The effect of label affinity on the sensitivity and specificity of a hapten radioimmunoassay: a comparison of three [125I]diphenylhydantoin radioligands with the 14C-labelled drug.

The effect on the sensitivity and specificity of a radioimmunoassay for diphenylhydantoin (DPH)has been investigated using three 125I-labelled tyrosine ester derivatives of DPH having different bridge lengths between the tyrosine moiety and the DPH moiety and 14C-labelled DPH. The results demonstrate that for a hapten which does not completely fill the antibody-binding sites, greatest sensitivity is achieved when the bridge of the iodine label is most dissimilar to that present in the original immunogen, when the hapten and label affinities are nearly equivalent. Greatest specificity is achieved with the label which most resembles the original immunogen. These results illustrate the difficulty of designing satisfactory labels for assays of both high specificity and sensitivity since minimal changes in label structure may produce greatly amplified changes in the subsequent affinity of the label for the antiserum.     

Recombinant avian oncoviruses. I. Alterations in the precursor to the internal structural proteins.

The synthesis of the gag precursor protein (Pr76) was studied in a number of recombinant avian oncoviruses, which were selected for recombination between the env and pol genes or the env and src genes. Such studies show that the electrophoretic mobility of the gag precursor protein of recombinant viruses (ΔPr76) was greater than that of the parental gene product (Pr76) in 16 of 24 cases. Viruses derived from recombination between endogenous (RAV-0) and exogenous viruses (RSV), as well as between two exogenous viruses, showed the ΔPr76 phenotype. In an mRNA-dependent rabbit reticulocyte translation system, 35 S RNA isolated from PR-RSV-C directed the synthesis of Pr76, while RNA isolated from a recombinant between PR-RSV-C and RAV-0 directed the synthesis of ΔPr76. These observations show that the synthesis of ΔPr76 is due to an alteration in the genome related to recombination. An analysis of the RNase T₁-resistant oligonucleotides demonstrated a crossover near the 5′ end of the genome (which may be within the gag gene) in two recombinant virus clones which synthesize ΔPr76 in infected cells; but no crossover was detected near the 5′ end of the genome in a third recombinant virus clone which synthesizes Pr76 in infected cells. Our data suggest that the synthesis of ΔPr76 is a consequence of recombination near the 5′ end of the genome.     

The dual effect of L-proline on spreading depression in the chicken retina

Evidence was presented for a glutamate agonistic effect of L-proline which promotes K+-based spreading depressions (SD) in chick retinas at relatively high concentrations (5 mM), in addition to an antagonistic effect which inhibits glutamate-based SDs at lower (2 mM) concentrations. Together these effects explain the observed biphasic effect of L-proline on the incidence of SD in the retina.     

Herpes simplex virus types 1 and 2: comparison of the defective genomes and virus-specific polypeptides.

Undiluted serial passage of HSV-1 or HSV-2 virions resulted in the synthesis of DNA which was resistant to cleavage by restriction endonuclease (Endo R.) HindIII. Defective DNAs of type 1 and 2 were observed to have similar biphasic renaturation kinetics and to have kinetic complexities of approximately 13% of the parental standard genome. Not more than 40% of the sequences found in defective HSV-2 DNA were found to be homologous to defective HSV-1 sequences. While an overproduction of an early polypeptide, VP175, was observed in cells infected with defective HSV-1, no overproduction of any polypeptide equivalent to VP175 was observed in cells infected with defective HSV-2. The results suggest that although the defective DNAs of HSV-1 and HSV-2 have some common physicochemical properties, their base sequences and genomic expression in the infected cell are different.     

Immunoreactive substance P in sympathetic ganglia: distribution and sensitivity towards capsaicin

The distribution of immunoreactive substance P was studied in sympathetic ganglia of guinea-pigs and rats. In both species the highest concentration was found in the coeliac-superior mesenteric ganglion. Guinea-pigs had three times higher concentrations of immunoreactive substance P than rats in all ganglia studied. Pretreatment of guinea-pigs with capsaicin (125 mg/kg; s.c.), an agent shown to deplete immunoreactive substance P only from primary sensory neurons, caused a 63–96% depletion of immunoreactive substance P in sympathetic ganglia and the splanchnic nerve. Superfusion of coeliac ganglia with capsaicin (3.3 × 10^?5M) in vitro led to a calcium-dependent release of immunoreactive substance P.The findings that capsaicin causes release and depletion of immunoreactive substance P from sympathetic ganglia support the hypothesis that substance P-containing fibers in sympathetic ganglia are primary sensory neurons.     

Assembly of a temperature-sensitive mutant of Rauscher murine leukemia virus at the cell surface induced by low temperature and by ligands.

