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Biomedical subjects

D Kowalski

Publications and source records attributed to D Kowalski.

90 records · Page 5Linked to original sources
PubMed1977

An endonuclease activity of venom phosphodiesterase specific for single-stranded and superhelical DNA.

A homogeneous preparation of venom phosphodiesterase from Crotalus adamanteus possesses an intrinsic endonuclease activity, specific for superhelical (form I) and single-stranded DNA. The phosphodiesterase degrades single-stranded T7 DNA by endonucleolytic cleavages. Duplex T7 DNA is hydrolyzed by the liberation of acid-soluble products simultaneously from the 3' and 5' termini but without demonstrable internal scissions in duplex regions. Since venom phosphodiesterase is known to hydrolyze oligonucleotides stepwise from the 3' termini, the cleavage at the 5' end of duplex T7 DNA is ascribed to an endonuclease activity. Form I PM2 DNA is nicked to yield first relaxed circles and then linear DNA which is subsequently hydrolyzed only from the chain termini. The linear duplex DNA intermediates consist of a discrete series of fragments (11 are usually resolved on agarose gels) with initial molecular weights ranging from 6.3 x 10(6) (the intact PM2 DNA size) to approximately 1 x 10(6). The cleavage of the form I molecule must, therefore, occur at a limited number of unique sites. The enzyme also cleaves nonsuperhelical, covalently closed circular PM2 DNA but at a 10(4) times slower rate. Both the endonuclease activity on form I DNA and the known exonuclease activity co-migrate on polyacrtkanude gels, are optimally active at pH 9, are stimulated by small concentrations of Mg2+, and are similarly inactivated by heat, reducing agents, and EDTA.

DNA, Single-Stranded↗
PubMed1976

Mung bean nuclease I. Physical, chemical, and catalytic properties.

A simplified purification procedure for mung bean nuclease has been developed yielding a stable enzyme that is homogeneous in regards to shape and size. The nuclease is a glycoprotein consisting of 29% carbohydrate by weight. It has a molecular weight of 39 000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme contains 1 sulfhydryl group and 3 disulfide bonds per molecule. It has a high content (12.6 mol %) of aromatic residues. Approximately 70% of the enzyme molecules contain a peptide bond cleavage at a single region in the protein. The two polypeptides, 25 000 and 15 000 daltons, are covalently linked by a disulfide bond(s). Both the cleaved and intact forms of the enzyme are equally active in the hydrolysis of the phosphate ester linkages in either DNA, RNA, or adenosine 3'-monophophate. The enzymatic activity of mung bean nuclease can be stabilized at pH 5 in the presence of 0.1 mM zinc acetate, 1.0 mM cysteine, and 0.001% Triton X-100. The enzyme can be inactivated and reactivated by the removal and readdition of Zn2+ or sulfhydryl compounds.

Amino Acids↗
PubMed1976

Mung bean nuclease I. Terminally directed hydrolysis of native DNA.

Under conditions which favor the duplex structure of DNA, mung bean nuclease catalyzes a limited number of double-strand cleavages (probably less than 50) in the interior of native T7 DNA. However, under conditions which are not as favorable to a tight helical structure, the large duplex polymers previously produced are completely degraded from their termini with a continuous accumulation of mono-, di-, and trinucleotides. The terminally directed activity is an intrinsic property of the enzyme molecule because (1) it is inactivated and reactivated in parallel with the single-strand activity and (2) the two activities coelectrophorese on analytical gels. Kinetic measurements indicate that the apparent Km for the terminally directed hydrolysis of native DNA is relatively high. The pH optimum for both the hydrolysis of denatured DNA and the terminally directed hydrolysis of native DNA becomes more acidic with increasing salt concentration. The relative preference for single-stranded structures increases as the pH becomes more basic.

DNA↗
PubMed1976

Chemical-enzymatic replacement of Ile64 in the reactive site of soybeen trypsin inhibitor (Kunitz).

All the reactive amino groups in soybean trypsin inhibitor (Kunitz) were protected by guanidination of 9 out of 10 lysyl residues with O-methylisourea and by carbamoylation of the NH2 terminal Asp with potassium cyanate. This derivative was converted to modified inhibitor (Arg63-Ile64 reactive site peptide bond hydrolyzed) by incubation with trypsin at pH 3. The NH2 terminal of Ile64 was allowed to react with phenyl isothiocyanate to produce inactive phenylthiocarbamoyl-modified inhibitor. Treatment with trifluoroacetic acid formed the anilinothiazolinone of Ile64 yielding des-Ile64-modified inhibitor. After renaturation and purification, this material coelectrophoresed with modified inhibitor but did not form a stable complex with trypsin. Incubation with tert-butyloxycarbonyl-(amino acid)-N-hydroxysuccinimide esters yielded [tert-butyloxycarbonyl-(amino acid64)]-modified inhibitor. The tert-butyloxycarbonyl protective group was removed in trifluoroacetic acid. After renaturation, active [amino acid64]-modified inhibitors were obtained for Ile64, Ala64, Leu64, and Gly64 replacements. The resynthesis of the reactive-site peptide bound by kinetic control dissociation of the trypsin-inhibitor complex yielded fully active [Ala64]-virgin inhibitor. Thus, soybean trypsin inhibitor (Kunitz) has been shown to tolerate the replacement of the P1' residue with retention of activity. The importance of P1' residues in the function of protein proteinase inhibitors is discussed.

