Physical and conformational properties of staphylokinase in solution.

The structure of staphylokinase has been analyzed by solution X-ray scattering, dynamic light scattering, ultracentrifugation and ultraviolet circular dichroism spectroscopy. Staphylokinase has a radius of gyration of 2.3 nm, a Stokes radius of 2.12 nm and a maximum dimension of 10 nm. The sedimentation coefficient is 1.71 S. These physical parameters indicate that the shape of staphylokinase is very elongated. The protein molecule consists of two folded domains of similar size. The mean distance of the centres of gravity of the domains is 3.7 nm. The mutual positions of the two domains are variable in solution. Thus, the molecule is shaped like a flexible dumbbell. About 18% of the amino acids of staphylokinase are organized in helical structures, 30% are incorporated in beta-sheets and 20% form turns.

Stalling of Escherichia coli RNA polymerase in the +6 to +12 region in vivo is associated with tight binding to consensus promoter elements.

Three synthetic promoters, PS1, PS2 and PS3, which differ in their core promoter elements, were studied in vivo and in vitro. Whereas an increased homology score correlates with higher rates of RNA polymerase binding, it does not correlate with activity in vivo. Permanganate probing in vivo reveals that PS1, which exhibits the lowest homology score, is rate-limited during the early phase of promoter-RNA polymerase interactions. By contrast, PS2 and PS3, with higher homology scores, are limited at a late step involving an open DNA region spanning from +6 to +12, indicating a stalling of RNA polymerase. These complexes disappear upon treatment of cells with rifampicin and are replaced by open complexes covering the start site. Because initiated complexes are selectively insensitive to rifampicin action, this confirms that RNA polymerase stalled at +6 to +12 has initiated RNA synthesis. Kinetic studies indicate that the enzyme is released slowly from this position and that this slow release appears to be responsible for the low promoter activity. For PS3, which exhibits the highest homology score and which binds RNA polymerase most efficiently, the release of the stalled complex is particularly slow. PS3 is found to be the weakest of the three promoters in vivo. These results support models in which promoter activity can be determined by various rate limiting steps, including those following the formation of open complexes and even the initiation of RNA synthesis.

Context-dependent effects of upstream A-tracts. Stimulation or inhibition of Escherichia coli promoter function.

Phased A-tract sequences were inserted in the upstream region of three synthetic promoters known to encompass different rate-limiting steps within the pathway of RNA polymerase-promoter interaction (Ellinger et al., accompanying paper). Promoter PS1, which is rate-limited in complex formation, was stimulated by A-tracts in vivo. Permanganate probing showed that the stimulation is due to an enhanced ability to compete for limiting RNA polymerase in vivo, leading to the increased formation of open complexes. By contrast, promoters PS2 and PS3, which are rate-limited in steps following open complex formation, were inhibited in vivo by A-tracts. Permanganate probing showed that the inhibition was accompanied by an A-tract-dependent accumulation of stalled initial transcribing complexes. A single A-tract was as effective as three. The phasing of the A-tracts with respect to the core promoter sequence was found to be important for promoter function. The position that caused maximal activation at one promoter caused maximal inhibition at another. These results suggest that the same molecular interaction gives rise to both inhibition and activation. This is likely to be due to facilitated RNA polymerase binding in the presence of A-tracts, which stimulates binding-limited promoters but inhibits promoter function in which polymerase escape and promoter clearance is rate limiting.

Prevalence of a common point mutation in the dihydropyrimidine dehydrogenase (DPD) gene within the 5'-splice donor site of intron 14 in patients with severe 5-fluorouracil (5-FU)- related toxicity compared with controls.

