Characterization of the N-glycans of recombinant bee venom hyaluronidase (Api m 2) expressed in insect cells.

Honeybee venom hyaluronidase (Api m 2) is a major glycoprotein allergen. Previous studies have indicated that recombinant Api m 2 expressed in insect cells has enzyme activity and IgE binding comparable with that of native Api m 2. In contrast, Api m 2 expressed in Escherichia coli does not. In this study, we characterized the carbohydrate side chains of Api m 2 expressed in insect cells, and compared our data with the established carbohydrate structure of native Api m 2. We assessed both the monosaccharide and the oligosaccharide content of recombinant Api m 2 using fluorophore-assisted carbohydrate electrophoresis and HPLC. To identify the amino acid residues at which glycosylation occurs, we digested recombinant Api m 2 with endoproteinase Glu-C and identified the fragments that contained carbohydrate by specific staining. Recombinant Api m 2 expressed in insect cells contains N-acetylglucosamine, mannose, and fucose, as well as trace amounts of glucose and galactose, and the oligosaccharide analysis is consistent with heterogeneous oligosaccharide chains consisting of two to seven monosaccharides. No sialic acid or N-acetylgalactosamine were detected. These results are similar to published data for native Api m 2, although some monosaccharide components appear to be absent in the recombinant protein. Analysis of proteolytic digests indicates that of the four candidate N-glycosylation sites, carbohydrate chains are attached at asparagines 115 and 263. Recombinant Api m 2 expressed in insect cells has enzymic activity and IgE binding comparable with the native protein, and its carbohydrate composition is very similar.     

Integrated photothermal flow cytometry in vivo

The capability of integrated flow cytometry to detect, in real time, moving cells in their natural states in vivo is demonstrated in a study of circulating red and white blood cells in lymph and blood flow of rat mesentery. This system combines dual pump-probe photothermal (PT) techniques, such as PT imaging, the PT thermolens method, and PT velocimetry, with high-resolution (up to 0.3 microm), high-speed (up to 1000 fps) transmission digital microscopy (TDM) and fluorescence imaging. All PT techniques are based on irradiation of cells in rat mesenteric microvessels with a spectrally tunable laser pulse (420 to 570 nm, 8 ns, 0.1 to 300 microJ) and on detection of temperature-dependent variations of the refractive index with a second continuous probe laser beam (633 nm, 1.4 mW). We focus on intravital monitoring of the integral PT response from single, moving, unlabeled cells (from 100 to 500 cells in one measurement). Potential in vivo applications of this new optical tool, called PT flow cytometry (PTFC), are discussed, including identification of selected cells with differences in natural absorptive properties and sizes, determination of laser-induced cell damage, estimation of flow velocity, and monitoring of circulating cells labeled with PT probes.     

Analysis of the butyrylcholinesterase gene and nearby chromosome 3 markers in Alzheimer disease

The K-variant of butyrylcholinesterase (BCHE-K) recently has been reported to be associated with Alzheimer disease (AD) in carriers of the epsilon4 allele of the apolipoprotein E (APOE) gene. We have re- examined the frequency of the BCHE-K allele in a large data set of both sporadic and familial cases of AD disease, and we have also examined the segregation of three genetic markers on chromosome 3 near BCHE . Our data neither support an association of BCHE-K with sporadic or familial AD, nor do they suggest the existence of another gene nearby on chromosome 3 as a common cause of familial AD.      

Effect of sarcomere length on step size in relaxed rabbit psoas muscle

Recent experiments have shown that shortening and stretching of sarcomeres in single activated and unactivated myofibrils occur in stepwise fashion (Yang et al. (1998) Biophys J 74: 1473-1483; Blyakhman et al. (2001) Biophys J 81: 1093-1100; Yakovenko et al. (2002) Am J Physiol Cell Physiol 283: 735-742). Here, we carried out measurements on single myofibrils from rabbit psoas muscle to investigate steps in unactivated specimens in more detail. Activated and unactivated myofibrils were released and stretched in ramp-like fashion. The time course of length change in the single sarcomere was consistently stepwise. We found that in the unactivated myofibrils, step size depended on initial sarcomere length, diminishing progressively with increase of initial sarcomere length, whereas in the case of activated sarcomeres, step size was consistently 2.7 nm.     

