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Biomedical subjects

K J Kramer

Publications and source records attributed to K J Kramer.

At least 37 records · Page 2Linked to original sources
PubMed1997

Proteinase-mediated insect resistance to Bacillus thuringiensis toxins.

Two Bacillus thuringiensis (Bt)-resistant strains of the Indianmeal moth, Plodia interpunctella, lack a major gut proteinase that activates Bt protoxins. The absence of this enzyme is genetically linked to larval survival on Bt-treated diets. When considered with previous data supporting the existence of receptor-mediated insect resistance to Bt, these results provide evidence that insect adaptation to these toxins occurs through multiple physiological mechanisms, which complicate efforts to prevent or manage resistance to Bt toxins in insect control programs.

Animals↗
PubMed1997

Isolation and characterization of a genomic clone for the gene of an insect molting enzyme, chitinase.

Genomic clones for a chitinolytic enzyme were isolated from a library of Sau 3A digested DNA from the tobacco hornworm, Manduca sexta, using a previously isolated chitinase cDNA clone as a probe [Kramer et al., Insect Biochem. Molec. Biol. 23, 691-701 (1993)]. Restriction enzyme mapping and Southern blot analysis of four genomic clones suggested that these are overlapping clones. Sequence analysis of the genomic clones and Southern blot analysis of total genomic DNA also suggest that the M. sexta genome has only one chitinase gene detectable by the cDNA probe. This gene is organized into at least 11 exons in a region spanning > 11 kb. The sequenced M. sexta chitinase gene has a series of exons corresponding to identifiable structural/functional regions of the protein. Similarities in structure and organization between the M. sexta chitinase gene and chitinase genes from other sources are described.

Amino Acid Sequence↗
PubMed1997

Insect chitinases: molecular biology and potential use as biopesticides.

Chitin, an insoluble structural polysaccharide that occurs in the exoskeletal and gut linings of insects, is a metabolic target of selective pest control agents. One potential biopesticide is the insect molting enzyme, chitinase, which degrades chitin to low molecular weight, soluble and insoluble oligosaccharides. For several years, our laboratories have been characterizing this enzyme and its gene. Most recently, we have been developing chitinase for use as a biopesticide to control insect and also fungal pests. Chitinases have been isolated from the tobacco hornworm, Manduca sexta, and several other insect species, and some of their chemical, physical, and kinetic properties have been determined. Also, cDNA and genomic clones for the chitinase from the hornworm have been isolated and characterized. Transgenic plants that express hornworm chitinase constitutively have been generated and found to exhibit host plant resistance. A transformed entomopathogenic virus that produces the enzyme displayed enhanced insecticidal activity. Chitinase also potentiated the efficacy of the toxin from the microbial insecticide, Bacillus thuringiensis. Insect chitinase and its gene are now available for biopesticidal applications in integrated pest management programs. Current knowledge regarding the molecular biology and biopesticidal action of insect and several other types of chitinases is described in this mini-review.

Amino Acid Sequence↗
PubMed1997

cDNAs for a chymotrypsinogen-like protein from two strains of Plodia interpunctella.

Gut proteinases are involved in the solubilization and activation of insecticidal toxins produced by Bacillus thuringiensis and may also be involved in resistance development. Approximately threefold lower chymotrypsin-like enzyme activity was observed in a Bt(entomocidus)-resistant strain of the Indianmeal moth, Plodia interpunctella, than that in the Bt-susceptible strain. Because chymotrypsin-like proteinases are involved in Bt protoxin activation in P. interpunctella, we compared cDNA sequences, mRNA expression levels, and genomic DNA for chymotrypsin-like enzymes in Bt-susceptible and Bt-resistant strains of P. interpunctella. To isolate cDNA coding for chymotrypsinogen-like proteinases, a probe was developed using polymerase chain reaction (PCR) amplification of a cDNA library from the Bt-susceptible strain using a vector primer and a degenerate primer corresponding to a conserved sequence in the active site of serine proteinases. This probe was used to screen cDNA libraries from resistant and susceptible strains. Predicted amino acid sequences from cDNA clones of each strain share similarity with sequences of chymotrypsin-like proteinases and are most similar to a chymotrypsin-like proteinase from the tobacco hornworm, Manduca sexta. cDNAs for putative chymotrypsinogen-like proteins, from both Bt-susceptible and Bt-resistant strains of P. interpunctella share an identical open reading frame of 846 nucleotides. The encoded proteins contain amino acid sequence motifs of serine proteinase active sites, disulfide-bridge cysteine residues, and both zymogen activation and signal peptides. A difference between these cDNAs was observed only in the untranslated region where a substitution of guanine for adenine occurred in the Bt-resistant strain. Southern and Northern blotting analyses indicated that there are no major differences in chymotrypsinogen-like genomic organization and mRNA expression in the two strains. These data suggest that chymotrypsinogen-like proteinase genes and their transcription are similar in the Bt-susceptible and Bt-resistant strains of P. interpunctella.

