Isolation of cuticular membranes from various conifer needles

Summary A method of isolating intact needle cuticles is presented. Cuticles were separated enzymatically from needles of Abies alba Mill., Picea abies (L.) Karst., Picea pungens Engelm., Pinus mugo Turra, and Taxus baccata L. Cuticle separation depended on the enzyme concentration, the developmental stage of the needles and the duration of incubation in the hydrolytic pectinase/cellulase solution. Cuticles could not be removed from needles older than 2 years. Scanning electron micrographs of enzymatically isolated cuticles are presented. The permeance coefficients for water and oxygen transport across the isolated cuticular membranes indicate their functional intactness. But permeance coefficients also show that isolation of cuticular membranes with chromic acid is an unacceptable method, since they are lo longer structurally or functionally intact following isolation by this method.     

Cuticular penetration of abscisic acid

Summary Penetration of 2-¹⁴C abscisic acid (ABA) through enzymatically isolated cuticles from tomato fruit and from the upper epidermis of apricot, pear and orange leaves was assessed. Penetration was linear with time, greater as the undissociated than the dissociated ion, and greater through dewaxed than non-dewaxed cuticles. Significantly less (3–6 times) (2-¹⁴C)ABA penetrated the tomato fruit cuticle than NAA or 2,4-D. The leaf cuticles were less permeable than the tomato fruit cuticle. There was no evidence that the ABA was altered during transfer across the cuticle.     

Expanding abdominal cuticle in the bug Rhodnius and the tick Boophilus

The abdominal cuticles of Rhodnius prolixus (fifth instar) and Boophilus microplus (adult female) expand dramatically and rapidly during feeding. In the unfed stage of both species the epicuticle of the abdomen is deeply folded, and when rapid stretching takes place the epicuticle unfolds and the underlying procuticle stretches so that the thickness of the cuticle is halved. The cuticles contained only trace amounts of protein soluble in water and aqueous KCl but substantial quantities were extracted with 7 M aqueous urea. The proteins were analysed for their amino acid composition and investigated by gel electrophoresis and isoelectric focusing.In solubility, amino acid composition, molecular weight distribution, and isoelectric points, the proteins isolated from both species resembled one another closely. They had higher molecular weights and higher isoelectric points than did the proteins from flexible, non-stretching cuticles and unlike them had high alanine and histidine and low aspartic acid and glutamic acid contents. Their amino acid composition was very similar to that of the whole cuticle. The proteins were not associated with neutral sugars. Both the Rhodnius and Boophilus cuticles had low chitin contents, 11·2 and 3·8% respectively (on a water-free basis). The composition of the cuticles and the properties of the proteins are discussed in relation to the stretching which they undergo.     

Separation, isolation and amino acid composition of uremic peptides

Gel filtration and thin layer chromatography were conducted on sera from uremic patients and normal subjects for the isolation of nitrogenous substances unique to uremia. Many ninhydrin-positive substances were found in greater amounts in uremic patients compared to normal subjects. Some of these ninhydrin-positive substances were also detected by staining with chlorine-tolidine. Amino acid analysis of these substances showed considerable qualitative and quantitative differences, perhaps reflecting interference with enzymatic activity by the uremic environment.     

大叶藤黄叶片角质层的酶分离技术

角质层运输特征的研究在生态学、环境和农用化学等方面都具有非常重要的意义 ,完好地分离角质层是进行这方面研究的前提。本文以生长在西双版纳热带雨林中的大叶藤黄为材料 ,探讨了角质层的酶分离技术。结果表明 ,30℃时用 2 % (w/v) ,pH 3 0的果胶酶和纤维素酶溶液浸泡大叶藤黄叶圆片 9h ,叶圆片边缘角质层分离 ,浸泡 5d可分离到完整的角质层 ,角质层在硼酸盐缓冲液 (0 0 1mol·L-1,pH 9 0 )中充分浸泡数日去除石炭酸 ,空气中干燥 ,室温下于聚乙烯的盒子保存备用     

Quantitative determination of catecholic degradation products from insect sclerotized cuticles