Deficiency of dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in 5-fluorouracil (5-FU) catabolism, has been linked to toxic side effects of 5-FU. The most prominent mutation of the DPD gene resulting in severe DPD deficiency is a G to A mutation in the GT 5'-splice recognition site of intron 14 (exon 14-skipping mutation). The corresponding mRNA lacks exon 14, and the enzymatic activity of the translated DPD protein is virtually absent. We developed a reverse transcription-PCR-based assay suitable for routine identification of the exon 14-skipping mutation and screened a control cohort of 851 Caucasian individuals as well as a cohort of 25 cancer patients reported by their physicians to have suffered from WHO grades 3-4 toxicity upon 5-FU chemotherapy. Within the control cohort, in total, eight heterozygotes were detected (0.94%): one heterozygote in 51 healthy donors, (1.96%); five heterozygotes in 572 hospital patients (0.87%); and two heterozygotes in 228 colorectal tumor patients (0.88%). Among the 25 patients with severe 5-FU-related toxicity, 5 (20%) were heterozygous and 1 (4%) was homozygous for the exon 14-skipping mutation. All six patients had experienced WHO grade 4 myelosuppression. Lethal outcome was seen in the homozygous and two of the heterozygous cases. We conclude that carriers of the DPD exon 14-skipping mutation are at significantly increased risk to experience life-threatening myelosuppression upon 5-FU treatment, even when the allelic status is heterozygous. These data lead us to suggest routine testing for the exon 14-skipping mutation before 5-FU treatment.

Complete nucleotide sequence of plasmid pGB3631, a derivative of the Streptococcus agalactiae plasmid pIP501.

The complete nucleotide (nt) sequence of plasmid pGB3631 (5842 nt), a deletion derivative of the Streptococcus agalactiae plasmid pIP501, was determined on both strands. Six open reading frames (ORFs) were found. Five ORFs were responsible for replication, copy-number control and resistance against MLS antibiotics.

Temperature-inducible gene expression in Bacillus subtilis mediated by the cI857-encoded repressor of bacteriophage lambda.

An efficient system to control the expression of cloned genes in Bacillus subtilis was established by introducing the Escherichia coli bacteriophage lambda cI857 repressor-pR promoter system into this host. A staphylokinase reporter gene (sak42D), which was fused to the lambda pR promoter was constitutively expressed in B. subtilis even when the cI857 gene was present on the same plasmid. S1 nuclease mapping of the transcription start point confirmed that the pR promoter was active in B. subtilis. Constitutive expression under pR-control in B. subtilis was, therefore, likely to result from a lack of repressor formation caused by the inefficiency of cI857 expression signals in the Gram+ host. This lack of repressor synthesis was overcome by fusing the cI857 gene to sak42D transcription and translation signals which have previously been shown to function efficiently in B. subtilis. Plasmids carrying the cI857 gene together with an alpha-amylase-encoding gene (amy) under pR-control mediated temperature-inducible amy expression at 37 degrees C and 42 degrees C. The high repression factor (greater than or equal to 1400) was comparable to the OR efficiencies reported in E. coli.

Expression and secretion of interferon-alpha 1 by Streptomyces lividans: use of staphylokinase signals and amplification of a neo gene.

A gene coding for mature human interferon, IFN-alpha 1, fused to the expression and secretion signals of a staphylokinase gene (sak) derived from Staphylococcus aureus phage 42D, was inserted into the Streptomyces promoter probe vector pIJ487. Streptomyces lividans transformed with the recombinant plasmid (pMG341) secreted biologically active IFN-alpha 1 into the culture medium. Expression of the IFN-alpha 1 gene was at least on the translational level directed by the sak signals since numerous upstream stop codons would have prevented the formation of a fusion protein. Long-term continuous chemostat cultivation under various limitation conditions was used to select clones with an IFN-alpha 1 yield increased about 60-100-fold (1-2 x 10(5) IU/ml). The increase in IFN-alpha 1 formation was accompanied by spontaneous amplification of the adjacent neo gene, but not of the remaining plasmid DNA. Examination of the DNA sequence around the endpoints of the amplified region revealed almost identical stem-loop structures followed by an octanucleotide direct repeat.

Bispecific antibody-mediated destruction of Hodgkin's lymphoma cells.

