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K H Ibsen

Publications and source records attributed to K H Ibsen.

At least 19 recordsLinked to original sources
PubMed1989

Expression of pyruvate kinase in astrocytes induced to differentiate in vitro.

Astrocytes maintained in a chemically defined media undergo differentiation and a parallel increase in pyruvate kinase specific activity. These changes are accompanied by a shift in the isoelectrofocusing pattern, but not by expression of pyruvate kinase M4, the characteristic adult rat brain isozyme. Thus, this chemically defined media lacks a substance required to induce pyruvate kinase M synthesis and this function can be uncoupled from other aspects of cellular differentiation. The uncoupling of pyruvate kinase maturation from cellular differentiation and the observation of only a single, 2.3 kilobase, pyruvate kinase mRNA molecule at different stages of the postnatal development of rat brain support the concept that the K- to M-isoform transformation is a post-transcriptional event. The effect of the individual components of this chemically defined medium on pyruvate kinase specific activity was studied by eliminating one component at a time. The increase in activity was found to be completely dependent upon fibroblastic growth factor and prostaglandin F2 alpha and was partially dependent on the simultaneous presence of insulin.

Aging↗
PubMed1989

Independent regulation of pyruvate kinase expression by cyclic AMP and prostaglandin F2 alpha in mouse mastocytoma cells.

P-815 mouse mastocytoma cells express the K isozyme of pyruvate kinase and the specific activity of this enzyme is increased in response to N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate, 8-bromoadenosine 3':5'-cyclic monophosphate, cholera toxin, and epinephrine, all of which also elevate the intracellular concentration of adenosine 3':5'-cyclic monophosphate. Prostaglandin F2 alpha also increases the cellular activity of this enzyme, but does not increase the adenosine 3':5'-cyclic monophosphate levels. Under all these conditions, the increase in enzymatic activity is accompanied by an equivalent increase in the pyruvate kinase protein level. However, neither the rate of enzyme synthesis nor the level of pyruvate kinase mRNA is elevated by N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate. On the other hand, it does increase the enzyme's half-life. In contrast, prostaglandin F2 alpha increases the rate of synthesis and the level of pyruvate kinase K mRNA, but has no influence on the rate of degradation. Therefore, these cells have two mechanisms which increase pyruvate kinase K levels. One operates via an increase in cAMP level and results in a decrease in the rate of degradation, whereas the other minimizes an upsurge in cAMP levels but still increases pyruvate kinase K activity by increasing its rate of synthesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Pyruvate kinase and total protein are regulated differently during growth of P-815 mastocytoma cells.

Total protein content of P-815 mastocytoma cells decreases and then increases in response to initiation of a new growth cycle. As the level increases, the rate of synthesis declines. Both events occur prior to any decrease in the rate of cellular proliferation. These temporal relationships indicate that the rate of protein synthesis reflects the intracellular concentration of protein rather than the cellular growth rate, as has been hypothesized. Pyruvate kinase protein metabolism differs from that of total protein in three ways: (a) accumulation does not stop, (b) the rate of synthesis does not decrease, and (c) only the rate of pyruvate kinase degradation is altered by a factor present in conditioned media. These observations suggest that there are specific mechanisms regulating pyruvate kinase at a post-transcriptional level.

Animals↗
PubMed1985

Studies on the association of the tetracyclines with mineralized tissue.

The course of work directed by Dr. Marshall R. Urist concerning the chemistry of the complexes between tetracyclines, bones, and the various other tissues is a landmark in hard tissue biology. These pioneer studies helped provide the biochemical basis of now standard methods of determining rates of turnover of mineralized tissues.

Animals↗
PubMed1984

Regulation of pyruvate kinase expression and growth in P-815 mastocytoma cells. II. Potential role of prostaglandins.

