bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneity in the structural dynamics of Sulfolobus solfataricus β‐glycosidase revealed by electron paramagnetic resonance and frequency domain fluorometry
Authors: Lozinsky Evgenia , Febbraio Ferdinando , Shames Alexander I. , Likhtenshtein Gertz I. , Bismuto Ettore , Nucci Roberto
Published: October 2002

The local and global dynamics of the Sulfolobus solfataricus β‐glycosidase were studied by electron spin resonance and time‐resolved fluorescence techniques. For electron paramagnetic resonance (EPR) investigations, the protein was covalently modified by the maleimido nitroxide spin label, which is specific for cysteine ‐SH groups, at position 344 and at position 101, where Ser‐101 was changed into a cysteine by site‐directed mutagenesis. The greater reactivity of exposed Cys‐101 suggested the exclusive modification of this amino acid compared with Cys‐344. The labeled proteins underwent temperature perturbation in the range 290–335 K and the values of the spin‐label rotation correlation frequencies (νc) ranged from 6 × 107 to 2 × 108 sec−1 for the protein labeled at position C344 and from 5.62 × 107 to 1.10 × 108 sec−1 for the protein labeled at C101. These rotation correlation values are related to the local dynamic characteristics of the protein matrix. The temperature dependence of rotation correlation frequencies expressed in terms of Arrhenius coordinates (log (νc) vs. 1/T) for the protein labeled at C344 exhibited a linear dependence but with a change in the slope at 311 K. For the protein labeled at C101, no change in the slope was observed at the same temperature. General dynamic information was deduced from the analysis of the fluorescence emission decay of the tryptophanyl residues that are present in each region of the protein structure. Fluorescence data analysis highlighted a bimodal distribution of fluorescence lifetimes arising from the contribution of two emitting groups: one consisting of closely clustered tryptophans responsible for the long‐lived emission component (7.1 nsec) and the other composed of tryptophans nearer to the protein surface, which can be associated to the short‐lived component (2.5 nsec). The temperature dependence of lifetime distribution parameters linked to the long‐lived and short‐lived components, expressed in Arrhenius coordinates, showed two different points in which the change in the slope occurred (i.e., 328 K and 338 K, respectively). The Arrhenius analysis of data provided the activation energy relative to the conformational changes characterizing the local and global movements running through the protein matrix.
Construction and characterization of protein libraries composed of secondary structure modules
Authors: Matsuura Tomoaki , Ernst Andreas , Plückthun Andreas
Published: October 2002

Only a minute fraction of all possible protein sequences can exist in the genomes of all life forms. To explore whether physicochemical constraints or a lack of need causes the paucity of different protein folds, we set out to construct protein libraries without any restriction of topology. We generated different libraries (all α‐helix, all β‐strand, and α‐helix plus β‐strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (α‐helix, β‐strand, and β‐turn) in various proportions, based primarily on the patterning of polar and nonpolar residues. We wished to explore that part of sequence space that is rich in secondary structure. The analysis of randomly chosen clones from each of the libraries showed that, despite the low sequence homology to known protein sequences, a substantial proportion of the library members containing α‐helix modules were indeed helical, possess a defined oligomerization state, and showed cooperative chemical unfolding behavior. On the other hand, proteins composed of mainly β‐strand modules tended to form amyloid‐like fibrils and were among the least soluble proteins ever reported. We found that a large fraction of members in non‐β‐strand–containing protein libraries that are distant from natural proteins in sequence space possess unexpectedly favorable properties. These results reinforce the efficacy of applying binary patterning to design proteins with native‐like properties despite lack of restriction in topology. Because of the intrinsic tendency of β‐strand modules to aggregate, their presence requires precise topologic arrangement to prevent fibril formation.
Distance‐scaled, finite ideal‐gas reference state improves structure‐derived potentials of mean force for structure selection and stability prediction
Authors: Zhou Hongyi , Zhou Yaoqi
Published: October 2002

The distance‐dependent structure‐derived potentials developed so far all employed a reference state that can be characterized as a residue (atom)‐averaged state. Here, we establish a new reference state called the distance‐scaled, finite ideal‐gas reference (DFIRE) state. The reference state is used to construct a residue‐specific all‐atom potential of mean force from a database of 1011 nonhomologous (less than 30% homology) protein structures with resolution less than 2 Å. The new all‐atom potential recognizes more native proteins from 32 multiple decoy sets, and raises an average Z‐score by 1.4 units more than two previously developed, residue‐specific, all‐atom knowledge‐based potentials. When only backbone and Cβ atoms are used in scoring, the performance of the DFIRE‐based potential, although is worse than that of the all‐atom version, is comparable to those of the previously developed potentials on the all‐atom level. In addition, the DFIRE‐based all‐atom potential provides the most accurate prediction of the stabilities of 895 mutants among three knowledge‐based all‐atom potentials. Comparison with several physical‐based potentials is made.
Construction of a fluorescent biosensor family
Authors: De Lorimier Robert M. , Smith J. Jeff , Dwyer Mary A. , Looger Loren L. , Sali Kevin M. , Paavola Chad D. , Rizk Shahir S. , Sadigov Shamil , Conrad David W. , Loew Leslie , Hellinga Homme W.
Published: October 2002

Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand‐mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand‐mediated hinge‐bending motion. Construction of cysteine mutations at these locations then permits site‐specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three‐dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.
Genetic analysis of response regulator activation in bacterial chemotaxis suggests an intermolecular mechanism
Authors: Re Sandra Da , Tolstykh Tatiana , Wolanin Peter M. , Stock Jeffry B.
Published: October 2002

Response regulator proteins of two‐component systems are usually activated by phosphorylation. The phosphorylated response regulator protein CheY∼P mediates the chemotaxis response in Escherichia coli. We performed random mutagenesis and selected CheY mutants that are constitutively active in the absence of phosphorylation. Although a single amino acid substitution can lead to constitutive activation, no single DNA base change can effect such a transition. Numerous different sets of mutations that activate in synergy were selected in several different combinations. These mutations were all located on the side of CheY defined by α4, β5, α5, and α1. Our findings argue against the two‐state hypothesis for response regulator activation. We propose an alternative intermolecular mechanism that involves a dynamic interplay between response regulators and their effector targets.
Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging
Authors: Bogin Oren , Levin Inna , Hacham Yael , Tel‐Or Shoshana , Peretz Moshe , Frolow Felix , Burstein Yigal
Published: October 2002

Previous research in our laboratory comparing the three‐dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224‐Lys254) and a short ion‐pair network (Lys257‐Asp237‐Arg304‐Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site‐directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K‐ and in the double mutant V224E/S254K‐CbADH, but not in the single mutant V224E‐CbADH. Both single amino acid replacements, M304R‐ and Q165E‐CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E‐CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R‐CbADH and solved its three‐dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.
Progesterone reverses the neuronal responses to hypoxia in rat nucleus tractus solitarius in vitro
Authors: Pascual Olivier , Morin‐Surun Marie‐Pierre , Barna Barbara , Denavit‐Saubié Monique , Pequignot Jean‐Marc , Champagnat Jean
Published: October 2002

The nucleus tractus solitarius (NTS) is a relay nucleus that integrates peripheral chemoreceptor input in response to hypoxia and hence influences the generation of respiratory rhythm. Several studies have shown that administration of progesterone stimulates ventilatory responses to hypoxia. There is some evidence that this steroid hormone can act at the level of the arterial peripheral chemoreceptors, whereas its action in the central nervous system remains unclear. To investigate a possible central involvement during hypoxia, we studied the effect of progesterone on neuronal activities recorded extra‐ and intracellularly in the NTS using brainstem slices. Central chemosensitivity was tested by comparing synaptic activity and intrinsic electro‐responsiveness of 38 neurones during normoxia and hypoxia. In more than two‐thirds of neurones recorded, hypoxia elicited a hyperpolarisation, a decrease in the input resistance and a decrease in spontaneous activity. In the remaining neurones (n= 12) hypoxia elicited a depolarisation and an increase in spontaneous activity. In all neurones tested, synaptic potentials evoked by stimulation of the tractus solitarius were decreased by hypoxia. While progesterone (1 μM) had no effect under normoxic conditions, it partially reversed all hypoxic neuronal responses. This effect developed over 2‐3 min and reversed within 5 min suggesting a non‐genomic mechanism of action. Taken together these results suggest that progesterone interacts with the hypoxia‐induced cellular signalling. We conclude that in the NTS, transmission of afferent signals is reduced by hypoxia and restored by progesterone administration. Such a mechanism may contribute to the stimulation of breathing in response to hypoxia observed following progesterone administration in vivo.
The sites of neural adaptation induced by resistance training in humans
Authors: Carroll Timothy J. , Riek Stephan , Carson Richard G.
Published: October 2002

Although it has long been supposed that resistance training causes adaptive changes in the CNS, the sites and nature of these adaptations have not previously been identified. In order to determine whether the neural adaptations to resistance training occur to a greater extent at cortical or subcortical sites in the CNS, we compared the effects of resistance training on the electromyographic (EMG) responses to transcranial magnetic (TMS) and electrical (TES) stimulation. Motor evoked potentials (MEPs) were recorded from the first dorsal interosseous muscle of 16 individuals before and after 4 weeks of resistance training for the index finger abductors (n= 8), or training involving finger abduction‐adduction without external resistance (n= 8). TMS was delivered at rest at intensities from 5 % below the passive threshold to the maximal output of the stimulator. TMS and TES were also delivered at the active threshold intensity while the participants exerted torques ranging from 5 to 60 % of their maximum voluntary contraction (MVC) torque. The average latency of MEPs elicited by TES was significantly shorter than that of TMS MEPs (TES latency = 21.5 ± 1.4 ms; TMS latency = 23.4 ± 1.4 ms; P < 0.05), which indicates that the site of activation differed between the two forms of stimulation. Training resulted in a significant increase in MVC torque for the resistance‐training group, but not the control group. There were no statistically significant changes in the corticospinal properties measured at rest for either group. For the active trials involving both TMS and TES, however, the slope of the relationship between MEP size and the torque exerted was significantly lower after training for the resistance‐training group (P < 0.05). Thus, for a specific level of muscle activity, the magnitude of the EMG responses to both forms of transcranial stimulation were smaller following resistance training. These results suggest that resistance training changes the functional properties of spinal cord circuitry in humans, but does not substantially affect the organisation of the motor cortex.
Frequency‐dependent shift from paired‐pulse facilitation to paired‐pulse depression at unitary CA3‐CA3 synapses in the rat hippocampus
Authors: Saviane Chiara , Savtchenko Leonid P. , Raffaelli Giacomo , Voronin Leon L. , Cherubini Enrico
Published: October 2002

