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Tryptophan (Trp) is an essential amino acid which metabolises via the kynurenine pathway to generate a number of bioactive substances referred to as kynurenines. Among those are 3-hydroxykynurenine (3-HKyn) and quinolinic acid, which are neurotoxic, as well as kynurenic acid (Kyna) and xanthurenic acid (XA), which, similarly to nicotinamide (NAm), show neuroprotective and anti-depressive effects. Routine exercise is known to modulate Trp metabolism in skeletal muscle and is thus believed to reduce the risk of depressive states in humans and laboratory animals. Analogously, it was hypothesised that exercise can influence Trp metabolism in horses as well. The aim of this study was to evaluate the influence of two different types of exercise on Trp metabolism in horses of the same breed. A total of 32 purebred Arabian horses were involved in the study. The 22 three-year-old racehorses were subjected to short-time intense exercise. Ten other horses were made to perform endurance competitions at a distance of 80 km. Blood samples were collected at rest and following the end of the exercise period. Plasma concentrations of Trp, kynurenine (Kyn), Kyna, 3-HKyn, XA and NAm were determined using Ultra-High Performance Liquid Chromatography-Electrospray Ionisation-Tandem Mass Spectrometry. Short-time intense exercise led to an increase in plasma concentrations of Kyn, Kyna and XA. The endurance effort induced an increase in Kyna and a decrease in Trp and NAm levels. Both types of exercise, short-time intensive exercise and endurance exercise induced an increase in Trp metabolites, especially Kyna, and did not induce an increase in Trp level. Thus, from a pathophysiological perspective of the kynurenine pathway’s influence on mental state, both types of exercise induced beneficial effects in horses.

The current predation threat of domestic horses is generally low, and horses do not know predators' frightening cues. We studied whether horses still recognise predation threats. The aim of the study was to analyse the emotional response of purebred Arabian horses (Arabian) and Polish Konik horses (Konik) to an Arabian panther (Panthera pardus nimr) (panther) growl and a grey wolf (Canis lupus) (wolf) howl. Panther vocalisation was known to Arabian ancestors, whereas ancestors of Konik knew wolf vocalisation. The response to the howls of golden jackals (Canis aureus) (jackal), which did not prey on equids, was also studied comparatively. Two groups of 10 adult horses of each breed were subject to predator sounds of one predator daily for 5 min during a turn out on pasture. The test was performed for 18 days in total. The sound of each predator was interchangeably featured from one loudspeaker for 3 days followed by four loudspeakers simultaneously to imitate a group of predators for 3 days. The horses' emotional agitation in response to the sounds was measured based on the parameters of heart rate variability (HRV) using telemetric devices. The results showed that the predators' sounds were identified by horses as stressful or neutral. Horses generally retained their anti-predator responses even in the current habitat, which typically lacks predation cues. The results are not always coherent and may demonstrate that the response is somewhat attenuated. The wolf howl elicited a stronger response in Koniks. The panther growl more strongly influenced Arabians, whereas the jackal howl minimally elicited an agitation in the horses. The differentiated response of the two horse breeds to the three predator species suggests that the response is an innate adaptation to the predation risk in the habitat of the breed ancestors. This response occurs regardless of the emotional arousal specific to a breed, and the frightening cue is not the sound per se but the possible attack of predators. Horses display a type of understanding of the sound meaning. Their HRV response seems to be adequate for the threat signalised by the sound.

The aim of this study was to compare the changes in hematological and biochemical parameters in race horses subjected to standardized field exercise tests. The tests were performed two times: in May, at the beginning of the racing season and then four months later. Ten horses were studied in May (5 Thoroughbreds and 5 Purebred Arabian horses). Eight horses were studied in September (4 Thoroughbreds and 4 Arabian horses). For each horse, venous blood samples were collected at rest, after warm-up, just after the end of each gallop, and 30 minutes after the end of the exercise. The concentration of blood lactic acid (LA) hemoglobin (Hb), plasma glucose (Glc), triacylglycerols (TG), glycerol, uric acid (UA), total protein, total thiobarbituric acid-reactive substances (TBARS), the activity of lactate dehydrogenase (LDH), kreatine kinase (CK) and aspartate aminotransferase (AST) and the total antioxidant status (TAS) were determined. The standardized field exercise test performed in September, in comparison to the first test, evoked the lowest increase in blood LA and plasma Glc and TG levels determined during exercise. The rapid return of elevated levels of LA, Hb and UA to their initial values was also observed in September, as compared to data obtained in May [ABSTRACT FROM AUTHOR]

Leptin is a tissue-derivative adipokine that regulates appetite, food intake and energy expenditure. It is still not clear how exercise affects plasma leptin concentration in horses. The aim of this study was to evaluate the influence of exercise intensity and duration on plasma leptin levels in working horses. A total of 38 horses were prospectively included in the study and grouped according to the type of exercise they performed: dressage (six stallions, group D), jumping (12 stallions, group J), race (12 Thoroughbred horses, six stallions and six mares, group R) and harness (10 light draft stallions, group H). Blood samples were taken both before and after routine exercise (immediately after the exercise, 30 min and 24 h after). Blood lactic acid (LA) and plasma concentration of leptin, cortisol, uric acid, triacylglycerols, glycerol and free fatty acids were determined. Immediately after exercise, group R had the highest level of LA, whereas groups D and J had the lowest levels. A significant increase in plasma leptin concentration was stated only in group H in samples taken immediately after the end of the exercise period and 30 min after the exercise period, as compared with the values obtained at rest. A significant increase in plasma cortisol concentration was found immediately after the end of the exercise period in groups R and H. Leptin exercise-to-rest ratio was significantly correlated with cortisol exercise-to-rest ratio (r=0.64; P

