Characterization of human cytokeratin 2, an epidermal cytoskeletal protein synthesized late during differentiation. Among the more than 30 different human proteins of the cytokeratin (CK) group of intermediate filament (IF) proteins, the significance of the epidermal polypeptide CK 2 (Moll et al., 1982, Cell 31, 11-24) has been repeatedly questioned in the literature. Here, we show, by in vitro translation and protein gel electrophoresis, that human epidermis from various body sites does indeed contain relatively large amounts of mRNA encoding a distinct polypeptide comigrating with native epidermal CK 2. We also report the isolation of a cDNA clone encoding the complete sequence of CK 2, which is a type II CK different from--but related to--epidermal CKs 1 and 5 on the one hand and corneal CK 3 on the other. The mRNA of approximately 2.6 kb encodes a polypeptide of 645 amino acids and M(r) 65,852, in good agreement with the value of 65.5 kDa previously estimated from gel electrophoresis. This human CK, the largest so far known, displays several features typical of CKs of stratified epithelia, including numerous repeats of glycine-rich tetrapeptides in the head and tail domains. Northern blot and in situ hybridizations have shown that CK 2 is expressed strictly suprabasally, usually starting in the third or fourth cell layer of epidermis, and this was confirmed at the protein level by immunohistochemistry using CK 2-specific antibodies. The protein has been detected as a regular epidermal component in skin samples from different body sites, albeit as a minor CK in "soft skin" (e.g., breast nipple, penile shaft, axilla), but not in foreskin epithelium and in other epithelia, in squamous metaplasias and carcinomas, or in cultured cell lines derived therefrom. We propose that CK 2 is a late cytoskeletal IF addition synthesized during maturation of epidermal keratinocytes which probably contributes to terminal cornification.


Regulation of U3 snRNA expression during myoblast differentiation. Differentiation of proliferating rat L6 myoblasts to syncytial multinucleated myotubes results in a significant downshift in the rate of U3 snRNA gene transcription, paralleling the decrease in rRNA synthesis previously documented. Coordinate production of U3 snRNA and rRNA during the differentiation process adds further support for a role of U3 snRNA in ribosome biogenesis. Despite the dramatic decrease in U3 snRNA transcription during differentiation, a corresponding drop in the cellular level of U3 snRNA does not occur. In myotubes, the amount of U3 snRNA is regulated at the post-transcriptional level in which there is a significant accumulation of U3 snRNA in the cytoplasm of myotubes. This intracellular redistribution of U3 snRNA may significantly affect the entire process of rRNA maturation or result from the decrease in ribosome production accompanying terminal differentiation of myoblasts.


Hypomethylation of human heterochromatin detected by restriction enzyme nick translation. Using the restriction enzymes MspI and HpaII in the nick translation procedure it has been shown that decondensation of the paracentric heterochromatin of chromosome 9 during human spermatogenesis is associated with hypomethylation of the DNA sequences in this domain. Somatic cells treated with 5'-azacytidine also showed decondensation of centromeric heterochromatin. In this instance, however, hypomethylation is detected both in the extended heterochromatin at the centromeres and in the euchromatin of the chromosome arms.


Protein kinase C associates with intermediate filaments and stress fibers. The subcellular distribution of protein kinase C (PKC) was determined by immunofluorescence using anti-PKC monoclonal antibodies (MAbs). The antibodies used were: (1) 1.9 MAb that is directed against an epitope in the catalytic domain of PKC, (2) 1.3 MAb that recognizes an isozyme of PKC (Mochly-Rosen, D., and Koshland, D. E., 1987, J. Biol. Chem. 262, 2291-2297; Mochly-Rosen, D., et al. 1987 Proc. Natl. Acad. Sci. USA 84, 4660-4664) and (3) MC-2a MAb that is directed against the beta-isozyme of PKC (Usuda, N., et al. 1991, J. Cell Biol. 112, 1241-1247). The cells used in this study were baby hamster kidney cells, vimentin+ and vimentin- clones of SW13 (a human adrenal carcinoma cell line), CEM (a human T cell line), U937 (a histiocytic myeloid cell line), and HL60 (a promyelocytic leukemia cell line). The 1.9 MAb was found to recognize a variety of subcellular components, viz., nucleus (nucleoplasm and nucleolus), cytoplasm, vimentin-type intermediate filaments (IF), stress fibers, and cell membrane. Among these components the beta-isozyme-specific MAbs (1.3 and MC-2a) recognized only the IF network, stress fibers, and edges of the cell membrane. Experiments with vimentin+ and vimentin- mutants of SW13 cells, double indirect immunofluorescence studies with anti-vimentin and anti-PKC antibodies, and drug studies confirmed that the IF network is the predominant cytoskeletal network labeled with all anti-PKC MAbs. Immunoblotting studies with the MC-2a MAb revealed that the observed staining of the IF network was not due to a cross-reaction of the MAb with IF proteins and that the MAb specifically recognizes PKC. These studies, while identifying the diverse cell components to which PKC binds, have demonstrated, for the first time, that PKC associates with the IF network in a variety of cell types. Additionally, the studies have confirmed the studies by others concerning the association of PKC with stress fibers.


Scattering of the silver-stained proteins of nucleolar organizer regions in Ishikawa cells by actinomycin D. Scattering of the silver-stained proteins of nucleolar organizer regions (Ag-NOR proteins) was produced by actinomycin D in Ishikawa cells. Scattering of Ag-NOR proteins was found only in cells treated with actinomycin D and various other agents had no effect. Scattering was dose-dependent up to 10(-2) micrograms/ml of actinomycin D, but it was not found at higher concentrations that caused marked inhibition of total DNA and RNA synthesis. Actinomycin D (10(-2) micrograms/ml) caused the following changes: (i) nucleolar segregation and (ii) emergence of dense fibrillar bodies in the nucleoplasm. Ag-NOR proteins were observed on the fibrillar centers and surrounding fibrillar components in control nucleoli, on the fibrillar and amorphous zones in segregated nucleoli, and on the dense fibrillar bodies emerging in the nucleoplasm. The scattering of Ag-NOR proteins was due to the argyrophilic nature of the dense fibrillar bodies. Actinomycin D (10(-1) micrograms/ml) also caused similar morphological alterations in the nucleolus and nucleoplasm, but Ag-NOR proteins were observed only on nucleolar remnants.


Reduction in heat shock gene expression correlates with increased thermosensitivity in senescent human fibroblasts. The expression of three major classes of heat shock genes was examined in human diploid cells at differing in vitro ages. Metabolic labeling of cellular proteins following a brief heat shock showed that the synthesis of heat shock proteins was significantly reduced in late-passage cells. Northern blot analyses revealed that the reduced expression of heat shock proteins in old cells correlated with a reduced accumulation of heat shock-specific transcripts. The attenuation of heat shock gene activity in senescent cells was not unique to thermal stress since exposure of cells to sodium arsenite (10-50 microM) elicited a similar response. The reduced expression of heat shock gene products correlated with an increased thermal lability in late-passage cells following acute hyperthermic (49 degrees C) exposure. The preinduction of heat shock genes protected cells against the lethal effects of acute hyperthermia and abolished the increased thermal lability observed in senescent cells. The reduced expression of the heat shock response demonstrates that old cells possess a diminished ability to withstand adverse environmental conditions and maintain homeostasis.


Absence of PDGF-induced, PKC-independent c-fos expression in a chemically transformed C3H/10T1/2 cell clone. The effect of platelet-derived growth factor (PDGF) on c-fos mRNA transcription was studied in the immortalized mouse embryo fibroblast C3H/10T1/2 Cl 8 (10T1/2) cells and the chemically transformed, tumorigenic subclone C3H/10T1/2 Cl 16 (Cl 16). In the 10T1/2 cells as well as the Cl 16 subclone, the dose-dependent PDGF stimulation of c-fos mRNA synthesis was similar in both logarithmically growing and confluent cultures. c-fos mRNA was induced severalfold by 12-O-tetradecanoylphorbol-13-acetate (TPA) in both 10T1/2 and Cl 16. Down-regulation of protein kinase C (PKC) activity by TPA pretreatment inhibited PDGF-stimulated c-fos mRNA expression in Cl 16 cells but did not affect this induction in the 10T1/2 cells. This inhibition was not a general phenomenon of 3-methylcholanthrene-mediated transformation of 10T1/2 cells since experiments with another transformed 10T1/2 cell clone, C3H/10T1/2 TPA 482, gave qualitatively the same results as the 10T1/2 cells. Receptor binding experiments showed that the nontransformed and transformed cells had a comparable number of PDGF receptors, 1.3 x 10(5) and 0.7 x 10(5) receptors per cell, respectively. Furthermore, cAMP-induced c-fos expression induced by forskolin is formerly shown to be independent of PKC down-regulation. In our experiments, forskolin induced c-fos expression in both clones. However, PKC down-regulation inhibited the forskolin-induced c-fos expression in Cl 16 cells. This apparently demonstrates cross talk between PKC and PKA in the c-fos induction pathway. The present results provide evidence for an impaired mechanism for activating c-fos expression through PKC-independent, PDGF-induced signal transduction in the chemically transformed Cl 16 fibroblasts compared to that in nontransformed 10T1/2 cells.


Parental imprinting of the human H19 gene. It has only recently become clear that genetic imprinting plays an important role in human embryogenesis and in processes leading to the development of pediatric cancers and other human diseases. Using a unique human tissue, the androgenetic complete hydatidiform mole, we established that the maternally inherited allele of the imprinted H19 gene is expressed. Our results also show that the paternal allele of the human IGF-II gene, a gene suspected to be parentally imprinted in humans, is expressed.


The jasmonate precursor, 12-oxo-phytodienoic acid, induces phytoalexin synthesis in Petroselinum crispum cell cultures. The pentacyclic biosynthetic precursor of jasmonic acid, 12-oxo-phytodienoic acid, was found to induce synthesis of the major flavonoid, apiin, in cell suspension cultures of Petroselinum crispum. The accumulation of apiin was preceded by an increase in the relative levels of poly (A)+ RNAs that code for the flavonoid biosynthetic enzymes phenylalanine ammonia lyase, 4-coumarate:CoA ligase and chalcone synthase, Poly (A)+ RNAs reached maximal levels at approximately 4-6 h after the addition of elicitor while flavonoids continued to accumulate in the cultures for at least 6 days. 12-Oxo-phytodienoic acid is the first pentacyclic precursor in the jasmonic acid biosynthetic chain which functions as a signal transducer for phytoalexin induction.


