Atmospheric Carbon and Transport - America (ACT-America) Data Sets: Description, Management, and Delivery.

The ACT-America project is a NASA Earth Venture Suborbital-2 mission designed to study the transport and fluxes of greenhouse gases. The open and freely available ACT-America data sets provide airborne in situ measurements of atmospheric carbon dioxide, methane, trace gases, aerosols, clouds, and meteorological properties, airborne remote sensing measurements of aerosol backscatter, atmospheric boundary layer height and columnar content of atmospheric carbon dioxide, tower-based measurements, and modeled atmospheric mole fractions and regional carbon fluxes of greenhouse gases over the Central and Eastern United States. We conducted 121 research flights during five campaigns in four seasons during 2016-2019 over three regions of the US (Mid-Atlantic, Midwest and South) using two NASA research aircraft (B-200 and C-130). We performed three flight patterns (fair weather, frontal crossings, and OCO-2 underflights) and collected more than 1,140 h of airborne measurements via level-leg flights in the atmospheric boundary layer, lower, and upper free troposphere and vertical profiles spanning these altitudes. We also merged various airborne in situ measurements onto a common standard sampling interval, which brings coherence to the data, creates geolocated data products, and makes it much easier for the users to perform holistic analysis of the ACT-America data products. Here, we report on detailed information of data sets collected, the workflow for data sets including storage and processing of the quality controlled and quality assured harmonized observations, and their archival and formatting for users. Finally, we provide some important information on the dissemination of data products including metadata and highlights of applications of ACT-America data sets.

A macrophage invasion mechanism for mycobacteria implicating the extracellular domain of CD43.

We studied the role of CD43 (leukosialin/sialophorin), the negatively charged sialoglycoprotein of leukocytes, in the binding of mycobacteria to host cells. CD43-transfected HeLa cells bound Mycobacterium avium, but not Salmonella typhimurium or Shigella flexneri. Quantitative bacteriology showed that macrophages (M(phi)) from wild-type mice (CD43(+/+)) bound M. avium, Mycobacterium bovis (bacillus Calmette-Guérin), and Mycobacterium tuberculosis (strain H37Rv), whereas M(phi) from CD43 knockout mice (CD43(-/)-) did not. Fluorescence microscopy demonstrated that the associated M. avium had been ingested by the CD43(+/+) M(phi). The inability of CD43(-/)- M(phi) to bind M. avium could be restored by addition of galactoglycoprotein (Galgp), the extracellular mucin portion of CD43. The effect of Galgp is not due to opsonization of the bacteria, but required its interaction with the M(phi) other mucins had no effect. CD43 expression by the M(phi) was also required for optimal induction by M. avium of tumor necrosis factor (TNF)-alpha production, which likewise could be reconstituted by Galgp. In contrast, interleukin (IL)-10 production by M. avium-infected M(phi) was CD43 independent, demonstrating discordant regulation of TNF-alpha and IL-10. These findings describe a novel role of CD43 in promoting stable interaction of mycobacteria with receptors on the M(phi) enabling the cells to respond specifically with TNF-alpha production.

Myocardial protection during ischemia by prior feeding with the creatine analog: cyclocreatine.

The ability of 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine), a synthetic creatine analog, to protect myocardium during global ischemia was assessed in isovolumic rat hearts using phosphorus-31 nuclear magnetic resonance spectroscopy. Wistar rats were fed a 1% cyclocreatine diet. After 2 weeks, cyclocreatine-fed (n = 8) and control (n = 7) rats were anesthetized, the heart was excised and retrograde perfusion was begun at 10 ml/min per g with 37 degrees C, phosphate-free buffer containing glucose and oxygen. Hemodynamic and spectroscopic data were obtained during baseline, ischemia and recovery periods (each 24 min). During ischemia, the heart of control rats developed a rigor-like increase in tonic pressure (ischemic contracture) not seen in the heart of cyclocreatine-fed rats (22 versus 1 mm Hg, p less than 0.01). This change was associated with significantly more adenosine triphosphate (ATP) at end-ischemia in the cyclocreatine group (1.6 versus 0.6 mumol/g, p less than 0.01) and delayed development of acidosis (p less than 0.001). With reperfusion, the heart of cyclocreatine-fed rats spontaneously defibrillated sooner than did the heart in control rats (178 versus 346 s, p less than 0.03). Diastolic pressure remained significantly elevated throughout recovery in control hearts compared with treated hearts (p less than 0.001). Prior feeding with cyclocreatine preserves myocardial adenosine triphosphate during ischemia, delays the development of acidosis and ischemic contracture and improves recovery of mechanical function on reperfusion.

