Expression of murine interleukin-2 cDNA in E. coli and biological activities of recombinant murine interleukin-2.

Murine interleukin-2 (MIL-2) cDNA was inserted into an expression vector carrying an Escherichia coli tryptophan promoter and was expressed in E. coli. Recombinant MIL-2 produced by E. coli supported the growth of murine CTLL-2 cells, but not that of human T-cell blasts. Recombinant MIL-2 strongly inhibited the binding of recombinant human IL-2 (HIL-2) to murine responder cells, but only very weakly inhibited the binding to human responder cells. Moreover, recombinant MIL-2 induced secondary alloantigen specific cytotoxic T lymphocytes (2 degrees CTL) from memory CTL and activated natural killer (NK) cells in murine systems in the same manner as recombinant HIL-2. The results suggest that the species hierarchy (that MIL-2 derived from native cell culture does not act on human T-cells) is due to the protein moiety, not the sugar moiety, and is to be ascribed to the difference in binding affinity of MIL-2 and HIL-2 to murine and human responder cells respectively, and that recombinant MIL-2 shares identical biological and immunological activities with recombinant HIL-2. Thus, MIL-2 might be a convenient tool for extensive studies of the pharmacological and physiological activities of IL-2 in murine models.

Role of unique consecutive glutamine repeats in active murine interleukin-2 molecule.

Murine interleukin-2 (MIL-2) cDNA deleted of 11 repeats of a CAG sequence, and that encoded from 33Met to 149Gln were inserted into an expression vector carrying an Escherichia coli tryptophan promoter and were expressed in E. coli, respectively. Recombinant MIL-2 deleted of 11 glutamine repeats (MIL-2(-Gln] supported the growth of murine CTLL-2 cells but recombinant MIL-2 initiated from 34Asp (34Asp-MIL-2) did not. The growth of human T-cell blasts was not supported by MIL-2(-Gln) or 34Asp-MIL-2. MIL-2(-Gln) had identical biological and immunological activities with intact MIL-2, but 34Asp-MIL-2 did not. These results suggest that the consecutive glutamine repeats do not play a role in the biological and immunological activities of MIL-2, but that the peptide sequence around them does, and the species hierarchy that MIL-2 does not act on human lymphocyte is not due to the presence of glutamine repeats in MIL-2.

High-level expression of human BSF-2/IL-6 cDNA in Escherichia coli using a new type of expression-preparation system.

BSF-2 (B cell stimulatory factor-2/IL-6) is a member of the lymphokine family and responsible for B cell differentiation. Expression plasmids of human BSF-2 cDNA were constructed using a trp promotor/operator and a trpA terminator. In an extract of Escherichia coli HB101 holding "direct" expression plasmid pBSF-2D, activity of BSF-2 was detected, but overproduction was not observed. A "fused" expression system was therefore developed to prepare the recombinant protein. In this system, cDNA was expressed as a fused protein with human IL-2 N-terminal peptide. In the case of the fused BSF-2 expression plasmid, pBSF-2F, inclusion bodies were observed and overproduction of the protein occurred. As this fused protein had a Phe-Arg-Ala sequence at the junction of hIL-2 and BSF-2, it was possible to process mature BSF-2 from the fused BSF-2 by treatment with kallikrein and aminopeptidase P. From 1 liter of E. coli culture, 45 mg of mature BSF-2 was purified; it had a relative biological activity equal to that of natural BSF-2 purified from T cells.

New approaches for the high-level expression of human interleukin-2 cDNA in Escherichia coli.

We constructed several expression plasmids of human IL-2 gene, some of which directed high-level synthesis of mature IL-2 protein in E. coli. In all the plasmids reported here, we installed the E. coli trp promoter and SD sequence upstream of the IL-2 cDNA. When DNA sequences containing the rho-independent transcription terminator such as those involved in the trpA and lpp gene were inserted downstream of the IL-2 cDNA sequence, the expression level of the IL-2 gene increased up to 5-fold. Moreover, the deletion of either the whole region including A-T and G-C tails or a part of the 3' non-coding sequence resulted in further increase of the expression of the IL-2 gene up to 500-fold. The mature IL-2 produced in E. coli exhibited biological and immunological activities indistinguishable from those of purified IL-2 from a human T cell line, Jurkat-111. The manipulations described here may be useful for the high-level expression of eukaryotic genes in E. coli.

