[Role of polymorphonuclear leukocytes (PMN) and active oxygen species in hyperthermia--enhancing effect of G-CSF on superoxide generation from PMN].

We examined in vitro the effect of G-CSF and temperature on superoxide (O2-) generation by Cypridina luciferin analog (CLA) dependent chemiluminescence. PMN significantly generated O2- at the concentration of G-CSF 25 ng/ml or more at 37 degrees C within the range of 0.1 from 1,000 ng/ml. O2- generation from PMN was remarkably enhanced, stimulated by opsonized zymosan (OZ) and phorbol myristate acetate (PMA), at 41 degrees C as compared with 37 degrees C. O2- generation was enhanced with the addition of 25 ng/ml of G-CSF at 41 degrees C as compared to without it at 41 degrees C. A significant enhancement of O2- generation from PMN was observed at 25 ng/ml G-CSF and 41 degrees C.


[Transcatheter arterial embolization therapy of metastatic bony tumor in hepatocellular carcinoma--a case report].

Metastatic bony tumor was found in a patient with liver metastasis of hepatocellular carcinoma, 5 years and 2 months after hepatectomy. Transcatheter arterial embolization therapy via lumbar artery was performed two times, at first with 20 mg of doxorubicin. The response was Progressive Disease by CT and bone scintigraphy, but recovery of performance status 4 to 2 was achieved in five days. In conclusion, transcatheter arterial embolization therapy may be a choice of therapy for metastatic bony tumor of hepatocellular carcinoma.


[Pharmacokinetics of the interferon inducer PHL-6 and interferon synthesis in target organs under various methods of drug administration].

The pharmacokinetics of PHL-6 and interferon synthesis dynamic in the target organs (tissues) of mice were studied during its and intraperitoneal administration. In the experimental setting, there was a direct correlation between the interferon production in the murine organs with single PHL-6 and distribution of 14C PHL-6. The highest radioactivity with its oral administration was detected in the liver and intestine. Interferon was actively synthesized in the intestine, liver and serum, showing the levels of 20000, 1024-2048 and 512-1024 IU/ml, respectively. The prolonged action of the drug was in good agreement with the low PHL-6 excretion from the body. It was also shown that almost the whole radiation dose 1 (greater than 98%) was excreted with feces and urine after single and chronic administrations of uniformly labeled PHL-6 which proved important clinical drug use.


Secretion of lysosomal and digestive enzymes into pancreatic juice under physiological and pathological conditions in rabbits.

To investigate the possible secretion of lysosomal enzymes into pancreatic juice during stimulation with a pancreatic secretagogue under both physiological and pathological conditions, we measured the amount of cathepsin B, a lysosomal enzyme, in the pancreatic juice during the infusion of 6 different concentrations of caerulein (0.02, 0.05, 0.2, 0.5, 1.0, and 2.0 micrograms/kg. hr). In one group of rabbits the pancreatic duct was only cannulated (free-flow group); in others the pancreatic duct was obstructed for 7 hours and secretin was infused at 0.2 CU/kg. hr (obstructed group). In addition, we evaluated the effect of the intraduodenal instillation of a liquid meal (2 g/kg) on the secretion of lysosomal enzymes into pancreatic juice. Caerulein stimulated the secretion of cathepsin B into pancreatic juice in a dose-dependent manner, as it did that of amylase, and at higher concentrations of caerulein (1.0 and 2.0 micrograms/kg. hr), both cathepsin B output and amylase output were decreased. There was a significant positive correlation between cathepsin B output and amylase output into pancreatic juice during stimulation with caerulein. Blockage of the pancreatic duct for 7 hours caused a significant rise in serum amylase levels and a redistribution of cathepsin B activity in the pancreatic subcellular fractions, as a result of which an increased amount of cathepsin B was recovered in the pellet obtained by 1000 x g centrifugation for 15 min, which contained many zymogen granules. These changes noted after short-term pancreatic duct obstruction are very similar to those previously noted in the early stage of diet-and caerulein-induced experimental pancreatitis, suggesting the colocalization of lysosomal enzyme and digestive enzymes. In the duct-obstructed animals, the secretion of cathepsin B stimulated by caerulein was significantly greater than in the free-flow group. Furthermore, the intraduodenal instillation of a liquid meal caused the secretion of cathepsin B into the pancreatic juice along with amylase. These results indicate that under physiological conditions, such as food intake, lysosomal enzymes are secreted into the pancreatic juice in response to stimulation by gut hormones in the same manner as classical pancreatic digestive enzymes. Moreover, zymogen colocalized with lysosomal enzymes in duct-obstructed animals is secreted into pancreatic juice in increased amounts together with digestive enzymes; this finding suggests that lysosomal enzymes play important pathophysiological roles in pancreatic juice and that acinar cells are altered to maintain cellular organization by secreting the potentially dangerous lysosomal enzymes. This pancreatic duct-obstructed rabbit model should be useful in clarifying the early events of acute pancreatitis.


Effect of 3-hour pancreatic duct obstruction on pancreatic lysosomal and digestive enzymes in rabbits.

We studied the effect of short-term (3 hours) pancreatic duct obstruction (PDO) on the exocrine pancreas and on the secretion of lysosomal enzymes into the pancreatic juice of rabbits during stimulation by pancreatic secretagogues. The following evaluations were made: serum amylase levels, pancreatic water content, pancreatic amylase, trypsinogen and cathepsin B content, and output of pancreatic enzymes and lysosomal hydrolases when stimulated by secretin and caerulein as well as the distribution of cathepsin B in subcellular fraction. PDO for 3 hours plus secretin infusion caused a significant rise in serum amylase levels, pancreatic water content, and pancreatic amylase and trypsinogen content due to congestion of digestive enzymes during PDO. There was also a redistribution of cathepsin B from the lysosomal fraction to the zymogen fraction. In normal rabbits and in those with only secretin infusion, caerulein stimulated the secretion of cathepsin B, into pancreatic juice. Just after PDO, the secretion of cathepsin B, amylase and trypsinogen significantly decreased. By 24 hours after PDO, the output of cathepsin B stimulated by caerulein and secretin had increased significantly. Amylase and trypsinogen output were also significantly increased at this stage, in both the secretin and caerulein fractions. These results indicate that the secretion of lysosomal enzymes into pancreatic juice is stimulated by gut hormones, such as caerulein, in the normal physiological state and in pathological states, such as PDO. These results also show augmented secretion of both lysosomal enzymes and pancreatic digestive enzymes in the recovery stage after PDO and their important roles at this stage.(ABSTRACT TRUNCATED AT 250 WORDS)


[Relationship between endothelial cells and extracellular matrix: investigation using the model of angiogenesis in vitro].

Using model of angiogenesis in vitro, the relationship between endothelial cells and extracellular matrix was studied. Endothelial cells of bovine brain microvascular vessels (BBECs), carotid artery (BCECs) and aorta (BAECs) were cultured on type I collagen gel and Matrigel. BBECs make tubular structures and BCECs and BAECs grow and make confluent monolayer on type I collagen gel. BCECs and BAECs make tubular structures when second layer of collagen gel was overlaid. BBECs, BCECs and BAECs rapidly make tubular structures on Matrigel. These morphological changes were not affected by basic fibroblast growth factor. The effect of extracellular matrices on the cell kinetics and morphology of cultured bovine carotid artery endothelial cells was studied. Endothelial cells show a cobble stone appearance on plastic and type I collagen gel. They proliferate and make capillary-like structures on reconstituted gels composed of type I collagen and basement membrane substrata. And that endothelial cells forming capillary-like structures were increased with the increase in the concentration of basement membrane substrata. Transmission electromicroscopic examination study revealed endothelial cells forming capillary-like structures have junctional complexes on type I collagen and its mixture with basement membrane substrata. But endothelial cells on basement membrane substrata have no junctional complexes. These results suggest that BBECs have more potent angiogenic ability than BCECs and BAECs. And that proliferation and morphogenesis of endothelial cells are regulated by extracellular matrices.


[The effect of nicardipine on angiogenesis in vitro].

We studied the effect of nicardipine, a calcium channel blocker, on the morphological change of endothelial cells in vitro. Cultured endothelial cells derived from bovine carotid artery make tubular structures between collagen gel layers. Tube formation of endothelial cells was suppressed by culture with 10(-9)-10(-5) M of nicardipine in a dose dependent manner. Migration of endothelial cells was also suppressed by the same dose of nicardipine. However, proliferation of endothelial cells was not enhanced. These findings suggest that nicardipine acts as an inhibitor of angiogenesis in vitro by inhibiting the migration of endothelial cells.


The detection of developmental problems in children.

General practitioners are ideally placed to assist in the identification of children whose development is slower than expected for age. In this article various methods of detecting developmental delay are discussed, and an algorithm is proposed for application in a busy office.


Biological markers of alcoholism.

The roles of two categories of biological markers--those relating to alcohol consumption and those relating to the risk of developing alcohol related problems--are reviewed. Platelet Mono-amino oxidase levels are low in individuals with a strong inheritance of their drinking problem (Cloninger Type II alcoholics). Elevated gamma-glutamyl transpeptidase (GGT) levels identify regular heavy drinkers with a sensitivity between 40-60%. Mean corpuscular volume (MCV) has a high specificity (95%) and is useful in detecting regular heavy drinkers in the ambulatory care population if other causes of an elevated MCV can be included.


Involvement of tyrosine kinase and protein kinase C in platelet-activating-factor-induced c-fos gene expression in A-431 cells.

In A-431 cells, platelet-activating factor (PAF) induces the expression of c-fos and TIS-1 genes in both the absence and the presence of cycloheximide in a structurally specific and receptor-coupled manner. We have now investigated the molecular mechanisms of this response, particularly in relation to the role of protein kinases. Pretreatment of cells with genistein or methyl-2,5-dihydroxycinnamate (tyrosine kinase inhibitors) or staurosporine (a protein kinase C inhibitor) for 20 min abolished the c-fos expression induced by PAF. Interestingly, when genistein was added 90 s after addition of PAF, no inhibition was observed. Similarly, staurosporine did not inhibit c-fos expression when added 8 min after PAF addition to the cells. These inhibitions were dose-dependent (IC50 for staurosporine was 180 nM, and for genistein 50 microM). Simultaneous addition of PAF and phorbol 12-myristate 13-acetate (PMA) did not give a synergistic effect on c-fos expression. Pretreatment of cells with PMA had no effect on [3H]PAF binding, but abolished the PAF-induced gene expression. PAF-stimulated gene expression was desensitized if cells were pretreated with PAF. Interestingly, epidermal growth factor was able to stimulate c-fos expression in PAF-desensitized cells, and thus indicated involvement of distinct mechanisms for the two stimuli. Forskolin, an activator of adenylate cyclase, did not induce c-fos expression and had no effect on the PAF response. Exposure of cells to PAF for as little as 1 min, followed by its removal, was sufficient to activate the gene expression and demonstrated the rapidity and the exquisite nature of the signalling involved in this process. It is concluded that activation of PAF receptor (a proposed G-protein-coupled receptor) causes rapid production of signals which induce the expression of c-fos gene and that this is mediated via tyrosine kinase and protein kinase C.


