The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis

The isolation and properties of a single site temperature sensitive protease mutant of Bacillus subtilis are described. Numerous criteria suggest that the mutation resides in the structural gene coding for a basic serine protease. The mutation has been mapped between aroD and lys-1 on the Bacillus subtilis chromosome. This protease exists as an intracellular and extracellular enzyme. The mutant cells are temperature sensitive for sporulation, antibiotic production, and the sporulation-specific alteration in DNA-dependent RNA polymerase β subunit. Several types of evidence indicate a direct involvement of this enzyme in a limited proteolytic cleavage of vegetative RNA polymerase β subunit, which produces the lower molecular weight β subunit found in sporulating cells. The derangement in this process is sufficient to account for the stoppage of sporulation at stage 0 when the mutant cells are grown at the non-permissive temperature.     

Okadaic acid mimics a nuclear component required for cyclin B-cdc2 kinase microinjection to drive starfish oocytes into M phase

G2-arrested oocytes contain cdc2 kinase as an inactive cyclin B-cdc2 complex. When a small amount of highly purified and active cdc2 kinase, prepared from starfish oocytes at first meiotic metaphase, is microinjected into Xenopus oocytes, it induces activation of the inactive endogenous complex and, as a consequence, drives the recipient oocytes into M phase. In contrast, the microinjected kinase undergoes rapid inactivation in starfish oocytes, which remain arrested at G2. Endogenous cdc2 kinase becomes activated in both nucleated and enucleated starfish oocytes injected with cytoplasm taken from maturing oocytes at the time of nuclear envelope breakdown, but only cytoplasm taken from nucleated oocytes becomes able thereafter to release second recipient oocytes from G2 arrest, and thus contains M phase-promoting factor (MPF) activity. Both nucleated and enucleated starfish oocytes produce MPF activity when type 2A phosphatase is blocked by okadaic acid. If type 2A phosphatase is only partially inhibited, neither nucleated nor enucleated oocytes produce MPF activity, although both do so if purified cdc2 kinase is subsequently injected as a primer to activate the endogenous kinase. The nucleus of starfish oocytes contains an inhibitor of type 2A phosphatase, but neither active nor inactive cdc2 kinase. Microinjection of the content of a nucleus into the cytoplasm of G2-arrested starfish oocytes activates endogenous cdc2 kinase, produces MPF activity, and drives the recipient oocytes into M phase. Together, these results show that the MPF amplification loop is controlled, both positively and negatively, by cdc2 kinase and type 2A phosphatase, respectively. Activation of the MPF amplification loop in starfish requires a nuclear component to inhibit type 2A phosphatase in cytoplasm.     

Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts

Previous independent studies suggested that type II cAMP-dependent protein kinase and the p34cdc2 protein kinase cell cycle regulator co-localize at centrosomes. In order to investigate whether there is an association of type II cAMP-dependent protein kinase with p34cdc2 in human fibroblasts, we used three different approaches. First, the regulatory subunits RI and RII were photoaffinity-labeled with 8-N3-[32P]cAMP, and anti-p34cdc2 immunoprecipitates were screened for the presence of either RI or RII regulatory subunits by one- or two-dimensional gel electrophoresis. Second, anti-RII alpha immunoprecipitates were screened for the presence of p34cdc2 by Western blot using three different affinity-purified antibodies recognizing different domains of human p34cdc2. Conversely, anti-p34cdc2 immunoprecipitates (three different antibodies), as well as the material retained on p13suc1-Sepharose Bio-Beads, which binds specifically p34cdc2, were screened for the presence of RII alpha. Finally, we have looked for cAMP-dependent protein kinase activity specifically inhibited by PKI in immunoprecipitates obtained from extracts treated with different anti-p34cdc2 antibodies. All these experiments gave concordant results and demonstrate that at least at G0/G1, human fibroblasts contain a complex of active type II cAMP-dependent protein kinase associated through its RII alpha subunit with p34cdc2.     

