The p53 tumor suppressor pathway is impaired in more than 90% of cervical cancers and cancer-derived cell lines as a result of infection by human papillomavirus (HPV). The HPV E6 oncoprotein forms complexes with p53 and promotes its degradation via the ubiquitin-dependent mechanism. In this study, we attempted to improve the clinical outcomes of this combined therapy by modifying the p53-targeted adenovirus to become radiation-responsive. The anti-tumor adenovirus was constructed by inserting a radiation-responsive expression cassette composed of the promoter of early growth response-1 (Egr-1) and the pro-apoptotic protein TRAIL. We showed that the addition of adenovirus containing Egr-1/TRAIL significantly increased cell death and apoptosis caused by radiotherapy. In mice bearing xenograft tumors, intratumoral administration of the Egr-1/TRAIL adenovirus followed by radiation significantly reduced tumor growth and enhanced tumor survival. Our Egr-1/TRAIL adenoviral gene product may offer a novel “one-two punch” tumor therapy for cervical cancers not only by potentiating radiation treatment but also by preserving p53 defect-specific tumor killing of the oncolytic adenovirus.

AIM: To demonstrate the oncologic outcomes of low rectal cancer and to clarify the risk factors for survival, focusing particularly on the type of surgery performed.

METHODS: Data from patients with low rectal carcinomas who underwent surgery, either sphincter-preserving surgery (SPS) or abdominoperineal resection (APR), at The First Affiliated Hospital of Sun Yat-sen University in China from August 1994 to December 2005 were retrospectively analyzed.

RESULTS: Of 331 patients with low rectal cancer, 159 (48.0%) were treated with SPS. A higher incidence of positive resection margins and a higher 5-year cumulative local recurrence rate (14.7% vs 6.8%, P = 0.041) were observed in patients after APR compared to SPS. The five-year overall survival (OS) was 54.6% after APR and 66.8% after SPS (P = 0.018), and the 5-year disease-free survival (DFS) was 52.9% after APR and 65.5% after SPS (P = 0.013). In multivariate analysis, poor OS and DFS were significantly related to positive resection margins, pT3-4, and pTNM III-IV but not to the type of surgery.

CONCLUSION: Despite a higher rate of positive resection margins after APR, the type of surgery was not identified as an independent risk factor for survival.

Small to moderate gains in Pig fertility can mean large returns in overall efficiency, and developing methods to improve it is highly desirable. High fertility rates depend on completion of successful pregnancies. To understand the molecular signals associated with pregnancy in sows, expression profiling experiments were conducted to identify differentially expressed genes in ovary and myometrium at different pregnancy periods using the Affymetrix Porcine GeneChipTM. A total of 974, 1800, 335 and 710 differentially expressed transcripts were identified in the myometrium during early pregnancy (EP) and late pregnancy (LP), and in the ovary during EP and LP, respectively. Self-Organizing Map (SOM) clusters indicated the differentially expressed genes belonged to 7 different functional groups. Based on BLASTX searches and Gene Ontology (GO) classifications, 129 unique genes closely related to pregnancy showed differential expression patterns. GO analysis also indicated that there were 21 different molecular function categories, 20 different biological process categories, and 8 different cellular component categories of genes differentially expressed during sow pregnancy. Gene regulatory network reconstruction provided us with an interaction model of known genes such as insulin-like growth factor 2 (IGF2) gene, estrogen receptor (ESR) gene, retinol-binding protein-4 (RBP4) gene, and several unknown candidate genes related to reproduction. Several pitch point genes were selected for association study with reproduction traits. For instance, DPPA5 g.363 T>C was found to associate with litter born weight at later parities in Beijing Black pigs significantly (p < 0.05). Overall, this study contributes to elucidating the mechanism underlying pregnancy processes, which maybe provide valuable information for pig reproduction improvement.

