Requirement for binding of catalytically active factor VIIa in tissue factor-dependent experimental metastasis.

Tissue factor (TF), the initiating cell surface receptor of the coagulation cascade, plays important roles in embryogenesis, angiogenesis, and tumor cell metastasis. It is controversial whether proteolytic function of TF complexed with its serine protease ligand VIIa is required for metastatic tumor dissemination. We show here in a model for TF-dependent experimental hematogenous metastasis, that TF supports metastasis by both proteolytic activity of the TF-VIIa complex and currently undefined functions of the cytoplasmic domain. We demonstrate that ligand binding of VIIa to TF is required for metastasis. Antimetastatic properties of covalently inactivated VIIa provide evidence that ligand binding is insufficient per se to support metastasis, emphasizing that proteolytic activity is necessary for the metastatic process. Ala or Asp mutations of cytoplasmic serine residues were introduced to preclude or mimic phosphorylation. In vivo analysis of these mutants suggests that local protease generation on the tumor cell surface does not serve simply to activate the cytoplasmic domain of TF by serine phosphorylation. Thus, extracellular functions of the catalytically active TF-VIIa complex cooperate with specific functions of the TF cytoplasmic domain to support the complex process of hematogenous tumor cell dissemination.     

Risk of recurrence of birth defects in Washington State

A population-based study was conducted using maternally-linked birth certificate records from Washington State for 1980–93 to evaluate the risk of birth defect occurrence among infants with pre-viously affected siblings, relative to infants whose siblings did not have birth defects. The risks of recurrence of similar and dissimilar defects were estimated, and the effects of change in paternity and/or city of residence were evaluated as proxies of genetic and environmental effects. At the first birth on record, 3322 women were identified in the linked certificates as giving birth to a child with a birth defect; 6620 women whose first birth did not result in an infant with a defect were randomly selected for comparison. Women with a malformed infant had an in-creased risk of having a malformed infant at the subsequent birth (Relative Risk = 1.9, [95% Confidence Interval (CI) = 1.5–2.4]), which did not vary by intervening changes in partner or residence. The risk of recurrence of the same general type of defect [RR = 11.7, 95% CI = 9.7–19.50] was much greater than that of occurrence of a dissimilar defect [RR = 1.5, 95% CI = 1.1–1.9]. This was consistent for all defect categories, and did not vary markedly by changes in partner or residence.     

Introduction: lipids and coronary disease--resolved and unresolved problems

To my mind, there are more unresolved problems regarding lipids and cardiovascular diseases than those which have been settled. While this may seem disappointing after 40 years of intensive research, the progress which has been made is remarkable and impressive. It is appropriate, therefore, to begin an introduction by outlining the issues which have been resolved and which are more or less internationally agreed. In doing so, I shall deliberately not digress into causes of coronary heart disease (CHD) other than lipids, even though other influences may be as or more important in relation to its pathogenesis.     

Comparative study between direct mild acid extraction and immunobead purification technique for isolation of HLA class I-associated peptides.

Identification of tumour-specific peptide(s) hidden within the groove of human leucocyte antigens is a crucial prerequisite for peptide vaccine therapy. Conventionally, the peptide(s) are isolated by mild acid extraction (MA) technique followed by sequential ultra-filtrations. Here we describe a new approach for peptide isolation using the immunobead purification (IB-P) technique in conjunction with reverse-phase high-performance liquid chromatography. The obtained data were validated by SDS-PAGE followed by the silver staining technique. The results can be summarised as follows: (1) Comparison of class I-associated peptides isolated from a bladder cell line before and after the correction of class I antigens by gene transfection followed by IB-P technique showed the presence of peptides only from the class I-corrected cells. The data were confirmed using the silver staining technique as a way of detecting individual peptide bands. (2) Whilst peptides could be isolated by both techniques, the MA method led to the isolation of peptides from both class I-negative and class I-positive Fen cell lysates. (3) The IB-P approach could be used for isolation of class I-associated peptides from a normal kidney tissue. The data showed the high efficiency of the IB-P approach for isolation of class I-associated peptides. Unlike the MA technique, where the presence of non- class I-associated peptides was a problem, the IB-P approach isolated only peptides associated with the class I antigens. In addition, the data showed the feasibility of extracting peptides from tissue fragments by the IB-P method. The approach presented here may assist the future development of peptide vaccine therapy in urological cancers.     

