Hb A2-Parkville or delta 47(CD6)Asp----Val, a new delta chain variant.

A new delta chain variant, Hb A2-Parkville [delta 47(CD6)Asp----Val], has been identified in a female of Italian parentage. The mobility of the variant is less than carbonic anhydrase towards the anode at alkaline pH.

Oestrogen sulfotransferase: molecular cloning and sequencing of cDNA for the bovine placental enzyme.

The female sex hormone, oestrogen, plays a central role in breast cell proliferation in both the normal and malignant state. It controls transcription from several genes, including that for the progesterone receptor, and in endometrial tissue, via this receptor, it controls the gene for the enzyme oestrogen sulfotransferase. This enzyme may control the level of the oestrogen receptor by sulfurylating free oestradiol. To study the mode of transcriptional control exercised by oestrogen, bovine oestrogen sulfotransferase cDNA has been cloned and the nucleotide sequence determined. The message, of which 1812 bases have been sequenced, contains an open reading frame of 885 bases which encode a protein of 295 amino acids and a maximum apparent molecular weight of 34,600. The deduced protein sequence is supported by existing peptide sequence data and appears to contain a steroid-binding region. Some physico-chemical characteristics of the enzyme appear to differ markedly from those previously reported.

Oestrogen sulfotransferase: isolation of a high specific activity species from bovine placenta.

During the course of a study of the control of expression of steroid-binding proteins in human mammary cancer oestrogen sulfotransferase was isolated from bovine placenta. By a combination of salt precipitation and ion-exchange and gel-permeation chromatography two forms of the enzyme were isolated. The forms, which apparently differ only in charge, have specific activities 100-300 times greater than has previously been reported for the enzyme. Partial peptide sequences of these enzymes are presented.

Relationship between flow cytometric parameters, steroid receptors, and menopausal status in breast cancers.

Of 163 human breast cancers examined, 68% contained detectable aneuploid populations, whilst 32% had apparently normal DNA distributions. A slightly higher incidence of aneuploidy was observed in pre-menopausal patients (83%) than in post-menopausal patients (66%). Also, pre-menopausal patients had slightly higher proportions of S-phase or cycling (S + G2 + M) cells. Estradiol receptor negative (ER-) tumours from post-menopausal patients were found to have the lowest incidence of aneuploidy (59%) and ER- tumours from pre-menopausal patients the highest (91%). There were no significant differences in the proportions of cycling nuclei when receptor status and menopausal status were considered. A weak relationship is shown to exist between flow cytometric data and two common prognostic indicators of breast cancer, namely receptor status and menopausal status.

Effects of dihydrotestosterone, 17 beta-estradiol and dexamethasone on mitogen--and allogeneic--induced lymphoblastogenesis.

In contrast to the in vivo action of sex steroids the steroidal hormones dihydrotestosterone (DHT) and estradiol (E2) produced a marked variable response on mitogen-induced lymphoblastogenesis. DHT and E2 at concentrations of 10(-11) mol l-1 incubated with mononuclear cell fraction in the presence of mitogens PHA, Con A and PWM caused inhibition, stimulation or no effect. Separation of T and B cell fractions and incubation with DHT and E2 likewise produced variations in response. However, dexamethasone (DEX) strongly inhibited proliferation of all cell fractions, particularly at the higher steroid concentrations. These results suggest that the reported effects of in vivo administered sex hormones on the immune system might possibly be mediated by a precursor cell of peripheral blood mononuclear cells, and that the in vitro system may not be a reproducible test for determining an individual's immunoendocrine status.

Human kappa-casein as a tumor marker: purification and properties.

To test the efficacy of kappa-casein as a marker of human malignancy, a protein human milk, ostensibly kappa-casein, has been purified and serum levels determined, by RIA, in normal subjects, breast cancer patients, and lactating women. The results do not support claims by other workers for this protein. Concurrent physicochemical characterization of the protein has shown that it is probably lactoferrin and this result casts some doubt on the earlier studies as it is a major, non-beta component of the casein fraction. It also indicates that caution should be exercised when homology between proteins is used as a guide to identity.