Pork quality is an economically important trait and one of the main selection criteria for breeding in the swine industry. In this genome-wide association study (GWAS), 455 pigs from a porcine Large White × Minzhu intercross population were genotyped using the Illumina PorcineSNP60K Beadchip, and phenotyped for intramuscular fat content (IMF), marbling, moisture, color L*, color a*, color b* and color score in the longissimus muscle (LM). Association tests between each trait and the SNPs were performed via the Genome Wide Rapid Association using the Mixed Model and Regression-Genomic Control (GRAMMAR-GC) approach. From the Ensembl porcine database, SNP annotation was implemented using Sus scrofa Build 9. A total of 45 SNPs showed significant association with one or multiple meat quality traits. Of the 45 SNPs, 36 were located on SSC12. These significantly associated SNPs aligned to or were in close approximation to previously reported quantitative trait loci (QTL) and some were located within introns of previously reported candidate genes. Two haplotype blocks ASGA0100525-ASGA0055225-ALGA0067099-MARC0004712-DIAS0000861, and ASGA0085522-H3GA0056170 were detected in the significant region. The first block contained the genes MYH1, MYH2 and MYH4. A SNP (ASGA0094812) within an intron of the USP43 gene was significantly associated with five meat quality traits. The present results effectively narrowed down the associated regions compared to previous QTL studies and revealed haplotypes and candidate genes on SSC12 for meat quality traits in pigs.

Hematological traits, which are important indicators of immune function in animals, have been commonly examined as biomarkers of disease and disease severity in humans and animals. Genome-wide significant quantitative trait loci (QTLs) provide important information for use in breeding programs of animals such as pigs. QTLs for hematological parameters (hematological traits) have been detected in pig chromosomes, although these are often mapped by linkage analysis to large intervals making identification of the underlying mutation problematic. Single nucleotide polymorphisms (SNPs) are the common form of genetic variation among individuals and are thought to account for the majority of inherited traits. In this study, a genome-wide association study (GWAS) was performed to detect regions of association with hematological traits in a three-generation resource population produced by intercrossing Large White boars and Minzhu sows during the period from 2007 to 2011. Illumina PorcineSNP60 BeadChip technology was used to genotype each animal and seven hematological parameters were measured (hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC) and red blood cell volume distribution width (RDW)). Data were analyzed in a three step Genome-wide Rapid Association using the Mixed Model and Regression-Genomic Control (GRAMMAR-GC) method. A total of 62 genome-wide significant and three chromosome-wide significant SNPs associated with hematological parameters were detected in this GWAS. Seven and five SNPs were associated with HCT and HGB, respectively. These SNPs were all located within the region of 34.6-36.5 Mb on SSC7. Four SNPs within the region of 43.7-47.0 Mb and fifty-five SNPs within the region of 42.2-73.8 Mb on SSC8 showed significant association with MCH and MCV, respectively. At chromosome-wide significant level, one SNP at 29.2 Mb on SSC1 and two SNPs within the region of 26.0-26.2 Mb were found to be significantly associated with RBC and RDW, respectively. Many of the SNPs were located within previously reported QTL regions and appeared to narrow down the regions compared with previously described QTL intervals. In current research, a total of seven significant SNPs were found within six candidate genes SCUBE3, KDR, TDO, IGFBP7, ADAMTS3 and AFP. In addition, the KIT gene, which has been previously reported to relate to hematological parameters, was located within the region significantly associated with MCH and MCV and could be a candidate gene. These results of this study may lead to a better understanding of the molecular mechanisms of hematological parameters in pigs.

Malignant peripheral nerve sheath tumors (MPNST) represent a group of highly heterogeneous human malignancies often with multiple histological origins, divergent differentiation patterns, and diverse immunohistochemical presentations. The differential diagnosis of MPNST from other spindle cell neoplasms poses great challenges for pathologists. This report provides a mini-review of these unique features associated with MPNST and also presents the first cases of MPNST with six differentiation patterns.

