bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteomic and Immunoblot Analyses of
Bartonella quintana
Total Membrane Proteins Identify Antigens Recognized by Sera from Infected Patients
Authors: Boonjakuakul Jenni K. , Gerns Helen L. , Chen Yu-Ting , Hicks Linda D. , Minnick Michael F. , Dixon Scott E. , Hall Steven C. , Koehler Jane E.
Published: February 2007

Bartonella quintana
is a fastidious, gram-negative, rod-shaped bacterium that causes prolonged bacteremia in immunocompetent humans and severe infections in immunocompromised individuals. We sought to define the outer membrane subproteome of
B. quintana
in order to obtain insight into the biology and pathogenesis of this emerging pathogen and to identify the predominant
B. quintana
antigens targeted by the human immune system during infection. We isolated the total membrane proteins of
B. quintana
and identified 60 proteins by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mass fingerprinting. Using the newly constructed proteome map, we then utilized two-dimensional immunoblotting with sera from 21
B. quintana
-infected patients to identify 24 consistently recognized, immunoreactive
B. quintana
antigens that have potential relevance for pathogenesis and diagnosis. Among the outer membrane proteins, the
v
ariably expressed
o
uter
m
embrane
p
rotein adhesins (VompA and VompB), peptidyl-prolyl
cis-trans
-isomerase (PpI), and
h
emin-
b
inding
p
rotein
E
(HbpE) were recognized most frequently by sera from patients, which is consistent with surface expression of these virulence factors during human infection.
Repression of Intracellular Virulence Factors in
Salmonella
by the Hha and YdgT Nucleoid-Associated Proteins
Authors: Silphaduang Umaporn , Mascarenhas Mariola , Karmali Mohamed , Coombes Brian K.
Published: February 2007

The Hha/YmoA family of nucleoid-associated proteins is involved in gene regulation in enterobacteria. In
Salmonella enterica
serovar Typhimurium, virulence genes required for intracellular growth are induced following host cell invasion but the proteins responsible for repressing these genes prior to host cell entry have not been fully identified. We demonstrate here that Hha is the major repressor responsible for silencing virulence genes carried in
Salmonella
pathogenicity island 2 prior to bacteria sensing an intracellular environmental cue.
A Riboswitch Regulates Expression of the Coenzyme B
12
-Independent Methionine Synthase in
Mycobacterium tuberculosis
: Implications for Differential Methionine Synthase Function in Strains H37Rv and CDC1551
Authors: Warner Digby F. , Savvi Suzana , Mizrahi Valerie , Dawes Stephanie S.
Published: February 2007

We observed vitamin B
12
-mediated growth inhibition of
Mycobacterium tuberculosis
strain CDC1551. The B
12
sensitivity was mapped to a polymorphism in
metH
, encoding a coenzyme B
12
-dependent methionine synthase. Vitamin B
12
-resistant suppressor mutants of CDC1551 containing mutations in a B
12
riboswitch upstream of the
metE
gene, which encodes a B
12
-independent methionine synthase, were isolated. Expression analysis confirmed that the B
12
riboswitch is a transcriptional regulator of
metE
in
M. tuberculosis
.
Role of PBP1 in Cell Division of
Staphylococcus aureus
Authors: Pereira S. F. F. , Henriques A. O. , Pinho M. G. , de Lencastre H. , Tomasz A.
Published: February 2007

We constructed a conditional mutant of
pbpA
in which transcription of the gene was placed under the control of an IPTG (isopropyl-β-
d
-thiogalactopyranoside)-inducible promoter in order to explore the role of PBP1 in growth, cell wall structure, and cell division. A methicillin-resistant strain and an isogenic methicillin-susceptible strain, each carrying the
pbpA
mutation, were unable to grow in the absence of the inducer. Conditional mutants of
pbpA
transferred into IPTG-free medium underwent a four- to fivefold increase in cell mass, which was not accompanied by a proportional increase in viable titer. Examination of thin sections of such cells by transmission electron microscopy or fluorescence microscopy of intact cells with Nile red-stained membranes showed a morphologically heterogeneous population of bacteria with abnormally increased sizes, distorted axial ratios, and a deficit in the number of cells with completed septa. Immunofluorescence with an antibody specific for PBP1 localized the protein to sites of cell division. No alteration in the composition of peptidoglycan was detectable in
pbpA
conditional mutants grown in the presence of a suboptimal concentration of IPTG, which severely restricted the rate of growth, and the essential function of PBP1 could not be replaced by PBP2A present in methicillin-resistant cells. These observations suggest that PBP1 is not a major contributor to the cross-linking of peptidoglycan and that its essential function must be intimately integrated into the mechanism of cell division.
Inactivation of the Mismatch Repair System in
Pseudomonas aeruginosa
Attenuates Virulence but Favors Persistence of Oropharyngeal Colonization in Cystic Fibrosis Mice
Authors: Mena Ana , Maciá María D. , Borrell Nuria , Moya Bartolomé , de Francisco Teresa , Pérez José L. , Oliver Antonio
Published: February 2007

