Detection of human papillomavirus in the oral cavities of persons with Fanconi anemia.

OBJECTIVE: We conducted a cross-sectional study to describe the prevalence and correlates of type-specific human papillomavirus (HPV) DNA in the oral cavities of persons with Fanconi anemia. MATERIALS AND METHODS: Oral swabs were collected from 67 participants with Fanconi anemia and tested for 27 HPV genotypes using polymerase chain reaction-based methods. RESULTS: Participants were a mean of 18.6 (standard deviation, 10.0) years of age (range 4-47 years). The prevalence of oral HPV infection was 7.5%, and the prevalence of high-risk HPV infection was 6.0%. HPV type 16 was not detected in any samples. Prevalence was higher in adults than in children (13.3% vs 2.7% in those ≥18 vs <18 years of age). Among adults, prevalence was higher in males than in females (25.0% vs 9.1%, respectively). CONCLUSIONS: Prevalence of oral HPV infection in persons with Fanconi anemia was comparable to estimates from other studies in the general population. However, in contrast to previous studies, we did not identify HPV type 16 (the type found in most HPV-related head and neck cancers) in any participants.

Evaluating the effect of spastin splice mutations by quantitative allele-specific expression assay.

BACKGROUND: mutations in the SPG4/SPAST gene are the most common cause for hereditary spastic paraplegia (HSP). The splice-site mutations make a significant contribution to HSP and account for 17.4% of all types of mutations and 30.8% of point mutations in the SPAST gene. However, only few studies with limited molecular approach were conducted to investigate and decipher the role of SPAST splice-site mutations in HSP. METHODS: a reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitative allele-specific expression assay were performed. RESULTS: we have characterized the consequence of two novel splice-site mutations (c.1493 + 1G>A and c.1414-1G>A) in the SPAST gene in two different families with pure HSP. The RT-PCR analysis revealed that both spastin mutations are indeed splice-site mutations and cause skipping of exon 12. Furthermore, RT-PCR data suggested that these splice-site mutations may cause leaky splicing. By means of a quantitative allele-specific expression assay, we could confirm that both splice-site mutations cause leaky splicing, as the relative expression of the exon 12-skipped transcript was reduced (21.1 ± 3.6 compared to expected 50%). CONCLUSIONS: our finding supports a "threshold-effect-model" for functional spastin in HSP. A higher level (78.8 ± 3.9%) of functional spastin than the expected ratio of 50% owing to leaky splicing might cause late age at onset of HSP. Remarkably, we could show that a quantitative allele-specific expression assay is a simple and effective tool to evaluate the role of most types of spastin splice-site mutations in HSP.

Compound heterozygosity in the SPG4 gene causes hereditary spastic paraplegia.

The SPG4 gene is frequently mutated in autosomal dominant form of hereditary spastic paraplegia (HSP). We report that the compound heterozygous sequence variants S44L, a known polymorphism, and c.1687G>A, a novel mutation in SPG4 cause a severe form of HSP in a patient. The family members carrying solely c.1687G>A mutation are asymptomatic for HSP. The reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the c.1687G>A mutation is a splice site mutation and causes skipping of the exon 15 of spastin. Furthermore, quantification of RT-PCR products by sequencing and quantification of allele-specific expression by pyrosequencing assay revealed that c.1687G>A is a leaky or hypomorphic splice site mutation. At the protein level, c.1687G>A mutation in SPG4 leads to E563K substitution. In ex vivo study, about 10% of cells expressing E563K mutant spastin showed filamentous expression pattern, suggesting a hypomorphic effect at the protein level. Collectively, our results suggest that S44L in association with c.1687G>A (E563K) drops the functional level of spastin below a threshold limit sufficient to manifest HSP.

Clinical signs, histology, and CD3-positive cells before and after treatment of dogs with chronic enteropathies.

