Efferents to the retina have multiple sources in teleost fish.

Multiple efferent systems project to the retina in three species of teleost fish investigated with the horseradish peroxidase technique. These animals are the first vertebrates shown to have more than one central nervous system structure projecting to the retina. The connections discovered may reflect a primitive organization of retina-brain interconnections.

Middle mississippian blastoid extinction event.

The Middle Mississippian blastoid (Phylum Echinodermata) extinction event (about 340 million years ago) was a rapid, habitat-specific extinction. Blastoids became rare or absent in shallow-water environments after the extinction, and this change was probably synchronous worldwide. Onshore-offshore habitat shifts have been recognized as an important historical trend among marine benthos. Unlike trends exhibited by other groups, blastoids appear to have repopulated shallow-water habitats after a period of diminished diversity and abundance.

The influence of creatine and a high glycemic carbohydrate on the growth performance and meat quality of market hogs fed ractopamine hydrochloride.

Crossbred barrows (n=128; 85±0.91kg) were randomly allotted to one of four dietary treatments. A pelleted corn-soybean diet containing 5ppm Paylean(®) (PAY) was tested against a negative control (NCON) diet formulated to meet or exceed the National Research Council's requirements for the growing pig, a pelleted corn-soybean diet containing 0.92% creatine and 2.75% dextrose (COMBO), and a pelleted corn-soybean diet containing a combination of 5ppm Paylean(®), 0.92% creatine, and 2.75% dextrose (PAYPLUS). No treatment differences were noted when comparing ADG (P=0.66) and hot carcass weight (P=0.75). Over the 27d test, barrows fed PAY and PAYPLUS produced loins with a larger (P<0.01) loin muscle area (LMA) than those fed NCON or COMBO diets. Barrows fed the NCON diet were fatter at the 10th-rib (P<0.01) than those animals fed the remaining dietary treatments. Dietary treatment did not affect the ultimate pH (P=0.87), Japanese color score (P=0.25) or Minolta L(∗) (P=0.61) and b(∗) (P=0.56) values of the loin. Loin chops from NCON, COMBO and PAYPLUS tended (P=0.07) to contain a higher intramuscular fat content than those from barrows fed PAY. Additionally, loin chops from the NCON and COMBO fed animals were more red (higher a(∗)-value) than those chops coming from animals fed the PAY diet (P<0.01).

Preclinical trials with combinations and conjugates of T101 monoclonal antibody and doxorubicin.

We investigated the potential for additive therapy for malignancy using an anti-human T-cell monoclonal antibody, T101, and the chemotherapy agent doxorubicin (DOX). We compared the efficacy of T101 alone, DOX alone, T101 and DOX covalently linked to dextran to form an immunoconjugate, T101 plus DOX mixed together and injected, T101 and DOX injected separately, and nonspecific murine IgG2A plus DOX mixed together. Inhibition of [3H]thymidine was examined in vitro, and the clinical efficacy of each treatment was tested on human T-cell tumors growing in athymic mice. In vitro experiments confirmed retention of immunoreactivity and cytotoxicity by the immunoconjugate, but it was not superior to DOX alone. In efficacy experiments, all therapeutic arms were superior to placebo treatment (P less than 0.05). However, the best results in the animal tumor model were obtained with T101 mixed with DOX, perhaps because of formation of weak complexes via hydrophobic bonds. This mixture was superior to all other treatments, both by growth curve analysis (P less than 0.05) and by analysis of complete regression of tumor (P less than 0.01). T101 mixed with DOX was superior to a mixture of nonspecific mouse immunoglobulin and DOX and superior to a combination of T101 injected i.v. and DOX injected i.p. The antitumor effect of T101 mixed with DOX was blocked by premodulating the target antigen with T101. These data suggest that further exploration into monoclonal antibody-anthracycline complexes is warranted.

Site-specific prodrug activation by antibody-beta-lactamase conjugates: regression and long-term growth inhibition of human colon carcinoma xenograft models.