Paired recordings between CA3 interconnected pyramidal neurons were used to study the properties of short‐term depression occurring in these synapses under different frequencies of presynaptic firing (n= 22). In stationary conditions (0.05‐0.067 Hz) pairs of presynaptic action potentials (50 ms apart) evoked EPSCs whose amplitude fluctuated from trial to trial with occasional response failures. In 15/20 cells, paired‐pulse ratio (PPR) was characterized by facilitation (PPF) while in the remaining five by depression (PPD). Increasing stimulation frequency from 0.05‐0.067 Hz to 0.1‐1 Hz induced low frequency depression (LFD) of EPSC amplitude with a gradual increase in the failure rate. Overall, 9/12 cells at 1 Hz became almost ‘silent’. In six cells in which the firing rate was sequentially shifted from 0.05 to 0.1 and 1 Hz, changes in synaptic efficacy were so strong that PPR shifted from PPF to PPD. The time course of depression of EPSC1 could be fitted with single exponentials with time constants of 98 and 36 s at 0.1 and 1 Hz, respectively. In line with the inversion of PPR at 1 Hz, the time course of depression of EPSC2 was faster than EPSC1 (7 s). Recovery from depression could be obtained by lowering the frequency of stimulation to 0.025 Hz. These results could be explained by a model that takes into account two distinct release processes, one dependent on the residual calcium and the other on the size of the readily releasable pool of vesicles.
Enhanced Interleukin-12 and CD40 Ligand Activities but ReducedStaphylococcus aureusCowan 1-Induced Responses Suggest a Generalized and Progressively Impaired Type 1 Cytokine Pattern for Human Schistosomiasis
Authors: Montenegro Silvia M. L. , Abath Frederico G. C. , Domingues Ana Lúcia C. , Melo Wlademir G. , Morais Clarice N. L. , Coutinho Eridan M. , Mahanty Siddhartha , Wynn Thomas A.
Published: October 2002

Whole-blood-cell cultures from schistosomiasis patients were stimulated with a variety of T-cell-dependent and T-cell-independent stimuli to determine whether the defect in type 1 cytokine expression observed following helminth infection is associated with alterations in interleukin-12 (IL-12) or CD40 ligand (CD40L) responsiveness. Cultures from uninfected individuals produced abundant gamma interferon in response toStaphylococcus aureusCowan 1 (SAC), while patients with intestinal and hepatosplenic disease displayed intermediate and weak responses, respectively. Importantly, the decrease in type 1 cytokine expression was not attributed to defects in IL-12- or CD40L-induced activity. Indeed, schistosomiasis patients displayed heightened responses and even produced more biologically active IL-12 when stimulated with SAC and CD40L than did uninfected controls. Finally, additional studies suggested only a partial role for IL-10, since intestinal patients were the only group that overproduced this downregulatory cytokine. Together, these studies demonstrate that the type 1 deficiency in chronic hepatosplenic schistosomiasis is not related to specific defects in IL-12, IL-10, or CD40L activity, although changes in the functional status of antigen-presenting cells appear to be involved.
The Growth Response of
Escherichia coli
to Neurotransmitters and Related Catecholamine Drugs Requires a Functional Enterobactin Biosynthesis and Uptake System
Authors: Burton Claire L. , Chhabra Siri Ram , Swift Simon , Baldwin Tom J. , Withers Helen , Hill Stephen J. , Williams Paul
Published: October 2002

The neurotransmitter norepinephrine (NE) stimulates the growth of low inocula of
Escherichia coli
in a minimal medium (SAPI) supplemented with serum (SAPI+serum) and induces the production of an “autoinducer” (AI) which, in turn, promotes
E. coli
growth in the absence of NE. Given the importance of NE, epinephrine, and their corresponding adrenergic agonists and antagonists in clinical medicine, we sought to investigate the molecular basis for these observations. Using a variety of NE precursors, metabolites, and therapeutic agents, we demonstrated that their ability to stimulate
E. coli
growth in SAPI+serum is dependent on the presence of a catechol (1,2-dihydroxybenzene) moiety with maximal activity requiring a two-carbon substituent incorporating a terminal primary amine. Serum contains the iron-binding glycoprotein, transferrin, and when SAPI+serum was supplemented with sufficient Fe
3+
to saturate transferrin, growth inhibition was relieved. Other metal cations, including Mg
2+
, Ca
2+
, and Zn
2+
, had no effect. These data suggested that the stimulation of
E. coli
growth by NE in SAPI+serum may involve the catecholate siderophore, enterobactin, a cyclic triester of 2,3-dihydroxybenzoylserine. Consistent with this hypothesis,
E. coli
strains with mutations in ferrienterobactin transport (
fepA
or
tonB
) or enterobactin biosynthesis (
entA
) did not respond to NE. Furthermore, NE induced expression of the ferrienterobactin receptor, FepA, during growth in SAPI+serum. The enterobactin degradation product, 2,3-dihydroxybenzoylserine (DBS) was as effective as NE in stimulating the growth of
E. coli
and mutations in
fepA
or
tonB
abolished the DBS-dependent growth stimulation. In contrast to NE, however, DBS stimulated the growth of the
entA
mutant. Moreover, after growth in an iron-limited M9 medium in the absence of NE, ethyl acetate extracts of the