BACKGROUND: Histopathology is widely used for the diagnosis of inflammatory bowel disease in dogs. Variations in lesions and unavailability of uniform grading systems limit the usefulness of histologic examination. HYPOTHESIS: CD3 cell numbers in chronic enteropathies of dogs correlate with clinical activity of the disease and with severity of histopathologic changes. ANIMALS: Nineteen client-owned dogs examined because of chronic diarrhea, vomiting, or both. METHODS: Samples of duodenal and colonic mucosa were collected endoscopically before and after treatment. Dogs that responded to a hypoallergenic diet were grouped as food-responsive diarrhea dogs (FRD, n=10). Dogs with no clinical improvement after 10 days of treatment then received prednisolone (immunosuppressive doses) and were grouped as steroid-responsive diarrhea dogs (SRD, n=9). Histopathologic assessment with a standardized grading system was performed retrospectively on the intestinal samples. Histologic score, total number of infiltrating cells, and CD3-positive cells were counted and compared with the clinical scoring. RESULTS: No statistically significant difference was detected among histologic grading, total number of cells in the lamina propria, and T-cell numbers in biopsies before and after treatment in either group (FRD and SRD). CONCLUSIONS AND CLINICAL IMPORTANCE: Currently used histopathologic grading scores, total numbers of cells, and numbers of CD3-positive cells did not allow differentiation between FRD and SRD and did not correlate with clinical response to therapy. Based on these results, new grading scores assessing other criteria than total cell numbers and CD3-positive cells should be evaluated in the future.

Upregulation of toll-like receptors in chronic enteropathies in dogs.

BACKGROUND: Inflammatory bowel disease (IBD) is thought to result from a dysregulated interaction between the host immune system and commensal microflora. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs), but their role in enteropathies in dogs is unknown. HYPOTHESIS: That there is a dysregulation of TLRs recognizing bacterial MAMPs in dogs with IBD. ANIMALS: Sixteen healthy beagles and 12 dogs with steroid-treated (ST) and 23 dogs with food-responsive (FR) diarrhea. METHODS: Prospective, observational study. mRNA expression of canine TLR2, 4, and 9 was evaluated by quantitative real-time RT-PCR in duodenal and colonic biopsies obtained before and after standard therapy. Samples from control dogs were taken at necropsy, with additional biopsies of stomach, jejunum, ileum, and mesenteric lymph node in 6 dogs. RESULTS: There were significant differences (P< or = .017) in expression of TLR2, 4, and 9 between the 6 sampled locations in healthy control dogs (lymph node > small intestine > or = colon). Before therapy, ST expressed more mRNA than control dogs for all 3 receptors (P < .05). There were no significant differences between pretreatment and posttreatment values, even though 32/35 dogs improved clinically. No associations were found when comparing receptor mRNA expression with either histology or clinical activity scores. CONCLUSIONS AND CLINICAL IMPORTANCE: Bacteria-responsive TLR2, 4, and 9 are upregulated in duodenal and colonic mucosa in IBD. This might lead to increased inflammation through interaction with the commensal flora. The absence of significant changes after therapy despite clinical improvement might point toward the existence of a genetic predisposition to IBD as described in human IBD.

Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect.

Several problems limit the application of gene transfer to correct the cystic fibrosis (CF) Cl(-) transport defect in airway epithelia. These include inefficient transduction with vectors applied to the apical surface, a low rate of division by airway epithelial cells, failure of transgene expression to persist, and immune responses to vectors or vector-encoded proteins. To address these issues, we used a feline immunodeficiency virus-based (FIV-based) vector. FIV vector formulated with a calcium chelator transduced fully differentiated, nondividing human airway epithelia when applied to the apical surface. FIV-based vector encoding the cystic fibrosis transmembrane conductance regulator cDNA corrected the Cl(-) transport defect in differentiated CF airway epithelia for the life of the culture (>3 months). When this approach was applied in vivo, FIV vector expressing beta-galactosidase transduced 1-14% of adult rabbit airway epithelia. Transduced cells were present in the conducting airways, bronchioles, and alveoli. Importantly, gene expression persisted, and cells with progenitor capacity were targeted. FIV-based lentiviral vectors may be useful for the treatment of genetic lung diseases such as CF. This article may have been published online in advance of the print edition.

Nuclear receptor and target gene mRNA abundance in duodenum and colon of dogs with chronic enteropathies.

Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR) and peroxisome proliferator-associated receptors alpha and gamma (PPARalpha, PPARgamma) are mediators of inflammation and may be involved in inflammatory bowel disease (IBD) and food responsive diarrhea (FRD) of dogs. The present study compared mRNA abundance of NR and NR target genes [multi drug-resistance gene-1 (MDR1), multiple drug-resistance-associated proteins (MRD2, MRD3), cytochrome P450 (CYP3A12), phenol-sulfating phenol sulfotransferase (SULT1A1) and glutathione-S-transferase (GST A3-3)] in biopsies obtained from duodenum and colon of dogs with IBD and FRD and healthy control dogs (CON; n=7 per group). Upon first presentation of dogs, mRNA levels of PPARalpha, PPARgamma, CAR, PXR and RXRalpha in duodenum as well as PPARgamma, CAR, PXR and RXRalpha in colon were not different among groups (P>0.10). Although mRNA abundance of PPARalpha in colon of dogs with FRD was similar in both IBD and CON (P>0.10), PPARalpha mRNA abundance was higher in IBD than CON (P<0.05). Levels of mRNA of MDR1 in duodenum were higher in FRD than IBD (P<0.05) or CON (P<0.001). Compared with CON, abundances of mRNA for MRP2, CYP3A12 and SULT1A1 were higher in both FRD and IBD than CON (P<0.05). Differences in mRNA levels of PPARalpha and MRP2 in colon and MDR1, MRP2, CYP3A12 and SULT1A1 in duodenum may be indicative for enteropathy in FRD and (or) IBD dogs relative to healthy dogs. More importantly, increased expression of MDR1 in FRD relative to IBD in duodenum may be a useful diagnostic marker to distinguish dogs with FRD from dogs with IBD.

P-glycoprotein expression in lamina propria lymphocytes of duodenal biopsy samples in dogs with chronic idiopathic enteropathies.

P-glycoprotein (p-gp) is a transmembrane protein functioning as a drug-efflux pump in the intestinal epithelium. Human patients with inflammatory bowel disease (IBD) who fail to respond to treatment with steroids express high levels of p-gp in lamina propria lymphocytes. The purpose of this study was to investigate p-gp expression in duodenal biopsy samples of dogs with chronic enteropathies and to evaluate the expression of p-gp after treatment with a known inducer of p-gp (prednisolone). Duodenal biopsy samples from 48 dogs were evaluated immunohistochemically with the mouse monoclonal antibody C219 for expression of p-gp in lamina propria lymphocytes. Biopsy samples were available from 15 dogs after treatment with prednisolone and 16 dogs after dietary therapy alone ("elimination diet"). Treatment with prednisolone resulted in an increase in p-gp expression (P=0.005). In contrast, dietary treatment alone produced no significant change in p-gp expression (P=0.59). A low p-gp score before initiation of steroid treatment was significantly associated with a positive response to treatment (P=0.01). These results indicate that lamina propria lymphocyte expression of p-gp is upregulated after prednisolone treatment in dogs with IBD, and that mucosal expression of p-gp may be of value in predicting the response to therapy.

Pharmacokinetics and clinical efficacy of cyclosporine treatment of dogs with steroid-refractory inflammatory bowel disease.

The usual treatment of dogs with inflammatory bowel disease (IBD) consists of administration of immunosuppressive doses of steroids. However, some dogs are refractory to steroid treatment and pose a significant challenge to the veterinarian. Because cyclosporine A (cyA) has been shown to be effective in steroid-resistant IBD in humans, the purpose of this study was to investigate the pharmacokinetics and clinical efficacy of PO cyA treatment in dogs with steroid-refractory IBD (n = 14). All dogs were treated with cyA 5 mg/kg PO q24h for a period of 10 weeks. A clinical activity score was assigned to assess severity of clinical signs before and after treatment. The total number of infiltrating lymphocytes and T cells in duodenal biopsies were assessed before and after treatment in 9 dogs. In addition, serum concentration of cyA was measured in 8 dogs over a 24-hour period. Pharmacokinetic profiles in dogs with IBD were similar to those of healthy dogs. Improvement of clinical signs was observed in 12 of 14 dogs with IBD. Median clinical activity score after treatment with cyA was significantly reduced from a median score of 9 to a median score of 5 (P = 0.001). T cell numbers in duodenal biopsies were significantly decreased after treatment from a median +/- 95% range in the villous region of 28 (19-30) cells/10,000 microm2 before versus 7 (0-10)/10,000 microm2 after treatment, P = 0.01; and from a median +/- 95% range number in the crypt region of 15 (6-23) cells/10,000 microm2 before versus 4 (0-9)/10,000 microm2 after treatment, P = 0.02, implying T cell lysis as a possible mechanism of action. In conclusion, based on this small study, cyA appears to be an effective alternative drug in dogs with IBD that are refractory to immunosuppressive doses of steroids.

