An osteonectin-like protein in the matrix of cultured osteogenic cell-line MC3T3-E1, which is associated with calcification.

Time-dependent changes of the [3H]-proline-labeled noncollagenous proteins synthesized by the osteogenic cell-line MC3T3-E1 were analyzed over a range starting from cell confluency to 13 days after confluency during which time cells formed a bone-like structure. It was found that the 40 kDa protein on SDS-polyacrylamide gel (SDS-PAGE) remarkably increased in the cell-matrix layer at about 9 days after cell confluence, just before calcification. This protein was highly purified and was found to contain high amounts of glutamic acid, glycine, and serine. An internal amino acid sequence of this protein was revealed to be K-X-M-A-P-E-E-X-P, which showed homology with the sequence of the EF-hand domain in osteonectin/SPARC (secreted protein, acidic, and rich in cysteine). This protein co-migrated with collagen in gel filtration, and ion-exchange chromatography. Furthermore, it showed high affinity to type I collagen.


Specific restriction fragment length polymorphism in liver mitochondrial DNA of the Chinese.

By use of restriction fragment length polymorphism analysis, we examined the liver mitochondrial DNA amplified by polymerase chain reaction from 60 Chinese subjects of 31 to 78 years of age. We found nine specific mtDNA polymorphisms that had never been reported before. Eleven subjects had an Alu I polymorphic site in the subunit 2 gene of NADH dehydrogenase, five had a Hae III polymorphic site in the cytochrome oxidase subunit 2 gene, and five had a Hinf I polymorphic site in the subunit 3 gene of cytochrome oxidase. No polymorphic site was found in the structural genes coding for subunits 1, 3, 4, 4L and 6 of NADH dehydrogenase, cytochrome b, and subunit 8 of ATP synthase. Detailed analysis of the RFLP data did not show age-dependent mtDNA polymorphisms. In addition, the analysis of the restriction patterns of all the mtDNAs revealed 12 mtDNA haplotypes in all the Chinese subjects examined. Among them, type 1 mtDNA was found to be the most predominant and comprised 63.3% of the total study subjects. The restriction patterns of type 1 mtDNA generated by all restriction enzymes were identical to those deduced from the Cambridge sequence of human mtDNA. About 8.3% of the subjects exhibited type 2 mtDNA, and 5% had types 3, 5 and 8 mtDNA, respectively. Each of the rest seven mtDNA types comprised about 2% of the samples. Moreover, type 1 mtDNA was found in the platelets of three white Americans. These findings suggest that type 2 to type 12 mtDNAs have come into existence through the generation or loss of specific polymorphic restriction sites in the mtDNA of the Chinese.


Yohimbine-precipitated clonidine withdrawal: an experimental model of the antihypertensive drug withdrawal syndrome.

In this study, a model of the clonidine withdrawal syndrome in normotensive rats was used to investigate the mechanisms and sites of the cardiovascular responses associated with this withdrawal. Clonidine (400 micrograms.kg-1.day-1), an alpha 2-adrenergic receptor agonist, was administered to rats via indwelling osmotic minipumps for 7 days. Withdrawal was precipitated by an intravenous injection of the alpha 2-adrenergic receptor antagonist yohimbine under alpha-chloralose anaesthesia, and the blood pressure and heart rate responses were recorded. Yohimbine (0.25, 0.50, and 1.0 mg/kg i.v.) in clonidine-treated rats provoked an immediate rise in blood pressure and heart rate. Similar injections in saline-treated rats produced slight hypotension and modestly increased the heart rate. Intracerebroventricular (i.c.v.) yohimbine injection (30 or 120 micrograms/kg in 10 microL volume) failed to elicit signs of withdrawal in clonidine-treated animals, but a subsequent intravenous injection of yohimbine (0.5 mg/kg) provoked brisk signs of withdrawal. hexamethonium (2 mg/kg) pretreatment did not abolish the increase in the heart rate, but it delayed the blood pressure increase. Pretreatment with atropine sulfate (1 mg/kg) did not block the yohimbine-induced increase in heart rate or blood pressure. This study demonstrates that yohimbine can effectively produce cardiovascular signs of withdrawal in rats chronically exposed to clonidine. The lack of i.c.v. yohimbine suggests that the antagonist-precipitated withdrawal may not have a central origin.(ABSTRACT TRUNCATED AT 250 WORDS)


A comparison of the effects of acute versus chronic administration of phenoxybenzamine on pressor responses elicited by the selective alpha 1-adrenoceptor agonist cirazoline in the pithed rat preparation.

The effects of nifedipine on the pressor responses to cirazoline were examined in the pithed rat preparation that had received either acute or chronic phenoxybenzamine treatment. Phenoxybenzamine was administered, i.v., to conscious rats, either acutely at 0.01, 0.03, and 0.1 mg/kg, 60 min prior to the commencement of the experiments or chronically at 0.1, 0.3, and 1.0 mg/kg, once daily for 7 days. Nifedipine was administered i.a. (1.0 mg/kg) after the animals had been pithed. The acute or chronic administration of phenoxybenzamine alone displaced the dose-response curve to cirazoline to the right in a dose-dependent manner, while reducing the slope function and maximum response to the agonist. The combined effects of acute phenoxybenzamine and nifedipine produced an additive inhibitory effect on the pressor response elicited by cirazoline, which was most apparent following the removal of receptor reserve by acute phenoxybenzamine. The inhibitory effects of nifedipine and chronically administered phenoxybenzamine were additive at the lower administered doses of the alkylating agent but, in contrast with the effects of acute phenoxybenzamine, the enhanced inhibitory effects of nifedipine were reduced following the removal of receptor reserve. These results indicate that the chronic administration of phenoxybenzamine reduces the additive inhibitory effects of nifedipine and phenoxybenzamine that were observed following the acute administration of phenoxybenzamine.


Suppression of spreading depression of Leão in neocortex by an N-methyl-D-aspartate receptor antagonist.

Spreading depression has been implicated in the pathophysiology of a number of diseases such as migraine, stroke and epilepsy. The characteristics of this phenomenon were explored in neocortex of anesthetized rats. Spreading depression was produced in 10 of 15 animals using mechanical, electrical and chemical stimulation. Mean amplitude of the DC shift was -9.3 mV, mean duration at any one electrode 65 sec and rate of spread 2-5 mm/min. Spreading depression was facilitated by focal interictal spike activity induced by penicillin and completely blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-aminophosphonovaleric acid (APV), providing further evidence that excitatory amino acid neurotransmission is a critical element in the development or propagation of the phenomenon.


Different sensitivities of human esophageal cancer cells to multiple anti-cancer agents and related mechanisms.

Mechanisms responsible for drug resistance in human esophageal cancer cell lines were investigated. Three cell lines established from human esophageal carcinoma (TE-1, SH-1, and TH) showed different sensitivities to vindesine, vincristine, cisplatin (CDDP), etoposide (VP-16), and pepleomycin. Both SH-1 and TH cell lines were twofold to sevenfold more resistant to pepleomycin, vindesine, and vincristine than TE-1 was. SH-1 showed twofold more resistance to CDDP than either TE-1 or TH did, and TH and TE-1 showed a 3-fold or 1.5-fold more resistance, respectively, to VP-16 than SH-1 did. The accumulation of tritiated vincristine in SH-1 and TH was approximately 50% that in TE-1. Two multidrug resistance reversal agents, cepharanthine and a synthetic dihydropyridine analogue (NK-252; Nikken Chemicals, Saitama, Japan), potentiated the cytocidal actions of vindesine against SH-1, TH, and TE-1 cells, with no apparent expression of P-glycoprotein in the three cell lines. The glutathione S-transferase pi gene was expressed in all three cell lines. DNA topoisomerase II levels were lowest in TE-1, followed by SH-1 and TH, although the accumulation of tritiated VP-16 was less in both TH and SH-1 than in TE-1. Differential sensitivities to anti-cancer drugs appear to be mediated through pleiotropic mechanisms.


An immunohistologic evaluation of C-erbB-2 gene product in patients with urinary bladder carcinoma.

Amplification or overexpression of the c-erbB-2 gene have been reported to correlate with poor patient prognosis in human breast, gastric, and ovarian cancer. Recently, the c-erbB-2 gene product was found to be expressed frequently in the urinary bladder carcinoma. In the current study, the presence of the c-erbB-2 gene product in urinary bladder carcinomas was compared with patient outcome to evaluate whether c-erbB-2 gene product could identify a subset of patients who are destined to have a poor prognosis. Immunohistologic study of the c-erbB-2 gene product was done in formaldehyde-fixed paraffin-embedded tissue specimens obtained from 88 transitional cell carcinomas of the human urinary bladder. Eighty-three patients who underwent complete tumor resection by total cystoprostatectomy (30 patients) or by bladder-preserving operations such as transurethral surgery (50 patients) or partial cystectomy (3 patients) entered a follow-up study. The other five patients did not enter the follow-up study because of lost follow-up (2 patients) or distant metastasis at the time of surgery. The c-erbB-2 gene product was expressed in 23 of 88 patients (26%), showing an increase in the expression rate corresponding to the advancement of tumor grade (P < 0.05) and tumor stage (P < 0.2). The 5-year disease-free survival rate was 48.5% for patients with c-erbB-2 negative tumors versus 9.7% for those with c-erbB-2 positive tumors (P < 0.01). The 5-year actuarial survival rate was 65.5% for patients with c-erbB-2 negative tumors versus 41.8% for those with c-erbB-2 positive tumors (P < 0.05). Multivariate analysis using Cox regression model showed that the c-erbB-2 gene product tissue status was a significant prognostic factor independent of grade and stage of the tumor. The results suggest that the c-erbB-2 gene product could be a tumor marker to identify a malignant subgroup in bladder carcinomas.


A kindred with multiple endocrine neoplasia type 2A associated with pruritic skin lesions.

A kindred affected by multiple endocrine neoplasia type 2A (MEN 2A), associated with symmetric, bilateral, scapular pruritic skin lesions (PSL), is reported. There were 21 members, including the propositus, in four generations. Screening of 10 family members showed a palpable thyroid tumor in 1 and positive results for a pentagastrin test in 7 others. Two of these patients had surgically confirmed pheochromocytoma. Two others had a biochemical diagnosis of pheochromocytoma but did not have surgery. Medullary thyroid carcinoma was confirmed in five patients and nodular C-cell hyperplasia in another. In five affected adults, PSL were observed in the interscapular region, crossing the midline in some. These lesions were characterized by hyperkeratosis and hyperpigmentation. In all the patients, the pruritus had been present long before the clinical or biochemical diagnosis. Skin biopsies were performed in two of these patients. No amyloid deposits were found (Congo red stain and electron microscopic examination were used). Two children (14 and 6 years old) in the fourth generation complained of scapular pruritus, although skin lesions were not apparent. Localized PSL must be sought when screening for MEN 2A.


Cytogenetics of multiple endocrine neoplasia syndrome. II. Chromosome abnormalities in an insulinoma and a glucagonoma from two subjects with MEN1.

Cytogenetic analysis of two pancreatic islet tumors, an insulinoma and a glucagonoma was ascertained in two subjects with multiple endocrine neoplasia type 1 (MEN1). The insulinoma had a modal peak at 84 chromosomes. Most cells were pseudotetraploid, and in all cells the normal chromosomes were represented in varied numbers, i.e., from 1 to 7 copies. The tumor had 5 characteristic and consistent marker chromosomes which were identified as deletions of chromosomes 1, 2, 7, 16, and 17. All metaphases had several double minute chromosomes (dmin) of variable size and possible intermediate structures between dmin and homogeneously staining chromosomal regions. The glucagonoma had a nearly equal proportion of normal metaphases and metaphases with structural and numerical abnormalities with no consistent trend.


Reduced intracellular drug accumulation in the absence of P-glycoprotein (mdr1) overexpression in mitoxantrone-resistant human MCF-7 breast cancer cells.

A mitoxantrone-resistant human MCF-7 breast cancer subline (MCF/MX) which is approximately 4000-fold resistant to mitoxantrone was isolated by serial passage of the parental wild-type MCF-7 cells (MCF/WT) in stepwise increasing concentrations of drug. MCF/MX cells were also approximately 10-fold cross-resistant to doxorubicin and etoposide but were not cross-resistant to vinblastine. Intracellular accumulation of radiolabeled mitoxantrone was markedly reduced in MCF/MX cells relative to that in the drug-sensitive MCF/WT cells. This decrease in intracellular drug accumulation into MCF/MX cells was associated with enhanced drug efflux, which was reversed when cells were incubated in the presence of sodium azide and 2, 4-dinitrophenol, suggesting an energy-dependent process. Incubation of MCF/MX cells with verapamil did not affect either the accumulation of mitoxantrone or the level of resistance in these cells. Furthermore, RNase protection and Western blot analyses failed to detect the expression of the mdr1 RNA or P-glycoprotein, a drug efflux pump known to be associated with the development of multidrug resistance in vitro. However, a polyclonal antibody directed against a synthetic peptide corresponding to the putative ATP binding domain of P-glycoprotein reacted with two (M(r) 42,000 and 85,000) membrane proteins from MCF/MX cells which were not found in MCF/WT. Functional assays and Western blot analysis for topoisomerase II revealed no differences in topoisomerase II activity or protein levels in MCF/MX cells. Thus, resistance in this cell line is apparently associated with enhanced drug efflux involving a pathway distinct from the mdr1-encoded multidrug transporter P-glycoprotein.


Selective inhibition of tumor cell growth by a recombinant single-chain antibody-toxin specific for the erbB-2 receptor.

A high percentage of human breast and ovarian tumors display amplified c-erbB-2 gene copies, leading to overexpression of the growth factor receptor. Its membrane location and elevated expression make the erbB-2 protein an appropriate target for a directed tumor therapy. We have used recombinant DNA technology to produce a single-chain antibody-exotoxin A (scFv-ETA) fusion protein which specifically binds the human erbB-2 receptor. The scFv portion is composed of the heavy- and light-chain variable domains of a monoclonal antibody which recognizes the extracellular domain of the human erbB-2 receptor. The bacterially produced scFv-ETA protein was shown to bind specifically to cells expressing the human erbB-2 protein. The scFv-ETA inhibits protein synthesis in erbB-2-expressing tumor cells at doses ranging from 2 to 200 ng/ml and is cytotoxic for these cells at equivalent doses. In athymic nude mice, administration of the scFv-ETA inhibited the growth of erbB-2-overexpressing human ovarian carcinoma cells.


Structural requirements of simple organic cations for recognition by multidrug-resistant cells.

We previously noted that a wide variety of drugs which are recognized by multidrug-resistant cells (MDR+) are positively charged. However, it remains unclear why and how such a large number of structurally different compounds can be distinguished by MDR+ cells. The majority of the diverse compounds subject to MDR are complex and thereby complicate definitive structure/function characterization of the P-glycoprotein-mediated MDR mechanism. Using a series of simple aromatic (alkypyridiniums) and nonaromatic (alkylguanidiniums) organic cations differing in their lipophilicity by stepwise additions of single alkyl carbons, we demonstrate by growth inhibition studies that a single aromatic moiety and a critical degree of lipophilicity (log P > -1) are required for recognition of these simple organic cations by MDR+ cells. Thus, MDR+ cells are not cross-resistant to the nonaromatic guanidiniums but do show cross-resistance to those aromatic pyridiniums with chain lengths > four. Resistance ratios, as determined by comparison of 50% inhibitory doses in MDR- versus MDR+ cells, increase as a function of increasing chain lengths of these latter simple aromatic compounds. Resistance to pyridinium analogues in MDR+ cells is reversible by co-treatment with nontoxic doses of verapamil. Preliminary uptake data with radioactive analogues further implicate the MDR mechanism of lowered drug accumulation in accounting for resistance to the pyridinium homologues. Utilization of these simple organic cations provides a rational basis for better defining the physical chemical properties of more complex compounds processed by the MDR mechanism and suggests a strategy for designing chemotherapeutic agents with reduced susceptibility to MDR.


