Multiple pathways for repair of oxidative DNA damages caused by X rays and hydrogen peroxide in Escherichia coli. The responses of Escherichia coli to X rays and hydrogen peroxide were examined in mutants which are deficient in one or more DNA repair genes. Mutant cells deficient in either exonuclease III (xthA) or endonuclease IV (nfo) had normal resistance to X rays, but an xthA-nfo double mutant showed a sensitivity increased over that of either parental strain. A DNA polymerase I mutant (polA) was more sensitive than the xthA-nfo mutant. Cells bearing mutations in all of the polA, xthA, and nfo genes were more sensitive to X rays than polA and xthA-nfo mutants. Similar repair responses were obtained by exposing these mutant cells to hydrogen peroxide, with the exception of the xthA mutant, which was hypersensitive to this agent. The DNA polymerase III mutant (polC(Ts)) was slightly more sensitive to the agents than the wild-type strain at the restrictive temperature. The sensitivity of the polC-xthA-nfo mutant to X rays and hydrogen peroxide was greater than that of polC but almost the same as that of the xthA-nfo mutant. From these results it appears that there are at least four repair pathways, the DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase III-, and exonuclease III/endonuclease IV-dependent pathways, for the repair of oxidative DNA damages in E. coli.


[Liatek HCV: the six-antigen test for confirmation of the presence of anti-HCV antibodies]. Fifty eight anti-HCV repeatable positive (the UBI HCV EIA, Microelisa test Organon Teknika) blood donors were tested using LIATEK-HCV EIA (Organon Teknika). The test was constructed using synthetic polypeptides renders possible to conduct separate investigation antibodies against structural and nonstructural antigens of HCV. In 51/58 (88 per cent) cases was agreement of the results with the UBI test, in 3/58 (5.1 per cent) results were negative, and in 4/58 (6.9 per cent) undetermined. In 90 per cent of the seropositive reactivity developed with four to six antigens. The most frequent "serological pattern" was anti: NS4, C1, C2, C3, C4 (31 per cent) and anti: NS4, NS5, C1, C2, C3, C4 (23.5 per cent).


Modulation of electrical activity in gastrointestinal smooth muscle by peptides. A rise in intracellular calcium is the predominant signal that leads to the activation of the contractile machinery in gastrointestinal smooth muscle. The primary sources of activating calcium are illustrated in Fig. 2. Voltage- and peptide-mediated release of intracellular calcium contribute to activation of some gastrointestinal smooth muscles. However, the primary source of activating calcium appears to be an influx of calcium across the plasma membrane. The degree of modulation of electrical activity by peptides varies depending upon the region of the gastrointestinal tract studied. Second messenger systems are undoubtly involved in the transduction pathway for receptor-mediated changes in ion channel activity in gastrointestinal smooth muscle. However, in comparison to other excitable cell types, little is known about the coupling mechanisms whereby peptide-receptor binding alters ion channel activity in gastrointestinal smooth muscle. This represents one of the challenging areas to be studied in the field of gastrointestinal smooth muscle. One disease in which a better appreciation of the regulation of ion channel activity could lead to therapeutic benefit is irritable bowel syndrome. A coupling of smooth muscle electrical activity to hypermotility in irritable bowel syndrome has been reported. CCK increases the level of spike activity which triggers hypermotility [40]. It would follow that inhibition of calcium influx should reduce spiking and, therefore, hypermotility. In fact, the calcium channel blockers nifedipine and nicardipine have been shown to decrease colonic motility in irritable bowel syndrome patients [62-64]. As our understanding of gastrointestinal smooth muscle ion channels expands, development of a gastrointestinal selective calcium channel blocker may be possible. This class of agents would be effective in the treatment of irritable bowel syndrome and potentially other peptide-related spastic smooth muscle disorders.


[Percutaneous alcoholization of a small hepatocarcinoma]. Twenty-nine small hepatocellular carcinomas (sHCCs) less than 5 cm in diameter were treated with percutaneous ethanol injection (PEI) under US guidance in 24 cirrhotic patients. The nodules were treated on an outpatient basis with 6-18 ethanol injections; the total amount of alcohol delivered to each lesion was 10-103 ml. Twenty-seven of the 29 HCCs (93.1%) showed no evidence viable neoplastic tissue at a dynamic CT scan combined with multiple fine-needle biopsies performed one month after the end of treatment; in 10 cases MR confirmed the presence of necrosis showing marked hypointensity of the lesions in T2-weighted images. None of the 27 necrotized sHCCs recurred locally during a 4-44 months' follow-up period (mean 18 months). Two lesions larger than 4 cm showed incomplete response to treatment. No complications occurred after a total number of 264 alcohol injections. The 1-year survival rate in the 16 patients with a follow-up longer than 12 months was 93.7%. PEI proved to be a safe and effective treatment for sHCCs. In particular, PEI can be viewed as a reliable alternative to surgery in the management of nodules less than 3 cm in diameter, considering the operative hazards and the high risk of new lesions occurring in resected livers.


Cation regulation of guinea pig cardiac nitrobenzylthioinosine binding sites. The effects of several ions on [3H]NBMPR specific binding were characterized in guinea pig cardiac membranes. Binding of [3H]NBMPR was enhanced by Mg2+ Ca2+, Na+ and K+ but inhibited by Mn2+, Cu2+ and Zn2+. The effects of Cu2+ and Zn2+ were observed in micromolar concentrations while the effects of the other cations were observed between 1 and 300 mM. The anions tested had no significant effect on the binding. Scatchard analyses of the [3H]NBMPR saturation equilibrium data showed that the enhancement by Na+ and Mg2+ was due to an increase in the number of binding sites number with no change in apparent affinity. The inhibition of the binding by Cu2+ and Zn2+ was due to a decrease in both the apparent affinity and the number of binding sites. The regulation by cations imply that [3H]NBMPR binding sites are physiological receptors that may be involved in postreceptor coupling.


Kappa agonist-induced reduction in dopamine release: site of action and tolerance. Kappa opioid agonists are known to inhibit dopamine release. We sought to determine the site of this action and the relationship of tolerance to this effect. Microdialysis perfusion of the nucleus accumbens in unanesthetized rats was used to monitor dopamine release, as well as DOPAC, HVA, and 5-HIAA efflux. Administration of the kappa agonist U-50488H (0.5-10 mg/kg, s.c.) resulted in a dose-related inhibition of basal dopamine release and a delayed reduction in HVA efflux. When added directly to the perfusion medium, U-50488H (10 microM) similarly reduced dopamine release and HVA efflux; however, a much higher concentration (1 mM) produced a transient increase in dopamine release. The more potent kappa agonist, spiradoline mesylate (5 mg/kg, s.c.) caused a more profound and long lasting reduction in dopamine release than that observed with U-50488H. Repeated injections of spiradoline (7 injections over 3 days at 5 and 10 mg/kg, s.c.) resulted in a persistent reduction in dopamine release with no further reduction in release being observed following an acute injection of spiradoline (1 mg/kg, s.c.). We conclude that kappa agonists act to inhibit dopamine release from the nucleus accumbens via a direct effect in that region, and that tolerance does not occur to this neurochemical effect. Thus, kappa agonists may prove useful in chronic conditions resulting from excessive dopamine release.


Blockade of electrical field stimulation-induced nonadrenergic noncholinergic (NANC) excitatory response of the guinea pig ileum. Determination of guinea pig ileal response to electrical field stimulation (EFS) before and after complete adrenergic and cholinergic blockade showed that the nonadrenergic noncholinergic excitatory (NANC-e) response contributed 40% to the total contractile response. NANC-e responses were reproducible to consecutive identical EFS. Tetrodotoxin significantly blocked the NANC-e response. The NANC-e responses obtained before and after treatment of the tissues with receptor antagonists of histamine (pyrilamine), substance P ([D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP) and serotonin (5-HT1 & 5-HT2) indicated that the response could be blocked by substance P and serotonin antagonists. Only the serotonin antagonist cyproheptadine produced a dose-dependent blockade. Both substance P and serotonin are suggested to play an important role in the NANC-e neurotransmission.


Effects of aprotinin on heparin activities and heparin neutralization with protamine. In order to investigate the new situation in which aprotinin is proposed as a novel approach to reducing post operative bleeding, specially in cardiopulmonary bypass (CPB) surgery during which heparin and protamine are commonly used, preliminary in vitro and in vivo studies have been performed. Aprotinin increases the anticoagulant heparin effects in vitro, and the hemorrhage time in vivo. But in addition to protamine, there are no statistically significant differences with heparin-protamine situation, indicating aprotinin does not disturb the neutralizing activities of protamine on heparin.


A case report of familial cyclic neutropenia. A 34-year-old female with cyclic neutropenia is reported. Family studies showed that her three sons and her mother were also involved. Oscillations in the blood neutrophil counts were almost regular, with a periodicity of 21 days. Numbers of colony-forming unit--granulocyte macrophage (CFU-GM) formed from the bone marrow cells of normal volunteers co-cultured with the patient's serum or mononuclear cell-conditioned medium (MNC-CM) were examined. Her serum prepared during the neutropenic phase inhibited the growth of CFU-GM, while her MNC-CM stimulated it. Human granulocyte colony-stimulating factor (hG-CSF) level in her serum was persistently high, with the peak occurring during the neutropenic phase. These results suggest that some inhibitory factors in the serum may be pathophysiologically important for cyclic neutropenia. To control infections, a pharmacological dose of hG-CSF was administered for 7 days around the early neutropenic phase. Her peripheral neutrophil counts oscillated from 1,200/mm3 to 17,000/mm3 with G-CSF, and from 150/mm3 to 1,800/mm3 without G-CSF.


[The fractionation of non-histone chromosomal proteins from rat kidney and liver and their DNA complexes by using the cation exchangers phosphocellulose and SE-Sephadex]. Results of the gradient chromatography of chromosomal nonhistone proteins of the rat liver and kidney and their complexes with DNA on the phosphocellulose and SE-Sephadex are presented. The profiles of elution of the preparation of the homologous tissues with NaCl linear gradient of 0.1-1.0 M are equal. In the fractions of 0.4-0.5 M NaCl, protein components are discovered only in the samples of kidney.


