Circular dichroism of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and various derivatives

We have recorded the near- and far-ultraviolet circular dichroism spectra of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and derivatives of these toxins. The far-ultraviolet spectra of various forms of diphtheria toxin were virtually identical, implying that no major changes in secondary structure accompany proteolytic nicking or dimerization of toxin, or binding of the endogenous dinucleotide, adenylyl-(3'-5')-uridine 3'-monophosphate (AdoPUrdP). Alpha-helix content was estimated to be 29%, as compared with 8% for fragment A. Near-ultraviolet spectra were identical between nicked and intact diphtheria toxin. A broad negative transition with a minimum at 304 nm was assigned to the intrachain disulfide bridge within the B moiety. Dimeric diphtheria toxin showed perturbations of aromatic residues. Binding of AdoPUrdP to monomeric diphtheria toxin or of adenylyl-(3',5')-uridine (AdoPUrd) to fragment A perturbed one or more tryptophans. The latter results correlate with evidence for involvement of a tryptophan in NAD binding. Native exotoxin A was estimated to have 16% alpha-helix, and the activated form of exotoxin A, 11%. An enzymically active, 31 kDa proteolytic fragment of exotoxin A showed similar alpha-helix content (7%) to that of diphtheria toxin fragment A.     

PMA induces the ligand-independent internalization of CR1 on human neutrophils

Phorbol myristate acetate (PMA) has been reported to confer on the C3b receptor (CR1) of neutrophils a capacity for phagocytosis of particles bearing C3b without the involvement of other membrane receptors. In the present study, we employed a monoclonal antibody, YZ-1, that is specific for CR1 to assess the effect of PMA on plasma membrane expression of CR1, total cellular CR1, and internalization of CR1 by neutrophils. PMA had a biphasic effect on the membrane expression of CR1 by purified neutrophils, with 4 ng/ml inducing a 60% increment in receptor expression, and higher concentrations causing up to a 70% decrement. PMA-dependent increases in CR1 expression were not accompanied by corresponding changes in total cellular CR1 and were preempted by treatment of cells with formyl-methionyl-leucyl-phenylalanine (FMLP). PMA-induced decreases in CR1 expression by neutrophils, as measured by binding of indirectly fluoresceinated or radiolabeled YZ-1, or of 125I-labeled dimeric C3b, were maximal with 20 to 30 ng/ml PMA, and occurred within 30 min of incubation at 37 degrees C. The PMA-dependent down-regulation of CR1 by neutrophils was not associated with a comparable decrease in total cellular CR1, and this response was observed to occur also with monocytes but not with peripheral blood lymphocytes. By tagging neutrophil CR1 with 125I-YZ-1 Fab and monitoring accessibility to Protease, intracellular CR1 (inaccessible) was discriminated from receptor on plasma membrane (accessible). Internalization of CR1 occurred within 5 min after addition of PMA to neutrophils, was dose dependent, and involved up to two-thirds of the tagged receptors. Therefore, PMA caused internalization of CR1 by neutrophils in the absence of ligand, indicating that this response was independent of a transmembrane signal generated by a C3b-CR1 interaction.     

Techniques for hand-rearing tree-shrews (Tupaia belangeri) from birth

In captivity, Tupaia belangeri (Thailand tree-shrew) frequently show aberrant patterns of maternal care which result in the death of offspring. In order to maximize the potential of the tree-shrew as an animal resource for experimental studies we have developed a program for hand-rearing tupaiidae from birth. Newborn tree-shrews were removed from mothers with a history of poor parental care to a nursery maintained under conditions of controlled relative humidity (70 ± 10%) and temperature (25 ± 1°C) on a 12 h dark: 12 h light cycle. The young tree-shrews were fed on a liquid formula until the eyes opened (Day 18–23) and for the subsequent 10 days on a transitional diet until they could feed themselves on solid food. Our hand-rearing protocol appears to conform to the natural weaning pattern of tupaiids. Food consumption during the liquid diet phase was linearly related to age and the increase in body weight in both sexes. The initial growth rate of male and female hand-reared tree-shrews was slower than that of maternally reared animals during the liquid diet phase. The growth rate accelerated subsequently and the body weight of hand-reared tree-shrews eventually reached that of maternally reared animals of the same sex. Various developmental changes occurred during the same period in artificially and naturally reared animals. In both hand-reared and maternally reared groups, the growth rate of male tree-shrews exceeded that of females from about Day 40 onward. The accelerated growth rate of male tree-shrews was in apparent association with an increase in androgen secretion at the onset of puberty. The high fecundity of tree-shrews in captivity reinforced with a program for hand-rearing the young make T belangeri a potential alternative to more conventional laboratory species in specific areas of biomedical research.     

Papovaviral sialoadenitis in athymic nude rats

A wasting disease was found in 32 athymic nude rats. The rats had parotid sialoadenitis with intranuclear inclusion bodies in ductal and acinar epithelial cells. Other common lesions included bronchitis, bronchiolitis and secondary bacterial pneumonia. Less commonly, rhinitis and Harderian adenitis were seen. Intranuclear inclusions were also seen in bronchial epithelium of 1 rat, Harderian gland acini of 1 rat and laryngeal glands of 2 rats. Viral particles, averaging 45 nm in diameter, sometimes in crystalline arrays, were found in the nucleus of parotid epithelial cells. By the use of the avidin-biotin-peroxidase complex (ABC) immunoperoxidase technique, antibodies to disrupted SV40 virus (the group specific antigen of the polyomavirus (miopapovavirus) genus of the papovavirus family) reacted with intranuclear inclusions and cytoplasm of parotid epithelium and inclusions in lung and Harderian gland. The viral antigen did not cross react with antibodies to mouse polyoma, mouse K or disrupted bovine papilloma viruses.     

