Delineation of type-specific regions on the envelope glycoproteins of human T cell leukemia viruses.

Two different approaches were used to map the type-specific regions on human T cell leukemia virus (HTLV) envelope glycoproteins. 1) Antibody reactivities of polymerase chain reaction-confirmed HTLV-I or HTLV-II carriers' sera were analyzed by Western blot assay with seven recombinant proteins containing different regions of HTLV-I or HTLV-II envelope proteins. 2) Rabbit antibodies elicited by nine HTLV-I Env synthetic peptides were used to react with the native HTLV envelope glycoproteins in an antibody-dependent cellular cytotoxicity (ADCC) assay. The results of the Western blot analysis showed that RP-B2, which contains amino acid residues 166 to 213 from HTLV-II exterior glycoprotein, was specifically reactive with 90.6% (48 of 53) of the HTLV-II carriers' sera but not with any of the HTLV-I carriers' serum (0 of 71). In contrast, RP-B, which contains amino acid residues 166 to 229 from HTLV-I exterior glycoprotein, was reactive with 85.1% (114 of 134) of the HTLV-I carriers' sera but not with any HTLV-II carrier serum (0 of 62). Furthermore, anti-HTLV-I Env synthetic peptide antibody-mediated ADCC identified several distinguishing HTLV-I ADCC epitopes in the middle region (amino acid residues 177 to 257) of the HTLV-I exterior glycoprotein. Therefore, HTLV type-specific epitopes reside mainly in a 69-amino acid sequence bounded by two cysteine residues (amino acids 157 and 225 for HTLV-I and 153 and 221 for HTLV-II), in the middle region of the exterior envelope glycoproteins.     

IFN-alpha induces autocrine production of IL-6 in myeloma cell lines.

IL-6 is a major tumor growth factor in human multiple myeloma. Myeloma cell lines, which have the same phenotypic characteristics and Ig gene rearrangements as the original fresh myeloma cells and whose growth is strictly dependent on exogenous IL-6 similar to fresh myeloma cells, have been reproducibly established. We show here that IFN-alpha stimulated the growth of five of six of these human myeloma cell lines by inducing an autocrine production of IL-6 in myeloma cells. Indeed, IFN-alpha induced IL-6 mRNA accumulation and IL-6 production in myeloma cells and the IFN-alpha-induced growth of these cells was inhibited by anti-IL-6 mAb. Moreover, IFN-alpha made possible the rapid emergence of autonomously growing myeloma cell sublines, which produced IL-6 as an autocrine growth factor. As IFN-alpha has a potential therapeutical interest for multiple myeloma, the present study opens up new directions for studying its effects on the myeloma clone in vivo.     

Binding and complement activation by C-reactive protein via the collagen-like region of C1q and inhibition of these reactions by monoclonal antibodies to C-reactive protein and C1q

Ligand-complexed C-reactive protein (CRP), like aggregated or complexed IgG, can react with C1q and activate the classical C pathway. Whereas IgG is known to bind to the globular region and not to the collagen-like region (CLR) of C1q, the site of interaction of C1q with CRP has not been defined. CRP-trimers were prepared by cross-linking and found to bind to C1q and to activate the C system. Heat-aggregated IgG (Agg-IgG) did not block the binding of CRP-trimers to C1q, nor did CRP-trimers block binding of Agg-IgG to C1q, suggesting that CRP and IgG bind at different sites. ELISA and Western blot analysis showed that CRP-trimers bound to the CLR, whereas Agg-IgG bound only to the globular region; similarly, anti-CLR mAb inhibited binding of CRP-trimers to C1q whereas anti-globular region mAb did not. Reactivity with CRP-trimers as well as with Agg-IgG was retained after reduction/alkylation and SDS treatment of C1q. A group of 22 anti-CRP mAb directed against at least six distinct native-CRP epitopes and eight distinct neo-CRP epitopes was tested for ability to inhibit the CRP-CLR interaction; one mAb, anti-native CRP mAb 8D8, with strong inhibitory activity was identified. Fab' of 8D8 blocked binding of CRP-trimers to intact C1q as well as CLR, and also inhibited CRP (CRP-trimers and CRP-protamine complexes) induced C activation, but had no effect on C1q binding or C activation by Agg-IgG. These results indicate that a conformation-determined region on CRP binds to a sequence-determined region on the CLR of C1q in an interaction which leads to C activation. Anti-CRP and anti-C1q mAb that specifically inhibit this interaction are described.     

Chemical modification of Bacillus thuringiensis subsp. thuringiensis (HD-524) trypsin-activated endotoxin: implication of tyrosine residues in lepidopteran cell lysis

A purified protein fraction from a solubilized and trypsin-digested extract of Bacillus thuringiensis subsp. thuringiensis (HD-524) fermentation powder was lytic to cells from several lepidopteran lines. Maximum yield was obtained by alkaline carbonate-thiocyanate solubilization of washed powder followed by trypsin digestion and Sephacryl (S-300) chromatography. The alkaline carbonate-solubilized fraction consisted predominantly of two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with MW of 144 +/- 0.9 kDa and 134 +/- 1.4 kDa. After trypsin treatment and column chromatography, the cytolytic fraction consisted of a major band with a MW of 60.0 +/- 1.8 kDa and a minor band of 69 +/- 0.9 kDa. Cells from Trichoplusia ni (TN368) were most susceptible to lysis with 50% of cells lysed at 3 micrograms/ml, followed by Spodoptera frugiperda cells (SF21AE) exhibiting 50% cell lysis at 5 micrograms/ml and Lymantria dispar cells (Ld652Y) showing 40% lysis at 10 micrograms/ml. Chemical modification of the polypeptides was performed to determine the role of certain amino acid residues in the cytolytic activity. The group-specific reagent tetranitromethane was used to nitrate and oxidize tyrosine and cysteine residues, respectively. Cysteine residues alone were also modified with p-hydroxymercuribenzoic acid. Lysine residues were modified with O-methylisourea. Of the three types of amino acid residues, only the modification of tyrosine resulted in reduced cell lysis.     

