The comparative haematology of cross-bred and indigenous east African goats of Tanzania and breeds reared in Denmark. Erythrocyte counts, haematocrit, haemoglobin concentration, red blood cell indices, total and differential leukocyte counts were determined in 202 cross-bred and 14 indigenous East African goats aged 6-12 months and also in 59 Norwegian dairy goats, of which 24 were 15-45 days old, 8 were 8 months old and 27 were over 3 years and pregnant. These were reared in Tanzania. Comparisons were made with 24 Dwarf and 57 Danish Landrace goats at 6-12 months of age and 76 adult pregnant Danish Landrace goats reared in Denmark. The purpose was to determine reference ranges for cross-bred and indigenous East African goats and to compare these with those of other breeds. The haemoglobin concentration, haematocrit, erythrocyte and white blood cells were lowest in the Norwegian kids. The highest values were observed in 6-12-month-old goats in all the breeds, whereafter they decreased to relatively constant adult levels. The mean corpuscular volumes were highest in kids followed by adult pregnant Norwegian and Danish Landrace goats, and lowest in 6-12 months old goats. East African and cross-bred goats had the smallest mean corpuscular volumes. The haemoglobin concentration, erythrocyte and leukocyte counts were highest in indigenous East African, followed by young Norwegian and cross-bred goats. The mean corpuscular haemoglobin concentration was highest in the cross-breds, while the mean corpuscular haemoglobin was higher in pregnant than in other goats. The age and breed differences were statistically significant.


Biochemical and physiological parameters and estimated work output in draught horses pulling loads for long periods. A study was undertaken in five draught horses of 648 +/- 33 kg body weight to find the effects of continuously pulling loads on their cardiovascular, respiratory and metabolic responses. A cart equipped with an odometer, for measuring distance, and a hydraulic dynamometer, for measuring draught force, was used. Heart and respiration rates and rectal temperatures were recorded. Blood samples for measuring arterial and venous pH and blood gases, haemoglobin, glucose and lactic acid concentrations and the serum activity of the enzymes creatine phosphokinase (CK), lactate dehydrogenase, aspartate aminotransferase and alkaline phosphatase were taken before exercise and immediately after each journey (morning and afternoon) of the daily work. Draught exercise, with loads which generated forces of between 0.57 and 0.59 kN, at speeds of 1.60 to 2.11 m/s, for 8 h daily for five consecutive days, with resting intervals of 10 min each hour, was well tolerated. Exercise tolerance was evaluated from the recovery from the changes observed in the biochemical and physiological parameters induced by the work. The analysis of these showed that, when the horses were subjected to prolonged periods of resting, their loss of fitness for work was shown by significant increases in the serum activity of muscle-derived enzymes and in blood lactate concentrations during the first day of work. However, over the following days the horses adapted to the work, so that the decreases in serum enzyme activities and blood lactate concentrations were reduced. Since similar observations have been described for racehorses, the determination of blood lactate concentrations and the serum activities of muscle-derived enzymes, specifically CK, seem to be good indicators of fitness in draught horses.


Effect of ageing and malnutrition on rat myocardium. I. The myocyte. The effects of ageing and starvation on the rat myocardium were studied by morphometric methods. Since cardiac muscle is a tissue with a high level of anisotropy, methods based on the concept of vertical planes were used to describe quantitative alterations in the rat myocyte both at the cellular and ultrastructural level. During starvation rapid and important changes were noted, particularly in the transverse dimension of cells and organelles. The most striking change, however, was the immediate dilatation of the myocyte T-system, reflecting an adaptive interaction between the intra- and extracellular environment. At the same time exocytosis of intracellular components into the extracellular space of the T-system was observed. The ratio of mitochondria to myofibrils decreased progressively during starvation. Such a decrease, in general, may reach a point when cellular energy supply becomes compromised. A comparison between different regions of the heart showed no differences and it can be concluded that the morphological changes during starvation are the same, and equally distributed, in both ventricles. The changes described in the aged rat heart point in the direction of a hypertrophy of the aged myocyte. This leads to a lower ratio between surface and volume which finds its representation at the subcellular level in a more spherical shape of nuclei and mitochondria. Unlike what is seen in malnutrition, the mitochondrial/myofibril ratio is higher in the older rat. From the morphological point of view, the atrophy of malnutrition and the hypertrophy of ageing are opposed, but in both there is a change in the relationship of the myocyte to its environment which directly influences the substrate exchange capacity. This tends to protect the myocyte in starvation but jeopardizes the older cell.


Effect of ageing and malnutrition on rat myocardium. II. The microvasculature. The modulating effects of ageing and malnutrition on rat myocardium were studied morphometrically with respect to the microvasculature. An increase in capillary density together with a decrease in capillary lumen cross-sectional area was noted during starvation. The important changes seen in the myocyte T-system were paralleled by a decreased diffusion distance for oxygen from the capillary lumen to the mitochondrion. The changes described in the aged rat heart point to an altered inter-relationship between parenchyma and vascularization with a lower capillary volume fraction and a greater diffusion distance from the capillary lumen to the mitochondrion; this is caused by hypertrophy of the aged myocyte. This reduction in capacity to exchange substrates is further reduced by the less developed T-system in the older myocyte.


Ag-NOR protein distribution correlates with patient survival in stage I endometrial adenocarcinoma. The relationship between argyrophil nucleolar organizer region (Ag-NOR) protein quantity and prognosis was studied in 33 cases of stage I endometrial adenocarcinoma. Ag-NOR protein quantity was measured by image analysis in silver-stained sections from paraffin-embedded samples of curettings. Patients had a minimum 10-year follow-up. Only 2 out of 25 patients exhibiting a mean Ag-NOR protein area of less than 3 microns2 died of cancer, whereas 5 of the 8 patients with a mean Ag-NOR protein area of more than 3 microns2 died of the disease. The present results demonstrate that the Ag-NOR protein value is closely related to patient survival in stage I endometrial carcinoma and that it is a reliable prognostic indicator in this type of carcinoma.


Comparative analysis of p53 protein immunoreactivity in prostatic, lung and breast carcinomas. In this study we analysed the expression of p53 protein in a total of 143 carcinomas immunohistochemically. These consisted of 34 prostatic adenocarcinomas, 59 lung and 50 breast carcinomas. In 28 cases, an average of 2-3 additional sections from different tumour areas were analysed. Forty-nine of the 143 carcinomas (34%) showed typical nuclear immunoreactivity by immunohistochemical staining with the p53 antibody CM-1. Two of the 34 prostatic carcinomas (6%) were p53 positive while 25 of the 59 lung carcinomas (43%) and 22 of the 50 breast carcinomas (44%) showed positivity for p53. By grade: 49% of grade III tumours, 36% of grade II and 5% of grade I tumours were p53 positive. There were significantly more p53-positive cases in grade II-III tumours than in grade I tumours (P = 0.001) when all tumours were taken into account. Further, there were significantly more p53-positive cases in grade III than in grade I-II tumours (P = 0.001). In lung tumours there were significantly more p53-positive cases in grade II-III tumours than in grade I tumours (P = 0.018). Similarly, there were significantly more p53-positive tumours in grade III breast tumours than in grade I-II tumours (P = 0.003). The low incidence of p53 positivity in prostate carcinomas suggests that mutations of the p53 gene are not as frequent in the neoplastic transformation of these tumours as in lung or breast carcinomas. The association of p53 positivity with tumours of higher grade suggests that p53 mutations lead to tumours of a more aggressive type. The analysis of tumours by multiple sections indicates that p53 positivity is not evenly distributed in tumour tissue. Therefore, analysis of additional tumour areas may reveal positivity some cases, which is not evident if only one section is studied.


Cell populations in the lesion of human cutaneous leishmaniasis: a light microscopical, immunohistochemical and ultrastructural study. To characterize the in situ cellular immune response in localized cutaneous leishmaniasis (LCL), the authors studied frozen skin biopsies from 50 patients with LCL due to Leishmania braziliensis guyanensis. A panel of 31 monoclonal antibodies was used, which defined the number and distribution of inflammatory cell subsets. Skin inflammatory infiltrates were composed of T cells (with a local CD4/CD8 ratio of 1.05 +/- 0.7 vs 1.48 +/- 0.3 in peripheral blood), macrophages and a smaller number of B cells, natural killer cells and granulocytes. Most of the T cells expressed activation markers (interleukin-2 and transferrin receptors, HLA-DR+) and an increase in T-cell-receptor gamma delta expression was noted. Analysis of the CD4+ subpopulations with newly available reagents showed that helper T cells (CD4+CD45RO+) exceeded the suppressor/inducer subset (CD4+CD45RA+) by 1.4:1. There were no differences between local immune variables from patients with primary infection (45 patients) and those with recurrence (5). In 7 patients, biopsies were analysed before and 1 month after specific treatment, and did not show significant differences except for a small increase of dermal CD1a+ (Langerhans) cells/mm2. The observed pattern of cellular skin infiltration suggests an immune-mediated tissue injury including T-cell-mediated cytotoxicity and delayed hypersensitivity reactions in addition to direct parasitic action.


Localization of thromboxane synthase in human tissues by monoclonal antibody Tü 300. Using the monoclonal antibody Tü 300 we localized thromboxane synthase, a secondary enzyme of the arachidonic acid cascade, employing the alkaline phosphatase anti-alkaline phosphatase method and indirect double labelling immunofluorescence in frozen sections of human tissues. Aside from platelets, the source of the antigen, all cells of the mononuclear phagocytic system were positive, including epithelioid cells and associated giant cells, starry sky macrophages, dendritic cells of T-cell areas, Langerhans cells and Kupffer cells. In addition, some epithelial cells such as epithelia of tonsillar crypts, reticular epithelia of the thymic cortex and ductular epithelia in liver, pancreas, female breast and salivary glands showed occasional focal reactivity for thromboxane synthase. We suggest that the mAb Tü 300 is a key marker for the macrophage system and the thromboxane generating system in normal and pathological conditions. It may detect functional activities of as yet unknown significance in some specialized epithelial cells.


