Daily expression of clock genes in whole blood cells in healthy subjects and a patient with circadian rhythm sleep disorder

In recent years, circadian rhythm sleep disorders in humans have been increasing. Clinical features characteristic of this disorder are well known, but the specific causes remain unknown. However, various derangements of circadian expression of the clock gene are a probable cause of this disease. We have attempted to elucidate the relationship between the expression of the clock genes in whole blood cells and the clinical features characteristic of this disorder. In this study, we indicate the daily expression of clock genes period (Per) 1, 2, 3, Bmal1, and Clock in whole blood cells in 12 healthy male subjects. The peak phase of Per1, Per2, and Per3 appeared in the early morning, whereas that of Bmal1 and Clock appeared in the midnight hours. Furthermore, in one patient case with circadian rhythm sleep disorder, we observed variations of the peak phase in clock genes by treatments such as light therapy, exercise therapy, and medicinal therapy. This study suggested that the monitoring of human clock genes in whole blood cells, which may be functionally important for the molecular control of the circadian pacemaker as well as in suprachiasmatic nucleus, might be useful to evaluate internal synchronization.     

Growth Inhibition by D-Mannosamine and Its Reversal by Glucose and Mannose in Bacillus subtilis

Growth of the Bacillus subtilis wild type strain was severelyinhibited by mannosamine. This inhibitory effect was reversedby the addition of glucose or mannose. Growth in peptone mediumwas accompanied by the consumption of the added glucose andby the accumulation of acetoin. Glucose utilization and acetoinproduction were markedly repressed in the cells grown in thepresence of mannosamine. Growth in the presence of mannosaminewas accompanied by a relative decrease in a protein with themolecular weight of 70,000 as revealed by SDS polyacrylamidegel electrophoresis. Our results indicate that mannosamine'sinhibition of growth is due primarily to low glucose utilizationalthough other cellular metabolic activity also may be involved. (Received August 15, 1983; Accepted April 2, 1984)     

Material properties of the ovine mitral valve anterior leaflet in vivo from inverse finite element analysis

We measured leaflet displacements and used inverse finite-element analysis to define, for the first time, the material properties of mitral valve (MV) leaflets in vivo. Sixteen miniature radiopaque markers were sewn to the MV annulus, 16 to the anterior MV leaflet, and 1 on each papillary muscle tip in 17 sheep. Four-dimensional coordinates were obtained from biplane videofluoroscopic marker images (60 frames/s) during three complete cardiac cycles. A finite-element model of the anterior MV leaflet was developed using marker coordinates at the end of isovolumic relaxation (IVR; when the pressure difference across the valve is approximately 0), as the minimum stress reference state. Leaflet displacements were simulated during IVR using measured left ventricular and atrial pressures. The leaflet shear modulus (G(circ-rad)) and elastic moduli in both the commisure-commisure (E(circ)) and radial (E(rad)) directions were obtained using the method of feasible directions to minimize the difference between simulated and measured displacements. Group mean (+/-SD) values (17 animals, 3 heartbeats each, i.e., 51 cardiac cycles) were as follows: G(circ-rad) = 121 +/- 22 N/mm2, E(circ) = 43 +/- 18 N/mm2, and E(rad) = 11 +/- 3 N/mm2 (E(circ) > E(rad), P < 0.01). These values, much greater than those previously reported from in vitro studies, may result from activated neurally controlled contractile tissue within the leaflet that is inactive in excised tissues. This could have important implications, not only to our understanding of mitral valve physiology in the beating heart but for providing additional information to aid the development of more durable tissue-engineered bioprosthetic valves.     

A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors

The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.     

Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein

Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.     

Diacylglycerol Kinase �� Augments C-Raf Activity and B-Raf/C-Raf Heterodimerization

