Requirement for three distinct lymphokines for the induction of cytotoxic T lymphocytes from thymocytes

The induction of cytotoxic T lymphocytes (CTL) from CTL precursors requires a combination of antigen and lymphokine signals. To investigate lymphokine requirements for CTL generation, we used an assay in which helper T cell and accessory cell-depleted spleen cells or whole thymocytes were cultured with lectin (Con A) and lymphokines. This culture was followed by assessment of lectin-dependent cytolysis. High concentrations of recombinant interleukin 2 (R-IL 2) (100 U/ml) alone were not sufficient for lectin-mediated CTL induction from thymocytes, whereas 20 to 100 U/ml of R-IL 2 alone could induce a significant lectin-mediated CTL response from accessory cell-depleted spleen cells. Using thymocytes as responders, we found purified or recombinant interferon-gamma (IFN-gamma) did not cause cytolytic activity either in the absence of or in the presence of R-IL 2. However, supernatant from Con A-stimulated rat spleen cells (rat Con A SN) in combination with R-IL 2 could induce cytolytic activity, suggesting that several factors are required for CTL induction. Con A SN was fractionated by gel filtration and the fractions were tested for ability to induce CTL. In the presence of a low level of R-IL 2 (5 U/ml), fractions with a Mr of approximately 31,000 could induce CTL, and this activity was referred to as CTL differentiation factor (CDF). The peak fractions containing CDF activity did not have detectable IL 1, IL 2, IFN-gamma, or CSF activity. However, by add-back experiments and the use of blocking antibodies, a monoclonal antibody against the IL 2 receptor or antibodies against murine IFN-gamma, we demonstrated that CTL induction from mature thymocytes (L3T4-, Lyt-2+) requires CDF activity in addition to IL 2 and IFN-gamma.     

A correlative study of the binding of dexamethasone in hypothalamic blocks in vitro with its ability to inhibit the release of bioactive corticotrophin-releasing factor

Rat hypothalamic blocks incubated in vitro were used to study the characteristics of binding of [3H]dexamethasone and other steroids to cytosolic binding sites. Cytosols prepared following incubation of the tissue with [3H]dexamethasone for 2 h contained specifically bound steroid in amounts that depended upon the concentration of potassium (but not sodium) ions in the extracting buffer. There was an increase in bound [3H]dexamethasone extracted as the potassium ion concentration increased up to 0.1 M, but not beyond. Dexamethasone, when added to hypothalami in vitro caused a biphasic inhibition of bioactive corticotrophin-releasing factor (CRF) release, and the extent of the second phase of inhibition was dose-related. 11-Epicortisol, when added in a 100-fold molar excess over dexamethasone was able to prevent the second phase of inhibition caused by the latter steroid, as in the binding studies it was able to cause a 50% reduction in the binding of [3H]dexamethasone. In the functional studies it was shown that 11-epicortisol was able to "rescue" the tissue from dexamethasone-mediated delayed inhibition of CRF secretion if added to the blocks 30 min (but not later) after the agonistic steroid.     

Physiology of F-Pilin Synthesis and Utilization

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to study the synthesis and turnover of F-pilin in membrane preparations of Escherichia coli K-12 under conditions which have been reported to affect the production of F-pili. Incorporation of [³⁵S]methionine into membrane F-pilin by cells in log phase was barely detectable at 25°C, but increased with temperature. The labeled pilin band was prominent in membranes from 37°C cultures and even more prominent if the growth temperature was raised to 42°C. The appearance of other tra products in the membranes was similarly temperature dependent. In cultures grown in glucose minimal medium at 37°C, the relative amount of membrane pilin and traT product synthesis remained unchanged from early log phase through early stationary phase; provision of glycerol or arabinose as a substitute carbon source had no obvious effect. Turnover of traT product and membrane F-pilin, as assessed in an Flac tra mutant strain which is incapable of elaborating pili, was not rapid. Both traT product and pilin subunits labeled in mid-log phase cells were still apparent in the membranes after growth of the cells to stationary phase. The relative amount of labeled pilin decreased with prolonged incubation in stationary phase, but the relative amount of traT product did not decrease even after the culture was incubated for 24 h. When wild-type Flac piliated cells were used, a similar result was obtained, but in this case, loss of F-pilin from the preparations could be acclerated by blending the cells. Although intermittent blending during culture growth caused a slow depletion of the labeled pilin pool, continuous blending resulted in the rapid disappearance of this pool from our preparations. Loss of other membrane polypeptides was not accelerated by our blending procedure, and blending did not affect the turnover of the pilin pool of the Flac tra mutant. Our data are consistent with a model in which pilin subunits are assembled transiently into pili, conserved by retraction, and made available for subsequent reassembly. Growth in 0.01% sodium dodecyl sulfate did not accelerate loss of pilin from the Flac strain compared with the Flac tra strain, and we suggest that in the presence of sodium dodecyl sulfate at this concentration, F-pili are not elaborated from cell surfaces.     

