A quantitative bacterial micro-assay for rapid detection of serum phenylalanine in dry blood-spots: Application in phenylketonuria screening

Phenylketonuria is an inherited metabolic disease, which is characterized by increased level of serum phenylalanine (Phe). The quantitative measurement of Phe in the serum is necessary to confirm the disease, and to distinguish phenylketonuria from other forms of hyperphenylalaninemia. In this study, we report a rapid and inexpensive micro-assay for simultaneous detection and quantitative measurement of serum Phe in dry blood-spots. Analysis of the standard curve showed a broad linear Phe range of 120-1800 micromol L(-1). Application of this method in conjunction with the standard Guthrie bacterial inhibition assay and high-pressure liquid chromatography in analyzing 34 samples from phenylketonuria patients and control samples produced comparable results, with the regression equation of Y= 0.994X + 0.996. The advantage of this method over the Guthrie bacterial inhibition assay is its ability to measure the serum Phe quantitatively without false positive results. The method was successfully applied to dried blood-spots as well as serum and whole blood samples. The cost per sample is about 20-50 US cents, which is much less than those of high-pressure liquid chromatography and enzymatic commercial kits. The method can be automated, which is suitable for neonatal and mass phenylketonuria screening, especially in developing countries, where funding is a limiting factor.     

A pilot C282Y hemochromatosis screening in Italian newborns by TaqMan technology

Hereditary hemochromatosis (HH) is a disorder of iron metabolism that leads to iron overload in middle age and can be caused by homozygosity for the C282Y mutation in the HFE gene. Preliminary studies have estimated the frequency of this mutation at 0.5-1% in Italy, but this has not been verified on a large sample. We analyzed 1,331 Italian newborns for the C282Y mutation in the HFE gene using dried blood spots (DBS) from the Neonatal Screening Center in Turin, Italy. The mutation was assessed using a semi-automatable 5'-nuclease assay (TaqMan technology). We detected 55 heterozygotes and no homozygotes in our sampling, resulting in an overall frequency of 2.1% +/- 0.6 for the C282Y allele. Differences in allele frequency were observed, and ranged from 2.7% +/- 1.3 in samples from Northern Italy, to 1.7% +/- 0.9 in samples from Central-Southern Italy. The low frequency of the at-risk genotype for iron overload suggests that genetic screening for HFE in Italy would not be cost effective. The present study, in addition to defining C282Y frequency, documents detection of the major HFE mutation on routine DBS samples from neonatal screening programs using a semi-automatable, rapid, reliable, and relatively inexpensive approach.     

Urine screening of five-day-old newborns: metabolic profiling of neonatal galactosuria

We determined urinary galactose and 4-hydroxyphenyllactic acid (4HPLA) in 4338 of 5-day-old newborns using a newly developed GC–MS screening method. Fifty-two infants were chemically diagnosed as having transient galactosuria based upon elevated urinary galactose levels (4.78–30.53 mg/mg creatinine, control 1.10±0.89 mg/mg creatinine). These infants did not excrete galactitol or galactonic acid into the urine, which is typical of hereditary galactosemia. Nearly 40% of the transient galactosuria was associated with immature infants (low birth weight or borne before 37 gestational weeks). Immature hepatic function is one explanation for neonatal transient galactosuria, but heterozygotes or the carriers of galactose degradation enzyme deficiencies were also suspected in some of the newborns, judging from the comparisons of urinary galactose and 4HPLA excretion between neonates and patients with galactosemia.     

Genetic analysis of human obesity in an Italian sample

An analysis of the genetic factors in obesity has been carried out on a sample of nuclear families from Aosta (N. Italy). The families consisted of the parents and sibs of all elementary school children considered to be obese during a preliminary screening and a similar sample of non-obese children and their nuclear families. The numbers of such families were 67 and 112, respectively. Several tests were applied in order to examine the genetic contribution to obesity, and in particular to investigate the presence of a dominant major gene. Our conclusions are that genetic factors are certainly present. Several analyses suggest the presence of a dominant major gene with weak effect.     

Genetic diversity and differentiation of indica and japonica rice detected by RFLP analysis

Summary Genetic diversity and differentiation in indica and japonica groups of the cultivated rice (Oryza sativa L.) were studied by assaying DNA restriction fragment length polymorphisms of 12 indica and 14 japonica rice lines digested with three restriction endonucleases. A total of 49 probes were selected to represent the entire RFLP map at intervals of 20–30 cM. It was shown that 95 of the 145 possible probe/enzyme combinations, involving 43 probes and all three enzymes, detected restriction fragment length variation, and the degree of polymorphism varied greatly from one probe/enzyme combination to another. These results demonstrate that indica rice is genetically more diverse than japonica type. Significant differentiation between the two rice groups was detected by 33 probes representing 11 of the 12 rice chromosomes. It was deduced that the processes leading to differentiation involved a combination of molecular events that include base substitutions and insertion/deletions.     

