Expression of preprotachykinin-A and neuropeptide-Y messenger RNA in the thymus.

The preprotachykinin-A gene, the common gene of mRNAs encoding both substance-P (SP) and neurokinin-A (NKA), was shown to be expressed in Sprague-Dawley rat thymus by detection of specific mRNA in gel-blot analyses. In situ hybridization revealed dispersed PPT-A-labeled cells in sections from rat thymus, with a concentration of grains over a subpopulation of cells in the thymic medulla. Also, neuropeptide-Y mRNA-expressing cells were found in the rat thymus, primarily in the thymic medulla. Rat thymic extracts contained SP-like immunoreactivity (SP-LI), and the major part of the immunoreactivity coeluted with authentic SP and SP sulfoxide standards. SP-LI was also detected in human thymus, which contained between 0.09-0.88 ng SP-LI/g wet wt. Evidence for translation of preprotachykinin-A mRNA in the rat thymus was obtained from the demonstration of NKA-LI in thymic cells with an epithelial-like cell morphology. Combined with previous observations on the immunoregulatory roles of tachykinin peptides and the existence of specific receptors on immunocompetent cells, the demonstration of intrathymic synthesis of NKA suggests a role for NKA-LI peptides in T-cell differentiation in the thymus.     

Simultaneous isolation and determination of prothymosin alpha, parathymosin alpha, thymosin beta 4, and thymosin beta 10

A method was described for the isolation of peptides from rat thymus. Frozen, powdered tissue was suspended in boiling buffer to inactivate endogenous proteinases, the suspension was homogenized, and the peptides were isolated by a two-step procedure including gel filtration and purification by HPLC. The recoveries from rat thymus were, in micrograms per gram of whole tissue, 60-80 for prothymosin alpha, 50-80 for thymosin beta 4, and 20-30 for thymosin beta 10. The procedure also yielded smaller quantities of a fourth peptide, designated parathymosin alpha. The quantities of these peptides in vertebrate tissues can be evaluated by applying radioimmunoassays for prothymosin alpha and thymosin beta 4 to the boiled tissue extract.     

The ubiquity of the highly phosphorylated nuclear protein P1

The present work shows that antibodies raised in rabbits against rat liver P1 confirmed the presence of P1 in lung, kidney, brain heart, muscle, intestine and thymus in rats. The antiserum reacted with P1 from human and monkey but not from bovine, pig and mouse P1 in spite of there being a close relationship in amino acid composition, electrophoretic properties and peptide mapping. Proteolytic digestion of rat P1 showed that only some of the peptides produced reacted with the antiserum, suggesting that conformational determinants may be dominating compared to sequential determinants in P1, or that only minor parts of P1 which exhibit sequential variation between species are immunoreactive.     

Effect of thymus polypeptide fractions on the development of rat thymus and spleen in organ culture

Peptides of the thymus--vilon, thymogen and thymalin, alone or in combination with concanavalin A, were used to investigate their effect on organotypic culture of thymus and spleen explants from 1- and 21-day old rats. Vilon, thymogen and thymalin in concentrations of 2 and 10 ng/ml and 5 ng/ml, resp., exerted stimulating effects in thymus and spleen tissue cultures from 21-day old rats as compared to the control explants. Vilon and thymogen showed inhibiting effect in the thymus tissue cultures from 1-day old rats as compared to the control explants. However, the peptides together with concanavalin A in concentration of 10 mkg/ml resulted in decreasing the action of concanavalin A alone. The polypeptide fractions of thymus and their synthetic analogs play different roles in the regulation of thymus and spleen development in rats of different age.     

Identification of urotensin II-related peptide as the urotensin II-immunoreactive molecule in the rat brain

Urotensin II (UII) has been reported as the most potent known vasoconstrictor. While rat and mouse orthologs of UII precursor protein have been reported, only the tentative structures of UII peptides of these animals have been demonstrated, since prepro-UII proteins lack typical processing sites for their mature peptides. In the present study, we isolated a novel peptide, UII-related peptide (URP), from the extract of the rat brain as the sole immunoreactive substance to anti-UII antibody; the amino acid sequence of the peptide was determined as ACFWKYCV. cDNAs encoding rat, mouse, and human precursor proteins for URP were cloned and revealed that the sequences of mouse and human URP peptides are the same as that for rat URP. Prepro-URP gene is expressed in several rat tissues such as those of the thymus, spleen, testis, and spinal cord, although with lower levels than the prepro-UII gene. In the human, the prepro-URP gene is expressed comparably to prepro-UII in several tissues except the spinal cord. URP was found to bind and activate the human or rat UII receptors (GPR14) and showed a hypotensive effect when administered to anesthetized rats. These results suggest that URP is the endogenous and functional ligand for UII receptor in the rat and mouse, and possibly in the human. We also describe the preparation of specific monoclonal antibodies raised against UII peptide and the establishment of a highly sensitive enzyme immunoassay system for UII peptides.     

