The curious ability of polyethylene glycol fusion technologies to restore lost behaviors after nerve severance.

Traumatic injuries to PNS and CNS axons are not uncommon. Restoration of lost behaviors following severance of mammalian peripheral nerve axons (PNAs) relies on regeneration by slow outgrowths and is typically poor or nonexistent when after ablation or injuries close to the soma. Behavioral recovery after severing spinal tract axons (STAs) is poor because STAs do not naturally regenerate. Current techniques to enhance PNA and/or STA regeneration have had limited success and do not prevent the onset of Wallerian degeneration of severed distal segments. This Review describes the use of a recently developed polyethylene glycol (PEG) fusion technology combining concepts from biochemical engineering, cell biology, and clinical microsurgery. Within minutes after microsuturing carefully trimmed cut ends and applying a well-specified sequence of solutions, PEG-fused axons exhibit morphological continuity (assessed by intra-axonal dye diffusion) and electrophysiological continuity (assessed by conduction of action potentials) across the lesion site. Wallerian degeneration of PEG-fused PNAs is greatly reduced as measured by counts of sensory and/or motor axons and maintenance of axonal diameters and neuromuscular synapses. After PEG-fusion repair, cut-severed, crush-severed, or ablated PNAs or crush-severed STAs rapidly (within days to weeks), more completely, and permanently restore PNA- or STA-mediated behaviors compared with nontreated or conventionally treated animals. PEG-fusion success is enhanced or decreased by applying antioxidants or oxidants, trimming cut ends or stretching axons, and exposure to Ca(2+) -free or Ca(2+) -containing solutions, respectively. PEG-fusion technology employs surgical techniques and chemicals already used by clinicians and has the potential to produce a paradigm shift in the treatment of traumatic injuries to PNAs and STAs.

[Kinetics of beta-lactamase inhibition by clavulanic acid]. / Cinetique de l'inhibition de beta-lactamases par l'acide clavulanique.

The mechanisms of action of 3 R-factors on beta-lactamases (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) (TEM-1 pI = 5.4, TEM-2 pI = 5.6 and Pitton's type 2 pI = 7.7) have been kinetically analyzed for clavulanic acid inactivation. Clavulanic acid appears as a competitive and irreversible inhibitor (Kcat inhibitor) reacting in two steps: a, formation of a reversible enzyme . inhibitor complex (characterized by a Ki); b, evolution of the reversible complex into a new derivative (covalent, stable and inactive) by monomolecular kinetics characterized by a k6 (or Kcat) related to half-life. The kinetic constants are: TEM-1: Ki = 0.8 micrometer, k6 = 0.027 s-1; TEM-2: Ki = 0.7 micrometer, k6 = 0.03 s-1; type 2: Ki = 0.6 micrometer, k6 = 0.046 s-1. These results justify the 'progressive irreversible' character of the inhibition generally described.

Inhibition kinetics of three R-factor-mediated beta-lactamases by a new beta-lactam sulfone (CP 45899).

A new beta-lactam sulfone, CP 45899, has been proved to be a time-dependent irreversible inhibitor of three R-factor-mediated beta-lactamases (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6): TEM-1 (pI = 5.4), TEM-2 (pI = 5.6) and Pitton's type 2 (pI = 7.7). This inhibition occurs in two principal steps: (1) formation of a reversible enzyme-inhibitor complex (characterized by a Ki); (2) evolution of this complex into one, or more, inactive protein(s) (kinact). With the three beta-lactamases CP 45899 shows, respectively, Ki of 0.9, 0.8 and 1.8 microM and kinact of 1.2 . 10(-3), 0.8 . 10(-3) and 1 . 10(-3) s-1; the turnover numbers are: 525, 2280 and 1220. These results are compared to those previously obtained with clavulanic acid.

Close amino acid sequence relationship between the new plasmid-mediated extended-spectrum beta-lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca.