A recombinant peptide model of the first nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator: comparison of wild-type and delta F508 mutant forms.

A series of recombinant peptides, each including the sequence proposed to be the first nucleotide-binding fold of cystic fibrosis transmembrane conductance regulator (CFTR), has been produced in an attempt to find a model peptide that would autologously fold into a soluble structure with native-like properties. The peptide NBDIF, which contains the 267-amino acid sequence of CFTR from 384 to 650, meets these requirements. The peptide was produced with a high expression bacterial plasmid pRSET, purified from inclusion bodies following solubilization with 6 M guanidine-HCl and refolded from 8 M urea. Competitive displacement of trinitrophenol-ATP by nucleotides reveals binding of ATP and related nucleotides with KDs in the low micromolar range; the KD for ATP gamma S is 1.0 +/- 0.4 microM and for ADP 8.8 +/- 3.1 microM. The native-like character of the model peptide's structure is further supported by the findings that the KD for the ATP analog, 5'-adenylimidodiphosphate, is fourfold lower than the KD for the methylene analog, 5'-adenylmethylenediphosphonate, and that ATP binding slows the trypsin proteolysis of NBDIF. The CD spectra of NBDIF and the parallel peptide containing the most common cystic fibrosis mutation, deletion of Phe 508, are essentially indistinguishable, both spectra indicating 28% alpha-helix and 23% beta-sheet, with insignificant differences in the amounts of beta-turns and random structure. Extensive investigation using multiple conditions with highly purified preparations of the model peptides demonstrates that they do not support ATP hydrolysis. These large recombinant peptides offer practical models for the investigation of the first nucleotide-binding domain of CFTR.

Rheological studies of the interaction of mucins with alginate and polyacrylate.

The associative interaction of purified ovine and porcine submaxillary mucins (OSM and PSM) with sodium alginate was evaluated by comparing the rheological properties of mixtures against those of pure alginate and mucin in dilute, semi-dilute, and concentrated solutions. These systems were investigated as models for the interaction of mucin with the extracellular alginate produced by Pseudomonas aeruginosa. In dilute solution, evidence for such interaction cannot be obtained because aggregate species exist both in the OSM-alginate mixtures as well as in pure OSM. However, in the semi-dilute regime, mixtures containing a higher proportion of mucin show systematically higher viscosities than those predicted by simple additivity. In concentrated solutions containing higher proportions of mucin, an enhanced elastic response is observed. These results demonstrate a substantial binding interaction of mucins with alginate. This property is not observed in mixtures containing a high proportion of alginate, suggesting that mucins possess relatively low numbers of interacting sites. Introduction of 3 mM Ca2+ ions to all mucin-alginate mixtures enhances the elasticity due to gelation of alginate. Finally, comparison of the rheological properties of PSM-alginate mixtures with those of PSM-polyacrylate mixtures indicates that the binding strength of alginate to mucin is significantly weaker than that of polyacrylate.

Light-scattering studies of fractionated ovine submaxillary mucins.

Static and dynamic light-scattering studies of solutions of ovine submaxillary mucin (OSM) glycoproteins, fractionated by exclusion chromatography on Sephacryl S-1000, are reported. These experiments yielded information regarding the structure and conformation of the glycoprotein chain, in the form of weight-average molecular weights, Mw, z-average radius of gyration, Rg,z, and z-average of the inverse hydrodynamic radius, (Rh-1)z. The values of (Rh-1)z are found to correlate very well with the S-1000 elution volume characteristics for four OSM fractions of different molecular weights. The structural parameters for these OSM fractions are, within experimental error, similar to those deduced for porcine submaxillary mucins (PSM) in earlier studies. The results suggest that, like PSM, the glycoprotein structure of OSM consists of linear chains constructed by covalently linking two or more elementary subunits together via disulfide bonds. In addition, the rigidity of the protein core of OSM is substantially greater than that observed for non-glycosylated-polypeptide random coils. Because (Rh-1)z, and hence, elution volume depends only on the molecular weight of the mucin protein core, the Mw calibration obtained for OSM should be applicable to the chromatography of other mucin glycoproteins.

