Comparison of the compact dry TC method with the standard method ISO 21149:2006 for determining aerobic colony counts in cosmetic emulsion.

OBJECTIVE: Compact Dry TC, a rapid method kit for determining aerobic colony counts, has been developed by Nissui Pharmaceutical Co. for food application. These plates are pre-sterilized and contain culture medium, a cold-soluble gelling agent and a colour redox indicator for rapid enumeration. In this study, the alternative method is compared with the standard method ISO 21149:2006 - Cosmetic - Microbiology - Enumeration and detection of aerobic mesophilic bacteria, for cosmetic emulsions application. METHODS: An oil-in-water (o/w) cosmetic emulsion was contaminated with a pool of bacterial strains (Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 9027). One millilitre of samples was spread on agar as described in ISO 21149. The colonies were enumerated after 3 days of incubation. At the same time, 1.2 mL samples were spread on Compact Dry TC kits. The kit was incubated at 35°C ± 1°C for 48 h, and the colonies were enumerated. Accuracy determination was carried out using six replicates at four levels of concentrations (10, 50, 100 and 250 CFU mL-1 ). The repeatability study was carried out using 12 replicates at four levels of concentrations (10, 50, 100 and 250 CFU mL-1 ). Variations relative to the analyst and to the batch of emulsion have been investigated. RESULTS: The linear correlation coefficients of Compact Dry TC Kit enumeration with standard method ISO 21149:2006 was 0.9999. In comparison study, no apparent differences were noted between the Compact Dry TC kit and the reference method ISO 21149, for the detection level of aerobic microorganisms. Relative accuracy, repeatability and intermediate precision studies were acceptable. In the repeatability study, the Shapiro-Wilk test has confirmed the normally distribution of the twelve assays. No significant variations in Compact Dry TC count results were observed with different analysts and different batches of emulsion. CONCLUSION: The results showed that the two compared methods 'Compact Dry TC' vs. 'conventional pour plate' performed equally well. Demonstration was achieved that the Compact Dry TC method may constitute a useful alternative tool for rapid enumeration of aerobic mesophilic bacteria in cosmetic emulsions.

Alterations in the chain dynamics of insoluble elastin upon proteolysis by serine elastases.

The high temperature dielectric relaxations of purified and elastolized ligamentum nuchae elastin in the dry state have been investigated by thermally stimulated depolarization current spectrometry, with an equivalent frequency comprised between 10(-2) and 10(-3) Hz. A main relaxation mode, located close to 150 degrees C and attributed to the dielectric manifestation of a glass transition, is found for all samples. After decomposition by the fractional polarization method, the analysis of the high temperature mode shows the existence of two relaxation mechanisms: a cooperative one, associated with flexible zones of the protein, and an isoenthalpic one, corresponding to more ordered and constrained zones. The activation parameters of the two mechanisms are dependent on the extent of elastolysis and on the nature of enzyme (pancreatic elastase vs leukocyte elastase). Both enzymes influence the dielectric behavior of elastin in a similar way: the activation enthalpy maximum of the relaxing units located in the flexible zones, characteristic of the cooperative length, decreases with increasing hydrolysis. Moreover, the isoenthalpic mechanism becomes cooperative at the highest extent of elastolysis, which highlights release of constraints in ordered zones. Nevertheless, the differences found between the two enzymatic hydrolyses are characteristic of distinct sites of cleavage in the elastin network.

Dielectric characterization of collagen, elastin, and aortic valves in the low temperature range.

The low temperature dielectric relaxation of porcine aortic valves and its main macromolecular proteins. i.e. elastin and collagen, have been investigated in the dry state and at low levels of hydration by thermally stimulated currents spectrometry, with an equivalent frequency of 10(-3) Hz. Two secondary relaxation modes, labeled gamma and beta with increasing temperature, are found for the three materials. Since the gamma-mode is independent upon hydration while the beta-mode is strongly plasticized by water, these relaxation modes have been attributed to localized motions of the polypeptidic chains containing apolar and polar residues, respectively. The deconvolution of the beta-mode by fractional polarization gives the experimental distribution of the dielectric relaxation times of the three materials, and allows us to deduce the activation parameters of each elementary process. These analyses shows the existence of compensation phenomena between the activation parameters, implying cooperative mechanisms. The occurrence of these phenomena with their characteristic parameters are used to specify the origin of the localized relaxation modes in collagen and elastin, and to assign the specific role of each protein in the aortic valves.

Characterisation of elastin and collagen in aortic bioprostheses.

Porcine aortic valves used as cardiac valve bioprostheses are well adapted to physiological functions in the short term, but they lack long-term durability. Several multi-step extractions have been performed to obtain a perfectly acellular matrix. A new physical methodology is proposed to evaluate the resulting fibrous protein damage after biochemical extraction (TRI-COL and SDS). Thermal analysis techniques are adapted to collagen and elastin characterisation in the solid state. The aortic tissue thermal transitions are determined by differential scanning calorimetry (DSC): elastin glass transition is observed around 200 degrees C, and collagen denaturation is observed around 230 degrees C. These parameters are characteristic of the elastin network arrangement and of collagen triple-helix stability. The technique of thermostimulated currents (TSC) is well suited to specify the chain dynamics of proteins. The low-temperature relaxations observed in both collagen and elastin are associated with localised motions, whereas the high-temperature modes are attributed to more delocalized motions of the chains. Therefore TSC and DSC spectrometries allow physical parameters specific to collagen and elastin to be obtained and their interaction in aortic tissues to be determined. According to the significant evolution of these parameters on SDS samples, the destabilizing effect of this detergent is highlighted.

Thermal analysis characterization of aortic tissues for cardiac valve bioprostheses.

