Reverse intrinsic activity of antagonists on G protein-coupled receptors. Biological effects observed with an antagonist are usually interpreted as the result of its ability to block receptor activation produced by an endogenous agonist. In this Principles article, Wolfgang Schütz and Michael Freissmuth show how considerable evidence has now been accumulated for G protein-coupled receptors that antagonists not only bind to the receptor, but also induce a conformational change that favours uncoupling of the receptor from its G protein. The spontaneous activity of the unliganded receptor (i.e. the receptor not occupied by any ligand) is a well-established phenomenon in reconstituted systems with purified components. However, its physiological relevance needs to be verified in a more physiological environment before biological effect of antagonists can be primarily ascribed to negative intrinsic activity.


Neurogenic versus vascular mechanisms of sumatriptan and ergot alkaloids in migraine. Sumatriptan and the ergot alkaloids are useful tools for deciphering drug mechanisms in migraine and related headaches. Both neuronal and vascular mechanisms have been proposed on the basis of actions of 5-HT at receptors resembling the 5-HT1D subtype. In this Viewpoint article, Michael A. Moskowitz argues that blockade of neural transmission and the neurogenic inflammatory response provides a mechanism by which sumatriptan and ergot alkaloids alleviate vascular headaches. He postulates, with similar arguments, that sumatriptan and ergot alkaloids may block headaches that develop from meningovascular inflammatory disorders such as from viral and bacterial meningitis and from the sequelae of head injury.


The genetic basis of malignant hyperthermia. Anaesthesia can induce skeletal muscle rigidity, hypermetabolism and high fever in humans genetically predisposed to malignant hyperthermia. If not immediately reversed, such episodes can lead to tissue damage and death. In swine with the corresponding condition, stress can induce death or lead to devalued meat products. Since muscle contraction is controlled by sarcoplasmic Ca2+, the abnormality, as reviewed here by David H. MacLennan, could reside in the skeletal muscle Ca(2+)-release channel gene, RYR1. Several observations support the view that a single RYR1 mutation is causal of malignant hyperthermia in all breeds of pigs and in at least some human families: the substitution of Cys for Arg615 as the sole deduced amino acid sequence change in a comparison of malignant hyperthermia and normal porcine RYR1 cDNAs; the linkage of this mutation to malignant hyperthermia in over 450 pigs in six breeds, including 338 meioses; and the appearance of the corresponding mutation, Cys for Arg614, across a species barrier, in a few human families, where it also cosegregates with malignant hyperthermia. Linkage of malignant hyperthermia to RYR1 is, however, not observed in all human families with malignant hyperthermia. Accordingly, other abnormal genes that may cause the condition are being sought.


[Hepatocellular adenoma after oral contraception]. Raised serum basic phosphatase was found incidentally in a woman aged 43 years. Investigation with biopsy revealed a hepatocellular adenoma. The tumour regressed after withdrawal of Neogentrol oral contraception which the patient had consumed for 17 years. The patient did not desire invasive treatment. Employment of oral contraceptive steroids for more than two years is associated with increased occurrence of hepatocellular adenomata. The hepatocellular adenoma is a clearly delimited, most frequently solitary, benign tumour with limited malignant potential but with a considerable risk of rupture with haemorrhage even after withdrawal of oral contraception. The hepatocellular adenoma has no malignant tumour vessels (in contrast to hepatocellular carcinoma) and it appears as a cold region on scintigraphy (in contrast to focal nodular hyperplasia), but the diagnosis can only be established with certainty by histological examination. The hepatocellular adenoma consists most frequently of large pale hepatocytes in trabeculae surrounded by a net of reticulin and separated by sinusoids. Biliary passage and portal spaces do not occur. The best treatment consists of excision of the tumour or embolisation. If invasive treatment is postponed, regular scanning should be performed to observe regression or progression of the tumour and oral contraception and pregnancy should be advised against on account of the risk of growth of the tumour.


Na,K-ATPase in crystalline form investigated by scanning force microscopy. Na,K-ATPase has been isolated in purified membrane fragments from kidney tissue and crystallized by phospholipase treatment to obtain two-dimensional, membrane-bound protein crystals. Scanning force microscopy has been used to identify and analyze the topography of the membrane fragments. Specific patterns in accordance with electron microscopic images have been found. In biological material under physiological conditions the scanning force is a crucial parameter for the resulting image at high resolution.


Urinary excretion of inorganic pyrophosphate by normal subjects and patients with renal calculi in north-western India and the effect of diclofenac sodium upon urinary excretion of pyrophosphate in stone formers. 24 h urinary pyrophosphate excretion was studied in 20 normal healthy subjects and 75 idiopathic stone formers from north-western regions of India. The mean 24-hour urinary excretion of pyrophosphate was significantly low in stone formers (50.67 +/- 2.16 mumol/24 h) as compared to that of normal subjects (71.46 +/- 5.46 mumol/24 h) (p less than 0.01). Diclofenac sodium, a non-steroidal anti-inflammatory agent, was administered 50 mg thrice daily for 1 week to 18 stone formers and 24-hour urinary pyrophosphate excretion was studied before and after drug therapy. The 24-hour urinary excretion of pyrophosphate increased from 54.32 +/- 21.40 to 78.31 +/- 28.03 mumol subsequent to diclofenac sodium therapy (p less than 0.01).


Retroperitoneal malignant fibrous histiocytoma. Report of two cases. We present 2 cases of malignant fibrous histiocytoma (MFH) of the retroperitoneum. Only 12-14% of all MFH occur in the retroperitoneum. Both patients were operated with the diagnosis of a kidney tumor. The surgical exposure showed a tumor arising from the retroperitoneum, infiltrating or surrounding the kidney. We would like to emphasize that large tumors of the retroperitoneum which resemble kidney tumors can also arise from mesenchymal tissue.


Hepatocellular carcinoma--image morphology in 40 patients. A retrospective analysis of image morphology in 40 patients with histologically proved hepatocellular carcinoma including two cases of fibrolamellar carcinoma is presented. Ultrasound and CT scan were done in all the patients. Angiogram was done in 15 of these patients to provide a surgical roadmap before hepatic resection. Detailed analysis of clinical features, biochemical parameters and image morphology is presented.


Carcinosarcoma of prostate. Immunohistochemical and ultrastructural observations. Carcinosarcoma of the prostate is a most uncommon disease, and only a few cases have been reported previously. We report the clinical and histopathologic findings in a seventy-three-year-old man with a prostatic carcinosarcoma. The mesenchymal component of the tumor was classified as malignant fibrous histiocytoma (MFH) by electron microscopy and immunohistochemical staining methods. This is to our knowledge the first case in which the stromal component of a prostatic carcinosarcoma has been classified as MFH.


Herpes virus infection of endothelium: new insights into atherosclerosis. Several pieces of evidence suggest that vascular endothelium may be a site of latent herpetic viral infection, and that activation of such infection might cause or aggravate atherosclerosis. The present studies which utilized HSV-1 infection of cultured endothelial monolayers, provide insights into two phenomena seemingly relevant in considerations of atherosclerosis. Thus, mechanisms are reported by which infected endothelium may be damaged by marginated inflammatory cells, and be transformed from an anticoagulant to a procoagulant tissue. First, granulocytes are attracted to, and avidly bind, endothelium infected for very brief periods. This interaction is associated with denudation of intact cells as well as actual cytolysis through release of PMN proteases and toxic oxygen species. Second, several potentially additive abnormalities of HSV-infected endothelium would seem to foster coagulation. These include: a) its loss of surface heparans and thrombomodulin; b) its inability to synthesize prostacyclin with associated incapacity to deter platelet adhesion; c) its disordered membrane lipid conformation which is likely associated with excessive surface thrombin generation; and d) its unique ability to generate and release tissue factor. We speculate that mechanical abrasion may reactivate latent herpes (HSV or CMV) infection in endothelial cells particularly those exposed to high shear forces--for instance, at vessel bifurcations. This may underlie the endothelial damage, clotting and atheroma formation commonly found at these sites.


Outbreaks of egg drop syndrome due to EDS-76 virus in quail (Coturnix coturnix japonica). Two outbreaks of the egg drop syndrome were observed in quail flocks maintained on a farm together with chickens. The decrease in egg production ranged from 10.6 per cent to 50.6 per cent, and the number of soft-shelled eggs increased with the decline in egg production. In both the outbreaks haemagglutination inhibiting antibodies to EDS-76 virus were detected. Fluorescent viral antigen specific to EDS-76 virus was also detected in the lining epithelial and glandular cells of the uterus.


Preliminary studies on congenital hypothyroidism in a family of Abyssinian cats. Congenital hypothyroidism was diagnosed in related Abyssinian cats. The disease appeared to be inherited as an autosomal recessive trait with affected homozygotes showing signs of reduced growth rate, shorter stature with kitten-like features, constipation and goitre. Hypothyroidism was confirmed by demonstrating low basal serum thyroxine levels which failed to increase after intravenous administration of thyroid stimulating hormone or thyrotropic releasing hormone. The radioiodide uptake of the thyroid glands was normal but a high proportion of the accumulated radioiodide was discharged after the administration of sodium perchlorate. It is concluded that the affected cats had a primary dyshormonogenesis: an organification (peroxidase) defect.


The phototoxic effect of erythrosin B on third-stage larvae of gastrointestinal nematodes in sheep. The phototoxic effect of erythrosin B on the infective third-stage larvae (L3) of naturally acquired mixed populations of ovine gastrointestinal nematodes was investigated. This xanthene dye was phototoxic when administered orally to parasitized lambs or applied directly to feces containing nematode ova. Phototoxicity was assessed by the lack of motility (non-swimming) exhibited by the L3 following their collection by Baermannization from cultured feces and exposure to fluorescent light for 360 min. When lambs were administered erythrosin B orally at dosages of O (control), 40, 60 and 80 mg dye kg-1 body weight daily for 10 consecutive days, the percentages of non-swimming L3 were 16%, 46%, 55% and 62%, respectively. However, erythrosin B phototoxicity did not persist after administration of the dye was discontinued and the percentage of non-swimming L3 declined to a level similar to that of the untreated controls within 2 days. The highest percentage of non-swimming L3 was observed when erythrosin B was added directly to feces containing nematode ova. A dose-response curve was evident from the successively higher percentages of non-swimming L3 with increasing concentrations of erythrosin B. Xanthene dyes have the potential to control parasites acquired by livestock on pasture by inducing a phototoxic reaction in the infective L3.