Sequence determination and genetic content of an 8.9-kb restriction fragment in the short unique region and the internal inverted repeat of Marek's disease virus type 1 DNA. The DNA sequence (8.9 kb) covering about 70% of the short unique region (Us) and part of the short inverted repeat of the Marek's disease virus type 1 GA strain was determined. Computer analysis of the sequence showed the presence of nine potential open reading frames (ORFs), consisting of more than 300 nucleotides in the Us region. Of these ORFs, four were found to be homologous to US10 (minor virion protein), US3 (protein kinase), US2, and US6 (gD) in the Us region of alpha-herpesvirus herpes simplex virus type 1. The protein kinase homologue is especially well conserved in alpha-herpesviruses. No counterpart of the nine MDV1 ORFs was found in the beta-herpes virus human cytomegalovirus and gamma-herpesvirus Epstein-Barr virus, suggesting that MDV1 is more similar to the alpha-herpesviruses. The junction of the Us region and the short inverted repeat was also determined by comparison between the sequences of the DNA fragments, including the terminal and internal repeats. Northern blot analysis showed that the Us region within the 8.9 kb sequence was transcriptionally active in MDV1-infected cells.


[The effect of insulin therapy on the level of natural killer cells of different immunological phenotypes (CD16+, CD56+ and CD57+) in the blood of patients with diabetes mellitus type I]. Flow cytometry with monoclonal antibody Leu-7 (CD57), Leu-11 (CD56) and Leu-19 (CD56) were used to study the content of different subpopulations of natural killer cells (NK-cells) in the blood of type I diabetes mellitus patients before and after insulin treatment and in healthy people. After a course of insulin therapy most patients showed restoration of the total cell number with antigens on their surface characteristic of NK-cells, especially CD56, that indicates the essential role of hypoinsulin in the depression of the NK-system. At the same time a small group of patients was distinguished who did not show such normalization. This may indicate participation of other mechanisms in the formation of natural immunity failure, in particular, the genetic.


[Circulating immune complexes and immunoglobulins in patients with an acute macrofocal myocardial infarct]. One of the pathogenetic mechanisms of development of acute myocardial infarction are immunologic changes of the humoral type. Indices of circulating immune complexes and A. M. G. immunoglobulins in acute myocardial infarction indicate the extension of involvement of the cardiac muscle, development of severe complications for the diagnosis and prognosis of acute myocardial infarction. An unfavourable sign is significant reduction of the level of circulating immune complexes and immunoglobulins in acute myocardial infarction.


Future prospects for treatment of hemoglobinopathies. Strategies for the treatment of sickle cell anemia and beta-thalassemia are founded on the knowledge that these disorders result from structural or functional defects in an adult gene for which an intact fetal counterpart exists. During the past decade, several pharmacologic agents have been investigated for their potential to ameliorate sickle cell anemia and beta-thalassemia by increasing the synthesis of fetal hemoglobin in adults. Progress in understanding globin gene regulation is now being combined with advances in retrovirus-mediated gene transfer, and the once-distant goal of providing gene therapy for hemoglobinopathies is rapidly approaching reality.


[Peroxisomal neurologic diseases and Refsum disease: very long chain fatty acids and phytanic acid as diagnostic markers]. Peroxisomal disorders are genetic metabolic diseases with generalized, multiple, or single functional disturbances of the peroxisome. According to the extent of the functional disturbances 3 groups of diseases can be differentiated: disorders with generalized loss of peroxisomal functions (Zellweger syndrome, ZS; neonatal adrenoleukodystrophy, NALD; infantile Refsum's disease), disorders with multiple enzymatic defects (e.g. rhizomelic chondrodysplasia punctata), and disorders with a single enzymatic defect in the peroxisome, the most important being adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN). Adult Refsum's disease, a genetic neurological disorder with phytanic acid accumulation, is due to a mitochondrial enzyme deficiency, but is often considered together with peroxisomal diseases because of phytanic acid (PHYT) accumulation in most peroxisomal diseases. The main clinical and pathological criteria of the major disorders and the biochemical parameters of their differentiation are presented. Elevated levels of very long chain fatty acids (VLCFA) and/or PHYT are the primary diagnostic markers for all peroxisomal disorders and adult Refsum's disease, respectively. Our investigations disclosed 30 ALD/AMN hemizygotes, 16 ALD/AMN heterozygotes, 8 cases of ZS/NALD and 7 patients with adult Refsum's disease. In addition, 15 cases of peroxisomal disorders were confirmed by biochemical investigations in autopsy material. With regard to peroxisomal disorders, therapeutic concepts exist only for ALD/AMN: corticosteroid substitution for adrenal insufficiency, dietary treatment, and bone marrow transplantation (BMT). Adult Refsum's disease can be treated successfully by dietary therapy. In case of dietary treatment and BMT, assay of VLCFA and/or PHYT is important for the biochemical evaluation of these therapies.


Management of severe atopic dermatitis.

Management of severe atopic dermatitis. The lack of knowledge concerning the pathophysiology of atopic dermatitis (AD) explains the absence of any specific treatment specially in severe atopic dermatitis. New treatments were recently suggested for the management of the disease. They all act on some component of the immune mechanisms which provoke the eczematous reactions. Among recent treatments proposed, I will discuss the use of cyclosporin A, puva therapy, thymopoietin and thymostimulin, antifungal therapy, alpha and gamma interferon, and treatment with interleukin 2.


Vasoactive intestinal polypeptide and substance P in the pathogenesis of atopic dermatitis. Neurogenic components are probably involved in the pathogenesis of atopic dermatitis (AD) and several neuropeptides have been implicated in the mechanisms underlying this disease. The aim of the present study was to evaluate by radio-immunoassay (RIA), the vasoactive intestinal polypeptide (VIP) and substance P (SP) content in whole-skin homogenates of AD lesions. RIA was performed using an antiserum, AH78, recognizing the carboxy-terminal fragment VIP (22-28) and a polyclonal antiserum directed against SP. VIP levels were markedly increased in lesional AD skin (5.62 +/- 1.25 pmol/g tissue) vis-à-vis controls (0.43 +/- 0.08 pmol/g tissue), whereas SP levels were significantly lower in lesional skin (0.25 +/- 0.03 pmol/g tissue) than in normal skin (0.97 +/- 0.24 pmol/g tissue). The results confirm that VIP and SP are relevant to the pathogenesis of AD and their imbalance might reflect diverse roles of these NP in the modulation of AD lesion.


[Pain reduction by trans-articular atlanto-axial screw fixation in patients with chronic polyarthritis]. To reduce the risk of cervical myelopathy, 32 patients suffering from rheumatoid arthritis (RA) and atlanto-axial dislocation (AAD) underwent a suboccipital fusion. All patients were interviewed with a pain questionnaire after a mean postoperative follow-up of 21 months (range 2-46). The results indicate a significant postoperative pain reduction as assessed by a visual numeric analogue scale, as well as a reduction of analgesic consumption.


Abnormal cutaneous neurosensitivity in atopic skin. Having found an inability of patients with atopic eczema to distinguish different levels of iontophoretically applied histamine concentrations, as shown by their diminished vascular reactions and itch responses, and reviewing this result in the light of our new findings of smaller flare reactions and weaker itch sensations following different concentrations of intradermally injected substance P, we have concluded that unmyelinated afferent skin nerve fibres in these patients seem to be affected by the pathophysiological mechanism of atopic eczema. We therefore suspect that a down-regulation of histamine receptors at nerve endings compensates for elevated histamine release from cutaneous mast cells in patients with atopic eczema.


An alpha-fetoprotein-producing carcinoma of the rectum. A rare alpha-fetoprotein (AFP)-producing carcinoma that originated in the rectum of a 54-year-old man is reported. High levels of AFP are found in the serum (5,126 ng/ml) and homogenate of the tumor tissue (2,600 ng/g). Two morphologic patterns were noted in the tumor. One was that of well differentiated tubular adenocarcinoma and the other was more polymorphous, in which the tumor cells grow in a solid-sheet with occasional glandular and papillary structures. Hyaline bodies and Schiller-Duval body-like structures were also found in the polymorphous area. There was a gradual transition between the well differentiated and the polymorphous area. AFP was positive in the polymorphous area and negative in the well differentiated area on immunohistochemical examination. These morphologic and immunohistochemical characteristics may suggest a differentiation toward embryonal carcinoma of the polymorphous component. Moreover, the polymorphous component showed marked vascular invasion and metastasis of liver as well as regional lymph nodes. AFP-producing carcinoma cells may be more aggressive than ordinary adenocarcinoma.


[Laser photocoagulation of experimentally produced choroidal neovascularization. 2. Comparison of effects by dye and argon blue-green lasers]. Choroidal neovascularization was produced in 18 eyes of 12 rhesus monkeys by application of intense krypton laser photocoagulation. Eight weeks or later, a total of 34 choroidal neovascularizations were treated either by dye laser at wavelengths of 577 or 630 nm or by argon blue-green laser. The treated lesions were evaluated clinically and histopathologically from 24 hours to 3 months after treatment. All the treated lesions were apparently healed by each of the 3 laser modalities. Histopathologically, all the treated choroidal neovascularizations were occluded 24 hours after treatment. At 2 weeks to 3 months after treatment, newly-formed choroidal vessels were present in 3 of 9 lesions (33%) after 577 nm dye laser treatment, in 8 of 9 lesions (89%) after 630 nm dye laser treatment, and in 5 of 8 lesions (62%) after argon blue-green laser. These results seemed to show the clinical superiority of 577 nm dye laser in treating choroidal neovascularization.


