Chimeras of Candida albicans Cdr1p and Cdr2p reveal features of pleiotropic drug resistance transporter structure and function

Members of the pleiotropic drug resistance (PDR) family of ATP binding cassette (ABC) transporters consist of two homologous halves, each containing a nucleotide binding domain (NBD) and a transmembrane domain (TMD). The PDR transporters efflux a variety of hydrophobic xenobiotics and despite the frequent association of their overexpression with the multidrug resistance of fungal pathogens, the transport mechanism of these transporters is poorly understood. Twenty-eight chimeric constructs between Candida albicans Cdr1p (CaCdr1p) and Cdr2p (CaCdr2p), two closely related but functionally distinguishable PDR transporters, were expressed in Saccharomyces cerevisiae. All chimeras expressed equally well, localized properly at the plasma membrane, retained their transport ability, but their substrate and inhibitor specificities differed significantly between individual constructs. A detailed characterization of these proteins revealed structural features that contribute to their substrate specificities and their transport mechanism. It appears that most transmembrane spans of CaCdr1p and CaCdr2p provide or affect multiple, probably overlapping, substrate and inhibitor binding site(s) similar to mammalian ABC transporters. The NBDs, in particular NBD1 and/or the ～150 amino acids N-terminal to NBD1, can also modulate the substrate specificities of CaCdr1p and CaCdr2p.     

Heat shock factor binds to heat shock elements upstream of heat shock protein 70a and Samui genes to confer transcriptional activity in Bombyx mori diapause eggs exposed to 5°C

To understand the molecular mechanisms of how 5 °C-incubation activates mRNA expression of Hsp70a and Samui genes in Bombyx mori diapause eggs, we first searched the 5′-upstream regions of the Hsp70a and Samui genes for heat shock elements (HSEs) and found two regions [Hsp70aHSE-1 (−95 to −58) and -2 (−145 to −121), and SamuiHSE-1 (−84 to −55) and -2 (−304 to −290)] corresponding to HSEs (repeats of nGAAn and/or nTTCn). We cloned four cDNAs encoding heat shock factor (HSF)-a2 (627 amino acids), -b (685 aa), -c (682 aa) and -d (705 aa), which were produced by alternative splicing. When we exposed diapause eggs to 5 °C beginning at 2 day post-oviposition to break diapause, HSFd mRNA only increased after chilling for 6–8 days, a pattern very similar to those of Hsp70a and Samui mRNAs. To examine further whether HSFd binds to the respective HSEs, we carried out a gel shift assay using HSFd protein expressed in a cell-free system and the isolated HSEs; migration of the respective digoxigenin(DIG)-labeled HSE-1 and -2 of Hsp70a and Samui was retarded by addition of HSFd; the retarded bands disappeared after addition of the corresponding unlabeled HSE-1 and -2 as competitors, but were not affected by addition of the respective mutated unlabeled HSE-1 and -2. These results indicated that HSFd protein binds to the respective HSEs and may activate mRNA expression of Hsp70a and Samui genes upon exposure of diapause eggs to 5 °C.     

Breed differences in allele frequency of the dopamine receptor D4 gene in dogs.

We previously reported that the dog dopamine receptor D4 (DRD4) gene is polymorphic as observed in humans, and four alleles were identified based on the number and/or order of the 12 and 39 bp sequences located in the homologous region of human DRD4. To assess the diversity of the DRD4 gene in dogs we examined the allelic variations in four breeds (beagle, golden retriever, Shetland sheepdog, and shiba) employing the polymerase chain reaction (PCR). As a result, we found three novel alleles and determined the DNA sequences of these alleles. The beagle shared four alleles, including 396, 435, 447a, and 447b, with the 435 (52.6%) and 447a (39.5%) alleles being common. The golden retriever had the 435 and 447a alleles, and the 435 allele was frequent (73.3%). In the Shetland sheepdog, the 435, 447a, and 498 alleles were observed, of which the 447a allele was most frequent (82.5%). The shiba had five alleles-447a, 447b, 486, 498, and 549-and the 447b allele was most common (55.4%). These findings suggest that the allele frequency varied among the four dog breeds, and analysis of the DRD4 polymorphism may therefore be useful for elucidating the relationships among dog breeds.     

TLR3-mediated synthesis and release of eotaxin-1/CCL11 from human bronchial smooth muscle cells stimulated with double-stranded RNA

Respiratory infections with RNA viruses, such as rhinovirus or respiratory syncytial virus, are a major cause of asthma exacerbation, accompanied by enhanced neutrophilic and/or eosinophilic inflammation of the airways. We studied the effects of dsRNA synthesized during RNA virus replication, and of its receptor, TLR3, on the synthesis of eosinophilic chemokines in bronchial smooth muscle cells (BSMC). Synthetic dsRNA, polyinosinic-cystidic acid (poly(I:C)), induced the synthesis of eosinophilic chemokines, eotaxin-1/CCL11 and RANTES/CCL5, from primary cultures of human BSMC, and IL-4 increased synergistically the synthesis of poly(I:C)-induced CCL11. A robust eosinophil chemotactic activity was released from BSMC stimulated with poly(I:C) and IL-4, which was mostly inhibited by preincubation with an anti-CCL11, but not with an anti-CCL5 Ab. Although the immunoreactivity of TLR3 was detectable on the cellular surface of BSMC by flow cytometric analysis, pretreatment with an anti-TLR3-neutralizing Ab failed to block the poly(I:C)-induced synthesis of CCL11. We have determined by confocal laser-scanning microscopy that the immunoreactivity of TLR3 was aggregated intracellularly in poly(I:C)-stimulated BSMC, colocalizing with fluorescein-labeled poly(I:C). The synthesis of CCL11 was prominently inhibited by the transfection of TLR3-specific small interfering RNA or by bafilomycin A1, an endosomal acidification inhibitor, further supporting the essential role played by intracellular TLR3 in the synthesis of poly(I:C)-induced CCL11 in BSMC. In conclusion, these observations suggest that, by activating intracellular TLR3 in BSMC, respiratory RNA virus infections stimulate the production of CCL11 and enhance eosinophilic inflammation of the airways in the Th2-dominant microenvironment.     

