Preparation of gfp-bcl-XL-contained recombinant adenovirus vector by the homologous recombination in bacteria

AIM: To prepare gfp-bcl-X_L-contained recombinant adenovirus(rAd-gfp-bcl-X_L).METHODS: Bcl-X_L gene was amplified from pEGFP-C₃-bcl-X_L, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-X_L. Then pAdTrack-CMV-bcl-X_L was linealinzed with PmeI and co-transformed into BJ5183 bacteria with adenovirus genomic plasmid of pAdEasy-1. The identified recombinant adenovirus plasmid was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles. The target gene was detected by PCR.RESULTS: There were about 35% positive recombinant bacterial clones after the co-transformation of pAdTrack-CMV-bcl-X_L and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-X_L into 293 cells. PCR test indicated that the recombinant Ad contained bcl-X_L gene. The titer of the purified rAd-gfp-bcl-X_L was 6.5×10¹² PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-X_L. This affords a good gene transfer vector for the gene therapy in human’s diseases.     

Preliminary study of plasma vascular endothelial growth factor (VEGF) during low- and high-dose radiation therapy of dogs with spontaneous tumors

High plasma vascular endothelial growth factor (VEGF) concentrations are associated with radiation resistance and poor prognosis. After an exposure to ionizing radiation in cell culture an early phase and a late phase of increased VEGF have been documented. The activation was dependent on the radiation dose. Therefore, the purpose of this study was to measure baseline plasma VEGF and changes in VEGF over the course of fractionated radiation therapy in dogs with spontaneous tumors. Dogs with tumors had a significantly higher pretreatment plasma VEGF than did dogs without tumors. Immediately after irradiation no increased plasma VEGF was observed. Over the course of radiation therapy there was an increased plasma VEGF in dogs treated with low doses per fraction/high total dose, whereas plasma VEGF remained stable in dogs irradiated with high doses per fraction/low total dose. The regulatory mechanisms are very complex, and therefore the value of plasma VEGF measurements as an indirect marker of angiogenesis induced by radiotherapy is limited.     

The change of dry matter intake, milk yield and bone turnover in primiparous cows compared to multiparous cows during early lactation

Data from six primiparous and nine multiparous Holstein cows were used to clarify the difference of Ca and P mobilization between primiparous and multiparous cows during early lactation. The dry matter intake (DMI) of primiparous cows was lower (P < 0.01) than those of multiparous cows. Milk yield was lower in primiparous cows at 7, 14, 21 (P < 0.01), and 28 days (P < 0.05) after parturition. There was no significant difference in milk Ca and P concentrations between primiparous and multiparous cows. There were no significant differences in plasma Ca and P concentrations between primiparous and multiparous cows. The plasma P level at 7 days postpartum in primiparous cows was lower (P < 0.05) than 28 days postpartum. The concentration of plasma osteocalcin (OC) measured as bone formation marker of primiparous cows was significantly higher than multiparous cows (P < 0.01) at 21 and 28 days postpartum. The urinary deoxypyridinoline (DPD) as bone resorption marker of primiparous cows tended to be higher (P < 0.10) than multiparous cows at 21 days after parturition and decreased to the same level as that of multiparae toward the peak lactation. These results show that Ca and P mobilization of primiparous cows are more active than multiparous cows.     

Results from the treatment of advanced stage squamous cell carcinoma of the nasal planum in cats, using a combination of intralesional carboplatin and superficial radiotherapy: a pilot study

Six cats with an advanced stage squamous cell carcinoma (SCC) of the nasal planum were treated with a combination of superficial radiotherapy and intralesional carboplatin therapy. This multimodality protocol was well tolerated by the majority of cats and resulted in complete responses in all cats (100%). Median follow‐up for all cats is 268 days, and the median time‐to‐recurrence, time‐to‐progression and overall survival have not yet been reached. Our study, although limited in number of animals and with a relatively short median follow‐up compared to other studies for this disease, suggests that a combination of radiotherapy and intralesional carboplatin is a useful treatment option for an advanced stage SCC of the nasal planum in cats and warrants further application of the multimodality approach presented here.     

