Angina pectoris and silent ischemia in the elderly: a management update.

Coronary artery disease accompanied by symptomatic and asymptomatic myocardial ischemia is a common entity in older patients. The pathophysiology of myocardial ischemia is related to an imbalance in myocardial demand and coronary perfusion. Treatment strategies for symptomatic myocardial ischemia include correction of aggravating medical conditions (eg, anemia or hypertension) and the use of nitrates, beta-adrenergic blockers, salicylates, and calcium-entry blockers, alone or in combination. Silent myocardial ischemia is also a prevalent condition in older individuals, with and without angina pectoris. Treatment regimens are similar to those used in symptomatic patients.


Purification and characterization of an agglutinin of the soft coral Sinularia species.

A D-galactose-specific agglutinin, named sinularian, has been isolated from the soft coral Sinularia sp. by affinity chromatography on acid-treated Sepharose 4B and by gel filtration on HPLC. Sinularian was a glycoprotein containing 11% sugar. It gave a single band corresponding to 78 kDa in SDS-PAGE, irrespective of a treatment with 2-mercaptoethanol. Sinularian agglutinated rabbit erythrocytes and murine leukemia cells but not sheep or human ABO erythrocytes. Its hemagglutinating activity was Ca(++)-independent. Sinularian promoted binding of macrophages to tumor cells.


Natural history of small untreated hepatocellular carcinoma in cirrhosis: a multivariate analysis of prognostic factors of tumor growth rate and patient survival.

We analyzed the growth pattern of tumor masses and the survival of 39 asymptomatic Italian patients with a total of 59 small (less than or equal to 5 cm in diameter) hepatocellular carcinomas arising from cirrhosis. The total length of the observation period ranged from 90 to 962 days, with an average of 364 +/- 229 (mean +/- S.D.). Doubling time ranged from 27.2 to 605.6 days (mean +/- S.D., 204.2 +/- 135; median = 171.6 days). Three different growth patterns were recognized: (a) tumors with no or very slow initial growth pattern (doubling time greater than 200 days), 10 cases (37%); (b) tumors with declining growth rate over time, 9 cases (33.4%); and (c) tumors with almost constant growth rate, 8 cases (29.6%). Using the stepwise discriminant analysis, we found a score based on albumin, alcohol intake, number of nodules, echo pattern and histological type that allowed a correct prediction of short doubling time (less than or equal to 150 days) in 55.6%, medium doubling time (151 to 300 days) in 60% and long doubling time (greater than 300 days) in 100% of cases. The estimated survival rate of the 39 patients, calculated by the Kaplan-Meier method was 81% at 1 yr, 55.7% at 2 yr and 21% at 3 yr. Stepwise discriminant analysis showed that a score based on sex, HBsAg status, alcohol consumption, ascites, gamma-glutamyltranspeptidase, prothrombin time, Child-Pugh class and all the sonographical parameters could predict 2-yr survival in 100% of cases. We conclude that great variability of growth patterns exists among and within small hepatocellular carcinomas. Prediction of subsequent growth rate is unreliable in most cases.(ABSTRACT TRUNCATED AT 250 WORDS)


Hereditary cystatin C amyloid angiopathy: identification of the disease-causing mutation and specific diagnosis by polymerase chain reaction based analysis.

Hereditary cystatin C amyloid angiopathy (HCCAA) is a dominantly inherited disease characterized by amyloidosis, dementia and fatal cerebral hemorrhage of young adults. A method for rapid and simple diagnosis of HCCAA is described. It is based upon oligonucleotide-directed enzymatic amplification of a 275-bp genomic DNA segment containing exon 2 of the cystatin C gene from a blood sample, followed by digestion of the amplification product with AluI. Loss of an AluI recognition site in the amplified DNA segment from HCCAA patients results in a deviating band-pattern at agarose gel electrophoresis, compared with that obtained from normal subjects or unaffected HCCAA family members. In a population of 9 patients with manifest HCCAA, 14 patients with other causes of brain hemorrhage and 16 healthy individuals, the diagnostic procedure displayed a sensitivity and specificity for HCCAA of 100%. Amplified DNA segments from 4 HCCAA patients of four different families were analyzed by nucleotide sequencing; the HCCAA-causing mutation in all families was found to be a single T----A substitution in the codon for amino acid residue 68 of cystatin C.


Genetic heterogeneity of early-onset familial breast cancer.

A gene for early-onset familial breast cancer has recently been mapped to the chromosome 17q12-23 region. In order to confirm the gene location, we have tested an extensive early-onset breast cancer family with 4 markers in this chromosome region. Linkage was negative with all 4 markers. This study suggests that there is genetic heterogeneity among early-onset breast cancer families.


Molecular analysis of the gene for the human vitamin-D-binding protein (group-specific component): allelic differences of the common genetic GC types.

DNA sequence analysis of the polymerase chain reaction products, including the coding region for amino acids 416 and 420, of the vitamin-D-binding protein (DBP, group-specific component, GC) shows allele-specific differences. The GC2 and GC1F phenotypes have an aspartic acid residue at amino acid position 416, whereas the GC1S phenotype has a glutamic acid at this position. In the GC2 phenotype, amino acid 420 is a lysine residue, and in the both common GC1 phenotypes, it is a threonine residue. The nucleotide exchanges involve a HaeIII (position 416) and a StyI (position 420) restriction site: the HaeIII restriction site is specific for the GC*1S allele and the StyI restriction site is specific for the GC*2 allele. We have tested 140 individual genomic DNA samples for the HaeIII site and 148 samples for the StyI site by restriction fragment length polymorphism (RFLP) analysis with a DBP-specific direct genomic DNA probe, and have compared these findings with the GC phenotype classification, by isoelectric focusing (IEF) of the corresponding plasma. The results of the HaeIII RFLP analysis and the IEF typing were in complete agreement. By using our DNA probe, we could disclose, in addition to the StyI site at amino acid position 420, two further StyI site downstream: one was specific for the GC*1S allele and another for the GC*1F allele. In 147 samples, there was agreement between the IEF GC typing and the analysis of the StyI restriction sites. In a single case, the observed result of the StyI-digest differed from the result expected after IEF classification: homozygous GC 1F-1F by IEF and heterozygous by StyI RFLP analysis. We discuss this finding as a recombination event or a possible silent allele in IEF typing. The GC polymorphism revealed by Southern blot analysis of StyI-digests provides an informative DNA marker system for chromosome 4q11-q13.


Reciprocal translocation between the proximal regions of the long arms of chromosomes 13 and 15 resulting in unbalanced offspring: characterization by fluorescence in situ hybridization and DNA analysis.

We describe two female siblings with similar clinical features consisting of hydrocephalus, scaphocephaly, hypotonia, mongoloid eye slant, blepharophimosis, micrognathia, supernumerary mouth frenula and mental retardation. Routine cytogenetic studies in the elder patient did not reveal any abnormality, and initially it was assumed that the syndrome had an autosomal recessive inheritance. However, a slightly larger chromosome 13 was seen in routine G-banded metaphases of the mother and the youngest of the two siblings. A shorter chromosome 15 was detected in the mother only. High resolution banding showed that the abnormal chromosome 13 contained an extra G-positive band at 13q12. The short chromosome 15 in the mother appeared to have a deletion of band q12. Fluorescence in situ hybridization using DNA markers specific to chromosomes 13 and 15 unequivocally showed that the mother was a carrier of a balanced reciprocal translocation t(13;15)(q12;q13), whereas the youngest sibling's karyotype was 46,XX,-13,+der(15)t(13;15)(q12;q13)mat, resulting in partial monosomy 13pter----q12 and partial trisomy 15pter----q13. The proband is thus trisomic for the critical region responsible for Prader-Willi syndrome and Angelman syndrome; this was confirmed by DNA analysis demonstrating one paternal and two maternal alleles from multiallelic marker loci mapping to 15q11-q13. This report illustrates the sensitivity and specificity offered by fluorescence in situ hybridization and its usefulness in the diagnosis and delineation of subtle chromosomal rearrangements.


A single base mutation in the gene for type III collagen (COL3A1) converts glycine 847 to glutamic acid in a family with Ehlers-Danlos syndrome type IV. An unaffected family member is mosaic for the mutation.

Ehlers-Danlos syndrome type IV, an inherited connective tissue disease, is usually caused by mutations in the gene for type III collagen. Here, we describe a glycine to glutamic acid substitution in a patient with this syndrome. Previous studies had shown that fibroblasts from the patient, his mother and brother secreted a reduced amount of type III collagen and also produced an overmodified form of the protein that was preferentially retained intracellularly. Peptide mapping experiments indicated that the mutation was located within cyanogen bromide peptide 9. This was supported by chemical cleavage analysis and sequencing of cDNA encoding this region. Allele-specific oligonucleotide hybridisation of genomic DNA confirmed that a G to A mutation converted Gly 847 to Glu. The mutation was present in two other affected family members and also in a third, who was clinically unaffected. Further analysis of this unaffected individual revealed reduced mutant:normal ratios in DNA obtained from both blood and hair samples, showing that she was mosaic for the mutation.


Structural gene aberrations in mucopolysaccharidosis II (Hunter).

A total of 14 unrelated German patients with X-linked iduronate-2-sulfatase (IDS) deficiency (Hunter syndrome, MPS II) showing variable clinical manifestations was screened for structural gene aberrations by Southern analysis. Using the IDS cDNA clone c2S15 as a probe, no Southern fragments could be detected in blots in the severely affected patient G-65 with respect to DNA digested by HindIII, PstI and TaqI, suggesting a total loss of the IDS structural gene. In this patient, the flanking loci DXS 297, DXS 296 and DXS 466 were tested. The locus DXS 466 is involved in the deletion, whereas both of the other loci are present. A normal 9.0-kb fragment disappeared and an aberrant fragment of 3.5 kb occurred in the HindIII blot of patient G-117. A normal Southern pattern was found in PstI and TaqI blots of this patient. This result can be interpreted as the generation of an additional HindIII restriction site by point mutation in an IDS gene intron.


Loss of heterozygosity on chromosome 11 in sporadic gastrinomas.

Gastrinomas are pancreatic endocrine neoplasms that arise either sporadically or are inherited as part of the multiple endocrine neoplasia type I syndrome (MENI). Loss of heterozygosity (LOH) in the region flanking the MENI gene at chromosome 11q13 has been documented in a few sporadic and familial pancreatic endocrine tumors, but not previously in sporadic gastrinomas. It has therefore been suggested that gastrinomas develop by a mechanism different from other tumors associated with the MENI syndrome. We report LOH on chromosome 11 in 5 of 11 sporadic gastrinomas. Four of these tumors have LOH for markers flanking the MENI region. Molecular evaluation of segments of chromosomes 3, 13, and 17 known to contain cloned or putative tumor suppressor genes fail to show LOH except at one locus in one tumor. These data suggest that a tumor suppressor DNA segment exists at 11q13 that may be involved in the development of sporadic gastrinomas.


