Silk worm Bm1 SINE RNA increases following cellular insults.

The effect of cell stresses upon the expression of the Bm1 short interspersed element (SINE) family in cultured silk worm cells is examined. Primer extension analysis shows that Bm1 repeats are transcribed by RNA polymerase III (Pol III) into cytoplasmic RNAs. Five consecutive T residues, which would normally terminate Pol III transcription, occur within the Bm1 consensus and are included within cDNA sequences representing these transcripts. In analogy to mammalian SINEs, the level of the Bm1 transcripts increases in response to either heat shock, inhibiting protein synthesis by cycloheximide or viral infection. The lifetime of Bm1 RNA increases following cell insults so that post-transcriptional events partially account for stress induced increases in its abundance. In the case of heat shock, the increase in Bm1 RNA follows the transient increase in hsp70 mRNA indicating that this response is temporally regulated to occur later in heat shock recovery. These results support the proposal that SINE RNAs serve a role in the cell stress response that predates the divergence of insects and mammals implying that SINEs are essentially a class of cell stress genes.

Stress induction of Bm1 RNA in silkworm larvae: SINEs, an unusual class of stress genes.

This study surveys the induction of RNA polymerase III (Pol III)-directed expression of short interspersed element (SINE) transcripts by various stresses in an animal model, silkworm larvae. Sublethal heat shock and exposure to several toxic compounds increase the level of Bm1 RNA, the silkworm SINE transcript, while also transiently increasing expression of a well-characterized stress-induced transcript, Hsp70 messenger RNA (mRNA). In certain cases, the Bm1 RNA response coincides with that of Hsp70 mRNA, but more often Bm1 RNA responds later in recovery. Baculovirus infection and exposure to certain toxic compounds increase Bm1 RNA but not Hsp70 mRNA, showing that SINE induction is not necessarily coupled to transcription of this particular heat shock gene. SINEs behave as an additional class of stress-inducible genes in living animals but are unusual as stress genes because of their high copy number, genomic dispersion, and Pol III-directed transcription.

Isolation and characterization of a novel lepidopteran-selective toxin from the venom of South Indian red scorpion, Mesobuthus tamulus.

BACKGROUND: Scorpion venom contains insect and mammal selective toxins. We investigated the venom of the South Indian red scorpion, Mesobuthus tamulus for the purpose of identifying potent insecticidal peptide toxins. RESULTS: A lepidopteran-selective toxin (Buthus tamulus insect toxin; ButaIT) has been isolated from this venom. The primary structure analysis reveals that it is a single polypeptide composed of 37 amino acids cross-linked by four disulfide bridges with high sequence homology to other short toxins such as Peptide I, neurotoxin P2, Lqh-8/6, chlorotoxin, insectotoxin I5A, insect toxin 15 and insectotoxin I1. Three dimensional modeling using Swiss automated protein modeling server reveals that this toxin contains a short alpha-helix and three antiparallel beta-strands, similar to other short scorpion toxins. This toxin is selectively active on Heliothis virescens causing flaccid paralysis but was non-toxic to blowfly larvae and mice. CONCLUSION: This is the first report of a Heliothine selective peptide toxin. Identification of diverse insect selective toxins offer advantages in employing these peptides selectively for pest control.

Characterization of recombinant Autographa californica nuclear polyhedrosis virus (NPV) expressing the beta-galactosidase gene in both Sf21 and Bm5 cells by Bombyx mori NPV p143 helicase gene.

Genomic DNA of recombinant AcNPV expressing beta-galactosidase was cotransfected with p143 helicase gene of BmNPV into Sf21 cells. Ac-Bm hybrid viruses capable of replicating in both Bm5 and Sf21 cells were isolated. Ac-Bm hybrid viruses expressing beta-galactosidase either at the highest (Ac-Bm hybrid virus-HE) or lowest (Ac-Bm hybrid virus-LE) level were chosen for the characterization of beta-galactosidase expression in Bm5 and Sf21 cells. Expression level of beta-galactosidase and replication of Ac-Bm hybrid virus-HE in Sf21 cells were nearly identical to those of recombinant AcNPV. Furthermore, replication of Ac-Bm hybrid virus-HE in Bm5 cells was similar to that of wild-type BmNPV, and Ac-Bm hybrid virus-HE clearly expressed beta-galactosidase in Bm5 cells. However, expression of beta-galactosidase by Ac-Bm hybrid virus-HE in Bm5 cells was significantly lower than that expressed in Sf21 cells. The titer of Ac-Bm hybrid virus-HE determined by plaque assays in Bm5 cells was similar to that determined in Sf21 cells, but the plaque size formed by Ac-Bm hybrid virus-HE in Bm5 cells was apparently smaller than that formed in Sf21 cells. In addition, expression levels and virus titers of Ac-Bm hybrid virus-LE in Sf21 and Bm5 were significantly lower than those of Ac-Bm hybrid virus-HE. Therefore, DNA sequences were determined for the region of the p143 gene controlling the host range in Ac-Bm hybrid viruses. The results showed that the deduced amino acid sequences of Ac-Bm hybrid virus-HE were almost identical to those of BmNPV. There were differences only in amino acids at positions 461 and 470, whereas those of Ac-Bm hybrid virus-LE were different at position 461, 470, 514, and 528 from those of BmNPV. In conclusion, our results clearly demonstrated that Ac-Bm hybrid virus-HE has an additional advantage of expanded host range for producing recombinant proteins.

