Clinical features, intestinal histopathology, and outcome in protein-losing enteropathy in Yorkshire Terrier dogs.

BACKGROUND: A poorly understood protein-losing enteropathy (PLE) disorder has been reported in Yorkshire Terrier dogs. OBJECTIVES: To describe clinical features, intestinal histopathology, and outcome in Yorkshire Terrier dogs with PLE, and to identify variables predictive of outcome. ANIMALS: Thirty client-owned Yorkshire Terrier dogs with PLE. METHODS: Retrospective study. Records of dogs with a diagnosis of PLE were reviewed. Intestinal histopathology was interpreted using the World Small Animal Veterinary Association gastrointestinal histopathology classification system. Discriminate analysis techniques were used to identify variables predictive of outcome. RESULTS: Females outnumbered males (20/30). Median age was 7 years (range 1-12). Common clinical signs were diarrhea (20/30), vomiting (11), ascites and abdominal distension (11), and respiratory difficulty (8). Histopathologic abnormalities included villous lymphatic dilatation, crypt lesions, villous stunting, and variable increases in cellularity of the lamina propria. All dogs were treated with glucocorticoids. Of 23 dogs with long-term follow-up, 9 had complete, and 3 had partial, resolution of signs, and 11 failed to respond to treatment. Median survival of responders was 44 months and of nonresponders was 12 months, with 4 dogs experiencing peracute death. Vomiting, monocytosis, severity of hypoalbuminemia, low blood urea nitrogen concentration, and villous blunting were predictive of survival <4 months. CONCLUSIONS: In addition to classic GI signs, Yorkshire Terriers with PLE often show clinical signs associated with hypoalbuminemia and low oncotic pressure. Lymphatic dilatation, crypt lesions, and villous stunting are consistent histopathologic findings. Clinical outcomes are variable, but many dogs experience remission of clinical signs and prolonged survival.

Risk factors for coughing in dogs with naturally acquired myxomatous mitral valve disease.

BACKGROUND: Cough often is reported as the primary clinical sign of congestive heart failure (CHF) in dogs with chronic degenerative myxomatous mitral valve disease (MMVD). Concurrent airway disease and compression of the left mainstem bronchus by a large left atrium also have been proposed as potential causes of coughing in these patients. OBJECTIVES: To investigate the association between the presence of coughing and different potential causes of cough, including CHF, abnormal radiographic airway pattern, and cardiomegaly in dogs affected by naturally acquired MMVD. ANIMALS: Two hundred six client-owned dogs. METHODS: Retrospective analysis performed on medical records of dogs affected by MMVD that underwent full cardiac evaluation, including echocardiographic examination and thoracic radiography. RESULTS: Univariate analyses showed that CHF is not a predictor of coughing (OR = 1.369; 0.723, 2.594), whereas abnormal radiographic airway pattern (OR = 3.650; 2.051, 6.496) and increased left atrial size observed radiographically (OR = 3.637; 1.904, 6.950) or echocardiographically (OR = 2.553; 1.436, 4.539) were significantly associated with coughing in dogs with MMVD. The same risk factors were significant in multivariate analyses. CONCLUSIONS AND CLINICAL IMPORTANCE: This study indicates that CHF is not significantly associated with coughing in dogs with MMVD. Instead, abnormal radiographic airway pattern and left atrial enlargement are associated with coughing in these patients. This important finding should be taken into account when considering diagnosis and clinical management of CHF in these dogs.

Modulation of aberrant CDK5 signaling rescues impaired neurogenesis in models of Alzheimer's disease.

Recent studies show that in Alzheimer's disease (AD), alterations in neurogenesis contribute to the neurodegenerative process. Neurodegeneration in AD has been associated with aberrant signaling through the cyclin-dependent kinase-5 (CDK5) pathway via its activators p35/p25; however, the role of CDK5 in the mechanisms of defective adult neurogenesis in AD is unknown. First, to study AD-like abnormal activation of CDK5 signaling in an in vitro model of neurogenesis, neuronal progenitor cells (NPCs) were infected with a viral vector expressing p35, and exposed to amyloid-ß protein (Aß(1-42)). These conditions resulted in impaired maturation and neurite outgrowth in vitro, and these effects were reversed by pharmacological or genetic inhibition of CDK5. Similarly, neurogenesis was impaired in a transgenic mouse model of AD that expresses high levels of amyloid precursor protein (APP), and this effect was reversed in transgenic mice crossed with a CDK5 heterozygous-deficient mouse line. A similar rescue effect was observed in APP transgenic mice treated with Roscovitine, a pharmacological inhibitor of CDK5. Taken together, these data suggest that the CDK5 signaling pathway has a critical role in maintaining the integrity of NPCs and neuronal maturation in the adult hippocampus. Moreover, potential therapeutic approaches could focus on modulating the aberrant activity of CDK5 to target the neurogenic and neurodegenerative alterations in AD.