An introduction to the possible role of central nervous system structures in neuroendocrine-immune systems interaction.

The involvement of different regions of the brain in the immune response was investigated with the aid of small electrolytic lesions. The lesions were placed in such a way that they covered different areas of the brain stem, basal ganglia, but also some parts of the frontal cortex. The cellular immune response as well as DNA synthesis with the aid of labelled precursors was measured. The results suggest the possibility of the existence of three circuits. One circuit represents catecholaminergic cell group A1-7 in reticular formation, nucleus parabrachialis and central ncl. amygdalae. The second circuit represents serotonergic rapheal groups B6.8, hypothalamus and ncl. basomedialis of amygdala. The third circuit represents ncl. amygdalae and the medial part of frontal cortex, namely the cingulate cortex area 1-2. The close correlation between the changes of the immune response and the CNS activity was also investigated in the experiments with immunosuppressed and immunostimulated animals by measuring of the turnover of some neurotransmitters but also by recording electrocephalographic activity.


Mouse-human chimeric antibody MH171 against the multidrug transporter P-glycoprotein.

We have developed a mouse-human chimeric antibody MH171, in which the antigen-recognizing variable regions of the mouse monoclonal antibody MRK17 are joined with the constant regions of human IgG1 antibodies. The MRK17 recognizes specifically the multidrug transporter P-glycoprotein and inhibits the growth of human multidrug resistant (MDR) tumor cells in vitro and in the xenograft nude mouse model system. The established chimeric MH171 antibody forms an apparently intact IgG composed of heavy and light chains covalently assembled via disulfide bonds in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and is specific to MDR cell lines with a similar affinity to the original mouse MRK17. MH171 also displays strong antibody-dependent cell-mediated cytotoxicity to the target cells in vitro, when human mononuclear cells are used as effector cells. The chimeric antibody against P-glycoprotein, MH171, should be a useful agent in the treatment of human drug-resistant tumors.


[Pancreaticopleural fistula].

We describe the case of a patient with a subdiaphragmatic collection and massive pleural effusion due to a pancreatico-pleural fistula. The fistulous tract was secondary to the extension of an intraabdominal pseudocyst through the diaphragmatic hiatus into the mediastinum, where it perforated into the right pleural cavity. Spontaneous closure of the fistula occurred after a three-week treatment with somatostatin.


Chaperonins groEL and groES promote assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase in Escherichia coli.

We have investigated the possible role of chaperonins groEL and groES in the folding and assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase (E1) in Escherichia coli. The mature E1 alpha subunit fused to maltose-binding protein (MBP) was coexpressed with mature E1 beta on the same vector in ES- and EL- mutant strains. Only small or trace amounts of active E1 component were obtained. Cotransformation of the ES- mutant host with a second vector overexpressing groEL and groES resulted in a greater than 500-fold increase in E1-specific activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the content of both MBP-E1 alpha and E1 beta polypeptides was markedly increased in the presence of overexpressed chaperonin proteins. The time course studies showed that the increase in E1-specific activity and subunit levels correlated with the increase in groEL and groES until the concentration of the chaperonins reached a saturating level in the cell. The functional MBP-E1 fusion protein from ES- double transformants were purified by amylose resin affinity chromatography. The MBP moiety was removed by subsequent digestion with Factor Xa endoprotease, followed by Sephacryl S-300HR chromatography. It was found that E1 alpha and E1 beta assembled into an active 160-kDa species, which was consistent with the alpha 2 beta 2 structure of E1. The present results demonstrate that chaperonins groEL and groES promote folding and assembly of heterotetrameric proteins of mammalian mitochondrial origin.


Substitution of the insulin receptor transmembrane domain with the c-neu/erbB2 transmembrane domain constitutively activates the insulin receptor kinase in vitro.

To examine the role of the transmembrane domain (TM) of the insulin receptor in insulin-induced receptor kinase activation, we prepared four mutated insulin receptors: 1) a Val938----Asp substitution (IR/TMv----D), 2) insertion of a 3-amino acid repeat (Val938-Phe939-Leu940) (IR/TM+3), or the entire TM was replaced by the corresponding domain of either the 3) platelet-derived growth factor (PDGF) receptor (IR/TMPDGFR) or 4) c-neu/erbB2 proto-oncogene product (IR/TMc-neu). Each mutant receptor was stably expressed in Chinese hamster ovary cells, assessed by fluorescence-activated cell sorting, insulin binding, and biosynthetic labeling. All mutant receptors exhibited normal affinity for insulin. Pulse-chase experiments showed that each proreceptor was processed into alpha- and beta-subunits, although the rate of IR/TMV----D conversion was reduced approximately 3-fold. With IR/TMPDGFR, IR/TMV----D, and IR/TM+3 basal and insulin-stimulated levels of autophosphorylation and tyrosine kinase activation were normal, both in wheat germ agglutinin (WGA)-purified receptor preparations and intact cells. By contrast, following WGA purification or isolation of crude membranes, IR/TMc-neu was a constitutively active autokinase and substrate kinase in vitro. However, in intact cells insulin-stimulated autophosphorylation and kinase activity appeared normal. We conclude that although there is considerable latitude in acceptable structure, residues within the insulin receptor transmembrane domain can play a functional role in regulation of insulin receptor tyrosine kinase activity.


Complete amino acid sequence of the human alpha 5 (IV) collagen chain and identification of a single-base mutation in exon 23 converting glycine 521 in the collagenous domain to cysteine in an Alport syndrome patient.

We have generated and characterized cDNA clones providing the complete amino acid sequence of the human type IV collagen chain whose gene has been shown to be mutated in X chromosome-linked Alport syndrome. The entire translation product has 1,685 amino acid residues. There is a 26-residue signal peptide, a 1,430-residue collagenous domain starting with a 14-residue noncollagenous sequence, and a Gly-Xaa-Yaa-repeat sequence interrupted at 22 locations, and a 229-residue carboxyl-terminal noncollagenous domain. The calculated molecular weight of the mature alpha 5(IV) chain is 158,303. Analysis of genomic DNA from members of a kindred with Alport syndrome revealed a new HindIII cleavage site within the coding sequence of one of the cDNA clones characterized. The proband had a new 1.25-kilobase HindIII fragment and a lack of a 1.35-kilobase fragment, and his mildly affected female cousin had both alleles. The mutation which was located to exon 23 was sequenced from a polymerase chain reaction-amplified product, and shown to be a G----T change in the coding strand. The mutation changed the GGT codon of glycine 521 to cysteine. The same mutation was found in one allele of the female cousin. The results were confirmed by allele-specific hybridization analyses.


Selective inhibitors of GTP synthesis impede exocytotic insulin release from intact rat islets.

To investigate whether GTP concentrations can be a regulatory step in exocytotic hormone secretion, we treated isolated rat islets with mycophenolic acid (MPA) or mizoribine, two selective inhibitors of de novo GTP synthesis. When islets were cultured overnight in purine-free medium containing the drug, MPA reduced GTP levels by up to 81 +/- 1%; guanine circumvented this block via the nucleotide "salvage" pathway. MPA concomitantly inhibited glucose (16.7 mM)-induced insulin secretion in batch-type incubations (or perifusions), by up to 68% at 50 micrograms/ml. Although the inhibition of secretion occurred over a similar concentration range as the reduction in total GTP content, the two variables were not directly correlated. However, the secretory effects also were prevented by adding guanine, but not hypoxanthine or xanthine, to the culture medium. Similar results for GTP content and insulin release were seen using mizoribine. Insulin content was modestly (-18%) reduced by MPA but indices of fractional release (release/insulin content) were also markedly impaired. Although MPA also reduced ATP levels more modestly (-39%) and increased UTP (+87%), these were not the cause of the secretory defect since adenine restored ATP and UTP nearly to normal, but did not alter the reduction in GTP content or insulin secretion. MPA also inhibited secretion induced by amino acid or by a phorbol ester but had virtually no effect on release induced by a depolarizing concentration of K+, suggesting that GTP depletion does not merely impede Ca+ influx or directly block Ca(2+)-activated exocytosis. However, a severe reduction of GTP content did not prevent the pertussis toxin-sensitive inhibition of insulin release induced by epinephrine, suggesting that the function of heterotrimeric GTP-binding proteins is not limited by ambient GTP concentrations. Although these studies do not elucidate the exact site(s) in the exocytotic cascade which depend on intact GTP stores, they do provide the first direct evidence that GTP is required (and can be rate limiting) for insulin release.


Site-directed mutagenesis of serine 40 of rat tyrosine hydroxylase. Effects of dopamine and cAMP-dependent phosphorylation on enzyme activity.

Rat tyrosine hydroxylase expressed with a baculovirus expression system contains covalent phosphate and has kinetic parameters consistent with those expected of phosphorylated enzyme (Fitzpatrick, P. F., Chlumsky, L. J., Daubner, S. C., and O'Malley, K. L. (1990) J. Biol. Chem. 265, 2042-2047). The phosphorylation site was identified as serine 40, by purifying the enzyme from cells grown in the presence of [32P]phosphate. Replacement of serine 40 with alanine by site-directed mutagenesis prevented phosphorylation but had little effect on the steady-state kinetic parameters at pH 7. Both wild type and S40A tyrosine hydroxylase were expressed in Escherichia coli; the kinetic parameters of the enzymes purified from bacteria were nearly identical to those of the enzymes expressed with the baculovirus system, although the bacterially expressed enzyme contained no covalent phosphate. Treatment of this wild type enzyme with cAMP-dependent protein kinase decreased the KBH4 value about 2-fold but had no effect on the Vmax value at pH 7. Treatment with a stoichiometric amount of dopamine decreased the Vmax value 15-fold and increased the KBH4 value 2-3-fold. Phosphorylation of the dopamine-bound enzyme increased the Vmax value 10-fold and decreased the KBH4 value 2-fold. The kinetic parameters of the dopamine-bound recombinant enzyme were identical to those of enzyme purified from PC12 cells. In contrast, the S40A enzyme was converted to a less active form by treatment with dopamine but was not affected by phosphorylating conditions. These results are consistent with a model in which the major effect of phosphorylation of serine 40 is to relieve tyrosine hydroxylase from the inhibitory effects of catecholamines.


Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland.

Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning. Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody. Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments. Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides. Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat. Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement. Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor. Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.


