Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.     

Probability of internal unwinding in the triple helix of collagen

Helix probability profiles are calculated for collagen models that take into account specific sequence and unwinding from the ends, by internal loops and by chain dissociation, using parameters from a previous study and the technique of the previous paper.² An analysis is made of the occurrence of imino acids in known collagen sequences and no significant difference is found from random occurrence. Studies of model sequences generated by random placement of the imino acids show no tendency for collagen to unwind via internal loops even when one end is covalently linked and a loop is permanently nucleated at that end.     

Livelihoods and Fisheries Governance in a Contemporary Pacific Island Setting

Inshore marine resources play an important role in the livelihoods of Pacific Island coastal communities. However, such reliance can be detrimental to inshore marine ecosystems. Understanding the livelihoods of coastal communities is important for devising relevant and effective fisheries management strategies. Semi-structured household interviews were conducted with householders in Langalanga Lagoon, Solomon Islands, to understand household livelihoods and resource governance in fishing-dependent communities. Households were engaged in a diverse range of livelihoods. Fishing, shell money production and gardening were the most important livelihoods. Proximity to an urban centre influenced how households accessed some livelihoods. Perceptions of management rules varied and different reasons were cited for why rules were broken, the most common reason being to meet livelihood needs. Current models of inshore small-scale fisheries management that are based on the notion of community-based resource management may not work in locations where customary management systems are weak and livelihoods are heavily reliant on marine resources. An important step for fisheries management in such locations should include elucidating community priorities through participatory development planning, taking into consideration livelihoods as well as governance and development aspirations.     

Parallel responses of bees to Pleistocene climate change in three isolated archipelagos of the southwestern Pacific

The impacts of glacial cycles on the geographical distribution and size of populations have been explored for numerous terrestrial and marine taxa. However, most studies have focused on high latitudes, with only a few focused on the response of biota to the last glacial maximum (LGM) in equatorial regions. Here, we examine how population sizes of key bee fauna in the southwest Pacific archipelagos of Fiji, Vanuatu and Samoa have fluctuated over the Quaternary. We show that all three island faunas suffered massive population declines, roughly corresponding in time to the LGM, followed by rapid expansion post-LGM. Our data therefore suggest that Pleistocene climate change has had major impacts across a very broad tropical region. While other studies indicate widespread Holarctic effects of the LGM, our data suggest a much wider range of latitudes, extending to the tropics, where these climate change repercussions were important. As key pollinators, the inferred changes in these bee faunas may have been critical in the development of the diverse Pacific island flora. The magnitude of these responses indicates future climate change scenarios may have alarming consequences for Pacific island systems involving pollinator-dependent plant communities and agricultural crops.     

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.     

Effect of Anakinra on the Gene Expression of Receptors Activated by the Peroxisome Proliferator in the Rat Brain in the Lithium Pilocarpine Model of Epilepsy

Journal of Evolutionary Biochemistry and Physiology - The role of neuroinflammation in the mechanisms of epileptogenesis has been widely discussed in recent years. One of the factors influencing...     

Quantification of gender specific thermal sensory and pain threshold before and after laser needle stimulation]

Quantitative thermal sensory and pain threshold testing (QST) was performed in 29 adult healthy volunteers (mean age 24.2 +/- 2.7 years; range: 18-29 years; 20 females, 9 males) using the Thermal Sensory Analyser TSA-II (Medoc Advanced Medical Systems, Ramat Yishai, Israel, and Minneapolis, Minnesota, USA) before and after laser needle acupuncture and placebo stimulation, respectively. Significant (p < or = 0,001; t-test) gender-specific differences were seen on cold pain threshold analysis. No significant changes in parameters of thermal sensory and pain thresholds were found before and after laser needle or placebo stimulation at acupuncture points for acute pain. However, a trend towards change in the median value of cold pain sensation after laser needle stimulation (p = 0.479; paired t-test; n.s.) was seen within the group of healthy females. The influence of stimulation of acupuncture points for chronic pain on the various parameters needs to be clarified in future studies.     

Targeting of cytochrome b2 into the mitochondrial intermembrane space: specific recognition of the sorting signal.

Cytochrome b2 contains 2-fold targeting information: an amino-terminal signal for targeting to the mitochondrial matrix, followed by a second cleavable sorting signal that functions in directing the precursor into the mitochondrial intermembrane space. The role of the second sorting sequence was analyzed by replacing one, two or all of the three positively charged amino acid residues which are present at the amino-terminal side of the hydrophobic core by uncharged residues or an acidic residue. With a number of these mutant precursor proteins, processing to the mature form was reduced or completely abolished and at the same time targeting to the matrix space occurred. The accumulation in the matrix depended on a high level of intramitochondrial ATP. At low levels of matrix ATP, the mutant proteins were sorted into the intermembrane space like the wild-type precursors. The results: (i) suggest the existence of one or more matrix components that specifically recognize the second sorting signal and thereby trigger the translocation into the intermembrane space; (ii) indicate that the mutant signals have reduced ability to interact with the recognition component(s) and then embark on the default pathway into the matrix by interacting with mitochondrial hsp70 in conjunction with matrix ATP; (iii) strongly argue against a mechanism by which the hydrophobic segment of the sorting sequence stops translocation in the hydrophobic phase of the inner membrane.     

True hermaphroditism in a 46,XY individual, caused by a postzygotic somatic point mutation in the male gonadal sex-determining locus (SRY): molecular genetics and histological findings in a sporadic case.

Recently, the gene for the determination of maleness has been identified in the sex-determining region on the short arm of the Y chromosome (SRY) between the Y-chromosomal pseudoautosomal boundary (PABY) and the ZFY gene locus. Experiments with transgenic mice confirmed that SRY is a part of the testis-determining factor (TDF). We describe a sporadic case of a patient with intersexual genitalia and the histological finding of ovotestes in the gonad, which resembles the mixed type of gonadal tissue without primordial follicle structures. The karyotype of the patient was 46,XY. By PCR amplification, we tested for the presence of PABY, SRY, and ZFY by using DNA isolated from peripheral blood leukocytes and for the presence of SRY by using DNA obtained from histological gonadal slices. The SRY products of both DNA preparations were further analyzed by direct sequencing. All three parts of the sex-determining region of the Y chromosome could be amplified from leukocytic DNA. The patient's and the father's SRY sequences were identical with the published sequence. In the SRY PCR product of gonadal DNA, the wild-type and two point mutations were present in the patient's sequence, simulating a heterozygous state of a Y-chromosomal gene: one of the mutations was silent, while the other encoded for a nonconservative amino acid substitution from leucine to histidine. Subcloning procedures showed that the two point mutations always occurred together. The origin of the patient's intersexuality is a postzygotic mutation of the SRY occurring in part of the gonadal tissue. This event caused the loss of the testis-determining function in affected cells.