Unusual repertoire of vocalizations in the BTBR T+tf/J mouse model of autism

BTBR T+ tf/J (BTBR) is an inbred mouse strain that displays social abnormalities and repetitive behaviors analogous to the first and third diagnostic symptoms of autism. Here we investigate ultrasonic vocalizations in BTBR, to address the second diagnostic symptom of autism, communication deficits. As compared to the commonly used C57BL/6J (B6) strain, BTBR pups called more loudly and more frequently when separated from their mothers and siblings. Detailed analysis of ten categories of calls revealed an unusual pattern in BTBR as compared to B6. BTBR emitted high levels of harmonics, two-syllable, and composite calls, but minimal numbers of chevron-shaped syllables, upward, downward, and short calls. Because body weights were higher in BTBR than B6 pups, one possible explanation was that larger thoracic size was responsible for the louder calls and different distribution of syllable categories. To test this possibility, we recorded separation calls from FVB/NJ, a strain with body weights similar to BTBR, and 129X1/SvJ, a strain with body weights similar to B6. BTBR remained the outlier on number of calls, displaying low numbers of complex, upward, chevron, short, and frequency steps calls, along with high harmonics and composites. Further, developmental milestones and growth rates were accelerated in BTBR, indicating an unusual neurodevelopmental trajectory. Overall, our findings demonstrate strain-specific patterns of ultrasonic calls that may represent different lexicons, or innate variations in complex vocal repertoires, in genetically distinct strains of mice. Particularly intriguing is the unusual pattern of vocalizations and the more frequent, loud harmonics evident in the BTBR mouse model of autism that may resemble the atypical vocalizations seen in some autistic infants.     

Effect of parenteral steroids on edema in replanted rat legs

The experiments described demonstrate that parenteral steroid therapy will decrease the amount of postoperative edema in the replanted leg of a rat.     

Long-term effects of grassland management on soil microbial abundance: implications for soil carbon and nitrogen storage

Grassland management intensification can significantly affect the structure and composition of important soil microbial groups such as bacteria and fungi. Changes to these microbial communities can greatly influence carbon (C) and nitrogen (N) cycling in grassland soils. Here we specifically address how microbial abundances might shift under the effect of multiple management practices and how this in turn might relate to changes in soil C and N storage. Soil samples were collected from a 23-year-old grassland experiment and real-time quantitative Polymerase Chain Reaction (PCR) was performed to address whether and how (1) chronic nutrient additions, (2) liming (i.e., the addition of CaCO₃ to soils), and (3) grazing by rabbits might affect archaeal, bacterial and fungal microbial groups. We found that liming additions significantly increased archaeal and bacterial abundance whilst strongly reducing fungal abundance. The addition of N-only (as NH₄NO₃) significantly reduced bacterial abundance while chronic grazing by rabbits resulted in positive effects on archaeal abundance. Despite long-term grassland management significantly affecting soil microbial abundances (and Fungal-to-Bacterial ratios), microbial changes were not related to either changes in soil C or N pools. Overall, our results suggest that (1) important microbial-‘soil functioning’ relationships may only be detected at lower taxonomic levels, and (2) liming-induced increases in soil pH determined significant shifts in soil microbial abundance, which could have important consequences for the delivery of multiple soil ecosystem services (i.e., nutrient regulation, C and N sequestration) from permanent grassland.     

Behavioral investigation of the coexistence of substance P,corticotropin releasing factor,and acetylcholinesterase in lateral dorsal tegmental neurons projecting to the medial frontal cortex of the rat

Colocalization of substance P (SP), corticotropin releasing factor (CRF), and acetylcholinesterase (AChE) was detected by retrograde tracing and immunocytochemical staining in the nucleus tegmentalis dorsalis lateralis (ntdl) projecting to the medial frontal cortex (MFC), septum, and thalamus of the rat. The histochemical results suggest that SP and CRF coexist within a subpopulation of ntdl cholinergic neurons that project to a number of forebrain regions including the MFC. Behavioral studies of the effects of SP, CRF, and the cholinergic agonist, carbachol, employed microinjections into the MFC of rats. SP and CRF did not elicit any behavioral effects when administered alone. Carbachol (1–5 μg/side) produced a stereotyped motor behavior, consisting of rapid forepaw treading while in an upright posture, resembling “boxing.” SP (1 μg/side) increased carbachol-induced “boxing.” CRF (1–10 ng/side) decreased carbachol-induced “boxing.” One possible functional significance of the coexistence of SP, CRF, and acetylcholinesterase, in neurons projecting to the medial frontal cortex in rats, appears to be a modulatory potentiation of cholinergic response by SP, and a modulatory inhibition of the cholinergic response by CRF.     

