Ran GTPase regulates Mad2 localization to the nuclear pore complex

In yeast and mammalian cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the nuclear pore complex (NPC) during interphase. Deletion of MAD1 or MAD2 did not affect steady-state nucleocytoplasmic distribution of a classical nuclear localization signal-containing reporter, a nuclear export signal-containing reporter, or Ran localization. We utilized cells with conditional mutations in the yeast Ran GTPase pathway to examine the relationship between Ran and targeting of checkpoint regulators to the NPC. Mutations that disrupt the concentration of Ran in the nucleus displaced Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells was correlated with activation of the spindle checkpoint. Our observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC.     

A new deletion of the mouse Y chromosome long arm associated with the loss of Ssty expression, abnormal sperm development and sterility

The mouse Y chromosome carries 10 distinct genes or gene families that have open reading frames suggestive of retained functionality; it has been assumed that many of these function in spermatogenesis. However, we have recently shown that only two Y genes, the testis determinant Sry and the translation initiation factor Eif2s3y, are essential for spermatogenesis to proceed to the round spermatid stage. Thus, any further substantive mouse Y-gene functions in spermatogenesis are likely to be during sperm differentiation. The complex Ssty gene family present on the mouse Y long arm (Yq) has been implicated in sperm development, with partial Yq deletions that reduce Ssty expression resulting in impaired fertilization efficiency. Here we report the identification of a more extensive Yq deletion that abolishes Ssty expression and results in severe sperm defects and sterility. This result establishes that genetic information (Ssty?) essential for normal sperm differentiation and function is present on mouse Yq.     

The gastroenteroinsular response to glucose ingestion during postexercise recovery

This study examined gastrointestinal hormone and peptide responses when glucose was ingested after prolonged exercise. Six endurance-trained male athletes ran on a treadmill for 2 h at 60% VO2 max. Immediately after the run, the athletes consumed 75 g of glucose in 250 ml of water (ExGLU) or flavored water as a placebo control (ExPL). On a separate visit, the athletes rested for 2 h and then consumed glucose (ConGLU). During the first 60 min of recovery from exercise alone (ExPL), plasma vasoactive intestinal peptide (VIP), gastrin, and glucagon-like peptide-1 (GLP-1) all increased significantly, whereas glucose, insulin, and gastric inhibitory polypeptide (GIP) were unchanged from the immediate postexercise value. When glucose was ingested after exercise (ExGLU), glucose, insulin, VIP, gastrin, GLP-1, and GIP were all increased (P < 0.01). However, when glucose was ingested after resting for 2 h (ConGLU), VIP levels were unaffected, although glucose, insulin, gastrin, GLP-1, and GIP levels increased (P < 0.05). The plasma glucose response was greater (P < 0.03) and the plasma insulin response lower (P < 0.004) during ExGLU compared with ConGLU. There was a significantly higher (P < 0.01) VIP response during the initial period of recovery in ExGLU than there was with both ExPL and ConGLU. Plasma VIP showed a modest negative correlation with circulating glucose (r = -0.35, P < 0.03) and insulin (r = -0.37, P < 0.03) during the ExGLU recovery period. In summary, when glucose is ingested after prolonged exercise, there is mild insulin resistance and a corresponding rapid transitory increase in plasma VIP. These data suggest that VIP may play an important glucoregulatory role when glucose is ingested during the immediate postexercise recovery period.     

Common motor mechanisms support body load in serially homologous legs of cockroaches in posture and walking

We studied the mechanisms underlying support of body load in posture and walking in serially homologous legs of cockroaches. Activities of the trochanteral extensor muscle in the front or middle legs were recorded neurographically while animals were videotaped. Body load was increased via magnets attached to the thorax and varied through a coil below the substrate. In posture, tonic firing of the slow trochanteral extensor motoneuron (Ds) in each leg was strongly modulated by changing body load. Rapid load increases produced decreases in body height and sharp increments in extensor firing. The peak of extensor activity more closely approximated the maximum velocity of body displacement than the body position. In walking, extensor bursts in front and middle legs were initiated during swing and continued into the stance phase. Moderate tonic increases in body load elicited similar, specific, phase dependent changes in both legs: extensor firing was not altered in swing but was higher after foot placement in stance. These motor adjustments to load are not anticipatory but apparently depend upon sensory feedback. These data are consistent with previous findings in the hind legs and support the idea that body load is countered by common motor mechanisms in serially homologous legs.     

