v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures.

To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage-independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage-independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development.

Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules.

To establish the role of the biliary epithelium in bile formation, we studied several aspects of biliary physiology in control rats and in rats with ductular cell hyperplasia induced by a 14-d extrahepatic biliary obstruction. Under steady-state conditions, spontaneous bile flow was far greater in obstructed rats (266.6 +/- 51.9 microliters/min per kg) than in controls (85.6 +/- 10.6 microliters/min per kg), while excretion of 3-hydroxy bile acids was the same in the two groups. Infusion of 10 clinical units (CU)/kg per h secretin produced a minimal choleretic effect in controls (+3.8 +/- 1.9 microliters/min per kg) but a massive increase in bile flow in the obstructed animals (+127.8 +/- 34.9 microliters/min per kg). Secretin choleresis was associated with an increase in bicarbonate biliary concentration and with a decline in [14C]mannitol bile-to-plasma ratio, although solute biliary clearance significantly increased. Conversely, administration of taurocholate (5 mumol/min per kg) produced the same biliary effects in control rats and in rats with proliferated biliary ductules. In the obstructed animals, the biliary tree volume measured during taurocholate choleresis (67.4 +/- 15.8 microliters/g liver) was significantly greater than that determined during the increase in bile flow induced by secretin (39.5 +/- 10.4 microliters/g liver). These studies indicate that, in the rat, the proliferated bile ductules/ducts spontaneously secrete bile and are the site of secretin choleresis. Furthermore, because the proliferated cells expressed phenotypic traits of bile ductular cells, our results suggest that whereas under normal conditions the biliary ductules/ducts in the rat seem to contribute little to bile formation, secretion of water and electrolytes is a property of biliary epithelial cells.

FIN13, a novel growth factor-inducible serine-threonine phosphatase which can inhibit cell cycle progression.

We have identified a novel type 2C serine-threonine phosphatase, FIN13, whose expression is induced by fibroblast growth factor 4 and serum in late G1 phase. The protein encoded by FIN13 cDNA includes N- and C-terminal domains with significant homologies to type 2C phosphatases, a domain homologous to collagen, and an acidic domain. FIN13 expression predominates in proliferating tissues. Bacterially expressed FIN13 and FIN13 expressed in mammalian cells exhibit serine-threonine phosphatase activity, which requires Mn2+ and is insensitive to inhibition by okadaic acid. FIN13 is localized in the nuclei of transiently transfected cells. Cotransfection of FIN13-expressing plasmids with a plasmid that expresses the neomycin resistance gene inhibits the growth of drug-resistant colonies in NIH 3T3, HeLa and Rat-1 cells. In transiently transfected cells, FIN13 inhibits DNA synthesis and results in the accumulation of cells in G1 and early S phases. Similarly, the induction of expression of FIN13 under the control of a tetracycline-regulated promoter in NIH 3T3 cells leads to growth inhibition, with accumulation of cells in G1 and early S phases. Thus, overexpression and/or unregulated expression of FIN13 inhibits cell cycle progression, indicating that the physiological role of this phosphatase may be that of regulating the orderly progression of cells through the mitotic cycle by dephosphorylating specific substrates which are important for cell proliferation.

Suppression of v-Src transformation in primary rat embryo fibroblasts.

