Transfection of the mouse ICAM-1 gene into murine neuroblastoma enhances susceptibility to lysis,reduces in vivo tumorigenicity and decreases ICAM-2-dependent killing

We determined the expression of intercellular adhesion molecules (ICAM) on neuro-2a cells in order to evaluate whether they were involved in cytolysis of murine neuroblastoma. Fluorescence-activated cell sorting analysis revealed that the control neomycin-resistance-genetransduced line (neuro-2a/LN) had poor expression of ICAM-1 (mean channel fluorescence, MCF=3.7). An ICAM-1-positive transfectant of neuro-2a (neuro-2a/ICAM-1⁺) (CMF=64.3) was generated to evaluate directly the role of this adhesion molecule in cytolysis. Neuro-2a/ICAM-1⁺ was more sensitive to LAK killing (69.7% at an effector-to-target ratio of 1001) compared to neuro-2a/LN (48.6%) (P<0.001). Blocking of neuro-2a/LN and neuro-2a/ICAM-1⁺ lysis with anti-ICAM-1 monoclonal antibodies (mAbs) did not account for all the LFA-1-dependent killing. These data indicate that even in neuro-2a/ICAM-1⁺ cells, other LFA-1 ligands participated in the effector-target interaction. Therefore, we examined these cell lines for ICAM-2 expression. Both neuro-2a/LN and neuro-2a/ICAM-1⁺ lines expressed ICAM-2 (MCF=16.4 and 16.5). ICAM-2 accounted for the majority of the LFA-1-dependent killing in the ICAM-1-negative target, neuro-2a/LN, while ICAM-1 played a primary role in the cytolysis of the ICAM-1⁺ transfectant. Inhibition of lysis in the presence of anti-ICAM-1 and ICAM-2 mAbs was comparable to that seen with the addition of anti-LFA-1 mAb, indicating that other LFA-1 ligands were not involved in this system. ICAM-1 expression was associated with decreased in vivo tumorigenicity; mice inoculated with neuro-2a/ICAM-1⁺ cells had a significantly longer survival compared to those receiving neuro-2a/LN cells (median survival time 35.5 versus 24.5 days) (P<0.001). It is important to note that ICAM-1 transfection of murine neuroblastoma did not alter its metastatic potential. We conclude that transfection of mouse neuroblastome with ICAM-1 increases its sensitivity to in vitro lysis and reduces its in vivo tumorgenicity. In ICAM-1-negative murine neuroblastoma cells, ICAM-2 plays a primary role in cell-mediated lysis.This work was supported in part by the Children's Cancer Research Fund, the Minnesota Medical Foundation, the Viking Children's Fund and NIH grants PO1-CA-21737, NO1-AI-85002. E. K. is a recipient of the Irvine McQuarrie Research Scholar Award and B. R. B. a recipient of the Edward Mallinkrodt Foundation Scholar Award     

High pressure conditions stimulate expression of chloramphenicol acetyltransferase regulated by the lac promoter in Escherichia coli

Abstract Recombinant plasmids with the chloramphenicol acetyltransferase (CAT) structural gene behind several kinds of promoters were tested for expression in Escherichia coli during growth at atmospheric pressure (0.1 MPa) and at high pressure (30 MPa). Expression of the CAT gene from the lac promoter was remarkably activated (approx. 78-fold) by high pressure in the absence of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). The stimulation of the CAT activity by the lac promoter at high pressure did not simply result from an increased plasmid copy number, because the CAT activities from the other promoters and β-lactamase activities were unaffected at high pressure.     

Human T Cell Leukemia Virus Type 1 (HTLV-1) Antibodies in Healthy Populations and Renal Transplanted Patients in the North-East of Brazil

The seroprevalence of human T cell leukemia virus type 1 (HTLV-1) infection was investigated in Brazilians (570): native inhabitants (298) and descendants from Japanese (272) living in Recife and its neighborhoods—North-east of Brazil. Furthermore, polytransfused renal transplanted patients (54) were also examined for the serological status to this virus. The seropositivity to HTLV-1, screened by enzyme-linked immunosorbent assay (ELISA), was low: 1.34% for the local population and 0.73% for the descendants from Japanese. However, the seropositivity for the renal transplanted patients was found to be 11.1%. This higher value suggests that this retrovirus infection seems to be of importance in this clinical condition.     