Treatment trends of retinoblastoma (RB) have gradually evolved from eye enucleation and external radiation to local treatment. Combined treatment with an oncolytic virus and chemotherapy is currently a new method in RB treatment. To investigate the therapeutic effect of oncolytic adenovirus SG600 in combination with vincristine (VCR) on retinoblastoma in vitro, the cell viability, cell cycle effects and apoptotic activity of HXO-RB44 cells treated with SG600, VCR or SG600 plus VCR were measured using a cell counting kit-8-based procedure and flow cytometry. Western blot analysis for Akt, p-Akt, p-p53 and p-Rb protein was performed to investigate the underlying mechanisms of combined therapy. The combination therapy exerted a synergistic antitumor effect via a type of G2/M and S phase arrest rather than the induction of apoptosis. The combination of VCR and SG600 further reduced Akt phosphorylation compared with cells treated with VCR alone, suggesting that SG600 could overcome chemoresistance, perhaps by down-regulating Akt in RB cells. An increase in the expression of p-p53 and decrease in p-Rb expression in HXO-RB44 after co-treatment might be associated with cell cycle block. Western blot examination revealed that VCR might enhance SG600 replication. These results suggest that viro-chemo combination therapy is a feasible and potentially promising approach for the treatment of retinoblastoma.

Fluctuations in serum autofluorescence (AF) intensity have recently been widely used as markers of certain diseases such as cancer. To determine the diagnostic value of serum AF intensity for liver fibrosis in rats, we induced liver fibrosis by subcutaneous injection of carbon tetrachloride into rats. The rat serum AF intensities were detected at the excitation wavelength of 337 nm and the emission wavelength of 512 nm. The degree of liver fibrosis was evaluated by Van Gieson’s staining. The relationship between serum AF intensity and the degree of liver fibrosis was analyzed by Spearman and Pearson Correlation. The diagnostic sensitivity and specificity of the serum AF was determined by analyzing the receiver operating characteristic (ROC) curves. Our results show that the serum AF intensity in the rat liver fibrosis model increased when compared with control rats eight weeks and twelve weeks post induction of liver fibrosis. However, there was no significant difference in serum AF intensity between fibrotic and control rats at four week post induction. Furthermore, serum AF intensity correlated positively with the severity of the degree of hepatic fibrosis. ROC analysis further suggested that serum AF intensity is a valid marker for staging fibrosis. Therefore, it may potentially be developed as a novel diagnostic tool for hepatic fibrosis.

To analyze the association between non-alcoholic fatty liver disease (NAFLD) and the incidence of diabetic nephropathy in patients with type 2 diabetes, the incidence of diabetic nephropathy was assessed in 413 type 2 diabetic patients, by testing the 24 h urinary albumin excretion rate (UAER). The NAFLD was diagnosed based on patient’s medical history and liver ultrasound. The difference in diabetic nephropathy incidence between patients with and without NAFLD was tested by χ2. Multivariate logistic regression analysis was used to assess the factors associated with diabetic nephropathy among type 2 diabetic patients. Total 363 out of 413 type 2 diabetic patients were enrolled in this study. The incidences of NAFLD and diabetic nephropathy in participants were approximately 56% (202/363) and 38% (137/363) respectively, and there was no significant difference in the prevalence of diabetic nephropathy between patients with and without NAFLD (37.1% vs. 38.5%, p = 0.787). The duration of diabetes (odds ratio [OR] 1.065, 95% confidence interval [CI] 1.014–1.120, p = 0.012), waist circumference (OR 1.077, 95% CI 1.040–1.116, p = 0.000), and fasting blood glucose (FBG; OR 1.136, 95% CI 1.023–1.1262, p = 0.017) were significantly associated with diabetic nephropathy, whereas sex, high blood pressure, total cholesterol (TC), triglyceride (TG), and ankle brachial pressure index (ABI) were not significantly associated with the disorder. The present results suggest that NAFLD is not related to the incidence of diabetic nephropathy in type 2 diabetes, but the duration of diabetes, waist circumference, and FBG are important factors for diabetic nephropathy in type 2 diabetes.

AIM: To investigate the histological origin of pseudomyxoma peritonei (PMP) in Chinese women.

METHODS: The clinical and pathological data were reviewed for 35 women with PMP, and specimens of the peritoneal, appendiceal and ovarian lesions of each patient were examined using the PV-6000 immunohistochemistry method. Antibodies included cytokeratin (CK)7, CK20, mucin (MUC)-1, MUC-2, carbohydrate antigen (CA)-125, estrogen receptor (ER), and progesterone receptor (PR).

