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Purpose: To establish if oxysterols stimulate differentiation of lens epithelial cells (LEC)., Methods: Primary cultures of lens epithelial cells were incubated with 7-ketocholesterol (7-keto), 25-hydroxycholesterol (25-OH) or cholesterol at 10 microg/ml for 10 days. Cells incubated with 100 ng/ml basic fibroblast growth factor (b-FGF) were used as positive controls for differentiation. The expression of the differentiation marker p57KIP2, proliferation marker PCNA (Proliferating Cell Nuclear Antigen) and fibers specific proteins gamma-crystallin, CP49, MIP26 following treatment with oxysterols was determined by western blot. Differentiation into fiber cells was further confirmed by counting the number of lentoid bodies formed following incubation with 7-keto., Results: LEC incubated with 7-keto presented higher levels of p57KIP2 and showed expression of fiber specific proteins such as MIP26 and CP49, compared to cells incubated with 25-OH or cholesterol. The differentiation marker p57KIP2 increased over time for cells incubated with 7-keto while there was a decline on the amount of the proliferation marker PCNA. The expression of the fiber specific proteins gamma-crystallin, MIP26 and CP49 was detected after 5 days of incubation with 7-keto. Differentiation was accompanied by a seven-fold increase in the number of lentoid bodies formed., Conclusions: Results show for the first time that 7-keto inhibits proliferation and stimulates differentiation of lens epithelial cells into fiber cells. The presence of 7-keto in the lens may disrupt the highly regulated differentiation program of LEC, compromising normal lens growth and transparency.

We previously showed that lens epithelial cells have a fully functional ubiquitin-proteasome pathway (UPP) and that ubiquitin-conjugating activity is up-regulated in response to oxidative stress. In this study we assessed the protein levels and activities of different components of the UPP in lens fibers. Calf lenses were dissected into four different regions: epithelial layer, outer cortex, inner cortex and nucleus. Relative levels of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s), endogenous ubiquitin conjugates, 19S and 20S proteasome subunits were determined by Western blotting. The activities of E1 and E2 were determined by thiol ester assays and the activities of the proteasome and isopeptidases were determined using ubiquitinated alpha-lactalbumin as a substrate. This work demonstrates that lens fibers, including those in the nuclear region, contain most, if not all, of the components for the UPP. Ubiquitin conjugation activity, proteasome activity and isopeptidase activity were also detected in all layers of the lens. The reduced ubiquitin conjugation activity in the inner regions of the lens appeared to be due to a decline in levels of a specific family of E2s, Ubc4 or Ubc5, which were shown to be the rate-limiting enzymes for the formation of high mass conjugates in the lens. Supplementation of Ubc4 or Ubc5 can partially restore the ubiquitin conjugation activity in the inner regions of the lens. Since Ubc4 and Ubc5 are involved in selectively ubiquitinating damaged or abnormal proteins, the decline in levels and activities of these E2s may be responsible for the accumulation of abnormal proteins in inner regions of the lens.

A decline in reduced glutathione (GSH) levels is associated with aging and many age-related diseases. The objective of this study was to determine whether other antioxidants can compensate for GSH depletion in protection against oxidative insults. Rabbit lens epithelial cells were depleted of > 75% of intracellular GSH by 25-200 microM buthionine sulfoximine (BSO). Depletion of GSH by BSO alone had little direct effect on cell viability, but resulted in an approximately 30-fold increase in susceptibility to H(2)O(2)-induced cell death. Experimentally enhanced levels of nonprotein sulfhydryls other than GSH (i.e., N-acetylcysteine) did not protect GSH-depleted cells from H(2)O(2)-induced cell death. In contrast, pretreatment of cells with vitamin C (25-50 microM) or vitamin E (5-40 microM), restored the resistance of GSH-depleted cells to H(2)O(2). However, concentrations of vitamin C > 400 microM and vitamin E > 80 microM enhanced the toxic effect of H(2)O(2). Although levels of GSH actually decreased by 10-20% in cells supplemented with vitamin C or vitamin E, the protective effects of vitamin C and vitamin E on BSO-treated cells were associated with significant ( approximately 70%) decreases in oxidized glutathione (GSSG) and concomitant restoration of the cellular redox status (as indicated by GSH:GSSG ratio) to levels detected in cells not treated with BSO. These results demonstrate a role for vitamin C and vitamin E in maintaining glutathione in its reduced form. The ability of vitamin C and vitamin E in compensations for GSH depletion to protect against H(2)O(2)-induced cell death suggests that GSH, vitamin C, and vitamin E have common targets in their actions against oxidative damage, and supports the preventive or therapeutic use of vitamin C and E to combat age- and pathology-associated declines in GSH. Moreover, levels of these nutrients must be optimized to achieve the maximal benefit.

The characteristics of [(125)I]Bolton-Hunter[Sar(9),Met(O(2))(11)]substance P ([(125)I]BH-SarSP) binding were investigated in membranes of human ascending, transverse, distal, and sigmoid colon circular muscle. Binding of [(125)I]BH-SarSP was of high affinity (K(D) = 68 nM) and low capacity (B(max) = 0.31 fmol/mg of wet weight tissue), and showed no regional differences. [(125)I]BH-SarSP binding was inhibited by SP approximately equal to [Pro(9)]SP > or = (2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine (CP99994) >> neurokinin (NK) A > or = neuropeptide gamma > [Lys(5),MeLeu(9),Nle(10)]-NKA(4-10) approximately (S)-N-methyl-N[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) butyl]benzamide (SR48968) >> senktide, suggesting binding to NK-1 sites. Most agonists seemed to bind to two sites. In autoradiographic studies, dense binding for [(125)I]BH-SarSP was associated with submucosal and longitudinal muscle blood vessels, and the submucosal margin of circular muscle (corresponding to interstitial cells of Cajal), with moderate binding over most of the circular muscle. In normal colon circular muscle strips, [Pro(9)]SP was almost ineffective, and SP caused contractions with pD(2) values of 5.3 to 5.7. No regional differences were observed in potency or efficacy. Responses to SP were inhibited by the NK-2 receptor antagonist SR48968, but not by NK-1 antagonist CP99994, indicating the involvement of NK-2 rather than NK-1 receptors. Atropine significantly inhibited contractions induced by SP, indicating a minor cholinergic component. Contractile responses to SP were considerably reduced in preparations from patients with diverticular disease, and marginally reduced in ulcerative colitis compared with control. This study clearly demonstrates an NK-1 binding site on human colon circular muscle, but its role in this tissue remains unclear and may not involve contractile mechanisms. The attenuated contractility in specimens with diverticular disease may reflect disease-related alterations of the tachykinin receptor system.