To understand the mechanism for resistance of primary cultures of rat embryo fibroblasts (REFs) to oncogene-induced transformation, we studied the transforming ability of a recombinant retrovirus, ZSV, containing v-src and neo genes in REFs and in the rat cell line F2408. The susceptibility of REFs to p60v-src transformation was markedly reduced when compared with that of F2408 cells, despite high levels of expression of functional p60v-src tyrosine kinase in the two systems. In hybrid cells obtained by somatic cell fusion between F2408 cells transformed by v-src and uninfected REFs, the transformed phenotype was suppressed despite persistent expression of p60v-src tyrosine kinase. On the other hand, hybrid cells between v-src transformed F2408 cells and uninfected F2408 cells retained the transformed phenotypes. These results indicate that primary cells possess an intracellular function(s) that cause suppression of the transformed phenotype induced by the v-src gene. In ZSV-infected REFs, tyrosine phosphorylation of cellular proteins, including p125 focal adhesion kinase, p70 paxillin and p130 was similar to that in the ZSV-infected F2408 cells, indicating that tyrosine phosphorylation of these proteins is not sufficient for the expression of transformed phenotype. On the other hand, cellular fibronectin and one of integrin receptors were downregulated in the ZSV-transformed F2408 cells but not in ZSV-infected REFs, suggesting that fibronectin and/or its receptor might play a role in suppressing v-src transformation in primary rat cells.

Bile acid structure and bile formation in the guinea pig.

The effects of intravenous infusions (1-4 mumol/min/kg) of 14 bile acids, cholic, deoxycholic, ursodeoxycholic, chenodeoxycholic, dehydrocholic, and their glycine and taurine conjugates, on bile flow and composition and on the biliary permeation of inert carbohydrates have been studied in the guinea pig bile fistula. Hydroxy bile acids were eliminated in bile without major transformation, except for conjugation (over 90%) when unconjugated bile acids were infused. During infusion of dehydrocholate and taurodehydrocholate, 77-100% of the administered dose was recovered in bile as 3-hydroxy bile acids, thus indicating that reduction of the keto group in position 3 was virtually complete. All bile acids produced choleresis at the doses employed: the strongest choleretic was deoxycholate (81.78 microliters/mumol), the weakest was taurodehydrocholate (10.2 microliters/mumol). Choleretic activity was directly and linearly related to bile acid hydrophobicity, as inferred by HPLC, both for similarly conjugated bile acids, and for bile acids having the same number, position, or configuration of the hydroxyl groups. In all instances, the rank ordering was: deoxycholate greater than chenodeoxycholate greater than cholate greater than ursodeoxycholate. During choleresis produced by any of the bile acids tested, bicarbonate concentration in bile slightly declined, but the calculated concentration in bile-acid-stimulated bile (45-57 mmol/l) was always higher than that measured in plasma (23-26 mmol/l). Biliary concentrations of cholesterol (20-68 mumol/l) and phospholipid (14-63 mumol/l) were very low during spontaneous secretion, and declined even further following bile acid choleresis. None of the infused bile acids consistently modified biliary excretion of cholesterol and phospholipid. Consistent with a previous observation from this laboratory, all hydroxy bile acids reversibly diminished [14C]erythritol and [14C]mannitol biliary entry during choleresis, while they increased or failed to modify that of [3H]sucrose and [3H]inulin. The rank ordering for the inhibitory effect on [14C]erythritol and [14C]mannitol permeation was: 3 alpha,7 alpha,12 alpha-trihydroxy greater than 3 alpha,7 alpha-dihydroxy greater than 3 alpha,7 beta-dihydroxy greater than 3 alpha,12 alpha-dihydroxy bile acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of low-dose phenobarbital on hepatic microsomal UDP-glucuronyl transferase activity.

To determine whether hepatic microsomal enzyme induction occurs in rats following administration of phenobarbital at doses similar to those used in humans (0.5 to 7.5 mg/kg), UDP-glucuronyl transferase (UDPGT) and cytochrome P-450 activities were measured in liver homogenate and microsomal preparations from control rats and rats treated for 6 days with phenobarbital at 1 and 3 mg per kg per day. While no significant increases in liver weight and protein content of homogenate and microsomal preparations were observed with either dose of the drug, both UDPGT and P-450 activities were enhanced significantly following administration of phenobarbital at 3 mg per kg per day. The activity of P-450 was increased by approximately 30% and that of UDPGT by 15-24 and 45-66%, respectively, employing bilirubin and p-nitrophenol as the acceptor substrate. The extent of induction of bilirubin or p-nitrophenol UDPGT was similar when measured with "native" enzyme or with enzyme activated by UDP-N-acetyl glucosamine, digitonin or deoxycholate. These data suggest that the discordant effects of phenobarbital on UDPGT and cytochrome P-450 previously reported in humans and rats may not be attributable solely to differences in the drug doses employed.

