Acetic acid tolerance in Drosophila is a prerequisite for ethanol tolerance

Acetic acid tolerance compared with ethanol tolerance of Drosophila simulans and six Drosophila melanogaster strains shows a curvilinear relation with apparent asymptotic hyperbolic profile. The upper limit of acetic acid tolerance is lower than that for ethanol. We compared strains which had pairwise identical alcohol dehydrogenase (ADH) coding regions but different genetic backgrounds. A positive regression existed for ethanol tolerance on ADH activity. Adh-null mutants with very low ethanol tolerances had appreciable acetic acid tolerances and as a consequence did not fit the curve. ADH-F and ADH-S strains selected for high ethanol tolerances had the ability to tolerate high ethanol concentrations even after selection had been relaxed for several years. These selected lines tolerated higher acetic acid concentrations than the non-selected original strains. We propose that intake of high concentrations of ethanol and oxidation into acetic acid induces esterification of ethanol and acetic acid into ethylacetate. This cannot take place after the intake of acetic acid only, which also gives a lower energy yield.     

Reverse gyrase is not a prerequisite for hyperthermophilic life

We disrupted the reverse gyrase gene from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. An apparent positive supercoiling activity that was observed in the host strain was not found in the disruptant strain. We found that a lack of reverse gyrase led to a retardation in growth that was more striking at higher temperatures. However, the disruption of the reverse gyrase gene did not lead to a lethal phenotype at 90 degrees C. This study provides experimental evidence that reverse gyrase is not a prerequisite for hyperthermophilic life.     

Protein unfolding is not a prerequisite for endoplasmic reticulum-to-cytosol dislocation

We examined the effects of protein folding on endoplasmic reticulum (ER)-to-cytosol transport (dislocation) by exploiting the well-characterized dihydrofolate reductase (DHFR) domain. DHFR retains the capacity to bind folate analogues in the lumen of microsomes and in the ER of intact cells, upon which it acquires a conformation resistant to proteinase K digestion. Here we show that a Class I major histocompatibility complex heavy chain fused to DHFR is still recognized by the human cytomegalovirus-encoded glycoproteins US2 and US11, resulting in dislocation of the fusion protein from the ER in vitro and in vivo. A folded state of the DHFR domain does not impair dislocation of Class I MHC heavy chains in vitro or in living cells. In fact, a slight acceleration of the dislocation of DHFR heavy chain fusion was observed in vitro in the presence of a folate analogue. These results suggest that one or more of the channels used for dislocation can accommodate polypeptides that contain a tightly folded domain of considerable size. Our data raise the possibility that the Sec61 channel can be modified to accommodate a folded DHFR domain for dislocation, but not for translocation into the ER, or that a channel altogether distinct from Sec61 is used for dislocation.     

Starvation is not a prerequisite for the formation of aerobic granules

Activated sludge with sludge volume index (SVI)₃₀ of 77 ml g⁻¹ and SVI₃₀ of 433 ml g⁻¹ was inoculated to start up reactors R1 and R2, respectively. In both R1 and R2, cycle time of 1 h and the influent chemical oxygen demand (COD) concentrations of 1,000 mg l⁻¹ were employed. Initial settling time of 2 min resulted in the loss of a substantial amount of biomass as wash-out and high effluent COD concentrations within the first week of operation. This implied that there was no starvation phase in each cycle of R1 and R2 during the first week of operation. However, aerobic granules with a size above 400 μm formed by day 7. Thus, it was concluded that starvation was not a prerequisite for the formation of aerobic granules. When cycle time was 1 h, the instability of aerobic granules was observed. When cycle time was prolonged to 1.5 h and granular sludge of 200 ml was used to start up reactor R3, the reactor R3 reached steady state within 1 week. SVI, size, and the morphology of granular sludge in R3 remained stable during the 47-day operation, which indicated that prolonged starvation time had positive effects on the stability of aerobic granules.     

Somaclonal variation for freezing tolerance in a population derived from norstar winter wheat

Summary Progeny of 66 plants regenerated from callus cultures derived from immature embryos of Norstar winter wheat were evaluated as seedlings for tolerance to controlled freezing. Greater freezing tolerance than the parent cultivar was observed in both R₂ and R₃ regenerate families. LT₅₀ values (predicted temperatures at which mean survival frequencies are 50%) for four families in the R₂ generation and three families in the R₃ were significantly lower than that of Norstar. In both R₂ and R₃ generations, most families did not differ significantly from the cultivar Norstar, by three separate measures of tolerance. Significant variation among families was observed in both R₂ and R₃ generations for survival, but not for plant height. Variation within family in the R₃ generation was also significant, though smaller than that among families. In the R₃ generation, eighteen families were significantly less freezing tolerant than Norstar according to LT₅₀, while thirteen were significantly less tolerant according to survival at a minimum temperature of-17 °C.NRCC No. 28387. This publication describes research performed as part of the program of the Plant Biotechnology Institute of the National Research Council of Canada through Plant Biotechnology Fund contract No. 31964-4-0021 awarded to T. H. H. Chen and M. D. Lazar, Alberta Research Council, Edmonton, Canada     

mRNA translation is not a prerequisite for small interfering RNA-mediated mRNA cleavage

