Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41

The V_H domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG^TM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The V_H-FLAG was composed of three isoforms (pI values of 4.6, 4.9, and 5.3) and the V_H molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the V_H isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the V_H domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its¹H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the V_H molecule, prevented dimerization of the V_H and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.     

Multiple extracellular domains of CCR-5 contribute to human immunodeficiency virus type 1 entry and fusion.

Human immunodeficiency virus type 1 (HIV-1) entry is governed by the interaction of the viral envelope glycoprotein (Env) with its receptor. The HIV-1 receptor is composed of two molecules, the CD4 binding receptor and a coreceptor. The seven-membrane-spanning chemokine receptor CCR-5 is one of the coreceptors used by primary isolates of HIV-1. We demonstrate that the mouse homolog of CCR-5 (mCCR-5) does not function as an HIV-1 coreceptor. A set of chimeras of human CCR-5 and mCCR-5 was studied for Env-induced cell fusion and HIV-1 infection. Using the HIV-1ADA envelope glycoprotein in a syncytium formation assay, we show that replacement of any fragment containing extracellular domains of mCCR-5 by its human counterparts is sufficient to allow Env-induced fusion. Conversely, replacement of any fragment containing human extracellular domains by its murine counterpart did not lead to coreceptor function loss. These results show that several domains of CCR-5 participate in coreceptor function. In addition, using a panel of primary nonsyncytium-inducing and syncytium-inducing isolates that use CCR-5 or both CXCR-4 and CCR-5 as coreceptors, we show that the latter dual-tropic isolates are less tolerant to changes in CCR-5 than strains with a more restricted coreceptor use. Thus, different strains are likely to have different ways of interacting with the CCR-5 coreceptor.     

Factors affecting the genetic engineering of plants by microprojectile bombardment

Since its development in the mid-1980s, microprojectile bombardment has been widely employed as a method for direct gene transfer into a wide range of plants, including the previously difficult-to-transform monocotyledonous species. Although the numerous instruments available for microprojectile-mediated gene delivery and their applications have been widely discussed, less attention has been paid to the critical factors which affect the efficiency of this method of gene delivery. In this review we do not wish to describe the array of devices used for microprojectile delivery or their uses which have already been definitively described, but instead wish to report on research developments investigating the factors which affect microprojectile-mediated transformation of plants.     

Dieback of Passion Fruit in Surinam

In Surinam, the commercial cultivation of the yellow passion fruit (Passiflora edulis f. flavicarpa) is difficult due to the occurrence of dieback. Symptoms referred to as dieback include a decrease in elongation of the shoot end internodes after a period of normal growth leading to wilting and death of the shoots. Fruits from plants showing dieback symptoms are much smaller than those from healthy plants. From shoots with dieback symptoms, three fungi were isolated including Colletotrichum gloeosporioides. However, inoculation experiments with these fungi on shoots of vigorously growing plants were negative, even after wound inoculation. It appeared that plants with dieback symptoms had a poorly developed root system, From these roots Fusarium solani was isolated, which appeared to be highly pathogenic to roots of the yellow passion fruit. After inoculation of the roots of 3-month-old plants, roots became infected and the aerial plant parts showed typical dieback symptoms. Plants with their root system reduced either by inoculating with F. solani or by clipping, and subsequently inoculated with C. gloeosporioides on the aerial parts 2 weeks later, showed dieback symptoms and infection by C. gloeosporioides in shoots with these symptoms. Thus, a badly functioning root system, for example caused by infection of F. solani leads to dieback and predisposes plants to infection by C. gloeosporioides. The latter fungus itself is not a primary pathogen of shoots of the yellow passion fruit in Surinam.     

Mechanism and contribution of the pentose phosphate cycle to glucose metabolism in epididymal fat tissue

1. Glucose 6-phosphate, fructose 6-phosphate and altroheptulose 7-phosphate are the major products formed non-oxidatively from ribose 5-phosphate by rat epididymal fat pad enzymes. 2. Arabinose 5-phosphate was detected among the reaction products and significant activity of the new enzyme of the L-type pentose pathway, D-glycero D-ido octulose 1,8-bisphosphate: D-altroheptulose 7-phosphotransferase was found. 3. The glucose moieties of glucose 1-phosphate, glucose 6-phosphate and glucose 1,6-bisphosphate were degraded and showed that epididymal fat pad enzymes relocate 14C from [2-14C]glucose into C-1, C-2, and C-3 of each hexose-phosphate. 4. The 14C-distribution patterns in the hexose-phosphates revealed that these intermediates were not in isotopic equilibrium and the rate of the transaldolase exchange reaction was relatively small. 5. The 14C-distribution data suggest that glucose 1-phosphate, rather than glucose 6-phosphate, is the first intermediate in the path of glycogen synthesis from glucose in this tissue. 6. The data provide the first proof of the mechanism of the pentose pathway in adipose tissue.     