Calf thymus deoxyuridine triphosphatase differs from rat spleen enzyme in molecular disposition

Antiserum against calf thymus dUTPase was raised in rats by injection of the partially purified enzyme. The antiserum did not react with dUTPase purified from rat spleen, while antibody against rat spleen dUTPase partially reacted with calf thymus enzyme. Native molecular weight of the calf thymus dUTPase was estimated at 46,000 daltons by gel filtration, and the denatured form of the enzyme was about 22,000, as judged by immunoblot analyses using the antibodies. These results indicate that the calf thymus dUTPase is composed of two identical subunits.     

Pathogenesis of autoimmunity after xenogeneic thymus transplantation

Thymus transplantation is a promising strategy to induce xenotolerance, but may also induce an autoimmune syndrome (AIS). The pathogenesis of this AIS was explored using nude rats as recipients. Thymus grafts consisted of fetal hamster thymic tissue with or without mixing with fetal rat tissue such as thymus, thyroid, salivary gland, and heart. All hamster thymus recipients died of AIS within 2-3 mo. In most recipients of xenothymus mixed with rat tissues such as thymus, thyroid, and salivary gland, but not heart, AIS was prevented, indicating an insufficient presence of rat epithelial cell Ags within the xenothymus. AIS could be transferred to control nude rats by whole splenocytes or by splenocyte subpopulations such as CD3(+), CD3(-), and B lymphocytes, but not by non-T, non-B cells from AIS animals. This transfer could be suppressed by cotransferring either CD4(+) or CD8(+) lymphocytes from euthymic rats, but not by splenocytes from recipients of syngeneic or xenogeneic thymus mixed with rat tissue, indicating a defective generation of regulatory lymphocytes. As for CD4(+) regulatory cells this defect was probably qualitative, because the percentages of CD4(+)CD25(+) or CD4(+)CD45RC(low) populations were normal after xenothymus transplantation. As for the CD8(+) regulatory cells, the defect was quantitative, as CD8(+) cell levels always remained low. The latter was related to the nonvascularized nature of thymus grafts. In conclusion, AIS after xenothymus transplantation in nude rats is due to a combination of insufficient intrathymic presence of host-type epithelial cell Ags and a defective generation of regulatory T lymphocytes.     

Starvation-sensitive UCP 3 protein expression in thymus and spleen mitochondria

To date, UCP 3 has only been associated with skeletal muscle and brown adipose tissue (BAT). Using RT-PCR/PCR methodology, we show that human spleen and human thymus contain UCP 3. In addition, using peptide antibodies, previously demonstrated to be selective for UCP 3, we show that UCP 3 protein is present in mitochondria isolated from rat thymus and mitochondria isolated from reticulocytes, monocytes and lymphocytes of rat spleen. UCP 3 protein expression is also starvation-sensitive. UCP 3 abundance is augmented in mitochondria isolated from thymus and mitochondria isolated from lymphocytes of the spleen from fasted rats when compared to fed controls. The results are consistent with a role for UCP 3 in developing lymphocytes, thymus atrophy and fatty acid utilisation in spleen and thymus.     

Cold acclimation and oxygen consumption in the thymus

Mitochondrial uncoupling protein 1 is usually associated with brown adipose tissue but has recently been discovered in rat and mouse thymus. We wished to establish whether there was a thermogenic role for UCP 1 in thymus and thus examined the effect of 5 weeks cold-acclimation on rat thymus tissue abundance, thymocyte oxygen consumption, thymus mitochondrial abundance, uncoupling protein 1 expression and function. We found that thymocytes from cold-acclimated rats had oxygen consumption rates 8 times less than those from rats held at room temperature and that thymocytes from cold-acclimated rats or rats kept at room temperature were noradrenaline insensitive. In addition, we found that thymus tissue or mitochondrial abundance was not increased after cold-acclimation. However uncoupling protein 1 expression per unit mass of mitochondria was increased after cold-acclimation, as determined by immunoblotting (approximately 1.7-fold) and GDP binding (approximately 1.5-fold). Consistent with our protein expression data, we also observed an increased, state 4 (approximately 1.5-fold), GDP-inhibitable (approximately 1.3-fold) and palmitate activatable (approximately 1.6-fold) oxygen consumption rates in isolated thymus mitochondria. However, extrapolation of our data showed that cold-acclimation only increased the amount of UCP 1 per gram of thymus tissue approximately 1.2-fold. Taken together, we conclude that UCP 1 does not have a thermogenic role in thymus.     