Osteosarcoma with features mimicking malignant fibrous histiocytoma. Three osteosarcomas (OS) with features resembling malignant fibrous histiocytoma (MFH) were selected and investigated to identify any clinico-pathological similarities. In all cases there was no significant difference from conventional OS on the radiography and laboratory data. The appearance of MFH-like features within the whole tumour tissue varied from 7% to 55%. It was composed of spindle-shaped cells arranged in short irregular fascicles and a storiform pattern admixed with osteoclast-like giant cells, but devoid of neoplastic osteoid. Such spindle-shaped cells had a poorly developed rough endoplasmic reticulum and expressed a strong alkaline phosphatase activity as well as vimentin. A series of allografts to athymic mice using the MFH-like tissues also showed histologically a proliferation of plump spindle-shaped cells with a storiform pattern lacking osteoclast-like giant cells, and intensely positive for alkaline phosphatase. These findings indicate that the MFH-like features are identified as modulated OS. The constituting cells are most likely to be poorly developed with possible phenotypic alteration in the maturation stage of osteoblastic cell lineage, but different from conventional MFH of bone as regards their distinct histochemical pattern.


Significance of fibrils in the formation of the Kimmelstiel-Wilson nodule. The pathogenesis of the nodular lesion in diabetic glomerulosclerosis is described in association with fibrils. Thirteen diabetic patients with glomerular nodular lesions and 9 diabetics without the nodules were examined by electron microscopy using periodic acid-thiocarbohydrazide-silver proteinate staining. In cases of nodular glomerulosclerosis, abundant fibrillar structures mixed with electron-dense material were detected within the nodule and the mesangial matrix. They were also occasionally observed along the subendothelial space of the glomerular capillary walls. On the cross-section, these fibrils, including the lucent periphery, were 34 nm wide. Immunohistologically, collagen V and collagen VI were detected in nodular lesions. In contrast, in cases of the diffuse type of glomerulosclerosis, the widened mesangium was composed of dense material, which resembled the original mesangial matrix. The above fibrils were not detected in the mesangium. These findings suggest that the accumulation of the peculiar fibrils in the glomerular mesangium is a major pathogenic factor in the formation of Kimmelstiel-Wilson nodules.


Analysis of tumour necrosis factor alpha-specific, lactose-specific and mistletoe lectin-specific binding sites in human lung carcinomas by labelled ligands. Receptor sites can be visualized by labelled ligands as an alternative to receptor-specific antibodies, as substantiated for two different receptor classes. Recombinant tumour necrosis factor alpha (TNF) was biotinylated via amino-groups and the resultant probe was applied to formalin-fixed, paraffin-embedded tissue sections of 94 primary bronchial carcinomas and to normal peripheral lung parenchyma. In addition, monoclonal antibodies specific for neuron-specific enolase (NSE) and TNF itself were used. The biotinylated beta-galactoside-specific mistletoe lectin, which exhibits dose-dependent immunomodulatory and toxic potency, and two probes that specifically detect certain types of sugar receptors were employed to illustrate further the feasibility of using ligands for receptor localisation. The tumours comprised 62 small cell lung carcinomas, 10 epidermoid carcinomas, 11 adenocarcinomas and 11 large cell anaplastic carcinomas. Expression of TNF-binding sites was found in 39 of the small cell lung carcinomas and in 13 of the non-small cell lung carcinomas. Binding capacity for the TNF-specific antibody was seen in similar proportions of small cell lung carcinomas and of non-small cell lung carcinomas. None of the normal lung parenchymas revealed significant staining. Binding capacities to mistletoe lectin were seen in all normal lung parenchymas and in nearly all cases of adenocarcinoma (10/11). A correlation between the expression of NSE and the binding capacities to TNF was detected. Endogenous lectins, specific for lactose or beta-GalNAc, were displayed in nearly one half of the small cell lung carcinoma cases (44% or 45% respectively) and in about 25% of the non-small cell lung carcinoma cases.


Mechanical properties of suture materials in vitro and after in vivo implantation in horses. Tensile testing of reproducible loops of size 5 braided polyester, size 2 polyglycolic acid, size 2 monofilament nylon, and size 2 polydioxanone sutures was performed in vitro and after subcutaneous implantation in horses for 7, 14, and 28 days. Maximum breaking strength, energy absorption, and percentage elongation to breakage point were measured. Size 5 braided polyester had the highest maximum breaking strength and energy absorption over 28 days. Polydioxanone had better mechanical performance over 28 days than did polyglycolic acid. On day 28, none of these parameters was measurable in polyglycolic acid. Monofilament nylon had the lowest breaking strength of the three materials tested on day 28.


Genomic organization and expression of the 3' end of the canine and feline enteric coronaviruses. The genomic organization at the 3' end of canine coronavirus (CCV) and feline enteric coronavirus (FECV) was determined by sequence analysis and compared to that of feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) of swine. Comparison of the latter two has previously revealed an extra open reading frame (ORF) at the 3' end of the FIPV genome, lacking in TGEV, which is currently designated ORF 6b. Both CCV and FECV possess 6b-related ORFs at the 3' ends of their genomes. The presence of ORF 6b in three of four viruses in this antigenic cluster strongly suggests that TGEV has lost this ORF by deletion. The CCV ORF 6b is collinear with that of FIPV, but the predicted amino acid sequences are only 58% identical. The FECV ORF 6b contains a large deletion compared to that of FIPV, reducing the collinear part to 60%. The sequence homologies were highest between CCV and TGEV on the one hand and between FECV and FIPV on the other. Previously, we showed that the expression product of the FIPV ORF 6b can be detected in infected cells by immunoprecipitation (Vennema et al., 1992). In the present study we have performed similar experiments with CCV and FECV. In infected cells both viruses produced proteins related to but different from the FIPV 6b protein.


Active protection against rotavirus infection of mice following intraperitoneal immunization. Active immunity to rotavirus has been demonstrated following oral inoculation with live virus but little is known about the effects of parenteral immunization. In this study, adult mice were immunized by intraperitoneal (ip) inoculation with live rotaviruses and later orally challenged with murine rotavirus (EDIM) to measure active immunity against infection. Three doses of EDIM (8 micrograms/dose) given intraperitoneally (ip) provided full protection against EDIM infection, whether administered with or without Freund's adjuvant. Only partial protection was found when the quantity of immunogen was reduced to < 2 micrograms/dose. Reduction of the number of doses from three to one (8 micrograms/dose), however, still resulted in protection of all mice. Significant protection was also observed after inoculation with one or three doses (2 micrograms/dose) of heterologous rotaviruses. Protection provided by the heterologous strains did not correlate with neutralizing antibody to EDIM, which indicated that neutralizing antibody to the challenge virus was not required for protection. uv-Inactivated EDIM also provided significant protection against EDIM, thus demonstrating that viral replication was not required for protection. These results suggest that parenteral immunization may be an effective method to vaccinate against rotavirus disease.


Mutations in the envelope protein of Japanese encephalitis virus affect entry into cultured cells and virulence in mice. The nucleotide sequences of the envelope protein of the Kamiyama 1 strain of Japanese encephalitis (JE) virus and a passaged mutant (Kamiyama 2 strain) were determined. Two amino acid differences, Ser-Phe at residue 364 and Asn-Ile at residue 367, distinguished Kamiyama 2 from Kamiyama 1. Six neutralization-resistant variants were selected from these two strains using a JE species-specific monoclonal antibody with neutralization and hemagglutination-inhibition reactivities. All variants had a single amino acid substitution at residue 52 and significantly reduced reactivity with other JE species-specific monoclonal antibodies. The variants derived from Kamiyama 2 strain showed reduced virulence in 3-week-old mice after peripheral inoculation but were as virulent as the parent virus when inoculated intracranially. These variants also showed altered early virus-cell interaction but not replication and reproduction in Vero cells. These findings indicate that the mutations at residues 52, 364, and 367 affect early virus-cell interaction in Vero cells and virulence in mice.


Involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin A. Brefeldin A (BFA) is a macrolide antibiotic that has multiple targets in vesicular transport and blocks membrane traffic between the cis- and trans-Golgi compartments, leading to the disruption of the trans-Golgi apparatus (for a review see Pelham, 1991, Cell 67, 449-451). Consequently, BFA interferes with the maturation of viral glycoproteins and suppresses the formation of infectious viruses that contain a lipid envelope. We report that this antibiotic strongly inhibits poliovirus replication even though this virus lacks a lipid envelope and does not encode any glycoproteins. Addition of BFA from the beginning of poliovirus infection blocks the synthesis of late proteins but has no effect on p220 cleavage, indicating that the input viral RNA is translated to produce active 2Apro. The presence of BFA at later times has no effect on poliovirus protein synthesis, indicating that this step is not a direct target for the antibiotic. Indeed, the target of BFA is viral RNA synthesis, because addition of the antibiotic at any time after poliovirus infection drastically reduces the incorporation of labeled uridine into poliovirus RNA. Both plus- and minus-stranded RNA syntheses are diminished when BFA is present from the beginning of infection, but plus-stranded RNA synthesis is more affected when the inhibitor is added at later times. The replication of poliovirus RNA takes place in close association with membrane vesicles that fill the cytoplasm of the infected cells. Little is known about the origin and function of these vesicles that form part of the viral replication complexes. Our findings suggest that the replication of poliovirus genomes may require the maturation of membranous vesicles from a vesicular compartment that is affected by BFA. The effects of BFA on late protein synthesis by other animal viruses varies according to the virus species examined. Among picornaviruses, rhinoviruses are sensitive to the antibiotic, whereas encephalomyocarditis virus is resistant. A negative-stranded RNA virus such as vesicular stomatitis is blocked by BFA, whereas vaccinia virus, a cytoplasmic DNA virus, is resistant.


Equine herpesvirus 5: comparisons with EHV2 (equine cytomegalovirus), cloning, and mapping of a new equine herpesvirus with a novel genome structure. A new equine herpesvirus, provisionally designated equine herpesvirus 5 (EHV5; Browning and Studdert (1987) J. Gen. Virol. 68, 1441-1447), was examined for the degree of genomic difference from equine herpesvirus 2 (EHV2) by Southern hybridizations. EHV5 and EHV2 whole genomic DNA probes were highly specific for homologous DNA only, indicating that significant genomic difference exists between the two viruses. Restriction endonuclease analysis of EHV5 strain 2-141 (EHV5.2-141) revealed that the genome is 179 kb and exists as a single isomer. Clones representing 82% of the genome were obtained and used to construct restriction maps for four restriction endonucleases. Hybridization experiments indicated that the EHV5.2-141 genome does not contain large terminal or internal repeats, although some evidence for very short repeated sequences in the genomic termini was obtained. Such a genome structure makes EHV5 unique among the equine herpesviruses but similar to the mouse, rat, and guinea pig cytomegaloviruses and the tupaiid herpesvirus. Sequence analysis of one of the genomic termini of EHV5.2-141 revealed the presence of a 30-bp sequence (pac-1; Deiss et al. (1986) J. Virol. 59, 605-618) which is highly conserved among herpesviruses.


