Sphingosine inhibits phorbol 12-myristate 13-acetate-, but not serum-induced, activation of Na+/H+ exchange in mammalian cells

Addition of serum to quiescent mammalian cells in culture initiates a series of events which culminates in DNA replication and cell division. One of the earliest events in this sequence of events is activation of Na+/H+ exchange, which can result in an increase in intracellular pH (pHin). The regulation of this change in activity is not known. Since treatment of 3T3 cells with activators of protein kinase C (kinase C) can result in an increased pHin, it has been hypothesized that serum stimulation of kinase C is responsible for activation of Na+/H+ exchange. Recently, sphingolipids have been discovered to inhibit kinase C both in vitro and in vivo. Therefore, we undertook the present study to ask whether or not inhibition of kinase C using sphingolipids prevents mitogen-induced alkalinization in 3T3 cells. Our results indicate that activators of kinase C stimulate Na+/H+ exchange in normal human fibroblasts (BoGi), but not in mouse embryo (3T3) cells. Addition of serum to BoGi cells, on top of saturating doses of phorbol 12-myristate 13-acetate (PMA), results in a further cytoplasmic alkalinization. Furthermore, sphingosine prevents the PMA-induced increase in pHin in BoGi cells, and phosphorylation of an 80 kDa protein in 3T3 cells, but not the serum-induced alkalinization in either BoGi or 3T3 cells. These data indicate that activation of kinase C does not participate in the physiological activation of Na+/H+ exchange in human fibroblasts or mouse embryo cells by serum.     

Identification of Candida albicans ALS2 and ALS4 and Localization of Als Proteins to the Fungal Cell Surface

Additional genes in the growing ALS family of Candida albicans were isolated by PCR screening of a genomic fosmid library with primers designed from the consensus tandem-repeat sequence of ALS1. This procedure yielded fosmids encoding ALS2 and ALS4. ALS2 and ALS4 conformed to the three-domain structure of ALS genes, which consists of a central domain of tandemly repeated copies of a 108-bp motif, an upstream domain of highly conserved sequences, and a domain of divergent sequences 3′ of the tandem repeats. Alignment of five predicted Als protein sequences indicated conservation of N- and C-terminal hydrophobic regions which have the hallmarks of secretory signal sequences and glycosylphosphatidylinositol addition sites, respectively. Heterologous expression of an N-terminal fragment of Als1p in Saccharomyces cerevisiae demonstrated function of the putative signal sequence with cleavage following Ala17. This signal sequence cleavage site was conserved in the four other Als proteins analyzed, suggesting identical processing of each protein. Primary-structure features of the five Als proteins suggested a cell-surface localization, which was confirmed by indirect immunofluorescence with an anti-Als antiserum. Staining was observed on mother yeasts and germ tubes, although the intensity of staining on the mother yeast decreased with elongation of the germ tube. Similar to other ALS genes, ALS2 and ALS4 were differentially regulated. ALS4 expression was correlated with the growth phase of the culture; ALS2 expression was not observed under many different in vitro growth conditions. The data presented here demonstrate that ALS genes encode cell-surface proteins and support the conclusion that the size and number of Als proteins on the C. albicans cell surface vary with strain and growth conditions.     

Possible role of Escherichia coli penicillin-binding protein 6 in stabilization of stationary-phase peptidoglycan.

Plasmids for high-level expression of penicillin-binding protein 6 (PBP6) were constructed, giving rise to overproduction of PBP6 under the control of the lambda pR promoter in either the periplasmic or the cytoplasmic space. In contrast to penicillin-binding protein 5 (PBP5), the presence of high amounts of PBP6 in the periplasm as well as in the cytoplasm did not result in growth as spherical cells or in lysis. Deletion of the C-terminal membrane anchor of PBP6 resulted in a soluble form of the protein (PBP6s350). Electron micrographs of thin sections of cells overexpressing both native membrane-bound and soluble PBP6 in the periplasm revealed a polar retraction of the cytoplasmic membrane. Cytoplasmic overexpression of native PBP6 gave rise to the formation of membrane vesicles, whereas the soluble PBP6 formed inclusion bodies in the cytoplasm. Both the membrane-bound and the soluble forms of PBP6 were purified to homogeneity by using the immobilized dye Procion rubine MX-B. Purified preparations of PBP6 and PBP6s350 formed a 14[C]penicillin-protein complex at a 1:1 stoichiometry. The half-lives of the complexes were 8.5 and 6 min, respectively. In contrast to PBP5, no DD-carboxypeptidase activity could be detected for PBP6 by using bisacetyl-L-Lys-D-Ala-D-Ala and several other substrates. These findings led us to conclude that PBP6 has a biological function clearly distinct from that of PBP5 and to suggest a role for PBP6 in the stabilization of the peptidoglycan during stationary phase.     