Adaptive control of dissolved oxygen concentration in a bioreactor

A new adaptive DO (dissolved oxygen) concentration control algorithm considering DO electrode dynamics with response time delay has been developed. A system model with two time-varying parameters was used to relate the DO concentration with two control variables: air flow rate and agitation speed. Parameters of this model were estimated on-line using a regularized constant trace recursive least-squares method. An extended Kalman filter was used to remove the effect of noises from the DO concentration measurements and thus to improve control performance. A discrete one-step ahead control scheme was adopted to determine control actions based on the parameter estimation results. Experimental results showed that the new adaptive DO concentration control algorithm performed better than other algorithms tested, a PID controller and adaptive algorithms without the DO electrode dynamics.     

An alternate method for estimation of cell growth kinetics from batch cultures

An alternative to estimation of cell growth kinetics via continuous culture experiments is proposed in this article. The method employed is based on batch culture experiments with very small inocula (initial cell concentrations being typically less than 5000 cells/mL). Such low initial cell concentrations result in extended exponential cell growth phase during which culture conditions remain unchanged, thereby permitting precise estimation of specific cell growth rates from batch experiments especially for fast-growing microorganisms such as Bacillus species. The effectiveness and utility of this approach are demonstrated via several experiments conducted with a wild-type strain (Bacillus subtilis TN106) and a recombinant strain (B. subtilis TN106[pAT5]). True establishment of exponential growth phase requires insignificant variance of most of the culture conditions during the initial growth phase. Satisfaction of this requirement is demonstrated for microbial systems investigated here. This approach is especially well suited for recombinant microorganisms containing segregationally unstable plasmids, since estimation of growth kinetics of these from continuous cultures is very difficult and highly unreliable due to continual reversion of recombinant ceils to plasmid-free host cells unless some selection pressure is applied at levels sufficient to keep the presence of plasmid-free cells minimal.     

IL-4 down-regulates the expression of J11d on murine B cells.

T cell-derived IL-4 has many effects on murine B cells, including the up-regulation of class II antigens and the induction of isotype switching. The development of memory B cells and the decreased expression of J11d antigens on these cells are influenced by T cells. In this report, we determined whether the decreased expression of J11d can also be mediated by T cell-derived lymphokines and, in particular, IL-4. We found that IL-4 can down-regulate the expression of J11d on both large and small B cells, that this effect becomes significant after 48 hr of culture and occurs at doses of IL-4 that are similar to those required to up-regulate murine class II MHC antigens encoded by I-region alpha genes (IA). Anti-IL-4 antibody completely blocks this effect but IFN-gamma does not. Other lymphokines (IL-1, IL-2, IL-3, IL-5, IL-6) neither induce a decrease in J11d nor alter the ability of IL-4 to down-regulate J11d expression. The decrease in J11d expression on B cells is not due to a preferential survival of cells expressing lower levels of J11d, although IL-4 has a more pronounced effect on these B cells. Finally, the down-regulation of J11d and up-regulation of IA by IL-4 occurs on all inducible B cells.     

Extracellular matrix (mesoglea) of Hydra vulgaris. II. Influence of collagen and proteoglycan components on head regeneration.

Hydra are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. As an extension of the previous study which indicated that mesoglea is a primitive basement membrane which has retained some characteristics of interstitial extracellular matrix, the present study was undertaken to analyze the role of mesoglea components during head regeneration in Hydra vulgaris. Studies were conducted that utilized drugs that affect collagen processing or secondary collagen structure (beta-aminoproprionitrile; 2,2'-dipydridyl; and cis-4-hydroxy-L-proline) and a drug that inhibits addition of glycosaminoglycan chains to proteoglycan core proteins (p-nitrophenyl-beta-D-xylopyranoside). These studies indicated that alterations in the structure of collagens or proteoglycans caused blockage of head regeneration in Hydra as monitored over a 48-hr period. Blockage of head regeneration was reversible once the drugs were removed, indicating that the drugs were not having a general toxic effect on the organism. Radiotracer studies also indicated that blockage of head regeneration was not simply due to a general depression of protein synthesis by the drugs. Various controls indicated that each drug was affecting mesoglea components under the conditions utilized in these studies. These observations indicate that preservation of normal mesoglea structure is required for Hydra head regeneration to proceed.     

Inositol 1,4,5-triphosphate phosphatase activity in membranes isolated from amphibian skeletal muscle [corrected

The hydrolysis of [3H]inositol 1,4,5-trisphosphate by a soluble fraction and by isolated transverse tubule and sarcoplasmic reticulum membranes from frog skeletal muscle was studied. Transverse tubule membranes displayed rates of hydrolysis several-fold higher than those of sacroplasmic reticulum and soluble fraction; Km and Vmax were 25.2 microM and 44.1 nmol/mg/min, respectively. Transverse tubule membranes sequentially hydrolyzed inositol trisphosphate to inositol bisphosphate, inositol 1-phosphate and inositol, indicating that these membranes have inositol bis- and monophosphatases in addition to inositol trisphosphatase.     

Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase

The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisCO) were cloned from the obligate autotroph Thiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30-bp intergenic region, the smallest described for the RuBisCO genes. The rbcL and rbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes for rbcL and rbcS found in other species demonstrated that the T. ferrooxidans genes have the closest degree of identity with those of Chromatium vinosum and of Alvinoconcha hessleri endosymbiont. Both T. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.