Isoenzyme Distribution Research Articles

Neuroendocrine phenotype in lung cancers: Comparison of immunohistochemistry with biochemical determination of enolase isoenzymes

The authors evaluated methods of recognition of neuroendocrine differentiation in lung cancer because this is thought to bear prognostic value. One hundred forty lung tumors were divided by immunohistochemical analysis using neuroendocrine markers (neuron-specific enolase, Leu7, chromogranin, neural cell adhesion molecules, SL11/14, and Leu19) into two groups of 51 neuroendocrine tumors positive for three neuroendocrine markers and 89 non-neuroendocrine tumors. Biochemical determination of enolase activity and isoenzyme distribution showed that the level of total enolase activity was similar between neuroendocrine and non-neuroendocrine tumors. alpha gamma and gamma gamma enolase isoenzyme percentages were higher in neuroendocrine tumors. A cut-off of gamma enolase % (alpha gamma/2 + gamma gamma) at 14% gave a sensitivity rate of 84% and specificity rate of 97% in separating neuroendocrine and non-neuroendocrine tumors, whereas immunohistochemical analysis using anti-gamma enolase had low specificity (68%) and immunohistochemical analysis with Leu 7 and chromogranin had high specificity (97%) and low sensitivity (37% and 60%) levels for neuroendocrine tumors. The best prediction of neuroendocrine differentiation was obtained using immunohistochemical analysis against neural cell adhesion molecules combined with biochemical estimation of enolase using gamma enolase of 14% and a gamma gamma isoenzyme more than 3%.

Distribution and Cellular Localization of Aspartate Aminotransferase Isoenzymes in Rice

Aspartate aminotransferase (EC 2.6.1.1, AAT) occurs as two forms in rice bran (from Oryza sativa cv. Koganemasari), AAT-1 and AAT-2. Immunotitrations with antisera against the AAT isoenzymes indicated an immunological distinction between AAT-1 and AAT-2. Sequential titration of total AAT activity showed that AAT-1 and AAT-2 comparised 82% and 18%of the total activity in the crude extract of rice bran, respectively. AAT-1 and AAT-2 comprised 45% and 55% of the total AAT activity in the crude extract of top tissues of the rice plant. AAT-1 was mainly in the cytosolic fraction of the rice plant cells. On the other hand, AAT-2 was the major isoenzyme of AAT in mitochondria and peroxisomes. Minute activity of AAT-1 was found in the membrane fractions of the mitochondria and peroxisomes and was solubilized with Triton X-100.

Altering creatine kinase isoenzymes in transgenic mouse muscle by overexpression of the B subunit.

To change the levels of expression and isoenzyme distribution of creatine kinase (CK) in muscle, transgenic technology was used to express the B subunit of CK in mouse muscle. Normally, mammalian skeletal muscle contains the MM dimer of CK. The BB dimer and MB heterodimer of CK can be found in brain and heart, respectively. Heterologous genes consisting of skeletal and cardiac muscle-specific actin promoters fused to the genomic coding region of the B form of CK were used to create transgenic mice. Lines were established from the three highest expressing founders. Analysis of skeletal muscle extracts revealed that all three lines had an increase in total CK activity measured under maximal velocity conditions. The highest expressing line, 7001, had a CK activity 150% that of control muscle. Nuclear magnetic resonance saturation transfer was used to measure the in vivo rate of the CK reaction. In 7001 hindlimb muscles, the CK catalyzed reaction was 200% that of control muscle. The elevation in CK activity in transgenic muscle was accompanied by significant changes in the composition of the cytosolic isoenzyme ratio of CK. In control, 100% of CK was MM, whereas 7001 had 60 +/- 18% MM, 32 +/- 10% MB, and 8 +/- 2% BB. There were no changes in ATP, phosphocreatine, Pi, or creatine levels in transgenic muscle compared with control. Immunofluorescence of myofibrils isolated from control and transgenic muscle revealed specific association of CK to the M line. Small amounts of MB CK were detected on myofibrils from transgenic mice. Transgenic mice expressing the B subunit of CK in muscle represent a first step toward altering CK isoforms so as to elucidate the specific roles of these isoforms in energy metabolism.

