Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity. Bacillus thuringiensis produces a variety of delta-endotoxins which bind to specific receptors in insect larval midguts. Following insertion into the membrane there is an alteration of ion flux culminating in osmotic lysis. Mutagenic oligonucleotides were used to define regions in one of these toxins involved in specificity and toxicity. One region is highly conserved among all toxins sequenced to date and many mutations resulted in loss of toxicity for three test Lepidoptera. The mutant toxins had lost the capacity to inhibit K(+)-dependent amino acid transport into larval midgut vesicles, but there was no effect on their ability to compete with wild type toxin for binding. The results are consistent with this amphiphilic helical region of the toxin being essential for toxicity. A second mutagenized region overlapped a portion of another potential amphiphilic helix. Mutations of only 2 residues, Ala-92 and Arg-93, resulted in loss of toxicity for two lepidopteran larvae but some activity remained for a third. The A92D mutant toxin competed with the wild type toxin for binding to vesicles prepared from midguts from the sensitive but not from the insensitive larvae. Decreased toxicity was also found when this mutation was transferred to two other related protoxin genes. A number of mutations of each of these residues was analyzed and selective loss of toxicity correlated with the absence of a positive charge. Despite being distal from the presumptive specificity domain, 1 or both of these residues must have an important role in the specific binding of toxins.


Dual mode of inhibition of purified DNA ligase I from human cells by 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate. 9-beta-D-Arabinofuranosyl-2-fluoroadenine (F-ara-A) is an analogue of adenosine and deoxyadenosine with potent anti-tumor activity. The mechanism of action for this compound has been elucidated as the inhibition of DNA and RNA synthesis, induction of DNA fragmentation, and genetic damage. This study demonstrated that DNA ligase I, an enzyme involved in DNA replication, is a target for the drug action. F-ara-adenine triphosphate (F-ara-ATP) at 80 microM inhibited the activity of DNA ligase I by more than 90%. In contrast, eight other related nucleoside analogues showed no effect on the enzyme activity at 200 microM. F-ara-ATP inhibited DNA ligation in two distinct ways. First, F-ara-ATP directly interacted with DNA ligase I and inhibited the formation of the ligase-AMP complex. This inhibition could not be reversed when free F-ara-ATP was eliminated from the treated enzyme; however, the addition of pyrophosphate, followed by gel filtration chromatography, restored enzyme activity, indicating that F-ara-ATP bound to the enzyme and altered the AMP-binding site. Secondly, the activity of DNA ligase I was inhibited when F-ara-ATP was incorporated into the 3' terminus of the DNA substrate. The dual mode of inhibition of DNA ligase I by F-ara-ATP indicates that its effect on DNA ligation may be important in the inhibition of DNA synthesis and the cytotoxicity of F-ara-A.


Role of G protein beta gamma subunits in the regulation of the plasma membrane Ca2+ pump. In Zajdela hepatoma cells (ZHC) the plasma membrane Ca2+ pump displayed no sensitivity to glucagon (19-29) (mini-glucagon), whereas in hepatocyte this metabolite of glucagon evoked a biphasic regulation of the Ca2+ pump system via a cholera toxin-sensitive G protein. Analysis of G protein subunits in ZHC membranes indicated the presence of cholera toxin-sensitive Gs alpha and G beta gamma proteins, whose functionality was manifested by GTP and NaF stimulation of adenylylcyclase activity, and pertussis toxin-catalyzed ADP-ribosylation of Gi alpha, respectively. However, immunoblotting experiments suggested a lower content in beta gamma subunits in ZHC as compared with hepatocyte plasma membranes. Complementation of ZHC or hepatocyte plasma membranes with purified beta gamma subunits from transducin (T beta gamma) caused inhibition of the basal activity of the Ca2+ pump at 10 and 300 ng/ml, respectively, and revealed (in ZHC) or increased (in hepatocytes) sensitivity of the system to mini-glucagon. After cholera toxin treatment of ZHC, T beta gamma no longer reconstituted the response of the Ca2+ pump to mini-glucagon, suggesting that the mechanism of beta gamma action is dependent on an association with the alpha subunit of a cholera toxin-sensitive G protein. It is concluded that G beta gamma subunits control both the basal activity of the plasma membrane Ca2+ pump and its inhibition by mini-glucagon.


Purification and properties of a secreted and developmentally regulated alpha-L-fucosidase from Dictyostelium discoideum. During its development the eukaryotic microorganisms Dictyostelium discoideum secretes an alpha-L-fucosidase (EC 3.2.1.51). In cells of the growth phase almost no alpha-L-fucosidase activity is detectable. The activity increases steadily up to the aggregation stage and accumulates also in the extracellular medium. The developmental regulation is mediated by pulsatile cAMP signals. The alpha-L-fucosidase was purified from extracellular medium. The isolation procedure started with concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulfate precipitation, followed by Sephacryl S-300 gel filtration and further purification by fast protein liquid chromatography on Mono Q, phenyl-Superose, and finally Superose 12. The purified preparation was found to be essentially free of activities of six other glycosidases also secreted by D. discoideum. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed one major band with an apparent molecular mass of 62 kilodalton. Gel filtration of the enzyme on a Superose 12 column was consistent with an active monomer. A monoclonal antibody was produced, which recognizes a carbohydrate epitope shared by all lysosomal enzymes in D. discoideum. The pH optimum of the alpha-L-fucosidase is at 3.7. The apparent Michaelis constant for p-nitrophenyl alpha-L-fucoside as substrate is 1.2 mM. The enzyme catalyzes preferentially the hydrolysis of alpha 1----6GlcNAc but also of alpha 1----2Gal and alpha 1----3Glc fucosyl linkages.


The variant human isovaleryl-CoA dehydrogenase gene responsible for type II isovaleric acidemia determines an RNA splicing error, leading to the deletion of the entire second coding exon and the production of a truncated precursor protein that interacts poorly with mitochondrial import receptors. Isovaleryl-CoA dehydrogenase (IVD) is a mitochondrial enzyme involved in leucine metabolism. Previous studies of fibroblasts from patients with isovaleric acidemia (IVA), an inherited defect in IVD, have revealed that IVD precursor protein produced by type II IVA cells is 3 kDa smaller than normal and is processed inefficiently to a mature form which is also 3 kDa smaller than normal. Using the polymerase chain reaction, we have identified a 90-base pair deletion encompassing bases 145-234 in type II IVD cDNA. This deletion is caused by an error in RNA splicing and predicts the in-frame deletion of 30 amino acids beginning with leucine 20 of the mature IVD. The rate of leader peptide cleavage by purified mitochondrial leader peptidases was similar for the variant and normal precursor IVDs expressed in vitro, and radiosequencing confirmed that both mature proteins contain identical amino termini. In vitro import studies showed that the efficiency of overall mitochondrial import of type II variant IVD precursor was approximately 30% of normal, as was its binding to the mitochondrial surface. Unlike its normal counterpart, the bound variant IVD precursor was readily released. These data suggest that binding of the variant protein to mitochondrial membrane receptors per se is hindered, resulting in the inefficient mitochondrial processing.


Adenovirus E1A represses the cyclic AMP-induced transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) in hepatoma cells. Adenovirus infection of hepatoma cells inhibited transcription of the phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) gene and virtually eliminated transcription of a chimeric gene which contained the PEPCK promoter linked to the structural gene for chloramphenicol acetyltransferase (CAT). This effect is due to the viral protein E1A, since adenovirus containing a deletion in the E1A gene did not repress transcription from the PEPCK promoter. Both the 243R and 283R products of the E1A gene were effective. The conserved region 1 (CR-1) domain of E1A was required for this effect. Treatment of hepatoma cells with 8-bromo-cAMP or transfection with plasmids coding for the catalytic subunit of protein kinase A, CAAT/enhancer binding protein alpha (C/EBP), or Jun, all potent inducers of PEPCK gene transcription, did not relieve the inhibition caused by E1A. This inhibition does not appear to be mediated by major enhancer elements and in the PEPCK gene since transcription from the PEPCK promoter containing block mutations in binding domains for C/EBP and cAMP regulatory element binding protein (CREB) was also inhibited by E1A. Transcription of chimeric genes containing two copies each of the major cAMP response domains (CRE-1 and P-3) linked to a neutral promoter and fused to the CAT structural gene was stimulated by the catalytic subunit of protein kinase A, but this effect was totally inhibited by E1A. The strong repressive effect of E1A on PEPCK gene transcription seems to involve an interruption of an obligatory interaction between factors which bind to the cAMP response element in the PEPCK promoter and the TATA box.


Antagonism of synaptosomal calcium channels by subtypes of omega-agatoxins. Venom of the funnel web spider Agelenopsis aperta inhibits the binding of 125I-omega-conotoxin GVIA (omega-CgTx) to calcium channels in chick brain synaptosomal membranes. Fractionation of the venom by liquid chromatography shows that this inhibitory activity is associated primarily with a diverse class of peptide toxins called omega-agatoxins (omega-Aga). Using binding inhibition as an assay, we purified and identified the novel, 76-amino acid toxin, omega-Aga-IIIA. Inhibition of 125I-omega-CgTx binding to chick synaptosomal membranes by omega-Aga-IIIA and omega-Aga-IIA is correlated with block of potassium-stimulated 45Ca entry into synaptosomes; omega-Aga-IA neither inhibits 125I-omega-CgTx binding nor 45Ca entry under identical conditions. omega-Aga-IIA and omega-Aga-IIIA are 20-30-fold more potent than omega-CgTx as antagonists of synaptosomal calcium channels. However, whereas omega-CgTx completely blocks 45Ca entry into synaptosomes at saturating concentrations, the omega-agatoxins maximally block only 60-70% of 45Ca entry. Pretreatment of synaptosomes with omega-Aga-IIIA occludes block of 45Ca entry by omega-CgTx. The results indicate that, while the omega-agatoxins bind to the entire population of omega-CgTx-sensitive calcium channels in chick synaptosomal membranes, they exert only a partial block of 45Ca flux. Such block could occur via two distinct mechanisms. Toxin binding may alter the kinetics of a homogeneous population of channels, resulting in lower overall conductance upon depolarization. Alternatively, the omega-agatoxins may bind to two distinct channel subtypes, only one of which is blocked as a result of toxin occupation.


