The effects of audience laughter on men''s and women''s responses to humor

Capsules are well-studied components of the bacterial surface that modulate interactions between the cell and its environment. Generally composed of polysaccharide, they are key virulence determinants in invasive infections in humans and other animals. Genetic determinants involved in capsule expression have been isolated from a number of organisms, but perhaps the best characterized is the kps cluster of Escherichia coli K1. In this review, the current understanding of the functions of the kps gene products is summarized. Further, a proposed mechanistic model for capsule expression is presented and discussed. The model is based on the premise that the numerous components of the kps cluster form a hetero-oligomeric complex responsible for synthesis and concurrent translocation of the capsular polysialic acid through sites of inner and outer membrane fusion. We view the ATP-binding cassette (ABC) transporter, KpsMT, to be central to the functioning of the complex, interacting with the biosynthetic apparatus as well as the extracytoplasmic components of the cluster to co-ordinate synthesis and translocation. The model provides the basis for additional experimentation and reflects emerging similarities among systems responsible for macromolecular export in Gram-negative bacteria.     

Chromosomal regions specific to pathogenic isolates of Escherichia coli have a phylogenetically clustered distribution

We studied the ancestry of virulence-associated genes in Escherichia coli by examining chromosomal regions specific to pathogenic isolates. The four virulence determinants examined were the alpha-hemolysin (hly) loci hlyI and hlyII, the type II capsule gene cluster kps, and the P (pap) and S (sfa) fimbria gene clusters. All four loci were shown previously to be associated with pathogenicity islands of uropathogenic E. coli isolates. The hly, kps, sfa, and pap regions each have an unexpected clustered distribution among the E. coli collection of reference (ECOR) strains, but all these regions were absent from a collection of diarrheagenic E. coli isolates. Strains in the ECOR subgroup B2 typically had a combination of at least three of the four loci, and all strains in subgroup D had a copy of the kps and pap clusters. In contrast, only four strains in subgroup A had either hly, kps, sfa, or pap, and no subgroup A strains had all four together. Strains of subgroup B1 were devoid of all four virulence regions, with the exception of one isolate that had a copy of the sfa gene cluster. This phylogenetic distribution of strain-specific sequences corresponds to the ECOR groups with the largest genome size, namely, B2 and D. We propose that the pathogenicity islands are ancestral to subgroups B2 and D and were acquired after speciation, with subsequent horizontal transfer into some group A, B1, and E lineages. These results suggest that the hly, kps, sfa, and pap pathogenicity determinants may play a role in the evolution of enteric bacteria quite apart from, and perhaps with precedence over, their ability to cause disease.     

Identification, genomic organization, and analysis of the group III capsular polysaccharide genes kpsD, kpsM, kpsT, and kpsE from an extraintestinal isolate of Escherichia coli (CP9, O4/K54/H5)

Group III capsular polysaccharides (e.g., K54) of extraintestinal isolates of Escherichia coli, similar to group II capsules (e.g., K1), are important virulence traits that confer resistance to selected host defense components in vitro and potentiate systemic infection in vivo. The genomic organization of group II capsule gene clusters has been established as a serotype-specific region 2 flanked by regions 1 and 3, which contain transport genes that are highly homologous between serotypes. In contrast, the organization of group III capsule gene clusters is not well understood. However, they are defined in part by an absence of genes with significant nucleotide homology to group II capsule transport genes in regions 1 and 3. Evaluation of isogenic, TnphoA-generated, group III capsule-minus derivatives of a clinical blood isolate (CP9, O4/K54/H5) has led to the identification of homologs of the group II capsule transport genes kpsDMTE. These genes and their surrounding regions were sequenced and analyzed. The genomic organization of these genes is distinctly different from that of their group II counterparts. Although kps(K54)DMTE are significantly divergent from their group II homologs at both the DNA and protein levels phoA fusions and computer-assisted analyses suggest that their structures and functions are similar. The putative proteins Kps(K54)M and Kps(K54)T appear to be the integral membrane component and the peripheral ATP-binding component of the ABC-2 transporter family, respectively. The putative Kps(K54)E possesses features similar to those of the membrane fusion protein family that facilitates the passage of large molecules across the periplasm. At one boundary of the capsule gene cluster, a truncated kpsM (kpsM(truncated) and its 5' noncoding regulatory sequence were identified. In contrast to the complete kps(K54)M, this region was highly homologous to the group II kpsM. Fifty-three base pairs 3' from the end of kpsM(truncated) was a sequence 75% homologous to the 39-bp inverted repeat in the IS110 insertion element from Streptomyces coelicolor. Southern analysis established that two copies of this element are present in CP9. These findings are consistent with the hypothesis that CP9 previously possessed group II capsule genes and acquired group III capsule genes via IS110-mediated horizontal transfer.     

