Neoplasia in Three Aye-Ayes (Daubentonia madagascariensis).

Tumours diagnosed in three aged captive aye-ayes (Daubentonia madagascariensis), held in two different institutions, are described. A cerebral glioblastoma was diagnosed based on histological and immunohistochemical findings in one of the animals following initial presentation with bilateral mydriasis, absent pupillary reflex, head tilt and ataxia. A second animal was humanely destroyed due to impaired locomotion associated with spondylosis and a post-mortem diagnosis of cholangiocarcinoma was made based on histology with further confirmation with immunohistochemical labelling for cytokeratin 7. A third aye-aye suffering from dental disease was diagnosed with an oral squamous cell carcinoma following an excisional biopsy from a non-healing wound in the lip. Due to progression of the neoplasia the animal was humanely destroyed and post-mortem examination revealed the presence on an additional unilateral phaeochromocytoma.

Spontaneous hepatocellular carcinoma in captive prosimians.

Spontaneous hepatocellular carcinoma has been reported as a relatively common neoplasm in prosimians; however, the cause is unknown. To investigate possible pathogenic mechanisms, the authors performed a review of all adult animals from a captive prosimian population that had postmortem examinations over the past 10 years. They performed a detailed histologic evaluation of all suspected proliferative liver lesions and diagnosed hepatocellular carcinoma in 14 of 145 lemurs (9.7%). Affected animals ranged between the ages of 6 and 40 years old. The tumors had an unusually aggressive growth pattern for animal species; metastasis to the lungs or mediastinum was evident in 7 of 14 animals. Thirty-one animals-9 with hepatocellular carcinomas and 22 age-matched controls without hepatic neoplasia-were tested to evaluate the relationship between hepatic iron stores (as well as other trace metals) and the presence of hepatocellular carcinoma. There was no difference between the hepatic iron, copper, or molybdenum in lemurs with hepatocellular carcinoma and those without, suggesting that iron is not a key element in the pathogenesis of liver tumor formation. Analysis of 22 serum samples from animals with and without liver tumors indicated no evidence of active infection with a hepadnavirus, the virus family that includes hepatitis B virus. Hepatitis C virus and aflatoxin B1 were considered as potential causes and ruled out owing to lack of associated histopathologic lesions. In conclusion, hepatocellular neoplasia is relatively common in captive prosimians, although previously suspected etiologies seem unlikely.

Neoplasia in prosimians: case series from a captive prosimian population and literature review.

Neoplastic diseases in prosimians have been sporadically reported in the literature. To provide a comprehensive review of prosimian neoplasia, a retrospective evaluation of neoplasia in a large captive prosimian colony and an extensive literature review were performed. Primates that belong to the Order Primata, Suborder Prosimii with histologic evidence of neoplasia were included. One hundred twenty-three cases of spontaneous neoplasia were identified in 101 prosimians from the Duke Lemur Center, and 124 cases were reported in 116 prosimians in the literature. Overall, this review compiled a total of 247 neoplasms in 217 prosimians. Of the 217 affected animals, 88 of 217 were males (41%), 100 of 217 were females (46%), and sex was not reported in 29 of 217 (13%). Ages ranged from 2 days to 36 years. Prosimian families represented were Lemuridae (80/217 [37%]), Cheirogaleidae (61/217 [28%]), Galagidae (44/217 [20%]), Lorisidae (28/217 [13%]), and Indriidae (4/217 [2%]). The most commonly affected species were the gray mouse lemur (Microcebus murinus) (28/217 [13%]), thick-tailed greater bush baby (Otolemur crassicaudatus) (23/217 [11%]), and black lemur (Eulemur macaco) (19/217 [9%]). Organ systems affected, in order of descending occurrence, were digestive (75/247 [30%]), reproductive (40/247 [16%]), hematopoietic (34/247 [14%]), integumentary (28/247 [11%]), endocrine (26/247 [11%]), and urinary (17/247 [7%]). The respiratory, nervous, musculoskeletal, and cardiovascular systems were infrequently affected. The most common neoplasms were hepatocellular (32/247 [13%]), lymphoma and/or leukemia (29/247 [12%]), biliary (15/247 [6%]), and mammary neoplasms (12/247 [5%]). This article should serve as a valuable reference for the types and relative frequencies of neoplasms that occur in prosimian species.