Haemoglobins of the shark, Heterodontus portusjacksoni. III. Amino acid sequence of the beta-chain.

The amino acid sequence of the beta-chain of the principal haemoglobin from the shark H. portusjacksoni has been determined. The chain has 141 residues, the same as that of mammalian alpha-chains and less than the 146 residues of mammalian beta-chains or the 148 residues of the alpha-chain from the tetrameric shark haemoglobin. The sequence was deduced from the sequences of peptides obtained by digestion of the globin or its cyanogen bromide fragments with trypsin, chymotrypsin, pepsin and papain. The difference in length of the beta-chain is most readily accounted for by the absence of the D helix. This small helical section is normally present in myoglobins and beta-globins but absent in alpha-chains. The deduction that it is absent from shark beta-chain is based on consideration of homology. The beta-chain shows the insertion of histidine beta2 and the deletions corresponding to residues A17 and AB1 relative to alpha-and myoglobin chains. The reactive thiol group in shark haemoglobin was shown by radioactive labelling to be residue 51 in the beta-chain, immediately preceding the E helix. The amino acid sequence of shark beta-chain shows 92 differences from human beta-chain, significantly more differences than shown by chicken or frog beta-chains, in line with its earlier time of divergence. If the tertiary structure of the shark beta-chain is the same as that of the horse then there are two changes in the alpha1beta2 contact site in oxyhaemoglobin and an additional one in deoxyhaemoglobin. When both alpha- and beta-chain contacts are considered there is a total of nine changes in residues involved in the alpha1beta2 contacts. There is no Bohr effect in shark haemoglobin, and of the residues normally involved in this effect the C-terminal histidine residue of the beta-chain is present, but the aspartyl (FG1) residue to which it is salt-linked is not, being replaced by a glutamyl residue.

Instability of DTNB-treated globin or haemoglobin.

Human haemoglobin or globin in its native form reacts with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with uptake of two 3-carboxylato-4-nitrothiophenol groups, one for each of the reactive thiols at the beta93 positions. Attempts to isolate the DTNB-treated globin by the acetone-HC1 method, which unfolds the protein chains, result in disulphide interchange and oxidation of almost all the uncoupled "masked" thiol groups. This modification is in marked contrast to the stability of haemoglobin or globin treated with reagents such as iodoacetic acid or N-ethylmaleimide that do not form disulphide bonds in blocking the thiol groups. The derivatized globin chains have been separated by urea-thiol buffer chromatography on carboxymethycellulose columns. Amino acid analysis and peptide mapping established the presence and location of disulphide bonds, whilst gel filtration in urea buffers and sodium dodecyl sulphate acrylamide gel electrophoresis defined the size of the products.

Haemoglobins of the shark, Heterodontus portusjacksoni II. Amino acid sequence of the alpha-chain.

The amino acid sequence of the alpha-chain of the principal haemoglobin from the shark, H. portusjacksoni has been determined. The chain has 148 residues and is acetylated at the amino terminal. The soluble peptides obtained by tryptic and chymotryptic digestion of the protein or its cyanogen bromide fragments were isolated by gel filtration, paper ionophoresis and paper chromatography. The amino acid sequences were determined by the dansyl-Edman procedure. The insoluble "core" peptide from the tryptic digestion contained 34 residues and required cleavage by several prosteases before the sequence was established. Compared with human alpha-chain there are 88 amino acid differences including the additional seven residues which appear on the amino terminal of the shark chain. There is also one deletion and one insertion. The chain contains no tryptophan but has four cysteinyl residues which is the highest number of such residues recorded for a vertebrate globin. In the alpha1beta1 contact sites there are four changes in the oxyhaemoglobin form and six deoxy form. Nine of the 16, alpha1beta1 contact sites show variation while three of the haem contact sites have changed in comparison to the residues known to be involved in these interactions in horse haemoglobin alpha-chain. Use of the sequence data to estimate a time of divergence of the shark from the main vertebrate line yielded the value of 410 +/- 46 million years. The data, in general, support the palaeontological view that bony fishes arose before the elasmobranchs.