Molecular characterization of the Bacillus anthracis main S-layer component: evidence that it is the major cell-associated antigen

Bacillus anthracis, the aetiological agent of anthrax, is a Gram-positive spore-forming bacterium. The cell wall of vegetative cells of B. anthracis is surrounded by an S-layer. An array remained when sap, a gene described as encoding an S-layer component, was deleted. The remaining S-layer component, termed EA1, is chromosomally encoded. The gene encoding EA1 (eag) was obtained on two overlapping fragments in Escherichia coli and shown to be contiguous to the sap gene. The EA1 amino acid sequence, deduced from the eag nucleotide sequence, shows classical S-layer protein features (no cysteine, only 0.1% methionine, 10% lysine, and a weakly acidic pi). Similar to Sap and other Gram-positive surface proteins, EA1 has three 'S-layer-homology’motifs immediately downstream from a signal peptide. Single- and double-disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of the wild-type bacilli. However, EA1 was more tightly bound than Sap to the bacteria. Electron microscopy studies and in vivo experiments with the constructed mutants showed that EA1 constitutes the main lattice of the B. anthracis S-layer, and is the major cell-associated antigen.     

Physical Activity,Obesity, and Cardiovascular Risk Factors in Children. The Belgian Luxembourg Child Study II

GUILLAUME, MICHÈLE, LEIF LAPIDUS, PER BJÖRNTORP, ANDRE LAMBERT. Physical activity, obesity, and cardiovascular risk factors in children. The Belgian Luxembourg Child Study II. Physical activity was measured in relation to cardiovascular (CV) risk factors in a randomly selected population of 1028 children from Province de Luxembourg in Belgium, a mainly rural area with a high prevalence of such risk factors among adults and children. Physical activity was estimated as participation in sport activities, a major indicator of leisure-time physical activity in schoolchildren, and physical inactivity was estimated as frequency and duration of television (TV) watching. Boys participated more frequently in sport activities than girls did (p=0. 001). A majority of the children watched TV daily. After age adjustment, bodyweight (girls, p<0. 012; boys, p<0. 027) and, in boys, body mass index (BMI) (p<0. 039) were related to days per week of TV watching. No significant relationships with other CV risk factors remained after adjustments for BMI. In analyses of independent contributions of age, TV watching, and sports activity on CV risk factors, age showed highly significant relationships. In boys, TV showed relationships with BMI (P<0. 04) and (borderline) with systolic blood pressure, independent of age and sports activity, whereas the latter was significantly related to subscapular skinfold (p<0. 04) and (borderline) with triceps skinfold and cholesterol. In girls, no significant independent contributions to risk factor associations were found. The father's education was directly associated with sports activities, whereas the mother being a housewife showed negative relationships to physical activity and positive to TV watching in their children, suggesting socioeconomic influence on the activity patterns of children. Furthermore, registrations suggested less physical activity in the most rural part of the area. It is concluded that children in this mainly rural area watch TV frequently. In boys, physical inactivity, measured both as TV watching and as registrations of sports activities, contributes independently to body fat mass. In girls, no contribution or weaker contributions of physical inactivity were found. This suggests that contributory factors leading to obesity might be different in girls and boys.     

Sperm nuclei analysis of a Robertsonian t(14q21q) carrier,by FISH,using three plasmids and two YAC probes

The meiotic segregation of chromosomes 14 and 21 was analysed in 1116 spermatozoa from an oligoasthenospermic carrier of a Robsertsonian translocation t(14q21q), and in 16 392 spermatozoa from a control donor, using two-colour fluorescence in situ hybridisation (FISH). Two YAC probes (cloned in yeast artificial chromosomes) specific for regions on the long arms of these chromosomes were co-hybridised. Of the spermatozoa, 12% were unbalanced, resulting from adjacent segregations. Chromosomes X, Y and 1 were also simultaneously detected in 1335 spermatozoa from the same carrier. Whereas gonosomal disomy rates were not significantly different from those of the control donors, disomy 1 were slightly but significantly increased to 0.7%. The diploidy rate was also slightly increased to approximately 1% in the translocation carrier.     

A High-Resolution Interval Map of the q21 Region of the Human X Chromosome

In a previous study, we have developed a panel of chromosomal rearrangements for the physical mapping of the q13-q21 region of the human X chromosome (Philippe et al., Genomics 17: 147-152, 1993). Here, we report the physical localization of 36 additional polymorphic markers by polymerase chain reaction analysis. The high density of chromosomal breakpoints in Xq21 allows us to map 58 DNA loci in 22 intervals. As a result, this segment of the X chromosome is saturated with approximately three sequence tagged sites per megabase of DNA, which will facilitate the construction of a YAC contig of this region.     

Ultrastructure of proteoglycans in the specific granules of guinea-pig basophilic leukocytes as demonstrated by cuprolinic blue staining

The ultrastructure of sulphate proteoglycans in basophil granules was examined using cytochemical procedures designed to stabilize and visualize these highly anionic macromolecules in situ. Unfixed or glutaraldehyde-prefixed guinea-pig spleen cells were submitted to fixation/staining in 2.5% glutaraldehyde, 0.2% cuprolinic blue (CB; a cationic phthalocyanin dye) and 0.2 or 0.3M MgCl₂ with or without glycosidase treatments. Abundant electron-dense precipitates were present throughout the granule matrix. The stained structures were often arranged in a quasi-crystalline typical banded pattern. Negative control basophils had no electrondense precipitates. Digestion with chondroitinase ABC destroyed the CB-positive electron-dense banded or filamentous patterns while sialidase treatment did not, but led to larger CB-positive filaments in the cytoplasm near the granules. Taking into account their high anionicity, as shown by the stability of dye binding in the presence of 0.3M MgCl₂, and their susceptibility to chondroitinase ABC, the CB-precipitates are assumed to be related to the sulphated proteoglycans previously characterized in basophil granules. The CB-positive crystalline or filamentous network of the granule matrix is also assumed to reflect the in situ location and organization of these intracellular proteoglycans and may be involved in maintaining the shape of the granule.     