Polyclonal antibodies against the polypeptide and carbohydrate epitopes of recombinant human choriogonadotropin beta-subunit. In our previous paper (Chen et al. (1991) J. Biol. Chem. 266, 4081-4087) we reported the preparation and characterization of recombinant human choriogonadotropin beta subunit (hCG beta) using the baculovirus-insect cell expression system. The rhCG beta was found to contain high mannose type N-linked carbohydrates and 3-4 serine-linked disaccharide chains. Despite the carbohydrate structural variation, the rhCG beta was similar to hCG beta in in vitro immunological and biological properties. In order to evaluate its in vivo immunological properties, rabbit antiserum against rhCG beta was produced. The antiserum was found to be almost identical to anti-hCG beta in binding to hCG beta as well as in its crossreactivity with human lutropin (hLH), hCG and human follitropin (hFSH) as indicated by radioimmunoassays using 125I-hCG beta as a tracer. Further characterization of the anti-rhCG beta antiserum revealed that there are three types of antibodies in terms of antigenic specificity present in the anti-rhCG beta antisera pool as shown by dot blot and radioimmunoassays. The carbohydrate-specific antibodies were separated by affinity chromatography using an ovalbumin-glycopeptide-Sepharose column. The antibodies held on the ovalbumin affinity adsorbent were specific for the high mannose type carbohydrates such as those present in rhCG beta, rhCG and thyroglobulin and failed to react with transferrin, alpha 1-acid glycoprotein and hCG alpha, all containing complex type carbohydrates. This was further supported by the fact that the recombinant unglycosylated hCG or periodate oxidized rhCG beta also did not show any reactivity with the carbohydrate specific antibodies. Two types of peptide epitopes seemed to be present in rhCG beta since when the flowthrough fraction from the ovalbumin-glycopeptide-affinity column was passed through the hCG beta-Sepharose column, the antibodies in the flowthrough from the latter column were specific to the unique antigenic determinants present only in the rhCG beta and not in hCG beta. The eluate from the hCG beta-Sepharose column contained the third type of antibodies, being the predominant ones, directed to the common epitopes between rhCG beta and hCG beta. The high mannose type specific antibodies are potentially useful in differentiating between the high mannose and complex type of N-linked carbohydrates present in a glycoprotein. Also, the antibody could provide an effective reagent in studying the intracellular processing of the N-linked oligosaccharides.(ABSTRACT TRUNCATED AT 400 WORDS)


Monoamines (serotonin and catecholamines) and their derivatives in infantile autism: age-related changes and drug effects. Levels of dopamine (DA) and its derivatives homovanillic acid (HVA), 3-4 dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3MT) and norepinephrine+epinephrine (NE + E), and serotonin (5HT) and its derivative 5-hydroxyindolacetic acid (5HIAA) were determined from the urine of 156 autistic children aged two to 12 years 6 months, and compared with those of age-matched mentally retarded non-autistic and normal controls. Very significant group and age effects were found for DA, HVA, 3MT, NE + E and 5HT. High HVA, 3MT, NE + E and 5HT levels were found in autistic and non-autistic children. The DA, HVA, 3MT, NE + E, 5HT and 5HIAA levels decreased significantly with age in the three groups. Significantly decreased levels of DA and HVA were observed in autistic children on haloperidol, compared with non-medicated autistic children. The results are discussed in relation to the hypothesis of a maturation defect of monoaminergic systems in autism.


Intellectual and physical outcome of children undernourished in early life is influenced by later environmental conditions. Thirty-five schoolchildren who share a common history of early undernutrition and who were reared after recovery by adoptive families (16), in institutional care (eight) or by their biological families (11) were assessed for physical and intellectual outcome. The adopted children had mean normal weight and height for age, but the children from institutions were significantly shorter. Adopted children had mean Full-scale, Verbal and Performance IQs in the normal ranges, with significant differences from the other two groups, mainly for the Verbal subscale. These results emphasize that the growth and development of early-undernourished children are not irreversibly fixed by the acute illness, but are highly sensitive and modifiable by early and stable environmental improvement.


Instability of the intelligence quotient-motor quotient relationship. The authors evaluated the stability over one- and two-year periods of the relationship between a cognitive measure (McCarthy General Cognitive Index) and a motor measure (McCarthy Motor Scale) to examine whether the cognitive-motor relationship changed significantly over time and whether children remained in the same service eligibility category, based on the cognitive referencing model. Results indicated substantial changes in the cognitive-motor relationship. Over a two-year period, more than 42 per cent of children changed their cognitive-motor relationship by at least 16 points. Children's eligibility for service, based on the cognitive referencing model, also fluctuated substantially over time. The authors conclude that the cognitive referencing model of triage is not an effective means of determining who should receive physical and occupational therapy services. Alternative methods are suggested.


The nature of the acetylcholine receptor in a Buccinum proboscis muscle examined by the sucrose-gap voltage clamp technique. 1. ACh dose-response curves for the radicular retractor muscle of Buccinum showed maximum force and membrane depolarisation of 3.3 mV at 50 mumol l-1 ACh. 2. PCh was found to be almost a full agonist for force and induced higher membrane depolarisations than ACh while BCh was only a partial agonist of very low potency. This suggests an AChR neither muscarinic nor nicotinic in mammalian terminology. 3. Neither muscarine nor nicotine had any direct agonistic effects on the muscle but pre-exposure to nicotine inhibited both force and membrane depolarisation induced by a subsequent dose of ACh. 4. The specific muscarinic and nicotinic antagonists atropine, d-tubocurarine and gallamine all inhibited ACh responses in a dose-dependent manner. 5. Single sucrose-gap recording showed that ACh induced a depolarisation resulting in a contracture. Double sucrose-gap voltage clamp recording showed that 10 mumol l-1 ACh induced an inward transmembrane current of ca 2 microA. Both ACh-induced depolarisation and inward current were abolished in Na-free media. 6. When clamped at a series of membrane voltages between natural Em and positive potentials the ACh-induced Na-dependent inward current declined as Em was reduced and was abolished at -10 mV. This current showed no reversal even at strong positive membrane voltages. 7. The AChR of this muscle appears to be neither exclusively nicotinic nor muscarinic but a hybrid and shows characteristics of voltage inactivation.


Na+/Ca2+ exchange mediation in the ouabain-induced contraction in human placental vessels. 1. Ouabain induced concentration-dependent contractions in segments of human placental arteries and veins, which were practically abolished in a Ca(2+)-free medium and not modified by the calcium antagonist nifedipine or the calcium agonist Bay K 8644. 2. Ouabain (10(-4) M) elicited a time-dependent enhancement of the 45Ca2+ uptake, which remained equal in presence of nifedipine or Bay K 8644. 3. The Na+/Ca2+ exchange blocker amiloride reduced both the contractions and the 45Ca2+ uptake induced by ouabain, whereas the Na ionophore monensin produced a parallel shift to the left of the concentration-response curve to ouabain. 4. These results suggest that ouabain-induced contractions in these vessels are dependent on the extracellular Ca2+, which mainly enters into the cell through the Na+/Ca2+ exchange system.


Effects of clonixin on the electrical activity of cardiac pacemaker cells. 1. The electrophysiological effects of clonixin, a non-steroidal analgesic, on cardiac pacemaker cells of spontaneously beating frog sinus venosus, were studied by intracellular recording of transmembrane potentials. 2. Results show that clonixin (Clx) between 1 x 10(-6) M and 3 x 10(-4) M, decreases the OS, APA, Vmax and frequency of primary and subsidiary cells, however pacemaker cells differ in their sensitivity to Clx. 3. At 2 x 10(-6) M, Clx completely blocked the spontaneous beating of primary cells. It is necessary to increase the Clx concentration about two orders of magnitude in order to attain a similar degree of blockade of subsidiary cells. 4. Previous or simultaneous superfusion with atropine does not modify Clx effects, thus a probable cholinergic mechanism of action for Clx is discarded. 5. When Clx concentrations were lower than 5 x 10(-4) M, their effects on both types of cells were partially reversed by a 100% increase of external calcium concentration. 6. BAY K-8644 which stimulates calcium influx through calcium L-type channels, reverted Clx effects on pacemaker cells. 7. It is suggested that Clx blocks calcium inward current which generates all or part of the upstroke of primary cells and subsidiary ones, respectively.


Differential effect of calcium and Bay K 8644 on the inhibitory action of estrogens in the rat uterus. 1. The effects of the estrogens estradiol (E2, 10(-7) to 3 x 10(-5) M) and diethylstilbestrol (DES, 10(-7) to 3 x 10(-6) M) on tonic contractions of the rat uterus induced by KCl and CaCl2 have been studied. 2. E2 and DES relaxed, in a dose-dependent way, the tonic contraction induced by KCl (60 mM) (IC50: 5.16 +/- 1.49 x 10(-6) and 4.51 +/- 0.03 x 10(-7) M); the tonic contraction induced by CaCl2 (3 mM) in the rat uterus incubated in depolarizing Krebs (127 mM of K+) have also been relaxed (IC50: 8.6 +/- 0.03 x 10(-7) and 2.56 +/- 0.07 x 10 M) by both drugs. 3. The CaCl2 (0.1 to 10 mM) counteracted the relaxing effect of E2 and DES, respectively, up to 28.13 +/- 10.2% and 34.71 +/- 11.5%, on KCl-induced contractions, and up to 126.36 +/- 19.35% and 95.8 +/- 16.3% on CaCl2-induced contractions. 4. Bay K 8644 (10(-10) to 10(-6) M) reversed the relaxing effect of E2 and DES, respectively, up to 42.49 +/- 2.28% and 43.31 +/- 3.59% on KCl-induced contractions, and up to 21.73 +/- 4.16% and 75.97 +/- 9.63% on CaCl2-induced contractions. 5. Propranolol (10(-6) M) did not modify the relaxing effect of E2 or DES on CaCl2-induced contractions.


Double stents for carcinoma of the esophagus invading the tracheo-bronchial tree. Extrinsic compression, neoplastic involvement of the trachea or left main bronchus, and esophago-airway fistula may cause airway obstruction and infection in patients with esophageal carcinoma. Further reduction of airway lumen may result from palliative treatment of dysphagia by radiation or esophageal stent insertion. In order to evaluate the extent of airway compromise, bronchoscopy was systematically performed in 39 consecutive patients with advanced carcinoma of the esophagus requiring esophageal endoprostheses. Airway obstruction observed in 10 patients (mean age, 60 years) resulted in the additional placement of a silicone stent in the trachea (five patients) or left main bronchus (five patients). Esophageal and airway procedures were performed under general anesthesia. All had squamous cell carcinoma of the middle third of the esophagus. Severe dyspnea at rest was documented in five patients prior to intervention. Esophago-tracheal fistula was present in five. Eight patients with associated, neoplastic invasion of the tracheo-bronchial tree required airway Nd:YAG laser therapy. The esophageal prosthesis contributed significantly to airway compromise in four patients. Symptomatic relief of dysphagia and dyspnea was obtained in all individuals. Mean survival was 121 days (range, 12 to 350 days). Complications were not serious, but included esophageal or tracheal stent migration in three patients.


Calcitonin enhances the effects of nimodipine on biogenic amines of rat brain. 1. Nimodipine affects the brain biogenic amines suggesting both inhibition of the striatal dopaminergic system and activation of serotonergic systems in various brain areas. 2. Salmon calcitonin affects serotonergic systems as nimodipine does. 3. It was hypothesized that these effects could be more than additive since they all have been ascribed to inhibition of neuronal calcium influx. 4. Biogenic amines and metabolites were determined in brain areas obtained from rats pretreated with saline or salmon calcitonin (10 MRC U/kg, s.c.) and treated with nimodipine (2.5-20 mg/kg, i.p.). 5. The effects of nimodipine and salmon calcitonin appeared to be merely additive.