Somatostatin gene transcription regulated by a bipartite pancreatic islet D-cell-specific enhancer coupled synergetically to a cAMP response element.

The insulin-, glucagon-, and somatostatin-producing cells in the pancreatic islets derive from a common precursor stem cell and differentiate sequentially during embryonic development, thereby providing an informative model for the study of the transcriptional mechanisms involved in the control of cell-specific gene expression. Relative to the early expression of the glucagon and insulin genes on embryonic days 10 and 12, respectively, the expression of the somatostatin gene is delayed (day 17). The relatively late expression of the somatostatin gene indicates the involvement of both negative and positive transcriptional control mechanisms. We show that the expression of the somatostatin gene in pancreatic islet cells is accomplished by the interplay of both positive and negative cis-regulatory DNA elements. We have characterized the functional properties of one of these positive control elements, the somatostatin gene upstream enhancer element (SMS-UE). The SMS-UE is a pancreatic islet D-cell-specific transcriptional regulator that acts synergistically with the cyclic AMP response element. Mutation-expression and cell-free transcription analyses show that the SMS-UE is a bipartite element with two interdependent functional domains. Our results indicate that the SMS-UE is part of a functional unit that includes other transcriptional control elements of the somatostatin gene proximal promoter, and that they act together to regulate the D-cell-specific transcription of the somatostatin gene in the islet cells of the pancreas.


Somatostatin gene upstream enhancer element activated by a protein complex consisting of CREB, Isl-1-like, and alpha-CBF-like transcription factors.

The expression of the genes encoding the hormones glucagon, insulin, somatostatin, and pancreatic polypeptide in the endocrine islets of the pancreas is regulated in a cell-specific manner, defining four distinct cellular phenotypes (A-, B-, D-, and F-cells, respectively). Binding of nuclear proteins to cognate DNA sequences within cis-acting regulatory elements mediates the transcriptional events that result in the cell-specific activation or repression of gene expression. In a parallel study, we describe the functional properties of the SMS-UE, a pancreatic islet D-cell specific enhancer element that regulates the expression of the somatostatin gene and contains two interdependent domains, A and B. In the studies described herein, we have characterized the nuclear proteins that recognize the SMS-UE. Domain A of the SMS-UE is a DNA enhancer sequence that is identical to that bound by the ubiquitously distributed CCAAT box-binding protein alpha-CBF, a transcription factor that regulates the expression of the human chorionic gonadotrophin alpha-subunit gene. The B-domain, on the other hand, binds an islet cell-specific protein with characteristics similar to those of Isl-1, a transcriptional activator protein that binds to the E2 enhancer of the rat insulin-1 gene. In addition, the SMS-UE binds transcription factor CREB but not CREM, the close homolog of CREB, on a site adjacent to, or overlapping, the 3' end of domain B. We show that the carboxyl-terminal bZIP domain of CREB binds to the cAMP response element of the somatostatin gene but is not sufficient for binding to the SMS-UE, and we present evidence suggesting that CREB.SMS-UE binding requires stabilization by a region of the protein located within the transactivation domain.


In vitro mutagenesis of potential N-glycosylation sites of arylsulfatase A. Effects on glycosylation, phosphorylation, and intracellular sorting.

The correct intracellular sorting of lysosomal enzymes such as arylsulfatase A depends on the presence of mannose 6-phosphate residues on high mannose type oligosaccharides. The arylsulfatase A cDNA contains three potential N-glycosylation sites, two of which are utilized. We have mutated one or two of the N-glycosylation sites and analyzed the glycosylation, phosphorylation, and intracellular sorting of the mutant arylsulfatase A polypeptides. The results show that each of the three glycosylation sites (I, II, and III) can be glycosylated, but glycosylation at sites I and II is mutually exclusive. In mutants with one oligosaccharide side chain at positions I, II, or III all side chains can acquire mannose 6-phosphate residues irrespective of their location. This demonstrates spatial flexibility of the phosphotransferase, which specifically recognizes lysosomal enzymes and initiates the addition of mannose 6-phosphate residues on oligosaccharide side chains. However, these mutants have different intracellular sorting efficiencies and seem to use different (mannose 6-phosphate receptor-dependent and -independent) sorting pathways.


Tetanus toxin potently stimulates tissue transglutaminase. A possible mechanism of neurotoxicity.

The observation that tetanus toxin (TT) contains two sequences that show homology to known transglutaminase (TGase) substrate sites suggested that the toxin and TGase might interact. This prediction was confirmed by two pieces of evidence. First, TT potently stimulated the enzymatic activity of TGase. The effect was maximal at physiological (micromolar) concentrations of the endogenous TGase regulators calcium and GTP. Second, TT and TGase displayed marked variations of their intrinsic fluorescence properties when they were coincubated, indicating the occurrence of binding between them. TT-TGase binding and TGase activation occurred at similar concentrations of TT and are probably causally related. The activation of TGase, an enzyme present in nerve endings that, when activated, can irreversibly cross-link cellular proteins, might mediate the neurotoxic action of TT.


Photoaffinity labeling of the organic cation/H+ exchanger in renal brush border membrane vesicles.

The brush border membrane of the proximal tubule contains two efflux pathways for organic cations from the cell to the tubular fluid: a P-glycoprotein and an organic cation/H+ exchanger. There is evidence that they transport many of the same substrates. Their structural relatedness is unknown and is the subject of this report. The experimental approach was to identify the exchanger with photoaffinity labeling reagents. The rationale was that if the P-glycoprotein and the organic cation/H+ exchanger transport many of the same substrates, then they might be photoaffinity labeled by the same reagents. [125I]Iodoarylazidoprazosin and [3H]azidopine are two reagents, which have been used, to photoaffinity label the P-glycoprotein. We found that several polypeptides were photolabeled in a time- and concentration-dependent manner. The photoincorporation into only two of these polypeptides (41 and 28 kDa) was blocked extensively by the presence of known substrates for the exchanger. The photoaffinity labeling of only the 41-kDa polypeptide was affected by treatment with the chemical reagents, N-ethylmaleimide and dithiothreitol, which are known to affect the exchanger reaction. The findings are consistent with the interpretation that a 41-kDa polypeptide is, or is a component of, the exchanger.


Heterozygosity for apolipoprotein E-4Philadelphia(Glu13----Lys, Arg145----Cys) is associated with incomplete dominance of type III hyperlipoproteinemia.

Apolipoprotein (apo) E-4Philadelphia is a double mutant of apoE in which residue 13 of the mature protein, glutamic acid (GAG), is replaced by lysine (AAG) and amino acid 145, arginine (CGT), is converted to cysteine (TGT). These mutations result in two restriction fragment length polymorphisms for the enzymes AvaI and BbvI, a smaller apparent molecular weight of apoE-4Philadelphia on sodium dodecyl polyacrylamide gels, and severe type III hyperlipoproteinemia (HLP) in a 24-year-old homozygous female (Lohse, P., Mann, W. A., Stein, E. A., and Brewer, H. B., Jr. (1991) J. Biol. Chem. 266, 10479-10484). In the current study, we have extended our analysis to include nine additional family members of the Philadelphia kindred spanning four generations. DNA and protein analysis demonstrated that the originally described propositus is a true homozygote for the epsilon-4Philadelphia allele and that six of the nine family members are heterozygous for the mutated allele and the normal epsilon-3 allele or, in one case, the epsilon-4 allele. Heterozygosity for apoE-4Philadelphia leads to the expression of a moderate form of type III HLP without clinical manifestations. These results are consistent with a dominant mode of inheritance of this dyslipoproteinemia. The simultaneous presence of unaffected individuals, heterozygotes, and a homozygote in the Philadelphia kindred makes it possible for the first time to demonstrate that the mutant apoE exhibits an incomplete or partial dominance of type III HLP. Heterozygosity for the normal epsilon-3 allele appears to have an influence on the expression of type III HLP, resulting in a phenotype intermediate between that of the two homozygous states.


Alterations of the c-erbB2 gene in human breast cancer.

DNA amplification, RNA overexpression and p185 protein expression of the c-erbB2 oncogene were investigated in 109 cases of breast cancer with the aim of evaluating any correlation between the different methods. A correlation between Southern blotting and immunohistochemical analysis of paraffin-embedded material was found. Thus, amplification of the c-erbB2 oncogene leads to overexpression of the p185 protein. By contrast, no statistical correlation could be shown between RNA overexpression, measured by Northern blotting, and immunohistochemical p185 membrane stainings. It is of special interest that most of the cases that are positive for Northern blotting and negative for immunochemistry are negative for Southern blotting as well. Contradictory findings between RNA overexpression and lack of immunohistochemical staining of p185 give rise to the assumption that a defective protein is encoded, which cannot be incorporated into the substructures of the tumour cell membrane. When screening for point mutations in the transmembrane domain of the c-erbB2 oncogene, no point mutation could be detected, either by using the endonuclease FokI, which cuts at position 2012 (the point mutation in the neu gene of the rat), or by direct sequencing.


Expression of Her2/neu oncogene product p185 in correlation to clinicopathological and prognostic factors of gastric carcinoma.

The expression of the Her2/neu gene product p185 was retrospectively analyzed in 58 patients with gastric carcinoma. The results were correlated to various clinicopathological and prognostic factors. Positive membrane staining for p185 could be detected in 38% of the patients (22/58). Membrane staining was significantly greater in well and moderately differentiated tumors of the intestinal type when compared with poorly differentiated lesions and carcinomas of the diffuse type (P less than 0.01). Positive membrane staining did not correlate with site and tumor stage, but T1 lesions had less membrane staining than more advanced primary tumors. Overall survival showed no difference between p185-positive and negative cases. Multivariate analysis defined a subgroup of curatively resected patients with stage III and IV disease that had a statistically significant poorer survival when p185 was overexpressed (P = 0.005). Overexpression of the Her2/neu product p185 appears to be associated with intestinal-type gastric carcinoma and may help in identifying a subset of patients at increased risk for shorter survival.


Selective inhibition of protein targeting to the apical domain of MDCK cells by brefeldin A.

Dipeptidyl peptidase IV (DPPIV) is mainly vectorially targeted to the apical surface in MDCK cells. BFA was found to abolish the apical targeting of DPPIV. This BFA effect could be achieved under conditions where the ER to Golgi transport and the total surface expression of DPPIV were essentially unaffected. BFA executed its effect during the transport from the trans-Golgi network (TGN) to the surface. The inhibition of apical targeting resulted in enhanced mis-targeting to the basolateral surface. The mistargeted DPPIV was transcytosed back to the apical domain only after BFA withdrawal. In contrast, the basolateral targeting of uvomorulin was unaffected by BFA. These results established that the apical targeting of DPPIV was selectively abolished by BFA.