Immunopathological study of eosinophils in eosinophilic granuloma of bone: evidence for release of three cationic proteins and subsequent uptake in macrophages. Eosinophils from two patients with eosinophilic granuloma of bone (EGB) were studied by combined immunohistochemical and immuno-ultrastructural methods with antibodies directed against three eosinophil granule proteins: major basic protein, eosinophil cationic protein, and eosinophil peroxidase. Immunohistostaining showed the presence and distribution of large numbers of eosinophils in the granuloma. Immuno-ultrastructural methods showed alterations of eosinophil fine structure associated with some steps in the release of granule proteins. No granule extrusion was seen, but rather cationic proteins diffused within cytoplasmic tubulo-vesicular structures. Furthermore, the three granule proteins were found within phagolysosomes of surrounding macrophages, suggesting an interaction between eosinophils and phagocytic cells at the destructive stage of EGB.


Composition and classification of human atherosclerotic lesions. Human atherosclerotic disease can be resolved into eight types of lesion, each characterized by its composition and structure and the absence or degree of intimal injury. The eight types have been arranged in the sequence in which they may progress in complexity from the initial change in childhood or youth to the clinical endpoints in older persons. While lesions at first increase primarily by intra- and extracellular accumulation of lipid, this in itself rarely accounts for symptomatic obstruction. Lipidic lesions become symptomatic primarily by means of successively superimposed deposits of thrombotic material. Non-homogeneity of hemodynamic forces within the length of an artery account for local differences in intima thickness (adaptive intimal thickening) and, in persons with risk factors, differences in susceptibility to lesion formation. According to the degree to which they can accumulate or retain lipid and bring about secondary mechanisms, specific locations of the arterial tree have been designated as atherosclerosis-resistant, atherosclerosis-prone and progression-prone.


Abnormal p53 immunoreactivity and prognosis in node-negative breast carcinomas with long-term follow-up. The expression of the p53 gene product was investigated immunocytochemically in a retrospective series of 164 formalin-fixed paraffin-embedded invasive breast carcinomas with pathologically proven negative lymph nodes. Overall, 78 tumors (48%) showed a variable degree of p53 immunoreactivity. Among these, 38 cases were low expressors (1-10% p53 immunoreactive tumor cells), 21 moderate expressors (10-50% immunoreactive cells) and 19 high expressors (> 50% immunoreactive cells). Abnormal p53 expression correlated significantly with tumor size, histological and nuclear grade, DNA ploidy, mitotic rate and proliferation index, and with the lack of estrogen receptors. Disease-free and adjusted survival analysis of the 124 node-negative patients with long term (more than 10 years) follow-up, however, did not reveal an independent prognostic role for p53 expression. These data suggest that the evaluation of p53 immunoreactivity may only play a role in a multiparametric prognostic assessment of node-negative breast carcinoma.


Comparison of various basement membrane components in benign and malignant peripheral nerve tumours. Immunohistochemical methods were used to analyse benign and malignant tumours of peripheral nerve tissue. We tested for the distribution of basement membrane (BM) components collagen IV, laminin, heparan sulphate proteoglycan, fibronectin, for S100 protein and for the presence of interstitial collagens III and V. Laminin was generally noted in association with Schwann cells, but collagen IV occurred with perineural cells. When tested for BM components, fibroblasts were notably non-reactive except for fibronectin. Three specific area-dependent BM patterns were observed in the benign tumours: (a) Schwann cell-like, in fascicular areas (Antoni A areas of schwannoma, central fibrous bundles of plexiform neurofibromas and central areas of cutaneous neurofibroma), (b) perineural cell-like (capsular structures of schwannoma) and (c) fibroblast-like (myxoid and fibrously transformed areas). Most malignant tissues showed a variably fragmentary focal deposition of laminin. Other BM components were present only in well-differentiated areas. Poorly differentiated tumours demonstrated fibronectin reactivity alone. Our results provide evidence that the specific staining pattern for BM components helps to differentiate the various cellular proliferations in neurogenic tumours. Schwann cells are not only distinguishable from perineural cells by S100 protein staining, but also by their specific BM staining. In addition, perineural cells can be separated from fibroblasts, which do not express BM material. The "tropism" of laminin in normal nerves and benign neural tumours--which persists in neurogenic sarcomas--indicates preferential Schwann cell differentiation in these cells.


Immunohistology of islet amyloid polypeptide in diabetes mellitus: semi-quantitative studies in a post-mortem series. Immunoreactivity for islet amyloid polypeptide (IAPP) in the islets of Langerhans of non-insulin-dependent diabetic patients and non-diabetic patients of a non-selected post-mortem series was studied with a new polyclonal IAPP antibody. Out of 133 patients examined, 124 exhibited immunoreactivity for IAPP. Immunoreactivity was localized intra- and extracellularly and was limited to the islets of Langerhans. No extracellular immunoreactivity was observed in amyloid-negative cases. Co-localization of insulin and IAPP in the same islet-cells was verified by double staining with monoclonal insulin and polyclonal IAPP antibodies. Of 100 patients with non-insulin-dependent diabetes mellitus (NIDDM) and islet amyloid, 98 exhibited IAPP-positive deposits and 71 exhibited intracellular immunoreactivity. Evaluation of intracellular immunoreactivity and degree of islet amyloid deposition in cases of overt NIDDM revealed an inverse relationship, in that intracellular IAPP immunoreactivity were reduced in patients with developing islet amyloid deposition. Our data are consistent with the hypothesis of primary beta-cell dysfunction leading to amyloid formation, with subsequent disturbance of beta-cell homeostasis.


Eccrine gland involvement in Krabbe's disease. Lysosomal storage inclusions were observed in skin eccrine gland secretory and myoepithelial cells in three cases of Krabbe's disease. In addition to storage there were numerous degenerative changes, occasionally resulting in cell necrosis. These findings suggest a generalized nature of the storage process in this lysosomal enzymopathy and point to high galactocerebroside turnover in eccrine gland epithelium. This knowledge may be of value in the biopsy diagnosis of Krabbe's disease.


Site-directed mutations in the potyvirus HC-Pro gene affect helper component activity, virus accumulation, and symptom expression in infected tobacco plants. Helper component (HC-Pro) is a virus-encoded nonstructural protein required for aphid transmission of potyviruses. In the tobacco vein mottling virus (TVMV) polyprotein, HC-Pro represents a 457 residue polypeptide from amino acid position 257 to 713. Previous sequence comparison studies have suggested that mutations of one or two specific amino acid residues in the HC-Pro protein might result in loss of aphid transmission activity. To test this hypothesis, the initial targets were the residues corresponding to these specific amino acids, a lys to glu change and an ile to val change at amino acid positions 307 and 482, respectively, of the TVMV polyprotein, as well as the combination of the two. Two additional mutations within the HC-Pro representing dipeptide changes thr-ser to ile-asp and thr-ala to leu-glu at amino acid positions (283/284) and (368/369), respectively, were also tested to further define the effects of mutations in this region on helper component activity. The mutations at positions 482 and (368/369) had no effect on aphid transmission activity, while mutation at position 307 completely abolished the activity. Except for the 482 mutation, all the mutations also affected symptomatology and virus accumulation in infected plants. Due to the very low concentrations of HC-Pro in plants infected with the (283/284) mutant, the effect of this dipeptide change on aphid transmission activity could not be assessed. The majority of the tested mutations fall within a putative zinc-finger motif postulated in the cysteine-rich N-terminus of HC-Pro. The possible role of this motif in the potyviruses is further discussed in the light of our present results with TVMV.


Myxoma virus M11L ORF encodes a protein for which cell surface localization is critical in manifestation of viral virulence. Myxoma virus (MYX) induces extensive immunosuppression in infected rabbits and is associated with high levels of mortality. The virus encodes multiple gene products designed to circumvent the cellular immune response to the viral infection. Deletion analysis has shown that the M11L open reading frame (ORF) is an important virulence factor which downregulates leukocyte infiltration of MYX-induced tumors. To investigate the role of the M11L protein in viral pathogenesis, we sequenced the MYX M11L ORF and showed that the sequence has motifs consistent with a 166-aa class III membrane-spanning molecule possessing a single transmembrane helix near the C-terminus and a 142-aa N-terminal extracellular domain that has six cysteine residues plus two consensus N-glycosylation sites. Transcription analysis indicates that M11L is expressed as an early gene, and surface immunofluorescence studies with anti-M11L antibodies reveal that M11L protein is transported to the infected cell surface. Immunoprecipitation analysis of an attenuated viral recombinant, vMYX-GF-delta M11L, indicates that an M11L variant protein with an altered C-terminus is synthesized at about 45% of wild type levels; however, it is not detectable on the cell surface, suggesting that proper M11L function requires localization at the infected cell membrane. We propose that M11L is a virulence factor whose function is to recognize an extracellular ligand essential for the cellular inflammatory response.


The nucleotide sequence of adenovirus type 11 early 3 region: comparison of genome type Ad11p and Ad11a. The early 3 region (E3) of two strains (genome type Ad11p and Ad11a) of human adenovirus serotype 11, causing persistent urinary and acute respiratory illnesses, respectively, has been identified and partially sequenced. The sequenced E3 regions of Ad11p and Ad11a were 1980 and 1966 bp long and encoded three complete ORFs, 18.5, 20.3, 20.6k within the Ad11p genome and 18.5, 20.3, 20.2k within the Ad11a genome. The sequence analysis of the 18.5k gene product demonstrated that a transmembrane domain and a cytoplasmic domain of Ad11p, Ad11a, and Ad35 was identical. Ad11p and Ad35 were homologous in the signal sequence. There was one amino acid mismatch between Ad11p and Ad11a, represented by an alanine instead of a proline. The endoplasmic reticulum lumenal domain, which binds to class I MHC, was relatively conserved between Ad11p and Ad11a with the exception of Glu80 and Glu104 in Ad11p, which were replaced by Gln80 and Lys104 in Ad11a. Within the 20.2k protein of Ad11a, the amino acid sequence Thr-Thr-Ser-His was deleted from a position immediately upstream the transmembrane region of the Ad11p 20.6k protein. The 9.0k E3 open reading frame (ORF) of Ad3 was deleted in the genomes of Ad11p and Ad11a. It is noteworthy that Ad11p and Ad35 which both cause persistent infection of the urinary tract display a remarkable similarity in several ORFs of the E3 region.