Transcription of BamHI-A region of the EBV genome in NPC tissues and B cells. Analysis by Northern blotting and sequencing of cDNA clones from a transcription library of a tumor biopsy from a nasopharyngeal carcinoma (NPC) patient showed that the BamHI-A region of the Epstein-Barr virus genome is abundantly and regularly transcribed in tumor tissues from NPC patients. The transcription occurred in a rightward direction terminating between coordinates 160,965 and 160,995, where two polyadenylation sites are located. Rightward transcription of this region also occurred in B lymphoid cells harboring the viral genome, albeit at a lower level than in the tumor tissues. Differential splicing yields a family of related transcripts displaying at least four splicing patterns. Different promoters may be utilized, further contributing to the diversity of this family of transcripts. A 2.8-kb unspliced transcript present in B95-8 cells was probably initiated from a TATA box located in position 158,204, while the other transcripts may utilize other promoters localized to other regions. All the transcripts encompass a putative open reading frame, BARFO, which is predicted to encode a basic protein of about 20 kDa. It shares 40% colinear amino acid sequence homology with the DNA binding region of a transcription factor, ICP4, specified by herpes simplex virus.


Interaction of nucleic acids with core-like and subcore-like particles of bluetongue virus. Bluetongue virus (BTV) core-like particles (CLPs) were synthesized by coexpression of VP3 and VP7 using a dual recombinant baculovirus. Purified CLPs were shown to bind single-stranded RNA in three different assay systems: gel retardation, nitrocellulose binding, and sucrose gradient sedimentation. CLPs showed equal affinity for BTV-specific and non-BTV RNA and also bound DNA. RNAase protection experiments demonstrated that bound RNA was accessible to immobilized ribonuclease, suggesting that the RNA was predominantly present on the outside of the CLPs. By using individually purified VP7 and VP3 in separate assays, the binding activity was shown to reside on VP3. These results indicate further functional homologies between BTV VP3 and the rotavirus inner-core VP2 protein.


Evolutionary relationships among the gnat-transmitted orbiviruses that cause African horse sickness, bluetongue, and epizootic hemorrhagic disease as evidenced by their capsid protein sequences. The amino acid sequences of four major capsid proteins of African horse sickness virus (serotype 4, AHSV-4) have been compared with those of Bluetongue virus of sheep. Epizootic hemorrhagic disease virus of deer, and the phylogenetic relationships established. Complete nucleotide sequence analysis of three RNA segments (L2, L3, and M6) of AHSV-4 and their encoded products, VP2, VP3, and VP5, together with previously published data for VP7 (Roy et al., 1991), have revealed that of the four capsid proteins the innermost protein, VP3, is the most conserved, and the outermost protein, VP2, is the most variable. Some 57-58% of the aligned BTV-10 and EHDV-1 VP3 amino acids are identical with those of AHSV-4. This compares to an identity of 79% between the BTV and EHDV VP3 sequences. For the VP7 proteins 64% of the aligned amino acids are identical between BTV-10 and EHDV-1, while they share 44-46% amino acid residues with the aligned VP7 protein of AHSV-4. By contrast, the VP2 proteins of the three viruses share only 19-24% identical amino acids. Various other comparative analyses of the proteins indicate that the VP2 species of the three orbiviruses are similar. Unlike VP2, the other outer capsid protein, VP5 is more conserved among the three viruses. On alignment, the VP5 of AHSV-4 has some 43-45% identical amino acids with that of BTV-10 and EHDV-1. Between BTV and EHDV, 62% of the aligned sequences are identical.


Amino acid sequence analysis of bovine rotavirus B223 reveals a unique outer capsid protein VP4 and confirms a third bovine VP4 type. The nucleotide and deduced amino acid sequence of the gene 4 of bovine rotavirus strain B223 is described. The open reading frame is predicted to encode a VP4 of 772 amino acids, shorter than described for any other rotavirus strain sequenced to date. B223 VP4 shows 70 to 73% similarity to other rotavirus VP4 proteins, demonstrating the presence of a unique VP4 type, and confirming a third VP4 allele in the bovine rotavirus population. Multiple sequence alignment with several other rotavirus strains created gaps in the sequence to account for a shorter VP4. The alignment shows a two contiguous amino acid deletions within the trypsin cleavage region of B223 VP4. Comparisons of two regions flanking the trypsin cleavage site, (aa 224 to 235, and aa 257 to 271) which show high homologies between strains, demonstrate that the region 5' to the trypsin cut site has a low homology (66%) to other rotavirus strains, although the region 3' to the trypsin cleavage site shows high homologies (86 to 93%) with other rotavirus strains. The lack of a conserved proline residue within the 5' flanking region suggests a possible altered local conformation of this site in B223 VP4. A second gap inserted into the VP4 of B223 on multiple sequence alignment is a three contiguous amino acid deletion at position 613-615 in the VP5* subunit. Previously defined biologic properties of this strain in relation to the determination of the amino acid composition of VP4 are discussed.


Mutations adjacent to the dimple of the canine parvovirus capsid structure affect sialic acid binding. The erythrocyte receptor on rhesus macaque erythrocytes used by canine parvovirus (CPV) for binding in hemagglutination (HA) was examined. Erythrocyte membrane proteins were electrophoresed and blotted to nitrocellulose and probed with [125I]-labeled CPV capsids, showing seven virus-binding proteins. Treatment of erythrocytes or isolated membranes with Clostridium perfringens neuraminidase virtually abolished virus binding. Binding was also affected by treatment with potassium periodate and inhibited by wheat germ agglutinin, but was not significantly affected by concanavalin A, peanut agglutinin, or soluble N-acetyl-neuraminlactose. A non-HA mutant of CPV failed to bind to erythrocytes or to blotted erythrocyte membrane proteins. The mutation was a single Arg-Lys difference of VP2 amino acid residue 377. The pH dependence of binding of the closely related feline panleukopenia virus was shown to result from a decreased binding in buffers with pH values of 6.8 or greater. The VP2 residues responsible for that difference have been shown to be 323 and 375. The sequences affecting binding were all adjacent to the dimple in the capsid, implicating that region of the capsid as the sialic acid binding site. The role of sialic acid in virus-host cell interactions was not defined, but the plaque sizes of the non-HA mutant and wild type CPV were indistinguishable.


Alternate poliovirus nonstructural protein processing cascades generated by primary sites of 3C proteinase cleavage. The post-translational regulation of picornavirus gene expression mediated by the cascade processing of viral proteins is not well understood. Both pulse-chase studies of infected cells and in vitro studies of the translation of poliovirus type 1 RNA transcribed from genomic cDNA clones indicate a specific cascade of polyprotein processing in which the P1, P2, and P3 precursor proteins are primary products of viral proteinase cleavage. We report the results of a short-time kinetic analysis of poliovirus type 1 protein processing in an in vitro translation system and in infected HeLa cells which indicate the existence of another, rapid pathway of polyprotein processing mediated by the activity of the 3C proteinase. The observed pathway is distinct from and in addition to the one previously known. The potential role of this alternative pathway of processing in the post-translational regulation of viral gene expression is discussed.


Studies on endoplasmic reticulum--Golgi complex cycling pathway in herpes simplex virus-infected and brefeldin A-treated human fibroblast cells. Brefeldin A (BFA), a fungal metabolite, significantly inhibited the release of herpes simplex virus type 1 (HSV-1) from infected human fibroblast cells. Electron micrographs of HSV-1-infected and BFA-treated human cells demonstrated the presence of enveloped particles trapped between outer and inner nuclear membranes. Analyses of viral glycoproteins B, C, and D (gB, gC, and gD) showed faster migrating, immature forms in BFA-treated cells when compared to the mature glycoproteins, as observed in the untreated control cells. The shift in mobilities of the glycoproteins in BFA-treated cells apparently was due to the disassembly of the Golgi complex when evaluated by an indirect immunofluorescence assay. The immature forms of gB, gC, and gD could not be detected on the surface of BFA-treated human fibroblast cells. Removal of BFA resulted in a reorganization of the Golgi complex and formation of fully glycosylated gB, gC, and gD. Moreover, the HSV-1 particles released from the treated cells after the removal of BFA completely restored the infectivity of the viral particles. Our results indicate that human fibroblast cells have an endoplasmic reticulum-Golgi cycling pathway.


The varicella-zoster virus immediate early protein, IE62, can positively regulate its cognate promoter. Varicella-Zoster virus (VZV) is a neurotropic alphaherpes virus closely related to herpes simplex virus (HSV). However, unlike its close relative HSV, VZV lacks a functional alpha-TIF (alpha-gene transinducing factor) that activates the transcription of immediate early genes during the initial events of the virus life cycle. Hence, in the absence of a functional alpha-TIF, the mechanism triggering the expression of immediate early genes in VZV at present remains unclear. Accumulating evidence indicates that the gene product of the putative immediate early gene ORF62 (IE62) plays a pivotal role in activating VZV genes of all three putative kinetic classes, namely immediate early (alpha), early (beta), and late (gamma) classes of VZV genes. In the present study, we show that IE62 can positively autoregulate its cognate promoter using a transient transfection assay, both in lymphocytes and in neural cells. In the same system, we can also demonstrate activation of the VZV IE62 promoter by HSV ICP4. By deletion analysis and oligonucleotide-directed site-specific mutagenesis we have localized specific regions in the IE62 promoter/upstream sequences that mediate inducibility by IE62 and HSV ICP4, and provide evidence that this promoter activation by these two proteins may be through different mechanisms. These data, taken together with the recent demonstration of the presence of IE62 in the VZ virion tegument (Kinchington, P.R., Hoagland, J.K., Arvin, A.M., Ruyechan, W.T., and Hay, J. 1992. J. Virol. 66, 359-366) provides a possible mechanism by which the triggering of VZV gene expression occurs in the absence of a functional alpha-TIF protein.


