[Intracranial venous sinus thrombosis in nephrotic syndrome]. A 3-year old child was admitted for a third relapse of nephrotic syndrome associated with intracranial hypertension related to dural sinus thrombosis (tomodensitometry). The treatment consisted in the association of low dose heparin and fresh frozen plasma. After a 3 year-follow-up, there was no neurologic sequelae, and the nephrotic syndrome was on complete remission. The radiologic features and the management of sinus thrombosis are discussed.


[Abdominal aortic aneurysm and Bourneville's tuberous sclerosis]. The authors report on a case of tuberous sclerosis diagnosed in the neonatal period on the basis of intracardiac tumor, rib anomalies and cerebral calcifications. At 4.5 months of age the infant presented acute abdominal pains which led to the discovery (ultrasound identification confirmed by CT scan) of a giant ectasia of the whole abdominal aorta. The infant died 2 days later from the rupture of this aortic aneurysm.


[Multifocal tuberculosis with cerebellar tuberculoma]. A 10-year old child was admitted for vomiting and a high grade fever. He had been previously immunized twice against tuberculosis. A cerebellar syndrome appeared, and the brain CT scan showed an intracerebellar mass. Disseminated lesions were detected on abdominal (splenic abscess) and thoracic (mediastinal lymph nodes) CT scans. The bone scintigraphy also showed multiple localisations. A craniotomy was performed that revealed a cerebellar tuberculoma which was completely removed. The child received an antituberculous treatment over 18 months and is currently well with a 5 year follow-up.


[Hematuric cystitis after bone marrow allograft: a case]. A 16-year-old boy experienced a severe macroscopic hemorrhagic cystitis 20 days after a partially mismatched allogeneic marrow transplantation for an acute lymphoblastic leukemia. The conditioning included cyclophosphamide and total body irradiation. After a conservative management, the hematuria spontaneously resolved after 3 months of gross hematuria. The authors review the literature, and stress the predisposing factors and clinical outcome of such complication. They advocate a conservative "watch-and-wait" approach including intensive bladder irrigation.


[Non traumatic acute suppurative mediastinitis]. Acute mediastinitis is uncommon. When it occurs, it usually follows an esophageal perforation or thoracic surgery. We report on a case of a 2-year-old girl with non traumatic mediastinitis secondary to a pharyngitis, due to Staphylococcus aureus. The anatomic pathways that connect the cervical region with the mediastinum explains how the infection can spread within the cervicothoracic spaces. Knowledge of this anatomy is also important in evaluating the possible causes of symptoms and signs in this area, and the possible complications of infections in these regions. The treatment associates antibiotics and drainage of the abscess by either surgical procedure or computed tomography guided selective needle aspirations.


[Blackfan-Diamond disease and malignancy: cause effect relationships?]. The authors report on a 24-year old patient with Blackfan-Diamond syndrome who developed a Hodgkin's disease. This patient became transfusion-dependent at the age of 10, after an initial period of corticosensitivity, and after failure of androgens. He developed hemochromatosis despite from parenteral chelation therapy. He died of infectious complications 4 months after the diagnosis of Hodgkin's lymphoma. A review of the literature shows an increased incidence of malignancies in Blackfan-Diamond syndrome (three cases of leukemia), and in similar disease (thalassemia and sickle cell disease), but not in other patients with hemosiderosis (primitive hemochromatosis, end-stage renal failure under dialysis). Etiopathogenic hypotheses are discussed.


[Primary cerebromeningeal hemorrhages in children (except newborn infants)]. The authors report their experience on 22 children admitted for spontaneous subarachnoid hemorrhages, to the pediatric and neurosurgery units of the University-Hospital of Saint-Etienne. There were seven cases of subarachnoid hemorrhages (five ruptured arterial aneurysms and one angioma) and 15 cases with associated intracranial hematomas (eight angiomas, one cavernoma, one aneurysm). Seven of 15 children with hematoma died, compared to two of seven children with subarachnoid hemorrhage. Half of the patients with hematomas and 20% of those with subarachnoid hemorrhage had sequellae. This rare pathology should be diagnosed in emergency, since brain damage occurs secondary to raised intracranial pressure. The respective roles of tomodensitometry, lumbar puncture and arteriography are discussed. The surgery should be performed in emergency, because the most frequent etiology is a vascular malformation, and because there is a risk of unexpected deterioration. When an underlying cause cannot be found neither by arteriography not by surgery, the follow-up should include a tomodensitometry and/or magnetic imaging.


[Iron deficiency in infants and children]. A study of the incidence of iron deficiency and its risk factors was carried out in 90 hospitalized or outpatient children and infants, 4 to 48 months old. Iron deficiency (serum iron concentration less than 10 mumol/l) was found in 70% of them and microcytosis (mean corpuscular volume less than 70 fl) in 10%. A good correlation was observed between hemoglobin concentration or mean corpuscular volume, and serum iron or ferritin concentrations. When risk factors were studied, 20% of the children were found to have insufficient meat or vegetable intakes. Only 45% of 6 month-old and 20% of 1 year-old infants received iron fortified milk formulas. However no significant correlation was found between the nutritional factors and the hematological data. Anemia and microcytosis were significantly more frequent in children born from immigrant parents as compared with native children, but there was no difference between these two groups for isolated iron deficiency. These results indicate that there is a need for a better prevention of iron deficiency in French infants and young children; a larger use of iron fortified milk formula until the age of 12 months is to be recommended.


[Congenital tubulopathy with magnesium loss]. The authors report on a 1-year old girl who presented with transient hypotonia and polydipsia related to renal-concentrating defect. Renal magnesium and calcium wasting were noted when the subject was 3.5 years old, in association with distal tubular acidosis and nephrocalcinosis. Hypocalcemia and hypomagnesiemia persisted when the patient was 9.5 years old. About 50 cases of tubular defects with renal magnesium loss have been reported in the literature and show that magnesium loss may be either isolated or associated with potassium and/or calcium wasting. This hereditary defect may be due to an alteration in magnesium reabsorption in the thick ascending limb of the loop of Henle.


[Comparative study of 24-hour calciuria and urinary calcium/creatinine ratio in children over 4 years of age]. The relationship between 24 hour urinary calcium excretion (U Ca/24 h) and urinary calcium/creatinine ratio (U Ca/creat) measured for morning and evening urine samples was studied in 56 children aged 4-15 years and hospitalized for benign conditions. Depending on the length of hospitalisation, 1 to 3 determinations of U Ca/24 h and U Ca/creat ratio were carried out for each child. Mean +/- SD U Ca/24 h was 0.05 +/- 0.058 mmol/kg. Mean U Ca/Cr, expressed in mmol/mmol, was 0.368 for total 24 h urine, 0.358 for the morning sample and 0.358 for the evening sample respectively. A good correlation was found between U Ca/24 h and 24 h U Ca/Cr ratio (r = 0.89), morning U Ca/creat (r = 0.83) and evening U Ca/creat ratio (r = 0.81) respectively. It is concluded that determination of the U Ca/Cr ratio for morning or evening urine samples is an efficient means of detecting hypercalciuria.


[Intervention in PMI after mothers in difficulties with their young children: a psychomotrician in the waiting-room]. The authors report on a 3-year experience of interventions by a psychomotrician in the waiting-room of a mother-child protection (PMI) centre in order to recognize and prevent mother-infant interaction difficulties. The main mission of the psychomotrician was to help mothers to discover the competences and the desire of communication of their infants. This experience appears to be positive, allowing the improvement of some mother-infant difficulties, and a better acceptance by the mothers of a child psychiatric consultation when it appears to be necessary.


[Development of the operative and non-operative treatment of acute intestinal intussusception in children]. A review of 152 cases of acute intestinal intussusception shows a dramatic evolution in its management within the last 15 years. Ultrasound is the main procedure utilized, and in the hands of a trained-radiologist, it allows a safe non-surgical treatment via barium or pneumatic reduction. Surgery is now limited to the rare cases of failure of the above treatment, and to advanced or complicated cases.


[Endoscopic extraction of a foreign body from the distal bronchus in the middle lobe, inaccessible by usual techniques, in a 3-year old child]. The authors report on the extraction of a piece of peanut impacted into the antero-internal bronchus of the right middle lobe, inaccesible to rigid bronchoscopy. After 4 months, this foreign body was visualised by flexible endoscope and removed by this technique under general anesthesia. We initially used a brush to pull the foreign body out of the distal bronchus and then used a biopsy forceps to remove it. Three months later, the chest-X-ray is normal and the child is asymptomatic.


Probing the conformations of eight cloned DNA dodecamers; CGCGAATTCGCG, CGCGTTAACGCG, CGCGTATACGCG, CGCGATATCGCG, CGCAAATTTGCG, CGCTTTAAAGCG, CGCGGATCCGCG and CGCGGTACCGCG. The self complementary DNA dodecamers d(CGCGAATTCGCG), d(CGCGTTAACGCG), d(CGCGTATACGCG), d(CGCGATATCGCG), d(CGCAAATTTGCG), d(CGCTTTAAAGCG), d(CGCGGATCCGCG) and d(CGCGGTACCGCG) have been cloned into the Smal site of plasmid pUC19. Radiolabelled polylinker fragments containing these inserts have been digested with nucleases and chemical agents, probing the structure of the central AT base pairs. The sequences AATT and AAATTT are relatively resistant to digestion by DNase I, micrococcal nuclease and hydroxyl radicals, consistent with the suggestion that they possess a narrow minor groove. Nuclease digestion of TTAA is much more even, and comparable to that at mixed sequence DNA. TpA steps in ATAT, TATA and GTAC are cut less well by DNAse I than in TTAA. DNasel cleavage of surrounding bases, especially CpG is strongly influenced by the nature of the central sequence.


Modular organization of related Archaeal plasmids encoding different restriction-modification systems in Methanobacterium thermoformicicum. Nucleotide sequence comparison of the related 13513-bp plasmid pFV1 and the 11014-bp plasmid pFZ1 from the thermophilic archaeon Methanobacterium thermoformicicum THF and Z-245, respectively, revealed a homologous, approximately 8.2 kb backbone structure that is interrupted by plasmid-specific elements. Various highly conserved palindromic structures and an ORF that could code for a NTP-binding protein were identified within the backbone structure and may be involved in plasmid maintenance and replication. Each plasmid contains at comparable locations a module which specifies components of different restriction-modification (R/M) systems. The R/M module of pFV1 contained, in addition to the genes of the GGCC-recognizing R/M system MthTI, an ORF which may be involved in repair of G-T mismatches generated by deamination of m5C at high temperatures.


Efficient large-scale sequencing of the Escherichia coli genome: implementation of a transposon- and PCR-based strategy for the analysis of ordered lambda phage clones. We have developed a strategy for efficient sequence analysis of the genome of E. coli K-12 using insertions of a Tn5-derived mini-transposon into overlapping ordered lambda phage clones to provide universal primer-binding sites, and PCR amplification of DNA segments adjacent to the insertions. Transposon-containing clones were selected by blue plaque formation on a dnaBamber lacZamber E. coli strain. Insertion points every 0.5-1 kb were identified by 'analytical PCR' and segments between the transposon inserts and phage arms were amplified by 'preparative PCR' using one biotinylated and one non-biotinylated primer. Single strands of amplified DNA fragments were coupled to Streptoavidin-coated paramagnetic beads (Dynabeads M280) through their biotin tails, purified magnetically, and used as templates for fluorescence-based automatic nucleotide sequencing.


Fidelity of replication of the leading and the lagging DNA strands opposite N-methyl-N-nitrosourea-induced DNA damage in human cells. Semi-conservative replication of double-stranded DNA in eukaryotic cells is an asymmetric process involving leading and lagging strand synthesis and different DNA polymerases. We report a study to analyze the effect of these asymmetries when the replication machinery encounters alkylation-induced DNA adducts. The model system is an EBV-derived shuttle vector which replicates in synchrony with the host human cells and carries as marker gene the bacterial gpt gene. A preferential distribution of N-methyl-N-nitrosourea (MNU)-induced mutations in the non transcribed DNA strand of the shuttle vector pF1-EBV was previously reported. The hypermutated strand was the leading strand. To test whether the different fidelity of DNA polymerases synthesizing the leading and the lagging strands might contribute to MNU-induced mutation distribution the mutagenesis study was repeated on the shuttle vector pTF-EBV which contains the gpt gene in the inverted orientation. We show that the base substitution error rates on an alkylated substrate are similar for the replication of the leading and lagging strands. Moreover, we present evidence that the fidelity of replication opposite O6-methylguanine adducts of both the leading and lagging strands is not affected by the 3' flanking base. The preferential targeting of mutations after replication of alkylated DNA is mainly driven by the base at the 5' side of the G residues.


Different binding site requirements for binding and activation for the bipartite enhancer factor EF-1A. The human transcription factor EF-1A binds to the purine-rich E1A core enhancer sequence in the adenovirus E1A and E4 and polyomavirus enhancer regions. The consensus binding site for EF-1A resembles that of members of the ets domain protein family. EF-1A activation of transcription requires a dimeric binding site. Analysis of binding sites containing point mutations revealed that EF-1A binding is determined by the core nucleotides of the binding site, while transcriptional activation is determined both by the core and some peripheral nucleotides that do not affect binding. We have purified EF-1A and analyzed its two constituent subunits, EF-1A alpha and EF-1A beta. EF-1A alpha (MW approximately 60kD) makes the primary DNA contacts. EF-1A beta (MW approximately 50 kD) forms a heteromultimeric complex with EF-1A alpha both in solution and on a dimeric binding site. Binding of both EF-1A subunits is necessary, but not sufficient, for transcriptional activation. We present immunochemical and functional evidence that EF-1A alpha is related to the murine ets-related protein GABP alpha and that EF-1A beta is related to the murine protein GABP beta.


Activation of human papillomavirus type 18 E6-E7 oncogene expression by transcription factor Sp1. The human papillomavirus 18 (HPV18) E6 and E7 proteins are considered to be primarily responsive for the transforming activity of the virus. In order to analyse the molecular mechanisms resulting in viral oncoprotein expression, it is necessary to identify the factors involved in the transcriptional regulation of the E6/E7 genes. Here we define by gel retardation experiments a sequence aberrant Sp1 binding site present in the promoter proximal part of the viral transcriptional control region (Upstream Regulatory Region, URR). Functional analyses employing transient reporter assays reveal that this Sp1 element is required for an efficient stimulation of the HPV18 E6/E7-promoter. Mutation of the Sp1 element in the natural context of the HPV18 URR leads to a strong decrease in the activity of the E6/E7-promoter in several cell lines. The magnitude of reduction varies between different cell types and is higher in cell lines of epithelial origin when compared with nonepithelial cells. Cotransfection assays using Sp1 expression vector systems further define the promoter proximal HPV18 Sp1 binding motif as a functional Sp1 element in vivo and show that its integrity is essential for the stimulation of the E6/E7-promoter by augmented levels of Sp1. These results indicate, that the cellular transcription factor Sp1 plays an important role for the stimulation of the E6/E7-promoter by the viral URR and represents a major determinant for the expression of HPV18 transforming genes E6 and E7.


Bound Lac repressor protein differentially inhibits the unwinding reactions catalyzed by DNA helicases. A partial duplex DNA substrate containing the Lac repressor binding site, within the duplex region, was constructed to examine the effect of bound Lac repressor on the unwinding reaction catalyzed by several DNA helicases. The substrate contained 90 base pairs of double-stranded DNA and, in the absence of Lac repressor, was effectively unwound by each of the seven helicases tested. The unwinding reactions catalyzed by Escherichia coli Rep protein, bacteriophage T4 Dda protein and E. coli DNA helicase I were not inhibited by the presence of bound Lac repressor. Both SV40 T antigen and E. coli helicase II were partially inhibited by bound repressor at the highest repressor concentrations tested. The helicase reactions catalyzed by E. coli DnaB protein and helicase IV were substantially inhibited by the presence of bound protein. When the length of the duplex region was increased to 323 base pairs the inhibition spectrum caused by bound Lac repressor on the unwinding reactions catalyzed by DnaB protein, helicase I and helicase II was essentially the same as that observed using the shorter partial duplex molecule. Inhibition of the unwinding reaction was due to the presence of bound Lac repressor as evidenced by the substantially weaker inhibition of helicase IV by Lac repressor in the presence of IPTG. In addition, we have shown that Rep protein displaces the bound repressor protein during the course of an unwinding reaction.


Effect of IDPN on the expression of POMC-derived peptides in rat motoneurones. Immunocytochemistry was used to detect beta-endorphin and alpha-melanotropin (alpha-MSH) in lumbar spinal motoneurones in rats treated with beta,beta'-iminodiproprionitrile (IDPN), a neurotoxicant that targets motoneurones or corn oil, which has no known neurotoxicity. After IDPN treatment most of the motoneurones were immunoreactive for both peptides but after corn oil treatment immunostaining was negligible. It is suggested that increased expression of the POMC-derived peptides may be part of the regenerative repertoire of the damaged motoneurone regardless of the cause of the lesion. Alternatively the peptides may simply accumulate in the motoneurones as a result of impaired axoplasmic transport.


CGRP antagonist activity of short C-terminal fragments of human alpha CGRP, CGRP(23-37) and CGRP(19-37). The activity of short C-terminal fragments of human alpha calcitonin gene-related peptide (CGRP), CGRP(19-37), and CGRP(23-37), and of the N-terminally acetylated (Ac) derivative, AcCGRP(19-37), has been investigated in the guinea pig isolated left atria (electrically driven) for their ability to antagonize the positive inotropic effect of human alpha CGRP. All the peptides tested produced a rightward displacement of the curve to the agonist without depressing the maximal response: apparent pA2 values were 5.39 and 4.81 for CGRP(19-37) and CGRP(23-37), respectively, as compared to 6.81 for CGRP(8-37). AcCGRP(19-37) was a more potent antagonist than the parent peptide, with an apparent pA2 value of 6.03.


Neurokinin A in the anterior pituitary of female rats: effects of ovariectomy and estradiol. The effect of acute and chronic ovariectomy and the substitutive treatment with 17-beta estradiol and/or progesterone on anterior pituitary levels of neurokinin A (NKA) was studied in female rats. Acute ovariectomy did not result in significant changes of NKA in the anterior pituitary gland as compared with the levels in diestrous intact rats, but a single injection of 5 micrograms of estradiol in ovariectomized rats significantly decreased NKA levels in the anterior pituitary gland. Progesterone was without effect and did not modify the decrease of NKA in the anterior pituitary gland induced by estradiol. In rats examined 11 to 17 days after ovariectomy, NKA in the anterior pituitary gland was significantly higher than in diestrous intact rats. In the hypothalamus, ovariectomy resulted in decreased levels of NKA in the median eminence-arcuate nucleus. Estradiol significantly reduced NKA stores in the anterior pituitary gland but increased them in the whole hypothalamus and in the median eminence-arcuate nucleus. Thus, estradiol seems to be a powerful regulator of NKA stores in the adenohypophysis and also in the hypothalamus.


Specific binding of 125I-LHRH agonist to hippocampal membranes: fluctuations during the estrous cycle. Specific binding of 125I-[D-Ala6-CH3-Leu7-Pro9,NHET]LHRH, a LHRH agonist, to hippocampal membranes prepared from ovariectomized female rates was examined. One high affinity binding site was observed with a Kd of 0.12 +/- 0.01 nM and an apparent Bmax of 13.0 +/- 3.8 fmol/mg. Luteinizing hormone-releasing hormone and the behaviorally active Ac-LHRH(5-10) were able to compete for the agonist binding site. Native LHRH had an apparent Ki of 1.73 nM, while AC-LHRH(5-10) was 30 times less potent. Competition studies examined over the rat estrous cycle revealed an eighteenfold decrease in apparent affinity during diestrus I and estrus compared with ovariectomized animals. Tissue from animals in proestrus had a Ki of 5.0 nM. Specific binding studies indicate that receptor concentration is highest in proestrus (6.11 +/- 0.90 fmol/mg) and significantly lower during estrus (2.4 +/- 0.29 fmol/mg). These data suggest that at least one fragment of native LHRH can interact with neuronal LHRH receptors and that these receptors, like those in the pituitary, can be modulated by circulating steroid hormones.


POMC-derived peptides in the neuromuscular system of streptozotocin-diabetic mice. Immunoreactivity for beta-endorphin and alpha-MSH/ACTH was demonstrated in intramuscular nerves in soleus, extensor digitorum longus, and diaphragm muscles of normal and streptozotocin-diabetic mice. There was a higher incidence of immunoreactive nerves in the muscles of the diabetic mice. Specific binding for [125I]ACTH was detected in a proportion of the muscle fibers, using autoradiography. There were significantly more fibers with specific [125I]beta-endorphin sites and specific [125]ACTH sites in some muscles in the diabetic mice. The increased expression of POMC-derived peptides and their receptors in the neuromuscular system of streptozotocin-diabetic mice may indicate early neuropathic change.


