Cdc42 and RhoA have opposing roles in regulating membrane type 1-matrix metalloproteinase localization and matrix metalloproteinase-2 activation

Proteolysis of the basement membrane and interstitial matrix occurs early in the angiogenic process and requires matrix metalloproteinase (MMP) activity. Skeletal muscle microvascular endothelial cells exhibit robust actin stress fibers, low levels of membrane type 1 (MT1)-MMP expression, and minimal MMP-2 activation. Depolymerization of the actin cytoskeleton increases MT1-MMP expression and MMP-2 activation. Rho family GTPases are regulators of actin cytoskeleton dynamics, and their activity can be modulated in response to angiogenic stimuli such as vascular endothelial growth factor (VEGF). Therefore, we investigated their roles in MMP-2 and MT1-MMP production. Endothelial cells treated with H1152 [an inhibitor of Rho kinase (ROCK)] induced stress fiber depolymerization and an increase in cortical actin. Both MMP-2 and MT1-MMP mRNA increased, which translated into greater MMP-2 protein production and activation. ROCK inhibition rapidly increased cell surface localization of MT1-MMP and increased PI3K activity, which was required for MMP-2 activation. Constitutively active Cdc42 increased cortical actin polymerization, phosphatidylinositol 3-kinase activity, MT1-MMP cell surface localization, and MMP-2 activation similarly to inhibition of ROCK. Activation of Cdc42 was sufficient to decrease RhoA activity. Capillary sprout formation in a three-dimensional collagen matrix was increased in cultures treated with RhoAN19 or Cdc42QL and, conversely, decreased in cultures treated with dominant negative Cdc42N17. VEGF stimulation also induced activation of Cdc42 while inhibiting RhoA activity. Furthermore, VEGF-dependent activation of MMP-2 was reduced by inhibition of Cdc42. These results suggest that Cdc42 and RhoA have opposing roles in regulating cell surface localization of MT1-MMP and MMP-2 activation.     

Comparative proteomic analysis of four Bacillus clausii strains: proteomic expression signature distinguishes protein profile of the strains

A comparative proteomic approach, using two dimensional gel electrophoresis and mass spectrometry, has been developed to compare and elucidate the differences among the cellular proteomes of four closely related isogenic O/C, SIN, N/R and T, B. clausii strains during both exponential and stationary phases of growth. Image analysis of the electropherograms reveals a high degree of concordance among the four proteomes, some proteins result, however, differently expressed. The proteins spots exhibiting high different expression level were identified, by mass-spectrometry analysis, as alcohol dehydrogenase (ADHA, EC1.2.1.3; ABC0046 isoform) aldehyde dehydrogenase (DHAS, EC 1.2.1.3; ABC0047 isoform) and flagellin-protein of B. clausii KSM-k16. The different expression levels of the two dehydrogenases were confirmed by quantitative RT-PCR and dehydrogenases enzymatic activity. The different patterns of protein expression can be considered as cell proteome signatures of the different strains.     

Novel use of a perfluorocarbon for supplying oxygen to aerobic submerged cultures

Summary A novel method for supplying oxygen by using a perfluorocarbon as an oxygen carrier in submerged cultures was demonstrated. Oxygen was transferred to the aqueous phase by liquid-liquid contacting in a spray column fermentor. Advantages of using such a system for supplying oxygen are discussed.Nomenclature F₁ recirculation rate of PP9 through nozzle - P_B back pressure at nozzle - SA_PP9 oxygen concentration in PP9 in aerator as a percent of saturation when air equilibrated at the end of the exponential phase of growth - T_F fermentation time to reach final cell concentration - V_A air flow rate through sparger - X_e cell concentration at the end of the exponential phase of growth - X_F final cell concentration - Ø_A gas (air) hold-up - Ø_L liquid (PP9) hold-up in aqueous system     

The effects of nonvolatile toxic substances on the yeast growth during ethyl alcohol fermentations

Summary Nonvolatile toxins accumulated during alcohol fermentation reduce the yeast growth rate, but not the maximum cell concentration. Specific growth rates decline exponentially according to the increase in toxins. The accumulation of salts and proteins seem to be responsible for the inhibition.To whom all correspondence should be addressed.     

Continuous ethanol fermentation ofZymomonas mobilis using soy flour as a protective agent

Biotechnology Letters - From the analysis of steady state data, an increase of 81.6% in ethanol concentration and fermentor productivity was obtained due to the addition of soy flour to a...     

Production of transgenic goats by pronuclear microinjection of in vitro produced zygotes derived from oocytes recovered by laparoscopy

Oocytes collected by laparoscopic ovum pick-up (LOPU) were successfully used to produce transgenic goats by pronuclear microinjection of in vitro zygotes. Estrus cycles of 109 donor goats were synchronized using intravaginal sponges impregnated with 60 mg of medroxyprogesterone acetate and treatment with 70 mg NIH-FSH-P1 and 300 IU eCG to stimulate follicular development. Follicles were aspirated under laparoscopic observation. In vitro maturation (IVM) of oocytes was performed in M199 supplemented with hormones, kanamycin and 10% estrus goat serum. Following IVM, oocytes were cocultured with capacitated semen in TALP supplemented with 20% estrus goat serum for 15-20 h. The resulting zygotes were microinjected with a linear DNA fragment. In total, 3293 follicles were aspirated (15.7+/-9 follicles aspirated per donor) and 2823 oocytes were recovered (13.4+/-8 oocytes per donor). A total of 1366 zygotes were microinjected and transferred into 219 recipient goats by midventral laparotomy (average 6.2 embryos per recipient). A total of 150 kids were born, of which 9 (6 M: 3 F) were confirmed to be transgenic by PCR and Southern blotting analyses. These results demonstrate that acceptable transgenesis rates can be obtained in goats by DNA microinjection of in vitro produced zygotes.     

Dispersal and habitat cuing of Eurasian red squirrels in fragmented habitats

Animal dispersal and subsequent settlement is a key process in the life history of many organisms, when individuals use demographic and environmental cues to target post-dispersal habitats where fitness will be highest. To investigate the hypothesis that environmental disturbance (habitat fragmentation) may alter these cues, we compared dispersal patterns of 60 red squirrels (Sciurus vulgaris) in three study sites that differ in habitat composition and fragmentation. We determined dispersal distances, pre- and post-dispersal habitat types and survival using a combination of capture–mark–recapture, radio-tracking and genetic parentage assignment. Most (75%) squirrels emigrated from the natal home range with mean dispersal distance of 1,014 ± 925 m (range 51–4,118 m). There were no sex-related differences in dispersal patterns and no differences in average dispersal distance, and the proportion of dispersers did not differ between sites. In one of the sites, dispersers settled in patches where density was lower than in the natal patch. In the least fragmented site, 90% of animals settled in the natal habitat type (habitat cuing) against 44–54% in the more strongly fragmented sites. Overall, more squirrels settled in the natal habitat type than expected based on habitat availability, but this was mainly due to individuals remaining within the natal wood. In the highly fragmented landscape, habitat cuing among emigrants did not occur more frequently than expected. We concluded that increased habitat fragmentation seemed to reduce reliable cues for habitat choice, but that dispersing squirrels settled in patches with lower densities of same-sex animals than at the natal home range or patch, independent of degree of fragmentation.