Cloning of a functional Burkitt's lymphoma polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) gene promoter by the polymerase chain reaction, and its interaction with inducible cellular factors.

The promoter of the human gene encoding the stress-responsive protein polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) was isolated from Burkitt's lymphoma cells by PCR. This promoter DNA segment (termed BiP670) or one of its 5' deletion derivatives was fused to the bacterial chloramphenicol acetyltransferase gene and introduced into HeLa cells for transient expression. BiP670 retained transcriptional activity at both the basal and Ca2+ ionophore A23187-inducible levels. However, there was no significant increase in promoter activity following a 5 h induction with 7 microM-A23187, and less than 5-fold induction at 15 h. In contrast, the steady-state mRNA level was induced by 18-fold at 5 h. The in vivo transactivation assays with BiP670 5' deletion derivatives indicate that the putative A23187-inducible element is located within a 70 bp DNA segment (i.e. spanning -39 to -107 bp upstream of the transcriptional initiation site). Using an in vitro gel mobility shift assay, A23187-inducible nuclear factors were identified from HeLa cell extracts. DNA-binding competition experiments also suggest that the 70 bp DNA segment contains a potential sequence motif for the binding of the A23187-inducible nuclear factors.


The effect of insulin-like growth factor II on glucose uptake and metabolism in rat skeletal muscle in vitro.

The effect of insulin-like growth factor II (IGF II) on the rates of lactate formation, glycogen synthesis and glucose transport in the presence of a range of concentrations of insulin were investigated using an isolated preparation of rat skeletal muscle. IGF II, at a concentration of 65 ng/ml, caused a small but significant increase in the rates of these processes at a basal physiological insulin concentration (10 muunits/ml), but was without effect in the presence of 1, 100, 1000 or 10,000 muunits of insulin/ml. Hence IGF II increased the insulin sensitivity of this tissue. This effect was removed if the incubation medium was supplemented with an equimolar concentration of IGF binding protein 1 (BP1). It is suggested that changes in the concentration of IGF II and/or BP1 may regulate glucose uptake and metabolism in skeletal muscle and have physiological significance in the control of blood glucose level.


Sequence requirements for processing of proinsulin in transfected mouse pituitary AtT20 cells.

To investigate the sequence requirements for proteolytic processing of prohormones at pairs of basic amino acids, normal and mutant proinsulins were expressed in the mouse pituitary corticotrophic cell line AtT20. The extent of processing was determined by h.p.l.c. analysis of insulin-like immunoreactivity secreted into the media of transfected cells. In this model system, normal proinsulin was efficiently processed to insulin. The mutant des-38-62-proinsulin, in which all but six amino acids of the C-peptide were deleted, was also processed to insulin but less efficiently than the wild-type. The mutant Lys64-Arg65 to Thr64-Arg65 was partially processed to insulin, while the mutant Arg31-Arg32 to Arg31-Gly32 was not processed at either site. These results indicate: (i) that a six-amino-acid spacer between the two pairs of basic amino acids in proinsulin is sufficient to permit processing at both sites; (ii) that the endoproteinase responsible for cleavage at the Lys64-Arg65 site will also recognize Thr64-Arg65; (iii) that the endoproteinase responsible for cleavage at the Arg31-Arg32 site will not recognize Arg31-Gly32; and (iv) that the change Arg31-Arg32 to Arg31-Gly32 affects processing at the Lys64-Arg65 site.


Cloning and properties of a cyanide hydratase gene from the phytopathogenic fungus Gloeocercospora sorghi.

The Cht gene encoding cyanide hydratase (CHT, EC 4.2.1.66), which detoxifies HCN and is thought to be important in fungal infection of cyanogenic plants, has been cloned from the phytopathogenic fungus Gloeocercospora sorghi. The gene was isolated by screening an expression library of G. sorghi using a CHT-specific antibody and using one of the positive cDNA clones as a probe in Southern hybridization to identify a 3.1 kb PstI genomic fragment. This PstI fragment expressed CHT activity when transformed into Aspergillus nidulans, a fungus that normally lacks CHT activity. Sequence analysis identified a single open reading frame of 1,107 base pairs which encodes a polypeptide of 40,904 daltons. The deduced amino acid sequence of CHT shares 36.5% identity to a nitrilase from the bacterium Klebsiella pneumoniae subsp. ozaenae.


Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages.

Prostaglandin H synthase is a key enzyme in the formation of prostaglandins and thromboxane from arachidonic acid. The recent cloning of a second prostaglandin H synthase gene, prostaglandin H synthase-2, which is distinct from the classic prostaglandin H synthase-1 gene, may dramatically alter our concept of how cells regulate prostanoid formation. We have recently shown that the enhanced production of prostanoids by lipopolysaccharide-primed alveolar macrophages involves the induction of a novel prostaglandin H synthase (J. Biol. Chem., (1992), 267, 14547-14550). We report here that the novel PGH synthase induced by lipopolysaccharide in alveolar macrophages is prostaglandin H synthase-2.


Common peptide epitopes in glycophorin and the endothelial sialoglycoprotein gp60.

Polyclonal anti-serum made against murine glycophorin gp3 (alpha gp) recognizes the endothelial albumin binding glycoprotein, gp60. In this study, we investigated the nature (peptide vs. carbohydrate) of the common epitope. First, a new technique was developed to remove oligosaccharides from glycoproteins that were first immobilized on filters and then subjected to beta-elimination. When greater than 90% of the glycans of gp60 were removed, alpha gp still recognized gp60 without apparent loss of affinity. Second, we used brefeldin A to accumulate unglycosylated glycophorin precursors in order to affinity-purify peptide-specific alpha gp immuno-globulins; these antibodies recognized gp60. Finally, alpha gp recognized from in vitro translations a 48 kDa putative polypeptide precursor of gp60. These different approaches indicate that gp60 and gp3 have at least one common epitope in their peptide backbones.


Alterations in the subcellular distribution of p21ras-GTPase activating protein in proliferating rat acinar cells.

Rat parotid acinar cells undergo transient proliferation in response to chronic administration of the beta-adrenergic agonist isoproterenol or epidermal growth factor (EGF). Treatment with these agents caused an increase in tyrosine phosphorylation of p21ras-GTPase activating protein (GAP). This phosphorylation event was accompanied by a redistribution of the protein from the plasma membrane to internal membrane compartments. Separation of subcellular membranes revealed increased GAP associated with a low density population of vesicles concomitant with growth stimulation as well as to the nuclear membrane, but not the nucleoplasm. Upon cessation of hyperplasia induced by isoproterenol, phosphorylated GAP present in the plasma membrane returned to control cell levels.


Soluble forms of E-selectin, ICAM-1 and VCAM-1 are present in the supernatants of cytokine activated cultured endothelial cells.

Soluble forms of the adhesion molecules ICAM-1, VCAM-1 and E-selectin have been detected in supernatants from cytokine activated cultured endothelial cells. The release has been quantified using two site enzyme immunoassays. The molecular weights of the released molecules have been determined by immunoprecipitation and are consistent with the generation of soluble forms by cleavage at a site close to the point of membrane insertion.


Regulation of the 202 gene expression by interferons in L929 cells.

Type I and II interferons (IFNs) stimulate the expression of the 202 and 2'-5' oligoadenylate synthetase (OASE) genes in L929, NIH 3T3 and LM-TK- fibroblastic cell lines. In two other cell lines, B16 melanoma and F9 teratocarcinoma, these cytokines induce OASE but not the 202 mRNA. In L929 cells, IFN-alpha induces the 202 mRNA at concentrations between 10 and 10(3) units/ml. To achieve maximal induction of the 202 mRNA, continuous exposure of L929 cells to IFN-alpha is necessary, whereas 30 minutes of exposure are sufficient to trigger maximal upregulation of the OASE transcript. The induction of the 202 mRNA is the consequence of both transcriptional and post-transcriptional events. Cycloheximide, a known inhibitor of protein synthesis, does not block the induction of 202 mRNA by IFN-alpha, demonstrating that new protein synthesis is not required for this effect. Protein kinase C, arachidonic acid metabolism via the cyclooxygenase or the lipoxygenase pathways and cAMP are not involved as second messengers in the induction of the 202 mRNA by IFN-alpha in L929 cells.


Cytosolic Ca(2+)-induced modulation of ion selectivity and amiloride sensitivity of a cation channel and beta agonist action in fetal lung epithelium.

The cytosolic Ca2+ concentration ([Ca2+]i) affects many cell functions, including the modulation of ion channel activity. In this study patch clamp experiments using primary cultures of fetal distal lung epithelium (FDLE) demonstrated that the elevation of [Ca2+]i modulated a 25pS amiloride-blockable non selective cation (NSC) channel's ion selectivity and sensitivity to amiloride. An elevation of [Ca2+]i from 0.1 microM to 1mM both increased open probability (Po) and decreased the ratio of the permeability to Na to the permeability to K (PNa/PK) from 1.96 +/- 0.11 (SEM, n = 6) to 0.88 +/- 0.04 (n = 6). Within the range of [Ca2+]i from 0.1 microM to 100 microM amiloride (0.5 microM) decreased Po, however amiloride (0.5 microM) no longer affected Po of the NSC channel when [Ca2+]i was increased to 1mM under physiologic membrane potentials. A beta adrenergic agonist (terbutaline, 10 microM) increased Po in cell-attached patches from almost 0 (Po less than 0.01; n = 9) to 0.39 +/- 0.09 (n = 9) and [Ca2+]i from 40 +/- 6nM (n = 9) to more than 1 microM. This suggested that amiloride-blockable NSC channel activity and ion permeability are modulated by changes in [Ca2+]i near physiologic membrane potentials and a beta adrenergic agonist increases [Ca2+]i to more than 1 microM (unlike other epithelial including adult alveolar cells) which is associated with activation the NSC channel.