P-815 mastocytoma cells increase the level of pyruvate kinase (PK) expression in response to chloroform-methanol extracts of conditioned media, butyrate, and dibutyryl cyclic AMP (but2cAMP) plus theophylline. The butyrate effect is indomethacin sensitive, suggesting a prostaglandin (PG) is the active signaling factor. Moreover, the chloroform-methanol extracts contain PGE2 and PGF2 alpha and additions of the latter enhance PK activity. PGE2 alone has little or no effect but acts synergistically with PGF2 alpha. These data show that PGF2 alpha can regulate PK levels. On the other hand, other factors may also be active, since the endogeneous and the but2cAMP plus theophylline effects are indomethacin insensitive. Most of the factors that increase PK activity also inhibit cellular growth; however, regulation of PK expression can be uncoupled from growth inhibition.

Animals↗
PubMed1984

Soluble suppressor factors elaborated in experimental malignant ascites.

Soluble suppressor factors in the sera of cancer patients inhibit lectin-stimulated lymphocyte proliferation. These factors, derived from human material, preclude easy corroboration by other investigators. To gain a general understanding of soluble suppressor factors and to avoid the necessary restrictions of human experimentation, an animal model was devised. Sprague-Dawley rats were injected ip with the Walker 256 carcinoma. The resultant ascites proved to be a stable, reproducible source of soluble suppressor factors. Ascites inhibited phytohemagglutinin (PHA)-induced blastogenesis of normal splenocytes by 98%. The possibility of a toxic effect was eliminated by vital staining of splenocytes and by examination in a specific lymphotoxin assay. Suppressor activity persisted after heating at 100 degrees C for 40 min. Extraction by lipid solvents revealed that the bulk of suppressor activity resides in the lipid phase. The active fraction of heat-treated ascites passed through an Amicon PM-10 filter. Thin-layer chromatography revealed the presence of prostaglandins E2 and F2 alpha. Tissue culture supernatants from short-term cultures derived from tumor-bearing animals revealed suppressor activity from thymus, spleen, and liver cultures (97, 91, and 71%, respectively). No suppressor activity was detected in cultures of cancer cells. This study has demonstrated in this animal model that prostaglandins play a major role in suppression of lectin-induced blastogenesis. All suppressor factors appear to be host derived. An understanding of the mechanism of release of these suppressor substances may open new avenues in the immunotherapy of cancer.

Animals↗
PubMed1984

Pyruvate kinase isoenzymes in human serum.

Pyruvate kinase activity in serum from presumably healthy individuals is labile, but can be maintained for a week when samples are stored frozen. Contrary to published data our electrophoretic and isoelectrofocusing studies with fresh sera show the presence of both K and M isoenzymes; the latter predominates. In freezer-stored serum the putative M isoenzyme showed a progressive decrease in pl value as well as a decreased electrophoretic mobility.

Adult↗
PubMed1983

Regulation of pyruvate kinase expression and growth in mastocytoma cells. I. Initial observations.

The specific activities of pyruvate kinase and phosphofructokinase but not lactate dehydrogenase increase as P-815 mastocytoma cells approach the stationary phase. During this growth period, the rates of uptake of labelled precursors into DNA, RNA and total protein decreases. On the other hand, the pyruvate kinase protein level changes in parallel with activity. Although the K-isozyme is the primary form of pyruvate kinase expressed, some M-type subunit is also present and both forms undergo an increase in specific activity. In addition, pyruvate kinase expression is also elevated by adding cAMP analogues with theophylline, butyrate or conditioned media. This increased level of expression is hypothesized to be a secondary event associated with a differentiation-like-induced expression of the mast cell phenotype.

Animals↗
PubMed1983

Erythrocyte membrane 2', 3'-cyclic nucleotide 3'-phosphodiesterase activity in multiple sclerosis.

The specific activities of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase, EC3.1.4.37) in erythrocyte membranes from five patients with active multiple sclerosis (MS) and five normals were compared. The CNPase assay used 2',3'-cyclic NADP as the substrate in an enzyme coupled reaction to produce fluorometrically detectable NADPH. The specific activity of CNPase in the MS and normal groups were 19.2 +/- 2.3 and 28.7 +/- 1.8 mumol/h/mg protein, respectively. The difference was significant with p less than 0.006, based on a one-tailed t-test.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1982

Deficiency and urinary losses of factor XII in adult nephrotic syndrome.

