Isozyme Profiles Research Articles

Tissue interactions and prostatic growth. I. Induction of adult mouse prostatic hyperplasia by fetal urogenital sinus implants.

A homologous chimeric prostate was produced by implantation of intact fetal urogenital sinus(es) (UGS) into the ventral prostate gland (VP) of an adult athymic mouse. A 10- to 20-fold overgrowth of the chimeric lobe of ventral prostate gland, as measured by glandular wet weight and by DNA content, was observed 4 to 9 wk following UGS implantation. The overgrowth was prostate-like as indicated by histologic composition and by responses to endogenous androgen, and was composed of both host and donor cells in about equal proportions as shown by glucose phosphate isomerase isozymic profiles. Unlike the canine model for prostatic hyperplasia, the mouse prostatic overgrowth occurred in the complete absence of exogenous sex steroids. The histoarchitecture of the chimeric VP and the isozymic detection of the contribution to the overgrowth by host cells have provided strong evidence that adult prostatic cells have been recruited to respond proliferatively by cellular interactions with fetal UGS. The demonstration of cellular interactions followed by reactivation of the fetal growth potential provides direct experimental evidence in support of McNeal's hypothesis that the reactivation of fetal growth potential may account for the development of human benign prostatic hyperplasia (BPH).

Trypanosoma cruzi: inoculation schedules and re-isolation methods select individual strains from doubly infected mice, as demonstrated by schizodeme and zymodeme analyses.

Groups of mice received double infections with the Y and F strains of Trypanosoma cruzi, the first inoculum of either strain being followed by a second inoculum of the other strain on day 5, 15, 30-40, or 60-65. Parasites were re-isolated from blood into culture, either directly or with an intermediate passage in gamma-irradiated mice, at intervals between 7 and 35 days after the second inoculation. Strain identification in the re-isolated material was by electrophoresis of kDNA fragments generated by the EcoRI restriction endonuclease and by electrophoresis for glucosephosphate isomerase isozymes. Both strains were identified in 22% of re-isolates originating from the experimental mice and only one of them was present in the remaining re-isolates, strain F being the most frequent. In some instances either Y or F was re-isolated from the same blood source, depending on whether culturing had been preceded or not by passage through a mouse. These results are certainly related to strain differences in the various aspects of host-parasite relationship and, possibly, growth rates in culture. The results demonstrate that: (1) more than one strain of T. cruzi can coexist in the same host; (2) the timing and method of parasite isolation from the vertebrate host act as selective factors, and further passages (in mice or cultures) may completely eliminate one (or more) strain from originally mixed trypanosome population, and (3) kDNA restriction "fingerprints" and isozyme profiles are simple, sensitive, and reliable techniques for strain identification both in single and mixed preparations.

Isozyme heterogeneity and numerical taxonomy of Trypanosoma cruzi stocks from Chile

Fifty-three stocks of Trypanosoma cruzi were isolated in Chile, 13 from patients, 32 from the domestic triatomine vector Triatoma infestans, and 8 from the silvatic and peridomestic vector T. spinolai. The majority of isolates from triatomine bugs were made by the direct culture of infected faeces. The 53 stocks and a single clone were characterized by a combination of starch-gel and cellulose acetate enzyme electrophoresis. Three groups of T. cruzi stocks were apparent from either simple visual comparisons of isozyme profiles or numerical taxonomy. The groups were designated Chilean zymodeme (Z) 1, which was similar to Brazilian Z1, Chilean Z2a, similar to Brazilian Z2 and Chilean Z2b, similar to Bolivian Z2 and with prominent heterozygous isozyme profiles. Chilean Z1 was isolated only from T. spinolai colonizing farm walls inhabited by the rodent Octodon degus. Chilean Z2a and Z2b were both isolated from domestic T. infestans and man, in some cases within the same household. Hardy-Weinberg equilibria were not found amongst a group of 22 stocks from a single locality and deviations from theoretical Hardy-Weinberg distributions were compatible with the absence of genetic exchange in the sampled population of T. cruzi.

