Ultrastructural changes in vitro of rat liver nucleoli in response to polyamines

Summary Structural alterations of the nucleoli of rat liver cells were noted when these nuclei were isolated with spermidine or spermine rather than magnesium. When 5–10 mM spermidine or spermine were used to isolate the nuclei, the nucleoli were a) larger, b) contained numerous and sometimes large lacunae, and c) were less aggregated and had prominent chromatin caps. These chromatin caps gave the nucleolus a ring-shaped appearance in the light microscope. These findings, coupled with physiological data that indicate that polyamines enhance nucleolar RNA polymerase activity (Russell et al., 1971), suggest that spermidine and spermine may be involved in the control of ribosomal RNA synthesis. To our knowledge, this is the first instance of the direct stimulation of ribosomal RNA synthesis during nuclear isolation.Supported by USPHS Grant NS-07934.     

Differential effects of polyamines on rat thyroid protein kinase activities

Ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis, has been shown to be regulated in thyroid by thyrotropin both in vivo and in vitro. Little, however, is known of the role of polyamines in thyroid cell function. Since studies in other tissues suggest that polyamines may influence protein phosphorylation, we studied the effect of the polyamines on various protein kinase activities in rat thyroid. Putrescine, spermidine, and spermine inhibit cyclic-AMP-dependent histone H1 kinase activity when measured in the cytosol fraction of rat thyroid; this effect is largely reproduced by NaCl concentrations of equivalent ionic strength. Both spermidine and spermine effect a 1.6-2.4-fold increase in cytosolic cyclic-AMP-independent (messenger-independent) casein kinase activity; stimulation by both polyamines is maximal at 5mM. A similar profile of stimulation is observed for messenger-independent casein kinase activity in crude nuclear preparations. Sodium chloride fails to stimulate both cytosolic and nuclear messenger-independent casein kinase activities at ionic strength equivalent to the spermine concentrations used. Spermine, but not putrescine, spermidine, or sodium chloride, inhibits calcium/phospholipid-dependent protein kinase C activity in cytosol extracts partially purified by DEAE chromatography. These findings suggest that regulation of protein kinase(s) by polyamines may represent a proximal locus (i) of action of thyrotropin-regulated ornithine decarboxylase activity in thyroid.     

Differential microvascular response to disuse in rat hindlimb skeletal muscles.

The aim of the study was to address discrepant findings in the literature regarding coupling between decreased functional demand during disuse and reduced capillarity. We previously reported [K. Tyml, O. Mathieu-Costello, and E. Noble. Microvasc. Res. 49: 17-32, 1995] that severe disuse of rat extensor digitorum longus (EDL) muscle caused by a 2-wk application of tetrodotoxin (TTX) on the sciatic nerve is not accompanied by capillary loss. Using the same animal model, the present study examined whether this absence of coupling could be explained in terms of 1) too short a duration of disuse and 2) muscle-specific response to disuse. Fischer 344 rats were exposed to either no treatment (control) or to 2- or 8-wk TTX applications. Fiber size, capillary density per fiber cross-sectional area, and capillary-to-fiber (C/F) ratio were determined by morphometry in the EDL muscle (control, 2- and 8-wk groups) and in the superficial portion of medial gastrocnemius (Gas) muscle (control, 2 wk). In both muscles, microvascular blood flow was evaluated by intravital microscopy [red blood cell velocity in capillaries (V(RBC))] and by laser Doppler flowmetry (LDF). Regardless of duration of TTX application or muscle type, TTX-induced disuse resulted in a significant reduction of fiber area (44-71%). However, capillary density increased in EDL muscle (both at 2 and 8 wk) but not in Gas muscle. C/F ratio decreased in EDL muscle at 8 wk (18%) and in Gas muscle (39%). This indicates that the effect on capillarity depended on duration of disuse and on muscle type. V(RBC) and LDF signal were significantly larger in EDL than in Gas muscle. Analysis of change in capillarity vs. V(RBC) suggested that the outcome of disuse may be modulated by blood flow. We conclude that the duration of skeletal muscle disuse per se does not dictate capillary loss, and we hypothesize that discrepant findings of coupling between functional demand and capillarity could be due to the presence/absence of flow-related angiogenesis superimposed on the capillary removal process during disuse.     

Multiple phosphorylation of pp30, a rat brain polyribosomal protein, sensitive to polyamines and corticotropin.