Amino Acid Sequence↗
PubMed1976

Chemical-enzymatic insertion of an amino acid residue in the reactive site of soybean trypsin inhibitor (Kunitz).

Modified (Arg63-Ile64 reactive-site peptide bond hydrolyzed) soybean trypsin inhibitor (Kunitz) with all reactive amino groups, except that of Ile64, protected was described in the preceding paper (Kowalski, D., and Laskowski, M., Jr. (1976), Biochemistry, preceding paper in this issue). Treatment of this inhibitor with tert-butyloxycarbonyl-Ala- and tert-butyloxycarbonyl-Ile-N-hydroxy-succinimide esters yields inactive endo-tert-butyloxycarbonyl-Ala63A-and endo-tert-butyloxycarbonyl-Ile63A-modified inhibitors. The tert-butyloxycarbonyl groups were removed by treatment of the proteins with trifluoroacetic acid. After renaturation and purification, the resultant endo-Ala63A- and endo-Ile63A-modified inhibitors co-electrophorese with modified inhibitor both on disc gels (pH 9.4) and sodium dodecyl sulfate gels (after reduction of disulfide bonds) and show end groups corresponding to the 63A residue. These derivatives fail to form stable complexes with trypsin, extending the previous observation (Kowalski, D., and Laskowski, M., Jr. (1972), Biochemistry 11, 3451) that acylation of the P1' residue in modified inhibitors leads to inactivation. However, the incubation of endo-Ala63A- and endo-Ile63A-modified inhibitors with trypsin at pH 6.5 leads to the synthesis of the Arg63-Ala63A and Arg63-Ile63A peptide bonds in 4% yield. This is very close to the yield anticipated from a semiquantitative theory for the value of the equilibrium constant for reactive-site peptide bond. An alternative chemical method of insertion is also described. Controlled treatment of modified inhibitor with the N-carboxyanhydride of Glu produced inactive endo-Glu63A-modified inhibitor. Incubation of this inactive derivative with trypsin at pH 6.5 leads to 16% synthesis of the Arg63-Glu63A peptide bond. The higher yield of single chain protein in this case is attributed to the influence of the negative charge of the Glu63A side chain. Thus, the insertion of an amino acid residue between the P1 and P1' residues in soybean trypsin inhibitor (Kunitz) converts a trypsin inhibitor into a trypsin substrate.

Amino Acid Sequence↗
PubMedDate not supplied

[Effect of hemodialysis on the concentration of beta 2-microglobulin and acute phase proteins in the serum of patients with chronic renal failure].

In 24 patients with terminal renal failure the concentrations were determined of beta 2-microglobulin, prealbumin, alpha 1-antitrypsin, ceruloplasmin, alpha 2-macroglobulin, antithrombin III, plasminogen, transferrin, C3c and Cr complement components in the serum before and after haemodialysis. A statistically significant rise of concentrations of these proteins was found. It is thought that this rise was due mainly to haemoconcentration, while the contact with dialysing membranes is less important.

Acute-Phase Proteins↗
PubMedDate not supplied

[Biochemical neoplasm markers in selected neurological diseases].

Using radioimmunoassay methods the authors assayed the concentration of biochemical neoplasm markers (BMN) in 106 patients with neurological diseases (M-56, F-50) in the serum, and in 20 cases in the cerebrospinal fluid. In certain cases these markers were present, and sometimes their concentration was raised: ferritin in multiple sclerosis from 200 to 1365 ng/ml, in ischaemic stroke up to 327.9 ng/ml, in Parkinson's disease up to 423 ng/ml in the serum. In some cases of other diseases the levels of carcinoembryonic antigen (CEA), acid prostatic phosphatase (PAP) and alpha-fetoprotein (AFP) were raised, similarly as that of human chorionic gonadotropin (HCG). Further studies are being conducted on BMN, including also other markers (CA 125, CA 199), with monoclonal antibodies, beta-endorphins and prostaglandins in neurological diseases, including multiple sclerosis. It is suggested (Nowak) that BMN may have an indirect role in the aetiology and pathogenesis of certain diseases of the nervous system and that they may have connections with prostaglandins.

Acid Phosphatase↗
PubMedDate not supplied

Studies on serum gastrin concentrations after test meal stimulation in patients suffering from bronchial asthma.