Antibody-directed catalysis (ADC) is a two-step method for the delivery of chemotherapeutic agents in which enzyme-antibody conjugate, prelocalized to antigen-bearing tumor cells, catalyzes the site-specific conversion of prodrug to drug. An ADC system consisting of F(ab')-beta-lactamase conjugates and a cephalosporin derivative of the oncolytic agent 4-desacetylvinblastine-3-carboxhydrazide was investigated. The ability of the system to mediate antitumor activity was compared with that of free drug given alone and with covalent drug-antibody conjugates in LS174T and T380 colon carcinoma xenografts in nude mice. Efficacy increased from moderate tumor growth inhibition by using free 4-desacetylvinblastine-3-carboxhydrazide to tumor regression and long-term stabilization with the ADC system. Labile covalent drug-antibody conjugates prepared from the same antibodies were less effective than ADC and required much higher antibody doses. The antigens KS1/4, carcinoembryonic antigen, and tumor-associated glycoprotein-72, TAG-72, present on the model cell lines, were chosen to investigate the effect of differences in subcellular location and expression heterogeneity on the efficacy of ADC delivery. Response was equivalent with the three tumor antigens. Hence, heterogeneous expression and membrane shedding of carcinoembryonic antigen and TAG-72, did not diminish the suitability of these antigens as targets for ADC therapy. In contrast, drug-antibody conjugate efficacy was more sensitive to subcellular location and heterogeneity. Thus, ADC is a highly effective form of immunochemotherapy in preclinical models, with applicability toward a variety of antigen targets.

Reduced antibody response to streptavidin through site-directed mutagenesis.

Streptavidin provides an effective receptor for biotinylated tumoricidal molecules, including radionuclides, when conjugated to an antitumor antibody and administered systemically. Ideally, one would like to administer this bacterial protein to patients repeatedly, so as to maximize the antitumor effect without eliciting an immune response. Therefore, we attempted to reduce the antigenicity of streptavidin by mutating surface residues capable of forming high energy ionic or hydrophobic interactions. A crystallographic image of streptavidin was examined to identify residues with solvent-exposed side chains and residues critical to streptavidin's structure or function, and to define loops. Mutations were incorporated cumulatively into the protein sequence. Mutants were screened for tetramer formation, biotin dissociation, and reduced immunoreactivity with pooled patient sera. Patient antisera recognized one minor continuous epitope with binding locus at residue E101 and one major discontinuous epitope involving amino acid residues E51 and Y83. Mutation of residues E51, Y83, R53, and E116 reduced reactivity with patient sera to <10% that of streptavidin, but these mutations were no less antigenic in rabbits. Mutant 37, with 10 amino acid substitutions, was only 20% as antigenic as streptavidin. Rabbits immunized with either streptavidin or mutant 37 failed to recognize the alternative antigen. Biotin dissociated from mutant 37 four to five times faster than from streptavidin. Residues were identified with previously undescribed impact on biotin binding and protein folding. Thus, substitution of charged, aromatic, or large hydrophobic residues on the surface of streptavidin with smaller neutral residues reduced the molecule's ability to elicit an immune response in rabbits.

Paraventricular organ of the lungfish Protopterus dolloi: morphology and projections of CSF-contacting neurons.

The morphology and projections of neurons in the paraventricular organ (PVO) were studied by means of silver impregnation after intraocular application of cobaltous lysine in the lungfish Protopterus dolloi. Cobalt-labeled neurons were found exclusively in the PVO in the dorsal and infundibular hypothalamus. These bipolar neurons possess one CSF-contacting process that protrudes into the ventricular lumen with a club-shape ending and a thick, ramifying process directed into the hypothalamic neuropil; the ependymofugal processes form intra- and extrahypothalamic projections. Impregnated fibers from paraventricular neurons cross in infundibular and hypothalamic commissures, the commissure of the posterior tuberculum, the postoptic, the habenular, and the anterior commissures. Projections to the infundibulum and the median eminence are relatively sparse; no fibers are labeled in the pituitary gland. Ascending projections to the forebrain are extensive. Major targets include the dorsal hypothalamus, the periventricular preoptic nuclei, the habenula, the subhabenular region, the anterodorsal thalamus, and the medial telencephalic hemisphere (septum). Most ascending fibers follow the medial forebrain bundle; others course in the fasciculus retroflexus and terminate in rostral parts of the ipsilateral habenula. Descending fibers run caudally along the ventral floor of the brainstem. They terminate in the neuropil of the mesencephalic tegmentum, ventral tectum, isthmic region, ventral portions of the reticular formation throughout the rhombencephalon, and extend into the spinal cord. Intraocular application of cobaltous lysine results in selective impregnation of neurons in the PVO and their ascending and descending projections, presumably via uptake of tracer from vascular circulation. These projections do not represent retinofugal or retinopetal projections. We provide conclusive evidence for the existence of a PVO in Protopterus. On the basis of PVO location and acetylcholinesterase histochemistry, we propose subdivisions of the infundibular hypothalamus corresponding to those in amphibians. Ascending PVO projections appear to be particularly well developed in lungfish compared with other species and may be related to specialized endocrine mechanisms in this group of vertebrates.

Central projections of the nervus terminalis and the nervus praeopticus in the lungfish brain revealed by nitric oxide synthase.

