Non-specific olfactory aversion induced by intrabulbar infusion of the GABA(A) receptor antagonist bicuculline in young rats.

On postnatal day 12, young rats show an aversion to an odor to which they had been exposed along with presentations of foot shock on postnatal day 11. The acquisition of this aversive learning involves and requires disinhibition of the mitral/tufted cells induced by centrifugal noradrenergic activation during somatosensory stimulation. This olfactory learning is established only for the odor to which the rat has been exposed during conditioning. Infusion of the GABA(A) receptor antagonist bicuculline at a high dose (2.0 nmol/each olfactory bulb) into the olfactory bulb in the presence of an odor is capable of developing olfactory aversive responses without somatosensory stimulation in young rats. The purpose of this study is to characterize the properties of bicuculline-induced aversive responses. In contrast to the odor specificity of aversive learning produced by odor-shock conditioning, bicuculline-induced aversive responses lack odor specificity. Namely, bicuculline infusion in the presence of a citral odor results, in a dose-dependent manner, in subsequent aversive responses to strange odors (benzaldehyde and vanillin) that have never been presented. Moreover, bicuculline infusion alone is sufficient to produce dose-dependent aversive responses to strange odors (citral, benzaldehyde and geraniol). From these results we suggest that disinhibition of mitral/tufted cells from granule cells by bicuculline infusion makes young rats aversive to strange odors non-specifically, as if the rats had learned the odor aversion as a result of odor exposure paired with foot shock. Different mechanisms of disinhibition of the mitral/tufted cells may underlie both the pharmacological manipulation and noradrenergic activation by somatosensory stimulation.

Early attachment of uncultured retinal pigment epithelium from aged donors onto Bruch's membrane explants.

Retinal pigment epithelium (RPE) transplantation might replace cells lost as a consequence of choroidal neovascular membrane excision in patients with age-related macular degeneration (AMD). Autologous transplantation of RPE cells harvested from a peripheral biopsy may overcome problems of immune rejection. To study the feasibility of autologous RPE cell transplantation, the authors examined the attachment of freshly harvested RPE cells from aged donors onto Bruch's membrane explants, debrided to (1) remove or (2) preserve the RPE basement membrane. Human retinal pigment epithelial sheets were harvested from adult donor eyes (N = 12, mean age 79.00 +/- 9.40 years) and, following incubation in collagenase, were mechanically fragmented into microaggregates. Microaggregates (approximately 120 000 cells) were seeded onto the paired explants (7 mm diameter) and incubated for 20 min, 1, 4, or 24 hr at 37 degrees C. The percent coverage of the debrided surface by microaggregates was determined by sampling the center of the explants with scanning electron microscopy. RPE microaggregate attachment to Bruch's membrane was significantly greater at all time points analysed in samples with intact basement membrane versus those with an exposed inner collagenous layer. Coverage of debridements retaining intact RPE basement membrane was 1.83 +/- 1.10% at 20 min, 3.54 +/- 2.14% at 1 hr, and 8.68 +/- 2.63% at 4 hr. Coverage of debridements lacking basement membrane was 0.10 +/- 0.04% at 20 min, 0.39 +/- 0.25% at 1 hr, and 0.63 +/- 0.42% at 4 hr. Based on their morphologic appearance, many cells were dying as early as 1 hr following seeding. To increase surface coverage, the authors seeded four times the above number of cells and incubated the specimens for 1 hr. Coverage on explants lacking RPE basement membrane showed no increase in the number of cells attached to the inner collagenous layer. There was a significant approximately three-fold increase in the number of cells attached in the presence of basement membrane. These results indicate that if RPE cells from aged human donors are used for transplantation, some modification of the Bruch's membrane surface or the cells must be considered for cell attachment and eventual cell survival.

Short-term study of allogeneic retinal pigment epithelium transplants onto debrided Bruch's membrane.