A cryptopromoter is activated in the proliferating cell nuclear antigen gene of growth arrested cells.

We have examined the regulation of the proliferating cell nuclear antigen gene (PCNA) in a hamster fibroblast cell line (tk-ts13) which is temperature sensitive for growth. These tk-ts13 cells, at the restrictive temperature, are growth arrested in the G1 phase of the cell cycle. The cells were stably transfected with a full length human PCNA gene, and the resulting cell lines (K525 cells) were analyzed. We find that, in growth arrested K525 cells, a cryptopromoter is activated in the transfected human PCNA gene. The cryptopromoter resides in intron 4 which is necessary for proper regulation of the PCNA gene. Removal of this intron leads to increased expression of PCNA in cells which have entered the G0 state. An Alu sequence residing in intron 4 is implicated as the promoter element which is active during growth arrest.


Reduced mRNA levels for the multidrug-resistance genes in cAMP-dependent protein kinase mutant cell lines.

We have previously shown that in Chinese hamster ovary (CHO) cells, a mutant cell line with a defective regulatory subunit (RI) for the cAMP-dependent protein kinase (Abraham et al: Mol. Cell. Biol., 7:3098-3106, 1987), and a transfectant cell line expressing the same mutant kinase, showed increased sensitivity to a number of drugs that are known to be substrates for the multidrug transporter (P-glycoprotein). In the current study we have investigated the mechanism by which cAMP-dependent protein kinase controls drug resistance. We report here that the sensitivity of the kinase defective CHO cell lines to multiple drugs results from decreased RNA levels for the multidrug-resistance gene. Similar results were obtained with mouse Y1 adrenal cells. Wild-type Y1 cells had high levels of P-glycoprotein due to expression of both the mdr1b and mdr2 genes, whereas the cAMP-dependent protein kinase mutant Kin 8 cells had decreased RNA levels for these genes. A Kin 8 transfectant with restored cAMP-dependent protein kinase activity recovered mdr expression, indicating a cause and effect relationship between the protein kinase mutations and mdr expression. No changes in nuclear run-off assays could be detected, suggesting a non-transcriptional mechanism of regulation. Wild-type Y1 cells are more drug sensitive despite having higher levels of P-glycoprotein than the mutant cells. This paradoxical result may be explained by the higher rate of synthesis of steroids by the wild-type Y1 cells, which appear to be inhibitors of P-glycoprotein transport activity.


Ubiquinone protects cultured neurons against spontaneous and excitotoxin-induced degeneration.

Ubiquinone is an endogenous quinone with pharmacological actions mainly related to its antioxidant properties. Here we report that ubiquinone protects cultured cerebellar granule cells against glutamate-induced neurotoxicity. In control cultures at 9 days of maturation in vitro (DIV), a 30-min exposure to 100 microM glutamate induced neuronal degeneration, as reflected by the great percentage (greater than 90%) of cells labeled with propidium iodide 24 h after the exposure. Glutamate-induced neuronal death was dramatically reduced in cultures treated daily with ubiquinone since the second DIV. In these cultures, glutamate failed to induce a "delayed" increase in the influx of 45Ca2+, an established parameter of excitotoxicity. Similarly, repeated addition of ubiquinone attenuated in a concentration-dependent manner the age-dependent degeneration of granule cells that is due to the toxic action of the endogenous glutamate progressively released into the medium. These results suggest that ubiquinone may be a useful drug in the therapy of acute and chronic neurodegenerative diseases related to hyperactivity of excitatory amino acid neurotransmission.


Adenosine receptor blockade augments interstitial fluid levels of excitatory amino acids during cerebral ischemia.

The excitotoxic hypothesis suggests that cerebral ischemic damage results in part from the accumulation of the excitatory and potentially toxic neurotransmitters glutamate and aspartate. Adenosine, which also increases during cerebral ischemia, is proposed to inhibit neurotransmitter release. The purpose of this study was to determine if adenosine receptor blockade exacerbates the accumulation of glutamate and aspartate during cerebral ischemia. Microdialysis probes, implanted bilaterally in the caudate nucleus of halothane-anesthetized rats, were used to (1) assess changes in interstitial fluid (ISF) glutamate, aspartate, adenosine, and adenosine metabolites; (2) measure local cerebral blood flow (H2 clearance); and (3) deliver 8-(p-sulfophenyl)theophylline (SPT), an adenosine receptor antagonist, locally to the brain. The probe on one side of the brain was perfused with artificial cerebrospinal fluid (CSF) containing 10(-3) M SPT, while the probe on the opposite side received only artificial CSF. Animals were exposed to 20 min of ischemia (carotid occlusion+arterial blood pressure = 50 mm Hg) followed by 60 min of reperfusion. Dialysate glutamate and aspartate increased during and after cerebral ischemia, but were increased to a greater extent in the presence of adenosine receptor blockade. Likewise, the increase in dialysate adenosine and adenosine metabolites was enhanced on the side of locally administered SPT. These data suggest that endogenous adenosine attenuates the accumulation of glutamate and aspartate during cerebral ischemia.


Measurement of amino acid compositions of glycoprotein systems by gas-phase hydrolysis and reversed-phase high-performance liquid chromatography.

Interest in glycoproteins and their compositions has increased in recent years. Work described in this report illustrates the use of an amino acid analysis protocol involving gas-phase hydrolysis and reversed-phase high-performance liquid chromatography of glycoprotein systems at microgram levels. In other amino acid analysis protocols the problem of losses of amino acids of glycoproteins has been documented. These losses were due to various reactions, referred to as browning or Maillard reactions, which yielded a residue from which amino acids were not recoverable. In our work, three glycoprotein systems are examined: ovalbumin, sICAM-1, and bovine serum albumin--which is naturally unglycosylated, but is spiked with about 30% saccharides. In all three cases, the compositional agreement between the molar ratio of amino acids determined empirically and that predicted is greater than 90%. Thus it is shown that the adverse effects of Maillard-type reactions are avoided, and the presence of carbohydrates causes negligible interferences with amino acid analysis performed under the conditions described herein.


Multilocus mapping of the X-linked hypophosphatemic rickets gene.

X-linked hypophosphatemic rickets (HYP), the most common form of familial hypophosphatemic (vitamin D-resistant) rickets, is an X-linked dominant disorder characterized by decreased renal tubular phosphate reabsorption and consequent hypophosphatemia. Despite the application of a wide variety of biochemical and cell biology techniques, controversy exists regarding whether a primary renal abnormality underlies the abnormal phosphate transport or if this defect is secondary to the effects of a hormonal/metabolic factor. Thus localization of the HYP gene and its ultimate cloning may be necessary to elucidate the pathophysiology of the disorder. In order to map the human HYP gene we investigated several new polymorphic probes for linkage to HYP and constructed a map of markers around the gene. The database used to ascertain linkage and perform mapping included 5 large HYP kindreds, 40 Centre d'Etudie Polymorphisms Humain reference pedigrees, and 19 kindreds which had been obtained for other disease linkage studies. Two point LOD scores (odds of linkage, log10) indicate that the probes DXS365, DXS257, DXS451, and DXS41 are tightly linked to the HYP locus. Indeed, there were no cross-overs between DXS365 and HYP with a peak LOD score of 13.98 [recombination fraction (theta) = 0.00]. Moreover, multipoint analysis reveals a probable locus order of: Xtel-DXS315-DXS43-DXS257-HYP-DXS41-DXS4 51-Xcen. The likelihood of HYP occurring between DXS257 and DXS41 is 407:1 over the next most likely position. DXS365 is located between DXS41 and DXS43 but could not be located with respect to HYP and DXS257. Regardless, we have located the HYP gene between the flanking markers DXS257 (telomeric) and DXS41 (centromeric) which are 3.5 centiMorgans apart. Thus, the results of this study will facilitate attempts to further localize and eventually clone the gene.


Hormone secretion in vitro by plurihormonal pituitary adenomas of the acidophil cell line.

Pituitary tumors producing GH and PRL are morphologically classified as monomorphous bihormonal acidophil stem cell adenomas (ASCAs) which cause hyperprolactinemia and two tumor types which are usually associated with acromegaly, the monomorphous plurihormonal mammosomatotroph adenomas (MSAs) and bimorphous mixed somatotroph-lactotroph adenomas. We studied 12 MSAs, 2 ASCAs, and 10 mixed adenomas in vitro to assess the secretory behavior of these tumors diagnosed by immunohistochemistry and electron microscopy. GH release by MSAs and all but one mixed tumor was greater than that of PRL; the opposite was true of the ASCAs. One mixed tumor which caused impotence and hyperprolactinemia contained predominantly lactotrophs and released greater amounts of PRL than of GH in vitro. All 12 MSAs and 6 of 10 mixed tumors released alpha-subunit of glycoprotein hormones. Incubation with GHRH increased release of GH and PRL by all tumors and of alpha-subunit when present; the responses of all hormones were parallel among MSAs whereas among mixed adenomas, GH and alpha-subunit had greater responses than PRL. TRH stimulated GH, PRL, and alpha-subunit release by MSAs in parallel; among mixed adenomas, PRL response was generally greater than that of GH or alpha-subunit. SRIH markedly reduced GH release by all MSAs; it inhibited GH and alpha-subunit release by mixed tumors more than it affected PRL. Bromocriptine inhibited GH, PRL, and alpha-subunit release by most MSAs and mixed tumors but did not inhibit GH or PRL release by ASCAs. This study demonstrates release of GH, PRL, and alpha-subunit by these morphologically classified plurihormonal tumors in vitro. Variable quantities of GH and PRL released by the different tumor types correlate with immunohistochemical and clinical data. The dynamic studies indicate that regulation of GH, PRL, and alpha-subunit release can be affected by GHRH, TRH, SRIH, and bromocriptine in these adenomas and suggest differences in receptor status. Our data strengthen the view that these three plurihormonal adenomas of the acidophil cell line are not only morphologically but also functionally different and warrant separation.


Clinical and genetic features of adrenocortical lesions in multiple endocrine neoplasia type 1.