Adenovirus transformation revertant resistant to retransformation by E1 but not by SV40-T and HPV16-E7 oncogenes. We have previously described a revertant cell line which expresses a dominant tumor suppressor phenotype to E1 but not to heterologous oncogenes such as c-myc, N-ras, or polyoma middle t (Sircar et al. (1988) Oncogene 3, 725-728). DNA tumor virus oncogenes have been suggested to transform cells via the common mechanism of sequestering the Rb-105 antioncoprotein. This paradigm would predict that our revertant cell line, which is resistant to retransformation by E1a, should also be resistant to the other members of the Rb-105 binding family of oncogenes. To test this hypothesis we transfected the revertant cell line with plasmids bearing SV40-T or the HPV16-E7 oncogenes. Because transformation was obtained by both oncogenes at efficiencies similar to the transformation of a related revertant cell line, the results suggest that the resistance phenotype is specific to E1a. This specificity was further confirmed by cell fusion experiments.


The complete nucleotide sequence of pepper mottle virus genomic RNA: comparison of the encoded polyprotein with those of other sequenced potyviruses. The complete nucleotide sequence of a pepper mottle virus isolate from California (PepMoV C) has been determined from cloned viral cDNAs. The PepMoV C genomic RNA is 9640 nucleotides excluding the poly(A) tail and contains a long open reading frame starting at nucleotide 168 and potentially encoding a polyprotein of 3068 amino acids. Comparison of the PepMoV C presumptive polyprotein with those of other sequenced members of the potyvirus group, including tobacco etch virus (TEV), tobacco vein mottling virus (TVMV), plum pox virus (PPV), and potato virus Y (PVY), allowed localization of putative protein cleavage sites. A similar analysis was used to determine the position of conserved viral protein-coding regions along the viral genomic RNA. These analyses confirm previous work indicating that genome organization is conserved among members of the genus Potyvirus. The localization of one PepMoV C gene product, the nuclear inclusion body protein a (NIa protein), was analyzed by expressing PepMoV cDNA deletion clones in bacteria and assaying for appearance of mature-sized coat protein, a cleavage product of the NIa protease. Comparative sequence analyses of the putative PepMoV polyprotein with those of TEV, TVMV, PPV, and PVY served to identify regions of the potyviral polyproteins which have diverged within the genus, as well as highly conserved protein features which may play an important functional role in the potyviral life cycle.


Complete nucleotide sequence of the cloned infectious genome of Junonia coenia densovirus reveals an organization unique among parvoviruses. We previously constructed a recombinant plasmid, pBRJ, encompassing an infectious Junonia coenia densovirus (JcDNV) genome (M. Jourdan et al. (1990). Virology 179, 403-409). We report here the complete viral sequence of pBRJ. The genome, 5908 nucleotides (nt) long, consists of an internal unique sequence flanked by long (517 nt) inverted terminal repeats. The first 96 bases of one extremity can fold into a typical Y-shaped hairpin structure. The opposite extremity is incomplete, lacking 88 nt. These terminal structures, similar to those of dependoviruses, human parvovirus B19 and Bombyx mori densovirus (BmDNV), strongly suggest a common mechanism of DNA replication for these parvoviruses. JcDNV genomic organization is unique among parvoviruses in that coding sequences are evenly distributed in the 5' half of both strands. On one strand, the major open reading frame (ORF1) encodes the four structural proteins. On the complementary strand, ORF2, ORF3 (included in ORF2), and ORF4 probably encode nonstructural proteins. JcDNV genome has little DNA homology with vertebrate parvoviruses and surprisingly even less with the two densoviruses presently sequenced. ORF1 contains the highly conserved PGY and G-rich regions and ORF2 the NTP-binding domain common to most structural and to all nonstructural vertebrate parvoviral ORFs, respectively. The single homology between JcDNV and BmDNV is unexpectedly located in JcDNV NTP-binding domain and BmDNV ORF2 assumed to encode structural polypeptides. Only a weak homology exists between JcDNV and Aedes DNV in their NTP-binding domain.


Mutational analysis of the coat protein gene of potato virus X: effects on virion morphology and viral pathogenicity. The role of the coat protein of potato virus X (PVX) was investigated by site-directed mutation of the coat protein gene. Mutant viruses with in-frame deletions in the 5' end of the coat protein gene were capable of systemically infecting plants, but produced virions with atypical morphology. Viruses with a frameshift mutation near the 5' end or with deletions in the central part of the coat protein gene failed to accumulate at detectable levels, even in the inoculated leaf. In protoplasts, mutants that infected systemically either had a wild-type phenotype or showed a small reduction in accumulation of genomic RNA. The other mutants, which did not accumulate in the inoculated leaf, were unaffected in genomic RNA accumulation 8 hr postinoculation, but at 16 hr and later they accumulated less genomic RNA than wild-type virus. None of the mutations had an effect on accumulation of negative-strand RNA. The data indicate that efficient accumulation and spread of PVX, even in the inoculated leaf, requires coat protein production and encapsidation of the viral RNA.


Replication capacities of natural and artificial precore stop codon mutants of hepatitis B virus: relevance of pregenome encapsidation signal. The emergence of hepatitis B virus variants unable to express HBe protein during late stage of viral infection may represent an important mechanism of viral persistence. The molecular mechanisms responsible for the elimination of HBe expression are nonsense or frameshift mutations or initiation codon mutations in part of its coding sequence, the precore region. So far only 2 of the 29 precore amino acid codons have been found mutated to stop codons in nature, although a total of 10 codons are convertible to stop codons by single nucleotide changes. Since the HBe-coding sequence is largely overlapped by the pregenome encapsidation signal (epsilon signal), a recently found cis-acting element required for the packaging of pregenomic RNA, the absence of other potential nonsense mutants could result from their impairment of the epsilon signal. Seven such potential stop codon mutants were constructed and tested for replication capacities by transfection into a hepatoma cell line. Five mutants were replication competent, but at levels lower than that of a prevalent natural stop codon mutant. The remaining two mutants were completely defective in DNA replication, which clearly explained why these two mutants are not found in nature. Northern blot analysis revealed wild-type levels of RNA transcription by these two mutants but complete lack of packaged pregenomic RNA. Additional studies lent further support to the importance of the epsilon signal in pregenome encapsidation and suggested relaxed sequence requirements for the computer-predicted hexanucleotide bulge region as compared to the hexanucleotide loop of the signal.


Transcription dependence of DNA packaging of bacteriophages T3 and T7. T3 and T7 phages package homologous DNA more efficiently than heterologous DNA and recombinant plasmids carrying DNA sequences necessary for DNA packaging (pac sequence). The pac sequence contains a promoter for phage RNA polymerase and transcription from the promoter is necessary for DNA packaging. T3 and T7 RNA polymerases are stringently specific for their own promoters. To examine the relationship between DNA packaging and transcription, we constructed a cleared in vitro system for packaging T3 or T7 DNA containing an ammonium sulfate fractionate of a high-speed supernatant of phage-infected cells. In the system, DNA packaging required GTP and was inhibited by the 3'-deoxy analog of GTP, ATP, or CTP. The DNA packaging activity paralleled the transcriptional activity, assayed by incorporation of [32P]UTP into acid-insoluble material. In the system, homologous DNA was packaged more efficiently than heterologous DNA, but heterologous DNA was packaged as efficiently as homologous DNA by the addition of heterologous phage RNA polymerase, demonstrating that the transcriptional specificity determines the DNA packaging specificity of T3 and T7.


Evidence for multiple species of vaccinia virus-encoded palmitylated proteins. When cells were infected with vaccinia virus in the presence of [3H]palmitic acid, radiolabel was incorporated into six viral proteins with apparent molecular weights of 92, 41, 37, 26, 17, and 14 kDa, all of which are expressed at late times during the infection cycle. The [3H]palmitate-labeled fatty acid moieties from the modified proteins were isolated, converted to p-nitrophenacyl derivatives, and subjected to reverse phase HPLC analysis which confirmed the identity of the fatty acid group as palmitic acid. Furthermore, the radiolabeled palmitate-protein bonds were sensitive to treatment with neutral hydroxylamine, suggesting that association of the fatty acid moieties with these proteins occurs via a thioester linkage. Previous studies by other investigators have identified the 37-kDa protein as the major antigen present in the outer membrane of extracellular enveloped virions, and demonstrated that the protein is modified by palmitic acid but is not glycosylated (G. Hiller and K. Weber J. Virol. (1985) 55, 651-659). Growth of vaccinia virus in the presence of tunicamycin indicated that the 41- and 26-kDa palmitylated proteins were also subject to modification by glycosylation, whereas like the 37-kDa protein, the 92-, 17-, and 14-kDa species did not appear to be glycosylated. Subcellular fractionation studies provided evidence that all of the viral palmitylated proteins were membrane-associated. Extraction of purified vaccinia virus with NP-40 and DTT demonstrated that the palmitylated proteins were associated with one of the viral membranes rather than the core of the virion. Viewing these results together with the previous reports of myristylated VV proteins (Franke et al. J. Virol. (1989) 63, 4285-4291), suggests that acylation of VV proteins represents a major modification pathway utilized by VV proteins during the assembly of progeny virions.


Phylogenetic analysis of alphaviruses in the Venezuelan equine encephalitis complex and identification of the source of epizootic viruses. We studied the evolution of alphaviruses in the Venezuelan equine encephalitis (VEE) complex using phylogenetic analysis of RNA nucleotide sequences from limited portions of the nsP4, E1, and 3' untranslated genome regions of representative strains. The VEE complex constituted a monophyletic group of viruses (descended from a common ancestor); some serologic VEE varieties such as subtype III formed monophyletic groups while subtype I did not. Subtype II Everglades and variety ID enzootic viruses formed a monophyletic group which also included all epizootic variety IAB and IC VEE isolates. Everglades virus diverged from this ID lineage (colonized North America) ca. 100-150 years ago, followed by divergence of variety IAB and IC epizootic viruses. Variety IAB viruses probably emerged from the variety ID lineage once during the early part of this century, while variety IC viruses evolved at least two times. These results identify the source of epizootic VEE viruses as the variety ID enzootic virus lineage which occurs in northern South America and Panama. Even if variety IAB and IC viruses are extinct, recent, multiple emergences of epizootic viruses from an enzootic lineage suggests that other epizootic VEE viruses may evolve again in the future. The close genetic relationship of subtype II Everglades virus to the variety ID lineage also implies the potential for emergence of equine-virulent VEE viruses in Florida.


