The dorsomedial hypothalamic nucleus revisited: 1998 update

This article reviews data that have accumulated since the early 1970s on the role of the dorsomedial hypothalamic nucleus (DMN) in neuroendocrine and autonomic homeostasis. Both the ventromedial hypothalamic nucleus (VMN) and the lateral hypothalamic area (LHA) project to the DMN, which in turn projects to the paraventricular nucleus of the hypothalamus (PVN), thus placing the DMN at an important nodal point of neuroendocrine/autonomic circuitries. The DMN is composed of cells and fibers containing neuropeptide Y (NPY), and the nutritional status (starvation-refeeding) is reflected in NPY levels of both VMN and DMN in Sprague-Dawley, Zucker (fa/fa), and corpulent rats (cp/cp JCR:LA). The DMN is involved in the final common pathway of corticotrophin-releasing hormone (CRH) secretion by the PVN, sympathetic nervous system outflow to the adrenal gland, and brown adipose tissue (BAT) thermogenesis. The DMN is also part of a "fear circuitry" regulating cardiovascular responses to stress such as myocardial blood flow and the tachycardia associated with the defense reaction. This appears to be mediated by a gamma amino butyric acid (GABA) mechanism. Although exhibiting reduced ponderal and linear growth and hypophagia and hypodipsia, the rat with DMN lesions (DMNL rat) has normal body composition, anabolic hormone levels, and intermediary metabolism, and it responds normally to numerous endocrine, nutritional, intra- and extracellular thirst and body weight-regulatory challenges. The DMNL rat shows normal efficiency of food utilization, but shows an attenuated response to the feeding-stimulatory effect of insulin. The only other lesion-induced abnormalities are hyperprolactinemia and a disrupted circadian corticosterone rhythm. The hyperprolactinemia in DMNL rats appears to be related to an attenuation of dopamine (DA). Rats with DMNL are capable of mating and can bear offspring, but there is a dramatic effect on litter size and other litter parameters that only improves when one parent is a DMNL rat. Antiaging effects produced by DMNL are evident in the prevention of age-associated microalbuminuria and kidney lesions, as well as, in prevention of the age-related decline in circulating insulin-like growth factor I (IGF-I). Recent evidence suggests that DMN, together with the VMN and the arcuate nucleus (ARC) of the hypothalamus, may be part of the circuitry that is responsive to the feedback signal from adipose tissue by the hormone leptin. The above findings and others suggest that the DMN plays a diverse role in physiological regulatory processes.     

Peroxisomal fatty acid oxidation capacity is more resistant to ischaemic and reperfusion injury than mitochondrial fatty acid oxidation capacity in feline hearts

Mitotic recombination induced by six alkylating agents has been studied in the wing-spot test of Drosophila melanogaster. The model mutagens chosen have different models of action at the DNA level. These are: the direct-acting small alkylating agent methylmethanesulfonate (MMS), the small promutagens N-dimethylnitrosamine (DMN) and N-diethylnitrosamine (DEN), the bifunctional cross-linking alkylating agents mitomycin C (MMC), chlorambucil (CLA) and monocrotaline (MCT). Flies of the standard cross (flr3 / TM3, Bds females and mwh males) were used to produce the larvae to be treated. Three-day old Drosophila larvae were exposed by chronic feeding for 48 h to three different concentrations of all six alkylating agents. Acute feeding for only 2 h was used in addition with DEN and MMC. Wings of the marker-heterozygous (mwh+ / + flr3) as well as of the balancer-heterozygous (mwh+ / TM3, Bds) progeny were analysed. The ranking of the compounds with respect to their genotoxic potency, based on mwh clone formation frequency in marker-heterozygous wings was: MMS > MNC > DMN > CLA approximately MCT > DEN. The ranking with respect to the induction of twin spots, which are produced by mitotic recombination exclusively, was: MMS > DMN > MMC > MCT > CLA > DEN. The quantitative determination of recombinagenic activity, based on mwh clone formation frequencies obtained in both types of wings, gave the following values: MMS, 93%; MCT, 87%; CLA, 80%; MMC, 73%; DMN, 67%; DEN, 22%. A clear relationship exists between the extent of N-alkylation of DNA and the efficiency of the monofunctional agents MMS and DMN as well of the bifunctional agents MCT, CLA and MMC to induce mitotic recombination. This contrasts with the ethylation of base oxygen atoms and the resulting lower efficiency of DEN to produce mitotic recombination.     