The effect of acute exercise was studied in a group of 42 clinically healthy young Standardbred trotters. These trotters had been divided into four groups according to their age. Their ages were from 1.5 to 3 years. Three jugular venous blood samples were collected via venipuncture from each horse. These samples were collected while (1) at rest, (2) after the end of the exercise and (3) 30 min after the end of the exercise. Exercise showed a significant increase in plasma leptin concentration (3.8 ± 0.31 at rest v. 4.3 ± 0.37 just after exercise and 4.4 ± 0.47 ng/ml after a 30-min rest; ANOVA P < 0.05). The difference between values obtained 30 min after exercise and at rest was significantly greater in 1.5-year-old horses than in those aged 2.5 years (+1.3 ± 0.43 v. +0.1 ± 0.15 ng/ml; ANOVA P < 0.05). The mean plasma leptin concentration was higher in fillies than in colts (4.9 ± 0.47 v. 3.5 ± 0.36 ng/ml; ANOVA P < 0.05). A positive correlation between the plasma concentrations of leptin and triacylglycerides measured just after exercise was detected (r = 0.65). The acute exercise significantly increased the plasma concentration of ghrelin that was measured just after exercise (1255 ± 55.9 v. 1127 ± 54.2 pg/ml; ANOVA P < 0.05). The exercise-induced age-related changes in the plasma ghrelin concentration were significantly lower in 2.5-year-old trotters than in 1.5-year olds. To sum up, the changes in plasma leptin and ghrelin concentrations during bouts of exertion tend to decrease with age and/or training of Standardbred foals.

Dissociative excitation of adenine (H5C5N5) into excited atomic fragments has been studied in the electron impact energy range from threshold to 400 eV. A crossed beam system coupled to a vacuum ultraviolet (VUV) monochromator is used to study emissions in the wavelength range from 90 to 200 nm. The beam of adenine vapor from a stainless steel oven is crossed at right angles by the electron beam and the resultant VUV radiation is detected in a mutually orthogonal direction. Excitation of the H Lyman series, the strongest features in the spectrum, is considered in detail. [ABSTRACT FROM AUTHOR]

A unique detector which is selectively sensitive to low energy metastable atoms, has been used to study the production of ground state N (2P) atoms following collisions of low energy (0–200 eV) electrons with molecular nitrogen. Time-of-flight techniques have revealed the existence of at least two distinct mechanisms yielding this dissociation product. Released kinetic energies in the dissociation have allowed positioning of the parent molecular states in the Franck–Condon region. This, together with excitation probability curves, has allowed probable parent states, such as B′ b′ and C′ 3Πu, to be identified making use of recent theoretical calculations. Both direct and pre-dissociation processes are shown to be involved. [ABSTRACT FROM AUTHOR]

Dissociative excitation of thymine following electron impact was studied in the energy range up to 430 eV. Emissions in the vacuum ultra-violet spectral region below 150 nm were studied and found to be dominated by the hydrogen Lyman series. Emission cross section data reveal that Lyman-α excitation displays a broad maximum at an electron impact energy of 160 eV. The probability of extracting other excited atoms from the parent molecule is found to be insignificant. Possible excitation and dissociation mechanisms in the parent molecule are discussed. [ABSTRACT FROM AUTHOR]

A novel detector is described in which solid nitrogen at 17 K is used as the most significant element. Metastable particles impinge on this element and immediately transfer their internal energy to the solid nitrogen producing photons, via excimer formation or otherwise, whose wavelength depends on the metastable being detected and the energy transfer process. The performance of the instrument for the detection of atomic oxygen and molecular nitrogen metastables is discussed.

Metastable N2 molecules produced by electron impact on N2 are detected using a unique solid nitrogen matrix detector. The time-of-flight system is shown to be selectively sensitive to a(1)Πg and (1)Σg (+) or (1)Γg metastable species. The latter species had been identified theoretically previously but was detected experimentally for the first time in the present investigation. Their identification and excitation as a function of electron energy from threshold to 300 eV are presented. Comparison is made with the data obtained by other techniques.

Elongase of very long chain fatty acids-4 (ELOVL4) is the only mammalian enzyme known to synthesize C28-C36 fatty acids. In humans, ELOVL4 mutations cause Stargardt disease-3 (STGD3), a juvenile dominant macular degeneration. Heterozygous Stgd3 mice that carry a pathogenic mutation in the mouse Elovl4 gene demonstrate reduced levels of retinal C28-C36 acyl phosphatidylcholines (PC) and epidermal C28-C36 acylceramides. Homozygous Stgd3 mice die shortly after birth with signs of disrupted skin barrier function. In this study, we report generation of transgenic (Tg) mice with targeted Elovl4 expression driven by an epidermal-specific involucrin promoter. In homozygous Stgd3 mice, this transgene reinstates both epidermal Elovl4 expression and synthesis of two missing epidermal lipid groups: C28-C36 acylceramides and (O-linoleoyl)-omega-hydroxy C28-C36 fatty acids. Transgene expression also restores skin barrier function and rescues the neonatal lethality of homozygous Stgd3 mice. These studies establish the critical requirement for epidermal C28-C36 fatty acid synthesis for animal viability. In addition to the skin, Elovl4 is also expressed in other tissues, including the retina, brain, and testes. Thus, these mice will facilitate future studies to define the roles of C28-C36 fatty acids in the Elovl4-expressing tissues.

G protein-coupled receptor (GPCR) pathways control glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAPs) for G(i) and G(q) α-subunits that control the intensity and duration of GPCR signaling. Herein we determined the role of Rgs16 in GPCR regulation of liver metabolism. Rgs16 is expressed during the last few hours of the daily fast in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominate. Rgs16 knock-out mice had elevated expression of fatty acid oxidation genes in liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma β-ketone levels compared with wild type mice. By contrast, transgenic mice that overexpressed RGS16 protein specifically in liver exhibited reciprocal phenotypes as well as low blood glucose levels compared with wild type littermates and fatty liver after overnight fasting. The transcription factor carbohydrate response element-binding protein (ChREBP), which induces fatty acid synthesis genes in response to high carbohydrate feeding, was unexpectedly required during fasting for maximal Rgs16 transcription in liver and in cultured primary hepatocytes during gluconeogenesis. Thus, RGS16 provides a signaling mechanism for glucose production to inhibit GPCR-stimulated fatty acid oxidation in hepatocytes.