Characterization of the hepatitis B virus X- and nucleocapsid gene transcriptional regulatory elements. The regulatory DNA sequence elements that control the expression of the hepatitis B virus X- and nucleocapsid genes in the differentiated human hepatoma cell lines, Huh7, Hep3B, PLC/PRF/5, and HepG2, the dedifferentiated human hepatoma cell line, HepG2.1, and the human cervical carcinoma cell line, HeLa S3, were analyzed using transient transfection assays. In this system, the hepatitis B virus enhancer I located between coordinates 1071 (-239) and 1238 (-72) increases transcription from the X-gene promoter located between coordinates 1239 (-71) and 1376 (+67) more than 30-fold in the differentiated hepatoma and the HeLa S3 cell lines. In the dedifferentiated hepatoma cell line, HepG2.1, the enhancer I sequence increases the level of transcription from the X-gene promoter approximately 10-fold. The enhancer I subregion between coordinates 1117 (-193) and 1204 (-106) appears to be important for enhancer function only in the differentiated hepatoma cell lines, whereas the enhancer I subregion between coordinates 1222 (-88) and 1238 (-72) is required for enhancer activity in each of the cell lines examined. In all of the cell lines, the X-gene minimal promoter element was within a 138-nucleotide sequence located between coordinates 1239 (-71) and 1376 (+67). The enhancer I sequence increases transcription from the nucleocapsid promoter approximately 3- to 10-fold in the Huh7, Hep3B, PLC/PRF/5, and HeLa S3 cell lines, whereas it had little influence on the level of transcription from this promoter in HepG2 and HepG2.1 cells. The minimal nucleocapsid promoter element was within a 105 nucleotide sequence located between coordinates 1700 (-85) and 1804 (+20). This indicates that the levels of transcription from the X- and nucleocapsid gene promoters are determined in a cell-type-specific manner, in part, by the hepatitis B virus enhancer I and the corresponding minimal promoter sequence.


Hepatitis B virus-DNA transfected myeloma cell-specific cytotoxic T cells in chronic hepatitis B patients. To study the mechanisms of hepatitis B virus (HBV)-induced chronic hepatitis (B-CH), we took chronic hepatitis B patients' peripheral blood lymphocytes (PBL) and examined their cytotoxic activities against human myeloma cells (ARH77) transfected by HBV-DNA. Two different transfected cells, one expressing HBV envelope antigens (S6) and the other expressing HBV core antigens (C4), were prepared and used as targets in the in vitro cytotoxic test. We found that PBL of B-CH patients had specific cytotoxic activity against these target cells (S6, 22.0 +/- 4.8%; C4, 21.6 +/- 4.8%), whereas no remarkable cytotoxic activity was observed in non-B chronic hepatitis patients as well as asymptomatic chronic HBV carriers. These specific cytotoxic activities were inhibited with anti-CD3 antibody, hence these killer cells belonged to T cells (cytotoxic T cells; CTL). The requirement of HLA class 1 antigens to exert these CTL activities was demonstrated by the absence of CTL activity with PBL obtained from HLA-nonidentical B-CH patients and by the inhibition of their activities with anti-HLA class 1 antibody. Thus, our results indicate that, at least two different CTL, one recognizing envelop antigen and the other recognizing core antigen, exist in chronic hepatitis B patients.


Membrane fusion of mumps virus with ghost erythrocytes and CV-1 cells. The octadecyl rhodamine (R18) fluorescent dequenching assay was used to examine membrane fusion between mumps virus and mammalian cells. Rapid fluorescent dequenching, indicative of membrane fusion, was observed when labeled mumps virus was mixed with either ghost erythrocytes or CV-1 cells. After 15 min a saturation limit of 18 virus per erythrocyte ghost and 6400 virus per CV-1 cell was observed. Fetuin was found to inhibit virus fusion, suggesting a role for sialic acid in virus binding to the cells. Two dequenching processes were observed of which the faster process is thought to be membrane fusion and the second process is thought to be probe proximal transfer.


Mutational analysis of the monopartite geminivirus beet curly top virus. Mutants of the monopartite geminivirus beet curly top virus have been screened for infectivity and symptom development in Nicotiana benthamiana and Beta vulgaris, for replication competence in N. benthamiana leaf discs, and for transmission by the leafhopper Circulifer tenellus. Disruption of open reading frame (ORF) V2 by the introduction of a termination codon resulted in symptomless infection of N. benthamiana associated with low levels of virus and reduced single-stranded (ss) DNA and prevented systemic infection of B. vulgaris. Reduced levels of ssDNA were produced by the mutant in N. benthamiana leaf discs, suggesting that V2 affects the synthesis or accumulation of this viral DNA form. Mutants in which ORF C2 had been truncated by the introduction of termination codons or by frame-shifting remained highly infectious and induced severe symptoms in both N. benthamiana and B. vulgaris. Similarly, a mutant containing a termination codon within ORF C3 was highly infectious and induced severe symptoms in N. benthamiana although infectivity in B. vulgaris was greatly reduced, symptoms were extremely mild, and virus levels were low. A synergistic effect of a double mutation in ORFs C2 and C3, manifested by the inability of mutants to systemically infect N. benthamiana and the production of reduced amounts of ssDNA in N. benthamiana leaf discs, suggests that both ORFs are functional in this host. A mutant containing a termination codon within the 5' terminus of ORF C4 produced severe symptoms in both N. benthamiana and B. vulgaris resembling those induced by wild-type virus. Comparison with the phenotypes of previously characterized ORF C4 mutants suggests that a conserved core sequence of this ORF is an important symptom determinant. ORF C2, C3, and C4 mutants produced virus particles and were transmitted by C. tenellus, eliminating agroinoculation as a contributory factor to the mutant phenotypes. Our results are compared with those derived from mutagenesis studies on related bipartite geminiviruses.


Expression of functional parvoviral NS1 from recombinant vaccinia virus: effects of mutations in the nucleotide-binding motif. The gene encoding the major replicative protein, NS1, of minute virus of mice (MVM) was transferred into a recombinant vaccinia virus vector in place of the vaccinia thymidine kinase gene. The NS1 gene was placed under control of a bacteriophage T7 promoter and expressed in cells coinfected with another recombinant vaccinia virus, vTF7-3, which encodes the T7 RNA polymerase. Expression of NS1 was further enhanced by the presence of a 5' untranslated region, derived from encephalomyocarditis virus, which allows efficient cap-independent translation. This system was used to produce and analyze wild-type NS1 and two mutant forms of the protein, NS1K405R and NS1K405M, in which the highly conserved lysine codon located in the putative purine triphosphate binding site of NS1 was changed to arginine and methionine, respectively. Full-length NS1 was expressed efficiently in both human and mouse cells infected with each of the three recombinant viruses, and in each case the NS1 was rapidly and efficiently translocated into the nucleus. Wild-type NS1 expressed in this way was biologically active. It was able to trans-activate an MVM P38 promoter located in a host chromosomal site, whereas the two mutant forms of NS1 showed no significant activity in this assay, and it was capable of resolving palindromic junction fragments cloned from multimeric MVM replicative form DNA molecules. These substrates, representing MVM genomic left-end:left-end and right-end:right-end fusions, were resolved in a DNA synthesis-dependent in vitro reaction supplemented with nuclear extracts containing recombinant wild-type NS1. Neither of the two mutant forms of the polypeptide had any detectable activity in this assay.


Polylactosaminoglycan modification of the respiratory syncytial virus small hydrophobic (SH) protein: a conserved feature among human and bovine respiratory syncytial viruses. We investigated the nature of the oligosaccharide modification of the glycosylated forms of the small hydrophobic integral membrane protein, SH (previously designated 1A), of respiratory syncytial (RS) virus. Analysis of SH protein expressed in cells infected with RS virus or with a recombinant vaccinia virus revealed two glycosylated SH protein species, SHg and SHp, which contained N-linked carbohydrate residues. SHp migrated diffusely on polyacrylamide gels, which suggested modification by polylactosaminoglycan oligosaccharides. Polylactosaminoglycan modification of SHp was established from three lines of investigation: (1) the synthesis of SHp in a cell line (IdID) conditionally defective in the ability to add specific carbohydrate residues to N- or O-linked oligosaccharide chains required the addition of galactose, which is a component of the N-acetyllactosamine repeating unit; (2) SHp was sensitive to digestion with endo-beta-galactosidase, which cleaves the beta 1-4 linkage between galactose and N-acetylglucosamine of the repeated N-acetyllactosamine subunit; and (3) SHp was selected by Datura stramonium lectin (Dsl), which has specificity for polylactosaminoglycans. The presence of SHp as a component of purified human subgroups A and B and bovine RS virus particles was demonstrated by Dsl affinity selection. In addition to SHp, nonglycosylated SHo was selected by Dsl affinity, indicating that SHp and SHo may associate to form complexes within infected cells and virus particles. To identify conserved amino acid residues among the human and bovine SH glycoproteins that may function as signals for polylactosaminoglycan modification, the nucleotide sequences of the SH protein genes of a human subgroup B virus (8/60) and a bovine virus (391-2) were determined and compared to those of a human subgroup A virus (A2), a subgroup B virus (18537), and a bovine virus (A51908). A comparison of the deduced amino acid sequences of the human and bovine RS virus SH proteins indicated that a central hydrophobic region and the presence of potential N-linked glycosylation sites on either side of the central hydrophobic region were conserved features that may be required for the polylactosaminoglycan modification of SH.


Hyperexpression of baculovirus polyhedrin and p10 is inversely correlated with actin synthesis. Polyhedrin and p10, two proteins encoded by Autographa californica M nuclear polyhedrosis virus, are hyperexpressed very late during normal infections. In this study we found that cytochalasin D, a drug that leads to increased actin synthesis in infected and uninfected host cells, delayed the amplified expression of polyhedrin and p10 when added to infected cells before hyperexpression was already in progress. Restoration of polyhedrin and p10 hyperexpression could be achieved by removal of the drug, but required new protein synthesis. An inverse correlation was observed between polyhedrin/p10 mRNA levels and actin mRNA levels at late and very late times during infection, regardless of whether cytochalasin D was added, removed, or never present. In comparison to mRNAs of polyhedrin and p10, the mRNA levels of the early/late viral gene 39K were much less affected by cytochalasin D and responded to drug removal more slowly. The results of these studies revealed an apparent correlation between the shut down of host actin genes and the amplified expression of polyhedrin and p10 in the presence and absence of cytochalasin D. The possibility that newly synthesized actin itself, either directly or indirectly, plays a negative regulatory role in the accumulation of polyhedrin and p10 mRNAs is discussed.


