Dysfunction of proprioceptive sensory synapses is a pathogenic event and therapeutic target in mice and humans with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by a varying degree of severity that correlates with the reduction of SMN protein levels. Motor neuron degeneration and skeletal muscle atrophy are hallmarks of SMA, but it is unknown whether other mechanisms contribute to the spectrum of clinical phenotypes. Here, through a combination of physiological and morphological studies in mouse models and SMA patients, we identify dysfunction and loss of proprioceptive sensory synapses as key signatures of SMA pathology. We demonstrate that SMA patients exhibit impaired proprioception, and their proprioceptive sensory synapses are dysfunctional as measured by the neurophysiological test of the Hoffmann reflex (H-reflex). We further show that loss of excitatory afferent synapses and altered potassium channel expression in SMA motor neurons are conserved pathogenic events found in both severely affected patients and mouse models. Lastly, we report that improved motor function and fatigability in ambulatory SMA patients and mouse models treated with SMN-inducing drugs correlate with increased function of sensory-motor circuits that can be accurately captured by the H-reflex assay. Thus, sensory synaptic dysfunction is a clinically relevant event in SMA, and the H-reflex is a suitable assay to monitor disease progression and treatment efficacy of motor circuit pathology.

Chromatographic resolution of tryptophan enantiomers with L-Leu-L-Leu-L-Leu peptide effects of mobile phase composition and chromatographic support.

Tryptophan enantiomers have been separated by zwitterion pair chromatography using L-leucine-L-leucine-L-leucine peptide as the zwitterion pairing agent. The peptide ligand is adsorbed onto an octadecylsilane support with excess ligand present in bulk solution. This article examines the roles of the hydrophobic matrix and the mobile phase components on tryptophan enantiomer binding and resolution. Capacity factors and enantioselectivites are given for both hydrophobic and hydrophilic matrices using mobile phases containing Leu-Leu-Leu peptide and/or salt. A decrease in selectivity upon the addition of mobile phase salt suggests that quadrupolar ion-pairing contributes to chiral recognition. Results indicate that binding is significantly reduced and separation is not achieved when Leu-Leu-Leu is coupled onto cross-linked or polymerized hydrophilic resins as well as onto macroporous polystyrene resin. However, resin-immobilized Leu-Leu-Asp-Leu-Leu-Leu, Leu-Leu-Glu-Leu-Leu-Leu, and Leu-Leu-Leu-Glu-Leu-Leu peptides, with ion-pairing sites designed to mimic the Leu-Leu-Leu-saturated C18 support, also do not resolve tryptophan enantiomers. This suggests the Leu-Leu-Leu structure is critical for enantiomer resolution. Because D- and L-tryptophan are separated in the absence of bulk Leu-Leu-Leu, chiral discrimination is believed to occur at the surface of the octadecylsilane support.

Combinatorial peptide on-resin analysis: optimization of static nanoelectrospray ionization technique for sequence determination.

The optimizations of static nanoelectrospray parameters to determine peptide or mimetic sequences released from resin were explored. Several different manufacturers of probe tips were utilized and a method was developed for the direct analysis of bead-bound peptides by nanoelectrospray. The method involved minimum sample handling to assure maximum recovery from individual beads. Parameters that were explored included an inside and outside wash of the probe tip, the distance from the probe housing to the probe tip, source temperature, drying gas flow, individual tips and presence of beads. The same soluble synthetic peptide was used in all comparisons, which had a molecular weight of 717 amu. The discovery of the sequence of a bead-bound peptide was achieved. The parameters that were found to effect signal were outside wash, presence of bead and distance. There was the need for pneumatic assist to initiate electrospray on some occasions, although this generally resulted in unsatisfactory performance.

Organization and evolution of olfactory receptor genes on human chromosome 11.

Olfactory receptors (OR) are encoded by a large multigene family including hundreds of members dispersed throughout the human genome. Cloning and mapping studies have determined that a large proportion of the olfactory receptor genes are located on human chromosomes 6, 11, and 17, as well as distributed on other chromosomes. In this paper, we describe and characterize the organization of olfactory receptor genes on human chromosome 11 by using degenerate PCR-based probes to screen chromosome 11-specific and whole genome clone libraries for members of the OR gene family. OR genes were identified by DNA sequencing and then localized to regions of chromosome 11. Physical maps of several gene clusters were constructed to determine the chromosomal relationships between various members of the family. This work identified 25 new OR genes located on chromosome 11 in at least seven distinct regions. Three of these regions contain gene clusters that include additional members of this gene family not yet identified by sequencing. Phylogenetic analysis of the newly described OR genes suggests a mechanism for the generation of genetic diversity.