Characterization of the murine cytomegalovirus genes encoding the major DNA binding protein and the ICP18.5 homolog. In several herpesviruses the genes for the major DNA binding protein (MDBP), a putative assembly protein, the glycoprotein B (gB), and the viral DNA polymerase (pol) collocate. In murine cytomegalovirus (MCMV), two members of this gene block, pol (Elliott, Clark, Jaquish, and Spector, 1991, Virology 185, 169-186) and gB (Rapp, Messerle, Bühler, Tannheimer, Keil, and Koszinowski, 1992, J. Virol., 66, 4399-4406) have been characterized. Here the two other MCMV genes are characterized, the gene encoding the MDBP and the ICP18.5 homolog encoding a putative assembly protein. Like in human cytomegalovirus (HCMV) the genes order is pol, gB, ICP18.5, and MDBP. The 4.2-kb MDBP mRNA is expressed first in the early phase, whereas the 3.0-kb ICP18.5 mRNA is a late transcript. The open reading frame of the MDBP gene has the capacity of encoding a protein of 1191 amino acids with a predicted molecular mass of 131.7 kDa. The MCMV ICP18.5 ORF is translated into a polypeptide of 798 amino acids with a calculated molecular mass of 89.1 kDa. Comparison of the amino acid sequences of the predicted proteins of MCMV with the respective proteins of HCMV, Epstein-Barr virus (EBV), and herpes simplex virus type-1 (HSV-1) reveals a striking homology ranging from 72% (HCMV), 50% (EBV), to 45% (HSV-1) for the MDBP sequence and from 74% (HCMV), 51% (EBV), to 49% (HSV-1) for the ICP18.5 sequence. These results establish the close relationship of the two cytomegaloviruses, and underline the usefulness of the murine model for studies on the biology of the CMV infection.


Rearrangement of viral sequences in cytopathogenic pestiviruses. Two cytopathogenic isolates of bovine viral diarrhea virus (cpBVDV) have been analyzed. For both viruses two regions of their genomic RNAs were found to be duplicated and rearranged. The viral genomes contain a small duplicated element (SD) derived from the genomic 5' end far downstream of its original context. This sequence is followed by a larger duplication which encompasses the region coding for the protein p80(LD), a molecular marker for cpBVDV. The SD element codes for the viral protease p20. In the case of the viruses analyzed here the aminoterminus of p80 is generated by autoproteolytic removal of the preceding SD-encoded protease. For one of the cpBVDV isolates a specific fusion protein (p28) could be identified which is composed of p20 and part of p10, another viral protein. Molecular characterization of the respective noncytopathogenic counterpart revealed that duplication and rearrangement of sequences as well as the expression of p28 and p80 are specific for the cytopathogenic virus.


Human cytomegalovirus penetrates host cells by pH-independent fusion at the cell surface. Biochemical, genetic, and morphological criteria were used to demonstrate that human cytomegalovirus penetrates permissive fibroblasts and nonpermissive Chinese hamster ovary (CHO) cells by pH-independent fusion between the virus envelope and the host cell plasma membrane and not by low pH-induced fusion within endosomes. Viral immediate early (IE) gene expression and infectivity were unaffected by conditions which block various stages of endocytosis or agents that alter the acidic pH of the endosome. IE gene expression was also evident in a mutant CHO cell line which is defective in endosomal acidification. Morphological analysis of the entry process at the electron microscopic level revealed viral particles in various stages of virion-plasma membrane fusion. In contrast, intact enveloped virions were not observed sequestered within coated pits or vesicular structures. Collectively, the data indicate that the entry pathway by which HCMV gains access to the cytoplasm of fibroblasts and CHO cells in order to initiate infection is via pH independent, virion envelope-plasma membrane fusion.


p26 protein and 33-nm particle associated with nucleocapsid of hepatitis C virus recovered from the circulation of infected hosts. Hepatitis C virus (HCV) has not yet been cultured or visualized. We attempted to recover HCV-associated particles from plasma of infected humans to assess the natural properties of the virus. Starting with 720 ml of donor plasma containing high titer of HCV core antigen ELISA activities, we identified HCV core antigen activity and viral RNA enriched in a potassium bromide density gradient fraction with a density of 1.115 g/ml. Icosahedron-shaped particles with an average diameter of 33 nm were liberated by treatment of the fraction with the detergent Tween 80. These particles were selectively visualized with an electron microscope using a grid coated with a murine monoclonal antibody directed to HCV core peptide and were also observed in aggregated forms with an immune electron microscope (IEM) with use of the anti-core antibody. An ultracentrifugation pellet of the above fraction was treated with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2ME) and run in SDS-PAGE. A protein that bound antibodies directed to the predicted core protein of HCV was found at a molecular size estimated as about 26,000 Da, significantly greater than the 191 amino acid residues predicted from the presumed core gene of HCV. It is possible that translation initiation and/or the COOH-terminal cleavage site for HCV core protein in vivo may differ from estimates derived from the amino acid sequence of the polyprotein precursor. The nucleocapsid could also be chemically altered in the infected cell, resulting in a gel mobility different from the native protein.


An amino-terminal fragment of SV40 T antigen transforms REF52 cells. An SV40 mutant, T147D, encodes only the amino-terminal 147 amino acids of large T antigen and does not make small t antigen. We show here that a retrovirus which expresses this mutant T antigen transforms rat REF52 cells as efficiently as a retrovirus that expresses both the wild-type large and small T antigens. This cell line had previously been refractory to transformation by mutants that make short, amino-terminal fragments of T antigen.


Expression of Epstein-Barr virus membrane antigen gp350/220 in E. coli and in insect cells. The Epstein-Barr virus open reading frame BLLF1 encodes the major envelope glycoproteins gp350 and gp220. Fragments of the gp350/220 gene were expressed in Escherichia coli in order to define regions of the polypeptide chain reacting with human sera. The C-terminal half of the protein was sufficient for recognition by all VCA-positive sera tested. A membrane anchor truncated version of gp350/220 was expressed in insect cells using the baculovirus system. Proteins of different sizes were specifically detected in the cells while a glycosylated 220-kDa protein was secreted. The insect cells were tested for their suitability as tools for performing monospecific immunofluorescence.


The restriction endonuclease map of Marek's disease virus (MDV) serotype 2 and collinear relationship among three serotypes of MDV. A BamHI, EcoRI, and XhoI restriction endonuclease map of Marek's disease virus (MDV) serotype 2 (MDV2) DNA was constructed by double-digest analyses of 28 cloned BamHI and 11 cloned EcoRI fragments of MDV2 DNA, followed by hybridization tests of these cloned BamHI DNA fragments with electrophoretically separated digests of MDV2-infected cell DNA. On this map, MDV2 genome consisted of two segments which have unique regions inserted between two inverted repeat regions as observed in MDV serotype 1 and 3 genomes. Further, the DNA homology among three serotypes of MDV was examined by hybridization under less stringent conditions using cloned BamHI fragments of MDV2 DNA. Most of the MDV2 fragments located within the unique regions hybridized with MDV serotype 1 and 3 DNAs, indicating the presence of the collinear relationship among three serotypes. In addition, MDV2 DNA fragments which hybridized with the DNA fragments encoding MDV1 gp57-65 (or A antigen) or MDV1 gp100, gp60, gp49 (or B antigen) were identified and these fragments of serotypes 1 and 2 found to be collinear.


Adenovirus containing a deletion of the early region 2A gene allows growth of adeno-associated virus with decreased efficiency. Efficient growth of adeno-associated virus (AAV) requires helper functions provided by a coinfecting adenovirus or herpesvirus. Earlier studies using adenoviruses having temperature-sensitive lesions in the early region 2A gene (E2A) produced contradictory evidence regarding the role of the E2A 72-kDa DNA-binding protein (DBP) in allowing efficient AAV growth. These disparate results may reflect varying levels of residual function in the temperature-sensitive DBP. We examined this issue using an adenovirus type 5 mutant (Add/802) that fails to produce any detectable DBP or any fragment of it. Our experiments show that AAV can carry out a full growth cycle in the complete absence of DBP. However, AAV DNA replication and rep and capsid protein synthesis were reduced several fold and the yield of infectious AAV was reduced by an order of magnitude. This appears to reflect mainly decreased post-transcriptional expression of AAV rep and capsid protein genes.


Major histocompatibility (B) complex control of the growth pattern of v-src DNA-induced primary tumors. Observations that the major histocompatibility (B) complex is a determinant of the growth pattern of Rous sarcoma virus (RSV)-induced tumors raised the question as to whether control is exerted at the level of a v-src-determined, i.e., transformation-specific, function. To investigate this point, the tumor size scores and tumor profile indices of v-src-induced tumors were compared in two lines of chickens congenic for B complex genotypes. The finding that the growth patterns of tumors, induced by v-src DNA inoculation at 6 weeks posthatch, differ in these two lines establishes that the B complex exerts control over tumor growth at the level of a v-src-determined function. The potential importance of this control, in terms of the naturally occurring case of an avian sarcoma virus infection, is suggested by the observation that the patterns of tumor growth in a given congenic line are similar whether the tumors are induced by v-src DNA or by RSV.


Terminal sequence conservation among the genomic segments of a group B rotavirus (IDIR strain). Terminal nucleic acid sequences were determined for all 11 segments of the IDIR strain of group B rotavirus. Consensus sequences were defined at both ends of the (+) RNA strands as 5' GGN(A/U)NA(A/U)(A/U)(A/U)---and---(A/U)NA(A/G)N(A/C)(C/A)CC3 '. The 5' and 3' terminal sequences of the (+) strand IDIR RNA were not complementary to one another. The IDIR terminal sequences and those of group A rotaviruses (GAR) were similar in that each of the (+) strands began with "GG" and ended with "CC." Otherwise, the IDIR terminal sequences did not match the consensus sequences that have been reported for the ends of the GAR genomic segments.


Characterization of human herpesvirus-6(U1102) and (GS) gp112 and identification of the Z29-specified homolog. Monoclonal antibody 2D10 (MAb 2D10) raised toward human herpesvirus-6(U1102) [HHV6(U1102)] immunoprecipitated three glycosylated peptides, M(r) 112,000, 64,000, and 58,000, designated as gp112 from U1102-infected lymphocytes. Pulse-chase experiments suggest that the M(r) 64,000 and 58,000 polypeptides are very likely generated by post-translational cleavage of the M(r) 112,000 polypeptide. MAb 2D10 neutralized virion infectivity in the presence of complement, suggesting that gp112 is located in the virion envelope. MAb 2D10 did not prevent the appearance of HHV6-specific cytopathic effect. MAb 2D10 was reactive with denatured gp112 in immunoblots. HHV6 isolates form two clusters (Schimer, Wyatt, Yamanishi, Rodriguez, and Frenkel, Proc. Natl. Acad. Sci. USA 88, 5922; Ablashi, Balachandran, Josephs, Hung, Krtueger, Kramarsky, Salahuddin, and Gallo, Virology 184, 545). MAb 2D10 reacted by immunofluorescence and immunoprecipitation with the prototypes of each cluster, GS and Z29. Whereas the proteins immunoprecipitated by MAb 2D10 from GS-infected lymphocytes had an electrophoretic pattern very similar to that of U1102 gp112, the homologous glycoprotein immunoprecipitated from Z29-infected lymphocytes consisted of three polypeptides with M(r) 102,000, 59,000, and 50,000. The data suggest a variation among HHV6 isolates as far as this glycoprotein is concerned.


Homology of the envelope glycoprotein B of human herpesvirus-6 and cytomegalovirus. The envelope glycoprotein B (gB) coding sequences of two strains of human herpesvirus-6 (HHV6 GS and Z29) were determined by sequencing a 2.5-kb open reading frame adjacent to the DNA polymerase sequence. The deduced primary translation product is 830 amino acids in length and is 96% conserved between the two divergent strains with no localized hypervariability noted. It contains the expected signal and transmembrane sequence motifs as well as a putative site of protease cleavage. There was 39% amino acid identity with the gB of human cytomegalovirus (CMV, strain AD169). HHV6-CMV gB peptide homology was evident through the entire sequence, but was especially strong in the amino-terminal portion of CMV gp55, which contains linear and conformational epitopes recognized by CMV-neutralizing antibodies. All 10 cysteine residues of HHV6 gB match corresponding residues of CMV gB. Sequence data suggest strong structural similarity and possible immunologic cross-reactivity of gB from the two viruses.


Molecular cloning of natural paramyxovirus copy-back defective interfering RNAs and their expression from DNA. Using the unique sequence organization of copy-back defective interfering (DI) RNAs of paramyxoviruses, Sendai virus (SV), and measles virus copy-back DI RNAs were PCR amplified and cloned, without having to separate them from their helper nondefective genomes. The cloning was designed so that T7 polymerase transcription of the plasmids would generate DI RNAs with the exact 5' and 3' ends. The SV DI clone, transcribed from the plasmid in BHK cells using T7 polymerase produced by a vaccinia virus recombinant, was encapsidated and replicated by the SV-L, P/C, and NP proteins expressed from cloned genes. Such experiments open the possibility of examining the cis-acting sequences involved in viral multiplication directly, without using indirect markers such as CAT activity.


Evidence of human polyomavirus BK and JC infection in normal brain tissue. Infection with the polyomaviruses JC and BK is ubiquitous in the human population and JCV is the only virus associated with the central nervous system disease progressive multifocal leukoencephalopathy. In the attempt to analyze the pathogenesis of polyomavirus infections we asked whether human polyomaviruses invade the brain during persistence. Brain autopsy material from 67 individuals with disorders other than PML was examined for the presence of polyomavirus DNA. Southern blot analysis demonstrated JCV-specific full-length virus genomes in healthy brain tissue in about 20% of the patients. Type-specific analysis with polymerase chain reaction and sequencing confirmed these data. Additionally, the presence of BKV DNA sequences covering an early gene fragment and the control region with flanking early and late protein coding sequences was detected. Cloning of the complete BKV genome from two cases supported the assumption that not only full-length JCV DNA was present in those tissue specimens but also BKV genomes. The data obtained demonstrate that dual infection of the brain with the polyomaviruses JCV and BKV is a common event and give strong evidence that both viruses frequently establish a latent CNS infection.


Serine protein kinase associated with varicella-zoster virus ORF 47. Varicella-zoster virus (VZV) ORF 47 lies in the unique long region of the VZV genome. Sequence homology studies have demonstrated that gene 47 possessed conserved protein kinase motifs. In this study, we investigated the properties of the ORF 47 product. First, a rabbit antiserum was raised against a protein generated from the fusion of the most antigenic ORF 47 domain with Escherichia coli beta-galactosidase. The high-titer antiserum reacted specifically with ORF 47 polypeptides translated in vitro. When incubated with VZV-infected cell lysate, the antiserum immunoprecipitated a phosphoprotein of M(r) 54,000, a size comparable with the predicted molecular mass. The precipitated viral protein was phosphorylated in a protein kinase assay; subsequent phosphoamino acid analysis indicated that the phosphotransferase associated with the ORF 47 protein was a serine protein kinase. Synthesis of the ORF 47 product in VZV-infected cell culture increased in the first and second days and plateaued after the third day of infection. The protein kinase activity associated with VZV ORF 47 had several distinctive biochemical properties: (i) its phosphotransferase activity was enhanced more by manganese than by magnesium, (ii) it utilized both ATP and GTP as donors of phosphate, and (iii) it phosphorylated both acidic and basic substrates. In summary, this report lends support to the computer homology data which predicted that VZV ORF 47 would encode a serine protein kinase.


Genetic mechanisms of antigenic variation in infectious bursal disease virus: analysis of a naturally occurring variant virus. The major immunogenic protein VP2 from a pathogenic field isolate (variant A virus) of infectious bursal disease virus (IBDV) was cloned and sequenced to examine antigenic variations. The VP2 open reading frame consists of 1509 nucleotides and codes for a 503 amino acid protein. Overall, the VP2 amino acid sequence of the variant A virus shares 98.6% identity with VP2 genes from other published IBDV strains. However, within the central region of VP2 (amino acids 222-334) lies a highly divergent area that we have termed the variable domain. Relative to five other IBDV isolates, a total of six amino acid changes occur within the variable domain of the variant A virus. At positions 284-288, a substitution of isoleucine to threonine, a decrease in the number of Chou and Fasman beta turns, and a switch from a hydrophilic to a hydrophobic region are found only in the variant A virus. Together these changes predict a decrease in antigenicity as determined by calculation of potential antigenic sites. This suggests that only minor changes within VP2 contributed to the emergence of a variant virus that can cause disease in immunized birds.


Quantitative assessment of poxvirus promoters in fowlpox and vaccinia virus recombinants. A comparison was undertaken of poxvirus promoters in vaccinia and fowlpox virus (FPV) recombinants using the level of beta-galactosidase expressed from the LacZ gene as a measure of promoter function. In this study a comparison was made of the vaccinia virus promoters, P 7.5 and P L11, the major late promoter of cowpox virus, P CPX (expressing the abundant inclusion body protein), and the FPV promoters, P E/L and P L. In vaccinia virus recombinants the FPV P E/L promoter expressed one-third to one-half the level of beta-galactosidase expressed by the P L11 promoter. In comparison with the P 7.5 promoter, the FPV P E/L promoter expressed four to five times the level of beta-galactosidase. In FPV recombinants beta-galactosidase activity expressed was equal for the P E/L and P CPX promoters. Levels expressed by P L11 and P L were one-half and one-fifth that level, respectively. The temporal regulation of the promoters was maintained in both vaccinia virus and FPV recombinants. The P E/L promoter of FPV has the TAAATG sequence characteristic of late poxvirus promoters at the transcription initiation site. In an attempt to enhance the utility of this promoter for the expression of foreign genes in FPV and vaccinia virus recombinants, the effect upon promoter function of changing the G of the ATG to A, T, or C was determined using transient expression assays with vaccinia virus. Substitution of A, T, or C for the G abolished promoter function. Because of its early/late function, the level of expression and the presence of the oppositely oriented late P L promoter, the FPV P E/L promoter will be valuable for the expression of foreign genes in poxvirus recombinants.


A HSV-1 variant (1720) generates four equimolar isomers despite a 9200-bp deletion from TRL and sequences between 9200 np and 97,000 np in inverted orientation being covalently bound to sequences 94,000-126,372 np. The genome structure of a spontaneously generated HSV-1 strain 17 variant, 1720, has been determined by restriction endonuclease and Southern blot analysis. The short segment of 1720 is unaltered compared to the parental strain 17 genome, whereas the long segment is extensively rearranged. Almost all of TRL (approximately 9.2 kb) has been deleted and consequently IRL is converted into unique sequence. Sequences from approximately 9200 nucleotide position (np) to 97,000 np are present in inverted orientation, covalently bound to sequences in the prototype orientation from approximately 94,000 np to the L/S junction at 126,372 np. Thus, sequences from 94,000 np to 97,000 np are now diploid, with one copy in the normal orientation and location, and the other at the long terminus as an inverted repeat; no inversion of the intervening unique sequences occurs about this novel inverted repeat. In contrast, normal inversions of the long and short segments occur to give four equimolar genomic isomers, indicating that the novel long terminus has gained an "a" sequence. The duplication of sequences between 94,000 np and 97,000 np results in a genome containing two copies of UL43 and one complete and one partial copy each of genes UL42 and UL44 encoding the 65 kD DNA-binding protein and glycoprotein C, respectively. The variant has been shown to grow normally in vitro following high multiplicity infection.


Overexpression and purification of enzymatically active recombinant integrase protein of Rous sarcoma virus. The carboxy-terminal domain of polymerase gene of Rous sarcoma virus was cloned into an expression vector under the control of lac regulatory elements, resulting in the plasmid pMF1413. Upon isopropyl-beta-D-thiogalactopyranoside induction, viral integration (IN) protein was expressed in large quantity in Escherichia coli. The expressed recombinant protein was prepurified by successive washing of the bacterial pellet with 0.1 M NaCl and detergents. Further purification was performed in high yield by standard chromatography methods. The purified enzyme revealed selective DNA cleaving activity on supercoiled plasmid with the LTR-LTR junction fragment. The reaction was metal ion dependent, with a preference for Mn2+ over Mg2+, and showed substrate specificity at 1 mM MnCl2.


Pharmacological study of the chicken's monocular optokinetic nystagmus: effects of GABAergic agonist and antagonists. When injected into the chicken open eye, the GABA-agonist THIP and the GABA-antagonists bicuculline and picrotoxin induced spontaneous eye movements in nasal-temporal (N-T) and in temporal-nasal (T-N) direction, respectively. These spontaneous movements were scarcely modulated by optokinetic stimulation, irrespective of the direction of stimulation. It is suggested that they are due to the suppression of directional selectivity of retinal ganglion cells. When injected into the closed eye, GABAergic drugs did not produce spontaneous nystagmus. THIP provoked a reduction of the N-T component, without modifying the T-N one, while GABA antagonists induced a significant increase in OKN performance, especially for the N-T direction of stimulation. In these conditions, picrotoxin also provoked an increase in the duration of both components of optokinetic after nystagmus, indicating a direct effect of the drug upon the velocity-storage system.


[The pathogenesis of central nervous system functional disordersafter exposure to microwave radiation]. A rate of lipid peroxidation, content of biogenic amines and cyclic nucleotides were studied in various brain structures of 168 rats under conditions of microwave irradiation within 24 min at 46 mW/cm2. Total irradiation of animals was shown to result in distinct inhibition of monoaminergic activity of brain, especially of hypothalamus, in impairment of metabolic reactions, in exhaustion of the lipid antioxidative system of brain cortex and in stimulation of the contrainsular apparatus functions.


