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L-Asparaginase (ASP), a chemotherapeutic agent used in the treatment of children with acute lymphoblastic leukaemia (ALL), is linked to thromboembolic complications secondary to an acquired deficiency of antithrombin III (ATIII). Fresh frozen plasma (FFP) is used to prevent and/or treat thrombotic complications in these children. However, the effect of FFP on plasma concentrations of ATIII and biochemical markers of activation of coagulation has never been tested. In this study, FFP (20 ml/kg) was administered to eight children with ALL receiving ASP in the consolidation phase of their treatment. Plasma samples were drawn pre-infusion, and following infusion at 1, 24, and 48 hr. Prior to the FFP infusions, plasma concentrations of prothrombin, fibrinogen, alpha 2-macroglobulin, heparin cofactor II, protein C, and protein S were similar to levels in healthy children. Only plasma concentrations of ATIII were significantly decreased (0.55 U/ml). Following FFP infusions, there was no statistical or clinically important increase in plasma concentrations of any coagulation protein at any time point. Pre-infusion plasma concentrations of markers of endogenous thrombin generation (thrombin-antithrombin III complexes (TAT)) and activation of the fibrinolytic system in response to activation of the coagulation system (D-dimer levels) were significantly increased. However, FFP had no statistical or clinically important effect on concentrations of these markers. We conclude that FFP administration for the prevention and treatment of acquired ATIII deficiency secondary to ASP has no demonstrable benefit on plasma levels of coagulation proteins and is unlikely to be of clinical benefit.

Recent studies have shown that fibroblast growth factors (FGFs) need to interact with cell-surface heparan sulfate proteoglycans (HSPGs) in order to bind to and activate FGF receptors. In this paper, three major heparin-binding proteins, histidine-rich glycoprotein (HRG) and antithrombin III (ATIII), which are constitutively present at high concentrations in plasma, and platelet factor 4 (PF4), which is released locally at high concentrations by degranulating platelets, were tested for their ability to act as modulators of FGF activity by competing with the FGFs for cell-surface HSPGs. HRGs from both chicken and human, and human PF4, were demonstrated to compete with each other and with acidic FGF (aFGF) and basic FGF (bFGF) for binding to BALB/c 3T3 cell-surface HSPGs, whereas ATIII did not compete. Thus, HRG, PF4, aFGF, and bFGF all interact with the same HS chains on the 3T3 cell surface, either binding to the same or binding to adjacent saccharide sequences on the chains. In terms of their relative binding affinity for cell-surface HSPGs, the hierarchy was shown to be PF4 > or = bFGF > aFGF = cHRG > hHRG. HRG was also shown to significantly inhibit both FGF-stimulated and endogenous 3T3 cell DNA synthesis. HRG also binds to extracellular matrices (ECM), originating from bovine corneal endothelial cells, in a heparin-inhibitable manner. Indeed, both HRG and PF4, at physiological concentrations, were shown to effectively inhibit the binding of 125I-aFGF and 125I-bFGF to ECM. In addition, HRG was able to displace biologically active bFGF from the ECM. On the basis of these findings, it is proposed that HRG and PF4 may act as positive regulators of FGF activity by displacing FGF from the ECM or basement membrane and making FGF available to responsive cells. Alternatively, they could act as negative regulators by masking HSPGs on responsive cells and preventing FGF receptor activation.

The deposition of 8-to-10-nm filaments into inclusion bodies is a fundamental cellular change that occurs in several degenerative processes of many tissues. However, little is known about the pathological filaments including whether the filaments assemble by the same mechanisms that govern the assembly of normal intermediate filaments. We have addressed this issue by studying the in vitro reassembly of the cytokeratin filaments that are deposited into experimental murine Mallory bodies (MBs) but have not yet become covalently crosslinked components of the MB. The reassembly process of both normal hepatocellular and MB-derived cytokeratins (CKs) was similar and characterized by a hierarchy of protofilament and protofibrils with a prominent axial periodicity of approximately 21 nm (normal hepatocellular CK, 20.7 +/- 2 nm; MB-derived CK, 20.1 +/- 2 nm). Purified MB-derived CK and normal hepatocellular CK comigrated in polyacrylamide gel electrophoresis indicating composition by similar CK isoforms. These results indicate that intermediate filaments formed from MB-derived CK are indistinguishable from filaments assembled from normal CK. On this basis, we conclude that the intermediate filaments that form inclusion bodies are not aberrantly assembled but become aggregated and post-translationally modified after their initial formation.