Nuclear transfer in cattle using colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells.

To assess the developmental potential of nuclear transfer embryos in cattle using mammary gland epithelial (MGE) cells derived from the colostrum, we compared the effectiveness of cloning using those cells and fibroblast cells derived from the ear. The fusion rate of the enucleated oocytes with fibroblast cells (75 +/- 4%) was significantly higher than that with MGE cells (56 +/- 7%, P<0.05). There were no significant differences in the cleavage rate (85 +/- 3% vs. 91+/- 2%) or in the developmental rate to the blastocyst stage (35 +/- 6% vs. 35 +/- 5%) using MGE cells vs. fibroblast cells as donor nuclei (P>0.05). After transfer of blastocysts derived from nuclear transfer embryos produced using MGE cells and fibroblast cells, 13% (4/31) and 16% (6/37) of recipient heifers were pregnant on Day 42 as assessed by ultrasonography, respectively. Two of the 4 and 4 of the 6 recipients of embryos with MGE cell- and fibroblast cell-derived nuclei, respectively, aborted within 150 days of pregnancy. Four live female calves were obtained from MGE cells or fibroblast cells. However, one died from internal hemorrhage of the arteria umbilicalis. The other three calves were normal and healthy. There were no differences in the pregnancy rate or calving rate when using MGE cells vs. fibroblast cells. Microsatellite DNA analyses confirmed that the cloned calves were genetically identical to the donor cows and different from the recipient heifers. We conclude that colostrum-derived MGE cells have the developmental potential to term by nuclear transfer, and the efficiency of development of those cloned embryos was the same as that of embryos obtained using fibroblast cells as donor nuclei, although there was a significant difference in the fusion rate. This method using MGE cells derived from colostrum, which is obtained easily and safely from live adult cows, is more advantageous for cloning with somatic cells.

[Study of serum phospholipase A2 activity in bronchial asthmatic patients].

We examined serum PLA2, lipids and platelets in 133 bronchial asthma patients who had not taken any glucocorticoids. Serum PLA2 was measured by radioimmunoassay. Serum PLA2 activity in the bronchial asthma patients was 384.2 +/- 114.7 (pmol/ml/min), and in the control group (healthy subjects) it was 271.7 +/- 53.9; this is a statistically significant difference (p < 0.01). Serum PLA2 of the mixed type was higher than that of the atopic type (p < 0.05) with respect to age, those subjects in their 60s showed statistically higher serum PLA2 values than those in their 20s and 30s (p < 0.05). The serum PLA2 level was statistically higher in a patient with an attack (409.1 +/- 113.9) than without an attack (342.6 +/- 94.3) (p < 0.05). However, there was no significant difference in lipid and platelet levels according to attack status. In conclusion, the serum PLA2 level is not only higher in asthma patients with attacks, but is also higher in asthma patients without attacks when compared to the control group. Therefore, the measurement of serum PLA2 is significant in predicting attacks.

[Retropharyngeal hematoma following a bruise of head and neck--a case report and review of literature].

Diseases affecting the retropharyngeal space are relatively uncommon, much less a case of retropharyngeal hematoma. However, a space-occupying lesion in this area can be life threatening and require emergency surgical intervention. There have been 46 cases of retropharyngeal hematoma reported in the English literatures from 1934 to 1989, but there has been no reported case in Japan. Recently we treated a 72-year-old male sustained retropharyngeal hematoma following a bruise of head and neck. The patients was received tracheostomy, antibiotic therapy and intravenous hyperalimentation, and this hematoma was resolved in about 2 weeks. The etiologies, diagnosis, therapies in our case and prognoses of the literature reviewed 47 cases of retropharyngeal hematoma were discussed comprehensively.

