The concentration of enkephalin-like material in the chick retina is light dependent

The enkephalin-like immunoreactivity in the retina of chicks has been studied using immunohistochemical and radioimmunoassay techniques. The histochemical experiments showed that the immunoreactivity was confined to a subpopulation of amacrine cells in the inner nuclear layer which projected processes into sublaminae 1 and 3-5 of the inner plexiform layer. The distribution of the immunoreactivity was markedly influenced by the ambient lighting conditions: it was reduced in the dark and restored by a period in the light. The reactivity was lost from both cell soma in the inner nuclear layer and from the processes. Radioimmunoassays showed that the quantity of enkephalin-like material was reduced by more than 60% after 12 h in the dark. Attempts to entrain a rhythm by keeping chicks on 12/12 h light/dark cycles for up to 4 days were largely unsuccessful. A rhythm may have been partially entrainable, but the major factor involved was light. These results highlight the lability of the neuropeptide in the retina and the need for controlled lighting conditions in studies of this kind. They also indicate that this system may be a fruitful model to explore two important issues: (i) it could allow studies of neuropeptide metabolism in a physiologically intact system; (ii) the role of particular amacrine cells in visual processing could be determined by depleting them of their neurotransmitter/neuromodulator.     

Evidence for an amacrine cell system in the ganglion cell layer of the rat retina

In the ganglion cell layer of the rat retina approx 50% of the cells with the Nissl morphology of neurons survive optic nerve section in infant and adult rats and cannot be retrogradely labelled with horseradish peroxidase. The number of neurons which can be retrogradely labelled with horseradish peroxidase from subcortical visual centres is similar to the number of axons in the optic nerve, and it is concluded that the small neurons do not send an axon into the optic nerve. The dendritic tree of the cells which have axons was demonstrated by filling the cells with horseradish peroxidase from the optic nerve. The dendritic structure of the cells which survive optic nerve section was shown by injecting horseradish peroxidase into the retina or impregnating with the Golgi method the cells which survive optic nerve section. A variety of amacrine cells were found in the ganglion cell layer which form branches in the lower part of the inner plexiform layer.It can be concluded that amacrine cells form a substantial number of the neurons in the ganglion cell layer.     

Electrophysiological evidence for an inhibitory accumbens-entopeduncular pathway in the rat

The effects of electrical stimulation of the nucleus accumbens (NA) on the activity of neurons of the entopeduncular nucleus (EP) were studied in the rat by extracellular single unit recordings. Many of the EP neurons were identified by antidromic activation from the lateral habenula (HBL). Stimulation of the NA induced inhibitions in 22.7% of the recorded EP cells. The data provide evidence for an inhibitory small projection from the NA to EP.     

Electrophysiological evidence for a somatotopic sensory projection to the striatum of the rat

The somatosensory projection to the striatum of the rat was investigated by electrophysiological mapping. Fifty-five units were encountered that responded to specific forms of stimulation delivered to particular parts of the body. Units that responded to stimuli delivered to the hindlimb, scrotum and tail were found in the most caudal regions of the striatum while those that responded to stimulation of the forelimb, head and neck were found in the anterior regions of the striatum. The results strongly suggest that there is in the striatum of the rat, a sensory map of the body that is organized on topographical principles.     

Electron microscopic evidence for scorpion toxin binding to synapses of rat brain cortex

The venom from the scorpion Centruroides noxius Hoffman was fractionated by Sephadex G-50 gel filtration. The toxic fraction (n.II) was coupled to Sepharose-4B and used for purification of specific horse immunoglobulins. The purified immunoglobulins were linked to ferritin with glutaraldehyde and repurified in the same affinity column. This complex bound to the postsynaptic membrane of rat brain tissue previously exposed to toxin.     

Neurotensin-like and somatostatin-like immunoreactivity within amacrine cells of the retina

