Sodium butyrate inhibits the synthesis of the transformation related protein p 53 in 3T6 mouse fibroblasts

Sodium butyrate, which blocks the cell cycle of many cell types in the G1 phase, strongly inhibits the synthesis of the transformation related, 53 kDa protein in 3T6 fibroblasts but much less so in SV 40 transformed mouse cells. By several criteria, this effect of the fatty acid appears to be indirect; p 53 synthesis takes place several hours after the butyrate-sensitive step in G1. The results are discussed in the light of a putative role of p 53 in growth control.     

Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit. We isolated a human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2, and we named this homolog hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits. Here we show that hHCP-6 becomes phosphorylated during mitosis and that its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-beta, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A.     

Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation

Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.     

Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the B alpha regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation- (L309 Delta) and phosphorylation- (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B', and B'-type regulatory subunits in NIH 3T3 fibroblasts and neuro-2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309 Delta, and T304D mutants, which are impaired in their ability to associate with the B alpha subunit, induces their loss. Silencing of B alpha subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal B alpha-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.     

An unusual presentation of a malignant jejunal tumor and a different management strategy

BACKGROUND: Malignant small bowel tumors are very rare and leiomyosarcoma accounts for less than 15% of the cases. Management of these tumors is challenging in view of nonspecific symptoms, unusual presentation and high incidence of metastasis. In this case report, an unusual presentation of jejunal sarcoma and management of liver metastasis with radiofrequency ablation (RFA) is discussed. CASE PRESENTATION: A 45-year-old male presented with anemia and features of small bowel obstruction. Operative findings revealed a mass lesion in jejunum with intussusception of proximal loop. Resection of bowel mass was performed. Histopathological findings were suggestive of leiomyosarcoma. After 3-years of follow-up, the patient developed recurrence in infracolic omentum and a liver metastasis. The omental mass was resected and liver lesion was managed with radiofrequency ablation. CONCLUSION: Jejunal leiomyosarcoma is a rare variety of malignant small bowel tumor and a clinical presentation with intussusception is unusual. We suggest that an aggressive management approach using a combination of surgery and a newer technique like RFA can be attempted in patients with limited metastatic spread to liver to prolong the long-term survival in a subset of patients.     

Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin

The Gram-negative type II secretion (T2S) system is a multiprotein complex mediating the release of virulence factors from a number of pathogens. While an understanding of the function of T2S components is emerging, little is known about what identifies substrates for export. To investigate T2S substrate recognition, we compared mutations affecting the secretion of two highly homologous substrates: heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae. Each toxin consists of one enzymatic A subunit and a ring of five B subunits mediating the toxin''s secretion. Here, we report two mutations in LT''s B subunit (LTB) that reduce its secretion from ETEC without global effects on the toxin. The Q3K mutation reduced levels of secreted LT by half, and as with CT (T. D. Connell, D. J. Metzger, M. Wang, M. G. Jobling, and R. K. Holmes, Infect. Immun. 63:4091-4098, 1995), the E11K mutation impaired LT secretion. Results in vitro and in vivo show that these mutants are not degraded more readily than wild-type LT. The Q3K mutation did not significantly affect CT B subunit (CTB) secretion from V. cholerae, and the E11A mutation altered LT and CTB secretion to various extents, indicating that these toxins are identified as secretion substrates in different ways. The levels of mutant LTB expressed in V. cholerae were low or undetectable, but each CTB mutant expressed and secreted at wild-type levels in ETEC. Therefore, ETEC''s T2S system seems to accommodate mutations in CTB that impair the secretion of LTB. Our results highlight the exquisitely fine-tuned relationship between T2S substrates and their coordinate secretion machineries in different bacterial species.Gram-negative bacteria have evolved a number of methods to secrete proteins into the extracellular milieu, with at least six specific secretion systems currently described (14, 30). Type II secretion (T2S), or the main terminal branch of the general secretory pathway, is a feature of a number of proteobacteria and has been shown to be required for pathogenesis and maintenance of environmental niches in a large number of species (5). The T2S system is a multiprotein complex of 12 to 15 components that spans the inner and outer membranes, allowing for the controlled release of certain folded proteins that have been directed to the periplasm through the Sec or Tat machinery (21). Aside from providing a means of exporting freely released virulence factors from plant, animal, and human pathogens (5), the T2S system has been shown to export surface-associated virulence factors (18), fimbrial components (46), outer membrane cytochromes (36), and a surfactant required for sliding motility in Legionella pneumophila (39), among other substrates.While an increasing number of studies have focused on understanding the structure and function of the components of the T2S system itself, little is known about what identifies a periplasmic protein as a substrate for secretion (21, 32). Because proteins secreted from the same bacterial species need not share any obvious structural homology, it is not even clear how much of a T2S substrate interacts with the secretion machinery (32). Analysis of two similar substrates that can each be secreted by the T2S systems of two distinct species would provide information about species-specific identification of T2S substrates and, by extension, the nature of the “secretion motif” identifying those substrates. Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae represent one such pair of substrates.ETEC and V. cholerae are enteric pathogens causing significant morbidity and mortality worldwide (33). The causative agents of traveler''s diarrhea and cholera, respectively, these two pathogens share a number of similarities, including the nature of their disease symptoms (38). Each pathogen secretes an AB₅ toxin important for colonization and the induction of water and electrolyte efflux from intestinal epithelial cells (1, 29). These toxins, LT and CT, are both encoded by two-gene operons. After sec-dependent transport to the periplasm, holotoxin formation occurs spontaneously (13), with one catalytic A subunit (LTA or CTA) assembling with five B subunits (LTB or CTB), which are responsible for the binding properties of the toxins. Export of fully folded and assembled LT or CT is then accomplished by the T2S system (34, 40). In ETEC, this system is encoded by gspC to -M (40), while in V. cholerae, these genes are found in the eps operon (34).LT and CT are very similar in structure, sharing approximately 80% sequence homology and 83% identity in the mature B subunit (16, 24). ETEC is thought to have acquired the genes for CT through horizontal transfer, with the toxins evolving over time to possess slight differences (45). As such, these toxins share the same primary host receptor, the monosialoganglioside G_M1, and catalyze the same ADP-ribosylation reaction within host cells (38). However, LT is able to bind other host sphingolipids in addition to G_M1 and to interact with sugar residues from the A-type blood antigen, which CT cannot bind (16, 41). Both LT and CT are able to associate with sugar residues in lipopolysaccharide (LPS) on the surface of E. coli cells (17). Binding to each of these substrates can be impaired by point mutation (26, 43).In this study, we report point mutations impairing the release of LT from ETEC and CT from V. cholerae. We analyzed the specificity of the defects in substrate recognition by comparing the effects of substituting charged and neutral residues in key regions of LTB and CTB. To confirm that the identified mutations resulted specifically in a secretion defect, we tested the effect of the mutations on (i) ligand binding by each toxin, (ii) toxin stability, and (iii) formation of secretion-competent B-subunit pentamers. By introducing comparable mutations into both toxins, including one previously reported to impair the secretion of CT (6), and exchanging toxin substrates between the two species, we have revealed species-dependent differences in T2S substrate recognition. Although wild-type LT and CT can be heterologously expressed and secreted from V. cholerae and ETEC, respectively, the substrate residues identified by the secretion machinery in each species are distinct. Together, our results demonstrate that highly homologous T2S substrates are recognized in different ways when secreted by two distinct systems.     