The inactivation of the mismatch repair (MMR) system of
Pseudomonas aeruginosa
modestly reduced in vitro fitness, attenuated virulence in murine models of acute systemic and respiratory infections, and decreased the initial oropharyngeal colonization potential. In contrast, the inactivation of the MMR system favored long-term persistence of oropharyngeal colonization in cystic fibrosis mice. These results may help in understanding the reasons for the low and high prevalences, respectively, of hypermutable
P. aeruginosa
strains in acute and chronic infections.
Reconstitution of the Biochemical Activities of the AttJ Repressor and the AttK, AttL, and AttM Catabolic Enzymes of
Agrobacterium tumefaciens
Authors: Chai Yunrong , Tsai Ching Sung , Cho Hongbaek , Winans Stephen C.
Published: February 2007

The
attKLM
operon encodes a lactonase (AttM) that hydrolyzes acylhomoserine lactone autoinducers, as well as two putative dehydrogenases (AttK and AttL). Here we show that AttK, AttL, and AttM collectively covert gamma-butyrolactone to succinate. Two metabolic intermediates, gamma-hydroxybutyrate and succinic semialdehyde, inactivated the AttJ repressor in vitro and induced
attKLM
transcription in vivo.
Interaction between Cell Division Proteins FtsE and FtsZ
Authors: Corbin Brian D. , Wang Yipeng , Beuria Tushar K. , Margolin William
Published: February 2007

FtsE and FtsX, which are widely conserved homologs of ABC transporters and interact with each other, have important but unknown functions in bacterial cell division. Coimmunoprecipitation of
Escherichia coli
cell extracts revealed that a functional FLAG-tagged version of FtsE, the putative ATP-binding component, interacts with FtsZ, the bacterial tubulin homolog required to assemble the cytokinetic Z ring and recruit the components of the divisome. This interaction is independent of FtsX, the predicted membrane component of the ABC transporter, which has been shown previously to interact with FtsE. The interaction also occurred independently of FtsA or ZipA, two other
E. coli
cell division proteins that interact with FtsZ. In addition, FtsZ copurified with FLAG-FtsE. Surprisingly, the conserved C-terminal tail of FtsZ, which interacts with other cell division proteins, such as FtsA and ZipA, was dispensable for interaction with FtsE. In support of a direct interaction with FtsZ, targeting of a green fluorescent protein (GFP)-FtsE fusion to Z rings required FtsZ, but not FtsA. Although GFP-FtsE failed to target Z rings in the absence of ZipA, its localization was restored in the presence of the
ftsA
* bypass suppressor, indicating that the requirement for ZipA is indirect. Coexpression of FLAG-FtsE and FtsX under certain conditions resulted in efficient formation of minicells, also consistent with an FtsE-FtsZ interaction and with the idea that FtsE and FtsX regulate the activity of the divisome.
Cross Talk between Type III Secretion and Flagellar Assembly Systems in
Pseudomonas aeruginosa
Authors: Soscia Chantal , Hachani Abderrahman , Bernadac Alain , Filloux Alain , Bleves Sophie
Published: February 2007

Pseudomonas aeruginosa
cytotoxicity is linked to a type III secretion system (T3SS) that delivers effectors into the host cell. We show here that a negative cross-control exists between T3SS and flagellar assembly. We observed that, in a strain lacking flagella, T3SS gene expression, effector secretion, and cytotoxicity were increased. Conversely, we revealed that flagellar-gene expression and motility were decreased in a strain overproducing ExsA, the T3SS master regulator. Interestingly, a nonmotile strain lacking the flagellar filament (Δ
fliC
) presented a hyperefficient T3SS and a nonmotile strain assembling flagella (Δ
motAB
) did not. More intriguingly, a strain lacking
motCD
genes is a flagellated strain with a slight defect in swimming. However, in this strain, T3SS gene expression was up-regulated. These results suggest that flagellar assembly and/or mobility antagonizes the T3SS and that a negative cross talk exists between these two systems. An illustration of this is the visualization by electron microscopy of T3SS needles in a nonmotile
P. aeruginosa
strain, needles which otherwise are not detected. The molecular basis of the cross talk is complex and remains to be elucidated, but proteins like MotCD might have a crucial role in signaling between the two processes. In addition, we found that the GacA response regulator negatively affects the T3SS. In a
gacA
mutant, the T3SS effector ExoS is hypersecreted. Strikingly, GacA was previously reported as a positive regulator for motility. Globally, our data document the idea that some virulence factors are coordinately but inversely regulated, depending on the bacterial colonization phase and infection types.
Transcriptional Regulation of the CO
2
-Concentrating Mechanism in a Euryhaline, Coastal Marine Cyanobacterium,
Synechococcus
sp. Strain PCC 7002: Role of NdhR/CcmR
Authors: Woodger Fiona J. , Bryant Donald A. , Price G. Dean
Published: February 2007

