Transforming growth factor-beta enhances calcitonin-induced cyclic AMP production and the number of calcitonin receptors in long-term cultures of human umbilical cord blood monocytes in the presence of 1,25-dihydroxycholecalciferol.

Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide, abundant in bone, that regulates both proliferation and differentiation of a wide variety of cells, but its role in osteoclast differentiation remains controversial. We have recently shown that long-term cultures of human cord blood monocytes, in the presence of 1,25 dihydroxycholecalciferol (1,25-(OH)2D3), give rise to cells that express two markers of the osteoclast phenotype, namely, the vitronectin receptor (VNR) and the calcitonin receptor (CTR). TGF-beta enhanced the proportion of cells expressing the VNR. In the present study, we investigated the effect of TGF-beta on the expression of CTR in cord blood monocytes cultured during 3 weeks in the presence of 1,25-(OH)2D3. When added within the first 2 weeks of culture, TGF-beta (500 pg/ml) significantly decreased the cell protein content. TGF-beta alone did not stimulate basal cAMP production. The 10 nM-sCT-stimulated cAMP production was enhanced by increasing TGF-beta concentrations from 50 pg/ml to 1,000 pg/ml: for 500 pg/ml TGF-beta, it was 294 +/- 28% vs. 140 +/- 25% for control cultures (p less than 0.01). The sCT dose-response curves showed a higher cAMP production from 10(-9) M to 10(-7) M of sCT in the presence of 500 pg/ml TGF-beta than in control cultures. The increase was 325 +/- 36% in the presence of TGF-beta and 195 +/- 13% in the absence of TGF-beta, for 10(-7) M sCT (p less than 0.01). This effect of TGF-beta on cAMP production was not observed either when it was added to monocyte cultures the last day or 2 hours before the end of the culture or in MCF7, a human breast cancer cell line that expresses CTR. [125I]-sCT binding studies performed on confluent cells showed similar Kd in control and TGF-beta-treated cells. By contrast, the CTR number was significantly increased in the presence of TGF-beta: 6.1 +/- 2 x 10(4) receptors per cell in control cultures and 28.8 +/- 8.1 x 10(4) receptors per cell in TGF-beta-treated cultures (p less than 0.05). It is thus suggested that TGF-beta increases the number of CTR of these cells that have other features of preosteoclasts. The role of this cytokine on the process of osteoclast differentiation and in bone resorption is thus emphasized.     

Affinity Purification of Human τ Proteins and the Construction of a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay for Human τ Detection

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.     

Pattern formation in one- and two-dimensional shape-space models of the immune system.

A large-scale model of the immune network is analyzed, using the shape-space formalism. In this formalism, it is assumed that the immunoglobulin receptors on B cells can be characterized by their unique portions, or idiotypes, that have shapes that can be represented in a space of a small finite dimension. Two receptors are assumed to interact to the extent that the shapes of their idiotypes are complementary. This is modeled by assuming that shapes interact maximally whenever their coordinates in the space-space are equal and opposite, and that the strength of interaction falls off for less complementary shapes in a manner described by a Gaussian function of the Euclidean "distance" between the pair of interacting shapes. The degree of stimulation of a cell when confronted with complementary idiotypes is modeled using a log bell-shaped interaction function. This leads to three possible equilibrium states for each clone: a virgin, an immune, and a suppressed state. The stability properties of the three possible homogeneous steady states of the network are examined. For the parameters chosen, the homogeneous virgin state is stable to both uniform and sinusoidal perturbations of small amplitude. A sufficiently large perturbation will, however, destabilize the virgin state and lead to an immune reaction. Thus, the virgin system is both stable and responsive to perturbations. The homogeneous immune state is unstable to both uniform and sinusoidal perturbations, whereas the homogeneous suppressed state is stable to uniform, but unstable to sinusoidal, perturbations. The non-uniform patterns that arise from perturbations of the homogeneous states are examined numerically. These patterns represent the actual immune repertoire of an animal, according to the present model. The effect of varying the standard deviation sigma of the Gaussian is numerically analyzed in a one-dimensional model. If sigma is large compared to the size of the shape-space, the system attains a fixed non-uniform equilibrium. Conversely if sigma is small, the system attains one out of many possible non-uniform equilibria, with the final pattern depending on the initial conditions. This demonstrates the plasticity of the immune repertoire in this shape-space model. We describe how the repertoire organizes itself into large clusters of clones having similar behavior. These results are extended by analyzing pattern formation in a two-dimensional (2-D) shape-space.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thermostability of soluble and immobilized alpha-amylase from Bacillus licheniformis

In view of a possible application of the alpha-amylase from Bacillus licheniformis as a time-temperature integrator for evaluation of heat processes,(11) thermal inactivation kinetics of the dissolved and covalently immobilized enzyme were studied in the temperature range 90-108 degrees C. The D-values (95 degrees C) for inactivation of alpha-amylase, dissolved in tris-HCl buffer, ranged from 6 to 157 min, depending on pH, ionic strength, and Ca(2+) and enzyme concentration. The z-value fluctuated between 6.2 and 7.6 degrees C. On immobilization of the alpha-amylase by covalent coupling with glutaraldehyde to porous glass beads, the thermoinactivation kinetics became biphasic under certain circumstances. For immobilized enzyme, the D-values (95 degrees C) ranged between 17 and 620 min, depending largely on certain environmental conditions. The z-value fluctuated between 8.1 and 12.9 degrees C. In each case of biphasic inactivation, the z-value of the stable fraction (with the higher D-values) was lower than the z-value of the labile fraction. (c) 1992 John Wiley & Sons, Inc.     