Epidemiological investigation showed that N-methylbenzylnitrosamine (NMBzA) has been associated with increased incidence of esophageal cancer (EC) in Linxian county, a high incidence area. In present study, our results indicate that NMBzA can induce amplification and over-expression of EGFr gene in human fetal esophageal epithelium (HFE) treated with NMBzA for 24 hours as shown by southern blot assay and immunohistochemistry. The papillary hyperplasia was induced in HFEs that cultured with NMBzA for 1 to 3 weeks. Amplification of c-myc and int-2 gene in HFEs treated by NMBzA for 1 week and 3 weeks was found, respectively. Deletions of p53 and Rb gene were found in human fetal esophageal carcinomas induced by NMBzA. Overexpression of p53 protein in human fetal esophageal carcinomas detected by immunohistochemical methods indicates that p53 gene mutation(s) may be occured. The HFE explants treated in vitro with NMBzA for 3 weeks were inoculated subcutanously into balb/c nude mice. No tumor was found in 5 months after inoculation, suggesting that only changes of oncogene(s) are insufficient to induce full transformation. Other genetic alterations (such as functional inactivation of Rb or/and p53 tumor suppressor genes) may be necessary in the further progression of malignant lesions.

<h4>Objective</h4>To discover the frequency of presentation, the aetiological distribution and clinical importance of a moderate rise in transaminases, which were found by chance in patients free from hepatic symptomology who were attending primary care clinics.<h4>Design</h4>Descriptive study of a crossover type.<h4>Setting</h4>La Victoria Health Centre in Valladolid.<h4>Patients</h4>The patients attended at any of the six general medical clinics between january and june 1993, in whom a moderate rise in transaminases was detected by chance and confirmed after a one-month interval.<h4>Measurements and main results</h4>Hepatic function tests were performed on 1,285 patients, in 212 of whom biochemical function was found to be disturbed. 137 of these 212 were studied in line with a diagnostic procedure worked out in collaboration with the Area specialist, after 75 had been eliminated for various reasons. The following aetiological distribution was observed: Viral 37.2% (Anti VHC + 30.7%, Ag HBs + 6.5%), ethylic 35.03%, metabolic 15.32% and medicinal 8.77%. Hemochromatosis was diagnosed in 2 patients; and a possible aetiology could not be established in 3. It was possible to perform a biopsy on 11 patients, with the suspected aetiology being confirmed in all of them.<h4>Conclusions</h4>The likelihood of the primary care doctor correctly diagnosing hepatic illnesses is high, given that we could establish an aetiology in 97% of cases. The most common aetiologies were viral and ethylic.

This study attempts to estimate the status of estrogen and progesterone receptors (ER and PR) in tissue of invasive breast carcinoma. Identical stereologic parameters of nuclear ultrastructure, of normal and malignant epithelium in the same breast were compared. Samples of normal and malignant epithelium of 30 surgically amputated breasts were analysed morphometrically. The concentration of ER and PR in the malignant epithelium was estimated by means of the dextran-coated charcoal (DCC) method. We measured the mean nuclear area (A), nuclear specific density (Sv/Vv) and nuclear numerical density (Nv) of epithelium. The comparison of such models of normal and malignant epithelium nuclear ultrastructure of the same breast demonstrated: a) the difference between Sv/Vv of nuclei very sensitively and specifically marks the ER status in malignant tissue (overall efficiency = 80%, p < 0.01), and b) the difference between A, Sv/Vv and Nv marks the concordant ER and PR status with identical overall efficiency. Minimal individual differences in size, surface structure and shape, observed between nuclei of malignant epithelium of positive concordant ER and PR status and nuclei of normal epithelium demonstrated that the highest morphological similarity is associated with biochemical similarity of cells exposed to the identical media of steroid hormones.