Expression of carcinoembryonic antigen and its predicted immunoglobulin-like domains in HeLa cells for epitope analysis.

Carcinoembryonic antigen (CEA) is a member of the immunoglobulin gene superfamily with one predicted variable domain-like region (N domain; 108 amino acids) and three sets of constant domain-like regions (A1B1, A2B2, and A3B3; 92 amino acids for A domains and 86 amino acids for B domains). In addition, CEA possesses two signal peptides, one at the amino terminus and one at the carboxyl terminus. Both are removed during posttranslational processing, with the one at the carboxyl terminus being replaced by a glycosylphosphatidylinositol (GPI) moiety. We have previously expressed the full length complementary DNA clone for CEA in Chinese hamster ovary cells and murine L cells, demonstrating proper processing of nascent polypeptide chains to mature, fully glycosylated CEA including the GPI anchor. Using the same full length CEA complementary DNA clone and the polymerase chain reaction, we have now constructed expression clones for secreted versions of the N domain, the A3B3 domain, and the A3 and B3 subdomains. The clones were expressed in HeLa cells using the beta-actin promoter. A stop codon was introduced at the end of the A3B3 and the A3 and B3 domains to allow secretion instead of retention on plasma membranes with the GPI anchor. Expressed products were purified to homogeneity by affinity chromatography using monoclonal antibodies specific for each domain and by reversed phase high pressure liquid chromatography. Purified domains were characterized by Western blotting, antibody binding and inhibition studies, amino-terminal sequence and amino acid analyses, and laser desorption/time of flight mass spectrometry. These analyses revealed that the monomeric N domain is of size 15,990, with a glycosylation mass of about 4100, in good agreement with two N-linked glycosyl units of about mass 2100. There is some evidence that the N domain forms dimers. The N domain reacted with antibodies specific for this domain with an affinity similar to that of intact CEA. The A3B3 domain had a mass of 34,462, with a glycosylation mass of 14,900, in good agreement with seven N-linked glycosylation sites of average mass 2100. The A3B3 domain reacted only with antibodies specific for this domain, with a slightly lower affinity than that of native CEA. The amino-terminal sequences of the N domain and A3B3 domain proteins demonstrated proper processing of the signal peptide.(ABSTRACT TRUNCATED AT 400 WORDS)

Non-Hodgkin's lymphomas of childhood and adolescence: results of a treatment stratified for biologic subtypes and stage--a report of the Berlin-Frankfurt-Münster Group.

PURPOSE: To prove the efficacy of a treatment stratified according to histology for children with non-Hodgkin's lymphoma (NHL), including acute B-cell leukemia (B-ALL). PATIENTS AND METHODS: From October 1986 to March 1990, 302 assessable patients, 0.6 to 17.8 years of age, with newly diagnosed NHL were enrolled onto study ALL/NHL-BFM 86. Fifty percent of patients had Burkitt-type lymphomas, including B-ALL; 24% had lymphoblastic lymphoma; 18% had diffuse large-cell lymphoma; and 8% had an NHL not further classified. Therapy group B included Burkitt's-type lymphomas, B-ALL, and most large-cell lymphomas including Ki-1 anaplastic large-cell lymphoma. Patients with stage I and II disease resected received three, while all others received six, 5-day therapy courses (dexamethasone, methotrexate [MTX] 0.5 g/m2 [5 g/m2 for stage IV and B-ALL], and intrathecal [IT] therapy in each course, plus ifosfamide, cytarabine, and etoposide alternating with cyclophosphamide and doxorubicin). Therapy for group non-B patients (lymphoblastic lymphoma and pleomorphic T-cell lymphoma [PTCL]) consisted of a Berlin-Frankfurt-Münster (BFM) acute lymphoblastic leukemia protocol, including cranial irradiation for advanced stage. Local therapy was restricted to patients with incomplete tumor regression. RESULTS: The probabilities of event-free survival (pEFS) at 7 years were 80% +/- 2% for the whole group, 81% +/- 3% for group B (n = 225), and 78% +/- 5% for group non-B (n = 77) with a follow-up duration of 3.6 to 7 years (median 5 years). Treatment results were comparable between NHL subtypes, except for PTCL, in which three of four patients suffered from relapse. Local disease manifestations were the most frequent site of failure. CONCLUSION: This therapy strategy provided patients of all NHL subtypes with an equally high chance to survive event-free, except patients with PTCL. With reduced systemic failure, local tumor control may become more important.