Prevalence of incomplete ossification of the humeral condyle in the limb opposite humeral condylar fracture: 14 dogs.

OBJECTIVE: The objectives of this study were to determine the frequency of incomplete ossification of the contralateral humeral condyle (IOHC) in mature dogs with unilateral, atraumatic humeral condylar fracture (HCF), and to determine the sensitivity of radiographs as a diagnostic tool for IOHC. METHODS: Computed tomography and radiographs were obtained for both elbows of 14 dogs with unilateral HCF. The images were evaluated by two boarded radiologists and the prevalence of IOHC in the limb contralateral to the HCF was identified. Sensitivity and specificity of the radiographic diagnosis of IOHC were determined. RESULTS: Incomplete ossification of the humeral condyle was present in six of 14 dogs, however IOHC was incomplete in three of the six affected dogs. Plain radiographs had a sensitivity of 0.83 (CI 95%: 0.36 to 0.99) and specificity of 1 (CI 95%: 0.60 to1). The Kappa coefficient between radiologists for radiographic examination was 0.714. Of the Spaniel breeds, four out of eight had IOHC in the limb contralateral to the HCF. CLINICAL SIGNIFICANCE: Computed tomography evaluation is more sensitive than radiographs for diagnosis of IOHC, particularly when assessing partial or incomplete IOHC. However, sensitivity of radiographic diagnosis is good and should be adequate in most cases. Clinical suspicion of IOHC in the contralateral limb to the unilateral HCF should be present; however overall frequency may not be as high as previously reported.

Cognitive deficits and degeneration of interneurons in HIV+ methamphetamine users.

The cellular basis for cognitive deficits in HIV+ patients with and without a history of methamphetamine (METH) use is unclear. We found that HIV+ METH users had more severe loss of interneurons that was associated with cognitive impairment. Compared with other markers, loss of calbindin and parvalbumin interneurons in the frontal cortex was the most significant correlate to memory deficits, suggesting a role in neurobehavioral alterations of HIV+ METH users.

Abeta vaccination effects on plaque pathology in the absence of encephalitis in Alzheimer disease.

The authors report a patient with Alzheimer disease (AD) without encephalitis who was immunized with AN-1792 (an adjuvanted formulation of Abeta-42). There were no amyloid plaques in the frontal cortex and abundant Abeta-immunoreactive macrophages, but tangles and amyloid angiopathy were present. The white matter appeared normal and minimal lymphocytic infiltration in the leptomeninges was observed. This case illustrates the effects of an Abeta-based immunization on AD pathogenesis in the absence of overt meningoencephalitis and leukoencephalopathy.

Amelioration of the cerebrovascular amyloidosis in a transgenic model of Alzheimer's disease with the neurotrophic compound cerebrolysin.

Increased production and reduced clearance of amyloid beta (Abeta) plays a central role in the pathogenesis of Alzheimer's disease (AD). We have recently shown that the neurotrophic peptide mixture Cerebrolysin (Cbl) has the ability of improving synaptic functioning and reducing amyloid deposition in a transgenic (tg) animal model of Alzheimer's disease (AD). Since in AD, potentially toxic Abeta aggregates accumulate not only around neurons but also in the blood vessels, then it is important to investigate whether bioactive compounds such as Cbl might have the capacity to ameliorate the age-related cerebral amyloid angiopathy (CAA) in tg models. To this end, tg mice expressing mutant human amyloid precursor protein (APP) under the Thy1 promoter were treated with Cbl or saline alone starting at 7 or 12 months of age for a total of three months. Neuropathological analysis with an antibody against Abeta showed that Cbl decreased amyloid deposition around the blood vessels in a time dependent manner. These effects were accompanied by a reduction in perivascular microgliosis and astrogliosis and increased expression of markers of vascular fitness such as CD31 and ZO-1. No lymphocytic infiltration was observed associated with Abeta in the vessels. Consistent with these findings, ultrastructural analysis showed that while in tg mice treated with saline alone there was an abundant accumulation of amyloid fibers in the vascular wall accompanied by thickening of the basal membrane and endothelial cell damage, in Cbl-treated mice there was considerable reduction in the subcellular alterations of endothelial and smooth muscle cells with preservation of basal membranes and intercellular junctions. Taken together, these results suggest that Cbl treatment might have beneficial effects in patients with cognitive impairment due to cerebrovascular amyloidosis by reducing Abeta accumulation and promoting the preservation of the cerebrovasculature.

