Analysis of protein glycosylation within the nematode Caenorhabditis elegans has revealed an abundant and unreported set of core chitobiose modifications (CCMs) to N-linked glycans. With hydrazine release, an array of glycomers and isobars were detected with hexose extensions on the 3- and 3,6-positions of the penultimate and reducing terminus, respectively. A full complement of structures includes a range of glycomers possessing a Galβ(1-4)Fuc disaccharide at the 3- and 6-positions of the protein-linked GlcNAc. Importantly, enzymatic (PNGase F/A) release failed to liberate many of these extended structures from reduced and alkylated peptides and, as a consequence, such profiles were markedly deficient in a representation of the worm glycome. Moreover, the 3-linked Galβ(1-4)Fuc moiety was notably resistant to a range of commercial galactosidases. For identification, the fragments were spectrum-matched with synthetic products and library standards using sequential mass spectrometry (MSⁿ). A disaccharide observed at the 3-position of penultimate GlcNAc, indicating a Hex-Fuc branch on some structures, was not further characterized because of low ion abundance in MSⁿ. Additionally, a Hex-Hex-Fuc trisaccharide on the 6-position of proximal GlcNAc was also distinguished on select glycomers. Similar branch extensions on 6-linked core fucosyl residues have recently been reported among other invertebrates. Natural methylation and numerous isobars complement the glycome, which totals well over 100 individual structures. Complex glycans were detected at lower abundance, indicating glucosaminyltransferase-I (GnT-I) and GnT-II activity. A range of phosphorylcholine (PC)-substituted complex glycans were also confirmed following a signature two-stage loss of PC during MSⁿ analysis, although the precursor ion was not observed in the mass profiles. In a similar manner, numerous other minor glycans may be present but unobserved in hydrazine-release profiles dominated by fucosylated structures. All CCM structures, including multiple isomers, were determined without chromatography by gas-phase disassembly (MSⁿ) in Paul and linear ion trap (IT) instruments.

To examine osmotic regulation during long-term acclimation to a hyperosmotic medium, hemolymph osmolality, [Na⁺] and total protein, tissue hydration, and free amino acid (FAA) pools in abdominal muscle, gills, central nervous tissue and hemolymph were quantified in the diadromous freshwater (FW) shrimp, Macrobrachium olfersii, during direct exposure to 21‰S seawater over a 20-day period. Hemolymph osmolality and [Na⁺] reach stable maxima within 24 h while total protein is unchanged. Muscle and nerve tissues rapidly lose water while gills hydrate; all tissues attain maximum hydration (+5%) by 5 days, declining to FW values except for gills. Total FAA are highest in muscle, reach a maximum by 2 days (+64%), declining to FW values. Gill FAA increase by 110% after 24 h, diminishing to FW values. Nerve FAA increase 187% within 24 h, and remain elevated. Hemolymph FAA decrease (−75%) after 24 h, stabilizing well below the FW concentration. During acclimation, muscle glycine (+247%), gill taurine (+253%) and proline (+150%), and nerve proline (+426%), glycine (+415%) and alanine (+139%) increase, while hemolymph leucine (−70%) decreases. Total FAA pools contribute 10–20% to intracellular (22–70 mmol/kg) and 0.5–2.4% to hemolymph (3–7 mOsm/kg) osmolalities during direct acclimation from FW. These data emphasize the modest participation of FAA pools in intracellular osmotic regulation during physiological adaptation by M. olfersii to osmotic challenge, accentuating the role of anisosmotic extracellular regulation, suggesting that, during the invasion of freshwater by the Crustacea, dependence on intracellular adjustment employing FAA as osmotic effectors, has become progressively reduced.

The reduction of circulating neutrophil migration to infection sites is associated with a poor outcome of severe sepsis. α-1-Acid glycoprotein (AGP) was isolated from the sera of severely septic patients by HPLC and acrylamide gel electrophoresis and identified by mass spectrometry. Both the isolated protein and commercial AGP inhibited carrageenin-induced neutrophil migration into the rat peritoneal cavity when administered i.v. at a dose of 4.0 μg per rat (95 pmol per rat). Analysis by intravital microscopy demonstrated that both proteins inhibited the rolling and adhesion of leukocytes in the mesenteric microcirculation. The inhibitory activity was blocked by 50 mg/kg aminoguanidine, s.c., and was not demonstrable in inducible nitric oxide synthase (iNOS) knockout mice. Incubation of AGP with neutrophils from healthy subjects induced the production of NO and inhibited the neutrophil chemotaxis by an iNOS/NO/cyclic guanosine 3,5-monophosphate-dependent pathway. In addition, AGP induced thel-selectin shedding by neutrophils. The administration of AGP to rats with mild cecal ligation puncture sepsis inhibited neutrophil migration and reduced 7-day survival from ≈80% to 20%. These data demonstrate that AGP, an acute-phase protein, inhibits neutrophil migration by an NO-dependent process and suggest that AGP also participates in human sepsis.