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Serum iron and ferritin concentrations were measured in 1,532 regular blood donors from South Wales who were undergoing HLA typing prior to registration on the British Bone Marrow and Platelet Donor Panel. Serum transferrin concentrations were determined for donors with serum iron concentrations > 24 mumol/l. There were 25 donors with transferrin saturations > 50% and 11 with transferrin saturations > 60%. There were five donors with serum ferritin concentrations > 200 micrograms/l (women) or > 300 micrograms/l (men). Two of the male donors had transferrin saturations > 50% and serum ferritin > 300 micrograms/l on repeat blood samples and are being treated by venesection. Donors with HLA-A3 did not differ from those without A3 in serum iron or ferritin concentrations. Even in the group of donors who were apparently homozygous for A3 there were neither abnormal serum iron nor ferritin concentrations. Although it is well established that measurements of transferrin saturation are required to detect homozygous haemochromatosis (HFE) in its earlier stages, the number of 'false-positive' results is likely to be unacceptably high for screening blood donors. Serum ferritin assays should identify donors with HFE and iron overload before the onset of liver damage. With two million regular donors and 300,000 new donors each year, a significant proportion of the U.K. population will be screened within 10 years. The assay of serum ferritin identifies donors with low levels of storage iron who are at risk of developing iron-deficiency anaemia. Furthermore, donation frequency may be increased for those donors with higher ferritin concentrations when blood supplies are low.

The membrane-intrinsic protein phospholamban (PLN), the regulatory protein of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, was chemically synthesized. The synthesis was accomplished by double couplings and efficient capping procedures, thus eliminating hydrophobic failure sequences. The crude peptide was purified by high-performance liquid chromatographic ion exchange and gel permeation chromatography in chloroform-methanol mixtures. Ion spray mass spectroscopy showed that the product had the correct molecular mass. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis runs produced the typical monomer-pentamer structural pattern. A predominantly helical CD spectrum was obtained in 0.075% C12E8 (67.9% helix, 1.8% beta, 12.2% turn, 18.1% random coil). Synthetic PLN was phosphorylated in detergent solutions by protein kinase A with a stoichiometry close to 1:1 (Pi to PLN monomer). Reconstitution of the isolated skeletal muscle SR Ca2+ ATPase in phosphatidylcholine membranes in the presence of PLN using the freezing and thawing technique yielded a preparation with lower Ca(2+)-dependent ATPase activity. The inhibition was mainly due to a decrease in the affinity (Km(Ca)) of the ATPase for Ca2+ and was partially reversed by PLN phosphorylation with protein kinase A. By contrast, addition of PLN to diluted intact SR vesicles uncoupled the Ca(2+)-transport reaction, suggesting an ionophoric effect of PLN. Because this effect was observed at very high PLN-to-SR vesicle ratios and was not influenced by PLN phosphorylation, its biological function is doubtful.

The current model for colorectal tumorigenesis defines four specific mutations (activation of a ras proto-oncogene and inactivation of the APC, p53 and DCC tumor-suppressor genes) that accumulate in a colonic epithelial cell as it progresses towards a carcinoma. However, further mutations must be needed for progression to malignancy because advanced adenomas have been observed with all four of these mutations. Loss of heterozygosity (LOH) for 11 loci spanning the distal portion of the long arm of chromosome 14 was studied in 89 sporadic colorectal adenocarcinomas and 25 adenomas. The overall rate of LOH in carcinomas was 53% (46/86 informative carcinomas). The smallest region of overlap (SRO) of deletions includes the markers D14S19 to D14S20. No LOH was seen in the 18 informative adenomas examined. There was a significant trend towards higher levels of LOH within the SRO in advanced Dukes' stages (P = 0.016). Since frequent loss of heterozygosity in a specific region of a chromosome may reflect the inactivation of a tumor-suppressor gene located there, these data suggest that a gene involved in the progression of colonic neoplasia may reside on the distal portion of the long arm of chromosome 14, and that its inactivation may be a critical event in this process.

