Activation of a Latent Measles Virus Infection in Hamster Cells

The characteristics of infectious measles virus released from latently infected hamster embryo fibroblast cells are described. Low levels of virus were released spontaneously when the cultures were incubated at 37 C; this phenomenon was observed 19 passages after the cells had been exposed to the virus and has continued through cell passage 45. The virus yield could be significantly increased by cocultivation of the hamster cells with BSC-1 cells or incubation of the latently infected cells at 33.5 C rather than at 37 C. Measles virus released after cocultivation demonstrated increased cytopathology in cell culture and reduced temperature sensitivity when compared to the virus released at 33.5 C. After cell passage 45, there was an increase in spontaneous release of virus. However, the viruses recovered by cocultivation or temperature release after cell passage 45 were nearly identical. These observations suggest a possible mechanism for measles virus activation in cells latently infected with this virus.     

Demonstration of Cholinesterase in Teeth

Human teeth have been studied by treatment with copper thio-choline, the method developed by Koelle for demonstrating activity of both specific and nonspecific cholinesterases. Freshly extracted teeth were collected and immediately sectioned on a cutting machine designed for calcified tissues. One series of teeth was sectioned sufficiently thin for microscopic study. Another series of teeth was bisected to expose the pulp chambers to the reagents. These teeth were divided into 5 experimental groups. The first group was treated with 10^-6M di-isopropylfluorophosphate (DFP) for 30 min at 37°C and then incubated with acetylthiocholine (AThCh) for 16 to 20 hr at 37°C in order to reveal the sites of activity of the specific enzyme, AChEase. The second group was incubated in a substrate of butyrylthiocholine (BuThCh) for 12 to 16 hr at 37°C to indicate the sites of the nonspecific ChEase. The third group was incubated in AThCh for 16 to 20 hr at 37°C without previous treatment by an inhibitor in order to reveal the presence and location of both the specific and nonspecific ChEase. The fourth and fifth groups were utilized as controls. Group 4 tissues were incubated without a substrate while those of group 5 were treated with DFP and then incubated with BuThCh. The specimens then were treated with ammonium sulfide to outline the sites of ChEase activity. The thin sections were mounted directly but the series of halved teeth were decalcified, embedded in paraffin, sectioned and then mounted. By these methods the presence and location of both specific and nonspecific ChEase activity were observed in human teeth. Concentration of specific ChEase was observed along the coronal aspect of the pulp chamber and along the course of the pulpal nerves. The nonspecific ChEase was observed throughout the pulpal tissue and appeared to be concentrated along the nerves and blood vessels. Neither series of control tissues exhibited any staining in the pulp tissue.     

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

Glycosylation of proteins is a key function of the biosynthetic‐secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell‐cell adhesion, blood‐group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein‐based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose‐1‐phosphate‐guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1‐domain polyphosphate kinase 2 (1D‐Ppk2) expressed in E. coli for the cell‐free production and regeneration of GDP‐mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP‐mannose is produced at various conditions, that is pH 7–8, temperature 25–35°C and co‐factor concentrations of 5–20 mM MgCl₂. The maximum reaction rate of GDP‐mannose achieved was 2.7 μM/min at 30°C and 10 mM MgCl₂ producing 566 nmol GDP‐mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane‐deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER‐associated lipid‐linked oligosaccharide (LLO) assembly. Thereby, in a one‐pot reaction, phytanyl‐PP‐(GlcNAc)₂‐Man₁ was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl‐PP‐(GlcNAc)₂‐Man₁ can serve as a substrate for the synthesis of LLO for the cell‐free in vitro glycosylation of proteins. A high‐performance anion exchange chromatography method with UV and conductivity detection (HPAEC‐UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP‐mannose regenerating cascade and can further be used to study coupling of the GDP‐mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell‐free production of LLOs as precursors for in vitro glycoengineering of proteins.     