Identification of functionally important regions of the muscular chloride channel CIC-1 by analysis of recessive and dominant myotonic mutations

Mutations in the muscular voltage-dependent Cl-channel, CIC-1, lead to recessive and dominant myotonia. Here we analyse the effects of one dominant (G200R) and three recessive (Y150C, Y261C, and M485V) mutations after functional expression in Xenopus oocytes. Glycine 200 is a highly conserved amino acid located in a conserved stretch in the putatively cytoplasmic loop between domains D2 and D3. Similar to several other dominant mutations the amino acid exchange G200R leads to a drastic shift by approximately 65 mV of the open probability curve to more positive voltages. As explored by co-expression studies, the shift is intermediate in heteromeric mutant/WT channels. Open channel properties such as single channel conductance, rectification or ion selectivity are not changed. Thus we identified a new region of the CIC-1 protein in which mutations can lead to drastic shifts of the voltage dependence. The recessive mutation M485V, which is located in a conserved region at the beginning of domain D10, leads to a drastic reduction of the single channel conductance from 1.5 pS for WT to approximately 0.3 pS. In addition, the mutant is strongly inwardly rectifying and deactivates incompletely at negative voltages. Ion-selectivity, however, is unchanged. These electrophysiological properties fully explain the recessive phenotype of the mutation and identify a new region of the protein that is involved in ion permeation and gating of the CIC-1 channel. The other two recessive mutations (Y150C and Y261C) had been found in a compound heterozygous patient. Surprisingly, expression of these mutants in oocytes yielded currents indistinguishable from WT CIC-1 when explored by two-electrode voltage clamp recording and patch clamping (either singly or both mutations co-expressed). Other mechanisms that are not faithfully represented by the Xenopus expression system must therefore be responsible for the myotonic symptoms associated with these mutations.     

Cloning of a lectin cDNA and seasonal changes in levels of the lectin and its mRNA in the inner bark of Robinia pseudoacacia

E. coli cells harboring pCG169 containing the secD secF locus possessed SecA protein almost entirely in an integral membrane form in which it displayed normal protein translocation activity. These results imply that integral membrane SecA is the catalytically active form of this enzyme and that products of the secD secF locus regulate SecA association with the inner membrane. Protease and biotinylation accessibility studies of right side-out and inside-out membrane vesicles derived from this strain revealed that SecA was exposed to the periplasmic surface of the inner membrane. These studies suggest a model of bacterial protein secretion, whereby insertion of SecA into the inner membrane and its association with SecY/E/G promotes assembly of active protein-conducting channels comprised in part of integral membrane SecA protein, and products of the secD secF locus regulate the channel assembly-disassembly reaction by modulating the SecA insertion-deinsertion step.     

Reconstitution of the protein insertion machinery of the mitochondrial inner membrane

We have reconstituted the protein insertion machinery of the yeast mitochondrial inner membrane into proteoliposomes. The reconstituted proteoliposomes have a distinct morphology and protein composition and correctly insert the ADP/ATP carrier (AAC) and Tim23p, two multi-spanning integral proteins of the mitochondrial inner membrane. The reconstituted system requires a membrane potential, but not Tim44p or mhsp70, both of which are required for the ATP-driven translocation of proteins into the matrix. The protein insertion machinery can thus operate independently of the energy-transducing Tim44p-mhsp70 complex.     

Total absence of protein 4.2 and partial deficiency of band 3 in hereditary spherocytosis

Unlike previously reported cases with total protein 4.2 deficiency due to mutations in the EPB42 gene, we describe a total deficiency in protein 4.2 with normal EPB42 alleles. Hereditary spherocytosis (HS) was observed in a Japanese woman (unsplenectomized) and her daughter (splenectomized). The mother showed a partial deficiency in band 3 and a proportional reduction in protein 4.2. She was heterozygous for a novel allele of the EPB3 gene, allele Okinawa, which contains the two mutations that define the Memphis II polymorphism (K56E, AAG-->GAG, and P854L, CCG-->CTG) and, additionally, the mutation: G714R, GGG-->AGG, located in a highly conserved position of transmembrane segment 9. The latter change was responsible for HS. In trans to allele Okinawa, the daughter displayed allele Fukuoka: G130R, GGA-->AGA, an allele known to alter the binding of protein 4.2 to band 3. The daughter presented with a more pronounced decrease of band 3, and lacked protein 4.2, resulting in aggravated haemolytic features. Although the father was not available for study, heterozygosity for allele Fukuoka has been documented in another individual who showed no clinical or haematological signs, and a normal content of band 3. We suggest that band 3 Okinawa binds virtually all the protein 4.2 in red cell precursors, band 3 Fukuoka being unable to do so, and that the impossibility of band 3 Okinawa incorporation into the membrane leads to degradation of the band 3 Okinawa protein 4.2 complex. In contrast, band 3 Fukuoka, free of bound protein 4.2, could then incorporate normally into the bilayer. Thus, protein 4.2 would not appear in the daughter's red cell membrane.     