The human genetic disorder ataxia-telangiectasia (A-T) is due to lack of functional ATM, a protein kinase which is involved in cellular responses to DNA double strand breaks (DSBs) and possibly other oxidative stresses, as well as in regulation of several fundamental cellular functions. Studies regarding responses in A-T cells to the induction of DSBs utilize ionizing radiation or radiomimetic chemicals, such as neocarzinostatin (NCS), which induce DNA DSBs. This critical DNA lesion activates many defense systems, such as the cell cycle checkpoints. The cell cycle is also regulated through a timed and coordinated degradation of regulatory proteins via the ubiquitin pathway. Our recent studies indicate that the ubiquitin pathway is influenced by the cellular redox status and that it is the major cellular pathway for removal of oxidized proteins. Accordingly, we hypothesized that the absence of a functional ATM protein might involve perturbations to the ubiquitin pathway as well. We show here that upon treatment with NCS, there was a transient 50-70% increase in endogenous ubiquitin conjugates in A-T and wt lymphoblastoid cells. Ubiquitin conjugation capabilities per se and levels of substrates for conjugation were also similarly enhanced in wt and A-T cells upon NCS treatment. We also compared the ubiquitination response in A-T and wt cells using H(2)O(2) as the stress, in view of preexisting evidence of the effects of H(2)O(2) on ubiquitination capabilities in other types of cells. As with NCS treatment, there was an approximately 45% increase in endogenous ubiquitin conjugates by 2-4 h after exposure to H(2)O(2). Both cell types showed a rapid 50-150% increase in de novo formed 125I-ubiquitin conjugates. As compared with wt cells, unexposed A-T cells had higher endogenous levels of conjugates and enhanced conjugation capability. However, A-T cells mounted a more muted ubiquitination response to the stress. The enhanced ubiquitin conjugation in unstressed A-T cells and attenuated ability of these cells to respond to stress are consistent with the A-T cells being under oxidative stress and with their having an 'aged' phenotype. The indication that ubiquitin conjugate levels and ubiquitin conjugation capabilities are enhanced upon oxidative stress without significant changes in GSSG/GSH ratios indicates that assays of ubiquitination provide a sensitive measure of cellular stress. The data also add support to the impression that potentiated ubiquitination response to mild oxidative stress is a generalizable phenomenon.

Hyperbaric oxygen therapy is used to treat various clinical conditions, but it also causes oxidative damage. The objectives of this study are to determine if increased vitamin C intake can prevent hyperbaric oxygen-induced damage and to determine interactions among vitamin C, glutathione and vitamin E in response to oxidative stress. The growth rates of unexposed guinea pigs fed 1.25 mg vitamin C/day were indistinguishable from that of guinea pigs fed 50 mg vitamin C/day. In contrast, hyperbaric oxygen exposure resulted in growth retardation in guinea pigs fed 1.25 mg vitamin C/day, but it had little effect on the growth rates of guinea pigs fed 50 mg vitamin C/day. Increased vitamin C intake also prevented hyperbaric oxygen-induced lipid peroxidation in the liver. In guinea pigs not exposed to hyperbaric oxygen, levels of vitamin C in tissues were closely related to vitamin C intake, but tissue levels of glutathione and vitamin E were not related to vitamin C intake. However, interactions between vitamin C and glutathione were observed upon chronic hyperbaric oxygen exposure. Chronic hyperbaric oxygen exposure resulted in >2-fold increases in the levels of glutathione in liver and lung of guinea pigs fed 1.25 mg vitamin C/day. In comparison, the oxidation-induced increases in glutathione were significantly attenuated in guinea pigs fed 50 mg vitamin C/day. These data show that increased intake of vitamin C can prevent or alleviate the hyperbaric oxygen-induced damage. The interactions between vitamin C and glutathione upon hyperbaric oxygen exposure indicate that there is a homeostatic regulation of antioxidant capacity in guinea pig tissues.

Neurokinin A (NKA) is potent in contracting the human detrusor muscle. Here, we have investigated whether these contractile responses are influenced by the presence of the mucosa, by the peptidase inhibitor phosphoramidon or by possible modulators, prostaglandins and nitric oxide. Contractile responses to neurokinin A were unaffected by indomethacin or N-omega-nitro-L-arginine, but were significantly reduced in strips containing mucosa. Phosphoramidon, an inhibitor of neutral endopeptidase 24.11 (neprilysin, CD10), was ineffective at 10 microM, but at 100 microM, significant increase in the maximum response was achieved by neurokinin A in detrusor strips with and without mucosa. In immunohistochemical studies, neutral endopeptidase immunoreactivity occurred in peripheral nerve trunks in the detrusor and in a fibrous meshwork in the subepithelial lamina propria. Our data indicate that neutral endopeptidase is present in bladder mucosa and detrusor, and support the concept that this metalloprotease and/or related enzymes are important in regulating the actions of tachykinins.

Lens development and response to peroxide stress are associated with dramatic changes in protein ubiquitination, reflecting dynamic changes in activity of the ubiquitin-activating enzyme (E1). Two isoforms of E1 (E1A and E1B) have been identified in lens cells although only one E1 mRNA, containing three potential translational start sites, has been detected. Novel, site-specific antibodies to E1 were generated and the hypothesis that the two isoforms of E1 are translated from alternative initiation codons of a single mRNA was tested. Antibodies raised against E1A-N peptide (Met(1)to Cys(23)of E1A) reacted only with E1A by immunoblot and immunoprecipitation. Antibodies raised against E1B-N peptide (Met(1)to Glu(25)of E1B or Met(41)to Glu(65)of E1A) and E1AB-C peptide (His(1030)to Arg(1058)of E1A or His(990)to Arg(1018)of E1B) reacted with both E1A and E1B. These results indicate that (1) E1A and E1B contain the same C-terminal residues; (2) E1A contains the N terminal sequence of E1B; and (3) E1B does not contain the N terminal sequence of E1A. The two isoforms of lens E1 are therefore translated from a single mRNA. Specifically, E1A is translated from the first initiation codon, and E1B translated from the second initiation codon. E1A and E1B were affinity-purified, and their ability to 'charge' ubiquitin carrier proteins (E2s) with activated ubiquitin was compared in a cell-free system. E1A and E1B were indistinguishable with respect to charging different E2s. However, E1 immunolocalization studies with human lens epithelial cells indicate that E1A and E1B are preferentially localized to the nucleus and cytosol, respectively. This observation suggests that E1A and E1B ubiquitinate different proteins and serve different functions in intact cells., (Copyright 2001 Academic Press.)

