Expression of carbonic anhydrase isozymes II and III in developing bovine parotid gland

Two cytosolic carbonic anhydrase isozymes (CA-II and CA-III) were studied by immunohistochemistry in bovine parotid glands during fetal development. In a 3-month-old fetus of crown-rump length (CRL) 17 cm, the expression of CA-II in undifferentiated epithelial cells was observed, whereas immunostaining for CA-III remained negative. At 26 cm CRL (4–5 months old), weak expression of CA-III in large ductal epithelial cells was noted. The accumulation of secreted granules in primary acinar cells was initially observed at this stage. In a newborn calf, anti-CA-II reactivity almost disappeared from most duct segments. The time-dependent expression and distribution of the isozymes in parotid glands may reflect different biological functions of these structurally closely related isozymes. Bovine parotid acinar cells of fetuses would thus appear to possess all the cellular structures and immunohistochemical properties at 4 and 5 months of gestation. CA-II subsequently disappeared from duct segments and nearly all acinar cells in adults were present at or just after birth.     

Comparative immunochemical studies of carbonic anhydrase III in horses and other mammalian species

1. Carbonic anhydrase III (CA-III) from different mammalian species (horse, cow, dog, cat, rat and rabbit) has been analyzed by the immunodiffusion technique with anti-equine CA-III serum. 2. Immunodiffusion demonstrated the absence of cross-reactivity between isozyme CA-I, CA-II, and CA-III. 3. Cross-reactions were observed between the CA-III from all the species examined except the rabbit. 4. Molecular weights and isoelectric points of CA-III from different species were determined by Western blotting.     

Long-term in vitro cell culture of Sinclair swine melanoma.

The melanoma of Sinclair swine exhibits several characteristics similar to human melanoma but demonstrates an unusually high incidence of spontaneous regression. A total of 66 finite cell lines derived from 21 swine melanotic lesions, both cutaneous and visceral, were studied in vitro over their life spans of up to 14 months. The growth characteristics of the cultures varied with the age of the swine from which the tumors were obtained. Cell cultures of tumors obtained from swine aged less than 2 months grew steadily in cluture with a population-doubling time of 120 to 180 hr until growth and division ceased after a maximum of 25 to 35 population doublings (6 to 8 passages). Cell cultures of tumors obtained from swine aged 3 months or older showed a biphasic growth pattern with an early slow growth rate (population-doubling time 120 to 160 hr), which shifted after 3 to 6 passages to a faster rate (80 to 110 hr population-doubling time) until termination of growth and division after a maximum of 75 to 85 population doublings (18 to 20 passages). The cultures were morphologically heterogeneous including cuboidal, spindle and dendritic cell types. Electron microscopy showed classic melanosomes only in the primary and passage 1 cultures although vesicular inclusions were numerous in later-passage cells. However, continued melanin synthesis was indicated by the spectroscopic characteristics of material obtained from medium of passage 8 cultures and by DOPA staining of cultures as advanced as passage 18.     

Long-term in vitro cell culture of sinclair swine melanoma

Summary The melanoma of Sinclair swine exhibits several characteristics similar to human melanoma but demonstrates an unusually high incidence of spontaneous regression. A total of 66 finite cell lines derived from 21 swine melanotic lesions, both cutaneous and visceral, were studied in vitro over their life spans of up to 14 months. The growth characteristics of the cultures varied with the age of the swine from which the tumors were obtained. Cell cultures of tumors obtained from swine aged less than 2 months grew steadily in culture with a population-doubling time of 120 to 180 hr until growth and division ceased after a maximum of 25 to 35 population doublings (6 to 8 passages). Cell culture of tumors obtained from swine aged 3 months or older showed a biphasic growth pattern with an early slow growth rate (population-doubling time 120 to 160 hr), which shifted after 3 to 6 passages to a faster rate (80 to 110 hr population-doubling time) until termination of growth and division after a maximum of 75 to 85 population doublings (18 to 20 passages). The cultures were morphologically heterogeneous including cuboidal, spindle and dendritic cell types. Electron microscopy showed classic melanosomes only in the primary and passage 1 cultures although vesicular inclusions were numerous in later-passage cells. However, continued melanin synthesis was indicated by the spectroscopic characteristics of material obtained from medium of passage 8 cultures and by DOPA staining of cultures as advanced as passage 18. This work was supported by a grant from the NIH, NCI (2 P01 CA 08023-11A1).     

