Structural characterization of a K-antigen capsular polysaccharide essential for normal symbiotic infection in Rhizobium sp. NGR234: deletion of the rkpMNO locus prevents synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-non-2-ulosonic acid.

Many early molecular events in symbiotic infection have been documented, although factors enabling Rhizobium to progress within the plant-derived infection thread and ultimately survive within the intracellular symbiosome compartment as mature nitrogen-fixing bacteroids are poorly understood. Rhizobial surface polysaccharides (SPS), including the capsular polysaccharides (K-antigens), exist in close proximity to plant-derived membranes throughout the infection process. SPSs are essential for bacterial survival, adaptation, and as potential determinants of nodulation and/or host specificity. Relatively few studies have examined the role of K-antigens in these events. However, we constructed a mutant that lacks genes essential for the production of the K-antigen strain-specific sugar precursor, pseudaminic acid, in the broad host range Rhizobium sp. NGR234. The complete structure of the K-antigen of strain NGR234 was established, and it consists of disaccharide repeating units of glucuronic and pseudaminic acid having the structure -->4)-beta-d-glucuronic acid-(1-->4)-beta-5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid-(2-->. Deletion of three genes located in the rkp-3 gene cluster, rkpM, rkpN, and part of rkpO, abolished pseudaminic acid synthesis, yielding a mutant in which the strain-specific K-antigen was totally absent: other surface glycoconjugates, including the lipopolysaccharides, exopolysaccharides, and flagellin glycoprotein appeared unaffected. The NGRDeltarkpMNO mutant was symbiotically defective, showing reduced nodulation efficiency on several legumes. K-antigen production was found to decline after rhizobia were exposed to plant flavonoids, and the decrease coincided with induction of a symbiotically active (bacteroid-specific) rhamnan-LPS, suggesting an exchange of SPS occurs during bacterial differentiation in the developing nodule.

Urinary proteins and red blood cell membrane negative charges in diabetes mellitus.

The nature and origin of proteinuria in diabetes mellitus have been investigated by measuring the urinary excretion of seven specific proteins of low (beta 2-microglobin, retinol-binding protein) or high molecular weight (albumin, transferrin, hemopexin and IgG). Using the Alcian Blue binding test, we also measured negative charges on red blood cell (RBC) membrane which according to recent studies might mirror the glomerular polyanion charge. A group of 190 diabetics was examined, including 90 patients with type I diabetes, 23 type II diabetics treated with diet and/or hypoglycaemic agents and 77 longstanding type II diabetics requiring insulin therapy. With the exception of beta 2-microglobulin all proteins measured were excreted in the urine of diabetics in significantly higher amounts than in controls. The assay of transferrin proved the most sensitive (58% positive) followed by albumin (49%), IgG (34%), hemopexin (28%) and retinol-binding protein (26%). Practically the same ranking was obtained when only type I diabetics were considered. RBC membrane negative charges were diminished in diabetics and negatively correlated with the urinary excretion of albumin (r = -0.61, n = 190). RBC charges were also negatively correlated with other urinary proteins of high molecular mass (r between - 0.5 and - 0.2) but presented no relation with urinary beta 2-microglobulin or retinol-binding protein. The loss of RBC charges in diabetics most likely reflects the concomitant depletion of the glomerular polyanion responsible for the increased glomerular leakage of high molecular mass plasma proteins. The preferential increase in transferrin excretion together with the progressive rise in the urinary excretion of IgG lead us to postulate that the loss of glomerular polyanion in diabetes is accompanied, from the early stage, by a progressive decrease in the size-selectivity of the glomerular filter. The urinary excretion of retinol-binding protein was weakly correlated with albuminuria (r = 0.26, n = 186). Eight % of diabetics showed an elevation of urinary retinol-binding protein without evidence of microalbuminuria, which clearly demonstrates that a proximal tubular impairment can occur independently of the glomerular alterations in the course of diabetic nephropathy.

Problems raised by the simultaneous reproducibility of positive allergic patch test reactions in man.