Analysis of 3' and 5' ends of N and NSs messenger RNAs of Toscana Phlebovirus. The 5' and 3' ends of N and NSs mRNAs, transcribed from the S segment of Toscana Phlebovirus, were analyzed by oligonucleotide primer extension and S1 nuclease mapping procedures. The results showed that both mRNAs acquired, at their 5' end, approximately 9-15 nucleotides not present in the viral template, suggesting an initiation transcription mechanism similar to the one described for influenza virus. Furthermore, the 3' ends of the two mRNAs were located in a sequence motif conserved in the S segment of two other Phleboviruses, the Rift Valley Fever and Sandfly Fever Sicilian viruses. This finding suggests the possible involvement of this sequence in the mechanism of transcription termination.


Characterization of the antibody response to three different versions of the HTLV-I envelope protein expressed by recombinant vaccinia viruses: induction of neutralizing antibody. Recombinant vaccinia viruses (RVV) designated RVV E1, RVV E2, and RVV E3, were constructed to express three different versions of the human T cell leukemia virus type I (HTLV-I) envelope proteins to determine which configuration elicits an optimal antibody response. RVV E1 expressed the native HTLV-I envelope proteins gp46 (surface protein) and gp21 (transmembrane protein), while RVV E2 expressed the envelope precursor with the proteolytic cleavage site deleted. The RVV E3 construct expressed only the external surface glycoprotein, gp46. Radioimmunoprecipitation and FACS analysis confirmed that the appropriate envelope proteins were expressed by RVV E1-, E2-, and E3-infected cells. Immunization studies were carried out using Balb/c, A/J, and C57BL/6 strains of mice. Balb/c mice responded poorly to immunization with all of the three RVV constructs. C57BL/6 mice produced neutralizing antibodies in response to immunization with all three constructs, whereas A/J mice developed neutralizing antibodies only when immunized with the RVV E1s construct. The results indicate that the humoral immune responses depend on the form of HTLV-I envelope proteins expressed by each RVV.


cis-acting elements for in trans complementation of replication-defective mutant of tobacco mosaic virus. We have shown that virus-encoded replicase components of tobacco mosaic virus (TMV)(130K/180K proteins) could complement a replication-defective mutant (LDR28) for viral replication in trans [Ogawa et al. Virology 185, 580-584 (1991)]. Using this trans-complementation system, we investigated the dispensability of regions of the 130K/180K protein genes as cis-acting elements for viral replication. A series of replication-defective mutants (LDRs) which had various deletions in the 130K/180K coding region were constructed. These were co-inoculated with a replication-competent mutant (LDCS29) into tobacco protoplasts. Accumulation of LDR-genomic RNA, CP mRNA, and CP was much increased by the removal of the 3' terminal one-third of the readthrough part of 180K protein gene (residues 4529-4937 of TMV-RNA) from LDR, suggesting that this region interferes with the viral replication in this trans-complementation system. In addition, accumulation of CP mRNA and CP was much decreased by the removal of the 5'terminal half of the 30K protein gene (residues 4938-5263 of TMV-RNA), suggesting the presence of an element to enhance the synthesis of CP mRNA in this region.


Structural and functional organization of the human endogenous retroviral ERV9 sequences. The human genome contains a variety of genetic elements similar in structure to retroviruses and retrotransposons. We report here the structural and functional organization of a novel human endogenous retroviral family (ERV9). Three polyadenylated RNAs, 8, 2, and 1.5 kb long, are detected by Northern blot in undifferentiated embryonal carcinoma NT2/D1 cells. Upon genomic cloning of an expressed ERV9 locus, we demonstrated that the three polyadenylated RNAs are originated by a single ERV9 locus by alternative usage of splicing and polyadenylation signals. DNA sequence analysis of different ERV9 LTRs have revealed that they are heterogeneous in length and that the length variability is due to the number of tandemly repeated subelements present in both U3 and U5 regions; moreover, the ERV9 LTRs are capable to drive expression of a reporter gene in transient expression assays. Finally, analysis of the ERV9 5' transcription start site has allowed us to define the U3-R-U5 organization of the ERV9 LTR.


Nucleotide sequence identifies Vilyuisk virus as a divergent Theiler's virus. The Vilyuisk virus, originally thought to be the cause of a degenerative neurological disease of inhabitants of Siberia, has been characterized by sequence analysis of its 5' noncoding and coat protein coding regions. In the 5' noncoding, leader, and VP4 regions, the nucleotide identity between the sequences of known strains of Theiler's virus and Vilyuisk virus is about 90%. In the VP1-encoding region, the similarity drops to about 66% compared to the 50% similarity between sequences of Theiler's virus and encephalomyocarditis virus. Using the known crystal structure of one Theiler's virus strain, it is shown that the sequence heterogeneities generally occur at exposed surface residues. Vilyuisk virus is the most divergent Theiler's virus known. A tissue culture-adapted isolate has been propagated and found to exhibit low neurovirulence in CD-1 mice.


The coat protein of white clover mosaic potexvirus has a role in facilitating cell-to-cell transport in plants. Functions of the coat protein of white clover mosaic potexvirus (WCIMV) were investigated using C-terminal deletion mutants. Whereas plants inoculated with RNA transcripts of a full-length wild-type clone of WCIMV produced typical infections, plants inoculated with transcripts of each mutant did not produce symptoms, and viral RNA species were not detected by Northern analysis. The mutants were able to replicate in protoplasts, although, relative to the wild-type RNA profile, the level of genomic RNA, but not subgenomic RNA, was reduced. These results indicate a role for the coat protein in efficient cell-to-cell transport in plants. Virus-like particles were detected in protoplast extracts inoculated with transcripts of a mutant in which the coat protein was truncated by 31 amino acids. This result suggests that the lack of detectable transport in plants was not due solely to a failure of the mutants to form virus particles. Possible roles for the coat protein in transport and replication are discussed. A 6-kDa open reading frame, internal to the coat protein gene, was shown by mutational analysis not to be essential for replication or transport.


The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes. A 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A]7) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and five potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and eight potential glycosylation sites, and appears to correspond to a 90-kDa nonstructural glycoprotein (GNS) identified in BEFV-infected cells (Walker et al. [1991] J. Gen. Virol. 72, 67-74). A database search indicated that both the G and GNS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with sigma virus and vesicular stomatitis virus serotypes.


Functional organization of enhancer (ENII) of hepatitis B virus. A new enhancer ENII, located in the X open reading frame and immediately upstream of the core gene promoter, has recently been identified in the genome of hepatitis B virus. We have studied the functional constituents of this new enhancer in different cell lines. ENII can be divided into two functional elements, A and B, corresponding to two major binding sequences for nuclear protein factors. Element A alone gave very low activity; however, it was a modulatory element important for cell-type specificity. Element B was shown to be the basic functional element of ENII, which retained about 70% of the enhancer activity of the complete ENII in HepG2 cells. Element B can be further dissected into three subunits, B1, B2, and B3, which act synergistically. A 52-bp sequence is identified as the core sequences of element B. A model for the mechanism of ENII function is proposed.


African swine fever virus-induced DNA polymerase is resistant to aphidicolin. African swine fever virus (ASFV) induces the synthesis of a virus-specific DNA polymerase, which is inhibited by phosphonoacetic acid and cytosine arabinoside. In contrast to all other alpha-like DNA polymerases of DNA viruses, ASFV-specific DNA polymerase is resistant to aphidicolin. Concentrations of the drug as high as 160 microM had no effect on virus production or plaquing efficiency. The resistance of ASFV DNA polymerase to aphidicolin was confirmed by analyzing the effect of the drug on viral DNA synthesis. A moderate inhibition of viral DNA synthesis was observed when aphidicolin was added immediately after virus adsorption but normal synthesis occurred, with a peak at 10 hr p.i., when the drug was added at 2 or 4 hr p.i. This suggests that a very early phase of ASFV DNA replication is sensitive to aphidicolin and is probably catalyzed by a different enzyme. An in vitro assay of DNA polymerase activity was used to assay the sensitivity of the virus-specific DNA polymerase to inhibitors. In correspondence to the results observed in vivo, phosphonoacetic acid strongly inhibited the enzyme activity, whereas aphidicolin had no effect. Resistance to aphidicolin was independent of the concentration of dCTP used in the assay. Three independent ASFV mutants resistant to phosphonoacetic acid showed the same resistance to aphidicolin as wild type virus.


Human coronavirus gene expression in the brains of multiple sclerosis patients. Total RNA extracted from both white and gray matter of brain tissue from multiple sclerosis (MS) patients and controls was analyzed using a reverse transcription-polymerase chain reaction for the presence of the nucleic acid of human coronavirus (HCV) 229E and OC43, the two strains characterized to date and associated with respiratory infections. HCV-229E viral RNA was detectable in the central nervous system tissue of 4 of 11 MS patients and in none of 6 neurological and 5 normal controls. No HCV-OC43 nucleic acid was detected in any of the specimens. These results suggest a neurotropism on the part of the 229E strain of human coronavirus and underline the importance of further studies on its tissue distribution. The fact that it was detected only in tissue from MS patients illustrates the need for continued studies on the possible role of coronaviruses in the etiology of MS.


Nucleotide sequence changes in the polymerase basic protein 2 gene of temperature-sensitive mutants of influenza A virus. Influenza A viruses bearing temperature-sensitive (ts) mutations are restricted in replication in the respiratory tract of animals and humans and are therefore attenuated. Nucleotide sequences were determined for the RNA segment coding for the polymerase basic protein 2 (PB2) from a panel of 12 influenza A/Udorn/307/72 (H3N2) ts viruses, previously characterized to have a ts mutation in the PB2 gene. Each of the viruses with a ts mutation in the PB2 gene had a single amino acid change located at position 65, 100, 112, 174, 298, 310, 386, 391, 556, or 658 of the PB2 protein. The sites of the single mutations were scattered throughout the length of the protein and occurred in regions that are highly conserved among the influenza A virus PB2 predicted amino acid sequences. Interestingly, the substitution of aspartic acid for asparagine at position 556 was found to lie within a region that has homology with cap-binding motifs of human and yeast proteins. Taken together, the findings of lesion sites in the A/Udorn/307/72 PB2 gene and the three reported amino acid changes at positions 265, 417, and 512 for A/AA/6/60, A/WSN/33, and A/FPV/Ros/34 ts PB2 genes, respectively, indicate that the PB2 gene can sustain a viable ts mutation at different sites. This information will allow us to construct cloned cDNA copies of the A/Udorn/307/72 PB2 gene mutagenized at specific sites. Different configurations of two or more ts mutations may be incorporated into the cDNA PB2 gene constructs. We have a host-range reassortant virus that should permit rescue of in vitro-produced transcripts of the PB2 gene into infectious virus. The rescue of these mutated PB2 RNA segments into an infectious influenza A virus may lead to the development of live attenuated reassortant virus vaccines that are satisfactorily attenuated, genetically stable, and immunogenic in humans.


