Phylogeny and the Evolution of the <Emphasis Type="Italic">Amylase</Emphasis> Multigenes in the <Emphasis Type="Italic">Drosophila montium</Emphasis> Species Subgroup

Abstract To investigate the phylogenetic relationships and molecular evolution of α-amylase (Amy) genes in the Drosophila montium species subgroup, we constructed the phylogenetic tree of the Amy genes from 40 species from the montium subgroup. On our tree the sequences of the auraria, kikkawai, and jambulina complexes formed distinct tight clusters. However, there were a few inconsistencies between the clustering pattern of the sequences and taxonomic classification in the kikkawai and jambulina complexes. Sequences of species from other complexes (bocqueti, bakoue, nikananu, and serrata) often did not cluster with their respective taxonomic groups. This suggests that relationships among the Amy genes may be different from those among species due to their particular evolution. Alternatively, the current taxonomy of the investigated species is unreliable. Two types of divergent paralogous Amy genes, the so-called Amy1- and Amy3-type genes, previously identified in the D. kikkawai complex, were common in the montium subgroup, suggesting that the duplication event from which these genes originate is as ancient as the subgroup or it could even predate its differentiation. Thc Amy1-type genes were closer to the Amy genes of D. melanogaster and D. pseudoobscura than to the Amy3-type genes. In the Amy1-type genes, the loss of the ancestral intron occurred independently in the auraria complex and in several Afrotropical species. The GC content at synonymous third codon positions (GC3s) of the Amy1-type genes was higher than that of the Amy3-type genes. Furthermore, the Amy1-type genes had more biased codon usage than the Amy3-type genes. The correlations between GC3s and GC content in the introns (GCi) differed between these two Amy-type genes. These findings suggest that the evolutionary forces that have affected silent sites of the two Amy-type genes in the montium species subgroup may differ.     

Molecular cloning of the protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis.

We determined the nucleotide sequence of a 1.9-kilobase fragment of Pseudomonas paucimobilis SYK6 chromosomal DNA that included genes encoding protocatechuate 4,5-dioxygenase, the enzyme responsible for the aromatic ring fission of protocatechuate. Two open reading frames of 417 and 906 base pairs were found that had no homology with previously reported sequences, including those encoding protocatechuate 3,4-dioxygenase. Since both open reading frames were indispensable for the enzyme activity, they should encode the subunits of protocatechuate 4,5-dioxygenase. We named these genes ligA and ligB. Protocatechuate 4,5-dioxygenase was efficiently expressed in Escherichia coli with the aid of the lac promoter, and the polypeptides of the ligA and ligB gene products were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid sequencing.     

Effects of infection with Angiostrongylus cantonensis on the circulating haemocyte population and the haematopoietic organ of the host snail M-line Biomphalaria glabrata

The number of circulating haemocytes, the size of the haematopoietic organ, and the size of haemocyte capsules around the parasite were studied in M-line Biomphalaria glabrata snails exposed to 100 or 400 first-stage larvae of Angiostrongylus cantonensis. The number of haemocytes in exposed snails increased significantly at 1 day post-exposure, decreased to control value, and then increased again. The decrease in number of circulating haemocytes is probably due to the removal of cells from the circulation to participate in encapsulation of larvae. The majority of circulating haemocytes in M-line B. glabrata are fully-spread granulocytes, which increase significantly in number in snails following exposure to A. cantonensis larvae. However, populations of partially-spread granulocytes, round cells, hyalinocytes and miscellaneous haemocytes were relatively constant. The size of capsules around the parasite increased during the 42-day interval of the experiment. The haematopoietic organ increased in size in response to infection.     

Detection of MPLW515L/K Mutations and Determination of Allele Frequencies with a Single-Tube PCR Assay

A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.     

Osteopontin negatively regulates parathyroid hormone receptor signaling in osteoblasts

Systemic hormonal control exerts its effect through the regulation of local target tissues, which in turn regulate upstream signals in a feedback loop. The parathyroid hormone (PTH) axis is a well defined hormonal signaling system that regulates calcium levels and bone metabolism. To understand the interplay between systemic and local signaling in bone, we examined the effects of deficiency of the bone matrix protein osteopontin (OPN) on the systemic effects of PTH specifically within osteoblastic cell lineages. Parathyroid hormone receptor (PPR) transgenic mice expressing a constitutively active form of the receptor (caPPR) specifically in cells of the osteoblast lineage have a high bone mass phenotype. In these mice, OPN deficiency further increased bone mass. This increase was associated with conversion of the major intertrabecular cell population from hematopoietic cells to stromal/osteoblastic cells and parallel elevations in histomorphometric and biochemical parameters of bone formation and resorption. Treatment with small interfering RNA (siRNA) for osteopontin enhanced H223R mutant caPPR-induced cAMP-response element (CRE) activity levels by about 10-fold. Thus, in addition to the well known calcemic feedback system for PTH, local feedback regulation by the bone matrix protein OPN also plays a significant role in the regulation of PTH actions.     

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studied using γ-³²P ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25 kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppresssed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.     

Control of Polymorphs on the Crystallization of Glycine Using a WWDJ Batch Crystallizer

The control of crystal polymorphs was investigated using a WWDJ batch crystallizer and glycine as a model compound. The WWDJ batch crystallizer is a newly developed crystallizer, which is equipped with a slurry sprinkler named Wall Wetter fixed on the shaft of an impeller and a double‐deck jacket. When a conventional crystallizer was used, the unstable α‐form crystals were always obtained. However, when the WWDJ batch crystallizer was used, the stable γ‐form crystals were obtained. The appearance of different polymorphs depends on the cooling rate during the crystallization. The γ‐form crystals were obtained by slow cooling, while the α‐form was obtained by rapid cooling. It means that the solvent‐mediated transformation of glycine crystal polymorphs can be controlled by changing the cooling rate in the WWDJ crystallizer. These results were obtained due to the fact that the WWDJ batch crystallizer accelerates the dissolution of metastable crystals and the growth of stable crystals.     

Peroxisome degradation requires catalytically active sterol glucosyltransferase with a GRAM domain

Fungal sterol glucosyltransferases, which synthesize sterol glucoside (SG), contain a GRAM domain as well as a pleckstrin homology and a catalytic domain. The GRAM domain is suggested to play a role in membrane traffic and pathogenesis, but its significance in any biological processes has never been experimentally demonstrated. We describe herein that sterol glucosyltransferase (Ugt51/Paz4) is essential for pexophagy (peroxisome degradation), but not for macroautophagy in the methylotrophic yeast Pichia pastoris. By expressing truncated forms of this protein, we determined the individual contributions of each of these domains to pexophagy. During micropexophagy, the glucosyltransferase was associated with a recently identified membrane structure: the micropexophagic apparatus. A single amino acid substitution within the GRAM domain abolished this association as well as micropexophagy. This result shows that GRAM is essential for proper protein association with its target membrane. In contrast, deletion of the catalytic domain did not impair protein localization, but abolished pexophagy, suggesting that SG synthesis is required for this process.