Neurotensin-like and somatostatin-like immunoreactivity was demonstrated in the pigeon retina, using both immunohistochemical and radioimmunoassay techniques.Immunohistochemical studies utilized both the indirect immunofluorescence and immunoperoxidase procedures with two well-characterized antisera to neurotensin and three well-characterized antisera to somatostatin. Specific immunoreactivity of each antiserum was established by absorption with either 10 μM synthetic neurotensin, somatostatin or leu⁵-enkephalin. Specific immunohistochemical staining for neurotensin and for somatostatin was observed within separate populations of multistratified amacrine cells. Neurotensin-like and somatostatin-like immunoreactivity were observed within somata located in the inner nuclear layer and within varicose processes ramifying in laminae 1, 3 and 4 of the inner plexiform layer. Immunoreactive somata and processes were observed throughout the retina and their density appeared to be greatest within central retinal regions. The somata-containing neurotensin-like and somatostatin-like immunoreactivity measured about 7 μm in diameter. The cell to cell spacing of neurotensin-like immunoreactive somata was approximately 30 μm and the cell to cell spacing of somatostatin-like immunoreactive somata was approximately 27 μm in central retinal regions. Within more peripheral retinal regions, immunoreactive cells were spaced farther apart.Radioimmunoassays utilizing well-characterized antisera to neurotensin and somatostatin demonstrate specific neurotensin-like and somatostatin-like immunoreactivity in acetic acid extracts of the retina. The concentration of immunoreactive neurotensin is 59 ± 7 fmoles per whole retina (mean ± S.E.M.) or 15.4 ± 2 fmoles per mg protein. The concentration of immunoreactive somatostatin is 2209 ± 440 fmoles per whole retina or 527 ± 76 fmoles per mg protein.These results demonstrate the existence of two additional neuropeptides within selected populations of retinal amacrine cells. The localization of several different neuropeptides within the retina suggests that neuropeptides play a specific role in retinal function.     

Visual checkerboard-evoked potentials from upper and lower retinal halves,and variation of check size

Human visually evoked potentials were studied with checkerboard reversal stimulation of the upper and lower halves of the visual field, using different check sizes and field diameters. Although attenuation of the overall amplitude was seen in the potentials with stimulation of the upper visual half-field, the steady-state response amplitude vs spatial frequency curves for upper and lower half-field stimulation were similar. Decrease of field size reduced the amplitude but did not change the shape of the amplitude vs spatial frequency curve with its peak of 14 min of arc check size. Dissimilar characteristics of half-field responses were seen with 2/sec reversal: stimulation of the lower visual half-field evoked wave forms with a positive component of 102 (112) msec peak latency, which was delayed by 21.5 (24) msec when the upper half-field was stimulated, using 56 (14) min checks.     

Reversal potential for glutamate responses in hippocampal pyramidal cells

L-Glutamic acid was iontophoresed onto hippocampal CA1 pyramidal neurons. Intracellular recordings revealed that glutamate reliably depolarized all neurons examined. The depolarization evoked by glutamate was voltage sensitivity and had an apparent reversal potential close to 0 mV.     

Evaluation of methods for chemically coupling foot-and-mouth disease virus to sheep red blood cells for immunological assays

Six methods of chemically coupling proteins to red blood cells were evaluated for their effectiveness in coupling foot-and-mouth disease virus (FMDV) to sheep red blood cells. The coupling agents tested were potassium periodate, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (ECDI), chromium chloride, glutaraldehyde, bis-diazotized benzidine (BDB) and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Of these, only the coupling methods using BDB and SPDP resulted in virus-red cell complexes that reacted with FMDV antiserum in passive hemagglutination and passive immune hemolysis assays. The BDB and SPDP methods were studied further to determine optimal coupling conditions, the kinetics of coupling and the effects of chemical couplers on viral integrity. Only the FMDV-red cell complexes formed with SPDP were suitable targets for detecting FMDV antibody producing lymphocytes in a hemolytic plaque assay.     

Heat induced ultrastructural injuries in lymphoid cells.

Exposing lymphoid cells to elevated temperatures in vitro causes both cell surface and intracellular structure alterations. In addition to commonly described thermal effects, i.e., destruction of nucleolus, chromatin, and polyribosomes, we have also observed several unique thermal-induced structural changes, including the loss of microvilli, formation of intracellular fibrous inclusion bodies, arrangement of mitochondrial cristae in a concentric fashion, and the non-random distribution of the organelles.     

Evidence for different receptor sites in mouse spleen cells for the Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins

Liposomes were reconstituted from phosphatidylcholine and Sendai virus glycoproteins HN or F and their interaction with mouse spleen cells was studied. Both the HN and F liposomes were able to stimulate chemiluminescence (CL), indicating that the glycoproteins were able to interact with the cell membrane independently of each other. The induction of CL in cells which had been pretreated with liposomes by monoclonal antibodies to either HN or F demonstrated that HN and F bind to the cells independently. The presence of F liposomes on the cell surface was also confirmed by immunoelectron microscopy. Cells pretreated with HN and F liposomes revealed a different pattern of CL when challenged with intact virus or the calcium ionophore A23187 indicating that HN and F bind to different receptor sites.     

