable membrane fragments from aerobically grown cells of Micrococcus denitrificans. This preparation contained flavins, cytochromes b, c, a and o, and catalyzed the synthesis of ATP coupled to the oxidation of NADH and succinate. The P:O ratios were about 1.0 for NADH and 0.4 for succinate oxidation. The electron-transfer pathways responsible for these oxidations were similar to, though not identical with, those of mammalian mitochondria in their construction and sensitivity to inhibitors. Oxidative phosphorylation by the membrane fragments was uncoupled by the usual uncouplers and energy-transfer inhibitors, though 2,4-dinitrophenol was much less effective and higher concentrations of oligomycin and tributyltin chloride were required for complete inhibition as compared with the mitochondrial system. Oleate also caused uncoupling, which was relieved by serum albumin. Treatment with high concentrations of LiCl yielded an essentially uncoupled preparation, but this treatment as well as many other procedures failed to yield soluble coupling factors. Unlike the mitochondrial ATPase activity, ATP hydrolysis by the membrane fragments was inhibited to about 50% by uncouplers and energy-transfer inhibitors. It seems that the bacterial preparation possessed two types of ATPase, one of which was sensitive to these reagents as well as to LiCl treatment and probably to high concentrations of ADP. The advantage of this preparation for the study of the mechanism of oxidative phosphorylation is discussed.     

A mutant with constitutive sexual organ development in<Emphasis Type="Italic"> Marchantia polymorpha</Emphasis> L.

In lower land plants, genes controlling the transition from vegetative growth to sexual reproduction have not yet been identified. In the dioecious liverwort Marchantia polymorpha, the transition to sexual reproduction accompanied by the formation of sexual organs on the gametophytic thallus is initiated under long-day conditions. By particle bombardment-mediated mutagenesis, we generated a mutant of M. polymorpha that constitutively forms sexual organs. This mutant is fully fertile, showing that the mutation does not affect formation of male or female sexual organs per se. Genetic analysis reveals that this phenotype is caused by mutation of a single autosomal locus, suggesting that this mutation defines or controls a gene regulating the transition to sexual reproduction in M. polymorpha.     

A general computational model of mitochondrial metabolism in a whole organelle scale

SUMMARY: A computational tool for mitochondrial systems biology has been developed as a simulation model of E-Cell2, a publicly available simulation system. The general model consists of 58 enzymatic reactions and 117 metabolites, representing the respiratory chain, the TCA cycle, the fatty acid beta-oxidation and the inner-membrane transport system. It is based on previously published enzyme kinetics studies in the literature; we have successfully integrated and packaged them into a single large model. The model can be easily extended and modified so that mitochondrial biologists/physiologists can integrate their own models and evaluate them in the context of the whole organelle metabolism. AVAILABILITY: The mitochondrial model is bundled up with E-Cell2 simulation system, which can be downloaded from http://www.e-cell.org. CD-ROMs are also available and are distributed at major conferences. SUPPLEMENTARY INFORMATION: All the kinetic data are available via http://www.e-cell.org     

Chemical and kinetic reaction mechanisms of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans

Quinohemoprotein amine dehydrogenase (QHNDH) possesses a cysteine tryptophylquinone (CTQ) prosthetic group that catalyzes the oxidative deamination of primary amines. In addition to CTQ, two heme c cofactors are present in QHNDH that mediate the transfer of the substrate-derived electrons from CTQ to an external electron acceptor. Steady-state kinetic assays yielded relatively small k(cat) values (<6 s(-1)), and the rate-limiting step appears to be the interprotein electron transfer from heme in QHNDH to the external electron acceptor. Transient kinetic studies of the CTQ-dependent reduction of heme in QHNDH by amine substrates yielded different rate constants for different substrates (72, 190, and 162 s(-1) for methylamine, butylamine, and benzylamine, respectively). Deuterium kinetic isotope effect (KIE) values of 5.3, 3.9, and 8.5 were observed, respectively, for the reactions of methylamine, butylamine, and benzylamine. These results suggest that the abstraction of a proton from the alpha-methylene group of the substrate, which occurs concomitant with CTQ reduction, is the rate-limiting step in the CTQ-dependent reduction of hemes in QHNDH by these amine substrates. In contrast, the reaction of 2-phenylethylamine with QHNDH does not exhibit a significant KIE ((H)k(3)/(D)k(3) = 1.05) and exhibits a much smaller rate constant of 16 s(-1). This suggests that for 2-phenylethylamine, the rate-limiting step in the single-turnover reaction is either hydrolysis of the imine reaction intermediate from CTQ or product release prior to intraprotein electron transfer. Analysis of the products of the reactions of QHNDH with chiral deuterated 2-phenylethylamines demonstrated that the enzyme abstracts the pro-S proton of the substrate in a highly stereospecific manner. Inspection of the crystal structure of phenylhydrazine-inhibited QHNDH suggests that Asp33(gamma) is the residue that performs the proton abstraction. On the basis of these results, kinetic and chemical reaction mechanisms for QHNDH are proposed and discussed in the context of the crystal structure of the enzyme.     

Circadian rhythm of atrioventricular conduction predicts long-term survival in patients with chronic atrial fibrillation

The R-R interval of the electrocardiogram during atrial fibrillation (AF) appears absolutely irregular. However, the Poincaré plot of the R-R interval reveals a sector shape of distribution that is unique to AF. Furthermore, the height of lower envelope (LE1.0) of the distribution and the degree of scatter above the envelope (scattering index) may reflect the refractoriness and concealment of atrioventricular (AV) conduction, respectively. We previously observed that both the LE1.0 and scattering index show clear circadian rhythms in patients with chronic AF and that the rhythms are blunted in those with congestive heart failure and chronic AF. In the present study, we examined if the blunted circadian rhythm of the AV conduction has prognostic value in patients with chronic AF. We studied a retrospective cohort of 120 patients who underwent 24h Holter monitoring at baseline. During an observation period of 33 +/- 16 mon, there were 25 deaths (21%) including 13 cardiac and 8 stroke deaths. All patients showed significant circadian rhythms in both LE1.0 and scattering index with acrophases occurring at night; however, patients dying subsequently from cardiac causes, but not those from fatal stroke were blunted in the circadian rhythms (the amplitudes were < 55% of those in surviving patients). Furthermore, the reduced circadian amplitude of scattering index was an increased risk for cardiac death even after adjustment of coexisting cardiovascular risks [adjusted relative risk (95% confidence interval) per 1-SD decrement, 4.24 (1.54-11.6)]. When patients were divided by the circadian amplitude of the scattering index of 36.5 msec (mean minus 1-SD), the 5yr cardiac mortality below and above the cutoff was 57 and 6%, respectively (log-rank test, p < 0.001). We conclude that the blunted circadian rhythm of AV conduction is an independent risk for cardiac death in patients with chronic AF.