Acid hydrolysates of cuticle from various insect species were quantitatively analyzed for five catecholic amino acid adducts. Four of the adducts are ketocatechols; in three of them the amino acid moiety, either lysine, glycine or beta-alanine, is connected via its amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone, in the fourth a tyrosine residue is connected to the same position via its phenolic group. The fifth adduct contains histidine linked via its imidazole-ring to the beta-position of the dopamine sidechain. The three ketocatecholic adducts containing alpha-amino acids were obtained in significant yields from adult cuticles of the locust Schistocerca gregaria, the cockroaches Blaberus craniifer and Periplaneta americana, and the beetles Pachynoda sinuata and Tenebrio molitor, but only in trace amounts from larval and pupal cuticles of T. molitor, pupal cuticles of the moths Manduca sexta and Hyalophora cecropia, and puparia of the blowfly Calliphora vicina. The beta-alanine-containing ketocatechol was not obtained from cuticle of locusts and T. molitor larvae and pupae, but it was present in the hydrolysates of the other cuticles. The beta-histidine-dopamine adduct was obtained from all the cuticles, the highest yield was obtained from adult P. sinuata and the lowest yield was from adult S. gregaria. The beta-histidine-dopamine adduct is derived from the product formed by reaction of p-quinone methides of N-acetyldopamine (NADA) or N-beta-alanyldopamine (NBAD) with histidine residues in the cuticular proteins. The ketocatecholic adducts are assumed to be degradation products of crosslinks formed when oxidized dehydro-NADA reacts with the cuticular proteins. The insect species investigated appear to use both pathways for sclerotization, but to widely differing extents; the dehydro-NADA pathway dominates in cuticles which are exposed to strong deforming forces, such as those of adult locusts and cockroaches, and the p-quinone methide pathway dominates in cuticle of lepidopteran pupae and blowfly puparia, which are not exposed to strong mechanical forces but have to be effectively protected against microbial and fungal attacks.     

Attenuation of UV radiation by plant cuticles from woody species

Transmittance spectra of isolated plant cuticles were measured in the wavelength range from 270 to 600 nm. The cuticles were enzymatically isolated from the leaves of 27 species (26 evergreen or deciduous woody, one succulent herbaceous) and from four species of fruits. With the exception of subtropical and tropical species all plants were cultivated in the field. The cuticles of the species studied strongly attenuated ultraviolet (UV) radiation at wavelengths < 400 nm while they were practically translucent in the visible range. Relatively broad transmittance minima occurred at wavelengths from 280 to 320 nm (UV-B). Spectral transmittances at 300 nm ranged from 0.004 (Ilex aquifolium) to 0.50 (Prunus avium) for leaf cuticles and from 0.00023 (Cydonia oblonga) to 0.005 (Mains domestica) for fruit cuticles. The constitutive UV protection by cuticular pigments may be supplemented, to varying degrees, by pigments located in the epidermal cell wall and protoplast. Thus, it is concluded that only a small fraction of incident UV-B radiation may actually reach the sensitive tissues of the leaves of non-herbaceous species and of fruits.     

CUTICLES FROM CHONDRUS CRISPUS (RHODOPHYTA)1

A simple, nondestructive physical process was developed for routinely isolating the outermost layers from female, male, and sporophyte fronds of Chondrus crispus Stack-house. Yields of pure cuticles from apical segments ranged from 0.74 to 2.35% on a dry weight basis after 5–7 d of culture. These undegraded cuticles were examined by electron microscopy (scanning and transmission electron microscopy), spectroscopy (infrared and X-ray), and chemical means. Cuticles isolated from female or male fronds were characterized by parallel arrays of electron-dense lamellae (typically 6–14) alternating with more electron-transparent regions. The thickness and uniformity of these lamellae provide the physical basis for the iridescence characteristic of C. crispus fronds. Sporophyte fronds are not iridescent. This phenomenon may be explained by the fewer electron-dense cuticular lamellae (usually three to seven) and the fact that these lamellae anastomose freely to form a thin cuticle with a highly irregular substructure. Elements detected by X-ray analysis, in addition to carbon and oxygen, included Mg, Br, S, and Ca in both gametophyte and sporophyte cuticles. Major features of FTIR spectra of all cuticles were absorbances due to proteins. A strong band, indicative of sulfate ester, occurred near 1250 cm^?1 in all cuticle preparations. Gametophyte, but not sporophyte, cuticles absorbed at 935, 846, and 800 cm^?1 consistent with the presence of kappa and/or iota carrageenan. Amino acid analyses showed that sporophyte and gametophyte cuticles were generally similar in gross composition. All contained proline as the principal residue together with significant amounts of cysteine, methionine, and lysine. Protein contents calculated from these analyses ranged from 37.6 to 44.4% on a dry weight basis as compared to 51.5–56.7% calculated from total nitrogen values. Up to 75% of the cuticle mass was solubilized by sodium dodecyl sulfate-β-mercaptoethanol. Three similar migrating bands were seen in female and male cuticle extracts on sodium dodecyl sulfate–polyacrylamide gel electrophoresis; however, none of the three weaker bands from sporophyte cuticles comigrated with those from gametophytes. Chloroform-methanol extraction removed < 3.3% of the cuticle mass, suggesting that lipids were minor components.     