The dose of petrolatum patch test material applied with the Leukotest or the Finn Chamber method varies significantly and is one important factor in the lack of reproducibility with present patch test methods. Ready-to-apply methods such as Epiquick and TRUE Test avoid this problem. In a right-side versus left-side comparative study of 19 Epiquick patches on 100 consecutive patients, the reproducibility was 95%. This is a much higher correlation than with previous comparative tests with both identical and different patch test systems.

pH influence of surfactant-induced skin irritation. A non-invasive, multiparametric study with sodium laurylsulfate.

Even though various experimental methods have been proposed for in vitro testing of detergents such as LSL (sodium laurylsulfate) no absolutely relevant clinical information can be inferred from them as to the irritancy of a given compound. In particular the relative importance of pH needs further assessment. This study reports on in vivo evaluation of skin function changes under given experimental conditions with SLS applied at 3 different pH values. There is a dramatic increase of transepidermal water loss (TEWL), i.e. a substantial reduction in the barrier function of the skin, when SLS is applied under occlusion for 48 H. The alkaline control solution (NaOH pH 9) induced low-grade, but significant TEWL increases, as compared to the other controls (distilled water pH 7; HCl pH 5), which had no influence on TEWL. The changes obtained with the controls were much lower than those observed with SLS. The barrier-function changes induced by the surfactant SLS could, however, promote transepidermal passage of acid and/or alkaline molecules, hence increasing toxic damage of the skin; yet no such effects could be observed, indicating that the main effects are due to detergency. Assessment of cutaneous blood flow values (CBFV) by laser Doppler velocimetry showed increased values after SLS. When pH-adjusted SLS solutions were compared, there was neither a difference in relation to pH nor did the control solutions induce any significant CBFV change. This study reveals that TEWL and CBFV are probably the most reliable methods to investigate acute irritancy by SLS. Accordingly, pH cannot be considered as a major contributive factor of irritancy when SLS solutions are applied under occlusion (48 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological modulation by cetirizine and atropine of the histamine- and methacholine-induced wheals and flares in human skin.

This study was planned to verify whether different methods for the measurement of skin reactivity, i.e. wheal and flare area, wheal thickness, skin capacitance and transepidermal water loss, were or were not able to discriminate between intradermally injected agonists (histamine and methacholine). For evaluating agonist/antagonist interactions, we adopted a cross-over, double-blind, placebo-controlled study designed to compare the effects of cetirizine and atropine. The intradermal injection of agonists elicited the appearance of wheal and flare reactions and, after histamine, the skinfold thickness was significantly increased. Skin capacitance and transepidermal water loss measurements reflected sweat gland activation after methacholine injection but were, respectively, not or less affected by histamine dry skin prick test or saline; hence, both methods appear very sensitive for in vivo testing of cholinomimetic agents. Cetirizine inhibited all the specific skin modifications induced by histamine challenge, wheals, flares and increased thickness, without affecting the methacholine-induced perspiration. This would further support the H1 specificity of this anti-H1 agent in vivo. However, at the agonist/antagonist ratios tested in view of the safety of the test persons, we were unable to objectivate methacholine blockade by atropine.

Histopathological discriminant criteria between lichenoid drug eruption and idiopathic lichen planus: retrospective study on selected samples.

The lesions observed in 15 idiopathic lichen planus (ILP) skin biopsies were compared with those occurring in 15 clinically relevant lichenoid drug eruptions (LDE). Various histopathological features were taken into account. Statistical analysis of results showed that none of these can be considered as a significant criterium allowing a pathognomonic differential diagnosis between ILP and LDE. Nevertheless, a strong suggestion of drug etiology can be inferred when the following histopathological signs ('LDE-related criteria') are present: focal parakeratosis, focal interruption of the granular layer, cytoid bodies in the cornified and granular layers, present in more than 50% of LDE and never in ILP.

[Invasive and non-invasive studies of the protective action of a silicon-containing cream and its excipient in skin irritation induced by sodium laurylsulfate]. / Etudes invasive et non invasive du pouvoir protecteur d'une crème siliconée et de son excipient vis-à-vis de l'irritation cutanée induite par le laurylsulfate de sodium.