In the mammalian lens, intracellular oxidants produced by photo-oxidative processes and exposure to toxic chemicals constitute stresses that produce cellular oxidative damage, result in changes in gene expression, and are causally related to cataract formation. Currently, it is believed that H(2)O(2) is the major oxidant to which the lens is exposed. In this report, we examine the activation and regulation of the oxidant-sensitive transcription factor, NF-kappa B, by H(2)O(2)-mediated oxidative stress in lens epithelial cells. Lens epithelial cells treated with H(2)O(2) demonstrated at 1 h a strong activation of NF-kappa B which returned to basal levels by 2 h. Under proteasome inhibition using both MG132 and lactacystin, H(2)O(2)-mediated activation of NF-kappa B was prevented, implicating the involvement of proteasome degradation of I kappa B proteins as being necessary for this activation. However, Western blot analysis demonstrated no degradation of I kappa B-alpha, -beta, or -epsilon associated with H(2)O(2)-mediated NF-kappa B activation. In comparison, when cells were treated with the cytokine TNF-alpha, NF-kappa B was strongly activated and degradation of both I kappa B-alpha and -beta was observed. These results clearly demonstrate that H(2)O(2)-mediated oxidative stress activates NF-kappa B in lens epithelial cells, which may subsequently lead to changes in gene expression. The results also reveal that different signaling pathways in the activation of NF-kappa B in lens epithelial cells are utilized by H(2)O(2) and TNF-alpha. These different pathways of NF-kappa B activation may be required to effect specific NF-kappa B-dependent gene expression in response to these different stimuli.

Understanding how oxidized proteins are removed is important since accumulation of such damaged proteins is causally related to cellular and organismic dysfunction, disease and aging. Previous work showed that activity of the ubiquitin-proteasome pathway (UPP) in lens cells increased during recovery from oxidative stress ( Shang et al., 1997b : J. Biol. Chem. 272, 23086-93). In this study we sought to determine if the up-regulation of the UPP during recovery from oxidative stress has a role in selective removal of oxidized proteins from the cells. In cells which were not exposed to peroxide, inhibition of the proteasome with MG132 or clasto-lactacystin beta-lactone had little effect on protein carbonyl levels. However, inhibition of the proteasome in the 20 microM peroxide-treated cells caused an approximate 60% increase in levels of protein carbonyl and an approximate 100% increase in levels of ubiquitin conjugates. The carbonyl-containing proteins that accumulated in the presence of the proteasome inhibitor co-localized with high molecular mass ubiquitin-protein conjugates. Furthermore, isolated carbonyl-containing proteins from H2O2-treated cells were ubiquitinated, and ubiquitin-conjugates were enriched with carbonyl-containing proteins. The diminished effect of proteasome inhibitors on protein carbonyl levels, together with the robust increase in ubiquitin-protein conjugates and accompanied increases in oxidized proteins, upon exposure to 60 microM H2O2 indicate that the proteasomal step of the UPP is more susceptible to oxidative inactivation than the ubiquitination step. In fact, oxidative stress is associated with a hyperactivation of the ubiquitin-activating enzyme. These data indicate that the UPP plays a role in removal of oxidatively damaged proteins from cells and that attenuation of the UPP activity may result in cytotoxic accumulation of damaged proteins, possibly including the ubiquitinated forms., (Copyright 2001 Academic Press.)

1. Bufokinin is a substance P-like neuropeptide and potent spasmogen isolated from the intestine of the cane toad Bufo marinus. In the present study, we investigated the effects of bufokinin on systemic blood pressure and heart rate in the anaesthetized toad and the distribution of bufokinin-like immunoreactivity in the toad vasculature. 2. Intravenous bufokinin caused a dose-dependent fall in systemic blood pressure (maximum fall 20 mmHg) with an ED50 of 2.9 pmol. At higher doses, the effect was prolonged and blood pressure did not return to baseline within 60 min. There was no significant change in heart rate associated with hypotension. 3. Bufokinin-like immunoreactivity was mapped in whole mounts of toad blood vessels and organs using a mouse polyclonal antibody BK3 (at 1:5000) and the avidin-biotin method. Bufokinin-immunoreactive fibres were associated with most blood vessels examined: a moderately dense perivascular network of varicose fibres was present around renal arteries, with sparser immunoreactive fibres in the ventral aorta, sciatic artery, anterior abdominal vein and hepatic portal vein. 4. Bufokinin-immunoreactive fibres, mainly following blood vessels, were seen in whole mounts of the urinary/bladder and tongue, but not in the air sac. In the heart ventricle, varicose fibres were found in the valve cusps, intracardiac ganglia, epicardium and myocardium close to the endocardium, but not in the rest of the myocardium. 5. The vasodepressor action of bufokinin and the presence of bufokinin-like immunoreactivity in varicose fibres in various vessels suggest a role for bufokinin in haemodynamic regulation and/or sensory nerve function in the toad. The lack of any reflex tachycardia in response to the falls in blood pressure was of note.

In this study, we have mapped the immunoreactivity and the binding sites for bufokinin, a tachykinin peptide from the toad intestine. Dense bufokinin-immunoreactive fibers were present at the myenteric plexus, but no cell bodies were stained, suggesting an extrinsic origin. Bufokinin nerve fibers were also associated with submucosal blood vessels and mesenteric arteries. Autoradiographic binding sites for [(125)I]Bolton-Hunter-bufokinin were densely localized over the intestinal circular and longitudinal muscle, submucosal blood vessels and the endothelium of mesenteric arteries. Mesenteric veins had minimal immunoreactivity and binding sites. In the anesthetized toad, topical application of bufokinin onto the mesentery caused a 2.7-fold increase in arterial blood flow, observed using intravital microscopy. This study supports a role for bufokinin as an endogenous spasmogen and hemodynamic regulator in the toad intestine.