Estimation of resistance to dimethylnitrosamine of normal hepatocytes, hyperplastic nodule cells and hepatoma cells of rats in primary culture

Normal hepatocytes, hyperplastic nodule cells and hepatoma cells of rats induced by 2-acetylaminofluorene treatment were transferred to primary culture. After exposure of the cells to dimethylnitrosamine (DMN) at concentrations of 10(-2) M, 10(-3) M, and 10(-4) M for 48 hr, the attached cells which remained on the dishes in a flattened shape were counted. Resistance to DMN was expressed as the percentage of the attached cells relative to the control without DMN treatment. The percentages increased in the order of normal hepatocytes, hyperplastic nodule cells and hepatoma cells.     

Intracerebroventricular CRF inhibits cold restraint-induced c-fos expression in the dorsal motor nucleus of the vagus and gastric erosions in rats

Acute exposure to cold-restraint induces vagal-dependent gastric erosions associated with activation of neurons in the dorsal motor nucleus of the vagus (DMN) in rats. The influence of intracerebroventricular (i.c.v.) injection of corticotropin-releasing factor (CRF) (10 micrograms) on c-fos expression in the brain and gastric erosions induced by 3 h cold-restraint was investigated in conscious rats. In cold-restraint exposed rats, CRF injected i.c.v. inhibited gastric erosions and the number of Fos positive neurons in the DMN by 93 and 72%, respectively, while Fos labelling in the nucleus tractus solitarius (NTS) was increased by 5-fold compared with vehicle group. c-fos expression was also induced in the central amygdala by i.c.v. CRF, unlike the vehicle-injected group exposed to cold-restraint. c-fos expression induced by cold-restraint in the raphe pallidus (Rpa) and paraventricular nucleus of the hypothalamus was not altered by i.c.v. CRF. These data indicate that central CRF-induced gastric protection results from the inhibition of DMN neuronal activity enhanced by cold-restraint. CRF action on DMN neurons may be related to the increase in the NTS and central amygdala inputs leading to inhibition of DMN neurons rather than to the decrease in the excitatory input from the caudal raphe projections to the DMN.     

Right ventricular diastolic filling: evaluation with velocity-encoded cine MRI

BACKGROUND AND PURPOSE: It has been reported that some angiotensin converting enzyme inhibitors can prevent stroke-prone spontaneously hypertensive rats from stroke at much higher doses than clinical doses used for hypertension therapy. This study was performed to investigate the prophylactic effectiveness of imidapril against stroke in comparison with enalapril. METHODS: Salt-loaded stroke-prone spontaneously hypertensive rats were orally given imidapril (0.5, 1, 2, and 5 mg/kg per day), enalapril (2 and 5 mg/kg per day), or hydralazine (5 mg/kg per day). Stroke signs were scored, and blood pressure, protein concentration, and N-acetyl-beta-D-glucosaminidase activity in urine were measured. After 2 weeks of medication, angiotensin converting enzyme activities in the aorta were measured 24 hours after dosing. RESULTS: In the control group, severe hypertension developed, and all rats died within 12 weeks because of stroke. Imidapril and enalapril dose-dependently decreased the stroke-related mortality, and both agents at 5 mg/kg per day showed excellent prophylaxis, although they did not inhibit hypertensive development. Imidapril at 0.5 mg/kg per day significantly prevented stroke to almost the same extent as enalapril at 2 mg/kg per day or hydralazine at 5 mg/kg per day. Imidapril dose-dependently suppressed the elevation of the two urinary indexes, which was followed by stroke. Imidapril inhibited enzyme activity in the aorta more strongly than did enalapril at the same dose. CONCLUSIONS: Imidapril prevented the incidence of stroke in stroke-prone spontaneously hypertensive rats at a dose of 0.5 mg/kg per day or more by amelioration of kidney dysfunction. Reduction of blood pressure is not necessary, although enzyme inhibition in the vasculature may partly relate to the effect.     