Effect of time post mortem on the concentration of endotoxin in rat organs: implications for sudden infant death syndrome (SIDS)

The aim of the study was to test the following hypotheses: (i) that endotoxin injected 40 min prior to death can be detected in rat organs post mortem and (ii) that endotoxin levels do not change with increasing time post mortem. Rats were injected with or without endotoxin in buffered saline, 40 min prior to being killed. Endotoxin levels in rat organs were assessed using a Limulus amoebocyte assay. The effect of storage time post mortem was assessed by following various storage regimes at 25 degrees C and 8 degrees C. Significant differences (P = < 0.001) in endotoxin levels of all samples tested were found between rats injected with and without endotoxin. A significant increase in detectable endotoxin was observed between 0 h and 6 h post mortem in rats injected with or without endotoxin. No difference in detectable endotoxin levels in the kidney, liver and spleen was observed from 30 h to 102 h post mortem in rats injected with or without endotoxin. In rats injected with endotoxin, detectable endotoxin levels in the heart were raised between 0 h and 6 h, 6 h and 54 h, and 30 h and 78 h. Endotoxin injected into rats 40 min prior to death can be detected post mortem. For rats injected with saline or endotoxin prior to death levels in the kidney, liver and spleen were not affected by storage at 8 degrees C for 30-102 h, after initial storage at room temperature for 6 h. Levels of endotoxin detected in the hearts of rats injected with saline were not affected by storage up to 102 h. In rats injected with endotoxin prior to death, detectable levels in the heart were significantly affected by increasing time in storage.     

Genomic organization and structure of the 3' region of human MUC3: alternative splicing predicts membrane-bound and soluble forms of the mucin.

The MUC3 gene encodes a large, glycosylated mucin produced by intestinal epithelial cells to form a protective barrier against the external environment. Recently published cDNA sequences for the carboxyl-terminal region of MUC3 polypeptide indicated that rodent Muc3 possesses two epidermal growth factor (EGF)-like domains, and putative transmembrane and cytoplasmic domains, whereas the sequence of human MUC3 predicted termination after the first EGF-like domain. Here we describe the complete genomic sequence encompassing the carboxyl terminal region of human MUC3, revealing the boundaries of 11 exons. RT-PCR and cDNA library cloning experiments indicate that the gene is alternatively spliced, yielding a major membrane-bound form as well as multiple soluble forms. Thus, this work indicates that both membrane-bound and soluble MUC3 mucin proteins are produced by alternative splicing of a single gene. A potentially important polymorphism involving a Tyr residue with a proposed role in signalling is described as well.     

Development of a Quantitative Bead Capture Assay for Soluble IL-7 Receptor Alpha in Human Plasma

Background

IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Rα (CD127) and common γ (CD132) chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. Previous reports showed the usefulness of CD127 as a prognostic marker in viral infections such as HIV, CMV, EBV and HCV. A soluble CD127 (sCD127) is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. Measuring sCD127 is important to define its role and may complement existing markers used in lymphopenic disease management. We describe a new quantitative assay for the measurement of sCD127 in plasma and report sCD127 concentrations in healthy adults.

Methodology/Principal Findings

We developed a quantitative bead-based sCD127 capture assay. Polyclonal CD127-specific antibodies were chosen for capture and a biotinylated monoclonal anti-CD127 antibody was selected for detection. The assay can detect native sCD127 and recombinant sCD127 which served as the calibrator. The analytical performance of the assay was characterized and the concentration and stability of plasma sCD127 in healthy adults was determined. The assay''s range was 3.2–1000 ng/mL. The concentration of plasma sCD127 was 164±104 ng/mL with over a log variation between subjects. Individual sCD127 concentrations remained stable when measured serially during a period of up to one year.

Conclusions/Significance

This is the first report on the quantification of plasma sCD127 in a population of healthy adults. Soluble CD127 plasma concentrations remained stable over time in a given individual and sCD127 immunoreactivity was resistant to repeated freeze-thaw cycles. This quantitative sCD127 assay is a valuable tool for defining the potential role of sCD127 in lymphopenic diseases.     

Identification of calmodulin and MlcC as light chains for Dictyostelium myosin-I isozymes

Dictyostelium discoideum express seven single-headed myosin-I isozymes (MyoA-MyoE and MyoK) that drive motile processes at the cell membrane. The light chains for MyoA and MyoE were identified by expressing Flag-tagged constructs consisting of the motor domain and the two IQ motifs in the neck region in Dictyostelium. The MyoA and MyoE constructs both copurified with calmodulin. Isothermal titration calorimetry (ITC) showed that apo-calmodulin bound to peptides corresponding to the MyoA and MyoE IQ motifs with micromolar affinity. In the presence of calcium, calmodulin cross-linked two IQ motif peptides, with one domain binding with nanomolar affinity and the other with micromolar affinity. The IQ motifs were required for the actin-activated MgATPase activity of MyoA but not MyoE; however, neither myosin exhibited calcium-dependent activity. A Flag-tagged construct consisting of the MyoC motor domain and the three IQ motifs in the adjacent neck region bound a novel 8.6 kDa two EF-hand protein named MlcC, for myosin light chain for MyoC. MlcC is most similar to the C-terminal domain of calmodulin but does not bind calcium. ITC studies showed that MlcC binds IQ1 and IQ2 but not IQ3 of MyoC. IQ3 contains a proline residue that may render it nonfunctional. Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain. The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme.