Supraspinal influences on recurrent inhibition in humans. Paralysis of descending control of Renshaw cells in patients with mental retardation. The recurrent inhibition of alpha motoneurons was studied in 8 mentally retarded subjects (age 16-35 years), six of whom also had non-pyramidal or extrapyramidal motor alterations, manifesting as rigid and inflexible voluntary and/or postural movements. Despite a similar degree of mental retardation (Raven spatial general intelligence test), the other 2 cases showed much more modest changes in motor behavior. At rest, recurrent inhibition on soleus motoneurons was normal in all patients. In the 6 cases exhibiting more severe motor abnormality, the changes in Renshaw cell excitability, which occur during postural or voluntary contractions in normal subjects, were not found. This expressed the lack of supraspinal influences on Renshaw cells in these patients. On the other hand, supraspinal modulating influences on Renshaw cells were virtually normal in the remaining 2 patients. The absence of excitability changes of recurrent inhibition to postural or voluntary movements is discussed in relation to the abnormality of motor behavior observed in these patients. In addition, since paralysis of adaptive changes of recurrent inhibition has so far only been described in spastic subjects, the present study demonstrates that the descending pathways, which control recurrent inhibition gain, are different from those which, when damaged, lead to spasticity. Finally, our results indicate that the changes in motor behavior often associated with mental retardation cannot be regarded merely as the consequence of defective motor learning.


Central motor conduction in relation to contra- and ipsilateral activation. Differences of central motor conduction between slight activation of the target muscle (ipsilateral: IL) and strong contraction of the contralateral (CL) muscle following magnetic motor cortex stimulation were studied in 18 controls; responses were recorded at abductor pollicis brevis (APB) and tibial anterior (TA). For APB a clearly faster response was obtained with IL activation (mean: 1.7 msec). The amplitude increased only slightly. The reverse was found for TA muscle: amplitude nearly doubled with IL activation, but the latency did not change. In both facilitation procedures a clear correlation was found between left and right normalised amplitude (cortex amplitude/M wave) for APB and TA, but not for the TA response with IL activation. This points to a different mechanism of enhancement for the ipsilateral activation of TA muscle. It is argued that a rise in excitability of spinal motoneurones is largely responsible for the increase in amplitude. In clinical practice contralateral activation as a method of facilitation seems appropriate in most cases: lack of EMG contamination of the baseline makes it easier to read the latency onset. Only in cases of a low TA response can IL activation give a better response.


Gonadotropin induces expression of c-fos and c-jun genes in rat ovaries. It has been shown that the expression of protooncogenes, c-fos and c-jun, induced by growth factors and hormones plays important roles in cellular proliferation, tissue differentiation and transcription of certain genes. Since gonadotropin stimulates ovarian steroidogenesis and cellular proliferation, we investigated whether gonadotropin affects the expression of c-fos and c-jun genes in rat ovaries. The expression of mRNA coding side chain cleavage enzyme (P450scc), the rate limiting enzyme in ovarian steroidogenesis was also studied. The effect of gonadotropin was examined in female rats whose gonadotrophs were medically ablated by GnRH agonist (TAP-144-SR). After intravenous administration of pregnant mare's serum gonadotropin (PMSG:30 IU/rat), their ovaries were dissected out at various time intervals and total RNA was extracted. Changes in the levels of c-fos, c-jun and P450scc mRNAs were determined by Northern blot analysis. The levels of c-fos and c-jun mRNAs increased rapidly and transiently with the peak levels at 15 min after PMSG administration. The levels of both mRNAs were decreased by 30 to 60 min. On the other hand, the levels of P450scc mRNA started to increase 60 min after PMSG. These results indicate that gonadotropin-induced increase in the expression of c-fos and c-jun genes may play important roles in mediating the action of gonadotropin on the ovaries.


Postprandial control of gallbladder contraction and exocrine pancreatic secretion in man. To explore the interactions between cholecystokinin (CCK) and the cholinergic system, we compared the effect of cholinergic or peptidergic CCK blockade on gallbladder contraction and pancreatic enzyme secretion using atropine and loxiglumide (a specific CCK antagonist) as pharmacological tools. Gallbladder contraction was measured by sonography and pancreatic secretion by a marker perfusion and aspiration technique. Graded doses of exogenous CCK8 induced dose-dependent contractions of the gallbladder and increasing enzyme outputs. Loxiglumide (10 mg kg-1 h-1) abolished the gallbladder response and prevented an increase in pancreatic enzyme secretion to CCK8. Atropine (5 micrograms kg-1 h-1), however, only reduced gallbladder contraction and enzyme output to CCK8. Gallbladder volumes decreased maximally to 12 +/- 4% after oral food, whereas enzyme output and plasma CCK levels increased 6- to 8-fold. Loxiglumide completely abolished gallbladder contraction and inhibited enzyme secretion by 30%. Atropine caused a small reduction in gallbladder volumes, but essentially blocked postprandial enzyme secretion. The results indicate that CCK is the major regulator of gallbladder contraction with the cholinergic system modulating the response, while the exocrine pancreas is crucially dependent on a cholinergic background with CCK modulating the secretory response.


The long-term inhibitory effect of a Ca2+ channel blocker, nisoldipine, on cytosolic Ca2+ and contraction in vascular smooth muscle. The mechanism of the long-term inhibitory effect of a dihydropyridine Ca2+ channel blocker, nisoldipine, on contraction and cytosolic Ca2+ level ([Ca2+]i) was examined in isolated rat aorta. Nisoldipine inhibited the [Ca2+]i and muscle tension induced by high K+. The inhibitory effects were antagonized by a Ca2+ channel activator, 100 nM Bay k8644, and by a high concentration of Ca2+ (6.5 mM). Ultraviolet light, which has been shown to decompose dihydropyridines, attenuated the effects of nisoldipine. After nisoldipine had been removed from muscle bath, the inhibitory effect faded away slowly. The residual inhibitory effects on [Ca2+]i and muscle tension were antagonized by Bay k8644, high Ca2+ and ultraviolet light. These results suggest that the inhibitory effect of nisoldipine is caused by a decrease in [Ca2+]i as a result of inhibition of L-type Ca2+ channels, and that the residual inhibitory effects are caused by the same mechanism as the inhibitory effects of nisoldipine, namely the tight binding of nisoldipine to Ca2+ channels even after washout.


Effects of philanthotoxin-343 on CA1 pyramidal neurons of rat hippocampus in vitro. A synthetic analog of philanthotoxin-433, philanthotoxin-343 (PhTX-343), was tested in hippocampal pyramidal neurons in vitro. PhTX-343 (2 microM) did not significantly change synaptic transmission mediated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptors in the CA1 region of hippocampus. However, PhTX-343 significantly suppressed both the synaptic N-methyl-D-aspartate receptor-induced current (NMDA) obtained in the presence of CNQX(6-cyano-7-nitroquinoxaline-2,3-dione)/picrotoxin (10 microM) and the directly evoked NMDA receptor-induced current to pressure ejection of NMDA in the presence of tetrodotoxin (0.5 microM). A short transient facilitation of both types of NMDA response was seen immediately after the beginning of PhTX-343 application. Our results suggest that at high concentration (2 microM) PhTX-343 inhibits the NMDA-gated current, while the early facilitation occurred during an initial low concentration of the compound. Both facilitative and depressive actions of PhTX-343 are localized at the postsynaptic membrane.


Lugol's iodine dye-enhanced endoscopy in patients with cancer of the oesophagus and head and neck. Lugol's iodine dye indicates the presence of unsuspected early oesophageal cancers during endoscopy at which such cancers fail to show the characteristic black colour change. We evaluated Lugol's iodine dye-enhanced endoscopy in 17 patients with oesophageal cancer. In a further 37 patients with head and neck cancer we examined the use of Lugol's iodine since these patients have a 29% risk of synchronous oesophageal cancer. The oesophagus was sprayed with Lugol's iodine (1.5%) during endoscopy. Any areas not turning black were biopsied. In 13 patients with oesophageal cancer discrete areas beyond the macroscopically obvious primary tumour showed no change in colour. Biopsy revealed cancer in all cases. Six synchronous cancers were found in the head and neck group, one of which was identified only by the use of Lugol's iodine. Lugol's iodine augmented the information gained about the oesophageal mucosa during endoscopy. It revealed unsuspected cancer which altered the management of patients with primary oesophageal cancer as well as those with head and neck cancer. We recommend the routine use of Lugol's iodine-enhanced endoscopy for surveillance of all 'at risk' oesophageal cases.


[The structural-functional architectonics of the enteral portion of the metasympathetic nervous system in representative lower vertebrates--the teleosts]. The character of neurons and interrelations among them in submucous plexus were found to change along the digestive tract in lower vertebrates. The plexus looks like a network of ganglion-like structures composed of different number of cells. Specific feature of the plexus involves the absence of a regular ganglionic structure which seems to characterise a transitory stage in formation of the submucous plexus in vertebrates.


[The formation of a hypokinetic syndrome of the digestive system under conditions of weightlessness]. In space flights of various duration, the gastropancreatic complex turned out to be the most liable part of the digestive system. The main changes occurring in the gastrointestinal tract involve an increase in the peptic gastric potential, pancreatic hyperenzymemia, change of the glycemic curve forms, a decrease in the liver and pancreas density.


Refsum disease: the presentation and ophthalmic aspects of Refsum disease in a series of 23 patients. Refsum disease (heredopathia atactica polyneuritiformis) was first described in 1946 and is a rare recessively inherited metabolic disease affecting phytanic acid metabolism. It causes retinitis pigmentosa, cataracts, a chronic polyneuropathy, cerebellar ataxia and cardiac arrhythmias amongst other clinical signs. By limiting dietary intake, plasma phytanic acid levels fall with an improvement in the neurological signs. The onset of retinitis pigmentosa usually precedes biochemical diagnosis by several years by which time the retinal damage is severe. A series of 23 patients have been reviewed. There was an average delay of 11 years (range 1-28 years) between the patient presenting to the ophthalmologist and being diagnosed as having Refsum disease. Although serial examinations have failed to show a definite change in the course of visual deterioration with treatment, early diagnosis is important to prevent the development of neurological disease.