A rat brain polyribosomal protein with an apparent Mr of 30 000, designated pp30, was further characterized. The protein was identified by its phosphorylation by an endogenous protein kinase sensitive to both corticotropin and spermine. Two-dimensional separation of a polyribosomal fraction was applied, combining non-equilibrium pH-gradient-gel electrophoresis in the first and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the second dimension. In this system, pp30 was separated into at least five defined phosphoprotein spots. Pulse-labelling with [gamma-32P]ATP followed by a chase for various time periods with excess unlabelled ATP resulted in a shift of the distribution of radioactivity and protein staining along the spots towards the anode. This suggests that the various spots of pp30 may represent multiple phosphorylation states. Limited proteolysis of the five spots with three different proteinases resulted in the same one-dimensional peptide maps with a given proteinase, indicating that all five spots represent different forms of a single phosphoprotein. Inhibition of the overall phosphorylation of pp30 by corticotropin or spermine was accompanied by a shift in the recovery of labelled phosphate towards spots nearer the cathode. Immunoblotting with monoclonal antibodies directed against ribosomal protein S6 stained only one band, a protein that had an apparent Mr of 34 000 and was clearly distinct from pp30.     

Differential response of rat limb bones to strenuous exercise

We examined the influence of a strenuous exercise regimen on tibial and metatarsal bones to show not only how the geometric, histological, and mechanical properties of immature bone respond to strenuous exercise but also how long bones within the same limb may respond differentially to exercise. Female Sprague-Dawley rats (8 wk old) were divided randomly into two groups: a sedentary control (n = 15) and an exercised group (n = 15). The exercise intensity was 80-90% of maximum oxygen capacity 5 days/wk for 10 wk. Mechanical properties of tibia and second metatarsus (MT) were determined with three-point bending, and contralateral bones were used for geometric and histological analyses. Length and middiaphyseal cross-sectional geometry of the exercised tibiae were significantly less than controls, but material properties were not different. The exercised tibiae had significantly lower structural properties (e.g., loads at the proportional limit and maximum and energy at failure load). The middiaphyseal dorsal cortex of exercised MT was significantly thicker than controls, but tensile stress at the proportional limit and elastic modulus of exercised MT were significantly less than controls. The average number of osteons and osteocytes per unit area of the tibial middiaphysis was significantly greater in the exercised group--especially in lateral and posterior cortices. The number of osteons and osteocytes per unit area in the MT, however, was significantly less in the exercised group. The differential effects of strenuous exercise on tibia and MT suggest that local loading and bone-specific responses have important roles in modulating the response of immature bone to strenuous exercise.     

Differential response of rat skeletal muscle glycogen metabolism to testosterone and estradiol.

Although reports on sex steroids have implicated them as promoting protein synthesis and also providing extra strength to the skeletal muscle, it remains unclear whether sex steroids affect glycogen metabolism to provide energy for skeletal muscle functions, since glycogen metabolism is one of the pathways that provides energy for the skeletal muscle contraction and relaxation cycle. The purpose of the current study was to show that testosterone and estradiol act differentially on skeletal muscles from different regions, differentially with reference to glycogen metabolism. To study this hypothesis, healthy mature male Wistar rats (90-120 days of age, weighing about 180-200 g) were castrated (a bilateral orchidectomy was performed to test the significance of skeletal muscle glycogen metabolism in the absence of testosterone). One group of castrated rats was supplemented with testosterone (100 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards). To test whether estradiol has any effect on male skeletal muscle glycogen metabolism 17beta-estradiol (5 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards) was administered to orchidectomized rats. To test whether these sex steroids have any differential effect on skeletal muscles from different regions, skeletal muscles from the temporal region (temporalis), muscle of mastication (masseter), forearm muscle (triceps and biceps), thigh muscle (vastus lateralis and gracilis), and calf muscle (gastrocnemius and soleus) were considered. Castration enhanced blood glucose levels and decreased glycogen stores in skeletal muscle from head, jaw, forearm, thigh, and leg regions. This was accompanied by diminished activity of glycogen synthetase and enhanced activity of muscle phosphorylase. Following testosterone supplementation to castrated rats, a normal pattern of all these parameters was maintained. Estradiol administration to castrated rats did not bring about any significant alteration in any of the parameters. The data obtained suggest a stimulatory effect of testosterone on skeletal muscle glycogenesis and an inhibitory effect on glycogenolysis. Estradiol did not play any significant role in the skeletal muscle glycogen metabolism of male rats.     

Short-term morphological response of the rat testis to administration of five chemotherapeutic agents.

