Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody.

Vasoactive intestinal peptide (VIP) labeled with 125I, [Tyr10-125I]VIP, can be hydrolyzed by immunoglobulin G (IgG) purified from a human subject, as judged by trichloroacetic acid precipitation and reversed-phase high-performance liquid chromatography (HPLC). The hydrolytic activity was precipitated by antibody to human IgG, it was bound by immobilized protein G and showed a molecular mass close to 150 kilodaltons by gel filtration chromatography, properties similar to those of authentic IgG. The Fab fragment, prepared from IgG by papain treatment, retained the VIP hydrolytic activity of the IgG. Peptide fragments produced by treatment of VIP with the antibody fraction were purified by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry and peptide sequencing. The scissile bond in VIP deduced from these experiments was Gln16-Met17. The antibody concentration (73.4 fmol per milligram of IgG) and the Kd (0.4 nM) were computed from analysis of VIP binding under conditions that did not result in peptide hydrolysis. Analysis of the antibody-mediated VIP hydrolysis at varying concentrations of substrate suggested conformity with Michaelis-Menton kinetics (Km). The values for Km (37.9 X 10(-9) M) and the turnover number kcat (15.6 min-1) suggested relatively tight VIP binding and a moderate catalytic efficiency of the antibody.

Mass spectrometric evidence for aldehyde adduction in carboxymyoglobin.

Lipid oxidation generates secondary oxidation products, which compromise myoglobin (Mb) redox stability. Although lipid oxidation-induced discoloration in carboxymyoglobin (COMb) is documented, the molecular basis for interactions between COMb and lipid oxidation products has not been investigated. Our objective was to characterize the adduction of 4-hydroxy-2-nonenal (HNE), a reactive lipid oxidation product, with COMb, utilizing mass spectrometry. Equine COMb and equine OxyMb (0.15mM) were incubated with HNE (1.0mM) at pH 7.4, 37°C (physiological condition) for 6h, and at pH 5.6, 4°C (typical meat storage condition) for 7days. The samples were analyzed in Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). MS spectra revealed that HNE formed mono-, di-, and tri-adducts with COMb at physiological conditions, whereas mono-, di-, tri-, and tetra-adducts were detected in OxyMb. This observation suggested a lower reactivity of COMb towards HNE at physiological conditions, compared to OxyMb. In contrast, at meat storage conditions, HNE formed mono- and di-adducts with both COMb and OxyMb, thus revealing a similar trend for aldehyde adduction in the cherry-red colored Mb redox forms. The present study is the first to report HNE adduction in COMb, and proteomic investigations are underway to determine the sites of HNE adduction in COMb.

Meat quality traits and proteome profile of woody broiler breast (pectoralis major) meat.

Woody breast meat has recently become prevalent in the broiler industry in both the United States and European Union. Recent publications have described the meat quality characteristics of woody breast meat as having hardened areas and pale ridge-like bulges at both the caudal and cranial regions of the breast. The present study investigated the meat quality (pH, color, cooking loss, and shear force) and protein quality characteristics (protein and salt-soluble protein content) in woody breast meat as compared to normal breast meat. In addition, the differences in the muscle proteome profiles of woody and normal breast meat were characterized. Results indicated that woody breast meat had a greater average pH (P < 0.0001) and cooking loss (P = 0.001) than normal breast meat, but woody breast meat did not differ in shear force (P > 0.05) in comparison to normal breast meat samples. The L*, a*, and b* values of woody breast fillets were greater than normal breast fillets (P < 0.0001 to L*; P = 0.002 to a*; P = 0.016 to b*). The woody breast meat had more fat (P < 0.0001) and moisture (P < 0.021) and less protein (P < 0.0001) and salt-soluble protein (P < 0.0001) when compared with normal breast fillets. Whole muscle proteome analysis indicated 8 proteins that were differentially expressed (P < 0.05) between normal and woody breast meat samples. The differences in muscle proteome between normal and woody breast meat indicated an increased oxidative stress in woody breast meat when compared to normal meat. In addition, the abundance of some glycolytic enzymes, which are critical to the regeneration of adenosine triphosphate (ATP) in postmortem muscles, was lower in woody breast meat than in normal breast meat. Proteomic differences provide additional information on the biochemical pathways and genetic variations that lead to woody breast meat. Further research should be conducted to elucidate the genetic and nutritional contributions to the proliferation of woody breast meat in the United States.