PURPOSE: To investigate the survival and behavior of retinal pigment epithelium (RPE) microaggregates transplanted onto hydraulically debrided Bruch's membrane and to compare results of using three different vehicles for cell delivery. METHODS: RPE microaggregates obtained from male cats were transplanted onto the tapetal area of female cats after native RPE was debrided. For the control, one of three vehicles was introduced into the debridements. Each transplant or control specimen was analyzed histologically and immunohistochemically. Transplanted male RPE cells were identified by in situ labeling of the cat Y chromosome. RESULTS: Histologically, significant numbers of condensed, darkly stained RPE nuclei were observed in all transplants compared with few TUNEL-positive RPE cells. Cellular retinaldehyde-binding protein was present up to day 7 in all RPE cells in transplants. In both transplant and control specimens, the antibody against the Ki-67 nuclear antigen labeled some RPE cells at day 3. TUNEL-positive outer nuclear layer nuclei were most frequently observed at day 1, but were much less frequent at 7 days in both transplant and control specimens. CONCLUSIONS: Transplanted RPE appeared to retain at least some markers of differentiation up to 7 days after surgery. Some proliferation of transplanted RPE cells was also seen. Apoptotic cell death of transplanted RPE, as judged by TUNEL staining was observed rarely. RPE transplants imposed no adverse effect on the overlying retina. RPE survival appeared to be similar with each of the three vehicles for cell delivery.

Reorganization of the uncrossed visual pathways as revealed by Fos-like immunoreactivity in rats with neonatal monocular enucleation.

To elucidate the neuronal characteristics of the functional expansion in the uncrossed visual pathways (UXVPs), resulting from early monocular enucleation in rats, the feasibility of stimulus-dependent induction of the immediate early gene c-fos was examined immunohistochemically. In the UXVPs of rats with monocular enucleation at birth, patterned visual stimuli induced Fos-like immunoreactive (FLI) neurons much more densely in wide areas of the superficial layer throughout the superior colliculus (SC), and in the striate and extrastriate areas of the visual cortex (VC). In the UXVPs of rats monocularly enucleated after maturity, however, only a few stimulus-dependent FLI neurons were scattered in the restricted portions of the SC and the VC.

Agrocybe cylindracea lectin is a member of the galectin family.

The complete amino acid sequence of Agrocybe cylindracea lectin was determined from the peptides obtained by chemical cleavages and enzymatic hydrolyses. The sequence shows 19.1% and 36.8% identity with those of human galectin-1 and Coprinus lectin-1, a fungal galectin, respectively. Seven residues, which are commonly found in carbohydrate recognizing domain (CRD) of galectins, were conserved. However, several insertions in the sequence, compared with those of human galectin-1 and Coprinus lectin-1, suggest that beta-strands S2, F3, and S4 and the loop structures between beta-strands F2 & S3 and F5 & S2 are different from those of galectins reported so far.

Facilitatory effect of ritanserin is mediated by dopamine D(1) receptors on olfactory learning in young rats.

The olfactory bulb is critically involved in early olfactory learning. In this study, we examined the effect of intrabulbar infusion of ritanserin, a 5-hydroxytryptamine(2) (5-HT(2)) receptor antagonist on a one-trial aversive olfactory learning in young rats. Ritanserin, a 5-HT(2) receptor antagonist, was continuously infused into the olfactory bulb of postnatal day-11 (PND 11) rat pups during a 30-min training session of pairing citral odor and foot shock. On the following day, the time spent in the part of the apparatus where the odor was present was measured as an index of odor aversion. Consistent with a previous study on olfactory preference learning, 1 nM ritanserin, but not 10 nM, blocked the olfactory aversive learning. We further examined the ability of 10 nM ritanserin to induce olfactory learning in the absence of the unconditioned stimulus foot shock. Pups that received intrabulbar infusion of 10 nM ritanserin in the presence of citral odor developed an aversion to the odor without foot shock. Since ritanserin has been shown to have an affinity for dopamine receptors, we examined the effect of dopamine antagonists on the ritanserin-induced aversive olfactory learning. Co-infusion of the dopamine D(1) receptor antagonist (+/-)-SKF-83566 with ritanserin dose-dependently prevented induced learning. In contrast, the D(2) receptor antagonist spiperone was without effect. These results extend the previous finding on the role of bulbar 5-HT(2) receptors in early olfactory learning and suggest that high concentration of ritanserin facilitates aversive olfactory learning through D(1) receptors in the olfactory bulb.