NIH/3T3 mouse fibroblasts, infected with a Rauscher murine leukemia virus temperature-sensitive mutant (ts25) defective in assembly of budding particles at 39°, produce virus at the cell surface when the temperature is shifted rapidly to 0°. Virus buds are not assembled within the first 10 min at 0° and gradually increase in number and degree of development over a 2-hr period. However, release of infectious virus could not be demonstrated at 0° and might, therefore, be an energy-dependent process. Significant budding activity was also induced at the nonpermissive temperature by incubating cells with 0.25% glutaraldehyde or with antiserum to the major virus envelope glycoprotein, gp70. Anti-gp70 serum may induce budding by promoting aggregation of gp70-containing molecular assemblies and, consequently, association of core components in some transmembrane fashion. Induction of virus assembly with glutaraldehyde might occur as a results of nonspecific cross-linking of membrane proteins. These results suggest that procedures commonly used to minimize ligand-induced redistribution of cell surface molecules, i.e., labeling at low temperatures or after mild aldehyde fixation, may not immobilize certain membrane-associated molecules.     

Inflammatory peptide in spinal cord: evidence that the mediator of antidromic vasodilatation is not substance P

Extracts from rat and bovine spinal cord were found to have 300-1000 times more cutaneous oedema-inducing activity than could be attributed to their substance P-like activity estimated by assay on guinea-pig ileum. The activity on both assay systems was reduced in cord extracts from rats pretreated as neonates with capsaicin. Results of assays of fractions obtained from gel-filtration chromatography indicated that an agent, in the molecular size range for peptides, was present in spinal cord extracts, which possessed some of the properties of substance P but was not identical to it.     

The isolation and preliminary characterization of temperature-sensitive transformation mutants of Moloney sarcoma virus.

Temperature-sensitive (ts) transformation mutants of Moloney murine sarcoma virus were isolated from uv-irradiated viral stocks using a selection and screening procedure based on the ability of MSV-transformed NRK cells to grow in methyl cellulose or agar suspension. Mutants isolated by this procedure formed colonies in agar 1000- to 10,000-fold more efficiently at 34° than at 39°. They also exhibited a transformed morphology and elevated hexose transport levels at 34°, but were phenotypically normal at 39°. Both morphology and hexose transport showed transformed → normal and normal → transformed conversion within 12–48 hr of a temperature shift from 34 to 39° and 39 to 34° respectively. In contrast, ts-transformed cells suspended in agar and incubated at 39° for 24 hr showed a 90% reduction in colony-forming ability when the plates were returned to 34°. Superinfection of nonproducer ts-transformed cells with leukemia virus resulted in the rescue of ts MSV. Rescued supernatants also contained a high proportion (10%) of wt MSV. Repeated cloning of ts mutants, either as virus or cells, did not significantly affect the proportion of ts or wt virus rescued. The ability of ts-transformed cells to express the transformed phenotype at 39° could be restored by wt MSV superinfection, but not by MLV superinfection.     

Peripheral somatic activation and spontaneous firing patterns of neurons in the periaqueductal gray of the cat

A total of 50 neurons in the periaqueductal gray (PAG) of the cat were studied. Sixty percent of the units were excited by nociceptive pinch stimulation. These units were also excited by non-nociceptive stimulation such as tapping, hair movement and/or joint rotation. Non-nociceptive stimulation excited 20% of the units, whereas nociceptive stimulation did not drive these units. Their receptive fields were large, and often encompassed the entire body. Spontanous discharges of PAG neurons revealed great irregularity.     

Autoradiographic mapping of somatostatin receptors in the rat central nervous system and pituitary

Somatostatin receptor-binding sites have been visualized by autoradiography in the rat central nervous system and the pituitary using the [Tyr3] derivative of the stable octapeptide somatostatin analogue SMS 201-995, code named 204-090 (sequence in text), which has been shown to label specifically high-affinity somatostatin receptors in brain homogenates. Receptors are particularly concentrated in the deeper layers of the cerebral cortex and large areas of the limbic system are rich in somatostatin receptors, in particular the hippocampus (CA1, CA2, dentate gyrus), most amygdaloid nuclei, the medial habenula and the septum. Parts of the olfactory, visual and auditory, as well as visceral and somatic sensory systems are heavily labelled, in particular the anterior olfactory nucleus and tubercle, the superior and inferior colliculi, the nucleus of the solitary tract, the substantia gelatinosa of the spinal cord and the spinal trigeminal nucleus. It is of interest that the central grey and locus coeruleus are also substantially labelled with [125I]204-090. Striatum has moderate amounts of somatostatin receptors, distributed in a patchy and heterogeneous way. Cerebellum and substantia nigra are virtually devoid of somatostatin receptors. The described receptors are likely to represent the molecular target for a variety of pharmacological actions of somatostatin in the central nervous system and they emphasize the role played by somatostatin as a neuropeptide in this organ.     