Comparison of different activators and diazotization procedures in the assay of bilirubin-UDP-glucuronyl transferase activity in rat liver.

The activity of bilirubin-UDP-glucuronyl transferase (UDPGT) in rat liver microsomes was assayed in standardized incubation mixtures employing two different procedures to quantitate formation of conjugated bilirubin: solvent extraction and diazotization with sulfanilic acid, or selective coupling with diazotized ethyl anthranilate at pH 2.7. Both of these procedures yielded equivalent UDPGT activities with several different microsomal preparations, averaging, respectively, 385 and 372 microU protein with untreated microsomes, 646 and 608 microU protein following activation with UDP-N-acetylglucosamine (3.07 mmol/l), and 1370 and 1478 microU protein after treatment with digitonin (0.65%). In rats pretreated with phenobarbital (75 mg/kg per 6 days), a 70-90% increase in UDPGT activity was observed with either diazotization procedure, irrespective of whether 'native', UDP-N-acetylglucosamine-or digitonin-activated enzyme was employed. These studies indicate that, when other conditions are standardized, equivalent information about UDPGT can be obtained with either of two widely employed diazotization reactions and a variety of enzyme activation procedures.

Bile secretion and its control in the newborn puppy.

To gain information about the ontogeny of neonatal biliary physiology, we determined gallbladder bile composition, hepatic bile flow and composition, and the choleretic effects of taurocholate, taurodehydrocholate, secretin, and glucagon in anesthetized puppies of 0-3 (n = 13), 7-21 (n = 9), and 28-42 (n = 8) days of age, and in fed (n = 4) and fasted (n = 4) adult dogs. Although gallbladder bile volume was similar in all puppies, chloride concentration in gallbladder bile declined with age (54.2, 33.2, 22.6, 15.9, and 5.8 mEq/liter in these respective groups), and so did bicarbonate concentration (43.5, 26.1, 17.6, 11.4, and 5.7 mEq/liter). In contrast, the concentrations of sodium (175.4, 189.2, 224.5, 251.6, and 279.4 mEq/liter) and bile acids (77.8, 137.3, 206.2, 219.6, and 280.7 mEq/liter) increased. Spontaneous bile flow rate in 0- to 3-day-old puppies averaged 0.194 microliter/min/g and increased to 0.365 microliter/min/g in puppies of 28-42 days of age. The latter value was not significantly different from that in fed adult dogs (0.344 microliter/min/g). 14C-erythritol bile-to-plasma ratio in spontaneously secreted bile increased with age (1.05, 1.08, 1.26, 1.48, and 1.70), and chloride concentration decreased (96.8, 85.8, 79.2, 74.3, and 60.1 mEq/liter). The choleretic activity of taurocholate (2 mumol/min/kg) was the same in all puppies (7.8 microliters/mumol) and adult animals (7.2 microliters/mumol). Taurodehydrocholate (2 mumol/min/kg) increased bile flow by a greater magnitude, yet its choleretic activity in the puppies (13.3 microliter/mumol) was the same as that in adult dogs (12.7 microliters/mumol). In all animals, total bile acid excretion accounted for 70-95% of the infused taurocholate or taurodehydrocholate.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple procedure to determine the biological titer of recombinant retroviral vectors.

