[Peptidoglycan modification of Staphylococcus aureus P18 strain after use of 6 active antibiotics on the bacterial wall]. / Modification du peptidoglycane de la souche de Staphylococcus aureus P18 après utilisation de six antibiotiques actifs sur la paroi bactérienne.

Six antibiotics active on the cell-wall were tested against a methicillin resistant Staphylococcus aureus P18 strain at the start of exponential growth rate. The concentration was four-fold the MIC determined in a non-hypersaline liquid medium. The peptidoglycan amino-acid content of the various cell wall samples was determined. An antibiotic-free S. aureus P18 and the methicillin-sensitive Cowan I. S. aureus strain were used a controls. Beta lactam antibiotic treatment, oxacillin and cephalothin, induced an increase of alanine content which suggests that only secondary-transpeptidation did not occur. Similar results were obtained using vancomycin. Amino-acid content was not modified by bacitracin, which is in agreement with the results expected. The results obtained for fosfomycin and cefamandole were not easy to interpret.

Asymptomatic association of hemoglobin Dunn (alpha 6[A4]Asp----Asn) and hemoglobin O-Arab (beta 121[GH4]Glu----Lys) in a Moroccan man.

We report on the association of Hb Dunn (alpha 6[A4]Asp----Asn) and Hb O-Arab (beta 121 [GH4]Glu----Lys) in a healthy Moroccan man. Hb Dunn had the same electrophoretic properties as Hb G-Philadelphia, but its percentage was lower. Its identification was based on sequence determination of the alpha T1 peptide. Bgl II and Eco RI mapping showed the presence of four alpha-genes. Hb O-Arab was easily recognized through its electrophoretic properties and was confirmed by the suppression of the Eco RI site located in exon 3 of the beta-gene. The percentages of the various hemoglobins showed that the doubly mutated hemoglobin Dunn/O-Arab has a normal stability and suggested that the Dunn mutation is carried by the alpha 1-gene. In cord blood [propositus's son], the output of the alpha Dunn gene was found equivalent to that existing in the adult.

Association in cis of beta +-thalassemia and hemoglobin S.

A Moroccan woman was investigated because of a typical beta-thalassemia trait associated with a low-percentage (11%) hemoglobin (Hb) variant. The beta-thalassemia trait was manifested by a microcytosis, a high HbA2 (above 6%), and an increase of the alpha/beta biosynthetic ratio (1.31). The variant was identified to HbS by amino acid analysis of the abnormal peptide (beta T1) and by DNA mapping with Sau I (Mst II) restriction endonuclease. No additional amino acid substitution was recorded in the beta s-chain. The reduction of beta-globin synthesis occurred exclusively at the expense of the beta s-chain. These results are consistent with the existence of a beta s mutation and a beta +-thalassemia in cis.

A new case of Hb Little Rock [beta 143(H21)His----Gln], a high affinity variant. Study during pregnancy.

A new case of Hb Little Rock [beta 143(H21)His----Gln] is described. This high affinity variant (P50 = 15 mm Hg in whole cells) has a nearly normal cooperativity. The abnormal beta chain is readily detectable using urea-Triton X-100 electrophoresis. The altered beta T-14 peptide was separated by high performance liquid chromatography. The proposita was followed during a pregnancy. Oxygen unloading was reduced, but normal oxygen loading was maintained in the fetus. There were neither placental abnormalities, nor intrauterine growth retardation.

Homozygous hemoglobin Knossos (alpha 2 beta 227(B9) Ala----Ser): a new variety of beta (+)-thalassemia intermedia associated with delta (0)-thalassemia.

Hb Knossos (beta 27 (B9) Ala----Ser) is a recently discovered hemoglobin variant endowed with beta-thalassemic properties (1,2) We present the first homozygous cases. The propositus, a 19-year-old man is originally from northeast Algeria, but is unrelated to other Algerians who have hemoglobin Knossos. He has a beta(+)-thalassemia intermedia syndrome, including microcytic, hypochromic anemia, enlargement of the spleen, and an increase in the number of reticulocytes. The reduction of beta-chain synthesis is pronounced (alpha/non alpha:2.76). Whole cells containing Hb Knossos have a dramatically low oxygen affinity (P50:38 mm Hg). The propositus also has homozygous delta(0)-thalassemia. The chromosome carrying these mutations is characterized by the DNA haplotype I.

