Some determinants of quality and yield in the isolation of adult heart cells from rat

We investigated the effect of changes in perfusate substrate and Ca content on the quality and yield of isolated adult rat heart cells. When 1 mM Ca was added to the recirculating perfusate 15 min after collagenase addition, the ATP level of cells in the heart 15 min later, and their morphology in histological section, was no different from when no Ca was added back. The cells subsequently isolated were of similar yield, but a greater percentage were rod-shaped, compared with cells isolated without Ca restoration to the perfusate. Increased yield could be obtained by including substrates in the perfusate in addition to glucose. Either fatty acids or amino acids were effective. We conclude that: (1) all cells in the heart are Ca tolerant at the end of enzyme perfusion; (2) the presence of substrates in addition to glucose can help cells survive the isolation process.     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Enrichment of DNRA bacteria in a continuous culture

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are competing microbial nitrate-reduction processes. The occurrence of DNRA has been shown to be effected qualitatively by various parameters in the environment. A more quantitative understanding can be obtained using enrichment cultures in a laboratory reactor, yet no successful DNRA enrichment culture has been described. We showed that a stable DNRA-dominated enrichment culture can be obtained in a chemostat system. The enrichment was based on the hypothesis that nitrate limitation is the dominant factor in selecting for DNRA. First, a conventional denitrifying culture was enriched from activated sludge, with acetate and nitrate as substrates. Next, the acetate concentration in the medium was increased to obtain nitrate-limiting conditions. As a result, conversions shifted from denitrification to DNRA. In this selection of a DNRA culture, two important factors were the nitrate limitation and a relatively low dilution rate (0.026 h⁻¹). The culture was a highly enriched population of Deltaproteobacteria most closely related to Geobacter lovleyi, based on 16S rRNA gene sequencing (97% similarity). We established a stable and reproducible cultivation method for the enrichment of DNRA bacteria in a continuously operated reactor system. This enrichment method allows to further investigate the DNRA process and address the factors for competition between DNRA and denitrification, or other N-conversion pathways.     

A template RNA entry channel in the fingers domain of the poliovirus polymerase

Positive-strand RNA viruses within the Picornaviridae family express an RNA-dependent RNA polymerase, 3D(pol), that is required for viral RNA replication. Structures of 3D(pol) from poliovirus, coxsackievirus, human rhinoviruses, and other picornaviruses reveal a putative template RNA entry channel on the surface of the enzyme fingers domain. Basic amino acids and tyrosine residues along this entry channel are predicted to form ionic and base stacking interactions with the viral RNA template as it enters the polymerase active site. We generated a series of alanine substitution mutations at these residues in the poliovirus polymerase and assayed their effects on template RNA binding, RNA synthesis initiation, rates of RNA elongation, elongation complex (EC) stability, and virus growth. The results show that basic residues K125, R128, and R188 are important for template RNA binding, while tyrosines Y118 and Y148 are required for efficient initiation of RNA synthesis and for EC stability. Alanine substitutions of tyrosines 118 and 148 at the tip of the 3D(pol) pinky finger drastically decreased the rate of initiation as well as EC stability, but without affecting template RNA binding or RNA elongation rates. Viable poliovirus was recovered from HeLa cells transfected with mutant RNAs; however, mutations that dramatically inhibited template RNA binding (K125A-K126A and R188A), RNA synthesis initiation (Y118A, Y148A), or EC stability (Y118A, Y148A) were not stably maintained in progeny virus. These data identify key residues within the template RNA entry channel and begin to define their distinct mechanistic roles within RNA ECs.     

Insights from Streptococcus pneumoniae glucose kinase structural model

Streptococcus pneumonia is the common cause of sepsis and meningitis. Emergence of multiple antibiotic resistant strains in the community‐acquired bacterium is catastrophic. Glucose kinase (GLK) is a regulatory enzyme capable of adding phosphate group to glucose in the first step of streptomycin biosynthesis. The activity of glucose kinase was regulated by the Carbon Catabolite Repression (CCR) system. Therefore, it is important to establish the structure‐function relation of GLK in S. pneumoniae. However, a solved structure for S. pneumoniae GLK is not available at the protein data bank (PDB). Therefore, we created a model of GLK from S. pnemoniae using the X‐ray structure of Glk from E. faecalis as template with MODELLER (a comparative modeling program). The model was validated using protein structure checking tools such as PROCHECK, WHAT IF and ProSA for reliability. The active site amino acid Asp114 in the template is retained in S. pneumoniae GLK model (Asp115). Solvent accessible surface area (ASA) analysis of the GLK model showed that known key residues playing important role in active site for ligand binding and metal ion binding are buried and hence not accessible to solvent. The information thus discussed provides insight to the molecular understanding of glucose kinase in S. pneumoniae.     