Immunological and conformation characterization of a phosphorylated immunodominant epitope on the paired helical filaments found in Alzheimer's disease.

The immunological recognition pattern of one of the most commonly used monoclonal antibodies, PHF-1, which detects the paired helical filaments of Alzheimer's disease, exhibits a high degree of similarity with the recognition of a polyclonal antibody, anti-T3P, raised against a synthetic phosphopeptide, GAEIVYKS(Phospho)PVVSGD, corresponding to amino acids 389-402 of the microtubule-associated protein tau. A panel of 16 synthetic non-phosphorylated and phosphorylated peptides, excised from different regions of tau and peptide analogs thereof, were used to show that PHF-1 is indeed directed against the T3 fragment. Circular dichroism spectroscopy shows that the phosphorylated peptide exhibits a limited propensity to form intramolecular beta-pleated sheets, and alteration is found in the reverse-turn structure that dominates the middle section of the molecule. The shift in the turn-forming amino acids may also allow a stacking procedure, may interfere with microtubule assembly, and, consequently, may be accountable for deposit formation.


The non specificity of specific nitric oxide synthase inhibitors.

L-NAME (Nw-Nitro-L-arginine methylester) and L-NMMA (NG- Monomethyl-L-arginine, monoacetate) are used widely as nitric oxide (NO) synthase inhibitors. Because of their functional groups (alcohols, amines and carboxylates), it appeared that they could interact with iron in a variety of systems. Using three in vitro models we observed these two compounds had inhibitory effects on cytochrome C reduction by ferrous iron, by ferrous iron accelerated by an unsaturated fatty acid or by epinephrine. This suggests that L-NAME and L-NMMA could have effects in iron containing systems found intracellularly apart from their inhibition of (NO) synthesis.


Effects of insulin and tyrosine kinase inhibitor on ion transport in the alveolar cell of the fetal lung.

We studied the effect of insulin and lavendustin-A (a tyrosine kinase inhibitor) on the short-circuit current (ISC) of primary cultures of fetal distal rat lung epithelium (FDLE). Insulin (2 microM) on the basolateral side of the monolayer increased ISC from 5.76 +/- 0.83 microA/cm2 (SEM, n = 7) to 7.23 +/- 1.00 microA/cm2 (p less than 0.01) under control conditions, and from 1.00 +/- 0.31 microA/cm1 to 1.53 +/- 0.34 microA/cm2 (p less than 0.05, n = 4) when amiloride (10 microM) was present on the apical side of the monolayer. Thus insulin increased both the amiloride-sensitive and insensitive ISC with the insulin-induced increase in ISC in the absence of amiloride (1.47 +/- 0.22 microA/cm2, n = 7) being significantly larger than that in the presence of 10 microM amiloride (0.53 +/- 0.14 microA/cm2, n = 4; p less than 0.025). Insulin's effect reached steady state in 1 hr. Lavendustin-A (10 microM), a tyrosine kinase inhibitor, applied to the apical side of the monolayer attenuated but did not completely block insulin's ability to increase in ISC; i.e., insulin increased ISC in lavendustin-A treated monolayers (0.63 +/- 0.09 microA/cm2, n = 5; p less than 0.0025) but the increase was significantly smaller than that without the pretreatment of lavendustin-A (p less than 0.05). In the presence of amiloride (10 microM) and lavendustin-A (10 microM) insulin was no longer able to increase ISC (change in ISC = 0.04 +/- 0.03 microA/cm2, n = 6), suggesting that lavendustin-A had blocked the insulin's effect on the amiloride-insensitive ISC. Lavendustin-A (10 microM) had no significant effect on the basal ISC in control and amiloride treated monolayers. Our studies demonstrate that insulin increases amiloride-insensitive ISC in FDLE via lavendustin-A sensitive tyrosine kinase and that insulin's action on the amiloride-sensitive ISC of FDLE is mediated through a lavendustin-A insensitive (and presumably tyrosine kinase-independent) pathway.


Tyrosine-protein kinase inhibition in human erythrocytes by polyphosphoinositides (PIP and PIP2).

In human erythrocytes Ser/Thr- and Tyr-phosphorylations of cytoplasmic domain of band 3 are catalyzed by casein kinase I and Tyr-protein kinase respectively, both distributed between cytosol and membrane structures. The results reported here show that purified cytosolic Tyr-protein kinase activity, assayed on added substrates such as poly(Glu,Tyr)4:1 and isolated chymotryptic fragments of band 3 cytoplasmic domain (cdb3), is potently inhibited by PIP and even more by PIP2. Similar inhibitory effects are displayed by these polyphosphoinositides also on the endogenous Tyr-phosphorylation of band 3, when they are added to the isolated native membranes, thus suggesting their involvement in regulating in-vivo Tyr-phosphorylation of membrane proteins.


A comparison of drug binding sites on mammalian albumins.

The fluorescent probes warfarin and dansylsarcosine are known to selectively interact with binding sites I and II, respectively, on human albumin. This paper investigates whether similar binding sites exist on bovine, dog, horse, sheep and rat albumins. Binding sites on albumins were studied by: (1) displacement of warfarin and dansylsarcosine by site I (phenylbutazone) and site II (diazepam) selective ligands; (2) the effects of non-esterified fatty acids (carbon chain lengths: C5-C20) and changes in pH (6-9) on the fluorescence of warfarin and dansylsarcosine; and (3) the ability of site selective ligands to inhibit hydrolysis of 4-nitrophenyl acetate. For bovine, dog, horse, human and sheep albumins the fluorescence of bound warfarin and dansylsarcosine was selectively decreased by phenylbutazone and diazepam, respectively. For these albumins medium chain fatty acids (C1-C12) reduced the fluorescence of dansylsarcosine (maximum inhibition with C9) whereas long chain acids (C12-C20) enhanced the fluorescence of warfarin (maximum increases with C12). In addition, changes in pH from 6 to 9 increased the fluorescence of warfarin and although site I ligands (warfarin/phenylbutazone) had no pronounced effects on 4-nitrophenyl acetate hydrolysis, site II ligands (dansylsarcosine/diazepam) significantly inhibited this reaction. Rat albumin behaved differently from the other albumins studied in that the C12-C20 fatty acids and changes in pH did not enhance the fluorescence of warfarin. Moreover, the differential effects of site I and site II ligands on the fluorescence of warfarin/dansylsarcosine and hydrolysis of 4-nitrophenyl acetate were less apparent with rat albumin. The results suggest bovine, dog, horse and sheep albumins have binding sites for warfarin and dansylsarcosine with similar properties to sites I and II on human albumin. By contrast, the warfarin binding site and to a lesser degree the dansylsarcosine site, of rat albumin have different characteristics from these sites on the other albumins studied.


Inhibition of platelet activation by tyrosine kinase inhibitors.

Protein tyrosine kinase (PTK) blockers (tyrphostins) inhibit in a dose-dependent fashion thrombin-induced aggregation and serotonin release with IC50 values in the 10-35 microM concentration range. The inhibition of thrombin-induced aggregation correlates with their potency in inhibiting phosphorylation of proteins on tyrosine residues. Using metabolically 32P-labelled human platelets, it was found that the tyrphostins have no effect on the decrease in [32P]phosphatidylinositol bisphosphate but prevent the replenishment of [32P]polyphosphoinositide. Tyrphostins decreased [32P]phosphatidic acid production induced by thrombin, although never by more than 50%, and only delayed the peak of diacylglycerol, suggesting that phospholipase C was still activated. Tyrphostins inhibited the thrombin-elicited early phosphorylation of p43 and p20, substrates for protein kinase C (PKC) and myosin light chain kinase, respectively, at short times of activation. This inhibition, however, was overcome after 1 min of stimulation with thrombin. Tyrphostin AG213 also inhibited platelet aggregation and tyrosine protein phosphorylation induced by phorbol myristate acetate (PMA), but did not inhibit pleckstrin phosphorylation. These results suggest that thrombin induces the phosphorylation of proteins on tyrosine residues which most probably results in the activation of phosphoinositide kinases. The ability of tyrphostins to inhibit phosphorylation of p43 and p20 when induced by thrombin but not when induced by PMA confirms that PTKs may be involved subsequent to PKC activation.


Inhibition of mouse glutathione transferases and glutathione peroxidase II by dicumarol and other ligands.

Dicumarol, often used as a specific inhibitor of DT diaphorase (NAD(P)H:(quinone-acceptor) oxidoreductase; EC 1.6.99.2), was found to potently inhibit GSH transferases (EC 2.5.1.18). Dicumarol exhibited an IC50 of 11 microM in inhibiting the conjugation of 1-chloro-2,4-dinitrobenzene (50 microM) by GSH transferase GT-8.7, the major hepatic class mu isoenzyme of CD-1 mice. The activities of GT-8.7 and of the class pi isoenzyme, GT-9.0, toward a carcinogenic substrate, 4-nitroquinoline 1-oxide (100 microM), were inhibited by dicumarol with IC50 values of 14 and 9 microM, respectively. Dicumarol also affected GSH peroxidase II activity, inhibiting the reduction of cumene hydroperoxide by GT-10.6, the predominant class alpha GSH transferase of mouse liver, with an IC50 of 14 microM. GSH peroxidase I (EC 1.11.1.9) and GSH peroxidase II activities were resolved by chromatography of liver and testis cytosols. While inhibiting GSH peroxidase II with IC50 of 9-10 microM, dicumarol did not affect the activity of the selenoenzyme, GSH peroxidase I. Whereas several other non-substrate ligands were more potent inhibitors of 1-chloro-2,4-dinitrobenzene conjugation, dicumarol effectively inhibited GSH transferase and GSH peroxidase II activities in the range of dicumarol concentrations frequently used for detection of DT diaphorase action. These results indicate that physiological consequences resulting from the use of supramicromolar concentrations of dicumarol should not be interpreted in terms of DT diaphorase inhibition alone.


[Bone marrow involvement in Whipple's disease].