Molecular cloning, expression, and chromosomal mapping of human chondroitin 4-sulfotransferase, whose expression pattern in human tissues is different from that of chondroitin 6-sulfotransferase.

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of the N-acetylgalactosamine residues of chondroitin. We previously reported the cloning of C4ST cDNA from mouse brain. We here report the cloning and expression of human C4ST cDNA. The cDNA was isolated from a human fetal brain cDNA library by hybridization with a DNA probe prepared from rat poly(A)(+) RNA used for the cloning of mouse C4ST cDNA. The cDNA comprises a single open reading frame that predicts a Type II transmembrane protein composed of 352 amino acids. The protein has an amino acid sequence homology of 96% with mouse C4ST. When the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that transfers sulfate to both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis indicated that human C4ST mRNAs (6.0 and 1.9 kb) are expressed ubiquitously in various adult human tissues. Dot blot analysis has shown that human C4ST is strongly expressed in colorectal adenocarcinoma and peripheral blood leukocytes, whereas strong expression of human chondroitin 6-sulfotransferase (C6ST) is observed in aorta and testis. These observations suggest that the expression of C4ST and C6ST may be controlled differently in human tissues. The C4ST gene was localized to chromosome 12q23.2-q23.3 by fluorescence in situ hybridization.

Gabaergic control of olfactory learning in young rats.

Olfactory learning in young rats correlates with neural plasticity in the olfactory bulb, and involves noradrenergic modulation of reciprocal dendrodendritic synapses between mitral cells and GABAergic granule cells. The purpose of this study was to examine, in vivo, the consequences of manipulating bulbar GABA transmission during training. In the first experiment, postnatal day 11 rat pups were trained in an olfactory associative learning task with citral odor and foot shock as the conditioned and unconditioned stimuli, respectively. The pups received continuous infusion of saline or the GABA(A) receptor agonist muscimol into the olfactory bulbs throughout a 30-min training session. The pups were then tested on postnatal day 12 for a preference for or an aversion to citral odor. Saline-infused control pups developed an aversion to citral odor. The GABA(A) receptor agonist muscimol impaired this aversive learning in a dose-dependent manner. In the second experiment, pups were exposed to the odor for 30 min while receiving continuous intrabulbar infusion of a low or high dose of the GABA(A) receptor antagonist bicuculline, without any other reinforcer. Depending on whether a low (0.2 nmol/bulb) or high (1.0 nmol/bulb) dose of bicuculline was infused, the pups showed a preference or an aversion for citral odor after infusion of low and high doses, respectively. These results indicate that disinhibition of mitral cells in the olfactory bulb is critical for olfactory learning in young rats, and suggest that the degree of disinhibition is an important determinant in acquiring either preference or aversion for the conditioned odor.

Isolation and amino acid sequence of a protein-synthesis inhibitor from the seeds of rye (Secale cereale).

A protein-synthesis inhibitor, designated RPSI, was isolated from the seeds of rye (Secale cereale) using gel filtration and S-Sepharose column chromatography. RPSI is a basic protein with an isoelectric point of over 10, and the concentration of protein required for 50% inhibition of protein synthesis (IC50) of purified RPSI was about ten-fold the concentration of ricin A-chain. The complete amino acid sequence of RPSI was discovered by analyzing the peptides and fragments obtained from the proteolytic digests and by the cyanogen bromide- and hydroxylamine-cleavages of RPSI. RPSI consists of 280 amino acid residues and has a molecular weight of 30,171. RPSI has only 21% sequence identity with that of ricin A-chain, but all five amino acid residues involved in the active site of ricin A-chain are conserved in RPSI.

