Jianxin Lu

Mutation in TRMU Related to Transfer RNA Modification Modulates the Phenotypic Expression of the Deafness-Associated Mitochondrial 12S Ribosomal RNA Mutations

The human mitochondrial 12S ribosomal RNA (rRNA) A1555G mutation has been associated with aminoglycoside-induced and nonsyndromic deafness in many families worldwide. Our previous investigation revealed that the A1555G mutation is a primary factor underlying the development of deafness but is not sufficient to produce a deafness phenotype. However, it has been proposed that nuclear-modifier genes modulate the phenotypic manifestation of the A1555G mutation. Here, we identified the nuclear-modifier gene TRMU, which encodes a highly conserved mitochondrial protein related to transfer RNA (tRNA) modification. Genotyping analysis of TRMU in 613 subjects from 1 Arab-Israeli kindred, 210 European (Italian pedigrees and Spanish pedigrees) families, and 31 Chinese pedigrees carrying the A1555G or the C1494T mutation revealed a missense mutation (G28T) altering an invariant amino acid residue (A10S) in the evolutionarily conserved N-terminal region of the TRMU protein. Interestingly, all 18 Arab-Israeli/Italian-Spanish matrilineal relatives carrying both the TRMU A10S and 12S rRNA A1555G mutations exhibited prelingual profound deafness. Functional analysis showed that this mutation did not affect importation of TRMU precursors into mitochondria. However, the homozygous A10S mutation leads to a marked failure in mitochondrial tRNA metabolisms, specifically reducing the steady-state levels of mitochondrial tRNA. As a consequence, these defects contribute to the impairment of mitochondrial-protein synthesis. Resultant biochemical defects aggravate the mitochondrial dysfunction associated with the A1555G mutation, exceeding the threshold for expressing the deafness phenotype. These findings indicate that the mutated TRMU, acting as a modifier factor, modulates the phenotypic manifestation of the deafness-associated 12S rRNA mutations.

Antitumor and immunomodulatory activity of polysaccharides from Sargassum fusiforme.

Sargassum fusiforme (Harv.) Setchel is an ingredient of Chinese herbal medicine that has been applied for thousands of years. This study was set up to evaluate the in vivo and in vitro anti-tumor potential of the polysaccharide (SFPS) from S. fusiforme and the immune response in tumor-bearing mice. SFPS was isolated by hot water extraction and ethanol precipitation. The mice inoculated with A549 cells were orally administrated with SFPS at the doses of 100 and 200mg/kg body weight for 28 days. The effects on the growth of tumor, serum TNF-α level, splenocyte proliferation, production of cytokines from peritoneal macrophages in A549-bearing mice were measured. Meanwhile, the cytotoxicity of SFPS on A549 cell line was also studied. Results showed that SFPS could not only significantly inhibit the growth of A549 lung adenocarcinoma in mice, but also remarkably promote IL-1 and TNF-α production from peritoneal macrophages, serum TNF-α level, and splenocytes proliferation in A549-bearing mice. The results indicate that SFPS has anti-tumor properties in vivo and in vitro, and improves the immune response in tumor-bearing mice. It could act as anti-tumor agent with immunomodulatory activity.

Mitochondrial 12S rRNA variants in 1642 Han Chinese pediatric subjects with aminoglycoside-induced and nonsyndromic hearing loss

