Apigenin inhibition of involucrin gene expression is associated with a specific reduction in phosphorylation of protein kinase Cdelta Tyr311

Apigenin is a plant-derived flavanoid that has significant promise as a skin cancer chemopreventive agent. In the present study, we examine the mechanism whereby apigenin regulates normal human keratinocyte differentiation. Expression of involucrin (hINV), a marker of keratinocyte differentiation, is increased by differentiating agents via a protein kinase Cdelta (PKCdelta), Ras, MEKK1, MEK3 cascade that increases AP1 factor level and AP1 factor binding to DNA elements in the hINV promoter. We show that apigenin inhibits this response. Apigenin suppresses the 12-O-tetradeconylphorbol-13-acetate-dependent increase in AP1 factor expression and binding to the hINV promoter and the increase in hINV promoter activity. Apigenin also inhibits the increase in promoter activity observed following overexpression of PKCdelta, constitutively active Ras, or MEKK1. The suppression of PKCdelta activity is associated with reduced phosphorylation of PKCdelta-Y311. The physiological importance of this phosphorylation event was confirmed by showing that the PKCdelta phosphorylation-defective mutant, PKCdelta-Y311F, is less able to increase hINV promoter activity. Activation of hINV promoter activity by the green tea polyphenol, (-)-epigellocathecin-3-gallate, is also inhibited by apigenin, suggesting that the two chemopreventive agents can produce opposing actions in keratinocytes. Additional studies show that the apigenin-dependent suppression of differentiation is associated with reduced cell proliferation but that there is no evidence of apoptosis.     

The physiological period length of the human circadian clock in vivo is directly proportional to period in human fibroblasts

Background

Diurnal behavior in humans is governed by the period length of a circadian clock in the suprachiasmatic nuclei of the brain hypothalamus. Nevertheless, the cell-intrinsic mechanism of this clock is present in most cells of the body. We have shown previously that for individuals of extreme chronotype (“larks” and “owls”), clock properties measured in human fibroblasts correlated with extreme diurnal behavior.

Methodology/Principal Findings

In this study, we have measured circadian period in human primary fibroblasts taken from normal individuals and, for the first time, compared it directly with physiological period measured in vivo in the same subjects. Human physiological period length was estimated via the secretion pattern of the hormone melatonin in two different groups of sighted subjects and one group of totally blind subjects, each using different methods. Fibroblast period length was measured via cyclical expression of a lentivirally delivered circadian reporter. Within each group, a positive linear correlation was observed between circadian period length in physiology and in fibroblast gene expression. Interestingly, although blind individuals showed on average the same fibroblast clock properties as sighted ones, their physiological periods were significantly longer.

Conclusions/Significance

We conclude that the period of human circadian behaviour is mostly driven by cellular clock properties in normal individuals and can be approximated by measurement in peripheral cells such as fibroblasts. Based upon differences among sighted and blind subjects, we also speculate that period can be modified by prolonged unusual conditions such as the total light deprivation of blindness.     

A method for measuring and modeling the physiological traits causing obstructive sleep apnea

There is not a clinically available technique for measuring the physiological traits causing obstructive sleep apnea (OSA). Therefore, it is often difficult to determine why an individual has OSA or to what extent the various traits contribute to the development of OSA. In this study, we present a noninvasive method for measuring four important physiological traits causing OSA: 1) pharyngeal anatomy/collapsibility, 2) ventilatory control system gain (loop gain), 3) the ability of the upper airway to dilate/stiffen in response to an increase in ventilatory drive, and 4) arousal threshold. These variables are measured using a single maneuver in which continuous positive airway pressure (CPAP) is dropped from an optimum to various suboptimum pressures for 3- to 5-min intervals during sleep. Each individual's set of traits is entered into a physiological model of OSA that graphically illustrates the relative importance of each trait in that individual. Results from 14 subjects (10 with OSA) are described. Repeatability measurements from separate nights are also presented for four subjects. The measurements and model illustrate the multifactorial nature of OSA pathogenesis and how, in some individuals, small adjustments of one or another trait (which might be achievable with non-CPAP agents) could potentially treat OSA. This technique could conceivably be used clinically to define a patient's physiology and guide therapy based on the traits.     

