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Biomedical subjects

R Kurkela

Publications and source records attributed to R Kurkela.

17 recordsLinked to original sources
PubMed2005

Enzymes as modulators in malignant transformation.

Experimental data suggest that sex steroids have a role in the development of breast and prostate cancers. The biological activity of sex steroid hormones in target tissues is regulated by several enzymes, including 17beta-hydroxysteroid dehydrogenases (17HSD). Changes in the expression patterns of these enzymes may significantly modulate the intracellular steroid content and play a pathophysiological role in malignant transformation. To further clarify the role of 17HSDs in breast cancer, we analyzed the mRNA expressions of the 17HSD type 1, 2, and 5 enzymes in 794 breast carcinoma specimens. Both 17HSD type 1 and 2 mRNAs were detected in normal breast tissue from premenopausal women but not in specimens from postmenopausal women. Of the breast cancer specimens, 16% showed signals for 17HSD type 1 mRNA, 25% for type 2, and 65% for type 5. No association between the 17HSD type 1, 2, and 5 expressions was detected. The patients with tumors expressing 17HSD type 1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients. The expression of 17HSD type 5 was significantly higher in breast tumor specimens than in normal tissue. The group with 17HSD type 5 overexpression had a worse prognosis than the other patients. Cox multivariate analyses showed that 17HSD type 1 mRNA, tumor size, and ERalpha had independent prognostic significance. Using an LNCaP prostate cancer cell line, we developed a cell model to study the progression of prostate cancer. In this model, androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in oxidative 17HSD activity was seen, whereas reductive activity seemed to increase. Since local steroid metabolism controls the bioavailability of active steroid hormones of target tissues, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of organs.

17-Hydroxysteroid Dehydrogenases↗
PubMed2004

Sex steroid hormone metabolism and prostate cancer.

The growth and function of the prostate is dependent on androgens. The two predominant androgens are testosterone, which is formed in the testis from androstenedione and 5alpha-dihydrotestosterone, which is formed from testosterone by 5alpha-reductases and is the most active androgen in the prostate. Prostate cancer is one of the most common cancers among men and androgens are involved in controlling the growth of androgen-sensitive malignant prostatic cells. The endocrine therapy used to treat prostate cancer aims to eliminate androgenic activity from the prostatic tissue. Most prostate cancers are initially responsive to androgen withdrawal but become later refractory to the therapy and begin to grow androgen-independently. Using LNCaP prostate cancer cell line we have developed a cell model to study the progression of prostate cancer. In the model androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in the oxidative 17beta-hydroxysteroid dehydrogenase activity was seen whereas the reductive activity seemed to increase. The changes suggest that during transformation estrogen influence is increasing in the cells. This is supported by the cDNA microarray screening results which showed over-expression of several genes up-regulated by estrogens in the LNCaP cells line representing progressive prostate cancer. Since local steroid metabolism controls the bioavailability of active steroid hormones in the prostate, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of the organ.

17-Hydroxysteroid Dehydrogenases↗
PubMed2004

17 beta-hydroxysteroid dehydrogenases--their role in pathophysiology.

17 beta-Hydroxysteroid dehydrogenases (17HSDs) regulate the biological activity of sex steroid hormones in a variety of tissues by catalyzing the interconversions between highly active steroid hormones, e.g. estradiol and testosterone, and corresponding less active hormones, estrone and androstenedione. Epidemiological and endocrine evidence indicates that estrogens play a role in the etiology of breast cancer, while androgens are involved in mechanisms controlling the growth of normal and malignant prostatic cells. Using LNCaP prostate cancer cell lines, we have developed a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition into more aggressive cells. Our data suggest that substantial changes in androgen and estrogen metabolism occur in the cells, leading to increased production of active estrogens during the process. In breast cancer, the reductive 17HSD type 1 activity is predominant in malignant cells, while the oxidative 17HSD type 2 mainly seems to be present in non-malignant breast epithelial cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach in treating estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered to be estrogen target tissues, such as the gastrointestinal tract.

17-Hydroxysteroid Dehydrogenases↗
PubMed2002

17 beta-hydroxysteroid dehydrogenases and cancers.

