Ex-Ex Primer: An experimentally validated tool for designing oligonucleotides spanning spliced nucleic acid regions from multiple species.

A comparative study of existing junction-primer-designing software revealed many limitations among them. Hence, we developed a new computational program, Ex-Ex Primer, which offers many improved, user-friendly features, and reliably creates junction primers and probes. This online suite can also be used to design primers/probes from other sites of nucleic acid recombination, insertion, deletion, or splicing, and regular probes/primers. The threshold for Tm difference between the complete junctional primer vs its partial sequence, which maps to one of the junctional regions, was changed based on an important observation made during the initial experimental validations. The tool is now thoroughly checked with RT-PCR and RT-qPCR experiments with more than 250 primer pairs over a few years. The junction-primer-designing features of the software are also better than other equivalent tools. Visualizing the exons and introns across transcripts, and enabling primer designing based on information from Ensembl, are some of the unique features of this tool. The primers suggested by the tool can be used to detect the expression of known transcripts, to test the existence of predicted DNA or RNA joints via hybridization-based techniques, or for validation and in silico analysis of RNA-Seq. URL: http://resource2.ibab.ac.in/exprimer/.

Etiological profile of diarrhea in solid organ transplant recipients at a tertiary care center in Southern India.

BACKGROUND: Diarrhea is one of the common gastrointestinal (GI) adverse events after solid organ transplantation. Diarrhea may be caused by infectious or non-infectious etiology. The infectious etiology of diarrhea varies according to the location and duration of diarrhea. Non-infectious etiologies include drugs, inflammatory bowel disease, neoplasia. The objective of this study was to evaluate the etiological profile of diarrhea in solid organ transplant recipients presenting to a tertiary care center in Southern India. METHODS: This was a retrospective analysis of prospectively collected data of all solid organ transplantation recipients referred to the Department of Medical Gastroenterology for evaluation of diarrhea from April 2012 till May 2014. All patients had stool evaluated by wet mount examination, modified acid fast (AFB) stain, trichrome stain, culture, and Clostridium difficile toxin assay. EDTA plasma was collected for quantitative Cytomegalovirus (CMV) detection by real-time PCR. If the diarrhea was acute (<2 wk), and no etiological agent was identified, empirical antibiotic therapy was instituted and followed up. If persistent or chronic diarrhea (>2-4 wk), endoscopic evaluation (upper GI endoscopy and/or colonoscopy with biopsies), depending on the clinical type of diarrhea was done. If no specific etiological diagnosis was established after endoscopic evaluation, breath test for SIBO and celiac serology were done. If no specific etiology was identified after the above investigations, dose of immunosuppressive drugs was reduced. If diarrhea responded to dose reduction, it was considered to be drug related. RESULTS: Fifty-eight episodes of diarrhea occurred in 55 solid organ transplant recipients during the study period. Renal transplant recipients constituted the majority (70%). Most (79%) of patients included in the study had their transplant > 6 mo ago. Infective diarrhea was the etiology in 46%, drug-related diarrhea in 29.3%. No specific etiology was identified in 22.4% of patients. Parasites accounted for 69% of all infective diarrhea. Stool evaluation was the main investigation in establishing diagnosis in acute diarrhea. Endoscopic evaluation was required in two thirds of patients to establish diagnosis in chronic diarrhea. CONCLUSION: GI infections and drug-related diarrhea were the common causes of diarrhea in solid organ transplant recipients. Parasites were the most common infectious etiology of diarrhea. Step-wise evaluation was able to identify the etiology in ~ 77% of patients. Overall, 98% of diarrheal episodes resolved.

Molecular fingerprinting of the Indian medicinal plant Strychnos minor Dennst.

