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PubMed2025

Increased yield of genetic diagnoses in inherited heart diseases using expanded genome and RNA-splicing analyses.

PURPOSE: The Australian Genomics Cardiovascular Disorders Flagship investigated genome sequencing as a first-line genetic test in 600 individuals with cardiomyopathy, primary arrhythmia syndromes, or congenital heart disease. Analysis of disease-specific virtual gene panels achieved a genetic diagnosis in 38% of participants. We sought to increase genetic diagnosis yields by analyzing lesser-evidenced disease genes, the mitochondrial genome, and by functional analysis of predicted splice-altering variants. METHODS: Genome sequences of 520 participants with cardiomyopathy or primary arrhythmia syndromes were reanalyzed in 572 cardiac genes and the mitochondrial genome. Participants with congenital heart disease were excluded. Variants predicted in silico to disrupt splicing were assessed with blood RNA and minigenes. RESULTS: A new genetic diagnosis was achieved in 4% (19/520) of participants, including deep intronic and mitochondrial genome variants. Ten participants had diagnostic variants in lesser evidenced disease genes; 9 had splicing variant pathogenicity functionally validated. Eleven participants had a newly identified variant of uncertain significance with high suspicion of pathogenicity, warranting clinical review. Our data supported the gene-disease association of 1 new cardiomyopathy gene, TBX20. CONCLUSION: Identifying new gene-disease relationships, maintaining contemporary gene panels, and integrating functional studies to refine splicing variant classifications increase genetic diagnoses for cardiomyopathies and primary arrhythmia syndromes.

Humans↗
PubMed2002

Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability, and allows identification of antigen-specific CD4+T cells in peripheral blood mononuclear cells.

Reliable, efficient systems for producing soluble HLA-DR molecules, suitable for multimerization and use as staining reagents, have proved elusive. We found that the addition of a flexible linker between peptide and N terminus of the DRB1*0101-chain (Crawford, F., Kozono, H., White, J., Marrack, P. and Kappler, J., Immunity 1998. 8: 675-682.), results in greater in vitro folding efficiency of Escherichia coli-expressed alpha- and beta-chains, and increases both the yield and stability of the DRA1*0101/DRB1*0101/peptide complexes. Although a 10-amino acid linker functioned efficiently for a 20mer epitope from HIV p24, a longer linker was required to produce a DR1 MHC class II tetramer with the influenza hemagglutinin epitope (HA(306-318)). The DR1-HA tetramer was able to stain positively over 98% of a specific clone (HA 1.7) with only a brief 30-min incubation. The tetrameric complexes detected clone cells diluted into PBMC, with high sensitivity, coupled with low background staining in CD4(+) cells. It was possible to detect antigen-specific CD4(+) T cells within a population of PBMC stimulated with the HA peptide. This demonstrates the potential to monitor CD4(+) T cell responses in peripheral blood in a number of clinical scenarios.

CD4-Positive T-Lymphocytes↗
PubMed1983

[Increased yields of insulin by recycling of non-combined insulin chains].

The yield of insulin by the combination of reduced A and B chains is increased from 15% to 33-34% if the non-insulin-containing fractions of the gel filtration are isolated and used for further combinations (recycling). Crude insulin recovered by gel filtration can be highly purified by preparative high-performance liquid chromatography. Lower yields of insulin were obtained on recombination of chains which had been recycled five times or more. Sulfur analyses show a loss of cystine sulfur. The occurrance of lanthionine and lysinoalanine indicates an alkaline destruction of cystine/cysteine during the repeated combination steps.

Amino Acids↗
PubMed1987

Pooled specimens for Chlamydia trachomatis: new approach to increase yield and cost efficiency.

Pooled specimens from the urethra and cervix accounted for 97% of 101 positive chlamydial isolations in 332 women, and this yield compared favourably with the individual yield from either the urethra (77%) or the cervix (88%). Pooling specimens caused no apparent increase in toxicity to the cell culture system. These results indicate the advantages, in terms of higher yield and no higher cost, of combining the urethral and cervical specimens in one container.