Affinity purification of von Willebrand factor using ligands derived from peptide libraries.

The chromatographic purification of vWF (von Willebrand Factor) from human plasma represents a challenge because it consists of multimers with molecular weights ranging from 0.5 to 10 million Daltons. Phage peptide library screening yielded a lead peptide (RLRSFY) that interacts with vWF. Conservative substitutions of terminal residues of the lead peptide led to a second peptide, RVRSFY, which was more efficient in the affinity chromatographic purification of vWF from protein mixtures. Adsorption isotherm measurements indicated multiple interactions between vWF and the immobilized peptide RVRSFY. Increases in peptide density on the chromatographic supports resulted in stronger association constants and higher maximum protein binding capacities. When the peptide density was lower than 32 mg/mL, there was no measurable interaction between vWF and immobilized peptide RVRSFY in HEPES buffer containing 0.5 M NaCl at pH 7. An increase in peptide density from 32 to 60 mg/mL increased the association constants from 0.9 x 10(6) to 2 x 10(6) (M-1). Divalent salts (calcium and magnesium chloride) were used to elute the retained vWF with 82.5% of the activity recovered. The interactions between vWF and the immobilized peptide RVRSFY are dominated by ionic attractions and also involve hydrophobic interactions at close contact. Finally, the purification of vWF from crude material PEG filtrate of a cryoprecipitate of human plasma is demonstrated using affinity chromatography with immobilized N-acetyl-RVRSFYK.

Chemically derived peptide libraries: a new resin and methodology for lead identification.

We have developed a new resin for peptide synthesis that can be used to synthesize and evaluate directly combinatorial peptide libraries for binding target proteins. Fidelity of the peptide synthesis using this hydrophilic resin is comparable to polystyrene-based resins. Peptide libraries synthesized on this resin were probed by a two color PEptide Library Immunostaining Chromatographic ANalysis (PELICAN) technique for sequences binding the serine protease Factor IX zymogen. This PELICAN technique readily distinguishes between beads interacting with the reagents for target detection (blue beads) from those beads specific for the target protein itself (red beads). Validation of the PELICAN technique, as well as purification of Factor IX from plasma, is demonstrated utilizing this resin.

How accurately can videokeratographic systems measure surface elevation?

INTRODUCTION: Surface topography, as opposed to dioptric topography, defines the corneal surface in simple terms without assumptions. Accordingly, it is important to know how well surface topography can be measured with current videokeratometric machines. PURPOSE: The purpose of this paper is to quantify the accuracy with which the TMS-1 Corneal Modeling System can measure the surface topography of calibrated spherical, elliptical, and bicurve surfaces. METHODS: The Computed Anatomy TMS-1 videokeratometer was used to measure three spherical, three elliptical, and two bicurve surfaces with known characteristics. Surface characteristics were either back-calculated from the dioptric files or directly obtained from the TMS-1 elevation file for each of 6400 points (256 points in each of 25 rings). The accuracy with which each method determined the true surface was quantified by calculating the root mean squared error (RMSE) of the 6400 measured surface elevations from the known surface elevation at each sampling point. RESULTS: (1) For spherical and elliptical surfaces, back-calculation of surface elevation from the dioptric file can be made with RMSE of 5 mu or less. (2) For spheres but not elliptical surfaces the TMS-1 elevation file defines the surface with RMSE 5 mu or less. (3) The surface area measured by placido-based videokeratometers varies with surface curvature. (4) RMSE in measured surface elevation increase as the distance from the videokeratometric axis increases. (5) For bicurves, the dioptric maps are smoothed by the TMS-1 over abrupt transitions and for large transitions never recover. Additionally, our back-calculation methods further smooth abrupt transitions, making the RMSE of the bicurve surface that is back-calculated from the dioptric file larger than the RMSE of the surface generated from the TMS-1 elevation file. CONCLUSIONS: Surface elevations can be back-calculated from dioptric files with RMSE of 5 microns or less for spheres and elliptical surfaces as long as there are no areas of abrupt transition. If areas of abrupt transition exist, the TMS-1 elevation file provides more accurate surface profile data.