E. coli entA
+

parent but not of the
entA
mutant contained AI, i.e., stimulated the growth of
E. coli
in SAPI+serum. Taken together, these data show that when low numbers of
E. coli
are inoculated into SAPI+serum, NE, DBS, and related catecholamines induce the enterobactin iron uptake system. This, in turn, facilitates iron sequestration from transferrin and indicates that the AI present in NE-conditioned SAPI+serum medium is enterobactin and its DBS breakdown products.
Cloning and Molecular Characterization of an Immunogenic LigA Protein of
Leptospira interrogans
Authors: Palaniappan Raghavan U. M. , Chang Yung-Fu , Jusuf S. S. D. , Artiushin S. , Timoney John F. , McDonough Sean P. , Barr Steve C. , Divers Thomas J. , Simpson Kenneth W. , McDonough Patrick L. , Mohammed Hussni O.
Published: October 2002

A clone expressing a novel immunoreactive leptospiral immunoglobulin-like protein A of 130 kDa (LigA) from
Leptospira interrogans
serovar pomona type kennewicki was isolated by screening a genomic DNA library with serum from a mare that had recently aborted due to leptospiral infection. LigA is encoded by an open reading frame of 3,675 bp, and the deduced amino acid sequence consists of a series of 90-amino-acid tandem repeats. A search of the NCBI database found that homology of the LigA repeat region was limited to an immunoglobulin-like domain of the bacterial intimin binding protein of
Escherichia coli
, the cell adhesion domain of
Clostridium acetobutylicum
, and the invasin of
Yersinia pestis
. Secondary structure prediction analysis indicates that LigA consists mostly of beta sheets with a few alpha-helical regions. No LigA was detectable by immunoblot analysis of lysates of the leptospires grown in vitro at 30°C or when cultures were shifted to 37°C. Strikingly, immunohistochemistry on kidney from leptospira-infected hamsters demonstrated LigA expression. These findings suggest that LigA is specifically induced only in vivo. Sera from horses, which aborted as a result of natural
Leptospira
infection, strongly recognize LigA. LigA is the first leptospiral protein described to have 12 tandem repeats and is also the first to be expressed only during infection. Thus, LigA may have value in serodiagnosis or as a protective immunogen in novel vaccines.
Infection Outcome and Cytokine Gene Expression inBrugia pahangi- Infected Gerbils (Meriones unguiculatus) Sensitized withBrucella abortus
Authors: Chirgwin Sharon R. , Elzer Philip H. , Coleman Sharon U. , Nowling Jena M. , Hagius Sue D. , Edmonds Matthew D. , Klei Thomas R.
Published: October 2002

Filarial infections have been associated with the development of a strongly polarized Th2 host immune response and a severe impairment of mitogen-driven proliferation and type 1 cytokine production in mice and humans. The role of this polarization in the development of the broad spectra of clinical manifestations of lymphatic filariasis is still unknown. Recently, data gathered from humans as well as from immunocompromised mouse models suggest that filariasis elicits a complex host immune response involving both Th1 and Th2 components. However, responses of a similar nature have not been reported in immunologically intact permissive models ofBrugiainfection.Brucella abortus-killed S19 was inoculated into theBrugia-permissive gerbil host to induce gamma interferon (IFN-γ) production. Gerbils were then infected withB. pahangi,and the effect of the polarized Th1 responses on worm establishment and host cellular response was measured. Animals infected with bothB. abortusandB. pahangishowed increased IFN-γ and interleukin-10 (IL-10) and decreased IL-4 and IL-5 mRNA levels compared with those in animals infected withB. pahangialone. These data suggest that the prior sensitization withB. abortusmay induce a down regulation of the Th2 response associated withBrugiainfection. This reduced Th2 response was associated with a reduced eosinophilia and an increased neutrophilia in the peritoneal exudate cells. The changes in cytokine and cellular environment did not inhibit the establishment ofB. pahangiintraperitoneally. The data presented here suggest a complex relationship between the host immune response and parasite establishment and survival that cannot be simply ascribed to the Th1/Th2 paradigm.
Mycobacterium tuberculosisin Chemokine Receptor 2-Deficient Mice: Influence of Dose on Disease Progression
Authors: Scott Holly M. , Flynn JoAnne L.
Published: October 2002