Cervix Uteri↗
PubMed1987

Growth yield increase linked to reductive dechlorination in a defined 3-chlorobenzoate degrading methanogenic coculture.

The microbially mediated reductive dehalogenation of aromatic compounds is potentially important in removal of chlorinated aromatic compounds from the environment. Thermodynamic data are presented which show that the reductive dechlorination of 3-chlorobenzoate to benzoate is exergonic, which led to the hypothesis that reductive elimination of chlorine from 3-chlorobenzoate yields biologically useful energy. In the present paper this hypothesis is tested. Experimental data were obtained with a defined 3-chlorobenzoate degrading methanogenic consortium. These data showed that (i) the molar growth yield of a defined 3-chlorobenzoate degrading consortium increased from 4.9 g protein per mol benzoate metabolized to 6.8 g protein per mol 3-chlorobenzoate when 3-chlorobenzoate replaced benzoate as energy source, and that (ii) the ATP level in starved consortium cells was twice as high when the cells were fed 3-chlorobenzoate than when fed benzoate. These observations show that the electrochemical potential between the redox partners of the H+/H2 (electron-donating) and 3-chlorobenzoate/benzoate (electron-accepting) couples is a potential source of energy and are consistent with the hypothesis that reductive dechlorination of aromatic compounds is coupled to a novel type of microbial chemotrophy.

Adenosine Triphosphate↗
PubMed1990

Increased yield of antibody-producing murine spleen cell hybridomas from fusions cultured in medium supplemented with mouse serum.

The number of monoclonal antibody-producing murine hybridomas were increased 7-10-fold in three separate fusions when supplementing the FBS-containing, or serum-free, growth factor-containing, culture medium with 5% normal mouse serum. The beneficial effect was seen in fusions of A/J and 129GIX+ spleen cells, from primary and secondary responses, and when using SP2/0 or P3X63Ag8U.1 as the fusion partner.

Animals↗
PubMed1997

Vibratory Stimulation of Ejaculation Yields Increased Motile Spermatozoa, Compared with Electroejaculation, in Squirrel Monkeys (Saimiri boliviensis).

Semen sample collection in squirrel monkeys has traditionally relied on electroejaculation (EE) via the rectum; how- ever, the yield is low when anesthesia is used. An alternative method has been advocated for men with spinal cord injury that relies on penile vibratory afferent stimulation. Vibratory stimulation (VS) physiologically coordinates emission and ejaculation without requiring anesthesia; this procedure was investigated in squirrel monkeys. During the breeding season, 10 donor males were evenly allotted to two groups; one received VS, and the other received EE. Three days later, treatments were reversed. For VS, a clinical unit was adapted to hold a 1.8-ml microcentrifuge tube as an artificial vagina. Unanesthetized, manually restrained animals were positioned in ventral recumbency. Vibratory stimulation consisted of a I-mm amplitude, 75-Hz stimulation for 1 min, rest for 1 min, then continued stimulation at 1.8-mm amplitude, 90 Hz for 1 min. For EE, animals were anesthetized by administration of Telazol(3.5 mg/kg of body weight, IM) and stimulated by EE via the rectum, using a 5- to 7.5-V, 60-Hz sine wave for 1 minute of 1 sec on/ I sec off and a probe with two longitudinal electrodes and a temperature monitor. Stimulation was repeated after 1 minute s rest. Semen samples were diluted, incubated, centrifuged, and evaluated for spermatozoal count and motility. Nine of 10 monkeys responded to VS with semen samples, all of which contained spermatozoa. Mean motility was 79.8% and total mean motile spermatozoa yield was 26.1 (range, 0.4 to 110) million. By use of EE, all monkeys produced semen samples, but only 6 samples contained spermatozoa, with mean motility of 70% and low yield of 0.534 (range, 0.038 to 2.2) million motile spermatozoa. In conclusion, penile VS was superior with regard to quantity of motile spermatozoa obtained. Additionally, no anesthesia was necessary for this relatively benign procedure.

