Effects of feeding rumen-protected methionine pre- and postpartum on reproductive outcomes of multiparous Holstein cows.

Our primary objective was to evaluate the effect of feeding rumen-protected Met (RPM) in the pre- and postpartum total mixed ration (TMR) on pregnancy per artificial insemination (AI) and pregnancy loss in multiparous Holstein cows. We also evaluated multiple secondary reproductive physiological outcomes before and after AI, including uterine health, ovarian cyclicity, response to synchronization of ovulation, and markers of embryo development and size. A total of 470 multiparous Holstein cows [235 at the University of Wisconsin (UW) and 235 at Cornell University (CU)] were used for this experiment. Experimental treatment diets were applied at the pen level (2 and 4 close-up pens at CU and UW, respectively, and 12 and 6 postfresh pens at CU and UW, respectively); thus, pen was the experimental unit, and cow was the observational unit. Cows were enrolled and randomly assigned to be fed the experimental treatment diets at approximately 4 wk before parturition until 67 d of gestation [147 d in milk (DIM)] after their first service. Close-up dry cow and replicated lactation pens were randomly assigned to treatment diets: RPM, prepartum = 2.83% (UW) and 2.85% (CU), postpartum = 2.58% (UW) and 2.65% (CU); and control (CON), prepartum = 2.30% (UW) and 2.22% (CU), postpartum = 2.09% (UW) and 2.19% (CU; Met as percentage of metabolizable protein). Vaginal discharge and uterine cytology (percentage of polymorphonuclear leucocytes) were evaluated at 35 ± 3 DIM. Cows received timed AI (TAI) at 80 ± 3 DIM after synchronization of ovulation with the Double-Ovsynch protocol. Ovarian cyclicity status, response to synchronization of ovulation, and luteal function were determined by measuring circulating concentrations of progesterone at 35 and 49 ± 3 DIM, 48 and 24 h before TAI, and 8, 18, 22, 25, and 29 d after TAI. Interferon-stimulated gene expression in white blood cells were compared on 18 d after TAI (CU only) and pregnancy-specific protein B concentrations at 22, 25, 29, 32, and 67 d after TAI. Pregnancy status was determined using pregnancy-specific protein B at 25 and 29 d after TAI, and by transrectal ultrasonography at 32, 39, and 67 d after TAI. Embryo and amniotic vesicle size were determined at 32 and 39 d after TAI. Pregnancy per AI (25 d: 64.7 vs. 64.0%, 32 d: 54.3 vs. 55.1% for CON and RPM, respectively) and pregnancy loss (25 to 67 d: 22.6 vs. 19.2% for CON and RPM, respectively) for synchronized cows did not differ. The proportion of cows with purulent vaginal discharge (CON = 7.7 vs. RPM = 4.6%) and cytological endometritis (CON = 20.8 vs. RPM = 23.6%) did not differ. Cyclicity status, ovarian responses to the synchronization protocol, and synchronization rate also did not differ. In addition, fold change for interferon-stimulated genes, concentrations of pregnancy-specific protein B, and embryo size were not affected by treatments. In conclusion, feeding RPM in the pre- and postpartum TMR at the amounts used in this experiment did not affect uterine health, cyclicity, embryo development, or reproductive efficiency in dairy cows.

Effects of feeding rumen-protected methionine pre- and postpartum in multiparous Holstein cows: Lactation performance and plasma amino acid concentrations.

