Cited2 is required both for heart morphogenesis and establishment of the left-right axis in mouse development

Establishment of the left-right axis is a fundamental process of vertebrate embryogenesis. Failure to develop left-right asymmetry leads to incorrect positioning and morphogenesis of numerous internal organs, and is proposed to underlie the etiology of several common cardiac malformations. The transcriptional modulator Cited2 is essential for embryonic development: Cited2-null embryos die during gestation with profound developmental abnormalities, including cardiac malformations, exencephaly and adrenal agenesis. Cited2 is also required for normal establishment of the left-right axis; we demonstrate that abnormal heart looping and right atrial and pulmonary isomerism are consistent features of the left-right-patterning defect. We show by gene expression analysis that Cited2 acts upstream of Nodal, Lefty2 and Pitx2 in the lateral mesoderm, and of Lefty1 in the presumptive floor plate. Although abnormal left-right patterning has a major impact on the cardiac phenotype in Cited2-null embryos, laterality defects are only observed in a proportion of these embryos. We have therefore used a combination of high-resolution imaging and three-dimensional (3D) modeling to systematically document the full spectrum of Cited2-associated cardiac defects. Previous studies have focused on the role of Cited2 in cardiac neural crest cell development, as Cited2 can bind the transcription factor Tfap2, and thus affect the expression of Erbb3 in neural crest cells. However, we have identified Cited2-associated cardiac defects that cannot be explained by laterality or neural crest abnormalities. In particular, muscular ventricular septal defects and reduced cell density in the atrioventricular (AV) endocardial cushions are evident in Cited2-null embryos. As we found that Cited2 expression tightly correlated with these sites, we believe that Cited2 plays a direct role in development of the AV canal and cardiac septa. We therefore propose that, in addition to the previously described reduction of cardiac neural crest cells, two other distinct mechanisms contribute to the spectrum of complex cardiac defects in Cited2-null mice; disruption of normal left-right patterning and direct loss of Cited2 expression in cardiac tissues.

Two novel reports of semidry stigmatic surface in Asteraceae

Research documents related to the morphology and function of style branches and stigmatic surface of Asteraceae are still rather few, and the literature reports are thus controversial. We report in the present study that the stigmatic surfaces of two non-related species of Asteraceae (Lessingianthus grandiflorus and Lucilia lycopodioides) have features of semidry stigmas. Sporodermis of both species was also analyzed so that we could understand how the stigmatic surface works during pollen deposition and rehydration. Stylar branches and pollen grains (sporodermis) were studied using scanning and transmission electron microscopy (SEM and TEM) and histochemistry techniques. The inner and marginal bands of stylar branches in these species display intermediary features between the dry and wet types of stigma: the cuticle characterizes the dry stigma and cells with secretory activity characterize the wet stigma; these showed differences from what has been described to the Asteraceae family, where stigmatic surface of species from several tribes is considered dry. Pollen grains are medium-size to large with exine ornamentation (echinate and echinolophate) and abundant secretion which latter characterizes pollenkitt. We can assume that two processes might help pollen grain hydration on stigmatic surface in Lessingianthus grandiflorus and Lucilia lycopodioides: (1) the presence of pollenkitt, as observed in the secretory content inside exine cavities and around pollen grains; and (2) the secretory activity of stigmatic surface cells, whose secretion accumulates among intercellular and subcuticular spaces and leads to cuticle disruption during the floral receptive phase. Our results suggest that ultrastructural and histochemical studies should be considered when describing stigmatic surface and that the ""semidry"" feature within Asteraceae should be investigated still more in detail, so that the taxonomic or adaptation value of this trait in the family can be verified. (C) 2010 Elsevier GmbH. All rights reserved.

