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Biomedical subjects

I Palva

Publications and source records attributed to I Palva.

At least 19 recordsLinked to original sources
PubMed1992

Randomised, placebo-controlled multicentre trial of clodronate in multiple myeloma. Finnish Leukaemia Group.

Osteolytic lesions and pathological fractures are common in multiple myeloma. Because clodronate inhibits osteoclastic resorption, we did a randomised, controlled trial in 350 patients from 23 hospitals. All patients received standard melphalan-prednisolone, and were randomised to receive clodronate 2.4 g daily or placebo for 24 months. The proportion of patients with progression of osteolytic bone lesions was twice as high in the placebo group (n = 168 at baseline) than in the clodronate group (n = 168 at baseline) in an intention-to-treat analysis (24 vs 12%, p = 0.026). Progression of vertebral fractures was lower in the clodronate group, but the difference was not significant (30 vs 40%). Serum calcium and urinary calcium excretion decreased significantly in both groups, but the changes were greater in the clodronate group. The percentage of patients feeling no pain increased more in the clodronate group (from 24 to 54%, p < 0.001) than in the placebo group (from 29 to 44%, p < 0.01). Side-effects were similar in both groups. We conclude that clodronate is an effective and safe adjunct in the management of multiple myeloma. The drug delays osteolytic bone lesions, reduces the degree of hypercalcaemia and hypercalciuria, and decreases pain.

Aged↗
PubMed1992

Expression of the Erwinia carotovora polygalacturonase-encoding gene in Bacillus subtilis: role of signal peptide fusions on production of a heterologous protein.

The pehA gene encoding an endopolygalacturonase (pectinase) of Erwinia carotovora subsp. carotovora has been cloned previously [Saarilahti et al., Mol. Microbiol. 4 (1990) 1037-1044]. We expressed pehA in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the alpha-amylase (Amy)-encoding gene, amyE, from Bacillus amyloliquefaciens. To test whether the location of the junction between the secretion vector and pehA affects the protein yield, we made four different junctions. Two constructs contained an intact Amy signal sequence, whereas the other two were fusions between the Amy signal sequence and the polygalacturonase (PG) signal sequence. There was approximately fourfold variation in the production efficiency of B. subtilis strains carrying the different constructs. The most efficient construct contained the N-terminal and hydrophobic regions of the Amy signal peptide joined to the C terminus of PG signal peptide. This construct produced, in a shake flask culture, 0.8 g of polygalacturonase per liter of growth medium. In a pulse-chase experiment, the signal peptide of the most efficient construct was rapidly cleaved while cleavage was slow in the other constructs. Our results suggest that fusions containing intact signal peptides, which are common when producing foreign proteins, are not necessarily the most efficient.

Amino Acid Sequence↗
PubMed1992

Improving the production of E. coli beta-lactamase in Bacillus subtilis: the effect of glucose, pH and temperature on the production level.

Bacillus subtilis has been considered a promising host for the production of foreign proteins. However, proteases released by the host organism can often cause rapid breakdown of secreted heterologous proteins. Here we report that the addition of 6% glucose and 100 mM potassium phosphate to the growth medium significantly reduces the degradation of E. coli TEM beta-lactamase secreted from B. subtilis, when applying an expression system based on B. amyloliquefaciens alpha-amylase. The yield of beta-lactamase was increased 10-20-fold when compared to the yield in Luria medium. The promoter of B. amyloliquefaciens alpha-amylase gene is repressed by glucose. However, here we show that the repression does not take place in a multicopy plasmid, thus enabling our approach to efficiently reduce the protease action by catabolite repression. We have also studied the role of pH and temperature on the beta-lactamase production in laboratory scale bioreactors. Low temperature and low pH are both favorable for a high level beta-lactamase production by the high copy plasmid construction.

Bacillus subtilis↗
PubMed1992

Bolus therapy with mitoxantrone and vincristine in combination with high-dose prednisone (NOP-bolus) in resistant multiple myeloma. Nordic Myeloma Study Group (NMSG).

In a phase II study, 58 patients with resistant multiple myeloma (MM) were treated with a combination chemotherapy (NOP-bolus regimen) consisting of mitoxantrone (16 mg/m2 for the first 25 patients and 12 mg/m2 for the subsequent 33), vincristine (2 mg), both as bolus injections on day 1 and prednisone (250 mg/d on d 1-4 and 17-20). In patients greater than 70 years of age, the mitoxantrone dose was reduced to 12 mg/m2 or 8 mg/m2, respectively. The treatment was repeated every 4 weeks. A response (greater than 50% reduction in M component) was obtained in 26% of the patients and a minor response (clinical improvement but less than 50% reduction in M component) in another 21%. Median response duration was 27 wk and median survival for all patients was 25 wk. There were no differences in response rate or duration between patients receiving the high or low mitoxantrone dose, but patients in the low-dose group had fewer serious infections.