Journal Article↗
PubMed2001

A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function.

The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Delta3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility. Histidine residues were appended to the C-terminus to simplify purification. The truncated proteins were highly expressed in Escherichia coli, could be released from the membrane using high salt conditions, and were purified from this fraction to specific contents up to 19 nmol P450/mg protein using a single Ni(2+)-agarose column. Gel filtration revealed that truncated P450 2B1 forms a monodisperse solution of hexamers in the absence of detergent and >95% monomers in 0.25% sodium cholate. All truncated proteins, including human 2B6, were active with 7-ethoxy-4-trifluoromethylcoumarin, and truncated 2B1 was shown to retain the native regio- and stereospecificity of testosterone hydroxylation. These data demonstrate that modification of the N-terminus yields high levels of properly folded P450s 2B with increased solubility, which are suitable for functional and structural analysis.

Amino Acid Sequence↗
PubMed1987

Increased yields of IL-2 in media conditioned by MLA 144 cells.

The gibbon leukaemia cell line MLA 144 produces IL-2 constitutively, and has been used as a source of IL-2 for the in vitro growth of T cells from a range of species. We have tried to increase the amount of IL-2 present in conditioned medium produced by this cell line, by manipulating the growth conditions and using compounds known to increase IL-2 production in other cell lines. We found that conditioned media containing a higher amount of IL-2 could be obtained from 72 h cultures of MLA 144 cells originally seeded at 4 X 10(5) cells/ml in DMEM containing 10% FBS and supplemented with 2-ME at 5 X 10(-5) M and PMA at 10 ng/ml.

Animals↗
PubMed1991

Increased yield of a lysozyme after self-cloning of the gene in Streptomyces coelicolor "Müller".

Streptomyces coelicolor "Müller" DSM3030 excretes a lysozyme comprising both beta-1,4-N-acetyl- and beta-1,4-N,6-O-diacetyl muramidase activities. The lysozyme is named Cellosyl. Gene libraries have been established using genomic DNA from the wild-type strain, S. coelicolor DSM3030, and from an overproducing mutant, S. coelicolor HP1, which exhibits about a twofold increase in lysozyme production. The lysozyme-encoding genes (cel) from both strains were detected by oligodeoxynucleotide hybridization. The nucleotide sequence of the cel genes isolated from both strains was shown to be identical. The different levels of lysozyme production could not be correlated with any mutations at the cel gene locus. The cel gene isolated from the wild-type strain could not be expressed in some other species of Streptomyces. However, self-cloning of the cel gene into S. coelicolor DSM3030 and HP1 resulted in a 2.5-fold increase in lysozyme production.

Amino Acid Sequence↗
PubMedDate not supplied

Purification of rat leydig cells: increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells.

Procedures for purification of Leydig cells have facilitated studies of their regulatory biology. A multistep procedure that includes a filtration with nylon mesh (100-micron pore size) to separate interstitial cells from the seminiferous tubules, combining centrifugal elutriation and Percoll density gradient sedimentation, has been used to obtain a 95% enrichment of rat Leydig cells. However, the number of recovered Leydig cells by this procedure represents only a small fraction of the 25 million, on average, that exist in the adult rat testis. The objective of this study was to test whether the yield of purified Leydig cells might be enhanced by substitution of unit-gravity sedimentation (S method) for the filter step (F method). We also asked whether a greater number of Leydig cell clusters, macrophages, or both would be recovered by this new method, and if the presence of Leydig cell clusters is associated with increased capacity for testosterone production in vitro. The number of purified Leydig cells was 1.9-fold higher for the S method than for the F method, with no differences in purity assessed by 3beta-hydroxysteroid dehydrogenase histochemical staining. Leydig cell clusters were also found in greater numbers with the S method both after collagenase dispersion and at the end of the purification. No differences were seen in testosterone production or in the number of macrophages present in the Leydig cells that were prepared by the 2 methods. These results indicate that the new method recovers greater numbers of Leydig cells by collecting clustered Leydig cells that are systematically eliminated when a filtration step is used.