Chromosome numbers and meiotic analysis in the pre-breeding of <Emphasis Type="BoldItalic">Brachiaria decumbens</Emphasis> (Poaceae)

A total of 44 accessions of Brachiaria decumbens were analysed for chromosome count and meiotic behaviour in order to identify potential progenitors for crosses. Among them, 15 accessions presented 2n = 18; 27 accessions, 2n = 36; and 2 accessions, 2n = 45 chromosomes. Among the diploid accessions, the rate of meiotic abnormalities was low, ranging from 0.82% to 7.93%. In the 27 tetraploid accessions, the rate of meiotic abnormalities ranged from 18.41% to 65.83%. The most common meiotic abnormalities were related to irregular chromosome segregation, but chromosome stickiness and abnormal cytokinesis were observed in low frequency. All abnormalities can compromise pollen viability by generating unbalanced gametes. Based on the chromosome number and meiotic stability, the present study indicates the apomictic tetraploid accessions that can act as male genitor to produce interspecific hybrids with B. ruziziensis or intraspecific hybrids with recently artificially tetraploidized accessions.     

Behavioural evidence of colour vision in free flying stingless bees

Colour vision was first demonstrated with behavioural experiments in honeybees 100 years ago. Since that time a wealth of quality physiological data has shown a highly conserved set of trichromatic colour receptors in most bee species. Despite the subsequent wealth of behavioural research on honeybees and bumblebees, there currently is a relative dearth of data on stingless bees, which are the largest tribe of the eusocial bees comprising of more than 600 species. In our first experiment we tested Trigona cf. fuscipennis, a stingless bee species from Costa Rica in a field setting using the von Frisch method and show functional colour vision. In a second experiment with these bees, we use a simultaneous colour discrimination test designed for honeybees to enable a comparative analysis of relative colour discrimination. In a third experiment, we test in laboratory conditions Tetragonula carbonaria, an Australian stingless bee species using a similar simultaneous colour discrimination test. Both stingless bee species show relatively poorer colour discrimination compared to honeybees and bumblebees; and we discuss the value of being able to use these behavioural methods to efficiently extend our current knowledge of colour vision and discrimination in different bee species.     

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association,Assembly, Maturation,and Function

Hydrogenases are metalloenzymes that catalyze 2H⁺ + 2e⁻ ↔ H₂. A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)⁺ as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.     

The distribution of a proctolin-like immunoreactive material in the terminal ganglion of the cockroach,Periplaneta americana L.

Summary Neurons with proctolin-like immunoreactivity were mapped in the terminal ganglion of Periplaneta americana. The effect of different fixation methods on the variability of immunostaining is described and discussed. The appearance of immunoreactive presynaptic terminals, described here for the first time in insects, points to a function of proctolin as neurotransmitter or neuromodulator in the central nervous system of P. americana besides its known role in the periphery. Proctolin-like immunoreactivity was shown in pre- and postsynaptic profiles. Synaptic contacts are described in detail.Supported by the Ministerium für Wissenschaft und Technik der DDR and by Sächsische Akademie der Wissenschaften zu Leipzig.The authors are indebted for excellent technical assistance to Mrs. Angelika Schmidt and Mr. Bernd Mäusezahl.     

RADseq approaches and applications for forest tree genetics

As tree species vary extensively in genome size, complexity, and resource development, reduced representation methods have been increasingly employed for the generation of population genomic data. By allowing rapid marker discovery and genotyping for thousands of genomic regions in many individuals without requiring genomic resources, restriction site-associated DNA sequencing (RADseq) methods have dramatically improved our ability to bring population genomic perspectives to non-model trees. The rapid recent increase in studies of trees utilizing RADseq suggests that it is likely to become among the most common approaches for generating genome-wide data for a variety of applications. Here we provide a practical review of RADseq and its application to research areas of tree genetics. We briefly review RADseq laboratory methods and consider analytical approaches for assembly, variant calling, and bioinformatic processing. To guide considerations for study design, we use in silico analyses of eight available tree genomes to illustrate how expected marker number and density vary across laboratory approaches and genome sizes, and to consider the ability of RADseq designs to query coding regions. We review the empirical use of RADseq for different research objectives, considering its strengths and limitations. Many studies have used RADseq data to perform genome scans for selection, although limited marker density and linkage disequilibrium will often compromise its utility for such analyses. Regardless of this limitation, RADseq offers a powerful and inexpensive technique for generating genome-wide SNP data that can greatly contribute to research spanning phylogenetic and population genetic inference, linkage mapping, and quantitative genetic parameter estimation for tree genetics.     

Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding

Background

Insulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis.