Extraretinal photoreceptors in the brain of the crayfish Cherax destructor.

Two clusters of red-brown pigmented cell somata lie among other cell somata along the anterior margin of the cerebral ganglion in the crayfish Cherax destructor. Electron micrographs show these cells to contain round electron dense pigment granules and that the cell membranes of two or more adjacent cells fold together to form rhabdom-like structures. The pigmented cells specifically bind a monoclonal antibody against the major species of opsin in R1-7 retinula cells of the compound eye of Cherax. When stimulated with light, the pigmented cells respond with a receptor potential-like depolarization. The axons of the pigmented cells terminate in the neuropil of the protocerebral bridge, together with neuronal elements that label with antibodies against serotonin and substance P. We suggest that the brain photoreceptors of the crayfish are important in the entrainment of circadian rhythms.     

Analysis of Adenosine Immunoreactivity, Uptake, and Release in Purified Cultures of Developing Chick Embryo Retinal Neurons and Photoreceptors

We have investigated the presence of endogenous adenosine and of mechanisms for adenosine uptake and release in chick embryo retinal neurons and photoreceptors grown in purified cultures in the absence of glial cells. Simultaneous autoradiographic and immunocytochemical analysis showed that endogenous adenosine and the uptake mechanism for this nucleoside colocalize in practically all the photoreceptors, but only in approximately 20% of the neurons. Approximately 25% of the neurons showed either immunocytochemical labeling or autoradiographic labeling, while greater than 50% of the neurons were unlabeled with both techniques. [3H]Adenosine uptake was saturable and could be inhibited by nitrobenzylthioinosine and dipyridamole and by pretreatment of the [3H]adenosine with adenosine deaminase. Although these observations indicate that the uptake is specific for adenosine, only 35% of accumulated radioactivity was associated with adenosine, with the remaining 65% representing inosine, hypoxanthine, and nucleotides plus uric acid. Adenosine as well as several of its metabolites were released by the cells under basal as well as K(+)-stimulated conditions. Potassium-enhanced release was blocked by 10 mM CoCl2 or in Ca2(+)-free, Mg2(+)-rich solutions. The results indicate that retinal cells that synthesize, store, and release adenosine differentiate early during embryogenesis and are therefore consistent with a hypothetical role for adenosine in retinal development.     

Marked Amine and Amine Metabolite Changes in Norrie Disease Patients with an X-Chromosomal Deletion Affecting Monoamine Oxidase

Urinary and plasma amines and amine metabolites were quantified in two individuals with Norrie disease resulting from a deletion in chromosomal region Xp11.3, recently reported to be associated with absence of the gene encoding monoamine oxidase (MAO)-A and nondetectable MAO-A activity in fibroblasts and MAO-B activity in platelets. Marked (four-to 100-fold) elevations in levels of urinary phenylethylamine, o-tyramine, and m-tyramine (which are preferential substrates for MAO-B) and marked reductions (90%) in levels of 3-methoxy-4-hydroxyphenylglycol (a deaminated metabolite of norepinephrine, a preferential substrate for MAO-A) in urine and plasma confirmed the presence of a systemic, functionally significant reduction in the activities of both MAO isozymes. The magnitude of these changes, which are equivalent to those found in subjects taking MAO-inhibiting antidepressants, suggests that early initiation of dietary and drug restrictions may be clinically important in these and other patients with X-chromosomal mutations involving MAO. These findings further support the proposition that the MAOA and MAOB genes are located in close proximity on the X chromosome. Negligible changes in the metabolites of dopamine and serotonin raise the possibility that other metabolic pathways are of importance for their production, that dietary or intestinal bacterial sources contribute substantially to the presence of these amine metabolites in urine, or both.     