Human formaldehyde dehydrogenase (FDH) and its assignment to chromosome 4.

A Cellogel procedure for electrophoretic characterization of formaldehyde dehydrogenase (FDH) is described. Isozyme profiles in 24 different human tissues indicate extreme quantitative heterogeneity and the existence of one major form and at least two minor forms of FDH in man. Biochemical and genetic analysis of the enzyme in Chinese hamster-human somatic cell hybrids has shown that the functional molecule of FDH is probably composed of two identical subunits and that its structural locus may be assigned to human chromosome 4.

Chagas' disease in the Amazon Basin IV

Of 151 Trypanosoma cruzi stocks from 117 different individual hosts collected in the states of Amazonas and Rondonia, 147 from 113 hosts were identified as zymodeme 1 (Z1). These included T. cruzi stocks from three marsupial species, two rodent species and three triatomine species although most were from the common opossum, Didelphis marsupialis. One T. cruzi stock from Rhodnius robustus was identified as Z1 with a Z3 PGM character, one from Sciurus sp. as Z3 and two from Monodelphis brevicaudata and Panstrongylus geniculatus as Z3 with a Z1 ASAT character. The ways in which stocks were isolated and grown up in vitro did not influence isozyme profile. These results support earlier evidence from Pará State that Z2 is absent from the Amazon basin and that the distribution of T. cruzi zymodemes in this region is quite different to that in endemic areas on the south of the continent.

Vertebrate hosts and vectors of Trypanosoma rangeli in the Amazon Basin of Brazil.

A total of 46 Trypanosoma rangeli stocks were isolated from naturally infected mammals and triatomine vectors. Twenty-two stocks were from the common opossum (Didelphis marsupialis), one from the brown "4-eyed" opossum (Metachirus nudicaudatus), one from the anteater (Tamandua tetradactyla), one from the coati (Nasua nasua), seven from Rhodnius pictipes and 14 from Rhodnius robustus. Two stocks were also isolated from recently fed sandflies (Lutzomyia sp., Shannoni group). The stocks were identified as T. rangeli on the basis of natural or experimental salivary gland infections in Rhodnius, inoculative (anterior station) transmission to mice, morphological parameters in parasitemic mice and comparisons of isozyme profiles with a known stock of T. rangeli isolated from man. Three other trypanosome stocks from D. marsupialis, T. tetradactyla and the three-toed sloth (Bradypus tridactylus) were morphologically similar to T. rangeli in culture but had quite different isozyme profiles and were not identified. It is concluded that T. rangeli is widely distributed in Amazonas, Pará and Rondonia States of Brazil, and probably extends into other regions where R. pictipes and R. robustus are known to occur. R. pictipes is light-attracted into houses and occasionally transmits Chagas' disease to man. It is likely that T. rangeli is also occasionally transmitted to man in the Amazon basin.

Electrophoretic Analysis of the Ticks Ornithodoros (Pavlovskyella) Erraticus and O. (P.) Sonrai (Acari: Argasidae)1

Horizontal starch gel electrophoresis was used to examine the isozyme profiles of 2 argasid tick species, Ornithodoros erraticus and Ornithodoros sonrai . Of the 9 loci examined, malate dehydrogenase ( Mdh ), hexokinase ( Hk ), and phosphoglucomutase ( Pgm ) proved to be species-diagnostic, showing no genetic overlap. The technique provides a means of monitoring colonies of the 2 species to check for cross-contamination and also clarifies the taxonomic status of the 2 forms.

