Nanopore sequencing of the glucocerebrosidase (GBA) gene in a New Zealand Parkinson's disease cohort.

INTRODUCTION: Bi-allelic mutations in the gene for glucocerebrosidase (GBA) cause Gaucher disease, an autosomal recessive lysosomal storage disorder. Gaucher disease causing GBA mutations in the heterozygous state are also high risk factors for Parkinson's disease (PD). GBA analysis is challenging due to a related pseudogene and structural variations (SVs) that can occur at this locus. We have applied and refined a recently developed nanopore DNA sequencing method to analyze GBA variants in a clinically assessed New Zealand longitudinal cohort of PD. METHOD: We examined amplicons encompassing the coding region of GBA (8.9 kb) from 229 PD cases and 50 healthy controls using the GridION nanopore sequencing platform, and Sanger validation. RESULTS: We detected 23 variants in 21 PD cases (9.2% of patients). We detected modest PD risk variant p.N409S (rs76763715) in one case, p.E365K (rs2230288) in 12 cases, and p.T408 M (rs75548401) in seven cases, one of whom also had p.E365K. We additionally detected the possible risk variants p.R78C (rs146774384) in one case, p.D179H (rs147138516) in one case which occurred on the same haplotype as p.E365K, and one novel variant c.335C > T or p.(L335 = ), that potentially impacts splicing of GBA transcripts. Additionally, we found a higher prevalence of dementia among patients with GBA variants. CONCLUSION: This work confirmed the utility of nanopore sequencing as a high-throughput method to identify known and novel GBA variants, and to assign precise haplotypes. Our observations may contribute to improved understanding of the effects of variants on disease pathogenesis, and to the development of more targeted treatments.

Detection of elevated serum beta-chemokine levels in seronegative Chinese individuals exposed to human immunodeficiency virus type 1.

The mutations in the CCR5 coding region, such as CCR5Delta32 and CCR5m303, that suppress the transmission of human immunodeficiency virus (HIV) type 1 do not exist in Chinese people. However, 9 Chinese subjects in Taiwan with histories of multiple sexual exposures to HIV remained uninfected, suggesting that certain anti-HIV factors do indeed exist. Experiments were therefore designed to investigate the immune mechanism that protects this cohort against HIV infection. Peripheral blood samples from these 9 subjects and 7 healthy people who had not been exposed to HIV were obtained for the quantitation of the levels for beta-chemokines and interleukin 16 (IL-16) in serum samples or secreted by peripheral blood lymphocytes. Significantly higher serum levels for nearly all 3 beta-chemokines, regulation on activation, normal T cell-expressed and secreted, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta (P<.05, P<.05, and P=.05, respectively), but not IL-16, were detected in the 9 HIV-uninfected subjects as compared with control subjects. The result suggests that among the host genes and cellular factors thus far identified, beta-chemokines are the major HIV-suppressive factors that protect Chinese people from infection with HIV.

Microarray profiling of gene expression patterns in bladder tumor cells treated with genistein.

Microarray technology was used to gain an insight into the molecular events of tumor cell growth inhibition mediated by the soy isoflavone genistein. For this, a susceptible bladder tumor line TCCSUP was treated with the inhibitory dose (50 microM) of genistein for various periods of time, followed by mRNA isolations, cDNA probe preparations, and hybridization individually to cDNA chips containing 884 sequence-verified known human genes. After analyzing the hybridization signals with a simple quantitative method developed by this study, we detected that egr-1, whose expression has been associated with proliferation and differentiation, was transiently induced and this expression pattern was later confirmed by RT-PCR. Thus, microarray technology is a reliable and powerful tool for profiling gene expression patterns in many biological systems related to cancer. We further detected many groups of genes with distinct expression profiles and most of them encode for proteins that regulate the signal transduction or the cell cycle pathways. These genes warrant further investigation as regards their roles in the susceptibility of the tumor cell line to the antitumor drug.

Influence of nucleotide polymorphisms in the CCR2 gene and the CCR5 promoter on the expression of cell surface CCR5 and CXCR4.