Factor XII was measured in the plasma and urine of 16 adult patients with nephrotic syndrome and 10 normal volunteers using a monospecific antibody to human factor XII. In addition plasma factor XII procoagulant activity was determined in both groups. Plasma immunoreactive factor XII level was significantly lower in the nephrotic group than the control group. This was associated with a significant reduction of plasma factor XII procoagulant activity. To examine the effect of albumin concentration on factor XII procoagulant activity purified human serum albumin was added in the in vitro system. Manipulation of albumin concentration failed to alter plasma factor XII activity. Considerable amounts of factor XII were recovered in the urine of all but one of the tested nephrotic patients while none were found in the urine of the control group. Plasma factor XII concentration was within normal limits in the single patient whose urine contained no detectable factor XII. Moreover, there was a significant negative correlation between plasma and urinary factor XII concentrations (r = -0.79; p less than 0.01). In addition, plasma factor XII concentration showed a significant correlation with serum albumin concentration (r = 0.63; p less than 0.05). It thus appears that renal losses of this plasma protein contribute to its low plasma concentration in our patients.

Adult↗
PubMed1982

Expression of multimolecular forms of pyruvate kinase in normal, benign, and malignant human breast tissue.

The levels of multimolecular forms of pyruvate kinase present in four normal human breast specimens, nine benign tissues, and 13 malignant breast carcinomas were determined. The different enzymatic forms were separated by isoelectrofocusing, quantitated photometrically, and characterized further by kinetic studies using phosphoenolpyruvate as the variable substrate in the presence of different effectors. A correlation between specific activity and malignancy was found. The mean specific activities (+/- S.E.) of the normal, benign, and malignant tissues were: 0.078 +/- 0.006, 0.36 +/- 0.072, and 3.50 +/- 0.696 IU/mg protein, respectively. A form of pyruvate kinase with an isoelectric point (pI) of 7.0 predominated in the breast tissues. The properties of this form were consistent with it being the K4 isozyme, known to be widely distributed in mammalian tissues. Higher pI forms were also found. The M4 isozyme, expressed by normal muscle and brain, has a pI value similar to the highest pI form found in the breast tissues. Therefore, the pI data alone suggest that the breast specimens also express some M-type subunits. This conclusion was not supported by the kinetic data. The higher pI forms are thought to be a posttranslationally modified K isozyme. Although this modified form is found in normal specimens, it seems more prevalent in neoplasms.

Breast Neoplasms↗
PubMed1981

Purification and properties of mouse pyruvate kinases K and M and of a modified K subunit.

The K4 and M4 isozymes of mouse pyruvate kinase were purified to homogeneity, and their physical, chemical, and kinetic properties were compared. The K isozyme is slightly larger, but a high degree of homology exists as evidenced by a similar amino acid composition, immunotitration value, and two-dimensional arginine peptide pattern after tryptic digestion. Also, the more active conformational form of the K isozyme has kinetic and chromatographic properties similar to those of the M isozyme. Only K subunit could be extracted with antibody from fresh spleen extracts, but this subunit can be cleaved to form a product with the mobility of the M subunit. The cleavage is accomplished by an endogenous enzyme and appears to be the first step in K-enzyme degradation. This product is called Kpm . KpmK hybrid could also be purified to homogeneity. This enzyme has the structure K2pmK2, and both types of subunit have activity. The Kpm form has a higher K0.5S value for phosphoenolpyruvate and a lower K0.5S value for ADP than does either the K or the M type. However, the Kpm and M subunits otherwise have very similar properties and it is speculated that the Kpm subunit is an M-type precursor.

Amino Acids↗
PubMed1977

Interrelationships and functions of the pyruvate kinase isozymes and their variant forms: a review.

The relationships among and the properties of the pyruvate kinase isozymes are reviewed, emphasizing their potential role in carcinogenesis. Particular consideration is given to evaluation of the concept that the three major nonreadily interconvertible forms are the products of distinct genes, the relationship of these forms to additional separable forms of pyruvate kinase, the types and possible functions of interconvertible forms of the major isozymes, and mechanisms affecting the genetic expression of the isozymes. Emphasis is placed upon the apparent derepression of the fetal isozyme in hepatomas and the influence of neoplasms and their extracts on the expression of pyruvate kinase in the liver of host animals.