In this report, we investigated the frequency and spectrum of mitochondrial 12S rRNA variants in a large cohort of 1642 Han Chinese pediatric subjects with aminoglycoside-induced and nonsyndromic hearing loss. Mutational analysis of 12S rRNA gene in these subjects identified 68 (54 known and 14 novel) variants. The frequencies of known 1555A>G and 1494C>T mutations were 3.96% and 0.18%, respectively, in this cohort with nonsyndromic and aminoglycoside-induced hearing loss. Prevalence of other putative deafness-associated mutation at positions 1095 and 961 were 0.61% and 1.7% in this cohort, respectively. Furthermore, the 745A>G, 792C>T, 801A>G, 839A>G, 856A>G, 1027A>G, 1192C>T, 1192C>A, 1310C>T, 1331A>G, 1374A>G and 1452T>C variants conferred increased sensitivity to ototoxic drugs or nonsyndromic deafness as they were absent in 449 Chinese controls and localized at highly conserved nucleotides of this rRNA. However, other variants appeared to be polymorphisms. Moreover, 65 Chinese subjects carrying the 1555A>G mutation exhibited bilateral and sensorineural hearing loss. A wide range of severity, age-of-onset and audiometric configuration was observed among these subjects. In particular, the sloping and flat-shaped patterns were the common audiograms in individuals carrying the 1555A>G mutation. The phenotypic variability in subjects carrying these 12S rRNA mutations indicated the involvement of nuclear modifier genes, mitochondrial haplotypes, epigenetic and environmental factors in the phenotypic manifestation of these mutations. Therefore, our data demonstrated that mitochondrial 12S rRNA is the hot spot for mutations associated with aminoglycoside ototoxicity.

Mitochondrial haplotypes may modulate the phenotypic manifestation of the deafness-associated 12S rRNA 1555A>G mutation

Mitochondrial 12S rRNA 1555A>G mutation is one of the important causes of aminoglycoside-induced and nonsyndromic deafness. Our previous investigations showed that the A1555G mutation was a primary factor underlying the development of deafness but was insufficient to produce deafness phenotype. However, it has been proposed that mitochondrial haplotypes modulate the phenotypic manifestation of the 1555A>G mutation. Here, we performed systematic and extended mutational screening of 12S rRNA gene in a cohort of 1742 hearing-impaired Han Chinese pediatric subjects from Zhejiang Province, China. Among these, 69 subjects with aminoglycoside-induced and nonsyndromic deafness harbored the homoplasmic 1555A>G mutation. These translated to a frequency of approximately 3.96% for the 1555A>G mutation in this hearing-impaired population. Clinical and genetic characterizations of 69 Chinese families carrying the 1555A>G mutation exhibited a wide range of penetrance and expressivity of hearing impairment. The average penetrances of deafness were 29.5% and 17.6%, respectively, when aminoglycoside-induced hearing loss was included or excluded. Furthermore, the average age-of-onset for deafness without aminoglycoside exposure ranged from 5 and 30years old, with the average of 14.5years. Their mitochondrial genomes exhibited distinct sets of polymorphisms belonging to ten Eastern Asian haplogroups A, B, C, D, F, G, M, N, R and Y, respectively. These indicated that the 1555A>G mutation occurred through recurrent origins and founder events. The haplogroup D accounted for 40.6% of the patients mtDNA samples but only 25.8% of the Chinese control mtDNA samples. Strikingly, these Chinese families carrying mitochondrial haplogroup B exhibited higher penetrance and expressivity of hearing loss. In addition, the mitochondrial haplogroup specific variants: 15927G>A of haplogroup B5b, 12338T>C of haplogroup F2, 7444G>A of haplogroup B4, 5802T>C, 10454T>C, 12224C>T and 11696G>A of D4 haplogroup, 5821G>A of haplogroup C, 14693A>G of haplogroups Y2 and F, and 15908T>C of Y2 may enhance the penetrace of hearing loss in these Chinese families. Moreover, the absence of mutation in nuclear modifier gene TRMU suggested that TRMU may not be a modifier for the phenotypic expression of the 1555A>G mutation in these Chinese families. These observations suggested that mitochondrial haplotypes modulate the variable penetrance and expressivity of deafness among these Chinese families.

A polysaccharide from Sargassum fusiforme protects against immunosuppression in cyclophosphamide-treated mice.