Purpose: In bladder, sensory afferent nerve fibers contain the "sensory neuropeptides" substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP), which interact with tachykinin NK-1 and NK-2 receptors and CGRP receptors, respectively. The purpose of this study was to examine the autoradiographic distribution of these three receptor types in the human bladder, to determine whether the anatomic location of the receptors was consistent with their known functional roles., Materials and Methods: Specimens of urinary bladder from 9 patients (58-74 years) were obtained at cystectomy. Frozen sections of dome were labeled with [125I]-Bolton-Hunter [Sar9,Met(O2)11]-SP (NK-1 receptors), [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10) (NK-2 receptors) and [125I]-rat CGRP-I. Binding sites were visualized using emulsion autoradiography., Results: NK-1 receptors were found over the endothelium of arterial blood vessels within the detrusor muscle and lamina propria, and over small vessels in the subepithelium. NK-2 receptors were seen over the detrusor muscle and very sparsely over blood vessels, whereas CGRP receptors were expressed densely over the smooth muscle layer of arteries and arterioles, and weakly over collecting venules. NK-1 and CGRP receptors were not observed over the detrusor muscle., Conclusions: Although the afferent nerves contain all three peptides, not all cell types express receptors for each peptide. The general distribution of receptors is in good agreement with the location of nerves, and with the known actions of SP and CGRP as vasodilator agents, and of NKA (but not SP or CGRP) in contracting the detrusor muscle.

Purpose: Recent epidemiologic studies suggest that differential risk for cataract in different areas of the lens may be related to intake of carotenoids, retinol, and tocopherol. Nevertheless, there is little information about differential localization of these nutrients in the lens. To determine the spatial distribution of fat-soluble nutrients within the lens, we determined levels of these nutrients in the epithelium/ outer cortex vs. inner cortex/nucleus., Methods: Concentrations of carotenoids, retinol, and tocopherol were determined in the epithelial/cortical (younger, more metabolically active tissue) and nuclear (older, less metabolically active) layers of human cataractous lenses (n = 7, 64-75 yr) by reverse-phase high-performance liquid chromatography (HPLC)., Results: Lutein/zeaxanthin was the only carotenoid, which was detected, in human lens. Consistent with prior reports, no beta-carotene or lycopene were detected. Concentrations of lutein/zeaxanthin, tocopherol, and retinol in epithelium/cortex tissue were approximately 3-, 1.8-, and 1.3-fold higher than in the older lens tissue. Specifically, the epithelial/cortical lens layer, comprising about half of the tissue, contains 74% of lutein/zeaxanthin (44 ng/g wet wt), 65% of alpha-tocopherol (2227 ng/g wet wt), and 60% of retinol (30 ng/g wet wt)., Conclusions: The data suggest that upon development and aging, there is differential localization of these nutrients. The data are also consistent with a protective role of these nutrients against oxidative damage in the epithelium and cortex of the human lens.

Levels of glutathione in liver and kidney, and other nutrients in plasma were evaluated in male and female ascorbate-requiring osteogenic disorder Shionogi (ODS) rats fed semipurified diets in which the concentrations of ascorbate were gradually decreased from 1965 to 180 mg/kg. Plasma ascorbate levels in ODS rats were unaffected when ascorbate levels in the diet were decreased from 1965 to 768 mg/kg. However, plasma ascorbate levels decreased progressively when levels of ascorbate in the diet were decreased from 527 to 180 mg/kg. Plasma ascorbate levels decreased up to 77% when the dietary ascorbate concentration decreased from 1965 to 180 mg/kg. Ascorbate levels in liver and kidney fell as much as 60-70% when the dietary ascorbate levels were reduced from 1965 to 180 mg/kg. However, the glutathione levels in these tissues were not affected. Plasma retinol and vitamin E levels were not affected by decreasing dietary ascorbate intake. Total cholesterol levels increased significantly in female rats as dietary ascorbate intake declined. Levels of glycated hemoglobin decreased significantly when dietary ascorbate levels decreased from 1965 to 527 mg/kg. This study suggests that levels of vitamin E, retinol and glutathione are not affected by decreased dietary intake of ascorbate under nonscorbutic conditions, whereas elevated ascorbate intake is associated with a decrease in levels of plasma cholesterol in female ODS rats. However, excessive intake of ascorbate may be associated with elevated glycation of hemoglobin. To achieve the maximal health benefit of ascorbate supplementation, further studies are necessary to define optimal ascorbate intakes.

The mammalian eye lens is composed of two distinct types of cells, epithelial cells and fiber cells. The fiber cells are generated throughout life via continuous differentiation of epithelial cells. Differentiation of lens cells involves dramatic changes in cellular components including altered activity of the ubiquitin dependent pathway. The concentration of high mass ubiquitin conjugates in the mitotically active-, differentiating-equatorial epithelial cells was 5-10 fold higher than that observed in mitotically quiescent, non-differentiated, central epithelial cells, even though there was a significant dilution of non-crystallin proteins due to an increase in level of crystallins in the differentiating cells. Similar observations were made when differentiation was modeled by exposure of lens epithelial explants to bFGF in culture. Activities of ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) in the differentiating equatorial epithelial cells were also up to 100% higher than those noted in non-differentiated central epithelial cells and E1 appears to be rate controlling for ubiquitinylation. Consistent with the higher concentrations of high mass ubiquitin conjugates, there was a trend of enhanced ability to execute ATP-dependent protein degradation in the differentiating equatorial epithelial cells as compared with degradation in the non-differentiated central epithelial cells. These data indicate that the ubiquitin dependent pathway is up-regulated during differentiation of lens cells. In the differentiated fibers, the concentration of high mass ubiquitin conjugates and relative activities of E1 and E2s were 50% lower than in the non-differentiated central epithelial cells. In comparison, the concentration of the 110 kDa E1 was unchanged in differentiated fibers. However, if the factor of dilution by the significant increase in the level of crystallins was taken into account, the level or activities of the components of ubiquitin pathway in the differentiated cells was higher than the level noted in non-differentiated cells. These data indicate that, as compared with other non-crystallin proteins, there is differential stabilization and/or synthesis of the 110 kDa E1 and some other components of the ubiquitin dependent pathway in differentiated fibers., (Copyright 1999 Academic Press.)