Expression of chimeric proteins encoded by the fused BLV reverse transcriptase: beta-galactosidase in Escherichia coli. The gene for bovine leukemia virus (BLV) reverse transcriptase was cloned in prokaryotic expression vector pUC8-2. After fusion of Escherichia coli lacZ gene to different parts of reverse transcriptase we detected expression of new proteins with molecular weights corresponding to the size of the hybrid genes. A coding region most probably responsible for about a hundred-fold decrease in expression of long fusion proteins has been identified. A few possible causes of this phenomenon were tested.


Distribution of nonmuscle actin during Xenopus laevis development. Using antisera specific for the N-terminal peptides of nonmuscle isoforms of actin we studied these proteins in the developing egg and embryo of Xenopus laevis. The antisera did not crossreact with muscle actin and did not react with the other nonmuscle isoform. By immunofluorescence we found a large maternal actin store in the oocyte, in the Balbiani body and the nucleoli, respectively. In the blastula, nonmuscle actin was preferentially expressed in the mitotic apparatus of the individual blastomeres. A gradual decrease in the amount of these isoforms in muscle tissue could be observed during development, apparently corresponding to an increase in the concentration of muscle specific isoforms. No differences between the distribution of the two isoforms could be found. The results would seem to indicate that development of the embryo is accompanied by a decline in the concentration of cytoskeletal actin isoforms down to an almost background level, accompanied by an increase in the concentration of the muscle specific isoforms. This points to potential function of nonmuscle actins in the early embryo.


Industrial experience with the detection of retroviruses. An important aspect of the virological assessment of cell substrates used to produce biologicals is their retroviral status. In this presentation we will attempt to document some of the practical experiences of industry when testing cell substrates for retroviruses using infectivity tests, reverse transcriptase and electron microscopy. We will also explore some important aspects of the experimental design of these retrovirus assay systems and review some results from the application of these methods in model, experimental systems.


Recent studies on retrovirus-like particles in Chinese hamster ovary cells. Highly concentrated (4000-7000-fold) culture fluids from CHO cells were analysed for the presence of retrovirus-like activity. Concentrates containing reverse transcriptase activity were detected and further purified by sucrose density gradient centrifugation. Particles banding at 1.13-1.16 g/ml were found to contain nucleic acid sequences and structural proteins related to those found in murine and other retroviruses. Analysis of the endonuclease gene has shown no intact open reading frames. Approximately 100-300 copies of the C-type sequences were present in the genome of CHO cell lines as well as in the DNA extracted from Chinese hamster liver, indicating that these sequences are present in the germ line of the species. Concentrates were analysed for infectivity by direct inoculation and co-cultivation with a series of detector cells. No evidence of infectivity was detected by reverse transcriptase, mink cell S+L- focus assay or by electron microscopic analysis of the inoculated detector cells after at least four passages in culture.


Differential expression of rat pancreatic islet beta-cell glucose transporter (GLUT 2), proinsulin and islet amyloid polypeptide genes after prolonged fasting, insulin-induced hypoglycaemia and dexamethasone treatment. The question posed by these studies was whether chronic adaptive changes in glucose-stimulated insulin secretion are accompanied by comparable changes in islet Beta-cell glucose transporter (GLUT 2) gene expression. Control, fasted (3-day), insulin-injected hypoglycaemic (5-day), and dexamethasone-treated (4-day) rats (n = 5 for each condition), were studied. After fasting significant decrements in proinsulin mRNA/microgram RNA (-32%, p < 0.05) and islet amyloid polypeptide mRNA/microgram RNA (-44%, p < 0.05) were observed, while there was no change in GLUT 2 mRNA/microgram RNA (-13%, p > 0.05). After insulin-induced hypoglycaemia, decrements in proinsulin mRNA/microgram RNA (-49%, p < 0.01) and islet amyloid polypeptide mRNA/microgram RNA (-44%, p < 0.01) were also observed, with no change in islet GLUT 2 mRNA/microgram RNA (-18%, p > 0.05). Dexamethasone treatment resulted in a marked stimulatory effect on proinsulin mRNA/microgram RNA (+236%, p < 0.001) and islet amyloid polypeptide mRNA/microgram RNA (+221%, p < 0.01), while again there was no change in islet GLUT 2 mRNA/microgram RNA (+0.3%, p > 0.05). Quantitative immunoblot analysis with a GLUT 2 specific antibody revealed no change in islet GLUT 2 protein with fasting, but a small decrease (-39 +/- 11%) in islet GLUT 2/microgram protein after insulin-induced hypoglycaemia. These results do not support the hypothesis that chronic changes in glucose-stimulated insulin secretion are accompanied by changes in GLUT 2 expression. In contrast to the lack of correlation with GLUT 2, there was a striking correlation between proinsulin and islet amyloid polypeptide mRNAs for all experimental conditions (r = 0.974, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Ion channels.

Ion channels. Insulin secretion by the pancreatic Beta cell is dependent upon transmembrane ion fluxes gated by the ATP-regulated potassium channel and the voltage regulated, L-type calcium channel. This work group examined major recent advances in the structure and modulation of ion channels and how those advances may pertain to the physiology of insulin secretion and the pharmacological treatment of Type 2 (non-insulin-dependent) diabetes mellitus. Structural studies have revealed that voltage gated ion channels are related, complex, and comprised of multiple components: sodium channels consist of three distinct subunits. L-type calcium channels, crucial to the insulin secretory response are structurally related to the sodium channel but contain additional subunits. Potassium channels are less closely related and appear to function as homotetramers. Modulation of ion channel activity is similarly complex: site specific phosphorylation by multiple protein kinases under the control of several intracellular second messenger systems may increase or decrease conductance. Subunit composition and relatively stable changes in the modal state of ion channels also appear to be critical to ion channel gating properties. Functional studies of the Beta-cell ATP-regulated potassium channel suggest two distinct nucleotide binding sites which link this channel to the metabolic state of the Beta cell. The multiple paths of ion channel modulation provide multiple targets for therapeutic intervention. Where detailed characterisation of ion channel structure has been achieved, those targets are being used for specific drug design. Such complete characterisation has not yet been achieved for Beta-cell ion channels and this presents a major goal for diabetes research.


Effects of estradiol on brain aminergic turnover of the female rainbow trout (Oncorhynchus mykiss) at the beginning of vitellogenesis. Brain serotonin and dopamine (DA) turnovers in the female rainbow trout were studied at the beginning of the vitellogenesis and related to blood estradiol (E2) levels; pituitary and plasma gonadotropin (GtH) were also assayed. Ovariectomy did not modify brain aminergic turnover. E2 replacement on ovariectomized fish increased hypothalamic DA turnover (increased DA and increased DA metabolites). E2 stimulated GtH synthesis (positive feedback) but did not enhance GtH release; hypothalamic E2-mediated aminergic inhibition upon release was suspected. Individual relations between blood E2 levels and catecholaminergic neurotransmitters were determined. A linear positive correlation (r = 0.82) was found for the hypothalamus, but not for the pituitary, the preoptic area, or the telencephalon. These data suggest that an activation of hypothalamic tyrosine hydroxylase (the limiting step of catecholamines synthesis) by E2 could develop as vitellogenesis proceeds.


Structural characterization of tachykinins (neuropeptide gamma, neurokinin A, and substance P) from a reptile, Alligator mississipiensis. An extract of the whole brain of the alligator (Alligator mississipiensis) contained very high concentrations of substance P-like immunoreactivity (405 pmol/g wet tissue) and neurokinin A-like immunoreactivity (514 pmol/g), as measured with antisera raised against the mammalian peptides. The primary structure of alligator substance P was established as: Arg-Pro-Arg-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. This sequence is the same as that of chicken substance P and shows one substitution (Arg for Lys3) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Neuropeptide gamma was the most abundant peptide and its primary structure was established as Asp-Ala-Gly-Tyr-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser- Phe-Val-Gly-Leu-Met-NH2. This sequence shows one substitution (Tyr for His4) compared with mammalian neuropeptide gamma. The second component was identical to mammalian neurokinin A. A peptide with the chromatographic properties of mammalian neuropeptide K was not identified in the extract.


Misreading of the argI message in Escherichia coli. It has previously been shown that either phenylalanine codon, UUU or UUC, could be misread as leucine during phenylalanine starvation, if the codons encoded residue 8 of the Escherichia coli argI gene product, ornithine transcarbamylase (OTC). However, no leucine misincorporation was detected when either of these same codons encoded residue 3. Here we report that leucine misincorporation can be directed by a UUU codon for residue 3 of OTC during phenylalanine starvation, if the argI gene has been mutated so that the codon preceding the UUU has been changed from the rarely used glycine codon GGG to the more commonly used GGC.


How can peptide vaccines work?

How can peptide vaccines work? Peptide antigens frequently induce antibodies which recognise the denatured form of a protein from which their sequences are derived. However, the ability to induce antibodies which crossreact with the native, fully folded form of the protein is less commonly observed. Although there is a growing number of examples in which this is the case, the ability to predict peptides having this property is extremely limited. Given the large surface areas involved in antibody/antigen interaction it is surprising that peptides could ever induce antibodies which would recognise the native protein well enough to have biological activity, such as the neutralization of infectivity. A mechanism is proposed to explain such observations which is compatible with many of the properties of antipeptide antibodies.


The conformational specificity of viral epitopes. Four types of antigenic sites found in viruses are discussed: cryptotopes, neotopes, metatopes and neutralization epitopes. The role played by conformation on the specificity of viral epitopes is illustrated in the case of tobacco mosaic virus and influenza virus. It appears that mechanisms reminiscent of induced fit contribute to the recognition of viral epitopes by antibodies.


Variation in O-antigens, niche-specific selection and bacterial populations. Bacterial populations usually consist of distinct clones, often apparently adapted to specific niches. A formal model is developed whereby niche-specific selection maintains the polymorphisms involved in clonal adaptation. Infrequent transfer of non-adaptive alleles to a clone is balanced by the selection for the resident adaptive allele. The model can account for the extensive polymorphism in surface antigens observed in bacteria, and also for the existence of sympatric clones of pathogenic species which differ in host range and/or mode of pathogenesis. Niche-specific selection combined with low levels of genetic transfer can also account for the high level of neutral variation in bacteria, and indirectly for their ability to respond rapidly to environmental changes.


Identification of Staphylococcus epidermidis using a 16S rRNA-directed oligonucleotide probe. Staphylococcus epidermidis is the most medically significant of the coagulase-negative staphylococci. An oligonucleotide probe (pSe) for identification of S. epidermidis was defined by comparing the sequences of the 16S rRNA variable region V6 from numerous coagulase-negative staphylococci. In order to increase the sensitivity of the detection, polymerase chain reaction amplification of the variable region with primers based on the conserved flanking sequences was applied. The detection limit of the polymerase chain reaction assay combined with pSe probe was shown to be 1 fg which corresponds to about one single bacterium. Additionally, a sensitive, non-radioisotopic system with chemiluminescence detection was tested.


DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities. A protocol was devised which permitted the extraction of DNA from deep marine sediments up to 503 m below the sea floor. These sediments have been laid down over the last 3 million years. 16S rRNA gene sequences were amplified from the DNA by the polymerase chain reaction. The details of the successful extraction and polymerase chain reaction methodology varied between samples from different depths. This emphasizes the attention to detail required to allow the diversity of bacteria in these deep sediments to be studied.


Platelet-induced thrombin generation time: a new sensitive global assay for platelet function and coagulation. Method and first results. A new sensitive test--platelet-induced thrombin generation time (PITT)--is described, in which the formation of thrombin in partially anticoagulated platelet-rich plasma (PRP) leads to aggregation immediately followed by coagulation of PRP. 0.6 ml PRP are rotated in a disk-shaped cuvette within the light beam of a photometer. In PITT, platelets stick to the cuvette wall and, mediated by a large PRP/surface/air interface at the cuvette wall, are activated and participate in thrombin formation which leads to aggregation and clotting. The times from onset of rotation until aggregation (Ta) and until coagulation (Tc) of the PRP samples are recorded. PITT was very sensitive and detected low concentrations of unfractionated heparin (0.01 IU/ml) in vitro. PITT parameters were significantly prolonged ex vivo 2 h after oral administration of acetylsalicylic acid (0.5 g) and after single subcutaneous injections of heparin (5,000 IU). Patients receiving phenprocoumon prophylaxis had markedly prolonged Ta and Tc values (longer than 20 min, n = 23). Patients with recent thrombotic episodes had markedly shorter values than healthy volunteers. PITT may become a very sensitive global test to detect mild hemorrhagic disorders, to monitor the effects of antithrombotic drugs and to detect patients with a risk of vascular occlusions.


Localization of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) in the rat to chromosome 4 and implications for the evolution of mammalian chromosomes. We have isolated a partial cDNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) in the rat. This cDNA hybridizes to a 6.1-kb RNA transcript from the human T84 epithelial cell line and a similarly sized transcript from the rat parotid gland. The nucleotide sequence of this cDNA shows 80.5% identity to the human CFTR cDNA sequence, and the deduced amino acid sequence of rat CFTR shows 75.5% identity to the amino acid sequence of human CFTR. We have used this cDNA to map the location of the gene encoding CFTR to rat chromosome 4. This result places CFTR within a syntenic group on rat chromosome 4 and on human chromosome 7 that includes the genes encoding interleukin 6 (IL6), erythropoietin (EPO), P-glycoprotein 1 (PGY1), and T cell receptor beta chain (TCRB). This group is divided between chromosomes 5 and 6 in the mouse. Mapping of CFTR to rat chromosome 4 shows that this syntenic group has been divided in the mouse lineage during the past 15 million years and further localizes that breakpoint to a sequence homologous to the human chromosome 7q21.1 and 7q32 region. Similarly, a group of five genes, CFTR, TCRB, HOX1, parathyroid hormone-like hormone (PTHLH), and Kirsten rat sarcoma 2 viral (v-Ki-ras2) oncogene homolog (KRAS2), is syntenic on rat chromosome 4 and mouse chromosome 6, but is divided between human chromosomes 7 and 12. These data suggest that the ancestral mammalian chromosome appeared as the present day rat chromosome 4, with all six genes grouped together, and that chromosomal breakages have occurred in the mouse and human lineages since the mammalian divergence.


Conservation of a 23-kDa human transplantation antigen in mammalian species. A group of transplantation antigens, referred to as tum- antigens, were identified in mouse tumor cells that had been mutagenized to produce variant cells and were recognized by clonal cytolytic T lymphocytes (CTL). Alterations in these variant cells that were recognized by CTL resulted from point mutations in the genes of specific proteins. We have isolated human and bovine cDNA clones that encode the homologs of the mouse tum- antigen P198. This 23.6-kDa protein is highly basic with a predicted pI of 11.55. p23/P198 is highly conserved across mammalian species, with > 94% identity (97% including conservative substitutions) among the human, bovine, and mouse deduced amino acid sequences. The nucleotide sequences of both the coding and 5'- and 3'-untranslated regions from human, bovine, and mouse are also highly conserved with > 88% identity in the coding regions. Hybridization of poly(A)+ RNA from various mammalian sources with cDNA and oligonucleotides specific for the coding region identified two mRNAs of 1.2 and 0.8 kb, whereas probes specific for the 3'-untranslated region between two consensus polyadenylation signals hybridized with the 1.2-kb, but not the 0.8-kb, mRNA. The abundance of the 1.2-kb mRNA relative to that of the 0.8-kb species varied depending upon the cell type. A single predominant transcription initiation site was mapped by primer extension. These studies indicate that this highly basic 23.6-kDa protein is encoded by two major mRNA species that differ only in the length of their 3'-untranslated regions and that the mechanism that gives rise to these two mRNAs, utilization of alternative polyadenylation sites, is conserved across species.(ABSTRACT TRUNCATED AT 250 WORDS)


A delayed-type hypersensitivity-inducing T-cell epitope of Semliki Forest virus mediates effective T-helper activity for antibody production. The rational development of peptide vaccines requires the identification of both B- and T-cell epitopes. In this study, potential T-helper cell epitopes of Semliki Forest virus (SFV) were identified on the basis of their ability to induce delayed-type hypersensitivity (DTH) in mice using recombinant SFV fragments produced as hybrid proteins with beta-galactosidase in Escherichia coli and synthetic peptides coupled to beta-galactosidase. Although the tested fragments spanned almost the entire amino acid sequence of the structural proteins of SFV, only one DTH-inducing region (located between amino acid 137 and 151 of the SFV E2 membrane protein) was identified. Peptides containing this E2 region stimulated lymph node cells from SFV-primed mice in vitro. The ability of the identified T-cell epitope to induce a specific T-helper response in mice was evaluated using synthetic peptides that contained combinations of the DTH-inducing region and different previously identified linear B-cell epitopes of E2. These peptides proved able to induce an antipeptide IgG response in mice in an H-2d-restricted fashion. One of the peptides was also able to induce high titres of IgG reactive with SFV-infected cells and protected 70-100% of the peptide-immunized mice after challenge with virulent SFV. Our findings suggest that DTH and T-helper activity are mediated by different doses of the same T-cell epitope.


Calcitonin gene-related peptide (CGRP) activates human neutrophils--inhibition by chemotactic peptide antagonist BOC-MLP. The effect of the neuropeptides substance P, neurokinin A and alpha-calcitonin gene-related peptide (CGRP) on human neutrophil granulocytes was investigated. Substance P induced secondary granule secretion at a concentration of 100 microM. CGRP induced a significant secretory response at 10 microM and thus appeared to be about 10 times more potent than substance P. Calcitonin and a fragment of CGRP, CGRP(8-37), had no effect on neutrophil degranulation. The chemotactic peptide antagonist BOC-MLP (100 microM) inhibited lactoferrin secretion mediated both by CGRP and chemotactic peptide FMLP almost completely, while secretion in response to tumour necrosis factor (TNF) was unaffected. Results from receptor binding studies showed that CGRP and N-formyl-methionyl-leucyl-phenylalanine (FMLP) do not compete for binding. This indicates that CGRP does not exert its effects by binding to the chemotactic peptide receptor. CGRP induced a rapid increase in the cytosolic-free calcium concentration and this increase was not, unlike that induced by FMLP, abolished by preincubation of the cells with pertussis toxin (1000 ng/ml). Therefore CGRP signal transduction in neutrophils appears to involve rapid changes in the cytosolic-free calcium concentration but not a pertussis toxin-sensitive G-protein. In summary, this is the first report to show that CGRP can directly activate neutrophil granulocytes, and this probably occurs via a cell surface receptor which is distinct from that of FMLP although both the CGRP and FMLP-mediated effects can be blocked by BOC-MLP.


Contribution of ICAM-1 and VCAM-1 to the morphological changes in monocytes bound to human venous endothelial cells stimulated with recombinant interleukin-4 (rIL-4) or rIL-1 alpha. The present study focused on the question of whether the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the surface of cultured human venous endothelial cells (EC), stimulated with recombinant interleukin-4 (rIL-4) or rIL-1 alpha, contributes to the stretching of human monocytes following their binding to EC. Stimulation of monolayers of venous EC with rIL-4 for 24 hr induced marked expression of VCAM-1 but not ICAM-1 on EC, increased the adhesiveness of EC for monocytes but did not promote stretching of EC-bound monocytes over the surface of EC. Stimulation of EC with rIL-1 alpha for 24 hr induced surface expression of both ICAM-1 and VCAM-1, enhanced the binding of monocytes to EC and increased the percentage of EC-bound monocytes with a stretched morphology about 2.7-fold. Anti-ICAM-1 but not anti-VCAM-1 mAb markedly reduced the percentage stretched monocytes on rIL-1 alpha-stimulated EC. We conclude that ICAM-1 but not VCAM-1 on cytokine-stimulated EC is essential for the stretching of EC-bound monocytes.


Hidden high-avidity anti-DNA antibodies occur in normal human gammaglobulin preparations. An ability to detect hidden high-avidity DNA-binding protein in human gammaglobulin samples was investigated. Using ion exchange chromatography on QAE-Sephadex A-50 a highly positive Farr assay DNA-binding fraction was reproducibly isolated from several commercial normal human gammaglobulin preparations. The estimated dissociation constant had a value of 1.04 x 10(-11) M thus confirming high avidity protein-DNA complex formation. Anti-DNA antibodies (Ab) ELISA data revealed that immunoglobulin (Ig) G, IgM and IgA participated in the protein-DNA interaction. Inhibitory experiments involving a number of polynucleotides, synthetic and natural polyanions demonstrated that both double-stranded DNA (dsDNA) and heat-denatured DNA but not RNA inhibited the protein-3H-DNA binding. Generally, the inhibiting effect was more pronounced when purine base-containing polynucleotides and polydeoxy- rather than polyribonucleotides were used. Synthetic polyanions and normal human sera (NHS) also markedly reduced the binding. The presence of hidden high-avidity DNA-binding antibodies in normal gammaglobulin preparations was suggested.