Simple non-invasive mapping of pain pathways in living humans, and the effects of acute non-invasively induced pain on substance P, oncogen C-fos Ab1, oncogen C-fos Ab2, dopamine, and acetylcholine. Most of the present knowledge on pain pathways is based on invasive animal experiments. In 1987, using the Bi-Digital O-Ring Test, Omura found that pain pathways can be evaluated non-invasively in living humans. In this paper, actual examples of such cases are reported. When mild pain was created by pinching different fingers of a normal human subject by placing a plastic clamp on the skin above a pain pathway, the indirect Bi-Digital O-Ring Test, through a fine electroconductive metal wire held by a third person, showed marked weakening of muscle tone of the third person resulting in the opening of the Bi-Digital O-Ring, which was preselected only when the minimum essential requirements to perform the Bi-Digital O-Ring Test were satisfied. The pain pathway thus detected had the following characteristics: For example, when the lateral side of the 5th finger of the left hand was pinched, the pathway was approximately along the ulnar nerve and then went to the dorsal root of the spinal cord around the area corresponding to the lower end of the 7th cervical vertebrae/upper end of the 1st thoracic vertebrae. At this level, the pain pathway goes to the opposite side (right side) then laterally up to the lower one-third of the medulla oblongata. It then turns horizontally from the right side to the left side at the same level, then goes up in the left side of the center of the medulla oblongata. At the left side it goes to the pons, and in the upper part of the pons it turns towards the midline. It then goes up and turns to the right side of the right cerebral cortex corresponding to the lateral side of the 5th finger.(ABSTRACT TRUNCATED AT 400 WORDS)


Cytoprotective effects of prostaglandins and a new potent protease inhibitor in acute pancreatitis. The redistribution of cathepsin B, a lysosomal enzyme, from the lysosomal pellet to the zymogen pellet in the subcellular fractionation, the colocalization of cathepsin B with digestive enzyme, and increased cellular, lysosomal, and mitochondrial fragility within acinar cells have been found during the early stages of caerulein-induced acute pancreatitis in rats. In the present study, the authors investigated the protective effects of prostaglandin E1 and E2, a combined therapy of these prostaglandins, and a new, synthetic, low molecular weight protease inhibitor, ONO3307, on the exocrine pancreas in this noninvasive model of experimental pancreatitis in vivo and in vitro. Prostaglandin E2, but not E1, prevented hyperamylasemia, congestion of amylase and trypsinogen in the acinar cells, redistribution of cathepsin B, and amylase and lactate dehydrogenase discharge from the dispersed acini. It also prevented cathepsin B leakage from the lysosomes and malate dehydrogenase leakage from the mitochondria in an almost dose-dependent manner, particularly at the dose of 100 micrograms/kg/hr continuous infusion. Furthermore, the combined therapy of prostaglandin E2 with ONO3307 strongly inhibited all the parameters tested in this study. This combination therapy seems to be the most effective against secretagogue-induced pancreatic injuries. These results indicate that cellular and subcellular organellar fragility seem to be closely involved in the pathogenesis of acute pancreatitis. Prostaglandin E2 seems to have important cytoprotective effects on the biologic membranes, such as a stabilizer of lysosomal or mitochondrial membranes. In addition, these findings also suggest the crucial roles of some unknown proteases in the etiology of acute pancreatitis, and indicate the clinical effectiveness of prostaglandins and this type of low molecular weight protease inhibitor for acute pancreatitis.


Signal transduction by T-cell receptors: mobilization of Ca and regulation of Ca-dependent effector molecules. There have been major advances over the last several years in understanding the molecular basis of signaling by the T lymphocyte (T-cell) antigen receptor. In this article we discuss the early phases of T-cell activation with an emphasis on receptor-associated signaling molecules, mobilization of Ca, and on the possible roles of Ca in signal transduction. Ligation of the extracellular domains of the T-cell receptor activates receptor-associated tyrosine kinases that can phosphorylate the gamma-isoform of phospholipase C, increasing its catalytic activity. This leads to production of inositol 1,4,5-trisphosphate, release of stored intracellular Ca, and activation of Ca-permeable plasma membrane channels. Many of the critical T-cell signal transducing enzymes such as phospholipase C and protein kinase C contain intrinsic Ca-binding domains, but for the most part the rise in cytoplasmic Ca is transduced by specialized Ca-binding proteins that lack catalytic domains. The Ca-binding proteins found in T-cells include members of both the EF-hand and annexin families, as well as other types of Ca-binding proteins. In T-cells, a number of important kinases, phosphatases, and cytoskeleton-modulating enzymes are functionally Ca dependent but have no Ca-binding domains and therefore must sense changes in the cytoplasmic Ca level through interactions with Ca-binding proteins.


Antisense oligonucleotides to CFTR confer a cystic fibrosis phenotype on B lymphocytes. Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed at low levels in nonepithelial cells. Recently, we demonstrated that CFTR is responsible for cell cycle-dependent adenosine 3',5'-cyclic monophosphate-responsive Cl- permeability in lymphocytes. Agonist responsiveness of cystic fibrosis (CF) lymphocytes was restored by transfection with plasmid containing wild type CFTR cDNA. CFTR mRNA is expressed in the B lymphoid cell line GM03299; however, quantitative reverse transcriptase-polymerase chain reaction indicates that the level of CFTR mRNA is at least 1,000 times lower than in T84 cells. CFTR protein could not be detected by Western blot or by immunoprecipitation of in vitro phosphorylated protein. However, antisense oligonucleotides representing codons 1-12 of CFTR caused a complete inhibition of cell cycle-dependent Cl-permeability [as determined by 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescence digital-imaging microscopy], thereby inducing normal cells to acquire a "CF phenotype." These studies provide direct evidence that a CFTR-associated Cl- permeability is present and measurable in lymphocytes, even though CFTR mRNA and protein are expressed at low levels.


Effects of okadaic acid indicate a role for dephosphorylation in pancreatic stimulus-secretion coupling. Okadaic acid completely inhibits phosphatase 2A at nanomolar concentrations, while complete inhibition of type 1 phosphatases occurs at 1 microM. Phosphatase 2B is significantly inhibited only at concentrations > 1 microM. In rat pancreatic acini, 1 microM okadaic acid shifted the cholecystokinin (CCK) dose-response curve for stimulating amylase release to the right without reducing maximal secretion. At 3 microM, okadaic acid inhibited maximal CCK-induced amylase release to 78 +/- 7% of control, whereas the inactive analogue 1-Nor-okadaone had no effect. Three lines of evidence indicate that this inhibition by okadaic acid occurs at a late step in stimulus-secretion coupling: 1) intracellular Ca2+ signaling in response to agonist stimulation was not appreciably altered by okadaic acid; 2) stimulation with phorbol ester plus thapsigargin (thus by-passing receptor activation), which gave 85 +/- 4% of maximal CCK-induced amylase release, was inhibited 66 +/- 4% by 3 microM okadaic acid; and 3) Ca(2+)-induced amylase secretion in streptolysin O-permeabilized cells was also reduced by 85 +/- 7%. Two-dimensional polyacrylamide gel electrophoresis of 32P-labeled acini and autoradiography demonstrated that okadaic acid dose dependently increased overall protein phosphorylation. Correspondingly, okadaic acid also led to an inhibition of CCK-induced dephosphorylation. These results show that okadaic acid inhibits pancreatic acinar secretion at a step after generation of intracellular messengers and indicate a role for protein dephosphorylation in stimulus-secretion coupling.


Anion channels for amino acids in MDCK cells. Large losses of amino acids by diffusion were previously observed in Madin-Darby canine kidney (MDCK) cells during volume regulation. Also, an outward rectifying anion channel was activated. Because this channel was not selective among anions, it was suggested that it could be permeable to amino acids. Its permeability to aspartate, glutamate, and taurine was studied using the patch-clamp technique in the inside-out configuration. Solutions containing 500 mM aspartate or glutamate were used on the cytoplasmic side of excised patches to detect single-channel currents carried by these anions. Permeability ratios were estimated in two different ways: 1) from the shift in reversal potential of current-voltage curves after anion replacement in the bath solution and 2) from comparisons of amplitudes of single-channel currents carried by tested anions and chloride, respectively. The values of aspartate-to-chloride and glutamate-to-chloride permeability ratios obtained with both methods were quite consistent and were of the order of 0.2 for both amino acids. Taurine in solutions at physiological pH 7.3 is a zwitterionic molecule and bears no net charge. To detect single-channel currents carried by taurine, solutions containing 500 mM taurine at pH 8.2 were used in inside-out experiments. Under these conditions 120 mM of negatively charged taurine was present in the solutions bathing the cytoplasmic side of excised patches. The permeability ratio estimated from the shift in reversal potential was 0.75. These results showed that some of the organic compounds released by cells during regulatory volume decrease could diffuse through this outwardly rectifying anionic channel.


Localization of the CHIP28 water channel in rat kidney. CHIP28 is an integral membrane protein that has been identified as the erythrocyte water channel and that is also expressed in the kidney. Antibodies against erythrocyte CHIP28 were used to localize this protein along the rat urinary tubule. By Western blotting, CHIP28 was detected in kidney plasma membrane and endosome fractions. With the use of immunocytochemistry, CHIP28 was located in brush-border and basolateral plasma membranes of the proximal tubule. The initial S1 segment was weakly stained, but the S2 and S3 segments were heavily labeled. Subapical vesicles were also positive. Apical and basolateral membranes of the long thin descending limb were strongly labeled, but ascending thin and thick limbs of Henle and distal convoluted tubules were negative. Some vasa recta profiles in the medulla were positive. CHIP28 is, therefore, present in membranes with a high constitutive water permeability, where it probably acts as a transmembrane water-conducting channel. Finally, a weak staining of apical and basolateral membranes of cortical collecting duct principal cells was detectable, suggesting a potential relationship of CHIP28 to the vasopressin-sensitive water channel.


Ca2+ sensitivity of contractile activation during muscarinic stimulation of tracheal muscle. The muscarinic agonists acetylcholine (ACh) and McN-A-343 act on a homogenous population of M3 receptors in canine tracheal smooth muscle; however, ACh is more effective at releasing stored Ca2+ and at stimulating inositol phosphate production. The effects of ACh and McN-A-343 on intracellular Ca2+ concentration ([Ca2+]i), myosin light chain (MLC) phosphorylation, active stress, and isotonic shortening velocity were compared to determine whether differences in their potency at stimulating second messenger pathways affected their ability to modulate the Ca2+ sensitivity of contractile filament activation. There were no differences in [Ca2+]i or isometric stress during the steady-state phase of submaximal contractions induced by ACh and McN-A-343. ACh produced slightly higher levels of MLC phosphorylation than McN-A-343; these levels were associated with much higher rates of isotonic shortening. This could indicate either an extremely high sensitivity of the shortening velocity to differences in MLC phosphorylation or that mechanisms other than MLC phosphorylation contribute to the regulation of shortening velocity. Results show that receptor-coupled pathways can modulate the relationship between [Ca2+]i and isotonic shortening velocity independently of the relationship between [Ca2+]i and isometric stress.


Effects of insulin on total RNA, poly(A)+ RNA, and mRNA in primary cultures of rat hepatocytes. The purpose of this study was to examine mechanisms involved in the regulation of protein synthesis in primary cultures of rat hepatocytes. Hepatocytes were maintained in a chemically defined serum-free medium in the presence or absence of insulin. The rate of protein synthesis in hepatocytes deprived of insulin between days 2 and 5 of culture was reduced to 67% of the rate observed in insulin-maintained controls. The decrease in protein synthetic rate was accompanied by a proportional fall in the content of both total RNA and poly(A)+RNA, suggesting that the capacity for protein synthesis was reduced in the absence of insulin. Both total RNA and poly(A)+ RNA contents and the protein synthetic rate were returned to control values after 3 days of insulin resupplementation. In addition, the effect of insulin on the expression of specific mRNAs was assessed by in vitro translation of total RNA followed by two-dimensional gel analysis of radiolabeled translation products. Only 13 of the greater than 150 spots discernible on the two-dimensional gels were altered in response to insulin. The mRNAs that were altered include examples of repression and stimulation of expression in response to insulin deprivation. Thus, in isolated rat hepatocytes, insulin regulates the capacity of both overall protein synthesis as well as the capacity for the synthesis of specific proteins.


