Drivers of geophagy by red brocket deer (Mazama americana) at Amazonian interior forest mineral licks.

Mineral licks are key ecological components of the Amazon rainforest, providing critical dietary functions for herbivorous and frugivorous mammals and birds, which help maintain the structure and function of the forest itself through seed and nutrient dispersal. One of the most frequent visitors of interior forest mineral licks in the Amazon is the red brocket deer (Mazama americana), a large-bodied ruminant frugivore and seed predator. While several hypotheses for the drivers of geophagy exist, including mineral supplementation, toxin adsorption, and habitat selection, robust data on geophagy for the red brocket deer for large numbers of mineral licks is nonexistent. We used soil data from 83 mineral licks in conjunction with camera trap data from 52 of those mineral licks and a mixed-effects modeling approach to test the three proposed hypotheses of geophagy for the red brocket deer. We found that consumed soils at mineral licks had elevated concentrations of almost all major and minor biologically active minerals measured, including Ca, Na, Mg, K, Cu, Zn, and Mn. Model results suggest that all three hypotheses hold true to some extent for the red brocket deer, with the greatest support for the mineral supplementation hypothesis, in particular with respect to Mg, Ca, Na, Cu, and Zn. This study provides critical information on the feeding ecology of the red brocket deer in the wild, and the first robust analysis of geophagy of an Amazonian mammal involving a large sample size of interior forest mineral licks.

Physical, landscape, and chemical properties of Amazonian interior forest mineral licks.

Mineral licks, sites where animals go to consume soil, are key resources for herbivorous birds and mammals in the Amazon, providing supplemental dietary nutrients and toxin adsorption functions. However, because they are often difficult to find, the properties of mineral licks are poorly understood. Here, we undertake the largest survey of Amazonian mineral licks to date to determine the landscape, physical, and chemical properties of these critical sites. We used a generalized linear mixed-effects modeling framework to assess how soil samples from 83 mineral licks differ from nearby control soils in a series of physical and chemical characteristics, then used Jaccard's index of similarity and a principal component analysis (PCA) to determine how those samples differed among themselves. We found that mineral licks were generally located in specific ranges of landscape variables. Soils from mineral licks had elevated concentrations of almost all minerals measured. There was very little similarity between consumed and control samples, and within each sample type. We suggest that these mineral licks have the potential to provide multiple services to visiting species, demonstrating their ecological importance. The high levels of dissimilarity between samples indicate that a large sample of mineral licks is needed to draw conclusions in studies pertaining to geophagy. We emphasize that studying mammal and bird visitation at these sites could provide critical conservation and physiological information on cryptic and understudied species of Amazonian herbivores.

Polyunsaturated fatty acids and neurotransmission in Caenorhabditis elegans.

Changes in PUFA (polyunsaturated fatty acid) metabolism can cause mental retardation and cognitive impairment. However, it is still unclear why altered levels of PUFAs result in neuronal dysfunction. Recent studies on the nematode Caenorhabditis elegans suggest that PUFA depletion may cause cognitive impairment by compromising communication among neurons. Pharmacological and electrophysiological experiments showed that animals devoid of most PUFAs release abnormally low levels of neurotransmitters. In addition, ultrastructural analysis revealed that synapses in these mutants are severely depleted of synaptic vesicles. The conclusion of these studies is that PUFAs are required to maintain a normal pool of synaptic vesicles at pre-synaptic sites, thus ensuring efficient neurotransmission.

Use of kidneys from hepatitis C seropositive donors shortens waitlist time but does not alter one-yr outcome.