RESULTS: Abundant colloidal mucinous tumors were observed in the peritoneum in all 35 cases. Thirty-one patients had a history of appendectomy, 28 of whom had mucinous lesions. There was one patient with appendicitis, one whose appendix showed no apparent pathological changes, and one with unknown surgical pathology. Ovarian mucinous tumors were found in 24 patients. The tumors were bilateral in 13 patients, on the right-side in nine, and on the left side in two. Twenty patients had combined appendiceal and ovarian lesions; 16 of whom had undergone initial surgery for appendiceal lesions. Four patients had undergone initial surgery for ovarian lesions, and relapse occurred in these patients at 1, 11, 32 and 85 mo after initial surgery. Appendiceal mucinous tumors were found in each of these four patients. Thirty-three of the 35 patients showed peritoneal lesions that were positive for CK20 and MUC-2, but negative for CK7, MUC-1, CA125, ER and PR. The expression patterns in the appendix and the ovary were similar to those of the peritoneal lesions. In one of the remaining two cases, CK20, CK7 and MUC-2 were positive, and MUC-1, CA125, ER and PR were negative. The ovaries were not resected. The appendix of one patient was removed at another hospital, and no specimen was evaluated. In the other case, the appendix appeared to be normal during surgery, and was not resected. Peritoneal and ovarian lesions were negative for CK20, MUC-2, CK7, MUC-1, CA125, ER and PR.

CONCLUSION: Most PMP originated from the appendix. Among women with PMP, the ovarian tumors were implanted rather than primary. For patients with PMP, appendectomy should be performed routinely. The ovaries, especially the right ovaries should be explored.

Wilms tumor (WT) occurs infrequently in adults. Even rarer is adult WT with extension by direct intravascular spread into the right side of the heart. The present report describes a partially differentiated WT with intracaval and intracardiac extension in a 54-year-old man. The morphologic and immunohistochemical findings confirmed the diagnosis.

Solitary fibrous tumor (SFT) is a rare spindle-cell neoplasm, especially in the thyroid. We report a case of primary solitary fibrous tumor of the thyroid gland in a 37 year-old Chinese man. The tumor was characterized by bland-looking spindle cells admixed with thin and thick collagen fibers. On immunohistochemistry study indicated that tumor cells were diffusely positive for CD34, Bcl-2 and CD99, and negative for Desmin, NSE, SMA, S-100, and CD68. The patient remains well 16 months after excision. The morphologic and immunohistochemical features of the thyroid SFTs are similar to their reported counterparts in other anatomic sites.

AIM: To investigate the relationship between human papillomavirus (HPV) infection and concurrent esophagus and gastric cardia cancer from the same patient (CC) and examine the significance of P16INK4A protein expression.

METHODS: Polymerase chain reaction was used to detect the presence of HPV type16 (HPV16). The expression of P16INK4A protein was detected using immunohistochemistry.

RESULTS: Among the CC specimens, HPV16-DNA was found in eight cases of esophageal squamous cell carcinoma (ESCC) and five cases of gastric cardia adenocarcinoma (GCA), respectively (47% vs 29%), and two of both ESCC and GCA. P16INK4A was highly expressed in both ESCC and GCA. In the HPV-associated positive CC, higher P16INK4A expression was observed in the GCA than in the ESCC (75% vs 25%, P < 0.05).

CONCLUSION: HPV16 as a correlated risk factor may play an important role in the development of ESCC and GCA. P16INK4A may be a screening index in the HPV-associated carcinoma of gastric cardia.

Collecting duct carcinoma (CDC) or Bellini duct carcinoma of the kidney is a rare, but highly aggressive renal epithelial malignancy, with an extremely poor prognosis. Modified cytokine-induced killer (mCIK) cells were injected into the pleural cavity to treat pleural metastasis of CDC. The patient, a 33-year-old male, was admitted to hospital for further treatment for severe pleural metastasis of CDC. We cured the pleural metastasis through intrapleural infusion with mCIK cells. After receiving this innovative treatment, the patient exhibited a positive response: the cough, dyspnea, chest distress and thoracalgia were evidently relieved, while the pleural fluid became clear after exhibiting haematodes and its level decreased significantly. The patient achieved partial success. This novel immunotherapy method is a promising treatment for patients with refractory pleural metastasis.