The series of genetic changes leading to malignancy in colorectal cancer is well reported. This includes mutational activation of the proto-oncogene Ki-ras and mutation/deletion of the p53 tumour suppressor gene. The frequency of these mutations was investigated in a panel of 52 colorectal cancer patients using a combination of immunocytochemistry and non-radioactive, digoxigenin-labelled in situ hybridisation. Sixty two per cent (32 of 52) of the study population were positive for p53 overexpression and 36% (19 of 52) positive for Ki-ras mutation. Twenty seven per cent (14 of 52) of the patients expressed both mutations. Mutation of either the p53 or the Ki-ras gene did not correlate with Dukes's stage, tumour differentiation or 5 year survival rate of the patients. Most of the rectal carcinoma specimens (11 of 15) showed p53 over-expression but the significance of this was not supported statistically. Thus detection of molecular changes is becoming more amenable to incorporation into routine histological carcinoma assessment because of the advent of non-radioactive labelling in in situ hybridisation and antibodies suitable for paraffin wax embedded specimens. The significance of these mutations in disease prognosis, however, remains questionable.

Androgen-induced growth factor (AIGF) is an autocrine growth factor for androgen-dependent SC-3 cells, which is induced by androgen stimuli. To elucidate the mechanism of the progression from hormone-dependent to -independent tumor, we transfected an expression vector of cDNA encoding AIGF into SC-3 cells and established a stable transfectant (A1) expressing AIGF. A1 cells showed enhanced DNA synthesis. This enhanced DNA synthesis was blocked by exposing the cells to AIGF antisense oligonucleotides, heparin, or suramin, indicating that enforced AIGF expression is responsible for the increase in DNA synthesis. However, A1 cells did not grow in serum-free medium unless stimulated with androgen. Recloning from A1 cells in semi-solid agar supplemented with fetal calf serum but without androgen quickly generated an autonomous subline that was able to grow rapidly in the serum-free medium irrespective of androgen stimulus. Mock-transfected SC-3 cells failed to form any colony under identical conditions. These results suggest that stable expression of AIGF alone is not sufficient for, but facilitates the conversion of SC-3 cells from androgen-dependent to -independent phenotype.

The present paper describes a mathematical model of the kinetics of the extrinsic coagulation cascade in vitro. The coagulation factors FI, FII, FV, FVII, FX, heparin and antithrombin III (ATIII) as well as soluble fibrin polymers are considered. The effect of single-factor deficiencies of the factors II, V, VII and X, diseases like hypo- and dysfibrinogenaemia, hepatic insufficiency, inhibited polymerisation by degradation products, heparin therapy with and without ATIII deficiency and coumarin therapy on prothrombin time can be portrayed. Physiology of coagulation is represented in a dynamic mathematical model as a differential equation system. The model is based on three reaction types: enzymatic cleavage, complex formation and polymerisation. The model was implemented in a continuous simulation program on a personal computer using the Pascal programming language. Unknown rate constants were estimated by chi 2 fit. Prothrombin time calculated by the model was compared to the training set of 20 plasma samples. In most but not all cases the model harmonized quite well with the coagulometric data.

Although osteoarthritis is characterized by a uniform pattern of clinical and radiological manifestations, it is a syndrome that can be produced by a variety of causative factors. Rare causes of osteoarthritis can be categorized as follows: 1) systemic metabolic disorders due to known biochemical and/or genetic abnormalities, such as hemochromatosis, ochronosis, Wilson's disease, Ehlers-Danlos syndrome (and probably the "idiopathic" joint hypermobility syndrome), sickle cell anemia, and thalassemia; 2) endocrine disorders, such as acromegaly, whose joint manifestations are now well-known, and hypothyroidism; 3) Paget's disease of bone, osteopetrosis (which induces changes in bone elasticity), and other systemic bone diseases; 4) dysplasias, which form a vast group including familial polyepiphyseal dysplasia, spondyloepiphyseal dysplasia congenita (especially its milder forms), Stickler's syndrome, osteo-onychodysplasia, Kniest's dysplasia, trichorhinopharyngeal syndrome, and a group of diseases that affect the epiphyses; 5) endemic forms of osteoarthritis, e.g., Mselini disease, Kashin-Beck disease, and Malnad disease, which are unknown in western Europe but have been reported to affect thousands of individuals in endemic areas. All these disorders are usually responsible for premature osteoarthritis, whose presentation sometimes bears the imprint of the causative abnormality but can be identical to that of common osteoarthritis. The effects of toxic substances (Kashin-Beck disease) or genetically-determined collagen II abnormalities (epiphyseal dysplasias) may explain the occurrence of these rare forms of premature osteoarthritis.

