Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162–170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.Abbreviations FR far-red light - R red light - WL white light - WL + FR white light supplemented with FR - HIR high-irradiance response - PAR photosynthetically active radiation - Pr, Pfr R- and FR-absorbing forms of phytochrome - Ptot total phytochrome - phyA (PhyA) gene (encoded protein) for phytochrome - WT wild type This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.M.; J.R. Cherry and R.D. Vierstra, (Department of Horticulture, University of Wisconsin-Madison, USA) are thanked for the provision of the transgenic tobacco line.     

Comparison of Representative Strains of Infectious Hematopoietic Necrosis Virus by Serological Neutralization and Cross-Protection Assays

Infectious hematopoietic necrosis virus (IHNV) is a pathogen of young salmon and trout. Viral epizootics among these fish in private and public rearing facilities have been a problem in the northwestern United States from California to Alaska, and an IHNV vaccine has been sought by the aquaculture experts. Since an IHNV vaccine must be designed to immunize against all viral serotypes, an analysis of IHNV serotypes was made. A large number of viruses from widely separated geographic locations and different fish species had already been placed in one of five electropherotypes by the migration of the virion proteins in sodium dodecyl sulfate-polyacrylamide gels. Also, there was evidence that some of these virus isolates had differences in virulence for chinook salmon, rainbow trout, or kokanee salmon. Previous serological studies with polyclonal rabbit antisera and three IHNV isolates indicated that there was only one serotype (B. B. McCain, J. L. Fryer, and K. S. Pilcher, Proc. Soc. Exp. Biol. Med. 137:1042-1046, 1971). A substantial number of new IHNV isolations have been made since that study, and thus a more extensive comparison was made of 10 different IHNV isolates representing the five electropherotypes. This report shows that the glycoprotein from a single isolate of IHNV can induce a protective immune response in vivo to the five IHNV electropherotypes. Plaque reduction neutralization assays indicated that there was only one serotype. Thus, despite the differences observed in the migration of the structural proteins for IHNV isolated from separate geographic locations and different fish species, only one neutralizing virus type was identified.     

Uridine Metabolism in the Goldfish Retina During Optic Nerve Regeneration: Whole Retina Studies

Accumulation of radioactivity from [3H]uridine in incubations of whole goldfish retinas is increased in the ipsilateral retina during a period of regeneration that follows unilateral optic nerve crush. Brief incubations to investigate the nature of enhanced labeling of the acid-soluble fraction showed a peak uptake 4 days following crush, with a gradual decrease to control levels by 21 days following crush. That nucleoside uptake may not mediate the effect is supported by the observation that the rate of uptake of 5'-deoxyadenosine, a nonmetabolizable nucleoside analog, is the same in post-crush (PC) and normal (N) retinal incubations. Following brief incubations of PC and N retinas with [3H]uridine, there is enhanced labeling in PC retinas relative to N retinas of recovered UMP, UDP, UTP, and uridine nucleotide sugars, whereas recovery of labeled uridine itself is slightly decreased. The results suggest that the increased accumulation of radioactivity in PC retinas following incubation with uridine reflects an increase in the activities of retinal uridine kinase and uridine nucleotide kinases.     

Shutoff on Neuroblastoma Cell Protein Synthesis by Semliki Forest Virus: Loss of Ability of Crude Initiation Factors to Recognize Early Semliki Forest Virus and Host mRNA's

A crude ribosomal wash containing the initiation factors of protein synthesis was isolated from mouse neuroblastoma cells 8 h after infection with Semliki Forest virus (SFV). The activity of this wash was compared with that of a wash from control cells in a cell-free protein-synthesizing “pH5” system, with early SFV mRNA (42S), late SFV mRNA (26S), encephalomyocarditis virus (EMC) mRNA, or neuroblastoma polyadenylated mRNA templates. A pronounced loss of activity (±80%) of the crude ribosomal wash from infected cells was observed with host mRNA (neuroblastoma polyadenylated mRNA) and early SFV mRNA, messengers which contain a cap structure at the 5′ terminus. However, these washes were only slightly less active in systems programmed with (noncapped) EMC mRNA and late SFV mRNA. Although late SFV mRNA (26S) is capped, the synthesis of late (= structural) proteins in infected lysates was insensitive to inhibition by cap analogs. Purified initiation factors eIF-4B (M_r, 80,000) and cap-binding protein (M_r, 24,000) from reticulocytes (but none of the others) were able to restore the activity of infected factors to about 90% of control levels in systems programmed with early SFV mRNA and host mRNA. These observations indicate that infection-exposed crude initiation factors have a decreased level of eIF-4B and cap-binding protein activity. However, after partial purification of these and other initiation factors from infected and control cells, we found no significant difference in activity when model assay systems were used. Furthermore, both eIF-4B and cap-binding protein from infected cells were able to restore the activity of these infection-exposed factors to the same level obtained when these factors isolated from control cells or reticulocytes were added. A possible mechanism for the shutoff of host cell protein synthesis is discussed.     

