Purification and characterization of a novel alpha-class glutathione transferase from human liver.

The importance of glutathione transferases (GST) as a major group of detoxification enzymes is well known. The human liver possesses these enzymes in high concentration and in a multiplicity of forms. We describe here a novel glutathione transferase isoenzyme isolated from liver using glutathione affinity chromatography, DEAE-sepharose and Mono-Q ion-exchange chromatography. The isoenzyme is a dimer of approximately 25 kDa with a blocked N-termini. Its kinetic and immunological properties indicate that it belongs to the alpha-class of GSTs. Its isoelectric point (8.0) is closely related to GST alpha (pI 7.8) and GST beta (pI 8.2) reported previously. More than 70% of the amino-acid sequence of this isoenzyme has been determined by automated Edman degradation procedure. The results suggest that this isoenzyme (which we term GST 8.0) may be a heterodimer of two, closely related, novel alpha-class GST subunits. Comparisons between the amino acid sequences of these two novel alpha-class subunits with those of the other alpha-class GST subunits already known indicate changes in a number of different residues localized in the electrophilic binding site. Further studies are needed to establish whether such differences are due to allelic polymorphism of the enzyme or to the existence of additional genes for alpha-class GSTs in human liver. These results are consistent with previous data which suggest that a multitude of different GSTs, especially of alpha class, are present in the human liver providing this tissue with an efficient mechanism of protection against xenobiotic and endogenous compounds.

Diving behaviour and haemoglobin function: the primary structure of the alpha- and beta-chains of the sea turtle (Caretta caretta) and its functional implications.

The amino acid sequence of the alpha- and beta-chains of haemoglobin (Hb) from the loggerhead sea turtle (Caretta caretta) has been determined. Comparison with that of human Hb shows differences in several residues involved in both alpha 1 beta 1 and alpha 1 beta 2 packing contacts. On the whole, in spite of the mutations, the essential characteristics of both interfaces seem to be maintained. The functional properties of the sea turtle Hb have been investigated at different temperatures and as a function of proton, chloride and organic phosphate concentrations. In addition to overall similarities shared with most of the vertebrate Hbs previously described, this molecule shows significant differences which could be related to the life behaviour of the turtle. In fact, while the shape of the Bohr-effect curve is well adapted for gas exchange during prolonged dives, the very small enthalpy change for O2 binding ensures that O2 delivery becomes essentially insensitive to the temperature changes of the environment. Moreover, and similarly to the case of emperor penguin Hb, the small alkaline Bohr effect appears to be only choride-linked, since the pH dependence of the O2 affinity is abolished in the absence of this ion. These functional characteristics are discussed on the basis of the primary structure of alpha- and beta-chains.

Structural studies on the loggerhead sea turtle (Caretta caretta) myoglobin.

The primary structure of myoglobin from the loggerhead sea turtle (Caretta caretta) has been determined; the protein consists of 153 amino acid residues. The ferric loggerhead sea turtle myoglobin has been crystallized in a form suitable for X-ray structural investigations. The crystals were grown at pH 8.0, in 0.05 M tris/HCl buffer, using 3.2 M ammonium sulfate as precipitating agent, at 4 degrees C, and belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell constants a = 37.2 A, b = 61.1 A, c = 75.2 A (one molecule, 17,000 M(r), in the asymmetric unit). A molecular replacement solution was found for the loggerhead sea turtle myoglobin crystals using sperm whale myoglobin structure as search model. The R-factor value, after molecular replacement, is 0.387, for the data in the 15-3.3 A resolution range. The results here reported are the basis for the first X-ray crystallographic investigation on a reptile myoglobin, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian (i.e. sperm whale) myoglobins.

Reinvestigation of the role of thiol groups of glyoxalase I purified from yeast (Saccharomyces cerevisiae).

Glyoxalase I has been purified to homogeneity from Saccharomyces cerevisiae and tested with two different thiol reagents, i.e., 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB). DTNB reacts with four thiol groups per molecule of enzyme and leads to a complete inhibition which is not reversed by addition of the disulfide-reducing agent dithiothreitol. On the other hand, CDNB slightly affects the glyoxalase-I activity and alkylates only one thiol residue/enzyme. In agreement, DTNB reacts with three thiol residues of the CDNB-reacted enzyme and no reactivation is observed after dithiothreitol treatment. The peptide containing the CDNB-reactive thiol group has been isolated and the sequence overlaps the segment 58-63 of the only known primary structure of glyoxalase I from Pseudomonas putida.