N-Cyclohexyl-N'-isopropylcarbodiimide was prepared and used for peptide couplings by various procedures. Reaction between Z-Val and Gly-OEt was used for yield studies and determination of the extent of the side reaction in solution. The value of this reagent for solid peptide synthesis was demonstrated by the synthesis of sequences comprising the 65-74 residues of acyl carrier protein. The efficiency of activation was determined by competition experiments. From all these studies the conclusion can be drawn that N-cyclohexyl-N'-isopropylcarbodiimide is comparable to or even better than the commonly used DCC for mediation of peptide bond formation in solid phase synthesis. By virtue of the solubility of N-cyclohexyl-N'-isopropylurea in dichloromethane, the carbodiimide seems to be a good candidate for practical execution of peptide bond formation by the standard carbodiimide procedure.

Deltorphins I and II (Tyr-D-Ala-Phe-Asp-Val-Val-Gly NH2 and Tyr-D-Ala-Phe-Glu-Val-Val-Gly NH2) display a high degree of delta-opioid receptor selectivity. Since they lack the intervening Gly3 residue found between the Tyr and Phe aromatic moieties in pentapeptide enkephalins, deltorphins I and II resemble a previously described series of cyclic tetrapeptides based on Tyr-c[D-Cys-Phe-D-Pen] (JOM-13). With the goal of development of structure-activity relationships for deltorphins and comparison with that of the cyclic tetrapeptides, ten analogs of deltorphin I were synthesized in which Phe3 was replaced with specific aromatic and nonaromatic amino acids with varying physicochemical properties. Results indicated that analogs containing the bicyclic aromatic amino acids 3-(1-naphthyl)-L-alanine [1-Nal; Ki(mu) = 767 nM, Ki(delta) = 7.70 nM], 3-(2-naphthyl)-L-alanine [2-Nal; Ki(mu) = 1910 nM, Ki(delta) = 49.2 nM], tryptophan [Ki(mu) = 1250 nM, Ki(delta) = 23.9 nM], and 3-(3-benzothienyl)-L-alanine [Bth; Ki(mu) = 112 nM, Ki(delta) = 3.36 nM] were fairly well tolerated at mu- and delta-receptors, though affinity was compromised to varying degrees relative to deltorphin I. Shortening the Phe side chain by incorporation of phenylglycine (Pgl) was detrimental to both mu (Ki = 4710 nM) and delta (Ki = 15.6 nM) binding, while extension of the side chain with homophenylalanine (Hfe) enhanced mu binding (Ki = 67.8 nM), leaving delta affinity unaffected (Ki = 2.64 nM). Substitution with nonaromatic amino acids valine and isoleucine led expectedly to poor opioid binding [Ki(mu) = > or = 10,000 nM for each, Ki(delta) = 160 and 94.7 nM, respectively], while peptides containing cyclohexylalanine (Cha) and leucine surprisingly retained affinity at both mu (Ki = 322 and 1240 nM, respectively) and delta (Ki = 10.5 and 12.4 nM, respectively) sites. In general, these trends mirror those observed for similar modification in Tyr-c[D-Cys-Phe-D-Pen].

