Six1 controls patterning of the mouse otic vesicle

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.     

Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton

Indian hedgehog (Ihh) is indispensable for development of the osteoblast lineage in the endochondral skeleton. In order to determine whether Ihh is directly required for osteoblast differentiation, we have genetically manipulated smoothened (Smo), which encodes a transmembrane protein that is essential for transducing all Hedgehog (Hh) signals. Removal of Smo from perichondrial cells by the Cre-LoxP approach prevents formation of a normal bone collar and also abolishes development of the primary spongiosa. Analysis of chimeric embryos composed of wild-type and Smo(n/n) cells indicates that Smo(n/n) cells fail to contribute to osteoblasts in either the bone collar or the primary spongiosa but generate ectopic chondrocytes. In order to assess whether Ihh is sufficient to induce bone formation in vivo, we have analyzed the bone collar in the long bones of embryos in which Ihh was artificially expressed in all chondrocytes by the UAS-GAL4 bigenic system. Although ectopic Ihh does not induce overt ossification along the entire cartilage anlage, it promotes progression of the bone collar toward the epiphysis, suggesting a synergistic effect between ectopic Ihh and endogenous factors such as the bone morphogenetic proteins (BMPs). In keeping with this model, Hh signaling is further found to be required in BMP-induced osteogenesis in cultures of a limb-bud cell line. Taken together, these results demonstrate that Ihh signaling is directly required for the osteoblast lineage in the developing long bones and that Ihh functions in conjunction with other factors such as BMPs to induce osteoblast differentiation. We suggest that Ihh acts in vivo on a potential progenitor cell to promote osteoblast and prevent chondrocyte differentiation.     

Genetic variations at urotensin II and urotensin II receptor genes and risk of type 2 diabetes mellitus in Japanese

Urotensin II is among the most potent vasoactive hormones known and the urotensin II (UTS2) gene is localized to 1p36-p32, one of the regions reported to show possible linkage with type 2 diabetes in Japanese. When we surveyed genetic polymorphisms in the UTS2 and urotensin II receptor (GPR14) gene, we identified two SNPs with amino acid substitutions (designated T21M and S89N and an SNP in the promotor region (-605G>A) of the UTS2 gene, and two SNPs in the non-coding region of the GPR14 gene. We then studied these three SNPs in the UTS2 gene and two SNPs in the GPR14 gene in 152 Japanese subjects with type 2 diabetes mellitus and two control Japanese populations. The allele frequency of 89N was significantly higher in type 2 diabetic patients than in both elderly normal subjects (P = 0.0018) and subjects with normal glucose tolerance (P = 0.0011), whereas the allele frequency of T21M and -605G>A in the UTS2 gene and those of two SNPs in the GPR14 gene were essentially identical in these three groups. Furthermore, in the subjects with normal glucose tolerance, 89N was associated with significantly higher insulin levels on oral glucose tolerance test, suggesting reduced insulin sensitivity in subjects with 89N. These results strongly suggest that subjects with S89N in the UTS2 gene are more insulin-resistant and thus more susceptible to type 2 diabetes mellitus development.     

Macrocyclic proteoglycan mimics. Potent inhibition of cell adhesion by a bundle of chondroitin sulfate chains assembled on the calix[4]resorcarene platform

Tailed calix[4]resorcarene macrocycle (tail=undecyl) can be used as a platform to assemble four glycosaminoglycan polysaccharide chains to give a new type of proteoglycan mimics. A tetra(chondroitin sulfate) derivative thus obtained from the reaction of macrocyclic octaamine and chondroitin sulfate lactone is readily immobilized on a tissue culture plastic (polystyrene) plate and inhibits fibronectin-mediated adhesion of BHK (baby hamster kidney) cells thereon remarkably strongly with 50% inhibition occurring at a 10 ng/mL or 40 pM concentration range.     

Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03

A lipolytic enzyme gene (lip8) was cloned from organic solvent-tolerant Pseudomonas aeruginosa LST-03 and sequenced. In the sequenced nucleotides, an open reading frame consisting of 1,173 nucleotides and encoding 391 amino acids was found. Lip8 is considered to belong to the family VIII of lipolytic enzymes whose serine in the consensus sequence of -Ser-Xaa-Xaa-Lys- acts as catalytic nucleophile. The gene was expressed in Escherichia coli and purified by a combination of ammonium sulfate fractionation and hydrophobic interaction and ion-exchange chromatographies to homogeneity on SDS-PAGE analysis. The optimum temperature and heat stability of Lip8 were not as high as those of Lip3 and LST-03 lipase, two other lipolytic enzymes from the same strain. Addition of glycerol to a solution containing Lip8 stabilized this enzyme. By measuring the activities against various triacylglycerols and fatty acid methyl esters having carbon chains of different lengths, Lip8 was categorized as an esterase which has higher activities against fatty acid methyl esters with short-chain fatty acids.     

Utility of Interphase FISH Panels for Routine Clinical Cytogenetic Evaluation of Chronic Lymphocytic Leukemia and Multiple Myeloma

Specific genetic abnormalities are of prognostic significance for patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM); however, routine cytogenetic analysis usually provides normal results. We utilized two probe panels for interphase fluorescence in situ hybridization (FISH) studies to enhance the ability to detect genetic abnormalities in samples that were referred for routine cytogenetic studies for possible diagnoses of CLL or MM. The CLL panel consisted of probes for 11q22.3 (ATM gene), 13q14 (D13S319), the centromere of chromosome 12 (D12Z3) and 17p13.1 (P53 gene). The MM panel included probes for 14q32 (IgH gene) and/or t(11:14)(q13;q32) (BCL1/IgH), 13q14 (D13S319) and 17p13.1 (P53 gene). FISH detected clonal aberrations not identified by conventional cytogenetics in an additional 8 of 23 (35%) samples referred for possible CLL and 7 of 42 (17%) samples with possible MM. The prognostic significance of the aberrations identified ranged from favorable, to intermediate, to poor. Our studies indicate that many samples referred for routine cytogenetics testing for CLL and MM yield normal results for both conventional and FISH testing, likely due to lack of definitive diagnosis in a percentage of cases. However, FISH is more sensitive for the detection of clinically significant chromosome abnormalities and should be the testing methodology of choice for these disorders.     

Effects of prolactin and growth hormone on plasma levels of lysozyme and ceruloplasmin in rainbow trout

In vivo and in vitro effects of prolactin (PRL) and growth hormone (GH) on plasma levels of lysozyme and ceruloplasmin were examined in the rainbow trout (Oncorhynchus mykiss). Hypophysectomy had no effect on the plasma lysozyme level. Implantation of PRL- or GH-containing cholesterol pellets increased the lysozyme level in a dose-related manner. After hypophysectomy and sham operation, plasma ceruloplasmin was elevated above the level in intact fish, suggesting inflammation caused by the surgery. PRL or GH treatment significantly attenuated the increased level of ceruloplasmin in the operated fish. Expression of lysozyme mRNA was detected in the leucocytes isolated from the peripheral blood by RT-PCR. In vitro administration of PRL or GH showed no effect on the proliferation of isolated leucocytes or on the total protein content; however, lysozyme activity in the medium increased in a dose-related manner. These results suggest that PRL and GH directly stimulate lysozyme production without affecting the proliferation of leucocytes, and the attenuated ceruloplasmin level increased in response to inflammation.     

(3Z)-2-Acetylamino-3-octadecen-1-ol as a potent apoptotic agent against HL-60 cells

