Genetic studies on capsid-length determination in bacteriophage T4. I. Isolation and partial characterization of second-site revertants of a gene 23 mutation affecting capsid length.

The T4 mutation ptg19-80 affects the mechanism of capsid-length determination. It is located in gene 23, which encodes the major structural protein of the capsid. The mutation results in the production of abnormal-length capsids in high frequencies. This paper describes the isolation and partial characterization of second-site revertants of ptg19-80. In the course of their analysis, it was discovered that ptg19-80 is itself a double mutation consisting of a gene 23 mutation (ptg19-80c), which causes the morphogenetic defect, and a suppressor mutation located near the lysozyme gene. Phenotypic characterization of nine pseudo-wild-type revertants of this double-mutation revealed that these revertants all produced lower frequencies of abnormal capsids than did ptg19-80. Seven of these revertants were shown to contain two suppressor mutations, one mapping in or near gene 22 and done mapping in or near gene 24. Both mutations were required for suppression. These suppressors displayed no discernible phenotype in the absence of ptg19-80c.     

Monitoring the integrity of self: biology of MHC-restriction of virus-immune T cells

All available evidence indicates that the cytotoxic thymus-derived lymphocyte (T cell), which is lytic for virus-infected target cells in vitro, is also the effector in cell-mediated immunity in vivo. Such T cell show two orders of specificity: for the virus in question, and for a particular self major histocompatibility complex (MHC) glycoprotein. Recirculating T cells amy thus be considered to survey the integrity of self, the self components involved being the cell-surface structures that are recognized as foreign during graft rejection. Virus-infected liver cells are apparently eliminated in much the same way as a transplanted organ. The necessary balance between self-tolerance (absence of autoreactivity) and self-monitoring effector T cell function seems to be established during the process of differentiation in thymus. The molecular nature of the underlying recognition events is, as yet, obscure.     

Evaluation of xylazine hydrochloride as the sole immobilizing agent in moose and caribou--and its subsequent reversal with idazoxan

Xylazine hydrochloride was used as the sole immobilizing agent in moose and caribou. The animals were free-ranging and immobilization was accomplished from a helicopter using powered darts. Following a period of immobilization during which radiotelemetry collars were fitted, the animals were revived using idazoxan (RX 781094) or its methoxy analogue RX 821002. Xylazine was administered at dose rates of approximately 3.0 mg/kg and 5.0 mg/kg to the moose and caribou, respectively. Moose received 430 +/- 27 mg of xylazine and a mean dose of 10 mg idazoxan (RX 781094). Caribou received 485 +/- 30 mg xylazine and a mean dose of 4 mg idazoxan (RX 821002). This technique gave adequate immobilization with rapid recovery of consciousness in both species.     

Calling song and selective phonotaxis in the field crickets,Gryllus firmus andG. pennsylvanicus (Orthoptera: Gryllidae)

The field cricket species, Gryllus firmusand G. pennsylvanicus,occur in a mosaic hybrid zone that roughly parallels the eastern slope of the Appalachian mountains in the northeastern United States. It is important to know what role, if any, the calling song plays in mate choice in sympatric and allopatric populations. In this report, we present results on the variability of calling song properties along transects across this hybrid zone. We also present the results of experiments on phonotactic selectivity of females from an allopatric population of G. firmus.The male calling song of allopatric G. firmuswas significantly slower in temporal rhythm (i. e., chirp and pulse repetition rates) and lower in pitch (i.e., dominant frequency) than that of allopatric G. pennsylvanicus.Calling song properties of males recorded in the hybrid zone varied considerably in temporal and spectral properties. In two-stimulus (choice) phonotaxis experiments, allopatric females of G. firmuspreferred synthetic calling songs with conspecific pulse repetition rates over songs that had lower and higher pulse rates. This preference persisted even when the sound pressure levels of alternative stimuli were unequal. Therefore, allopatric females of G. firmuscan discriminate between conspecific and heterospecific calling songs. Whether or not this same selectivity is present in sympatric populations remains unclear. Investigations of phonotactic selectivity in other allopatric and sympatric populations of both species are currently under way.     