A DC-specific cytolytic T lymphocyte line is OKT8+1

A human cytotoxic T lymphocyte (CTL) line, A9, was generated by limiting dilution and was selected because of its apparent DC specificity. A9 is 100% OKT3+, 90% OKT4+, and 10% OKT8+, but by negative selection the CTL present are entirely OKT8+. These OKT8+ CTL are totally inhibitable by Genox 3.53, an anti-DC1 monoclonal antibody (mAb), and Leu-10, an anti-DC subgroup mAb, but are not inhibitable by a panel of anti-HLA-DR mAb. These CTL are also inhibitable by anti-OKT3 and anti-LFA-2 but not by OKT4 or OKT8 mAb. These findings extend previous studies that showed that OKT8+ CTL recognize HLA-A,B antigens, whereas OKT4+ CTL recognize HLA-DR and SB antigens. It is possible that an as yet undefined T cell surface molecule is involved in DC recognition.     

Characterization of a Nerve Growth Factor-Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule-Associated Protein 2 and pp250

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.     

Double-strand cleavage and strand joining by the replication initiator protein of filamentous phage f1

The replication initiator protein (gene II protein (gpII] of bacteriophage f1 is a multifunctional protein that plays central roles in initiation and termination of phage DNA replication. It introduces a nick at a specific site on the (+)-strand of supercoiled replicative form DNA. The 3'-hydroxyl end of the nick serves as the primer for (+)-strand rolling-circle replication. Upon completion of a round of synthesis, gpII cleaves and circulaizes the displaced single strand. When Mn2+ is included in the buffer instead of Mg2+, gpII cleaves both strands. In this paper, we investigate the mechanism of the Mn2+-dependent double-strand cleavage activity of gpII. This reaction, unlike nicking in the presence of Mg2+, does not require superhelicity. The reaction proceeds in two kinetic steps: first nicking of the (+)-strand, and then cleavage of the (-)-strand. The nucleotide sequence requirement for nicking is reduced compared to that in the presence of Mg2+. The product of the double-strand cleavage has an unusual structure. The left end is a telomere-like hairpin since the (+)- and (-)-strands are joined, as demonstrated by base sequencing. The right end has a onebase 3'-overhang. This reaction probably reflects the cleavage-joining activity of gpII in the termination event.     

Discrepancies between MIC and MLC values of amphotericin B against isolates ofAspergillus species

There is little information addressing the phenomena of discrepancy between minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC) values of amphotericin B (AMB) to clinical isolates of fungi. This study assessed in vitro activity of AMB against 70 clinical isolates of aspergilli: 30 strains ofAspergillus fumigatus, 20 strains ofAspergillus flavus and 20 strains ofAspergillus niger. Susceptibility tests were accomplished using a macro broth dilution procedure, with special emphasis on ascertainment of MLCs. AMB exhibited low MIC values against all clinical isolates. While we did not identify any AMB resistant isolates among 70Aspergillus spp. studied as judged by MIC levels, analysis of the data demonstrated a clear discrepancy between the MIC and MLC levels of AMB obtained against clinical isolates ofAspergillus spp. The MLC values of AMB were significantly higher than the MIC values with MIC 50 and MIC 90 of 0.29 and 0.5 µg/ml, respectively, at the second reading time, and MLC 50 and MLC 90 of 2.31 and 9.24 µg/ml, respectively (p<0.001). Additionally, minimal lethal concentrations in 36/70 (51%) of aspergillal isolates studied produced drug concentrations above those which can usually be sustained in patient plasma or tissue.     

Asparaginase synthesis by semi-continuous fermentor cultures of Vibrio succinogenes

Summary Vibrio succinogenes produces an asparaginase that does not hydrolyze glutamine, is not immunosuppressive, and has antitumor activity. Fermentor cultures initiated by small inocula exhibit a pattern of increasing enzyme activity consistent with induction during exponential phase. Semi-continuous cultures permit the harvesting of fully induced cells.     

DNA polymerase activity in muscle cultures

Nuclei within myotubes do not synthesize DNA for replication. Accordingly, cultures of myotubes display low levels of DNA polymerase activity. The coincidental decline in DNA polymerase activity and increased formation of multinucleated myotubes during culture does not prove that the loss of capacity to synthesize DNA is a consequence of fusion. Tne experiments described demonstrate that myogenic cells prevented from fusing have low levels of DNA polymerase activity. This is consistent with the notion that, in myogenic cultures, there is a population of mononucleated cells, the myoblasts, which have withdrawn from the mitotic cycle before fusion.