Expression of carbonic anhydrase isoenzyme-like immunoreactivity in the limbus spiralis of the human fetal cochlea

The distribution of carbonic anhydrase isoenzymes (CA I, II. III, V) was analyzed in the 14-, 15- and 16-week-old human tetal cochleae using immunohistochemical methods. The interdental cells in the limbus spiralis were strongly labelled with antibodies against CA I and III and the spiral ligament was also positive for CA I and III. The stria vascularis and organ of Corti were however negative for CA I, II, III and V. These results suggest that the interdental cells in the human fetus may be related to fluid and ion transport of endolymph especially in the early stages of development.

Protein kinase C in the rat retina: immunocharacterization of calcium-independent δ, ϵ and ζ isoenzymes

Using isoenzyme-specific antibodies, subtypes of protein kinase C were determined in isolated retinae of dark adapted and light-exposed rats by SDS-PAGE-Western blotting. In addition to the previously observed α and β-subspecies, the rat retina also expressed the δ, ϵ- and ζ-isoenzymes of protein kinase C. Exposure of the animals to physiological or high levels of light does not elicit changes in the pattern or in the distribution of the different isoenzymes in the cytosolic or particulate fractions. This study demonstrates, for the first time, the presence of three calcium-independent protein kinase C isoenzymes in the rat retina.

Glutathione S-transferase activity and isoenzyme distribution in ovarian tumour biopsies taken before or after cytotoxic chemotherapy

A study involving the measurement of glutathione S-transferase activities and isoenzyme distributions in human ovarian tumours has been carried out. These tumours have been obtained either at initial debulking surgery, prior to cytotoxic chemotherapy, or at second look laparotomy following chemotherapy. The response rates of these two groups to chemotherapy differ markedly, with patients who have relapsed following initial chemotherapy showing a reduction in response rates to subsequent chemotherapy. Analysis of these data show no statistically significant differences between the glutathione S-transferase activity or isoenzyme distribution in these two groups of patients. Significant differences were observed in the glutathione-S-transferase activities (GST) between tumours and normal ovaries. GST activities in pre-chemotherapy tumours (n = 33, P = 0.01) and post-chemotherapy tumours (n = 20, P = 0.001) where significantly higher than the GST activity in normal ovaries (n = 15). One feature was the expression of the basic isoenzyme which is expressed more in normal ovaries than in tumours. No differences in these parameters were observed in normal peritoneal tissue taken from patients before or after chemotherapy. These data do not support the hypothesis that changes in glutathione S-transferase enzyme activity or isoenzyme expression are major determinants of response to chemotherapy in ovarian tumours.

Rat Plasma Clearance Rate and Organ Distribution of β-Hexosaminidase Isoenzymes from Human Serum

beta-Hexosaminidase (EC 3.2.1.30) is markedly increased in human serum in liver disease, chronic alcoholism, and pregnancy. Knowledge of the clearance rate of plasma/serum beta-hexosaminidase is necessary to evaluate this increase. We studied the plasma clearance of beta-hexosaminidase isoenzymes (purified from human serum and placenta) after their infusion into rat circulation. A recently developed enzyme immunoassay method was used to measure the human beta-hexosaminidase isoenzymes; this method relies on both immunoreactivity and enzyme activity, so intact human isoenzymes were measured. In comparison with the placental isoenzymes (t1/2 less than 2 min), the serum forms showed a considerably slower clearance (t1/2 = 2-4 h). However, desialylation of the serum forms resulted in their rapid clearance (t1/2 less than 2 min). The organ localization of the enzyme eliminated from the circulation was investigated 24 h after infusion. Placental and native serum isoenzymes accumulated mainly in the liver and the spleen. Desialylated serum forms were almost exclusively localized to the liver.