The differential staining pattern of the X chromosome in the embryonic and extraembryonic tissues of postimplantation homozygous tetraploid mouse embryos. (C57BL x CBA)F1 hybrid female mice were mated with hemizygous Rb(X.2)2Ad males to distinguish the paternal X chromosome. Homozygous tetraploids were produced by blastomere fusion at the 2-cell stage, and 161 of these were transferred to recipients and analysed on the 10th day of gestation. 59 implants contained resorptions and 76 contained either an embryo and/or extraembryonic membranes. 38 (20, XXXX and 18, XXYY) were analysed to investigate their X-inactivation pattern. Embryonic and yolk sac endodermally- and mesodermally-derived samples were analysed by G-banding and by Kanda analysis. In the XX and XY controls, the predicted pattern of X-inactivation was observed, though 12.2% of metaphases in the XX series displayed no X-inactivation. In the XY series the Y chromosome was seen in a high proportion of metaphases. In the XXXX tetraploids, 8 cell lineages were recognized with regard to their X-inactivation pattern, though most belonged to the following 3 categories: (XmXm)XpXp, Xm(XmXp)Xp and XmXm(XpXp). The other categories were only rarely encountered. In the embryonic and mesodermally-derived tissue the ratio of these groups was close to 1:2:1, whereas in the endodermally-derived tissue it was 1:4.11:4.88, due to preferential paternal X-inactivation. A significant but small proportion of all 3 tissues analysed displayed no evidence of X-inactivation. Indirect evidence suggests that this represents a genuine group because of the high efficiency of the Kanda staining. The presence of the Xm(XmXp)Xp category is consistent with the expectation that X-inactivation occurs randomly in 2 of the 4 X chromosomes present. The presence of small numbers of preparations with no evidence of X-inactivation and other unexpected categories suggests that these are probably selected against during development.


Identification of a repetitive element in the snail Biomphalaria glabrata: relationship to the reverse transcriptase-encoding sequence in LINE-1 transposons. BamHI-digested Biomphalaria glabrata DNA contains a repetitive 2.0-kb fragment which is readily discernible by ethidium bromide staining. We present evidence that this repetitive element is related at both the nucleotide and amino acid levels to long interspersed nuclear element (LINE)-like transposons. Although comparable elements have been described in several invertebrates, this is the first report of a molluscan homologue. In common with LINE transposons, an open reading frame in the B. glabrata element shows significant homology to reverse transcriptase--a feature believed to allow the dissemination of these elements in the eukaryotic genome.


Prostate specific antigen: clinical use in the diagnosis and management of prostate cancer. Prostate specific antigen (PSA) has replaced prostatic acid phosphatase as the preeminent clinical tumor marker in the management of patients with prostate cancer. Serum PSA levels have been shown to be proportional to clinical and pathologic stage of prostate cancer and in particular to correlate closely with prostate cancer volume. Serum levels of PSA thus serve as a useful adjunct in the initial clinical staging of men with prostate cancer. Serum PSA values provide a unique and valuable tool in monitoring prostate cancer progression over time and its response to surgical, radiation, and/or hormonal therapies. Unfortunately, its lack of specificity for prostate cancer leaves many questions unanswered as to the efficacy and advisability of using PSA as a screening test for this cancer.


Omission of exon 12 in cystic fibrosis transmembrane conductance regulator (CFTR) gene transcripts. Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA transcripts isolated from both expressing and "non-expressing" cell types of normal individuals exhibit differential splicing to a variable extent in a region encoding the putative nucleotide binding fold of the CFTR polypeptide. Sequence analysis of the aberrant fragments obtained after cDNA polymerase chain reaction amplification confirmed the in-frame joining of exons 11 and 13. The proportion of alternative splicing is reproducible and constant in a given individual. The omission of exon 12 in a significant proportion of transcripts supports the hypothesis that a minimal amount of correctly expressed CFTR is sufficient for the maintenance of a clinically normal phenotype.


Incidence and expression of the N1303K mutation of the cystic fibrosis (CFTR) gene. The N1303K mutation was identified in the second nucleotide binding fold of the cystic fibrosis (CF) gene last year. We have gathered data from laboratories throughout Europe and the United States of America in order to estimate its frequency and to attempt to characterise the clinical manifestations of this mutation. N1303K, identified on 216 of nearly 15,000 CF chromosomes tested, accounts for 1.5% of all CF chromosomes. The frequency of the N1303K allele varies significantly between countries and ethnic groups, being more common in Southern than in Northern Europe. This variation is independent of the delta F508 allele. It was not found on UK Asian, American Black or Australian chromosomes. N1303K is associated with four different linked marker haplotypes for the polymorphic markers XV-2c, KM.19 and pMP6d-9. Ten patients are homozygous for this mutation, whereas 106 of the remainder carry one of 12 known CF mutations in the other CF allele. We classify N1303K as a "severe" mutation with respect to the pancreas, but can find no correlation between this mutation, in either the homozygous or heterozygous state, and the severity of lung disease.


Relationships between %Hb F or %G gamma and the haplotypes in the beta-globin gene cluster in the normal adult Japanese and Korean populations. Haplotypes or subhaplotypes in the beta-globin gene cluster were examined in 31 normal Japanese and 21 Korean families. Major haplotypes were VII, V, and I, which shared a common subhaplotype [+----], and occurred in 70% of the Japanese and 68% of the Koreans. Three haploypes which shared a common subhaplotype [-+-++], bearing the XmnI site 5' to the G gamma-globin gene, occurred in 13% of the Japanese and 7.3% of the Koreans. One subhaplotype [-++-+], which is closely linked to the A gamma T-globin gene, had an incidence of 6.6% in the Japanese and 20% in the Koreans. Rare subhaplotypes [---++], and [- +] which has been observed mainly in Melanesians and Polynesians, and a very rare subhaplotype [-----] were observed. The present study suggests that high or low G gamma-globin chain production is closely related to subhaplotypes [-+-++] and [+----], respectively, among the normal adult population.


%Hb A2, %Hb F, %G gamma values and the haplotypes in the beta-globin gene cluster in Japanese adults with elevated Hb F. The percentages of minor adult hemoglobin (%Hb A2) in hemolysates and G gamma-globin chain (%G gamma) in fetal Hb (Hb F) of 15 individuals with elevated Hb F levels (2.0-11%) among 11,000 healthy Japanese adults were examined. Most of them might be carriers for the determinants of hereditary persistence of fetal hemoglobin. Subjects with less than 1.3% Hb A2, some of whom might be also carriers for delta-thalassemia determinants, had high G gamma values (54-70%). Those homozygous for a subhaplotype [+-----] 5' to the delta-globin gene had low to mid G gamma values (7-49%), while those homozygous for [-++-++] possessed high G gamma values (60-85%), but varied Hb F values (3.1-11%). Those heterozygous for the presence of the XmnI site 5' to (-158 bp to the cap site of) the G gamma-globin gene had mid to high G gamma values (53-65%). Factors for the high or low G gamma-globin gene expression in the Japanese adult with elevated Hb F level should be highly associated with a subhaplotype [-++-++] or [+-----], respectively.


The XmnI site 5' to the G gamma-globin gene polymorphism and its relationship to %Hb F and %G gamma in normal Japanese and Korean adults. The ratio of fetal hemoglobin to total hemoglobin (%Hb F), the ratio of G gamma to total gamma globin (%G gamma), and the polymorphism of the XmnI site at -158 base pairs from the cap site of the G gamma-globin gene were examined in normal unrelated Japanese (n = 113) and Korean (n = 44) adults. The frequency of the presence of the XmnI site was 0.15 in the Japanese and 0.16 in the Korean population. There were statistically significant differences in the %G gamma values of the Japanese between those +/+ and those +/- or -/- at the XmnI site (p less than 0.01). The Korean %G gamma values showed a statistically significant difference (p less than 0.01) between those +/- and those -/-. The presence of the XmnI site was significantly associated with the elevation of G gamma-globin chain synthesis, but this relationship was not necessarily absolute. The absence of the XmnI site in an adult with a gamma-globin gene triplication (G gamma AG gamma A gamma/G gamma A gamma) more or less reduced the level of G gamma-globin chain synthesis, but the presence of the XmnI site in an adult with a gamma-globin gene deletion (GA gamma/G gamma A gamma) had no effects on the proportion of the two gamma-globin chains.


Enzyme-linked immunosorbent assay for riboflavin carrier protein and modified protein: application for epitope analysis. Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards the conformational epitopes and antibodies to disulphide bond reduced carboxymethylated riboflavin carrier protein (RCM-RCP) to the sequential epitopes. Taking advantage of this premise and in order to map the epitopes of RCP recognized by the antibodies, enzyme-linked immunosorbent assays were validated for RCP and RCM-RCP using the Avidin-Biotin system. The usefulness of these assays were illustrated when antigenicity of peptides derived from RCM-RCP following trypsinization were examined. Two major (T1,T2) and one minor peptide (T3) fractions were obtained when the tryptic peptides were fractionated on DEAE-cellulose. RCP has a blocked N-terminal. Tryptic peptides (T1 and T2) on microsequencing revealed the absence of an N-terminal amino acid, indicating that these fragments emanate from the N-terminal region of RCP. In support of this observation is the finding that antipeptide antibody to cRCP (10-24) of cRCP interacted with T1 as well as T2 indicating the presence of the sequential epitope (10-24) of cRCP in these fragments. In RCP-ELISA, only T2 displaced RCP and peptides T1 and T2 displaced RCM-RCP in RCM-RCP ELISA. Differences in the ability of these fragments (T1 and T2) to displace RCP and RCM-RCP reflect the subtle changes in the spatial structures of these epitopes in RCP and RCM-RCP.


Hormonal modulation of biosynthesis of prostatic specific antigen, prostate specific acid phosphatase and prostatic inhibin peptide. Hormonal modulation of in vitro biosynthesis of three prostatic secretory proteins, prostate specific acid phosphatase (PSAP), prostate specific antigen (PSA) and prostatic inhibin peptide (PIP) by human benign hyperplasia (BPH) tissue was studied. LH and inhibins caused increase in the synthesis of all three proteins whereas FSH enhanced the synthesis of PIP and PSA only but decreased PSAP synthesis. Prolactin and thyroid releasing hormone decreased synthesis of PIP and PSAP. However, PSA synthesis was enhanced by TRH and was decreased by prolactin. Estradiol caused significant increase in PSA and PSAP but no discernible changes in PIP synthesis were noticed. Testosterone caused an increase in PIP, PSA and PSAP. These data indicate that biosynthesis of PIP, PSA and PSAP by BPH tissue is under multihormonal regulation.