Generation of an expression library in the baculovirus expression vector system.

The construction and screening of a small cDNA library consisting of 2 x 10(4) clones in the baculovirus expression vector system are described. This library consists of antibody heavy chain sequences isolated from the spleen of a mouse immunized with tetanus toxoid fragment C. A portion of this library was used to produce a pool of recombinant baculoviruses which were screened for production of antibody fragments reactive to tetanus toxoid without prior expression in Escherichia coli. The pool of 30 clones was found to contain at least 6 different populations of antibody indicating that diversity existed within the library. Positive clones were isolated from the baculovirus system and confirmed as being capable of producing a tetanus reactive antibody by expression as a beta-lactamase fusion protein in E. coli. One of these clones was returned to the baculovirus system using a different transfer vector, and tetanus binding reconfirmed. The results presented here show that the concept of the construction and screening of a baculovirus expression library is feasible even with 'difficult' proteins, such as antibody heavy chain fragments, and that the baculovirus expression vector system has the potential to produce cDNA expression libraries which can be screened directly for the desired protein.

Adsorption of endogenous polyphenols relieves the inhibition by fruit juices and fresh produce of immuno-PCR detection of Escherichia coli O157:H7.

The immuno-polymerase chain reaction (PCR) approaches facilitate rapid (8 h) detection of Escherichia coli O157:H7 in contaminated dairy products and ground beef samples with detection sensitivities approaching 1 colony forming unit (cfu) g-1 ml-1. However, no PCR products were obtained when the method was applied to identify E. coli O157:H7 in tainted apple juice. Enzyme-linked immuno-assay (ELISA) results suggested non-specific binding of endogenous polyphenols (ubiquitous in plant products) to antibodies present on the surface of the immunobeads, making the latter unavailable for capturing the target bacteria Treatment of the test sample, prior to IMS, with a synthetic fining agent, polyvinylpyrrolidone, restored the full function and sensitivity of the immuno-PCR. The study demonstrates the suitability of the improved method as a generic strategy for rapid screening of fruit juices and plant produce for E. coli O157:H7.

Nucleotide sequence of the variable region of the heavy and light chains of a monoclonal IgG antibody reactive to herbicides, terbutryn and prometryn.

Members of the triazine family of herbicides are reliable indicators of contamination of the ground water or soil with pesticide residues. To facilitate better detection of the chemical residues using improved immunoassay procedures, several monoclonal antibodies against triazine herbicides have been developed. K1F4 is a hybridoma secreting monoclonal (IgG) antibody reactive to terbutryn and prometryn, two members of the triazine family. We have cloned the genes encoding the variable regions of the heavy and light chains of this monoclonal antibody and report the nucleotide sequence here.

A high-throughput assay format for determination of nitrate reductase and nitrite reductase enzyme activities.

The authors describe a microplate-based high-throughput procedure for rapid assay of the enzyme activities of nitrate reductase and nitrite reductase, using extremely small volumes of reagents. The new procedure offers the advantages of rapidity, small sample size-nanoliter volumes, low cost, and a dramatic increase in the throughput sample number that can be analyzed simultaneously. Additional advantages can be accessed by using microplate reader application software packages that permit assigning a group type to the wells, recording of the data on exportable data files and exercising the option of using the kinetic or endpoint reading modes. The assay can also be used independently for detecting nitrite residues/contamination in environmental/food samples.

Development of a recombinant baculovirus expressing an insect-selective neurotoxin: potential for pest control.

Recombinant nuclear polyhedrosis viruses (NPVs) expressing insect-selective toxins, hormones, or enzymes could enhance their insecticidal properties. We have constructed a recombinant, polyhedrin-positive Autographa californica NPV (AcNPV) that is orally infectious and expresses an insect-selective toxin (AaIT), isolated from the scorpion Androctonus australis, under the control of the p10 promoter. Bioassays with the recombinant baculovirus on 2nd instar larvae of Heliothis virescens demonstrated a significant decrease in the time to kill (LT50 88.0 hours) compared to wild-type AcNPV (LT50 125 hours). Production of AaIT was confirmed by western blot analysis of larval hemolymph from infected H. virescens, and bioassays with larvae of Sarcophaga falculata.