Cyanobacterial photosynthesis occurs in radically diverse habitats and utilizes various forms of a CO
2
-concentrating mechanism (CCM) featuring multiple inorganic carbon (C
i
) transporters. Cyanobacteria from dynamic environments can transform CCM activity depending on C
i
availability, and yet the molecular basis for this regulation is unclear, especially in coastal strains. LysR family transcription factors resembling the Calvin cycle regulator CbbR from proteobacteria have been implicated in the expression of C
i
transporter genes in freshwater cyanobacteria. Our survey of related factors revealed a group of divergent CbbR-like sequences confined to freshwater and coastal or offshore cyanobacteria. Inactivation of the single gene (termed
ccmR
) from this variable cluster in the euryhaline (coastal) strain
Synechococcus
sp. strain PCC 7002 led to constitutive expression of a high-affinity CCM. Derepression of HCO
3
−
transporter gene transcription, including that of BicA, a recently discovered HCO
3
−
transporter (G. D. Price et al., Proc. Natl. Acad. Sci. USA
101
:18228-18233, 2004), was observed. A unique CcmR-regulated operon containing
bicA
plus 9 open reading frames encoding likely Na
+
/H
+
antiporters from the CPA1 and Mnh families was defined that is essential for maximal HCO
3
−
-dependent oxygen evolution. The promoter region required for C
i
-regulated transcription of this operon was defined. We propose that CcmR (and its associated regulon) represents a specialization for species inhabiting environments subject to fluctuating C
i
concentrations.
Complete Genome Sequence of the Prototype Lactic Acid Bacterium
Lactococcus lactis
subsp.
cremoris
MG1363
Authors: Wegmann Udo , O'Connell-Motherway Mary , Zomer Aldert , Buist Girbe , Shearman Claire , Canchaya Carlos , Ventura Marco , Goesmann Alexander , Gasson Michael J. , Kuipers Oscar P. , van Sinderen Douwe , Kok Jan
Published: February 2007

Lactococcus lactis
is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of
Lactococcus lactis
subsp.
cremoris
MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category “carbohydrate metabolism and transport,” by far the largest category of novel proteins in comparison with
L. lactis
subsp.
lactis
IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the “lateral gene transfer hot spot” in the genome of
Streptococcus thermophilus
. Although the parent of
L. lactis
MG1363 was used to demonstrate lysogeny in
Lactococcus
,
L. lactis
MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the
L. lactis
MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.
Lipolytic Enzymes inMyxococcus xanthus
Authors: Moraleda-Muñoz Aurelio , Shimkets Lawrence J.
Published: February 2007

The genome ofMyxococcus xanthusencodes lipolytic enzymes in three different families: patatin lipases, α/β hydrolases, and GDSL lipases. One member of each family was characterized. The protein encoded by MXAN_3852 contains motifs characteristic of patatins. MXAN_5522 encodes a protein with the G-X-S-X-G motif characteristic of the lipase subfamily of α/β hydrolases. MXAN_4569 encodes a member of the GDSL family of lipolytic enzymes. Strains with deletions of MXAN_5522 and MXAN_4569 undergo faster development and earlier myxospore formation than the wild-type strain. The MXAN_5522 mutation results in spore yields substantially higher than those seen for wild-type cells. Gene expression analysis using translationallacZfusions indicates that while all three genes are expressed during development, only MXAN_5522 and MXAN_4569 are expressed during vegetative growth. The proteins encoded by these genes were overexpressed using a T7 RNA polymerase transcription (pET102/D-TOPO) system inEscherichia coliBL21 Star (DE3) cells. The substrate specificities of the purified enzymes were investigated usingp-nitrophenyl esters with chain lengths from C2to C16. These enzymes preferentially hydrolyzed esters of short-chain fatty acids, yielding the highest activity withp-nitrophenyl acetate.
H-NS Antagonism inShigella flexneriby VirB, a Virulence Gene Transcription Regulator That Is Closely Related to Plasmid Partition Factors
Authors: Turner Elizebeth C. , Dorman Charles J.
Published: February 2007

The VirB protein ofShigella flexneriis a positive regulator of the major virulence operons of this enteroinvasive intracellular pathogen. VirB resembles no other transcription factor but is strongly homologous to plasmid partition proteins. We found that the binding of the VirB protein to the promoter region of theicsBvirulence gene induced hypersensitivity to cleavage by DNase I over a region to which the H-NS repressor protein binds and completely abolished the protection of this sequence from DNase I by H-NS. In the absence of H-NS, the VirB protein had no additive effect on the ability of theicsBpromoter to form an open transcription complex, indicating that VirB is not involved in the recruitment of RNA polymerase to the promoter or in open complex formation. Similarly, VirB did not stimulate promoter function in an in vitro transcription assay but acted as an antagonist of H-NS-mediated repression. A sequence located upstream of theicsBpromoter and related tocis-acting elements involved in plasmid partitioning was required for promoter derepression by VirB. Alterations to one heptameric motif within this DNA sequence attenuated VirB binding and derepression oficsBtranscription.
Mycoplasma hyopneumoniae
mhp379 Is a Ca
2+
-Dependent, Sugar-Nonspecific Exonuclease Exposed on the Cell Surface
Authors: Schmidt Jonathan A. , Browning Glenn F. , Markham Philip F.
Published: February 2007