Antithrombin III (ATIII) is a member of the serine protease inhibitor (serpin) family. As a step towards a better understanding of the heparin-binding mechanism of mammalian ATIIIs, the amino acid sequence of porcine ATIII was established by sequence analysis of the peptides derived from cyanogen bromide cleavage and enzymatic digestion with lysyl endopeptidase, V8 protease, and trypsin. Porcine ATIII was found to consist of 431 amino acid residues, with a calculated molecular weight of 48,930 without carbohydrate. Its molecular weight with 16.4% carbohydrate was estimated as 56,955, which is in good agreement with the value determined by SDS-PAGE. Porcine ATIII showed high sequence similarity to other mammalian ATIIIs, including the reactive site, heparin-binding basic amino acid residues, and disulfide bonds. The most notable feature of porcine ATIII was that it possesses only three carbohydrate chains, at Asn136, 156, and 193, whereas other mammalian ATIIIs have four, additional chain being at Asn97; this is replaced by Asp in porcine ATIII. In the case of human ATIII, the chains are at Asn96, 135, 155, and 192. The heparin-binding affinities of human and porcine ATIIIs were compared using an immobilized heparin column. Porcine ATIII eluted from the column with a peak at an NaCl concentration of 924 mM while human ATIII eluted at 838 mM NaCl. Neuraminidase treatment of each ATIII enhanced the heparin-affinity to the same extent. These results suggest that in spite of the high degree of amino acid sequence identity between porcine and human ATIIIs (91% identical), porcine ATIII has a higher heparin-binding affinity than human, because it lacks a carbohydrate chain at Asp97.

Although it is widely accepted that tumor suppressor genes play an important role in the genesis and progression of human cancer, little is known about genetic events that accumulate during multistage lung carcinogenesis. Thus, to determine a subset of tumor suppressor genes that are involved in the genesis and progression of non-small cell lung carcinoma (NSCLC), 22 brain metastases and 23 stage I primary lung tumors were examined for allelic losses at 40 loci on 10 chromosomes including the loci of 5 tumor suppressor genes, APC, WT1, RB, p53, and DCC. The incidence of allelic losses on chromosomes 3p, 13q, and 17p was high (> 60%) in both primary tumors and brain metastases. In brain metastases, a high incidence of allelic losses (> 60%) was also observed at loci on chromosomes 2q, 18q, and 22q, and the incidence of allelic losses on these chromosomes in brain metastases was significantly higher than that in primary tumors (P < 0.05). In two cases of brain metastases with corresponding primary lung tumors, sequential accumulation of allelic losses during progression of primary lung tumors was observed on several chromosomes including chromosomes 2q and 18q. These results indicate that, besides loss of heterozygosity for chromosomes 3p, 13q, and 17p, loss of heterozygosity for chromosomes 2q, 18q, and 22q also occurs frequently in advanced NSCLCS. Thus, it is possible that loss of heterozygosity on chromosomes 2q, 18q, and 22q occurs late in the progression of NSCLC and/or causes phenotypic alterations of NSCLC cells into more aggressive ones.

<h4>Background/aims</h4>Basement membrane disruption is a characteristic of invasive carcinoma. Gelatinases (also called type IV collagenases) that degrade type IV collagen are believed to play an important role in tumor invasion and metastasis. Intraductal neoplasm (IDN) of the pancreas is a unique tumor that grows intraductally with an infrequent stromal invasion. However, in IDN, basement membrane anomaly and gelatinase expression have not been clarified. The aim of this study was to examine the relationships between basement membrane disruption and the expression of gelatinase A in relation to the biological behavior of IDN or pancreatic duct cell carcinoma (DCC).<h4>Methods</h4>Disruption of basement membrane and expression of gelatinase A were immunohistochemically investigated in 14 cases of IDN and 16 cases of DCC.<h4>Results</h4>Basement membrane was intact in all cases of IDN, whereas focal to severe disruption was observed in DCC. Stromal expression of gelatinase A was higher in DCC than in IDN, and the intensity of expression was related to the degree of disruption of the basement membrane in DCC.<h4>Conclusions</h4>Stromal expression of gelatinase A and disruption of basement membrane, common characteristics of invasive cancer, were not prominent in IDN. This may be related to the benign biological behavior of IDN.

