Oligoribonucleotide initiators for herpes simplex virus DNA synthesis in vivo and in vitro.

Replicating herpes simplex virus (HSV) DNA, labeled with [³H]thymidine or [³H]uridine, was isolated from infected primary rabbit kidney cells. Nascent DNA strands are covalently linked to oligoribonucleotide stretches. The chain length of this RNA segment amounts to approximately 36 nucleotides wich are located at the terminus of the replicating DNA. The extent of RNA initiator synthesis seems to be closely correlated with the rate of HSV-DNA synthesis. The half life of the RNA initiator was estimated as 35 min. Evidence is presented by comparative studies regarding the characteristics of the naturally occurring RNA initiator, that cordycepin (3′-dAdo) is an inhibitor of the synthesis of the RNA initiator: (1) identical maximal chain length of the 3′-dAMP-RNA, (2) identical half-life of the 3′-dAMP-RNA segment, and (3) susceptibility to RNases and alkali. The 3′-dAMP-RNA segment is not covalently linked to HSV-DNA. HSV-Induced DNA polymerases utilizes not only deoxyribooligomers $\text{[}\text{d(pA)}{}_{\mathrm{<mtext>8</mtext>}}\text{]}$ as initiators for the DNA template-dependent DNA polymerization. The initiating activity of both the DNA and the RNA segment is abolished when it carries a 3′-dAMP moiety at its 3′-terminus.     

Variation of DNA polymerase and RNA polymerase activities in cells infected with herpes simplex virus type 1.

Infection of rabbit kidney cells with herpes simplex virus (HSV) leads to pronounced alterations of the different species of DNA-dependent DNA and DNA-dependent RNA polymerases. The activity of distinct polymerase species was determined; it was ruled out first that deoxyribonucleases or ribonucleases had an influence on these determinations and second that the activities of isoenzymes modified the evaluation of the activity of a distinct enzyme species. The three different DNA-dependent DNA polymerases (form a and β and HSV-induced enzyme) were separated by velocity sedimentation through sucrose density gradients and were asscyayed using activated DNA as template. DNA polymerase a and HSV polymerase were resolved by different assay conditions. The cellular polymerase a decreases by 80% 10 hr after infection with HSV, while the cellular polymerase β shows a slight increase during this period. The activity of HSV-induced DNA polymerase at the beginning of infection is virtually zero; it increases to a maximum 8 hr postinfection. The activity of DNA-dependent RNA polymerase I declines dramatically, by 68%, after infection with HSV while the alterations of the activities of RNA polymerases II and III are less pronounced.     

9-beta-D-Arabinofuranosyladenine as a tool to study herpes simplex virus DNA replication in vitro.

9-β-d-Arabinofuranosyladenine (araAdo) strongly suppresses herpes simplex virus (HSV) DNA synthesis in intact cell systems. After incubation with araAdo, two HSV-DNA fractions can be isolated by neutral isopycnic CsCl density gradient centrifugation, a light fraction with a buoyant density of 1.726 g/cm³, and a heavy fraction with a density of 1.738 g/cm³. After recentrifugation in neutral CsCl gradients, the light and heavy fractions are detected at a density of 1.729 and 1.741, respectively. Analysis of the two HSV-DNA fractions by hydroxylapatite chromatography and by digestion with nuclease S₁ revealed that the light fraction consists predominantly of native DNA and the heavy fraction of denatured DNA. AraAdo is incorporated into HSV-DNA and has been shown to be present in terminal positions at the 3′-hydroxyl position. The HSV-DNA pieces, containing incorporated araAdo, are of low molecular weight (less than 2.6 × 10⁶) and are not assembled to higher molecular weight aggregates.     