Tumorigenic suppression of a human cutaneous squamous cell carcinoma cell line in the nude mouse skin graft assay.

The development of human squamous cell carcinomas has been associated with a number of genetic alterations involving chromosome 11, including cytogenetic and allelic deletions as well as amplification of genes in the 11q13 region. To determine the relevance of chromosome 11 in the formation of tumors of stratified squamous epithelial origin, we have introduced, via microcell fusion, a normal human chromosome 11 into the cutaneous squamous cell carcinoma cell line A3886TGc2. The ability of chromosome 11 to modulate the tumorigenicity of A3886TGc2 was evaluated first by inoculating cells s.c. in nude mice. All hybrids remained tumorigenic but exhibited longer tumor latencies than the parent, a result previously observed by other laboratories. We then tested our epidermally derived hybrids in the more physiologically relevant environment of the nude mouse skin graft system. The tumorigenic phenotype of three of four chromosome 11 hybrids placed into nude mouse skin grafts was completely suppressed. Polymerase chain reaction amplification of DNA from normal skin present at the suppressed graft sites failed to detect the introduced human cells. This information indicates that the normal skin is of mouse origin and suggests that the chromosome 11 microcell hybrids did not differentiate in vivo, but most likely failed to survive. We propose that external environmental factors present at the site of inoculation modulate the tumorigenic potential of these cells.


Expression and CpG methylation of the insulin-like growth factor II gene in human smooth muscle tumors.

Previously we have shown that expression of the insulin-like growth factor II (IGF-II) gene in 36 normal smooth muscle tissues (myometria) and 26 benign smooth muscle tumors (leiomyomas) was detectable by Northern blot analysis but that the RNA levels were low. In 9 of 20 malignant smooth muscle tumors (leiomyosarcomas) IGF-II gene expression was also low or absent, while in 11 of 20 the IGF-II gene was abundantly expressed. In 32 of these tissues we have now studied the DNA methylation state of the IGF-II gene. For the analysis of overall methylation of the gene the restriction endonucleases HpaII and MspI were used. In normal smooth muscle and in leiomyomas the IGF-II gene appeared to be methylated. In leiomyosarcomas with low IGF-II gene expression the DNA was partly demethylated. In leiomyosarcomas with abundant IGF-II gene expression overall methylation of the DNA tended to be low. In addition, we have studied the methylation state of one particular CpG site in the IGF-II gene with the restriction endonuclease AvaII. The results of the latter analysis confirm the analysis with HpaII and MspI. In conclusion, in malignant smooth muscle tumors the data indicate an inverse correlation between CpG methylation and expression of the IGF-II gene.


Transcriptional repression of the neu protooncogene by estrogen stimulated estrogen receptor.

Both neu gene overexpression and loss of estrogen receptor (ER) expression have been found to correlate with a poor prognosis in human breast cancer. Studies of breast tumor specimens have suggested that these two factors are not independent, leading us to hypothesize that there is a causal relationship between loss of ER and overexpression of neu. In this report, we confirm that ER can negatively regulate the expression of the neu gene protein product, p185neu, in two ER positive but not an ER negative breast cancer cell line(s). We have produced sublines which stably express human ER from a previously ER negative human breast cancer cell line. We demonstrate that the expression of ER in these cell lines is sufficient to confer the ability to respond to estradiol by down-regulating neu expression at both the protein and RNA levels. Utilizing neu promoter-chloramphenicol acetyltransferase constructs in transient cotransfection assays, we have also shown that this regulation occurs at the transcriptional level and requires the presence of both ER and estradiol. Furthermore, utilizing promoter deletion constructs, we provide evidence that a 140-base pair region of the neu promoter is required for this transcriptional regulation. When placed in a heterologous promoter, this 140-base pair region allows transcriptional repression by estradiol stimulated ER; thus, it represents an estrogen responsive region within the neu promoter. Finally, we have used gel mobility shift analysis to demonstrate an alteration in the nuclear factor(s) binding to this promoter region in estradiol stimulated versus estradiol deprived breast cancer cells. This study provides the first evidence that the inverse clinical correlation between neu and ER expression may be due to transcriptional repression of neu by estradiol stimulated ER.


A new functional role for P-glycoprotein: efflux pump for benzo(alpha)pyrene in human breast cancer MCF-7 cells.

We propose that the cellular burden of certain carcinogens may be mitigated by P-glycoprotein (P-gp), the putative drug efflux pump. In a series of multidrug resistant human breast cancer MCF-7 cells with increasing P-gp expression we examined this hypothesis using benzo(alpha)pyrene, a widely distributed environmental and dietary carcinogen. We found that multidrug resistant cells were cross-resistant to benzo(alpha)pyrene and the rates of efflux for benzo(alpha)pyrene were higher in multidrug resistant cells than in wild type cells. Evidence supporting the involvement of P-gp included the inhibition of azidopine binding to P-gp benzo(alpha)pyrene and the inhibition of benzo(alpha)pyrene efflux by Adriamycin and verapamil. Our findings suggest that P-gp may play a role in the cellular defense to carcinogens. The expression of P-gp and the modulation of its function may affect the susceptibility of normal tissues to transformation by carcinogens.


Evidence for the involvement of a potential second tumor suppressor gene on chromosome 17 distinct from p53 in malignant astrocytomas.

Molecular analysis of malignant astrocytomas demonstrated three distinct groups of tumors with chromosome 17p abnormalities, which include (a) deletion of the p53 locus (17p13.1) and mutations in the remaining allele, (b) deletion of the p53 locus but no detectable mutations in the remaining allele, and (c) deletions not including the p53 locus but mutations in one of the alleles. Furthermore, deletion mapping analysis demonstrated allelic loss of genes distal to D17S28/D17S5 markers (17p13.3) in group C tumors. The loss of heterozygosity of genes on chromosome 17 without detectable mutation (group B) or deletion (group C) in the p53 gene implies the presence of a second tumor suppressor gene in the telomeric region of 17p, the homozygous functional inactivation of which may play a role, either alone or in conjunction with p53, in the initiation and/or progression of astrocytic neoplasms.


Cytotoxic T lymphocytes and immune surveillance.

Cytotoxic T lymphocytes recognize peptide fragments of cytoplasmic proteins presented by class I MHC molecules. Recently, these peptides have been shown to be short, usually nonamers, and their binding to particular class I molecules is now well understood. There is also detailed understanding of how proteins are processed to generate antigenic peptides and some knowledge of the T lymphocyte receptors involved. CTL have been shown to be important in controlling virus infections, including those that cause tumours. CTL have also been demonstrated against other tumours, and their activity may lead to the abnormalities of MHC class I molecule expression that are frequently observed on tumours. The overall role of CTL is probably to survey body cells for abnormalities, including those induced by virus infection and mutation, and to destroy potentially harmful cells.


Thyroid hormone receptors and their role in development.

That most major physiological actions of thyroid hormones could be mediated via hormonal regulation of gene expression has been known for more than 25 years. The localization of TR in the cell nucleus, first reported almost 20 years ago, confirmed this concept. But it is only since the cloning of the TR gene and its identification as the c-erbA oncogene, accomplished 6 years ago, that we have begun to understand the details of the interaction between the hormone and its receptor and between the receptor and its target gene. Perhaps the most significant concept to emerge from the molecular studies is that the TR belongs to the superfamily of nuclear receptors for steroid hormones and morphogens such as retinoids. It highlights the evolutionary conservation of a major network of cellular signalling and intracellular regulatory pathways and which has helped bring us closer to a unified concept of the action of many growth and developmental hormones. Several important questions to be solved in the future become obvious from this brief review of the role of thyroid hormone in regulating developmental processes. Among these is the explantation for the high degree of tissue specificity of hormonal regulation of gene expression. The discovery of differential expression of the two major TR genes and the generation of multiple isoforms of the receptor by alternate splicing go some way to answering this question, but this is clearly not the sole factor determining tissue specificity. It will be most important to find out more about the interaction between the receptor and other transcription factors or nuclear proteins, some of which may be tissue specific and others expressed ubiquitously. The autoinduction of TR during amphibian metamorphosis, described above, emphasizes the intriguing question of how receptor genes are regulated during development. We know little as yet about the promoters and regulatory factors involved in this process. Finally, we have also described the recent recognition of genetic defects in TRs that underlie thyroid hormone linked diseases in humans. Future studies on the molecular genetics of receptors will enhance the importance in clinical practice of receptor linked diseases.