A water-soluble polysaccharide (SFPS) isolated from Sargassum fusiforme was purified by DEAE-52 cellulose anion-exchange and Sephadex G-200 gel filtration chromatography. The high performance gel permeation chromatography (HPGPC) analysis showed that the average molecular weight (Mw) of SFPS was 299 kDa. The SFPS was composed of D-fucose, L-xylose, D-mannose and D-galactose in a molar ratio of 5.9:1.0:2.3:2.2. The results showed that SFPS stimulated proliferation and the cytokines (IL-2, IL-6 and IFN-γ) secretion of splenic lymphocytes in cyclophosphamide-induced immunosuppressed mice. SFPS markedly increased the phagocytic rates and cytokines (IL-2, IL-6 and TNF-α) secretion of peritoneal macrophages. Administration of SFPS significantly raised spleen index. It could act as an efficacious adjacent immunopotentiating therapy or an alternative means in lessening chemotherapy-induced immunosuppression, and also can be utilized as immunostimulants for food and pharmaceutical industries.

Mitochondrial tRNAThr G15927A mutation may modulate the phenotypic manifestation of ototoxic 12S rRNA A1555G mutation in four Chinese families

Objective To investigate the role of mitochondrial modifiers in the development of deafness associated with 12S rRNA A1555G mutation. Methods Four Chinese families with nonsyndromic and aminoglycoside-induced deafness were studied by clinical and genetic evaluation, molecular and biochemical analyses of mitochondrial DNA (mtDNA). Results These families exhibited high penetrance and expressivity of hearing impairment. Penetrances of hearing loss in WZD31, WZD32, WZD33, and WZD34 pedigrees ranged from 50 to 67% and from 39 to 50%, respectively, when aminoglycoside-induced hearing loss was included or excluded. Matrilineal relatives in these families developed hearing loss at the average of 14, 13, 16, and 15 years of age, respectively, when aminoglycoside-induced deafness was excluded. Mutational analysis of entire mtDNA in these families showed the homoplasmic A1555G mutation and distinct sets of variants belonging to haplogroup B5b1. Of these, the tRNAThr G15927A mutation locates at the fourth base in the anticodon stem (conventional position 42) of tRNAThr. A guanine (G42) at this position of tRNAThr is highly conserved from bacteria to human mitochondria. The lower levels and altered electrophoretic mobility of tRNAThr were observed in cells carrying A1555G and G15927A mutations or only G15927A mutation but not cells carrying only A1555G mutation. The abolished base pairing (28C-42G) of this tRNAThr by the G15927A mutation caused a failure in tRNA metabolism, worsening the mitochondrial dysfunctions altered by the A1555G mutation. Conclusion The G15927A mutation has a potential modifier role in increasing the penetrance and expressivity of the deafness-associated 12S rRNA A1555G mutation in those Chinese pedigrees.

Protective effect of the polysaccharide from Ophiopogon japonicus on streptozotocin-induced diabetic rats.

The present study was designed to investigate the effect of OJP1, a polysaccharide isolated from the roots of Ophiopogon japonica, on blood lipid metabolism, antioxidant activity, as well as its protective effect on the liver and kidneys in diabetic rats. Results showed that OJP1 significantly reduced the MDA concentration and increased the activity of both GPx and SOD in the serum, liver and kidneys of diabetic rats. Moreover, the values of TG, TC, LDL-C and HDL-C in diabetic rats were significantly reversed by OJP1 treatment. Biochemical and histopathological analyses also showed that OJP1 can alleviate liver and kidneys injury in diabetic rats. The mRNA expression of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) in diabetic rats decreased significantly after administration of OJP1. Altogether, these results suggest that OJP1 possess potent antioxidant activity and can protect the liver and kidneys from the injurious effects of diabetes.

Mitochondrial ND5 T12338C, tRNACys T5802C, and tRNAThr G15927A variants may have a modifying role in the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation in three Han Chinese pedigrees†‡

