Chilling tolerance in Arabidopsis involves ALA1, a member of a new family of putative aminophospholipid translocases.

The lipid composition of membranes is a key determinant for cold tolerance, and enzymes that modify membrane structure seem to be important for low-temperature acclimation. We have characterized ALA1 (for aminophospholipid ATPase1), a novel P-type ATPase in Arabidopsis that belongs to the gene family ALA1 to ALA11. The deduced amino acid sequence of ALA1 is homologous with those of yeast DRS2 and bovine ATPase II, both of which are putative aminophospholipid translocases. ALA1 complements the deficiency in phosphatidylserine internalization into intact cells that is exhibited by the drs2 yeast mutant, and expression of ALA1 results in increased translocation of aminophospholipids in reconstituted yeast membrane vesicles. These lines of evidence suggest that ALA1 is involved in generating membrane lipid asymmetry and probably encodes an aminophospholipid translocase. ALA1 complements the cold sensitivity of the drs2 yeast mutant. Downregulation of ALA1 in Arabidopsis results in cold-affected plants that are much smaller than those of the wild type. These data suggest a link between regulation of transmembrane bilayer lipid asymmetry and the adaptation of plants to cold.

Defective major histocompatibility complex class I expression in a sarcomatoid renal cell carcinoma cell line.

We studied major histocompatibility complex (MHC) class I expression in 12 tumor cell culture lines established from patients with metastatic renal cell carcinoma (RCC). In one of these cell culture lines, UOK 123, we found no surface expression of beta 2-microglobulin (beta 2m) and MHC class I by flow cytometry. Immunofluorescence staining using three different monoclonal antibodies to beta 2m revealed no detectable beta 2m in the endoplasmic reticulum (ER), Golgi apparatus, cytoplasm, or on the cell surface. There was no evidence of folded class I molecules inside or on the surface of the cells; however, the ER stained intensively for unfolded class I molecules. Transient expression of beta 2m by UOK 123 after infection with a recombinant vaccinia virus containing the gene for beta 2m resulted in normal expression of both beta 2m and class I (HLA-A, B, C) determinants assessed by flow cytometry analysis. No expression of class I or beta 2m was seen with the recombinant vaccinia vector carrying a control gene. The inability of class I molecules to reach the cell surface is due to the requirement of beta 2m for proper folding and presentation of the class I MHC complex. The failure to assemble and express MHC class I complex on the cell surface renders these cells incapable of antigen presentation to cytotoxic T cells and provides a mechanism for escape from immune recognition by the tumor.

Use of interleukin-7, interleukin-2, and interferon-gamma to propagate CD4+ T cells in culture with maintained antigen specificity.

In contrast to CD8+ T cells, it has been difficult to establish consistently satisfactory conditions for the bulk culture of antitumor CD4+ T cells in either mice or humans. This difficulty is not limited to tumor antigen, since similar problems are encountered growing CD4+ T cells that recognize alloantigen, tetanus, or candida. Four basic findings are reviewed in this article, stemming from work with identical results in both human and mouse. (a) Although CD4+ T cells initially proliferate after exposure to appropriate antigen-presenting cells (APCs), such proliferation is not sustained; however, expansion of CD4+ T cells can be achieved with the addition of recombinant interleukin-2 (rIL2) or rIL-7. (b) Specific CD4+ responses to antigens are often better sustained with exogenous IL-7 than with exogenous IL-2. (c) Adding rIL-7 or rIL-2 permits sustained CD4+ T-cell proliferation, but subsequent culture restimulation is complicated by high background reactivity to APCs whether APCs are antigen pulsed or not; this problem can be overcome by addition of exogenous interferon-gamma (IFN-gamma) to the CD4+ cultures. (d) In certain instances proliferation is paradoxically impaired by reexposure to specific antigen, possibly reflecting apoptosis; this problem is also overcome by addition of rIFN-gamma to culture. We conclude that combinations of exogenous IL-7, IL-2, and IFN-gamma with APC restimulation can be used to sustain antigen-specific CD4+ T cells in culture. Using these techniques, antitumor CD4+ T cells were propagated from the peripheral blood of two tumor-bearing patients.

Ulcer risk in a duodeno-gastric antireflux operation in dogs.

Duodenogastric reflux is a possible pathogenetic mechanism in some type I gastric ulcers. An antireflux operation is therefore a logical procedure but involves the risk of marginal ulceration. To examine this risk, the following study was performed: 18 dogs were divided into 3 groups of 6. One group had an antireflux operation performed (AR), one had AR plus parietal cell vagotomy (AR + PCV), and the third was a control group that was given daily injections of 40 mg repository histamine. All control dogs developed ulcers after 7-84 days, mean 37 days, of histamine. Three dogs in the AR group developed ulcers spontaneously 55-92 days postoperatively, whereas none of the AR + PCV group developed ulcers spontaneously 72-108 days postoperatively. After histamine injection two of the remaining three AR dogs developed ulcers after 3-4 days of stimulation, and three of the six AR + PCV dogs developed ulcers after 4-5 days of stimulation. It is concluded that the AR operation is heavily ulcer-prone and that PCV does not protect sufficiently against ulceration.