Selective collision-induced fragmentation of ortho-hydroxybenzyl-aminated lysyl-containing tryptic peptides.

RATIONALE: In protein studies that employ tandem mass spectrometry the manipulation of protonated peptide fragmentation through exclusive dissociation pathways may be preferred in some applications over the comprehensive amide backbone fragmentation that is typically observed. In this study, we characterized the selective cleavage of the side-chain Cζ-Nε bond of peptides with ortho-hydroxybenzyl-aminated lysine residues. METHODS: Internal lysyl residues of representative peptides were derivatized via reductive amination with ortho-hydroxybenzaldehyde. The modified peptides were analyzed using collision-induced dissociation (CID) on an Orbitrap tandem mass spectrometer. Theoretical calculations using computational methods (density functional theory) were performed to investigate the potential dissociation mechanisms for the Cζ-Nε bond of the derivatized lysyl residue resulting in the formation of the observed product ions. RESULTS: Tandem mass spectra of the derivatized peptide ions exhibit product peaks corresponding to selective cleavage of the side-chain Cζ-Nε bond that links the derivative to lysine. The ortho-hydroxybenzyl derivative is released either as a neutral moiety [C7H6O1] or as a carbocation [C7H7O1](+) through competing pathways (retro-Michael versus Carbocation Elimination (CCE), respectively). The calculated transition state activation barriers indicate that the retro-Michael pathway is kinetically favored over CCE and both are favored over amide cleavage. CONCLUSIONS: The application of ortho-hydroxybenzyl amination is a promising peptide derivatization scheme for promoting selective dissociation pathways in the tandem mass spectrometry of protonated peptides. This can be implemented in the rational development of peptide reactive reagents for applications that may benefit from selective fragmentation paths (including crosslinking or MRM reagents).

Substituent effects on the gas-phase fragmentation reactions of protonated peptides containing benzylamine-derivatized lysyl residues.

Motivated by the need for chemical strategies designed to tune peptide fragmentation to selective cleavage reactions, benzyl ring substituent influence on the relative formation of carbocation elimination (CCE) products from peptides with benzylamine-derivatized lysyl residues has been examined using collision-induced dissociation (CID) tandem mass spectrometry. Unsubstituted benzylamine-derivatized peptides yield a mixture of products derived from amide backbone cleavage and CCE. The latter involves side-chain cleavage of the derivatized lysyl residue to form a benzylic carbocation [C(7)H(7)](+) and an intact peptide product ion [(MH(n))(n+) - (C(7)H(7))(+)]((n-1)+). The CCE pathway is contingent upon protonation of the secondary ε-amino group (N(ε)) of the derivatized lysyl residue. Using the Hammett methodology to evaluate the electronic contributions of benzyl ring substituents on chemical reactivity, a direct correlation was observed between changes in the CCE product ion intensity ratios (relative to backbone fragmentation) and the Hammett substituent constants, σ, of the corresponding substituents. There was no correlation between the substituent-influenced gas-phase proton affinity of N(ε) and the relative ratios of CCE product ions. However, a strong correlation was observed between the π orbital interaction energies (ΔE(int)) of the eliminated benzylic carbocation and the logarithm of the relative ratios, indicating the predominant factor in the CCE pathway is the substituent effect on the level of hyperconjugation and resonance stability of the eliminated benzylic carbocation. This work effectively demonstrates the applicability of σ (and ΔE(int)) as substituent selection parameters for the design of benzyl-based peptide-reactive reagents which tune CCE product formation as desired for specific applications.

Influence of operator site geometry on transcriptional control by the YefM-YoeB toxin-antitoxin complex.