Isolated from an Escherichia coli strain MEN-1 is a plasmid-mediated beta-lactamase that confers resistance to methoxy imino third-generation cephalosporins. The protein purified to homogeneity was digested by trypsin, chymotrypsin and endoproteinase Asp-N. Amino acid sequence determinations of the resulting peptides gave rise to the alignment of the 263 residues of the beta-lactamase. From amino acid sequence comparison MEN-1 was found to share more than 72% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca. Therefore, MEN-1 is the first transferable extended-spectrum beta-lactamase which is not directly derived from the widespread TEMs or SHV-1 penicillinases with which it presents less than 39% identity.

Characterization and amino acid sequence analysis of a new oxyimino cephalosporin-hydrolyzing class A beta-lactamase from Serratia fonticola CUV.

Serratia fonticola CUV produces two isoenzymes (forms I and II) with beta-lactamase activity which were purified by a five-step procedure. The isoenzymes had identical kinetic parameters and isoelectric point (pI = 8.12). They were characterized by a specific activity towards benzylpenicillin of 1650 U/mg. The beta-lactamase hydrolyzed benzylpenicillin, amoxycillin, ureidopenicillins, first- and second-generation cephalosporins. Carboxypenicillins and isoxazolylpenicillins were hydrolyzed to a lesser extent. Towards cefotaxime and ceftriaxone (third-generation cephalosporins), the S. fonticola enzyme exhibited catalytic efficiencies much higher than those of MEN-1 and extended-spectrum TEM derivative beta-lactamases. The beta-lactamase from S. fonticola was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam. The purified isoenzymes were digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino acid sequence determinations of the resulting peptides allowed the alignment of 267 amino acid residues (Swiss-Prot, accession number P 80545) for form I beta-lactamase. Form II is five residues shorter than form I at its N-terminus. From amino acid sequence comparisons, S. fonticola CUV beta-lactamase was found to share more than 69.3% identity with the chromosomally encoded beta-lactamases of Klebsiella oxytoca, Proteus vulgaris, Citrobacter diversus and the plasmid-mediated enzymes MEN-1 and Toho-1. Therefore, the oxyimino cephalosporin-hydrolyzing beta-lactamase of S. fonticola belongs to Ambler's class A. Contribution of the serine at ABL 237 in the broad-spectrum activity of these beta-lactamases is discussed.

Chromosomally encoded cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A.

Proteus vulgaris RO104 strain produces a chromosomally encoded beta-lactamase that confers resistance to various beta-lactam antibiotics including methoxyimino third-generation cephalosporins. The beta-lactamase hydrolyzes first- and second-generation cephalosporins efficiently and cefotaxime to a lesser extent. Catalytic activity is inhibited by low concentrations of clavulanic acid and sulbactam. By its broad-spectrum substrate profile, beta-lactamase of Proteus vulgaris RO104 belongs to the group 2e defined by Bush. The protein purified to homogeneity by a four-step procedure was characterized by a pI of 8.31 and a specific activity of 1200 U/mg. The beta-lactamase was digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino-acid sequence determinations of the resulting peptides allowed the alignment of the 271 amino-acid residues of the protein which did not contain any cysteine residue. From amino-acid sequence comparisons, Proteus vulgaris RO104 beta-lactamase was found to share about 68% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca D488 and E23004. Therefore, the cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A.

Clinical inhibitor-resistant mutants of the beta-lactamase TEM-1 at amino-acid position 69. Kinetic analysis and molecular modelling.