17 beta-Hydroxysteroid dehydrogenases (17HSDs) catalyze the interconversions between active 17 beta-hydroxysteroids and less-active 17-ketosteroids thereby affecting the availability of biologically active estrogens and androgens in a variety of tissues. The enzymes have different enzymatic properties and characteristic cell-specific expression patterns, suggesting differential physiological functions for the enzymes. Epidemiological and endocrine evidence indicate that estrogens play a key role in the etiology of breast cancer while androgens are involved in mechanisms controlling the growth of prostatic cells, both normal and malignant. Recently, we have developed, using LNCaP prostate cancer cell lines, a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition to more aggressive cells, able to grow in suspension cultures. Our results suggest that substantial changes in androgen and estrogen metabolism occur in the cells during the process. These changes lead to increased production of active estrogens during transformation of the cells. Data from studies of breast cell lines and tissues suggest that the oxidative 17HSD type 2 may predominate in human non-malignant breast epithelial cells, while the reductive 17HSD type 1 activity prevails in malignant cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach to treat estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered as estrogen target tissues such as colon. Our data show that the abundant expression of 17HSD type 2 present in normal colonic mucosa is significantly decreased during colon cancer development.

17-Hydroxysteroid Dehydrogenases↗
PubMed2001

Ligand affinity, homodimerization, and ligand-induced secondary structural change of the human vitamin d receptor.

The intrinsic tryptophan fluorescence signal of the full-length nuclear receptor hVDR was used to directly determine the dissociation constants, K(d), of two ligands yielding K(d) = 32 nM for 1alpha,25(OH)(2)D(3) and K(d) = 322 nM for 25(OH)D(3). Ligand binding was accompanied by a conformational change in the alpha-helical part of hVDR as revealed by CD spectroscopy. In addition, the presence of calcitriol was found to be a necessary prerequisite for the homodimerisation of hVDR which was monitored using fluorescence anisotropy. We conclude that the observed ligand-induced structural change of hVDR is conditional for dimerisation of the protein.

Calcifediol↗
PubMed1998

Androgen-sensitive human prostate cancer cells, LNCaP, produce both N-terminally mature and truncated prostate-specific antigen isoforms.

To characterize prostate-specific antigen (PSA) produced by cancer cells, different isoforms of PSA secreted by the human prostate cancer cells, LNCaP, were purified. LNCaP-PSA production was induced by synthetic androgen, R1881. LNCaP-PSA was separated into four pools. The molecular mass of LNCaP-PSA isoforms in these pools was 34 kDa under reducing conditions and 29 kDa under nonreducing conditions on SDS/PAGE. pI of LNCaP-PSA isoforms varied from 6.8 to 8.2. Pool A had the highest specific activity, 37 nmol/(min x mg). All the pools formed stable complexes with alpha1-antichymotrypsin and alpha2-macroglobulin. The pools contained 10-60% of N-terminally correctly processed LNCaP-PSA isoforms. According to the molecular modelling, the addition or deletion of two or four N-terminal amino acids could affect the three-dimensional structure and thereby remarkably reduce the enzyme activity of LNCaP-PSA.

Amino Acid Sequence↗
PubMed1997

Human prostate-specific glandular kallikrein is expressed as an active and an inactive protein.

A polymorphism in the human prostate-specific glandular kallikrein (hKLK2) gene was described by direct sequencing (by PCR) of genomic DNAs isolated from prostatic cancer tissue, benign prostatic hyperplasia tissue, and blood leukocyte specimens. Results showed two forms of human prostate-specific glandular kallikrein protein (hK2), a consequence of a change from C to T at base 792 in the hK2 coding region. Producing the two forms as recombinant proteins in insect cells demonstrated that Arg226-hK2 (CC genotype) is an active protein and Trp226-hK2 (TT genotype) is inactive. Polymorphism studies of 36 patients with prostatic diseases identified only 1 with the TT genotype. The same kind of polymorphism was not detected in the human prostate-specific antigen (hKLK3) gene. Arg226-hK2 possessed only trypsin-like enzyme activity, whereas recombinant human prostate-specific antigen (hPSA) had only chymotrypsin-like activity. Monoclonal and polyclonal antibodies raised against hPSA purified from seminal plasma detected both active and inactive hK2. Thus, because inactive as well as stable hK2 protein may be present, a lack of trypsin-like activity in hPSA standards is not enough to confirm that the materials are free of hK2 contamination.