Simultaneous quantification of DNA and RNA in tissue sections. A comparative analysis of the Methyl Green-Pyronin technique with the Gallocyanin chromalum and Feulgen procedures using image cytometry

Summary For simultaneous cytophotometric measurement of DNA and RNA, the standardized Methyl Green-Pyronin Y technique is an obvious choice. It is, however, first necessary to correlate the uptake of Pyronin Y to the staining intensity of RNA.The material consisted of paraffin sections of formalin- or Carnoy-fixed rat liver. The sections were pretreated with water, buffer, deoxyribonuclease, ribonuclease, or both enzymes in sequence, and stained with the standardized Methyl Green-Pyronin Y procedure, Gallocyanin chromalum, or the Feulgen analtion. Sections stained directly without pretreatment served as controls. Staining intensities were measured with an image analyser for cell nuclei, nucleoli and cytoplasm.After deoxyribonuclease treatment, nuclear staining intensity with Methyl Green, Gallocyanin chromalum, and Schiff's reagent dropped nearly to zero. The same was seen for both nucleoli and cytoplasm with Pyronin Y and Gallocyanin chromalum after ribonuclease treatment. Staining intensity of Pyronin Y correlated directly with that of Gallocyanin chromalum for nucleoli and cytoplasm. After ribonuclease treatment, a direct correlation was found between the nuclear staining intensity of Methyl Green and nuclear absorption of Gallocyanin chromalum.We conclude that the standardized Methyl Green-Pyronin Y stain is reliable for the simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine.     

Methoxychlor-induced atresia in the mouse involves Bcl-2 family members, but not gonadotropins or estradiol

Methoxychlor (MXC) is an organochlorine pesticide that increases the rate of ovarian atresia. To date, little is known about the mechanism by which MXC induces atresia. Because Bcl-2 (an antiapoptotic factor), Bax (a proapoptotic factor), gonadotropins, and estradiol are important regulators of atresia in the ovary, the purpose of this study was first to examine whether MXC-induced atresia occurred through alterations in Bcl-2 or Bax, and second, to examine the effect of MXC on gonadotropins, estradiol, and their receptors. CD-1 mice were dosed with 8-64 mg kg(-1) day(-1) MXC or vehicle (sesame oil). Ovaries were subjected to analysis of antral follicle numbers, Bcl-2, Bax, estrogen receptor, and follicle-stimulating hormone receptor levels. Blood was used to measure gonadotropins and estradiol. In some experiments, mice that overexpressed Bcl-2 or mice that were deficient in Bax were dosed with MXC or vehicle and their ovaries were analyzed for atresia. MXC caused a dose-dependent increase in the percentage of atretic antral follicles compared with controls at the 32 and 64 mg kg(-1) day(-1) doses of MXC. MXC treatment did not result in changes in Bcl-2 levels, but it did result in an increase in Bax levels in antral follicles. MXC treatment did not affect gonadotropin or estradiol levels, nor did it affect the levels of follicle-stimulating hormone or estrogen receptors. Mice that overexpressed Bcl-2 or mice that were deficient in Bax were protected from MXC-induced atresia. These data suggest that MXC induces atresia through direct effects on the Bax and Bcl-2 signaling pathways in the ovary.     

Characterization of cyclicity and hormonal profile with impending ovarian failure in a novel chemical-induced mouse model of perimenopause

4-Vinylcyclohexene diepoxide (VCD) causes early, gradual ovarian failure in mice because it specifically targets small pre-antral ovarian follicles. The period between loss of these follicles and ovarian failure is analogous to perimenopause in women. We sought to characterize the period of onset of ovarian failure in VCD-treated mice in regard to estrous cycle length and hormonal changes. Female C57Bl/6 mice (age, 28 days) were dosed daily for 15 days with VCD (160 mg/kg intraperitoneally) to cause early ovarian failure or with vehicle only (control animals). Cycle length was monitored by vaginal cytology. Plasma levels of 17beta-estradiol (E2), progesterone (P4), and follicle-stimulating hormone (FSH) in control and VCD-treated animals were measured during proestrus of cycles 1 through 12. Cycle length (mean, 5.8 days) did not differ between groups for cycles 1 through 4. In contrast, cycle length during cycles 5 through 12 was increased (mean length, 10.9 days; P < 0.05 versus control) in VCD-treated animals, which also showed an apparent increase in plasma FSH levels. Plasma E2 and P4 at proestrus did not differ between groups during any cycle. Ovarian failure in VCD-treated mice was confirmed by histological evaluation on day 156 after onset of dosing, whereas control animals were still cycling. Therefore, despite compromised cycle length in VCD-treated mice, peak ovarian steroid production in preovulatory follicles at proestrus is adequate. These results demonstrate that the VCD-treated mouse can serve as an appropriate model to mimic hormonal changes during the perimenopausal transition in women.     

Complexity of heart rate fluctuations in near-term sheep and human fetuses during sleep.