Tumorigenicity of SV40-transformed human and monkey cells in immunodeficient mice

Human cells transformed in vitro by SV40 to the anchorage-independent state rarely form tumors in nude mice and therefore constitute an important exception to the otherwise tight correlation between anchorage independence and cellular tumorigenicity. In this paper we explore a number of possible explanations for this unusual situation. We find that the phenomenon is not restricted to human cells but includes monkey cells as well. The nontumorigenic phenotype of the primate SV40 transformants is highly stable. We are unable, through selection of ever more anchorage-independent lines, to generate a primate SV40 transformant which will grow as a tumor in even the most immunologically crippled animals. One tumor was obtained from SV80 (an SV40-transformed human cell line) following injection into a mouse deficient in both T and B cell functions. However, cell lines derived from this tumor are not significantly more tumorigenic than the SV80 parent. This low incidence of tumor formation is not due to the fact that the primate cells are transformed by nononcogenic defective viral genomes nor to a nutritional inadequacy of the host animal for the growth of human cells. Although a T cell-independent mechanism may be the major mechanism involved in tumor suppression, it is unlikely that this completely accounts for the general lack of tumor growth by most of these cells. It appears that the interaction of SV40 (a primate virus) with primate cells may be intrinsically less oncogenic than its interaction with rodent cells.     

Multiplicity-dependent induction of viral capsid antigen in Raji cells superinfected with Epstein-Barr virus

A quantitative analysis of Epstein-Barr virus (EBV)-induced early antigen (EA) and viral capsid antigen (VCA) syntheses was carried out in Raji cells superinfected with purified, concentrated P3HR-1 EBV. When the cells were exposed to the virus and assessed by immunofluorescence and immunoprecipitation, EA induction occurred significantly (17%) but not VCA (less than 1%), at a low-input multiplicity of infection (MOI) of 10 EBV DNA copies/cell. In contrast, at a high MOI of 500 EBV DNA copies/cell, the majority of cells were positive for both EA (82%) and VCA (61%). The latter VCA synthesis was accompanied by the replication of EBV DNA. Kinetic studies showed that EA induction was directly proportional to the dilution of the infecting virus, while VCA was made following three-hit kinetics. The implications of these results are discussed in relation to the heterogeneous nature of P3HR-1 EBV and a possible role of EA in VCA synthesis.     

Biosynthesis of J-chain in human lymphoid cells producing immunoglobulins of various isotypes

The relationship between synthesis, secretion, and subcellular localization of J-chain, IgM, IgA, and IgG was investigated in cultures of PWM-stimulated human PBL and in lymphoblastoid cell lines. Cells were examined for surface, cytoplasmic, and secreted immunoglobulins (Igs) and J-chain by immunofluorescence and radioimmunoassay (RIA). By these techniques, J-chain was detected in cells that produce polymeric or monomeric Igs. In PWM-stimulated PBL the synthesis of J-chain paralleled the production of Igs. In both PWM-stimulated (for 2 days) and unstimulated PBL, equal proportions of free and disulfide-linked J-chain were found. Increased amounts of intracellular J-chain were produced at later stages in PWM-stimulated PBL and J-chain occurred mostly in a free form. In tissue culture fluids, J-chain was not secreted in a free form but was always disulfide-linked to polymeric Igs. In lymphoblastoid cell lines, J-chain was present in a disulfide-linked form in IgM and IGA producers, but in IgG cells and in an IgM cell line (DAUDI) that did not secrete IgM but expressed it on the cell membrane, intracellular J-chain was present in free form. Although various proportions of polymeric and monomeric IgA were seen in culture fluids from IgA-secreting cell lines, intracellular IgA occurred mostly in a monomeric form. Further studies revealed that the ability to produce polymers was not equally distributed among all cells and might vary according to their content of J-chain and stage of maturation. Subcellular fractionation and subsequent analyses for J-chain and Ig in PWM-stimulated PBL and in IgM or IgG-producing cell lines revealed that these proteins were associated with fractions that contained ribosomes, cell sap, and low molecular weight RNA. In lysates of IgG and J-chain producing cells grown in the presence of 3H-labeled amino acids, intracellular J-chain was not disulfide-linked to IgG.     