Animals↗
PubMed1976

Electrofocusing and kinetic studies of adult and embryonic chicken pyruvate kinases.

Chicken embryos less than 15 days old contain only the K isozyme of pyruvate kinase, which appears to exist in vivo as an R,T conformational set with pI values of 7.2 and 6.6, respectively. Sets of lower pI and higher pI K-isozyme variants also are obtained. Whole embryos of 15 days or more of development show progressively increasing amounts of higher pI, lower K0.5S enzymatic variants. Tissue distribution and kinetic properties suggest that the highest pI form (pH 8.8-9.0) is an M-isozyme analogue. The intermediate forms are postulated to be hybrids. Adult liver extracts contain only the embryonic K isozyme; no evidence for an L-isozyme analogue was obtained. All major forms of the enzymes are compared with respect to saturation by phosphoenolpyruvate in the absence of effector and in the presence of fructose 1,6-diphosphate, alanine, serine, phenylalanine, tryptophan, and/or Mg-ATP.

Aging↗
PubMed1976

Inhibition of chicken pyruvate kinases by amino acids.

Alanine, serine, and phenylalanine behave as inhibitors competitive with phosphoenolpyruvate for the activated forms of the chicken pyruvate kinases. On the other hand, phenylalanine and alanine behave as K-type inhibitors and serine behaves as a heterotropic activator of pyruvate kinase variants which undergo homotropic activation. Tryptophan lowers the Vm and tends to yield complex plots with all variants studied. Kinetic patterns obtained in the presence of phenylalanine also show some characteristics not generally associated with a competitive mechanism. These observations are related to data previously obtained using the rat isozymes and are used to formulate a mechanism which explains the effects of the amino acids. This mechanism hypothesizes that all the effector amino acids bind to the phosphoenolpyruvate site; however, amino acids with nonpolar side chains also interact with a nonpolar region of the T conformer and thereby stabilize it. It is further proposed that there are two such nonpolar regions on the various pyruvate kinases--the one which reacts with the nonbulky side chains, and another which reacts only with relatively bulky side chains. The stabilizing effect of this second nonpolar interaction imparts inhibitory characteristics which are not competitive in nature. Serine and perhaps other polar compounds may also bind at the phosphoenolpyruvate site, but because of their polarity exert a repulsive force at the same nonpolar site with which the nonbulky nonpolar amino acids interact. This repulsion stabilizes the R conformation. Presumably the homotropic activating effects of phosphoenolpyruvate operate via this same mechanism. The data are also used to support a specific sequential-concerted mechanism for the homotropic activating effect of phosphoenolpyruvate. According to this mechanism, phosphoenolpyruvate adds sequentially to the first two subunits. This interaction causes the respective subunits to convert to the R conformation but, once two subunits are in R conformation, the remaining two subunits convert in concert.

Alanine↗
PubMed1975

Extraction of a factor from Ehrlich ascites tumor cells that increases the activity of the fetal isozyme of pyruvate kinase in mouse liver.

Isoelectrofocusing studies of mouse tissue extracts show mice to have a pyruvate kinase isozyme pattern very similar to that of the rat. Moreover, electrofocusing or kinetic assays conducted on liver extracts from normal mice and from mice bearing Ehrlich ascites tumors show that the latter have a higher proportion of the fetal K-isozyme of pyruvate kinase. Serial injection of the supernatant remaining after centrifugation of homogenized tumor cells at 100,000 x g, or of the phenolic extracts from the latter, produced a similar shift in the liver isozyme pattern involves both a decrease in L-isozyme activity and an increase in K-isozyme activity. However, only the increase in activity of the K-isozyme appears to be a specific response to injection of the extracts. The presence of a specific factor in these extracts was confirmed by the observation that similar extracts prepared from normal adult tissues did not increase activity of the K-isozyme. On the other hand, phenolic extracts from fetal mice did increase K-isozyme activity as did injections of serum from tumor-bearing mice or of the cell-free ascites fluid. Evidence is presented supporting the concept that the factor is proteinaceous in a nature, and that it acts by deprepressing synthesis of the K-isozyme.

Animals↗