A malaria invasion receptor, the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum recognizes the terminal Neu5Ac(alpha 2-3)Gal- sequences of glycophorin A. Plasmodium falciparum malaria parasites invade human erythrocytes by means of a parasite receptor for erythrocytes, the 175-kD erythrocyte binding antigen (EBA-175). Similar to invasion efficiency, binding requires N-acetylneuraminic acid (Neu5Ac) on human erythrocytes, specifically the glycophorins. EBA-175 bound to erythrocytes with receptor-like specificity and was saturable. The specificity of EBA-175 binding was studied to determine if its binding is influenced either by simple electrostatic interaction with the negatively charged Neu5Ac (on the erythrocyte surface); or if Neu5Ac indirectly affected the conformation of an unknown ligand, or if Neu5Ac itself in specific linkage and carbohydrate composition was the primary ligand for EBA-175 as demonstrated for hemagglutinins of influenza viruses. Most Neu5Ac on human erythrocytes is linked to galactose by alpha 2-3 and alpha 2-6 linkages on glycophorin A. Soluble Neu5Ac by itself in solution did not competitively inhibit the binding of EBA-175 to erythrocytes, suggesting that linkage to an underlying sugar is required for binding in contrast to charge alone. Binding was competitively inhibited only by Neu5Ac(alpha 2-3)Gal-containing oligosaccharides. Similar oligosaccharides containing Neu5Ac(alpha 2-6)Gal-linkages had only slight inhibitory effects. Binding inhibition assays with modified sialic acids and other saccharides confirmed that oligosaccharide composition and linkage were primary factors for efficient binding. EBA-175 bound tightly enough to glycophorin A that the complex could be precipitated with an anti-glycophorin A monoclonal antibody. Selective cleavage of O-linked tetrasaccharides clustered at the NH2 terminus of glycophorin A markedly reduced binding in inhibition studies. We conclude that the Neu5Ac(a2,3)-Gal- determinant on O-linked tetrasaccharides of glycophorin A appear to be the preferential erythrocyte ligand for EBA-175.


Targeted disruption of the ABP-120 gene leads to cells with altered motility. The actin-binding protein ABP-120 has been proposed to play a role in cross-linking F-actin filaments during pseudopod formation in motile Dictyostelium amebas. We have tested this hypothesis by analyzing the phenotype of mutant cell lines which do not produce ABP-120. Two different transformation vectors capable of targeted disruption of the ABP-120 gene locus have been constructed using a portion of an ABP-120 cDNA clone. Three independent cell lines with different disruption events have been obtained after transformation of amebas with these vectors. The disruption of the ABP-120 gene by vector sequences results in either the production of a small amount of truncated ABP-120 or no detectable protein at all. The phenotypes of two different clones lacking ABP-120, generated in strains AX3 and AX4, have been characterized and show identical results. ABP-120- cells tend to remain rounder before and after cAMP stimulation, and do not reextend pseudopods normally after rapid addition of cAMP. In addition, ABP-120- cells translocating in buffer exhibit defects in both the rate and extent of pseudopod formation. The amount of F-actin cross-linked into the cytoskeleton after cAMP stimulation of ABP-120- cells is reduced at times when ABP-120 has been shown to be incorporated into the cytoskeleton, and this correlates temporally with the absence of reextension of pseudopods after cAMP stimulation. The instantaneous velocity is significantly reduced both before and after cAMP stimulation in the ABP-120- cells, and the cells show decreased chemotactic efficiency compared to ABP-120+ controls. This phenotype is consistent with a role for ABP-120 in pseudopod extension by cross-linking actin filaments as proposed by the "cortical expansion model" (Condeelis, J., A. Bresnick, M. Demma, C. Dharmawardhane, R. Eddy, A. L. Hall, R. Sauterer, and V. Warren. 1990. Dev. Genet. 11:333-340).


Transforming growth factor beta 1 is a powerful modulator of platelet-derived growth factor action in vascular smooth muscle cells. We have studied the effect of transforming growth factor beta 1 (TGF-beta 1) on vascular smooth muscle cell (SMC) mitogenesis and expression of thrombospondin and other growth related genes. We found that TGF-beta 1 treatment of vascular SMC induced a prolonged increase in steady-state mRNA levels of thrombospondin as well as alpha 1 (IV) collagen. The increase began at approximately 2 h, peaked by 24 h, and remained considerably elevated 48 h after growth factor addition. There was a corresponding increase in thrombospondin protein as well as increased expression of several other secreted polypeptides. The increase in thrombospondin contrasted sharply with that observed for platelet-derived growth factor (PDGF) which induced a rapid and transient increase in thrombospondin mRNA level. Although TGF-beta 1 was able to directly enhance expression of thrombospondin as well as the growth-related genes c-fos and c-myc, and induced c-fos expression with identical kinetics as PDGF, it was unable to elicit [3H]thymidine incorporation into DNA in three independent smooth muscle cell strains. However, TGF-beta 1 was able to strongly increase the mitogenic response of SMC to PDGF. Addition of both TGF-beta 1 and PDGF to SMC also caused a synergistic increase in the expression of thrombospondin as well as c-myc. Interestingly, in one other smooth muscle cell strain, a weak and delayed mitogenic response to TGF-beta 1 alone was observed. Our results strongly suggest that induction of thrombospondin expression by TGF-beta 1 and by PDGF occurs by distinct mechanisms. In addition, that TGF-beta 1 can enhance PDGF-induced mitogenesis may be due to the ability of TGF-beta 1 to directly induce the expression of thrombospondin, c-fos, c-myc, and the PDGF beta-receptor.


Parathyroid hormone stimulates protein kinase C but not adenylate cyclase in mouse epidermal keratinocytes. Intact human parathyroid hormone, hPTH [1-84], and the hPTH [1-34] fragment stimulated membrane-associated protein kinase C (PKC) activity in immortalized (but still differentiation-competent) murine BALB/MK-2 skin keratinocytes. Unexpectedly, the hormone and its fragment did not stimulate adenylate cyclase. The failure of PTH to stimulate adenylate cyclase activity was not due to the lack of a functioning receptor-cyclase coupling mechanism because the cells were stimulated to synthesize cyclic adenosine monophosphate (cyclic AMP) by the beta-adrenergic drug isoproterenol. Thus, skin keratinocytes seem to have an unconventional PTH receptor that is coupled to a PKC-activating mechanism but not to adenylate cyclase. These observations suggest that normal and neoplastic skin keratinocytes respond to the PTH-related peptide that they make and secrete.


Identification of basic fibroblast growth factor sensitivity and receptor and ligand expression in human colon tumor cell lines. Basic fibroblast growth factor (bFGF) has been shown to be mitogenic to many different eukaryotic cell lines of mesodermal and neuroectodermal origin. Addition of exogenous bFGF to the chemically defined media of five characterized human colon tumor cell lines, cultured in the absence of epidermal growth factor (EGF), resulted in stimulation of growth from 24% to 146% in four of five cell lines, as measured by a colorimetric MTT assay. A positive dose-response relationship was observed when colon cells were treated with bFGF concentrations from 1 pM to 1 nM. bFGF showed a cumulative effect with EGF in stimulating the proliferation of colon tumor cells. The growth-inhibitory effect of exogenous transforming growth factor-beta (TGF-beta) on these cells was abolished by bFGF. When colon tumor cells were examined on immunoblots with a fibroblast growth factor (FGF) receptor-specific antibody, bands were detected at apparent molecular weights of 131 and 145 kDa. Conditioned media and cell lysates from the same human colon tumor cell lines were immunoprecipitated with a bFGF-specific antibody. An immunoreactive band was detected that comigrated with authentic human recombinant bFGF (16 kDa). Furthermore, preabsorption of anti-bFGF antibody with authentic ligand blocked immunodetection of the 16 kDa band on immunoblots. Documentation of a bFGF response, receptor, and ligand expression in human colon tumor cell lines is novel, and may represent a more widespread role for FGF that extends to epithelial cells and tumors of endodermal germ layer origin. The expression of both ligand and receptors by these cells indicates that bFGF could be involved in their growth regulation at the autocrine level.


Subtypes of betaglycan and of type I and type II transforming growth factor-beta (TGF-beta) receptors with different affinities for TGF-beta 1 and TGF-beta 2 are exhibited by human placental trophoblast cells. Transforming growth factor-beta is likely to be an important factor controlling placental activities, including growth, differentiation, invasiveness, hormone production, and immunosuppression. We have used a chemical cross-linking technique with either 125I-TGF-beta 1 or 125I-TGF-beta 2 and bis(sulfosuccinimidyl) suberate (BS3) to characterize TGF-beta binding components on human placental cells in primary culture. Trophoblast-enriched primary cultures exhibited a predominant affinity-labelled complex characteristic of membrane-anchored betaglycan (formerly termed the Type III TGF-beta receptor) and relatively low levels of the Type I and Type II TGF-beta receptor complexes. The results from affinity labelling saturation and competition experiments with TGF-beta 1 and TGF-beta 2 suggest the existence of two distinct subtypes of betaglycan: one subtype has a lower capacity and higher affinity, binds both TGF-beta 1 and TGF-beta 2, yet has a preferential affinity for TGF-beta 2; the second subtype has a higher capacity and lower affinity and binds TGF-beta 1 exclusively. In contrast, mesenchymal cell-enriched placental primary cultures possessed only one subtype of the betaglycan component that binds the two TGF-beta isoforms with similar affinities and capacities as observed on most cell lines. These experiments demonstrate that the betaglycan component which exhibits a higher affinity for TGF-beta 2 than for TGF-beta 1, that we had observed previously on term placental membranes, is actually present on trophoblast cells. In addition to the two distinctive betaglycan subtypes, subtypes of the Type I and II TGF-beta receptors were detected on the trophoblast-enriched cultures. In competition experiments, when 125I-TGF-beta 1 was used as the radiotracer, the Type I and II TGF-beta receptors show a much higher affinity for TGF-beta 1 than for TGF-beta 2, as observed with other cell types. However, when 125I-TGF-beta 2 was used, low abundance subtypes of both the Type I and II receptors that show similar affinities for TGF-beta 1 and TGF-beta 2 were also revealed.