Homologous and heterologous mitogenic desensitization of Swiss 3T3 cells to phorbol esters and vasopressin: role of receptor and postreceptor steps

Prolonged treatment of Swiss 3T3 cells with phorbol 12,13 dibutyrate (PDB) rendered the cells refractory to subsequent mitogenic stimulation by both PDB and vasopressin. In contrast, the cells retained full responsiveness to a wide variety of other mitogens. An early response to vasopressin and phorbol esters, inhibition of (125I)-labeled epidermal growth factor [(125I)-EGF] binding, was also substantially decreased in PDB pretreated cells. The cross desensitization was not produced by vasopressin; this ligand induced homologous but not heterologous desensitization. Exposure of Swiss 3T3 cells to PDB caused a down regulation of (3H)-PDB receptors but did not reduce the binding of vasopressin to refractory cells. The time-course (t1/2 = 7 h) and dependence on PDB concentration (half maximal at 20 nM) for this phorbol ester receptor loss paralleled the induction of the mitogenic desensitizations to both PDB and vasopressin. However, the time-course of recovery revealed an important dissociation between receptor presence and mitogenic response. When Swiss 3T3 cultures, which had been pretreated with PDB, were washed to remove this ligand and incubated in its absence for 24 h, both (3H)-PDB receptors and PDB or vasopressin inhibition of (125I)-EGF binding were almost completely restored to control levels. However the homologous and heterologous mitogenic desensitizations showed a very different reversal time. After a 24-h recovery period PDB-treated refractory cells were still unable to synthesize DNA in response to PDB or vasopressin. The mitogenic desensitizations were however completely reversible; after a 48-h incubation in the absence of PDB the cells responded fully to the mitogenic actions of PDB or vasopressin. This finding suggests that a further postreceptor step was also desensitized by prolonged PDB treatment. The presence of a low level of cycloheximide during the PDB pretreatment blocked induction of this postreceptor refractoriness. We propose that this refractory postreceptor step selectively blocks both PDB and vasopressin stimulation of DNA synthesis and may represent the point at which the mitogenic pathways of phorbol esters and vasopressin converge.     

The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase

A 110-kDa protein present in chicken intestinal brush-border microvilli is believed to laterally link the actin filament bundle that forms the structural core of the microvilli with the microvillar plasma membrane. We have purified a 110-kDa protein to greater than 95% homogeneity by extraction of brush borders with solution containing 0.6 M KCl and 5 mM ATP, followed by gel filtration chromatography, sedimentation as a complex with exogenous actin, and hydroxylapatite chromatography. The 110-kDa protein-calmodulin complex bound F-actin in the absence but not the presence of ATP and had K+,EDTA-ATPase (0.2 mumol/min/mg) and Ca2+-ATPase (0.2 mumol/min/mg) activities and Mg2+-ATPase activity (0.03 mumol/min/mg) that was not activated by F-actin. The actin-binding and ATPase activities of the complex were similar to those of purified brush-border myosin. However, immunoblot analysis showed no reactivity between the 110-kDa protein and polyclonal antibody against purified chicken brush-border myosin. Also, peptide maps of 110-kDa protein and myosin obtained by limited proteolysis with chymotrypsin and Staphylococcus aureus V8 protease had few, if any, peptides in common. Immunoblot analysis also showed that myosin heavy chain was stable under the conditions of the preparation.     

The envelope gene and long terminal repeat sequences contribute to the pathogenic phenotype of helper-independent Friend viruses.

Friend murine leukemia virus (F-MuLV) and Friend mink cell focus-inducing virus (Fr-MCF) are helper-independent murine retroviruses which induce a rapidly fatal erytholeukemia in NIH Swiss mice. Amphotropic clone 4070 (Ampho) is a murine retrovirus which does not cause leukemia in these animals. Mice inoculated with Ampho, an Fr-MCF/Ampho pseudotype, or F-MuLV developed leukemia in 0, 50, and 100% of animals, respectively. To identify the F-MuLV and Fr-MCF sequences responsible for leukemia, we constructed hybrid viral genomes between these viruses and Ampho, using subgenomic fragments of molecularly cloned viral DNA. Transfection of these hybrid viral DNAs into fibroblasts produces recombinant retroviruses. These new viruses are assayed in vivo for their ability to cause leukemia. Recombinant viruses constructed between the Ampho genome and the Fr-MCF envelope gene do not cause leukemia. Similarly, viruses constructed by using either the Fr-MCF long terminal repeat U3 region or the F-MuLV long terminal repeat U3 region and the remainder of the Ampho genome do not cause leukemia. However, if the Fr-MCF envelope gene plus the Fr-MCF U3 region are joined to Ampho, the resulting virus causes erythroleukemia in 14% of mice. Recombinant viruses made between the Fr-MCF envelope gene, the F-MuLV U3 region, and the remainder of the Ampho genome cause erythroleukemia in 38% of mice. This study demonstrates that both the envelope gene of Fr-MCF and the U3 regions of Fr-MCF and F-MuLV contain sequences which contribute to the leukemic phenotype of helper-independent Friend viruses.