Objectives were to evaluate the effect of feeding rumen-protected methionine (RPM) in pre- and postpartum total mix ration (TMR) on lactation performance and plasma AA concentrations in dairy cows. A total of 470 multiparous Holstein cows [235 cows at University of Wisconsin (UW) and 235 cows at Cornell University (CU)] were enrolled approximately 4 wk before parturition, housed in close-up dry cow and replicated lactation pens. Pens were randomly assigned to treatment diets (pre- and postpartum, respectively): UW control (CON) diet = 2.30 and 2.09% of Met as percentage of metabolizable protein (MP) and RPM diet = 2.83 and 2.58% of Met as MP; CU CON = 2.22 and 2.19% of Met as percentage of MP, and CU RPM = 2.85 and 2.65% of Met as percentage of MP. Treatments were evaluated until 112 ± 3 d in milk (DIM). Milk yield was recorded daily. Milk samples were collected at wk 1 and 2 of lactation, and then every other week, and analyzed for milk composition. For lactation pens, dry matter intake (DMI) was recorded daily. Body weight and body condition score were determined from 4 ± 3 DIM and parturition until 39 ± 3 and 49 DIM, respectively. Plasma AA concentrations were evaluated within 3 h after feeding during the periparturient period [d -7 (±4), 0, 7 (±1), 14 (±1), and 21 (±1); n = 225]. In addition, plasma AA concentrations were evaluated (every 3 h for 24 h) after feeding in cows at 76 ± 8 DIM (n = 16) and within 3 h after feeding in cows at 80 ± 3 DIM (n = 72). The RPM treatment had no effect on DMI (27.9 vs. 28.0 kg/d) or milk yield (48.7 vs. 49.2 kg/d) for RPM and CON, respectively. Cows fed the RPM treatment had increased milk protein concentration (3.07 vs. 2.95%) and yield (1.48 vs. 1.43 kg/d), and milk fat concentration (3.87 vs. 3.77%), although milk fat yield did not differ. Plasma Met concentrations tended to be greater for cows fed RPM at 7 d before parturition (25.9 vs. 22.9 µM), did not differ at parturition (22.0 vs. 20.4 µM), and were increased on d 7 (31.0 vs. 21.2 µM) and remained greater with consistent concentrations until d 21 postpartum (d 14: 30.5 vs. 19.0 µM; d 21: 31.0 vs. 17.8 µM). However, feeding RPM decreased Leu, Val, Asn, and Ser (d 7, 14, and 21) and Tyr (d 14). At a later stage in lactation, plasma Met was increased for RPM cows (34.4 vs. 16.7 µM) consistently throughout the day, with no changes in other AA. Substantial variation was detected for plasma Met concentration (range: RPM = 8.9-63.3 µM; CON = 7.8-28.8 µM) among cows [coefficient of variation (CV) > 28%] and within cow during the day (CV: 10.5-27.1%). In conclusion, feeding RPM increased plasma Met concentration and improved lactation performance via increased milk protein production.

LPA1 receptor and chronic stress: Effects on behaviour and the genes involved in the hippocampal excitatory/inhibitory balance.

The LPA1 receptor, one of the six characterized G protein-coupled receptors (LPA1-6) through which lysophosphatidic acid acts, is likely involved in promoting normal emotional behaviours. Current data suggest that the LPA-LPA1-receptor pathway may be involved in mediating the negative consequences of stress on hippocampal function. However, to date, there is no available information regarding the mechanisms whereby the LPA1 receptor mediates this adaptation. To gain further insight into how the LPA-LPA1 pathway may prevent the negative consequences of chronic stress, we assessed the effects of the continuous delivery of LPA on depressive-like behaviours induced by a chronic restraint stress protocol. Because a proper excitatory/inhibitory balance seems to be key for controlling the stress response system, the gene expression of molecular markers of excitatory and inhibitory neurotransmission was also determined. In addition, the hippocampal expression of mineralocorticoid receptor genes and glucocorticoid receptor genes and proteins as well as plasma corticosterone levels were determined. Contrary to our expectations, the continuous delivery of LPA in chronically stressed animals potentiated rather than inhibited some (e.g., anhedonia, reduced latency to the first immobility period), though not all, behavioural effects of stress. Furthermore, this treatment led to an alteration in the genes coding for proteins involved in the excitatory/inhibitory balance in the ventral hippocampus and to changes in corticosterone levels. In conclusion, the results of this study reinforce the assumption that LPA is involved in emotional regulation, mainly through the LPA1 receptor, and regulates the effects of stress on hippocampal gene expression and hippocampus-dependent behaviour.