Comparison of biological effects of a sustained delivery system and nonencapsulated LH-RH antagonist SB-75 in rats. Recently, we developed long-acting microcapsules and microgranules of the LH-RH antagonist SB-75. In this study, we compared the inhibitory effects of a single injection of encapsulated and nonencapsulated LH-RH antagonist SB-75 on gonadotropin and testosterone secretion. The resulting serum SB-75 levels were also measured by RIA. Microgranules containing 4% of this antagonist in poly(DL-lactide-co-glycolide) were administered IM at two different doses (30 and 60 mg/rat) to male rats. Other groups of rats were injected SC with equivalent doses of nonencapsulated SB-75 (1.25 and 2.5 mg/rat). The administration of microgranules at a dose of 60 mg/rat produced a significant elevation of serum SB-75 until day 76, and serum testosterone and LH levels were suppressed below the detection limit of the RIA for a period of 70 days. An equivalent dose of nonencapsulated SB-75 acetate (2.5 mg/rat) produced a significant elevation of SB-75 levels for 20 days and decreased testosterone to castration values and LH levels for merely 21 days. In rats treated with 30 mg microgranules of SB-75 or an equivalent dose of SB-75 acetate (1.25 mg/rat), serum testosterone and LH were suppressed to a similar extent, but for only 2 weeks. In another study, the effect of a single SC injection of 1.25 mg/rat of antagonist SB-75 on pituitary LH-RH receptors was determined, 7 and 60 days after administration. SB-75 produced a significant (p < 0.01) downregulation of membrane receptors for LH-RH 7 days after administration.(ABSTRACT TRUNCATED AT 250 WORDS)


In situ demonstration of Epstein-Barr virus in intravenous drug abusers with generalized lymphadenopathy. We have studied by the in situ hybridization method the presence of Epstein-Barr virus (EBV) DNA genome in lymph node tissues from 11 patients with persistent generalized lymphadenopathy. Using a biotinylated EBV DNA probe, we demonstrated EBV nucleic acid in scattered germinal centre cells in eight of the 11 cases. Our results suggest that EBV is not a determinant factor in the pathogenesis of this lymphadenopathy, but support its possible implication in B cell malignant transformation in cases of AIDS-associated lymphoma.


Molecular properties of the vasoactive intestinal peptide receptor in aorta and other tissues. The molecular weight of the vasoactive intestinal peptide (VIP) receptor was assessed in bovine aorta, and rat liver, lung, and brain by covalent cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor in all four tissues was found to be a single polypeptide of approximate M(r) 54,000, contradicting previous claims for substantial heterogeneity in the molecular weight of this receptor. Guanine nucleotides inhibit cross-linking of 125I-VIP to its receptor, and cross-linking with ethylene glycolbis(succinimidylsuccinate) provides further evidence for complex formation between VIP, its receptor and a guanine nucleotide-binding regulatory protein (G-protein). The precise mechanism of receptor-G-protein coupling may differ between the aorta and other tissues.


Inhibition of angiotensin converting enzyme by the metalloendopeptidase 3.4.24.15 inhibitor c-phenylpropyl-alanyl-alanyl-phenylalanyl-p-aminobenzoate. Inhibitors of metallopeptidases may represent new alternatives in the treatment of cardiovascular disease. Recent investigations have linked the hypotensive properties of the metalloendopeptidase 3.4.24.15 (MEP 24.15) inhibitor c-phenylpropyl-alanyl-alanyl-phenylalanyl-para-aminobenzoate (cFP-A-A-F-pAB) to the attenuation of bradykinin metabolism. However, since angiotensin converting enzyme (ACE) is widely recognized to contribute to the metabolic clearance of bradykinin, we characterized the specificity of cFP-A-A-F-pAB towards ACE. We also determined whether cFP-A-A-F-pAB inhibits the conversion of angiotensin I (Ang I) to Ang II by pulmonary ACE. The ACE activity toward the synthetic substrate hippuryl-histidine-leucine (Hip-His-Leu) was measured in vitro using both a purified lung preparation and pooled rat serum. The ACE activity was inhibited at increasing concentrations of the MEP 24.15 inhibitor. Kinetic analysis revealed that cFP-A-A-F-pAB competitively inhibited pulmonary ACE with a Ki of 0.19 microM. In rat serum, cFP-A-A-F-pAB also competitively inhibited ACE. The hydrolysis of Ang I into Ang II by pulmonary ACE was inhibited to a similar extent by both cFP-A-A-F-pAB and the ACE inhibitor MK 422. These findings are the first to show that the MEP 24.15 inhibitor cFP-A-A-F-pAB also inhibits ACE. We suggest that the reported hypotensive actions of cFP-A-A-F-pAB may be due to the reduction in both bradykinin metabolism and Ang II generation arising from the blockade of ACE.


Mobilization of calcium from intracellular stores as one of the mechanisms underlying the antiopioid effect of cholecystokinin octapeptide. In enzymatically dissociated brain cells prepared from neonatal rats, KCl produced a significant increase in [Ca2+]i and this increase could be prevented by verapamil or nifedipine, known to block voltage-sensitive calcium channels. The opioid receptor agonists ohmefentanyl (OMF, mu agonist), [D-Pen2,D-Pen5]enkephalin (DPDPE, delta agonist), and 66A-078 (kappa agonist) produced a marked suppression of the Ca2+ influx induced by high K+ depolarization. The suppressive effect of OMF, DPDPE, and 66A-078 on the high K(+)-induced increase in [Ca2+]i was markedly reversed by their respective antagonists beta-funaltrexamine (beta-FNA), ICI174864, and nor-binaltorphimine (nor-BNI). Cholecystokinin octapeptide (CCK-8), at concentrations of 0.3, 3.0, and 30 nM, dose-dependently mobilized Ca2+ from intracellular stores. While CCK-8 30 nM did not affect significantly the increase of [Ca2+]i following high K+, it did reverse the suppression of the high K(+)-induced increase in [Ca2+]i by the mu agonist OMF and the kappa agonist 66A-078, but not that by the delta agonist DPDPE. The results suggested that while opioid ligands suppress [Ca2+]i by blocking voltage-operated Ca2+ influx, the antiopioid effect of CCK-8 seems to be operated via mobilization of Ca2+ from intracellular stores.


Partial characterization of IR-alpha-MSH peptides found in melanoma tumors. Our previous work indicated that IR-alpha-MSH (immunoreactive alpha-melanocyte-stimulating hormone) plasma levels are three times as high in melanoma patients with progressing disease than in disease-free patients, and that the melanoma tumor itself may be the source of IR-alpha-MSH. Further identification of the material in tumor extracts has been carried out in this study, and the results presented here show that the immunoreactivity is associated with a major fraction of about 16 kDa and another of 5-9 kDa. Significant amounts of the immunoreactive material were also found in human melanoma cells but not in culture supernatants. The presence of this material may be related to the melanogenic status of the tumor cells. We have estimated the intracellular IR-alpha-MSH to be within a 0.4 to 2.3 nM range in melanoma tumor cells. We have investigated the melanogenic effect of the IR-alpha-MSH material and its relationship to alpha-MSH. Purified extracts both from metastases and cultured cells were found to promote frog skin darkening as well as tyrosinase activity in Cloudman S91 melanoma cells. The IR material could also displace labeled alpha-MSH from its binding sites in human melanoma cells. Our data clearly indicate that melanoma cells engage in an autocrine production of alpha-MSH-like bioactive peptides by melanoma cells, of larger mol.wt., which are able to bind to MSH receptors. These peptides may be involved in the regulation of melanogenesis and possibly in the growth and proliferation of melanoma cells by an autocrine/paracrine mechanism.


[Leukocytic cytomegalic antigen. A new diagnostic method of cytomegalovirus infection after transplantation]. The occurrence of cytomegalovirus (CMV) viremia after transplantation is predictive of visceral lesions. Three-hundred and sixty blood samples were collected from 21 transplant recipients and examined. Direct CMV antigen detection was positive in 41 samples (11.4 percent), rapid viral isolation in 24 samples (6.7 percent) and conventional cell culture in 9 cases (2.5 percent). Direct detection of CMV antigen in blood leucocytes is as specific as, and more sensitive and rapid than isolation in culture. In 50 percent of secondary infections the antigenaemia assay and serology were equally sensitive, and antigenemia appeared earlier in 2 primary infections.


Interaction of opioid peptides and other drugs with multiple kappa receptors in rat and human brain. Evidence for species differences. Previous experiments resolved four kappa binding sites in guinea pig brain termed kappa 1a, kappa 1b, and kappa 2b. The present study was undertaken to examine the occurrence of kappa receptor subtypes in rat and human brain. [3H]U69,593 and [3H]bremazocine were used to label kappa 1 and kappa 2 binding sites, respectively, present in brain membranes depleted of mu and delta binding sites by pretreatment with the irreversible ligands, BIT and FIT. Low levels of [3H]U69,593 binding precluded a detailed quantitative study of kappa 1 binding sites in these species. Quantitative examination of [3H]bremazocine binding resolved two kappa 2 binding sites in both rat and human brain whose ligand selectivity patterns differed from that of the guinea pig. These observations suggest that there may be considerable variation in the ligand recognition site of kappa receptor subtypes among mammalian species.


Homology modeling of a heme protein, lignin peroxidase, from the crystal structure of cytochrome c peroxidase. A 3-dimensional model of lignin peroxidase (LiP) was constructed based on its sequence homology with other peroxidases, particularly cytochrome c peroxidase, the only protein with a known crystal structure in the peroxidase family. The construction of initial conformations of insertions and deletions was assisted by secondary structure predictions, amphipathic helix predictions, and consideration of the specific protein environment. A succession of molecular dynamics simulations of these regions with surrounding residues as constraints were carried out to relax the bond lengths and angles. Full protein molecular dynamics simulations with explicit consideration of bound waters were performed to relax the geometry and to identify dynamically flexible regions of the successive models for further refinement. Among the important functionally relevant structural features predicted are: (i) four disulfide bonds are predicted to be formed between Cys3 and Cys15, Cys14 and Cys285, Cys34 and Cys120 and Cys249 and Cys317; (ii) a glycosylation site, Asn257, was located on the surface; (iii) Glu40 was predicted to form a salt bridge with Arg43 on the distal side of the heme and was considered as a possible origin for the pH dependence of compound I formation; and (iv) two candidate substrate binding sites with a cluster of surface aromatic residues and flexible backbones were found in the refined model, consistent with the nature of known substrates of LiP. Based on these predicted structural features of the model, further theoretical and experimental studies are proposed to continue to elucidate the structure and function of LiP.


