Thermal stability of lysozyme Langmuir-Schaefer films by FTIR spectroscopy.

Fourier transform infrared spectroscopy has been applied to study the thermal stability of multilayer Langmuir-Schaefer (LS) films of lysozyme deposited on silicon substrates. The study has confirmed previous structural findings that the LS protein films have a high thermal stability that is extended in a lysozyme multilayer up to 200 degrees C. 2D infrared analysis has been used here to identify the correlated molecular species during thermal denaturation. Asynchronous 2D spectra have shown that the two components of water, fully and not fully hydrogen bonded, in the high-wavenumber range (2800-3600 cm-1) are negatively correlated with the amine stretching band at 3300 cm-1. On the grounds of the 2D spectra the FTIR spectra have been deconvoluted using three main components, two for water and one for the amine. This analysis has shown that, at the first drying stage, up to 100 degrees C, only the water that is not fully hydrogen bonded is removed. Moreover, the amine intensity band does not change up to 200 degrees C, the temperature at which the structural stability of the multilayer lysozyme films ceases.

Rescue of impaired angiogenesis in spontaneously hypertensive rats by intramuscular human tissue kallikrein gene transfer.

Angiogenesis represents a compensatory response targeted to preserve the integrity of tissues subjected to ischemia. The aim of the present study was to examine whether reparative angiogenesis is impaired in spontaneously hypertensive rats (SHR), as a function of progression of hypertension. In addition, the potential of gene therapy with human tissue kallikrein (HK) in revascularization was challenged in SHR and normotensive Wistar-Kyoto rats (WKY) that underwent excision of the left femoral artery. Expression of vascular endothelial growth factor and HK was upregulated in ischemic hindlimb of WKY but not of SHR. Capillary density was increased in ischemic adductor muscle of WKY (from 266+/-20 to 633+/-73 capillaries/mm(2) at 28 days, P<0.001), whereas it remained unchanged in SHR (from 276+/-20 to 354+/-48 capillaries/mm(2), P=NS), thus compromising perfusion recovery as indicated by reduced plantar blood flow ratio (0.61+/-0.08 versus 0.92+/-0.07 in WKY at 28 days, P<0.05). In separate experiments, saline or 5x10(9) pfu adenovirus containing the HK gene (Ad.CMV-cHK) or the beta-galactosidase gene (Ad.CMV-LacZ) was injected intramuscularly at 7 days after the induction of ischemia. Ad.CMV-cHK augmented capillary density and accelerated hemodynamic recovery in both strains, but these effects were more pronounced in SHR (P<0.01). Our results indicate that native angiogenic response to ischemia is impaired in SHR, possibly as a result of defective modulation of endothelial cell mitogens. Supplementation with kallikrein, one of the growth factors found to be deficient in SHR, restores physiological angiogenic response utilitarian for tissue healing. Our discoveries may have important implications in vascular medicine for therapeutic benefit.

Role of calcitonin gene-related peptide and kinins in post-ischemic intestinal reperfusion.

The involvement of kinins, calcitonin gene-related peptide (CGRP), and tachykinins during mesenteric post-ischemic reperfusion was studied in anesthetized rats by using antagonists for bradykinin (BK) B1, BK B2, CGRP1, or tachykinin NK1 receptor, or by capsaicin-induced desensitization. B1, B2, or CGRP1 receptor antagonists or desensitization attenuated the transient hypotension and plasma protein and leukocyte infiltration of intestinal wall observed during post-ischemic reperfusion. These effects were abolished by the combination of B2 and CGRP1 blockade as well as by B2 antagonism in capsaicinized rats, while NK1 blockade was ineffective. Our results suggest that kinins and CGRP contribute to systemic vasodilatation and microvascular leakage during mesenteric reperfusion. Pharmacological blockade of these systems could help preventing hypotension and intestinal injury consequent to reperfusion.

Local delivery of human tissue kallikrein gene accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia.