Extracellular activity of NBAD-synthase is responsible for colouration of brown spots in Ceratitis capitata wings.

After the emergence of the Ceratitis capitata imago, the pale and folded wings are expanded and sclerotized to acquire the definitive form and to stabilize the cuticle. The wings of this fly show a specific pattern of brownish and black spots. Black spots are pigmented by melanin, whereas there was scarce information about the development of the brownish spots. N-beta-alanydopamine (NBAD) is the main tanning precursor in C. capitata body cuticle, and we hypothesized that it may be responsible for the colouration of the brownish spots. We determined the topology and timing of NBAD synthesis and deposition to attain the species-specific colouration pattern. We demonstrated that during the first hours the colour of the brownish spots was principally determined by the tanning of the hairs. Haemolymph circulation through the veins is required to tan the wings. We confirmed that soon after wing spreading, most of the wing epidermal cells disappeared. Thus, the tanning of the brown spots was accomplished when the wing lamina was devoid of cells. NBAD synthase (NBAD-S; Ebony protein in D. melanogaster) activity in wings was detected in pharate adults and lasted several days after the emergence, even after the end of the tanning process. This observation is in contrast to epidermal NBAD-S activity in the body, where it was nearly undetectable 48 h post emergence. Our results indicate that NBAD-S was exported and deposited into the extracellular matrix of the brown spot areas before cell death and that tanning occurs through gradual export of NBAD precursors (dopamine and b-alanine) from veins.

maLPA1-null mice as an endophenotype of anxious depression.

Anxious depression is a prevalent disease with devastating consequences and a poor prognosis. Nevertheless, the neurobiological mechanisms underlying this mood disorder remain poorly characterized. The LPA1 receptor is one of the six characterized G protein-coupled receptors (LPA1-6) through which lysophosphatidic acid acts as an intracellular signalling molecule. The loss of this receptor induces anxiety and several behavioural and neurobiological changes that have been strongly associated with depression. In this study, we sought to investigate the involvement of the LPA1 receptor in mood. We first examined hedonic and despair-like behaviours in wild-type and maLPA1 receptor null mice. Owing to the behavioural response exhibited by the maLPA1-null mice, the panic-like reaction was assessed. In addition, c-Fos expression was evaluated as a measure of the functional activity, followed by interregional correlation matrices to establish the brain map of functional activation. maLPA1-null mice exhibited anhedonia, agitation and increased stress reactivity, behaviours that are strongly associated with the psychopathological endophenotype of depression with anxiety features. Furthermore, the functional brain maps differed between the genotypes. The maLPA1-null mice showed increased limbic-system activation, similar to that observed in depressive patients. Antidepressant treatment induced behavioural improvements and functional brain normalisation. Finally, based on validity criteria, maLPA1-null mice are proposed as an animal model of anxious depression. Here, for we believe the first time, we have identified a possible relationship between the LPA1 receptor and anxious depression, shedding light on the unknown neurobiological basis of this subtype of depression and providing an opportunity to explore new therapeutic targets for the treatment of mood disorders, especially for the anxious subtype of depression.

Cyclosporine A for severe atopic dermatitis in children. efficacy and safety in a retrospective study of 63 patients.

BACKGROUND: Cyclosporine A (CSA) is an immunosuppressant agent widely used in severe atopic dermatitis (AD). However, experience in children is limited. OBJECTIVES: To assess the efficacy and adverse events of CSA therapy in children. METHODS: Retrospective study of children with severe AD treated with CSA between January 2009 and December 2015. RESULTS: Data from 63 patients were collected. Mean age at the beginning of treatment was 8.4 years (±3.6). The median starting dose was 4.27 (±0.61) mg/kg/day. After 4 weeks of treatment, the outcome was excellent in 35% of cases, good in 29% and poor in 36% of the patients. The response was better in patients without eosinophilia (P < 0.05). The median duration of treatment was 4.6 months (range 1.5-21.6). Side-effects were frequent but mild, being more common in patients after longer treatment periods (P < 0.05). Mean time of follow-up was 19.4 months (±12.7). Prolonged remission (>6 months) was observed in 13 patients (20%). LIMITS: This is a retrospective review. The follow-up period is limited. CONCLUSIONS: Our data confirm that CSA is efficacious and acts rapidly in the majority of children with severe AD. CSA therapy can provide sustained remission in some patients. CSA seems to be well tolerated in children, but strict monitoring is mandatory.