Amino Acid Sequence↗
PubMed1996

Characterization of products from the reactions of N-acetyldopamine quinone with N-acetylhistidine.

When insects harden or sclerotize their exoskeletons, quinones of N-acetylated catecholamines such as N-acetyldopamine (NADA) undergo nucleophilic addition reactions with amino acids such as histidine in cuticular proteins. To determine the products that might form when this type of reaction occurs during cuticle sclerotization, the reactions between electrochemically prepared NADA quinone and N-acetylistidine (NAcH), a protein model nucleophile, have been investigated at pH 7. Two major products, 6-[N-(N-acetylhistidyl)]-N-acetyldopamine and 2-[N-(N-acetylhistidyl)]-N-acetyldopamine, were purified by semipreparative reversed-phase liquid chromatography and identified by mass spectrometry and nuclear magnetic resonance spectroscopy. The relative molar ratio of the C(6) mono-addition adduct to the C(2) mono-addition adduct is 87:13. UV/vis spectroscopic analysis shows that both products have an absorption maximum at 284 nm. Cyclic voltammetry shows that these adducts are oxidized less readily than NADA.

Benzoquinones↗
PubMed1996

Projuvenoids: synthesis and biological evaluation of sulfenylated, sulfinylated, and sulfonylated carbamates.

Applying the proinsecticide principle developed earlier for neurotoxic carbamate insecticides, a series of new N-sulfenylated, N-sulfinylated, and N-sulfonylated derivatives of fenoxycarb were synthesized and evaluated for juvenile hormone mimicking activity. Laboratory evaluations of the compounds using Pieris brassicae and Sitophilus oryzae, as well as field experiments using Bemisia tabaci, showed that several symmetrical biscarbamates with either a sulfenyl or sulfinyl bridge possessed higher activity than the parent carbamate. From the unsymmetrical compounds containing biologically inert derivatizing moieties, one of the sulfenylated biscarbamates also showed improved activity against P. brassicae. The changes in the biological activity of the sulfur-containing derivatives compared to that of the parent compound are attributed to the modified physicochemical characteristics, i.e., increased lipophilicity facilitating penetration, transport, as well as protection of the compound from metabolism.

Animals↗
PubMed1996

Certified reference material for the quality control of cadmium, copper nickel and zinc determination in estuarine water (CRM 505).

Analyses of estuarine water samples are routinely carried out by a number of laboratories to monitor the level of environmental contamination by toxic substances, e.g. heavy metals. A series of interlaboratory studies has been organised by the Community Bureau of Reference, BCR (now renamed Standards, Measurements and Testing Programme) to improve the quality of the determinations of selected trace metals in estuarine water samples. The improvement achieved allowed to produce a reference material (CRM 505) which was certified for its content of Cd, Cu, Ni and Zn. Further improvements were considered to be necessary for the certification of other trace elements, e.g. Pb. This material was collected in the Tagus Estuary, carefully prepared (filtered and acidified) and its homogeneity and long term stability were verified. This paper presents the work performed for the certification of Cd, Cu, Ni and Zn. Indicative values are reported for As, Co, Fe, Mn, Pb and V.

Journal Article↗
PubMed1996

Interlaboratory studies to improve the quality of mercury determination in seawater.