As cancer survival rates improve, there is increasing concern about the adverse effects of chemotherapeutic agents on male fertility. Five chemotherapeutic agents (amethopterin, AP or methotrexate; doxorubicin, DX; cytoxan or cyclophosphamide, CP; cisplatinum, CDDP; and 5-fluorouracil, 5-FU) which belong to three different categories of chemotherapeutic agents (antimetabolite, antibiotic, alkylating agent, alkylating agent, antimetabolite, respectively) were given systemically to adult rats to determine the short-term morphological patterns of response in the testis, and the testes were examined by light microscopy. Morphological patterns of response were found to be highly characteristic for each agent, and some shared morphological responses were evident. All except one chemotherapeutic agent (5-FU) caused spermatogonial damage. Among the defects seen were probable degenerating meiotic spermatocytes (CDDP), presence of micronuclei (DX), "arrested" spermatid development (5-FU), and abnormally shaped step 15 spermatids (5-FU). Damage that could be due to the effect of an agent on the Sertoli cell was failure of sperm release (5-FU, CDDP, DX, and AP), increase in the Sertoli cell lipid (5-FU), and malorientation of step 8 spermatids (5-FU, DX). The varied patterns of damage observed are a possible explanation of why the reproductive recovery potential in cancer patients undergoing chemotherapy is variable and drug-specific.     

Differential conjugation of polyamines to calf nuclear and nucleolar proteins

Nuclei and nucleoli isolated from calf liver contain acid-precipitable putrescine, spermidine and spermine conjugates. The polyamines are released upon peptide bond hydrolysis. All of the nuclear putrescine conjugate and a major portion of the polyamine conjugates are localized within the nucleolus. Nuclei and nucleoli also contain, in proportions consistent with the nucleolar/nuclear protein ratio, the putative conjugating enzyme, transglutaminase, as well as amine acceptor substrates to which radiolabeled putrescine can be conjugated by endogenous enzyme. Extraction of the isolated organelles with saline solutions of increasing ionic strength showed a differential distribution of the polyamine derivatives: all the covalently linked putrescine was associated with the less soluble components of the chromatin residue, while the spermidine and spermine conjugates were associated with several salt-extractable protein fractions as well as tightly bound to the chromatin pellet. Mono-gamma-glutamyl putrescine was detected after proteolytic digestion of the 600 mM NaCl fraction, further suggesting the enzymatic action of transglutaminase(s) in the conjugation process.     

Differences between tissues in response of S-adenosylmethionine decarboxylase to administration of polyamines.

Different reduced forms of xanthine oxidase, labelled specifically in the cyanide-labile site with 33S, were prepared and examined by electron paramagnetic resonance. Coupling of this isotope to molybdenum(V) was quantified with the help of computer simulations and found to differ markedly from one reduced form to another. The xanthine Very Rapid signal shows strong, highly anisotropic, coupling with A(33S)av. 1.27 mT. For this signal, axes of the g- and A(33S)-tensors are rotated relative to one another. One axis of the A-tensor is in the plane of gxx ang gyy, but rotated by 40 degrees relative to the gxx axis, whereas the direction of weakest coupling to sulphur deviates by 10 degrees from the gzz axis. In contrast with this signal, only rather weaker coupling was observed in different types of Rapid signal [A(33S)av. 0.3--0.4 mT], and in the Inhibited signal coupling was weaker still [A(33S)av. 0.1--0.2 mT]. Clearly, there must be substantial differences in the structures of the molybdenum centre in the different signal-giving species, with the sulphur atom perhaps in an equatorial type of ligand position in the Very Rapid species but in a more axial one in the other species. Structures are discussed in relation to the mechanism of action of the enzyme and the nature of the proton-accepting group that participates in turnover.     

Involvement of polyamines in plant response to abiotic stress

Environmental stresses are the major cause of crop loss worldwide. Polyamines are involved in plant stress responses. However, the precise role(s) of polyamine metabolism in these processes remain ill-defined. Transgenic approaches demonstrate that polyamines play essential roles in stress tolerance and open up the possibility to exploit this strategy to improve plant tolerance to multiple environmental stresses. The use of Arabidopsis as a model plant enables us to carry out global expression studies of the polyamine metabolic genes under different stress conditions, as well as genome-wide expression analyses of insertional-mutants and plants over-expressing these genes. These studies are essential to dissect the polyamine mechanism of action in order to design new strategies to increase plant survival in adverse environments.     

Differential response of chick liver and brain membranes to short ethanol treatment

The effect of 60 hr ethanol ingestion on lipid composition of liver and brain membranes from 2-day-old chicks was investigated. Analysis of hepatic membrane cholesterol shows that ethanol induced a slight increase in microsomes exclusively due to free cholesterol while mitochondria was not affected. In brain, both fractions showed a clear increase in their cholesterol content, while a high decrease was observed in myelin. Free cholesterol was also the main responsible for the changes found in brain. The ethanol-treated animals showed an alteration in their phospholipid composition exclusively in brain microsomes and myelin. Despite all these changes, the values of cholesterol/phospholipid molar ratio in both liver and brain membranes remained unaltered after short ethanol treatment. Our results indicate that neonatal chick brain membranes appears to be especially sensitive to the presence of ethanol.     