Mente Activa® Improves Impaired Spatial Memory in Aging Rats.

OBJECTIVES: Aging is accompanied by a decline in several aspects of the cognitive function, having negative personal and socioeconomic impacts. Dietary supplements could be beneficial for preventing age-related cognitive decline. In this context, we examined whether the nutritional supplement Mente Activa® has beneficial effects on aging-related cognitive deficits without inducing side effects. METHODS: Mente Activa® was administered to old rats (n= 30 treated rats and n= 30 control rats) during 5 months, and the Morris water maze was used to test the learning capacities of the animals. The first assessment was conducted before the nutritional intervention (age of 18-19 months), to determine the baseline of the performance of animals on this test, and the second assessment was performed at the end of the treatment (23-24 moths). In order to examine possible secondary effects of this nutritional supplement, plasma, heart anatomy and liver parameters were evaluated. RESULTS: Our data indicate that supplemented rats showed less escape latency, distance swum, higher use of spatial search strategies, and crossed the former platform location with higher frequency than control rats. These effects were specific of the treatment, indicating that this nutritional supplement has a beneficial effect on spatial memory. On the other hand, the regular intake of Mente Activa® did not induce any negative effects in plasma parameters and heart size. CONCLUSIONS: Aged rats under a sustained dietary intake of the nutritional supplement Mente Activa® displayed improved learning and memory abilities compared to the non-treated rats. These results suggest the therapeutic potential and safety of use of Mente Activa® for age-related cognitive deficits, particularly, in the onset of the first cognitive dysfunction symptoms.

Carbon dots obtained using hydrothermal treatment of formaldehyde. Cell imaging in vitro.

Highly photoluminescent carbon dots have been prepared in a one step procedure by hydrothermal treatment of formaldehyde at 180 °C. They show green fluorescence under UV light exposure and emission spectra are centered at 440 nm. Fluorescence lifetimes comprise between 0.7 and 2.70 ns, when the synthesis process lasted for 1-7 days. TEM images of nanoparticles showed a homogeneous size/shape distribution. When the thermal treatment process was carried out for a long time (30 days) formation of aggregates occurred. Carbon dots were further analyzed using (1)H and (13)C-NMR, Raman and FTIR spectroscopy techniques and XPS. Cell imaging of nanoparticles was carried out by using mouse MC3T3-E1 pre-osteoblasts as a model. The nanoparticles were selectively localized in the cytoplasm without further functionalization and could be realized by cellular phagocytosis, so that the fluorescence of these can be used for live cell imaging in vitro.

A comparative analysis of intraperitoneal versus intracerebroventricular administration of bromodeoxyuridine for the study of cell proliferation in the adult rat brain.

Bromodeoxyuridine (BrdU) is the most widely used marker to detect proliferative cells in the adult brain. Here we analyse whether the route of administration of the tracer influences the number of labelled cells. For the intraperitoneal (ip) administration of BrdU, we performed two daily injections during 7 days, and for an intracerebroventricular (icv) delivery, it was continuously infused into one lateral ventricle for a 7 days period as well. After ip administration, cells labelled with BrdU were seen in the subventricular zone of the striatal wall of the lateral ventricle, the hippocampus and the neurohemal circumventricular organs. Also, the habenula and large myelinated tracts, such as the fornix and the corpus callosum, showed many BrdU-positive nuclei. Labelled nuclei were scarce in the parenchymal regions of the rest of the brain. In contrast, a significant increase in the number of BrdU-positive nuclei was observed in the parenchyma of the periventricular zones after icv administration of the marker, thus showing a greater availability of the tracer when it was administered directly into the ventricular cerebrospinal fluid. We suggest that the availability of BrdU in the vicinity of proliferating cells may depend on the permeability of the brain vessels to nucleosides in each location. By using double immunocytochemistry we found that neurons, astrocytes, oligodendrocytes, tanycytes and microglia had incorporated the tracer, demonstrating their proliferation capacity.