Species loss of stoneflies (Plecoptera) in the Czech Republic during the 20th century

1. Rapid expansion and intensification of anthropogenic activities in the 20th century has caused profound changes in freshwater assemblages. Unfortunately, knowledge of the extent and causes of species loss (SL) is limited due to the lack of reliable historical data. An unusual data set allows us to compare changes in the most sensitive of aquatic insect orders, the Plecoptera, at some 170 locations in the Czech Republic between two time periods, 1955–1960 and 2006–2010. Historical data (1890–1911) on assemblages of six lowland rivers allow us to infer even earlier changes. 2. Regional stonefly diversity decreased in the first half of the 20th century. Streams at lower altitudes lost a substantial number of species, which were never recovered. In the second half of the century, large‐scale anthropogenic pressure caused SL in all habitats, leading to a dissimilarity of contemporary and previous assemblages. The greatest changes were found at sites affected by organic pollution and a mixture of organic pollution and channelisation or impoundment. Colonisation of new habitats was observed in only three of the 80 species evaluated. 3. Species of moderate habitat specialisation and tolerance to organic pollution were most likely to be lost. Those with narrow specialisations in protected habitats were present in both historical and contemporary collections. 4. Contemporary assemblages are the consequence of more than a 100 years of anthropogenic impacts. In particular, streams at lower altitude and draining intensively exploited landscapes host a mere fragment of the original species complement. Most stonefly species are less frequently present than before, although their assemblages remain almost intact in near‐natural mountain streams. Our analyses demonstrate dramatic restriction of species ranges and, in some cases, apparent changes in altitudinal preference throughout the area.     

Evidence of hexaploid karyotype in shortnose sturgeon

A karyotype analysis by several staining techniques was carried out on triplicate samples of the shortnose sturgeon, Acipenser brevirostrum. The chromosome number was found to be 2n = 372 +/- 6. A representative karyotype of 374 chromosomes was composed of 178 metacentrics/submetacentrics and 196 telocentrics/acrocentrics and microchromosomes. The signals of fluorescent in situ hybridization (FISH) with a HindIII satellite DNA probe were visible on 14 chromosomes. The signals obtained with a PstI satellite DNA probe appeared on 12 chromosomes. The FISH with a 5S rDNA probe revealed fluorescent signals on 6 chromosomes. These last results, compared with 2 signals in species with about 120 chromosomes and 4 in species with 240, support the hypothesis that A. brevirostrum is a hexaploid species, probably of hybrid origin. Based on these results, we propose a model explaining speciation events occurring in sturgeons by hybridization, genome duplication, and diploidization.     