SUMMARY

The analysis of longitudinal data to study changes in variables measured repeatedly over time has received considerable attention in many fields. This paper proposes a two-level structural equation model for analyzing multivariate longitudinal responses that are mixed continuous and ordered categorical variables. The first-level model is defined for measures taken at each time point nested within individuals for investigating their characteristics that are changed with time. The second level is defined for individuals to assess their characteristics that are invariant with time. The proposed model accommodates fixed covariates, nonlinear terms of the latent variables, and missing data. A maximum likelihood (ML) approach is developed for the estimation of parameters and model comparison. Results of a simulation study indicate that the performance of the ML estimation is satisfactory. The proposed methodology is applied to a longitudinal study concerning cocaine use.

In longitudinal studies, investigators often measure multiple variables at multiple time points and are interested in investigating individual differences in patterns of change on those variables. Furthermore, in behavioral, social, psychological, and medical research, investigators often deal with latent variables that cannot be observed directly and should be measured by 2 or more manifest variables. Longitudinal latent variables occur when the corresponding manifest variables are measured at multiple time points. Our primary interests are in studying the dynamic change of longitudinal latent variables and exploring the possible interactive effect among the latent variables.

Much of the existing research in longitudinal studies focuses on studying change in a single observed variable at different time points. In this article, we propose a novel latent curve model (LCM) for studying the dynamic change of multivariate manifest and latent variables and their linear and interaction relationships. The proposed LCM has the following useful features: First, it can handle multivariate variables for exploring the dynamic change of their relationships, whereas conventional LCMs usually consider change in a univariate variable. Second, it accommodates both first- and second-order latent variables and their interactions to explore how changes in latent attributes interact to produce a joint effect on the growth of an outcome variable. Third, it accommodates both continuous and ordered categorical data, and missing data.

The occurrence of generic Escherichia coli, E. coli O157, Salmonella, and Campylobacter in cattle manure, beef carcasses, catch basin water, and soils receiving manure application was determined in 21 Alberta feedlots. In cattle manure, generic E. coli (98%, 2069/2100) and Campylobacter (76%, 1590/2100) were frequently detected; E. coli O157 (7%, 143/2100) and Salmonella (1%, 20/2100) were less frequently detected. Samples from beef carcasses in the cooler following Hazard Analysis Critical Control Point interventions yielded only 1 isolate each of generic E. coli and Campylobacter (1/1653) and no Salmonella (0/1653). Catch basin water specimens were positive for generic E. coli in both the spring (62%, 13/21) and the fall (52%, 11/21). Other bacteria were detected only in the spring water specimens, including E. coli O157 (29%, 6/21), Salmonella (5%, 1/21), and Campylobacter (52%, 11/21). Generic E. coli was frequently isolated from soil specimens (30%, 27/88), but E. coli O157 was not found in soil samples obtained in the spring and was only occasionally detected in the fall samples (9%, 3/32). Salmonella were occasionally found in the soil specimens collected in the spring (3%, 2/56), but not in the fall season (0/32). Campylobacter jejuni was frequent in cattle manure (66%, 1070/1623), but rare in carcass and environmental samples. E. coli O157 and Salmonella were rarely detected in cattle or the environment. Generic E. coli and Salmonella were rarely detected on carcasses.

Background

Abberant aryl hydrocarbon receptor (AhR) expression and AhR pathway activation are involved in gastric carcinogenesis. However, the relationship between AhR pathway activation and gastric cancer progression is still unclear. In present study, we used 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD), a classic and most potent ligand of AhR, to activate AhR pathway and investigated the effect of AhR pathway activation on human gastric cancer AGS cell invasion and explored the corresponding mechanism.