The heterozygous form of 4.1(-) hereditary elliptocytosis [the 4.1(-) trait].

Using clinical, morphological, genetic, and biochemical criteria, we studied ten white and North African families with hereditary elliptocytosis (HE). In four families, elliptocytic individuals displayed a highly significant reduction of band 4.1, which was recorded using two electrophoretic procedures. The 4.1a/4.1b ratio was also significantly reduced, as is usually observed in suspensions enriched in young red cells. This form of HE was invariably associated with the following characteristics: absence of clinical signs; numerous, smooth and well-elongated elliptocytes; dominant transmission; and, when investigated, normal osmotic fragility. Its frequency, among all forms of HE, is about one third as a first estimate, at least in whites and North Africans. In the other six families studied, elliptocytic subjects presented normal 4.1 bands. Again, the 4.1a/4.1b ratio was decreased, reflecting the red cell age-dependent changes in these two components. In three of these families, elliptocytosis was accompanied by clinical signs of variable intensity, and the mode of inheritance could not be unequivocally determined. Therefore, HE with a partially reduced band 4.1 defines a homogeneous variety of HE that can be isolated from other forms of HE. We suggest that it be termed the 4.1 (-) trait, so as to correspond with a previously proposed terminology.

The Hb F composition in a Moroccan family with beta zero-thalassaemia and Hb O-Arab.

We report on a Moroccan family in which the proposita displays a picture of beta-thalassaemia intermedia, associated with heterozygous Hb O-Arab (beta 121 Glu----Lys) and a beta zero-thalassaemia trait. Hb O-Arab was ascertained by the disappearance of the Eco RI restriction site that normally overlaps the beta-globin gene codon 121. The proposita further presents high proportions of Hb F (12.1%) and of G gamma chains (68.6%). The transmission of the proband's haemoglobin markers was analyzed (the proband's husband displaying normal haemoglobin). The beta zero-thalassaemia and O-Arab genes underwent mutual exclusion. A high Hb F (9.28%) level was found in one child, in association with the beta zero-thalassaemia trait, while another child carrying the latter trait displayed normal levels of Hb F. This situation suggests that a heterocellular HPFH determinant is involved. However, there was no means to establish whether the high Hb F proportion in the mother results solely from the beta zero-thalassaemia -Hb O-Arab association or whether an additional HPFH determinant is present. No DNA deletion was detectably associated with the high proportion of Hb F. In this family, the G gamma percentage was high whenever the beta zero-thalassaemia gene was present, regardless of total Hb F percentage. This observation is consistent with the view that the control of the G gamma percentage in the adult is linked to the beta-locus.

Thalassemia-like abnormalities of the red cell membrane in hemoglobin E trait and disease.

In recent studies, we observed a decrease of KMapp, an abnormally biphasic kinetics of the red cell membrane neutral phosphatase and an increased binding of hemoglobin to the membrane in various forms of beta-thalassemia. Since the gene encoding the beta chain (beta E chain) of hemoglobin E (HbE) is endowed with some thalassemic characteristics, we studied the erythrocyte membrane in 25 individuals with Hb E trait or disease. The apparent Michaelis-Menten constant for p-nitrophenylphosphate (the artificial substrate used) was significantly decreased, as in beta-thalassemia. However, the kinetics was monophasic in all the heterozygotes and in four of the homozygotes. It was biphasic only in the three other homozygotes. Vmax was also significantly reduced, a fact that is masked, when not reversed in beta-thalassemia, owing to the rejuvenation of the red cell population. In 5 mM phosphate buffer (pH 8.00), the binding of Hb E to the erythrocyte ghosts was increased in the homozygotes. In the heterozygotes, Hb A binding was also increased, as is the case in beta-thalassemia. This latter fact suggests that the membrane binding site(s) of hemoglobin is (are) altered. We found a highly significant increase of Hb F in EE subjects. The present study extends to the red cell membrane the beta-thalassemic phenotype associated with the beta E gene.