The authors present the case of a 57 years old woman who had atypical Whipple's disease characterized solely by migratory arthralgia, weight loss and unexplained anemia which had lasted for 5 years. Once the diagnosis of Whipple's disease was established on abdominal lymph node biopsy, the retrospective study of a bone marrow biopsy revealed the presence of PAS-positive sickle-formed particles within medullary macrophages. Review of the literature attests the rarity of ante-mortem diagnosis of Whipple's disease in bone marrow preparations. We suggest that bone marrow biopsies stained with the PAS technique be employed in clinical settings of chronic migratory arthralgia and unexplained anemia.


Quinolinehydrazones as inhibitors of retroviral reverse transcriptase.

The inhibitory activity of a series of 2- and 4-quinolinehydrazones on retroviral reverse transcriptase has been studied on enzymes from M-MuLV, RAV-2, and on a crude lysate of HIV-1, assuming the first two enzymes as potential models of the third. The highest activity is mainly found in lipophilic, water soluble 4-quinolinehydrazones. The inhibitory activity of these compounds decreases in changing from the M-MuLV to the RAV-2, and HIV-1 enzymes, in this order.


[Growth behavior of Enterobacteriaceae in BRILA-MUG-broth and in different modifications of broth].

Both substances, brilliant green and bile, inhibit the growth of gram-positive bacteria in culture media and selectively enrich gram-negative bacteria. Therefore, the brilliant green-lactose-bile broth (BRILA) and the BRLA broth supplemented with tryptophan and methyl-umbelliferyl-beta-D-glucuronide (BRILA-MUG) contain brilliant green as well as bile. Because BRILA-MUG broth as a selective enrichment and differentiating medium of faecal coliform and total coliform bacteria, E. coli and coliforms, respectively, is recommended for testing samples of surface water according to the EC guidelines for bathing waters (no. 76/160 EWG), the question arose of the optimal combination of components in the BRILA-MUG broth. As the described investigations show, the addition of buffer substances did not improve the culture properties of the BRILA-MUG broth. However, the original BRILA broth was improved by supplementing it with buffer substances such as CaCO3 or Na2HPO4. The same effect of culture improvement was obtained by removing brilliant green. This modification of BRILA broth is practically identic with the well-known MacConkey broth. On the other hand, the modification of omitting bile from the original BRILA broth causes a remarkable impairment of the culture properties lowering bacterial counts per ml by 3-5 log. The observations suggest that brilliant green inhibits both, gram-positive bacteria as well as the gram-negative Enterobacteriaceae. Therefore, it is a selective substance of doubtful usefulness.


Identification of antigenic regions of the human Ro 60 kDa protein using recombinant antigen and synthetic peptides.

Autoantibodies reacting with the human Ro 60 kDa protein are present in anti-Ro/SS-A positive sera from patients with several different connective tissue diseases including Sjögren's syndrome and systemic lupus erythematosus. To investigate the humoral immune response to this protein, the pattern of antibody recognition of recombinant Ro 60 kDa proteins encoded by both full-length and deletion clones was analysed by immunoblotting. An antigenic region recognized by nearly all anti-Ro 60 kDa positive sera was found to reside in the middle part of the protein. In addition, some sera reacted with two other antigenic regions located in the amino-terminal and carboxyl-terminal part of the protein. For further mapping, overlapping peptides covering the most frequently recognized region of the protein were synthesized by solid-phase methods and used as antigens in ELISA. Three major patterns of reactivity to Ro 60 kDa peptides were found. These results not only indicate the presence of an immunodominant region but also heterogeneity in the autoimmune human response to the Ro 60 kDa antigen.


Neurochemical effects of electrically and chemically induced seizures: an in vivo microdialysis study in the rat hippocampus.

This study examined the effects of electroconvulsive shock (ECS) on interstitial concentrations of serotonin (5-HT), its metabolite 5-hydroxyindoleacetic acid (5-HIAA), acetylcholine and choline, and the dopamine metabolite homovanillic acid (HVA) in the hippocampus of freely moving rats using online brain microdialysis. The effects of ECS on 5-HT, 5-HIAA, and HVA were compared to the effects of seizures induced by the convulsant agent flurothyl. Interstitial concentrations of 5-HT increased several fold in response to ECS and this increase was accompanied by a significant increase in the concentration of HVA. Acetylcholine and choline concentrations were also increased significantly by ECS. The ECS-induced increase in interstitial 5-HT was markedly reduced when the voltage-dependent sodium channel blocker tetrodotoxin (1 mumol/L) was added in the perfusion solution, indicating that the observed increase was of neuronal origin. Interstitial concentrations of 5-HT also increased in response to flurothyl-induced seizures and this increase was accompanied by a significant increase in the concentration of HVA. These results provide direct in vivo evidence that interstitial concentrations of 5-HT increase several fold in response to both ECS- and flurothyl-induced seizures. These observations are discussed in relation to the hypothesized role of 5-HT in ECS-induced memory deficits.


Antigenic mapping of a human lambda light chain: correlation with three dimensional structure.

Although the amino acid sequence and three-dimensional structure of human immunoglobulin light chains have been known for more than 15 years, the location of antigenic markers characteristic of lambda chains has not been determined. Here, we use a set of synthetic overlapping peptides to completely model the sequence of the lambda chain Mcg and test these for the binding of rabbit and goat antisera specific for lambda chain determinants. We assess peptide contributions to lambda-antigenic reactivity and also to identify a portion of C-region where conformational factors contribute to the antigenicity. Specific determinants occur both in the constant and variable (first and third framework) domains of the molecule. The fourth framework of the variable region, a segment specified by the joining gene, is also recognized and cross-reacts antigenically with the homologous region of T cell receptor beta chains. Major lambda specific determinants are localized in the N- and C-terminal segments, which are linear and devoid of major conformational folding. Other segments that are strongly antigenic, such as the third framework of the V region (residue 78-93) and a segment of the constant region (residues 177-192), show strong conformational dependence in antigenicity.


Induced resistance of trypsin to sodium dodecylsulfate upon complex formation with trypsin inhibitor.

The stabilities of trypsin and soybean trypsin inhibitor in sodium dodecylsulfate (SDS) were examined by SDS-polyacrylamide gel electrophoresis (PAGE). Both samples contained several bands, all of which migrated to positions corresponding to the appropriate molecular weight or less, even when the samples were unheated, suggesting that both the trypsin and trypsin inhibitor are susceptible to SDS-induced denaturation. When they were mixed together prior to addition of SDS-PAGE sample buffer (1% SDS), a new smearing band appeared which corresponded to a molecular weight of around 46,000, suggesting that these proteins form a stable complex in SDS. This was confirmed by electroblotting and sequence analysis, which indicated that this band contains both the trypsin and inhibitor sequences. At a fixed concentration of the inhibitor, increasing concentrations of the trypsin resulted in an increase in the intensity of the complex band. When the mixture was heated for 10 min in 1% SDS, the complex band disappeared in a temperature-dependent manner. The melting temperature determined under the experimental conditions used was about 35 degrees C. Similar results were obtained with Bowman-Birk trypsin inhibitor, except that the complex with the above inhibitor had a higher melting temperature, around 41 degrees C, suggesting that the Bowman-Birk inhibitor/trypsin complex is more stable than the soybean inhibitor/trypsin complex.


Immunological evidence for a conformational difference between recombinant bovine rhodanese and rhodanese purified from bovine liver.

Rhodanese has been utilized as a model enzyme for the study of protein structure-function relationships. The enzyme has recently been cloned and the recombinant enzyme is now available for investigation. However, prior to use in structure-function studies, the recombinant enzyme must be shown to have the same structure and activity as the bovine liver enzyme used in the previous studies. An immunological study of the conformations of these enzyme conformers is described. Three antibodies (two monoclonal and one polyclonal, site-directed antibody) were shown to detect distinct and nonoverlapping epitopes. The epitopes of the monoclonal antirhodanese antibodies (R207 and MAB11) were mapped to the same CNBr digest fragment of the amino terminal domain of rhodanese, and the epitope of the site-directed antibody prepared against the interdomain tether sequence of rhodanese (PAT-T1) was mapped to that region of rhodanese (residues 142-156). The rhodanese conformers were studied by monitoring the accessibility of the epitopes recognized by each antibody in each conformer using an indirect ELISA. None of the antibodies could detect its epitope on the purified liver enzyme. Two of the antibodies (R207 and PAT-T1) could also not detect their epitopes on the recombinant enzyme. However, MAB11 did detect a conformational difference between the natural and recombinant rhodanese conformers, indicating the conformational difference is localized in the first 73 amino acids of rhodanese. This difference presumably reflects the difference in the histories of the two enzymes and may be due to differences in enzyme folding, differences in the purification procedures, and differences in storage conditions--all of which could influence the final conformation of the enzyme.


High-level expression of recombinant human FK-binding protein from a fusion precursor.

The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L in Escherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a approximately 30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed in E. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to [3H]FK-506. The fusion construct, CKS-FKBP, was also found to bind [3H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein.


Both ends of Escherichia coli ribosomal protein S13 are immunochemically accessible in situ.

To investigate the structure of Escherichia coli ribosomal protein S13 in 30S ribosomal subunits, we have previously generated 22 S13 specific monoclonal antibodies and mapped their specific epitopes to the S13 sequence. The availability of these S13 epitopes in situ has been further examined by incubating these monoclonal antibodies with 30S ribosomal subunits and analyzing formation of monoclonal antibody-linked ribosome dimers by sucrose gradients centrifugation. We have found that none of the 22 monoclonal antibodies makes ribosome dimers individually as do typical antisera. However, one monoclonal antibody, designated AS13-MAb 2, reacts with 30S ribosomal subunits to form immunocomplexes sedimenting faster than subunit monomers. When AS13-MAb 2 is paired with any one of three monoclonal antibodies directed to the S13 C-terminal epitopes, dimer formation is observed. Other pairs of monoclonal antibodies directed to distinct S13 epitopes have been tested similarly for dimer formation. Monoclonal antibody AS13-MAb 22, directed to the N-terminal region of 22 residues, also causes subunits to form typical dimers, but only if paired with one of the three monoclonal antibodies directed to the S13 C-terminal region. The close proximity of the epitopes recognized by AS13-MAbs 2 and 22 has been established by the mutual competition between the antibodies binding to intact 30S subunits. These results corroborate our previous observation, using polyclonal antibodies, that S13 has more than one epitope exposed on 30S subunits. Our finding that sequences on both ends of the S13 molecule are immunochemically accessible provides information about the molecular organization of S13 in situ.