Results

To determine whether AhR pathway can be activated in AGS cells, we examined the expression of CYP1A1, a classic target gene of AhR pathway, following TCDD exposure. RT-PCR and western blot analysis showed that both CYP1A1 mRNA and protein expression were increased in a dose-dependent manner following TCDD treatment and AhR antagonist resveratrol (RSV) could reverse this TCDD-induced CYP1A1 expression. To determine whether TCDD treatment of AGS cells results in an induction of MMP-9 expression, we detected MMP-9 mRNA using RT-PCR and detected MMP-9 enzymatic activity using gelatin zymography. The results showed that both MMP-9 mRNA expression and enzymatic activity were gradually increased with the concentration increase of TCDD in media and these changes could be reversed by RSV treatment in a dose-dependent manner. To examine whether AhR activation-induced MMP-9 expression and activity in AGS cells results in increased migration and invasion, we performed wound healing migration assay and transwell migration and invasion assay. After TCDD treatment, the migration distance and the migration and invasion abilities of AGS cells were increased with a dose-dependent manner. To demonstrate AhR activation-induced MMP-9 expression is mediated by c-Jun, siRNA transfection was performed to silence c-Jun mRNA in AGS cells. The results showed that MMP-9 mRNA expression and activity in untreated control AGS cells were very weak; After TCDD (10 nmol/L) treatment, MMP-9 mRNA expression and activity were significant increased; This TCDD-induced MMP-9 expression and activity increase could be abolished by c-Jun siRNA transfection.

Conclusion

AhR pathway activation enhances gastric cancer cell invasiveness likely through a c-Jun-dependent induction of MMP-9. Our results provide insight into the mechanism and function of the AhR pathway and its impact on gastric cancer progression.

In the title compound, C16H13N5O2S, the five non-H atoms of the urea linkage adopt a planar configuration owing to the presence of an intra­molecular N—H⋯O hydrogen bond. The maximum deviation from planarity is 0.022 (2) Å. The thia­diazole and pyridine heterocyclic rings are close to being coplanar, with a dihedral angle of 6.7 (2)° between their mean planes. Inter­molecular N—H⋯O hydrogen bonds link two neighbouring mol­ecules into centrosymmetric R 2 2(8) dimers. Four C atoms and the attached H atoms of the benzene ring are disordered over two positions of equal occupancy.

Background

Streptococcus pneumoniae infection starts from colonization of the host respiratory tract where interaction with host respiratory tract epithelial cells occurs. To investigate pneumococcal genes that are involved in the early stage of interaction with host epithelial cells, transcriptional responses of an encapsulated pathogenic pneumococcal strain TIGR4 upon exposure to human lung epithelial cells A549 for 0.5 h and 1 h time periods were investigated by using TIGR (JCVI) microarray technology. Gene expression changes were validated by quantitative real-time PCR (qRT-PCR) analysis.

Findings

We observed different transcriptional profiles at two incubation time periods in which most gene expressions were down-regulated at 0.5 h but up-regulated at 1 h. Many genes associated with ribonucleotide biosynthesis were down-regulated at both time points, whereas the genes associated with cell envelope, energy metabolism, transport and protein synthesis were mostly up-regulated at 1 h. Furthermore, these profiles were compared to the transcriptomes of a TIGR4-derived strain in response to human macrophages for the same time periods. We found one set of genes that exhibited similar expression changes upon exposure to both types of host cells, including cell envelope-associated bgaA (SP0648) and nanA (SP1693), and uncharacterized gene clusters such as SP1677–SP1680 and SP1688–SP1690.

Conclusion

These data indicate that at the early stage of interaction with host epithelial cells, a complex gene regulation and expression change occur in bacteria. Some of them might play an essential role during pathogen-host interactions and for the establishment of infection.

