Autocrine CCL3 and CCL4 Induced by the Oncoprotein LMP1 Promote Epstein-Barr Virus-Triggered B Cell Proliferation

Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.     

Revision of the genus Thermistis Pascoe 1867, with descriptions of three new species (Coleoptera: Cerambycidae: Lamiinae: Saperdini)

The genus Thermistis Pascoe 1867 is revised. T. croceocincta conjunctesignata Rondon & Breuning 1971 is upgraded to a species and newly recorded from China and Myanmar. T. xanthomelas Holzschuh 2007 is newly recorded from Vietnam, Laos and Myanmar. T. sulphureonotata Pu 1984 is newly recorded from Vietnam and Laos. Three new species are described from China: T. hainanensis Lin & Yang n. sp. from Hainan Island, T. kaiyuni Chou & Kurihara n. sp. from Taiwan Island and T. cheni Lin & Chou n. sp. from Yunnan and Sichuan provinces. Photographs of habitus and terminalia and a key to the eleven valid species of Thermistis are presented.     

Diatoms, silicic acid and biogenic silica dynamics along the salinity gradient of the Scheldt estuary (Belgium/The Netherlands)

The Scheldt estuary (Belgium/The Netherlands) was sampled along the entire salinity gradient from 2003 to 2005 for silicic acid (DSi), biogenic silica (BSi), suspended particulate matter (SPM) and pigments. Net DSi consumption and/or release within the estuary were investigated by comparing measured DSi concentrations with (fully-transient) model simulations of the concentrations that would have been obtained in case of conservative transport. The DSi consumption was at maximum in May due to diatoms of presumably marine origin blooming in the lower estuary. DSi consumption decreased rapidly in July, probably because of the grazing pressure of copepods also of marine origin, and DSi was released from late summer onwards. Multiple regression analyses showed that most of the BSi did not follow the dynamics of the living diatoms but rather that of the SPM. They also suggested that diatoms were more silicified in the upper estuary than in the lower estuary. Phytoliths were not expected to contribute significantly to the BSi pool. As BSi dynamics strongly differed from those of diatoms and DSi, this study highlighted the importance of taking BSi into account when investigating estuarine silica dynamics. This study also revealed the fundamental role of the coupling between the biogeochemical and ecological functioning of the lower estuary and that of the adjacent coastal zone. This contrasts with the classical consideration that estuaries act as one-way filters for dissolved and particulate material of riverine origin.     

Discovery of 3-phenyl-1H-5-pyrazolylamine derivatives containing a urea pharmacophore as potent and efficacious inhibitors of FMS-like tyrosine kinase-3 (FLT3)

Preclinical investigations and early clinical trials suggest that FLT3 inhibitors are a viable therapy for acute myeloid leukemia. However, early clinical data have been underwhelming due to incomplete inhibition of FLT3. We have developed 3-phenyl-1H-5-pyrazolylamine as an efficient template for kinase inhibitors. Structure–activity relationships led to the discovery of sulfonamide, carbamate and urea series of FLT3 inhibitors. Previous studies showed that the sulfonamide 4 and carbamate 5 series were potent and selective FLT3 inhibitors with good in vivo efficacy. Herein, we describe the urea series, which we found to be potent inhibitors of FLT3 and VEGFR2. Some inhibited growth of FLT3-mutated MOLM-13 cells more strongly than the FLT3 inhibitors sorafenib (2) and ABT-869 (3). In preliminary in vivo toxicity studies of the four most active compounds, 10f was found to be the least toxic. A further in vivo efficacy study demonstrated that 10f achieved complete tumor regression in a higher proportion of MOLM-13 xenograft mice than 4 and 5 (70% vs 10% and 40%). These results show that compound 10f possesses improved pharmacologic and selectivity profiles and could be more effective than previously disclosed FLT3 inhibitors in the treatment of acute myeloid leukemia.     

Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model.     

Cardiometabolic risks during anabolic hormone supplementation in older men

Objective:

To determine the cardiometabolic risks of testosterone and growth hormone (GH) replacement therapy to youthful levels during aging.

Design and Methods:

A double‐masked, partially placebo controlled study in 112 men 65‐90 years‐old was conducted. Transdermal testosterone (5 g vs. 10 g/day) using a Leydig Cell Clamp and subcutaneous recombinant GH (rhGH) (0 vs. 3 vs. 5 μg/kg/day) were administered for 16‐weeks. Measurements included testosterone and IGF‐1 levels, body composition by DEXA, and cardiometabolic risk factors (upper body fat, blood pressure, insulin sensitivity, fasting triglycerides, HDL‐cholesterol, and serum adiponectin) at baseline and after 16 weeks of treatment.