Hydrocortisone induces an increase of amylase content in individual zymogen granules from rat pancreas

This study was performed to evaluate the effects of different doses of hydrocortisone (1, 10 and 25 mg/kg/day) administered for 1, 3 and 8 days on pancreatic enzyme storage in rats. The enzyme content in both pancreas homogenates and in individual isolated zymogen granules (ZGs) was measured using standard biochemical assays and flow cytometry, respectively. Hydrocortisone did not alter the total amount of pancreatic DNA but increased the pancreas enzyme content in a time-dose-dependent way. Amylase activity was significantly increased after hydrocortisone administration at day +8 when 10 mg/kg/day was used, and from the first day of treatment when 25 mg/kg/day was administered. A significant increase in trypsin activity was also observed in response to 25 mg/kg/day of hydrocortisone but only from the third day of treatment onwards. As compared with control rats, chronic administration of either 1 or 10 mg/kg/day of hydrocortisone did not alter significantly either the size or the percentage of the two ZG subpopulations (Z1 and Z2) identified in the pancreas by flow cytometry; in addition, no significant changes were observed in the mean amylase content per individual granule, although its mean concentration increased in rats treated with 10 mg/kg/day for 3 and 8 days. Nevertheless, when 25 mg/kg/day of hydrocortisone were administered for 1 and 3 days, a significant increase in the proportion of Z1 ZGs was observed, which may be related to the formation of new and smaller ZGs. When a very high dose of hydrocortisone (25 mg/kg/day) was used, an overall increase in the pancreatic enzyme content related to an increase in the mean amylase content per individual ZG was observed; this effect was apparent from the first day of treatment in the Z1 subset of ZGs and from day +3 in the Z2 subpopulation. Only a high concentration of hydrocortisone was able to alter the enzyme storage process in individual zymogen granules, but they maintain a normal enzyme load at lower hydrocortisone doses.     

Phase II study of irinotecan and etoposide in patients with metastatic non-small-cell lung cancer

Melatonin production in the chick pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT; EC 2.3.1.87), the penultimate enzyme in melatonin synthesis. In contrast to the external regulation of pineal rhythms in mammals by the suprachiasmatic nucleus, rhythmic changes in AA-NAT activity in cultured chick pineal cells are controlled by an oscillator located in the pineal cells themselves. Here we present evidence that the chick pineal clock generates a rhythm in the abundance of AA-NAT mRNA in cultured cells that parallels the rhythm in AA-NAT activity. In contrast, elevating cAMP by forskolin treatment markedly increases AA-NAT activity without producing strong changes in AA-NAT mRNA levels, and lowering cAMP by norepinephrine treatment decreases enzyme activity without markedly decreasing mRNA. These results suggest that clock-controlled changes in AA-NAT activity occur primarily through changes at the mRNA level, whereas cAMP-controlled changes occur primarily through changes at the protein level. Related studies indicate that the clock-dependent nocturnal increase in AA-NAT mRNA requires gene expression but not de novo protein synthesis, and that AA-NAT mRNA levels are suppressed at all times of the day by a rapidly turning over protein. Further analysis of the regulation of chick pineal AA-NAT mRNA is likely to enhance our understanding of the molecular basis of vertebrate circadian rhythms.     

Recovery of blood mononuclear cell calcineurin activity segregates two populations of renal transplant patients with different sensitivities to cyclosporine inhibition

