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In Saccharomyces cerevisiae, DNA polymerase delta (POLIII), the product of the CDC2 (POL3) gene, possesses, in its N-terminal half, the well conserved 3-domain 3' to 5' exonuclease site. Strains selectively mutagenized in this site display a mutator phenotype detected as a drastically increased spontaneous forward mutation rate to canavanine resistance or as an elevated reversion rate to lysine prototrophy. Assays on a partially purified extract of the mutant giving the largest mutator effect indicate that the 3' to 5' exonuclease activity is reduced below the detection limit whereas the DNA polymerizing activity has wild-type level. Therefore, our results provide experimental support for the hypothesis that the exonucleolytic proofreading activity associated with DNA polymerase delta resides on the DNA polymerase delta subunit and enhances the fidelity of DNA replication in yeast.

The transcriptional enhancers of retroviruses that lack an oncogene are important determinants of their oncogenicity. However, no specific cellular transcriptional activator has yet been found to determine the oncogenicity for any of these viruses. The SL3-3 enhancer factor 1 (SEF1) cellular transcriptional activators are expressed preferentially in T lymphocytes. In the SL3-3 murine leukemia virus enhancer, two different sequences can bind SEF1 activators. We show that mutation of the SEF1 binding sites disrupts the disease potential of SL3-3 murine leukemia virus, implying that SEF1 transcriptional activators are required for tumor induction by SL3-3. The SEF1 site mutations did not appear to affect the pathogenicity of SL3-3 by impairment of virus multiplication, but rather by a specific defect in the ability of neoplastic transformation.

Serpins encompass a superfamily of proteinase inhibitors that regulate many of the serine proteinases involved in inflammation and hemostasis. In vitro, many serpins are catalytically inactivated by proteinases that they do not inhibit, leading to the concept of proteolytic down-regulation of serpin inhibitory capacity. The extent to which down-regulation of serpin activity occurs in vivo is debated, since little is known of the rates at which the process occurs. To address this debate, we have measured the rates of inactivation of three serpins, alpha 1-proteinase inhibitor (alpha 1PI), alpha 1-antichymotrypsin (alpha 1ACT), and antithrombin III (ATIII), by three human matrix metalloproteinases (MMPs-1, -2, and -3) thought to be involved in tissue destruction and repair. Our object was to establish a working kinetic model which can be used to predict whether serpin inactivation by these proteinases is likely to occur in vivo. We determined the rates of inactivation of these three serpins by each of the MMPs and compared these to rates of inhibition of the MMPs by an endogenous inhibitor, alpha 2-macroglobulin. An equation designed to predict the extent of substrate hydrolyzed by an enzyme in the presence of an enzyme inhibitor gave the following predictions of the inactivation in vivo: (i) ATIII is unlikely to be inactivated by the MMPs. (ii) MMP-2 (72-kDa gelatinase/type IV collagenase) is unlikely to inactivate any of the three serpins. (iii) MMP-1 (tissue collagenase) will inactivate alpha 1PI and alpha 1ACT only when its concentration saturates that of its controlling inhibitors. (iv) MMP-3 (stromelysin) may inactivate small amounts of alpha 1PI and more significant amounts of alpha 1ACT, even in the presence of its controlling inhibitors. Any physiologic or pathologic inactivation of these serpins by these MMPs that occurs in vivo will probably be due to MMP-3, and will likely only take place in tissues and inflammatory loci where the concentration of MMP inhibitors is depressed.