Solvent effects on the viscoelastic behavior of porcine submaxillary mucin.

Rheological methods have been used to investigate the intermolecular interactions of porcine submaxillary mucins (PSM) in solution. PSM is a high molecular weight glycoprotein consisting of a linear, semi-flexible protein backbone to which a large number of oligosaccharides (1-5 saccharide units) are attached as side chains. Concentrated aqueous solutions of PSM containing different amounts of guanidine hydrochloride (GdnHCl) were subjected to both controlled stress and controlled strain rheological analyses. In the absence of GdnHCl, PSM solutions exhibit viscoelastic properties characteristic of a gel: the storage modulus, G', is much larger than the loss modulus, G", at all deformation frequencies, and the compliance is 100% recoverable at small stresses, indicative of strong intermolecular interactions. In 3.0 M aqueous GdnHCl, PSM forms a viscoelastic solution, with G" > G' at all frequencies and a relatively small recoverable compliance, pointing to disruption of the intermolecular interactions by the chaotropic salt. Intermediate behavior is observed in 1.5 M GdnHCl, characteristic of a marginal gel: G' approximately G" and greater than 50% recoverable compliance. In dilute solution, PSM behaves viscoelastically as a typical polyelectrolyte. However, concentrated solutions are turbid, the turbidity decreasing as GdnHCl is added, indicating that extensive intermolecular association accompanies the gelation process. The results show that although PSM is secreted in nature as a viscous solution, it can form gels that are similar to those of tracheobronchial and gastric mucins, and suggest common features to the gelation mechanism, with the strength of the gel correlated with the length of the oligosaccharide side chains.

Effects of lipid on the structure and rheology of gels formed by canine submaxillary mucin.

Rheological experiments have shown that canine submaxillary mucin (CSM) forms gels in aqueous solution at low ionic strength and in 6M GdnHCl. Examination of specimens of intact CSM and also its subunits prepared by reduction and carboxymethylation showed that the presence of lipid increases the gel-forming capability, probably as a result of enhancement of the intermolecular hydrophobic interactions. The rheological evidence for gelation is that substantially larger values of the oscillatory storage modulus, G' (omega), and the dynamic complex viscosity, eta*(omega), are observed for lipid-containing CSM. This is backed up by electron micrographs of freeze fractured specimens, where we observe a network morphology in which the cross-links are formed as a result of non-bonded interactions between a number of CSM chains. The intermolecular interactions responsible for gelation probably involve hydrophobic association between the interdigitated oligosaccharides, and/or between the non-glycosylated regions of the protein core, and can occur even in a highly chaotropic medium (6M GdnHCl). In contrast to previous experiments with porcine submaxillary mucin and human tracheobronchial mucin, which form microphase-separated gels in aqueous solution, CSM solutions undergo macroscopic phase separation into polymerrich (gel) and polymer-poor (sol) phases. These data point to stronger hydrophobic interactions in lipid-containing CSM.

Alterations of energy metabolism in the spontaneously hypertensive rat: a 31P nuclear magnetic resonance study.

We quantified high-energy phosphate metabolites in hypertensive hypertrophied and normal myocardium and monitored temporal changes using the non-invasive 31P nuclear magnetic resonance (NMR) spectroscopy. Hearts from 18 month spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY) were perfused with a phosphate-free buffer at 10 cc/min per g and paced at 240 beats/min on a modified Langendorff apparatus. Perfusion pressure, left ventricular pressure (LVP) and dP/dt were recorded and successive 31P NMR spectra were collected during a 24-min baseline period (oxygenated buffer), anoxia (N2-bubbled and glucose-free buffer) until a 70% fall in LVP occurred, and recovery. An aminomethylphosphonate standard, located within the LVP balloon, permitted absolute quantification of myocardial phosphate moieties (including inorganic phosphate (Pi), creatine phosphate (CP) and ATP). During perfusion, SHR hearts demonstrated higher coronary resistance but no significant differences in LVP or dP/dt. Spontaneously hypertensive rat hearts had lower CP, ATP and CP/Pi ratio and showed a faster fall in cardiac function during anoxia, associated with parallel rates of changes in the phosphate moieties.

