Electron microscope tomography of Balbiani Ring hnRNP substructure

Three-dimensional (3-D) reconstructions, by electron microscope tomography, of selectively stained, contrast enhanced Balbiani Ring (BR) hnRNP granules reveal a complex spatial arrangement of RNA-rich domains. This particulate substructure was examined by volume rendering computer graphics. Modeling the arrangement of RNA-rich domains is made difficult by apparent structural flexibility and/or heterogeneity of composition. Formulation of a consensus 3-D arrangement of RNA-rich domains will require an expanded data base of reconstructed BR granules and the development of new image manipulation and analysis techniques. This study demonstrates the potential for ultra-structural cell biology of combining several new techniques: selective nucleic acid staining, electron spectroscopic imaging to enhance contrast, electron microscope tomography and volume rendering computer graphics.Abbreviations BR Balbiani Ring - EMT electron microscope tomography - ESI electron spectroscopic imaging - hnRNP heterogeneous nuclear ribonucleoprotein - OA-B osmium ammine-B - kb kilobases by P.B. Moens     

The dynamics of sparse random networks

Recurrent neural networks with full symmetric connectivity have been extensively studied as associative memories and pattern recognition devices. However, there is considerable evidence that sparse, asymmetrically connected, mainly excitatory networks with broadly directed inhibition are more consistent with biological reality. In this paper, we use the technique of return maps to study the dynamics of random networks with sparse, asymmetric connectivity and nonspecific inhibition. These networks show three qualitatively different kinds of behavior: fixed points, cycles of low period, and extremely long cycles verging on aperiodicity. Using statistical arguments, we relate these behaviors to network parameters and present empirical evidence for the accuracy of this statistical model. The model, in turn, leads to methods for controlling the level of activity in networks. Studying random, untrained networks provides an understanding of the intrinsic dynamics of these systems. Such dynamics could provide a substrate for the much more complex behavior shown when synaptic modification is allowed.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.     

Changes in maximum oxygen uptake during prolonged training, overtraining, and detraining in horses

Tyler, Catherine M., Lorraine C. Golland, David L. Evans,David R. Hodgson, and Reuben J. Rose. Changes in maximum oxygenuptake during prolonged training, overtraining, and detraining inhorses. J. Appl. Physiol. 81(5):2244-2249, 1996.Thirteen standardbred horses were trained asfollows: phase 1 (endurance training, 7 wk),phase 2 (high-intensity training, 9 wk),phase 3 (overload training, 18 wk), andphase 4 (detraining, 12 wk). Inphase 3, the horses were divided intotwo groups: overload training (OLT) and control (C). The OLT groupexercised at greater intensities, frequencies, and durations than groupC. Overtraining occurred after 31 wk of training and was defined as asignificant decrease in treadmill run time in response to astandardized exercise test. In the OLT group, there was a significantdecrease in body weight (P < 0.05).From pretraining values of 117 ± 2 (SE)ml ^· kg^{1 ·} min¹,maximal O₂ uptake(O_{2 max}) increased by15% at the end of phase 1, and when signs of overtraining werefirst seen in the OLT group,O_{2 max} was 29%higher (151 ± 2 ml ^· kg^{1 ·} min¹in both C and OLT groups) than pretraining values. There was nosignificant reduction inO_{2 max} until after 6 wk detraining whenO_{2 max} was 137 ± 2 ml ^· kg^{1 ·} min¹.By 12 wk detraining, meanO_{2 max} was134 ± 2 ml ^· kg^{1 ·} min¹,still 15% above pretraining values. When overtraining developed, O_{2 max} was notdifferent between C and OLT groups, but maximal values forCO₂ production (147 vs. 159 ml ^· kg^{1 ·} min¹)and respiratory exchange ratio (1.04 vs. 1.11) were lower in the OLTgroup. Overtraining was not associated with a decrease inO_{2 max} and, afterprolonged training, decreases inO_{2 max} occurredslowly during detraining.

