Unusual cysteine modifications in natural product biosynthesis.

l-Cysteine is a highly reactive amino acid that is modified into a variety of chemical structures, including cysteine sulfinic acid in human metabolic pathways, and sulfur-containing scaffolds of amino acids, alkaloids, and peptides in natural product biosynthesis. Among the modification enzymes responsible for these cysteine-derived compounds, metalloenzymes constitute an important family of enzymes that catalyze a wide variety of reactions. Therefore, understanding their reaction mechanisms is important for the biosynthetic production of cysteine-derived natural products. This review mainly summarizes recent mechanistic investigations of metalloenzymes, with a particular focus on recently discovered mononuclear non-heme iron (NHI) enzymes, dinuclear NHI enzymes, and radical-SAM enzymes involved in unusual cysteine modifications in natural product biosynthesis.

Biallelic mutations in LSS in autosomal-recessive mutilating palmoplantar keratoderma.

Mutilating palmoplantar keratoderma (PPK) is a heterogeneous genetic disease that poses enormous challenges to clinical diagnosis and genetic counselling. Lanosterol synthase (LSS) gene encodes LSS involved in the biosynthesis pathway of cholesterol. Biallelic mutations in LSS were found to be related to diseases such as cataracts, hypotrichosis and palmoplantar keratoderma-congenital alopecia syndrome. The aim of this study was to investigate the contribution of the LSS mutation to mutilating PPK in a Chinese patient. The clinical and molecular characteristics of the patient were evaluated. A 38-year-old male patient with mutilating PPK was recruited in this study. We identified biallelic variants in the LSS gene (c.683C > T, p.Thr228Ile and c.779G > A, p.Arg260His). Immunoblotting revealed that the Arg260His mutant showed a significantly reduced expression level while Thr228Ile showed an expression level similar to that of the wild type. Thin layer chromatography revealed that mutant Thr228Ile retained partial enzymatic activity and mutant Arg260His did not show any catalytic activity. Our findings show the correlation between LSS mutations and mutilating PPK.

Biallelic Variants in Lanosterol Synthase (LSS) Cause Palmoplantar Keratoderma-Congenital Alopecia Syndrome Type 2.

Palmoplantar keratoderma-congenital alopecia syndrome type 2 is an autosomal recessive disorder with an unknown genetic basis. In this study, we identified biallelic variants in the LSS gene in two unrelated palmoplantar keratoderma-congenital alopecia syndrome type 2 cases (c.3G>A, p.Met1? and c.1025T>G, p.Ile342Ser in patient 1; c.1522G>T, p.Gly508Trp and c.428+42T>A in patient 2) presenting with additional clinical features, including early-onset cataracts, pseudoainhum, and agenesis of the corpus callosum. LSS encodes lanosterol synthase (LSS), which functions in the cholesterol biosynthesis pathway by converting (S)-2,3-oxidosqualene to lanosterol. The c.3G>A variant resulted in an alternative translation initiation at residue Met81, producing an N-terminal truncated protein (LSS-ΔN80), as shown by immunoblotting. The c.428+42T>A variant introduced a potential splicing site, leading to a premature stop codon. Ex vivo studies revealed downregulation of LSS in both patients. Remarkably decreased lanosterol levels were found in vitro in three LSS variants, LSS-ΔN80, p.Ile342Ser, and p.Gly508Trp, suggesting a loss of enzymatic activity. Transmission electron microscopy and immunofluorescence showed abnormal cornified envelope formation in the stratum corneum of the patients. Taken together, our findings indicate LSS as a causative gene for palmoplantar keratoderma-congenital alopecia syndrome type 2, which emphasizes the importance of the cholesterol synthesis pathway in human skin cornification.

Interactive effects of dung deposited onto urine patches on greenhouse gas fluxes from tropical pastures in Kenya.