Cell receptor-independent infection by a neurotropic murine coronavirus. The cellular receptors for a coronavirus, mouse hepatitis virus (MHV), have been recently identified as one or more members of the carcinoembryonic antigen (CEA) family. The neurotropic JHM strain of MHV (MHV-JHM) possesses a highly fusogenic surface (S) glycoprotein. This protein is now shown to promote the spread of MHV into cells lacking the specific CEA-related MHV receptor. Resistant cells are recruited into MHV-induced syncytium with consequent production of progeny virus. Cell-to-cell spread of virus via membrane fusion without the requirement for specific cell surface receptor offers a novel way for virus to spread within infected hosts.


A regulatory role for the 32K protein in proteolytic processing of cowpea mosaic virus polyproteins. We have studied the regulation of proteolytic processing of the polyproteins encoded by cowpea mosaic virus M-RNA and B-RNA. For that purpose mutations were introduced in full-length cDNA clones of these RNAs. RNA transcripts were translated in rabbit reticulocyte lysate and the effect of mutations on the processing was analysed. These studies revealed that the 32K protein is released from the 200K B-polyprotein by an intramolecular cleavage and remains associated with the 170K protein, probably by interaction with the 58K domain of the 170K protein. In this complex the conformation of the 170K protein is such that further cleavages are very slow. This complex carries out the processing of the Gln/Met site in the M-polyprotein. The 170K protein produced by a B-RNA mutant that lacks the 32K coding region was efficiently processed into 110K, 87K, 84K, 60K, 58K and 24K cleavage products. Thus, the 32K protein regulates the B-polyprotein processing by slowing it down and, on the other hand, enhances trans cleavage of M-polyproteins at a Gln/Met site.


Processing of VPg-containing polyproteins encoded by the B-RNA from cowpea mosaic virus. To study the processing of putative VPg precursors the expression of specific mutant transcripts derived from a full-length cDNA clone of cowpea mosaic virus (CPMV) B-RNA was examined in a rabbit reticulocyte lysate system. This study revealed that the 170K protein produced by a B-RNA mutant that lacks the 32K coding region was efficiently processed by mainly intramolecular cleavages at three different sites into three sets of proteins of 60K + 110K, 84K + 87K, and 58K + 112K. Further cleavage of the 60K protein into 58K and VPg has not been observed in this in vitro system. The 84K protein can be further processed by an intramolecular cleavage reaction via two alternative pathways, either into 26K (VPg + 24K) and 58K proteins or into 24K and 60K proteins. VPg can be released from the 112K (VPg + 110K) precursor either directly or via the 26K intermediate. Immunoblot analysis showed that the 112K protein is present in CPMV-infected plant cells indicating that the in vitro observations may hold true in vivo.


The nucleotide sequence of apple stem grooving capillovirus genome. The complete nucleotide sequence of apple stem grooving virus (ASGV) genome has been determined. The genome is 6496 nucleotides in length excluding a 3'-terminal poly(A) tail and contains two overlapping open reading frames (ORFs). ORF1 begins at nucleotide position 37 and is terminated at position 6341, encoding a protein with a molecular weight of 241 kDa. ORF2, which is in a different reading frame within ORF1, begins at position 4788 and can encode a 36-kDa protein. The 241-kDa protein contains two consensus sequences associated with the RNA-dependent RNA polymerase and the NTP-binding helicase. Comparisons of amino acid sequences around these conserved motifs with other RNA viruses revealed that ASGV has extensive similarities with apple chlorotic leaf spot, tymo-, carla-, and potexviruses, and is a member of the sindbis-like supergroup. ASGV coat protein is found to be located in the C-terminal region of the 241-kDa polyprotein. The 36-kDa protein encoded by ORF2 contains the consensus sequence Gly-Asp-Ser-Gly found in the active site of several cellular and viral serine proteases.


The 3'-nucleotide sequence of an ordinary strain of potato virus S. The sequence of 1346 nucleotides of the 3'-terminal region of an ordinary strain of potato virus S was determined from cloned cDNA. This portion of the viral RNA contains two major open reading frames (ORFs), encoding the coat protein of M(r)33K and a protein of M(r)11K 3' to the coat protein, both of which showed high homology, 93% and 79%, respectively, with the equivalent proteins of the Andean strain. A major block of amino acids that differ between the ordinary and Andean strains was present in both the coat protein and the 11K. These blocks may be responsible for the substantial differences between the ordinary and Andean strains at the symptom and aphid transmissibility levels.


Expression of biologically active HIV glycoproteins using a T7 RNA polymerase-based eucaryotic vector system. Bacteriophage T7 RNA polymerase and a derivative containing a nuclear localization signal were transiently expressed in CV-1 cells and were shown to localize to the cytoplasm and nucleus, respectively. A vector was constructed containing T7 promoter and transcription terminator sequences flanking a picornaviral 5' untranslated sequence for cap-independent translation and a polyA signal. Expression of the HIV-1 envelope glycoproteins in this vector system gave high levels of specific transcripts and translation products, independent of the subcellular localization of T7 RNA polymerase. The synthesis of HIV glycoproteins was also completely independent of the coexpression of the HIV rev protein, which is normally required for the expression of HIV structural proteins. In addition, a polyA signal was not required, whereas the presence of the picornaviral 5' untranslated region was necessary for efficient expression. Different possibilities to account for these findings are discussed. The HIV glycoproteins synthesized in this system were normally processed and assembled; they could induce syncytium formation and complement an env-deletion mutant of HIV-1.


Multiple cis-acting DNA elements that regulate transcription of the adenovirus 12 E1A gene. To delineate cis-acting elements for adenovirus (Ad) 12 E1A gene transcription, we transfected HeLa and NIH3T3 cells with DNAs having various deletions in the 5'-upstream region linked to the chloramphenicol acetyltransferase gene. Deletions in the regions between nucleotide (nt) positions 54 and 166, and 167 and 200, with respect to the left end of the viral genome at nt position 1, caused a two- to three-fold reduction in transcription. Transcription decreased to an almost undetectable level with loss of the region between nt positions 201 and 282. The effect of these mutations was almost consistent between both cell lines. The region between nt positions 77 and 94 stimulated transcription when situated upstream of the simian virus 40 early promoter in either orientation. Transcription was stimulated about ninefold in the presence of the DNA that encodes the product of the 13S, but not the 12S mRNA of the Ad12 E1A gene. These results indicate that transcription of the Ad12 E1A gene is regulated by multiple cis-acting elements and is stimulated by its own gene product.


Neutralizing monoclonal antibodies against alpha and beta subunits of the Ustilago maydis virus encoded toxin. The toxins secreted by Ustilago maydis are encoded by dsRNA viruses. The KP6 toxin encoded by subtype P6 consists of two polypeptides alpha and beta, which are not covalently bound. Neutralizing monoclonal antibodies (MoAbs) were raised against each subunit. Some of the anti-beta MoAbs identify different epitopes in the antigen. The MoAbs were used to affinity purify alpha and beta polypeptides from culture media and to detect the precursor of the mature toxin.


Nucleotide and deduced amino acid sequence of the canine adenovirus type 1 proteinase. The DNA sequence of an open reading frame (ORF) corresponding to the canine adenovirus type 1 (Can1) proteinase gene was determined. A total of 1171 base pairs were sequenced from the downstream end of the HindIII-A Can1 genomic fragment, including adjacent regions corresponding to the carboxy-terminal portions of the hexon and the DNA-binding proteins. The predicted Can1 proteinase consists of 206 residues (23,325 D) of which 68% are identical and 83% are similar to the sequence of the human Ad2 proteinase. Alignment with the Ad2 proteinase identified a number of conserved residues that could form part of the catalytic triad of the enzyme.


The phase of PVEP in Maxwellian view: influence of contrast, spatial and temporal frequency. The temporal phase of the pattern reversal VEP has been investigated using stimulation with laser interference fringes in Maxwellian view. VEP phase was almost constant as function of spatial frequency (2-30 c/deg, 6.5 r/sec, 51 subjects). The phase function however shows a small phase increase at low spatial frequencies consistent with the existence of multiple temporal mechanisms. Contrast variation yields a smaller phase increase with decreasing contrast than conventional stimulation. The phase is linear as function of reversal rate (2-31 r/sec) for low and high spatial frequencies. The indication of more than one temporal mechanism has also been found when the test field diameter was varied. With decreasing test field size the phase increases at 6.5 r/sec, but decreases at 18 r/sec (for 12 c/deg).


Pharmacological study of the chicken's monocular optokinetic nystagmus: involvement of the ON retinal channel evidenced by the glutamatergic separation of ON and OFF pathways. The chicken's monocular optokinetic nystagmus (OKN), recorded by the magnetic search coil technique, displays a directional asymmetry, the temporal-nasal (T-N) stimulation being more efficient than the nasal-temporal (N-T) one to evoke the reflex. The intravitreal administration of APB, a glutamate agonist which selectively blocks the ON retinal channel strongly reduced the eye monocular OKN; it also induced spontaneous eye movements in the T-N direction. The intravitreal injection of PDA another glutamate analog, which reduces the OFF channel, while increasing the activity of the ON channel, induced a large increase in OKN velocity gain, especially for a N-T stimulation at the lowest drum speeds. These results indicate the main involvement of the ON retinal channel in the OKN genesis and the inhibitory effect of the OFF channel upon this oculomotor reflex.


The development of maximum displacement limits for discrimination of motion direction in infancy. The development of visual motion mechanisms has been studied in infants by using forced-choice preferential looking to measure maximum displacement limits (dmax) for the detection of coherent motion in random-dot patterns. The motion consisted of a sequence of coherent displacements at intervals of 20 msec. Between 8 and 15 weeks, dmax for discrimination of coherent from incoherent motion, and for discrimination of opposite directions of coherent motion, increased with age; in both conditions, dmax for the oldest infants was less than a third of the value found in adults. For 10-11-week-old infants, and for an adult subject, reducing stimulus contrast from 88 to 48% had no effect on dmax for direction discrimination, which indicates that the rise in dmax with age is not simply a result of improving contrast sensitivity. When the displacement interval was increased from 20 to 40 msec, dmax increased significantly in 8-11-week-olds, but remained unchanged in 12-15-week-olds. These results show that while directional mechanisms are present in the visual system by 8 weeks, they operate over a restricted velocity range. The upper limit of this velocity range increases with age. After about 12 weeks, the increase is mainly due to changes in the spatial properties of motion mechanisms; however, in younger infants changes in their temporal properties are also important.