Characterizing gastrointestinal transit time in four lemur species using barium-impregnated polyethylene spheres (BIPS).

Differences in dietary profiles and gastrointestinal (GI) morphologies observed across lemur species suggest that there may be variation in patterns of digesta flow through the GI tract related to the method of digesta processing. Using radio-opaque barium-impregnated polyethylene spheres (BIPS), we characterized such patterns in four lemur species: Varecia variegata (VV), Eulemur fulvus (EF), Propithecus verreauxi (PV), and Hapalemur griseus (HG) (n = 2 per species). After an initial radiograph was taken under light sedation, the animals were fed the BIPS together with a small meal. A combination of 30 small (1.5 mm) and 10 large (5 mm) BIPS was administered. Radiographs were then taken on a species-dependent basis up to 48 hr post-dosage. For small BIPS, the gastric transit time (GTT; time of first exit of BIPS from stomach) was 0.25-2 hr for VV, EF, and HG, and approximately 10 hr for PV. The oro-rectal transit time (ORTT; time of first appearance in the rectum) was < 2 hr for VV and EF, and 24.0 hr for PV and HG. The intestinal transit time (ITT, measured as ORTT - GTT) was < 1.5 hr for VV and EF, and approximately 14 hr and 22 hr for PV and HG, respectively. These data suggest that the GTT of digesta as measured with BIPS was rapid for VV, EF, and HG. For VV and EF, the ORTT and ITT were also rapid, while for HG they were much slower. PV was characterized by delayed GTT, and a more rapid ITT compared to HG. Thus, patterns of flow for PV and HG, despite similar ORTT, differed in that HG emptied BIPS more rapidly and ITT was slower. The flow of BIPS did not differ for VV and EF. These data reveal new information in addition to the total tract transit time, and complement existing knowledge regarding anatomy and diet.

Use of total dietary fiber across four lemur species (Propithecus verreauxi coquereli, Hapalemur griseus griseus, Varecia variegata, and Eulemur fulvus): does fiber type affect digestive efficiency?

In vivo digestibility and transit of two experimental diets were compared across four lemur species for which gastrointestinal morphology and preliminary data on physiology differ:Varecia variegata (VV), Eulemur fulvus (EF), Propithecus verreauxi (PV), and Hapalemur griseus (HG). Since free-ranging groups consume varied amounts of slowly fermentable insoluble fiber (IF) and rapidly fermentable soluble fiber (SF), differences in digestibility may be related to variation in the fiber types consumed. To investigate this, two diets were designed to provide 28% of dry matter (DM) as total dietary fiber (TDF). The ratio of IF/SF (g/g) differed across the diets (12.15:1 for the IF diet, and 3.76:1 for the IF/SF diet). The DM digestibility (DMD) of both diets differed across species: DMD was lower for EF and VV (approximately 56-58%), and higher for PV (72%) and HG (76%). The fiber digestibility results were as follows: TDF digestibility was similar for VV and EF (23% and 28%), higher for PV (56%), and highest for HG (66%). IF digestibility was lower for VV and EF (20% and 28%), and higher for PV and HG (53% and 62%). The transit times (TTs) of the two markers Cr and Co were similar (approximately 3.5 hr for VV and EF, 25 hr for PV, and 30 hr for HG). The mean retention times (MRTs) showed the same trend. The results from these captive groups suggest there are large differences in digestive efficiency that are likely related to the varied fiber composition of the free-ranging diet, and the amount of time the digesta are retained in the gut.

The GreenScreen genotoxicity assay: a screening validation programme.

