The conversion of 3-hydroxyanthranilic acid to cinnabarinic acid by the nuclear fraction of rat liver

Although several authors have implicated 3-hydroxyanthranilic acid (3-OHA) as an intermediate in tryptophaniacin pathway in animals (Kaplan, 1961), alternative pathways of metabolism of this compound have not been fully explored. Madhusudanan Nair obtained an enzyme from spinach leaves which could convert 3-OHA to cinnabarinic acid (private communication). Viollier and Süllmann (1950) reported the conversion of 3-OHA to an unidentified red compound by rat liver homogenates. The present investigation describes the identification of this product as cinnabarinic acid (2-amino-3-H-isophenoxazine-3-one-1,9-dicarboxylic acid). Cinnabarinic acid is known to occur in nature along with cinnabarin is olated from the fungus Polystictus sanguineus (Gripenberg et al., 1957; Gripenberg, 1958).

Intracellular distribution of coenzyme Q in rat liver

Coenzyme Q was found to be distributed in rat liver cell fractions. Mitochondria accounted for only 40–60% of the total. The presence of coenzyme Q in nuclei, isolated by several methods, could always be correlated with the presence of oxidative enzymes. It has been established that coenzyme Q is a constituent of microsomes. Administered coenzyme Q10-C14 was preferentially taken up by mitochondrial and microsomal fractions. Exogenous coenzyme Q appears to be rapidly metabolized.

Oxidation of vitamin A1 and vitamin A2 aldehydes to the corresponding acids by enzymes from pig and rat livers

VITAMIN A is stored in rat liver largely as its ester with small amounts of the alcohol, but is transported in the normal circulating blood in the latter form1. Although it was generally believed that the alcohol form is the more physiological state of the vitamin, since the work of Dowling and Wald2, it is being recognized that vitamin A acid and not the alcohol may be nearer to the 'active vitamin A'. If this were to be so, it would be important to demonstrate that a mechanism exists in the rat for the production of vitamin A acid from vitamin A alcohol through the intermediate, the aldehyde. Regarding the formation of the aldehyde, it has been well established that the alcohol dehydrogenase can bring about the conversion of vitamin A alcohol to retinene3. The presence of an enzyme in rat and pig liver catalysing the oxidation of retinene1 and retinene2 to the corresponding acids has been demonstrated in the present work and the partially purified enzyme preparation shown to be completely devoid of alcohol dehydrogenase activity.

Distribution of coenzyme Q in rat liver cell fractions

COENZYME Q (CoQ), which is widely distributed in animal, plant and microbial sources, has been implicated in electron transport1 and generally assumed to be associated with mitochondria. However, it has also been found in non-mitochondrial fractions of green leaves, although it appears to be concentrated in mitochondria2. A similar distribution has now been demonstrated in rat liver cell fractions.

Model For Regulation Of Delta-Aminolevulinate Synthetase Induction In Rat-Liver

A reciprocal relationship exists between the cytochrome P-450 content and d-aminolaevulinate synthetase activity in adult rats. In young rats the basal d-aminolaevulinate synthetase activity is higher and the cytochrome P-450 content is lower compared with the adult rat liver. Administration of allylisopropylacetamide neither induces the enzyme nor causes degradation of cytochrome P-450 in the young rat liver, unlike adult rat liver. Allylisopropylacetamide fails to induce d-aminolaevulinate synthetase in adrenalectomized–ovariectomized animals or intact animals pretreated with successive doses of the drug, in the absence of cortisol. The cortisol-mediated induction of the enzyme is sensitive to actinomycin D. Allylisopropylacetamide administration degrades microsomal haem but not nuclear haem. Haem does not counteract the decrease in cytochrome P-450 content caused by allylisopropylacetamide administration, but there is evidence for the formation of drug-resistant protein-bound haem in liver microsomal material under these conditions. Phenobarbital induces d-aminolaevulinate synthetase under conditions when there is no breakdown of cytochrome P-450. On the basis of these results and those already published, a model is proposed for the regulation of d-aminolaevulinate synthetase induction in rat liver.