Utilization of hepatitis C seropositive kidney donors remains controversial. We examined the use of hepatitis C seropositive donors for renal transplantation. Data for creatinine, liver function tests, cold ischemia time, and graft and patient survival were analyzed from 20 hepatitis C seropositive recipients receiving cadaveric renal allografts from seropositive donors and were compared with 20 hepatitis C seropositive recipients receiving allografts from seronegative donors. Recipients receiving a kidney from a hepatitis C seropositive donor were on the waitlist for 9.9 +/- 1.8 months, compared with 17.8 +/- 3.3 months for those receiving a kidney from a seronegative donor (p < 0.05). There were no significant differences in graft or patient survival. Incidences of acute cellular rejection and acute tubular necrosis were similar. There were no significant differences in creatinine, alanine aminotransferase, alkaline phosphatase, or bilirubin values. While there was a significant difference in aspartate aminotransferase at 2 wk and 6 months, these differences were of questionable clinical importance. In conclusion, donor seropositivity for hepatitis C should not preclude renal transplantation into a hepatitis C seropositive recipient and utilization of these organs decreases waitlist time for hepatitis C seropositive recipients.

Requirements of multiple domains of SLI-1, a Caenorhabditis elegans homologue of c-Cbl, and an inhibitory tyrosine in LET-23 in regulating vulval differentiation.

SLI-1, a Caenorhabditis elegans homologue of the proto-oncogene product c-Cbl, is a negative regulator of LET-23-mediated vulval differentiation. Lack of SLI-1 activity can compensate for decreased function of the LET-23 epidermal growth factor receptor, the SEM-5 adaptor, but not the LET-60 RAS, suggesting that SLI-1 acts before RAS activation. SLI-1 and c-Cbl comprise an N-terminal region (termed SLI-1:N/Cbl-N, containing a four-helix bundle, an EF hand calcium-binding domain, and a divergent SH2 domain) followed by a RING finger domain and a proline-rich C-terminus. In a transgenic functional assay, the proline-rich C-terminal domain is not essential for sli-1(+) function. A protein lacking the SH2 and RING finger domains has no activity, but a chimeric protein with the SH2 and RING finger domains of SLI-1 replaced by the equivalent domains of c-Cbl has activity. The RING finger domain of c-Cbl has been shown recently to enhance ubiquitination of active RTKs by acting as an E3 ubiquitin-protein ligase. We find that the RING finger domain of SLI-1 is partially dispensable. Further, we identify an inhibitory tyrosine of LET-23 requiring sli-1(+) for its effects: removal of this tyrosine closely mimics the loss of sli-1 but not of another negative regulator, ark-1. Thus, we suggest that this inhibitory tyrosine mediates its effects through SLI-1, which in turn inhibits signaling upstream of LET-60 RAS in a manner not wholly dependent on the ubiquitin-ligase domain.

The amino-terminal domain of the golgi protein giantin interacts directly with the vesicle-tethering protein p115.

Giantin is thought to form a complex with p115 and Golgi matrix protein 130, which is involved in the reassembly of Golgi cisternae and stacks at the end of mitosis. The complex is involved in the tethering of coat protomer I vesicles to Golgi membranes and the initial stacking of reforming cisternae. Here we show that the NH(2)-terminal 15% of Giantin suffices to bind p115 in vitro and in vivo and to block cell-free Golgi reassembly. Because Giantin is a long, rod-like protein anchored to the membrane by its extreme COOH terminus, these results support the idea of a long, flexible tether linking vesicles and cisternae.

Positive and negative tissue-specific signaling by a nematode epidermal growth factor receptor.

The major determinants of receptor tissue tyrosine kinase (RTK) signaling specificity have been proposed to be Src homology 2 (SH2) binding sites, phosphotyrosine-containing oligopeptides in the cytoplasmic domain of the receptor. The Caenorhabditis elegans epidermal growth factor receptor homologue LET-23 has multiple functions during development and has eight potential SH2-binding sites in a region carboxyl terminal to its kinase domain. By analyzing transgenic nematodes for three distinct LET-23 functions, we show that six of eight potential sites function in vivo and that they are required for most, but not all, of LET-23 activity. A single site is necessary and sufficient to promote wild-type fertility. Three other sites activate the RAS pathway and are involved only in viability and vulval differentiation. A fifth site is promiscuous and can mediate all three LET-23 functions. An additional site mediates tissue-specific negative regulation. Putative SH2 binding sites are thus key effectors of both cell-specific and negative regulation in an intact organism. We suggest two distinct mechanisms for tissue-specific RTK-mediated signaling. A positive mechanism would promote RTK function through effectors present only in certain cell types. A negative mechanism would inhibit RTK function through tissue-specific negative regulators.

