Biomedical subjects

Jun Shimada

Publications and source records attributed to Jun Shimada.

At least 19 recordsLinked to original sources

PubMed2006

Application of data mining approaches to drug delivery.

Computational approaches play a key role in all areas of the pharmaceutical industry from data mining, experimental and clinical data capture to pharmacoeconomics and adverse events monitoring. They will likely continue to be indispensable assets along with a growing library of software applications. This is primarily due to the increasingly massive amount of biology, chemistry and clinical data, which is now entering the public domain mainly as a result of NIH and commercially funded projects. We are therefore in need of new methods for mining this mountain of data in order to enable new hypothesis generation. The computational approaches include, but are not limited to, database compilation, quantitative structure activity relationships (QSAR), pharmacophores, network visualization models, decision trees, machine learning algorithms and multidimensional data visualization software that could be used to improve drug delivery after mining public and/or proprietary data. We will discuss some areas of unmet needs in the area of data mining for drug delivery that can be addressed with new software tools or databases of relevance to future pharmaceutical projects.

Computer Simulation↗

PubMed2006

Efficacy of a novel oral carbapenem, tebipenem pivoxil (TBM-PI), against experimental otitis media caused by penicillin resistant Streptococcus pneumoniae in chinchilla.

An animal model of otitis media using chinchillas was developed to evaluate the efficacy of tebipenem pivoxil (TBM-PI) against experimental otitis media. Chinchillas inoculated via the transbullar approach with Streptococcus pneumoniae serogroup 6 were included in the efficacy study with TBM-PI, amoxicillin (AMX) or untreated as controls. TBM-PI resulted in survival rate of 83%, compared with 25% survival for AMX and 0% survival for controls (p<0.01). Quantitative cultures in the middle ear effusions at day 5 of the TBM-PI group yielded 3.5+/-2.4log(10)CFUs/ml. TBM-PI is a promising antibiotic for the treatment of acute otitis media.

Administration, Oral↗

PubMed2006

High prevalence of Streptococcus pneumoniae with mutations in pbp1a, pbp2x, and pbp2b genes of penicillin-binding proteins in the nasopharynx in children in Japan.

OBJECTIVE: To evaluate the resistances of Streptococcus pneumoniae to beta-lactams developed by stepwise alterations in high-molecular-weight penicillin-binding proteins (PBPs) with a reduced binding affinity of beta-lactams. Among the numerous mutations in pbp genes that alter the affinity for beta-lactams, the decreased affinity of PBP1A, 2X and 2B is especially important in the development of resistances to beta-lactams. STUDY DESIGN: Retrospective review. METHODS: In this study, we investigated the mutations in pbp1a, pbp2x, and pbp2b genes evaluated by polymerase chain reaction (PCR) in 866 pneumococcal isolates collected from the nasopharynx of Japanese children with acute otitis media. RESULTS: 210 strains (24.3%) exhibited no mutations in the three pbp genes. 333 strains (38.5%) had mutations in the three pbp genes, 78 (9.0%) in two pbp genes, whereas 245 (28.3%) displayed mutations in only one pbp gene. Among the 656 strains with mutations in pbp genes, 620 (94.5%) strains had mutations in pbp2x. The annual prevalence of antimicrobial-resistant S. pneumoniae showed a gradual increase in strains with mutations in the three pbp genes and a parallel decrease in strains without mutations. CONCLUSIONS: PCR-based genotyping can characterize the antimicrobial resistances in pneumococci along with minimal inhibitory concentrations (MICs). Physicians should pay attention to the recent increase in antimicrobial-resistant S. pneumoniae when treating pediatric acute otitis media.

Acute Disease↗

PubMed2006

Antimicrobial resistance in Haemophilus influenzae isolated from the nasopharynx among Japanese children with acute otitis media.

