The LINC Complex Assists the Nuclear Import of Mechanosensitive Transcriptional Regulators.

Mechanical forces play pivotal roles in directing cell functions and fate. To elicit gene expression, either intrinsic or extrinsic mechanical information are transmitted into the nucleus beyond the nuclear envelope via at least two distinct pathways, possibly more. The first and well-known pathway utilizes the canonical nuclear transport of mechanoresponsive transcriptional regulators through the nuclear pore complex, which is an exclusive route for macromolecular trafficking between the cytoplasm and nucleoplasm. The second pathway depends on the linker of the nucleoskeleton and cytoskeleton (LINC) complex, which is a molecular bridge traversing the nuclear envelope between the cytoskeleton and nucleoskeleton. This protein complex is a central component in mechanotransduction at the nuclear envelope that transmits mechanical information from the cytoskeleton into the nucleus to influence the nuclear structure, nuclear stiffness, chromatin organization, and gene expression. Besides the mechanical force transducing function, recent increasing evidence shows that the LINC complex plays a role in controlling nucleocytoplasmic transport of mechanoresponsive transcriptional regulators. Here we discuss recent findings regarding the contribution of the LINC complex to the regulation of intracellular localization of the most-notable mechanosensitive transcriptional regulators, ß-catenin, YAP, and TAZ.

Inner Nuclear Membrane Protein, SUN1, is Required for Cytoskeletal Force Generation and Focal Adhesion Maturation.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is composed of the inner nuclear membrane-spanning SUN proteins and the outer nuclear membrane-spanning nesprin proteins. The LINC complex physically connects the nucleus and plasma membrane via the actin cytoskeleton to perform diverse functions including mechanotransduction from the extracellular environment to the nucleus. Mammalian somatic cells express two principal SUN proteins, namely SUN1 and SUN2. We have previously reported that SUN1, but not SUN2, is essential for directional cell migration; however, the underlying mechanism remains elusive. Because the balance between adhesive force and traction force is critical for cell migration, in the present study, we focused on focal adhesions (FAs) and the actin cytoskeleton. We observed that siRNA-mediated SUN1 depletion did not affect the recruitment of integrin ß1, one of the ubiquitously expressed focal adhesion molecules, to the plasma membrane. Consistently, SUN1-depleted cells normally adhered to extracellular matrix proteins, including collagen, fibronectin, laminin, and vitronectin. In contrast, SUN1 depletion reduced the activation of integrin ß1. Strikingly, the depletion of SUN1 interfered with the incorporation of vinculin into the focal adhesions, whereas no significant differences in the expression of vinculin were observed between wild-type and SUN1-depleted cells. In addition, SUN1 depletion suppressed the recruitment of zyxin to nascent focal adhesions. These data indicate that SUN1 is involved in the maturation of focal adhesions. Moreover, disruption of the SUN1-containing LINC complex abrogates the actin cytoskeleton and generation of intracellular traction force, despite the presence of SUN2. Thus, a physical link between the nucleus and cytoskeleton through SUN1 is required for the proper organization of actin, thereby suppressing the incorporation of vinculin and zyxin into focal adhesions and the activation of integrin ß1, both of which are dependent on traction force. This study provides insights into a previously unappreciated signaling pathway from the nucleus to the cytoskeleton, which is in the opposite direction to the well-known mechanotransduction pathways from the extracellular matrix to the nucleus.

Blonanserin treatment in patients with methamphetamine-induced psychosis comorbid with intellectual disabilities.

OBJECTIVE: Methamphetamine (MA) use has recently been associated with high levels of psychiatric hospitalization and serious social dysfunction. MA use causes frequent psychotic symptoms, which can be treated with antipsychotics. However, people with intellectual disabilities (ID) are vulnerable to adverse effects resulting from treatment with antipsychotic medications. METHOD: We report two cases of MA-induced psychosis (MAP) in patients with ID who were treated with the antipsychotic blonanserin. RESULTS: In both the cases presented, symptoms of psychosis were improved by switching medications from other antipsychotic drugs to blonanserin. Despite the presence of ID in these patients, no significant adverse effects, such as sedation, were detected after treatment with blonanserin. CONCLUSION: Blonanserin may be an effective and well-tolerated pharmacotherapeutical treatment for patients with MAP comorbid with ID. However, further work is necessary to validate this claim.