The association of hemoglobin Knossos and hemoglobin Lepore in an Algerian patient.

We report on a 54 years-old male patient from North-Eastern Algeria who combines two hemoglobin variants that are associated with thalassemia-like disorders: Hb Lepore and Knossos (beta 27 Ala----Ser) (1, 2). A beta-thalassemia intermedia picture gradually developed and finally required splenectomy at the age of 53. Total absence of Hb A2 indicated that the beta Knossos gene is most probably flanked with a delta(0)-thalassemia gene. No DNA deletion additional to the Lepore deletion was found. Hb F was elevated (12.3%) with 24% G gamma Hb F. In whole cells, Hb Knossos, representing 70% of total hemoglobin, displayed a decreased affinity for oxygen (P50 = 35 mm Hg), a fact presumably accounting for the relatively good tolerance of the condition.

Alpha-globin gene deletions associated with alpha A and alpha G Philadelphia in an Algerian family that includes two Hb G homozygotes.

An Algerian family with a high degree of consanguinity and including two homozygotes for Hb-G Philadelphia is presented. Whether homozygotes or heterozygotes, all subjects displayed microcytosis (with various degrees of poikilocytosis) and a moderately depressed alpha-globin chain synthesis. Hb H and Heinz bodies were absent. DNA mapping revealed the presence of a 3.7 kb deletion resulting from the rightward type of recombination event between alpha 2 and alpha 1 genes on both the alpha A/ and the alpha G/ chromosomes. Such data indicate that the -alpha A/ and -alpha G/ haplotypes are involved and suggest that the -alpha G/ haplotype, which is very rare in Algeria, has an African Black origin. In subjects with genotype (-alpha A/-alpha G) or (-alpha G/-alpha G), the output of the remaining alpha genes is sufficiently high to avoid the appearance of Hb H. This situation contrasts with that reported in an Algerian patient, who had a (-alpha A/-alpha A) genotype but who was producing Hb H (Whitelaw et al. 1980). The data collected from this family suggest that the -alpha A/ haplotypes are heterogeneous in Algerians.

[Apolipoproteins and glycosylated hemoglobin in the young insulin-dependent diabetic]. / Apolipoprotéines et hémoglobine glycosylée chez le jeune diabétique insulino-dépendant.

Apolipoproteins A and B, total cholesterol, triglycerides, HDL, LDL, VLDL cholesterol and glycosylated hemoglobin levels were measured in 81 children and adolescents with juvenile-onset diabetes. There were 41 males and 40 females aged 7 to 22 years. They were separated into 3 groups depending on severity: G1 (well controlled diabetes), G2 (partly controlled diabetes), G3 (poorly controlled diabetes). In boys, triglycerides levels were significantly high in group G1, apolipoprotein B, total cholesterol, triglycerides, VLDL cholesterol levels and Alc glycosylated hemoglobin were raised in group G3. Furthermore, there was a positive correlation between the percentage of Alc glycosylated hemoglobin and LDL cholesterol level. In girls, triglycerides levels were significantly high in group G1, as were apolipoprotein B, triglycerides and VLDL cholesterol levels in group G2 and apolipoprotein B, triglycerides, LDL cholesterol, VLDL cholesterol levels and the percentage of Alc glycosylated hemoglobin in group G3. A positive correlation between the percentage of Alc glycosylated hemoglobin and LDL cholesterol level was also found.

The genetic abnormalities involving red cell membrane protein 4.1 with or without elliptocytosis.