DNA↗
PubMed1995

Differential androgen regulation of rat prostatic acid phosphatase transcripts.

Steady-state levels of the mRNAs encoding rat prostatic acid phosphatase (rPAP) were measured from the ventral prostate of rats after castration and testosterone replacement. The longest and the most abundant mRNA molecule (4.9 kb) of the rPAP gene was quite resistant to the hormonal status of the prostate, whereas its medium-sized transcript (2.3 kb) and the shortest one (1.5 kb) were up-regulated by androgens. For comparison, we also analyzed the prostatic mRNA levels of rat lysosomal acid phosphatase (rLAP), whose gene expression is not considered to be hormonally dependent. Only modest variation was observed in the rLAP transcript after androgen withdrawal and replacement. Corresponding protein level investigations are analogous with our regulation studies, particularly in the case of 1.5-kb rPAP mRNA.

Acid Phosphatase↗
PubMed1995

Expression of active, secreted human prostate-specific antigen by recombinant baculovirus-infected insect cells on a pilot-scale.

We have expressed human prostate-specific antigen (PSA) on a pilot-scale in Spodoptera frugiperda Sf9 insect cells using recombinant baculovirus system. Infected cells secreted PSA into culture medium at a concentration of 2-4 mg per liter. PSA was expressed both in active and inactive forms which were separated in a final purification step using cation-exchange chromatography eluted with a low salt gradient. The N-terminus of active PSA was correctly cleaved; two amino acids of the propeptide remained, however, at the N-terminus of the inactive PSA. Purified recombinant PSA showed a chymotrypsin-like activity with the synthetic substrate MeO-Suc-Arg-Pro-Tyr-pNA, but did not have a trypsin-like activity when Pro-Phe-Arg-pNA was used. The molecular mass of active PSA was 31.0 kDa in reduced SDS-PAGE, 26.0 kDa in nonreduced SDS-PAGE and 26.5 kDa in ion spray mass spectrometry. The active protein formed complexes with alpha 1-antichymotrypsin (ACT) and alpha 2-macroglobulin (alpha 2M) in vitro similar to the commercial PSA purified from human seminal fluid.

Amino Acid Sequence↗
PubMed1993

Rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization.

Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system. Recombinant protein was secreted into the medium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells. About 20% of the cell protein produced was rPAP. Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the signal peptide is recognized and properly cleaved in insect cells. The enzyme was purified by using L-(+)-tartrate affinity chromatography. The purified protein had a high specific activity of 2620 mumol.min-1.mg-1 with p-nitrophenyl phosphate at the substrate, and it also showed phosphotyrosine phosphatase activity. The molecular mass of the recombinant rPAP was 155 kDa. Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase. The active enzyme is a trimer of subunits differing only in glycosylation. When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space group P3(1)21) with cell dimensions a = 89.4 A and c = 152.0 A. The observed diffraction pattern extends to a resolution of at least 3 A.

Acid Phosphatase↗
PubMed1993

Pilot-scale production of murine monoclonal antibodies in agitated, ceramic-matrix or hollow-fiber cell culture systems.

The purpose of this research was to compare three bioreactor systems for the pilot-scale production of monoclonal antibodies (MAbs). We needed to produce gram quantities of murine MAbs against human prostatic acid phosphatase for use in fragmentation, radiolabeling and in vivo radio-imaging studies. The stable hybridoma cell line secreting IgG1 antibodies was chosen for production. Of the available bioreactor systems, we chose to test an agitating 30-liter bioreactor in repeated batch mode, a ceramic-matrix bioreactor in both repeated batch and continuous perfusion modes and a hollow-fiber bioreactor in continuous perfusion mode. The highest cultured MAb yield, 151 +/- 126 mg/day (mean +/- SD, n = 22), was achieved in the 30-liter bioreactor in repeated batch mode with the MAb being harvested in a large volume of medium, giving a reactor productivity of 4.3 +/ 3.4 mg/liter/day (mean +/- SD, n = 22). The most concentrated MAb was harvested from the continuously perfused hollow-fiber bioreactor, which had the highest reactor productivity, 307 +/ 142 mg/liter/day (mean +/- SD, n = 47) and an average rate of MAb production of 55.3 +/- 25.7 mg/day (mean +/- SD, n = 47). Taking the use of serum into consideration, the cost of MAb production was lowest in the continuously fed and harvested ceramic-matrix and hollow-fiber cell culture systems. A compact blood glucose meter proved to be a novel and suitable device for the rapid monitoring of glucose concentrations in hybridoma cultures.

