Alan W. Steggles

A splicing mutation in the cytochrome b5 gene from a patient with congenital methemoglobinemia and pseudohermaphrodism

We have analyzed reticulocyte and leukocyte mRNAs isolated from a patient with congenital methemoglobinemia and pseudohermaphrodism. The cytochrome b5 cDNA sequences were amplified using specific oligonucleotide primers and the polymerase chain reaction (PCR). DNA sequencing indicated that there was a 16-bp deletion in the cDNA leading to a new, in-frame stop signal and resulting in a truncated protein of 45 amino acids. Genomic DNA was analyzed, and the molecular lesion was shown to be an AG→GG alteration in the 3′ splicing junction of intron 1. The splice site alteration leads to the usage of the nearest AG as an alternative splice site, resulting in a 16-bp deletion in the mRNA. All of the studies on reticulocyte mRNA and genomic DNA indicated that the patient was homozygous for the lesion.

Identification and partial purification of hamster microsomal cytochrome P-450 isoenzymes

We have identified and partially purified three forms of cytochrome P-450 from hamster liver microsomes. Phenobarbital (PB) treatment induced three major polypeptides with relative mobilities (Mr) of 47,000, 50,000 and 51,500. The 47,000 polypeptide was assigned as epoxide hydrolase, since it was also enhanced by trans-stilbene oxide (TSO) treatment. Two polypeptides (Mr = 48,500 and 53,500) were induced by both 3-methylcholanthrene (3-MC) and beta-naphthoflavone (BNF) treatments. Treatment with Aroclor 1254 induced three polypeptides (Mr = 48,500, 50,000 and 53,500), indicating the induction of both drug- and carcinogen-inducible cytochrome P-450s. Liver microsomal benzo[a]pyrene hydroxylase activity was not affected significantly by any of these inducers. In contrast, it was induced 2- to 3-fold in lung microsomes by 3-MC, BNF or Aroclor 1254 treatment. Benzphetamine N-demethylase and 7-ethoxycoumarin O-deethylase activities, expressed as nmoles of product formed per min per mg of liver microsomal protein, were increased 3- to 4-fold by either PB or Aroclor treatment. The activity of 7-ethoxycoumarin O-deethylase was the only one enhanced significantly by 3-methylcholanthrene or beta-naphthoflavone treatment in liver microsomes. Pregnenolone-16-alpha-carbonitrile (PCN) and TSO did not alter any of these activities. The major polypeptides induced by PB (Mr = 50,000) and 3-MC (Mr = 48,500 and 53,500 respectively) were partially purified, to a specific content of 6-10 nmoles P-450/mg of protein and were active in catalyzing N-demethylation of benzphetamine, hydroxylation of benzo[a]pyrene, and O-deethylation of 7-ethoxycoumarin with different substrate specificity. None of these isoenzymes immuno-cross-reacted with antibodies prepared against rabbit cytochrome P-450LM2 or P-450LM4.

Levels of gastrin-cholecystokinin-like immunoreactivity in the brains of genetically obese and non-obese rats.

Levels of gastrin-cholecystokinin-like immunoreactivity were measured in three brain regions (cortex, diencephalon, brainstem) and the pituitary gland in groups of genetically obese Zucker rats and their non-obese littermates. The obese animals had significantly increased body weights and significantly lowered brain weights. However, levels of gastrin-cholecystokinin-like immunoreactivity were not different between the two groups in any of the regions measured. These results contrast with a recent report [11] in which ob/ob mice were found to have decreased levels of cholecystokinin in their brains.

Differential expression of the mRNAs for the soluble and membrane-bound forms of rabbit cytochrome b5

Total RNA was extracted from a variety of rabbit tissues and reverse transcribed for use in the polymerase chain reaction technique. Using primers designed to amplify the membrane-bound liver cytochrome b5 cDNA, products of two sizes were observed. Both hybridized strongly to a radiolabelled liver cytochrome b5 probe. Sequencing confirmed that the two types of cDNA product encoded the membrane-bound and the soluble forms of b5. Messenger RNA corresponding to the soluble cytochrome was detected in the lung, gallbladder and the adrenal gland, as well as in reticulocytes and bone marrow. This was an unexpected finding since the protein has been isolated only from erythrocytes. In contrast, membrane-bound cytochrome b5 mRNA was detected in all tissues tested, suggesting that the corresponding protein is ubiquitous in tissue distribution.

Amino acid substitutions in the membrane-binding domain of cytochrome b5 alter its membrane-binding properties

The structure-function relationships of the 43-amino-acid membrane-binding domain of cytochrome b5 have been examined in two mutant forms of the protein. In one mutant, two tryptophans in the membrane-binding domain, at positions 108 and 112, were replaced by leucines, and in the second mutant, in addition, aspartic acid 103 was also replaced by leucine. The fluorescence emission spectra of the three proteins and their degree of quenching by brominated lipids indicate that the mutations are not producing major conformational changes or allowing a deeper degree of penetration of the domain into the bilayer. The hydrophobicities of the three proteins were compared, by determining strengths of self-association and membrane affinities, and it was found that the protein with two additional leucines was much less hydrophobic and the one with three additional leucines was much more hydrophobic than the native cytochrome. It appears that small changes in amino acid composition, which produce no gross changes in the structure of the membrane-binding domain, will nevertheless produce very large changes in the strengths of self- and membrane-association. These differences in self-association had profound effects on the times required for membrane-association to reach equilibrium.

