OBJECTIVE: To study the types and characteristics of TUBB1 mutation in children with congenital hypothyroidism (CH) and thyroid dysgenesis (TD) in Shandong, China. METHODS: Mutations of the whole coding region of the TUBB1 gene were analyzed for 289 children with CH and TD in Shandong. Whole-genome DNA was extracted from peripheral blood leukocytes. PCR multiplication was performed for the whole coding region of the TUBB1 gene. Sanger sequencing was performed for the PCR products, and a biological information analysis was performed. RESULTS: Among the 289 children with CH and TD, 4 (1.4%) were found to have a c.952C>T(p.R318W) heterozygous mutation in the TUBB1 gene, resulting in the change of tryptophan into arginine at codon 318 of TUBB1 protein. This mutation was evaluated as "potentially pathogenic" based on the classification criteria and guidelines for genetic variation by American College of Medical Genetics and Genomics. CONCLUSIONS A novel mutation is detected in the exon of the TUBB1 gene in children with CH and TD in Shandong, suggesting that the TUBB1 gene may be a candidate pathogenic gene for CH children with TD.
Child ; China ; Congenital Hypothyroidism ; genetics ; DNA Mutational Analysis ; Humans ; Mutation ; Thyroid Dysgenesis ; genetics ; Tubulin ; genetics

OBJECTIVE: To explore the clinical characteristics and genetic etiology of a patient with adolescent-onset hypomyelinated leukodystrophy with atrophy of basal ganglia and cerebellum (H-ABC). METHODS: A patient who was diagnosed with H-ABC in March 2018 at the First Affiliated Hospital of Nanjing Medical University was selected as the study subject. Clinical data was collected. Peripheral venous blood samples of the patient and his parents were collected. The patient was subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. RESULTS: The patient, a 31-year-old male, had manifested with developmental retardation, cognitive decline and abnormal gait. WES revealed that he has harbored a heterozygous c.286G>A variant of the TUBB4A gene. Sanger sequencing confirmed that neither of his parents has carried the same variant. Analysis with SIFT online software indicated the amino acid encoded by this variant is highly conserved among various species. This variant has been recorded by the Human Gene Mutation Database (HGMD) with a low population frequency. The 3D structure constructed by PyMOL software showed that the variant has a harmful effect on the structure and function of the protein. According to the guidelines formulated by the American College of Medical Genetics and Genomics (ACMG), the variant was rated as likely pathogenic. CONCLUSION The c.286G>A (p.Gly96Arg) variant of the TUBB4A gene probably underlay the hypomyelinating leukodystrophy with atrophy of basal ganglia and cerebellum in this patient. Above finding has enriched the spectrum of TUBB4A gene variants and enabled early definitive diagnosis of this disorder.
Male ; Humans ; Adolescent ; Adult ; Magnetic Resonance Imaging ; Basal Ganglia/pathology* ; Cerebellum ; Atrophy/pathology* ; Mutation ; Tubulin/genetics*

OBJECTIVETo observe the effect of recombinant adenovirus-mediated wild-type p53 gene on the number and proteins of centrosome in K562 cells. To explore the possibility of application of wild-type p53 gene therapy in the treatment of chronic myeloid leukemia.

METHODSThe recombinant adenoviruses carrying wild-type p53 gene (Ad5 wtp53), mutant p53 gene (Ad5 mtp53) or the green fluorescent protein (GFP) gene was repeatedly amplified and co-infected into K562 cells with cation polybrene. The optimal infection titer and infection time of the recombinant adenoviruses were determined by MTT assay, p53 mRNA and protein expression were determined by RT-PCR and Western blot respectively. The centrosomal structural protein gamma-tubulin and the spindle protein alpha-tubulin were marked simultaneously by indirect immunofluorescence staining, and the expression of the centrosomal gamma-tubulin protein, the mitosis and the number of centrosome were observed under the laser confocal microscopy.

RESULTSInfection efficiency with recombinant adenoviruses was facilitated by polybrene in K562 cells, and 4 microg/ml polybrene was chosen. The optimal adenovirus infection titer was 20,000 MOI and the optimal infection time was 72 hours. p53 mRNA and P53 protein can be expressed in K562 cells by Ad5wtp53 and Ad5mtp53. Both the expression of the centrosomal gamma-tubulin protein and the number of centrosomes were decreased after Ad5wtp53 infection.

CONCLUSIONThere is sustained expression of P53 protein in K562 cells after its infection by Ad5wtp53. Wild-type P53 protein can lead to the down-regulation of the number of centrosomes and the expression of centrosomal gamma-tubulin protein in K562 cells.

