Estimation of Steroids and Steroid Metabolism in a Lower Vertebrate (Lampetra planeri Bloch)

Pregnenolone, androstenedione and testosterone were identified by RIA in tissue homogenates of the pronephric region, the opisthonephros, the gonads and in plasma samples from male and female immature and mature adult brook lampreys. Additionally, hydroxysteroid dehydrogenase activity was determined spectrophotometrically in homogenates from the same tissues of mature and spent adult brook lampreys employing pregnenolone, testosterone or 3β,17β-dihydroxy-5β-androstane as substrates. The steroid levels show differences corresponding to developmental stages, tissues and sex. Remarkable quantities of testosterone were measured in the testicular tissue homogenates, in homogenates obtained from the pronephric region and in the serum.     

Cell injury and regeneration of human epithelium in organ culture

Human esophageal, tracheal, and pancreatic ductal fragments were collected at autopsy after a postmortem interval of 12 hours or less and maintained in explant organ culture for 30 days. The viability and growth of the explants was assessed by morphology, LDH enzyme release, and cellular outgrowth. The viability and growth of the bronchial explant epithelium was directly related to the postmortem interval. Esophageal epithelial regeneration followed the desquamation of the superficial cell layers. Pancreatic epithelia appeared to grow more slowly and with less outgrowth than the other tissues. Epithelial cell growth along the explant surface and onto the culture dish appeared to proceed through the well-characterized process that follows cell injury, i.e., flattening, migration, replication, and differentiation. Thus, sufficient numbers of viable epithelial cells capable of regeneration were present in routine autopsy epithelium, but there was considerable variation from tissue to tissue and case to case. The most effective and accurate approach to follow when evaluating and predicting the growth and viability of these explants is by using a combination of morphologic, enzymatic and biologic assays. Errors in the interpretation of viability are possible when only one assay method is utilized. These tissues grown in explant organ culture are suitable for studies on the mechanism and response of epithelia to cell injury, recovery and wound healing.Abbreviations 4F-1G 4% formaldehyde, 1% glutaraldehyde - HIFBS heat inactivated fetal bovine serum - IA immediate autopsy - LDH lactate dehydrogenase - OsO₄ osmium tetroxide - RA routine autopsy     

Homoiogenetic Neural Induction in Xenopus Chimeric Explants

We previously raised monoclonal antibodies specific for epidermis (7) and neural tissue (8) of Xenopus for use as markers of tissue differentiation in induction experiments (8). Here we have used these monoclonal antibodies to examine homoiogenetic neural induction, by which cells induced to differentiate to neural tissues can in turn induce competent ectoderm to do the same. Presumptive anterior neural plate excised from late gastrulae of Xenopus laevis was conjugated with competent ectoderm from the initial gastrula of Xenopus borealis , either side by side or with their inner surfaces together. The chimeric explants enabled us to distinguish induced neural tissues from inducing neural tissues. In both types of explant, neural tissues identified by the neural tissue-specific antibody, NEU-1, were induced in the competent ectoderm by the presumptive anterior neural plate. The results suggest that homoiogenetic neural induction does occur in Xenopus embryos.     

Quantitative analysis of composite umbilical cord tissue health using a standardized explant approach and an assay of metabolic activity

Background

Umbilical cord (UC) tissue can be collected in a noninvasive procedure and is enriched in progenitor cells with potential therapeutic value. Mesenchymal stromal cells (MSCs) can be reliably harvested from fresh or cryopreserved UC tissue by explant outgrowth with no apparent impact on functionality. A number of stem cell banks offer cryopreservation of UC tissue, alongside cord blood, for future cell-based applications. In this setting, measuring and monitoring UC quality is critical.

Age-dependent lipid peroxidation in neonatal rat lung tissue.

A unique, age-specific pulmonary lipid peroxidation has been found to occur after incubation of neonatal rat lung homogenates in the absence of any added factors. As measured by the formation of malondialdehyde, lipid peroxidation was not detectable in rat lung homogenates prepared from animals immediately after birth but appeared by the second day and reached a maximum at 5 days of age. The effect gradually disappeared by 20 to 21 days after birth. The addition of NADPH did not enhance lipid peroxidation in the sensitive age group nor did it initiate lipid peroxidation when added to lung homogenates from either 1-day-old or adult rats. The activities and concentrations of various endogenous antioxidants were measured in neonatal lung tissue. When measured in lung tissue obtained from rats during the sensitive age period, no concomitant deficiencies of glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase, reduced glutathione, or a-tocopherol were observed. With the exception of α-tocopherol, none of these factors inhibited malondialdehyde formation when added to homogenized lung tissue prepared from 5-day-old rats. α-Tocopherol did inhibit malondialdehyde formation in 5-day-old rat lung homogenates but at a concentration much greater than the endogenous concentration found in adult rat lungs. The 21-day neonatal age period during which malondialdehyde is produced following incubation of lung tissue is similar to the 3-week period immediately after birth reported to be the time of maximum proliferation of rat lung fibroblasts, type 1 pneumocytes and type II pneumocytes.     

