A death involving probenecid.

A death following deliberate ingestion of approximately 75 g of probenecid in a 36-year-old man is described. Tissue concentrations of probenecid were highest in serum (710 mg/L) and liver (550 mg/kg). Probenecid was also detected in vitreous and bile. Ethanol was also detected in blood at 0.13 g/100 mL.

Prolactin and breast cancer risk.

A study of 424 women was undertaken to determine whether there was an association between serum prolactin levels and breast cancer; whether prolactin levels would reflect degrees of risk of developing breast cancer; and whether associations between known risk factors for breast cancer and serum prolactin concentrations could be demonstrated. Prolactin levels higher than the median value in control subjects were found to be associated with a more than two-fold increase in the risk of breast cancer (relative risk, 2.1; confidence interval [CI], 1.0-4.5). Moreover, a relative risk of 1.7 (CI, 0.9-3.3) for a group of women with benign epithelial hyperplasia (high risk of developing breast cancer), and a relative risk of 1.0 (CI, 0.6-1.8) for a group with benign fibrocystic disease (low risk of developing breast cancer), provided supportive evidence that prolactin plays a role in the development of breast cancer. A considerable fall in the concentration of prolactin at menopause was noted, so those women who have an early menopause have a reduced period of exposure to high concentrations of prolactin. Similarly, there was a considerable reduction in prolactin concentration after the first pregnancy. Finally, our results showed that, in premenopausal women, a high intake of saturated fats was associated with a high prolactin concentration. Our study supports the concept that parity, menstrual status, and saturated fat consumption influence a woman's exposure to prolactin and therefore the risk of developing breast cancer.

Molecular and functional characterization of amylin, a peptide associated with type 2 diabetes mellitus.

The 37-amino acid peptide called amylin is a major component of the islet amyloid deposited in the pancreases of persons with type 2 diabetes mellitus. We report the isolation of a partial cDNA clone and a phage lambda genomic clone of the coding region of the amylin gene. The DNA sequence encodes a protein sequence identical to that of amylin isolated from the amyloid found in the diabetic pancreas and shows that amylin is likely to be synthesized as a precursor peptide, now named proamylin. We have demonstrated that the amylin gene is present on chromosome 12 and that it is probably transcribed in the islets of Langerhans. The sequences of the genes for amylin and the calcitonin gene-related peptides (CGRPs) show strong similarity, especially over their 5' coding regions, where both peptides have a conserved intramolecular disulfide bridge, and also over their 3' coding regions, where the presence of a glycine codon strongly suggests that the carboxyl-terminal residue of amylin, like that of CGRP, is amidated. To examine the functional relevance of these posttranslational modifications, the biological activity of amylin synthesized with or without the disulfide bridge and/or amidation was measured. It was found that both features are necessary for full biological activity, thereby confirming the functional importance of those regions of the molecule whose sequences are conserved at both protein and genetic levels.

Amylin and the amylin gene: structure, function and relationship to islet amyloid and to diabetes mellitus.

