Conservation and diversification of Msx protein in metazoan evolution

Msx (/msh) family genes encode homeodomain (HD) proteins that control ontogeny in many animal species. We compared the structures of Msx genes from a wide range of Metazoa (Porifera, Cnidaria, Nematoda, Arthropoda, Tardigrada, Platyhelminthes, Mollusca, Brachiopoda, Annelida, Echiura, Echinodermata, Hemichordata, and Chordata) to gain an understanding of the role of these genes in phylogeny. Exon-intron boundary analysis suggested that the position of the intron located N-terminally to the HDs was widely conserved in all the genes examined, including those of cnidarians. Amino acid (aa) sequence comparison revealed 3 new evolutionarily conserved domains, as well as very strong conservation of the HDs. Two of the three domains were associated with Groucho-like protein binding in both a vertebrate and a cnidarian Msx homolog, suggesting that the interaction between Groucho-like proteins and Msx proteins was established in eumetazoan ancestors. Pairwise comparison among the collected HDs and their C-flanking aa sequences revealed that the degree of sequence conservation varied depending on the animal taxa from which the sequences were derived. Highly conserved Msx genes were identified in the Vertebrata, Cephalochordata, Hemichordata, Echinodermata, Mollusca, Brachiopoda, and Anthozoa. The wide distribution of the conserved sequences in the animal phylogenetic tree suggested that metazoan ancestors had already acquired a set of conserved domains of the current Msx family genes. Interestingly, although strongly conserved sequences were recovered from the Vertebrata, Cephalochordata, and Anthozoa, the sequences from the Urochordata and Hydrozoa showed weak conservation. Because the Vertebrata-Cephalochordata-Urochordata and Anthozoa-Hydrozoa represent sister groups in the Chordata and Cnidaria, respectively, Msx sequence diversification may have occurred differentially in the course of evolution. We speculate that selective loss of the conserved domains in Msx family proteins contributed to the diversification of animal body organization.     

Phylogenetic analysis of and oligonucleotide probe development for eikelboom type 021N filamentous bacteria isolated from bulking activated sludge

Fifteen filamentous strains, morphologically classified as Eikelboom type 021N bacteria, were isolated from bulking activated sludges. Based on comparative 16S ribosomal DNA (rDNA) sequence analysis, all strains form a monophyletic cluster together with all recognized Thiothrix species (88.3 to 98.7% 16S rDNA sequence similarity) within the gamma-subclass of Proteobacteria. The investigated Eikelboom type 021N isolates were subdivided into three distinct groups (I to III) demonstrating a previously unrecognized genetic diversity hidden behind the uniform morphology of the filaments. For in situ detection of these bacteria, 16S rRNA-targeted oligonucleotide probes specific for the entire Eikelboom type 021N-Thiothrix cluster and the Eikelboom type 021N groups I, II, and III, respectively, were designed, evaluated, and successfully applied in activated sludge.     

CSF hypocretin-1/orexin-A concentrations in patients with subarachnoid hemorrhage (SAH)

The aim of this study was to examine the role of the hypothalamic hypocretin/orexin system in complications of delayed ischemic neuronal deficit (DIND) resulting from symptomatic vasospasm in patients with aneurysmal subarachnoid hemorrhage (SAH). CSF hypocretin-1/orexin-A levels were measured in 15 SAH patients. DIND complications occurred in seven patients with symptomatic vasospasm. Hypocretin-1/orexin-A levels were low in SAH patients during the 10 days following the SAH event. CSF hypocretin-1/orexin-A levels were lower in patients with DIND complications than in those who did not develop DIND. A significant transient decline in CSF hypocretin-1/orexin-A levels was also observed at the onset of DIND in all patients with symptomatic vasospasm. The reduced hypocretin/orexin production observed in SAH patients may reflect reduced brain function due to the decrease in cerebral blood flow. These results, taken together with recent experimental findings in rats that indicate hypocretin receptor 1 (orexin 1 receptor) mRNA and protein are elevated following middle cerebral artery occlusion, suggest that a reduction in hypocretin/orexin production in SAH and DIND patients is associated with alterations in brain hypocretin/orexin signaling in response to ischemia.     

