Transfectomas expressing both secreted and membrane-bound forms of chimeric IgE with anti-viral specificity.

Because of the lack of a cell line expressing on surface and secreting human IgE of known Ag specificity, the construction of a transfectoma line possessing such properties would be useful for studying the roles of surface IgE and the effects of anti-IgE antibodies on IgE-producing B cells. Toward this goal, the human genomic DNA segment encompassing the two exons encoding the membrane anchor peptide of epsilon-chain and their flanking regions was sequenced. Hybrid epsilon and kappa genomic DNA comprising the C regions of human epsilon- and kappa-chains and the H and L chain V regions of the murine mAb BAT123, which reacts with the gp120 envelope protein of HIV-1, were constructed. Mammalian expression vectors containing these fusion genes were used to transfect murine myeloma Sp2/0 cells, and transfectants stably expressing on surface and secreting into culture medium chimeric IgE were obtained. The chimeric IgE showed identical Ag-binding properties as the murine mAb BAT123. Acting in concert with the specific peptide Ag polyvalently coupled to a protein carrier, the chimeric antibody could induce histamine release from human blood basophils. These results demonstrate the potential utility of the transfectoma cells and the chimeric IgE in studying the roles of membrane-bound IgE and effects of anti-IgE antibodies on IgE-producing B cells.     

Elucidation of the subunit orientation in CCT (chaperonin containing TCP1) from the subunit composition of CCT micro-complexes.

A collection of chaperonin containing TCP1 (CCT) micro-complexes that are comprised of subsets of the constitutively expressed CCT subunits have been identified. These CCT micro-complexes have mol. wts ranging from 120 to 250 kDa and are present in cells at lower abundance (<5%) as compared with intact CCT. Biochemical characterization of these microcomplexes has shown that several are comprised of two different types of CCT subunit. Furthermore, it was observed that each subunit associates with only one or two other different types of subunit, suggesting that each subunit has fixed partners. This observation, together with CCT gene counting being concordant with the 8-fold structural symmetry, is consistent with predictions derived from analysis of the primary structures of these subunits concerning inter-subunit interactions, and implies a unique topology of the subunits constituting the torodial ring in CCT. The series of subunit-subunit association patterns determined from CCT micro-complexes has provided information to infer, from the 5040 (7!factorial) combinatorial possibilities, one probable subunit orientation within the torodial ring.     

Ability of modified forms of phenylalanine tRNA to stimulate guanosine pentaphosphate synthesis by the stringent factor-ribosome complex of E. coli

tRNA(Phe) of E. coli, modified at its 4-thiouridine ((4)Srd) and 3-(3-amino-3-carboxypropyl)uridine (nbt(3)Urd) residues, was tested for its ability to induce (p)ppGpp synthesis. The (4)Srd residue was derivatized with the p-azido-phenacyl group, cross-linked to Cyd(13), and the borohydride reduction product of the cross-link was prepared. The nbt(3)Urd residue was derivatized with the N-(4-azido-2-nitrophenyl)glycyl group. None of these derivatives had more than a minor effect on the affinity of the tRNA for the stringent factor-ribosome complex, and no effect at all on the maximum velocity of (p)ppGpp synthesis, either at 2 or 82 mM NH(4)Cl. These two regions of the tRNA which are on opposite faces of the tRNA molecule do not appear to be structurally important for recognition by the stringent factor-ribosome complex. They may provide useful sites, therefore, for the introduction of photoaffinity or fluorescent probes with which to study tRNA-stringent factor recognition.     

Covalent cross-linking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid.

