Antioxidant concentration effect on stability of Brazil nut (Bertholletia excelsa) crude oil.

Shelled and broken Brazil nuts easily lose quality, if not properly stored. Pressing is an alternative use of these nuts and the crude oil stability was studied. Our previous studies demonstrated that TBHQ (200 mg kg-1) was very efficient to prevent rancidity development in oils bottled in brown and clear glass. As TBHQ has higher price than other phenolic antioxidants like BHT and BHA, an oven test (at 63 degrees C) was conducted to determine the economical and best concentration of TBHQ for Brazil nut crude oil. An assay at ambient temperature was conducted in brown and clear glass flasks with and without the economical concentration of TBHQ calculated (83 mg kg-1) for 90 days. Acid, peroxide, and iodine indices and the absorptivity at 232 nm were determined. TBHQ, even at this low dosage, was very efficient in both brown and clear glass flasks. Peroxide value increased from 11.5 meq O2 kg-1 to average 15 and 35, in TBHQ and control samples after 90 days. The absorptivity at 232 nm remained at 1.3 in samples with TBHQ while the control increased to 1.6.

Ultraviolet spectrophotometric evaluation of corn oil oxidative stability during microwave heating and oven test.

The effect of microwave heating on the oxidative stability of corn oil was determined by absorptivity in the UV spectrum and by peroxide and acid values. Oil samples with antioxidants BHA/BHT (1:1; 200 mg kg(-)(1)), with and without citric acid, were heated in a microwave oven (800 W, 2450 MHz) for 0-36 min. Absorptivity at 232 and 270 nm increased during microwave exposure. Control values of absorptivity at 232 nm increased from 3.568 to 12.874 after 36 min of heating. Peroxide value showed a significant difference in the initial stage of heating (0-6 min), but after this time, the peroxide value decreased due to the instability of hydroperoxides at high temperatures. Control 232 nm absorptivities after 6 days in the oven test were similar to those after 32-36 min of microwave heating. Effective antioxidants in the oven test did not show any protection during microwave heating. UV spectrophotometry is a suitable tool for microwave oxidation monitoring.

Comparative Study of FISH, PCR and Cytogenetics on 25 Patients Submitted to Bone Marrow Transplantation (BMT) for Chronic Myelogenous Leukemia (CML); Which Tests can we Use in Routine Analysis Post BMT?

Chronic Myeloid Leukaemia (CML) shows an excellent response to allogeneic bone marrow transplantation (BMT) with a 60-80% long term disease free survival in recipients of unmanipulate marrow. The most frequent cause of treatment failure is leukaemic relapse, due to the re-emergence of malignant recipient clones. Clinical and haematological relapse is usually preceded by molecular evidence of relapse. Early detection of molecular relapse may allow intervention with immunotherapy such as donor lymphocyte infusions (DLI). This study was undertaken to compare results from two centres who employ either Fluorescent In Situ Hybridisation (FISH) or polymerase chain reaction (PCR) analysis of DNA polymorphisms as their routine method of detecting residual host cells following BMT for CML in order to establish (1) if these methods are equivalent for routine laboratory use in reporting of chimaerism results to the referring clinician, and (2) if these methods are beneficial for indicating new and early therapeutic strategies. FISH analyses for the X and Y chromosomes (in sex mismatched patients) and/or FISH for BCR and ABL loci were compared with short tandem repeat PCR (STR-PCR) and conventional karyotyping in serial analyses in 25 patients submitted to BMT for Philadelphia positive (Ph) CML. Comparison of all results on samples assessed between 1 and 13 years post BMT indicated that FISH and PCR, performed on the same bone marrow samples displayed similar results in more than 90% of patients in first 3 years after BMT which increased to a concordance rate of 100% in long term survivors. In contrast, comparison of FISH or PCR versus cytogenetic analysis indicated a low concordance rate, with less than 50% of samples showing similar results during all the follow-up period. Eighty percent of recipients (22 patients) had evidence of mixed chimaerism following BMT (initial level of positivity 1-6% recipient cells) during the follow-up period. This low percentage of recipient cells remained stable in 7 patients, while 9 patients reverted to a donor profile. All 16 patients are in haematological remission. In addition the 3 patients with complete donor chimaerism remain in remission. In the remaining 6 patients, a progressive increase in recipient cells occurred (progressive mixed chimaerism, PMC), and was followed by haematological relapse. We conclude that FISH and PCR can be used to monitor CML patients post BMT and transient or stable low level mixed chimaerism is not associated with leukaemia relapse, but PMC is predictive of imminent relapse and its detection may help to illucidate the timing of early intervention with donor lymphocyte infusion.