Cancer pain in the marital system: a study of patients and their spouses.

Forty married patients with metastatic cancer, who were receiving opioid medication for cancer pain, were interviewed for this study. They were asked about their pain and its treatment, their beliefs regarding cancer pain, their concerns about opioid analgesics, their mood state, and the nature of their interaction with their spouse in relation to these issues. The spouses of these cancer patients were interviewed separately about the same issues. For example, patients who were concerned about medication side effects tended to suffer high levels of pain before requesting additional analgesics. Spouses are shown in this study to be an important support for the patient and an essential source of information regarding the patient's pain and its management. For example, although spouses were generally accurate in their estimates of the patients' pain levels, in the case of relatively stoic patients, who may underreport their pain levels, the spouses' estimates were higher than the patients'. The results also indicate that patients underestimate the distress their pain causes to their spouses and that spouses tend to downgrade their own support to the patients. Implications and limitations of these findings are discussed.

Immigrant earnings differentials and birth-year effects for men in Canada: post-war-1972.

"This paper investigates immigrant earnings differentials for males in Canada and how these earnings have changed over time leading up to 1972 with workers' year of birth. The paper uses the 1973 Job Mobility Survey, which contains a direct measure of work experience reported independent of age. Thus, using age as a birth-year index, it is found that cross-sectional earnings differentials of immigrant men have widened since the later 1960s relative to those of native-born workers." (SUMMARY IN FRE)

Lipid oxidation-induced oxidation in emu and ostrich myoglobins.

Emu and ostrich are ratites gaining increasing popularity as sources of low-fat meats. Secondary products of lipid oxidation, such as 4-hydroxy-2-nonenal (HNE), compromise myoglobin redox stability in a species-specific manner. However, the molecular basis of lipid oxidation-induced oxidation in ratite myoglobins has not been investigated. Therefore, our objective was to characterize lipid oxidation-induced oxidation in ratite myoglobins, in comparison with beef myoglobin. At physiological condition (pH7.4, 37 °C) HNE accelerated (P<0.05) oxidation of emu, ostrich, and beef oxymyoglobins. Autoxidation and HNE-induced oxidation were greater (P<0.05) in ostrich oxymyoglobin than in emu and beef oxymyoglobins. Mass spectrometric analyses revealed that HNE formed mono-adduct with both emu and ostrich myoglobins after 6h of incubation. Tandem mass spectrometry demonstrated that HNE adducted histidine 36 in ostrich myoglobin, whereas histidines 34 and 36 were adducted in emu myoglobin. The results indicate that primary structure of ratite myoglobins influences their redox stability in the presence of prooxidants.

Amino acid sequence of myoglobin from emu (Dromaius novaehollandiae) skeletal muscle.

The objective of the present study was to characterize the primary structure of emu myoglobin (Mb). Emu Mb was isolated from Iliofibularis muscle employing gel-filtration chromatography. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry was employed to determine the exact molecular mass of emu Mb in comparison with horse Mb, and Edman degradation was utilized to characterize the amino acid sequence. The molecular mass of emu Mb was 17,380 Da and was close to those reported for ratite and poultry myoglobins. Similar to myoglobins from meat-producing livestock and birds, emu Mb has 153 amino acids. Emu Mb contains 9 histidines. Proximal and distal histidines, responsible for coordinating oxygen-binding property of Mb, are conserved in emu. Emu Mb shared more than 90% homology with ratite and chicken myoglobins, whereas it demonstrated only less than 70% sequence similarity with ruminant myoglobins.

Characterization of bison (Bison bison) myoglobin.

Bison is an alternate meat species gaining increased popularity in North America. Although previous investigations reported that bison meat discolors faster than beef, the molecular basis of this observation has not been investigated. Therefore, the objective of the present study was to determine the redox stability, thermostability, and primary structure of bison myoglobin (Mb), in comparison with beef Mb. Purified bison and beef myoglobins were analyzed for autoxidation, lipid oxidation-induced oxidation, and thermostability. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry was utilized for determining the exact molecular mass of bison Mb, whereas Edman degradation was employed to determine the amino acid sequence. Bison and beef myoglobins behaved similarly in autoxidation, lipid oxidation-induced oxidation, and thermostability. The observed molecular mass of bison and beef myoglobins was 16,949 Da, and the primary structure of bison Mb shared 100% similarity with beef and yak myoglobins. Noticeably, the amino acid sequence of bison Mb was different from other ruminant myoglobins, such as water-buffalo, sheep, goat, and red-deer. The present study is the first to report the primary structure of bison Mb. Same primary structure and similar biochemical attributes of bison and beef myoglobins suggested that the observed rapid discoloration in bison meat could not be attributed to biochemistry of bison Mb.