Can prenatal exposure to a 900 MHz electromagnetic field affect the morphology of the spleen and thymus,and alter biomarkers of oxidative damage in 21-day-old male rats?

We investigated the effects of a 900 Megahertz (MHz) electromagnetic field (EMF), applied during the prenatal period, on the spleen and thymus of 21-day-old male rat pups. Pregnant Sprague-Dawley rats were divided into control and EMF groups. We applied 900 MHz EMF for 1 h/day to the EMF group of pregnant rats. Newborn male rat pups were removed from their mothers and sacrificed on postnatal day 21. Spleen and thymus tissues were excised and examined. Compared to the control group, thymus tissue malondialdehyde levels were significantly higher in the group exposed to EMF, while glutathione levels were significantly decreased. Increased malondialdehyde and glutathione levels were observed in splenic tissue of rats exposed to EMF, while a significant decrease occurred in superoxide dismutase values compared to controls. Transmission electron microscopy showed pathological changes in cell morphology in the thymic and splenic tissues of newborn rats exposed to EMF. Exposure to 900 MHz EMF during the prenatal period can cause pathological and biochemical changes that may compromise the development of the male rat thymus and spleen.     

Multiple species of ornithine decarboxylase in rat tissues: Effects of dexamethasone

Several species of ornithine decarboxylase were separated by chromatography of rat thymus and kidney extracts on DEAE-Sepharose CL-6B. One major and one minor species were absent from thymus of rats two hours after hormone treatment but otherwise, the elution profile was identical to thymus from control animals. The elution patterns of ODC activity in kidneys of rats treated 2.5 or 5 hours before sacrifice with dexamethasone differ from that of control kidney and from each other. Enzyme from kidneys early after hormone treatment is eluted earlier than enzyme from control tissue, while at 5 hours, the enzyme is eluted much later than in the control. This suggests that the hormone-induced activity is subsequently modified.     

Cold acclimation and oxygen consumption in the thymus

Mitochondrial uncoupling protein 1 is usually associated with brown adipose tissue but has recently been discovered in rat and mouse thymus. We wished to establish whether there was a thermogenic role for UCP 1 in thymus and thus examined the effect of 5 weeks cold-acclimation on rat thymus tissue abundance, thymocyte oxygen consumption, thymus mitochondrial abundance, uncoupling protein 1 expression and function. We found that thymocytes from cold-acclimated rats had oxygen consumption rates 8 times less than those from rats held at room temperature and that thymocytes from cold-acclimated rats or rats kept at room temperature were noradrenaline insensitive. In addition, we found that thymus tissue or mitochondrial abundance was not increased after cold-acclimation. However uncoupling protein 1 expression per unit mass of mitochondria was increased after cold-acclimation, as determined by immunoblotting (∼ 1.7-fold) and GDP binding (∼ 1.5-fold). Consistent with our protein expression data, we also observed an increased, state 4 (∼ 1.5-fold), GDP-inhibitable (∼ 1.3-fold) and palmitate activatable (∼ 1.6-fold) oxygen consumption rates in isolated thymus mitochondria. However, extrapolation of our data showed that cold-acclimation only increased the amount of UCP 1 per gram of thymus tissue ∼ 1.2-fold. Taken together, we conclude that UCP 1 does not have a thermogenic role in thymus.     

Organ specific expression of ras oncoproteins during growth and development of the rat

Summary Expression of proteins encoded by the ras proto-oncogenes was examined in extracts from normal rat organs using anti-ras p21 antibodies generated against synthetic peptides. The highest level of ras p21 was found in brain (cerebrum) and was predominantly of c-Ha-ras origin. Levels of brain ras p21 did not vary from the newborn period of 3 months of age. Moderate levels of ras p21s were detected in lung, spleen and thymus. In contrast to the p21 in brain, these levels varied with the age of the rats and were encoded by other members of ras proto-oncogene family (Ki-ras or N-ras). This organ specific expression of different ras genes might be related to developmental control of gene expressions.     

Substance P-like immunoreactivity in capsaicin-sensitive structures of the rat thymus

Unlike in mouse and hamster, the thymus of rats or guinea pigs contains measurable amounts of substance P-like immunoreactivity (SP-LI), which, in a HPLC system, eluted as authentic SP or SP sulfoxide. Ontogenetic study showed that in rats the SP-LI content of the thymus increased up to 60 days from birth, and decreased thereafter. Capsaicin, but not 6-hydroxydopamine (6-OHDA) pretreatment completely depleted thymic SP-LI content in both newborn and adult rats. Animals treated with capsaicin as newborns, but not as adults, showed lower thymus weights as compared to controls. Rats pretreated with capsaicin as adults underwent partial time-dependent recovery of thymic SP-LI content. Somatostatin-like immunoreactivity (SST-LI) of rat thymus, eluting in part as authentic SST, was unaffected both by capsaicin or 6-OHDA pretreatment. Taken together, these findings demonstrate the existence of capsaicin-sensitive structures containing SP in the rat thymus. The possible function(s) that capsaicin-sensitive structures could exert in the thymus, among which a trophic action, mediated by the efferent function of sensory neurons, remain(s) to be established.     