Retroviral vectors are widely used to deliver genetic material to live cells both in experimental and clinical settings. The ability of these vectors to transduce target cells is an important aspect of their clinical applicability and one of the factors determining their transduction efficiency is vector functional titer. Current methods for titrating retroviral vectors involve measuring the number of target cells in culture transduced by a given volume of vector solution. In this report, we describe a new procedure which allows one to estimate the actual number of infectious particles capable of transducing a permissive cell type. Vector biological titer is calculated from the fractional decline in transduction efficiency observed when a given volume of vector solution is sequentially added to multiple dishes containing permissive cells. Values determined this way are greater than those obtained from a single transduction experiment, with the difference being inversely proportional to the degree of cell permissiveness for vector entry. The present procedure is simple, reliable and expeditious. It will be useful to standardize vector biological titers determined in different laboratories, and help implement strategies for efficient gene delivery protocols.

Postnatal changes in hepatic microsomal enzyme activities in the puppy.

The in vitro activities of glucose-6-phosphatase (G6P), UDP-glucuronyl transferase (GT) and P-450 were measured in liver homogenate and/or microsomal suspensions from puppies, 0-42 days of age (n = 26), and adult dogs (n = 3). For each of these enzymes, an age-related increase in the in vitro activity was observed, with the lowest value detected at birth. By the 28th-42nd day of postnatal life, P-450-specific activity was 350 and 85%, G6P 225 and 188%, p-nitrophenol GT 430 and 105% and bilirubin GT 317 and 123% of that seen in 0-hour-old puppies and adults dogs, respectively. The age-related changes in G6P and GT were observed when native enzymes or enzymes activated by deoxycholate or UDP-N-acetylglucosamine (GT) were used. However, the ratio between activated and native p-nitrophenol GT activity decreased as a function of age, and UDP-N-acetylglucosamine failed to activate bilirubin GT in puppies of 0-42 days of age. Total liver protein also increased with age, and hepatic water content was significantly higher in 0- to 42-day-old puppies (76.3%) than in adult dogs (71.4%). Thus, differences between puppies and adult animals were not the same when protein content or enzyme activities were expressed per unit of wet or dried liver weight. Phenobarbital, injected intraperitoneally at 15 mg/kg/day for 6 consecutive days to 8- to 13-day-old puppies (n = 3), produced induction of P-450 (235% of age-matched controls) and bilirubin GT activity (160%), diminished G6P activity (81%), and failed to modify p-nitrophenol GT activity (102%). These studies indicate that, in the puppy, (1) the in vitro activities of P-450, G6P and GT are immature at birth and develop during postnatal life; (2) as in other species, bilirubin and p-nitrophenol may be conjugated in the dog liver by two functionally distinct GT.

Role of ductular bile water reabsorption in canine bile secretion.

To quantitate ductular and/or ductal bile water reabsorption and to define its role in hepatic bile formation, spontaneous and taurocholate-stimulated bile flow and composition were measured in anesthetized dogs postprandially (n = 4) and after a 24-hour (n = 4) and a 48-hour (n = 4) fasting period. Spontaneous bile flow in fed dogs (0.344 +/- 0.116 microliter/min/gm liver tissue) was significantly higher than that in 24-hour (0.179 +/- 0.046 microliter/min/gm) and 48-hour fasted animals (0.096 +/- 0.085 microliter/min/gm). One 48-hour fasted dog was virtually cholestatic (spontaneous bile flow 0.004 microliter/min/gm), and another 48-hour fasted animal had a mean bile flow rate as low as 0.036 microliter/min/gm. Calculated ductular bile water reabsorption (carbon 14-erythritol biliary clearance minus bile flow) was the same in the three groups of animals (0.166 +/- 0.056, 0.208 +/- 0.033, and 0.204 +/- 0.055 microliter/min/gm, respectively). Administration of sodium taurocholate (0.5 to 2.0 mumol/min/kg) revealed intact canalicular secretory capacity in all dogs, and no change in bile water reabsorption rate was observed during choleresis. Bile chloride and bicarbonate concentrations during spontaneous secretion were significantly lower in 48-hour fasted dogs when compared with fed animals. In the two 48-hour fasted dogs with a very low rate of spontaneous bile flow, chloride and bicarbonate concentrations in hepatic bile were as low as those seen in gallbladder bile (5 to 10 mEq/L) and increased during taurocholate choleresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Biliary excretion of inorganic electrolytes: its role in hepatic bile formation.

