Volume comparisons in the cerebellar complex of primates. II. Cerebellar nuclei

Volumes of medial, interposed, and lateral cerebellar nuclei (MCN, ICN, and LCN) were measured in Insectivora, Scandentia, and Primates, including man. The relative size of the nuclei was expressed in size indices. Insectivora had by far the smallest cerebellar nuclei. The simians, in general, had larger cerebellar nuclei than the prosimians, but there was considerable overlap. From Insectivora to man, the MCN was the least progressive and the LCN the most progressive. The indices are expected to reflect the relative size of the three longitudinal zones of the cerebellum (vermis/MCN, pars intermedius/ICN, hemisphere/LCN). They, together with those of the ventral pons and cerebellum (part I), are discussed in relation to the predominant locomotor pattern of a species, and with reference to evolutionary trends in primate phylogeny.     

Biochemical evidence for multiple independent emetine resistance genes in Chinese hamster cells.

Hybridization-complementation studies indicated that mutations in multiple genes can render Chinese hamster cells resistant to the alkaloid translation inhibitor emetine. Two of the genes, emtA and emtB, recognized in Chinese hamster lung and ovary cell lines, respectively, are known to affect the ribosomes of the cells directly. Although mutations in a third gene, emtC, affect the translation apparatus of Chinese hamster peritoneal cells in vitro (Wasmuth et al., Mol. Cell. Biol. 1:58-65, 1981), the molecular product of the emtC locus remains to be determined. To study the molecular basis for genetic complementation among emetine-resistant Chinese hamster cell mutants, we analyzed ribosomal proteins elaborated by complementing, emetine-sensitive hybrid clones (EmtB X EmtA and EmtB X EmtC) and by emetine-resistant clones that segregated from the hybrids. The electrophoretic forms of ribosomal protein S14 (the emtB gene product) elaborated by these clones indicated that the EmtA and EmtC phenotypes are independent of the emtB locus and that the emtA and emtC loci are not chromosomally linked to emtB.     

The novel flightless-I gene brings together two gene families, actin- binding proteins related to gelsolin and leucine-rich-repeat proteins involved in Ras signal transduction

The Drosophila melanogaster gene flightless-I, involved in gastrulation and muscle degeneration, has Caenorhabditis elegans and human homologues. In these highly conserved genes, two previously known gene families have been brought together, families encoding the actin- binding proteins related to gelsolin and the leucine-rich-repeat (LRR) group of proteins involved in protein-protein interactions. Both these gene families exhibit characteristics of molecular changes involving replication slippage and exon shuffling. Phylogenetic analyses of 19 amino acid sequences of 6 related protein types indicate that actin- associated proteins related to gelsolin are monophyletic to a common ancestor and include flightless proteins. Conversely, comparison of 24 amino acid sequences of LRR proteins including the flightless proteins indicates that flightless proteins are members of a structurally related subgroup. Included in the flightless cluster are human and mouse rsp-1 proteins involved in suppressing v-Ras transformation of cells and the membrane-associated yeast (Saccharomyces cerevisae) adenylate cyclase whose analogous LRRs are required for interaction with Ras proteins. There is a strong possibility that ligands for this group could be related and that flightless may have a similar role in Ras signal transduction. It is hypothesized that an ancestral monomeric gelsolin precursor protein has undergone at least four independent gene reorganization events to account for the structural diversity of the extant family of gelsolin-related proteins and that gene duplication and exon shuffling events occurred prior to or at the beginning of multicellular life, resulting in the evolution of some members of the family soon after the appearance of actin-type proteins.      

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

Pooled-Peptide Epitope Mapping Strategies Are Efficient and Highly Sensitive: An Evaluation of Methods for Identifying Human T Cell Epitope Specificities in Large-Scale HIV Vaccine Efficacy Trials

