Analysis of relative levels of production of pertussis toxin subunits and Ptl proteins in Bordetella pertussis

Pertussis toxin is transported across the outer membrane of Bordetella pertussis by the type IV secretion system known as the Ptl transporter, which is composed of nine different proteins. In order to determine the relative levels of production of pertussis toxin subunits and Ptl proteins in B. pertussis, we constructed translational fusions of the gene for alkaline phosphatase, phoA, with various ptx and ptl genes. Comparison of the alkaline phosphatase activity of strains containing ptx'- or ptl'-phoA fusions indicated that pertussis toxin subunits are produced at higher levels than Ptl proteins, which are encoded by genes located toward the 3' end of the ptx-ptl operon. We also engineered strains of B. pertussis by introducing multiple copies of the ptl genes or subsets of these genes and then examined the ability of each of these strains to secrete pertussis toxin. From these studies, we determined that certain Ptl proteins appear to be limiting in the secretion of pertussis toxin from the bacteria. These results represent an important first step in assessing the stoichiometric relationship of pertussis toxin and its transporter within the bacterial cell.     

Immunohistochemical comparison of p16 expression in actinic keratoses and squamous cell carcinomas of the skin.

There are approximately 200,000 new cases of cutaneous squamous cell carcinoma diagnosed each year in the United States, with between 1300 and 2300 deaths per year from metastatic disease. The tumor suppressor p16, encoded by the CDKN2/INK4a locus, has been reported mutated in >or=24% of squamous cell carcinomas. Mutations of the p16 gene have also been found in actinic keratoses, the first identifiable lesion in the continuum from normal skin to squamous cell carcinoma. We hypothesized that there may be an appreciable difference in expression of p16 between normal skin, actinic keratoses, squamous cell carcinoma in situ, and invasive squamous cell carcinoma. Ten actinic keratoses, 10 in situ squamous cell carcinomas, and 10 invasive squamous cell carcinomas were examined using the immunoperoxidase method with antigen retrieval for anti-p16(INK4a) antibody. All 10 actinic keratoses were positive for weak to moderate p16 staining in the lower third to lower half of the epidermis (especially the basal keratinocytes). This staining was significant when compared with the lack of staining seen in normal skin controls. Twenty percent of in situ squamous cell carcinomas had moderate to strong staining in only the lower half to lower two thirds of the epidermis, whereas 70% of the in situ squamous cell carcinomas exhibited full-thickness p16 staining, with no staining in the dermis. Thirty percent of invasive squamous cell carcinomas had full-thickness staining of the in situ component of the lesion, and 100% of invasive squamous cell carcinomas exhibited moderate to strong staining of the invasive component of the lesion. These findings indicate correlation between the increased expression of p16 during the progression of skin from actinic keratosis to in situ squamous cell carcinoma to invasive squamous cell carcinoma. These data may lend further support to the view of the actinic keratosis as a precursor lesion to squamous cell carcinoma.     

Membrane localization of the S1 subunit of pertussis toxin in Bordetella pertussis and implications for pertussis toxin secretion

Pertussis toxin is secreted from Bordetella pertussis with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters. The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known. To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins. In wild-type B. pertussis, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes. In mutants of B. pertussis that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes. Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of B. pertussis. First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100. Second, recombinant S1 previously has been shown to localize to the outer membrane of Escherichia coli (J. T. Barbieri, M. Pizza, G. Cortina, and R. Rappuoli, Infect. Immun. 58:999-1003, 1990). Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins. By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit. Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of B. pertussis. Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system.     

Tyrosine kinase activity associated with the CD7 antigen: correlation with regulation of T cell integrin function

Rapid up-regulation of the functional activity of integrin adhesion receptors is a hallmark of T cell activation. Monoclonal antibody engagement of the CD7 antigen on human T cells results in an increase in β1 and β2 integrin-mediated adhesion within minutes. This suggests that CD7 is capable of transducing intracellular signals, and is consistent with other indirect studies implicating CD7 as a signaling receptor on T cells. In this report, we have explored the intracellular mechanism by which CD7 modulates integrin functional activity. First, CD7-mediated up-regulation of T cell adhesion was found to be unique when compared to phorbol ester stimulation and CD3/T cell receptor cross-linking, based on differences in the kinetics of activation-dependent integrin-mediated adhesion and lack of increase in CD2 functional activity. Second, up-regulation of integrin activity mediated by CD7 cross-linking was completely inhibited by the tyrosine kinase inhibitor herbimycin A. Third, antiphosphotyrosine immunoblotting demonstrated that antibody engagement of CD7 results in a rapid but transient increase in tyrosine phosphorylation in human T cells. Finally, CD7 immunoprecipitates contain in vitro kinase activity, as demonstrated by phosphorylation of a predominant band of 80 kDa and multiple other bands. Phosphoamino acid analysis of the 80-kDa substrate revealed phosphorylation on tyrosine as well as serine and threonine residues. Together, our results suggest that CD7 is associated with tyrosine kinase activity and that this tyrosine kinase activity correlates with the ability of CD7 to regulate T cell integrin functional activity.     

Brief original scientific report: a new surgical approach for the treatment of left pulmonary and hepatic hydatid disease

Background

The combination of pulmonary and hepatic hydatid cysts is frequently encountered, and poses a challenge in terms of surgical accessibility. The surgical treatment of the two locations by the same incision (thoracotomy with phrenotomy) has been proposed, but always from the right side. However, applying this technique to the left side seems to be more difficult and unusual. We herein describe a new left-sided technique that was used to treat two patients with pulmonary and hepatic hydatid cysts.

Methods

The first patient was 14-year-old; he had bilateral pulmonary hydatid cysts and one type I cyst of the left lobe of the liver. The second patient was a 10-year-old female who had a hydatid cyst of the upper left lobe with one type III cyst of hepatic segments 2 and 3.

Results

Both patients were operated on via a left lateral thoracotomy through the sixth intercostal space. They underwent cystectomy for the left pulmonary hydatid cysts, followed by padding, and then the hepatic cyst was treated by Lagrot’s method via a radial phrenotomy. The postoperative course was uneventful in both cases, with postoperative hospital stays of 3 and 5 days, respectively.

Conclusion

This combined treatment of pulmonary and hepatic hydatid cysts by the left-sided thoracic approach is feasible and provides a good outcome. It should be indicated under the same conditions of accessibility and feasibility applied for the right thoracic side.     

The Evolving Role of Midwives as Laborists

This article examines the history and present state of the midwife as laborist. The role of the midwife and obstetrician laborist/hospitalist is rapidly evolving due to the need to improve patient safety and provide direct care due to reduced resident work hours, as well as practice demands experienced by community providers and other factors. Models under development are customized to meet the needs of different communities and hospitals. Midwives are playing a prominent role in many laborist/hospitalist practices as the first‐line hospital provider or as part of a team with physicians. Some models incorporate certified nurse‐midwives/certified midwives as faculty to residents and medical students. The midwifery laborist/hospitalist practices at Baystate Medical Center in Springfield, Massachusetts, are presented as an example of how midwives are functioning as laborists. Essential components of a successful midwife laborist program include interdisciplinary planning, delineation of problems the model should solve, establishment of program metrics, clear practice guidelines and role definitions, and a plan for sustained funding. This article is part of a special series of articles that address midwifery innovations in clinical practice, education, interprofessional collaboration, health policy, and global health.