Screening of thermotolerant yeasts as producers of superoxide dismutase

Abstract A screening procedure for highly thermostable yeast superoxide dismutase was developed. Growth yields at various temperatures were estimated for ten mesophilic and thermotolerant strains, belonging to the genera Saccharomyces, Kluyveromyces and Pichia . Higher yields at 45°C were obtained for K. lactis 90-3 and 90-4. A correlation between the ability to grow at higher temperature and the thermostability of the superoxide dismutase enzyme synthesized was observed. A comparison of the operational stability of the superoxide dismutase of all tested strains suggests that the enzyme of K. lactis strains was more thermostable than that of the other tested microorganisms.     

Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain

BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Like other cytotoxicity-mediating effectors, BteA uses its multifunctional N-terminal domain to target phosphatidylinositol (PI)-rich microdomains in the host membrane. Despite their structural similarity, T3SS effectors exhibit a variable range of membrane interaction modes, and currently only limited structural information is available for the BteA membrane-targeting domain and the molecular mechanisms underlying its function. Employing a synergistic combination of structural methods, here we determine the structure of this functional domain and uncover key molecular determinants mediating its interaction with membranes. Residues 29–121 of BteA form an elongated four-helix bundle packed against two shorter perpendicular helices, the second of which caps the domain in a critical ‘tip motif’. A flexible region preceding the BteA helical bundle contains the characteristic β-motif required for binding its cognate chaperone BtcA. We show that BteA targets PI(4,5)P₂-containing lipoprotein nanodiscs and binds a soluble PI(4,5)P₂ analog via an extensive positively charged surface spanning its first two helices, and that this interaction is weaker for PI(3,5)P₂ and abolished for PI(4)P. We confirmed this model of membrane-targeting by observation of BteA-induced changes in the structure of PI(4,5)P₂-containing phospholipid bilayers using small-angle X-ray scattering (SAXS). We also extended these results to a larger BteA domain (residues 1–287), confirming its interaction with bilayers using calorimetry, fluorescence and SAXS methods. This novel view of the structural underpinnings of membrane targeting by BteA is an important step towards a comprehensive understanding of cytotoxicity in Bordetella, as well as interactions of a broad range of pathogens with their respective hosts.     

In vivo hormonal environment leads to differential susceptibility of the corpus luteum to apoptosis in vitro

We evaluated the involvement of the in vivo hormonal environment on the ability of the rat corpus luteum (CL) to undergo apoptosis. Gel electrophoretic DNA fragmentation analysis revealed no apoptosis in CL isolated either the 2 last days of pregnancy (Days 21 and 22) or throughout the 4 days following parturition, suggesting that the number of cells undergoing apoptosis at the same time is not sufficient to allow for visualization of DNA breakdown. In contrast, CL incubated in serum-free medium underwent significant apoptosis, as evaluated by chromatin condensation and DNA fragmentation, regardless of their developmental stage in pregnancy. However, CL obtained on Day 7 of pregnancy and on Day 4 postpartum demonstrated higher sensitivity to apoptosis in vitro, but lactation reduced significantly the capacity of the CL to undergo apoptosis when maintained in culture. These data suggest that the exposure of the CL to different hormonal environments throughout pregnancy and after parturition is responsible for the differential susceptibility to apoptosis observed in vitro. We have previously shown that progesterone is a direct factor for survival of the CL. Prolactin stimulates luteal progesterone production; therefore, we examined whether prolactin prevents apoptosis in luteal cells independently of its stimulatory action on progesterone production. We used a luteal cell line (GG-CL) that expresses the prolactin receptor but does not produce progesterone. These cells undergo apoptosis under conditions of serum starvation, and addition of prolactin to the culture medium significantly reduced DNA fragmentation. These results indicate that the extent of luteal cell death induced by incubation of CL under serum-free conditions depends on the hormonal environment to which this endocrine gland is exposed in vivo. These results also indicate an important role for lactation in preventing apoptosis, which is further supported by the antiapoptotic activity of prolactin observed in luteal cells.     

Combined association,segregation and aggregation analysis on case-control family data

Recently genetic epidemiologists have begun using case-control family study designs to investigate the role of genetic and environmental risk factors in disease etiology. The objective of these studies is to assess the association of environmental factors with the disease trait; to characterize the disease genes using segregation analysis; and to quantify the residual familial aggregation after controlling for environmental and genetic factors. Typically these objectives are achieved by conducting separate studies and analysis. This paper describes an estimating equation based approach for a combined association, segregation and aggregation analysis on data from case-control family studies. Simulations indicate that the method performs well in a variety of settings. The method is illustrated using simulated family history data made available to participants in a recent Genetic Analysis Workshop.     