A fluorometric procedure for measuring the neuraminidase activity: its application to the determination of this activity in influenza and parainfluenza viruses

A fluorometric procedure for quantitating the amount of N-acetylneuraminic acid enzymatically released by the neuraminidase activity from N-acetylneuraminyl-lactose (sialyl-lactose) has been developed. The liberated lactose is hydrolyzed with beta-galactosidase, and the released galactose is oxidized with galactose dehydrogenase and NAD+; finally, the NADH produced is measured by fluorometry (excitation at 340 nm and analysis of emitted light at 465 nm). The fluorometric assay is about 10-fold more sensitive than the spectrophotometric procedure that measures NADH at 340 nm. It readily measures amounts as little as 2 nmol of sialic acid, and does not require the use of radioactive isotopes. Interferences due to sucrose or other substances, which cause errors in some cases with the use of the periodate-thiobarbiturate method for neuraminidase activity determination, are avoided. The procedure reported here provides a sensitive, rapid, and relatively simple method (feasible with commercialized reagents) for measuring the neuraminidase activity not only in purified samples from different sources but also directly in biological materials such as viruses. The technique has been tested with some viruses recently isolated belonging to Orthomyxoviridae or Paramyxoviridae families, known to be rich in neuraminidase. Reciprocally, this method can also be employed for determining the sialic acid concentration in acylneuraminyl-lactose-containing compounds when using purified neuraminidase for hydrolysis.     

Chlorinated tyrosine derivatives in insect cuticle

A method for quantitative measurement of 3-monochlorotyrosine and 3,5-dichlorotyrosine in insect cuticles is described, and it is used for determination of their distribution in various cuticular regions in nymphs and adults of the desert locust, Schistocerca gregaria. The two chlorinated tyrosine derivatives were present in all analyzed regions in mature adult locusts, the highest concentrations were found in the sclerotized cuticle of femur and tibia, but significant amounts were also present in the unsclerotized arthrodial membranes. Small amounts of the two amino acids were obtained from pharate, not-yet sclerotized cuticle of adult femur and tibia, the amounts increased rapidly during the first 24 h after ecdysis and more slowly during the next two weeks. Control analyses using stable isotope dilution mass spectrometry have confirmed that the chlorinated tyrosines are not artifacts formed during sample hydrolysis. Mono- and dichlorotyrosine are also present in cuticular samples from other insect species, such as the beetle, Tenebrio molitor, the moth Hyalophora cecropia, the cockroach Blaberus craniifer, and the bug Rhodnius prolixus, but not in the sclerotized puparial cuticle of the blowfly, Calliphora vicina, or in sclerotized ootheca from the cockroach, Periplaneta americana. Cuticular sclerotization and formation of chlorotyrosines occur simultaneously in locust legs; sclerotized cuticles tend to have a higher content of chlorotyrosines than unsclerotized cuticles, but it is concluded that the chlorotyrosines are not just a by-product from the sclerotization process.     

Differential effects of Mg2+ on various hydrolytic and synthetic activities of multifunctional glucose-6-phosphatase

The adaptive synthesis of fatty acid synthetase in the livers of rats fed a fat-free diet following 48 hr of fasting has been studied using immunochemical methods. The development of fatty acid synthetase activity during adaptive synthesis occurs about 3 hr following feeding, whereas the synthesis of material precipitable by anti-fatty acid synthetase serum, as judged by the incorporation of ³H-labeled amino acids into the immunoprecipitate, commenced within 1 hr. Extracts of liver of rats fed a fat-free diet for 1–3 hr following fasting contain increasing amounts of material which competes with purified fatty acid synthetase for antibody binding sites, even though they have no fatty acid synthetase activity. This suggests the presence of enzymatically inactive precursors of fatty acid synthetase in the liver extracts. The incorporation of [¹⁴C]pantothenate into fatty acid synthetase during adaptive synthesis follows the same pattern as the development of enzyme activity, indicating that these enzymatically inactive precursors of fatty acid synthetase may represent an apoenzyme which is converted to the enzymatically active holoenzyme by the incorporation of the 4′-phosphopantetheine prosthetic group. The subcellular site of synthesis of fatty acid synthetase was shown to be in the pool of polysomes that are not membrane bound, rather than in the rough endoplasmic reticulum.     