Neuroendocrine phenotype in lung cancers. Comparison of immunohistochemistry with biochemical determination of enolase isoenzymes.

The authors evaluated methods of recognition of neuroendocrine differentiation in lung cancer because this is thought to bear prognostic value. One hundred forty lung tumors were divided by immunohistochemical analysis using neuroendocrine markers (neuron-specific enolase, Leu7, chromogranin, neural cell adhesion molecules, SL11/14, and Leu19) into two groups of 51 neuroendocrine tumors positive for three neuroendocrine markers and 89 non-neuroendocrine tumors. Biochemical determination of enolase activity and isoenzyme distribution showed that the level of total enolase activity was similar between neuroendocrine and non-neuroendocrine tumors. alpha gamma and gamma gamma enolase isoenzyme percentages were higher in neuroendocrine tumors. A cut-off of gamma enolase % (alpha gamma/2 + gamma gamma) at 14% gave a sensitivity rate of 84% and specificity rate of 97% in separating neuroendocrine and non-neuroendocrine tumors, whereas immunohistochemical analysis using anti-gamma enolase had low specificity (68%) and immunohistochemical analysis with Leu 7 and chromogranin had high specificity (97%) and low sensitivity (37% and 60%) levels for neuroendocrine tumors. The best prediction of neuroendocrine differentiation was obtained using immunohistochemical analysis against neural cell adhesion molecules combined with biochemical estimation of enolase using gamma enolase of 14% and a gamma gamma isoenzyme more than 3%.

Altered isoenzyme distribution in the non-infarcted myocardium in rats with myocardial infarction

Altered isoenzyme distribution in the non-infarcted myocardium in rats with myocardial infarction

Expression of carbonic anhydrase isoenzymes in the developing endolymphatic sac of the human fetus and the mouse embryo

The distribution of carbonic anhydrase isoenzymes (CA) was analyzed in the developing endolymphatic sac (ES) of the mouse embryo and human fetus using immunohistochemical method. The primordial ES epithelium was labelled with CA I and CA II, but was weakly labelled with CA III and CA V. In the thirteenth and fifteenth gestational day (GD) mice, the ES epithelium was positive for CA I and CA II. After seventeenth GD, the ES epithelium was however weakly positive for CA I and CA II. In the 11 and 12 week old human fetus, the ES epithelium was strongly labelled with CA I and CA II. In the 16 week old human fetus, the ES epithelium was however weakly positive for CA I and CA II. These results suggest that the fetal ES has an activity of CA and plays a role in the otoconial formation especially in the early stage during evolution.

Immunohistochemical distribution of isoenzymes of glutathione transferase in adult rat adrenal gland before and after hypophysectomy

The distribution of glutathione transferase subunits 1, 2, 3, 4, 7 and 8 in the different cells of the female and male rat adrenal and the effects of hypophysectomy on these isoenzymes were studied using immunohistochemical methods. All these glutathione transferase subunits, with the exception of subunit 1, were present in the adrenal. Each subunit showed, however, its own characteristic distribution pattern. After hypophysectomy, increased staining for these isoenzymes was generally observed, and this effect was also cell-specific. Staining for subunit 2 increased in intensity in the zona fasciculata and reticularis after hypophysectomy, whereas a decrease was observed in the zona glomerulosa. Staining for subunit 8 was increased in the borderline between the capsule and zona glomerulosa, as well as in medullary chromaffin cells after hypophysectomy. The Mu subunits 3 and 4 increased markedly in fascicular and reticular cells after hypophysectomy and staining for subunit 3 was also increased in the medullary cells. A slight, but more general, increase was observed for subunit 7. We conclude from these experiments that the increases in glutathione transferase subunits observed in the rat adrenal after hypophysectomy are due to increased protein synthesis and/or increased protein stability and not to a selective destruction of cells lacking, or with low levels of, the isoenzymes.