The covalent cross-linking of unmodified Escherichia coli N-acetylvalyl-tRNA to the 16S RNA of Escherichia coli ribosomes upon near-UV irradiation previously reported by us [Schwartz, I., & Ofengand, J. (1978) Biochemistry 17, 2524--2530] has been studied further. Up to 70% of the unmodified tRNA, nonenzymatically bound to tight-couple ribosomes at 7 mM Mg2+, could be cross-linked by 310--335-nm light. Covalent attachment was solely to the 16S RNA. It was dependent upon both irradiation and the presence of mRNA but was unaffected by the presence or absence of 4-thiouridine in the tRNA. The kinetics of cross-linking showed single-hit behavior. Twofold more cross-linking was obtained w-th tight-couple ribosomes than with salt-washed particles. Puromycin treatment after irradiation released the bound N-acetyl[3H]valine, demonstrating that the tRNA was covalently bound at the P site and that irradiation and covalent linking did not affect the peptidyl transferase reaction. Cross-linking was unaffected by the presence of O2, argon, ascorbate (1 mM), or mercaptoethanol (10 mM). Prephotolysis of a mixture of tRNA and ribosomes in the absence of puly(U2,G) did not block subsequent cross-linking in its presence nor did it generate any long-lived chemically reactive species. There was a strong tRNA specificity. E. coli tRNA1Val and tRNA1Ser and Bacillus subtilis tRNAVal and tRNAThr could be cross-linked, but E. coli tRNA2Val, 5-fluorouracil-substituted tRNA1Val, tRNAPhe, or tRNAFMet could not. By sequence comparison of the reactive and nonreactive tRNAs, the site of attachment in the tRNA was deduced to be the 5'-anticodon base, cmo5U, or ,o5U in all of the reactive tRNAs. The attachment site in 16S RNA is described in the accompanying paper [Zimmerman, R. A., Gates, S. M., Schwartz, I., & Ofengand, J. (1979) Biochemistry (following paper in this issue)]. The link between tRNA and 16S RNA is either direct or involves mRNA bases at most two nucleotides apart since use of the trinucleotide GpUpU in place of poly(U2,G) to direct the binding and cross-linking of N-acetylvalyl-tRNA to the P site did not affect either the rate or yield of cross-linking. Both B. subtilis tRNAVal (mo5U) and E. coli tRNA1Val (cmo5U) gave the same rate and yield of cross-linking when directed by the trinucleotide GpUpU. Therefore, the presence of the charged carboxyl group in the cmo5U-containing tRNA apparently does not markedly perturb the orientation of this base with respect to its reaction partner in the 16S RNA. The cross-linking of AcVal-tRNA only takes place from the P site. At 75 mM KCl and 75 mM NH4Cl, less than 0.4% cross-linking was found at the A site, while 55.5% was obtained at the P site. However, when the salt concentration was lowered to 50 mM NH4Cl, 5% cross-linking to the A site was detected, compared to 49% at the P site. Thus, a simple change in the ionic strength of the incubation mixture was able to alter the affinity labeling pattern of the ribosome.     

Generation and characterization of monoclonal antibodies to the putative CD4-binding domain of human immunodeficiency virus type 1 gp120.

A panel of seven monoclonal antibodies against the relatively conserved CD4-binding domain on human immunodeficiency virus type 1 (HIV-1) gp120 was generated by immunizing mice with purified gp120. These monoclonal antibodies reacted specifically with gp120 in an enzyme-linked immunosorbent assay and Western blots (immunoblots). By using synthetic peptides as antigens in the immunosorbent assay, the epitopes of these seven monoclonal antibodies were mapped to amino acid residues 423 to 437 of gp120. Further studies with radioimmunoprecipitation assays showed that they cross-reacted with both gp120 and gp160 of diverse HIV-1 isolates (HTLV-IIIB, HTLV-IIIRF, HTLV-IIIAL, and HTLV-IIIWMJ). They also bound specifically to H9 cells infected with HTLV-IIIB, HTLV-IIIRF, HTLV-IIIAL, HTLV-IIIZ84, and HTLV-IIIZ34 in indirect immunofluorescence studies. In addition, they blocked effectively the binding of HIV-1 to CD4+ C8166 cells. Despite the similarity of these properties, the monoclonal antibodies differed in neutralizing activity against HTLV-IIIB, HTLV-IIIRF, and HTLV-IIIAL, as demonstrated in both syncytium-forming assays and infectivity assays. Our findings suggest that these group-specific monoclonal antibodies to the putative CD4-binding domain on gp120 are potential candidates for development of therapeutic agents against acquired immunodeficiency disease syndrome.     