Functional properties of sunflower seed meal obtained by ethanol extraction.

The objective of this work was to determine the functional properties of sunflower seed meal var. Anhandy obtained through ethanol intermittent oil extraction in four concentrations (99 degrees GL, 96 degrees GL, 93 degrees GL and 90 degrees GL). Meal nitrogen solubility and dispersibility, and oil absorption capacities were evaluated. The highest protein solubility (70%) was obtained in 93 degrees GL extraction meal. 99 degrees GL and 90 degrees GL extracted meals showed the best water absorption performances (11.4 ml H2O/g protein), while 96 degrees GL meal was the best in oil absorption (7.3 ml free oil absorbed/g protein). The highest nitrogen dispersibility was found in 96 degrees GL and 99 degrees GL meals (1.6% dispersed nitrogen or ca. 27% yield). Nitrogen solubility essays in salt solutions indicated that pH 11 was the best; however, the yield was even lower than in aqueous solutions. Meals obtained with more concentrated ethanol-water solutions were indicated for further processing to concentrates and isolates.

Sunflower seed protein concentrates and isolates obtention from ethanol oil extraction meals--(technical note).

The objective of this work was to study and identify the necessary processing steps for obtaining good quality sunflower seed protein concentrate and isolate when the oil is extracted with ethanol. This work is part of a research project on using ethanol as renewable solvent for sunflower seed oil recovery and possible further processing of the meal. Both 99 degrees GL and 90 degrees GL ethanol were employed in the extractions to produce the concentrate. Isolates were obtained by treating the concentrate with NaOH and HCl solutions and final rinsing with acidified water. Both products were light in color and almost free from chlorogenic acid.

Molecular cloning of RhD cDNA derived from a gene present in RhD-positive, but not RhD-negative individuals.

The Rh blood group system plays a major role in immune and nonimmune hemolytic states. Although an Rh cDNA has been previously cloned, there is no information on which Rh antigenic protein it encodes. Using polymerase chain reaction (PCR) amplification, we have identified this original Rh clone, here designated Rh21, and an additional Rh cDNA clone, Rh13, that is 96% nucleotide- and 92% amino acid-identical to Rh21, with the substitutions scattered throughout the sequence. A molecular genetic approach was used to match this Rh clone with an Rh specificity. The mRNA transcript for Rh13 was present in reticulocytes from RhD-positive individuals, but was absent from the reticulocytes of RhD-negative individuals. Using conventional screening of genomic libraries, as well as PCR cloning, partial genomic clones for these two Rh cDNAs were obtained. Based on PCR analysis and Southern blots, the Rh21 gene was present in all individuals, but an intact Rh13 gene was only present in RhD-positive and not RhD-negative individuals. Thus, by correlating the presence of Rh mRNA and gene sequences with individual Rh phenotypes, we were able to establish that the new Rh13 cDNA clone represents the RhD protein.

Mapping of epitopes, glycosylation sites, and complement regulatory domains in human decay accelerating factor.