YefM-YoeB is among the most prevalent and well-characterized toxin-antitoxin complexes. YoeB toxin is an endoribonuclease whose activity is inhibited by YefM antitoxin. The regions 5' of yefM-yoeB in diverse bacteria possess conserved sequence motifs that mediate transcriptional autorepression. The yefM-yoeB operator site arrangement is exemplified in Escherichia coli: a pair of palindromes with core hexamer motifs and a center-to-center distance of 12 bp overlap the yefM-yoeB promoter. YefM is an autorepressor that initially recognizes a long palindrome containing the core hexamer, followed by binding to a short repeat. YoeB corepressor greatly enhances the YefM-operator interaction. Scanning mutagenesis demonstrated that the short repeat is crucial for correct interaction of YefM-YoeB with the operator site in vivo and in vitro. Moreover, altering the relative positions of the two palindromes on the DNA helix abrogated YefM-YoeB cooperative interactions with the repeats: complex binding to the long repeat was maintained but was perturbed to the short repeat. Although YefM lacks a canonical DNA binding motif, dual conserved arginine residues embedded in a basic patch of the protein are crucial for operator recognition. Deciphering the molecular basis of toxin-antitoxin transcriptional control will provide key insights into toxin-antitoxin activation and function.

Recruitment of the ParG segregation protein to different affinity DNA sites.

The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. In addition to its multifunctional role in segrosome assembly, the ParG protein of multiresistance plasmid TP228 is a transcriptional repressor of the parFG partition genes. ParG is a homodimeric DNA binding protein, with C-terminal regions that interlock into a ribbon-helix-helix fold. Antiparallel beta-strands in this fold are presumed to insert into the O(F) operator major groove to exert transcriptional control as established for other ribbon-helix-helix factors. The O(F) locus comprises eight degenerate tetramer boxes arranged in a combination of direct and inverted orientation. Each tetramer motif likely recruits one ParG dimer, implying that the fully bound operator is cooperatively coated by up to eight dimers. O(F) was subdivided experimentally into four overlapping 20-bp sites (A to D), each of which comprises two tetramer boxes separated by AT-rich spacers. Extensive interaction studies demonstrated that sites A to D individually are bound with different affinities by ParG (C > A approximately B >> D). Moreover, comprehensive scanning mutagenesis revealed the contribution of each position in the site core and flanking sequences to ParG binding. Natural variations in the tetramer box motifs and in the interbox spacers, as well as in flanking sequences, each influence ParG binding. The O(F) operator apparently has evolved with sites that bind ParG dissimilarly to produce a nucleoprotein complex fine-tuned for optimal interaction with the transcription machinery. The association of other ribbon-helix-helix proteins with complex recognition sites similarly may be modulated by natural sequence variations between subsites.

Prolonged response times characterize cognitive performance in multiple sclerosis.

Cognitive impairment is amongst the main symptoms affecting multiple sclerosis (MS) and should be comprehensively and accurately assessed. To study the added value of a computerized neuropsychological battery enabling the measurement of response times in the cognitive domains, 58 randomly selected MS patients and 71 age-, gender- and education-matched healthy subjects were evaluated. Construct and discriminant validity were assessed for the standard Neuropsychological Screening Battery for Multiple Sclerosis (NSBMS) and the Mindstreams Computerized Cognitive Battery (MCCB). The MCCB demonstrated good construct validity in comparison with the NSBMS in memory (P < 0.001), executive function (P < 0.001), attention (P < 0.05) and information processing (P < 0.05) domains. In addition, it showed high discriminant validity most prominently for executive function, attention and motor skills (P < 0.001). Response times measured by the computerized battery were longer in all cognitive domains and varied with cognitive load, demonstrating that response time deficits in MS are associated with particular task demands. We conclude that in MS prolonged response times on a range of cognitive tasks signify abnormal conduction within demyelinative tracts.

Involvement of glycine and GABAA receptors in the pathogenesis of spinal myoclonus: in vitro studies in the isolated neonatal rodent spinal cord.