The leukaemia oncogene v-erbA: a dominant negative version of ligand dependent transcription factors that regulates red cell differentiation?

The v-erbA oncogene of avian erythroblastosis virus alters the growth properties and arrests differentiation of chick erythroid progenitor cells. The v-erbA protein is a mutated, ligand independent version of the c-erbA/T3R alpha chick receptor for T3, a ligand dependent transcriptional regulator. In reconstituted systems using idealized hormone responsive elements, over-expressed v-erbA acts as a dominant repressor of transcription mediated by liganded c-erbA/T3R alpha. This property seems to account for at least part of the phenotype of AEV transformed erythroid cells and for the transcriptional repression of some erythrocyte specific genes. However, v-erbA is likely to interfere with regulatory circuits other than those directly regulated by T3 receptors. Aspects of this hypothesis are discussed in the context of available evidence for the role of T3 and other hormones in erythroid progenitor cells proliferation/differentiation.


Pharmacologic controversy of CNS stimulants in Gilles de la Tourette's syndrome.

The controversy of the use of stimulants in Gilles de la Tourette's syndrome (GTS) can only be understood by examining the relationship between GTS and attention deficit-hyperactivity disorder (ADHD), because the relationship between the two disorders is complex, and stimulants are used in the one (ADHD) and may be contraindicated in the other (GTS). This relationship therefore has to be viewed from several perspectives, including clinical, genetic, neurobiochemical, neurophysiological, and treatment strategies, to highlight the complexities involved, and reasons for controversy. The present review will examine these relationships, and thus the evidence for and against the treatment of GTS with stimulants.


[Heart insufficiency treated with beta-blocking agents. Comparison of responders and non-responders].

In the last few years several Authors reported positive results using beta-blocking therapy in chronic heart failure. They also observed a group of patients who did not improve after this treatment. The aim of our study was comparing hemodynamic, neurohumoral and heart rate responses to beta-blockers in 2 groups of patients: Group A, patients who improved and Group B, patients who did not improve their clinical conditions after beta-blockade. We studied 26 patients with chronic heart failure of different origin: coronary artery disease and idiopathic dilative cardiomyopathy. They were treated with atenolol 50 mg/die for at least 1 year. Patients were divided into 2 groups: Group A including patients who increased the exercise time and the peak VO2 (> or = 2 ml/min/kg) and Group B, patients who did not increase exercise time and peak VO2 during beta-blockers. In Group A patients we observed a correlation between heart rate and end-diastolic volume by scintigraphy. We did not observe this correlation in Group B patients. The ejection fraction, evaluated by scintigraphy, significantly increased in Group A, while in Group B did not change. In both groups of patients we observed that plasmatic norepinephrine decreased significantly. In our opinion, this reduction is not due to an amelioration of organ clearance, because it was observed even in patients who did not show an increase of cardiac index and of ejection fraction. We suggest that the decrease of plasmatic norepinephrine might be the effect of beta-blockade and that it is not related to clinical response after treatment.


Improved isovolumetric relaxation in canine reperfused myocardium after beta 1 adrenergic stimulation.

beta 1 Adrenergic stimulation with the partial agonist xamoterol improves the systolic recovery of reperfused ("stunned") myocardium. The effects of this agent on isovolumetric relaxation and diastolic ventricular filling were evaluated. In 15 open chest dogs, the distal left circumflex coronary artery was occluded for 15 min and then reperfused. Eight dogs served as placebo controls, and seven received xamoterol (100 micrograms.kg-1 intravenously) at 10 min reperfusion. Diastole was divided into an isovolumetric relaxation phase and a subsequent filling phase, and wall excursion velocities were calculated. In the control group isovolumetric wall thinning velocity in the reperfused myocardium recovered only slowly, from -5.67(SD 5.29) mm.s-1 at 10 min reperfusion to -4.84(6.35) mm.s-1 at 30 min reperfusion and 2.99(7.97) mm.s-1 at 8 h reperfusion. Xamoterol increased isovolumetric wall thinning velocity in the reperfused myocardium, from -7.78(6.25) mm.s-1 at 10 min reperfusion to 3.43(6.09) mm.s-1 at 30 min reperfusion, and 12.04(5.66) mm.s-1 at 8 h reperfusion, whereas wall thinning velocity during the filling phase was unaffected. At 8 h reperfusion the difference between the groups was still significant. When compared with the effects on systolic wall thickening velocity, the impairment of isovolumetric wall thinning velocity during occlusion and the improvement due to xamoterol were both more marked. These functional differences were not reflected by the two indices of global diastolic function, peak negative dP/dt and relaxation constant tau. beta 1 Adrenergic stimulation with xamoterol improves isovolumetric relaxation in reperfused myocardium more markedly than systolic contraction, but it has no direct effect during diastolic filling.


Transmural heterogeneity of postjunctional alpha 2 adrenergic coronary vasoconstriction in hypoperfused cat left ventricle.

The aim was to analyse the influence of coronary postjunctional alpha 1 and alpha 2 adrenergic vasoconstriction in hypoperfused myocardium with special emphasis on transmural distribution of blood flow. The left coronary artery was hypoperfused by means of a clamped shunt line. Sequential selective postjunctional alpha 1 (doxazosin) and alpha 2 (SK&F 104078) adrenergic antagonism was established following beta adrenergic antagonism with propranolol. Regional myocardial blood flow was measured with radiolabelled microspheres during equal coronary perfusion pressures. Experimental subjects were nine pentobarbitone anaesthetised open chest cats. Left coronary hypoperfusion decreased endocardial blood flow, whereas epicardial flow was unaltered. In this situation alpha 1 adrenergic antagonism did not affect myocardial blood flow. Subsequent alpha 2 antagonism impaired endocardial blood flow, whereas epicardial blood flow was augmented. Mean coronary vascular resistance declined following combined alpha 1 and alpha 2 adrenergic antagonism. Coronary alpha adrenergic vasoconstriction is localised mainly epicardially in hypoperfused myocardium and counteracts endocardial hypoperfusion distal to a fixed coronary stenosis. Furthermore, this vasoconstriction is of the postjunctional alpha 2 adrenergic subtype.


Long-term effects of syrup medications for recurrent otitis media on the dental health of 6- to 8-year-old children.

The dental response to repeated antimicrobial and antihistamine medications was studied by comparing the dental health of 64 adenoidectomized children 5 years after surgery to that of 212 untreated controls. Annual dental recordings starting from the age of 3 years were obtained from health care centers. As expected, the proportion of children who had several (> or = 11) syrup medications was significantly higher (p < 0.001) in the adenoidectomized than in the control group. Sucrose-containing syrup medications were prescribed twice as often for the children of the adenoidectomized as for the control group (p < 0.001). However, the average amount of antimicrobial syrup medications prescribed was 19.2 +/- (SD) 13.0 per child for the adenoidectomized as compared to 8.5 +/- 8.3 for the control children (p < 0.001). The dmf value of the adenoidectomized children at the age of 3 years (mean +/- SEM: 0.5 +/- 0.1) was significantly (p < 0.005) lower than that of the controls (1.1 +/- 0.2). The difference was still significant (p < 0.01) at the age of 4 years, but disappeared thereafter. In conclusion, the antibacterial syrup medication seemed to be associated with a significant decline in dental caries at first. The simultaneous use of antihistamines was, however, thought to delay normal tooth maturation, so that after discontinuation of the antimicrobial medication, accelerated formation of new carious lesions took place.