Kinetics of parvovirus replication,cytopathic effects,and mitotic arrest in synchronized bovine fetal spleen cells

Initiation of autonomous parvovirus replication depends on the S phase of host cells actively traversing the cell cycle. The parvoviruses Lu III and H-1 inhibit synchronized cells from entering mitosis, implying that parvoviruses rapidly shut down cell cycle traverse during G2 phase. Bovine parvovirus (BPV) did not inhibit the entry into mitosis of hydroxyurea synchronized bovine fetal spleen cells. Mitotic indexes of infected cultures were as much as 60-fold higher than those of mock-infected controls. Mitosis in control and infected cultures peaked at 10 hr after infection corresponding to the end of the BPV eclipse period. Cytopathic changes, including morphological alteration of mitotic chromosomes, were detected in mitotic cells from infected cultures by light and electron microscopy. Arrest of BPV-infected cells in mitosis may explain these results. Not all infected cells were killed in mitosis, since some developed intranuclear inclusions and became pycnotic as nucleated, interphase cells. Inclusion formation was coincident with viral morphogenesis in interphase nuclei at 16 to 18 hr postinfection, late in the viral replication cycle. The cell cycle stage at which parvovirus-infected cells are arrested and cytopathic events ensue may be determined by the cellular progression rate from S phase through G2 and M.     

Localization of IgG1-producing cells within a hyperstimulated lymph node of mice infected with an intestinal nematode parasite

In the coeliac lymph node of IgG₁ hypergammaglobulinaemic BALB/c mice infected with the intestinal nematode, Nematospiroides dubius, the bulk of in vitro production of biosynthetically-labelled IgG₁ appeared to be concentrated in the cortical regions of the node and not the medulla. This was demonstrated by incubating segments of the grossly enlarged lymph node with [³H]leucine and assessing [³H]IgG₁ production by a protein A-Sepharose binding method.     

Early protein synthesis in activated human peripheral blood mononuclear cells

Though B-cell division and Ig synthesis in response to pokeweed mitogen (PWM) require interaction with T-cells and monocytes, it is not clear which earlier events in B-cell activation share this requirement, and which are the result of direct interaction of mitogen with the B-cell. Having previously shown that the acceleration of lecithin synthesis in human B-cells at 16-20 hr requires both T-cells and monocytes, we now examine whether B-cells require similar interactions to increase their protein synthetic rate, another important activation event. At 21-24 hr of PWM stimulation, the stimulation index (SI) for incorporation of [35S]methionine into protein was 2.1 +/- 0.4 for unfractionated cells, 1.7 +/- 0.1 for B-cells, 2.5 +/- 0.1 for T-cells, and 3.4 +/- 0.5 for monocytes. Thus monocytes contributed substantially to early mitogen-induced protein synthesis by human peripheral blood mononuclear cells. When the monocyte/B-cell fraction (MB) and T-cell fraction (T) were mixed at various ratios in PWM-stimulated cultures, synergy was apparent at MB:T ratios of 1:1 and 1:2, indicating cell interactions augmented early mitogen-driven protein synthesis in at least one of these cell types. However, much or all of this synergy could be attributed to T-cells, whose protein synthetic response was augmented by B-cells and monocytes. In contrast, the early increase in B-cell protein synthesis appeared to be independent of cell interactions, since their SI of 1.7 was not influenced by varying the proportion of M- or T-cells over a 50-fold range. These contrasting results between two contemporary events fits the hypothesis that one (accelerated phospholipid synthesis) requires a first signal plus one or more cell interaction signals, whereas the other (accelerated protein synthesis) requires only the first signal.     

Interferon action against reovirus: activation of interferon-induced protein kinase in mouse L929 cells upon reovirus infection

Experiments are presented which indicate that reovirus infection results in an activation of the interferon (IFN)-induced protein kinase in mouse L929 cells. This is indicated by (i) the phosphorylation in vivo of a 67,000 M_r (67K) polypeptide, which is characteristic of the IFN-induced protein kinase, within a few hours after reovirus infection of IFN-treated mouse L929 cells, and (ii) the phosphorylation in vitro of the 67K polypeptide as well as the α-subunit of exogenously added initiation factor eIF-2 in extracts of IFN-treated reovirus-infected cells without the addition of double-stranded RNA which is required in similar extracts from uninfected cells. In NIH 3T3 cells, which are deficient in (2′,5′)oligoadenylate-activated endonuclease, the replication of reovirus is inhibited by IFN treatment, though EMC virus replication is insensitive. It is suggested that this kinase activation observed upon reovirus infection may play a role in the antiviral action of IFN against reovirus.