Lungfishes possess two cranial nerves that are associated with the olfactory system: the nervus terminalis enters the telencephalon with the olfactory nerve, and the nervus praeopticus enters the diencephalon at the level of the optic nerve. We investigated the central projections of the nervus terminalis and the nervus praeopticus in the Australian lungfish (Neoceratodus forsteri) and in the African lungfish (Protopterus dolloi) by NADPH-diaphorase histochemistry (nitric oxide synthase; NOS) and compared them with the projections of the nervus terminalis of the frog (Xenopus laevis). In Neoceratodus, NOS-positive fascicles of the nervus terminalis divide and project with a ventral component through the septum and with a dorsal component through the pallium; fibers of both trajectories extend caudally beyond the anterior commissure and join the lateral forebrain bundle. In the nervus praeopticus, about 300 fibers contain NOS; they innervate the preoptic nucleus and continue their course through the diencephalon; many fibers cross in the commissure of the posterior tuberculum. In Protopterus, ganglion cells of the nervus terminalis and of the nervus praeopticus contain NOS. NOS-positive fibers of the nervus terminalis project through the septal region but not through the pallium. Several major fascicles cross in the rostral part of the anterior commissure, where they are joined by a small number of NOS-containing fibers of the nervus praeopticus. Both nerves innervate the preoptic nucleus. The number and pathways of the fascicles of the nervus terminalis are not always symmetric between the two sides. The nervus terminalis fascicles remain in a ventral position, whereas the nervus praeopticus gives rise to the more dorsal fascicles. Many fibers of the two nerves extend throughout the diencephalon and cross in the commissure of the posterior tuberculum. These findings demonstrate many similarities but also significant differences between the contributions of the nervus terminalis and the nervus praeopticus to forebrain projections in the two lungfishes. They support the view that the nervus praeopticus is part of a nervus terminalis system comparable to that in frogs and other nonmammalian vertebrates.

NADPH-diaphorase in the central nervous system of the larval lamprey (Lampetra planeri).

The distribution of nitric oxide synthase (NOS) in the lamprey brain was studied by using reduced nicotinamide-adenine-dinucleotide phosphate (NADPH)-diaphorase histochemistry to further elucidate the evolution of neurons synthesizing nitric oxide. Intense labeling of fibers and/or neurons was found in portions of the lamprey central nervous system, such as the olfactory system, the pineal organ, the habenular region, the nervus stato-acousticus (N. VIII), the brainstem, and the spinal cord, and also in the adenohypophysis. Labeled giant cells located at the floor of the 3rd and 4th ventricle were recognized as reticulospinal neurons. Mauthner and Müller cells were identified according to morphological criteria. Eight pairs of Müller cells and one pair of Mauthner cells were labeled by NADPH histochemistry. None of these cells had, as yet, been described to display NOS activity in any vertebrate. The massive staining of these cells and the apparent lack of labeling, e.g., in teleost fishes, may be a histochemical correlate to already known differences of functions served by these cells in different species. In addition, our results suggest that the nitric oxide (NO) system has appeared early in vertebrate evolution.

Dorsomedial telencephalon of lungfishes: a pallial or subpallial structure? Criteria based on histology, connectivity, and histochemistry.

The dorsomedial telencephalon of lepidosirenid lungfishes has been interpreted in two divergent ways: earlier investigators regarded it as a subpallial (septal) structure; more recently, it has been reinterpreted as the medial pallium (hippocampus). To resolve this question, we identified parameters that are conclusive in their association with either the medial pallium or the septum in anamniotes. The present study examines the position of ependymal thickenings and the distribution of acetylcholinesterase (AchE) in the cerebral hemispheres of the African lungfish Protopterus, the Australian lungfish Neoceratodus, and the amphibian species Xenopus and Ambystoma. In addition, projections from the hypothalamus (paraventricular organ) to the telencephalon are investigated in Protopterus. Ependymal specializations are located dorsally and ventrally in the lateral ventricles of amphibians, but laterally and medially in lungfishes. In Protopterus, the paraventricular organ projects to the medial telencephalic hemisphere, but not to the dorsal roof. High levels of AchE are present in restricted neuropil regions of the medial hemisphere and in the ventral and ventrolateral telencephalon, but they are lacking in the dorsal roof. Intensely AchE-stained neuronal cell bodies are located in the ventral telencephalon (rostrally) and the dorsomedial telencephalon (at mid-level). In Neoceratodus, AchE staining is pronounced in the septal area, but absent in the pallium. The terminal nerve proper lacks AchE stain in Protopterus; nerve fibres of the preoptic nerve are AchE-positive in both lungfish species. In Xenopus, AchE staining of fibers and terminals is restricted to the subpallium (medial septum, tuberculum olfactorium, striatum, nucleus accumbens, and medial amygdala); cell bodies are AchE positive in parts of the subpallium and rostral pallium. Comparison of cytological, histochemical, and "connectional" parameters substantiates the interpretation that the dorsomedial telencephalon of lungfishes represents a subpallial, but not a "medial pallial" structure. The dorsomedial part of the lepidosirenid telencephalon corresponds to the septum in the most plesiomorphic living lungfish, Neoceratodus forsteri, but it differs considerably from the dorsomedial telencephalon (medial pallium) in amphibians.

