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The first X-ray Extended Range Technique (XERT)-like experiment at the Australian Synchrotron, Australia, is presented. In this experiment X-ray mass attenuation coefficients are measured across an energy range including the zinc K-absorption edge and X-ray absorption fine structure (XAFS). These high-accuracy measurements are recorded at 496 energies from 8.51 keV to 11.59 keV. The XERT protocol dictates that systematic errors due to dark current nonlinearities, correction for blank measurements, full-foil mapping to characterize the absolute value of attenuation, scattering, harmonics and roughness are measured over an extended range of experimental parameter space. This results in data for better analysis, culminating in measurement of mass attenuation coefficients across the zinc K-edge to 0.023–0.036% accuracy. Dark current corrections are energy- and structure-dependent and the magnitude of correction reached 57% for thicker samples but was still large and significant for thin samples. Blank measurements scaled thin foil attenuation coefficients by 60–500%; and up to 90% even for thicker foils. Full-foil mapping and characterization corrected discrepancies between foils of up to 20%, rendering the possibility of absolute measurements of attenuation. Fluorescence scattering was also a major correction. Harmonics, roughness and bandwidth were explored. The energy was calibrated using standard reference foils. These results represent the most extensive and accurate measurements of zinc which enable investigations of discrepancies between current theory and experiments. This work was almost fully automated from this first experiment at the Australian Synchrotron, greatly increasing the possibility for large-scale studies using XERT.

The most accurate measurements of the mass attenuation coefficient for metals at low temperature for the zinc K-edge from 9.5 keV to 11.5 keV at temperatures of 10 K, 50 K, 100 K and 150 K using the hybrid technique are reported. This is the first time transition metal X-ray absorption fine structure (XAFS) has been studied using the hybrid technique and at low temperatures. This is also the first hybrid-like experiment at the Australian Synchrotron. The measured transmission and fluorescence XAFS spectra are compared and benchmarked against each other with detailed systematic analyses. A recent method for modelling self-absorption in fluorescence has been adapted and applied to a solid sample. The XAFS spectra are analysed using eFEFFIT to provide a robust measurement of the evolution of nanostructure, including such properties as net thermal expansion and mean-square relative displacement. This work investigates crystal dynamics, nanostructural evolution and the results of using the Debye and Einstein models to determine atomic positions. Accuracies achieved, when compared with the literature, exceed those achieved by both relative and differential XAFS, and represent a state-of-the-art for future structural investigations. Bond length uncertainties are of the order of 20–40 fm.

Contains fulltext : 291547.pdf (Publisher’s version ) (Open Access) OBJECTIVES: Genome-wide association studies have revealed over 200 genetic susceptibility loci for prostate cancer (PCa). By combining them, polygenic risk scores (PRS) can be generated to predict risk of PCa. We summarize the published evidence and conduct meta-analyses of PRS as a predictor of PCa risk in Caucasian men. PATIENTS AND METHODS: Data were extracted from 59 studies, with 16 studies including 17 separate analyses used in the main meta-analysis with a total of 20,786 cases and 69,106 controls identified through a systematic search of ten databases. Random effects meta-analysis was used to obtain pooled estimates of area under the receiver-operating characteristic curve (AUC). Meta-regression was used to assess the impact of number of single-nucleotide polymorphisms (SNPs) incorporated in PRS on AUC. Heterogeneity is expressed as I(2) scores. Publication bias was evaluated using funnel plots and Egger tests. RESULTS: The ability of PRS to identify men with PCa was modest (pooled AUC 0.63, 95% CI 0.62-0.64) with moderate consistency (I(2) 64%). Combining PRS with clinical variables increased the pooled AUC to 0.74 (0.68-0.81). Meta-regression showed only negligible increase in AUC for adding incremental SNPs. Despite moderate heterogeneity, publication bias was not evident. CONCLUSION: Typically, PRS accuracy is comparable to PSA or family history with a pooled AUC value 0.63 indicating mediocre performance for PRS alone. 01 april 2023