Differential response of rat brown and white adipose tissue to environmental or nutritional stress.

1. In vivo fatty acid synthesis by brown adipose tissue was enhanced in rats exposed to cold (5 degrees C) or altitude (4300 m) for 7 days but was unaltered in rats exposed to heat (35 degrees C) for an equivalent period. In vivo fatty acid synthesis by white adipose tissue was depressed by cold exposure while altitude and heat exposure had no effect. 2. In vitro, CO2 production and lipid synthesis were elevated in brown adipose tissue from rats fasted for 4 days. Refeeding (4 days) such rats reversed these effects, leading to depressed values relative to those of control rats. In contrast, these metabolic events in white adipose tissue were decreased by fasting and increased compared to controls during subsequent refeeding.     

Differential expression of GABAA receptor alpha-subunits in rat brain during development.

Unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor have been expressed in E. coli and used to generate polyclonal antisera specific for these subunits. The antibodies identify proteins by SDS-polyacrylamide gel electrophoresis and western blotting of molecular size 51 kDa, 53 kDa, 59 kDa and 55 kDa, respectively, which show differential patterns of expression during development. Whereas the alpha 2 and alpha 3 subunits are present at early stages, the expression of alpha 1 and alpha 3 subunits is low at birth and increases with age. This differential expression could be correlated with previous studies examining the developmental expression of BZ1 and BZ2 benzodiazepine binding sites.     

Biogenic aldehyde metabolism in rat brain. Differential sensitivity of aldehyde reductase isoenzymes to sodium valproate

The effects of inhibitors of aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) on the formation of 3-methoxy-4-hydroxyphenethylene glycol from normetanephrine have been studied in rat brain homogenates. The reaction pathway was shown to be unaffected by several inhibitors of the major (high Km) form of aldehyde reductase such as sodium valproate. Two isoenzymes of aldehyde reductase have been separated and characterized from rat brain. The minor (low Km) isoenzyme is shown to be relatively insensitive to sodium valproate and exhibits a similar inhibitor-sensitivity profile to that obtained for methoxyhydroxyphenethylene glycol formation. The low Km isoenzyme is therefore implicated in catecholamine metabolism. The metabolism of succinic semialdehyde and xylose by rat brain cytosol has also been examined. Aldose metabolism may also be attributed to the action of the low Km reductase, but the existence of a separate succinic semialdehyde reductase is postulated. The possible roles of aldehyde reductases in brain metabolism and the relationship between these enzymes and aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) are discussed.     

Phosphatidylinositol-4-phosphate kinase from rat brain. Activation by polyamines and inhibition by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol-4-phosphate (PtdIns-P) kinase was purified approximately 30-fold from rat brain cytosol. No contaminating activity of PtdIns kinase or of phosphomonoesterase and phospholipase C using PtdIns-P or PtdIns-P2 as substrate could be detected in the enzyme preparation. The PtdIns-P kinase activity was severalfold higher when PtdIns-P/PtdEtn vesicles rather than PtdIns-P alone were used as substrate. This might be due to increased accessibility of the enzyme for the vesicular substrate, further indicated by the lower activity obtained when PtdCho or PtdIns, phospholipids with bulky head groups, was also present in the vesicles. The product PtdIns-P2 was a competitive inhibitor with respect to PtdIns-P and 50% inhibition of enzyme activity was observed at the same product concentration regardless of whether the substrate-product mixture was presented in vesicular or micellar form, or the substrate and product were added in separate vesicles. The polyamines spermine and spermidine enhanced PtdIns-P kinase activity severalfold. Spermine also caused a shift in the MgCl2 saturation curve from sigmoidal to hyperbolic, lowering the Mg2+ concentration required for optimum kinase activity to the physiological range. Myelin basic protein enhanced the enzyme activity when PtdIns-P/PtdEtn vesicles were used as substrate, whereas it was inhibitory when PtdIns-P was added alone. The possible role of polyamines and the product PtdIns-P2 in the regulation of PtdIns-P kinase activity is discussed.     

Differential reaction of condensed and diffuse chromatin to polyamines. I. Reaction of interphase nuclei chromatin to putrescine]

Condensation of the interphase nuclei chromatin under putrescine treatment was studied in cultured human fibroblasts 46, XX: 47, XXX: 49, XXXXY, and aneuploid cells of the Chinese hamster. The effect was tested separately for diffuse and condensed chromatin. Putrescine treatment did not affect the percentage of cell nuclei with X-chromatin bodies in the human cell strains while significantly increasing the percentage of nuclei with coarse chromatin network and chromocenters. In cultured Chinese hamster cells, putrescene did not change the percentage of nuclei with identified chromocenters and no significant condensation of diffuse chromatin was observed either.