Comparative measurements of bone mineral density and bone contrast values in canine femora using dual-energy X-ray absorptiometry and conventional digital radiography.

BACKGROUND: Aseptic loosening due to bone remodelling processes after total hip replacement is one common cause for revision surgery. In human medicine, dual-energy X-ray absorptiometry (DEXA) is the gold standard for quantitative evaluation of bone mineral density, whereas in veterinary medicine conventional radiography is used for follow-up studies. Recently, a method has been described using digital X-ray images for quantitative assessment of grey scale values of bone contrast. Therefore, the aim of the present study was to evaluate the correlation of bone mineral density (BMD) measured by DEXA with grey scale values (GV) measured in digital X-ray images (RX50, RX66) ex vivo. RESULTS: The measured GV in the chosen X-ray settings showed on average a good correlation (r = 0.61) to the measured BMD with DEXA. Correlation between the two X-ray settings was very good (r = 0.81). For comparisons among regions of interests (ROIs) a difference of 8.2% was found to be statistically significant, whereas in the case of RX50 and RX66 differences of 5.3% and 4.1% were found to be statistically significant. CONCLUSIONS: Results indicate that measuring absolute changes in bone mineral density might be possible using digital radiography. Not all significant differences between ROIs detectable with DEXA can be displayed in the X-ray images because of the lower sensitivity of the radiographs. However, direct comparison of grey scale values of the periprosthetic femur in one individual patient during the follow-up period, in order to predict bone remodelling processes, should be possible, but with a lesser sensitivity than with DEXA. It is important that the same X-ray settings are chosen for each patient for follow-up studies.

Biomechanical comparison of a new expandable intramedullary nail and conventional intramedullary nails for femoral osteosynthesis in dogs.

Intramedullary nailing of diaphyseal femoral fractures is a commonly used treatment method in dogs because of its biological and biomechanical advantages compared to bone plating. To achieve adequate resistance of the intramedullary nail against torsional and axial compressive forces, additional application of transcortical screws is needed. As these interlocking screws represent a frequent cause of post-operative complications, a new expandable intramedullary nail (EXPN) was developed, which was designed to provide adequate fracture stabilisation without the need for transcortical fixation. The evaluation of the biomechanical properties of the new EXPN with regard to torsional, compressive and bending stability as well as direct comparison to the biomechanical properties of conventional Steinmann (STMN)- and interlocking (ILN) nails was carried out with different biomechanical test arrangements. No significant statistical differences regarding the torsional and bending resistance between the EXPN and ILN group were seen, which indicates that rotatory as well as bending stability of the innovative EXPN is similar to the conventional ILN. Nevertheless, the percentage deviation between the attempted and successfully reached physiological compressive forces was significantly higher (p = 0.045) in the EXPN group compared to the ILN group, which indicates that the compressive stability of the innovative EXPN might be weaker compared to the ILN. In summary, the new EXPN represents an interesting alternative to conventional intramedullary nails. However, in direct comparison to conventional interlocking nails, the EXPN has shown weaknesses in the neutralization of axial compressive forces, which indicates that at least biomechanically the interlocking nail seems advantageous. Further in-vitro and in-vivo investigations are required before clinical use can be recommended.

Technical validation of a new microfluidic device for enrichment of CTCs from large volumes of blood by using buffy coats to mimic diagnostic leukapheresis products.

Diagnostic leukapheresis (DLA) enables to sample larger blood volumes and increases the detection of circulating tumor cells (CTC) significantly. Nevertheless, the high excess of white blood cells (WBC) of DLA products remains a major challenge for further downstream CTC enrichment and detection. To address this problem, we tested the performance of two label-free CTC technologies for processing DLA products. For the testing purposes, we established ficollized buffy coats (BC) with a WBC composition similar to patient-derived DLA products. The mimicking-DLA samples (with up to 400 × 106 WBCs) were spiked with three different tumor cell lines and processed with two versions of a spiral microfluidic chip for label-free CTC enrichment: the commercially available ClearCell FR1 biochip and a customized DLA biochip based on a similar enrichment principle, but designed for higher throughput of cells. While the samples processed with FR1 chip displayed with increasing cell load significantly higher WBC backgrounds and decreasing cell recovery, the recovery rates of the customized DLA chip were stable, even if challenged with up to 400 × 106 WBCs (corresponding to around 120 mL peripheral blood or 10% of a DLA product). These results indicate that the further up-scalable DLA biochip has potential to process complete DLA products from 2.5 L of peripheral blood in an affordable way to enable high-volume CTC-based liquid biopsies.