Acid Phosphatase↗
PubMed1990

Time-resolved immunofluorometric assay of human prostate-specific antigen.

Assay of human serum prostate-specific antigen (PSA) is gaining importance in diagnosis and follow-up of prostatic cancer. In this time-resolved immunofluorometric assay of PSA, strip-wells were coated with a polyclonal antibody against PSA. To prepare the label, a monoclonal antibody displaying high affinity towards PSA was purified and derivatized with diethylenetriaminepentaacetic acid. With use of this derivative, seven to eight Eu atoms could be combined with one antibody molecule with no decrease in immunoreactivity. The minimum detectable concentration of PSA was 0.12 microgram/L. In 60 of 63 women studied, the PSA concentration in serum was less than 0.2 microgram/L. The increase in PSA in serum of asymptomatic men 51 years old or older, as compared with that for younger subjects, was possibly a result of "occult" prostatic hyperplasia. Most of the patients with prostatic hyperplasia or prostatic cancer had higher PSA concentrations than did subjects of ages less than 50 years or asymptomatic age-matched healthy subjects. Results compared favorably with those by an established technique relying on the use of radioactive label. Our method for measuring PSA in human serum is convenient, inexpensive, and well compatible with present clinical practice.

Aged↗
PubMed1988

Preparation of F(ab')2 fragments from monoclonal mouse IgG1 suitable for use in radioimaging.

Monoclonal antibodies of IgG1 subclass raised against purified human prostate-specific acid phosphatase were subjected to different procedures to produce F(ab')2 fragments suitable for radioimaging of prostatic cancer, following derivatization and labeling with radionuclides. The main aim was to obtain highly purified fragments with preserved immunological activity. Optimized pepsin digestion led to the formation of mainly F(ab')2 and Fab fragments, and, following Sephacryl S-200 gel filtration, the yield of pure F(ab')2 fragments was 24 +/- 11% of the theoretical maximum. After papain digestion in the absence of thiols, no formation of Fab fragments was observed, and the F(ab')2 fragments formed could be efficiently separated from Fc fragments by chromatofocusing or ion exchange chromatography. The yield of F(ab')2 fragments from papain digestion was 50 +/- 5% of the theoretical maximum. Both the above procedures gave F(ab')2 fragments with immunoreactivity and affinity identical to those of the precursor IgG1, despite the fact that isoelectric focusing profiles of the two F(ab')2 preparations differed, suggesting different digestion sites.

Acid Phosphatase↗
PubMed1978

[Free fatty acids of wood mushrooms].

The total lipid content and percentages of free fatty acids in the lipids of nine wood mushrooms were determined. The total lipid content varied from 9% to 16% of the dry matter. Between 15.9 and 46.5% of the total lipids consisted of free fatty acids. The fatty acid composition of the free fatty acids of six mushrooms was analyzed quantitatively. The main component of the free acids was linoleic acid, which accounted for 30.3...60.7% of the free acids.

Basidiomycota↗
PubMed1976

[Dry matter losses in mushroom (Lactarius rufus) by blanching].

According to recommended international standards edible fungi are blanched before salting and freezing. A study was conducted on the solution losses of Lactarius rufus due to blanching. Weight losses, changes of dry matter, raw fat, total nitrogen, amino nitrogen and ash contents as well as the pH value were determined when various methods of blanching were used. 3 min blanching at 95-100 degrees C was able to inactivate catalase and peroxydase while 6 min blanching was needed for inactivating polyphenoloxydase totally. After blanching there were 1/10 - 1/100 of spores left. During the 3 min blanching in water five times the quantity of mushrooms the losses of dry matter were about 10%; when doubling the quantity of blanching water the losses increased to 2-3 fold. The doubling of blanching time had no significant influence on the losses. The soluble dry matter content of blanched mushrooms was less than 50% of that of the fresh. Total nitrogen of fresh mushrooms was equal to that of the blanched but the amino nitrogen decreased to one tenth by blanching. The mineral element content of blanched mushrooms was about the half of that of the fresh. Blanching caused a slight decrease in the pH value. The necessity of the blanching of all edible fungi before freezing was discussed.

Basidiomycota↗