Androstenedione interferes in luteal regression by inhibiting apoptosis and stimulating progesterone production

Androgens, in concert with lactogenic hormones, contribute to the maintenance of function of the corpus luteum (CL) in pregnant rats. Whereas some of the androgenic actions in the CL are clearly mediated by intracrine conversion to estrogen, pure androgenic effects are also implicated in the regulation of this transient endocrine gland. In this report, we have established, to our knowledge for the first time, the expression of androgen receptor (AR) mRNA and protein throughout gestation in the rat CL. We have found that the AR remains expressed in the CL of gestation on Day 4 postpartum and becomes expressed in the newly formed CL after postpartum ovulation. An AR immunoreactive protein was identified in the CL of pregnancy as well as in prostate and epididymis, which were used as positive controls. The luteal AR protein had mainly nuclear localization, yet some diffuse cytoplasmic staining was also observed. Moreover, we have established that androstenedione, the main circulating androgen in pregnant rats, significantly reduces the decline in luteal weight observed during postpartum structural regression. This effect was correlated with a decrease in the number of cells undergoing apoptosis and with enhanced levels of circulating progesterone. In addition, in vivo administration of androstenedione delayed the occurrence of DNA fragmentation in postpartum CL incubated in serum-free conditions. Finally, we have shown that the interference with apoptosis in vitro elicited by androstenedione is accompanied by an increased capacity of the CL to secrete progesterone. In summary, the results of this study have established that the rat CL expresses AR throughout pregnancy and after parturition, and they have defined a potential role for androstenedione in opposing postpartum luteal regression through inhibition of apoptosis and stimulation of progesterone production.     

Ploidy and nuclear area as a predictive factor of histologic grade in primary breast cancer

OBJECTIVE: To compare ploidy and nuclear area with histologic grade in breast cancer using cytologic samples. STUDY DESIGN: Fine needle aspirates from 85 patients with primary breast cancer were analyzed to identify ploidy and nuclear area. The Feulgen technique was used to stain the material. We used the SAMBA 4000 image analysis system (Grenoble, France) for analyzing ploidy and nuclear area. Each patient underwent a biopsy, and the histologic grade was analyzed. RESULTS: A significant association was found between ploidy and nuclear area, between histologic grade and nuclear area, and between ploidy and histologic grade. As ploidy became aneuploid and polyploid and nuclear area became larger, histologic grade became higher. CONCLUSION: A reliable and rapid evaluation of variables for breast cancer can be achieved using cytologic preparations by measuring ploidy and nuclear area of malignant cells with an image analysis system. Ploidy and nuclear area have a significant association with histologic grade.     

Expressions of amyloid precursor protein, synaptophysin and presenilin-1 in the different areas of the developing cerebellum of rat

This study reveals the expressions of Alzheimer's disease-related amyloid precursor protein, presenilin-1, and a presynaptic marker protein, synaptophysin, in the archi-, paleo- and neocerebellum during the postnatal development of the rat. The Western blot results demonstrate a gradual increase in the soluble amyloid precursor protein level in the archicerebellum during the first 3 weeks, while in the neo- and paleocerebellum the levels reach a plateau as early as the 1st week. Immunohistochemically, the protein is present in the deep part of the external granule cell layer and the internal granule cell layer in the newborn animal, while in 3-week-old animals the staining appears mainly in the perikarya and dendrites of the Purkinje cells. The level of synaptophysin increases progressively from postnatal day 7 up to 3 weeks in the archi- and paleocerebellum, and up to 6 weeks in the neocerebellum. Immunohistochemically, the amyloid precursor protein staining appears first in the inner part of the molecular layer and in the internal granule cell layer. In a 3-week-old animal, synaptophysin staining is present in all areas of the cerebellar molecular layer and in the internal granule cell layer. The presenilin-1 immunohistochemical reaction appeared equally in the archi-, paleo- and neocerebellum. Much of the staining is present in the glial cells and Purkinje cells. Less immunoreactivity is observed in the Golgi cells and granule cells. It is concluded that the postnatal expressions of soluble and membrane-bound amyloid precursor protein, synaptophysin and presenilin-1 are regulated differently during the ontogenetical development of the archi-, paleo- and neocerebellum of rat. Further, the amyloid precursor protein and presenilin-1 may be present in cells which do not degenerate in Alzheimer's disease.     

Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease

Microglia are the principal immune cells of the brain. In Alzheimer disease, these brain mononuclear phagocytes are recruited from the blood and accumulate in senile plaques. However, the role of microglia in Alzheimer disease has not been resolved. Microglia may be neuroprotective by phagocytosing amyloid-beta (Abeta), but their activation and the secretion of neurotoxins may also cause neurodegeneration. Ccr2 is a chemokine receptor expressed on microglia, which mediates the accumulation of mononuclear phagocytes at sites of inflammation. Here we show that Ccr2 deficiency accelerates early disease progression and markedly impairs microglial accumulation in a transgenic mouse model of Alzheimer disease (Tg2576). Alzheimer disease mice deficient in Ccr2 accumulated Abeta earlier and died prematurely, in a manner that correlated with Ccr2 gene dosage, indicating that absence of early microglial accumulation leads to decreased Abeta clearance and increased mortality. Thus, Ccr2-dependent microglial accumulation plays a protective role in the early stages of Alzheimer disease by promoting Abeta clearance.     

Reduction in interaction between cGMP and cAMP in dog ventricular myocytes with hypertrophic failure

Baseline function and signal transduction are depressed in hearts with hypertrophic failure. We tested the hypothesis that the effects of cGMP and its interaction with cAMP would be reduced in cardiac myocytes from hypertrophic failing hearts. Ventricular myocytes were isolated from control dogs, dogs with aortic valve stenosis hypertrophy, and dogs with pacing hypertrophic failure. Myocyte function was measured using a video edge detector. Cell contraction data were obtained at baseline, with 8-bromo-cGMP (10(-7), 10(-6), and 10(-5) M), with erythro-9-(2-hydroxy-3-nonyl)adenine [EHNA; a cAMP phosphodiesterase (PDE(2)) inhibitor] plus 8-bromo-cGMP, or milrinone (a PDE(3) inhibitor) plus 8-bromo-cGMP. Baseline percent shortening and maximal rates of shortening (R(max)) and relaxation were slightly reduced in hypertrophic myocytes and were significantly lower in failing myocytes (R(max): control dogs, 95.3 +/- 17.3; hypertrophy dogs, 88.2 +/- 5.5; failure dogs, 53.2 +/- 6.4 mum/s). 8-Bromo-cGMP dose dependently reduced myocyte function in all groups. However, EHNA (10(-6) M) and milrinone (10(-6) M) significantly reduced the negative effects of cGMP on cell contractility in control and hypertrophy but not in failing myocytes (R(max) for control dogs: cGMP, -46%; +EHNA, -21%; +milrinone, -19%; for hypertrophy dogs: cGMP, -40%; +EHNA, -13%; +milrinone, -20%; for failure dogs: cGMP, -40%; +EHNA, -29%; +milrinone, -32%). Both combinations of EHNA-cGMP and milrinone-cGMP significantly increased intracellular cAMP in control, hypertrophic, and failing myocytes. These data indicated that the cGMP signaling pathway was preserved in hypertrophic failing cardiac myocytes. However, the interaction of cGMP with the cAMP signaling pathway was impaired in these failing myocytes.     

PGF2alpha induced differential expression of genes involved in turnover of extracellular matrix in rat decidual cells

In the rat, the decidual tissue is an important component for maternal recognition of pregnancy. Decidualization can be induced by either the implantation of the blastocyst or by artificial stimuli. The process of decidua formation or decidualization, is characterized by growth and differentiation of endometrial stromal cells. Prostaglandin F2alpha (PGF2α) has been shown to be involved in inhibition of implantation, alteration of embryo development, induction of luteal regression, and the mediation of pregnancy loss induced by microorganism infections. In order to establish a direct role for PGF2α in decidual function, we have evaluated its effects on the expression of an extensive array of genes using primary decidual cell culture. Upon treatment with PGF2α sixty genes were significantly down-regulated whereas only six genes were up-regulated (from a total of 1176 genes studied). Interestingly, the majority of the genes inhibited by PGF2α are either directly or indirectly involved in the turnover of the extracellular matrix (ECM). Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (tPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase, all of which were significantly up-regulated by PGF2α. The results strongly suggest that the abortificient role of elevated levels of PGF2α after implantation is due, in large part, to inhibition of genes involved in the normal turnover of the extracellular matrix necessary for decidual formation.