Cloning of the gene coding for chitobiase ofSerratia marcescens

Summary A λ phage DNA library ofSerratia marcescens was constructed and a clone carrying the gene coding for chitobiase (E.C.3.2.1.29) was isolated and characterized. Deletion analysis limited the cloned region to 4.5 kb that is capable of efficient expression of chitobiase.Escherichia coli cells harboring a plasmid carrying the cloned gene express chitobiase constitutively. The molecular weight of the protein is about 95000 daltons. In exponentially growingE. coli cells the chitobiase enzyme was found to be secreted into the periplasm.     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

The decolorization of the polymeric dye Poly-Blue (polyvinalamine sulfonate-anthroquinone) by lignin degrading fungi

Summary In this work we have investigated the decolorization of the polymeric dye Poly-B411 by several fungi. Only fungi with known lignin degrading ability were able to decolorize the dye. Pleurotus ostreatus sp. florida decolorized the dye both in solid and liquid media. Decolorizing ability developed in the absence of the dye but only when the fungus had been previously cultivated on lignin containing substrates.The work was supported by a grant from the Charles Wolfson Trust     

Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes.

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.     

Chemical Detection of Microbial Prey by Bacterial Predators

A motile, predacious bacterium which degraded Pythium debaryanum was strongly attracted to substances released into the medium by the fungus. A nonpredacious bacterium was not attracted to these substances. The predator bacterium was specifically attracted to cellulose and its oligomers which are known to be components of the cell wall of Pythium. Ethanol inhibited chemotaxis of the bacterium without affecting either its motility or its ability to degrade cellulose. A second predacious bacterium was isolated for the alga, Skeletonema costatum. The role of chemoreception in the detection of microbial prey by bacterial predators in natural habitats is discussed.     

In Vivo Expression of Inducible Nitric Oxide Synthase in Cerebellar Neurons

Abstract: In the CNS, nitric oxide (NO) functions as both neuromodulator and neurotoxic agent. In vivo neuronal expression of NO synthase (NOS) has been attributed to constitutive NOS—both the neuronal and the endothelial types. The other class of NOS—the inducible NOS (iNOS)—is known to mediate toxic effects of NO in various tissues. In this study, we show for the first time that direct intracerebellar injection of endotoxin and cytokine (lipopolysaccharide and interferon-γ) induced in vivo neuronal expression of the iNOS gene, as demonstrated by fluorescent in situ hybridization and immunohistochemical staining analyzed by confocal laser-scanning microscopy. This raises the possibility that neuronal iNOS might contribute significantly to the vulnerability of the brain to various insults.     

External Protons Enhance the Activity of the Hyperpolarization-activated K Channels in Guard Cell Protoplasts of Vicia faba

Hyperpolarization-activated K channels (K _H channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell mode, we characterized the effect of varying the external pH between 4.4–8.1 on the activity of the K _H channels in isolated guard cell protoplasts from Vicia faba leaves. Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the K _H channels by 30–150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by ∼60% between pH 8.1 and 4.4 with an apparent pK _a of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing of these charges was ∼30 ? and their apparent dissociation constant was 10^−4.6. The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence of all four transition rate constants. Received: 26 April 1996/Revised: 29 June 1996     

Elements within the beta-lactoglobulin gene inhibit expression of human serum albumin cDNA and minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice.

Two new beta-lactoglobulin (BLG)/human serum albumin (HSA) hybrid gene vectors were constructed and tested for expression in COS-7 cells and in transgenic mice. The HSA sequences were inserted between the second and sixth BLG exons. Transient transfection experiments with these vectors as well as a series of additional vectors with either the BLG 5'- or 3'- intragenic sequences revealed that sequences within BLG exon 1/intron 1/exon 2 abrogated BLG- directed HSA expression in vitro, regardless of the presence of HSA introns or the origin of the 3' polyadenylation signal. In contrast, the same BLG expression cassette enabled the efficient expression of HSA cDNA or minigene in the mammary gland of transgenic mice with subsequent secretion of the corresponding protein into the milk of 56 and 82%, respectively of the mouse strains at levels up to 0.3 mg/ml. Previous attempts to express HSA cDNA inserted into exon 1 of the BLG gene had failed [Shani,M., Barash,I., Nathan,M., Ricca,G., Searfoss,G.H., Dekel,I., Faerman,A., Givol,D. and Hurwitz,D.R. (1992) Transgenic Res. 1, 195- 208]. The new BLG expression cassette conferred more stringent tissue specific expression than previously described BLG/HSA constructs [Barash,I, Faerman,A., Ratovitsky,T, Puzis,R., Nathan,M., Hurwitz,D.R. and Shani, M. (1994) Transgenic Res. 3, 141-151]. However, it was not able to insulate the transgenes from the surrounding host DNA sequences and did not result in copy number dependent expression in transgenics. Together, the in vitro and in vivo results suggest both positive and negative regulatory elements within the BLG intragenic sequences evaluated. The new BLG construct represents an extremely valuable vector for the efficient expression of cDNAs in the mammary gland of transgenic animals.