BACKGROUND: Human tissue kallikrein (HK) releases kinins from kininogen. We investigated whether adenovirus-mediated HK gene delivery is angiogenic in the context of ischemia. METHODS AND RESULTS: Hindlimb ischemia, caused by femoral artery excision, increased muscular capillary density (P:<0.001) and induced the expression of kinin B(1) receptor gene (P:<0.05). Pharmacological blockade of B(1) receptors blunted ischemia-induced angiogenesis (P:<0.01), whereas kinin B(2) receptor antagonism was ineffective. Intramuscular delivery of adenovirus containing the HK gene (Ad. CMV-cHK) enhanced the increase in capillary density caused by ischemia (969+/-32 versus 541+/-18 capillaries/mm(2) for control, P:<0.001), accelerated blood flow recovery (P:<0.01), and preserved energetic charge of ischemic muscle (P:<0.01). Chronic blockade of kinin B(1) or B(2) receptors prevented HK-induced angiogenesis. CONCLUSIONS: HK gene delivery enhances the native angiogenic response to ischemia. Angiogenesis gene therapy with HK might be applicable to peripheral occlusive vascular disease.

Participation of kinins in the captopril-induced inhibition of intimal hyperplasia caused by interruption of carotid blood flow in the mouse.

1. In the rat balloon injury model, angiotensin-converting enzyme (ACE) inhibitors prevent vascular remodelling by inhibiting angiotensin II generation and kinin breakdown. We investigated if ACE inhibition also prevents the structural vascular responses to disruption of carotid artery blood flow and if kinin potentiation plays a role in such a protection. 2. Morphometric analysis of the structural alterations caused by ligation of the left carotid artery was performed 14 days after surgery in J129Sv wild-type mice (B(2)(+/+)) drinking normal tap water or water containing captopril (120 mg kg(-1) per day). In addition, the effect of captopril on vascular remodelling was tested in B(2)(+/+) given the bradykinin (BK) B(1) receptor antagonist des-Arg(9)-[Leu(8)]-BK (DALBK, 50 nmol kg(-1) per day, intraperitoneally) or the BK B(2) receptor antagonist D-Arg, [Hyp(3),Thi(5)D-Tic(7),Oic(8)]-BK (icatibant, 1 micromol kg(-1) per day, intraperitoneally), and in B(2) receptor gene knockout mice (B(2)(-/-)). 3. Interruption of blood flow resulted in carotid artery intimal hyperplasia and media thickening in untreated B(2)(+/+), these responses being partially suppressed by captopril. The inhibition of intimal thickening exerted by captopril was reduced in B(2)(+/+) given DALBK or icatibant (P<0.05 for both comparisons) as well as in B(2)(-/-) (P<0.05). Neither antagonism of kinin receptors nor disruption of the B(2) receptor gene altered the suppressive effect of captopril on media thickening. The protection of vascular wall structure was independent of the reduction in blood pressure by captopril. 4. These results demonstrate that kinins participate in the inhibitory effect of captopril on intimal hyperplasia via B(1) and B(2) receptor signalling. Our findings may have important implications in treating vascular remodelling evoked by altered shear stress conditions.

Role of the bradykinin B2 receptor in the maturation of blood pressure phenotype: lesson from transgenic and knockout mice.

The binding of bradykinin (BK) to its B2 receptor results in a wide spectrum of biological effects including vasodilation, smooth muscle contraction and relaxation, pain, and inflammation. In order to gain a better insight into the physiological function of this potent vasoactive peptide, murine models have been created by the use of gene insertion or deletion. The results of studies using these strategies are revisited in the present article. In transgenic mice harboring the human BK B2 receptor cDNA (cHBKR), expression of the transgene was identified in the aorta, brain, heart, lung, liver, kidney, uterus and prostate gland by RT-PCR Southern blot analysis. These mice displayed an exaggerated hypotensive response to intra-aortic injection of BK, whereas the blood pressure of knockout mice, homozygous for targeted disruption of the endogenous gene, was insensitive to BK. Two transgenic mouse lines expressing the human BK B2 receptor showed a significant reduction of systolic tail-cuff blood pressure (84 +/- 1 mm Hg, n = 28; 80 +/- 1 mm Hg, n = 24; P < 0.001) compared with the control littermates (97 +/- 1 mm Hg, n = 52). Systolic blood pressure was elevated in BK B2 receptor knockout mice (124 +/- 1 mm Hg, n = 38). In heterozygous mice, systolic blood pressure was similar to that of controls until 5 month-old, then it raised to the elevated levels of knockout mice at 7 months of age. Together these data indicate that kinins acting through the B2 receptor play a role in the development of the blood pressure phenotype.

Heparin down-regulates the phorbol ester-induced protein kinase C gene expression in human endothelial cells: enzyme-mediated autoregulation of protein kinase C-alpha and -delta genes.

