Spatial summation of thermal sensitivity is limited to small areas: Comparisons of the forehead, forearm, abdomen, and foot.

The purpose of the present study was to examine if spatial summation in thermal sensitivity exists when stimulating areas larger than about 1% of body surface area (BSA) (approximately 200 cm2). We hypothesized that spatial summation would exist within a limited area and the effect would be insignificant for over the 1%BSA. Fifteen young males participated in this study and we measured their warmth and hot sensation thresholds on the four body regions (the forehead, forearm, abdomen, and instep) using the three sizes of radiant film heaters (10 × 10, 15 × 15, and 20 × 20 cm2 heating film area). The heating panel was kept at a distance of 10 cm from the skin and the surface temperature of the heating panel increased by 1 °C·s-1. The results showed that warmth and hot sensation thresholds were higher for the 100 cm2 condition than the 225 or 400 cm2 conditions (P < 0.05), but no differences were found between the 225 and 400 cm2 conditions. Secondly, the instep was most insensitive to the gradual increase of radiant heat among the four body regions for all three stimulating film sizes, even though the hot threshold was lowest for the instep because the initial foot temperature was lower than other skin temperatures. In summary, spatial summation in thermal sensitivity was found for the 100 and 225 cm 2 conditions, but not for the 225 and 400 cm2 conditions. These results suggest that spatial summation exists but limited to small stimulating areas, smaller than approximately 1% BSA.

Comparison of anthracyclines used for induction chemotherapy in patients with FLT3-ITD-mutated acute myeloid leukemia.

This retrospective analysis compared anthracyclines (as part of an induction regimen) in 128 newly diagnosed FLT3-ITD-mutated AML patients. Induction regimens comprised high-dose daunorubicin (HD-DN; 90 mg/m2/d × 3d; n = 44), standard-dose daunorubicin (SD-DN; 45 mg/m2/d × 3d; n = 51), or idarubicin (IDA; 12 mg/m2/d × 3d; n = 33) in combination with cytarabine (100-200 mg/m2/d × 7d). Fifty-three patients showing persistent leukemia on interim bone marrow examination received a second course of induction chemotherapy comprising 2 days of daunorubicin (45 mg/m2/d) or IDA (8 or 12 mg/m2/d) in addition to 5 days of cytarabine. Complete remission (CR) rates were 77.3%, 56.9%, and 69.7% for HD-DN, SD-DN, and IDA, respectively (P = 0.101; HD-DN vs. SD-DN, P = 0.036; HD-DN vs. IDA, P = 0.453; IDA vs. SD-DN, P = 0.237). The HD-DN showed higher overall survival (OS) and event-free survival (EFS) than SD-DN and IDA: the differences between HD-DN and SD-DN (P = 0.009 for OS and P = 0.010 for EFS) were statistically significant. Results of in vitro studies using FLT3-ITD-mutated cell lines supported these findings. In conclusion, HD-DN improved the CR rate, OS, and EFS of FLT3-ITD-mutated AML patients. HD-DN also tended to yield better outcomes than IDA, though the difference was not significant. The superiority of HD-DN over IDA should be confirmed in future studies.

Induction of immunoglobulin transcription factor 2 and resistance to MEK inhibitor in melanoma cells.

Primary or acquired resistance to MEK inhibitors has been a barrier to successful treatment with MEK inhibitors in many tumors. In this study, we analyzed genome-wide gene expression profiling data from 6 sensitive and 6 resistant cell lines to identify candidate genes whose expression changes are associated with responses to a MEK inhibitor, selumetinib (AZD6244). Of 62 identified differentially expressed genes, we selected Immunoglobulin Transcription Factor 2, also known as transcription factor 4 as a potential drug resistance marker for further analysis. This was because the ITF-2 expression increase in resistant cell lines was relatively high and a previous study has suggested that ITF-2 functions as an oncogene in human colon cancers. We also established an AZD6244 resistant cell line (M14/AZD-3) from an AZD6244 sensitive M14 cell line. The expression of the ITF-2 was elevated both in primary AZD6244 resistant cell line, LOX-IMVI and acquired resistant cell line, M14/AZD-3. Targeted silencing of ITF-2 by siRNA significantly enhanced susceptibility to AZD6244 in resistant cells. Wnt/ß-catenin pathway was activated through direct interaction of p-ERK and GSK3ß. Our results suggest that up-regulation of the ITF-2 gene expression is associated with cellular resistance to MEK inhibitors, and activation of Wnt signaling pathway through interaction of p-ERK and GSK3ß seems to be a mechanism for increase of ITF-2.

Establishment and characterization of hypomethylating agent-resistant cell lines, MOLM/AZA-1 and MOLM/DEC-5.

