Pegfilgrastim for the management of neutropenia during neoadjuvant chemotherapy with docetaxel, cisplatin, and 5-fluorouracil in esophageal cancer patients.

BACKGROUND: Neoadjuvant docetaxel, cisplatin, and 5-fluorouracil (DCF) therapy is a new standard for locally advanced esophageal squamous cell carcinoma. The optimal timing of pegfilgrastim with the DCF regimen to prevent febrile neutropenia (FN) remains controversial. The effectiveness of concomitant pegfilgrastim administration with continuous 5-fluorouracil (5-FU) infusion in the DCF regimen was therefore assessed. METHODS: All patients who received neoadjuvant DCF for esophageal cancer were retrospectively assessed. Patients who had been scheduled to receive pegfilgrastim on days 3-5 (early group) or days 7-9 (regular group) of the DCF regimen were included. Uni- and multivariate analyses were used to assess risk factors for FN. RESULTS: Eighty-eight patients were included in the analysis. The 26 patients in the early group received pegfilgrastim as scheduled. In the 62 patients of the regular group, 51 received pegfilgrastim at a median of 7 days after starting DCF chemotherapy. However, 11 patients in the regular group could not receive pegfilgrastim. Twenty-two patients of the regular group and 2 patients of the early group developed FN after the first session of DCF. Early administration of pegfilgrastim and grade 4 neutropenia were significantly associated with onset of FN, with multivariate analysis identifying early administration of pegfilgrastim as an independent preventive factor and grade 4 neutropenia as a risk factor, after adjusting for sex and age. CONCLUSION: Early pegfilgrastim administration is a safe approach that reduces the incidence of FN in DCF therapy. Using pegfilgrastim with continuous 5-FU infusion in the DCF regimen represents a reasonable option to prevent FN.

The role of brain-localized gamma and alpha oscillations in inattentional deafness: implications for understanding human attention.

Introduction: The processes involved in how the attention system selectively focuses on perceptual and motor aspects related to a specific task, while suppressing features of other tasks and/or objects in the environment, are of considerable interest for cognitive neuroscience. The goal of this experiment was to investigate neural processes involved in selective attention and performance under multi-task situations. Several studies have suggested that attention-related gamma-band activity facilitates processing in task-specific modalities, while alpha-band activity inhibits processing in non-task-related modalities. However, investigations into the phenomenon of inattentional deafness/blindness (inability to observe stimuli in non-dominant task when primary task is demanding) have yet to observe gamma-band activity. Methods: This EEG experiment utilizes an engaging whole-body perceptual motor task while carrying out a secondary auditory detection task to investigate neural correlates of inattentional deafness in natural immersive high workload conditions. Differences between hits and misses on the auditory detection task in the gamma (30-50 Hz) and alpha frequency (8-12 Hz) range were carried out at the cortical source level using LORETA. Results: Participant auditory task performance correlated with an increase in gamma-band activity for hits over misses pre- and post-stimulus in left auditory processing regions. Alpha-band activity was greater for misses relative to hits in right auditory processing regions pre- and post-stimulus onset. These results are consistent with the facilitatory/inhibitory role of gamma/alpha-band activity for neural processing. Additional gamma- and alpha-band activity was found in frontal and parietal brain regions which are thought to reflect various attentional monitoring, selection, and switching processes. Discussion: The results of this study help to elucidate the role of gamma and alpha frequency bands in frontal and modality-specific regions involved with selective attention in multi-task immersive situations.

Characterization of nucleolar localization and exclusion signals in terminal deoxynucleotidyltransferase interacting factor 2/estrogen receptor α-binding protein.

Terminal deoxynucleotidyltransferase interacting factor 2/estrogen receptor α-binding protein (TdIF2/ERBP) is a multifunctional nucleolar protein. The nucleolar localization of TdIF2/ERBP is important for its functions because it promotes ribosomal RNA transcription. However, signal sequences that direct TdIF2/ERBP to the nucleolus are not well characterized. We examined the TdIF2/ERBP sequence using truncation and mutation analyses to determine whether the nucleosome binding and C-terminal domains of TdIF2/ERBP possess nucleolar localization signals (NoLSs). In these domains, four NoLSs that could direct the mCherry protein to the nucleolus were detected. In addition, a short stretch of hydrophobic residues (VLLVL) in the center of TdIF2/ERBP acted as a nucleolar exclusion signal, which reduced the nucleolar accumulation of mCherry-NoLS fusion proteins. These results would contribute to improving the prediction of NoLSs from protein sequences. The short, transferrable localization signals would be valuable tools for understanding the association between localization and functions of nucleolar proteins. Abbreviations TdIF2: terminal deoxynucleotidyltransferase interacting factor 2; ERBP: estrogen receptor α-binding protein; EGFP: enhanced green fluorescent protein; NLS: nuclear localization signal; NoLS: nucleolar localization signal; NoES: nucleolar exclusion signal; DAPI: 4',6-diamidino-2-phenylindole.

