Obstetric aspects in women with facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, and congenital myopathies.

OBJECTIVE: To increase the knowledge about pregnancy and delivery in women with certain muscle diseases, which is important for obstetric management and family planning of affected women. DESIGN: The obstetric histories of patients with facioscapulohumeral (FSH) muscular dystrophy, limb-girdle (LG) muscular dystrophy, and congenital myopathies (CM) were retrospectively evaluated using questionnaires and medical reports. PATIENTS: The condition of 27 patients with different myopathies (FSH muscular dystrophy, n = 11; LG muscular dystrophy, n = 9; and CM, n = 7 [subdivided into 5 patients with central core disease, 1 patient with cytoplasmic bodies, and 1 patient with unspecified myopathy]) were ascertained from January 1, 1992, to December 31, 1994, through departments of neurology and human genetics, and the German self-support group for muscle diseases. Fifty-eight gestations resulting in 52 live births were reviewed. RESULTS: Miscarriages were reported in 3 of 26 gestations in 11 patients with FSH dystrophy, whereas 3 of 15 pregnancies in patients with LG dystrophy were terminated. Preterm births occurred in 2 patients with FSH dystrophy and in 3 patients with CM. Operative deliveries (vaginal operation or cesarean section) were performed in 6 of 23 gestations in patients with FSH dystrophy (1 emergency section), in 5 of 12 patients with LG dystrophy (2 emergency sections), and in 3 of 17 deliveries in patients with CM. Patients with FSH dystrophy generally coped well with their muscle disease in pregnancy and after delivery; however, 4 women were stated to have difficulties in caring for their families. The situation differed in LG dystrophy, where most women (5 of 9) experienced worsening of weakness in pregnancy and required assistance after delivery. In the patients with CM, 3 women experienced a deterioration during pregnancy, and 4 patients reported difficulties after delivery. CONCLUSIONS: No deleterious outcome of pregnancy and labor was observed in this series of patients with muscular dystrophy or CM, although operative deliveries were more frequent. A significant aggravation of symptoms in gestation is more likely to occur in patients with early-onset and progressive myopathy than in those with stable disease courses.

Lytic effect of two fluoroquinolones, ofloxacin and pefloxacin, on Escherichia coli W7 and its consequences on peptidoglycan composition.

Examination of biochemical changes in Escherichia coli W7 after exposure to ofloxacin or pefloxacin revealed distinct concentration-dependent responses. At levels close to the MIC, extensive filamentation was followed by a lytic event, which involved an active protein synthesis. This lysis was correlated with changes in the peptidoglycan composition, particularly a decrease in the average glycan chain length, involving the action of the autolysines. At higher concentrations, no lysis occurred and the growth was totally inhibited as well as the protein synthesis. The peptidoglycan composition exhibited an increase in the average glycan chain length, suggesting an apparent reduced activity of the lytic transglycosylase. These results show that exposure to low concentrations of quinolones leads to the induction of lysis and peptidoglycan modifications which might contribute to the bactericidal effects of quinolones.

Growth pattern of the murein sacculus of Escherichia coli.

The mechanism by which the murein sacculus of Escherichia coli is being enlarged during growth was investigated by pulse and pulse-chase labeling with [3H]diaminopimelic acid. Changes in the composition of the sacculus during aging were analyzed in detail by high performance liquid chromatography separation of the muropeptide subunits released after complete muramidase digestion. After pulses as short as 10 s, a group of novel phosphorylated muropeptides was detected. The kinetics of their appearance is consistent with these structures being derived from the undecaprenylphosphate-linked growing points of murein. A complex maturation process of murein took place including a rapid decay of pentapeptide side chains and a 10-fold increase in tripeptidyl moieties. In addition, the total degree of cross-linkage increased from 16 to 25%, partly due to a 3-fold increase in the formation of LD-A2pm-A2pm cross-links. In pulse-chase experiments the cross-linkage started to decrease after a maximum at about 35 min of chase. The kinetics in the distribution of the radioactivity among acceptor and donor part in the major cross-bridges Tetra-Tetra and Tetra-Tri differed from each other substantially, indicating that the latter structure is completely cleaved within three generations, whereas only 40% of Tetra-Tetra is cleaved during the same time. Furthermore, the attachment of the lipoprotein to murein was delayed by about one generation. It is proposed that these findings reflect an inside-to-outside growth mechanism of the murein sacculus of E. coli.

Separation and quantification of muropeptides with high-performance liquid chromatography.

About 80 different muropeptides, the subunits which comprise the polymer murein of Escherichia coli, were resolved by high-performance liquid chromatography. The muropeptides were released from isolated murein by complete digestion with muramidase from Chalaropsis spec. The separation method is based on reversed phase chromatography of the sodium borohydride-reduced compounds using ODS (C18) columns and a linear gradient elution with sodium phosphate buffer and methanol as organic modifier. The effect of temperature, pH, ionic strength, and the steepness of the gradient and of different support materials on the separation of the muropeptides was investigated. The new method represents a major improvement over previous methods with respect to resolution, sensitivity, and speed. Analytical as well as preparative separations can be realized. Quantitative analysis of murein composition is achieved by a linear gradient from 50 mM sodium phosphate, pH 4.31, to 75 mM sodium phosphate, pH 4.95, containing 15% methanol for 135 min on a 250 X 4.6 mm 3-micron Hypersil ODS column at 55 degrees C using a flow rate of 0.5 ml/min. With uv detection at 205 nm about 20 micrograms of murein per analysis is sufficient. The detection limit per compound is about 5 ng. A method for the evaluation of the analytical data allowing a convenient comparison of different muropeptide pattern is described.