Effect of serum concentration on hybridoma viable cell density and production of monoclonal antibodies in CSTRs and on shear sensitivity in air-lift loop reactors

The death rate of hybridoma cells, grown in a continuous culture, has been studied in a small air-lift loop reactor as a function of reactor height and injected gas flow rate. The first-order death-rate constant was found to be proportional to the reciprocal height and to the gas flow rate, in accordance with the hypothetical killing volume model for insect cells in bubble columns. Furthermore, the effect of the serum concentration on viable cell concentration and cell productivity has been investigated in a continuous culture. A serum component became growth limiting when the serum concentration was decreased from 2% to 1%. No effect of the serum concentration on specific cell productivity could be measured. Samples from this culture were also studied in the air-lift loop reactor to determine the effect of serum concentration on the shear sensitivity. The cells' shear sensitivity increased with decreasing serum concentration. The protective effect of serum was found to be physical as well as physiological.     

Tissue distribution of constitutive and induced soluble peroxidase in rat. Purification and characterization from lacrimal gland.

A thorough search for a soluble peroxidase in 31 different tissues of rat indicated the presence of a constitutive activity only in lacrimal, preputial and submaxillary gland. An induced soluble peroxidase activity was also detected in the lactating mammary gland and in the estrogen-induced uterine secretory fluid. The lacrimal gland was the richest source of the enzyme. No peroxidase activity was detected in the lactating mammary gland of mouse and hamster nor in the preputial gland of mouse and uterine fluid of hamster. The three constitutive and two induced soluble peroxidases of rat had a native molecular mass of 73 kDa by gel filtration and they showed a similar mobility in native PAGE. Lactoperoxidase of cow's milk and solubilized rat membrane-bound peroxidases of uterus, intestine and bone marrow showed in native PAGE a mobility which was distinctly different from that of rat soluble peroxidases. As the lacrimal gland of rat was the richest source of soluble peroxidase, the enzyme was purified from this gland to apparent homogeneity; SDS/PAGE then showed a single band of molecular mass 75 kDa which was similar to that obtained by gel filtration. Peroxidase also purified from preputial and submaxillary gland, as well as commercial lactoperoxidase, had a similar molecular mass on SDS/PAGE to purified lacrimal peroxidase. The visible spectrum of lacrimal peroxidase was similar to that of lactoperoxidase but different from membrane-bound peroxidase of rat neutrophils. On isoelectric focussing, purified lacrimal peroxidase resolved into about 14 multiple forms spanning a pI range of 6.5-3.5 while lactoperoxidase focussed at the cathode. Evidence presented suggests that the multiple forms are possibly due to differences in glycosylation. Immunodiffusion, immunoprecipitation and Western blot using antilacrimal peroxidase serum showed a similar interacting species for all five soluble peroxidases of rat while membrane-bound peroxidases showed no interaction. Although in immunodiffusion, the antiserum failed to cross-react with lactoperoxidase it did interact with lactoperoxidase on Western blot. The results indicate that the various constitutive and induced soluble peroxidases of rat tissues are similar to lacrimal peroxidase but are distinctly different from the known membrane-bound peroxidases of rat. However the lacrimal peroxidase shows both similarities as well as dissimilarities with bovine lactoperoxidase. This soluble peroxidase system of rat could be useful to study tissue-specific regulation of gene expression at the molecular level.     

The 70-kilodalton heat-shock proteins of the SSA subfamily negatively modulate heat-shock-induced accumulation of trehalose and promote recovery from heat stress in the yeast, Saccharomyces cerevisiae.

In the yeast, Saccharomyces cerevisiae, the disaccharide trehalose is a stress-related metabolite that accumulates upon exposure of cells to heat shock or a variety of non-heat inducers of the stress response. Here, we describe the influence of mutations in individual heat-shock-protein genes on trehalose metabolism. A strain mutated in three proteins of the SSA subfamily of 70-kDa heat-shock proteins (hsp70) overproduced trehalose during heat shock at 37 degrees C or 40 degrees C and showed abnormally slow degradation of trehalose upon temperature decrease from 40 degrees C to 27 degrees C. The mutant cells were unimpaired in the induction of thermotolerance; however, the decay of thermotolerance during recovery at 27 degrees C was abnormally slow. Since both a high content of trehalose and induced thermotolerance are associated with the heat-stressed state of cells, the abnormally slow decline of trehalose levels and thermotolerance in the mutant cells indicated a defect in recovery from the heat-stressed state. A similar albeit minor defect, as judged from measurements of trehalose degradation during recovery, was detected in a delta hsp104 mutant, but not in a strain deleted in the polyubiquitin gene, UB14. In all our experiments, trehalose levels were closely correlated with thermotolerance, suggesting a thermoprotective function of trehalose. In contrast, heat-shock proteins, in particular hsp70, appear to be involved in recovery from the heat-stressed state rather than in the acquisition of thermotolerance. Cells partially depleted of hsp70 displayed an abnormally low activity of neutral trehalase when shifted to 27 degrees C after heat shock at 40 degrees C. Trehalase activity is known to be under positive control by cAMP-dependent protein kinases, suggesting that hsp70 directly or indirectly stimulate these protein-kinase activities. Alternatively, hsp70 may physically interact with neutral trehalase, thereby protecting the enzyme from thermal denaturation.