Mycoplasma hyopneumoniae
mhp379 is a putative lipoprotein that shares significant amino acid sequence similarity with a family of bacterial thermostable nucleases. To examine the nuclease activity of mhp379, the gene was cloned and expressed in
Escherichia coli
following the deletion of the amino-terminal signal sequence and prokaryotic lipoprotein cleavage site and mutagenesis of the mycoplasma TGA tryptophan codons to TGG. The recombinant fusion protein yielded a 33-kDa thrombin cleavage product, corresponding in size to the mature mhp379 protein. Exonuclease activity was indicated by agarose gel electrophoresis analysis of the reaction products that were released when different nucleic acid substrates were used. Endonuclease activity was also indicated by the digestion of closed circular plasmid DNA. The recombinant mhp379 fusion protein completely digested single-stranded DNA, double-stranded DNA (dsDNA), and RNA. The optimal reaction conditions were determined with a novel nuclease assay based on the enhancement of fluorescence of SYBR green I bound to dsDNA. Optimal activity was observed in the presence of calcium ions at a concentration of 15 mM and a pH of 9.5. No nuclease activity was observed in the absence of calcium ions. Mycoplasmas do not have the ability to synthesize nucleic acid precursors, and thus, nucleases are likely to be important in the acquisition of precursors for the synthesis of nucleic acids. Homologs of an ATP-binding cassette (ABC) transport system were identified immediately downstream of the gene encoding mhp379, and two homologs of
M. pneumoniae
lipoprotein multigene family 2 were also identified immediately upstream. Homologs of mhp379 were identified in the sequenced genomes of a number of mycoplasma species, and in most cases the homologous ABC transport system was identified immediately downstream of the homologous gene; in several cases a homolog of
M. pneumoniae
lipoprotein multigene family 2 was also identified immediately upstream. These observations suggest that mhp379 comprises part of a conserved ABC transport operon in mycoplasmas and that the exonuclease activity of mhp379 may be associated with the conserved function of the ABC transport system in the import of nucleic acid precursors. This is the first study to identify the gene and characterize the activity of a mycoplasma exonuclease.
Characterization of PPT
Ns
, a Cyanobacterial Phosphopantetheinyl Transferase from
Nodularia spumigena
NSOR10
Authors: Copp J. N. , Roberts A. A. , Marahiel M. A. , Neilan B. A.
Published: February 2007

The phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds, including fatty acids, polyketides, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by transfer of a phosphopantetheinyl moiety. The diverse PPT superfamily can be divided into two families based on specificity and conserved sequence motifs. The first family is typified by the
Escherichia coli
acyl carrier protein synthase (AcpS), which is involved in fatty acid synthesis. The prototype of the second family is the broad-substrate-range PPT Sfp, which is required for surfactin biosynthesis in
Bacillus subtilis
. Most cyanobacteria do not encode an AcpS-like PPT, and furthermore, some of their Sfp-like PPTs belong to a unique phylogenetic subgroup defined by the PPTs involved in heterocyst differentiation. Here, we describe the first functional characterization of a cyanobacterial PPT based on a structural analysis and subsequent functional analysis of the
Nodularia spumigena
NSOR10 PPT. Southern hybridizations suggested that this enzyme may be the only PPT encoded in the
N. spumigena
NSOR10 genome. Expression and enzyme characterization showed that this PPT was capable of modifying carrier proteins resulting from both heterocyst glycoplipid synthesis and nodularin toxin synthesis. Cyanobacteria are a unique and vast source of bioactive metabolites; therefore, an understanding of cyanobacterial PPTs is important in order to harness the biotechnological potential of cyanobacterial natural products.
Effect of Salt Shock on Stability of λ

imm434

Lysogens
Authors: Shkilnyj Paul , Koudelka Gerald B.
Published: February 2007

The affinities of the bacteriophage 434 repressor for its various binding sites depend on the type and/or concentration of monovalent cations. The ability of bacteriophage 434 repressor to govern the lysis-lysogeny decision depends on the DNA binding activities of the phage's
c
I repressor protein. We wished to determine whether changes in the intracellular ionic environment influence the lysis-lysogeny decision of the bacteriophage λ

imm434

. Our findings show that the ionic composition within bacterial cells varies with the cation concentration in the growth media. When λ

imm434

lysogens were grown to mid-log or stationary phase and subsequently incubated in media with increasing monovalent salt concentrations, we observed a salt concentration-dependent increase in the frequency of bacteriophage spontaneous induction. We also found that the frequency of spontaneous induction varied with the type of monovalent cation in the medium. The salt-dependent increase in phage production was unaffected by a
recA
mutation. These findings indicate that the salt-dependent increase in phage production is not caused by activation of the SOS pathway. Instead, our evidence suggests that salt stress induces this lysogenic bacteriophage by interfering with 434 repressor-DNA interactions. We speculate that the salt-dependent increase in spontaneous induction is due to a direct effect on the repressor's affinity for DNA. Regardless of the precise mechanism, our findings demonstrate that salt stress can regulate the phage lysis-lysogeny switch.
The SXT/R391 Family of Integrative Conjugative Elements Is Composed of Two Exclusion Groups
Authors: Marrero Joeli , Waldor Matthew K.
Published: February 2007

Conjugative elements often encode entry exclusion systems that convert host cells into poor recipients for identical or similar elements. The diversity of exclusion systems within families of conjugative elements has received little attention. We report here the most comprehensive study to date of the diversity of exclusion determinants within a single family of conjugative elements. Unexpectedly, our analyses indicate that there are only two exclusion groups among the diverse members of the SXT/R391 family of integrative conjugative elements.
Transposon Mutagenesis Identifies Sites Upstream of the
Neisseria gonorrhoeae pilE
Gene That Modulate Pilin Antigenic Variation
Authors: Kline Kimberly A. , Criss Alison K. , Wallace Anne , Seifert H. Steven
Published: February 2007

