Identification and elimination of ion suppression in the quantitative analysis of sirolimus in human blood by LC/ESI-MS/MS

Ion suppression can negatively affect the performance characteristics of LC/ESI-MS/MS based methods, and we wished to identify sources of ion suppression in an assay for quantitating sirolimus in human whole blood. We first compared the peak areas of sirolimus and ascomycin added to human blood samples treated with and without extraction using octadecyl silyl (ODS)-silica gel after protein precipitation, and we found that water-soluble compounds cause the ion suppression for both drugs. Post-column infusion studies indicated that compounds retained in the sample after ODS extraction and protein precipitation caused ion suppression. MS analysis of these compounds suggested they were hydroxyl group-possessing phosphocholines, and this was confirmed using purified lysophosphatidylcholine variants. Therefore, we included a HybridSPE treatment step after the ODS extraction into the preanalytical workflow to remove phosphocholines, and this successfully eliminated the observed ion suppression for determining sirolimus concentration in human whole blood by LC/ESI-MS/MS. Sirolimus is a highly lipophilic molecule, and this study demonstrates the impact that preanalytical extraction and purification steps can have on a laboratory's ability to accurately detect and quantitate this and other lipophilic drugs.     

Accumulation of Diphosphorylated Butirosin A Derivatives by Phosphatase-negative Mutants of Bacillus vitellinus

It has been reported that Bacillus vitellinus, a butirosin A-producing bacterium, has two enzymes: butirosin A-3′-phosphotransferase, catalyzing phosphorylation of butirosin A, and phosphatase, catalyzing dephosphorylation of butirosin A-3′-phosphate.

A phosphatase-negative mutant, P-15, was derived from a potent butirosin A producer, BA-44, by NTG treatment. The mutant, P-15, was found to produce a butirosin A derivative when it was grown in a medium containing a relatively high concentration of inorganic phosphate. This compound was isolated and confirmed to be butirosin A-6′-N-diphosphate by physico-chemical analysis and ¹³C-NMR spectrometry.

Furthermore, a mutant strain, AP-165, was derived from the phosphatase-negative mutant, P-15. This strain, AP-165, was isolated as a nonproducer on an agar piece of bouillon medium, but was found to accumulate a major product, 6′-deamino-6′-hydroxybutirosin A-6′-O-diphosphate, and a minor one, 6′-deamino-6′-hydroxybutirosin A-6′-O-monophosphate.

It seems likely that these phosphorylated compounds are possible intermediates of butirosin A biosynthesis in B. vitellinus.     

Biosynthesis of Enduracidin: Origin of Enduracididine and Other Amino Acids

The biosynthetic origin of the amino acid moieties of enduracidin was investigated by feeding experiments with labeled compounds. Results of the incorporation and the distribution of radioactivity into the antibiotic revealed that glycine, l-serine, l-threonine, l-alanine, L-aspartic acid, l-ornithine and l-citrulline were incorporated into the corresponding amino acid moieties. Unique amino acids, enduracididine and its isomer with an imidazolidine ring, were derived from l-arginine, but not histidine. K₁ (4-hydroxyphenylglycine) and K₂ (3,5-dichloro-K₁) moieties were derived from l-tyrosine. ³⁶Cl-Sodium chloride was incorporated into the antibiotic in the early stage of fermentation.     

2′-N-Acetylation of Butirosin A by Mutants of Streptomyces fradiae

To reveal the mechanism of reducing sugar-induced polymerization of proteins, monomeric lysozymes were isolated at various stages of storage from whole lysozyme (WL) being kept with glucose at 75% relative humidity and 50°C for up to 30 days, and their chemical properties were investigated and compared with the corresponding WL. The impairment of Lys, Arg, and Trp residues was observed in all the isolated monomeric lysozymes (IM) as well as in the WL.

When the IM were stored for another 10 days without glucose, they polymerized and had an additional impairment of Arg and Lys but not Trp residues. All IM exhibited almost the same polymerization rate, but the sum of additional losses of Lys and Arg residues varied. The IM was also found to cross-link untreated lysozymes even in the absence of glucose.