Primary Structural Documentation of the Major Urinary Protein of the Indian Commensal Rat (Rattus rattus) Using a Proteomics Platform

Purpose: A number of proteome studies have been performed recently to identify pheromone-related protein expression and their molecular function using genetically modified rodents' urine. However, no such studies have used Indian commensal rodents; interestingly, in a previous investigation, we confirmed the presence of volatile molecules in commensal rodents urine and these molecules seem to be actively involved in pheromonal communication. Therefore, the present study aims to identify the major urinary protein [MUP] present in commensal rat urine, which will help us to understand the protein's expression pattern and intrinsic properties among the rodents globally. Experimental Design: Initially, the total urinary proteins were separated by 1-D and 2-D electrophoresis and then subsequently analyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight and Mass Spectrometer (MALDI-TOF/MS). Furthermore, they were then fragmented with the aid of a Tandem Mass Spectrometer (TOF/TOF) and the identified sequences aligned and confirmed using similarity with the deduced primary structures of members of the lipocalin superfamily.Results: The SDS-PAGE protein profiles showed distinct proteins with molecular masses of 15, 22.4, 25, 28, 42, 50, 55, 68, and 91 kDa. Of these proteins, the 22.4 kDa protein was considered to be target candidate. When 2D electrophoresis was carried out, about similar to 50 spots were detected with different masses and various pI ranges. The 22.4 kDa protein was found to have a pI of about 4.9. This 22.4 kDa protein spot was digested and subjected to mass spectrometry; it was identified as rat MUP. The fragmented peptides from the rat MUP at 935, 1026, 1192, and 1303 m/z were further fragmented with the aid of MS/MS and generated de novo sequence and this confirmed this protein to be the MUP present in the urine of commensal rats.Conclusion: The present investigation confirms the presence of MUP with a molecular mass of 22.4 kDa in the urine of commensal rats. This protein may be involved in the binding of volatile molecules and opens up a discussion about how volatile and non-volatile molecules in the commensal rats' urine may contribute chemo-communication.

Redistribution of subcellular calcium in rat liver on administration of vanadate

Mitochondria isolated from the livers of rats administered with sodium meta-, ortho-, or polyvanadate, but not vanadyl sulphate, exhibited enhanced Ca2+ — stimulated respiration and uptake of calcium. These effects were shown also by mitochondria isolated from livers perfused with polyvanadate. The concentration of acid-soluble calcium decreased significantly in the mitochondrial fraction on vanadate treatment, while that in the cytosol showed a corresponding increase. Phenoxybenzamine, an antagonist to a-adrenergic receptors, effectively inhibited vanadate-induced Ca2+ mobilization, but surgical sympathectomy was without effect. This is the first demonstration of vanadate mimicking agr-adrenergic agonists in vivo.

Increase in alpha-glycerophosphate dehydrogenase and other oxidoreductase activities of hepatic mitochondria on administration of vanadate to the rat

Liver mitochondria isolated from vanadate-administered rats showed increased (20-25%) rates of oxidation of both NAD(+)-linked substrates and succinate. Respiratory control index and ADP/O were unaffected by the treatment. Dormant and uncoupler-stimulated ATPase activity also was not affected by vanadate administration. Membrane-bound, electron-transport-linked dehydrogenase activities (both NAD(+)- and succinate-dependent) increased by 15-20% on vanadate treatment. Mitochondrial alpha-glycerophosphate dehydrogenase activity increased by 50% on vanadate administration. The above effects of vanadate on oxidoreductase activities could be prevented by the prior administration of antagonists to alpha-adrenergic receptors. Substrate-dependent H2O2 generation by mitochondria also showed an increase on vanadate administration.