High-accuracy X-ray mass attenuation coefficients were measured from the first X-ray Extended Range Technique (XERT)-like experiment at the Australian Synchrotron. Experimentally measured mass attenuation coefficients deviate by ∼50% from the theoretical values near the zinc absorption edge, suggesting that improvements in theoretical tabulations of mass attenuation coefficients are required to bring them into better agreement with experiment. Using these values the imaginary component of the atomic form factor of zinc was determined for all the measured photon energies. The zinc K-edge jump ratio and jump factor are determined and results raise significant questions regarding the definitions of quantities used and best practice for background subtraction prior to X-ray absorption fine-structure (XAFS) analysis. The XAFS analysis shows excellent agreement between the measured and tabulated values and yields bond lengths and nanostructure of zinc with uncertainties of from 0.1% to 0.3% or 0.003 Å to 0.008 Å. Significant variation from the reported crystal structure was observed, suggesting local dynamic motion of the standard crystal lattice. XAFS is sensitive to dynamic correlated motion and in principle is capable of observing local dynamic motion beyond the reach of conventional crystallography. These results for the zinc absorption coefficient, XAFS and structure are the most accurate structural refinements of zinc at room temperature.

We present a new technology for analyzing the molecular structure and in particular subtle conformational differences in Ni complexes using X-ray absorption spectroscopy (XAS), enabling tighter and more robust constraints of structure and dynamic bond lengths. Self-absorption and attenuating effects have a large impact in fluorescence X-ray absorption spectroscopy (XAS), compromising accuracy and insight in structural and advanced analyses. We correct for these dominant systematic effects. We investigate nickel(II) complexes, that is, bis(N-n-propyl-salicylaldiminato) nickel(II), "n-pr", and bis(N-i-propyl-salicylaldiminato) nickel(II), "i-pr", in 15 mM solutions with 0.1% w/w Ni. One is "square-planar" and one is "tetrahedral", with identical coordination numbers. We identify two key sources of uncertainty and provide robust estimates for them, reflecting the quality of the data, and provide meaningful estimates of χr2 suitable for hypothesis testing. We apply significance and model testing for fluorescence data, with direct uncertainty estimates. Two new peaks are revealed in the X-ray absorption fine structure (XAFS) at k ≈ 4.4 and 5.4 A-1. The high intrinsic accuracy of our processed data allows these features to be well modeled and yields deeper potential insight. Three important notions in the field are addressed: resolvability of shell radii, estimation of the number of independent data points in least-squares or Bayesian analysis, and the effect of uncertainties on the determined structure and the determinability of key structural parameters. Conventional XAFS fitting requires a kmin and a kmax. The origin of these limits is explained from the data, in a quantitative manner. Being able to distinguish the isomers spectroscopically and structurally places strong demands on the data, the uncertainties, and the model interpretation, and this article reports success in this subtle structural identification. Two nearby shells-the innermost two shells-are identified quantitatively, well below the conventional aliasing limit. This illustrates the application of new technology to gain new insight.

An approach to investigate XAS data without standard interpolation of experimental data and with minimal loss of information content has been developed. The additional physical insight accorded by the correct propagation of experimental uncertainty has been used to determine newly refined structures for the innermost co-ordination shell of Ni(II) coordination complexes., Accurate experimental XAFS (X-ray absorption fine-structure) data including uncertainties are required during analysis for valid comparison of results and conclusions of hypothesis testing on structural determinations. Here an approach is developed to investigate data without standard interpolation of experimental data and with minimal loss of information content in the raw data. Nickel coordination complexes bis(i-n-propylsalicylaldiminato)nickel(II) (i-pr) and bis(N-n-propylsalicylaldiminato)nickel(II) (n-pr) are investigated. The additional physical insight afforded by the correct propagation of experimental uncertainty is used to determine newly refined structures for the innermost co-ordination shell. Two sets of data are investigated for each complex; one optimized for high point accuracy and one optimized for high point density. Clearly both are important and in this investigation the quality of the physical insight from each is directly provided by measured and propagated uncertainties to fairly represent the relevant accuracies. The results provide evidence for an approximate tetrahedral geometry for the i-pr Ni complex that is more symmetric than previously concluded, with our high point accuracy data yielding ligand lengths of 2.017 ± 0.006 Å and 2.022 ∓ 0.006 Å for Ni—N and Ni—O bonds, respectively, and an even more skewed square-planar (i.e. rhombohedral) arrangement for the n-pr complex with corresponding bond lengths of 2.133 ± 0.004 Å and 1.960 ∓ 0.003 Å. The ability to distinguish using hypothesis testing between the subtle differences in XAFS spectra arising from the approximate local tetrahedral and square-planar geometries of the complexes is also highlighted. The effect of standard interpolation on experimental XAFS spectra prior to fitting with theoretical model structures is investigated. While often performed as a necessary step for Fourier transformation into position space, this will nonetheless skew the fit away from actual data taken, and fails to preserve the information content within the data uncertainty. The artificial effects that interpolation imposes on χr 2 are demonstrated. Finally, a method for interpolation is introduced which locally preserves the χr 2 and thus information content, when a regular grid is required, e.g for further analysis in r-space.