Mast cell degranulating (MCD) peptide analogs with reduced ring structure.

Mast cell degranulating (MCD) peptide, a component of bee venom, is a 22 amino acid peptide with two disulfide bridges. In this first structure-activity study of MCD peptide, three analogs were synthesized and tested: two analogs shortened by omitting sequences 6-10 and 8-13, respectively, and one analog lacking the disulfide bridge between cysteine residues 5 and 19. These analogs were synthesized by solid-phase methods and were compared to MCD peptide in two assays for inflammation: histamine release from mast cells and superoxide anion release from neutrophils. All three analogs produced histamine release, although with only about one fifth of the activity of MCD peptide. Superoxide anion-releasing activity, however, did not parallel histamine release. MCD peptide did not release superoxide anion, while the 6-10 and 8-13 deletion analogs were strong and weak stimulants, respectively, of this anion. CD spectra showed that the secondary structures of the three analogs were very similar to that of MCD peptide, so that a change in secondary structure cannot completely explain the changes in releasing activities. Charge differences between the two deletion analogs and MCD peptide may explain some of the differences in activity. This is the first demonstration that the various activities of MCD peptide can be separated, and provides a lead through which the purported antiinflammatory activity of MCD peptide may possibly be explored in the future.


Aberrant expression of the simple epithelial type II keratin 8 by mouse skin carcinomas but not papillomas.

Keratins have been demonstrated to be suitable markers of changes taking place during epithelial neoplasia. Therefore, we analyzed 18 mouse skin tumors (nine papillomas and nine squamous cell carcinomas), induced either by two-stage carcinogenesis with 7,12-dimethylbenz[a]anthracene(DMBA)/12-O-tetradecanoylphorbol-13-acetat e or complete carcinogenesis with DMBA, by immunofluorescence with a monoclonal antibody to keratin (K) 8 (TROMA-1). Immunoperoxidase staining and immunoblotting were also used on selected tumor samples to further explore for the presence of K8. All of the papillomas tested were negative for the presence of K8, whereas the carcinomas were positive. The level of K8 expression in carcinomas showed a positive correlation with the degree of malignancy. Northern blot analysis using a K8 cDNA probe suggested that control of K8 expression in mouse skin tumors occurs at the transcriptional level. Double-label immunofluorescence staining using TROMA-1 and RK13 antibodies demonstrated that K8 did not generally colocalize with K13, a keratin normally found in internal stratified epithelial but aberrantly expressed in mouse epidermal tumors. Furthermore, tumors expressing high levels of K8 showed a reduced expression of K13. Histological examination of immunoperoxidase-stained tumors demonstrated that K8-positive cells were mainly found in anaplastic areas, whereas K13 foci were restricted to well-differentiated regions. Our results demonstrate that K8 expression is a marker of late stages of carcinoma progression in the mouse skin carcinogenesis model.


Expression of the transin, c-fos, and c-jun genes in rat transplantable osteosarcomas and malignant fibrous histiocytomas.

The expression of the transin, c-fos, and c-jun genes was assessed in transplantable osteosarcomas and malignant fibrous histiocytomas, as well as in pancreatic duct adenocarcinomas and hepatocellular carcinomas of rats and hamsters. Northern blot analysis revealed that both an undifferentiated osteosarcoma of spontaneous origin (SOS) and 4-hydroxyaminoquinoline 1-oxide (4-HAQO)-induced malignant fibrous histiocytomas with metastatic potential to the lung showed remarkably increased expression of transin mRNA transcripts. This was not the case for the other tumors. Interestingly, levels of transin mRNA were lower in lung metastatic lesions than in primary subcutaneous SOS tumors. The primary SOS and MFH expressed both c-fos and c-jun genes in conjunction with the transin gene, whereas the non-transin expressers, a 4-HAQO-induced osteosarcoma (COS) and the pancreatic duct adenocarcinomas, demonstrated one or the other, but not both. These results suggest a possible involvement of transin expression in the progression of spontaneous osteosarcomas and 4-HAQO-induced malignant fibrous histiocytomas in rats. Expression of the c-fos and c-jun genes may play a regulatory role in this process.


p53 mutations are absent from carcinogen-induced mouse liver tumors but occur in cell lines established from these tumors.

Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined, by single-strand conformation polymorphism analysis of polymerase chain reaction products, a total of 93 carcinogen-induced liver tumors from mice of three different strains (C3H/He, C57BL/6J, and B6C3F1) for the presence of p53 aberrations. Hepatoma lines, established from 12 liver tumors, were also included in the analysis. While structural aberrations of the p53 gene were not detected in any of the primary mouse liver tumors analyzed, single-base substitutions occurred at different locations within the p53 gene in three of the cell lines during in vitro propagation. One hepatoma line carried two point mutations on separate alleles. All four mutations were either G:C----C:G or C:G----G:C transversions. Mutations at codon 61 of the c-Ha-ras gene, which were frequent in primary liver tumors from C3H/He and B6C3F1 mice, were not detected in the hepatoma lines. Our data indicate (i) that c-Ha-ras but not p53 mutations play an important role during the early stages of mouse hepatocarcinogenesis and (ii) that p53 mutations confer a selective growth advantage to the mutated hepatoma cells in vitro.


T-cell receptors: germline polymorphism and patterns of usage in demyelinating diseases.

The genomic organization of the T-cell receptor (TCR) gene complexes accounts for many central aspects of T-cell immunobiology, including specificity and diversity. Recent data indicate that polymorphism of TCR genes is present within a species and may influence the immune phenotype of an individual. Such polymorphism has been detected by RFLP, by the presence of large regions of insertion or deletion of germline DNA, and by allelic variability of individual gene segments that are expressed. In addition to allelic variation of TCR genes, immune responses may also be influenced by the repertoire of the TCR molecules that are expressed by responding T-cell populations. In some situations, pathogenic T-cell responses may involve expression of limited numbers of TCR gene families. This is true, for example, in experimental allergic encephalomyelitis, an autoimmune nervous system disease mediated by T-cells reactive to myelin basic protein. In the human disease counterpart, multiple sclerosis, a more complex pattern of T-cell recognition to the putative autoantigen is generally present, although in some individuals a restricted response may occur. Specific therapies targeted to certain TCR molecules represents a promising approach to chronic inflammatory diseases in humans. The efficacy of such therapies will be determined in part by the TCR repertoire expressed in individual disease situations and by the potential for plasticity in the pathogenic T-cell response that may exist.


Long-term consequences of interleukin-6 overexpression in transgenic mice.

With the aim of using interleukin-6 (IL-6)-inducible promoters to express transgenes, we investigated the long-term consequences of high levels of IL-6 in mice. As a first step, we generated transgenic mice constitutively expressing the murine IL-6 at a level sufficient to induce IL-6-responsive genes. These mice were analyzed with respect to the indirect and direct consequences of elevated IL-6 expression over a time period of about 2 years. Although biologically active IL-6 was expressed from the transgene and different alterations could be documented (less immature B cells in bone marrow, expression of IL-6-inducible liver genes), the mice appeared healthy and could easily be used for breeding. Only in mice older than 18 months did we find a high incidence of lymphomas associated with different tissues. These results indicate that the side effects of long-term treatment with IL-6 are relatively moderate, and that IL-6 might be used to mediate the expression of heterologous genes in the context of functional studies.


Human interferon regulatory factor 1: intron-exon organization.

Interferon (IFN) regulatory factor 1 (IRF-1) is a transcriptional regulatory protein that mediates the transcriptional activation of the IFN-alpha and IFN-beta genes by viruses and IFNs. To characterize the mechanisms that govern the level of IRF-1 in cells, we isolated the IRF-1 gene and characterized the structure of its intronic and exonic domains and of its regulatory promoter region. A human placental genomic library was screened with an IRF-1 cDNA probe, and two clones that contained the IRF-1 gene and its 5' regulatory region were obtained. We used these clones to determine the complete nucleotide sequence for the IRF-1 gene, finding that the IRF-1 gene spanned 7.72 kb of DNA and included 10 exons and 9 introns. When the deduced amino acid sequences were compared among different species, the most conserved exons were exons 2, 3, and 4, in which the putative DNA binding domain for the IRF-1 protein is located.


Concurrent measurement of serotonin metabolism and single neuron activity changes in the lateral hypothalamus of freely behaving rat.

To further investigate the activity of serotonin neurons in relation to feeding behavior, the metabolic activity of the serotonergic system and single neuron activity changes in the lateral hypothalamic area (LHA) were investigated concurrently in freely behaving rats. The extracellular concentration of 5-hydroxyindoleacetic acid (5-HIAA), a metabolic product of serotonin in the LHA, began to increase concomitantly with the early stage of nocturnal eating. The increased 5-HIAA returned to the basal level within 3 or 4 h. In conjunction with the increase in serotonin metabolism, activity of 12 out of 30 LHA neurons (40%) increased, whereas it decreased in 7 (23%), and in 11 (37%) it showed no change. An intracerebroventricular injection of lisuride suppressed the increased activity in 7 of the 12 neurons, but had no effect on the others. These results suggest that the concurrent increase in serotonin metabolism and neuron activity changes in the LHA may occur in the early portion of the nocturnal eating period, and may be important in controlling feeding behavior.


Self-reported prevalence of disability after subarachnoid haemorrhage, with special emphasis on return to leisure and work.

The prevalences of motor and language impairments and of disabilities in activities of daily living (ADL), leisure and work were investigated in a consecutive series (n = 296) of long-term survivors of subarachnoid haemorrhage (SAH). Motor and language impairments were present in 17 and 20%, respectively. The majority reported independence in self-care (91%) and instrumental (80%) ADL, but among the self-care independent, 23% reported need of personal assistance. Leisure disability occurred in 48% and vocational disability in 40%. Hence, disabilities are more common after SAH than is indicated by occurrences of motor and language impairments. It is concluded that the discrepancy between the prevalences of impairments and of disabilities may be to a great extent caused by coping difficulties in relation to socio-demographic and geographic circumstances. The findings indicate a need for rehabilitative follow-up for virtually all SAH-patients.


Synthetic peptides as vaccines.

The use of synthetic peptides as an alternative approach to vaccination is currently being pursued. This is particularly true for viral and parasitic diseases in which no vaccines are yet available, most notably the acquired immune deficiency syndrome.


A histologic study of nonmorphogenetic forms of hereditary hearing impairment.