The nuclear-cytoplasmic relationship in ‘mosaic‘ skeletal muscle fibers from mouse chimaeras

In mouse chimaeras, individual skeletal muscle fibers typically contain populations of myonuclei derived from both cell lines. This 'mosaic' circumstance has provided an opportunity to investigate directly whether the mammalian myofiber syncytium is functionally subdivided into territories, each preferentially influenced by products encoded by the local myonucleus, or whether the multiple nuclei direct the synthesis of products that achieve a uniform distribution throughout the fiber. Chimaeras were produced in which one cell line was derived from an embryo homozygous for gpi-1a, whereas the other was homozygous for the gpi-1b; each allele specifies electrophoretically distinguishable isozymes of the cytosolic enzyme glucosephosphate isomerase (GPI-1). Microtechniques capable of measuring the proportion of each isozyme expressed within small samples of individual muscle fibers have been established, permitting the comparison of the relative quantitative distributions of the GPI-1 isozyme types along the length of individual chimaera fibers. From individual mosaic fibers, all samples yielded identical isozyme profiles, demonstrating that GPI-1 is not sequestered adjacent to the nucleus directing its synthesis; rather, it achieves a homogeneous distribution throughout the mosaic syncytium. The GPI-1 gene locus encodes only the GPI-1 monomer, whereas the functional enzyme detected in our analysis is a dimer that results from the aggregation of monomers in the cytoplasm. The quantitative distribution of dimer types within each mosaic fiber was consistent with random aggregation amongst all monomers represented in the final isozyme pattern, a result requiring that monomers or earlier precursors were mixed in the myofiber cytoplasm prior to assembly of the enzymatically active dimer. Thus, both the final distribution of enzyme dimers within fibers and the patterns of monomer aggregation suggest that there are no subdivisions related to the spatial separation of the genotypically distinct myonuclei within mosaic muscle fibers.

Effect of an Ozone Injury Retardant Chemical on Isozyme Profiles from Alfalfa Callus in Vitro

Plant ozone injury retardant (EDU or ethylenediurea) at 1.0 ppm inhibited growth of callus of alfalfa cultivars Williamsburg (ozone-sensitive) and MSB-CW5An2(ozone-insensitive) germplasm of Medicago sative. The presence of EDU(0.1 ppm) in growth medium increased the number of protein and peroxidase isozyme bands in alfalfa cultivar stem callus and ozone modified their intensities. Protein profiles of MSB stem callus from media containing EDU or exposed to ozone were unchanged. Marked differences were observed between the peroxidase profiles of ozonated and control ozone-insensitive stem callus from media containing EDU. Protein profiles of ozonated ozone-insensitive leaf callus differed slightly from controls.

Expression of adenosine deaminase and purine nucleoside phosphorylase isozymes in lymphohemopoietic precursor cells and normal and neoplastic lymphoid populations

Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) isozyme profiles were analyzed in rat lymphoid cell populations at differing developmental stages. Day 12 fetal liver and Day 16 fetal thymocytes, both of which are composed of large, basophilic precursor cells, had a single ADA isozyme whereas newborn thymocytes, young adult thymocytes, and all peripheral lymphocytes contained a primary and two secondary ADA isozymes. In contrast, PNP isozyme profiles were virtually identical in all tissues examined, one primary and one secondary isozyme band. A 5-day organ culture of Day 16 fetal thymus resulted in the expression of the primary and two secondary ADA isozyme bands; this finding indicates that secondary ADA isozyme expression during thymocyte development occurs in the differentiated progeny of Day 16 fetal thymocytes which originally expressed only the primary ADA isozyme band. Thus, T-lymphocyte differentiation in the rat is accompanied by the appearance of secondary ADA isozymes whereas PNP isozymes remain essentially unchanged. Gross virus-induced thymic lymphomas such as fetal liver and fetal thymus expressed only the primary isozyme band. Moreover, the thymic lymphomas and Day 16 fetal thymocytes both displayed identical, unique cell-surface antigenic phenotypes. These results support a thymocyte precursor origin for Gross virus-induced thymic lymphomas in the rat.