Confirmation of cultivated Porphyra tenera (Bangiales, Rhodophyta) by polymerase chain reaction restriction fragment length polymorphism analyses of the plastid and nuclear DNA

Polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) analysis of the plastid ribulose‐1,5‐bisphosphate carboxylase (RuBisCo) spacer region was developed for a more reliable and rapid species identification of cultivated Porphyra in combination with PCR‐RFLP analysis of the nuclear internal transcribed spacer (ITS) region. From the PCR‐RFLP analyses of the plastid and nuclear DNA, we examined seven strains of conchocelis that were used for cultivation as Porphyra tenera Kjellman but without strict species identification. The PCR‐RFLP analyses suggested that two strains, C‐32 and 90‐02, were cultivated P. tenera and that the other five strains, C‐24, C‐28, C‐29, C‐30 and M‐1, were Porphyra yezoensis f. narawaensis Miura. To identify species more accurately and to reveal additional genetic variation, the two strains C‐32 and 90‐02 were further studied by sequencing their RuBisCo spacer and ITS‐1 regions. Although RuBisCo spacer sequences of the two strains were identical to each other, each of their ITS‐1 sequences showed a single substitution. The sequence data again confirmed that the two strains (C‐32 and 90‐02) were cultivated P. tenera, and suggested that the two strains showed some genetic variation. We concluded that PCR‐RFLP analysis of the plastid and nuclear DNA is a powerful tool for reliable and rapid species identification of many strains of cultivated Porphyra in Japan and for the collection of genetically variable breeding material of Porphyra.     

Growth of Ecklonia cava (Laminariales, Phaeophyta) sporophytes transplanted to a locality with different temperature conditions

Transplanting experiments were carried out to determine whether the small type sporophytes with short stipe of Ecklonia cava Kjellman (Laminariales, Phaeophyta) growing in a locality with warm temperatures, change into larger type with a long stipe when transplanted to a locality with cooler temperatures. Juvenile E. cava sporophytes, having a stipe shorter than 5 cm long were collected from Tei in Tosa Bay (southern Japan) (seawater temperature 15–29°C) and transplanted to Nabeta Bay (central Japan) (seawater temperature 13–25°C), where larger type E. cava sporophytes characterized by long stipe (ca 1 m) grow. They were attached to artificial reefs at the sea bottom (9 m depth) in Nabeta Bay to monitor their growth. For comparison, juvenile E. cava sporophytes of almost similar size growing in Nabeta Bay were also transplanted in the same way to the same experimental site. Observations of growth of sporophytes from Tei and Nabeta were carried out monthly for 2 years from November 1995 to October 1997. The transplanted Tei and Nabeta sporophytes showed an increase in stipe length and diameter from winter to spring, whereas almost no increase was observed during summer and autumn. At the end of the study period, the stipe of Nabeta sporophytes reached 25.6 cm in length and 17.0 mm in diameter, whereas that of Tei sporophytes reached 11.1 cm in length and 11.2 mm in diameter. The primary blade length was 16.0 cm in Nabeta sporophytes, whereas it was 5.5 cm in Tei sporophytes. Thus, Tei sporophytes still remained smaller than Nabeta sporophytes even under the same environmental conditions.     

Research note: Identification of a cDNA homologous to the cell-cycle-controlling cdc2 gene in Acroslphonia duriuscula (Acrosiphoniales, Chlorophyta)

Cyclin‐dependent kinases, the most notable of which is cdc2, are key regulators of the cell cycle and are highly conserved in evolution. We have cloned and analyzed one cDNA containing an open reading frame of 337 amino acids from a multinucleate, multicellular green alga, Acrosiphonia duriuscula (Ruprecht) Collins. The deduced protein, named Adcdc2‐1, showed 51% and 54% amino acid sequence identity to yeast cdc2/cdc28 and human cdc2, respectively. Several domains that characterize the cdc2‐related kinases were identified from this sequence, although the PSTAIRE motif was replaced with PPTTIRE. Furthermore, this protein has conserved Tyr and Thr residues that are sites of phosphorylation in cdc2‐related kinases and are important for regulating kinase activity. The present results suggest that the universal cdc2 is conserved in algae with unique structural characteristics.     