Genomic DNA of Strychnos minor (Dennst) of sixteen geographical regions were amplified using thirteen primers. The analyses of products of Radom Amplified Polymorphic DNA (RAPD) revealed polymorphism among the samples. The polymorphic information content (PIC) was maximum for the primer of OPB-04 (0.40) followed by the primer OPB-20 (0.39). Clustering analysis by Jaccard's coefficient index exhibited similarity matrix and distance matrix. The amplified products of the 13 primers collectively showed similarity index ranged from 0.654 to 0.986. The phylogenetic trees were constructed by UPGMA cluster analysis. Significant variations in the amplified genomic DNA suggest genetic variability of population but low inter-population genetic segregation.

Removal of malachite green from aqueous solutions using a modified chitosan composite.

The synthesis of a novel composite adsorbent prepared from coir pith activated carbon (CPAC), chitosan and sodium dodecyl sulphate (SDS, an anionic surfactant) is reported. The characterisation of the composite was done using SEM, XRD, UV-visible and IR spectroscopy studies. The effectiveness of the composite was made for the removal of a toxic cationic dye, malachite green (MG) from waste water based on adsorption studies. The reaction conditions for the adsorption studies were optimized based on initial dye concentration, dose rate, reaction time, pH and temperature. Langmuir and Freundlich isotherm models were adopted to study the mechanism of adsorption. The adsorption process was found to follow pseudo second order kinetics. The results of the present study indicate that the CPAC based composite could be an effective low cost adsorbent for the removal of MG from waste water.

Isolation, structure elucidation and anticancer activity from Brevibacillus brevis EGS 9 that combats Multi Drug Resistant actinobacteria.

Actinobacteria is the most widely distributed organism in the mangrove environment and produce a large amount of secondary metabolites. A new environmental actinobacterial stain exhibited strong antimicrobial activity against vancomycin and methicillin resistant actinobacteria. The active producer strain was found to be as Brevibacillus brevis EGS9, which was confirmed by its morphological, biochemical characteristics and 16S rRNA gene sequencing. It was deposited in NCBI GeneBank database and received with an accession number of KX388147. Brevibacillus brevis EGS9 was cultivated by submerged fermentation to produce antimicrobial compounds. The anti-proliferative agent was extracted from Brevibacillus brevis EGS9 with ethyl acetate. The bioactive metabolites of mangrove actinobacteria was identified by Liquid chromatography with mass spectrometry analysis. The result of the present investigation revealed that actinobacteria isolated from mangroves are potent source of anticancer activity. The strain of Brevibacillus brevis EGS9 exhibited a potential in vitro anticancer activity. The present research concluded that the actinobacteria isolated from mangrove soil sediment are valuable in discovery of novel species.

Screening of novel actinobacteria and characterization of the potential isolates from mangrove sediment of south coastal India.

The importance of the current research is to investigate the different types of samples from the various mangrove sediments; as source of actinobacteria from the mangrove wet soil. Potential isolate screening by antimicrobial activity and identified actinobacteria was characterized based on cultural morphology, physiological and biochemical characteristics. Three different types of media were used to isolate actinobacteria from various geographical region of mangrove soil sediment and the genotype locus was recognized by 16S rDNA. Totally 144 actinobacteria isolates were recovered from 10 samples using three media. The most active culture media in the isolation of actinobacteria were ISP2 and Glycerol Yeast Extract Agar. Among 144 isolates, 38 isolates (26.38%) exhibited antimicrobial activity. Out of 38 isolates, potentially active 2 cultures were further supported for morphological and biochemical characterization analysis. Most of the isolates were produced pharmaceutically important enzymes such as protease, amylase, lipase, cellulose and also revealed antimicrobial activity against tested microorganism. The enriched salt, pH and temperature tolerance of the actinobacteria isolates to discharge commercially valuable primary and secondary bioactive metabolites. The present results functionally characterize novel mangrove actinobacteria and their metabolites for commercial interest in pharmaceutical industry.

HPTLC fingerprint profile, in vitro antioxidant and evaluation of antimicrobial compound produced from Brevibacillus brevis-EGS9 against multidrug resistant Staphylococcus aureus.

