Malformation uropathies and multiple malformation syndromes

The authors, from their experience emphasize the associated malformations' frequency in major congenital urinary tract malformations (26,9%). It is essential to recognize in these multiple defects some certified syndromes - inherited or not. The most associations are still unknown, nevertheless the genetic counselling require an accurate diagnosis.     

An estimate on the effect of point mutation and natural selection on the rate of amino acid replacement in proteins

We outline a method for estimating quantitatively the influence of point mutations and selection on the frequencies of codons and amino acids. We show how the mutation rate, i.e., the rate of amino acid replacement due to point mutation, can be affected by the codon usage as well as by the rates of the involved base exchanges. A comparison of the mutation rates calculated from reliable values of codon usage and base exchange probabilities with those that would be expected on the basis of chance reveals a notable suppression of replacements leading to tryptophan, glutamate, lysine, and methionine, and particularly of those leading to the termination codons. If selection constraints are neglected and only mutations are taken into account, the best agreement between expected and observed frequencies of both codons and amino acids is obtained for alpha = 1.13-1.15, where (Formula: see text). The "selection values" of codons and amino acids derived by our method show a pattern that partially deviates from others in the literature. For example, the selection pressure on methionine and cysteine turns out to be much more pronounced than expected if only the discrepancies between their observed and expected occurrences in proteins are considered. To estimate to what extent randomly occurring amino acid replacements are accepted by selection, we constructed an "acceptability matrix" from the well-established matrix of accepted point mutations. On the basis of this matrix "acceptability values" of the amino acids can be defined that correlate with their selection values. We also examine the significance of mutations and selection of amino acids with respect to their physicochemical properties and functions in proteins. The conservatism of amino acid replacements with respect to certain properties such as polarity can be brought about by the mutational process alone, whereas the conservatism with respect to other relevant properties--among them all measures of bulkiness--obviously is the result of additional selectional constraints on the evolution of protein structures.     

Effect of physical environment on the conformation of ricin. Influence of low pH.

The molecular properties of ricin (the toxic lectin from Ricinus communis seeds, RCA II or RCA 60) were evaluated by analytical ultracentrifugation, viscosimetry, c.d., fluorescence and equilibrium dialysis. Measurements of sedimentation (S0(20,W) = 4.60 S) and viscosity (eta = 2.96 X 10(-2) dl/g) indicated that, at neutral pH, the ricin molecule is very compact. Various transitions were explored, and a pH-triggered change in the ricin conformation was observed between pH 7 and 4. In this range, the sedimentation coefficient, far-u.v. c.d. and fluorescence altered simultaneously without unfolding. Below pH 7 the change in the ricin conformation was accompanied by a decrease in the affinity of ricin for galactosides, and at pH 4.0 by an alteration in its binding capacity. These effects of low pH are discussed in relation to the physical conditions encountered by ricin molecules during their entry into living cells.     

Construction of recombination-deficient strains of Pseudomonas aeruginosa

Summary The rec-102 mutation had pleiotropic effects in Pseudomonas aeruginosa: low recombination proficiency in conjugation and transduction; high UV sensitivity; inability to induce pyocin R2 by mitomycin C; and increased susceptibility to mitomycin C and nalidixic acid. The rec-102 locus was mapped by R68.45-mediated conjugation in the 45 min region of the PAO chromosome, between argF and thr-9001. By selection for a marker in this region, rec-102 can be introduced into a P. aeruginosa strain of interest using an R68.45 rec-102 donor. The recombination-deficient strains constructed in this way were phenotypically similar to Escherichia coli recA mutants.     

Binding of β-lactam antibiotics to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R39

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl₂ buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10⁻⁶, 1.5×10⁻⁶ and 0.63×10⁻⁶s⁻¹ (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [¹⁴C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [¹⁴C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10⁻⁵s⁻¹ and 0.8×10⁻⁴s⁻¹ respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.     

Studies on the biosynthesis of gramicidin S in a cell-free system from Bacillus brevis. Further attempts to elucidate its mechanism of synthesis

1. The pH optima for the incorporation of (14)C-labelled amino acids into gramicidin S by an 11000g cell-free extract from Bacillus brevis have been determined. The pH optima for leucine, proline, phenylalanine, ornithine and valine were 7.5-7.7, 7.5-7.7, 7.7-7.9, 7.7-7.9 and 8.0-8.2 respectively. Hence the greatest difference in pH optima existed between leucine and valine, where it was 0.5pH unit. 2. The 11000g cell-free extract incorporated into gramicidin S only the l-isomers of valine, proline and ornithine. However, both isomers of leucine are utilized and the experiments indicate that a leucine racemase exists in the 11000g cell-free extract. With phenylalanine the l-isomer is utilized much more effectively than the d-isomer. This is noteworthy since it is the d-isomer that occurs in gramicidin S. The experiments indicate that conversion of the l-isomer into the d-form takes place at a stage beyond that of the free amino acid.     

Human monocytes convert leukotriene B4 to two dihydro-leukotriene B4-metabolites

Human monocytes metabolize LTB4 by an additional pathway different from omega-oxidation. Reverse-phase high performance liquid chromatography showed four metabolites: 20-COOH-LTB4, 20-OH-LTB4 and two metabolites less polar than LTB4 with an UV maximum at 232 nm. Gas-chromatography mass-spectrometry showed nearly identical mass spectra for both metabolites. The main mass fragments of the two metabolites were increased by two mass units compared to LTB4. Our findings suggest that LTB4 had been reduced to a known and a new dihydro-metabolite of LTB4. Both metabolites together amounted to 85% of total metabolites. The remaining 15% were omega-oxidation products. Thus, the major pathway of LTB4 metabolism by human monocytes is reduction to dihydro-LTB4.     

Gating in iodate-modified single cardiac Na+ channels

Summary Elementary Na⁺ currents were recorded at 19°C during 220-msec lasting step depolarizations in cell-attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the modifying influence of iodate, bromate and glutaraldehyde on single cardiac Na⁺ channels.Iodate (10 mmol/liter) removed Na⁺ inactivation and caused repetitive, burst-like channel activity after treating the cytoplasmic channel surface. In contrast to normal Na⁺ channels under control conditions, iodate-modified Na⁺ channels attain two conducting states, a short-lasting one with a voltage-independent lifetime close to 1 msec and, likewise tested between –50 and +10 mV, a long-lasting one being apparently exponentially dependent on voltage. Channel modification by bromate (10 mmol/liter) and glutaraldehyde (0.5 mmol/liter) also included the occurrence of two open states. Also, burst duration depended apparently exponentially on voltage and increased when shifting the membrane in the positive direction, but there was no evidence for two bursting states. Chemically modified Na⁺ channels retain an apparently normal unitary conductance (12.8±0.5 pS). Of the two substates observed, one of them is remarkable in that it is mostly attained from full-state openings and is very short living in nature; the voltage-independent lifetime was close to 2 msec. Despite removal of inactivation, open probability progressively declined during membrane depolarization. The underlying deactivation process is strongly voltage sensitive but, in contrast to slow Na⁺ inactivation, responds to a voltage shift in the positive direction with a retardation in kinetics. Chemically modified Na⁺ channels exhibit a characteristic bursting state much shorter than in DPI-modified Na⁺ channels, a difference not consistent with the hypothesis of common kinetic properties in noninactivating Na⁺ channels.