Modulation of anti-acetylcholine receptor antibody specificities and of experimental autoimmune myasthenia gravis by synthetic peptides. Synthetic peptides corresponding to selected sequences from the nicotinic acetylcholine receptor (AChR) were employed to identify possible antigenic determinants within the receptor which can modulate the anti-AChR response and experimental autoimmune myasthenia gravis (EAMG). Immunization of rabbits with peptides T alpha 73-89, T alpha 351-368, T delta 354-367 and H alpha 351-368, prior to AChR inoculation, affected the course of EAMG in six out of eight rabbits. These six protected rabbits survived three inoculations of AChR and survived for at least five months after the third injection with AChR, whereas control rabbits died following one or two injections of AChR. The survival of peptide-preimmunized rabbits injected with AChR seemed to correlate with the antibody specificities in immunoblots. Following AChR inoculation there was a shift in reactivity, from a subunit-restricted response, to reactivity with all subunits of the receptor. This shift was delayed in protected rabbits. This may indicate that the reactivity with the entire Torpedo receptor molecule represents a loss of tolerance to AChR which culminates in the autoimmune disease, EAMG.


Immunoglobulin heavy and light chain gene sequences of a human CD5 positive immunocytoma and sequences of four novel VHIII germline genes. To analyse the V genes expressed by an IgM lambda CD5-positive immunocytoma heavy and light chain V region genes were cloned and sequenced. The heavy chain is composed of a previously undescribed VHIII gene joined to an unknown D gene and to JH4. The light chain V region is composed of a V lambda II gene rearranged to J lambda 1. In an attempt to clone the germline counterpart of the VHIII gene expressed in the immunocytoma PCR amplifications of genomic DNA were carried out and four previously unknown VHIII genes were identified. As several independent clones for the heavy and light chain V region genes were sequenced the rate of somatic mutation of the V genes was calculated to be below 2 x 10(-5)/bp/cell division.


Tumor necrosis factor-alpha stimulates both apical and basal production of HIV in polarized human intestinal HT29 cells. The human colon epithelial cell line HT29 can be infected by selected strains of the human immunodeficiency virus (HIV) [9]. In the present study, it is shown that tumor necrosis factor-alpha (TNF-alpha) is a potent stimulator of HIV replication in chronically infected differentiated HT29 cells, but not in undifferentiated cells. The polarity of HIV production upon TNF-alpha stimulation has been studied in polarized monolayers of differentiated HT29 cells grown on porous-bottomed dishes. It is shown that the cytokine induced a dramatic increase of HIV production through the two opposite sides of the monolayer, i.e. the apical and basolateral plasma membrane domains. The effect of TNF-alpha was mainly localized at the level of viral mRNA synthesis as demonstrated by in situ hybridization. These data support the concept that cytokines released as a result of intestinal inflammatory responses could promote HIV replication and contribute to the gastrointestinal disease in HIV-infected patients.


Different response of astrocytes and Bergmann glial cells to portacaval shunt: an immunohistochemical study in the rat cerebellum. The present study was performed in order to follow the response of rat cerebellum astroglial cells (Bergmann glial cells and astrocytes) to long-term portacaval shunt (PCS), by means of glial fibrillary acidic protein (GFAP) and vimentin immunoreactivities. Bergmann glia accumulated GFAP in response to PCS, whereas astrocytes decreased GFAP immunoreactivity when compared to control rats. The increase of GFAP occurs in cells located in the cerebellar layer where glutamate is mainly released. Since the vimentin content remained unaltered in response to PCS, when compared to control rats, it can be concluded that only the GFAP filaments are affected by PCS. Nevertheless, GFAP immunoreactivity presents regional differences in the cerebellar astroglial population, and the factors responsible for these variations are still unknown.


Heterogeneity in gap junction expression in astrocytes cultured from different brain regions. Heterogeneity among astrocytes suggests that their role in the central nervous system is more complex than is commonly recognized. This paper describes just such a functional difference, comparing gap junctions in astrocytes derived from two brain regions. Astrocytes, both in situ and in culture, employ gap junctions as a means of intercellular communication. Recent evidence utilizing cultured rat cortical and striatal astrocytes has shown that these channels consist of subunits of connexin 43, the same protein as that composing cardiac gap junctions. Here we report that astrocytes cultured from neonatal rat hypothalamus contain a greater number of functional channels than astrocytes from the striatum, a difference reflected in both connexin 43 protein and mRNA. Specifically, in hypothalamic astrocytes the level of connexin 43 protein was approximately four times that found in comparable cultures from the striatum, as determined by immunoblotting. Complementary results from immunocytochemical experiments using an antibody specific for connexin 43 reveal significantly greater fluorescence in astrocytes cultured from the hypothalamus as compared to those from the striatum. Northern blot analysis showed that connexin 43 mRNA levels were also approximately 4-fold greater in the hypothalamic cultures, consistent with the difference seen by immunoblotting. Finally, dye coupling studies using confluent cultures consistently showed that within 1 min Lucifer Yellow injected into striatal astrocytes spread to immediately surrounding cells while in hypothalamic astrocytes dye often spread to apparent third or fourth order neighbors within the same time period. Thus, the higher level of connexin 43 expression seen in hypothalamic astrocytes results in cells with greater numbers of functional channels.(ABSTRACT TRUNCATED AT 250 WORDS)


Solid-phase synthesis of bovine pancreatic trypsin inhibitor (BPTI) and two analogues. A chemical approach for evaluating the role of disulfide bridges in protein folding and stability. The linear sequence of bovine pancreatic trypsin inhibitor (BPTI) has been assembled by stepwise Fmoc solid-phase peptide synthesis on a polyethylene glycol-polystyrene (PEG-PS) graft support with p-alkoxybenzyl ester anchoring. Similar methods were used to prepare two analogues, the first with all six half-cystine (Cys) residues replaced by alpha-amino-n-butyric acid (Abu), and the second with replacement of Abu at four Cys positions while retaining the native pairing between positions 14 and 38. Following cleavage from the support, the linear molecules (reduced form) were purified by semipreparative reversed-phase high performance liquid chromatography (HPLC). The native structure of BPTI was then formed by oxidation of a dilute solution of the protein at pH 8.7 in the presence of oxidized glutathione. The BPTI analogue with one disulfide bridge was obtained following treatment with dimethyl sulfoxide (DMSO)-pH 6 buffer (1:9). Overall yields of homogeneous proteins were 2-4%, and further characterization was provided by amino acid analysis, sequencing, ion electrospray mass spectrometry, analytical HPLC, and capillary zone electrophoresis (CZE). Purified synthetic BPTI with the native sequence was indistinguishable from natural material by the analytical and biophysical criteria applied, including circular dichroism (CD) spectra and inhibition of trypsin action. Studies are in progress to evaluate conformational features of the analogues which respectively lack two, or all three, of the native disulfide bridges.


Human astroglial but not microglial cells synthesize alpha 2-macroglobulin in vitro. Alpha 2-macroglobulin (alpha 2M) is a serum proteinase inhibitor with a broad specificity. At present its role in human brain is unknown, but recent data report its presence in the CNS, particularly at glial level. Previous studies from our group demonstrated the synthesis and secretion of alpha 2M in different glial cultures derived from an astrocytoma and a glioblastoma. In the present study a human fetal astroglial cell line and two microglial established cell lines are examined for the presence of alpha 2M by using polyclonal antibodies in ELISA and immunofluorescence assays. While we observed a strong specific positivity in the cytoplasm and in the culture medium of the GFAP, vimentine positive cells, no positivity was detected in FcR, lysozyme positive microglial cells. Since interaction of proteinases and proteinase inhibitors appear to play a crucial role in the development of neuroimmunological competence, these data suggest a dissociation of macro and micro-glia immune functions.


Solid phase peptide synthesis of lipopeptide vaccines eliciting epitope-specific B-, T-helper and T-killer cell response. Lymphocyte subpopulations involved in the self and nonself recognition processes are antibody producing cells, T-helper cells and T-killer cells. By using lipopeptide adjuvants and lipopeptide-antigen conjugates each of the major pathways of immune response can be specifically addressed on the molecular level of minimized synthetic lipopeptide vaccines. The immunologically active principle of the lipopeptide constructs is the synthetic N-terminus of bacterial lipoprotein, tri-palmitoyl-S-glycerylcysteine, which can be covalently linked to B-, T-helper and CTL epitopes. Methods of multiple peptide synthesis based on Merrifield's solid-phase synthesis allow the economic production of the high numbers of overlapping lipopeptides required for the complete immunological screening of viral proteins.


Expression of the carbohydrate epitope 3-fucosyl-N-acetyl-lactosamine (CD15) in the vertebrate cerebellar cortex. The distribution of the carbohydrate epitope CD15 was investigated on paraffin sections of the brains of man and mammals (monkey, dog, rabbit, rat, mouse, dolphin), reptile, bird and fish by means of immunohistochemistry. This paper demonstrates a differential expression of the CD15 epitope in the cerebella of these various vertebrates. CD15 positivity was found on glial cells and neuronal structures. In adult brains two major distribution patterns were distinguished: one with very intense labelling of the molecular layer, for which the rat is representative, the other with very low immunoreactivity in this layer (mouse). Amongst the rodents (mouse, rat and rabbit), as well as the monkey and human, the positivity in the molecular layer could be attributed to Bergmann fibres of the Golgi epithelial cells. A typical parasagittal band pattern, present in the mouse molecular layer for CD15, which is absent in rat and rabbit molecular layer, is present during human cerebellar development. CD15 positivity on neuronal structures is found on parallel fibres in the developing human, on the lower stellate cells in the dog, and in climbing fibres of the dolphin and, presumably, the catfish too. Moreover, within the parrot cerebellum, large CD15-positive mossy fibre-like endings are found just at the infraplexiform layer.