The solution structure of mucous glycoproteins: proton NMR studies of native and modified ovine submaxillary mucin.

The solution structure of native and systematically modified ovine submaxillary mucin (OSM) has been probed by proton NMR spectroscopic methods. Most of the resonances in the spectra have been tentatively assigned to the peptide and O-linked disaccharide, alpha-N-acetylneuraminic acid 2----6 alpha-N-acetylgalactosamine protons. On the basis of the observed chemical shifts, spectral resolution, and behavior of the exchangeable protons it is concluded the mucin possesses internal segmental flexibility and exists in solution as a random coil peptide. No long-lived interresidue peptide or carbohydrate hydrogen bonds were detected. The removal of (i) the C8 and C9 carbons of the sialic acid residue, (ii) the entire sialic acid residue, and (iii) the complete disaccharide side chain resulted in no significant changes in peptide core conformation. A limited set of proton spin coupling constants and nuclear Overhauser enhancements has been obtained for the threonine glycopeptide side chains in native and modified mucin. The results are consistent with the previously reported conformations for the (1----6) linkage in oligosaccharides and the threonyl glycosidic linkage in glycopeptides. The OSM disaccharide may exist as a extended linear structure with rotational freedom about the GalNAc C5-C6 bond, while the threonine glycosidic linkage appears to be sterically constrained, although multiple conformations about the threonine C beta-O gamma bond may be allowed. The small chemical shift perturbations detected in the glycosylated threonine methyl protons and the GalNAc carbons upon removal of the terminal sialic acid residue are consistent with this model.

Biophysical approaches to salivary mucin structure, conformation and dynamics.

Our understanding of the origins of the physical and biochemical properties of mucous glycoproteins is incomplete and not with out controversy. Recent molecular biological and biophysical studies revealing the architecture and solution structure and dynamics of a series of salivary mucins, invaluable toward resolving many of these questions, are discussed. Mucins are very large, structurally heterogeneous, and highly expanded molecules with the carbohydrate playing a key role in maintaining the extended mucin conformation.

Amino group environments and metal binding properties of carbon-13 reductively methylated bovine alpha-lactalbumin.

13C NMR spectroscopy has been used to study the amino group environments and metal binding properties of 13C reductively methylated bovine alpha-lactalbumin. Bovine alpha-lactalbumin is a Ca2+ metalloprotein containing 12 lysyl amino groups and a free amino terminus. All 13 amino groups can be 13C-dimethylated without altering Ca2+ binding or biological activity. pH titrations (chemical shift vs. pH) of this dimethylated protein reveal unique behavior for each of the 13 amino groups. The pKa values for the lysyl amino groups range from 9.1 to 10.8 while the pKa for the N-terminal amino group is 8.3. This relatively high pKa (by 1 pH unit) for the N-terminal supports its interaction in an ion pair as proposed by Warme et al. [Warme, P. K., Momany, F. A., Rumball, S. V., Tuttle, R. W., & Scheraga, H. A. (1974) Biochemistry 13, 768-782]. Carbon-13 NMR studies further show that the removal of Ca2+ from the high-affinity binding site results in a conformational change, with the disruption of the N-terminal ion pair interaction (pKa decreased to 7.4). The study of Zn2+ binding to Ca2+-saturated protein suggests that Zn2+ binds initially at a low-affinity Ca2+ site while maintaining the N-terminal ion pair interaction. The further addition of Zn2+ leads to the disruption of this ion pair forming a presumed apoprotein-like conformation. Finally on the basis of the specific effects of added Mn2+ on the 13C NMR spectra of the methylated protein, a low-affinity divalent metal binding site is proposed about 7.5 A from the amino terminus.

Structure and dynamics of mucin-like glycopeptides. Examination of peptide chain expansion and peptide-carbohydrate interactions by stochastic dynamics simulations.

