Rhythmic expression of circadian clock genes in human leukocytes and beard hair follicle cells

Evaluating individual circadian rhythm traits is crucial for understanding the human biological clock system. The present study reports characterization of physiological and molecular parameters in 13 healthy male subjects under a constant routine condition, where interfering factors were kept to minimum. We measured hormonal secretion levels and examined temporal expression profiles of circadian clock genes in peripheral leukocytes and beard hair follicle cells. All 13 subjects had prominent daily rhythms in melatonin and cortisol secretion. Significant circadian rhythmicity was found for PER1 in 9 subjects, PER2 in 3 subjects, PER3 in all 13 subjects, and BMAL1 in 8 subjects in leukocytes. Additionally, significant circadian rhythmicity was found for PER1 in 5 of 8 subjects tested, PER2 in 2 subjects, PER3 in 6 subjects, and BMAL1 in 3 subjects in beard hair follicle cells. The phase of PER1 and PER3 rhythms in leukocytes correlated significantly with that of physiological rhythms. Our results demonstrate that leukocytes and beard hair follicle cells possess an endogenous circadian clock and suggest that PER1 and PER3 expression would be appropriate biomarkers and hair follicle cells could be a useful tissue source for the evaluation of biological clock traits in individuals.     

Subversion of cellular autophagy by Anaplasma phagocytophilum

Anaplasma phagocytophilum , the causative agent of human granulocytic anaplasmosis, is an obligatory intracellular pathogen. After entry into host cells, the bacterium is diverted from the endosomal pathway and replicates in a membrane-bound compartment devoid of endosomal or lysosomal markers. Here, we show that several hallmarks of early autophagosomes can be identified in A. phagocytophilum replicative inclusions, including a double-lipid bilayer membrane and colocalization with GFP-tagged LC3 and Beclin 1, the human homologues of Saccharomyces cerevisiae autophagy-related proteins Atg8 and Atg6 respectively. While the membrane-associated form of LC3, LC3-II, increased during A. phagocytophilum infection, A. phagocytophilum -containing inclusions enveloped with punctate GFP-LC3 did not colocalize with a lysosomal marker. Stimulation of autophagy by rapamycin favoured A. phagocytophilum infection. Inhibition of the autophagosomal pathway by 3-methyladenine did not inhibit A. phagocytophilum internalization, but reversibly arrested its growth. Although autophagy is considered part of the innate immune system that clears a variety of intracellular pathogens, our study implies that A. phagocytophilum subverts this system to establish itself in an early autophagosome-like compartment segregated from lysosomes to facilitate its proliferation.     

RAGE mediates vascular injury and inflammation after global cerebral ischemia

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in a diverse range of pathological conditions. To analyze the roles of RAGE and its decoy receptor, endogenous secretory RAGE (esRAGE), in the global cerebral ischemia, three different mouse cohorts, wild-type, RAGE^−/−, and esRAGE transgenic (Tg) mice were subjected to bilateral common carotid artery occlusion (BCCAO). RT-PCR and immunohistochemical analysis revealed that expression of RAGE was induced in the vascular cells at 12 h, and then in the neurons and glia from 3 to 7 days in the hippocampus after BCCAO. The numbers of surviving neurons in the hippocampal CA1 region were significantly higher in RAGE^−/− and esRAGE Tg mice than those in wild-type mice in the periods between 24 h and 7 days after BCCAO. Lower levels of 3-nitrotyrosine (3-NT) and higher levels of endothelial nitric oxide synthase (eNOS), together with enlarged vascular areas were observed in RAGE^−/− and esRAGE Tg mice at 12 h after BCCAO. In the later periods, expressions of glia-derived inflammatory mediators TNFα and inducible nitric oxide synthase (iNOS) were reduced in RAGE^−/− and esRAGE Tg mice. These results suggest that RAGE may contribute to delayed neuronal death after global cerebral ischemia by enhancing vascular injury and deleterious glia-mediated inflammation.     