Polymorphisms in the CCR2 gene (CCR2-64I) and the CCR5 promoter (pCCR5-59029G) have been correlated with slower HIV-1 disease progression. How these polymorphisms influence the rate of AIDS progression has remained unclear. We have therefore investigated whether these nucleotide polymorphisms will reduce the expression levels of surface CCR5 and CXCR4, and thus lead to slower AIDS progression. For this, a cohort of Chinese volunteers in Taiwan was subjected to the determination of CCR2 and pCCR5 genotypes followed by analysis of the surface CCR5 and CXCR4 expression on five cell types derived from peripheral blood mononuclear cells by flow cytometry. Several significant associations were detected between genotypes and expression levels of the proteins. The most important finding was that an increased number of CD4(+) cells expressing CCR5 correlated with pCCR5-59029A homozygosity without the interference of both the CCR2-64 and the CCR5 delta 32 (deleted 32 bp) mutations (P: = 0.0453), which is consistent with the previous data on the association of the genotype to AIDS progression. Since different genetic polymorphisms co-exist in human beings, the rate of AIDS progression as well as the risk of rheumatoid arthritis may be governed by the interplay of the array of nucleotide changes and their affected proteins.

Femtosecond-pulse cavity-dumped solid-state oscillator design and application to ultrafast microscopy.

The construction, modeling, and performance characteristics of a new resonator design for ultrafast cavity-dumped oscillators are presented. An acousto-optic Bragg cell was incorporated at the end of the longer arm of a Ti:sapphire oscillator rather than in the shorter arm as in several recent studies. The new arrangement improves the pulse intensity stability of the oscillator and significantly reduces the effort required in construction. The experimental findings are supported by comparison of the stability regions of the laser cavities based on the two different designs. To demonstrate the potential of cavity-dumped oscillators for spatially resolved ultrafast spectroscopy studies, the pulse duration is characterized at the focal plane of two achromatic high-N.A. oil-immersion objectives with different amounts of flat-field correction. Transform-limited pulse widths as short as 15 fs are obtained. To our knowledge, this is the shortest pulse duration measured with true high-N.A. (N.A. > 1) focusing conditions.

Characterization of a desiccation-related protein in lily pollen during development and stress.

This work characterizes a lily (Lilium longiflorum Thunb. cv. Snow Queen) anther (LLA) protein associated with desiccation. Peptide mapping analysis revealed that the abundant LLA-23 doublet contained similar polypeptides, having an isoelectric point of 6.1. Immunoblots of pollen protein from developing anther/pollen confirmed that the LLA-23 protein accumulated only at the later stage of pollen maturation and that the levels remained steady in mature and vital pollen. The accumulation of LLA-23 proteins was correlated with desiccation that naturally occurred in pollen. Subcellular fractionation of pollen proteins revealed that the protein was located in the cytoplasmic fraction. Premature drying of developing pollen confirmed that the concomitant accumulation of LLA-23 was associated with desiccation. Peptide sequence analysis demonstrates similarities between the lily LLA-23 and a family of water-deficit/ripening-induced proteins including LP3 of pine, DS2 of potato, and Asr of tomato and pummelo. In addition, the concomitant accumulation of LLA-23 can be experimentally manipulated by methyl jasmonate (Me-JA) and salicylic acid (SA) as well as by mannitol and methyl viologen. The LLA-23 represents a novel member of the water-deficit/ripening-induced proteins.

Aplasia cutis congenita: report of one case.

We present a female newborn with a skin defect on the vertex. Aplasia cutis congenita was diagnosed. No associated anomaly was found. There was no family or drug history noted. At one-year follow-up, the lesion tended to contract and became epithelialized. We report this case and also review the literature, and then we suggest a checklist for aplasia cutis congenita.

Enhancement of gastric mucus phospholipid secretion by an antiulcer agent, ebrotidine.