In the present study, in vitro antimicrobial activity of Brevibacillus brevis EGS9 against multi drug resistant Staphylococcus aureus (MDRSA) and to investigate the antimicrobial, antioxidant activity and HPTLC finger print profile of Brevibacillus brevis EGS9. Primary screening was done using by cross streak method against multi drug resistant Staphylococcus aureus. The bioactive metabolites were extracted from Brevibacillus brevis EGS9 using ethyl acetate extraction. Ethyl acetate extract showed significant antimicrobial activity against Escherichia coli (20.2 ± 0.1) mm, Candida albicans (19.2 ± 0.3) mm and Bacillus cereus (18.6 ± 0.2) mm respectively. Forty three UTI bacterial strains were isolated from mid-urine samples of 50 males and 50 females. Escherichia coli were more predominant (48%) followed by Klebsilla pneumonia (29%), Pseudomonas aeruginosa (17%), Staphylococcus aureus (4%) and Enterobacter faecalis (6%). The ethyl acetate extract was examined to evaluate antibacterial properties against isolated UTIs bacterial pathogens. The results were revealed that the maximum zone was measured in Escherichia coli (18.1 ± 0.4) mm and minimum zone of inhibition was shown against Pseudomonas aeruginosa (10.6 ± 0.3) mm. Based on the results obtained, the extract of Brevibacillus brevis EGS9 exhibited dose dependent manner of antioxidant activity. The DPPH scavenging activity of lowest concentration at 25 µg/ml and high concentration at 1000 µg/ml was measured at 2.4% and 39.5% respectively. HPTLC finger print profile was showed the active compounds present in crude extract, which may responsible for the antioxidant prospective. These results showed that, the significant antimicrobial properties against pathogen; this work will be helpful to explore the active compound identification in the field of pharmaceutical research and able to produce new drug molecules against pathogens.

An efficient removal of crystal violet dye from waste water by adsorption onto TLAC/Chitosan composite: A novel low cost adsorbent.

A composite of Typha latifolia activated carbon (TLAC) (a novel, low cost absorbent) and chitosan (TLAC/Chitosan composite) was prepared. The composite was characterised using IR spectra, XRD, FESEM and Pore size studies. Its effectivity was tested for the removal of crystal violet dye from aqueous solutions. The effect of pH, dose rate and initial dye concentration was evaluated. The adsorption isotherm, kinetics and thermodynamic parameters were studied. Langmuir and Freundlich isotherm models were found fit effectively for the dye adsorption data in the present study. The adsorption followed pseudo-second order kinetics. The evaluated thermodynamic parameters show a spontaneous and exothermic reaction. Overall, this study indicates TLAC/Chitosan composite as an effective adsorbent for the removal of crystal violet dye from aqueous solutions.

Synthesis and characterization of metallic nanoparticles impregnated onto activated carbon using leaf extract of Mukia maderasapatna: Evaluation of antimicrobial activities.

In the present research, in vitro antimicrobial activity of metallic nanoparticles impregnated on activated carbon (MNPI-AC) was investigated. Activated carbon (AC) was successfully prepared from Fishtail palm Caryota urens seeds by using two surface modification process (i) sulphuric acid treated Caryota urens seeds (SMCUS) (ii) ultrasonic assisted Caryota urens seeds (UACUS). Mukia maderasapatna plant extract was used as reducing agent for the synthesis of metallic nanoparticles. The characterization studies of MNPI - AC were performed by using a UV-visible spectrophotometer and Fourier Transform Infrared Spectroscopic (FT-IR) analyses. Different active functional groups were identified by FTIR studies which were responsible for impregnation of metallic nanoparticles on a surface of AC. The antimicrobial activity of MNPI - AC was examined against four bacterial strains: 2 g positive (Staphylococcus aureus and Staphylococcus epidermidis) and 2 g negative (Pseudomonas aeruginosa and Escherichia coli) and one fungal strain (Candida albicans). Among different MNPs, Pb-AC (UACUS) shows that higher zone of inhibition. These results in the literature showed that MNPI - AC are to be effective for deactivation and inactivation of microbes in an efficient manner.