Transmission of human cytomegalovirus from infected uterine microvascular endothelial cells to differentiating/invasive placental cytotrophoblasts

Analysis of placentas infected with human cytomegalovirus (CMV) suggested that viral transmission could involve differentiating/invasive cytotrophoblasts in villi that attach the placenta to the uterine wall. To parse the cellular components in this process, we developed a coculture system of polarized uterine microvascular endothelial cell (UtMVEC) infection with an endothelial cell-tropic pathogenic strain of CMV. Then we evaluated the potential role of neutrophils and endothelial cells in the spread of infection to differentiating cytotrophoblasts. As shown by immunocytochemistry and analysis of viral replication, CMV preferentially infected endothelial cells via apical membranes and disrupted cell junction proteins, thereby altering paracellular permeability and cell polarity. Neutralizing antibodies to CMV glycoprotein B, an envelope component that facilitates virion penetration, blocked plaque formation in polarized UtMVEC. Neutrophils transmitted CMV infection to UtMVEC, which in turn infected cytotrophoblasts. However, neutrophils did not directly infect cytotrophoblasts. These findings implicate endothelial cells from the uterine microvasculature as a potential source for CMV infection of endovascular cytotrophoblasts of the anchoring villi. Possibly the cytokine/chemokine milieu in the pregnant uterus could attract immune cells that infect endothelial cells in hybrid fetal-maternal vessels. In turn, these cells could infect endovascular cytotrophoblasts, one possible initiation point of a cascade that results in retrograde placental CMV infection.     

Intergenerational instability of the CAG repeat of the gene for Machado- Joseph disease (MJD1) is affected by the genotype of the normal chromosome: implications for the molecular mechanisms of the instability of the CAG repeat

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by unstable expansion of a CAG repeat in the MJD1 gene at 14q32.1. To identify elements affecting the intergenerational instability of the CAG repeat, we investigated whether the CGG/GGG polymorphism at the 3' end of the CAG repeat affects intergenerational instability of the CAG repeat. The [expanded (CAG)n-CGG]/[normal (CAG)n- GGG] haplotypes were found to result in significantly greater instability of the CAG repeat compared to the [expanded (CAG)n- CGG]/[normal (CAG)n-CGG] or [expanded (CAG)nGGG]/[normal (CAG)n-GGG] haplotypes. Multiple stepwise logistic regression analysis revealed that the relative risk for a large intergenerational change in the number of CAG repeat units (< -2 or > 2) is 7.7-fold (95% CI: 2.5-23.9) higher in the case of paternal transmission than in that of maternal transmission and 7.4-fold (95% CI: 2.4-23.3) higher in the case of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes than in that of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n- GGG]/[normal (CAG)n-GGG] haplotypes. The combination of paternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes resulted in a 75.2-fold (95% CI: 9.0-625.0) increase in the relative risk compared with that of maternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n- GGG]/[normal (CAG)n-GGG] haplotypes. The results suggest that an inter- allelic interaction is involved in the intergenerational instability of the expanded CAG repeat.      

Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia

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Humoral immune response to thymidylate synthase in colon cancer patients after 5-FU chemotherapy

Thymidylate synthase (TYMS), the critical enzyme for DNA synthesis and a target for chemotherapy, was recently characterized as an oncogene and a potential target for specific immunotherapy. Here we report TYMS-specific antibody response in a fraction of colon cancer patients. Humoral immune response to TYMS is induced by chemotherapy using TYMS inhibitors, such as 5-fluorouracil (5-FU), and may be associated with tumor burden. Therefore, TYMS may serve as a useful serological biomarker for monitoring the course of disease and treatment in cancer patients.