The effect of interval vs. continuous exercise on plasma leptin and ghrelin concentration in young Standardbred horses was studied. The experiment was conducted on 27 trotters, in the age between 2 and 3 years. They were divided into two groups according to the type of exercise. Blood samples were collected through jugular venipuncture in the following experimental conditions: at rest, immediately after exercise and 30 minutes after the end of the effort. Plasma leptin and ghrelin concentrations were determined using RIA tests. The continuous exercise induced an increase in plasma leptin concentration whereas the interval type of exercise did not influence the level of this hormone (3.47 +/- 0.78 vs. 4.07 +/- 0.94 and 2.31 +/- 0.15 vs. 2.36 +/- 0.21 ng/mL, respectively). The plasma ghrelin concentration measured after the continuous exercise, significantly increased (720 +/- 27.4 vs. 814 +/- 13.8; p < or = 0.05) whereas concentration of this hormone assessed after the interval exercise, significantly dropped (982 +/- 56.5 vs. 842 +/- 35.6 pg/mL; p < or = 0.05). The changes in plasma ghrelin concentration measured after the end of the effort correlated inversely with blood lactic acid concentration. In conclusion, the obtained results showed that medium-intensive type of exercise, such as trot, interval or continuous, slightly affected plasma leptin level but significantly affected plasma ghrelin concentration in young Standardbred trotters.

Purpose: Complement factor H (Cfh) is a key regulator of the alternative complement pathway. A Cfh variant (Y402H) increases the risk for AMD. The purpose of this study was to develop a pathophysiologically relevant animal model of AMD based on this genetic risk factor., Methods: The authors generated chimeric Cfh transgenic mouse lines using two constructs consisting of the human CFH sequence for SCR6-8 (with either 402Y or 402H), flanked by the mouse sequence for SCR1-5 and SCR9-20. They tested the expression of the transgenic mRNA and protein molecules and examined the mice at 12 to 14 months of age for clinical and histologic retinal changes., Results: Nuclease protection assay and qRT-PCR analysis demonstrated transgenic mRNA expression in the liver and in the posterior segment of the eye. Western blot analysis showed that the transgenic proteins are present in the circulation at levels comparable to those of mouse Cfh. The chimeric proteins were found to be functional, as demonstrated by their ability to restore physiological serum levels of complement component C3 in Cfh KO mice. Clinical examination showed subretinal drusen-like deposits. Histology demonstrated an accumulation of subretinal cells that stained with a macrophage/microglia marker. Basal laminar deposits, long-spaced collagen, and increased numbers of lipofuscin granules were seen on electron microscopy. Immunohistochemistry showed a thicker sub-RPE band of C3d staining., Conclusions: Chimeric Cfh proteins led to AMD-like characteristics in mice. This may represent a good model for studying the role of complement and other components of the immune system in early AMD.

Recent studies have established that retinal health depends on the presence of polyunsaturated C28-C36 fatty acids, in addition to docosahexaenoic acid (DHA, C22:6n-3). Initially characterised 20 years ago, these C28-C36 fatty acids are found as sn-1 acyl components of retinal phosphatidylcholines (PCs), which have DHA in the sn-2 position. This unique PC species is found in both rod- and cone-dominant retinas, mainly in the photoreceptor outer segments where the majority of phototransduction reactions take place. In bovine photoreceptor outer segments, this species is a significant component of lipid membranes. Its C28-C36 fatty acids account for 10 mol % of total PC fatty acids. Polyunsaturated C28-C36 fatty acids are synthesised in the retina, in contrast to eicosapentaenoic acid (EPA, C20:5n-3) and DHA which in humans are predominantly of dietary origin. Synthesis of C28-C36 fatty acids appears to be exclusively catalysed by elongase of very-long-chain fatty acids-4 (Elovl4). Mutations in Elovl4 cause Stargardt disease-3, a juvenile autosomal dominant macular degeneration. A mouse genetic model of the disease carries a human pathogenic 5 bp deletion in the mouse Elovl4 gene. It demonstrates early selective deficiency of retinal C28-C36 acyl PCs, followed later by reduced electroretinographic signals and increased accumulation of toxic N-retinylidene-N-retinylethanolamine (A2E).

Stargardt disease-3 (STGD3) is a juvenile dominant macular degeneration caused by mutations in elongase of very long chain fatty acid-4. All identified mutations produce a truncated protein which lacks a motif for protein retention in endoplasmic reticulum, the site of fatty acid synthesis. In these studies of Stgd3-knockin mice carrying a human pathogenic mutation, we examined two potential pathogenic mechanisms: truncated protein-induced cellular stress and lipid product deficiency. Analysis of mutant retinas detected no cellular stress but demonstrated selective deficiency of C32-C36 acyl phosphatidylcholines. We conclude that this deficit leads to the human STGD3 pathology.

The first time observed excitation spectrum of the C(1)1(5(1)P(1))<--X(1)0+(5(1)S(0)) transition in CdHe van der Waals molecules is reported. Vibrational spectrum in the UV region (2286.0-2296 A) was excited in a continuous molecular-jet-expansion beam of CdHe seeded in helium using an in-house-built nitrogen-dye laser system. The excitation spectrum exhibits two vibrational components (v'<--v''=0) highly broadened by means of unresolved rotational structure and some additional contributions of "hot-bands" components (v'<--v''=1). The last effect is due to an extremely small separation of the vibrational levels in the ground X(1)0+ state of the CdHe molecule, where v'=0 vibrational level is separated from v''=0 by merely 6.0 cm(-1). It follows therefore that even in an extremely cold environment (T(v) approximately 10K) of a jet-expansion beam the population of v''=1 level is feasible, due to some residual collisions, and hence the v'<--v'=1 transitions are highly probable. The assignment of vibrational bands and numerical analysis of the spectrum was based and obtained with the aid of a rigorous computer simulation of the C(1)1<--X(1)0+ transition including the impact of rotational structure and hot-bands contributions. As a result we obtained optical potential parameters of the C(1)1(5(1)P(1)) state of CdHe molecule which are further discussed in terms of our recent (and only existing) experimental results regarding the X(1)0+, B1(5(3)P(1)) and A0+(5(3)P(1)) states of CdHe as well as in terms of ab initio calculations results.