Lactate dehydrogenase, creatine kinase, and their isoenzymes in pleural effusions

Lactate dehydrogenase (LD; EC 1.1.1.27) and creatine kinase (CK; EC 2.7.3.2) are widely distributed cytoplasmic enzymes. LD has five and CK has three isoenzymes distributed in different proportions in various tissues. The amounts of LD and CK and the distribution of isoenzymes in different body fluids are not thoroughly characterized. We have measured the total LD and CK concentrations and their isoenzyme distribution in pleural aspirates and in serum from 22 patients with benign conditions and from 14 patients with malignant effusions. In malignant pleural fluid, the mean total LD was 662 U/L; in benign conditions, it was nearly 5840 U/L with large variations (91-43 400 U/L) according to clinical diagnosis, the highest values being reached in inflammatory lesions. The mean total CK concentration in pleural fluid was close to the serum value in both groups of patients, as was the pleural CK isoenzyme distribution. The LD isoenzyme distribution in pleural effusions differed from that in serum in both groups, with LD-4 and -5 being the main isoenzymes in their pleural fluid specimens (greater than 42% of total LD). The total LD concentration correlated somewhat (r = 0.57) with the total pleural protein content. In conclusion, the pleural LD isoenzyme distribution, both in benign and malignant conditions, differs from that in serum, having shifted towards more anaerobic and embryonic isoenzymes (LD-4 and -5). Moreover, the greater the concentration of pleural total LD, the greater the proportion of LD-4 and -5. These data suggest that visceral or parietal pleural cells are rich in LD isoenzymes 4 and 5.

Distribution of glutathione S-transferase isoenzymes in primary epithelial tumors of the ovary

Rahilly M, Al Nafussi A, Harrison DJ. Distribution of glutathione S-transferase isoenzymes in primary epithelial tumors of the ovary. Int J Gynecol Cancer 1991; 1: 268–274. The glutathione S-transferases play a central role in the detoxification of electrophilic xenobiotics, including cytotoxic drugs and carcinogens, by conjugation with reduced glutathione. There are four major classes of human GST: pi, alpha, mu and microsomal GST. GST pi has been implicated in the progression of carcinogenesis in both animal and human studies. This study examined constitutive GST isoenzyme expression in 60 primary epithelial tumors of the ovary, including benign, borderline and frankly malignant categories. GST pi was the predominant isoenzyme, being strongly expressed in all tumor types. The intensity of GST pi staining was independent of grade in all categories of carcinoma. Thus GST pi has no potential role as a marker of differentiation or tumor progression in epithelial ovarian tumors. GST alpha, which localizes to steroid-producing cells in normal ovary, was useful in demonstrating enzymatically active stromal cells in ovarian carcinomas with functioning stroma. GSTs, particularly the alpha and microsomal isoenzymes, have been implicated in both intrinsic and acquired resistance to cancer chemotherapy. Alpha, mu and microsomal GSTs displayed a heterogeneous staining pattern in all tumor types. This heterogeneity of expression may contribute to intrinsic drug resistance in ovarian carcinomas.

Beneficial effects of diltiazem on the natural history of hypertensive diabetic cardiomyopathy in rats