Relationship between ultrasonographic nerve morphology and severity of diabetic sensorimotor polyneuropathy.

BACKGROUND AND PURPOSE: In the current study, the aim was to characterize the nerve ultrasound cross-sectional areas (CSAs) of type 2 diabetic patients with diabetic sensorimotor polyneuropathy (DSP) of different severities. METHODS: A hundred symptomatic DSP patients and 40 age-matched healthy controls were prospectively recruited. DSP severity was ascertained through the Toronto Clinical Scoring System (TCCS). Nerve electrophysiology and ultrasound were performed on both lower limbs and the non-dominant upper limb. RESULTS: The sural nerve was inexcitable in 19.1% of mild, 40.0% of moderate and 69.0% of severe DSP groups. In contrast, CSAs were measurable in all nerves of DSP patients and were significantly larger compared to controls. Patients with severe DSP had significantly larger ulnar, peroneal, tibial and sural nerves compared to mild DSP patients. By receiver operating characteristic curve analysis, the cut-off value for the sural nerve at 2 mm(2) was a good discriminator (area under the curve 0.88) between the presence and absence of DSP (sensitivity 0.90; specificity 0.74) but performed less well in discriminating between the severity of DSP (cut-off 2.75 mm(2); area under the curve 0.62; sensitivity 0.59; specificity 0.73). Significant correlations were demonstrated between TCSS scores, most neurophysiology parameters and CSAs of the ulnar, peroneal, tibial and sural nerves. CONCLUSION: Nerve ultrasound in DSP reveals enlarged CSAs and these changes worsen with increasing disease severity, thus serving as a useful diagnostic tool especially when neurophysiology is unrevealing.

Inflammasome activity is essential for one kidney/deoxycorticosterone acetate/salt-induced hypertension in mice.

BACKGROUND AND PURPOSE: Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1ß and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1ß and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. EXPERIMENTAL APPROACH: Wild-type and inflammasome-deficient ASC(-/-) mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry. KEY RESULTS: 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro-caspase-1, and the cytokine, pro-IL-1ß, as well as protein levels of active caspase-1 and mature IL-1ß. Following treatment with 1K/DOCA/salt, ASC(-/-) mice displayed blunted pressor responses and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice. CONCLUSIONS AND IMPLICATIONS: Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1ß pathway as a potential therapeutic target in hypertension.

Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke.

Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen-glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1ß and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1ß and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.

Variability in the segregating generation of eggplant for earliness and yield.

In the past, very little efforts have been taken for development of inbred lines of brinjal through the exploitation of genetic variability present in the exotic hybrids. F2 generation obtained from the selfing of F1 hybrid provides all possible variations. So, selection with particular objectives in F2 generation is very much effective and selfing of those selected genotypes generation after generation helps to develop inbred lines (similar to the parental lines of the exotic hybrids). These inbreds with desired characters including high yield potential can be used as High Yielding Variety (HYV) as well as the parents for hybrid variety. To increase the genetic yield potential, the maximum utilization of the desirable characters for synthesizing of any ideal genotypes is essential. Variability in brinjal is expected to be immense as the fruits vary greatly in shape and size. The present investigation was undertaken at Department of Horticulture, Agricultural College and Research Institute, Madurai during 2011 to determine variability in segregants of eggplant (Solanum melogena L.). The crosses L5 x T4 (Palamedu Local x EP 65) and L4 x T1 (Alagarkovil Local x Annamalai) had the highest mean with high variability for individual fruit weight and fruit yield per plant. These crosses were best for using as a base population for further improvement in fruit yield and fruit weight as they had high heritability and genetic advance. Favorable low mean with high variability occurred for days to first flowering (earliness) in the crosses L5 x T2 (Palamedu Local x KKM 1) and L4 x T2 (Alagarkovil Local x KKM 1). Direct selection may be executed considering these genotypes for selection towards the development of early in flowering and high yielding brinjal variety.

Neuroprotection in stroke by complement inhibition and immunoglobulin therapy.