Calorie restriction (R) is the only known method to delay the aging process and extend mean and maximal lifespan in rodents. R has been shown to delay the age-related accumulation of damaged proteins and to protect organisms from various stresses which can produce damaged proteins. Such stresses include irradiation, heat shock, and oxidative stress. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) has been associated with the degradation of abnormal and/or damaged proteins. We examined the effect of diet and oxidative stress on activities of the UPP in supernatants from livers taken from 23-month-old Emory mice which had been exposed to an in-vivo injection of paraquat. Paraquat induces oxidative stress by generating superoxide radicals. In livers from non-stressed animals, steady-state levels of endogenous ubiquitin conjugates, de novo conjugate formation, and E1 and E2 activities were significantly lower in R animals than in control (C) animals. However, after exposure to paraquat, levels of endogenous ubiquitin conjugates were significantly higher in R versus C animals, and de novo conjugate formation and E1 and E2 activities in R animals rose to levels which were indistinguishable from levels of these activities noted in C animals. R was associated with an increased ability to degrade beta-lactoglobulin by the UPP after an oxidative stress was imposed. Ability to degrade beta-lactoglobulin by the C or R livers in non-stressed animals was not significantly different. Taken together, these data indicate that oxidative stress in R animals is associated with enhanced levels of ubiquitin conjugates and that this enhancement may be due to an increase in UPP activity. These data also indicate that the ability to form ubiquitin conjugates and the UPP system does not change with oxidative stress in C animals. The latter is consistent with prior reports that suggests that older C animals may already be in a state of enhanced oxidative stress and that activities of the UPP provide a sensitive indicator of levels of cellular redox status.

The ubiquitin-proteasome pathway (UPP) regulates critical cell processes, including the cell cycle, cytokine-induced gene expression, differentiation, and cell death. Recently we demonstrated that this pathway responds to oxidative stress in mammalian cells and proposed that activities of ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) are regulated by cellular redox status (i.e., GSSG:GSH ratio). To test this hypothesis, we altered the GSSG:GSH ratio in retinal pigment epithelial cells with the thiol-specific oxidant, diamide, and assessed activities of the UPP. Treatment of cells with diamide resulted in a dose-dependent increase in the GSSG:GSH ratio resulting from loss of GSH and a coincident increase in GSSG. Increases in the GSSG:GSH ratio from 0.02 in untreated cells to > or = 0.5 in diamide-treated cells were accompanied by dose-dependent reductions in the levels of endogenous Ub-protein conjugates, endogenous E1-ubiquitin thiol esters, and de novo ubiquitin-conjugating activity. As determined by the ability to form E1-ubiquitin and E2s-ubiquitin thiol esters, E1 and E2s were both inhibited by elevated GSSG:GSH ratios. Inhibition of E1 was associated with the formation of E1-protein mixed disulfides. Activities of E1 and E2s gradually recovered to preoxidation levels, coincident with gradual recovery of the GSSG:GSH ratio. These data support S-thiolation/dethiolation as a mechanism regulating E1 and E2 activities in response to oxidant insult. Ubiquitin-dependent proteolytic capacity was regulated by the GSSG:GSH ratio in a manner consistent with altered ubiquitin-conjugating activity. However, ubiquitin-independent proteolysis was unaffected by changes in the GSSG:GSH ratio. Potential adaptive and pathological consequences of redox regulation of UPP activities are discussed.

Calorie restriction (R), the only known method to delay the aging process and extend mean and maximal lifespan, has been shown to delay the age-related decline in protein degradation. There are several proteolytic pathways. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) is frequently associated with degradation of damaged abnormal and/or regulatory proteins. We examined the effect of aging and R on supernatants of livers taken from young (4.5 months) and old (23 months) Emory mice. Aging was associated with increased levels of endogenous ubiquitin conjugates, enhanced ability to form high molecular weight conjugates and ubiquitin activating (E1) and ubiquitin conjugating (E2) activity in the control (C) liver supernatants. The age-related increase in levels of endogenous ubiquitin conjugates in liver appears to be primarily due to increased E1 and E2 activities. R prevented the age-related increase in E1 and E2 activity, and thus prevented the age-related increase in levels of ubiquitin conjugates. In spite of the age-related increase in ubiquitin conjugates, no age-related changes in ubiquitin-dependent proteolytic pathway were observed in the C animals. R was associated with an enhanced ability (130%) to degrade beta-lactoglobulin by the ubiquitin-dependent proteolytic pathway in livers from 4.5-month-old animals relative to age-matched C livers. However, rates of the ubiquitin-dependent degradation of beta-lactoglobulin in the 23-month-old C and R animals were indistinguishable. There were no age- or diet-related differences in the ability to degrade another substrate, oxidized ribonuclease (RNase).

Dietary calorie restriction extends both mean and maximum life span and retards age-related diseases, including eye lens cataract in Emory mice. The beneficial effects of calorie restriction have been hypothesized to reflect enhanced tissue antioxidant capacity. As a test of this hypothesis, we reared male and female Emory mice on control (C) or 40% calorie-restricted (R) diets. We then determined activities of total superoxide dismutase (T-SOD), Cu/Zn-SOD, Mn-SOD, glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT) in eye lens, liver and kidney of young (4.5 or 6 months), mature (11 or 12 months) and old (22 months) animals. Effects of diet, age and sex were evaluated by multi-factor ANOVA. Only kidney GR activities (mean +/- S.E.M.) were significantly enhanced with the R diet (R, 61 +/- 2 vs. C, 54 +/- 3 U/mg protein; P = 0.03). More frequently, we noted reduced antioxidant enzyme activity in R as compared with C animals, including reduced activities of T-SOD in lens, liver and kidney, Cu/Zn-SOD in liver and kidney, liver Mn-SOD and liver CAT (P < 0.05). Effects of age on antioxidant enzyme activity in C mice included age-dependent decreases in lens and kidney CAT and in liver Mn-SOD. There was also an age-dependent increases in liver and kidney Cu/Zn-SOD and liver GR. None of these age-dependent alterations in antioxidant enzyme function were attenuated in tissues of mice fed the R diet. Values for liver CAT were significantly lower in females than in males (P = 0.05). These results indicate that antioxidant enzyme activities in Emory mouse tissues are influenced by diet, age and sex. However, it is unlikely that increased lifespan and attenuation of cataract (and perhaps other age-dependent debilities), which are associated with the R diet in the Emory mouse, are due to enhanced antioxidant enzyme capabilities.