The purpose of our study was to evaluate the protective effect of a new silicone-containing barrier-cream (Anthydro) and its vehicle (Anthydro without silicone) in the prevention of cutaneous irritation by detergents. We therefore planned a study in several stages, using an anionic surfactant well known for its irritant properties: sodium laurylsulphate (SLS) in aqueous solution. In a first series of experiments, the protective effect of the Anthydro cream against SLS was studied by invasive methods on guinea-pigs in order to determine histologically the protective effect of the cream when a 10 p. 100 SLS solution was applied on the skin under occlusion during 24 hours (Square chambers, Van der Bend). Typical and reproducible lesions were apparent, and the protected sites were compared with the unprotected sites. In parallel, we used non-invasive methods (conductivity, transepidermal water loss and cutaneous blood flow) to determine in humans the protective effect of Anthydro cream in comparison with unprotected sites after application during 24 hours of patches soaked with a 5 p. 100 SLS solution on the forearms of 13 adult and healthy volunteers (Silver patch tests, Van der Bend). In both experiments the Anthydro cream was effective in reducing the SLS-induced cutaneous irritation. In a second series of experiments, the Anthydro barrier-cream was compared with its "base" (Anthydro without silicone) in terms of effectiveness, following the same experimental procedure (invasive on guinea-pigs, and non-invasive on humans). The base was shown to be effective in protecting against irritation. However, the histological lesions were less intense when the skin was protected by Anthydro than by its vehicle. Concerning the non-invasive methods in humans, no significant statistical differences appeared in the measurement of various parameters between the sites protected by Anthydro and the sites protected by the vehicle. These results lead us to suggest the existence of an essentially mechanical protective effect in which the silicone plays a very small part in terms of effectiveness. These experimental results necessitates further investigations to be extrapolated to occupational conditions without tests performed in industries and well-conducted epidemiological investigations.

Variations in the quantities of petrolatum applied in patch testing.

When using patch tests to objectify contact allergy in patients, many different materials are used in different clinics. Whatever the material used, there are presumably differences in the quantities of allergens dispersed in petrolatum applied in the patch tests by the clinicians. Therefore, we designed an experiment in order to evaluate whether such differences do indeed exist. The results presented here clearly show that the quantities of petrolatum applied varies considerably in the patch tests (i.e. Leukotest Beiersdorf) from dermatologist to dermatologist. In addition, we found that for an individual dermatologist differences occur from day to day. It is our conclusion that in the interest of the patients and in the interest of multicentric studies, standardization of patch test materials is of great importance, especially in regards to the quantities of petrolatum applied.

Radiation sensitivity of Bloom's syndrome lymphocytes during S and G2 phases.

In order to study chromosome sensitivity of Bloom's syndrome (BS) cells in relation to the replication stage, gamma-ray irradiation was performed immediately before adding bromodeoxyuridine (BrdU) to lymphocyte cultures of one BS patient and of one control. It was found that BS cells are much more sensitive to the irradiation than control cells at the end of S and at G2 phases. The rate of induction of chromosome breaks is significantly increased and that of chromatid breaks and exchanges is also increased, though to a lesser degree. Our results also favor the existence of a cell subpopulation in BS characterized by a slow cycle, a high spontaneous chromosome aberration rate, and a high radiation sensitivity.

Detailed cell cycle analysis in human lymphocytes; application to gamma-irradiated cells.

A method based on BrdU incorporation for analyzing in detail the kinetics of the cell cycle is described. The S phase has been subdivided into five subphases, each recognizable by their BrdU incorporation pattern at metaphase. The method can be useful for the study of abnormal cell cycles, and may have particular application in mutagenesis studies concerning the various subphases of the S phase, without using synchronization techniques. An application of the method is described, showing that gamma-irradiation, during the course of the S phase, leads to a lack of cells which were in early S phase at the time of irradiation. This finding can be related either to a higher lethality at this stage of the cell cycle or to a delay in completion of DNA replication after irradiation.

[Tentative classification of radial figures and an evaluation of the consequences of their radiation induction]. / Tentative de classification des figures radiales et estimation des conséquences de leur induction par l'irradiation.

A classification is proposed of the tri- and quadriradials, based on theoretical and observed anomalies induced by radiation. Mitotic consequences of radials during S-phase are analysed, and it is shown that the rate of induction of unbalanced rearrangements is very much higher than that of balanced ones. After extrapolation to germ cells, it can be assumed that irradiation during S-phase will induce a high risk of chromosome imbalance for progeny which will be restricted almost entirely to the first generation.