Force detection in cockroach walking reconsidered: discharges of proximal tibial campaniform sensilla when body load is altered

We examined the mechanisms underlying force feedback in cockroach walking by recording sensory and motor activities in freely moving animals under varied load conditions. Tibial campaniform sensilla monitor forces in the leg via strains in the exoskeleton. A subgroup (proximal receptors) discharge in the stance phase of walking. This activity has been thought to result from leg loading derived from body mass. We compared sensory activities when animals walked freely in an arena or on an oiled glass plate with their body weight supported. The plate was oriented either horizontally (70-75% of body weight supported) or vertically (with the gravitational vector parallel to the substrate). Proximal sensilla discharged following the onset of stance in all load conditions. In addition, activity was decreased in the middle third of the stance phase when the effect of body weight was reduced. Our results suggest that sensory discharges early in stance result from forces generated by contractions of muscles that press the leg as a lever against the substrate. These forces can unload legs already in stance and assure the smooth transition of support among the limbs. Force feedback later in stance may adjust motor output to changes in leg loading.     

Characterization of two mutations associated with epimerase-deficiency galactosemia, by use of a yeast expression system for human UDP-galactose-4-epimerase.

UDP-galactose-4-epimerase (GALE) is a highly conserved enzyme that catalyzes the interconversion of UDP-galactose and UDP-glucose. Impairment of this enzyme in humans results in one of two clinically distinct forms of epimerase-deficiency galactosemia-one benign, the other severe. The molecular and biochemical distinction between these disorders remains unknown. To enable structural and functional studies of both wild-type and patient-derived alleles of human GALE (hGALE), we have developed and applied a null-background yeast expression system for the human enzyme. We have demonstrated that wild-type hGALE sequences phenotypically complement a yeast gal10 deletion, and we have biochemically characterized the wild-type human enzyme isolated from these cells. Furthermore, we have expressed and characterized two mutant alleles, L183P-hGALE and N34S-hGALE, both derived from a patient with no detectable GALE activity in red blood cells but with approximately 14% activity in cultured lymphoblasts. Analyses of crude extracts of yeast expressing L183P-hGALE demonstrated 4% wild-type activity and 6% wild-type abundance. Extracts of yeast expressing N34S-hGALE demonstrated approximately 70% wild-type activity and normal abundance. However, yeast coexpressing both L183P-hGALE and N34S-hGALE exhibited only approximately 7% wild-type levels of activity, thereby confirming the functional impact of both substitutions and raising the intriguing possibility that some form of dominant-negative interaction may exist between the mutant alleles found in this patient. The results reported here establish the utility of the yeast-based hGALE-expression system and set the stage for more-detailed studies of this important enzyme and its role in epimerase-deficiency galactosemia.     

A p90rsk Mutant Constitutively Interacting with MAP Kinase Uncouples MAP Kinase from p34cdc2/Cyclin B Activation in Xenopus Oocytes

The efficient activation of p90^rsk by MAP kinase requires their interaction through a docking site located at the C-terminal end of p90^rsk. The MAP kinase p42^mpk1 can associate with p90^rsk in G₂-arrested but not in mature Xenopus oocytes. In contrast, an N-terminally truncated p90^rsk mutant named D2 constitutively interacts with p42^mpk1. In this report we show that expression of D2 inhibits Xenopus oocyte maturation. The inhibition requires the p42^mpk1 docking site. D2 expression uncouples the activation of p42^mpk1 and p34^cdc2/cyclin B in response to progesterone but does not prevent signaling through p90^rsk. Instead, D2 interferes with a p42^mpk1-triggered pathway, which regulates the phosphorylation and activation of Plx1, a potential activator of the Cdc25 phosphatase. This new pathway that links the activation of p42^mpk1 and Plx1 during oocyte maturation is independent of p34^cdc2/cyclin B activity but requires protein synthesis. Using D2, we also provide evidence that the sustained activation of p42^mpk1 can trigger nuclear migration in oocytes. Our results indicate that D2 is a useful tool to study MAP kinase function(s) during oocyte maturation. Truncated substrates such as D2, which constitutively interact with MAP kinases, may also be helpful to study signal transduction by MAP kinases in other cellular processes.