The local anesthetics procaine and piperocaine blocked induction of the plasmid-determined enzyma... more The local anesthetics procaine and piperocaine blocked induction of the plasmid-determined enzymatic activities involved in the metabolism of n-alkanes in Pseudomonas putida. Procaine reversibly inhibited existing alkane hydroxylase activity. Induction of a soluble aliphatic amidase activity was not affected. These results support the hypothesis that induction of the plasmid-determined alkane metabolic system in P. putida involves a membrane component(s).
We are studying the molecular mechanism of cellular protein localization. The availability of gen... more We are studying the molecular mechanism of cellular protein localization. The availability of genetic techniques, such as gene fusion in Escherichia coli, has made this subject particularly amenable to study in this prokaryote. We have constructed a variety of strains in which the gene coding for an outer membrane protein is fused to the gene coding for a normally cytoplasmic enzyme, beta-galactosidase. The hybrid protein produced by such strains retain beta-galactosidase activity; this activity serves as a simple biochemical tag for studying the localization of the outer membrane protein. In addition, we have exploited phenotypes exhibited by certain fusion strains to isolate mutants that are altered in the process of protein export. Genetic and biochemical analyses of such mutants have provided important insights into the mechanism of protein localization.
The plasmid-determined inducible alkane hydroxylase of Pseudomonas putida resolved into particula... more The plasmid-determined inducible alkane hydroxylase of Pseudomonas putida resolved into particulate and soluble fractions. Spinach reductase and spinach ferredoxin could replace the soluble hydroxylase component. Two alkane hydroxylase mutants show in vitro complementation (S. Benson and J. Shapiro, J. Bacteriol., 123: 759-760, 1975): one, alk-7, lacks an active soluble component and the other, alk-181, lacks an active particulate component. Together with previous results on a particulate alcohol dehydrogenase enzyme (Benson and Shapiro, J. Bacteriol., 126: 794-798, 1976), these results allowed us to assay three plasmid-determined inducible activities: soluble alkane hydroxylase (alkA+), particulate alkane hydroxylase (alkB+), and particulate alcohol dehydrogenase (alkC+). Growth tests and in vitro complementation assays revealed three groups of plasmid mutations that block expression of alkane hydroxylase activity: alkA, which so far includes only the alk-7 mutation; alkB, which in...
New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-l... more New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype. Several mutations mapped in a new gene, secD. These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins. The secD gene is closely linked to tsx on the E. coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby. A plasmid carrying secD+ was identified and used to show that the mutations are recessive. The secD gene may code for a component of the cellular export machinery.
In vitro complementation assays have been used to demonstrate the induction of alkane hydroxylase... more In vitro complementation assays have been used to demonstrate the induction of alkane hydroxylase proteins in mutants lacking the ability to convert n-alkanes to their primary alcohols. Purified heptane is an effective inducer in a mutant lacking detectable hydroxylase activity.
Summary The outer membrane forms the exterior layer of the cell envelope of Gram negative bacteri... more Summary The outer membrane forms the exterior layer of the cell envelope of Gram negative bacteria and differentiates these from Gram positive and acid fast bacteria. It serves as the first selective permeability barrier of the cell by limiting access of components to the periplasm. It also serves as the site of recognition for many agents including bacteriophages, colicins, and antibodies. The membrane bilayer is asymmetric with phospholipids present in the inner leaflet and lipopolysaccharides in the outer leaflet. Proteins in the outer membrane link the outer membrane to the cell wall and function as channels through which substances cross the lipid bilayer. These channels can be specific or nonspecific. Large molecules and those with charges use specific mechanisms to move across the outer membrane. Lipopolysaccharides are glycolipid macromolecules that are unique to Gram negative bacteria. They protect the cell from hydrophobic sbstances such as detergents. They are highly immunogenic, used to taxonomically classify bacterial strains, and endotoxins. The bulk permeability of Gram negative bacteria is largely determined by the composition of the membrane proteins and the structure of the lipopolysaccharides.
