Spleen and Liver Pigmented Macrophages of Rana Esculenta L. A New Melanogenic System?

The present study reports the results of a morpho‐functional analysis of spleen pigmented cells from Rana esculenta L. and comparison with liver melanin‐synthesizing cells, belonging to the macrophage cell lineage. Cytological and cytochemical analyses show that parenchymal pigmented cells of the spleen, like those of the liver, are positive to peroxidase and lipase reactions and have phagocytic properties. The observation of premelanosomes in various stages of differentiation, together with the demonstration of dopa oxidase activity in the melanosome proteins, indicate that spleen pigmented macrophages have endogenous melanogenic ability as do liver pigmented macrophages. Attempts to demonstrate tyrosine‐hydroxylase activity in melanosome protein extracts from frog spleen and liver, using the same protocol as for mammalian tyrosinases, gave negative results. As regards the dopa oxidase activity revealed, some of its properties differ from the typical behaviour observed for tyrosinases from different sources. Peroxidase activity is shown in spleen and liver melanosome proteins with p‐phenylenediamine‐pyrocatechol (PPD‐PC), and not with typical peroxidase substrates. Suitable inhibition tests revealed that dopa oxidase and peroxidase activities might be supported by two different proteins. Liver melanosome extracts display a very strong laccase (dimethoxyphenol‐oxidase) activity but spleen extracts do not. Differences observed in the enzymatic properties of the spleen and liver melanosomes suggest that pigmented macrophages may undergo tissue‐specific differentiation. These preliminary data show that the melanin pathway of pigmented macrophages is different from that of melanocytes and may pave the way to identification of a new melanogenic pathway in vertebrates.     

Expression pattern in the antennae of a newly isolated lepidopteran Gq protein alpha subunit cDNA.

From the antennae of the moth Mamestra brassicae, we have identified a lepidopteran G protein alpha subunit belonging to the Gq family, through immunological detection in crude antennal extract and antennal primary cell cultures, followed by molecular cloning. The complete cDNA sequence (1540 bp) contains an open reading frame encoding a protein of 353 amino acids. This deduced sequence possesses all of the characteristics of the Gq family and shares a very high degree of amino-acid sequence identity with vertebrate (80% with mouse or human Gqalpha) and invertebrate subunits (varying between 60 and 87% for Gqalpha from organisms as diverse as sponge and Drosophila). The expression pattern of the Gq subunit in adult antennae was associated with the olfactory sensilla suggesting a specific role in olfaction. These data provide molecular evidence for a component of the phosphoinositide signaling pathway in moth antennae: this G protein alpha subunit may be involved in the olfaction transduction process through interaction with G-protein-coupled receptors, stimulating the phospholipase C mediated second messenger pathway.     

Melanogenesis in the pigment cells of Rana esculenta L. liver: evidence for tyrosinase-like activity in the melanosome protein fraction

This work demonstrates the presence of a tyrosinase-like activity in the pigment cells of frog Rana esculenta L. liver. The activity was evidenced in the protein melanosome fraction extracted with differential centrifugation methods. The study of this activity, carried out with spectrophotometric methods, indicates 1) the system presents the characteristics of an allosteric enzyme; 2) the grade of cooperativity shows oscillations going from negative cooperativity toward the substrate, evident in the warmest months of the year, to an absence of cooperativity in the coldest months of the year; and 3) the levels of activity of the system also vary according to season, with the highest levels appearing in the coldest months of the year. Given that this extracutaneous system of pigment cells, different from melanocytes, is able to carry out melanogenesis, we suggest its inclusion in the classification of pigment cells.     

Seasonal variations of Rana esculenta L. skin tyrosinase

Various enzymes are known to be involved in melanin biosynthesis, but the key role appertains to tyrosinase. In amphibians this enzyme displays peculiar characteristics: i) it requires an activation process; ii) its level of enzymatic activity in the animal skin changes depending on the season. In this work, by using chymotrypsin, subtilisin and SDS as putative activators, we studied the activation process of the skin pro-tyrosinase of Rana esculenta L. in different seasons over a period of two years. We found that chymotrypsin and subtilisin were able to yield an active enzyme, but not SDS. The maximum levels of tyrosinase activity were recorded in winter and the minimum in summer. We detected tyrosinase activity in the melanosomal fraction, where the enzyme form was least sensitive to proteolytic activation, probably corresponding to a "mature" tyrosinase. The enzyme forms found in the microsomal and soluble fractions were more sensitive to proteolytic activation, probably corresponding to "immature" tyrosinase. On SDS-PAGE, the tyrosinase activity assays showed a dopa-positive band at 200 kDa and a second aggregated band with a still higher molecular mass. The significance of these results in frog melanogenesis regulation is discussed.     