An antiaggregation gene therapy strategy for Lewy body disease utilizing beta-synuclein lentivirus in a transgenic model.

Current experimental gene therapy approaches for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) include the use of viral vectors expressing antiapoptosis genes, neurotrophic factors and dopaminergic system enzymes. However, since increasing evidence favors a role for alpha-synuclein accumulation in the pathogenesis of these disorders, an alternative therapy might require the transfer of genes that might block alpha-synuclein accumulation. beta-Synuclein, the nonamyloidogenic homologue of alpha-synuclein, has recently been identified as a potential candidate. Thus, in vivo transfer of genes encoding beta-synuclein might provide a novel approach to the development of experimental treatments for PD and DLB. To assess this possibility and to better understand the mechanisms involved, a lentiviral vector expressing human (h) beta-synuclein (lenti-beta-synuclein) was tested in a transgenic (tg) mouse model of halpha-synuclein aggregation. This study showed that unilateral intracerebral injection of lenti-beta-synuclein reduced the formation of halpha-synuclein inclusions and the accumulation of halpha-synuclein in synapses and ameliorated the neurodegenerative alterations in the tg mice. Both in vivo and in vitro coimmunoprecipitation and immunoblot experiments show that the mechanisms of beta-synuclein neuroprotection involve binding of this molecule to halpha-synuclein and Akt, resulting in the decreased aggregation and accumulation of halpha-synuclein in the synaptic membrane. Together, these data further support a role for beta-synuclein in regulating the conformational state of alpha-synuclein and suggest that this gene transfer approach might have potential for the development of alternative therapies for PD and DLB.

Elevated ovarian expression and serum concentration of alpha inhibin in the luteal phase during follicular development in the squirrel monkey (Saimiri boliviensis) compared to the human.

The goal of the present investigation was to determine in the squirrel monkey the source and pattern of inhibin, a hormone known to effect reproductive steroid levels via pituitary and ovarian mechanisms. Since this seasonally polyestrous species is known to have elevated serum levels of reproductive steroids compared to other primates, the levels of ovarian alpha subunit mRNA expression and serum total alpha inhibin, estradiol, progesterone, and luteinizing hormone were measured and compared to human levels. Expression of the alpha subunit was robust in monkey luteal tissue compared to expression in human luteal tissue. Squirrel monkey serum inhibin peaked 4 days after the luteinizing hormone surge and correlated with progesterone changes. These luteal serum levels of inhibin were greater than 12 times higher than the human levels yet bio-LH activities were less than in the human during the luteal phase. Inhibin concentrations during the nonbreeding season were generally half the levels measured in the breeding season and undetectable in ovariectomized animals. However, exogenous FSH stimulation induced a marked rise in inhibin, which correlated with an estradiol rise. In conclusion, abundant alpha inhibin subunit expression in the luteal ovary of the squirrel monkey and loss of serum delectability in ovariectomized animals indicates that the principle source of inhibin in the squirrel monkey is the ovary. Elevated serum inhibin levels during the luteal phase concurrent with ovulatory-size follicular development is unique among species studied thus far. Possible simultaneous inhibin production from both follicular and luteal tissue may be responsible for the exceptionally high inhibin levels.

4.5 kb of the rat tyrosine hydroxylase 5' flanking sequence directs tissue specific expression during development and contains consensus sites for multiple transcription factors.

To delineate DNA sequences responsible for developmentally correct expression of the rat tyrosine hydroxylase (TH) gene, we analyzed a line of transgenic mice expressing high levels of human placental alkaline phosphatase (AP) under control of 4.5 kb of 5' flanking DNA from the rat TH gene in embryos and adults. Several regions, such as the accessory olfactory bulb, which were not thought to synthesize TH protein or do so only transiently, were shown to express TH protein using an improved method of antigen retrieval for TH immunohistochemistry. Many of these regions had been shown to express TH-driven reporter genes in transgenic mice. In the central nervous system, AP was detected in essentially all TH-expressing cell groups throughout development and in adults. In the peripheral nervous system, transgene expression paralleled endogenous TH expression in the developing adrenal medulla and sympathetic ganglia but not in transiently TH-positive cells in dorsal root ganglia. Peripheral expression in the adult adrenal medulla was very weak and absent in sympathetic ganglia. The specificity with which the 4.5 kb region directs transgene expression in embryos is comparable to that observed with longer 5' flanking promoter regions, implying that this region contains the control elements for appropriate expression during development. Sequence analysis of the region demonstrates a GT dinucleotide repeat, an element that resembles the neural restrictive silencer element (NRSE), which restricts transcription of neuronal genes in non-neuronal cells, and consensus sites for three families of transcription factors, Ptx1/3, Nurr1 and Gli1/2, which are required for the early differentiation of mesencephalic neurons.

