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Strategies to promote intervertebral disc (IVD) regeneration have been hindered by the lack of knowledge of IVD fundamental cellular/molecular components. One of the key points to be addressed is the characterization of nucleus pulposus (NP) cell population(s). This study establishes an improved method for bovine NP (bNP) cell isolation, whose procedure is still not consensual among the literature, allowing a thorough characterization of cell (sub)populations that exist in the young NP.bNP was digested with distinct enzymes (collagenase-type-I, collagenase-type-II, and collagenase-type-XI) at different concentrations (0.5, 1.0, and 2.0 mg/mL), for 4 and 19 h. Cell yield, viability/apoptosis, and morphology were analyzed by flow cytometry and imaging flow cytometry. Identification of cell subpopulations within NP and its phenotype was investigated by assessing expression of CD29, CD44, CD45, CD34, CD146, and Brachyury.It was found that bNP cells present a similar morphology independently of the digestive enzyme used. However, cell yield was greatly improved by Coll-XI (2 mg/mL) treatment for a short digestion period. Interestingly, three subpopulations, with different sizes and auto-fluorescence, were consistently identified by flow cytometry. And crucially, differential expression of cell markers was found among these subpopulations.This study demonstrated that collagenase-type-XI is an efficient enzyme that is used for digesting bNP. And most importantly, three phenotypically distinct subpopulations of cells where identified within the bNP. Such knowledge is key for a better understanding of NP cell biology and its potential endogenous regenerative capacity.

In order to analyze the RNA populations present in different cells of very early embryos, we have developed a protocol to purify these large blastomeres using counterflow centrifugal elutriation (CCE). This procedure employs ethanol fixation to stabilize the cells against shear forces encountered during CCE. Using this method, we fractionated the three different blastomere types of the 16-cell sea urchin embryo, the micromeres, mesomeres, and macromeres, achieving 96, 94, and 96% mean purities, respectively. We show here that intact RNA is recovered with equal efficiency from each blastomere preparation. Using this method, we have identified several RNAs that are distributed non-uniformly among these cells.

S-Adenosyl-L-homocysteine hydrolase from mouse leukemic cells L1210 was purified to evident homogeneity with no concomitant enzymatic activities. The isoelectric point of the enzyme is 5.65 and the optimum temperatures for the synthesis and hydrolysis are 52 °C and 47 °C, respectively. The respective KM values for adenosine and S-adenosyl-L-homocysteine (SAH) are 3.8 and 4.4 μmol l-1. The optimum pH value for the synthesis of SAH is 6.95-7.20 and for the hydrolysis 7.15-7.80. The enzyme is competitively inhibited by 9-(S)-(2,3-dihydroxypropyl)adenine and inactivated with both enantiomers of eritadenine and 3-(adenin-9-yl)-2-hydroxypropanoic acid.

1. A purified fraction of ribonuclease-resistant RNA isolated from rat liver whole cells and nuclei was chromatographed on cellulose columns, and a double-stranded RNA fraction isolated. 2. The double-stranded structure of the RNA was firmly defined by its behaviour on cellulose, its symmetrical base composition, immunoprecipitin reaction, and its characteristic biphasic heat denaturation curve. 3. A nuclear origin for double-stranded RNA was established. It can be synthesized in the presence of actinomycin D. No significant quantities could be detected in the cytoplasm.

The cytoplasm of cells infected with rovirus contains 8 of the 10 polypeptides coded by this virus in free form in amounts equivalent to 1 to 10% of the progeny virus yield. All of these polypeptides exist in monomeric form except polypeptides μ1 and μ2 which occur as dimers, the form in which they are present in virions. One of these polypeptides, σ3, a constituent of the outer capsid shell, binds strongly to double-stranded RNA; it can be isolated in high yield in pure form by passing extracts of infected cells through columns of poly(IC)-cellulose and eluting. Another polypeptide, σ2A, a nonstructural polypeptide, binds strongly to single-stranded RNA and appears to exist in infected cells complexed with RNA. It can be isolated in high yield in pure form by passing extracts of infected cells, suitably treated to dissociate these complexes, through columns of poly(A)-cellulose and eluting. The possible significance of the highly specific nucleic acid affinities of these two reovirus-coded polypeptides is discussed.