Studies of pathogenetic mechanisms of myoclonus and spinal spasticity have established a strong association between deficient inhibitory glycinergic transmission and pathologic rigidity and tremor. Consistent with known cases in the clinical literature, electrophysiologic data from animal models of myoclonus implicate dysfunction of segmental spinal cord circuitry. The present study sought to further explore pathogenetic mechanisms at the circuit level. In vitro preparations of isolated spinal cord from neonatal rodents allowed for stable recordings of individual cells as well as populations of motoneurons. Blockade of glycine receptors enhanced 5- to 15-Hz sinusoidal oscillations that were synchronous in entire populations of motoneurons as well as along multiple segments of the spinal cord. Oscillations at motoneurons were mediated largely by non-NMDA excitatory synaptic inputs. Blockade of GABAA receptors, and not GABAB receptors, abolished sinusoidal oscillations, suggesting a critical role for GABAA receptors in the premotoneuronal circuitry responsible for generation or transmission of the sinusoidal oscillations. These data offer new insights into possible pathogenetic mechanisms of spinal myoclonus and may help guide future research leading to specific therapies for hyperkinetic movement disorders of spinal origin.

Hemispatial and directional performance biases in motor neglect.

Motivationally responsive motor deficits may occur in isolation or as part of more general neglect syndromes. We describe three patients with two discrete syndromes of isolated motor neglect, differentiated primarily by their performance in motor tasks enacted within or toward the contralesional hemispace. The lesions in our patients likely disrupted attentional interactions with two separable sensorimotor processing subsystems. Physiologic data support the existence of a parietal-lateral premotor circuit that processes information encoded in spatial coordinates referenced to the extrapersonal environment and of a basal ganglionic-mesial premotor circuit that processes information mostly encoded in egocentric skeletomotor coordinates. The correlation of ischemic lesions resulting in hemispatial and directional biases in motor neglect with disruption of known physiologic subsystems may provide the basis for rational cognitive rehabilitation of these higher-order motor deficits. These observations are supported by recent PET studies that document the presence of specific attentional-motoric interactions within discrete processing components of a distributed sensorimotor attentional network.

The nigrostriatal dopaminergic system in familial early onset parkinsonism with parkin mutations.

Nigrostriatal dopaminergic function and cerebral energy metabolism were measured with PET in two brothers with early-onset parkinsonism caused by mutation of the parkin gene. Energy metabolism did not differ, but the nigrostriatal dopaminergic pattern was clearly different than that of sporadic PD. Thus parkinsonism in these two patients was shown to be pathophysiologically different than PD.

The effect of botulinum toxin injections to the calf muscles on freezing of gait in parkinsonism: a pilot study.

BACKGROUND: Freezing of gait (FOG) is a common and very disabling parkinsonian symptom, which is poorly understood and responds unsatisfactorily to medical treatment. We recently reported a unique patient with Parkinson's disease (PD) who had significant alleviation of FOG shortly after she was injected with botulinum toxin type A (BTX-A) for foot dystonia (Giladi et al. 1997). OBJECTIVE: To assess the effect of BTX-A injections into the calf muscles of parkinsonian patients on FOG. METHOD: BTX-A was injected in an open fashion into the calf muscles of 10 parkinsonian patients (age 55-75 years) with FOG as a predominant symptom. Response of FOG was assessed subjectively by the patient from worsening (-1) to marked improvement (+3). One patient was injected in a single blind fashion with saline or BTX-A after he had an initial good response. RESULTS: Seven patients reported different rates of improvement of FOG severity in 15 out of 17 therapeutic sessions. Four patients (40%) reported marked improvement (+3) of FOG in 5 sessions. Two patients reported no effect in two sessions. The mean duration of improvement was 6 weeks (range 1-12 weeks) with definite deterioration afterwards. The patient who was injected in a single blind fashion did not respond to saline injections but improved significantly with BTX-A treatment. CONCLUSIONS: We observed a clear temporal relationship between BTX-A injections into the calf muscles of parkinsonian patients and improvement of FOG. A double blind placebo controlled prospective study is needed before any conclusions can be drawn about the role of BTX-A injection in FOG.

Hand rhythmic tapping and timing in Parkinson's disease.