Human chorionic gonadotropin. V. Tissue specificity of binding and partial characterization of soluble human chorionic gonadotropin-receptor complexes.

An in vivo human chorionic gonadotropin (hCG)-receptor complex was solubilized from the subcellular fraction of ovarian and testicular tissues of rats that had been injected with 125-I-labeled hCG. The soluble hCG-receptor complex was partially characterized by Sepharose 6B chromatography in the presence of the nonionic detergent, Emulphogene, and was shown to have a molecular size of about 65 A. By this method it was also shown that the in vivo uptake of radioactivity by rat gonadal tissues represents 125-I-hCG and not the dissociated subunits or degradation products of the hormone. A soluble hCG-receptor complex isolated in vitro in approximately the same yield from both rat testicular and ovarian homogenates was shown to be the same size. The hCG-receptor appears to be specifically located in gonadal tissue; a corresponding hCG-receptor complex was not obtained from liver or kidney that incorporated significant levels of 125-I-hCG administered in vivo. Furthermore, a desialyzed hCG-receptor complex was obtained from rat testis but not liver; desialyzed hCG, like other desialyzed glycoproteins, is nonspecifically bound by rat liver homogenates. The binding of hCG and luteinizing hormone (LH) by rat testis receptor exhibits a high degree of specificity. Other glycoprotein hormones without LH activity, such as follicle-stimulating hormone and thyroid-stimulating hormone, and glycoproteins such as fetuin or alpha1-acid glycoprotein do not bind to the hCG/LH receptors. Desialyzed hCG was 2 times more effective in competing for binding to rat testis receptors than "native" hCG, indicating that caution must be exercised when the radioligand receptor assay is utilized to assay hCG preparations varying in sialic acid content.     

Split-seed: a new tool for maize researchers

Until recently, immature embryos have been a choice tissue for manipulation in culture for regeneration and production of transgenic maize plants. The utility of this explant has been compromised by low output, genotype dependence and time-consuming incubation in tissue culture. We have developed a new explant, the split-seed, which addresses these limitations by formally treating each seed as though it were a “dicot”. By splitting maize seed longitudinally, three different tissues: the scutellum, the coleoptilar-ring and the shoot apical meristems are simultaneously exposed. The cells of these tissues can be made competent to enhance the regeneration, given that the molecular networks resulting from exposure of the split-seed to hormones is likely to be different from whole seed and, in turn, affects the in vitro response. Using this explant, callus induction frequency exceeded 92% and the regeneration frequency was 76%. The mean number of shoots regenerated via callus was 11 shoots per callus clump and 28 shoots per explant at first sub-culture. All of the regenerated plants survived and were 95% fertile. The large numbers of fertile plants produced were regenerated in 6–8 weeks. Finally, the incidence of regenerated plants varies as a function of growth regulator profile.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Contact smear cytologic preparations of organ culture explant tissue

The effects of cytologic sampling by contact-smear on the viability and morphology of epithelial tissues maintained as explants in organ culture were studied. Tracheal and pancreatic tissues from Syrian golden hamsters were maintained for 30 days in culture, and sampled cytologically at weekly intervals. Analysis of sequential Papanicolaou-stained smears demonstrates that explant tissues can be evaluated cytologically over time in vitro without apparent effects on either the tissue viability, histochemistry or morphology. Microscopic examination further reveals that cellular samples retain diagnostically significant cytomorphologic features of the in vivo tissues. These observations indicate that this technique is useful for understanding the long-term response to carcinogens of epithelial tissue maintained in explant organ culture.     