De huidige hypothese voor de verbeterde effectiviteit van chemohyperthermie (Synergo®) is een verhoogde permeabiliteit van het urotheel door een verwarmde omgeving. Hierdoor zou er een diepere penetratie van de chemotherapie worden gerealiseerd.

An inhibition enzyme immunoassay (IEIA), using a human monoclonal antibody (K14) reactive with gp41 of HIV-1, was evaluated for its applicability to the serology of HIV-1 infections. Using panels of serum samples from seronegative and confirmed HIV-1-seropositive individuals, it was shown that all the HIV-1-positive samples in a panel from The Netherlands and 97% of the HIV-1-positive samples from Tanzania were identified by this IEIA. Six per cent of the IEIA-positive samples from Tanzania could not be confirmed in other assays. Testing of serial dilutions of serum samples from African individuals with confirmed HIV-1, HIV-2 or HIV(ANI70) infections in the K14 IEIA, indicated that a HIV-1-specific assay based on this principle may be developed.

The epidemiologic characteristics of prostate cancer (PCa) have been recognized for several decades. It is of great importance to understand the factors responsible for prostate carcinogenesis, why some carcinomas remain "clinically silent" during life, whereas other tumors progress to present clinically and may lead to PCa-related death. A better understanding of these mechanisms in molecular genetic terms should point to more rational approaches to disease prevention, intervention and treatment. The aim of this review is to provide a comprehensive overview of the current state of knowledge regarding the molecular alterations of PCa.

Knowledge concerning differentiation of neuroendocrine (NE) cells during development of the human prostate is rather fragmentary. Using immunohistochemistry combined with a morphometric method, we investigated the distribution and density of NE cells in the developing human prostate, with special emphasis on the topographical relationship of NE cells with the developing gland.Consecutive sections from a total of 42 human prostates taken during autopsy of fetuses (12-38 weeks of gestation), prepubertal males, and young adults were immunostained for chromogranin A and serotonin. Computer-assisted image analysis was used to assess the total number of cells in the different parts of the branching glandular anlage, i.e., budding tips and acini/ducts. Next, the number of NE cells was counted manually. The NE cell density (NE cell index) was then determined.NE cells could first be detected in the prostate from 13 weeks of gestation. By 21 weeks of gestation, all prostates contained NE cells. NE cells were mainly confined to the acinous/ductal regions, while most of the budding tips lacked NE staining. NE cell indexes of individuals were highly variable, mostly in the youngest age group.In the normal prostate, NE cell density probably remains constant in acini/ducts from fetuses to young adulthood. The presence of neuroendocrine cells in well-developed glandular structures at such an early fetal age and their absence in the less differentiated budding tips possibly indicates that differentiation of NE cells is associated with glandular maturation. NE cells occur preferentially in the acinous/ductal region, implying a paracrine function during secretory differentiation of exocrine epithelial cells.

P-Cadherin is a member of the cadherin family of cell surface glycoproteins that mediate Ca2+-dependent cell-cell adhesion and is expressed in a differential fashion in normal epithelial tissues. The expression of P-cadherin in human prostate cancer development has not been investigated previously. By immunohistochemistry, we show that P-cadherin expression is restricted to the cell-cell border of basal epithelial cells in 30 normal prostate samples. This staining is down-regulated in prostatic intraepithelial neoplasia and is absent in all 25 of the well to poorly differentiated prostate cancer specimens analyzed. To examine potential P-cadherin-regulatory elements, we sequenced the 5'-flanking region of this gene. Similar to the mouse gene, the human P-cadherin promoter is TATA-less, contains an Sp-1 binding site and, analogous to the human E-cadherin sequence, demonstrates a GC-rich region characteristic of a CpG island. Cytosine methylation of this region occurs in P-cadherin-negative prostate cancer cell lines but not in cell lines expressing this gene. In vivo, a lack of expression in 12 clinical prostate cancer specimens is not associated with methylation of the P-cadherin promoter. These results demonstrate that the expression of the basal cell marker P-cadherin is lost in prostate cancer development and that in vivo mechanisms other than cytosine methylation regulate this consistent loss of expression.