To define the role of inorganic electrolyte secretion in hepatic bile formation, the effects of secretin, glucagon, and differently structured bile acids on bile flow and composition were studied in the dog, guinea pig, and rat. In the dog and guinea pig, secretin (2.5-10 clinical units X kg-1 X 30 min-1) increased bile flow and bicarbonate concentration in bile, a finding consistent with the hypothesis that the hormone stimulates a bicarbonate-dependent secretion possibly at the level of the bile ductule-duct. In the rat, secretin (5-15 CU X kg-1 X 30 min-1) failed to increase bile secretion. Glucagon (1.25-300 micrograms X kg-1 X 30 min-1) increased bile flow in all the three species, and produced no changes in biliary bicarbonate concentrations in the dog and rat. In the guinea pig, however, glucagon choleresis was associated with an increase in bicarbonate concentration in bile, similar to that observed with secretin. The choleretic activities of various bile acids (taurocholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, and ursodeoxycholic acid, infused at 30-360 mumol X kg-1 X 30 min-1) were similar in the rat (6.9-7.2 microL/mumol), but different in the guinea pig (11-31 microL/mumol). In the latter species, the more hydrophobic the bile acid, the greater was its choleretic activity. In all instances, bile acid choleresis was associated with a decline in the biliary concentrations of chloride, but with no major change in bicarbonate levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Permeation patterns of polar nonelectrolytes across the guinea pig biliary tree.

The biliary permeation of polar nonelectrolytes was studied in anesthetized, bile duct-cannulated guinea pigs with functional cholecystectomy and nephrectomy. During spontaneous secretion, the steady-state bile-to-plasma ratio (B/P) of [14C]urea, [14C]erythritol, [14C]mannitol, [3H]sucrose, and [3H]inulin was 1.02, 0.90, 0.38, 0.12, and 0.04, respectively. Differently structured hydroxy bile acids, but not taurodehydrocholate, reversibly diminished [14C]erythritol and [14C]mannitol B/P during choleresis, and with some of them, particularly taurocholate and glycochenodeoxycholate, the biliary clearance of either solute declined below precholeretic levels. For any given hydroxy bile acid, the degree of B/P diminution was directly related to the molecular radii of these two inert carbohydrates. All bile acids failed to decrease [14C]urea, [3H]sucrose, and [3H]inulin B/P. On the contrary, most of them irreversibly increased [3H]sucrose and [3H]inulin permeability. These results suggest that in the guinea pig 1) hydroxy bile acids diminish the size or rearrange the architecture of the canalicular membrane "aqueous pores" through which [14C]erythritol and [14C]mannitol enter the canaliculus, and 2) solutes of the size of or smaller than [14C]mannitol enter bile primarily through a transcellular route, whereas [3H]sucrose, and [3H]inulin permeate mainly via a transjunctional shunt pathway. These studies indicate that [14C]erythritol and [14C]mannitol cannot be used to estimate canalicular bile flow in this species.

Glucagon induces biliary protein excretion in guinea pigs.