A yeast (Saccharomyces cerevisiae) DNA repair reporter assay termed the GreenScreen assay (GSA) is described. This is a novel, cost-effective genotoxicity screen, developed to provide a pre-regulatory screening assay for use by the pharmaceutical industry and in other applications where significant numbers of compounds need to be tested. It provides a higher throughput and a lower compound consumption than existing eukaryotic genotoxicity assays and is sensitive to a broad spectrum of mutagens and, importantly, clastogens. We describe a simple, robust assay protocol and a validation study. The end-point of the test reflects the typically eukaryotic chromosomes and DNA metabolizing enzymes of yeast. The capacity for metabolic activation (MA) in yeast is limited compared with the mammalian liver or its extracts, but the assay does detect a subset of compounds that would require MA in existing genotoxicity tests. The GSA detects a different spectrum of compounds to bacterial genotoxicity assays and thus, together with an in silico structure-activity relationship (SAR) screen, and possibly a high throughput bacterial screen, would provide an effective preview of the regulatory battery of genotoxicity tests.

Fecal inoculum can be used to determine the rate and extent of in vitro fermentation of dietary fiber sources across three lemur species that differ in dietary profile: Varecia variegata, Eulemur fulvus and Hapalemur griseus.

To estimate fermentative capacity among lemur species, four fiber substrates were tested across three species, Eulemur fulvus, Hapalemur griseus and Varecia variegata. The substrates, cellulose, beet pulp, citrus pulp and citrus pectin, ranged in composition from completely insoluble fiber (IF) to completely soluble fiber (SF), respectively. The lemurs consumed a nutritionally complete biscuit formulated for primates [85 g/100 g diet dry matter (DM)] and locally available produce (15 g/100 g diet DM). Feces were then collected and used to inoculate fermentation tubes prefilled with fiber substrates and an anaerobic growth medium. Dry matter disappearance (DMD), and acetate, propionate, butyrate, and total short-chain fatty acid (SCFA) production were measured in tubes subjected to 6, 12, 24 or 48 h of fermentation. Results were fitted to a logistic growth model. The maximal production (MP) time at which production or disappearance is at one-half maximum (t(50)) and the fermentation rate at 3 h were calculated. The maximal disappearance of DM differed among substrates (citrus pectin > citrus pulp > beet pulp; P < 0.0001) and species (E. fulvus > H. griseus > V. variegata; P < 0.001). V. variegata reached t(50) for acetate and total SCFA production faster than H. griseus or E. fulvus (P < 0.02). Three-hour production rates of acetate and total SCFA were also greater for V. variegata for citrus pulp and citrus pectin (P < 0.01). Few species differences were observed for beet pulp. Results provide evidence for differences in fermentative capacity and suggest that fiber solubility and fermentability should be considered when assessing the nutritional management of lemurs.

Description of the gastrointestinal tract of five lemur species: Propithecus tattersalli, Propithecus verreauxi coquereli, Varecia variegata, Hapalemur griseus, and Lemur catta.

The objective of this project was to better define the similarities and differences in gastrointestinal morphology present in lemur species. Measurements of the gastrointestinal tract of lemurs were obtained at necropsy from the captive population at Duke University Primate Center. Measurements of body length and weight, as well as gastrointestinal length, were recorded from five prosimian species: Propithecus tattersalli, Propithecus verreauxi, Varecia variegata, Hapalemur griseus, and Lemur catta. Photographs and measurements were used to obtain illustrations. Preliminary results suggest differences in gastrointestinal morphology among lemur species that coincide with differences in diet. Distinct sacculations in either the cecum or the colon were present for H. griseus, L. catta, P. verreauxi, and P. tattersalli, but not for V. variegata. The Propithecus specimens possessed a much greater ratio of gastrointestinal length to body length than the other three species. A short, blunt cecum and a shortened and sacculated colon were unique characteristics of the H. griseus specimens. These differences correlate well with a dietary shift from consumption of large amounts of structural plant cell wall (Propithecus sp.) to consumption of variable or moderate amounts (H. griseus, L. catta, and V. variegata). They also suggest that captive groups would benefit from further diet refinement in captivity.