In multiple endocrine neoplasia type 1 (MEN-1), benign enlargement of the adrenal cortex has been found in about one third of necropsy cases. To elucidate the clinical and genetic characteristics of the MEN-1 adrenal lesion, we have investigated 33 MEN-1 patients. Twelve individuals (37%) demonstrated adrenal enlargement, which was bilateral in 7 of them. Histopathology revealed diffuse and nodular cortical hyperplasia, adenomas, and a single case of adrenocortical carcinoma. The apparently benign adrenal enlargements were not associated with presently ascertainable biochemical disturbances in the hypothalamic-pituitary-adrenocortical axis, and they were without radiological signs of progression during follow-up. The individual developing unilateral adrenocortical carcinoma showed rapid adrenal expansion, feminization, and an abnormal urinary steroid profile after 4 yr of observation for bilateral minor adrenal enlargements. Pancreatic endocrine tumors were significantly overrepresented and present in all MEN-1 individuals with adrenal involvement. In agreement with findings in sporadic cases, the MEN-1 adrenocortical carcinoma genome showed loss of constitutional heterozygosity for alleles at 17p, 13q, 11p, and 11q. The benign adrenal lesions retained heterozygosity for the MEN-1 locus at chromosome 11 q 13. Despite its prevalence and malignant potential, the pituitary-independent adrenocortical proliferation does not appear to be a primary lesion in MEN-1, but might represent a secondary phenomenon, perhaps related to the pancreatic endocrine tumor.


Upregulation of intercellular adhesion molecule-1 (ICAM-1) expression in primary cultures of human brain microvessel endothelial cells by cytokines and lipopolysaccharide.

The expression of intercellular adhesion molecule-1 (ICAM-1) by human cerebral endothelium was studied in primary cultures of human brain microvessel endothelial cells following treatment with bacterial lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma). Surface expression of ICAM-1 was examined with the immunogold silver staining technique. Intact cerebral endothelial cells constitutively express low levels of ICAM-1. Stimulation with LPS and cytokines induces upregulation of ICAM-1 which is minimal with IFN-gamma and maximal with LPS or a combination of IFN-gamma and TNF-alpha. Upregulation of ICAM-1 expression is concentration- and time-dependent, is observed as early as 4 h following incubation and persists for up to 72 h in the continuous presence of LPS or cytokines. The ICAM-1 expression is not reversed by 3 days after removal of the LPS or cytokines. These findings may be relevant to the interactions between leukocytes and brain microvessel endothelial cells in inflammatory and demyelinating diseases of the CNS.


Characterization of a rat retinal endothelial cell culture and the expression of P-glycoprotein in brain and retinal endothelium in vitro.

Retinal vascular endothelia form one aspect of the blood-retinal barrier and, like the blood-brain barrier, control the passage of molecules and cells into the parenchyma. To facilitate comparative in vitro studies, rat retinal endothelial cells have been cultured and characterised. Using immunocytochemical techniques, retinal endothelium was positive for von Willebrand's factor, tight junction-associated polypeptide (ZO-1) and the transferrin receptor. The cells also expressed high-affinity uptake of acetylated low-density lipoprotein. Using the monoclonal antibodies JSB-1 and C219, the product of the multidrug resistance gene, P-glycoprotein, was found to be expressed on primary cultures of both brain and retinal endothelium.


Langerhans cells, inflammation markers and human papillomavirus infections in benign and malignant epithelial tumors from transplant recipients.

Organ transplant recipients frequently develop warts which progress toward premalignant or malignant lesions after a rather long grafting period. The local immune responses of such lesions (warts, condyloma acuminata, actinic keratoses, Bowen, basal and squamous cell carcinomas) was studied in 32 frozen skin specimens taken from 15 male transplant recipients and compared to similar lesions from the normal population. We studied the expression of T cell subsets, Langerhans cell phenotype, HLA class 1 (beta 2-microglobulin), HLA class 2 (DR antigen), and intercellular adhesion molecule 1 (ICAM 1). The presence of HPV infection was also considered, using in situ hybridization with biotinylated probes in order to examine the correlation with immunological markers. In the dermis, the lesions from grafted patients showed a moderate to intense inflammatory reaction of HLA-DR-positive cells. Most of these cells were CD4+ and CD8+ without any predominance of a single T cell subset. In the epidermis, most lesions were characterized by a reduced number of CD1-positive cells; this was concomitant with a decrease or a loss of beta 2-microglobulin expression by epithelial cells. HLA-DR antigen was not expressed by keratinocytes or tumoral cells in any specimen; ICAM 1 antigen was observed in a few cases. The expression of these markers was similarly modified with or without the presence of HPV DNA. Conversely, most lesions from non-immunocompromised patients, except warts, showed intense inflammatory reactions, with a predominance of CD4-positive cells and large foci of ICAM 1-positive cells. Expression of activation markers by keratinocytes occurred mainly in condylomas and squamous cell carcinomas. In the normal population, HPV infection was only detected in papilloma lesions. These data indicate, in lesions from grafted patients, a lack of effective immune response with partial inhibition of activation markers expressed by keratinocytes. It is conceivable that immunosuppressive treatment with solar exposure may also be responsible for the local immune deficiency and thus for the conversion of benign warts toward malignant lesions in grafted patients.


The effects of interferon-beta on phorbol ester or calcium ionophore-induced intercellular adhesion molecule-I expression in epidermal carcinoma cells.

Keratinocyte intercellular adhesion molecule (ICAM)-I expression is induced by interferon (IFN)-gamma. It has been previously reported that IFN-beta suppresses IFN-gamma-induced ICAM-I expression in A431 cells, a human squamous cell carcinoma cell line. In this study, the suppression mechanisms were investigated at the post second messenger level. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore (A23187) induce ICAM-I expression in A431 cells. ICAM-I expression induced by either was not suppressed with cotreatment with IFN-beta. Furthermore, IFN-beta did not inhibit the translocation of protein kinase C (PKC) by TPA. It appears that the pathways involved in ICAM-I expression induced by activation of PKC or increased in intracellular Ca++ are not affected by IFN-beta.


Quantitative image cytometry of hepatocytes expressing gamma-glutamyl transpeptidase and glutathione S-transferase in diethylnitrosamine-initiated rats treated with phenobarbital and/or phthalate esters.

Image cytometry was used to quantify the volume of liver expressing two histochemical markers associated with neoplasia, gamma-glutamyl transpeptidase (GGT) and the placental isozyme of glutathione S-transferase (GST-P). Rats were treated with diethylnitrosamine (DENA) followed by phenobarbital (PB), di(2-ethylhexyl)phthalate (DEHP), or di-n-octyl-phthalate (DOP) for 26 weeks. In one series, PB-treated rats were given 2.0%, 0.5%, or 0.1% DEHP in the feed. GGT expression was detected diffusely throughout the liver parenchyma in several treatment groups so that any enhanced expression in altered foci (AF) and nodules (N) was not apparent. GST-P was detected only in AF and N. GST-P may represent a second genetic alteration, as GST-P+ AF and N also expressed GGT but not the reverse. The peroxisome proliferator DEHP inhibited expression of GGT or GST-P in livers of either DENA-treated or DENA+PB-treated rats. With GST-P the reduction was correlated to a reduced number of AF and N. In contrast, DEHP's stereoisomer, DOP, was as effective as PB in promoting expression of both markers. We conclude that image cytometry of hepatocytes expressing GST-P can be used in the bioassay of the carcinogenic potential of chemicals that affect liver proliferation.


Serotonin storage and chromogranins: an experimental study in rat gastric endocrine cells.

Chromogranins (Cg) and secretogranins (Sg) are acidic proteins localized in the secretory granules of a large variety of endocrine cells collectively named APUD cells (amine precursor uptake and decarboxylation). To examine the possible function of Cg/Sg as amine storage proteins, enteroendocrine cells of the rat gastric antral mucosa, i.e., serotonin-containing enterochromaffin (EC)-cells, gastrin (G)-, and somatostatin (D)-cells, were investigated immunohistochemically in serial semi-thin sections of controls and after intervention in serotonin synthesis. CgA and CgB immunoreactivity was determined semiquantitatively by optical density measurements. Experiments included inhibition of serotonin synthesis by p-chlorophenylalanine (pCPA), exogenous application of the serotonin precursor 5-hydroxytryptophan (5-HTP), and a combination of both treatments. The cellular distribution of Cg and the density of its immunoreactivity were closely related to the primary content of serotonin and the ability to store serotonin after 5-HTP application. Thus, Cg may act as amine-binding proteins in enteroendocrine cells, binding most probably being due to ionic interactions between Cg and the biogenic amines. EC- and G-cells, however, differed in their amine-handling properties and in the response of their Cg immunoreactivity after intervention in serotonin synthesis. We conclude, therefore, that the physiological function of Cg as amine storage proteins is restricted to endocrine cells with an endogenous content of amines. In other endocrine cells, exhibiting only a potential amine production, APUD may be considered as a kind of supravital staining without physiological significance.


Anti-hantavirus antibodies in human sera in Czechoslovakia.

By indirect immunofluorescence using antigens of hantavirus Hantaan and CG 18-20 on Vero E6 cells were examined 5,827 samples of sera from 5,299 probands of Czechoslovakia. In 49 persons (0.94%) were found antibodies of titres 1:32 and higher. Two groups of elderly farm workers showed a cluster of positive individuals amounting to 9.9% and 29.4% respectively. The ratio of positivities in some other, randomly and specifically selected groups was deep below 1%. The partial results for the group of farmers were confirmed by RIA test. Occasionally antibodies only to one of the hantavirus serotypes, at other time, to both were found. The authors discuss the findings of antibodies of the two serotypes in humans as related to the evidenced existence of two hantavirus antigen serotypes in animals.


Characterization and toxicity to mosquito larvae of four Bacillus sphaericus strains isolated from Brazilian soils.

Four Bacillus sphaericus strains, S1, S2, S5, and L2, isolated from Brazilian soils, were found to be toxic to larvae of the mosquitoes Culex pipiens and Anopheles stephensi at a level similar to that of strain 2362 which is now used operationally. Like strain 2362, the four strains belonged to the serotype H5 and produced major proteins of apparent molecular weights of 125, 110, 56, and 43 kDa. These latter two proteins were immunologically related to toxins of the same molecular weight as B. sphaericus 2362. Although the four Brazilian strains were very similar to strain 2362, gas chromatography analysis of the fatty acids revealed that these strains were different from strain 2362 and from each other, except for a possible similarity between strains S1 and S5.


Genomic variations in mosquitocidal strains of Bacillus sphaericus detected by M13 DNA fingerprinting.

The genomic variation of Bacillus sphaericus reference and local strains belonging to different serotypes was examined by DNA fingerprinting. A phage M13 DNA probe detected a number of variable fragments in the restriction digests of total strain DNAs. The patterns of band distribution showed a certain homology among mosquitocidal strains, expressed by similarity index D and might be a reliable criterion for assessing the level of genomic similarity between closely related strains. An important advantage of DNA fingerprinting is the differentiation of one bacterial strain from another, both expressing common phenotype and possessing highly similar genomic portions. The strain variation revealed by the M13 probe will be useful for characterization of individual strains within a serotype. It could help as well to solve some uncertain cases based on the results obtained by other methods of identification.