This study was done to determine glucagon's effect on protein biliary excretion in anesthetized, bile duct-cannulated guinea pigs. Glucagon (1.4 nmol.min-1.kg-1) induced choleresis and increased protein biliary concentration from 0.12 +/- 0.04 to 0.20 +/- 0.6 mg/ml and protein output from 22.8 +/- 3.8 to 54.5 +/- 16.1 micrograms.kg-1.min-1. Protein biliary excretion increased during the first 10 min of glucagon infusion and progressively declined thereafter. Biochemical analysis of biliary protein revealed that the increase could be accounted for primarily by an increase in the lysosomal enzymes acid phosphatase and beta-glucuronidase. Biliary excretion of the canalicular membrane enzymes 5'-nucleotidase and alkaline phosphatase only modestly increased, whereas that of [14C]sucrose, a marker of paracellular fluid transport, was unaffected. On the other hand, glucagon enhanced biliary entry of horseradish peroxidase in a fashion similar to that observed with total endogenous protein. These effects were mediated by the adenosine 3',5'-cyclic monophosphate (cAMP) system, since infusion of dibutyryl-cAMP at 0.5 mumol.kg-1.min-1 increased bile flow and biliary protein excretion in a time-dependent manner, as observed with glucagon. Glucagon's failure to sustain enhanced protein biliary output was not due to declining hepatic concentrations of cAMP or to depletion of hepatocellular lysosomal enzymes. These studies provide evidence that glucagon stimulates biliary excretion of protein in guinea pigs that can be accounted for by biliary discharge of enzyme originating from the canalicular membrane and, primarily, from the lysosomal compartment. Although the precise mechanism(s) underlying these effects remains to be elucidated, it is suggested that the increase in canalicular membrane enzyme excretion is due to glucagon's effect on exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Variable transduction efficiency of murine leukemia retroviral vector on mammalian cells: role of cellular glycosylation.

To elucidate the cellular tropism of Moloney murine leukemia virus (MuLV), we have studied the transduction efficiency of a recombinant MuLV vector carrying the beta-galactosidase reporter gene on a variety of rodent cells. Under optimal conditions for in vitro cell transduction, primary cultures of adult rat fibroblasts derived from various organs were very poorly transduced by the ecotropic MuLV vector (0.02-0.12%) when compared to immortalized cells such as the F2408 (42%) and 3Y1 (defined as 100%) lines. Primary cultures of fibroblasts from neonatal (3.7%) or embryonic rat tissues (4.6%) and primary cultures of rat mammary epithelial cells (3-4%) were somewhat more susceptible. Immortalization of rodent fibroblasts with Polyomavirus Large T. SV40 Large T, and E6-E7 genes of human papilloma virus resulted in a modest or minimal increase in transduction efficiency, and introduction of the transforming genes v-Src, v-Ras, and v-Raf was in most instances associated with a decrease in MuLV vector entry. Variability of transduction efficiency was not related to differences in cellular growth rate and treatment of MuLV vectors in vitro with deoxyribonucleoside triphosphates and treatment of cells in culture with protease inhibitors failed to modify cellular entry of the MuLV vector. On the other hand, inhibition of cellular glycosylation with swansonine, 1-deoxymannojirimycin and, primarily, tunicamycin enhanced entry of the ecotropic vector by up to 220-fold, particularly into cells which were otherwise highly resistant. These findings demonstrate major differences in transduction efficiency of the ecotropic MuLV vector on rodent cells and indicate that cellular glycosylation plays a critical role in determining MuLV cellular tropism.

Cellular aging is a critical determinant of primary cell resistance to v-src transformation.