Both p21ras and protein kinase C (PKC) are believed to function downstream of plasma membrane-associated tyrosine kinases in cellular signal transduction pathways. However, it has remained controversial whether they function in the same pathway and, if so, what their relative position and functional relationship in such a pathway are. We investigated the possibilities that p21ras and PKC function either upstream or downstream of each other in a common linear pathway or that they function independently in colinear signal pathways. Either decreased expression of endogenous normal ras in fibroblasts transfected with an inducible antisense ras construct or overexpression of a mutant ras gene reduced the capacity of the phorbol ester tetradecanoyl phorbol acetate to trigger expression of the tetradecanoyl phorbol acetate-responsive and ras-dependent reporter gene osteopontin (OPN). PKC depletion decreased basal OPN mRNA levels, and the overexpression of ras restored OPN expression to the level of non-PKC-depleted cells. We propose a model in which ras and PKC function in distinct and interdependent signaling pathways.

Cancer is a genetic disease; tumor cells differ from their normal progenitors by genetic alterations that affect growth-regulatory genes. There exist 2 classes of such cancer genes: the oncogenes, which function as positive growth regulators, and the tumor suppressor genes, which function as negative growth regulators. Oncogenes are widely conserved among diverse forms of life and are active in transmitting growth signals from the cell periphery to the cell nucleus. These signaling functions can be disturbed by many types of genetic change; the result of an altered growth signal is often cancer. Tumor suppressor genes have an attenuating effect on cell growth that is lost as a result of inactivating mutations or deletion of the gene; in DNA virus-transformed cells, it is abrogated by neutralization of the tumor suppressor protein through a viral gene product. Tumor suppressor genes were first recognized in inherited cancers; defects in a tumor suppressor transmitted through the germ line can lead to increased tumor incidence in the offspring. Tumor suppressors also play important roles in nonheritable cancer, however; many tumors in humans show defects in tumor suppressor genes. Most cancers harbor multiple genetic changes in oncogenes as well as tumor suppressor genes. Oncogenes induce aberrant growth through a gain in function; tumor suppressor genes contribute to oncogenesis through a loss of function. Both types of mutation work together to produce cancer; the changes are not constant but increase in number as the tumor develops from benign to more and more malignant. Cancer results from the accumulation of genetic changes. Oncogenes and tumor suppressor genes provide important insights into the regulation of cell growth. This knowledge can now be used to develop gene-specific therapies for cancer.

The integrase (IN) protein of the human immunodeficiency virus (HIV) is required for specific cleavage of the viral DNA termini, and subsequent integration of the viral DNA into target DNA. To identify the various domains of the IN protein we generated a series of IN deletion mutants as fusions to maltose-binding protein (MBP). The deletion mutants were tested for their ability to bind DNA, to mediate site-specific cleavage of the viral DNA ends, and to carry out integration and disintegration reactions. We found that the DNA-binding region resides between amino acids 200 and 270 of the 288-residues HIV-1 IN protein. The catalytic domain of the protein was mapped between amino acids 50 and 194. For the specific activities of IN, cleavage of the viral DNA and integration, both the DNA-binding domain and the conserved amino-terminal region of IN are required. These regions are dispensable however, for disintegration activity.

Antibiotics of the beta-lactam class may cause coagulation defects and bleeding. It has been suggested that N-methyltetrazolethiol (NMTT), a common side chain group at the 3'-position of the cephem or 1-oxacephem frame, could be responsible for the hypoprothrombinemic effect of the antibiotics and that it could inhibit the liver vitamin K-epoxide reductase activity. Flomoxef (6315-S) is a new oxacephem antibiotic which differs from latamoxef because it has [1-(2-hydroxethyl)-1H-tetrazol-5-yl] thiomethyl (HTT) as a side chain at the 3'-position of cephem group instead of NMTT and an extensive modification of 7 beta-acylamino side chain. The present study was carried out to study its effects on vitamin K-dependent blood coagulation parameters in human volunteers. Ten adult patients (6 men and 4 women), suffering from chronic bronchitis, entered into the study. Each patient received ten 1 g i.m. injections of flomoxef at 12-hourly intervals. Apparently, the treatment with this oxacephem antibiotic had no significant effect. PT, PTT and fibrinogen remained in the normal range in all patients and factors II+VII+X, protein C, protein S and AT III were not depleted. The trend was similar both in men and women. Based on the results of the present study, we conclude that flomoxef is an antibiotic that does not exhibit an effect on blood coagulation, even in males.(ABSTRACT TRUNCATED AT 250 WORDS)

