Production of staphylococcal enterotoxins C1 and C2 and thermonuclease throughout the growth cycle

Synthesis of enterotoxins C1 and C2 and thermonuclease throughout the growth cycle was investigated with Staphylococcus aureus type strains FRI137 and FRI361 and S. aureus isolates M5 (C1) and L2 (C2) of animal origin. Both enterotoxins were produced during the exponential growth phase or at the beginning of the stationary phase. The minimal incubation time (7 to 12 h) and the lowest population (10(7) to 2 x 10(9) CFU/ml) associated with detectable enterotoxin (1 to 6.5 ng/ml) were related to the total amount of toxin produced after 24 h. Thermonuclease was detected in all samples whenever enterotoxins were detected. Furthermore, strain FRI137 produced thermonuclease earlier and at lower cell populations than it did enterotoxin C1. Patterns of enterotoxin and thermonuclease synthesis did not correlate. The concentration of toxins increased throughout the growth cycle, while the concentration of thermonuclease remained constant during the last hours of the growth cycle.     

A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis.

Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal-ion-directed site-specificity of hydroxyl radical detection.

A wide variety of .OH detectors are in use for determination of biological .OH production. The chemical generation of .OH is site-specific with respect to the metal-binding site, and thus .OH detectors with metal-binding properties may affect the biological damage and bias .OH detection. The present study shows that both salicylate and phenylalanine, added as low molecular weight .OH indicators, decreased Cu(II) binding to erythrocyte ghosts. In a cell-free system, Cu(II) complexed to both salicylate and phenylalanine. Phenylalanine is a stronger Cu(II) chelator than salicylate, both when competing for Cu(II) bound to ghosts and when competing directly with each other. When OH radicals were generated by ascorbate and Cu(II), the amount of .OH detected as dihydroxybenzoates was proportional to the amount of .OH produced. However, when phenylalanine was added to this system, the efficiency of .OH detection by salicylate strongly decreased, concomitant with the transfer of Cu(II) binding from salicylate to the amino acid. This decrease was larger than that predicted by calculations for random competition of the two detectors for .OH. Deoxyribose and mannitol, which do not bind copper appreciably, competed poorly with salicylate for the .OH. Hydroxylation of phenylalanine, on the other hand, was only slightly affected by the presence of salicylate and unaffected by deoxyribose and mannitol. These results suggest that the detection of .OH by low molecular weight .OH indicators was related to the relative affinity of the detectors for the catalyzing metal, and thus partially site-specific. Furthermore, glutamate, which does not contain an aromatic ring but binds Cu(II) with considerable affinity, competed strongly with salicylate for the .OH, indicating that metal-binding properties rather than the presence of an aromatic ring were the cause of the deviation from random competition. The results indicate that .OH indicators with metal-binding properties affect the distribution of catalytic metal ions in a biological system, causing a shift of free radical damage and localizing a site-specific reaction of .OH on these detectors, with a resulting positive bias in the apparent .OH production.     

One step shoot tip multiplication and rooting of Digitalis thapsi L.

Shoot tips from seedlings of Digitalis thapsi L. were cultured on Murashige and Skoog's medium and the effect of various auxins (2,4-D, NAA and IAA) were analyzed alone or in combination with cytokinis (BA and kinetin). Shoot multiplication and direct rooting of the new shoots were obtained after four weeks of culture in MS medium without hormones, but callus formation and the appearance of abnormal phenotypes were frequent. The addition of auxins to the cultures prevented the formation of callus but not the appearance of variant phenotypes. Both drawbacks could be avoided by combination of NAA or IAA with BA or kinetin. The best results for shoot multiplication and direct rooting were obtained with 0.5 mg l^-1 NAA and 0.1 or 0.5 mg l^-1 kinetin.Abbreviations BA 6-benciladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - NAA naphtalene acetic acid - MS Murashige and Skoog     

Genetic variation of recent Alu insertions in human populations

The Alu family of intersperesed repeats is comprised of ovr 500,000 members which may be divided into discrete subfamilies based upon mutations held in common between members. Distinct subfamilies of Alu sequences have amplified within the human genome in recent evolutionary history. Several individual Alu family members have amplified so recently in human evolution that they are variable as to presence and absence at specific loci within different human populations. Here, we report on the distribution of six polymorphic Alu insetions in a survey of 563 individuals from 14 human population groups across several continents. Our results indicate that these polymorphic Alu insertions probably have an African origin and that there is a much smaller amount of genetic variation between European populations than that found between other populations groups. Present address: Department of Pathology, Stanley S. Scott Cancer Center, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112 Correspondence to: M.A. Batzer     

