Autoradiography of nucleoside uptake into the retina

A selective uptake mechanism for some nucleosides and related substances was found in retinae of light adapted rabbits and fish. After the intravitreal injection in vivo of [³H]adenosine, [³H]inosine, [³H]guanosine and certain related compounds, the distribution of radioactivity was studied by autoradiography. Retinae were also incubated in [³H]adenosine and [³H]inosine and then were similarly processed.In rabbits, the accumulation of radioactivity from [³H]adenosine and [³H]guanosine was predominantly into glial cells, but also into neurons. [³H]Inosine labelled glia almost exclusively. However, the adenosine analog, [³H]methylphenylethyl-adenosine, resulted in well-defined neuronal labelling in this species. In fish, a few photoreceptor cell bodies exhibited strong radioactivity with the nucleosides, presumably representing incorporation into nucleic acids of replicating cells. Labelling was also seen in horizontal cells, amacrine cells and ganglion cells after the injection of either [³H]adenosine, [³H]guanosine or [³H]inosine.To some extent, the selective accumulation of radioactivity is likely to be due to cell replication, but in most neurons, other factors must be responsible. Judging from what is known about the actions of adenosine in central nervous tissue, signal transmission in the retina could be such a factor.     

Possible Mechanism of Dantrolene Stabilization of Cultured Neuroblastoma Cell Plasma Membranes

Abstract: Some reports have suggested that dantrolene interacts directly with the membrane bilayer. We investigated effects of dantrolene on changes in membrane properties induced by compound 48/80 (C48/80), a membrane stimulator. The addition of C48/80 for 1 min elicited a rapid, dose-dependent Ca²⁺ influx, which was reduced to 14% by the absence of external Ca²⁺. Dantrolene inhibited the C48/80-induced increase in Ca²⁺ permeability of plasma membranes in a concentration-dependent manner (0.33–10 µ M , IC₅₀ value was 5 µ M ). We next examined C48/80-induced changes in structural and dynamic membrane properties by electron spin resonance (ESR). The ratio h ₀/ h ₋₁ was determined to evaluate membrane fluidity. C48/80 increased the membrane fluidity in a concentration-dependent manner (0.1–0.56 mg/ml). Dantrolene (10 µ M ) itself did not change the membrane fluidity, but it significantly reduced the C48/80-induced increase in membrane fluidity (0.56 mg/ml). Moreover, the C48/80-induced increase in fluidity was dependent on extracellular Ca²⁺. We conclude that dantrolene protects neuroblastoma cell plasma membrane from C48/80-induced membrane perturbation, which causes Ca²⁺ influx and an increase in membrane fluidity. These findings strongly suggest that dantrolene directly stabilizes the neuronal plasma membrane.     

Neurotensin. Immunohistochemical localization in central and peripheral nervous system and in endocrine cells and its functional role as neurotransmitter and endocrine hormone

The present study attempts to compile information on the possible physiologic role of the endogenous peptide neurotensin (NT) as a hormone and/or neurotransmitter. The methodological approach is immunohistochemical localization of NT in the entero-endocrine system as well as in the central and peripheral nervous systems. The results found in the three systems are first related to the pharmalogical and physiological findings in the literature. Subsequently their significance is discussed for each organ separately before attempting a final overall interpretation. Briefly, the present study reveals the following essential findings: The occurrence and distribution of NT-IR entero-endocrine cells (N-cells) in different mammals including man, as well as in representative members of all classes of vertebrates and higher invertebrates, are analyzed and evaluated morphometrically. The NT-IR cells in all investigated species are demonstrated to be of the open type. The innervation of paravertebral and prevertebral ganglia by NT-IR fibers is described; at least a portion of these fibers is thought to originate in NT-IR perikarya of the substantia intermedia of the spinal cord. The involvement of these NT-IR fibers in the regulation of systemic blood flow (hypertension) is suggested. The existence of NT-IR innervation of the gastro-intestinal tract is considered to be a general phenomenon. This notion is reaffirmed by phylogenetic investigation of the NT-IR enteric nerves. The pharmacological effects of NT in different portions of the gastro-intestinal tract, reported in the literature are related to the immunohistochemical localization of NT. In light of the present results, some of the effects of NT which were previously considered to be of an endocrine or paracrine nature - such as contraction of the guinea-pig ileum - are interpreted as effects of NT of neuronal origin. The specific NT-IR innervation of target cells in the exocrine pancreas (vascular smooth muscle, acinar cells) is demonstrated, and participation of NT-IR nerve fibers in regulation of the secretion of pancreatic juice is postulated. The innervation of the heart (coronary vasculature, myocardium, conduction system) by NT-IR fibers is demonstrated in various mammals and for the first time also in man. The cardiac NT-IR nerve fibers are thought to be the cytological substrate for different NT effects on heart action (coronary vasoconstriction, positive inotropy and chronotropy) reported in the literature.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of target proteins of clinical immunity to Plasmodium falciparum in a region of low malaria transmission