Characterization of inflammatory responses to eccentric exercise in humans

Eccentric exercise commonly results in muscle damage. The primary sequence of events leading to exercise-induced muscle damage is believed to involve initial mechanical disruption of sarcomeres, followed by impaired excitation-contraction coupling and calcium signaling, and finally, activation of calcium-sensitive degradation pathways. Muscle damage is characterized by ultrastructural changes to muscle architecture, increased muscle proteins and enzymes in the bloodstream, loss of muscular strength and range of motion and muscle soreness. The inflammatory response to exercise-induced muscle damage is characterized by leukocyte infiltration and production of pro-inflammatory cytokines within damaged muscle tissue, systemic release of leukocytes and cytokines, in addition to alterations in leukocyte receptor expression and functional activity. Current evidence suggests that inflammatory responses to muscle damage are dependent on the type of eccentric exercise, previous eccentric loading (repeated bouts), age and gender. Circulating neutrophil counts and systemic cytokine responses are greater after eccentric exercise using a large muscle mass (e.g. downhill running, eccentric cycling) than after other types of eccentric exercise involving a smaller muscle mass. After an initial bout of eccentric exercise, circulating leukocyte counts and cell surface receptor expression are attenuated. Leukocyte and cytokine responses to eccentric exercise are impaired in elderly individuals, while cellular infiltration into skeletal muscle is greater in human females than males after eccentric exercise. Whether alterations in intracellular calcium homeostasis influence inflammatory responses to muscle damage is uncertain. Furthermore, the effects of antioxidant supplements are variable, and the limited data available indicates that anti-inflammatory drugs largely have no influence on inflammatory responses to eccentric exercise. In this review, we compare local versus systemic inflammatory responses, and discuss some of the possible mechanisms regulating the inflammatory responses to exercise-induced muscle damage in humans.

Two distinct regions in 2q24.2-q24.3 associated with idiopathic epilepsy

P>Approximately 50% of all carriers of 2q21-q31 deletions present epileptic seizures. The band 2q24 constitutes the smallest commonly deleted segment in these patients, and contains the voltage-gated sodium channel genes SCN1A and SCN2A, associated with Dravet syndrome and benign familial neonatal-infantile seizures, respectively. A further putative locus involving epilepsy in the region was previously identified through disruption of the SLC4A10 gene by translocation. In the course of performing high-resolution DNA copy number analyses on syndromic mentally impaired individuals, we encountered three patients with overlapping deletions in chromosome region 2q24. Two of these patients exhibited epileptic seizures in addition to mental deficiency. The deletion in one of the epileptic patients did not include the SCN cluster, demonstrating that a less severe form of epilepsy maps to an adjacent genomic region. This second region comprises about 3 Mb and contains the candidate gene SLC4A10, providing further support for the potential role of this gene in epilepsy.

Psychosocial sequelae of the 1989 Newcastle earthquake .2. Exposure and morbidity profiles during the first 2 years post-disaster

Background. A sample of 1089 Australian adults was selected for the longitudinal component of the Quake Impact Study, a 2-year, four-phase investigation of the psychosocial effects of the 1989 Newcastle earthquake. Of these, 845 (78%) completed a survey 6 months post-disaster as well as one or more of the three follow-up surveys. Methods. The phase I survey was used to construct dimensional indices of self-reported exposure to threat the disruption and also to classify subjects by their membership of five 'at risk' groups (the injured; the displaced; owners of damaged small businesses; helpers in threat and non-threat situations). Psychological morbidity was assessed at each phase using the 12-item General Health Questionnaire (GHQ-12) and the Impact of Event Scale (IES). Results. Psychological morbidity declined over time but tended to stabilize at about 12 months post-disaster for general morbidity (GHQ-12) and at about 18 months for trauma-related (IES) morbidity. Initial exposure to threat and/or disruption were significant predictors of psychological morbidity throughout the study and had superior predictive power to membership of the targeted 'at risk' groups. The degree of ongoing disruption and other life events since the earthquake were also significant predictors of morbidity. The injured reported the highest levels of distress, but there was a relative absence of morbidity among the helpers. Conclusions. Future disaster research should carefully assess the threat and disruption experiences of the survivors at the time of the event and monitor ongoing disruptions in the aftermath in order to target interventions more effectively.

Psychosocial sequelae of the 1989 Newcastle earthquake .3. Role of vulnerability factors in postdisaster morbidity

Background. This paper examines the contributions of dispositional and non-dispositional factors to post-disaster psychological morbidity. Data reported are from the 845 participants in the longitudinal component of the Quake Impact Study. Methods. The phase 1 survey was used to construct dimensional indices of threat and disruption exposure. Subsequently, a range of dispositional characteristics were measured, including neuroticism, personal hopefulness and defence style. The main morbidity measures were the General Health Questionnaire (GHQ-12) and Impact of Event Scale (IES). Results. Dispositional characteristics were the best predictors of psychological morbidity throughout the 2 years post-disaster, contributing substantially more to the variance in morbidity (12-39%) than did initial exposure (5-12%), but the extent of their contribution was greater for general (GHQ-12) than for post-traumatic (IES) morbidity. Among the non-dispositional factors, avoidance coping contributed equally to general and post-traumatic morbidity (pr = 0.24). Life events since the earthquake (pr = 0.18), poor social relationships (pr = -0.25) and ongoing earthquake-related disruptions (pr = 0.22) also contributed to general morbidity, while only the latter contributed significantly to post-traumatic morbidity (pr = 0.15). Conclusions. Medium-term post-earthquake morbidity appears to be a function of multiple factors whose contributions vary depending on the type of morbidity experienced and include trait vulnerability, the nature and degree of initial exposure, avoidance coping and the nature and severity of subsequent events.