Gene conversion mediates the variation of virulence-associated surface structures on pathogenic microorganisms, which prevents host humoral immune responses from being effective. One of the best-studied gene conversion systems is antigenic variation (Av) of the pilin subunit of the
Neisseria gonorrhoeae
type IV pilus. To identify
cis-
acting DNA sequences that facilitate Av, the 700-bp region upstream of the pilin gene
pilE
was targeted for transposon mutagenesis. Four classes of transposon-associated mutations were isolated, distinguishable by their pilus-associated phenotypes: (i) insertions that did not alter Av or piliation, (ii) insertions that blocked Av, (iii) insertions that interfered with Av, and (iv) insertions that interfered with pilus expression and Av. Mutagenesis of the
pilE
promoter did not affect the frequency of Av, directly demonstrating that pilin Av is independent of
pilE
transcription. Two stretches of sequence upstream of
pilE
were devoid of transposon insertions, and some deletions in these regions were not recoverable, suggesting that they are essential for gonococcal viability. Insertions that blocked pilin Av were located downstream of the RS1 repeat sequence, and deletion of the region surrounding these insertions completely abrogated pilin Av, confirming that specific sequences 5′ to
pilE
are essential for the recombination events underlying pilin Av.
YycH and YycI Interact To Regulate the Essential YycFG Two-Component System in
Bacillus subtilis
Authors: Szurmant Hendrik , Mohan Michael A. , Imus P. Michael , Hoch James A.
Published: February 2007

The YycFG two-component system is the only signal transduction system in
Bacillus subtilis
known to be essential for cell viability. This system is highly conserved in low-G+C gram-positive bacteria, regulating important processes such as cell wall homeostasis, cell membrane integrity, and cell division. Four other genes,
yycHIJK
, are organized within the same operon with
yycF
and
yycG
in
B. subtilis
. Recently, it was shown that the product of one of these genes, the YycH protein, regulated the activity of this signal transduction system, whereas no function could be assigned to the other genes. Results presented here show that YycI and YycH proteins interact to control the activity of the YycG kinase. Strains carrying individual in-frame deletion of the
yycI
and
yycH
coding sequences were constructed and showed identical phenotypes, namely a 10-fold-elevated expression of the YycF-dependent gene
yocH
, growth defects, as well as a cell wall defect. Cell wall and growth defects were a direct result of overregulation of the YycF regulon, since a strain overexpressing YycF showed phenotypes similar to those of
yycH
and
yycI
deletion strains. Both YycI and YycH proteins are localized outside the cytoplasm and attached to the membrane by an N-terminal transmembrane sequence. Bacterial two-hybrid data showed that the YycH, YycI, and the kinase YycG form a ternary complex. The data suggest that YycH and YycI control the activity of YycG in the periplasm and that this control is crucial in regulating important cellular processes.
The Crystal Structure of
Bacillus subtilis
YycI Reveals a Common Fold for Two Members of an Unusual Class of Sensor Histidine Kinase Regulatory Proteins
Authors: Santelli Eugenio , Liddington Robert C. , Mohan Michael A. , Hoch James A. , Szurmant Hendrik
Published: February 2007

YycI and YycH are two membrane-anchored periplasmic proteins that regulate the essential
Bacillus subtilis
YycG histidine kinase through direct interaction. Here we present the crystal structure of YycI at a 2.9-Å resolution. YycI forms a dimer, and remarkably the structure resembles that of the two C-terminal domains of YycH despite nearly undetectable sequence homology (10%) between the two proteins.
Escherichia coli abg
Genes Enable Uptake and Cleavage of the Folate Catabolite
p
-Aminobenzoyl-Glutamate
Authors: Carter Eric L. , Jager Lindsey , Gardner Lars , Hall Christel C. , Willis Stacey , Green Jacalyn M.
Published: February 2007

Escherichia coli
AbgT was first identified as a structural protein enabling the growth of
p
-aminobenzoate auxotrophs on exogenous
p
-aminobenzoyl-glutamate (M. J. Hussein, J. M. Green, and B. P. Nichols, J. Bacteriol. 180:6260-6268, 1998). The
abg
region includes
abgA
,
abgB
,
abgT
, and
ogt
; these genes may be regulated by AbgR, a divergently transcribed LysR-type protein. Wild-type cells transformed with a high-copy-number plasmid encoding
abgT
demonstrate saturable uptake of
p
-aminobenzoyl-glutamate (

K
T

= 123 μM); control cells expressing vector demonstrate negligible uptake. The addition of metabolic poisons inhibited uptake of
p
-aminobenzoyl-glutamate, consistent with this process requiring energy.
p-
Aminobenzoyl-glutamate taken in by cells expressing large amounts of AbgT alone is not rapidly metabolized to a form that is trapped in the cell, as the addition of nonradioactive
p-
aminobenzoyl-glutamate to these cells results in a rapid loss of intracellular label. The addition of nonradioactive
p
-aminobenzoate has no effect. The
abgA
,
abgB
, and
abgAB
genes were cloned into the medium-copy-number plasmid pACYC184;
p
-aminobenzoate auxotrophs transformed with the clone encoding
abgAB
demonstrated enhanced ability to grow on low levels of
p
-aminobenzoyl-glutamate. When transformed with complementary plasmids encoding high-copy levels of
abgT
and medium-copy levels of
abgAB
,
p
-aminobenzoate auxotrophs grew on 50 nM
p
-aminobenzoyl-glutamate. Our data are consistent with a model of
p
-aminobenzoyl-glutamate utilization in which AbgT catalyzes transport of
p
-aminobenzoyl-glutamate, followed by cleavage to
p
-aminobenzoate by a protein composed of subunits encoded by
abgA
and
abgB.
While endogenous expression of these genes is very low under the conditions in which we performed our experiments, these genes may be induced by AbgR bound to an unknown molecule. The true physiological role of this region may be related to some molecule similar to
p
-aminobenzoyl-glutamate, such as a dipeptide.
Cytosolic Proteins Contribute to Surface Plasminogen Recruitment ofNeisseria meningitidis
Authors: Knaust Andreas , Weber Martin V. R. , Hammerschmidt Sven , Bergmann Simone , Frosch Matthias , Kurzai Oliver
Published: February 2007

