Corresponding patterns of geographic variation among populations ofSilene latifolia (=S. alba =S. pratensis) (Caryophyllaceae)

Morphological and biochemical data were analysed from 30 greenhouse-grown populations of EuropeanSilene latifolia. Six separate character sets (flavones, seed, pollen, capsules, male and female flower morphology) were used in the analyses. There was broad-scale congruence between trends of geographic variation in most character sets, with the populations being assigned to western (or southern and western) and eastern clusters. The eastern and western clusters abut along a transition zone that runs roughly from Belgium to the northern Balkans; this zone represents a region of relatively rapid change and contains populations intermediate between the eastern and western clusters. Variation in flower morphology was weak and discordant with variation in the other character sets. The origin and maintenance of the variation pattern is discussed in terms of migrational history and hybrid zones.     

Differences in blastema-associated proteins according to position along body axis in regenerating planarians

To test the hypothesis that neoblasts in different positions in regenerating pieces of planarians may be differentially responsive to diffusible stimuli through differentially expressed membrane receptors, we compared membrane surface proteins in blastemas induced at various positions along an anterior-posterior axis in Dugesia gonocephala (Dugès). The proteins were biotinylated and identified by molecular weight in SDS-PAGE (sodium-dodecylsulfate polyacrilamide gel electrophoresis). This SDS-PAGE pattern was then compared with that of N-glycosylated proteins incorporating ³H-mannose. One 20 kDa glycoprotein present in all blastemas at 3 d was absent from more anterior blastemas at 6 d irrespective of whether those blastemas were at the cephalic or the caudal end of the regenerating piece. The expression of this protein appears to be determined by the position of the blastema along the body axis rather than by its prospective fate.     

Lectinomics I. Relevance of exogenous plant lectins in biomedical diagnostics

This review focuses on utilization of plant lectins as medical diagnostic reagents and tools. The lectin-related diagnostic is aimed at detection of several diseases connected to alteration of the glycosylation profiles of cells and at identification of microbial and viral agents in clinical microbiology. Certain lectins, proposed for or used as diagnostic tools could even recognize those cellular determinants, which are not detected by available antibodies. Broad information is presented on the lectinomics field, illustrating that lectin diagnostics might become practical alternative to antibody-based diagnostic products. In addition, the rising trend of lectin utilization in biomedical diagnostics might initiate a development of innovative methods based on better analytical technologies. Lectin microarray, a rapid and simple methodology, can be viewed as an example for such initiative. This technology could provide simple and efficient screening tools for analysis of glycosylation patterns in biological samples (cellular extracts, tissues and the whole cells), allowing thus personalized detection of changes associated with carbohydrate-related diseases.     

Stereochemistry of Cervicarcin

The relative stereochemistry of cervicarcin, an antitumor antibiotic, was determined as shown in 1, which represents the absolute stereochemistry also.     

In vitro and in vivo efficacy of anti‐chikungunya virus monoclonal antibodies produced in wild‐type and glycoengineered Nicotiana benthamiana plants

Chikungunya virus (CHIKV) is a mosquito‐transmitted alphavirus, and its infection can cause long‐term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti‐CHIKV monoclonal antibody (mAb) produced in wild‐type (WT) and glycoengineered (?XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ?XFT carried a single N‐glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N‐glycans including the typical plant GnGnXF₃ glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ?XFT plant‐produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ?XFT‐produced mAbs. This is the first report of the efficacy of plant‐produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.     

Could the glycosylation analysis of seminal plasma clusterin become a novel male infertility biomarker?

Male infertility is becoming a rapidly growing problem around the world, mainly in the highly developed countries. Seminal proteome composition seems to be one of the crucial factors of the proper course of fertilization ‐ clusterin (CLU) is among the most important ones. CLU, as one of the crucial seminal plasma glycoproteins, plays a very important role in sperm capacitation and immune tolerance in the female reproductive tract. CLU is also known as a sensitive marker of oxidative stress. It has six n ‐glycosylation sites and also exhibits chaperone activity. An analysis of changes in the profile and degree of CLU glycosylation may shed some new light on the molecular mechanisms of the fertilization process and may be used as an additional diagnostic marker of male fertility. This study constitutes a review of the recently available literature concerning human seminal CLU, including changes in its glycosylation, analyzed in the context of human reproduction.     

Multivariate data analysis of growth medium trends affecting antibody glycosylation

Use of multivariate data analysis for the manufacturing of biologics has been increasing due to more widespread use of data-generating process analytical technologies (PAT) promoted by the US FDA. To generate a large dataset on which to apply these principles, we used an in-house model CHO DG44 cell line cultured in automated micro bioreactors alongside PAT with four commercial growth media focusing on antibody quality through N-glycosylation profiles. Using univariate analyses, we determined that different media resulted in diverse amounts of terminal galactosylation, high mannose glycoforms, and aglycosylation. Due to the amount of in-process data generated by PAT instrumentation, multivariate data analysis was necessary to ascertain which variables best modeled our glycan profile findings. Our principal component analysis revealed components that represent the development of glycoforms into terminally galacotosylated forms (G1F and G2F), and another that encompasses maturation out of high mannose glycoforms. The partial least squares model additionally incorporated metabolic values to link these processes to glycan outcomes, especially involving the consumption of glutamine. Overall, these approaches indicated a tradeoff between cellular productivity and product quality in terms of the glycosylation. This work illustrates the use of multivariate analytical approaches that can be applied to complex bioprocessing problems for identifying potential solutions.     

Condensation reactions catalyzed by α-N-acetylgalactosaminidase from Aspergillus niger yielding α-N-acetylgalactosaminides

Abstract

Extracellular α-N-acetylgalactosaminidase from Aspergillus niger catalyzed glycosylation yielding a series of 2-acetamido-2-deoxy-α-D-galactobiosides using 2-acetamido-2-deoxy-D-galactopyranose as a glycosyl donor. The isomers α-D-GalpNAc-(1→6)-D-GalpNAc, α-D-GalpNAc-(1→3)-D-GalpNAc and α-D-GalpNAc-(1→6)-D-GalfNAc were isolated and spectrally characterized. The purified enzyme was further used for the glycosylation of free amino acids (serine and threonine) and their N-(tert-butoxycarbonyl)-protected analogs to synthesize the Tn antigen (GalpNAc-α-O-Ser/Thr) and its N-(tert-butoxycarbonyl)-protected derivatives.     

How independent are TSE agents from their hosts?

Central to understanding the nature TSE agents (or prions) is how their genetic information is distinguished from the host. Are TSEs truly infectious diseases with host-independent genomes, or are they aberrations of a host component derived from the host genome? Recent experiments tested whether glycosylation of host PrP affects TSE strain characteristics. Wild-type mice were infected with 3 TSE strains passaged through transgenic mice with PrP devoid of glycans at 1 or both N-glycosylation sites. Strain-specific characteristics of 1 TSE strain changed but did not change for 2 others. Changes resulted from the selection of mutant TSE strains in a novel replicative environment. In general the properties of established TSEs support the genetic independence of TSE agents from the host, and specifically the primary structure of PrP does not directly encode TSE agent properties. However sporadic TSEs, challenge this independency. The prion hypothesis explains emerging TSEs relatively successfully but poorly accounts for the diversity and mutability of established TSE strains, or how many different infectious conformations are sustained thermodynamically. Research on early changes in RNA expression and events at the ribosome may inform the debate on TSE agent properties and their interaction with host cell machinery.