Purpose: Autosomal dominant Stargardt disease-3 (STGD3) is caused by mutations in elongase of very long chain fatty acids-4 (ELOVL4). The goal of this study was to generate and characterize heterozygous and homozygous knockin-mice that carry a human STGD3 pathogenic mutation in the mouse Elovl4 gene., Methods: Recombinant Stgd3-knockin mice were generated using a DNA construct which introduced a pathogenic five-base pair deletion and two point mutations in exon 6 of the Elovl4 gene. Stgd3-mouse genotypes were confirmed by Southern blot analysis and expression of wild-type (wt) and mutated Elovl4 mRNAs assayed by nuclease protection assay. The retinal phenotype of heterozygous Stgd3 mice was characterized by morphological studies, elecroretinographic (ERG) analysis and assay of lipofuscin accumulation. Homozygous Stgd3 mice were examined for both retinal and gross morphology. They were also analyzed for skin morphology and skin barrier function, and for epidermal lipid content using high performance liquid chromatography (HPLC) combined with mass spectrometry (MS)., Results: The Stgd3 allele codes for a truncated mouse Elovl4 protein, which also contains the same aberrant 8-amino acid C-terminus encoded by the human pathogenic STGD3 allele. Heterozygous Stgd3 mice expressed equal amounts of both wt and mutant Elovl4 mRNAs in the retina, showed no significant changes in retinal morphology, but did show accumulation of lipofuscin and reduced visual function. Homozygous Stgd3 mice were born with an expected Mendelian frequency, without any initial gross anatomical or behavioral abnormalities. By 6-12 h postpartum, they became dehydrated and died. A skin permeability assay detected a defect in epidermal barrier function. Homozygous mutant epidermis expressed a normal content of mutated Elovl4 mRNA and contained all four epidermal cellular layers. HPLC/MS analysis of epidermal lipids revealed the presence of all barrier lipids with the exception of the complete absence of acylceramides, the critical lipids for barrier function of the skin., Conclusions: The generated Stgd3-knockin mice are a genetic model of human STGD3 and reproduce features of the human disease: accumulation of lipofuscin and reduced visual functions. Homozygous Stgd3 mice showed a complete absence of acylceramides from the epidermis. Their absence suggests a role for Elovl4 in acylceramide synthesis, and in particular, a role in the synthesis of the unique very long chain C30-C40 fatty acids present in skin acylceramides.

The aim of this study was to compare changes in epinephrine (E), norepinephrine (NE) and dopamine (DA) levels in blood plasma of two racehorses breeds: Arabian and Thoroughbred during moderate intensity exercise performed in the same conditions. The increase in plasma E level just after exercise was higher in Thoroughbreds than in Arabians. During the whole test, the Arabians showed the higher levels of NE and DA as compared to those found in Thoroughbreds.

The objective of the present study was to compare changes of blood parameters induced by the same work and performed in the same conditions in two racehorse breeds, Thoroughbred and Purebred Arabian. The effect of moderate-intensity exercise was studied in 20 stallions--ten Thoroughbreds, aged 2-3 years and the same number of Purebred Arabians, 3-4 years old. All the horses were administrated the same effort test consisting in 1200 m gallop at a speed typical of the daily training sessions. Three jugular venous blood samples were collected for each horse: at rest, just after the end of the gallop and after 30-minute rest. In the gathered blood, a hemoglobin (Hb) concentration was determined as well as plasma level of glucose (Glc), triacylglycerols (TG), glycerol, free fatty acids (FFA), total plasma proteins (TP) and the activity of creatine kinase (CK), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST). In the Arabian horses, an increase in levels of TP, glycerol, FFA and CK activity measured just after exercise was higher than that in Thoroughbreds. Similarly, after a 30-minute rest, a post-exercise rise of TP, AST, glycerol and FFA proved to be higher in the Arabian horses compared to that in the Thoroughbreds. Only TG plasma concentration measured 30 minutes following the effort was significantly lower in the Arabian horses than in Thoroughbreds. It can be concluded that the Thoroughbred horses adapted better to the effort test applied in this study as compared to the Purebred Arabian horses. The parameters related to lipid metabolism proved to be the most sensitive indicators of breed differences in relation to moderate-intensity exercise.

A special xenon matrix detector has been used to study the production of S(1S) following controlled electron impact on thiophosgene (Cl2CS) targets over an electron energy range from threshold to 400 eV. Time-of-flight spectroscopy has been used to measure S(1S) fragment kinetic energies. Fragments with energies in excess of 1 eV have been observed. The absolute cross section for S(1S) production reaches a maximum of [1.05+/-0.35] x 10(-18) cm2 at approximately 125 eV impact energy. Two different fragmentation processes, involving triplet and singlet excited states of the parent Cl2CS molecule, have been identified.

Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. Rom1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and rom1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and rom1. The critical threshold for the combined abundance of rds and rom1 is approximately 60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.

The retinal degeneration slow or rds gene encodes rds/peripherin, an integral membrane glycoprotein in the outer segments of rod and cone photoreceptors. Mice homozygous for a null mutation in rds fail to develop outer segments and undergo subsequent degeneration of photoreceptors by the apoptotic pathway. Mutations in the human RDS gene are responsible for several forms of inherited blindness including autosomal-dominant retinitis pigmentosa and macular degeneration. Here, we examined the effects of ectopic Bcl-2 expression in transgenic photoreceptors on the rate of retinal degeneration in rds mutant mice. We observed an approximately twofold preservation of photoreceptors compared with nontransgenic rds mutant mice at 3 months. Immunoblot analysis showed similar levels of Bcl-2 in 2-, 3-, and 4-week-old transgenic mice. Expression of Bcl-2 in the rds mouse did not lead to outer segment formation and did not induce cell death. These results suggest that Bcl-2 expression may be an effective therapeutic strategy in humans with mutations in RDS or other genes that affect the integrity of photoreceptor outer segments.

Mice homozygous for the retinal degeneration slow (rds) mutation completely lack photoreceptor outer segments. The rds gene encodes rds/peripherin (rds), a membrane glycoprotein in the rims of rod and cone outer segment discs. rds is present as a complex with the related protein, rom1. Here, we generated transgenic mice that express a chimeric protein (rom/D2) containing the intradiscal D2 loop of rds in the context of rom1. rom/D2 was N-glycosylated, formed covalent homodimers, and interacted non-covalently with itself, rds, and rom1. The rds.rom/D2 interaction was significantly more stable than the non-covalent interaction between rds and rom1 by detergent/urea titration. Analysis of mice expressing rom/D2 revealed that rds is 2.5-fold more abundant than rom1, interacts non-covalently with itself and rom1 via the D2 loop, and forms a high order complex that may extend the entire circumference of the disc. Expression of rom/D2 fully rescued the ultrastructural phenotype in rds+/- mutant mice, but it had no effect on the phenotype in rds-/- mutants. Together, these observations explain the striking differences in null phenotypes and frequencies of disease-causing mutations between the RDS and ROM1 genes.

