Anthony S. Serianni

A nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant Alaskan beetle Upis ceramboides

Thermal hysteresis (TH), a difference between the melting and freezing points of a solution that is indicative of the presence of large-molecular-mass antifreezes (e.g., antifreeze proteins), has been described in animals, plants, bacteria, and fungi. Although all previously described TH-producing biomolecules are proteins, most thermal hysteresis factors (THFs) have not yet been structurally characterized, and none have been characterized from a freeze-tolerant animal. We isolated a highly active THF from the freeze-tolerant beetle, Upis ceramboides, by means of ice affinity. Amino acid chromatographic analysis, polyacrylamide gel electrophoresis, UV-Vis spectrophotometry, and NMR spectroscopy indicated that the THF contained little or no protein, yet it produced 3.7 ± 0.3 °C of TH at 5 mg/ml, comparable to that of the most active insect antifreeze proteins. Compositional and structural analyses indicated that this antifreeze contains a β-mannopyranosyl-(1→4) β-xylopyranose backbone and a fatty acid component, although the lipid may not be covalently linked to the saccharide. Consistent with the proposed structure, treatment with endo-β-(1→4)xylanase ablated TH activity. This xylomannan is the first TH-producing antifreeze isolated from a freeze-tolerant animal and the first in a new class of highly active THFs that contain little or no protein.

Carbon-13-enriched carbohydrates. Preparation of aldononitriles and their reduction with a palladium catalyst

Abstract Cyanide condenses with aldoses at 25° in aqueous solution between pH 7.0–9.0 to produce aldononitriles in high yield. These nitriles may be reduced catalytically over palladium-barium sulfate (5%) at pH 4.2 ± 0.1 and 25° to yield the corresponding aldoses in 60–90% yield, depending on the structure of the nitrile. 1-Amino-1-deoxyalditols are produced in approximately 10% yield, and their formation is favored when hemiacetal formation is hindered in the parent aldose. Generally, the product epimeric aldoses can be separated from contaminating by-products and from each other by ion-exchange and adsorption chromatography. This procedure has been applied to the preparation of [1- 13 C]-enriched pentoses and hexoses.

Carbon-13-enriched carbohydrates. Preparation of erythrose, threose, glyceraldehyde, and glycolaldehyde with 13C-enrichment in various carbon atoms

Abstract Two-, three-, and four-carbon aldononitriles were prepared, and catalytically reduced with palladium-barium sulfate (5%) to the coresponding aldoses in high yields at pH 1.7 ± 0.1 and atmospheric pressure. Carbon-13-enriched glycolaldehyde, glyceraldehyde, erythrose, and threose were prepared with enrichment in various carbon atoms, permitting unequivocal assignment of chemical shifts for all carbons and determination of the proportions of cyclic hemiacetals and linear gem -diol forms in solution. Carbon-carbon and carbon-hydrogen coupling-constants for the furanose ring and linear hydrates of these short-chain aldoses are reported and discussed.

Glycerol metabolism in a freeze-tolerant arctic insect: an in vivo 13C NMR study

SummaryFreeze-tolerance in larvae ofGynaephora groenlandica is enhanced by the accumulation of glycerol in the winter. Since summer larvae remain freeze-tolerant despite the lack of glycerol, we investigated glycerol metabolism as a function of acclimation and body temperature using non-invasive13C NMR spectroscopy. Major constituents of hemolymph isolated from cold- and warm-acclimated larvae were identified with the aid of standard NMR spectra and confirmed by TLC and GLC. Spectra obtained on live, warm-acclimated larvae showed the presence of lipids, glycogen, glucose, trehalose and amino acids. Similar spectra of cold-acclimated or previously frozen larvae showed the additional presence of glycerol. In vitro time-lapse13C spectra ofd-[1-13C]glucose added separately to hemolymph or extracted fat body tissue showed that glycerol is synthesized from glucose in the fat body tissue and distributed to the peripheral tissue via hemolymph. In vivo time-lapse13C spectra of cold- and warm-acclimated larvae were obtained after injection withd-[1-13C]glucose to monitor the production of labeled metabolic intermediates and end-products. [13C]Glycerol was produced between −30°C and 30°C but accumulated only below 5°C. Above 5°C glycerol was degraded and the13C label incorporated mainly into glycogen. The mechanism underlying temperature control of glycerol biosynthesis and degradation may provide a clue to the role of glycerol in enhancing freeze-tolerance in these insects.

Comparative overwintering physiology of Alaska and Indiana populations of the beetle Cucujus clavipes (Fabricius): roles of antifreeze proteins, polyols, dehydration and diapause.

