Ventricular extrasystoles with syncopal episodes, perodactyly, and Robin in sequence in three generations: a new inherited MCA syndrome?

We observed the combination of the Robin sequence with perodactyly (hypoplasia and/or agenesis of the distal phalanx of the toes) and cardiac arrhythmia (ventricular extrasystoles occurring as bigemini or multifocal tachycardia with syncopal episodes) in 6 relatives in 3 generations. This familial association has not been reported before and probably represents a previously unrecognized heritable malformation syndrome.


Protease inhibitors as a model for NCL disease, with special emphasis on the infantile and adult forms.

An animal model of NCL disease has been developed with the use of protease inhibitors. Young rats received a continuous infusion of various specific protease inhibitors or of physiological saline into the lateral ventricle of the brain using osmotic mini-pumps. Treatment lasted for 2 weeks, at which time animals were sacrificed and the brains were removed and processed for light or electron microscopic analysis. The thiol protease inhibitors leupeptin and E64C, but not saline or the serine protease inhibitor aprotinin, caused a massive accumulation of ceroid-lipofuscin (CL) in brain cells that bore a strong morphological resemblance to the CL in the infantile and adult forms of NCL disease, and bore similarity to the CL of the late infantile and juvenile forms. Leupeptin also caused the death of cerebellar Purkinje cells, as is seen in the infantile and adult forms of NCL. Further evidence is presented in support of the hypothesis (Ivy et al.: Science 226:985-987, 1984) that decreased or defective lysosomal thiol proteases or their substrates may underly the pathogenesis of at least the infantile and adult forms of NCL disease. Administration of protease inhibitors to the brains of young rats provides an important model for studying the cellular mechanisms of ceroid-lipofuscino-genesis.


Altered protein patterns in brains of children with neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative diseases characterized by massive intralysosomal accumulation of storage materials. We have studied the protein patterns in 5 NCL, 5 control, and one Alzheimer disease brains. When protein patterns in NCL and control brain gray matter homogenates were examined by SDS-PAGE, NCL brains showed an absence or greatly reduced amounts of the Mr 160-180 kDa component and reduced amounts of the Mr 29-36 kDa component. Concomitantly, an increase in several components with Mrs of 45-50 kDa was noted. The 180 kDa polypeptide appears to be a glycoprotein because it was bound to the lectins concanavalin A and Ulex europaeus. Recently, the abnormal processing of amyloid protein precursor (APP) and its potential role in NCL have been suggested. Possible defects in tissue proteases and protease inhibitors may be considered responsible for the presence of these amyloid beta protein precursor fragments. To examine this possibility we are using polyclonal antibodies to the C terminal 672-695 (APP) and monoclonal antibodies to inter-alpha-trypsin inhibitor. Polypeptides with molecular weights of approximately 35-38 kDa were detected in the NCL brain, but not in controls in both cases. These findings suggest abnormal protein processing in NCL brain tissue, disturbances in protein and glycoconjugate metabolism, impaired lysosomal function (i.e., metabolic enzyme and/or proteases/proteinase inhibitor abnormalities), and the involvement of improperly processed APP.


Regional variability of colonocyte growth and differentiation in the rat.

The location of stem cells and the direction of colonocyte migration in the normal rat colonic crypt were investigated using a serial bromodeoxyuridine (BrdU) and [3H]thymidine labeling protocol. The results demonstrate that in the distal colon the stem cells are located in the crypt base and that cells migrate up toward the luminal surface. In the proximal colon, however, the stem cells are located in the midcrypt, and the colonocytes migrate in two directions, up toward the luminal surface and down toward the crypt base.


Temporary cerebral ischemia. Effects of pentastarch or albumin on reperfusion injury.

Recent investigations have proposed that, after temporary ischemia, pentastarch may reduce microvascular permeability and reperfusion injury. However, this hypothesis has not been tested in the brain. Accordingly, after 180 min of temporary middle cerebral artery occlusion, the effect of pentastarch or albumin on blood-brain barrier permeability and cerebral injury was investigated in isoflurane-anesthetized rats. One of the following was maintained for the final 60 min of occlusion and throughout reperfusion: control-hematocrit was not manipulated; pentastarch-hematocrit was decreased to approximately 30% with pentastarch; or albumin-hematocrit was decreased (approximately 30%) with albumin. Part A (n = 21): 30 min of reperfusion was allowed, and blood-brain barrier permeability was determined with the indicator dye Evans Blue. Part B (n = 14): in different animals, 120 min of reperfusion was allowed, and cerebral injury (2,3,5-triphenyltetrazolium chloride stain) and edema (specific gravity) were assessed. Part C (n = 4): in different animals, the blood-brain barrier was evaluated by electron microscopy. Evans Blue (micrograms per gram brain tissue, mean +/- SD) was greater in the control (20.8 +/- 9.0) and albumin (15.5 +/- 7.3) groups versus the pentastarch (4.7 +/- 2.7) group (P less than 0.05). Brain injury (percent of hemisphere ipsilateral to occlusion) was less and specific gravity greater in the pentastarch (33 +/- 8 and 1.040 +/- 0.003 respectively) versus the albumin group (45 +/- 6 and 1.035 +/- 0.003). This study supports the hypothesis that during temporary cerebral ischemia, pentastarch decreases brain injury and edema.(ABSTRACT TRUNCATED AT 250 WORDS)


Allergy to cefazolin: study of in vivo cross reactivity with other betalactams.

We present two cases of hypersensitivity to cefazolin (one anaphylaxis). The skin prick tests with cefazolin at therapeutic concentration were positive. For the in vitro study we used conjugates of cefazolin with human serum albumin, obtaining a positive result in the histamine release test in both patients. In one case, we demonstrated anticefazolin-specific IgE antibodies by RAST. The controls did not react to any of these tests. Finally, we carried out skin tests and challenge tests with other betalactam antibiotics, including benzylpenicillin, amoxicillin, aztreonam, cefuroxime and cefotaxime, showing in both patients good tolerance to all these drugs.


Wilderness injuries and illnesses.

To determine injury and illness patterns and occurrence rates during wilderness recreation. Prospective injury and illness surveillance study. Wilderness areas throughout the Western hemisphere. All students and instructors on National Outdoor Leadership School courses over a five-year period. A single fatality occurred, resulting in a death rate of 0.28 per 100,000 person-days of exposure. Injuries occurred at a rate of 2.3 per 1,000 person-days of exposure. Sprains and strains and soft tissue injuries accounted for 80% of the injuries. The illness rate was 1.5 per 1,000 person-days of exposure. Sixty percent of illnesses were due to nonspecific viral illnesses or diarrhea; hygiene appeared to have a significant impact on the incidence of these illnesses. Thirty-nine percent of the injuries and illnesses required evacuation (1.5 per 1,000 person-days of exposure). The injury and illness patterns indicate that wilderness medical efforts should concentrate on wilderness hygiene and management of musculoskeletal injuries and soft tissue wounds. The data also indicate that wilderness activities can be conducted relatively safely, but the decision to participate should be individualized, with an understanding of risks versus benefits.


Expression of intermediate filament proteins in the adult human vestibular labyrinth.

The immunohistochemical detection of intermediate filament proteins, cytoskeletal constituents that allow the characterization of tissues, was investigated in frozen sections of the chemically fixed, nondecalcified, adult human vestibular labyrinth. Cytokeratins (CKs) were detected in all epithelia (including the sensory epithelia), although substantial differences in the degree of staining between individual cells occurred. The expression of CKs 7, 8, 18, and 19 as detected with our subunit-specific monoclonal antibodies in the vestibular epithelia is typical of "simple" epithelia and is identical to the CK subtypes found in the human cochlea. Although immunostaining for CK 7 was very weak and was limited to certain vestibular wall cells, the other CKs demonstrated a pronounced and rather uniform distribution throughout the different epithelia. All epithelia (including the sensory epithelia) displayed expression of vimentin, thus demonstrating co-expression with CKs. Vimentin was also present in the subepithelial connective tissue fibroblasts and mesothelial lining of the vestibular labyrinth. Neurofilament proteins were detected in all neuronal structures. The intense staining for CKs in the maculae and cristae implies that these sensory organs are rigid structures, a finding that may possibly be of importance in the mechanoelectrical transduction process for the sense of equilibrium.


Primary salivary gland amyloidosis causing sicca syndrome.

Sicca syndrome (SS), consisting of xerostomia and xerophthalmia, may be caused by various disease processes. We present a unique case of SS secondary to primary amyloidosis. Amyloidosis is a rare but definite cause of SS and should be included in the differential diagnosis of any patient who presents with sicca symptoms. A literature review comparing amyloidotic patients with SS and patients with amyloidosis only demonstrates that both of these groups of patients present similarly with regard to symptoms. However, the majority of patients with SS present with sicca symptoms initially in addition to symptoms of amyloidosis. These SS patients also present with proteinuria and negative serology test results. Therefore, patients presenting with sicca symptoms, proteinuria, and negative serologic findings should be suspect for amyloidosis. The importance of distinguishing the diagnosis of Sjögren's syndrome from SS in these patients cannot be overemphasized. There is a significantly higher incidence of developing a lymphoma in Sjögren's syndrome patients. This has important implications for the head and neck surgeon treating these patients.


Epithelial-myoepithelial carcinoma of salivary glands.

Epithelial-myoepithelial carcinomas comprise approximately 1% of all salivary gland neoplasms. They are preponderantly tumors of the parotid glands with a relatively low mortality but a decided locoregional aggressiveness. Histopathologically, the carcinomas are characterized by a dual cell population of epithelial (ductal) cells and myoepithelial cells. These cells vary in their dominance and phenotypic expression.


Glomerular and tubular proteinuria in type 1 (insulin-dependent) diabetic patients with and without retinopathy.

We compared the urinary excretion of albumin, transferrin, N-acetyl-beta-D-glucosaminidase and alpha-1-microglobulin in 78 Type 1 (insulin-dependent) diabetic patients: 39 with retinopathy and 39 without. The two groups were matched for age, sex and duration of diabetes. The patients with retinopathy had increased excretion (median and range) of albumin [1.7(0.3-399.1) versus 1.0(0.3-116.6) mg/mmol creatinine, P less than 0.05], transferrin [114.2 (4.1-37126.2) versus 33.4 (1.0-4176.7) micrograms/mmol creatinine, P less than 0.01] and N-acetyl-beta-D-glucosaminidase [23.8 (1.1-119.1) versus 15.0 (0.1-65.1) mumol/h/mmol creatinine, P less than 0.05] but not alpha-1-microglobulin. Transferrin excretion correlated with albumin excretion. The prevalence of increased transferrin excretion (transferrinuria) was greater than that of microalbuminuria in patients both with and without retinopathy (P less than 0.01 in both cases). Urinary transferrin seems likely to be predominantly of glomerular origin and merits prospective longitudinal evaluation as a potential index of the microangiopathic process.


