New insights into the biosynthesis of mycobacterial lipomannan arising from deletion of a conserved gene

Genetic construction of a mutant strain (designated MSMEG4245) of Mycobacterium smegmatis, defective in a broadly conserved gene for a putative glycosyltransferase of the glycosyltransferase-C superfamily, results in a phenotype marked by the virtual absence of the phosphatidylinositol-containing lipomannan and lipoarabinomannan, replaced instead by a novel truncated form of lipomannan. The normal spectrum of phosphatidylinositol mannosides, long presumed precursors of these lipoglycans, was retained. Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry of the mutated form of lipomannan shows a family of phosphatidylinositol-anchored lipomannans with from only 5 to 20 Manp residues as compared with lipomannan from the wild type strain consisting of 21-34 Manp residues but with few changes in the branching pattern. Thus, MSMEG4245 is apparently a key mannosyltransferase, required for the proper elongation of lipomannan to its normal state and subsequent synthesis of lipoarabinomannan. The corresponding ortholog in Mycobacterium tuberculosis H37Rv has been identified as Rv2174. This previously unrecognized feature of the biosynthesis of lipomannan/lipoarabinomannan allows a significant revision of structural and biosynthetic schemata and provides a molecular basis of selectivity in biosynthesis, as conferred by the MSMEG4245 gene.     

Glycomic mapping of pseudomucinous human ovarian cyst glycoproteins: identification of Lewis and sialyl Lewis glycotopes

Expression of sialyl Lewis x (sLe(x)) and sialyl Lewis a (sLe(a)) on cell-surface glycoproteins endows cells with the ability to adhere to E-, P-, and L-selectins present on endothelia, platelets, or leukocytes. Special arrangements of these glycotopes in cancers are thought to play a key role in metastasis. Previous studies have mostly described membrane-bound sLe(x) and sLe(a) activities. In this report, the major O-glycans of the secreted human ovarian cyst sialoglycoproteins from a Le(a+) nonsecretor individual (human ovarian cyst sample 350) were characterized by MS/MS analyses and immuno-/lectin-chemical assays. The results showed that HOC 350 carries a large number of epitopes for sLe(x), sLe(a), and Le(a) reactive antibodies. Advanced MS/MS sequencing coupled with mild periodate oxidation and exoglycosidase digestions further revealed that the O-glycans from HOC 350 are mostly of core 1 and 2 structures, extended and branched on the 3-arm with both type I and type II chains, complete with variable degrees of terminal sialylation and/or fucosylation to yield the sLe(x) or sLe(a) epitopes. Thus, the underlying core and peripheral backbone structures are similar to that of a previously proposed composite structural model for nonsialylated human ovarian cysts O-glycans, but with some notable distinguishing structural features in addition to sialylation.     

Energetics and dynamics of SNAREpin folding across lipid bilayers

Membrane fusion occurs when SNAREpins fold up between lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy-versus-distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (approximately 2-4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k(B)T) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers (40-50 k(B)T).     

Association of ischemic stroke onset time with presenting severity,acute progression,and long-term outcome: A cohort study

BackgroundPreclinical data suggest circadian variation in ischemic stroke progression, with more active cell death and infarct growth in rodent models with inactive phase (daytime) than active phase (nighttime) stroke onset. We aimed to examine the association of stroke onset time with presenting severity, early neurological deterioration (END), and long-term functional outcome in human ischemic stroke.Methods and findingsIn a Korean nationwide multicenter observational cohort study from May 2011 to July 2020, we assessed circadian effects on initial stroke severity (National Institutes of Health Stroke Scale [NIHSS] score at admission), END, and favorable functional outcome (3-month modified Rankin Scale [mRS] score 0 to 2 versus 3 to 6). We included 17,461 consecutive patients with witnessed ischemic stroke within 6 hours of onset. Stroke onset time was divided into 2 groups (day-onset [06:00 to 18:00] versus night-onset [18:00 to 06:00]) and into 6 groups by 4-hour intervals. We used mixed-effects ordered or logistic regression models while accounting for clustering by hospitals. Mean age was 66.9 (SD 13.4) years, and 6,900 (39.5%) were women. END occurred in 2,219 (12.7%) patients. After adjusting for covariates including age, sex, previous stroke, prestroke mRS score, admission NIHSS score, hypertension, diabetes, hyperlipidemia, smoking, atrial fibrillation, prestroke antiplatelet use, prestroke statin use, revascularization, season of stroke onset, and time from onset to hospital arrival, night-onset stroke was more prone to END (adjusted incidence 14.4% versus 12.8%, p = 0.006) and had a lower likelihood of favorable outcome (adjusted odds ratio, 0.88 [95% CI, 0.79 to 0.98]; p = 0.03) compared with day-onset stroke. When stroke onset times were grouped by 4-hour intervals, a monotonic gradient in presenting NIHSS score was noted, rising from a nadir in 06:00 to 10:00 to a peak in 02:00 to 06:00. The 18:00 to 22:00 and 22:00 to 02:00 onset stroke patients were more likely to experience END than the 06:00 to 10:00 onset stroke patients. At 3 months, there was a monotonic gradient in the rate of favorable functional outcome, falling from a peak at 06:00 to 10:00 to a nadir at 22:00 to 02:00. Study limitations include the lack of information on sleep disorders and patient work/activity schedules.ConclusionsNight-onset strokes, compared with day-onset strokes, are associated with higher presenting neurologic severity, more frequent END, and worse 3-month functional outcome. These findings suggest that circadian time of onset is an important additional variable for inclusion in epidemiologic natural history studies and in treatment trials of neuroprotective and reperfusion agents for acute ischemic stroke.