Quantitative and Electrophoretic Analysis of Lactate Dehydrogenase during Tail Regeneration in the Scincid Lizard Mabuya Carinata

Electrophoretic separation of lactate dehydrogenase (L-lactate; NAD-oxidoreductase, EC 1.1.1.27) isozymes together with the quantitative evaluation of muscle-type (A) and heart-type (B) subunits were carried out during different phases of tail regeneration in the regenerate as well as liver and skeletal muscle of the Scincid lizard Mabuya carinata. Both the electrophoretic pattern of isozymes and the subunit ratio (ratio of B-type to A-type subunits) indicate increased anaerobiosis during the period of wound healing in all three tissues. Blastemic and early differentiation phases are marked by an aerobic pattern of metabolic process, as revealed by the increased proportion of B subunits as well as the appearance of well-defined, fastmoving (anodic) LDH 1 and LDH 2 isozymes. From the differentiation phase onward there was a gradual reversal of this pattern, which finally attained, with the completion of regeneration, the original anaerobic pattern of isozymic profile and subunit ratio characteristic of the corresponding tissues of normal animals (with intact tails). These changes in LDH activity are discussed in terms of the overall subcellular metabolic adaptations pervading the animal body in response to the regenerative process.

Processing of snake venom l-amino acid oxidase during intracellular transport

The isoelectrofocusing patterns of l-amino acid oxidase (LAO) from venom gland homogenates and of the secreted venom of Vipera palaestinae have been compared. The LAO isozyme profile of actively synthesizing gland spans over a wider range of pIs (4.8–6.0) and includes more variants as compared with the profile of the secreted venom. A basic shift of the isoelectrofocusing pattern of LAO obtained by treatment of the gland homogenate or the venom with neuraminidase indicates that sialic acid residues are responsible for the changes in the electronegativity of the isozymes. Analyses of subcellular fractions show that the microsomal fraction of the venom gland homogenate exhibits the highest multiplicity of molecular forms of LAO, whereas the fraction including the secretory granules has an isozyme profile similar to the venom. Double labelling experiments show that the newly synthesized LAO include isozymes which span over a wide range of pIs, whereas later on labelling of the more acidic isozymes is prominent. The results obtained may suggest that the sialic acid residues which are attached to LAO during its transport serve as “markers” for secretion.

In vitro comparison of embryonic myoblasts and myogenic cells isolated from regenerating adult rat skeletal muscle

Myogenic cells from regenerating adult rat muscle were compared in culture with embryonic myoblasts. No differences were found in their growth rates or fusion characteristics. Embryonic and regenerating cells fused with one another to form mosaic myotubes. Both showed the same increase in creatine kinase activity and shift in isozyme profile following fusion. These results support the view that myogenic cells from regenerating muscle are essentially the same as embryonic myoblasts.

Altered myosin isozyme patterns from pressure-overloaded and thyrotoxic hypertrophied rabbit hearts.

Cardiac hypertrophy, induced by pressure overload, leads to a depression in the rate of force development, velocity of shortening, tension-dependent heat generation, and myosin ATPase activity, whereas cardiac hypertrophy, induced by thyroxine administration, leads to an increase in these parameters. These changes have been attributed, in part, to structural changes in myosin. In this study, we have investigated changes in the relative content of myosin isozymes and differences in primary structure of the isozymes in pressure-overloaded and thyrotoxic cardiac hypertrophy in the rabbit. Three myosin isozymic forms (V1 = fastest, V2 = intermediate, V3 = slowest mobility) were observed in pyrophosphate polyacrylamide gels from normal hearts with the V3 component being the predominant species. In the pressure-overloaded model, the V1 and V2 components disappeared or were present in reduced amounts leaving the V3 more predominant. The most striking difference was the isozymic profile produced in thyrotoxic hearts where the V1 became the predominant component and V2 and V3 the minor components. alpha-Chymotryptic digestion of myosin heavy chains produced characteristic, reproducible peptide patterns for each of the animal models, as did fluorographic analyses of alpha-chymotryptic digests of 14C-iodoacetamide (IAA)-labeled SH1 peptides of myosin. Our results suggest that altered proportions of myosin isozymes may be responsible for altered cardiac performance.

Induction of sarcomas in athymic mice.