Alleviation of non-islet cell tumour hypoglycaemia by growth hormone therapy is associated with changes in IGF binding protein-3.

The hypoglycaemia caused by non-islet cell tumours is often associated with an increase in plasma insulin-like activity. In many cases there is a relative if not always absolute increase in plasma insulin-like growth factor II (IGF-II). Growth hormone (GH) secretion is almost invariably depressed as is the plasma insulin response to oral glucose. Despite the high concentration of IGFs (i.e. IGF-I and IGF-II) normally found in the plasma of healthy people their potential hypoglycaemic effect is not manifest due to the tightness with which they are bound to specific binding proteins (IGFBPs). Plasma levels of the major binding protein (IGFBP-3), which is GH-dependent, were depressed in three patients with tumour induced hypoglycaemia. Treatment with biosynthetic GH restored IGFBP-3 to levels which were approximately equimolar to total plasma IGF concentrations, alleviated the hypoglycaemia and restored the plasma insulin responses to oral glucose. We suggest that after GH treatment IGF-II is sequestered by stimulated IGFBP-3 in association with a pre-existing acid-labile subunit to form high molecular weight complexes which prevent IGF-II gaining access to tissues receptors through which it exerts its hypoglycaemic effects.


Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms.

The population architecture of sulfidogenic biofilms established in anaerobic fixed-bed bioreactors was characterized by selective polymerase chain reaction amplification and fluorescence microscopy. A region of the 16S rRNA common to resident sulfate-reducing bacteria was selectively amplified by the polymerase chain reaction. Sequences of amplification products, with reference to a collection of 16S rRNA sequences representing most characterized sulfate-reducing bacteria, were used to design both general and specific hybridization probes. Fluorescent versions of these probes were used in combination with fluorescence microscopy to visualize specific sulfate-reducing bacterial populations within developing and established biofilms.


Cytotoxic effects of hexavalent chromium in cultured murine macrophages.

The in vitro effect of hexavalent chromium (CrVI) on mouse peritoneal macrophages was investigated. Our results demonstrate the existence of a threshold concentration of 2.5 microM of CrVI above which chromium affects the viability of macrophages in a concentration dependent manner. Morphological changes were seen with increasing concentrations of chromium. Evidence is presented that protein production (virus-induced interferon) in murine peritoneal macrophages was impaired by hexavalent chromium at low concentrations, while random migration and phagocytic activity were only affected at high concentrations of CrVI.


Effects of streptozotocin diabetes on amylase release and cAMP accumulation in rat parotid acinar cells.

Rat parotid responses to sympathetic nerve stimulation in vivo are impaired 2-4 weeks after the induction of streptozotocin diabetes. In this study, the effects of experimental diabetes of similar duration and severity on noradrenaline-stimulated amylase release and cAMP accumulation were examined in vitro. Amylase levels were significantly lower in acinar cells isolated from diabetic animals than in controls, and cellular amylase increased after treatment of the diabetic animals with either thyroxine (T4) or insulin. Diabetes and T4 had no apparent affect on amylase release measured as a percentage of the total. In contrast, giving insulin resulted in a significant reduction in maximal secretion (20.4 +/- 2.4% compared with 43.6 +/- 7.6%). Similar results were observed when amylase release was stimulated with forskolin. Basal cAMP levels were unaffected by diabetes or T4 (7.8 +/- 2.3 pmol/mg protein), but stimulated cAMP levels were significantly greater in diabetic acinar cells than in controls. Insulin reversed the effects of diabetes on cAMP accumulation, whereas T4 had no effect. Thus, diabetes (2-4 weeks) and insulin in vivo appear to have paradoxical effects on parotid amylase release and cAMP accumulation in vitro. Further, the effects of diabetes appear to be unrelated to thyroid status.


Amylase mRNA synthesis and ageing in rat parotid glands following isoproterenol-stimulated secretion.

In the parotid, as well as in other exocrine glands, secretory protein synthesis declines with age. However, whether this decline in the steady-state rate of protein synthesis reflects the reduced digestive activity of the animal or actual cellular alterations that affect synthesis is unknown. Here the ability to synthesize amylase and its mRNA during the period of enhanced protein synthesis following secretion induced by isoproterenol was compared in acinar cells of 2-and 24-month-old rats. In unstimulated glands, rates of synthesis of total protein and amylase, as well as amounts of amylase mRNA, were significantly less in the older rats than in their younger counterparts. After stimulation with isoproterenol, which induced the secretion of about 50% of stored proteins, rates of synthesis of total protein, as well as amylase, were increased by about 2.5 x the unstimulated rates in both age groups. However, the amount of amylase mRNA did not increase in parallel with the increase in the rate of amylase protein synthesis in both young and old rats. The molecular size of the mRNA was the same in stimulated and unstimulated glands of both age groups. Thus, it appears that parotid acinar cells from old rats can be stimulated to synthesize secretory proteins at an increased rate. It remains to be determined what causes the reduced rate of protein synthesis in unstimulated glands in old rats.


Histological evaluation of the effect of transseptal fibre resection on the rate of physiological migration of rat molar teeth.

Twenty-seven female Sprague-Dawley rats were given lead acetate as a vital stain, and rates of alveolar bone formation, representative of drift, were measured histologically. Teeth around which the transseptal fibre system had been destroyed drifted less quickly on both functioning (p less than 0.01) and non-functioning (p less than 0.001) sides than those with intact transseptal fibres. Both horizontal and vertical components of physiological drift were equally affected by destruction of the transseptal fibre system. It was felt that the transseptal fibres probably exerted their primary effect on the tooth, with bone remodelling around the drifting tooth being affected secondarily.


bFGF enhances the development of the collateral circulation after acute arterial occlusion.

An adequate collateral circulation is crucial to tissue survival subsequent to proximal major arterial occlusion. The precise mechanism of collateral blood vessel development and the biochemical mediators involved in this process are unknown. To evaluate the influence of a number of agents on the development of the collateral circulation, we developed a rat model of severe hind limb ischaemia. The recovery of blood flow after acute arterial occlusion was increased by exogenous basic fibroblast growth factor and heparin, and decreased by protamine. Erucamide (cis-13-docosenamide), an angiogenic lipid, had no effect on collateral blood flow. These results indicate that basic fibroblast growth factor and heparin are potential therapeutic agents in the treatment of peripheral vascular disease.


Ca(2+)-dependent and Ca(2+)-independent mechanisms modulate whole-cell cationic currents in human neutrophils.

We used whole-cell, voltage-clamp methodology to study the activation and inhibition of cationic currents in neutrophil. Cationic channels involved were impermeable to N-methyl-D-glucamine and to choline, but permeable to Na+, K+, Cs+, tris(hydroxymethyl)amino-ethane, and tetraethylammonium. N-formyl-L-methionyl-L-leucyl-L-phenylalanine, the Ca(2+)-ionophore A23187, and phorbol myristate acetate activated the cationic current. Activated currents showed voltage dependence and outward rectification. The Ca(2+)-chelator 1,2 bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate markedly inhibited A23187-induced currents, but only partially decreased phorbol ester- or chemoattractant-induced currents. Dibutyryl cAMP diminished only the chemoattractant-induced currents. The adenosine analogs 5'N-ethylcarboxamidoadenosine and N6-cyclohexyladenosine blocked the currents induced by all agents. Thus, we conclude that activation and inhibition of cationic channels in human neutrophils involve both Ca(2+)-dependent and Ca(2+)-independent mechanisms.


Characterization of a rat variant alpha-fetoprotein.

The rat adult liver and hepatocyte lines express an 1.7-kb variant alpha-fetoprotein (AFP) mRNA which differs from the 2.2-kb fetal AFP transcript in sequence in the 5' region. Here we report the characterization of a variant AFP cDNA, ARFP9, which is 1349 bp in length and encodes a 325 amino acid polypeptide. Nucleotides 225 to the 3' end (1125 bp) in ARFP9 corresponds exactly to nucleotide 873 to the 3' end of the fetal AFP mRNA. However, the first 224-bp of ARFP9, which is located in the 7th intron (designated the V exon) of the rat AFP gene, is not present in the 2.2-kb fetal AFP transcript. The size of the V exon is about 266-bp. In vitro expression experiments showed that the variant AFP is an unglycosylated intracellular protein of 37 kDa. Methyl-isobutyl-xanthine (MIX) stimulated expression of the fetal AFP mRNA but inhibited expression of the variant AFP mRNA, suggesting that in the rat, the two AFP transcripts are developmentally and differentially regulated.


Kinins mediate kallikrein-induced endothelium-dependent relaxations in isolated canine coronary arteries.

ACE inhibitors elicit the release of endothelium-derived relaxing factors in perfused isolated canine arteries (Mombouli and Vanhoutte, J. Cardiovasc. Pharmacol. 1991, 18: 926-927); this action is antagonized by bradykinin-receptor antagonists suggesting that it is mediated by local kinin generation. The effects of exogenous tissular kallikrein (porcine) were examined in vitro in the isolated canine coronary artery. Isometric tension was measured in blood vessel rings (with and without endothelium) contracted with prostaglandin F2 alpha. The kallikrein elicited relaxations in rings with, but not in those without, endothelium. This response was augmented by the angiotensin converting enzyme inhibitor perindoprilat, and it was antagonized by the selective B2-kinin receptor antagonist HOE 140 and aprotinin, an inhibitor of tissular kallikrein. These data suggest that in the canine coronary artery, kallikrein causes relaxations that may be mediated by kinins generated from endogenous kininogens present in the vascular wall.


Alternative splicing products of the tenascin gene distinguish rat liver fat storing cells from arterial smooth muscle cells and skin fibroblasts.

Fat storing-(Ito-)cells (FSC) transform into a myofibroblast-like cell type during liver fibrogenesis. A similar development can be observed in cell culture. At the moment, a definite marker to differentiate transformed FSC from smooth muscle cells (SMC) is not available. We recently found that FSC, SMC and skin fibroblasts (SF) synthesize tenascin, a novel matrix protein. As it is reported that various tissues express different tenascin forms by the mechanism of alternative pre-mRNA splicing, we analyzed the tenascin transcripts in these cell types. Total RNA extracted from cultured FSC, SMC and SF, analyzed by Northern blot hybridization, showed a 7.2 kb transcript in FSC, a 8.7 kb mRNA in SMC, whereas SF produced both messages. As the splicing pattern of FSC in primary culture did not change after passaging, this differential expression of tenascin mRNA might provide a tool to identify myofibroblast-like cells derived from FSC. The important fibrogenic mediator transforming growth factor-beta (TGF-beta) increased tenascin gene expression in each cell type. In SMC, TGF-beta additionally induced the production of the 7.2 kb transcript. Determination of tenascin transcripts will allow to examine the purity of FSC cultures and facilitate a better identification of the cells involved in liver fibrosis.