The target molecules of antibodies against falciparum malaria remain largely unknown. Recently we have identified multiple proteins as targets of immunity against Plasmodium falciparum using African serum samples. To investigate whether potential targets of clinical immunity differ with transmission intensity, we assessed immune responses in residents of low malaria transmission region in Thailand. Malaria asymptomatic volunteers (Asy: n = 19) and symptomatic patients (Sym: n = 21) were enrolled into the study. Serum immunoreactivity to 186 wheat germ cell-free system (WGCFS)-synthesized recombinant P. falciparum asexual-blood stage proteins were determined by AlphaScreen, and subsequently compared between the study groups. Forty proteins were determined as immunoreactive with antibody responses to 35 proteins being higher in Asy group than in Sym group. Among the 35 proteins, antibodies to MSP3, MSPDBL1, RH2b, and MSP7 were significantly higher in Asy than Sym (unadjusted p < 0.005) suggesting these antigens may have a protective role in clinical malaria. MSP3 reactivity remained significantly different between Asy and Sym groups even after multiple comparison adjustments (adjusted p = 0.033). Interestingly, while our two preceding studies using African sera were conducted differently (e.g., cross-sectional vs. longitudinal design, observed clinical manifestation vs. functional activity), those studies similarly identified MSP3 and MSPDBL1 as potential targets of protective immunity. This study further provides a strong rationale for the application of WGCFS-based immunoprofiling to malaria vaccine candidate and biomarker discovery even in low or reduced malaria transmission settings.     

Molecular cloning of cDNA from foot-and-mouth disease virus C1-Santa Pau (C-S8). Sequence of protein-VP1-coding segment

cDNA segments copied from the RNA of foot-and-mouth disease virus (FMDV) C₁-Santa Pau (isolate C-S8) have been cloned in plasmid pBR322. A 998-bp DNA fragment, that includes the region coding for capsid protein VP1, the carboxy terminus of VP3, and the amino terminus of precursor protein p52 has been sequenced. Comparison of the nucleotide sequence with those from FMDV O₁K, A₁₀61, a₁₂ and C₃ Indaial (Kurz et al., Nucl. Acids Res. 9 (1981) 1919–1931; Kleid et al., Science 214 (1981) 1125–1129; Boothroyd et al., Gene 17 (1982) 153–161; Makoff et al., Nucl. Acids Res. 10 (1982) 8285–8295) indicates extensive variability between the corresponding gene segments, including short insertions and deletions. Base transversions are more frequent than transitions within the VP1 coding segment, but not in the sequence coding for the amino-terminal end of p52. The nucleotide sequence divergence is reflected in variability in both the primary and the predicted higher-order structures of the encoded VP1s.     

Effects of p-fluorophenylalanine on the induction of mutations in bacteriophage T4: I. 5-Bromouracil mutagenesis

The amino acid analogue p-fluorophenylalanine (PFPA) was found to have no mutagenic activity in the r system of bacteriophage T4. However, under standard conditions for 5-bromouracil (5-BU) mutagenesis, PFPA depressed the induced frequencies for both forward and reverse mutations. When the folate antagonist sulphanilamide (SU) was omitted from the mutagenic treatment medium or when it was replaced by Trimethoprim (TM), another folate antagonist, this depressive effect was abolished. It was proposed that PFPA alleviated the inhibitory action of SU.     

Fluoxetine, 17-β estradiol or folic acid combined with intra-lateral septal infusions of neuropeptide Y produced antidepressant-like actions in ovariectomized rats forced to swim

Folic acid is antidepressant, either alone or combined with several antidepressant drugs. However, the antidepressant-like actions of folic acid combined with intra-lateral septal (LSN) infusions of neuropeptide Y (NPY) in the forced swimming test (FST) have not been tested before. Thus, systemic injections of fluoxetine (20.0 mg/kg, P < 0.05; s.c.) or 17-β estradiol (10.0 μg/rat, P < 0.05; s.c.) or oral administrations of folic acid (50.0 mg/kg, P < 0.05; 75.0 mg/kg, P < 0.05) or NPY intra-LSN (3.0 μg, P < 0.05; 3.5 μg, P < 0.05) reduced immobility of ovariectomized Wistar rats. Subthreshold doses of: folic acid (25.0 mg/kg) or 17-β estradiol (5.0 μg/rat, P < 0.05) or fluoxetine (15.0 mg/kg, P < 0.05; s.c.) combined with subthreshold doses of NPY (2.5 μg/rat, P < 0.05; intra-LSN) and these combinations produced antidepressant-like actions; which were canceled by BIBP 3226 (a NPY-Y1 receptor antagonist). It is concluded that folic acid produced antidepressant-like effects probably through the participation of the NPY Y1 receptors found in the lateral septal nuclei.     