Mass spectrometric investigations on lactate adduction to equine myoglobin.

Research focused on determining the fundamental mechanisms by which lactate influences color stability has not considered a direct effect of lactate on myoglobin. Thus, the objective of this study was to use Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry to examine lactate adduction to myoglobin. Equine oxymyoglobin and equine carboxymyoglobin (0.15mM) were incubated with sodium lactate (200mM) at 4 degrees C, pH 5.6 in 50mM sodium citrate buffer or at 37 degrees C, pH 7.4 in 50mM sodium phosphate buffer, simulating typical meat storage and physiological conditions, respectively. Controls consisted of myoglobin plus a volume of deionized water equivalent to that used to deliver the lactate treatments. No peaks corresponding to lactate-Mb adducts could be detected in the mass spectra of samples incubated up to 360min at pH 7.4, 37 degrees C or 8days at pH 5.6 and 4 degrees C. Our results suggest that lactate did not form covalent adducts with equine oxy- and carboxy-myoglobin.

Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain.

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the nucleotide binding site by the substrate photoaffinity analogue [gamma-32P]-8-azido-dATP. The alpha and beta polypeptides of photolabeled terminal transferase were resolved by high-performance liquid chromatography. The beta polypeptide was digested with trypsin and fractionated by reverse-phase chromatography. Two 32P-containing fractions were isolated and subjected to amino acid sequence analysis. Peptides were identified as Ile209-Lys232 (B26) and Val233-Lys239 (B27). Peptide B26 was further resolved into two overlapping species; one contained an additional lysine residue at the N-terminus which resulted from tryptic cleavage between Lys207 and Lys208. In order to ensure that the sequenced peptides corresponded to the photolabeled species, we devised an anion-exchange procedure to isolate photolabeled peptides from the mixture. Analysis of photolabeled peptides from terminal transferase alpha beta using DEAE-cellulose chromatography followed by reverse-phase HPLC confirmed that the photolabeled species were peptides B26 and B27. Peptide B26, the major photolabeled species, contained a conserved octapeptide region found in several eucaryotic DNA polymerases. In addition, peptide B27 was flanked by a sequence that has been implicated in triphosphate binding in other proteins. Structure predictions, based on sequence data, place the two peptides identified by photolabeling in spatial proximity consistent with the participation of both in the nucleotide binding domain.

Children's processing of prosodic cues for phrasal interpretation.

Using synthetic speech, word duration and fundamental frequency (F0) contours were parametrically manipulated to examine processes of phrasal interpretation by adult and child (5 and 7 years old) listeners. From an adult male voice, versions of the phrase "pink and green and white" were resynthesized to produce stimuli suggesting two possible interpretations: [(pink and green) and white] and [pink and (green and white)]. For each stimulus, listeners pointed to a picture to indicate which interpretation was intended. All subjects used duration and (to a lesser extent) intonation as perceptually salient cues for phrasal interpretation. The manner in which subjects processed this information was evaluated by comparing subjects' performance with the predictions of three different information processing models: a nonindependent cue-evaluation model, and two independent cue-evaluation models (an additive model, and the multiplicative, fuzzy logical model). Performance was best described by the fuzzy logical model, which assumes independent cue evaluation and generates a classification function characterized by cue trading relations. The results suggest that, similar to adults, children as young as 5 years of age rely on acoustic-prosodic information for syntactic phrase interpretation, and they process this information in an adultlike manner.

Terminal deoxynucleotidyltransferase. Alignment of alpha- and beta-subunits of the core enzyme along the primary translation product.