A calcium-dependent cation channel in mast cell granule membranes reconstituted into a lipid bilayer.

Secretory granules isolated from rat peritoneal mast cells were reconstituted into a lipid planar bilayer and membrane current was measured. A novel calcium- and voltage-dependent cation channel with single-channel conductances of 80 pS for fully open and 60 pS for sub-conducting states was detected. This channel opened only when potential of the cis compartment, which corresponds to the cytoplasmic side in the cell, was positive with respect to the trans compartment and only when Ca concentration of the cis compartment was higher than 10(-7) M. The open channel probability further increased, depending on the concentration of Ca. The physiological role of this channel remains to be determined.


Human autoantibody binding to multiple conformations of DNA.

Systemic lupus erythematosus and rheumatoid arthritis in humans are characterized by circulating and tissue fixed autoantibodies reactive with self antigens including nucleic acids and other nuclear components. Native calf thymus DNA (B-form), DNA.RNA hybrid (A-form), and left handed DNA (Z-form) were reactive with autoantibodies derived from SLE sera. Inhibition studies suggest that antibodies are recognizing multiple conformations presented by altogether different polymers and A- or Z-DNA might be the immunogenic stimulus for the production of antibodies cross reactive with native DNA.


Induction of propranolol metabolism by the azo dye sudan III in rats.

Effects of the azo dye sudan III, an inducer of cytochrome P450 isozymes belonging to the CYP1A subfamily, on propranolol (PL) in vitro and in vivo metabolism were investigated in rats. The kinetic parameters of the activity for each metabolic pathway were determined in liver microsomes from control and sudan III-treated rats. Sudan III pretreatment increased extensively PL 4-hydroxylase, 5-hydroxylase and N-desisopropylase activities at high but not at low PL concentrations. On the other hand, kinetic parameters of 7-hydroxylase activity were not affected by sudan III pretreatment. Sudan III pretreatment decreased blood concentrations of PL after intraportal infusion of PL at high doses (12.5 and 20 mg/kg), but not at a low dose (5 mg/kg). These observations were consistent with data obtained from the in intro studies showing that sudan III pretreatment induced low-affinity but not high-affinity cytochrome P450 isozymes involved in PL metabolism in rat liver microsomes.


Clinical value of the assessment of gynaecological tumour angiogenesis by transvaginal colour Doppler.

Angiogenesis occurs in the body in only a few physiological conditions, but it regularly precedes carcinogenesis. Neovascularisation is a term well known to the pathologist, but with the advent of colour Doppler it is now of interest to those using ultrasonography. Since morphological criteria alone are insufficient to characterise space occupying lesions, visualisation of newly formed vessels and consequently, their blood flow characteristics, seems to provide a clearer distinction in vivo between benign and malignant gynaecological tumours. Very low resistance indices in newly formed vessels, as a predictor of malignancy, have already been established. The most recent area of investigation covering the differences between centrally and peripherally placed vessels offers additional criteria for diagnosing gynecological malignancy.


Northern and Southern blot analysis of human RNA and DNA in autopsy material.

Fresh biopsy material for molecular biological investigations is not obtainable from all relevant normal human tissues. We studied the feasibility of using RNA and DNA from autopsies for Northern and Southern blot analysis. Tissue samples from seven organs were obtained from 10 autopsies performed 21-118 h postmortem. Extracted RNA and DNA were examined by Northern and Southern blot analysis using oligo-labelled human DNA probes recognizing gene transcripts of 2-5 kb. The results indicated that, in general, Northern blot analysis was feasible with the applied probes when the tissue was obtained less than two days postmortem. Histological examination showing slight or no autolysis and the presence of ribosomal bands after gel electrophoresis were both indicative parameters of RNA preservation. DNA was appropriate for Southern blotting when the tissue was obtained less than three to five days postmortem, depending on the organ from which the DNA was extracted.


Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex.

Nitric oxide (NO) is an important messenger both systemically and in the CNS. In digital Ca2+ imaging and patch-clamp experiments, clinically available nitroso compounds that generate NO are shown to inhibit responses mediated by the NMDA subtype of the glutamate receptor on rat cortical neurons in vitro. A mechanism of action for this effect was investigated by using the specific NO-generating agent S-nitrosocysteine. We propose that free sulfhydryl groups on the NMDA receptor-channel complex react to form one or more S-nitrosothiols in the presence of NO. If vicinal thiol groups react in this manner, they can form a disulfide bond(s), which is thought to constitute the redox modulatory site of the receptor, resulting in a relatively persistent blockade of NMDA responses. These reactions with NO can afford protection from NMDA receptor-mediated neurotoxicity. Our results demonstrate a new pathway for NO regulation of physiological function that is not via cGMP, but instead involves reactions with membrane-bound thiol groups on the NMDA receptor-channel complex.


Differential wound activation of members of the phenylalanine ammonia-lyase and 4-coumarate:CoA ligase gene families in various organs of parsley plants.

We analyzed the developmental regulation and the activation by wounding of several stress-related genes in various parsley organs. The genes encode phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL), two enzymes of general phenylpropanoid metabolism; a flavonoid specific enzyme, chalcone synthase (CHS); a furanocoumarin specific enzyme, bergaptol O-methyltransferase (BMT); and a pathogenesis-related protein (PR 1). All genes or gene families exhibited high levels of expression in roots and during certain stages of leaf development. PAL, 4CL and CHS were preferentially expressed in young leaves, BMT and PR 1 in old leaves. An appreciable increase in CHS mRNA levels was observed in wounded leaves. By contrast, root wounding led to a decrease in the existing CHS mRNA levels. A biphasic response (a decrease followed by an increase) to wounding was seen for BMT and PR 1 mRNAs in roots and for BMT mRNA in attached leaves. Using gene-specific oligonucleotide probes to measure the expression rates of three of the four PAL genes and of the two 4CL genes separately we observed a differential behavior of the individual family members under many of the conditions tested. While PAL-3 was preferentially activated in wounded leaves and 4CL-1 in wounded roots, PAL-2 and 4CL-2 were primarily responsible for the high constitutive expression levels in roots and flowering stems respectively. Despite the differential expression of their individual members, the PAL and 4CL gene families displayed very similar changes in the overall patterns of expression, reflecting their closely related functions in phenylpropanoid metabolism.


Lymphoblastoid cell adhesion mediated by a dimeric and polymeric endogenous beta-galactoside-binding lectin (galaptin).

Glutaraldehyde-polymerized human splenic galaptin, a beta-galactoside-binding lectin, was demonstrated to have enhanced hemagglutinating and asialofetuin binding activity relative to native dimeric galaptin when these lectins were present in solution. The polymerized lectin consisted primarily of 2-, 4- and 12-membered species after reductive alkylation. Both forms of galaptin bound, at 4 degrees C, to saturable B lymphoblastoid cell surface receptors. Estimates obtained by Scatchard analyses, with the binding data expressed in terms of 14.5 kDa subunit molarity, were 5 x 10(7) binding sites/cell with affinity constant Ka = 2.2 x 10(5) M for dimeric galaptin and 17 x 10(7) binding sites/cell with Ka = 3.4 x 10(5) M-1 for polymeric galaptin. Both forms of galaptin adsorbed to polystyrene with high efficiency; however, only plastic-adsorbed polymeric galaptin mediated adhesion of lymphoblastoid cells. Cell adhesion was inhibited by lactose. Plastic-adsorbed polymeric galaptin bound asialofetuin more efficiently than dimeric galaptin. Asialofetuin binding was inhibited 65% and 30-50% by lactose for plastic-adsorbed polymeric and dimeric galaptin, respectively. Native fetuin bound to the adsorbed dimeric galaptin in a lactose-insensitive manner. These data indicate that cell surface receptor-galaptin interaction is carbohydrate specific whereas polystyrene-adsorbed galaptin may demonstrate protein-protein interactions with soluble ligands.


TP53 tumor suppressor gene: a model for investigating human mutagenesis.

More than 350 independent point mutations of the TP53 gene, found in a wide variety of human cancers, were compiled and analysed. From this study, we confirm the presence of four hot-spot regions which colocalize with some highly conserved domains of the protein. We also define a new hot-spot region which is observed predominantly in lung tumors. Analysis of the mutational events suggests the direct involvement of environmental carcinogens in lung tumors and hepatocarcinomas, and spontaneous mutagenesis generating essentially CpG transitions in most of the remaining ones. Furthermore, we demonstrate in this work that the TP53 gene is an informative model with which to study the molecular mechanisms of mutagenesis in the human genome.


Cytogenetic analysis of renal angiomyolipoma.

Chromosome analysis of a benign, unilateral, renal angiomyolipoma revealed the karyotype 44,XX,-8, -12, -14, -21, +der (8q14q), + der(12)(12pter----12q14-15::12q24----12q14-15::+ ++21q21----21qter). This indicates that structural changes of 12q13-15, which are so frequently observed in the common malignant and benign lipogenic tumors, may also occur in the rarer variants of these neoplasms.


Cytogenetic analysis of 57 primary prostatic adenocarcinomas.

Cytogenetic analysis after short-term culture in vitro of primary tumor samples was attempted in 82 patients with prostatic cancer. Tumor material was obtained by radical prostatectomy or transurethral resection. Successful cytogenetic studies were performed on 57 tumors of which five were well, 30 moderately, and 22 poorly differentiated adenocarcinomas. Only normal karyotypes were found in 24 tumors. Structural nonclonal aberrations were detected in 18 and clonal karyotypic abnormalities in 15 tumors. The most common clonal numerical aberration was loss of the Y chromosome; a missing Y was found in six tumors, in three of these as the sole anomaly. Clonal structural chromosomal rearrangements, usually accompanied by numerical changes, were detected in 12 tumors. The rearrangements involved 18 of the 22 autosomes and the X chromosome. Chromosomes 1, 7, and 10 were most frequently affected. Deletions, duplications, inversions, insertions, and balanced as well as unbalanced translocations were represented. The breakpoints in chromosome 1 were scattered along both the short and long arms with no obvious clustering, whereas those in chromosomes 7 and 10 were clustered at bands 7q22 (two deletions and two duplications in four different tumors) and 10q24 (two translocations, one deletion, and one inversion in four tumors). One additional tumor displayed a derivative chromosome 10 with a breakpoint in 10q23, and one had monosomy 10. Altogether, these abnormalities resulted in loss of 10q24----qter in five tumors. Monosomy 8 and rearrangements of the short arm of chromosome 8 leading to loss of 8p21----pter were seen in four tumors. Double minute chromosomes were found in two tumors.