Within aMycobacterium tuberculosis-induced granuloma, lymphocytes and macrophages work together to control bacterial growth and limit the spread of infection. Chemokines and chemokine receptors are involved in cell migration and are logical candidates for a role in granuloma formation. In the present study we addressed the role of CC chemokine receptor 2 (CCR2) inM. tuberculosisinfection. In previous studies (W. Peters et al., Proc. Natl. Acad. Sci. USA 98:7958-7963, 2001), CCR2−/−mice were found to be highly susceptible to a moderate or high dose of H37Rv administered intravenously (i.v.). We have expanded those studies to demonstrate that the susceptibility of CCR2−/−mice is dose dependent. After low-dose aerosol or i.v. infection of CCR2−/−mice withM. tuberculosis, there was a substantial delay in cell migration to the lungs and delayed expression of gamma interferon and inducible nitric oxide synthase. The CCR2−/−mice had a severe and prolonged deficiency in the number of macrophages in the lungs and an early increase in the number of neutrophils. Despite these deficiencies in cell migration, the CCR2−/−mice did not have increased bacterial loads in the lungs compared to wild-type (C57BL/6) mice and successfully formed granulomas. This finding is in contrast to CCR2−/−mice infected with a high dose ofM. tuberculosisadministered i.v. These results indicate that with low-dose infection, a delay in immune response in the lungs does not necessarily have detrimental long-term effects on the progression of the disease. The fact that CCR2−/−mice survive with substantially fewer macrophages in the low-dose models implies that the immune response to low-doseM. tuberculosisinfection in mice is more robust than necessary to control the infection. Finally, these data demonstrate that, in cases of infectious disease in knockout models, clear phenotypes may not be evident when one is solely evaluating bacterial numbers and survival. Functional assays may be necessary to reveal roles for components of the multifactorial immune system.
Neisseria gonorrhoeae
Porin P1.B Induces Endosome Exocytosis and a Redistribution of Lamp1 to the Plasma Membrane
Authors: Ayala Patricia , Vasquez Brandi , Wetzler Lee , So Magdalene
Published: October 2002

The immunoglobulin A (IgA) protease secreted by pathogenic
Neisseria
spp. cleaves Lamp1, thereby altering lysosomes in a cell and promoting bacterial intracellular survival. We sought to determine how the IgA protease gains access to cellular Lamp1 in order to better understand the role of this cleavage event in bacterial infection. In a previous report, we demonstrated that the pilus-induced Ca
2+
transient triggers lysosome exocytosis in human epithelial cells. This, in turn, increases the level of Lamp1 at the plasma membrane, where it can be cleaved by IgA protease. Here, we show that porin also induces a Ca
2+
flux in epithelial cells. This transient is similar in nature to that observed in phagocytes exposed to porin. In contrast to the pilus-induced Ca
2+
transient, the porin-induced event does not trigger lysosome exocytosis. Instead, it stimulates exocytosis of early and late endosomes and increases Lamp1 on the cell surface. These results indicate that
Neisseria
pili and porin perturb Lamp1 trafficking in epithelial cells by triggering separate and distinct Ca
2+
-dependent exocytic events, bringing Lamp1 to the cell surface, where it can be cleaved by IgA protease.
Primary Dendritic Cells PhagocytoseCryptococcus neoformansvia Mannose Receptors and Fcγ Receptor II for Presentation to T Lymphocytes
Authors: Syme Rachel M. , Spurrell Jason C. L. , Amankwah Ernest K. , Green Francis H. Y. , Mody Christopher H.
Published: October 2002

Different “professional” antigen-presenting cells (APC) have unique characteristics that favor or restrict presentation of microbial antigens to T cells, depending on the organism.Cryptococcus neoformansis a pathogenic yeast that presents unique challenges to APC, including its large size, its rigid cell wall, and its ability to stimulate T cells as a mitogen. T-cell proliferation in response to theC. neoformansmitogen (CnM) requires phagocytosis and processing of the organisms by accessory cells prior to presentation of CnM to T cells. Because of the requirement for uptake of the organism and more limited costimulatory requirements of mitogens, macrophages might be the most likely cellular source for the accessory cell. However, the present study demonstrates that a transiently adherent cell that was CD3−, CD14−, CD19−, CD56−, HLA-DR+, and CD83+with a dendritic morphology, rather than monocyte-derived or tissue (alveolar) macrophages, was the most efficient APC for presentation of CnM. A large number of these cells bound and internalized the organism, and only a small number of dendritic cells were required for presentation of the mitogen to T cells. Further, the mannose receptor and Fcγ receptor II were required for presentation ofC. neoformans, as blocking either of these receptors abrogated both uptake ofC. neoformansand lymphocyte proliferation in response to CnM. These studies demonstrate the surprising fact that dendritic cells are the most efficient accessory cells for CnM.
Merozoite Surface Protein 1-Specific Immune Response Is Protective against Exoerythrocytic Forms ofPlasmodium yoelii
Authors: Kawabata Yuko , Udono Heiichiro , Honma Kiri , Ueda Masakatsu , Mukae Hiroshi , Kadota Jun-ichi , Kohno Shigeru , Yui Katsuyuki
Published: October 2002