A minichromosome-like structure in wheat embryos

A crude nuclear fraction of resting wheat embryos was used as the source of putative plant minichromosomes: unique DNA sequences the size of genes and flanked by telomere-type repeats. Preliminary separation of low-molecular-weight DNA species from chromosomal DNA (Hirt's method), velocity sedimentation, and isopycnic centrifugation were followed by PCR amplification of minichromosome-like sequences. The most abundant PCR product was cloned and sequenced. In addition to telomeric repeats (defined by a PCR primer), which were the expected sequences, the linear DNA molecule (637 pb) contained an ARS-like element, RAP1-binding site, and two relatively long ORFs. The whole sequence seems to represent a naturally occurring plant minichromosome.     

1.56 Å structure of mature truncated human fibroblast collagenase

The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 Å resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded β-sheet and three α-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase. © 1994 Wiley-Liss, Inc.     

Identification of N-terminal helix capping boxes by means of 13C chemical shifts

Summary We have examined the ¹³C and ¹³C chemical shifts of a number of proteins and found that their values at the N-terminal end of a helix provide a good predictor for the presence of a capping box. A capping box consists of a hydrogen-bonded cycle of four amino acids in which the side chain of the N-cap residue forms a hydrogen bond with the backbone amide of the N3 residue, whose side chain in turn may accept a hydrogen bond from the amide of the N-cap residue. The N-cap residue exhibits characteristic values for its backbone torsion angles, with and clustering around 94±15° and 167±5°, respectively. This is manifested by a 1–2 ppm upfield shift of the ¹³C resonance and a 1–4 ppm downfield shift of the ¹³C resonance, relative to their random coil values, and is mainly associated with the unusually large value of . The residues following the N-cap residue exhibit downfield shifts of 1–3 ppm for the ¹³C resonances and small upfield shifts for the ¹³C ones, typical of an -helix.     

Interactions between nitrate and ammonium during uptake by carob seedlings and the effect of the form of earlier nitrogen nutrition

Seedlings of carob ( Ceratonia siliqua L. cv. Mulata) were used in two sets of experiments in order to evaluate; (1) the reciprocal effects of each nitrogen form on net uptake of nitrate and ammonium, and (2) the effect of earlier nitrogen nutrition on ammonium versus nitrate uptake. In the former group of experiments we studied the kinetics of nitrate and ammonium uptake as well as the interference of each of the two forms with net uptake of ammonium and nitrate by both nitrogen depleted and nitrogen fed carob seedlings. On the whole, nitrogen depletion led to increase in both affinity and V_max of the system for both forms of nitrogen, at the same time as the effects of nitrate on uptake of ammonium and vice versa were concentration dependent. In the second group of experiments the effects of earlier nitrogen nutrition on nitrate and ammonium uptake were characterized, and in this case we observed that: (a) if only one form of N was supplied, ammonium was taken up in greater amounts than nitrate; (b) the presence of ammonium enhanced nitrate uptake; (c) ammonium uptake was inhibited by nitrate; (d) there was a significant effect of the earlier nitrogen nutrition on the response of the plants to a different nitrogen source. The latter was evident mainly as regards ammonium uptake by plants grown in ammonium nitrate. The interactions between nitrate and ammonium uptake systems are discussed on the basis of the adaptation to the nitrogen source during early growth.     

Polymorphism of the tumor necrosis factor beta gene in systemic lupus erythematosus: TNFB-MHC haplotypes

