Direct microsensor measurement of nitric oxide production by the osteoclast.

Nitric oxide (NO) triggers marked osteoclast retraction which closely resembles that due to Ca2+. The effect of Ca2+ has been attributed to a stimulated release of NO. Here, we show for the first time, by direct measurement with a microsensor, that osteoclasts do indeed produce NO and that this production is enhanced by a high Ca2+. We also show that the Ca2+ ionophore, A23187, mimics the latter. Furthermore, osteoclasts on dentine produce more NO than osteoclasts on glass and NO release from dentine-plated osteoclasts is much less sensitive to stimulation by Ca2+. Finally, the microsomal Ca2+ store-depleting agent, thapsigargin, attenuates NO release only from osteoclasts on glass, suggesting that stored Ca2+ has the dominant effect in modulating NO release from non-resorbing cells. NO is a powerful inhibitor of bone resorption: a direct demonstration of its production is therefore strong evidence for a role in modulating osteoclast function.     

Continuous bioprocessing: The real thing this time?: 10th Annual bioProcessUK Conference,December 3–4, 2013, London,UK

The Annual bioProcessUK Conference has acted as the key networking event for bioprocess scientists and engineers in the UK for the past 10 years. The following article is a report from the sessions that focused on continuous bioprocessing during the 10^th Annual bioProcessUK Conference (London, December 2013). These sessions were organized by the ‘EPSRC Centre for Innovative Manufacturing in Emergent Macromolecular Therapies’ hosted at University College London. A plenary lecture and workshop provided a forum for participants to debate topical issues in roundtable discussions with industry and academic experts from institutions such as Genzyme, Janssen, Novo Nordisk, Pfizer, Merck, GE Healthcare and University College London. The aim of these particular sessions was to understand better the challenges and opportunities for continuous bioprocessing in the bioprocessing sector.     

Inbreeding under a cyclical mating system

Summary General recursion formulae for the coefficient of inbreeding under a cyclical mating system were derived in which one male and one female are selected from each of the n families per generation (population size N = 2 n). Each male is given the family number of his sire in each generation, while his mate comes from another family, varying systematically in different generations. Males of the r-th family in generations 1, 2, 3,..., t = n–1 within each cycle mate with females from families r+1, r+2, r+3,..., r+t to produce generations 2, 3, 4,..., t+1=1, respectively. The change in heterozygosity shows a cyclical pattern of rises and falls, repeating in cycles of n–1 generations. The rate of inbreeding oscillates between <-3% to >6% in different generations within each cycle, irrespective of the population size. The average rate of inbreeding per generation is approximately 1/[4 N-(Log₂N+1)], which is the rate for the maximum avoidance of inbreeding. The average inbreeding effective population size is approximately 2 N–2.     

Characterization of a heterogeneous camel milk whey non-casein protein

A milk protein, occurring in the whey fraction, has been characterized from camel milk. Determination of the primary structure reveals the existence of two related types of chain with residue differences in at least the N-terminal region. A fragment representing an N-terminal part of the protein was also recovered (heterogeneous at the same positions). The absence of cysteine residues in the protein shows that no disulphide bridges are present. The pattern of fragments and a parent protein resembles that for casein and its fragments, showing that fragments and a multiplicity of forms may be typical for different milk proteins.     

Use of anti-idiotypic antibodies as probes for the interaction of TSH subunits with its receptor

TSH is a heterodimeric glycoprotein hormone, whose dissociated subunits are without biological activity. This has precluded the assessment of the relative contribution of each subunit to hormone action. We have raised anti-idiotypes to monoclonal antibodies specific, respectively, for the alpha and beta hTSH subunits. The anti-beta anti-idiotype inhibited 125I-hTSH binding to the beta subunit-specific monoclonal quantitatively, whereas 125I-hTSH binding to the alpha subunit-specific monoclonal was not inhibited by anti-alpha anti-idiotypes, suggesting that only the former is an "internal image" anti-idiotype. Neither of the two anti-idiotypes nor equimolar mixtures thereof inhibited 125I-bTSH binding to thyroid membranes, even though radiolabelled anti-idiotypes showed saturable binding to thyroid plasma membrane which was inhibited 41-65% by bTSH. Each anti-idiotype alone caused 9% inhibition (compared to 50% by NRIgG) of thyroid plasma membrane adenylate cyclase. Equimolar mixtures (125 micrograms/ml IgG of each anti-idiotype) induced enzyme activity equivalent to 85% of that of 250 mU/ml of TSH. The TSH-like action of the two anti-idiotypes was also reflected in their capacity to increase (450% by 250 micrograms/ml IgG compared to normal rabbit IgG) the uptake of 131I into isolated thyrocytes and to promote the organization of such cells into follicular structures. At 250 micrograms/ml, anti-beta anti-idiotype promoted the organization of small follicles and only at a concentration of 500 micrograms/ml did it enhance 131I uptake.     

Primary structure of the hemoglobin α-chain of rose-ringed parakeet (Psittacula krameri)

The structure of the hernoglobin α-chain of Rose-ringed Parakeet was determined by sequence degradations of the intact subunit, the CNBr fragments, and peptides obtained by digestion with staphylococcal Glu-specific protease and trypsin. Using this analysis, the complete α-chain structure of 21 avian species is known, permitting comparisons of the protein structure and of avian relationships. The structure exhibits differences from previously established avian α-chains at a total of 61 positions, five of which have residues unique to those of the parakeet (Ser-12, Gly-65, Ser-67, Ala-121, and Leu-134). The analysis defines hemoglobin variation within an additional avian order (Psittaciformes), demonstrates distant patterns for evaluation of relationships within other avian orders, and lends support to taxonomic conclusions from molecular data.     

Primary structure of hemoglobin β-chain fromColumba livia (gray wild pigeon)

Primary structure of -chain of pigeon is presented. It was determined by amino acid sequence analysis of intact -chain and its peptides obtained by the enzymatic and chemical cleavage. Comparison of amino acid sequence of the chain with other available data shows 14 Ile, 61 Lys, and 113 Ile as residues specific to pigeon. One important replacement at ₁₁ contact is 55 MetSer.     

Microtubule-associated protein tau. A component of Alzheimer paired helical filaments

Microtubule-associated protein tau was purified from bovine brain microtubules by either (1) phosphocellulose chromatography, (2) heat treatment at pH 6.4, (3) heat treatment at pH 2.7, (4) heat treatment at pH 2.7 followed by extraction with perchloric acid and precipitation with glycerol, or (5) by precipitation with ammonium sulfate followed by extraction with perchloric acid. All of these tau preparations reacted specifically with antibodies to Alzheimer paired helical filaments. Affinity purified antibodies to tau labeled both Alzheimer neurofibrillary tangles and plaque neurites but not amyloid in Alzheimer brain tissue sections and labeled paired helical filament polypeptides on Western blots. Human brain tau and paired helical filament polypeptides co-migrated on sodium dodecyl sulfate-polyacrylamide gels. These results suggest that tau is a major component of Alzheimer paired helical filaments.     

Isolation of the first trypsin inhibitor from the genusAspergillus

The fungusAspergillus flavipes was grown on a Czapeck sucrose medium; the biomass so obtained was treated with high concentration of sucrose to release intracellular metabolites. Sephadex G-75 chromatography of the latter yielded a pure protein having anti-trypsin activity in vitro.