Background: Certain hereditary spastic paraplegia (HSP)-related proteins possess hairpin domains and regulate the morphology of the endoplasmic reticulum (ER) network. Results: Protrudin possesses a hairpin domain and interacts with HSP-related proteins. Conclusion: Protrudin regulates ER morphology and function. Significance: Mutant protrudin produced in certain individuals with HSP is prone to form microaggregates that induce ER stress.

ABSTRACT. Using RT-PCR and semi-quantitative PCR, mRNA expression for canine interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-5, IL-10, tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β in peripheral blood mononuclear cells (PBMCs) was examined in dogs with or without demodicosis. mRNA expression for IFN-γ as well as TNF-α in dogs with demodicosis (localized (LD) and generalized (GD)) was slightly lower than those in dogs without demodicosis (healthy controls). Expression of IL-5 mRNA in dogs with demodicosis was higher than that in control dogs, but there were no significant differences in IL-4 and IL-10 mRNA expression levels among the three groups. On the other hand, expression levels of TGF-β mRNA in dogs with GD were higher than those in control dogs and dogs with LD. The expression levels of IL-5 and TGF-β mRNA decreased in all three dogs with GD which showed resolution of the clinical signs. Taken together, these results suggest that the Th2-like response in PBMCs from dogs with demodicosis is up-regulated, and that subsequent increased expression of IL-5 and TGF-β mRNA in dogs with GD is reversible after treatment. Therefore, these cytokines, particularly IL-5, might be a useful clinical index of the clinical course in demodicosis. Also, increased TGF-β mRNA expression might be a key factor for revealing the difference in the mechanism of onset between LD and GD.

Protrudin is a membrane protein that regulates polarized vesicular transport. Now, we have identified a novel isoform of protrudin (protrudin-L) that contains an additional seven amino acids between the FFAT motif and the coiled-coil domain compared with the conventional isoform (protrudin-S) as a result of alternative splicing of a microexon (exon L). Protrudin-L mRNA was found to be mostly restricted to the central nervous system in mice, whereas protrudin-S mRNA was detected in all tissues examined. With the use of a splicing reporter minigene that produces two distinct fluorescent proteins in a manner dependent on the splicing pattern of protrudin transcripts, we found that most neurons express protrudin-L, whereas astrocytes express both protrudin isoforms and oligodendrocytes express only protrudin-S. Protrudin-L associated to a greater extent with vesicle-associated membrane protein-associated protein (VAP) than protrudin-S. Expression of protrudin-L in hippocampal neurons of protrudin-deficient mice also promoted neurite outgrowth more efficiently than protrudin-S. Our results suggest that protrudin-L is a neuron-specific protrudin isoform that promotes axonal elongation and contributes to the establishment of neuronal polarity.

We studied on the infection of domestic cat and dog fleas with Bartonella henselae by polymerase chain reaction (PCR) . A total of 62 fleas (36 Ctenocephalidis felis from cats, 24 C. felis from dogs and 2 Ctenocephalidis canis from dogs) , stored in 70% ethanol, were analyzed by PCR for B. henselae specific DNA. Of the 62 fleas, C. felis from cats and dogs were positive for B. henselae specific DNA in 12 of the 36 (33.3%) and in 5 of the 24 (20.8%) , respectively, and C. canis from dogs was positive in 2 of the 2 (100%). Our results demonstrated that pet fleas were infected with B. henselae, and suggest that flea transmission of B. henselae between cats or dogs may occur, and direct transmission of B. henselae from pet fleas to human may cause cat scratch disease.

Alternative splicing gives rise to diversity of the proteome, and it is especially prevalent in the mammalian nervous system. Indeed, many factors that control the splicing process govern nervous system development. Among such factors, SRRM4 is an important regulator of aspects of neural differentiation including neurite outgrowth. The mechanism by which SRRM4 regulates neurite outgrowth has remained poorly understood, however. We now show that SRRM4 regulates the splicing of protrudin gene (Zfyve27) transcripts in neuronal cells. SRRM4 was found to promote splicing of protrudin pre-mRNA so as to include a microexon (exon L) encoding seven amino acids in a neuron-specific manner. The resulting protein (protrudin-L) promotes neurite outgrowth during neurogenesis. Depletion of SRRM4 in Neuro2A cells impaired inclusion of exon L in protrudin mRNA, resulting in the generation of a shorter protein isoform (protrudin-S) that is less effective at promoting neurite extension. SRRM4 was found to recognize a UGC motif that is located immediately upstream of exon L and is necessary for inclusion of exon L in the mature transcript. Deletion of exon L in Neuro2A or embryonic stem cells inhibited neurite outgrowth. Our results suggest that SRRM4 controls neurite outgrowth through regulation of alternative splicing of protrudin transcripts.

Tyrosine kinase inhibitors of epidermal growth factor receptor (EGFR) improve the survival of patients with lung adenocarcinoma, and determine the EGFR mutation status before treatment is necessary. In contrast to biopsy samples, cytological specimens are obtained less invasively and are useful for EGFR mutation analyses. Recently, novel antibodies against two major EGFR mutations were developed: SP111, which is specific for the E746-A750 deletion in exon 19; and SP125, which is specific for the L858R mutation. To the best of our knowledge, no study has evaluated cytological specimens using the two novel antibodies, thus their specificity and sensitivity were examined in surgical resection, and cytological lung adenocarcinoma samples in the present study. Previous screening for EGFR mutation status by molecular testing identified delE746-A750 in 3 cases and the L858R mutation in 7 cases; the other cases did not have the L858R or the delE746-A750 mutation. Using a four-grade scoring system (score 0 to 3+), the immunohistochemistry (IHC) and immunocytochemistry (ICC) results were compared with those of molecular testing. Using a score of ≥2 as positive, IHC and ICC using SP111 demonstrated sensitivities of 100 and 33.3%, and specificities of 100 and 100%, respectively. IHC and ICC using SP125 revealed sensitivities of 100 and 71.4%, and specificities of 100 and 100%, respectively. Therefore, screening for EGFR mutations by ICC may facilitate therapeutic decision-making, particularly in medical centers that are unable to perform molecular testing.

Auditory brainstem response (ABR) is a useful method in evaluating auditory function in human. To investigate the ABR threshold is more effective than to pursue the trends in each component of ABR. In this study, tone burst sound stimuli were employed and the ABR threshold shift caused by kanamycin administration was investigated in dogs. In a series of monitoring of ABR against short-period auditory lesions, changes in the ABR waveform after intravenous administration of kanamycin were detected. These changes returned gradually and were reversible. The changes in ABR against long-period auditory function disorder were perceived by an increase in the ABR threshold. The ABR threshold shift occurred earlier in the high frequency sounds than in the lower frequency sounds. This is why amino glycoside antibiotics damage the cochlear hair cells in the basal layer and lead to the loss of hearing selectively for high frequency tones. These findings suggest that tracing of the ABR threshold by tone bursts could provide information that has a specificity for frequency in hearing tests and is a useful method in clinical veterinary medicine or/and toxicological tests.

Peripheral blood samples from dogs infected with Babesia gibsoni were analyzed by flow cytometry for parasitized erythrocytes after staining with hydroethidine. Cells identified as positive by flow cytometry were erythrocytes infected with B. gibsoni. Analysis of 26 samples by flow cytometry for % parasitemia revealed a correlation coefficient of 0.97 in comparison to the conventional method of light microscopy.