The kinetic parameters of three IRT (Inhibitor-Resistant-TEM-derived-) beta-lactamases (IRT-5, IRT-6 and IRT-I69) were determined for substrates and the beta-lactamase inhibitors: clavulanic acid, sulbactam and tazobactam, and compared with those of TEM-1 beta-lactamase. The catalytic behaviour of the beta-lactamases towards substrates and inhibitors was correlated with the properties of the amino acid at position ABL69. The three IRT beta-lactamases contain at that position a residue Ile, Leu and Val, amino acids whose side-chain are branched. Molecular modelling shows that the methyl groups of Ile-69 (C gamma 2) and Val-69 (C gamma 1) produced steric constraints with the side chain of Asn-170 as well as the main chain nitrogen of Ser-70, a residue contributing to the oxyanion hole. We suggest that hydrophobicity could be the main factor responsible for the kinetic properties of Met69Leu (IRT-5), as no steric effects could be detected by molecular modelling. Hydrophobicity and steric constraints are combined in Met69Ile and Met69Val, IRT-I69 and IRT-6, respectively.

Repetitive intrathecal injections of poliovirus replicons result in gene expression in neurons of the central nervous system without pathogenesis.

Poliovirus-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. In the current study, a replicon encoding green fluorescent protein (GFP) was encapsidated into authentic poliovirions, using established procedures. Intrathecal delivery of encapsidated replicons encoding GFP to the CNS of mice transgenic for the human poliovirus receptor did not result in any functional deficits as judged by behavioral testing. Histological analysis of the CNS of mice given a single intrathecal injection of poliovirus replicons encoding GFP revealed no obvious pathogenesis in neurons (or other cell types) within the CNS. The expression of GFP was confined to motor neurons throughout the neuroaxis; a time course of expression of GFP revealed that expression was detectable 24 hr postinoculation and returned to background levels by 120 hr postinoculation. A procedure was devised to allow repetitive inoculation of replicons within the same animal. Behavioral testing of animals that had received 6 to 13 independent inoculations of replicons revealed no functional deficits. Histological analysis of the CNS from animals that had received 6 to 13 sequential inoculations of replicons revealed no obvious abnormalities in neurons or other cell types in the CNS; expression of GFP was demonstrated in neurons 24 to 72 hr after the final inoculation of the replicon. Furthermore, there was no obvious inflammatory response in the CNS after the multiple inoculations. These studies establish the safety and efficacy of replicons for gene delivery to the CNS and are discussed with respect to use of replicons as new therapeutic strategies for spinal cord injuries and/or neurological diseases.

Single amino acid substitution between SHV-1 beta-lactamase and cefotaxime-hydrolyzing SHV-2 enzyme.

SHV-2 beta-lactamase was purified from an overproducing variant of a clinical isolate of Escherichia coli resistant to cefotaxime. Pure protein was digested by trypsin and Lys-C endoproteinase. Proteolytic peptides, isolated by reverse-phase HPLC, were submitted to manual Edman degradation and aligned by homology with the sequence of SHV-1 beta-lactamase. A putative amino acid sequence was deduced. Structural comparison revealed that SHV-2 differed from SHV-1 by only one amino acid, Gly----Ser, at position 213 of the mature protein.

Structural study of hemoglobin Hazebrouck, beta 38(C4)Thr----Pro. A new abnormal hemoglobin with instability and low oxygen affinity.

A new beta-variant has been detected and structurally defined in a French male, with a life-long history of hemolytic anemia. This variant is moderately unstable and has a low oxygen affinity. The abnormal hemoglobin was not detected by standard electrophoretic procedures. It moved slightly slower than Hb A during isoelectric focusing (IEF). Two minor fractions were also seen; the first migrated just cathodal to Hb F, as did partially oxidized Hb A or hemichrome derivatives of some unstable hemoglobins; the second in the position of free alpha-chains. The abnormal beta-chain was readily separated from both beta A- and alpha A-chains by acid-urea-Triton globin chain electrophoresis. Structural study was conducted simultaneously by fingerprinting and high-performance liquid chromatography (HPLC) of tryptic peptides. A new mutation beta 38(C4)Thr----Pro was found, which was named Hb Hazebrouck.

Reduced effects of monocular deprivation on a population of A-laminae neurons in the cat's dorsal lateral geniculate nucleus.