A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog.

The let-23 gene encodes a Caenorhabditis elegans homolog of the epidermal growth factor receptor (EGFR) necessary for vulval development. We have characterized a mutation of let-23 that activates the receptor and downstream signal transduction, leading to excess vulval differentiation. This mutation alters a conserved cysteine residue in the extracellular domain and is the first such point mutation in the EGFR subfamily of tyrosine kinases. Mutation of a different cysteine in the same subdomain causes a strong loss-of-function phenotype, suggesting that cysteines in this region are important for function and nonequivalent. Vulval precursor cells can generate either of two subsets of vulval cells (distinct fates) in response to sa62 activity. The fates produced depended on the copy number of the mutation, suggesting that quantitative differences in receptor activity influence the decision between these two fates.

LET-23-mediated signal transduction during Caenorhabditis elegans development.

We are using Caenorhabditis elegans vulval induction to study intercellular signaling and its regulation. Genes required for vulval induction include the LIN-3 transforming alpha-like growth factor, the LET-23 epidermal growth factor (EGF)-receptor-like transmembrane tyrosine kinase, the SEM-5 adaptor protein, LET-60 Ras, and the LIN-45 Raf serine/threonine kinase. Inactivation of this pathway results in a failure of vulval differentiation, the "vulvaless" phenotype. Activation of this pathway either by overexpression of LIN-3, a point mutation in the LET-23 extracellular domain, or hyperactivity of LET-60 Ras results in excessive vulval differentiation, the "multivulva" phenotype. In addition to searching for new genes that act positively in this signaling pathway, we have also characterized genes that negatively regulate this inductive signaling pathway. We find that such negative regulators are functionally redundant: mutation of only one of these negative regulators has no effect on vulval differentiation; however, if particular combinations of these genes are inactivated, excessive vulval differentiation occurs. The LIN-15 locus encodes two functionally redundant products, LIN-15A and LIN-15B, that formally act upstream of the LET-23 receptor to prevent its activity in the absence of inductive signal. The LIN-15A and B proteins are novel and unrelated to each other. The unc-101, sli-1, and rok-1 genes encode a distinct set of negative regulators of vulval differentiation. The unc-101 gene encodes an adaptin, proposed to be involved in intracellular protein trafficking. The sli-1 gene encodes a protein with similarity to c-cbl, a mammalian proto-oncogene not previously linked with a tyrosine kinase-Ras-mediated signaling pathway. LIN-3 and LET-23 are required for several aspects of C. elegans development--larval viability, P12 neuroectoblast specification, hermaphrodite vulval induction and fertility, and three inductions during male copulatory spicule development. Fertility and vulval differentiation appear to be mediated by distinct parts of the cytoplasmic tail of LET-23, and by distinct signal transduction pathways.

Long-term exposure to opioid antagonists up-regulates prodynorphin gene expression in rat brain.

We investigated the effect of long-term administration of opioid antagonists on the regulation of prodynorphin gene expression in rat brain. Intracerebroventricular (i.c.v.) injections for seven days of nor-binaltorphimine (nor-BNI), the highly selective kappa opioid antagonist, naloxone and its longer acting analog naltrexone, both relatively selective antagonists for the mu opioid receptor, markedly raised prodynorphin mRNA levels in rat hypothalamus, hippocampus and striatum. Peptides, namely immunoreactive-dynorphin A (ir-dyn A), were unaffected after chronic treatment with all antagonists, in the same tissues. These results, taken together with our previous observations, suggest that chronic opioid antagonists, acting on kappa and mu opioid receptors, clearly up-regulate prodynorphin gene expression in discrete rat brain regions, activating its biosynthesis. Moreover, our data support the hypothesis that the endogenous opioid system plays a role in the mechanisms underlying the development of opiate tolerance.