Survey of medical training in cytopathology carried out by the journal Cytopathology

Anshu, A. Herbert, B. Cochand‐Priollet, P. Cross, M. Desai, R. Dina, J. Duskova, A. Evered, A. Farnsworth, W. Gray, S. S. Gupta, K. Kapila, I. Kardum‐Skelin, V. Kloboves‐Prevodnik, T. K. Kobayashi, H. Koutselini, W. Olszewski, B. Onal, M. B. Pitman, ?. Marin?ek, T. Sauer, U. Schenck, F. Schmitt, I. Shabalova, J. H. F. Smith, E. Tani, L. Vass, P. Vielh and H. Wiener
Survey of medical training in cytopathology carried out by the journal Cytopathology This report of the Editorial Advisory Board of Cytopathology gives the results of a survey of medical practitioners in cytopathology, which aimed to find out their views on the current situation in undergraduate and postgraduate training in their institutions and countries. The results show that training in cytopathology and histopathology are largely carried out at postgraduate level and tend to be organized nationally rather than locally. Histopathology was regarded as essential for training in cytopathology by 89.5% of respondents and was mandatory according to 83.1%. Mandatory cytopathology sections of histopathology were reported by 67.3% and specific examinations in cytopathology by 55.4%. The main deficiencies in training were due to its variability; there were insufficient numbers of pathologists interested in cytology and a consequent lack of training to a high level of competence. Pathologists without specific training in cytopathology signed out cytology reports according to 54.7% of responses, more often in centres where training was 3–6 months or less duration. Although 92.2% of respondents thought that specialist cytology should not be reported by pathologists without experience in general cytopathology, that practice was reported by 30.9%, more often in centres with small workloads. The survey report recommends that 6–12 months should be dedicated to cytopathology during histopathology training, with optional additional training for those wanting to carry out independent practice in cytopathology. Formal accreditation should be mandatory for independent practice in cytopathology. When necessary, temporary placements to centres of good practice should be available for trainees intending to practise independently in cytopathology. There should be adequate numbers of pathologists trained in cytopathology to a high level of competence; some of their time could be released by training cytotechnologists and trainee pathologists to prescreen cytology slides and assess adequacy of fine‐needle aspiration samples when immediate diagnosis was not required. The survey demonstrated a clear need for European and international guidelines for training in cytopathology.     

Determination of total phenylalanine and galactose from a sample of dry blood on paper filter: its application on neonatal screening

Neonatal screening programs for metabolic disorders are recommended especially for phenylketonuria and congenital hypothyroidism. The present study was designed to adapt, develop and evaluate a SUMA method for total galactose (Gal) and phenylalanine (Phe) measurement on filter paper blood specimens. A single 5 mm disk with blood was deproteinized with methanol-acetone and eluted with distilled water. Ten microliters of the extract was transferred to one well of a ultramicroELISA plate, and the reaction solution was added to determine Phe level. The remaining extract was used for the GAL determinations. The method showed good linearity in a 0-50 mg/dl concentration range for Phe and 0-60 mg/dl for Gal. The detection limit was 0.14 mg/dl for Phe and 0.9 mg/dl for Gal. Reproducibility was assessed with filter paper blood specimens containing Gal and Phe at low, middle and high levels. Intraassay coefficients of variation were 10%, 7.5%, 6.22%, and 8.5%, 7%, 5%, respectively, whereas interassay coefficients of variation were 9.54%, 6%, 7% and 6%, 4.6%, 5.6%, respectively. In 1,000 samples from newborns, four samples of Phe and two samples of Gal showed a concentration below the threshold set for each assay. This method provides a rapid means to survey for a low incidence disease (i.e., galactosaemia: incidence, 1/30,000), in existing phenylketonuria analysis programs, where an incidence of 1/10,000), easily justifies the cost of mass screening.     

Two cases of benign methylmalonic aciduria detected during a pilot study of neonatal urine screening

Two cases of benign methylmalonic aciduria (MMAuria) were found among 9780 neonatal screenings using the previously described screening method consisting of urease digestion, ethanol deproteinization and gas chromatography-mass spectrometry. Combining this screening method with the stable isotope dilution technique showed very specific and sensitive measurements of methylmalonic acid in urine. The concentrations of urinary methylmalonic acid were measured at several ages. The levels of urinary methylmalonic acid in two patients varied from 0.27 to 3.04 mol/mol creatinine (control<0.01 mol/mol creatinine). Methylcitrate and homocystine were not increased in the patient's urine or blood. Blood propionylcarnitine was also at normal levels. The urinary methylmalonate excretions were decreased to the levels of about 50% of the start point after vitamin B12 treatment in one patient, but the other patient showed no change. No clinical abnormalities were observed during these periods.     

Directionality of lambda plasmid DNA replication carried out by the heritable replication complex

There are two ‘pathways’ of replication of λ plasmids in Escherichia coli. One pathway requires the assembly of a new replication complex before replication and the second pathway is based on the activity of the replication complex inherited by one of two daughter plasmid copies after a preceding replication round. Such a phenomenon was postulated to occur also in other replicons, including Saccharomyces cerevisiae autonomously replicating sequences. Here we investigated directionality of λ plasmid replication carried out by the heritable and newly assembled replication complexes. Using two-dimensional agarose gel electrophoresis and electron microscopy we demonstrated that in both normal growth conditions and during the relaxed response to amino acid starvation (when only replication carried out by the heritable complex is possible), bidirectionally and undirectionally replicating plasmid molecules occurred in host cells in roughly equal proportions. The results are compatible with the hypothesis that both complexes (heritable and newly assembled) are equivalent.     