The generation of nerve cells for the cat's dorsal lateral geniculate nucleus takes place between embryonic day (E) 22 and E32. Neurons generated before E28 exhibit a full range of soma sizes and morphological features, whereas neurons generated after E28 have only smaller somata and a more limited array of morphological features. We measured the effects of monocular deprivation on neurons in the cat's dorsal lateral geniculate nucleus that were generated at different times, with birthdates defined by injections of 3H-thymidine. Whereas populations of nerve cells generated before E28 exhibit changes in cell size that are, on average, typical of those seen in monocularly deprived cats, populations of nerve cells generated after E28 are, on average, less affected by visual deprivation.

Anterograde transneuronal degeneration in the ectomamillary nucleus and ventral lateral geniculate nucleus of the chick.

The effects of anterograde transneuronal atrophy were studied in two visual nuclei of the chick--the ectomamillary nucleus (EMN), which shows marked degenerative changes following enucleation, and the ventral lateral geniculate nucleus (GLv), which shows less severe changes following enucleation. The chicks were enucleated on the day of hatching and killed between 2 and 81 days later. Reconstructions of the EMN and GLv revealed that enucleation retarded the growth of these two nuclei. The volume of the control EMN and GLv, ipsilateral to the removed eye, continued to increase after eye removal. The experimental EMN did not increase in volume during this time while the experimental GLv increased in volume but at a slower rate than the control GLv. The volume of the experimental GLv remained smaller than the control volume. In order to determine whether the volumetric changes were due to arrest of cellular growth or to atrophy of the neurons, a morphometric study was carried out in the two nuclei. Measurements of the cross-sectional area of EMN neurons revealed a 20% decrease in soma area in the experimental EMN in comparison with those in the control EMN. Since neurons in the control EMN did not increase in area after hatching, it was concluded that the changes were due to atrophy rather than arrest of neuron growth. Furthermore, there was a 35% neuron loss in the EMN. The GLv, which is composed of two laminae, consistently showed a greater decrease in soma cross-sectional area and neuron loss in its neuropil lamina (comparable to the transneuronal effects in the EMN) than in its lamina interna. Thus, in both nuclei, eye removal led to neuron loss and a decrease in soma cross-sectional area when compared with the contralateral (control) nucleus.

Morphology of normal and deafferented neurons in the chick ectomamillary nucleus.

Neurons in the ectomamillary nucleus (EMN) undergo both atrophy and cell death following eye removal at hatching. It is not known whether all EMN neurons are affected uniformly by transneuronal atrophy or whether cell loss is an artifact due to misidentification of atrophied neurons as glia. In a preliminary morphological study, four types of neurons were found in the EMN by using the rapid Golgi method: A large multipolar neuron (type I); two medium-sized spindle-shaped neurons, one possessing many dendritic branches (type II) and the other possessing few dendritic branches (type III); and a small round neuron (type IV). Horseradish peroxidase (HRP) was then injected into two of the EMN projection fields in enucleated chicks in order to label retrogradely as many EMN neurons as possible. Types, I, II, and III neurons were identified both in the control and experimental EMN. The three types of backfilled neurons showed different degrees of transneuronal atrophy ranging from 12 to 47%. The type IV neuron, which could not be backfilled, was inferred to atrophy by 33%. Substantial differences in transneuronal atrophy, therefore, exist among the different types of neurons within the same nucleus. Since no glialike neurons could be retrogradely labeled it was concluded that there is a true neuron loss in the EMN following eye removal rather than mistaken identification of neurons as glia.

Birthdates of neurons in the retinal ganglion cell layer of the ferret.