Adult↗
PubMed1992

S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence.

The surface (S)-layer protein of Lactobacillus brevis was isolated, purified, and characterized. The S-layer protein is the major protein of the cell, with an apparent molecular mass of 46 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunogold electron microscopy with polyclonal antiserum against the isolated 46-kDa protein was used to confirm the surface location of this protein. N-terminal amino acid sequences of the intact 46-kDa protein and its tryptic peptides were determined. The gene of the S-layer protein was amplified from the genome of L. brevis by polymerase chain reaction with oligonucleotides, synthesized according to the N-terminal amino acid sequences, as primers. The polymerase chain reaction fragments containing the entire S-layer gene and its regulatory regions were sequenced. Nucleic acid sequence analysis revealed one open reading frame with a capacity to encode a protein of 48,159 Da. From the regulatory region of the gene, two subsequent promoters and a ribosome binding site, showing typical features of prokaryotic consensus sequences, were found. The coding region contained a characteristic gram-positive-type signal peptide of 30 amino acids. Removal of the signal peptide results in a polypeptide of 435 amino acids, which is in excellent agreement with the size of the S-layer protein determined by SDS-PAGE. The size and the 5' end analyses of the S-layer transcripts confirmed the monocistronic nature of the S-layer operon and the functionality of the two promoters found.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of the secY gene from Lactococcus lactis and identification of conserved regions by comparison of four SecY proteins.

Sec Y is an integral membrane protein which participates in the translocation of proteins through the bacterial cell membrane. We have cloned the sec Y gene of Lactococcus lactis, and found its deduced protein sequence, 439 amino acids long, to be similar in length to the previously determined Sec Y proteins of Escherichia coli, Bacillus subtilis and Mycoplasma capricolum. Comparison of the L. lactis Sec Y to the 3 other Sec Y proteins revealed 90 conserved amino acid residues (21%). Nearly half of the conserved residues are clustered in 2 of the 10 transmembrane segments, and in 2 of the 6 cytoplasmic regions. Some of the conserved regions are apparently responsible for the interactions of Sec Y with signal sequences, and the proteins SecE and SecA.

Amino Acid Sequence↗
PubMed1991

Production of pectin methylesterase from Erwinia chrysanthemi B374 in Bacillus subtilis.

The gene coding for pectin methylesterase (PME) of Erwinia chrysanthemi B374 (pme) was cloned by a polymerase chain reaction. The pme gene was expressed in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the alpha-amylase gene from B. amyloliquefaciens. The cultivation of B. subtilis cells carrying the cloned pme resulted in efficient secretion of PME into the culture medium based on enzymatic and sodium dodecyl sulphate-poly-acrylamide gel electrophoresis characterizations. The NH2-terminal sequence analysis of the secreted PME revealed two different NH2-termini. Heterologous processing was probably due to a second putative signal peptidase cleavage site at the joint region between the PME and alpha-amylase signal peptide.

Bacillus subtilis↗
PubMed1991

Refractory myelomatosis treated with mitoxantrone in combination with vincristine and prednisone (NOP-regimen): a phase II study. The Nordic Myeloma Study Group (NMSG)

In a phase II study, patients with refractory myelomatosis were treated with a combination chemotherapy (NOP regimen): mitoxantrone (bolus injection of 4 mg/m2 on days 1-4), vincristine (continuous infusion of 0.4 mg/24 h on days 1-4) and prednisone (250 mg/d on days 1-4 and 17-20). The treatment was repeated every 4 weeks. Ninety-two patients were treated after they were found refractory to treatment with melphalan and prednisone (and occasionally vincristine) (n = 50) or more intensive treatment regimens (n = 42) including anthracyclines (n = 18). Response (greater than or equal to 50% reduction of M protein) was obtained in 23 patients and minor response (clinical improvement but less than 50% reduction in M protein) in 22 patients. The median duration of the response was 7.5 months. Equal response rates were observed irrespective of the type of previous treatment. The major toxicity was myelosuppression with severe granulocytopenia and infections. However, the frequency decreased throughout the cycles. The NOP treatment is recommended in refractory myelomatosis, especially in patients refractory to other intensive regimens. Patients in a poor clinical condition or with thrombocytopenia before treatment should have a reduced mitoxantrone dose in the first treatment cycles.