Tumor-specific granulocyte/macrophage colony-stimulating factor and interferon γ secretion is associated with in vivo therapeutic efficacy of activated tumor-draining lymph node cells

In this study, cytokine release by tumor-draining lymph node cells sensitized in vitro (IVS-TDLN) was examined and correlated with therapeutic efficacy in adoptive immunotherapy. Mice bearing immunologically distinct MCA 207 and MCA 205 sarcoma tumors were utilized in criss-cross experiments. IVS-TDLN obtained from mice bearing 10-day subcutaneous (s. c.) tumors mediated immunologically specific regression of established 3-day pulmonary metastases, but demonstrated non-specific cytolytic reactivity against both tumors in a 4-h⁵¹Cr-release assay. By contrast, these IVS-TDLN cells were found specifically to secrete granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon (IFN) when restimulated in vitro with irradiated tumor cells. To determine the predictive value of tumor-specific cytokine release with in vivo therapeutic efficacy, a kinetic analysis of antitumor activities of TDLN obtained from animals bearing MCA 207 tumors for increasing lengths of time was performed. IVS-TDLN cells from mice bearing day-7, -10 and-14 s. c. tumors manifested tumor-specific release of GM-CSF and IFN, and mediated significant antitumor reactivity in vivo. In contrast IVS-LN cells from day-0 and day-21 tumor-bearing animals did not release significant amounts of GM-CSF and IFN, and were not therapeutically efficacious in vivo. Day-4 IVS-TDLN released high levels of GM-CSF and IFN non-specifically, and were not therapeutic in adoptive immunotherapy at doses effective for day-7 and day-14 IVS-TDLN cells. In other experiments, IVS cells generated from different lymph node groups in animals bearing 10-day established s. c. tumors were examined and found to have unique profiles of cytokine release. In these studies, the ability of IVS cells to release specifically both cytokines as opposed to one was associated with greater therapeutic efficacy on a per cell basis. Our findings suggest that the tumor-specific releases of GM-CSF and IFN are useful parameters to assess the in vivo therapeutic efficacy of immune lymphocytes.     

Isolation of a Drosophila Gene Encoding a Head-Specific Guanylyl Cyclase

Abstract: We have isolated and characterized a new guanylyl cyclase gene ( dgcl) in Drosophila. The deduced amino acid sequence (683 amino acids) most closely resembled the mammalian solubletype guanylyl cyclase α subunit. The cyclase catalytic domain was highly conserved between the mammalian and Drosophila guanylyl cyclases. The dgcl mRNA was detected in wild-type heads but not in bodies, and its level was reduced in the mutant eyes absent (eya) , indicating that dgcl is preferentially expressed in the CNS and in the eye. The enriched distribution in the eye suggests that dgcl may have a role in phototransduction.     

Micronucleus assays on 5-fluorouracil and 6-mercaptopurine with mouse peripheral blood reticulocytes.

As part of the 5th collaborative study of the Collaborative Study Group for the Micronucleus Test (CSGMT), the sensitivity and advantages of the micronucleus assay using mouse peripheral blood cells were evaluated using 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP). The peripheral blood cells were collected from a tail vein of CD-1 male mice just before and 24-120 h after intraperitoneal injection. At 24-h intervals. The maximum incidence of micronucleated reticulocytes (MNRETs) at 50 mg/kg 5-FU was observed 96 h after injection; at 100 mg/kg, the peak was delayed to 120 h, and followed severe bone marrow depression. With 6-MP, maximum MNRETs were observed 48 h after treatment at all doses tested. At dose levels higher than 50 mg/kg, severe bone marrow depression was observed after maximum MNRETs. Though the appearance patterns of MNRETs and the bone marrow depression were different between 5-FU and 6-MP, the positive response of both chemical could be detected with this assay system as well as with the micronucleus test using femoral bone marrow cells.