Measurement of Postmortem 1,5-anhydroglucitol in Vitreous Humor for Forensic Diagnosis.

In forensic diagnosis, postmortem blood glucose is known to be susceptible to change after death. However, the 1,5-anhydroglucitol (1,5-AG) concentrations in plasma and cerebrospinal fluid (CSF) reflect the mean blood glucose level for a short period of time. In this study, we compared the postmortem 1,5-AG concentrations in vitreous humor and CSF in 47 subjects to evaluate the utility of this concentration in the vitreous humor for forensic diagnosis. The postmortem 1,5-AG concentrations in vitreous humor (mean±SD: 20.2 ± 8.7 µg/mL) and CSF (16.8 ± 8.7 µg/mL) did not differ significantly and showed a strong correlation (r(2) = 0.87, p < 0.01). These results suggest that the vitreous humor 1,5-AG concentration provides useful information on the antemortem blood glucose level, in addition to the HbA1c value and the CSF 1,5-AG concentration.

Differential patterns of blood oxygenation in the prefrontal cortex between patients with methamphetamine-induced psychosis and schizophrenia.

Despite some slight differences in symptomatology, differential diagnosis of methamphetamine-induced psychosis (MAP) versus schizophrenia can be challenging because both disorders present a large overlap in their clinical symptoms. However, a recent study has shown that near-infrared spectroscopy (NIRS) performed during a cognitive task can be a powerful tool to differentiate between these two disorders. Here, we evaluated verbal fluency task performance during NIRS in 15 patients diagnosed with MAP and 19 with schizophrenia matched for age and sex. We used prefrontal probes and a 24-channel NIRS machine to measure the relative concentrations of oxyhaemoglobin every 0.1 s during the task. For each patient, the neurocognitive function and clinical psychopathology were evaluated using the Positive and Negative Symptom Scale (PANSS), and the Brief Assessment of Cognition in Schizophrenia (BACS). Oxyhaemoglobin changes in the prefrontal cortex were significantly higher in the MAP group compared to those in the schizophrenia group, particularly in the right dorsolateral prefrontal cortex. In contrast, we found no significant difference in PANSS and BACS scores. Our findings suggest that NIRS measurement could be applied to differentiate patients with MAP from those with schizophrenia, even in cases where clinical symptoms are similar.

Raspberry ketone promotes the differentiation of C3H10T1/2 stem cells into osteoblasts.

The decrease in the bone mass associated with osteoporosis caused by ovariectomy, aging, and other conditions is accompanied by an increase in bone marrow adipose tissue. The balance between osteoblasts and adipocytes is influenced by a reciprocal relationship. The development of modalities to promote local/systemic bone formation by inhibiting bone marrow adipose tissue is important in the treatment of fractures or metabolic bone diseases such as osteoporosis. In this study, we examined whether raspberry ketone [4-(4-hydroxyphenyl)butan-2-one; RK], which is one of the major aromatic compounds of red raspberry and exhibits anti-obesity action, could promote osteoblast differentiation in C3H10T1/2 stem cells. Confluent C3H10T1/2 stem cells were treated for 6 days with 10-100 µg/mL of RK in culture medium containing 10 nM all-trans-retinoic acid (ATRA) or 300 ng/mL recombinant human bone morphogenetic protein (rhBMP)-2 protein as an osteoblast-differentiating agent. RK in the presence of ATRA increased alkaline phosphatase (ALP) activity in a dose-dependent manner. RK in the presence of rhBMP-2 also increased ALP activity. RK in the presence of ATRA also increased the levels of mRNAs of osteocalcin, α1(I) collagen, and TGF-ßs (TGF-ß1, TGF-ß2, and TGF-ß3) compared with ATRA only. RK promoted the differentiation of C3H10T1/2 stem cells into osteoblasts. However, RK did not affect the inhibition of early-stage adipocyte differentiation. Our results suggest that RK enhances the differentiation of C3H10T1/2 stem cells into osteoblasts, and it may promote bone formation by an action unrelated to adipocyte differentiation.