One of the difficulties in developing an effective malaria vaccine is the antigenic change of the parasite during the life cycle. It is desirable that vaccine-induced protective immunity be effective at different stages of parasite development. Merozoite surface protein 1 (MSP1) is a candidate vaccine antigen against blood-stage malaria, but it is also expressed in the exoerythrocytic forms. It was not known, however, whether the anti-MSP1 immune response is effective against the liver-stage malaria parasite. We generated a recombinant protein of MSP1 fused to heat-shock cognate protein 70 (hsc70) and studied its vaccination effect. When C57BL/6 mice were immunized with the fusion protein prior to challenge infection withPlasmodium yoeliisporozoites, the onset of parasitemia was delayed or no parasitemia was observed. To determine whether this was due to the protective immunity against liver-stage parasites,P. yoelii-specific rRNA in the infected liver was quantitated by real-time reverse transcription-PCR analysis. The level of parasite-specific rRNA was reduced in mice immunized with the fusion protein of MSP1 and hsc70 but not with hsc70 alone, indicating that MSP1-specific immunity can be protective against the exoerythrocytic form of the parasite. Furthermore, the adoptive transfer experiments of immune lymphocytes and serum into naive mice suggested that the protective immunity was dependent on cellular and not humoral immunity. Finally, the vaccine-induced protection was also observed in A/J, C3H, and BALB/c mice, suggesting that MSP1-specific protective immunity at the exoerythrocytic stage can be induced in animals over a wide range of genetic backgrounds.
Local Delivery of the Viral Interleukin-10 Gene Suppresses Tissue Inflammation in MurinePneumocystis cariniiInfection
Authors: Ruan Sanbao , Tate Chandra , Lee Janet J. , Ritter Thomas , Kolls Jay K. , Shellito Judd E.
Published: October 2002

The relationship between tissue inflammation and clearance of the opportunistic pathogenPneumocystis cariniiis poorly understood. We asked whether the anti-inflammatory cytokine interleukin-10 (IL-10) is released during the host response to infection withP. cariniiand whether local delivery of the IL-10 gene could suppress tissue inflammatory responses without compromising clearance of infection. Control and CD4-depleted mice were inoculated withP. carinii, and at serial intervals after inoculation, lung tissue was assayed for IL-10 by enzyme-linked immunosorbent assay. We found that IL-10 was released in lung tissue in control mice and was present in higher concentrations in CD4-depleted mice with progressive infection. Control and CD4-depleted mice were then pretreated with 109PFU of intratracheally administered adenoviral vector containing the viral IL-10 gene or the luciferase gene followed by inoculation withP. carinii. Pretreatment with viral IL-10 did not alter clearance of infection in control mice or severity of infection in CD4-depleted mice but did decrease tissue inflammation. We then asked whether gene transfer of viral IL-10 could decrease tissue inflammation during immune reconstitution. In these experiments, immunodeficientscidmice were inoculated withP. cariniiand were heavily infected after 4 weeks. When these mice are immunologically reconstituted by intravenous administration of spleen cells from normal mice, a hyperinflammatory reaction developed in lung tissue, associated with high mortality. In comparison to control mice, mice treated with viral IL-10 prior to reconstitution showed significantly decreased lung wet weight, bronchoalveolar lavage fluid (BALF) lactate dehydrogenase, and BALF neutrophils. In contrast, infection intensity, as measured by PCR forP. cariniirRNA, was unchanged between the IL-10 and luciferase groups. Survival was also improved in the IL-10-treated mice. We conclude that release of IL-10 is part of the host response to infection withP. cariniiand that gene therapy with viral IL-10 can lessen excessive tissue inflammation without altering pathogen clearance. In the setting of immune reconstitution andP. cariniipneumonia, pretreatment with the viral IL-10 gene decreases excessive tissue inflammation and improves survival. These results are relevant to acute respiratory failure after initiation of antibiotic treatment for humanP. cariniipneumonia and to immune reconstitution syndromes in human immunodeficiency virus-positive patients started on highly active antiretroviral therapy.
Contributions of the N- and C-Terminal Domains of Surfactant Protein D to the Binding, Aggregation, and Phagocytic Uptake of Bacteria
Authors: Hartshorn Kevan L. , White Mitchell R. , Crouch Erika C.
Published: October 2002

Collectins play important roles in host defense against infectious microorganisms. We now demonstrate that the serum collectins mannose-binding lectin (MBL) and conglutinin have less ability to bind to, aggregate, and enhance neutrophil uptake of several strains of gram-negative and gram-positive bacteria than pulmonary surfactant protein D (SP-D). Collectins are composed of four major structural domains (i.e., N-terminal, collagen, and neck and carbohydrate recognition domains). To determine which domains of SP-D are responsible for its greater bacterial binding or aggregating activity, activities of chimeric collectins containing the N-terminal and collagen domains of SP-D coupled to the neck recognition domains and carbohydrate recognition domains (CRD) of MBL or conglutinin (SP-D/Congneck+CRDand SP-D/MBLneck+CRD) were tested. The SP-D/Congneck+CRDand SP-D/MBLneck+CRDchimeras bound to and aggregated the bacteria more strongly than did wild-type MBL or conglutinin. SP-D/MBLneck+CRDalso enhanced neutrophil uptake of bacteria more so than MBL. Hence, the SP-D N-terminal and/or collagen domains contribute to the enhanced bacterial binding and aggregating activities of SP-D. In prior studies, SP-D/Congneck+CRDand SP-D/MBLneck+CRDhad increased ability to bind to influenza virus compared not only with that of conglutinin or MBL but with that of wild-type SP-D as well. In contrast, the chimeras had either reduced or unchanged ability to bind to or aggregate bacteria compared to that of wild-type SP-D. Hence, although replacement of the neck recognition domains and CRDs of SP-D with those of MBL and conglutinin conferred increased viral binding activity, it did not favorably affect bacterial binding activity, suggesting that requirements for optimal collectin binding to influenza virus and bacteria differ.
Role of Interferon-Stimulated Gene Factor 3γ and Beta Interferon in HLA Class I Enhancement in Synovial Fibroblasts upon Infection withChlamydia trachomatis
Authors: Rödel Jürgen , Vogelsang Heinz , Prager Katrin , Hartmann Matthias , Schmidt Karl-Hermann , Straube Eberhard
Published: October 2002