Differential induction of the c-fos promoter through distinct PDGF receptor-mediated signaling pathways. The multiple isoforms of PDGF induce fibroblastic mitogenesis through two distinct PDGF receptors, alpha and beta. The molecular mechanisms by which these alpha and beta PDGF receptors regulate gene expression are poorly understood. We present data which indicates that differential induction of c-fos gene expression by PDGF isoforms occurs through distinct PDGF alpha and beta receptor-mediated signaling pathways. Comparison of PDGF-AA with PDGF-BB stimulation showed that PDGF-BB induced prolonged expression of the c-fos gene in BALB/c-3T3 cells, but that PDGF-AA induced more potent activation of the serum response element (SRE) in transient transfection assays. PDGF-AA, which binds alpha but not beta PDGF receptors, could only induce the SRE through a protein kinase C (PKC)-dependent pathway, whereas PDGF-BB, which binds both alpha and beta PDGF receptors, could also induce the SRE through a PKC-independent pathway. These results suggest that PDGF alpha receptors activate the PKC-dependent signaling pathway while PDGF beta receptors also activate a PKC-independent pathway. In addition, we found that PDGF-BB could induce another c-fos promoter element within the -90 to +10 region, suggesting that the more potent mitogenic effect and prolonged c-fos gene expression induced by PDGF-BB may result from cooperativity between more than one c-fos promoter elements.


Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells. Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.


Detection of cytomegalovirus antibodies by an enzyme-linked immunosorbent assay using recombinant polypeptides of the large phosphorylated tegument protein pp150. Parts of the large phosphorylated tegument protein, pp150, of human cytomegalovirus (HCMV) were expressed in bacteria. The resulting fusion proteins were tested in a Western blot (immunoblot) assay for reactivity with a monoclonal antibody against pp150, with a polyspecific rabbit antiserum, and with human reconvalescent-phase sera. Those fusion proteins that performed well in the Western blot assay were used as antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against HCMV. Five different recombinant beta-galactosidase fusion proteins were evaluated by ELISA using 62 seropositive and 38 seronegative human serum samples. Of all the proteins tested, one peptide representing 162 amino acids of pp150 was superior to the others with regard to sensitivity and specificity. All sera known to be positive for antibodies against HCMV were identified by combining the results of the ELISAs with the different pp150 fusion proteins. Therefore, it appears that peptides from a single protein of HCMV might be sufficient to identify HCMV-seropositive individuals by recombinant ELISA.


MIC quality control guidelines for Haemophilus susceptibility tests using cefdinir (FK482), cefepime, cefetamet, cefpirome, ceftibuten, fleroxacin, temafloxacin, clarithromycin, RP59500, and trospectomycin. A multilaboratory study was performed to establish broth microdilution MIC quality control (QC) guidelines for 10 investigational drugs which previously demonstrated significant activity against Haemophilus influenzae. MIC QC ranges for H. influenzae ATCC 49247 with Haemophilus test medium were determined by using multiple contemporary lots of Haemophilus test medium and the National Committee for Clinical Laboratory Standards' recommended numbers of replicate tests. On the basis of these results, QC ranges (generally modal MIC +/- one log2 dilution) are proposed for cefdinir, cefepime, cefetamet, cefpirome, ceftibuten, fleroxacin, temafloxacin, clarithromycin, RP59500, and trospectomycin. The proposed QC guidelines for clarithromycin and temafloxacin were recently accepted by the National Committee for Clinical Laboratory Standards.


Amplification of human papillomavirus type 16 transforming genes from cervical cancer biopsies and lymph nodes of Hungarian patients. We have used a polymerase chain reaction (PCR) to examine cervical cancer biopsy specimens and pelvic lymph nodes for the presence of human papillomavirus type 16 (HPV 16) DNA. Of the 75 cervical specimens tested, 36 (48%) were positive for HPV 16 in the PCR. A total of 65 pelvic lymph nodes removed during radical surgery on 35 women were also analyzed. Lymph nodes originating from 19 patients whose cervical biopsy specimens were negative for HPV 16 seemed to lack HPV 16 sequences. For 16 women with positive PCR results for cervical biopsy specimens, 9 of 10 lymph node metastases were positive in the PCR, while 11 of their 36 histologically negative lymph nodes were also shown to contain HPV 16 DNA.


Selective detection of Mycoplasma fermentans by polymerase chain reaction and by using a nucleotide sequence within the insertion sequence-like element. A new assay using the polymerase chain reaction to amplify a 206-nucleotide specific gene sequence within the insertion sequence-like element of Mycoplasma fermentans has been developed. The unique insertion sequence-like element exists in multiple copies in the M. fermentans genome. The assay selectively amplifies DNA from all strains of M. fermentans tested. In contrast, DNA from other species of human and nonhuman mycoplasmas, common tissue culture-contaminating mycoplasmas, and bacteria, as well as human, monkey, and mouse tissues do not produce the amplified DNA products specific for M. fermentans.


Susceptibility testing of clinical isolates of Enterococcus faecium and Enterococcus faecalis. We collected 103 clinical Enterococcus faecium isolates from across Canada, performed standard broth microdilution susceptibility testing, and compared these results with results from the MicroScan Pos MIC Type 6 panel (Baxter Health Care Corp., West Sacramento, Calif.) and the AMS-Vitek Gram-Positive Susceptibility card (Vitek Inc., St. Louis, Mo.). High-level aminoglycoside resistance to gentamicin and streptomycin was detected by a single-concentration agar method with 1,000 micrograms of each aminoglycoside per ml. In addition, we tested the effect of the lower calcium content in broth media as recommended in National Committee for Clinical Laboratory Standards (NCCLS) guideline M7-A2 on the activity of the highly calcium-dependent agent daptomycin. Of the 103 E. faecium isolates, there were 4 and 30 isolates with high-level gentamicin resistance (HLGR) and high-level streptomycin resistance (HLSR), respectively. An additional 39 (37 with HLGR and 36 with HLSR) E. faecium isolates were tested by both the MicroScan and the AMS-Vitek systems. The AMS-Vitek card demonstrated sensitivities of 95 and 82% for detecting HLGR strains and HLSR strains, respectively. The MicroScan panel demonstrated improved sensitivities for detecting HLGR (42 to 97%) and HLSR (64 to 84%) when readings were performed manually instead of being generated automatically. Ampicillin resistance (MIC, greater than or equal to 16 micrograms/ml) was detected in 23 of the 103 E. faecium isolates. Only 14 and 20 of these were detected by the MicroScan panels and AMS-Vitek cards, respectively. beta-Lactamase activity was not detected in any isolates. The lower calcium content in broth media recommended by NCCLS guideline M7-A2 markedly reduced the in vitro activity of daptomycin against Enterococcus spp.


Rotavirus serotype G3 predominates in horses. Foal fecal group A rotavirus strains were characterized by electropherotype, serotype, and subgroup and shown to be distinctly different from rotaviruses of other mammals. Of 86 strains that were electropherotyped, 98% had similar profiles, with gene segments 3 and 4 close together and segments 7, 8, and 9 widely spaced. Of 70 strains that had sufficient detectable VP7 antigen to be serotyped by enzyme-linked immunosorbent assays (ELISAs), 63% were serotype G3 (39% were subtype G3A and 24% were subtype G3B), 4% were serotype G13, and 33% were untypeable. Serotypes G1, G2, G4, G5, G6, G9, G10, and G14 were not detected, although G5 and G14 strains have been identified among cultivable equine strains. Of 50 strains that had sufficient detectable VP6 antigen to be subgrouped by ELISAs, only 12% were able to be assigned to either subgroup I or II, with the remaining 88% belonging to neither subgroup.


Isolation of an avianlike group A rotavirus from a calf with diarrhea. An atypical group A rotavirus (993/83) was isolated from a 3-day-old German calf with diarrhea. It differed from 35 conventional German bovine rotavirus isolates analyzed previously with respect to subgroup (strain 993/83 was non-subgroup I and non-subgroup II), serotype (strain 993/83 showed a two-way cross-reaction with serotype 7 and a one-way cross-reaction with serotype 3), and electropherotype (strain 993/83 showed comigrating gene segments 10 and 11). Isolate 993/83 reacted with only one of four monoclonal antibodies that recognized a common VP6 epitope(s). In addition, VP6 and VP2 of isolate 993/83 showed one-dimensional peptide maps that differed substantially from the peptide maps of VP6 and VP2 from all bovine rotavirus isolates. By RNA-RNA hybridization, the 993/83 probe failed to react with a panel of mammalian rotavirus strains, including bovine rotaviruses. It hybridized, however, to genomic RNA of an avian rotavirus strain. Isolate 993/83 could thus represent a candidate for a natural interspecies transmission of rotavirus between different classes of vertebrates.


Rapid detection and identification of avian infectious bronchitis virus. A rapid and sensitive method for the detection and unambiguous typing of infectious bronchitis virus (IBV) is described. RNA was isolated from IBV-infected allantoic fluid and was transcribed into cDNA. This cDNA was amplified by the polymerase chain reaction. The polymerase chain reaction products were subsequently analyzed on an agarose gel. The presence of IBV-specific RNA in the allantoic fluid then allowed the amplification of a 438-bp DNA fragment from the nucleocapsid (N) gene. For the typing of IBV isolates, we used amplified double-stranded DNA as a template in a sequencing reaction. We report 360 bases of the N gene of 18 IBV isolates. The sequence of the N gene was different between serologically indistinguishable IBV strains and may be a valuable tool in epidemiologic studies. A phylogenetic tree that was based on the sequences obtained did not agree with trees that were based on other parts of the sequence, illustrating the high frequency of recombination between IBV strains.


Glycoprotein glycosylation and the immunosuppressive effects of human pregnancy serum. Pregnancy serum contains a factor or factors which suppress T lymphocyte proliferation, although the identity of the factor(s) is still unclear. We have demonstrated that the immunosuppressive activity of pregnancy sera can be destroyed by treatment with periodate which oxidises protein-linked oligosaccharides. Similar effects have been noted with uromodulin, a potent immunosuppressive glycoprotein initially isolated from pregnancy urine. We find, however, that uromodulin is present in both pregnancy and non-pregnancy sera, and that removal of uromodulin from pregnancy serum by lectin affinity chromatography is not associated with loss of activity, ruling out this glycoprotein as the immunosuppressive factor. The possible role of protein-linked oligosaccharides of other serum glycoproteins in causing the pregnancy-related immunosuppression is discussed.


