Identification of protein kinase disruptions as suppressors of the calcium sensitivity of S. cerevisiae Δptp2 Δmsg5 protein phosphatase double disruptant

The double disruptant of the S. cerevisiae protein phosphatase (PPase) genes, PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes calcium-sensitive growth (Ca^s). Previous study using Fluorescent-activated cell sorting (FACS) analysis showed that this growth defect with calcium occurs at G1–S transition in the cell cycle. We discovered that six non-essential protein kinase (PKase) disruptions (Δbck1, Δmkk1, Δslt2/Δmpk1, Δmck1, Δssk2 and Δyak1) suppressed the Ca^s-phenotype of the Δptp2 Δmsg5 double disruptant. Bck1p, Mkk1p and Slt2p are components of the mitogen-activated protein kinase (MAPK) cascade of cell wall integrity pathway (Slt2 pathway), and Mck1p is its down regulator. Ssk2p is the MAPK kinase kinase of the high-osmolarity glycerol (HOG) pathway, while Yak1p is a negative regulator for the cAMP-dependent PKA pathway. FACS analysis revealed that only the disruption of Δssk2 and Δyak1 but not Δbck1, Δmkk1, Δslt2 and Δmck1 was able to suppress the delayed G1–S transition, suggesting that suppression of the growth defect is not always accompanied by suppression of the G1–S transition delay. The discovery of these PKases as suppressors revealed that in addition to the previously anticipated Slt2 pathway, HOG, Yak1p and Mck1p regulatory pathways may also be involved in the calcium sensitivity of the Δptp2 Δmsg5 double disruptant.     

Delta1 family members are involved in filopodial actin formation and neuronal cell migration independent of Notch signaling

Delta family proteins are transmembrane molecules that bind Notch receptors and activate downstream signaling events in neighboring cells. In addition to serving as Notch ligands, Notch-independent roles for Delta have been suggested but are not fully understood. Here, we demonstrate a previously unrecognized role for Delta in filopodial actin formation. Delta1 and Delta4, but not Delta3, exhibit filopodial protrusive activity, and this activity is independent of Notch signaling. The filopodial activity of Delta1 does not depend on the PDZ-binding domain at the C-terminus; however, the intracellular membrane-proximal region that is anchored to the plasma membrane plays an important role in filopodial activity. We further identified a Notch-independent role of DeltaD in neuronal cell migration in zebrafish. These findings suggest a possible functional link between Notch-independent filopodial activity of Delta and the control of cell motility.     

Light and electron microscopic studies on experimental nocardia-toxicosis in mice

An exotoxin (HS-6) produced by Nocardia otitidiscaviarum isolated from certain lesions of cutaneous nocardiosis of a male 82-year-old patient induced severe injuries in the pancreas, liver, stomach, small intestine, heart, thymus and kidney of male ICR mice. Mice given Nocardia-free preparation of HS-6 at a dose of 1 mg/kg of body weight developed several autophagic vacuoles in the pancreas and liver within 20 min after the i.p. injection. Thereafter, the autophagic vacuoles increased in number and size with time. About 24 hr after the administration of HS-6, the liver showed marked accumulation of fat droplets in the cytoplasm of the hepatocytes. Although they contained abundant autophagic vacuoles in the regions of RER, there were no lipomatoses in the acinar cells of the pancreas, those of the chief cells and smooth muscle cells of the stomach, Paneth cells, goblet cells, smooth muscle cells of the small intestine, and plasma cells in the digestive tract. Biochemical examinations revealed that HS-6 had no significant effect on the protein synthesis of reticulocytes. Inoculation of the Nocardia into the mouse peritoneal cavities caused marked granulomatoses in the pancreas, liver and regional lymph nodes, but did not develop autophagic vacuoles in RER regions of these organs.     

Evidence that the Entamoeba histolytica Mitochondrial Carrier Family Links Mitosomal and Cytosolic Pathways through Exchange of 3′-Phosphoadenosine 5′-Phosphosulfate and ATP

Entamoeba histolytica, a microaerophilic protozoan parasite, possesses mitosomes. Mitosomes are mitochondrion-related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. The biological roles of Entamoeba mitosomes have been a long-standing enigma. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. Sulfate activation cooperates with cytosolic enzymes, i.e., sulfotransferases (SULTs), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. Notably, cholesteryl sulfate plays an important role in encystation, an essential process in the Entamoeba life cycle. These findings identified a biological role for Entamoeba mitosomes; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. Here, we demonstrated that the E. histolytica mitochondrial carrier family (EhMCF) has the capacity to exchange 3′-phosphoadenosine 5′-phosphosulfate (PAPS) with ATP. We also confirmed the cytosolic localization of all the E. histolytica SULTs, suggesting that in Entamoeba, PAPS, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for SULTs. In contrast, ATP, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. Taking our findings collectively, we suggest that EhMCF functions as a PAPS/ATP antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic SULTs for the production of sulfolipids.     

