Purification to homogeneity of the major "4S" PAH binding protein from "non responsive" DBA/2N mouse liver by affinity chromatography.

DBA/2N is a genetically non responsive inbred strain of mice in which administration of polycyclic aromatic hydrocarbons (PAHs) does not induce microsomal monooxygenase activity. DBA/2N mouse liver cytosol contains a polycyclic aromatic hydrocarbon-binding protein that sediments, in a sucrose gradient, at 4S ("4S" PAH-BP). Its binding kinetic and physicochemical properties indicate that this protein is practically indistinguishable from the "4S" PAH-BP identified and characterized in liver cytosol of rats and other PAH responsive rodents including C57 B1/6J mice. "4S" PAH-BP was purified to homogeneity from DBA/2N mouse liver by ammonium sulfate fractionation of the cytosol, followed by Sephadex G-200 chromatography and, finally, affinity chromatography using 1-aminopyrene-Sepharose 6B. This procedure yielded about 50 micrograms of protein from 50-60 g of mouse liver, with a recovery of 18%. "4S" PAH-BP as a complex with 3H-(benzo-a-pyrene) was more than 99% pure. A single band was seen on polyacrylamide gel electrophoresis under non denaturing conditions. H-BaP comigrated with the protein band. 3H-BaP bound to the protein was displaced by PAHs with a specificity identical to that obtained using crude cytosol. On electrophoresis in SDS gels, the purified protein migrated as a single protein band with an apparent molecular weight of 40,000.

"4S" polycyclic aromatic hydrocarbon binding protein. Its role as a benzo(a) pyrene cytosolic carrier to the microsomes of DBA/2N mouse liver.

Rodent liver cytosol and other biological systems contain two proteins that bind polycyclic aromatic hydrocarbons (PAH) in a non covalent manner and that sediment at a different rate when centrifuged on sucrose gradient. The role of the smaller protein ("4S" PAH-BP) was studied. When DBA/2N mouse liver homogenate was incubated with 3H-BaP, most of the radioactivity was found in the microsomal subcellular fraction. The cytosol binding activity apparently decreased but reincubation of the cytosol with the radioactive ligand completely restored "4S" PAH-BP activity. The microsomal uptake of 3H-BaP can be studied in a reconstituted system in which microsomes are incubated with radioactive benzo(a)pyrene in the presence of crude cytosol. In these conditions the microsomal uptake rate of 3H-BaP increased with the temperature and at 37 degrees C ten minutes were required to reach the plateau. When cytosol was substituted by HEDG buffer, the amount of radioactivity found in the microsomes decreased drastically. 0.2 microM was the benzo(a)pyrene concentration required to saturate the microsomes. When microsomes were incubated with ammonium sulfate cytosolic fractions or with homogeneously purified "4S" PAH-BP, the 3H-BaP uptake was restored and reached the maximum with 3 micrograms/ml of purified protein. The radioactive benzo(a)pyrene bound to microsomes was oxidated in the presence of NADPH regenerating system. The oxidated products were discharged from microsomes only when "4S" PAH-BP was either present during the incubation or added at its end. Thus, this protein is able to transfer benzo(a)pyrene to the microsomal metabolization sites and to facilitate the release of oxidized products and, presumably, bind them.

[Biological tumor markers]. / I biomarcatori tumorali.

The classification of tumor markers can at present be only provisional since in this field new acquisitions are reported daily. Nevertheless, during the last few years, a number of monographs and chapters in oncology texts have become available for students and physicians in which certain parameters (sensitivity, specificity, predictive value of individual markers) are illustrated, thus allowing for a more accurate evaluation of their diagnostic efficacy and offering indications as to which of these markers are most useful in relation to the site of the tumor. Although tumor markers are present throughout the organism, the above review is limited to those found in blood. Clinical results obtained to far have already lead to the conclusion that tumor markers are useful above all for the diagnostic and therapeutic follow-up of patients rather than for the diagnosis of tumors. The present tendency is for the association of several markers since a higher percentage of positive results is achieved both for diagnostic purposes and for the detection of residual foci after apparently successful treatments and for the precocious detection of recurrences, as well as for the evaluation of the efficacy of ongoing therapies.

Diagnosis of hemophilia A in a female subject by using restriction fragment length polymorphisms linked to the factor VIII gene.

A 6-year-old girl, daughter of a male patient with moderate hemophilia A (factor VIII 3%), was referred to our Center because she also had very low levels of factor VIII (4%). The proband's brother has mild hemophilia A (7%); the mother (29%) is a possible carrier, no other case of hemophilia A being reported in her family. Hence, the factor VIII deficiency found in the girl is consistent either with a carrier state with extreme lyonization in favour of the hemophilic gene or with homozygosity for the hemophilia gene. To distinguish these possibilities, we studied the segregation of three restriction fragment length polymorphisms (RFLPs) linked to the factor VIII gene in the 4 members of the family. Employing one intragenic (FVIII-BclI) and two extragenic (St14-TaqI and Dx13-BglII) RFLPs, we showed that the proband has inherited from both parents the defective gene, being therefore homozygous for the hemophilia gene.