Amylin, the major peptide component of the islet amyloid commonly found in the pancreases of patients with type 2 (non-insulin-dependent) diabetes mellitus (NIDDM), is a recently discovered islet polypeptide. This peptide has many structural and functional features suggesting that it is a novel hormone, which may control carbohydrate metabolism in partnership with insulin and other glucoregulatory factors. Amylin is synthesised in, and probably secreted from, the beta-cells of the islets of Langerhans, where it has recently been immunolocalised to secretory granules. DNA cloning studies indicate that in the human and the rat, amylin is generated from a precursor, preproamylin, which displays a typical signal peptide followed by a small prohormone-like sequence containing the amylin sequence. The presence of the signal peptide suggests that amylin is secreted and plays a physiological role. Amylin is probably generated by proteolytic processing similar to that for proinsulin and other islet prohormones. The human amylin gene encodes the complete polypeptide precursor in two exons which are separated by an intron of approx. 5 kb, and is located on chromosome 12. Amylin is a potent modulator of glycogen synthesis and glucose uptake in skeletal muscle, and is capable of inducing an insulin-resistant state in this tissue in vitro, and perhaps also in the liver in vivo. In normal metabolism, amylin could act in concert with insulin as a signal for the body to switch the site of carbohydrate disposal from glycogen to longer-term stores in adipose tissue, by making skeletal muscle relatively insulin-resistant, whilst at the same time leaving rates of insulin-stimulated carbohydrate metabolism in adipose tissue unaltered. Several lines of evidence now implicate elevated amylin levels in the pathogenic mechanisms underlying NIDDM, and suggest to us that the obesity which frequently accompanies this syndrome is a result of, rather than a risk factor for, NIDDM. Following the beta-cell destruction which occurs in type 1 (insulin-dependent) diabetes mellitus (IDDM), it is probable that amylin secretion disappears in addition to that of insulin. As patients with insulin-treated IDDM frequently experience problems with hypoglycaemia, and as amylin acts to modulate the action of insulin in various tissues, it is possible that amylin deficiency may contribute to morbidity in insulin-treated IDDM, perhaps through the loss of a natural damping mechanism which guards against hypoglycaemia under conditions of normal physiology.

Genes expressed during conidiation in Neurospora crassa: characterization of con-8.

The filamentous fungus Neurospora crassa, by a series of defined changes, differentiates from a mycelium composed of branching hyphae to form dormant spores, called conidia. Several genes of unknown function (con genes) that are preferentially expressed during this period have been cloned. Transcription of these genes has been examined in conidiation-defective mutants, and the results obtained revealed that con-6, con-8, con-10, con-11 and con-13 are most likely to play a unique role during conidiation, con-8 is expressed early during conidial differentiation. Genomic and cDNA sequence analyses with con-8 clones identified one open reading frame, interrupted by two introns, which encodes a weakly acidic 18.4 kDa polypeptide containing 176 amino acid residues, con-8 is unusual in that it is transcribed as two mRNA species, 1.0 and 1.25 kb in length. S1 nuclease mapping and primer extension analyses identify one major initiation site, one major polyadenylation site, and demonstrated the existence of heterogeneity at the messenger's 5' and 3' ends.

Molecular analysis of a Neurospora crassa gene expressed during conidiation.

The asexual developmental pathway in the life cycle of the filamentous fungus Neurospora crassa culminates in the formation of spores called conidia. Several clones of genomic Neurospora DNA have been isolated that correspond to mRNA species expressed during conidiation and not during mycelial growth (V. Berlin and C. Yanofsky, Mol. Cell. Biol. 5:849-855, 1985). In this paper we describe the characterization of one of these clones, named pCon-10a. This clone contains two genes, con-10 and con-13, which are induced coordinately during the later stages of conidiation. The two genes are separated by 1.4 kilobases of DNA; they are located on linkage group IV and are transcribed from the same strand of DNA. The molecular organization and sequence of one of these genes, con-10, and its flanking regions are presented. Full-length cDNA clones for con-10 also were isolated and sequenced, and transcription-initiation and polyadenylation sites were defined. The con-10 gene contains an open reading frame interrupted by two small introns and encodes an 86-amino-acid residue polypeptide that is both hydrophilic and weakly acidic. Expression of the con-10 gene in various mutants defective at different stages of conidiation indicates that it plays a role after aerial hyphal development. Possible functions, organization, and regulation of conidiation-specific genes are discussed.

Transformation of Arthrobacter and studies on the transcription of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli.

We report the development of a plasmid-mediated transformation system for Arthrobacter sp. NRRLB3381, using the Streptomyces cloning vector pIJ702. Our procedure gives a transformation frequency of 10(3)/micrograms of plasmid DNA. In addition we have explored the expression of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli, and shown that the ermA promoter is recognized in S. lividans not E. coli. The relationship between Arthrobacter, Streptomyces and E. coli promoters is discussed.