Amoeboid and ramified microglia: their interrelationship and response to brain injury. Rio-Hortega's hypothesis that transiently appearing amoeboid microglia might become ramified microglia in the adult and that the latter could differentiate into brain macrophages in the event of brain damage could not be proved because of inherent limitations in existing techniques. The present investigation used a novel method of labelling the rat supraventricular amoeboid microglia with an enduring fluorescent marker, rhodamine B isothiocyanate, introduced intraperitoneally. Observation of their subsequent development showed that they became transformed into the ramified microglia. Both the amoeboid and ramified microglia were OX-42 positive, indicating their macrophage/monocyte lineage. Other microglia in the cerebral neocortex, which were also OX-42 positive, were not derived from any of the rhodamine-labelled cells. Rhodamine-labelled microglia did not migrate toward the site of a superficial cerebral injury. Following a deep lesion reaching the corpus callosum, greatly increased numbers of labelled amoeboid microglia were frequently observed at or near the lesion site. Large rhodamine-labelled cells, which were OX-42 positive, appeared at all lesioned sites, and such were most likely blood derived monocytes. The antigenicity of the ramified microglia became elevated when rhodamine B isothiocyanate was present intracellularly and even more so with the presence of a nearby intracerebral stab wound.


Changes of host cell infiltration into Meth A fibrosarcoma tumor during the course of regression induced by injections of a BCG nucleic acid fraction. MY-1, which consists of DNA and RNA extracted and purified from Mycobacterium bovis strain BCG, causes the regression of various experimental syngeneic tumors when injected intratumorally. In order to identify the host cells involved in the antitumor mechanism(s) of MY-1, we examined Meth A tumors inoculated intradermally to BALB/c mice, which were given multiple injections of MY-1 following tumor inoculation. Histological and immunohistochemical examinations were performed at several time points. On day 4 after inoculation, the MY-1-treated tumors were heavily infiltrated with a heterogeneous population of mononuclear cells with low density nuclei. The MY-1-injected tumors contained asialo-GM1-positive cells and Mac-1-positive cells, which indicated that the infiltrating mononuclear cells were natural killer cells and macrophages. On day 14 after inoculation, the tumors were infiltrated with a large number of L3T4-positive cells and fewer Lyt-2-positive cells, both of which were more abundant in the MY-1-treated tumors than in the control tumors. The observed sequence of host cell infiltration corresponded well with our previous studies which have indicated that the antitumor mechanism of MY-1 is divided into two phases, i.e. the early phase when natural killer cells and macrophages inhibit tumor growth, and the late phase when L3T4-positive cells act to induce tumor regression via a delayed-type hypersensitivity against tumor cells.


The effects of the antifungal azoles itraconazole, fluconazole, ketoconazole and miconazole on cytokine gene expression in human lymphoid cells. The antifungal azole drugs itraconazole (itra; R51,211), fluconazole (flu; UK-49,858), ketoconazole (keto) and micronazole (mico) have been investigated for their suppressive influence on the gene expression of the immunoregulatory cytokines IL2, IL4, IL9, GM-CSF, TNF-alpha, IFN-gamma, as well as both chains of the IL2 receptor in human PBMC and of the cytokines in the human keratinocyte cell line HaCat 17.5. The results obtained in Northern blot analysis were compared with the effects of the established immunosuppressant drug CSA and the new immunosuppressive drug FK 506, as well as the cytokine TGF-beta, which is also immunosuppressive. While 1 microgram/ml CSA and 0.1 microgram/ml FK 506 completely suppressed PHA-stimulated accumulation of mRNA for IL2, IL4, IL9, GM-CSF, TNF-alpha and IFN-gamma in PBMC, flu, keto and TGF-beta failed to inhibit any (except TNF-alpha blocked by TGF-beta). Itra and mico did suppress accumulation of mRNA, but unlike CSA and FK 506, only at high doses (10 micrograms/ml) and after extended incubation (24 h). None of the drugs nor TGF-beta suppressed the expression of the IL2R-alpha and IL2R-beta genes or TNF-alpha-stimulated cytokine gene expression in keratinocytes. Itra and mico, 1 mg/ml (achievable serum level), caused only slight inhibition of the cytokines in PBMC after 6 and 24 h of incubation. These results demonstrate that the mode of action of the azoles is different from CSA and FK 506.(ABSTRACT TRUNCATED AT 250 WORDS)


Fibrinolysis activity promotes tumor invasiveness of B16 melanoma cell lines through a reconstituted gel matrix. We studied the role of the fibrinolytic function in the invasiveness of murine melanoma B16F1 and F10 cells using a reconstituted matrix on a filter in a modified Boyden chamber. The main species of plasminogen activators (PAs) synthesized in cell lysates and released into conditioned media by these cells was found to be tissue-type PA (t-PA). The invasiveness of these cell lines was enhanced by adding plasminogen to the gel matrix. This enhancing effect of plasminogen was markedly suppressed by adding anti-t-PA IgG and plasmin inhibitors into the gel matrix, but less affected by anti-urokinase-type PA (u-PA) IgG, offering more evidence to the hypothesis that the activation of the fibrinolytic system by PAs plays an important role in the invasiveness of murine melanoma B16 cell lines, and indicating that t-PA contributed more than u-PA to the invasive potential of these cells into the pericellular matrix.


Restoration with phorbol ester of a locomotor defect in human leukaemic lymphocytes: a visual study of chronic lymphocytic leukaemia cells. Visual assays were used to study the effect of phorbol myristate acetate (PMA) on the locomotion of lymphocytes from 14 patients with chronic lymphocytic leukaemia (CLL). Previous reports had shown that CLL cells from blood were defective in locomotor capacity and that adding PMA to the cells did not restore locomotion in a short-term (30 min) assay, but did stimulate unusual non-locomotor, multipolar, morphologies in a small proportion of the cells. Here we describe experiments in which CLL cells were cultured for 48 h in PMA. Many of the cells acquired locomotor morphologies with front-tail polarity which was unlike the short-term multipolar morphology. These cultured cells were also capable of locomotion and invaded collagen gels. Autoradiography suggested that after culture in PMA, the locomotory cells were the most active cells in 3H-uridine uptake. A computer analysis suggested that the cells in locomotor morphology were the same cells that increased in size. These findings suggest that, to acquire locomotor capacity, CLL lymphocytes require an appropriate signal to allow the cells to enter the cell cycle. During G1 the lymphocytes develop the capacity for locomotion. Long-term, but not short-term, culture in PMA provides such a signal but the mechanism by which it does so is unclear.


NH2-terminal modification of the phosphatase 2A catalytic subunit allows functional expression in mammalian cells. Functional expression of recombinant wild-type phosphatase 2A catalytic subunit has been unsuccessful in the past. A nine-amino-acid peptide sequence (YP-YDVPDYA) derived from the influenza hemagglutinin protein was used to modify the NH2 and/or COOH terminus of the phosphatase 2A catalytic subunit. Addition of the nine-amino-acid sequence at the NH2 terminus allowed recombinant phosphatase 2A expression as a predominantly cytosolic phosphatase 2A enzyme. The 12CA5 monoclonal antibody that recognizes the nine-amino-acid hemagglutinin peptide sequence was used to immunoprecipitate the epitope-tagged phosphatase 2A catalytic subunit. Assay of the immunoprecipitated epitope-tagged phosphatase 2A demonstrated an okadaic acid-sensitive dephosphorylation of [32P] histone H1 and [32P]myelin basic protein similar to that measured with the wild-type enzyme. Functional phosphatase activity could be demonstrated for the NH2-terminal modified phosphatase 2A catalytic subunit following transient expression in COS cells or stable expression in Rat1a cells. In contrast, the COOH-terminal-modified phosphatase 2A catalytic subunit was very poorly expressed. The NH2-, COOH-modified subunit, having the nine-amino-acid hemagglutinin peptide sequence encoded at both termini of the polypeptide, was also expressed as a functional phosphatase 2A enzyme. Thus, NH2-terminal modification of the phosphatase 2A catalytic subunit results in a functional plasmid-expressed enzyme. The unique nine-amino-acid epitope-tag sequence also provides a method to easily resolve the recombinant phosphatase 2A from the endogenous wild-type gene product and related phosphatases expressed in cells.


Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The beta subunit modulates potassium activation of the Na,K-pump. In order to understand the molecular mechanism of ouabain resistance in the toad Bufo marinus, Na,K-ATPase alpha and beta subunits have been cloned and their functional properties tested in the Xenopus laevis oocyte expression system. According to sequence comparison between species, alpha 1, beta 1, and beta 3 isoforms were identified in a clonal toad urinary bladder cell line (TBM 18-23). The sequence of the alpha 1 isoform is characterized by two positively charged amino acids (Arg, Lys) at the N-terminal border of the H1-H2 extracellular loop and no charged amino acid at the C terminus, a pattern distinct from the ouabain-resistant rat alpha 1 isoform. The coexpression of alpha 1 beta 1 or alpha 1 beta 3 TBM subunits in the Xenopus oocyte resulted in the expression of identical maximum Na,K-pump currents with identical inhibition constant for ouabain (Ki) (alpha 1 beta 1: 53 +/- 3 microM; n = 7 vs. alpha 1 beta 3: 57 +/- 3.0 microM; n = 8) but distinct potassium half activation constant (K1/2) (alpha 1 beta 1: 0.87 +/- 0.08 mM, n = 16; alpha 1 beta 3: 1.29 +/- 0.07 mM, n = 17; p less than 0.005). We conclude that (i) the TBM alpha 1 isoform is necessary and sufficient to confer the ouabain resistant phenotype; (ii) the beta 3 or beta 1 subunit can associate with the alpha 1 equally well without affecting the ouabain-resistant phenotype; (iii) some specific sequence of the beta subunit can modulate the activation of the Na,K-pump by extracellular potassium ions.


Characterization of putative glycoinositol phospholipid anchor precursors in mammalian cells. Localization of phosphoethanolamine. A number of mammalian cell surface proteins are anchored by glycoinositol phospholipid (GPI) structures that are preassembled and transferred to them in the endoplasmic reticulum. The GPIs in these proteins contain linear ethanolamine (EthN)-phosphate (P)-6ManManManGlcN core glycan sequences bearing an additional EthN-P attached to the Man residue (Man 1) proximal to GlcN. The biochemical precursors of mammalian GPI anchor structures are incompletely characterized. In this study, putative [3H]Man-labeled GPI precursors were obtained by in vitro GDP-[3H] Man labeling of HeLa cell microsomes and by in vivo [3H]Man labeling of class B and F Thy-1 negative murine lymphoma mutants known to accumulate incomplete GPIs. The high performance liquid chromatography-purified in vitro and accumulated in vivo GPI products were structurally analyzed by nitrous acid deamination, hydrofluoric acid, trifluoroacetic acid hydrolysis, biosynthetic labeling, and exoglycosidase treatment. The data were consistent with a biosynthetic scheme in which Man and EthN-P are added stepwise to the developing glycan. Several additional points were demonstrated: 1) putative mammalian GPI precursors contain incomplete core glycans corresponding to those in previously characterized trypanosome GPI precursors. 2) The proximal EthN-P found in mature mammalian GPI anchor structures is added to Man 1 prior to incorporation of Man 2 and Man 3. 3) Glycans in the incomplete GPIs that accumulate in classes B and F lymphoma mutants consist of Man2- and Man3GlcN in which EthN-P is linked to Man 1. 4) Distal EthN-P linked to the 6-position of Man, characteristic of the complete GPI core, is found both in a subsequent GPI species with the glycan sequence EthN-P-6ManMan(EthN-P----)ManGlcN and in a more polar GPI product.