CONCLUSION: High prevalence of penicillin-binding protein (PBP) gene mutated (PGM) strains of H. influenzae should be taken into account when treating otitis media in children. OBJECTIVE: To evaluate prevalence of ss-lactamase nonproducing ampicillin-resistant (BLNAR) strains of Haemophilus influenzae with mutations in ftsI gene encoding penicillin-binding protein 3 (PBP3) among children with otitis media. METHODS: A total of 644 nasopharyngeal isolates of H. influenzae was collected from pediatric acute otitis media (AOM) patients with or without otitis media with effusions (OME) at the clinics of Otolaryngology-Head and Neck Surgery, Wakayama Medical University Hospital and six affiliated hospitals in Wakayama prefecture between January 1999 and December 2003. Minimal inhibitory concentrations (MICs) of ampicillin (AMP), cefditoren (CDN), cefdinir (CFD), cefaclor (CCL), cefpodoxime (CPD), and cefcapene (CFPN) were determined by the microbroth dilution method according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS). Types of mutations in PBP3 gene (ftsI) were evaluated by a polymerase chain reaction (PCR)-based genotyping method. ss-Lactamase gene (bla) was also identified by PCR. RESULTS: ss-Lactamase-producing (BLP) strains with the bla gene were identified in 16 (2.5%) of isolates. PGM strains were identified in 279 (43.3%) isolates. There were 242 (37.6%) PGM1-nonBLP strains with mutations in variable mutated locus of ftsI, 35 (5.4%) PGM2-nonBLP strains with mutations in highly mutated locus of ftsI, 2 (0.3%) BLP-PGM strains with mutations in ftsI and producing ss-lactamase. BLP-nonPGM strains producing ss-lactamase without mutations in ftsI were identified in 14 (2.2%) isolates. MICs of PGM1-nonBLP strains to AMP were 0.5-2.0 microg/ml. The MIC(90) of CDN to the PGM1-nonBLP strains was lowest (0.06 microg/ml). Proportions of PGM1-nonBLP strains rapidly increased during 1999 to 2002 and then decreased in 2003. In contrast, PGM2-nonBLP strains increased in 2003.

Acute Disease↗

PubMed2006

Antimicrobial resistance of Haemophilus influenzae isolated from the nasopharynx of Japanese children with acute otitis media.

CONCLUSION: A high prevalence of penicillin-binding protein gene-mutated (PGM) strains of Haemophilus influenzae should be taken into account when treating otitis media in children. OBJECTIVE: To evaluate the prevalence of beta-lactamase-non-producing ampicillin-resistant strains of H. influenzae with mutations in the ftsI gene encoding penicillin-binding protein 3 (PBP3) among children with otitis media. MATERIAL AND METHODS: A total of 644 nasopharyngeal isolates of H. influenzae were collected from pediatric acute otitis media patients with or without otitis media with effusion at the clinics of the Department of Otolaryngology-Head and Neck Surgery, Wakayama Medical University Hospital and 6 affiliated hospitals in Wakayama Prefecture between January 1999 and December 2003. MICs to ampicillin (AMP), cefdinir (CFD), cefaclor (CCL), cefpodoxime (CPD) and cefcapene (CFPN) were determined by a microbroth dilution method according to the recommendations of the National Committee for Clinical Laboratory Standards. Types of mutations in the PBP3 gene (ftsI) were evaluated by means of a polymerase chain reaction (PCR)-based genotyping method. The beta-lactamase gene (bla) was also identified by means of PCR. RESULTS: Beta-lactamase-producing (BLP) strains having the bla gene were identified in 16 isolates (2.5%). PGM strains were identified in 279 isolates (43.3%). There were 242 PGM1-non-BLP strains (37.6%) with mutations in the variable mutated locus of ftsI, 35 PGM2-non-BLP strains (5.4%) with mutations in the highly mutated locus of ftsI and 2 BLP-PGM strains (0.3%) with mutations in ftsI that produced beta-lactamase. BLP-non-PGM strains producing beta-lactamase without mutations in ftsI were identified in 14 isolates (2.2%). MICs of PGM1-non-BLP strains to AMP were 0.5-2.0 microg/ml. The MIC90 of CDN to the PGM1-non-BLP strains was the lowest (0.06 microg/ml). The proportion of PGM1-non-BLP strains increased rapidly during 1999-2002 and then decreased in 2003. In contrast, the proportion of PGM2-non-BLP strains increased in 2003.