Analyses of protein extracts of human breast cancers: changes in glycoprotein content linked to the malignant phenotype.

The protein and glycoprotein composition of Triton X-100 extracts of breast biopsies from 17 women with benign breast disease and from 11 women with invasive breast carcinoma were investigated using electrophoresis in SDS-containing gradient polyacrylamide gels, followed by Coomassie Blue (CB) staining and the binding of radio-iodinated wheat germ agglutinin (WGA). Patterns were analysed after the CB-step for differences in protein composition, and after the WGA-step for differences in glycoprotein composition. Tissue extracts from patients with benign breast disease have less CB stained bands than similar extracts from the cancer patients. A particular consistent change was the appearance of an extra band at 58 Kdaltons in the cancer extracts. In contrast to the CB results, WGA detected less major bands, in the 40-60 Kd region, in the cancer extracts than at similar locations in benign extracts. Analysis of blood sera using the above techniques suggested that certain serum proteins could account for some of the WGA changes, but not the changes after CB staining. However, residual contamination of the specimens by blood proteins seemed unlikely because of the washing procedure used, unless these components were very strongly associated with the tissue. Differential synthesis of serum proteins by benign and malignant breast tissue may also explain some of our findings. Examination of the histopathology adjacent to the extracted tissue suggested that the degree of reduction in WGA-binding may be related to the extent of local invasiveness. Other animal and human studies suggest that reduced glycosylation of tumour-associated proteins may be linked to increased malignancy. The current findings may reflect a general pattern of change in tumour glycoprotein composition linked to malignant expression.

Reduced heat resistance of mutant spores after cloning and mutagenesis of the Bacillus subtilis gene encoding penicillin-binding protein 5.

Part of the gene encoding penicillin-binding protein 5 from Bacillus subtilis 168 was cloned in Escherichia coli with a synthetic oligonucleotide as a hybridization probe. The gene was designated dacA by analogy with E. coli. The nucleotide sequence was determined, and the predicted molecular mass was 45,594 daltons (412 amino acids). A comparison of the predicted amino acid sequence with that of the E. coli penicillin-binding protein 5 indicated that these enzymes showed about 25% identity. The B. subtilis dacA gene was mutated by integration of a plasmid into the structural gene by homologous recombination. A comparison of the mutant and control strains revealed that (i) the mutant lacked detectable penicillin-binding protein 5, (ii) the D-alanine carboxypeptidase activity of membranes isolated from the mutant was only 5% of that measured in membranes from the control strain, (iii) the mutant cells showed apparently normal morphology only during exponential growth, and after the end of exponential phase the cells became progressively shorter, (iv) the mutant sporulated normally except that the forespore occupied about two-thirds of the mother cell cytoplasm and, during its development, migrated towards the center of the mother cell, and (v) purified mutant spores were 10-fold less heat resistant but possessed normal refractility and morphology. Preliminary chemical analysis indicated that the structure of the cortex of the mutant was different.

Extragenital donovanosis: three cases from Western Australia.

Three unusual cases of extragenital donovanosis are described. The presenting features were ulceration of the anterior abdominal wall, discharging sinuses of the neck, and an abscess of bone, respectively. Two cases were diagnosed recently and were the only instances of extragenital infection in a series of 47 patients with confirmed donovanosis diagnosed at a public health laboratory in Perth, Western Australia, between 1979 and the beginning of 1985. The third case, which occurred in 1977, was found in the records of a major teaching hospital.

Donovanosis in Western Australia.

We report and discuss a series of 47 consecutive patients with donovanosis that were diagnosed in a public health laboratory in Western Australia during slightly more than six years. Most came from the tropical northern parts of Western Australia, there was a preponderance of women in the series, and vulval lesions were the most common manifestation of the disease. Two men had extragenital lesions, though each was eventually found to have concomitant genital lesions.

An erythromycin-resistance gene from an erythromycin-producing strain of Arthrobacter sp.