Improved demonstration of immunohistochemical prognostic markers for survival in follicular lymphoma cells.

Follicular lymphoma (FL) is one of the most common forms of the low-grade non-Hodgkin's lymphoma in adults, with a characteristic translocation, t(14;18)(q32;q21) that deregulates the expression of the BCL2 gene. The clinical course of FL patients is variable, whereby a subset of patients survive for long periods even without relapses, whereas the majority have frequent relapses with shorter survival. We have analyzed a series of 186 FLs, studying the correlation between clinical outcome and the tumor cell expression of a set of immunohistochemical markers, using an automated procedure for tissue microarrays to reduce the subjectivity of scoring. The results identified several markers associated with differences in overall survival (OS) in univariate analyses, such as Cyclin E, Mdm2, CD10, p21, IgD, Bcl-xL, CD30, and E2F6. Cases with a higher level of expression of Cyclin E, Mdm2, p21, IgD, Bcl-xL, CD30, and E2F6 were associated with a significantly shorter OS. On the other hand, strong CD10 expression was linked to a significantly better outcome. A Cox model was then constructed, integrating the Follicular Lymphoma International Prognostic Index (FLIPI) score and a restricted selection of three immunohistochemical markers: Cyclin E, Mdm2, and CD10 expression. A potentially useful finding is that the integrated FLIPI plus immunohistochemical model can be used to identify a subset of 26 patients (almost 20% of the total series), with a survival probability of 100% at 5 years. This not only confirms that a group of FL cases may have a very good clinical course, but also indicates that this group can be identified using this integrated clinical and immunohistochemical approach.

A sensitive method to analyse the effect of putative regulatory ligands on the release of glycoprotein from primary cultures of dispersed bovine subcommissural organ cells.

The subcommissural organ (SCO) releases into the cerebrospinal fluid (CSF) large glycoproteins that polymerize forming the Reissner's fibre (RF), which is involved in CSF circulation and homeostasis. We obtained high purity primary cultures of bovine secretory SCO cells and measured glycoprotein release by a reliable and sensitive ELISA method. We also analysed the effect of regulatory ligands known to control the secretory activity of the SCO. Cells cultured for short time (4h) released a high amount of glycoproteins that decreased with time. In young cultures, ATP increased and serotonin inhibited secretion rate. By contrast the acetylcholine agonist carbachol and high potassium did not evoke any detectable change in SCO glycoprotein release. These results support not only the suitability of the methodological approach but an important role of both ATP and serotonin in regulating SCO secretory activity as well.

IGF-I stimulates neurogenesis in the hypothalamus of adult rats.

In the brain of adult rats neurogenesis persists in the subventricular zone of the lateral ventricles and in the dentate gyrus of the hippocampus. By contrast, low proliferative activity was observed in the hypothalamus. We report here that, after intracerebroventricular treatment with insulin-like growth factor I (IGF-I), cell proliferation significantly increased in both the periventricular and the parenchymal zones of the whole hypothalamus. Neurons, astrocytes, tanycytes, microglia and endothelial cells of the local vessels were stained with the proliferative marker 5-bromo-2'-deoxyuridine (BrdU) in response to IGF-I. Conversely, we never observed BrdU-positive ciliated cubic ependymal cells. Proliferation was intense in the subventricular area of a distinct zone of the mid third ventricle wall limited dorsally by ciliated cubic ependyma and ventrally by tanycytic ependyma. In this area, we saw a characteristic cluster of proliferating cells. This zone of the ventricular wall displayed three cell layers: ciliated ependyma, subependyma and underlying tanycytes. After IGF-I treatment, proliferating cells were seen in the subependyma and in the layer of tanycytes. In the subependyma, proliferating glial fibrillary acidic protein-positive astrocytes contacted the ventricle by an apical process bearing a single cilium and there were many labyrinthine extensions of the periventricular basement membranes. Both features are typical of neurogenic niches in other brain zones, suggesting that the central overlapping zone of the rat hypothalamic wall could be considered a neurogenic niche in response to IGF-I.