1. Rat gastric mucosal cells, subjected to phospholipid labeling by incubating the cell suspension in DMEM with [3H]choline, were exposed to different concentrations (0-150 microM) of H2-receptor antagonists, ebrotidine and ranitidine, and the phospholipid secretory responses were evaluated. 2. In the absence of the drugs, the secretion of choline-containing phospholipids over a 1 hr period averaged 3.97% of the total cellular labeled phospholipids. Ebrotidine caused a dose-dependent increase in the rate of phospholipid secretion which was most pronounced at 1 hr and persisted for at least 2 hr. The maximal effect was attained at 120 microM ebrotidine giving a 36% increase in phospholipid secretion. 3. The phospholipid secretory response to ebrotidine was accompanied by an increase in gastric mucosal cell cAMP level which reached a maximum value of 2.1-fold over that of controls at 1 hr. Ranitidine, in contrast, neither evoked increase in cAMP level nor caused any stimulation in phospholipid secretion. 4. The results indicate that the gastroprotective properties of ebrotidine are associated with the ability of the drug to elicit a rapid stimulation in gastric mucus phospholipid secretion, and that ranitidine does not possess such property.

Identification of buccal mucosal mucin receptor.

A receptor for salivary mucin was isolated from epithelial cell membrane of buccal mucosa by a procedure involving membrane solubilization with octylglucoside followed by affinity chromatography on Sepharose-bound wheat germ agglutinin. The receptor protein displayed an apparent molecular weight of 97kDa and exhibited binding specific to mucin in a concentration-dependent manner. The receptor, furthermore, showed a requirement for carbohydrate chains in mucin for binding, as their removal caused a marked (89%) reduction in binding capacity. Scatchard analysis revealed a linear plot with a single class of high affinity binding (Kd = 1.1 microM; Bmax = 7.68nmol/mg protein. The results provide for the first time evidence for the existence in buccal mucosa of a specific receptor for salivary mucin.

Effect of ebrotidine on the synthesis and secretion of gastric sulfomucin.

1. The effect of a new antiulcer agent, ebrotidine, on the synthesis and secretion of sulfomucin in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in DMEM containing [3H]proline, [3H]glucosamine and [35S]Na2SO4 as markers for mucin synthesis, glycosylation and sulfation, in the presence of 0-150 microM ebrotidine. 2. The drug, while showing no discernible effect on the apomucin synthesis, evoked a dose-dependent increase in mucin glycosylation and sulfation, which at 100 microM ebrotidine, attained its maximum of 2.4 and 2.7-fold stimulation, respectively. 3. The analysis of mucin secretory responses revealed that ebrotidine caused a concentration-dependent enhancement in sulfomucin secretion which attained its maximum increase of 3.3-fold at 100-120 microM ebrotidine. Furthermore, the sulfomucin elaborated in the presence of ebrotidine exhibited a higher content of a large molecular-weight mucus glycoprotein form, the assembly of which is intimately associated with the sulfation event. 4. The results suggest that the ability of ebrotidine to enhance gastric sulfomucin synthesis and secretion may play an important role in the gastroprotective mechanism of action of this agent.

Glycosulfatase activity of Porphyromonas gingivalis a bacterium associated with periodontal disease.

Porphyromonas gingivalis, a bacterium implicated in the pathogenesis of periodontal disease, was found to elaborate an extracellular glycosulfatase enzyme. Upon purification by low temperature acetone fractionation, an active enzyme at 60% acetone was obtained which on SDS-PAGE gave a protein band of 37kDa. The glycosulfatase effectively caused desulfation of galactosyl- and lactosylceramide sulfates (pH 5.0) which contain the sulfate ester groups at C-3 of galactose, a well as proteoglycans (pH 5.7-6.2) of gingival tissue which are rich in N-acetylgalactosamine-4-sulfate, but not the sulfated salivary mucin with the sulfate groups at C-6 of galactose and C-6 of N-acetylglucosamine. The results demonstrate for the first time that P. gingivalis displays glycosulfatase activity and that the disruptive action of this enzyme may be a major factor in the etiology of periodontal disease.

Nitecapone effect on the synthesis and secretion of gastric sulfomucin.