Rds/peripherin is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks. In mammals, it is thought to stabilize the disk rim through heterophilic interactions with the related nonglycosylated protein roml. Glycosylation of rds/peripherin at asparagine 229 is widely conserved in vertebrates. In this study, we investigated the role of rds/peripherin N-glycosylation. We generated transgenic mice that expressed only S231A-substituted rds/peripherin in their retinas. This protein was not glycosylated but formed covalent dimers with itself and with glycosylated rds/peripherin. Nonglycosylated rds/peripherin also interacted noncovalently with rom1 homodimers to form a heterooligomeric complex. The glycosylated rds/peripherin..rom1 complex bound to concanavalin A-Sepharose, suggesting that the glycan is not directly involved in the interaction between these proteins. In double transgenic mice expressing normal and S231A-substituted rds/peripherin, the mRNA-to-protein ratios were similar for both transgenes, indicating no effect of N-glycosylation on rds/peripherin stability. Finally, expression of nonglycosylated rds/peripherin in transgenic mice rescued the phenotype of outer segment nondevelopment in retinal degeneration slow (rds-/-) null mutants. These observations indicate that N-glycosylation of rds/peripherin is not required for its normal processing, stability, or in vivo function.

The inherited retinal dystrophies represent a large and heterogenous group of hereditary neurodegenerations, for many of which, the molecular defect has been defined. However, the mechanism of cell death has not been determined for any form of retinal degeneration. The retinal degeneration slow (rds-/-) mutation of mice is associated with nondevelopment of photoreceptor outer segments and gradual death of photoreceptor cell bodies, attributed to the absence of the outer segment protein rds/peripherin. Here, we examined the effects of a transgene encoding normal rds/peripherin that had integrated into the X-chromosome in male and female rds-/- mutant retinas. In 2-month-old transgenic males and homozygous-transgenic females on rds-/-, we observed virtually complete rescue of both the outer segment nondevelopment and photoreceptor degeneration. In contrast, hemizygous-transgenic rds-/- female littermates showed patchy distributions of the transgene mRNA, by in situ hybridization analysis, and of photoreceptor cells that contain outer segments. This pattern is consistent with random inactivation of the X-chromosome and mosaic expression of the transgene. Surprisingly, we observed significant photoreceptor cell loss in both transgene-expressing and nonexpressing patches in hemizygous female retinas. These observations were supported by nuclease protection analysis, which showed notably lower than predicted levels of transgene mRNA in retinas from hemizygous females compared with male and homozygous female littermates. This phenotype suggests an important component of non-cell-autonomous photoreceptor death in rds-/- mutant mice. These results have significance to both the etiology and potential treatment of human inherited retinal degenerations.

Purpose: In this study, the authors present the biochemical, morphologic, and physiological analyses of a transgenic mouse model for retinal degeneration slow (RDS)-mediated retinitis pigmentosa caused by a proline 216 to leucine (P216L) amino acid substitution in rds/peripherin., Methods: The authors assembled a mutant rds transgene that encodes rds/peripherin with a P216L substitution. Transgenic mice were generated on wild-type (+/+), heterozygous (rds-/+), and homozygous (rds-/rds-) null genetic backgrounds. These mice were analyzed biochemically, by light and electron microscopy, and by electroretinography., Results: In P216L-transgenic mice on a +/- background, the authors observed expression-level-dependent photoreceptor degeneration and outer-segment shortening. Expression of the P216L transgene on an rds-/+ background resulted in more severe photoreceptor degeneration and outer-segment dysplasia than seen in nontransgenic rds-/+ mutants. Severely dysplastic outer segments were detectable in P216L transgenics on an rds-/rds-null background. The reduction in b-wave amplitudes by electroretinography were well correlated with the degree of photoreceptor degeneration, but not outer-segment dysplasia in these different rds mutants., Conclusions: The phenotype in P216L-transgenic mice on an rds-/+ genetic background probably is caused by a combination of two genetic mechanisms: a direct dominant effect of the P216L substituted protein, and a reduction in the level of normal rds/peripherin. The expression pattern of the normal and mutant genes in these animals is similar to that predicted for humans with RDS-mediated autosomal-dominant retinitis pigmentosa. These mice may thus be considered an animal model for this disease.

We have isolated and characterized three homologs of mammalian rds/peripherin from Xenopus retinae. One (xrds38) is likely the Xenopus ortholog, while the other two (xrds36 and -35) are more distant relatives. By immunocytochemical analysis of retinal sections, xrds38 is distributed in both rod and cone photoreceptors, while xrds36 and xrds35 are present in rods only. At the EM level, xrds38 is present specifically in the rims and incisures of rod and cone outer segment discs. All are N-glycosylated and form covalent dimers. Immunoprecipitation analysis showed that in rods, these three proteins interact to form heterotetrameric or higher-order complexes. The pattern of sequence conservation among the xrds proteins, mammalian rds/peripherin, and mammalian rom-1 suggest that the central portion of the intradiscal D2 loop contains the interacting structural elements.

To identify unique gene products that could be crucial for circadian and light-dark regulations, using the technology of differential display, we have investigated mRNAs that were isolated from rat pineals and retinas collected either during subjective day (D) or subjective night (N). A subpopulation of about 50 mRNAs were amplified from pineal using designed primers and reverse transcriptase-polymerase chain reaction. Thirty five of the mRNAs were expressed equivalently in both D and N samples. From the 15 mRNAs showing differential expression, four amplified products were selected, based on higher expression during the subjective night (N310, N320, N383, N420) and each was subcloned and sequenced. Of the four cDNAs studied, only N310 was determined to have significant identity to a known cell adhesion protein, called F3. This protein is highly expressed in developing mouse neurons and is related to chicken contactin. Each differentially displayed product identified is being examined further in order to understand their potential significance in the circadian regulation of pineal and retinal activities.