Acute Disease↗

PubMed2005

A recombinant P4 protein of Haemophilus influenzae induces specific immune responses biologically active against nasopharyngeal colonization in mice after intranasal immunization.

Outer membrane protein P4, together with P6, is highly conserved among all typeable and nontypeable strains of Haemophilus influenzae (H. influenzae). Thus, the protein is an attractive antigen for the inclusion in a vaccine against nontypeable H. influenzae (NTHi). However, the ability of P4 to induce antibodies protective against NTHi infections is still controversial. In this study, we investigated the specific mucosal immune responses against NTHi induced by intranasal immunization with the lipidated form of recombinant P4 protein (rP4) and non-fatty acylated recombinant P6 protein (rP6) with or without cholera toxin (CT) in BALB/c mice model. Intranasal immunization with either rP4+CT, a mixture of rP4 and rP6+CT, or rP4 and rP6 without CT elicited anti-rP4 specific IgG antibody in serum of mice. Intranasal immunization with either rP4+CT or a mixture of rP4, rP6+CT elicited anti-rP4 specific IgA antibody in nasopharyngeal washing (NPW), while intranasal immunization with rP4 and rP6 without CT did not induced anti-rP4 specific IgA antibody responses in NPWs. Sera from mice intranasally immunized with rP4+CT and a mixture of rP4, rP6+CT also showed bactericidal activity. Significant clearance of NTHi in nasopharynx was seen 3 days after the inoculation of live NTHi in mice intranasally immunized with rP4+CT. The current findings suggested that P4 would be a useful antigen as the component of the vaccine to induce protective immune responses against NTHi. The use of an intranasal vaccine composed of the different surface protein antigens is an attractive strategy for the development of a vaccine against NTHi.

Adjuvants, Immunologic↗

PubMed2005

Current status of antimicrobial susceptibility of clinical isolates of Streptococcus pyogenes in Japan: report of a countrywide surveillance study.

The growing number of macrolide-resistant strains of Streptococcus pyogenes is an increasing problem worldwide. In this study, we evaluated 62 clinical isolates of S. pyogenes obtained from the upper respiratory tract. Susceptibilities to penicillins, cephalosporins, fluoroquinolones, macrolides, and carbepenems were determined by minimal inhibitory concentrations (MICs). Expressions of macrolide-resistance genes (mefA, ermB, and ermTR) were also examined, by polymerase chain reaction (PCR). All strains were susceptible to beta-lactams. On the other hand, of the 62 S. pyogenes isolates, 6.5%, 6.5%, 6.5%, and 3.2% of the strains were resistant to azithromycin (AZM), roxithromycin (RXM), clarithromycin (CAM), and telithromycin (TEL), respectively. Four (6.5%) strains had a type of macrolide-resistance gene; there were two strains with ermB and two strains with ermTR, and these four strains were resistant to AZM, CAM (one strain was intermediately resistant), and RXM. Strains having ermB were resistant to TEL (MIC, > or = 8 microg/ml), while strains having ermTR were susceptible to TEL. Physicians and researchers need to take into consideration the macrolide resistance of some strains of S. pyogenes.

Anti-Bacterial Agents↗

PubMed2005

Evaluation of mutations in penicillin binding protein-3 gene of non-typeable Haemophilus influenzae isolated from the nasopharynx of children with acute otitis media.