The DCC tumor suppressor gene has been shown to be frequently deleted or its expression reduced or absent in colorectal, gastro-intestinal, pancreatic, prostatic, and breast carcinomas, and glioblastomas. By allelotype analysis using the DCC-flanking polymorphic marker D18S5 we have previously shown that allelic deletions at 18q21 occur in 40% of male germ cell tumors (Murty et al., 1994). In order to further understand the role of DCC gene in germ cell tumorigenesis, we evaluated deletions by loss of heterozygosity (LOH) and mRNA expression by RT-PCR in tumor tissues and cell lines. Analysis of 61 paired normal-tumor DNAs using the probes D18S5, JOSH 4.4 (a polymorphism within the DCC locus) and a (CA)n polymorphism in an intron of DCC revealed that 45% of GCTs had allelic deletions. In addition, two homozygous deletions were found in the DCC gene among 91 (61 used in the LOH analysis and an additional 30) tumor DNAs when screened with the cDNA probes (pDCC 1.65, pDCC 1.9 and pDCC 1.0). By RT-PCR analysis of four normal testes, nine GCT cell lines and 14 tumor tissues, DCC gene expression was detected in all four normal testes, while four (45%) GCT cell lines and one (7%) tumor specimen showed lack of expression. In addition, DCC expression was highly reduced in three (21%) tumor tissues. The high frequency of LOH at 18q21 was characteristic of seminomas as well as all subsets of non-seminomas in primary as well as metastatic states. Frequent allelic loss in all histologic subsets, homozygous deletions, and loss of expression of DCC suggest that suppression of this gene's function is an early event in GCT development.

Hereditary hemochromatosis is a prevalent inherited disorder with an estimated frequency of homozygosity of 0.2 to 0.45% in Caucasians. The disease is characterized by progressive iron overload until a massive accumulation of body iron occurs. Undetected, the disorder eventually can produce either cirrhosis, diabetes mellitus, cardiac disease, arthritis, or hepatocellular carcinoma or a combination of these manifestations. Early diagnosis and treatment prevents organ damage and normalizes life expectancy. Screening studies to detect hemochromatosis are most effectively accomplished by measurement of the serum iron and total iron binding capacity. Treatment is most effectively performed by frequent phlebotomy until body stores are empty and then 3 to 4 times yearly for life. The basic defect of hemochromatosis appears to increase iron absorption, decrease iron excretion, and produce preferential deposit of iron in hepatic parenchymal cells rather than Kupffer cells. The genetic abnormality of hemochromatosis is located on chromosome 6 in close association with the gene for HLA antigens. Recent speculation postulates that tumor necrosis factor may be involved in the etiology of this disease because of its location on chromosome 6 and its effect upon iron transport.

We report here the use of multiplex fluorescent polymerase chain reaction (PCR) for quantitative allele loss detection using microsatellites with 2-5 base pair repeat motifs. Allele loss of APC, DCC, p53 and RB1 in colorectal tumours has been reported previously using a variety of methods. However, not all workers used intragenic markers. We have used microsatellite polymorphisms which map within, or are closely linked to, these tumour-suppressor gene loci in order to determine whether these loci are indeed the targets for alteration in colorectal cancer. In addition, we have assayed two other tumour-suppressor genes, WT1 and NF1, to see whether they play a role in colorectal carcinogenesis. The putative metastasis-suppressor gene, NM23, was also investigated since there have been conflicting reports about its involvement in colorectal carcinogenesis. Allele loss was detected at the DCC (29%), p53 (66%), RB1 (50%) and NF1 (14%) loci and in the APC/MCC region (50%), but not at the WT1 or NM23 loci. These rapid, and mostly gene-specific, fluorescent multiplex PCR assays for allele loss detection could be modified to devise a single molecular diagnostic test for the important lesions in colorectal cancer.

A motif essential for the transcriptional activation function 2 (AF-2) present in the E region of retinoic acid receptor (RAR) alpha and 9-cis retinoic acid receptor (RXR) alpha has been characterized as an amphipathic alpha-helix whose main features are conserved between transcriptionally active members of the nuclear receptor superfamily. This conserved motif, which can activate autonomously in the absence of ligand in animal and yeast cells, can be swapped between nuclear receptors without affecting the ligand dependency for activation of transcription, thus indicating that a ligand-dependent conformational change is necessary to reveal the AF-2 activation potential within the E region of the nuclear receptor. Interestingly, we show that the precise nature of the direct repeat response element to which RAR/RXR heterodimers are bound can affect the activity of the AF-2s of the heterodimeric partners, as well as the relative efficiency with which all-trans and 9-cis retinoic acids activate the RAR partner.