In hypothalamic cells cultured in serum-free medium, the quantity of tyrosine hydroxylase mRNA increases after treatment with an activator of the protein kinase A pathway (8-bromoadenosine cyclic AMP, 3-isobutyl-1-methylxanthine, or forskolin) or an activator of protein kinase C (12-O-tetradecanoylphorbol 13-acetate or sn-1,2-diacylglycerol). The tyrosine hydroxylase mRNA level decreases in the cells after inhibition of protein kinase C with calphostin C or after depletion of protein kinase C by extended phorbol ester treatment. These data suggest that both protein kinase pathways regulate tyrosine hydroxylase gene expression in hypothalamic cells. As simultaneous activation of both pathways has less than an additive effect on the tyrosine hydroxylase mRNA level, they appear to be interrelated. Compared with the rapid and dramatic increase of the tyrosine hydroxylase mRNA level in pheochromocytoma cells, activation of the protein kinase A or protein kinase C pathway in the cultured hypothalamic cells induces slow changes of a small magnitude in the amount of tyrosine hydroxylase mRNA. The slow regulation of tyrosine hydroxylase gene expression in hypothalamic dopaminergic neurons corresponds to the relatively high stability of tyrosine hydroxylase mRNA (half-life = 14 +/- 1 h) in these cells.

Using an in vitro incubation system, the role of the cyclic AMP-dependent protein kinase A (PKA) pathway in the regulation of the in situ activity of tyrosine hydroxylase (TH) was studied in the hypothalamuses of young and aged ovariectomized rats. Hypothalamic tissue was incubated for 60 min in medium containing 3-hydroxybenzylhydrazine dihydrochloride, a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor, and various agents that modify the activity of the PKA pathway. At the end of the incubation, the tissue was homogenized and analyzed for DOPA and TH mass. The in situ molar activity of TH was expressed as the moles of DOPA accumulating in the tissue per mole of TH per hour. Forskolin, an activator of adenylyl cyclase and the cyclic AMP agonist, (Sp)-cyclic adenosine 3',5'-monophosphothioate, significantly (P < .01) increased the in situ molar activity of TH in the hypothalamic dopaminergic (DAergic) neurons of both young and aged rats. Theophylline, a phosphodiesterase inhibitor, did not affect the TH molar activity in the hypothalamuses of aged animals but did significantly (P < .001) increase its activity in those of young rats. When vasoactive intestinal peptide was evaluated, the TH molar activity was significantly (P < .005) increased in the hypothalamuses of young rats but not in those of aged rats. It was suggested that the deficiency of DA secretion by hypothalamic DAergic neurons of aged rats may be the result of insufficient activation of PKA caused by failure of transduction of an extracellular signal to activate adenylyl cyclase and produce cyclic AMP.

The effects of lead nitrate [Pb(NO(3))(2)] on tyrosine hydroxylase (TH) mass, TH mRNA, and catecholamine (CA) secretion by primary cultures of hypothalamic and adrenal cells of 18- to 22-day-old rat fetuses were investigated. Two-week-old hypothalamic and 4-week-old adrenal cell cultures were incubated for 24 h or 14 days with various concentrations (10(-10) to 10(-3)M) of Pb(NO(3))(2). In general, a 24-h exposure to low concentrations of Pb (10(-10) to 10(-7)M) suppressed the secretion of dihydroxyphenylalanine (DOPA) by hypothalamic dopaminergic cells and especially norepinephrine and epinephrine by adrenal cells; conversely, high concentrations (10(-4) and 10(-3)M) were stimulatory. This biphasic, concentration-dependent action of Pb on CA secretion occurred irrespective of the gestational age of the cell donor. Long-term (14 days) incubation of hypothalamic cells with Pb had an effect similar to that seen with a short-term incubation except that 10(-3)M Pb inhibited DOPA secretion. For adrenal cells, a long-term incubation with 10(-10) and 10(-9)M Pb inhibited CA secretion, whereas 10(-8)M was stimulatory. The TH mRNA content of the cells was not affected by Pb(NO(3))(2), regardless of its concentration. However, a 14-day incubation with low concentrations (10(-10) and 10(-9)M) of Pb resulted in an increase in TH mass in hypothalamic cells compared to controls, whereas high concentrations (10(-4) and 10(-3)M) markedly suppressed the level of TH. The effects of Pb on TH in adrenal cells were similar to those in hypothalamic cells. In conclusion, the actions of Pb(NO(3))(2) on CA secretion are biphasic: low concentrations are inhibitory, and high concentrations are stimulatory. Time of exposure does not appear to be a significant factor since a 24-h incubation with Pb was as effective as was a 14-day incubation. Thus, Pb(NO(3))(2) has a paradoxical effect on catecholaminergic cells, and in the absence of knowledge of the concentration of Pb, it is not possible to predict the toxic consequences of lead poisoning.

We investigated the involvement of second messenger systems in the control by pituitary cytotropic factor (CTF) of tyrosine hydroxylase (TH) expression in primary cultures of hypothalamic cells. Forskolin, an activator of adenylyl cyclase, as well as Sp-cAMP[S] [(Sp)-cyclic adenosine 3',5'-monophosphothioate], a cAMP agonist, and theophylline, an inhibitor of phosphodiesterase activity, stimulate the secretion of dihydroxyphenylalanine (DOPA) and dopamine (DA), suggesting a role for cAMP-dependent protein kinase in the secretion of catecholamines by hypothalamic dopaminergic cells. When cells were cultured with either CTF or forskolin for 14 days, a progressive increase in the secretion of DOPA and DA was observed throughout the period of incubation. At the end of the 2-week culture period, the amount of TH in the cells, determined by immunoblot analysis, was appreciably increased compared to controls. When the cells were analyzed immunocytochemically for TH, the TH-positive cells that had been incubated with CTF or forskolin for 2 weeks were found to have neurites that appeared larger than those of TH-positive cells in the controls. The diameters of the perikarya of TH-positive cells in cultures incubated with CTF also appeared larger than the controls. After incubation of hypothalamic cells with CTF for 96 h, the amount of TH mRNA in the cultures was significantly increased. When membranes isolated from PC12 cells were incubated for 10 min with 50 microM forskolin, the specific activity of adenylyl cyclase was increased 20-fold; CTF had no effect on adenylyl cyclase activity of PC12 cell membranes. Yet, CTF significantly (P less than 0.001) stimulated the secretion of DOPA and DA by PC12 cells. When hypothalamic cells were incubated with both forskolin and CTF, using doses of each that stimulated maximal secretion, the secretion of DOPA and DA was equal to sum of the secretions with each stimulant alone. These additive actions of forskolin and CTF and the failure of CTF to activate adenylyl cyclase in membranes of PC12 cells suggest that forskolin and CTF stimulate catecholamine secretion by hypothalamic dopaminergic cells through different mechanisms, perhaps through different protein kinases. When hypothalamic cells were incubated with CTF and W-7 [N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide], an inhibitor of calmodulin, the secretion of DOPA was significantly (P less than 0.001) less than that in cultures that were not incubated with W-7. The findings of this study suggest that TH expression in hypothalamic dopaminergic cells is controlled by redundant protein kinases, including cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase.