Cytogenetic abnormalities in renal oncocytic neoplasms.

We have performed cytogenetic studies on five renal oncocytic neoplasms (three grade 2 tumors and two grade 1 tumors) identified histologically by light microscopy. One grade 1 tumor failed to produce mitotic cells. The other four tumors exhibited both normal and abnormal cell lines. Numerical abnormalities were found in both the single grade 1 and two of the grade 2 tumors whereas structural abnormalities were limited to grade 2 tumors. Aneuploidy of chromosome 12 was observed in both grade 1 and 2 tumors. Grade 2 tumors showed more extensive numerical change than the grade 1 tumors. Abnormalities of chromosome 3 characteristic of renal cell carcinoma were not found in any tumor in this series. A combination of C-banding and HaeIII endonuclease banding was used to identify an ambiguous marker. In our four cases and in the cases previously reported, loss of a sex chromosome, abnormalities of chromosomes 1 and 22, and trisomy 12 are findings most often observed in renal oncocytoma.


A complex genetic rearrangement in a t(10;14)(q24;q11) associated with T-cell acute lymphoblastic leukemia.

The t(10;14)(q24;q11) is observed in the leukemia cells of 5-10% of cases of T-cell acute lymphoblastic leukemia (T-ALL). Recently, molecular analyses of a number of these translocations revealed simple reciprocal translocations between the T-cell receptor delta chain gene (TCRD) and a region of 10q24. We have characterized, at the molecular level, a t(10;14)(q24;q11) in a patient with T-ALL. The translocation in this case, in contrast to the previous cases, is part of a complex genetic rearrangement. In addition to a reciprocal translocation between the D delta 3 gene segment of TCRD and a region of 10q24, a local inversion occurred within TCRD, involving the D delta 2 and V delta 2 gene segments. As a consequence, the entire joining and constant regions and most of the diversity regions of TCRD are located on the derivative 14 chromosome, whereas the joining and constant regions of TCRA are positioned on the derivative 10 chromosome. The chromosome 10 breakpoint in our patient, as in other t(10;14), clusters within a 9 kb breakpoint region. The occurrence of seven breakpoints within a localized region of chromosome 10 implies the existence of a nearby gene whose activation may have conferred a selective advantage on the leukemia cells. Moreover, as in the previous cases, the translocation in the present study exhibits recombination signal sequences or signal-like sequences adjacent to the breakpoint junction. The presence of such motifs suggests the involvement of the recombinase enzyme system in the generation of this genetic alteration.


Karyotype and T-cell receptor expression in T-lineage acute lymphoblastic leukemia.

The relationship between karyotype and expression of the T-cell receptor (TCR) proteins was examined in 19 patients with T-lineage acute lymphoblastic leukemia (T-ALL). All patients expressed CD3 molecules in the cytoplasm or on the cell membrane. Patients were classified according to TCR expression thus: no TCR expression (TCR-), six cases; cytoplasmic expression of TCR beta chain (cTCRB) only, six cases; membrane expression of TCR alpha and beta chains (mTCRAB), five cases; membrane expression of TCR gamma and delta (mTCRGD), two cases. A chromosomally abnormal clone was detected in 15 cases. The most common site of chromosomal change was at 14q11 (seven cases), the chromosomal band to which TCRA and TCRD have been mapped; as a deletion (two cases); or as a translocation with reciprocal breakpoints in bands containing the TCRG (7p15); TCRB (7q35); or putative oncogenes HOXII (10q24), RBTN2 (11p13) or MYC (8q24) genes. Breakpoints were also seen in 6q (three cases), 9p (two cases), or 11q23 (two cases). The following observations were made: All four chromosomally normal cases lacked TCR expression (TCR-). Breakpoints at 14q11 were found in one of six TCR- cases, four of six cTCRB cases, and two of five mTCRAB cases. Abnormalities of 6q and of 9p were seen only in cases with full TCR expression (mTCRAB or mTCRGD).


Karyotype instability and altered differentiation of rat sarcoma cells after retroviral infection.

The karyotypic and phenotypic stability of cultured rat fibrosarcoma cells was challenged by infection with Moloney murine sarcoma virus (MoMuSV). After transformation, the spindle-like morphology of the parental HH-16 cl.2/1 cells had altered to a rounded phenotype, which was maintained in tumors produced by inoculating transformed cells into congenic animals. In contrast to the parental cells, transformed cells lacked cables of cytokeratins 14-16 and 19 and showed reduction of the mesenchymal marker protein vimentin. Additionally, the morphologically altered cell clones tf-1 to tf-3 had lost growth arrest in the presence of dexamethasone. The DNA of the transformed cells contained between four and six randomly integrated proviral copies. Karyotypic alterations were manifested by reduction of morphologically intact chromosomes in the MoMuSV-transformed cells together with increase of structural aberrations. Three additional markers were identified in the virus-transformed cell clones. Karyotypic instability induced by MoMuSV infection appeared closely related to reduction of the cellular differentiation status, although only cells of clone tf-1 had increased metastatic potential.


Chromosome aberrations in 35 primary ovarian carcinomas.

Cytogenetic analysis was performed on short-term cultures of primary ovarian carcinomas from 62 patients. Cytogenetic analysis was successful in 59 cases. Clonal chromosome aberrations were detected in 35 tumors. Only numerical changes or a single structural change were found in five carcinomas: trisomy 12 was the sole anomaly in two tumors, one tumor had the karyotype 50,XX, + 5, + 7, + 12, + 14, a fourth tumor had a balanced t(1;5), and the fifth tumor had an unbalanced t(8;15). The fact that four of these five carcinomas were well differentiated suggests that simple karyotypic changes are generally characteristic of these less aggressive ovarian tumors. The majority of the cytogenetically abnormal tumors (n = 30) had complex karyotypes, with both numerical and structural aberrations and often hypodiploid or near-triploid stemlines. The numerical imbalances (comparison with the nearest euploid number) were mostly losses, in order of decreasing frequency -17, -22, -13, -8, -X, and -14. The structural aberrations were mostly deletions and unbalanced translocations. Recurrent loss of genetic material affected chromosome arms 1p, 3p, 6q, and 11p. The breakpoints of the clonal structural abnormalities clustered to several chromosome bands and segments: 19p13, 11p13-15, 1q21-23, 1p36, 19q13, 3p12-13, and 6q21-23. The most consistent change (16 tumors) was a 19p + marker, and in 12 of the tumors the 19p + markers looked alike.


Specific metaphase and interphase detection of the breakpoint region in 8q24 of Burkitt lymphoma cells by triple-color fluorescence in situ hybridization.

Triple fluorescence in situ hybridization with a plasmid DNA library from sorted human chromosomes 8 in combination with bacteriophage clones flanking the breakpoint in 8q24 of the Burkitt lymphoma cell line J1 was used for the specific delineation of this breakpoint in individual tumor cells. With this approach, tumor-specific breakpoints in translocation chromosomes can be detected at all stages of the cell cycle with high specificity.


Clonal aberrations of chromosomes X, Y, 7 and 10 in normal kidney tissue of patients with renal cell tumors.

By means of G-banding techniques, chromosome aberrations were studied in short-term cultures of normal renal parenchymal cells from 45 patients with renal cell carcinoma. Clonal chromosomal aberrations were detected in 29 patients; loss of the Y chromosome as well as trisomy X, 5, 7, 9, 10, 12, and 18 was found. Chromosomes 7 and 10 were involved preferentially. Results of fluorescence in situ hybridization with chromosome 7- and 10-specific DNA probes on non-cultured normal kidney cells suggested that the aberrations developed in vivo.


Trisomy 2 as the sole chromosomal abnormality in a hepatoblastoma.

Short-term cultures of a fine-needle aspirate from a hepatoblastoma were analyzed cytogenetically. Trisomy 2 was found as the sole abnormality, yielding the karyotype 47,XY, + 2/46,XY. Because trisomy for all or part of chromosome 2 has been described, although together with other aberrations, in seven of the 11 hepatoblastomas hitherto reported, the finding of + 2 as the only anomaly in the present case strongly indicates that additional chromosome 2 material is of pathogenetic significance in this tumor type.


Interstitial loss of the same region of 5q in multiple adenomas and a carcinoma derived from an adenomatous polyposis coli (APC) patient.

Accumulation of genetic alterations in oncogenes and tumor suppressor genes causes the transformation of a normal cell into a malignant cell. Recently, Fearon and Vogelstein (Cell 61:759-767, 1990) reported on a model for the genetic pathway in development of colorectal neoplasia. To investigate genetic alterations in colorectal carcinomas, we examined allelic losses on some chromosomes in adenomas and carcinomas derived from patients with adenomatous polyposis coli (APC). We found evidence for an interstitial deletion of 5q. Loss of heterozygosity (LOH) of 5q around the APC locus was observed in both adenoma and carcinoma in one case. The fact that the same region of chromosome 5 was lost in five adenomas and one carcinoma derived from the same patient suggests that a somatic interstitial deletion may be caused not by random mechanisms but by a specific mechanism.


The NM23 gene maps to human chromosome band 17q22 and shows a restriction fragment length polymorphism with BglII.

The NM23-Hl gene is a putative tumor suppressor gene that may be important in the metastasic process. Recent genetic and immunological data indicate that the NM23-Hl gene encodes a protein with nucleoside diphosphate (NDP) kinase activity. The mapping of NM23-Hl by panels of rodent-human somatic cell hybrids and in situ hybridization showed that the gene is located in human chromosome band 17q22. A two-allele polymorphism with BglII was demonstrated.


Ring chromosomes in a retroperitoneal lipoma of childhood.

We report here the presence of ring chromosomes in a retroperitoneal lipoma from a 12-year-old male. Ring chromosomes are strongly associated with the pathological subtype "atypical lipoma" in adults. In contrast, however, the tumour described here possesses no atypical features.


Ph-positive chronic myeloid leukemia with loss of the segment distal to M-bcr.

We describe a patient with chronic myeloid leukemia (CML) and a 22q- but no 9q+ chromosome. Southern blot analysis showed a BCR rearrangement. The patient soon developed profound and superficial thrombophlebitis in arms and legs and died from pulmonary embolisation 15 days after diagnosis. Five CML cases with a deletion of 22q but no known translocation of 22q11----qter have been described earlier. The present patient is the first such case, however, in whom a BCR rearrangement has been demonstrated.


Localization of the fibroblast growth factor receptor-4 gene to chromosome region 5q33-qter.