The polymerase chain reaction and direct sequencing were used to determine the nature of the mutations in the antithrombin III (AT3) gene in seven unrelated patients with familial antithrombin III (ATIII) deficiency and recurrent venous thrombosis. Three novel mutations were found, two associated with a type I deficiency state (Pro80-->Thr and His120-->Tyr) manifesting reduced synthesis of ATIII. The other novel lesion (Met251-->Ile) was associated with a dysfunctional ATIII protein (type II ATIII deficiency) and is predicted to interfere either with a heparin-induced conformational change in the ATIII molecule or with docking to thrombin. A novel polymorphism (Tyr158-->Cys) was also found to occur in several individuals of Scandinavian origin.

The aim of the present study was to investigate loss of heterozygosity (LOH) or microsatellite instability in chronic myeloid leukaemia (CML) blast crisis at genomic locations which are known or postulated to harbour tumour suppressor genes. We studied 48 patients in blast crisis of myeloid (n = 31), lymphoid (n = 15), megakaryocytic (n = 1), or mixed lineage (n = 1) phenotype by comparing constitutional DNA extracted from buccal epithelial cells or chronic phase leucocytes with DNA obtained from blast crisis leucocytes. Twelve variable number tandem repeat loci from six different chromosomes were amplified by polymerase chain reaction using labelled primers, and fractionated on polyacrylamide gels. After autoradiography, length as well as intensity of the amplified products were compared between constitutional and blast crisis samples. LOH was scored as complete, partial or none in informative patients. Complete LOH was found in one patient at 8p22 and another at 13q14; partial LOH was detected in three patients at 11p13 and/or 11p15. No LOH was found at 6q27, 8p21, 18q21, 22q11-12 and 22q13 in any patient. Furthermore, no consistent difference in allelic length was observed in 517 paired amplifications indicating no microsatellite instability. We conclude that the Rb gene at 13q14, the Wilms tumour gene at 11p13, the DCC gene at 18q21, the neurofibromatosis 2 gene at 22q11-13 and uncloned tumour suppressor genes at 6q27, 8p21-22 and 11p15, as well as genes responsible for microsatellite instability, are unlikely to be involved in the progression of CML to blast crisis in the majority of patients.

The translocation from chromosome 8 to chromosome 21, t(8;21), associated with acute myeloid leukemia results in production of an AML1/MTG8(ETO) fusion transcript. The product of the AML1 gene contains an evolutionarily conserved 128-amino acid region referred to as the "Runt domain," which is necessary for binding to DNA at the PEBP2 site. A fragment of the AML1 protein containing mainly the Runt domain and the antisense oligonucleotide complementary to the fusion transcript strongly inhibited the growth and induced differentiation of cell lines derived from acute myeloid leukemia containing t(8;21). These results indicate that the transcriptional regulation through the PEBP2 site is critically important for growth and differentiation of t(8;21) leukemic cells and that the product of the chimeric gene is responsible for the maintenance of the leukemic phenotype.

This review examines selected areas of contemporary prostate cancer research in terms of the impact of prostatic cellular and histopathological heterogeneity. Prostate tumor progression is accompanied by dysregulation of multiple growth factor networks as well as disruption of normal patterns of cell-cell interactions. Molecular and cytogenetic studies demonstrate that prostate cancer results from the accumulation of several different genetic defects. No single event predominates, but modifications in tumor suppressor genes or functional elimination of the suppressor gene product are more common than activation of known oncogenes. Intratumor heterogeneity is also detectable at the genetic level. This further complicates efforts to correlate modifications at specific loci with progression or outcome. The development of new in vitro and in vivo systems for the study of human prostate cancer should increase our understanding of this complex disease. In each approach, knowledge of the histopathology of the normal and neoplastic prostate is essential.