Splice loss and mode conversion in a multimode fiber.

Splice loss and mode conversion have been studied theoretically for a multimode fiber. Equations have been derived using electromagnetic theory for calculating splice loss and mode conversion in the general multimode fiber. Numerical examples are given for a square-law index fiber in both arc-fusion splice and V-groove splice cases. The influence of the refractive-index profile at the splice on splice loss and mode conversion coefficient is shown. The change in baseband frequency response due to mode conversion at splice is calculated.

Transmission characteristics of splices in graded-index multimode fibers.

Losses and baseband bandwidth changes caused by a splice in a graded-index multimode fiber have been studied theoretically. Splices are classified into three categories, and their influence on mode conversion coefficients and baseband frequency response has been investigated. Transmission characteristics for splices in cascade are also investigated.

[Effect of dibutyryl cyclic AMP on the GTP cyclohydrolase activity and tetrahydrobiopterin content of pheochromocytoma PC12h cells].

Effect of dibutyryl cyclic AMP (dBcAMP)-treatment of pheochromocytoma PC12h cells, a subclonal line of PC12, on the GTP cyclohydrolase activity and biopterin (BP) content of the cells was studied. Both the enzyme activity and BP content of the cells were transiently increased by dBcAMP treatment with both the maximal activity and content at 1 to 2 days after the treatment was started. The effect of dBcAMP was not detectable at concentrations lower than 10 microM and reached the plateau level at 500 microM in a dose-dependent manner. EC50 values of dBcAMP for increasing the enzyme activity and BP content were the same, approximately 200 microM. The dBcAMP-induced increase in both the GTP cyclohydrolase activity and BP content of the PC12h cells was completely blocked by cycloheximide, a protein synthesis inhibitor, suggesting that the effect of dBcAMP is mediated by the synthesis of some protein, most probably GTP cyclohydrolase itself.

Excess loss caused by a lossy outer layer in multimode optical fibers.

Theoretical and experimental investigations are made on an excess loss arising due to a lossy outer layer attached around a cladding in multimode step-index fibers. Equations for evaluating the excess loss are derived using the electromagnetic theory for LP modes. Experiments are made on fibers with a pure fused silica jacket around a cladding, focusing on the wavelength dependence of the excess loss. It is found that the loss increases as the wavelength becomes long and that the measured values are in good agreement with the calculated ones.

Excess loss caused by an outer layer in multimode step-index fibers: experiment.

Experiments have been performed to evaluate the increase in loss due to an outer layer around the cladding in a multimode step-index fiber composed of core and cladding. Liquid has been used to simulate the outer layer. Measured excess loss for each mode group is in agreement with our previously developed electromagnetic theory. Total excess loss for all guided modes existing in the fiber has also been measured by varying the mode power distribution, the refractive index of the outer layer, the cladding thickness, and the wavelength. Results are found to be in good agreement with the theory, and it is concluded that the theory is correct.

Transmission characteristics of graded-index optical fibers with a lossy outer layer.

Changes in transmission characteristics caused by an outer layer have been investigated for graded-index fibers. Equations have been derived using the WKB method for calculating baseband frequency response and excess loss in a general type of the graded-index fiber. Numerical examples have been given for a square-law fiber, mainly with cladding thickness and index difference between outer layer and cladding as parameters. Measurement of the excess loss has been carried out for fibers with various cladding thickness and outer layer-cladding index differences. The measured loss is in agreement with the theoretical value.

Reflection properties of splices in graded-index optical fibers.

The reflection properties of splices, such as fusion splices and V-groove splices, in graded-index multimode optical fibers were investigated theoretically and experimentally. Equations have been derived for calculating reflection in fusion splices and V-groove splices. Several reflection characteristics in splices were measured using a time-domain reflectometer. The measured results are discussed using the deived equations