CD30 is a molecule that is overexpressed on the surface of Hodgkin's lymphoma cells. Therefore, CD30 represents a potential candidate for immunotherapy. In this study, we report the in vitro results of two bispecific molecules (BSMs) that target CD30 to trigger molecules expressed on myeloid effector cells. The first BSM is composed of the Fab' fragment of a CD30-specific antibody, Ki-4, chemically linked to the Fab' fragment of the humanized CD64 (FcgammaRI)-specific antibody, H22 (H22xKi-4). In the second BSM, the H22 Fab' is replaced with the Fab' fragment of the CD89 (FcalphaR)-specific, antibody, A77 (A77xKi-4). Both BSMs were able to bind specifically to lymphoma cell lines expressing CD30. In addition, the H22xKi-4 and A77xKi-4 BSMs were shown to bind cells expressing CD64 and CD89, respectively. Both BSMs mediated potent, dose-dependent antibody dependent cell-mediated cytotoxicity (ADCC) of CD30-expressing tumor cell lines when human monocytes were used as effector cells. In addition, freshly prepared polymorphonuclear leukocytes (PMNs) and effector cells in whole blood were able to mediate the ADCC of targets in conjunction with the A77xKi-4 BSM in some, but not all, experiments. Furthermore, we examined the ability of monocyte-derived macrophages (MDMs) to phagocytose CD30-expressing tumor cell lines in conjunction with the BSM. MDM-mediated phagocytosis was significantly enhanced in the presence of both BSMs. These results demonstrate that targeting lymphoma cells via CD30 to the myeloid high affinity Fc receptor for IgG and to the Fc receptor for IgA results in potent in vitro anti-tumor activity.

Functional significance of NH2- and COOH-terminal regions of staphylokinase in plasminogen activation.

Structure/function relationships in the activation of plasminogen with staphylokinase were studied using mutants of recombinant staphylokinase (Sak42D). Deletion of up to 10 NH2-terminal amino acids (Sak42D delta N10) did not affect plasminogen activation, but removal of 11 amino acids completely abolished the ability to activate plasminogen. Elimination of potential plasmin cleavage sites in the NH2-terminal region yielding mutants Sak42D(K8H,K10H,K11H) and Sak42D(K6H,K8H,K11H) did not alter the rate of the exposure of a proteolytically active site (amidolytic activity) in equimolar mixtures with plasminogen, but destroyed the plasminogen activator properties of these muteins. Deleting two residues following the preferred processing site at position 10 (Sak42 delta (K11,G12)) resulted in a mutein also inactive in plasminogen activation. Removal of the COOH-terminal Lys136, yielding Sak42D delta C1, or of Lys135 and Lys136 in Sak42D delta C2 resulted in proteins with strongly reduced plasminogen activation capacity. In contrast, substitution of Lys135 and Lys136 with Ala in Sak42D(K135A,K136A) did not affect activation. Cyanogen bromide cleavage of Sak42D(M26L,E61M,D82E) produced a 61 amino acid NH2-terminal and a 65 amino acid COOH-terminal fragment which did not activate plasminogen, but bound to plasminogen with affinity constants Ka of 4.0 x 10(5) M-1 and 1.4 x 10(7) M-1, respectively (as compared to a Ka of 1.1 x 10(8) M-1 for Sak42D). These results indicate that Lys11 and the COOH-terminal region of staphylokinase play a key role in the activation of plasminogen.

Synergistic cytotoxic effect of human recombinant tumor necrosis factor alpha combined with dihydroambazone.

Dihydroambazone (DHA) is a water-soluble derivative of the experimental anticancer drug ambazone. In vitro, a combination of DHA and human recombinant tumor necrosis factor alpha (TNF) exerted a strong synergism of cytotoxicity against both mouse melanoma B16K cells and the TNF-sensitive mouse fibroblast line L-M (S). Furthermore, in a colony-forming assay with B16K cells a combination of TNF and DHA inhibited colony-formation much more severely than either drug alone. An increased antiproliferative efficiency was also confirmed in vivo against established subcutaneous melanoma B16 tumors of C57BL/6 mice.

Synergistic cytotoxicity of human recombinant tumour necrosis factor alpha combined with microtubule effectors.