Intrasynaptosomal Sequestration of [3H]Amphetamine and [3H]Methylenedioxyamphetamine: Characterization Suggests the Presence of a Factor Responsible for Maintaining Sequestration

We examined the incorporation of [3H]methylenedioxyamphetamine ([3H]MDA) and [3H]amphetamine into rat brain synaptosomes. Saturation studies, using increasing concentrations of nonradioactive ligand, revealed that [3H]-MDA interacted with two saturable sites that were sensitive to boiling of the tissue. Eadee-Scatchard plots of [3H]MDA saturation data were curvilinear; nonlinear curve-fitting analysis of these data suggested the presence of high- and low-affinity [3H]MDA sites of association: KD high = 295 nM, Bmax high = 32 pmol/mg of protein; KD low = 45 microM, Bmax low = 5.2 nmol/mg of protein. Association of [3H]MDA to the low-affinity site was dependent on the presence of isotonic sucrose in the incubation medium. The high capacities of these sites argue against a bimolecular interaction of [3H]MDA with monovalent protein binding sites. [3H]MDA incorporation was reduced under conditions that disrupt the integrity of plasma membranes, such as sonication, incubation in hypotonic media, and incubation in the presence of the detergent digitonin. These data indicate that [3H]MDA incorporation into synaptosomes may represent an internalization and sequestration phenomenon. [3H]MDA incorporation was also reduced by preincubation of the synaptosomal preparation at 37 degrees C or in hypotonic buffer at 4 degrees C, a result suggesting that this sequestration is maintained by an intrasynaptosomal component that is lost under the preincubation conditions described above. [3H]MDA incorporation was pH dependent (maximal at pH 7.5) and temperature sensitive (maximal incorporation occurred at 21 degrees C and was substantially reduced at 37 degrees C). [3H]Amphetamine was also incorporated into synaptosomes, and this incorporation was sensitive to the same physical manipulations of the tissue preparation as [3H]MDA incorporation. The synaptosomal sequestration of both [3H]MDA and [3H]amphetamine was inhibited by permeant cations, such as sodium and potassium, a result suggesting that the proposed intrasynaptosomal component that maintains the sequestration is anionic. Preliminary pharmacological profiles of [3H]MDA and [3H]amphetamine sequestration were identical. The rank order of inhibitor potencies for the incorporation of both ligands was desipramine greater than amphetamine greater than MDA greater than methylphenidate. This order of potency does not correspond to the lipophilicity of the test drugs. The synaptosomal incorporation and sequestration of [3H]MDA, [3H]methylenedioxymethamphetamine, and [3H]amphetamine described in the present report may be important in the molecular mechanism of action of monoamine release induced by the amphetamines.     

Short-term stimulatory effect of Sertoli cell conditioned medium on Leydig cell steroidogenesis is not mediated by inhibin

Addition of concentrated rat Sertoli cell conditioned medium (rSCCM) to isolated Leydig cells from immature rats stimulated steroid production more than 13-fold within 4 h. LH-stimulated steroidogenesis was not enhanced by addition of rSCCM. The biological activity of the concentrated rSCCM was higher after incubation of Sertoli cells with FSH, whereas FSH alone did not stimulate steroid production. This effect of rSCCM was not due to inhibin, since highly purified 32 kDa rat inhibin, in doses equivalent to those present in rSCCM, had no effect on steroidogenesis during the 4 h incubation period. Furthermore, inhibin could be separated from the Leydig cell stimulating factor by anion-exchange chromatography. These results indicate a short-term paracrine control of Leydig cell steroidogenesis by Sertoli cell derived factors, which differ from inhibin.     

Linker insertion mutagenesis of herpesviruses: inactivation of single genes within the Us region of pseudorabies virus.

We describe a technique for the systematic inactivation of nonessential genes within the genome of a herpesvirus without the requirement for phenotypic selection. This technique is based on the insertion of an oligonucleotide containing translational stop codons at a random site within a large cloned viral DNA fragment. Mutant virus is then reconstituted by cotransfection with overlapping viral clones, together comprising the entire viral genome, as described previously (M. van Zijl, W. Quint, J. Briaire, T. de Rover, A. Gielkens, and A. Berns, J. Virol. 62:2191-2195, 1988). This technique was used to construct, in a single experiment, a set of 13 viable pseudorabies virus strains with oligonucleotide insertions within all known genes of the Us region except for the gp50 gene, which proved essential for virus growth in cell culture. The growth rate in porcine kidney cells of mutants of all nonessential Us genes was similar to that of the parental virus, with the exception of a mutant of the recently identified protein kinase gene.     