The reactivity of sulfhydryl groups of bovine cardiac troponin C

Bovine cardiac troponin C (cTnC) contains 2 cysteine residues, Cys-35 located in the nonfunctional Ca2+-binding loop I and Cys-84 in the N-terminal segment of the central helix. We have studied the reactivity of Cys residues in cTnC with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). The latter compound fluoresces only when reacted with the protein. The reaction with DTNB followed second order kinetics with respect to DTNB, the rate constants being 3.37 s-1 M-1 and 1.82 s-1 M-1 in the presence and absence of Ca2+, respectively. These rates are much slower than the rate of reaction with Cys-98 of skeletal TnC (sTnC) or with the urea-denatured cTnC, indicating that both Cys residues are partly buried within the structure of the protein. The increase in reactivity was induced by binding of Ca2+ to the single low affinity Ca2+ binding site (site II). The fluorescence increase upon reaction of cTnC with CPM in the absence of Ca2+ could be fitted with a single exponential equation indicating that both cysteine residues are equally available to the reagent. The reaction in the presence of Ca2+ was biphasic. Analysis of CNBr fragments of cTnC labeled with CPM under various conditions indicated that in the presence of Ca2+ the reactivity of Cys-84 is increased while that of Cys-35 is slightly decreased. This finding is consistent with the model of Herzberg et al. (Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644) and the data of Ingraham and Hodges (Ingraham, R. H., and Hodges, R. S. (1988) Biochemistry 27, 5891-5898), suggesting that the Ca2+-induced conformational change in the N-terminal half of TnC involves separation of the helix C from the central helix, thereby increasing the accessibility of Cys-84. The slow overall kinetics, however, indicates that the structure in the vicinity of Cys residues is relatively compact regardless of Ca2+. We interpret the increase in reactivity towards CPM as consistent with a Ca2+-induced exposure of a hydrophobic pocket in the vicinity of Cys-84.     

Comparison of antimicrobial resistance patterns between clinical and sewage isolates in a regional hospital in Taiwan

Aims:  To compare bacterial populations and antimicrobial resistance patterns between clinical and sewage isolates from a regional hospital in northern Taiwan. The dissemination of antibiotic-resistant bacteria from hospital compartments to the hospital sewage treatment plant was examined.
Methods and Results:  A total of 1020 clinical isolates and 435 sewage isolates were collected between July and September 2005. The percentages of Gram-negative bacteria from the clinical and sewage isolates were 87·2% and 91·0%, respectively ( P  =   0·033). Escherichia coli were the leading bacterial isolates in both groups. Antimicrobial susceptibility testing showed a significant difference ( P  <   0·001) in resistance to ampicillin (85·6% vs 94·1%), ampicillin/sulbactam (31·7% vs 55·4%), cefazolin (29·2% vs 71·5%) and cefuroxime (20·7% vs 61·9%) between clinical and sewage coliform isolates, respectively.
Conclusions:  The sewage isolates had higher antimicrobial resistance rates than the clinical isolates from the same hospital.
Significance and Impact of the Study:  The low efficacy of the hospital sewage treatment may contribute to the dissemination of multidrug resistant bacteria from this hospital compartments to the environment. Practices which limit the disposal of antimicrobial agents into the wastewater system may be the possible measure to prevent the selection of multidrug-resistant bacteria from sewage treatment plants.     

Biosynthesis of spectinomycin: heterologous production of spectinomycin and spectinamine in an aminoglycoside-deficient host, Streptomyces venezuelae YJ003

Aims:  To obtain spectinomycin and spectinamine by heterologous expression into the biosynthetic deoxysugar (desosamine) gene-deleted host Streptomyces venezuelae YJ003.
Methods and Results:  The 17-kb spectinomycin biosynthetic gene cluster from Streptomyces spectabilis ATCC 27741 was heterologously expressed into Streptomyces venezuelae YJ003. Furthermore, the speA , speB and spcS2 encoded in the spectinomycin biosynthetic gene cluster of cosmid pSPC8 were also heterologously characterized to be responsible for the production of spectinamine.
Conclusions:  The results of this study indicated that pSPC8 contains all the genes necessary for the biosynthesis of spectinomycin. We also concluded that SpeA, SpeB and SpcS2 are sufficient for the biosynthesis of spectinamine. We also verified that SpeB and SpcS2 show dual character in the biosynthetic pathway of spectinomycin in Streptomyces spectabilis .
Significance and Impact of the Study:  This is the report regarding the expression of a biosynthetic gene cluster that gives rise to the production of aminoglycoside antibiotics in Streptomyces venezuelae YJ003. Therefore, this work may serve as a foundation for further research on spectinomycin biosynthesis and other aminoglycosides.