The visually related posterior pretectal nucleus in the non-percomorph teleost Osteoglossum bicirrhosum projects to the hypothalamus: a DiI study.

This study was done to elucidate the ancestral (plesiomorphic) condition for visual pathways to the hypothalamus in teleost fishes. Three patterns of pretectal organization can be discerned morphologically and histochemically in teleosts. Their taxonomic distribution suggests that the intermediately complex pattern (seen in most teleost groups) is ancestral to both the elaborate pattern (seen in percomorphs) and the simple pattern (seen in cyprinids). The pretectal nuclei involved can be demonstrated with acetylcholinesterase histochemistry selectively and reliably in different species of teleosts, suggesting that the same-named nuclei are homologous in representatives of the three different patterns. Whereas there are visual pathways to the hypothalamus in both the elaborate (percomorph) and the simple (cyprinid) patterns, different pretectal and hypothalamic nuclei are involved. Thus visual hypothalamic pathways in these two patterns would not appear to be homologous. In this study, circuitry within the third, i.e., the intermediately complex, pattern is investigated. It is demonstrated that visual pathways project via the pretectum to the hypothalamus in Osteoglossum bicirrhosum and that they are very similar to the visual pathways in the elaborate pattern. This suggests that the circuitry in the intermediately complex pattern, as represented by Osteoglossum, is plesiomorphic (evolutionarily primitive) and the circuitry in both the simple pattern (seen in cyprinids) and the elaborate pattern (seen in percomorphs) is apomorphic (evolutionarily derived) for teleosts.

Distribution of FMRFamide-like immunoreactivity in the amphibian brain: comparative analysis.

FMRFamide is a small neuropeptide present in particular neurons of the basal forebrain and midbrain of the vertebrate groups studied, especially fishes and mammals. In order to assess interspecies variation, the distribution of FMRFamide-like immunoreactivity was studied in the brains of 13 species of amphibian. Although FMRFamide-immunoreactive (IR) terminals occurred throughout much of the brain, IR cell groups were noted in circumscribed regions of the CNS. In the eight anuran species studied, two major populations of labeled perikarya were observed: one in the septopreoptic area and another one in the caudal portion of the diencephalon. The rostrocaudal extent of both and the number of labeled somata in each neuronal group displayed species-specific differences. In urodeles and gymnophiones, labeled perikarya were located in the diencephalon, but there were remarkable species differences in the number of such cells. It is discussed whether sex or season of collection may account for some of the differences observed. The distribution of FMRFamide-IR perikarya, fibers, and pathways in the brain of anurans, urodeles, and gymnophiones was compared. The existence of FMRFamide perikarya in the anterior preoptic neuropil and medial septum appeared to be a feature common to all anurans; labeled neurons in the dorsal thalamus, however, may be present only in the (viviparous) gymnophione Typhlonectes compressicauda. Cerebrospinal fluid contacting FMRFamide neuronal cell bodies and fibers were observed in each of the three taxonomic orders. The data are compared with those previously obtained for other groups of vertebrates.

Misinterpretation of interaction effects: a reply to Rosnow and Rosenthal.

The structural model of analysis of variance for multiway tables, with main effects and interaction parameters, is overspecified. Only the set of estimable functions of the cell means is useful. The individual estimates of the parameters are artificial as regards the underlying scientific process. Rosnow and Rosenthal (1989a) stated that it is "absolutely necessary" (pg 146) that interactions should be interpreted by examining the usual estimates of the interaction parameters. This is incorrect.

Functional subdivisions of the olfactory system correlate with lectin-binding properties in Xenopus.

Soybean agglutinin (SBA) is known to selectively label a portion of neurons in amphibian and mammalian primary olfactory systems. Hitherto, no other distinctive features have been found to correlate with the two neuronal populations. Investigating SBA-HRP binding in olfactory mucosa and CNS of Xenopus, we noted that labelled and unlabelled structures can readily be assigned to different olfactory subsystems. The SBA negative one is utilized to detect air-borne odors, whereas major SBA-positive structures serve a role in the perception of water dissolved molecules. Some labelled fibers by-pass the olfactory bulb, traverse the telencephalon and innervate prosencephalic structures. They are considered to be aberrant olfactory nerve fibers, rather than being part of the terminal nerve.