Ontogeny of peptides in human hypothalamus in relation to sudden infant death syndrome (SIDS). The brains of mammals are not mature at birth, in particular in humans. Growth and brain development are influenced by the hormonal state in which the hypothalamus plays the major regulatory role. The maturation of the hormonal patterns leads to the physiological establishment of chronological variations as revealed by the circadian variations of both hypothalamic peptides and pituitary hormones (as illustrated for hypothalamic-pituitary-thyroid axis by the determination of thyro-stimulating hormone (TSH) and thyrotropin-releasing hormone (TRH) circadian rhythms in the rat (Jordan et al., 1989)). It has been established that hypothalamic peptide variations are regulated by hormonal feed-back and amine systems, with the maturation of the latter also being dependent upon the whole functional maturation of the brain. Though these systems have been studied in the rat, very little information is currently available with regard to the human brain. The only biochemical or immunohistochemical information published to date concerns either the fetus or the adult. We have studied four main peptidergic systems (somatostatin-releasing inhibiting factor (SRIF), thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hormone (LHRH) and delta sleep inducing peptide (DSIP) in post-mortem adults and infants and in sudden infant death syndrome (SIDS) brains either by autoradiography and/or immunochemistry of radioimmunology. From a technical point of view, human brain studies display certain pitfalls not present in animal studies. These may be divided into two subclasses: ante- and post-mortem. Ante-mortem problems concern mainly sex, laterality, nutritional and treatment patterns while post-mortem problems concern post-mortem delay and conditions before autopsy and hypothalamic dissection. This might induce dramatic changes in morphological, immunochemical and autoradiographic evaluations. The matching of pathological subjects with controls is particularly difficult in the case of SIDS because of the rapid changes which take place in physiological regulatory processes during the first year of life. Thus, the treatment of hypothalamic tissue samples both for immunochemistry, radioimmunology and autoradiographic studies required techniques which must be rigorously controlled. For example, SRIF studies were carried out with three different antibodies, which gave similar results. The use of different technical procedures as well as different antibodies is discussed. These types of differences might explain, at least in part, the discrepancy observed until now. As previously described in the fetus (Bugnon et al., 1977b; Bouras et al., 1987), we confirmed that in the infant hypothalamic SRIF immunoreactive cell bodies are present in the paraventricular and suprachiasmatic nuclei and in the periventricular area.(ABSTRACT TRUNCATED AT 400 WORDS)


Hormonal influences on morphology and neuropeptide gene expression in the infundibular nucleus of postmenopausal women. Neuronal hypertrophy occurs in a subpopulation of neurons in the infundibular nucleus of post-menopausal women. The hypertrophied neurons contain NKB, SP and estrogen receptor gene transcripts. Although associated with reproductive aging, post-menopausal neuronal hypertrophy is not a sign of central nervous system degeneration. Quite the opposite, because the hypertrophy is accompanied by marked increases in tachykinin gene expression, reflecting increased neuronal activity. We have proposed that infundibular neurons containing NKB, SP and estrogen receptor mRNAs participate in the hypothalamic circuitry regulating estrogen negative feedback on gonadotropin release in the human. In addition, there is evidence to suggest that the hypertrophied tachykinin neurons may be involved in the initiation of menopausal flushes. Because menopause affects a well characterized system, and has consistent and substantial changes in hormone levels, we have had the rare opportunity to correlate changes in hormone secretion with structural and neurochemical changes in the human hypothalamus. We suspect that future studies of the hypothalami of post-menopausal women will continue to be a fruitful avenue for investigating neuroendocrine regulation in the human.


[Effect of chlorpromazine on K+-dependent regulation of aldosterone biosynthesis in guinea pig adrenal cortex]. The effects of chlorpromazine, a calmodulin inhibitor, on K(+)-dependent regulation of aldosterone biosynthesis were studied in sections and dispersed adrenocortical cells of guinea pigs. K ions in concentrations 5-11 mM significantly (p < 0.01) stimulated aldosterone and protein biosynthesis and protein phosphorylation in sections and cells of the guinea pig adrenal cortex. Using the radioautography of P-labeled polypeptides separated in polyacrylamide gel, the authors have demonstrated K+ enhancement of phosphate incorporation in the proteins with molecular masses of 40 and 75 kD in the cytosols and of 75 kD proteins in the mitochondria. Chlorpromazine in concentration 50 microM reduced K(+)-stimulated steroidogenesis and protein phosphorylation, simultaneously increasing labeled phosphate incorporation in the proteins in low K+ concentrations, not stimulating steroidogenesis. K(+)-stimulated protein biosynthesis was virtually unchanged in the presence of chlorpromazine. Increased K+ concentration in the medium resulted in reduction of cGMP concentration in adrenal cortex sections. The nucleotide level grew in the presence of chlorpromazine in elevated concentrations of K+ and reduced if the ion concentrations were low (1-3 mM). The mechanisms of chlorpromazine effect on K(+)-dependent steroidogenesis changes, protein phosphorylation and cGMP levels in the adrenocortical tissue are discussed, as is the role of calmodulin in K(+)-dependent regulation of aldosterone biosynthesis.


Receptor localization in the human hypothalamus. In summary, we have illustrated and discussed the applicability of different techniques to the study of neurotransmitter receptors in the human brain. Because of the availability of these techniques it is possible today to examine in detail the alterations in density produced by different physiological or pathological conditions. The use of these techniques will in the future, without any doubt, be of help in understanding the chemical neuroanatomy of the human hypothalamus.


Risk assessment for aflatoxin B1: a modeling approach. The data generated by Yeh et al. on hepatitis B virus, aflatoxin, and primary hepatocellular carcinoma (PHC) in Southern Guangxi, China was used to evaluate the cancer potency of aflatoxin. We examined model fits to these data to explore whether hepatitis B virus (HBV) and aflatoxin intake act together to affect PHC rates in an additive, multiplicative, or interactive fashion, using relative and excess risk model forms. Purely additive models fit the data poorly. Fitted models were checked for plausibility by comparing predictions for the U.S. population with actual PHC incidence rates in the United States, and parameter stability was evaluated. The multiplicative relative risk and the interactive excess risk models provided satisfactory descriptions of the Yeh et al. data and U.S. PHC rates. There is about an eight-fold difference in the potency estimate for aflatoxin under the multiplicative relative risk (5.7 (mg/kg-day)-1) and interactive excess risk models (45.6 mg/kg-day)-1). The assumptions and limitations of the various models are discussed.


Diffusion coefficient for O2 in plasma and mitochondrial membranes of rat cardiomyocytes. Oxygen diffusion in plasma membranes and mitochondrial (Mt) membranes of ventricular myocytes isolated from adult rat hearts was measured using O2-induced fluorescence quenching of pyrene-butyric acid. The diffusion coefficient for oxygen (DO2 in 10(-6).cm2 x s-1) of the plasma membrane was 2.91 +/- 0.05 (37 degrees C) in the control group, which was significantly higher than that of Mt membrane (2.23 +/- 0.11) (P < 0.001). The DO2 of the plasma membrane was reduced to 2.50 +/- 0.08 (P < 0.001) in myocytes isolated from heart after 15 min reperfusion following no-flow ischemia for 30 min, while that of the Mt membrane remained almost unchanged. Administration of hydrogen peroxide and hypochlorous acid to the isolated myocytes also reduced the plasma membrane DO2. The decrease in the plasma membrane DO2 correlated with that in the fraction of rod-shaped myocytes. We conclude that the plasma membrane is more susceptible to reperfusion injury than the Mt membrane, but these membranes do not limit O2 uptake in the heart because of the high absolute values of DO2.


Molecular biologic studies of Hodgkin's disease. There has been intense interest in applying the techniques of molecular biology to further our understanding of Hodgkin's disease. Numerous studies of the status of lymphocyte antigen receptor genes have been performed, with conflicting results. There is more uniformity in the results of Epstein-Barr viral (EBV) studies; evidence of EBV can be identified in the Reed-Sternberg cells of approximately 40% to 50% of cases. There is no consensus on whether the t(14;18) is present in tissues involved by Hodgkin's disease.


Wilms' tumor: a paradigm, a parallel, and a puzzle. Wilms' tumor, a childhood tumor of the kidney, parallels retinoblastoma in several ways. Both malignancies occur in the very young, involve paired organs, arise from embryonal cells, can develop unilaterally or bilaterally, and can occur in hereditary and non-hereditary forms. However, Wilms' tumor rarely occurs in family clusters and may involve the loss of function of multiple genes.


Hereditary colorectal cancers.

Hereditary colorectal cancers. Individuals with one of the highest known risks of developing colorectal cancer are members of hereditary colorectal cancer families. Familial adenomatous polyposis (FAP) and hereditary nonpolyposis colon cancer (HNPCC) are the most commonly recognized hereditary colorectal cancer syndromes. Surveillance, family identification, and education are of major importance. Genetic screening holds further promise.


Herpes simplex virus induces unscheduled DNA synthesis in virus-infected cervical cancer cell lines. We evaluated herpes-simplex-virus-type-2(HSV2)-induced unscheduled DNA synthesis in virus-infected cervical cancer (HeLa, CaSki, C-33A, and SiHa) cells. HSV2 replication was approximately 100-fold more efficient in the HeLa cells than in less susceptible C-33A and SiHa cells. In dual parameter flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation, HSV2-infected HeLa cells showed a rapid increase in the proportions of DNA-synthesizing G1- and S-phase cells, whereas in C-33A and SiHa cells, the proportions of DNA-synthesizing G1- and early S-phase cells were increased late in the infection. Blocking of HSV2 replication by phosphonoformate inhibited virus-induced changes in HeLa cells, but not in C-33A and SiHa cells. Anti-BrdU antibodies exhibited a coarse globular nuclear staining pattern in the C-33A cells, while the other cells showed speckled and/or fine globular nuclear fluorescence. Anti-ICP8 (HSV-specified major DNA-binding protein) antibodies revealed that, in C-33A cells, ICP8 remained in the cytoplasm, whereas in the other cells, speckled or globular nuclear fluorescence was found. Our results showed that HSV2 induced the unscheduled synthesis of cellular DNA, which was host-cell-dependent, and in virus infected C-33A cells, it may be attributable to both viral and cellular proteins.