Fluid shear stress applied by orbital shaking induces MG-63 osteosarcoma cells to activate ERK in two phases through distinct signaling pathways.

Fluid shear stress (FSS) induces a series of biochemical responses in osteoblasts, and this "mechanoresponse" regulates their survival, proliferation and differentiation. However, the events in cells immediately after FSS application are unclear, and how biochemical signals from soluble factors modify the mechanoresponses is largely unknown. We used the orbital shaking method, instead of the frequently used parallel plate method, to examine activation of ERK and AKT by FSS for detailed tracking of its temporal transition. We found that ERK activation by orbital shaking was biphasic. The early phase was independent of Ca2+, PI3-kinase, and Rho kinase but required RAF activity. The late phase was dependent on Ca2+ but not RAF. These results suggest that the superior time-resolving capability of the orbital shaking method to separate the previously unrecognized Ca2+-independent early phase of ERK activation from the late phase. We also found that a certain combination of serum starvation and medium renewal affected ERK activation by FSS, suggesting that a soluble factor(s) may be secreted during serum starvation, which modified the phosphorylation level of ERK. These findings revealed novel aspects of the osteoblastic mechanoresponses and indicated that the orbital shaking method would be a useful, complementary alternative to the parallel plate method for certain types of study on cellular mechanoresponses.

Live-cell imaging of HP1α throughout the cell cycle of mouse C3H10T1/2 cells and rhythmical flickering of heterochromatin dots in interphase.

Heterochromatin protein 1 alpha (HP1α) localizes to heterochromatin in interphase and shows dynamic molecular behavior in living cells. We previously reported that during mitosis, the majority of HP1α diffused into the cytoplasm but some remained in centromere heterochromatin. Here, we further characterize the molecular behavior of HP1α throughout the cell cycle. Time-lapse imaging of DsRed-HP1α through two successive cell divisions indicated that interphase can be divided into four phases. HP1α forms heterochromatin dots in early G1, which are maintained without any apparent changes (Phase 1). However, the HP1α dots begin to diffuse into the nucleoplasm and start flickering with a rhythmical cycle (Phase 2). Then, the HP1α dots diffuse further towards the periphery of the nucleus (Phase 3), and uniformly diffuse throughout the entire nucleus (Phase 4). Rhythmical flickering of HP1α dots in the middle of interphase may be useful for following cell cycle progression in mouse living cells.

Identification of novel nuclear protein interactions with the N-terminal part of filamin A.

Since certain missense mutations in the N-terminal part of filamin A (FLNA) cause inherited skeletal malformation, we screened for proteins that bind to this part of FLNA. We identified two nuclear proteins that are specifically associated with the N-terminal region of FLNA. This suggests more extensive nuclear function of filamin than expected.

Differential responses of mitotic spindle pole formation to microtubule-stabilizing agents epothilones A and B at low concentrations.

We previously reported that the microtubule-stabilizing agent docetaxel induced formation of fragile acentrosomal spindle poles but that structurally related paclitaxel did not. In the present study, we examined whether the microtubule-stabilizing agents epothilones A and B, which are structurally similar, affect the centrosome/spindle pole architecture.We investigated mitotic processes in epothilone A or B-treated human MDA-MB-435 cells, in which the centrosomes, spindle poles and mitotic micro-tubules were simultaneously visualized by GFP-Aurora A kinase. Fluorescence microscopy of metaphase cells indicated that several chromosomes were misaligned away from the metaphase plate when treated with IC(50) concentrations of epothilone A or B (4.5 or 2 nM, respectively), suggesting that microtubule dynamics was impaired. Interestingly, epothilone B induced formation of acentro-somal spindle poles, but this effect was not observed for epothilone A. Live-cell imaging showed that aster-like structures ectopically arose around the nuclear envelope at the onset of mitosis in epothilone B-treated cells and that one of these asters became an acentrosomal spindle pole. Aster-like structures also arose in the presence of epothilone A, but they were merged into centro-some-derived spindle poles during prometaphase and completely disappeared until metaphase. These results indicate that the centro-some/spindle pole integrity is strongly affected by epothilone B but is not greatly affected by epothilone A. Our findings show that the two epothilones cause different cellular responses at equipotent concentrations and suggest that they have different mechanisms of activity in cells.

A multi-fluorescent MDA435 cell line for mitosis inhibitor studies: simultaneous visualization of chromatin, microtubules, and nuclear envelope in living cells.