RNA interference constitutes a major means of eliminating mRNAs, yet how the small interfering RNAs (siRNA) within the RNA-induced silencing complex (RISC) finds its homologous target in the cell remains unknown. An attractive hypothesis is that RNA interference is linked to translation which allows RISC ready access to every translated mRNA. To test whether translation could direct siRNAs to mRNAs, chemical and biological inhibitors of translation and their effects on mRNA cleavage were tested. Our results show that mRNA degradation by siRNAs is not dependent on mRNA translation.     

Arabidopsis HDA6 is required for freezing tolerance

The control of energy homeostasis within the hypothalamus is under the regulated control of homeostatic hormones, nutrients and the expression of neuropeptides that alter feeding behavior. Elevated levels of palmitate, a predominant saturated fatty acid in diet and fatty acid biosynthesis, alter cellular function. For instance, a key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. To determine these mechanisms we used an immortalized, clonal, hypothalamic cell line, mHypoE-44, to demonstrate that palmitate directly alters the expression of molecular clock components, by increasing Bmal1 and Clock, or by decreasing Per2, and Rev-erbα, their mRNA levels and altering their rhythmic period within individual neurons. We found that these neurons endogenously express the orexigenic neuropeptides NPY and AgRP, thus we determined that palmitate administration alters the mRNA expression of these neuropeptides as well. Palmitate treatment causes a significant increase in NPY mRNA levels and significantly alters the phase of rhythmic expression. We explored the link between AMPK and the expression of neuropeptide Y using the AMPK inhibitor compound C and the AMP analog AICAR. AMPK inhibition decreased NPY mRNA. AICAR also elevated basal NPY, but prevented the palmitate-mediated increase in NPY mRNA levels. We postulate that this palmitate-mediated increase in NPY and AgRP synthesis may initiate a detrimental positive feedback loop leading to increased energy consumption.     

Docking is not a prerequisite but a temporal constraint for fusion of secretory granules

We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic β cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.     

Cytosolic proline is required for basal freezing tolerance in Arabidopsis

The amino acid proline accumulates in many plant species under abiotic stress conditions, and various protective functions have been proposed. During cold stress, however, proline content in Arabidopsis thaliana does not correlate with freezing tolerance. Freezing sensitivity of a starchless plastidic phosphoglucomutase mutant (pgm) indicated that localization of proline in the cytosol might stabilize the plasma membrane during freeze–thaw events. Here, we show that re-allocation of proline from cytosol to vacuole was similar in the pyrroline-5-carboxylate synthase 2–1 (p5cs2–1) mutant and the pgm mutant and caused similar reduction of basal freezing tolerance. In contrast, the starch excess 1–1 mutant (sex1-1) had even lower freezing tolerance than pgm but did not affect sub-cellular localization of proline. Freezing sensitivity of sex1-1 mutants affected primarily the photosynthetic electron transport and was enhanced in a sex1-1::p5cs2–1 double mutant. These findings indicate that several independent factors determine basal freezing tolerance. In a pgm::p5cs2–1 double mutant, freezing sensitivity and proline allocation to the vacuole were the same as in the parental lines, indicating that the lack of cytosolic proline was the common cause of reduced basal freezing tolerance in both mutants. We conclude that cytosolic proline is an important factor in freezing tolerance of non-acclimated plants.     

Accumulation of cyclin E is not a prerequisite for passage through the restriction point

The restriction point (R) is defined as the point in G(1) after which cells can complete a division cycle without growth factors and divides G(1) into two physiologically different intervals in cycling cells, G(1)-pm (a postmitotic interval with a constant length of 3 to 4 h) and G(1)-ps (a pre-DNA-synthetic interval with a variable length of 1 to 10 h). Cyclin E is a G(1) regulatory protein whose accumulation has been suggested to be critical for passage through R. We have studied cyclin E protein levels in individual cells of asynchronously growing cell populations, with respect to both passage through R and entry into S phase. We found that the postmitotic G(1) cells that had not yet reached R were negative for cyclin E accumulation. On the other hand, cells that had passed R were found to accumulate cyclin E at variable times (1 to 8 h) after passage through R and 2 to 5 h before entry into S. These kinetic data rule out the hypothesis that passage through R is dependent on the accumulation of cyclin E but suggest, instead, the converse, that passage through R is a prerequisite for cyclin E accumulation. Furthermore, we found that most of the cyclin E protein is downregulated within 1 to 2 h after entry into S.     