Adult↗
PubMed1991

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.

Base Sequence↗
PubMed1991

Secretion of TEM beta-lactamase with signal sequences isolated from the chromosome of Lactococcus lactis subsp. lactis.

With TEM beta-lactamase as a reporter gene, a set of expression-secretion-promoting fragments were isolated from the chromosome of Lactococcus lactis subsp. lactis. The fact that only translocated beta-lactamase renders cells resistant to ampicillin allowed direct ampicillin selection with an Escherichia coli vector (pKTH33). The clones showing the greatest ampicillin resistance were subcloned onto a replicon capable of replication in lactic acid bacteria (pVS2), and the nucleotide sequences of the relevant fragments were determined. The structure of the secretion-promoting fragments in general resembled that of gram-positive true signal sequences, with a strongly positively charged N terminus, a long hydrophobic core, and a putative signal peptidase recognition site. The promoterlike sequences preceding the signal sequences matched well with those of previously published lactococcal promoters. In addition to E. coli, the functioning of these expression-secretion cassettes was studied in three gram-positive hosts: Bacillus subtilis, L. lactis, and Lactobacillus plantarum. Efficient expression and secretion of TEM beta-lactamase into the culture medium of each gram-positive host was obtained. Furthermore, when a strain of L. lactis subsp. lactis showing increased sensitivity to lysozyme was compared with a standard laboratory strain, threefold-higher secreted enzyme activities were detected.

Amino Acid Sequence↗
PubMed1990

Production and secretion of pertussis toxin subunits in Bacillus subtilis.

Pertussis toxin (PT) is a major component of today's acellular whooping cough vaccines. The use of acellular vaccines is predicted to increase sharply in the near future. There is therefore a need to produce PT in a way that makes its purification as easy as possible. Our approach was to express all five PT subunits individually in Bacillus subtilis. We have used vectors containing the promoter and signal sequences of the alpha-amylase gene of Bacillus amyloliquefaciens followed by an insert encoding the appropriate PT-subunit. All PT-subunits were secreted and found in the culture supernatant. The level of expression varied considerably: S1 and S5 were produced in large quantities whereas much smaller amounts of S2, S3 and S4 were found. The subunits were also present in the membrane fraction of the respective strains.

Amino Acid Sequence↗
PubMed1990

Structural analysis of the pehA gene and characterization of its protein product, endopolygalacturonase, of Erwinia carotovora subspecies carotovora.

A clone producing a polygalacturonase (EC 3.2.1.15) in Escherichia coli was isolated from a genomic library of Erwinia carotovora subspecies carotovora constructed in PUC18. The DNA segment carrying the corresponding structural gene, named pehA, contained an open reading frame (ORF) encoding a 402-amino-acid (aa) polypeptide with an Mr of 42,849. In E. carotovora the polygalacturonase was synthesized with a 26-aa cleavable signal peptide. The mature 376-aa PehA had a calculated Mr of 40,064 and a pl of 10.19. The pH optimum of the enzyme was about 5.5 and the temperature optimum was in the range 35-45 degrees C. Analysis of the reaction products of polygalacturonic acid hydrolysis indicated that the PehA protein is an endopolygalacturonase. No similarity was observed between the aa sequences of PehA and other pectic enzymes of erwinias. However, substantial similarity was detected within the C-terminal portions of PehA and a previously described tomato polygalacturonase, suggesting that the bacterial and eukaryotic polygalacturonases may have a common origin.

Amino Acid Sequence↗
PubMed1990

Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase.

We have cloned the operon coding for the Bacillus subtilis S complex, which has been proposed to be a component in protein secretion machinery. A lambda gt10 library of B. subtilis was screened with antiserum directed against the Staphylococcus aureus membrane-bound ribosome protein complex, which is homologous to the B. subtilis S complex. Two positive overlapping lambda clones were sequenced. The S-complex operon, 5 kilobases in size, was shown to contain four open reading frames and three putative promoters, which are located upstream of the first, the third, and the last gene. The four proteins encoded by the operon are 42, 36, 48, and 50 kilodaltons in size. All of these proteins were recognized by antisera separately raised against each protein of the S. aureus membrane-bound ribosome protein and B. subtilis S complexes, thus verifying the S-complex identity of the lambda clones. Sequence analysis revealed that all four proteins of the B. subtilis S complex are homologous to the four subunits of the human pyruvate dehydrogenase (PDH). Also, the N terminus of the 48-kilodalton protein was found to have 70% amino acid identity with the N-terminal 211 amino acids, determined so far, from the E2 subunit of B. stearothermophilus PDH. Furthermore, chromosomal mapping of the S-complex operon gave a linkage to a marker gene located close to the previously mapped B. subtilis PDH genes. Thus, the S complex is evidently identical to the B. subtilis PDH, which has been shown to contain four subunits with molecular weights very similar to those of the S complex. Therefore, we propose that the S complex is not a primary component of protein secretion.

