Evaluation of a radioreceptor assay to assess exogenous estrogen activity in serum of patients with breast cancer.

A radioreceptor assay (RRA) for the determination of total estrogen activity, was set up and used to assess the possible presence of exogenous molecules with estrogen activity in serum; a comparison was made with the specific radioimmunoassay (RIA) for the endogenous estrogen 17-B estradiol (17-B-E2). The assay was first performed on sera from healthy people taking estrogens in the form of oral contraceptives or lotions for local application whose total estrogenic activity in the blood was assumed to be abnormal. The assay was then performed on serum from 98 patients with early breast cancer and 20 patients with metastasis, not undergoing hormone therapy. A higher estrogen activity was found in 2.5% of sera compared to the activity found using the RIA method which is specific for endogenous estrogen 17-B-E2, the RRA/17-B-E2 ratio being higher than 3. Increased estrogen activity was found in 10% serum samples from digoxin treated cardiopathic patients, with an RRA/17-B-E2 ratio ranging from 4.4 to 20. The RRA assay could prove useful for showing up exogenous estrogen activity from various sources (drugs, food) in sera of people in whom estrogen stimulation could be potentially dangerous (i.e. in patients with hormone-sensitive tumors). This exogenous activity could support a certain degree of neoplastic stimulation and, therefore, unfavourably condition the patients' therapeutic response.     

Biochemical alterations in 7,12-dimethylbenz[a]anthracene-induced mammary tumors from rats subjected to caloric restriction

Caloric restriction reduces the incidence and progression of a broad spectrum of neoplastic diseases, yet little is known about the biochemical and molecular mechanisms involved. Profiles of enzyme activities of importance in cellular energy utilization were examined in 7,12-dimethylbenz[a]anthracene-induced (DMBA) mammary adenocarcinomas from rats fed ad libitum or calorically restricted diets. The diets provided equal nutrients except for fewer carbohydrate-derived calories; graded caloric restriction was 10, 20, 30 and 40%. The specific activities of hexokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, malic enzyme and fructose-1,6-bisphosphatase were all elevated to varying degrees in both large palpable and small, non-palpable tumors from calorically restricted hosts compared to activities in tumors from ad libitum-fed rats. Phosphofructokinase activity was increased in palpable tumors from calorically restricted hosts but markedly reduced in non-palpable tumors. These results suggest adaptive or compensatory alterations in tumor enzyme profiles in response to the altered nutritional state of the host.     

Successful pregnancy in a woman with congenital factor XIII deficiency treated with substitutive therapy. Report of a second case

A syndrome of marked fetal wastage is associated with congenital factor XIII deficiency in adult women. A previously unreported case of a woman with factor XIII deficiency is described, in which substitutive treatment with normal plasma or placental factor XIII concentrate permitted two normal pregnancies. Factor XIII activity was maintained above 1-2% with intermittent infusion of 300 ml to 450 ml of plasma every 14 days or of 500 units of concentrate every 21 days. This case confirms the only other case so far reported in which factor XIII substitutive therapy was able to permit a normal pregnancy in a woman with factor XIII deficiency and seems to suggest factor XIII to be involved in the process of annidation.     

Generation and characterization of peptide-specific antibodies that inhibit von Willebrand factor binding to glycoprotein IIb-IIIa without interacting with other adhesive molecules. Selectivity is conferred by Pro1743 and other amino acid residues adjacent to the sequence Arg1744-Gly1745-Asp1746