Blood flow regulation in the cerebral microvasculature with an arcadal network: a numerical simulation

Blood flow regulation in the cerebral microvasculature with an arcadal network was investigated using a numerical simulation. A mathematical model for blood flow in the arcadal network, based on in vivo data of cat cerebral microvasculature and flow velocity was developed. The network model consists of 45 vessel segments and 25 branching points. To simulate microvascular response to blood flow, non-reactive (solid), cerebral arteriole-like, or skeletal muscle arteriole-like responses to wall shear stress were taken into account. Numerical calculation was carried out in the flow condition where the inlet (arterial) pressure was changed from 60 to 120 mmHg. Flow-rate in each efferent vessel and the mean flow-rate over all efferent vessels were evaluated for assessment of blood supply to the local area of cerebral tissue. The simulation demonstrated the wall shear stress-induced vasodilation in the arcadal network worked to maintain the blood flow at a constant level with pressure variable in a wide range. It is suggested that an individual microvessel (segment) should join in the regulatory process of flow, interacting with other microvessels (cooperative regulation).     

Ancient DNA analysis of the Japanese sea lion (Zalophus californianus japonicus Peters, 1866): preliminary results using mitochondrial control-region sequences

In this study, we successfully extracted ancient DNA from skeletal remains of the Japanese sea lion-a species that is practically extinct-from archaeological sites and determined a partial sequence of its mitochondrial DNA control region. A molecular phylogenetic tree constructed by the neighbor-joining (NJ) method showed that the sequences from Japanese sea lions clustered together, with a high bootstrap value, and that this cluster was closest to the California sea lion cluster. The distinctly divergent cluster of Japanese sea lions reflected the morphological classification of these animals as a distinct species of the genus Zalophus; however, proximity to the California sea lion cluster simultaneously implied conformation with the traditional classification of these animals as a subspecies of Zalophus californianus. The average amount of nucleotide substitution between the Japanese and California sea lions was 7.02%. The Japanese and California sea lions were estimated to have diverged 2.2 million years ago, i.e., in the late Pliocene Epoch. This is the first report on a genetic analysis of the Japanese sea lion.     

Essential role of mouse Dead end1 in the maintenance of spermatogonia

Dead end is a vertebrate-specific RNA-binding protein implicated in germ cell development. We have previously shown that mouse Dead end1 (DND1) is expressed in male embryonic germ cells and directly interacts with NANOS2 to cooperatively promote sexual differentiation of fetal germ cells. In addition, we have also reported that NANOS2 is expressed in self-renewing spermatogonial stem cells and is required for the maintenance of the stem cell state. However, it remains to be determined whether DND1 works with NANOS2 in the spermatogonia. Here, we show that DND1 is expressed in a subpopulation of differentiating spermatogonia and undifferentiated spermatogonia, including NANOS2-positive spermatogonia. Conditional disruption of DND1 depleted both differentiating and undifferentiated spermatogonia; however, the numbers of A_single and A_paired spermatogonia were preferentially decreased as compared with those of A_aligned spermatogonia. Finally, we found that postnatal DND1 associates with NANOS2 in vivo, independently of RNA, and interacts with some of NANOS2-target mRNAs. These data not only suggest that DND1 is a partner of NANOS2 in undifferentiated spermatogonia as well as in male embryonic germ cells, but also show that DND1 plays an essential role in the survival of differentiating spermatogonia.     

Characterization of Senescence-Accelerated Mouse Prone 6 (SAMP6) as an Animal Model for Brain Research

The senescence-accelerated mouse (SAM) was developed by selective breeding of the AKR/J strain, based on a graded score for senescence, which led to the development of both senescence-accelerated prone (SAMP), and senescence-accelerated resistant (SAMR) strains. Among the SAMP strains, SAMP6 is well characterized as a model of senile osteoporosis, but its brain and neuronal functions have not been well studied. We therefore decided to characterize the central nervous system of SAMP6, in combination with different behavioral tests and analysis of its biochemical and pharmacological properties. Multiple behavioral tests revealed higher motor activity, reduced anxiety, anti-depressant activity, motor coordination deficits, and enhanced learning and memory in SAMP6 compared with SAMR1. Biochemical and pharmacological analyses revealed several alterations in the dopamine and serotonin systems, and in long-term potentiation (LTP)-related molecules. In this review, we discuss the possibility of using SAMP6 as a model of brain function.