Summary Bacteria are unicellular organisms that have a variety of sizes, shape, and envelope stru... more Summary Bacteria are unicellular organisms that have a variety of sizes, shape, and envelope structures. The minimal requirements are cytoplasm, a cell membrane that surrounds the cytoplasm, and a DNA chromosome. A few have internal structures such as vacuoles and storage bodies but none have true organelles. The cell envelope can be as simple as a single membrane. However, it is generally a multilayered structure that includes a cytoplasmic membrane, a cell wall, and additional structures exterior to the cell wall. The nature of the bacterial envelope determines whether the strain is a Gram positive, Gram negative, or acid fast organism. Gram positive bacteria have thick cell walls, Gram negative bacteria thin cell walls plus a second exterior membrane, and acid fast bacteria have a thin cell wall plus a thick layer of specialized lipids. Cell walls function as an exoskeleton that define the overall cell shape. Outer membranes protect Gram negative cells from detergents and enzymes but limit permeability. They contain specialized channel-forming proteins that provide the means for small molecules to diffuse across this barrier. The outer leaflet of the outer membrane contains lipopolysaccharides. Lipopolysaccharides are unique glycolipids that form a barrier that protects the cell from hydrophobic agents. They are endotoxins, highly immunogenetic, and the surface components recognized by serotyping antibodies. Bacteria can have a variety of surface appendages. These include flagella for cell movement, fimbriae for adherence, and pili for genetic exchanges. Many bacteria surround themselves with a thick polysaccharide coat (the glycocalyx) in the form of a capsule or as slime. This layer helps protect the cells from dehydration and contributes to the pathogenicity of many pathogens.
Drug Discovery and Traditional Chinese Medicine, 2001
... 12 ANTIBACTERIAL SYNERGY IN RUBRICINE: AN EXTRACT FROM THE ROOTS OF ARNEBIA EUCHROMA A CHINES... more ... 12 ANTIBACTERIAL SYNERGY IN RUBRICINE: AN EXTRACT FROM THE ROOTS OF ARNEBIA EUCHROMA A CHINESE MEDICINAL HERB SPENCER A ... is treated with rubricine, we observe two responses, a rapid killing effect that reduces the number of colony forming units ...
We describe the isolation and characterization of mutations in ompF that alter the pore propertie... more We describe the isolation and characterization of mutations in ompF that alter the pore properties of the OmpF porin. The selection makes use of the fact that maltodextrins larger than maltotriose are too large to diffuse through the normal OmpF pore. By demanding growth on maltodextrins (Dex+) in the absence of the LamB protein, which is normally required for the uptake of these large sugars, we are able to obtain ompF mutations. These include transversions, transitions and small deletions. We obtained almost exclusively ompF mutations in spite of the fact that analogous alterations in ompC can result in similar phenotypes. Fifteen independent point mutations identify residues R42, R82, D113 and R132 of the mature peptide as important in pore function. The alterations result in uncharged amino acids being substituted for charged amino acids. Growth tests, antibiotic sensitivities and rates of [14C]maltose uptake suggest that the alterations result in an increased pore size. Small deletions of six to 15 amino acid residues in the region between A108 and V133 of mature OmpF dramatically alter outer membrane permeability to hydrophobic antibiotics and detergents as well as conferring a Dex+ phenotype. We suggest that these mutations affect both the pore function and interactions with other outer membrane components. A model of OmpF protein structure based on general rules for folding membrane proteins and these mutations is presented.
Strain Pop3299 contains the lamB::lacZ42-12 gene fusion that encodes a hybrid protein that is eff... more Strain Pop3299 contains the lamB::lacZ42-12 gene fusion that encodes a hybrid protein that is efficiently exported to the cellular envelope of Escherichia coli. As a result of this efficient export, this strain is killed by the inducer maltose and unable to grow on minimal media supplemented with lactose. In an attempt to isolate mutants in which export of the hybrid protein is altered, we selected Lac+ mutants of strain Pop3299 on lactose tetrazolium media. Unlike mutants previously isolated on lactose minimal media, all the mutants we obtained carried large deletions within the lamB::lacZ gene fusion. Thus, it appears that the type of selection employed affects the type of mutations obtained. We have analyzed the nucleotide sequences of representative mutants, and demonstrate a correlation between the deletion size and the export-related maltose and lactose phenotypes. In addition, we demonstrate that the deletions do not appear to arise from regions of micro-homology.