Chlamydia trachomatisinfection can cause reactive arthritis that is associated with the persistence of chlamydial organisms in the joint. Fibroblasts of the synovial membrane represent host cells forChlamydiaduring articular infection. In this study we investigated the expression of HLA class I molecules in synovial fibroblasts following infection withC. trachomatisD. The expression of HLA class I heavy chain (HLA-I) was up-regulated in infected cultures as shown by reverse transcription-PCR and immunoblotting. The increase in cell surface expression of HLA-I and β2microglobulin on infected fibroblasts was demonstrated by flow cytometric analysis. Suppression of enhanced production of interferon-stimulated gene factor 3γ (ISGF3γ) in infected cell cultures by antisense oligonucleotide treatment reduced the level of HLA-I. Blocking antibodies to beta interferon (IFN-β) inhibited theChlamydia-induced enhancement of both ISGF3γ and HLA-I. These findings show that the up-regulation of HLA-I in synovial fibroblasts infected withC. trachomatisis caused by the induction of IFN-β, which in turn stimulates the synthesis of ISGF3γ, a transcription factor participating in the regulation of the HLA-I gene. The IFN-β-mediated expression of HLA-I onChlamydia-infected cells may be a regulatory factor in the immune response in chlamydial infections.
Inhibition of Endoplasmic Reticulum-Associated Degradation in CHO Cells Resistant to Cholera Toxin,
Pseudomonas aeruginosa
Exotoxin A, and Ricin
Authors: Teter Ken , Holmes Randall K.
Published: October 2002

Many plant and bacterial toxins act upon cytosolic targets and must therefore penetrate a membrane barrier to function. One such class of toxins enters the cytosol after delivery to the endoplasmic reticulum (ER). These proteins, which include cholera toxin (CT),
Pseudomonas aeruginosa
exotoxin A (ETA), and ricin, move from the plasma membrane to the endosomes, pass through the Golgi apparatus, and travel to the ER. Translocation from the ER to the cytosol is hypothesized to involve the ER-associated degradation (ERAD) pathway. We developed a genetic strategy to assess the role of mammalian ERAD in toxin translocation. Populations of CHO cells were mutagenized and grown in the presence of two lethal toxins, ETA and ricin. Since these toxins bind to different surface receptors and attack distinct cytoplasmic targets, simultaneous acquisition of resistance to both would likely result from the disruption of a shared trafficking or translocation mechanism. Ten ETA- and ricin-resistant cell lines that displayed unselected resistance to CT and continued sensitivity to diphtheria toxin, which enters the cytosol directly from acidified endosomes, were screened for abnormalities in the processing of a known ERAD substrate, the Z form of α1-antitrypsin (α1AT-Z). Compared to the parental CHO cells, the rate of α1AT-Z degradation was decreased in two independent mutant cell lines. Both of these cell lines also exhibited, in comparison to the parental cells, decreased translocation and degradation of a recombinant CTA1 polypeptide. These findings demonstrated that decreased ERAD function was associated with increased cellular resistance to ER-translocating protein toxins in two independently derived mutant CHO cell lines.
Clinical and Immune Impact of
Mycobacterium bovis
BCG Vaccination Scarring
Authors: Jason Janine , Archibald Lennox K. , Nwanyanwu Okey C. , Kazembe Peter N. , Chatt Julie A. , Norton Elizabeth , Dobbie Hamish , Jarvis William R.
Published: October 2002