Effects of acute hypoxemia on insulin-like growth factors and their binding proteins in fetal sheep. It has been proposed that insulin-like growth factor I (IGF-I) regulates fetal growth and differentiation. Plasma IGF-I concentrations correlate positively with fetal nutrient availability and newborn birth weights. To explore the hypothesis that hypoxemia decreases fetal growth by decreasing fetal IGF-I availability, we instrumented 14 fetal sheep with vascular catheters. At least 4 days after surgery, 10 fetuses were made acutely hypoxemic by infusing nitrogen into the maternal trachea for 3 h. Fetal blood oxyhemoglobin saturation decreased from 53 +/- 6 (SD) to 31 +/- 9%. Concomitantly, plasma IGF-I concentrations decreased from 91 +/- 11 to 67 +/- 10 ng/ml and IGF-I binding protein-1 concentration increased significantly, as assessed by ligand and Western blot analysis. Fetal IGF-I concentrations remained below control values throughout a subsequent recovery period (68 +/- 12 ng/ml at 6 h). In four control fetuses and in the ewes, plasma IGF-I concentrations were not significantly different from control values (97 +/- 18 and 181 +/- 18 ng/ml, respectively). These data support the hypothesis that decreases in fetal oxygen availability may decrease fetal growth by decreasing IGF-I production and availability.


Immunolocalization of antioxidant enzymes and isozymes of glutathione S-transferase in normal rat lung. Polyclonal antisera to manganese and copper-zinc superoxide dismutases, catalase, glutathione peroxidase (GPx), and isozymes of glutathione S-transferase (liver and placental isolates, GST-L and GST-P, respectively) were used to localize these enzymes in normal rat lung by immunostaining. Light-microscopic results, using an immunoperoxidase technique, were expanded on by electron-microscopic immunogold localization. The findings were consistent with previous biochemical work. However, both GPx and GST-P were predominantly localized to extracellular connective tissue of the lung. These findings demonstrate the basal antioxidant enzyme phenotypes for parenchymal lung tissue at light- and electron-microscopic levels. Significant components of enzymatic defense to oxidant stress are heterogeneously distributed throughout rat lung tissue including both epithelial cell surfaces and the extracellular matrix.


CFTR channels in immortalized human airway cells. The cystic fibrosis (CF) gene codes for CF transmembrane regulator (CFTR), a small-conductance linear Cl- channel, but numerous studies have identified a larger conductance, rectifying Cl- channel as the adenosine 3',5'-cyclic monophosphate (cAMP)-regulated channel that is defective in airway cells. We examined Cl- conductance in a bronchial epithelial cell line that expresses CFTR, 16HBE14o-, (CFTR+) and in an airway cell line that does not, 9HTEo-/S, (CFTR-). Ionomycin or hypotonic Ringer increased iodide efflux from both cell lines; however, forskolin increased iodide efflux or whole cell Cl- currents only in CFTR+ cells. Forskolin-stimulated whole cell currents were linear, voltage independent, and blocked by iodide. Cell-attached and outside-out patches from confluent CFTR+ but not CFTR- cells revealed 6-pS channels having linear current-voltage relations, permselectivity Cl > I (partial block by external iodide), and little or no inhibition by 5-nitro-2-(3-phenylpropylamino)-benzoate. The number of active channels per patch increased from 0.6 to 3.0 after forskolin. Channels closed after excision with tau = 4 s, but activity could be prolonged with ATP or protein kinase A plus ATP. Channels were modeled with one open and four closed states and show apparent cooperativity in gating. Rectifying Cl- channels previously implicated in CF were not seen in cell-attached recordings from either cell line but were abundant in excised patches from both cell lines. Thus CFTR channels are the pathway for cAMP-mediated Cl- conductance in these human airway cells, Ca2+ and swelling-induced channels do not require CFTR, and CFTR-cells display a CF phenotype.


[Cerebrovascular circulation in patients with oligomenorrhea]. Brain hemodynamics was studied in 76 patients with oligomenorrhea aged 18 to 35 from the data of rheoencephalography. Disorders of the regional tone of brain vessels were different and related to the time of the menstrual cycle disorder; this may be explained by the degree of changes in various neuromediator mechanisms of brain circulation and reproductive system function regulation.


A morphological study of the rabbit corneal assay. We present a morphological study of the rabbit corneal assay. The effects of corneal pockets filled with methylcellulosis carriers with and without basic fibroblast growth factor (bFGF) were studied 4, 7, 11, 14 and 21 days following surgery. In the controls, strongly vacuolated keratocytes are present at the borders and especially at the bottom of the pocket. A large number of such cells can also be found in the region between the pocket and the epithelium. These cells obviously undergo necrosis. A few vacuolated keratocytes with poorly structured cytoplasm can even be found in the stroma between the pocket and the limbus. There are also changes in the epithelium. It is thinner than normal and the cells are longitudinally oriented. Construction of the pocket sometimes results in a fissure extending a variable distance from the bottom of the pocket to the limbus. Basic FGF induced blood-vessel growth from the limbus into the cornea. At least until day 7 this is accompanied by high vulnerability of the vessels, and changes in the pattern and structure of the extracellular matrix. Neutrophilic granulocytes, monocytes and a few mast cells migrate in front of the vessels. As in the controls, vacuolated and necrotic keratocytes are present within the stroma. From day 11 onward, many more vessels can be found in front of than behind the pocket, often located adjacent to vacuolated keratocytes. We suggest that an angiogenic substance placed in the corneal pocket is not the only angiogenic stimulus in the test, but acts together with many stimuli caused by the surgical manipulations.


Distribution and possible origins of substance P-containing nerve fibers in the rat liver. The distribution and possible origins of substance P-containing nerve fibers in the rat liver were investigated by immunohistochemistry and nerve transection. Nerve fibers with substance P-like immunoreactivity formed a more complex network than previously known in the walls of portal vein branches. Substance P-immunoreactive fibers were seen not only in and around the walls of the hepatic artery, but also in close association with the hepatic veins and bile ducts. Transection of the greater splanchnic nerves and/or the vagus nerves indicated that substance P-immunoreactive fibers in the walls of the portal and hepatic veins enter the liver via both nerves, and that those associated with the hepatic artery and bile ducts stem from the greater splanchnic nerves. The widespread distribution of hepatic substance P and its complex innervation pattern within the liver suggest that it is involved in a variety of physiological processes in this organ.


[Pharmacology of low molecular weight hydroxyethyl starch]. In the last ten years, the practice of plasma volume expansion has changed significantly. Most clinicians have put a stop to the use of fresh frozen plasma because of growing concerns about hepatitis and AIDS transmission. Today, natural and synthetic colloids and crystalloids are used to a great extent. Although clinical practice varies from one institution to another, the most widely observed change was a major increase in the administration of human serum albumin (HSA). As a result, the cost of plasma volume expansion became so high that it justified finding safe and cheaper alternatives to HSA. Low molecular weight, hydroxyethylstarch (HES) are the synthetic colloids which are closest to HSA. HES are modified natural polymers whose physico-chemical properties are defined by their molecular weight and molar substitution ratio. Average molecular weights of these poly-dispersed solutions are approximately 200 to 250 kd (in weight) and 60 kd (in number). Hydroxyethylation, which slows down hydrolysis by alpha-amylase, is best quantified by the molar substitution ratio between the proportions of hydroxyethyl-ether and glucose. HES have pharmacokinetic properties which are independent of molecular weight and directly related to the molar substitution ratio. The two HES available in France are Elohes and Lomol, Elohes, at a concentration of 6%, has a colloid-osmotic effect close to that of plasma. It induces an initial plasma volume expansion greater than that of the infused volume, and has a long lasting effect (24 h) related to its molar substitution ratio (0.62). Lomol, at a concentration of 10%, is hyperoncotic.(ABSTRACT TRUNCATED AT 250 WORDS)


Relationship between colloid osmotic pressure and plasma protein concentration in cattle, horses, dogs, and cats. The relationship between colloid osmotic pressure (COP) and protein concentration was investigated for purified proteins and plasma samples obtained from cattle, horses, dogs, and cats. At equivalent concentrations, bovine albumin exerted a COP that exceeded that of gamma-globulins by a mean factor of 4.4. Similar relationships between COP and protein were observed in the other species. Consequently, for a given total protein concentration, COP was dependent on the albumin/gamma-globulins ratio. A commonly used nomogram for estimating COP from protein concentration, the Landis-Pappenheimer equation, did not provide reliable results for plasma samples from these species.


Measurement of HIV-1 reverse transcriptase by a nonradioactive assay system. Reverse transcriptase activity was measured by incorporation of dUMP linked to digoxigenin into a suitable template-primer molecule. Incorporation was monitored by using peroxidase-conjugated Fab fragments directed against digoxigenin. The standard assay measuring incorporation of radiolabeled nucleotides into acid-precipitable material was compared with this new immunochemical assay with regard to its usefulness for testing inhibitors of reverse transcriptase.


Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction. The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.


Uncultivated cyanobacteria, Chloroflexus-like inhabitants, and spirochete-like inhabitants of a hot spring microbial mat. Analysis of 16S rRNA sequences retrieved as cDNA (16S rcDNA) from the Octopus Spring cyanobacterial mat has permitted phylogenetic characterization of some uncultivated community members, expanding our knowledge or diversity within this microbial community. Two new cyanobacterial 16S rRNA sequences were discovered, raising to four the number of cyanobacterial sequence types known to occur in the mat. None of the sequences found is that of the cultivated thermophilic cyanobacterium Synechococcus lividus. A new 16S rRNA sequence characteristic of green nonsulfur bacteria and their relatives was discovered, raising to two the number of such sequences known to exist in the mat. Both are unique among the 16S rRNA sequences of cultivated members of this group, including an Octopus Spring isolate of Chloroflexus aurantiacus and Heliothrix oregonensis, whose sequences we report herein. Two spirochete-like 16S rRNA sequences were discovered. One can be placed in the leptospira subdivision of the spirochete group, but the other has such a loose affiliation with the spirochete group that it might actually belong to an as yet unrecognized subdivision or even to a new eubacterial line of descent.