[Depression of the mononuclear phagocyte system caused by high doses of narcotics]. We evaluated in human monocytes the effect of high doses of alfentanyl on the expression of vimentin filaments, the phagocytic activity and the membrane display of HLA-DR molecules in the subjects undergoing surgery. The study was performed on 30 patients, ASAI-II. The patients received 100 mcg/kg i.v. of Alfentanil and the maintenance of anaesthesia was made with Alfentanil (2-3 mcg/kg/min.). The patients were randomized in two groups. The patients were ventilated with N2O:O2 (1:1) (Group I) or air: O2 (1:1) (Group II). After surgery, all patients of the Group II received Naloxone (0.2-0.4 mg). Central venous blood samples were obtained before induction, one and two hours after induction of anaesthesia and at the end of surgery. Separation of monocytes was performed according to Boyum technique. CD35 and HLA-DR molecules and vimentin filaments were studied by indirect immunofluorescence method using monoclonal antibodies. Percentage of positive cells were read with a cytofluorometer. The phagocytic function of monocytes was determined by ingestion of latex particles. Cortisol and ACTH plasma levels were determined by RIA. High doses of Alfentanyl depress phagocytic function and membrane display of CD35 and HLA-DR molecules in monocyte and induce marked changes in the organization of vimentin filaments in these cells in patients undergoing surgery. This monocytic depression was more marked in the patients ventilated with N2O. In our results there was uninhibition of ACTH and cortisol plasma levels responses to surgical stress by Alfentanil administration. Since the effects of Alfentanil were reversed by Naloxone, an opioid receptor mechanism seems to mediate these events.


Antimicrobial effect of a dental varnish, in vitro. The effects of a polymer based antimicrobial releasing varnish Cervitec were investigated against different gram-positive and gram-negative bacterial strains as well as a yeast using the agar diffusion inhibitory test (ADT-test in vitro). As positive controls a 1% chlorhexidine gel and 1% aqueous solution of thymol and a placebo polymer varnish without active agents were employed. The test experimental varnish containing 1% chlorhexidine and 1% thymol showed antimicrobial activity against all gram-positive and gram-negative microorganisms tested including one Candida strain. The positive controls were similar in effect compared to the test varnish. No antimicrobial effect was observed with the placebo varnish without active ingredients. Toothpicks and dental floss treated with the test varnish showed an antimicrobial effect against S. mutans even after storing in room temperature up to 12 months. The results from this study support earlier laboratory studies that chlorhexidine and thymol diffuse out of the experimental varnish and that the varnish is active against various of oral pathogens. The possibility to use toothpicks or dental floss impregnated with the varnish with the aim to become chemotherapeutically active against periodontal diseases as well as against dental caries, is promising and should be tested in vivo.


SPECT imaging of the benzodiazepine receptor: autoradiographic comparison of receptor density and radioligand distribution. SPECT imaging of benzodiazepine (BZ) receptor using the antagonist [123I]iomazenil in nonhuman primates was correlated with ex vivo autoradiography and in vitro receptor autoradiography of postmortem tissue sections taken at the time of maximal brain uptake of the radiotracer. Cryostat sections prepared from brain tissue blocks at the orientation of the SPECT imaging plane showed high activity in gray matter regions, with gray to white matter ratios of greater than 30:1. After at least one week decay of 123I, these same tissue sections were used to localize and quantify the distribution of BZ receptors with the standard technique of in vitro receptor autoradiography using [125I]iomazenil. The ex vivo autoradiographic distribution of activity was highly correlated (r = 0.89) with the distribution of BZ receptors, although a few brain regions showed reproducible discrepancies between ex vivo and in vitro results. The ex vivo autoradiograms provided quantitative data from a realistic "biological phantom," which may be used to assess the accuracy of image reconstruction and to investigate differences between the distribution of an intravenously (i.v.) administered tracer and that of its target in brain tissue.


Serotonergic signalling between thyroid cells: protein kinase C and 5-HT2 receptors in the secretion and action of serotonin. Parafollicular (PF) cells of the thyroid gland are neural crest derivatives, which costore the neurotransmitter, 5-hydroxytryptamine (5-HT) with calcitonin. PF cells are located adjacent to follicular (F) cells within the basement membrane of thyroid follicles. It has been proposed that 5-HT serves an intercellular signalling function in the thyroid and that F cells are its target. This proposal was tested by using cell lines derived from PF (medullary thyroid carcinoma [MTC]) and F (FRTL-5) cells to study the mechanisms that mediate the secretion and action of 5-HT. Secretion of 5-HT by MTC cells was evoked by thyroid stimulating hormone, thyrotropin (TSH), elevated extracellular calcium (increases [Ca2+]e), or by agents that increase intracellular cAMP (increases [cAMP]i). When protein kinase C (PKC) was down-regulated by prolonged treatment of MTC cells with phorbol 12-myristate 13-acetate (PMA), or PKC was inhibited by staurosporin, the TSH- or PMA-evoked secretion of 5-HT was blocked; however, interference with PKC function did not affect 5-HT secretion evoked by increases [Ca2+]e or increases [cAMP]i. In the putative targets, FRTL-5 cells, 5-HT increased the turnover of phosphoinositides (PI), cytosolic calcium (increases [Ca2+]i), increases [cAMP]i, and biphasically modified the effect of TSH on cAMP. All of these 5-HT effects were inhibited by 5-HT2 receptor antagonists (spiperone and ketanserin) and by pertussis toxin (PTx), suggesting that the actions of 5-HT are mediated by 5-HT2 receptors, which are coupled to a G protein. This suggestion was supported by the following additional observations: FRTL-5 membranes bound the 5-HT2 agonist, [125I]2,5-dimethoxy-4-iodophenylisopropylamine ([125I]-DOI), and anti-idiotypic antibodies, which recognize 5-HT2 receptors. [125I]-DOI binding was inhibited by guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) and the antibodies were displaced by spiperone. Data are consistent with the hypothesis that 5-HT serves as a PF to F cell messenger.


Multiple metabotropic glutamate receptors regulate hippocampal function. Selective activation of metabotropic glutamate receptors with trans-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) stimulates phosphoinositide hydrolysis and elicits three major physiological responses in area CA1 of the hippocampus. These include direct excitation of pyramidal cells, blockade of synaptic inhibition, and decreased transmission at the Schaffer collateral-CA1 pyramidal cell synapse. Physiological effects of trans-ACPD are thought to be mediated by activation of phosphoinositide hydrolysis. However, it is now clear that multiple metabotropic glutamate receptor subtypes exist, some of which are not coupled to phosphoinositide hydrolysis. Thus, we performed a series of studies aimed at determining whether the physiological effects of trans-ACPD in the hippocampus are mediated by activation of the predominant phosphoinositide hydrolysis-linked glutamate-receptor. We report that L-2-amino-3-phosphonopropionic acid (L-AP3), an antagonist of trans-ACPD-stimulated phosphoinositide hydrolysis, does not inhibit the physiological effects of trans-ACPD in area CA1 at concentrations that maximally inhibit trans-ACPD-stimulated phosphoinositide hydrolysis in this region. Furthermore, 1S,3S-ACPD activates the phosphoinositide hydrolysis-linked glutamate receptor but does not reduce evoked field excitatory postsynaptic potentials (EPSPs) in area CA1. However, we report that the physiological effects of 1R,3S- and 1S,3R-ACPD are consistent with the hypothesis that these effects are mediated by activation of a metabotropic glutamate receptor. Thus, our data are consistent with the hypothesis that the physiological effects of trans-ACPD in area CA1 of the hippocampus are mediated by metabotropic glutamate receptors that are distinct from the AP3-sensitive phosphoinositide hydrolysis-linked glutamate receptor.


[Classical fowl plague and milder influenza infections in birds and mammals]. Wild waterfowl are currently considered the largest reservoir of the various haemagglutinin (H) and neuraminidase (N) subtypes of influenza virus. Until now thirteen different H-types and nine different N-types have been detected in these populations. In the first instance, virus transmission from fowl to other animal species and to man is not causing disease problems. However, small changes at the molecular level of a given HN-subtype recently caused a dramatic increase in virulence for chickens. Genes fragments coding for haemagglutinin or neuraminidase can be exchanged between viruses which propagate in the same individual. This phenomenon-'genetic reassortment'-is of major epidemiological significance when it occurs in pigs. New influenza epidemics in the human population consistently originate in areas where waterfowl, pigs and human beings live close together. At the moment, the virological and serological diagnosis of influenza A infections is based ELISAs for antigen and antibody detection. Both ELISAs employ a monoclonal antibody directed against a conserved antigenic determinant of the influenza A nucleoprotein. The use of these tests can simplify the diagnosis of and screening for influenza A infections, particularly in those species which harbour several H- and N-subtypes.


Magnification mismatches between micrographs: corrective procedures and implications for structural analysis. Quantitative structural analysis from electron micrographs of biological macromolecules inevitably requires the synthesis of data from many parts of the same micrograph and, ultimately, from multiple micrographs. Higher resolutions require the inclusion of progressively more data, and for the particles analyzed to be consistent to within ever more stringent limits. Disparities in magnification between micrographs or even within the field of one micrograph, arising from lens hysteresis or distortions, limit the resolution of such analyses. A quantitative assessment of this effect shows that its severity depends on the size of the particle under study: for particles that are 100 nm in diameter, for example, a 2% discrepancy in magnification restricts the resolution to approximately 5 nm. In this study, we derive and describe the properties of a family of algorithms designed for cross-calibrating the magnifications of particles from different micrographs, or from widely differing parts of the same micrograph. This approach is based on the assumption that all of the particles are of identical size: thus, it is applicable primarily to cryo-electron micrographs in which native dimensions are precisely preserved. As applied to icosahedral virus capsids, this procedure is accurate to within 0.1-0.2%, provided that at least five randomly oriented particles are included in the calculation. The algorithm is stable in the presence of noise levels typical of those encountered in practice, and is readily adaptable to non-isometric particles. It may also be used to discriminate subpopulations of subtly different sizes.


Three-dimensional reconstruction of single particles negatively stained or in vitreous ice. The random-conical reconstruction method has been highly successful in three-dimensional imaging of macromolecules under low-dose conditions. This article summarizes the different steps of this technique as applied to molecules prepared with negative staining or vitreous ice, and sketches out the current directions of development. We anticipate that by using new instrumental developments, transfer function correction and computational refinement techniques, a resolution in the range of 7-10 A could ultimately be achieved.


Quantitative energy-filtered electron microscopy of biological molecules in ice. The theoretical and experimental bases for quantitative electron microscopy of frozen-hydrated specimens are described, with special considerations of energy filtration to improve the images. The elastic and inelastic scattering from molecules in vacuum and in ice are calculated, and simple methods to approximate scattering are introduced. Multiple scattering calculations are used to describe the scattering from vitreous ice and to predict the characteristics of images of frozen-hydrated molecules as a function of ice thickness and accelerating voltage. Energy filtration is predicted to improve image contrast and signal-to-noise ratio. Experimental values for the inelastic scattering of ice, the energy spectrum of thick ice, and the contrast of biological specimens are determined. The principles of compensation for the contrast transfer function are presented. Tobacco mosaic virus is used to quantify the accuracy of interpreting image intensities to derive the absolute mass, mass per unit length, and internal mass densities of biological molecules. It is shown that compensation for the contrast transfer function is necessary and sufficient to convert the images into accurate representations of molecular density. At a resolution of 2 nm, the radial density reconstructions of tobacco mosaic virus are in quantitative agreement with the atomic model derived from X-ray results.