We have investigated, in young and aged rats, tuberoinfundibular dopaminergic (TIDA) and nigrostriatal neurons of the brain, and the role of the testes and anterior pituitary (AP) on 1) the in situ activity of tyrosine hydroxylase (TH), 2) translation of TH mRNA, as reflected by the mass of TH, and 3) transcription of the TH gene, as revealed by the mass of TH mRNA. The median eminence (ME) and corpus striatum (CS) were used as the sources of proximal neurites of the TIDA neurons and nigrostriatal neurons, respectively. The arcuate-periventricular nuclei of the ventral hypothalamus were used as the source of perikarya of the TIDA neurons, and substantia nigra (SN) nuclei of the midbrain were used as the source of perikarya of nigrostriatal neurons. The in situ activity of TH was calculated using the rate of accumulation of dihydroxyphenylalanine after pharmacological inhibition of dihydroxyphenylalanine decarboxylase activity. TH mass and TH mRNA were measured using an immunoblot assay and an S1 nuclease protection assay, respectively. Compared to intact animals, orchidectomized young and aged male rats had significantly (P less than 0.001) increased in situ activity of TH in the ME and CS, but not the SN. Orchidectomy also caused a significant (P less than 0.01) increase in the quantity of TH in the ME and a 2- to 3-fold increase in TH in the CS and SN. In contrast to castration, AP grafts, implanted in the lateral ventricles of the brain, caused a significant (P less than 0.001) increase only in TH activity in the ME. No effect of AP grafts was seen on TH activity of the CS or SN. AP grafts had no effect on the amount of TH in the ME, CS, or SN. In the TIDA neurons of young males and in the nigrostriatal neurons of young and aged castrates, the amount of TH mRNA was not different from that in intact males. AP grafts had no effect on TH mRNA in these dopaminergic neurons. These studies show that TIDA neurons and nigrostriatal neurons share some common aspects in their regulation and are dissimilar in others. AP grafts stimulate TH activity in young as well as aged TIDA neurons, but not in nigrostriatal neurons. Castration leads to an increase in TH mass in both TIDA and nigrostriatal neurons. The increase in TH mass is not associated with an increase in the amount of TH mRNA. It is suggested that castration results in increased translation of TH mRNA, but not transcription of the TH gene.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of cerebroventricular anterior pituitary (AP) grafts on brain tyrosine hydroxylase (TH) expression in the median eminence (ME), substantia nigra (SN), and corpus striatum (CS) has been investigated in young and aged female rats. TH expression was studied using the following indices: in situ TH activity, TH mass, and quantity of TH mRNA. The rate of synthesis of dihydroxyphenylalanine (DOPA) was evaluated by measuring its accumulation in the ME, SN, and CS. TH mRNA and TH mass were quantified by an S1 nuclease protection assay and an immunoblot assay, respectively. Viability of the grafts was demonstrated by histological examination and by their ability to secrete PRL into the cerebrospinal fluid (CSF). Liver grafts served as controls. In castrated young and castrated aged animals with AP grafts, the PRL concentrations in the CSF were 204 +/- 49 (mean +/- SE) and 345 +/- 83 ng/ml, respectively, compared to control values of 14 +/- 9 and 23 +/- 9. In intact aged animals, the concentration of PRL was 729 +/- 180 ng/ml in CSF of rats with AP grafts and 223 +/- 62 ng/ml in the controls. DOPA synthesis in the ME of castrated young rats and castrated aged rats with AP grafts was significantly (P less than 0.01) greater than that in controls with liver grafts. AP grafts did not stimulate DOPA synthesis in the ME of intact aged animals. The synthesis of DOPA in the SN and CS was not affected by AP grafts, regardless of the status of the animal. The amount of TH mRNA and the quantity of TH were not influenced by the AP grafts in any of the animal models. However, the in situ molar activity of TH in the ME was significantly greater in castrated young rats and castrated aged rats bearing AP grafts than in animals with liver grafts. The in situ molar activity of TH in the ME of castrated young rats was greater than that of castrated or intact aged animals. It is concluded that AP grafts secrete a substance that stimulates DOPA synthesis in the ME of young as well as aged castrated animals, but not in the nigrostriatal system. This stimulation is due to increased catalytic activity of a fixed number of TH molecules rather than an increase in the mass of TH.

A novel, simplified procedure for the removal of DNA template from newly synthesized RNA is described. The method avoids the use of DNase and involves extraction of the RNA transcription products at acid pH using either phenol or phenol: chloroform:guanidinium thiocyanate mixture. Both procedures are as effective as DNase digestion in removing DNA template, but are free of the hazard of RNA degradation, are highly reproducible and remove proteins at the same time.