Determinations of pollutants in various matrices are routinely performed by a number of organizations for monitoring contamination levels of the marine environment. Projects for improving the quality of determinations of trace metals in seawater have been successfully concluded within the scope of the BCR-Programme (now renamed Standards, Measurements and Testing Programme) of the European Commission; however, the determination of total mercury could not be considered at this stage both because of the poor analytical performances observed and the unsuitability of the containers (polyethylene) used for the seawater certified reference material (CRM 579). A specific project has therefore been organized to improve the state of the art of Hg determination in seawater and to investigate the best way of storing a candidate CRM. Two interlaboratory studies have been concluded and a certification campaign has now started. The materials have been collected in the Southern Bight of the North Sea, carefully prepared (filtered and acidified) and their homogeneity and stability were verified. This paper presents the results of the two interlaboratory studies.

Journal Article↗
PubMed1996

alpha-Amylase inhibitors from wheat: amino acid sequences and patterns of inhibition of insect and human alpha-amylases.

Four alpha-amylase inhibitors, WRP24, WRP25, WRP26, and WRP27, were purified from wheat flour by preparative, reversed-phase high performance liquid chromatography. All have polypeptide molecular masses of about 14 kDa and are members of the cereal superfamily of protease and alpha-amylase inhibitors. Sedimentation velocity analysis indicated that WRP25 and WRP27 are monomeric proteins, whereas WRP24 is a dimer. WRP24 is identical in N-terminal amino acid sequence to the well characterized 0.19 dimeric inhibitor from wheat kernels. WRP25 and WRP26 differ in sequence from each other at only three positions and represent previously unseparated forms of the 0.28 wheat inhibitor. WRP27 is a previously uncharacterized inhibitor and is more similar in sequence to the 0.28 inhibitor than to the 0.19 inhibitor. WRP25 and WRP26 inhibited alpha-amylases from the rice weevil, red flour beetle, and the yellow meal worm, but did not inhibit human salivary alpha-amylase. WRP24 inhibited the human as well as the insect alpha-amylases, but inhibited one of the two rice weevil alpha-amylases much more strongly than the other. WRP27 was notable in that, of the enzymes tested, it strongly inhibited only the rice weevil alpha-amylases. We observed that the growth rate of red flour beetle larvae was slowed when purified WRP24 was included in the diet at a level of 10%. Addition of WRP24 to corn starch resulted in greater weight loss of red flour beetle adults than occurred on control diets. Our results support the hypothesis that these alpha-amylase inhibitors provide wheat seeds with a selective evolutionary advantage since the inhibitors can slow the growth of insect pests that attack cereal grains.

Amino Acid Sequence↗
PubMed1996

Cloning, expression, and hormonal regulation of an insect beta-N-acetylglucosaminidase gene.

Chitinolytic enzymes such as beta-N-acetylglucosaminidases are major hydrolases involved in insect molting. By screening a Manduca sexta (tobacco hornworm) cDNA library with an antibody against beta-N-acetylglucosaminidase from molting fluid of M. sexta pharate pupae, several putative cDNA clones for this enzyme were isolated. The longest of the cDNA clones has an insert of approximately 3 kb, and the complete nucleotide sequence was determined. Because this clone is missing the initiation codon and nucleotides corresponding to the leader peptide, the mRNA 5'-end sequence was determined by PCR (polymerase chain reaction) amplification and cycle sequencing. The sequence of the encoded protein from positions 23 to 35 is identical to the NH2-terminal sequence of one of the beta-N-acetylglucosaminidases isolated from pharate pupal molting fluid. The amino acid sequence is similar to those of silkworm, human, mouse, bacterial, and several other beta-N-acetylglucosaminidases. Two highly conserved regions in the amino acid sequence were found in all members of this family. Southern blot analysis suggested that the number of genes in the Manduca genome closely related to the cDNA clone may be as few as one. The beta-N-acetylglucosaminidase gene is expressed most abundantly in epidermal and gut tissues on days 6 and 7 of fifth instar larvae. Injection of 20-hydroxyecdysone induced expression of the beta-N-acetylglucosaminidase gene, whereas topical application of the juvenile hormone analog, fenoxycarb, suppressed the inductive effect of molting hormone.