Struvite (MgNH4PO4.6H2O) crystals, the major mineral component of infectious urinary calculi, were produced in vitro by growth of a clinical isolate of Proteus mirabilis in artificial urine. P. mirabilis growth and urease-induced struvite production were monitored by phase contrast light microscopy and measurements of urease activity, pH, ammonia concentrations, turbidity, and culture viability. In the absence of pyrophosphate, struvite crystals appeared within 3-5 h due to the urease-induced elevation of pH and initially assumed a planar or 'X-shaped' crystal habit (morphology) characteristic of rapid growth. When pyrophosphate was present, initial precipitation and crystal appearance were significantly impaired and precipitates were largely amorphous. When crystals did appear (usually after 7 or 8 h) they were misshapen or octahedral in shape indicative of very slow growth. X-ray diffraction and Fourier transform infrared spectroscopy (FTIR) identified all crystals as struvite. Trace contaminates of carbonate-apatite (Ca10(PO4)6CO3) or newberyite (MgHPO4.H2O) were produced only in the absence of pyrophosphate. P. mirabilis viability and culture pH elevation were unaffected by the addition of pyrophosphate, whereas urease activity and ammonia concentrations were marginally reduced. Struvite could also be produced chemically by titration of the artificial urine with NH4OH. If pyrophosphate was present during titration, the same inhibitory effect on crystal growth occurred, so it is unlikely that urease inhibition is important. Lowering of pyrophosphate concentration from 13-0.45 mumol/l did not reduce its inhibitory activity so it is unlikely to act by chelating free Mg2+. We propose that pyrophosphate inhibits struvite growth principally through direct interference with the chemical mechanisms involved in crystal nucleation and growth, because of its effectiveness at very low concentrations.

Loss of tumor suppressor genes is involved in the mechanism of tumorigenesis of many solid tumors. We tested 9 hepatitis B virus (HBV)-positive and 10 HBV-negative hepatocellular carcinomas for loss of somatic heterozygosity using 14 polymorphic probes mapping to chromosomes 4, 11, 13, and 17. Losses were found on all chromosome arms tested. The highest frequency of loss was observed at the D13S1 locus (67%) at band 13q12. Losses were also observed at three other loci on 13q. Twenty-one % of informative cases showed loss on 17p using the probe pYNZ22 which maps near the p53 locus. Losses on 4q were infrequent with 17% found at one locus and no loss at two others. The retinoblastoma gene and the locus on 17p were only inactivated in our HBV-negative tumors, although the numbers were too small for statistical significance. For all loci tested, we found no significant differences in the frequency of losses with HBV status, ethnic background, cirrhosis, grade of tumor, or presence of hemochromatosis.

We have isolated a cDNA clone encoding the human S3 ribosomal protein from a normal human colon cDNA library. The clone was identified as one of many that detected genes whose level of expression was increased in adenocarcinoma of the colon relative to normal colonic mucosa. Increased levels of the S3 transcript were present in the tumors of all eight patients examined. Moreover, the S3 mRNA was also more abundant in 7 of 10 adenomatous polyps, the presumed precursor of carcinoma. Additional studies demonstrated that increased levels of mRNAs encoding several other ribosomal proteins, including S6, S8, S12, L5, and P0, were present in colorectal tumors and polyps. These results suggest that there is increased synthesis of ribosomes in colorectal tumors and that this increase is an early event in colon neoplasia.

Eighteen cytidines are changed to uridines in the coding sequence of transcripts for cytochrome c oxidase subunit 2 (cox2) in maize mitochondria. The temporal relationship of editing and splicing was examined in cox2 transcripts by sequence analysis of spliced and unspliced cDNAs. Cloned cDNAs of unspliced cox2 transcripts ranged from clones with no edited nucleotides to completely edited forms, while spliced cDNAs were nearly completely edited. Incompletely edited transcripts in the nascent pool of unspliced transcripts represent intermediates of the editing process. These results indicate that editing proceeds without a strong directional bias and suggest that RNA editing is a posttranscriptional process.

Virus-specific cytotoxic T lymphocytes (CTL) recognize virus-derived peptides presented by major histocompatibility complex (MHC) class I molecules on virus-infected cells. Such peptides have been isolated from infected cells and were compared to synthetic peptides. We found previously the Kd- or Db-restricted natural influenza nucleoprotein peptides to coelute on reversed phase high performance liquid chromatography columns with certain peptidic by-products present in synthetic peptide preparations. Here we show by extensive biochemical and immunological comparison that the natural peptides in all respects behave as the surmised synthetic nonapeptides, and thus, must be identical to them. The absolute amounts of these natural peptides contained in infected cells could be determined to be between 220 and 540 copies by comparing with defined amounts of pure synthetic nonapeptides. The comparison of the natural Kd-restricted peptide with published synthetic peptides known to contain other Kd-restricted CTL epitopes suggested a new MHC allele-specific T cell epitope forecast method, based on the defined length of nine amino acid residues and on critical amino acid residues at the second and the last position.