New Limonoids from Melia toosendan.

The root bark of Melia toosendan afforded four new limonoids with a C-19/C-29 bridged acetal structure, together with two known limonoids, 12α-hydroxyamoorastatone and its 12-acetate. The new compounds were elucidated as 1-O-acetyltrichilin H and 29-O-substituted amoorastatone derivatives, named neoazedarachins A, B and D, by spectroscopic and chemical means. Their antifeedant activity was also studied.

Isolation and Some Properties of the Antimicrobial Peptide (Pa-AMP) from the Seeds of Pokeweed (Phytolacca americana).

An antimicrobial peptide, designated Pa-AMP, was purified by gel filtration on Sephadex G-75 followed by S-Sepharose, Cosmosil-SP, and reverse-phase HPLC from the seeds of pokeweed (Phytolacca americana). Pa-AMP is a basic peptide having an isoelectric point of over 10 and its extinction coefficient at 280 nm of 1% aqueous solution was 7.7. Pa-AMP has a molecular mass of 4 kDa and 3.4 kDa on tricine SDS-PAGE under nonreducing and reducing conditions, respectively. The N-terminal amino acid of Pa-AMP was blocked. The concentrations of peptide required for 50% inhibition (IC50) of the growth of plant pathogenic fungi, Gram-positive, and Gram-negative bacteria were 3 to 41 µg/ml. Differing from other peptides, Pa-AMP inhibited the growth of some Gram-negative bacteria.

Purification and carbohydrate-binding specificity of Agrocybe cylindracea lectin.

A lectin was isolated from fruiting bodies of Agrocybe cylindracea by two ion-exchange chromatographies and gel filtration on Toyopearl HW55F. The lectin was homogeneous on polyacrylamide gel electrophoresis and its molecular mass was determined to be 30000 by gel filtration, and 15000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, signifying a dimeric protein. Its carbohydrate-binding specificity was investigated both by sugar-hapten inhibition of hemagglutination and by enzyme-linked immunosorbent assay. The inhibition tests showed the affinity of the lectin to be weakly directed toward sialic acid and lactose, and the enhanced affinity toward trisaccharides containing the NeuAc alpha2,3Gal beta-structure. Importantly, the lectin strongly interacted with glycoconjugates containing NeuAc alpha2,3Gal beta1,3GlcNAc-/GalNAc sequences.

A lectin from leaves of Neoregelia flandria recognizes D-glucose, D-mannose and N-acetyl D-glucosamine, differing from the mannose-specific lectins of other monocotyledonous plants.

A mannose-specific lectin was isolated from leaves of Neoregelia flandria, an ornamental plant that belongs to Bromeliaceae, a family of monocotyledons. The amino acid composition and molecular mass of the lectin were similar to those of mannose-specific lectins from other monocotyledons. However, in a test to examine the inhibition of hemagglutination, it became apparent that the isolated lectin recognized D-glucose and N-acetyl D-glucosamine in addition to D-mannose, unlike mannose-specific lectins from the monocotyledons that have been reported to date.

Substrate specificity and transglucosylation catalyzed by cycad beta-glucosidase.

Initial rates of transglucosylation with diglucosides and diglucose-azoxyglycosides as acceptor by cycad beta-glucosidase were tentatively obtained. The formation of beta-1,3 glucosidic linkage was predominant, except for neocycasin A (beta-laminaribioside of methylazoxymethanol, MAM) as an acceptor. With neocycasin A as an acceptor, beta-1,4 and beta-1,6 glucosidic linkages were formed but beta-1,3 linkage was not. Whereas with laminaribiose as acceptor, laminaritriose and triose with beta-1,6 linkage were formed, but triose with beta-1,4 linkage was not. On the other hand, with other diglucoses and neocycasin B (beta-gentiobioside of MAM) as acceptor, all the linkages formed were beta-1,3 glucosidic. The aglycone of azoxyglycosides, MAM, affected the kind of linkages formed in the trisaccharides. When initial rates of the linkage formation of the transglucosylation at 100 mM acceptor were compared with the hydrolysis rates obtained by Lineweaver-Burk plot, the order of formation rates of the di- and tri-glucosides by transglucosylation was the same as obtained for the hydrolysis parameter, kcat/Km. Km values for various substrates could be grouped according to the kind of the linkages (beta-1,3, beta-1,4, and beta-1,6) first split by the enzyme.