Immunological and molecular characterization of plasminogen activator inhibitors 1 and 2 in baboon (Papio anubis) placental tissues

Two major plasminogen activator inhibitors (PAI-1 and PAI-2) increase in the peripheral circulation during pregnancy in humans. PAI-1 is of vascular endothelial origin whereas PAI-2 is produced primarily by human placental tissues. This study was undertaken to determine a) if PAI-1 and PAI-2 are also present in the baboon and b) their association with pregnancy. Citrated plasma was obtained from pregnant baboons sequentially at 15 +/- 3-day intervals between Days 30 and 140 of pregnancy. PAI activity increased significantly (p less than 0.05) at Day 120 (15.3 IU/ml) and 140 (21.8 IU/ml) of gestation and returned to baseline (2.6 IU/ml) 48 h post cesarean section. Placental tissues obtained at cesarean section during the third trimester were either placed in explant culture, fixed for immunocytochemistry, or frozen for RNA extraction. Western blot analysis of tissue culture media (TCM) indicated that the polyclonal antibody to PAI-1 reacted with a major band (Mr 47 000) in TCM from placental tissues while the PAI-2 antibody reacted primarily with a doublet (Mr 67 000 and 69 000) in these same media. PAI-1 was immunocytochemically localized primarily in the chorioamniotic tissue (CAM-D) and PAI-2 was found predominantly in placental villi. Slot blot hybridization with cDNAs to PAI-1 and PAI-2 indicated that the mRNA for PAI-2 was found primarily in placental villi, whereas the mRNA for PAI-1 was present in all three tissue compartments.(ABSTRACT TRUNCATED AT 250 WORDS)     

The occurrence of soluble and membrane-bound non-specific lipid transfer protein (sterol carrier protein 2) in rat tissues

The occurrence and subcellular distribution of the non-specific lipid transfer protein (nsL-TP; sterol carrier protein 2) in rat tissues was investigated by the immunoblotting technique using the affinity purified antibody against rat liver nsL-TP. Highest levels of the protein were found in the homogenates of liver, lung and adrenals, whereas it could hardly be detected in brain. In other tissues (i.e., testis, kidney, heart and intestine) the protein was present at intermediate concentrations. Analysis of subcellular fractions obtained by differential centrifugation demonstrated that in all tissues except for the liver, nsL-TP was predominantly present in the particulate fractions. In the particulate fractions of all tissues, an immunoreactive 58 kDa-protein was detected. Density centrifugation of a nuclear-free homogenate from liver and testis indicated that the 58 kDa-protein did, and nsL-TP did not, cofractionate with catalase. This suggests that in these tissues the bulk of nsL-TP is extraperoxisomal. Membrane-bound nsL-TP in testis was sensitive to trypsin treatment, suggesting that it is exposed to the cytosol. Release of nsL-TP by washing the membranes with 0.1 M Na2CO3 (pH 11.5), indicated that nsL-TP is a periferal protein. It was shown by chromatofocussing that nsL-TP extracted from membrane fractions was more basic than nsL-TP present in the cytosol fraction from rat liver (isoelectric point of 8.7).     

Direct somatic embryogenesis of Agave fourcroydes Lem. through thin cell layer culture

Here, we describe a new protocol for the induction of direct somatic embryogenesis of Agave fourcroydes through thin cell layer (TCL) culture technology. The protocol was optimized for the main factors known to affect the process, including the type of explant (stem or leaf tissue), type and concentration of exogenous growth regulators (α-naphthalene acetic acid [NAA], 2,4-diclorophenoxyacetic acid [2,4-D], 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid [picloram], and 3,6-dichloro-2-methoxybenzoic acid [dicamba]), and the influence of plant genotype. Thin tissue segments cut transversally (tTCLs) from stems of in vitro-cultured plants gave the best embryogenic response when cultured with 2.26 μM dicamba (92.22 embryos/explant) or 2.07 μM picloram (81.72 embryos/explant). It was interesting to observe that the embryogenic capacity of these tissues was affected by the presence of 6-benzylaminopurine (BA) in the culture medium in which the explant donor plantlets were maintained. Thirteen clonal lines (each derived from a different parental plant), compared for their embryogenic competence under the same culture conditions, produced very different embryogenic responses that varied from very high (117 embryos/explant) to null. The histological analysis revealed that the amount of meristematic tissue present in the tTCLs varied according to the region of the stem (apical, middle, or basal) from which they originated. The cells of the vascular procambium became competent and developed into cell lines that formed embryos, either by a unicellular or a multicellular pathway. Mature embryos germinated in half-strength Murashige and Skoog medium without growth regulators and 85% of regenerated plants was successfully acclimatized in a greenhouse.     