Hypertensive diabetic rats develop a cardiomyopathy characterized by systolic and diastolic ventricular dysfunction, myocardial hypertrophy and fibrosis, pulmonary congestion and a very high mortality rate. Alterations in contractile proteins and sarcoplasmic reticular calcium (Ca2+) transport in diabetic myocardium and their partial reversal with verapamil suggest that calcium channel blockade may prevent death from congestive heart failure in hypertensive diabetic rats.A large group of rats with renovascular hypertension and streptozotocin diabetes were divided into four gruups: untreated animals (Group 1) and animals treated with 100 (Group 2), 300 (Group 3) or 600 (Group 4) mg/kg per day of sustained release diltiazem mixed in their food. Treatment was begun shortly after the onset of hypertension and diabetes. Mortality rates after 4 months were 59% (19 of 32), 53% (17 of 32), 27% (7 of 26) and 35% (12 of 34) in Groups 1,2,3 and 4, respectively; the mortality rate in age-matched control rats was 5% (1 of 19). The reductions in mortality rates in Groups 3 and 4 were statistically significant.Diltiazem did not change systolic blood pressure, serum glucose concentration, heart rate or left ventricular mass. There was a trend to decreased left ventricular interstitial fibrosis and perivascular fibrosis in diltiazem-treated animals. Ventricular collagen concentration was similar in untreated hypertensive diabetic and control rats; levels were higher in hypertensive diabetic rats that died than in those that survived. There was a trend to decreased collagen concentration as diltiazem dose increased.Myosin isoenzyme distribution was not changed in Groups 3 and 4 (in comparison with Group 1). In all hypertensive diabetic groups, rats that died had a higher blood pressure, heart rate, relative left ventricular mass, lung weight and lung water than did survivors. The mortality rate was two to three times higher among rats with an initial blood pressure ≥180 mm Hg. The beneficial effects of diltiazem on survival were most significant among rats with severe hypertension.

Changes in expression and distribution of lactate dehydrogenase isoenzymes in the developing chick retina

We have investigated the expression and distribution of lactate dehydrogenase (LDH) isoenzymes in the developing and adult chicken retina. Non-denaturing polyacrylamide gel electrophoresis was used to follow the appearance and expression of the five main LDH isoenzymes in tissue homogenates. Immunohistochemistry was used to define the distribution of the aerobic heart type LDH and the anaerobic muscle type LDH in paraffin sections of embryonic and adult chick retina. The electrophoretic results show that the expression of the anaerobic isoenzyme increases in the retina as development proceeds. Immunoreactivity against the anaerobic isoenzyme localizes to the inner plexiform layer and other regions of the inner chick retina. The aerobic isoenzyme immunoreactivity is localized to the inner segments of the photoreceptor cells as well as the ganglion cell layer. Both antibodies bind weakly in other layers of the retina with the notable exception that the anaerobic protein does not localize in the photoreceptor cell inner segments. These results provide further evidence for the anaerobic nature of the adult inner retina. As retinal development proceeds, the expression of the anaerobic isoenzyme in the retina increases.

Contractile and calcium regulating capacities of myocardia of different sized mammals scale with resting heart rate.

The purpose of this study was to determine if selected biochemical parameters representing the contractile and calcium regulating systems of cardiac muscle scaled among mammals having inherently different resting heart rates (RHR). Eight mammalian species with RHR ranging from 51 to 475 beats per minute (bpm) were studied. The oxidative capacity of the myocardium is highly correlated with the RHR. The hypothesis of the present study was that the capacities of the energy utilizing processes of contraction and calcium regulation would also be correlated to the functional demand imposed on the muscle as represented by the RHR. Myosin (M) and myofibrillar (MF) ATPase activities, myosin isoenzyme distribution and sarcoplasmic reticulum (SR) ATPase activity were determined. Animals with RHR above 300 bpm express V1 myosin while animals with lower RHR express primarily V3. M and MF ATPase activities correlated with RHR, but the major difference in activities occurred at the 'threshold' RHR of about 300 bpm at which the switch from V3 to V1 appears to occur. SR ATPase activity per mg of microsomal protein was for the most part constant among different mammals, but the SR ATPase activity per g of heart tissue was significantly correlated with RHR as slower beating hearts tended to yield less SR protein per unit mass. We conclude that both the contractile and calcium regulating systems are scaled to the functional parameter of RHR among different mammals. The contractile system uses a slow myosin ATPase isoform at low resting heart rates whereas above the postulated threshold RHR of about 300 bpm a switch in gene expression to a fast myosin ATPase isoform occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic and diagnostic significance of creatine kinase isoenzymes

Creatine kinase isoenzyme content is frequently used to assess the state of differentiation of muscle and neural tissue and following release into plasma as diagnostic markers for acute myocardial infarction, skeletal muscle disease, and neurologic injury. The establishment of thrombolytic therapy as the standard of care for acute myocardial infarction and new information on the tissue distribution of creatine kinase isoenzymes has necessitated the development of more rapid assays for the diagnosis of infarction and expanded the potential use of these isoenzymes as markers for other disease states.