Urine is necessary to provoke bladder inflammation in protamine sulfate induced urothelial injury

Purpose: The bladder is normally impermeable to possible hostile environmental factors and toxic urinary wastes. Any disruption of the permeability barrier would permit the leakage of urine constituents into the underlying cells layers and subsequent inflammation. Protamine sulfate, which increases urothelial permeability, is used in experimental models of cystitis. We examined whether protamine sulfate alone could cause bladder inflammation or if the association of protamine sulfate and urine is needed for this condition. Materials and Methods: Female Wistar rats (Center for the Development of Experimental Models for Medicine and Biology, Federal University of Sao Paulo, Sao Paulo, Brazil) had the bladder catheterized and instilled with protamine sulfate (10 mg) or sterile saline for 30 minutes. To exclude urine other groups of rats underwent bilateral nephrectomy and the same procedure was used. One day after instillation the bladders were removed for histopathology. Edema and vascular congestion were graded from 0-none to 3-severe. Polymorphonuclear and mast cells were counted. The Kruskal-Wallis test was performed for statistical analysis. Results: Intravesical instillation of protamine sulfate in nonnephrectomized rats led to inflammation, in contrast to findings in rats instilled with saline. On the other hand, nephrectomized rats showed no inflammatory changes following the instillation of protamine sulfate or saline. The mast cell count was similar in all groups. Conclusions: Bladder inflammation in this experimental model of urothelial injury was not due to protamine sulfate alone. The association of protamine sulfate and urine was necessary to trigger the inflammatory cascade. Thus, urine indeed has an important role in the development of bladder inflammation in an environment of higher urothelial permeability.

Chemical treatment of Escherichia coli .1. Extraction of intracellular protein from uninduced cells

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetetraacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. (C) 1997 John Wiley & Sons, Inc.

Numerical and experimental study of a homogenizer impinging jet

High-pressure homogenization is a key unit operation used to disrupt cells containing intracellular bioproducts. Modeling and optimization of this unit are restrained by a lack of information on the flow conditions within a homogenizer value. A numerical investigation of the impinging radial jet within a homogenizer value is presented. Results for a laminar and turbulent (k-epsilon turbulent model) jet are obtained using the PHOENICS finite-volume code. Experimental measurement of the stagnation region width and correlation of the cell disruption efficiency with jet stagnation pressure both indicate that the impinging jet in the homogenizer system examined is likely to be laminar under normal operating conditions. Correlation of disruption data with laminar stagnation pressure provides a better description of experimental variability than existing correlations using total pressure drop or the grouping 1/Y(2)h(2).

A synthesis of the findings from the Quake Impact Study: A two year investigation of the psychosocial sequelae of the 1989 Newcastle earthquake

This paper summarises the major findings from the Quake Impact Study (QIS), a four-phase longitudinal project that was conducted in the aftermath of the 1989 Newcastle (Australia) earthquake. A total of 3,484 subjects participated in at least one component of the QIS, comprising a stratified sample of 3,007 drawn from community electoral rolls and 477 from specially targeted supplementary samples (the injured, the displaced, the owners of damaged businesses, and the helpers). Subjects' initial earthquake experiences were rated in terms of weighted indices of exposure to threat and disruption. Psychological morbidity was measured at each phase using the General Health Questionnaire (GHQ-12) and the Impact of Event Scale (IES). Selected findings and key conclusions are presented for each of six areas of investigation: service utilisation during the first 6 months post-disaster; patterns of earthquake experience and short-term (6-month) psychosocial outcome; earthquake exposure and medium term (2-year) psychosocial outcome; vulnerability factors and medium-term psychosocial outcome: specific community groups at increased risk (e.g., the elderly and immigrants from non-English-speaking backgrounds); the effects of stress debriefing for helpers. Threshold morbidity (i.e., likely caseness) rates are also presented for a broad range of subgroups. In addition to presenting an overview of the QIS, this paper synthesises the major findings and discusses their implications for future disaster management and research from a mental health perspective.