Lack of mutagenic activity of dimethylformamide.

Different test systems have been utilized to evaluate the mutagenic and carcinogenic properties of dimethylformamide (DMF), an aliphatic amide used as a solvent in chemical industry. The Ames test was performed on different strains of Salmonella typhimurium, whereas the ability of DMF to induce structural aberrations in eukaryotic chromosomes was tested by in vitro observations on human lymphocytes and in vivo experiments on mouse bone marrow. Furthermore, male mice were treated with DMF for the induction of sperm abnormalities. The negative results obtained in all test systems as well as the absence of positive reports in man or in experimental animals with respect to induction of cancers suggest strongly that DMF is devoid of mutagenic or carcinogenic properties.

Risk of chromosomal disease due to radiation. Tentative estimate from the study of radiation-induced translocations in human fibroblasts.

A sample of 214 reciprocal 2-break translocations observed in fibroblasts, both after accidental 'in vivo', and experimental 'in vitro' gamma-irradiation, was studied. The distribution of the breaks along the chromosomes does not seem at random. The minimal possible imbalance that these translocations could induce by malsegregation, if they existed in germ cells, was estimated. These imbalances were compared with the chromosomal trisomies and monosomies known to be compatible with life after birth in man. It is concluded that about 2/5 of the radiation-induced translocations might induce a viable trisomy and/or monosomy. This result, similar to that previously obtained in human lymphocytes, indicates the validity of the extrapolation from one tissue to another, and hopefully to germ cells.

[Non random position of metaphasic chromosomes. II. Effect of condensation on the position of X chromosomes]. / Position non aléatoire des chromosomes métaphasiques. II. Effet de la condensation sur la position des chromosomes X.

A study is reported on the location of X chromosomes in human female lymphocytes and fibroblasts at mitosis. By the use of BrdU, incorporated during either early or late S-phase, the late and the early replicating Xs were identified, so that it could be determined which was more peripheral in the metaphase plate. In lymphocytes, when BrdU is incorporated during late S-phase, the late-replicating X, which is the less condensed, is the more peripheral. On the contrary, when BrdU is incorporated during early S-phase, the early replicating X, which is the less condensed, is the more peripheral. On the other hand, in fibroblasts grown on coverslips, the late-replicating X is always peripheral, regardless of the time of BrdU treatment. These results confirm the peripheral location of the late-replicating X and demonstrate the presence of artifacts, depending on chromosome condensation, occurring during the procedures of slide preparation.

[Non random position of metaphasic chromosomes. III. Position of chromosomes involved in constitutional translocations]. / Position non aléatoire des chromosomes métaphasiques. III. Position des chromosomes dans les translocations constitutionnelles.

Autosome-autosome and sex chromosome-autosome constitutional translocations are analyzed in order to determine whether the affected chromosomes have a particular position in the spread metaphase. Except for one t (10;15) and for several t (X;autosome), the rearranged chromosomes and their normal homologues seem to have random positions. In so far as it has been demonstrated that the metaphase chromosome's positions reflect those of the interphase chromosomes, it is concluded that no specific ordering, transmissible cell generation after generation, exists. The position of the chromosomes would be determined after anaphase migration and would remain unchanged until the subsequent metaphase. Therefore, the rearrangements do not impose any particular topological constraints to the involved chromosomes. The only exceptions may concern the sex chromosomes and those carrying nucleolar organizers.

[Non random position of metaphasic chromosomes. IV. Study of translocations t(7;14)(p14;q12) and t(7;14)(q35;q12)]. / Position non aléatoire des chromosomes métaphasiques. IV. Etude des translocations t(7;14)(p14;q12) et t(7;14)(q35;q12).

A study is reported on the distances between rearranged chromosomes and between their normal homologues in the case of t(7;14)(p14;q12) and of t(7;14)(q35;q12), in lymphocytes from patients with ataxia-telangiectasia (AT) and in non-affected persons. The study shows that rearranged chromosomes, in t(7;14)(p14;q12) are closer together, in non-AT persons, than their normal homologues. This is interpreted as the result of recent, repeated, and poorly transmitted mutations, in non-AT persons. On the contrary, this translocation, probably not eliminated in AT patients, becomes the most frequent.