Combinations of human recombinant tumour necrosis factor alpha (rhTNF alpha) with each of four different agents disturbing the microtubule system of the cellular cytoskeleton were tested for synergistic cytotoxic action against murine melanoma B16K and L-M(S) cells. In addition to the known microtubule effectors colchicine, vincristine, and taxol, the influence of the fluorenone-azo-methine derivative alpha-diphenylene-N-(p-[bis-(beta-hydroxyethyl-amino]-phenyl)- nitrone (DHPN) on the rhTNF alpha cytotoxicity was studied. Applying a novel computer-based isobole method [Suehnel J (1990) Antiviral Res 13:23-40] concentration ranges of synergistic, zero, and antagonistic interaction were found after in vitro combination of rhTNF alpha with each of the drugs tested in a 72-h cytotoxicity assay. In contrast, a 24-h exposure of B16K cells to these combinations still did not inhibit in vitro colony formation to a greater extent than either drug alone. A preliminary in vivo experiment revealed an increased antitumour effect after treatment of established subcutaneous melanoma B16 tumours with a combination of rhTNF alpha and DHPN.

Circadian variation of dihydropyrimidine dehydrogenase mRNA expression in leukocytes and serum cortisol levels in patients with advanced gastrointestinal carcinomas compared to healthy controls.

PURPOSE: The activity of dihydropyrimidine dehydrogenase (DPD) - the rate-limiting enzyme in fluorouracil (5-FU) catabolism - has been reported to vary according to the time of day. On the basis of this data, so-called chronomodulated chemotherapy regimens with variable-rate infusions of 5-FU have been investigated in the treatment of advanced colorectal cancer. Recent results suggest lower toxicity of 5-FU by chronomodulated application. However, the pattern of circadian DPD activity levels have been shown to vary considerably. METHODS: We, therefore, studied the circadian changes in mRNA expression of DPD in leukocytes of ten patients with advanced gastrointestinal carcinomas prior to chronomodulated 5-FU-based salvage therapy and in 5five healthy controls. Simultaneously, we measured serum cortisol levels (SCL) to evaluate the endogenous circadian hormone rhythm. RESULTS: SCL displayed a consistent circadian rhythm with the mean peak value of serum cortisol at 8 a.m. and the mean trough value at 11 p.m. both in patients and in controls. However, mean minimum-maximum serum cortisol differences of SCL were significantly lower in patients compared to controls. In the 5fivehealthy controls, a trend towards a circadian rhythm of DPD mRNA expression was observed with the peak of expression at 5 a.m. which was significantly different from the trough at 2 p.m. ( P<0.005 Mann-Whitney-Wilcoxon test). When each control was studied separately, only two individuals showed circadian variations that could be fitted to a cosine wave ( P=0.001, P=0.014, Cosinor analysis). In contrast, DPD mRNA expression in patients with advanced gastrointestinal carcinomas did not demonstrate any consistent circadian rhythm. Pairwise comparisons of groups of DPD mRNA levels at different times of the day did not show significant differences. CONCLUSIONS: In conclusion, our analysis of DPD mRNA expression in leukocytes from healthy controls demonstrates first evidence for a circadian DPD mRNA expression periodicity. In patients with advanced gastrointestinal carcinomas, however, this rhythm seems to be disturbed although circadian endogenous cortisol secretion pattern is maintained.

Immunoelectron microscopy of Bacillus subtilis cells secreting human interferon alpha 1 or staphylokinase.

Post-embedding labelling techniques with colloidal gold-IgG or -protein A complexes were used to determine the subcellular location of IFN alpha 1 and staphylokinase secreted from Bacillus subtilis GB500 cells. Both proteins were present in the cytoplasma and the cell envelope pointing to a posttranslational mode of translocation across the cytoplasmic membrane. 5- to 10-fold higher concentrations of gold particles per 0.1 micron 2 were found on the cell envelope and clustering was observed suggesting preferential regions for secretion sites. Several control experiments ensured the specificity of the labelling data.

Expression and subcellular location of native and mutant hTNF alpha proteins in Escherichia coli.