Programmed DNA rearrangement of a cyanobacterial hupL gene in heterocysts.

Programmed DNA rearrangements that occur during cellular differentiation are uncommon and have been described in only two prokaryotic organisms. Here, we identify the developmentally regulated rearrangement of a hydrogenase gene in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120. Heterocysts are terminally differentiated cells specialized for nitrogen fixation. Late during heterocyst differentiation, a 10.5-kb DNA element is excised from within the hupL gene by site-specific recombination between 16-bp direct repeats that flank the element. The predicted HupL polypeptide is homologous to the large subunit of [NiFe] uptake hydrogenases. hupL is expressed similarly to the nitrogen-fixation genes; hupL message was detected only during the late stages of heterocyst development. An open reading frame, named xisC, identified near one end of the hupL DNA element is presumed to encode the element's site-specific recombinase. The predicted XisC polypeptide is homologous with the Anabaena sp. strain PCC 7120 site-specific recombinase XisA. Neither XisC nor XisA shows sequence similarity to other proteins, suggesting that they represent a different class of site-specific recombinase.

RNA recognition by an isolated alpha helix.

A 17 amino acid peptide containing the arginine-rich region of the HIV Rev protein binds specifically to Rev response element (RRE) RNA. Even though it is highly charged, the peptide forms an alpha helix in solution, but only when its N- and C-termini are modified to provide favorable electrostatic interactions with the helix macrodipole. Binding affinity for IIB RNA (the primary binding site within the RRE) increases with alpha helix content, whereas nonspecific binding affinity is independent of helix content. Binding of mutant peptides demonstrates that one threonine, one asparagine, and four arginine side chains are important for sequence-specific recognition. Transactivation of the HIV LTR using Tat-Rev peptide hybrids and the RRE IIB site indicates that the peptide adopts an alpha-helical conformation in vivo. The results suggest that interactions with the RNA backbone may help to orient the alpha helix in the major groove of RNA.

Postdural puncture headache: comparison of 25-gauge Whitacre and Quincke needles.

BACKGROUND AND OBJECTIVES: To evaluate the influence of the shape of the needle tip on postdural puncture headache (PDPH) independent of the needle diameter, a 25-gauge Whitacre and a 25-gauge Quincke needle were compared. METHODS: In a prospective, randomized, double-blind fashion, the study was carried out on 400 patients who received spinal anesthesia for operations of the lower extremities. The 25-gauge Whitacre needle (group 1) and the 25-gauge Quincke needle (group 2) were randomly assigned to the patients, 200 in each group. Patients were interviewed postoperatively on days 1, 3, 5, and 7 using a standardized questionnaire. Only postural headache was defined as PDPH. The intensity of both postural and nonpostural headache were quantified using a 4-point rating scale and a visual analog pain scale (VAS). Statistical analysis was performed with parametric and nonparametric tests when appropriate, p < or = 0.05 was considered as significant. RESULTS: There were no differences in age and sex distribution between the two groups. Significantly more patients in group 2 (8.5%) complained of PDPH than in group 1 (3%, p < or = 0.02). Duration of PDPH ranged from 1-3 days (median: 1) in group 1, and from 1-9 days (median: 3) in group 2. This difference closely approached significance (p = 0.058). The mean maximal intensity of PDPH was comparable in both groups. Severe PDPH occurred only in two patients of group 2. One of them required a blood patch. With respect to the nonpostural headache, no significant differences were seen. CONCLUSIONS: The use of a conical tipped Whitacre needle results in significantly less PDPH compared to a standard Quincke spinal needle of the same size.

Contribution of the nucleus of the optic tract to optokinetic nystagmus and optokinetic afternystagmus in the monkey: clinical implications.