Contrast sensitivity as a function of spatial frequency, viewing distance and eccentricity with and without spatial noise. Using computer graphics and a two-alternative forced-choice method we measured threshold contrast as a function of viewing distance, spatial frequency, and eccentricity for gratings with and without added, white two-dimensional spatial noise. Our experiments showed that in spatial noise contrast sensitivity was independent of viewing distance as long as contrast sensitivity was lower with noise than without. With increasing spatial frequency (f) the grating area (A) was reduced in order to keep the relative grating size (Af2) constant. At all spatial frequencies the test gratings thus had the same amount of detail and contour. Noise spectral density was reduced in direct proportion to grating area in order to keep the physical signal-to-noise ratio constant. An increase in spatial frequency was thus accompanied with reductions in grating area and noise spectral density similar to those produced by a corresponding increase in viewing distance. In agreement, contrast detection in spatial noise was found to be independent of spatial frequency as long as contrast sensitivity was lower with noise than without. The effect of increasing eccentricity on visual performance can be compensated for by reducing the viewing distance (M-scaling). Hence, without M-scaling the effect of increasing eccentricity is similar to that of increasing viewing distance. In agreement, we found that contrast sensitivity in spatial noise was independent of eccentricity as long as contrast sensitivity was lower with noise than without.


Effects of contrast substitutions upon motion detection in spatially random patterns. We report the effects of contrast changes between frames in a random-square kinematogram. When the contrasts of both frames are too low to permit directional discrimination, increasing the contrast of either the first or the second frame alone makes directional discrimination possible. However, at suprathreshold contrasts for motion detection, increasing the contrast of either the first or the second frame alone makes discrimination more difficult. We conclude that motion detection is a special case of contrast discrimination, in agreement with the Reichardt model of motion detection.


Scotopic visual efficiency: constraints by optics, receptor properties, and rod pooling. We studied the influence of optics, photoreceptor properties, and rod pooling on scotopic contrast sensitivity by comparing the performance of an ideal discriminator to that of human observers. Comparisons of human and ideal contrast sensitivities indicated that preretinal factors and summation area were not sufficient to explain the shape of the human CSF. Spatial pooling of rods was explored as a possible explanation of this discrepancy. Our highest efficiency, expressed in terms of a human/ideal contrast sensitivity ratio, was about 1:3 (0.33) for a contrast discrimination task.


The role of color in the motion system.

The role of color in the motion system. We have examined the ability of observers to determine the direction of movement of a variety of colored plaid patterns. When the two plaid components are of unequal spatial frequency or of unequal luminance or chromatic contrast, observers judge the direction of movement incorrectly. These errors are correlated with a misjudgement of the speeds of the two components. Our results provide support for an initial decomposition into oriented components followed by a subsequent component-to-pattern recombination of moving equiluminant and colored plaids. At equal multiples of threshold contrast a moving luminance grating is about 8 times more powerful than a moving equiluminant grating in determining the apparent direction of motion of a plaid. When both are present, luminance and color do not interact linearly. Color and motion must be processed in parallel in at least partially separate pathways.


Head-centric visual localization with lateral body tilt. The aim of the present investigation was to decide whether subjects can correctly judge the head-centric orientation of a display in the dark in spite of the counterrolling of their eyes caused by head tilt. Four subjects were asked to set two luminous points orthogonally to the median plane of their heads, with head and body upright or tilted 90 degrees to the left or right side. The luminous points could be rotated in a frontal plane about a stationary fixation light situated straight ahead. Subjects viewed this display monocularly with either eye. In the same three body positions, ocular counterroll was determined photographically. The settings turned out to be neither accurately oculocentric nor head-centric, and there were considerable inter- and intrapersonal variances. However, on average, display adjustments were more head-centric: mean difference of ocular counterroll between left-ear-down and right-ear-down conditions was 13.2 deg, whereas the mean difference of the subjective head-centric orientations was only 3.8 deg.


Predictive smooth pursuit eye movements near abrupt changes in motion direction. The stimulus-response characteristics of predictive smooth pursuit eye movements near the time of predictable, abrupt changes in target motion direction were studied. Expectations about the speed and direction of target motion both before and after the direction change affected specific components of the predictive pursuit responses. We propose that, when the direction of target motion is expected to change, the cessation of motion in one direction and the initiation of motion in a new direction are separately anticipated and that predictive pursuit movements are summated responses to these two events.


Two-stage analysis of the motion of 2-dimensional patterns, what is the first stage? The sum of two differently orientated moving sinusoidal gratings of similar spatial frequency, contrast, and velocity appears as a single coherent "plaid" pattern. The visual system is thought to analyse the motion of plaids in two stages, first analysing the motion of the (1-D) components, and then calculating a speed and direction which is consistent with those 1-D motions. We find that the direction of motion of a plaid (components 1.6 c/deg orientated +60 degrees and -60 degrees) can be discriminated at velocities so low that the direction of motion of its components is not discriminable. This finding is not consistent with the "two-stage" hypothesis in the form that it is usually expressed. We suggest that mechanisms sensitive to the motion of local elements in the pattern, such as edges, could also contribute to the first stage of the analysis of plaid motion.


Analysis of the motion of 2-dimensional patterns: evidence for a second-order process. The sum of two differently orientated moving sinusoidal gratings of similar spatial frequency, contrast, and velocity appears as a single coherent "plaid" pattern. The visual system is thought to analyse the motion of plaids in two stages, first analysing the motion of the (1-D) components, and then calculating a speed and direction which is consistent with those 1-D motions. We studied the apparent direction of motion of plaids made by adding two components that had the same spatial frequency and contrast, and were symmetrically oriented about the vertical axis. The gratings moved in jumps, and we studied the effect of varying the size of the jump, the angle between the component gratings, and the temporal interval between the jumps, on the perceived direction of motion. When the size of the jumps was increased to 3/8 of their spatial period, the perceived direction of motion of the plaid pattern reversed, although if one component were presented alone, its direction of movement did not reverse. Reversed motion of this type was consistently obtained if the angle between the components was greater than about 140 degrees, if the interval between jumps was at least 25 msec, and if the spatial frequency of the component gratings was less than about 4 c/deg. When the angle between the components was smaller, or the time between jumps was greater, most observers saw normal motion in the direction predicted by the two-stage hypothesis. When the spatial frequency was raised, observers saw no consistent motion.(ABSTRACT TRUNCATED AT 250 WORDS)


A comparison between colour and luminance contrast in a spatial linking task. We report experiments which compare the ability of subjects to employ colour vs luminance contrast as a basis for discriminating the degree of collinearity of random element string pairs. The purpose of the study was to determine the extent to which spatial integration mechanisms could utilize colour contrast. In order to probe directly the processes of spatial integration per se, it was necessary to control for any differences in the efficiency with which the visual system utilized colour and luminance contrast to locate the positions of the individual elements in the test stimuli. To do this we first established the "equivalent" luminance contrast of an isochromatic stimulus which produced equal performance to an isoluminant stimulus in a 2 element per string alignment task. This equated the colour defined and luminance defined stimuli for local positional acuity. We then measured performance for both isoluminant and equivalent luminance contrast stimuli for strings consisting of 2, 4, 8 and 16 elements. This tested for any differences in the processes of spatial integration. For both unmasked stimuli and stimuli embedded in luminance noise, there was no consistent trend favouring either luminance or colour contrast as the number of elements in the stimuli was increased. We conclude that the visual system is able to employ colour contrast as efficiently as luminance contrast for collinearity judgements, thus implicating a general role for colour vision in spatial integration tasks.


Nonlinear processes in spatial-frequency channel models of perceived texture segregation: effects of sign and amount of contrast. Observers rated the degree of segregation between two textures, each composed of the same two element types but in differing arrangements (a checkerboard arrangement in the middle region of the pattern and a striped arrangement in the top and bottom regions). The two element types in a given pattern were either both solid squares or both center-surround elements. In center-surround elements the average luminance equaled the background luminance. The two elements types were identical in size but differed in sign and/or amount of contrast. Discrepancies between the observers' ratings of perceived segregation and the predictions of simple (linear) spatial-frequency and orientation channels models of texture segregation suggested adding nonlinear processes to the model. Complex channels (a rectification-type nonlinearity between two linear-filtering stages) can explain why some patterns made of center-surround elements segregate even though there is little energy at the spatial frequencies that differentiate the two textures. Complex channels cannot, however, explain the very poor segregation of "same-sign-of-contrast" patterns (where the luminances of the two element types were both far above or both far below the background). This second result might arise from a local nonlinearity preceding the channels and might be ascribed to retinal light adaptation except that it occurs at contrasts less than or equal to 25%! Alternatively, it might arise from normalization, which may result from intracortical inhibition. Some preliminary quantitative predictions were computed from two models, one incorporating complex channels and an early local nonlinearity, the other complex channels and normalization. With suitable choices of parameters, either model could account for the results.


A reduction in stimulus duration can improve wavelength discriminations mediated by short-wave cones. Virtually all visual discriminations become less accurate when either the luminance or the duration of the stimulus is reduced. An exception is found for wavelength discriminations near 460 nm, where an increase in either luminance or duration can cause the threshold to rise. For flashes of 100 msec or less, the critical variable is the total energy of the flash (i.e. the product of retinal illuminance and flash duration), and wavelength discrimination is optimal at an intermediate value; higher stimulus energy causes discrimination to deteriorate. To explain these findings we suppose that discrimination in this region of the spectrum is mediated by a channel that draws opposed signals from the short-wavelength cones and from some combination of the middle- and long-wavelength cones, and that high stimulus energies cause saturation of this channel.


"Effortless" texture segmentation and "parallel" visual search are not the same thing. If a region composed of one set of texture elements is effortlessly perceived on a background composed of another set, the texture segmentation is said to occur "preattentively". If the time required to find a target item among distractor items is independent of the number of distractor items, the visual search is said to occur with all items processed "in parallel". Effortless, preattentive texture segmentation and parallel visual search are sometimes assumed to be equivalent measures of the same parallel processing stage in the visual system. The demonstrations presented here show that this is not the case. Parallel processing can occur with stimuli that do not support effortless texture segmentation and vice versa.