Two multistep extractions were achieved on porcine aortic tissues to obtain acellular matrices used for cardiac bioprostheses. The evaluation of structural modifications and the possible damage of extracellular matrix fibrous proteins were investigated by means of thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Protein-water interactions and degradation temperatures were determined by TGA. DSC was used to characterize protein thermal transitions (glass transition and denaturation), which provided information on the dynamic structure of the aortic tissue components. Sodium dodecyl sulfate (SDS) extraction had a destructuring effect, while Triton and cholate treatments did not affect the structural integrity of either elastin and collagen. A DSC comparison showed that SDS destabilizes the collagen triple helical domain and swells the elastin network.

[Collagens of the internal saphenous vein, normal and varicose, as a function of age]. / Collagènes de la veine saphène interne, normale et variqueuse, en fonction de l'âge.

The normal venous vein contains more collagen in the young subject than in the ageing subject. This is confirmed by the decrease of thickness of aged venous wall indicating that the fibrosis present in the aged subject is atrophic in nature. Collagen content of the varicose vein wall remains practically unchanged with age: the varicose vein is a dysplasic vein.

Collagen of the normal and the varicose human saphenous vein: a biochemical study.

Various types of collagen (I, III, IV, V) were identified in normal and varicose human saphenous veins using pepsin digestion and cyanogen bromide digestion followed by SDS-polyacrylamide gel electrophoresis. Varicose veins were found to have a higher collagen content than normal veins. This is consistent with the morphological fibrosis which has regularly been described. No essential differences were found in the collagen composition of dilated and apparently healthy portions of varicose veins.

Interstitial collagens and ageing in human aorta.

Types I and III collagen were identified in four segments of human aorta using pepsin and cyanogen bromide digestion followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Collagen is not uniformly distributed in the different segments of the arterial wall. Collagen type I is always the major collagen present. With ageing collagen type III decrease in quantity from the heart to the distal portion of the aorta. Histologically the elastic tissue is more altered in the lower abdominal section of aorta than in the arch. This study allowed a correlation between morphological observations and biochemical changes.

[Lathyrism and collagens I and III (author's transl)]. / Lathyrisme et collagènes de type I et III.

The administration of lathyrogenic agents (BAPN) at weak and prolonged doses (9 weeks, 1 g/kg/day) induced in the rat simultaneous alterations of aortic and cutaneous connective tissues comparable to the modifications observed during ageing in man. The lathyrogens produced this effect of inhibiting lysine-oxydase, the enzyme responsible for cross-linking collagen and elastin which leads to an increase in extractable collagen in the tissues. But BAPN did not appear to affect synthesis of collagen either in amount or in types I and III collagen ratio.

[Lathyrism and collagens I and III (author's transl)]. / Lathyrisme et collagènes de type I et III.

The administration of lathyrogenetic agents (BAPN) at weak and prolonged doses (9 weeks, 1 g/kg/day) induced in the rat simultaneous alterations of aortic and cutaneous connective tissues comparable to the modifications observed during ageing in man. The lathyrogens produced this effect by inhibiting lysine-oxydase, the enzyme responsible for cross-linking collagen and elastin which leads to an increase in extractable collagen in the tissues. But BAPN did not appear to affect synthesis of collagen either in amount or in types I and III collagen ratio.

Lathyrism: aortic and cutaneous collagen in the rat.

In the rat the administration of lathyrogenic toxic agents at low doses over prolonged periods of time caused simultaneous alterations in aortic and cutaneous tissues. BAPN caused an increase in soluble collagen in the skin, but in aorta there was no increase in neutral salt soluble collagen, but an increase in the amount that could be solubilized with pepsin. Type III collagen was found to represent 7% in the control aorta samples and this level was not affected by BAPN. In skin Type III accounts for 11% of the extractable collagen in controls and 14% in lathyritic animals. Biochemical changes observed in this study show evidence for collagenous lesions by increases in solubility in both skin and aorta but in neither tissue was the percentage of Type III altered by lathyrism.

Age dependent biochemical changes in dermal connective tissue. Relationship to histological and ultrastructural observations.

A biochemical study of skin biopsies of young normal subjects and two groups of aged subjects, one active and the other confined to bed, was made and the results compared with histological observations. Skin biopsies were incubated with 3H proline and 14C glucosamine and connective tissue components were sequentially extracted. In each extract the specific activity of total and collagenous protein was determined. With age a decrease was observed in both the amount of protein and the 3H proline incorporation of collagenous extracts. A parallel increase in 14C glucosamine incorporation was also noticed in extracts containing glycosaminoglycans and structural proteins. These results agree in part with the changes observed by light and electron microscopy, which revealed an increase in ground substance and fragmentation of collagen bundles in the dermis of older subjects.

[Gallium sulfate and skin allografts in the mouse]. / Sulfate de gallium et allogreffes cutanées chez la souris

Survival of skin allografts in mice are prolonged when these animals are injected subcutaneously with gallium sulfate before and after the grafting. The average survival is then 17, 2 days whereas it is 12,5 days for controls. For mice treated with an immunodepressive drug such as cortisone acetate average survival is 13,4 days; it is 17 days when cortisone has been added to the metallic salt.

[Value and duration of the bone localization of gallium in the mouse]. / Importance et durée de la localisation osseuse du gallium chez la souris

Gallium was administered subcutaneously to mice, and was found to be localized essentially in the skeleton. It was measured in the bones of animals given daily injections of gallium sulfate. There was little or no mortality. The amount of gallium found rose with the number of injections, and was greater when higher doses were injected. Elimination was very low after cessation of treatment. Results were used to study the action of gallium on cutaneous allografts in mice.