Adenoviridae ; genetics ; Centrosome ; metabolism ; Genes, p53 ; genetics ; Genetic Vectors ; Humans ; K562 Cells ; Transfection ; Tubulin ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVE: To explore the genetic basis for a fetus with dysgenesis of corpus callosum and other brain malformations. METHODS: Whole exome sequencing was carried out for the fetus and its parents. Suspected pathogenic variants were verified by Sanger sequencing. RESULTS: A novel de novo missense variant c.758T>A (p.L253Q) of the TUBB2B gene was identified, which was unreported previously. Based on the guidelines from the American College of Medical Genetics, the c.758T>A variant was predicted to be likely pathogenic. Bioinformatics analysis predicted that the leucine at position 253 was highly conserved among various species, and the c.758T>A variant may impact the formation of hydrogen bonds between Leu253 and Asp249 and Met257 residues, which in turn may affect the combination of GTP/GDP and function of the TUBB2B protein. CONCLUSION The c.758T>A variant of the TUBB2B gene probably underlay the fetal malformations in this Chinese family. Above discovery has enriched the spectrum of TUBB2B gene variants and provided a basis for genetic counseling and prenatal diagnosis.
Brain ; Female ; Fetus/abnormalities* ; Humans ; Malformations of Cortical Development/genetics* ; Pregnancy ; Prenatal Diagnosis ; Tubulin/genetics* ; Whole Exome Sequencing

OBJECTIVETo analyze the fungal composition in Massa Medicata Fermentata based on culture dependent method and independent PCR-SSCP technique.

METHODFungi were directly isolated from Massa Medicata Fermentata samples. The obtained strains were identified according to morphology and DNA sequence. Meanwhile the total fungal DNA was extracted from Massa Medicata Fermentata samples, the cultural independent PCR-SSCP technique based on β-tubulin gene were used to identify the mycobiota.

RESULTAccording to cultural method, Aspergillus flavus and Rhizopus oryzae were present in Massa Medicata Fermentata samples, while A. flavus and A. niger were present in fried Massa Medicata Fermentata samples. In contrast, 5 species were obtained by PCR-SSCP technique, A. flavus was overlapped with fungal taxa derived from culture dependent method; A. ambiguu and A. s ivoriensis were dominant with relative abundance of 57% and 35% respectively, while the relative abundance of A. flavus was as low as 4%. None species was obtained from fried Massa Medicata Fermentata samples.

CONCLUSIONPCR-SSCP based on β-tubulin gene could distinguish fungi into species, culture dependent method combined with culture independent method could better understand the fungal composition associated with Massa Medicata Fermentata fermentation.

Fermentation ; Fungi ; isolation & purification ; Medicine, Chinese Traditional ; Polymerase Chain Reaction ; methods ; Polymorphism, Single-Stranded Conformational ; Tubulin ; genetics

OBJECTIVETo investigate the changes of the mRNA expressions of myocardial cytoskeletal proteins in endotoxemic rats.

METHODSThirty-seven Wistar rats were randomized into two groups with injection of 10 mg/kg lipopolysaccharide (LPS) or normal saline through the femoral vein. The cardiac function of the rats was monitored continuously for 24 h, and the morphological changes of the cardiac myocytes were observed with HE staining and electron microscope. The mRNA levels of myocardial cytoskeletal proteins including actin, tubulin and desmin were determined by RT-PCR.

RESULTSNo significant difference was found in the number of CD3(+)T lymphocytes in the TILs between different groups. After the immunotherapy, the peLPS injection resulted in significant impairment of the cardiac function and myocardial microstructure of the rats with reduced heart rate and left ventricular systolic pressure (LVSP). The mRNA expression of actin in the cardiac myocytes measured by fluorescence optical density was reduced significantly 8 h after LPS injection, and that of tubulin was decreased significantly 24 h after LPS treatment; desmin mRNA expression showed no significant variation after LPS injection.

CONCLUSIONLPS can significantly impair the cardiac function of the rats possibly by inducing damages of the myocardial cytoarchitecture and causing changes in the mRNA expressions of such cytoskeletal proteins as actin and tubulin.

Actins ; genetics ; metabolism ; Animals ; Cytoskeletal Proteins ; genetics ; metabolism ; Endotoxemia ; chemically induced ; metabolism ; Female ; Lipopolysaccharides ; Myocardium ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Tubulin ; genetics ; metabolism

AIMTo perform screening, related to A-kinase anchoring proteins 4 (AKAP4) and tubulin proteins, in spermatozoa with absent or severely reduced motility in order to detect the status of the fibrous sheath and the axonemal structure.

METHODSAn immunocytochemical study of tubulin, used as a positive control, and AKAP4 was carried out to detect the presence and the distribution of these proteins in different sperm samples. The morphological characteristics of sperm were studied by transmission electron microscope (TEM) and the results were elaborated using a formula reported in previous studies. PCR was carried out on DNA extracted from peripheral blood lymphocytes to analyse partial sequences of the Akap4 and Akap3 genes.

RESULTSImmunolabelling of tubulin and AKAP4 showed different patterns, which led us to divide the patients into groups. In group I, the absence of AKAP4 and tubulin was revealed, although these patients did not show alterations in the Akap4/Akap3 binding site. TEM evaluation highlighted that a high presence of necrosis was associated with total sperm immotility. In group II, a regular AKAP4 and tubulin signal was present, although motility was reduced and TEM analysis revealed the presence of immaturity. In group III, in which a weak AKAP4 label associated with normal tubulin staining and reduced motility was observed, a severe disorganization of the fibrous sheath was highlighted by TEM.