Evidence for the presence of carbonic anhydrase III in the myoid cells of the bovine thymus. Immunocytochemical and ultrastructural study

An immunohistochemical study was carried out to detect the localization of carbonic anhydrase III (CA-III) in the bovine thymus. It was found that the CA-III activity was localized in the cells forming small clusters dispersed in the medullary region. By ultrastructural observation, these cells were identified as myoid cells.     

Evidence for the presence of carbonic anhydrase III in the myoid cells of the bovine thymus

Summary An immunohistochemical study was carried out to detect the localization of carbonic anhydrase III (CA-III) in the bovine thymus. It was found that the CA-III activity was localized in the cells forming small clusters dispersed in the medullary region. By ultrastructural observation, these cells were identified as myoid cells.     

Acylation and carbamylation of equine muscle carbonic anhydrase (CA-III) upon reaction with p-nitrophenyl esters and carbamoyl phosphate

Equine muscle carbonic anhydrase (CA-III) behaves like ubiquitin in undergoing extensive acylation of N epsilon-lysine residues upon reacting with p-nitrophenyl esters. The enzyme undergoes extensive carbamoylation of lysine residues when reacted with carbamoyl phosphate. The modification of from 6 to 7 lysine residues results in the production of a series of more anodic electrophoretic components. The derivatization of the lysine residues leads to a marked decrease in the enzyme's ability to hydrate CO2. The equine CA-III possesses both acid and alkaline phosphatase activities in contrast to the rabbit which possesses only the former type.     

Fatty acid synthesis in vitro in the liver and adipose tissue of rats of various ages.

In white male Wistar rats aged 2, 4, 6, 9, 12, 18 and 32 months, receiving a standard laboratory diet (25 cal% proteins, 22 cal% fat and 53 cal% glycides) we assessed the total amount of fatty acids in the carcass and in vitro lipogenesis in liver and adipose tissue by the means of glucose-14C(U). The amount of body fat increased with age; the highest rate of increase in the percentage of body fat occurs 2 to 4 months after birth. Incorporation of 14C of glucose into total liver lipids attains maximum values in the group of rats age 2 to 4 months. By the age of 6 months lipogenesis decreased to a level observed in all the remaining age groups. In adipose tissue, the incorporation of 14C into total lipids decreased between the age of 2 to 4 months, a further decrease was observed around the age of 6 months. No appreciable changes were noted in any of the older age groups.     

Role of thyroid hormones in the normal and glucocorticosteroid hormone-induced evolution of carbamoyl-phosphate synthase (ammonia) activity in axolotl liver.

1. In axolotl liver, the activity of carbamoyl-phosphate synthase (ammonia), expressed per mg liver protein, decreases to a minimum at 5 months of age, then increases to a maximum at 8 months of age which is followed by a decrease again. The initial decrease between 3 and 5 months of age appears to be largely due to an increase in non-carbamoyl-phosphate synthase protein and the following increase between 5 and 8 months of age to a relative increase of carbamoyl-phosphate synthase protein. 2. Treatment of the animals with triiodothyronine causes an increase in carbamoyl-phosphate synthase activity, the extent of which is dependent upon hormone concentration and age of the animal. After 8 months of age no increase of enzyme occurs upon thyroid hormone treatment, although metamorphosis occurs. 3. Glucocorticosteroid hormones stimulate carbamoyl-phosphate synthase activity 2-to 3-fold in animals older than 6 months. However, in animals younger than 6 months, low concentrations of thyroid hormone, insufficient to induce metamorphosis, are necessary as permissive agents. 4. The stimulatory effects of high concentrations of thyroid hormones (T3) on carbamoyl-phosphate synthase appear to be mediated via a stimulatory effect on glucocorticosteroid biosynthesis. 5. The natural rise in enzyme activity between 5 and 8 months of age seems to be due to a rise in the concentration of circulating glucocorticosteroid hormones.     

Peculiarities of urea distribution in tissues of rats of different age

The content of urea was studied in protein-free filtrates of the liver kidneys, skeletal muscles, myocardium, spleen, brain tissues and blood serum as well as in urine of 1, 3, 6, 12 and 24-month rats. It is shown that at the age of 6 months the content of urine in most tissues under study is significantly decreased (by 42-73%), at the age of 12 months in the spleen and at the age of 24 months in the brain tissues as compared to the one-month animals. The level of urine decrease in the liver and brain tissues of 24-month animals is less pronounced than in other tissues, that corresponds to age peculiarities of their protein metabolism. A decrease of blood consumption per weight unit and a relative increase in the amount of nitrogen excreted with urine are observed with ageing. The arginase activity in the liver decreases essentially only in 3-month animals. A conclusion is drawn that peculiarities of food consumption and the character of changes in the urea content in tissues and urine are adaptation manifestation of an age decrease in the intensity of nitrogen metabolism and protein demand of the organism.     