Selection of a Pseudomonas cepacia strain constitutive for the degradation of trichloroethylene. Tn5 insertion mutants of Pseudomonas cepacia G4 that were unable to degrade trichloroethylene (TCE), toluene, or phenol or to transform m-trifluoromethyl phenol (TFMP) to 7,7,7-trifluoro-2-hydroxy-6-oxo-2,4-heptadienoic acid (TFHA) were produced. Spontaneous reversion to growth on phenol or toluene as the sole source of carbon was observed in one mutant strain, G4 5223, at a frequency of approximately 1 x 10(-4) per generation. One such revertant, G4 5223-PR1, metabolized TFMP to TFHA and degraded TCE. Unlike wild-type G4, G4 5223-PR1 constitutively metabolized both TFMP and TCE without aromatic induction. G4 5223-PR1 also degraded cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, and 1,1-dichloroethylene and oxidized naphthalene to alpha naphthol constitutively. G4 5223-PR1 exhibited a slight retardation in growth rate at TCE concentrations of > or = 530 microM, whereas G4 (which was unable to metabolize TCE under the same noninducing growth conditions) remained unaffected. The constitutive degradative phenotype of G4 5223-PR1 was completely stable through 100 generations of nonselective growth.


Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora. Portions of the 16S rRNA from closely related species of the genus Bifidobacterium that are found in the human intestinal microflora were sequenced in order to design species-specific oligonucleotide probes. Five oligonucleotide probes ranging from 16 to 19 bases in length and complementary to 16S rRNA sequences from Bifidobacterium adolescentis, B. bifidum, B. breve, B. infantis, and B. longum were synthesized. With crude high-molecular-weight RNA preparations as targets, these probes showed the desired species specificity, even down to a 1-nucleotide difference. For the practical evaluation of these probes, their specificity and sensitivity were tested against seven strains of the same species and 54 strains of heterologous bacteria with fixed whole cells as targets. The probes for B. adolescentis, B. breve, and B. longum showed efficient and specific hybridization. Although the probes for B. bifidum and B. infantis cross-reacted with a few bacterial strains not isolated from humans, these probes showed species specificity for human intestinal bacteria. These 16S rRNA probes should prove valuable for the identification and detection of human intestinal Bifidobacterium species.


[Target epitopes of monoclonal antibodies against ABH structures]. Comparing the reactivities of murine and human monoclonal antibodies directed against the antigens of A, B, and H blood groups by using different techniques (agglutination, inhibition by synthetic oligosaccharides and salivary antigens, tissue immunofluorescence, the use of an anti-idiotype antibody) enabled us to show a high heterogeneity of anti-A, anti-AB antibodies and, to some extend, anti-B and anti-H antibodies. Despite this diversity, the determination of the antibody specificity thanks to synthetic antigens made it possible to distinguish several groups of anti-A, B, AB, and H antibodies and to establish a classification that takes also their reactivity towards salivary and tissue antigens into account. On the contrary, few correlations were found between these tests and the ability of antibodies to agglutinate red blood cells of the different ABO-system variants.


Individual differences in basal cisternal cerebrospinal fluid 5-HIAA and HVA in monkeys. The effects of gender, age, physical characteristics, and matrilineal influences. We examined the effects of gender, age, weight, length, body shape (ectomorphy), and matrilineal influences on cisternal cerebrospinal fluid 5-hydroxyindoleacetic acid (CSF 5-HIAA) and homovanillic acid (HVA) in 78 socially living adult and adolescent vervet monkeys. CSF 5-HIAA and the 5-HIAA:HVA ratio were higher (by 27% and 18%, respectively) in females. In both sexes, CSF 5-HIAA and the 5-HIAA:HVA ratio increased with age. Neither weight nor length were independently related to CSF 5-HIAA or HVA; however, shape correlated with CSF 5-HIAA and HVA in males (higher in thin, long subjects). Male offspring had CSF 5-HIAA concentrations and 5-HIAA:HVA ratios that were significantly closer to their mothers than did age-matched, maternally unrelated males. Repeated measures of CSF 5-HIAA and HVA in another 22 males living in unvarying settings showed that individual differences in these measures persisted over time. The data underscore the impact of gender, age, and matrilineal relationships on individual differences in CSF monoamine metabolites and highlight the importance of controlling for age and gender in neuropharmacological investigations of clinical populations.


The lpr and gld genes in systemic autoimmunity: life and death in the Fas lane. The single gene lpr and gld models of spontaneous systemic autoimmunity have attracted much attention in recent years. Here, Philip Cohen and Robert Eisenberg describe the fascinating recent findings that the lpr and gld [corrected] phenotypes result from defects in the Fas gene and, perhaps, in the ligand for fas, respectively.


CD40 and its ligand, an essential ligand-receptor pair for thymus-dependent B-cell activation. The discovery of CD40 and its ligand, gp39, have created new perspectives on the activation of resting B cells by TH cells. Here, Randolph Noelle, Jeffrey Ledbetter and Alejandro Aruffo describe how these molecules were characterized and, from recent findings, examine their relevance to immune activation.


Detection of retinoic acid receptor mRNA in rat tissues by reverse transcriptase-polymerase chain reaction. The presence of retinoic acid receptor (RAR) alpha, beta and gamma mRNA was examined in 16 different kinds of rat tissue using the highly sensitive reverse transcriptase-polymerase chain reaction technique. The data demonstrated that each tissue expressed at least two types of RAR mRNA. Among the three types of RAR mRNA, RAR alpha was widely expressed in all types of organ and was the dominant form expressed in the gastrointestinal tract. RAR beta mRNA was not present in the intestine and spleen. In addition, RAR beta mRNA levels were high in the heart, lung, brain, testis and epididymis. RAR gamma mRNA was abundant in both male and female reproductive systems, as well as epidermal tissues. The prevalence of each RAR mRNA in the tissues suggests the diverse biological roles of these receptors.


Serum neopterin levels in patients with hepatocellular carcinoma. Increased concentrations of neopterin have been found in conditions causing a stimulation of cellular immunity, including various malignancies. In liver diseases, serum or urinary neopterin levels have been studied in acute viral hepatitis, chronic hepatitis, fatty liver and liver cirrhosis. In the present study neopterin serum levels have been measured in 16 patients with hepatocellular carcinoma (HCC), in 32 patients with liver cirrhosis, and in 28 healthy subjects as controls. Mean values of serum neopterin were significantly increased (p < 0.01) in patients with HCC (15.89 +/- 6.34 nmol/l) when compared with those of normal subjects (4.74 +/- 2.13 nmol/l), but no difference was observed between patients with HCC (associated or not with liver cirrhosis) and patients with liver cirrhosis. Neopterin concentrations are not affected by liver cirrhosis aetiology, nor by its clinical severity, and are not correlated to the values of serum alpha-fetoprotein, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl-transferase, and gamma-globulin. The results show that there is a consistent overlap of values in patients with HCC and liver cirrhosis; macrophage activation seems to be a feature of chronic liver diseases, irrespective of HCC development.


The use of detergent-based aqueous two-phase systems for the isolation of extracellular proteins: purification of a lipase from Pseudomonas cepacia. The partitioning of a variety of extracellular lipases, both pro- and eucaryotic, in detergent-based aqueous two-phase systems was examined. The results revealed that all procaryotic lipases showed a clear preference for the detergent-rich coacervate phase. In contrast, all eucaryotic lipases were significantly excluded from this phase, most probably caused by their glycosylation. The potential of such detergent-based systems for the isolation of extracellular lipases directly from cell-free culture broth was analyzed using the bacterium Pseudomonas cepacia (DSM 50181). This strain was identified after a limited screening for lipase activity. About 76% of the lipase could be extracted into the coacervate phase in just one purification step, leading to a four-fold concentration of lipase and a purification factor of 24.


Entrapment of protein protease inhibitors in red blood cells. Aprotinin and alpha 1-proteinase inhibitor have been encapsulated in human red blood cells (RBC) by a dialysis technique that involves transient hypotonic haemolysis followed by isotonic resealing. Both protease inhibitors can be encapsulated to a considerable extent. These molecules are released only by haemolysis of the cells and that excludes the possibility of using loaded erythrocytes for a slow release of the inhibitor(s) in the blood stream. However, the stability of the two inhibitors, the evidence for the binding of aprotinin to RBC components, and the results showing inhibition of endogenous proteolytic activity indicate that the inhibitors may be valuable in blocking, at least partially, undesired intraerythrocytic proteolytic reactions.


Pretreatment natural killer antigen density correlates to clinical response in tumor patients receiving long-term subcutaneous recombinant interleukin-2 and recombinant interferon-alpha. We evaluated density of the natural killer (NK) cell-associated CD56 antigen on circulating NK cells of 47 patients with advanced renal cell carcinoma. Patients received a combination of low-dose subcutaneous recombinant interleukin-2 (rIL-2) and recombinant interferon-alpha (rIFN-alpha) as home therapy. Antigen density of CD56 before therapy was 2.2-fold higher (P < 0.005) in patients who subsequently achieved a complete or partial remission when compared with patients who presented with progressive disease on therapy. After a 6-week treatment cycle, NK cells of treatment responders expressed significantly (2.1-fold; P < 0.005) more CD56 antigens than NK cells in nonresponding patients. These results suggested a potential role of both pre- and posttreatment NK antigen density levels as a biologic correlate to treatment response.


The basal forebrain cholinergic system in the raccoon. The distribution of neurons displaying choline acetyltransferase (ChAT) immunoreactivity was examined in the raccoon basal forebrain using a rabbit antiserum and a monoclonal antibody. Alternating sections were used for Nissl staining. ChAT-positive neurons were arranged in a continuous mass extending from the medial septum to the caudal pole of the pallidum. Based upon spatial relations to fibre tracts, the clustering of neuronal groups, and cytological criteria, the basal forebrain magnocellular complex can be subdivided into several distinct regions. Although clear nuclear boundaries were often absent, the ChAT-positive neurons were divided into: the nucleus tractus diagonalis (comprising pars septi medialis, pars verticalis and pars horizontalis); nucleus praeopticus magnocellularis; substantia innominata; and the nucleus basalis of Meynert. Comparison with Nissl-stained sections indicated the presence of varying proportions of non-cholinergic neurons clustered or arranged loosely within these basal forebrain subdivisions. These data provide a structural basis for studies concerned with the topographical and physiological aspects of the raccoon basal forebrain cholinergic projections and its comparison with the basal forebrains of other species.


