Competitive yeast fermentation with selective flocculation and recycle

Continuous fermentations were carried out involving competition between two strains of Saccharomyces cerevisiae. One of the strains has a lower specific growth rate and is very flocculent, whereas the fastergrowing strain is nonflocculent. The product stream from the chemostat was fed into an inclined settler where the flocculent strain was partially separated from the nonflocculent strain as a result of the higher sedimentation rate of the flocculent cells. The underflow from the inclined settler, which was concentrated and enriched with flocculent cells, was recycled to the chemostat. When no recycle was used, the fastergrowing, nonflocculent yeast rapidly overtook the culture. With selective recycle, however, the experiments demonstrated that the slower-growing flocculent yeast could be maintained as the dominant species. A theoretical development is also presented in order to describe the competition between two strains in the bioreactor-settler system. The concept of selective recycle via selective flocculation and sedimentation offers a possible means of maintaining unstable recombinant microorganisms in continuous fermentations.     

Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis

Spermiogenesis in Caenorhabditis elegans involves the conversion of spherical, sessile spermatids into bipolar, crawling spermatozoa. In males, spermiogenesis is induced by mating, while in hermaphrodites, spermiogenesis occurs before the first oocytes are fertilized. Alternatively, spermiogenesis can be induced in vitro by treatment with monensin triethanolamine, or pronase. Treatment with the calmodulin inhibitors, trifluoperazine, chlorpromazine, or W7, also induces spermiogenesis in vitro with a half maximal effect at 20 microM. Upon initial activation, spermatids extend long, thin spikes and undergo extensive cellular movements. Eventually, a single motile pseudopod forms through the restructuring of one or more of these spikes. These transient spikes can be prolonged in vitro by removing triethanolamine as soon as the spermatids first form spikes. Spermatids from spe-8 and spe-12 spermatogenesis-defective (spe) mutants activate in vivo with male but not hermaphrodite sperm activator. In vitro, the mutant spermatids arrest spermiogenesis at the spike stage when activated with pronase, but form normal spermatozoa if subsequently or initially treated with monensin or triethanolamine. We present a model of spermiogenesis in which the mutant defects and the action of the pharmacological agents are ordered relative to one another.     

Characteristics of the pulmonary transport functions for heat and dye in pulmonary edema and orthostasis

The aim of this study was to investigate whether changes in the distribution of pulmonary blood flow and disturbances of the pulmonary microcirculation can be detected by use of inflow-outflow indicator-dilution measurements. In 18 anesthetized (N2O-piritramide) mongrel dogs 221 thermal-indocyanine green dye indicator dilution kinetics were recorded in the pulmonary artery and aorta after central venous indicator injection. The lagged normal density function was used as a model for the pulmonary transport functions for heat and dye. The parameters of the lagged normal density function were computed by a non-linear least squares procedure by iterative convolution. After baseline measurements, in nine dogs, pulmonary edema was induced by central venous application of oleic acid. In nine other dogs, measurements were performed before and after postural changes. Our data show that both the microvascular injury caused by oleic acid edema and the perfusion heterogeneity caused by orthostasis can be detected by the indicator dilution technique since the both relative dispersion and skewness of the transport functions for heat and dye were significantly increased after these interventions.     

High-dose mode of death in Tribolium

This study reports the first demonstration within a single insect genus (Tribolium) of both the acute, or lethal-midlethal, dose-independent pattern of mortality, and the hyperacute, dose-dependent pattern, after appropriate doses of ionizing radiation. This demonstration provides resolution of apparently contradictory reports of insect responses in terms of doses required to cause lethality and those based on survival time as a function of dose. A dose-dependent mortality pattern was elicited in adult Tribolium receiving high doses, viz., 300 Gy or greater; its time-course was complete in 10 days, before the dose-independent mortality began. Visual observations of heavily-irradiated Tribolium suggested neural and/or neuromuscular damage, as had been previously proposed by others for lethally-irradiated wasps, flies, and mosquitoes. Results of experiments using fractionated high doses supported the suggestion that the hyperacute or high-dose mode of death is the result of damage to nonproliferative tissues. Relative resistance of a strain to the hyperacute or high-dose mode of death is not necessarily correlated with resistance to the midlethal mode, which is believed to be the result of damage to the proliferative cells of the midgut.