In vitro studies have shown that the immunosuppressive property of cyclosporine (CsA) depends on its ability to inhibit the phosphatase activity of calcineurin, a critical enzyme for T cell activation. Here we sought to investigate whether measurement of calcineurin activity in peripheral blood mononuclear cells (PBMC) from 30 renal transplant patients given CsA as a part of their immunosuppressive regimen would help in optimizing CsA therapy. We first documented that in PBMC from these patients complete inhibition of calcineurin phosphatase activity by in vitro addition of CsA occurs at concentrations that are easily achieved in vivo for a dose as low as 3 mg/kg/day orally, which corresponds to trough CsA blood levels of 100-150 ng/ml. However, ex vivo, at a blood CsA trough level of 250 ng/ml, calcineurin activity in PBMC was only inhibited from 40% to 70% as compared with controls. Patients on higher doses of CsA had a further inhibition of baseline calcineurin activity, although a complete suppression was never reached. A significant correlation was found between trough CsA concentration and the basal calcineurin activity (r=0.48; P=0.0085). To clarify the relationship between the daily exposure of patients to CsA and changes in the enzyme activity of calcineurin, we then correlated the pharmacokinetic profile of CsA in these patients with different CsA dosing (<4, 4-6, >6-8, >8 mg/kg/day) with the profile of calcineurin activity at different intervals from dosing. Each of the above CsA doses suddenly reduced calcineurin activity, with a nadir at 2 hr after maximum blood concentration. The degree of the inhibition was not a function of peak CsA blood levels. In all patients, CsA blood level returned to basal values 10 hr after dosing. By contrast, only in 50-70% of patients (depending on the dose) did calcineurin activity return to baseline at the same time point after dosing. In summary we have shown that (1) inhibition of calcineurin activity measured ex vivo in PBMC taken from CsA-treated transplanted recipients reflects the blood CsA trough level; (2) after CsA the time-course of inhibition of enzyme activity is relatively independent from CsA pharmacokinetics; (3) the rate of recovery of calcineurin activity 10 hr after CsA dosing segregates two populations of transplanted recipients -- one with complete recovery of the enzyme activity and another that never returns to the baseline calcineurin level.     

Response to therapy in demyelinating motor neuropathy

We reviewed results of immunotherapy in patients with demyelinating motor neuropathy (DMN), and found that patients over 50 years of age at onset responded poorly, and younger patients responded variably to intervention. We suggest that patients with DMN be given a guarded prognosis, particularly if >50 years of age at onset.     

Early investigations on the effect of methyl mercuric chloride upon DMN-acute hepatotoxicity

Acute toxicity induced by DMN was partially prevented by previously administering methyl mercuric chloride (MMC), a chemical inhibitor of the drug metabolizing enzyme system (DMES). We have studied the early changes occurring during the course of DMN-intoxication, namely disaggregation of polysomal profiles and necrosis, evaluated morphologically and by the release of S-GPT.     

A short-term assessment of tumor-promotion activity in the livers of rats treated with two genotoxic methylating agents: dimethylnitrosamine and methylnitrosourea

Induction of replicative DNA synthesis (RDS) and mitoinhibitory effects were studied in the hepatocytes of F344 rats exposed in vivo to the methylating agents dimethylnitrosamine (DMN, hepatocarcinogen) and methylnitrosourea (MNU, non-hepatocarcinogen). Cytotoxicity and chromosome aberrations (CA) in rat liver were also investigated to clarify the cause of changes in RDS and mitoinhibitory effects, respectively. The animals were killed at different intervals (up to 14 days) after a single oral dose, or 1 day after 7 or 14 days of repeated oral doses. The hepatocytes were isolated and cultured with Williams' medium E to assess their RDS, mitoinhibitory effects and CA. Mitoinhibitory effects were investigated by monitoring their effect on epidermal growth factor-induced replicative DNA synthesis (EGF-induced RDS) in rat hepatocytes. Hepatotoxic effects were assessed by measuring aspartate transaminase and alanine transaminase in the plasma and by histopathological examination. In the single-dose study, DMN (20 mg/kg body weight (bw)) induced both RDS and hepatotoxicity. MNU (50 mg/kg bw) induced RDS without causing hepatotoxicity, and thus was classified as a mitogen. In the repeated-dose study, DMN (4 mg/kg bw) induced both RDS and hepatotoxicity, but MNU (10 mg/kg bw) induced neither. Both inhibition of EGF-induced RDS and induction of CA were observed in the hepatocytes of rats treated with DMN, but were not observed with MNU in both single and repeated dose studies. The mitoinhibitory effect of DMN persisted for 14 days after the single dose and time dependently increased for 14 days after repeated administration. This mitoinhibitory effect correlated positively with CA. The mitoinhibitory effect was thought to be attributable to the DNA-damaging effect that induces CA. We concluded that the differences which we found in this study between DMN and MNU contribute to the differences in their hepatocarcinogenicity. Our findings suggested that both cell proliferative and mitoinhibitory properties play an important role in tumor promotion, and measuring them may provide an ancillary index that is useful in predicting hepatocarcinogenicity.     