Two hypomethylating agents (HMAs), azacitidine and decitabine, have demonstrated clinical activities in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML); however, potential problems include development of acquired resistance. HMA-resistant patients have very poor prognosis and this cohort of patients constitutes an important area of research. To understand the mechanisms underlying HMA-resistance and to overcome it, we established an azacitidine-resistant cell line, MOLM/AZA-1 and a decitabine-resistant cell line, MOLM/DEC-5 using MOLM-13. For cytogenetic characterization, we performed microarray-based comparative genomic hybridization (array-CGH), which identified a total of 15 copy number alterations (CNAs). Among these CNAs, eight regions in HMA-resistant cell lines showed CNA patterns distinct from the parental MOLM-13 genome. Single nucleotide polymorphism (SNP) microarray was also performed to obtain a more reliable interpretation of the identified CNAs, and all HMA-resistance-specific CNAs except one detected by array-CGH were successfully validated. In addition to CNAs, copy neutral loss of heterozygosity and mosaic loss events were identified in HMA-resistant cell lines. In our resistant cell lines, MDR-1 was not overexpressed, while DNMT3b was upregulated. Azacitidine and decitabine did not inhibit DNMT1, DNMT3a, or DNMT3b in both HMA-resistant cell lines, while they inhibited the enzymes in parental MOLM-13. We also developed mouse xenograft models using MOLM/AZA-1 and MOLM/DEC-5. Our in vitro and in vivo models of HMA-resistant cell lines will provide clues for the elucidation of molecular mechanisms related to the development of resistance to HMA and tools for the application of novel therapeutics for AML and MDS.

PoCRIP1, Paralichthys olivaceus cysteine-rich intestinal protein 1: molecular characterization, expression analysis upon Edwardsiella tarda challenge and a possible role in the immune regulation.

Cysteine-rich intestinal protein (CRIP) is a LIM domain protein containing a zinc-finger motif and plays a role in the regulation of the inflammatory immune response. In the present study, we isolated a CRIP1 cDNA, designated PoCRIP1, from an olive flounder Paralichthys olivaceus intestine cDNA library by EST analysis. The PoCRIP cDNA consists of 421 bp with a polyadenylation signal sequence, AATAAA, and a poly(A) tail; it encodes a polypeptide of 76 amino acids containing a double zinc-finger motif (Cys(2)HisCys and Cys(4) sequences). The deduced amino acid sequence of PoCRIP1 showed 75.3-94.7% homology with CRIP1s of other species, including mammals. The PoCRIP1 transcript was highly expressed in the intestine and pyloric ceca and moderately expressed in the gill, heart, kidney, liver, muscle, spleen, skin, and stomach of normal conditioned flounder. Inducible expression of the PoCRIP1 transcript was observed in flounder challenged with Edwardsiella tarda, an economically important pathogen for aquaculture of flounder. Over-expression of PoCRIP1 augmented p65-driven flounder IL-6 promoter activity in HINAE cells. These results suggest that PoCRIP1 may function in the immune response of the flounder through the regulation of cytokine expression.

Cloning and functional characterization of the p65 subunit of NF-κB from olive flounder (Paralichthys olivaceus).

NF-κB is a master transcription factor found in almost all cell types that responds to diverse cellular stimuli by activating the expression of stress response genes, including immune-related genes. cDNA encoding the p65 subunit of olive flounder (Paralichthys olivaceus) NF-κB (Po-p65) was isolated through an EST analysis of an olive flounder cDNA library, a screen of BAC library, and rapid amplification of cDNA ends (RACE). The cDNA for Po-p65 encodes a polypeptide 626 amino acids in length containing a well-conserved Rel-homology domain (RHD). The primary sequence of Po-p65 showed strong homology with p65 from perch and zebrafish (82.7 and 64.4%, respectively), and shared 43.4-42.1% homology with p65 from other species, including mammals, while the N-terminal RHD of Po-p65 showed strong identity (95.6-67.8%) with that of other species. Po-p65 mRNA expression was detected in all flounder tissues examined. The over-expression of full-length Po-p65 (Po-p65f), but not of a Po-p65 C-terminus deletion mutant (Po-p65ΔC), stimulated κB element-driven reporter (κB-luc) activity in a dose-dependent manner and regulated the expression of p65 target genes, including TNF-α and IκB-α, in HINAE olive flounder cells. Po-p65f translocated to the nucleus following stimulation with poly I:C in HINAE cells. Together, these results suggest that Po-p65 is evolutionarily and functionally conserved in flounder and mammals and may provide clues to the detailed molecular mechanism(s) underlying immune response regulation in flounder.

Hypoxia induces the PDZ domain-containing syntenin in the marine teleost Paralichthys olivaceus.

Syntenin is a scaffolding PDZ domain-containing protein with diverse biological activities, including organization of protein complexes in the plasma membrane, regulation of B-cell development, intracellular trafficking, synaptic transmission, and cancer metastasis. In the present study, we isolated and characterized the cDNA of the olive flounder Paralichthys olivaceus syntenin, designated PoSyntenin. The full-length CDS of PoSyntenin with 5'- and 3'-UTR sequences is 2618bp long and consists of a 909bp open reading frame preceded by a 161bp 5'-UTR and followed by a 1551bp 3'-UTR. The PoSyntenin cDNA encodes a polypeptide of 302 amino acids containing two PDZ domains, which shares 61-80% homology with those of other species, including humans. Expression of the PoSyntenin mRNA was detectable from 1day post-hatching and constitutively in the brain, spleen, intestine, stomach, eye, liver, kidney, and gill of normal conditioned fish. Expression of the PoSyntenin mRNA was upregulated in the eye, liver, kidney, spleen, brain, gill, and intestine of flounder under hypoxia and was increased by treatment with the hypoxia-mimic CoCl(2) (a HIF-1 inducer) in HINAE cells. Taken together, these results suggest that PoSyntenin is a hypoxia target gene that has a potential role in the hypoxia response mechanism of fish.