We investigated how the complexity of fetal heart rate fluctuations (fHRF) is related to the sleep states in sheep and human fetuses. The complexity as a function of time scale for fetal heart rate data for 7 sheep and 27 human fetuses was estimated in rapid eye movement (REM) and non-REM sleep by means of permutation entropy and the associated Kullback-Leibler entropy. We found that in humans, fHRF complexity is higher in non-REM than REM sleep, whereas in sheep this relationship is reversed. To show this relation, choice of the appropriate time scale is crucial. In sheep fetuses, we found differences in the complexity of fHRF between REM and non-REM sleep only for larger time scales (above 2.5 s), whereas in human fetuses the complexity was clearly different between REM and non-REM sleep over the whole range of time scales. This may be due to inherent time scales of complexity, which reflect species-specific functions of the autonomic nervous system. Such differences have to be considered when animal data are translated to the human situation.     

Linkage relationships of several insecticide resistance factors in the house fly (Musca domestica L.)

Summary Strains of the house fly resistant to parathion, malathion, Isolan, and DDT were crossed with a susceptible strain which carried the recessive marker, stubby-wing (stw). F₁ populations were composed of normal-winged insecticide-resistant flies. When the F_1's were backcrossed to the stw parent, the resultant normal-winged progeny were resistant and the stw progeny were susceptible. Thus the major factors for resistance in the strains studied were all located on the same chromosome.Similar cross-over ratios were observed with parathion and malathion-resistant strains and populations of resistant stw flies were established. Low ali-esterase, characteristic of the parent resistant strains, was also present in the resistant stw strains.No crossing-over occurred between stw and DDT-resistance. Resistance to DDT, present in the malathion-resistant strain, was not introduced with malathion-resistance into the stw strain. Therefore, the factors for DDT and malathion resistance, although linked, are genetically distinct.

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Zusammenfassung Stubenfliegenstämme, die Resistenzfaktoren gegen Parathion, Malathion, Isolan^R (1-isopropyl-3-methyl-5-pyrazolyl dimethylcarbamate) und DDT tragen, wurden mit einem anfälligen Stamm gekreuzt, der das rezessive Markierungsgen, stummelflügelig (stw), aufweist. Die F₁-Populationen jeder Kreuzung bestanden aus normalflügeligen, insektizidresistenten Fliegen. Wenn die F₁-Fliegen mit den stw-Eltern rückgekreuzt wurden, waren die normalflügeligen Nachkommen resistent gegen alle Insektizide und die stw-Nachkommen anfällig. Also sind die Hauptresistenzfaktoren der untersuchten Stämme alle in dem gleichen Chromosom lokalisiert.Für die Faktoren, welche die Resistenz gene Parathion und Malathion kontrollieren, wurden ähnliche crossing over-Verhältnisse beobachtet, und es wurden Populationen von stw-Fliegen aufgebaut, die resistent gegen diese Insektizide sind. Niedrige Ali-Esterase-Aktivität, die für die parathion- und malathionresistenten Ausgangsstämme charakteristisch ist, war auch in den resistenten stw-Stämmen vorhanden, so daß das Gen für niedrige Ali-Esterase-Aktivität identisch oder eng mit den Resistenzgenen verbunden ist.Eine unzulängliche Trennung zwischen anfälligen und isolanresistenten Fliegen schloß die Möglichkeit der crossing over-Messung des Faktors für Isolanresistenz aus. Zwischen stw und DDT-Resistenz wurde kein crossing over beobachtet. Hohe Kreuzungsresistenz gegen DDT, die in einem malathionresistenten Stamm vorhanden ist, wurde mit dem Faktor für Malathionresistenz nicht in den stw-Stamm eingeführt. Die Faktoren für die Resistenz gegen DDT und Malathion sind demnach genetisch unterschiedlich.

     

DNP-Enhanced MAS NMR: A Tool to Snapshot Conformational Ensembles of α-Synuclein in Different States

Intrinsically disordered proteins dynamically sample a wide conformational space and therefore do not adopt a stable and defined three-dimensional conformation. The structural heterogeneity is related to their proper functioning in physiological processes. Knowledge of the conformational ensemble is crucial for a complete comprehension of this kind of proteins. We here present an approach that utilizes dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of sparsely isotope-labeled proteins in frozen solution to take snapshots of the complete structural ensembles by exploiting the inhomogeneously broadened line-shapes. We investigated the intrinsically disordered protein α-synuclein (α-syn), which plays a key role in the etiology of Parkinson’s disease, in three different physiologically relevant states. For the free monomer in frozen solution we could see that the so-called “random coil conformation” consists of α-helical and β-sheet-like conformations, and that secondary chemical shifts of neighboring amino acids tend to be correlated, indicative of frequent formation of secondary structure elements. Based on these results, we could estimate the number of disordered regions in fibrillar α-syn as well as in α-syn bound to membranes in different protein-to-lipid ratios. Our approach thus provides quantitative information on the propensity to sample transient secondary structures in different functional states. Molecular dynamics simulations rationalize the results.