CONCLUSION: Younger children tend to harbor more resistant strains because they are exposed to these pathogens more often through contacts with siblings or attendance at day-care centers and are frequently treated with antibiotics. The high prevalence of BLNAR strains should be taken into account in the treatment of AOM in young children. OBJECTIVE: Non-beta-lactamase-producing ampicillin-resistant (BLNAR) strains with mutations in penicillin-binding protein (PBP) genes of Haemophilus influenzae have been prevalent recently among younger children. MATERIAL AND METHODS: We investigated mutations in the ftsI gene encoding PBP-3 of H. influenzae isolated from the nasopharynx of children with acute otitis media (AOM) using polymerase chain reaction (PCR). RESULTS: Strains containing the bla gene (beta-lactamase-producing ampicillin-resistant) were identified in 4.7% of cases. Strains with mutations in the ftsI gene (BLNAR) were identified in 23.3% of cases. Strains without mutations in the ftsI gene and that did not contain the bla gene (non-beta-lactamase-producing ampicillin-susceptible) were identified in 70.7% of cases. Strains with both expression of the bla gene and mutations in the ftsI gene (beta-lactamase-producing amoxicillin clavulanate-resistant) were identified in 1.3% of cases. The MICs of ampicillin against the strains evaluated in this study were 0.5-2.0 microg/ml. Cefditoren-pivoxil had the lowest MIC90 against the strains (0.06 microg/ml). Strains with mutations in the ftsI gene (BLNAR) were broadly identified among young children.

Acute Disease↗

PubMed2005

Increase of macrolide-resistant Streptococcus pneumoniae-expressing mefE or ermB gene in the nasopharynx among children with otitis media.

OBJECTIVE: To evaluate prevalence of macrolide resistant strains and the genotypes of the resistance among Streptococcus pneumoniae isolated from the nasopharynx of children with otitis media. STUDY DESIGN: Retrospective review. METHODS: A total of 858 S. pneumoniae isolates were collected from the nasopharynx of pediatric patients with acute otitis media at the clinics of Otolaryngology-Head and Neck Surgery, Wakayama Medical University Hospital and six affiliated hospitals in Wakayama prefecture between January 1998 and December 2002. The antibiotic susceptibility patterns were analyzed for penicillin, erythromycin, and clindamycin according to the National Committee for Clinical Laboratory Standards. Macrolide resistance genes of mefE and ermB were determined by polymerase chain reaction of all S. pneumoniae. RESULTS: Of 858 clinical isolates, 259 (30.1%) were strains without ermB or mefE gene, 279 (32.5%) carrying mefE, 292 (34.0%) carrying ermB, and 28 (3.4%) carrying both genes. There was a strong correlation between phenotypes and the presence of macrolide resistance genes. The macrolide resistance genes were especially frequently identified among penicillin-resistant S. pneumoniae. Strains carrying ermB gene gradually increased from 25% in 1998 to 45% in 2002, with a concurrent decrease in strains carrying mefE from 36% in 1998 to 1999 to 19% in 2002. Strains having mefE were frequently identified among children younger than 2 years old. The current finding suggested that high-level ermB-mediated macrolide resistance in S. pneumoniae is increasing at an alarming rate in pediatric patients with otitis media, especially among young children. Physicians should pay close attention to such macrolide-resistant bacterial pathogens in the antimicrobial treatment of pediatric patients with otitis media.

Bacterial Proteins↗

PubMed2004

Treatment and outcome of severe and non-severe acute otitis media.