Two imported malaria cases from Switzerland.

Two cases of falciparum malaria diagnosed in Italy, are described. The two patients, father and son, live in Geneva 3 kilometers away from the International airport. In July 1989, they left Switzerland for a short visit to Italy. Seven days after their arrival in Italy, they developed symptoms of malaria, subsequently microscopically confirmed. They had not been abroad for the past 25 years and therefore they had probably contracted the infection from an imported tropical mosquito carried to Geneva from the airport to or nearby their house.

[Statistic relation between seropositivity to Toxoplasma gondii and several risk factors in a sample of pregnant women]. / Relazione statistica tra sieropositività a Toxoplasma gondii ed alcuni fattori di rischio in un campione di donne gravide.

We analysed, in the period 1984-1986, the serum of 1733 pregnant women. The relation between the positivities and some probable factors of risk, alimentary habits and touch with tame animals has had valued by statistical method. The analysis shows no increase of risk in the group used to eat underdone meats compared with the group in touch tame animals.

[In vitro evaluation of the antibiotic effect of imipenen on bacterial strains isolated mostly from compromised patients]. / Valutazione in vitro dell'attività antibiotica dell'imipenem su ceppi batterici isolati in prevalenza da pazienti compromessi.

The antibacterial activity in vitro of Imipenem (N-formimidoyl-thienamycin) was studied on 237 recently-isolated nosocomial bacterial strains and compared with Aztreonam, Cefotaxime, Ceftazidime, Ceftriaxone, Piperacillin, Amikacin and Netilmicin through the determination of susceptibility by the Kirby-Bauer method. Taking into account that 65% bacterial strains, out of the examined ones, came from antibiotic high-pressure departments (45% from bone-marrow transplanted patients in the Hematological Department and 20% from Intensive Care Unit patients), the results obtained in vitro show an activity of Imipenem on Gram-negative bacteria as much as a 92.7% susceptibility and a 75.7% on Gram-positive bacteria; when compared with the activity of the other antibiotics, Imipenem shows a really excellent activity.

[Comparison between 2 separation methods of IgM and IgG in serologic diagnosis]. / Confronto tra due metodi di separazione delle IgM dalle IgG nella diagnostica sierologica.

The concentrations of the IgG and IgM fractions have been determined on thirty whole sera and after treatment with protein A, or after chromatographic separation. The analysis of the differences has been effected with Student's t test. No statistically significant differences have been noted between the two methods of separation as far as IgM fraction is concerned, but, on the contrary, significant differences are present for the separation of IgG fraction.

Marrow transplantation in patients with advanced thalassemia.

In a study of the outcome of marrow transplantation in patients with advanced thalassemia, 40 patients with homozygous beta-thalassemia who were 8 to 15 years of age (median, 10) received HLA-identical allogeneic marrow after treatment with busulfan and cyclophosphamide. Twenty-eight of the 40 patients were alive and free of disease 260 to 939 days after transplantation, and 2 patients were alive with thalassemia 372 and 1133 days after transplantation. The actuarial probabilities of survival and of disease-free survival at two years were 75 percent and 69 percent, respectively. Ten patients (25 percent) died. Three died of cardiac failure, interstitial pneumonitis, or septicemia within 14 days of transplantation. Three died of infectious complications associated with acute graft-versus-host disease at 46 to 97 days, and two died of infectious complications of chronic graft-versus-host disease at 249 and 290 days. Two patients had transplant rejection and died with marrow aplasia 115 and 192 days after transplantation. One patient had rejection after four months and while the marrow was aplastic underwent a successful second transplantation; the patient was alive without thalassemia 624 days after the first transplantation. The actuarial probability of grade 2 or higher acute graft-versus-host disease in the 32 patients with initial sustained engraftment was 35 percent. Three patients had chronic graft-versus-host disease, which was fatal in two and still active on day 710 in the third. We conclude that bone marrow transplantation can potentially save patients with advanced thalassemia from an otherwise inexorable progression to death from the complications of blood transfusions. The ultimate outcome in this group of patients must await a longer follow-up.

Ultrastructural and cytochemical study on the epithelium lining ductuli efferentes in Equus asinus.