[Pathologic changes in the organs in chickens after infection with the turkey herpesvirus THV-BIO-I]. The objective of our work was to investigate the dynamics of pathological lesions of chicken organs after infection with high doses of turkey herpesvirus THV-BIO-I. This virus strain is commonly used in form of the Marvak vaccine against Marek's disease of poultry in Czechoslovakia. High doses of the vaccine are used in practice with respect to the epizootological situation. The incidence of pathological lesions in the organs of Brown Leghorn chickens was investigated in a five-week experiment. One-day chickens were infected intramuscularly with the HVT strain at the doses of approximately 10(2), 10(3) and 10(4) PFU in 0.2 ml of infective inoculum per chick. The body weights of ten chickens of each group were recorded at intervals of 1, 2, 3 and 5 weeks after infection, serological examination was performed for precipitating antibodies to MDV and the feather was examined for MDV-antigen. Bursae Fabricii and spleens were weighed. Thymus, bursae Fabricii, spleens, peripheral nerves (n. ischiadicus and pl. brachialis) and gonads were sampled for histopathological examination. Neither maternal nor post-infection antibodies were found in any chick. Cytolytic lesion severity of lymphoid organs was scored using the scale of immunosuppression degrees (0-4). Morphological criteria were published in a previous paper (Halouzka and Jurajda, 1991b). The differences observed in the weights of bursa Fabricii and spleen between the infected and control chickens were not statistically significant. The observed lymphoid infiltrations in the skin, gonads, nerves and other tissues following the HVT infection are well-known and correlate with the infection dose.(ABSTRACT TRUNCATED AT 250 WORDS)


From genes to complex structures of bluetongue virus and their efficacy as vaccines. Bluetongue virus-like and core-like structures consisting of multiproteins in different molar ratios, have been synthesized using baculovirus multiple expression vectors. These particles lacking genetic materials, mimic the single- and double-shelled authentic virus particles and have been shown to be highly immunogenic and protective for sheep challenged with infectious virus. The formation of virus-like particles, using this new technology, offers a novel approach to vaccine development.


A study of the basis of virulence variation of bovine rotaviruses. Rotaviruses are enteric pathogens of cattle but sub-clinical infections are common. Virulence variation has been identified with bovine rotaviruses and some rotaviruses replicated without clinical signs in non-immune calves. The rotavirus genome is composed of eleven segments of double-stranded RNA and the fourth largest segment codes for a non-glycosylated surface protein, VP4, which has been linked with virulence. In the present study the biological basis of rotavirus virulence variation was studied in vivo and compared with the known properties of the fourth gene. Calves were inoculated orally with a virulent rotavirus or a rotavirus of low virulence which multiplied but failed to cause diarrhoea. They were taken for necropsy at intervals of 2 days after inoculation. Clinical signs, virus in faeces and the percentage of infected small intestinal epithelium were determined. Damage to the small intestine was assessed by measurement of villus heights and crypt-cell production rates. Virulence was associated with a greater level of colonization of the small intestinal epithelium, greater enterocyte damage and preferential infection of the upper small intestine. The fourth gene determines the ability of rotaviruses to spread in vitro and the finding that virulence was associated with greater colonization in vivo raises the possibility that this gene may have an important role in rotavirus virulence.


Infectious bursal disease of poultry: antigenic structure of the virus and control. The present knowledge of genome organisation, structural basis of pathogenicity and antigenicity of infectious bursal disease virus (IBDV) are briefly reviewed. The current situation of IBDV infection in various countries is stated and recommendations for improved vaccination schemes are given.


[Ophthalmopathy caused by precision work of sorters of precious stones]. A total of 440 female workers aged 17 to 50, whose work records ranged from 1 to 29 years, engaged in grading the diamonds by the color, shape, size, and quality (a total of 24 to 33 positions) were examined. A random sample of 110 subjects was singled out; this sample was divided into 2 equal groups with or without asthenopic complaints. The refraction, absolute accommodation volume, and relative accommodation reserves were under study. Comparison of these two groups of workers has shown that subjects with precision ophthalmopathy show a trend to a higher incidence of myopia, reduction of the absolute accommodation volume by 1.6 diopters and of the relative accommodation reserves by 1.3 diopters.


Proteins encoded in the 5' region of the pestivirus genome--considerations concerning taxonomy. The first protein encoded within the pestivirus open reading frame is a nonstructural protein which removes itself from the polyprotein by autoproteolytic cleavage. The following nucleocapsid protein ends just before a putative signal sequence preceding three glycosylated proteins. All three glycoproteins are part of the viral envelope and exist in the form of disulfide-linked dimers. Pestiviruses have recently been reclassified as members of the family Flaviviridae which now comprises three genera, namely flavivirus, hepatitis C virus group and pestivirus. All members of the family have certain characteristics in common like the overall genome organization and the strategy of gene expression. Major differences exist, however, between the genera; the most obvious ones concern proteins encoded in the 5' region of the respective genomes.


Characterisation of p20 gene sequences from a border disease-like pestivirus isolated from pigs. A pestivirus, isolated from pigs with haemorrhagic lesions, was antigenically more similar to border disease (BD) virus than to either hog cholera (HC) or bovine viral diarrhoea (BVD) viruses. After reverse transcription the genome at the 5' end, along with the same region from a BD isolate from sheep, was amplified by the polymerase chain reaction and cloned. The region of the p20 gene was sequenced and compared with published data for BVD and HC viruses. A number of motifs were conserved in the amino acid sequences of all the viruses. The pig isolate had a greater degree of homology in this region with the BD isolate (87%) than with BVD (73%) or HC (74%) viruses. This further confirms the BD-like nature of the virus.


Infection of ovine fetal brain cell cultures with cytopathogenic and non-cytopathogenic bovine viral diarrhoea virus. The in vitro cell tropism of non-cytopathogenic (ncp) and cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) was studied in primary dissociated brain cell cultures derived from ovine fetuses of different gestational ages. The cell types infected were identified by double immunofluorescence using antibodies against BVDV and cell type-specific markers. In cultures infected with ncp BVDV viral antigen was present in neurofilament (NF 200 kDa)-positive neurons, glial fibrillary acidic protein (GFAP)-positive astrocytes and fibronectin-expressing cells. Estimation of the percentages of individual cell types infected with ncp BVDV indicated a tropism for NF 200-positive neurons. In cultures infected with cp BVD virus cytopathic changes were observed beginning at 40 hours post infection. Viral antigen was present in vacuolated NF 200-, GFAP- and fibronectin-positive cells. In comparison with non-infected control cultures a considerable reduction of the number of the different cell types was seen.


Spatial visualization of progressive states of maturing lentivirus. Progressive states of maturing lentivirus: maedia visna virus (MVV) and human immunodeficiency virus (HIV), respectively, have been visualized by 2-D electron microscopy and by 3-D electron microscopic tomography. A major fraction of MVV and a low percentage of HIV appear as immature particles 4 to 5 days post virus infection. Upon budding the gag-precursor material is densely packed inside the external envelope. After virus release the major portion of precursors is assembled within an approximately 25 nm thick layer directly attached to the envelope. Structural maturation of the core is different for the two viruses. Pleomorphic cores are observed in mature MVV in contrast to structurally defined cores of HIV. The latter are principally cone-shaped, spanning the entire diameter of the virion with a 40 to 60 nm wide free end and an approximately 20 nm narrow end attached to the envelope with a core-envelope-link.


Animal immunodeficiency viruses.

Animal immunodeficiency viruses. Feline immunodeficiency virus (FIV) has morphological, physical and biochemical characteristics similar to human immunodeficiency virus (HIV), the cause of AIDS in man. However, it is antigenically and genetically distinct from HIV; an antigenic relatedness with equine infectious anaemia virus has been demonstrated. FIV has been molecularly cloned and sequenced. Diagnostic tests are commercially available and attempts at preparing inactivated, subunit and molecularly engineered vaccines are being made in different laboratories. During FIV infection a transient primary illness can be recognized, with fever, neutropenia and lymphadenopathy. After a long period of clinical normalcy a secondary stage is distinguished with signs of an immunodeficiency-like syndrome. The incubation period for this stage can be as long as 5 years, during which gradual impairment of immune function develops. Many FIV-infected cats are presented for the first time showing vague signs of illness: recurrent fevers, emaciation, lack of appetite, lymphadenopathy, anaemia, leucopenia and behavioural changes. Later, the predominant clinical signs observed are chronic stomatitis/gingivitis, enteritis, upper respiratory tract infections, and infections of the skin. Neoplasias, neurological, immunological and haematological disorder are seen in a smaller proportion. The immunodeficiency-like syndrome is progressive over a period of months to years. Concomitant infection with feline leukaemia virus has been shown to accelerate the progression of disease. In vitro, phenotypic mixing between FIV and an endogenous feline oncovirus (RD114) has been demonstrated which leads to a broadening of the cell spectrum of the lentivirus. Bovine immunodeficiency virus (BIV) has been isolated only once, and all attempts to obtain additional isolates have failed; it has been recovered from the leucocytes of cattle with persistent lymphocytosis, lymphadenopathy, lesions in the central nervous system, progressive weakness and emaciation. As with the feline representative, BIV also was found to possess a lentivirus morphology and to encode a reverse transcriptase with Mg++ preference; it replicates and induces syncytia in a variety of embryonic bovine tissues in vitro. Antigenic analyses have demonstrated a conservation of epitopes between the major core protein of BIV and HIV. The original isolate has been molecularly cloned and sequenced. Besides the three large open reading frames (ORFs) comprising the gag, pol, and env genes common to all replication-competent retroviruses, five additional small ORFs were found. Numerous point mutations and deletions were found, mostly in the env-encoding ORF. These data suggest that, within a single virus isolate, BIV displays extensive genomic variation.(ABSTRACT TRUNCATED AT 400 WORDS)


Genomic heterogeneity of small ruminant lentiviruses detected by PCR. In order to detect a large spectrum of small ruminant lentiviruses, primers for PCR were chosen in conserved parts of the LTR and GAG genes of Icelandic Visna virus 1514 and of the POL gene of caprine arthritis-encephalitis virus. This set of primers was tested in six different caprine arthritis-encephalitis virus (CAEV)- and Maedi-Visna virus isolates of Dutch, American and Swiss origin. The LTR primers allowed the detection of the corresponding fragments of all isolates. The GAG primers allowed amplification of the corresponding fragments of all but the Swiss Maedi-Visna virus strain OLV. Using the POL primers, one Maedi-Visna- and two caprine arthritis-encephalitis virus strains were detected after one round of amplification. Sequencing of the GAG and POL amplification products and comparison to Icelandic Visna virus and CAEV strain CO revealed total heterogeneity of 38% for the GAG- and 28% for the POL fragment. The virus strains studied fall into two groups which are more closely related to one another than to Icelandic Visna virus.