Replicative functions of the SV40(cT)-3 mutant defective for nuclear transport of T antigen

The SV40(cT)-3 mutant is defective in transport of SV40 large tumor antigen (T-ag) to the nucleus. Several properties of T-ag associated with SV40 lytic infection and attributed to its nuclear localization were examined to determine whether biologically significant levels of the mutant T-ag (cT-ag) that were immunologically undetectable were transported to the nucleus in SV40(cT)-3-infected TC-7 cells. SV40(cT)-3 was defective in regulation of T-ag synthesis and initiation of viral DNA synthesis. These defects were presumably due to the lack of nuclear transport of cT-ag, since cT-ag was capable of interacting with the SV40 origin of viral DNA synthesis in a solution binding assay. The level of fatty acid acylation, a modification specific for the cell surface associated T-ag, was not affected by the cT mutation. The cT mutation sufficiently suppressed the nuclear transport of wild-type (WT) T-ag in SV40(cT)-3-infected COS-1 cells to result in the cessation of WT-T-ag-stimulated SV40(cT)-3 viral DNA synthesis. These results are discussed with respect to the recent findings that SV40(cT)-3 is fully competent for the transformation of established cell lines and the induction of cellular DNA synthesis in quiescent cells.     

The effect of the association of Gossypol and Lonidamine on the energy metabolism of Ehrlich ascites tumor cells

The effect of the association of Gossypol and Lonidamine on the energy metabolism of Ehrlich ascites tumor cells has been investigated. The action of the drug on tumor cells was studied by addition of the drugs to cells harvested from Swiss male mice. The results may be summarized as follows: (1) Low concentrations of Gossypol increase the rate of oxygen consumption by uncoupling oxidative phosphorylation. High concentrations result in an inhibition of oxygen consumption with a mechanism that must be regarded as not directly related to the uncoupling activity. (2) Gossypol, at concentrations at which it exerts an uncoupling activity, stimulates mitochondrial ATPase which in turn increases the aerobic and anaerobic rates of lactate production. The decrease of glycolysis at high concentrations of Gossypol does not depend on the inhibition of enzymes of the glycolytic pathway, but must be ascribed to cell death. (3) The association of a low concentration of Gossypol with Lonidamine brings about a further inhibition of oxygen consumption. Moreover, Lonidamine abolishes the stimulation of glycolysis induced by Gossypol and lowers lactate production to values that are quite similar to those found with Lonidamine alone. (4) It may be concluded that the association of Gossypol and Lonidamine results in a very effective decrease of the energy requirements of cancer cells.     

The nature of polypeptides larger in size than the major surface antigen components of hepatitis b and like viruses in ground squirrels, woodchucks, and ducks

The relationships of various polypeptides associated with hepatitis B surface antigen (HBsAg), ground squirrel hepatitis surface antigen (GSHsAg), woodchuck hepatitis surface antigen (WHsAg), and duck hepatitis B surface antigen (DHBsAg) were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tryptic peptide mapping. Analysis of independent antigen isolates by SDS-PAGE resulted in bands consistently observed at 24,000, 28,000, 32,000, 43,000, and 50,000 Da with HBsAg; at 22,000, 25,000, 35,000, 37,000, 39,000, and 42,000 Da with GSHsAg and WHsAg; and at 18,500, 30,000, and 38,500, Da with DHBsAg. Comparison of the major polypeptide pair from the mammalian viruses by tryptic peptide mapping suggests more than a single point of glycosylation or other post-translational modification(s) in some paired comparisons and/or heterogeneity in glycosylation in others. Comparison of the major component of each mammalian virus (HBsAg p24, GSHsAg p22, or WHsAg p22), or the major polypeptide of DHBsAg (p18.5), with their respective larger polypeptides by peptide mapping indicated that one or more of the larger components in each virus shares extensive homology with the appropriate major component. Further, these larger components possess additional spots, interpreted as additional primary sequences, which were not found in the map of the appropriate major component. Collectively, the results suggest that a number of surface antigen-associated polypeptides may be partially encoded for by the pre-S gene region known to exist in hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV), and likely to exist in ground squirrel hepatitis virus (GSHV) and duck hepatitis B virus (DHBV) DNA.     

An attenuated mutant of Venezuelan encephalitis virus: Biochemical alterations and their genetic association with attenuation

A temperature-sensitive mutant of Venezuelan encephalitis virus derived by chemical mutagenesis from the hamster-virulent 68U201 wild-type (wt) strain was previously found to be attenuated. This mutant, is 126, replicated in infected hamsters and elicited production of protective antibodies. Further phenotypic differences between is 126 and 68U201 wt have been characterized in an attempt to localize the genetic basis of the mutant's attenuated virulence. The mutant was shown to differ from the parent virus with respect to virion surface structure-dependent characteristics: temperature lability, plaque sizes in Vero cells, and binding properties to hydroxylapatite. The surface difference was identified by isoelectric focusing of the virion envelope glycoproteins as an alteration in the E₁ glycoprotein. The common genetic basis of all these phenotypic differences was demonstrated by the isolation of independently arising, stable genetic revertants of ts 126, which exhibited characteristics identical in every respect to 68U201 wt. It appears from these studies that the mutation which gave rise to the is 126 mutant virus occurred in the structural gene coding for the E₁ envelope glycoprotein and that the resultant phenotypic alteration in this glycoprotein is genetically associated with the mutant's lack of virulence.     