It appears that many forms of syndromic and nonsyndromic hereditary hearing impairment are secondary to either neuroepithelial or cochleosaccular dysfunction. Making this distinction can be difficult in human temporal bone specimens; however, this added knowledge may ultimately provide prognostic and therapeutic information in hearing habilitation. Fundamental studies using animal models of different types of hereditary deafness may also prove useful in this respect.


[Proposal for a nutritional study using a survey of anthropometric data on school-age children from the Molise countryside].

The present work is a preliminary study, preceding a larger one in the Molise countryside ; its aim is to set out methodological and operative basis to estimate the nutrition state of children aged 6-10 years, through a survey of anthropometric data. The list of considered parameters is here related. From the analysis of the different parameters it comes out that the tested population shows a trend to have a body weight too high compared to its height. That involves a more thick-set and less long-limbed body structure. Although the sample is small in number, the results enable us to believe that carrying on the study on a vast scale is suitable.


High level expression on a chimeric anti-ganglioside GD2 antibody: genomic kappa sequences improve expression in COS and CHO cells.

We report a flexible strategy for the high level expression of a recombinant human monoclonal antibody (mAb) in Chinese hamster ovary (CHO) cells, initially using COS monkey kidney cell transfections to evaluate rapidly modifications to immunoglobulin (Ig) DNA constructs. Using sequential transfections with two amplifiable markers, we generated CHO cell lines and clones that secrete 80-110 micrograms/10(6) cells/24 hours of a mouse-human chimeric IgG1 kappa mAb. This cellular productivity is considerably greater than most murine hybridomas and transfected myelomas. Our data also demonstrate that genomic kappa sequences can improve mAb expression in COS and CHO cells. As a paradigm, we focused our expression studies on a human chimeric form of 3F8, a murine mAb that binds to ganglioside GD2 on neuroblastoma and melanoma tumor cells.


Growth and production kinetics of human x mouse and mouse hybridoma cells at reduced temperature and serum content.

The growth and production kinetics of a mouse hybridoma cell line and a human-mouse heterohybridoma were analyzed under conditions of reduced temperature and serum content. The mouse hybridoma P24 had a constant cell specific production rate and RNA content, while the heterohybridoma 3D6-LC4 showed growth associated production kinetics and an increased RNA content at higher growth rates. This behaviour of 3D6-LC4 cells can be explained by the unusual cell cycle kinetics of this line, which can be arrested in any phase under growth limiting conditions, so that a low growth rate does not result in a greater portion of high producing G1-phase cells. Substrate limitation changes the cell cycle distribution of this cell line to a greater extent than low temperature or serum content, which indicates that this stress factor exerts a greater physiological control than assumed.


Mathematical modelling and simulation of a recycle dialysis membrane reactor in a reversed micellar system.

A mathematical framework was developed for the evaluation of a recycle dialysis membrane reactor (RDMR). The lipase-catalyzed hydrolysis of olive oil in an AOT-iso-octane reversed micellar system was employed as a model. Three specific operational strategies have been considered, namely batch, fed-batch, and fed-batch--bleed. Simulation shows the conversion of substrate to be strongly dependent on efficient use of the substrate, since the permeability coefficients of both substrate and product are quite similar. Sensitivity analyses were performed to assess the influences of various parameters (membrane area, substrate feed rate, solvent bleed rate and permeability) on the performance of the reactor in different modes of operation. The analyses presented are useful to assist the optimization of the operational strategy used for the RDMR system.


Surfactant interference on lipase catalysed reactions in microemulsions.

The hydrolysis of palm oil with lipase as a catalyst was carried out in two different microemulsion systems. One system was based on a nonionic surfactant, pentaethylene glycol monododecyl ether (C12EO5) and the other system was based on an anionic surfactant, sodium bis(2-ethylhexyl)sulphosuccinate (AOT). The yield of free fatty acid produced by the reaction was found to be much lower in the C12EO5 system compared to the AOT system. Radiochromatography showed that the low yield was due to enzymatic esterification on the nonionic surfactant. Kinetic measurements showed that the reaction rate is about ten times faster in the AOT based microemulsion than in the nonionic system. Differences in the microemulsion structure and interfacial tensions in the two systems were found to be of no significant importance for explaining this difference. Kinetic data of mixed surfactant microemulsions indicated that the observed difference in the reaction rates of the AOT and the C12EO5 microemulsion systems was a consequence of the C12EO5 surfactant competing with the substrate for the active site of the enzyme. The reason why C12EO5 surfactant inhibited the reaction, and AOT surfactant did not, was found to be related to differences in the structures of the hydrophobic part of the surfactant.


Hepatitis C virus antibodies and hepatitis C virus RNA in Chinese blood donors determined by ELISA, recombinant immunoblot assay and polymerase chain reaction.

Antibodies to hepatitis C virus (anti-HCV) were determined in Chinese blood donors from the city of Wuhan by a second generation ELISA screening test and a confirmatory recombinant immunoblot assay (RIBA II). Two materials of 281 and 222 sera were sampled under similar conditions in 1989 and 1990, respectively. The first collection of sera was sent to Sweden in lyophilized form, the second directly as fresh unfrozen sera. A high proportion (7.1%) of the lyophilized sera reacted positively in the anti-HCV screening assay, but only seven (2.5%) were positive by the RIBA confirmatory test. In four of these sera HCV-RNA could be detected by polymerase chain reaction (PCR) analysis. In the second material of fresh sera six reacted positively in the screening anti-HCV ELISA, but only one was RIBA positive and four were RIBA indeterminate. None of these sera was positive for HCV-RNA. Thus, the overall prevalence of anti-HCV among the 503 Chinese blood donors, as identified by RIBA, was 1.6%, and of HCV-RNA by PCR 0.8%. The confirmed antibody prevalence is higher than reported from the Western world. Caution about using data from the screening ELISA only, especially if sera have been handled in an unorthodox way, is emphasized.


Long-term psychological consequences of childhood frontal lobe lesion in patient DT.

Patient DT was examined 26 years after she acquired focal frontal lobe damage at 7 years of age. This report focused on several aspects of psychological outcome, including the empirical study of social development into early adulthood. Standardized measures of empathy, psychosocial development, and personality were analyzed, along with a moral judgment interview and patterns of adult social behavior. Results indicated that DT has a very limited capacity for empathic understanding, inadequate identity development, difficulties in vocational adjustment, and a concrete level of moral reasoning. Her social behavior and profile of test scores suggest that social development and adaptation have been arrested at early adolescent levels. We conclude that early frontal lobe damage has profound effects on social development, and that the frontal lobes provide a crucial neural substrate for social maturation.


Developmental impact of frontal lobe injury in middle childhood.

Executive function of frontal lobe systems, like intelligent behavior, appears to demonstrate two major features: it is adaptive and goal-directed. Disruption of executive function following injury to the frontal lobes in childhood might be predicted to impact the trajectory of normal cognitive, behavioral, and social development. Case studies of two children injured at different ages showed the primary developmental impact to involve the behavioral and social realms. While cognitive development may be suspected to also be considerably influenced, assessment of changes in cognitive abilities by traditional psychometric means was found to be problematic. Psychometric changes were found to occur over a prolonged period of time, emphasizing the importance of documenting observations and chronicling postinjury events.


The oocyte nucleus isolated in oil retains in vivo structure and functions.

We describe a technique for isolating the nucleus of the giant amphibian oocyte under paraffin oil. The method precludes the losses of small solutes and proteins that accompany isolation of nuclei into aqueous media. An individual oocyte is blotted, placed under oil, punctured near the animal pole and then squeezed to gently extrude the nucleus into the oil, thereby avoiding exposure to any aqueous environment. Light and electron microscopy of the oil-isolated nucleus demonstrate that its in vivo morphology is preserved. We also describe techniques that facilitate the study of nuclear functions under oil. Oil-isolated oocyte nuclei retain many in vivo functions for several hours, including size-selective envelope permeability, RNA synthesis and the ability to break down in response to cdc2/cyclin meiotic maturation promoting factor.


In search of retrotransposons: exploring the potential of the PCR.

A rapid and universal procedure for isolating reverse transcriptase encoding elements from diverse mammalian genomes using PCR is described. We have designed short, degenerate primers to conserved amino acid domains of retroviral reverse transcriptase. These primers amplify a region, predicted to be 342-396 base pairs for most mammalian retroviruses, that spans several conserved domains of reverse transcriptase. The region encoded by the amplified PCR product contains a number of highly conserved amino acids that aid in identification of either degenerate reverse transcriptase or reverse transcriptase from new, undescribed elements. Additionally, these primers allow the amplification of a piece of DNA large enough to be used for phylogenetic analysis. The primers have been used successfully to isolate a region of three related reverse transcriptases from two mammalian taxa. The generality of this approach is discussed.


Utility of internal markers to improve the accuracy of cystic fibrosis genotype analysis.

DNA diagnostic tests often utilize restriction endonuclease digestion of PCR-amplified portions of genes under analysis. When partial digestion occurs, the resulting patterns may lead to error in diagnosis. To overcome such potential errors in cystic fibrosis testing, we have developed internal markers that can increase the precision and reliability of genotype assignments.


A nonradioactive micro-assay for released reverse transcriptase activity of a lentivirus.

A nonradioactive micro-assay procedure for detection of released reverse transcriptase activity from cells infected with equine infectious anemia virus is described. This procedure utilizes biotinylated-dUTP in conjunction with a streptavidin-alkaline phosphatase conjugate. Detection of alkaline phosphatase is by autoradiography of the chemiluminescence produced during enzymatic dephosphorylation of Lumi Phos 530. This method, as with reverse transcriptase micro-assays employing 32P-labeled nucleotides, is suited to the processing of numerous samples, while having the advantages of safety and stability normally associated with nonradioactive methods of detection. Sensitivity is comparable to a reverse transcriptase micro-assay using 32P-dTTP.


The use of synthetic peptide combinatorial libraries for the identification of bioactive peptides.

The systematic preparation of synthetic peptide combinatorial libraries (SPCLs), each composed of tens of millions of peptides that can be screened in existing diagnostically or pharmacologically relevant in vitro assay systems, is reviewed. The identification of optimal peptide sequences has been achieved through the screening in solution of SPCLs, each element of which is composed of more than 100,000 nonsupport-bound peptides in equimolar representation, along with an iterative synthesis and screening process. Examples are presented in which an SPCL, composed in total of 52,128,400 acetylated hexa-peptides, is used along with an iterative selection process to precisely identify the antigenic determinant of a peptide recognized by a monoclonal antibody using competitive enzyme-linked immunosorbent assay. This same library was also used to develop highly potent antimicrobial peptides in bacterial growth inhibition assays. A separate non-acetylated SPCL was used to screen and identify high affinity peptide ligands using an opiate radio-receptor binding assay.