Our polymerase chain reaction cloning of novel tyrosine kinases expressed in the K562 chronic myeloid leukemia cells has revealed a novel fibroblast growth factor receptor, FGFR4. We have here mapped the FGFR4 gene by analysis of somatic cell hybrids and in situ hybridization to the 5q33-qter chromosomal region. This finding is of interest in that the FGFR4 gene is expressed in several leukemia cell lines and the 5q33-qter region is involved in nonrandom chromosomal translocations in acute myelogenous leukemias and Ki-I lymphomas.


Chromosome aberrations in an alpha-fetoprotein-producing hepatoblastoma.

The cytogenetic findings of an alpha-fetoprotein (AFP)-producing hepatoblastoma in a 14-month-old girl are reported. Ten of 36 tumor cells were chromosomally abnormal, yielding the composite karyotype 47,XX,2q +, t(3;5)(p25;q31),dup(4)(q12q26), +20. Three of the chromosomal abnormalities (2q+, break at 4q12, +20) have been reported previously and might be of pathogenetic significance.


Recognition of G-U mismatches by tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III).

The coordination complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III) [Rh(DIP)3(3+)], which promotes RNA cleavage upon photoactivation, has been shown to target specifically guanine-uracil (G-U) mismatches in double-helical regions of folded RNAs. Photoactivated cleavage by Rh(DIP)3(3+) has been examined on a series of RNAs that contain G-U mismatches, yeast tRNA(Phe) and yeast tRNA(Asp), as well as on 5S rRNAs from Xenopus oocytes and Escherichia coli. In addition, a "microhelix" was synthesized, which consists of seven base pairs of the acceptor stem of yeast tRNA(Phe) connected by a six-nucleotide loop and contains a mismatch involving residues G4 and U69. A U4.G69 variant of this sequence was also constructed, and cleavage by Rh(DIP)3(3+) was examined. In each of these cases, specific cleavage is observed at the residue which lies to the 3'-side of the wobble-paired U; some cleavage by the rhodium complex is also evident in several structured RNA loops. The remarkable site selectivity for G-U mismatches within double-helical regions is attributed to shape-selective binding by the rhodium complex. This binding furthermore depends upon the orientation of the G-U mismatch, which produces different stacking interactions between the G-U base pair with the Watson-Crick base pair following it on the 5'-side of U compared to the Watson-Crick pair preceding it on the 3'-side of U. Rh(DIP)3(3+) therefore serves as a unique probe of G-U mismatches and may be useful both as a model and in probing RNA-protein interactions as well as in identifying G-U mismatches within double-helical regions of folded RNAs.


On the mechanism of a mammalian neuronal type nicotinic acetylcholine receptor investigated by a rapid chemical kinetic technique. Detection and characterization of a short-lived, previously unobserved, main receptor form in PC12 cells.

The mammalian nicotinic acetylcholine receptor in PC12 cells has many properties characteristic of the neuronal receptors involved in key chemical reactions that are responsible for signal transmission between cells of the nervous system. This report describes initial investigations of the mechanism of this receptor using a rapid chemical kinetic technique with a time resolution of 20 ms, which represents a 250-fold improvement over the best time resolution (5 s) employed in previous studies. Carbamoylcholine, a stable analogue of the neurotransmitter acetylcholine, was the activating ligand used, and the concentration of open transmembrane receptor-channels in PC12 cells was measured by recording whole-cell currents at pH 7.4, 21-23 degrees C, and a transmembrane voltage of -60 mV. Two receptor forms that account for 80% and 20% of the receptor-controlled current were detected; the main receptor form, accounting for 80% of the whole-cell current, desensitized completely before the first measurements had been made in previous studies. Only the main receptor form has been investigated so far using the new method. The constants of a mechanism that accounts for the concentration of the open transmembrane receptor-channel over a 100-fold range of carbamoylcholine concentration were evaluated: the dissociation constant of the site controlling channel opening (K1 = 2.0 mM), the channel-opening equilibrium constant (phi -1 = 5.0), and the dissociation constant of an inhibitory site to which carbamoylcholine binds (KR = 6.5 mM). These evaluated constants allow one to calculate Po, the conditional probability that at a given concentration of carbamoylcholine the receptor-channel is open. Po was also determined in the presence of 2 mM carbamoylcholine by an independent method, the single-channel current-recording technique, and the agreement between the Po values obtained in two independent ways is within experimental error. This result indicates that the time resolution of the chemical kinetic technique employed was sufficient to evaluate the constants pertaining to the active state of the receptor, which forms a transmembrane channel, before its conversion to desensitized receptor forms with different properties. Previous kinetic measurements with a time resolution of 5 s showed that many compounds, such as anesthetic-like molecules, nerve growth factor, and substance P, modify the function of the neuronal receptor in PC12 cells or react specifically with the neuronal but not with the muscle receptor, for example, some toxins.(ABSTRACT TRUNCATED AT 400 WORDS)


On the mechanism of the gamma-aminobutyric acid receptor in the mammalian (mouse) cerebral cortex. Chemical kinetic investigations with a 10-ms time resolution adapted to measurements of neuronal receptor function in single cells.

The gamma-aminobutyric acidA (GABA) receptor belongs to a superfamily of proteins involved in chemical reactions that regulate signal transmission between cells of the nervous system and is the target of some of the agents most frequently used in medicine to control disorders of the central nervous system. In contrast to the nicotinic acetylcholine receptor, which initiates signal transmission and is the best characterized member of the superfamily, the GABA receptor forms anion-specific transmembrane channels and inhibits signal transmission. The chemical kinetic experiments described here, in which fast chemical reaction techniques were used, indicate that both receptor proteins may operate by the same mechanism. Also described is the use of a chemical kinetic technique with a 10-ms time resolution that we have developed for making measurements with single cells isolated from specific areas of the nervous system, in this case the cerebral cortex of embryonic mice. A flow device was used to equilibrate receptors on the cell surface with GABA, and the concentration of open transmembrane channels in the cells was then measured by recording the whole-cell currents at pH 7.2, 21-23 degrees C, and a transmembrane voltage of -70 mV. Two different receptor forms, A alpha and A beta, were detected in cerebral cortical cells. Although the ratio of A alpha to A beta varied from cell to cell, on average 35% and 65% of the receptor-controlled current was associated with receptor forms A alpha and A beta, respectively. At saturating concentrations of GABA, the rate coefficients of desensitization, alpha and beta, associated with these two forms have maximal values of 4.4 and 0.7 s-1, respectively. The constants of a mechanism that accounts for the open transmembrane channels of both receptor forms were evaluated over a 50-fold range of GABA concentration. The dissociation constant of the site controlling channel opening was 40 microM for A alpha and 320 microM for A beta. The channel-opening equilibrium constant, phi-1, was 3.5 for A alpha and 20 for A beta. The evaluated constants allow one to calculate Po, the conditional probability that at a given concentration of GABA the receptor-channel is open. Po could also be determined in the presence of 100 microM GABA by an independent method in which different assumptions are made in the interpretation of the experimental results, the single-channel current-recording technique. The value of Po obtained (0.56) was in good agreement with the Po value (0.61) calculated for receptor form A alpha from chemical kinetic measurements at 100 microM GABA.(ABSTRACT TRUNCATED AT 400 WORDS)


Synthesis and photochemistry of photolabile N-glycine derivatives and effects of one on the glycine receptor.

Three photolabile precursors of glycine containing a photosensitive 2-nitrobenzyl moiety attached to the amino group have been synthesized. When exposed to ultraviolet radiation between 308 and 350 nm, the compounds photolyze to release glycine, an important inhibitory neurotransmitter in the central nervous system. The identification of glycine as a photolysis product was determined by two different methods: separation of the photolyzed sample by thin-layer chromatography followed by a reaction with ninhydrin, and recognition of derivatized glycine using the Waters pico-tag method in conjunction with high-performance liquid chromatography. The photolysis of these compounds at 22 degrees C has been investigated, and the rate of decay of a transient intermediate in the reaction, which is assumed to reflect product release, has been measured. For N-(alpha-carboxy-2-nitrobenzyl)glycine this decay rate was found to be 940 s-1 at pH 6.8 and 600 s-1 at pH 7.5. Additionally, this compound was found to exhibit biological activity upon photolysis; cultured mouse spinal cord cells containing neuronal glycine receptors were used to detect the glycine liberation. The approach adopted here is useful in demonstrating the utility of photolabile precursors of neurotransmitters that have the protecting group linked to the neurotransmitter through the amino group. The rapid photolysis of such compounds to release free neurotransmitter is valuable in gaining access to chemical kinetic studies of neurotransmitter receptors. Previously, such studies have been limited because the available methods for neurotransmitter delivery did not give a sufficiently high time resolution.


Disulfide bond-coupled folding of bovine pancreatic trypsin inhibitor derivatives missing one or two disulfide bonds.

The disulfide bond-coupled folding and unfolding mechanism (at pH 8.7, 25 degrees C in the presence of oxidized and reduced dithiothreitol) was determined for a bovine pancreatic trypsin inhibitor mutant in which cysteines 30 and 51 were replaced with alanines so that only two disulfides, between cysteines 14 and 38 and cysteines 5 and 55, remain. Similar studies were made on a chemically-modified derivative of the mutant retaining only the 5-55 disulfide. The preferred unfolding mechanism for the Ala30/Ala51 mutant begins with reduction of the 14-38 disulfide. An intramolecular rearrangement via thiol-disulfide exchange, involving the 5-55 disulfide and cysteines 14 and/or 38, then occurs. At least five of six possible one-disulfide bond species accumulate during unfolding. Finally, the disulfide of one or more of the one-disulfide bond intermediates (excluding that with the 5-55 disulfide) is reduced giving unfolded protein. The folding mechanism seems to be the reverse of the unfolding mechanism; the observed folding and unfolding reactions are consistent with a single kinetic scheme. The rate constant for the rate-limiting intramolecular folding step--rearrangements of other one-disulfide bond species to the 5-55 disulfide intermediate--seems to depend primarily on the number of amino acids separating cysteines 5 and 55 in the unfolded chain. The energetics and kinetics of the mutant's folding mechanism are compared to those of wild-type protein [Creighton, T. E., & Goldenberg, D. P. (1984) J. Mol. Biol. 179, 497] and a mutant missing the 14-38 disulfide [Goldenberg, D. P. (1988) Biochemistry 27, 2481]. The most striking effects are destabilization of the native structure and a large increase in the rate of unfolding.


Purification of the vesamicol receptor.

