Cell differentiation inducers derived from thalidomide

5-Hydroxy- and 4-amino-2-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione (5HPP-33 and 4APP-33, respectively) have been shown to possess cell differentiation-inducing activity toward human leukemia cell line HL-60.     

Interleukin-1beta targets interleukin-6 in progressing dextran sulfate sodium-induced experimental colitis

Inflammatory bowel disease (IBD) is an immunologically mediated disorder that is characterized by chronic, relapsing, and inflammatory responses. Dextran sulfate sodium (DSS)-induced experimental colitis in mice has been recognized as a useful model for human IBD and interleukin (IL)-1beta is a key cytokine in the onset of IBD. The purpose of the present study was to clarify which pro-inflammatory mediators are targeted by IL-1beta in mice with DSS-induced colitis. First, we found that DSS markedly induced IL-1beta production in both dose- and time-dependent manners (P < 0.05 and P < 0.01, respectively) in murine peritoneal macrophages (pMphi), while that of tumor necrosis factor-alpha was insignificant. Further, the expressions of mRNA and protein for IL-1beta were increased in colonic mucosa and pMphi from mice that received drinking water containing 5% DSS for 7 days (P < 0.01, each). In addition, the expressions of IL-6, granulocyte macrophage-colony stimulating factor, inducible nitric oxide synthase, and cyclooxygenase-2 mRNA were also time dependently increased (P < 0.01, each). Furthermore, administration of rIL-1beta (10 microg/kg, i.p.) significantly induced the expressions of IL-1beta and IL-6 mRNA in colonic mucosa from non-treated mice (P < 0.01). Anti-mIL-1beta antibody treatments (50 microg/kg, i.p.) attenuated DSS-induced body weight reduction and shortening of the colorectum (P < 0.05, each), and abrogated the expressions of IL-1beta and IL-6 mRNA in colonic mucosa (P < 0.01, each). Our results evidently support the previous findings that IL-1beta is involved in the development of DSS-induced experimental colitis in mice, and strongly suggest that IL-1beta targets itself and IL-6 for progressing colonic inflammation.     

Principles of two-photon excitation microscopy and its applications to neuroscience

The brain is complex and dynamic. The spatial scales of interest to the neurobiologist range from individual synapses (approximately 1 microm) to neural circuits (centimeters); the timescales range from the flickering of channels (less than a millisecond) to long-term memory (years). Remarkably, fluorescence microscopy has the potential to revolutionize research on all of these spatial and temporal scales. Two-photon excitation (2PE) laser scanning microscopy allows high-resolution and high-sensitivity fluorescence microscopy in intact neural tissue, which is hostile to traditional forms of microscopy. Over the last 10 years, applications of 2PE, including microscopy and photostimulation, have contributed to our understanding of a broad array of neurobiological phenomena, including the dynamics of single channels in individual synapses and the functional organization of cortical maps. Here we review the principles of 2PE microscopy, highlight recent applications, discuss its limitations, and point to areas for future research and development.     

Improved Golgi-like visualization in retrogradely projecting neurons after EGFP-adenovirus infection in adult rat and monkey.

An adenovirus vector was generated using a neuron-specific promoter synapsin I and enhanced green fluorescent protein (EGFP) reporter (AdSynEGFP). In addition, two modifications were identified that resulted in robust and reliable retrograde transport and EGFP expression after injection of the virus into three different brain regions in adult rats (medial prefrontal cortex, posterior thalamic nuclear group, and CA1). These are postinjection survival times of 14 days and addition of high concentrations of NaCl (>or=600 mM) to the injection buffer. These modifications resulted in obvious improvement in the intensity of the EGFP signal and in the number of labeled cells. Use of anti-EGFP in immunofluorescence or immunoperoxidase processing further enhanced the signal so that Golgi-like filling of dendritic spines and axon collaterals was routinely achieved. Effectiveness of the AdSynEGFP for Golgi-like filling was confirmed in one rhesus monkey with injections in visual area V4. Because of the long-term viability of the infected neurons (at least up to 28 days in rats and 22 days in monkey), this AdSynEGFP is suitable for use in microcircuitry studies in combination with other fluorescently tagged elements, including anterogradely labeled extrinsic projections. The native EGFP signal (without antibody enhancement) may be sufficient for studies involving cultured cells or slices.     

Behavioral responses to the alarm pheromone of the ant Camponotus obscuripes (Hymenoptera: Formicidae)

The alarm pheromone of the ant Camponotus obscuripes (Formicinae) was identified and quantified by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Comparisons between alarm pheromone components and extracts from the major exocrine gland of this ant species revealed that the sources of its alarm pheromone are Dufour's gland and the poison gland. Most components of Dufour's gland were saturated hydrocarbons. n-Undecane comprised more than 90% of all components and in a single Dufour's gland amounted to 19 microg. n-Decane and n-pentadecane were also included in the Dufour's gland secretion. Only formic acid was detected in the poison gland, in amounts ranging from 0.049 to 0.91 microl. This ant species releases a mixture of these substances, each of which has a different volatility and function. When the ants sensed formic acid, they eluded the source of the odor; however, they aggressively approached odors of n-undecane and n-decane, which are highly volatile. In contrast, n-pentadecane, which has the lowest volatility among the identified compounds, was shown to calm the ants. The volatilities of the alarm pheromone components were closely related to their roles in alarm communication. Highly volatile components vaporized rapidly and spread widely, and induced drastic reactions among the ants. As these components became diluted, the less volatile components calmed the excited ants. How the worker ants utilize this alarm communication system for efficient deployment of their nestmates in colony defense is also discussed herein.     