Validated hydrophilic interaction LC-MS/MS method for determination of N-methyl-2-pyrrolidinone residue: Applied to a depletion study of N-methyl-2-pyrrolidinone in swine liver following intramuscular administration of drug N-methyl-2-pyrrolidinone formulation

A hydrophilic interaction high-performance liquid chromatography coupled with tandem mass spectrometry method for determination of N-methyl-2-pyrrolidinone in swine liver was developed and validated. After the fortification of N-methyl-d(3)-2-pyrrolidinone-d(6) as the deuterium-labeled internal standard, N-methyl-2-pyrrolidinone in swine liver was extracted by acetonitrile and the supernatant was led through a C18+WAX mixed-mode SPE cartridge for removal of the matrix interferences. The final eluate was acidified by formic acid and then injected onto a 3μm 15cm×2.1mm TX column for hydrophilic interaction chromatographic analysis. Mass spectrometry detection was carried on a PE Sciex API 4000 triple quadrupole mass spectrometer using positive turbo-ion spray ionization mode. The MRM transitions were 100→58 for N-methyl-2-pyrrolidinone and 109→62 for N-methyl-d(3)-2-pyrrolidinone-d(6). Solvent calibration standards could be readily used for quantitative analysis of N-methyl-2-pyrrolidinone with excellent precision and accuracy, although there are endogenous levels of N-methyl-2-pyrrolidinone in many blank matrices. The true recovery was nearly 100% and the MRM signal of N-methyl-2-pyrrolidinone was suppressed about 30% because of the matrix effect. Nevertheless, N-methyl-d(3)-2-pyrrolidinone-d(6) completely compensated the ion-suppression effect and the injection-to-injection variation. The detection limit was 5ngg(-1) swine liver. The validated method was applied to a depletion study of N-methyl-2-pyrrolidinone in swine liver following intramuscular administration of a drug N-methyl-2-pyrrolidinone formulation.     

Multi-scale characterization of swine femoral cortical bone

Multi-scale experimental work was carried out to characterize cortical bone as a heterogeneous material with hierarchical structure, which spans from nanoscale (mineralized collagen fibril), sub-microscale (single lamella), microscale (lamellar structures), to mesoscale (cortical bone) levels. Sections from femoral cortical bone from 6, 12, and 42 months old swine were studied to quantify the age-related changes in bone structure, chemical composition, and mechanical properties. The structural changes with age from sub-microscale to mesoscale levels were investigated with scanning electron microscopy and micro-computed tomography. The chemical compositions at mesoscale were studied by ash content method and dual energy X-ray absorptiometry, and at microscale by Fourier transform infrared microspectroscopy. The mechanical properties at mesoscale were measured by tensile testing, and elastic modulus and hardness at sub-microscale were obtained using nanoindentation. The experimental results showed age-related changes in the structure and chemical composition of cortical bone. Lamellar bone was a prevalent structure in 6 months and 12 months old animals, resorption sites were most pronounced in 6 months old animals, while secondary osteons were the dominant features in 42 months old animals. Mineral content and mineral-to-organic ratio increased with age. The structural and chemical changes with age corresponded to an increase in local elastic modulus, and overall elastic modulus and ultimate tensile strength as bone matured.     

Comparative immunohistolocalization of carbonic anhydrase isozymes I, II and III in the equine and bovine digestive tract

Summary Immunohistochemical localizations of carbonic anhydrase isozymes (CA-I, CA-II and CA-III) in equine and bovine digestive tracts were studied. In the horse, epithelial cells in both the oesophagus and non-glandular part of the stomach lacked all three isozymes. In contrast, surface epithelial and parietal cells in the glandular region of the stomach showed reactivity for CA-II. In the small intestine, absorptive columnar cells covering the villi in the duodenum were positive for CA-II. The epithelium of the jejunum and ileum lacked all three isozymes. In the large intestine, CA-II was detected in the columnar cells in the upper part of the crypt. In cattle, epithelial cells of the oesophagus showed reactions for CA-I and CA-III but not for CA-II. Although the absorptive epithelial cells of the small intestine lacked CA-I, CA-II and CA-III, those of the upper part of large intestine crypts were heavily stained for all three isozymes.     

Immunohistochemical study of carbonic anhydrase III in the extraocular muscles of human embryos.