Numerical investigations of stress shielding in total hip prostheses.

Aseptic loosening of the prosthesis is still a problem in artificial joint implants. The loosening can be caused by, among other factors, resorption of the bone surrounding the prosthesis owing to stress shielding. In order to find out the influence of the prosthesis type on post-operative stress shielding, a static finite element analysis of a femur provided with the conventional uncemented stem BICONTACT and of one with the femoral neck prosthesis SPIRON was carried out. Strain energy densities and maximal principal strain distributions were calculated and compared with the physiological situation. Here, stress shielding was demonstrated in both periprosthetic femora. To determine the areas of the stress shielding, the bone in each FE model was subdivided into three regions of interest (ROI): proximal, diaphyseal, and distal. The numerical computations show stress shielding in the proximal ROI of both periprosthetic femora. Diaphyseally, the femoral neck prosthesis SPIRON, in contrast to the conventional uncemented long-stem prosthesis BICONTACT, causes no decrease in the strain distribution and thus no stress shielding. Distally, no change in the load distribution of either periprosthetic femur could be found, compared with the physiological situation.

Strain adaptive bone remodelling: influence of the implantation technique.

Total hip arthroplasties (THA) can be performed with cemented and uncemented femoral components. Aseptic loosening of the joint replacement still illustrates a problem for both implantation techniques. This loosening can be caused, among other factors, by resorption of the bone surrounding the implant due to stress shielding. In order to analyse the absolute influence of the implantation technique on the bone degeneration in the periprosthetic femur, the strain adaptive bone remodelling after THA was investigated in a three-dimensional finite element (FE) simulation of a femur provided with a cemented and uncemented BICONTACT (Aesculap, Tuttlingen, Germany) femoral component. For this, a bone density evolution theory was implemented in the FE code MSC.MARC. In these static FE simulations, the muscle and hip resultant forces represent the maximum loading situation in the normal walking cycle. To describe the mechanical properties of the bone, an isotropic material law dependent upon density was used. The situation directly after implantation without any bone ingrowth was simulated. The cemented femoral component was bonded to the bone by a homogenous cement mantle. The numerical results show that proximally, the bone resorption areas surrounding the BICONTACT stem are heavily dependent upon anchoring technique. Furthermore, no significant bone remodelling is calculated in the distal periprosthetic femur in both models.

Problems with ultrasonic measurements of shear modules of structured media.

The elastic constants of linearly elastic, isotropic or anisotropic bone material are required for many numerical simulations. These constants are often measured ultrasonically, but this can lead to mistakes, especially if shear modules of spongiosa are considered. The reason is that spongiosa is a structure composed of trabeculae, each of which acts as a kind of beam which allows longitudinal, shear and also bending waves to propagate; the bending waves are as fast as the longitudinal waves and are indistinguishable from the shear waves. Furthermore, mistakes in measuring Young's modulus cannot be avoided in every case. Several numerical simulations of wave propagations in homogeneous media, and especially in periodically and irregularly structured media, were carried out via the application of explicit finite element codes. Results showing the above-mentioned effects are presented and discussed. These can help to explain in detail why mistakes may occur during ultrasonic measurements.

Influence of Hip Prosthesis Size and Its Coating Area on Bone Remodeling.

We developed a numerical model to describe the bone remodeling process in periprosthetic bone tissues and validated it by means of dual energy X-ray absorptiometry methods with different types of hip implants. In this paper, we applied the numerical model to investigate the influence of implant size and of the size of the porous coated area on bone remodeling in a periprosthetic human femur in an effort to define properties of implants, which would reduce bone remodeling after total hip arthroplasty. Two different sizes of a newly designed implant and three different coating area sizes were investigated in this paper. The results show that the smaller the implant, the less bone remodeling occurs. Reducing prosthesis size by 2mm from all sides has decreased bone remodeling by 14.4%. Extending the coating area on both, lateral and medial parts of the implant, has decreased bone remodeling in the lateral part of the femur and increased it in the medial part. In conclusion, depending on these results, the oversized hip replacement would cause more bone resorption in the femur. Concerning the coating area, the manufacturer must find a compromise between the small coating area with less bone remodeling in the medial part of the femur as well as less primary stability and the bigger coating area with less bone remodeling in the lateral part of the femur, but with higher bone remodeling in its medial part and more primary stability.