Eurasia Journal of Mathematics, Science and Technology Education
This paper reports on teachers’ experiences of a 10-week, wiki-based, predominantly mobile scienc... more This paper reports on teachers’ experiences of a 10-week, wiki-based, predominantly mobile science education intervention for secondary school students. It was designed to shift traditional pedagogical approaches towards more inquiry-based approaches by combining the strengths of wikis and mobile smart devices. The four participating Macau science teachers, with shared responsibility for 250 students, noted greater student engagement and reported some shifts towards more constructivist pedagogy and more active student learning. Evidence emerged of the value of personalization, collaboration, online feedback, and authentic learning, but the lack of mobile optimization of the wiki platform negatively impacted the degree of personalization, collaboration and authenticity. Lessons were learned about the need for educational technology interventions to carefully consider the intersection between software and hardware; the reasons for the recent decline of wikis as educational tools; and ...
When students begin a course, they generally enter with expectations of what will and will not ha... more When students begin a course, they generally enter with expectations of what will and will not happen. On the other hand, faculty members also enter a classroom with perceptions of what students expect from a course. Having a mismatch between these sets of expectations is natural, but can prove detrimental to learning. This paper compares results from a student survey of expectations, and a mirrored faculty survey of faculty perceptions of student expectations. The results section examines a survey of 286 instructors, and compares a subset of these from the University of Maryland's College of Computing, Mathematical and Natural Sciences to the expectations of 816 STEM students and 57 Computer Science (CS) students. Faculty fail to predict student expectations for various course elements, successfully approximating only 8 out of 22 elements for CS students, and 10 out of 22 elements for the aggregated students.
INTRODUCTION Protein localization is the process by which a protein is exported from its site of ... more INTRODUCTION Protein localization is the process by which a protein is exported from its site of synthesis in the cytoplasm to any one of a number of different cellular compartments. During the last decade, this aspect of cellular biogenesis has been the focus of considerable research. Despite obvious differences in the subcellular structure of prokaryotic and eukaryotic cells, all cells seem to use similar mechanisms of protein export. For example, experiments using recombinant DNA technology have shown that the intragenic information specifying export in a eukaryotic gene (ovalbumin, insulin) can be recognized by Escherichia coli and vice versa (alkaline phosphatase [PhoA]; β -lactamase [Bla]) (Fraser and Bruce 1978; Talmadge et al. 1980; Roggenkamp et al. 1981, Muller et al. 1982). This conservation of export mechanisms has fostered exchange of information among scientists working in areas as diverse as eukaryotic cell biology and prokaryotic molecular genetics. In this paper we ...
The local anesthetics procaine and piperocaine blocked induction of the plasmid-determined enzyma... more The local anesthetics procaine and piperocaine blocked induction of the plasmid-determined enzymatic activities involved in the metabolism of n-alkanes in Pseudomonas putida. Procaine reversibly inhibited existing alkane hydroxylase activity. Induction of a soluble aliphatic amidase activity was not affected. These results support the hypothesis that induction of the plasmid-determined alkane metabolic system in P. putida involves a membrane component(s).
We are studying the molecular mechanism of cellular protein localization. The availability of gen... more We are studying the molecular mechanism of cellular protein localization. The availability of genetic techniques, such as gene fusion in Escherichia coli, has made this subject particularly amenable to study in this prokaryote. We have constructed a variety of strains in which the gene coding for an outer membrane protein is fused to the gene coding for a normally cytoplasmic enzyme, beta-galactosidase. The hybrid protein produced by such strains retain beta-galactosidase activity; this activity serves as a simple biochemical tag for studying the localization of the outer membrane protein. In addition, we have exploited phenotypes exhibited by certain fusion strains to isolate mutants that are altered in the process of protein export. Genetic and biochemical analyses of such mutants have provided important insights into the mechanism of protein localization.
The plasmid-determined inducible alkane hydroxylase of Pseudomonas putida resolved into particula... more The plasmid-determined inducible alkane hydroxylase of Pseudomonas putida resolved into particulate and soluble fractions. Spinach reductase and spinach ferredoxin could replace the soluble hydroxylase component. Two alkane hydroxylase mutants show in vitro complementation (S. Benson and J. Shapiro, J. Bacteriol., 123: 759-760, 1975): one, alk-7, lacks an active soluble component and the other, alk-181, lacks an active particulate component. Together with previous results on a particulate alcohol dehydrogenase enzyme (Benson and Shapiro, J. Bacteriol., 126: 794-798, 1976), these results allowed us to assay three plasmid-determined inducible activities: soluble alkane hydroxylase (alkA+), particulate alkane hydroxylase (alkB+), and particulate alcohol dehydrogenase (alkC+). Growth tests and in vitro complementation assays revealed three groups of plasmid mutations that block expression of alkane hydroxylase activity: alkA, which so far includes only the alk-7 mutation; alkB, which in...