Changes in Cholecystokinin receptor binding in rat brain after food deprivation

Levels of cholecystokinin (CCK) receptor binding in 7 brain regions were measured in two groups of adult male rats using iodinated CCK-8 as the radioligand. One group was deprived of food for 72 h prior to sacrifice and the other group had food available ad libitum. The deprivation resulted in a 13% decrease in body weight. In comparison to the ad libitum rats, the deprived group had a significantly lower level of CCK receptor binding in the olfactory bulb, and significantly higher levels of binding in the caudate nucleus, hypothalamus and midbrain. No significant differences were noted in samples of cerebral cortex, hippocampus or hindbrain. These results demonstrate that levels of CCK receptor binding can be altered by an acute change in the metabolic state of the animal.

Obesity- and sex-related alterations in growth hormone messenger RNA levels

The secretion of growth hormone (GH) is abnormal in genetically obese Zucker rats. Measurements of pulsatile GH release and circulating GH levels in lean (Fa/?) and obese (fa/fa) rats have shown that both are reduced in the latter. We have studied pituitary GH gene expression in order to understand the role of GH synthesis in this abnormality. Obese animals have lower pituitary GH mRNA levels than lean controls. Within each genotype a sex difference was observed with the female animals having lower GH mRNA levels than the males. It is unlikely that the GH abnormality is due to a generalized pituitary defect because prolactin mRNA levels were the same in all four groups of rats.

The human cytochrome b5 gene and two of its pseudogenes are located on chromosomes 18q23, 14q31-32.1 and 20p11.2, respectively

Using very high stringency hybridization conditions for the Southern blot hybridization analysis of hamster-human cell hybrid DNA, we were able to map the human cytochrome b5 gene and two of its pseudogenes (psgb51 and psgb52) unambiguously to chromosomes 18, 14, and 20. These localizations were confirmed and extended to 18q23, 14q31-32.1, and 20p11.2 by using a combination of nonisotopic in situ hybridization of chromosomal spreads and the polymerase chain reaction analysis of DNA samples isolated from somatic cell hybrids retaining deletions or translocations of chromosome 18.

Upper genital tract abnormalities in the syrian hamster as a result ofin utero exposure to diethylstilbestrol

Prenatal exposure to diethylstilbestrol (DES) causes a significant increase in the carcinogenic response of the hamsters reproductive tract to subsequent DES treatment. Uteri from DES treated females from untreated- and DES treated mothers (C.D & D.D) have abnormal hyperplasia with characteristic finger-like structures projecting into the lumen of the uteri. Inside these papillae along with the rest of the stroma are cystic glands. We found that these glands had no openings into the uterine lumen and that they “begin” and “end” in the stroma. In addition there are two types of cells lining the cystic gland i.e., pale cells and acidophilic cells. Capillary beds surround the cystic glands. We have named these uterine structures “cystadenomatous papilloma”. In addition, we found a spectrum of hyperplastic abnormalities in C.D and D.D uteri and carcinomain situ in D.D uteri. Similar neoplasms have been described in human pathology. Ultrasound observations have demonstrated thatin utero exposure to DES may result in uterine hypoplasia and because it appears to be similar to the changes seen in prenatally DES treated (D.C) hamsters, cross sectional areas of C.C (untreated control), D.C, C.D and D.D uteri were compared. Our results show that later in life uterine hypoplasia also occurs in the 100 days old D.C hamsters and since D.C uteri present hyperplasia with cystic structures, our data support the hypothesis thatin utero DES-treated human females may later in life develop benign and malignant lesions in their reproductive tract. Much of these data corresponds to what has been found in humans, and consequently warrants further investigation into the use of Syrian hamster as a model to understand the uterine abnormal morphogenesis in regards to hypoplasia, adenocarcinoma and carcinoma development in the human.

The quantitation of liver cytochrome P450—LM2 mRNA in rabbits of different ages and after phenobarbital treatment

Using very young (neonate or 0 day), 50-, 100-, 300- and 830-day-old rabbits we have studied changes in liver drug-metabolizing activities. Total liver microsomal cytochrome P450 content, benzphetamine N-demethylase and 7-ethoxycoumarin O-deethylase activities all decline with increasing age. When the rabbits of different age groups were pretreated with phenobarbital, liver microsomal enzyme activities increased in each group as compared with its control, but the total induced activities still decreased with increasing age. Using a specific assay for translatable cytochrome P450-LM2 mRNA, we show that the age-dependent decrease in control and phenobarbital-induced enzyme activities is due to a decrease in the levels of translatable mRNA specific for cytochrome P450-LM2. This is the first report on an age-dependent decrease in the level of a specific translatable mRNA. We do not know whether this decrease is due to decreased mRNA synthesis, or increased mRNA degradation.