Analysis of DNA methylation in single circulating tumor cells.

Direct analysis of circulating tumor cells (CTCs) can inform on molecular mechanisms underlying systemic spread. Here we investigated promoter methylation of three genes regulating epithelial-to-mesenchymal transition (EMT), a key mechanism enabling epithelial tumor cells to disseminate and metastasize. For this, we developed a single-cell protocol based on agarose-embedded bisulfite treatment, which allows investigating DNA methylation of multiple loci via a multiplex PCR (multiplexed-scAEBS). We established our assay for the simultaneous analysis of three EMT-associated genes miR-200c/141, miR-200b/a/429 and CDH1 in single cells. The assay was validated in solitary cells of GM14667, MDA-MB-231 and MCF-7 cell lines, achieving a DNA amplification efficiency of 70% with methylation patterns identical to the respective bulk DNA. Then we applied multiplexed-scAEBS to 159 single CTCs from 11 patients with metastatic breast and six with metastatic castration-resistant prostate cancer, isolated via CellSearch (EpCAMpos/CKpos/CD45neg/DAPIpos) and subsequent FACS sorting. In contrast to CD45pos white blood cells isolated and processed by the identical approach, we observed in the isolated CTCs methylation patterns resembling more those of epithelial-like cells. Methylation at the promoter of microRNA-200 family was significantly higher in prostate CTCs. Data from our single-cell analysis revealed an epigenetic heterogeneity among CTCs and indicates tumor-specific active epigenetic regulation of EMT-associated genes during blood-borne dissemination.

Characterization of a xenograft model of human ovarian carcinoma which produces ascites and intraabdominal carcinomatosis in mice.

We have used in vivo and in vitro procedures to select a subpopulation of cells from the human ovarian carcinoma cell line, NIH:OVCAR-3, with the capacity to grow i.p. in female nude athymic mice. After i.p. injection of these cells, animals develop metastatic spread similar to that of clinical ovarian cancer. Disease progression is characterized by the development of massive ascites, extensive invasive i.p. tumors, and pulmonary metastases. The malignant ascites cells are transplantable, manifest cytoplasmic androgen and estrogen receptors, and express the ovarian cancer associated antigen CA125 (116,000 units/ml of ascites supernatant). The cells also have the same chromosome markers which were present in the original cell line, NIH:OVCAR-3. Survival following i.p. passage of ascites is dependent on tumor cell inoculum ranging from a median survival of 39 days with 40 million cells to 84 days for 11.5 million transplanted cells. The characteristics of this unique in vivo model make it well suited for the evaluation of new drugs and novel experimental therapies in ovarian cancer. In addition, this in vivo model, together with ovarian cancer cell lines, may prove particularly useful for the study of pharmacological ways to specifically increase the cytotoxicity of anticancer agents in tumor cells while not increasing toxicity in normal tissues. The presence of hormone receptors should facilitate the experimental evaluation of hormonal therapy in ovarian cancer.

Radiation survival parameters of antineoplastic drug-sensitive and -resistant human ovarian cancer cell lines and their modification by buthionine sulfoximine.

The optimum integration of chemotherapy and irradiation is of potential clinical significance in the treatment of ovarian cancer. A series of human ovarian cancer cell lines have been developed in which dose-response relationships to standard anticancer drugs have been determined, and the patterns of cross-resistance between these drugs and irradiation have been established. By stepwise incubation with drugs, sublines of A2780, a drug-sensitive cell line, have been made 100-fold, 10-fold, and 10-fold more resistant to Adriamycin (2780AD), melphalan (2780ME), and cisplatin (2780CP). Two additional cell lines, NIH:OVCAR-3nu(Ag+) and NIH:OVCAR-4(Ag+), were established from drug-refractory patients. 2780ME, 2780CP, OVCAR-3nu(Ag+), and OVCAR-4(Ag+) are all cross-resistant to irradiation, with DOS of 146, 187, 143, and 203, respectively. However, 2780AD remains sensitive to radiation, with a DO of 111, which is similar to that of A2780 (101). Glutathione (GSH) levels are elevated in 2780ME, 2780CP, OVCAR-3nu(Ag+), and OVCAR-4(Ag+) to 4.58, 6.13, 12.10, and 15.14 nmol/10(6) cells as compared to A2780, with 1.89 nmol/10(6) cells. However, the GSH level in 2780AD is only minimally higher than that in A2780 (2.94 nmol/10(6) cells). Buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly increases the radiation sensitivity of 2780ME (changing the DO from 143 to 95) and 2780CP to a lesser extent, suggesting that intracellular GSH levels may play an important role in the radiation response of certain neoplastic cells. These results suggest that the sequential use of irradiation following chemotherapy with melphalan and cisplatin may be less effective than a combined modality approach, which integrates radiation and chemotherapy prior to the development of drug resistance and cross-resistance to irradiation.