Wi-Sun Ryu and colleagues investigate the association of stroke onset time with presenting severity, early neurological deterioration (END), and long-term functional outcome in ischemic stroke.     

Immunoreactive glucagons purified from dog pancreas, stomach and ileum

Previous studies have shown that pig intestine contains a 69 amino acid glucagon (glicentin) as well as a 37 amino acid glucagon (oxyntomodulin). In pig pancreas the 29 amino acid glucagon predominates. Since glucagon is thought to be expressed from a single gene in mammals, these differences in molecular forms indicate differential posttranslational processing of the glucagon precursor by different tissues. In the current study glucagon immunoreactivity (IR) was separately purified from dog pancreas, stomach mucosa and ileum mucosa. Purification and sequence analysis of the different tissue glucagons show that dog pancreas and stomach mucosa contain glucagon-29 while ileum mucosa contains glucagon-37 and glucagon-69. The latter is the major form present with glucagon-37 accounting for only 10-20% of the total ileum glucagon content. The N-terminal 32 amino acid portion of dog glucagon-69 differs at 6 sites from pig glucagon-69: RSLQDTEEKSRSFSAPQTEPLNDLDQMNEDKR... The C-terminal glucagon-37 is identical to pig oxyntomodulin.     

A phase 1/2 clinical trial of enzyme replacement in fabry disease: pharmacokinetic, substrate clearance, and safety studies

Fabry disease results from deficient alpha-galactosidase A (alpha-Gal A) activity and the pathologic accumulation of the globotriaosylceramide (GL-3) and related glycosphingolipids, primarily in vascular endothelial lysosomes. Treatment is currently palliative, and affected patients generally die in their 40s or 50s. Preclinical studies of recombinant human alpha-Gal A (r-halphaGalA) infusions in knockout mice demonstrated reduction of GL-3 in tissues and plasma, providing rationale for a phase 1/2 clinical trial. Here, we report a single-center, open-label, dose-ranging study of r-halphaGalA treatment in 15 patients, each of whom received five infusions at one of five dose regimens. Intravenously administered r-halphaGalA was cleared from the circulation in a dose-dependent manner, via both saturable and non-saturable pathways. Rapid and marked reductions in plasma and tissue GL-3 were observed biochemically, histologically, and/or ultrastructurally. Clearance of plasma GL-3 was dose-dependent. In patients with pre- and posttreatment biopsies, mean GL-3 content decreased 84% in liver (n=13), was markedly reduced in kidney in four of five patients, and after five doses was modestly lowered in the endomyocardium of four of seven patients. GL-3 deposits were cleared to near normal or were markedly reduced in the vascular endothelium of liver, skin, heart, and kidney, on the basis of light- and electron-microscopic evaluation. In addition, patients reported less pain, increased ability to sweat, and improved quality-of-life measures. Infusions were well tolerated; four patients experienced mild-to-moderate reactions, suggestive of hypersensitivity, that were managed conservatively. Of 15 patients, 8 (53%) developed IgG antibodies to r-halphaGalA; however, the antibodies were not neutralizing, as indicated by unchanged pharmacokinetic values for infusions 1 and 5. This study provides the basis for a phase 3 trial of enzyme-replacement therapy for Fabry disease.     

Germline mutations in BMPR1A/ALK3 cause a subset of cases of juvenile polyposis syndrome and of Cowden and Bannayan-Riley-Ruvalcaba syndromes

Juvenile polyposis syndrome (JPS) is an inherited hamartomatous-polyposis syndrome with a risk for colon cancer. JPS is a clinical diagnosis by exclusion, and, before susceptibility genes were identified, JPS could easily be confused with other inherited hamartoma syndromes, such as Bannayan-Riley-Ruvalcaba syndrome (BRRS) and Cowden syndrome (CS). Germline mutations of MADH4 (SMAD4) have been described in a variable number of probands with JPS. A series of familial and isolated European probands without MADH4 mutations were analyzed for germline mutations in BMPR1A, a member of the transforming growth-factor beta-receptor superfamily, upstream from the SMAD pathway. Overall, 10 (38%) probands were found to have germline BMPR1A mutations, 8 of which resulted in truncated receptors and 2 of which resulted in missense alterations (C124R and C376Y). Almost all available component tumors from mutation-positive cases showed loss of heterozygosity (LOH) in the BMPR1A region, whereas those from mutation-negative cases did not. One proband with CS/CS-like phenotype was also found to have a germline BMPR1A missense mutation (A338D). Thus, germline BMPR1A mutations cause a significant proportion of cases of JPS and might define a small subset of cases of CS/BRRS with specific colonic phenotype.     

An amperometric cellobiose dehydrogenase-based biosensor can be used for measurement of cellulase activity

The hemoflavoenzyme cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium has been used in an amperometric redox polymer-based biosensor. Used in conjugation with a FIA system this biosensor can replace colorimetric assays for measuring cellobiose liberated from cellulose in a series of cellulase-containing samples. The biosensor gave the same result as the Somogyi-Nelson method in a less time-consuming and laborious manner. The two methods showed about the same precision.