A Novel SPAST Mutation Results in Spastin Accumulation and Defects in Microtubule Dynamics.

BACKGROUND: Haploinsufficiency is widely accepted as the pathogenic mechanism of spastic paraplegia type 4 (SPG4). However, there are some cases that cannot be explained by reduced function of the spastin protein encoded by SPAST. OBJECTIVES: To identify the causative gene of autosomal dominant hereditary spastic paraplegia in three large Chinese families and explore the pathological mechanism of a spastin variant. METHODS: Three large Chinese hereditary spastic paraplegia families with a total of 247 individuals (67 patients) were investigated, of whom 59 members were recruited to the study. Genetic testing was performed to identify the causative gene. Western blotting and immunofluorescence were used to analyze the effects of the mutant proteins in vitro. RESULTS: In the three hereditary spastic paraplegia families, of whom three index cases were misdiagnosed as other types of neurological diseases, a novel c.985dupA (p.Met329Asnfs*3) variant in SPAST was identified and was shown to cosegregate with the phenotype in the three families. The c.985dupA mutation produced two truncated mutants (mutant M1 and M87 isoforms) that accumulated to a higher level than their wild-type counterparts. Furthermore, the mutant M1 isoform heavily decorated the microtubules and rendered them resistant to depolymerization. In contrast, the mutant M87 isoform was diffusely localized in both the nucleus and the cytoplasm, could not decorate microtubules, and was not able to promote microtubule disassembly. CONCLUSIONS: SPAST mutations leading to premature stop codons do not always act through haploinsufficiency. The truncated spastin may damage the corticospinal tracts through an isoform-specific toxic effect.

Knockdown of BmTCP-1ß Delays BmNPV Infection in vitro.

The molecular mechanism of silkworm resistance to Bombyx mori nucleopolyhedrovirus (BmNPV) infection remains unclear. The chaperonin containing t-complex polypeptide 1 (TCP-1) is essential for the folding of tubulin and actin to produce stable and functional competent protein conformation. However, little is known about this protein in silkworm. In the present study, a gene encoding the TCP-1ß protein in silkworm was characterized, which has an open reading fragment of 1,611 bp encoding a predicted 536 amino acid residue-protein with a molecular weight of approximately 57.6 kDa containing a Cpn60_TCP1 functional domain. The sequence conservation is 81.52%. The highest level of BmTCP-1ß mRNA expression was found in the midgut, while the lowest was in the hemolymph. To further study the function of BmTCP-1ß, expression was knocked down with siRNA in vitro, resulting in significant downregulation of the selected cytoskeletal-related genes, actin and tubulin, which was also confirmed by overexpression of BmTCP-1ß in BmN cells using the pIZT/V5-His-mCherry insect vector. Moreover, knockdown of BmTCP-1ß significantly prolonged the infection process of BmNPV in BmN cells, which was also verified by overexpression of BmTCP-1ß in BmN cells. Based on the results of the present study, we concluded that BmTCP-1ß plays a vital role in BmNPV infection by regulating the expression of tubulin and actin. Taken together, our work provides valuable data for the clarification of the molecular mechanism of silkworm resistance to BmNPV infection.

Utilizing VEGF165b mutant as an effective immunization adjunct to augment antitumor immune response.

Compelling evidence has shown that blocking VEGF via monoclonal antibodies may be beneficial in that it not only inhibits tumor angiogenesis but also reduces immune suppression and promotes T cell infiltration into tumors. Herein, we determined whether our recently generated VEGF165b mutant could be used as a co-immunization adjunct to augment the peptide cancer-vaccine- induced immune response in a mouse model of breast cancer. When co-immunized mVEGF165b with the peptide-based cancer vaccine (MUC1, a T-cell epitope dominant peptide vaccine from Mucin1), the VEGF antibody titers increased approximately 600,000-fold in mice. Moreover, the anti-VEGF antibody also reduced the frequency of regulatory T cells (Tregs) in both preventive and therapeutic scenarios. Mechanistically, the decrease of the Tregs population was associated with a remarkably increased MUC-1-specific IFN-γ-producing CD8+ T cells and anti-MUC1 humoral response. Finally, this combination co-immunization produced a superior antitumor response and significantly prolonged survival of tumor-bearing mice. In conclusion, our findings suggest that mVEGF165b may be an ideal immunization adjunct to enhance the immune efficacy of peptide-based tumor vaccines by overcoming immune tolerance.