β1-Integrin is up-regulated via Rac1-dependent reactive oxygen species as part of the hypertrophic cardiomyocyte response

β₁-Integrin mediates cardiomyocyte growth and survival and its proper regulation is essential for the structural and functional integrity of the heart. β₁-Integrin expression is enhanced in hypertrophy, but the mechanism and significance of its up-regulation are unknown. Because reactive oxygen species (ROS) are important mediators of myocardial remodeling we examined their role in regulated β₁-integrin expression. Hypertrophy was induced in neonatal cardiomyocytes by endothelin-1 (ET-1), which activated the regulatory NADPH oxidase subunit Rac1, evoked ROS, and enhanced fetal gene expression and cardiomyocyte size. ET-1 also enhanced cell adhesion and FAK phosphorylation and inhibited oxidative stress-induced cardiomyocyte apoptosis. Further, ET-1 increased β₁-integrin mRNA and protein expression via Rac1-ROS-dependent MEK/ERK and EGF receptor-PI3K/Akt activation as shown by adenoviral dominant-negative Rac1 or overexpression of copper/zinc-superoxide dismutase. The relevance of regulated β₁-integrin expression was examined in cardiomyocytes, in which targeting siRNA impeded the ET-1-induced β₁-integrin up-regulation. In these cells, ET-1-induced cell adhesion, FAK phosphorylation, and hypertrophic response were significantly blunted, whereas its antiapoptotic effect was predominantly unchanged, suggesting at least partial dissociation of prohypertrophic and prosurvival signaling elicited by ET-1. In conclusion, β₁-integrin up-regulation in response to ET-1 is mediated via Rac1-ROS-dependent activation of prohypertrophic pathways and is mandatory for ET-1-induced FAK activation, cell adhesion, and hypertrophic response.     

Microcomputed tomography used to link head morphology and observed feeding behavior in cichlids of Lake Malaŵi

Cichlids inhabiting the Great Lakes of Africa have radiated extremely rapidly, with Lake Malaŵi harboring some 850 species. This rapid radiation may be linked to the diversity in behaviors, sexual selection, and phenotypic plasticity. To determine the relationships between morphology and behaviors, microcomputed tomography (microCT) was used to observe internal morphological structures. Observed morphological adaptations were linked with observed behavior of cichlids in Lake Malaŵi with respect to the various available food resources. Many of these adaptations have parallels, sometimes into the finest details, in other drainage systems and can thus be considered as variations of how cichlids in general respond to environmental opportunities and challenges. Variations in the structure and teeth of the pharyngeal jaws and the oral jaws allowed for fine tuning of specializations, so that various species can utilize the same source without direct competition. We suggested that high‐resolution X‐ray computed tomography will permit scientists to infer life history and behavior characters of rare or extinct taxa from a detailed examination of morphology and linkages between morphology and behavior found in extant species.     

Identification of roots in lava tube caves using molecular techniques: implications for conservation of cave arthropod faunas

Lava tube cave ecosystems on the volcanic islands of Hawai‘i support communities of rare and highly specialized cave arthropods. In these cave ecosystems, plant roots, both living and dead, provide the main energy source for cave animals. Loss of deep-rooted plants over caves will affect populations of cave-adapted animals living below. Furthermore, the loss of native plant species will likely eliminate host specific cave animals. Thus, identification of plant roots currently found in caves is necessary for the development of effective management actions that encourage the growth of appropriate deep-rooted plant species, thereby protecting the underlying cave ecosystem. We used molecular techniques to identify plant roots found within cave ecosystems on the islands of Maui and Hawai‘i. Sequences of the internal transcribed spacer (ITS) regions and the 5.8S gene of nuclear ribosomal DNA from cave roots were compared to sequences of known plant species either collected on the surface over the footprint of each cave or to sequences accessioned in GenBank. Roots in the cave ecosystem studied on Maui belonged to two alien tree species: Eucalyptus tereticornis and Grevillea robusta. Within the Hawai‘i cave ecosystem, roots of two plant species were identified: the alien tree G. robusta and the native vine Cocculus orbiculatus. The Maui cave ecosystem supports populations of at least 28 species of arthropods, including eight that are blind obligate cave inhabitants. The Hawai‘i cave ecosystem supports 18 arthropod species, of which three are cave-adapted. Creating protected reserves around biologically significant caves, controlling, and preventing the introduction of harmful invasive plant species within the cave footprint, and encouraging the establishment of deep-rooted native plant species is essential for the continued survival of the unique ecosystems found within Hawaiian lava tube cave systems.