On basis of the results obtained hitherto, it is suggested that the glucose-induced polymerization of lysozymes proceeds through the following paths. At the first step, some bifunctional agents (BF), probably α-dicarbonyl compounds, generated from the reaction between ?-amino groups of lysine residues and glucose, attach to Arg, Lys, and Trp residues through one of their two functional sites. At the second step, some of those proteins with BF attached polymerize by binding of the other unoccupied functional site with the remaining Lys and Arg (not Trp) residues of the other protein molecules. The other of the proteins with BF attached polymerize through the combination between the other unoccupied functional sites themselves with no loss of amino acid residues.     

Functional conservation of clock output signaling between flies and intertidal crabs

Intertidal species have both circadian and circatidal clocks. Although the behavioral evidence for these oscillators is more than 5 decades old, virtually nothing is known about their molecular clockwork. Pigment-dispersing hormones (PDHs) were originally described in crustaceans. Their insect homologs, pigment-dispersing factors (PDFs), have a prominent role as clock output and synchronizing signals released from clock neurons. We show that gene duplication in crabs has led to two PDH genes (β-pdh-I and β-pdh-II). Phylogenetically, β-pdh-I is more closely related to insect pdf than to β-pdh-II, and we hypothesized that β-PDH-I may represent a canonical clock output signal. Accordingly, β-PDH-I expression in the brain of the intertidal crab Cancer productus is similar to that of PDF in Drosophila melanogaster, and neurons that express PDH-I also show CYCLE-like immunoreactivity. Using D. melanogaster pdf-null mutants (pdf(01)) as a heterologous system, we show that β-pdh-I is indistinguishable from pdf in its ability to rescue the mutant arrhythmic phenotype, but β-pdh-II fails to restore the wild-type phenotype. Application of the three peptides to explanted brains shows that PDF and β-PDH-I are equally effective in inducing the signal transduction cascade of the PDF receptor, but β-PDH-II fails to induce a normal cascade. Our results represent the first functional characterization of a putative molecular clock output in an intertidal species and may provide a critical step towards the characterization of molecular components of biological clocks in intertidal organisms.     

Experimental infection of Dirofilaria immitis in raccoon dogs

Canine heartworm (Dirofilaria immitis) is a nematode that naturally parasitizes in the pulmonary arteries and the right ventricle of domestic dogs (Canis familiaris) as final hosts. Japanese raccoon dogs (Nyctereutes procyonoides viverrinus) also are known to be susceptible to infection by the parasite. However, prevalence of this infection among free-ranging raccoon dogs is low and so is the worm burden. To examine the susceptibility of the raccoon dog to D. immitis infection, 3 raccoon dogs and 2 beagles were inoculated 4 times with 25 third-stage larvae (L3s) of D. immitis at 3-wk intervals. Worms were recovered from 2 raccoon dogs and both domestic dogs. The average percentage of recovery (2.3%) of the raccoon dogs was almost 10 times lower (24.5%) than that of the domestic dogs, but there was no significant difference in the body length of worms recovered from 2 types of hosts. To examine microfilaremia, 2 raccoon dogs were infected with 100 L3s. Microfilaremia was observed for 180 days postinoculation (PI) but disappeared at about 300 days PI. The raccoon dog was mildly susceptible to infection with D. immitis, but surviving worms developed and matured normally.     

Mechanisms of plasmin-catalyzed inactivation of factor VIII: a crucial role for proteolytic cleavage at Arg336 responsible for plasmin-catalyzed factor VIII inactivation

Plasmin not only functions as a key enzyme in the fibrinolytic system but also directly inactivates factor VIII and other clotting factors such as factor V. However, the mechanisms of plasmin-catalyzed factor VIII inactivation are poorly understood. In this study, levels of factor VIII activity increased approximately 2-fold within 3 min in the presence of plasmin, and subsequently decreased to undetectable levels within 45 min. This time-dependent reaction was not affected by von Willebrand factor and phospholipid. The rate constant of plasmin-catalyzed factor VIIIa inactivation was approximately 12- and approximately 3.7-fold greater than those mediated by factor Xa and activated protein C, respectively. SDS-PAGE analysis showed that plasmin cleaved the heavy chain of factor VIII into two terminal products, A1(37-336) and A2 subunits, by limited proteolysis at Lys(36), Arg(336), Arg(372), and Arg(740). The 80-kDa light chain was converted into a 67-kDa subunit by cleavage at Arg(1689) and Arg(1721), identical to the pattern induced by factor Xa. Plasmin-catalyzed cleavage at Arg(336) proceeded faster than that at Arg(372), in contrast to proteolysis by factor Xa. Furthermore, breakdown was faster than that in the presence of activated protein C, consistent with rapid inactivation of factor VIII. The cleavages at Arg(336) and Lys(36) occurred rapidly in the presence of A2 and A3-C1-C2 subunits, respectively. These results strongly indicated that cleavage at Arg(336) was a central mechanism of plasmin-catalyzed factor VIII inactivation. Furthermore, the cleavages at Arg(336) and Lys(36) appeared to be selectively regulated by the A2 and A3-C1-C2 domains, respectively, interacting with plasmin.     

The effects of light on sex determination in gametophytes of the fern <Emphasis Type="Italic">Ceratopteris richardii</Emphasis>

The sexuality of homosporous fern gametophytes is usually determined by antheridiogen, a pheromone that promotes maleness. In this work the effect of photomorphogenically active light on antheridiogen-induced male development was examined for gametophytes of Ceratopteris richardii. Although blue light did not affect sensitivity to Ceratopteris antheridiogen (A_Ce) in wild-type gametophytes, it was found that the gametophytes of the her1 mutant, which are insensitive to A_Ce, developed into males when grown under blue light in the presence of A_Ce. Thus, we conclude that another A_Ce-signal transduction pathway activated by blue light exists latently in the gametophytes of C. richardii. Red light, on the other hand, suppressed male development. Because simultaneous red and blue light-irradiation did not promote male development in the her1 gametophytes, the action of red light seems to dominate that of blue light. The results of experiments with a photomorphogenic mutant also suggested that phytochrome may be involved in the action of red light.     

Expression mapping of cytotoxic T-lymphocyte antigen-2α gene transcripts in mouse brain

Cytotoxic T-lymphocyte antigen-2alpha (CTLA-2alpha), an inhibitor peptide homologous to the proregion of mouse cathepsin L, was originally discovered and expressed in mouse-activated T-cells and mast cells. Expressed recombinant CTLA-2alpha is shown to exhibit selective inhibition to cathepsin L-like cysteine proteinases. However, its in vivo targets in mammalian tissues are yet to be identified. We carried out in situ hybridization studies to examine the expression pattern of CTLA-2alpha mRNA and determine the specific cell types synthesizing CTLA-2alpha in the mouse brain. CTLA-2alpha mRNA was detected in various neuronal populations within the telencephalon in cerebral cortices, olfactory system, septum, basal ganglia, amygdala and highest levels were observed in the hippocampus. Within the diencephalon high density of positive cells was found in mediodorsal and lateral posterior thalamic nuclei and medial habenular nucleus (MHb). In the hypothalamus, high density of CTLA-2alpha mRNA labeling was seen in the suprachiasmatic nucleus (Sch), optic tract, arcuate nucleus, and median eminence. The fasciculus retroflexus and its termination in the mesencephalic interpeduncular nucleus were also densely labeled. Other mesencephalic expression sites were the superior colliculus, periaqueductal gray, paramedian raphe nucleus, and inferior colliculus. In the rhombencephalon, strong labeling was detected in the pontine, vestibular, and reticular nuclei. Intense expression was also noted within cerebellar cortex in Purkinje neurons and at a moderate level in granule cell layer, stellate, and basket cells. A possible function of this novel inhibitor peptide in relation to learning, memory, and diseases is discussed.