Five PDGF B amino acid substitutions convert PDGF A to a PDGF B-like transforming molecule. We used site-directed mutagenesis to determine the minimum number of PDGF B residues needed to convert PDGF A to a potently transforming PDGF B-like molecule. Substitution of two PDGF B subdomains, 106-115 and 135-144, were found to be critical. These substitutions were sufficient to broaden the ability of PDGF A to activate beta as well as alpha platelet-derived growth factor (PDGF) receptors and increase its transforming efficiency to that of PDGF B. Within subdomain I, either PDGF B residues Arg-109 and Asn-115 or Arg-109, Leu-110, and Arg-113, in combination with subdomain II PDGF B residues Asn-136, Arg-137, and Arg-142 were identified as being essential. Those mutants with transforming ability comparable with PDGF B showed significantly lower efficiencies of beta receptor triggering. Thus, our studies identify a small number of PDGF B amino acids indispensable for beta PDGF receptor interaction and suggest that a low level of beta PDGF receptor activation is sufficient to dramatically increase PDGF transforming efficiency in NIH 3T3 cells.


Alteration of phosphotyrosine-containing proteins at the early stage of erythroid differentiation of mouse erythroleukemia (MEL) cells. Following treatment of mouse erythroleukemia (MEL) cells with dimethyl sulfoxide and other typical erythroid inducing agents, the profile of cellular phosphotyrosine-containing proteins was drastically altered. We found that the level of almost all of the phosphotyrosine-containing proteins was either decreased or disappeared at a very early stage of differentiation. Addition of sodium orthovanadate (Na3VO4), a specific inhibitor of phosphotyrosine phosphatases, prevented the alteration as well as erythroid differentiation. Mutant MEL cells, which are resistant to differentiation by dimethyl sulfoxide, were apparently insensitive to the alteration. These results indicate that dephosphorylation of phosphotyrosine residues in cellular proteins is coupled with a reaction(s) which is responsible for triggering differentiation of MEL cells.


The amyloid beta-protein precursor promoter. A region essential for transcriptional activity contains a nuclear factor binding domain. A manifestation of Alzheimer's disease is the presence of amyloid depositions in brains of afflicted individuals. A major component of these depositions is the amyloid beta-protein, which is a truncated form of the larger amyloid beta-protein precursor (APP). To investigate the regulation of APP gene expression, the APP promoter and selected deletions were placed 5' to the reporter gene chloramphenicol acetyltransferase. The promoter deletions were transfected into different cell lines that showed variant levels of endogenous APP transcripts. Transient transfection assays showed that 96 base pairs 5' to the transcriptional start site are sufficient for cell type-specific promoter activity. A nuclear factor that binds to this region in a sequence-specific manner was identified by mobility shift electrophoresis, DNase footprinting, and methylation interference. The DNase-protected region covers about 25 base pairs on both strands (position -31 to -55). Mutations within this domain revealed a sequence of 12 base pairs that is crucial for factor binding. This sequence overlaps with the consensus sequences for transcription factors AP-1 and AP-4. However, competition experiments suggest that the nuclear factor that binds to the APP promoter is distinct from both AP-1 and AP-4. Factor binding to the characterized recognition sequence is observed in nuclear extracts originating from human, mouse, and rat cells, suggesting a high degree of conservation.


Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase. The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels of the cloned liver GH receptor bound to anti-phosphotyrosine antibody, suggesting that the cloned GH receptor is tyrosyl phosphorylated in vivo. GH-GH receptor complexes purified from transfected COS-7 cells using anti-GH antibody incorporated 32P when incubated with [gamma-32P]ATP, indicating association of tyrosine kinase activity with cloned liver GH receptor. The level of phosphorylation of the GH receptor was very low, as compared with the endogenous GH receptor in 3T3-F442A cells, suggesting that tyrosine kinase activity is not intrinsic to the cloned GH receptor but rather resides with a kinase present at low levels in the COS-7 cells. To test whether a higher level of GH receptor phosphorylation would be observed when the GH receptor was expressed in a different cell line, GH receptor cDNAs were stably transfected into mouse L and CHO cells, which have few or no endogenous GH receptors, and RIN5-AH cells, which do express endogenous GH receptors. In vivo tyrosyl phosphorylation of the cloned GH receptor in mouse L cells and in vitro phosphorylation of the cloned GH receptor in both L and CHO cells were higher than in transfected COS-7 cells but still substantially lower than in untransfected 3T3-F442A cells. Significantly increased 32P incorporation into tyrosyl residues in GH receptors in the in vitro kinase assay was demonstrated for GH receptors isolated from the transfected RIN5-AH cells. These studies show that the cloned liver GH receptor can be tyrosyl phosphorylated when expressed in a variety of cell types. The finding that the level of phosphorylation of GH receptor appears to vary with cell type is consistent with the cloned liver GH receptor being a substrate for an associated tyrosine kinase and with the amount of such a GH receptor-associated tyrosine kinase being cell type-specific.


Novel amino-terminal propeptide configuration in a fibrillar procollagen undergoing alternative splicing. We isolated overlapping cDNAs from embryonic libraries of the sea urchin Strongylocentrotus purpuratus coding for a fibrillar procollagen (2 alpha chain) with a predicted molecular mass of about 320 kDa. The deduced primary structure of the echinoid chain consists of a 265-amino acid carboxyl-propeptide, a triple helical domain made of 337 uninterrupted Gly-X-Y repeats, and an unusually long amino-propeptide. Aside from a 10-cysteine globular region, a collagenous sequence, and a nonhelical segment, this protein domain includes a novel 4-cysteine motif repeated several times. Interestingly, preliminary evidence indicates that different combinations of the 4-cysteine repeats are encoded by alternatively spliced transcripts. Irrespective of this, the sea urchin 2 alpha procollagen chain represents the longest fibrillar molecule identified to date by cDNA cloning experiments in both vertebrate and invertebrate organisms.


Nerve growth factor promotes the activation of phosphatidylinositol 3-kinase and its association with the trk tyrosine kinase. We investigated the involvement of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in the initiation of signal transduction by nerve growth factor (NGF) in the rat pheochromocytoma PC12 cell line. PtdIns 3-kinase catalyzes the formation of phosphoinositides with phosphate in the D-3 position of the inositol ring and previously has been found to associate with other activated protein tyrosine kinases, including growth factor receptor tyrosine kinases. Anti-phosphotyrosine immunoprecipitates had PtdIns 3-kinase activity that reached a maximum (9 times the basal activity) after a 5-min exposure of PC12 cells to NGF (100 ng/ml). Since NGF activates the tyrosine kinase activity of gp140trk, the protein product of the trk proto-oncogene, we also examined the association of PtdIns 3-kinase with gp140trk. Anti-gp140trk immunoprecipitates from NGF-stimulated PC12 cells had increased PtdIns 3-kinase activity compared to that of unstimulated cells, and larger increases were detected in cells overexpressing gp140trk, indicating that PtdIns 3-kinase associates with gp140trk. NGF produced large increases in [32P]phosphatidylinositol 3,4-bisphosphate and [32P]phosphatidylinositol 3,4,5-trisphosphate in PC12 cells labeled with [32P]orthophosphate, indicating an increase in PtdIns 3-kinase activity in intact cells. Using an anti-85-kDa PtdIns 3-kinase subunit antibody, we found that NGF promoted the tyrosine phosphorylation of an 85-kDa protein and two proteins close to 110 kDa. These studies demonstrate that NGF activates PtdIns 3-kinase and promotes its association with gp140trk and also show that NGF promotes the tyrosine phosphorylation of the 85-kDa subunit of PtdIns 3-kinase. Thus, PtdIns 3-kinase activation appears to be involved in differentiation as well as mitogenic responses.


Microvascular leakage induced by substance P in rat urinary bladder: involvement of cyclo-oxygenase metabolites of arachidonic acid. 1. Intravenous administration of substance P (SP) or of the NK1 selective agonist [beta-Ala4, Sar9, Met (O2)11] SP-(4-11) increased vascular permeability in the urinary bladder of urethane-anaesthetized rats, providing evidence for an NK1 receptor-mediated inflammatory response. 2. BW 755C, a dual inhibitor of arachidonate cyclo-oxygenase and lipoxygenase, significantly reduced the plasma extravasation induced by SP, but did not modify the effect of [beta-Ala4, Sar9, Met (O2)11] SP-(4-11). 3. SP-induced microvascular leakage was also inhibited by systemic pretreatment with indomethacin or with the prostaglandin receptor antagonist SC-19220, while it was unaffected by the selective 5-lipoxygenase inhibitor BW A4C or the leukotriene antagonist FPL 55712. 4. Pretreatment of rats with the mast cell degranulating agent compound 48/80 significantly attenuated the inflammatory effect of SP. Indomethacin administration to 48/80-pretreated animals failed to produce further inhibition. 5. These findings indicate that intravascular SP promotes plasma exudation in rat urinary bladder through an NK1-mediated effect on venular permeability and the release of cyclo-oxygenase metabolites of arachidonic acid. The latter effect largely derives from the interaction of the neuropeptide with mast cells.


Factors influencing survival of patients with heterotaxy syndrome undergoing the Fontan procedure. This study was undertaken to determine those factors that may influence survival in patients with heterotaxy syndrome undergoing the Fontan procedure. The Fontan procedure remains the preferred palliative procedure for patients with heterotaxy syndrome. Although the mortality rate has improved for patients without this syndrome undergoing the Fontan procedure, it remains high for patients with heterotaxy syndrome. The medical records of 20 consecutive pediatric patients with asplenia (n = 12) and polysplenia (n = 8) who underwent the Fontan procedure between January 1, 1986 and December 31, 1990 were reviewed. Anatomic and hemodynamic data were collected, as well as data on types of surgical palliative procedures and on outcome of the Fontan procedure. There were two early and two late deaths for a total mortality rate of 20% in the patients with heterotaxy syndrome, as compared with 8.5% for the patients without this syndrome who underwent the Fontan procedure during the same time period. Factors that significantly increased the risk of the Fontan procedure in these patients were 1) preoperative findings of greater than mild atrioventricular valve regurgitation, b) hypoplastic pulmonary arteries, and c) mean pulmonary artery pressure greater than or equal to 15 mm Hg after 6 months of age. Systemic and pulmonary venous anomalies coupled with single-ventricle anatomy were not significant risk factors for determining a poor outcome of the Fontan procedure. This study suggests that the outcome of the Fontan procedure in patients with heterotaxy syndrome may be improved by early protection of the pulmonary vascular bed, despite the existence of other cardiac anomalies.