Effects of replacing the promoter of the immediate early gene with the promoter of Drosophila heat-shock gene HSP70 on the growth and virulence of pseudorabies virus. We investigated whether altering control of expression of an essential gene of pseudorabies virus (PRV) influences virus replication and virulence. The PRV immediate early (IE) gene was selected as a target, and its promoter was replaced with the promoter of the heat-shock gene HSP70 of the fruit fly Drosophila. The HSP70 promoter was selected because it is well characterized and can be induced in a broad range of eukaryotic cell lines at temperatures around 42 degrees C. Overlap recombination was used to construct the NIA3-HSP mutant virus. When stocks of the recombinant virus were titrated at 42 degrees C, virus titres were 100 times higher than titres obtained at 37 degrees C. Once replication began, however, the rate of growth of the mutant NIA3-HSP was equal at both temperatures. When wild-type virus was titrated at both temperatures, titres were identical. Mice that were infected with the mutant virus had a longer mean-time-to-death than those infected with the wild-type virus. Thus, the mutant virus was considered to be less virulent. We conclude that replication and virulence of PRV can be modified by altering control of expression of the viral IE gene.


Equine lentivirus, comparative studies on four serological tests for the diagnosis of equine infectious anaemia. Serological diagnosis of equine infectious anemia is of necessity group-reactive, i.e. based on viral core protein p26, because viral envelope components as well as the host's immune response to them undergo rapid antigenic change. Since 1970 the agar gel-immunodiffusion test ("Coggins-test") has been the diagnostic method of choice. Recently, ELISA tests have been introduced for faster and theoretically more sensitive serodiagnosis, while Western blots have been used to clarify doubtful results obtained in Coggins-tests. A commercial competitive ELISA was found to give practically equivalent results to the Coggins-test. The sensitivity of this market product is intentionally kept marginal in order to avoid false-positive "reactor horses". Another commercial ELISA, non-competitive, gave inconsistent results, creating great turmoil among horse owners when falsely positive. Caution is also indicated when interpreting Western blots. Sera of strongly positive horses gave as many as eleven bands, of medium positives fewer bands, and of the weakest reactors solely the p26 band. Single p26 banding was, however, also encountered in 5% healthy horses, in two of them consistently over time, which are accordingly considered non-specific. In order to be interpreted as positive, a Western blot for this equine lentivirus must band with its core protein plus at least one glycoprotein, similar to the recommended criterion for a positive reading of serum samples from AIDS patients.


An investigation of the effect of antisense RNA gene on bovine leukaemia virus reproduction in cell culture. A possible approach to control of bovine lymphoproliferative disease caused by bovine leukaemia virus (BLV) may be the development of an "antiviral information immunity" based on the effect of anti-sense RNA (asRNA). A numbers of constructs were obtained, under control of various promotors (herpesvirus thymidine kinase, T-antigen SV40 promoter), carrying as DNA against gene X, the expression product of which is a transactivator of viral transcription from the BLV LTR promotor. As a model system for the analysis of antiviral activity of constructs developed, cloned continuous cell lines of BLV-producing FLK cells were used. The level of BLV expression in cells transfected with the constructs was determined by various parameters. Differences were detected in different clones obtained from non-transfected cells, as well as variation between transfected clones, as measured by reverse transcriptase, competitive radio-immunoassay for BLV p24, the viral particle count on agar membrane, and the tumorigenicity for nude mice. The differences in inhibition of expression of BLV genes and their products may be explained in terms of the site of integration of asDNA and the number of integrated copies.


Target recognition by bovine mononuclear, MHC-unrestricted cytotoxic cells. A population of bovine non B/non T, cytotoxic lymphocytes with natural killer activity against virus-infected and non-infected embryonic kidney cells was functionally characterized. The data obtained in experiments of flow cytometry and immuno-peroxidase staining show that a CD2-, CD4-, CD8-, TcR gamma delta-, CD3+, CD45+, FcR+ lymphoid killer cell does exist within bovine peripheral blood leucocytes. This population can detect the down-regulation of class I MHC antigens or the expression of embryonic forms thereof, as shown by experiments of 17-hour 51Cr release and binding to target cells. This model was tested in vitro in experiments on virus-infected bovine kidney cells. The emerging picture was substantially in agreement with the "missing self" theory as a possible option for target cell recognition. In this respect, the profound alteration of MHC Class I expression could represent a major early event, recognized on virus-infected cells by the immune system.


Role of different genes in the virulence and pathogenesis of Aujeszky's disease virus. In this study the role of different genes located in the unique short region of the genome of Aujeszky's disease virus was examined. Inactivation of the genes encoding the protein kinase (PK), gp63, and gI reduced virulence of the virus for pigs, in contrast to inactivation of the genes encoding the 28 kDa protein, and gX. There was no correlation between virulence and virus multiplication in vitro or in the oropharynx in vivo. The morphogenesis of the PK mutant was altered. The gI mutant replicated to normal titres in the oropharynx and could be recovered from the trigeminal ganglia but not from other parts of the central nervous system, suggesting that gI facilitates the spread of the virus from neuron to neuron. All mutants induced neutralizing antibody and complete or partial protection against a challenge infection. PK and gp63 were required for the induction of complete protection, although these proteins are reportedly not targets for neutralizing antibody or cytotoxic T cells.


Round table on control of Aujeszky's disease and vaccine development based on molecular biology. A summary is given on the 4 topics which were discussed during the round table and which represent current knowledge on the molecular biology of Aujeszky's disease (pseudorabies) virus. They include a review on 1. the genome and gene products of the virus; 2. the viral genes associated with virulence; 3. the immunological role of the viral gene products and 4. studies intended to compare the efficacy of several commercially available vaccines and to establish a possible correlation between antibodies against individual structural viral proteins and degree of protection. It was concluded that gI deleted vaccines appear to be the best choice for use in intensive vaccination programmes towards eradication of Aujeszky's disease virus. However, there remains a need for development of more potent vaccines which induce strong humoral and cell mediated immune responses and afford complete protection, virological protection included. It is often observed that live vaccine strains which are completely avirulent lose much capacity to replicate and spread within the vaccinated animal. It is, however, not excluded that a certain degree of dissemination may be needed to be fully efficacious. Loss of virulence may thus be accompanied by too much loss of immunogenicity. An improved genetic stability of live vaccine strains when they are obtained by genetic manipulation, possibly justifies a more widespread dissemination of the vaccine strain in the body compared to that with conventionally developed strains or compared to what is presently allowed.


Interactions of bovine and caprine herpesviruses with the natural and the foreign hosts. Bovine herpesvirus 1 (BHV1) and caprine herpesvirus 1 (CapHV1) are useful models to study virus-host interactions, as well as pathogenicity and latency, when comparing the outcome of infection in the natural and the foreign hosts. Molecular seroepidemiological analyses revealed that cross-reacting antibodies were mainly induced by glycoprotein gI (gB analogue), by the major capsid protein and by nonstructural proteins, whereas the most virus-specific antibodies were elicited by glycoproteins gIII and gIV. These glycoproteins, especially gIII (gC analogue), might therefore play an important role in the virus-host-interactions. As a basis for further studies, we re-evaluated observations concerning experimental infections with BHV1 and CapHV1 in the natural and the foreign hosts. All parameters indicated that both viruses were able to infect either host, but that the pathogenicity was restricted to the natural host. Latent virus could be reactivated exclusively from cows infected with BHV1. It was possible neither to reactivate BHV1 from goats, nor to reactivate CapHV1 from either species. The experiments indicated that the outcome of infection in the natural and the foreign host is dependent on host and viral factors, whereby gIII is only one important virus component involved. Further investigations in the host and host cell range of BHV1 and CapHV1 will help to clarify the role of factors responsible for virus-host-interactions.


Molecular biology of bovine herpesvirus type 4. Bovine herpesvirus type 4 (BHV-4) is a ubiquitous virus of cattle. Its genome is a 144 +/- 6 kb double-stranded DNA consisting of a unique central part (L-DNA) flanked at both ends by tandem repeats called polyrepetitive DNA (prDNA or H-DNA). The overall arrangement of genes has been obtained by the analysis of homologies between short BHV-4 DNA sequences and corresponding genes of Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS). The gene expression is temporally regulated. Glycoprotein precursor p (gp10/gp17) is expressed as gamma 1 polypeptide. Glycoproteins gp1, gp8, gp11 and their precursors are gamma 2 proteins. The analysis of strain variations allows the definition of two types of strains, based on the DNA patterns: the Movar 33/63-like and the DN 599-like strains. Only the M40 strain, isolated in India, fails to fit this classification. The genomic variations have been compiled to build a dendrogram showing three levels of divergence between BHV-4 strains or isolates. The available molecular data indicate that the BHV-4 genome shares much similarity with the DNA of EBV and HVS, two representative members of the gammaherpesvirinae. BHV-4 may therefore be classified in the subfamily gammaherpesvirinae.


Cell-mediated cytolysis of lymphoblastoid cells expressing Marek's disease virus-specific phosphorylated polypeptides. Cell-mediated immune responses against Marek's disease virus (MDV)-antigens were examined using reticuloendotheliosis virus (REV)-transformed lymphoblastoid cell line CU91 and three cell lines derived from CU91. CU210 was established by establishing a latent MDV infection in CU91. Transfection of CU210 with pNL1, a selectable plasmid or with pNL1 and the cloned BamHI A fragment of MDV DNA resulted in the establishment of CU212 and CU211, respectively. CU211 expressed a MDV-specific phosphorylated polypeptide, while CU210 and CU212 were negative for MDV antigens. Only CU211 was lysed by MDV-specific effector cells. All cell lines were lysed by syngeneic REV-specific effector cells, although high levels of expression of the phosphorylated protein reduced the level of REV-specific lysis.


Virus inactivation of fresh frozen plasma by a solvent detergent procedure: biological results. In order to increase the safety of blood products, we have developed a procedure for the virus inactivation of fresh frozen plasma. Several batches have been prepared and with the first 10 batches, each of them composed of 60 litres of plasma, we have determined a set of biological parameters. Virus inactivation was realised using TnBP (1%) and Octoxynol 9 (1%). After their elimination with castor oil using chromatography on insolubilized C18 resin, glycine was added and the pH of the plasma was adjusted to 7.4. Plastic bags were aseptically filled with a mean volume of 200 ml of plasma. The mean levels of coagulation factors were all over 0.7 U/ml and their recovery from initial plasma was nearly the same as total protein except for factor VIII:C. The net loss in factor VIII:C was 16%, when including the dilution of plasma. In vivo and in vitro tests demonstrated that in the final product there were no activated factors. As in fresh frozen plasma, the protein concentration was over 50 g/l and the potassium level lower than 5 mmol/l. According to these results, virus-inactivated plasma has the same qualities of fresh frozen plasma and could now replace it.