Characterization of two new point mutations in the low density lipoprotein receptor genes of an English patient with homozygous familial hypercholesterolemia.

Two new point mutations have been detected in the low density lipoprotein (LDL) receptor gene of a patient with a clinical diagnosis of homozygous familial hypercholesterolemia (FH). The patient is a compound heterozygote, in whom the mutant allele inherited from his English father has a single base substitution of A for G in exon 3, changing the codon for residue 80 in the mature protein from glutamic acid to lysine. The mutant allele inherited from his mother, who is of Irish origin, has a single base pair deletion in the codon for residue 743 in exon 15 that causes a frameshift and introduces a new stop codon in the adjacent position. The glu80 to lys mutation results in a transport-defective phenotype and a mature protein that migrates abnormally slowly on nonreduced SDS-PAGE, but normally under reducing conditions; this was confirmed by site-directed mutagenesis and expression in vitro. The deletion in exon 15 results in a null phenotype in which the putative truncated receptor protein cannot be detected in cultured skin fibroblasts and the amount of mRNA derived from the allele is reduced. The glu80 to lys mutation was found in a further five unrelated individuals in a sample of 200 FH patients from the London area and in 11 from a sample of 77 FH patients from Manchester. Haplotype analysis suggested that all the patients had inherited this allele from a common ancestor. The deletion in exon 15 was not found in the London sample, nor in any unrelated individuals in the Manchester sample.


Genetic and dietary interactions in the regulation of HMG-CoA reductase gene expression.

Inbred strains of mice exhibit large genetic variations in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. A tissue-specific genetic variation between the strains BALB/c and C57BL/6, resulting in about 5-fold higher levels in hepatic reductase activity in strain C57BL/6, was examined in detail. The activity difference between these two strains could be explained entirely by differences in hepatic reductase mRNA levels. In genetic crosses, the variation segregated as a single major Mendelian element. Surprisingly, the mode of inheritance was recessive since F1 mice exhibited the BALB/c levels of enzyme activity. Despite the fact that the rates of hepatic sterol synthesis also differed between the strains by a factor of about five, the altered hepatic reductase expression did not significantly influence plasma lipoprotein levels. The response to a high cholesterol, high fat diet between the strains was remarkably different. Thus, in BALB/c mice, both hepatic reductase activity and mRNA levels were affected only slightly, if at all, by cholesterol feeding, while in strain C57BL/6 mice both were reduced more than 10-fold by cholesterol feeding. Several lines of evidence, including analysis of cis-acting regulatory elements, the nonadditive mode of inheritance, and genetic studies of the HMG-CoA reductase gene locus on mouse chromosome 13, support the possibility that the variation in reductase expression is not due to a mutation of the structural gene but, rather, is determined by a trans-acting factor controlling reductase mRNA levels. The variation provides a striking example, at the molecular level, of the importance of dietary-genetic interactions in the control of cholesterol metabolism.


Diabetes incidence in users and non-users of antihypertensive drugs in relation to serum insulin, glucose tolerance and degree of adiposity: a 12-year prospective population study of women in Gothenburg, Sweden.

As part of a prospective population study in Gothenburg, Sweden, women aged 50 years were subjected to an intravenous glucose tolerance test on entry to the study and followed up for 12 years. Manifest diabetes was the only end-point registered in this part of the study. Of 352 initially non-diabetic women, 17 (4.8%) subjects developed diabetes, with a fourfold increased risk in women taking antihypertensive drugs (diuretics or beta-blockers, or both) compared with women who were not taking such medication. The increased risk was observed independently of initially measured glucose metabolism variables and degree of adiposity, although the incidences were higher overall if the use of antihypertensive drugs was combined with fasting hyperinsulinaemia and adiposity. This study provides further evidence to support the view that diuretics and beta-blockers are precipitators of type 2 diabetes mellitus.


Dural arteriovenous malformation of the transverse sinus with sinus occlusion: report of a case.

We report a case of dural arteriovenous malformation (AVM) of the transverse sinus with sinus occlusion. This 49-year-old man developed right parietal lobe dysfunction with acute onset. Computed tomography and magnetic resonance imaging (MRI) showed a non-hemorrhagic venous infarct in the subcortical white matter of the right parietal lobe, and diffusely dilated subcortical veins. Thrombosis of the right internal jugular bulb was also revealed on MRI. Cerebral angiography showed a dural AVM in the posterior fossa with occlusion of the right transverse sinus and retrograde venous drainage into the superior sagittal sinus, causing diffuse engorgement of the superficial cortical and the deep intramedullary veins. The focal neurologic deficits in this case were due to a non-hemorrhagic venous infarct in the subcortical white matter of the right parietal lobe secondary to retrograde cortical venous drainage.


Pseudoathetosis as a presenting symptom of spinal multiple sclerosis.

We report on a 38-year-old female patient with acute proprioceptive sensory impairment and pseudoathetosis in the four limbs, particularly in the fingers of both hands. She had great difficulty in buttoning, unbuttoning, using chopsticks and writing, because she was no longer able to feel her fingers in space. There was a profound loss of position and vibration sensation in all limbs, especially in both hands. Pseudoathetoid movement, a rare presentation of multiple sclerosis, was observed in the outstretched hands and extended fingers. T1- and T2-weighted magnetic resonance imaging (MRI) of the spinal cord revealed an inactive intramedullary lesion in the C2-C5 segments. Gadolinium-enhanced MRI revealed an active lesion in the posterior columns of the cervical cord at the C3 vertebral level, which is very likely responsible for pseudoathetoid movement. Based on the disseminated lesions in the spinal cord verified by MRI the four neurologic manifestations, and the abnormal somatosensory-evoked potentials, we made a definitive diagnosis of multiple sclerosis of the spinal cord.


Brachial plexus neuropathy associated with scrub typhus: report of a case.

We report on a 20-year-old man who had scrub typhus with the unusual neurologic complication of brachial plexus neuropathy. The clinical features of fever, headache, pneumonitis, eschar, high Weil-Felix OX-K agglutination and Rickettsia tsutsugamushi immunofluorescence titers confirmed the diagnosis of scrub typhus. Brachial plexus neuropathy was proven by an electrophysiologic examination. He had a nearly complete recovery after adequate medical treatment.


Therapeutic effect of guar gum in patients with non-insulin-dependent diabetes mellitus.

Diets with a high-fiber content have been shown to produce some beneficial effects on metabolic factors in subjects with NIDDM. However, some controversies still exist. In this report, the long-term effect of guar gum (Guarina) on both glycemic and blood lipid profiles was assessed in a randomized, double-blind and cross-over study on 16 (seven male and nine female) subjects with NIDDM. Each subject received placebo (P) and Guarina (G) treatment for two eight-week periods separated by a four-week period to facilitate wash-out. Fasting plasma glucose levels showed significant improvement during G treatment but not during P treatment (151.7 +/- 7.9 vs 168.6 +/- 12.2 mg/dl, p less than 0.01 by paired Student's t test). Hemoglobin Alc levels decreased significantly during G treatment but not during P treatment (6.9 +/- 0.2 vs 7.2 +/- 0.8%, p less than 0.001). Fasting insulin concentrations also showed significant lowering during G treatment but not during P treatment (18.3 +/- 2.1 vs 23.1 +/- 2.9 U/ml, p less than 0.005). Other variables, including serum total cholesterol, triglyceride, HDLc, LDLc, sodium, potassium, chloride, magnesium and calcium levels showed no significant changes during G or P treatment. Ten out of the 16 patients (62.5%) suffered from side effects; these included abdominal cramps (one case), diarrhea (seven cases) and skin itching (one case). In conclusion, guar gum effectively lowers fasting plasma glucose and HbAlc levels in subjects with NIDDM. Hyperinsulinemia could also be ameliorated. The effectiveness and side effects of guar gum treatment should be cautiously evaluated in each NIDDM subject.


Motor-evoked potentials in diabetes mellitus.

By using magnetic stimulation of the motor cortex and in the cervical region, conduction time in the central motor pathway was measured in 35 patients with diabetes mellitus (DM) and in 41 control subjects. Motor-evoked potentials (MEPs) were recorded from the contralateral thenar muscles. Central conduction time (CCT) was obtained by subtracting the latency of the spinal MEP from that of the scalp MEP. To compare central and peripheral nerve functions, the motor nerve conduction velocity (MCV) of the median nerve was also tested in patients with DM. In the 35 cases of DM, the mean latency of the cortical MEPs showed a significant increase, compared with normal controls; the mean CCT was also significantly prolonged in the patient group. There was a good correlation between central motor abnormalities and the duration of DM, as well as with impairment of the peripheral nervous system. Latencies of greater than two standard deviations were defined as abnormal. The abnormal rate in patients with DM was 29% for latency of the cortical MEP, 20% for latency of the cervical MEP and 37% for the CCT. In addition, there was a 35% abnormal rate for distal latency of the median MCV and a 40% abnormal rate for the median MCV. These findings support the theory that the metabolic disturbance in DM affects both the central and peripheral nervous systems in man. MEP studies provide objective measurements of central motor pathways.


Reduction of herniated temporal lobe in patients with severe head injury and uncal herniation.

Thirty-two patients with severe head injury (Glasgow coma scale 5-7/15) and uncal herniation were treated surgically from January 1988 to June 1990. Reduction of the herniated temporal lobe in addition to classical surgical procedures (craniotomy with evacuation of hematoma and resection of the contused brain) was performed in 10 patients (group A). The remaining 22 patients (group B) were treated similarly but without reduction of the herniated temporal lobe. In group A, there was one operative mortality. Eight patients made a rapid and complete recovery of pupil size and light reflex within two days after the operation, and one recovered within two months after the operation. The recovery of motor strength was also rapid in these nine patients. After three to 20 months (average, 11 months) of follow-up, the outcome was good in two patients, and the other seven were moderately disabled. In group B, 12 patients (55%) died after the operation. When compared with group A, the surviving 10 patients made a slower and less satisfactory recovery of oculomotor nerve function and motor status, and had a worse outcome after four to 25 months (average, 14 months) of follow-up (moderately disabled, six patients; severely disabled, two patients; vegetative, two patients). From my experience, it appears that the procedure of reduction is simple and seems to result in a more rapid and complete recovery of oculomotor nerve function and motor status, and it may even contribute to a better overall outcome.


Effects of timolol and acetazolamide on intraocular pressure elevation following argon laser iridotomy.