The present study determined the temporal and spatial patterns of genesis for neurons of different sizes in the retinal ganglion cell layer of the ferret. Fetal ferrets were exposed to tritiated thymidine on embryonic days E-22 through E-36. One to 3 months after birth, they were perfused and their retinae dissected, and autoradiographs were prepared from resin-embedded sections throughout the entire flattened retinal ganglion cell layer. Soma size differences in conjunction with separate retrograde labeling and calbindin immunocytochemical studies were used as criteria for identifying different retinal ganglion cell subtypes in juvenile and adult ferrets. Neurons of different sizes in the ganglion cell layer were generated at different stages during development. Medium sized cells were generated primarily between E-22 and E-26; the largest cells were generated between E-24 and E-29; small cells were generated between E-26 and E-32; and very small cells were generated between E-29 and E-36. The former three groups were interpreted to be three subtypes of retinal ganglion cells, while the latter group was interpreted to be displaced amacrine cells. This temporal order of the genesis of ganglion cell classes is consistent with the spatial ordering of their fibers in the mature optic chiasm and tract, and it is consistent with the developmental change in decussation pattern recently shown in the optic pathway of embryonic ferrets. The spatial pattern of genesis suggests that ganglion cells of a particular class are added to the ganglion cell layer in a centroperipheral fashion initiated in the dorsocentral retina nasal to the area centralis. No evidence was found for a wave of ganglion cell addition that proceeded in a spiralling pattern around the area centralis, as has been reported in the cat.

Development of parvalbumin immunoreactivity in the chick Edinger Westphal nucleus.

To determine when the calcium-binding protein parvalbumin appears during development, neurons in the chick Edinger Westphal nucleus were examined for parvalbumin immunoreactivity at a variety of embryonic stages. Parvalbumin immunoreactivity appeared on embryonic day 14 (E14, Hamburger and Hamilton stage 40) in predominantly lateral Edinger Westphal neurons. Cytochrome oxidase activity within the nucleus was examined throughout development, as an indicator of physiological activity, and expression of cytochrome oxidase was compared with that of parvalbumin. Cytochrome oxidase activity was found to be uniformly high in all parts of the Edinger Westphal nucleus throughout development. Either the Edinger Westphal nucleus in physiologically active quite early in its development or other energy demands mask the correlation of cytochrome oxidase with electrical activity. Cytochrome oxidase was expressed well before parvalbumin immunoreactivity appeared. Voltage-activated calcium currents were characterized in E12 Edinger Westphal neurons. In both amplitude and composition, E12 calcium currents resemble those of E16 neurons, excluding the possibility that calcium currents appear de novo during or just prior to the appearance of parvalbumin. Both cytochrome oxidase activity and calcium currents are observed in Edinger Westphal neurons well before the appearance of parvalbumin during development. These findings do not exclude the possibility that physiological activity affects the expression of parvalbumin since other factors such as changing patterns of synaptic activity or the appearance of calcium conducting NMDA receptors have yet to be examined. However, they raise the possibility that additional factors such as an intrinsic developmental program or a change in the neuron's basal intracellular calcium requirements may also be involved.

Cefotaxime-hydrolysing activity of the beta-lactamase of Klebsiella oxytoca D488 could be related to a threonine residue at position 140.

The chromosomally encoded beta-lactamase of Klebsiella oxytoca D483 strain, active against all third-generation cephalosporins but ceftazidime, was purified to homogeneity. The pure protein was digested by trypsin, Staphylococcus aureus V8 protease or proteinase Asp-N. Amino acid sequences of the HPLC-separated proteolytic peptides were determined by manual Edman degradation. Overlapping fragments gave the alignment of the 263 residues of the beta-lactamase which presented 90% homology with the beta-lactamase of the K. oxytoca E23004 strain and about 40% homology with the other enzymes of the structural class A. The cefotaximase activity might result from interaction of a threonine residue at position 140 (position 165 in the numbering of Ambler) with the oxyimino group of the antibiotic.

An additional ionic bond suggested by molecular modelling of TEM-2 might induce a slight discrepancy between catalytic properties of TEM-1 and TEM-2 beta-lactamases.