The D-JH complex is an intermediate to the complete immunoglobulin heavy-chain V-region gene

We have examined the organization of the immunoglobulin JH segments in three clones derived from a single Abelson murine leukemia virus-transformed cell. Cloning and nucleotide sequence analyses of the JH-containing fragments have revealed the rearrangement from the preformed D-JH complex to the complete VH-D-JH gene, which was accompanied by the expression of the intra-cytoplasmic mu chain. In one case a JH segment downstream to the preformed D-JH was used to create a new VH-D-JH gene. Upon the D-JH and VH-D-JH rearrangements the intervening D segments were deleted from the chromosome. One of the expressed VH genes suffered from a large deletion of the 3' portion (including the 95th cysteine residue) of the VH segment. We discuss the possible mechanism of the allelic exclusion.     

Adenylyl cyclase-cAMP system inhibits thrombin-induced HSP27 in vascular smooth muscle cells

We previously reported that thrombin stimulates the induction of heat shock protein (HSP) 27 via p38 mitogen-activated protein (MAP) kinase activation in aortic smooth muscle A10 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on the thrombin-stimulated induction of HSP27 in A10 cells. Forskolin, a direct activator of adenylyl cyclase, reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the HSP27 accumulation. Furthermore, dibutyryl-cAMP (DBcAMP), a permeable analog of cAMP, significantly suppressed the accumulation of HSP27. On the other hand, calphostin C, an inhibitor of protein kinase C (PKC), reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. Moreover, forskolin reduced the p38 MAP kinase phosphorylation induced by the 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, and significantly suppressed the TPA-stimulated accumulation of HSP27. These results indicate that adenylyl cyclase-cAMP system has an inhibitory role in thrombin-stimulated HSP27 induction in aortic smooth muscle cells, and the effect seems to be exerted on the thrombin-induced PKC- p38 MAP kinase signaling pathway.     

Expression of parsley flavone synthase I establishes the flavone biosynthetic pathway in Arabidopsis thaliana

Arabidopsis thaliana lacks the flavone biosynthetic pathway, probably because of a lack or low activity of a flavone synthase. To establish this biosynthetic pathway in Arabidopsis, we subjected this model plant to transformation with the parsley gene for flavone synthase type I (FNS-I). Transgenic seedlings expressing FNS-I were cultured in liquid medium with or without naringenin, and plant extracts were then analyzed by high-performance liquid chromatography. In contrast to wild-type seedlings, the transgenic seedlings accumulated substantial amounts of apigenin, which is produced from naringenin by FNS-I, and the apigenin level correlated with the abundance of FNS-I mRNA in three different transgenic lines. These results indicate that the FNS-I transgene produces a functional enzyme that catalyzes the conversion of naringenin to apigenin in Arabidopsis. These FNS-I transgenic lines should prove useful in investigating the in vivo functions of enzymes that mediate the synthesis of the wide variety of flavones found in other plants.     

Morphological and Body Color Variation in Thai <Emphasis Type="Italic">Macaca fascicularis fascicularis</Emphasis> North and South of the Isthmus of Kra

Long-tailed macaques (Macaca fascicularis fascicularis) are widely distributed in Southeast Asia and are morphologically and genetically (Tosi et al. in International Journal of Primatology 23:161–178, 2002) distinguishable on either side of the Isthmus of Kra (ca. 10.5°N). We compared the somatometry and body color of 15 local populations of long-tailed macaques in Thailand distributed over areas from 6.5°N to 16.3°N and also a Thai rhesus macaque population at 17.2°N. Limb proportions and body color variation follow the geographical trend. However, contrary to a previous report, body size does not decrease with latitude in the northern group and also in the southern (southerly distributed) rhesus macaque. Relative tail length (RTL) and color contrast in yellow between the back and thigh are the sole traits that distinctively separate the 2 groups: the southern group has a long relative tail length (RTL >125%) and small color contrast, whereas the northern group has a short RTL (<120%) and large color contrast. The southern rhesus macaques appear to have somatometric and body color traits that follow the geographical trend in long-tailed macaques, though they maintain their distinctive species-specific traits of shorter RTL (ca. 55%), shorter relative facial length, and a bipartite body color pattern. Researchers assume that the northern group of long-tailed macaques and the southern rhesus macaques had undergone partial introgression with each other. Montane refugia present during the glacial period are localities in which introgression occurred in long-tailed macaques.