Maitotoxin triggers the cortical reaction and phosphatidylinositol-4,5-bisphosphate breakdown in amphibian oocytes

Maitotoxin, a potent marine toxin extracted from peredinians, was found to mimic fertilization in Xenopus oocytes and to trigger the breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2, the precursor of inositol 1,4,5-trisphosphate], an increase of intracellular pCa and the cortical reaction, including the exocytosis of cortical granules and a wave-like propagation of contraction in the animal hemisphere. All these effects of maitotoxin required the presence of external calcium. Moreover, the toxin considerably increased Ca2+ influx in amphibian oocytes arrested at first meiotic prophase, due to the permanent activation of voltage-dependent Ca2+ channels. Nevertheless it is doubtful that maitotoxin acts primarily as a Ca2+ ionophore or at the level of Ca2+ channels. Indeed no stimulation of Ca2+ uptake was observed in metaphase-II-arrested oocytes, although maitotoxin readily triggered the breakdown of PtdIns(4,5)P2 as well as the cortical reaction in such cells. On the other hand, PtdIns(4,5)P2 breakdown was not reduced in oocytes microinjected with EGTA, although the calcium chelator prevented the oocytes from undergoing the cortical reaction. Taken together, these findings support the view that the toxin might act primarily by increasing PtdIns(4,5)P2 phosphodiesterase activity.     

p34cdc2 is located in both nucleus and cytoplasm; part is centrosomally associated at G2/M and enters vesicles at anaphase.

The cdc2+ gene product p34cdc2 is located immunocytochemically in both the nucleus and cytoplasm of human cells. It is uniformly distributed throughout the cytoplasm and is irregularly distributed in the nucleus. Part of p34cdc2 is associated with the centrosome and centrosomal staining increases late in the cell cycle and at the onset of mitosis. This distribution is corroborated by cell fractionation which also indicates that slower migrating forms of p34cdc2 are found in isolated centrosomes and in Triton-insoluble fractions. We propose that one role of the p34cdc2 protein kinase is to modify the centrosome bringing about formation of the mitotic spindle. At anaphase p34cdc2 becomes associated with vesicles in the middle of the cell between the reforming nuclei. A similar location is found for p13suc1 and we suggest that the vesicular localization plays a role in p34cdc2 kinase inactivation at the end of mitosis.     

Latitudinal clines: an evolutionary view on biological rhythms

Properties of the circadian and annual timing systems are expected to vary systematically with latitude on the basis of different annual light and temperature patterns at higher latitudes, creating specific selection pressures. We review literature with respect to latitudinal clines in circadian phenotypes as well as in polymorphisms of circadian clock genes and their possible association with annual timing. The use of latitudinal (and altitudinal) clines in identifying selective forces acting on biological rhythms is discussed, and we evaluate how these studies can reveal novel molecular and physiological components of these rhythms.     

ORIGINAL ARTICLE: Epithelioid hemangioendothelioma in the right auricle of an adult,male Rhesus macaque (Macaca mulatta)

Abstract Background A 9‐year‐old, male castrate, Rhesus macaque was euthanized following a prolonged history of chronic renal failure. Results Necropsy revealed a proliferative lesion within the right cardiac auricle composed of neoplastic epithelioid cells which infiltrated the myocardium and frequently exhibited intracytoplasmic luminae. Cells multifocally exhibited strong cytoplasmic immunoreactivity for Factor VIII‐related protein (von Willebrand’s factor). Conclusions The histological characteristics of this tumor are consistent with a diagnosis of epithelioid hemangioendothelioma, an intermediate‐grade vasoformative neoplasm which has to our knowledge not previously been reported in the heart of a non‐human species.     

Sexual signaling: climatic carry-over

A long term study of warblers in the Himalayas reveals a surprising contrast in the effects of warm springs as opposed to warm summers on a signaling trait, emphasizing the need to consider year-round influences of the environment on morphological variation.     

LKB1 Regulates Pancreatic β Cell Size, Polarity, and Function

Pancreatic β cells, organized in the islets of Langerhans, sense glucose and secrete appropriate amounts of insulin. We have studied the roles of LKB1, a conserved kinase implicated in the control of cell polarity and energy metabolism, in adult β cells. LKB1-deficient β cells show a dramatic increase in insulin secretion in vivo. Histologically, LKB1-deficient β cells have striking alterations in the localization of the nucleus and cilia relative to blood vessels, suggesting a shift from hepatocyte-like to columnar polarity. Additionally, LKB1 deficiency causes a 65% increase in β cell volume. We show that distinct targets of LKB1 mediate these effects. LKB1 controls β cell size, but not polarity, via the mTOR pathway. Conversely, the precise position of the β cell nucleus, but not cell size, is controlled by the LKB1 target Par1b. Insulin secretion and content are restricted by LKB1, at least in part, via AMPK. These results expose a molecular mechanism, orchestrated by LKB1, for the coordinated maintenance of β cell size, form, and function.