Activation of the complement system occurs in a variety of neuroinflammatory diseases and neurodegenerative processes of the CNS. Studies in the last decade have demonstrated that essentially all of the activation components and receptors of the complement system are produced by astrocytes, microglia, and neurons. There is also rapidly growing evidence to indicate an active role of the complement system in cerebral ischemic injury. In addition to direct cell damage, regional cerebral ischemia and reperfusion (I/R) induces an inflammatory response involving complement activation and generation of active fragments, such as C3a and C5a anaphylatoxins, C3b, C4b, and iC3b. The use of specific inhibitors to block complement activation or their mediators such as C5a, can reduce local tissue injury after I/R. Consistent with therapeutic approaches that have been successful in models of autoimmune disorders, many of the same complement inhibition strategies are proving effective in animal models of cerebral I/R injury. One new form of therapy, which is less specific in its targeting of complement than monodrug administration, is the use of immunoglobulins. Intravenous immunoglobulin (IVIG) has the potential to inhibit multiple components of inflammation, including complement fragments, pro-inflammatory cytokine production and leukocyte cell adhesion. Thus, IVIG may directly protect neurons, reduce activation of intrinsic inflammatory cells (microglia) and inhibit transendothelial infiltration of leukocytes into the brain parenchyma following an ischemic stroke. The striking neuroprotective actions of IVIG in animal models of ischemic stroke suggest a potential therapeutic potential that merits consideration for clinical trials in stroke patients.

Identification and localisation of ATP P2X receptors in rat midbrain.

P2X receptors that are gated by extracellular ATP are among the few known examples of ligand-gated cation-selective channels. There have been seven cloned proteins identified to date as members of the P2X receptor family in a wide range of tissues from the peripheral and central nervous systems and from many species. To determine the distribution of the P2X subtypes in the rat midbrain, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting was combined with immunolocalisation using confocal microscopy. Subtypes P2X1-6 were detected in the periaqueductal gray area and the ependymal layer bordering the ventricle with a widespread distribution.

Influence of grafting a smaller target muscle on the magnitude of naturally occurring trochlear motor neuron death during development.

About half of the motor neurons produced by some neural centers die during the course of normal development. It is thought that the size of the target muscle determines the number of surviving motor neurons. Previously, we tested the role of target size in limiting the number of survivors by forcing neurons to innervate a larger target (Sohal et al., '86). Results did not support the size-matching hypothesis because quail trochlear motor neurons innervating duck superior oblique muscle were not rescued. We have now performed the opposite experiment, i.e., forcing neurons to innervate a smaller target. By substituting the embryonic forebrain region of the duck with the same region of the quail before cell death begins, chimera embryos were produced that had a smaller quail superior oblique muscle successfully innervated by the trochlear motor neurons of the duck. The number of surviving trochlear motor neurons in chimeras was significantly higher than in the normal quail but less than in the normal duck. The smaller target resulted in some additional loss of neurons, suggesting that the target size may regulate neuron survival to a limited extent. Failure to achieve neuron loss corresponding to the reduction in target size suggests that there must be other factors that regulate neuron numbers during development.

Synapse formation on quail trochlear neurons transplanted in duck embryos before naturally occurring motor neuron death.

About half of the trochlear motor neurons in duck and quail embryos die during normal development. In a previous study the role of target muscle in controlling the number of surviving motor neurons was investigated by reducing the number of neurons innervating the muscle. This was accomplished by removing the midbrain of the duck embryo and grafting in its place the midbrain of the quail embryo before motor neuron death begins. It was observed that the number of surviving trochlear motor neurons in the quail-duck chimera embryos was not significantly different from that of the normal quail. The present investigation was undertaken to determine whether trochlear motor neurons in the chimera embryos received afferent synapses. Brains of duck, quail and chimera embryos on days 16 and 20 were processed for electron microscopical observations. Synapses formed on motor neurons of the chimera embryos. Surprisingly, synapses on motor neurons of quail differed from those of duck, both qualitatively and quantitatively. Synapses on the motor neurons of the chimera embryos developed in a fashion similar to that for the duck motor neurons. Our failure to rescue trochlear motor neurons in the chimera embryos suggests that the developing motor neurons may respond to a larger target muscle only if they received a normal complement of afferent synaptic input.