Characterization of the diffusion of non-electrolytes across plant cuticles: properties of the lipophilic pathway

Systemic crop protection products are commonly sprayed onto foliage, whereupon the active substances must penetrate into the leaves in order to become biologically active. Penetration of the plant cuticle is the rate-limiting step. The diffusion of organic non-electrolytes within cuticles is a purely physical process that can be described and analysed in the same way as is done for diffusion in synthetic polymer membranes. Solute mobilities in cuticles vary considerably between plant species. For a given species they decrease with increasing solute size, and this size selectivity holds for all of the plant species investigated so far. Wax extraction from leaf cuticles increases the mobility of solutes tremendously, but size selectivity is not affected. Furthermore, diffusion within plant cuticles is extremely temperature dependent. An analogous increase in solute mobility can be achieved by using accelerators, which enhance the fluidity of the polymer matrix and of the waxes. The effects of temperature and plasticizers on the diffusion of non-electrolytes in wax and the cutin matrix have been used to characterize the nature of the lipophilic pathway. The 'free volume' theory can be used to explain the influence of the size and shape of the solute, and its dependence on temperature. The physico-chemical nature of the diffusion pathway has been shown, by thermodynamic analysis, to be identical for a wide range of solute lipophilicities. This approach also explains the mode of action and the intrinsic activity of plasticizers.     

Leaf cuticles behave as asymmetric membranes : evidence from the measurement of diffusion potentials

Cuticles were isolated enzymatically from the leaves of two maple species (Acer saccharum Marsh and A. platanoides L.) and from orange (Citrus aurantium L.). The cuticles were placed in a plastic cuvette and different concentrations of KCl were perfused over the physiological inner and outer surfaces while the electrical potential (E¹⁰) that developed across the cuticles and was caused by ion diffusion was measured. E¹⁰ was always positive, indicating that the permeability of K⁺ was always greater than that of Cl^-. Measured E¹⁰ in cuticles did not fit the Goldman equation, whereas, E¹⁰ measured during KCl diffusion across selected artificial membranes fit the equation. The magnitude of E¹⁰ in cuticles and artificial membranes also was dependent on ionic strength, decreasing as ionic strength increased. These observations are explained by combining classical transport equations with equations that describe the equilibrium ion distribution between ionic double layers in the cuticle or membranes and the bathing solution.     

套袋与未套袋苹果果实表皮及心部真菌种群结构与变化动态

2011年在山东海阳,2013年在蓬莱和栖霞选取当地常规管理的苹果园,采用传统组织分离法对套袋苹果、未套袋苹果的果实表皮及心部组织进行真菌分离并进行形态学和分子生物学鉴定。对不同时期采集的套袋及未套袋果实表皮与心部的真菌种类、菌落数量、组织分离率、多样性、相似性系数、相对分离频率等指标进行统计分析。结果表明,两个年度内从果实表皮共分离获得真菌43属,从心部获得真菌31属,相对分离频率较高的真菌包括：链格孢Alternaria spp.、枝状枝孢Cladosporium cladosporioides、镰孢菌Fusarium spp.、青霉菌Penicillium spp.等。研究结果表明：果实套袋后,在果实表皮定殖真菌的种类减少,多样性降低,而数量增加,组织带菌率升高;随果实生长,套袋果实表皮上的真菌种类与未套袋果实表皮真菌种类的相似度逐渐降低;7、8月份,套袋果实表皮上真菌种类明显减少,至8月底,套袋果实表面只有链格孢等少数几种优势真菌定殖。果实套袋后,定殖于果实心部的真菌种类和数量有所波动,套袋果和未套袋果上所分离真菌的相似度有所降低;5月底自果实心部分离真菌的种类和数量与9月底所分离真菌的种类和数量差异不大。     