New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-l... more New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype. Several mutations mapped in a new gene, secD. These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins. The secD gene is closely linked to tsx on the E. coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby. A plasmid carrying secD+ was identified and used to show that the mutations are recessive. The secD gene may code for a component of the cellular export machinery.
In vitro complementation assays have been used to demonstrate the induction of alkane hydroxylase... more In vitro complementation assays have been used to demonstrate the induction of alkane hydroxylase proteins in mutants lacking the ability to convert n-alkanes to their primary alcohols. Purified heptane is an effective inducer in a mutant lacking detectable hydroxylase activity.
Summary The outer membrane forms the exterior layer of the cell envelope of Gram negative bacteri... more Summary The outer membrane forms the exterior layer of the cell envelope of Gram negative bacteria and differentiates these from Gram positive and acid fast bacteria. It serves as the first selective permeability barrier of the cell by limiting access of components to the periplasm. It also serves as the site of recognition for many agents including bacteriophages, colicins, and antibodies. The membrane bilayer is asymmetric with phospholipids present in the inner leaflet and lipopolysaccharides in the outer leaflet. Proteins in the outer membrane link the outer membrane to the cell wall and function as channels through which substances cross the lipid bilayer. These channels can be specific or nonspecific. Large molecules and those with charges use specific mechanisms to move across the outer membrane. Lipopolysaccharides are glycolipid macromolecules that are unique to Gram negative bacteria. They protect the cell from hydrophobic sbstances such as detergents. They are highly immunogenic, used to taxonomically classify bacterial strains, and endotoxins. The bulk permeability of Gram negative bacteria is largely determined by the composition of the membrane proteins and the structure of the lipopolysaccharides.
Summary Bacteria are unicellular organisms that have a variety of sizes, shape, and envelope stru... more Summary Bacteria are unicellular organisms that have a variety of sizes, shape, and envelope structures. The minimal requirements are cytoplasm, a cell membrane that surrounds the cytoplasm, and a DNA chromosome. A few have internal structures such as vacuoles and storage bodies but none have true organelles. The cell envelope can be as simple as a single membrane. However, it is generally a multilayered structure that includes a cytoplasmic membrane, a cell wall, and additional structures exterior to the cell wall. The nature of the bacterial envelope determines whether the strain is a Gram positive, Gram negative, or acid fast organism. Gram positive bacteria have thick cell walls, Gram negative bacteria thin cell walls plus a second exterior membrane, and acid fast bacteria have a thin cell wall plus a thick layer of specialized lipids. Cell walls function as an exoskeleton that define the overall cell shape. Outer membranes protect Gram negative cells from detergents and enzymes but limit permeability. They contain specialized channel-forming proteins that provide the means for small molecules to diffuse across this barrier. The outer leaflet of the outer membrane contains lipopolysaccharides. Lipopolysaccharides are unique glycolipids that form a barrier that protects the cell from hydrophobic agents. They are endotoxins, highly immunogenetic, and the surface components recognized by serotyping antibodies. Bacteria can have a variety of surface appendages. These include flagella for cell movement, fimbriae for adherence, and pili for genetic exchanges. Many bacteria surround themselves with a thick polysaccharide coat (the glycocalyx) in the form of a capsule or as slime. This layer helps protect the cells from dehydration and contributes to the pathogenicity of many pathogens.
Drug Discovery and Traditional Chinese Medicine, 2001
... 12 ANTIBACTERIAL SYNERGY IN RUBRICINE: AN EXTRACT FROM THE ROOTS OF ARNEBIA EUCHROMA A CHINES... more ... 12 ANTIBACTERIAL SYNERGY IN RUBRICINE: AN EXTRACT FROM THE ROOTS OF ARNEBIA EUCHROMA A CHINESE MEDICINAL HERB SPENCER A ... is treated with rubricine, we observe two responses, a rapid killing effect that reduces the number of colony forming units ...