Cep85 Relays Plk1 Activity to Phosphorylated Nek2A for Its Timely Activation in Centrosome Disjunction.

Timely centrosome separation is critical for accurate chromosome separation. It is initiated by Nek2A at the onset of mitosis, but the mechanism for the strict requirement of phosphorylated Nek2A for its own activation remains unclear. In this study, we have found that Plk1 interacts with Cep85 and forms a ternary complex with Cep85-Nek2A. Nek2A binding, but not its kinase activity, is pre-required for Cep85 to be phosphorylated by Plk1. Nek2A-dependent Cep85 phosphorylation, in turn, leads to the dissociation of phosphorylated Cep85 exclusively from phospho-Nek2A, thereby increasing the freed phospho-Nek2A activity. Both kinases are also required for phosphorylating endogenous Cep85 in cells, and timely phosphorylation of Cep85 and Nek2A is crucial for initiating centrosome disjunction at G2/M. Overall, our study has uncovered a previously unrecognized role of Plk1 and Nek2A and identified Cep85 as a missing piece directly relaying Plk1 activity to Nek2A for its activation in centrosome disjunction.

Activation of hypothalamic astrocytes suppresses feeding without altering emotional states.

Emerging evidence shows that hypothalamic astrocytes react to and counteract energy surfeit produced by high-fat diet (HFD) feeding. However, the functional role of astrocytes in the control of energy states and the underlying molecular mechanism(s) during physiological conditions remain largely underexplored. In the present study, by taking advantage of spatiotemporally precise optogenetic approaches, real-time measurements of extracellular adenosine, and behavioral assays, we find that optogenetic stimulation of astrocytes localized in the medial basal hypothalamus (MBH) suppresses food intake in a frequency dependent manner with high frequency, but not low frequency, stimulation of astrocytes reducing food intake. Furthermore, stimulation of MBH astrocytes diminishes orexigenic ghrelin or fasting-induced hyperphagia without effecting anxiety-related behavior. Consistent with a frequency dependent role for MBH astrocytes in feeding behavior, optogenetic stimulation of MBH astrocytes increases extracellular levels of adenosine in a frequency dependent manner. Collectively, our results provide new insights into the role of astrocytes in physiological functions during naturally occurring behaviors, such as feeding. GLIA 2016;64:2263-2273.

Caffeine increases food intake while reducing anxiety-related behaviors.

The objective of this study was to determine the effects of different doses of caffeine on appetite and anxiety-related behavior. Additionally, we sought to determine if withdrawal from chronic caffeine administration promotes anxiety. In this study, we utilized rodent open field testing and feeding behavior assays to determine the effects of caffeine on feeding and anxiety-related behavior (n = 8 mice; 4-8 weeks old). We also measured 2 h and 24 h food intake and body-weight during daily administration of caffeine (n = 12 mice; 4-8 weeks old). To test for caffeine withdrawal induced anxiety, anxiety-related behavior in rodents was quantified following withdrawal from four consecutive days of caffeine administration (n = 12 mice; 4-8 weeks old). We find that acute caffeine administration increases food intake in a dose-dependent manner with lower doses of caffeine more significantly increasing food intake than higher doses. Acute caffeine administration also reduced anxiety-related behaviors in mice without significantly altering locomotor activity. However, we did not observe any differences in 24 h food intake or body weight following chronic caffeine administration and there were no observable differences in anxiety-related behaviors during caffeine withdrawal. In conclusion, we find that caffeine can both increase appetite and decrease anxiety-related behaviors in a dose dependent fashion. Given the complex relationship between appetite and anxiety, the present study provides additional insights into potential caffeine-based pharmacological mechanisms governing appetite and anxiety disorders, such as bulimia nervosa.