(2R,3Z)-, (2R,3E)-, (2S,3Z) and (2S,3E)-2-Acetylamino-3-octadecen-1-ol, and (2R)- and (2S)-2-acetylamino-octadecan-1-ol were prepared using the Wittig olefination of Garner's aldehyde (N-Boc-N,O-isopropylidene-L- or D-serinal) from L- or D-serine. The apoptotic activities of these saturated and unsaturated 2-acetylaminoalcohols were examined in human leukemia HL-60 cells using MTT assay. Among the newly synthesized compounds, the cis-isomers were the most potent. Despite their simple structures, (2R,3Z)- and (2S,3Z)-2-acetylamino-3-octadecen-1-ol showed high and comparable apoptotic activities compared with N-acetyl-D-erythro-sphingosine (D-e-C2-Cer, a well-known inducer of apoptosis). Their apoptotic activities were in the order D-e-C2-Cer approximately L-e-C2-Cer approximately (2R,3Z)- approximately (2S,3Z)->(2R,3E)- approximately (2S,3E)- approximately (2R)- approximately (2S)-derivative. Qualitative analysis of DNA fragmentation caused by these compounds was conducted using agarose gel electrophoresis, and typical DNA fragmentation was found in the cases of (2R,3Z)- and (2S,3Z)-isomers such as C2-Cer, but not trans and saturated isomers. The morphological features of the cells, the proteolytic processing of pro-caspase-3, and the cleavage of PARP as a result of exogenous treatment with (2R,3Z)- and (2S,3Z)-isomers indicated that cell death induced by these compounds was apoptosis. These observations suggest that these newly synthesized compounds, (3Z)-2-Acetylamino-3-octadecen-1-ol, have similar characteristics and apoptosis-inducing activities against HL-60 cells with C2-Cer.     

Caspase-1 and -3 mRNAs are differentially upregulated in motor neurons and glial cells in mutant SOD1 transgenic mouse spinal cord: a study using laser microdissection and real-time RT-PCR

Amyotrophic lateral sclerosis is characterized by selective motor neuron degeneration. An apoptotic pathway is thought to be involved. It is difficult, however, to analyze the molecular pathogenic mechanism in single motor neurons because of complexity in the neural tissue, which consists of multiple lineages of cells neighboring motor neurons. We quantified the caspase-1 and -3 mRNA in single motor neurons and neighboring glial cells isolated from the spinal ventral horn of mutant SOD1 transgenic (Tg) mice and littermates. Motor neurons and neighboring glial cells were isolated from spinal sections by laser microdissection, and the mRNAs were quantified by RT-PCR. In the Tg mice, caspase-1 mRNA was first upregulated in motor neurons and second in glial cells. The caspase-3 mRNA was increased in motor neurons following the caspase-1 mRNA. These results indicated that caspase-1 and -3 mRNAs are differentially upregulated in motor neurons and glial cells of the Tg mice, and that mRNAs in isolated cells can be accurately assessed using our procedures.     

Ca2+-dependent modulation of intracellular Mg2+ concentration with amiloride and KB-R7943 in pig carotid artery

It has long been recognized that magnesium is associated with several important diseases, including diabetes, hypertension, cardiovascular, and cerebrovascular diseases. In the present study, we measured the intracellular free Mg2+ concentration ([Mg2+]i) using 31P nuclear magnetic resonance (NMR) in pig carotid artery smooth muscle. In normal solution, application of amiloride (1 mm) decreased [Mg2+]i by approximately 12% after 100 min. Subsequent washout tended to further decrease [Mg2+]i. In contrast, application of amiloride significantly increased [Mg2+]i (by approximately 13% after 100 min) under Ca2+-free conditions, where passive Mg2+ influx is facilitated. The treatments had little effect on intracellular ATP and pH (pHi). Essentially the same Ca2+-dependent changes in [Mg2+]i were produced with KB-R7943, a selective blocker of reverse mode Na+-Ca2+ exchange. Application of dimethyl amiloride (0.1 mM) in the presence of Ca2+ did not significantly change [Mg2+]i, although it inhibited Na+-H+ exchange at the same concentration. Removal of extracellular Na+ caused a marginal increase in [Mg2+]i after 100-200 min, as seen in intestinal smooth muscle in which Na+-Mg2+ exchange is known to be the primary mechanism of maintaining a low [Mg2+]i against electrochemical equilibrium. In Na+-free solution (containing Ca2+), neither amiloride nor KB-R7943 decreased [Mg2+]i, but they rather increased it. The results suggest that these inhibitory drugs for Na+-Ca2+ exchange directly modulate Na+-Mg2+ exchange in a Ca2+-dependent manner, and consequently produce the paradoxical decrease in [Mg2+]i in the presence of Ca2+.