The response of an established line of rat liver cells to thyroid hormone

The response of an established line of non-transformed adult rat liver epithelial cells (ARL 15) to thyroid hormone (T3) (3,5,3'-triiodothyronine) was characterized. Exposure of confluent monolayers to 1.10(-8) M T3 for 3 days increased O2 consumption (QO2) between 14-58%, ouabain-sensitive Rb+ uptake 26%, (Na+ + K+)-ATPase activity 32%, alpha-glycerophosphate dehydrogenase activity 103% and cytochrome oxidase activity 208%. The ARL 15 cells, maintained in continuous culture, therefore, exhibit the hallmarks of an authentic physiological response to thyroid hormone.     

Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes

The molecular processes involved in the transduction of small staphylococcal plasmids by a generalized transducing phage, phi 11, have been analysed. The plasmids are transduced in the form of linear concatemers containing only plasmid DNA; plasmid-initiated replication is required for their generation but additive interplasmid recombination is not. Concatemers are probably generated by the interaction of one or more phage functions with replicating plasmid DNA. Insertion of any restriction fragment of the phage into the plasmid causes an approximately 10(5)-fold increase in transduction frequency, regardless of the size or genetic content of the fragment. The resulting transducing particles (Hft particles) contain mostly pure linear concatemers composed of tandem repeats of the plasmid::phage chimera, and their production requires active plasmid-initiated replication. The high frequency of transduction is a consequence of homologous recombination between the linear chimeric and phage concatemers, which has the effect of introducing an efficient pac site into the former. Following introduction into lysogenic recipient bacteria, the transducing DNA is first converted to the supercoiled form, then processed to monomers by a mechanism that requires the active participation of the plasmid replication system.     

Neuronal cell adhesion molecules and cytotactin are colocalized at the node of Ranvier

Immunocytochemical methods were used to show that Ng-CAM (the neuron-glia cell adhesion molecule), N-CAM (the neural cell adhesion molecule), and the extracellular matrix protein cytotactin are highly concentrated at nodes of Ranvier of the adult chicken and mouse. In contrast, unmyelinated axonal fibers were uniformly stained by specific antibodies to both CAMs but not by antibodies to cytotactin. Ultrastructural immunogold techniques indicated that both N-CAM and Ng-CAM were enriched in the nodal axoplasm and axolemma of myelinated fibers as well as within the nodal regions of the myelinating Schwann cell. At embryonic day 14, before myelination had occurred, small-caliber fibers of chick embryos showed periodic coincident accumulations of the two CAMs but not of cytotactin, with faint labeling in the axonal regions between accumulations. Cytotactin was found on Schwann cells and in connective tissue. By embryonic day 18, nodal accumulations of CAMs were first observed in a few medium- and large-caliber fibers. Immunoblot analyses indicated that embryonic to adult conversion of N-CAM and a progressive decrease in the amount of Ng-CAM and N-CAM occurred while nodes were forming. Sciatic nerves of mouse mutants with defects in cell interactions showed abnormalities in the distribution patterns and amount of Ng-CAM, N-CAM, and cytotactin that were consistent with the known morphological nodal disorders. In trembler (+/Tr), intense staining for both CAMs appeared all along the fibers and the amounts of N-CAM in the sciatic nerve were found to be increased. In mice with motor endplate disease (med/med), Ng-CAM and N-CAM, but not cytotactin, were localized in the widened nodes. Both trembler and med/med Schwann cells stained intensely for cytotactin, in contrast to normal Schwann cells which stained only slightly. All of these findings are consistent with the hypothesis that surface modulation of neuronal CAMs mediated by signals shared between neurons and glia may be necessary for establishing and maintaining the nodes of Ranvier.     

Differential contributions of Ng-CAM and N-CAM to cell adhesion in different neural regions