Preferential expression of the Drosophila rutabaga gene in mushroom bodies, neural centers for learning in insects.

Seven lines were isolated with P element insertions in the cytogenetic vicinity of the learning and memory gene, rutabaga, from an enhancer detector screen designed to mark genes preferentially expressed in mushroom bodies. Six of these lines performed poorly in learning and memory tests, and several failed to complement an existing rutabaga allele. Molecular cloning revealed that the P elements were inserted in the putative promoter of the rutabaga gene. RNA in situ hybridization and immunohistochemistry demonstrated that the expression of the rutabaga gene, which encodes a Ca2+/calmodulin-responsive adenylyl cyclase, is markedly elevated in the mushroom bodies of normal flies and that the insertion elements compromised its expression in the new rutabaga mutants. The reisolation of a known learning and memory gene, but with a heretofore unknown expression pattern, strongly supports the postulate that mushroom bodies are principal sites mediating olfactory learning and memory.


Role of the floor plate in axonal patterning in the zebrafish CNS.

To determine the role of the floor plate (FP) in CNS development, I have used labeling techniques, including immunolabeling, to analyze cyclops mutant embryos, which lack the FP. Except for the anterior brain, the mutant phenotype is almost exclusively confined to the vicinity of the ventral CNS midline. In the midbrain, the number of ventral neurons is reduced and cell patterning is disturbed. In contrast, the neuronal arrangement in the spinal cord is almost normal, including in particular both primary and secondary motoneurons. Longitudinal axonal bundles are disorganized in both the brain and spinal cord. Laser ablating the FP in wild-type embryos locally phenocopies cyclops axonal disturbances, and transplanting wild-type FP precursor cells into mutants locally rescues the disturbances. These results demonstrate a significant role for the FP in pathfinding and fasciculation by axons in situ, especially during their longitudinal courses.


PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity.

Expression of oncogenic ras in PC12 cells causes neuronal differentiation and sustained protein tyrosine phosphorylation and activity of extracellular signal-regulated kinases (ERKs), p42erk2 and p44erk1. Oncogenic N-ras-induced neuronal differentiation is inhibited by compounds that block ERK protein tyrosine phosphorylation or ERK activity, indicating that ERKs are not only activated by p21ras but serve as the primary downstream effectors of p21ras. Treatment of PC12 cells with nerve growth factor or fibroblast growth factor results in neuronal differentiation and in a sustained elevation of p21ras activity, of ERK activity, and of ERK tyrosine phosphorylation. Epidermal growth factor, which does not cause neuronal differentiation, stimulates only transient (< 1 hr) activation of p21ras and ERKs. These data indicate that transient activation of p21ras and, consequently, ERKs is not sufficient for induction of neuronal differentiation. Prolonged ERK activity is required: a consequence of sustained activation of p21ras by the growth factor receptor protein tyrosine kinase.


Protein phosphatases modulate the apparent agonist affinity of the light-regulated ion channel in retinal rods.

Ion channels directly activated by cGMP mediate the light response in retinal rods. Several components of the enzyme cascade controlling cGMP concentration are regulated, but there are no accepted mechanisms for modulation of the response of the channel to cGMP. Here we report evidence that in excised patches an endogenous protein phosphatase converts the channel from a state with low cGMP sensitivity to a state with almost 3 orders of magnitude higher sensitivity in the predicted physiological range of cGMP concentration. The action of this endogenous phosphatase was blocked by specific serine/threonine phosphatase inhibitors (microcystin-LR, okadaic acid, and calyculin A). An increase in apparent agonist affinity also was produced by addition of purified protein phosphatase 1. In contrast, protein phosphatase 2A decreased apparent agonist affinity, suggesting that two phosphorylation sites may regulate the agonist sensitivity of the channel in a reciprocal manner. This regulation may be involved in fine-tuning the light response or in light or dark adaptation.


Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of the trk proto-oncogene with src homology 2 domains.

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.


Human lung mast cells and pulmonary macrophages produce tumor necrosis factor-alpha in sensitized lung tissue after IgE receptor triggering.

Tumor necrosis factor (TNF) is considered to play a key role in the pathogenesis of allergic disorders. We examined TNF production in human lung fragments after IgE receptor triggering at mRNA and protein levels. IgE receptor triggering was performed by sensitizing lung fragments with monoclonal human IgE and then exposing them to anti-human IgE antibody. Cytotoxic activity against L929 cells appeared in the culture supernatant of lung fragments 2 h after IgE receptor triggering and increased for up to 4 h. This cytotoxic activity was completely neutralized by anti-human TNF antibody. Northern blot analysis demonstrated that 1.8-kb TNF mRNA transcripts in sensitized lung fragments were expressed as early as 1 h after IgE receptor triggering and continued up to 4 h. Immunohistochemical analysis revealed TNF localization in tissue mast cells, alveolar macrophages, tissue macrophages, and bronchial epithelial cells. Double staining with anti-TNF antibody and alcian blue clearly identified that lung mast cells are one of the TNF-positive cell types in the pulmonary tissue. With immunoelectron microscopy, TNF immunoreactivity was detected in the rough endoplasmic reticulum and the perinuclear spaces in tissue macrophages, and in the cytosol and the perinuclear spaces in bronchial epithelial cells. In addition, IgE was detected on the cell surface of mast cells, tissue macrophages, and alveolar macrophages. These results suggest that TNF is released from mast cells and pulmonary macrophages through IgE receptor triggering and may play a key role in the allergic reaction in human airway.


The expression of leukocyte-endothelial adhesion molecules is increased in perennial allergic rhinitis.

Accumulating evidence supports the importance of leukocyte-endothelial cell adhesion molecule (CAM) expression as an initiating process in tissue inflammation. To investigate the relevance of CAM expression to allergic airways inflammation, nasal biopsies from patients with perennial allergic rhinitis (n = 8) and from nonatopic healthy volunteers (n = 8) were immunostained with monoclonal antibodies directed against the CAMs, intercellular adhesion molecule-1 (ICAM-1), endothelial cell adhesion molecule-1 (ELAM-1), and vascular cell adhesion molecule-1 (VCAM-1). The endothelial staining of these CAMs was related to the number of vessels within each biopsy, delineated by a monoclonal antibody against Ulex europaeus-1 lectin bound to endothelial cells, and to the number of tissue leukocytes staining for one of the ligands of ICAM-1, the beta 2 integrin, lymphocyte function-associated antigen (LFA-1). Expression of CAMs was related to the number of infiltrating neutrophils, eosinophils, and lymphocytes identified immunohistochemically within the biopsies. ICAM-1 was the most prominent CAM present on the endothelium of the normal nasal mucosa, with less expression of ELAM-1 and only minimal or absent expression of VCAM-1. In perennial rhinitis, both ICAM-1 (P less than 0.05) and VCAM-1 (P less than 0.01) expression on endothelial cells were increased and were positively correlated in their level of expression (P less than 0.002). The number of tissue LFA-1-positive cells was significantly greater (P less than 0.05) in the biopsies from the perennial rhinitics (median, 27.3/mm2) than from the healthy controls (median, 5.3 cells/mm2). LFA-1 expression significantly correlated with the number of ICAM-1-positive vessels (P less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)


Interleukin-8 and RANTES inhibit basophil histamine release induced with monocyte chemotactic and activating factor/monocyte chemoattractant peptide-1 and histamine releasing factor.

The objective of this study was to investigate the effect of interleukin-8 (IL-8) and RANTES on basophil histamine release induced with monocyte chemoattractant peptide-1 (MCP-1) and crude histamine releasing factor (HRF). IL-8 induced low levels of histamine release (8.5 +/- 0.5%) from basophils obtained from only six of 20 donors at high concentrations (10(-6) M). RANTES induced histamine release (16 +/- 2%) from basophils of four of 15 donors at 10(-7) M concentration. However, both IL-8 and RANTES inhibited MCP-1 and HRF-induced histamine release from basophils dose-dependently at concentrations of 10(-9) to 10(-7) M. Basophils from all donors showed a significant inhibitory response (greater than 15%). The maximal inhibition of MCP-1 and HRF by IL-8 was 28 +/- 4% and 48 +/- 8%, respectively. The maximal inhibition of MCP-1 and HRF by RANTES was 26 +/- 4% and 43 +/- 6%, respectively. Peripheral blood mononuclear cell-derived HRF was purified into three distinct peaks by reverse-phase high performance liquid chromatography. Peak I contained MCP-1 as judged by binding to an immunoaffinity column that was prepared with anti-MCP-1 antibody. IL-8 inhibited histamine release induced with all three peaks of HRF. The inhibition of histamine release by IL-8 was significantly higher in normal subjects than in allergic patients (59 +/- 9% versus 31 +/- 7%, P less than 0.05). Both IL-8 and RANTES inhibited cytokine-induced histamine release only and did not affect histamine release by anti-IgE, FMLP, and C5a.(ABSTRACT TRUNCATED AT 250 WORDS)


The effect of T-cell depletion on enhanced basophil histamine release after in vitro incubation with live influenza A virus.

A number of mechanisms participate in virus-induced asthma. Previously, we described enhanced basophil histamine release (HR) during an experimentally induced rhinovirus infection and after in vitro incubation of peripheral blood mononuclear cells (PBMC) with influenza virus. This study extends our previous observations and examines the effect of influenza A virus on basophil leukotriene C4 (LTC4) release as well as the effect of T-cell depletion on virus-enhanced basophil HR. PBMC were isolated from ragweed-allergic subjects and incubated with live influenza A virus or control medium (allantoic fluid). After incubation with influenza A, ragweed antigen (AgE) stimulated LTC4 and HR were enhanced (P less than 0.05). To further define the role of T cells in virus-enhanced basophil secretion, PBMC were isolated and divided into two aliquots. In one aliquot, T cells were removed by magnetic bead separation of mouse monoclonal anti-CD3-coated lymphocytes. T-cell-depleted and nontreated PBMC suspensions were incubated with influenza A or control medium, collected, and challenged with AgE to release histamine. Basophil HR was enhanced in the virus-treated group of PBMC that had not undergone T-cell depletion. In contrast, virus incubation did not enhance HR in the T-cell-depleted fraction. Finally, preliminary analysis of the supernate from virus-treated leukocytes indicates the presence of interferon-gamma. These findings suggest that T cells, and their cytokine products, play an integral role in the process by which viruses enhance basophil HR.