Characterization of histamine-releasing activity: role of cytokines and IgE heterogeneity. Histamine-releasing factors (HRFs) are a group of cytokines that cause histamine release (HR) from basophils and mast cells. The concept of the priming effect of cytokines and the heterogeneity of IgE involved in the HRF-induced HR have been emphasized in recent years. In this study, we performed a series of experiments to elucidate the above-mentioned hypotheses. The stock HRF were obtained by stimulating mononuclear cells (MNC) with phytohemagglutinin (PHA). Maximal activity was observed 36 hr after culture. By gel filtration, HRF was eluted with a peak activity ranging from 12 to 18 KD. A large portion (75%) of HRF activity could be neutralized by a combination of antibodies against interleukin 1 (IL-1), IL-3, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha). The stimulation of basophils with 100 ng/ml each of IL-3, IL-6, IL-7, GM-CSF, or TNF-alpha alone caused 10% HR; however, when the cells were pretreated with 10 ng/ml of either IL-3, IL-6, IL-7, IL-8, TNF-alpha, or GM-CSF and then stimulated with anti-IgE, a marked increase in HR was regularly observed. The combination of 100 ng/ml each of IL-1, IL-3, IL-8, GM-CSF, and TNF-alpha could induce only about 20% HR; furthermore, such combinations did not have an additive or synergistic priming effect on anti-IgE-induced HR compared to the effect of single cytokines. Stripping of surface-bound IgE with lactic acid markedly reduced the capacity of basophils to release histamine in response to MNC-HRF and anti-IgE. Passive sensitization of IgE-stripped basophils with high-HRF responders' serum could restore their responsiveness to both MNC-HRF and anti-IgE, but passive sensitization with low-HRF responders' serum could restore responsiveness to anti-IgE only. Moreover, passage of MNC-HRF through high-, but not low-HRF, responders' IgE-Sepharose columns significantly reduced the HR activity of MNC-HRF. Finally, although the eluant could induce only 10% HR, the majority of its HR activity could be restored by the addition of effluent but not by the mixture of IL-1, IL-3, IL-8, GM-CSF, and TNF-alpha, suggesting the presence of a complex interaction among those cytokines. In summary, MNC-HRF contained at least two types of HRF activity; one was IgE dependent and the other was IgE independent.(ABSTRACT TRUNCATED AT 400 WORDS)


Tyrosine-specific protein phosphorylation in response to anti-CD3 antibody is diminished in old mice. Antiphosphotyrosine immunoblots were used to characterize phosphotyrosine-containing proteins (PY-PPNs) in anti-CD3 stimulated murine T lymphocytes. Activation led to increased phosphorylation of three PY-PPNs (MWs of 120, 80, and 40 kD) within 2 minutes, and maximal phosphoprotein levels were sustained for at least 30 min. There was a progressive decline with age in the responses of these three PY-PPNs to anti-CD3 stimulation, and some 20-23-month-old mice were virtually nonresponsive. A second group of PY-PPNs was present in unstimulated cells and not affected by anti-CD3 treatment or by age. Responses to Con A and to an antibody to the T-cell receptor were also found to be lower in old mice. Our data show that the pattern of tyrosine-specific phosphorylation in normal murine T cells is similar but not identical to patterns previously defined in murine tumor T-cell lines, and that T cells in old mice have defects in tyrosine-specific phosphorylation that could contribute to their diminished responsiveness to mitogenic stimuli.


Sera and leukocyte elastase-type protease and antiprotease activity in healthy and atherosclerotic subjects of various ages. Serum and granulocyte elastase-type protease activities were determined simultaneously with their main plasma proteinase inhibitors such as alpha-1-antitrypsin and alpha-2-macroglobulin in healthy control and atherosclerotic (ATS) subjects. The age-related associations of these parameters were also investigated. Serum elastase-type protease activity increased, but not statistically significantly, with aging in both control and ATS subjects. The enhancement of elastase-type protease activity in sera of ATS patients was significantly (p less than .02) greater than control subjects only in the case of the elderly. The granulocytes' elastase activity was significantly greater in granulocytes derived from both middle-aged and elderly ATS patients (p less than .03 and p less than .06) compared to age-matched control subjects. Alpha-1-antitrypsin was not significantly lower, whereas alpha-2-macroglobulin was significantly lower in sera of ATS subjects compared to age-matched control subjects (p less than .01). The conclusion is that increased elastase-type activity and decreased antiproteinase activity should be considered as potential factors in atherosclerotic arterial wall damage. The similarity of the results in the elderly and the ATS subjects suggest that atherosclerosis is an early aging process.


[The effects of glucocorticoids on angiogenesis in vitro]. Glucocorticoids administration presumably affects the vascular system and causes avascular necrosis of the femoral head. However, the mechanism by which this occurs is still unknown. In order to clarify the action of glucocorticoids on the vascular system, we investigated the effects of dexamethasone on an angiogenesis model in vitro. The angiogenesis model was obtained by co-culturing vessel fragments and myofibroblastic cells from rat epididymal fat pads. In this model, myofibroblastic cells induce capillary formation by producing an endothelial cell growth factor and collagen. Dexamethasone at physiological doses inhibited significantly capillary growth by suppressing the collagen synthesis by myofibroblastic cells. However, dexamethasone had no effect on endothelial cells. These results indicate that glucocorticoids are related to the pathogenesis of avascular necrosis of the femoral head by inhibiting the repair of the vascular system in vivo.


Thrombin cleavage of apolipoprotein Bh of rabbit LDL: structural comparisons with human apolipoprotein B-100. Rabbit plasma low density lipoprotein (LDL) contains one major apolipoprotein of apparent molecular weight of 320 kDa, designated apolipoprotein (apo) Bh, while another component termed apoB1 of apparent molecular weight of 220 kDa is found in chylomicrons. The fragments generated by thrombin digestion of the protein moieties of rabbit and human LDL were separated by polyacrylamide gradient gel electrophoresis and compared. As in the human species, the enzyme produced limited cleavage patterns of rabbit LDL apoB. Within the first 2 h, two fragments (Tr1 and Tr2, with apparent molecular weights 280,000 and 44,000, respectively) appeared. Longer incubations led to the production of two additional peptides, Tr3 and Tr4 (apparent molecular weights 180,000 and 96,000, respectively). Ten monoclonal antibodies, developed against rabbit LDL and designated P01 to P10, were found to react with rabbit apoB. Some also cross-reacted with human apoB. Epitope mapping, performed with these antibodies, showed that Tr3 and Tr4 were derived from the further degradation of Tr1. The rabbit is one of the most frequently used animals in atherosclerosis research. Its LDL receptor has been characterized and there exists a strain of homozygous LDL receptor-deficient rabbits referred to as WHHL rabbits. Despite this, little has been done to characterize the structure of rabbit apoB; only a short region has been sequenced and shown to be the carboxyl-terminal region, the rabbit apoB1. The molecular weight of human apoB (550,000) is much larger than rabbit apoBh. In both species, a primary and secondary thrombin cleavage occur, but the size of the fragments produced is very different between the two species. Identification of the thrombolytic fragments of the rabbit apoB have afforded the opportunity to compare the structures of both apoB species.


Disease inhibition by major histocompatibility complex binding peptide analogues of disease-associated epitopes: more than blocking alone. Peptide analogues of disease-associated epitopes were studied for inhibition of experimental allergic encephalomyelitis (EAE) and adjuvant arthritis (AA) in Lewis rats. EAE- and AA-associated analogues were selected as competitors because of their in vitro inhibitory activity on proliferation of encephalitogenic and arthritogenic T cells. Although the EAE-associated competitor had a superior major histocompatibility complex (MHC) binding affinity, the AA-associated competitor was a better inhibitor of the in vitro proliferation of arthritogenic T cells. Furthermore, although in vivo EAE was inhibited by both competitors, AA was only inhibited by the AA-associated competitor. Remarkably, in contrast to what was expected of a regular MHC competitor peptide, the AA-associated peptide analogue also prevented AA upon immunization before disease induction and appeared to induce T cell responses that crossreacted with the original disease-associated epitope. Therefore, it is concluded that antigen-specific regulatory mechanisms were involved in synergy with MHC competition. The integration of both qualities into a single "competitor-modulator" analogue peptide may lead to the development of novel, more effective, disease-specific immunomodulatory peptides.


Septin: a factor in plasma that opsonizes lipopolysaccharide-bearing particles for recognition by CD14 on phagocytes. We have previously reported that lipopolysaccharide (LPS) binding protein (LBP) opsonizes endotoxin (LPS) for recognition by CD14 on phagocytes. Here we show that normal human plasma contains high titers of an activity that also binds LPS (Re, 595) and mediates recognition by CD14. Opsonization of LPS-coated particles with plasma enables the particles to be bound by phagocytes. Further, opsonization with plasma also enables subnanogram-per-milliliter concentrations of LPS to induce dramatic alterations in the function of leukocyte integrins on polymorphonuclear leukocytes and to induce secretion of tumor necrosis factor by monocytes, suggesting that opsonization by factors in plasma may be important in responses of cells to endotoxin. The opsonic activity in plasma appears distinct from LBP since it is not blocked by neutralizing antibodies against LBP. Surprisingly, the opsonic activity of plasma is not present in a single protein species, but at least two species must be combined to observe activity. Further, the opsonic activity of plasma for LPS is blocked by addition of protease inhibitors, suggesting that proteolytic activity or activities are required for opsonization. These properties are suggestive of the action of a protease cascade, but opsonic activity of plasma is not affected by blockade or depletion of either the complement or clotting cascades. We propose the name "septin" to describe this novel LPS-opsonizing activity in plasma.


The immunosuppressive and toxic effects of FK-506 are mechanistically related: pharmacology of a novel antagonist of FK-506 and rapamycin. FK-506 inhibits Ca(2+)-dependent transcription of lymphokine genes in T cells, and thereby acts as a powerful immunosuppressant. However, its potential therapeutic applications may be seriously limited by several side effects, including nephrotoxicity and neurotoxicity. At present, it is unclear whether these immunosuppressive and toxic effects result from interference with related biochemical processes. FK-506 is known to interact with FK-binding protein-12 (FKBP-12), an abundant cytosolic protein with cis-trans peptidyl-prolyl isomerase activity (PPIase) activity. Because rapamycin (RAP) similarly binds to FKBP-12, although it acts in a manner different from FK-506, by inhibiting T cell responses to lymphokines, such an interaction with FKBP-12 is not sufficient to mediate immunosuppression. Recently, it was found that the complex of FKBP-12 with FK-506, but not with RAP, inhibits the phosphatase activity of calcineurin. Here, we used L-685,818, the C18-hydroxy, C21-ethyl derivative of FK-506, to explore further the role of FKBP-12 in the immunosuppressive and toxic actions of FK-506. Although L-685,818 bound with high affinity to FKBP-12 and inhibited its PPIase activity, it did not suppress T cell activation, and, when complexed with FKBP-12, did not affect calcineurin phosphatase activity. However, L-685,818 was a potent antagonist of the immunosuppressive activity of both FK-506 and RAP. Moreover, L-685,818 did not induce any toxicity in dogs and rats or in a mouse model of acute FK-506 nephrotoxicity, but it blocked the effect of FK-506 in this model. Therefore, FK-506 toxicity involves the disruption of biochemical mechanisms related to those implicated in T cell activation. Like immunosuppression, this toxicity is not due to the inhibition of the PPIase activity of FKBP-12, but may be linked to the inhibition of the phosphatase activity of calcineurin by the drug FKBP-12 complex.