The enzyme NBAD-synthase plays diverse roles during the life cycle of Drosophila melanogaster.

This report shows the biochemical characterization and life cycle-dependent expression of Drosophila melanogaster N-beta-alanyldopamine synthase (NBAD-synthase or Ebony protein). This enzyme not only catalyzes the synthesis of NBAD, the main sclerotization and pigmentation precursor of insect brown cuticles, but also plays a role in brain neurotransmitter metabolism. In addition to the epidermis expression our immunodetection experiments show the novel localization of NBAD-synthase in different regions of the adult brain, in the foregut of pharate adult and, surprisingly, in the epidermis of the trachea during embryogenesis. These results demonstrate that NBAD-synthase is a versatile enzyme involved in different, previously unknown, time- and tissue-dependent processes.

The role of N-beta-alanyldopamine synthase in the innate immune response of two insects.

Insects trigger a multifaceted innate immune response to fight microbial infections. We show that in the yellow mealworm, Tenebrio molitor, septic injuries induce the synthesis of N-beta-alanyldopamine (NBAD), which is known as the main sclerotization precursor of insect brown cuticles. We demonstrate that NBAD synthase is induced in the epidermis of the mealworm and of the Medfly, Ceratitis capitata, by infection with Escherichia coli. Our results indicate that synthesis of NBAD seems to be a novel component of the overall innate immune response in insects.

CFTR gene analysis in Latin American CF patients: heterogeneous origin and distribution of mutations across the continent.

BACKGROUND: Cystic Fibrosis (CF) is the most prevalent Mendelian disorder in European populations. Despite the fact that many Latin American countries have a predominant population of European-descent, CF has remained an unknown entity until recently. Argentina and Brazil have detected the first patients around three decades ago, but in most countries this disease has remained poorly documented. Recently, other countries started publishing their results. METHODS: We present a compilation and statistical analysis of the data obtained in 10 countries (Argentina, Brazil, Chile, Colombia, Costa Rica, Cuba, Ecuador, Mexico, Uruguay and Venezuela), with a total of 4354 unrelated CF chromosomes studied. RESULTS: The results show a wide distribution of 89 different mutations, with a maximum coverage of 62.8% of CF chromosomes/alleles in the patient's sample. Most of these mutations are frequent in Spain, Italy, and Portugal, consistent with the origin of the European settlers. A few African mutations are also present in those countries which were part of the slave trade. New mutations were also found, possibly originating in America. CONCLUSION: The profile of mutations in the CFTR gene, which reflects the heterogeneity of its inhabitants, shows the complexity of the molecular diagnosis of CF mutations in most of the Latin American countries.

[Primary adenocarcinoma of the urinary bladder: our experience]. / Adenocarcinoma vesical primario: nuestra experiencia.

Adenocarcinoma of the bladder is an uncommon neoplasm. Depending on its origin it is classified in: primary, secondary and urachal. Generally it grows to the density of the wall, so its clinical appearence is delayed, with the subsequent delayed diagnosis and although an agressive treatment is performed, it frequently has a very bad prognosis. Since there are very few publications of this kind of neoplasm in the literature the lines of actuation in this pathology are not well established. We report the eleven cases of adenocarcinoma neoplasm of the bladder treated in our centre and review the literature.

Immunocytochemical characterisation of the wall of the bovine lateral ventricle.

