Growth-promoting effects of acidic and basic fibroblast growth factor on rabbit articular chondrocytes aging in culture

Rabbit articular chondrocytes have a limited growth potential in vitro. After four passages in culture, chondrocytes have accomplished more than 50% of their life span. At this stage of culture, they are considered to be senescent-like, since a dramatic decrease in proliferative capacity and enhanced cell size and protein content are observed. These aged cells are, however, still able to respond to fibroblast growth factor (FGF). The addition of either acidic or basic FGF (10 ng/ml) to culture medium permitted an enhanced proliferation. The attenuation of FGF mitogenic activity during aging was not observed for both fractions. Moreover, when treated with acidic or basic FGF, aged chondrocytes had a smaller size and a lower protein content. The acidic FGF was less potent than the basic FGF in delaying the evolution of aged chondrocytes to senescence.     

Conserved residues of tumour necrosis factor and lymphotoxin constitute the framework of the trimeric structure

Four distinct areas of primary sequence conservation between known tumour necrosis factor and lymphotoxin polypeptides from various species can be recognized. When these amino acid sequences are highlighted in the three-dimensional structure, all are found in the same region, constituting the framework of the trimeric structure.     

Anaerobic dechlorination of 2,4-dichlorophenol in freshwater sediments in the presence of sulfate.

In the presence of added sulfate, 2,4-dichlorophenol and 4-chlorophenol were transformed stoichiometrically to 4-chlorophenol and phenol, respectively, in anaerobic freshwater lake sediments between 18 and 40 degrees C. The concomitantly occurring sulfate reduction reduced the initial sulfate concentration from 25 mM to about 6 to 8 mM and depressed methane formation.     

Assay of L-3-hydroxyacyl-coenzyme A dehydrogenase with substrates of different chain lengths

A method for assaying L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which permits rate measurements with L-3-hydroxyacyl-CoA substrates of various chain lengths at physiological pH is described. The method is based on a coupled assay system in which 3-ketoacyl-CoA compounds formed by the dehydrogenase are cleaved by 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in the presence of CoASH. The advantages of this assay method are its irreversibility and elimination of product inhibition. The assay procedure was used to determine the kinetic parameters (Km, Vmax) of pig heart L-3-hydroxyacyl-CoA dehydrogenase with several substrates of various chain lengths. The data obtained show the enzyme to be most active with medium-chain substrates whereas Km values for medium-chain and long-chain substrates are almost equal but much lower than those previously reported.     

Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pH's where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.     

A general pre-steady-state solution to complex kinetic mechanisms

We have developed a general method for solving transient kinetic equations using Laplace transforms. Laplace transforms can be used to transform systems of differential equations that describe pre-steady-state kinetics to systems of linear algebraic equations. The general form of the pre-steady-state solution is (formula; see text) where I(t) is the time dependence of the physically observed property of the system, n is the number of intermediates, lambda i are the observed rate constants (reciprocals of the relaxation times), t is time, and Ii are the amplitude coefficients associated with each observed rate constant. We have written a program in compiled BASIC to run on a personal computer to evaluate Ii and lambda i. The program will evaluate the rate constants and coefficients of a mechanism with eight intermediates and seven relaxation times in 4 s on an 8-MHz PC-XT equipped with a math coprocessor. The most complex mechanism that we have solved, a mechanism containing 20 intermediates and 19 relaxation times, required approximately 5 min. We believe that this method will be useful to evaluate the differences in transient properties of complex biochemical mechanisms.     

Biosynthesis of fusarochromanone and its monoacetyl derivative by Fusarium equiseti.

One fluorescent compound previously named TDP-2 was isolated and purified from a rice culture of Fusarium equiseti (Alaska 2-2). Mass spectral and nuclear magnetic resonance data indicated that it is a C-3'-N-acetyl derivative of fusarochromanone, a newly discovered mycotoxin. Time course studies of synthesis of these two compounds on autoclaved rice and Czapek-Dox medium enriched with soybean peptone indicated that fusarochromanone was converted to TDP-2 in the cultures. A high concentration of peptone in the liquid medium may stimulate both fusarochromanone synthesis and its conversion to TDP-2.     

Disulfide and secondary structures of recombinant human granulocyte colony stimulating factor

Molecular characteristics and secondary structures of recombinant methionyl human granulocyte colony stimulating factor produced by genetically engineered Escherichia coli are described. Limited radiolabeling of the protein with tritiated iodoacetate and determination of the labeled residue revealed that this recombinant protein contains only one free cysteine at position 17 which is not essential for activity. The free cysteine is inaccessible to modification unless the molecule is unfolded under denaturing conditions. The molecule forms two disulfide bridges which were assigned as Cys(36)-Cys(42) and Cys(64)-Cys(74) based on the results of isolation and characterization of disulfide-containing peptides obtained from a subtilisin digest of the intact protein. CD analyses and secondary structure prediction suggest that the molecule is abundant in alpha-helical structures.     

Cell swelling induced by the permeant molecules urea or glycerol induces immediate high amplitude thyrotropin and prolactin secretion by perifused adenohypophyseal cells

The permeant molecules, urea and glycerol, evoked a prompt secretory burst of TSH and PRL when added to the extracellular medium of acutely dispersed anterior pituitary cells. Secretion of both hormones was proportional to the concentration of urea or glycerol between 26 and 104 mM (r greater than 0.89, P less than 0.001). Equivalent concentrations of the impermeant molecule, mannitol, did not induce secretion. The acute TSH and PRL secretory responses to TRH, hyposmolarity, and permeant molecules were qualitatively indistinguishable. These data support our hypothesis that cell swelling and resultant plasmalemma expansion is a potent inducer of hormone secretion. Since the secretory response to permeant molecules was not reduced in a Ca2+-free medium containing 0.1 mM EGTA, an increase in Ca2+ transport across the plasmalemma to raise cytosol Ca2+ concentration does not appear involved.     

Molecular dynamics of antitumor ether-linked phospholipids in model membranes: a spin-label study

We have synthesized a spin-labeled derivative of ET-18-OCH3, a known antitumor ether-linked phospholipid. The spin-labeled analog was shown to be as potent as ET-18-OCH3 in inhibiting 3H-thymidine uptake of HL60 leukemic cells. Electron spin resonance (ESR) studies showed that the mobility of this ether-linked phospholipid in the membrane is more restricted when compared to its ester-linked counterparts. It is probable that the absence of the bulky carbonyl oxygens allows closer packing of the two alkyl chains in the ether-linked phospholipid, thereby reducing the angular amplitude of the motion of the alkyl chains. These findings may be of importance in elucidating mechanisms by which the antitumor ether-linked phospholipids perturb the structure of cellular membranes.