We report here on the clinical, genetic, and molecular characterization of three Han Chinese pedigrees with aminoglycoside‐induced and nonsyndromic hearing loss. Clinical evaluation revealed the variable phenotype of hearing impairment including severity, age‐at‐onset, audiometric configuration in these subjects. The penetrance of hearing loss in WZD8, WZD9, and WZD10 pedigrees were 46%, 46%, and 50%, respectively, when aminoglycoside‐induced deafness was included. When the effect of aminoglycosides was excluded, the penetrance of hearing loss in these pedigrees were 23%, 31%, and 37.5%, respectively. Mutational analysis of the complete mitochondrial genomes showed the homoplasmic A1555G mutation and distinct sets of mitochondrial DNA variants belonging to haplogroups D4b2b, B5b1, and F2, respectively. Of these, the tRNACys T5802C, tRNAThr A15924C, and ND5 T12338C variants are of special interest as these variants occur at positions which are highly evolutionarily conserved nucleotides of tRNAs or amino acid of polypeptide. These homoplasmic mtDNA variants were absent among 156 unrelated Chinese controls. The T5802C and G15927A variants disrupted a highly conserved A‐U or C‐G base‐pairing at the anticodon‐stem of tRNACys or tRNAThr, while the ND5 T12338C mutation resulted in the replacement of the translation‐initiating methionine with a threonine, and also located in two nucleotides adjacent to the 3′ end of the tRNALeu(CUN). Thus, mitochondrial dysfunctions, caused by the A1555G mutation, would be worsened by these mtDNA variants. Therefore, these mtDNA mutations may have a potential modifier role in increasing the penetrance and expressivity of the deafness‐associated 12S rRNA A1555G mutation in those Chinese pedigrees.

Maternally inherited hearing loss is associated with the novel mitochondrial tRNASer(UCN) 7505T>C mutation in a Han Chinese family

Mutations in mitochondrial DNA (mtDNA) have been found to be one of the most important causes of sensorineural hearing loss. We report here a clinical, genetic, molecular and biochemical characterization of a Han Chinese pedigree with maternally transmitted nonsyndromic hearing impairment. Seven of nine matrilineal relatives exhibited a variable severity and age-at-onset (8 years old) of hearing loss. Mutational analysis of mtDNA identified the novel homoplasmic tRNA(Ser(UCN)) 7505T>C mutation and other 37 variants belonging to haplogroup F1. The 7505T>C mutation, which is absent in 449 Chinese controls, is located at a highly conserved base-pairing (10A-20U) of tRNA(Ser(UCN)). The abolishment of 10A-20U base-pairing likely alters the tRNA(Ser(UCN)) metabolism. Functional significant of this mutation was supported by approximately 65% reductions in the level of tRNA(Ser(UCN)) observed in the lymphoblastoid cell lines carrying the 7505T>C mutation, compared with the wild-type cell lines. This reduced tRNA level is below the proposed threshold to support a normal respiration in lymphoblastoid cells. Furthermore, the highly conserved tRNA(Ala) 5587T>C and Cytb C93Y variants may have a modifying role of deafness expression associated with the 7505T>C mutation. However, genotyping analysis of nuclear modifier gene TRMU and the prominent deafness-cause gene GJB2 failed to detect any mutations in the member of this family. These data strongly indicate that the novel tRNA(Ser(UCN)) 7505T>C mutation is involved in maternally transmitted hearing loss. However, other genetic, epigenetic or environmental factors may contribute to the phenotypic variability of this family. Our findings will be helpful for counseling families of maternally inherited hearing loss.

Sargassum fusiforme polysaccharide activates nuclear factor kappa-B (NF-κB) and induces cytokine production via Toll-like receptors.

This study was designed to investigate the mechanism of macrophage activation by the Sargassum fusiforme polysaccharide (SFPS). As a result, SFPS significantly enhanced cytokines and nitric oxide (NO) productions in peritoneal macrophages, and stimulated macrophages to produce the cytokines and NO through the induction of their genes expression. The pretreatment of peritoneal macrophages with special antibodies [Toll-like receptors (TLRs) antibody] significantly blocked SFPS-induced tumor necrosis factor alpha (TNF-α) and NO production. Furthermore, pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-κB, effectively suppressed SFPS-induced TNF-α and interleukin 1β (IL-1β) secretion in peritoneal macrophages, indicating that SFPS stimulated macrophages to produce cytokines through the NF-κB pathway and the result was further confirmed by the experiment of Western blotting (WB) and confocal laser scanning microscope (CLSM). Taken together, these results suggest that SFPS-mediated induction of cytokines and NO production in macrophages is mediated, at least in part, by TLRs/NF-κB signaling pathway.