We have shown that lipid peroxidation stimulates collagen-alpha 1 (I) gene transcription in cultured cells. Because iron is a transitional metal known to induce lipid peroxidation, we investigated whether hepatic lipid peroxidation modulates collagen gene expression in iron-overloaded rats. In this animal model of hemochromatosis, we show colocalization with iron in the hepatic acinar zone 1 of both lipid peroxidation and increased collagen-alpha 1 (I) transcripts, using immunohistochemistry for malondialdehyde-protein adducts and in situ hybridization, respectively. Iron overload stimulated the expression of the cytokine transforming growth factor-beta (TGF-beta) in acinar zone 1, in spite of the minor degree of hepatocellular necrosis and inflammation. The formation of reactive aldehydes and TGF-beta, both inducers of collagen gene expression, may play a role in the stimulation of hepatic collagen production in iron overload. These mechanisms could be a link between iron overload and fibrosis in genetic hemochromatosis.

<h4>Background</h4>This prospective study was intended to determine in a homogeneous population of children whether hemofiltration, performed during cardiopulmonary bypass rewarming, is able to improve hemodynamics and biologic hemostasis variables, to reduce postoperative blood loss, time to extubation, and plasma cytokines, and complement fragments.<h4>Methods</h4>Thirty-two children undergoing surgical correction of tetralogy of Fallot were randomly assigned to a hemofiltration or control group. Hemofiltration was performed with a polysulphone hemofilter during rewarming of cardiopulmonary bypass. Plasma clotting factors, D-dimers, antithrombin-III, complement fragments C3a and C5a, interleukin-1 beta, interleukin-6, interleukin-8, and tumor necrosis factor-alpha were measured before and after hemofiltration. Systemic mean arterial pressure, left atrial pressure, time to extubation, and postoperative blood loss were monitored.<h4>Results</h4>In the hemofiltration group, significant reductions in 24-h blood loss (250 (176-356) vs. 319 (182-500) ml/m2, median (minimum-maximum), time to extubation (15 (9-22) vs. 19 (11-24) h), plasma concentrations of C3a, C5a, interleukin-6, and tumor necrosis factor-alpha were observed compared to control. Arterial oxygen tension on admission to the intensive care unit was significantly greater in the hemofiltration group (136 +/- 20 vs. 103 +/- 25 mmHg, mean +/- SD). Significant increases in mean arterial pressure, clotting factors, and antithrombin-III were noted for the hemofiltration group. No intergroup difference was observed in left atrial pressure, platelets count, D-dimers, interleukin-8, and duration of stay in the intensive care unit.<h4>Conclusions</h4>Hemofiltration during cardiopulmonary bypass in children improves hemodynamics and early postoperative oxygenation and reduces postoperative blood loss and duration of mechanical ventilation. Hemofiltration is able to remove some major mediators of the inflammatory response.

Estrogen receptors (ERs) and progesterone receptors (PRs) were determined by an immunocytochemical assay (ICA) on fine needle aspirates (FNAs) from patients with primary, recurrent and metastatic mammary carcinoma, and the results were compared to those with the biochemical dextran-coated charcoal (DCC) method performed on the surgical sample in order to compare the two methods. The aspirates were suspended in a buffered saline solution, cytocentrifuged onto glass slides and immunocytochemically stained according to the protocol of commercial kits employing monoclonal antibodies specific for ER and PR. Immunocytochemical staining of malignant cells was evaluated on the basis of the percentage of stained cells; 10% staining was taken as the cutoff value. Fine needle aspiration biopsies (FNABs) from 107 breast carcinomas were analyzed immunocytochemically for ER and 31 of them for PR, also. The overall concordance between ICA and DCC was 88% for ER and 87% for PR. The sensitivity, specificity, and positive and negative predictive value of ICA on FNAs as compared to conventional DCC were 87%, 90, 97% and 63%, respectively, for ER and 85%, 100%, 100% and 56% for PR. These findings suggest that estrogen immunocytochemical assays and progesterone immunocytochemical assays on FNAs in breast cancer patients are reliable techniques for evaluating receptor status and can be useful in assessing ER and PR whenever surgical biopsy is not indicated and when information about ER and PR status is required at the time of the clinical diagnosis.