On the existence of receptors to the pheromonal steroid, 5 alpha-androst-16-en-3-one, in porcine nasal epithelium

The binding of the odorant, 5 alpha-androst-16-en-3-one, to porcine nasal tissues, has been investigated using methods normally employed for studying both cytosolic and membrane-bound receptors. 5 alpha-Androst-16-en-3-one was generally taken up more avidly by homogenates of olfactory (nervous) tissue than by respiratory tissue, but binding to the former was only partially prevented by prior heating or by excess ligand, suggesting some degree of specific binding. At low protein concentration, saturable binding was noted but these data were not reproducible. The binding of a non-odorant, DHA, was only 2% that of 5 alpha-androst-16-en-3-one. Using agarose gel electrophoresis, some evidence was obtained for binding protein(s) to the odorous 16-adrostene in porcine respiratory tissues, that were absent from previously heated tissue. Experiments with SDS-treated, or cell-membrane-enriched preparations, of nasal epithelium did not show improved binding of 5 alpha-androst-16-en-3-one. We conclude that the extreme hydrophobicity of 5 alpha-androst-16-en-3-one is probably responsible for the high degree of non-specific binding noted and for variability in results. This is discussed in relation to other known odorous ligand/receptors in olfactory tissue, particularly that of 5 alpha-androstan-3-one.     

Evaluation of peanut (Arachis hypogaea L.) leaflets from mature zygotic embryos as recipient tissue for biolostic gene transfer

Leaflets from mature peanut embryos are a useful recipient tissue for biolistic DNA transfer. Fertile plants were regenerated from leaflets from genotypes representing all botanical types of peanut. Regeneration frequency was strongly influenced by genotype. NPT II and GUS chimaeric gene fusions, driven by the CaMV 35S promoter, were expressed transiently following biolistic delivery to unexpanded leaflets. Bombardment conditions affecting transient expression frequency were determined using a prototype of the Bio Rad PDS 1000/He helium-powered particle acceleration apparatus. Stably transformed calli were derived routinely from leaflet tissue bombarded with the NPT II gene and subsequently cultured on kanamycin. Several plants have been regenerated from treated explants under kanamycin selection. Thus far, none of these has been stably transformed. The occurrence of escapes suggests that kanamycin is an inefficient selective agent for the recovery of transgenic peanuts from this explant. Experiments designed to regenerate plants using published regeneration protocols from stably transformed calli, devoid of primary explant tissue, have been unsuccessful.     

Adaptation of the phosphotungstate method for the determination of vitamin C contents in animal and human tissues

The usefulness of phosphotungstate reagent for vitamin C determination in tissue homogenates has been confirmed. An optimal homogenization medium was selected: 1.8 M solution of HPO3 in 1.3 M CH3COOH. With this medium the analytical curve (at 700 nm) demonstrated the right linearity, correlation and recovery coefficients were appropriately high (0.999 and 99.8%) and the values of intraserial and interserial variation coefficient were low (< 5% and < 10%, respectively). It makes this method sensitive, easily repeatable, and useful for vitamin C determination in animal and human tissues, including neoplastic ones.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes reduction of glucose transporting activities in the plasma membranes of adipose tissue and pancreas from the guinea pig.

Toxicity from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure results in severe metabolic imbalances leading to a loss of fat stores in many animal species, a phenomenon known as the wasting syndrome. In this paper, we report that TCDD treatment at very low doses (0.03-1 micrograms/kg, single intraperitoneal injection) causes a profound reduction of glucose uptake by guinea pig adipose tissue, pancreas and brain. This effect of TCDD is dose-dependent, with a dose as small as 0.03 micrograms/kg resulting in a significant decrease. In adipose tissue, the decrease begins within 6 h of treatment and persists at least 28 days. The Vmax of glucose transport was decreased by TCDD treatment, whereas the Km was unchanged. Liver behaves oppositely to adipose tissue. At early stages of treatment (6-12 h) glucose uptake was depressed, while at later stages (24-96 h) it was increased. In situ (explant tissue culture) treatment with TCDD yields similar trends as in vivo studies for glucose uptake in all three tissues. In adipose tissue culture TCDD starts reducing glucose uptake after 30 minutes. The inhibitory potencies of three dioxin congeners on adipose glucose transporting activities follows the same order of their toxicities in vivo. TCDD's effect on glucose transport is sensitive to cytochalasin B, a specific inhibitor of glucose transporter proteins. Based on these observations and the importance of glucose transporters to cellular energy maintenance, we conclude that at least in guinea pigs the reduction of glucose transporters in various tissues is one of the major causes for TCDD-induced wasting syndrome, which is so prominent in this species.     

