Targeting apoptosis by hydroxymethylacylfulvene in combination with gamma radiation in prostate tumor cells

Hydroxymethylacylfulvene (HMAF) is a novel agent with alkylating activity and is a potent inducer of apoptosis that is currently undergoing Phase II clinical trials for prostate cancer. This study explored the pro-apoptosis and anti-proliferative potential of HMAF in combination with gamma radiation in human prostate tumor cell lines. Apoptosis was assessed based on the generation of fragmented DNA, a terminal transferase flow cytometry assay, and cell morphology. In each of the tumor cell lines examined, radiation alone induced a marginal level of apoptosis, even after a prolonged 48-h incubation after exposure. In contrast, HMAF alone was a potent inducer of apoptosis in prostate tumor cells but not in normal cells. Marked levels of apoptosis in tumor cells were also observed for the combination of HMAF with gamma radiation. When drug treatment preceded irradiation, at least additive levels of apoptosis were observed in both androgen-responsive and androgen-independent cells. The combined treatment with ionizing radiation and HMAF reduced the radiation dose needed for the same level of clonogenic survival up to 2.5-fold. The potentiation of apoptosis and reduction in the clonogenic survival of tumor cells occurred at HMAF concentrations lower than that which reduced survival to 10% and at doses up to 6 Gy. No potentiation of apoptosis or clonogenic inhibition was noted in normal cells. These results suggest that the combination of HMAF with gamma radiation may have clinical utility for treatments of prostate cancer.     

Corelease and differential exit via the fusion pore of GABA, serotonin, and ATP from LDCV in rat pancreatic beta cells

The release of gamma-aminobutyric acid (GABA) and ATP from rat beta cells was monitored using an electrophysiological assay based on overexpression GABA(A) or P2X2 receptor ion channels. Exocytosis of LDCVs, detected by carbon fiber amperometry of serotonin, correlated strongly (approximately 80%) with ATP release. The increase in membrane capacitance per ATP release event was 3.4 fF, close to the expected capacitance of an individual LDCV with a diameter of 0.3 microm. ATP and GABA were coreleased with serotonin with the same probability. Immunogold electron microscopy revealed that approximately 15% of the LDCVs contain GABA. Prespike "pedestals," reflecting exit of granule constituents via the fusion pore, were less frequently observed for ATP than for serotonin or GABA and the relative amplitude (amplitude of foot compared to spike) was smaller: in some cases the ATP-dependent pedestal was missing entirely. An inward tonic current, not dependent on glucose and inhibited by the GABA(A) receptor antagonist SR95531, was observed in beta cells in clusters of islet cells. Noise analysis indicated that it was due to the activity of individual channels with a conductance of 30 pS, the same as expected for individual GABA(A) Cl- channels with the ionic gradients used. We conclude that (a) LDCVs accumulate ATP and serotonin; (b) regulated release of GABA can be accounted for by exocytosis of a subset of insulin-containing LDCVs; (c) the fusion pore of LDCVs exhibits selectivity and compounds are differentially released depending on their chemical properties (including size); and (d) a glucose-independent nonvesicular form of GABA release exists in beta cells.     

IL-21 is highly produced in Helicobacter pylori-infected gastric mucosa and promotes gelatinases synthesis

Helicobacter pylori (Hp) infection is associated with gastric inflammation and ulceration. The pathways of tissue damage in Hp-infected subjects are complex, but evidence indicates that T cell-derived cytokines enhance the synthesis of matrix metalloproteinases (MMP) that contribute to mucosal ulceration and epithelial damage. In this study, we have examined the role of the T cell cytokine IL-21 in Hp-infected gastric mucosa and evaluated whether IL-21 regulates MMP production by gastric epithelial cells. We show that IL-21 is constitutively expressed in gastric mucosa and is more abundant in biopsy specimens and purified mucosal CD3(+) T cells from Hp-infected patients compared with normal patients and disease controls. We also demonstrate that IL-21R is expressed by primary gastric epithelial cells, as well as by the gastric epithelial cell lines AGS and MKN28. Consistently, AGS cells respond to IL-21 by increasing production of MMP-2 and MMP-9, but not MMP-1, MMP-3, MMP-7, or tissue inhibitors of MMP. Analysis of signaling pathways leading to MMP production reveals that IL-21 enhances NF-kappaB but not MAPK activation, and inhibition of NF-kappaB activation reduces IL-21-induced MMP-2 and MMP-9 production. Finally, we show that treatment of Hp-infected gastric explants with anti-IL-21 reduces epithelial cell-derived MMP-2 and MMP-9 production. These data indicate that IL-21 is overexpressed in Hp-infected gastric mucosa where it could contribute to increased epithelial gelatinase production.     