Detection of eyeshine by flashlight fishes of the family Anomalopidae. Flashlight fishes of the family Anomalopidae live in clear tropical waters and are nocturnally active. They have luminescent organs located just below the pupils of their eyes. The relation of the luminescent organ to the pupil of the anomalopid eye is similar to that of the illumination and pupil system of the ophthalmoscope and identical to that of some photoretinoscopes. Indeed, one species of flashlight fish, Anomalops katoptron, actually moves its luminous organ away from its pupils in the process of occluding its light organ, making a retinoscopic-like movement. By photographing the eyeshine of a number of fishes with a photoretinoscope and by analyzing the optics of light organs of fish of the family Anomalopidae as well as the optics of reflecting eyes, we show under what light conditions and ranges flashlight fishes may reasonably be able to detect eyeshine from other fishes in the environment. Further, we suggest that flashlight fishes may be able to communicate with each other by altering the accommodation of their eyes. In such a communication system, the sender radiates no energy and communicates only with the interrogating receiver of the information. To our knowledge, this utilization of eyeshine, both for detection and for communication, is unique in the animal kingdom.


Effects of lateral masking and spatial precueing on gap-resolution in central and peripheral vision. The data made available by this experiment add two points to the information obtained by previous studies on eccentric vision and lateral masking. First, they demonstrate a clear effect of target-mask similarity on visual gap-resolution in eccentric vision: increasing target-mask similarity systematically decreases resolution performance. Second, spatial precueing has no effect on gap-resolution performance. One implication of this experiment is that with the present stimulus configurations, visual resolution should decrease much more strongly with eccentricity than it does with classical isolated or bar-masked optotypes.


Quantal noise and decision rules in dynamic models of light adaptation. To evaluate some of the consequences of including probabilistic processes (e.g. quantal noise) in a computable model of light-adaptation dynamics, we considered the behavior of a general class of models. These models contain four stages: (1) early noise; (2) a deterministic filtering and gain-changing stage; (3) late noise; (4) a decision rule that is either an ideal (signal-known-exactly) detector or a peak-trough detector. With the ideal detector and without late noise, the observer's sensitivity as a function of mean luminance and temporal frequency is not affected by the filtering and gain-changing stage. Consequently, if the early noise is entirely quantal fluctuations, sensitivity will always be a square-root function of mean luminance and a uniform (flat) function of temporal frequency. This latter prediction is contradicted by all known data; either the ideal-detector is the wrong decision rule or sensitivity is almost always limited by sources of noise other than quantal fluctuations. With the peak-trough detector, however, with or without late noise, the observer's sensitivity as a function of temporal frequency does reflect the sensitivity of the low-level filtering and gain-changing stage. Late noise is needed, however, if the observer's sensitivity as a function of mean luminance is to go through both a square-root and a Weber region. Comparing these conclusions to similar work on the spatial frequency dimension highlights differences between the spatial and temporal frequency domains. Finally, on the basis of these analyses and evidence from the literature, we question whether quantal fluctuations limit visual sensitivity under any condition.


Plan and operation of the NHANES I Epidemiologic Followup Study, 1987. This report describes the plan and operation for the 1987 data collection wave of the Epidemiologic Followup to the first National Health and Nutrition Examination Survey (NHANES I). Tracing and data collection were conducted on 11,750 persons 25-74 years of age at NHANES I who were not known to be deceased in the 1982-84 and 1986 data collection waves of the NHANES I Epidemiologic Followup Study.


Sample design: Third National Health and Nutrition Examination Survey. This report presents a detailed description of the sample design for the Third National Health and Nutrition Examination Survey, 1988-94, including a brief description of research that led to the choice of the final design. The National Health and Nutrition Examination Survey (NHANES) is one of the major surveys of the National Center for Health Statistics, Centers for Disease Control. Information on the health and nutritional status of the noninstitutionalized population of the United States is collected through the NHANES household interviews and standardized physical examinations.


[Changes in hemostasis in persons exposed to a change in atmospheric pressure]. In eight healthy volunteers--four professional and four amateur divers--in a hyperbaric chamber simulated diving was implemented, corresponding to the most frequently encountered conditions of diving activities in Czechoslovakia. Throughout the trial indicators of haemostasis were investigated. Under the selected model conditions changes of the investigated parameters developed which can be considered indicators of a latent process of disseminated coagulation and the so-called syndrome of silent bubbles.


[Air embolism].

[Air embolism]. Air embolism is a dramatic event which threatens the patient's life during invasive therapeutic and diagnostic procedures. Immediately after establishment of the diagnosis treatment in a hyperbaric chamber should be started. The prognosis depends to a certain extent on how quickly the patient is transferred into a hyperbaric chamber. Treatment with hyperbaric oxygen is based on fundamental physical and physiological principles of the action of compressed air and the supply of a large amount of oxygen in a dissolved form into the ischaemic tissue beyond the obstruction. Installation of hyperbaric chambers in hospitals would make successful and prompt treatment of all cases of embolism possible, as well as of some other diseases indicated for hyperbaric oxygen therapy.


[Hyperbaric oxygenation in the treatment of necrotizing fasciitis]. The authors present the results of treatment provided to 11 patients with necrotizing fasciitis who were after a surgical operation and after administration of antibiotics treated in a hyperbaric chamber. A total of 8 patients (82%) recovered completely. The authors discuss the theory of action of hyperbaric oxygen on microorganisms and draw attention to the possible reduction of mortality of this serious disease when using hyperbaric oxygenation. The latter is considered an important auxiliary method which supplements surgical treatment, rational antibiotic therapy and in particular careful intensive care.


[Treatment of ischemic cerebrovascular accidents with hyperbaric oxygenation]. The authors present eleven year's experience with comprehensive treatment of patients who suffered an ischemic cerebrovascular attack. This comprises so-called conventional treatment of cerebrovascular attacks and also hyperbaroxia, i.e. administration of oxygen under high partial pressure. The authors used a therapeutic pressure of 0.2 MPa for 90 minutes in individual exposures. Of 31 treated patients 23 improved, i.e. 76%; eight improved slightly, eight significantly and seven substantially. The condition of the remainder did not change. Hyperbaroxia should therefore be used everywhere where this method is available.


[Estimation of energy utilization in patients based on catabolism of endogenous proteins]. The authors describe their own methodical procedure used for estimation of energy requirements of patients with a defined nutrition. They ascribe importance to the quantitative assessment of the degree of catabolism of endogenous proteins the value of which they transform mathematically to calculate the catabolic factor with which they multiply the basal energy output calculated according to Harris-Benedict equations. At the same time they take into account also the thermogenic action of nutrients. In 28 patients with gastroenterological diseases they confronted their own procedure with indirect calorimetry and theoretical calculations using Jabor's catabolic factor and Nejedly's formula. Differences between values of their own and other theoretical calculations, as compared with the results of indirect calorimetry, nor the results of Jabor's and Nejedly's calculation as compared with those of the authors did not reach the level of statistical significance. With regard to the assembled results the authors consider the suggested procedure for estimation of energy requirements as feasible, in particular when indirect calorimetry is not available.


[Rapid continuous stimulation of the atrium (overdrive) in the treatment of refractory supraventricular tachycardia]. In 71 patients, divided into four groups by the type of supraventricular tachycardia (SVT) during electrophysiological examination, 918 stimulations were implemented by the method of rapid continuous stimulation of the atria (overdrive) in order to interfere with a SVT paroxysm. In addition to characteristics of tachycardia the authors evaluated parameters of overdrive stimulation, i.e. the duration of the stimulation cycle (CL STIM), the ratio CL STIM/CL SVT, the number of stimuli required to terminate tachycardia (N STIM) and their mutual relations. In the group of atrioventricular reciprocal tachycardias (WPW, n = 17) the effectiveness was 50.4%, CL SVT 334 +/- 43 ms, the ratio CL STIM/CL SVT 78.3 +/- 12%, the median of N STIM 14 +/- 6. In the group of AV nodal tachycardias (AVNR, n = 26) the effectiveness is 53.1%, CL SVT 356 +/- 70 ms, CL STIM/CL SVT 77 +/- 8.6%. In the group of atrial tachycardias (AT, n = 5) the effectiveness was 62.3%, CL SVT 348 +/- 24 ms, CL STIM/CL SVT 73.7 +/- 7.5%, N STIM 6 +/- 4. In the group of atrial flutter (AFL, n = 23) the effectiveness was 9.2%, CL SVT 226 +/- 29 ms, CL STIM/CL SVT 84.5 +/- 8.2%, N STIM 22.5 +/- 9. The effectiveness of overdrive stimulation in AFL is significantly lower than in other groups of SVT. The regression correlation in the entire group of tachycardias for calculation of a suitable duration of CL STIM--0.855 x CL SVT--28 (ms), the median number of stimuli N STIM 14 +/- 7.(ABSTRACT TRUNCATED AT 250 WORDS)


[Hypercalciuria].

[Hypercalciuria]. Hypercalciuria is the cause of almost 20% of all secondary osteoporoses and in 23% these cases are associated with calcium urolithiasis. It is therefore necessary to search for these patients actively because their treatment with hydrochlorothiazide and amiloride is easy and highly effective. We must not be satisfied with the finding of hypercalciuria as the only cause of demineralization of bone, as several causes may combine in a single patient. Comprehensive treatment of osteopenia associated with hypercalciuria is relatively shorter and more successful than in other forms of secondary osteopenias.


[Anti-idiotype antibodies to specific HLA. Incidence after bone marrow transplantation. Specificity and binding activity]. Anti-idiotype antibodies against HLA specificities anti-HLA-A1, -A2, -A9, -A10, -B7, -B8, -B12 were detected in sera of 13 patients after transplantation of bone marrow and in five patients who had no transplantation but many blood transfusions. They had a multispecific character, inhibited concurrently 4-5 of the mentioned HLA antibodies. HLA antibodies were not present in sera of these patients and did not develop even after treatment with dithiothreitol. It seems therefore that anti-idiotypic antibodies have also a IgG structure. The bond of anti-idiotype antibodies with HLA specificities was firm and maximal: they were not released after 24 hours incubation at room temperature and did not form bonds with other HLA antibodies with the same specificity. The authors discuss the importance of anti-idiotype antibodies against HLA specificities for the outcome of bone marrow transplantation and therapeutically successful thrombocyte transfusion.