Improved linkage map and thirty new microsatellite markers for rat Chromosome 10

An improved linkage map for rat Chromosome (Chr) 10 with two F₂ populations was constructed. Thirty new microsatellite markers were generated from a Chr 10-specific, small-insert genomic library and mapped to rat Chr 10. Among them were the rat homologs for the mouse gene for light and heavy chains of myeloperoxidase and human neurofibromatosis 1. Eight newly generated markers (D10Mco62, D10Mco63, D10Mco64, D10Mco65, D10Mco67, D10Mco68, D10Mco70, and D10Mco74) were mapped to the region of the rat Chr 10 blood pressure QTL. The availability of such markers may be instrumental in the search for genes responsible for the hypertension. Received: 13 July 1998 / Accepted: 9 September 1998     

Two blood pressure/cardiac mass quantitative trait loci on Chromosome 3 in Dahl rats

Interval mapping was used to identify putative quantitative trait loci (QTL) for blood pressure and cardiac mass on Chromosome (Chr) 3 in F₁(S × R) × S population of 150 rats raised on an 8% NaCl diet. Two genetic markers 95.7 cM apart, D3Wox3 and D3Mco5 (tightly linked to Edn3), showed ``suggestive' linkage to blood pressure (LOD = 2.0 and 1.8 respectively). In addition, D3Wox3 showed ``suggestive' linkage to heart weight (LOD = 2.5), and D3Mco5 showed ``suggestive' linkage to body weight–adjusted heart weight (LOD = 2.1). Congenic rats (designated S.R-Edn3) were constructed by introgressing the R-rat Edn3 allele (and flanking loci) into the S strain. On a 2% NaCl diet, S.R-Edn3 rats had lower blood pressure (21.4 mm Hg, P= 0.0005) and heart weight (59 mg, P= 0.0038) compared with S rats, confirming the existence of a blood pressure QTL on Chr 3 near Edn3 even though QTL linkage analysis of blood pressure did not achieve stringent statistical criteria for significance. The results of the congenic experiment and the large distance between the two putative QTL suggest the presence of at least two independent blood pressure/cardiac mass QTL detectable on Chr 3 in the Dahl rat model of genetic hypertension. Received: 24 April 1998 / Accepted: 29 September 1998     

Degradation of Chlorobenzenes at Nanomolar Concentrations by Burkholderia sp. Strain PS14 in Liquid Cultures and in Soil

The utilization of 1,2,4,5-tetrachloro-, 1,2,4-trichloro-, the three isomeric dichlorobenzenes and fructose as the sole carbon and energy sources at nanomolar concentrations was studied in batch experiments with Burkholderia sp. strain PS14. In liquid culture, all chlorobenzenes were metabolized within 1 h from their initial concentration of 500 nM to below their detection limits of 0.5 nM for 1,2,4,5-tetrachloro- and 1,2,4-trichlorobenzene and 7.5 nM for the three dichlorobenzene isomers, with 63% mineralization of the tetra- and trichloroisomers. Fructose at the same initial concentration was, in contrast, metabolized over a 4-h incubation period down to a residual concentration of approximately 125 nM with 38% mineralization during this time. In soil microcosms, Burkholderia sp. strain PS14 metabolized tetrachlorobenzene present at 64.8 ppb and trichlorobenzene present at 54.4 ppb over a 72-h incubation period to below the detection limits of 0.108 and 0.09 ppb, respectively, with approximately 80% mineralization. A high sorptive capacity of Burkholderia sp. strain PS14 for 1,2,4,5-tetrachlorobenzene was found at very low cell density. The results demonstrate that Burkholderia sp. strain PS14 exhibits a very high affinity for chlorobenzenes at nanomolar concentrations.     

Multiphasic Kinetics of Transformation of 1,2,4-Trichlorobenzene at Nano- and Micromolar Concentrations by Burkholderia sp. Strain PS14

The transformation of 1,2,4-trichlorobenzene (1,2,4-TCB) at initial concentrations in nano- and micromolar ranges was studied in batch experiments with Burkholderia sp. strain PS14. 1,2,4-TCB was metabolized from nano- and micromolar concentrations to below its detection limit of 0.5 nM. At low initial 1,2,4-TCB concentrations, a first-order relationship between specific transformation rate and substrate concentration was observed with a specific affinity (a⁰_A) of 0.32 liter · mg (dry weight)⁻¹ · h⁻¹ followed by a second one at higher concentrations with an a^o_A of 0.77 liter · mg (dry weight)⁻¹ · h⁻¹. This transition from the first-order kinetics at low initial 1,2,4-TCB concentrations to the second first-order kinetics at higher 1,2,4-TCB concentrations was shifted towards higher initial 1,2,4-TCB concentrations with increasing cell mass. At high initial concentrations of 1,2,4-TCB, a maximal transformation rate of approximately 37 nmol · min⁻¹ · mg (dry weight)⁻¹ was measured, irrespective of the cell concentration.