[Detection of a steady increase in endonuclease activity in dog liver cell nuclei after long term arterial hypotension and in post-resuscitation period]. Presence of Ca2+, Mg(2+)-dependent endonuclease activity was found in dog liver cell nuclei. Specific characteristics of chromatin autolysis were also studied in liver cell nuclei after 2-4 hrs long arterial hypotension as compared with that within the later restoration period 1-3 months. The rate of DNA acid-soluble fraction accumulation correlated directly with the arterial hypotension duration. Quantitative evaluation of the liver tissue Ca2+, Mg(2+)-dependent endonuclease activity was undertaken under conditions of hemorrhagic shock. The enzymatic activity was not normalized both after death and in the postresuscitation period. Analysis of the chromatin autolysis and of alterations in the enzymatic activity during postresuscitation period enabled to suggest that the isozyme spectrum of Ca2+, Mg(2+)-dependent endonuclease was altered.


[Cyclic adenosine monophosphate and myocardial lipids in fluothane anesthesia depending on the oxygen content in the inhalation mixture]. Content of cyclic adenosine monophosphate (cAMP), which involved in regulation of heart metabolism, was altered in myocardium depending on oxygen concentration in the inhalation mixture. Increase of oxygen concentration during gas anesthesia stimulated lipolysis and elevated cAMP content, while these parameters were lowered after decrease of oxygen content in the mixture.


[Formation of cell membrane signal ATP by activated neutrophils and macrophages: connection with superoxide production and formyl peptide receptor status]. Formation of plasmatic membrane signal ATR in target cells containing growth peptide factors have been found in various mammalian tissues including neutrophils activated by formyl-peptide. Maximal development of the plasmatic membrane ATP was observed within 3 days in neutrophils of rats with septicopyemia. Production of superoxide anion in neutrophils of these rats was the highest also within 3 days and constituted 3.7 nmol/mg of protein as compared with 1.4 nmol/mg in controls; within the subsequent periods dynamics of the superoxide anion content correlated with the rate of plasmatic membrane signal ATP synthesis. The state of receptors to formyl-peptide was altered as follows: content of their binding sites in neutrophils was distinctly decreased at the beginning of septicopyemia and continued to decrease within later periods but the receptors affinity was not altered. Thus, production of superoxide anion was not related directly to amount of receptors to formyl-peptide, while it correlated with formation of the plasmatic membrane signal ATP. The phenomenon of the signal ATP synthesis in neutrophils occurred mainly at concentrations of formyl-peptide activating phagocytosis. Other immunocompetent cells-macrophages produced also the plasmatic membrane signal ATP, 28.1 nmol/mg of protein, after stimulation with formyl-peptide and their ATP-formating activity was the highest among the target cells studied. This suggests that plasmatic membrane signal ATP is involved in membrane transduction of a signal to activation of neutrophils and macrophages.


Virus inactivation during intravenous immunoglobulin production. Effects of time, temperature, pH and stabilizers (i.e. medium) on inactivation of lipid-enveloped model viruses, Semliki Forest and vesicular stomatitis viruses in the production process of intravenous immunoglobulin were investigated on a laboratory scale. The lowering of pH, the raising of temperature and the increasing of incubation time improved the inactivation effect. However, small changes in pH and stabilizer concentrations did not influence the results. Inactivation was not linear and a clear tailing off could be seen. Therefore, for complete virus inactivation incubation times longer than 20 h are necessary. Inactivation took place much more rapidly in intravenous immunoglobulin solution than in intramuscular immunoglobulin solution. Processing steps such as freeze-dying in the presence of ethanol or storage of intramuscular immunoglobulin in the liquid state at pH7 only partially inactivated these viruses.


[The combined treatment of patients with cerebral venous dystonia]. The authors proposed and tried a method of complex treatment cerebral venous dystonias (hypertensive type) including along with traditional drug treatment also intranasal administration of novodrin and electrostimulation of the mastoid zone of the scalp. Of 36 patients with clinical disorders of the venous cerebral circulation (hypertensive type evaluated rheoencephalographically) after closed head injuries 24 patients showed a recovery or significant improvement, 10--an improvement and 2--remained unchanged.


[The intracellular second messenger system and its regulatory effects in children with a bronchial asthma attack complicated by heart failure]. Components of the intracellular mediators system: calmodulin in leukocytes, Ca2+ and cyclic nucleotides in leukocytes and blood plasma were studied in children with attack of bronchial asthma and heart failure. Alterations in content of these biologically active substances correlated with clinical manifestations of bronchial asthma: severity of the disease, duration of the attack, contractile activity of myocardium. Calmodulin, Ca2+ and cyclic nucleotides were demonstrated to be involved in development of the asthmatic attack. Alterations in the system calmodulin-Ca2+ were related to adaptation and contributed to realization of regulating effects responsible for a decrease of impairments in tissues.


[A comparative study of the physico-chemical properties of lysine oxidase from Trichoderma sp. and Trichoderma viride Y 244-2]. Some physicochemical properties of L-lysine oxidase from two strains of Trichoderma were studied. Content of metals and cofactors (Se, Zn, Cu, Fe, Co, Mn, Mo), amino acid analysis, secondary structure were estimated. The enzyme molecule from Trichoderma sp was found to contain both FAD and PQQ cofactors.


[Dynamic features of rapid changes of blood phosphoinositides in patients with chronic alcoholism]. The time-course of rapid changes in blood phosphoinositide content was studied in 48 patients with Stage II chronic alcoholism of and in healthy volunteers. Content of phosphoinositides was estimated within short intervals 30, 60, 120 and 180 sec after the samples were obtained. In the blood of the patients with alcoholism as compared with healthy volunteers the time-course of rapid changes in phosphoinositide content exhibited a higher amplitude and failed to return to the initial level within 3 min. Alterations in content of the phospholipid correlated with duration of alcohol abuse. The data obtained suggest that homeostasis of main biochemical regulators is impaired after alcohol consumption.


[Method of producing fusarin C in perlite-liquid culture medium]. For researching the biosynthesis labelled Fusarin C(Fc) by Fuscarium moniliforme, a more quick and convenient method of Fusarin C production and purification were established, and a good liquid culture medium consisted of different kinds organic matters (hydroxy proline, sucrose and glycerin), inorganic salts and perlite replaced corn grit medium. The perlite-liquid culture medium inoculated with the strain of F. moniliforme yields 936mg Fc/kg organic matter with in 14 days of incubation at 28 degrees C. As compared with the corn grit medium, the amount of Fc from perlite-liquid medium was more than that from corn grit medium (831mg Fc/kg corn grit). In all experiments both thin-layer chromatography and high-pressure liquid chromatography were used to confirm the presence of Fc. parameters which were important for the optimal biosynthesis of Fc included hydroxy proline and sucrose concentrations, incubated time/temperature and amount of perlite. The 40g of sucrose/L liquid culture was optimal concentration for Fusarin C production. Of three contained N-matter tested, hydroxy proline was the best sources of N-atom for Fusarin C. Under the absence of hydroxy proline, the Fc wasn't synthesized in perlite-liquid culture medium by F. moniliforme. A culture time/temperature study of Fc production was done, and the optimal Fc amounts was synthesized after incubation for 14 days at 28 degrees C on perlite-liquid culture medium.


[Transposition of Tn917 in Bacillus pumilus]. Transposition Tn917 was introduced into Bacillus pumilus 289 by protoplast transformation with plasmid pTV32. The temperature-sensitive replication property of pTV32 was maintained in B. pumilus. Tn917 was transposed efficiently in B. pumilus with 4.8 x 10(-4) transposition rate. The yield of auxotrophs was about 0.65% in all insertional mutants. It indicated a prospects for the use of Tn917 as a tool for insertional mutagenesis and genetic manipulation in B. pumilus.


[The biochemical indices of the blood serum in experimental hepatitis A in tamarins]. Serum biochemical parameters were studied in 42 healthy wild-caught adult tamarins (S. mystax), males and females, to determine the normal values. Blood samples were drawn repeatedly, and the serum was tested for aspartate aminotransferase, alanine aminotransferase, isocitric dehydrogenase, serum glucose, serum urea, triglyceride, cholesterol, albumin, and total protein. The results indicated that serum chemistry values were similar to those reported as normal for both humans and other Callitrichidae species. The study of serum biochemical parameters in tamarins with experimental hepatitis A indicated that serum enzyme activities alone reflected the hepatic damage, while other biochemical parameters were of no real clinical importance. The experimental results showed the levels of serum urea to be indicative of the pathological involvement of the kidneys in experimental hepatitis A in some cases.


[Dyslipidemia and an elevated lipid level in the cells in experimental herpetic infection and their correction with antiviral chemotherapeutic agents]. The influence of herpes simplex virus on lipid exchange and accumulation by blood vessels cells was studied. In acute herpes infection in rabbits, typical dyslipidemia characterised by a rise in the content of total cholesterol, low and very low density lipoprotein cholesterol and triglycerides in the absence of manifest changes in concentration of high density lipoprotein was detected. HSV infection of smooth muscle cell culture of human embryo aorta was accompanied by increased accumulation of free lipids in the cells. The use of antiherpetic preparations during the infection led to correction of the lipid spectrum of the infected animals and was accompanied by normalization of intracellular lipid contents. A possible pathogenetic role of HSV in atherogenesis which may be connected with at least two processes: the development of lipidemic disturbances and formation of pathologic lipid depot in the arterial wall, is discussed.


Hep G2 cell line as a human model for sulphate conjugation of drugs. 1. The objective of this study was to examine the usefulness of the hepatoma cell line Hep G2 as a model for human sulphoconjugation of drugs, in particular stereoselective conjugation. 2. Using the substrates p-nitrophenol and dopamine, we found sulphation activities consistent with the presence of both the phenol (P) and the monoamine (M) form of the human phenolsulphotransferases in these cells. 3. The Kmapp was 3.0 microM for the sulphation of p-nitrophenol. This activity was inhibited selectively by 2,6-dichloro-4-nitrophenol, IC50 6 microM. The Kmapp was 39 microM for the sulphation of dopamine. This activity was selectively inhibited by elevated temperature. 4. The chiral adrenergic drugs (+/-)-terbutaline and (+/-)-4-hydroxypropranolol were both sulphated stereoselectively with Kmapp and Vmaxapp values for each enantiomer virtually identical to previous observations with human liver cytosol. 5. In a direct comparison, the estimated activity of the P form of phenolsulphotransferase in the Hep G2 cell line was 30% of that in human liver, whereas, surprisingly, the activity of the M form of phenolsulphotransferase was 4.5 times higher in the Hep G2 cells than in the liver.


[Incomplete thrombosis of the inferior vena cava, a cause of chronic recurrent lung embolism. Diagnostic possibilities and case report]. Detection of complete inferior vena cava thrombosis is relatively simple because of the clinical symptoms and the pathological results of cw Doppler sonography in the region of the common femoral vein. Drainage volume detectable by means of venous occlusion plethysmography is considerably reduced during the acute phase, but normalizes with increasing degree of collateralization. In case of incomplete inferior vena cava thrombosis clinical signs, cw. Doppler sonography and venous occlusion plethysmography are not very relevant. Therefore, imaging technique is additionally required. Basing on a case report, the article demonstrates that ultrasound-B-method and echocardiography, or preferable endoechocardiography, may contribute to a more reliable diagnosis. Cavography is still the gold standard if invasive therapy is planned.