The vesamicol receptor (VR) present in cholinergic synaptic vesicles isolated from the electric organ of Torpedo was solubilized in cholate detergent and stabilized with glycerol and a phospholipid mixture. The receptor was purified in 7% yield by hydroxylapatite, wheat germ lectin affinity, DEAE anion-exchange, and size exclusion chromatographies based on a [3H]vesamicol binding assay. A final specific binding of 4400 pmol/mg of protein was obtained. The cholate-solubilized VR complex exhibited variable aggregation states with particle molecular masses of 210-3500 kDa in different experiments. The purified VR exhibited very heterogeneous electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with very diffuse protein staining at about 240 kDa. No "classical" polypeptide or glycopeptide band was detected. One form of the SV1 epitope, which is characteristic of cholinergic synaptic vesicle proteoglycan, copurified precisely with the VR. The SV2 epitope, which is found in most neuronal and endocrine secretory vesicles, also closely purified with the VR. Substantially purified VR retained both enantioselectivity for (-)-vesamicol and a linked AcCh-binding site. This confirms the allosteric model for the VR in the AcCh transporter. The physicochemical properties of the VR and copurification of it with the SV1 epitope strongly suggest that the VR is associated with cholinergic vesicle proteoglycan. A second proteoglycan that is not associated with the VR but which carries the SV1 and SV2 epitopes also was observed.


Calcium-dependent interaction of chlorpromazine with the chloroplast 8-kilodalton CF0 protein and calcium gating of H+ fluxes between thylakoid membrane domains and the lumen.

Earlier work suggested that Ca2+ ions in the chloroplast thylakoid lumen interact with thylakoid membrane proteins to produce a proton flux gating structure which functions to regulate the expression of the energy-coupling H+ gradient between localized and delocalized modes [Chiang, G., & Dilley, R. A. (1987) Biochemistry 26, 4911-4916]. In this work, one of the phenothiazine Ca2+ antagonists, chlorpromazine, was used as a photoaffinity probe to test for Ca(2+)-dependent binding of the probe to thylakoid proteins. [3H]Chlorpromazine photoaffinity-labels thylakoid polypeptides of Mr 8K and 6K, with generally much less label occurring in other proteins (some experiments showed labeled proteins at Mr 13K-15K). More label was incorporated in circumstances where it is expected that Ca2+ occupies the putative H+ flux gating site, compared to when the gating site is not occupied by calcium. The photoaffinity labeling of the 8-kDa protein was also influenced by the energization level of the thylakoids (less labeling under H+ uptake energization). The 8-kDa protein was identified by partial amino acid sequence data as subunit III of the thylakoid CF0 H+ channel complex. The partial amino acid sequence of the 6-kDa protein (19 residues were determined with some uncertainties) was compared to data in the GCG sequence analysis data base, and no clear identity to a known sequence was revealed. Neither the exact site of putative Ca2+ binding to the CF0 proteolipid nor the site of covalent attachment of the chlorpromazine to the CF0 component has been identified. Evidence for gating of energy-linked H+ fluxes by the hypothesized Ca(2+)-CF0 gating site came from the correlation between Ca(2+)-dependent binding of chlorpromazine to the CF0 8-kDa protein with inhibition of light-driven H+ uptake into the lumen but no inhibition of H+ uptake into sequestered membrane domains. When conditions favored a delocalized delta mu H+ coupling mode, less chlorpromazine was bound to the CF0 structure, and much larger amounts of H+ ions were accumulated in the lumen. The data support the hypothesis that Ca2+ ions act in concert with the 8-kDa CF0 protein (and perhaps another protein, the 6-kDa polypeptide?) in a gating mechanism for regulating the expression of the energy-coupling H+ gradient between localized or delocalized coupling modes.


Fluorescence-detected assembly of the signal recognition particle: binding of the two SRP protein heterodimers to SRP RNA is noncooperative.

Protein-RNA and protein-protein interactions involved in the assembly of the signal recognition particle (SRP) were examined using fluorescence spectroscopy. Fluorescein was covalently attached to the 3'-terminal ribose of SRP RNA following periodate oxidation, and the resulting SRP RNA-Fl was reconstituted into a fluorescent SRP species that was functional in promoting translocation of secretory proteins across the membrane of the endoplasmic reticulum. Each of the two protein heterodimers purified from SRP elicited a substantial change in fluorescein emission upon association with the modified RNA. The binding of SRP9/14 to singly-labeled SRP RNA-Fl increased fluorescein emission intensity by 41% at pH 7.5 and decreased its anisotropy from 0.18 to 0.16. The binding of SRP68/72 increased the fluorescein anisotropy from 0.18 to 0.23 but did not alter the emission intensity of SRP RNA-Fl. These fluorescence changes did not result from a direct interaction between the dye and protein because the fluorescein remained accessible to both iodide ions and fluorescein-specific antibodies in the complexes. The spectral changes were elicited by specific SRP RNA-protein interactions, since (i) the SRP9/14- and SRP68/72-dependent changes were unique, (ii) an excess of unlabeled SRP RNA, but not of tRNA, blocked the fluorescence changes, and (iii) no emission changes were observed when SRP RNA-Fl was titrated with other RNA-binding proteins. Each heterodimer bound tightly to the RNA, since the Kd values determined spectroscopically and at equilibrium for the SRP9/14 and the SRP68/72 complexes with SRP RNA-Fl were less than 0.1 and 7 +/- 3 nM, respectively. The binding affinity of SRP68/72 for SRP RNA-Fl was unaffected by the presence of SRP9/14, and hence the binding of the heterodimers to SRP RNA is noncooperative in the absence of SRP54 and SRP19. The SRP protein heterodimers therefore associate randomly and independently with SRP RNA to form domains in the particle that are distinct both structurally and functionally. Any cooperativity in SRP assembly would have to be mediated by SRP54 and/or SRP19.


Ruthenium red affects the intrinsic fluorescence of the calcium-ATPase of skeletal sarcoplasmic reticulum.

We have studied the effect of Ruthenium red on the sarcoplasmic reticulum Ca(2+)-ATPase. Ruthenium red does not modify the Ca2+ pumping activity of the enzyme, despite its interaction with cationic binding sites on sarcoplasmic reticulum vesicles. Two pools of binding sites were distinguished. One pool (10 nmol/mg) is dependent upon the presence of micromolar Ca2+ and may therefore represent the high-affinity Ca2+ transport sites of the Ca(2+)-ATPase. However, Ruthenium red only slightly competes with Ca2+ on these sites. The other pool (15-17 nmol/mg) is characterized as low-affinity cation binding sites of sarcoplasmic reticulum, distinct from the Mg2+ site involved in the ATP binding to the Ca(2+)-ATPase. The interaction of Ruthenium red with these low-affinity cation binding sites, which may be located either on the Ca(2+)-ATPase or on surrounding lipids, decreases tryptophan fluorescence level of the protein. As much as 25% of the tryptophan fluorescence of the Ca(2+)-ATPase is quenched by Ruthenium red (with a dissociation constant of 100 nM), tryptophan residues located near the bilayer being preferentially affected.


Complete determination of disulfide bonds localized within the short consensus repeat units of decay accelerating factor (CD55 antigen).

Decay accelerating factor (DAF) has 4 SCR (short consensus repeat) units. Each SCR unit consists of approx. 60 amino acids characterized by having four conserved cysteine residues and several other highly conserved residues which include proline, tryptophan, tyrosine/phenylalanine and glycine. To determine the disulfide-bonding pattern, we used the urine form of DAF. After thermolysin and trypsin digestion, we isolated seven disulfide-linked peptides by HPLC purification. Because all of the cysteine residues are disulfide-bonded, DAF should contain eight disulfide bonds. After subtilisin and trypsin digestion, we isolated the eighth disulfide-bonded peptides by HPLC purification. From sequence analyses of these peptides, we could identify all disulfide bonds in the 4 SCR units of DAF as being between the first and the third and between the second and the fourth half-cystines within each SCR unit.


In vivo potentiation of ricin toxicity by monensin delivered through liposomes.

Monensin, a carboxylic ionophore, which is known to raise intravesicular pH, was intercalated in liposomes and its effect on the toxicity of ricin in mice was studied. The toxicity of ricin in vivo was found to be significantly enhanced by the administration of monensin intercalated in liposomes (liposomal monensin). The observed enhancement of the toxicity of ricin by monensin was highly dose-dependent and was maximal when ricin was injected within 60 min of monensin injection. The survival time was found to be reduced in the range of 8-20 h, depending on the dose of ricin used, by liposomal monensin. Stability of liposomes containing monensin as inferred from the release of entrapped calcein or FITC-dextran under both in vivo and in vitro conditions was comparable to that observed for liposomes without monensin. Liposomal monensin remains in circulation for 2 h and was cleared from the blood stream after 4 h. In contrast, 15 min was required for the clearance of monensin when administered in free form. Studies on the distribution of liposomal monensin and 125I-ricin in various tissues have revealed that monensin is mainly localized in the liver and spleen which are also the major sites for ricin accumulation. Our observation on the substantial enhancement of ricin toxicity in vivo by liposomal monensin strongly supports the potential usefulness of the latter as a potentiating agent in the enhancement of the toxicity of immunotoxin or hormonotoxin for selective elimination of cancer cells.


Nucleotide sequence of rat vascular cell adhesion molecule-1 cDNA.

Vascular cell adhesion molecule 1 (VCAM-1) is an inducible transmembrane protein which is expressed by vascular endothelium following cytokine activation. VCAM-1 mediated the adhesion of certain blood leukocytes and tumor cells via the interaction with its counter-receptor, the integrin VLA4. When initially cloned from interleukin-1 (IL-1) stimulated human umbilical vein endothelial cells, VCAM-1 was reported to contain six immunoglobulin-like domains. However, subsequent cDNA clones and structural analysis of the human gene evealed an alternatively spliced seventh immunoglobulin domain. This seven domain form appears to be the predominant transcript in IL-1 activated endothelium. In this report, the cloning and nucleotide sequence of rat VCAM-1 is described.


Regulation of chloride transport in cultured normal and cystic fibrosis keratinocytes.

Cultured normal (N) cystic fibrosis (CF) keratinocytes were evaluated for their Cl(-)-transport properties by patch-clamp-, Ussing chamber- and isotopic efflux-measurements. Special attention was paid to a 32 pS outwardly rectifying Cl- channel which has been reported to be activated upon activation of cAMP-dependent pathways in N, but not in CF cells. This depolarization-induced Cl- channel was found with a similar incidence in N and CF apical keratinocyte membranes. However, activation of this channel in excised patches by protein kinase (PK)-A or PK-C was not successful in either N or CF keratinocytes. Forskolin was not able to activate Cl- channels in N and CF cell-attached patches. The Ca(2+)-ionophore A23187 activated in cell-attached patches a linear 17 pS Cl- channel in both N and CF cells. This channel inactivated upon excision. No relationship between the cell-attached 17 pS and the excised 32 pS channel could be demonstrated. Returning to the measurement of Cl- transport at the macroscopic level, we found that a drastic rise in intracellular cAMP induced by forskolin did in N as well as CF cells not result in a change in the short-circuit current (Isc) or the fractional efflux rates of 36Cl- and 125I-. In contrast, addition of A23187 resulted in an increase of the Isc and in the isotopic anion efflux rates in N and CF cells. We conclude that Cl(-)-transport in cultured human keratinocytes can be activated by Ca2+, but not by cAMP-dependent pathways.