Use of an enzyme-linked immunosorbent assay (ELISA) for screening of hybridoma antibodies against cell surface antigens

A micro-ELISA for screening of antibodies from hybridoma cultures against surface antigens of human melanoma is described. The technique employs alkaline phosphatase-conjugated protein A and target cells attached to poly-l-lysine-coated microtiter plates. The micro-ELISA is equally sensitive as the radioimmunoassay. Mild glutaraldehyde treatment of cells did not lead to an appreciable loss of antigen activity. The fixed cells can be stored at 4°C for at least 6 weeks. It is concluded that the ELISA is superior to the radioimmunoassay in the following aspects: (1) exclusion of radioactive hazards, (2) speed of performance, and (3) lower costs.     

Immunocytochemical demonstration of β-lipotropin in mouse pituitary cells and neurons cultured in vitro

Specific antiserum directed against human β-lipotropin was used to demonstrate the presence of β-lipotropin immunoreactivity in dissociated cell cultures of mouse pituitary, hypothalamus, spinal cord and dorsal root ganglia.A large population of pituitary cells and hypothalamic neurons were positive for β-lipotropin immunoreactivity, while only 10–20% of the neurons from spinal cord and dorsal root ganglia were stained. The specificity of the β-lipotropin immunoreaction was confirmed by blocking the reaction by prior absorption of the antiserum with added β-lipotropin.     

The impact of stress experience on heart rate and task performance in the presence of a novel stressor

The impact of prior stress experience on heart rate and task performance in the presence of a novel stressor was studied. During a training phase 42 female students were required to solve arithmetical problems under conditions of either no stressor, delayed auditory feedback, or distraction. Subsequently in a testing phase all subjects performed the same task in the presence of delayed auditory feedback. Training in the presence of stress resulted in decreased heart rate and better performance in the testing phase. These data were interpreted as supporting the hypothesis that training under stress-inducing conditions increases stress resistance which generalizes to a novel stressor and which leads subjects to experience the novel stressor as being less stressful.     

Pathologic study of parathyroid glands in tertiary hyperparathyroidism

Tertiary hyperparathyroidism is defined as persistent parathyroid hyperfunction developing from the secondary hyperplasia that occurs after restoration of renal function by dialysis or kidney transplantation. Controversy continues as to whether parathyroid adenoma or hyperplasia accounts for the autonomous hyperfunction. A review of 128 parathyroids from 41 patients with tertiary hyperparathyroidism revealed marked hyperplasia in 39 patients (95 per cent), with a predominance of chief cells, an abundance of oxyphil cells, and 10- to 40-fold increases in parathyroid mass. This hyperplasia was considered to be the predominant morphologic feature of tertiary hyperparathyroidism. Adenomas, found only in two patients (5 per cent), seem to be rare. Diffuse, moderately enlarged hyperplastic glands were found predominantly in patients with transplants, whereas nodular, markedly enlarged hyperplastic parathyroids were observed more frequently in patients treated by dialysis. In spite of the different patterns of hyperplasia and the different gland sizes in these two groups of patients, the grades of hypercalcemia were similar. The results of ultrastructural studies indicate that the majority of parenchymal cells in diffuse, and some cellular areas in nodular, hyperplasia may consist of cells with high secretory activity that do not respond normally to hypercalcemia. It is concluded that both increased parenchymal mass and cellular differentiation, leading to hyperactivity, account for tertiary hyperparathyroidism.     

Senescence in vitro and ionising radiations—the human diploid fibroblast model

The influence of ionising radiations on ageing is still controversial. Since Hayflick established the concept that diploid cells have finite lifespan in vitro, human diploid fibroblast (HDF) cultures have been recognised as a potent experimental model for cytogerontological investigations. In this study HDF cultures in phase II were exposed to acute irradiation with either X-rays on fast neutrons. The replicative potentials and labelling indices with [³H]thymidine were measured post irradiation until the cultures ceased growth in phase III. Cell mortality was measured by cloning. The apparent loss in replicative potential of irradiated mass cultures was wholly attributable to the loss of viable clonogenic cells. The current concept of precocious clonal senescence in vitro as a late effect of irradiation in clonogenic survivors is not supported by the present experiments. Instead, our results suggest that exposure to a single dose of ionising radiations either causes total replicative incapacitation (killing) of HDF cells and their progeny early after irradiation or leaves their replicative potentials unperturbed.     