To study the effect of hypotensive agents on intraocular pressure elevation following argon laser iridotomy, 0.5% timolol maleate topically and acetazolamide 125 mg orally were given in 39 eyes, one hour prior to laser iridotomy, with 29 eyes serving as the control. The mean pressure two hours after laser iridotomy was 18.9 +/- 7.2 mmHg in the control group and 12.8 +/- 3.9 mmHg in the pretreated group. Ocular pressure was elevated from the baseline pressure of the prelaser status in two eyes (5%) only in the timolol-acetazolamide treated group and in 16 eyes (55%) in the control group. The pressure elevation two hours after laser iridotomy was significantly less in the timolol-acetazolamide pretreated group.


T lymphocyte changes in open heart surgery.

To investigate the effect of open heart surgery on T lymphocytes and their subpopulations, 20 patients, who had undergone moderate- to high-dose fentanyl anesthesia and a cardiopulmonary bypass (CPB), were studied using flow cytometry techniques and monoclonal antibodies during and after surgery. The ages of these patients ranged from four to 61 years with eight being male and 12 being female. The disease entity consisted of four with coronary, six with congenital and 10 with valvular heart disease. No cyanotic patients were included in this study. Peripheral blood samples were collected before anesthesia, immediately before the surgical incision, on the first postoperative day (POD1) and on the second postoperative day (POD2), respectively. We found no significant changes in the percentage of total T cells (T3), or helper (T4) and suppressor (T8) T cells during anesthesia before the surgical incision. On POD1, all T lymphocyte subset percentages decreased significantly when compared to pre-operative values (total T cells: 58.4 +/- 12.6 vs 24.4 +/- 8.4, helper T cells: 33.3 +/- 10.1 vs 15.4 +/- 6.3, suppressor T cells: 23.0 +/- 6.4 vs 10.0 +/- 4, all p less than 0.001) but returned to preoperative levels on POD2. Throughout the study period, there were no significant changes in the T helper cell to T suppressor cell ratio. In spite of the transient decrease in T lymphocytes and their subpopulations, no clinical evidence of infection was noted in any patient.(ABSTRACT TRUNCATED AT 250 WORDS)


Correlation between left ventricular systolic function and dipyridamole thallium SPECT redistribution patterns in coronary artery disease.

The dipyridamole thallium-201 single photon emission computed tomography (SPECT) and resting gated blood pool ventriculography were sequentially conducted in 31 consecutive patients with angiographically proven coronary artery disease. The functional significance of various thallium redistribution patterns was assessed. The patients with an entirely complete (CR) or partial (PR) redistribution pattern had a higher global left ventricular ejection fraction than those with combined PR and no redistribution (NR), while patients with an entirely CR pattern did not have a statistically better ejection fraction than those with PR. Furthermore, myocardial segments with normal perfusion (N) and those with CR or PR had a higher regional ejection fraction than those with NR in the infero-apical area and the septal area. The regional ejection fraction was statistically higher in patients with CR than in those with PR in the septal area and higher, though not statistically significant, in the infero-apical area. The functional difference between groups with N and CR was not significant. Thus, we conclude that the redistribution patterns of dipyridamole thallium SPECT are closely correlated with systole function. Myocardium, in the presence of redistribution, will have a better functional performance in coronary artery disease.


Effect of estrogen on the serum level of thyroglobulin.

Thyroglobulin has been widely accepted as a useful parameter for monitoring the recurrence of differentiated thyroid carcinoma. In thyroid cell cultures, it has been reported that estrogen may stimulate the tgb gene which controls synthesis of thyroglobulin by the thyroid follicular cell. Since the serum estrogen levels of women fluctuate during the menstrual cycle, pregnancy and menopause, thyroglobulin may be unreliable in the follow-up of recurrence of differentiated thyroid carcinoma in females if it can be affected by estrogen in vivo. In order to clarify the effect of estrogen on thyroglobulin, the sera of 16 men, 42 women and 20 children were collected and divided into seven groups. Their serum estradiol and thyroglobulin levels were 17.7 +/- 1.8 pg/mL (mean +/- SEM) and 17.1 +/- 1.6 ng/mL, respectively in 20 children, 26.7 +/- 4.1 pg/mL and 12.9 +/- 1.2 ng/mL, respectively in 16 males, 151.6 +/- 20.4 pg/mL and 13.7 +/- 4.2 ng/mL, respectively in five females on the 10th to 14th days of the menstrual cycle, 16.7 +/- 1.8 pg/mL and 15.2 +/- 2.1 ng/mL, respectively in 12 postmenopausal females, 530.0 +/- 131.0 pg/mL and 14.2 +/- 1.8 ng/mL, respectively in eight pregnant women in the first trimester, 3613.9 +/- 1014.2 pg/mL and 12.0 +/- 1.6 ng/mL, respectively in seven pregnant women in the second trimester and 9246.0 +/- 694.5 pg/mL and 16.4 +/- 2.0 ng/mL, respectively in 10 pregnant women in the third trimester. The serum estradiol levels were significantly different (p less than 0.005) among these seven groups, but the serum thyroglobulin levels were not (p = 0.25). Therefore, serum thyroglobulin levels are not affected by estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)


Holter monitoring in patients with Wolff-Parkinson-White syndrome: with special reference to intermittent pre-excitation.

To characterize the intermittency of pre-excitation in patients with Wolff-Parkinson-White syndrome (W-P-W), we studied the ambulatory electrocardiograms (ECGs) of 48 patients (32 men and 16 women with a mean age of 38.5 years) with W-P-W. As documentation, at least one ECG for each patient had been performed during the follow-up period at our clinics. All cardiovascular drugs were discontinued for at least two days before Holter ECG monitoring. Through a careful beat-to-beat analysis of the QRS complex morphology, we were able to classify these patients into three groups: persistent pre-excitation in 26 (54.2%); intermittent pre-excitation in 17 (35.4%); and no pre-excitation in five (10.4%). There was no significant difference in sex, frequency of arrhythmias or mean heart rate among the three groups. Among the 17 showing intermittent pre-excitation, type A W-P-W was more prevalent (12 cases), and the ratio of pre-excitation to normal beats tended to decrease with age. Conduction normalized gradually with an increase in heart rate in 12 patients and suddenly in five other cases. There were two patients who showed a period of 2:1 pre-excitation intermingled with 1:1 pre-excitation and 1:1 normalized beats. These changes occurred with equal frequency both during the daytime and at night. These findings suggest that intermittent pre-excitation is a rather frequent phenomenon in patients with W-P-W. It occurs more frequently in type A patients and in older patients. Therefore, it may be an intermediate phase between manifest and concealed W-P-W.


Detection of mass lesions in the collapsed lung by ultrasonography.

Ultrasonography is useful in the detection of mass lesions in the collapsed lung, using the collapsed lungs as a "sonic window". Twenty-four patients suspected of having a tumor causing lung collapse, as shown on their chest radiographs, were examined by ultrasonography. Eighteen out of 24 patients were found to have mass lesions in their collapsed lungs. Thoracic computed tomography (CT) was also performed in 12 of these 18 patients; of those, 11 showed compatibility with sonographic findings in the detection of mass lesions in their collapsed lungs. The remaining six of these 24 patients with no mass lesions detected by ultrasonography were proven to have collapsed lung due to sputum impaction (n = 2) and lung cancer (n = 4). The fact that four patients had lung cancer that was not detectable by ultrasonography, might have been due to relatively small mass lesions at deep locations (main or intermediate bronchus) and narrowing of the "sonic window" (partial lung collapse). Though it has some limitations, ultrasonography is helpful in detecting mass lesions in collapsed lungs. Sono-guided fine needle aspiration biopsy (SGFNAB) can also be performed simultaneously, smoothly and without any major complications. In our series, SGFNAB was performed in eight out of 18 patients to make a cytopathologic diagnosis. We recommend this safe, convenient, and noninvasive method to screen for lesions in the collapsed lung, especially when bronchoscopic examination is impossible.


Localization of obscure gastrointestinal bleeding by technetium 99m-labeled red blood cell scintigraphy.

When a bleeding source from the gastrointestinal (GI) tract cannot be identified with conventional diagnostic studies, it is known as GI bleeding of an obscure origin. In the past three years, in vivo Technetium 99m-labeled red blood cell scintigraphy (RBC scan) has been added to our armamentarium for the diagnosis of obscure GI bleeding. Out of a total of 26 cases, the bleeders could be detected in 12 or 46.2% by RBC scan. The time required ranged from 15 minutes to 24 hours (median, one hour). In 14 patients with active bleeding during the scan period, 11 had positive scans (sensitivity, 78.6%). In 12 patients with inactive bleeding, 11 had negative scans (specificity, 91.7%). Angiography was conducted in nine cases, with all showing negative findings; however, six of them had a positive focus by RBC scan. Laparotomy was performed in seven scan-positive patients, and in three scan-negative patients because of a positive Meckel's scan (two cases) or recurrent bleeding (one case). Of the 12 scan-positive patients, incorrect localization was noted in two patients due to rapid transit of the labeled RBC in the small bowel. False localization could be prevented by shortening the sequential imaging interval. It is concluded that an RBC scan is a very sensitive and safe tool for detection of GI bleeding of an intermittent nature, because the bleeder can be monitored for 24 hours after a single injection. It can be used as a preangiographic screening test and to guide the surgeon in surgical planning or decision-making.(ABSTRACT TRUNCATED AT 250 WORDS)


Mechanics of acutely shortened canine diaphragm muscle in vitro.

Acute changes in lung volume, as might be seen in an airway-obstructed patient, may lead to different amounts of shortening of the various inspiratory muscles, of which the diaphragm is the most important. The purpose of this study is to investigate the twitch and contractile characteristics of acutely shortened canine diaphragm muscle in vitro during isometric contraction. We found that with diaphragmatic muscle shortening from Lo (optimal length, defined as being that length at which peak twitch tension was generated) to 70% Lo, the time-to-peak tension, half relaxation time and twitch tension were significantly reduced. The force-frequency curves, obtained from different muscle lengths, disclosed that shorter muscles generate disproportionately less force during low-frequency stimulation. These factors may, at least in part, contribute to the vulnerability to fatigue of those with shorter diaphragmatic muscle length.


Conservative treatment of neonatal hydronephrosis.