1. The role of the pretectal NOT and the DTN in producing horizontal OKN and OKAN were studied using electrical stimulation and lesions. Positive stimulation sites lay in NOT, DTN, and in a fiber bundle in the pulvinar that is presumably a cortical input to NOT. 2. When the region of NOT was electrically stimulated in darkness, horizontal nystagmus was evoked with ipsilateral slow phases. Eye velocity rose slowly to a steady-state level and was followed by afternystagmus at the end of stimulation. The time constant of rise of stimulus-induced nystagmus was similar to the slow rise of slow-phase eye velocity during OKN. The saturation velocity of the induced nystagmus and the falling time constant of the stimulus afternystagmus were the same as those of OKAN. This suggests that electrical stimulation of NOT and DTN had elicited the slow component of OKN, i.e., that component produced by the velocity storage mechanism in the vestibular system. 3. Consistent with this postulate, activity induced by NOT stimulation could enhance, prolong, or block the slow component of OKN and OKAN depending on whether slow phases were to the same or opposite side. Stimulus-induced activity also interacted with vestibular nystagmus as would OKN and OKAN. 4. Unilateral lesions of NOT and DTN caused a loss of OKAN and the slow rise in OKN to the ipsilateral side. Steady-state velocities of OKN were reduced. The initial jump of OKN slow-phase velocity was the same or somewhat less after lesions but was not lost. 5. Partial lesions of a fiber bundle in the lateral pulvinar caused a transient change in OKN and OKAN, consistent with the idea that it carries activity for the slow component from the cortex to NOT. A lesion of the MRF, just rostral to the superior colliculus, caused a transient loss of the rapid component of OKN. This region appears to carry activity responsible for the initial jump in slow-phase velocity at the onset of stimulation. 6. We conclude that: (a) NOT and probably DTN lie in the indirect pathway that produces the slow component of horizontal OKN and OKAN to the ipsilateral side in the rhesus monkey. This pathway activates the velocity storage mechanism in the vestibular nuclei. (b) At the level of NOT, the pathway responsible for the slow component of OKN and OKAN is anatomically distinct from the pathway responsible for rapid changes in eye velocity at the onset of OKN.(ABSTRACT TRUNCATED AT 400 WORDS)

Serum levels of mepivacaine-HCl during continuous axillary brachial plexus block.

In order to find out whether repeated doses of plain mepivacaine are appropriate for long-lasting microvascular surgery, a catheter-induced axillary plexus block was performed in 17 patients by using 400 mg of mepivacaine-HC1 every two hours. All patients underwent replantation surgery of one or more amputated fingers. Duration of operation varied from 8 to 24 hours. In all cases, there was adequate analgesia and muscle relaxation for the surgical procedure. Serum levels of mepivacaine were determined before, and 15 and 60 minute after the initial injection. With each following injection of mepivacaine, blood samples were taken at the same time schedule. Within the first eight hours, after four 400 mg doses of mepivacaine were given, all serum levels remained below the level of 5 to 6 micrograms/ml, which is said to be the lower level for mild cerebral toxic reactions in venous blood (blood/plasma distribution 0.92 +/- 0.04). After this time, when more than 1600 mg of mepivacaine had been administered, two patients exceeded the level of 6 micrograms/ml. The highest serum concentration observed in any of the patients was 7.0 micrograms/ml. This was 15 minutes after the 12th injection of 400 mg of mepivacaine and an overall dosage of 4800 mg. None of the patients showed evident signs of central nervous system or cardiovascular toxicity.

Computing high order wave aberration coefficients from variations of best focus for small artificial pupils.

Van den Brink (1962) measured the variation of best focus of vertical bar targets through a 1.4 mm aperture across the pupil of the eye. He expressed his results in contour maps of isodioptric lines across the pupil. The relative focus of a portion of the pupil is a function of the curvature of the wave front within the aperture. The curvature, in turn, is proportional to the second derivative of the wave aberration polynomial. Thus it is possible to make estimates of some of the coefficients of the wave aberration polynomial from Van den Brink's focusing data. This has been done for two states of accommodation. The results indicate that the wave aberration polynomial of the measured eye contains some significant terms of a power higher than hitherto measured.

Sweat gland differentiation in mammary adenoid cystic carcinoma.

A breast tumor is described which presented as an exophytic mass, and which by both light and electron microscopic examination had a biphasic histologic composition. In the superficial area adjacent to the epidermis, it showed tubular differentiation similar to a cutaneous tubular apocrine adenoma and salivary basal cell adenoma, and, in the deeper portion, it had the characteristic features of adenoid cystic carcinoma. Their possible interrelationships are discussed, and mammary adenoid cystic carcinoma is briefly reviewed.