Individual neurons can express both the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) at their cell surfaces. To determine how the functions of the two molecules may be differentially controlled, we have used specific antibodies to each cell adhesion molecule (CAM) to perturb its function, first in brain membrane vesicle aggregation and then in tissue culture assays testing the fasciculation of neurite outgrowths from cultured dorsal root ganglia, the migration of granule cells in cerebellar explants, and the formation of histological layers in the developing retina. Our strategy was initially to delineate further the binding mechanisms for each CAM. Antibodies to Ng-CAM and N-CAM each inhibited brain membrane vesicle aggregation but the binding mechanisms of the two CAMs differed. As expected from the known homophilic binding mechanism of N-CAM, anti-N- CAM-coated vesicles did not co-aggregate with uncoated vesicles. Anti- Ng-CAM-coated vesicles readily co-aggregated with uncoated vesicles in accord with a postulated heterophilic binding mechanism. It was also shown that N-CAM was not a ligand for Ng-CAM. In contrast to assays with brain membrane vesicles, cellular systems can reveal functional differences for each CAM reflecting its relative amount (prevalence modulation) and location (polarity modulation). Consistent with this, each of the three cellular processes examined in vitro was preferentially inhibited only by anti-N-CAM or by anti-Ng-CAM antibodies. Both neurite fasciculation and the migration of cerebellar granule cells were preferentially inhibited by anti-Ng-CAM antibodies. Anti-N-CAM antibodies inhibited the formation of histological layers in the retina. The data on perturbation by antibodies were correlated with the relative levels of expression of Ng-CAM and N-CAM in each of these different neural regions. Quantitative immunoblotting experiments indicated that the relative Ng-CAM/N-CAM ratios in comparable extracts of brain, dorsal root ganglia, and retina were respectively 0.32, 0.81, and 0.04. During culture of dorsal root ganglia in the presence of nerve growth factor, the Ng-CAM/N-CAM ratio rose to 4.95 in neurite outgrowths and 1.99 in the ganglion proper, reflecting both polarity and prevalence modulation. These results suggest that the relative ability of anti-Ng-CAM and anti-N-CAM antibodies to inhibit cell-cell interactions in different neural tissues is strongly correlated with the local Ng-CAM/N-CAM ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nerve growth factor enhances expression of neuron-glia cell adhesion molecule in PC12 cells

The neuron-glia cell adhesion molecule (Ng-CAM) has been identified in mammalian brain tissue and PC12 pheochromocytoma cells as Mr 200,000 and Mr 230,000 species, respectively. When PC12 cells were treated with nerve growth factor (NGF), the amount of Ng-CAM at the cell surface was increased approximately threefold, whereas the amount of the neural cell adhesion molecule (N-CAM) remained unchanged. An NGF-inducible large external glycoprotein (NILE) has been previously identified by its enhanced expression in NGF-treated PC12 cells. Ng-CAM and NILE are similar in molecular weight, expression during development, and responsiveness to NGF in PC12 cells, suggesting that the two molecules are related. In addition, antibodies to Ng-CAM and NILE cross-reacted and the molecules had similar peptide maps after limited proteolysis. Moreover, antibodies to Ng-CAM inhibited fasciculation of neurites, a functional property shared with NILE. The results show that cell adhesion molecules can respond selectively to growth factors and suggest that NILE is, in fact, mammalian Ng-CAM.     

Site-restricted expression of cytotactin during development of the chicken embryo

The sequential appearance of the extracellular matrix (ECM) protein, cytotactin, was examined during development of the chicken embryo by immunohistochemical techniques. Although cytotactin was identified as a molecule that mediates glia-neuron interactions, preliminary immunohistochemical localization of the molecule suggested that it was an ECM protein with a widespread but nonetheless more restricted distribution than either fibronectin or laminin. In the present study, it was found that cytotactin is first present in the gastrulating chicken embryo. It appears later in the basement membrane of the developing neural tube and notochord in a temporal sequence beginning in the cephalic regions and proceeding caudally. Between 2 and 3 d of development, the molecule is present at high levels in the early neural crest pathways (surrounding the neural tube and somites) but, in contrast to fibronectin and laminin, is not found in the lateral plate mesoderm or ectoderm. At later times, cytotactin is expressed extensively in the central nervous system, in lesser amounts in the peripheral nervous system, and in a number of nonneural sites, most prominently in all smooth muscles and in basement membranes of lung and kidney. Cytotactin appears in adult tissues with distributions that are similar to those seen in embryonic tissues. The findings raise the possibility that certain ECM proteins contribute to pattern formation in embryogenesis as a result of their restricted expression in a spatiotemporally regulated fashion at some sites but not at others.     

Correlation between ventilation and brain blood flow during hypoxic sleep

Ventilation and brain blood flow (BBF) were simultaneously measured during carbon monoxide (CO) inhalation in awake and sleeping goats up to HbCO levels of 40%. Unilateral BBF, which was continuously measured with an electromagnetic flow probe placed around the internal maxillary artery, progressively increased with CO inhalation in the awake and both sleep stages. The increase in BBF with CO inhalation during rapid-eye-movement (REM) sleep (delta BBF/delta arterial O2 saturation = 1.34 +/- 0.27 ml X min-1 X %-1) was significantly greater than that manifested during wakefulness (0.87 +/- 0.14) or slow-wave sleep (0.92 +/- 0.13). Ventilation was depressed by CO inhalation during both sleep stages but was unchanged from base-line values in awake goats. In contrast to slow-wave (non-REM) sleep, the ventilatory depression of REM sleep was primarily due to a reduction in tidal volume. Since tidal volume is more closely linked to central chemoreceptor function, we believe that these data suggest a possible role of the increased cerebral perfusion during hypoxic REM sleep. Induction of relative tissue alkalosis at the vicinity of the medullary chemoreceptor may contribute to the ventilatory depression exhibited during this sleep period.     

Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. I. Protective cross-immunity between transplantable tumor lines

Dimethylbenz[a]anthracene (DMBA)-induced transplantable fibrosarcomas in B2 homozygous chickens (SC line) grow progressively in normal chickens, but are rejected by chickens immunized previously with irradiated tumor cells and Corynebacterium parvum. Tumor-immune chickens resist challenge by the immunizing tumor lines as well as by some, but not all, fibrosarcoma lines. The pattern of cross-reactivity between four DMBA-induced transplantable tumor lines was examined in detail. Ability to reject a tumor challenge correlated very well (p less than 0.001) with the presence of delayed-type hypersensitivity (DTH) to that tumor. Immunization with one of two of the DMBA-induced lines tested also caused rejection of transplantable tumors developed from methylcholanthrene-induced and benzo(a)pyrene-induced primary fibrosarcomas. Although immunization with tumor caused DTH to chicken embryo fibroblasts (CEF), immunization with CEF failed to cause protective immunity or DTH to tumors. Presence of protective immunity, where tested, also correlated with the ability of spleen cells from immune donors to inhibit tumor growth in Winn tests. Humoral immunity exhibited even greater cross-reactivity than did cellular immunity. Distinct patterns of cross-reactivity were nevertheless observed with respect to the serum antibodies as detected in ELISA. Two of these patterns were also observed in several sera from primary tumor-bearing chickens, both including reactivity with CEF. Such reactivity was absent from normal chicken sera.     

Cathepsin B and D activity in stimulated peritoneal macrophages

Summary Highly sensitive and specific synthetic substrates were used to quantitate cathepsin B and D activity in peritoneal macrophages in response to stimulation in vivo with mineral oil and thioglycollate. After intraperitoneal instillation of mineral oil the activity of cathepsin B increased significantly (to 15 300 units/mg protein versus 7 340 in saline controls), reaching values approaching those found in alveolar macrophages (18 400 units/mg protein). Significantly greater stimulation of enzyme activity was obtained after intraperitoneal instillation of thioglycollate (23 600 units/mg protein). Cathepsin D activity also increased significantly after both mineral oil and thioglycollate. However, the increase was moderate (from 806 to about 1 200 units/mg protein), remaining still more than six times lower-than in alveolar macrophages. The data are the first to demonstrate that cathepsin B activity can be stimulated in vivo in peritoneal macrophages by instillation of agents that induce acute inflammation. They also point to a differential control of expression of cathepsin B and D activity in both peritoneal and alveolar macrophages in spite of the common lysosomal origin of the two enzymes.Abbreviations Cbz -N-benxyloxycarbonyl - 2NA 2-naphthylamine - EDTA ethylenediamine tetraacetate - DMSO dimethylsulfoxide - PBS phosphate-buffered saline - PM peritoneal macrophage - AM alveolar macrophage     

Transport of chlorine in the proximal tubule. Its effects on water-electrolyte absorption

Several studies in rat kidney have established that an appreciable fraction of proximal absorption is passive in nature and occurs across the highly conductive paracellular pathway. Passive absorption is generally ascribed to the transepithelial Cl- distribution, luminal Cl- activity (alpha lCl) being higher than plasma Cl- activity (alpha pCl). The inequality alpha lCl greater than alpha pCl generates a transepithelial diffusion potential, lumen positive, which taken together with the chemical potential differences of Cl- and Na+ across the epithelium gives rise to transepithelial electrochemical potential differences for Cl- and Na+ favoring their absorption. The alpha lCl greater than alpha pCl distribution is traditionally ascribed to preferential bicarbonate absorption. We argue that HCO3- absorption alone cannot generate a non equilibrium transepithelial Cl- distribution. Other mechanisms are necessary. Our measurements in amphibian proximal tubule demonstrate that the intracellular Cl- activity, alpha cCl, is higher than the theoretical value predicted for equilibrium. This distribution is the result of two basolateral coupled transport processes (Cl-/HCO3- exchange and Cl-/Na+ cotransport). It contributes to the exit of Cl- from cell to lumen (by passive diffusion and K+/Cl- cotransport), yielding alpha lCl values higher than the theoretical value for equilibrium with regard to plasma. Thus, a small transcellular flux of Cl- (without solvent) proceeds from interstitium to lumen. It compensates the dissipative tendency of a much higher paracellular Cl- absorptive flux (in association with water) on the transepithelial Cl- gradient. The result is a steady-state luminal Cl- distribution above equilibrium, along the major part of the proximal tubule.