Discrimination performance and resolution capacity of uncrossed visual pathways in one-eyed albino rats.

Our recent study has demonstrated that adult rats with one eye removed at birth (OEB) relearn a discrimination between alternating black and white stripes, 30 mm wide each, oriented horizontally and vertically faster than control rats monocularly enucleated in adulthood (OET), when relearning is carried out after lesions of the visual cortex and transections of the optic tract contralateral to the remaining eye. Yet, we could not obtain the corresponding results in the acquisition of the discrimination following the same surgical treatments: OEBs did master the discrimination, whereas OETs did not. We hypothesized that a large discrepancy in OETs' performance between the acquisition and relearning occurred because the stripes were close to the limit of the resolution capacity of the uncrossed visual pathways (UXVPs), and hence that a better performance of OEBs was to indicate an increase in the resolution capacity, which resulted from reorganization of the UXVPs brought about by monocular enucleation at birth. To test the hypothesis we tried to approximate the limit of the resolution capacity of the UXVPs in OEBs and OETs using seven different sized test stripes ranging from 20 to 5 mm in width after both OEBs and OETs had relearned the discrimination of the 30 mm training stripes following the same surgical treatments mentioned above. It was found that the median width of the smallest stripes for OETs to discriminate was 10 mm, and that of OEBs 7.5 mm. Although OETs could not discriminate the smallest stripes which OEBs could, they were able to discriminate stripes one third smaller than those hypothesized. Based on these findings the possibility was discussed that the acquisition as well as the relearning of the discrimination of the 30 mm stripes mediated by the UXVPs in OEBs and OETs might not be influenced by the resolution capacity, but mostly, if not entirely, by the size of the visual field.

Effects of monocular enucleation at birth upon learning of a vertical-horizontal discrimination in hooded rats.

Previously we have demonstrated that adult albino rats with one eye removed at birth (OEB) relearn a black-white discrimination faster than those monocularly enucleated at maturity (OET) when relearning is conducted after lesioning of the visual cortex contralateral to the remaining eye (Type A experiment). This faster relearning phenomenon is considered to be one behavioral expression of the functioning of the expanded uncrossed visual pathways (expanded UXVPs) resulting from monocular enucleation at birth. However, neither OEBs nor OETs were able to master the discrimination when the experiment was conducted without previous learning following the same surgical treatment (Type B experiment). We hypothesized that this occurs because the cues to discriminate might be close to the threshold of discrimination for either the normal UXVPs or the expanded UXVPs. In order to gain insight into the hypothesis, the present study was undertaken using hooded rats as subjects which genetically possess larger and presumably more efficient functioning UXVPs. The questions addressed were as follows: 1) Whether or not the UXVPs can mediate a vertical-horizontal discrimination in OEBs and OETs. 2) If they can, is there any difference in the upper limit of discrimination capacity between the normal UXVPs and the expanded UXVPs? Three experiments were carried out. In the Type A experiment OEBs relearned discrimination of the 10-mm stripes [0.44 cycles/degree (c/d)] faster than OETs (Experiment 1), yet in the Type B experiment neither OEBs nor OETs were capable of acquiring that discrimination (Experiment 2). However, they could originally master the discrimination equally well when the width of stripes was broadened to 30 mm (0.15 c/d). And when the width of stripes was systematically reduced thereafter, the width of the smallest stripes for the expanded UXVPs to discriminate was found to be 6 mm (0.73 c/d) and that for the normal UXVPs 10 mm (0.44 c/d) [Experiment 3]. These findings were discussed in relation to the hypothesis advanced on our previous data in albino rats.