Xylem feeding by spittlebug nymphs: some observations by optical and cryo-scanning electron microscopy.

The feeding of spittlebug nymphs (Philaenus spumarius) from mature xylem vessels was studied by optical and cryo-analytical scanning electron microscopy. Feeding did not produce xylem embolisms and vessels remained liquid-filled during the day. Saliva secreted by the insect forms a hardened lining (salivary sheath) between the stylet bundle and the plant tissues. This sheath is continuous through the hole made by the stylets as they enter a vessel, and it extends into the vessel and along its periphery beyond the breach. The sheath is heterogeneous, with a thin outer layer adjoining the plant tissues and a thicker layer that contacts the stylet bundle. Both layers give positive histochemical reactions for proteins and, in fresh tissues, contain a red, strongly autofluorescent pigment, possibly condensed tannin derived from the plant (which is lost during tissue preparation), and other phenyl propanoid compounds, which are retained and which may produce the intense reaction of the periodic-acid-Schiff's-positive inner layer. It is concluded that the salivary sheath allows the insects to feed from functioning vessels without embolizing them or losing xylem fluid to the surrounding tissues. These findings and others in the entomological literature indicate low daytime tensions in the xylem conduits of the host plants.

Expression and activity of the newt Msx-1 gene in relation to limb regeneration.

The Msx-1 homeobox gene is expressed in various contexts during vertebrate development, including the progress zone of the avian and mouse limb bud. Expression of mouse Msx-1 in a cultured myogenic cell line conferred a transformed phenotype and inhibited fusion into myotubes. It has been proposed that Msx-1 expression is required to maintain certain cells in a proliferating and undifferentiated state and may be associated with the ability to regenerate limbs. Urodele amphibians such as the newt regenerate their limbs by formation of a growth zone or blastema, and we have isolated and sequenced newt Msx-1 (NvMsx-1) from a limb blastemal cDNA library. NvMsx-1 expression was detectable in RNA preparations from both limb and tail and their regeneration blastemas, although cultured cells established from limb blastemal mesenchyme gave negative results. When either COS cells or cultured newt blastemal cells were cotransfected with an expression vector for NvMsx-1 and reporter plasmids containing multiple homeobox protein binding sites, NvMsx-1 repressed reporter expression. If NvMsx-1 was expressed together with a marker enzyme in cultured newt blastemal cells, no significant difference in DNA synthesis was observed relative to control transfectants. When myogenic mononucleate cells were transfected with NvMsx-1 and subsequently exposed to low serum to promote fusion, the fraction of Msx-1 positive cells in myotubes was comparable to a control transfected population analysed in the same culture. These results indicate that although Msx-1 expression could be important for limb regeneration, it does not exert a cell-autonomous effect on proliferation or myogenic differentiation of cultured blastemal cells.

Molecular characterization of plastid pyruvate kinase from castor and tobacco.

Clones encoding two different forms of plastid pyruvate kinase (PKp; EC 2.7.1.40) have been isolated from both castor and tobacco seed cDNA libraries. One form, designated PKpA, from castor was described in a previous report, and the tobacco homologue of PKpA has now been isolated. In addition, a second cDNA, designated PKpG, has been identified and sequenced in both species. Western blot analysis, using antibodies raised against protein overexpressed from these clones, indicates that they encode the two predominant polypeptides of plastid pyruvate kinase from developing castor endosperm. In castor, both PKpA and PKpG are encoded by single genes. In the allotetraploid Nicotiana tabacum, there are two copies of each, one derived from each of the progenitors of this species. The expression of the genes for PKpA and PKpG was examined in various tissues from both castor and tobacco. In castor, both forms are expressed in developing and germinating endosperm and in the root but neither is expressed in the leaf. In tobacco, both forms are expressed in developing seeds but in mature tissues, PKpA is most abundant in roots and PKpG in leaves.

Neurogenesis in the olfactory epithelium.

The initial step in the recognition of odors occurs when individual odorant molecules enter the nasal cavity and interact with the sensory endings of neurons located in the olfactory epithelium. These neurons are unique in several respects. First, they are directly accessible to the external environment. Second, perhaps because of their exposure to toxic substances in the environment, they degenerate and are replenished continuously from a population of stem cells at the base of the epithelium. Third, the sensory neurons born in the olfactory epithelium of adults retain the ability to differentiate and establish synaptic contact with target cells in the mature olfactory bulb. These unique features, which are conserved phylogenetically (for example, see Blaustein et al), provide substantial rationale for studying neuronal genesis and differentiation in the olfactory epithelium. We will summarize much of what is currently known about the development of the olfactory epithelium, the birth and differentiation of olfactory receptor neurons, and the molecular correlates of these events.