Mice with targeted deletion of eNOS develop hyperdynamic circulation associated with portal hypertension

Systemic vasodilation is the initiating event of the hyperdynamic circulatory state, being most likely triggered by increased levels of vasodilators, primarily nitric oxide (NO). Endothelial NO synthase (eNOS) is responsible for this event. We tested the hypothesis that gene deletion of eNOS and inducible NOS (iNOS) may inhibit the development of the hyperdynamic circulatory state in portal hypertensive animals. To test this hypothesis, we used mice lacking eNOS (eNOS-/-) or eNOS/iNOS (eNOS/iNOS-/-) genes. A partial portal vein ligation (PVL) was used to induce portal hypertension. Sham-operated animals were used as a control. Hemodynamic characteristics were tested 2 wk after surgery. As opposed to our hypothesis, PVL also caused significant reduction in peripheral resistance in eNOS-/- compared with sham animals (0.33 +/- 0.02 vs. 0.41 +/- 0.03 mmHg. min x kg body wt x ml(-1); P = 0.04) and in eNOS/iNOS-/- animals with PVL compared with that of the sham-operated group (0.44 +/- 0.02 vs. 0.54 +/- 0.04; P = 0.03). This demonstrates that, despite gene deletion of eNOS, the knockout mice developed hyperdynamic circulation. Compensatory vasodilator molecule(s) are upregulated in place of NO in the systemic and splanchnic circulation in portal hypertensive animals.     

Conformation of oligopeptides with L-leucyl-L-leucyl-L-alanyl and L-methionyl-L-methionyl-L-leucyl repeating units

The conformation of oligopeptides with hydrophobic side chains, Nps-(L -Leu-L -Leu-L -Ala)_n-OEt and Nps-(L -Met-L -Met-L -Leu)_n-OEt(n = 1–6), in the solid state, obtained either by evaporation of the solvent or by precipitation with diethyl ether from a 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) solution, has been studied with ir spectroscopy and x-ray powder-diffraction measurements. The conformation of these peptides in the HFIP solution has been studied by CD spectroscopy. Due to a strong preference of the amino acids to form an α helix, the peptides begin forming α helices at the dodecapeptide in the HFIP solution, and in the solid state by evaporation. In the solid state, with precipitation, the α-helical conformation is first observed at the octadecapeptide and the lower peptides assume a β structure. The conformational change, from the α helix to the β structure of the peptides with 12 to 15 amino acid residues, during the precipitation process, is due to a strong tendency of the amino acids to form the β-structure in rather short peptide lengths.     

Fruit Availability and Habitat Use by Chimpanzees in the Kalinzu Forest,Uganda: Examination of Fallback Foods

We studied seasonal change in habitat use by chimpanzees in the Kalinzu Forest, Uganda. The forest comprises various types of vegetation. For each vegetation type, we compared number of chimpanzees (per km²) that used the vegetation with fruit availability in different census periods. We estimated the number of chimpanzees by nest count and fruit availability via density of fallen fruit. The mixed mature forest contained a large amount of fruit during the high-fruiting season, but it decreased rapidly in the low-fruiting season. The number of chimpanzees also decreased in mixed mature forest in approximate proportion with fruit availability. In the Parinari-dominated mature and secondary forests, both fruit availability and number of chimpanzees were very low throughout the study. In the Musanga-dominated secondary forest, the number of chimpanzees increased toward the low-fruiting season, though the fruit availability decreased slightly. A multiple regression analysis showed that various fruits had significant effects on the number of chimpanzees during the high-fruiting season, while only Musanga leo-errerae had a significant effect during the low-fruiting season. The results suggest that the fruit of Musanga leo-errerae functions as a fallback food, and a combination of different vegetation types supports the chimpanzees in the Kalinzu Forest.     

Paradoxical gain‐of‐function mutant of the G‐protein‐coupled receptor PROKR2 promotes early puberty

The human genome encodes ~750 G‐protein‐coupled receptors (GPCRs), including prokineticin receptor 2 (PROKR2) involved in the regulation of sexual maturation. Previously reported pathogenic gain‐of‐function mutations of GPCR genes invariably encoded aberrant receptors with excessive signal transduction activity. Although in vitro assays demonstrated that an artificially created inactive mutant of PROKR2 exerted paradoxical gain‐of‐function effects when co‐transfected with wild‐type proteins, such a phenomenon has not been observed in vivo. Here, we report a heterozygous frameshift mutation of PROKR2 identified in a 3.5‐year‐old girl with central precocious puberty. The mutant mRNA escaped nonsense‐mediated decay and generated a GPCR lacking two transmembrane domains and the carboxyl‐terminal tail. The mutant protein had no in vitro signal transduction activity; however, cells co‐expressing the mutant and wild‐type PROKR2 exhibited markedly exaggerated ligand‐induced Ca²⁺ responses. The results indicate that certain inactive PROKR2 mutants can cause early puberty by enhancing the functional property of coexisting wild‐type proteins. Considering the structural similarity among GPCRs, this paradoxical gain‐of‐function mechanism may underlie various human disorders.