Calcium-dependent alterations in dendritic architecture of hippocampal pyramidal neurons.

Dendritic arbor formation and the underlying mechanisms are crucial for the functional connectivity and plasticity of neurons. We used a focal electric field to locally raise calcium levels in individual dendritic shafts of isolated hippocampal pyramidal neurons, in order to develop an accessible system for studying dendritic branch formation, and to test the role of calcium as an intrinsic signal that may participate in arborization. Filopodia were induced in a manner temporally and spatially related to induced calcium rises. Certain filopodia also thickened and were transformed into dendritic branches. These results suggest that calcium-mediated signaling can induce branching in dendrites, and describe an accessible system for studying the intracellular machinery that drives dendritic arborization.

Mutagenicity of ethyl carbamate to lacZ- transgenic mice.

The mutagenicity of the rodent carcinogen ethyl carbamate (EC, urethane) has been assessed using the lacZ- transgenic mouse mutation assay (Muta Mouse). In two separate experiments a single i.p. dose of 900 mg/kg urethane followed by a 14-16 day expression period yielded statistically significant (approximately 2-fold) increases in mutation frequency (MF) in the lung and liver of the mice. In the second experiment MF in the spleen and bone marrow was also assessed and statistically significant increases in MF were observed in each case. Concomitantly conducted bone marrow micronucleus assays gave 15.8- and 8-fold increases respectively in the incidence of micronucleated polychromatic erythrocytes in the two studies.

Laminin directs growth cone navigation via two temporally and functionally distinct calcium signals.

During development, growth cones navigate to their targets via numerous interactions with molecular guidance cues, yet the mechanisms of how growth cones translate guidance information into navigational decisions are poorly understood. We have examined the role of intracellular Ca2+ in laminin (LN)-mediated growth cone navigation in vitro, using chick dorsal root ganglion neurons. Subsequent to contacting LN-coated beads with filopodia, growth cones displayed a series of stereotypic changes in behavior, including turning toward LN-coated beads and a phase of increased rates of outgrowth after a pause at LN-coated beads. A pharmacological approach indicated that LN-mediated growth cone turning required an influx of extracellular Ca2+, likely in filopodia with LN contact, and activation of calmodulin (CaM). Surprisingly, fluorescent Ca2+ imaging revealed no LN-induced rise in intracellular Ca2+ in filopodia attached to their parent growth cone. However, isolation of filopodia by laser-assisted transection unmasked a rapid, LN-specific rise in intracellular Ca2+ (+73 +/- 11 nM). Additionally, a second, sustained rise in intracellular Ca2+ (+62 +/- 8 nM) occurred in growth cones, with a distinct delay 28 +/- 3 min after growth cone filopodia contacted LN-coated beads. This delayed, sustained Ca2+ signal paralleled the phase of increased rates of outgrowth, and both events were sensitive to the inhibition of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) with 2 microM KN-62. We propose that LN-mediated growth cone guidance can be attributed, in part, to two temporally and functionally distinct Ca2+ signals linked by a signaling cascade composed of CaM and CaM-kinase II.

Afferent innervation influences the development of dendritic branches and spines via both activity-dependent and non-activity-dependent mechanisms.

The present investigation uses an in vitro co-culture system to study the role of afferent innervation in early development and differentiation of hippocampal neurons. Our experiments indicate that the formation of two key morphological features, dendritic branches and dendritic spines, is induced by afferent innervation. Hippocampal neurons develop multiple dendritic branches and spines only when extensively innervated by living axonal afferents. No morphological changes occurred when hippocampal neurons were plated on other cell surfaces such as fixed axons or astrocytes. Furthermore, afferents exerted their effect locally on individual dendrites that they contacted. When one portion of the dendritic arbor of a neuron was contacted by afferents and the other portion was not, morphological effects were restricted to the innervated dendrites. Innervation of some of the dendrites on a neuron did not produce global effects throughout the neuron. Afferent-induced dendritic branching is independent of activity, since branch induction was unaffected by chronic application of TTX or glutamate receptor blockers. In contrast, the formation of dendritic spines is influenced by activity. The number of developing spines was reduced when TTX or a cocktail of three glutamate receptor blockers was applied. Blockade of individual AMPA, NMDA, or metabotropic glutamate receptors did not affect the number of spines. These results, taken together, demonstrate that afferents can have a prominent influence on the development of postsynaptic target cells via both activity-dependent and non-activity-dependent mechanisms, indicating the presence of multiple signals. Accordingly, this suggests an important interplay between pre- and postsynaptic elements early in development.