Mucins and other highly O-linked glycoproteins have been found to exist in random-coil conformations with peptide chain dimensions about 3-fold more expanded than found for deglycosylated mucins or denaturated proteins. We have examined the origin of the peptide chain expansion in mucins by stochastic dynamics simulations which include a treatment of solvation energy effects based on solvent-accessible surface area and polarizability [GB/SA; Still, C. W., et al. (1990) J. Am. Chem. Soc. 112, 6127]. The glycopeptides studied contained pairs of threonine residues (flanked by alanine residues) which were O-glycosylated by the di- and monooligosaccharide side chains alpha-NeuNAc(2-6)alpha-GalNAc and alpha-GalNAc. These glycopeptides serve as simple models for native and asialo ovine submaxillary mucin. Computer stochastic dynamic simulations show a significant decrease in end-to-end distance and radius of gyration (32% and 33%, respectively) upon complete removal of carbohydrate from the glycopeptide AAA(NeuNAc-(2-6)GalNAc)-T(NeuNAc(2-6)GalNAc)-TAAA. These changes are consistent with the extrapolations of the mucin chain dimension data to glycopeptides of this size. The simulations have identified two potentially strong peptide-carbohydrate hydrogen bonds that can influence the orientation of O-linked GalNAc. With two contiguous glycosylated sites, the lowest energy conformation obtained is characterized by a GalNAc amide proton hydrogen bond to the carbonyl of the peptide residue C-terminal to the site of glycosylation. This conformation differs from the glycopeptide conformations predicted for glycopeptides with single or widely spaced glycosylation sites. The results suggest that the experimentally determined mucin peptide chain dimensions can be fully accounted for by short-range (+/- 3 residue) intramolecular steric and hydrogen bond interactions resulting from the clustering of glycosylated residues.

Structure and dynamics of porcine submaxillary mucin as determined by natural abundance carbon-13 NMR spectroscopy.

Nearly all of the resonances in the 13C NMR spectrum of porcine submaxillary mucin glycoprotein (PSM) have been assigned to the peptide core carbons and to the carbons in the eight different oligosaccharide side chains that arise from the incomplete biosynthesis of the sialylated A blood group pentasaccharide (alpha-GalNAc(1-3) [alpha-Fuc(1-2)]-beta-Gal(1-3) [alpha-NeuNGl(2-6)]- alpha-GalNAc-O-Ser/Thr). By use of these assignments, a nearly complete structural analysis of intact PSM has been performed without resorting to degradative chemical methods. Considerable structural variability in the carbohydrate side chains was observed between mucins obtained from different animals, while no variability was observed between glands in a single animal. The dynamics of the PSM core and carbohydrate side chains were examined by using the carbon-13 nuclear magnetic resonance relaxation times and nuclear Overhauser enhancements of each assigned carbon resonance. The peptide core of PSM exhibits internal segmental flexibility that is virtually identical with that of ovine submaxillary mucin (OSM), whose carbohydrate side chain consists of the alpha-NeuNAc(2-6)alpha-GalNAc disaccharide. The longer oligosaccharide side chains of PSM, therefore, have no significant effect on peptide core mobility compared to the shorter side chains of native OSM or asialo-OSM. Although the dynamics of the shorter carbohydrate side chains shared by both OSM and PSM appear to be identical, the A and H blood group structures in PSM have reduced mobilities, indicating that the glycosidic linkages of the terminal sugars in these determinants are relatively inflexible. These results differ from most reports of glycoprotein dynamics, which typically find the terminal carbohydrate residues to be undergoing rapid internal rotation about their terminal glycosidic bonds. The results reported here are consistent with previous studies on the conformations of the A and H determinants derived from model oligosaccharides and further indicate that the conformations of these determinants are unchanged when covalently bound to the mucin peptide core. In spite of their carbohydrate side-chain heterogeneity, mucins appear to be ideal glycoproteins for the study of O-linked oligosaccharide conformation and dynamics and for the study of the effects of glycosylation on polypeptide conformation and dynamics.

Carbon-13 NMR studies of native and modified ovine submaxillary mucin.