1. The effect of a new antiulcer agent, nitecapone, on the synthesis and secretion of sulfomucin in gastric mucosa was investigated using mucosal segments incubated in the presence of [3H]proline, [3H]glucosamine and [35S]sulfate. 2. The drug, while showing no discernible effect on the apomucin synthesis, evoked a dose-dependent increase in mucin glycosylation and sulfation, which at 225 microM nitecapone, attained its maximum of 1.8 and 2.2-fold stimulation, respectively. 3. The analysis of mucin secretory responses revealed that nitecapone caused a concentration-dependent enhancement in sulfomucin secretion attaining maximum increase of 1.5-fold at 150 microM nitecapone. 4. The stimulatory effect of nitecapone on sulfomucin secretion was accompanied by 1.4-fold increase in mucosal cAMP level, and showed sensitivity to protein kinase A inhibitors, thus pointing towards the involvement of protein kinase A in mediation of gastric sulfomucin secretory responses to nitecapone. 5. The ability of nitecapone to enhance sulfomucin synthesis and secretion could be of importance to the gastroprotective action of this agent.

Effect of Helicobacter pylori lipopolysaccharide on the synthesis of sulfated gastric mucin.

The effect of H. pylori lipopolysaccharide on the synthesis and secretion of sulfated mucus glycoprotein in gastric mucosa was investigated. The lipopolysaccharide, while showing no discernible effect on the apomucin synthesis, exerted a profound inhibitory effect on the process of mucus glycoprotein glycosylation and sulfation, and evoked a rapid (within 15 min) inhibition (65%) in both mucin glycosylation and sulfation at its optimal concentration of 100 micrograms/ml. The data on mucin secretory responses indicated that the added lipopolysaccharide caused a 57% stimulation in sulfated mucin secretion within 15 min followed thereafter by inhibition, which reached a maximum of 32% by 45 min. The high molecular weight mucin form predominated in the initial secretion, while prolonged exposure to the lipopolysaccharide caused a significant increase in the low molecular weight mucin form. The results suggest that H. pylori lipopolysaccharide exerts detrimental effect on mucus glycoprotein sulfation and assembly.

Role of sulfation in post-translational processing of gastric mucins.

1. Gastric mucosal segments were incubated in MEM supplemented with various sulfate concentrations in the presence of [3H]glucosamine, [3H]proline and [35S]Na2SO4, with and without chlorate, an inhibitor of 3'-phosphoadenosine-5'-phosphosulfate formation. 2. Incorporation of glucosamine and sulfate depended upon the sulfate content of the medium and reached a maximum at 300 microM sulfate. Introduction of chlorate into the medium, while having no effect on protein synthesis as evidenced by proline incorporation, caused, at its optimal concentration of 2 mM, a 90% decrease in mucin sulfation and a 40% drop in glycosylation. 3. At low sulfate content in the medium and in the presence of chlorate, the incorporation of sulfate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulfate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulfation in its carbohydrate chain. 4. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and that sulfate availability is essential for the formation of the high molecular-weight mucin polymer.

Helicobacter pylori lipopolysaccharide effect on the synthesis and secretion of gastric sulfomucin.

The effect of H. pylori lipopolysaccharide on the synthesis and secretion of sulfated mucin in gastric mucosa was investigated using mucosal segments incubated in the presence of [3H]proline, [3H]glucosamine and [35S]Na2SO4. The lipopolysaccharide, while showing no discernible effect on the apomucin synthesis was found to inhibit the process of mucin glycosylation and sulfation, which at 100 micrograms/ml lipopolysaccharide reached the optimal inhibition of 65%. The analysis of mucin secretory responses revealed that the lipopolysaccharide by first 15 min caused a 57% stimulation in sulfomucin secretion followed thereafter by inhibition, which reached maximum of 32% by 45 min. The results suggest that colonization of gastric mucosa by H. pylori may be detrimental to the process of gastric sulfomucin synthesis and secretion.

Glycosulfatase activity of Helicobacter pylori toward gastric mucin.