Upon oxidative stress cells show an increase in the oxidized glutathione (GSSG) to reduced glutathione (GSH) ratio with a concomitant decrease in activity of the ubiquitinylation pathway. Because most of the enzymes involved in the attachment of ubiquitin to substrate proteins contain active site sulfhydryls that might be covalently modified (thiolated) upon enhancement of GSSG levels (glutathiolation), it appeared plausible that glutathiolation might alter ubiquitinylation rates upon cellular oxidative stress. This hypothesis was explored using intact retina and retinal pigment epithelial (RPE) cell models. Exposure of intact bovine retina and RPE cells to H2O2 (0.1-1.7 micromol/mg) resulted in a dose-dependent increase in the GSSG:GSH ratio and coincident dose-dependent reductions in the levels of endogenous ubiquitin-activating enzyme (E1)-ubiquitin thiol esters and endogenous protein-ubiquitin conjugates and in the ability to form de novo retinal protein-125I-labeled ubiquitin conjugates. Oxidant-induced decrements in ubiquitin conjugates were associated with 60-80% reductions in E1 and ubiquitin-conjugating enzyme (E2) activities as measured by formation of ubiquitin thiol esters. When GSH levels in RPE cells recovered to preoxidation levels following H2O2 removal, endogenous E1 activity and protein-ubiquitin conjugates were restored. Evidence that S thiolation of E1 and E2 enzymes is the biochemical link between cellular redox state and E1/E2 activities includes: (i) 5-fold increases in levels of immunoprecipitable, dithiothreitol-labile 35S-E1 adducts in metabolically labeled, H2O2-treated, RPE cells; (ii) diminished formation of E1- and E2-125I-labeled ubiquitin thiol esters, oligomerization of E225K, and coincident reductions in protein-125I-labeled ubiquitin conjugates in supernatants from nonstressed retinas upon addition of levels of GSSG equivalent to levels measured in oxidatively stressed retinas; and (iii) partial restoration of E1 and E2 activities and levels of protein-125I-labeled ubiquitin conjugates in supernatants from H2O2-treated retinas when GSSG:GSH ratios were restored to preoxidation levels by the addition of physiological levels of GSH. These data suggest that the cellular redox status modulates protein ubiquitinylation via reversible S thiolation of E1 and E2 enzymes, presumably by glutathione.

We have examined the development of the laminar and areal distribution of cortical commissural neurons in a marsupial mammal, the wallaby Macropus eugenii. In this species, commissural axons approach the major cerebral commissure, the anterior commissure, via either the internal capsule or the external capsule and first cross the midline at postnatal day 14 (P14). By retrogradely labelling these axons with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) at P15, we show here that the cell bodies of these neurons are restricted to a region of cortex adjacent to the rhinal fissure. Most of these labelled neurons are located in the compact cell zone of the cortical plate, with only a few labelled cells found in the zone of loosely packed cells deep to this layer. Over the subsequent 66 days, commissural neurons are found progressively more dorsally, rostrally, and caudally, so that, by P80, they are present throughout the extent of the neocortex. At this age, they are mainly pyramidal in morphology and form a single band within the deeper part of layer 5 of the developing cortex. From P80 to adulthood, the distribution of commissural neurons has been assessed in the visual cortex by using retrograde transport of horseradish peroxidase. At P80, labelled neurons with immature pyramidal morphology are present throughout the occipital cortex; as in DiI material, somata are located in deep layer 5. At P165, previously shown to be the age when commissural axon numbers peak, widespread labelling is present in the occipital region, with labelled cells now found in two bands corresponding to layers 3 and 5. After this age, neurons become more restricted in distribution, so that, by adulthood, commissural neurons are no longer apparent throughout area 17 but are restricted to a localised region around the area 17/18 boundary. Within this region, labelling is still present in layers 3 and 5 but is more dense in layer 3. The gradual restriction of commissural fields seen here in the wallaby is similar to that reported in the neocortex in many eutherians. These findings also support studies in eutheria, suggesting that subplate neurons do not appear to play a major role in commissural development.

Relations between the ubiquitin pathway and cellular stress have been noted, but data regarding responses of the ubiquitin pathway to oxidative stress are scanty. This paper documents the response of this pathway to oxidative stress in lens cells. A brief exposure of lens epithelial cells to physiologically relevant levels of H2O2 induces a transient increase in activity of the ubiquitin-dependent pathway. Ubiquitin conjugation activity was maximal and increased 3. 5-9.2-fold over the activity noted in untreated cells by 4 h after removal of H2O2. By 24 h after removal of H2O2, ubiquitin conjugation activity returned to the level noted in untreated cells. In parallel to the changes in ubiquitin conjugation activity, the activity of ubiquitin-activating enzyme (E1), as determined by thiol ester formation, increased 2-6.7-fold during recovery from oxidation. Addition of exogenous E1 resulted in an increase in ubiquitin conjugation activity and in the levels of ubiquitin carrier protein (E2)-ubiquitin thiol esters in both the untreated cells and the H2O2-treated cells. These data suggest that E1 is the rate-limiting enzyme in the ubiquitin conjugation process and that the increases in ubiquitin conjugation activity which are induced upon recovery from oxidation are primarily due to increased E1 activity. The oxidation- and recovery-induced up-regulation of E1 activity is primarily due to post-synthetic events. Substrate availability and up-regulation of E2 activities also appear to be related to the enhancement in ubiquitinylation upon recovery from oxidative stress. The oxidation-induced increases in ubiquitin conjugation activity were associated with an increase in intracellular proteolysis, suggesting that the transient increase in ubiquitinylation noted upon recovery from oxidative stress may play a role in removal of damaged proteins from the cells.