Residues of Heat-Labile Enterotoxin Involved in Bacterial Cell Surface Binding

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of traveler''s diarrhea worldwide. One major virulence factor released by this pathogen is the heat-labile enterotoxin LT, which upsets the balance of electrolytes in the intestine. After export, LT binds to lipopolysaccharide (LPS) on the bacterial surface. Although the residues responsible for LT''s binding to its host receptor are known, the portion of the toxin which mediates LPS binding has not been defined previously. Here, we describe mutations in LT that impair the binding of the toxin to the external surface of E. coli without altering holotoxin assembly. One mutation in particular, T47A, nearly abrogates surface binding without adversely affecting expression or secretion in ETEC. Interestingly, T47A is able to bind mutant E. coli expressing highly truncated forms of LPS, indicating that LT binding to wild-type LPS may be due primarily to association with an outer core sugar. Consequently, we have identified a region of LT distinct from the pocket involved in eukaryotic receptor binding that is responsible for binding to the surface of E. coli.Enterotoxigenic Escherichia coli (ETEC), a common etiologic agent behind traveler''s diarrhea, is also a significant cause of mortality worldwide (38). Many strains of ETEC elaborate a virulence factor called heat-labile enterotoxin or LT (34). LT is an AB₅ toxin, consisting of a single A subunit, LTA, and a ring of five B subunits, LTB (33). LTB mediates the toxin''s binding properties, and LTA ADP ribosylates host G proteins, increasing levels of cyclic AMP and causing the efflux of electrolytes and water into the intestinal lumen (27, 35). Each subunit of LT is translated separately from a bicistronic message and then transported to the periplasm, where holotoxin assembly spontaneously occurs (16). Subsequent export into the extracellular milieu is carried out by the main terminal branch of the general secretory pathway (31, 36).LT binds eukaryotic cells via an interaction between LTB and host gangliosides, primarily the monosialoganglioside GM₁ (35). The binding site for GM₁, situated at the interface of two B subunits, has been identified by crystallography (26). GM₁ binding can be strongly impaired by a point mutation in LTB that converts Gly-33 to an aspartic acid residue (37). LT is highly homologous to cholera toxin (CT), both in sequence and structure (7, 35), contributing to ETEC''s potentially cholera-like symptoms (39).Previous work in our lab has demonstrated that LT possesses an additional binding capacity beyond its affinity for host glycolipids: the ability to associate with lipopolysaccharide (LPS) on the surface of E. coli (20). LPS, the major component of the outer leaflet of the gram-negative outer membrane, consists of a characteristic lipid moiety, lipid A, covalently linked to a chain of sugar residues (30). In bacteria like E. coli, this sugar chain can be further divided into an inner core oligosaccharide of around five sugars, an outer core of four to six additional sugars, and in some cases a series of oligosaccharide repeats known as the O antigen. Lipid A itself cannot inhibit binding of soluble LT to cells containing full-length or truncated LPS, indicating that the LT-LPS interaction involves sugar residues on the surface of E. coli (19). The addition of the inner core sugar 3-deoxy-d-manno-octulosonic acid (Kdo) is the minimal lipid A modification required for LT binding, although longer oligosaccharide chains are preferred, and expression of a kinase that phosphorylates Kdo abrogates binding by LT (19). Competitive binding assays and microscopy with fluorescently labeled ETEC vesicles show that binding to GM₁ and LPS can occur at the same time, revealing that the binding sites are distinct (20, 23). In contrast to LT''s ability to bind to the surface of ETEC, CT (or LT, when expressed heterologously) cannot bind Vibrio cells, presumably because Kdo is phosphorylated in Vibrio spp. (5).As a result of the LT-LPS surface interaction, over 95% of secreted LT is found associated with E. coli outer membrane vesicles (OMVs), rather than being secreted solubly (20). OMVs are spherical structures, 50 to 200 nm in diameter, that are derived from the outer membrane but also enclose periplasmic components (24). As such, active LT is found both on the surface of an OMV and within its lumen (21). ETEC releases a large amount of OMVs (40), and these vesicles may serve as vehicles for delivery of LT to host cells.Recent work by Holmner et al. has uncovered a third binding substrate for LT: human blood group A antigen (17, 18). This interaction was noted previously as a novel binding characteristic of artificially constructed CT-LT hybrid molecules, but it has now been shown to occur with wild-type LT as well (17, 18). LTB binding to sugar residues in the receptor molecule occurs at a site that is separate from the GM₁-binding pocket, in the same region we proposed was involved in LPS binding (17, 19). While the severity of cholera disease symptoms has been linked to blood type (14), the effects of blood type on ETEC infection are less clear. However, it has been demonstrated that LT can use A antigen as a functional receptor in cultured human intestinal cells (11, 12), and one recent cohort study found an increased prevalence of ETEC-based diarrhea among children with A or AB blood type (29).We set out to generate a mutation in LT that reduces its LPS binding without adversely affecting its expression, secretion, or toxicity. In this work, we present the discovery of point mutations in LTB that impair its interactions with the bacterial surface. Examination of these mutations reveals an LPS binding pocket which shares residues with the blood sugar pocket. Binding studies of mutants to bacteria with truncated LPS provide a better understanding of the roles that inner and outer core sugars play in toxin binding, and expression, secretion, and toxicity studies demonstrate which mutant is a particularly good candidate for future research. These binding mutants may lead to further discovery of the role that surface binding plays in the pathogenesis associated with ETEC infection.