Terminal deoxynucleotidyltransferase exists in multiple Mr forms, all apparently generated from a single polypeptide of 62kDa. On isolation and purification, the smallest catalytically active protein of this enzyme consists of two subunits, alpha (12kDa) and beta (30kDa). Recently a complementary-DNA nucleotide sequence has been reported for a portion of the enzyme from human lymphoblast. We have pinpointed the locations of the alpha- and beta-subunits within the elucidated nucleotide sequence. From these data, the portions of the nucleotide sequence coding for the catalytically important area of the transferase can be estimated. Here the amino acid sequence of a number of tryptic peptides from calf alpha- and beta-subunits is presented. Because of the striking homology between the amino acid sequence of the calf enzyme and that predicted for human lymphoblast enzyme, it is possible for us to conclude that the alpha-subunit was generated from the C-terminus of the precursor protein and the beta-subunit was non-overlapping and proximal.

Identification of a novel serum amyloid A protein in BALB/c mice.

Four serum amyloid A protein (SAA) genes and two SAA gene products, SAA1 and SAA2, were identified in BALB/c mice. Using analytical isoelectric focusing we have identified a quantitatively significant new member of the SAA family and designated it 'SAA5'. This protein has characteristics never before described for any SAA molecule. In the highly conserved region between amino acids 33 and 44, identical in all SAAs from all species examined, SAA5 had four amino acid substitutions. In addition, the induction of SAA5 by lipopolysaccharide had different kinetics from that of the other mouse SAAs. Our data suggest that the mouse SAA gene family is more complex in composition and regulation than previously surmised.

Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: identification of peptides in the DNA binding domain.

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp221-Lys231 (peptide B8) and Cys234-Lys249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an alpha-helical array of 39 A which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase beta that has been implicated in the binding of DNA template.

Duration and fundamental frequency correlates of phrase boundaries in productions by children and adults.

Two experiments examined how children and adults produce acoustic correlates for phrase boundaries during speech. Adult and children (ages 5 and 7) were asked to describe groupings of colored blocks, first in a relatively spontaneous manner and next under more structured conditions designed to elicit grouping information concerning "which blocks go together." In the spontaneous condition, adults gave elaborate descriptions of block positions and color, whereas children produced short descriptions such as "pink, green, white;" "pink, green, and white;" and "pink and green and white." In the structured condition, all subjects produced utterances corresponding to three syntactic bracketings of the phrase "pink and green and white:" [pink and (green and white)]; [(pink and green) and white]; and [pink and green and white]. Acoustic analyses indicated that adults reliably control both duration and fundamental frequency (F0) to signal phrase boundaries, whereas children of both age groups demonstrate little evidence of either type of information being used. We interpret these finding as suggesting that children as old as age 7 do not produce prosodic cues for this type of standing ambiguity in their everyday speech. Possible reasons for this lack of phrase boundary prosodic correlates are examined.

Mouse serum amyloid A protein. Complete amino acid sequence and mRNA analysis of a new isoform.

Four serum amyloid A protein (SAA) genes and two gene products, apo-SAA1 and apo-SAA2 were identified in BALB/c mice (type A). SJL/J mice (type B) are thought to be defective in apo-SAA2 expression. A unique variant of mouse apo-SAA was identified in SJL/J mice by isoelectric-focusing analysis of high-density lipoprotein from endotoxin-treated mice. Complete amino-acid-sequence analysis of this quantitatively major form of SJL/J apo-SAA (pI 5.9) showed it to be identical with the apo-SAA2 isoform from BALB/c mice, except for the substitution of aspartic acid for alanine at position 101. Isoform-specific analysis of mRNA from liver of BALB/c and SJL/J mice and their F1 hybrid progeny (CSJLF1/J) mice revealed further differences in the 3' untranslated regions of the genes, not only encoding apo-SAA2 and apo-SAA pI 5.9, but also apo-SAA1. The SAA genes of SJL/J mice thus differ from BALB/c in exon 4. Additional minor isoforms corresponding to apo-SAA2 (pI 6.3) in SJL/J mice and apo-SAA (pI 5.9) in BALB/c mice were identified. We propose that, when analysing a multigene family such as SAA, thorough analysis at the protein level should complement molecular-biological approaches where the use of a too-limited repertoire of probes can obscure complexities.

Human serum amyloid A protein. Complete amino acid sequence of a new variant.