Activation of nonselective cation channels by physiological cholecystokinin concentrations in mouse pancreatic acinar cells. The activation of the nonselective cation channels in mouse pancreatic acinar cells has been assessed at low agonist concentrations using patch-clamp whole cell, cell-attached patch, and isolated inside-out patch recordings. Application of acetylcholine (ACh) (25-1,000 nM) and cholecystokinin (CCK) (2-10 pM) evoked oscillatory responses in both cation and chloride currents measured in whole cell experiments. In cell-attached patch experiments we demonstrate CCK and ACh evoked opening of single 25-pS cation channels in the basolateral membrane. Therefore, at least a component of the whole cell cation current is due to activation of cation channels in the basolateral acinar cell membrane. To further investigate the reported sensitivity of the cation channel to intracellular ATP and calcium we used excised inside-out patches. Micromolar Ca2+ concentrations were required for significant channel activation. Application of ATP and ADP to the intracellular surface of the patch blocked channel opening at concentrations between 0.2 and 4 mM. The nonmetabolizable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP, 0.2-2 mM), also effectively blocked channel opening. The subsequent removal of ATP caused a transient increase in channel activity not seen with the removal of ADP or AMP-PNP. Patches isolated into solutions containing 2 mM ATP showed channel activation at micromolar Ca2+ concentrations. Our results show that ATP has two separate effects. The continuous presence of the nucleotide is required for operation of the cation channels and this action seems to depend on ATP hydrolysis. ATP can also close the channel and this effect can be demonstrated in excised inside-out patches when ATP is added to the bath after a period of exposure to an ATP-free solution. This action does not require ATP hydrolysis. Under physiological conditions hormonal stimulation can open the nonselective cation channels and this can be explained by the rise in the intracellular free Ca2+ concentration.


The CD45 tyrosine phosphatase regulates phosphotyrosine homeostasis and its loss reveals a novel pattern of late T cell receptor-induced Ca2+ oscillations. CD45 is a transmembrane tyrosine phosphatase implicated in T cell antigen receptor (TCR)-mediated activation. In T cell variants expressing progressively lower levels of CD45 (from normal to undetectable), CD45 expression was inversely related to spontaneous tyrosine phosphorylation of multiple proteins, including the TCR zeta chain, and was directly correlated with TCR-driven phosphoinositide hydrolysis. The Ca2+ response in these cells was altered in an unexpected fashion. Unlike wild-type cells, stimulated CD45- cell populations did not manifest an early increase in intracellular Ca2+, but did exhibit a delayed and gradual increase in mean intracellular Ca2+. Computer-aided fluorescence imaging of individual cells revealed that CD45- cells experienced late Ca2+ oscillations that were not blocked by removal of extracellular Ca2+. CD45 revertants had the signaling properties of wild-type cells. Thus, CD45 has a profound influence on both TCR-mediated signaling and phosphotyrosine homeostasis, and its loss reveals a novel role for this tyrosine phosphatase in Ca2+ regulation.


Demonstration of peptidoglycan-associated Brucella outer-membrane proteins by use of monoclonal antibodies. A monoclonal antibody (3D6) was produced which reacted only with Brucella sonicated cell extracts that had been lysozyme-treated after sonication. The monoclonal antibody (mAb) reacted with the three major outer-membrane proteins (OMPs) of B. melitensis B115 in Western blots. A large number of reactive bands ranging from 12 to 43 kDa were present in lysozyme-treated Escherichia coli and Yersinia enterocolitica sonicated cell extracts. In a latex agglutination inhibition immunoassay, mAb 3D6 showed better reactivity with purified peptidoglycan (PG) of B. melitensis B115 than with that of Escherichia coli. This mAb was also used in immunogold electron microscopy with whole Brucella cells and sections. No binding was observed on whole cells and immunogold labelling in sections was observed close to the outer membrane, in the periplasmic space and in the cytoplasm. These findings indicate that mAb 3D6 is specific for PG subunits. Immunoblot analysis of B. melitensis B115 rough sonicated cell extracts after SDS-PAGE, with or without lysozyme treatment, was performed using mAbs specific for Brucella OMPs of molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa, for PG and for rough lipopolysaccharide (R-LPS) and smooth lipopolysaccharide (S-LPS). mAbs specific for the 25-27, 31-34 and 36-38 kDa OMPs reacted with three to six bands. All of them except the band of lowest molecular mass reacted with the PG-specific mAb and not with R-LPS- and S-LPS-specific mAbs.(ABSTRACT TRUNCATED AT 250 WORDS)


Pharmacological models of generalized absence seizures in rodents. A number of animal models of generalized absence seizures in rodents are described. These include absence seizures induced by gamma-hydroxybutyrate (GHB), low dose pentylenetetrazole, penicillin, THIP, and AY-9944. All of these models share behavioral and EEG similarity to human absence seizures and show pharmacologic specificity for antiabsence drugs such as ethosuximide and trimethadione. Moreover, the absence seizures induced by these agents are exacerbated by GABAergic agonists, a property unique to experimental absence seizures. These models are predictable, reproducible, and easy to standardize. They are useful both in studying mechanisms of pathogenesis of absence seizures as well as in screening for antiabsence activity of potential antiepileptic drugs.


Argentophilic intracytoplasmic inclusions in multiple system atrophy. Argentophilic intracytoplasmic glial inclusions were recently reported in olivo-ponto-cerebellar atrophy (OPCA). We examined the brains of 3 cases of OPCA [2 with striato-nigral degeneration (SND) and 1 without SND], 1 case of pure autonomic failure (PAF) without pathology of OPCA or SND, as well as 36 controls including 2 cases of Holmes' type cerebellar cortical atrophy and 2 cases of Joseph's disease. Although the inclusions were tubulin-positive, the immunoreactivity was different from that of the dendrites. Electron microscopically, the microtubular structures composing the inclusion were fuzzy with granular material. These findings may indicate that the microtubules composing the inclusions are modified. Inclusion-bearing cells appeared to be oligodendrocytes while many of them had larger and lighter nuclei than those of normal-looking oligodendrocytes without the inclusions. The inclusions were widely distributed in a characteristic fashion beyond the typical lesions of OPCA, SND and PAF. The distribution pattern was essentially the same in the case of PAF and 3 cases of OPCA irrespective of the presence or absence of OPCA or SND lesions. In contrast, argentophilic inclusions were not observed in other types of spinocerebellar degeneration, in Holmes' type cerebellar cortical atrophy or in Joseph's disease. It is suggested, in line with other studies, that the inclusion may be specific to OPCA and related disorders which include PAF and a useful marker to distinguish OPCA from other neurodegenerative diseases.


Growth charts only marginally improved maternal learning from nutrition education and growth monitoring in Lesotho. A study done in Lesotho in 1985-1986 assessed whether growth charts increased the impact of nutrition education and growth monitoring on maternal learning about weaning practices and diarrhea. Seven hundred and seventy six mothers were given three monthly sessions of group nutrition education along with growth monitoring of children and individual counseling. Growth charts, which were taught to one of two groups, fostered learning but only on issues related to diarrhea and only among new clinic attendants, mothers with less than secondary schooling and mothers of malnourished children. These benefits, however, were small (differences less than 10%) compared with the overall impact of the nutrition education and growth monitoring intervention (increases between baseline and post-intervention were greater than 50% for some questions). Our findings suggest that well-designed clinic-based nutrition education and growth monitoring can have a significant impact on maternal nutrition knowledge. Teaching growth charts to mothers may not be necessary for obtaining such results in programs conducted under ideal conditions. More research is needed to determine under what circumstances, for what purposes and for whom growth charts may be beneficial.


Carbohydrate intake determines pancreatic acinar amylase activity and release despite chronic alcoholemia in rats. Adverse effects observed in alcoholic rats are often attributed to alcohol per se. Alcoholic liver damage, however, can be avoided by modulating nutritional factors despite high blood alcohol concentrations. Hence, we examined the effect of blood alcohol concentration on pancreatic enzyme activity and release. Three liquid diets containing 36 and 18% of total energy derived from alcohol and protein, respectively, were fed. Each alcohol diet contained 11, 21 or 31% of energy from carbohydrate, and the fat concentration was appropriately adjusted. The control groups of rats (fed an isoenergetic liquid diet without alcohol) and the alcoholic groups of rats were maintained for 2 wk. The three groups of alcoholic rats consumed 13.3 +/- 2.3, 13.3 +/- 2.2 and 13.2 +/- 1.9 g/kg of alcohol daily, and their corresponding blood alcohol levels were 41.5 +/- 4.3, 55.4 +/- 8.9 and 44.6 +/- 2.2 mmol/L. Pancreatic acinar amylase activity in alcoholic rats was proportional to carbohydrate ingested, despite high blood alcohol concentrations; chymotrypsin and trypsin activities were unchanged. Acinar enzyme activities in control rats were similar. Furthermore, cholecystokinin-octapeptide-stimulated amylase release in alcoholic rats corresponded with the amylase concentration in acini, whereas stimulated trypsin output was unaltered in both control and alcoholic rats. These results demonstrate that neither alcohol ingestion nor high blood alcohol concentration affects the activities of pancreatic proteases and that the changes in the activity and release of amylase are related to the intake of carbohydrate.


[Studies on the function of mast cells infiltrating in nasal polyps]. The function of nasal polyp mast cells has not been elucidated despite the large number of these cells observed in tissues. We examined these mast cells histochemically, immunohistologically and functionally. Ninety-three percent of collagenase dispersed cells in a nasal polyp were formalin-sensitive. These dispersed cells released histamine in reaction to calcium ionophore A23187 in a dose dependent manner, but not in response to C5a, Compound 48/80 or Substance P. From these results, dispersed mast cells from nasal polyps were considered to be analogous to dispersed mast cells from the human lung and nasal mucosa but not those from human skin. On the other hand, in the reaction with anti-human IgE, dispersed mast cells from a non allergic nasal polyp could not be seen to release histamine. In only 2 of 7 patients, could histamine release in response to Japanese red cedar antigen, from mast cells sensitized passively with the serum of Japanese red cedar pollinosis, seen. Using small tissue samples from polyps, histamine was released by anti-human IgE in allergic patients but not in non allergic patients. Immunohistologically in allergic nasal polyps, some IgE positive mast cells could be seen, whereas in non allergic polyps these cells were absent. These observations suggest that mast cells which had accumulated in nasal polyps both with and without allergy were capable of functional histamine release, whereas in the nasal polyps of allergy patients but not in non-allergic patients these cells are involved in IgE mediated reactions.