Mutations in the Caenorhabditis elegans let-23 EGFR-like gene define elements important for cell-type specificity and function.

The Caenorhabditis elegans let-23 gene is a genetically characterized member of the epidermal growth factor receptor (EGFR) tyrosine kinase family. Mutations in let-23 can produce five phenotypes in the nematode. Alleles of let-23 include null alleles, reduction-of-function alleles and alleles that disrupt function in some cell types and not others. We have sequenced some of these mutations to identify sequences and regions important for overall let-23 function and for let-23 function in specific cell types. Our data indicate that in vivo, the receptor's C-terminus can be partitioned into at least three domains that each contribute to receptor function in different cell types. In particular, we find distinct domains that mediate hermaphrodite fertility and vulval induction. Our data also demonstrate for the first time that a single, conserved residue in the ligand binding domain is critical for function in vivo and that mutations in the extracellular cysteines characteristic of the EGFR family can lead to a partial or a complete reduction of receptor function.

Alterations in vasoactive intestinal polypeptide-related peptides after pentylenetetrazole-induced seizures in rat brain.

The possible involvement of vasoactive intestinal polypeptide-related peptides in pentylenetetrazol (PTZ)-induced seizures in rats was investigated. The chemoconvulsant PTZ was administered (45 mg/kg i.p.) either acutely or chronically for three days. The detailed time course of changes in VIP-(1-28) and VIP-(22-28) was examined in several rat brain areas 5 and 20 min and 24 h after acute treatment and after three days chronic treatment. Ir-VIP levels dramatically decreased in all areas 5 min after PTZ injection, remained low after 20 min and progressively increased back to control values after 24 h and after three days of repeated treatment (except for the cortex). Chromatographic analysis of extracts prepared from PTZ-treated rats revealed a concomitant decrease in VIP-(1-28) and increase in VIP-(22-28). Thus VIP-(22-28) might be a product of the internal cleavage of the precursor VIP-(1-28) after its neuronal release; alternatively, VIP-(22-28) might be generated by post-transcriptional processing of VIP-(1-28), and thus be an 'independent' neuropeptide. The results suggest that VIP-(1-28)/VIP-(22-28)-containing neurons might be involved in PTZ-induced seizures in rat brain, and that VIP-(22-28) might play a role in these experimental seizures.

Substance P is diminished and vasoactive intestinal peptide is augmented in psoriatic lesions and these peptides exert disparate effects on the proliferation of cultured human keratinocytes.

An involvement of neurogenic components in the pathogenesis of psoriatic lesions has been suggested and neuropeptides are thought to play a modulatory role in cutaneous inflammation. In this study, we evaluated the immunoreactivity of the neuropeptides vasoactive intestinal polypeptide (VIP) and substance P (SP) in the skin of patients with chronic plaque psoriasis, by immunohistochemistry and radioimmunoassay. No differences were observed, by immunohistochemistry, in the expression and localization of VIP and SP between psoriatic and normal skin. Using the radioimmunologic technique on whole skin homogenates, VIP levels were significantly increased in psoriatic lesions as compared to normal skin. By contrast, SP levels were significantly lower in lesional and non-lesional psoriatic skin than in normal skin. In addition, we examined the effect of VIP and SP on the proliferation of cultured normal human keratinocytes. VIP (1-28) (1 nM-1 microM) as well as VIP fragments (10-28) (1 nM-1 microM) and (22-28) (1 nM-1 microM) stimulated the proliferation of keratinocytes in a dose-dependent manner, whereas the VIP fragment (1-12) (1 nM-1 microM) was ineffective. The VIP antagonist (N-Ac-Tyr1, D-Phe2)-GRF (1-29)-NH2 (0.1 microM) significantly inhibited the VIP effect on keratinocytes. On the other hand, SP (0.1 microM) not only failed to stimulate keratinocyte growth, but also blocked the VIP-induced stimulation of these cells. The imbalance of cutaneous VIP and SP and their disparate effects on the proliferation of normal human keratinocytes in culture would suggest that these peptides are involved in the pathogenesis of psoriasis and may exert different modulatory activities in the mechanisms underlying the psoriatic lesion.