Endothelin-1 upregulation mediates aging-related cardiac fibrosis.

Endothelin-1 (ET-1) plays a major role in regulating myocardial fibrosis in several pathological conditions, such as hypertension and diabetes. Aging is an independent risk factor for myocardial fibrosis. We hypothesized that ET-1 upregulation may be a basis of enhanced collagen synthesis in the senescent fibroblasts resulting in cardiac fibrosis with aging. To examine this hypothesis, we cultured mouse cardiac fibroblasts to passage-30 (P30). ß-Galactosidase activity and several other aging markers were markedly increased in P30 (vs. P3) fibroblasts, indicating that these cells were indeed undergoing senescence. Importantly, ET-1 expression was markedly upregulated in P30 (vs. P3) fibroblasts. Of note, estrogen receptor-α (ER-α), an important negative regulator of ET-1, was downregulated in P30 fibroblasts. We also studied aged (130-weeks old, female) mice hearts, and observed that ET-1 was upregulated and ER-α was downregulated in these hearts (vs. 6-week old mice hearts, female). Similar observations were made in the fibroblasts isolated from aged mice hearts. ET-1 upregulation with aging was also seen in ≈70-year old (vs. ≈30-year old) human heart sections. In concert with ET-1 upregulation, the expression of fibronectin and collagens was found to be markedly increased in P30 cardiac fibroblasts in culture, fibroblasts isolated from the aged mice hearts, and in aged human hearts. Interestingly, inhibition of ET-1 in the senescent P30 fibroblasts by 2 different strategies (the use of siRNA and the use of endothelin converting enzyme inhibitors) markedly suppressed expression of fibrosis signals. Further, treatment with synthetic ET-1 enhanced fibronectin and collagen expression in P3 cardiac fibroblasts. These observations in mice and human hearts suggest that aging-related cardiac fibrosis is, at least partially, dependent on the upregulation of ET-1.

Overexpression of CyclinA2 ameliorates hypoxia-impaired proliferation of cardiomyocytes.

Hypoxia is a primary mediator for cell survival, and has been reported to inhibit cardiomyocyte proliferation in fetal and neonatal hearts. CyclinA2 is a key regulator of cell proliferation. Whether CyclinA2 affects cardiomyocyte proliferation in hypoxic conditions remains unexamined. This study was designed to investigate the roles of CyclinA2 expression on hypoxia-impaired cardiomyocyte proliferation. Cardiomyocytes were isolated from neonatal rats and randomly separated into six groups: Control, hypoxia, enhanced green fluorescent protein (EGFP)-Adv, EGFP-Ccna2, EGFP-Adv + hypoxia and EGFP-Ccna2 + hypoxia. The cells in the control group were cultured in a general cell incubator; the cells in the hypoxia group were placed in a hypoxic chamber for 12 h; the cells in the EGFP-Adv and EGFP-Ccna2 groups were separately transfected with EGFP-adenovirus capsids or EGFP-adenovirus capsids with CyclinA2 cDNA for 18 h, and then placed in a general incubator for an additional 12 h; the cells in the EGFP-Adv + hypoxia and EGFP-Ccna2 + hypoxia groups were separately transfected with EGFP-adenovirus capsids or EGFP-adenovirus capsids with CyclinA2 cDNA for 18 h, and then placed in a hypoxia chamber for an additional 12 h. CyclinA2 expression was measured using immunochemical staining and western blot analysis, and cardiomyocyte proliferation was measured using the cell counting kit 8. GFP fluorescence indicated a high transfection efficiency (>80%), and immunochemical staining showed that CyclinA2 was mainly distributed in the nucleus. CyclinA2 expression was downregulated following exposure to hypoxia for 12 h. Cardiomyocyte proliferation was also significantly decreased following exposure to hypoxia for 12 h. However, compared with the EGFP-Adv group, CyclinA2 expression and cardiomyocyte proliferation was markedly increased in the EGFP-Ccna2 group. Furthermore, compared with the EGFP-Adv + hypoxia group, CyclinA2 expression and cell proliferation were markedly increased in the EGFP-Ccna2 + hypoxia group. These findings indicate that CyclinA2 upregulation improves cardiomyocyte proliferation in hypoxic conditions.