Alpha-Hydroxylation of lignoceric and nervonic acids in the brain. Effects of altered thyroid function on postnatal development of the hydroxylase activity

Rat brain postnuclear preparations catalyzed the alpha-hydroxylation of nervonic acid with an apparent Km of 3 muM. Evidence has been presented which suggests that nervonic acid in the brain is hydroxylated by the same enzyme system which hydroxylates lignoceric acid. The hydroxylase activity in brains of normal (euthyroid) rats increased rapidly from a low in the period immediately following birth to a maximum at the 23rd day and then declined to a low level characteristic of the mature brain. Neonatal hypothyroidism retarded the development of the activity and shifted its peak to the 39th day after birth. Conversely, neonatal hyperthyroidism accelerated the entire developmental pattern and shifted the peak to the 16th day after birth. The hydroxylase activity in mouse brain was also increased by thyroid hormone administration from the 13th through the 18th day after birth. Unlike normal mice, the low activity in jimpy mice was not affected by this treatment. It is concluded that thyroid hormones play an important role in the control of brain fatty acid alpha-hydroxylation. The stimulation of alpha-hydroxy fatty acid synthesis in response to hyperthyroidism during the early postnatal period may be one of the major effects of thyroid hormones in accelerating myelination of the central nervous system.     

Protein phosphatase-1 and -2a activities in cultured fetal chick neurons: differential regulation by insulin and insulin-like growth factor-I

In this study, we examined the developmental expression and regulation by insulin and insulin-like growth factor-I (IGF-I) of protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) in cultured fetal chick neurons. Protein phosphatase activities were measured using 32P-labeled phosphorylase-a or 32P-labeled S6 kinase substrate peptide. In cell extracts from day 1-5 cultures, 40-45% of spontaneous protein phosphatase activity was due to PP-1. PP-2A accounted for the remaining 55-60% of enzyme activity. Spontaneous PP-1 activity increased by 100% in day 2 cultures and remained constant thereafter. PP-2A activity increased by 48% in day 2 cultures, with minimal increases in enzyme activity in later cultures. Under the assay conditions employed, at all times in culture a significant proportion (45-50%) of PP-1 was in an inactive form that could be reactivated by trypsin. PP-2A activity was not influenced by trypsin. Insulin stimulated neuronal PP-1 activity in day 4 and 5 cultures, but had no effect in earlier cultures. The activation of PP-1 by insulin was rapid, with a maximal effect (30-40% increase over basal levels) at 5 min with 10 ng/ml insulin. Insulin did not alter total (trypsin-released) PP-1 activity, the content of PP-1 catalytic subunit, or PP-2A activity at any time in culture. In contrast to insulin, IGF-I had no effect on PP-1 activity at any time in culture, but significantly increased PP-2A activity in day 5 cultures. Maximal stimulation of PP-2A activity by IGF-I was observed at 10 min, with an EC50 of 5 ng/ml. These results indicate that chick forebrain neurons contain both PP-1 and PP-2A activities and that neuronal PP-1 and PP-2A activities are differentially regulated by insulin and IGF-I. We conclude that although insulin and IGF-I share many steps in signal transduction, these growth factors have distinct actions on neuronal phosphatase activity that may impact on differences in their neurotropic actions.     

Drug metabolism in hepatocyte sandwich cultures of rats and humans

Adult hepatocytes from rat and man were maintained for 2 weeks between two gel layers in a sandwich configuration to study the influence of this culture technique on the preservation of basal activities of xenobiotic-metabolizing phase I and phase II enzymes. The response of these enzyme activities to an enzyme inducer was investigated using rifampicin (RIF). Basal levels of cytochrome P-450 (CYP) isozymes were characterized by measuring ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and the specific oxidation of testosterone (T). In hepatocytes from untreated rats, CYP isozyme levels, including the major form CYP 2C11, increased during the first 3 days in culture. After this period of recovery, the levels of CYP 2C11, CYP 2A1, and CYP 2B1 decreased, whereas CYP 3A1 increased. In contrast to these dynamic changes, CYP activities such as CYP 1A2 and the major isozyme CYP 3A4 were largely preserved until day 9 in cultures of human hepatocytes. In measuring phase II activities, a distinct increase in glucuronosyltransferase (UDP-GT) activity toward p-nitrophenol (PNP) was found for rat and human hepatocytes over 2 weeks in culture. Sulfotransferase (ST) activity toward PNP showed an initial increase, with a maximum at day 7 and day 9 in culture, respectively, and then decreased until day 14. Glutathione S-transferase (GST) activity decreased constantly during the time of culture. Effects of the enzyme-inducing drug rifampicin on phase I and phase II enzymes were investigated using cultured human hepatocytes. Rifampicin treatment (50 micromol/L) for 7 days resulted in a 3.7-fold induction of CYP 3A4 at day 9 in culture. ECOD activity was increased sixfold and phase II ST activity increased twofold compared to the initial value at day 3. No effect of rifampicin on CYP 3A was found in cultures of rat hepatocytes. These results demonstrate that rat and human hepatocytes preserve the major forms of CYP isozymes and phase II activities and respond to inducing drugs such as rifampicin. The novel hepatocyte sandwich culture is suitable for investigating drug metabolism, drug-drug interactions and enzyme induction.     