BACKGROUND: Dysrhythmia is one of the features frequently associated with the motor disturbance in Parkinson's disease (PD). The mechanism responsible for this phenomenon is not known. OBJECTIVES: To assess the rhythmic movements of the hand in PD patients in general and in parkinsonian subtypes. METHODS: Fifty-one PD patients (32 males) with mean age 66.3 +/- 9.1 years (6.6 years of symptoms) and 36 healthy controls (age 64.9 +/- 13.2, range 40-85) were studied. Subjects were asked to tap with their dominant or less affected arm on a digitized switch board at their most comfortable pace (16 s), fastest tapping speed (12 s), and at different frequencies provided by a metronome. The mean rhythm and the tap-to-tap variation were compared. Performance of the PD patients and control subjects were compared, as there were different subtypes of PD patients. Patients were subclassified into: tremor predominant (TP) (14 patients), freezing predominant (FP) (11 patients), akinetic-rigid (AR) (12 patients) and an unclassified group (UC) (14 patients). Results. There was no significance difference between patients and controls in the self-chosen, most comfortable tapping rate or in the tap-to-tap variation of the self-paced task. PD patients tapped at a significantly slower rate than controls when asked to tap at their fastest rate (4.39 +/- 1.32 vs. 5.14 +/- 1.31 Hz; p < 0.01). This difference was the result of an especially slow performance of the TP and AR subgroups (3.85+/-1.20 and 3.88+/-1.46, respectively; p < 0.01 compared to the control group). TP was the only subgroup to show an increased tap-to-tap variation at their fastest tapping rate compared to the control group (0.070 +/- 0.057 vs. 0.029 +/- 0.025 s, respectively, p < 0.05). The TP subgroup also showed hastening when they followed an externally given rhythm of 2.5 Hz and they tapped at 2.73 +/- 0.36 Hz p < 0.05). CONCLUSIONS: Externally driven and self-paced tapping are preserved in patients with PD, when examined at their best 'on' state. The tremor predominant subgroup seems to have specific pacing disturbances.

D-lactate dehydrogenase. Substrate specificity and use as a catalyst in the synthesis of homochiral 2-hydroxy acids.

This note compares the substrate specificity of D-lactate dehydrogenase (D-LDH, EC 1.1.1.28) to that of L-lactate dehydrogenase (L-LDH, EC 1.1.1.27), illustrates three procedures that use D-LDH in synthesis and two methods for recycling NADH, and provides experimental details illustrating the use of D-LDH in organic synthesis.

Hereditary juvenile-onset craniocervical predominant generalized dystonia with parkinsonism.

OBJECTIVE: To report a unique hereditary, juvenile onset, craniocervical predominant, generalized dystonia and parkinsonism affecting four members of one family. FAMILY DESCRIPTION: A father and three of his four daughters presented to us over the past 30 years with a similar picture of generalized dystonia, starting in the craniocervical region in the second or third decade of life. They later developed moderate parkinsonism, mainly manifesting bradykinesia, rigidity and abnormal postural reflexes. Biochemical and genetic tests excluded Wilson's disease, Huntington's disease and Oppenheim's dystonia. CONCLUSION: This is a new type of familial dystonia-parkinsonism where the craniocervical dystonic symptoms are most prominent in the early stages while parkinsonism becomes the predominant problem later in life. A search for the genetic mutation in this family is underway.

Recognition of saccharides by the OpcA, OpaD, and OpaB outer membrane proteins from Neisseria meningitidis.