Results:

Some cardiometabolic factors improved (total and trunk fat, triglycerides, HDL‐cholesterol) and others worsened (systolic blood pressure, insulin sensitivity index [QUICKI], adiponectin). Cardiometabolic risk composite scores (CRCSs) improved (?0.69 ± 1.55, P < 0.001). In multivariate analyses, QUICKI, triglycerides, and HDL‐cholesterol contributed 33%, 16%, and 14% of the variance in CRCS, respectively. Pathway analyses indicated that changes in fat and lean mass were related to individual cardiometabolic variables and CRCS in a complex manner. Changes in BMI, reflecting composite effects of changes in fat and lean mass, were more robustly associated with cardiometabolic risks than changes in fat mass or LBM individually.

Conclusions:

Testosterone and rhGH administration was associated with diverse changes in individual cardiometabolic risk factors, but in aggregate appeared not to worsen cardiometabolic risk in healthy older men after 4‐months. The long‐term effects of these and similar anabolic therapies on cardiovascular events should be investigated in populations with greater functional limitations along with important health disabilities including upper body obesity and other cardiometabolic risks.

     

Characterization of Genomic Inheritance of Intergeneric Hybrids between Ascocenda and Phalaenopsis Cultivars by GISH,PCR-RFLP and RFLP

BackgroundThe intergeneric hybrids between Ascocenda John De Biase ‘Blue’ and Phalaenopsis Chih Shang''s Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study. In order to confirm the inheritance in hybrid progenies, genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analysis were conducted to confirm the intergeneric hybridization status.Methods/ResultsGISH analysis showed the presence of both maternal and paternal chromosomes in the cells of the putative hybrids indicating that the putative hybrid seedlings were intergeneric hybrids of the two parents. Furthermore, twenty-seven putative hybrids were randomly selected for DNA analysis, and the external transcribed spacer (ETS) regions of nrDNA were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and RFLP analyses to identify the putative hybrids. RFLP analysis showed that the examined seedlings were intergeneric hybrids of the two parents. However, PCR-RFLP analysis showed bias to maternal genotype.ConclusionsBoth GISH and RFLP analyses are effective detection technology to identify the intergeneric hybridization status of putative hybrids. Furthermore, the use of PCR-RFLP analysis to identify the inheritance of putative hybrids should be carefully evaluated.     

Probing the polarity and water environment at the protein-peptide binding interface using tryptophan analogues

7-Azatryptophan and 2,7-diazatryptophan are sensitive to polarity changes and water content, respectively, and should be ideal for studying protein-protein and protein-peptide interactions. In this study, we replaced the tryptophan in peptide Baa (LKWKKLLKLLKKLLKLG-NH₂) with 7-azatryptophan or 2,7-diazatryptophan, forming (7-aza)Trp-Baa and (2,7-aza)Trp-Baa, to study the calmodulin (CaM)-peptide interaction. Dramatic differences in the (7-aza)Trp-Baa and (2,7-aza)Trp-Baa fluorescence properties between free peptide in water and calmodulin-bound peptide were observed, showing a less polar and water scant environment at the binding interface of the peptide upon calmodulin binding. The affinity of the peptides for binding CaM followed the trend Baa (210±10 pM)<(7-aza)Trp-Baa (109±5 pM)<(2,7-aza)Trp-Baa (45±2 pM), showing moderate increase in binding affinity upon increasing the number of nitrogen atoms in the Trp analogue. The increased binding affinity may be due to the formation of more hydrogen bonds upon binding CaM for the Trp analogue with more nitrogen atoms. Importantly, the results demonstrate that (7-aza)Trp and (2,7-aza)Trp are excellent probes for exploring the environment at the interface of protein-peptide interactions.     

Acidogenic growth model of embryogenic cell suspension culture and qualitative mass production of somatic embryos from triploid bananas

A young male flower-derived embryogenic suspension cell population of AAA ‘Pei Chiao’, ‘Dwarf Cavendish’, and AAB ‘Raja’ was used for developing an acidogenic growth model . We hypothesized that a close relationship exists between the self-regulated pH medium and the corresponding changes in the growth phases. Studies have reported that a pH below 4.6 may prevent the embryogenic cells from undergoing polar growth. Controlling pH up to a level 4.6 within 2 days during the changes of pre-embryogenic cells (PECs) and proembryogenic masses into embryogenic determined cells (EDCs) uniformly resulted in unequal cell division. The hydrogen ion buffer 2-N-morpholino-ethanesulfonic acid at 10 g L⁻¹ was added to MA2 and MA3 media, showing the medium pH of MA3 up to 5.0, thus maintaining a relatively stable pH in AAA ‘Pei-Chiao’ and AAB ‘Raja’ cells that autoregulate acidification, significantly increasing the number of somatic embryos. When the proliferation and globularization phases were acidified to pH 3.5 ± 0.2, cells were released to free single cells of PECs and EDCs after 21 days. This study provides possible explanation that PECs deposit callose on their cell walls as a possible protector from strong acidic condition. Regulation at pH 5.0 ± 0.2 resulted the production efficiency achieved was 0.9 million somatic embryos per 1 mL of the settled cell volume.