Accumulation of damaged proteins is a major age-related change in lenses of virtually all species and is associated with lens opacification. Proteolytic removal of the damaged proteins may play an important role in maintaining the transparency of the lens. In many tissues, selective removal of abnormal or damaged proteins occurs via a ubiquitin-dependent proteolytic pathway. Ubiquitin, an 8.5 kDa polypeptide, selectively binds to proteins to form ubiquitin-protein conjugates. This ubiquitin-protein conjugate is, in most cases, a signal for protein degradation. In this work, age-related changes in rat lens in the following aspects were detected: (a) levels of the ubiquitin-protein conjugates, (b) some of the enzymes involved in ubiquitin conjugation in rat lenses, and (c) ability to respond to oxidative damage. Endogenous ubiquitin-protein conjugates were detected in epithelium, cortex and nucleus of lenses from young and old rats. The levels of endogenous high molecular weight (HMW) ubiquitin-protein conjugates in each developmental zone of the lenses from young rats were higher than that in the counterparts of lenses from old animals. Peroxide-treatment generally resulted in elevated levels of endogenous HMW ubiquitin-protein conjugates although masses of bulk proteins remain unchanged. The increases in ubiquitin-protein conjugates in the epithelial sections of young and old lenses upon oxidative stress were comparable. In the cortex of young lenses, there was a significant oxidation-related increase in ubiquitin-protein conjugates. There was a similar trend but diminished response in the cortex of old lenses. Nuclear fibers from young lenses also showed an oxidation-induced increase in the level of ubiquitin-protein conjugates. This response was not observed in nuclear fibers of old lenses. The ability to form HMW-ubiquitin conjugates with exogenous 125I-labeled ubiquitin in the lens also increased upon oxidative stress. The extent of the increase in the de-novo ubiquitin conjugating activity upon exposure to oxidation in old lens was much smaller than in young lens. Ubiquitin-activating enzyme (E1), and ubiquitin conjugating enzymes (E2(17k), E2(20k) and E2(25k) were detected by thiol ester assays or Western blot analysis. No significant age-related changes in the levels of E1, E2(17k), E2(20k) and E2(25k) were detected. The activity of E1 and E2(17k) increased upon exposure to H2O2. These data indicate that lens has the ability to increase ubiquitin conjugation activity in response to oxidative stress and this ability is attenuated upon aging. The age-related decrease in the ability to mount a ubiquitin-dependent response upon oxidation may contribute to the accumulation of damaged proteins in the old lenses.

Prior studies suggest that whisker afferents have but one central projection pattern, despite their association with differing peripheral receptors that predict central morphology in other systems. Target factors in barrelettes are thought to dictate afferent projection patterns; yet, barrelettes differ in their size, shape and development. We tested the hypothesis that whisker afferents have differing morphologies that are predicted by peripheral and central factors. Branching patterns and collaterals of 78 Neurobiotin-stained afferents were compared in rats. Fibers from one whisker had precisely somatotopic projections but highly varied morphologies. For the entire sample, analysis of variance revealed significant intrafiber variance in collateral number and arbor shape that was attributed to the target subnucleus. Significant interfiber variance did not reflect response adaptation rate, direction sensitivity, whisker row origin or parent fiber bifurcation in the trigeminal root. Instead, we found the following. 1) Mandibular fibers had more elongated arbors than maxillary axons. In subnuclei interpolaris and principalis, mandibular fibers had larger arbors with more boutons/collateral than maxillary axons; in oralis and interpolaris, mandibular fibers had fewer collaterals than those of the maxillary division. 2) Upper lip whisker axons had more boutons than those from the B-D row in all subnuclei. 3) Rostral whisker are afferents had larger arbors and more boutons than those from middle or caudal arcs due to significant arc effects in interpolaris and oralis. Thus, whisker afferents are not structurally uniform, and some morphological features are predictable. Intrafiber variance is attributed to the central target; interfiber variance reflects maxillary versus mandibular origin, upper lip origin and whisker rostrocaudal arc.

There is a growing consensus that altered proteins are more susceptible to degradation than native proteins. The enhancement of degradation of damaged proteins may be of significance since it prevents the accumulation of damaged proteins in cells. Several proteolytic pathways have been discovered in the lens. These include ATP-independent, ATP-dependent and ATP/ubiquitin-dependent proteolytic pathways. However, the extent of involvement of these proteolytic pathways in degradation of damaged proteins is not well described. alpha-Crystallin was oxidized by exposure to 0.03-3.2 mol.OH (mol protein)-1. Modifications to the oxidized alpha-crystallin and proteolytic susceptibility of the oxidized alpha-crystallin were studied. Exposure to > 0.32 mol.OH per mole of subunit produced aggregates and fragments of alpha-crystallin. Changes in isoelectric points of the proteins were observed after exposure to 0.64 mol.OH (mol protein)-1. The extent of loss of tryptophan and sulfhydryl groups was related to the level of .OH-exposure. Carbonyl content increased progressively with increasing oxidation. When incubated with a supernatant of bovine lens epithelial cells, the .OH-modified proteins were proteolytically degraded up to three times faster than untreated alpha-crystallin. ATP stimulated the degradation of native alpha-crystallin and alpha-crystallin which was exposed to 1.6 mol.OH (mol subunit protein)-1 (alpha 1.6). Sixty-seven per cent and 100% of the ATP-dependent degradation of native alpha-crystallin and alpha 1.6 was ubiquitin-dependent, respectively. The data indicate that alpha-crystallins oxidized by .OH are recognized and degraded rapidly by cytoplasmic proteolytic systems in bovine lens epithelial cells. Both ATP-independent and ATP/ubiquitin-dependent proteolytic pathways are involved in the degradation of native and oxidized alpha-crystallin.

Roles for ubiquitin (an 8.5 kDa polypeptide) involve its conjugation to proteins as a signal to initiate degradation and as a stress protein. We investigated ubiquitin conjugation and ubiquitin-dependent proteolytic activities in cultured bovine lens epithelial cells (BLECs) upon oxidative challenge. A 44% decrease in intracellular glutathione confirmed oxidative stress upon incubation with 1 mM H2O2. After 30 min incubation, endogenous high-molecular-mass ubiquitin conjugates decreased 73%, and intracellular proteolysis decreased about 50%. In the supernatants of the oxidatively treated BLECs, the ability to form high-molecular-mass ubiquitin conjugates with exogenous 125I-labelled ubiquitin decreased 28%, and ATP-dependent degradation of oxidized alpha-crystallin decreased 36%. When the H2O2-treated BLECs were allowed to recover for 60 min, intracellular proteolysis returned to the level of control cells. There was also a subsequent transient enhancement of intracellular proteolysis and a simultaneous recovery of endogenous high-molecular-mass ubiquitin conjugates. In parallel cell-free experiments, conjugating activity with exogenous 125I-labelled ubiquitin and ATP-dependent degradation of oxidized alpha-crystallin increased 35% and 72% respectively compared with non-oxidatively treated BLECs. ATP-independent proteolysis showed little response to exposure or removal of H2O2. These results indicate that (1) the rate of intracellular proteolysis in BLECs is associated with the level of endogenous high-molecular-mass ubiquitin conjugates and (2) oxidative stress may inactivate the ubiquitin conjugation activity with coordinate depression of proteolytic capability. Enhancement in ubiquitin conjugation and proteolytic activities during recovery from oxidative stress may be important in removal of damaged proteins and restoration of normal function of BLECs. The inactivation of ubiquitin-dependent proteolysis by oxidation may be involved in the accumulation of altered proteins and other adverse sequelae in the oxidatively challenged aging lens.