Although thiazolidinediones (TZDs) were found to be ligands for peroxisome proliferators-activated receptorγ (PPARγ), the mechanism by which TZDs exert their anticancer effect remains unclear. Furthermore, the effect of TZDs on metastatic and angiogenesis potential of cancer cells is unknown. Our results in this paper show that rosiglitazone inhibited SGC-7901 gastric cancer cells growth, caused G1 cell cycle arrest and induced apoptosis in a dose-dependent manner. The effects of rosiglitazone on SGC-7901 cancer cells were completely reversed by treatment with PPARγ antagonist GW9662. Rosiglitazone inhibited SGC-7901 cell migration, invasiveness, and the expression of MMP-2 in dose-dependent manner via PPARγ-independent manner. Rosiglitazone reduced the VEGF induced angiogenesis of HUVEC in dose-dependent manner through PPARγ-dependent pathway. Moreover, rosiglitazone did not affect the expression of VEGF by SGC-7901 cells. Our results demonstrated that by PPARγ ligand, rosiglitazone inhibited growth and invasiveness of SGC-7901 gastric cancer cells and angiogenesis in vitro via PPARγ-dependent or -independent pathway.

Abstract

New bioactive proteins need to be screened from various microorganisms for the increasing need for industrial and pharmaceutical peptide, proteins, or enzymes. A novel polymerase chain reaction (PCR) method, restriction site-dependent PCR (RSD-PCR), was designed for rapid new genes cloning from genomic DNA. RSD-PCR strategy is based on these principles: (i) restriction sites disperse throughout genomes are candidacy for universal pairing; (ii) a universal primer is a combination of a 3′-end of selected restriction sites, and a 5′-end of degenerated sequence. A two-round PCR protocol was designed and optimized for the RSD-PCR: amplify the single strand target template from genomic DNA by a specific primer and amplify the target gene by using the specific primer and one of the universal RSD-primers. The optimized RSD-PCR was successfully applied in chromosome walking using specific internal primers, and cloning of new genes using degenerated primers derived from NH2-terminal amino acid sequence of protein.

The glpTQ operon of Haemophilus influenzae type b (Hib) and nontypeable H. influenzae (NTHi) strains is highly conserved, except for a 1.4-kb glpTQ intergenic region that was found in most Hib strains. The presence of this intergenic region results in divergent glpTQ transcriptional profiles for Hib and NTHi where Hib strains appear to have evolved an alternative promoter for glpQ expression. Based on the intergenic region's low G+C content, we speculate that this DNA fragment was acquired by lateral transfer.

The mig gene of Streptococcus dysgalactiae, a major bovine mastitis pathogen, encodes two plasma protein-binding receptors, α2-macroglobulin (α2-M) and immunoglobulin G (IgG). In this study, the mig gene from one S. dysgalactiae isolate was cloned and expressed in Escherichia coli. The IgG receptor region encoded by mig was conserved in 16 S. dysgalactiae strains. An isogenic mig mutant was constructed by allele replacement mutagenesis of the wild-type gene in S. dysgalactiae. The IgG-binding activity was lost in the mig mutant strain, whereas the α2-M receptor activity was still expressed but was detected only in the culture supernatant. In flow cytometry phagocytosis and bacterial-colony-counting bactericidal assays, the wild-type strain was found to be significantly more resistant to phagocytosis and killing by bovine neutrophils (PMNs) than the mig mutant strain when bacteria were preincubated with bovine serum. We therefore speculate that the Mig protein of S. dysgalactiae plays a role in virulence of the bacteria by binding to the plasma protein α2-M or IgG and thus preventing phagocytosis by bovine PMNs.

During apoptotic cell death, cell surface ligands initiate phagocytosis of the dying cell. Clearance of these apoptotic cells is thought to occur without an immune response. Since a number of autoantigens are located at the cell surface or within apoptotic blebs, we examined whether exposure of mice to syngeneic apoptotic cells by the intravenous route could induce autoantibody production. Normal mice injected with syngeneic apoptotic thymocytes developed antinuclear autoantibodies and anticardiolipin and anti-ssDNA antibodies. The autoantibody levels were generally lower than those observed in MRL/Faslpr mice and were transient. Surprisingly, six out of six immunized mice demonstrated immunoglobulin G deposition in the glomeruli several months after immunization. These findings indicate that systemic exposure to apoptotic cells can induce an immune response in normal mice, and may help to explain antigen selection and initiation of the immune response in diseases characterized by increased rates of apoptosis such as AIDS and, possibly, systemic lupus erythematosus.