The role of T3 surface molecules in the activation of human T cells: a two-stimulus requirement for IL 2 production reflects events occurring at a pre-translational level

The human T cell leukemia Jurkat was used as a model to examine the requirements of T cell activation. These studies demonstrated that antibodies reactive with the T cell-specific T3 antigen were insufficient to result in the activation of Jurkat cells, determined by the secretion of IL 2. IL 2 production occurred only in the presence of a second stimulus, the phorbol ester PMA. With the use of an IL 2-specific cDNA probe, the appearance of IL 2 RNA, similarly, occurred only when cells were stimulated with both anti-T3 antibodies and PMA. These results demonstrate a two-stimulus requirement for gene expression in human T cells.     

A new method for the quantitative determination of monosaccharides, amino sugars and N-acetylneuraminic acid and of 6-deoxyhexose (fucose) in the presence of other sugars

1. Monosaccharides, amino sugars and N-acetylneuraminic acid were determined by using an original colorimetric assay procedure, based on the detection of formaldehyde released after periodate oxidation. A range of these compounds was investigated by this method and they were all found to obey Beer's law within the concentration range 0-0.6mumole/ml. 2. A simple method for the determination of 6-deoxyhexose concentration in the presence of other monosaccharides is also described. 3. The optimum pH for the release of formaldehyde from sugars by periodate oxidation was 7.0-7.5. 4. The methods described have considerable advantages over existing assay systems and their particlar value in automatic colorimetry, where the use of concentrated acids is undesirable, is discussed.     

Dynamic adhesion and separation of cells in vitro. II. Interactions of cells with hydrophilic and hydrophobic surfaces

Experiments made on the passage of cells through untreated and siliconized glass beads, and on the adhesion and spread of cultured cells on glass and Teflon surfaces show that, in the absence of serum and in its presence in low concentrations, cell adhesion and spread is sensitive to substratum wettability. On the other hand, in the presence of 100% serum, no differences in adhesive parameters are detectable. It is concluded that arguments correlating cell adhesion to surface wettability, and, by inference, surface free energy, are unsubstantiated in 100% serum, which may well approximate to the in vivo situation. The results also show no correlation between parameters of cell adhesion and cell separation, and thereby support the hypothesis that these are different processes.     

Adenosine Triphosphate and Other Requirements for the Utilization of Glucose by Agents of the Psittacosis-Trachoma Group.

Weiss, Emilio (Naval Medical Research Institute, Bethesda, Md.). Adenosine triphosphate and other requirements for the utilization of glucose by agents of the psittacosis-trachoma group. J. Bacteriol. 90:243-253. 1965.-The agent of meningopneumonitis cultivated in the allantoic cavity of chick embryos and purified by differential centrifugations was employed for most of the studies of the requirements for glucose utilization. The evolution of C(14)O(2) from glucose-1-C(14) was used as the criterion of metabolic activity in most experiments. The rate of glucose utilization increased somewhat during the first hour of incubation at 34.4 C and became approximately constant during the second hour. Changes in glucose concentration from 1 to 5 mm did not appreciably affect metabolic activity. More vigorous CO(2) production was obtained when the ratio of K(+)-Na(+) was >1 and, under certain conditions, when the concentration of inorganic phosphate was relatively high (0.05 m). Glucose utilization was entirely dependent on added adenosine triphosphate (ATP) and Mg(++). The effect of ATP was greatly reduced when the microorganisms were partially disrupted with sonic energy. Adenosine diphosphate (ADP) could be substituted for ATP, but the activity was reduced to less than 20%. ATP was not required when glucose-6-phosphate was substituted for glucose. With ADP and glucose, glucose-6-phosphate was an effective competitor of glucose utilization. Nicotinamide adenine dinucleotide phosphate (NADP) enhanced CO(2) production from carbon 1, but not from other carbons, with glucose and, especially, glucose-6-phosphate as substrates. ATP and NADP produced the above-described effects only when their concentrations were comparable to those of the substrates. These concentrations always exceeded the amount of CO(2) produced (0.05 to 0.5 mumole/mg of agent protein). The concentration of NADP could be reduced when oxidized glutathione was added. Diphosphothiamine had no effect on CO(2) production. Qualitatively similar results were obtained with the agent of trachoma purified from yolk sac. These experiments furnish evidence that agents of the psittacosistrachoma group, despite their enzymatic capabilities, require an exogenous source of energy.     