In many types of cells, modified proteins are selectively and rapidly removed by various proteolytic systems. In eye lens, as in most cells, there appears to be a multiplicity of proteolytic pathways, including ubiquitin-dependent, ATP-dependent and ATP-independent pathways. Each of these appears to be involved in the degradation of alpha-crystallins. The objective of this study was to determine if oxidized beta- and gamma-crystallins would be selectively degraded and which proteolytic systems might be involved. beta- and gamma-crystallins were oxidized by exposure to 137Cs radiation under N2O. This system generates *OH primarily. Oxidation of beta- and gamma-crystallins was indicated by decreased protein sulfhydryl content and tryptophan fluorescence, as well as by increased levels of carbonyl and high molecular weight aggregates with increasing radiation dose. gamma-crystallin was more susceptible than beta-crystallin to oxidation based on loss of native crystallin, increase in aggregates and fragmentation products, and loss of tryptophan. Low molecular mass polymers (dimers) appear to be the precursors of high mass aggregates induced upon oxidation. At a specific level of oxidative insult interchain covalent bonds in addition to disulfides were more extensive in the polymers of gamma-crystallin as compared to beta-crystallin. Except for beta-crystallin irradiated with 1 krad, the degradation rate of crystallins using both reticulocyte and bovine lens epithelial cells (BLEC) proteolytic systems increased in proportion to the extent of oxidation. Proteolysis of oxidized gamma-crystallins increased 1775% and 900%, respectively, using reticulocytes and BLEC supernatants as the source of proteases.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to prepare human-mouse chimeric antibody against encephalitis type B virus, hybridoma 51-8 cells secreting monoclonal antibody against the virus were used as material for isolating heavy-chain variable region gene of the McAb. High molecular weight DNA of the hybridoma cell was partially digested by BamHI, and then constructed a gene library containing 2 x 10(7) pfu with lambda EMBL-3 as vector. With cDNA of heavy-chain variable region of the monoclonal antibody as probe, nine positive plaques were screened from 360,000 plaques, which have been proven that they contained a fragment of heavy-chain variable region genes by dot hybridization and Southern hybridization. Four recombinants among the nine positive plaques were further distinguished with J11 probe containing J3, J4 and heavy-chain enhancer. After cutting by EcoR1, there was a 3.8 kb fragment in three recombinants as similar to that in liver cell and Sp2/0 cell, but not in the fourth recombinant (lambda 8a4) which contained a 4.5 kb fragment that did not present in liver cell and Sp2/0 cell. The results showed that the inserts in former 3 recombinants were non-rearranged fragment of heavy-chain variable region genes, but the insert in lambda 8a4 contained a rearranged functional variable region gene. The 4.5 kb fragment could not be hybridized with a probe containing J1 and J2, but contained VH, J3 or/and J4 and enhancer, that further proved it was a functional variable region gene. Therefore, the 4.5 kb fragment was isolated and subcloned in pUC 19, and its physical map was made.(ABSTRACT TRUNCATED AT 250 WORDS)

Eight monoclonal antibodies reactive with the matrix protein of human immunodeficiency virus type 1 (HIV-1), p17gag, were isolated from rats which had been immunized with solubilized HIV-1 lysate. The epitope specificities of these antibodies were determined with a series of synthetic peptides representing overlapping regions of p17. Six of the antibodies were mapped to three distinct regions of p17, while two antibodies (G11g1 and G11h3) reacted only with intact recombinant p17, suggesting that they were directed against conformational or discontinuous epitopes. All the antibodies bound to HIV-infected cells which had been permeabilized with acetone, but only G11g1 and G11h3 reacted with live HIV-infected cells. Specificity studies with diverse virus strains demonstrated that these two antibodies recognized distinct epitopes, one which was group specific for HIV-1, and one which was shared with HIV type 2 and simian immunodeficiency virus. Binding competition studies indicated that these epitopes were proximal in native p17. Despite their reactivity with intact cells, these two antibodies did not possess appreciable virus-neutralizing activity. These results indicate that a form of p17 is expressed on the surfaces of live HIV-infected cells which is accessible to some, but not all, antibodies against p17. These cell surface molecules may play a role in the generation of antibodies against p17gag that are characteristic of early stages of HIV infection, and they may act as natural targets for the immune system and as potential targets for immunotherapy of HIV-infected cells.

With the guanidinium isothiocyanate method, total RNA was isolated from hybridoma cells that secrete monoclonal antibody against Brucella melitenses. Poly (A)+ RNA was obtained by oligo (dT)-cellulose affinity chromatography. Reverse transcriptase reaction was performed with a primer 3'A-T-A-G-G-T-G-A-C-C 5' that is complement to the codons of No. 122-125 amino acid residues in 5' terminus of constant region. The size of synthesized ds-cDNA is about 300bp, that is consistent with the length of variable region genes of heavy chain. The ds-cDNA was inserted into plasmid pUC19 with dC: dG tailing method, and the inserted plasmid was used to transform E. coli HB101. It has been proved that the insert was a variable region gene of heavy chain by clone hybridization in situ, size of insert and Southern blot.

A 8.3 μ/ml 6-thioguanine (6TG)-resistant strain was isolated from a rat tetraploid cell line by step-by-step selection in 6TG-medium. In the 6TG-resistant cell population 51% of the cells were tetraploid and 35% of the cells were hypertetraploid, i.e., one chromosome more than a tetraploid. The 6TG-resistant strain grew very well in RPMI 1640 medium with intervals of three days between subcultures. The 6TG-resistant cells all have a homogeneously staining region (HSRs) in one of the X chromosomes which do not stain after chromosome C-banding. They also possess a higher NORs activity and much lower frequency of sister chromatid exchanges (SCE). When the 6TG-resistant RCT cells were subcultured in 6TG-free medium for three days, their SCE frequency did not change. 5'-bromodeoxyuridine (BrdU) significantly suppressed the NORs activity for both 6TG-resistant cells and 6TG-sensitive cells (P<0.001).