UNLABELLED: To determine outcomes in acute otitis media (AOM) according to severity of disease and to assess different initial treatment regimens, 308 with AOM were enrolled and divided into severe (n = 277; 89.9%) and non-severe (n = 31; 10.1%) groups based on symptoms and tympanic membrane changes. Children in the severe group were initially managed with amoxicillin (AMPC) whereas children in the non-severe group were initially managed without antibiotics. Children were monitored on days 1, 5, 10, 14 and 28. Five outcome measures were assessed: disappearance of symptoms at day 5, resolution of tympanic membrane changes by day 28, disappearance of middle ear effusions by day 28, recurrence of acute symptoms prior to day 28, and need to change treatment regimens. Children with severe disease were more often male (57% versus 36%, P < 0.05) and more often colonized with pathogens (77% versus 55%, P < 0.05 than children with non-severe disease. The two groups were similar with respect to age and day care attendance. Despite differences in initial treatment regimens between the two groups, symptoms improved at the same rate for severe and non-severe disease, 94% by day 5. In contrast, tympanic membranes returned to normal in 69% of the severe and 81% of the non-severe groups by day 28; however, as early as day 5, 10% of the severe and 55% of the non-severe groups demonstrated normal tympanic membranes. Middle ear effusions similarly disappeared more slowly in the severe group, 52% versus 74% by day 14 and 76% versus 84% by day 28. Recurrence rates of acute symptoms occurred with equal frequency in the severe, 15%, and non-severe groups, 10%. Failure of the symptoms or the tympanic membranes to improve led to antibiotic changes in 59.9% of the severe group and to the addition of antibiotics in 51.6% of the non-severe group. Children in the severe group who failed to improve with an initial course of amoxicillin were younger (40.2 months versus 45.8 months, P < 0.05), had higher tympanic membrane scores (4.5 versus 4.1, P < 0.05), and were more often colonized with penicillin-resistant Streptococcus pneumoniae (33.8% versus 6.5%, P < 0.01) than children who responded to AMPC. In a similar manner, children with non-severe disease who failed to improve without antibiotics were younger (40.7 months versus 54.8 months, P < 0.05) and more often colonized with pathogens (75.0% versus 33.4%, P < 0.05). CONCLUSION: Severe disease occurred more often among males and among children colonized with pathogens. Response to treatment was impaired in younger children and in children colonized with pathogens, especially penicillin-resistant Streptococcus pneumoniae.

Acute Disease↗

PubMed2004

The novel antipsychotic aripiprazole is a partial agonist at short and long isoforms of D2 receptors linked to the regulation of adenylyl cyclase activity and prolactin release.

Aripiprazole is a novel antipsychotic with a unique mechanism of action, which differs from currently marketed typical and atypical antipsychotics. Aripiprazole has been shown to be a partial agonist at the D(2) family of dopamine (DA) receptors in biochemical and pharmacological studies. To demonstrate aripiprazole's action as a partial D(2) agonist in pituitary cells at the molecular level, we retrovirally transduced the short (D(2S)) and the long (D(2L)) form of the human DA D(2) receptor gene into a rat pituitary cell line, GH4C1. [(3)H]-raclopride saturation binding analyses revealed a B(max) value approximately four-fold higher at D(2S) receptor-expressing GH4C1 cells than at D(2L) receptor-expressing GH4C1 cells, while a K(d) value was similar. Aripiprazole inhibited forskolin-stimulated release of prolactin in both D(2S) and D(2L) receptor-expressing GH4C1 cells, whereas the maximal inhibition of prolactin release was less than that of DA. Similarly, aripiprazole partially inhibited forskolin-induced cAMP accumulation in both D(2) receptor-expressing cells. Aripiprazole antagonized the suppression attained by DA (10(-7) M) in both D(2) receptor-expressing cells and, at the maximal blockade of cAMP, yielded residual cAMP levels equal to those produced by aripiprazole alone. These results indicate that aripiprazole acts as a partial agonist at both D(2S) and D(2L) receptors expressed in GH4C1 cells. These data may explain, at least in part, the observations that aripiprazole shows a novel antipsychotic activity with minimal potential for adverse events including no significant increase of serum prolactin levels in clinical studies.

Adenylyl Cyclases↗

PubMed2004

Commitment and nucleation in the protein G transition state.

An accurate characterization of the transition state ensemble (TSE) is central to furthering our understanding of the protein folding reaction. We have extensively tested a recently reported method for studying a protein's TSE, utilizing phi-value data from protein engineering experiments and computational studies as restraints in all-atom Monte Carlo (MC) simulations. The validity of interpreting experimental phi-values as the fraction of native contacts made by a residue in the TSE was explored, revealing that this definition is unable to uniquely specify a TSE. The identification of protein G's second hairpin, in both pre and post-transition conformations demonstrates that high experimental phi-values do not guarantee a residue's importance in the TSE. An analysis of simulations based on structures restrained by experimental phi-values is necessary to yield this result, which is not obvious from a simplistic interpretation of individual phi-values. The TSE that we obtain corresponds to a single, specific nucleation event, characterized by six residues common to all three observed, convergent folding pathways. The same specific nucleus was independently identified from computational and experimental data, and "Conservation of Conservation" analysis in the protein G fold. When associated strictly with complete nucleus formation and concomitant chain collapse, folding is a well-defined two state event. Once the nucleus has formed, the folding reaction enters a slow relaxation process associated with side-chain packing and small, local backbone rearrangements. A detailed analysis of phi-values and their relationship to the transition state ensemble allows us to construct a unified theoretical model of protein G folding.

