Caudal Brainstem Raphe Nuclei: Neural Substrate for their Involvement in the Expression of Some Biological Rhythms

Nucleus raphe pallidus (RPa) lies ventrally in the caudal brainstem, where it is coextensive rostrally with the nucleus raphe magnus (RMg) and caudally with the nucleus raphe obscurus (ROb). Retrograde neuronal tracing studies of our laboratory, carried out in rats and presented elsewhere, with fluorogold, true-blue or fast-blue, iontophoretically injected or by crystalline deposit, along the RPa extent, displayed many labeled pericaria at the preoptic area (POA), as well as lateral (LH) and dorsomedial (DMH) hypothalamus; paraventricular nucleus (PVN) and dorsal (DR) and median (MnR) raphe nuclei among others structures. In addition, RPa, which projects to the intermediolateral column, has been demonstrated to bear relation to many of the somatic-visceral functions also reported for POA. Iontophoretic injections of PHA-L, an anterograde tracer, in the POA subnuclei, presented terminal and varicose labeled fibers in RPa, as well as in the RMg, ROb, paraventricular thalamic (PVA), PVN and supraoptic nucleus (SO), LH, subparaventricular zone (sPVZ) and locus coeruleus (LC). Interestingly, POA, PVA, PVN, LH and SO have been described as retino- and suprachiasmatic-recipients. Taken together, these neuronal connections between brainstem raphe nuclei and POA, the similarity of functions to which they are related, as well as connections with other retino-suprachiasmatic-recipient structures, suggest that these caudal brainstem raphe nuclei could be part of the output system for the expression of some biological rhythms.     

A whole genome RNAi screen of Drosophila S2 cell spreading performed using automated computational image analysis

Recent technological advances in microscopy have enabled cell-based whole genome screens, but the analysis of the vast amount of image data generated by such screens usually proves to be rate limiting. In this study, we performed a whole genome RNA interference (RNAi) screen to uncover genes that affect spreading of Drosophila melanogaster S2 cells using several computational methods for analyzing the image data in an automated manner. Expected genes in the Scar-Arp2/3 actin nucleation pathway were identified as well as casein kinase I, which had a similar morphological RNAi signature. A distinct nonspreading morphological phenotype was identified for genes involved in membrane secretion or synthesis. In this group, we identified a new secretory peptide and investigated the functions of two poorly characterized endoplasmic reticulum proteins that have roles in secretion. Thus, this genome-wide screen succeeded in identifying known and unexpected proteins that are important for cell spreading, and the computational tools developed in this study should prove useful for other types of automated whole genome screens.     

Tidal flushing of ammonium from intertidal sediments of Ria Formosa,Portugal

Ammonium concentrations were determined in near-bottom water and intertidal surface sediments collected in February and July 1993 at five stations of Ria Formosa, a shallow meso-tidal coastal lagoon in southern Portugal. At each station, samples were taken a few minutes before tidal inundation, and subsequently 2, 10, 15 and 20 minutes thereafter. Ammonium concentration in near-bottom waters increased dramatically in the first 2 minutes followed by a decrease during the 18 minutes of flooding (maximum range 10.3-2.2 M). The highest levels in the flooding water were concomitant with a decrease of extractable ammonium recorded in the upper sediment layer (2 cm). Laboratory experiments indicated that ammonium is easily extracted from the sediment solids by physical perturbation, as one would expect when tidal water flushes over the intertidal area. This perturbation results in the export of ammonium from the sediment, by pulse mechanisms of short time intervals. On a daily scale this amount is two orders of magnitude higher than transport resulting from molecular diffusion.     

Creating opportunities for science PhDs to pursue careers in high school education

The United States is confronting important challenges at both the early and late stages of science education. At the level of K–12 education, a recent National Research Council report (Successful K–12 STEM Education) proposed a bold restructuring of how science is taught, moving away from memorizing facts and emphasizing hands-on, inquiry-based learning and a deeper understanding of the process of science. At higher levels of training, limited funding for science is leading PhDs to seek training and careers in areas other than research. Might science PhDs play a bigger role in the future of K–12 education, particularly at the high school level? We explore this question by discussing the roles that PhDs can play in high school education and the current and rather extensive barriers to PhDs entering the teaching profession and finally suggest ways to ease the entrance of qualified PhDs into high school education.In many K–12 classrooms, science is presented as a series of textbook facts; students are not exposed to scientific methods of inquiry and lose interest in science. At the very opposite end of the science training pipeline, life science PhDs and postdocs in the United States are experiencing difficulties in finding university jobs, a situation that will likely persist in the coming decade if research funding fails to grow; we cannot expect all PhD graduates to become principal investigators (PIs) at academic institutions.Might these two problems add up to a solution (or at least a partial solution)? Is there a place for graduates of PhD training programs in teaching K–12 science, particularly at the high school (HS) level (the focus of this article)? We argue that the answer is “yes” and that more PhDs, even if their numbers are small compared with the entire teaching pool, could have a catalytic effect on reinvigorating precollege science education. This topic is not new; the National Research Council (NRC) issued two thoughtful reports on attracting science and math PhDs to secondary school education more than a decade ago (Committee on Attracting Science and Mathematics Ph.D.s to Secondary School Teaching, National Research Council, 2000 ; Committee on Attracting Science and Mathematics PhDs to K-12 Education: From Analysis to Implementation, Division of Policy and Global Affairs, National Research Council, 2002 ). Their recommendations were not implemented, however, and the reports have largely been forgotten. Little has changed since then; the roadblocks, both in perception and logistics, that discouraged a PhD from becoming a HS teacher in the year 2000 still exist. Since the NRC reports were released, the topic of a HS teaching career option for a PhD has rarely been discussed or debated in our scientific community. We feel that it is time to reopen this discussion. The focus of this article is on PhDs entering the high school system, but much of this discussion also pertains to graduates of science master degree programs and to individuals with scientific training becoming involved in all levels of K–12 education. Our goal is to make students, postdocs, and senior scientists aware of the value of high school teaching for certain individuals as well as for our nation''s educational system. We also consider how changes at the local level (including the perception of K–12 teaching within research universities), as well as at the policy level of teacher accreditation, might facilitate this career path.     