Glucocorticoids stimulate transcription of the rat phenylethanolamine N-methyltransferase (PNMT) gene in vivo and in vitro.

1. Phenylethanolamine N-methyltransferase (PNMT) is regulated by glucocorticoid hormones. This study investigates the ability of glucocorticoids to modulate transcription of the rat PNMT gene in vivo and in vitro. 2. In the adrenal glands of hypophysectomized (HPX'd) rats, the synthetic glucocorticoid dexamethasone (DEX) stimulates production of PNMT mRNA. Quantitative hybridization reveals that the levels of PNMT mRNA increase approximately threefold in total and poly(A)+RNA after 4 days of DEX treatment of HPX'd rats, a level which is maximal for this treatment. 3. ACTH, the hormonal stimulus of glucocorticoid biosynthesis in the adrenal cortex, enhances PNMT mRNA production to levels comparable to that achieved with DEX in this system. The steroid responsiveness of PNMT message production is specific for glucocorticoids. DEX also increases PNMT mRNA in the brain stem, although the magnitude and speed of response are lower than observed in the adrenal gland. 4. Additional confirmation of the inductive ability of glucocorticoids is demonstrated by the increase in PNMT immunoprecipitated following translation in vitro of adrenal RNAs from DEX-treated rats. Furthermore, the PNMT mRNA signal obtained by in situ hybridization histochemistry in adrenal sections and in primary cultures of dispersed rat adrenal medullae reveals that DEX effects on PNMT mRNA can be elicited both in vivo and in vitro. 5. Specifically, glucocorticoids exert their effects on expression of PNMT mRNA by elevating the rate of PNMT gene transcription: a 2.3-fold increase in PNMT transcription persists for 18 hr following DEX treatment of HPX'd rats. In summary, this study establishes that glucocorticoids directly and rapidly stimulate transcription of the rat PNMT gene.


A combined evaluation of biochemical and morphological changes during human neuroblastoma cell differentiation.

1. The effects of retinoic acid, gamma-interferon, cytosine arabinoside, nerve growth factor, tumor necrosis factor, and 12-O-tetradecanoylphorbol 13-acetate on the human neuroblastoma cell line, LAN-5, were studied. Intracellular levels of acetylcholinesterase, neuron-specific enolase, catecholamines and related neurotransmitters, vasointestinal peptide, and substance P were evaluated after induction. 2. Cell morphology was strongly affected by retinoic acid, gamma-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate. The main effects of retinoic acid and gamma-interferon were the loosening of cell clusters and the extension of long neurites; cytosine arabinoside induced cell body swelling and marked neuritogenesis. Following 12-O-tetradecanoylphorbol 13-acetate treatment, the cells became small, round, and neuritic. Conversely, modifications induced by nerve growth factor and tumor necrosis factor were mild. Cell proliferation rate was reduced by retinoic acid, gamma-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate, while nerve growth factor and tumor necrosis factor were devoid of effects. 3. Acetylcholinesterase activity was significantly stimulated by retinoic acid and by gamma-interferon. Neuron-specific enolase activity was unaffected by all treatments except 12-O-tetradecanoylphorbol 13-acetate, which enhanced it by 1.6-fold. 4. The cellular catecholamine and related metabolite content was lowered by retinoic acid and gamma-interferon, while cytosine arabinoside and, even more, 12-O-tetradecanoylphorbol 13-acetate showed a stimulatory activity on their intracellular accumulation. 5. Finally, the cell-associated vasointestinal peptide level was strikingly increased by gamma-interferon and, to a lesser extent, by retinoic acid, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate. 6. It is concluded that the most relevant biochemical changes associated with LAN-5 cells differentiation involve the repertoire of neurotransmitters and neuropeptides. These events vary in quality and in quantity, likely due to the pattern complexity of gene expression triggered by each inducer in determining the diversity of neuronal phenotypes.


Microtubule-associated protein 2 (MAP2)-immunoreactive neurons in the retina of Bufo marinus: colocalisation with tyrosine hydroxylase and serotonin in amacrine cells.

Neuron populations in the retina of the toad, Bufo marinus, were labelled with a monoclonal antibody raised against microtubule-associated protein 2 (MAP2). A subpopulation of cones, probably corresponding to the blue-sensitive small single cones, large diameter amacrine cells in the most proximal row of the inner nuclear layer and some large ganglion cells in the ganglion cell layer were labelled. Double labelling experiments were carried out to establish the colocalisation of MAP2 with known putative transmitter substances of the anuran amacrine cells. MAP2 was colocalised in a subpopulation of serotonin-immunoreactive and in all tyrosine hydroxylase-immunoreactive amacrine cells. The results indicate, that the MAP2 content in the neurons of the anuran retina can be correlated with other well-defined neurochemical and/or physiological properties.


Characterization of a RFamide-positive subset of ganglionic cells in the hydrozoan planular nerve net.

The complexity of the hydrozoan planular nervous system was examined. Using a whole-mount technique with indirect immunofluorescence, the spatial pattern of ganglionic cells showing RFamide-like immunoreactivity was visualized. RFamide antiserum bound a subset of ganglionic cells in the anterior and upper middle regions of the planula and a few ganglionic cells in the upper tail region. Labeled cells consisted of bipolar and multipolar neurons. Stained processes from these cells formed a three-dimensional nerve net that followed the contour of the mesoglea; such fibers were striking in terms of their large numbers, long lengths, and organization into distinct bundles. Labeled fibers were seen to contact other ganglionic cells, sensory cells, epithelio-muscle cells, the mesoglea, and the outside free surface. All stained cell bodies and fibers were found in the ectoderm. Using the same technique the reappearance of RFamide-positive ganglionic cells in epithelial tissue of chimeric grafts of planulae was observed. Interstitial cells capable of forming RFamide-positive ganglionic cells underwent extensive anterior-posterior migrations in the grafts, moved into the epithelial tissue, and differentiated into RFamide-positive ganglionic cells. Stained repopulated ganglionic cells always formed in the same position in the epithelial tissue as was observed in control planulae suggesting that the expression of RFamide-like substances may be position dependent in the planula.


Immunocytochemical localization of muscarinic acetylcholine receptors in the rat endocrine pancreas.

Immunocytochemical application of the antimuscarinic acetylcholine receptor antibody M35 to pancreas tissue revealed the target areas for the parasympathetic nervous system. Immunoreactivity in the endocrine pancreas was much higher than that in the exocrine part. Moreover, the endocrine cells at the periphery of the islets of Langerhans displayed the highest level of immunoreactivity. Based on these findings in the mantle of the islets, two types of islets have been distinguished: type-I islets with intensely stained mantle cells, and type-II islets with a much lower concentration of these cells. On average, type-I islets were larger (244.8 microns +/- 6.1 SEM) than type-II islets (121.5 microns +/- 3.8 SEM). M35-immunoreactivity was present on the majority of D cells, which were characterized by their immunoreactivity to somatostatin [of 446 D cells 356 (79.8%) were M35-immunopositive]. However, only a small proportion of the intensely stained mantle cells belonged to the D cell population. Therefore, it is concluded that the majority of the intensely stained mantle cells represent glucagon-secreting A and/or pancreatic polypeptide-secreting F cells. The intensity of M35-immunoreactivity at the periphery and central core of the islets paralleled the density of cholinergic innervation, suggesting a positive correlation between the intensity of cholinergic transmission and the number of muscarinic acetylcholine receptors at the target structures. The present study further revealed some striking parallels for the muscarinic acetylcholine receptor characteristics between the (endocrine) pancreas and the central nervous system.


Bethanechol and a G-protein activator, NaF/AlCl3, induce secretory response in Paneth cells of mouse intestine.