The Escherichia coli SRP and SecB targeting pathways converge at the translocon

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.     

UGA suppressor that maps within a cluster of ribosomal protein genes

A suppressor of UGA mutations (supU) maps near or within a cluster of ribosomal protein genes at 72 min on the Salmonella typhimurium genetic map. The suppressor is relatively inefficient, and its activity is abolished by rpsL (formerly strA) mutations. The suppressor is dominant to a wild-type supU allele. The map position of this suppressor suggests that it may owe its activity to an alteration of ribosome structure.     

A GTP-dependent "push" is generally required for efficient protein translocation across the mitochondrial inner membrane into the matrix

Mitochondrial biogenesis requires translocation of numerous preproteins across both outer and inner membranes into the matrix of the organelle. This translocation process requires a membrane potential (DeltaPsi) and ATP. We have recently demonstrated that the efficient import of a urea-denatured preprotein into the matrix requires GTP hydrolysis (Sepuri, N. B. V., Schülke, N., and Pain, D. (1998) J. Biol. Chem. 273, 1420-1424). We now demonstrate that GTP is generally required for efficient import of various preproteins, both native and urea-denatured. The GTP participation is localized to a particular stage in the protein import process. In the presence of DeltaPsi but no added nucleoside triphosphates, the transmembrane movement of preproteins proceeds only to a point early in their translocation across the inner membrane. The completion of translocation into the matrix is independent of DeltaPsi but is dependent on a GTP-mediated "push." This push is likely mediated by a membrane-bound GTPase on the cis side of the inner membrane. This conclusion is based on two observations: (i) GTP does not readily cross the inner membrane barrier and hence, primarily acts outside the inner membrane to stimulate import, and (ii) the GTP-dependent stage of import does not require soluble constituents of the intermembrane space and can be observed in isolated mitoplasts. Efficient import into the matrix, however, is achieved only through the coordinated action of a cis GTP-dependent push and a trans ATP-dependent "pull."     

Cytoplasmic loop three of cystic fibrosis transmembrane conductance regulator contributes to regulation of chloride channel activity

To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three point-mutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Chinese hamster ovary cell lines stably expressing wild-type CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the protein. Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26 degrees C) or incubated in the presence of 10% glycerol. For all mutants, the open probability (P0) of the CFTR channels was significantly altered. S945L-CFTR and G970R-CFTR showed a severe reduction in the P0, whereas the H949Y mutation doubled the P0 relative to wild-type. The changes in P0 predominantly resulted from an alteration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had current-voltage relationships that were not completely linear over the range +/-80 mV, but showed slight outward rectification. The fact that CL3 mutations can have subtle effects on channel conductance indicates that this region may be physically close to the inner mouth of the pore.     

Inhibition of breast cancer in nude mouse model by anti-angiogenesis

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pell. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning.     

Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a

Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens.     

The oligomycin sensitivity conferring protein of rat liver mitochondrial ATP synthase: arginine 94 is important for the binding of OSCP to F1

The oligomycin sensitivity conferring protein (OSCP) is an essential subunit of the mitochondrial ATP synthase (F0F1) long regarded as being directly involved in the energetic coupling of proton transport to ATP synthesis. To gain insight into the function of OSCP, mutations were made in a highly conserved central region of the subunit, and the recombinant proteins were studied using several biochemical assays. Rat liver OSCP was expressed to high levels in Escherichia coli, solubilized from inclusion bodies, renatured, and purified to homogeneity. The recombinant protein was able to reconstitute oligomycin-sensitive ATPase activity to inner membrane vesicles depleted of F1 and OSCP, and bound to F1 with a stoichiometry of 1:1. A novel fluorescence anisotropy assay was developed to study the affinity of binding of F1 to OSCP, providing a Kd value of 51 +/- 11 nM. Two highly conserved, charged residues (E91 and R94) which lie within the central region of OSCP were mutated, and the recombinant proteins (E91Q, R94Q, and R94A) were purified to homogeneity and judged by CD spectroscopy to have structures similar to that of the wild-type protein. Both R94 mutants demonstrated little or no binding to F1, while the E91Q bound in a manner identical to that of wild-type OSCP. Significantly, all three mutant proteins were able to reconstitute F1 with membranes and to confer oligomycin sensitivity to the same extent as wild-type OSCP. These results demonstrate that a single tight binding site exists on isolated rat liver F1 for OSCP, and implicate arginine 94 as playing a critical role in this site. In addition, these results indicate that this tight binding site is not required for conferral of oligomycin sensitivity to the reconstituted F0F1 complex.     

Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion

Escherichia coli biotin ligase is a cytoplasmic protein which specifically biotinylates the biotin-accepting domains from a variety of organisms. This in vivo biotinylation can be used as a sensitive signal to study protein secretion and membrane protein insertion. When the biotin-accepting domain from the 1.3S subunit of Propionibacterium shermanii transcarboxylase (PSBT) is translationally fused to the periplasmic proteins alkaline phosphatase and maltose-binding protein, there is little or no biotinylation of PSBT in wild-type E. coli. Inhibition of SecA with sodium azide and mutations in SecB, SecD, and SecF, all of which slow down protein secretion, result in biotinylation of PSBT. When PSBT is fused to the E. coli inner membrane protein MalF, it acts as a topological marker: fusions to cytoplasmic domains of MalF are biotinylated, and fusions to periplasmic domains are generally not biotinylated. If SecA is inhibited by sodium azide or if the SecE in the cell is depleted, then the insertion of the MalF second periplasmic domain is slowed down enough that PSBT fusions in this part of the protein become biotinylated. Compared with other protein fusions that have been used to study protein translocation, PSBT fusions have the advantage that they can be used to study the rate of the insertion process.     

Mathematical treatment of the kinetics of binding protein dependent transport systems reveals that both the substrate loaded and unloaded binding proteins interact with the membrane components

Binding-protein-dependent transport systems in Gram-negative bacteria are multicomponent systems in which a soluble periplasmic binding protein of high substrate binding affinity establishes the major substrate recognition site. Usually, there are two membrane proteins which are thought to interact with the substrate loaded form of the binding protein to allow transport of substrate to occur. Transport is against the concentration gradient and needs energization by an ATP hydrolyzing polypeptide. Overall transport is considered mainly unidirectional owing to the high energy of ATP hydrolysis coupled to transport. We dissected the overall transport process into three individual steps: (i) reversible binding of substrate to the binding protein; (ii) reversible binding of the binding protein to the membrane components forming the translocation complex; (iii) irreversible transport of substrate through the membrane and dissociation of the binding protein from the complex. Two models were considered. In the first, only the substrate-loaded binding protein interacts with the membrane components, while in the second model both the loaded and the unloaded form of the binding protein interact with the membrane components. The mathematical analysis of the second model revealed that the substrate concentration KM at half-maximal rate of transport approaches KD of the binding protein when the last step of transport becomes low and when the concentration of binding protein in the periplasm becomes large. This is usually observed in real systems. Under the same conditions, in model 1 KM approaches zero and is hence considerably smaller than KD. This has never been observed in any real system. In addition, the dependence of the overall rate of transport on the concentration of binding protein in the periplasm follows a sigmoidal curve only when model 2 is considered. The sigmoidal behavior becomes more pronounced when the substrate concentration is low and it is less pronounced when the last step in overall transport is low. This phenomenon has been observed with the Escherichia coli maltose transport system. Thus, at least for the maltose transport system, it seems likely that both the loaded and the unloaded forms of the binding protein interact with the membrane components. We propose that this should generally be considered in binding-protein-dependent transport systems.     

GTP hydrolysis is essential for protein import into the mitochondrial matrix

Protein import into the innermost compartment of mitochondria (the matrix) requires a membrane potential (delta psi) across the inner membrane, as well as ATP-dependent interactions with chaperones in the matrix and cytosol. The role of nucleoside triphosphates other than ATP during import into the matrix, however, remains to be determined. Import of urea-denatured precursors does not require cytosolic chaperones. We have therefore used a purified and urea-denatured preprotein in our import assays to bypass the requirement of external ATP. Using this modified system, we demonstrate that GTP stimulates protein import into the matrix; the stimulatory effect is directly mediated by GTP hydrolysis and does not result from conversion of GTP to ATP. Both external GTP and matrix ATP are necessary; neither one can substitute for the other if efficient import is to be achieved. These results suggest a "push-pull" mechanism of import, which may be common to other post-translational translocation pathways.     

Tim9, a new component of the TIM22.54 translocase in mitochondria

We have identified Tim9, a new component of the TIM22.54 import machinery, which mediates transport of proteins into the inner membrane of mitochondria. Tim9, an essential protein of Saccharomyces cerevisiae, shares sequence similarity with Tim10 and Tim12. Tim9 is located in the mitochondrial intermembrane space and is organized into two distinct hetero-oligomeric assemblies with Tim10 and Tim12. One complex contains Tim9 and Tim10. The other complex contains Tim9, Tim10 and Tim12 and is tightly associated with Tim22 in the inner membrane. The TIM9.10 complex is more abundant than the TIM9.10.12 complex and mediates partial translocation of mitochondrial carriers proteins across the outer membrane. The TIM9.10.12 complex assists further translocation into the inner membrane in association with TIM22.54.