Characterization of poly(ADP-ribosyl)ated domains of rat pachytene chromatin

Poly(ADP-ribosyl)ation of nuclear proteins was several-fold higher in the pachytene spermatocytes than in the premeiotic germ cells of the rat. Among the histones of the pachytene nucleus, histone subtypes H2A, H1 and H3 were poly(ADP-ribosyl)ated. Based on the immunoaffinity fractionation procedure of Malik, Miwa, Sugimara & Smulson [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] we have fractionated DNAase-II-solubilized chromatin into poly(ADP-ribosyl)ated chromatin (PAC) and non-poly(ADP-ribosyl)ated chromatin (non-PAC) domains on an anti-[poly(ADP-ribose)] IgG affinity matrix. Approx. 2.5% of the pachytene chromatin represented the PAC domains. A significant amount of [alpha-32P]dATP-labelled pachytene chromatin (labelled in vitro) was bound to the affinity matrix. The DNA of pachytene PAC domains had internal strand breaks, significant length of gaps and ligatable ends, namely 5'-phosphoryl and 3'-hydroxyl termini. On the other hand, the PAC domains from 18 h regenerating liver had very few gaps, if any. The presence of gaps in the pachytene PAC DNA was also evident from thermal denaturation studies. Although many of the polypeptides were common to the PAC domains of both pachytene and regenerating liver, the DNA sequences associated with these domains were quite different. A 20 kDa protein and the testis-specific histone H1t were selectively enriched in the pachytene PAC domains. The pachytene PAC domains also contained approx. 10% of the messenger coding sequences present in the DNAase-II-solubilized chromatin. The pachytene PAC domains, therefore, may represent highly enriched DNA-repair domains of the pachytene nucleus.

Studies on follicular atresia: role of gonadotropins and gonadal steroids in regulating cathepsin-D activity of preovulatory follicles in the rat

Preovulatory follicular atresia was studied using pregnant mare serum gonadotropin (PMSG)-primed rats (15 IU/rat) which were deprived of hormonal support either by allowing the metabolic clearance of the PMSG or by injecting a specific PMSG antiserum (PMSG a/s). Atresia was monitored by an increase in lysosomal cathepsin-D activity and a decrease in the receptor activity of the granulosa cells (GC) isolated from the preovulatory follicles. It was shown that the increase in lysosomal activity and the decrease in receptor activity seen at 96 h after PMSG (or PMSG plus PMSG a/s) could be arrested both by follicle stimulating hormone (FSH) and luteinizing hormone (LH). Injection of cyanoketone or clomiphene citrate together with FSH/LH prevented this 'rescue' suggesting a role for estrogens in the regulation of atresia. Although the administration of estradiol-17 beta (20 micrograms/rat) together with PMSG a/s could show a 'rescue effect' in terms of reduction in cathepsin-D activity the gonadotropin receptor activities of these granulosa cells were not restored. The injection of dihydrotestosterone (DHT) to 48 h PMSG-primed rats induced atresia as noted by an increase in cathepsin-D activity. However, the exogenous administration of FSH along with DHT prevented this atretic effect suggesting that DHT is not having a direct effect on atresia. Determination of androgen: estrogen content of the granulosa cells and an analysis of the individual profile of androgen and estrogen revealed that the increase in cathepsin-D activity could be correlated only with the decrease in GC estrogen content. This along with the observation that GC showed a loss of estrogen synthesis well before the increase in cathepsin-D activity strongly points out that the lack of estrogen rather than an increase in androgen is the principle factor responsible for the atresia of preovulatory follicles in the rat.

Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.

Nonlinear measures of heart rate time series: influence of posture and controlled breathing

In this study, we investigated measures of nonlinear dynamics and chaos theory in regards to heart rate variability in 27 normal control subjects in supine and standing postures, and 14 subjects in spontaneous and controlled breathing conditions. We examined minimum embedding dimension (MED), largest Lyapunov exponent (LLE) and measures of nonlinearity (NL) of heart rate time series. MED quantifies the system's complexity, LLE predictability and NL, a measure of deviation from linear processes. There was a significant decrease in complexity (P<0.00001), a decrease in predictability (P<0.00001) and an increase in nonlinearity (P=0.00001) during the change from supine to standing posture. Decrease in MED, and increases in NL score and LLE in standing posture appear to be partly due to an increase in sympathetic activity of the autonomous nervous system in standing posture. An improvement in predictability during controlled breathing appears to be due to the introduction of a periodic component. (C) 2000 published by Elsevier Science B.V.