Prolonged exposure to a low-dose of bisphenol A increases spontaneous motor activity in adult male rats.

We investigated the effects of bisphenol A (BPA), an environmental endocrine-disrupting chemical, on spontaneous motor activity in adult male rats. The rats were implanted intraperitoneally with mini-osmotic pumps containing either BPA (50 µg/kg body weight per day) in sesame oil (BPA-treated group) or sesame oil only (vehicle-treated group). Spontaneous motor activity during a 24-h period was measured over 5 days from day 9 to day 13 after implantation using an animal movement analysis system. Spontaneous motor activity during the last 2 h of the dark phase and during the first 1-h of the light phase was increased in the BPA-treated group. Total spontaneous motor activity during the 12-h light phase, but not the 12-h dark phase, was higher in the BPA-treated group than in the vehicle-treated group. These findings suggest that BPA may induce hyperactivity in adult male rats during the 12-h light phase, especially during the 2 h immediately preceding sleep-onset and 1 h immediately following sleep-onset.

Genistein inhibits cell invasion and motility by inducing cell differentiation in murine osteosarcoma cell line LM8.

BACKGROUND: One of the problems associated with osteosarcoma is the frequent formation of micrometastases in the lung prior to diagnosis because the development of metastatic lesions often causes a fatal outcome. Therefore, the prevention of pulmonary metastases during the early stage of tumor development is critical for the improvement of the prognosis of osteosarcoma patients. In Japan, soy is consumed in a wide variety of forms, such as miso soup and soy sauce. The purpose of this study is to investigate the effect of genistein, an isoflavone found in soy, on the invasive and motile potential of osteosarcoma cells. METHODS: LM8 cells were treated for 3 days with various concentrations of genistein. The effect of genistein on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2'-deoxyuridine (BrdU) incorporation study. The assays of cell invasion and motility were performed using the cell culture inserts with either matrigel-coated membranes or uncoated membranes in the invasion chambers. The expression and secretion of MMP-2 were determined by immunohistochemistry and gelatin zymography. The subcellular localization and cellular level of ß-catenin were determined by immunofluorescence and Western blot. For examining cell morphology, the ethanol-fixed cells were stained with hematoxylin-eosin (H&E). The expression of osteocalcin mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Genistein dose-dependently inhibits cell proliferation. Genistein-treated cells were less invasive and less motile than untreated cells. The expression and secretion of MMP-2 were lower in the genistein-treated cultures than in the untreated cultures. ß-Catenin in untreated cells was located in the cytoplasm and/or nucleus, while in genistein-treated cells it was translocated near to the plasma membrane. The level of ß-catenin was higher in genistein-treated cells than in untreated cells. Treatment of LM8 cells with genistein induced morphological changes, markedly decreased the formation of multilayer masses of cells, and markedly increased the expression of osteocalcin mRNA. CONCLUSIONS: Genistein decreased invasive and motile potential by inducing cell differentiation in LM8 cells. Genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects.

Identification of human brain from a tissue fragment by detection of neurofilament proteins.