Altered cortical glutamatergic and GABAergic signal transmission with glial involvement in depression.

Abnormalities in L-glutamic acid (glutamate) and GABA signal transmission have been postulated to play a role in depression, but little is known about the underlying molecular determinants and neural mechanisms. Microarray analysis of specific areas of cerebral cortex from individuals who had suffered from major depressive disorder demonstrated significant down-regulation of SLC1A2 and SLC1A3, two key members of the glutamate/neutral amino acid transporter protein family, SLC1. Similarly, expression of L-glutamate-ammonia ligase, the enzyme that converts glutamate to nontoxic glutamine was significantly decreased. Together, these changes could elevate levels of extracellular glutamate considerably, which is potentially neurotoxic and can affect the efficiency of glutamate signaling. The astroglial distribution of the two glutamate transporters and L-glutamate-ammonia ligase strongly links glia to the pathophysiology of depression and challenges the conventional notion that depression is solely a neuronal disorder. The same cortical areas displayed concomitant up-regulation of several glutamate and GABA(A) receptor subunits, of which GABA(A)alpha1 and GABA(A)beta3 showed selectivity for individuals who had died by suicide, indicating their potential utility as biomarkers of suicidality. These findings point to previously undiscovered molecular underpinnings of the pathophysiology of major depression and offer potentially new pharmacological targets for treating depression.

Product-mediated regulation reveals the polymorphic nature of the yeast assimilatory nitrate reductase.

Nitrite functioned as an effective inducer of nitrate reductase, the enzyme responsible for the reduction of nitrate in the nitrate assimilation pathway in Candida utilis. Nitrite-induced synthesis of nitrate reductase in C. utilis was repressed by various metabolites of nitrate, including ammonia. Readily-assimilable sources of nitrogen such as ammonia and glutamate exerted a stronger repression on nitrate reductase induction than did less-readily assimilable hydrazine and hydroxylamine. Nitrite-mediated induction of nitrate reductase appeared more sensitive to repression by nitrate metabolites than was nitrate-mediated induction. Based on the inducer-specific differences in the sensitivity of the enzyme to repression by various intermediary metabolites and on other properties, it is proposed that the C. utilis nitrate reductase is either polymorphic or utilizes alternative receptor(s) for binding various gratuitous inducers including nitrite in initiating the induction pathway.

Ability to assimilate nitrogenous oxides is limited to a few species of yeasts.

Several species of yeasts, Saccharomyces cerevisiae, Debaryomyces hansenii, Candida utilis, C. albicans, C. pelliculosa and C. tropicalis, were tested for their ability to utilise different inorganic nitrogen compounds as sole sources of nitrogen, and the results are presented as a function of their growth. All of them grew well on reduced nitrogenous compounds such as ammonium sulphate (ammonia) and L-asparagine, while only C. utilis and C. pelliculosa grew, at a slower rate, on oxidised nitrogenous compounds, nitrate and nitrite. The differences observed in the ability of various yeasts to assimilate the inorganic nitrogen compounds are believed to reflect their individuals abilities to regulate the uptake rates of potentially toxic compounds and keep them in a bound/transient state until reduced to less toxic and more-readily assimilable forms.

A simple micromethod for rapid kinetic analyses of yeast enzymes in situ.

A simple approach for rapid assay of enzymes in large numbers of samples of whole cells of yeast is described. The technique involves treatment of yeast cells with toluene dissolved in ethanol (1:4, v/v; TE), rendering them selectively permeable to small molecules, while letting the enzymes remain intracellular and catalytically active but accessible to exogenously added assay reagents. The permeated cell preparations permit rapid assay of nitrate reductase activity in situ [P. V. Choudary and G. Ramananda Rao (1976) Current Sci. 45, 324-327]. Investigation of enzyme kinetic parameters such as pH optima, temperature dependence, and substrate saturation, which involves several samples and needs to be investigated very rapidly and accurately, is greatly facilitated by permeated cells prepared from a very small culture of cells, eliminating the need for elaborate preparations of cell extracts. By using permeabilized cell preparations, the need to supplement the reaction mixture with expensive cofactors can also be obviated to a large extent. Further, the enzyme activity displays a broader range of tolerance in situ to variations in reaction conditions compared to that in vitro. Permeabilized cell preparations thus seem to present the enzymes in the form of relatively less disintegrated functional complexes, making them a valuable tool in biochemical and cell biological investigations, and facilitate the correlation of in vitro data with the in vivo phenomena more confidently.

Rapid and sensitive immunomagnetic separation-polymerase chain reaction method for the detection of Escherichia coli O157:H7 in raw milk and ice-cream.