The relationship of calbindin-containing neurons with substance P, Leu-enkephalin and cholecystokinin fibres: an immunohistochemical study in the rat thalamus. In the rat thalamus, immunoreactivity for the calcium binding protein calbindin (Cb) is mostly confined to neuronal cell bodies, sometimes revealing proximal dendrites, of the midline, intralaminar and posterior regions. Substance P (SP)-, cholecystokinin (CCK)- and Leu-enkephalin (L-ENK)-immunoreactive (ir) elements in the thalamus are fibre-like structures, intermingled with punctate elements probably representing axonal arborizations and their synaptic boutons. These peptidergic fibres are unevenly distributed in several thalamic domains, including the areas that contain Cb-ir neurons. The relationship between Cb-ir cell bodies and these three different peptidergic systems of thalamic innervation was studied with immunohistochemistry. Single-labelling experiments on adjacent sections and double immunostaining on the same section were performed. A considerable overlap between Cb-ir perikarya and SP-ir fibres was found in most thalamic nuclei. In particular, in the intralaminar nuclei and posterior complex, SP-ir punctate elements were frequently observed in close proximity to Cb-ir cell bodies and dendrites. On the other hand, no consistent topographical correspondence between Cb-ir perikarya and CCK- or L-ENK-ir fibres was evident. Altogether, the present data suggest a selective anatomical and, possibly, functional relationship between SP and Cb in at least a subpopulation of rat thalamic neurons.


A paradigm for examining toxicant effects on viability, structure, and axonal transport of neurons in culture. N1E.115 murine neuroblastoma cells differentiating in serum-free medium were used to develop a paradigm for testing neurotoxicity in vitro. The paradigm was designed to test the effects of toxicants on four different aspects of cell function or structure: 1. Viability as shown by the retention of cellular radiolabel (51Cr); 2. Growth and maintenance of neurites as reflected by the incidence and average length of these processes; 3. Gross structure of neurites; and 4. Velocity and flux of rapid anterograde and retrograde axonal transport as judged by video-enhanced differential interference contrast microscopy. To evaluate this paradigm, colchicine and vinblastine were used as neurotoxicants with a well-understood mechanism of action. These agents were only weakly cytotoxic according to the Cr-release assay, but were able to interfere with neurite outgrowth at nanomolar concentrations. Neurites that were elaborated in the presence of vinblastine and colchicine were often disfigured by numerous swellings packed with organelles. In established neurites, micromolar concentrations of vinblastine inhibited organellar motility with great rapidity, blocking all signs of transport within 20 min. The effect of colchicine was slower and less complete, but still impressive. We suggest that this four-part analysis represents a highly sensitive in vitro test for neurotoxicity, and a means of analyzing the relation between abnormalities of transport and structural damage of nerve cells.


Molecular motors in axonal transport. Cellular and molecular biology of kinesin. Neurons require a large amount of intracellular transport. Cytoplasmic polypeptides and membrane-bounded organelles move from the perikaryon, down the length of the axon, and to the synaptic terminals. This movement occurs at distinct rates and is termed axonal transport. Axonal transport is divided into the slow transport of cytoplasmic proteins including glycolytic enzymes and cytoskeletal structures and the fast transport of membrane-bounded organelles along linear arrays of microtubules. The polypeptide compositions of the rate classes of axonal transport have been well characterized, but the underlying molecular mechanisms of this movement are less clear. Progress has been particularly slow toward understanding force-generation in slow transport, but recent developments have provided insight into the molecular motors involved in fast axonal transport. Recent advances in the cellular and molecular biology of one fast axonal transport motor, kinesin, have provided a clearer understanding of organelle movement along microtubules. The availability of cellular and molecular probes for kinesin and other putative axonal transport motors have led to a reevaluation of our understanding of intracellular motility.


Organelles in fast axonal transport. What molecules do they carry in anterograde vs retrograde directions, as observed in mammalian systems? The present minireview describes experiments carried out, in short-term crush-operated rat nerves, using immunofluorescence and cytofluorimetric scanning techniques to study endogenous substances in anterograde and retrograde fast axonal transport. Vesicle membrane components p38 (synaptophysin) and SV2 are accumulating on both sides of a crush, but a larger proportion of p38 (about 3/4) than of SV2 (about 1/2) is recycling toward the cell body, compared to the amount carried with anterograde transport. Matrix peptides, such as CGRP, ChRA, VIP, and DBH are recycling to a minor degree, although only 10-20% of surface-associated molecules, such as synapsins and kinesin, appear to recycle. The described methodological approach to study the composition of organelles in fast axonal transport, anterograde as compared to retrograde, is shown to be useful for investigating neurobiological processes. We make use of the "in vivo chromatography" process that the fast axonal transport system constitutes. Only substances that are in some way either stored in, or associated with, transported organelles can be clearly observed to accumulate relative to the crush region. Emphasis in this paper was given to the synapsins, because of diverging results published concerning the degree of affiliation with various neuronal organelles. Our previously published results have indicated that in the living axons the SYN I is affiliated with mainly anterogradely fast transported organelles. Therefore, some preliminary, previously unpublished results on the accumulations of the four different synapsins (SYN Ia, SYN Ib, SYN IIa, and SYN IIb), using antisera specific for each of the four members of the synapsin family, are described. It was found that SYN Ib clearly has a stronger affiliation to anterogradely transported organelles than SYN Ia, and that both SYN IIa and SYN IIb are bound to some degree to transported organelles.


Axon-myelin transfer of phospholipids and phospholipid precursors. Labeling of myelin phosphoinositides through axonal transport. Previous studies have provided evidence for axon-to-myelin transfer of intact lipids and lipid precursors for reutilization by myelin enzymes. Several of the lipid constituents of myelin showed significant contralateral/ipsilateral ratios of incorporated radioactivity, indicative of axonal origin, whereas proteins and certain other lipids did not participate in this transfer-reutilization process. The present study will examine the labeling of myelin phosphoinositides by this pathway. Both 32PO4 and [3H]inositol were injected monocularly into 7-9-wk-old rabbits and myelin was isolated 7 or 21 days later from pooled optic tracts and superior colliculi. In total lipids 32P counts of the isolated myelin samples showed significant contralateral/ipsilateral ratios as well as increasing magnitude of contralateral-ipsilateral differences during the time interval. Thin-layer chromatographic isolation of the myelin phosphoinositides revealed significant 32P-labeling of these species, with PIP and PIP2 showing time-related increases. This resembled the labeling pattern of the major phospholipids from rabbit optic system myelin in a previous study and suggested incorporation of axon-derived phosphate by myelin-associated enzymes. The 32P label in PI, on the other hand, remained constant between 7 and 21 days, suggesting transfer of intact lipid. This was supported by the labeling pattern with [3H]inositol, which also showed no increase over time for PI. These results suggest axon-myelin transfer of intact PI followed by myelin-localized incorporation of axon-derived phosphate groups into PIP and PIP2. The general topic of axon-myelin transfer of phospholipids and phospholipid precursors is reviewed.


Does nerve impulse activity modulate fast axonal transport? The possibility that the amount of newly synthesized material made available for fast axonal transport is regulated by nerve impulse activity was examined in an in vitro preparation of bullfrog dorsal root ganglia (DRG) and sciatic nerve. Under conditions that precluded effects of impulse activity on either uptake or incorporation of precursor, patterned stimulation of the sciatic nerve (1 out of every 2 s) produced a frequency- and time-dependent decrease in the amount of radiolabeled protein accumulating at a nerve ligature. The response to patterned stimulation was significantly greater than that to continuous stimulation when the same number of stimuli were delivered. In unligated nerve preparations, patterned stimulation decreased the amplitude of the transport profile with no concomitant change in the wave front distance. Nerve stimulation produced no observable ultrastructural alterations within neuronal cell bodies of the DRG. We propose that the physiological significance of these results is not that nerve impulse activity decreases fast axonal transport, but that the amount of transport increases during periods of electrical quiescence. According to this hypothesis, activity-dependent macromolecules of the axolemma and nerve terminals are replenished during periods when the neuron is firing less frequently. These findings are discussed in light of reports that chronic in vivo stimulation increases the amount of fast-transported, radiolabeled protein (Chan et al., 1989) and that TTX-blockade of neuronal activity has no effect on protein transport (Edwards and Grafstein, 1984; Riccio and Matthews, 1985).


Acrylamide-induced alterations in axonal transport. Biochemical and autoradiographic studies. Alterations in the axonal transport of proteins, glycoproteins, and gangliosides in sensory neurons of the sciatic nerve were examined in adult male rats exposed to acrylamide (40 mg ip/kg body wt/d for nine consecutive days). Twenty-four hours after the last dose, the L5 dorsal root ganglion (DRG) was injected with either [35S]methionine to label proteins or [3H]glucosamine to label glycoproteins and gangliosides. The downflow patterns of radioactivity for [35S]methionine-labeled proteins and [3H]glucosamine-labeled gangliosides were unaltered by acrylamide treatment. In contrast, the outflow pattern of labeled glycoproteins displayed a severely attenuated crest with no alteration in velocity, suggesting a preferential transfer with the unlabeled stationary components in the axolemma. Retrograde accumulation of transported glycoproteins and gangliosides was unaltered for at least 6 h; however, by 24 h, there was a 75% decrease in the amount of accumulated material. The accumulation of [35S]methionine-labeled proteins was not altered. Autoradiographic analysis revealed an acrylamide-induced paucity of transported radiolabeled glycoproteins selectively in myelinated axons with no effect on "nonmyelinated" axons. The pattern of transported proteins was similar in both control and acrylamide-exposed animals. These results suggest a preferential inhibition of glycosylation or axonal transport of glycoproteins in neurons bearing myelinated axons. More importantly, it suggests that interpretations of axonal transport data must be made with the consideration of alterations in selective nerve fibers and not with the tacit assumption that all fibers in the nerve population are equally affected.


Regeneration of an adult peripheral nerve preparation in culture. The methods used to maintain the vagus nerve from the adult rat in culture and how regeneration is studied in this preparation are described. A hypothesis is presented on the triggering of the cell body reaction. It is suggested that this reaction is initiated by proteins synthesized in nonneuronal cells at the site of a nerve lesion. These proteins, referred to as regenerins, reach the nerve cell body by retrograde axonal transport, where they initiate the regeneration process.