Very high frequency of reversion to guanidine resistance in clonal pools of guanidine-dependent type 1 poliovirus.

We have carefully examined the frequency of guanidine-resistant revertants in six different clonal pools of guanidine-dependent mutants of type 1 poliovirus. The mutation frequency was (6.5 +/- 6.3) x 10(-4) (with all amino acid substitutions occurring at position 227). The minimal corrected base substitution frequency per single nucleotide site in the codon for amino acid 227 was (2.1 +/- 1.9) x 10(-4).     

Molecular and serologic analysis of IgG1 deficiency caused by new forms of the constant region of the Ig H chain gene deletions

Selective IgG1 deficiency is a rare disease. We report a familial form of IgG1 deficiency, in which IgG1 was undetectable in a 5-yr-old girl with a history of asthma and respiratory tract infections. Her father had an IgG1 level that was one-third of the mean amount found in normal healthy controls. The defect in the proband was caused by a homozygous deletion of the structural gene for C gamma 1. A Southern blot analysis demonstrated that the maternal haplotype contained a deletion encompassing C gamma 1, C psi epsilon 1, C alpha 1, C psi gamma, and C gamma 2, whereas the deletion on the paternal haplotype was confined to the C gamma 1 gene. Neither of these deletions has previously been reported. IgG1 normally constitutes the dominant isotype for antibodies directed against protein Ag, including viral proteins. We have analyzed the immune response to a number of different protein and polysaccharide Ag in the patient and her parents. In the proband, antiviral antibodies were restricted to the IgG3 and IgG4 subclasses. However, the total amount of IgG directed against several viruses was below the concentration found in normal seropositive individuals. The father and the paternal grandfather, both with low serum IgG1 levels, also had asthma, thus indicating a possible causal relationship.     

Induction of lysis by T cell receptor gamma delta+/CD3+ T lymphocytes via CD2 requires triggering via the T11.1 epitope only

The requirements for activation of the lytic machinery through CD2 of TCR gamma delta+/CD3+ cells were examined, by utilizing bispecific heteroconjugates containing anti-CD2 mAb cross-linked to anti-DNP. Contrary to the CD2 activation requirements in TCR alpha beta+/CD3+ cells, cytotoxic activity in TCR gamma delta+/CD3+ clones and TCR-/CD3- NK cell clones can be induced by heteroconjugates containing a single anti-CD2 (OKT11.1) mAb. Activation of TCR gamma delta+/CD3+ cells via CD2 is independent of heteroconjugates binding to CD16 (Fc gamma RIII), because heteroconjugates prepared from Fab fragments induced equal levels of lysis. Moreover, anti-CD16 mAb did not inhibit triggering via CD2 in TCR gamma delta+/CD3+ cells. In TCR-/CD3- NK cells, however, induction of cytotoxicity via CD2 is co-dependent on interplay with CD16. Anti-CD3 mAb blocked the anti-CD2 x anti-DNP heteroconjugate-induced cytotoxicity of TCR gamma delta+/CD3+ cells, indicating a functional linkage between CD2 and CD3 on these cells. We conclude that induction of lysis via CD2 shows qualitatively different activation requirements in TCR gamma delta+/CD3+, TCR alpha beta+/CD3+ CTL and TCR-/CD3- NK cells.     

Cytologic and ultrastructural findings of a peculiar alteration in cervical cells from patients with human papillomavirus infections

Inclusion bodies were previously found in Papanicolaou smears of patients infected by the human papillomavirus (HPV). To ascertain their origin, 12 biopsies from patients with colposcopic evidence of papillomatous lesions were studied by cytology and electron microscopy. In several instances, koilocytotic atypias and electron-dense masses included within epithelial cells were observed. Some of these epithelial cells appeared to be surrounded by other cells in a sort of concentric arrangement. The electron-dense masses were composed of intermediate filaments, vacuoles and electron-dense material. They seemed to be dyskeratotic cells. They can be compared with apoptotic bodies and may be related to a disturbance of the involucrin expression caused by the HPV infection.