The plasmid-mediated TEM-1 and TEM-2 beta-lactamases are the most commonly encountered among Gram-negative bacteria. They belong to molecular class A, and differ by one amino acid at position 39:TEM-1 have a glutamine and TEM-2 a lysine. Kinetic parameters (kcat and Km) and catalytic efficiency (kcat/Km) of TEM-1 and TEM-2 beta-lactamases are slightly, but significantly different. For all antibiotics except methicillin and cefazolin, the catalytic efficiency values of TEM-2 are clearly greater than that of TEM-1. Molecular modelling of TEM-2, when compared to that of TEM-1, showed an additional ionic bond between Lys-39 and Glu-281.

Val-237 for Ala substitution in the TEM-2 beta-lactamase dramatically alters the catalytic efficiencies towards carbenicillin and ticarcillin.

The mutant 554 of TEM-2 beta-lactamase was selected for a decrease in the resistance to carbenicillin of an Escherichia coli K12 carrier. The amino acid sequence of the mutant beta-lactamase was determined by manual Edman degradation analysis of proteolytic peptides. A single substitution Val for Ala was localized at position 237. The mutant exhibited only 2% of the catalytic efficiency of the wild-type enzyme towards carbenicillin and ticarcillin, whereas it retained 30-60% of the hydrolytic activity towards other penicillin and cephalosporin substrates. Carfecillin, the phenyl ester of the side-chain carboxyl group of carbenicillin, was hydrolysed as a good substrate. This suggests that the behaviour of the mutant enzyme towards carbenicillin may result from ionic rather than steric constraints. A molecular model of the Val-237 TEM-2 mutant suggests possible electrostatic interaction between Glu-171 and the carboxylic group of the side chain of carbenicillin.

Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to beta-lactamase inhibitors.

The clinical isolate Escherichia coli PEY was highly resistant to amoxycillin, ticarcillin and piperacillin associated to beta-lactamase inhibitors such as clavulanic acid, sulbactam, tazobactam and brobactam but susceptible to cephalosporins, aztreonam and imipenem. The susceptibility to mecillinam indicated that this phenotype was not related to hyperproduction of the TEM-1 beta-lactamase. E. coli PEY produced a new plasmid-mediated inhibitor-resistant beta-lactamase of pI 5.2, which was named IRT-4. The determination of the amino acid sequence (Swiss-Prot accession number, P00810) of the purified protein indicated that IRT-4 differed from TEM-1 by two substitutions: Leu for Met-69 (ABL numbering) and Asp for Asn-276. A Met-69-Leu variant of TEM-1, obtained by site-directed mutagenesis, has been described as resistant to clavulanate. The Asp for Asn-276 substitution has not been reported previously. The side chains of Asp-276 and Arg-244 were expected to interact. Determinations of 50% inhibitory concentrations of beta-lactamase inhibitors and substrate profile of IRT-4 suggested that such an ionic bond was implicated in the alteration of the mechanistic process of TEM-1 beta-lactamase.

Oxacillin-hydrolyzing beta-lactamase involved in resistance to imipenem in Acinetobacter baumannii.

Acinetobacter baumannii strain A148, a clinical isolate resistant to imipenem (MIC = 32 mg l-1), synthesized two beta-lactamases with pIs 6.3 and > 9.2. The pI 6.3 enzyme hydrolyzed the penicillins, including isoxazoylpenicillins, first-, second- and, to a lesser extent, third-generation cephalosporins. It was inhibited by chloride ions and by the penem beta-lactamase inhibitor BRL 42715. Clavulanate was a weak inhibitor and EDTA did not affect the beta-lactamase activity. This enzyme also hydrolyzed imipenem with a catalytic efficiency (Kcat/Km) of 1500 mM-1 s-1. Moreover, this purified beta-lactamase produced a positive microbiological clover-leaf test with imipenem. Therefore, the pI 6.3 beta-lactamase was considered to be involved in the imipenem resistance of A. baumannii strain A148.