Overexpression of protein kinase C-alpha and protein kinase C-delta has been shown to modulate a number of biological effects, including the cell growth and differentiation. We hypothesized that heparin, a potent antimitogenic drug, could affect the cell proliferation by inhibiting the expression of specific protein kinase C genes. Heparin, markedly but not completely, inhibited the serum-stimulated protein kinase C-alpha and -delta mRNA expression. Protein kinase C inhibition or down-regulation significantly decreased the serum-induced protein kinase C isoenzyme gene expression. Heparin failed to inhibit the residual effect of serum that was resistant to the above-mentioned treatments. Phorbol 12-myristate 13-acetate elicited an increase of protein kinase C isoenzyme gene expression that was completely prevented by protein kinase C inhibition or down-regulation. Heparin dose-dependently counteracted and ultimately abolished the increase in the protein kinase C isoenzyme gene expression elicited by phorbol 12-myristate 13-acetate. These results suggest that the inhibition of an autoregulatory role wielded by protein kinase C on the protein kinase C-alpha and -delta gene expression might represent a possible mechanism by which glycosaminoglycans modulate the cell growth.

Plasma and platelet ascorbate pools and lipid peroxidation in insulin-dependent diabetes mellitus.

BACKGROUND: As diabetes mellitus represents a situation in which production of peroxides is increased, the aim of this study was to investigate the relationship between plasma and platelet levels of ascorbic acid (AA)/dehydroascorbic acid (DHA) and those of malonyldialdehyde (MDA), an indirect marker of lipoperoxides, both assayed using high-performance liquid chromatography (HPLC), in 59 patients with insulin-dependent diabetes mellitus (IDDM) compared with 51 healthy control subjects matched for sex, age, smoking habits, as well as for dietary intake of energy, alcohol and vitamin C. RESULTS: Mean plasma and platelet MDA were significantly higher in the patients affected with IDDM than in control subjects. Moreover, the diabetic group was characterized by a huge decrease in plasma AA [8.45 +/- 5.5 mumol L-1 (SD) vs. 33.4 +/- 7.6 mumol L-1, P = 0.0001], mirrored by a significant increase in plasma DHA (11.9 +/- 3.9 mumol L-1 vs. 3.9 +/- 2.5 mumol L-1, P = 0.0001). No detectable DHA was observed in the platelets from both diabetic and control subjects, whereas AA was significantly increased in platelets from diabetic patients compared with control subjects (42.6 +/- 7.4 vs. 34.8 +/- 5.1 nmol 10(-9) platelets, P = 0.0001). Platelet AA in the diabetic group was significantly inversely correlated with glycated haemoglobin (r = -0.34; P = 0.04) and directly with plasma AA (r = 0.39; P = 0.02), the sum of plasma AA + DHA (r = 0.44; P = 0.009) and with platelet MDA (r = 0.38; P = 0.02). CONCLUSION: (a) The ratio plasma AA/DHA is significantly lowered in IDDM in association with an increase in MDA levels; (b) only AA is detected in platelets, being augmented in the diabetic group; (c) plasma ascorbate depletion does not reflect platelet levels of AA; and, finally, (d) metabolic control, as well as intracellular lipoperoxides, modulates platelet AA in IDDM.

Renovascular hypertension in bradykinin B2-receptor knockout mice.