Animals↗
PubMed1990

The improving prospects for yield increase by genetic engineering in antibiotic-producing Streptomycetes.

Molecular genetics has spawned a dramatic expansion of the biotechnology industry in the direction of the products of single genes. On the other hand, antibiotics--some of the classical products of biotechnology--result from the concerted action of many genes, and it is therefore less straightforward to apply the new techniques to antibiotic production. Studies of cloned genes for antibiotic biosynthesis are now providing information that should allow the application of a combination of traditional and recombinant DNA methodology to the improvement of yield in antibiotic-producing Streptomyces species.

Anti-Bacterial Agents↗
PubMed1992

Increased yield of myeloid progenitor cells in bone marrow harvested for autologous transplantation by pretreatment with recombinant human granulocyte-colony stimulating factor.

Pretreatment with haemopoietic cytokines prior to marrow harvest may result in improved quality of bone marrow harvested for autologous bone marrow transplantation (BMT). Such improvements may reduce the risk for graft failure and decrease time to engraftment. Patients undergoing autologous BMT received recombinant human G-CSF (rhG-CSF) immediately prior to marrow harvest. rhG-CSF was administered as daily subcutaneous injections for 5 days at 5 micrograms/kg body weight. Comparison of bone marrow samples before and after rhG-CSF treatment showed an increased bone marrow cellularity and a ninefold increase in the number of marrow leucocytes per volume aspirated. The mean marrow myeloid:erythroid ratio increased from 2.6 to 4.0. The mean numbers of immature (CD38 positive) and proliferating (CD71 positive) myeloid cells increased significantly from 41.6 to 50.8% and from 17.0 to 34.8%, respectively. Other subsets studied, including CD34 positive stem cells, were unchanged. The relative numbers of day 7 and 14 granulocyte-macrophage colony-forming units (day 7/14 GM-CFU) were unchanged. Long-term marrow cultures revealed that the numbers of 'long-term culture initiating cells' were unchanged after rhG-CSF treatment in spite of the ninefold increase in cellularity. To date, five of the patients have been transplanted with autologous marrow harvested after rhG-CSF treatment. Time to trilineage engraftment was unchanged compared with historical controls. We conclude that pretreatment with rhG-CSF prior to marrow harvest may improve the graft by increasing the total number of myeloid lineage restricted progenitor cells, resulting in stable but not accelerated myeloid engraftment of autologous marrow.

Bone Marrow Transplantation↗
PubMed1997

Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli.

Accumulation of soluble recombinant hemoglobin (rHb1.1) in Escherichia coli requires proper protein folding, prosthetic group (heme) addition, and subunit assembly. This served as a new model system for the study of the effects of temperature, protein synthesis rates, and protein accumulation rates on protein solubility in E. coli. Fermentation expression of rHb1.1 at 30 degrees C from cultures containing a medium or high globin gene dosage (pBR-based or pUC-based plasmids with rHb1.1 genes under the control of the tac promoter) was compared. A medium gene dosage resulted in rHb1.1 accumulating to approximately 7% of the soluble cell protein, of which 78% was soluble. A high globin gene dosage resulted in a > or = 3-fold increase in total globin to 23 to 24% of the soluble cell protein, but 70% was insoluble. Accumulation of insoluble rHb1.1 began immediately upon induction. The proportion of rHb1.1 from the high globin gene dosage that accumulated as insoluble globin was affected by reducing (i) the inducer concentration and (ii) the temperature. Reducing the inducer concentration reduced globin synthesis up to eightfold but increased the proportion of soluble rHb1.1 to 93%. In contrast, total globin protein synthesis was barely affected by reducing the temperature from 30 to 26 degrees C, while soluble globin accumulation increased > 2-fold to approximately 15% of the soluble cell protein. The contrast between the effects of reducing rates of protein synthesis and accumulation and those of reducing temperature suggests that lower temperature stabilizes one or more folding intermediates. We propose a simplified physical model which integrates protein synthesis, folding, and heme association. This model shows that temperature-dependent apoglobin stability is the most critical factor in soluble rHb1.1 accumulation.

