Sodium periodate stimulation of human lymphocytes : A comparison between normal and chronic lymphocytic leukemia lymphocytes

Lymphocytes from healthy donors and from patients with chronic lymphocytic leukemia (CLL) were stimulated to divide with sodium periodate. The time of maximal response of normal lymphocytes to sodium periodate (NaIO₄) was earlier than that observed to phytohemagglutinin (PHA), but the magnitude was lower. In comparison, CLL lymphocytes responded to NaIO₄ more extensively and earlier than to PHA.     

Postnatal gender-dependent maturation of cellular cysteine uptake

Background. In view of the functional capacity of glutathione synthesis in premature infants, and because the availability of cysteine is one the rate limiting steps in glutathione synthesis, we hypothesized that the low glutathione levels in premature infants may be due to immaturity of the active cellular uptake of cysteine.

Objective. To document in cells from newborn infants the effect of maturity and gender on cysteine uptake and consequently on glutathione levels.

Methods. Incorporation of L-[35S] cysteine was measured in leukocytes from cord blood and from tracheal aspirates (TAC) of newborn infants of varying (gestational as well as postnatal) ages and gender. Cysteine uptake was correlated with glutathione in TAC.

Results. The maturity of newborn girls positively influences cysteine uptake, which is responsible for 78% of the variation in their glutathione content. However, in newborn boys, gestational and postnatal ages did not influence the cysteine uptake.

Discussion. Cysteine uptake appears to be the limiting step explaining the reported gender-related differences in glutathione as well as the low levels of this central antioxidant found in premature infants. The immature cysteine uptake found in cells from premature infants raises questions about the bioavailability of this conditionally essential amino acid in regimens of parenteral nutrition for human neonates.     

A sensitive,fluorometric method for the assay of microsomal hydroxylase: N-demethylation of p-chloro-N-methylaniline

A fluorometric method for the assay of microsomal hydroxylase activity is described. N-Demethylation of p-chloro-N-methylaniline yields p-chloroaniline, which is coupled with fluorescamine, extracted with ethylacetate, and measured fluorometrically. This method can determine low levels of N-demethylase activity.     

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H_I and H_IV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca²⁺ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca²⁺ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca²⁺ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca²⁺. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.     

L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.     

Characterization of ferredoxins on a nanomole scale

Two method are described for the characterization of ferredoxins. First, mapping of tryptic peptides from 2 to 3 nmol of carboxymethylated ferredoxin by two-dimensional thin-layer electrophoresis and chromatography. Second, gel electrophoresis of tryptic digests of apoferredoxins. The latter method discriminates between ferredoxins of closely related species.     

Cloning of murine transformed cell lines in suspension culture with efficiencies near 100%

Addition of 3 × 10⁶ thymus cells from either syngeneic, allogeneic or xenogeneic animals increases the cloning efficiencies of murine thymomas (EL-4, WC-2), B-lymphomas (McPC 1748, 38C-13), Abelson-virus transformed cell lines (F and K), mastocytomas (P815), myelomas (AbPC22, X63-AG8, 5563, MOPC 104 E, RFC 5, W 3469) and hybrids of myelomas and normal B-lymphocytes (Sp-1), all adapted to tissue culture, to near 100%. Thymus cells also increase the efficiencies of growth initiation in primary in vitro cultures of myeloma tumor cells (S117) transplanted in vivo, and of cells fused between the azaguanine-resistant X63-AG8 myeloma cell line and normal, LPS-stimulated B-lymphocyte blasts.