Plasminogen recruitment is a common strategy of pathogenic bacteria and results in a broad-spectrum surface-associated protease activity.Neisseria meningitidishas previously been shown to bind plasminogen. In this study, we show by several complementary approaches that endolase, DnaK, and peroxiredoxin, which are usually intracellular proteins, can also be located in the outer membrane and act as plasminogen receptors. Internal binding motifs, rather than C-terminal lysine residues, are responsible for plasminogen binding of theN. meningitidisreceptors. Recombinant receptor proteins inhibit plasminogen association withN. meningitidisin a concentration-dependent manner. Besides binding purified plasminogen,N. meningitidiscan also acquire plasminogen from human serum. Activation ofN. meningitidis-associated plasminogen by urokinase results in functional activity and allows the bacteria to degrade fibrinogen. Furthermore, plasmin bound toN. meningitidisis protected against inactivation by α2-antiplasmin.
Delayed cord clamping in preterm infants delivered at 34–36 weeks’ gestation: a randomised controlled trial
Authors: Ultee C A , van der Deure J , Swart J , Lasham C , van Baar A L
Published: February 2007

Background:

Even mild iron deficiency and anaemia in infancy may be associated with cognitive deficits. A delay in clamping the cord improves haematocrit levels and results in greater vascular stability and less need for packed cell transfusions for anaemia in the first period after birth. Follow-up data on haemoglobin levels after the neonatal period were not available.

Objective:

To provide neonatal and follow-up data for the effects of early or delayed clamping of the cord.

Methods:

37 premature infants (gestational age 34 weeks, 0 days–36 weeks, 6 days) were randomly assigned to one of two groups in the first hour after birth, and at 10 weeks of age. In one group the umbilical cord was clamped within 30 seconds (mean (SD) 13.4 (5.6)) and in the other, it was clamped at 3 minutes after delivery. In the neonatal period blood glucose and haemoglobin levels were determined. At 10 weeks of age haemoglobin and ferritin levels were determined.

Results:

The late cord-clamped group showed consistently higher haemoglobin levels than the early cord-clamped group, both at the age of 1 hour (mean (SD) 13.4 (1.9) mmol/l vs 11.1 (1.7) mmol/l), and at 10 weeks (6.7 (0.75) mmol/l vs 6.0 (0.65) mmol/l). No relationship between delayed clamping of the umbilical cord and pathological jaundice or polycythaemia was found.

Conclusion:

Immediate clamping of the umbilical cord should be discouraged.
Morphological approach to tumours of the testis and paratestis
Authors: Emerson Robert E , Ulbright Thomas M
Published: February 2007

Most neoplastic scrotal masses ultimately prove to be germ cell tumours and are recognisable with routine haematoxylin and eosin-stained sections. The differential diagnosis may be focused, even before reviewing histological sections, by knowledge of patient age, medical history, tumour site (testicular vs paratesticular) and gross findings. Some cases may prove to be diagnostically challenging, including rare tumours, a common tumour with an unusual pattern, a metastatic tumour, or a neoplasm with features that mimic another tumour. Several morphological patterns are seen with some frequency and these generate recurring sets of differential diagnostic considerations. These common patterns include testicular tumours with a predominant diffuse arrangement of cells with pale to clear cytoplasm, tumours with a glandular/tubular pattern, tumours with a microcystic pattern and tumours composed of oxyphilic cells. Intratubular proliferations of atypical cells, paratesticular glandular and/or papillary tumours, or tumours with spindle cell morphology can also be challenging to diagnose correctly. In some problematic cases, immunohistochemical staining may be useful to resolve these differential diagnoses.
Continuing education for pathology laboratory technologists: a needs analysis in a Singapore teaching hospital
Authors: Tan Kong-Bing , Thamboo Thomas Paulraj , Lim Yaw-Chyn
Published: February 2007

Background/Aims:

Continuing education is important to laboratory technologists. It helps them keep pace with the advances in medicine and pathology and thus to provide quality service. A survey was conducted to assess the attitudes of pathology laboratory technologists towards different educational activities.

Methods:

Relevant continuing education activities include lectures, case discussions, journal clubs, short courses, scientific conferences and higher educational courses. Technologists were asked for their views on these various activities, scoring each item 1–5 out of 5.

Results:

The response rate was 66% (19/29); respondents’ work experience ranged from <3 years to >20 years. Short courses was rated the most highly; 89% of respondents considered them good/excellent. General medical knowledge and the methods and rationale of key histopathology procedures were the preferred lecture topics. Most respondents (58%) were interested (4–5/5) to have their own journal club. 84% of respondents were fairly keen/keen (4–5/5) to participate in conferences. 74% responded that they were fairly keen/keen to consider pursuing higher education. 84% were fairly keen/keen to upgrade themselves and to assume higher responsibilities.

