Do individual allocyclic chromosomes in metaphase reflect their interphase domains?

Summary Individual S phase allocyclic chromosomes have been analyzed in Bloom syndrome lymphocytes, in cells with an r(9), and in hypotetraploid Ehrlich mouse ascites cells treated with 1-methyl-2-benzyl hydrazine. On the basis of the following observations, we conclude that such chromosomes more or less reflect their domains in interphase: (1) The S phase allocyclic chromosomes have the same structure as S phase prematurely condensed chromatin (PCC) in fused cells; in other words they form limited areas of chromatin dots; (2) the allocyclic chromosome is the only chromosome in a metaphase plate which synthesizes DNA simultanneously with interphase nuclei; (3) the size of the allocyclic chromosomes is related to the size of the corresponding metaphase chromosome; and (4) the S phase allocyclic chromosomes resemble closely the chromosome domains in interphase made visible with biotinylated human DNA. A variety of evidence shows that most allocyclic chromosomes are simply left behind in their cycle, which presumably is caused by a deletion or inactivation of a hypothetical coiling center situated on each chromosome arm.     

Hemicellulolytic and cellulolytic functions of the domains of a β-mannanase cloned from Caldicellosiruptor saccharolyticus

Various combinations of the four domains of the multifunctional mannanase from Caldicellosiruptor saccharolyticus have been cloned and expressed in Escherichia coli. The four domains comprise two catalytic domains (1 and 4), and two putative cellulose binding domains (2 and 3). Each of the six gene products (Man1, Man123, Man1234, Man23, Man234 and Man4) was partially purified by heat treatment.The enzymes Man1234, Man123 and Man1 exhibited activity on mannans, and Man1234, Man234 and Man4 exhibited activity on xylan and carboxymethylcellulose (CMC). For the complete enzyme (Man1234) all activities were of the same order of magnitude. Activities were additive against a mixture of mannan and xylan or mannan and CMC (but not xylan and CMC). The expression product Man23 exhibited activity on none of the substrates tested, nor did its presence influence thermostability or significantly reduce the K_m value for any of the substrates. However, when expressed in combination with domains 1 or 4 it greatly increased their activity.We conclude that domain 1 catalyses mannan hydrolysis and domain 4 catalyses xylan and CMC hydrolysis at the same active site: domains 2 and 3 have no obvious function, since they do not reduce substrate K_m nor affect thermostability. However, their effect on rates of substrate hydrolysis may indicate a role influencing the conformation of the adjacent catalytic domains.     

WAVE binds Ena/VASP for enhanced Arp2/3 complex–based actin assembly

The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex–based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP''s ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.     

Temporal variation of β-diversity and assembly mechanisms in a bacterial metacommunity

The turnover of community composition across space, β-diversity, is influenced by different assembly mechanisms, which place varying weight on local habitat factors, such as environmental conditions and species interactions, and regional factors such as dispersal and history. Several assembly mechanisms may function simultaneously; however, little is known about how their importance changes over time and why. Here, we implemented a field survey where we sampled a bacterial metacommunity consisting of 17 rock pools located at the Swedish Baltic Sea coast at 11 occasions during 1 year. We determined to which extent communities were structured by different assembly mechanisms using variation partitioning and studied changes in β-diversity across environmental gradients over time. β-Diversity was highest at times of high overall productivity and environmental heterogeneity in the metacommunity, at least partly due to species sorting, that is, selection of taxa by the prevailing environmental conditions. In contrast, dispersal-driven assembly mechanisms were primarily detected at times when β-diversity was relatively low. There were no indications for strong and persistent differences in community composition or β-diversity between permanent and temporary pools, indicating that the physical disturbance regime is of relatively minor importance. In summary, our study clearly suggests that there are temporal differences in the relative importance of different assembly mechanisms related to abiotic factors and shows that the temporal variability of those factors is important for a more complete understanding of bacterial metacommunity dynamics.     

Comorbidity among HHT patients and their controls in a 20 years follow-up period

Background

Hereditary Haemorrhagic Telangiectasia (HHT) is an autosomal dominant genetic disorder with a wide variety of clinical manifestations due to the presence of multiple arteriovenous malformations in various tissues and organs.