The Ras/B-Raf/C-Raf/MEK/ERK signaling cascade is critical for the control of many fundamental cellular processes, including proliferation, survival, and differentiation. This study demonstrated that small interfering RNA-dependent knockdown of diacylglycerol kinase η (DGKη) impaired the Ras/B-Raf/C-Raf/MEK/ERK pathway activated by epidermal growth factor (EGF) in HeLa cells. Conversely, the overexpression of DGKη1 could activate the Ras/B-Raf/C-Raf/MEK/ERK pathway in a DGK activity-independent manner, suggesting that DGKη serves as a scaffold/adaptor protein. By determining the activity of all the components of the pathway in DGKη-silenced HeLa cells, this study revealed that DGKη activated C-Raf but not B-Raf. Moreover, this study demonstrated that DGKη enhanced EGF-induced heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf. DGKη physically interacted with B-Raf and C-Raf, regulating EGF-induced recruitment of B-Raf and C-Raf from the cytosol to membranes. The DGKη-dependent activation of C-Raf occurred downstream or independently of the already known C-Raf modifications, such as dephosphorylation at Ser-259, phosphorylation at Ser-338, and interaction with 14-3-3 protein. Taken together, the results obtained strongly support that DGKη acts as a novel critical regulatory component of the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade via a previously unidentified mechanism.The Ras/Raf/MEK³/ERK signaling pathway is critical for the transduction of the extracellular signals to the nucleus, regulating diverse physiological processes such as cell proliferation, differentiation, and survival (1, 2). The binding of extracellular ligands, such as growth factors and cytokines, to cell surface receptors activates Ras. The Raf serine/threonine kinase transmits signals from activated Ras to the downstream protein kinases, MEK1 and MEK2, subsequently leading to activation of ERK1 and ERK2.In mammals, the Raf kinase consists of three isoforms, A-Raf, B-Raf, and C-Raf (Raf-1). It is clinically known that both B-Raf and C-Raf mutations are associated with human cancers (3–5). Knock-out mouse studies demonstrated that each individual Raf isoform has distinct functions, although the three Raf isoforms have high homology in the amino acid sequence (6). The mechanisms underlying C-Raf activation are complicated and thus are not completely understood (3). In response to extracellular signals, C-Raf is initially recruited from cytosol to the plasma membrane and undergo conformational changes by binding directly to the active Ras (7). In addition, other modifications and factors are required for the sufficient activation of C-Raf. For example, dephosphorylation of Ser-259 and phosphorylation of Ser-338, Tyr-341, Thr-491, and Ser-494 are critical for the activation of C-Raf (8–11). Feedback phosphorylation of C-Raf by ERK was also reported to be important for the modulation of C-Raf activity (12, 13). C-Raf activity is regulated by the interaction with 14-3-3 protein (14). Moreover, the heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf, has been reported to play an essential role in the activation of the MEK-ERK signaling pathway (15–17). Although B-Raf and C-Raf are the central regulatory components in the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade involved in a variety of pathophysiological events, the activation mechanisms of C-Raf by B-Raf are still unclear.Diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol to generate phosphatidic acid. DGK has been recently recognized as an emerging key regulator in a wide range of cell signaling systems (18–20). To date, 10 mammalian DGK isozymes have been identified. They characteristically contain two or three protein kinase C-like C1 domains and a catalytic region and are subdivided into five subtypes according to their structural features (18–20). Their structural variety and distinct expression patterns in tissues allow us to presume that each DGK isozyme has its own biological functions. Indeed, recent studies have revealed that individual DGK isozymes play distinct roles in cell functions through interactions with unique partner proteins such as protein kinase C (21, 22), Ras guanyl nucleotide-releasing protein (23, 24), phosphatidylinositol-4-phosphate 5-kinase (25), chimerins (26, 27), AP-2 (28), and PSD-95 (29).DGKη belongs to the type II DGKs containing a pleckstrin homology domain at the N terminus and the separated catalytic region (19, 30). Two alternative splicing products of DGKη have been identified as DGKη1 and -η2 (31). DGKη2 possesses a sterile α-motif (SAM) domain at the C terminus, whereas DGKη1 does not. This study demonstrated that the expression levels of DGKη1 and -η2 were regulated differently by glucocorticoid, and that they were translocated from the cytoplasm to endosomes in response to stress stimuli as osmotic shock and oxidative stress (31). However, the physiological roles of DGKη remain unknown.This study showed that siRNA-dependent knockdown of DGKη inhibits cell proliferation of the HeLa cells. In addition, DGKη is required for the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade activated by epidermal growth factor (EGF). Intriguingly, DGKη regulates recruitment of B-Raf and C-Raf from cytosol to membranes and their heterodimerization. Moreover, this study demonstrated that DGKη activates C-Raf but not B-Raf in an EGF-dependent manner. The data show DGKη as a novel key regulator of the Ras/B-Raf/C-Raf/MEK/ERK signaling pathway.