Utility of Multistep Protocols in the Analysis of Sentinel Lymph Nodes in Cutaneous Melanoma: An Assessment of 194 Cases.

CONTEXT.­: Currently, no universal protocol exists for the assessment of sentinel lymph nodes (SLNs) in cutaneous melanoma. Many institutions use a multistep approach with multiple hematoxylin-eosin (H&E) and immunohistochemical stains. However, this can be a costly and time- and resource-consuming task. OBJECTIVE.­: To assess the utility for multistep protocols in the analysis of melanoma SLNs by specifically evaluating the Calgary Laboratory Services (CLS) protocol (which consists of 3 H&E slides and 1 S100 protein, 1 HMB-45, and 1 Melan-A slide per melanoma SLN block) and to develop a more streamlined protocol. DESIGN.­: Histologic slides from SLN resections from 194 patients with diagnosed cutaneous melanoma were submitted to the CLS dermatopathology group. Tissue blocks were processed according to the CLS SLN protocol. The slides were re-reviewed to determine whether or not metastatic melanoma was identified microscopically at each step of the protocol. Using SPSS software, a decision tree was then created to determine which step most accurately reflected the true diagnosis. RESULTS.­: We found with Melan-A immunostain that 337 of 337 negative SLNs (100%) were correctly diagnosed as negative and 55 of 56 positive nodes (98.2%) were correctly diagnosed as positive. With the addition of an H&E level, 393 of 393 SLNs (100%) were accurately diagnosed. CONCLUSIONS.­: We recommend routine melanoma SLN evaluation protocols be limited to 2 slides: 1 H&E stain and 1 Melan-A stain. This protocol is both time- and cost-efficient and yields high diagnostic accuracy.

Increasing incidence rates, distribution and histological characteristics of primary gastrointestinal non-Hodgkin lymphoma in a North American population.

BACKGROUND: The incidence of primary extranodal non-Hodgkin lymphoma (NHL) of the gastrointestinal (GI) tract has been on the rise. OBJECTIVES: To determine the incidence of primary GI NHL and distribution according to site and histological type in a large North American adult population over a 10-year period. METHODS: All diagnoses of GI NHL made between January 1999 and January 2009 were reviewed using a regional pathology database. Patients ≥18 years of age living within health region boundaries were included. Age- and sex-adjusted incidence rates of GI NHL according to GI site and histological type over a 10-year period were calculated and compared. RESULTS: A total of 149 cases of primary GI NHL were identified during the study period. Age- and sex-adjusted yearly incidence rates ranged from 0.13 per 100,000 in 1999, to 2.39 per 100,000 in 2007. Histological distribution (47% diffuse large B cell lymphoma, 24% extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue type, 8% follicular and 5% mantle cell) and site distribution (47% stomach, 26% small bowel, 17% colon) were obtained with increasing annualized incidence rates for each of these sites over time. Remaining cases included multiple GI sites of involvement (9%) and esophagus (0.7%). DISCUSSION: Population-based GI NHL incidence rates in the present study were higher than those described elsewhere in North America and Europe. Nearly one-half showed high-grade (diffuse large B cell lymphoma) histology at diagnosis. Incidence rates for the colon exceed those described in other studies worldwide. CONCLUSION: Because the majority of GI NHL are diagnosed on endoscopic biopsy, clinicians and pathologists must be vigilant of this entity.

DmsD, a Tat system specific chaperone, interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis.

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein-protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a "node" in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.

An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins.

Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, coimmunoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system.

Twin-arginine translocase may have a role in the chaperone function of NarJ from Escherichia coli.

NarJ is a chaperone involved in folding, maturation, and molybdenum cofactor insertion of nitrate reductase A from Escherichia coli. It has also been shown that NarJ exhibits sequence homology to a family of chaperones involved in maturation and cofactor insertion of E. coli redox enzymes that are mediated by twin-arginine translocase (Tat) dependent translocation. In this study, we show that NarJ binds the N-terminal region of NarG through Far Western studies and isothermal titration calorimetry, and the binding event occurs towards a short peptide sequence that contains a homologous twin-arginine motif. Fractionation experiments also show that the interaction of NarJ to the cytoplasmic membrane exhibits Tat-dependence. Upon further investigation through Far Western blots, the interactome of NarJ also exhibits Tat-dependence. Together the data suggest that the Tat system may play a role in the maturation pathway of nitrate reductase A.

Folding forms of Escherichia coli DmsD, a twin-arginine leader binding protein.

Escherichia coli DmsD interacts with the twin-arginine leader sequence of the catalytic sub-unit (DmsA) of DMSO reductase. DmsD was purified as a mixture of a number of different folding forms including: dimer (A); monomer (B); a minor thiol oxidized form; a heterogeneously folded or multi-conformational monomer form which displayed a ladder of bands on native-PAGE (D); and proteolytically degraded and aggregated forms. Polyacrylamide gel electrophoresis (PAGE), under denaturing and non-denaturing conditions, was used to examine the folding and stability of DmsD. Additionally, the biophysical methods of dynamic light scattering, circular dichroism, fluorescence, and mass spectroscopy were also used. Form D could be converted to form B by treatment with 4M urea, which is the concentration at which form B begins to denature. Forms A/B could be converted to D by incubation at pH 5.0. Forms A/B and D all had twin-arginine leader binding activity.