The adhesion of the pathogen Neisseria meningitidis to host cell surface proteoglycan, mediated by the integral outer membrane proteins OpcA and Opa, plays an important part in the processes of colonization and invasion by the bacterium. The precise specificities of the OpcA and Opa proteins are, however, unknown. Here we use a fluorescence-based binding assay to show that both proteins bind to mono- and disaccharides with high affinity. Binding of saccharides caused a quench in the intrinsic fluorescence emission of both proteins, and mutation of selected Tyr residues within the external loop regions caused a substantial decrease in fluorescence. We suggest that the intrinsic fluorescence arises from resonance energy transfer from Tyr to Trp residues in the beta-barrel portion of the structure. OpcA bound sialic acid with a Kd of 0.31 microM and was shown to be specific for pyranose saccharides. The binding specificities of two different Opa proteins were compared; unlike OpcA, neither protein bound to monosaccharides, but both bound to maltose, lactose, and sialic acid-containing oligosaccharides, with Kd values in the micromolar range. OpaB had a 10-fold higher affinity for sialic acid-containing ligands than OpaD as a result of the mutation Y165V, which was shown to restore this specificity to OpaD. Finally, the OpcA- and Opa-dependent adhesion of meningococci to epithelial cells was shown to be partially inhibited by exogenously added sialic acid and maltose. The results show that OpcA and the Opa proteins can be thought of as outer membrane lectins and that simple saccharides can modulate their recognition of complex proteoglycan receptors.

Phenotypic heterogeneity and disease course in three murine strains with mutations in genes encoding for alpha 1 and beta glycine receptor subunits.

Impaired glycinergic inhibition causes human hyperekplexia, and may be involved in the pathogenesis of movement disorders associated with uremia, spinal cord lesions, DDT poisoning, and tetanus. Three autosomal recessive mutant mouse strains with single-gene mutations affecting either the alpha 1 (spasmodic and oscillator) or beta (spastic) subunits of the glycine receptor were studied. Serial videotaped examinations assessed the severity of hyperkinetic features. Homozygote oscillator mice appeared normal until postnatal day (P) 11-14, when decreased exploratory movements, spastic gait, stimulus-induced myoclonic bouts, rigidity, and tremor were noticeable. All symptoms gradually worsened until death by P21-P23. In contrast, spastic and spasmodic mice were most severely affected by the 3rd-5th week of life and had a lessening of symptom severity in adulthood. Within each mutant strain, there was marked interanimal variation of severity of the other motor abnormalities, possibly because of stochastic variability in developmental processes. These mutants represent good animal models for elucidation of molecular and cellular issues regarding the glycine receptor and for the study of pathogenetic mechanisms of movement disorders.

Locomotor modulation of disynaptic EPSPs from the mesencephalic locomotor region in cat motoneurons.

Locomotor modulation of disynaptic EPSPs from the mesencephalic locomotor region in cat motoneurons. J. Neurophysiol. 80: 3284-3296, 1998. When low-frequency tetanization of the mesencephalic locomotor region (MLR) produce fictive locomotion in unanesthetized, decerebrate cats, each MLR stimulus produces a distinctive cord dorsum potential (CDP) and oligosynaptic excitatory postsynaptic potentials (EPSPs) in many lumbosacral motoneurons. The average segmental latency from the initial CDP wave [mean delay from stimulus: 4.3 +/- 0.9 (SD) ms] to the onset of detectable MLR EPSPs was 1.6 +/- 0.4 ms, suggesting a disynaptic segmental connection. In gastrocnemius/soleus, flexor hallucis longus, flexor digitorum longus, tibialis anterior, and posterior biceps-semitendinosus motoneurons (35/38 cells), MLR EPSPs either appeared or were enhanced during the phase of fictive stepping in which the target motoneurons were depolarized and the motor pool was active (the phase), with parallel changes between EPSP amplitudes and membrane depolarization. In contrast, MLR stimulation produced small (1/10) or no EPSPs in extensor digitorum longus (EDL) motoneurons, with no phase enhancement (4/10) or oligosynaptic inhibitory postsynaptic potentials during the phase (5/10). Eight of 10 flexor digitorum longus (FDL) cells exhibited membrane depolarization in the early flexion phase of fictive stepping, and five of these showed parallel enhancement of disynaptic MLR EPSPs during early flexion. Three cases were studied when the FDL motor pool exhibited exclusively extensor phase firing. In these cases, the disynaptic MLR EPSPs were enhanced only during the extensor phase, accompanied by membrane depolarizations. We conclude that the last-order interneurons that produce disynaptic MLR EPSPs may well participate in producing the depolarizing locomotor drive potentials (LDPs) found in hindlimb motoneurons during fictive locomotion. However, the absence of linkage between MLR EPSP enhancement and LDP depolarizations in EDL motoneurons suggests that other types of excitatory interneurons also must be involved at least in some motor pools. We compared these patterns with the modulation of disynaptic EPSPs produced in FDL cells by stimulation of the medial longitudinal fasciculus (MLF). In all seven FDL motoneurons tested, disynaptic MLF EPSPs appeared only during the extension phase, regardless of when the FDL motoneurons were active. The fact that the modulation patterns of MLR and MLF disynaptic EPSPs is different in FDL motoneurons indicates that the two pathways do not converge on common last-order interneurons to that motor pool.