Somatic hybridization of sexually incompatible petunias: Petunia parodii,Petunia parviflora

Summary Somatic hybrid plants were regenerated following the fusion of leaf mesophyll protoplasts of P. parodii with those isolated from a nuclear-albino mutant of P. parviflora. Attempts at sexual hybridization of these two species repeatedly failed thus confirming their previously established cross-incompatibility. Selection of somatic hybrid plants was possible since protoplasts of P. parodii would not develop beyond the cell colony stage, whilst those of the somatic hybrid and albino P. parviflora produced calluses. Green somatic hybrid calluses were visible against a background of albino cells/calluses, and upon transfer to regeneration media gave rise to shoots. Shoots and the resultant flowering plants were confirmed as somatic hybrids based on their growth habit, floral pigmentation and morphology, leaf hair structure, chromosome number and Fraction 1 protein profiles. The relevance of such hybrid material for the development of new, and extensively modified cultivars, is discussed.     

Purification of an NADH-(dichlorophenol-indophenol) oxidoreductase from Bacillus stearothermophilus.

The crystals of the entomocidal protein of Bacillus thuringiensis are admixed with proteinases that in the course of their dissolution cause gradual degradation of the "genuine" crystal-forming protein components (i.e. the primary biosynthetic products) to products of lower molecular weight. This phenomenon might explain at least partially the contradictory data on the molecular parameters of the crystal-forming proteins. Preliminary inactivation of the proteinases adsorbed on the crystals allowed us to eliminate this source of the artefacts and to gain more reliable data on the protein composition of the crystals formed by various strains of B. thuringiensis. It has been shown that the crystals formed by all serotypes of B. thuringiensis, with the exception of the serotype V, contain only one protein with a mol. wt. of 145000, 135000 or 130000, depending on the strain. The majority of the strains that belong to the serotype V form crystals consisting of two proteins with mol. wts. of 135000 and 130000, but some of them also have a third component with a mol. wt. of 65000.     

Electron transfer reactions of cytochrome c confined within erythrocyte ghosts

We have prepared and characterized resealed erythrocyte ghosts in which the only discernible pigment is cytochrome c. The resealed ghosts have the normal orientation and are free of 'leaky' species; they are stable and can be maintained at 4 degrees C for many days without lysis. The internal cytochrome c participates in redox reactions with both soluble and insolubilized cytochrome c present externally, and with external cytochrome b5. No reaction was observed with plastocyanin, cytochrome c oxidase or NADPH-cytochrome c reductase. A study has been made of the reaction of the internal cytochrome c with the low molecular weight reductants, ascorbate and glutathione. Complex kinetics are observed with both reagents: with ascorbate the results are best explained by assuming the existence, in the membrane, of a redox-active species able to undergo dedimerization. A protein bound disulfide bond would satisfy the requirement.     

Use of radio telemetry for studying upriver migration of adult Atlantic salmon (Salmo salar)

This paper describes and evaluates a 50 mHz radio telemetry system for studying river movements of adult Atlantic salmon ( Salmo salar ). In fresh water for most applications radio telemetry is preferable to ultrasonic telemetry, because the receiving element (antenna) can be above water, and radio signals are scarcely affected by turbulent, weedy or ice-covered water. Within the range of 10–200 mHz higher frequencies are preferred, since the efficient antenna size is inversely proportional to frequency, and attenuation of signals is independent of frequency. Transmitters were cylindrical (6.5–9.6 cm long * 1.9 cm diam) with a 0.5 wavelength antenna trailing from one end. Each emitted pulsed signals on one of 20 crystal-controlled channels between 49.100 and 49.385 mHz. Transmitters were placed in the stomachs of salmon and the antenna trailed out the last gill slit. Receivers were portable 20-channel manual or automatic scan models, and antennas were 48 cm diam capacitor tuned loops. Some salmon regurgitated transmitters. Two salmon were recaptured and showed no ill effects from carrying transmitters for 32 and 42 days. Pulse rate had little effect on known transmitter life under natural conditions. Known tag life was variable, but averaged 70 days for transmitters with 1000 mah batteries. The range of transmission of transmitters to a receiving system in an airplane at 410 m altitude was about 10 km, and to a boat about 1 km. Range to a land vehicle was variable depending on obstructions. From the airplane transmitters can be located within a radius of about 50 m.