Peripheral origin of olfactory nerve fibers by-passing the olfactory bulb in Xenopus laevis.

After DiI injections into the diencephalon of Xenopus, two types of retrogradely labelled cells were found in the nasal area: (i) receptor cells in the olfactory epithelium and (ii) a small cell group located between the main olfactory epithelium and the vomeronasal system. These results reveal an extensive extrabulbar olfactory projection of olfactory receptor cells. Fibers of these cells do not terminate in the olfactory bulb but innervate targets in the diencephalon directly. The other type of retrogradely labelled cells, apparently, are not part of any epithelium. They resemble similar cell groups which have previously been regarded as part of the nervus terminalis system in other vertebrates.

Interspecific variation of isthmo-optic projections in poikilothermic vertebrates.

Retinopetal projections from isthmic and caudal mesencephalic regions were studied in a number of poikilothermic vertebrates. The tested animals were elasmobranchs, bony fishes, amphibians, and reptiles. Isthmo-optic projections were absent in elasmobranchs, sturgeons, most teleosts, all anuran and urodel amphibians, and in anolis lizards. Tegmental projections to the retina were found in a few fishes and in most reptiles. Interspecific variations regarding three morphological parameters were as follows: (i) isthmic projections to the contralateral eye originate from one or from two distinct nuclei; (ii) major interspecific differences exist regarding numbers and aggregation densities of cells in the nuclei of origin; (iii) the percentage of retinopetal fibres that innervate the ipsilateral retina varies significantly between species.

The nervus terminalis in larval and adult Xenopus laevis.

Nervus terminalis (nt) projections were studied by HRP injections into one nostril in adult Xenopus and in Xenopus tadpoles. Central nt targets are: medial septum, preoptic nucleus, nucleus of the anterior commissure, and hypothalamus (mainly ipsilaterally). In Xenopus tadpoles, additional fibers reach the ipsilateral dorsal thalamus and the mesencephalic tegmentum, bilaterally; furthermore, hypothalamic projections are bilateral. Xenopus tadpole nt connections resemble those of adult urodeles more closely than the projections of frogs. However, Xenopus tadpoles lack nt innervation of the medial septum.

Expression of S100 protein in the vestibular nuclei during compensation of unilateral labyrinthectomy symptoms.

In adult guinea pigs, unilateral labyrinthine lesions were inflicted by chloroform injections into the middle ear. Immunoreactivity for S100 protein (S100) in the vestibular nuclei was studied during compensation of lesion-induced postural asymmetry symptoms, i.e., nystagmus, asymmetrical head position. 1 h after unilateral labyrinthectomy, increased levels of astroglial S100 immunoreactivity were found in the superior vestibular nucleus and in the medial/lateral vestibular nucleus border region on the side contralateral to the deafferentation. Bilaterally, the astrocytic S100 immunoreaction increased in the lateral vestibular nuclei around Deiters neurons. Maximal expression of S100 was noted 3 h after the lesion. Subsequently, it diminished. Our data reveal that transsynaptically altered neuronal activity induces an astrocytic reaction which provides increased levels of S100 to the local neuropil. Calcium and zinc binding S100 proteins may play a functional role for the neuroplasticity during vestibular compensation.

Comparative neurology of the optic tectum in ray-finned fishes: patterns of lamination formed by retinotectal projections.

Retinotectal projections were studied in 33 different species of Actinopterygii, the ray-finned fishes, with horseradish peroxidase and cobalt tracing techniques. The distribution of retinorecipient layers in the contralateral optic tectum was analyzed. In addition, the degree of differentiation of the stratum periventriculare, and the presence of ipsilateral retinotectal projections was examined. Retinofugal fibers are labeled in the stratum opticum (SO), stratum fibrosum et griseum superficiale (SFGS), stratum griseum centrale (SGC), stratum album centrale (SAC) and stratum periventriculare (SPV). Some species lack the projection to the SO, others lack the projection to the SGC, and a third group of fishes lack both projections. Five different patterns of retinorecipient tectal strata are distinguished. These patterns correlate with the species' taxonomic position. Evolutionary trends of tectal lamination and retinotectal innervation are described. The retinotectal projection patterns provide a useful indicator of phylogenetic relationships. Some of our data suggest different relationships between actinopterygian species than hitherto believed.