A general method for the induction and screening of antisera for cDNA-encoded polypeptides: antibodies specific for a coronavirus putative polymerase-encoding gene

A prokaryotic vector, pGE374, containing the recA and lacZ genes, out-of-frame, was used for the expression of cDNA derived from the putative polymerase-encoding gene of the coronavirus mouse hepatitis virus strain A59 (MHV-A59). The pGE374/viral recombinant vector generates a tripartite bacterial/viral protein composed of a segment of the RecA protein at the N terminus, the coronaviral sequences in the middle, and an enzymatically active beta-galactosidase at the C terminus. Rabbits immunized with such recombinant proteins generated antibodies to the MHV-A59 portion of the tripartite protein. Because the MHV-A59 polymerase proteins have been difficult to identify during infection, we used a novel method to demonstrate the viral specificity of the antiserum. The viral cDNA was excised from the expression vector, and transferred to a pGem vector, downstream from and in-frame with a portion of the cat gene. This construct contained a bacteriophage RNA polymerase promoter that enabled the cell-free synthesis of a fusion protein that was used to verify that antibodies were generated to the expressed viral DNA. This strategy was shown to successfully result in the specific generation of antibodies to the encoded information of the viral cDNA. Furthermore, this method has general applicability in the generation and characterization of antibodies directed against proteins encoded in cDNAs.     

Prion protein PrPc interacts with molecular chaperones of the Hsp60 family.

Prions mediate the pathogenesis of certain neurodegenerative diseases, including bovine spongiform encephalopathy in cattle and Creutzfeldt-Jakob disease in humans. The prion particle consists mainly, if not entirely, of PrPSc, a posttranslationally modified isoform of the cellular host-encoded prion protein (PrPc). It has been suggested that additional cellular factors might be involved in the physiological function of PrPc and in the propagation of PrPSc. Here we employ a Saccharomyces cerevisiae two-hybrid screen to search for proteins which interact specifically with the Syrian golden hamster prion protein. Screening of a HeLa cDNA library identified heat shock protein 60 (Hsp60), a cellular chaperone as a major interactor for PrPc. The specificity of the interaction was confirmed in vitro for the recombinant proteins PrPc23-231 and rPrP27-30 fused to glutathione S-transferase with recombinant human Hsp60 as well as the bacterial GroEL. The interaction site for recombinant Hsp60 and GroEL proteins was mapped between amino acids 180 and 210 of the prion protein by screening with a set of recombinant PrPc fragments. The binding of Hsp60 and GroEL occurs within a region which contains parts of the putative alpha-helical domains H3 and H4 of the prion protein.     

Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide -II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the -II tubulin fragment, but it showed interaction with the Affigel-conjugated -I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.Abbreviations PAA poly (L-aspartic acid) - HMW-MAPs high molecular weight microtubule associated proteins     

Risk of breast cancer in relation to the interval since last full term pregnancy.

OBJECTIVE--To examine whether the risk of breast cancer is increased by a recent term pregnancy. DESIGN--Population based case-control study. SETTING--Eight areas in the United States. SUBJECTS--Cases were 2279 multiparous women residents of the eight areas aged 25-49 who were diagnosed as having breast cancer during 1980-2. Controls were 2357 multiparous women selected from the same areas by random digit dialing. MAIN OUTCOME MEASURE--Relative risk of developing breast cancer according to the time interval since last full term pregnancy. RESULTS--The distribution of intervals since the last term pregnancy was similar in cases and controls. Adjusted for age, parity, and age at first term pregnancy, the odds ratios observed for categories of years since the last full term pregnancy were: 0-2 years, odds ratio 1.16 (95% confidence interval 0.84 to 1.59); 3-6 years, odds ratio 1.21 (0.95 to 1.54); 7-9 years, odds ratio 1.04 (0.84 to 1.38); > or = 10 years, odds ratio 1.00 (reference). CONCLUSIONS--Among multiparous women aged 25-49 years there was no association between the risk of breast cancer and the time interval since the last full term pregnancy.     

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.