Simultaneous determination of procaine and para-aminobenzoic acid by LC-MS/MS method

A sensitive high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA). N-Acetylprocainamide (NAPA) was used as an internal standard for procaine and PABA analysis. This assay method has also been validated in terms of linearity, lower limit of detection, lower limit of quantitation, accuracy and precision as per ICH guidelines. Chromatography was carried out on an XTerra MS C(18) column and mass spectrometric analysis was performed using a Quattro Micro mass spectrometer working with electro-spray ionization (ESI) source in the positive ion mode. Enhanced selectivity was achieved using multiple reaction monitoring (MRM) functions, m/z 237-->100, m/z 138-->120, and m/z 278-->205 for procaine, PABA and NAPA, respectively. Retention times for PABA, procaine and NAPA were 4.0, 4.7 and 5.8min, respectively. Linearity for each calibration curve was observed across a range from 100nM to 5000nM for PABA, and from 10nM to 5000nM for procaine. The intra- and inter-day relative standard deviations (RSD) were <5%.     

A cis-element in the 5' untranslated region of the preproinsulin mRNA (ppIGE) is required for glucose regulation of proinsulin translation

Insulin production in pancreatic beta cells is predominantly regulated through glucose control of proinsulin translation. Previously, this was shown to require sequences within the untranslated regions (UTRs) of the preproinsulin (ppI) mRNA. Here, those sequences were found to be sufficient for specific glucose-regulated proinsulin translation. Furthermore, an element 40-48 bp from the 5' end of the ppI mRNA specifically bound a factor present in islets of Langerhans. Glucose-responsive factor binding to this cis-element exhibited temporal and glucose-concentration-dependent patterns that paralleled proinsulin biosynthesis. Mutating this cis-element abolished the ability of ppI mRNA UTRs to confer glucose regulation upon translation. Like the rat 5'UTR, the human ppI 5'UTR conferred glucose regulation of translation. However alternative splicing of the human 5'UTR that disrupts the cis-element abolished glucose-regulated translation. These data indicate that glucose regulation of cis-element/trans-acting factor interaction is a key component of the mechanism by which glucose regulates insulin production.     

Drug-delivering nerve conduit improves regeneration in a critical-sized gap

Autologous nerve grafts are the current “gold standard” for repairing large nerve gaps. However, they cause morbidity at the donor nerve site and only a limited amount of nerve can be harvested. Nerve conduits are a promising alternative to autografts and can act as guidance cues for the regenerating axons, without the need to harvest donor nerve. Separately, it has been shown that localized delivery of GDNF can enhance axon growth and motor recovery. FK506, an FDA approved small molecule, has also been shown to enhance peripheral nerve regeneration. This paper describes the design of a novel hole-based drug delivery apparatus integrated with a polytetrafluoroethylene (PTFE) nerve conduit for controlled local delivery of a protein such as GDNF or a small molecule such as FK506. The PTFE devices were tested in a diffusion chamber, and the bioactivity of the released media was evaluated by measuring neurite growth of dorsal root ganglions (DRGs) exposed to the released drugs. The drug delivering nerve guide was able to release bioactive concentrations of FK506 or GDNF. Following these tests, optimized drug releasing nerve conduits were implanted across 10 mm sciatic nerve gaps in a BL6 yellow fluorescent protein (YFP) mouse model, where they demonstrated significant improvement in muscle mass, compound muscle action potential, and axon myelination in vivo as compared with nerve conduits without the drug. The drug delivery nerve guide could release drug for extended periods of time and enhance axon growth in vitro and in vivo.     

A field portable method for the semi‐quantitative estimation of dehydration tolerance of photosynthetic tissues across distantly related land plants

Desiccation tolerant (DT) plants withstand complete cellular dehydration, reaching relative water contents (RWC) below 30% in their photosynthetic tissues. Desiccation sensitive (DS) plants exhibit different degrees of dehydration tolerance (DHT), never surviving water loss >70%. To date, no procedure for the quantitative evaluation of DHT extent exists that is able to discriminate DS species with differing degrees of DHT from truly DT plants. We developed a simple, feasible and portable protocol to differentiate between DT and different degrees of DHT in the photosynthetic tissues of seed plants and between fast desiccation (< 24 h) tolerant (FDT) and sensitive (FDS) bryophytes. The protocol is based on (1) controlled desiccation inside Falcon tubes equilibrated at three different relative humidities that, consequently, induce three different speeds and extents of dehydration and (2) an evaluation of the average percentage of maximal photochemical efficiency of PSII (F_v/fm) recovery after rehydration. Applying the method to 10 bryophytes and 28 tracheophytes from various locations, we found that (1) imbibition of absorbent material with concentrated salt‐solutions inside the tubes provides stable relative humidity and avoids direct contact with samples; (2) for 50 ml capacity tubes, the optimal plant amount is 50–200 mg fresh weight; (3) the method is useful in remote locations due to minimal instrumental requirements; and (4) a threshold of 30% recovery of the initial F_v/fm upon reaching RWC ≤ 30% correctly categorises DT species, with three exceptions: two poikilochlorophyllous species and one gymnosperm. The protocol provides a semi‐quantitative expression of DHT that facilitates comparisons of species with different morpho‐physiological traits and/or ecological attributes.     

Mutation in a flexible linker modulates binding affinity for modular complexes

Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which β-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4–ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.     

Development of the catecholaminergic innervation of the song system of the male zebra finch

Catecholamines (CA) have been proposed to have neuromodulatory actions, particularly on attention and learning, in a number of neural systems. Because several of the interconnected brain nuclei that mediate song learning and production in the adult male zebra finch (Taeniopygia guttata) contain these neurotransmitters, we investigated the appearance of the catecholaminergic innervation of the song nuclei of male zebra finches during posthatch development, specifically during the period in which song learning occurs. We studied the development of immunoreactivity for tyrosine hydroxylase (TH) in the song nuclei HVc, RA, NIf, LMAN, and Area X in young males aged 20, 35, and 60 days as well as in adults (>90 days). We also visualized catecholamines directly in Area X using CA histofluorescence. Both TH immunoreactivity and CA histofluorescence were initially low in Area X relative to their levels in the surrounding parolfactory lobe (LPO), and then increased during development to become more intense than in LPO by days 60–90. Similarly, TH immunoreactivity in HVc was initially low relative to that in the surrounding neostriatum, then increased during development to become more intense than that in the surround by day 60. TH immunostaining also increased markedly in NIf, RA, and LMAN over the same period. These results show that the levels of catecholamines and their major synthetic enzyme increase in song nuclei during development and thus raise the possibility that these transmitters contribute to the development of the song system or to song learning. © 1996 John Wiley & Sons, Inc.