Nanoarchaeal 16S rRNA gene sequences are widely dispersed in hyperthermophilic and mesophilic halophilic environments

The Nanoarchaeota, proposed as the fourth sub-division of the Archaea in 2002, are known from a single isolate, Nanoarchaeum equitans, which exists in a symbiotic association with the hyperthermophilic Crenarchaeote, Ignicoccus. N. equitans fails to amplify with standard archaeal 16S PCR primers and can only be amplified using specifically designed primers. We have designed a new set of universal archaeal primers that amplify the 16S rRNA gene of all four archaeal sub-divisions, and present two new sets of Nanoarchaeota-specific primers based on all known nanoarchaeal 16S rRNA gene sequences. These primers can be used to detect N. equitans and have generated nanoarchaeal amplicons from community DNA extracted from Chinese, New Zealand, Chilean and Tibetan hydrothermal sites. Sequence analysis indicates that these environments harbour novel nanoarchaeal phylotypes, which, however, do not cluster into clear phylogeographical clades. Mesophilic hypersaline environments from Inner Mongolia and South Africa were analysed using the nanoarchaeal-specific primers and found to contain a number of nanoarchaeal phylotypes. These results suggest that nanoarchaeotes are not strictly hyperthermophilic organisms, are not restricted to hyperthermophilic hosts and may be found in a large range of environmental conditions.     

Effect of Food Restriction on Ghrelin in Normal‐Cycling Female Rats and in Pregnancy

Objective: Ghrelin is a 28‐amino‐acid acylated peptide that was recently identified as the endogenous ligand for the growth hormone secretagogue receptor. Previous studies have shown that ghrelin potently increases growth hormone release and food intake. The aim of this study was to clarify the physiological implications of ghrelin in the regulation of energy balance, by assessing the effect of undernutrition throughout 21 days in normal‐cycling and pregnant rats on ghrelin. Research Methods and Procedures: We have determined ghrelin levels by radioimmunoassay and gastric ghrelin mRNA expression by Northern blot analysis during 21 days of chronic food restriction (30% of ad libitum available diet) in normal‐cycling female rats and in pregnancy. Results: Our results show that chronic food restriction led to an increase in plasmatic ghrelin levels in normal‐cycling female rats. In pregnancy, ghrelin plasmatic levels were enhanced particularly during the latter part of gestation (19 and 21 days) compared with pregnant rats with free access to food. Gastric ghrelin mRNA expression showed a similar expression pattern, being higher in the food‐restricted group than in the group fed ad libitum, in normal‐cycling as well as in pregnant rats. Discussion: These observations indicate that ghrelin plasmatic levels and ghrelin gastric mRNA are up‐modulated during undernutrition in normal‐cycling rats and in pregnancy. These findings suggest that increased ghrelin levels may have a role in mediating the physiological responses to undernutrition and could represent an adaptative response to prevent long‐lasting alterations in energy balance and body weight homeostasis.     

Lipid factor (bVLF) from bovine vitreous body evokes in EGFR-T17 cells a Ca2+-dependent K+ current associated with inositol 1,4,5-trisphosphate-independent Ca2+ mobilization

Bovine vitreous lipid factor (bVLF) is a complex phospholipid isolated from bovine vitreous body with strong Ca(2+)-mobilizing activity. In this study, the effects of bVLF on membrane potential were investigated in EGFR-T17 fibroblasts with the whole-cell patch clamp technique on monolayer cells, as well as with the fluorescent dye bis-oxonol as membrane potential-sensitive probe on monolayer and suspension cells. bVLF induced a transient hyperpolarization characterized by an initial peak and subsequent return to resting membrane potential levels within 1-2 min. The increase of [Ca(2+)](i) was concomitant with an outward current responsible for the hyperpolarizing response. Results with: (a) high [K(+)](o) media; (b) the monovalent cation ionophore gramicidin; and (c) substitution of K(+) with Cs(+) in the intracellular solution were consistent with the involvement of K(+) channels. The bVLF-induced hyperpolarization was blocked by the K(+) channel blockers, quinine and tetraethylamonium chloride, and partially affected by 4-aminopyridine. The calcium ionophore ionomycin caused a similar hyperpolarization as bVLF. When intracellular calcium was buffered by adding BAPTA to the pipette solution, bVLF-activated outward current was prevented. Moreover, the hyperpolarization response was strongly reduced at low doses (3 nM) of specific Ca(2+)-activated K(+) channel blockers, charybdotoxin and iberiotoxin. Based on these observations we conclude that bVLF hyperpolarizes the cells via the activation of a Ca(2+)-dependent K(+) current. In addition, it was observed that bVLF did not have a significant effect on intercellular communication measured by a single patch-electrode technique. Thus, membrane potential changes appeared to belong to the earliest cellular responses triggered by bVLF, and are closely associated with phosphatidic acid-dependent [Ca(2+)](i) mobilization.     

