Insights into class D beta-lactamases are revealed by the crystal structure of the OXA10 enzyme from Pseudomonas aeruginosa.

BACKGROUND: beta-lactam antibiotic therapies are commonly challenged by the hydrolytic activities of beta-lactamases in bacteria. These enzymes have been grouped into four classes: A, B, C, and D. Class B beta-lactamases are zinc dependent, and enzymes of classes A, C, and D are transiently acylated on a serine residue in the course of the turnover chemistry. While class A and C beta-lactamases have been extensively characterized by biochemical and structural methods, class D enzymes remain the least studied despite their increasing importance in the clinic. RESULTS: The crystal structure of the OXA10 class D beta-lactamase has been solved to 1.66 A resolution from a gold derivative and MAD phasing. This structure reveals that beta-lactamases from classes D and A, despite very poor sequence similarity, share a similar overall fold. An additional beta strand in OXA10 mediates the association into dimers characterized by analytical ultracentrifugation. Major differences are found when comparing the molecular details of the active site of this class D enzyme to the corresponding regions in class A and C beta-lactamases. In the native structure of the OXA10 enzyme solved to 1.8 A, Lys-70 is carbamylated. CONCLUSIONS: Several features were revealed by this study: the dimeric structure of the OXA10 beta-lactamase, an extension of the substrate binding site which suggests that class D enzymes may bind other substrates beside beta-lactams, and carbamylation of the active site Lys-70 residue. The CO2-dependent activity of the OXA10 enzyme and the kinetic properties of the natural OXA17 mutant protein suggest possible relationships between carbamylation, inhibition of the enzyme by anions, and biphasic behavior of the enzyme.

The molecular puzzle of two-component signaling cascades.

Two-component systems constitute prevalent signaling pathways in bacteria and mediate a large variety of adaptative cellular responses. Signaling proceeds through His-Asp phosphorelay cascades that involve two central partners, the histidine protein kinase and the response regulator protein. Structural studies have provided insights into some design principles and activation mechanisms of these multi-domain proteins implicated in the control of virulence gene expression in several pathogens.

Maturation of peripheral nerves in preterm infants: proprioceptive and motor nerve conductions of tibial nerve.

Premature birth induces a profound change in the environmental factors affecting nerve maturation. The proprioceptive sensory and motor nerve conduction velocities (NCV) of the posterior tibial nerve, which reflect peripheral nerve maturation, have been measured in 3 groups of newborns. Two groups of premature (PT) babies, studied when they reached the expected date of birth (group I, gestational age (GA) at birth, 28-31 weeks, n = 8; group II, GA at birth, 32-35 weeks, n = 6) were compared to 9 normal full-term (FT) newborns. As previously shown, the motor NCV of PT babies at a post-conceptional age close to term is similar to that of FT newborns: group I, 22.70 +/- 2.95 m/s (mean +/- SD); group II, 25.90 +/- 4.61 m/s; FT, 25.48 +/- 4.09 m/s. The proprioceptive sensory NCV was significantly lower in group I (21.59 +/- 4.39 m/s) than in group II (31.89 +/- 4.15 m/s) and FT newborns (32.22 +/- 6.56 m/s) (p less than 0.01). Such a delay in maturation could be responsible for the subtle clinical dysfunctions often observed in PT babies.

[Somatosensory evoked potential of the lower extremity in the premature neonate]. / Potentiel évoqué somesthésique du membre inférieur chez le nouveau-né prématuré.

The morphology of SEP is a good index of cortical maturation. Cerebral SEP is elicited by stimulation of the posterior tibial nerve at 38 wk post-conceptional age in 37 neonates distributed in 3 groups: I = gestational age (GA) at birth less than 31 wk; II = GA 23-36 wk; III = full-term neonates (FTN). A somatosensory response was identified in only 21 cases (56.8%). The presence of SEP in 3 groups, ie I, II, III, was 45.5%, 53.8% and 69.2% respectively. The morphological characteristics studied (latencies, amplitude and rising time) in these neonates were different: the peak latencies of major positive wave (P1) and rising time were not significantly different between I and III. These parameters were statistically different between II and III. SEP development is linked to some morphological factors-height, and growth-but also to the maturation of the central nervous system (myelinisation and central pathway organization). These results indicate that until 38-39 wk post-conceptional age, the maturation of the central somatosensory pathway is variable at different birth periods; this suggest the possible role of extra-uterine factors of in the acceleration of neurological development.

6-(hydroxyalkyl)penicillanates as probes for mechanisms of beta-lactamases.

6-(Hydroxyalkyl)penicillanates have proven helpful as probes for the mechanisms of beta-lactamases, enzymes of resistance for beta-lactam antibiotics. The present report summarizes the concepts on design, syntheses and use of these molecules in mechanistic studies of beta-lactamases.