Paneth cells located at the bottom of intestinal crypts may play a role in controlling the bacterial milieu of the intestine. Using morphometry to clarify the secretory mechanism of the Paneth cells, we studied the ultrastructural changes in mouse Paneth cells produced following intra-arterial perfusion with Hanks' balanced salt solution containing a cholinergic muscarinic secretagogue (bethanechol), a neuroblocking agent (tetrodotoxin), or a G-protein activator (NAF/AlCl3). Bethanechol (2 x 10(-4) mol/l) induced Paneth-cell secretion. Many Paneth cells massively exocytosed their secretory material into the crypt lumen; the enhanced secretion caused degranulation and vacuole formation. However, tetrodotoxin (2 x 10(-6) mol/l) did not prevent the bethanechol-enhanced secretion by the Paneth cells. NaF (1 x 10(-2) mol/l) and AlCl3 (1 x 10(-5) mol/l) induced massive exocytosis of the Paneth cells; the exocytotic figures were similar to those observed in mice stimulated by bethanechol. G-protein activation was followed by a sequence of intracellular events, resulting in exocytosis.


Immunocytochemical and circadian biochemical analysis of neuroactive amino acids in the pineal gland of the rat: effect of superior cervical ganglionectomy.

Semiquantitative immunocytochemistry by immuno-gold techniques revealed differences in the spatial distribution of glutamate, glutamine, and taurine within the pineal gland, with greatest labeling over pinealocytes, glia, and endothelia, respectively. At the subcellular level, glutamate labeling tended to be highest over pinealocyte synaptic ribbons and mitochondria, and lowest over lipid inclusions. Pineal levels of glutamate, glutamine and taurine, as measured by high performance liquid chromatography, did not vary over a light: dark cycle. Superior cervical sympathetic denervation, which abolishes pineal melatonin synthesis, resulted in a nearly 50% reduction in pineal glutamate levels, but had no effect on levels of glutamine and taurine. Other amino acids (alanine, arginine, aspartate, serine) were reduced by 23%-33% following sympathectomy. These data suggest an important role for glutamate in pinealocyte function(s) possibly related to the noradrenergic innervation of the gland.


Evidence for projections from medullary nuclei onto serotonergic and dopaminergic neurons in the midbrain dorsal raphe nucleus of the rat.

The anterograde tracer Phaseolus vulgaris-leucoagglutinin was injected into the medial nucleus of the solitary tract and into the rostral dorsomedial medulla. A sequential two-color immunoperoxidase staining was accomplished in order to demonstrate the co-distribution of presumed terminal axons with chemically distinct neurons in the dorsal raphe nucleus of the midbrain central gray, i.e., B7 serotonergic and A10dc dopaminergic neurons. Black-stained efferent fibers from the medial nucleus of the solitary tract and the rostral dorsomedial medulla intermingled with brown-stained serotonergic (5-hydroxytryptamine-immunoreactive) or dopaminergic (tyrosine hydroxylase-immunoreactive) neurons. Light microscopy revealed that the black-stained efferent axons exhibited numerous en passant and terminal varicosities that were often found in close apposition to brown-stained serotonergic and dopaminergic somata, and to proximal and distal dendrites and dendritic processes. The close association of immunoreactive elements suggests the presence of axo-somatic and axo-dendritic synaptic contacts of medullary fibers with serotonergic and dopaminergic neurons in the dorsal raphe nucleus. These projections could be involved in the modulation of dorsal raphe neurons, depending on the autonomic status of an animal.


Ovarian innervation develops before initiation of folliculogenesis in the rat.

Sympathetic neurotransmitters have been shown to be present in the ovary of the rat during early postnatal development and to affect steroidogenesis before the ovary becomes responsive to gonadotropins, and before the first primordial follicles are formed. This study was undertaken to determine if development of the ovarian innervation is an event that antedates the initiation of folliculogenesis in the rat, Rattus norvegicus. Serial sections of postnatal ovaries revealed a negligible frequency of follicles 24 h after birth (about 1 primordial follicle per ovary). Twelve hours later there were about 500 follicles per ovary, a number that more than doubled to about 1300 during the subsequent 12 h, indicating that an explosive period of follicular differentiation occurs between the end of postnatal days 1 and 2. Electron microscopy demonstrated that before birth the ovaries are already innervated by fibers containing clear and dense-core vesicles. Immunohistochemistry performed on either fetal (day 19) or newborn (less than 15h after birth) ovaries showed the presence of catecholaminergic nerves, identified by their content of immunoreactive tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. While some of these fibers innervate blood vessels, others are associated with primordial ovarian cells, thereby suggesting their participation in non-vascular functions. Since prefollicular ovaries are insensitive to gonadotropins, the results suggest that the developing ovary becomes subjected to direct neurogenic influences before it acquires responsiveness to gonadotropins.


Vasoreactivity of the intracranial internal carotid artery.

The vasoreactivity of the intracranial segment of the internal carotid artery to transmitters, present in the perivascular sympathetic, parasympathetic and sensory nerves, as well as to other vasoactive agents of relevance for headache, was tested in man and monkey. The total arterial segment from both species is equipped with contractile receptors for noradrenaline, serotonin, prostaglandin F2 alpha, ergotamine and sumatriptan. Further, the total arterial segment dilated upon exposure to calcitonin gene-related peptide in both species. Other vascoactive transmitters, acetylcholine, substance P and neurokinin A, caused only weak dilatation, restricted to the proximal extracavernous segment in the monkey. The findings are discussed in relation to the pathogenesis and treatment of cluster headache.


Homeotic genes of the Bithorax complex repress limb development in the abdomen of the Drosophila embryo through the target gene Distal-less.

Homeotic genes encode transcription factors that are thought to specify segmental identity by regulating expression of subordinate genes. Limb development is repressed in the abdominal segments of the Drosophila embryo by the hometic genes of the Bithorax complex (BX-C). Localized expression of the homeobox gene Distal-less (DII) is required for leg development in thoracic segments. We have identified a minimal cis-regulatory enhancer element that directs DII expression in the larval leg primordia. We present evidence that the BX-C proteins repress DII expression in abdominal segments by binding to a small number of specific sites in this element. Mutating these sites eliminates BX-C protein binding and renders the element insensitive to BX-C-mediated repression in vivo. Repression of limb development in the abdomen appears to be controlled at the DII enhancer. Thus DII may serve as a downstream target gene through which the homeotic genes control abdominal segment identity in the Drosophila embryo.


D type cyclins associate with multiple protein kinases and the DNA replication and repair factor PCNA.

Human cyclin D1 has been associated with a wide variety of proliferative diseases but its biochemical role is unknown. In diploid fibroblasts we find that cyclin D1 is complexed with many other cellular proteins. Among them are protein kinase catalytic subunits CDK2, CDK4 (previously called PSK-J3), and CDK5 (also called PSSALRE). In addition, polypeptides of 21 kd and 36 kd are identified in association with cyclin D1. We show that the 36 kd protein is the proliferating cell nuclear antigen, PCNA. Cyclin D3 also associates with multiple protein kinases, p21 and PCNA. It is proposed that there exists a quaternary complex of D cyclin, CDK, PCNA, and p21 and that many combinatorial variations (cyclin D1, D3, CDK2, 4, and 5) may assemble in vivo. These findings link a human putative G1 cyclin that is associated with oncogenesis with a well-characterized DNA replication and repair factor.


Role of the production of natural killer cell stimulatory factor (NKSF/IL-12) in the ability of B cell lines to stimulate T and NK cell proliferation.