Molecular and serological analyses of two bovine rotaviruses (B-11 and B-60) causing calf scours in Australia. Fecal specimens from 78 calves involved in outbreaks of calf diarrhea which occurred in three farms in Victoria, Australia, in 1988 were analyzed for rotaviruses. Thirty-eight samples were positive for group A virus antigen by enzyme-linked immunosorbent assay, and 20 of these contained viral double-stranded RNAs that could be detected by polyacrylamide gel electrophoresis. Two major electropherotypes could be observed, and a representative isolate of each electropherotype (isolates B-11 and B-60) was successfully adapted to grow in MA104 cells. Sequencing of the VP7 genes directly from RNA transcripts of fecal and cell culture-adapted viruses demonstrated that no base changes occurred in this gene upon adaptation to growth in MA104 cells. Sequencing also revealed that the VP7 protein of B-60 was closely related to G serotype 6 (G6) strains, whereas the B-11 sequence was significantly different from all previously published sequences except the recently reported VP7 sequences of bovine isolates 61A and B223, particularly across the antigenic regions A, B, and C. The other strains most closely related to B-11 by VP7 amino acid sequence analysis were G4 porcine strains BMI-1 and BEN-144 and G8 human strain 69M. Serotyping of B-11 and B-60 gave results that were in good agreement with the sequencing data. Hyperimmune typing sera clearly identified B-60 as a member of G6, whereas the B-11 strain reacted to moderate titers only with antisera to some G10 strains. Antiserum raised against B-11 neutralized some strains of G10 cross-reacted with porcine G4 type isolates BMI-1 and BEN-144 but not with other G4 strains or with rotaviruses of other mammalian G serotypes. Northern blot hybridization showed that B-11 was closely related to the recently reported bovine G10 strain B223, and they both possessed a similar segment 4 that was different from that of either UK bovine or NCDV rotavirus.


Thyrotropin-releasing hormone selectively reverses lorazepam-induced sedation but not slowing of saccadic eye movements. To investigate preliminary reports that benzodiazepine-induced sedation may be reversed by thyrotropin-releasing hormone (TRH), we examined the effect of TRH or saline placebo on two variables which are sensitive to benzodiazepine agonists: changes in sedation and saccadic eye movements. Lorazepam 10 micrograms/kg i.v. increased self-ratings of sedation and reduced self-ratings of alertness and these changes were almost completely reversed by TRH. In contrast the slowing of saccadic eye movements by lorazepam was not reversed by TRH. The effects of TRH do not appear to be due to a direct antagonism at the benzodiazepine receptor, since flumazenil reverses changes in both variables. Moreover ligand binding studies reveal that TRH has very low affinity at this receptor. These clinical data provide the first demonstration that it is possible to distinguish between the effects of benzodiazepines on saccadic eye movements and psychological self-ratings.


U-54494A reduces 4-AP-induced afterdischarges of CA1 pyramidal cells in the hippocampal slice of the rat. The anticonvulsant activity of U-54494A was studied in a 4-aminopyridine (4-AP) epilepsy model using extracellular recordings in in vitro hippocampal slices. Field potentials were evoked by stimulation of Schaffer collaterals, and recorded from the CA1 region of the hippocampus after infusion of 4-AP in the absence and presence of U-54494A. The number and the total area of after discharges (AD) in the presence of 4-AP were significantly decreased by increasing concentrations of U-54494A. In contrast, U-54494A did not significantly change the latency, duration, or area of the evoked PS in this paradigm. Phenytoin, a standard anticonvulsant, decreased the PS area without affecting either the PS latency or duration, or the AD number or area in the same paradigm. These present results provide more evidence that U-54494A is a novel and effective anticonvulsant that may be useful in the treatment of paroxysmal activity, without having generalized depressive effects.


In vivo sodium chemical shift imaging.

In vivo sodium chemical shift imaging. The shift reagents thulium(III) 1,4,7,10-tetraazacyclododecane N,N',N",N"'tetramethylenephosphonate (TmDOTP5-), and dysprosium(III)triethylenetetramine-hexaacetate (DyTTHA3-) are compared in this work for their uses in sodium chemical shift imaging (NaCSI). In a series of experiments using phantoms we evaluated the relative contributions of bulk magnetic susceptibility (BMS) effects and hyperfine shifts to the induced 23Na chemical shift for these two shift reagents. The ratios of BMS effects to hyperfine shifts suggest that TmDOTP5- should be a more effective shift reagent than DyTTHA3- for 23Na NMR spectroscopy as well as NaCSI. The dependence on pH and free Ca2+ concentration of the 23Na NMR frequency shift induced by TmDOTP5- was evaluated. It was found that TmDOTP5- produces good spectral resolution under physiologic conditions. Examples presented from in vivo NaCSI experiments using TmDOTP5- to study diffusion in the posterior chamber of the rabbit eye and to monitor the rate of clearance of aqueous fluid from the anterior chamber demonstrate the effectiveness of this new shift reagent and of the NaCSI technique for in vivo studies.


Inflammatory pseudotumor of the liver masquerading as a metastasis in a child treated for nephroblastoma. A 3 1/2-year-old boy presented with a palpable hepatic tumor thought on clinical and radiological grounds to be a metastasis but which was found to be an inflammatory pseudotumor on histological examination. Eighteen months previously he had received chemotherapy and radiotherapy for a stage IV Wilms' tumor, which had been surgically excised 4 months after commencing treatment. This case illustrates the importance of obtaining a histological diagnosis in the management of patients with malignant tumors.


Extrapulmonary and pulmonary small-cell carcinoma: tumor biology, therapy, and outcome. Extrapulmonary small-cell cancer is a distinct clinicopathological entity from small-cell anaplastic carcinoma of the lung. Approximately 1,000 cases have been projected annually in the United States, which represents an overall incidence of between 0.1% and 0.4% of all cancer. Not surprisingly then, little information is available regarding the treatment of this disease, which presents a challenge to the clinician when it is regionally confined. The majority of patients with extrapulmonary small-cell neoplasms have only been treated with local modalities of therapy, surgery, radiation, or a combination of both. Prolonged survival is not infrequent, which is in contrast to the experience for small-cell lung cancer and surprising given our current systemic approach to patients with this disease. This report will summarize the similarities and differences in biology, natural history, and clinical characteristics of patients with extrapulmonary small-cell cancer and small-cell anaplastic carcinoma of the lung. The histogenesis of small-cell cancer is briefly reviewed. A general therapeutic approach to patients with small-cell lung cancer is reported. Lastly, recommendations for therapy of patients with regionally confined extrapulmonary small-cell cancer by primary site are outlined.


Screening indices for cytomegalovirus retinitis in patients with human immunodeficiency virus. Cytomegalovirus retinitis, the major cause of blindness in patients with the acquired immunodeficiency syndrome, can be arrested by early detection and treatment. We identified 9 screening indices for early CMV retinitis: T-4/T-8 ratio less than 0.11, T-4 count less than 30, T-8 count less than 500, leukocytes less than 4,200, platelets less than 240,000, hemoglobin less than 11.6, hematocrit less than 35, less than 6 for a diagnostic profile which incorporates these 7 indices, and less than 2 for the IA-sum which compounds T-cell inversion and anemia. A threshold value was determined for each index, identified by retrospective review of charts of 15 patients with CMV retinitis and 30 without. Sensitivity, specificity, positive predictive value, negative predictive value, and receiver operating characteristic curve area was calculated. The best screening index was the T-cell ratio, which had the highest sensitivity (0.93), negative predictive value (0.94-0.99) and receiver operating characteristic curve area (0.89), and a positive predictive value among the highest (0.50-0.82).


Familial adenomatous polyposis: case report and review of extracolonic manifestations. FAP, if left untreated, results in colorectal cancer. Appropriate early surgical intervention is of utmost importance. With the reduction in mortality from colorectal carcinoma, an increasing number of patients with FAP need lifelong follow-up to screen for extracolonic manifestations. Today, the major causes of death in patients with FAP who are cured of colorectal cancer, or have had a colectomy before its development, are desmoid tumors and periampullary carcinomas. In family members at risk, screening with flexible sigmoidoscopy should be initiated in adolescence; symptoms of diarrhea and rectal bleeding would warrant an even earlier examination. The upper gastrointestinal tract should be assessed endoscopically at the time the diagnosis of FAP is made. If any polyps are detected, a biopsy is essential. If no gastric or duodenal polyps are found, repeated examinations at 3-5 year intervals probably suffice in asymptomatic patients. Surgically, colectomy will be necessary. It is usually deferred until late adolescence, when it is thought that the patient will be mature enough to handle the emotional aspects of the operation as well as the possible future morbidity due to the procedure. The presence or absence of rectal polyps as well as the site and depth of any invasive rectal carcinoma will determine the appropriate surgical procedure. Alternatives must be well understood by the physician and discussed carefully with the patient preoperatively.


Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Thyroid hormones and retinoic acid function through nuclear receptors that belong to the steroid/thyroid-hormone receptor superfamily. Thyroid hormone receptors (TRs) and retinoic acid receptors (RARs) require auxiliary nuclear proteins for efficient DNA binding. Here we report that retinoid X receptors RXR alpha is one of these nuclear proteins. RXR alpha interacts both with TRs and with RARs, forming heterodimers in solution that strongly interact with a variety of T3/retinoic acid response elements. Transfection experiments show that RXR alpha can greatly enhance the transcriptional activity of TR and RAR at low retinoic acid concentrations that do not significantly activate RXR alpha itself. Thus, RXR alpha enhances the transcriptional activity of other receptors and its own ligand sensitivity by heterodimer formation. Our studies reveal a new subclass of receptors and a regulatory pathway controlling nuclear receptor activities by heterodimer formation.


Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Cellular responsiveness to retinoic acid and its metabolites is conferred through two structurally and pharmacologically distinct families of receptors: the retinoic acid receptors (RAR) and the retinoid X receptors (RXR). Here we report that the transcriptional activity of RAR and RXR can be reciprocally modulated by direct interactions between the two proteins. RAR and RXR have a high degree of cooperativity in binding to target DNA, consistent with previous reports indicating that the binding of either RAR or RXR to their cognate response elements is enhanced by factors present in nuclear extracts. RXR also interacts directly with and enhances the binding of nuclear receptors conferring responsiveness to vitamin D3 and thyroid hormone T3; the DNA-binding activities of these receptors are also stimulated by the presence of nuclear extracts. Together these data indicate that RXR has a central role in multiple hormonal signalling pathways.


Clinical outcome in aggressively treated meningeal gliomatosis. We reviewed 11 consecutive patients with an antemortem cytologic diagnosis of meningeal gliomatosis. In three patients, meningeal gliomatosis was diagnosed before surgical resection of the glioma. Three of five patients with anaplastic astrocytoma and one with oligodendroglioma improved with treatment. Patients with glioblastoma multiforme did not respond and had a median survival from the diagnosis of meningeal gliomatosis of only 8 weeks. Meningeal gliomatosis can be an early finding in gliomas. Treatment response is probably related to tumor histology and grade.