Mapping carcass and meat quality QTL on Sus Scrofa chromosome 2 in commercial finishing pigs

Quantitative trait loci (QTL) affecting carcass and meat quality located on SSC2 were identified using variance component methods. A large number of traits involved in meat and carcass quality was detected in a commercial crossbred population: 1855 pigs sired by 17 boars from a synthetic line, which where homozygous (A/A) for IGF2. Using combined linkage and linkage disequilibrium mapping (LDLA), several QTL significantly affecting loin muscle mass, ham weight and ham muscles (outer ham and knuckle ham) and meat quality traits, such as Minolta-L* and -b*, ultimate pH and Japanese colour score were detected. These results agreed well with previous QTL-studies involving SSC2. Since our study is carried out on crossbreds, different QTL may be segregating in the parental lines. To address this question, we compared models with a single QTL-variance component with models allowing for separate sire and dam QTL-variance components. The same QTL were identified using a single QTL variance component model compared to a model allowing for separate variances with minor differences with respect to QTL location. However, the variance component method made it possible to detect QTL segregating in the paternal line (e.g. HAMB), the maternal lines (e.g. Ham) or in both (e.g. pHu). Combining association and linkage information among haplotypes improved slightly the significance of the QTL compared to an analysis using linkage information only.     

<Emphasis Type="Italic">α</Emphasis>-Amylase inhibitor-1 gene from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> expressed in <Emphasis Type="Italic">Coffea arabica</Emphasis>plants inhibits α-amylases from the coffee berry borer pest

Background  

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US70 billion per year. There are 115 species in the < i > Coffea < /i > genus, but only two, < i > C. arabica < /i > and < i > C. canephora < /i > , are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer ( < i > Hypotheneumus hampei < /i > ), is responsible for worldwide annual losses of around US70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.     

Anomalous cross-linking by mechlorethamine of DNA duplexes containing C-C mismatch pairs

Nitrogen mustards such as mechlorethamine have previously been shown to covalently cross-link DNA through the N7 position of the two guanine bases of a d[GXC].d[GYC] duplex sequence, a so-called 1,3 G-G-cross-link, when X-Y = C-G or T-A. Here, we report the formation of a new mechlorethamine cross-link with the d[GXC].d[GYC] fragment when X-Y is a C-C mismatch pair. Mechlorethamine cross-links this fragment preferentially between the two mismatched cytosine bases, rather than between the guanine bases. The cross-link also forms when one or both of the guanine bases of the d[GCC].d[GCC] fragment are replaced by N7-deazaguanine, and, more generally, forms with any C-C mismatch, regardless of the flanking base pairs. Piperidine cleavage of the cross-link species containing the d[GCC].d[GCC] sequence gives DNA fragments consistent with alkylation at the mismatched cytosine bases. We also provide evidence that the cross-link reaction occurs between the N3 atoms of the two cytosine bases by showing that the formation of the C-C cross-link is pH dependent for both mechlorethamine and chlorambucil. Dimethyl sulfate (DMS) probing of the cross-linked d[GCC].d[GCC] fragment showed that the major groove of the guanine adjacent to the C-C mismatch is still accessible to DMS. In contrast, the known minor groove binder Hoechst 33258 inhibits the cross-link formation with a C-C mismatch pair flanked by A-T base pairs. These results suggest that the C-C mismatch is cross-linked by mechlorethamine in the minor groove. Since C-C pairs may be involved in unusual secondary structures formed by the trinucleotide repeat sequence d[CCG]n, and associated with triplet repeat expansion diseases, mechlorethamine may serve as a useful probe for these structures.