Identification of a 55-kDa ezrin-related protein that induces cytoskeletal changes and localizes to the nucleolus.

Normal and transformed human cells when stained for ezrin, an F-actin-binding ERM (ezrin/radixin/moesin) family protein, revealed a faint and intense immunofluorescence, respectively. Surprisingly, nuclear staining that was assigned to the nucleolus by confocal laser and immunoelectron microscopy was detected in both cell types and was more prominent in normal cells due to the absence of glistering cytoplasmic fluorescence. By Western analysis the nuclear fraction was seen to have a 55-kDa ezrin-reactive protein that did not react to the antibodies raised against the C-terminus of the protein, suggesting that it may correspond to an endogenously cleaved N-terminus of the protein. Transfections of cells with a cDNA encoding full-length ezrin tagged with green fluorescent protein (GFP) at its N-terminus indeed resulted in two GFP-tagged products corresponding to full-length and 55-kDa endogenously cleaved forms. Transfection with a cDNA encoding approximately 55 kDa of the ezrin N-terminus (N-ezrin) showed that it can translocate to the nucleus. N-ezrin transfected cells exhibited irregular cell edges and collapse of actin fibers. Similar changes were seen following microinjection of anti-p81/ezrin antibody, suggesting that N-ezrin may function as a dominant negative competitor of ezrin. These data demonstrate the existence of an N-terminal cleavage form of ezrin that localizes to the nucleolus and that its overexpression induces cytoskeletal changes.     

Analysis of morphological variability in the Indian germplasm of Allium sativum L.

Allium sativum germplasm collected from different parts of India was subjected to critical analysis of 14 easily noticeable morphological traits. Variability encountered in quantitative as well as qualitative, bulb and plant traits was documented. While analysing this variability, an attempt was made to highlight the genotype-environment interaction, its impact on the expression of morphological traits in this apomict and evaluate the different collections for intraspecific phylogeny.     

Effects of α-Tubulin K40 Acetylation and Detyrosination on Kinesin-1 Motility in a Purified System

Long-range transport in cells is achieved primarily through motor-based transport along a network of microtubule tracks. Targeted transport by kinesin motors can be correlated with posttranslational modifications (PTMs) of the tubulin subunits in specific microtubules. To directly examine the influence of specific PTMs on kinesin-1 motility, we generated tubulin subunits that were either enriched in or lacking acetylation of α-tubulin lysine 40 (K40) or detyrosination of the α-tubulin C-terminal tail. We show that K40 acetylation does not result in significant changes in kinesin-1’s landing rate or motility parameters (velocity and run length) across experimental conditions. In contrast, detyrosination causes a moderate increase in kinesin-1’s landing rate. The fact that the effects of detyrosination are dampened by prior K40 acetylation indicates that the combination of PTMs may be an important aspect of the functional output of microtubule heterogeneity. Importantly, our results indicate that the moderate influences that single PTMs have on kinesin-1 in vitro do not explain the strong correlation between specific PTMs and kinesin-1 transport in cells. Thus, additional mechanisms for regulating kinesin-1 transport in cells must be explored in future work.     

Hsp70 family member, mot-2/mthsp70/GRP75, binds to the cytoplasmic sequestration domain of the p53 protein

Hsp70 family member mot-2/mthsp70/GRP75/PBP74 was shown to bind to the tumor suppressor protein p53. In this study, by in vivo coimmunoprecipitation of mot-2 with p53 and its deletion mutants, the mot-2 binding site of p53 was mapped to its C-terminal amino acid residues 312-352, a region of p53 that includes its cytoplasmic sequestration domain. These data demonstrate that cytoplasmic sequestration and inactivation of p53 by mot-2 occurs by its binding to the cytoplasmic sequestration domain. Therefore, perturbation of mot-p53 interactions can be employed to abrogate cytoplasmic retention of wild-type p53 in tumors.     

Emergence of deciduous teeth in Punjabi children, north India.