Objective

To study the need for hospital admittance in a group of HHT patients and matched controls during a 20?years follow-up period commencing in 1995.

Methods

All HHT patients in the County of Funen, Denmark, were included. For each patient, three age and sex matched controls were identified at the time of enrolment. Data on all hospitalisations were extracted from the national health registers and compared with clinical records. The hospitalisations were grouped as HHT relevant or not HHT relevant based on the discharge diagnosis (International Classification of Diseases, ICD10) and with particular focus on infections, bleedings and thromboembolic events.Patients with HHT were compared with controls concerning the first time incidence of each discharge diagnosis.

Results

We included 73 HHT patients and 219 controls of which one control was lost to follow-up. HHT-patients had significantly more hospitalisations per person caused by infections in joints and bones, but not caused by infections in general. Bleeding episodes were, as expected, more frequent among the HHT-patients. The study revealed a similar incidence of abscesses and thromboembolisms, including in the central nervous system, among the HHT patients and controls.

Conclusions

Based on this study Danish HHT patients had an increased comorbidity of infections in joints and bones and of bleeding episodes. However, the incidence of thromboembolisms, cerebral abscesses and other conditions commonly considered related to HHT was comparable between the patients and the controls. The patients included in this study were closely monitored at a highly specialised HHT Centre where they received relevant diagnostic evaluation, treatment and counselling. Since this is assumed to benefit the overall health of the patients, it may explain why these patients were less prone to comorbidity than other studies have suggested.

     

α-Actinin-4/FSGS1 is required for Arp2/3-dependent actin assembly at the adherens junction

We have developed an in vitro assay to study actin assembly at cadherin-enriched cell junctions. Using this assay, we demonstrate that cadherin-enriched junctions can polymerize new actin filaments but cannot capture preexisting filaments, suggesting a mechanism involving de novo synthesis. In agreement with this hypothesis, inhibition of Arp2/3-dependent nucleation abolished actin assembly at cell-cell junctions. Reconstitution biochemistry using the in vitro actin assembly assay identified α-actinin-4/focal segmental glomerulosclerosis 1 (FSGS1) as an essential factor. α-Actinin-4 specifically localized to sites of actin incorporation on purified membranes and at apical junctions in Madin-Darby canine kidney cells. Knockdown of α-actinin-4 decreased total junctional actin and inhibited actin assembly at the apical junction. Furthermore, a point mutation of α-actinin-4 (K255E) associated with FSGS failed to support actin assembly and acted as a dominant negative to disrupt actin dynamics at junctional complexes. These findings demonstrate that α-actinin-4 plays an important role in coupling actin nucleation to assembly at cadherin-based cell-cell adhesive contacts.     

CWN-1 functions with DSH-2 to regulate C. elegans asymmetric neuroblast division in a β-catenin independent Wnt pathway

In Caenorhabditis elegans, Wnt signaling regulates many asymmetric cell divisions. During embryogenesis, the C. elegans Dishevelled (Dsh) homolog, DSH-2, regulates asymmetric neuroblast division of the ABpl/rpppa blast cell. Dsh is a key intracellular component of both β-catenin dependent and β-catenin independent Wnt pathways. In C. elegans, most of the well-characterized asymmetric cell divisions regulated by Wnts are dependent on β-catenin. In the ABpl/rpppa neuroblast division, however, we determined that DSH-2 regulates cell polarity through a β-catenin independent Wnt pathway. We also established that the C. elegans Wnt homolog, cwn-1, functions to regulate asymmetric division of the ABpl/rpppa blast cell. Our results indicated that cwn-1 does not act alone in this process, and it functions with another redundant ligand that appears not to be a Wnt. Finally, we show widespread requirements for DSH-2 during embryogenesis in the generation of many other neurons. In particular, DSH-2 function is necessary for the correct production of the embryonic ventral cord motor neurons. This study demonstrates a role for DSH-2 and Wnt signaling in neuronal specification during C. elegans embryogenesis.     