Incidence and diagnostic significance of minor pathologic changes in the adult pancreas at autopsy: a systematic study of 112 autopsies in patients without known pancreatic disease

In 112 unselected autopsies of adult patients without known pancreatic disease (except adult-onset diabetes mellitus), the pancreas was examined to establish the incidence and degree of such minor pancreatic lesions as lipomatosis, fibrosis, alterations of ducts and ductal epithelium, inflammatory infiltrates, focal necrosis, acinar dilation, and vascular changes. Each lesion was then tested for statistically significant correlations with the age of the patient and a number of clinical conditions, including cholelithiasis, adult-onset diabetes mellitus, adiposity, generalized severe atherosclerosis, chronic alcoholism, severe bacterial infection prior to death, and generalized malignant tumor. This was done in the hope of finding associated or predisposing factors for the pancreatic lesions. The results show, in addition to the unexpectedly high incidence of the various pancreatic lesions, a clear increase of lipomatosis, fibrosis, and both ductal and ductal epithelial alterations with increasing age; these conditions were accompanied by a steady decrease in the mean weight of the gland, starting at the age of about 40 years, except in cases of advanced lipomatosis. The latter condition was associated with adult-onset diabetes mellitus. Severe generalized atherosclerosis was correlated with lipomatosis and fibrosis, but the two latter conditions were found together only rarely. Acute (terminal) lesions, including focal necrosis and acinar dilation, were associated with severe bacterial disease prior to death. Other statistically significant correlations were rare, indicating the lack of specificity of these minor pancreatic lesions rather than offering a clue as to their pathogenesis. The diagnostic significance and the relations of these lesions to clinically relevant chronic pancreatitis are discussed briefly.     

Evidence of transient effect by lignocaine on alveolar macrophage morphology

Alveolar macrophages were extracted from rat lung by pulmonary lavage. The lavage fluid consisted of Joklik's minimum essential medium alone, containing lignocaine, or lignocaine with the addition of 10% foetal calf serum. Although the presence of lignocaine increased the macrophage yield 3-fold, in the absence of foetal calf serum the resulting macrophage population exhibited gross cytoplasmic vacuolation. The presence of foetal calf serum prevented this vacuolar formation.There was no difference in either the osmolarity or pH of the media and although the vacuolation failed to produce changes in a cell viability assay, the presence of the morphological change is obviously undesirable when functional studies are to be carried out.     

Defective viral RNAs in Aedes albopictus C6/36 cells persistently infected with Semliki Forest virus

A persistent infection of Semliki Forest virus (SFV) has been established in Aedes albopictus C6/36 cells. Only a small number of cells survived the initial infection with this RNA virus and gave rise to a persistently infected culture which produced continuously small amounts of infectious virus. To investigate whether defective viral RNA is involved in establishing and maintaining a persistent infection, the intracellular viral RNA was analyzed early and late after infection by blot hybridizations. Several defective viral RNAs were detected with a common sequence corresponding to the 3′ end of the viral genome during and after the establishment of the persistent infection. These defective viral RNAs resemble the defective interfering RNAs in vertebrate cells generated during serial undiluted passages of standard SFV. The defective viral RNAs are rarely released from cells as virions. The rapid generation of defective viral RNAs may be important for the establishment of a persistent infection in mosquito cells.     

Induction of late cutaneous reactions by skin-blister fluid from allergen-tested and normal skin