Mast cell heterogeneity in man: unique functional properties of skin mast cells in response to a range of polycationic stimuli. Human mast cell heterogeneity was assessed by histochemical and detailed functional criteria using mast cells isolated from foreskin, uterine myometrium and lung parenchyma. The skin mast cells were histochemically distinct from their counterparts in the other two tissues by being predominantly insensitive to blockage of dye-binding following formalin fixation (ca. 80%). Functionally, a wide range of structurally diverse polycationic compounds induced selective histamine release from the skin mast cells (ca. 10% at top concentrations) although these cells were less responsive to immunological ligands and calcium ionophores when compared with the uterine and lung cells. The basic compounds, polyarginine and histone, proved to be more generalised histamine liberators as compared with their structural analogues, polylysine and protamine sulphate, probably by virtue of their high content of arginine residues and hydrophobic nature (histone). Studies with the anaphylatoxin, C3a, and its analogues 21R and C3ades Arg on skin mast cells emphasized the importance of basic amino acids for histamine-liberating peptides. Skin mast cells also proved more susceptible than their uterine counterparts to lysis by the detergents, Triton X-100 and Tween 20, suggesting that fundamental differences in membrane structure and/or fluidity might account for functional heterogeneity within the human mast cell population.


1-beta-D-arabinosylcytosine and 5-azacytidine induce internucleosomal DNA fragmentation and cell death in thymocytes. Incubation of mouse thymocytes with arabinosylcytosine or 5-azacytidine induced dose-dependent internucleosomal DNA cleavage followed by cell death. This process was RNA and protein synthesis-dependent, since DNA fragmentation and cell death was inhibited by actinomycin D and cycloheximide. The results suggest that the cytidine analogs induce apoptosis, a programmed cell death, in thymocytes. The DNA cleavage induced by arabinosylcytosine and 5-azacytidine was inhibited by deoxycytidine and cytidine, respectively, suggesting that phosphorylation of these antimetabolites is required to induce DNA cleavage. DNA fragmentation was unaffected by the addition of aphidicolin or 3-aminobenzamide, indicating that DNA cleavage is not due to the inhibition of DNA synthesis or repair. Other antimetabolites including methotrexate, fluoropyrimidines and thiopurines failed to induce DNA fragmentation. Arabinosylguanine induced DNA fragmentation similar to that produced by the cytidine analogs, suggesting similarity to the selective sensitivity of T lymphocytes to deoxyguanosine toxicity. The precise mechanism by which DNA cleavage is induced remains unclear, but the present study shows that certain antimetabolites act on cells not only by inhibiting proliferation, but by inducing apoptosis with internucleosomal DNA fragmentation.


Age-related expression patterns of the CD15 epitope in the human lateral geniculate nucleus (LGN). The age-related distribution of the trisaccharide epitope 3-fucosyl-N-acetyl-lactosamine (CD15) was evaluated in the human lateral geniculate nucleus (LGN). Coronal paraffin sections from individuals between the 12th week of gestation to 99 years of age were processed for immunohistochemistry using monoclonal antibodies against the CD15 epitope. CD15 immunoreactivity was present in the neuropil from the 14th week of gestation with a graded pattern along the anteroposterior and mediolateral axes of the LGN. Immunoreactivity then became preferentially located within the future cell layers, shortly before cellular segregation was visible in Cresyl Violet stained sections. Maximal CD15 expression occurred from the 22nd week of gestation until the beginning of visual experience (second week of postnatal life). During the subsequent period the spatial pattern of CD15 expression changed. Whereas immunoreactivity in the cell layers gradually disappeared, CD15 positive astrocytes became transiently concentrated in the intercellular layers. The staining within the interlaminar region was best developed at about one year of postnatal life. The adult pattern was found at around 10 years of age, when the LGN appeared almost unstained. Two stages of CD15 expression can thus be separated. The first is characterized by neuropil staining and is synchronized with the time profile of neuronal maturation and of formation of non-stabilized contacts. CD15 is at this time possibly correlated with structural instability and increased vulnerability but at the same time with a high degree of plasticity. The second, peri- and postnatal stage is characterized by CD15 positive astrocytes. These appeared when CD15 in the neuropil disappeared. This loss of CD15 expression in the neuropil occurs during the phase of experience-dependent establishment of the mature interconnectivity and probably heralds loss in plasticity. The time-related expression pattern of CD15 is therefore compatible with the idea that CD15 levels reflect different degrees of developmental determination of retino-geniculate interaction.


Expression of the CD15 epitope in the human magnocellular basal forebrain system. The distribution of the carbohydrate epitope 3-fucosyl-N-acetyl-lactosamine (CD15) has been immunocytochemically evaluated in coronal paraffin sections through the magnocellular basal forebrain system--the nucleus basalis of Meynert, the nucleus of the diagonal band of Broca and the medial septal nucleus--of 202 human brains. The brains derived from differently aged controls (n = 54) and from patients suffering from organic brain diseases (n = 129) or psychiatric disorders (n = 19). In 30 cases dementia was clinically diagnosed. CD15 first appeared around birth when it became localized on singular astrocytes. The astrocyte number and process density steadily increased, and at approximately 12 years the typical adult-type pattern was acquired. Considerable variations in the expression patterns were noted with regard to the astrocyte number, the intensity in immunostaining and the process relations of CD15-positive astrocytes with the magnocellular neurons. In the light of these variations, and of conflicting additional changes in other areas of most diseased brains, it was difficult to correlate different intensities and patterns to specific diseases. The results, however, provide evidence for an increase in CD15 expression and in process network density of astrocytes in the lateral part of the nucleus basalis of Meynert in cases of Huntington's disease.


Critical role of HLA-DR beta 1 residue 58 in multiple polymorphic epitopes recognized by xenogeneic and allogeneic antibodies. In a previous study, we identified glutamic acid at position 58 in DR (beta 1*1101) as critical for the epitopes recognized by the DRw11-specific mAb GS88.2, as well as the I-LR1 mAb that recognizes a polymorphic epitope on DR(alpha,beta 1*1101) and some DP molecules. The purpose of this study was to determine whether other polymorphic residues contribute to these epitopes and whether DR beta glutamic acid or alanine 58 and DP beta glutamic acid 56, the analogous position in DP beta, contribute to epitopes recognized by other anti-class-II mAb and allosera. Site-directed mutagenesis and transfection were used to produce cells bearing wild-type or mutant class II molecules that were analyzed with mAbs by flow cytometry and with human allosera by absorption and subsequent microcytotoxicity assays. These studies demonstrate that the residue at DR beta position 58 plays a central role in at least three different mAb epitopes and an epitope recognized by anti-DRw11 allosera. Substitution of glutamic acid for alanine at position 58 of eight DR beta chains caused gain of binding of four mAbs to all of the mutant molecules, except DR(alpha,beta 4*0101). These data suggest that the side chains of DR beta 58 and DP beta 56 point outward from the alpha-helix and directly contact antibody.


Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia. A thermostable lipase from Pseudomonas cepacia has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification included treatment of the culture supernatant with acrinol, hydrophobic interaction chromatography, and gel filtration. The enzyme was a monomeric protein with M(r) of 36,500 and pI of 5.1. The optimal pH at 50 degrees C and optimal temperature at pH 6.5 were 5.5-6.5 and 55-60 degrees C, respectively, when olive oil was used as the substrate. Simple triglycerides of short and middle chain fatty acids (C < or = 12) were the preferred substrates over those of long chain fatty acids. The enzyme cleaved all the ester bonds of triolein, with some preference for the 1,3-ester bonds. The enzyme retained all its activity even after incubation at 75 degrees C (pH 6.5) for 30 min. Further, the activity was not impaired during 21 h storage at pH 6.5 in 40% water-miscible solvents including methanol, ethanol, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, and dioxane. The addition of dimethylsulfoxide or acetone to the assay mixture in the range of 0-35% stimulated the enzyme, whereas benzene or n-hexane had an inhibitory effect. These properties together with the N-terminal amino acid sequence confirmed that the enzyme differs from the known Pseudomonas sp. lipases.


Proliferation and differentiation of fetal rat intestinal epithelial cells in primary serum-free culture. It has been a subject of controversy whether fibroblastic cells are necessary for the proliferation of intestinal epithelial cells in primary culture. To answer this question, we have developed a serum-free primary culture system which allows reproducible and quantitative assays of proliferation and differentiation of fetal rat intestinal epithelial cells in the absence of fibroblastic cells. Pure intestinal epithelial tissues were obtained from 16.5-day fetal rats without contamination of mesenchymal cells, and were successfully cultured on a collagen gel in a medium consisting of Ham's F12, bovine serum albumin, epidermal growth factor (EGF), insulin, cholera toxin, transferrin and hydrocortisone. The epithelial nature of the cultured cells was confirmed by the presence of cytokeratin in the cells. Under optimal culture conditions, intestinal epithelial cells readily attached to the substratum in a day, and proliferated rapidly in vitro, increasing their number about 10 times in the first 5 days. EGF, insulin, cholera toxin, transferrin and hydrocortisone synergistically induced the epithelial proliferation, and lack of any one of them resulted in a significant reduction of the proliferation. In contrast, fetal bovine or horse serums, which have been widely used to supplement culture media, severely inhibited the epithelial proliferation. Histological examination showed that the epithelial cells formed simple cuboidal epithelia with basally-located nuclei when cultured on collagen gels. The intestinal epithelial nature of the cells was affirmed by the presence of villin on their luminal surface. Ultrastructurally, cells were connected by tight junctions and desmosomes at the subluminal region, and microvilli were projecting on the luminal surface, indicating that the cells in primary culture retained some characteristics of absorptive epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Tenascin in bone morphogenesis: expression by osteoblasts and cell type-specific expression of splice variants. The extracellular matrix glycoprotein, tenascin, is associated in vivo with mesenchyme undergoing osteogenesis and chondrogenesis, but is absent from mature bone and cartilage matrix. The expression of tenascin by osteoblastic cells in vitro has been investigated by immunoblotting and immunocytochemistry. Tenascin was secreted into the medium and deposited in the matrix by human and rat osteoblast-like cell lines, as well as by primary osteoblast-enriched cultures from chick embryo calvarial bones. In primary osteoblast-enriched cultures, extracellular tenascin was found only in cell aggregates expressing the osteoblast marker alkaline phosphatase. Chicken osteoblast cultures synthesized almost exclusively the largest tenascin subunit, whereas fibroblast cultures from periostea of chicken calvariae synthesized approximately equal amounts of all three subunits. In situ hybridization studies of developing chicken bones, using a cDNA probe that hybridizes to all chicken tenascin splice variants, showed specific labelling of both osteogenic and chondrogenic regions of developing endochondral bones. In contrast, a cDNA probe specific for the large tenascin splice variant showed specific hybridization in osteogenic but not chondrogenic regions. Within osteogenic regions, tenascin mRNA was expressed by osteoblasts. A comparison of in situ hybridization and immunohistochemical studies demonstrated that tenascin mRNA and protein were codistributed in osteogenic regions of endochondral and membrane bones, whereas protein was retained in regions of differentiating cartilage where mRNA was no longer detectable. The results presented here demonstrate that tenascin is synthesized by osteoblasts. Moreover, within developing bones, there are at least three different cell type-specific patterns of expression of tenascin splice variants.