Expression of tyrosine hydroxylase (TH) in cultured cells of the ventral hypothalamus-midbrain of fetal rats has been investigated. TH mRNA and TH were quantified by an S1 nuclease protection assay and an immunoblot assay, respectively. Dihydroxyphenylalanine (DOPA) and dopamine secretion were evaluated using their rates of accumulation in the culture medium. The rate of accumulation of DOPA was 2-3 times that of dopamine. Inhibitors of TH activity caused a dose-dependent reduction in DOPA secretion. During an 11-week culture of dissociated cells, TH mRNA increased from 1.6 to 2.8 attomole/well between the first and fourth week of culture, remained steady to the ninth week, and then declined. TH increased from 12 to 105 fmol/well between the first and seventh week and then declined. DOPA secretion increased until the sixth week and then remained steady to the tenth week. An extract of rat pituitaries stimulated DOPA secretion by the cultures in a dose-dependent manner. This activity, attributed to a cytotropic factor (CTF), was inactivated by heating for 10 min in a boiling water bath, but was unaffected by trypsin digestion. Incubation with CTF for 24, 48, 72, and 96 h resulted in a day by day increase in the secretion of DOPA. After 96 h of culture with CTF, the amount per well of TH mRNA, but not TH, was significantly (P less than 0.01) greater than the control value. Pituitary CTF is probably not PRL, since rat PRL did not appreciably affect DOPA secretion or the amount of TH mRNA or TH in the cells. Withdrawal of CTF from CTF-stimulated cells resulted in a marked reduction in DOPA secretion as well as a decrease in TH mRNA. These results support the hypothesis that the pituitary gland contains a cytotropic factor that stimulates TH expression in fetal brain cells of the hypothalamus-midbrain.

Using a solution hybridization-S1 nuclease protection assay, we quantitatively studied tyrosine hydroxylase mRNA (mRNATH) in catecholaminergic cells of the substantia nigra, hypothalamus, superior cervical ganglion, and adrenal of male rats from early neonatal life to old age. Throughout this time, the lowest level of mRNATH in any tissue was found in the youngest animals, and their development was associated with an increase in the quantity of mRNATH. However, the extent of the increase as well as the pattern of change was dependent on the tissue. The amount of mRNATH in the substantia nigra of 1-day-old pups was 162 +/- 7 attomoles (mean and S.E.M.), increasing to 877 +/- 39 amol at 14 days of age. Then, the amount fell to 480 +/- 25 amol at 6 weeks of age, but changed little between 6 weeks and 23 months of age. In the hypothalamus of 1-day-old pups, the quantity of mRNATH was 24 +/- 3 amol, increasing to 60 +/- 6 amol at 2 weeks and changed little thereafter. mRNATH in the superior cervical ganglion increased gradually until 10 months of age; at which time the amount was 3 times that of neonatal animals. In the adrenal, mRNATH increased continuously throughout the period of observation. The amount of mRNATH in the adrenal of 23-month-old animals was 25 times that in the adrenal of 4-day-old pups. These data suggest that tyrosine hydroxylase gene expression does not diminish in aged rats.

The role of prolactin and of estradiol and progesterone in the control of the biosynthetic and secretory activity of TIDA neurons has been investigated in the following animal models: young female rats, aged female rats, and young male rats. The indices of TIDA neuronal function employed were a) mass of TH in neurites in the ME, b) total in situ activity of TH in the ME, c) in situ molar activity of TH in the ME, and d) secretion of dopamine into hypophysial portal blood. It was found that prolactin in high concentration in the circulation and in the CSF had little, if any, effect on the mass of TH in the ME. However, a high concentration of prolactin in either the circulation or in the CSF stimulated significantly the in situ TH activity in the ME whether expressed in terms of total activity per ME or activity per mole of TH. The stimulation of TH activity with prolactin was prevented by immunoneutralization of circulating prolactin. A high concentration of prolactin in the CSF was as effective in stimulating TH activity in the ME of rats with intact pituitary glands as in hypophysectomized rats. In addition to prolactin, treatment of animals with intact pituitaries with a combination of estradiol and progesterone markedly stimulated the total in situ activity of TH of the ME as well as the in situ molar activity of TH of the ME, but neither estradiol nor progesterone alone had an effect on TH activity. Hypophysectomy abolished the stimulatory action of estradiol and progesterone on TH activity of the ME. In addition to the in situ activity of TH in the ME, estradiol-progesterone treatment stimulated the secretion of dopamine into hypophysial portal blood. Neither estradiol nor progesterone alone affected dopamine secretion by TIDA neurons. We conclude that exposure to high concentrations of prolactin or to both estradiol and progesterone stimulate the biosynthetic and secretory activity of TIDA neurons. These hormones are effective in old rats and well as young rats and in males as well as females.

Pump-and-probe methods of laser spectroscopy with time resolution were used to study fluorescence and excitation spectra arising from transitions involving the (1) product operator(u)(4(1)P), (3) product operator(g)(4(3)P), and X(1)Sigma(+)(g)(4(1)S) states for the Zn(2) excimer. An analysis of the vibrational bands yielded vibrational constants, relative energies, and relative internuclear separations for the (1) product operator(u) and (3) product operator(g) states.

During germination of lupin seeds, the levels of in-vivo tRNA aminoacylation increase in different ways, depending on the species of tRNA. Column chromatography of tRNA on reverse-phase-chromatography (RPC-5) has shown the presence of 4 peaks of isoleucyl-tRNA, 5 of leucyl-tRNA, 5 of lysyl-tRNA, 2 of tyrosyl-tRNA, and 4 of valyl-tRNA. Cochromatography of periodate treated and control tRNA preparations, labeled with radioactive amino acids, indicates identical aminoacylation in vivo of isoaccepting tRNAs during plant development. One isoacceptor of isoleucine tRNA changes its elution profile after periodate treatment.

Transfer RNAs were isolated from plants representing mono- and dicotyledons: wheat embryos and lupin seeds. The two tRNA preparations were compared by polyacrylamide-gel-electrophoresis mapping. Transfer RNAs extracted from the separated spots were tested for acceptance of 11 amino acids. Comparison of electrophoretic mobility of respective tRNA species indicates the overall similarity of tRNA populations in the two plants studied. Especially, isoaccepting tRNAs for glycine, tyrosine and valine and some of isoacceptors of tRNAArg, tRNAAsp, tRNALeu, tRNALys and tRNAPhe occupy identical or closely similar positions on both polyacrylamide-gel maps. However, some tRNA isoacceptors from one population have no counterparts in the second one, which may indicate differences in their primary structures.