Models, Molecular↗

PubMed2004

Factors associated with clinical outcomes in acute otitis media.

Acute otitis media (AOM) is a common disease in childhood. If predictors of outcome in AOM were known, it would be possible to individualize therapy. Our aim was to identify factors that predict the outcome in AOM. We enrolled 368 children with AOM (ages, 10 to 86 months). The severity of symptoms and the severity of tympanic membrane changes were graded with a scoring system. Nasopharyngeal colonization with middle ear pathogens was determined on day 1. Three outcomes were assessed: persistence of symptoms at day 5, persistence of tympanic membrane changes at day 28, and recurrence of acute symptoms prior to day 28. Persistence of symptoms at day 5 was associated with younger age (35 versus 44 months; p < .001), higher symptom score on day 1 (3.5 versus 2.9; p < .05), and colonization with Streptococcus pneumoniae (61% versus 41%; p < .05). Persistence of tympanic membrane changes at day 28 was associated with younger age (39 versus 45 months; p < .01), higher tympanic membrane score on day 1 (4.1 versus 3.6; p < .01), and nasopharyngeal colonization with S. pneumoniae, especially drug-resistant S. pneumoniae (33% versus 13%; p < .05). Recurrence of acute symptoms prior to day 28 occurred in 14% of the children. Streptococcus pneumoniae was the only pathogen associated with an increased recurrence rate (23%) as compared to the group without pathogens (7%; p < .05). Age, severity of disease at presentation, and nasopharyngeal colonization patterns were proven to be important determinants of outcome in AOM.

Child↗

PubMed2003

Side-chain dynamics and protein folding.

The processes by which protein side chains reach equilibrium during a folding reaction are investigated using both lattice and all-atom simulations. We find that rates of side-chain relaxation exhibit a distribution over the protein structure, with the fastest relaxing side chains located in positions kinetically important for folding. Traversal of the major folding transition state corresponds to the freezing of a small number of side chains, belonging to the folding nucleus, whereas the rest of the protein proceeds toward equilibrium via backbone fluctuations around the native fold. The postnucleation processes by which side chains relax are characterized by very slow dynamics and many barrier crossings, and thus resemble the behavior of a glass.

Amino Acids↗

PubMed2003

End-stage kidney at the onset of nephrotic syndrome in a 4-year-old girl.

A previously healthy Japanese girl aged 4 years suddenly developed idiopathic nephrotic syndrome (INS) associated with acute renal failure (ARF). Despite prompt initiation of hemodialysis and prednisolone treatment, her ARF did not recover. A percutaneous renal biopsy performed within a month from the onset of INS revealed lesions of end-stage kidney, which is unusual for the acute stage of INS. Although the etiology of her ARF remains speculative, an acute exacerbation of an underlying chronic disease, such as non-nephrotic focal segmental glomerulosclerosis might be the cause.

Acute Kidney Injury↗

PubMed2002

Transcriptional activation of endoglin and transforming growth factor-beta signaling components by cooperative interaction between Sp1 and KLF6: their potential role in the response to vascular injury.