Cytokine expression in an ex vivo culture system of duodenal samples from dogs with chronic enteropathies: modulation by probiotic bacteria.

There is evidence that probiotics have immune-modulating effects on intestinal inflammation during chronic enteropathies (CE). In an ex vivo culture system we investigated the influence of probiotics on mRNA and protein expression levels of cytokines in intestinal samples from dogs suffering from CE. Duodenal samples of client-owned dogs with CE (group CE; n = 12) were collected during diagnostic endoscopy. Additional duodenal samples of gastrointestinally healthy dogs (group C; n = 4) from an unrelated study were available. Based on histopathological analyses, no pathological changes or only mild to moderate eosinophilic and/or lymphoplasmacytic duodenitis were diagnosed. Tissue samples were cultured: (1) with cell culture medium alone (negative control), (2) with a probiotic cocktail (PC), constituted of three Lactobacilli spp. from healthy canine fecal isolates, (3) with the individual strains of PC, and (4) with a placebo powder. Viability of intestinal tissue and probiotic bacteria before and after culture was evaluated. The mRNA abundance of interleukin (IL)-10, IL-12p40, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta1 was analyzed by real-time polymerase chain reaction (RT-PCR). Protein concentrations of IFN-gamma and IL-10 were measured in culture supernatant by ELISA. Results of RT-PCR were expressed as 2(-2DeltaCrossing Point) x 100 after normalization with beta-actin. There was a loss of about 1 log CFU/mL of probiotic bacteria during the incubation period. Viability of tissue was maintained as confirmed by non-significant release of lactate dehydrogenase. In C, addition of PC increased IL-10 mRNA levels (P < 0.1). In CE, PC increased mRNA and protein levels of IL-10 (P < 0.05). On the mRNA level, the ratio of TNFalpha-/IL-10, IFN-gamma/IL-10, and IL-12p40/IL-10 decreased after addition of PC (P < 0.05). The results demonstrate favorable effects of PC on regulatory cytokines relative to inflammatory cytokines that might contribute to reduction of intestinal inflammation.

Novel mutations in the Atlastin gene (SPG3A) in families with autosomal dominant hereditary spastic paraplegia and evidence for late onset forms of HSP linked to the SPG3A locus.

Hereditary spastic paraplegias (HSP) comprise a genetically and clinically heterogeneous group of neurodegenerative disorders characterised by progressive spasticity and hyperreflexia of the lower limbs. Autosomal dominant hereditary spastic paraplegia linked to the SPG3A locus on chromosome 14q11-21 accounts for approximately 10% of autosomal dominant hereditary spastic paraplegia (ADHSP). It is caused by mutations in the SPG3A gene encoding the protein atlastin. To date, only five disease-causing mutations in the SPG3A gene have been described. We analysed 13 SPG4-negative families for mutations in the SPG3A gene and identified a mutation in 38% (5/13). Two of the mutations are novel, c.481G>C (p.A161P) and c.740A>C (p.H247P). One of the novel mutations was found both in a family with early onset of symptoms and in a late onset family. Furthermore, we report on numerous polymorphisms detected in the SPG3A gene.

Mutation analysis of the spastin gene (SPG4) in patients in Germany with autosomal dominant hereditary spastic paraplegia.

Hereditary spastic paraplegias (HSP) comprise a genetically and clinically heterogeneous group of neurodegenerative disorders characterized by progressive spasticity and hyperreflexia of the lower limbs. Autosomal dominant hereditary spastic paraplegia 4 linked to chromosome 2p (SPG4) is the most common form of autosomal dominant hereditary spastic paraplegia. It is caused by mutations in the SPG4 gene encoding spastin, a member of the AAA protein family of ATPases. In this study the spastin gene of HSP patients from 161 apparently unrelated families in Germany was analyzed. The authors identified mutations in 27 out of the 161 HSP families; 23 of these mutations have not been described before and only one mutation was found in two families. Among the detected mutations are 14 frameshift, four nonsense, and four missense mutations, one large deletion spanning several exons, as well as four mutations that affect splicing. Most of the novel mutations are located in the conserved AAA cassette-encoding region of the spastin gene. The relative frequency of spastin gene mutations in an unselected group of German HSP patients is approximately 17%. Frameshift mutations account for the majority of SPG4 mutations in this population. The proportion of splice mutations is considerably lower than reported elsewhere.