Identification and molecular characterization of two novel mutations in COL1A2 in two Chinese families with osteogenesis imperfecta.

Osteogenesis imperfecta (OI, also known as brittle bone disease) is caused mostly by mutations in two type I collagen genes, COL1A1 and COL1A2 encoding the pro-α1 (I) and pro-α2 (I) chains of type I collagen, respectively. Two Chinese families with autosomal dominant OI were identified and characterized. Linkage analysis revealed linkage of both families to COL1A2 on chromosome 7q21.3-q22.1. Mutational analysis was carried out using direct DNA sequence analysis. Two novel missense mutations, c.3350A>G and c.3305G>C, were identified in exon 49 of COL1A2 in the two families, respectively. The c.3305G>C mutation resulted in substitution of a glycine residue (G) by an alanine residue (A) at codon 1102 (p.G1102A), which was found to be mutated into serine (S), argine (R), aspartic acid (D), or valine (V) in other families. The c.3350A>G variant may be a de novo mutation resulting in p.Y1117C. Both mutations co-segregated with OI in respective families, and were not found in 100 normal controls. The G1102 and Y1117 residues were evolutionarily highly conserved from zebrafish to humans. Mutational analysis did not identify any mutation in the COX-2 gene (a modifier gene of OI). This study identifies two novel mutations p.G1102A and p.Y1117C that cause OI, significantly expands the spectrum of COL1A2 mutations causing OI, and has a significant implication in prenatal diagnosis of OI.

Identification of a novel genetic locus on chromosome 8p21.1-q11.23 for idiopathic basal ganglia calcification.

Idiopathic basal ganglia calcification (IBGC) is a neurodegenerative disorder that is characterized by basal ganglia and extrabasal ganglia calcification, and usually inherited in an autosomal dominant pattern. To date, two genetic loci for IBGC were identified on chromosomes 14q and 2q, but further genetic heterogeneity clearly exists. In this study, a large Chinese family with autosomal dominant IBGC was characterized. Linkage analysis excluded the 14q13 and 2q37 loci. The large family was then characterized by genome-wide linkage analysis to identify a novel genetic locus for IBGC. Significant linkage was identified with markers on chromosome 8p21.1-q11.23 with a maximum LOD score of 4.10. Fine mapping defined the new genetic locus within a 25 Mb region between markers D8S1809 and D8S1833. Future studies of the candidate genes at the 8p21.1-q11.23 locus may lead to identification of a disease-causing gene with IBGC.

Identification of a novel splicing mutation in the growth hormone (GH)-releasing hormone receptor gene in a Chinese family with pituitary dwarfism.