[Inhibitory activity of mite IgG4, antibody on antigen-induced histamine release from human peripheral blood leukocytes]. Several reports have yielded conflicting results on the role of IgG4 antibody on the surfaces of target cells in immediate type allergy. This study was performed to elucidate whether IgG4 antibody inhibits IgE-mediated histamine release from target cells after antigenic stimulation, and whether it has reaginic activity. Serum was obtained from patients with nasal allergy receiving specific immunotherapy for housedust and mites. IgE and IgG4 were enriched affinity chromatographically using monoclonal antibodies to IgE and IgG4, respectively, from the pooled sera. Both fraction revealed high antibody activity to Dermatophagoides Farinae antigen. Peripheral blood leukocytes from three non-allergic donors were passively sensitized with 100 or 300 micrograms of IgG4 according to the method of Levy and Osler with a slight modification. Minimal or no histamine release was observed from leukocytes after challenge with both mite antigen and anti-IgG4 monoclonal antibodies. Furthermore, to investigate the reaginic activity of IgG4, leukocytes from patients with nasal allergy were stimulated with anti-IgG4 antibodies. The leukocytes of only three out of twenty patients released up to 10% histamine regardless of the IgG4 concentration, while the other patients' leukocytes released minimal amounts of histamine. Two of the three above-mentioned non-allergic donors were passively sensitized with 100 or 300 micrograms of IgG4 either one hour after sensitization with 100 ngs of IgE or simultaneously with the same amount of IgE. After sensitization with 100 ngs of IgE, one showed high-grade histamine release after challenge with 0.5 micrograms/ml mite antigen and the other showed middle-grade release with 0.1 micrograms/ml mite. With the presence of 300 micrograms of IgG4, histamine release was significantly inhibited in both donors regardless of the manner of sensitization.(ABSTRACT TRUNCATED AT 250 WORDS)


Activation and differentiation of myelomonocytic cells in rheumatoid arthritis and healthy individuals--evidence for antagonistic in vitro regulation by interferon-gamma and tumor necrosis factor alpha, granulocyte monocyte colony stimulating factor and interleukin 1. We analyzed expression of HLA-DR and CD14 molecules on myelomonocytic cells and its regulation by various inflammatory cytokines in 6 patients with rheumatoid arthritis (RA) and 4 healthy individuals who had undergone bone marrow aspiration. At start of the bone marrow culture there was a significantly higher number of HLA-DR and CD14 positive bone marrow mononuclear cells in patients with RA than in normals. In addition, RA bone marrow mononuclear cells expressed an up to 10-fold higher mean density of both molecules than normal bone marrow mononuclear cells during the whole culture period of up to 14 days. The effect of the cytokines interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF alpha), granulocyte monocyte colony stimulating factor (GM-CSF) and interleukin 1 (IL-1) on the expression of CD14 or HLA-DR was different: IFN-gamma strongly upregulated HLA-DR expression and down-regulated CD14 expression while TNF alpha, GM-CSF and IL-1 mainly stimulated CD14 expression on bone marrow mononuclear cells. Our data suggest that RA bone marrow mononuclear cells exhibit an activated phenotype and that TNF-alpha GM-CSF and IL-1 mainly stimulate the differentiation of bone marrow macrophages whereas IFN-gamma activates them.


Analysis of lymphocyte phenotype and T cell receptor genotype in Felty's syndrome. Natural killer (NK) cells and CD3+ large granular lymphocytes (LGL) were investigated in patients with Felty's syndrome (FS), rheumatoid arthritis (RA) and healthy controls. In most patients with FS, NK cell number and activity were decreased. CD3+ LGL were unchanged. However, in one patient a marked expansion of CD3- CD16+ CD56+ (NK) cells was seen and in a second, an expansion of CD3+ LGL. In FS there was also an increase in HLA- DR+ and CD8+ but not gamma delta+ T cells. Three of 11 patients with FS studied demonstrated a dominant rearrangement of the T cell receptor beta gene constant region consistent with oligoclonal T cell expansion.


Treatment of recurrent gliomas with eflornithine. Oral eflornithine in combination with intravenous mitoguazone (methylbisguanylhydrazone) has shown activity against recurrent anaplastic gliomas. Eflornithine alone, however, has not been evaluated against recurrent gliomas. This study compared the antitumor activity of oral eflornithine with that of oral eflornithine combined with intravenous mitoguazone in the treatment of patients with recurrent or progressive glioblastoma multiforme as well as nonglioblastoma anaplastic gliomas. During the 1st year of therapy with eflornithine alone, the drug was given at a dose of 3.6 g/m2 on days 1-14, 22-35, and 43-56 every 8 hours; cycles were repeated every 63 days until progression. For the 2nd year, the drug was given on days 1-14, 29-42, and 57-70, with 84 days between cycles. For the 1st and 2nd years of eflornithine-mitoguazone therapy, eflornithine was given at 1.8 g/m2 on the same schedule. Mitoguazone was given intravenously at 200 mg/m2 on the final day of each 2-week sequence of eflornithine therapy. Response was determined by evaluating changes in the size of contrast-enhanced neuroimages. Because of two cases of lethal hepatic necrosis, the initial random allocation of patients to the eflornithine-mitoguazone arm was stopped after 23 patients had been accrued. Ninety-eight patients were entered in the eflornithine arm; 80 patients (36 glioblastoma multiforme patients and 44 anaplastic glioma patients) were assessable for response. Antitumor activity (partial response, minor response, and stable disease) was seen in 45% of the patients with anaplastic gliomas, for a median of 49 weeks, but in only 17% of patients with glioblastoma multiforme (median not attained). Twenty-one (20%) of the patients with anaplastic glioma and 33% of the patients with glioblastoma multiforme were removed from the study before completing the first 8-week course of therapy because of neurological deterioration and tumor progression by the 5th week of treatment. This study suggests that eflornithine alone is an effective palliative therapy for recurrent anaplastic gliomas. Additional studies are needed to confirm our finding.


Influence of testicular carcinoma on ipsilateral spermatogenesis. A histological review of radical orchiectomy specimens was performed to assess the impact of testicular cancer on spermatogenesis. Slides from 28 patients with testicular cancer were available for review, consisting of 14 pure seminomas, 12 embryonal carcinomas and 2 mixed tumors. For each specimen tubules adjacent (less than 3 mm.) to the tumor and distant (more than 3 mm.) from the tumor were evaluated. This study indicates that marked impairment of ipsilateral spermatogenesis is associated with testicular carcinoma, particularly in the vicinity of the tumor. The quality of distant spermatogenesis appears to be influenced by tumor type and not by elevation of known serum tumor markers, such as human chorionic gonadotropin and alpha-fetoprotein, nor by the presence of carcinoma in situ.


Endodermal sinus tumor of vulva (a case report). An unusual case of endodermal sinus tumor (EST) of the ovary at an extragonadal site-vulva, in an unmarried female of 25 yr is reported. The patient presented only with a vulval swelling on the right side without any other signs or symptoms. The internal genital organs mainly the ovaries were normal.


Detection of clinically significant prostate cancer by transrectal ultrasound-guided systematic biopsies. Systematic biopsies are a useful, sensitive means to detect carcinoma of the prostate. However, multiple biopsies pose a risk for detecting clinically insignificant prostate cancer, that is those cancers less than 0.5 cc in volume, which occur in approximately 32% of all white men more than 50 years old. Systematic biopsies were positive for cancer in 442 of 816 patients and 60 (14%) demonstrated only a minute focus of cancer (3 mm. or less) in 1 of the 6 biopsy specimens. In 27 patients with these minute foci who underwent radical prostatectomy a wide range of cancer volumes was observed; 30% of these 27 cancers were less than 0.5 cc (15% less than 0.2 cc) and may not have required therapy. Thus, the overall risk of detecting an insignificant cancer is 4.0% with systematic biopsies. Performance of confirmatory biopsies in patients with a minute focus (3 mm. or less) of cancer on initial systematic biopsies resulted in cancers less than 0.5 cc being removed in only 1 of 10 radical prostatectomies (10%, none was less than 0.2 cc). Thus, with the addition of confirmatory biopsies the risk of detecting insignificant cancer is 1.4%. Conservative management is recommended for patients without significant cancer on repeat biopsies in whom initial biopsies have revealed only a minute focus of cancer in 1 of the biopsy cores. We believe that concern is also warranted for patients who have 3 mm. or less of cancer demonstrated by several nonsystematic biopsies directed at a suspicious hypoechoic lesion in whom the digital rectal examination is normal.


Quantitative estimation of tissue prostate specific antigen, deoxyribonucleic acid ploidy and cytological grade in fine needle aspiration biopsies for prognosis of hormonally treated prostatic carcinoma. The prognostic value of deoxyribonucleic acid (DNA) flow cytometry, cytological grading and the direct assay of prostate specific antigen (PSA) in the material of fine needle aspirates was studied in 67 consecutive patients with newly detected prostatic carcinoma. All patients were hormonally treated (castration in 27 and luteinizing hormone-releasing hormone agonist or parenteral estrogens in 40). The patients were followed for a minimum of 2 years. PSA was analyzed in the biopsy material by a direct radioimmunoassay and related to the total amount of DNA. In parallel biopsies DNA ploidy using flow cytometry and cytological grade were established. Patients with a geometric mean value of greater than or equal to 0.12 microgram. PSA/microgram. DNA had a progression rate of 7%, compared to 59% for those with less than 0.12 microgram. PSA/microgram. DNA. In Cox multivariate analysis cytology and tissue PSA content were the most important factors in expressing the difference for interval to progression in hormonally treated patients.


Comparative response of nestling European starlings and red-winged blackbirds to an oral administration of either dimethoate or chlorpyrifos. Red-winged blackbird (Agelaius phoeniceus; blackbird) and European starling (Sturnus vulgaris; starling) nestlings were dosed with either 2.0 mg/kg body mass chlorpyrifos, 50.0 mg/kg body mass dimethoate, or a propylene glycol carrier in situ. Four growth measurements (body mass, culmen, tarsus, wing) were recorded from nestlings to determine if these organophosphorus compounds caused perturbations in development at sublethal concentrations. Blackbird nestlings were more sensitive to chlorpyrifos than starling nestlings were more sensitive to dimethoate than blackbird nestlings. This was in contrast to reported adult LD50 values where the reverse was true. Blackbird nestlings were more tolerant of a substantially higher concentration of dimethoate than the adult LD50. The sensitivity of starling nestlings to dimethoate was similar to adults. In contrast, juveniles of both species were more sensitive to chlorpyrifos than adults. After the initial 24 hr, surviving nestlings dosed with either chemical recovered and continued their development. Exposure to dimethoate caused significant depression in starling body mass during the initial 24 hr period. Survivors obtain body mass equal to controls within 48 hr post dosing. The research presented here demonstrates that the simple supposition that passerine nestlings are typically more sensitive to toxins than adults does not always hold true. It also indicates that sensitivity relationships among adults do not necessarily apply to their nestlings.


Keratitis in free-ranging koalas (Phascolarctos cinereus) on Magnetic Island, Townsville. Seventy free-ranging koalas (Phascolarctos cinereus) from Magnetic Island (Queensland, Australia) underwent an ocular examination, blood collection and serological examination for Chlamydia psittaci antibodies, and an examination of their teeth and genitalia. In 12 koalas long-standing unilateral keratitis was noted and in another 10 animals long-standing bilateral keratitis was observed. All animals were seronegative for Chlamydia psittaci and apart from some nasal discharge and a variety of assorted medical findings there was no sign of chlamydial infection. These ocular findings probably represent a new disease of unknown etiology.