A gene (ermA) coding for a presumed erythromycin-resistance (ErR) determinant from an Er-producing Arthrobacter sp. strain (NRRLB3381) was isolated from a gene bank in phage vector lambda 2001 by probing with a Streptomyces ErR gene. Strongly hybridizing fragments were subcloned and the appropriate segments sequenced. The ermA gene is 76 mol% G + C in content and specifies a protein of 340 aa with an Mr of 37454. S1 nuclease mapping and primer extension identified the putative promoter, which resembles the consensus sequence of Escherichia coli promoters particularly in the -10 region. A potential ribosome-binding site (RBS) (AGGAG) was also located. Unexpectedly, the majority of in vivo ermA transcripts detected were only 245 nt long, suggesting that expression of ErR may be regulated post-transcriptionally. Substantial homology is observed between the predicted aa sequences of the ermA-coded protein and the products of three other ErR determinants, from organisms that do not produce Er.

Rat adrenocortical carcinoma 494 autophosphorylating protein kinase, autophosphorylating protein kinase 500. Purification, biochemical and immunological characterization, and substrate specificity.

A novel autophosphorylating protein kinase, autophosphorylating protein kinase 500, independent of cyclic AMP, cyclic GMP, calcium, and calmodulin was purified from rat adrenocortical carcinoma 494 by ammonium sulfate fractionation followed by the chromatographic steps of DEAE-cellulose, gel filtration, cyclic AMP-epoxy Sepharose, and phosphocellulose. Sometimes two additional chromatographic purification steps of chromatofocusing and gel filtration were necessary for complete purification. The enzyme was homogeneous as evidenced by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sucrose density sedimentation studies indicated that Mr of the enzyme was 490,000, while ultracentrifugal analysis demonstrated a value of 481,400 (+/-7%). The protein was composed of two identical subunits each with Mr = 250,000. The enzyme molecule was slightly asymmetric with frictional and sedimentation coefficients of 1.28 and 18.20, respectively, and a Stokes radius of 66 A. Isoelectric focusing electrophoresis revealed a single peak with pI 4.6, indicating acidity of the protein. The enzyme self phosphorylated one or more of its serine residues. The reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 mM Mn2+ or 10 mM Mg2+) were essential for optimum activity. Autophosphorylating protein kinase 500 did not phosphorylate the commonly used exogenous substrates such as histones, casein, phosvitin, or protamine. Analysis of autophosphorylating protein kinase 500 with rabbit anti-autophosphorylating protein kinase 500 IgG by immunoelectrophoresis and crossed immune electrophoresis demonstrated single arcs of precipitation, confirming the biochemical demonstration of enzyme purification and homogeneity. Indirect immunofluorescence studies revealed an intracytoplasmic localization of the enzyme in cultured and freshly isolated adrenocortical carcinoma 494 cells. Both cell types revealed an intensity of perinuclear enzyme fluorescence, but an absence of the enzyme in the nuclei or nucleoli. The anti-autophosphorylating protein kinase 500 IgG blocked the self-catalyzed phosphorylation of autophosphorylating protein kinase 500, providing immunological support of the biochemical results that autophosphorylation is an intrinsic characteristic of the enzyme. When autophosphorylating protein kinase 500 was incubated with membrane-bound ribosomes, it phosphorylated a Mr = 31,000 protein. This phosphorylation was blocked by the anti-autophosphorylating protein kinase 500 IgG.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship of calcium and membrane guanylate cyclase in adrenocorticotropin-induced steroidogenesis.

Chlorpromazine, when incubated with isolated adrenal cells, inhibited the ACTH-stimulated formation of cGMP and corticosterone production. It also inhibited the ACTH-stimulated membrane guanylate cyclase, but did not affect the binding of ACTH to the membrane receptors. cGMP-induced steroidogenesis was not affected by the drug. These data indicate that chlorpromazine interferes with adrenal steroid metabolism at a site between the hormone receptor and guanylate cyclase and also show that guanylate cyclase is composed of separate receptor and catalytic components. Furthermore, based on the premise that chlorpromazine exerts its inhibitory action by blocking the binding of a calcium receptor protein, such as calmodulin, to the receptor-coupled guanylate cyclase, it is proposed that the interaction of calcium, presumably through a calcium-binding protein, is essential for ACTH-dependent guanylate cyclase.

