Biomedical subjects

Michiko Saito

Publications and source records attributed to Michiko Saito.

9 recordsLinked to original sources

PubMed2006

A diphtheria toxin receptor deficient in epidermal growth factor-like biological activity.

Targeted cell ablation in animals is a powerful method for analyzing the physiological function of cell populations and generating various animal models of organ dysfunction. To achieve more specific and conditional ablation of target cells, we have developed a method termed Toxin Receptor mediated Cell Knockout (TRECK). A potential shortcoming of this method, however, is that overexpression of human heparin-binding epidermal growth factor-like growth factor (hHB-EGF) as a diphtheria toxin (DT) receptor in target cells or tissues may cause abnormalities in transgenic mice, since hHB-EGF is a member of the EGF growth factor family. To create novel DT receptors that are defective in growth factor activity and resistant to metalloprotease-cleavage, we mutated five amino acids in the extracellular EGF-like domain of hHB-EGF, which contains both DT-binding and protease-cleavage sites. Two of the resultant hHB-EGF mutants, I117A/L148V and I117V/L148V, possessed little growth factor activity but retained DT receptor activity. Furthermore, these mutants were resistant to metalloprotease-cleavage by 12-O-tetradecanoylphorbol-13-acetate stimulation, which is expected to enhance DT receptor activity. These novel DT receptors should be useful for the generation of transgenic mice by TRECK.

Amino Acid Sequence↗

PubMed2006

A transcriptional enhancer that directs telencephalon-specific transgene expression in mouse brain.

Telencephalin (TLCN, intercellular adhesion molecule-5 [ICAM-5]) is a cell adhesion molecule belonging to the immunoglobulin superfamily, which plays important roles in dendritic morphogenesis and synaptic plasticity. The expression of TLCN is restricted to neurons in the most rostral brain segment, telencephalon. Here, we examined a transcriptional regulatory mechanism underlying the telencephalon-specific expression of TLCN. TLCN gene is located in the ICAM gene cluster containing ICAM-1, ICAM-4, and TLCN (ICAM-5) within 30 kb on the mouse chromosome 9. The nucleotide sequence of the 5'-flanking region of mouse TLCN gene is highly homologous to that of human and dog orthologs, suggesting the presence of important regulatory elements for its transcription. To determine the telencephalon-specific enhancer region, we generated several lines of transgenic mice that harbor transgenes consisting of different length of the 5'-flanking region of mouse TLCN gene (3.9, 1.5, 1.1, and 0.2 kb) fused to humanized renilla green fluorescent protein cDNA as a fluorescent reporter. Consequently, we identified a crucial region between 1.1 and 0.2 kb upstream of the transcription start site that directs the telencephalon-specific expression. This enhancer was applied to the Cre/loxP-mediated conditional expression system to generate several transgenic lines with different patterns of recombination in the telencephalon and will be further used as a powerful tool for genetic manipulation in the telencephalic neurons.

5' Flanking Region↗

PubMed2005

A novel phenylalanine-based targeting signal directs telencephalin to neuronal dendrites.

Neurons sort out a variety of functional molecules to appropriate subcellular destinations. Telencephalin (TLCN; intercellular adhesion molecule-5) is a cell adhesion molecule specifically localized to somatodendritic membranes in the telencephalic neurons. Here, we established a new in vivo strategy to analyze neuronal sorting mechanisms by ectopic expression of molecules of interest in the cerebellar Purkinje cells of transgenic mice. By using this system, we identified a novel dendritic targeting determinant in the cytoplasmic tail region of TLCN. A full-length TLCN ectopically expressed in the Purkinje cells was localized exclusively to dendrites but not to axons. In contrast, a deletion of cytoplasmic C-terminal 12 amino acids (residues 901-912) or a point mutation of Phe905 to Ala abrogated the dendrite-specific targeting with appearance of the truncated and point-mutated TLCN in both axons and dendrites. Furthermore, an addition of the C-terminal 17 amino acids (residues 896-912) of TLCN to an unrelated molecule (CD8) was sufficient for its specific targeting to dendrites in several types of neurons. Because the C-terminal region of TLCN does not contain any canonical dendritic targeting sequences such as the tyrosine-based motif or the dileucine motif, this study suggests a novel mechanism of protein trafficking to the dendritic compartment of neurons.

Amino Acid Motifs↗

PubMed2005

Location of micropyles and early embryonic development of the two-spotted cricket Gryllus bimaculatus (Insecta, Orthoptera).

Early embryogenesis of the two-spotted cricket Gryllus bimaculatus was examined by scanning electron microscopy and several fluorescence staining methods, with special reference to these four issues: (i) the location of micropyles; (ii) the transfer of the female pronucleus following meiosis; (iii) the timing of cellularization; and (iv) the process of the germ primordium formation. Between two and four micropyles lie in the mid-ventral region of the egg. The egg nucleus is at the mid-dorsal periphery of the new laid egg, and meiosis resumes and is completed there. The female pronucleus moves to the mid-ventral side, and fertilization occurs there. Energid starts to proliferate and migrates to the periphery of the egg, initiating blastoderm formation. Actin caps surround each superficial nucleus. Cellularization occurs during the blastoderm stage. At a late blastoderm stage, nuclei aggregate in both the posterolateral patch-like regions of the egg to form a germ primordium. The germ primordium looks like a pair of dumbbells. Both the patches shift towards the ventral side and fuse into a germ primordium. The germ primordium contracts to produce a clearly delineated germ band. Observations on distribution patterns of F-actin indicate that, all through the process, the germ primordium retains that unity, and is not separated into two parts.

Actin Cytoskeleton↗

PubMed2004

Aberrant transcription of unrearranged T-cell receptor beta gene in mouse brain.

