Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K _L a and of mixing (via pH measurements) in bioreactors up to 8 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.     

Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro

Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1-cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids—glutamine, taurine, and glycine—were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium-5. Moreover, addition of another eight amino acids—asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM-6)—had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM-5 and ?6 (chemically defined, protein-free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley-Liss, Inc.     

Genes encoding homologues of three consecutive enzymes in the butyrate/butanol-producing pathway of Clostridium acetobutylicum are clustered on the Clostridium difficile chromosome

Abstract Screening of a Clostridium difficile ψEMBL3 gene library with antisera raised against C. difficile culture supernatant identified several clones expressing a 31-kDa protein. A 1.8-kb Hin dIII fragment subcloned from one of the clones was sufficient for expression of the 31-kDa polypeptide. Southern blot analysis showed a region homologous to this fragment to be present in all of 13 different C. difficile strains tested. Sequence analysis of the 1.8-kb fragment revealed three adjacent open reading frames. A database search showed that these three open reading frames appeared to encode homologues of three consecutive enzymes in the butanol/butyrate-producing pathway of Clostridium acetobutylicum (crotonase, β-hydroxybutyryl coenzyme A dehydrogenase and thiolase).     

The adaptive significance of self-medication

Not all pharmacists are human; other species also use medicinal substances to combat pathogens and other parasites. Self-medicating behaviour is a topic of rapidly growing interest to behaviourists, parasitologists, ethnobotanists, chemical ecologists, conservationists and physicians. Although most of the pertinent literature is anecdotal, several studies have now attempted to test the adaptive function of particular self-medicating behaviours. We discuss the results of these studies in relation to simple hypotheses that can provide a framework for future tests of self-medication.     

Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae.

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype.Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.     

Population density and genetic diversity are positively correlated in wild felids globally

Aim

Insights into the biological and evolutionary traits of species, and their ability to cope with global changes, can be gained by studying genetic diversity within species. A cornerstone hypothesis in evolutionary and conservation biology suggests that genetic diversity decreases with decreasing population size, however, population size is difficult to estimate in threatened species with large distribution ranges, and evidence for this is limited to few species. To address this gap, we tested this hypothesis across multiple closely related species at a global scale using population density which is a more accessible measure.

Location

Global.

Time Period

Contemporary.

Major Taxa Studied

Wild felids in their natural habitats.

Methods

We obtained data from published estimates of population density assessed via camera trap and within-population genetic diversity generated from microsatellite markers on 18 felid species across 41 countries from 354 studies. We propose a novel method to standardize population density estimates and to spatially join data using K-means clustering. Linear mixed-effect modelling was applied to account for confounding factors such as body mass, generation length and sample size used for the genetic estimates.

Results

We found a significant positive correlation between population density and genetic diversity, particularly observed heterozygosity and allelic richness. While the confounding factors did not affect the main results, long generation length and large sample size were significantly associated with high genetic diversity. Body mass had no effect on genetic diversity, likely because large-bodied species were over-represented in our data sets.

Main Conclusions

Our study emphasizes how recent demographic processes shape neutral genetic diversity in threatened and small populations where extinction vortex is a risk. Although caution is needed when interpreting the small population density effect in our findings, our methodological framework shows promising potential to identify which populations require actions to conserve maximal genetic variation.     

Quantitative Evaluation of Erotactin Secretion in Eggs of Hormosira banksii (Fucales,Phaeophyceae)

The release of the C₁₁-hydrocarbon spermatozoid attractant hormosirene by eggs of the Australian fucoid Hormosira banksii has been determined quantitatively using a closed-loop extraction technique. The eggs secrete erotactin for more than 2 weeks with an exponentially decreasing rate. The initial secretion rate was estimated to be 75 fmol h^?1 egg^?1 and the total amount of hormosirene produced per egg to be 2.35 pmol (0.35 ng). The results were used to calculate the threshold range for spermatozoid chemotaxis in the pheromone gradient around eggs.     

Evaluating perinatal mortality rates: effects of referral and case mix.

OBJECTIVE--To evaluate perinatal mortality rates as a method of auditing obstetric and neonatal care after account had been taken of transfer between hospitals during pregnancy and case mix. DESIGN--Case-control study of perinatal deaths. SETTING--Leicestershire health district. SUBJECTS--1179 singleton perinatal deaths and their selected live born controls among 114,362 singleton births to women whose place of residence was Leicestershire during 1978-87. MAIN OUTCOME MEASURE--Crude perinatal mortality rates and rates adjusted for case mix. RESULTS--An estimated 11,701 of the 28,750 women booked for delivery in general practitioner maternity units were transferred to consultant units during their pregnancy. These 11,701 women had a high perinatal mortality rate (16.8/1000 deliveries). Perinatal mortality rates by place of booking showed little difference between general practitioner units (8.8/1000) and consultant units (9.3-11.7/1000). Perinatal mortality rates by place of delivery, however, showed substantial differences between general practitioner units (3.3/1000) and consultant units (9.4-12.6/1000) because of the selective referral of high risk women from general practitioner units to consultant units. Adjustment for risk factors made little difference to the rates except when the subset of deaths due to immaturity was adjusted for birth weight. CONCLUSION--Perinatal mortality rates should be adjusted for case mix and referral patterns to get a meaningful result. Even when this is done it is difficult to compare the effectiveness of hospital units with perinatal mortality rates because of the increasingly small subset of perinatal deaths that are amenable to medical intervention.     

Evidence of Commingling in Human Eating Behavior

This investigation tested whether distributions of certain aspects of eating behavior were consistent with the notion of a “mixture model;” that is, two or more distinct Commingled component distributions, consistent with the possibility of major gene action. Undergraduates (n=901) completed self-report trait measures of hunger, disinhibition, and dietary restraint. Variables were residualized for gender and age and transformed to remove skewness. Residualized transformed distributions were tested for departure from unimodality with Hartigan's (14) dip statistic. The distributions of all three aspects of eating behavior were significantly non-unimodal. Next, component multivariate normal distributions were estimated via maximum likelihood. Likelihood ratio tests were employed to compare nested models. A mixture of four distributions with unequal variance-covariance matrices tit significantly better than any more parsimonious model. In sum, these data strongly suggest that the distributions of several measures of eating behavior are composed of four component distributions. This finding is consistent with the possibility of major gene effects for eating behavior.     

A 610 kb YAC clone harbors 7 cM of tomato (Lycopersicon esculentum) DNA that includes themale sterile 14 gene and a hotspot for recombination

Pollen development requires both sporophytic and gametophytic gene expression. We are using a map-based cloning technique to isolate sporophytic genes which, when mutant, cause pollen abortion and a male sterile (ms) phenotype in tomato (Lycopersicon esculentum). We have genetically characterized onems locus (ms14) using RFLP analysis and identified flanking markers. High-resolution genomic physical mapping indicates that thems14 locus is located in a ～300 kb region. We have identified a YAC clone with an insert size of ～610 kb that contains thems14-linked markers, reflects the organization of the physical map and therefore most probably contains thems14 gene. In addition, we present evidence that the relationship between physical and genetic distance in this chromosomal region changes abruptly from ～105–140 kb/cM to less than 24 kb/cM, and suggest that the TG393-TG104 region is a hotspot for recombination.