Two spin-labeled derivatives of the ion conductive peptide alamethicin were synthesized and used to examine its binding and state of aggregation. One derivative was spin labeled at the C-terminus and the other, a leucine analogue, was spin labeled at the N-terminus. In methanol, both the C and N terminal labeled peptides were monomeric. In aqueous solution, the C-terminal derivative was monomeric at low concentrations, but aggregated at higher concentrations with a critical concentration of 23 microM. In the membrane, the C-terminal label was localized to the membrane-aqueous interface using 13C-NMR, and could assume more than one orientation. The membrane binding of the C-terminal derivative was examined using EPR, and it exhibited a cooperativity seen previously for native alamethicin. However, this cooperativity was not the result of an aggregation of the peptide in the membrane. When the spectra of either the C or N-terminal labeled peptide were examined over a wide range of membrane lipid to peptide ratios, no evidence for aggregation could be found and the peptides remained monomeric under all conditions examined. Because electrical measurements on this peptide provide strong evidence for an ion-conductive aggregate, the ion-conductive form of alamethicin likely represents a minor fraction of the total membrane bound peptide.

The properties of heparin and hirudin to inhibit thrombin from binding to the freshly-excised rabbit aorta wall were compared in vitro. When aorta segments were incubated with 125I-thrombin (4.4 +/- 0.4 nM) in the presence of heparin or hirudin, both anticoagulants inhibited 125I-thrombin binding to the endothelium in a concentration-dependent manner (IC50: 0.1 USP U heparin/ml; 0.1 ATU hirudin/ml). Endothelium-bound 125I-thrombin was displaced by either heparin (50% liberated at 4.1 U/ml) or hirudin (0.4 U/ml). Using de-endothelialized aortas, heparin inhibited thrombin binding by the exposed subendothelium (IC50: 1.8 U/ml) whereas hirudin was without effect. Neither heparin nor hirudin was able to significantly liberate thrombin bound to the exposed subendothelium. These observations suggest that both heparin and hirudin mask the binding site on thrombin to the endothelial cell membrane. A separate site on thrombin must bind to the subendothelium because only heparin inhibits binding. Thrombin, although bound reversibly to the endothelium, is bound irreversibly to the exposed subendothelium due, probably, to reaction with endogenous extracellular antithrombin activities (e.g. antithrombin-III, protease nexin-1).

Normal eukaryotic cells do not initiate mitosis until DNA replication has been completed. This requirement can be bypassed by exposing cells to certain chemicals. We report here that chemically induced premature mitosis is not readily achieved in all mammalian species. Although hamster cells underwent premature mitosis following treatment with caffeine, the protein phosphatase inhibitor okadaic acid, and the protein kinase inhibitors 2-aminopurine and 6-dimethyl-aminopurine, the mouse and human cells examined in this study displayed little or no response to any of these compounds. Differences in cell permeability or metabolism could not account for the species specificity of these drugs, because other biochemical and mitosis-promoting activities were apparent in human cells. Cell-type specificity can be explained, however, by the timing of cyclin B synthesis and p34cdc2/cyclin B complex formation during the cell cycle. Synthesis of cyclin B and formation of a p34cdc2/cyclin B complex, both of which are required for initiation of mitosis, were prevalent in hamster cells arrested in S phase but were absent or barely detectable in arrested human cells. In hamster cells, the hyperphosphorylated form of p34cdc2 was complexed with cyclin B and underwent tyrosine dephosphorylation during caffeine-induced premature mitosis. These findings indicate that the onset of mitosis is regulated somewhat differently among mammalian cell types and that these differences affect the vulnerability of cells to drug-induced mitotic aberrations and cytogenetic damage.