Biochemical mechanisms of stone alteration carried out by filamentous fungi living in monuments

Biochemical weathering mechanisms carried out by Penicillium frequentans and Cladosporium cladosporoides on unaltered sandstone, granite and limestone were studied using FTIR, X-ray diffraction, atomic absorption and flame photometry. Strains belonging to both fungal species, isolated from the façades of two Spanish Cathedrals, were used.Large amounts of oxalic, citric and gluconic acids were produced by P. frequentans in broth cultures. These metabolites caused extensive deterioration of clay silicates, micas and feldspars from both sandstone and granite and also of calcite and dolomite from limestone, as a result of high cation release and organic salts formation such as calcium, magnesium and ferric oxalates and calcium citrates. Comparatively, the biodegradative effect brought about by C. cladosporoides was much less than that caused by P. frequentans. Neither organic acids nor organic salts were formed by C. cladosporiodes samples.It is concluded that filamentous fungi are able to cause an extensive weathering of stone, due principally to organic acid excretion, although other metabolites participate to a lesser extent in these deteriorative processes. Ecological adaptative mechanisms, such micronutrients uptake and trivalent cations chelation (Fe³⁺ and Al³⁺) are derived from fungal growth on stone monuments.     

Some aspects of the regeneration process carried out by means of pluripotent stem cells

This work is devoted to the vital topic of regeneration by stem cells. Cells-predecessors and differentiated cells can divide a limited number of times (Alberts et al., 1994) and are not capable of providing tissue regeneration throughout the ontogenesis. The tissue renewal during such a long period is impossible without participation of a specialized system responsible for regeneration. The given system is submitted by stem cells which are capable of being differentiated in all types of somatic cells and in a line of germ cells, and also have ability to self-renew during the whole life of an organism. Results of our research suggest that stem cells make up a universal mechanism of regeneration which has been formed during evolution.     

alpha-Hemolysin-induced dephosphorylation of EGF receptor of A431 cells is carried out by rPTPsigma

Earlier we have shown that the epidermal growth factor receptor was unable to retain its phospho Tyr signal after the assembly of staphylococcal alpha-hemolysin (alpha-HL). However, the nature of the protein tyrosine phosphatase (PTPase) or its identity is not known. In this report, we demonstrate that the alpha-HL elevates the activity of receptor like protein tyrosine phosphatase sigma (rPTPsigma). The alpha-HL induced dephosphorylation is prominent only in intact A431 cells. The PTPase activity is not inhibited if the alpha-HL treatment precedes PTPase inhibitor treatments. The anti-EGFr immunoprecipitates have exhibited higher PTPase activity after alpha-HL treatment of A431 cells. Interestingly, PTPase activity of anti-EGFr immunoprecipitates from the A431 cells expressing the antisense message of rPTPsigma has not increased despite alpha-HL treatment, confirming the role of rPTPsigma in the dephosphorylation of EGFr. The studies presented here will be useful in understanding the process of signal modulation by the assembly of alpha-HL.     

Novel mechanism of bacteriocin secretion and immunity carried out by lactococcal multidrug resistance proteins

A natural isolate of Lactococcus lactis was shown to produce two narrow spectrum class II bacteriocins, designated LsbA and LsbB. The cognate genes are located on a 5.6-kb plasmid within a gene cluster specifying LmrB, an ATP-binding cassette-type multidrug resistance transporter protein. LsbA is a hydrophobic peptide that is initially synthesized with an N-terminal extension. The housekeeping surface proteinase HtrA was shown to be responsible for the cleavage of precursor peptide to yield the active bacteriocin. LsbB is a relatively hydrophilic protein synthesized without an N-terminal leader sequence or signal peptide. The secretion of both polypeptides was shown to be mediated by LmrB. An L. lactis strain lacking plasmid-encoded LmrB and the chromosomally encoded LmrA is unable to secrete either of the two bacteriocins. Complementation of the strain with an active LmrB protein resulted in restored export of the two polypeptides across the cytoplasmic membrane. When expressed in an L. lactis strain that is sensitive to LsbA and LsbB, LmrB was shown to confer resistance toward both bacteriocins. It does so, most likely, by removing the two polypeptides from the cytoplasmic membrane. This is the first report in which a multidrug transporter protein is shown to be involved in both secretion and immunity of antimicrobial peptides.     

Genetic analysis of thirty-three platelet polypeptides detected in two-dimensional polyacrylamide gels.

Two-dimensional gel electrophoresis followed by silver-staining was utilized to visualize platelet polypeptides for genetic analysis. A subset of 33 polypeptides that were most suited for scoring was selected. Families consisting of father-mother-child trios were studied. Thirty-six polypeptides of a total of 1,413 scored in children's gels exhibited the combination of a normal and a variant polypeptide. The observed index of heterozygosity of 2.55% is comparable to our previously reported findings for red cell proteins.