Determination of risperidone and 9-hydroxyrisperidone in plasma, urine and animal tissues by high-performance liquid chromatography. A high-performance liquid chromatographic method has been developed for the simultaneous determination of the new antipsychotic risperidone and its major metabolite 9-hydroxyrisperidone in plasma, urine and animal tissues. The alkalinized plasma samples were extracted with ethyl acetate and further purified prior to reversed-phase chromatography with ultraviolet detection at 280 nm. The method could also be applied to urine samples and animal tissue homogenates. Quantification limits were 2 ng/ml for plasma and urine and 10 ng/g for animal tissue. The method was applied to pharmacokinetic studies in experimental animals, human volunteers and patients.


Smooth muscle dilatation in the human uterine artery induced by substance P, vasoactive intestinal polypeptide, calcitonin gene-related peptide and atrial natriuretic peptide: relation to endothelium-derived relaxing substances. The relaxatory influences of substance P (SP), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP) were investigated in human uterine arteries precontracted by noradrenaline in vitro. SP, VIP, CGRP and ANP all relaxed isolated uterine arteries with intact endothelium. When tested on vessels devoid of their endothelium VIP and SP had no effect on smooth muscular tone, while ANP and CGRP still induced unchanged vasodilatation. These results suggest an involvement of an endothelium-derived relaxing substance in the mechanisms by which VIP and SP induce relaxation of the isolated human uterine artery. On the other hand, ANP and CGRP seem to act on the same vessel preparation in vitro independently of the vascular endothelium. Both addition of noradrenaline and exchange of sodium against potassium in the organ chambers resulted in smooth muscle contraction irrespective of the integrity of the endothelium.


Dot blot immunoassay for detection of human semen. A sensitive and specific dot blot immunoassay based on prostate specific antigen (PSA) antibodies and a radiolabelled Protein A detection system was developed for detection of trace amounts of human semen in stains. The method was found highly sensitive and detected semen stain extracts diluted 10,000 times. Semen stains up to five years old were successfully detected by the method. The assay is found to be highly suitable for semen detection in forensic analysis.


DNA fingerprints of Helicobacter pylori before and after treatment with omeprazole. To test whether a hypoacidic environment may potentially "stress" Helicobacter pylori DNA, encouraging the emergence of strain variation. This hypothesis was tested by inducing prolonged hypoacidity with omeprazole, a potent antisecretory drug. The genomic DNA of H pylori was studied by electrophoretic separation of restriction endonuclease fragments followed by rRNA gene hybridisation in seven patients infected with H pylori before and after treatment with omeprazole 20-40 mg daily for six to eight weeks. DNA was isolated and purified using the guanidium thiocyanate reagent method. DNA samples were digested with Hae III, electrophoresed, vacublotted, and hybridised using a biotinylated cDNA probe prepared from 16S and 23S rRNA from H pylori NCTC 11638. Isolates were compared using their ribopatterns (DNA fingerprints). A total of 26 isolates were obtained; all DNA isolates were cut by Hae III, which was the enzyme that gave the best resolved hybridisation patterns for analysis. No two patients harboured the same strain. The isolates from two patients showed evidence of subtypic variation; one patient had two distinct strains and four patients had their own indistinguishable strains before and after treatment with omeprazole. For each patient, the paired ribopatterns of H pylori DNA were not affected by treatment with omeprazole for six to eight weeks. The H pylori genome is relatively stable when exposed to the conditions of prolonged hypoacidity that result from treatment with omeprazole.


Evaluation of three techniques for differential diagnosis of prostatic needle biopsy specimens. To determine whether acidic mucin staining, lectin histochemistry using Wisteria floribunda agglutinin, and immunohistochemistry using the monoclonal antibody EAB 903 are of benefit in distinguishing between hyperplastic and carcinomatous prostatic glandular tissue in needle biopsy specimens. Formalin fixed, paraffin wax embedded prostatic needle biopsy specimens of benign and malignant tissue were examined. Alcian blue-periodic acid Schiff staining was performed on 33 benign and 34 malignant cases. Wisteria floribunda agglutinin (WFA) binding sites were demonstrated by the avidin-biotin peroxidase (ABC) technique with and without neuraminidase pretreatment on 34 benign cases and 32 malignant cases. EAB903 anticytokeratin antibody binding sites were demonstrated using both an indirect immunoperoxidase (IIP) technique and an avidin-biotin peroxidase complex method on seven benign and 31 malignant cases. Acidic mucin staining was found in 17 of 34 malignant glands and was weakly positive in five of 33 benign glands. WFA positivity before neuraminidase pretreatment was present in 29 of 32 malignant glands and in 19 of 34 benign glands. After neuraminidase all benign and malignant cases showed positivity. EAB 903 positivity was seen in 11 of 31 malignant glands using the IIP technique and in two of 31 malignant glands using the ABC technique. In seven benign cases there was positivity in all glands using the IIP method with predominant basal cell staining in three and superficial cell staining in four. In benign cases using the ABC method two cases were negative. None of the three methods studied showed sufficient sensitivity and specificity to allow their recommendation for routine diagnostic use.


Monoamine oxidase inhibitors phenelzine and brofaromine increase plasma serotonin and decrease 5-hydroxyindoleacetic acid in patients with major depression: relationship to clinical improvement. We have examined the effects of two monoamine oxidase (MAO) inhibitors with different mechanisms of action--phenelzine and brofaromine--on peripheral serotonergic (5-hydroxytryptamine [5-HT]) measures, sensitive to the inhibition of MAO-A (intra- and extracellular 5-HT and related metabolites in blood). Both drugs increased the concentration of 5-HT in platelet-free plasma (254%, p less than 0.001) in patients with depressive illness (DSM-III-R) after 6 weeks of daily treatment. Platelet 5-HT was also increased significantly in both drug treatment groups but more marked in the patient group treated with phenelzine. The acid/amine ratio at 6 weeks was 30% of pretreatment values (p less than 0.000) and individual variability correlated significantly with the Hamilton Rating Scale for Depression. Plasma 5-HT increased more markedly in responders than in nonresponders and a significant inverse relationship surfaced between plasma 5-HT and the Hamilton Rating Scale for Depression. The results support other reports of comparable antidepressant efficacy for brofaromine and phenelzine, both inhibitors of MAO-A in humans. The consistent relationship we found between the biochemical and clinical changes again suggests and supports a key role of 5-HT in the antidepressant effect of these MAO inhibitors.


Galanin receptors in the brain of a teleost: autoradiographic distribution of binding sites in the Atlantic salmon. The distribution of galanin (GAL) binding sites in the brain of the Atlantic salmon (Salmo salar) was investigated by means of radioligand binding in conjunction with autoradiography by using high-performance liquid chromatography (HPLC) characterized radio-iodinated porcine galanin ([125I]GAL). On slide-mounted sections of frozen salmon brain homogenate, [125I]GAL (4 nM) bound rapidly and reversibly to a single population of sites with a Kd of 1.0 +/- 0.08 nM (n = 3) and Bmax of 2.38 +/- 0.19 fmol/mg wet tissue. Specific [125I]GAL binding was found in cellular regions, in fiber tracts, and in neuropil areas throughout the brain, except for in the olfactory bulb, pineal organ, and cerebellum. Autoradiographic microdensitometric measurements revealed high total [125I]GAL binding in the ventral hypothalamus (inferior lobes; around 7-12 fmol/mg tissue), the dorsal spinal cord (between 6 and 12 fmol/mg tissue), sublayers of the optic tectum (around 8 fmol/mg), torus semicircularis (around 7 fmol/mg), and glomerular complex (around 6 fmol/mg). Intermediate densities of [125I]GAL binding (3-5 fmol/mg tissue) were found in the pituitary, telencephalon, dorsolateral thalamic nucleus, and raphe nuclei and in association with the forebrain bundles. Except for in the optic tectum, there is a good concordance of [125I]GAL binding sites and GAL-immunoreactive fiber projections in most brain areas of the salmon. The wide distribution of GAL binding sites provides further evidence that a GAL-like substance might be involved in a diversity of brain functions of teleosts. The topographic distribution of target sites in the hypothalamo-hypophyseal axis indicates that GAL-like substances may have both direct and indirect effect on pituitary functions while in extrahypothalamic areas, functional implications by GAL may include involvement in somatosensory, central gustatory, olfactory, and visual functions. This study provides evidence for the presence of a specific GAL receptor in the brain of the Atlantic salmon. Together the distribution of GAL binding and GAL-like molecules provide a covering delineation of the GAL neuronal system in the brain of the Atlantic salmon. Comparisons with mammals suggest that the GAL receptor molecule has been well preserved during evolution and that GAL-like substances may be present, and even possess similar functional properties, throughout the vertebrate phylogeny.


Postnatal development of area 17 callosal connections in Tupaia. The goal of the present study was to investigate the pattern of maturation of callosal projecting neurons in a well-studied mammalian visual system with unique structural and functional properties. Studies of the distribution pattern of interhemispheric connections in the adult tree shrew primary visual cortex reveal not only a high concentration of labeled neurons along the area 17/18 border, as in standard experimental animals such as the cat and monkey, but also numerous callosal projecting neurons in the adjacent dorsal part of area 17, which largely corresponds to the binocular visual field (Kretz and Rager, Exp. Brain Res. 82:271, '90). Callosal projections were anatomically traced in 11 tree shrews (Tupaia belangeri) at various ages between postnatal day 7 (7, 9, 10, 13, 15, 17, 19, and 26 days old) and adulthood (107 days old). In each animal, four injections of wheat germ agglutinin conjugated to horseradish peroxidase were made in a standard configuration into the striate cortex of one hemisphere. In young tree shrews only 7 and 9 days old, heavily labeled terminal axon structures could be seen in the white matter and in layer VI of the opposite hemisphere. Only a few labeled neurons, however, were detected in layer III. The small number of labeled neurons indicated that early in postnatal development, only a few callosal axons had invaded the upper cortical layers. By 10 days of age, the number of supragranular neurons was increasing and the maximal value was counted in a 13-day-old tree shrew. A sharp decline in the number of labeled supragranular neurons was noticed--about 94% in our case--between days 13 and 15. In animals more than 15 days old, the distribution pattern and the density of the neurons looked like the pattern seen in the adult Tupaia brain. The labeled cells were mostly concentrated in layers II and III. The majority of neurons resembled typical pyramidal cells. However, some of the neurons in sublayer IIIc had elongated cell bodies oriented parallel to the laminar boundaries. In contrast to the supragranular cells found in all stages investigated, small populations of labeled cells in layer VI were observed in 9- to 17-day-old tree shrews only. In young postnatal animals 7 to 13 days old, a peculiar cell type was labeled on the ipsilateral side. In coronal sections these cell bodies formed a continuous band that extended from the ventricular wall to the subcortical white matter. These cells might belong to a population of cells still in migration.