The prostate gland consists of a complex ductal system lined with exocrine basal and luminal cells, and neuroendocrine epithelial cells. This paper reviews the histologic and molecular cell biologic characteristics of these cells, in normal adult tissue, during prostate morphogenesis, and in the development of benign and malignant neoplastic conditions. Expression of differentiation markers, as well as proliferation and apoptosis markers, growth factors and associated receptors, and abnormalities in genes and chromosomes are reviewed. Accumulating data indicate that (1) pluripotent immortal stem cells are located in the basal cell compartment of the prostate; (2) there is a subpopulation of epithelial cells in the prostate gland (intermediate cells) that have both structural and functional characteristics common to basal and luminal cells, which may be identified in various conditions; and prostate NE cells may have the same common origin as other exocrine cells, and share the same differentiation pathway. A stem cell model is proposed in which both exocrine and endocrine cells are derived from a subpopulation of basal cells (stem cell) that give rise to luminal cells through intermediate cells (pluripotent amplifying cells). These cells are also probably highly implicated in the early development of prostate benign and malignant neoplasia.

Histologic grade and tumor volume are markers of malignant phenotype. More objective markers, however, have been sought for needle biopsy specimens. The aim of this study was to evaluate how immunohistochemical expression of the potential prognostic markers E-cadherin and p53 in biopsy specimens relates to the expression of these markers in prostatectomy specimens. Therefore, we analyzed 47 prostatectomy specimens and their preoperative biopsy specimens. Fixation of surgical specimens and the immunohistochemical assay for both E-cadherin and p53 expression was optimized. All paraffin blocks containing areas of carcinoma were submitted for immunohistochemical analysis. The prevalence of abnormal p53 immunoreactivity was only 11%. In addition, abnormal p53 expression was virtually restricted to cases that were already identified as having a poor prognosis on the basis of the large volume and the high grade of their carcinomas. In 28% of the cases, we found abnormal immunoreactivity for E-cadherin. These cases revealed considerable heterogeneity in topographic distribution of abnormal expression. The level of sensitivity to the detection of abnormal E-cadherin expression or abnormal p53 in the prostatectomy specimen was 15% and 60%, respectively. In view of the inherent heterogeneity of E-cadherin expression and the low prevalence of abnormal p53 expression, we question the use of these markers for prognostic purposes in needle biopsy specimens. Unless representative sampling by needle biopsy can be assured, the use of E-cadherin expression will be of most value in prostatectomy specimens.

We have induced tumors by feeding guinea pigs with a diet containing 25 or 30% dried bracken fern for 100 or 150 days. A high incidence of bladder tumors was obtained. All but one animal had preneoplastic or neoplastic lesions after 4 months; after one year, 24 or 25 exposed animals had carcinoma. Bladder tumors obtained were essentially pure transitional cell carcinomas, although 4 cases (7% of the exposed animals and 10% of the 39 transitional cell carcinoma observed) showed areas of focal squamous metaplasia. Immunohistological detection of cytokeratins 10, 13, and 18 confirmed the transitional nature of these tumors. Tumor development can be followed by ultrasonography and cytology. Bladder tumors arose through several steps. Dysplasia and preneoplastic hyperplasia were seen after 4 months and papillary carcinomas appeared after 6 months, whereas muscle-invasive carcinomas required 1 year. Thus this model reproduces the full spectrum of preneoplastic and neoplastic bladder lesions observed in humans. Interestingly, when tumors were induced in older guinea pigs, none of them progressed to a muscle-invasive stage. This phenomenon should provide the opportunity to study the molecular mechanisms associated with these two different growth patterns, a major issue in understanding human bladder tumor progression.