Myelin proteolipid protein expressed in COS-1 cells is targeted to actin-associated surfaces

The compact myelin sheath represents one of the largest expanses of membrane-membrane contact in the body and, in the central nervous system, requires the myelin proteolipid protein (PLP) for assembly, To determine whether the molecular properties of PLP promote membrane adhesion and direct its subcellular localization in the absence of oligodendrocyte-specific targeting mechanisms, PLP was expressed in COS-I fibroblasts, Immunofluorescence staining indicated that PUP was translated effectively, transited the rough endoplasmic reticulum and Golgi apparatus, was delivered to the cell surface, and was endocytosed, In the plasma membrane, the PLP distribution was patchy and only sporadically coincided with sites of membrane-membrane contact between PLP-expressing cells, PLP was not randomly distributed, however, but correlated closely with microfilament locations in leading edge membranes and microvilli, as demonstrated by phalloidin double labeling, Our results indicate that even in non-myelinating cells, PLP can be concentrated in membranes associated with movement and growth, and suggest possible roles for the actin cytoskeleton in PLP localization, As PLP, DM20, and the DM20-like M6 protein all associate with actin-enriched membranes, this may be a common feature of PLP/DM20 gene family members. (C) 1997 Wiley-Liss, Inc.

The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins

The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3 Delta 977-994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3 Delta 977-994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.

Histology of dart tag insertion sites in the epaulette shark

Samples of dermal and epidermal tissues of epaulette sharks Hemiscyllium ocellatum were examined histologically to assess damage caused by tagging. Tissues from around tag sites were removed at time intervals ranging from 100 min to 284 days post-tagging. These samples showed acute and chronic responses to tagging. Acute responses consisted of localized tissue breakdown and haemorrhaging, and occurred within the first few hours after tag insertion. At 10 h post-tagging, an intermediate response was apparent. This phase was characterized by further haemorrhaging and red and white blood cell movement into the wound area. The chronic response observed in the 10-284-day post-tagging samples was characterized by fibrous tissue formation to sequester the tag. This tissue presumably protects the adjacent musculature from further trauma produced by movement of the tag and provides a continuous barrier between the internal and external milieu. Tissue repair appeared to progress consistently in all specimens and no secondary infections at the tag site were seen. Tagging produced only localized tissue disruption and did not appear to be detrimental to the long term health of individual sharks. Our findings show that spaghetti style dart tagging is an acceptable method for marking individuals (40-75+ cm total length) of this species. (C) 1997 The Fisheries Society of the British Isles.

Cumulative sedimentation analysis of Escherichia coli debris size

A new method to measure Escherichia coil cell debris size after homogenization is presented. It is based on cumulative sedimentation analysis under centrifugal force, coupled with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis of sedimented proteins. The effects that fermentation and homogenization conditions have on the resulting debris distributions were investigated using this method. Median debris size decreased significantly from approximately 0.5 mu m to 0.3 mu m as the number of homogenization passes increased from 2 to 10. Under identical homogenization conditions, uninduced host cells in stationary phase had a larger debris size than exponential cells after 5 homogenizer passes. This difference was not evident after 2 or in passes, possibly because of confounding intact cells and the existence of a minimum debris size for the conditions investigated. Recombinant cells containing protein inclusion bodies had the smallest debris size following homogenization. The method was also used to measure the size distribution of inclusion bodies. This result compared extremely well with an independent determination using centrifugal disc photosedimentation (CDS), thus validating the method. This is the first method that provides accurate size distributions of E. coli debris without the need for sample pretreatment, theoretical approximations (e.g. extinction coefficients), or the separation of debris and inclusion bodies prior to analysis. (C) 1997 John Wiley & Sons, Inc.

Absence of galectin-3 does not affect the development of experimental tongue carcinomas in mice

Background: Galectin-3 is a lectin that presents pivotal roles in tumor biology and there are no studies evaluating their expression in dysplasias and carcinomas developed from tongue carcinogenesis models. Aims: To investigate the role of galectin-3 in the development of tongue carcinomas using a mouse model of oral carcinogenesis. Methods: Galectin-3-deficient (gal3(-/-)) and wild-type (gal3(+/+)) mice were challenged with 4-nitroquinoline-1-oxide in drinking water for 16 weeks and killed at different times. Tongues were removed and the number of dysplasias and carcinomas was counted. An immunohistochemical study for galectin-3 was performed only in the tongue from gal3(+/+) mice. Results: In both groups, a reduction of dysplasias and an increase of carcinomas from week 16 to week 32 (p > 0.05) were observed. A predominance of high cytoplasmic and nuclear galectin-3 expression was observed in carcinomas (64.7%) and dysplasias (55.5%), respectively (p > 0.05). The perilesional areas always presented a statistical cytoplasmic and nuclear galectin-3 overexpression. Conclusions: Absence of galectin-3 did not directly affect the process of carcinogenesis and a cytoplasm shift of galectin-3 seems to be associated with development of tongue carcinomas. (C) 2010 Elsevier Inc. All rights reserved.