N-acetylglucosaminyltransferase regulates the expression of neolactoglycolipids including sulfoglucuronylglycolipids in the developing nervous system

Lactosylceramide N-acetylglucosaminyltransferase (GlcNac-Tr) in the synthesis of lactotriosylceramide (LcOse3Cer) was characterized in the nervous system. The microsomal membrane GlcNAc-Tr required a divalent metal ion, preferably Mn2+, and a nonionic detergent. The pH optimum was around 7.0. The enzyme also transferred GlcNAc to neolactotetraosylceramide (nLcOse4Cer), GM1, and asialo-GM1, but not to other glycolipids. The Km value for lactosylceramide was 21 microM (Vmax = 91 pmol/mg/h), and that for nLcOse4Cer was 35 microM (Vmax = 112 pmol/mg/h). The GlcNAc-Tr for the glycolipids appears to be separate from that for oligosaccharides. The developmental expression of GlcNAc-Tr, both in the cerebral cortex and cerebellum, correlated well with the tissue levels of LcOse3Cer, nLcOse4Cer, sulfoglucuronylglycolipids (SGGLs), and other neolacto series glycolipids (nLSGs). In the cerebral cortex, the specific activity of GlcNAc-Tr decreased sharply from a maximum level at embryonic day 15, and by postnatal day 10 onward, it was undetectable. In the adult cerebral cortex, although significant activities of other glycosyltransferases involved in the subsequent steps of the synthesis of SGGLs were present, the absence of GlcNAc-Tr stymied the formation of LcOse3Cer and therefore the synthesis of nLSGs, including SGGLs. In the cerebellum, the GlcNAc-Tr specific activity declined from the day of birth to postnatal day 3, but later, the activity increased and reached a maximum at postnatal day 15, which correlated with the increasing synthesis of nLSGs. The results indicate that lactosylceramide GlcNAc-Tr is the key regulatory enzyme controlling the differential expression of all nLSGs in the developing nervous system.     

Establishment of regional differences in brush border enzymatic activities during the development of fetal mouse small intestine

In order to study the establishment of regional differences in brush border enzymic activities during the development of fetal mouse small intestine we have followed (1) the differentiation of microvilli by morphometry, and (2) the developmental pattern of three brush border enzymes (lactase, glucoamylase and alkaline phosphatase). From day 16 to day 19 of gestation, the height of duodenal microvilli increases 2.4 times on the absorptive cells located near the tip of the villi. During the same period in the upper half of the duodenal villi, the number of microvilli per square micrometer rises by a factor of 2.4 and the microvillous surface area increases by a factor of 5.2. The differentiation of ileal microvilli follows a similar pattern but they are always shorter and less numerous than those of the duodenum. Lactase activity appears at 18 days of gestation; the other two brush border enzymes are first detected at 16 days of gestation. Afterwards all three enzyme activities increase rapidly and a decreasing gradient of activity is established from the proximal to the distal segment of the small intestine. Hence, the structural development of the microvilli and the appearance of brush border enzyme activities occur simultaneously and a proximo-distal gradient is already established at 16 days of gestation.     