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Résumé Les résultats de nombreuses études des réactions des insectes adultes de différents groupes à l'irradiation s'opposent quant à l'importance de la dose provoquant la létalité et quant aux modalités de la mort. Les diptères et les guèpes impliquent des doses très élevées,-des centaines de Gy-, ne présentent aucune période caractérisant la mort par irradiation, et décèdent de plus en plus tôt avec l'augmentation des doses. Beaucoup d'autres insectes succombent à des doses (milétalelétale) beaucoup plus faibles,-de quelques Gy à des dizaines-, et quelle que soit la dose meurent au bout d'un temps voisin.Au cours de cette étude, nous avons pu observer que ces deux types de mortalité peuvent être provoqués chez le même genre d'insecte (Tribolium), avec des doses convenables d'irradiation . Un syndrôme caractéristique a été provoqué avec des doses très élevées, de 300 Gy ou plus,-à ces doses la mort est obtenue en 10 jours après l'irradiation. L'absence de syndrôme caractéristique se produit avec des doses inférieures ou égales à 80 Gy; la mort a lieu alors entre 10 et 16 jours en fonction de la dose.Les différences entre les deux types de décès indiquent deux processus de mort par irradiation. La manifestation d'une désorientation et d'une perte de coordination motrice chez les Tribolium fortement irradiés suggère des altérations neurales et/ou neuromusculaires comme cause/s de ce type de mort provoquée par des doses élevées. L'implication de tissus sans prolifération a été confortée par les résultats d'expériences utilisant de hautes doses fractionnées. Le type de mort milétal est considéré après de nombreuses observations indépendantes, comme le résultat d'atteintes à la prolifération des cellules de l'intestin moyen.Les données contradictoires sur les réactions des insectes aux irradiations proviennent d'abord de l'absence de connaissances sur le lieu des dégâts. Les diptères sont connus maintenant, après différentes études, comme perdant complètement l'aptitude au renouvellement cellulaire, et présentent ainsi un type de mort avec dose élevée. Beaucoup d'autres insectes adultes ont un renouvellement cellulaire de l'intestin moyen limité, et ainsi présentent le type de mort milétal. Le type de mort, dit haute dose, peut être induit dans cette dernière catégorie d'insectes par une irradiation suffisamment forte, et, dans le cas du Tribolium le déroulement de la mort se produit alors de deux façons bien distinctes.

     

Crosslinking of elongation factor Tu to tRNA(Phe) by trans-diamminedichloroplatinum (II). Characterization of two crosslinking sites on EF-Tu

In a preceding paper [(1987) Nucleic Acids Res. 15, 5787-5801], we have used trans-diamminedichloroplatinum (II) to induce reversible RNA-protein crosslinks within the ternary EF-Tu/GTP/Phe-tRNA(Phe) complex and have identified two crosslinking sites on the tRNA. The aim of the present paper is to determine the crosslinking sites on EF-Tu. Two tryptic peptides located in domain I could be identified, a major one (residues 45-74) and a minor one (residues 117-154). The use of Staphylococcus aureus V8 protease led to the isolation of two major peptides (residues 56-68 and 64-68) and one minor peptide (118-124). These results are discussed in the light of the current knowledge of the topography of the EF-Tu/tRNA complex.     

Crystal structure of thermitase from Thermoactinomyces vulgaris at 2.2 A resolution

The crystal structure of thermitase from Thermoactinomyces vulgaris has been determined by x-ray diffraction at 2.2 A resolution. The structure was solved by a combination of single isomorphous replacement and molecular replacement methods. The structure was refined to a conventional R factor of 0.24 using restrained least square procedures CORELS and PROLSQ. The tertiary structure of thermitase is similar to that of subtilsin BPN'. The greatest differences between these structures are related to the insertions and deletions in the sequence.     