Decay accelerating factor (DAF, CD55) is a glycophospholipid-anchored membrane protein that protects cells from complement-mediated damage by inhibiting the formation and accelerating the decay of C3/C5 convertases. DAF deletion mutants lacking each of the four short consensus repeats (SCR) or the serine/threonine-rich region (S/T) were created by site-directed mutagenesis. These deletion mutants were expressed by stable transfection in Chinese hamster ovary cells for the purpose of mapping important structural and functional sites in DAF. The epitopes on DAF for 16 murine mAb were mapped by immunoprecipitation studies as follows: SCR1, 6; SCR2, 3; SCR3, 3; SCR4, 3; S/T, 1. Testing of 13 mAb showed complete blocking of DAF function only by 1C6 and 1H4, both directed at SCR3. The single N-linked glycosylation site was confirmed at a location between SCR1 and SCR2, and the multiple O-linked oligosaccharides were localized to the S/T region. Functional activity of DAF mutants was assessed by the ability of these transfected constructs to protect Chinese hamster ovary cells from cytotoxicity induced by rabbit antibody plus human complement. Removal of SCR1 had no effect on DAF function, but individual deletion of SCR2, SCR3, or SCR4 totally abolished DAF function. Surprisingly, deletion of the S/T region totally abrogated DAF function, but this could be restored by a fusion construct placing the four SCR domains of DAF onto the HLA-B44 molecule, implying that the O-glycosylated S/T region serves as an important but nonspecific spacer projecting the DAF functional domains above the plasma membrane. Overall, the creation of DAF deletion mutants has elucidated important structure-function relations in the DAF molecule.

Further evidence consistent with Yqh as an indicator of risk of gonadal blastoma in Y-bearing mosaic Turner syndrome.

An 8-year-old girl with some features of Turner syndrome and karyotype 45X/46XY had developed a bilateral gonadoblastoma in her rudimentary ovaries. Her normal Y chromosome showed the characteristic distal fluorescence, as seen in her father's. Another mosaic, this time 45X/46XidicY, and also with some Turner features had rudimentary ovaries, but no gonadoblastoma had developed at age 14. The nature of her idicY, which showed no fluorescent distal Yq and had one of the centromeres inactivated, was confirmed by in situ hybridisation with a Yp-specific probe. Using primers from a human Yp-specific sequence, we amplified DNA extracted from paraffin-embedded ovarian tissue from both cases, and from a normal testicle and a normal ovary as controls. The finding of the expected Y-derived PCR product in the rudimentary gonads from these mosaic patients indicates the presence of their Y chromosome in both. We discuss the validity of the findings, and the possible role of sequences in or near the fluorescent part of Yq in the origin of gonadoblastoma in Y-bearing mosaic Turner syndrome.

Frequency of deletion 508 among Irish cystic fibrosis patients.

We estimate the incidence of cystic fibrosis in Ireland to be at least 1 case per 1838 live births. We have analysed DNA from 44 Irish CF patients for the presence of deletion 508, using the polymerase chain reaction. The deletion was found in 76% of their chromosomes, and approximately 58% of the patients are homozygous for this deletion. Our results are not significantly different from those found in Canadian or UK patient populations, in which frequencies are higher than those found in Southern European countries.

Structure of the gene for human complement protein decay accelerating factor.

Decay accelerating factor (DAF) is a glycophospholipid-anchored membrane protein that is part of the regulators of complement activation (RCA) gene family located on human chromosome 1, band q32. These proteins, beginning at their amino terminus, consist largely of multiple copies of an approximately 60 amino acid short consensus repeat (SCR). A DAF cDNA clone was used to identify overlapping bacteriophage genomic clones. The human DAF gene spans approximately 40 kb and consists of 11 exons. The length of these exons and introns varies considerably, with the exons ranging from 21 to 956 bp and the introns ranging from approximately 0.5 to 19.8 kb. SCR I, II, and IV are all encoded by single exons; however, SCR III is encoded by two separate exons, with the splice junction occurring after the second nucleotide of the codon for the glycine residue at position 34 of the consensus sequence. This feature has also been found in CR1, CR2, membrane cofactor protein, and murine factor H. Following the SCR in DAF is a 76 amino acid serine/threonine-rich domain encoded on three separate exons. Exon 10 encodes the Alu family sequence that has been found as an insert in a minor class of DAF cDNA, thus indicating that this mRNA arises by standard alternative splicing. The last DAF exon, which comes after the largest intron of 19.8 kb, encodes the hydrophobic carboxy terminus and the 3'UT region. The nature of the signal that directs posttranslational attachment of a glycophospholipid anchor to DAF is not known, but that signal is apparently spread over three exons and greater than 20 kb. An analysis of the DAF gene provides additional evidence for the common evolutionary heritage of the RCA gene family. The exon/intron structure of this gene will facilitate experiments aimed at understanding the functions of the various domains of DAF.

Sequential sampling in clinical cytogenetics: a quality control viewpoint.