Characterization of a cis-diamminedichloroplatinum(II)-resistant human ovarian cancer cell line and its use in evaluation of platinum analogues.

Human ovarian cancer cell lines with stable cisplatin resistance have been developed by chronic exposure of the parent cisplatin-sensitive A2780 line to increasing concentrations of cisplatin. 2780CP8 (CP8 refers to this cell line's growth in medium containing 8 microM cisplatin) has several clonal cytogenetic abnormalities but lacks homogeneously staining regions or double-minute chromosomes. It has a significantly greater monolayer growth rate, cloning efficiency in agarose, and total glutathione content compared to the A2780 line, but similar activities of several glutathione-dependent enzymes. The 2780CP8 subline is 7.3-fold resistant to cisplatin compared to the A2780 line, as well as cross-resistant to irradiation and melphalan. It is not cross-resistant to Adriamycin, but this develops with increased cisplatin resistance (14-fold) obtained by further cisplatin exposure of 2780CP8. Of the cisplatin analogues tested which are of current clinical interest, carboplatin, iproplatin, and tetraplatin, only the latter is more cytotoxic than cisplatin in the A2780 and 2780CP8 lines. The 2780CP8 subline is also cross-resistant to these analogues in the relative order carboplatin greater than iproplatin greater than tetraplatin (most to least cross-resistant). Treatment of a highly cisplatin resistant cell line (2780CP70) with either melphalan or cisplatin was associated with a significant increase in [3H]thymidine incorporation into DNA in the presence of 10 mM hydroxyurea compared with the parent sensitive cell line which showed essentially no capacity to repair DNA damage by these drugs. A2780 and its cisplatin-resistant cell lines may thus be useful in studying drug resistance mechanisms, in screening new drugs for activity (especially against drug resistant tumors), and in formulating induction and salvage therapies for ovarian cancer.

[Determination of the elastic properties of the compact bone in the femur of dogs]. / Ermittlung der elastischen Eigenschaften der Kompakta im Femur des Hundes.

Total hip endoprotheses are a good possibility for treatment degenerative wear and pathologic damage of the hip joint in man as well as in dogs. However, aseptic loosening of the protheses, especially in the area of the shaft, is still a problem in conventional total hip endoprotheses. The purpose of the present study was to use the finite-element-analysis (FEA), to enhance endoprothetic design and to prevent loosening of protheses. In order to simulate the femur of the dog for numerical analysis, a material law for the compacta in the femur of the dog was developed. The elastic properties of the compacta were experimentally determined by using compression tests of bone samples of euthanised dogs. The results show constant denseness and a constant axial elastic modul in the compacta.

Phase II trial of interferon-beta-serine in metastatic renal cell carcinoma.

Interferon-beta-serine (IFN-beta-ser) is a muteine, recombinant IFN that is tolerated at a dose fivefold to 10-fold higher than IFN-alfa and interacts with the same cell membrane receptor as IFN-alfa. We hypothesized that at high doses IFN-beta-ser might induce a higher response rate than IFN-alfa in metastatic renal cell carcinoma. We undertook a phase II trial of IFN-beta-ser in patients with metastatic renal cell carcinoma. Patients were treated three times each week by a 2-hour intravenous infusion. Doses were escalated weekly (.25 to 5.5 mg, 1 mg = 180,000,000 U) until the maximum-tolerated treatment dose (MTTD) was determined. The MTTD is defined as one dose level less than that which caused grade 3 toxicity and was subsequently administered three times weekly for at least 4 weeks. Twenty-nine patients were entered, and 25 were assessable for response and toxicity. The performance status was 0-1 in all patients and only one patient received previous chemotherapy. The MTTD dose was 2.5 mg (range, 0.5 to 5.5 mg per treatment), although in 10 patients, doses were later deescalated because of cumulative toxicity. Initial dose-limiting toxicity and cumulative toxicity were fatigue, malaise, and fever in most patients. Hepatic transaminitis, neutropenia, and elevation of serum creatinine were also observed but were not dose-limiting. There was one complete response (CR) and four partial responses (PRs). All responses but one occurred in pulmonary metastases. The median time to response was 26 days (range, 17 to 102 days). These data demonstrate that IFN-beta-ser given in high doses exhibits significant antitumor activity in renal cell carcinoma; however, the objective response rate is 20%. This is no higher than previous IFN studies; therefore, we reject the hypothesis than IFN-beta-ser at high doses may induce a greater response rate than IFN-alfa. However, we did observe more responses than were seen in a similar trial undertaken with lower dose IFN-beta serine in renal cell carcinoma.