Six patients (three males and three females), mean age 35.2 years (range 31-43 years), with extensive venous thrombosis were studied. Initial laboratory data indicated that all patients had normal antithrombin III (ATIII), four patients had low protein C (PC), three patients had low protein S (PS) and two patients had low plasminogen. Four patients had high fibrinogen and all patients had reduced tissue-type plasminogen activator activity, elevated tissue plasminogen activator inhibitor and low fibrinolytic activity. All patients were treated with danazol, 5-7 mg/kg orally once daily. In all patients there was significant elevation of ATIII, PC, PS, and plasminogen, reduction in plasma fibrinogen and PAI and enhancement of fibrinolysis. During the 12-36 months period of follow-up, there were no symptoms or signs that suggested recurrence of thrombosis. Apart from weight gain of 5-10 kg and disturbed menstrual cycle in two women, no major side effects were seen. These data suggest that danazol is potentially useful therapy that may increase levels of natural anticoagulants in patients with thrombotic illnesses in which ATIII, PC and PS are low or normal. Further studies are needed to confirm these observations.

Sequence analysis of the 5'-flanking region of the gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) revealed several putative cis-regulatory elements. To delineate the function of these regulatory elements, various deletion mutants of the 5'-flanking region were connected to the reporter gene chloramphenicol acetyltransferase (CAT) and promoter activity was measured in transient transfection assays. Transfection experiments performed with the sequence extending from -508 to +59 produced a high-level transient expression of the CAT gene in BALB/c 3T3-SV-T2 and NIH 3T3 cells. Removal of the sequence from +16 to +59 which includes the second transcription start point at +43, a TATA-like box and 5'-untranslated sequences abolished the promoter activity. Deletion analysis of 5'-upstream sequences revealed that the region from positions -55 to +59 is sufficient to mediate a high CAT activity comparable to the level obtained with the construct -508/+59. Within this region are found a CAAT box, a TATA-like box and two putative GC boxes. A functional analysis of the promoter showed that the sequence from -55 to +59 is sufficient to respond to stimulation by serum.

To assess whether an abnormality in transferrin receptor expression or regulation could represent an underlying metabolic defect in the reticuloendothelial (RE) system in hemochromatosis, monocytes were analyzed for the expression of the transferrin receptor using a monoclonal antibody (Act II) to the transferrin receptor (CD71) and flow cytometric analysis. Hemochromatosis patients (n = 14), and normal volunteers with no clinical evidence of iron overload (n = 14) were studied. A significant inverse relationship was observed for the relationship between the expression of transferrin receptor on monocytes and log(hepatic iron concentration) in hemochromatosis patients (r = -0.59, P less than .02) and also for the relationship between the expression of transferrin receptor and log(serum ferritin) in normal volunteers (r = -0.90, P less than .001). There was no significant difference in the mean expression of monocyte transferrin receptor between hemochromatosis patients and normal volunteers. However, the expression of the transferrin receptor in hemochromatosis patients was disproportionately higher than would be predicted from the relationship between serum ferritin and transferrin receptor expression in normal volunteers. The inverse relationship of the monocyte transferrin receptor relative to body iron stores in hemochromatosis is consistent with observations in other tissues, and suggests that non-transferrin iron metabolism, including ferritin, requires further investigation in the RE cell in hemochromatosis.

Eight unrelated patients with recurrent thromboembolism, a family history of thrombosis, and plasma antithrombin III (ATIII) activity/antigen levels consistent with a diagnosis of heterozygous type I ATIII deficiency were studied by polymerase chain reaction/direct sequencing of ATIII gene exon-coding regions. Frameshift mutations of one base and two bases, respectively, were found to have occurred in two unrelated patients at the same GAG codon (Glu 245) within exon 4 of the ATIII gene. A literature search showed six further hitherto unrecognized deletion "hotspots" in four other human genes. These deletion-prone sites exhibited sufficient sequence homology with each other to derive a consensus sequence (T G A/G A/G G A/C), suggesting that deletion in human genes may not only be non-random but also sequence-directed.