We have previously used human Epstein Barr virus (EBV)-transformed B lymphoblastoid cell lines for the identification and purification of a novel cytokine, natural killer cell stimulatory factor (NKSF/IL-12), that has pleiotropic effects on human lymphocytes. B cell lines are also routinely employed as feeder cells for the culture of T and natural killer (NK) cells. In this report we describe the ability of two NKSF/IL-12 producing B cell lines (RPMI-8866 and Cess) and two nonproducing lines (Raji and Daudi) to stimulate the proliferation of T and NK cells in 8-day PBL cultures. We demonstrate, using an anti-NKSF/IL-12 neutralizing monoclonal antibody, that the endogenous production of NKSF/IL-12 in these cultures can significantly enhance the proliferation and cytotoxic activity of T and NK cells. We also report that the addition of exogenous rNKSF/IL-12 can greatly increase the number of T and NK cells obtained from the cultures following stimulation by the B cell lines. Aside from the possible practical applications, the enhanced proliferation of T and NK cells consistently observed in the presence of endogenously produced NKSF/IL-12 or exogenously added rNKSF/IL-12 in this system may further our understanding of the role of this cytokine during an in vivo immune response.


Selective activation of murine V beta 8.2 bearing T cells by Pseudomonas exotoxin A.

We have determined that Pseudomonas aeruginosa exotoxin A (PE) can selectively stimulate the proliferation of V beta bearing T lymphocytes. Murine thymocytes were fractionated by selective agglutination with peanut agglutinin (PNA) and the PNA- thymocytes, which represent mature thymocytes, were shown to be responsive to PE stimulation. In addition, mature peripheral T lymphocytes (nylon wool nonadherent splenocytes) were also observed to respond to PE stimulation. Both CD4+ and CD8+ splenic T lymphocyte populations proliferated in response to PE. Flow microfluorimetry analysis of PNA- thymocytes stimulated with PE indicated that V beta 8.2 bearing T cells were preferentially expanded. Thus, our data indicate that PE represents a microbial super antigen which stimulates murine thymocytes which bear the V beta 8.2 element of the T cell receptor.


Acetylated alpha-tubulin in the pollen tube microtubules.

Three monoclonal alpha-tubulin antibodies YL 1/2 (Kilmartin et al., 1982), 6-11B-1 (Piperno and Fuller, 1985) and DM1A (Blose et al., 1984) were used in indirect immunofluorescence (IIF) microscopy of the microtubule (MT) cytoskeleton in tobacco (Nicotiana tabacum) pollen tubes. The majority of pollen tube MTs contain tyrosinated alpha-tubulin recognized by YL 1/2. Acetylated alpha-tubulin revealed by 6-11B-1 was detected in the generative cell and in the kinetochore fibers, in polar spindle regions, and in the cell plate of the phragmoplast during generative cell division. In addition, small fragments of acetylated microtubules were seen in the older parts of the pollen tube grown on a taxol medium. The interaction of pollen tube MTs with mAb 6-11B-1 suggested that acetylation of alpha-tubulin correlates well with the putative arrays of stable MTs.


Infectious pancreatic necrosis virus internalization and endocytic organelles in CHSE-214 cells.

The early events that take place during the internalization of infectious pancreatic necrosis virus (IPNV) into Chinook salmon embryo cells (CHSE-214) were analyzed ultrastructurally. Endocytic tracers were employed in order to characterize the organization of endocytic organelles in CHSE-214 cells, as well its relation to the IPNV penetration. Results demonstrate that IPNV appear internalized within vesicular compartments which are located peripherally in CHSE-214 cells. Despite the high rate of infectious multiplicity few virus particles were detected inside the cells. Endocytic tracer labelling of tubulovesicular elements and endosomes of host cells showed a well developed endocytic apparatus. Results suggest that endocytosis may be involved during the initiating events in the productive IPNV infection.


Somatostatin receptors.

The neuropeptide somatostatin (SRIF) is a neurotransmitter in the brain that exerts physiological actions including the modulation of Ca2+ and K+ conductances, neuronal cell firing, neurotransmitter release, and certain behaviors such as locomotion and cognitive functions. SRIF induces its biological effects by interacting with cell surface receptors. Recent studies have revealed that subtypes of SRIF receptors exist in the brain and other tissues. The SRIF1 receptor can be distinguished by its high affinity for the agonist MK 678, is coupled to G proteins, and mediates the stimulatory effects of SRIF on a delayed rectifier K+ current in brain neurons. Furthermore, MK 678, when applied to the nucleus accumbens, evokes locomotor activity, and SRIF1 receptors in this brain region selectively mediate the stimulation of this behavioral response to SRIF. SRIF1 receptors are unevenly distributed in the brain, with high levels in the dentate gyrus of the hippocampus, the locus coeruleus, the neostriatum, and the inner layers of the cerebral cortex. This receptor subtype has characteristics similar to the recently cloned SRIF receptor, SSTR2. A second SRIF receptor subtype has been identified in the brain and is referred to as the SRIF2 receptor. It has no affinity for MK 678, can be selectively labeled with smaller structural analogs of the peptide CGP 23996, and has characteristics similar to the recently cloned receptor subtype SSTR1. SRIF2 receptors are not efficiently coupled to G proteins and have a distinct but overlapping distribution in brain with SRIF1 receptors. No clear biological function has been identified for SRIF2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Tendon injuries of the foot and ankle.

Tendon injuries are often caused by direct trauma or overuse. Pathology may consist of inflammatory lesions external to the tendon sheath or inflammation of either the peritenon, sheath, or tendon. This article reviews the diagnosis and treatment of injuries to the peroneal, peroneus brevis, peroneus longus, anterior tibial, flexor hallucis longus, and posterior tibial tendons.


Cloning and genetic analyses of two highly polymorphic, moderately repetitive nuclear DNAs from Phytophthora infestans.

Randomly selected clones from a Phytophthora infestans partial genomic library were characterized by hybridizing individual clones to Southern blots of total genomic DNA digested with the restriction enzyme EcoRI. Among 59 clones that were screened on seven different central-Mexican isolates, five revealed a unique banding pattern for each isolate tested. Two of these clones were tested further; the banding patterns produced by both were somatically stable when probed to DNA from 63 single-zoospore (asexual) progeny from five different "parent" isolates. For one probe, RG57, each band appeared to represent a unique genetic locus in three different crosses, and each locus segregated for the presence or absence of a band. No bands were found to be allelic, but two pairs of cosegregating loci were identified. Genetic analyses of the other probe (RG7) revealed many more pairs of cosegregating bands and some bands which were allelic. When these probes were hybridized to DNA from the other five species in Phytophthora group IV, probe RG57 hybridized strongly to DNA from P. colocasiae, P. phaseoli and P. mirabilis, but weakly or not at all to that of P. hibernalis and P. ilicis. Probe RG7 hybridized fairly strongly to DNA from all six species. Because the sequence recognized by probe RG57 appears to be evolutionarily conserved, and is dispersed, moderately repetitive and highly polymorphic, it could be very useful in additional studies on the genetics and population biology of P. infestans.


Mitochondrial DNA of Schizophyllum commune: restriction map, genetic map, and mode of inheritance.

Mitochondrial DNA (mtDNA) found in the basidiomycete Schizophyllum commune (strain 4-40) is a circular molecule 49.75 kbp in length. A physical map containing 61 restriction sites revealed no repeat structures. Cloned genes from Neurospora crassa, Aspergillus nidulans, and Saccharomyces cerevisiae were used in Southern hybridizations to locate nine mitochondrial genes, including a possible pseudogene of ATPase 9, on the restriction map. A probe from a functional ATPase 9 gene identified homologous fragments only in the nuclear genome of S. commune. Restriction fragment length polymorphisms (RFLPs) between mtDNA isolated from different strains of S. commune were used to show that mitochondria do not migrate with nuclei during dikaryosis.


Transmission of mitochondrial DNA in Ustilago violacea.

Mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) were used as genetic markers for following mitochondrial transmission in the basidiomycete Ustilago violacea. Yeast-like cells of opposite mating types (a1 and a2) were mated on 2% water agar and were treated with alpha-tocopherol to induce formation of dikaryotic hyphae. Upon depletion of the alpha-tocopherol, the hyphae budded off haploid cells with parental nuclear genotypes. These cells were examined for mitochondrial RFLP phenotype. In progeny expressing the a1 mating type, mitochondria from either parent were observed equally frequently. In progeny with the a2 mating type, mitochondria were almost exclusively (94%) from the a2 parent.