Immunofluorescent analysis of creatine kinase in cultured astrocytes by conventional and confocal microscopy: a nuclear localization. The subcellular localization of creatine kinase (CK) was examined in primary cultures of astrocytes with immunofluorescent labeling methods and detection by both standard fluorescence microscopy and confocal laser-scanning microscopy. With conventional microscopy, the pattern of CK staining was uniform throughout the cell cytoplasm and appeared to stain the nuclear region intensely. Staining of CK in the nuclear region co-localized with the DNA-specific Hoechst nuclear stain. CK produced a diffuse cytoplasmic staining pattern that was different from the staining pattern produced by the cytoskeletal proteins glial fibrillary acidic protein and tubulin, both of which showed a filamentous cytoskeletal network that excluded the nucleus. To examine the structural details of CK in the nuclear region, serial optical sections were taken through the cell monolayer with a confocal microscope. The cells were immunostained for CK, and the CK-staining pattern was compared with the staining pattern produced by propidium iodide, which is specific for DNA in RNase-treated samples and stains total nucleic acid in untreated samples. CK staining was present within the nucleus in each section taken through the monolayer. The nucleolus did not stain for CK. The pattern of CK staining in the nucleus (and cytoplasm) was distinctly different from the staining pattern of either DNA or total nucleic acid. Nuclear CK appeared to have a granular, particulate pattern, which is suggestive of a nucleoplasmic distribution.


Immunohistochemical and ultrastructural characterization of tonofilament and hemidesmosome abnormalities in a case of epidermolysis bullosa herpetiformis (Dowling-Meara). A neonate with epidermolysis bullosa herpetiformis (EBH) (Dowling-Meara) had an undescribed ultrastructural and immunohistochemical abnormality. The objective was to clarify the ultrastructural and immunohistochemical abnormalities in EBH to gain further insight into the pathogenesis of this disorder. Tissue from the patient was studied with routine histochemistry, electron microscopy, and immunohistochemistry. Excessive clumping of tonofilaments on electron microscopic examination, anomalous hemidesmosomes, and immunohistochemical evidence of aberrant keratin expression by basal epidermal cells was found. This case of EBH provides further evidence for primary abnormalities involving cytoskeletal-membrane attachment plaque formation in this rare disorder.


Erythema multiforme: pathomechanism of papular erythema and target lesion. Skin lesions of erythema multiforme show time-dependent changes from early papular erythema to the late target lesion which consists of a peripheral elevated erythematous area and a central depressed area. We investigated the pathomechanism of erythema multiforme, by examining the papular erythema and target lesion separately. In the early papular erythema, a small number of polymorphonuclear leukocytes and nuclear debris were seen intermingled with mononuclear cells around the slightly swollen blood vessels, on which immunoglobulin and complement components were deposited. Circulating immune complex levels were occasionally elevated. Sera from the patients generated high levels of reactive oxygen species and nitroblue tetrazolium test revealed positive reaction on the infiltrating cells around the blood vessels. These findings suggest that the papular erythema develops via incomplete type III allergic reaction, followed by damage through reactive oxygen species. In the target lesion, the activity of histamine-N-methyltransferase, which is the major histamine-degrading enzyme, was markedly decreased in the peripheral elevated erythematous area and it was recovering in the central clearing area. ICAM-1 and HLA-DR antigens were expressed on the surfaces of the keratinocytes. An increased number of epidermal Langerhans cells and CD4 cell infiltration were observed in the peripheral elevated erythematous area, while a decreased number of epidermal Langerhans cells and CD8 cell infiltration in the central depressed area were observed. These findings suggest that impaired histamine metabolism and cellular allergic reactions play important roles in the development of the target lesion.


Ultrastructure of Golgi-impregnated and gold-toned neurons in the central nucleus of the inferior colliculus in the cat. The combined Golgi/electron microscope technique was used to analyse the cytoarchitecture and the fine structure of the central nucleus of the inferior colliculus in the cat. The analysis of Golgi-impregnated sections discriminates three major neuronal types, according to somato-dendritic morphology, and to presence or absence of dendritic spines. Two major types (spiny and aspiny) might be further subdivided into large, medium-large, medium-small and small, whilst the third ("mixed") type is subdivided into large and medium-large types. The large, medium-large and medium-small cells of the major types appear to be efferent (relay) neurons, whilst the small spiny, and especially the small aspiny cells are interneurons. In agreement with previous data, the subdivision of the relay neurons in disc-shaped and stellate cells is confirmed but the disc-shaped neurons are further subdivided in typical and atypical. The dendritic fields of the latter neurons correspond greatly but not exclusively to the fibrodendritic laminae of the central nucleus. In addition to the axonal ramification of the local circuit neurons, the axons of most (if not all) types of relay cells emit a moderate to scant, rarely--a substantial number of collaterals. The collaterals of the large spiny neurons (atypical disc-shaped cells) occasionally innervate also the cell of origin. Parallel to the light microscopic discriminations of the different neuronal types, the electron microscopic observations confirm that the ultrastructural characteristics might be very distinct. Especially evident are the differences between the large neuronal types, concerning the amount and arrangement of the granular endoplasmic reticulum, and the mode of the perikaryal, dendritic, and axonal innervation by various synaptic bouton types. Along with the unequivocal discrimination of the neurons in the central nucleus of the inferior colliculus according to the dendritic orientation, we suggest also a more detailed classification of the neuronal types according to the perikaryal size, fine dendritic morphology, and ultrastructural characteristics. Further hodological experiments, combined with the presently explored technique, will help to clarify the complicated synaptic events in the central nucleus of the inferior colliculus.


Influence of spinal cord hemisection on the configurational changes in motor and primary afferent neurons and the chemical messenger alterations in the rat lumbar segments. Cholera toxin B subunit (CTB), a retrograde transport marker, was injected into the rat gastrocnemius muscles, and changes in CTB-labeling pattern of motoneurons and primary afferent neurons at the level L4 and L5 after spinal cord hemisection of the L1 level were observed in conjunction with the alterations of chemical messengers such as serotonin (5-HT), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (Gal), Met-enkephalin (Enk), and neuropeptide Y (NPY). Motoneurons at the L4 and L5 levels on the lesioned side exhibited significant shrinkage of their dendritic arbors without apparent loss of their number throughout all stages from 1 to 12 weeks after the hemisection of the spinal cord. Postoperatively, central processes of neuron of the dorsal root ganglia (DRG) on the lesioned side increased progressively compared to that on the contralateral side with the passage of time. The percentage of CTB-labeled neurons in the DRG has been consistently smaller in number on the lesioned side after the operation, and the difference between sides became more apparent during the later postoperative stages. 5-HT-containing fibers in the anterior and posterior horns on the lesioned side showed a significant decrease in the number, while no apparent changes were observed in the distribution of nerve fibers containing CGRP, SP, Gal, Enk, and NPY.


Maturation of neurons in neocortical slice cultures: A light and electron microscopic study on in situ and in vitro material. Using light and electron microscopic methods, we investigated the development and morphology of neurons in neocortical slice cultures. Slices taken from the visual cortex of 6-day-old rats and cultivated for 14 or 20 days were compared with in situ material of corresponding age (P 20 and P 26). Maturation and differentiation of pyramidal and non-pyramidal cells kept in vitro were found to have progressed considerably. In the light microscope the neurons exhibited a morphological appearance strikingly similar to that of the neurons of the neocortex in situ at the same age. The fine structure of the tissue in vitro also had a mature appearance, corresponding in most respects to the material in situ. Synapses and dendritic spines were well-developed. Sometimes a spine apparatus was contained in the sections and occasionally a myelinated fiber could be seen. GABA-immunoreactive cells making symmetric synaptic contacts were also present. Despite these similarities, some quantitative differences could be observed. In slice cultures, only 52% of the synapses were located on spines (78% in situ). In vitro, a larger proportion of synapses (30%) showed a postsynaptically concave curvature than was the case in situ (12%). The areal density of synapses in vitro reached only about 70% of that in situ. This was probably a side-effect of the larger size of dendritic and axonal profiles on electron micrographs of in vitro-material. The most striking difference was that large synapses and synapses containing a large amount of synaptic vesicles were considerably more frequent in vitro than in situ.


Evidence for the existence of a projection from the dorsal column nuclei to the substantia nigra in the cat. Discrete injections of horseradish peroxidase in the substantia nigra of cats resulted in moderate to scant retrograde neuronal labeling of the contralateral dorsal column nuclei (nucleus gracilis and nucleus cuneatus). A moderate number of degenerating synaptic boutons emanating from the axons of stereotaxically lesioned dorsal column nuclei were identified by electron microscopy in the neuropil of the contralateral substantia nigra zona compacta and its dorsal division-nucleus parabrachialis pigmentosus. The present findings furnish evidence for a moderate, entirely crossed afferent projection of the dopaminergic neurons of the mesencephalic tegmentum, arising in the dorsal column nuclei.


Subicular efferents to histaminergic neurons in the posterior hypothalamic region of the rat studied with PHA-L tracing combined with histidine decarboxylase immunocytochemistry. The projections from the subiculum to histaminergic cells in the posterior hypothalamic region of the rat were studied by means of anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L) combined with histidine decarboxylase (HDC)-immunohistochemistry. PHA-L was injected at various loci along the dorsoventral and proximodistal axes of the subiculum. This resulted in labeling of the fornix and of terminal plexuses at various locations in the diencephalon and the mammillary body. Following deposition of PHA-L in the proximal part of the dorsal subiculum, labeled fibers in the posterior hypothalamus were confined to the mammillary nuclei, whereas after injections of PHA-L in the distal part of the dorsal subiculum and the entire ventral subiculum, labeled fibers were also present in clusters of histaminergic cells located around the mammillary nuclei. The density of the PHA-L labeled fibers within these clusters increased from low to moderate in association with a shift of the injection sites from dorsal to ventral and from proximal to distal parts of the subiculum, i.e., the highest fiber labeling was seen after injections of PHA-L in the distal part of the ventral subiculum. In the latter experiments, PHA-L labeled fibers reached HDC-immunoreactive neurons in the tuberal magnocellular nucleus, the deepest layer of the caudal magnocellular nucleus, the two bridges of histaminergic cells in the posterior hypothalamus, and the histaminergic neurons scattered in the supramammillary region. A few labeled fibers invaded the postmammillary caudal magnocellular nucleus. The presence of varicosities on the PHA-L labeled fibers in close proximity to the cell bodies and dendrites of the histaminergic neurons suggest the existence of synaptic contacts.