Amino Acid Sequence↗
PubMed1989

Do treatment protocols improve end results? A study of survival of patients with multiple myeloma in Finland.

OBJECTIVE: To determine whether patients with multiple myeloma treated in three consecutive clinical trials of chemotherapy of the Finnish Leukaemia Group during 1979-85 had a more favourable prognosis than both patients treated in the trial area before the trials and those treated in the rest of Finland. DESIGN: Comparison of the time trend in survival of patients living in the trial area with that of patients living in the rest of Finland. SETTING: Trial area covered 17 of the 21 main hospital districts in Finland (serving more than two thirds of patients with multiple myeloma). PATIENTS: 663 Men and 690 women in the trial area and 318 men and 307 women in the reference area aged under 71 in whom multiple myeloma was diagnosed during 1959-85. In the trial area the disease was diagnosed in 455 men and 493 women in 1959-78 and in 208 men and 197 women in 1979-85; in the reference area it was diagnosed in 234 men and 227 women in 1959-78 and in 84 men and 80 women in 1979-85. MAIN OUTCOME MEASURES: Five year cumulative relative survival rates during 1959-85, annual relative survival rates in the first seven years of follow up during 1979-85, and fitted annual relative survival rates in the first five years of follow up during 1959-85. RESULTS: During the first two years of follow up the annual relative survival rates did not differ between the two areas, but in the next five years of follow up patients in the trial area did better than those in the reference area. For cases diagnosed in 1979-85 the difference in the five year cumulative relative survival rates between patients in the trial area and those in the reference area was 10%, those in the trial area doing better. Generalised proportional hazards regression analysis of the first five years of follow up showed that the patients in the trial area had a survival advantage over those in the reference area. The model with the best fit included year of follow up, time of diagnosis, the joint effect of year of follow up and time of diagnosis, and the joint effect of area and time of diagnosis. CONCLUSION: The patients in the trial area benefited from the clinical trials, which suggests that the use of a treatment protocol improves the end results of treatment. In other words, the results favour a systematic treatment schedule in preference to a schedule determined by the free choice of a clinician.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1989

Production of diphtheria toxin CRM228 in B. subtilis.

The gene coding for a nontoxic diphtheria toxin (DT), tox228, was isolated from lysogenic Corynebacterium diphtheriae and cloned into pBR322. A mature form of the tox228 gene, lacking its signal sequence, was expressed in Bacillus subtilis using a B. amyloliquefaciens alpha-amylase secretion vector. To test the possibility of producing partially deleted DT molecules, which could be used for cell-directed toxin conjugates, a truncated form lacking 151 amino acids from the C-terminus of the DT was generated by oligonucleotide mutagenesis. Both the truncated and intact DT were efficiently secreted into the culture medium. During prolonged cultivation, the truncated form was less stable than the intact DT molecule.

Amino Acid Sequence↗
PubMed1988

Quantification of alpha-amylase mRNA in Bacillus subtilis by nucleic acid sandwich hybridization.

A novel hybridization test for quantification of mRNA from bacterial cells lysed directly in the culture medium was developed and optimized. The method uses two adjacent probes from a DNA fragment of interest in a sandwich hybridization. An unlabeled probe is immobilized on a solid support to capture homologous nucleic acids in the test solution. Hybrid detection is performed with the other, labeled, probe, which can bind to the filter only as a sample-mediated hybrid. We used the Bacillus amyloliquefaciens alpha-amylase gene and its mRNA as models for characterizing gene expression in Bacillus subtilis. To confirm the specificity of mRNA hybridization, nondenatured nucleic acid samples were allowed to react simultaneously with two filters: the mRNA-specific filter containing anti-sense single-stranded DNA and the background filter containing single-stranded DNA of the mRNA sense, respectively. We also developed a rapid enzymatic lysis procedure for B. subtilis, allowing complete degradation of late stationary phase cells grown in rich culture medium, while retaining mRNA molecules of high integrity. The applicability of this rapid lysis procedure and the hybridization method for mRNA analyses in long-term fermentations was demonstrated.

Bacillus subtilis↗