The composition of the murein of Escherichia coli.

Escherichia coli murein, the polymer from which the shape-maintaining structure of the cell envelope is made, shows unexpected complexity. The separation of murein building blocks with high performance liquid chromatography reveals about 80 different types of muropeptides. Their behavior in high performance liquid chromatography and their chemical structure are described. The complexity of E. coli murein is due to the free combination of seven different types of side chains (L-Ala-D-Glu-R with R = -OH, -m-A2pm, -m-A2pm-D-Ala, -m-A2 pm-Gly, -m-A2pm-D-Ala-D-Ala, -m-A2pm-D-Ala-Gly, -m-A2pm-Lys-Arg) with two types of cross-bridges (D-Ala-m-A2pm, -m-A2pm-m-A2pm). The novel type of cross-bridge, A2pm-A2pm, contains an L,D-peptide bond, as shown by Edman degradation and chemical analysis of the reaction products. The A2pm-A2pm cross-bridge is assumed to play a role in the adaptation of the cross-linkage of murein to different growth conditions of the cell. The structural data of E. coli murein agree best with a model of a thin, however multilayered, murein sacculus.

Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope.

Cell envelopes of Salmonella typhimurium and Escherichia coli were disrupted in a French pressure cell and fractionated by successive cycles of sedimentation and floatation density gradient centrifugation. This permitted the identification and isolation of several membrane fractions in addition to the major inner membrane and murein-outer membrane fractions. One of these fractions (fraction OML) accounted for about 10% of the total cell envelope protein, and is likely to include the murein-membrane adhesion zones that are seen in electron micrographs of plasmolyzed cells. Fraction OML contained inner membrane, murein, and outer membrane in an apparently normal configuration, was capable of synthesizing murein from UDP-[3H]N-acetylglucosamine and UDP-N-acetylmuramylpentapeptide and covalently linking it to the endogenous murein of the preparation, and showed a labeling pattern in [3H]galactose pulse-chase experiments that was consistent with its acting as an intermediate in the movement of newly synthesized lipopolysaccharide from inner membrane to outer membrane. The fractionation procedure also identified two new minor membrane fractions, with characteristic protein patterns, that are usually included in the region of the major inner membrane peak in other fractionation procedures but can be separated from the major inner membrane fraction and from contaminating flagellar fragments by the subsequent floatation centrifugation steps.

UDP-N-acetylmuramylpentapeptide as acceptor in murein biosynthesis in Escherichia coli membranes and ether-permeabilized cells.

Two widely used in vitro systems of Escherichia coli capable of synthesizing murein were evaluated by using high-pressure liquid chromatography for murein analysis. Comparison of the composition of murein synthesized by either a membrane preparation or ether-treated cells with native murein revealed that both in vitro systems failed to synthesize murein that was identical to murein formed in vivo. Furthermore, neither system attached the lipoprotein to the murein. Ether-treated cells, however, were superior to the membrane preparation in catalyzing the formation of the remarkable A2pm-A2pm cross-linkage. In both systems an atypical transpeptidation reaction was found to take place in which exogenously supplied UDP-N-acetylmuramylpentapeptide was directly linked to the murein without participation of the bactoprenol lipid carrier. The direct transpeptidation yields preferentially trimeric peptide bridges with the UDP-linked muramylpentapeptide serving as the acceptor.

Sequences of the active-site peptides of three of the high-Mr penicillin-binding proteins of Escherichia coli K-12.

The amino acid compositions of the radioactive peptides obtained from trypsin digestion of [14C]benzylpenicillin-labeled penicillin-binding proteins (PBPs) 1A, 1B, and 3 of Escherichia coli have been obtained. Complete digestion of these peptides with a combination of aminopeptidase M and carboxypeptidase Y showed that benzylpenicillin was bound to a serine residue in each of these proteins. Comparison of the compositions of the penicillin-labeled peptides with the complete amino acid sequences of PBPs 1A, 1B, and 3 showed that the acylated serine occurs near the middle of each of the proteins, within the conserved sequence Gly-Ser-Xaa-Xaa-Lys-Pro. The sequence around the acylated serine of these high Mr PBPs shows little similarity to that around the acylated serine of the low-Mr PBPs (D-alanine carboxypeptidases) or of the class A or class C beta-lactamases, except that in all of these enzymes which interact with penicillin the acylated serine residue occurs within the sequence Ser-Xaa-Xaa-Lys.

Murein structure and lack of DD- and LD-carboxypeptidase activities in Caulobacter crescentus.

High-pressure liquid chromatography of a muramidase digest of murein sacculi from Caulobacter crescentus showed that the absence of D-alanine carboxypeptidase activity in the cells was reflected by a very high content of pentapeptide in the murein. Approximately half of the pentapeptide side chains were shown to contain glycine, which replaced D-alanine as the terminal amino acid.