Several mutant hTNF alpha genes were constructed by deletion and stepwise reconstitution of regions coding for C-terminal sequences. The mutant hTNF alpha proteins behaved differently from native hTNF alpha when expressed in Escherichia coli. They were either sensitive to proteolytic degradation or formed insoluble aggregates depending on the strains and conditions used for expression. By contrast, native hTNF alpha was always present in a soluble form and had a tendency to associate with the cytoplasmic membrane. It was even transported to the periplasmic space in E. coli as shown by both cell fractionation and immunoelectron microscopy. The different behaviour of mutant hTNF alpha proteins probably results from a disturbance of protein folding.

Isolation of a putative nicotinic acetylcholine receptor from the central nervous system of Locusta migratoria.

The alpha-bungarotoxin binding component from locust central nervous tissue was solubilized and purified by affinity chromatography on alpha-bungarotoxin Sepharose 4B. On sucrose density gradients containing Triton X-100, the toxin binding site sedimented with an apparent sedimentation coefficient of about 10 S. As revealed by sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified receptor protein was composed predominantly of 65,000 molecular weight polypeptides.

Comparison of mRNA expression levels determined with TaqMan and competitive template RT-PCR.

Two methods for measurement of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) mRNA expression were compared. Although the relative mRNA levels compared to beta-actin measured with competitive template RT-PCR were different from the data obtained with a TaqMan based PCR, a significant correlation between the two assays was found.

[The use of phage lambda promotors for expressing genes of secreted proteins in Bacillus subtilis cells]. / Ispol'zovanie promotorov faga lambda dlia ékspressii genov sekretiruemykh belkov v kletkakh Bacillus subtilis.

At was shown with the help of promoterless alpha-amylase and staphylokinase genes that lambda PR and lambda PL promoters could be used in Bacillus subtilis. Promoters strength was compared to promoter of alpha-amylase gene, this enabled to order the promoters in a row: PAA greater than lambda PR greater than lambda PL. The lambda PR promoter region was controlled by temperature in E. coli cells only, but not in B. subtilis, therefore, the active lambda C1857 gene product was not produced in B. subtilis cells. The lambda PR promoter is used by B. subtilis at a later growth stage than PAA and the lambda PL promoter at a still later stage than lambda PR. The data enables lambda PR to be considered as quite useful for Bacilli.

[Vector plasmids and the strategy of molecular cloning for streptococci]. / Vektornye plazmidy i strategiia molekuliarnogo klonirovaniia dlia streptokokkov.

The experiments on elaboration of gene engineering methodology for streptococci are described. Two vectors were constructed for DNA cloning in streptococci on the basis of plasmids pSM19035 and pIP2501. Some of the plasmids occurred to be valuable vectors for molecular cloning in bacilli. Peculiarities of the transformation mechanism in streptococci were found to impede the molecular cloning. The recombination technique of cloning was successfully used in the streptococcal system.

High yield production and purification of recombinant staphylokinase for thrombolytic therapy.

Recombinant plasmids were constructed in which the signal sequence of the sak42D and the sakSTAR staphylokinase genes were replaced by an ATG start codon and which express staphylokinase under the control of a tac promoter and two Shine-Dalgarno sequences in tandem. Induction of transfected E. coli TGl cells in a bacterial fermentor produced intracellular staphylokinase representing 10 to 15% of total cell protein. Gram quantities of highly purified recombinant staphylokinase were obtained from cytosol fractions by chromatography, at room temperature, on SP-Sepharose and on phenyl-Sepharose columns, with yields of 50 to 70 percent. The material, at a dose of 4 mg/kg, did not produce acute reactions or affect body weight in mice. Intravenous administration of 10 mg SakSTAR over 30 minutes in five patients with acute myocardial infarction induced complete coronary artery recanalization, without associated fibrinogen degradation. However, neutralizing antibodies appeared in the plasma of all patients within 12 to 20 days. Thus, the present expression and purification method for recombinant staphylokinase yields large amounts of highly purified mature protein (approximately 200 mg per liter fermentation broth) suitable for a more detailed clinical investigation of its potential as a thrombolytic agent.