The ultrastructure and cytochemistry of the epithelium lining ductuli efferentes in adult Equus asinus was studied. The epithelium is constituted of randomly distributed ciliated and non-ciliated cells extending from the basement membrane to the lumen. A third cellular type appearing to be in a degenerating state may also occur with variable frequency and is basally located. Mainly "non-ciliated cells" display ultrastructural and cytochemical features which can be related to strong resorptive activity; lysosomes and pigment granules, lipofuscinic in nature, are also present. Pigment masses exhibiting the same morphological and histochemical features fill the cytoplasm of degenerating cells. The results are compared with those obtained by previous authors in other Mammals and in Birds and are related to the functions commonly attributed to ductuli efferentes. The origin of degenerating cells, which are peculiar of Equidae, is discussed.

Clinical and endocrinological investigations on superovulation induced in heifers by human menopausal gonadotrophin.

After an i.v. injection of human menopausal gonadotrophin (hMG), three components of the disappearance time in heifers were found for immunoreactive LH (half-life 13.8--1020 min) and immunoreactive FSH (half-life 21.3--1090 min). When heifers were treated daily with hMG from Days 9, 10 or 11 of the cycle for 3 or 5 days, a total dose of 1350 i.u. FSH induced 10.2 +/- 2.5 (mean +/- s.d.) corpora lutea (CL) in 6 animals and 13 persistent follicles in 4 animals, while a total dose of 2100 i.u. FSH induced 14.3 +/- 1.5 CL in 6 heifers and there were no persistent follicles. Nine heifers treated with a single i.m. injection of 1500 i.u. PMSG exhibited 11.4 +/- 8.6 CL with 17 persistent follicles in 7 animals. Progesterone concentrations rose significantly faster and the oestradiol drop was more rapid after oestrus in heifers treated with hMG than in those treated with PMSG. These results demonstrate that stimulation of the ovarian follicles of heifers is more homogeneous when hMG is used.

[Circulating immune complexes in a population of subjects seropositive for rheumatoid factor]. / Immunocomplessi circolanti in una popolazione di soggetti sieropositivi per il fattore reumatoide.

The level of immune complexes has been detected with the C1q binding test in 179 subjects positive by Latex agglutination test (titre greater than 1/20). The average of C1q binding was 5.36%, significantly higher than in 30 normal blood donors. A positive correlation was observed between the percent of C1q binding and, respectively, the titre of the latex test and the erythrocyte sedimentation rate. Of the 36 patients with rheumatoid arthritis, 16 (45.7%) showed an high level of C1q binding activity (greater than 5%), as well as 4 of the 8 patients with chronic hepatitis (50%) and 11 of the 31 patients with infectious diseases. No immune complexes were detected among the 11 healthy latex positive subjects.

Behavior of ovarian and testicular interstitial cells during ontogenesis in the horse. Morphological and histochemical studies "in vivo" and "in vitro".

The results of a study on interstitial cells of the horse gonads from foetal life to puberty are reported. The morphological (also ultrastructural) histochemical, histophysical and histoenzymological findings both in the organ and in monolayer cultures, clarify the problem of the ontogenesis of these cells showing that: --foetal interstitial cells give origin to "xanthochrome" cells; --"xanthochrome" cells in the prepuberal gonad are continuously renewed; --the same type of cells which in th prepuberal period undergo lipochromic degeneration, differentiate at puberty into Leydig cells in the testis and probably into thecal cells in the ovary.

[Determination of residual antibiotic activity in cerebro-spinal fluid (author's transl)]. / Determinazione dell'attività antibiotica residua nel liquor.

A microbiological method for the determination of residual antibiotic activity in CSF is described. This disc-diffusion method, utilizing Bacillus stearotermophilus as test organism, is highly sensitive for many antibiotics, including those crossing the blood-brain barrier.

Experimental arthrosis from intra-articular vitamin A injection in the rabbit. Morphological and biochemical study: considerations on the pathogenesis of human arthrosis in relation to the experimental model.

The key to the pathogenesis of arthrosis lies in the mechanism responsible for the initial lesions. In this experimental work the possibility of producing arthrosic changes is demonstrated by activating lysosomal chondrocytic enzymes by the intra-articular injection of Vitamin A in rabbits. On the basis of the experimental results the authors discuss the possible role that activation of the lysosomal hydrolytic enzymes might play in producing primary and secondary arthrosis in humans. They advance the hypothesis that this mechanism may be the common final step in the degradation of the articular cartilage, whatever the aetiological factor.