Liver transplantation for acute or chronic liver failure by hepatitis non-A, non-B and hepatitis C viral infections: a postoperative follow-up. Orthotopic liver transplantation (OLT) was performed for liver failure related to hepatitis non-A, non-B (HNANB) or hepatitis C (HCV) infections in 12 patients. Of those, 8 patients had chronic and 4 acute hepatic failure. To determine the incidence of recurrent infection, the clinical course, histological findings and serological HCV markers (HCV-RNA and detection of anti HCV antibodies, respectively) were comparatively studied in these patients. Recurrent infection was apparent in 5 of 6 patients transplanted for liver cirrhosis attributable to chronic HCV infection and with HCV-RNA detectable in serum. The clinical course of infection after OLT varied considerably. Chronic active hepatitis, progressing to liver cirrhosis 13 months postoperatively and an acute hepatitis, resolving spontaneously were seen in one case each. Recurrent infection led to chronic persistent hepatitis in the remainder. None of the patients with acute liver failure experienced recurrent infection. HCV-RNA was detectable in all the patients after OLT, with HCV-RNA present pretransplant, however the presence of HCV-RNA in serum was not necessarily associated with clinical illness.


Parathyroid adenomectomy under local anesthesia with intra-operative monitoring of UcAMP and/or 1-84 PTH. Because 80% of patients with primary hyperparathyroidism have a single adenoma and because most adenomas are now visualized by ultrasonography, we have attempted to remove these suspected single adenomas under local anesthesia with intra-operative monitoring of urinary cAMP (UcAMP) and 1-84 parathyroid hormone (PTH) serum levels. In the last 2 years, 45 patients (mean age 65 years) with primary hyperparathyroidism underwent surgery with local anesthesia when a single adenoma was strongly suspected by ultrasonography. Patients with equivocal or misleading ultrasonography, e.g., those with associated thyroid or multiglandular pathology and those who were non-cooperative, were excluded from this procedure. UcAMP and 1-84 PTH were determined prior to the incision, at the time of removal of the adenoma, and at regular intervals until 120 minutes after the operation. Results were available 45 min to 60 min after sampling for PTH and 60 min to 80 min for UcAMP. Forty-two adenomas were removed through a 2 cm to 3 cm skin incision in a mean time of 25 minutes, with no adverse effect, no morbidity, and minimal discomfort. The 42 patients were normocalcaemic on follow-up. The monitorings always predicted the success of the operation. In the 3 remaining patients, because the monitorings remained elevated at the end of the procedure, the patients underwent classical bilateral neck dissection under general anesthesia. This new approach can be safely accomplished with short operative time and hospital stay. The absence of general anesthesia is reassuring for the patients who are reluctant to undergo general anesthesia.(ABSTRACT TRUNCATED AT 250 WORDS)


The DNA-binding protein P52 of human cytomegalovirus reacts with monoclonal antibody CCH2 and associates with the nuclear membrane at late times after infection. Monoclonal antibody CCH2 is commonly used for the detection of human cytomegalovirus (HCMV) infected cells in tissue sections as well as in cultured cells. The specificity of CCH2 was determined by screening a recombinant lambda-gt11 cDNA gene bank from HCMV-infected fibroblasts. By sequencing a reactive clone, the antigen was identified to be the non-structural DNA binding protein p52 of HCMV (UL44 reading frame). The viral insert from the lambda clone was recloned in bacterial expression vectors. For this, a new vector, pRos-RS, was constructed. The resulting clones were tested in immunoblot analyses. They were reactive with CCH2 as well as with reconvalescent sera positive for antibodies against HCMV, by this proving the specificity of CCH2. Using this monoclonal antibody in confocal microscopy, the subcellular localization of p52 in infected cells was analyzed. In these analyses, p52 was found to be nuclear and to be associated with the nuclear membrane at late times after infection.


Genotype-specific in vitro amplification of sequences of the wild type 3 polioviruses from Mexico and Guatemala. The extensive nucleotide sequence heterogeneity among independent genotypes of wild polioviruses permits the systematic design of genotype-specific molecular reagents. We have prepared two sets of polymerase chain reaction (PCR) primer pairs specific for the genotype of wild poliovirus type 3 recently endemic to Mexico and Guatemala. Nucleotide sequences of a representative wild type 3 virus isolated in Mexico in 1989 differed from the corresponding Sabin 3 (Leon 12 a1b) sequences at 167 of 900 positions within the VP1 region. From the sequence data, wild virus-specific primer pairs were designed to complement regions of high mismatch (greater than 33%) with Sabin 3 templates. Primer binding sites were spaced along the genome so that the predicted amplification products (142 bp and 163 bp) could be easily resolved electrophoretically from the products generated with our Sabin strain-specific primers (Sabin 1: 97 bp; Sabin 2: 71 bp; Sabin 3: 53 bp). RNAs of all wild type 3 poliovirus isolates from Mexico and Guatemala obtained over a 13-year period (1977-1990) served as efficient templates for amplification of the 142-bp and 163-bp products. Genomic templates derived from vaccine-related polioviruses and most heterologous wild polioviruses were inactive under equivalent reaction conditions. Amplifications generating a 114-bp product with a broadly reacting primer pair, matching highly conserved sequences in the 5'-noncoding region, provided a positive control for the presence in samples of poliovirus (or enterovirus) RNAs. Selective amplification of wild Mexico-Guatemala type 3 poliovirus sequences was obtained with either primer set in reactions containing large stoichiometric excesses (up to 10(6)-fold) of vaccine-related RNAs. We have used wild genotype-specific PCR primer sets to facilitate identification of wild polioviruses present in both clinical and environmental samples.


Comparative sequence analyses of the cognate structural protein VP6 genes of five US bluetongue viruses. The S3 segment (the small segment 3), encoding the structural protein, VP6, from the five United States (US) prototype bluetongue virus (BTV) serotypes were amplified by the Clamp-R method and cloned as full-length entities. The complete nucleotide sequence of each cognate gene segment was determined. Each cognate S3 segment of BTV-10, 11, 13 and 17 was 1049 nucleotides long and contained an open reading frame (ORF) capable of encoding a 325-amino acid protein. However, the S3 segment of BTV-2, which also contained 1049 nucleotides, had a longer 5'-non-coding region of 99-nucleotide and contained an ORF capable only of encoding a 301-amino acid protein. Comparative analyses of the predicted amino acid sequences of S3 segments of BTV-2, 10, 11, 13 and 17 revealed that VP6 was unusually high in glycine and contained few aromatic amino acids, but a high concentration of charged amino acids, which is a characteristic of a hydrophilic protein. Phylogenetic analyses indicated that BTV-11, 13 and 17 were more closely related than the other two US BTV serotypes. BTV-2 was the most distantly related.


Identification and expression of the UL1 gene product of equine herpesvirus 1. Sequences encoding the UL1 gene of equine herpesvirus type 1 (EHV-1) are conserved in the genome of EHV-1 defective interfering particles (DIPs) that mediate oncogenic transformation and persistent infection. The UL1 protein was identified by in vitro transcription/translation and hybrid-arrest translation analyses which employed a UL1/pGEM-3Z construct designated pGEML1. SDS-PAGE analyses of in vitro translation products synthesized from UL1-specific RNA revealed that the UL1 ORF encodes a 30 kDa protein which corresponds in size to the 258 amino acid protein predicted by DNA sequence analyses. This result was confirmed by arresting translation of the in vitro transcribed UL1 RNA with an oligodeoxynucleotide complementary to UL1 coding sequences. The UL1 protein is a homolog of the predicted protein encoded by the ORF2 gene of varicella-zoster virus, but UL1 has no homolog in herpes simplex virus type 1. The UL1 protein contains a region conforming to a 'PEST' (Proline, Glutamic acid, Serine, and Threonine) sequence, which is commonly found in proteins with half-lives of less than two hours.


Sequence and analysis of the BamHI "D" fragment of Shope fibroma virus: comparison with similar regions of related poxviruses. Differences observed in the virulence of two related leporipoxviruses are closely tied to a particular region of their genomes. For the virulent poxvirus of this pair, malignant rabbit fibroma virus (MV), this region is the BamHI "C" fragment, which is 10.7 kb. For the avirulent poxvirus, Shope fibroma virus, SFV, this region is the corresponding BamHI "D" fragment, which is 13.1 kb. As part of our attempt to understand the virulence of these two viruses, we sequenced these two DNA fragments. The sequence for the BamHI "C" fragment of MV is reported elsewhere (Strayer et al., 1991). We report here the sequence for SFV's BamHI "D" fragment and resultant open reading frames, and compare both DNA and open reading frame structures to those of MV and other known poxviruses. The BamHI "D" fragment of SFV contains 12 open reading frames of 100 amino acids or more, arranged similarly to orf's in MV and vaccinia. Striking similarities between SFV and MV are seen in certain parts of this restriction fragment, including substantial stretches of DNA in which the two viruses are identical. Clear homologies exist between these leporipox virus genomes and those of other related poxviruses. To understand the pathogenesis of virus infection, one must appreciate the structure of those viral genes that play important roles in infection.


[Future developmental perspectives in rehabilitation and after care of the elderly]. It has now become imperative to organize a differentiated system for geriatric rehabilitation in the Federal Republic of Germany. However, the increasing predominance of diagnostic and therapeutic principles suggests that the holistic vision once associated with "geriatrics" may become of secondary importance as a result of the transfer of treatment strategies from the intensive care sector. The complexity of the everyday person-environment interfaces of chronically ill elderly persons in need of nursing services stands in contrast to the way the success of rehabilitation is still understood in terms of clinical criteria. With respect to future developments, ideas for geriatric rehabilitation and, in particular, follow-up care are being developed that are defined in terms of criteria that have to be newly developed--wide-ranging rather than narrowly conceived criteria--related to rehabilitation objectives that also have to be redefined, namely quality of life, effectiveness and a broad promotion of health. An understanding of geriatric rehabilitation practice defined in this way relating to everyday situations can be expected to yield a high degree of quality assurance in view of the assumed criteria of broadly defined success.