We evaluated whether kinins exert a protective action against the development of two-kidney, one clip (2K1C) hypertension, a model characterized by an activated renin-angiotensin system in the ischemic kidney and increased expression of the bradykinin (BK) B2 receptor in the contralateral kidney. BK B2-receptor knockout (B2-/-), wild-type (B2+/+), and heterozygous (B2+/-) mice underwent clipping of the left renal artery, with the other kidney remaining untouched. Basal systolic blood pressure (SBP, via tail-cuff plethysmography) was higher in B2-/- mice than in B2+/- or B2+/+ mice (121+/-2 versus 113+/-2 and 109+/-1 mm Hg; P<0.05 for both comparisons). SBP did not change from basal values after sham operation, but it increased in mice that underwent clipping. The increase in SBP was greater in 2K1C B2-/- mice than in B2+/- or B2+/+ mice (28+/-2 versus 14+/-2 and 14+/-2 mm Hg, respectively, at 2 weeks; P<0.05 for both comparisons). Blockade of the BK B2 receptor by Icatibant enhanced the pressure response to clipping in B2+/+ mice (29+/-2 mm Hg at 2 weeks). Intra-arterial mean blood pressure (MBP) was higher in 2K1C than in respective sham-operated mice, with the MBP difference being higher in B2-/- mice (32 and 38 mm Hg, at 2 and 4 weeks, respectively), and higher in B2+/+ mice given Icatibant (30 and 32 mm Hg) than in B2+/+ mice without Icatibant (17 and 18 mm Hg). At 4 weeks, acute injection of an angiotensin type 1 receptor antagonist normalized the MBP of 2K1C hypertensive mice. A tachycardic response was observed 1 week after clipping in B2-/- and B2+/- mice, but this effect was delayed in B2+/+ mice. However, the HR response to clipping in B2+/+ mice was enhanced by Icatibant. Within each strain, heart weight to body weight ratio was greater in 2K1C hypertensive mice than in sham-operated control animals (B2-/-: 5.7+/-0.1 versus 5.2+/-0.1; B2+/+: 5.1+/-0.1 versus 4.5+/-0.1; P<0.01 for both comparisons). The clipped kidney weight to nonclipped kidney weight ratio was consistently reduced in mice with 2K1C hypertension. Our results indicate that kinins acting on the BK B2 receptor exert a protective action against excessive blood pressure elevation during early phases of 2K1C hypertension.

Transferrin and transferrin receptor gene expression and iron uptake in hepatocellular carcinoma in the rat.

Iron plays an important role in cell growth and metabolism. In preneoplastic liver nodules, a rise in the number of transferrin receptors (Tf-R) is associated with decreased endocytosis of the Fe2-Tf/Tf-R complex. Because nodules are precursors of hepatocellular carcinoma (HCC), the question arises whether changes in iron uptake by nodules persist in HCC. Current work showed up-regulation of Tf messenger RNA (mRNA) production in preneoplastic nodules, 12 to 37 weeks after initiation, and down-regulation in atypical nodules (at 45 and 50 weeks) and HCCs, induced in rats by the "resistant hepatocyte" model. Tf-R gene expression increased in nodules and HCCs. Tf-R numbers increased, without changes in affinity constant, in HCC. Iron uptake was higher in HCC than in normal liver, 5 to 40 minutes after injection of 59Fe2-Tf, with preferential accumulation in cytosol of tumor cells and in microsomes of normal liver. Purification through Percoll gradient of mitochondria plus lysosomes allowed the identification in liver and HCC of an endosomal compartment sequestering injected 125I-Tf. This subfraction was not seen when 59Fe2-Tf was injected into rats, and 59Fe was found in particulate material of both tissues. Liver and HCC exhibited comparable basal activities of plasma membrane NADH oxidase, an enzyme involved in iron uptake and cell growth. Stimulation of this activity by Fe2-Tf was higher in HCC than in normal liver. These results indicate that Tf expression may be a marker of preneoplastic liver progression to malignancy. Differently from nodules, HCC may sequester relatively high iron amounts, necessary for fast growth, both through the endocytic pathway and the reduced form of nicotinamide adenine dinucleotide (NADH) oxidase system.

Opioid peptide gene expression in the primary hereditary cardiomyopathy of the Syrian hamster. I. Regulation of prodynorphin gene expression by nuclear protein kinase C.

Prodynorphin gene expression was investigated in adult ventricular myocytes isolated from normal (F1B) or cardiomyopathic (BIO 14.6) hamsters. Prodynorphin mRNA levels were higher in cardiomyopathic than in control myocytes and were stimulated by treatment of control cells with the protein kinase C (PKC) activator 1, 2-dioctanoyl-sn-glycerol. Both chelerythrine and calphostin C, two PKC inhibitors, abolished the stimulatory effect of the diglyceride and significantly reduced prodynorphin gene expression in cardiomyopathic myocytes. Nuclear run-off experiments indicated that the prodynorphin gene was regulated at the transcriptional level and that treatment of nuclei isolated from control cells with 1, 2-dioctanoyl-sn-glycerol increased prodynorphin gene transcription, whereas chelerythrine or calphostin C abolished this transcriptional effect. Direct exposure of nuclei isolated from cardiomyopathic myocytes to these inhibitors markedly down-regulated the rate of gene transcription. The expression of PKC-alpha, -delta, and -epsilon, as well as PKC activity, were increased in nuclei of cardiomyopathic myocytes compared with nuclei from control cells. The levels of both intracellular and secreted dynorphin B, a biologically active product of the gene, were higher in cardiomyopathic than in control cells and were stimulated or inhibited by cell treatment with 1,2-dioctanoyl-sn-glycerol or PKC inhibitors, respectively.

c-myc amplification in pre-malignant and malignant lesions induced in rat liver by the resistant hepatocyte model.