[Cell surface adhesion molecules: structure, function, clinical aspects]. Intercellular and cell-matrix interactions play a crucial role in several physiological and pathological processes. These relationships are based on different cell adhesion molecules. Although adhesion receptors exhibit structural diversity, cell adhesion mechanisms are basically similar to each other. Details of receptor functions (e.g. the exact mechanisms of signal transduction) is poorly understood. In this paper authors summarize data on different cell adhesion antigens and also describe a few important biological features to collect more data on the complex interactions of these molecules.


[Malignant hyperthermia].

[Malignant hyperthermia]. Malignant hyperthermia (MH) is a life threatening complication following anaesthesia with potent inhalational agents and suxamethonium. The signs of MH are caused by increased metabolism and secondary stimulation of the sympathetic nervous system due to uncontrolled, high intracellular concentrations of calcium in skeletal muscle. The hyperthermia is secondary to the increased energy turnover. Calcium release from the sarcoplasmic reticulum is increased due to a low threshold for release and prolonged opening of the calcium channel upon normal stimulation. The gene encoding the calcium channel is localized on chromosome 19 in humans, and a substitution of cysteine for arginine has recently been described in one family with MH.


Free radical formation in single crystals of 2'-deoxyguanosine 5'-monophosphate tetrahydrate disodium salt: an EPR/ENDOR study. Single crystals of 2'-deoxyguanosine 5'-monophosphate were X-irradiated at 10 K and at 65 K, receiving doses between 4.5 and 200 kGy, and studied using K-band EPR, ENDOR, and field-swept ENDOR (FSE) spectroscopy. Evidence for five base-centered and more than nine sugar-centered radicals was found at 10 K following high radiation doses. The base-centered radicals were the charged anion, the N10-deprotonated cation, the C8 H-addition radical, a C5 H-addition radical, and finally a stable radical so far unidentified but with parameters similar to those expected for the charged cation. The sugar-centered radicals were the H-abstraction radicals centered at C1', C2', C3', and C5', an alkoxy radical centered at O3', a C5'-centered radical in which the C5'-O5' phosphoester bond appears to be ruptured, a radical tentatively assigned to a C4'-centered radical involving a sugar-ring opening, as well as several additional unidentified sugar radicals. Most radicals were formed regardless of radiation doses. All radicals formed following low doses (4.5-9 kGy) were also observed subsequent to high doses (100-200 kGy). The relative amount of some of the radicals was dose dependent, with base radicals dominating at low doses, and a larger relative yield of sugar radicals at high doses. Above 200 K a transformation from a sugar radical into a base radical occurred. Few other radical transformations were observed. In the discussion of primary radicals fromed in DNA, the presence of sugar-centered radicals has been dismissed since they are not apparent in the EPR spectra. The present data illustrate how radicals barely traceable in the EPR spectra may be identified due to strong ENDOR resonances. Also, the observation of a stable radical with parameters similar to those expected for the charge guanine cation is interesting with regard to the nature of the primary radicals stabilized in X-irradiated DNA.


Infiltrating neutrophils differ from circulating neutrophils when stimulated with C5a, NAP-1/IL-8, LTB4 and FMLP. In this study we report on functional characteristics of pustule as well as blood polymorphonuclear neutrophils (PMN) in a patient suffering from relapsing bullous staphyloderma. Large numbers of viable PMN from newly formed pustules as well as from the peripheral blood were investigated. During the course of disease chemotactic migration, enzyme degranulation, superoxide-anion generation and leukotriene B4 production were determined simultaneously. The results revealed C5a- and NAP-1/IL-8-specific dysfunction of pustule PMN as compared with blood PMN. In contrast, FMLP-elicited functional activities of pustule PMN were only slightly affected. Our findings provide evidence that in inflamed tissue invading PMN are regulated by in situ generated mediators. C5a produced by staph, aureus-induced activation of the alternative pathway of the complement cascade represents a predominant regulatory factor in situ. Furthermore, the results substantiate previous observations concerning different modulation of C5a and f-met-peptide receptors on human PMN.


Polymerase II promoter activation: closed complex formation and ATP-driven start site opening. Studies on bacterial RNA polymerases have divided the initiation pathway into three steps, namely (i) promoter binding to form the closed complex; (ii) DNA melting to form an open complex, and (iii) messenger RNA initiation. Potassium permanganate was used to detect DNA melting by mammalian RNA polymerase II in vitro. Closed complexes formed in a rate-limiting step that was stimulated by the activator GAL4-VP16. Adenosine triphosphate was then hydrolyzed to rapidly melt the DNA within the closed complex to form an open complex. Addition of nucleoside triphosphates resulted in the melted bubble moving away from the start site, completing initiation.


Seroepidemiology of viral infections among intravenous drug users in northern California. Intravenous drug users are frequently exposed to parenterally transmitted viral infections, and these infections can spread to the general population through sexual activity. We investigated the prevalence of serologic markers for human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type I/II (HTLV-I/II), hepatitis B virus (HBV), and hepatitis C virus (HCV) in intravenous drug users and their sexual contacts. Of 585 drug users from northern California tested for these serologic markers, 72% were reactive for the antibody to HCV, 71% for the antibody to hepatitis B core antigen, 12% for HTLV-I/II antibodies, and 1% for the HIV-1 antibody. The prevalence of serologic markers for these four viruses correlated with the duration of intravenous drug use, the ethnic group, and the drug of choice. More than 85% of subjects infected with either HCV or HBV were coinfected with the other virus. All persons reactive to HTLV-I/II antibodies had antibodies for either HBV or HCV. Of 81 sexual contacts tested, 17% had evidence of HBV infection while only 6% were reactive for HTLV-I/II antibodies and 4% for the antibody to HCV. None of this group was infected with HIV-1. We conclude that HTLV-I/II and HCV are inefficiently transmitted to sexual contacts while HBV is spread more readily. Programs designed to discourage the sharing of drug paraphernalia, such as needle and syringe exchanges, should decrease the risk of parenterally spread viral infections in intravenous drug users and thus slow the spread of these infections to the general population.


Neurofibromatosis in infantile autism and other types of childhood psychoses. Recent findings have suggested that the simultaneous occurrence of neurofibromatosis and childhood psychosis might be more than a coincidence. In this study of 341 children with infantile autism and other types of childhood psychosis seen as inpatients in two university clinics of child psychiatry in a 25-year period, only one case (0.3%) of concomitant occurrence of the disorders was found, which is a frequency no higher than expected by chance.


Excessive life changes during childhood and their effects on mental and physical health in adulthood. To evaluate the long-lasting effects of excessive life changes during childhood, a group of children sent during the war years 1939-1945 from Finland to Sweden was examined. Their physical and mental health were compared with those of a control group. Information was gathered by postal questionnaire and from various official records both in Finland and Sweden. A clinical interview enhanced the findings of the study. The groups did not differ greatly from each other. Somatically the control group suffered from chronic illnesses more than the group of war children. This particularly applied to cardiovascular diseases; the control group also received more medication free of charge, i.e. medication for chronic diseases, the expenses of which are covered by Public Health Care. As regards mental illnesses the groups were similar. The group of war children was more capable than the control group at recognizing and expressing their feelings.


Birth order, development and personality. First borns tend to be different, some of the time, than children born into the other birth orders. This seems due to parental treatment. Parents are often overly anxious about their first child, and may be more restrictive with the first child than with later children. It may be that this is especially true if the first child is female, given the tendency of parents to be more protective of females than of males. Also, the first child has more time alone with the parents than subsequent children, by virtue of having no siblings until the second child is born. From the above factors, it appears that first borns grow up more fearful than later born children but also more intellectually oriented. These are only tendencies and would not be absolutely true in every instance. But, the birth order does seem to initiate a tendency for things to be as stated. As a result, first borns achieve much more than the other birth orders, perhaps due to early adult-oriented styles learned when they had only the parents and no other siblings. If this speculation is correct, the same finding should hold for only children as well. The greater anxiety, achievement, and creativity of some first borns means their lives may be very different as a result of having been the first born child in the family.


School refusal in Japan.

School refusal in Japan. In Japan, school refusal began to attract the attention of psychiatrists from about 1960, and has been one of the main focuses of contemporary child and adolescent psychiatry because of the increase in the number of patients. It is now one of the most popular diagnosis in the clinical practice of child and adolescent psychiatry in this country. School refusal, however, is not listed as a diagnostic classification of a syndrome or disease in such international classifications of psychiatry as the ICD-9, ICD-10 draft, DSM-III, or DSM-III-R. It is an important task for child and adolescent psychiatry in Japan to determine the position of school refusal in relation to the international diagnostic standards. In this paper, (1) We described the actual conditions of school refusal in Japan. (2) We argued that it was reasonable and clinically useful to retain the concept of school refusal as a clinical entity unique to Japan.


The Finnish family competence study. Childbearing attitudes in pregnant nulliparae. In this paper, the childbearing and childrearing attitudes of 1,443 primigravid women are characterized in relation to their level of knowledge of childbirth. Mothers with little childbirth knowledge needed extra support in the transition to parenthood. Their attitude toward pregnancy was more negative and they were less interested in obtaining childbirth information. The level of childbirth knowledge was measured by means of a questionnaire during the mother's first visit to the maternity health care clinic. The knowledge had some prognostic value in identifying the mothers who acutely needed support in coping with the experiences of pregnancy and in the transition to parenthood.


Hopelessness in children and adolescents. An overview. Most research on hopelessness has focused on adult populations. However, with the recent publication of Kazdin's Hopelessness Scale for Children, there has been increasing attention directed toward hopelessness in children. This report reviews research on hopelessness, and in both adults and children, emphasizes findings which support Beck's cognitive trial view of depression. Also discussed is the neglected topic of hopelessness within the academic setting, including attributions, motivation and delayed gratification. This report suggests ways to prevent and treat hopelessness in the school-aged child, focusing primarily on cognitive-behavioral techniques.


Interferences in psychosocial development of seriously health-impaired and physically disabled children. Educational implications. Improvements in neonatal care and medical intervention have dramatically increased the number of seriously health-impaired and physically disabled children who now survive to enroll in elementary and secondary schools. Investigation into the post-secondary status of those populations reveals that few of them are employed or living independently. Some researchers have recently found that the disappointing status of these individuals correlates more significantly with psychosocial factors than with intelligence or severity of the disability. This paper describes the circumstances which typically surround children with serious chronic illness or physical disability in the context of Erikson's stages of psychosocial development and suggests strategies for educators regarding the social development of such individuals during the elementary and secondary school years.