Discussion:

The results indicate a positive outlook of technologists towards continuing education. The planning of future lecture topics has taken into account their preferences, and talks on the pursuit of higher education have been organised. It is hoped that these measures and others, will help boost the usefulness of continuing education and enhance the work environment.
Extrahepatic biliary atresia with cartilaginous metaplasia in the gallbladder remnants: Figure 1
Authors: Altamirano E , Drut R
Published: February 2007

Extrahepatic biliary atresia (EBA) is a chronic inflammatory process leading to fibrous obliteration of the biliary tree. Cartilaginous metaplasia within the remaining walls of the biliary main ducts in EBA has been reported once, although as cartilage heterotopia. A similar finding is presented here, but in the gallbladder of an infant with the full-blown manifestations of EBA; it is proposed that the alteration results from metaplasia of local connective tissue. Also of note is the complete disappearance of smooth muscle cells in the walls of the biliary tree in EBA, a fact which seems to be missing in the description of the pathology in these cases.
Role of cADPR in sodium nitroprusside-induced opossum esophageal longitudinal smooth muscle contraction
Authors: Campbell R. K. , Wells R. W. , Miller D. V. , Paterson W. G.
Published: February 2007

Nitric oxide (NO) relaxes most smooth muscle, including the circular smooth muscle (CSM) of the esophagus, whereas in the adjacent longitudinal smooth muscle (LSM), it causes contraction. The second messenger pathways responsible for this NO-induced LSM contraction are unclear, given that these opposing effects of NO are both cGMP dependent. In intestinal LSM, but not CSM, cADP ribose (cADPR)-dependent pathways participate in Ca2+ mobilization and muscle contraction; whether similar differences exist in the esophagus is unknown. The purpose of this study was to determine whether cADPR plays a role in the NO-mediated contraction of opossum esophageal LSM. Standard isometric tension recordings were performed using both LSM and CSM strips from opossum distal esophagus that were hung in 10-ml tissue baths perfused with oxygenated Krebs solution. cADPR produced concentration-dependent contraction of LSM strips with an EC50 of 1 nM and peak contraction of 57 ± 18% of the 60 mM KCl-induced contraction. cADPR had no effect on CSM strips at concentrations up to 10−6 M. The EC50 of cADPR caused contraction (18 ± 2% from initial resting length) of isolated LSM cells. Sodium nitroprusside (SNP; 300 μM) induced contraction of LSM strips that averaged 67 ± 5% of the KCl response. cADPR antagonists 8-bromo-cADPR and 8-amino-cADPR, as well as ryanodine receptor antagonists ryanodine and tetracaine, significantly inhibited the SNP-induced contraction. In conclusion, in the opossum esophagus, 1) cADPR induces contraction of LSM, but not CSM, and 2) NO-induced contraction of LSM appears to involve a cADPR-dependent pathway.
Intestinal inflammation downregulates smooth muscle CPI-17 through induction of TNF-α and causes motility disorders
Authors: Ohama Takashi , Hori Masatoshi , Momotani Eiichi , Iwakura Yoichiro , Guo Fengling , Kishi Hiroko , Kobayashi Sei , Ozaki Hiroshi
Published: February 2007

Motility disorders are frequently observed in intestinal inflammation. We previously reported that in vitro treatment of intestinal smooth muscle tissue with IL-1β decreases the expression of CPI-17, an endogenous inhibitory protein of smooth muscle serine/threonine protein phosphatase, thereby inhibiting contraction. The present study was performed to examine the pathophysiological importance of CPI-17 expression in the motility disorders by using an in vivo model of intestinal inflammation and to define the regulatory mechanism of CPI-17 expression by proinflammatory cytokines. After the induction of acute ileitis with 2,4,6,-trinitrobenzensulfonic acid, CPI-17 expression declined in a time-dependent manner. This decrease in CPI-17 expression was parallel with the reduction of cholinergic agonist-induced contraction of smooth muscle strips and sensitivity of permeabilized smooth muscle fibers to Ca2+. Among the various proinflammatory cytokines tested, TNF-α and IL-1β were observed to directly inhibit CPI-17 expression and contraction in cultured rat intestinal tissue. Moreover, both TNF-α and IL-1β inhibited CPI-17 expression and contraction of smooth muscle tissue isolated from wild-type and IL-1α/β double-knockout mice. However, IL-1β treatment failed to inhibit CPI-17 expression and contraction in TNF-α knockout mice. In β-escin-permeabilized ileal tissues, pretreatment with anti-phosphorylated CPI-17 antibody inhibited the carbachol-induced Ca2+sensitization in the presence of GTP. These findings suggest that CPI-17 was downregulated during intestinal inflammation and that TNF-α plays a central role in this process. Downregulation of CPI-17 may play a role in motility impairments in inflammation.
Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP- and Ca2+-induced endocytosis by recruitment of AP2 and clathrin
Authors: Musch Mark W. , Arvans Donna L. , Walsh-Reitz Margaret M. , Uchiyama Kazuhiko , Fukuda Mitsunori , Chang Eugene B.
Published: February 2007

