The effect of oxygen on the survival of non-Saccharomyces yeasts during mixed culture fermentations of grape juice with Saccharomyces cerevisiae

AIMS: The effect of oxygen on the survival of Torulaspora delbrueckii and Kluyveromyces thermotolerans during mixed culture fermentations in grape juice with Saccharomyces cerevisiae was investigated. METHODS AND RESULTS: Fermentations were carried out in two simple fermentation systems differing in the availability of oxygen. At low available oxygen conditions, T. delbrueckii and K. thermotolerans began to die off after two days of mixed culture fermentation. In filtrates from 2-day-old mixed cultures, single cultures of T. delbrueckii and K. thermotolerans survived and actively produced ethanol to concentrations of approx. 65 and 70 g l-1, respectively, at low available oxygen conditions. Oxygen clearly increased the survival time and decreased the death rate of T. delbrueckii and K. thermotolerans in mixed cultures, whereas it did not affect the growth and survival of S. cerevisiae. CONCLUSION: Our results show that the deaths of T. delbrueckii and K. thermotolerans in mixed cultures at low available oxygen conditions are not due to toxic metabolites produced by the yeasts but rather to the lack of oxygen. Furthermore, they indicate that T. delbrueckii and K. thermotolerans are less tolerant to low available oxygen conditions than S. cerevisiae. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reveals new knowledge on the mechanisms underlying the succession of yeasts during wine fermentations. This knowledge may be of importance when creating defined, mixed starter cultures for the controlled production of wines with a wide range of flavour compositions.     

Application of the zona-free manipulation technique to porcine somatic nuclear transfer

The recent demonstration of a successful zona-free manipulation technique for bovine somatic nuclear transfer (NT) that is both simpler and less labor intensive is of considerable benefit to advance the applications of this technology. Here, we describe that this method is also applicable to porcine somatic NT. Porcine cumulus oocyte complexes were matured in TCM-199 medium before sequential removal of the cumulus and zonae. Zona-free oocytes were bisected using a microknife, and the halves containing the metaphase plate (as determined by Hoechst 33342 staining) were discarded. Each half cytoplast was agglutinated to a single granulosa cell (primary cultures grown in 0.5% serum for 2-5 days prior to use) in phytohaemagglutinin-P. Subsequently, each half cytoplast-granulosa cell couplet was simultaneously electrofused together and to another half cytoplast. Reconstructed embryos were activated in calcium ionophore A23187 followed by DMAP and were then individually cultured in microwells in NCSU-23 medium. On day 7 after activation, blastocyst yield and total cell numbers were counted. Of 279 attempted reconstructed NT embryos, 85.0 +/- 2.8% (mean +/- SEM; n = 5 replicates) successfully fused and survived activation. The blastocyst rate (per successfully fused and surviving embryo) was 4.8 +/- 2.3% (11/236; range, 0-12.8%). Total blastocyst cell count was 36.0 +/- 4.5 (range, 18-58 cells). The blastocyst rate and total cell numbers of parthenogenetically activated and zona-free control oocytes propagated under the same conditions was 11.6 +/- 3.9% (35/335 embryos; n = 3 replicates) and 36.8 +/- 5.2, respectively. Developmentally halted embryos that could still be evaluated on day 7 possessed 54.4 +/- 2.3% (53/96 embryos; n = 3 replicates) anucleate blastomeres, the latter representing 53.5 +/- 6.6% of the blastomeres in such embryos. In conclusion, blastocyst yield was independent of activation efficiency and was likely reduced by insufficient nuclear remodeling, reprogramming, imprinting, or other effects. The data also suggest that fragmentation was a considerable problem that could conceivably contribute to halted development in a high proportion of embryos. The results indicate that the zona-free manipulation technique can be successfully applied to pig somatic NT. Although such zona-free early cleavage stage embryos cannot be transferred to recipients at present, this technique permits simplification of the NT technique for application in basic research, until pig nonsurgical blastocyst transfer becomes a realistic option.     

Dose-response relationship for parathyroid adenoma after exposure to ionizing radiation in infancy

Several authors have suggested that there is an excess risk of hyperparathyroidism, adenomas or hyperplasia after exposure to ionizing radiation. There is still, however, some uncertainty about this association, because these diseases are often asymptomatic and escape clinical detection if not specially searched for. This study is based on a pooled Swedish cohort of 27,925 persons with skin hemangiomas. The majority received radiation treatment in infancy between 1920 and 1965 in Stockholm and Gothenburg. The mean age at treatment was 6 months and the median thyroid dose was 0.20 Gy (range 0-28.5 Gy). Record linkage with the Swedish Cancer Register for the period 1958-1997 gave 43 cases of parathyroid adenoma in the cohort. Analyses of excess relative risk (ERR) models were performed using Poisson regression methods. Clinical records were scrutinized to determine if the childhood radiation exposure was known (biased cases) at the time of diagnosis. Seven of the cases of parathyroid adenoma were classified as biased cases. The standardized incidence ratio (SIR) was 2.10 (95% confidence interval 1.52-2.82) when all cases were included and 1.76 (95% CI 1.23-2.43) with the biased cases excluded. A linear dose-response model with stratification for sex fitted the data best. The ERR per gray was 3.84 (95% CI 1.56-8.99) with all cases and 1.56 (95% CI 0.36-4.45) with the biased cases excluded. There was a significant difference in the ERR per gray between the two subcohorts, probably because of different diagnostic activity in the regions. Our findings confirm that there is a dose-response relationship for radiation-induced parathyroid adenomas.     