Lipopolysaccharide heterogeneity in Pasteurella haemolytica isolates from cattle and sheep. Lipopolysaccharide (LPS) from 40 isolates of Pasteurella haemolytica, comprising 23 serotype A1, seven serotype A2, one serotype T4, one serotype T10 and eight untypable isolates, obtained from diseased and healthy cattle or sheep, was characterized by SDS-PAGE and Western blotting. Ten different SDS-PAGE LPS profiles, five smooth and five rough, were identified among the biotype A and untypable isolates and designated LPS types 1-10. LPS types 1 and 2 were smooth, had similar O-antigen banding-patterns but differed in the low-molecular-mass or core-oligosaccharide regions; type 3 LPS was rough but had a core-oligosaccharide region similar to that of LPS type 1. No similarities were observed between these LPS types and types 6, 7 and 9, which were smooth, and types 4, 5, 8 and 10, which were rough. Most serotype A1 isolates (19/23) were of LPS type 1, whereas two isolates each had LPS of types 2 and 3. The majority (5/7) of serotype A2 isolates possessed type 3 LPS, whereas the remaining two isolates each had LPS of types 4 and 5. There was much greater heterogeneity within the untypable group of isolates, which comprised LPS of types 1 and 9 (two isolates each), and 6, 7, 8 or 10 (one isolate each). Western blotting analysis demonstrated that LPS types 1 and 2 had immunologically identical O-antigen side-chains but differed in their core-oligosaccharide regions, whereas the core-oligosaccharide region of rough LPS type 3 was immunologically very similar to that of LPS type 1. The other LPS types were immunologically unrelated to these three LPS types. The majority (20/23) of serotype A1 isolates originated from cattle and possessed LPS types 1 or 2, different from most (5/7) of the serotype A2 isolates which originated from sheep and possessed LPS of types 3 or 4. However, two of the three ovine serotype A1 isolates had the same type 3 LPS as occurred in most of the ovine serotype A2 isolates, suggesting a possible correlation between LPS type and host specificity. This study has demonstrated that LPS diversity within different serotypes of P. haemolytica is greater than was previously thought and that certain LPS types might be host-specific.


Targeting of specific domains of diphtheria toxin by site-directed antibodies. Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224-237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513-526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominantly against a narrow region within the peptide, consisting of only 5-6 aa residues.(ABSTRACT TRUNCATED AT 250 WORDS)


Antibodies recognizing a variety of different structural motifs on meningococcal Lip antigen fail to demonstrate bactericidal activity. The neisserial Lip antigen is a conserved antigen associated with the pathogenic Neisseria species, and is composed of multiple repeats of a consensus pentapeptide. A series of monoclonal antibodies reacting with meningococcal Lip antigen were subjected to epitope mapping, using solid-phase synthetic peptides based on the consensus repeat sequence. The antibodies were found to recognize different continuous epitopes based on the consensus sequence. One monoclonal antibody was utilized in affinity chromatography to obtain purified Lip antigen and the antigen was used for immunization of mice. The resulting antisera did not recognize Lip antigen on Western blots but reacted specifically with Lip antigen in immune precipitation experiments, indicating that the predominant polyclonal immune response was directed against conformational epitopes. Despite the diversity of both continuous and conformational epitopes recognized by the antibodies produced, none of the antibodies demonstrated the ability to promote complement-mediated bactericidal activity. Thus despite its initial apparent promise as a potential vaccine candidate the case for the inclusion of Lip antigen in vaccine formulation cannot be supported at present.


Isolation and characterization of adhesin-defective TnphoA mutants of septicaemic porcine Escherichia coli of serotype O115:K-:F165. Non-enterotoxigenic porcine Escherichia coli strains belonging to the serogroup O115 have been associated with septicaemia and diarrhoea. Putative factors important in the pathogenicity of E. coli of serogroup O115 include fimbrial antigen F165, haemagglutination (MRHA), lipopolysaccharide, serum resistance, capsule and production of aerobactin. Using TnphoA transposon insertion mutagenesis, two classes of mutants were obtained from E. coli of serotype O115:F165 with respect to the phenotypic expression of fimbrial antigen F165 and MRHA of sheep erythrocytes: class I, F165-MRHA-, serum resistant; class II, F165+MRHA-, serum resistant. In a chicken lethality model, class I mutants were either virulent or of intermediate virulence, while class II mutants were of intermediate virulence. Alkaline phosphatase activity of class I and class II TnphoA mutants showed similar environmental regulation to that of fimbrial antigen F165. Moreover, class I and class II mutants were mutated in the prs-like locus, and lacked a 18.5 kDa and/or a 17.5 kDa fimbrial band.


Influence of prophylaxis on proximal venous thrombus formation after total hip arthroplasty. Deep vein thrombosis (DVT) is the most frequent complication after total hip arthroplasty, and proximal DVT is more likely to produce clinical pulmonary emboli than distal DVT. The authors have assessed the incidence and anatomic location of phlebographically proven fresh DVT following total hip arthroplasty in 855 patients over 39 years of age. Eight different regimens (2 warfarin, 3 aspirin, 1 dextran, 1 external pneumatic compression, and 1 dextran combined with external pneumatic compression) were evaluated and compared with an historical placebo control group from the same institution. No significant difference (P < .05) existed in proximal DVT incidence between the placebo group and any of the three aspirin groups, the dextran group, the external pneumatic compression group, or the dextran combined with external pneumatic compression group. In contrast, warfarin, given both in traditional and low-dose regimens, provided a significant reduction in proximal DVT compared with the placebo group (P < .001; statistical power, 0.84 and 0.99, respectively). The low-dose regimen had 10 times fewer bleeding complications than the traditional regimen. All prophylaxis regimens should be evaluated for both proximal and distal DVT formation, as well as for overall incidence. Low-dose warfarin offers the best protection against proximal thrombi of the agents studied, and it is also safer than traditional dosages of warfarin.


Potent inhibitors of histamine release, two novel triterpenoids from the Okinawan marine sponge Penares incrustans. Two novel triterpenoids with an unusual 14-carboxyl group, penasterone [1] and acetylpenasterol [2], were isolated from the Okinawan marine sponge Penares incrustans. The structures of 1 and 2 were established mainly on the basis of nmr spectroscopic data. The relative stereochemistry of 1 was confirmed by single-crystal X-ray diffraction analysis. Compounds 1 and 2 potently inhibited histamine release from rat peritoneal mast cells induced by anti-IgE in a dose-dependent manner.


Neuroenteric cyst of the optic nerve: case report with immunohistochemical study. A 36 year old man developed slowly progressive unilateral visual loss due to a cystic lesion of the intraorbital optic nerve. Pathologically the lesion was an epithelium lined cyst entirely within the atrophic nerve. The cyst lining consisted of columnar epithelium partly pseudostratified and ciliated with evidence of mucin secretion, and was immunoactive for cytokeratin but not glial fibrillary acidic protein. This lesion is considered to be similar to neuroenteric cysts that have been reported elsewhere in the nervous system, and the immunocytochemical results are consistent with a heterotopia derived from the primitive ectoderm of the stomatodeum.


Functional properties and axon terminations of interneurons in laminae III-V of the mammalian spinal dorsal horn in vitro. 1. The functional organization of interneurons in spinal laminae III-V was studied in an isolated preparation of hamster dorsal horn with sensory innervation from an excised skin patch. Morphological details of 40 neurons were visualized by intracellular injection of horseradish peroxidase. Active and passive membrane properties, synaptic responses to cutaneous nerve volleys, and responses to innocuous mechanical stimuli were determined for 25 cells with identified axons. 2. Neurons were classified into two types: 1) cells with local axons, branching in proximity to the cell soma and dendrites, that produced numerous synaptic boutons (740 +/- 504/axon; mean +/- SD), often arranged in clusters and 2) neurons with deep axons that usually bifurcated into rostral and caudal daughter branches up to 2.5 mm long, giving off collaterals ventral to the cell body and dendrites and forming significantly fewer boutons (155 +/- 140/axon) than local axon cells. A majority of boutons of local axon and deep axon cells, 89 and 83%, respectively, were of the en passant type. 3. Dendritic trees of local axon cells were relatively compact dorsoventrally (119 +/- 42 microns) and mediolaterally (128 +/- 45 microns), but were elongated rostrocaudally (404 +/- 121 microns). In comparison, dendritic trees of deep axon cells radiated significantly farther dorsoventrally (218 +/- 88 microns) and mediolaterally (180 +/- 34 microns), but exhibited comparable rostrocaudal spread (413 +/- 128 microns). There was no correlation between dorsoventral and mediolateral dendritic spread and mediolateral soma location for either cell type. However, for medially situated deep axon cells the rostrocaudal dendritic spread was up to 180% greater than for those located laterally. For nearly one-half of all cells (49%; 17/35) dendritic processes extended dorsally into lamina II. 4. Local axon cells had resting membrane potentials that were more negative than deep axon cells (-59.5 +/- 6.1 and -53.6 +/- 4.7 mV, respectively), but the amplitude and duration of action potentials generated by the two types were similar. Neuronal input resistance (RN) and membrane time constant (tau m) varied widely from cell to cell, but were not significantly different for local axon (77.4 +/- 46.8 M omega, 13.4 +/- 9.5 ms) and deep axon cells (46.5 +/- 19.2 M omega, 6.6 +/- 3.0 ms). 5. Volleys in myelinated afferent fibers activated fast rising excitatory postsynaptic potentials (EPSPs) that exhibited later, more slowly rising potentials with multiple components in a majority of deep axon (89%) and local axon (72%) neurons.(ABSTRACT TRUNCATED AT 400 WORDS)