As a part of our efforts to create a pre-made multi-fluorescent cell library for various cytological assays, we made a triple-fluorescent cell line in which microtubules, chromosomes, and nuclear envelopes were visualized for simultaneous observation of spindle structure and chromosome distribution in living cells. Pilot experiments with microtubule-disturbing drugs showed the advantages of this cell line in mitosis inhibitor studies.

Dynamic behavior of FCHO1 revealed by live-cell imaging microscopy: its possible involvement in clathrin-coated vesicle formation.

The intracellular behavior of human FCHO1 protein was investigated by live-cell imaging microscopy. The fluorescence intensity of green fluorescent protein (GFP)-FCHO1 fluctuated periodically in a perinuclear region approximately every 100 s, reminding us of the periodic fluctuations of clathrin reported in our recent work. The periodicity of FCHO1 was temporally correlated with that of clathrin, suggesting that FCHO1 is involved in clathrin-coated vesicle formation.

Live imaging of spindle pole disorganization in docetaxel-treated multicolor cells.

Treatment of cells with docetaxel at low concentrations induces aberrant bipolar spindles of which two centrosomes stay at only one pole, and also induces multipolar spindles. To gain insight into the relations between centrosome impairment and structural defects of the spindle, live-cell imaging was performed on a human MDA Auro/imp/H3 cell line in which centrosomes/mitotic spindles, nuclear membrane and chromatin were simultaneously visualized by fluorescent proteins. In the presence of docetaxel at IC(50) concentration, the centrosomes did not segregate, and multiple aster-like structures ectopically arose around the disappearing nuclear membrane. Those ectopic structures formed an acentrosomal pole opposing to the two-centrosomes-containing pole. In late metaphase, one pole often fragmented into multiple spindle poles, leading multipolar division. These results suggest that spindle pole fragility may be induced by centrosome impairment, and collapse of the pole may contribute to induction of aneuploid daughter cells.

Rhythmic cycle of clathrin-coated pit formation at the trans-golgi network in human MDA-MB-435 cells.

We studied the in vivo dynamics of enhanced green fluorescent protein-tagged clathrin light chain a (GFP-CLCa) at the trans-Golgi network (TGN) in MDA-MB-435 cells. The intensity of fluorescence signals of GFP-CLCa periodically increased and decreased at the TGN approximately every 100 s. This suggests that the formation of clathrin-coated pits occurs synchronously and periodically at the TGN.

Genomic structure and 5'-flanking sequences of rat N-acetylglucosaminyltransferase I gene and regulatory role of its transcriptional diversity.

It has been known that mouse, rat, and human N-acetylglucosaminyltransferase I (GnT-I) genes produce at least two transcripts, which differ in their 5'-untranslated region (5'-UTR) length, and the longer transcript is preferentially expressed in brains. However, the physiological meaning of this brain-specific expression pattern was unknown. We cloned the rat GnT-I gene and analyzed its structure. It consisted of five exons, and four of them coded only 5'-UTRs. A putative NF-kappaB binding site was found in the 5'-flanking sequence for the transcript that was previously shown to be induced by inflammation. The unusually long 5'-UTR of the major GnT-I transcript in rat brain was shown to inhibit protein production from the following coding sequence in COS7 cells. Comparison of the GnT-I protein/mRNA ratio in rat brain and liver showed that GnT-I mRNA in the brain was translated 3.8-times less efficiently than in the liver. Implications are discussed of these results in regulation of GnT-I expression in rat brain.

Cytotoxic activity of Bacillus thuringiensis Cry proteins on mammalian cells transfected with cadherin-like Cry receptor gene of Bombyx mori (silkworm).

Cry1Aa, an insecticidal protein produced by Bacillus thuringiensis, has been shown to bind to cadherin-like protein, BtR175, in Bombyx mori (silkworm) midgut. We previously reported three variant alleles of BtR175 (BtR175a, b and c). When transiently expressed in COS7 cells, all the three BtR175 variants bound to Cry1Aa. We stably expressed BtR175b in HEK293 cells. These BtR175b-expressing cells swelled and died in the presence of activated Cry1Aa in a dose- and time-dependent manner, showing that BtR175b itself can impart Cry1Aa-susceptibility to mammalian cells. These cells were more susceptible to Cry1Aa than to Cry1Ab and Cry1Ac. Since dispersed B. mori midgut cells were reported to be highly susceptible to Cry1Ac, this result suggested that other Cry1Ac-specific receptor(s) were simultaneously working with BtR175 in the midgut cells. Advantages are also discussed of applying these transfected mammalian cells to toxicity assays of mutant Cry proteins.