Hereditary non-polyposis colorectal carcinoma (HNPCC) syndrome is characterized by early onset and multiple cancers of predominantly the proximal colon and occasionally other organs. The mode of transmission is compatible with autosomal dominant inheritance but the location and characteristics of the putative susceptibility gene are unknown. We performed linkage analyses with the aim of proving or excluding the existence of a susceptibility locus on 18q. This hypothesis was based on the frequent involvement of the DCC gene in colorectal carcinoma and on the previously reported linkage between HNPCC and the Kidd blood group locus (JK) also on 18q. Seven HNPCC families were tested with eight polymorphisms, including three from within DCC. The DCC locus could be excluded as the HNPCC susceptibility locus in five families in which the two point logarithm-of-odds scores were -3.66, -3.63, -4.12, -7.90, and -3.74 at the recombination fraction of 0.00. In the remaining two families linkage could be neither excluded nor confirmed. The added pairwise logarithm-of-odds score for all seven families was -22.65 at the recombination fraction of 0.00. Multipoint analyses of linkage in the seven families suggested exclusion of some 60 cM in the region DCC-D18S18-D18S22-D18S7 as the site for HNPCC susceptibility locus. In addition to DCC, the excluded portion comprises JK.

Five women with premature ovarian failure were studied in a randomized cross-over design to compare the biochemical effects of transdermal to oral estradiol administration when used in doses appropriate for endometrial preparation in a donor oocyte program. Patients randomly received increasing dosages of oral micronized or transdermal estradiol for 4 week, with progesterone added in the last 2 weeks, to mimic a normal hormonal cycle. Serum samples were assayed throughout treatment and compared to those from normally cycling premenopausal controls. In general, serum estradiol remained within the normal range in both treatment groups, whereas peak serum estrone levels were 10-fold higher in the orally treated group than those in the transdermally treated group. Serum levels of sex hormone-binding globulin, thyroid binding globulin, and renin substrate were all significantly elevated by day 14 in the orally treated patients and unchanged in the transdermal subjects. While plasminogen was unaltered by either route of administration, antithrombin-III levels fell with both treatments. Changes in gonadotropin levels were similar in both groups, with suppression of FSH by the end of the simulated cycles, but not into the normal premenopausal range. In conclusion, both estrogen replacement regimens provided near-normal serum estradiol profiles. However, despite the relatively high doses necessary to mimic a hormonally normal cycle, the transdermal route did not significantly alter the hepatic parameters studied, suggesting that this route of administration may have less adverse hepatic effects.

Reported is a case of successful pulmonary thromboembolectomy and inferior caval vein interruption in a patient with subacute massive pulmonary thromboembolism (PTE). He had been complicated by the right atrial and deep vein thrombosis due to type I familial antithrombin III (ATIII) deficiency. Pulmonary embolectomy and caval interruption was performed 20 days after the initial attack and followed by anti-coagulation therapy. Thrombi weighed 50 gm were removed mainly from the left pulmonary artery. The right atrial thrombus which had got smaller by the preoperative fibrinolytic therapy could not be found with right atriotomy. Respiratory function at rest and slightly elevated pulmonary arterial pressure was normalized post-operatively. Although the PTE in this patient was considered to be subacute by the histological findings of the fibrin thrombus removed, tapering of pulmonary arteries in PAG and obstructive intimal hypertrophy of the small vessels in the lungs suggests the chronic nature of the PTE (post-thrombotic obstruction of pulmonary arteries) caused by the hyper-coagulable state. In patients with familial ATIII deficiency, 50% of normal ATIII activity can be expected because they are heterozygotes, so the cardio-pulmonary bypass should be run with special caution to the heparin dosage. Caval vein interruption and warfarin therapy are recommended for prophylaxis of recurrent PTE.

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.