We have studied 20 caucasian individuals including 3 independent persons and 17 persons belonging to 6 unrelated families. These subjects are normal or elliptocytic. On biochemical grounds, families with hereditary elliptocytosis (HE) can be divided in two groups. (i) Three families in which HE is associated with a marked reduction (approximately 30%) of the band 4.1 percentage, upon scanning of SDS-polyacrylamide gels. This deficiency is associated with the absence of obvious clinical symptom and a dominant genetic transmission. (ii) Three HE families display a normal amount of band 4.1. Among two of them, HE is concomitant with moderate clinical manifestations and with a genetic transmission that seems to be morphologically recessive. In the last family, HE is transmitted as a dominant trait and is clinically silent. Finally, in the 3 isolated persons, HE is asymptomatic but the mode of genetic transmission could not be ascertained. One of them has a decreased amount of band 4.1 similar to that observed in the first group. To date, the band 4.1 deficiency is probably the best example of a relatively frequent specific molecular defect associated with a specific morphological abnormality. In one family with this type of HE, we have fortuitously discovered, at the heterozygous state, a variant of protein 4.1, shortened by about 8 500 daltons, involving both subcomponents 4.1a and 4.1b and morphologically silent.

Kinetic alterations of the red cell membrane phosphatase in alpha- and beta-thalassemia.

We studied the red cell membrane neutral phosphatase, which is part of the Na+K+ ATPase, in several types of oxidative hemolytic anemias. We used an artificial substrate, the p-nitrophenylphosphate. In controls and in patients heterozygous for various unstable hemoglobins (Hb Hope, Hb Köln, or Hb Hammersmith), the kinetics were of the Michaelis-Menten type. On the contrary, in nearly all patients with alpha- or beta-thalassemia, the kinetics displayed an abnormally biphasic character. The apparent Michaelis constant (KMapp) was significantly decreased. The biphasic character correlated with the imbalance of globin chain synthesis. The beta-mercaptoethanol markedly increased Vmax in controls, but had little effect on the biphasic kinetics. Omission of K+ abolished the biphasic kinetics. The abnormal kinetics failed to appear with another artificial substrate, the 4-methylumbelliferylphosphate, nor did it appear with ATP, the natural substrate. In vitro, H2O2 treatment of normal and thalassemic red cells was unable to induce or exaggerate, respectively, the biphasic kinetics, but generated alterations of a different nature. We suggest that the various kinetic alterations of the phosphatase in thalassemic syndromes originate from the imbalance of globin chain synthesis. However, the involvement of an oxidative process remains to be demonstrated.

Alterations of globin chain synthesis and of red cell membrane proteins in congenital dyserythropoietic anemia I and II.

Red cell membrane proteins were investigated in two unrelated children with congenital dyserythropoietic anemia (CDA) I and two siblings with CDA II. The CDA I patients displayed globin chain synthesis imbalance, with reduction of the non alpha/alpha ratio. One of the CDA II patients presented the reverse alteration. Whenever globin chain synthesis was unbalanced, the membrane p-nitrophenylphosphatase had an abnormally biphasic kinetics, consistent with substrate excess inhibition, as is observed in alpha- or beta-thalassemic syndromes. One CDA I patient displayed a decrease of electrophoretic band 4.1 along with an ectopic phosphorylated protein at the level of band 4.2. In CDA II and, to a lesser extent, in CDA I, the in vitro endogenous phosphorylation of band 2 + 2.1 was sharply reduced.

A shortened variant of red cell membrane protein 4.1.

In a healthy 32-yr-old woman with normal red cell morphology, a shortened variant of cytoskeletal membrane protein 4.1 is described at the heterozygous state. One haploid set of protein 4.1 migrates below protein 4.2 and displays a reduction in mass of approximately 8500 with regard to the normal haploid set. The shortening corresponds to a deletion of about 75 amino acids and concerns both subcomponents a and b of protein 4.1. It seems to involve some phosphorylation site(s). It was transmitted to the proposita's son (who inherited elliptocytosis with band 4.1 deficiency from his father). To our knowledge, the present abnormality is the first unequivocal variant of erythrocyte membrane protein 4.1 recognized up to now.

The screening of alpha- and beta-globin chain variants with isoelectric focusing.

A simple isoelectric focusing technique is presented for the screening of alpha- and beta-globin chain variants on polyacrylamide gel in the presence of 8 M urea. No detergent was used. Variants focused at discrete pH values, depending on the charge variation (-2, -1, 0, +1 or +2) introduced by the mutation. A linear relationship was evidenced between the charge variation and the shift of the apparent isoelectric point of the variants. The practical interest of this procedure is illustrated.