Chronic opiate agonists down-regulate prodynorphin gene expression in rat brain.

The effects of long-term administration of opioid agonists on the regulation of prodynorphin gene expression in rat brain were investigated. Chronic intracerebroventricular treatment with the synthetic opioid agonist acting on the kappa receptor, U-50,488H, and the classic mu agonist morphine markedly decreased prodynorphin mRNA levels in hypothalamus, hippocampus and striatum of tolerant rats. Levels of ir-Dynorphin A remained unchanged except in two cases. Chronic exposure to opiates thus appears to induce modifications of the endogenous opioid system, as regards gene expression regulation.

Skin levels of vasoactive intestinal polypeptide in atopic dermatitis.

Atopic dermatitis (AD) can be exacerbated by various factors, including emotional stress, scratching and sweating. The aim of the present study was to evaluate the hypothesis that the inflammatory reaction in AD is also neurogenic. For this purpose, the levels of vasoactive intestinal polypeptide were measured radioimmunologically in whole-tissue homogenates of lesional skin of 13 patients with atopic dermatitis. Radioimmunoassay was performed using an antiserum, AH78, recognizing the carboxy-terminal fragment vasoactive intestinal polypeptide (22-28). Vasoactive intestinal polypeptide immunoreactivity was detected in relatively low amounts in control skin (0.428 +/- 0.08 pmol/g tissue), whereas a marked increase in the peptide was observed in lesional skin of patients with atopic dermatitis (5.62 +/- 1.25 pmol/g tissue). These results seem to suggest that vasoactive intestinal polypeptide could have a pathogenetic relevance in skin lesions of atopic dermatitis.

Distribution and characterization of VIP-related peptides in the rat spinal cord.

The possible existence in the rat spinal cord of a peptide related to VIP, VIP(22-28), has been evaluated. VIP contains paired basic aminoacid residues at which posttranslational cleavage of these peptides might occur. The lumbo-sacral region of rat spinal cord had the most VIP(22-28)-like immunoreactivity (ir-VIP(22-28]. Chromatographic analysis of spinal extracts showed that ir-VIP(22-28) consisted of two major peaks, one eluting as authentic VIP(1-28) and the other as VIP(22-28). HPLC confirmed these results, revealing the presence of intact VIP(1-28) and two or more less hydrophobic peptides, one of which corresponded to authentic VIP(22-28). The other two components found have not yet been identified. Further studies are necessary to provide information on the biological significance of VIP(22-28).

Morphine affects prodynorphin gene expression in some areas of rat brain.

The effect of a chronic morphine treatment on prodynorphin gene expression has been studied. Morphine has been intraperitoneally administered twice daily for seven days into rats. RNAs from hippocampus and striatum have been extracted and analyzed with probes complementary to the prodynorphin mRNA. A marked reduction in mRNA levels was detected in hippocampus, following chronic morphine treatment; in striatum results showed either a slight decrease or no substantial changes in mRNA levels. These results indicate that the chronic morphine is able to induce modifications in the homeostasis of the endogenous opioid gene expression, at least in some areas of the rat brain. In addition, our data support the hypothesis that a tolerance to opiates might involve alterations in functions of brain pathways which utilize the opioid peptidergic system.