In patients suffering from bronchial asthma serum gastrin concentrations were measured before the feeding of a standard meal and at 15, 30, 60, 90, and 120 minutes after. All subjects studied were divided into 2 main groups: I. Those not receiving corticosteroids and II. Those undergoing long-term treatment with corticosteroids. Results obtained from these two groups were compared to those found in 32 healthy persons. In the group I a statistically significant increase in serum gastrin concentrations was seen in subjects with the atopic form of bronchial asthma, before test meal ingestion and 15, 30 and 60 min after, where as in subjects with non atopic form of bronchial asthma an increase in serum gastrin concentration was found before the feeding of test meal and 15, 60, 90 and 120 min after. In all subjects suffering from both forms of bronchial asthma and belonging to group II, serum gastrin concentrations were significantly elevated in comparison with healthy persons. This increase in serum gastrin level was greater in subjects with non atopic for of bronchial asthma. Patients having contracted complications associated with corticosteroids administration presented serum gastrin levels higher than those without complications. Oral administration of corticosteroids resulted in more intensive stimulation of gastrin release than parenteral administration.

Administration, Oral↗
PubMedDate not supplied

[Relation between serum levels of C-terminal fragment, beta 2 microglobulin and creatinine in patients with chronic nephropathies at different stages of the disease].

The serum concentrations of the C-terminal fragment of parathyroid hormone, beta 2-microglobulin and creatinine were determined in 40 patients with chronic nephropathies at various stages of renal function impairment. Statistically significant positive correlations were found between the concentrations of creatinine and the C-terminal fragment (r = 0.6656, p less than 0.001), creatinine and beta 2-microglobulin (r = 0.7752, p less than 0.001) and beta 2-microglobulin and the C-terminal fragment (r = 0.6391, p less than 0.001). The authors believe that the concentration of the C-terminal fragment i the serum in chronic renal failure is not reflecting the degree of secondary hyperparathyroidism but is only an index of renal function failure.

Adult↗
PubMedDate not supplied

[ACTH in the plasma and cerebrospinal fluid in patients with multiple sclerosis].

In 1984 and at the beginning of 1985 the authors carried out radioimmunoassays (SORIN-CIS kit) the plasma levels of ACTH in 116 multiple sclerosis patients (m-52, f-64) and in 10 cases this radioimmunoassay was done in the cerebrospinal fluid (m-5, f-5). The control group comprised 90 patients with ischialgia and neuroses. The normal value in the plasma was from 0 to 80.86 pg/ml, and in the fluid it was from 0 to 77.08 pg/ml. In multiple sclerosis patients the plasma ACTH level was from 0 to 286.9 pg/ml, in the cerebrospinal fluid from 0 to 89 pg/ml. The values of ACTH were significantly higher in multiple sclerosis patients, mainly in males. In the fluid the level of ACTH was significantly higher in the studied patients. No significant differences in ACTH levels were found between males and females with multiple sclerosis, and in the control group this level was higher in females. Raised ACTH level was found mainly in multiple sclerosis with lung duration of the disease (10 years) at the time of exacerbations. The authors continue studies on the axis hypothalamus-hypophysis-adrenals, on various hormones, prostaglandins, beta-endorphin, biochemical markers, cAMP, cCMP, arylosulphatase A and B MBC etc.

Adrenocorticotropic Hormone↗
PubMedDate not supplied

[Growth hormone levels in the blood serum and cerebrospinal fluid of patients with multiple sclerosis].

In 1984 the authors determined by radioimmunoassay (ORIPI-RIA kit) the serum growth hormone levels in 35 patients with multiple sclerosis (20 M, 15 F) aged from 20 to 54 years, and in 10 cases the determination was carried out in the cerebrospinal fluid (CSF). The control group comprised 40 patients with ischialgia or neuroses. The normal range of GH values in the serum determined with this kit was from 0 to 80 microIU/ml, and the laboratory normal range was from 0 to 10 microIU/ml. The normal GH value for the CSF is being established (calculations based on mean values). No significant differences were observed in the GH concentrations in the serum between the patients and the control group, and between males and females in either group. No significant differences were found also in the GH level in the CSF of males and females with multiple sclerosis. Treatment with corticosteroids received by over 80% of the patients (at least 6 months before the study) caused no significant rise in GH concentration. However, an increasing tendency was observed of GH concentration in patients which requires confirmation in a greater number of cases.

Adult↗
PubMedDate not supplied

[Prolactin in the blood serum of patients with multiple sclerosis].

The authors determined in 1984 by radioimmunoassay (INEP kit) the serum prolactin concentration in 76 multiple sclerosis patients (33 males and 34 females) aged from 21 to 53 years, with disease duration from 2 to 30 years. The control group comprised 110 healthy subjects. Normal prolactin values were 150-750 mIU/ml in females and 150-500 mIU/ml in males. In about one-third of the patients raised prolactin level was found which failed to correlate with the age of the patients and with increased beta-endorphin level in them. The authors suppose that hyperprolactinaemia may be of importance in certain clinical signs of multiple sclerosis. Further investigations on prolactin are conducted with reference to the circadian rhythm of prolactin secretion and dynamic tests.

Adult↗