Natural abundance 13C NMR spectroscopy has been used to study the solution structure and dynamics of the ovine submaxillary mucin (OSM). Results at both 45.3 and 67.9 MHz show the extremely viscous mucin to possess sufficient internal segmental flexibility to allow high-resolution 13C NMR studies. Essentially all of the resonances in the spectra have been assigned to individual carbons of the carbohydrate disaccharide side chain alpha- NeuNAc2 ----6 alpha-Gal-NAc-Ser/Thr and to the protonated carbons of the major peptide residues. Spin-lattice relaxation times and nuclear Overhauser enhancements reveal that the internal mobility of the mucin is unaffected by large changes in molecular weight and hence bulk viscosity. On the basis of the relaxation measurements the peptide and carbohydrate side chain mobilities increase stepwise from the glycosylated peptide residue alpha-carbons to the terminal sialic acid (NeuNAc) side-chain C9 carbon. Removal of the terminal sialic acid C8 and C9 side-chain carbons as well as the complete removal of the NeuNAc residue does not alter the dynamics of the peptide core. However, the removal of carbons C8 and C9 from the NeuNAc residue produces an increase in its ring mobility or conformational flexibility. Complete removal of sialic acid produces an increase in the mobility or flexibility of the GalNAc ring and reduces the chemical shift sensitivity of the GalNAc ring carbons to the different serine and threonine linkages. The pKa value for the sialic acid carboxyl group in the intact mucin is 2.0, while it increases to 2.4 after the removal of the NeuNAc C8 and C9 side-chain carbons. This change in pKa confirms the intramolecular hydrogen bond interaction of the C8 hydroxyl with the C2 carboxyl group in the alpha-NeuNAc residue as previously suggested by Jennings and Bhattacharjee [ Jennings , H.J., & Bhattacharjee , A.K. (1977) Carbohydr . Res. 55, 105-112]. The relaxation time values and temperature dependence of the chemical shift of the NeuNAc C7 carbon suggest that this group is also involved in an intramolecular interaction. Overall the 13C NMR results indicate that the relatively simple mucous glycoprotein, OSM, is a highly extended and internally flexible molecule which in solution possesses little secondary structure.

A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins.

A new approach for removing O-glycosidically linked carbohydrate side chains from glycoproteins is described. Periodate oxidation of the C3 and C4 carbons in peptide-linked N-acetylgalactosamine (GalNAc) residues generates a dialdehyde product which, under mild alkaline conditions, undergoes a beta-elimination which releases carbohydrate and leaves an intact peptide core. The pH and time dependence, and intermediates of the elimination, have been extensively followed by carbon-13 NMR spectroscopy and amino acid analysis using ovine submaxillary mucin (OSM) as the substrate. The deglycosylation of OSM is complete and provides apomucin in high yield with an amino acid composition identical to the starting material. Carboxymethylated OSM when deglycosylated by this method gives an apomucin with an apparent molecular weight of ca. 700 x 10(3). The molecular weight is the same as that calculated for the peptide core of the starting mucin, demonstrating the absence of peptide core cleavage. This contrasts with the use of trifluoromethanesulfonic acid (TFMSA), which generates apomucin products of lower molecular weights. Oligosaccharide side chains substituted at C3 of the peptide-linked GalNAc residue are resistant to the oxidation and elimination. Glycoproteins containing these more complex side chains can be deglycosylated by pretreatment with TFMSA under mild (0 degree C) conditions, which removes peripheral sugars (while leaving the peptide-linked GalNAc residue intact), followed by oxidation and beta-elimination. Studies on the deglycosylation of porcine submaxillary mucin and human tracheobronchial mucin indicate that this approach provides more efficient removal of carbohydrate and less peptide core degradation than a more vigorous (25 degrees C) treatment with TFMSA alone. 13C NMR spectroscopic studies and carbohydrate analysis of the deglycosylation intermediates of the human mucin indicate that certain sialic acid containing and N-acetylglucosamine-containing oligosaccharides have elevated resistance to TFMSA treatment at 0 degrees C. By the use of neuraminidase, repeated mild TFMSA treatments, and multiple oxidations and beta-eliminations, the human mucin can be nearly completely deglycosylated. It is expected that all mucins and most glycoproteins containing O-glycosidic linkages can be readily and nearly completely deglycosylated using this combined approach.

Low probe concentration can cause problems in multilocus DNA fingerprinting (cautionary notes III).