Psychological aspects of precocious puberty. An overview. During the last three decades the psychological consequences of precocious puberty have received considerable attention. The nature and shortcomings of relevant studies are described. It is emphasized that specialized counselling and guidance of families with children who have to cope with precocious puberty is important, since it often contributes to an appropriate behaviour adjustment without the development of psychiatric symptomatology. Guidelines for medical service and psychological management are provided.


Down's syndrome and infant gaze. Gaze behavior of Down's syndrome and nondelayed infants in interactions with their mothers. The study reported here compared the gaze behavior of infants with Down syndrome (DS) and nondelayed infants during interactions with their mothers. The subjects were 10 DS infants and 11 nondelayed infants. Five of the DS infants and 6 of the nondelayed infants were 4 months old; the rest were 9 months old. The results support the expectation that infants with DS gazed at their mothers longer than did nondelayed infants during face-to-face play, and also indicate that all the infants visually attended to their mothers less at 9 months than at 4 months of age. It is conjectured that the increased gaze of the infants with DS may well facilitate attachment in the 1st year of life.


Sexual differentiation in children's drawings of pregnancy. An analysis is reported of drawings of pregnancy by children of both sexes, with the hypothesis that it would be possible to determine sex-related characteristics of the drawings. Clear sexual differentiation in male and female children's representations of pregnancy was revealed by to features: the location of the baby inside the body of the mother and the tendency in some of the boys to "masculinize" the figure, of the expectant mother.


Body composition of rural Beninese women in different seasons assessed by skinfold thickness and bioelectrical-impedance measurements and by a deuterium oxide dilution technique. Body composition of 24 rural Beninese women was assessed in a pre- and a postharvest season by using skinfold-thickness measurements, bioelectrical-impedance analysis (BIA), and deuterium oxide (D2O) dilution. Fat mass assessed by D2O dilution was 12.3 +/- 3.3 kg; by skinfold-thickness measurements, 13.8 +/- 3.3 kg; and by BIA, 14.1 +/- 2.9 kg. Fat mass assessed by D2O was significantly lower (P less than 0.05) than fat mass assessed by the other two methods. Body weight in the postharvest season was 0.8 +/- 1.6 kg higher (P less than 0.05) than during the preharvest season. All three methods demonstrated that there were no significant changes in fat mass but that fat-free mass increased significantly (P less than 0.05) from pre- to postharvest season.


Linoleate inhibits EPA incorporation from dietary fish-oil supplements in human subjects. Thirty healthy male subjects were randomly allocated into one of two treatment groups: group H subjects were maintained on a high-linoleic acid (LA) diet with the saturated fatty acids kept low and those in group L were kept on a low-LA, low saturate diet. After a 3-wk run-in period, subjects consumed a fish-oil supplement containing 1.6 g eicosapentaenoic acid (EPA)/d and 0.32 g docosahexaenoic acid as triglycerides for a further 4 wk. The diets alone resulted in a significant change in neutrophil phospholipid LA (H = 12.7 +/- 2.9%, L = 9.0 +/- 0.9%; P less than 0.05). Dietary supplementation with fish oil resulted in a significant increase in EPA in neutrophil phospholipids that was greater in group L (mean 2.0 +/- 0.4%) than group H (mean 1.5 +/- 0.3%; P less than 0.005). Reductions in arachidonic acid concentrations in neutrophil membranes were not different, nor could differences in leukotriene B4 production be detected. Our study indicates that the ingestion of n-6 fatty acids within the diet is an important determinant of EPA incorporation into neutrophil membranes.


Physical properties of dietary fiber that influence physiological function: a model for polymers along the gastrointestinal tract. The quantitative measurement of dietary fiber does not recognize its diverse actions on nutrient absorption, sterol metabolism, fermentation in the colon, and stool weight. These differences in action are more likely due to differences in physical characteristics along the gastrointestinal tract. This paper explores such physical characteristics and attempts to classify dietary fiber in a more physical manner. This approach recognizes the diverse and variant action of each dietary fiber, which may be modified as a result of processing and cooking without changing in any way the quantitative measurement of dietary fiber. The general principles developed in this paper could also be applied to other polymeric materials passing along the gastrointestinal tract.


Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots. Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hybridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, beta 2-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFMPs were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMPs. RFMPs can be used as genetic markers, because their alleles segregate in a Mendelian manner. Unlike most other methods for detecting DNA sequence polymorphisms, a genomic DNA blot made from one gel can be hybridized consecutively with many (30 or more) different probes.


Characterization of the fetal inflammatory response to cytomegalovirus placentitis. An immunohistochemical study. The histopathologic features of cytomegalovirus placentitis, an established cause of chronic villitis, are well documented. However, the immunologic features of the fetal inflammatory response to placental cytomegalovirus infection are largely unknown. The characterization of the fetal-derived inflammatory cell reaction may be important in our understanding of both the intrauterine as well as the antenatal immunological response of the neonate to this important viral infection. We examined formalin-fixed placentas from four cases of confirmed congenital cytomegalovirus infection using an in situ DNA probe to cytomegalovirus, and a variety of antibodies to leukocyte antigens, including anti-CD68, CD45RO, CDw75, CD74, IgG, IgM, and IgA. All four placentas showed marked hyperplasia of fetal-derived placental macrophages, termed Hofbauer cells. A lymphocytic villitis was present in all placentas, which was characterized by positive staining in all cases with T-cell antibodies. There was no evidence of positive staining of lymphocytes using B-cell antibodies in any of the cases. Two placentas showed plasmacellular villitis, which stained positively for both IgG- and IgM-secreting cells, that was present as early as the second trimester of gestation. No IgA positivity of plasma cells was observed. These data are presented in light of current theories of fetal viral immunity.


Adenomyoepithelioma of the breast. A report of two cases. We describe two cases of adenomyoepithelioma of the breast. The two tumors reported herein were well-circumscribed lesions that showed ducts lined by inner epithelial and outer myoepithelial cells and solid areas made up of fasciculated spindle myoepithelial cells. This dual differentiation was corroborated by immunohistochemical studies and electron microscopy.


An ultrastructural comparison of sinusoids in hepatocellular carcinoma, adenomatous hyperplasia, and fetal liver. Transmission and scanning electron microscopic examinations were performed to evaluate the fine-structural differences in sinusoids of hepatocellular carcinoma, adenomatous hyperplasia of the liver, and fetal livers. In cancerous sinusoids, thickened endothelial cells with loss of sieve plates were conspicuous features. In the sinusoids of adenomatous hyperplasia, scanning electron microscopy showed apparent sieve plates, and basement membrane formation (capillarization) was less conspicuous than in carcinoma. In fetal livers, occasional large gaps as well as small fenestrations in the endothelium were noted. Basement membrane materials were not seen. It was concluded that the sinusoids of hepatocellular carcinoma lost the fine-structural nature of normal sinusoids and showed much more prominent capillarizations than the sinusoids of adenomatous hyperplasia, and that the fine structures of cancerous sinusoids were also different from those of the sinusoids in fetal livers.


Growth rate analysis of lung metastases appearing 18 years after resection of cutaneous adenoid cystic carcinoma. Case report and review of the literature. Growth rate analysis of lung metastases of a cutaneous adenoid cystic carcinoma, which appeared 18 years after the resection of the primary tumor from the scalp is presented. The doubling times of the metastases were long compared with that of other lung metastases. They were 22 months for the metastasis in the right lung and 70 months for the metastasis in the left lung, with a shortening of the doubling time in the left side to 10.4 months in the last 4 months of observation. Backward extrapolation showed that the metastases to the lung were disseminated before the diagnosis and surgical resection of the primary tumor. To our knowledge, this is the third reported case of lung metastases from a cutaneous adenoid cystic carcinoma out of 25 documented cases. We present a review of the literature and discuss the clinical implications of our findings.


Heart allograft involvement with Epstein-Barr virus-associated posttransplant lymphoproliferative disorder. We describe a 60-year-old man who underwent heart transplant and died 5 months later. At autopsy, the patient was found to have posttransplant lymphoproliferative disorder (PTLD), which was not suspected ante mortem. The PTLD involved the lung, lymph nodes, spleen, and kidney and the intima of right coronary artery of the transplanted heart. Epstein-Barr virus infection was demonstrated on direct gel and dot blot after polymerase chain reaction amplification. In situ hybridization for Epstein-Barr virus DNA confirmed the presence of Epstein-Barr virus-infected lymphocytes in the intima of the right coronary artery of the cardiac allograft. To our knowledge, this case represents the first report of involvement of a heart allograft by PTLD in which Epstein-Barr virus infection of the atypical cells is documented. Although primary allograft involvement by PTLD has rarely been reported in heart transplant recipients, recognition that this may occur is important, since the lymphoid infiltrate of PTLD must be distinguished from the lymphoid infiltrate of acute rejection to avoid inappropriate therapy.


Signet-ring cell carcinoma of the prostate. We report an intriguing case of signet-ring cell carcinoma of the prostate, ie, a rare histopathologic pattern of prostatic carcinoma. We present the results of histologic, immunohistologic, and electron microscopic examinations in our case, and we compare the findings with those of cases that have been previously reported. Although the histopathologic findings in the rare cases of signet-ring cell carcinoma of the prostate thus far reported have varied greatly, the following conclusions can be made: (1) signet-ring cell carcinoma of the prostate is a high-grade carcinoma; (2) a prostatic primary should be considered in cases of metastatic signet-ring cell carcinoma, especially when results of gastrointestinal studies are unelucidating; and (3) staining for mucin, lipid, prostate-specific antigen, prostate-specific acid phosphatase, and carcinoembryonic antigen is variable.


Cytomegalovirus colitis presenting as hematochezia and requiring resection. Cytomegalovirus infection is one of the most prevalent viral infections affecting recipients of cardiac allografts. Of the various severe systemic manifestations, those in the gastrointestinal tract have a unique way of presenting, specifically in the colon where a process related to cytomegalovirus infection that involves all layers, with dilatation as a prominent clinical feature, has been suggested. We report herein a case of patient with a heart allograft who had severe episodes of rejection that responded to boosting doses of steroids. Because of persistent fever, diarrhea, hematochezia, and computed tomographic findings of the abdomen that showed a highly abnormal appearance of the ascending and transverse segments of the colon, this patient subsequently underwent celiotomy. The involved segment of the colon was found to have severe inflammation with mucosal necrosis; a subtotal colectomy was done. The abundant cytomegalovirus inclusions found in the vascular endothelium of the removed damaged segment of the colon suggest that cytomegalovirus may have been a causal factor in this form of colitis. The patient recovered uneventfully.