The cytoarchitecture of the walls of the bovine lateral ventricles was investigated by the use of immunocytochemistry. We defined three types of walls. Type 1 lined regions of white matter and had ciliated cuboidal ependyma, a few subependymal cells and a narrow subjacent glial layer. Type 2 lined the striatum and possessed ependymal cells with conspicuous basal processes that extended through a wide subependyma containing many subependymal cells and a wide subjacent glial network. Type 3 lined the rostral horn and displayed ependymal cells with the longest basal processes and wider subependymal and glial layers. Ependymal cells of type 2 and 3 walls were labelled with antibodies against S-100beta protein, vimentin, GFAP, BLBP and nestin. Anti-betaIII-tubulin stained small cells in the subependyma and inside the GFAP- and vimentin-positive subjacent glial network. Anti-PCNA-positive nuclei were abundant in the subependymal and glial layers of type 2 and 3 walls. DiI in vitro tracing studies revealed small bipolar cells in the glial layer at a distance from the site of the label deposit. These results suggest that neurogenesis takes place in adult bovine subependyma mostly in the walls of the striatum and the rostral horn, and that young neuroblasts may migrate in a rostro-ventral direction through the glial network.

Neurogenesis in explants from the walls of the lateral ventricle of adult bovine brain: role of endogenous IGF-1 as a survival factor.

Previous studies have shown the existence of proliferating cells in explants from bovine (Bos Taurus) lateral ventricle walls that were maintained for several days in vitro in the absence of serum and growth factors. In this study we have characterized the nature of new cells and have assessed whether the insulin-like growth factor-1 (IGF-1) receptor regulates their survival and/or proliferation. The explants were composed of the ependymal layer and attached subependymal cells. Ependymal cells in culture were labelled with glial markers (S-100, vimentin, GFAP, BLBP, 3A7 and 3CB2) and did not incorporate bromodeoxiuridine when this molecule was added to the culture media. Most subependymal cells were immunoreactive for beta III-tubulin, a neuronal marker, and did incorporate bromodeoxiuridine. Subependymal neurons displayed immunoreactivity for IGF-1 and its receptor and expressed IGF-1 mRNA, indicating that IGF-1 is produced in the explants and may act on new neurons. Addition to the culture media of an IGF-1 receptor antagonist, the peptide JB1, did not affect the incorporation of bromodeoxiuridine to proliferating subependymal cells. However, JB1 significantly increased the number of TUNEL positive cells in the subependymal zone, suggesting that IGF-1 receptor is involved in the survival of subependymal neurons. In conclusion, these findings indicate that neurogenesis is maintained in explants from the lateral cerebral ventricle of adult bovine brains and that IGF-1 is locally produced in the explants and may regulate the survival of the proliferating neurons.

Analysis and quantification of the secretory products of the subcommissural organ by use of monoclonal antibodies.

Bovine Reissner's fiber (RF) glycoproteins were used as antigen for the production of polyclonal and monoclonal antibodies (Mabs). We also produced Mabs against intracellular secretory glycoproteins of the bovine subcommissural organ (SCO). These Mabs were used for immunodetection of secretory proteins in situ (structural and ultrastructural immunocytochemistry), in blots, and in solutions. Three different antigen-mediated ELISA were designed to evaluate the affinity of the Mabs, to study the nature of the epitopes, and for competition test among Mabs. Two double antibody sandwich ELISA were designed to detect and quantify soluble secretory materials in different samples, to study coexistence of epitopes, and to elucidate whether epitopes for Mabs are repeated or not in the RF-glycoproteins. Twenty-three Mabs recognizing the bovine RF- and SCO-glycoproteins in solutions (ELISA) as well as in tissue sections, were obtained. Nineteen of these Mabs also recognized the pig SCO, 11 the rabbit SCO, 6 the dog SCO, and 5 the rat SCO. None of the Mabs recognized the SCO of non-mammalian species. The different types of ELISA demonstrated that: (1) the epitopes reside in the proteinaceous moiety of the secretion, (2) they coexist in the same molecular forms and, with few exceptions, they did not overlap, (3) they were not repeated in the secretory molecule(s). Three Mabs were used for immunoblotting of RF; one of them revealed the same band pattern as that shown by an anti-RF serum. It is concluded that all Mabs raised in our laboratory are directed against non-repeated sequences of RF-glycoproteins that have not been conserved in vertebrate phylogeny.