Induction and characterization of the major surfactant apoprotein during rabbit fetal lung development

Antibodies directed against the major apoprotein associated with rabbit lung surfactant were used to characterize the induction and cellular localization of this protein during rabbit fetal lung development. In lung tissues from rabbits of 26 days gestational age and older, discrete epithelial type II cells were stained positively using the peroxidase antiperoxidase technique. The content of the major protein in homogenates of fetal lung tissue was analyzed using an immunoblotting technique. A protein of about 29 kDa, pI less than or equal to 5.6, was first detectable in fetal lung tissue on day 24 of gestation. The 29-36 kDa, mature form of the surfactant apoprotein was first detectable in lung homogenates from 30-day gestational age fetal rabbits. Treatment of homogenates of day 26 and 31 fetal lung tissues with endoglycosidase F, yielded, in both cases, an immunoreactive triplet with more neutral isoelectric points than the proteins in the untreated homogenates. By immunoblot analysis, we found that only the 29-36 kDa, mature form of the surfactant apoprotein was present in lamellar bodies purified from lung tissues of fetuses of 28 and 31 days and from day 2 neonates. These findings are suggestive that only the mature, 29-36 kDa form of the surfactant apoprotein is associated with lamellar bodies during fetal lung type II cell differentiation in vivo.     

The sequential enzymatic determination of DNA and RNA

Enzymatically small quantities of native DNA and RNA in crude tissue homogenates can readily be specifically determined using ethidium bromide as a fluorescent indicator. The ethidium bromide-polynucleotide complexes of DNA and RNA serve as effective substrates for deoxyribonuclease and ribonuclease, respectively. Optimal divalent metal cation requirements were determined for a common reaction medium that is compatible for both the DNase and RNase reactions. To a single reaction mixture, that contains a biological sample, sequential addition of DNase and RNase produces a specific and rapid decrease in fluorescence that is proportional to the respective amounts of DNA and RNA present. Levels of DNA and RNA found in six different tissues of the rat were determined enzymatically by this method and compared to that obtained by alternate techniques. Enzymatically determined values were highly reproducible and correlate well with those values obtained by more time-consuming, conventional methods. Most enzymatically determined RNA levels in tissues, however, were significantly greater than those levels obtained spectrophotometrically. Advantages of the enzymatic procedure for analysis of tissue polynucleotide content are: (1) rapid determination of both DNA and RNA within a single sample aliquot allowing maximum use of available sample; (2) extensive fractionation and extraction of the tissue are not required; (3) it is especially useful when quantities of tissue are limited; and (4) sensitivity to 0.05 and 0.25 μg of DNA and RNA, respectively. Reaction conditions developed for the assay also provide for a highly sensitive means to continuously monitor DNA hydrolysis; DNase activity is directly proportional to the amount of enzyme added.     

A sensitive cyclic nucleotide phosphodiesterase assay for transient enzyme kinetics

A rapid and sensitive method was developed for the quantitative determination of alpha-tocopherol in tissues and plasma of rats and mice. Tissue and plasma were extracted in acetone and chromatographed on a reverse-phase C18 column with 2% water in methanol. Fluorescence and ultraviolet detection were used for tissue and plasma alpha-tocopherol levels, respectively. Extraction of tissues and plasma was found to be more complete in acetone than in other solvent systems analyzed. The average recovery of alpha-tocopherol added to tissue samples was 97%. As little as 0.1 g of tissue or 0.1 ml plasma can be accurately used for analysis. The method is sensitive to 0.05 micrograms alpha-tocopherol/g tissue.     

Effect of steroid hormones on sulfated oviductal glycoprotein secretion by oviductal explants in vitro

Explants of rabbit ampullary and isthmic tissue were cultured 4 days with and without exogenous steroids, and the sulfated oviductal glycoprotein (SOG) concentration in the explant culture supernatants was determined. Tissues cultured with progesterone plus estrogen secreted significantly more SOG than control tissues, whereas tissues cultured with estrogen alone did not. Ampullary tissues cultured with progesterone plus estrogen secreted significantly more SOG than control tissues on Days 2 and 3, whereas SOG secretion by isthmic tissues was significantly above control secretion on Day 4. Ampullary and isthmic tissues differed significantly in their secretory capacity. Maximum ampullary SOG secretion was approximately 650 ng SOG/mg tissue/day. Maximum isthmic SOG secretion was approximately 30 ng SOG/mg tissue/day. These findings suggest that the oviduct is composed of discrete functional regions that provide support to gametes and developing embryos through the unique secretory characteristics of each region.