Antibody-binding sites were mapped on all overlapping nonapeptides of the major core protein p24 of human immunodeficiency virus type 1 (HIV-1) using murine monoclonal antibodies (MAbs) and sheep and rabbit polyclonal antibodies raised against HIV-1/H9 (strain IIIB) viral lysate and antibodies obtained from humans infected with HIV-1. The binding sites were mapped to various distinct regions of this protein. After superimposition of the antibody-binding sites on a proposed model of p24 of HIV-1, these sites appeared to be located on the surface of the protein on loops, turns and coils of p24 but, unexpectedly, not on the major part of the predicted 'puff'. Little reaction was found with the inaccessible anti-parallel beta-barrel. These results are the first experimental evidence for the validity of the structure proposed for p24 of HIV-1.

A human Epstein-Barr virus-transformed lymphoblastoid B-cell line was generated from peripheral blood mononuclear cells (PBMC) of an asymptomatic human immunodeficiency virus type I (HIV-1) seropositive donor, which produces a human monoclonal antibody K14 (IgG1), reactive with an epitope on the transmembrane part (gp41) of the envelope glycoprotein of HIV-1. This monoclonal antibody reacts with a lysate of HIV-1-infected H9 cells, gradient purified HIV-1, and a vaccinia recombinant HIV-1 gp160 protein, but not with HIV-2 antigens in an enzyme-linked immunosorbent assay (ELISA). When used as an immobilized ligand in an immune affinity column, K14 selectively purifies gp41 from a HIV-1-infected H9 cell lysate. Although no reactivity was observed in ELISA with a panel of partially overlapping synthetic nonapeptides spanning the whole length of HIV-1 gp41, it was shown to react with recombinant envelope proteins, provided that they did contain amino acids 643-692: deletion of this part resulted in the disappearance of the reactivity. Testing of an extensive panel of the sera from HIV-1 seropositive or seronegative donors from Europe and Africa, including a selected group of donors before and after HIV-1 seroconversion, in a competition ELISA with horseradish peroxidase-conjugated K14, showed that the epitope recognized on gp41 is immunodominant and conserved. K14 does not neutralize HIV-1 infectivity or virus-mediated cell fusion, and does not mediate antibody-dependent cellular cytotoxicity.

The rod visual pigment, rhodopsin, and its illuminated form, opsin, were used to induce experimental autoimmune uveoretinitis in rats. Rhodopsin appears to be more pathogenic than opsin. A dose of 250 micrograms rhodopsin injected in Freund's complete adjuvant and pertussis adjuvant induces nongranulomatous inflammation with higher frequency, which starts earlier and is more severe than that induced by opsin. Two weeks postinjection, the mean score of rhodopsin-injected animals is more than twice as high as that of opsin-injected animals. The high pathogenicity of rhodopsin appears to be related to the biochemical integrity of the protein and depends on its state of illumination. The levels of the immune responses (both cellular and humoral) measured at day 10 postinjection do not account for the pronounced difference in pathogenicity between rhodopsin and opsin. The developmental patterns of severe uveoretinitis induced by rhodopsin or opsin were histologically evaluated and appear to be similar. In both cases we observed dense mononuclear and polymorphonuclear cell infiltrations in the retina and anterior uvea. Only in the severe stages does the choroid become involved. However, rhodopsin causes more pronounced involvement of the ciliary body, pars plana, and anterior chamber. The inflammation finally results in total elimination of the photoreceptor cell layer.

With the aid of specific antisera to the retinal proteins S-antigen and alpha-transducin and to the rhodopsin apoprotein opsin, we have labeled various cell populations in the pineal organ, parapineal organ, habenular nucleus, and subcommissural organ in two teleost species: the rainbow trout and the European minnow. Although these proteins are associated with photoreceptor functions, not only photoreceptor cells but also the majority of parenchymal cells in the pineal organ were immunoreactive. Immunoreactive cells with dendrite- and axonlike processes were observed also in the parapineal organ and the habenular nucleus. Furthermore, S-antigen-immunoreactive, long, axonal processes were observed in the pineal organ and could be traced from the pineal organ to the habenular nucleus and to the pretectal area. In the light of recent HRP electron microscopical and immunocytochemical studies we propose (1) that not only the classical pineal photoreceptor cells of poikilothermic vertebrates but also other types of CSF-contacting neurons may be the phylogenetic ancestors of mammalian pinealocytes, and (2) a close interrelationship between the pineal organ and the limbic system, effectuated by the direct projections from pineal photoreceptors/CSF-contacting neurons/pinealocytes to the habenular nucleus, and by displaced "pinealocytelike" elements scattered in the habenular nucleus.