Delayed-type cutaneous hypersensitivity to melanoma antigens and its implication in active specific immunotherapy in cancer

Summary In this study, we explored whether soluble tumor-cell surface-associated antigens (TAA) might be derived from autochthonous as well as allogeneic sources as immunogens for active specific immunotherapy. Using two popular cell membrane-bound antigen extraction techniques (3 M KCl and isotonic-hypotonic NaCl), we examined the immunogenic potential of such TAA and the specificity of immunologic host reactivity through a delayed-type cutaneous hypersensitivity reaction (DTH) as a guideline for their immunogenic potential in a human malignant melanoma model system. We found that either extraction technique could provide soluble TAA from both autochthonous and allogeneic sources capable of eliciting DTH. While evidence of positive DTH with autochthonous TAA reaffirms the immunogenicity of such TAA, the specificity of host reactivity against TAA derived from allogeneic sources is extremely difficult to establish, even with TAA partially purified by column chromatography in Sephadex G-200. Patients exhibited reactivity to other TAA derived from tumors of different histologies and often to more than one component isolated by column chromatography. Furthermore, when a group of melanoma patients was tested against a panel of melanoma antigens in any random combination, DTH to allogeneic TAA was seen in an unpredictable order and with inconsistent frequency. We conclude, therefore, that while autochthonous antigen immunizations may be justified, more careful studies will be necessary to define the antigenic profile of a given tumor (individual specificity vs shared specificity), establish specificity of alloantigens, and devise suitable methods for testing immunologic specificity for alloantigens, before rational immunotherapy with allogeneic tumor antigens will be feasible.     

Heavy metal composition of polysomal fractions following cadmium challenge

Endogeneous levels of zinc and copper were found to be 1.2±0.1×10⁻² and 0.3±0.1×10⁻² μg/A260 unit, respectively, in polysomal fractions from control animals; cadmium, however, was undetectable. In experimental animals (injected with cadmium) zinc, copper, and cadmium were found in polysomal fractions isolated by two different methods. One hour after a cadmium injection there was a rise in both the zinc and copper content of the polysomal fractions, which then declined steadily to below control levels by 16 h. Neither zinc nor cadmium were dialyzable from these fractions by a TRIS buffer; however, addition of 0.01M EDTA to the buffer resulted in removal of 75% of the zinc and all of the detectable cadmium. The addition of cadmium (CdCl₂) to control supernatants (adjusted to the cadmium concentration present in supernatants 6 h after in vivo exposure) resulted in metal binding to polysomal fractions in levels comparable to those observed after in vivo exposures to the metal. When cadmium was added in the form of cadmium thionein, a smaller fraction of the metal was isolated with the polysomal fraction. Cadmium bound to polysomal fractions in vivo (24 h after exposure) was sensitive to release by protease digestion, but insensitive to release by ribonuclease digestion.     

Intracellular phosphate pools show compartmentalization of intracellular pH in turtle urinary bladder

The urinary bladder of the fresh water turtle is capable of acidification and Na transport, in vitro, and it has been extensively used as a model of distal nephron of the kidney. In the course of measuring intracellular pH of stripped turtle bladder mucosa with phosphorus nuclear magnetic resonance, we observed the consistent presence of two inorganic phosphorus resonances under aerobic conditions, indicating the existence of a pH gradient possibly between cytosol and mitochondrion. This pH gradient was collapsed by addition of N₂ and could be restored by reintroduction of oxygen. These observations demonstrate the existence of a spontaneous pH gradient between cytosol and mitochondria of turtle bladder epithelial cells.     

Phosphorus-31 relaxation times of 2,3-diphosphoglycerate in intact human erythrocytes

The reciprocals of the spin-lattice relaxation times (T₁s) of the 2-P and 3-P nuclei of 2,3-diphosphoglycerate (DPG) increased linearly as percent DPG bound was raised in model hemoglobin solutions. The 2-P T₁ was slightly greater in intact erythrocytes than in model solutions under similar experimental conditions. The change in the 3-P T₁ with cellular deoxygenation was anomalous indicating that this nucleus should not be used to estimate DPG binding inside intact erythrocytes.     

Granulosis virus in the intestinal lumen of Neoaplectana carpocapsae: Retention of infectivity after treatment with formaldehyde or high pH

High pH values (>11.0) cause the dissolution of occlusion bodies of the granulosis virus (GV) of Pseudaletia unipuncta and subsequent inactivation of the virus within 24 hr. The GV is also inactivated within 48 hr by 0.04% formaldehyde. The GV is found in the intestinal lumen of infective third stage nematodes (dauer juveniles) of Neoaplectana carpocapsae when development occurs in GV-infected hosts. The GV in these dauer juveniles retains its infectivity even when the nematodes are placed into an alkaline solution with pH values of 11.1 or 12.1 or in 0.04% formaldehyde up to 336 hr. However, significant loss of infectivity of GV occurs when the nematodes are in formaldehyde but not at high pH values. The dauer juveniles are ensheathed by the second stage cuticle. This cuticle probably protects the GV in the intestinal lumen of the nematode from the high pH and formaldehyde.