Recording the free-living behaviour of small-bodied, shallow-diving animals with data loggers

1. Time-depth data recorders (TDRs) have been widely used to explore the behaviour of relatively large, deep divers. However, little is known about the dive behaviour of small, shallow divers such as semi-aquatic mammals. 2. We used high-resolution TDRs to record the diving behaviour of American mink Mustela vison (weight of individuals 580-1275 g) in rivers in Oxfordshire (UK) between December 2005 and March 2006. 3. Dives to > 0.2 m were measured in all individuals (n = 6). Modal dive depth and duration were 0.3 m and 10 s, respectively, although dives up to 3 m and 60 s in duration were recorded. Dive duration increased with dive depth. 4. Temperature data recorded by TDRs covaried with diving behaviour: they were relatively cold (modal temperature 4-6 degrees C across individuals) when mink were diving and relatively warm (modal temperature 24-36 degrees C across individuals) when mink were not diving. 5. Individuals differed hugely in their use of rivers, reflecting foraging plasticity across both terrestrial and aquatic environments. For some individuals there was < 1 dive per day while for others there was > 100 dives per day. 6. We have shown it is now possible to record the diving behaviour of small free-living animals that only dive a few tens of centimetres, opening up the way for a new range of TDR studies on shallow diving species.     

Design of a novel quantitative PCR (QPCR)-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wld s ) gene

Background

The strain of MeCP2^tm1.1Bird mice is a broadly used model for Rett syndrome. Because males carrying the invalidated MeCP2 locus are sterile, this strain has to be maintained in a heterozygous state. All animals therefore have to be genotyped at every generation to discriminate those carrying the invalidated allele (+/- females and y/- males) from those that do not. This is conveniently carried out by PCR on tail genomic DNA but because the primer pairs described initially for this purpose yield very similar size DNA bands on the WT and the KO alleles, this requires to carry out two independent PCR reactions on tail DNA preparations from all animals.

Results

After cloning and sequencing the PCR fragment amplified on the KO allele, we tested several sets of primers that were designed to yield PCR fragments of different sizes on the KO and WT alleles.

Conclusion

We have thus identified a set of three primers that allows for efficient genotyping of the animals by a single PCR reaction. Furthermore, using of this set of primers also resolves a recurrent problem related to the tendency of one of the initial primers to give rise to a non specific band because of its capacity to anneal at both ends of a repeated genomic element which we have identified as MurvyLTR.     

Living on the edge: Exploring the role of coastal refugia in the Alexander Archipelago of Alaska

Although islands are of long‐standing interest to biologists, only a handful of studies have investigated the role of climatic history in shaping evolutionary diversification in high‐latitude archipelagos. In this study of the Alexander Archipelago (AA) of Southeast Alaska, we address the impact of glacial cycles on geographic genetic structure for three mammals co‐distributed along the North Pacific Coast. We examined variation in mitochondrial and nuclear loci for long‐tailed voles (Microtus longicaudus), northwestern deermice (Peromyscus keeni), and dusky shrews (Sorex monticola), and then tested hypotheses derived from Species Distribution Models, reconstructions of paleoshorelines, and island area and isolation. In all three species, we identified paleoendemic clades that likely originated in coastal refugia, a finding consistent with other paleoendemic lineages identified in the region such as ermine. Although there is spatial concordance at the regional level for endemism, finer scale spatial and temporal patterns are less clearly defined. Demographic expansion across the region for these distinctive clades is also evident and highlights the dynamic history of Late Quaternary contraction and expansion that characterizes high‐latitude species.     

Tolerance of wheat cultivars to root‐lesion nematode (Pratylenchus thornei) assessed by normalised difference vegetation index is predictive of grain yield

Tolerant wheat cultivars yield well when sown in fields infested with the root‐lesion nematode Pratylenchus thornei, which is present in 67% of fields in the subtropical grain region of eastern Australia. Wheat breeding programmes require accurate phenotyping to select germplasm with superior tolerance to P. thornei. This study investigated normalised difference vegetation index (NDVI) as a phenotypic tool to predict the tolerance of wheat cultivars on low and high P. thornei population densities. Three, 2‐year field experiments used a resistant and a susceptible wheat cultivar in the first year to develop low and high P. thornei populations. In the second year, 36 wheat cultivars were sown on these plots. A NTech Greenseeker was used to determine the NDVI of each plot at regular times during the season and grain yield was measured at crop maturity. There was an inverse relationship between P. thornei population densities and the NDVI for intolerant wheat cultivars. Regression analysis showed a highly predictive response between the yield tolerance index and NDVI with R² ranging from 0.85 (n = 36) to 0.93 (n = 36) for the three experiments. The area under the disease progress curve with respect to NDVI was highly predictive of yield tolerance (R² = 0.92; n = 36) when there were high populations (9,091 P. thornei/kg), but not when populations were low (578 P. thornei/kg). Tolerant cultivars can be identified by NDVI when sown on soil containing high populations (>2,500 P. thornei/kg) by measurement at approximately 1,000 degree days after sowing. Greenseeker is a valuable tool for wheat breeders to select germplasm with tolerance of P. thornei.