A radioimmunoassay is developed for bovine opsin using a rabbit antiserum against bovine rod outer segment membranes. The assay is specific for opsin. Rhodopsin, bacteriorhodopsin and hemoglobin do not show cross-reaction. It can be carried out rapidly, has a sensitivity of 0.01 pmol bovine opsin and gives accurate results, even in the presence of a large excess of rhodopsin. Under the conditions described, the assay can be used to measure bovine opsin and rhodopsin in each other's presence by running a sample before and after illumination, with a sensitivity 2000-times higher than with spectrophotometric methods. The opsin content of rather crude preparations such as bovine retina homogenates can be accurately determined. Rabbit and mouse opsin can also be assayed with a reasonable degree of accuracy using the same rabbit antiserum.

With the use of antisera against bovine retinal S-antigen and bovine opsin the authors demonstrate that in cerebellar medulloblastomas certain tumor cells display immunocytochemical properties characteristic of retinal photoreceptors and pinealocytes. S-antigen-like and opsin-like immunoreactions occur in nine of 28 medulloblastomas investigated. All tumors displaying S-antigen-like immunoreactive neoplastic cells also contain opsin-like immunoreactive cells; however, the opsin-like immunoreactive cells were less frequent than the S-antigen-like immunoreactive cells throughout all positive cases. The immunoreactive cells displayed several long processes. Generally, both S-antigen and opsin-like immunoreactive cells considerably vary in number among individual tumors. The results indicate that certain neoplastic cells of medulloblastoma are capable of expression of photoreceptor-specific proteins and, thus, may be closely related to tumor cells of retinoblastoma and pineocytomas previously shown to bind antisera against retinal S-antigen and opsin. No S-antigen and opsin-like immunoreaction was found in malignant teratomas and germinomas of the pineal gland, oat cell tumors, astrocytomas, ependymomas, oligodendrogliomas, glioblastomas, gangliogliomas, gangliocytoma, ganglioneuroblastomas, neuroblastomas, and esthesioneuroblastoma.

A rod-specific antiserum was used to immunolabel elements within the retina and pineal of the adult Djungarian hamster and Welsh Mountain sheep. In the retina immunostaining was localized to the outer segments and perikarya of photoreceptor cells, while in the pineal limited numbers of labelled pinealocytes were scattered throughout the gland. An enzyme-linked immunosorbent assay (ELISA) was then used to obtain a quantitative measure of rod opsin in total eye and pineal extracts from the Djungarian hamster. Total rod opsin (+/- SEM) in the eye was measured by absorbance spectroscopy (1.88 +/- 0.10 nmoles opsin/eye) and by using the ELISA (1.75 +/- 0.02 nmoles opsin/eye). The opsin content from a total of 56 pineals gave a mean value of 0.34 +/- 0.01 pmoles opsin/pineal. Since a functional photopigment should be coupled in a 1:1 ratio to a chromophore, we investigated whether we could identify 11-cis and/or all-trans retinaldehydes in the pineal extracts by quantitative extraction and HPLC analysis as the oximes. No evidence of 11-cis or all-trans retinaloxime could be found, the chromatograms were indistinguishable from those produced by extracts of cortical brain tissue. We conclude that the opsin present within the adult hamster pineal is not coupled to the common vertebrate retinaldehyde chromophore, and as a result, is unlikely to be part of a functional photopigment.

The retinal proteins opsin, alpha-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocytochemistry we have employed antibodies directed against these "photoreceptor proteins" in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and alpha-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina, in the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.

A versatile, multispecies enzyme-linked immunosorbent assay for the rod visual pigment (rhod)opsin has been developed. For this quantitative inhibition assay a monospecific polyclonal antiserum is used which is elicited in rabbits against bovine rod outer-segment membranes. Detergent concentrations as high as 1.0% can be used in the assay with only a slight loss in sensitivity. The assay allows quantitative determination of the apoprotein opsin with a detection level of about 0.04 pmol per sample by using standards prepared by illumination of spectrophotometrically determined amounts of rhodopsin. The antiserum shows considerable cross-reactivity with opsin from several species (mouse, rat, quail, monkey and man). The high degree of monospecificity and cross-reactivity of the antiserum already allowed quantitation of opsin content in crude eye extracts of mouse, rat and quail with a sensitivity comparable to that of bovine opsin. Similar types of multispecies immunoassays for quantitation of highly conserved membrane proteins can be developed using the described approach, requiring only a monospecific antiserum elicited against an easily accessible species and crude tissue extracts both for coating and as a source of the inhibitory antigen.