[Regulation of purine biosynthesis. I. Isolation of add:: MudJ (lacZ, Kanr) insertions and genetic mapping]. Report here is the isolation of adenosine deaminase deficient mutants and genetic mapping. Engineering transposon MudJ (lacZ, Kanr) was used for mutagenesis and six add:: MudJ were obtained among 20,000 Kanr transductants. Adenosine deaminase activity of these mutants were assayed and all are negative. Cotransduction analysis of add::MudJ indicated that add is 70% linked to pmi(31') and 37% linked to zxx1900::Tn10d-tet insertion which is 10% linked to purR(30'). Three points cross showed that add is located between pmi and Tn10d-tet insertion. Therefore the gene order is purR-zxx1900::Tn10d-tet-add-pmi.


[Ultrastructural changes in tumor cell nuclei during spontaneous regression of induced mouse sarcoma virus tumors]. Experimental induction of tumours by mouse sarcoma virus inoculation into the Balb/c mice is a suitable model for following up the mechanisms involved in the regression processes, that is, degradation of tumour cells. Using the electron microscope the analysis of the nucleus of tumour cells in different phases of regression (from 8 to 23 day after inoculation) was performed in order to observe the mechanisms of degradation of tumour cells. It has been concluded that the changes of apoptosis of tumour cells occurred most frequently.


[Association of liver cirrhosis, hepatitis B virus infection and hepatocellular carcinoma--immunohistochemical study]. In order to assess frequency and importance of the presence of alpha fetoprotein (AFP) and AlAChy as well as hepatitis B virus (HBV) infection antigens in the tissue of hepatocellular carcinoma (HCC) and in the liver tissue outside a tumour, there have been performed retrospective light-microscopic and immunohistochemical examinations of the liver tissue in 65 cases of HCC (39 autopsy and 26 biopsy cases). The finding of AlAChy in 100% of autopsy cases of HCC and 80.7% of biopsy cases of HCC compared with the presence of AFP in 33% of autopsy and 61.5% of biopsy cases point out that AlAChy is a more sensitive marker than AFP in diagnosing HCC. The presence of HBV infection antigens in 43.5% of autopsy cases and 23.5% of biopsy cases of HCC point to an important role of HBV infection in development of HCC in the studied population.


[Malignant fibrous histiocytoma of the bone]. Five cases of malignant bone fibrous histiocytoma diagnosed by bioptic material are presented. The authors state that the diagnosis of these rare bone malignant neoplasms do not present a larger diagnostical problem. However, some differential diagnostic problems are often met compared to other bone neoplasms, especially in osteosarcoma, so in these cases are recommended that together with routine histologic examinations in establishment of diagnosis should be also used enzymohistochemical methods, especially alkaline and acid phosphatase. Also, it is necessary to use immunohistochemical methods with corresponding markers which the authors have used in establishment of diagnosis of their cases. Based on the analysis of the presented cases the authors support the theory of J. Brooks on the histogenesis of these tumours within his theory of differentiation of the mesenchimal tissue which is easily recognized in recidives and metastases of soft tissue sarcomas by phenotypes organized at the lower level of differentiation.


[The ECoG of rats with genetic catalepsy]. Electrographic study was carried out in Wistar rats and the rats of genetical catalepsy (GC) strain. In contrast to Wistar rats epileptiform activity was observed in ECoG of GC rats being enhanced at the transition to a cataleptic state. Analysis of spectra and coherence of EEG revealed the presence of interhemispheric brain asymmetry in all the rats. In some frequency bands in GC rats inversion of interhemispheric asymmetry was found, which had been characteristic for Wistar strain. The highest interhemispheric synchronization of biopotentials was observed in the frontal cortical areas in GC rats and in the occipital areas in those of Wistar strain.


[Does the destruction of the catecholaminergic neurons in newborn rat pups influence the modulatory function of the cholinergic system?]. On outbred ratlings aged 21-31 days the influence was studied of the destruction of catecholaminergic (CA) system on the reactions of the neurones of the cortical somatosensory zone, elicited by the stimulation of the ischiatic nerve and modulation of these reactions after stimulation of the basal nuclei area (the source of the neocortex cholinergic innervation) and acetylcholine (ACh) microiontophoretic application. It is shown that destruction of CA system in newborn ratlings increases the reactivity of the somatosensory cortical neurones in 21-31 days old animals to sensory stimulation; it does not influence the efficiency of modulating action of the cholinergic system of the forebrain and leads to the increase of modulating influence of the applicated ACh. It is postulated that as the result of perinatal destruction of CA brain system, in the neocortex a specific morpho-functional organization is formed of structures and processes at which the modulating function of the forebrain cholinergic system turns out, by quantitative criterion, at least, to be compensated.


[The assessment of the subjective second by using the "individual minute" test]. Correlation of the two characteristics of the "individual minute" test with age and sex of the subjects was examined in 151 persons. Close correlation was observed between the first (counting in the course of 60 s) and the second (counting time up to 60 s) modes of testing, but not between the two modes of testing and age and sex. Results are discussed from the viewpoint of the existence of the common system of time interval estimation the method of their quantum organization being used in different investigation modes.


[The pathological dominant and the stable pathological state]. The pathological dominant is considered at a model of alcohol motivation which is in competitive relations with drinking motivation. The steady pathological state is considered at a model of seizure readiness in rats with audiogenic epileptiform seizures. The alcohol motivation might be ceased by the activation of competitive systems (thirst motivation), but the level of seizure readiness might be lowered intrasystemically, for example, by the habituation mechanisms.


[The reinforcing function of the emotions]. In the paper are summarised the results of investigations of the central mechanisms of reinforcing properties of emotions which have been carried out during the last years in Pavlov Physiological Department of the Institute for Experimental Medicine. An attempt is made to outline a perspective of the future development in this domain of the brain physiology which is a key one for the theory of conditioning.


[The contribution of Academician L. A. Orbeli to the theory of higher nervous activity]. In the paper are discussed the main directions of the investigation of the higher nervous activity, fruitfully developed by academician L. A. Orbeli and his school. These directions are the following: the evolutionary approach, the study of the influence of the sympathetic nervous and endocrine systems, of the role of the extracortical brain structures, especially the hypothalamus and the cerebellum, and the significance of pain and efferent systems for the higher nervous activity.


[Study results and prospects in the pathogenesis of neuroses]. An attempt is made to highlight the modern state of investigations concerning the experimental neuroses, to show new branches in studying pathogenesis of neuroses. A new concept is substantiated on a participation of the cerebral circulatory hypoxia at neuroses. Is demonstrated a high efficiency of prophylaxis and therapy of neuroses using the anti-oxidants of anti-hypoxic action.


[Movement-related brain potentials in 6- to 7-year-old children]. Spatial distribution and component structure of movement-related brain potentials (MRBP) were studied in 6-7-years-old children. The structure of Bereitschaftspotentials was found to be complex and heterogeneous the latency of P200 component being shorter than that in adults. MRBP recorded contained specific for children late negative wave that had been called postaction negativity, or PAN. The amplitudes of almost all MRBP components in children under study was the highest in the frontal areas. The results obtained may reflect neurophysiological changes associated with motor development.


[The outlook for the experimental study of the network functions of cortical cells during learning]. The main outcome of the experiments described in the paper is an idea on the gnostic cortical microset. Multineuronal activity recorded from the motor cortex of cats with a conditioned response to time and the following cross-correlation analysis revealed a strict distribution of interneuronal connections within the microsystems (between the adjacent neurons) and variable connections between the remote neurons during the active waiting stage of two minute interval. Additional analysis of the narrow (0.5 ms) peaks of the histograms allowed to form a view on the synaptic interaction in time. It was found that there was different temporal distribution of the spikes in the peak obtained due to correlograms of neuronal pairs. Some cortical neurons demonstrated a visible synaptic activation at the end of the waiting period when other signs of the temporary behaviour were absent. Pharmacological testing functional interneuronal connections with acetylcholine and Ca(2+)-suppressing drug EGTA have raised a question on the neurochemical specificity of the intra- and extracortical synapses.


[The mapping of cortical interaction in the asthenic neurosis syndrome]. Topographic mapping method was used to study the spatial-temporal organization of neocortical electrical activity at norm and asthenic neurosis. Patients with asthenic syndrome had shown right hemisphere dominance by the number of cortical interconnections in theta-band and interhemispheric discordance by coherence function.


[The functional coupling of the mechanisms for the selection of significant signals and the motor reactions they induce]. The data are described in favour of forming the integrative image of the significant signals in the mammalian brain. This image is created as a summary of the significant at this moment information, selected in the specialized areas of the cortex. The hypothesis is founded on the investigations of the cortico-striatal interrelations. They suggest that the results of the cortical analysis, which are dispersed through the specialized cortical fields, enter the striatum via the corticofugal pathways and transform into an integrative form in its neuronal network. The experimental data show that the striatal activity recorded during instrumental behaviour does not contain motor programs or commands, but reflects the peculiarity of the sensory environment, in which the movements are realized.


[The comparative psychology of primates]. The comparative study of cognitive activity of primates was carried out during individual and group training. In macaques, capuchins, and chimpanzees was studied the function of abstraction during the recognition of complex visual images and the transition from 2-dimensional plane images to 3-dimensional volume objects. Common features and distinctions were revealed for intellectual characteristics of different species of primates. The formation of intragroup competitive behaviour was observed in marmosets, macaques, and baboons in the process of training.


[The participation of the cholinergic system of the nucleus accumbens in the differentiation of sound signals in dogs]. Chronic experiments were carried out on five dogs with a defensive instrumental reflex (IR) associated with the maintenance of the flexor posture. Was studied the influence of carbocholine bilateral microinjections (0.05-0.10 mcg) into the nucleus accumbens (NAC) on the realization of the IR and on the behavioural differentiation of acoustic signals in a defensive situation. Cholinergic system of the NAC was shown to participate in both the motor and sensory mechanisms connected with the realization of motor responses to defensive and differentiation signals. Activation of the cholinergic system of the NAC led to an "improvement" of behavioural signal differentiation probably due to an increase of attention to significant stimuli. The cholinergic system of the NAC participated in the regulation of voluntary movements. Its influence was mainly excitatory and had unspecific and prolonged character.