Finger tremor after carbon disulfide-based pesticide exposures.

Index finger tremor accompanying voluntary movement was studied in 19 age-matched control subjects and in 19 grain industry employees chronically exposed to carbon disulfide-based fumigants. Visual judgments of tremor amplitude made by neurologists during clinical examinations equaled the sensitivity of computerized tremor amplitude measurements. Tremor frequency variations detectable only with computerized measurement were present in grain workers with and without increased tremor amplitudes. Frequency differences discriminated between normal subjects and 74% of the grain workers. The distribution of tremor frequency power in the grain workers was often sequestered at 5 to 7 Hz, reminiscent of tremor in idiopathic Parkinson's disease. These findings suggest that the measurement of subtle tremor frequency changes may provide an early indication of chronic carbon disulfide poisoning.

The use and development of retroviral vectors to deliver cytokine genes for cancer therapy.

In this review, we describe technical advancements of retroviral vectors to address issues of safety, titer, and clinical scale manufacturing to produce high-quality retroviral vector preparations that have made direct intratumoral administration of cytokine encoding recombinant vectors a feasible cancer therapy in the clinic. We also review possible further advances in retroviral vector design, which may prove important in expanding these clinical applications.

Transduction of murine cerebellar neurons with recombinant FIV and AAV5 vectors.

Our data demonstrate that vectors derived from recombinant feline immunodeficiency virus (rFIV) and adeno-associated virus type 5 (rAAV5) transduce cerebellar cells following direct injection into the cerebellar lobules of mice. Both recombinant viruses mediated gene transfer predominantly to neurons, with up to 2500 and 1500 Purkinje cells transduced for rAAV5 or rFIV-based vectors, respectively. The vectors also transduced stellate, basket and Golgi neurons, with occasional transduction of granule cells and deep cerebellar nuclei. rAAV5 also spread outside the cerebellum to the inferior colliculus and ventricular epithelium, while rFIV demonstrated the ability to undergo retrograde transport to the physically close lateral vestibular nuclei. Thus, AAV5 and FIV-based vectors show promise for targeting neurons affected in the hereditary spinocerebellar ataxias. These vectors could be important tools for unraveling the pathophysiology of these disorders, or in testing factors which may promote neuronal survival.

Prevention of work-related psychological disorders. A national strategy proposed by the National Institute for Occupational Safety and Health (NIOSH).

The National Institute for Occupational Safety and Health (NIOSH) recognizes psychological disorders as a leading occupational health problem. This document, developed by a NIOSH working group led by the present authors, represents a first attempt to fashion a comprehensive national strategy to protect and promote the psychological health of workers. Roles are identified for industry, labor, government, and academia. Key initiatives include (a) steps to improve working conditions and employee mental health services and (b) increased research and surveillance to advance understanding of the problem. Because work-related psychological disorders appear to be a rapidly developing problem lacking complete definition in terms of scope and etiology, this strategy is not to be considered a final statement of NIOSH policy.

Behavioral science activities at the Centers for Disease Control and Prevention. A selected overview of exemplary programs.

Behavioral research and surveillance activities are conducted across the Centers for Disease Control and Prevention (CDC). This article highlights activities in 4 program areas: violence against women, tuberculosis elimination, HIV prevention, and occupational health. The unique constraints and opportunities of each organization and program focus have shaped the way research has developed in each of these areas. Behavioral scientists also face many common challenges at CDC. Despite the difficulties of integrating behavioral research into an institution that historically has focused on biomedical and epidemiological research, behavioral scientists have made important contributions to public health. Many opportunities remain for psychologists to translate theory and operationalize constructs for use in solving important public health problems.

Two-step purification of the outer membrane transporter and activator protein ShlB from Escherichia coli using internally His6-tagged constructs.

ShlB from Serratia marcescens was isolated and purified from a porin-deficient Escherichia coli BL21 strain using a combination of detergent extraction, affinity and ion-exchange chromatography. An internal histidine affinity tag was introduced that did not interfere with activity. At each stage of the purification scheme biological activity of the ShlB protein was assessed. Using this scheme, several His(6)-tagged mutants of ShlB were purified to electrophoretic homogeneity.