The Michaelis constants of a nonchromogenic substrate may be determined using a chromogenic substrate

A general method is presented for the determination of the KM and Vmax for a nonchromogenic substrate in an experimental system where a chromogenic and a nonchromogenic substrate compete for the active site of a single enzyme. Entire progress curves of absorbance versus time for the transformation of the chromogenic substrate must be obtained in the absence and presence of the nonchromgenic substrate. Two quantities may then be extracted from the entire kinetic curves: the value of a delta Area, the area bounded by the kinetic traces of absorbance versus time in the presence and absence of the nonchromogenic substrate; and the value of delta t(5%), the time required to transform 5% of the chromogenic substrate in the presence of the nonchromogenic substrate minus the corresponding time required in its absence. The values of KM and Vmax for the nonchromogenic substrate may be obtained from the dependencies of delta Area and delta t(5%) upon the concentration of the nonchromogenic substrate. The ability of this procedure to yield the correct values of KM and Vmax was demonstrated using beta-lactamase, beta-galactosidase, alkaline phosphatase, and 19 chromogenic/nonchromogenic substrate pairs. This method is equally valid in the absence or presence of competitive product inhibition and should be applicable to any enzyme-catalyzed, strongly exergonic reaction.     

Caldesmon is present in human and pig erythrocytes

Caldesmon, a major actin- and calcium-dependent calmodulin-binding protein, is now considered as an essential inhibitory factor of the actomyosin machinery in smooth muscle cells as well as in non-muscle cells. Since its structure seems to vary with the cell in a same species and because platelet and erythrocyte have a common embryonic origin, we have used the affinity purified antibody raised against the platelet caldesmon to determine whether this protein is present in erythrocyte. Using the immunoblotting technique, we show here that, in whole erythrocytes, only a single polypeptide crossreacts with this polyclonal antibody. A 71-72 kDa Mr has been calculated from SDS-PAGE. It is therefore different from those of the gizzard (Mr 145-150 kDa) or the platelet (Mr 80 kDa) proteins. Furthermore, we also give evidence that it is not adducin since this newly discovered calcium-dependent calmodulin-binding protein of erythrocyte, does not crossreact with the polyclonal affinity purified antibody raised against platelet caldesmon.     

Phosphorylation of peripherin, an intermediate filament protein, in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons

Peripherin, an intermediate filament protein, described recently, is expressed in well defined neuronal populations. We studied the phosphorylation, in vivo, of this protein in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons labelled with [32P]-orthophosphate. The autoradiograms of proteins separated on two-dimensional polyacrylamide gels were compared with the Coomassie-blue stainings. The results show that peripherin occurs as a mixture of phosphorylated and non-phosphorylated isoforms, and that these forms coexist in both differentiated and non-differentiated cells. We demonstrate by cleavage at the unique tryptophan residue, a characteristic shared by most other intermediate filament proteins (IFP), that the phosphorylation sites are located on the amino-terminal half of peripherin as it is for vimentin and desmin. These results are discussed in relation to the organization of the filamentous network constituted by peripherin.     

Histamine stimulates calcium-mediated protein phosphorylation in a colonic epithelial cell line

Protein phosphorylation responses in intact enterocytes were examined by stimulating 32Pi-labeled T84 cell monolayers with histamine and resolving proteins by two-dimensional gel electrophoresis. Histamine increases 32P-incorporation into two acidic proteins of Mr 83,000 and of Mr 29,000, designated p83 and p29. Labeling of p83 and p29 is also increased in cells exposed to ionomycin, but not in cells exposed to vasoactive intestinal peptide under conditions resulting in cAMP-mediated secretion and cAMP-stimulated protein phosphorylation. When T84 cell fractions are incubated with [gamma-32P]ATP, labeling of p83 is stimulated by Ca++, but not by cAMP. Thus, histamine stimulates Ca++-mediated protein phosphorylation during the regulation of Cl- secretion.