Serum amyloid A protein (SAA), an acute-phase reactant and apolipoprotein of high-density lipoprotein, is a polymorphic protein with six reported isoforms. These are the products of three genes, i.e., cDNA pA1, cDNA pSAA82 and genomic DNA SAAg9, the last two being allelic variants at a single locus. We have identified an individual with additional novel SAA isoforms on isoelectric-focusing analysis. By using 3-bromo-3-methyl-2-(2'-nitrophenylsulphenyl)-indolenine (BNPS-skatole) cleavage of the protein at tryptophan residues we obtained the complete amino acid sequence of a novel isoform. Additional cleavage by endoproteinase Asp-N allowed verification of the tryptophan residues and complete amino acid sequence of both isoforms. The suitability of this approach to the rapid sequencing of SAA was demonstrated. Sequence analysis and quantification suggest that these isoforms are the result of the first confirmed allelic variation at the SAA1 locus. We designate the protein products of this allele SAA1 beta (pI 6.1) and SAA1 beta des-Arg (pI 5.6).

Cleavage of vasoactive intestinal peptide at multiple sites by autoantibodies.

Vasoactive intestinal peptide (VIP) fragments generated by autoantibodies purified from the blood of two human beings were separated and sequenced. Based on the identity of these fragments, seven peptide bonds cleaved by the antibodies were identified. Six of the seven scissile bonds are clustered in the region of VIP spanning residues 14-22 and were cleaved by antibodies from both human subjects. The seventh scissile bond is located at residues 7-8 and was cleaved by antibodies from one of the subjects. The scissile bonds link amino acid residues with different size, charge, and hydrophobicity. The hydrolytic activity of the antibodies was selective in that they failed to hydrolyze polypeptides unrelated in sequence to VIP (insulin and atrial natriuretic peptide). These observations demonstrate substrate specific hydrolysis by naturally occurring antibodies and expand the range of peptide bonds hydrolyzed by these antibodies.

State-of-the-art biomolecular core facilities: a comprehensive survey.

A survey of 124 protein and/or nucleic acid chemistry facilities has provided a basis for estimating the resources needed to establish a facility, the financial support needed to keep it operating, and the technical capabilities it might reasonably be expected to achieve. Based on these data, an average core facility occupied 870 ft2, was staffed by three full-time personnel, and was equipped with 4-5 major instrument systems. Because user fees generated an average of about $101,000/year in income compared with an average operating budget of about $197,000/year, even a facility that charged user fees would, on average, still require an annual subsidy of about $96,000. Although most government and industrial core facilities did not assess user fees, at least 83 of the 124 respondents did have a preestablished schedule of service charges that enabled a compilation to be made of the average cost of providing a number of typical facility analyses and syntheses. The greater than 100-fold range in charges assessed in core facilities for seemingly identical services was shown to result from the equally large range in the degree of subsidization of these laboratories. Although an average facility might be expected to offer four or five of the following six major services--amino acid sequencing, amino acid analysis, HPLC peptide isolation, peptide synthesis, fragmentation of proteins and DNA synthesis--less than 10% of the responding laboratories provided mass spectrometry, capillary zone electrophoresis, or RNA synthesis. With the exception of peptide synthesis, which had an average turn-around time of about 24 days, all other major services had turn-around times that averaged in the range of 4-9 days. Additional data are summarized regarding average sample throughput in core laboratories and the amount of protein that is needed for hydrolysis/amino acid analysis and sequencing.

Activation of the MAPK signal cascade by the neural cell adhesion molecule L1 requires L1 internalization.

L1-mediated axon growth involves intracellular signaling, but the precise mechanisms involved are not yet clear. We report a role for the mitogen-activated protein kinase (MAPK) cascade in L1 signaling. L1 physically associates with the MAPK cascade components Raf-1, ERK2, and the previously identified p90(rsk) in brain. In vitro, ERK2 can phosphorylate L1 at Ser(1204) and Ser(1248) of the L1 cytoplasmic domain. These two serines are conserved in the L1 family of cell adhesion molecules, also being found in neurofascin and NrCAM. The ability of ERK2 to phosphorylate L1 suggests that L1 signaling could directly regulate L1 function by phosphorylation of the L1 cytoplasmic domain. In L1-expressing 3T3 cells, L1 cross-linking can activate ERK2. Remarkably, the activated ERK localizes with endocytosed vesicular L1 rather than cell surface L1, indicating that L1 internalization and signaling are coupled. Inhibition of L1 internalization with dominant-negative dynamin prevents activation of ERK. These results show that L1-generated signals activate the MAPK cascade in a manner most likely to be important in regulating L1 intracellular trafficking.