Apoproteins↗
PubMed1990

Milk yield increase after anthelmintic treatment of dairy cattle related to some parameters estimating helminth infection.

On 81 farms blood samples were taken from adult dairy cattle, on pasture in October 1986 and after stabling in December 1986, to measure antibody titres against the nematodes Dictyocaulus viviparus, Cooperia spp. and Ostertagia spp., and the trematode Fasciola hepatica, and serum pepsinogen values. Faecal samples, collected in October, were examined to confirm the presence of parasites by means of egg counts and larval identifications. From December until the end of the stabling period, dry cows were either treated with albendazole or left untreated in alternate sequence of calving date. Treated cows produced 132.9 kg milk per cow per lactation more than untreated cows (P less than 0.01). Fat and protein percentage were not significantly influenced by anthelmintic treatment. The mean herd milk yield response to treatment varied from -889 to +1231 kg milk per cow per lactation. There was a significant between-herd variation in antibody titres against nematodes and in pepsinogen values. However no significant correlations between these parameters and the mean herd milk yield response to treatment were found.

Albendazole↗
PubMed1991

Increased yields of IgG2a- and IgG3-secreting hybridomas after fusion of B cells from mice with autoimmune diseases.

Hybridoma technology has made the production of antigen-specific monoclonal antibodies feasible and almost routine, but the production of certain biologically desirable antibody isotypes has remained difficult. Three strains of autoimmune mice (MRL/l, NZB, and BXSB) were compared to a normal strain (BALB/c), in fusions with a BALB/c myeloma (NS-1) in order to study the rescue of relevant isotypes with the desired antigenic specificities. Mice from these four strains were immunized with colon carcinoma cells, and the hybridoma supernatants from thirty fusions were analyzed for (1) reactivity with cell surface determinants on the immunizing cell line; and (2) Ig class and subclass isotypes. We found that compared to BALB/c mice, MRL/l mice produced greater numbers, and NZB and BXSB mice comparable numbers, of cell surface-reactive hybridoma clones per fusion. MRL/l mice produced the largest number and highest percentage of cell-surface reactive IgG2a (22.4%) and IgG3 (10.6%) producing clones, followed by NZB mice which produced predominantly IgG2a clones (12.3%). BXSB mice, which have latent autoimmune disease, showed no significant difference from normal BALB/c controls (IgG2a:0.7% and IgG3:1.9% vs. IgG2a:4.8% and IgG3:4.8%). The increase in IgG2a and IgG3 clones derived from MRL/l mice was age-dependent, correlating with the age at which abnormal proliferation of T cell and splenic enlargement occurs (2-4 months). We conclude that MRL/l mice are useful for generating monoclonal antibodies of the IgG2a or IgG3 isotype, provided fusions are performed at the time of maximal lymphoproliferation.

Animals↗
PubMed2001

Increased yield and activity of soluble single-chain antibody fragments by combining high-level expression and the Skp periplasmic chaperonin.

The success of recombinant antibody fragments as diagnostic reagents and therapeutic agents depends on the availability of sufficient functional material. We have produced a bacterial expression vector that combines high-level expression driven by a modified Shine-Dalgarno sequence with the periplasmic chaperonin Skp. Using this vector, we are able to obtain higher yields of soluble antibody fragments from cultures without the need for supplementation of the culture medium during expression. The fragments produced in the presence of the Skp show improved antigen binding activity compared to when the chaperonin is absent.

Animals↗