Oestrogen receptor assay and morphology of breast cancer.

The associations between oestrogen receptors, several morphological features and prognosis were studied in 53 cases of primary breast carcinoma. There was no significant association between oestrogen receptors and any morphological property studied (histological grade, cellularity, fibrosis, elastosis, necrosis, lymphatic permeation, follicular hyperplasia, encapsulation, vascular invasion, tubular areas, histiocytosis, lymphocytic cell infiltration, number of invaded lymph nodes, tumour size, site of recurrence). Disease-free interval and survival were longer in patients having cancers with positive oestrogen receptors. Good prognostic signs were low histological grades, tubular areas and absence of lymphatic invasion within the primary tumour.

Cultural, morphological, cell membrane, enzymatic, and neoplastic properties of cell lines derived from a Hodgkin's disease lymph node.

A neoplastic cell line (designated HuT11) has been established in continuous culture from an involved lymph node of a patient with Stage IIA Hodgkin's disease of the mixed cellularity type. The HuT11 line has been morphologically heterogeneous, consisting of mononucleate lymphoid-like cells, polygonal epithelioid cells, and mono-, bi-, and multinucleate giant cells. Four clones initiated from isolated binucleate giant cells of the HuT11 line also have been successfully established as continuous cell lines. The cloned lines have been morphologically distinct and more homogeneous, although typical giant cells have consistently appeared throughout the long-term culture of each. The HuT11 lines have grown as monolayers in McCoy's Medium 5A supplemented with 10% fetal calf serum, with generation times of 12 to 14 hr and high saturation densities. Cytogenetic studies showed that early and later passages of HuT11 cells were aneuploid, and all cell lines were successfully heterotransplanted in the hamster cheek pouch. Repeated indirect immunofluorescence examinations have shown each cell line to be negative for Epstein-Barr virus nuclear antigen. Indirect immunofluorescence tests in which monospecific immunoglobulins were used revealed positive membrane reactions for the gamma (heavy)-chain and kappa (light)-chain of human immunoglobulin G in approximately 20% of viable cells in each line; however, direct immunofluorescence with anti-human immunoglobulin G F(ab')2 reagent failed to confirm these reactions. Rosette tests for B- and T-lymphocyte and macrophage membrane receptors yielded negative results. All cell lines were strongly phagocytic for latex particles and neutral red dye. Cytochemical stains of the monolayers revealed abundant esterase, fluoride-resistant nonspecific esterase, acid phosphatase, and leucine aminopeptidase activities, while lysozyme assays were negative. Although some properties of the HuT11 lines have suggested a macrophage derivation, an undifferentiated lymphoid cell origin of the Hodgkin's neoplastic cell remains a possibility.

Changes in the collagenolytic activity released by primary VX-2 carcinoma cultures as a function of tumor growth.

Serum-free media of minced tissue cultures of VX-2 rabbit carcinoma contained a specific collagenolytic activity capable of releasing soluble radioactive peptides from [14C]-labeled collagen fibrils. It was also capable of reducing the viscosity of acid-soluble collagen solutions by cleaving the tropocollagen (TC) molecules primarily at one site to TCA (75%) and TCB (25%) fragments. Three chromatographic fractions were separated by gel filtration: F1, (MW 85-110,000) present in larger amounts in early cultures of younger tumor tissue; F2, (MW-35-40,000) the major component with maximum production in the day 3 media of younger and advanced tumor tissues; F3, (MW 18-22,000) the minor component. Early cultures of younger tumor tissue contained a latent collagenase and were subject to trypsin activation suggesting the presence of inactive enzyme precursors or an enzyme-inhibitor complex.