The microtubule cytoskeleton in human phagocytic leukocytes is a highly dynamic structure.

The microtubule cytoskeleton of human leukocytes has been difficult to study, in part, due to the lack of a reliable protocol for the indirect immunofluorescence staining of microtubules in these cells. We report here the development of a simple and reliable immunocytochemical labeling protocol for the examination of microtubules in leukocytes including monocytes, neutrophils, and eosinophils. The dynamic properties of microtubules in both monocytes and neutrophils were examined by indirect immunofluorescence staining of cells following exposure to nocodazole. Nocodazole-induced depolymerization is extremely rapid in both cell types, as is the regrowth of microtubules following removal of the nocodazole. Rapid reorganization of the microtubule cytoskeleton was also observed in neutrophils undergoing chemotactic stimulation. Bundling of microtubules was observed in both monocytes and neutrophils isolated from patients undergoing taxol infusion chemotherapy. The taxol-induced bundles were transient in nature as they were absent from samples collected 48 h following the completion of the taxol infusion. These results demonstrate the unique dynamic properties of leukocyte microtubules and indicate that they can be altered in vivo. The development of this staining protocol should allow for the further analysis of leukocyte microtubules as related to the normal functional response of these cells and form the basis for correlating alterations in microtubule dynamics with the effects of taxol on leukocyte function.

Phase II studies of recombinant human tumor necrosis factor alpha in patients with malignant disease: a summary of the Southwest Oncology Group experience.

From June 1988 to November 1990 the Southwest Oncology Group initiated nine protocols for the phase II evaluation of recombinant human tumor necrosis factor alpha (rhuTNF alpha) in cancer patients. Patients with diverse metastatic malignancies including breast, colon, gastric, pancreatic, endometrial, and bladder cancers, as well as multiple myeloma and various sarcomas received 150 micrograms/m2 of rhuTNF alpha daily for 5 days every other week. Of 147 patients entered in the study, 127 were eligible and were evaluated for toxicity and response. Of 124 patients known to have completed treatment, 92 (74%) went off study for progression, 21 (17%) for toxicity, and 12 (10%) for other causes, mainly that of worsening medical condition. Thirteen percent of patients experienced grade 4 or fatal toxicity. The most serious toxicities were pulmonary failure and coagulopathies. The predominant grade 3 toxicities were symptomatic (chills, fever, malaise, headache, myalgia, and nausea or vomiting). Only one partial remission was seen in a patient with metastatic bladder cancer lasting 4 months (rate 0.8%, exact 95% confidence interval 0-4%). At the study dose and schedule, rhuTNF alpha does not appear to have significant antitumor activity. The biological basis for this finding is discussed.

Induction of progesterone receptor with 17 beta-estradiol in human ovarian cancer.

We utilized a xenograft model of human ovarian cancer to study the ability of estrogen to induce progesterone receptor. Tumor cytosol from 17 beta-estradiol treated oophorectomized animals, but not oophorectomized controls, contained a [3H]ORG 2058 binding moiety of sedimentation coefficient 6-9S. This component showed specificity for the progestagens: progesterone, ORG 2058, and R5020 and for the antiprogestagen cyproterone acetate. At 1000-fold molar excess, 5 alpha-dihydrotestosterone competed partially for these sites while diethylstilbestrol, dexamethasone, and the antiandrogen, SCH 16423, were ineffective competitors. The dissociation constant for this progestagen binding entity was 0.14 nM using [3H]ORG 2058 as labeled ligand and R5020 as competitor. In addition, saturation analysis demonstrated that approximately 400 fmol of progestagen specific binding capacity was available per mg of cytosol protein. These data suggest that estrogen can induce progesterone receptor in human ovarian carcinoma.