     

Cloning and comparative mapping of a gene from the commonly deleted region of DiGeorge and Velocardiofacial syndromes conserved in C. elegans

We have identified and cloned a gene, ES2, encoding a putative 476 amino acid protein with a predicted M _r of 52,568. The gene is localized within the DiGeorge/Velocardiofacial syndrome locus on 22q11.2 and is deleted in all the patients in which a deletion within 22q11 could be demonstrated, with the exception of one patient. ES2 is expressed in all the tissues studied. Sequence comparison showed identity with five ESTs and at the amino acid level the sequence was highly similar to, and collinear with, a hypothetical C. elegans protein of unknown function. Mutation analysis was performed in 16 patients without deletion, but no mutation has been found. The cDNA sequence is conserved in mouse and is localized on MMU16B1-B3, known to contain a syntenic group in common with HSA 22q11.2. Received: 25 March 1996 / Accepted: 15 May 1996     

Association of mouse fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity.

Previously, we demonstrated induction of a unique macrophage prothrombinase during infection of BALB/cJ mice by mouse hepatitis virus strain 3 (MHV-3). By immunologic screening, a clone representing this prothrombinase was isolated from a cDNA library and sequenced. The sequence identified this clone as representing part of a gene, musfiblp, that encodes a fibrinogen-like protein. Six additional clones were isolated, and one clone, p11-3-1, encompassed the entire coding region of musfiblp. Murine macrophages did not constitutively express musfiblp but, when infected with MHV-3, synthesized musfiblp-specific mRNA. musfiblp mRNA induction was earlier and significantly greater in BALB/cJ than A/J macrophages. Prothrombinase activity was demonstrated when musfiblp was expressed from p11-3-1 in RAW 264.7 cells. These data suggest that musfiblp encodes the MHV-induced prothrombinase.     

Mutations in the processing site of the precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit: effects on import,processing, assembly and stability

The small subunit (SSU) of Rubisco is synthesized in the cytosol in a precursor form. Upon import into the chloroplast, it is proteolytically processed at a Cys-Met bond to yield the mature form of the protein. To assess the importance of the Met residue for recognition and processing by the stromal peptidase, we substituted this residue with either Thr, Arg or Asp. The mutant precursor proteins were imported into isolated chloroplasts, and the products of the import reactions were analyzed. Mutants containing Thr or Arg residues at the putative processing site were processed to a single peptide, comigrating with the wild-type protein. N-terminal radio-sequencing revealed that these mutants were processed at the Cys-Thr and the Cys-Arg bond, respectively. After import of the Asp-containing mutant, four processed forms of the protein were observed. Analysis of the most abundant one, co-migrating with the wild-type protein, demonstrated that this species was also a product of correct processing, at the Cys-Asp bond. All the correctly processed peptides were found to be associated with the holoenzyme of Rubisco, and remained stable within the chloroplast, like the wild-type protein. The results of this study, together with previous ones, suggest that proper recognition and processing of the SSU precursor are more affected by residues N-terminal to the processing site than by the residue on the C-terminal side of this site.     

Bioreactors for surface-immobilized cells

Surface immobilization of plant cells avoids the problem of hydrodynamic or shear stress, which tends to be characteristic of suspended cells cultured in typical, mechanically agitated bioreactor systems. Surface immobilization also promotes the natural tendency for plant cells to aggregate, which may improve the synthesis and accumulation of secondary metabolites. In addition, exchange of medium is made simple in surface-immobilized systems, and extracellular secondary products are easily recovered on a continuous basis. However, problems related to regulation of the thickness of the immobilized cell layer, maintenance of the biomass in a productive condition, and vacuolar retention of secondary products have yet to be resolved satisfactorily. This review focusses on two surface-immobilization technologies, differing primarily in the nature and the configuration of the inert support. Prototypes of these designs have been applied to a variety of plant cell systems at bioreactor volumes up to 20 litres. Results obtained with several alternative technologies are also summarized.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - SIPCB surface-immobilized plant cell bioreactor National Research Council of Canada publication no. 38460