Apical membrane sodium hydrogen exchanger 3 (NHE3), a major pathway for non-nutrient-dependent intestinal Na+absorption, is tightly regulated by second messenger systems that affect its functional activity and membrane trafficking. However, the events and components involved in NHE3 regulation are only partially understood. We report that the adaptor protein synaptotagmin I (Syt I) plays a pivotal role in cAMP- and Ca2+-induced cargo recognition of NHE3 and initiation of its endocytosis. Both mouse small intestine (jejunum) and Caco-2BBe Syt I coimmunoprecipitated with NHE3, particularly following increases in cellular cAMP or Ca2+. Following short interfering RNA (siRNA) suppression of Syt I expression, cAMP- and Ca2+-induced inhibition of NHE3 activity were still observed but NHE3 endocytosis was blocked, as assessed by22Na influx and apical membrane biotin labeling, respectively. Similar effects on NHE3 inhibition and endocytosis were observed by siRNA suppression of either the μ-subunit of the adaptor protein 2 (AP2) complex or the heavy chain of clathrin. Coimmunoprecipitation analyses of NHE3 with these adaptor proteins revealed that cAMP- and Ca2+-induced NHE3-Syt I interaction preceded and was required for recruitment of AP2 and the clathrin complex. Confocal microscopy confirmed both the time sequence and protein associations of these events. We conclude that Syt I plays a pivotal role in mediating cAMP- and Ca2+-induced endocytosis of NHE3 (but not in inhibition of activity) through cargo recognition of NHE3 and subsequent recruitment of AP2-clathrin assembly required for membrane endocytosis.
Proteomic analysis of hepatic iron overload in mice suggests dysregulation of urea cycle, impairment of fatty acid oxidation, and changes in the methylation cycle
Authors: Petrak Jiri , Myslivcova Denisa , Man Petr , Cmejla Radek , Cmejlova Jana , Vyoral Daniel , Elleder Milan , Vulpe Christopher D.
Published: February 2007

Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease, and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis, and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload and the cellular processes that result in the observed pathological changes. In addition, the variability in outcome and the compensatory response that likely modulates the effect of increased iron levels are not understood. To provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we studied hepatic iron overload induced by carbonyl iron-rich diet in mice and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely the urea cycle, fatty acid oxidation, and the methylation cycle. This pattern of changes likely reflects compensatory and pathological changes associated with liver iron overload and provides a window into these processes.
Role of caveolae in the pathogenesis of cholesterol-induced gallbladder muscle hypomotility
Authors: Xiao Zuoliang , Schmitz Frank , Pricolo Victor E. , Biancani Piero , Behar Jose
Published: February 2007

Muscle cells from human gallbladders (GB) with cholesterol stones (ChS) exhibit a defective contraction, excess cholesterol (Ch) in the plasma membrane, and lower binding of CCK-1 receptors. These abnormalities improved after muscle cells were incubated with Ch-free liposomes that remove the excess Ch from the plasma membrane. The present studies were designed to investigate the role of caveolin-3 proteins (Cav-3) in the pathogenesis of these abnormalities. Muscle cells from GB with ChS exhibit higher Ch levels in the plasma membrane that were mostly localized in caveolae and associated with parallel increases in the expression of Cav-3 in the caveolae compared with that in GB with pigment stones (PS). The overall number of CCK-1 receptors in the plasma membrane was not different between muscle cells from GB with ChS and PS, but they were increased in the caveolae in muscle cells from GB with ChS. Treatment of muscle cells from GB with ChS with a Gαi3protein fragment increased the total binding of CCK-1 receptors (from 8.3 to 11.2%) and muscle contraction induced by CCK-8 (from 11.2 to 17.3% shortening). However, Gαq/11protein fragment had no such effect. Moreover, neither fragment had any effect on muscle cells from GB with PS. We conclude that the defective contraction of muscle cells with excessive Ch levels in the plasma membrane is due to an increased expression of Cav-3 that results in the sequestration of CCK-1 receptors in the caveolae, probably by inhibiting the functions of Gαi3proteins.
Chronically administered retinoic acid has trophic effects in the rat small intestine and promotes adaptation in a resection model of short bowel syndrome
Authors: Wang Lihua , Tang Yuzhu , Rubin Deborah C. , Levin Marc S.
Published: February 2007

Following the loss of functional small bowel surface area, the intestine undergoes a compensatory adaptive response. The observation that adaptation is inhibited in vitamin A-deficient rats following submassive intestinal resection suggested that vitamin A is required for this response and raised the possibility that exogenous vitamin A could augment adaptation. Therefore, to directly assess whether chronically administered retinoic acid could stimulate gut adaptation in a model of short bowel syndrome and to address the mechanisms of any such effects, Sprague-Dawley rats were implanted with controlled release retinoic acid or control pellets and then subjected to mid-small bowel or sham resections. At 2 wk postoperation, changes in gut morphology, crypt cell proliferation and apoptosis, enterocyte migration, the extracellular matrix, and gene expression were assessed. Retinoic acid had significant trophic effects in resected and sham-resected rats. Retinoic acid markedly inhibited apoptosis and stimulated crypt cell proliferation and enterocyte migration postresection. Data presented indicate that these proadaptive effects of retinoic acid may be mediated via changes in the extracellular matrix (e.g., by increasing collagen IV synthesis, decreasing E-cadherin expression, and reducing integrin β3levels), via affects on Hedgehog signaling (e.g., by reducing expression of the Hedgehog receptors Ptch and Ptch2 and the Gli1 transcription factor), by increasing expression of Reg1 and Pap1, and by modulation of retinoid and peroxisome proliferator-activated receptor signaling pathways. These studies are the first to demonstrate that retinoic acid can significantly enhance intestinal adaptation and suggest it may be beneficial in patients with short bowel syndrome.