Ten healthy volunteers (five atopic, five nonatopic) and seven patients suffering from allergic bronchial asthma and rhinitis/conjunctivitis as well as showing dual reactions after intradermal or bronchial allergen challenge were investigated. Using the suction blister technique, we obtained skin-blister fluid (SBF) from dual skin reactions 30, 60, 180, and 300 min after allergen injection and from normal untested skin. The biologic activity of SBF was tested by intradermal reinjection of the fluid into the donor. SBF taken from dual skin reactions 30 or 60 min after allergen injection produced late cutaneous reactions (LCRs) quite similar to those induced by the allergen. SBF taken from LCR areas 180 or 300 min after antigen testing had much weaker effects, similar to SBF from untested skin. A possible content of allergen extract in SBF from allergen-tested skin areas was not responsible for the observed effects as demonstrated in passive cutaneous anaphylaxis experiments in monkeys. High doses of SBF from untested skin were able to induce LCRs similar to but weaker than LCRs produced by SBF taken at early phases from dual skin reactions. Similar volumes of autologous heparin-plasma or serum did not induce LCRs. It is concluded that during the initial phase of dual skin reactions, factors are formed that are able to induce LCRs. The generation of these mediators seems to be caused at least in part by the extravasation of plasma.     

Cortical potential correlates of selective attention in multidimensional scaling

Cortical evoked potentials were recorded from the vertex of subjects performing a similarity rating task. Proximity data were analysed according to INDSCAL (Carroll and Chang, 1970). Brain activity was averaged separately for different stimulus attributes and different stages of the judgement process. The amplitudes of the two positive deflections of the averaged evoked potential, peaking around 160 and 330 msec after stimulus onset respectively, were systematically influenced by two factors: The weight, which the evoking attribute received in the overall dissimilarity judgement, and the position of the evoking attribute in the stimulus train. The results support the hypothesis that differences in the perception of similarity are due to differences in attentional set. Reference is made to the ‘additive-difference model’ of scaling behaviour (Tversky and Krantz, 1970) and Broadbent's (1970) distinction between stimulus set and response set attention.     

The effect of denervation of Merkel cells in cats

The morphology of Merkel cells in sinus hair follicles of the upper lip (vibrissae with a circular blood sinus), and in touch domes of the glabrous skin of the nose, was investigated in cats up to 12 weeks after resection of the infraorbital nerve. Even 12 weeks after denervation there was neither an ultrastructural alteration of Merkel cells nor a reduction of their number in sinus hair follicles. Also in touch domes the Merkel cells did not disappear.     

Cytogenetics of neoplasia of Xiphophorus

Certain laboratory hybrid Xiphophorus fish develop heritable neoplasia. Neoplasia can also be induced in some hybrids by treatment with carcinogens. In either case the neoplasms are mediated by a “tumor gene,” that is normally controlled by linked or nonlinked “regulating genes” or both. Light and electron microscopic studies on these two types of tumors reveal important differences. The heritable tumor is a compact mass of tissue formed of densely packed melanized melanocytes often with nuclear pockets and projections. A large number of cells undergoing a process of nuclear fragmentation has been observed. The induced tumors, on the other hand, contain fewer melanized melanocytes and show a comparative increase in the amount of connective tissue. The nuclear pockets and projections commonly found in the melanocytes of the heritable tumors are not present in the melanocytes of the induced melanoma. The most important difference observed was the presence of different types of structural chromosome aberrations in the induced tumor and a total absence in the heritable ones. These anomalies seem to be epiphenomenal, and the real genetic change is subchromosomal.     

Neutralization of adenovirus infectivity and cytotoxin in various cell cultures

The neutralization of human adenovirus 5 and 11 by homologous and heterologous rabbit antisera was determined by CPE inhibition in various cell cultures (HeLa, HEL, Vero, secondary kidney cells from cercopithecus, rabbit, mouse), or in HeLa cells made impermissive by IUdR inhibition. The results concerning sensitivity and specificity were similar in all cases. Crude and purified virus showed similar neutralization. Immunofluorescence neutralization in HeLa cell cultures gave similar results; this method is suitable for demonstrating subtle immunological relations between adenovirus types.The neutralization of the early cytopathic factor (‘cytotoxin’) showed a pattern of cross-reactivity different from the virion; the cytotoxin was found to be active in part of the cell cultures only. It is concluded from the results that the virus function(s) blocked by antibody appear to be identical for the replicative cycle in infection and for the initiation of the abortive infection in non-permissive cells. Hence, either kind of cells may be used for neutralization tests.