Immunocytochemistry with a rod-specific antiserum was used to study the post-hatch development (2 days–300 days) of photoreceptor elements within the pineal of the Japanese quail. At all ages staining was restricted to limited numbers of pinealocytes scattered throughout the gland. An enzyme-linked immunosorbent assay (ELISA), with the same rod-specific antibody, was then used to obtain a quantitative measure of rod opsin in total eye and pineal extracts in both the developing retina and pineal. The opsin content of both tissues shows a marked increase during the first 30 days after hatch and then plateaued to 0.84±0.02 nmoles opsin in the eye and 2.20±0.11 pmoles opsin equivalents in the pineal. The increase in opsin in the retina may be associated with continued post-hatch development of the photoreceptors. We then attempted to demonstrate the presence of the rhodopsin chromophore within pineal and retinal extracts using HPLC analysis. In both retinal and pineal extracts, 11-cis retinaldehyde was identified and a light-induced shift from the 11-cis to the all-trans isomer was clearly shown. This analysis also allowed us to calculate the total content of 11-cis and all-trans retinaldehyde (derived from both rod and non-rod photoreceptors) of the eye and pineal (eye: 1.7±0.2 nmoles; pineal: 4.6±0.5 pmoles). In the quail eye, the total amount of retinaldehyde is more than twice the amount of rod-like opsin. This probably reflects the large contribution of cones in the quail retina; the cone pigments will contribute to the retinaldehyde content but are not recognized by the rodspecific antibodies. In the pineal, we also found more than double the concentration of retinaldehyde than we would have predicted from the amount of rod-like opsin. These results, coupled with our immunocytochemical findings, suggest that the quail pineal contains at least two classes of photoreceptor, some ‘rod-like’, others ‘non rod-like’.

In 020/A mice, homozygous for the retinal degeneration slow (rds) gene, the photoreceptor cells fail to develop outer segments, and in the absorption spectra of retinal extracts the rhodopsin peak is lacking. Application of an enzyme-linked immunoassay using antisera against bovine opsin shows, however, that opsin is present in the homozygous mutant retina (0.010 nmol/eye) at 3% of the level of the normal retina (0.38) nmol/eye) of Balb/c mice. In the retina of heterozygous mice the opsin level (0.19 nmol/eye) is about half of the normal. Detection of opsin in the rds mutant retina demonstrates the functional basis for the reported electroretinographic response and light-mediated reduction in cyclic nucleotide levels in this mutant.

We present the first evidence that purified rhodopsin can induce experimental autoimmune uveoretinitis (EAU) in monkeys. Injection of a highly purified lipid-free rhodopsin preparation provokes severe chorioretinitis with concomitant anterior uveitis. The onset of disease is earlier, its frequency is higher, and the inflammation is considerably more severe than in EAU induced under similar conditions by opsin. The first inflammatory cells are observed in the ciliary body and pars plana. Within a few days the inflammation extends into the anterior chamber, choroid, and retina. Retinitis predominates in the central area, while chorioretinitis is observed in the periphery, both accompanied by damage to and elimination of the photoreceptor cells. The monkeys develop high cellular and humoral immune responses against rhodopsin and opsin. The cellular response maximum just precedes the onset of EAU. This may indicate that cellular immunity has an important role in the pathogenesis of rhodopsin-induced EAU.