We have investigated by restriction fragment analysis genomic abnormalities involving the c-myc gene in DNA isolated from adenomas and hepatocellular carcinomas (HCCs). Adenomas and HCCs were induced by the "resistant hepatocyte" protocol in diethylnitrosamine-initiated male F344 rats. Southern-blot analysis of EcoRI-restricted DNA from normal liver, early and late adenomas, 12 weeks (EAs) and 30 weeks (LAs) after initiation, and HCCs, showed 2 bands of 18 and 3.2 kb hybridizing with c-myc, in all tissues. c-myc amplification occurred in almost all HCCs, and in the majority of EAs and LAs. These results were confirmed by dilution analysis. c-myc amplification was also seen in adenomas and HCCs by Southern analysis with HindIII-restricted DNA, and in HCCs by differential PCR. c-myc mRNA increase occurred in all adenomas and HCCs, but it was higher in the lesions showing gene amplification. Moreover, a 13-kb DNA extraband, hybridizing with c-myc, was found in the HindIII-restricted DNA from HCCs, but not in normal liver and adenomas, and a 7.1-kb extra band was present in EcoRI-digested DNA from one LA. EcoRI-restricted DNA from some adenomas exhibited a decrease in intensity of the 18-kb fragment, and an increase in intensity of the 3.2-kb fragment. No alteration in banding pattern occurred in the beta-actin gene in adenomas. These results provide evidence of amplification and some other rearrangements involving the c-myc gene, in pre-malignant and malignant liver lesions, induced by the RH protocol, and suggest a role of c-myc rearrangement in the progression of adenomas to malignancy.

The BN rat strain carries dominant hepatocarcinogen resistance loci.

The phylogenetically distant F344 and BN rat strains and their (BN x F344) F1 hybrids were compared for susceptibility to hepatocarcinogenesis using the 'resistant hepatocyte' model. Quantitative stereological analysis of frequency (number/liver) and size (mean volume and volume fraction) of placental form glutathione S-transferase (GST-P)-positive lesions was carried out at 8, 15 and 32 weeks after diethylnitrosamine initiation. The number/liver of GST-P-positive lesions at any time point was slightly higher in BN and (BN x F344) F1 rats than in F344 rats, but not statistically different. However, mean volume and volume fraction of GST-P positive lesions were much higher in F344 than in both BN and (BN x F344) F1 rats at any time point, with a difference of up to > 10-fold. GST-P-positive lesions exhibited a significantly higher labeling index and much lower remodeling in male F344 than in BN and (BN x F344) F1 rats. HCCs were present at 54-57 weeks after initiation in 77% of male F344 and in no (BN x F344) F1 rats and at 70 weeks HCCs were observed in 100% of male F344 and in 23% of (BN x F344) F1 rats. These results suggest that the BN rat strain is resistant to hepatocarcinogenesis and that its resistance is genetically transmitted as a dominant character to F1 hybrids of the BN strain with the F344 susceptible strain.

Correlation between S-adenosyl-L-methionine content and production of c-myc, c-Ha-ras, and c-Ki-ras mRNA transcripts in the early stages of rat liver carcinogenesis.

gamma-Glutamyltranspeptidase (GGT)-positive and glutathione S-transferase (placental-GST-P) positive foci were induced in male Wistar rats by initiation with diethylnitrosamine (DENA), followed by selection and phenobarbital (PB). GGT- and GST-P-positive foci occupied 20-46% and 27-68% of liver parenchyma, respectively, 5-9 weeks after initiation. A high DNA synthesis was found in GGT-positive foci. Decrease in S-adenosyl-L-methionine (SAM) level and SAM/S-adenosylhomocysteine (SAH) ratio, and overall DNA hypomethylation occurred in the liver during the development of enzyme altered foci (EAF). These parameters underwent very small and transient changes in the liver of uninitiated rats at the 5th week, when EAF occupied 0.7-1.4% of the liver. At the 9th week, high RNA transcripts of c-myc, c-Ha-ras, and c-Ki-ras were found in the liver of initiated rats, but not in that of uninitiated rats. Immunohistochemical evaluation of c-myc gene product showed overexpression in GST-P-positive cells. SAM treatment of initiated rats caused inhibition of EAF growth, recovery of SAM/SAH ratio and DNA methylation, and decrease in protooncogene expression proportional to the dose and length of treatment. Liver SAM/SAH ratio was positively correlated with DNA methylation, and negatively correlated with transcript levels of the three protooncogenes. Thus, decrease in SAM/SAH ratio and DNA hypomethylation are early features of hepatocarcinogenesis promotion in rats fed a diet containing adequate lipotrope amounts, paralleled by overexpression of growth-related genes and rapid growth. Re-establishment of a physiologic SAM level makes it possible to inhibit protooncogene expression and EAF growth and to prevent late liver lesion development.