We developed a method for identifying human brain from a tissue-like fragment by detection of neurofilament protein (NF) using enzyme-linked immunosorbent assay (ELISA). NF was extracted from 0.1 g of organ/tissue homogenized with Tris-HCl buffer (pH 7.2) containing urea, phenylmethylsulfonyl fluoride (PMSF), EDTA and, EGTA. It was necessary to dilute the extract at more than 2(3)-fold to avoid immunosuppression by urea. Positive reaction was always obtained for NF-H in 2(3)-fold diluted extract of brain tissue, however, NF-L and NF-M were not always detected when a brain fragment contained gray matter. Human cerebral white matter could be easily distinguished from other organs/tissues by detecting any of the NF-subunits. Brains of human and some animals could be discriminated by detecting NF-L or NF-M, although the species specificity of NF-H was not good. Our findings suggested that detection of NF-H was more useful than NF-L and NF-M for identifying a brain from a tissue-like fragment. The present ELISA method for NF-H could identify human brain specimens under the following conditions: putrefied at 4 degrees C for up to 3 weeks, dried at 37 degrees C for at least 4 months, heated at 50 degrees C for at least 4 weeks. Our results showed that our method is useful for identification of brain tissue in forensic stain analysis. Two practical cases are described.

A new 39-plex analysis method for SNPs including 15 blood group loci.

A novel 39-plex typing system for single nucleotide polymorphisms (SNPs) has been developed. This multiplex approach has the advantage of being able to type 38 autosomal SNPs and one sex-discriminating base exchange site on the X and Y chromosomes rapidly and simultaneously. The SNP loci on the autosomes, which we examined, contain 15 loci distributed on blood type genes: three on RhCE, two each on Km and Gc, and one each on Duffy, AcP1, Tf, MN, GPT, EsD, PI, and Kidd genes. Thirty-seven genomic DNA fragments containing a total of 38 SNPs and one sex-discriminating site were amplified in one multiplex PCR reaction. Following the reaction, single nucleotide primer extension reaction was performed by dividing these SNP loci into five groups. The SNP type of each of the 39 loci was determined at one time by capillary electrophoresis using the newly designed multi-injection method. The combined PD (power of discrimination) of this typing system was (1-1.1) x 10(-14), and the MEC (mean exclusion chance) was 0.9990. We applied this system to forensic cases, including 16 paternity testing cases (13 non-exclusion and three exclusion cases) and one personal identification case. For the paternity testing cases, the highest Essen-Möller's W-value was 0.9999995. The pM (matching probability) of the personal identification case was 2.22 x 10(-17). These data showed that this system was an excellent tool for use in forensic cases of paternity testing and personal identification.

Allele frequencies of single nucleotide polymorphisms in the second exon of the myoglobin gene among the Japanese.

The difference in the allele frequencies of two single nucleotide polymorphisms (SNPs) in the second exon of the myoglobin gene between Japanese and other populations is reported. These SNPs are the substitutions of (A79G) and (T109C), and they were investigated by a single polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis followed by direct sequencing. The substitutions were always linked and two alleles were found in the samples used: the A-T allele with no substitution at positions (79A) and (109T) and the G-C allele with substitutions of (79G) and (109C). The frequencies of these alleles were 0.755 and 0.245, respectively, and they were found to be in Hardy-Weinberg equilibrium. The distribution of alleles in the Japanese population was significantly different from that reported among whites, blacks, and Hispanics (p < 0.0001).

Typing of Y chromosome single nucleotide polymorphisms in a Japanese population by a multiplexed single nucleotide primer extension reaction.

We have developed a new method for typing single nucleotide polymorphisms (SNPs) on the human Y chromosome based on a multiplexed single nucleotide primer extension. This method has the advantage that several SNPs are typed rapidly and simultaneously. We examined 15 different SNP loci on Y chromosome, M9, M105, M122, M125, M128, M130, SRY465, IMS-JST006241, IMS-JST006841, IMS-JST002611, IMS-JST003305, IMS-JST008425, IMS-JST021354, IMS-JST021355 and IMS-JST055457, in 159 Japanese males. From the typing results of these 15 loci, we found 13 haplotypes. Gene diversity for each locus ranged from 0.025 to 0.486 and the haplotype diversity was estimated to be 0.838. This method could be readily applied for personal identification and paternity testing.