Biomedical subjects
V L Pascali
Publications and source records attributed to V L Pascali.
The use of immobilized pH gradients for the detection of human polymorphisms in the forensic identification of bloodstains.
Some polymorphic proteins (alpha 1-antitrypsin, orosomucoid, transferrin, group specific component, plasminogen) and enzymes (phosphoglucomutase, acid phosphatase, estrase D) were determined in bloodstain extracts by isoelectric focusing with carrier ampholytes (CA) and with immobilized pH gradients (IPGs) rehydrated with CA. IPGs yield superior results for typing of genetics markers in bloodstains since phenotypes are better distinguished and the bands are straighter and sharper in the presence of contaminants. Also, the sensitivity of IPGs with CA is similar to isoelectric focusing (IEF) with CA. A new variant, ACP*B1, found in Negroid west African populations and not found in Caucasians is described. Such a variant can only be determined by IPGs since its isoelectric point (pI 5.95) is close to that of the ACP*B (pI 6.05) variant.
Errors in sizing bands of hypervariable DNA profiles on autoradiograms: are they Gaussian?
The accuracy of procedures for sizing hypervariable restriction fragments by Southern blot analysis (SBA) has been tested under three different experimental conditions: (i) intrablot serial analyses: three heterozygous DNA profiles were tested 14 times each in the same gel electrophoresis; (ii) intralaboratory analyses: we replicated three profiles (six autoradiographic bands) in over 100 SBA experiments; (iii) interlaboratory analyses: 15 serial measurements produced in a recent collaborative study (Forensic Sci. Int. 1991, 49, 1-15) were taken into account. In these three cases, a typical U-shaped correlation curve between molecular size and coefficient of variation was found. We explain decrease of accuracy at both extremities of the gels in terms of: (i) enlarged shapes of bands and mean electrophoretic resolution at the cathode; (ii) diffusion and blurring of bands at the anodal edge. The three populations of data were subjected to a chi 2 test and to the Kolmogorov-Smirnov test in order to verify their compliance with normal distribution. Twenty-three out of 27 tests indicated no significant deviation from the assumption of Gaussian distribution. We recommend the adoption of tests for normality to validate the use of symmetric confidence intervals for calculating gene frequencies and asserting a match between adjacent bands.
New phenomenon of correlation between measurements errors pertaining to heterozygous DNA profiles.
Explore the source record for details and available documents.
Efficient, small scale electroelution of high molecular weight DNA from agarose gels by a miniature vertical electrophoresis cell.
A miniature, vertical electrophoretic system is described that allows quick elution and efficient harvesting of double-stranded, high molecular weight DNA fragments from agarose gels. A gel slice containing the band of interest is excised from the agarose gel after submarine electrophoresis and placed into an electrophoresis cell, assembled by inserting a truncated micropipette tip into a 2.0 mL polypropylene tube. The tip supports the agarose slice and is connected to dialysis tubing. Platinum wire electrodes are placed over the tip and at the bottom of the test tube. DNA molecules are quickly eluted into the dialysis bag (60 s to 10 min for bands ranging from 2 to 23 kb) and then easily recovered. The method is highly efficient and allows considerable time saving. Of special interest is its applicability to small starting amounts of DNA (less than 0.5 micrograms per band). Purity of the DNA is comparable, if higher, to that obtainable by conventional electroelution on dialysis bags and DEAE-cellulose membrane electrophoresis. The system is simple enough to be used as a routine method for eluting multiple bands in short times.
Estimating allele frequencies of hypervariable DNA systems.
Several polymorphisms of human DNA have been shown to be hypervariable due to the recurrence of a variable number of tandem repeats (VNTRs) in the lengths of allelic restriction fragments. The recurrence of allelic variants in this novel class of polymorphisms seems to comply well with a model of continuous random variables. Based on this assumption, we have compiled some simple algorithms for classification of continuous data and estimation of classes of relative frequencies and have implemented these routines for the management of databases storing hypervariable single locus DNA genetic systems. The algorithms are compiled in BASIC language and can be incorporated in task-oriented computer programs. Three procedures are discussed, based in turn on: (a) using predetermined, arbitrary classes; (b) point estimations of frequencies for single fragments using error measurements associated with the kilobase value assignment; (c) estimates of phenotype frequencies according to error measurements. Error measurements are obtained from a statistic of values pertaining to several restriction fragments (genomic controls) repeatedly tested in different experiments. Problems related to these approaches are discussed.
Characterization of genetic variants of human serum transferrin by isoelectric focusing: comparison between conventional and immobilized pH gradients, and application to a protocol for paternity testing.
The polymorphism of transferrin (Tf) is currently being studied by isoelectric focusing in carrier ampholyte-generated pH gradients, carrier ampholyte-separator pH gradients or in immobilized pH gradients. Details for obtaining reproducible results with each of the three procedures are outlined. The effectiveness of pretreatment of serum samples with ferrous/ferric salts is discussed, and incubation times optimized after spectrophotometric measurement of the monoferric Tf conversion. Most of the presently available commercial batches of carrier ampholytes do not reliably discriminate the six common TfC subtypes. Resolution of C1, C3 and C2 was achieved by adding 20 to 90 mM HEPES slab gels prepared with various carrier ampholytes. Isoelectric focusing in carrier ampholyte-separator pH gradients cannot be recommended as a standard typing procedure because the results strongly depend on the batch of carrier ampholytes. Tf subtype resolution was only achieved by using isoelectric focusing in immobilized pH gradients with pH slopes reliably reproducible from one experiment to another. Two major shortcomings of immobilized pH gradients are a marked tendency to protein precipitation at the application site and an interaction of proteins with the charged matrix. A protocol for Tf subtyping in immobilized pH gradients is described, based on prior desialylation of samples instead of pretreatment with iron. Sample entry into the matrix was optimized by addition of 5 mM Tris to the gels, and initially running them at low voltage. Recommendations are provided for the application of Tf typing for paternity testing.