Escherichia coli O157:H7 in spiked samples of raw milk and ice-cream was enriched in tryptic soy broth for 4 h, captured by immunomagnetic separation, subjected to amplification by polymerase chain reaction of parts of the verotoxin genes (SLT-I and SLT-II), and detected by agarose gel electrophoresis. Using this method, as few as 1 cfu Esch. coli O157:H7/g food could be detected in < 10 h.

Comparison of different primers for rapid detection of Salmonella using the polymerase chain reaction.

Salmonella is the leading cause of food-borne diarrhoeas in the US. In recent years polymerase chain reaction (PCR) has become the method of choice for rapid and sensitive detection of Salmonellae in contaminated foods. As a result, several different primer sets have been reported for use in PCR-based assay systems. In order to identify an optimal primer set from among the wide range of primers reported in the literature, we synthesized five different pairs and evaluated their relative performance in PCR under uniform assay conditions using a common panel of the target (Salmonella) and non-target (non- Salmonella) bacterial strains. Of the five sets of primers tested, the one designed on the basis of a 199 bp repeat sequence of S. weltevreden[Jitrapakdee et al. (1995) Molecular and Cellular Probes 9, 375-382] gave optimal results with most bacterial strains examined.

A unified discrete model of immune response.

In this paper we propose a unified model of immune response in terms of discrete automata describing the concentrations of the cells constituting the immune network. The model of normal immune response proposed by Kaufman, Urbain and Thomas and that of auto-immune response proposed by Weisbuch, Atlan and Cohen are special cases of this unified model. Moreover, this model also describes the immune response in patients infected by the human immunodeficiency virus (HIV), the virus that is known to cause Acquired Immune Deficiency Syndrome (AIDS).

Partial purification and properties of the assimilatory nitrate reductase of the food yeast Candida utilis.

The addition of nitrate, EDTA and dithiothreitol to the enzyme extraction buffer resulted in improved stability of the assimilatory nitrate reductase activity from the food yeast Candida utilis at both 4 degrees C and -10 degrees C. By incorporating this critical step in the following sequence the yeast NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) was purified approximately 68-fold by protamine sulphate precipitation, calcium gel adsorption, ion exchange chromatography and gel filtration. Both NADPH-nitrate reductase and NADH-nitrate reductase activities remained in constant association and ratio (2:3) during the entire course of purification. The enzyme showed an absolute requirement of NADPH or NADH for its activity. Maximal enzyme activity was obtained with 10-120 micrograms protein in a 10 min assay at 30 degrees C at pH 6.5, with an apparent Michaelis constant of 0.69 mM for nitrate as substrate. The enzyme is a molybdoflavo-protein involving sulphydryl groups, and is highly sensitive to free reducing agents, heavy metal ions and electron-transfer inhibitors. The results also suggested possible involvement of a second metal ion, perhaps iron, which was hypothesized to participate in the electron transfer scheme catalysed by this enzyme.

Role of murine cytochrome P-450 2F2 in metabolic activation of naphthalene and metabolism of other xenobiotics.

Despite their substantially lower levels relative to hepatic tissue, pulmonary cytochrome P-450 (CYP) monooxygenases play an important role in the metabolic activation of substrates that cause lung injury. The target- and species-selective toxicity of a number of pulmonary toxicants has been attributed to the presence and distribution of activating enzymes with high kcat in target airways of susceptible species. However, experimental demonstration of these concepts and quantitative assessment of the contribution of individual CYP isoforms is lacking. This study was undertaken to characterize the catalytic activities of CYP2F2 with naphthalene, a murine Clara cell toxicant, as well as with other xenobiotics that either undergo metabolic activation to cytotoxic intermediates or that function as "isoform-selective" substrates. Recombinant CYP2F2 was produced using the baculovirus expression vector system in Spodoptera frugiperda and Trichoplusia ni cells, accounting up to approximately 20% of the total cellular protein. Incubations containing naphthalene, recombinant CYP2F2, NADPH-cytochrome P-450 oxidoreductase, and NADPH-regenerating system metabolized naphthalene with a high degree of stereoselectivity to 1R, 2S-naphthalene oxide (66:1 enantiomeric ratio). The Km and kcat values, along with the specificity constant, for naphthalene metabolism by recombinant CYP2F2 were 3 microM, 104 min-1, and 5.8 x 10(5) M-1 s-1, respectively. Recombinant CYP2F2 also metabolized ethoxyresorufin, pentoxyresorufin, p-nitrophenol, and 1-nitronaphthalene at easily detectable levels. The results from this work suggest that CYP2F2 1) plays a key role in the species- and cell-selective toxicity of naphthalene and 2) efficiently metabolizes a number of other substrates, including the lung toxicant 1-nitronaphthalene.