SUMMARY The beetle Cucujus clavipes is found in North America over a broad latitudinal range from North Carolina (latitude ∼35°N) to near tree line in the Brooks Range in Alaska (latitude, ∼67°30′ N). The cold adaptations of populations from northern Indiana (∼41°45′ N) and Alaska were compared and, as expected, the supercooling points (the temperatures at which they froze) of these freeze-avoiding insects were significantly lower in Alaska insects. Both populations produce glycerol, but the concentrations in Alaska larvae were much higher than in Indiana insects (∼2.2 and 0.5 mol l–1, respectively). In addition, both populations produce antifreeze proteins. Interestingly, in the autumn both populations have the same approximate level of hemolymph thermal hysteresis, indicative of antifreeze protein activity, suggesting that they synthesize similar amounts of antifreeze protein. A major difference is that the Alaska larvae undergo extreme dehydration in winter wherein water content decreases from 63–65% body water (1.70–1.85 g H2O g–1 dry mass) in summer to 28–40% body water (0.40–0.68 g H2O g–1 dry mass) in winter. These 2.5–4.6-fold reductions in body water greatly increase the concentrations of antifreeze in the Alaska insects. Glycerol concentrations would increase to 7–10 mol l–1 while thermal hysteresis increased to nearly 13°C (the highest ever measured in any organism) in concentrated hemolymph. By contrast, Indiana larvae do not desiccate in winter. The Alaska population also undergoes a diapause while insects from Indiana do not. The result of these, and likely additional, adaptations is that while the mean winter supercooling points of Indiana larvae were approximately –23°C, those of Alaska larvae were –35 to– 42°C, and at certain times Alaska C. clavipes did not freeze when cooled to –80°C.

(13C)-substituted sucrose: 13C-1H and 13C-13C spin coupling constants to assess furanose ring and glycosidic bond conformations in aqueous solution.

Abstract Sucrose (β- d -fructofuranosyl α- d -glucopyranoside, 1), methyl α- d -fructofuranoside (2), and methyl β- d -fructofuranoside (3) have been prepared by chemical and / or enzymic methods with single sites of 13C-substitution at C-1, C-2, C-3, and C-6 of the fructofuranosyl ring . 1H (500 MHz) and 13C (75 and 125 MHz) NMR spectra of 1–3 have been obtained, yielding 1H1H, 13C13C spin coupling constants that were used to assess furanose ring and glycoside bond conformations is aqueous (2H2O) solution. Results show that the conformational mobility of the furanosyl ring in 3 is altered when incorporated into 1. Furthermore, 13C13C and 13C1H spin couplings across the glycosidic linkage suggest a ψ torsion angle different from that observed in the crystal (φ appears similar). Interplay between the strength of the exoanomeric effect and hydrogen bonding in solution may be responsible, in part, for the apparent conformational flexibility of 1. In addition, spin couplings in 2 and 3 have been compared to those measured previously in α- d -threo-pentulofuranose (4) and β- d -threo-pentulofuranose (5), respectively, as a means to study the effect of glycosidation and hydroxymethyl substitution on the solution conformation of the 2-ketofuranose ring. The conversion of 4 to 2 is accompanied by minimal conformational change, whereas a significant change accompanies the conversion of 5 to 3, showing that the effect of substitution on ring conformation depends highly on ring configuration before and after substitution.Sucrose (beta-D-fructofuranosyl alpha-D-glucopyranoside, 1), methyl alpha-D-fructofuranoside (2), and methyl beta-D-fructofuranoside (3) have been prepared by chemical and/or enzymic methods with single sites of 13C-substitution at C-1, C-2, C-3, and C-6 of the fructofuranosyl ring. 1H (500 MHz) and 13C (75 and 125 MHz) NMR spectra of 1-3 have been obtained, yielding 1H-1H, 13C-1H, and 13C-13C spin coupling constants that were used to assess furanose ring and glycoside bond conformations in aqueous (2H2O) solution. Results show that the conformational mobility of the furanosyl ring in 3 is altered when incorporated into 1. Furthermore, 13C-13C and 13C-1H spin couplings across the glycosidic linkage suggest a psi torsion angle different from that observed in the crystal (phi appears similar). Interplay between the strength of the exoanomeric effect and hydrogen bonding in solution may be responsible, in part, for the apparent conformational flexibility of 1. In addition, spin couplings in 2 and 3 have been compared to those measured previously in alpha-D-threo-pentulofuranose (4) and beta-D-threo-pentulofuranose (5), respectively, as a means to study the effect of glycosidation and hydroxymethyl substitution on the solution conformation of the 2-ketofuranose ring. The conversion of 4 to 2 is accompanied by minimal conformational change, whereas a significant change accompanies the conversion of 5 to 3, showing that the effect of substitution on ring conformation depends highly on ring configuration before and after substitution.