[Importance of reference registries in bone tumor diagnosis. Experiences with the Swiss Bone Tumor Register]. Bone tumors are extremely rare. It is for this reason that in numerous places reference registries have been set up, actually cover the whole area which within the German-speaking countries. They are intended to enable more precise diagnoses on the base of their larger numbers of cases. The bone tumor registry by the Swiss Society of Pathology at the Department of Pathology of the University of Basel has been started in 1972. Today it includes about 6500 numbers of patients. Of the 4500 numbers concerning tumors of the skeleton excluding the cranium, 3000 have been sent as reference cases from Switzerland and from abroad. The importance of this kind of registry is demonstrated by the fact that in 1100 of these cases the diagnosis had to be changed by the registry, 106 times from "malignant" to "benign", and 124 times from "benign" to "malignant". The complex of problems is displayed by examples from several groups of tumors. In conclusion, the importance of a close collaboration between clinician, radiologist, and pathologist for putting up a correct diagnosis is stressed because it is the crucial condition for an optimal therapy, in particular for malignant tumors.


A point mutation in the thymidine kinase gene is responsible for acyclovir-resistance in herpes simplex virus type 2 sequential isolates. A number of HSV-2 isolates, sequentially recovered from ulcerative ano-genital lesions of an AIDS patient during a prolonged treatment with acyclovir (ACV), have been studied at the molecular level. All of them were highly resistant to ACV (ACV-r) and shown to be virtually deficient in thymidine kinase (TK) activity. The ACV-r phenotype was demonstrated to be due to the production of truncated TK polypeptide. Structural alteration of this gene, as shown in one isolate, was caused by a chain-terminating mutation that originated from a cytidine deletion at position 520 of the TK open reading frame. This mutation generated a TGA stop codon 27 nucleotides downstream. An additional isolate was also recovered following ACV discontinuation and after a cycle of treatment with foscarnet. This isolate had lost the ACV-r trait and was characterized by a wild type TK sequence and by the production of a functional enzyme. Data presented confirm that a prolonged treatment with acyclovir can easily select ACV-r HSV-2 isolates carrying a TK- phenotype caused by a frameshift mutation. Although recovered from lesions tributary of different myelomers, these isolates may belong to the same strain that has undergone multiple cycles of reactivation and has possibly mutated during its axonal route to the skin.


Differential expression of HPV types 6 and 11 in condylomas and cervical preneoplastic lesions. Tumor biopsies from exophytic and flat condylomas at different locations on the genital epithelium (10 cases) and in cervical intraepithelial neoplasia (CIN) grades 1-2 (6 cases) were analysed for HPV types 6 and 11 DNA and RNA. The presence of mRNA species which could encode the E6, E7, E1M, E2, E2C, E4, E5 and L1 proteins was determined using the RNA polymerase chain reaction (PCR) technique with primers that flank previously mapped or predicted splice sites. The state of the viral DNA in the tumor biopsies was established by Southern blot analysis. We could detect the various mRNA species in biopsies from condylomas associated with both HPV types. The size of the RNA PCR products were in agreement with the previously mapped splice sites of mRNAs recovered from an experimental condyloma induced by HPV11. The major viral transcript encoding the E1i--E4 protein was detected in all the tumor biopsies. From the rare transcripts the rate of detection of mRNA species encoding the E1M, E2C proteins was the highest. In 2 of 6 CIN biopsies analysed only the major viral transcript was detected. The overall results of this study suggest that early gene products of HPV types 6 and 11 may be important in the induction of cellular proliferation and condylamatous differentiation but these possibly may not be required for the development of the HPV-associated cervical neoplasia.


The soluble form of two N-terminal domains of the poliovirus receptor is sufficient for blocking viral infection. By means of deleting a C-terminal portion of the open reading frame of the poliovirus receptor cDNA, and by vaccinia virus-mediated overexpression we have produced a protein corresponding to the first two N-terminal Ig-like domains of the poliovirus receptor. This protein that lacked the third Ig-like domain, the transmembrane region and most of the intracellular C-terminal tail was detected in the medium of vaccinia virus infected cells. The properties of the truncated PVR cDNA were further characterized by in vitro translation and modification. The molecular weight of the unmodified protein was found to be 27 kDa; translation in the presence of dog pancreas microsomes led to an increase in molecular weights which we attribute to N-glycosylation. Upon incubation with poliovirus at 37 degrees C, the vaccinia-virus generated protein specifically reduced infectivity of poliovirus. Sucrose gradients of poliovirus particles derived after incubation with the protein showed the induction of a slower sedimenting particle (135S). Our experiments suggest that the two N-terminal domains of the poliovirus receptor in soluble form are sufficient for the conversion of poliovirus into a non-infectious particle.


The complete nucleotide sequence of the JS strain of human parainfluenza virus type 3: comparison with the Wash/47885/57 prototype strain. The nucleotide sequence of the JS strain of human parainfluenza virus type 3 (PIV3) was determined from a series of 14 overlapping cDNA clones and was compared to that of the previously sequenced prototype PIV3 strain, Wash/47885/57 (Galinski, 1991). Overall, there were 630 (4%) nucleotide differences between the two viruses. 15462 nucleotides comprised the JS genome in contrast to 15463 which constituted the genome of the prototype virus. This was accounted for by a single nucleotide deletion in the 5' non-coding region of the JS phosphoprotein gene. Four nucleotide substitutions were found in the leader region at the 3' end of the viral genome at positions 24, 28, 42 and 45, whereas no differences were found in the 44 base trailer region. All of the transcription start and stop signals and intergenic sequences were conserved between the two viruses with the exception of the transcription stop signal of the matrix (M) gene where there was a nucleotide transposition between bases 7 and 8. A comparison of all of the nucleotide differences in the 3' and 5' non-coding regions of each gene showed a variability of 9.8% and 10.5%, respectively. The 3' non-coding regions of the nucleocapsid (NP) and M genes were completely conserved in contrast to the polymerase (L) gene in which 25% of the nucleotides were different. Differences were observed in the 5' non-coding regions of each gene and ranged from 5.9% for the hemagglutinin neuraminidase (HN) gene to 14.6% for the M gene. An analysis of the amino acid differences in each open reading frame revealed that of all the genes, the coding region of the M gene was the most highly conserved (1.1% amino acid variability), while the phosphoprotein (P) gene was the most variable (5.8% amino acid variability). As these two viruses are wild type strains, these differences in nucleotide and amino acid sequence are compatible with efficient replication in vivo.


[The structural organization and neurochemical mechanisms of the participation of the nucleus accumbens in the interaction of the limbic and motor systems and in the regulation of motor behavior]. The review of own and literature data devoted to structural and neurochemical organization of the nucleus accumbens (Acc) as well as spatial organization of their projection fibers in comparison with nucleus caudatus (neostriatum) has been presented. The facts concerned with correlations revealed between both the cell clusters and histochemical compartments as well as the compartmental organization of afferent and efferent striatal connections were analyzed. The presented data propose the existence of sensorimotor and limbic parts of the dorsal and ventral striatum, which are involved in the parallelly functioning systems. The common and different signs of these two systems and its role in the regulation of the movement behaviour has been proposed. A lot of attention also was payed to the Acc and the neostriatum interaction. The many pathways by which Acc can influence neostriatum functions and therefore the motor activity controlled by the neostriatum are discussed. It was shown that the Acc can influence on the striatal synaptic DA release. The sign of this influence depended upon DA/glutamic acid interactions in the Acc. It was stressed that the influence of Acc on striatal DA-ergic system is very likely mediated via kainate/quisqualate (but not NMDA) inputs of the neostriatum. The balance of DA-ergic mechanisms of neostriatum and Acc as important basis of animals adequate behaviour in conditioning situation was proposed. The disbalance of these mechanisms could leads to pathology.


[The dynamic and regional characteristics of the high-resolution EEG structure in voluntary activity]. For revealing the dynamics of interrelations between EEG components having stable tuning to individually determined frequencies a dynamical high resolution EEG structure analysis was used. There were three successive experimental stages: rest, motor reproduction of time interval and tapping. Significant increase of EEG components intercorrelations for the right frontal area, minimal number of significant relationships between EEG and movement temporal characteristics, and the occurrence of hemispheric asymmetry in the number of EEG-movement correlations were observed for time reproduction task. This task specifics and the possibilities of used approach for precise functional states differentiation are discussed.


[The conditioned-reflex mechanism of the development of tolerance for and dependence on narcotic agents]. At the beginning the term "tolerance" determinations, given in different source of literature, have been considered. Further, the history of conditioned of tolerance and abuse creation from psycho-active drugs have been discussed. The hart of the article contains the critical points of the current representation of neurophysiological theory in the field of behavior, interaction mechanisms between live systems and drugs. The point of view, dominating in pharmacology science that tolerance is the direct result of drug substances intervention into the organism, has been opposed. Separation of primary and secondary physiological effects of drugs, allowed to the authors to conclude that the dominant role belongs to the state living system and to the presence of necessities during the motivation creations for the second drug use and to the tolerance changing.


[The behavioral effects of the destruction of the nucleus basalis magnocellularis in the cat forebrain]. Behavioural experiments were carried out on cats by the method modelling complicatedly organized non-automatized behaviour with elements of generalization and abstraction. By the results of fulfillment of test tasks by animals with partial destruction of the macrocellular basal nucleus the conclusion is made on the participation of this formation in the structural-functional ensuring of complex integrative forms of activity-thinking and learning. The suggested mechanism of drawing of the basal nucleus in gnostic and cognitive processes in the non-specific ensuring of the system of structures directly participated in thinking and learning.


[Maternal behavior after the administration of morphine or naloxone to pregnant female rats and the pain sensitivity and brain mu-opioid receptors in the progeny]. Morphine or naloxone injected twice a day (10 mg/kg/day) to rat females from 15 to 18 days of gestation had no effect on their litter size or body weight of pups. Time necessary for the female to bring pups into the nest from the opposite end of the cage, that is a characteristic of maternal care and negatively correlated with the mean body weight of the pup in the litter, did not change after treatment with drugs during gestation. Newborns treated with mu-opioid receptor ligands during intrauterine development had an elevated number of 3H-naloxone binding sites in the brain. However, the number of 3H-naloxone binding sites on the 9 and 16 days of life, as well as pain thresholds under electric stimulation of the tail at a month age were equal in these rats and offsprings of the intact or saline treated mothers.


[A factor analysis of behavioral organization in mice]. Laboratory mice with Robertsonian translocation Rb(8,17)1Iem, mice of CBA substrain with the same translocation as well as CBA normal karyotype mice were investigated in several behavioural tests, their results being analysed by means of factor analysis. The genetic differences were revealed in the patterns of factor loadings, in particular in factors "feeding behaviour structure" and "weight loss". The behaviour of mice during logic problem solving (the ability to extrapolate the direction of food stimulus movement was tested) proved to be genotype-dependent in respect to connections with exploratory behaviour indices. Tendencies towards stereotypic or variable (plastic) behaviour patterns were revealed in CBA mice and mice with Rb(8,17)1Iem mice respectively.