Primary cell cultures are in general resistant to the transforming effect of a single oncogene, a finding considered consistent with the multistage theory of carcinogenesis. In the present studies, we examined whether cellular age, differentiation stage, and/or tissue origin of primary cells plays a role in determining their response to v-src transformation. To study the role of cellular age, rat mammary fibroblasts were isolated from a 50-day-old female rat and infected with a recombinant retrovirus carrying a v-src gene after 2, 7, 14, 21, and 28 days of continuous growth. To determine whether cellular differentiation is important, fibroblasts were isolated from embryos at 12 and 16 days of gestation, from newborns, and from a 30-day-old rat and similarly infected. Finally, the role of primary-cell histogenesis was assessed by infecting primary cultures of fibroblasts isolated from the mammary gland, dermis, and lungs of a mature rat. When compared to 3Y1 cells, all preparations of primary cultures exhibited considerable resistance to v-src transformation. However, whereas primary cells isolated from different tissues responded similarly to the transforming effect of the oncogene, major differences were observed when cells were transduced at different stages of their in vitro life span. v-src was capable of inducing formation of foci and growth in soft agar in early-passage cells but failed to do so in primary cultures infected after 14 days of continuous passaging. Similarly, both the number of foci and the number of colonies in soft agar decreased with tissue donor age. The differential response of young and senescing cells could not be explained by mutations in v-src provirus, by differences in functional v-src expression, or by growth stimulation or suppression via paracrine mechanisms. Furthermore, v-src cooperated with an immortalizing gene, like simian virus 40 large T, polyomavirus large T, E6 and E7 of human papillomavirus, or an activated p53 mutant, to induce anchorage-independent growth of primary cultures but failed to do so with cytoplasmic transforming genes, like v-abl, v-ras, or v-raf, which did not confer indefinite division potential. These studies indicate that cellular aging is a critical determinant of primary-cell resistance to v-src transformation. It is suggested that v-src requires a nuclear auxiliary function for transformation which is present in early-passage cells, particularly when these cells are derived from embryonic tissue, but is lost as cells approach replicative senescence. This auxiliary function is provided by nuclear oncogenes but not cytoplasmic transforming genes.

The intrahepatic biliary epithelium in the guinea pig: is hepatic artery blood flow essential in maintaining its function and structure?

To determine whether hepatic artery blood flow is essential in maintaining the function and structure of bile ductules/ducts, the acute effects of hepatic artery ligation on bile secretion and hepatic ultrastructure were examined in anesthetized, bile duct-cannulated guinea pigs. Sixty minutes after hepatic artery ligation, spontaneous bile flow (5.08 +/- 0.4 microliter per min per gm liver) was virtually the same as that before hepatic artery ligation (5.31 +/- 0.3 microliter per min per gm), as were the choleretic effects of 10 CU per kg per 30 min secretin (7.14 +/- 0.9 vs. 7.21 +/- 0.9 microliter per min per gm), 300 micrograms per kg per 30 min glucagon (6.72 +/- 0.9 vs. 6.59 +/- 0.8 microliter per min per gm) and 60 mumoles per kg per 30 min glycochenodeoxycholate (6.43 +/- 0.6 vs. 6.45 +/- 0.6 microliter per min per gm). The failure of hepatic artery ligation to affect bile secretory function could not be attributed to the existence of collateral arterial blood flow to the liver. First of all, hepatic artery ligation resulted in diminishing significantly hepatic venous, but not portal, oxygen content. More importantly, in isolated guinea pig livers, perfused through the portal vein alone, secretin, glucagon and glycochenodeoxycholate produced changes in bile flow and composition similar to those seen in vivo. Electron microscopy showed no major ultrastructural changes of hepatic parenchyma and biliary epithelium 2 hr after hepatic artery ligation, or 2 hr after perfusing the liver through the portal vein alone save for some portal edema in the latter instance.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile secretory apparatus in the newborn dog: relationship between structural and functional immaturities.

In the dog, bile secretion is not fully mature at birth and develops during postnatal life. To try to establish morphologic correlates to the physiologic deficiencies, we examined the ultrastructure of hepatic parenchyma and biliary epithelium in a newborn puppy and in 3 puppies of 1, 3, and 7 days of age. At birth, the hepatocytes contain much glycogen and fat droplets, a small smooth endoplasmic reticulum and Golgi apparatus, rare autophagic vacuoles, and numerous lysosomes. The sinusoidal microvilli are short, and submembrane vesicles are few and small. The bile canaliculus is not dilated, but few and short microvilli and no pericanalicular vesicles are seen. The biliary epithelial cells are normal in size, but the luminal surface of the bile ductule has no microvilli and numerous blebs. These morphologic features change with maturation and, by the first week of life, the fine structure of the hepatocytes, bile ductular cells, and biliary passages resemble that observed in the adult liver. These findings provide morphologic support for the concept that, in the dog, the bile secretory apparatus is immature at birth and develops during postnatal life.