A Chinese family with autosomal recessive pituitary dwarfism was identified and the proband was evaluated by MRI and hormonal analysis, which revealed pituitary dwarfism with a complete growth hormone deficiency. MRI showed a pituitary gland with a small anterior pituitary of 2.2mm and evidence of hypoplastic pituitary. Linkage analysis with markers spanning 17 known genes for dwarfism revealed linkage of the family to the growth hormone-releasing hormone receptor (GHRHR) gene. Mutational analysis of all exons and exon-intron boundaries of GHRHR was carried out using direct DNA sequence analysis. A novel homozygosis mutation, a G to A transition located in the splice donor site at the beginning of intron 8 (IVS8+1G>A), was identified in the proband. The two other patients in the family are homozygous, whereas the living mother of the proband is heterozygous for the IVS8+1G>A mutation. The mutation was not found in 100 normal chromosomes from healthy Chinese individuals of Han nationality. An in vitro splicing assay using HeLa cells transfected with expression vectors containing the normal or the mutant GHRHR minigenes consisting of genomic fragments spanning exons 7-9 showed that the IVS8+1G>A mutation caused abnormal splicing, which is predicted to give rise to truncation or frameshift, leading to severely truncated GHRHR proteins. These results provide strong evidence that the splicing mutation IVS8+1G>A of GHRHR is a cause of pituitary dwarfism in the Chinese family.

[Effects of 50 Hz magnetic fields on gene expression in MCF-7 cells].

OBJECTIVE: To investigate whether 50 Hz magnetic fields (MF) can change the gene expression profile in MCF-7 cells and to screen MF responsive genes. METHODS: In vitro cultured MCF-7 cells were continuously exposed or sham-exposed to 0.4 mT of 50 Hz MF for 24 hours. Affymetrix Human Genome Genechips (U133A) were applied to analyze gene expression profiles in MF exposed and sham-exposed MCF-7 cells and the data were processed with Genechip data analysis software MAS 5.0 and DMT 3.0. Real-time RT-PCR assay was employed to examine the differentially expressed genes. RESULT: Thirty differentially expressed genes were screened with 100 % consistency change calls in the MF exposed MCF-7 cells. Six independent real-time RT-PCR analyses showed that SCNN1A, METTL3 and GPR137B were slightly but statistically significantly changed in MCF-7 cells after exposure to 50 Hz MF (P<0.05), while other analyzed genes exhibited slight up-and down-fluctuations in expressions and no increase or decrease in each gene expression reached statistical significance (P>0.05). CONCLUSION: The present study identified three 50 Hz MF responsive genes in MCF-7 cells and the biological consequences of expression changes in these MF responsive genes need to be further investigated.0.4 mT 50 Hz MF exposure for longer duration might induce DNA double-strand breaks in human lens epithelial cells in vitro.

Effect of pharmacologic preconditioning with tetrandrine on subsequent ischemia/reperfusion injury in rat liver.

The effect of tetrandrine (TET) pretreatment of Wistar rats subjected to warm hepatic ischemia/reperfusion (I/R) was investigated. After 50 minutes of ischemia in the left and median lobes of the liver and 24 hours of reperfusion (I/R group), the rats were killed. The TET+I/R group rats were pretreated with TET (50 mg/kg body weight IP) 30 minutes prior to the onset of ischemia. Blood samples were taken for measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). Tissue was taken from the ischemic lobes for measurement of superoxide dismutase (SOD), malonyldialdehyde (MDA), and myeloperoxidase (MPO); determination of the wet/dry weight (W/D) ratio; and histologic studies. The results showed that ALT, AST, and LDH levels in serum were increased in the I/R group; tissue MDA generation, MPO activity, and the W/D ratio were also increased, accompanied by decreased SOD activity. The serum ALT, AST, and LDH levels, as well as the tissue MPO level and W/D ratio, were lower in the TET+ I/R group than in the I/R group; and the SOD level was higher in the TET+IR group than in the I/R group. Moreover, the serum ALT and AST, tissue MDA, and W/D ratio in the TET+I/R group were higher, and the SOD was lower than in the sham group. The histologic examination showed protection against liver damage in the TET+I/R group. The results demonstrated that pretreatment with TET could somewhat protect the liver against I/R injury but does not prevent it. The simultaneous decrease of both lipid peroxide generation and polymorphonuclear neutrophil infiltration in the ischemic liver may explain the acquisition of tolerance following administration of TET.