Substrate recognition ability differs among various prokaryotic tRNase Zs

There exists a significant difference in pre-tRNA preference among prokaryotic tRNase Zs. This is an enigma, because pre-tRNAs should form the common L-shaped structure and tRNase Zs should form the common structure based on the alphabeta/betaalpha-fold. To address this issue, we examined six different eubacterial and archaeal tRNase Zs including two newly isolated tRNase Zs for cleavage of 18 different pre-tRNA substrates. Two Thermotoga maritima, one Thermus thermophilus, one Bacillus subtilis, one Thermoplasma acidophilum, and one Pyrobaculum aerophilum enzymes were tested. To our surprise, the newly isolated proteins T. maritima and T. thermophilus showed the weak tRNase Z activity, even though their primary amino acid sequences are, on the whole, quite different from those of the typical tRNase Zs. We confirmed that substrate recognition ability is quite different among those tRNase Zs. In addition, we found that the optimal conditions as a whole differ significantly among the enzymes. From these results, we provided several clues to solve the enigma by showing the potential importance of the 74th-76th nucleotide sequence of pre-tRNA, the flexible arm length of tRNase Z, the divalent metal ion species, and the histidine corresponding His222 in T. maritima tRNase Z.     

Characterization and cDNA cloning of dipeptidyl peptidase IV from the venom of Gloydius blomhoffi brevicaudus

Dipeptidyl peptidase activity was investigated in snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis and Crotalus atrox. The strongest dipeptidyl peptidase IV (DPP IV) activity was found in venom from G. blomhoffi brevicaudus. The substrate specificity, susceptibility to inhibitors, and pH optimum of the partially purified enzyme were similar to those of known DPP IVs from bacteria and eukaryotes. The G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on amino acid sequences highly conserved in known DPP IVs. Two cDNA species encoding DPP IV were obtained, and designated as DPP IVa and DPP IVb. This is the first study to report the primary structure of DPP IV from a reptile. The deduced amino acid sequences for DPP IVa and DPP IVb both consist of 751amino acid residues and are highly homologous to each other. A putative catalytic triad for serine proteases, Ser-616, Asp-694, and His-726, is present. It is of particular interest that the deduced NH(2)-terminal sequence associated with the characteristic signal peptide is identical to that determined from the purified DPP IV. This indicates that the signal peptide of snake venom DPP IV is not cleaved off during biosynthesis, unlike those of other snake venom proteins.     

Identification and characterization of canine growth differentiation factor-9 and its splicing variant

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor-β (TGF-β) superfamily, is expressed exclusively in the oocyte within the ovary and plays essential roles in the ovarian function in mammals. However, a possible involvement of GDF-9 in canine ovarian physiology that has a unique ovulation process among mammals has not been studied. Interestingly, we have isolated two types of cDNA clones generated by an alternative splicing from a canine ovarian total RNA. The predominant long form cDNA shares a common precursor structure with GDF-9s in other species whereas the minor short form cDNA has a 172 amino acid truncation in the proregion. Using a transient expression system, we found that the long form cDNA has a defect in mature protein production whereas the short form cDNA readily produces mature protein. However, mutations at one or two N-glycosylation sites in the mature domain of the short form GDF-9 caused a loss in mature protein production. These results suggest that the prodomain and N-linked glycosylation of the mature domain regulate proper processing and secretion of canine GDF-9. Based on the biological functions of GDF-9, these characteristics of canine GDF-9 could be causatively linked to the unique ovulation process in the Canidae.     

The gene expression landscape of thermogenic skunk cabbage suggests critical roles for mitochondrial and vacuolar metabolic pathways in the regulation of thermogenesis

Floral thermogenesis has been described in several plant species. Because of the lack of comprehensive gene expression profiles in thermogenic plants, the molecular mechanisms by which floral thermogenesis is regulated remain to be established. We examined the gene expression landscape of skunk cabbage (Symplocarpus renifolius) during thermogenic and post-thermogenic stages and identified expressed sequence tags from different developmental stages of the inflorescences using super serial analysis of gene expression (SuperSAGE). In-depth analysis suggested that cellular respiration and mitochondrial functions are significantly enhanced during the thermogenic stage. In contrast, genes involved in stress responses and protein degradation were significantly up-regulated during post-thermogenic stages. Quantitative comparisons indicated that the expression levels of genes involved in cellular respiration were higher in thermogenic spadices than in Arabidopsis inflorescences. Thermogenesis-associated genes seemed to be expressed abundantly in the peripheral tissues of the spadix. Our results suggest that cellular respiration and mitochondrial metabolism play key roles in heat production during floral thermogenesis. On the other hand, vacuolar cysteine protease and other degradative enzymes seem to accelerate senescence and terminate thermogenesis in the post-thermogenic stage.     

Representation of a mixture of pheromone and host plant odor by antennal lobe projection neurons of the silkmoth Bombyx mori

Pheromone-source orientation behavior can be modified by coexisting plant volatiles. Some host plant volatiles enhance the pheromonal responses of olfactory receptor neurons and increase the sensitivity of orientation behavior in the Lepidoptera species. Although many electrophysiological studies have focused on the pheromonal response of olfactory interneurons, the response to the mixture of pheromone and plant odor is not yet known. Using the silkmoth, Bombyx mori, we investigated the physiology of interneurons in the antennal lobe (AL), the primary olfactory center in the insect brain, in response to a mixture of the primary pheromone component bombykol and cis-3-hexen-1-ol, a mulberry leaf volatile. Application of the mixture enhanced the pheromonal responses of projection neurons innervating the macroglomerular complex in the AL. In contrast, the mixture of pheromone and cis-3-hexen-1-ol had little influence on the responses of projection neurons innervating the ordinary glomeruli whereas other plant odors dynamically modified the response. Together this suggests moths can process plant odor information under conditions of simultaneous exposure to sex pheromone.