Hatching asynchrony and brood reduction in Tengmalm's owl <Emphasis Type="Italic">Aegolius funereus</Emphasis>: the role of temporal and spatial variation in food abundance

Hatching asynchrony is the consequence of birds initiating incubation before clutch completion. It has been suggested that variation in hatching asynchrony in owls is extensive, and therefore they should be excellent objects to study the effects of spatio-temporal variation in food abundance on this phenomenon. We examined how abundance and predictability of food affected hatching asynchrony in Tengmalm's owl Aegolius funereus (Linnaeus), which mainly feeds on voles which fluctuate in 3- to 4-year cycles in northern Europe. Hatching span averaged 6-7 days (range 0-13 days) and increased with clutch size. Food supply did not directly influence levels of hatching asynchrony but it influenced indirectly via marked among-year changes in clutch size. During the decrease phase of the vole cycle the proportion of hatchlings producing fledglings decreased with asynchrony, suggesting that chick mortality was most common among asynchronous broods when food became scarce. This finding is consistent with Lack's brood reduction hypothesis, i.e. that if food becomes scarce during the nestling period the youngest nestlings would die first without endangering the survival of the whole brood.     

Automated detection of remote homology

The classification of a newly identified protein as a member of a superfamily is important for focusing experiments on its most likely functions. Such classification, often performed by hand, has now been fully automated. This sophisticated new approach takes into account not only alignment scores but also a number of other computable attributes, such as functional sites deduced from sequence conservation patterns.     

Going to the roots of the stem cell controversy

The purpose of this paper is to describe the scientific background to the current ethical and legislative debates about the generation and use of human stem cells, and to give an overview of the ethical issues underlying these debates.
The ethical issues discussed are 1) stem cells and the status of the embryo, 2) women as the sources of ova for stem cell production, 3) the use of ova from other species, 4) slippery slopes towards reproductive cloning, 5) the public presentation of stem cell research and 6) the evaluation of scientific uncertainty and its implications for public policy.     

Characterization of a novel testicular form of human hormone-sensitive lipase

Hormone-sensitive lipase (HSL) is an esterase and lipase, which are essential for spermatogenesis. Two HSL mRNAs are expressed in human testis. A long form is encoded by a testis-specific exon and nine exons common to testis and adipocyte HSL. Here we show that the short-form 3.3-kb mRNA possesses a unique 5' end that is transcribed from a novel testis-specific exon. The corresponding protein is similar to the 775-amino-acid-long adipocyte HSL. Immunohistochemistry experiments on human testis sections revealed that the long form is strictly expressed in haploid germ cells whereas the short form is expressed in interstitial and tubular somatic cells as well as premeiotic germ cells.     

Ligand Binding and Polymerization Characteristics of Human Milk Folate Binding Protein Depend on Concentration of Purified Protein and Presence of Amphiphatic Substances

Two folate binding proteins are present in human milk; one of 27 kDa is a cleavage product of the other one (100 kDa) which possesses a hydrophobic membrane anchor. A drastic change of radioligand binding characteristics and appearance of aggregated weak-radioligand affinity forms on gel filtration occurred at low concentrations of both proteins in the absence of Triton X-100 or other amphiphatic substances, e.g. cetyltrimethylammonium and phospholipids. These findings are consistent with a model predicting association between unliganded and liganded monomers resulting in weak-ligand affinity dimers. Amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers become hydrophilic in the liganded state) thereby preventing association between these monomeric forms prevailing at low concentrations of the protein. Bio-Gel P-300 chromatography of the 27 kDa protein revealed a pronounced polymerization tendency, which diminished with decreasing protein concentrations, however, not in the presence of cetyltrimethylammonium. The data could have some bearings on observations indicating that naturally occurring amphiphatic substances, cholesterol and phospholipids, are necessary for the important clustering of membrane folate receptors.     

The bradykinin BK2 receptor mediates angiotensin II receptor type 2 stimulated rat duodenal mucosal alkaline secretion

Background  

This study investigates bradykinin and nitric oxide as potential mediators of AT2-receptor-stimulated duodenal mucosal alkaline secretion. Duodenal mucosal alkaline secretion was measured in methohexital- and α-chloralose-anaesthetised rats by means of in situ pH-stat titration. Immunohistochemistry and Western blot were used to identify the BK2 receptors.     

Rapid automatic detection and alignment of repeats in protein sequences

Many large proteins have evolved by internal duplication and many internal sequence repeats correspond to functional and structural units. We have developed an automatic algorithm, RADAR, for segmenting a query sequence into repeats. The segmentation procedure has three steps: (i) repeat length is determined by the spacing between suboptimal self-alignment traces; (ii) repeat borders are optimized to yield a maximal integer number of repeats, and (iii) distant repeats are validated by iterative profile alignment. The method identifies short composition biased as well as gapped approximate repeats and complex repeat architectures involving many different types of repeats in the query sequence. No manual intervention and no prior assumptions on the number and length of repeats are required. Comparison to the Pfam-A database indicates good coverage, accurate alignments, and reasonable repeat borders. Screening the Swissprot database revealed 3,000 repeats not annotated in existing domain databases. A number of these repeats had been described in the literature but most were novel. This illustrates how in times when curated databases grapple with ever increasing backlogs, automatic (re)analysis of sequences provides an efficient way to capture this important information.