From February 1990 to January 1991, 19 cases of hydronephrosis in children of less than one year of age were managed at Mackay Memorial Hospital. In the majority of these patients, there were evident causes such as ureteropelvic junction stenosis, ureterovesical reflux or a posterior urethral valve for which definite therapeutic measures were performed. However, some cases had no obvious origins and the hydronephrosis was speculated to be from nonobstructive or physiologic dilatation of the kidneys. The conventional tools, such as intravenous pyelogram or renal ultrasound, which comprise the mainstay of diagnosis, provide limited information on renal functional status. Recent introduction of the Tc-99m diethylene triamine penta-acetic acid (DTPA) diuretic renal scan has enabled us to distinguish between obstructive and nonobstructive hydronephrosis and helps us to determine whether or not surgery is necessary. In the past year, eight patients with hydronephrosis of less than one year of age were diagnosed as nonobstructive after a series of evaluations using renal ultrasound, voiding cystourethrography (VCUG) and Tc-99m DTPA diuretic renal scan. Follow-up studies by echography or DTPA renal scan revealed spontaneous resolution of the dilated collecting systems in these cases and confirms our belief that some hydronephrosis in neonates and infants may resolve spontaneously and may just be a manifestation of physiologic change during development. The value of the Tc-99m DTPA diuretic renal scan in the diagnosis of obstructive uropathy is discussed.


Helicobacter pylori, gastritis and duodenitis in the healing process of duodenal ulcer.

The occurrence of antral gastritis, duodenitis, gastric metaplasia and Helicobacter pylori (H. pylori) were compared between 63 endoscopically proven duodenal ulcer (DU) patients and 34 non-ulcer dyspepsia (NUD) patients with no ulcer history and no ulcer present as documented by endoscopy. The DU group showed a significantly higher rate of active antral gastritis (89% vs 41% p less than 0.05), a higher antral H. pylori carrying rate (76% vs 27% p less than 0.01), a higher rate of active chronic duodenitis (75% vs 32% p less than 0.05), and a higher rate of gastric metaplasia in the duodenal bulb (68% vs 27% p less than 0.05) than the NUD group. The H. pylori carrying rate in the bulb was 16% in the DU group and 0% in the NUD group. The difference is evident, although it is statistically insignificant. All 10 cases carrying H. pylori in the duodenum in the DU group had active chronic duodenitis with gastric metaplasia. Further evaluation of the variables (rate of active antral gastritis and duodenitis and the carrying rate of H. pylori in the antrum and bulb) showed no difference between different ulcer stages (active, healing, or scarred). The above findings strongly suggest a close relation between H. pylori and duodenal ulcer. However, the low occurrence rate of the bacteria in the bulb can only indicate a partial etiologic role of the bacteria in DU. No improvement in antral gastritis, duodenitis and H. pylori occurrence, despite the healing of an ulcer, is in agreement with the naturally high recurrence rate of duodenal ulcers.


Infantile osteopetrosis: report of two cases.

Two cases of infantile osteopetrosis are reported. Both were males aged four and eight months at presentation. They presented with osteosclerotic change of the bone, leukoerythroblastic anemia, optic atrophy, hepatosplenomegaly and frequent infection. The histology of the bone showed thickened bone trabeculae with little osteoclastic activity, although in one patient the number of osteoclasts increased, while in the other they did not. One received a bone marrow transplant (BMT) but died from disseminated cytomegaloviral infection, pulmonary hemorrhage and sepsis. The post-transplant marrow histology showed evidence of engraftment and osteoclastic activity. The other only received a course of prednisolone, which was of little help. His condition has followed a natural course with progressive visual impairment and marrow failure. Our cases suggest that infantile osteopetrosis should be taken into consideration in dealing with infants who present with early marrow failure and that patients of infantile osteopetrosis should receive BMT. BMTs appear to be the only cure. They should be given as early as possible to avoid major consequences and severe infection.


Purification of radioiodinated human insulin by high performance liquid chromatography for a sensitive radioimmunoassay.

The optimal sensitivity of a radioimmunoassay depends on the purity of the radiolabeled antigen. The conventional purification methods are not complete and are time consuming. The combination of a Sep-pak C18 cartridge and high performance liquid chromatography (HPLC) for the purification of 125I-labeled insulin in our study revealed that the Sep-pak cartridge can serve as the preliminary step to remove unreacted radioactive iodide, the reactants, and labeled but presumably damaged materials unadsorbed to the cartridge. The fractions eluted from the Sep-pak containing high radioactivity and high immunoreactivity to the antibody were chosen for further purification by HPLC to eliminate undesirable radiolabeled substances with a lesser immunoreactivity. The purified radiolabeled insulin was used to develop a sensitive radioimmunoassay with detecting limits of 0.03 microU/mL per tube.


Bilateral severance of thumbs: report of two cases.

Management of hand injuries is a common practice but bilateral amputation of thumbs is very rare. We report on two cases of both thumbs amputated by a similar machine. In case 1, the man's left thumb was closed with a composite skin graft because the severance was through the middle of the nail. A wrap-around procedure was performed to reconstruct the right thumb which was infected after a primary V-Y advancement flap. The final results were acceptable. Replantation of detached thumbs, if possible, achieves the best functional recovery, which was well demonstrated in Case 2. Although the interphalangeal joints were arthrodesed, the patient was greatly satisfied with the results because of excellent appearance, adequate strength and nearly normal sensation. Prevention of bilateral thumb injuries is important. The paper-cutting machine on which these accidents occurred should be improved in order to prevent further similar catastrophes.


Lymphokinetic monitoring of a liver transplant recipient treated with OKT3.

Orthotopic liver transplantation was performed on a 48-year-old female patient with end-stage liver disease due to primary biliary cirrhosis. A course of OKT3 (5 mg/kg intravenously for 14 days) was given as immunosuppressive therapy. Lymphocyte population, T lymphocyte subsets and the lymphokine level in the peripheral blood were serially measured. Total T cells disappeared in the peripheral circulation within 10 minutes after an injection of OKT3. Helper T cells and suppressor T cells were also suppressed during the course of treatment. The ratio of T4/T8 declined from 2.5 to around 1.3. The serum interleukin-2 and the interleukin-2 receptor did not change markedly during treatment. The serum neopterin level increased after discontinuation of OKT3. The anti-mouse OKT3 antibody increased after the ninth day of OKT3 treatment. OKT3 showed potent T cell suppressive activity.


Simultaneous bilateral tubal pregnancies after in vitro fertilization and embryo transfer: report of a case.

Ectopic pregnancies continue to be a major complication of in vitro fertilization and embryo transfer (IVF-ET). A case of bilateral simultaneous tubal pregnancy after IVF-ET is described. The patient underwent ovum pick-up (OPU) through a laparotomy with concomitant pelvic surgery. Embryo transfer (ET) was performed two days after OPU; this resulted in bilateral tubal pregnancies, diagnosed and treated one month apart. There are several possible causal mechanisms for the increased rate of ectopic pregnancies following IVF-ET. It is important to recognize that care in the transfer technique, with respect to the catheter position and limiting the volume of transfer medium to 20 microL, and an awareness of previous occlusion of the tubal ostia, or of a salpingectomy before IVF-ET, can help to minimize this complication rate. Two important points are the possibility of a simultaneous bilateral tubal pregnancy after IVF-ET, and the necessity of carefully examining both adnexa at the time of surgery for an ectopic pregnancy. Early and accurate diagnosis of a simultaneous bilateral ectopic pregnancy can prevent the necessity of a second operation and reduce maternal morbidity and mortality.


Dietary alpha-linolenic acid deficiency and early uterine development in female rats.

Feeding rats a purified diet containing peanut oil with a low alpha-linolenic acid [18:3(n-3)] content resulted in lower amounts of (n-3) polyunsaturated fatty acids, mainly docosahexaenoic acid [22:6(n-3)], greater amounts of docosapentaenoic acid [22:5(n-6)] in uterus phospholipids, and altered postnatal uterus development when compared with rats fed a diet containing peanut and rapeseed oils. Maximal differences in uterine growth, as measured by uterine weight, protein and DNA content, occurred between d 24 and 30 postpartum and disappeared near the end of sexual development (d 40). The induction of the progesterone receptor was not affected, and serum estradiol concentrations were not significantly reduced in deficient animals. Moreover, growth response of the uterus to low doses of 17 beta-estradiol (less than 5 micrograms/kg) was significantly reduced in ovariectomized animals fed the diet containing only peanut oil. However, the maximal response of the uterus, observed with higher 17 beta-estradiol doses (5-50 micrograms/kg), was not affected. Because the two diets used differed in the content of alpha-linolenic acid, it is likely that alpha-linolenic acid deficiency in animals fed the diet containing only peanut oil was the cause of the affected uterine development.


Work-related injuries to the foot. Data from an occupational injury/illness surveillance system.

In 1988, a total of 990 work-related injuries to the foot of employees from private-sector companies were characterized in an occupational injury/illness surveillance system maintained by a network of occupational health centers. The mean age of the worker with a foot injury was 34.2 years (+/- 12.0), with 83% occurring among men; 22.3% of the cases were fractures or sprains/strains. Jobs involving extensive manual material handling or vehicular operations were the most often listed occupations among those with work-related foot injuries. Across occupational groups, being struck by an object accounted for 58.4% of the foot injuries. Regardless of industry group, metal items and vehicles were related to 50.7% of all work-related foot injuries. Specifically, foot injuries were found to be associated with being struck by boxes, metals, or vehicles, or to being caught in, under, or between vehicles or machinery. A peak of work-related injuries involving the foot is observed during the summer months.


Comparative study of interaction mode of diazepines with human serum albumin and alpha 1-acid glycoprotein.

The binding of nine diazepines to human serum albumin (HSA) and to alpha 1-acid glycoprotein (AGP) was investigated by means of fluorescence and circular dichroism (CD) spectroscopies. The binding parameters of diazepam obtained from fluorescence agreed with those obtained from CD measurements. Diazepines have one tight binding site on both HSA and AGP. The binding parameters (nK) of the diazepine: HSA systems are slightly higher than those of diazepine:AGP systems. The relationship between the binding parameters for these two serum proteins and the physicochemical parameters of diazepines was studied by multiple regression analysis to elucidate the binding mode. Moreover, the effects of long-chain fatty acids and cesium chloride on the binding of diazepines to HSA and AGP were also studied. The driving force for the binding of diazepines to both proteins appears to be hydrophobic interaction. In addition, steric effects and electrostatic interactions may also contribute to the binding of diazepines to HSA and AGP, respectively.


Synthesis and antiulcer activity of N-substituted N'-[3-[3-(piperidinomethyl)phenoxy]propyl]ureas: histamine H2-receptor antagonists with a potent mucosal protective activity.

As an aim toward developing new antiulcer agents, new N-substituted N'-[3-[3-(piperidinomethyl)phenoxy]propyl]ureas were synthesized and evaluated for histamine H2-receptor antagonistic, gastric antisecretory, and gastric mucosal protective activities. A QSAR study showed that the most favorable N-substituents were electron-donating straight-chain alkyl groups of short length such as ethyl group from the viewpoint of dual action, i.e., gastric antisecretory and mucosal protective actions. Among the ureas studied, compounds 4, 5, and 8-10 were selected as candidates for further study.