A glycosulfatase activity toward sulfated gastric mucus glycoprotein was identified in the extracellular material elaborated by H. pylori, a bacteria implicated in the etiology of gastric disease. Upon acetone precipitation, an active enzyme fraction at 64% acetone was obtained which on SDS-PAGE gave a major 30kDa protein band. The H. pylori glycosulfatase exhibited maximum activity (314.8 pmol/mg protein/h) at pH 5.7 in the presence of Triton X-100 and CaCl2, and was capable of removal of the sulfate ester groups situated at C-6 of N-acetylglucosamine, galactose and glucose. However, the enzyme was ineffective toward galactosylceramide and lactosylceramide sulfates which contain the sulfate ester group on C-3 of galactose. The results suggest that H. pylori is capable of overcoming the interference by sulfated mucus glycoprotein with its colonization of gastric mucosa.

Glycosaminoglycan patterns in gingival proteoglycans of rat with age.

Among the potential biochemical indices that are closely associated with craniofacial development are the proteoglycans. Gingival segments from the palate of 4-, 6-, 8-, 12- and 18-week-old rats were incubated for 4 h in medium containing [3H]-glucosamine and [35S]-Na2SO4, and subjected to proteoglycan isolation and glycosaminoglycan analysis. Two distinct proteoglycan fractions differing in the degree of sulphation were obtained by ion-exchange chromatography. The incorporation of both labels in the undersulphated fraction increased with age; there was a pronounced decrease with age in the sulphated proteoglycan fraction. The undersulphated proteoglycans showed an age-dependent decrease in hyaluronic acid, and increase in dermatan sulphate and chondroitin 4- and 6-sulphates. Gel filtration of the sulphated proteoglycan fraction yielded high and low molecular-weight proteoglycans, the glycosaminoglycans of which were particularly rich (61-76%) in dermatan sulphate. Smaller quantities of chondroitin 4- and 6-sulphates, and heparan sulphate were also present. All glycosaminoglycans showed a decrease in content with age. The findings suggest a possible correlation between gingival proteoglycan/glycosaminoglycan patterns and development.

Role of sulfation in the processing of gastric mucins.

The role of sulfation in the processing of mucus glycoprotein in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in MEM at various medium sulfate concentrations in the presence of [35S]Na2SO4, [3H]glucosamine and [3H]proline, with and without chlorate an inhibitor of PAPS formation. The results revealed that the mucin sulfation attained maximum at 300 microM medium sulfate concentration. Introduction of chlorate into the incubation medium, while having no effect on the protein synthesis as evidenced by [3H]proline incorporation, caused at its optimal concentration of 2 mM a 90% decrease in mucin sulfation and a 40% drop in mucin glycosylation. Evaluation of mucin molecular forms distribution indicated the predominance of the high molecular mucin form in the intracellular fraction and the low molecular mucin from in the extracellular fraction. Increase in medium sulfate caused an increase in the high molecular weight mucin form in both fractions, and this effect was inhibited by chlorate. Also, higher medium sulfate concentrations led to a higher degree of sulfation in the high molecular weight mucin form, the effect of which was inhibited by chlorate. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and is required for mucin polymer formation. Hence, the disturbances in mucin sulfation process could be detrimental to the maintenance of gastric mucus coat integrity.

Identification of gastric mucosal mucus glycoprotein sulfotransferase.

Sulfation of mucus glycoproteins, reaction catalyzed by Golgi resident sulfotransferase, is an important event in posttranslational processing of gastric mucins. Here we report the purification of mucus glycoprotein sulfotransferase enzyme from the microsomal fraction of rat gastric mucosa. The enzyme was released from the membrane with 0.5% Triton X-100 and precipitated from the 100,000xg supernatant with 90% ice-cold acetone. The enzyme activity (44.7 pmol/mg/45 min) in the precipitate was enriched nearly 10-fold compared to Triton X-100 extract of microsomal membrane (4.2 pmol/mg/45 min). On SDS-PAGE, the enzyme gave a single 43 kDa protein band, which was active towards mucin, but did not catalyze the sulfation of galactosylceramide. The study is the first to report the characteristics of a sulfotransferase enzyme specific for gastric mucin.