Expression of the Genes for the alpha- and beta-Subunits of Pyrophosphate-Dependent Phosphofructokinase in Germinating and Developing Seeds from Ricinus communis.

Various tissues from both germinating and developing castor seeds (Ricinus communis L.) have been analyzed for the level of expression of the genes for the alpha- and beta-subunits of pyrophosphate-dependent phosphofructokinase (PFP). In tissues in which PFP is expressed, there is a single mRNA species of approximately 2 kilobases for each of the subunits. In germinating endosperm, the gene for the alpha-subunit is expressed at an earlier time after imbibition than that for the beta-subunit, whereas in developing castor seed endosperm, both genes are highly and coordinately expressed. During seedling development, there is tissue-specific expression of the two genes. Tissues in which there is a high level of mRNA correspond with tissues in which both subunits of PFP can be detected. The differential expression of the two subunit genes in germinating endosperm does not result in the presence of the alpha-subunit polypeptide in the absence of the beta-subunit polypeptide. Southern analysis of castor genomic DNA indicates the presence of a single gene for both the alpha- and beta-subunits of PFP in contrast with potato, in which there are at least two genes for each subunit.

The dependence of motoneurons on their target muscle during postnatal development of the mouse.

Motoneurons seem to require contact with their target muscle even after embryogenesis is complete, but the consequences of target-deprivation during postnatal development are poorly understood. To examine the fate of motoneurons separated from their targets postnatally, we labeled the motoneurons that innervate the biceps brachii muscle with the retrograde tracer Fluorogold and then separated them from their muscle by amputating the forelimb. Fluorogold was subsequently found within motoneurons, as well as within much smaller cells that were identified as microglia. The number of labeled microglial cells steadily increased with time following limb amputation, while the number of labeled motoneurons declined. The magnitude of this response depended on the age of the animal: the younger the animal at the time of the amputation, the greater the number of labeled microglia and the more extensive the neuronal loss. To ensure that the response to amputation was caused by target deprivation, rather than by the injury itself, the nerve to the biceps muscle was cut or crushed. In this way, axons were transected but target access was only temporarily denied. After the nerve was cut, motoneurons began to reinnervate the muscle within 3 weeks but, just as after amputation, the spinal cord subsequently contained labeled microglia and a reduced number of motoneurons. In contrast, after nerve crush, reinnervation began within 4 d and there was no evidence of motoneuron death. Our results demonstrate that target-deprivation causes motoneurons to be lost in an age- and time-dependent manner, and indicate a critical period after axotomy during which motoneurons must reinnervate their target in order to survive. Further, we provide evidence that microglial cells may phagocytose dying motoneurons. The approach we used would provide a convenient assay for testing candidate motoneuron growth factors in animals where in vivo studies of the embryo are difficult.

Purification of the alliin lyase of garlic, Allium sativum L.

1. Alliin lyase (EC 4.4.1.4) was purified up to sevenfold from garlic-bulb homogenates. The enzyme was unstable to storage at -10 degrees , particularly in dilute concentrations, but the addition of glycerol (final concentration 10%, v/v) stabilized the activity completely for at least 30 days. 2. The purified enzyme had an optimum pH for activity at 6.5. The addition of pyridoxal phosphate stimulated the reaction rate and the stimulation became more marked as the purification proceeded. 3. Hydroxylamine (10mum) and cysteine (0.5mm) inhibited the enzyme activity by more than 80%. Spectral studies indicated that cysteine reacted with pyridoxal phosphate bound to the protein. 4. The K(m) values for S-methyl-, S-ethyl-, S-propyl-, S-butyl- and S-allyl-l-cysteine sulphoxides were determined. With S-allyl-l-cysteine sulphoxide the K(m) was 6mm and the V(max.) was greater than those with the other substrates tested. 5. The thioether analogues of the substrates were competitive inhibitors for the lyase reaction. The K(i) decreased with increasing chain length of the alkyl substituent. With S-ethyl-l-cysteine sulphoxide as substrate the K(i) was 33, 8 and 5mm respectively for S-methyl-, S-ethyl- and S-propyl-l-cysteine. 6. The addition of EDTA or Mg(2+), Mn(2+), Co(2+) or Fe(2+) stimulated the reaction rate. Other bivalent cations either had no effect or gave a strong inhibition. In the presence of EDTA no further increase of activity was observed with added Mg(2+).