Immobilized pH gradients in ultrathin-layer (100 micron) gels: avoiding capillarity by a 'squeezing-sealing mold' technique.
Several procedures for casting 100 microns ultrathin immobilized pH gradients are described. When acrylate/glass molding cassettes (Pascali et al., Electrophoresis 1987, 8, 371-373) are used two main problems are encountered: (i) a tendency of polymerization solutions, at the beginning of delivery, to spread across the glass surfaces with troublesome effects on the gradient stratification, and (ii) the raising of steep menisci at both extremities of the pH intervals, originating from capillarity phenomena and resulting in nonuniform gradients with bowed electrophoretic patterns. The first shortcoming was acceptably solved by increasing the density of sucrose gradients, and pouring them into prewarmed molding cassettes. The detrimental effect of menisci could be overcome by using a 'squeezing-sealing mold' technique. A molding cassette was endowed with a continuous, squared spacer frame, the upper side being open by inserting a wedged clip. A slight excess of polymerization solution was first dispensed into the cassette and squeezed away on removal of the clip. By completely excluding air from the molding cassette, uniform and well reproducible ultrathin gels could be cast. A major advantage of ultrathin immobilized pH gradient gels is the drastically shorter focusing time.
Determination of neutral haemoglobin variants by immobilized pH gradient, reversed-phase high-performance liquid chromatography and fast-atom bombardment mass spectrometry: the case of a Hb Torino alpha 43 (CE1) Phe----Val.
A neutral haemoglobin variant was identified by the combined use of different analytical methods. Isoelectric focusing on immobilized pH gradients (under denaturing conditions) allowed to detect and alpha chain variant. HPLC of tryptic digest showed that its amino-acid variation resided on T alpha 6 peptide (position 41-56). In the fast-atom bombardment (FAB) positive mass spectrum of the tryptic digest, the protonated molecular ion of the T alpha 6 peptide occurred 48 mass units lower than the normal T alpha 6 fragment, corresponding to a Phe----Val substitution. To a partial sequence determination (aminopeptidase digestion of the T alpha 6 peptide, followed by amino acid determination and FAB-MS analysis of the digestion-generated mixture) the substitution appeared to be on Phe43 (CE1). This variant is already known as Hb Torino. The procedure here described proved to be fast and simple, and feasible whenever neutral variants are supposed to occur.
Is sternal bone marrow needle biopsy still a hazardous technique? Report of three further fatal cases.
Three fatal cases of death following sternal bone marrow needle biopsy have come to the authors' attention in the past 10 years. The hazards of the technique may be related to the incongruous stylet guard-adjustment for the thickness and hardness of the sternal wall. All three cases involved suits for professional malpractice. Controversial aspects of these cases involving physician liability are discussed.
Unusual transferrin TF*D mutants in some Congo populations.
Transferrin allele (TF) distribution was investigated in three Bantu and one Babenga Pygmy groups of Congo, by isoelectric focusing on polyacrylamide (PIEF). In comparison with other Black populations, the four groups had a twofold higher TF*D frequency. Three different TF*D mutants were observed, the commonest type of which was identical by PIEF to a *D1 Caucasian allele. The classification of the other two is discussed. The homozygous TF D1 pattern was present in Babenga. The polymorphism of transferrin confirmed the uniqueness of anthropological traits in such Congo populations.
Vitamin D-binding protein in the perinatal period.
The molecular polymorphism and quantitative levels of serum vitamin D-binding protein (DBP) were ascertained in a group of preterm, full-term and hypocalcaemic full-term newborn infants. The serum concentration of DBP is not influenced by phenotype and increases with fetal age from the 32nd-33rd week of gestation until the 35th week of gestation. Neither different DBP allele distribution nor abnormal quantitative levels found among 29 hypocalcaemic full-term newborn infants.
[Transferrin and infertility: genetic-clinical study. Preliminary note].
Explore the source record for details and available documents.
Group-specific component subtypes along the Italian Peninsula.
Nine areas in Italy were analyzed for the frequencies of the Gc system (group-specific component). No significant discrepancy was found among their frequencies as they resulted after isoelectric focusing (IEF). When more accurately analyzed, gene frequencies revealed a microheterogeneity affecting one sample (city of Bari) against eight tightly clustered values.
Transferrin subtypes in cystic fibrosis.
The molecular polymorphism and quantitative data of serum transferrin (Tf) were ascertained in a group of fibrocystic patients, their parents and controls. Quantitative rates of pre-albumin, retinol binding globulin (RBG) and alpha-1-glycoprotein were also investigated as a reference for the evaluation of Tf quantitative data. Neither different allele distributions nor abnormal electrophoretic patterns were observed among CF patients. A slight lowering of Tf, pre-albumin and RBG, probably due to malnutritional condition in CF subjects was found.
Antitrypsin and Gc polymorphisms in some populations of Congo: an unusual, highly frequent mutant, PIS, in Bateke and Babenga.
Four population samples from Congo (Beti and Bateke from Cuvette area, Bateke and Babenga from 'Pool' area) were typed for the electrophoretic mutants of protease inhibitor (PI) and group-specific component (Gc), as part of a general survey on the genetic structure of these ethnic groups. An unusual PI mutant (tentatively called PIS*) had a singularly high frequency in the Bateke and the Babenga. Data shown here will help in ascertaining whether genetic differentiation could exist in Congolese populations.