Cold-induced mitochondrial degradation and cryoprotectant synthesis in freeze-tolerant arctic caterpillars

SummaryThe larvae ofGynaephora groenlandica, a long-lived moth endemic to the high arctic, are perennially freeze-tolerant and able to increase their freeze-tolerance by synthesizing glycerol. Cold-induced mitochondrial changes were correlated (using electron microscopy, DNA staining, cytochrome c assay, and oxygen uptake) with glycerol production (using NMR spectroscopy) in larvae under different acclimations and in the field. Hypometabolism in summer- or warm-acclimated larvae led to glycerol accumulation. Extended exposure to near-zero or freezing temperatures caused mitochondrial degradation and glycerol accumulation. Rapid freezing of warm-acclimated larvae did not result in mitochondrial breakdown. Mitochondrial reconstitution upon warm-acclimation occurred much more rapidly (<1 week) than did degradation (>2 months). Concomitant with mitochondrial breakdown was reduced oxidative metabolism, but the cytochrome c concentration remained independent of acclimation temperature. The adaptive response to cold by mitochondrial degradation and glycerol accumulation byG. groenlandica may be linked to diapause in other species of ectotherms.

DL-Apiose substituted with stable isotopes: synthesis, n.m.r.-spectral analysis, and furanose anomerization

The branched-chain pentose DL-apiose has been synthesized in good yield by a new and simple chemical method that can be adapted to prepare (1-13C)-, (2-13C)-, (1-2H)- and/or (2-2H)-enriched derivatives. N.m.r. spectra (1H- and 13C-) have been interpreted with the aid of selective (13C)- and (2H)-enrichment, and 2D and 13C[13C]-n.m.r. spectra. The solution composition of DL-(1-13C)apiose in 2H2O, determined by 13C-n.m.r. spectroscopy, has been found to differ from that determined previously by 1H-n.m.r. spectroscopy. Several 13C-1H and 13C-13C couplings have been measured and interpreted in terms of apiofuranose ring conformation. Ring-opening rate-constants of the four apiofuranoses [3-C-(hydroxymethyl)-alpha- and -beta-D-erythrofuranose, and 3-C-(hydroxymethyl)-alpha- and -beta-L-threofuranose] have been determined by 13C-saturation-transfer n.m.r. spectroscopy, and compared to those obtained previously for the structurally related tetrofuranoses.

A thermal hysteresis-producing xylomannan glycolipid antifreeze associated with cold tolerance is found in diverse taxa

The presence of large-molecular-mass, thermal hysteresis (TH)-producing antifreezes (e.g., antifreeze proteins) has been reported in numerous and diverse taxa, including representative species of fish, arthropods, plants, fungi, and bacteria. However, relatively few of these antifreeze molecules have been chemically characterized. We screened diverse species by subjecting their homogenates to ice-affinity purification and discovered the presence of a newly identified class of antifreeze, a xylomannan-based TH-producing glycolipid that was previously reported in one species of freeze-tolerant Alaskan beetle. We isolated xylomannan-based antifreeze glycolipids from one plant species, six insect species, and the first frog species to be shown to produce a large-molecular-mass antifreeze. 1H NMR spectra of the ice-purified molecules isolated from these diverse freeze-tolerant and freeze-avoiding organisms were nearly identical, indicating that the chemical structures of the glycolipids were highly similar. Although the exact functions remain uncertain, it appears that antifreeze glycolipids play a role in cold tolerance.

The role of endogenous antifreeze protein enhancers in the hemolymph thermal hysteresis activity of the beetle Dendroides canadensis

Antifreeze proteins (AFPs) lower the freezing point of water by a non-colligative mechanism, but do not lower the melting point, therefore producing a difference between the freezing and melting points termed thermal hysteresis. Thermal hysteresis activity (THA) of AFPs from overwintering larvae of the beetle Dendroides canadensis is dependent upon AFP concentration and the presence of enhancers of THA which may be either other proteins or low molecular mass enhancers. The purpose of this study was to determine the relative contributions of endogenous enhancers in winter D. canadensis hemolymph.Winter hemolymph collected over four successive winters (1997-1998 to 2000-2001) was tested. The first three of these winters were the warmest on record in this area, while December of the final year was the coldest on record. Protein and low molecular mass enhancers raised hemolymph THA 60-97% and 35-55%, respectively, based on hemolymph with peak THA for each year collected over the four successive winters. However, the hemolymph AFPs were not maximally enhanced since addition of the potent enhancer citrate (at non-physiologically high levels) resulted in large increases in THA. (13)NMR showed that glycerol was the only low molecular mass solute present in sufficiently high concentrations in the hemolymph to function as an enhancer. Maximum THA appears to be approximately 8.5 degrees C.