Acetylglucosaminidase↗
PubMed1996

Catecholamine-containing proteins from the pharate pupal cuticle of the tobacco hornworm, Manduca sexta.

Three catecholamine-containing proteins from tanning, pharate pupal, abdominal cuticle of the tobacco hornworm, Manduca sexta, have been purified to apparent homogeneity and characterized. These proteins have apparent molecular masses of 32, 41 and 48 kDa and were shown by liquid chromatography-electrochemical analysis, after heating in 1 M acetic acid at 110 degrees C, to release nu-beta-alanylnorepinephrine (NBANE). NBANE is a hydrolysis product of N-beta-alanyldopamine (NBAD) that is bonded covalently at the beta-side-chain carbon to amino acid residues of cuticular proteins. Amino acid compositional analysis revealed that MS-PCP32 (32 KDa) has a high content of alanine (25.6%), valine (13.1%), proline (10.8%) and glycine (10%), a low level of phenylalanine, and no detectable tyrosine or methionine. In contrast, MS-PCP41 (41 kDa) had a much higher content of glycine (31.2%) and substantial levels of serine (9.2%), proline (10.2%) and glutamate/glutamine (10.6%), whereas tyrosine and phenylalanine were not detected. Two of the three purified proteins showed apparent similarities to each other in nu-terminal amino acid sequences, and to several other known insect cuticular proteins. Proteins MS-PCP41 and MS-PCP48 had the characteristic GGX triplet repeat, which is found in a variety of cuticular proteins and may be important for protein folding appropriate for cuticular functions. Therefore, a diversity of cuticular proteins with different amino acid sequences and properties apparently are secreted into the presumptive pupal exocuticle. These then can form adducts and possible cross-links with NBAD through its quinonoid intermediates during cuticular sclerotization.

Amino Acid Sequence↗
PubMed1996

Luminal proteinases from Plodia interpunctella and the hydrolysis of Bacillus thuringiensis CryIA(c) protoxin.

The ability of proteinases in gut extracts of the Indianmeal moth, Plodia interpunctella, to hydrolyze Bacillus thuringiensis (Bt) protoxin, casein, and rho-nitroanilide substrates was investigated. A polyclonal antiserum to protoxin CryIA(c) was used in Western blots to demonstrate slower protoxin processing by gut enzymes from Bt subspecies entomocidus-resistant larvae than enzymes from susceptible or kurstaki-resistant strains. Enzymes from all three strains hydrolyzed N-alpha-benzoyl-L-arginine rho-nitroanilide, N-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, and N-succinyl-ala-ala-pro-leucine rho-nitroanilide. Zymograms and activity blots were used to estimate the apparent molecular masses, number of enzymes, and relative activities in each strain. Several serine proteinase inhibitors reduced gut enzyme activities, with two soybean trypsin inhibitors, two potato inhibitors, and chymostatin the most effective in preventing protoxin hydrolysis.

Animals↗
PubMed1996

A recombinant baculovirus 42-kilodalton C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 protects Aotus monkeys against malaria.

The immunogenicity and protective efficacy of baculovirus recombinant polypeptide based on the Plasmodium falciparum merozoite surface protein 1 (MSP-1) has been evaluated in Aotus lemurinus griseimembra monkeys. The MSP-1-based polypeptide, BVp42, corresponds to the 42-kDa C-terminal processing fragment of the precursor molecule. Immunization of Aotus monkeys with BVp42 in complete Freund's adjuvant resulted in high antibody titers against the immunogen as well as parasite MSP-1. Fine specificity studies indicated that major epitopes recognized by these antibodies localize to conserved determinants of the 19-kDa C-terminal fragment derived from cleavage of the 42-kDa processing fragment. Effective priming of MSP-1-specific T cells was also demonstrated in lymphocyte proliferation assays. All three Aotus monkeys immunized with BVp42 in complete Freund's adjuvant showed evidence of protection of protection against blood-stage challenge with P. falciparum. Two animals were completely protected, with only one parasite being detected in thick blood films on a single days after injection. The third animal had a modified course of infection, controlling its parasite infection to levels below detection by thick blood smears for an extended period in comparison with adjuvant control animals. All vaccinated, protected Aotus monkeys produced antibodies which inhibited in vitro parasite growth, indicating that this assay may be a useful correlate of protective immunity and that immunity induced by BVp42 immunization is mediated, at least in part, by a direct effect of antibodies against the MSP-1 C-terminal region. The high level of protection obtained in these studies supports further development of BVp42 as a candidate malaria vaccine.