The differentiation of extraocular muscles was studied immunohistochemically in externally normal human embryos (Carnegie stages 13-23), using antibodies to carbonic anhydrase (CA) III and beta-enolase as the markers of type 1 and type 2 muscle fibers, respectively. At stage 18, some myoblasts were immunoreactive to beta-enolase antibodies, however, CA-III immunoreactivity was not observed around the optic vesicle. At stage 20, CA-III immunoreactivity appeared in some muscle fibers of extraocular muscles. From stage 21 to stage 23, CA-III-immunoreactive fibers increased and almost equalled the number of beta-enolase-immunoreactive fibers. These findings suggest that CA-III-immunoreactive type 1 fibers appear in the late stage of myogenesis compared with beta-enolase-immunoreactive type 2 fibers.     

Opposite changes with age in liver and muscle in the mitochondrial and soluble glucose-1,6-bisphosphate and 6-phosphogluconate dehydrogenase

Glucose-1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, was markedly decreased in liver of adult rats (2 months of age) as compared to young rats (1-2 weeks of age). This regulator was found to be present in both the mitochondrial and soluble fractions of liver. Its concentration in both these fractions was decreased with age. Concomitant to the decrease in Glc-1,6-P2, which is a potent inhibitor of 6-phosphogluconate dehydrogenase, the activity of this enzyme was markedly increased with age in both the mitochondrial and soluble fractions. However, the increase in this enzyme's activity was more pronounced in the mitochondrial fraction. The mitochondrial enzyme was more susceptible to inhibition by Glc-1,6-P2 as compared to the soluble enzyme, and this may explain the greater enhancement in its activity with age in this fraction. The tibialis anterior muscle exhibited changes with age opposite to those found in liver; Glc-1,6-P2 concentration, in both the mitochondrial and soluble fractions of muscle increased with age, and this increase was accompanied by a concomitant reduction in the activity of the mitochondrial and soluble 6-phosphogluconate dehydrogenase. Similar to liver, the mitochondrial enzyme was more affected by age, as it also exhibited a greater susceptibility to inhibition by Glc-1,6-P2.     

Downregulation of telomerase in rat during the aging process.

Telomerase is an RNA-dependent DNA polymerase that maintains the tandem arrays of telomeric repeats at the eukaryotic chromosome ends. Because of its ability to replenish lost telomeric sequences, telomerase is thought to be required for cell proliferation. At present, very little information on the role of telomerase in aging is available. In the present study, we tested the telomerase activity of Fischer 344 rat testis and liver at 6, 12, 18 and 24 months of age. As the testis is an androgen-dependent tissue, we also investigated the changes of testosterone and mRNA levels of androgen receptor in this tissue. Our results show that the telomerase activity of Fischer 344 rat testis significantly reduced at 24 months of age compared to 6 months of age, and that the mRNA level of telomerase protein component 1 (TLP-1) show a corresponding decrease with the telomerase activity. Interestingly, this down-regulation was not observed in the liver. The testosterone level in testis increased until 18 months of age, but reduced by 50% at 24 months of age. Our conclusions are that the telomerase activity is age-dependent and its change is a tissue-specific phenomenon.     

Collagenase of hepatocytes and sinusoidal liver cells in the reversibility of experimental cirrhosis of the liver

In order to explore the cellular source(s) and the behaviour of the collagenolytic activity previously described in rat liver homogenates, in the reversibility of experimental cirrhosis of the liver, enriched suspensions of hepatocytes and of sinusoidal liver cells were obtained by a procedure which employs low EDTA concentrations and no bacterial collagenase. Cell suspensions were prepared from three different groups of animals: 1) normal controls, 2) rats with CCl4-induced cirrhosis of the liver, and 3) rats with swine serum-induced cirrhosis of the liver. Animals were sacrificed in each group upon completion of treatment and also after 3, 6 and 12 months. In each liver wet weight and collagen concentration were determined, and collagenolytic activity of both enriched cell suspensions was measured separately. In addition, histological studies of liver tissue and ultrastructural examination of cell suspensions were performed by standard procedures. Enriched suspensions of both normal hepatocytes and sinusoidal liver cells display Ca2(+)-dependent collagenolytic activities. Both cell suspensions obtained from each of the two types of cirrhotic livers show normal or slightly increased average levels of collagenase activity at the time of treatment discontinuation, when average liver collagen content ranges from 6 to 10-fold over normal, suggesting that the normal collagenase/collagen ratio is disturbed and that collagenolytic activity is deeply decreased in relation to the actual liver collagen load.(ABSTRACT TRUNCATED AT 250 WORDS)