Data on deciduous tooth emergence of 312 children aged 4 to 31 months of Punjabi parentage are presented. Probit analysis was used to derive the median age of tooth emergence. Female children are found to be advanced with respect to tooth emergence than their male counterparts. While comparing the present data with those from other populations it is found that, in general, the mean number of emerged teeth in Punjabi children is more at most ages, with lower median age of eruption for most teeth. Magnitude of interage variability in the eruption times is noticed to be maximum in the 16-17 and 20-21 months age groups. The findings of the study suggest that number of teeth can be used as a parameter for the estimation of age.     

Brown adipose tissue activity controls triglyceride clearance

Brown adipose tissue (BAT) burns fatty acids for heat production to defend the body against cold and has recently been shown to be present in humans. Triglyceride-rich lipoproteins (TRLs) transport lipids in the bloodstream, where the fatty acid moieties are liberated by the action of lipoprotein lipase (LPL). Peripheral organs such as muscle and adipose tissue take up the fatty acids, whereas the remaining cholesterol-rich remnant particles are cleared by the liver. Elevated plasma triglyceride concentrations and prolonged circulation of cholesterol-rich remnants, especially in diabetic dyslipidemia, are risk factors for cardiovascular disease. However, the precise biological role of BAT for TRL clearance remains unclear. Here we show that increased BAT activity induced by short-term cold exposure controls TRL metabolism in mice. Cold exposure drastically accelerated plasma clearance of triglycerides as a result of increased uptake into BAT, a process crucially dependent on local LPL activity and transmembrane receptor CD36. In pathophysiological settings, cold exposure corrected hyperlipidemia and improved deleterious effects of insulin resistance. In conclusion, BAT activity controls vascular lipoprotein homeostasis by inducing a metabolic program that boosts TRL turnover and channels lipids into BAT. Activation of BAT might be a therapeutic approach to reduce elevated triglyceride concentrations and combat obesity in humans.     

Biochemical and molecular analyses of copper-zinc superoxide dismutase from a C4 plant Pennisetum glaucum reveals an adaptive role in response to oxidative stress

Cadherin 23 (CDH23) is an important constituent of the hair cell tip link in the organ of Corti. Mutations in cdh23 are associated with age-related hearing loss (AHL). In this study, we proposed that the Cdh23(nmf308/nmf308) mice with progressive hair cell loss had specific morphological changes and suffered a base to apex gradient and age-related hearing loss, and that mutations in cdh23 were linked to AHL. The Cdh23(nmf308/nmf308) mice produced by the N-nitrosourea (ENU) mutagenesis program were used as an animal model to study AHL and progressive hair cell loss. RT-PCR was performed to confirm the cdh23 mutation in Cdh23(nmf308/nmf308) mice and genetic analysis was used to map the specific mutation site. Distortion product otoacoustic emission (DPOAE) assay and acoustic brainstem evoked response (ABR) threshold analysis were carried out to evaluate the AHL. Cochlear histology was examined with scanning electron microscope (SEM) and transmission electron microscope (TEM), as well as the nuclear labeling by propidium iodide staining; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activities were examined to evaluate cell apoptosis. Genetic mapping identified the candidate gene linking AHL in Cdh23(nmf308/nmf308) mice as cdh23. A mutation in exon3 (63 T>C) was screened as compared with the sequence of the same position of the gene from B6 (+/+) mice. The cochleae outer hair cells were reduced from 5-10% at one month to 100% at three months in the basal region. DPOAE and ABR exhibited an increasing threshold at high frequencies (≥16kHz) from one month of age. Morphological and cellular analysis showed that Cdh23(nmf308/nmf308) mice exhibited a time course of histological alterations and cell apoptosis of outer hair cells. Our results suggest that the cdh23 mutation may be harmful to the stereociliary tip link and cause the hair cell apoptosis. Due to the same cdh23 mutations in human subjects with presbycusis (Petit et al., 2001; Zheng et al., 2005), the Cdh23(nmf308/nmf308) mouse is an excellent animal model for investigating the mechanisms involved in human AHL.     

Active ras triggers death in glioblastoma cells through hyperstimulation of macropinocytosis

Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death.