Animals↗
PubMed1996

Assessment of the role of naturally acquired antibody levels to Plasmodium falciparum merozoite surface protein-1 in protecting Papua New Guinean children from malaria morbidity.

We investigated the prevalence and magnitude of naturally acquired humoral immune response to the major merozoite surface protein (MSP-1) in a malaria-endemic population in Papua New Guinea. A prospective longitudinal study in 0.5-15-year-old children was conducted for one year to examine the relationship between acquired immune response to MSP-1 and subsequent susceptibility to clinical disease. The prevalence and concentration of antibodies to both N-(195A) and C-terminal (BVp42) regions of MSP-1 as well as to the parasite-derived MSP-1 increased with age, with the highest prevalence and concentration of antibodies being detected for the parasite-derived MSP-1 molecule and the C-terminal region of MSP-1. As malaria morbidity decreases with age, a significant negative correlation was observed between antibody levels to both 195A and BVp42 and the incidence rate of clinical malaria. When age and past exposure were corrected for, only antibody concentrations against BVp42 and to a lesser extent parasite-derived MSP-1 were significantly associated with protection from clinical malaria and severe parasitemia. The reduction in the incidence rate of clinical malaria observed in individuals with high antibody concentration to MSP-1 may be due to antibodies directed against epitopes within the C-terminal region of MSP-1.

Adolescent↗
PubMed1995

On-column reduction of catecholamine quinones in stainless steel columns during liquid chromatography.

The chromatographic behavior of quinones derived from the oxidation of dopamine and N-acetyldopamine has been studied using liquid chromatography (LC) with both a diode array detector and an electrochemical detector that has parallel dual working electrodes. When stainless steel columns are used, an anodic peak for the oxidation of the catecholamine is observed at the same retention time as a cathodic peak for the reduction of the catecholamine quinone. In addition, the anodic peak exhibits a tail that extends to a second anodic peak for the catecholamine. The latter peak occurs at the normal retention time of the catecholamine. The origin of this phenomenon has been studied and metallic iron in the stainless steel components of the LC system has been found to reduce the quinones to their corresponding catecholamines. The simultaneous appearance of a cathodic peak for the reduction of catecholamine quinone and an anodic peak for the oxidation of the corresponding catecholamine occurs when metallic iron in the exit frit reduces some of the quinones as the latter exits the column. This phenomenon is designated as the "concurrent anodic-cathodic response." It is also observed for quinones of of 3,4-dihydroxybenzoic acid and probably occurs with o- or p-quinones of other dihydroxyphenyl compounds. The use of nonferrous components in LC systems is recommended to eliminate possible on-column reduction of quinones.

Animals↗
PubMed1994

Altered protoxin activation by midgut enzymes from a Bacillus thuringiensis resistant strain of Plodia interpunctella.

Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of the Indianmeal moth, Plodia interpunctella (Hubner), resistant to B. thuringiensis subspecies entomocidus (HD-198) was slower than that by midgut proteinases from the susceptible parent strain or a strain resistant to B. thuringiensis subspecies kurstaki (HD-1, Dipel). Midgut extracts from entomocidus-resistant insects exhibited five-fold lower activity toward the synthetic substrate alpha-N-benzoyl-DL-arginine rho-nitroanilide than extracts from susceptible or kurstaki-resistant insects. Midgut enzymes from susceptible or kurstaki-resistant insects converted the 133 kDa CryIA(c) protoxin to 61-63 kDa proteins, while incubations with entomocidus-resistant enzymes resulted in predominantly products of intermediate size, even with increased amounts of midgut extract. The 61-63 kDa proteins were only produced by entomocidus-resistant midgut extracts after long term incubations with the protoxin. The data suggest that altered protoxin activation by midgut proteinases is involved in some types of insect resistance to B. thuringiensis.

Animals↗