Dual effect of insulin on in vitro leptin secretion by adipose tissue

Although it is widely accepted that insulin stimulates leptin secretion, a dual action was observed using a validated in vitro system, i.e., an early (less than 48 h) inhibitory action, followed later (48-96 h) by a clear-cut stimulation. While the inhibitory phase was observed at every glucose concentration tested (from 1 to 25 mM), the stimulatory phase required the presence of physiological or supraphysiological glucose concentrations. In fact, leptin secretion was virtually eliminated in the presence of glucose uptake inhibitors. This dual effect of insulin was not due to modifications of the ob mRNA levels, suggesting that it depends entirely on posttranslational mechanisms. In conclusion, insulin appears to induce an early inhibition of leptin secretion by the adipose cell, followed later by a stimulatory effect secondary to the metabolic changes triggered by the insulin-induced increase in glucose uptake.     

Short-term fasting abolishes the sex-related difference in GH and leptin secretion in humans

We studied growth hormone (GH) and leptin secretion in eight male (age 29.3 +/- 1.2 yr, body mass index 22.2 +/- 0.5 kg/m(2)) and seven female normal subjects (28.0 +/- 0.8 yr, 20.1 +/- 0.7 kg/m(2)) before and after 36 h of fasting. In the fed state, 8-h mean GH and leptin concentrations were higher in females (P < 0.05 and P < 0. 0001, respectively). Fasting increased GH and decreased leptin in both sexes. There was significant interaction between gender and fasting (P < 0.05 for GH and P < 0.005 for leptin). Females showed a slighter increase in GH but a more marked decrease in leptin, so that there was no significant gender-related difference in GH and leptin after fasting. Fasting did not modify insulin-like growth factor (IGF) I, IGF binding protein (IGFBP)-3, acid-labile subunit, or GH binding protein; increased IGFBP-1 and free fatty acids (P < 0.0001) but decreased glucose (P < 0.001) and insulin levels (P < 0.05). In males, insulin levels were higher (P < 0.05) in the fed state and underwent deeper reduction after fasting (interaction P < 0.03). In conclusion, GH and leptin secretions are higher in women than in men in the fed but not in the fasting condition, which abolishes these gender-related differences in humans.     

Molecular cloning and initial characterization of African green monkey (Cercopithecus aethiops) corticotropin releasing factor receptor type 1 (CRF1) from COS-7 cells

We report the expression of endogenous CRF1 in COS-7 cells (African green monkey origin). Cloning of the coding region of CRF1 gene identified three alternatively spliced isoforms with nucleotide and predicted amino acid sequences corresponding to the membrane bound alpha and c and soluble e isoforms. DNA sequencing of the main isoform CRF1alpha showed homologies of 99%, 97% and 91% with the rhesus monkey, human and rodent genes, respectively; the deduced protein sequence differed in only one amino acid with rhesus monkey and human. Western blot analysis with antibodies against human CRF1 demonstrated immunoreactive proteins with MW of 37, 52, 70 and 80-85 in crude membrane or cytoplasm preparation; two additional species of 40 and 60 kDa were detected only in the cytoplasmic fraction. On immunocytochemistry CRF1 was localized to both the cell surface and intracellularly. The receptor was functional, e.g., addition of CRF to COS-7 cells inhibited cell proliferation and stimulated release of arachidonic acid; nevertheless, it was poorly coupled to cAMP production (its stimulation was minimal in native cells). In conclusion, COS cells that are routinely used for the study of transfected CRF receptors do express endogenous CRF1 mRNA with splicing behavior similar to that reported in human and rodent cells, and translated into functional CRF1 receptors.     

Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes

Triphosphate tunnel metalloenzymes (TTMs) are present in all kingdoms of life and catalyze diverse enzymatic reactions such as mRNA capping, the cyclization of adenosine triphosphate, the hydrolysis of thiamine triphosphate, and the synthesis and breakdown of inorganic polyphosphates. TTMs have an unusual tunnel domain fold that harbors substrate- and metal co-factor binding sites. It is presently poorly understood how TTMs specifically sense different triphosphate-containing substrates and how catalysis occurs in the tunnel center. Here we describe substrate-bound structures of inorganic polyphosphatases from Arabidopsis and Escherichia coli, which reveal an unorthodox yet conserved mode of triphosphate and metal co-factor binding. We identify two metal binding sites in these enzymes, with one co-factor involved in substrate coordination and the other in catalysis. Structural comparisons with a substrate- and product-bound mammalian thiamine triphosphatase and with previously reported structures of mRNA capping enzymes, adenylate cyclases, and polyphosphate polymerases suggest that directionality of substrate binding defines TTM catalytic activity. Our work provides insight into the evolution and functional diversification of an ancient enzyme family.     

Inositol 1,4,5-trisphosphate-independent Ca(2+) mobilization triggered by a lipid factor isolated from vitreous body.

A complex phospholipid from bovine vitreous body with a strong Ca(2+)-mobilizing activity has been recently isolated to homogeneity by our group. In this work, a sequential analysis of its transmembrane signaling pathway has been undertaken to characterize the intracellular mechanisms responsible for the Ca(2+) rise. The results show that this phospholipid induces, in a dose-dependent manner (ED(50) of around 0.25 microgram/ml), a Ca(2+) mobilization from inositol 1,4,5-trisphosphate-insensitive intracellular stores, with no participation of extracellular Ca(2+). Upon repeated administration, it shows no signs of autologous desensitization, does not induce heterologous desensitization of the L-alpha-lysophosphatidic acid (LPA) receptor but is desensitized by the previous administration of LPA. The Ca(2+)-mobilizing activity requires a membrane protein, is blocked after preincubation of the cells with pertussis toxin and phorbol esters, as well as by U73122 (an inhibitor of phospholipases C/D), R59022 (a diacylglycerol kinase inhibitor), and D609 (which inhibits phosphatidylcholine-specific phospholipase C). Upon administration of this phospholipid, the intracellular levels of phosphatidic acid (PA) rise with a time course that parallels that of the Ca(2+) mobilization, suggesting that PA could be responsible for this Ca(2+) signal. Exposure to AACOCF(3) (a specific inhibitor of phospholipase A(2)) does not modify the Ca(2+) rise, ruling out the possibility that the PA generated could be further converted to LPA by the action of phospholipase A(2). Based on the experimental data obtained, a signaling pathway involving a phosphatidylcholine-specific phospholipase C coupled to diacylglycerol kinase is proposed. This compound may represent a new class of bioactive lipids with a putative role in the physiology of the vitreous body.     

Retinal organoids derived from rhesus macaque iPSCs undergo accelerated differentiation compared to human stem cells

PurposeTo compare the timing and efficiency of the development of Macaca mulatta, a nonhuman primate (NHP), induced pluripotent stem cell (rhiPSC) derived retinal organoids to those derived from human embryonic stem cells (hESCs).ResultsGeneration of retinal organoids was achieved from both human and several NHP pluripotent stem cell lines. All rhiPSC lines resulted in retinal differentiation with the formation of optic vesicle‐like structures similar to what has been observed in hESC retinal organoids. NHP retinal organoids had laminated structure and were composed of mature retinal cell types including cone and rod photoreceptors. Single‐cell RNA sequencing was conducted at two time points; this allowed identification of cell types and developmental trajectory characterization of the developing organoids. Important differences between rhesus and human cells were measured regarding the timing and efficiency of retinal organoid differentiation. While the culture of NHP‐derived iPSCs is relatively difficult compared to that of human stem cells, the generation of retinal organoids from NHP iPSCs is feasible and may be less time‐consuming due to an intrinsically faster timing of retinal differentiation.ConclusionsRetinal organoids produced from rhesus monkey iPSCs using established protocols differentiate through the stages of organoid development faster than those derived from human stem cells. The production of NHP retinal organoids may be advantageous to reduce experimental time for basic biology studies in retinogenesis as well as for preclinical trials in NHPs studying retinal allograft transplantation.