Clinical evaluation of netilmicin in the field of internal medicine.

An open clinical trial was carried out to evaluate therapeutic efficacy and safety of netilmicin. Forty hospitalized adult patients suffering from complicated urinary tract infections (UTI) (19), lower respiratory tract infections (20), septicemia (3) and soft tissue infection (1) due to in vitro susceptible microorganisms were admitted to the study. Twenty-nine of these patients had severe underlying diseases interfering with host defenses. Twenty-nine out of the 40 patients (73%) were cured and 6 (15%) had a favorable clinical response giving an overall satisfactory clinical response of 88%. Eradication of causative microorganisms was obtained in 29 (73%) patients, and three substitutions during or at the end of treatment were observed. Netilmicin was well tolerated: neither clinical abnormalities of otovestibular function nor hepatic or hematological alterations were found, while only two patients developed a mild and transient increase of BUN and creatinine serum levels.

[Changes in carcinoembryonic antigen in patients with breast cancer]. / Comportamento dell'antigene carcinoembrionale in pazienti con cancro della mammella.

Serum carcinoembryonic antigen (CEA) level was evaluated by radioimmunoassay technique (Kit-Sorin) in 13 patients (12 female and 1 male) with breast carcinoma. Serial determinations were carried out before and after surgery and during chemotherapy. Data indicate that CEA levels are correlated with the size and the staging of the neoplasm as well as with the efficacy of the therapeutic approach.

[Determination of various fractions of complement (Cl, C3c, C3a, C4) in a population of healthy neonates]. / Determinazione di diverse frazioni del complemento (C1, C3c, C3a.,C4) su di una popolazione neonatale sana.

The authors have studied plasma levels of various complement fractions (C1,C3c,C3att.,C4) in 127 newborns. Referring to gestation period the studied subjects were divided as follows: 25 newborns (less than 37 weeks), 78 newborns (37-41 weeks), 21 newborns (more then 41 weeks). The mean values of various fractions results higher in the newborns with higher gestation period. Considering the gestational ages, the mean values obtained for each complement fraction are not different from those considered "normals". The range of single value turned out very wide. This fact should lead to prudence in the diagnostic valuation.

Estrogen and progesterone binding proteins in human colorectal cancer. A preliminary characterization of estradiol receptor.

Estradiol receptor (ER) and progesterone receptor (PgR) were assayed in tumors from 20 patients with primary colorectal cancer. Ten of 20 tumors contained high affinity sites for 17 beta-estradiol and progesterone. The highest concentration of ER was 56 fmol/mg of protein. The ER dissociation constant ranged from 1.6 X 10(-10) M (mean 4.6 +/- 2.6). The highest concentration of PgR was 42 fmol/mg of protein. The PgR dissociation constant ranged from 3 X 10(-9) to 9 X 10(-9) M (mean 5.65 +/- 2.1). Four out of 20 specimens analyzed were from male patients and all resulted negative for both receptors. Sixty per cent of ER positive tumors were also PgR positive, whereas only 20% of ER negative were PgR positive. Sucrose gradient centrifugation showed that cytoplasmic ER of colorectal cancer sedimented at 3 S in the absence of protease inhibitors and at 4.5 S in the presence of 1 mM phenylmethylsulphonyl fluoride (PMSF) both in low and in high ionic strength. When chromatographed on Sephadex G-200 almost all ER was quantitatively recovered in the included fractions. Molecular weights of ER eluted from Sephadex G-200 ranged from 90,000 to 50,000 daltons. Elution profile and molecular weight heterogeneity suggest that, in spite of the presence of PMSF, there is a limited proteolysis of ER. Partially purified colorectal cancer ER did not bind to sepharose-heparin. The isoelectric point of ER was 6.4-6.5.

[Current status of simple radial immunodiffusion for determination of minimal quantities of IgA and IgM]. / Attualita' della immunodiffusione radiale semplice per la determinazione di minime quantita' di IgA e IgM.

IgA and IgM determination has been performed in the blood of the umbilical cord of 120 healthy newborn infants at different gestational ages. The utilization of the "Tripartigen Platelets" and "L.C. Partigen" has shown the extreme sensibility of the latter in determining minimum quantities of IgA and IgM.

Pathogenicity of the intercellular antibodies of pemphigus and their periodic removal from the circulation by plasmapheresis.

The authors are convinced that the intercellular antibodies of pemphigus are pathogenic. Consequently periodic plasmapheresis in two patients with pemphigus has been performed to remove the antibodies from the circulation. After larger plasma-exchanges there was a decrease of the antibody titre and a parallel improvement of the clinical condition.