The interferon gamma, enzyme-linked immunospot (IFN-γ ELISpot) assay is widely used to identify viral antigen-specific T cells is frequently employed to quantify T cell responses in HIV vaccine studies. It can be used to define T cell epitope specificities using panels of peptide antigens, but with sample and cost constraints there is a critical need to improve the efficiency of epitope mapping for large and variable pathogens. We evaluated two epitope mapping strategies, based on group testing, for their ability to identify vaccine-induced T-cells from participants in the Step HIV-1 vaccine efficacy trial, and compared the findings to an approach of assaying each peptide individually. The group testing strategies reduced the number of assays required by >7-fold without significantly altering the accuracy of T-cell breadth estimates. Assays of small pools containing 7–30 peptides were highly sensitive and effective at detecting single positive peptides as well as summating responses to multiple peptides. Also, assays with a single 15-mer peptide, containing an identified epitope, did not always elicit a response providing validation that 15-mer peptides are not optimal antigens for detecting CD8+ T cells. Our findings further validate pooling-based epitope mapping strategies, which are critical for characterizing vaccine-induced T-cell responses and more broadly for informing iterative vaccine design. We also show ways to improve their application with computational peptide:MHC binding predictors that can accurately identify the optimal epitope within a 15-mer peptide and within a pool of 15-mer peptides.     

Model-based strategy for cell culture seed train layout verified at lab scale

Cell culture seed trains—the generation of a sufficient viable cell number for the inoculation of the production scale bioreactor, starting from incubator scale—are time- and cost-intensive. Accordingly, a seed train offers potential for optimization regarding its layout and the corresponding proceedings. A tool has been developed to determine the optimal points in time for cell passaging from one scale into the next and it has been applied to two different cell lines at lab scale, AGE1.HN_AAT and CHO-K1. For evaluation, experimental seed train realization has been evaluated in comparison to its layout. In case of the AGE1.HN_AAT cell line, the results have also been compared to the formerly manually designed seed train. The tool provides the same seed train layout based on the data of only two batches.     

Expeditious synthesis and chromatographic resolution of (+)- and (-)-trans-1-benzylcyclohexan-1,2-diamine hydrochlorides

The hitherto unknown (-)- and (+)-1-benzylcyclohexan-1,2-diamine hydrochlorides 4a. HCl and 4b. HCl were synthesized by means of diastereoselective alpha-iminoamine rearrangement with subsequent imine reduction and hydrogenolysis. The relative trans-configuration as well as the absolute (1S,2R) and (1R,2S) configurations of 4a and 4b, respectively, were elucidated on the basis of an X-ray analysis of 3b. HCl. The enantiomeric excess (ee) values of the title compounds (>99%) were determined by chiral HPLC on a Chirex (D) Penicillamine column.     

Adaptive,model-based control by the Open-Loop-Feedback-Optimal (OLFO) controller for the effective fed-batch cultivation of hybridoma cells

Although fed-batch suspension culture of animal cells continues to be of industrial importance for the large scale production of pharmaceutical products, existing control concepts are still insufficient. Changes in cell metabolism during cultivation and between similar cultivations, the complexity of the cell metabolism, and the lack of on-line state variables restrict the transfer of available control strategies established in bioprocess engineering. A process control strategy designed to achieve optimized process control must account for all these difficulties and fit sophisticated requirements toward adaptability and flexibility. The combination of a fed-batch process and an Open-Loop-Feedback-Optimal (OLFO) control provides a new approach for cell culture process control that couples an efficient cultivation concept to a capable process control strategy. The application of an adaptive, model-based OLFO controller to a hybridoma cultivation and experimental results are presented.     

Application of the fluorogenic probe technique (TaqMan PCR) to the detection of Enterococcus spp. and Escherichia coli in water samples

A recent PCR detection technique (TaqMan) based on the 5'-3'-exonuclease activity of the Taq DNA polymerase was applied to the detection of indicator organisms in water samples. In this technique, an increasing fluorescence signal is measured online which enables direct assessment of results after PCR without additional detection steps. The test is completed within about 5 h. Two sets of primers and probes were designed and tested: a genus-specific assay for the detection of Enterococcus spp. based on 23S rRNA sequence and an Escherichia coli-specific assay based on the uidA gene sequence. Specificity of the assays was confirmed by testing strains of target bacteria and potential interfering microorganisms. Application of the tests to 55 natural water samples showed the need of an overnight enrichment step to achieve compliance with detection limits of existing regulations. Compared with a parallel microbiological examination of the samples, agreement was 96% with the Enterococcus assay and 98% with the E. coli assay. The rapidity and feasibility of the method point to benefits in drinking water analysis, particularly in emergency situations and, thus, to improved public health management.