With the aid of specific antisera to the retinal proteins S-antigen and alpha-transducin and to the rhodopsin apoprotein opsin, we have labeled various cell populations in the pineal organ, parapineal organ, habenular nucleus, and subcommissural organ in two teleost species: the rainbow trout and the European minnow. Although these proteins are associated with photoreceptor functions, not only photoreceptor cells but also the majority of parenchymal cells in the pineal organ were immunoreactive. Immunoreactive cells with dendrite- and axonlike processes were observed also in the parapineal organ and the habenular nucleus. Furthermore, S-antigen-immunoreactive, long, axonal processes were observed in the pineal organ and could be traced from the pineal organ to the habenular nucleus and to the pretectal area. In the light of recent HRP electron microscopical and immunocytochemical studies we propose (1) that not only the classical pineal photoreceptor cells of poikilothermic vertebrates but also other types of CSF-contacting neurons may be the phylogenetic ancestors of mammalian pinealocytes, and (2) a close interrelationship between the pineal organ and the limbic system, effectuated by the direct projections from pineal photoreceptors/CSF-contacting neurons/pinealocytes to the habenular nucleus, and by displaced "pinealocytelike" elements scattered in the habenular nucleus.

Opsin-like immunoreactivity was observed in the retinae and pineal organs of the mouse, rat and guinea pig, and the pineal organ of the cat. In the retina the immunoreaction was restricted to photoreceptor cells, which displayed immunostaining in their perikarya and outer and inner segments. Distinct pinealocytes endowed with characteristic processes were labelled in the pineal organs of the mouse and cat. However, in the cat the number of immunoreactive pinealocytes was very limited. In the pineal organs of the rat and guinea pig immunoreaction was very weak and diffuse. No immunoreaction was observed when the antibody was preabsorbed with purified bovine (rhod)opsin. These findings are in accord with the results of previous studies indicating molecular similarities between retinal photoreceptors and pinealocytes in mammals.

With the use of antisera against bovine retinal S-antigen and bovine opsin the authors demonstrate that in cerebellar medulloblastomas certain tumor cells display immunocytochemical properties characteristic of retinal photoreceptors and pinealocytes. S-antigen-like and opsin-like immunoreactions occur in nine of 28 medulloblastomas investigated. All tumors displaying S-antigen-like immunoreactive neoplastic cells also contain opsin-like immunoreactive cells; however, the opsin-like immunoreactive cells were less frequent than the S-antigen-like immunoreactive cells throughout all positive cases. The immunoreactive cells displayed several long processes. Generally, both S-antigen and opsin-like immunoreactive cells considerably vary in number among individual tumors. The results indicate that certain neoplastic cells of medulloblastoma are capable of expression of photoreceptor-specific proteins and, thus, may be closely related to tumor cells of retinoblastoma and pineocytomas previously shown to bind antisera against retinal S-antigen and opsin. No S-antigen and opsin-like immunoreaction was found in malignant teratomas and germinomas of the pineal gland, oat cell tumors, astrocytomas, ependymomas, oligodendrogliomas, glioblastomas, gangliogliomas, gangliocytoma, ganglioneuroblastomas, neuroblastomas, and esthesioneuroblastoma.

We have studied the clinicopathological features of experimental autoimmune uveoretinitis (EAU) induced in Lewis rats by injection of different doses of rhodopsin and its illuminated form opsin. Rhodopsin consistently appears to be more pathogenic than opsin. Injected in Freund's complete adjuvant and pertussis adjuvant 50 micrograms of rhodopsin induces a frequency of severe EAU similar to 250 micrograms of opsin. Intensity, frequency and location of ocular inflammation are markedly dose dependent. At high dose (100-250 micrograms), rhodopsin induces severe bilateral uveoretinitis in all animals, which starts with acute inflammation of the anterior eye segment at day 10-12 followed by chorioretinitis (predominantly retinitis) which results in complete elimination of the photoreceptor cells. At low dose (20 micrograms), rhodopsin induces mild transient inflammation in 60% of the animals, mainly consisting of mild posterior retinitis which starts at day 20 and leads to a typical multiple focal destruction of the photoreceptor cells. Intermediate doses cause an intermediate type of disease. Omission of pertussis adjuvant lowers the frequency of severe disease at low doses of rhodopsin, delays its onset and changes its features. The last characteristic has been observed in particular at intermediate doses (50-100 micrograms). In these cases, EAU usually starts by cell infiltration of the vitreous, while the anterior segment is only mildly affected. Without pertussis adjuvant the pathogenicity of opsin is low. Even in both adjuvants severe EAU can only be evoked by a high dose of opsin. Although there exists a marked difference in uveitogenicity between rhodopsin and opsin, the immunogenicity is similar and seems not to be correlated with their pathogenicity.