Effect of S-adenosyl-L-methionine on the development of preneoplastic foci and the activity of some carbohydrate metabolizing enzymes in the liver, during experimental hepatocarcinogenesis.

gamma-Glutamyltranspeptidase (GGT)-positive foci and glutathione-S-transferase, placental (GST-P)-positive lesions occupied 36% and 54% of liver parenchyma, respectively, in Wistar rats 8 weeks after initiation with diethylnitrosamine, followed by selection. The administration of S-adenosyl-L-methionine (SAM, 384 mumol/kg/day) caused 77% and 42% falls in the percentage of GGT-positive and GST-P-positive lesions, respectively. There also occurred a 46% decrease in labeling index of GGT-positive foci, in SAM-treated rats. These changes were associated with decrease in liver pyruvate kinase (PK), lactate dehydrogenase and glycerol-3-phosphate dehydrogenase. SAM did not affect these enzymatic activities in normal and uninitiated controls, but it caused a consistent increase in initiated rats. Enolase, fructose-biphosphatase and malic enzyme (ME) activities increased in the liver of initiated rats. SAM did not modify significantly these enzymatic activities, either in control or in initiated rats. Glucose-6-phosphate dehydrogenase (G6PDH) was 113% higher in the liver of initiated rats than in uninitiated controls. SAM treatment did not significantly affect this enzymatic activity in uninitiated rats, but caused a great decrease in initiated ones. As expected, there occurred a marked rise in GGT activity in the liver of initiated rats, with respect to controls. SAM caused an increase in GGT activity in normal and uninitiated controls, but it caused a 77% fall in GGT activity in initiated rats, coupled with a 380% rise in remodeling of GGT-positive lesions. Histochemical determination of G6PDH and ME activities showed that in the absence of SAM many preneoplastic lesions expressed higher G6PDH and ME activities than surrounding liver. SAM did not affect ME-positive lesions, while it caused a decrease in the number of G6PDH-positive lesions. Immunohistochemical determination of PK activity, isoenzyme L, showed a decrease in GST-P-positive lesions. Many of these lesions were no longer recognizable as lesions expressing a low PK activity, in SAM-treated rats. However, a relatively small number of GST-P-positive lesions expressing a low PK activity were still present in these rats. These data suggest that glucose channelled into triacylglycerol and pyruvate synthesis decreases in rat liver, during the development of preneoplastic foci, while the production of reducing equivalents and pentose phosphates increases, thus favoring DNA synthesis and detoxification reactions. Decrease in DNA synthesis, in SAM-treated rats, is paralleled by a partial reversion of carbohydrate metabolic features to those present in normal liver.

Alterations of ornithine decarboxylase gene during the progression of rat liver carcinogenesis.

Liver nodules and carcinomas, developing in F344 rats initiated with diethylnitrosamine, exhibit high ornithine decarboxylase (ODC) activity and DNA synthesis. ODC-related RNAs of 1.8, 2.1 and 2.6 kb are produced by normal rat liver. Early preneoplastic nodules, developing 10 weeks after initiation, showed overproduction of 1.8 and 2.1 kb RNAs, while the 2.6 kb RNA was barely detectable. Rises in the 1.8, 2.1 and 2.6 kb RNAs occur in late nodules (30 weeks after initiation) and in carcinomas. The comparison of different tissues for relative increase in ODC activity, RNA levels and DNA synthesis showed that these parameters behaved in the same way: highest increases occurred in early nodules and carcinomas. These observations suggest that overexpression of ODC gene and alterations in regulatory mechanisms of ODC gene expression may be implicated in the progression of preneoplastic lesions to malignancy. Southern blot analysis of PstI DNA digests revealed the presence of ODC gene rearrangement in two carcinomas and in one late nodule. However, the role of this phenomenon in the progression of preneoplastic lesions is unclear, due to the possibility that ODC pseudogenes are involved instead of or in addition to ODC gene.