We have observed that there is some resemblance between the problems of sampling in industrial quality control and the process of diagnosis of mixed cell populations in cytogenetics. This resemblance enabled us to draw from the methodology of the former science to solve some diagnostic problems in the latter. We considered which of the several sampling procedures available for quality control would be more efficient and more suitable in clinical cytogenetics, concluding that 'sequential sampling' combines both features. We also studied the effect that some abnormalities due to technical factors had on sample size required to reach a diagnosis with a given level of confidence. We give a set of tables for sequential sampling in diagnostic laboratories, illustrating their use in discriminating between mosaicism and pseudomosaicism in prenatal diagnosis and in the diagnosis of the fragile X syndrome. Finally, we discuss the merits of sequential sampling as shown here, comparing it with the current method used in cytogenetics to exclude chromosomal mosaicism.

Molecular cloning and chromosomal localization of human membrane cofactor protein (MCP). Evidence for inclusion in the multigene family of complement-regulatory proteins.

Membrane cofactor protein (MCP), a regulatory molecular of the complement system with cofactor activity for the factor I-mediated inactivation of C3b and C4b, is widely distributed, being present on leukocytes, platelets, endothelial cells, epithelial cells, and fibroblasts. MCP was purified from a human T cell line (HSB2) and the NH2-terminal 24-amino acid sequence obtained by Edman degradation. An oligonucleotide probe based on this sequence was used to identify a clone from a human monocytic (U937) cDNA library. Nucleotide sequencing showed a 43-bp 5'-untranslated region, an open reading frame of 1,152 bp, and a 335-bp 3'-untranslated region followed by a 16-bp poly(A) track. The deduced full-length MCP protein consists of a 34-amino acid signal peptide and a 350-amino acid mature protein. The protein has, beginning at the NH2 terminus, four approximately 60-amino acid repeat units that match the consensus sequence found in a multigene family of complement regulatory proteins (C3b-receptor or CR1, C3d-receptor or CR2, decay-accelerating factor, C4-binding protein, and factor H), as well as several other complement and non-complement proteins. The remainder of the MCP protein consists of 25 amino acids that are rich in serine and threonine (probable site of heavy O-linked glycosylation of MCP), 17 amino acids of unknown significance, and a 23-amino acid transmembrane hydrophobic region followed by a 33-amino acid cytoplasmic tail. The MCP gene was localized to human chromosome 1, bands 1q31-41, by analysis of human x rodent somatic cell hybrid clones and by in situ hybridization. This same genetic region contains the multigene family of complement-regulatory proteins, which is thereby enlarged to include the functionally and structurally related MCP.

A computer model for the study of segregation in reciprocal translocation carriers: application to 20 new cases.

We report on a computer program that, given the breakpoints and the chromosomes involved in a translocation, generates all the possible imbalanced gametes, calculates their corresponding imbalances, and arranges them in order of increasing imbalance. When compared to current, more cumbersome criteria from the literature, both methods agreed on 196 cases of 199 (greater than 98%). When compared to observed data from families with aneuploid offspring, both our program and the other reported methods yield a rate of accurate prediction of 87%. The use of the program is illustrated in 20 new translocations from our laboratory. The possible influence of crossing over in meiosis I in altering the gamete that is most likely to be passed to aneuploid live births is discussed.

The fragile X syndrome: the patients and their chromosomes.

We have reviewed recent publications, mostly from 1980 onwards, concerned with the problem of identifying patients with the fragile X chromosome and mental retardation, considering the two practical sides of the problem, that is, identification by their external appearance and by chromosomal studies. We conclude that this condition covers a large range of physical findings which occur in varying degrees in people with the chromosome marker. We have tried to clarify the existent criteria that have to be considered for an accurate cytogenetic diagnosis.

Tables for the cytogenetic study of fragile X chromosomes for diagnostic purposes.

The frequency of expression of the fragile X chromosome varies from patient to patient. Many cases have been reported showing frequencies of less than 2%. With such low frequencies, the risk of erroneous diagnosis is great unless the appropriate number of cells is studied. We present here Tables based on the binomial distribution relating the frequency of expression of the fragile X in a patient with the sample size required to obtain a probability P of correct diagnosis according to different criteria.