The power of multilocus DNA fingerprinting depends on the reliability with which the uniqueness of an individual's profile can be demonstrated. This cautionary note stresses the importance of the probe concentration in this procedure. In case of a probe shortage, DNA fragments rich in tandem repeats have the potential to impede hybridization in other parts of the gel, and thus interfere with bands that are part of a DNA fingerprinting profile.

DNA preparation and efficient microsatellite analysis from insect hemolymph.

A simple and time-saving method for DNA preparation for efficient microsatellite analysis is described. The method is based on thermal treatment of only 1-5 microL of insect hemolymph in a Chelex 100-suspension. Since hemolymph withdrawal does not harm the insects, analysis of mating systems, population structure and phylogenetic reconstruction can be conducted with minimal experimental influence.

Proton nuclear magnetic resonance spectroscopy and ligand binding dynamics of the Escherichia coli L-arabinose binding protein.

The L-arabinose binding protein (ABP) from Escherichia coli was studied by proton nuclear magnetic resonance spectroscopy (1H NMR). Distinct spectral changes occur when ABP binds its natural ligand, L-arabinose, which involve resonances in the aromatic ring current shifted methyl, bulk methyl, methylene, aromatic, and amide proton regions of the spectra. Several amide resonances can be "protected" from deuterium exchange if L-arabinose is bound to ABP prior to deuterium oxide dialysis. On the basis of the pH dependence of their chemical shifts, two low-field resonances have been tentatively assigned to C2 protons of two of the three histidines present in ABP. These histidyl residues have pK values of 8.0 and 8.6 which support their involvement in ionic interactions observed earlier in the crystallographic analysis. One histidyl residue shows a small chemical shift change upon the addition of arabinose. When ABP binds D-galactose, changes in the spectra occur which are different than those observed when L-arabinose is bound. Binding of L-arabinose and D-galactose to the binding protein (ABP) was considered by equilibrium binding and fluorescence emission spectroscopy. ABP binds L-arabinose and D-galactose with high affinities (Kd's at 6 degrees C of 1.3 x 10(-7) and 1.9 x 10(-7) M, respectively), and both enthalpy and entropy contribute to the ABP-ligand association. When excited at 285 nm, ABP has a fluorescence emission maximum of 340 nm which is quenched and blue shifted (to 337 nm) upon binding L-arabinose. ABP binding D-galactose produced a similar emission shift but no fluorescence quenching.

Site-specific core 1 O-glycosylation pattern of the porcine submaxillary gland mucin tandem repeat. Evidence for the modulation of glycan length by peptide sequence.

The sequence-specific O-linked core 1 ([R1, R2]-beta-Gal(1-3)-alpha-GalNAc-O-Ser/Thr) glycosylation pattern has been quantitatively determined for 30 of the 31 Ser/Thr residues in the 81-residue porcine submaxillary gland mucin tandem repeat. This was achieved by Edman amino acid sequencing of the isolated tandem repeat after selective removal of non-C3-substituted, peptide-linked GalNAc residues by periodate oxidation and subsequent trimming of the remaining oligosaccharides to peptide-linked GalNAc residues by mild trifluoromethanesulfonic acid/anisole treatment. The sequencing reveals 61% (range, 12-95%) of the peptide alpha-N-acetylgalactosamine (GalNAc) residues to be substituted by core 1 chains, a value in agreement with the carbon-13 NMR analysis of the native mucin. Residues with the lowest C3 substitution were typically clustered in regions of sequence with the highest densities of (glycosylated) serine or threonine. This suggests that the porcine beta3-Gal, core 1, transferase is sensitive to peptide sequence and/or neighboring core GalNAc glycosylation in vivo, in keeping with earlier in vitro enzymatic glycosylation studies (Granovsky, M., Blielfeldt, T., Peters, S., Paulsen, H., Meldal, M., Brockhausen, J., and Brockhausen, I. (1994) Eur. J. Biochem. 221, 1039-1046). These results demonstrate that the O-glycan structures in mucin domains are not necessarily uniformly distributed along the polypeptide core and that their lengths can be modulated by peptide sequence. The data further suggest that hydroxyamino acid spacing may contribute to the regulation of glycan length, thereby, providing a mechanism for maintaining an optimally expanded, protease resistant, mucin conformation.