Effect of antifoam addition on gas-liquid mass transfer in stirred fermenters

The influence of three well-known antifoaming agents (polypropylene glycol, silicone and soybean oil) on gas-liquid mass transfer in stirred tanks is studied, both in model and in fermentation media. The effect of antifoam concentration, ionic strength, viscosity, agitation speed and gas flow rate are investigated. It is found that antifoam addition at low concentrations markedly decreases the gas-liquid volumetric mass transfer coefficient, k_La, for the three antifoam agents tested. Although the major effect is on the film coefficient k_L, some effect is also detected on the specific area, a. It is found that the influence of viscosity and antifoam addition are not cumulative: each tends to attenuate the other's effect on mass transfer. Both for silicone and for soybean oil, but not for PPG in the concentration range studied, there is an antifoam concentration above which further antifoam addition starts to improve k_La.     

Primary cells derived from Tuberous Sclerosis Complex patients show autophagy alteration in the haploinsufficiency state

Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field.     

Effects of central administration of lead, cadmium and other divalent cations on body temperature in mice

The intracerebroventricular administration of lead or cadmium to mice produced a marked decrease in body temperature. The maximum decreases in body temperature produced by the central administration of the heavy metals (1.97±0.3°C for lead and 2.87±0.6°C for cadmium) were similar to those obtained after acute peripheral administration of these compounds. The decreases in body temperature obtained after central administration of calcium, magnesium or zinc were significantly smaller than those produced by the heavy metals. The results indicate that the central nervous system is implicated in the hypothermia induced by these divalent cations and suggest that the alteration of membrane ionic permeability by these substances at hypothalamic sites may be related to the control of body temperature.     

Biomineralization of Carbonates by Marinococcus albus and Marinococcus halophilus Isolated from the Salar de Atacama (Chile)

We studied the precipitation of carbonates in 17 strains of moderately halophilic, Gram-positive cocci belonging to two species: Marinococcus halophilus and Marinococcus albus, isolated from the Salar de Atacama (Chile). They were cultivated in solid and liquid laboratory media for 42 days at salt concentrations (wt/vol) of 3%, 7.5%, 15%, and 20%. The bioliths precipitated were studied by X-ray diffraction and scanning electron microscopy. M. halophilus formed crystals at each of the salt concentrations, with a maximum number of strains capable of precipitating carbonates at 7.5% and 15% salt concentrations. M. albus did not precipitate at 20% and showed a maximum at 7.5%. This behavior is similar to that of other Gram-positive bacteria and differs from that found in Gram-negative bacteria. The bioliths precipitated were spherical, generally isolated, with a size of 10–100 μm, varying with salinity. They were of magnesium calcite (CO₃ Ca_1-x Mg_x) with Mg content increasing with increasing salinity and Mg/Ca molar ratio of the culture medium. These results demonstrate the active role played by M. halophilus and M. albus in the precipitation of carbonates. Received: 3 November 1998 / Accepted: 9 March 1999     

Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts

The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease. This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T. cruzi (ID50 = 35 microM). When added to intact cells, MPNO inhibited the growth of T. cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T. cruzi trypomastigotes (ID50 = 20 microM). At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts. Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO. Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography. Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.     

Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera.

Formation of a novel structure, the aggresome, has been proposed to represent a general cellular response to the presence of misfolded proteins (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883-1898; Wigley, W.C., R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino, and P.J. Thomas. 1999. J. Cell Biol. 145:481-490). To test the generality of this finding and characterize aspects of aggresome composition and its formation, we investigated the effects of overexpressing a cytosolic protein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP-250 within the aggresome appeared not to be ubiquitinated. In vivo time-lapse analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region where they remain as distinct but closely apposed particulate structures. Overexpression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting that the minus-end-directed motor activities of cytoplasmic dynein are required for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtubules. However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by soluble, nonubiquitinated proteins as well as by integral transmembrane ubiquitinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins.     

The rational modulation of autophagy sensitizes colorectal cancer cells to 5-fluouracil and oxaliplatin

Colorectal cancer (CRC) is the third most common and deadliest cancer globally. Regimens using 5-fluorouracil (5FU) and Oxaliplatin (OXA) are the first-line treatment for CRC, but tumor recurrence is frequent. It is plausible to hypothesize that differential cellular responses are triggered after treatments depending on the genetic background of CRC cells and that the rational modulation of cell tolerance mechanisms like autophagy may reduce the regrowth of CRC cells. This study proposes investigating the cellular mechanisms triggered by CRC cells exposed to 5FU and OXA using a preclinical experimental design mimicking one cycle of the clinical regimen (i.e., 48 h of treatment repeated every 2 weeks). To test this, we treated CRC human cell lines HCT116 and HT29 with the 5FU and OXA, combined or not, for 48 h, followed by analysis for two additional weeks. Compared to single-drug treatments, the co-treatment reduced tumor cell regrowth, clonogenicity and stemness, phenotypes associated with tumor aggressiveness and poor prognosis in clinics. This effect was exerted by the induction of apoptosis and senescence only in the co-treatment. However, a week after treatment, cells that tolerated the treatment had high levels of autophagy features and restored the proliferative phenotype, resembling tumor recurrence. The pharmacologic suppression of early autophagy during its peak of occurrence, but not concomitant with chemotherapeutics, strongly reduced cell regrowth. Overall, our experimental model provides new insights into the cellular mechanisms that underlie the response and tolerance of CRC cells to 5FU and OXA, suggesting optimized, time-specific autophagy inhibition as a new avenue for improving the efficacy of current treatments.     

Rapid purification and molecular modeling of AaIT peptides from venom of Androctonus australis

As recombinant viruses expressing scorpion toxins are moving closer toward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological activity in nontarget organisms. In the past, we purified a large amount of Androctonus australis anti-insect toxin (AaIT) present in the venom of A. australis with an analytical reversed-phase column by repeated runs of crude sample. We now report 20 times improved efficiency and speed of the purification by employing a preparative reversed-phase column. In just two consecutive HPLC steps, almost 1 mg of AaIT was obtained from 70 mg crude venom. Furthermore, additional AaIT was obtained from side fractions in a second HPLC run. Recently discovered insect selective toxin, AaIT5, was isolated simultaneously from the same venom batch. It shows different biological toxicity symptoms than the known excitatory and depressant insect toxins. AaIT5 gave 100% mortality with a dose of less than 1.3 μg against fourth-instar tobacco budworms Heliothis virescens 24 h after injection. During the purification process, we implemented mass spectrometry in addition to bioassays to monitor the presence of AaIT and AaIT5 in the HPLC fractions. Mass spectrometric screening can unambiguously follow the purification process and can greatly facilitate and expedite the downstream purification of AaIT and AaIT5 eliminating the number of bioassays required. Further, electrospray ionization was compared with matrix-assisted desorption/ionization and evaluated as a method of choice for mass spectrometric characterization of fractions from the venom purification for it provided higher mass accuracy and relative quantitation capability. Molecular models were built for AaIT5, excitatory toxin AaIT4, and depressant toxin LqhIT2. Three-dimensional structure of AaIT5 was compared with structures of the other two toxins, suggesting that AaIT5 is similar to depressant toxins. Arch. Insect Biochem. Physiol. 38:53–65, 1998. © 1998 Wiley-Liss, Inc.