Two isolectins from leaves of winged bean, Psophocarpus tetragonolobus (L.) DC.

Two isolectins were isolated from leaves of winged bean and characterized. They differed from each other in terms of their immunological properties, hemagglutinating activities, sugar inhibition patterns, and amino acid compositions. Both lectins were acidic and one of them (L-I), which was inactive toward trypsinized human type O erythrocytes, was similar to one of green shell lectins (WGS-1); which resembled basic seed lectin in its immunological properties. The amino-terminal sequence of L-I was homologous to that of WGS-1. The amino acid composition of L-I was similar to that of basic seed lectin, but the extent of the homology between amino-terminal sequences was low when L-I and basic seed lectin were compared. Examination by ELISA revealed that L-I and WGS-1 were distinct from the basic lectins of seeds and tuberous roots. L-I had a disulfide bridge between two subunits and it exhibited high hemagglutinating activity toward human type A erythrocytes, as compared to its activity toward other erythrocytes. By contrast, the properties of a second acidic lectin from winged bean leaves (L-II) were very similar to those of acidic lectins from seeds and tuberous roots, and the similarities extended as far as the immunological properties.

Long-term callosal lesions and learning of a black-white discrimination by one-eyed rats.

We know from our previous studies that mature rats with monocular enucleation at birth (OEBs), as well as animals enucleated at maturity (OETs), were unable to learn a black-white discrimination when they were trained after lesions of the visual cortex contralateral to the remaining eye. Since it is well known that synaptic reorganization takes place in the adult rat brain through reactive synaptogenesis following deafferentation, we wondered if long-term callosal lesions in OEBs and OETs would bring out such synaptic reorganization in the visual cortex and, consequently, affect the outcome of the discrimination mentioned above. In the present study, two experiments were carried out: in Experiment 1 the previous experiment was replicated in that OEBs and OETs of 3 months of age were trained on the discrimination 10 days following unilateral visual cortex lesions; in Experiment 2, effects of callosal lesions made 10 weeks earlier either at 3 weeks of age or 13 weeks of age were investigated. The results were: 1) the findings of the previous experiment were confirmed; 2) the long-term callosal lesions facilitated the acquisition of discrimination in OEBs but not in OETs; 3) the facilitative effects were more prominent in OEBs with callosal lesions at 3 weeks of age than in those at 13 weeks of age. The findings were discussed in relation to possible synaptic reorganization produced in the visual cortex ipsilateral to the remaining eye following callosal lesions made 10 weeks earlier and also in relation to reorganization of the uncrossed visual pathways resulting from monocular enucleation at birth.

Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin.

Poly(A)-rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17-kDa lectin polypeptide upon translation in a wheat-germ cell-free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2-kDa signal peptide. A second post-translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA. Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal-sequence and a C-terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments.

Differential effects of unilateral optic tract transections and visual cortical lesions upon a pattern discrimination in albino rats with removal of one eye at birth.

Previously we have demonstrated that adult rats with one eye removed at birth (OEB) relearn a black-white discrimination faster than control rats monocularly enucleated at maturity (OET), when relearning is conducted after lesions of the visual cortex contralateral to the remaining eye. This faster relearning phenomenon is considered to be one behavioral expression of the functioning of the expanded uncrossed visual pathways resulting from monocular at birth. The present study was concerned with the question of whether the same phenomenon can be observed in the discrimination between alternating black and white stripes oriented horizontally and vertically. Two experiments were carried out. In the first experiment, which is a replication of one of our previous studies, relearning was conducted after the visual cortical lesions contralateral to the remaining eye. The results were consistent with those of the previous one in which neither OEBs nor OETs were found able to relearn the discrimination. In the second experiment, relearning was conducted after transections of the optic tract contralateral to the remaining eye. It was shown that under this condition both OEBs and OETs could relearn the discrimination, and furthermore, that OEBs restored the habit faster than OETs. Possible mechanisms underlying the difference in the results from the two experiments were discussed.