Drop foot after high tibial osteotomy: a prospective study of aetiological factors.

Drop foot is not uncommon after high tibial osteotomy for genu varum. The authors report their results of a prospective study of 16 patients operated on between May 1990 and May 1991. All patients had medial femoro-tibial osteoarthritis with a constitutional genu varum. They all had a subtraction valgus high tibial osteotomy fixed by a blade plate. The experimental protocol included clinical review, antero-lateral compartment pressure measurements, intra- and post-operative electromyography, assessment of the post-operative drainage, serum estimation of muscle enzymes and post-operative arteriography. From their own results and a literature review, the authors consider successively the different aetiological factors for post-operative drop foot. Certain deficits occur due to direct trauma on the nerve during high osteotomy of the fibula, by local high pressure due to poor haemostasis or ineffective drainage. In addition, there are several related phenomena. The pneumatic tourniquet sensitises the nerve to trauma, and stretching of the nerve during correction of the deformation depends on the local anatomical factors and their marked variation. In order to diminish the frequency of these post-operative complications, the authors suggest limiting the surgical approach, and limiting as far as possible the traumatic manoeuvres on the nerve by using a tibial resection jig, which allows correction without forced manoeuvres. Finally, the authors discuss the benefits of using a pneumatic tourniquet.

Value of somatosensory evoked potentials in saphenous entrapment neuropathy.

Neuralgia of the saphenous nerve (SN) is a rare clinical syndrome simulating a vascular disorder of the lower extremities. In four cases, the presenting complaint was persistent pain on the medial aspect of the knee. Examination revealed tenderness over the site of exit of the SN form the femoral canal. Femoral nerve motor conduction, quadriceps H-reflex and EMG of the leg muscles were normal. The sensory nerve action potential of the SN in the leg was not obtained in some patients, even in the unaffected leg. SEP were therefore preferred for diagnosis and performed at the infrapatellar and descending branches of the right and left SN and recordings from the Cz'-Fz electrode. Latency and amplitude differences were evaluated and compared with a control group of healthy subjects. An alteration in the SEP from one branch was observed on the painful side. Posterior tibial responses were normal. In one case, pain resolved immediately after neurolysis, confirming SN entrapment above the femoral canal, before its division. Pain resolved in two other cases and persisted in the last after medical treatment. SEP studies are valuable in the diagnosis of an isolated lesion of the SN.

The high resolution crystal structure for class A beta-lactamase PER-1 reveals the bases for its increase in breadth of activity.

The treatment of infectious diseases by beta-lactam antibiotics is continuously challenged by the emergence and dissemination of new beta-lactamases. In most cases, the cephalosporinase activity of class A enzymes results from a few mutations in the TEM and SHV penicillinases. The PER-1 beta-lactamase was characterized as a class A enzyme displaying a cephalosporinase activity. This activity was, however, insensitive to the mutations of residues known to be critical for providing extended substrate profiles to TEM and SHV. The x-ray structure of the protein, solved at 1.9-A resolution, reveals that two of the most conserved features in class A beta-lactamases are not present in this enzyme: the fold of the Omega-loop and the cis conformation of the peptide bond between residues 166 and 167. The new fold of the Omega-loop and the insertion of four residues at the edge of strand S3 generate a broad cavity that may easily accommodate the bulky substituents of cephalosporin substrates. The trans conformation of the 166-167 bond is related to the presence of an aspartic acid at position 136. Selection of class A enzymes based on the occurrence of both Asp(136) and Asn(179) identifies a subgroup of enzymes with high sequence homology.

Maturation of peripheral nerves in preterm infants. Motor and proprioceptive nerve conduction.

The peripheral nerve maturation (proprioceptive and motor nerve conduction velocities (PNCV and MNCV] was studied in 3 groups of newborn babies. Two groups of premature babies (PT), studied when they reached the expected date of birth (group I, gestational age (GA) at birth 27-31 weeks, n = 13, group II, GA at birth 32-35 weeks, n = 9), were compared to 10 normal full-term newborns (FT). The MNCV of PT babies was similar to that of FT babies: group I 22.8 +/- 3.3 m/sec (X +/- S.D.), group II 24.9 +/- 4.3 m/sec, FT 25.7 +/- 3.9 m/sec. PNCV was significantly lower in group I (18.1 +/- 5.9 m/sec) than in group II (28.3 +/- 6.4 m/sec) and in FT babies (32.0 +/- 7.4 m/sec) (P less than 0.001). Such a delay in maturation could be partly responsible for the neurological impairment often observed in PT babies.