The World Health Organization recommends
Mycobacterium bovis
BCG vaccination in areas of high tuberculosis prevalence. BCG's clinical and immune effects, not necessarily
Mycobacterium tuberculosis
specific, are unclear. BCG vaccine scarring often is used as a surrogate marker of vaccination or of effective vaccination. We evaluated BCG scarring status in relation to clinical findings and outcome in 700 hospitalized Malawians, of whom 32 had
M. tuberculosis
bloodstream infections (BSI) (10 of whom had cellular immune studies done) and of whom 48 were infants <6 months old and therefore recently vaccinated (19 of whom had immune studies). In the patients ≥6 months old, scarring was not related to the presence of pulmonary symptoms (35 versus 30%), chronic cough or fever, mortality, or
M. tuberculosis
BSI. In
M. tuberculosis
BSI patients, scarring was unrelated to mortality, vital signs, or clinical symptoms but those with scarring had higher proportions of memory and activated T cells and more type 2-skewed cytokine profiles. Infants with either BCG scarring (
n
= 10) or BCG lesional inflammation (
n
= 5) had no symptoms of sepsis, but 18 of 33 infants without BCG vaccination lesions did. Those with BCG lesions had localized infections more often than did those without BCG lesions. These infants also had lower median percentages of lymphocytes spontaneously making interleukin-4 (IL-4) or tumor necrosis factor alpha (TNF-α) and lower ratios of T cells spontaneously making IL-4 to T cells making IL-6. Thus, we found that, in older patients, BCG vaccine scarring was not associated with
M. tuberculosis
-specific or nonspecific clinical protection. Those with
M. tuberculosis
BSI and scarring had immune findings suggesting previous
M. tuberculosis
antigen exposure and induction of a type 2 cytokine pattern with acute reexposure. It is unlikely that this type 2 pattern would be protective against mycobacteria, which require a type 1 response for effective containment. In infants <6 months old, recent BCG vaccination was associated with a non-
M. tuberculosis
-specific, anti-inflammatory cytokine profile. That the vaccinated infants had a greater frequency of localized infections and lesser frequency of sepsis symptoms suggests that this postvaccination cytokine pattern may provide some non-
M. tuberculosis
-specific clinical benefits.
Yersinia pseudotuberculosis
Harbors a Type IV Pilus Gene Cluster That Contributes to Pathogenicity
Authors: Collyn François , Léty Marie-Annick , Nair Shamila , Escuyer Vincent , Ben Younes Amena , Simonet Michel , Marceau Michaël
Published: October 2002

Fimbriae have been shown to play an essential role in the adhesion of pathogenic gram-negative bacteria to host cells. In the enteroinvasive bacterium
Yersinia pseudotuberculosis
, we characterized a previously unknown 11-kb chromosomal locus involved in the synthesis of type IV pili. The locus consists of 11 open reading frames forming a polycistronic unit and encoding putative Pil proteins, PilLMNOPQRSUVW. When introduced into
Escherichia coli
, the
Y. pseudotuberculosis
operon reconstituted bundles of filaments at a pole on the bacterial surface, demonstrating that the
pil
locus was functional in a heterogenous genetic background. Environmental factors regulated transcription of the
Y. pseudotuberculosis
operon; in particular, temperature, osmolarity, and oxygen tension were critical cues. Deletion of the type IV pilus gene cluster was associated with a reduction of
Y. pseudotuberculosis
pathogenicity for mice infected orally. Forty-one percent of
Y. pseudotuberculosis
strains isolated from human or animal sources harbored the type IV pilus locus. Therefore, the
pil
locus of
Y. pseudotuberculosis
might constitute an “adaptation island,” permitting the microorganism to colonize a vast reservoir.
Mycobacterium tuberculosisUptake by Recipient Host Macrophages Is Influenced by Environmental Conditions in the Granuloma of the Infectious Individual and Is Associated with Impaired Production of Interleukin-12 and Tumor Necrosis Factor Alpha
Authors: Li Yong-jun , Petrofsky Mary , Bermudez Luiz E.
Published: October 2002

Transmission ofMycobacterium tuberculosisfrom one individual to another usually is associated with episodes of coughing. The bacteria leave the environment of the lung cavity of the infected person and travel in droplets to reach the recipient's respiratory tract. Therefore, at the time that the bacteria encounter alveolar cells (macrophages and epithelial cells) in the new host, they express virulence determinants that are regulated by the environmental conditions in the infected person. To determine if those environmental conditions encountered in the lung cavity (hyperosmolarity, acidic pH, and low oxygen tension, among others) would influence the uptake ofM. tuberculosisby the recipient's alveolar macrophages,M. tuberculosisH37Rv was incubated under several conditions for different periods of time, washed at 4°C, and used to infect human monocyte-derived macrophages. While increased osmolarity had no effect onM. tuberculosisuptake compared to the uptake of bacteria grown on 7H10 Middlebrook medium, both acidic pH and anaerobiosis increased the uptake of the H37Rv strain four- to sixfold. Using anti-CD11b receptor blocking antibodies or mannoside to inhibit the uptake ofM. tuberculosisby macrophages, we determined that while uptake ofM. tuberculosiscultured on 7H10 medium was inhibited 77% ± 6% in the presence of anti-CD11b antibody, the antibody had no effect on the uptake ofM. tuberculosisincubated at pH 6.0 and was associated with 27% inhibition ofM. tuberculosispreviously exposed to anaerobic conditions. The mannose receptor was also not involved with invasion after exposure to acidic conditions, and mannoside resulted in only 32% inhibition of uptake by macrophages ofM. tuberculosisexposed to anaerobiosis. Uptake by macrophages also resulted in the secretion of significantly lower amounts of interleukin-12 and tumor necrosis factor alpha than that by macrophages infected with a strain cultured under laboratory conditions.M. tuberculosiscultured under the pH and oxygen concentration found in the granuloma expresses a large number of proteins that are different from the proteins expressed by bacteria grown under laboratory conditions. The results suggest thatM. tuberculosisin vivo may be adapted to gain access to the intracellular environment in a very efficient fashion and may do so by using different receptors from the complement and mannose receptors.