A randomized prospective trial of acyclovir and immune globulin prophylaxis in liver transplant recipients receiving OKT3 therapy. The use of OKT3 therapy is a major risk factor for opportunistic infections in liver transplant recipients. In the last 2 years, we prospectively randomized 100 patients receiving OKT3 therapy into either a control group (n = 50) or a prophylaxis group (n = 50). Prophylaxis consisted of six doses of intravenous immune globulin over 4 weeks and oral acyclovir for 3 months after OKT3 therapy. The two groups were comparable with respect to demographic, immunologic, and clinical characteristics. The regimen of prophylaxis resulted in (1) a significant reduction in the incidence of herpetic and Epstein-Barr viral infections; (2) no change in the incidence of cytomegalovirus infections; (3) a significant decrease in the incidence of fungal infections; and (4) fewer deaths due to sepsis. The incidence of viral and fungal infections was higher after OKT3 induction than after rescue therapy. Our conclusion is that opportunistic infections are frequent after OKT3 therapy in hepatic allograft recipients. Treatment with intravenous immune globulin and oral acyclovir is safe and effective in preventing non-cytomegaloviral and fungal infections in this setting, thus conferring a survival advantage with fewer deaths due to sepsis.


Chemical cleavage of plasmid DNA by Cu(II), Ni(II) and Co(III) desferal complexes. It is demonstrated that the Cu(II), Co(III) and Ni(II) complexes of a siderophore chelating drug desferal cleave DNA, in contrast to the corresponding Fe(II) complex which does not bring about DNA scission. Hydroxy radical scavengers inhibit the cleavage reaction.


Evidence for heat-stable liver cytosol substance(s) capable of causing oxidative activation of fructose 1,6-bisphosphatase. The endogenous fructose 1,6-bisphosphatase (FBPase) in chicken liver extract undergoes a drastic increase in activity if the pH of the extract is in the alkaline range. Greater and more consistent activation occurs when purified FBPase, placed inside dialysis sack, is incubated in liver extract. Maximal activation (over 16-fold) is accompanied by the disappearance of 4 highly reactive sulfhydryl groups (SH) per molecule of enzyme. The activating effect of the extract remains essentially unchanged after heating to 100 degrees C. Activation can be reversed by dithiothreitol. These data show the existence in liver cytosol of heat-stable substance(s) capable of activating FBPase presumably by forming disulfide bonds with the enzyme's highly reactive SH groups.


Inhibition of ligand binding and antiproliferative effects of tumor necrosis factor and lymphotoxin by soluble forms of recombinant P60 and P80 receptors. Tumor necrosis factor (TNF) is one of the mediators of inflammatory responses. Recently, the cDNA for two distinct receptors of TNF with predicted molecular masses of 60 kDa and 80 kDa, respectively, were isolated. In this report, we compare the inhibitory effects of these two forms of recombinant soluble TNF receptors (extracellular domains) on the ligand binding and on the antiproliferative effects of TNF and lymphotoxin (LT) in a human histiocytic lymphoma cell line (U-937). Our results show that the soluble form of the p60 receptor is approximately 100-fold more potent than the soluble form of the p80 receptor in inhibiting both the antiproliferative effects of TNF as well as in blocking TNF binding to U-937 cells. In contrast, the antiproliferative effects of LT and its binding to cells is inhibited equally by both the p60 and p80 forms of the soluble receptor. Thus, overall our results indicate that the two soluble receptors differ in their ability to inhibit TNF and LT. The impotance of these soluble receptors in blocking the harmful effects of TNF and LT is discussed.


Assembly of a hybrid from the alpha subunit of Na+/K(+)-ATPase and the beta subunit of H+/K(+)-ATPase. Messenger RNA for the alpha subunit of Torpedo californica Na+/K(+)-ATPase was injected into Xenopus oocytes together with that of the beta subunit of rabbit H+/K(+)-ATPase. The Na+/K(+)-ATPase alpha subunit was assembled in the microsomal membranes with the H+/K(+)-ATPase beta subunit, and became resistant to trypsin. These results suggest that the H+/K(+)-ATPase beta subunit facilitates the stable assembly of the Na+/K(+)-ATPase alpha subunit in microsomes.


Two high molecular mass proteases from sea urchin sperm. Two-types of high molecular mass proteases have been purified from sea urchin sperm using DEAE-Sephacel, hydroxylapatite and Superdex 200 column chromatography. Both proteases showed similar hydrolyzing activities toward synthetic peptides, but they differed in the molecular mass and peptide composition. One was probably identical to a proteasome (multicatalytic proteinase), judging from its molecular mass (650 kDa) and polypeptide composition. The other one was composed of several polypeptides with molecular masses ranging from 24 kDa to 125 kDa and its molecular mass was estimated as 950 kDa by gel filtration. These two proteases, however, were closely related to each other. Immunological studies revealed that the 950-kDa protease comprised at least five subunits of the 650-kDa protease.


Monoclonal antibody to GABA binding protein, a possible GABAB receptor. A monoclonal antibody has been raised against a partially purified preparation for the GABAB receptor. The antibody recognized a protein of about 80 kDa in bovine brain synaptic membrane. Immunoabsorbent agarose beads conjugated with the antibody were able to remove, without visible changes in electrophoresed profiles of total proteins, over 90% of the baclofen suppressive GABA binding activity (designated herein, GABAB receptor binding activity) in the solubilized synaptic membrane fraction. Moreover, the addition of GB-1 antibody directly inhibited the GABA binding activity in the crude synaptic membrane fraction. These results indicate that the monoclonal antibody obtained here recognizes the GABA binding protein, or more specifically a GABAB receptor.


Increased expression of the 67kDa-laminin receptor gene in human small cell lung cancer. Gene expression of the precursor of the 67kDa-laminin receptor was examined by Northern analysis using 11 established human lung cancer cell lines and 25 lung cancer tissues obtained by operation. As a result, one transcript, the size of which was 1.2 kb was shown in all cell lines and tissues examined. An increased level of mRNA was demonstrated in cell lines which proliferated rapidly and in small cell lung cancer cell lines. It was also indicated that gene expression of the laminin receptor was up-regulated especially in small cell lung cancer tissue. In this context, 67kDa-laminin receptor appears to be a marker for biological aggressiveness of human lung cancer.


Effect of high K+ exposure on phosphoinositide metabolism in frog skeletal muscle. Using [3H]myo-inositol labeled frog skeletal muscles, we have studied the effect of high K+ exposure on phosphoinositide metabolism. After 12 hours labeling, 80mM K+ exposure induced a time-dependent change. The labeling associated with phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PIP) gradually increased and decreased, respectively. The labeled phosphatidylinositol 4,5-bisphosphate (PIP2) first decreased, and then recovered. An accumulation of the labeling in inositol phosphates was shown. In shortening the labeling to 30 min, 15 min high K+ exposure was found to only increase the labeling in all fractions. Taken together, these results show that high K+ exposure can activate the turnover of phosphoinositides, which is consistent with the hypothesis that the metabolism of phosphoinositides may regulate excitation- contraction (e-c) coupling.


Investigation of sulfur containing amino acids at the lipoxygenase active site using a platinum complex. Inactivation of native soybean lipoxygenase-1 was observed upon preincubation with (NEt4)[PtCl3(P(Bun)3)]. Removal of the platinum complex(es) from the inactivated enzyme by treatment with sodium diethyldithiocarbamate (Naddtc) which reverses methionine but not cysteine binding, restores most of the activity. Linoleic acid, an enzyme substrate, protects it from inactivation. The quenching of the fluorescence of the putative active site tryptophans which accompanies inactivation disappears after Naddtc reactivation. The (NEt4)[PtCl3(P(Bun)3)]-inactivated enzyme iron(II) cannot be oxidized at variance with that of the native or Naddtc reactivated enzyme, as checked by EPR spectroscopy. These results show that at least one methionine is close to the iron binding site in soybean lipoxygenase-1.


Histamine and calcium are independently regulated intracellular mediators of lymphocyte mitogenesis. In addition to cytosolic calcium ([Ca2+]i), intracellular histamine has been implicated as a mediator of mitogenesis in normal mouse spleen cells stimulated by the plant lectin, concanavalin (Con) A. We have linked the growth-promoting action of this amine with its binding to distinct intracellular sites, designated HIC, in microsomes and nuclei and shown that the proliferative response of lymphocytes can be blocked by antagonizing the binding of histamine to HIC by N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine.HCl (DPPE), or by depleting intracellular histamine levels by the specific irreversible inhibitor of histidine decarboxylase, alpha-FMH. We now demonstrate that, at a concentration which completely inhibits both 3H-histamine binding to HIC and 3H-thymidine incorporation into DNA, DPPE fails to block the acute (30 seconds) rise in [Ca2+]i in spleen cells exposed to Con A. Conversely, the calcium channel antagonist, verapamil, suppresses the Con A-induced rise in [Ca2+]i at a concentration which correlates with its inhibition of thymidine incorporation into DNA, but does not prevent histamine synthesis or bind to HIC. Thus, in Con A-stimulated lymphocytes, intracellular histamine and calcium appear to be independently regulated, but essential, mediators of the proliferative response.


Sequence and genomic structure of the human adult skeletal muscle sodium channel alpha subunit gene on 17q. The amino acid sequence of the sodium channel alpha subunit from adult human skeletal muscle has been deduced by cross-species PCR-mediated cloning and sequencing of the cDNA. The protein consists of 1836 amino acid residues. The amino acid sequence shows 93% identity to the alpha subunit from rat adult skeletal muscle and 70% identity to the alpha subunit from other mammalian tissues. A 500 kb YAC clone containing the complete coding sequence and two overlapping lambda clones covering 68% of the cDNA were used to estimate the gene size at 35 kb. The YAC clone proved crucial for gene structure studies as the high conservation between ion channel genes made hybridization studies with total genomic DNA difficult. Our results provide valuable information for the study of periodic paralysis and paramyotonia congenita, two inherited neurological disorders which are caused by point mutations within this gene.


Effects of HS-142-1, a novel non-peptide ANP antagonist, on diuresis and natriuresis induced by acute volume expansion in anesthetized rats. In this study we used HS-142-1, a novel non-peptide antagonist for the atrial natriuretic peptide (ANP) receptor, to clarify the possible physiological significance of ANP in acute hypervolemia. Substantial volume expansion in anesthetized rats induced a strong diuresis and natriuresis. These renal responses were significantly blocked by HS-142-1 at a dose of 3.0 mgkg-1 i.v. This observation suggests that ANP and its guanylyl cyclase-coupled receptor are, under the present conditions, physiologically involved that appears to be responsible for the renal responses in the volume homeostasis.