N-acetyl-β-d-glucosaminidase in the localisation of urinary tract infection in patients with spinal cord injury

This study evaluates the pattern of urinary N-acetyl-beta-D-glucosaminidase (NAG) isoenzyme excretion in patients with spinal cord injury (SCI) and its use as a diagnostic tool in localising the site of urinary tract infection (UTI). NAG-B excretion in 27 control SCI patients (mean 207.78 units) was significantly higher than in 10 normal controls (mean 12.6 units) p less than 0.001). The relative isoenzyme distribution as represented by NAG-B/Total NAG percentage is however similar in both groups, 24.27 and 20.38% respectively. NAG-B excretion in 6 SCI patients with upper UTI was not significantly higher than in 12 SCI patients with lower UTI. NAG-B/Total NAG percentage was significantly different between these two groups (35.3% and 24.98% respectively, p less than 0.05). There was no significant difference in NAG-B excretion or NAG-B/Total NAG percentage between control SCI patients and those with lower UTI. The results indicate that there is a non-selective increase in urinary NAG excretion in control SCI patients and those with lower UTI. In SCI patients with upper urinary UTI there is a selective increase in NAG-B excretion. The overlap in enzyme values between the different groups suggests that the test may not be clinically useful in localising the site of UTI.

A comparative research of zymograms of lactate dehydrogenase in different tissues of 25 species of vertebrates

The distribution and characteristics of LDH isoenzymes in different tissues of 25 species of vertebrate were comparatively analysed by discontinuous polyacrylamide gel disk electrophoresis, combined with experiments on heat and urea inhibition. The result showed that in different tissues ofCtenopharyngodon idellus, Carassius, ayratus, Trichiurus haumete andMiichthys miiuy in addition to the distribution of LDH isoenzymes, composed of A and B subunits, a E4 isoenzyme band, moving very fast to the positive pole, was detected in the eye tissue ofTrichiurus haumele and a F4 isoenzyme hand, moving very slow to the positive pole, was found in liver and kidney ofCtenopharyngodon idellus. The A4 isoenzyme band was found in different tisseues ofPleuronichthys cornutus, inhabiting on the bottom of the ocean. From the amphition to the mammal, with the exception ofHyta arboreu immaculata, Chinnemys reevesii, Trionyx sinensis, Gallus gallus domesticus andCorvus macrorhynchos, which have special electrophoretic behaviours. In different tissues of the other animals distributed the five kinds of tetrameter of isoenzyme bands composed of A and B subunits. Meanwhile, the C4 isocnzyme band was detected in the testicle tissues of animals. The C4 bandof Rattus norvegicus caraco andLepus timidus located between the bands of LDH4 and LDHS and the C4 band ofErinaceus europaeus, a primitical animal, located to the farther side of LDHS and moving very slow to the positive pole. The distribution characteristics of LDH isoenzymes of different classes of vertebrates were compared and it was suggested that no parallism existed between the changes of LDH isoenzyme zymograms and phylogeny of vertebrates.

The diagnostic significance of lactate dehydrogenase isoenzymes in urinary cytology

Lactate dehydrogenase (LDH) isoenzyme distribution was examined in 106 urine samples being tested cytologically for evidence of bladder cancer; the samples were selected to have less than 20 leucocytes and erythrocytes per high power field and the LDH pattern determined by electrophoresis. The Papanicolaou stained-smears showed 68 negative, 17 suspicious and 21 positive. The LDH M-fraction of the urinary supernatant in cytologically positive cases was significantly greater than in negative cases, although the latter included a few false negative samples. Some of the false negatives gave positive results for the LDH M-fraction; these results suggest that the determination of LDH isoenzymes in the urine is useful in diagnosing urinary tract cancers, including early stage, and for follow-up of patients with bladder cancers after surgical resection.