The activity of ENU, iPMS and MMS in male mouse germ cells using the Muta Mouse positive selection transgenic mutation assay.

The alkylating agents ethyl nitrosourea (ENU), isopropyl methanesulphonate (iPMS) and methyl methanesulphonate (MMS) are potent male rodent germ cell mutagens. The mutagenic activity of these compounds in male mouse germ cells has been evaluated using the Muta Mouse positive selection transgenic mutation assay. Both ENU (150 mg/kg) and iPMS (100 mg/kg) gave increased mutant frequencies in testicular DNA recovered 50 days after dosing. During the course of the mutation assays on iPMS its activity as a dominant lethal mutagen was confirmed by mating the treated animals with virgin (non-transgenic) females on day 10 post-dosing. Ova analysis on animals exposed to iPMS confirmed earlier reports that the dose level used caused sterility in mice 40 days after dosing. This sterility was shown to be due to aspermia in the treated mice at day 50 post-dosing. These collected findings indicate that at day 50 post-dosing with iPMS, mutations in testicular DNA can be observed in sterile animals. MMS (100 mg/kg) was not mutagenic to either testicular DNA or epididymal sperm DNA, 10 or 50 days, respectively, after dosing.

Functional compartmentalization of the neuronal growth cone: determining calcium's place in signaling cascades.

The growth cone is generally regarded as the basic unit of neuronal organization concerned with development of connections within the nervous system. The discussion below illustrates that the growth cone itself can be subdivided into distinct units of organization. It is this functional compartmentalization which enables the growth cone to read the molecular terrain it traverses and to convert this information into precise motor events. Our discussion will focus on the flow of information from the environment to the growth cone. In particular, we will follow signaling events from their remote processing within filopodia to the biological equivalent of a central processing unit in the core of the growth cone.

Developmental regulation of plasticity along neurite shafts.

Although it is becoming increasingly clear that structural dynamics on neurite shafts play important roles in establishing neuronal architecture, the underlying mechanisms are unknown. The present study investigates local induction of filopodia along the shafts of neurites, a process that, by analogy to the growth cone, can represent the first stage in the generation of a new neuronal process. We show that filopodia can be induced reliably along the neurite shaft in response to a localized electric field stimulus that evokes large local intracellular calcium increases. Neither induction of filopodia nor a local rise in intracellular calcium occurred in calcium-free medium. Although calcium induction of neurite filopodia is highly reliable, forming in response to more than 90% of attempts, it is developmental state-dependent, since neurite filopodia could not be induced in neurons previously defined as "stable state." We have found two distinct changes in stable state neurons that can decrease the ability to induce new neurites. The first is a reduced calcium response: Field stimulation produced large local rises (280 nM) in intracellular calcium in growing neurons, whereas the identical stimulus produced smaller changes (148 nM) in stable state neurons. Second, stable state neurons change so that even when the stimulus intensity was increased to elicit a calcium response that would have been sufficient to induce filopodia in growing neurites, neurite filopodia were still not induced. Thus, intracellular calcium plays a key role in structural changes along the shafts of neurites. Furthermore, developmental changes in both calcium homeostatic components and in calcium responsiveness (i.e., the sensitivity of cellular components that modulate neurite morphology) underlie shifts from plasticity to stability of neuronal architecture in this system.

Involvement of nitric oxide in the elimination of a transient retinotectal projection in development.