Neuronal compartments and axonal transport of synapsin I. Studies on the transport kinetics and the posttranslational modification of synapsin I in mouse retinal ganglion cells were performed to obtain an insight into the possible factors involved in forming the structural and functional differences between the axon and its terminals. Synapsin I, a neuronal phosphoprotein associated with small synaptic vesicles and cytoskeletal elements at the presynaptic terminals, is thought to be involved in modulating neurotransmitter release. The state of phosphorylation of synapsin I in vitro regulates its interaction with both synaptic vesicles and cytoskeletal components, including microtubules and microfilaments. Here we present the first evidence that in the mouse retinal ganglion cells most synapsin I is transported down the axon, together with the cytomatrix proteins, at the same rate as the slow component b of axonal transport, and is phosphorylated at both the head and tail regions. In addition, our data suggest that, after synapsin I has reached the nerve endings, the relative proportions of variously phosphorylated synapsin I molecules change, and that these changes lead to a decrease in the overall content of phosphorus. These results are consistent with the hypothesis that, in vivo, the phosphorylation of synapsin I along the axon prevents the formation of a dense network that could impair organelle movement. On the other hand, the dephosphorylation of synapsin I at the nerve endings may regulate the clustering of small synaptic vesicles and modulate neurotransmitter release by controlling the availability of small synaptic vesicles for exocytosis.


Rhodamine-labelled phalloidin stains components in the chromosomal spindle fibres of crane-fly spermatocytes and Haemanthus endosperm cells. In crane-fly spermatocytes and Haemanthus endosperm, all metaphase and anaphase chromosomal spindle fibres were stained with rhodamine-labelled phalloidin. In crane-fly spermatocytes, each kinetochore was stained with rhodamine-labelled phalloidin at diakinesis of prophase and after colcemid caused metaphase spindles to depolymerize. Since phalloidin stains actin filaments, the distributions of rhodamine-labelled phalloidin-stained material in crane-fly spermatocytes and Haemanthus endosperm suggest that actin filaments might interact with microtubules to produce forces that move chromosomes during cell division, either directly or via an intermediate motor molecule.


Relationships between the rapid axonal transport of newly synthesized proteins and membranous organelles. Rapid axonal transport is generally viewed as being exactly analogous to the secretory process in nonneuronal cells. The cell biology of rapid axonal transport is reviewed, the central concern being to explore those aspects that do not fit into the general secretory model and which may thus represent specific neuronal adaptations. Particular attention is paid to the relationship between the transport of newly synthesized proteins and of the membranous organelles that act as carriers. Sites in the transport sequence at which the behavior of axonal transport may differ from the secretory model are at the initiation of axonal transport at the trans-side of the Golgi apparatus, within the axon where molecules are deposited from the moving phase to a stationary phase, and at nerve terminals or axonal lesions where transport reversal takes place.


Organization and slow axonal transport of cytoskeletal proteins under normal and regenerating conditions. The organization of the axonal cytoskeleton was investigated by analyzing the solubility and transport profile of the major cytoskeletal proteins in motor axons of the rat sciatic nerve under normal and regenerating conditions. When extracted with the Triton-containing buffer at low temperature, 50% of tubulin and 30% of actin were recovered in the insoluble form resistant to further depolymerizing treatments. Most of this cold-insoluble form was transported in slow component a (SCa), the slower of the two subcomponents of slow axonal transport, whereas the cold-soluble form showed a biphasic distribution between SCa and SCb (slow component b). Changes in slow transport during regeneration were studied by injuring the nerve either prior to (experiment I) or after (experiment II) radioactive labeling. In experiment I where the transport of proteins synthesized in response to injury was examined, selective acceleration of SCb was detected together with an increase in the relative proportion of this component. In experiment II where the response of the preexisting cytoskeleton was examined, a shift from SCa to SCb of the cold-soluble form was observed. The differential distribution and response of the two forms of tubulin and actin suggest that the cold-soluble form may be more directly involved in axonal transport.


Transport of receptors.

Transport of receptors. The axonal transport of neurotransmitter receptors is thought to be a common phenomenon in many neuronal systems. The "machinery" for receptor (protein) "assembly" is found in the cell bodies of neurons and the "manufacture" of receptors takes place there. These receptors are then "shipped" to their ultimate destinations by a transport process. This is an axonal transport mechanism in the case of presynaptic receptors. Some form of transport process may also exist to send receptors out into the dendritic arborizations of neurons, although the latter is more difficult to verify. Axonal transport has been demonstrated, in the peripheral nervous system, for many different neurotransmitter receptors. In the central nervous system, the results are less clear, but indicate the presence of a transport mechanism for catecholamine, acetylcholine, and opiate sites. One important component then, in the development of receptors, is the transportation to terminal membrane sites where they are ultimately incorporated and available for interaction with neurotransmitters and drugs.


Improved indirect fluorescence immunocytochemical method using counterstains. Immunocytochemistry in recent years has provided powerful tools for research in neurobiology. One of the more popular techniques is the indirect fluorescence technique in which fluorescein isothiocyanate (FITC) or tetrahodamine isothiocyanate (TRITC) is used. Although widely used, this technique has two disadvantages: (1) localization may be difficult in relation to background morphology, and (2) the fluorescence fades. The study reported here describes a modification of an indirect immunocytochemical technique using FITC, TRITC and 7-amino-4-methyl-coumarin-3-acetic acid (AMCA) which enhances localization and significantly prolongs fluorescence. Evans blue was used as a counterstain. The results show that FITC and AMCA stained cells are seen against a background of clearly distinguishable cells after counterstaining with Evans blue. However, Evans blue is not compatible with TRITC. In addition, the fluorescence life of the FITC is extended from several days to several weeks with Evans blue. These results clearly indicate that Evans blue can be used to improve indirect fluorescence immunocytochemical techniques.


DiI as a marker for cellular transplantation into solid organs. Research in cellular transplantation is frequently compromised by an inability to identify transplanted cells engrafting into orthotopic sites if they exhibit normal morphology and no unique antigenic markers. A method is described for using the fluorescent dye DiI as a marker for cell transplantation studies. This dye is not metabolized or exchanged between cells in vitro or in vivo and enables identification of engrafted cells by fluorescence microscopy, flow cytometry or fluorescence-activated cell sorting. Applications are described in autologous hepatocellular and thyroid follicular cell transplantation.


Synthesis of digoxigenin-labeled cDNA as a test for mRNA integrity. The nucleotide analogue digoxigenin-11-dUTP is widely used as a nonradioactive marker in a broad range of techniques including dot blots, Southern and Northern blots, colony and plaque screenings and in situ hybridizations. In this report, we describe the incorporation of this molecule into a cDNA synthesized by reverse transcriptase as a novel application for digoxigenin. This reaction can be performed as a useful control to check the integrity of a bulk mRNA preparation in the construction of a cDNA library, since the ability of mRNA to direct the synthesis of long molecules of first-strand cDNA is a sign of its integrity.


Flavonoid-specific staining of Arabidopsis thaliana. Crop yields may be threatened by increases in UV-B radiation resulting from depletion of the ozone layer. In higher plants, the presence of flavonols provides a protective mechanism, and we report a novel staining procedure for the visualization of such protectants in plant tissue. It is shown that the proposed technique provides sensitive and specific fluorescence of flavonoids in chlorophyll-bleached tissue of Arabidopsis thaliana.


Development of a rapid method for detecting bacterial cells in situ using 16S rRNA-targeted probes. A rapid method for the identification of bacterial cells using 16S rRNA-directed, fluorescently tagged oligonucleotide probes has been developed. The parameters evaluated for their effect on labeling intensity included storage time, type of fixative, time of fixation, treatment time with methanol:formaldehyde and treatment time with borohydride. The results of tests using a variety of microorganisms, both Gram-positive and Gram-negative, are presented. Using this method, cells are spotted onto slides and stored desiccated until hybridized. This method may be especially applicable to environmental samples, which comprise diverse cell types and frequently require storage prior to examination.


Cisplatinum dose dependent response in germ cell cancer evaluated by tumour marker modelling. This study presents an analysis on longitudinal tumour marker series in twenty-two patients with non-seminomatous germ cell cancers treated with cisplatinum (DDP) based combination chemotherapy. Series of alphafoetoprotein (AFP), human chorionic gonadotrophin (HCG) and lactate dehydrogenase (LDH) were analyzed applying a dynamic mathematical marker model. The model analysis provided quantitated values for growth rate and treatment response in the marker producing cells. The analysis showed that LDH had to be above 2,000 U/l to be a trustworthy tumour marker. HCG producing cells tended to grow faster than AFP producing cells, and were 3-5-fold more sensitive to the chemotherapy given than AFP producing cells. Treatment response versus DDP dose appeared to be bi-phasic, but with no significant change in treatment efficiency within the given range of DDP doses.


Hepatic DNA methylation in young, middle-aged, and senescent rats: the effect of mitogen-induced cell proliferation. We have investigated the effect of a single dose of the hepatomitogen lead nitrate on the 5-methyldeoxycytidine (5-mdcyd) content and the HpaII, MspI, and HaeIII restriction patterns of hepatic DNA from young, middle-aged, and senescent rats. It was found that (i) the methylation pattern of genomic DNA changed significantly with age and (ii) the methylation patterns were differentially affected by the liver mitogen in the three cell populations here considered.


Tenascin expression in basal cell carcinoma. Tenascin (hexabrachion, cytotactin) is an extracellular matrix glycoprotein whose expression is strongly increased in hyperproliferative skin diseases, as shown by immunohistochemistry with polyclonal sera. In this study we describe a new monoclonal antibody (T2H5) against human tenascin. The specificity of T2H5 was validated by sequential immunoprecipitation of tenascin with polyclonal sera. T2H5 was used to analyse the presence of tenascin in basal cell carcinoma. Using Western blotting, at least two forms of tenascin were found, with approximate molecular weights of 210 and 300 kDa. In cultured human skin fibroblasts only the high molecular weight form was found.


Electron microscopy of alpha 2-macroglobulin with a thiol ester bound ligand. In order to covalently bind the hydrolyzed thiol ester groups of the human alpha 2-macroglobulin (alpha 2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M(r) = 13,000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed alpha 2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of alpha 2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in alpha 2M. They also suggest that the four thiol esters are located near the center of the alpha 2M molecule.


Substance P induces intracellular calcium increase and translocation of protein kinase C in epidermis. Substance P is a neuropeptide present in, and released from, peripheral C nerve endings. The presence of substance P-positive nerve fibres in the epidermis has been reported. We investigated the effect of substance P on the transmembrane signalling system of pig epidermal keratinocytes. Treatment of pig epidermis with substance P resulted in an increase in inositol 1,4,5-trisphosphate (IP3), and in intracellular free calcium. The treatment also resulted in translocation of protein kinase C from a cytosol to a membrane fraction. Substance P, however, did not affect the beta-adrenergic- or histamine (H2)- adenylate cyclase responses of the epidermis. Neither forskolin-induced, nor cholera toxin-induced cyclic AMP accumulation were affected by substance P treatment. These results consistent with the view that substance P stimulates phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis of keratinocytes, resulting in IP3-Ca2+ and diacylglycerol-protein kinase C signal activation. Although protein kinase C is known to affect the epidermal adenylate cyclase system, no evidence for such 'cross-talk regulation' was detected in keratinocytes by substance P treatment.