赖氨酸采食量对早期断奶仔猪生长发育及胰腺内分泌机能的影响

将55头21日龄断奶的雄性仔猪随机分为5组，各组仔猪饲喂赖氨酸含量为0．60％、0．80％、1．00％、1．20％和1．40％的试验日粮21d。42日龄结束饲养试验时进行葡萄糖灌注试验，观察赖氨酸采食量对早期断奶仔猪生长发育和胰腺内分泌的影响。结果表明：早期断奶仔猪体增重与赖氨酸采食量为强正相关，根椐体增重指标，21～42日龄阶段，仔猪的赖氨酸采食量不应低于3．16g／d，日粮赖氨酸含量不应低于1.40％。赖氨酸严重缺乏(采食量小于1．45g／d)时，胰岛素的分泌量下降，对高血糖应激的反应能力降低。     

Distribution of alpha‐neoendorphin,ACTH (18–39) and beta‐endorphin (1–27) in the alpaca brainstem

Using an immunocytochemical technique, we have studied in the alpaca brainstem the distribution of immunoreactive structures containing prodynorphin (alpha‐neoendorphin)‐ and pro‐opiomelanocortin (adrenocorticotrophin hormone (18–39) (ACTH), beta‐endorphin (1–27))‐derived peptides. No peptidergic‐immunoreactive cell body was observed. Immunoreactive fibres were widely distributed, although in most of the brainstem nuclei the density of the peptidergic fibres was low or very low. In general, the distribution of the immunoreactive fibres containing the peptides studied was very similar. A close anatomical relationship occurred among the fibres containing alpha‐neoendorphin, ACTH or beta‐endorphin (1–27), suggesting a functional interaction among the three peptides in many of the brainstem nuclei. The number of fibres belonging to the prodynorphin system was higher than that of the pro‐opiomelanocortin system. A moderate/low density of immunoreactive fibres was observed in 65.11% (for alpha‐neoendorphin (1–27)), 18.18% (for ACTH) and 13.95% (for beta‐endorphin) of the brainstem nuclei/tracts. In the alpaca brainstem, a high density of immunoreactive fibres was not observed. The neuroanatomical distribution of the immunoreactive fibres suggests that the peptides studied are involved in auditory, motor, gastric, feeding, vigilance, stress, respiratory and cardiovascular mechanisms, taste response, sleep‐waking cycle and the control of pain transmission.     

Genetic risk factors for osteochondrosis in various horse breeds

Osteochondrosis (OC) is an injury to cartilage canals with a following necrosis in the growth cartilage, from there it can develop to osteochondrosis dissecans (OCD). Due to its high impact in the equine industry, new insights into predisposing factors and potential high‐risk genetic variants are warranted. This article reviews advancements in quantitative and molecular genetics in refining estimation of genetic parameters and identifying predisposing genetic loci. Heritabilities were highest for hock OC with estimates at 0.29–0.46 in Hanoverian warmblood and Norwegian trotters, whereas in Thoroughbreds only very low genetic variation seemed to be present in hock OC lesions. Whole genome scans using the Illumina Equine SNP50 or SNP70 Beadchip were performed in Thoroughbred, Standardbred, French and Norwegian trotter, Hanoverian and Dutch warmblood. Validation studies in Spanish Purebred and Hanoverian warmblood horses corroborated OC risk loci on ECA 3, 14, 27 and 29. Particularly, a strong association with hock‐OCD was found for a single nucleotide polymorphism (SNP) on horse chromosome (ECA) 3 upstream to the LCORL gene. Gene expression and microRNA analyses may be helpful to understand pathophysiological processes in equine OC and to connect OCD‐associated genomic regions with potential candidate genes. Furthermore progress in elucidating the underlying genetic variants and pathophysiological changes in OC may be expected from whole genome DNA and RNA next‐generation sequencing studies.     

Canine GM2‐Gangliosidosis Sandhoff Disease Associated with a 3‐Base Pair Deletion in the HEXB Gene

Background

GM2‐gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either β‐hexosaminidase A (Hex‐A) and β‐hexosaminidase B (Hex‐B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency.

Objectives

To characterize the phenotype and genotype of GM2‐gangliosidosis disease in an affected dog.

Animals

One affected Shiba Inu and a clinically healthy dog.

Methods

Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes.

Results

A 14‐month‐old, female Shiba Inu presented with clinical signs resembling GM2‐gangliosidosis in humans and GM1‐gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex‐A and Hex‐B activities in both tissues. Genetic analysis identified a homozygous, 3‐base pair deletion in the HEXB gene (c.618‐620delCCT).

Conclusions and Clinical Importance

Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2‐gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2‐gangliosidosis seen in this dog is the Sandhoff type. Because GM1‐gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1‐ and GM2‐gangliosidosis should be considered to make a definitive diagnosis.