Neonatal B lymphocyte subpopulations and method of delivery.

We studied four groups of healthy term newborn infants: (1) 11 infants born by vaginal delivery; (2) 11 infants born by elective cesarean section; (3) 10 infants born by emergency cesarean section with labor, and (4) 10 infants born by complicated vaginal delivery. Total and differential leukocyte counts, cortisol blood level, and B lymphocyte subpopulations (SmIg, sIgD, sIgM, CD19, CD20, CD21, CD23) were evaluated in cord blood samples from the four infant groups. Furthermore, the Pentothal blood level was measured in infants born by elective cesarean section and in their mothers at delivery. Higher total and differential leukocyte counts and cortisol blood levels were observed in group 1 and 4 infants as compared with group 2 and 3 infants. A significant correlation was observed between cortisol blood level and leukocyte counts. The percentages of positivity to cell surface markers of B lymphocyte subpopulations were significantly higher in infants born by elective cesarean section. A negative significant correlation of thiopentone with sIgM and CD21 was observed. These data indicate a significant influence of method of delivery and of thiopentone on B lymphocyte subpopulations.


Surface-reactive biomaterials in osteoblast cultures: an ultrastructural study.

The tissue/biomaterial interface reactions of three biomaterials selected as candidates for hard tissue replacement were studied at the electron microscopical level after incubation with enzymatically isolated rat bone cells. An electron-dense layer was routinely observed between hydroxyapatite, coral, cytodex polymer and the neighbouring cells. This layer was visible before bone formation occurred, and was collagen free. The ultrastructural features revealed a needle-shaped filamentous layer continuous with coral material, whereas hydroxyapatite or cytodex/tissue interface was granular in appearance. These different structures may indicate reactive surfaces, depending on the composition of the substrate.


Reduction in blood loss and blood use after cardiopulmonary bypass with high-dose aprotinin versus autologous fresh whole blood transfusion.

Ninety patients undergoing cardiac surgery were randomly divided into three groups of 30 patients to compare the effects on bleeding and transfusion requirements of either intraoperative infusion of high-dose aprotinin (GpI) or reinfusion of autologous fresh whole blood (GpII) versus a control group (GpIII). Standardized anesthetic, perfusion, and surgical techniques were used. Platelet counts, hemoglobin concentration, hematocrit, fibrinogen, and Ivy-Nelson bleeding times determined at fixed times perioperatively did not differ among the three groups. The total loss from the chest drains was significantly reduced in GpI (328 +/- 28 mL; mean +/- SEM) as compared with the loss in GpII and GpIII (775 +/- 75 mL and 834 +/- 68 mL, respectively). There was a threefold difference in the total hemoglobin loss (GpI, 14.2 +/- 1.7 g; GpII, 50.1 +/- 5.0 g; GpIII, 45.0 +/- 5.2 g). GpI patients also received less banked blood: 250 +/- 53 mL versus 507 +/- 95 mL in GpII and 557 +/- 75 mL in GpIII. No GpI patient required transfusion of platelets or fresh frozen plasma. Fresh whole autologous blood transfusions had no significant hemostatic effect and failed to reduce the homologous blood requirement. Conversely, high-dose aprotinin reduced blood loss and transfusion requirements.


High-dose aprotinin in cardiac surgery: three years' experience in 1,784 patients.

The effect of the proteinase-inhibitor aprotinin on blood loss and homologous blood requirement in cardiac surgery was investigated. In a prospective study, 902 adult patients were treated with high-dose aprotinin (total greater than 5 x 10(6) kallikrein inactivator units [KIU]; group A), while 882 patients without aprotinin administration served as the controls (group C). Both groups were operated on between January 1987 and October 1989, and included patients with primary coronary artery bypass grafting (n = 525 group C, n = 560 group A), valve replacement (n = 292 group C, n = 264 group A), or combined procedures (n = 65 group C, n = 78 group A), as well as cardiac reoperations (n = 91 group C, n = 110 group A). The average blood loss 36 hours postoperatively in the aprotinin group was 679 +/- 419 mL, compared with 1,038 +/- 671 mL in the control group (P less than 0.05). Total homologous blood requirement was also significantly less in group A (942 +/- 1,630 mL) compared with group C (1,999 +/- 2,283 mL) (P less than 0.05), a reduction of 53%. Serum creatinine concentrations did not show intergroup differences on the first postoperative day (group A, 1.2 +/- 0.7; group C, 1.3 +/- 0.5 mg/dL) or on discharge from the intensive care unit (ICU). Thus, impairment of renal function as a consequence of aprotinin treatment was not observed. Three patients developed signs of mild circulatory depression after injection of aprotinin, which responded promptly to vasopressor therapy.(ABSTRACT TRUNCATED AT 250 WORDS)


pH transients due to monosynaptic activation of GABAA receptors in rat hippocampal slices.

Extracellular pH transients were evoked in rat hippocampal brain slices by activation of a monosynaptic inhibitory pathway following pharmacological blockade of glutaminergic transmission. Repetitive stimulation in stratum radiatum near the recording site in stratum pyramidale evoked an immediate alkaline shift which was potentiated by pentobarbital and blocked by picrotoxin but not by 2-hydroxy-saclofen. Benzolamide, a poorly permeant inhibitor of carbonic anhydrase (CA), and prontosil-dextran 5000, a macromolecular CA inhibitor, abolished the alkaline transients evoked by stimulation and by exogenous GABA. Thus an extracellular CA is involved in regulating interstitial pH in brain, and the stimulation-induced alkaline transients are caused by net influx of CO2 into CA1 neurons in response to efflux of bicarbonate across postsynaptic GABAA receptor channels.


Enkephalin-immunoreactive nociceptive neurons in the cat spinal cord.

In this combined electrophysiological-ultrastructural study in the cat spinal cord, we detected enkephalin-like immunoreactivity using internally radio-labelled monoclonal antibodies in functionally characterized neurons which had been filled intracellularly with horseradish peroxidase. Of the 4 neurons included in this study, two were positive for enkephalin immunoreactivity; one was a nociceptive specific neuron in lamina I, the other a wide dynamic range neuron in lamina V. The other two cells were devoid of immunoreactivity for enkephalin; one was a wide dynamic range neuron and the other was a non-nociceptive neuron. These results thus provide a morphological substrate within the spinal dorsal horn for the release of an endogenous opioid following administration of substance P or noxious cutaneous stimulation.


Is the nucleus corticalis of teleosts a new cholinergic central nervous system for vertebrates?

Acetylcholinesterase histochemistry suggests that the pretectal nucleus corticalis of teleost fish may be cholinergic. Because immunohistochemical analysis with antibodies against choline acetyltransferase provides a more reliable means of recognizing cholinergic central nervous structures, we investigated transverse brain sections of the African cichlid fish, Hemichromis lifalili and Hemichromis guttatus, with standard immunohistochemical techniques and found the nucleus corticalis to contain choline acetyltransferase. This supports the hypothesis that teleosts (unlike all other known vertebrates) have a cholinergic second-order sensory (i.e. visual) circuit.


FK 506 associated disorders in liver transplantation.

Immunosuppressive regimens are usually required for patients receiving organ transplants. The development of a post-transplant lymphoproliferative disorder is an infrequent complication of such therapy. FK 506 is a new immunosuppressant agent that has recently been used in patients receiving organ transplantation. This report describes a 20 month old Saudi child who developed post-transplant lymphoproliferative disorder while receiving FK 506 following liver transplantation. Such a complication has been recognized with cyclosporine but has not been well addressed as yet with FK 506. The child also developed progressive renal complications. There was also a difficulty in interpreting the results for IgM antibodies to different viruses. The overall features of progressive renal toxicity and those of lymphadenopathy, hepatosplenomegaly, fever, neutropenia and thrombocytopenia reversed following discontinuation of FK 506 therapy. It is concluded that all the above complications, though reversible, may well be linked to the new immunosuppressant agent FK 506.


Computer simulation of the pacemaker oscillations of thalamocortical cells.

Computer simulations of the spontaneous pacemaker oscillations observed in thalamocortical (TC) cells were performed using a biophysical model that included the low voltage activated, Ca2+ current, IT, and the mixed Na+/K+, inward rectifying current, Ih. The simulations accurately reproduced the waveform and voltage-dependence of the pacemaker oscillations, hence the transition from tonic firing to burst firing which is a peculiar feature of the electrical activity of TC cells both in vivo and in vitro. The results provide further support to the suggestion that IT and Ih are essential for the presence of the pacemaker oscillations, and indicate that our model is a useful tool for the study of spontaneous, low-frequency oscillatory activities in TC cells.


Differential lectin reactivities of alpha-fetoprotein in hepatocellular carcinoma: diagnostic value when serum alpha-fetoprotein levels are slightly raised.

The specificity and sensitivity of alpha-fetoprotein (AFP) binding to Concanavalin-A (Con-A) and Lens culinaris agglutinin (LCA) in 26 South African blacks with advanced symptomatic hepatocellular carcinoma (HCC) but only slightly raised serum AFP concentrations (20-500 ng/mL) was compared with that in patients with similar serum AFP levels from diseases that might be mistaken clinically for HCC (seven 'benign' liver disease [BLD] patients and six with metastatic liver disease [MLD] from gastrointestinal tumours). Con-A-Sepharose-4B affinity chromatography did not differentiate between the different groups: fucosylation rations for the HCC patients were 0.81 +/- 0.60, compared with 0.63 +/- 0.27 and 0.54 +/- 0.32 in patients with BLD and MLD, respectively. Electrophoresis of AFP serum and fraction in the presence or absence of Con-A and LCA showed an increase in the AFP C2 band. Rank correlation analysis of the AFP L2 and L3 bands combined could distinguish between patients with HCC and other hepatic diseases (P less than 0.05).


[Immunoenzyme analysis of antigen-specific determination of HBsAG-containing circulating immune complexes (CIC HBsAG/IgM and CIC HBsAG/IgG) in blood serum of patients with HBV-infection].