A diffusion chamber for assessing efficacy of natural anti-fouling defenses in marine organisms

A slow-release diffusion chamber is here described for use in assessing the effectiveness of potentially bioactive compounds as anti-fouling agents. Its settlement surface is a porous 15 mm thick slab of Portites lobata skeleton, through which an aqueous extract of a given marine organism is permitted to diffuse. The chamber is placed at an underwater site for 30 days at a time, and settlement of sessile, benthic organisms, particularly algae, is permitted to occur. Comparative settlement levels were compared between chambers containing an extract and a seawater control. An extract of Sinularia flexibilis (Octocorallia, Alcyonacea) was assessed using such chambers and found to be an effective natural anti-fouling agent or defense mechanism against Ectocarpus sp. and pennate diatoms, reducing their cover by ∼ 50% when compared to experimental conditions using seawater as a control. Intra-chamber variability in extract concentration was found to be minimal. No significant differences were found in extract concentrations between replicate chambers through time. The chamber is a highly cost-effective and efficient tool by which such assessments can be made and easily replicated. It is recommended that coral which has recently died be collected and used as a source of substratum for this purpose.     

Accuracy of administrative claims data for polypectomy

Background:

The frequency of polypectomy is an important indicator of quality assurance for population-based colorectal cancer screening programs. Although administrative databases of physician claims provide population-level data on the performance of polypectomy, the accuracy of the procedure codes has not been examined. We determined the level of agreement between physician claims for polypectomy and documentation of the procedure in endoscopy reports.

Methods:

We conducted a retrospective cohort study involving patients aged 50–80 years who underwent colonoscopy at seven study sites in Montréal, Que., between January and March 2007. We obtained data on physician claims for polypectomy from the Régie de l’Assurance Maladie du Québec (RAMQ) database. We evaluated the accuracy of the RAMQ data against information in the endoscopy reports.

Results:

We collected data on 689 patients who underwent colonoscopy during the study period. The sensitivity of physician claims for polypectomy in the administrative database was 84.7% (95% confidence interval [CI] 78.6%–89.4%), the specificity was 99.0% (95% CI 97.5%–99.6%), concordance was 95.1% (95% CI 93.1%–96.5%), and the kappa value was 0.87 (95% CI 0.83–0.91).

Interpretation:

Despite providing a reasonably accurate estimate of the frequency of polypectomy, physician claims underestimated the number of procedures performed by more than 15%. Such differences could affect conclusions regarding quality assurance if used to evaluate population-based screening programs for colorectal cancer. Even when a high level of accuracy is anticipated, validating physician claims data from administrative databases is recommended.Population-based screening programs for colorectal cancer rely heavily on the performance of colonoscopy as either the initial examination or as the follow-up to a positive screening by virtual colonography, double-contrast barium enema or fecal occult blood testing. Colonoscopy is the only screening examination accepted at 10-year intervals among people at average risk without significant polyps found. It allows direct visualization of the entire colon and rectum and permits removal of adenomatous polyps, precursors of colorectal cancer. The frequency of polypectomy is an important indicator of quality assurance for colorectal cancer screening programs.In the province of Quebec, physicians are reimbursed for medical services by the Régie de l’Assurance Maladie du Québec (RAMQ), the government agency responsible for administering the provincial health insurance plan. Physicians receive additional remuneration for performing a polypectomy if they include the procedure code in their claim.Data from physician claims databases are commonly used in health services research,^1–7 even though the data are collected for administrative purposes and physician reimbursement. Procedure codes in physician claims databases are presumed to have a very high level of agreement with data in medical charts.⁸ A physician making a claim will need to submit the diagnostic code and, when applicable, the procedure code. Studies that rely on physician claims databases can be divided into those that examine the diagnostic codes entered and those that examine the procedure codes entered. Few studies have attempted to validate procedure codes, and often not as the primary study objective.^9–14We conducted a study to determine the level of agreement between physician claims for polypectomy and documentation of the procedure in endoscopy reports.