Oct-1 functions as a transactivator in the hormonal induction of β-casein gene expression

An adverse environmental experience of the growing fetus leads to permanent changes in the structure and contractile function of the heart; however, the mechanisms are incompletely understood. To examine if a maternal low protein (LP) diet can modulate the gene and protein expression of the Ca²⁺-cycling proteins in the neonatal heart, we employed a rat model in which pregnant dams were fed diets containing either 180 (normal) or 90 g (low) casein/kg diet for 2 weeks before mating and throughout pregnancy. A significant reduction in the L-type Ca²⁺-channel mRNA level in the LP group was detected at 1, 7, and 14 days of age. Although ryanodine receptor (RyR) mRNA levels progressively declined in the aging heart in both groups, the RyR mRNA levels were consistently higher in the LP group. A reduction in RyR protein content was seen only in the hearts of the LP group at 7 days of age. The Na⁺-Ca²⁺-exchanger (NCX) mRNA level was also markedly increased at all ages. Although an increase in sarco(endo)plasmic reticulum ATPase 2a (SERCA) 2a mRNA was only detected in the LP group at 7 days of age, corresponding protein level was depressed. On the other hand, an initial decrease (at 1 day of age) followed by an increase (at 14 and 28 days of age) in phospholamban (PLB) mRNA levels was detected. Although PLB protein level was also depressed at 1 day of age in the LP group, a marked increase was seen at 7 days of age. Moreover, the ratio of serine 16 and threonine 17 phosphorylated PLB to non-phosphorylated PLB was reduced at 7 days of age in the hearts of offspring of the LP group. These data suggest that maternal LP diet can induce alterations in the gene expression and protein levels of the Ca²⁺-cycling proteins in the neonatal heart.     

Abnormal actin binding of aberrant β-tropomyosins is a molecular cause of muscle weakness in TPM2-related nemaline and cap myopathy

NM (nemaline myopathy) is a rare genetic muscle disorder defined on the basis of muscle weakness and the presence of structural abnormalities in the muscle fibres, i.e. nemaline bodies. The related disorder cap myopathy is defined by cap-like structures located peripherally in the muscle fibres. Both disorders may be caused by mutations in the TPM2 gene encoding β-Tm (tropomyosin). Tm controls muscle contraction by inhibiting actin-myosin interaction in a calcium-sensitive manner. In the present study, we have investigated the pathogenetic mechanisms underlying five disease-causing mutations in Tm. We show that four of the mutations cause changes in affinity for actin, which may cause muscle weakness in these patients, whereas two show defective Ca2+ activation of contractility. We have also mapped the amino acids altered by the mutation to regions important for actin binding and note that two of the mutations cause altered protein conformation, which could account for impaired actin affinity.     

15-Deoxy-Δ-prostaglandin J2 impairs the functions of histone acetyltransferases through their insolubilization in cells

The cyclopentenonic prostaglandin 15-deoxy-Δ^12,14-PG J₂ (15d-PGJ₂) is a metabolite derived from PGD₂. Although 15d-PGJ₂ has been demonstrated to be a potent ligand for peroxisome proliferator activated receptor γ (PPARγ), the functions are not fully understood. In order to examine the effect of 15d-PGJ₂ on histone acetyltransferases (HATs), several lines of cell including mouse embryonic fibroblast (MEF) cells were exposed to 15d-PGJ₂. Three types of HAT, p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF), selectively disappeared from the soluble fraction in time- and dose-dependent manners. Inversely, HATs in the insoluble fraction increased, suggesting their conformational changes. The decrease in the soluble form of HATs resulted in the attenuation of NF-κB-, p53-, and heat shock factor-dependent reporter gene expressions, implying that the insoluble HATs are inactive. The resultant insoluble PCAF and p300 seemed to be digested by proteasome, because proteasome inhibitors caused the accumulation of insoluble HATs. Taken together, these results indicate that 15d-PGJ₂ attenuates some gene expressions that require HATs. This inhibitory action of 15d-PGJ₂ on the function of HATs was independent of PPARγ, because PPARγ agonists could not mimick 15d-PGJ₂ and PPARγ antagonists did not inhibit 15d-PGJ₂.     