A complex enzyme immunoassay (ELISA) has been designed for antigen-specific determination of HBsAg-containing circulating immune complexes (CIC HBsAg/IgM and CIC HBsAg/IgG) in human blood sera in parallel with registration of free HBsAg and specific antibodies to viruses of hepatitis A, B and D. It is shown that effective formation of HBsAg-containing CIC serologically is registered predominantly as a mutually incompatible marker with detection of free HBsAg (in 70-85% of the cases). CIC HBsAg/IgM and CIC HBsAg/IgG may be registered both in parallel and as mutually exclusive markers. Effective formation of HBsAg-containing CIC in the presence of anti-HBsAg occurs in case of a mild course of viral hepatitis of epidemic and sporadic type, while in severe forms of VH-free HBsAg is predominantly detected thus pointing either to ineffective formation of HBsAg-containing CIC or to their continuous registration with demonstration of the effect of delay of witching of anti-HBsM over to anti-HBsG (or CIC HBsAg/IgM to CIC HBsAg/IgG). It was also found that in case of epidemic VH in Tajik SSR (1987) serologically marked as VH both A and B convalescent phase was characterized by parallel disappearance (or lowering of the titer levels) of HBsAg-containing CIC and class M antibodies to both hepatitis A (anti-HAV M) and B (anti-HBcM, anti-HBsM) along with the containing parallel registration of relevant G-antibodies (anti-HAV G/anti-HBcG). This observation requires further studies both in terms of close association of viruses of hepatitides A and B and with regards to possible antigenic mimicry.


[Interactions of interferon with other immunomodulators in the regulation of human natural killer cell activity].

The cytotoxic activity of natural killer (NK) cells isolated from peripheral blood of 20 healthy donors and 34 patients with multiple sclerosis (MS) against labelled with H3-uridine target cells K-562 before and after their 1 hr treatment with reaferon (RF), T-activin (TA), myelopid (MP), opioid preparation dalargin (DL) as well as with combinations of TA, MP and DL with RF was studied in 14 hrs cytotoxic test. It has been shown that combination of RF with TA, MP and DL changed the regulatory action of these peptides on NK cell activity in healthy donors in vitro. The same combination of the preparations in patients with MS caused another changes in regulation of NK activity by them because NK cells in MS patients had had initially changed sensitivity to action of these regulatory polypeptides.


Support of human cord blood progenitor cells on human stromal cell lines transformed by SV40 large T antigen under the influence of an inducible (metallothionein) promoter.

We describe the development of a human bone marrow (BM) culture system which allows study of the interaction of stromal cell lines (SCL) and highly purified hematopoietic progenitor cells. Normal BM stromal cells were electroporated with a plasmid containing the simian virus 40 (SV40) large T antigen (SV40 T Ag) under the control of a synthetic metallothionein promoter (MT4); this construct is designated MT4 SV40 T Ag. SCL in which the rate of proliferation could be controlled by altering the zinc (Zn) concentration were characterized, demonstrating that the SCL were heterogeneous with respect to G-CSF and GM-CSF production. Suppression of SCL proliferation on removal of Zn made it possible to use these lines in coculture with purified CD34+ progenitor cells from umbilical cord blood. The ability to control proliferation of SCL has allowed us to maintain the survival and expansion of colony-forming cells in culture for up to 2 months. These lines have enabled us to test for stromal cell characteristics at a clonal level and provided us with a tool to analyze the events leading to lineage commitment and hematopoietic differentiation, as demonstrated by suppression of hematopoiesis by an antibody directed against the c-kit molecule.


P-selectin mediates Ca(2+)-dependent adhesion of activated platelets to many different types of leukocytes: detection by flow cytometry.

Previous studies have shown that thrombin-activated platelets interact through the P-selectin with neutrophils and monocytes. To identify other types of leukocytes capable of such an interaction, eosinophils, basophils, and lymphocytes were isolated from whole blood. Binding of these cells to activated platelets was examined in a double immunofluorescence assay and the results show that activated platelets not only bind to neutrophils and monocytes, but also to eosinophils, basophils, and subpopulations of T lymphocytes. Using monoclonal antibodies (MoAbs) specific for subsets of T cells, we could further demonstrate that the T cells which bind activated platelets are natural killer (NK) cells and an undefined subpopulation of CD4+ and CD8+ cells. All these interactions were dependent on divalent cations and were completely inhibited by an MoAb against P-selectin. Thus, P-selectin mediates the binding of activated platelets to many different types of leukocytes. Studies with leukocytes treated with proteases or neuraminidase have shown that the structures recognized by P-selectin are glycoproteins carrying sialic acid residues. Because the loss of binding of activated platelets to neuraminidase-treated neutrophils was almost complete, but only partial to treated eosinophils, basophils, and monocytes, the latter cell types may have different P-selectin ligands in addition to those present on neutrophils. We found that two previously identified ligands for P-selectin, the oligosaccharides Le(x) and sialyl-Le(x), had little or no inhibitory effect on adhesion of activated platelets to leukocytes and that binding was not inhibited by MoAbs against these oligosaccharides. In addition, there was no correlation between the expression of Le(x) on several cell types and their capacity to bind activated platelets. In contrast, the expression of sialyl-Le(x) on cells was almost perfectly correlated with their ability to bind activated platelets. Thus, while Le(x) cannot be a major ligand for P-selectin, a possible role for sialyl-Le(x) in P-selectin-mediated adhesion processes cannot be dismissed. Finally, activated platelets were found to bind normally to monocytes and neutrophils of patients with paroxysmal nocturnal hemoglobulinuria (PNH) and to neutrophils from which phosphatidyl inositol (PI)-linked proteins had been removed by glycosylphosphatidyl inositol-specific phospholipase C (GPI-PLC) digestion. This suggests that at least part of the P-selectin ligands on these cells are not GPI-anchored.


Platelet dense granule membranes contain both granulophysin and P-selectin (GMP-140).

We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.


Infection of hematopoietic progenitor cells by human cytomegalovirus.

The susceptibility of hematopoietic progenitor cells to infection by human cytomegalovirus (HCMV) was investigated using several strains of HCMV, including the recombinant strain RC256. RC256 is derived from the laboratory strain Towne and contains the Escherichia coli LacZ gene coding for beta-galactosidase (beta-gal) regulated by an early HCMV promoter. Expression of LacZ allowed the detection of HCMV in individual hematopoietic cells. Clonogeneic bone marrow (BM) progenitors, including CD34+ cells, could be infected with HCMV and would then form normal hematopoietic colonies. By polymerase chain reaction (PCR) amplification of DNA, HCMV could be detected in both erythroid and myeloid colonies. LacZ activity was observed predominantly in cells of myelomonocytic lineage. When cells derived from HCMV-infected progenitors were cocultivated with permissive human fibroblasts, infectious virus expressing LacZ was recovered. Although no characteristic HCMV cytopathology was observed in BM colonies, high virus to cell ratios resulted in a moderate inhibition of colony formation. Since infected hematopoietic progenitors can harbor HCMV for weeks and through several differentiation steps in culture, we postulate that in vivo these cells may serve as a reservoir of latent virus and contribute to HCMV dissemination.


Response assessment in chronic lymphocytic leukemia after fludarabine plus prednisone: clinical, pathologic, immunophenotypic, and molecular analysis.

The goals of this study were to evaluate the response to treatment in chronic lymphocytic leukemia (CLL) according to clinical, pathologic, immunophenotypic, and molecular features, as well as to address the clinical significance of each finding. One hundred fifty-nine CLL patients with either advanced Rai stage III or IV (81 patients) or progressive Rai stage 0 to II (78 patients) were treated with fludarabine (30 mg/m2/d intravenously every day for 5 days) plus prednisone (30 mg/m2/d orally daily for 5 days). Thirty-six patients were previously untreated. The response rates were 12% complete response (CR), 30% nodular complete response (nCR), and 18% partial response (PR). In all patients who achieved a complete response (both CR and nCR) less than 30% of nucleated cells were lymphocytes on marrow aspirate differential analysis; however, nCR patients had residual nodular and/or interstitial lymphocyte involvement on marrow biopsy examination. There was no evidence of leukemic infiltration on marrow biopsy examination in CR patients. With a median follow-up of 35 months, comparison of time to progression in the CR and nCR groups at 2 years showed a projected 87% versus 55% progression-free survival (P less than .03). Residual disease assessment by flow cytometry using simultaneous dual-color staining on blood and marrow lymphocytes was also performed on each patient. Residual disease was determined by the expression of CD5 on B lymphocytes and the monoclonality of surface light-chain expression. After six courses of fludarabine plus prednisone, no residual disease was detected by flow cytometry in 89% of the CRs, 51% of the nCRs, and 19% of the PRs. Clinical residual disease in PR patients with no residual disease detectable by flow cytometry was limited to lymph-adenopathy. Time to progression at 2 years was longer in CR and nCR patients having no residual disease detected by flow cytometry (84% v 39% 2-year progression-free survival, P less than .001). Posttreatment lg gene rearrangement analysis using JH, J kappa, and C lambda probes demonstrated no rearranged bands and a return to the germline configuration in five of seven CRs and two of eight nCRs studied. The molecular studies were concordant with the dual-parameter immunophenotype results and none of the patients who reverted to a germline DNA pattern after treatment have experienced relapse. The absence of detectable minimal residual disease by bone marrow biopsy, dual-color flow cytometry, and lg gene rearrangement analysis is achieveable in CLL with fludarabine and is predictive of the response duration.


Erythropoietin rapidly induces tyrosine phosphorylation in the human erythropoietin-dependent cell line, UT-7.

UT-7 is a human megakaryoblastic cell line capable of growing in interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (Epo) (Cancer Res 51:341, 1991). We used this cell line and a selected Epo-dependent subcell line (UT-7/Epo) to study the early signal transduction events induced by Epo. When UT-7 cells were exposed to Epo, tyrosine phosphorylation of several proteins (with molecular weight equivalent to that of p85, p110, and p145) was observed. Protein phosphorylation occurred in both a dose- and time-dependent manner. p85 showed a marked increase in phosphotyrosine content within 30 seconds; maximal phosphorylation was observed at 1 minute. Subsequently, tyrosine phosphorylation of p110 and p145 was observed, beginning at 1 minute and reaching plateau at 5 minutes. The degree of phosphorylation of these three proteins gradually decreased thereafter. In addition, in UT-7/Epo cells, Epo induced tyrosine phosphorylation of other proteins that were not observed in Epo-induced UT-7 cells. The concentration of Epo required to induce tyrosine phosphorylation was in the same range of concentration required to stimulate cell growth. Epo was also able to activate p21ras as measured by exchange of guanosine diphosphate for guanosine triphosphate. These data show that tyrosine phosphorylation and P21ras activation are early signals in the Epo-induced mitogenic pathway.