Two distinct gene classes have been implicated in colorectal carcinogenesis. Tumour promoter genes (oncogenes, dominant oncogenes) produce an excessive positive stimulus to cell proliferation. The ras family of oncogenes are an example. Acquired mutations of the c-k-ras gene are commonly found in colonic adenomas and carcinomas. Tumour suppressor genes (anti-oncogenes, recessive oncogenes) normally constrain or regulate cell proliferation. Loss of this function through gene deletion or mutation is oncogenic. Inherited tumour suppressor gene mutations have now been identified in several of the familial cancer syndromes. Acquired tumour suppressor gene mutations are found in both sporadic and hereditary cancers. Together with the tumour promoter genes they provide the genetic basis for the cellular changes occurring during carcinogenesis. The retinoblastoma gene was the first human tumour suppressor gene to be characterized and exemplifies the class. More recently, linkage studies in the hereditary cancer syndromes and the detection of specific deletions in sporadic tumours have helped to identify several new tumour suppressor genes. At least four of these (MCC, APC, p53 and DCC) apparently contribute to sporadic colorectal carcinogenesis. Germ line APC mutations produce the inherited colorectal cancer syndrome familial adenomatous polyposis (FAP). Detection of these mutations using linked markers has already found clinical application in the screening of families with this disease. In the future, genetic diagnosis of hereditary non-polyposis colorectal cancer (HNPCC) and the recognition of those genetically susceptible to sporadic colorectal cancer may become possible. At the same time, as our understanding of the genes involved improves, new avenues for treatment and prevention of colorectal cancer may emerge.

The natural anticoagulant pathway involving heparan sulfate proteoglycan and antithrombin III (ATIII) was studied in serial biopsies from 90 cardiac allograft recipients. The ATIII component of this pathway was identified immunocytochemically on venous endothelium and arterial smooth muscle cells and intima of normal donor hearts and stable allografts. Unstable grafts lacked vascular ATIII and contained fibrin deposits. Neither stable nor unstable grafts had ATIII-reactive capillary endothelium. Grafts with absent vascular ATIII could (1) result in death, (2) revert to an arterial/venous ATIII distribution or (3) develop ATIII-reactive capillary endothelium. The development of ATIII-reactive capillaries was associated with a survival advantage, and such reactivity seemed to be promoted by heparin.

To demonstrate that human alpha-thrombin is effectively inactivated by human antithrombin III (AT) during the production of a fibrin clot we measured the amount of alpha-thrombin activity which can be recovered from a clot generated from purified human proteins. We discovered that 0.05-0.07% of the original alpha-thrombin activity is recovered from a fibrin clot produced from a reaction mixture where the initial concentrations of AT and alpha-thrombin were chosen at a ratio (17.5) to allow complete conversion of fibrinogen to fibrin. These results indicated that alpha-thrombin is successfully inactivated by AT during the production of a fibrin clot. Further, when an amount of alpha-thrombin equal to that recovered from a fibrin clot is introduced into a solution of fibrinogen and AT identical to that utilized to produce the clot only 4% of the fibrinogen is converted to fibrin. These results suggest that i) when a fibrin clot is dissolved during fibrinolytic therapy little active alpha-thrombin should be released from the clot and ii) this amount of thrombin is insufficient to catalyze rethrombosis without proposing de novo production of thrombin. The action on factors XI, VIII, and V of the small amount of thrombin released upon thrombolysis, however, may provide the stimulus for de novo production of sufficient thrombin to catalyze rethrombosis.