Comparison of pregnancy rates following gamete intrafallopian transfer (GIFT) under general anesthesia with thiopental sodium or propofol.

To determine whether the pregnancy rate for gamete intrafallopian transfer (GIFT) patients who received thiopental sodium was different from the pregnancy rate for patients who received propofol for the induction of general anesthesia. Retrospective review of clinical records. Private outpatient infertility clinic. Two hundred eighty-two consecutive GIFT procedures performed on 230 patients with infertility. Patients undergoing GIFT were divided into two groups on the basis of whether they received thiopental or propofol for induction of anesthesia. Clinical pregnancy following each GIFT procedure was assessed by multiple-serum beta human chorionic gonadotropin and ultrasound determinations. The pregnancy rates of 24.6% and 25.8% for the thiopental and propofol groups, respectively, were not significantly different. The pregnancy rate following GIFT for patients given propofol for induction of general anesthesia did not differ from that for patients given thiopental.


Large systemic collateral arteries developing late after total repair of tetralogy of Fallot.

A 22-year-old man with tetralogy of Fallot successfully underwent a corrective operation. Twenty years after total correction, large systemic collateral arteries (SCA) developed. The SCA arose from the right subclavian and internal mammary arteries. The SCA were not present at the time of total repair and thoracotomy had not been performed on this side. A closed-tube thoracostomy for postoperative pneumothorax may be responsible for the development of the large SCA.


[Alterations in neuro-development in the 1st year of life in newborn infants weighing 2000 grams or less at birth].

The infants with low weight at birth have great chance of exhibiting early death or neurodevelopmental sequelae. We prospectively evaluated the neurological and psychological development during the first year of life in 118 infants who weighted less than 2,000 g at birth. All patients were examined with the Amiel Tison and Grenier test (neurologic evaluation), Gesell test (psychological) and Brunette-Lezine test (Psychomotor scale). Mean gestational age was 33 weeks (SD = 3.1) and birth mean weight was 1495 g (SD = 291). Seventy per cent were delivered by cesarean section. Neurological abnormalities were found in 20 infants (alteration of muscular tone in all and motor deficit in 50%). Twenty five per cent had abnormal Gesell test and 17% psychomotor retardation (13% mild and 4% severe).


Contrast transesophageal echocardiographic demonstration of coronary artery fistula within left atrial appendage thrombus in mitral stenosis.

Coronary neovascularization and fistula formation arising from the left circumflex artery demonstrated by coronary angiography is a specific sign for the presence of left atrial appendage thrombus in patients with mitral stenosis. However, the fistula drainage site in the left atrium in relation to the thrombus cannot be ascertained by the angiographic method. We performed transesophageal echocardiography simultaneously with coronary angiography in five patients with severe mitral stenosis and left atrial appendage thrombus. The angiography showed coronary neovascularization and fistula arising from the left circumflex artery in three patients. In these three patients, the transesophageal echocardiography confirmed the presence of a coronary fistula by identifying contrast exuding from the surface of the thrombus. Thus we have shown for the first time the usefulness of contrast transesophageal echocardiography in imaging the exact drainage site of coronary artery fistula from left atrial appendage thrombus.


Ultrahigh-resolution scanning electron microscopy and its application to medical researches.

By the development of an ultrahigh-resolution SEM, the resolution of SEM was markedly improved. Concerning specimen preparation, the osmium-DMSO-osmium method, which is effective for revealing intracellular structures, and the carbon plate method for observing smaller objects such as viruses and biological macromolecules were devised in recent years. With the improvement of instrumental resolution and specimen preparation techniques, the SEM has become a powerful tool for studying ultrastructure in biomedicine.


The effectiveness of early intervention: a critical review.

The following principles are now clearly defined in the management of children with developmental delay: (1) Multidisciplinary teams are more effective than individual therapeutic approaches. (2) The whole development of the child needs to be considered rather than a single deficient area alone. (3) Home-based programmes are more effective in the young preschool child than centre-based programmes alone. (4) Parent involvement in partnership with professionals is essential for sustained progress. (5) Maximum effectiveness is achieved when parental skills are increased. (6) Programmes commencing earlier in preschool years are more effective than those that commence late. This concept has been recently challenged and evidence supports benefits for the disadvantaged rather than the disabled. White also contends that there is 'simply not enough information to be confident about the long-term impact of early intervention with handicapped children and evidence in support of many of the commonly held positions about mediating variables (e.g. parental involvement, age at start) is either non-existent or contradictory. Early intervention is clearly effective in offering parental support, fostering parent/child relationships and diminishing anxiety even for those programmes that have not at present been proven in altering the developmental disability. Programmes that involve high cost, disrupt total family functioning, deflect scarce resources away from more proven areas of effectiveness should be discouraged, and they should never cause guilt in either parent or professional when they seem ineffective. Future research should include investigation of outcomes other than cognitive and physical functioning alone. We should be warned from the somewhat crisp statement of Mark Twain: 'There is something fascinating about science. One gets such wholesale returns of conjecture out of such a trifling investment in fact'.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunorecognition of ganglioside epitopes: correlation between affinity and cytotoxicity of ganglioside antibodies.

Cell-surface gangliosides have immunomodulatory effects that are presumed to play a role in tumour growth, progression, metastasis and therapy. To study the epitopes of gangliosides on human malignant melanomas and to search for monoclonal antibodies (Mabs) with superior immunological effector functions, 19 ganglioside antibodies were established. Specificity and affinity of nine antibodies of IgG3 isotype were evaluated by enzyme linked immunosorbent assay and thin layer chromatography with a panel of purified gangliosides. All antibodies recognised the ganglioside GD3, but their epitope specificity divided them into five groups. Their affinity constants for ganglioside GD3 ranged from 4.7 x 10(6) to 2.3 x 10(8), with 2 x 10(7) for Mab R-24. Two antibodies possessed a higher affinity for GD2 than for GD3. The functional properties of the antibodies were investigated in vitro. Differences in the degree of tumour lysis by complement fixation correlated with the affinity constants. Every ganglioside antibody differed in epitope recognition, affinity and cytotoxicity. Therefore some of these antibodies might even be more useful in the immunotherapy of malignant melanoma than Mab R-24.


Octreotide and interferon alfa: a new combination for the treatment of malignant carcinoid tumours.

24 patients with malignant carcinoid tumours received octreotide and interferon alfa (IFN-alpha). All the patients initially received octreotide 50-100 micrograms, twice daily. When progressive symptoms or increasing biochemical markers were observed, the daily dose was raised to a median 300 micrograms. If the initial dose proved ineffective or if no improvement was seen after escalation, IFN-alpha was added (median 9 MU subcutaneously per week). After the addition of IFN-alpha, 17 of the 22 patients (77%) with elevated urinary 5-hydroxyindoleacetic acid showed a significant (> 50%) reduction. Only 1 patient progressed and 4 had continuously stable biochemical disease. No significant reduction in tumour size was noted; in 5 patients, the tumour continued to grow despite decreasing hormone levels. 18 patients had carcinoid syndrome when IFN-alpha was added in 10 (56%) symptoms ameliorated. Thus, the addition of IFN-alpha is beneficial for patients with malignant carcinoid tumours that progress and/or who do not respond to octreotide.


Treatment of metastatic carcinoid tumour with recombinant interferon alfa.

14 patients with metastatic carcinoid tumour were treated with recombinant interferon alfa 6-30 x 10(6) IU weekly for 3-25 (median 6.5) months. A decrease in the 24-h urinary 5-hydroxyindoleacetic acid (5-HIAA) level to less than 50% of the pretreatment value was observed in 5 of the 10 cases with an elevated urinary 5-HIAA level. In 4 of the 5 remaining patients, the 5-HIAA level decreased 30-50% from the pretreatment value. 5 of the 9 evaluable patients with carcinoid syndrome experienced symptomatic relief, but none became symptom-free. Severe toxicity was not observed. The median time to progression was 4.5 months, and, in patients with a greater than 50% decrease in 24-h urinary-5-HIAA, it was 17 months. Objective regression in tumour size could not be demonstrated in any of the patients.


Antitumour effect and symptomatic control with interferon alpha 2b in patients with endocrine active tumours.

26 patients with progressive neuroendocrine tumours received 3 x 10(6)U/m2 interferon alfa (IFN-alpha 2b) subcutaneously thrice weekly, until progression, as outpatients with moderate toxicity. 4/16 carcinoids and none out of 10 endocrine pancreatic tumours showed objective regression. Another 17 patients (68%) had no change. For a median of 34 weeks symptom control was excellent: 9 of 17 patients had major relief from pain, 11 of 13 from diarrhoea, and 7 of 7 from flushing. Thus, low-dose INF-alpha 2b given thrice weekly might be as effective as daily treatment with higher dosages. Treatment was only administered to patients with progression or major symptoms and this did not seem to adversely affect remission quality and survival.


ABEP as primary chemotherapy for Hodgkin's disease.

20 untreated Hodgkin's disease patients and 1 patient relapsing after radiotherapy (17 stage IIB-IV and 4 stage I-IIA) were given doxorubicin, bleomycin, etoposide and prednisone on a 21-day cycle. The response rate was 95% and 16 patients (76%) achieved complete remission. 4 patients have relapsed 2, 5, 22 and 50 months after treatment. Survival was 100% at a median follow-up of 35 months. However, due to dyspnoea on exertion in 2 patients, bleomycin will be abandoned, and the occurrence of two second malignancies questions the role of etoposide as a leukaemogenic agent.


Progress, problems and priorities in quality of life research.

Considerable progress has been made in the methodology of quality of life research and we now need to judge the clinical utility of this approach in clinical trials. Some of the logistic problems in implementing these studies are formidable, however, and this has contributed to a slow uptake of quality of life research in clinical practice. Pragmatic guidelines are now needed to help overcome problems of implementation and analysis. Further adaptation and refinement of instruments will be needed to meet new challenges in the field, but we must be selective in our research initiatives and ensure that quality of life becomes translated into improved patient care.