Isolation and characterization of entomopox virions from virus-containing inclusions of Amsacta moorei (Lepidoptera: Arctiidae)

Preparations of Amsacta moorei entomopox virions were obtained from virus-containing inclusions (VCI) by using a carbonate-thioglycolate solution (pH 10.7–11.5). The virions possessed a uniform coat (“halo”) surrounding the viral envelope and exhibited an RNA polymerase activity. The “halo” could be removed by prolonged exposure to the carbonate-thioglycolate solution. Virions obtained by this treatment, however, possessed low infectivity and no detectable RNA polymerase activity. Removal of the “halo” by trypsin resulted in virions which possessed RNA polymerase activity and relatively high infectivity.Preparations of particles with and without the “halo” were similar in percent DNA, protein per OD₂₆₀, number of particles per OD₂₆₀, and RNA polymerase activity. Particles without the “halo,” however, were less dense (1.262 g/cm³) in CsCl than those with the “halo” (1.282 g/cm³) and 15–45 times more infective.Parallel studies of “nonhaloed” Amsacta virions (trypsin-treated) and vaccinia virions showed that both viruses contained similar amounts of protein per OD₂₆₀, but Amsacta virions contained only 36% of the DNA found in vaccinia.     

M protein instability and lack of H protein processing associated with nonproductive persistent infection of HeLa cells by measles virus

Persistent infections such as subacute sclerosing panencephalitis (SSPE) which do not produce infectious virus particles (nonproductive persistence) are often accompanied by a reduced steady-state amount of the viral matrix (M) protein and/or reduced hemadsorption activity. The possible causes of these aberrations associated with nonproductive persistence were investigated by following changes in the viral proteins with time in pulse-chase experiments. Three HeLa cell lines persistently infected with measles virus; K11, K11A, and HG111; were compared to each other and to acutely infected HeLa cells. K11 produces infectious virions at a low level (productive persistence). K11A and HG111 are both nonproductive persistently infected cell lines derived from K11. K11A cells have a reduced steady-state amount of viral M protein and reduced hemadsorption activity. HG111 cells have reduced hemadsorption but a normal level of viral M protein. As such, these cell lines serve as good model systems for the study of nonproductive persistent infection associated with SSPE. The reduced amount of M protein in K11A was found to result from rapid degradation of the protein. Degradation of the protein resulted from changes in the protein itself rather than from cellular changes. The hemagglutination (H) protein was found to be present at a low level in K11A cells. In addition, in both K11A and HG111 cells, conversion of the sugar moiety of the H glycoprotein from the high mannose form to the complex sugar form did not take place. Such modification usually occurs concomitant with transport of glycoproteins onto the cell surface. As such, lack of processing could preclude the appearance of functional H proteins on the cell surface. This could account for the reduced hemadsorption activity in these cells. The roles that these changes may play in the generation of nonproductive persistence are discussed.     

Lymphokine-mediated fusion and migration inhibition of alveolar macrophages

Guinea pigs were shown to produce a lymphokine termed macrophage fusion factor (MFF) which mediated the fusion of 70–80% of guinea pig or rabbit alveolar macrophages, but not guinea pig peritoneal macrophages. In the conventional migration inhibitory factor (MIF) assay, guinea pig aveolar macrophages were inhibited in their migration and large numbers of giant cells were present. There appeared to be a correlation between the titer of MFF and migration inhibition of alveolar macrophages but not with MIF titer as expressed on the peritoneal macrophage. Guinea pig MFF production was erratic and its absence from lymphokine supernatant fluids correlated with an absence of migration inhibitory activity for the alveolar macrophage. Guinea pig MIF production was more constant and high titers were invariably present. Rabbit crude lymphokine supernatant fluids containing MFF also inhibited the migration of their alveolar macrophages when measured at 24 and 48 hr during the MIF assay. Extensive numbers of giant cells were observed in the cell fan whenever migration inhibition was present. α-l-Fucose, which is known to block the receptor sites of MIF, failed to block giant cell formation in either the MFF or the MIF assay and also failed to block migration inhibition of the alveolar macrophages. The results suggest that lymphokines other than MIF can inhibit the migration of alveolar macrophages in the standard MIF assay and that the lymphokine responsible for migration inhibition and fusion of alveolar macrophages is the same lymphokine, MFF.