Movement of Cations through Cuticles of Citrus aurantium and Acer saccharum: Diffusion Potentials in Mixed Salt Solutions

We examined some biophysical mechanisms of ion migration across leaf cuticles enzymatically isolated from Acer saccharum L. and Citrus aurantium L. leaves. Diffusion potential measurements were used to calculate the permeabilities of Cl^-, Li⁺, Na⁺, and Cs⁺ ions all as a ratio with respect to the permeability of K⁺ in cuticles. In 2 millimolar ionic strength solutions the permeability sequence from high to low was K = Cs > Na > Li » Cl. When the outer and inner surfaces of cuticles were bathed in artificial precipitation and artificial apoplast, respectively, diffusion potentials ranging from −52 to −91 millivolts were measured (inside negative). The Goldman equation predicted that the measured potentials were enough to increase the driving force on the accumulation of heavy metals by a factor of 4 to 7. Other ions migrate with forces 3 to 10 times less than predicted by the Goldman equation for concentration differences alone. Our analysis showed that Ca²⁺, and perhaps Mg²⁺, might even be accumulated against concentration gradients under some circumstances. Their uptake was apparently driven by the diffusion potentials created by the outward migration of monovalent salts. We feel that future models predicting leaching of nutrients from trees during acid rain events must be modified to account for the probable influence of diffusion potentials on ion migration.     

STRUCTURE OF THE PEAR LEAF CUTICLE WITH SPECIAL REFERENCE TO CUTICULAR PENETRATION

Study of the pear leaf cuticle (Pyrus communis L. ‘Bartlett‘), in both intact and enzymatically isolated forms, has revealed that the cuticular membrane is separated from the underlying epidermal cell wall by a layer of pectic substances which extend into but not through the membrane. A layer of embedded birefringent waxes occurs towards the outer surface of the cuticular membrane. Platelet-like epicuticular waxes are deposited on the outer surface. The upper cuticular membrane is astomatous. The lower epidermis is stomatous, and the outer cuticular membrane is continuous with that lining the substomatal cavity. The lower cuticular membrane is also generally thicker than the upper, and both the upper and lower cuticular membranes are thicker over veinal than over mesophyll tissue. The birefringence frequently is discontinuous over anticlinal walls and over veinal tissue. The lower cuticle appears to contain fewer embedded waxes (as indexed by birefringence) than the upper. Enzymatic isolation of the cuticular membrane from the underlying tissues does not appear to cause any discernible change in structure as viewed with a light microscope. These findings are discussed in light of current knowledge concerning penetration of foliar applied substances into the leaf.     

Gas permeability of plant cuticles

Cuticles from the adaxial surface of Citrus aurantium L. leaves and from the pericarp of Lycopersicon esculentum L. and Capsicum annuum L. were isolated enzymatically and their oxygen permeability was determined. Isolated cuticles were mounted between a gaseous and an aqueous compartment with the physiological outer side of the membrane facing the gaseous compartment. Permeability for oxygen was characterized by permeability (P) and diffusion (D) coefficients. P and D were independent of the driving force (gradient of oxygen concentration) across the cuticle, thus, Henry's law was obeyed. P values for the diffusion of oxygen varied between 3·10^-7 (Citrus), 1.4·10^-6 (Capsicum), and 1.1·10^-6 (Lycopersicon) m·s^-1. Extraction of soluble lipids from the cuticles increased the permeability. By treating the cutin matrix and the soluble lipids as resistances in series, it could be demonstrated that the soluble lipids were the main resistance for oxygen permeability in Citrus cuticles. However, in Lycopersicon and Capsicum, both the cutin matrix and the soluble lipids determined the total resistance. P values were not affected by either the proton concentration (pH 3–9) or the cations (Na⁺, Ca²⁺) present at the morphological inner side of the cuticles. It is concluded that the water content of cuticles does not affect the permeability properties for oxygen. Partition coefficients indicated a high solubility of oxygen in the cuticle of Citrus. The data suggest a solubility process in the cuticle of Citrus with respect to oxygen permeation.Abbreviations CM cuticular membrane - MX cutin polymer matrix - SCL soluble cuticular lipids     