We describe the isolation and characterization of mutations in ompF that alter the pore propertie... more We describe the isolation and characterization of mutations in ompF that alter the pore properties of the OmpF porin. The selection makes use of the fact that maltodextrins larger than maltotriose are too large to diffuse through the normal OmpF pore. By demanding growth on maltodextrins (Dex+) in the absence of the LamB protein, which is normally required for the uptake of these large sugars, we are able to obtain ompF mutations. These include transversions, transitions and small deletions. We obtained almost exclusively ompF mutations in spite of the fact that analogous alterations in ompC can result in similar phenotypes. Fifteen independent point mutations identify residues R42, R82, D113 and R132 of the mature peptide as important in pore function. The alterations result in uncharged amino acids being substituted for charged amino acids. Growth tests, antibiotic sensitivities and rates of [14C]maltose uptake suggest that the alterations result in an increased pore size. Small deletions of six to 15 amino acid residues in the region between A108 and V133 of mature OmpF dramatically alter outer membrane permeability to hydrophobic antibiotics and detergents as well as conferring a Dex+ phenotype. We suggest that these mutations affect both the pore function and interactions with other outer membrane components. A model of OmpF protein structure based on general rules for folding membrane proteins and these mutations is presented.
Strain Pop3299 contains the lamB::lacZ42-12 gene fusion that encodes a hybrid protein that is eff... more Strain Pop3299 contains the lamB::lacZ42-12 gene fusion that encodes a hybrid protein that is efficiently exported to the cellular envelope of Escherichia coli. As a result of this efficient export, this strain is killed by the inducer maltose and unable to grow on minimal media supplemented with lactose. In an attempt to isolate mutants in which export of the hybrid protein is altered, we selected Lac+ mutants of strain Pop3299 on lactose tetrazolium media. Unlike mutants previously isolated on lactose minimal media, all the mutants we obtained carried large deletions within the lamB::lacZ gene fusion. Thus, it appears that the type of selection employed affects the type of mutations obtained. We have analyzed the nucleotide sequences of representative mutants, and demonstrate a correlation between the deletion size and the export-related maltose and lactose phenotypes. In addition, we demonstrate that the deletions do not appear to arise from regions of micro-homology.
Eurasia Journal of Mathematics, Science and Technology Education
This paper reports on teachers’ experiences of a 10-week, wiki-based, predominantly mobile scienc... more This paper reports on teachers’ experiences of a 10-week, wiki-based, predominantly mobile science education intervention for secondary school students. It was designed to shift traditional pedagogical approaches towards more inquiry-based approaches by combining the strengths of wikis and mobile smart devices. The four participating Macau science teachers, with shared responsibility for 250 students, noted greater student engagement and reported some shifts towards more constructivist pedagogy and more active student learning. Evidence emerged of the value of personalization, collaboration, online feedback, and authentic learning, but the lack of mobile optimization of the wiki platform negatively impacted the degree of personalization, collaboration and authenticity. Lessons were learned about the need for educational technology interventions to carefully consider the intersection between software and hardware; the reasons for the recent decline of wikis as educational tools; and ...
When students begin a course, they generally enter with expectations of what will and will not ha... more When students begin a course, they generally enter with expectations of what will and will not happen. On the other hand, faculty members also enter a classroom with perceptions of what students expect from a course. Having a mismatch between these sets of expectations is natural, but can prove detrimental to learning. This paper compares results from a student survey of expectations, and a mirrored faculty survey of faculty perceptions of student expectations. The results section examines a survey of 286 instructors, and compares a subset of these from the University of Maryland's College of Computing, Mathematical and Natural Sciences to the expectations of 816 STEM students and 57 Computer Science (CS) students. Faculty fail to predict student expectations for various course elements, successfully approximating only 8 out of 22 elements for CS students, and 10 out of 22 elements for the aggregated students.
INTRODUCTION Protein localization is the process by which a protein is exported from its site of ... more INTRODUCTION Protein localization is the process by which a protein is exported from its site of synthesis in the cytoplasm to any one of a number of different cellular compartments. During the last decade, this aspect of cellular biogenesis has been the focus of considerable research. Despite obvious differences in the subcellular structure of prokaryotic and eukaryotic cells, all cells seem to use similar mechanisms of protein export. For example, experiments using recombinant DNA technology have shown that the intragenic information specifying export in a eukaryotic gene (ovalbumin, insulin) can be recognized by Escherichia coli and vice versa (alkaline phosphatase [PhoA]; β -lactamase [Bla]) (Fraser and Bruce 1978; Talmadge et al. 1980; Roggenkamp et al. 1981, Muller et al. 1982). This conservation of export mechanisms has fostered exchange of information among scientists working in areas as diverse as eukaryotic cell biology and prokaryotic molecular genetics. In this paper we ...
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