We investigated the Nco I restriction fragment length polymorphism (RFLP) of the tumor necrosis factor beta (TNFB) gene in 173 patients with systemic lupus erythematosus (SLE), 192 unrelated healthy controls, and eleven panel families, all of German origin. The phenotype frequency of the TNFB*1 allele was significantly increased in patients compared to controls (63.6% vs 47.1%, RR = 1.96, p <0.002). The results of a two-point haplotype statistical analysis between TNFB and HLA alleles show that there is linkage disequilibrium between TNFB*1 and HLA-A1, Cw7, B8, DR3, DQ2, and C4A DE. The frequency of TNFB*1 was compared in SLE patients and controls in the presence or absence of each of these alleles. TNFB*1 is increased in patients over controls only in the presence of the mentioned alleles. Therefore, the whole haplotype A1, Cw7, B8, TNFB*1, C4A DE, DR3, DQ2 is increased in patients and it cannot be determined which of the genes carried by this haplotype is responsible for the susceptibility to SLE. In addition, two-locus associations were analyzed in 192 unrelated healthy controls for TNFB and class I alleles typed by serology, and for TNFB and class II alleles typed by polymerase chain reaction/oligonucleotide probes. We found positive linkage disequilibrium between TNFB*1 and the following alleles: HLA-A24, HLA-B8, DRB1*0301, DRB1*1104, DRB1*1302, DQA1*0501, DQB1*0201, DQB1*0604, and DPB1*0101. TNFB*2 is associated with HLA-B7, DRB1*1501, and DQB1*0602.This study was supported by grants from the Federal Ministry of Research and Technology (BMFT/DFVLR, 01 VM 8608/9), the German Academic Exchange Service (DAAD, 322/501/014/0), and SFB (217).This work is part of the doctoral thesis of M. P. Bettinotti.     

Plasma and liver selenium levels in the rat during supplementation with 0.5, 2, 6, and 15 ppm selenium in drinking water

Plasma and liver selenium of Wistar rats were determined after 1, 3, and 6 mo supplementation with 0.5, 2, 6, or 15 ppm selenium as sodium selenite in drinking water. Plasma selenium was not different from control values at additional intake of 0.5 ppm but increased above usual levels at higher intakes. A highly significant correlation was observed between the total quantity of selenium ingested and plasma selenium after 1 mo treatment (r=0.99,p<0.01), but was less pronounced after 3 and 6 mo (0.94,p<0.05, and 0.78,p<0.05, respectively). The decrease in plasma selenium with time of treatment was more pronounced at higher intakes. There was also a highly significant correlation between total selenium intake and liver selenium concentration (r=0.99,p<0.01) after 1 mo of treatment, but this time liver selenium did not change with time, and the correlation remained highly significant throughout the investigation. Liver selenium therefore appears as a more sensitive and more representative measure of selenium intake than plasma selenium. Most supplements did not affect body weight and survival of animals, except when the diet was supplemented with 15 ppm for 6 mo; however, alterations in biochemical parameters concerning lipid status and hepatic function were observed at levels above 2.0 ppm.     

Organic matter and nutrient cycling within an endemic birch stand in the Etna massif (Sicily):Betula aetnensis Rafin

At altitudes between 1300 m to 2100 m in the Etna massif (Sicily), an endemic species of theBetula genus,Betula aetnensis Rafin, grows in a well-defined microclimatical context. Aboveground biomass and nutrient content studies within one stand revealed no significant differences from the otherBetula species, normally found in colder more temperate climate regions.Throughout the studied sites, biomass production, nutrient cycling and various structural or physiological characteristics (leaf area index) varied very little.Other researches indicate that the originality ofBetula aetnensis lies more in the histological or anatomical characteristics of its water conducting system which enables the species to adapt to Mediterranean-climate summer droughts in the Etna massif.

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Riassunto Sull'Etna, tra 1300 e 2100 m d'altitudine, in una zona microclimaticamente ben definita del versante nordorientale, si rinviene laBetula aetnensis Rafin.Dallo studio della fitomassa e della mineralomassa aerea del bosco di Monte Baracca, è emerso che non vi sono differenze notevoli con le altre specie indagate del genereBetula, più caratteristiche dei climi temperati e freddi.La produzione di biomassa, cosi come la gestione degli elementi nutritivi, è molto simile ai diversi popolamenti già indagati, cosi come certe caratteistiche strutturali e fisiologiche (leaf area index).L'originalità dellaBetula aetnensis è da ricercarsi nel vantaggio che ne ricava, a livello endogeno, sfruttando le caratteristiche istologiche ed anatomiche del suo apparato conduttore, che le consentono un efficace ed eccellente adattamento alle condizioni di siccità estive particolari del clima mediterraneo del vulcano.