Biomacromolecules of Algae and Plants and their Fossil Analogues

A review of our current understanding of resistant biomacromolecules derived from present and past algae and higher plants is presented. Insight in the nature of recent and fossil macromolecules is strongly hampered by the difficulties in obtaining the material in pure and unaltered form. For the extant material, avoiding artificial condensation and structural alteration as a result of chemical isolation and purification of biomacromolecules requires constant attention. To date, several types of sporopollenin seem to occur. One type is characterised by oxygenated aromatic building blocks, in particular p-coumaric acid and ferrulic acid. The other type is thought to consist predominantly of an aliphatic biopolymer. In this review it is concluded that extant sporopollenin consists of the aromatic type, whereas the aliphatic component of fossil sporopollenin is due to early-diagenetic oxidative polymerization of unsaturated lipids. The cuticles of most higher plants contain the aliphatic biopolyester cutin. Additionally, cuticles of drought-adapted, mostly CAM plants, seem to contain the non-hydrolysable aliphatic biopolymer cutan. Only a very few algae are able to biosynthesize resistant, (fossilisable) cell walls: some Chlorophyta, Eustigmatophyta and Prasinophyta produce the aliphatic biopolymer algaenan. Some Dinophyta are also capable of producing algaenan cell walls. Additionally, some taxa produce highly resistant cyst-walls with a high proportion of aromatic moieties. For the morphologically well-preserved fossil material, contamination by organic particles other than the target taxon is hard to eliminate and can contribute to either the aliphatic or aromatic signal. Furthermore, post-mortem migration of aliphatic moieties into, and their condensation onto the macromolecule might occur, e.g. by oxidative polymerization. These phenomena hamper the evaluation of the aliphatic signature of fossil plant material and may for example explain the preservation of initially cutin-based cuticles or non-algaenan containing algae. The extent to which migration and in situ formation of aromatic moieties plays a role in modifying resistant algal macromolecules, notably under elevated temperature and/or pressure conditions, still remains an open question.     

Amber from the Alpine Triassic of Lunz (Carnian,Austria): a classic palaeobotanical site

Amber from the Triassic (Carnian) of Lunz, a locality which has produced a rich and famous fossil flora, was analysed using UV‐B‐fluorescence, attenuated total reflectance Fourier transform infrared spectroscopy (ATR‐FTIR) and pyrolysis gas chromatography mass spectroscopy (Py‐GC‐MS). The amber is classified as a class Ib resinite based on its chemical composition, which is characterized by bicyclic products derived from a regular labdatriene structure and the absence of succinic acid. The presence of diterpenoids and absence of triterpenoids is clear evidence of a gymnosperm origin for the Lunz amber, and the prevalence of sesquiterpenoids and diterpenoids point to a conifer family as a possible botanical source. In search of amber attached to identifiable floral remains we have screened new and historical collections of the macroflora from a number of localities of the Lunz area using UV‐B, and describe a striking yellow UV‐B‐fluorescence of cuticles of pteridosperms, ginkgophytes and conifers. This varies with the diagenetic state of the sediment and may be lost altogether. Detectable amounts of fluorescent compounds were observed in gymnosperm taxa, but not in any ferns and only very weakly in horsetails. This underlines that fluorescent compounds are derived from gymnosperm plants, mostly likely arising secondarily from cyclic hydrocarbons by desaturation during diagenesis as a parallel process in amber as well as in cuticles.     

Tyrosine-derived cross-linking amino acids in the sheath of Haemonchus contortus infective larvae

The sheath or second-molt cuticle (2M) was isolated from in vitro exsheathed Haemonchus contortus infective larvae (L3[2M]). Acid hydrolysates of 2-mercaptoethanol (2ME)-soluble and 2ME-insoluble cuticular proteins were analyzed by high performance liquid chromatography for tyrosine-derived cross-linking amino acids. Dityrosine and isotrityrosine were identified by their chromatographic behavior, absorbance spectra, and other chemical characteristics in both the 2ME-soluble and 2ME-insoluble fractions. Dityrosine and isotrityrosine were found in greater amounts in the 2ME-insoluble proteins. When intact 2M cuticles were labeled with 125I prior to acid hydrolysis, radiolabel was recovered in tyrosine but not dityrosine or isotrityrosine indicating that the tyrosine cross-links are not susceptible to iodination in the intact protein. The results are consistent with a hypothesis that tyrosine-derived cross-links are important components of H. contortus 2M cuticular proteins.