CORRELATED BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES IN NITROGEN-STARVED EUGLENA GRACILIS1

Growth of Euglena gracilis Z Pringsheim under photoheterotrophic conditions in a nitrogen-deprived medium resulted in progressive loss of chloroplastic material until total bleaching of the cells occurred. Biochemical analysis and ultrastructural observation of the first stages of the starvation process demonstrated an early lag phase (from 0 to 9 h) in which cells increased in size, followed by a period of cell division, apparently supported by the mobilization of some chloroplastic proteins such as the photosynthetic CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. The degradation of the enzyme started after 9 h of starvation and was preceded by a transient concentration of this protein in pyrenoidal structures. Protein nitrogen and photosynthetic pigments as well as number of chloroplasts per cell decreased during proliferation through mere distribution among daughter cells. However, after 24 h, when cell division had almost ceased, there was a slow but steady decline of photosynthetic pigments. This was paralleled by observable ultrastructural changes including progressive loss of chloroplast structure and accumulation of paramylon granules and lipid globules in the cytoplasm. These findings reinforce the role of chloroplastic materials as a nitrogen source during starvation of E. gracilis in a carbon-rich medium. The excess of ribulose-1,5-bisphosphate carboxylase/oxygenase acts as a first reservoir that, once exhausted, is superseded by the generalized disassembly of the photosynthetic structures, if the adverse environment persists more than 24 h.     

Brucella-Salmonella lipopolysaccharide chimeras are less permeable to hydrophobic probes and more sensitive to cationic peptides and EDTA than are their native Brucella sp. counterparts.

A rough (R) Brucella abortus 45/20 mutant was more sensitive to the bactericidal activity of polymyxin B and lactoferricin B than was its smooth (S) counterpart but considerably more resistant than Salmonella montevideo. The outer membrane (OM) and isolated lipopolysaccharide (LPS) of S. montevideo showed a higher affinity for these cationic peptides than did the corresponding B. abortus OM and LPS. We took advantage of the moderate sensitivity of R B. abortus to cationic peptides to construct live R B. abortus-S-LPS chimeras to test the activities of polymyxin B, lactoferricin B, and EDTA. Homogeneous and abundant peripheral distribution of the heterologous S-LPS was observed on the surface of the chimeras, and this coating had no effect on the viability or morphology of the cells. When the heterologous LPS corresponded to the less sensitive bacterium S B. abortus S19, the chimeras were more resistant to cationic peptides; in contrast, when the S-LPS was from the more sensitive bacterium S. montevideo, the chimeras were more susceptible to the action of peptides and EDTA. A direct correlation between the amount of heterologous S-LPS on the surface of chimeric Brucella cells and peptide sensitivity was observed. Whereas the damage produced by polymyxin B in S. montevideo and B. abortus-S. montevideo S-LPS chimeras was manifested mainly as OM blebbing and inner membrane rolling, lactoferricin B caused inner membrane detachment, vacuolization, and the formation of internal electron-dense granules in these cells. Native S and R B. abortus strains were permeable to the hydrophobic probe N-phenyl-1-naphthylamine (NPN). In contrast, only reduced amounts of NPN partitioned into the OMs of the S. montevideo and B. abortus-S. montevideo S-LPS chimeras. Following peptide exposure, accelerated NPN uptake similar to that observed for S. montevideo was detected for the B. abortus-S. montevideo LPS chimeras. The partition of NPN into native or EDTA-, polymyxin B-, or lactoferricin B-treated LPS micelles of S. montevideo or B. abortus mimicked the effects observed with intact cells, and this was confirmed by using micelle hybrids of B. abortus and S. montevideo LPSs. The results showed that LPS is the main cause of B. abortus' resistance to bactericidal cationic peptides, the OM-disturbing action of divalent cationic chelants, and OM permeability to hydrophobic substances. It is proposed that these three features are related to the ability of Brucella bacteria to multiply within phagocytes.     

A microtitre plate-based assay for the screening of β-lactams

A.R. QUESADA, A. CAÑEDO, M.A. MORENO AND J.L. FERNÁNDEZ-PUENTES. 1996. A simple, rapid, sensitive and automatizable method for the detection and quantification of bacterial cell wall inhibitors has been developed. The procedure is characterized by the use of a micro-organism hypersensitive to β-lactam antibiotics that contains an inducible cytosolic β-galactosidase; this enzyme is released when the micro-organism cell wall is disrupted by the antibiotic action, and then measured by the use of a chromogenic substrate. The present method allows the detection of β-lactam traces in other non-β-lactam antibiotics, and has been successfully applied in the detection of small amounts of β-lactams in biological fluids such as milk and Actinomycetes fermentation broths. The easy automatization of this method makes it specially suitable for the screening of new antibiotics of natural origin.