Nitric oxide synthesis in the CNS endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues.

Inhibition of nitric oxide production by arginine analogues was examined in three cell systems; macrophages, CNS tissue and endothelial cells. Nitric oxide production was assessed indirectly using in vitro assays measuring nitrite production (macrophages), cGMP elevation (CNS) and acetylcholine-induced relaxation of aortic ring segments (endothelium). NG-monomethyl-L-arginine and NG-amino-L-arginine possessed similar inhibitory activity in all three assays, while NG-nitro-L-arginine displayed a striking selectivity for inhibition of brain and endothelial cell nitric oxide synthesis, with IC50 values of 0.05 microM in the CNS versus 200 microM in macrophages. These results suggest that distinct enzymes are responsible for nitric oxide synthesis in different cell types, and indicate that it may be possible to selectively modulate nitric oxide production in vivo.     

The degradative fate of ubiquitin-protein conjugates in nucleated and enucleated cells.

Covalent ligation of multiple copies of ubiquitin to proteins is known to target intracellular proteins for degradation by large molecular weight cytosolic proteinase(s). Ubiquitin protein conjugates are found in cytosolic cell compartments suggesting that ubiquitination may have multiple roles. We have detected ubiquitinated proteins in the lysosomal apparatus of normal fibroblasts and fibroblasts treated with lysosomal proteinase inhibitors. In contrast rabbit reticulocytes lack lysosomes. We present here direct evidence for ubiquitination of mitochondrial proteins during rabbit reticulocyte maturation. In addition ubiquitination appears to be associated with the terminal differentiation of human keratinocytes. These results suggest that: 1. ubiquitin-protein conjugates may be degraded lysosomally 2. organellar proteins may be degraded by the ubiquitin system 3. ubiquitination is involved in the programmed elimination of proteins and organelles from several cell types during differentiation.     

Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment

Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.     

Anti-Thy-1 plus complement-treated, cultured bone marrow cells resemble fetal thymocytes in killer cell function and marker expression

Anti-Thy-1.2 plus complement treated bone marrow cells were tested after short-term culture for their ability to lyse allogeneic target cells. Significant lytic activity was generated after 9 days, and required both CAS and splenic or PEC feeders as culture supplements. Allogeneic as well as syngeneic-specific cytotoxic cells were generated polyclonally under such conditions, and could be separated by using limiting dilution protocols. When 65 clones were tested for lytic activity toward three targets bearing H-2k, H-2d, and H-2b haplotypes, respectively, only two clones lysed all three targets; 53 clones showed specificity toward one target only. Targets low in class I H-2 expression were lysed only minimally compared with high H-2 expressors. Allogeneic-kill by C57BL/6 bone marrow cells grown on AKR feeder cells was destroyed by treating effectors with anti-Thy-1.2, but not anti-Thy-1.1, antibody plus complement, suggesting 1) a de novo generation of surface Thy-1 during culture and 2) that effectors were derived from bone marrow, but not feeder, populations. Partial inhibition of kill occurred by treatment of effectors with anti-asialo-GM1 (approximately 80%), anti-Lyt-2 (approximately 60%), or anti-Ly-5.1 (approximately 30%) antibodies plus complement; treatment of effectors with anti-L3T4 or anti-NK-1.1 antibodies plus complement had no effect. When precursor populations were treated with either anti-Thy-1.2 alone or a combination of anti-Thy-1.2 and anti-Lyt-2 antibodies plus complement, killers were easily demonstrated. However, the addition of an anti-asialo-GM1 antibody plus complement treatment before culture abolished function. The characteristics of these effectors showed a resemblance to those described previously for day 14 to 17 fetal thymocytes, designated pCTL.