Endoglin is an endothelial membrane glycoprotein involved in cardiovascular morphogenesis and vascular remodeling. It associates with transforming growth factor-beta (TGF-beta) signaling receptors to bind TGF-beta family members, forming a functional receptor complex. Arterial injury leads to up-regulation of endoglin, but the underlying regulatory events are unknown. The transcription factor KLF6, an immediate-early response gene induced in endothelial cells during vascular injury, transactivates TGF-beta, TGF-beta signaling receptors, and TGF-beta-stimulated genes. KLF6 and, subsequently, endoglin were colocalized to vascular endothelium (ie, expressed in the same cell type) following carotid balloon injury in rats. After endothelial denudation, KLF6 was induced and translocated to the nucleus; this was followed 6 hours later by increased endoglin expression. Transient overexpression of KLF6, but not Egr-1, stimulated endogenous endoglin mRNA and transactivated the endoglin promoter. This transactivation was dependent on a GC-rich tract required for basal activity of the endoglin promoter driven by the related GC box binding protein, Sp1. In cells lacking Sp1 and KLF6, transfected KLF6 and Sp1 cooperatively transactivated the endoglin promoter and those of collagen alpha1(I), urokinase-type plasminogen activator, TGF-beta1, and TGF-beta receptor type 1. Direct physical interaction between Sp1 and KLF6 was documented by coimmunoprecipitation, pull-down experiments, and the GAL4 one-hybrid system, mapping the KLF6 interaction to the C-terminal domain of Sp1. These data provide evidence that injury-induced KLF6 and preexisting Sp1 may cooperate in regulating the expression of endoglin and related members of the TGF-beta signaling complex in vascular repair.

Animals↗

PubMed2002

Intranasal immunization with recombinant outer membrane protein P6 induces specific immune responses against nontypeable Haemophilus influenzae.

OBJECTIVE: Nontypeable Haemophilus influenzae (NTHi) is one of the leading causative pathogens for otitis media. The outer membrane protein P6 of NTHi is highly conserved among the strains and is an attractive candidate for a preventive vaccine. However, for the production of a relatively small amount P6 containing lipopolysaccharides, the development of a recombinant version of this protein is required. This study was designed to investigate the specific mucosal immunity induced by intranasal immunization of recombinant P6 (rP6) with cholera toxin (CT). METHODS: BALB/c mice were immunized with of rP6 (30 microg) and CT (2 microg) intranasally every 2 days for 2 weeks. Anti-rP6 specific IgG, IgA and IgM antibodies and the subclass of anti-rP6 specific IgG antibody were determined by enzyme linked immunosorbent assay (ELISA). Anti-rP6 specific IgA in nasopharyngeal washings were also determined by ELISA. Nasopharyngeal clearance of inoculated NTHi after the intranasal immunization were assessed. All statistical differences between the two groups were assessed by ANOVA parametric test. RESULTS: Intranasal immunization with rP6 and CT evoked rP6-specific mucosal IgA immune response as well as the systemic IgG immune response against rP6 and enhanced nasopharyngeal clearance of inoculated live NTHi. CONCLUSION: These results indicate the good immunogenicities of rP6 to induce specific immune responses against NTHi. Intranasal immunization with rP6 will be an effective approach to protect infections of NTHi.

Analysis of Variance↗

PubMed2002

The ensemble folding kinetics of protein G from an all-atom Monte Carlo simulation.

Protein G is folded with an all-atom Monte Carlo simulation by using a Gō potential. When folding is monitored by using burial of the lone tryptophan in protein G as the reaction coordinate, the ensemble kinetics is single exponential. Other experimental observations, such as the burst phase and mutational data, are also reproduced. However, more detailed analysis reveals that folding occurs over three distinct, three-state pathways. We show that, because of this tryptophan's asymmetric location in the tertiary fold, its burial (i) does not detect certain intermediates and (ii) may not correspond to the folding event. This finding demonstrates that ensemble averaging can disguise the presence of multiple pathways and intermediates when a non-ideal reaction coordinate is used. Finally, all observed folding pathways eventually converge to a common rate-limiting step, which is the formation of a specific nucleus involving hydrophobic core residues. These residues are conserved in the ubiquitin superfamily and in a phage display experiment, suggesting that fold topology is a strong determinant of the transition state.

Binding Sites↗