High dose cisplatin and high dose carboplatin in refractory ovarian cancer.

High dose cisplatin (40 mg/m2 qd x 5) and high dose carboplatin (400 mg/m2 qd x 2) were administered to advanced ovarian cancer patients who were refractory to standard therapy which included standard dose cisplatin regimens. Cisplatin was administered in 250 ml of 3% saline with 61 per day of saline hydration while carboplatin was administered in 500 ml D5W by continuous infusion for 48 hours. Objective responses were observed in 6/19 (32%) patients treated with high dose cisplatin and an additional 8 patients (42%) had minor responses or stable disease. The preliminary response rate in an ongoing phase II trial of high dose CBDCA is 33% (4/12 patients). There have been no responses to high dose CBDCA in patients who were resistant to high dose cisplatin. The dose limiting toxicity of high dose cisplatin is a peripheral neuropathy while high dose carboplatin results in severe, but reversible, myelosuppression. High dose carboplatin was less emetogenic than cisplatin and did not produce renal toxicity or peripheral neuropathy. In addition, in vitro cytotoxicity studies in cisplatin sensitive and resistant human ovarian cancer cell lines demonstrated a steep dose response relationship with both cisplatin and carboplatin and extensive cross resistance between these two platinum analogs. Studies are currently in progress to determine the efficacy and toxicity of high dose cisplatin or high dose carboplatin combined with alkylating agents in previously untreated advanced ovarian cancer patients.

Failure of beta-amyloid protein fragment 25-35 to cause hippocampal damage in the rat.

Considerable excitement has been generated as of late over reports that fragments of the amyloid precursor protein can be neurotoxic both in vivo and in vitro. In this brief report we study the neurotoxicity of the fragment corresponding to amino acids 25-35 of the beta-amyloid protein in the hippocampus in vivo. Under the conditions studied, we do not observe any evidence of consistent, dose-related damage above that seen with vehicle alone.

DNA methyltransferase genes of Bacillus subtilis phages: comparison of their nucleotide sequences.

The phi 3T DNA methyltransferase (Mtase) and most of the SP beta Mtase genes have been sequenced. With the exception of their promoters, no difference was found between the phi 3T and SP beta Mtase genes which code for an enzyme with a Mr of 50 507, consisting of 443 amino acids (aa). Comparison of the deduced aa sequence of the phi 3T/SP beta type Mtase (target specificity: GGCC and GCNGC) with that of the previously established sequence of the SPR Mtase (Buhk et al., 1984) which has the target specificity GGCC and CCGG, reveals strong similarities between these two types of enzymes. There is, however, one striking difference: both the phi 3T/SP beta and the SPR enzymes contain at different positions inserts of 33 aa, which have no homology to each other. We suggest that the methylation specificity unique to each of the two types of Mtases (GCNGC in phi 3T/SP beta; CCGG in SPR) depends on these inserts, while the GGCC-specific modification potential common to all Mtases is determined by structures conserved in both types of enzymes. A DNA fragment of non-modifying phage Z, which shows homology to both flanks of the SPR Mtase gene, was also sequenced. This segment can be described as a derivative of SPR DNA, in which the Mtase gene and sequences at its 5' end have been deleted, with the deletion extending between two direct repeats of 25 bp.

Restriction and modification in Bacillus subtilis: nucleotide sequence, functional organization and product of the DNA methyltransferase gene of bacteriophage SPR.

Bacillus subtilis phage SPR codes for a DNA methyltransferase (Mtase) which methylates the 5' cytosine in the sequence GGCC and both cytosines in the sequence CCGG. A 2126-bp fragment of SPR DNA containing the Mtase gene has been sequenced. This fragment has only one significant open reading frame of 1347 bp, which corresponds to the Mtase gene. Within the sequence the Mtase promoter has been defined by S1 mapping. The size of the SPR Mtase predicted from the deduced amino acid composition is 49.9 kDal. This is in agreement with both the Mr of the purified enzyme and with that of the SPR Mtase gene product identified here by minicell technique. Base changes leading to mutants affected in Mtase activity were localized within the Mtase gene.