Three new species,four new records and an updated checklist of Habenaria (Orchidaceae) from Rio Grande do Sul,Brazil

Three new species of Habenaria (Orchidaceae) section Nudae from Rio Grande do Sul, southern Brazil, are described and illustrated: Habenaria australis, H. kleinii and H. sobraliana. These are the first records of H. sect. Nudae for the state. Based on the examination of living and dried specimens, a total of 33 species and 400 collections of Habenaria were recorded for Rio Grande do Sul. Based on this survey, an updated checklist of the genus Habenaria for Rio Grande do Sul has been compiled. Four species are known only from this state, while seven other species are restricted to southern Brazil, Argentina and Uruguay. Habenaria hieronymi, previously known only from Argentina, is recorded for the first time from Brazil, and H. brachyphyton, H. ekmaniana and H. melanopoda are new records for Rio Grande do Sul.     

Expression and Functional Characterization of Membrane-Integrated Mammalian Corticotropin Releasing Factor Receptors 1 and 2 in Escherichia coli

Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors'' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2β -PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2β expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of E. coli bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2β product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2β were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2β membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our E. coli expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.     

A standardized kinesin nomenclature

In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.     

A reinvestigation of the Gn-RH (gonadotrophin-releasing hormone) systems in the goldfish brain using antibodies to salmon Gn-RH

Summary The organization of Gn-RH systems in the brain of teleosts has been investigated previously by immunohistochemistry using antibodies against the mammalian decapeptide which differs from the teleostean factor. Here, we report the distribution of immunoreactive Gn-RH in the brain of goldfish using antibodies against synthetic teleost peptide.Immunoreactive structures are found along a column extending from the rostral olfactory bulbs to the pituitary stalk. Cell bodies are observed within the olfactory nerves and bulbs, along the ventromedial telencephalon, the ventrolateral preoptic area and the latero-basal hypothalamus. Large perikarya are detected in the dorsal midbrain tegmentum, immediately caudal to the posterior commissure. A prominent pathway was traced from the cells located in the olfactory nerves through the medial olfactory tract and along all the perikarya described above to the pituitary stalk. In the pituitary, projections are restricted to the proximal pars distalis. A second immunoreactive pathway ascends more dorsally in the telencephalon and arches to the periventricular regions of the diencephalon. Part of this pathway forms a periventricular network in the dorsal and posterior hypothalamus, whereas other projections continue caudally to the medulla oblongata and the spinal cord. Lesions of the ventral preoptic area demonstrate that most of the fibers detected in the pituitary originate from the preoptic region.     

Antinociceptive and anti-inflammatory effects of electroacupuncture on experimental arthritis of the rat temporomandibular joint

This study investigated the antinociceptive and anti-inflammatory effects of electroacupuncture (EA) on zymosan-induced acute arthritis of the rat temporomandibular joint (TMJ). Male Wistar rats were injected with saline or zymosan (control group; 2?mg) into the left TMJ. Low frequency EA (10?Hz, 30?min) was performed at acupoints (LI4, LI11, ST36, ST44) or sham points 2?h after or 1?h before zymosan administration. Mechanical hypernociception was accessed by the electronic Von Frey method after zymosan administration. Rats were sacrificed 6?h after zymosan administration and the joint was removed for histopathological analysis, myeloperoxidase activity assessment, vascular permeability observations, and immunohistochemical verification of inflammatory mediators. The results showed that EA inhibited zymosan-induced hypernociception, compared with the control group and with the sham group (p?< 0.05). The results showed that EA inhibited inflammatory parameters such as neutrophil migration, vascular permeability, and tumour necrosis factor α (TNF-α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) expression in the TMJ compared with the sham group (p < 0.05). Histopathological analysis showed that EA significantly inhibited edema and periarticular infiltration (p?< 0.05) compared with the control and sham groups. EA at acupoints produced antinociceptive and anti-inflammatory effects on zymosan-induced arthritis in the rat TMJ.     

Heterologous expression of leader-less <Emphasis Type="Italic">pga</Emphasis> gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis>: intracellular production of prokaryotic enzyme

Background  

Penicillin G acylase of Escherichia coli (PGA_Ec) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of β-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGA_Ec in this host and studied yeast production capacity and enzyme authenticity.