We have generated antibodies against a synthetic peptide corresponding to the sequence of human von Willebrand factor (vWF) between residues Glu1737-Ser1750 which includes the Arg-Gly-Asp sequence common to several adhesive molecules. Two anti-peptide antibodies, one polyclonal, and one monoclonal reacted with native vWF and inhibited its binding to platelet glycoprotein (GP) IIb-IIIa, but showed negligible cross-reactivity with fibrinogen, fibronectin, and vitronectin, three other molecules that contain the sequence Arg-Gly-Asp and bind to platelets. The structural bases for the specificity of the two antibodies were evaluated by testing the ability of peptides homologous to the parent sequence, but with single amino acid substitutions, to neutralize the binding of the two antibodies to vWF. The substitution of Pro1743, the residue immediately adjacent to the Arg-Gly-Asp sequence on the amino-terminal side, with Phe resulted in a peptide that failed to interact with either antibody. Thus, Pro1743 is important for maintaining a peptide conformation recognized by two antibodies specific for the GP IIb-IIIa-binding domain of vWF. Other residues important for optimal peptide reactivity with the polyclonal antibody were Ser1742, Arg1744, and Gly1745, whereas Gly1741, Gly1745, and Asp1746, but not Arg1744, were important for reactivity with the monoclonal antibody. The epitopes of both antibodies, therefore, included at least 2 of the residues in the sequence Arg-Gly-Asp considered the common cell-binding site of adhesive molecules that interact with GP IIb-IIIa. Nevertheless, both antibodies reacted only with vWF. These studies demonstrate that peptide-specific antibodies, unlike the promiscuous GP IIb-IIIa receptor, can recognize distinctive structural characteristics of the cell-binding domain of adhesive molecules imposed by residues adjacent to the sequence Arg-Gly-Asp.     

Chemical composition and antimicrobial activity of 2 Peruvian plants]

In this work we determined the hydrocarbon , sterol and triterpenoid composition of two plants used as antibacterial drug in traditional peruvian medicine, Berberis rariflora and Chenopodium multifidum. In both plants the presence of unsaturated hydrocarbons is very low, whereas the presence of odd hydrocarbons is considerable. The delta 7 sterols are more abundant than delta 5 sterols. We found also a great presence of stigmasterol derivatives in both plants, whereas lanosterol derivates are more abundant in B. rariflora. The microbiological tests shows for both plants an evident antibacterial activity against Gram(+) bacteria.     

von Willebrand factor. A reduced and alkylated 52/48-kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein Ib

We have purified a reduced and alkylated tryptic fragment of von Willebrand factor (vWF) which migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 52/48-kDa doublet, but behaved as a single 46-kDa species after partial deglycosylation. After extensive treatment with denaturants, the 52/48-kDa polypeptide retained its ability to inhibit ristocetin-induced platelet aggregation in the presence of native vWF, as well as aggregation induced by desialylated vWF alone. Therefore, the 52/48-kDa polypeptide interacts with the platelet glycoprotein Ib receptor even in the absence of ristocetin. Both the 52/48- and the 46-kDa species inhibited ristocetin-induced binding of the intact molecule to platelets, but did not affect thrombin-induced binding. Determination of the NH2-terminal sequence of both members of the doublet gave identical results: VTLNPSDPEHCQ. This provided additional evidence that differences between the doublet constituents were only of carbohydrate composition and established the position of this peptide within the vWF polypeptide chain of approximately 2050 amino acid residues as beginning with the residue tentatively designated 449. These studies suggest that native conformation is not necessary for binding of vWF to platelets at the glycoprotein Ib receptor and that a linear amino acid sequence following residue 449 defines a domain responsible for this interaction.     

Cloning and characterization of the gene encoding an esterase from Spirulina platensis

The gene encoding a 23 kDA serine esterase from the cyanobacterium Spirulina platensis has been identified, cloned, characterized and expressed in Escherichia coli. The primary structure of the esterase deduced from the DNA sequence displayed 32% sequence identity with the carboxylesterase (esterase II) encoded by estB of Pseudomonas fluorescens; the highest degree of homology is found in a stretch of 11 identical or highly conserved amino acid residues corresponding to the GXSXG consensus motif found in the catalytic site of many serine proteases, lipases and esterases.     

Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells. The use of pulse labelling directly in production medium

During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of ³⁵S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII against cleavage, probably by binding to rFVIII. This revised version was published online in July 2006 with corrections to the Cover Date.