CONCLUSIONWhile the role of AKAP4 in sperm motility is unclear, absent or weak AKAP4-labelling seems to be associated with absent or weak sperm motility.

A Kinase Anchor Proteins ; Humans ; Infertility, Male ; genetics ; physiopathology ; Male ; Microscopy, Electron ; Polymerase Chain Reaction ; Protein Precursors ; genetics ; metabolism ; Semen ; physiology ; Sperm Motility ; Spermatozoa ; physiology ; ultrastructure ; Tubulin ; genetics ; metabolism

OBJECTIVETo investigate whether the alterations of microtubule and microfilament expression are responsible for the neurotoxicity of carbon disulfide.

METHODSWistar rats were administered with carbon disulfide by gavage at a dosage of 300 or 500 mg/kg for continuous 12 weeks (five times per week). Spinal cords of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and ultracentrifuged to yield a pellet and a corresponding supernatant fraction. Then, the contents of alpha-tubulin, beta-tubulin, and beta-actin in both fractions were determined by immunoblotting. In the meantime, their mRNA levels in spinal cords were quantified using reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSIn the supernatant fraction, the contents of beta-tubulin and beta-actin in both treated groups increased significantly (P < 0.01) the content of beta-tubulin increased by 141% and 158% respectively, and the content of beta-actin increased by 19% and 32% respectively. In the pellet fraction, the content of beta-tubulin in both groups increased by 107%(P < 0.01) and 118%(P < 0.01) respectively, and the others keep unaffected. In the meantime, the levels of of mRNA expression of beta-tubulin and beta-actin gene were elevated consistently in CS(2)-treated groups (P < 0.01) the levels of mRNA expression of beta-tubulin increased by 207% and 212% respectively, and the levels of mRNA expression of beta-actin increased by 94% and 91% respectively.

CONCLUSIONCarbon disulfide intoxication results in alternations of microtubule and microfilament expression, and the alternations might be related to its neurotoxicity.

Actins ; genetics ; metabolism ; Animals ; Carbon Disulfide ; poisoning ; Disease Models, Animal ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Spinal Cord ; drug effects ; metabolism ; Tubulin ; genetics ; metabolism

In order to further study the potential interaction between PrP protein and the tubulin and identify the binding region in PrP with tubulin, native tubulin was extracted from rabbit brian tissues, while various recombinant PrP proteins were expressed and purified. The possible molecular interaction between various PrP fusion proteins and tubulin was tested by pull-down and immunoprecipitation assays. Remarkable molecular interaction between the full length PrP and tubulin was observed by pull-down and immunoprecipitation assays. Subsequently, the binding regions within PrP with tubulin were firstly mapped in the aa 23 -- 91 region within N-terminus of PrP peptide. The studies of the association of PrP with tubulin may further provide insight into the unresolved mechanism of active transport of PrP protein in neurons and possible cellular pathways by which prion causes disease.
Animals ; Binding Sites ; genetics ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Humans ; Immunoprecipitation ; Prions ; genetics ; isolation & purification ; metabolism ; Protein Binding ; Rabbits ; Recombinant Fusion Proteins ; genetics ; metabolism ; Tubulin ; metabolism

Pigs are increasingly recognized as "natural" hosts of infection by human respiratory viruses because of their similarities to humans in terms of lung physiology, airway morphology, cell types, and distribution of cell receptors in the respiratory tract. We wished to explore the mechanisms of infection by respiratory viruses and screening of drug that could be used to treat respiratory-system diseases. Hence, we developed a model of well-differentiated porcine airway epithelial cells (PAECs) derived from pig-lung tissue and cultured them with serum-free medium under an air-liquid interface condition in vitro. We identified the PAEC model using scanning electron microscopy, electrophysiology, and immunohistology. To evaluate application of gene therapy of adeno-associated virus (AAV)6 on the PAEC model, we generated recombinant adeno-associated virus 6-green fluorescent protein (rAAV6-GFP) using the three-plasmid transfection method and infected PAECs from the apical surface with rAAV6-GFP. Results demonstrated that the PAEC model comprised a multilayer epithelial structure containing ciliated mucous secretory cells, with basal cells located directly beneath the multilayer. rAAV6-GFP could infect PAECs from the apical surface and efficiently transduce PAECs to mediate the long-term expression of the exogenous gene. Establishment of a model of well-differentiated PAECs in vitro could lay a solid foundation for the study of infection by respiratory pathogens, as well as the screening and gene therapy of agents used to treat diseases of the respiratory system.
Animals ; Cell Differentiation ; Dependovirus ; genetics ; Epithelial Cells ; cytology ; metabolism ; Green Fluorescent Proteins ; genetics ; HEK293 Cells ; Humans ; Lung ; cytology ; Membrane Potentials ; Mucins ; metabolism ; Swine ; Transduction, Genetic ; Tubulin ; metabolism