J Myron Crawford

Yale University, New Haven, CT, United States

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Publications (4)5.43 Total impact

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    ABSTRACT: An antibacterial protein was purified from acidified gill extract of a bivalve mollusk, the American oyster (Crassostrea virginica). Protein isolation was best accomplished by briefly boiling the tissues in a weak acetic acid solution. Adding protease inhibitors while boiling did not have a major effect on activity recovery. In contrast, use of only protease inhibitors (without boiling) resulted in virtually no recovery of this activity. The amino acid sequence of this antibacterial protein was identified as a histone H2B and was designated cvH2B. cvH2B had potent activity against gram-negative bacteria, including the human pathogens Vibrio parahaemolyticus and Vibrio vulnificus, which commonly reside in oyster tissues. We estimated that the concentration of this protein was well within the concentration that was inhibitory to these bacterial pathogens in vitro. This is the first report of the antimicrobial function of histone H2B from any mollusk.
    Marine Biotechnology 12/2009; 12(5):543-51. DOI:10.1007/s10126-009-9240-z · 3.15 Impact Factor
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    The Yale journal of biology and medicine 03/2008; 80(4):195-211.
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    ABSTRACT: Conclusions The genomics “revolution” has succeeded in sequencing the human and many other genomes and was made possible by key discoveries in molecular biology (e.g., restriction enzymes) and the amazing rate at which major biotechnological breakthroughs were and are continuing to be made in this broad field. As anticipated, the ever-growing knowledge about the human and other genomes as well as the new and very powerful genomics biotechnologies are giving rise to impressive achievements that span from taxonomy to criminal investigations to uncovering genes associated with human disease. Realizing the importance of spurring a similar revolution in proteomics, NIH has funded biotechnology centers and grants to develop more powerful proteomics technologies. Generally, however, the expectations for proteomics have exceeded the clinical accomplishments. Some of the driving forces for a proteomics “revolution” are the renewed appreciation that the biological effector molecule generally is the protein and not its encoding mRNA; the inability of predicting the occurrence of many important protein post-translational modifications (PTM) such as phosphorylation (which is thought to occur on as many as one-third of human proteins and often plays a key role in modulating protein function) from genomics data; and the frequent lack of agreement between mRNA vs. protein expression data (e.g., see [22]).Several challenges stand in the way of a true proteomics “revolution,” and they are illustrated in human plasma, which is the most complex human proteome but also the most useful as it potentially contains virtually the entire human proteome due to tissue “leakage” and is the most readily available clinical specimen. While there are probably only a relatively modest number of true plasma proteins (e.g., about 500 secreted by the liver and intestines), each is present in an average of perhaps 100 forms due to differential glycosylation, splicing, proteolytic processing, and PTMs [23]. Added to these 50,000 protein variants are perhaps almost 21,000 other human proteins [24] that may leak into the plasma and may each be present in about 50 variant forms (e.g., five resulting from alternative splicing/promoter usage and 10 from the addition of > 200 different PTMs), thus adding another 1,000,000 potential “plasma” proteins that are then mixed with perhaps another 10 million different immunoglobulin sequences [23]. Adding considerably to the challenge is the 10 order of magnitude range in protein concentrations in plasma, which is many orders of magnitude above the dynamic range of any current biotechnology such as iTRAQ, DIGE, or immunological platforms. Three approaches used to try to address the wide dynamic range of individual plasma proteins are depletion of abundant proteins such as serum albumin, enrichment (by immunological or other means) of classes of proteins of interest (e.g., phosphoproteins), and multi-dimensional/multistep approaches used to fractionate plasma prior to proteomic analysis. One need not look very far into the Keck Web pages to discern that while genomics can interrogate the relative level of expression of approximately 38,000 human transcripts on a single array and a single chip can analyze 1 million human SNPs, proteomics is limited to the range of about 500 to 700 (with optimal iTRAQ samples) to 1,000 to 2,000 proteins/sample (with optimal DIGE samples). We believe that to reach the expectations anticipated for proteomics, this several order of magnitude difference between the capabilities of contemporary genomic and proteomics technologies must be closed, and currently, it is not clear if any of the available proteomics technologies have the inherent capability to do so. While we believe that closing this biotechnological gap is one of the most difficult of all biotechnological challenges, we also believe the rewards for doing so will prove to be well worth the needed effort and funding. In our opinion, however, no government agency has yet made the high level of sustained commitment that will be needed to bring the goal of “routine” interrogation of the human proteome within reach.
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    ABSTRACT: An antimicrobial peptide was purified from acidified gill extract of a bivalve mollusk, the American oyster (Crassostrea virginica), by preparative acid-urea--polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. The 4265.0 Da peptide had 38 amino acids, including 6 cysteines. It showed strongest activity against Gram-positive bacteria (Lactococcus lactis subsp. lactis and Staphylococcus aureus; minimum effective concentrations [MECs] 2.4 and 3.0 microg/ml, respectively) but also had significant activity against Gram-negative bacteria (Escherichia coli D31 and Vibrio parahemolyticus; MECs 7.6 and 15.0 microg/ml, respectively). Comparison of the amino acid sequence with those of other known antimicrobial peptides revealed that the novel peptide had high sequence homology to arthropod defensins, including those from other bivalves, the mussels Mytilus edulis and Mytilus galloprovincialis. This is the first antimicrobial peptide to be isolated from any oyster species and we have named it American oyster defensin (AOD).
    Biochemical and Biophysical Research Communications 01/2006; 338(4):1998-2004. DOI:10.1016/j.bbrc.2005.11.013 · 2.28 Impact Factor