Verification of 14C and O2 derived primary organic production measurements using an enclosed ecosystem

The stimulus for these experiments came from a recent seriesof papers which have suggested that the ¹⁴C technique may underestimateprimary production by as much as 10-fold. We evolved the followingstrategy to attempt to verify the ¹⁴C technique in nearshorewaters: (i) to examine the validity of in vitro (i.e., bottleincubation) measurements by comparing observed in situ oxygenchanges in a large enclosed natural ecosystem against thosedetermined in vitro and if no evidence of containment effectswere indicated, (ii) compare ¹⁴C and oxygen measurements insimultaneous in vitro incubations. The first step essentiallytests the containment problem, the second the physiologicaland calibration problems of the ¹⁴C technique. The first experimentwas run with nitrate as the main source of inorganic nitrogen,the second with ammonia. PQs for converting the ¹⁴C measurementsto oxygen values were calculated from the equation PQ = PQ_c+ 2/(C/NO₃) where PQ_c is the ‘carbon PQ’ (takenas 1.25) and (C/NO₃) is the molar carbon to nitrate assimilationratio. Although there appear to be some minor residual problemsin the interpretation of the data when nitrate was the dominantnitrogen source, the overriding conclusions were: first, thatthe close agreement between the changes in in situ and in vitrodissolved oxygen concentration during the photoperiod gave noevidence for any notable containment effect upon photosynthesis.Secondly, the in vitro rates of ¹⁴CO₂-determined photosyntheticproduction and gross photosynthetic oxygen production agreed,within the precision of the two techniques. The experiment furtherdemonstrated the need to determine soluble as well as particulateorganic production and to pay attention to the potential effectof the nitrogen nutrient upon the PQ. Thus it was concludedthat our data give no evidence for marked errors in the ¹⁴C-techniqueof measuring primary organic production for coastal waters. ^{Present address: Department of Marine Microbiology, Instituteof Botany, University of Gothenburg, Carl Skottsbergs Gata 22,S-413 19 Gothenburg, Sweden.}     

Fertilization and early development in Beroe ovata

Fertilization in the clear egg (1 mm in diameter) of the ctenophore Beroe ovata and, in particular, the positioning and movements of pronuclei, and their relationship to the larval oral-aboral axis have been observed. Fertilization can take place anywhere on the egg surface. The sperm pronucleus remains at its entry site and becomes surrounded by a specialized zone (30–50 μm in diameter) beneath the surface referred to as the sperm pronuclear zone or SPZ and devoid of large cortical granules. Polyspermy has been observed to be frequent; each pronucleus is surrounded by its own SPZ. Only the egg pronucleus migrates with a continuous velocity (averaging 18 μm/min) and moves beneath the surface directly toward the immobile sperm pronucleus. In polyspermic eggs, the egg pronucleus can probe several SPZ, each containing a single sperm nucleus, before it finally enters one SPZ and fuses with the chosen sperm pronucleus. These migrations of the egg pronucleus occur over several millimeters and take hours, but the mechanism underlying the motion or how the egg pronucleus decides which SPZ to enter is not yet known. Under our experimental conditions the mitotic apparatus and the first cleavage plane which defines the oral-aboral axis of the larva (see Reverberi (1971). “Experimental Embryology of Marine and Fresh-Water Invertebrates.” North-Holland, Amsterdam. for review) pass through the point of sperm entry. During fertilization and cleavage, movements of a cortical autofluorescent material are clearly seen. This material is segregated into micromeres as cleavage progresses.     

Calmodulin during development and metamorphosis in urodelan amphibians

Calmodulin isolated and purified to homogeneity from young larvae is very similar to that obtained from adult Pleurodeles waltlii and these proteins are almost identical to previously described vertebrate calmodulins. During P. waltlii development, an increase in total individual calmodulin content is observed after the heart beating stage. In dorsal axial muscle, calmodulin level which is very high at the beginning of larval life (premetamorphosis) decreases strikingly in the first part of prometamorphosis. Such an evolution is observed in Ambystoma mexicanum too. Then, a significant increase occurs during metamorphosis. In contrast, calmodulin level in P. waltlii cardiac ventricular muscle increases continuously from hatching to the end of metamorphic climax. Thyroxine treatment which promotes precocious metamorphosis in P. waltlii and experimental metamorphosis in neotenic A. mexicanum, induces a rapid and significant increase in muscle calmodulin concentration.     

A new approach for using cofactor dependent enzymes: example of alcohol dehydrogenase

The use of enzymes requiring a cofactor as substrate in organic synthesis is still a problem since the cofactors are expensive. This study deals with a new approach consisting of using fragments of NAD+. Three fragments of NAD(H) are examined. The activities of NMN+ and NMNH are greatly improved by the addition of adenosine in ethanol oxidation and in cyclohexanone reduction, respectively. Nicotinamide mononucleoside is not active in the ethanol oxidation but the addition of AMP promotes this reaction.     

Peptidase N of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa possesses a peptidase N activity analogous to those described in Escherichia coli and Salmonella typhimurium . This activity resides in a protein with an M _r value of 85 000. Part of this peptidase activity appears to be associated with the cytoplasmic membrane. The K _M value for this peptidase bound to the cytoplasmic membrane is in the range of 0.5 mM.