Lysosome inhibitors enhance the ability of cyclic AMP-elevating agents to induce the LDL receptor in human vascular smooth muscle cells. In human vascular smooth muscle cells cyclic AMP elevation by forskolin increases synthesis of the LDL receptor by a mechanism which appears independent of sterol control. This increased receptor synthesis is further enhanced by chloroquine. Both forskolin and prostaglandin E1 increase the number of cell surface LDL receptors indicating that prostaglandins could exert physiological control over LDL metabolism. This effect is enhanced synergistically by chloroquine. The stimulation by forskolin of LDL receptor synthesis and expression leads to increased metabolism of apo-B and increased hydrolysis of LDL-borne cholesteryl ester. These effects of cyclic AMP on the activity of the LDL pathway are enhanced more than additively by preincubation with the reversible lysosomal inhibitor NH4Cl. Thus cyclic AMP causes up-regulation of the LDL receptor pathway resulting in increased rates of LDL metabolism but this effect can be damped or masked in cell culture by a cyclic AMP-sensitive lysosomal event, probably the acute stimulation of lysosomal cholesterol ester hydrolase.


Extracellular ATP stimulates increases in Na+/K+ pump activity, intracellular pH and uridine uptake in cultures of mammalian cells. Using 3T3 and 3T6 mouse fibroblasts and A431 epidermoid carcinoma cells, we previously observed that extracellular ATP and ADP were mitogens and they synergized with other growth factors (Huang, N., Wang, D. and Heppel, L. A. (1989) Proc. Natl. Acad. Sci. USA 86, 7904-7908). We now report that ATP and ADP stimulated Na+ entry, intracellular alkalinization and Na+/K+ pump activity, which are early events that had been proposed to play a central role in DNA synthesis. In addition, ATP, ADP and AMPPNP stimulated uridine uptake by a pathway involving arachidonic acid metabolism. In A431 cells, activation of protein kinase C also contributed to ATP-dependent stimulation of uridine uptake. Concentrations of indomethacin and pertussis toxin which inhibited uridine uptake also blocked arachidonic acid metabolism and DNA synthesis. ATP acted as a competence factor. Interestingly, ATP did not have to be continuously present to stimulate uridine uptake. It was equally effective even when it was washed away after brief treatment of cells.


Effect of D2O on maize plasmalemma ATPase and electron transport coupled proton pumping. Heavy water (D2O) has been used as a putative inhibitor of the plasma membrane H(+)-ATPase and the plasma membrane redox system. Concentrations above 50% D2O inhibited H+ secretion and the plasma membrane redox system of Zea mays L. roots. Inhibition of H+ secretion by vanadate was reduced in presence of D2O. The plasma membrane of roots was transiently depolarized after the addition of heavy water in concentrations above 5%. The repolarization of the plasma membrane that takes place while the H+ secretion is still reduced by heavy water indicates that, despite the overall inhibiting effect of D2O, the plant is still able to regulate the membrane potential.


Increased sensitivity of an adriamycin-resistant human small cell lung carcinoma cell line to mitochondrial inhibitors. The energy metabolism of an atypical multidrug resistant human small cell lung carcinoma cell line (GLC4/ADR) was studied. The glycolytic rate was 30% reduced and the glucose-6-phosphate dehydrogenase activity 2-fold increased in GLC4/ADR compared to the parental sensitive line (GLC4). Although mitochondrial respiration activities were similar in both cell lines, GLC4/ADR was more sensitive to the antimitochondrial drugs doxycycline and oligomycin, while cross-resistance was observed for the glycolytic inhibitor 2-deoxyglucose and for the antimitochondrial drug rhodamine-123. Continuous incubation with doxycycline induced a dramatic reduction of mitochondrial mRNAs in both cell lines, whereas a strong reduction of the nuclear-coded mRNA for subunit IV of cytochrome c oxidase was induced in GLC4/ADR only. Incubation with doxycycline had an additive effect on the cytotoxicity of adriamycin in both cell lines. Thus, a form of collateral sensitivity to antimitochondrial drugs may exist in atypical multidrug resistant cell lines.


Regulation of intracellular Mg2+ by superoxide in amnion cells. Changes of intracellular free Mg2+ concentration ([Mg2+]i) in human amnion cells induced by superoxide anion were determined using a highly Mg(2+)-sensitive fluorescent dye Mg(2+)-fura2 or Mg(2+)-indol. Superoxide anion, produced by addition of xanthine oxidase to hypoxanthine, induced decrease of [Mg2+]i. The decrease was significantly inhibited by an anion channel blocker, 4,4'diisothiocyano-2,2' disulfonic acid stilbene (DIDS). Superoxide dismutase (SOD), injected into cells by cell fusion, also inhibited the change of [Mg2+]i, but catalase did not. Superoxide anion induced prompt increase of intracellular pH (pHi) as well as decrease of [Mg2+]i and subsequently activated the increase of intracellular free Ca2+ ([Ca2+]i) and the release of arachidonate. In contrast to superoxide anion, NH4Cl which induces increase of pHi in amnion cells increased [Mg2+]i. The elevation of basal level of [Mg2+]i by Mg(2+)-ionophore inhibited the change of [Ca2+]i and the release of arachidonate induced by superoxide anion. These results suggest that superoxide anion, transported through anion channels into cells, decreases [Mg2+]i directly, not due to a pH-effect and that the decrease of [Mg2+]i may regulate biological functions of the cells via increase of [Ca2+]i.


Significant existence of deleted mitochondrial DNA in cirrhotic liver surrounding hepatic tumor. To understand the role of mitochondria in carcinogenesis, we compared the amount of deleted mtDNAs between human hepatic tumors and surrounding cirrhotic portion of the liver of ten patients by using polymerase chain reaction (PCR). Multiple mtDNA deletions were detected in cirrhotic portion, but no deletions were detected in the tumor portion. Direct sequencing of the fragments revealed a 7,079-bp deletion (nucleotide position 8,992-16,072) involving no direct repeated sequences and a 7,436-bp deletion (position 8,649-16,084) involving a 12-bp directly repeated sequence of 5'-CATCAACAACCG-3' exists in both the ATP6 gene and the D-loop region. These mtDNA mutations could be one of the endogenous factors that induce somatic mutations in nuclear genome and etiologically contribute to human carcinogenesis.


Biochemical characterization of binding sites for dihydropyridine and omega-conotoxin in brain of adult chicken. Binding studies using the calcium channel blockers omega-conotoxin and dihydropyridine revealed a rather equal amount of binding sites in brain from adult chicken. The omega-conotoxin binding sites could be solubilized using digitonin, without substantial loss, whereas a great decrease in dihydropyridine binding sites was observed, indicating that both types of binding sites have different sensitivity to solubilization. In contrast to ion exchange chromatography where both binding sites comigrated, glycoprotein affinity chromatography led to a different partition of the binding sites in the flow through and eluate fractions. Our results indicate that both types of calcium channel blockers bind to different targets in adult chicken.


The internal calcium store in airway muscle: emptying, refilling and chloride. Possible new directions for drug development. This review examines the ionic mechanisms underlying acetylcholine (Ach) depolarization of airway smooth muscle and suggests that multiple mechanisms are involved. Increased chloride and nonspecific cation conductance, and decreased or rapidly inactivating potassium conductances seem to be involved. Chloride ions also seem to play an important role in determining whether Ca2+ remains inside or is replenished in the sarcoplasmic reticulum (SR). The physiological role of Ach-induced depolarization is analysed and is suggested to be the promotion of the refilling of Ca2+ stores, partly through a direct refilling of SR-Ca2+ stores by way of an L-type Ca2+ channel. This refilling is promoted by Ca2+ channel agonists and is independent of the transmembrane potential. Ca(2+)-release by a variety of agonists leads to depolarization and stable membrane oscillations which depend on the action of the Ca(2+)-store uptake mechanisms in order to function. These oscillations may play a role in prolonged bronchoconstriction. Better knowledge of the control mechanisms of Cai2+ is likely to reveal new targets for the therapy of asthma and provide a better understanding of the function of airway smooth muscle.


Calcium channels and their involvement in cardiovascular disease. The widespread distribution of L-type Ca2+ channels in the cardiovascular system makes that system a natural 'target' for drugs which inhibit L-type Ca2+ channel activity. Now that tissue-dependent differences in the chemical composition of the calcium antagonist binding sites have been recognized it may be possible to develop drugs with enhanced tissue selectivity. The search for new compounds should not be restricted to improvements in tissue selectivity, however. Some of the ancillary properties of the L-type Ca2+ channel inhibitors--including their ability to protect against lipid peroxidation--should not be lost because these ancillary properties may contribute significantly to their usefulness as therapeutic agents.


The anticollagenolytic potential of lymecycline in the long-term treatment of reactive arthritis. We sought to determine the antiinflammatory properties of lymecycline in the long-term treatment of reactive arthritis (ReA). Quantitative assay of collagenase activity by densitometry after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Therapeutic levels of lymecycline do not directly inhibit the activity of human neutrophil interstitial collagenase, but can prevent the oxidative activation of latent human neutrophil collagenase. This non-antimicrobial, anticollagenolytic property of lymecycline may contribute to its therapeutic efficacy in the treatment of patients with ReA.


Interactions between anticollagen antibodies and chondrocytes. To investigate the interactions between anticollagen antibodies and living chondrocytes. Mouse monoclonal anti-type II collagen (anti-CII) antibodies, rabbit anti-human CII, and rat anti-CII, anti-CIV, anti-CV, anti-CVI, and anti-CIX were studied in vitro to determine their ability to bind to the plasma membrane of living bovine chondrocytes. Mouse monoclonal anti-CII, rabbit anti-CII, and rat anti-CII, anti-CV, and anti-CIX were shown to bind in vitro to the plasma membrane of bovine chondrocytes. Antibody binding was not observed with anti-CIV, or with chondrocytes previously incubated with bacterial collagenase. A significant increase in chondrocyte caseinase and collagenase secretion was observed following sequential incubation with the monoclonal antibodies and a source of activating cytokines. These results suggest that collagen autoantibodies may exert some of their pathogenic effects on cartilage through interactions with resident chondrocytes, leading to modulation of the rate of secretion of cartilage matrix-degrading enzymes.