Fluorescence cytometry of microtubules and nuclear DNA during cell-cycle and reverse-transformation.

Synchronized CHO-K1 cells and their dibutyryl c-AMP treated counterparts have been characterized by means of static and flow fluorescence cytometry at the level of nuclear DNA and cytoplasmic microtubules. In order to confirm earlier findings on synchronized population, Carnoy fixed and hydrolyzed, several new findings are here reported at the level of single intact cell. The fluorescence intensity of DAPI-stained glutaraldehyde fixed 2C cells correlates well with the average absorbance of the corresponding Feulgen-stained cells, thereby appearing also to be a measure of chromatin condensation during the G1 phase. In the early part of G1, the drastic alteration in anti-beta tubulin immunostaining is shown to parallel microtubule depolymerization induced by calcium or colcemide. The known 1-2 h lengthening of the G1 period after reverse-transformation appears to correlate with a similar delay in the abrupt chromatin decondensation. The above results are discussed in terms of the role of microtubules and nuclear morphometry (and their coupling) in the control of cell cycle progression of transformed vs. fibroblast-like cells.

Comparative effects of L-methionine, S-adenosyl-L-methionine and 5'-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.

Male Wistar rats, initiated with diethylnitrosamine (DENA), were subjected to a selection treatment, according to the "resistant hepatocyte" model, followed or not followed by phenobarbital (PB). Rats received, for 3 weeks after selection, 4 i.m. doses (96 mmol/kg) of L-methionine, S-adenosyl-L-methionine (SAM), or 5'-methylthioadenosine (MTA), a SAM catabolite formed during polyamine synthesis or by spontaneous splitting of SAM at physiologic temperature and pH. They were then killed. In some rats, SAM and MTA treatments were started 20 weeks after initiation. The animals were killed 3 weeks later and persistent (neoplastic) nodules (PN) were collected. Some rat groups received 1/2 and 1/4 of the above SAM and MTA doses, or 1/8 of the above MTA dose. SAM and MTA, but not methionine, caused a dose-dependent decrease in number and surface area of gamma-glutamyltranspeptidase (GGT)-positive foci, and in labeling index (LI) of focal cells, coupled with remodeling. SAM and MTA liver contents, SAM/S-adenosylhomocysteine (SAH) ratio and overall methylation of liver DNA were low during the development of GGT-positive foci. SAM, but not methionine, caused a dose-dependent recovery of SAM content and DNA methylation, and a partial reconstitution of liver MTA pool. Exogenous MTA only induced the reconstitution of MTA pool, without affecting SAM level and DNA methylation. Recovery of SAM and MTA pool and DNA methylation was found in the rats subjected to SAM plus MTA, indicating the absence of inhibition of DNA methyltransferases in vivo by MTA. MTA also inhibited liver reparative growth in partially hepatectomized rats, without modifying SAM content and DNA methylation of regenerating liver (RL). A high activity of ornithine decarboxylase (ODC) was found in the liver, during the development of preneoplastic foci, and in PN. This activity was inhibited by SAM and MTA treatments. Although MTA was more effective than SAM, the decrease in ODC activity was coupled with a larger fall in DNA synthesis in SAM-treated than in MTA-treated rats. Thus the antipromotion effect of SAM could not merely depend on its (spontaneous) transformation into MTA. Although MTA production may play a role in the SAM antipromotion effect, other mechanisms could be involved. A role of DNA methylation in the inhibition of growth by SAM is suggested. MTA is a potential chemopreventive agent for liver carcinogenesis.

Effects of lectins on cytoskeleton and morphology or cultured chick embryo fibroblasts.

The microfilaments and microtubules of cultured chick embryo skin fibroblasts were studied in the presence of exogenous lectins by an indirect immunofluorescence technique. Lectin treatment induced modifications in the arrangement of myosin, actin and tubulin, determined depolymerization of the proteins and caused changes in cell shape and size. The results suggest that the interaction between lectins and their specific membrane receptors triggers a series of changes in the cytoskeletal pattern via transmembrana as yet unknown mechanisms and that these are responsible for the alterations in cell shape and size.