Early postnatal hyperthyroidism alters hippocampal circuitry and improves radial-maze learning in adult mice

Inbred mice show strain-specific differences in the hippocampal mossy fiber projection. These differences are most pronounced in the portion of the projection that forms synaptic connections with the basal dendrites of the CA3 pyramidal neurons [intra- and infrapyramidal mossy fiber (IIP-MF) projection]. We have previously demonstrated that the extent of the IIP-MF subfield is positively correlated with the capacity to perform a spatial radial-maze task and that an experimentally induced enlargement of the IIP-MFs, by means of postnatal thyroxine treatment, predicted the ability of adult two-way avoidance learning. In the present study, we tested whether this treatment would also influence radial-maze performance. Forty-five male mouse pups from the inbred strain DBA/2 (chosen because of scanty IIP-MF projection and poor radial-maze learning) were divided into three groups that received daily injections of either 2 micrograms L-thyroxine, an alkaline vehicle solution, or physiological saline. Treatment lasted from postnatal days 0 to 11. At the age of 3 months, these animals were tested in an eight-arm radial maze. The extent of their IIP-MF projections was measured by means of planimetry on Timm-stained sections. Thyroxine-treated animals made significantly fewer errors and had larger IIP-MF projections as compared to both control groups. Within each group, the individual variability of the IIP-MF projection was significantly and positively correlated with performance. We conclude that experimentally modified IIP-MF projections mediate processes underlying spatial working memory. It would appear that the hippocampal circuitry alterations induced by postnatal hyperthyroidism can counteract a hereditary lack of talent, albeit only partially and in selected populations.     

DNA transfection of a gene repressing aryl hydrocarbon hydroxylase induction

High mol. wt genomic DNA from a genetically dominant aryl hydrocarbonhydroxylase (AHH)-deficient mutant cell line derived from themouse hepatoma cell line Hepa-1 was used to transfect the parentcell line. AHH-deficient transfectants were recovered followingsingle-step selection in medium containing the carcinogen benzo(a)pyrene.The transfectants arose at a frequency of 2 x 10^–7. Thisfrequency was at least 4-fold greater than the frequency ofspontaneous forward mutation in this cell line. In another setof experiments, dominant mutant DNA was co-transfected alongwith the selectable plasmid pSV2ecogpt into parental Hepa-1cells. The frequency of co-transfection was determined to be3 x 10^–8. This frequency was 150 times greater than thatexpected on the basis of coincident but unrelated spontaneousmutation and plasmid uptake. Both types of transfectants werejudged, following somatic cell hybridizations, to possess thedominant phenotype of the mutant cell line, demonstrating thata trans-acting dominant negative regulator of AHH was transferredin these experiments. DNA transfection should therefore providea means for the molecular cloning of the gene that encodes thedominant regulator.     

Biological mechanisms and cardiovascular effects of omega-3 fatty acids

The mechanisms and cardiovascular effects of omega-3 fatty acids are reviewed. Omega-3 polyunsaturated fatty acids are the major ingredient found in commercially available fish oil products. The incidence of many diseases, including coronary heart disease, diabetes mellitus, and psoriasis, is lower in Eskimos, who ingest diets rich in omega-3 fatty acids, compared with European controls. Potential mechanisms by which these fatty acids cause their many physiologic effects include competing with omega-6 fatty acids for prostaglandin and leukotriene pathways and enhancing cell membrane fluidity by virtue of the high degree of unsaturation. Numerous studies have documented longer bleeding times and decreased platelet aggregation in subjects ingesting omega-3 fatty acids. Omega-3 fatty acids may reduce serum cholesterol concentrations by decreasing the synthesis of very low density lipoprotein and, therefore, low-density lipoprotein. Blood viscosity is significantly and uniformly lower in subjects receiving omega-3 fatty acids compared with controls. Potential risks of supplementation with fish oils include hypervitaminosis A and D, vitamin E deficiency, increased bleeding times, decreased platelets, and ingestion of contaminated fish. Supplementation with moderate amounts of omega-3 fatty acids appears to be relatively safe. Possible adverse effects include nausea, diarrhea, and a "fishy" taste. Properly controlled, long-term clinical trials are needed to determine whether supplementation with omega-3 fatty acids would be therapeutically beneficial in various patient populations and disease states.     

Pharmacokinetic and pharmacodynamic studies with 4′-epi-doxorubicin in nasopharyngeal carcinoma patients

Summary The plasma pharmacokinetic profile of 4-epidoxorubicin (epirubicin) was investigated in 28 patients with nasopharyngeal carcinoma (NPC) after single i.v. rapid infusions. All patients had normal liver and renal functions. Plasma concentrations of the parent compound were specifically determined by a high-performance liquid chromatographic (HPLC) method, with UV detection at 254 nm. Plasma levels of the compound were fitted to a three-compartment open model; a triexponential decrease in plasma concentrations with a long terminal plasma halflife (44.8±21.2 h) was observed in 27 patients. The respective mean (±SD) serum concentration at 72 h and the AUC, plasma clearance, and terminal elimination rate constant in complete responders were 7.67±1.98 ng/ml, 4,002±3,080 ng· h/ml, 26.6±12.9 l/h·m², and 0.009±0.007 l/h, whereas those in nonresponders were 4.96±1.8 ng/ml, 1,88±652.8 ng·h/ml, 44.4±15 l/h·m², and 0.017±0.006 l/h, respectively; these differences were significant (P(0.05). Epirubicin produced a 52% response rate, including 6 patients with a complete response, 8 with a partial response, 11 with no change, and 2 with progressive disease. No relationship could be found between the various pharmacokinetic parameters and either leukopenia, age, or sex. These observations strongly suggest that plasma clearance may be one of the determining factors affecting the response or nonresponse of NPC patients to epirubicin, and a dose adjustment according to plasma clearance would probably increase the response rate.