Dung and urine patches on grasslands are hotspots of greenhouse gas (GHG) emissions in temperate regions, while its importance remains controversial for tropical regions as emissions seem to be lower. Here we investigated N2O, CH4 and CO2 emissions from urine and dung patches on tropical pastures in Kenya, thereby disentangling interactive and pure water, dung or urine effects. GHG fluxes were monitored with automated chambers for 42-59 days covering three seasons (short rainy season, long rainy season, dry season) for six treatments (Control; +1 L water; +1 kg dung; 1 L urine; 1 L water +1 kg dung; 1 L urine +1 kg dung). Cumulative CO2 emissions did not differ among treatments in any of the seasons. Water or urine addition alone did not affect CH4 fluxes, but these were elevated in all dung-related treatments. Scaled up on the total area covered, dung patches halve the CH4 sink strength of tropical pastures during the dry season, while during the rainy season they may turn tropical pastures into a small CH4 source. For N2O, both dung and urine alone and in combination stimulated emissions. While the N2O emission factor (EFN2O) from dung being constant across seasons, the EFN2O for urine was greater during the short rainy season than during the dry season. Combined application of urine + dung was additive on EFN2O. While the mean dung EFN2O in our study (0.06%) was similar to the IPCC Guidelines for National GHG Inventories EFN2O for dry climate (0.07%), the urine EFN2O we measured (0.03-0.25%) was lower than the IPCC value (0.32%). In addition, the IPCC Guidelines assume a urine-N: dung-N ratio of 0.66:0.34, which is higher than found for SSA (<0.50:0.50). Consequently, IPCC Guidelines still overestimate N2O emissions from excreta patches in SSA.

The optimization of sintering treatment on bovine-derived bone grafts for bone regeneration: in vitro and in vivo evaluation.

Modifications of sintering temperature and treatment time of bovine-derived bone grafts affect their physicochemical properties and further influence biological activity. Three different temperature sintered bovine-derived bone grafts: group I (300 °C 3 h), group II (300 °C 3 h plus 530 °C 6 h), and group III (300 °C 3 h plus 1000 °C 2 h) and Bio-Oss® were characterized and then compared in vitro for their effects on bone marrow stromal cells (BMSCs) migration, proliferation, and differentiation as estimated by cell migration assay, Alkaline phosphatase (ALP) activity assay, and Alizarin red staining. Further, the four bone grafts were implanted into the calvarial defects of rabbits to evaluate bone regeneration and graft degradation. The four deproteinized bovine-derived bone grafts displayed different surface topography. Group II displayed the highest potential of attracting cells. Both groups I and II markedly promote BMSCs differentiation. After 6 and 12 weeks, defects grafted with groups I and II displayed a significant higher bone fraction than defects grafted with group III and Bio-Oss®. Bone graft remnants remained in all four groups. Taken together, sintering at 300 °C for 3 h and sintering at 300 °C for 3 h with an addition of 530 °C for 6 h of bovine-dervied bone grafts displayed potential use in bone regeneration. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:272-281, 2020.

Natural product incarvillateine aggravates epileptic seizures by inhibiting GABAA currents.

A natural monoterpene alkaloid incarvillateine isolated from the plant Incarvillea sinensis is known to relieve inflammatory and neuropathic pain. However, the molecular target for the action of incarvillateine remains elusive. Here, we report that incarvillateine exacerbates epileptic seizures by inhibiting subtypes of γ-Aminobutyric acid type A (GABAA) receptors. Two-electrode voltage clamp recordings of α1ß3γ2, α2ß3γ2, α3ß3γ2 and α5ß3γ2 subtypes expressed in Xenopus oocytes revealed that incarvillateine inhibited the GABAA currents with IC50 of 25.1 µM, 43.1 µM, 105.1 µM and 93.7 µM, respectively. Whole-cell patch clamp recordings of hippocampal slices confirmed that incarvillateine inhibited spontaneous inhibitory postsynaptic currents (IPSCs), and miniature IPSCs and tonic currents. Moreover, inhibition of GABAA currents and spontaneous IPSCs by incarvillateine persisted even in the presence of blockers of adenosine receptors. In addition, incarvillateine enhanced epileptic discharges induced by Mg2+-free artificial cerebrospinal fluid (ACSF) in hippocampal slices. Furthermore, intracerebral ventricular injections of incarvillateine increased the severity of seizures induced by kainic acid in a dose-dependent manner. Taken together, our data demonstrate that incarvillateine aggravates seizures by inhibition of GABAA currents and GABAergic synaptic transmissions.

[Transcatheter delivery of recombinant adenovirus vector containing exogenous aquaporin gene in treatment of Sjögren's syndrome].

Sjögren's syndrome is a kind of autoimmune disease, whose main clinical symptoms are dry mouth, dry eye and chronic parotid glandular inflammation. The conservative treatments include artificial tears or saliva,oral administration of corticosteroids,and immunosuppressantsl with limited effectiveness. Along with the development of molecular biology, vast attentions are being paid to researches on gene therapy for Sjögren's syndrome, hopefully to bring gospel to patients with Sjögren's syndrome. This article reviews the recent research progresses on transcatheter delivery of recombinant adenovirus vector with aquaporin gene in experimental treatment of Sjögren's syndrome.