Food deprivation is not a prerequisite for the amoebal to plasmodial transition in Physarum polycephalum

The effect of food supply on the onset of asexual and sexual plasmodium formation in Physarum polycephalum was studied. Asexual differentiation occurs readily in amoebae carrying the matAh mating type allele. The density at which these amoebae begin to differentiate is influenced by the ind locus, which controls the production of a diffusible inducer. The alleles ind-1 and ind-2 are known. Strains carring the ind-1 allele begin plasmodium formation at a low amoebal density (rapid differentiation), while strains carring the ind-2 allele differentiate at a higher amoebal density (slow differentiation). The onset of differentiation is characteristic of the strain and did not change with a 20-fold variation in the number of food bacteria available. Sexual differentiation occurs between compatible amoebal strains. For a given pair of amoebal strains the onset of plasmodium formation occurs at a characteristic cell density that is determined by the genetic backgrounds of the strains. The ind locus is one of the genes that influences this cell density. Plasmodia are formed at a lower cell density in crosses involving compatible amoebae carrying the ind-1 allele than they are in crosses with strains carrying the ind-2 allele. As was found for asexual differentiation, an approximate 20-fold variation in the food supply did not affect the initiation of sexual plasmodium formation. These results suggest that in most cases starvation does not trigger the differentiation of amoebae into plasmodia. The time of onset of plasmodium formation is determined largely by genetic factors.     

Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.     

In vitro establishment is not a sufficient prerequisite for transformation by activated ras oncogenes

Activated ras genes transform REF52 cells only at low frequencies and adenovirus early region 1A collaborates with ras oncogenes to convert REF52 cells to a tumorigenic phenotype. While failure to transform did not result from an absence of ras gene expression, E1A appeared to enhance expression of transfected ras genes by approximately tenfold. However, enhanced ras expression alone does not account for collaboration by E1A since overexpression of T24 Ha-ras p21 induced morphological crisis and cell growth arrest rather than stable transformation. These results indicate that E1A contributes complementing biochemical activities that enable ras genes to transform REF52 and suggest that the role of E1A in primary cell transformation may extend beyond facilitating in vitro establishment.     

Expression of a cold-responsive Lt-Cor gene and development of freezing tolerance during cold acclimation in wheat (Triticum aestivum L.).

Time-courses of the development of freezing tolerance and the expression of a cold-responsive gene wlt10 were monitored during cold acclimation in wheat (Triticum aestivum L.). Bioassay showed that cold acclimation conferred much higher freezing tolerance on a winter cultivar than a spring cultivar. Northern blot analysis showed that the expression of wlt10 encoding a novel wheat member of a cereal-specific LT-COR protein family was specifically induced by low temperature. A freezing-tolerant winter cultivar accumulated the mRNA more rapidly and for a longer period than a susceptible spring cultivar. The increase in the amount of mRNA was temporary but the peak occurred at the time when the maximum level of freezing tolerance was attained. The mRNA accumulated more in the leaves than in the roots, and different light/dark regimes modulated the level of mRNA accumulation. Genomic Southern blot analyses using the nulli-tetrasomic series showed that the wlt10 homologues were located on the homologous group 2 chromosomes.     

Understanding spermatogenesis is a prerequisite for treatment

Throughout spermatogenesis multiplication, maturation and differentiation of germ cells results in the formation of the male gamete. The understanding of spermatogenesis needs detailed informations about the organization of the germinal epithelium, the structure and function of different types of germ cells, endocrine and paracrine cells and mechanisms, intratesticular and extratesticular regulation of spermatogenesis. Normal germ cells must be discriminated from malformed, apoptotic and degenerating germ cells and tumor cells.     

Programmed cell death is not a necessary prerequisite for fusion of the fetal mouse palate

It has been widely accepted that programmed cell death (PCD) is an essential event in palatogenesis and that its failure can result in cleft palate, one of the most common birth defects in the human. However, some conflicting results have been reported concerning the timing of cell death occurring in the fusing palate and therefore the role of PCD in palatal fusion is controversial. In order to clarify whether cell death is indispensable for mammalian palatogenesis, we cultivated the palates of day-13 mouse fetuses in vitro and prevented cell death by treating them with the inhibitors of caspases-1 and -3 or with aurintricarboxylic acid which inhibits the activity of caspase-activated DNase. Even when cell death was almost completely inhibited, palatal fusion took place successfully. Histological examination revealed that in the absence of apoptotic cell death, the medial edge epithelia of opposing palatal shelves adhered to each other and subsequently, the midline epithelial seam was disrupted and disappeared to bring about mesenchymal confluence across the palate. It seems that cell death is not a necessary prerequisite for palatal fusion but it may help to efficiently eliminate unnecessary cells which failed to migrate or differentiate properly.