The importance of the last strand at the C-terminus in βB2-crystallin stability and assembly

Congenital cataract is the leading cause of childhood blindness worldwide. Investigations of the effects of inherited mutations on protein structure and function not only help us to understand the molecular mechanisms underlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lens abnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E and R188H mutations on βB2-crystallin structure and stability using a combination of biophysical, cellular and molecular dynamic simulation analysis. Both V187 and R188 are located at the last strand of βB2-crystallin Greek-key motif 4. All of the three mutations promoted βB2-crystallin aggregation in vitro and at the cellular level. These three mutations affected βB2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal domain, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, while R188H perturbed the dynamic oligomeric equilibrium by dissociating the dimer and stabilizing the tetramer. Our results highlighted the importance of the last strand in the structural integrity, folding, assembly and stability of β-crystallins. More importantly, we proposed that the perturbation of the dynamic equilibrium between β-crystallin oligomers was an important mechanism of congenital hereditary cataract. The selective stabilization of one specific high-order oligomer by mutations might also be deleterious to the stability and folding of the β-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance of β-crystallins are achieved by the precisely regulated oligomeric equilibrium.     

The dioxin receptor controls β1 integrin activation in fibroblasts through a Cbp–Csk–Src pathway

Recent studies have suggested a regulatory role for the dioxin receptor (AhR) in cell adhesion and migration. Following our previous work, we report here that the C-terminal Src kinase-binding protein (Cbp) signaling pathway controls β1 integrin activation and that this mechanism is AhR dependent. T-FGM AhR ?/? fibroblasts displayed higher integrin β1 activation, revealed by the increased binding of the activation reporter 9EG7 anti-β1 mAb and of a soluble fibronectin fragment, as well as by enhanced talin-β1 association. AhR ?/? fibroblasts also showed increased fibronectin secretion and impaired directional migration. Notably, interfering Cbp expression in AhR ?/? fibroblasts reduced β1 integrin activation, improved cell migration and rescued wild-type cell morphology. Cbp over-expression in T-FGM AhR ?/? cells enhanced the formation of inhibitory Csk–Cbp complexes which in turn reduced c-Src p-Tyr⁴¹⁶ activation and focal adhesion kinase (FAK) phosphorylation at the c-Src-responsive residues p-Tyr⁵⁷⁶ and p-Tyr⁵⁷⁷. The c-Src target and migration-related protein Cav1 was also hypophosphorylated at p-Tyr¹⁴ in AhR ?/? cells, and such effect was rescued by down-modulating Cbp levels. Thus, AhR regulates fibroblast migration by modulating β1 integrin activation via Cbp-dependent, Src-mediated signaling.     

Identification of a Karyopherin β1/β2 Proline-Tyrosine Nuclear Localization Signal in Huntingtin Protein

Among the known pathways of protein nuclear import, the karyopherin β2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin β2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a β sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin β1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.     

The β-barrel assembly machinery (BAM) is required for the assembly of a primitive S-layer protein in the ancient outer membrane of Thermus thermophilus

The ancient bacterial lineage Thermus spp has a primitive form of outer membrane attached to the cell wall through SlpA, a protein that shows intermediate properties between S-layer proteins and outer membrane (OM) porins. In E. coli and related Proteobacteria, porins are secreted through the BAM (β-barrel assembly machinery) pathway, whose main component is BamA. A homologue to this protein is encoded in all the Thermus spp so far sequenced, so we wondered if this pathway could be responsible for SlpA secretion in this ancient bacterial model. To analyse this hypothesis, we attempted to get mutants on this BamA^th of T. thermophilus HB27. Knockout and deletion mutants lacking the last 10 amino acids were not viable, whereas its depletion by means of a BamA antisense RNA lead defective attachment to the cell wall of its OM-like envelope. Such defects were related to defective folding of the SlpA protein that was more sensitive to proteases than in a wild-type strain. A similar phenotype was found in mutants lacking the terminal Phe of SlpA. Further protein–protein interaction assays confirmed the existence of specific binding between SlpA and BamA^th. Taking together, these data suggest that SlpA is secreted through a BAM-like pathway in this ancestral bacterial lineage, supporting an ancient origin of this pathway before the evolution of the Proteobacteria.