During the course of serial passage of 50 human xenografts in the athymic mouse over a period of 5 years we have observed two cases of induction of sarcomas in the murine stromal tissue associated with the human xenografts. Both times the growth of the murine sarcomas overtook that of the human xenograft. This change was monitored by analysis of the lactate dehydrogenase isozyme profile and histology of each passage of the human xenografts in the athymic mice. The two murine sarcomas were subsequently established in tissue culture. The sarcoma cell lines were found to be malignant by morphological and growth characteristics and were tumorigenic. They contained large amounts of murine leukemia virus when assayed for reverse transcriptase activity by infection of mouse SC-1 cells and BALB/c and NIH Swiss fibroblasts with filtered supernates, and some type C virus particles were observed by electron microscopy in tumor tissues. However, we were unable to demonstrate the presence of murine sarcoma virus by in vitro transformation of fibroblasts or sarcoma formation in vivo will cell free filtrates. Preliminary biochemical data indicate that the sarcomas are extremely high in plasma membrane ATP phosphohydrolase.

Phosphoglycerate kinase isozyme profile in reproductive cells and tissues of cattle: sperm specificity of PGK-B1.

Phosphoglycerate kinase isozyme profile in reproductive cells and tissues of cattle: sperm specificity of PGK-B1.

Lactate dehydrogenase activity in mouse lung following 1,1-dichloroethylene: index of airway injury.

Lactate dehydrogenase (LD) activity and its isozyme profile in mouse lung homogenate was affected by oral administration of 1,1-dichloroethylene (1,1-DCE). Following 100 mg 1,1-DCE/kg, LD-3 increased significantly. After 200 mg 1,1-DCE/kg, LD-5 increased whereas LD-1 and LD-2 decreased, with a resultant higher M:H ratio than controls. In contrast, elevated LD activity in serum following 1,1-DCE was predominantly associated with striking increases in total activity and changes in isozyme patterns resulting in a decrease in the M:H ratio. LD activity in liver and erythrocytes were unaffected by 1,1,-DCE administration. Although total activity in kidney was decreased, no changes were detected in the isozyme profile. Pulmonary damage induced by 1,1-DCE was reflected in significant increases in total activity and all isozymes in bronchopulmonary lavage fluids. Thus, detection of lung-derived LD activity in lung lavage fluids can be a useful index of pulmonary airway injury.

2,4,5-T effects on cardiac and serum Lactic Dehydrogenase (LDH) and Creatine Kinase (CK) isozymes I. Maternal enzyme activities and isozyme profiles

2,4,5-T effects on cardiac and serum Lactic Dehydrogenase (LDH) and Creatine Kinase (CK) isozymes I. Maternal enzyme activities and isozyme profiles

2,4,5-T effects on cardiac and serum Lactic Dehydrogenase (LDH) and Creatine Kinase (CK) isozymes II. Neonatal enzyme activities and isozyme profiles

2,4,5-T effects on cardiac and serum Lactic Dehydrogenase (LDH) and Creatine Kinase (CK) isozymes II. Neonatal enzyme activities and isozyme profiles

Creatine kinase activity in normal and Duchenne muscular dystrophy fibroblasts.

Cultured human skin fibroblasts from 9 patients with Duchenne muscular dystrophy (DMD) and 8 normal age- and sex-matched controls were examined for creatine kinase (CK) activity. Both the normal and the DMD fibroblasts were found to have significant levels of CK activity (approximately 10 x 10(-3) IU per milligram of fibroblast protein). The control cells had slightly higher CK activity than the DMD lines, but this difference was not significant (0.2 less than P less than 0.1). The MM (muscle) isozyme, the BB (brain) isozyme, and the MB (hybrid) isozyme, of CK were found to be present in fibroblasts. The isozymes were separated by electrophoresis and the relative amount of each was determined for both normal and DMD cells. In normal fibroblasts, approximately 48% of the total CK activity was of the MM type, 40% was of the BB type, and 12% was of the MB type with no significant differences apparent between normal and DMD groups. The presence in human fibroblasts of significant levels of CK activity with a characteristic isozyme profile is an important consideration for studies of this "marker" enzyme in the pseudohypertrophic muscle of DMD.