Purification and characterization of a calf thymus protein active on lipid metabolism

1. This work concerns the purification of a calf thymus protein that increases the binding of human 125I-labeled low density lipoprotein (LDL) on both human skin fibroblasts and a special line of rat liver cells, BRL 3A. 2. It was found that the thymus gland affects cholesterol metabolism via an activation of the LDL receptor pathway. 3. Moreover, the thymus protein active on the LDL receptor pathway has a different amino acid composition and molecular weight from other well-characterized thymic peptides.     

Isolation and partial characterization of prothymosin alpha from porcine tissues

Prothymosin alpha, an immunoactive polypeptide of 12 kDa, has been isolated from porcine thymus, spleen, lung and kidney. It lacks aromatic and sulfur-containing amino acids and has a high content of glutamic and aspartic acids. Tryptic digestion of porcine thymus prothymosin alpha yielded peptides which on separation, amino acid analysis and alignment with the known sequence of prothymosin alpha from rat and man showed that the amino terminal portion of the molecule is conserved and the few differences present are confined to the carboxy terminal.     

Immunochemical characterization of neurotensin-like peptides in chicken

Using radioimmunoassay and 3 region specific antisera toward bovine neurotensin (NT), the NT-like peptides in chicken have been shown to differ from NT but to strongly resemble its COOH-terminal region. Three substances were identified, one of which resembled NT biologically and appeared to share 7 or 8 of its COOH-terminal residues. The two other peptides were smaller than NT but seemed to possess 4–6 residue homologies with it. Tissue distribution studies indicated that the chicken pancreas and thymus had unusually high levels of this material (>200 fold that in rat) and that the 3 substances were distributed differently in tissues. Chromatographic studies showed that the peptides obtained from brain, intestine, thymus, and pancreas were similar. These results, demonstrating evolutionary conservation of the COOH-terminal region of NT, are in keeping with the known importance of this region for biological activity. These findings also suggest the existence of an NT-family of peptides serving multiple biological roles.     

Identification of a low-Mr acidic nuclear protein as prothymosin alpha

We have purified to homogeneity a 15-kDa perchloric acid (PCA)-soluble protein from rat thymus nuclei. This highly acidic protein showed a Mr of ca. 30 kDa in acetic acid/urea gels, probably due to oligomer formation. Sequence analysis of internal tryptic and thermolytic peptides revealed that the purified protein is, in fact, prothymosin alpha, a very hydrophilic polypeptide, which has been previously classified as a thymic or immunomodulating hormone. We found that prothymosin alpha is a rather abundant nuclear protein in rat thymus; its concentration is comparable to that of a well-characterized nonhistone protein HMG-14. The subcellular localization and physicochemical properties of prothymosin alpha suggest that its function is related to those of other long polyacidic regions containing nuclear proteins.     

A thymus factor influences the in vitro testosterone secretion of Leydig cells in the rat

Thymus extracts obtained from 15-day-old rats were fractionated through molecular sieve chromatography, and the fractions assayed in vitro by changes produced in the testosterone secretion of Leydig cells obtained from adult rat testes. Fractions corresponding to 27-28000 mol wt of the thymus extract diminish the testosterone secretion of Leydig cells stimulated with hCG. No changes in the basal testosterone secretion were produced by the presence of the thymus fractions. The inhibitory effect is dose related and persists during 180 min of incubation. Fractions of the same mol wt obtained from liver, heart and spleen do not modify the testosterone secretion of Leydig cells. The inhibitory activity of the thymus factor disappears after heat or trypsin treatment. Further fractioning in preparative flat bed electrofocusing makes manifest that the inhibitory activity is focused at pH 4.7. The data demonstrate the existence in rat thymus of a factor, probably of protein nature, which modifies the in vitro hCG response of a testis cell suspension.     

Synthesis of lipids in the thymus of irradiated rats

Changes in the synthesis of total lipids from 14C-glucose and 3H2O and in triacylglycerol concentration in the thymus of rats X-irradiated with a single dose of 14.35 or 6.0 Gy were investigated. Both lipid synthesis and TAG concentration were found to increase in the irradiated rat thymus.