Recurrent annular erythema associated with anti-SS-B/La antibodies: analysis of the disease-specific epitope. We have found that anti-SS-B/La antibodies are present in a group of patients with a characteristic recurrent annular erythema, and immunological abnormalities. The presence of a disease-specific epitope for this entity has been examined by comparing immunological reactivity of anti-SS-B/La antibodies between these patients and patients with classical Sjögren's syndrome who have anti-SS-B/La antibodies but do not have this characteristic erythema. In immunoblotting using three different antigen sources, all the sera from both groups exhibited the same reaction patterns. V8 protease mapping showed that the sera could be divided into three groups by the difference in reactivity to two main digested peptide fragments. However, no significant relationship of the reactivity in peptide mapping between the two patient groups was observed. Further studies are necessary to elucidate the role of anti-SS-B/La antibodies in disease pathogenesis in these patients, in particular by more precise peptide mapping using recombinant SS-B/La antigen proteins.


Cytophagic histiocytic panniculitis: a report of four cases. Four patients presented with a clinical picture of spiking fever, erythematous nodular subcutaneous skin lesions, anaemia and leucopenia. Inconstant features were weight loss (3/4), splenomegaly (3/4), thrombocytopenia (3/4), raised liver enzymes (3/4), hepatomegaly (2/4) and serosal effusions (2/4). The histopathological findings of lobular histiocytic panniculitis with 'bean-bag' cells were characteristic of cytophagic histiocytic panniculitis (CHP). No infectious agent could be detected, and there was no histological evidence of malignant neoplasia. Unlike most previously described patients with CHP, three of our four patients responded to treatment with immunosuppressive or cytostatic drugs.


Association of proteins in acidic solutions--a case study with beta-globulin. The investigation of the effect of acid pH on the structure of beta-globulin indicated several transitions as a function of pH. Upon reducing the pH from 7.0, the beta-globulin molecule underwent an expansion due to hydration up to pH 5.0, and a further increase in H+ concentration resulted in unfolding. This is a single step cooperative denaturation as indicated by the viscosity profile. At extreme acid pH values (below pH 2.0) the protein associates or folds to a different conformational motif as shown by blue shift of ultraviolet fluorescence emission maximum and decrease in reduced viscosity values by more than 30% due to an entropically driven hydrophobic interaction. The conformational analysis of beta-globulin showed a decrease up to pH 3.0, followed by an increase in the ordered structure at low pH values indicating that the low pH values stabilized this new conformation. These results are discussed in view of the molten globule structure of proteins.


Effects of N-terminal extension peptides on the structure and stability of bovine pancreatic trypsin inhibitor studied by 1H n.m.r. Four N-terminal extended species of the wild-type bovine pancreatic trypsin inhibitor (WT-BPTI), Arg-BPTI (1-BPTI), Met-Glu-Ala-Glu-BPTI (4-BPTI), Ser-Ile-Glu-Gly-Arg-BPTI (5-BPTI) and Gly-Ser-Ile-Glu-Gly-Arg-BPTI (6-BPTI) have been studied by 1H n.m.r. The overall structure of the protein is largely unaffected by the addition of extension peptides. pH titration effects on the C-terminal Ala 58 H beta chemical shift indicate that the structure of 1-BPTI at neutral pH is very similar to that of the WT protein, with a salt bridge between the main chain terminal charges. A salt bridge interaction is prevented by addition of the longer extension peptides. Temperature stabilities are measured by high temperature hydrogen isotope exchange and by microcalorimetry. The stability of 1-BPTI is equal to that of WT-BPTI. A slight decrease in stability is observed for longer extensions, following the order WT-BPTI = 1-BPTI < 5-BPTI = 6-BPTI < 4-BPTI. Small changes in chemical shift are observed for 30 invariant resonances in 4-, 5- and 6-BPTI and for a subset of this group in 1-BPTI. These protons are distributed over about half of the BPTI molecule. The size of the chemical shift changes for many resonances follow the same ranking as the temperature stability. The chemical shift effects are attributed to charge and dielectric effects from extension peptides that probably share a common orientation on the surface of BPTI.


Interferon alpha therapy in patients with chronic type C hepatitis: changes of serum ALT, anti-HCV & HCV-RNA. After the discovery of type C hepatitis virus, the studies on this virus are extensively progressing. The treatment of this viral infection is also widely progressing. Among many agents, recombinant interferon alpha therapy is generally accepted as an effective single agent. To evaluate the efficacy of interferon and to observe the changes of serum aminotransferase (ALT), antibody to hepatitis C virus (anti-HCV) and HCV ribonucleic acid (HCV-RNA), we treated 10 patients with chronic type C hepatitis for 6 months. Patients were randomly divided into 2 groups: 5 patients in group A received interferon and the other 5 in group B received no therapy. Interferon was administered at a dose of 3 million units (MU) daily for the first month and thrice weekly for the following 5 months, and followed up for 2 years. In group A, serum ALT returned to normal in 4: 3, starting at the first month and one at the 3rd month of therapy and maintained normal throughout the follow-up period. In contrast, serum ALT level persistently fluctuated in 4 patients in group B. In one patient, serum ALT returned to normal one and a half years later. Regardless of therapy, serum anti-HCV titer remained unchanged in all patients. However, HCV-RNA, using polymerized chain reaction (PCR), became undetectable in all responded patients and in one untreated patient whose serum ALT returned to normal spontaneously. This study suggested that interferon alpha therapy in patients with chronic type C hepatitis may be clinically effective. Our study also indicated that the detection of HCV-RNA by PCR is useful to predict the prognosis of chronic type C hepatitis.


Prevalence of antibodies to hepatitis C virus in patients with various types of liver diseases. Hepatitis C virus (HCV) is known to be a major cause of non-A, non-B hepatitis (NANBH) and is thought to be an important causative agent of serious liver disease. Recently the role of HCV in the development of various liver disease is suggested. Sera from 222 patients with various liver diseases had been kept frozen at -20 degrees C until the test. Anti-HCV was detected using the ABBOTT HCV EIA Test System (ABBOTT Co., America) following the manufacturer's instructions. The assay uses a recombinant HCV antigen (C 100-3) synthesized in yeast. HCV antibodies (anti-HCV) were detected in 35 (31.5%) of 111 HBsAg-negative patients. The prevalence rate of anti-HCV was 61.9% (13 out of 21 patients) in chronic hepatitis, 29.1% (14 out of 48) in liver cirrhosis, 26.3% (5 out of 19) in hepatocellular carcinoma and 13% (3 out of 23) in acute hepatitis was far less (3 out of 111 patients, 2. 7%) than that of HBsAg-negative patients (p < 0.01). In this group, anti-HCV was detected in 2 (5.1%) out of 39 liver cirrhosis, 1 (1.9%) out of 52 chronic hepatitis, among them 47 were biopsy-proven chronic active hepatitis, and none of 20 hepatocellular carcinoma. These data suggest that, in Korea, 1) coinfection of HCV and HBV is infrequent, 2) HCV might be an important cause of HBsAg-negative chronic hepatitis, 3) HCV is seemed to be a less likely important factor associated with liver cirrhosis or hepatocellular carcinoma in HBsAg-negative patients, but further prospective study with a large population is necessary.


Meningothelial hamartoma of the scalp. A case report with immunohistochemical studies. An unusual and, apparently, hitherto undescribed congenital lesion of the scalp which proved to be heterotopic meningothelial tissue is reported, and its clinical, morphological, and theoretical implications are reviewed. A 14-month-old male infant exhibited a soft tissue lesion on the midline of the parietal scalp since birth. The lesion had grown in size since birth. Histological examination showed an admixture of mature adipose tissue, bands consisting of bundles and small nodules of dense collagen, both enclosed and bordered by rests and strands of meningothelial cells. A network of vessel-like channels lined by plump hyperchromatic cells with spindle-shaped nuclei and occasionally multinucleated giant cells was one of the prominent features. Immunohistochemically, these cells were positive for vimentin, but staining was negative for EMA and all other antibodies tested. A fibrocollagenous stalk via bony defect showed no arachnoid cell rests. The authors believe that the herein described lesion and the hamartoma of the scalp described by Suster and Rosai (1990) may represent varying morphological expressions of a pathogenetically related process. Precautions appropriate to the possibility of intracranial extension must be taken at the time of surgery.


Cerebrospinal fluid protein variations in common to Alzheimer's disease and schizophrenia. Analysis of silver stained two-dimensional (2D) gels of cerebrospinal fluid (CSF) from 27 patients with schizophrenia (SCZ) and 10 patients with Alzheimer's disease (AD) revealed an increase in the relative amount of a polypeptide of 18,000M(r) and isoelectric point of 6.5 when compared to the appropriate controls. This protein was identified by its electrophoretic characteristics and by immune analysis of Western blots as an isoform of alpha-2 haptoglobin, provisionally identified as alpha-2FS haptoglobin. Alzheimer's disease versus control CSF samples showed a 6.8-fold increase in the percent mean density value of this haptoglobin isoform (n = 10 AD vs 11 control; P > 0.025) while a 4.4-fold increase was observed in the schizophrenic patients (n = 17 SCZ vs 10 control; P > 0.001). Two additional polypeptides (proteins '127' and '128') of 40,000 M(r) and isoelectric points 5.7 and 5.9, respectively, described previously by this laboratory, were found in the CSF of 27% of schizophrenics, 23% of the Alzheimer's disease patients, and 4% of the controls in the current study. The presence of proteins 127 and 128, as well as the increased concentrations of alpha-2 haptoglobin in the CSF of Alzheimer's disease and schizophrenic patients, may be useful as diagnostic biological markers. They may also indicate a common pathophysiology between these diseases.


Comparison of electrophoretic distribution patterns of ribosomal RNA gene restriction fragments and of ribosomal subunit proteins of Lactococci, Streptococci, and Pediococci. Comparison of electrophoretic distribution patterns of ribosomal RNA gene restriction fragments and of ribosomal subunit proteins are equally effective procedures for detecting differences and similarities in the Lactococci, Streptococci and Pediococci examined. Electrophoretic distribution patterns of ribosomal subunit proteins may be a useful tool in taxonomic studies.