Differential modulation of disynaptic cutaneous inhibition and excitation in ankle flexor motoneurons during fictive locomotion.

1. Intracellular recording from extensor digitorum longus (EDL) and tibialis anterior (TA) alpha-motoneurons during fictive locomotion was used to examine patterns of modulation of oligosynaptic postsynaptic potentials (PSPs) produced by electrical stimulation of the cutaneous superficial peroneal (SP) and medial plantar (MPL) nerves in unanesthetized, decerebrate adult cats. 2. In all 20 EDL motoneurons studied, electrical stimulation of the SP nerve with single pulses at about twice threshold for the most excitable fibers in the nerve (2xT) produced either no synaptic potentials or relatively small oligosynaptic excitatory or inhibitory PSPs (EPSPs or IPSPs), both at rest and during the extension phase of fictive stepping. However, at the onset of the flexion phase large, presumably disynaptic IPSPs (central latencies 1.7-2.0 ms) appeared in the SP responses. These IPSPs usually decreased in amplitude later in the flexion phase despite maintained membrane depolarization. 3. In most (7/8) TA motoneurons, SP stimulation produced oligosynaptic EPSPs at rest and during the extension phase of fictive stepping. These EPSPs were suppressed during flexion in a majority of TA cells studied (5/8) but no clearly disynaptic IPSPs were found in any TA motoneuron. 4. In most EDL and TA motoneurons, stimulation of the MPL nerve produced oligosynaptic EPSPs at rest and during the extension phase, most with latencies in the presumably disynaptic range (< or = 2.0 ms). When present, these MPL EPSPs were suppressed throughout the flexion phase of stepping in almost all EDL (18/ 20) and TA (6/8) motoneurons examined. 5. The available evidence suggests that these modulation effects during fictive stepping are due primarily to convergence of control information from the spinal central pattern generator (CPG) for locomotion onto segmental interneurons in the oligosynaptic cutaneous pathways. 6. These observations extend the evidence for precise differential control of transmission through cutaneous reflex pathways in the cat hindlimb by the locomotor CPG. Taken together with earlier evidence about locomotor modulation of cutaneous PSPs in flexor digitorum longus (FDL) motoneurons, the data suggest that cutaneous information from the dorsal surface of the foot, carried in part by the SP nerve, projects to digit motoneurons (FDL and EDL) through discrete sets of last-order interneurons that also receive powerful excitation from the locomotor CPG during flexion. In contrast, the last-order interneurons that convey excitatory information from the SP nerve to at least some TA motoneurons are inhibited by the CPG during flexion. 7. Another contrast resides in the fact that oligosynaptic cutaneous excitation from the plantar surface of the foot, via the MPL nerve, is suppressed in FDL, EDL, and TA motoneurons during the flexion phase of locomotion. The available information is consistent with the possibility that MPL effects may be delivered to these motor nuclei by common interneurons. 8. We suggest an interneuronal circuitry that could account for these observations and discuss possible functional implications of modulation of these sensory pathways during locomotion.