The nervous system and the immune system share several functional molecules involved in various cell-cell interaction events. In this study, we used in situ hybridization to identify immune molecules that are expressed by a restricted population of neurons in the mouse brain and found that mRNA for the beta subunit of T-cell receptor (TCRbeta) was predominantly and strongly localized to neurons in deep layers of the cerebral neocortex and weakly expressed in the thalamus. Developmentally, TCRbeta mRNA expression started at embryonic day 15 in the thalamic nuclei and at postnatal day 1 in the cerebral neocortex. The level of TCRbeta mRNA in the neocortex subsequently increased until postnatal day 21, and it remained high in the adult. Detailed analysis revealed that only the Cbeta2 segment of TCRbeta, not the Cbeta1 or Vbeta segments, was expressed by the brain neurons. By the 5' rapid amplification of cDNA ends method, we determined a brain-specific transcription start site in the Jbeta2 region locus, not in the Vbeta region locus. Furthermore, we confirmed that the aberrant transcription around the Jbeta2 region took place only in neurons and lymphocytes in transgenic mice. These results demonstrate that the transcriptional machinery for unrearranged TCRbeta expression is shared by the nervous and immune systems and raise a possibility of gene rearrangement in neurons under certain circumstances.

Alternative Splicing↗

PubMed2004

Improved parameter estimation for variance-stabilizing transformation of gene-expression microarray data.

A gene-expression microarray datum is modeled as an exponential expression signal (log-normal distribution) and additive noise. Variance-stabilizing transformation based on this model is useful for improving the uniformity of variance, which is often assumed for conventional statistical analysis methods. However, the existing method of estimating transformation parameters may not be perfect because of poor management of outliers. By employing an information normalization technique, we have developed an improved parameter estimation method, which enables statistically more straightforward outlier exclusion and works well even in the case of small sample size. Validation of this method with experimental data has suggested that it is superior to the conventional method.

Algorithms↗

PubMed2003

Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse.

In the mouse olfactory system, each olfactory sensory neuron (OSN) expresses only one odorant receptor (OR) gene in a monoallelic and mutually exclusive manner. Such expression forms the genetic basis for OR-instructed axonal projection of OSNs to the olfactory bulb of the brain during development. Here, we identify an upstream cis-acting DNA region that activates the OR gene cluster in mouse and allows the expression of only one OR gene within the cluster. Deletion of the coding region of the expressed OR gene or a naturally occurring frame-shift mutation allows a second OR gene to be expressed. We propose that stochastic activation of only one OR gene within the cluster and negative feedback regulation by that OR gene product are necessary to ensure the one receptor-one neuron rule.

Alleles↗

PubMed2003

Liver regeneration in heparin-binding EGF-like growth factor transgenic mice after partial hepatectomy.

BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family, is synthesized in the form of a membrane-anchored precursor (proHB-EGF), which subsequently is processed proteolytically to mature HB-EGF. This study describes the effects of HB-EGF on liver regeneration after 70% partial hepatectomy in proHB-EGF transgenic mice with liver-specific expression. METHODS & RESULTS: No significant differences in liver/body weight ratios and in bromodeoxyuridine (BrdU)-labeling index (the ratios of BrdU-positive hepatocyte nuclei) were found between adult transgenic and wild-type mice. However, in regenerating liver after partial hepatectomy, transgenic mice had higher liver/body weight ratios than wild-type mice and at 120 hours reached a level equal to that before partial hepatectomy. The BrdU-labeling index was about 5 times higher in the livers of transgenic mice compared with the wild type (51.5% vs. 10.2%, respectively; P < 0.01) at 48 hours after partial hepatectomy. Activation of microtubule-associated protein kinase after partial hepatectomy was higher and earlier in the transgenic mice as compared with the wild-type mice. Soluble HB-EGF was increased in the liver (at 8 min) after partial hepatectomy, indicating that the shedding of proHB-EGF occurred after partial hepatectomy. CONCLUSIONS: The transgenic expression of HB-EGF accelerates the proliferation of hepatocytes after partial hepatectomy, suggesting that HB-EGF functions as a hepatotrophic factor in vivo.

Animals↗

PubMed2002

[Antibiotic susceptibility and beta-lactamase activity of pathogenic microbes isolated in Miyagi Prefecture between December in 1999 and February in 2000].

We determined beta-lactamase activity and antimicrobial susceptibility of 556 strains consisting of 10 species isolated in four medical institutions and one microbiological laboratory of Miyagi Prefecture in Japan between December in 1999 and February in 2000. beta-Lactamase determined by nitrocefin method was positive in 68% of S. aureus, in 15% of H. influenzae and in 100% of M. catarrhalis. Penicillinase/cephalosporinase determined by acidometry was positive in 9%/10% of E. coli, in 17%/2% of K. pneumoniae, in 16%/58% of E. cloacae, in 43%/78% of S. marcescens, and in 4%/32% of P. aeruginosa, respectively. Of a total of 298 strains of Enterobacteriaceae and P. aeruginosa, 25 strains (14 strains of E. coli, 10 strains of K. pneumoniae and one strain of S. marcescens) produced class A beta-lactamase, two strains of E. cloacae produced class B beta-lactamase, and 12 strains (one strain of E. coli, four strains of E. cloacae, six strains of S. marcescens and one strain of P. aeruginosa) produced class C beta-lactamase. According to NCCLS standard, three strains (one strain of E. coli and two strains of K. pneumoniae) of ESBL-positive microbes were detected. beta-Lactamase-negative ampicillin-resistant (BLNAR) strains of H. influenzae were found in 10/40 (25.0%) of the strains tested.

Enterobacteriaceae↗