Radiology of skeletal tuberculosis

The process of hepatic fibrosis, and the changes in contents of hepatic hyproxyproline (HYP) and serum procollagen type III peptide (PIIINP) were examined in two rat models for hepatic fibrosis, i.e. bile duct ligation/scission (BDL/s)- and dimethylnitrosamine (DMN)-induced models. In addition, an expression of type III collagen mRNA in the liver of BDL/s model was also examined. In BDL/s model, hepatic fibrosis started at 2 weeks after operation (WAO) and cirrhosis with prominent bile duct hyperplasia was detected at and after 5 WAO. Serum PIIINP content measured using a modified double armed inhibition enzyme-linked immunosorbent assay (ELISA) method proposed by us started to increase at 1 WAO and continued to increase thereafter. Hepatic HYP content measured colorimetrically started to increase at 3 WAO and it continued to increase until 7 WAO. An expression of type III collagen mRNA in the liver was enhanced at and after 2 WAO, especially at 4 and 5 WAO. In DMN model, marked hepatic fibrosis was detected at 1 week after the last DMN administration (WAA), and the degree of fibrosis was apparently reduced at 4 WAA. Serum PIIINP content prominently increased at 1 WAA and decreased at and after 3 WAA. Hepatic HYP content showed a marked increase at 1 WAA and decreased thereafter. The present results indicated that the sequences of hepatic fibrosis, hepatic HYP content and serum PIIINP content were well correlated with each other in both BDL/s and DMN models. In conclusion, ELISA system for the detection of serum PIINP content is considered to be reliable method for assessment of cirrhotic liver, and the present two rat models for liver fibrosis/cirrhosis seems to be a good tool for researching antifibrotic agents.     

Studies on the mechanism of dimethylnitrosamine-induced acute liver injury in mice

Male and female adult C3H- +/+, C3H-gld/gld.lpr/lpr (gld.lpr) and CBA-lprcg/lprcg (lprcg) mice were given a single i.p. dose of 30 mg/kg dimethylnitrosamine (DMN). Liver tissues were collected from mice killed 6, 12, 24 and 36 hrs post treatment, and the progression of the lesions was characterized morphologically and by the TUNEL method. DMN induced centrilobular hepatic injury accompanied with acute hemorrhage, and all mice died 36 to 48 hrs after the dosing. At 12 hrs after DMN administration, centrilobular hepatocytes revealed nuclear chromatin clumping. At 24 hrs, hepatocyte nuclei became fragmented to form apoptotic cells. Ultrastructurally, chromatin was condensed into a compact granular mass or crescent granular cap at the nuclear periphery. At 36 hrs, the number of apoptotic cells increased and they protruded into the sinusoid or were engulfed by the neighboring hepatocytes. A TUNEL-positive signal preceded the morphological changes and a few normal appearing centrilobular hepatocytes were positive 6 hrs post dosing. Endothelial damage was seen immunohistochemically at 24 hrs by disruption of type IV collagen and factor VIII-related antigen, resulting in massive hemorrhage in the centrilobular to mid zone. No inflammatory reactions were observed throughout the degeneration. The findings indicate that a single i.p. administration of DMN induced severe and fatal toxicity in liver tissues in mice which resembled human fulminant hepatitis. However, as gld-lpr and lprcg mice defective in apoptosis through the Fas system also showed similar severe liver damage, the Fas/Fas ligand system is not involved in DMN-induced liver apoptosis. No other organs or tissues were damaged, and the control mouse liver was intact.     

cDNA cloning and gene expression of phenylalanine ammonia-lyase in Lithospermum erythrorhizon

Two cDNA clones (LEPAL-1 and LEPAL-2) encoding phenylalanine ammonia-lyase were isolated from cell suspension cultures of Lithospermum erythrorhizon. Northern kinetic studies showed that LEPAL-1 mRNA contents markedly increased one day after inoculation of the cells into fresh medium, then decreased to the steady-state level. The course of mRNA accumulation paralleled that of PAL enzyme activity. The rapid induction of PAL activity seems to reflect the induction of dihydroechinofuran biosynthesis, while shikonin was produced at the steady-state level of PAL activity. The course of LEPAL-2 mRNA accumulation seemed to be similar to, but much lower than that of LEPAL-1. In the intact plant, both genes are expressed mainly in the root, the organ in which shikonin is exclusively produced and accumulated. Genomic Southern blot analyses showed that both genes are present in the genome as single copies.