Relationship of early neural tube appearance to myeloschisis in a chick embryo model.

Myeloschisis, a form of neural tube defect involving the spinal cord, was induced in chicks by creating a window in the eggshell of the fertilized egg at 26 h after placement in an incubator. The embryos were stained and photographed through the window at 48 to 60 h after commencement of incubation and the neural tube was determined to be normal, delayed, irregular, or abnormally open for the developmental stage of the embryo. The eggs were then reincubated until 14 days of age. At that time the chicks were determined to have normal spinal cords or myeloschisis by gross examination, dissection, and histology. The appearance of the neural tube at 48 to 60 h was then correlated with the appearance of the spinal cord at 14 days of age. It was determined that in the chick embryo model of myeloschisis induced by windowing, a normal neural tube at the time of neural tube closure results in a 2.3% incidence of myeloschisis, a widely open neural tube a 47% incidence of myeloschisis, an irregular neural tube a 37% incidence of myeloschisis, and delayed closure of the neural tube a 14% incidence of myeloschisis. Thus, no appearance of an early neural tube can be said to always correlate with myeloschisis or a normal outcome. Additionally, it appears that within a single model there may be multiple mechanisms of production of myeloschisis.

Influence of reduced neuron pool on the magnitude of naturally occurring motor neuron death.

The present investigation was undertaken to examine the role of peripheral competition in survival of motor neurons during development. A loss of approximately half of the trochlear motor neurons in duck and quail occurs during the course of normal embryogenesis. The number of motor neurons in the nucleus of quail prior to the onset of cell death is identical to the final number of survivors in the nucleus of duck embryos (about 1,300 neurons). In the present study competition at the peripheral target was decreased by reducing the number of trochlear motor neurons initially projecting to their target muscle. This was accomplished by substituting the midbrain of duck embryos with the same neural tissue from quail embryos. Midbrain transplantation was performed before motor axon outgrowth and normal cell death begin. The development of the motor neurons and their sole target of innervation, the superior oblique muscle, was examined by using a variety of techniques. The source of the grafted motor neurons and of a reduction in the size of the motor neuron pool was confirmed from histological sections and cell counts. The grafted motor neurons projected their axons into the appropriate peripheral target, which was determined by the use of HRP tracing technique. Counts of muscle fibers, motor endplates, and acetylcholine receptors and measurement of total muscle protein indicated that the size of the superior oblique muscle in the chimera embryos was similar to that of the normal duck but significantly larger than the muscle in quail embryos. Electrophysiological observations indicated that the grafted trochlear motor neurons made functional connections with the superior oblique muscle. Counts of the trochlear motor neurons after the period of cell death indicated an average of 1,310 neurons in the nucleus of duck, 772 in quail, and 690 in the chimera embryos. The number of motor neurons in the chimera embryos is not significantly different from that in the normal quail. In other words, in spite of reduced peripheral competition trochlear motor neuron death of normal magnitude occurred. Lack of increased cell survival in our study suggests that trochlear motor neurons do not compete for survival at the peripheral target.

Increased motor neuron projection during development does not increase the number of neuromuscular synapses.

We investigated in developing embryos whether the total number of neuromuscular synapses is determined by the muscle or by the number of innervating motor neurons. The superior oblique muscle of duck embryos was hyperinnervated by preventing the naturally occurring death of trochlear motor neurons using immunoglobulin G from patients with acquired myasthenia gravis. In spite of a significant increase in the number of motor neurons innervating the muscle, a corresponding increase in the number of neuromuscular synapses did not occur. These results suggest that the total number of synapses in a muscle is independent of the number of innervating motor neurons and that it is determined intrinsically by the muscle itself.