The adult pattern of axonal connections from the eye to the brain arises during development through the refinement of a roughly ordered set of connections. In the chick visual system, refinement normally results in the loss of the ipsilateral retinotectal connections. Inhibition of nitric oxide synthesis reduced the loss of these transient connections. Because nitric oxide is expressed by tectal cells with which retinal axons connect and because reduction of nitric oxide synthesis by tectal cells resulted in a change in the connections of retinal axons, nitric oxide probably serves as a messenger from tectal cells back to retinal axons during development.

Correlation of nitric oxide synthase expression with changing patterns of axonal projections in the developing visual system.

A precise pattern of connections between the retina and central visual nuclei in the brain is established during development. Activity-dependent presynaptic mechanisms and NMDA receptor-mediated postsynaptic mechanisms are thought to play important roles in this developmental process. A model proposed for production of the newly described neurotransmitter, nitric oxide, involves presynaptic activity and activation of postsynaptic NMDA receptors. If present in the developing visual system, nitric oxide could represent a form of retrograde communication from postsynaptic to presynaptic cells that mediates the formation of the proper pattern of connections. This study used the diaphorase histochemical technique to detect the presence of nitric oxide synthase (NOS), the enzyme responsible for the production of nitric oxide, in the developing chick optic tectum. Results from this study showed that NOS is present in the developing tectum and that its expression coincides temporally with innervation by retinal axons. NOS expression reaches a peak at the time that refinement of the initial pattern of connections is occurring. WGA/HRP labeling of retinal axons confirmed that processes of NOS-positive cells in the tectum extend well into the area of the ingrowing retinal axons. Histochemical results from eyeless chick embryos indicate that NOS expression is dependent on the presence of retinal axons, which suggests that retinal axons synapse on cells that express nitric oxide. Northern blot analysis using a cDNA probe to NOS from rat brain verified the histochemical results. These results are consistent with nitric oxide having a role in development of the proper pattern of connections in the chick retinotectal system.

Subtractive immunization techniques for the production of monoclonal antibodies to rare antigens.

Traditional techniques for the production of monoclonal antibodies usually result in generation of monoclonal antibodies to immunodominant molecules. To enhance the production of monoclonal antibodies to rare or less immunodominant antigens, subtractive immunization techniques have been employed. This study compared the ability of several subtractive immunization techniques to suppress the immune system to a given antigen. Neonatal tolerization, chemical immunosuppression with cyclophosphamide, a combination of the two and various permutations of these techniques were compared. The results from this study indicated that chemical immunosuppression with cyclophosphamide was the most effective subtractive immunization technique and that the cyclophosphamide regime employed was a critical determinant in the success of chemical immunosuppression.

Elimination of the transient ipsilateral retinotectal projection is not solely achieved by cell death in the developing chick.

During development of the projection from the retina to the brain in the chick, a transient ipsilateral retinotectal projection forms and disappears. This disappearance is coincident with a wave of ganglion cell death in the retina. The contribution of cell death to the disappearance of this projection, as opposed to another mechanism such as axon retraction, was examined. Retinal ganglion cells with a projection to the ipsilateral tectum were retrogradely labeled by injection of long-lasting fluorescent dyes into the tectum prior to the onset of ganglion cell death. Large injections of fast blue labeled approximately 1800 ganglion cells in the ipsilateral retina before the period of cell death began. If the injected embryos were allowed to survive past the peak period of ganglion cell death, the average number of labeled ganglion cells in the ipsilateral retina was reduced by somewhat more than half. It is possible that the remaining labeled ganglion cells projected only to nontectal visual nuclei and were labeled by fast blue that had diffused out of the tectum. This was tested by repeating the study using very localized injections of 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate or fluorescent microspheres into the tectum. These small injections confirmed that cells with transient projections to the ipsilateral tectum survived past the elimination of this projection. Thus, ipsilaterally projecting ganglion cells have, at most, a slightly greater propensity for death than the average ganglion cell, and elimination of the transient ipsilateral retinotectal projection in chick can be explained only, in part, by the mechanism of cell death.(ABSTRACT TRUNCATED AT 250 WORDS)