Maternal transmission of mitochondrial DNA in interspecific hybrids of Populus.

Restriction fragment analysis was conducted to investigate the mode of inheritance of mitochondrial (mt) DNA in F1 progeny of two P. deltoides x P. deltoides, three P. deltoides x P. nigra, and two P. deltoides x P. maximowiczii controlled crosses, and in Populus x canadensis by using 16 restriction endonucleases and two heterologous probes of cloned mtDNA fragments of maize. Five restriction fragment length polymorphisms (RFLPs) of mtDNA differentiated P. deltoides from P. nigra, whereas three RFLPs of mtDNA separated P. deltoides from P. maximowiczii. In all cases, F1 progeny of P. deltoides x P. nigra, and P. deltoides x P. maximowiczii, crosses had mtDNA restriction fragments of only their maternal P. deltoides parents. P. x canadensis had mtDNA restriction fragments of only P. deltoides. F1 progeny of intraspecific P. deltoides crosses also had the same mtDNA fragments as their maternal parent. The results clearly demonstrate uniparental-maternal inheritance of the mitochondrial genome in F1 interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii.


Lipid lowering effects of high linoleate and high alpha-linolenate diets in rats and mice. Consequence of long-term feedings.

Diets high in linoleate (safflower oil) or high in alpha-linolenate (perilla oil) were fed to rats for 11 months, and the effects of the diets on plasma and tissue lipids were compared. The plasma levels of total cholesterol (Cho), phospholipids (PL) and triacylglycerol (TG) were significantly lower in the high alpha-linolenate group than in the high linoleate group, the differences being more than 30% in the levels of total Cho and TG. The diets had differential effects on the lipid contents of major tissues: the TG level in muscle was higher but both the TG level in depot fat and the PL level in muscle were lower in the high alpha-linolenate group than in the high linoleate group. In order to clarify whether or not the hypolipidemic effect of the high alpha-linolenate diet was due to changes in the distribution of lipids among tissues, whole body lipids were estimated in mice fed these diets for 5 months. The whole body Cho content was significantly lower, by 28%, in the high alpha-linolenate group compared with the high linoleate group, but the total lipid content, PL and neutral lipids were similar between the groups. Our results indicate that the high alpha-linolenate diet has a more potent cholesterol lowering effect in plasma and body tissue than the high linoleate diet; interestingly, whole body TG levels are similar but tissue distributions of TG are different between the two dietary groups.


Studies on antiulcer drugs. V. Synthesis and antiulcer activity of aralkylbenzazoles.

A series of 2-alkylamino-5- or 6-aralkyl-substituted benzazoles were synthesized and tested for histamine H2-receptor antagonist and anti-stress ulcer activities. These new compounds showed little or no histamine H2-receptor antagonist activity in contrast to imidazo[1,2-a]pyridine analogues (I). On antiulcer assay, however, some pyridine derivatives (II) exerted higher activity than the reference compounds, sofalcone, sucralfate and cimetidine. The structure-activity relationships of these compounds are discussed.


Corroding characteristics of Eikenella corrodens.

Fourteen strains of Eikenella corrodens isolated from human oral cavity were studied to determine corroding characteristics. Nine out of the 14 strains produced corroding colonies under anaerobic culture condition. One of them produced corrosion even in an aerobic culture. No morphological differences in surface structures were observed between corroding and non-corroding strains of E. corrodens by transmission electron microscopy. The morphology of corroding colonies of E. corrodens was then examined by scanning electron microscopy. The surface of the corroding-colony center was smoothly convex. A boundary line was clear between the smooth center and the surrounding corrosion region. Double or triple frill-like structures surrounded the center portion with small convexities. Spreading bacterial masses were observed in the outer portion of the colony. Morphological observations of the corroding colony edge indicated that a surface translocation termed "twitching motility" or "gliding motility" occurs in the outer portion and plays a role in its colonization of periodontal regions.


QT intervals at heart rates from 50 to 120 beats per minute during 24-hour electrocardiographic recordings in 100 healthy men. Effects of atenolol.

Conflicting reports about changes in QT intervals suggest that QT values should be compared at similar heart rates. Relations between QT and RR intervals were determined after measurement of QT values by Holter recording at heart rates of 50, 60, 70, 80, 90, 100, 110, and 120 beats per minute in 100 healthy young men. Fifteen men underwent a second recording during acute treatment with the beta-blocking agent atenolol. At heart rates between 80 and 120 beats per minute, the QT interval was significantly longer (from 9 to 16 msec), and at a heart rate of 50 beats per minute significantly shorter (26 msec), than values calculated from Bazett's formula. Sleep prolonged QT values by 18 msec at a heart rate of 60 beats per minute and by 21 msec at a heart rate of 50 beats per minute compared with the waking state. Atenolol lengthened QT intervals significantly (by 11-14 msec) at heart rates between 90 and 110 beats per minute and shortened them (by 12 msec) at a heart rate of 60 beats per minute. During sleep, QT intervals were the same before and after atenolol. The method of plotting QT against RR intervals after measurement of QT values at similar stable spontaneous heart rates before and after intervention allows changes in autonomic state and heart rate to be taken into account. By this method, QT values can be compared without distortion effects caused by correction formulas.


Reduction of infarct size with coronary venous retroperfusion.

The purpose of this study was to determine whether coronary venous retroperfusion with pressure-controlled intermittent coronary sinus occlusion (PICSO) alone and in combination with coronary venous substrate enhancement using L-glutamate would decrease ischemic damage after surgical revascularization for an acute coronary occlusion. In 40 pigs, the second and third diagonal vessels were occluded with snares for 90 minutes followed by 30 minutes of cardioplegic arrest and 180 minutes of reperfusion with the coronary snares released. During the period of coronary occlusion, 10 pigs received PICSO using a balloon-tipped triple-lumen catheter in the coronary sinus; 10 pigs received PICSO plus oxygenated blood transfused retrograde via the PICSO catheter (7 ml/min), 10 pigs received PICSO plus an oxygenated blood L-glutamate (13 mM) solution, and 10 pigs received neither PICSO, blood, nor L-glutamate through the coronary sinus (unmodified). Hearts treated with PICSO had higher wall motion scores (1.27 +/- 0.33 for unmodified, 2.40 +/- 0.40* for PICSO, 2.45 +/- 0.20* for PICSO plus blood, 2.85 +/- 0.30* for PICSO plus L-glutamate; *p < 0.05 from unmodified where 4 is normal to -1 is dyskinesia), lower area of necrosis-to-area of risk ratio using histochemical staining techniques (73 +/- 4% for unmodified, 27 +/- 4 for PICSO; 18 +/- 2* for PICSO plus blood, 12 +/- 1* PICSO plus L-glutamate; *p < 0.05 from unmodified), significantly less tissue acidosis (pH) compared with the unmodified group (pH, -0.41 +/- 0.13 for unmodified, -0.16 +/- 0.03* for PICSO, -0.19 +/- 0.02* for PICSO plus blood, -0.20 +/- 0.08* for PICSO plus L-glutamate; *p < 0.05 from unmodified). Coronary venous retroperfusion with PICSO alone and in combination with coronary venous substrate enhancement using L-glutamate significantly decreases ischemic damage during urgent surgical revascularization.


Effect of antibody to leukocyte adhesion molecule CD18 on recovery of neonatal lamb hearts after 2 hours of cold ischemia.

Prior studies have shown that leukocytes are important in reperfusion injury after normothermic ischemia, and recently discovered leukocyte adhesion molecules that bind to endothelial ligands may be involved in this process. No studies on the role of leukocyte adhesion molecules in hypothermic ischemia are available. We assessed the effect of preischemic administration of monoclonal antibody to the leukocyte adhesion molecule CD18 on recovery of isolated blood-perfused neonatal lamb hearts arrested for 2 hours with 15 degrees C K+ cardioplegic solution by comparing nine antibody-treated and nine control hearts. At 30 minutes after reperfusion, antibody-treated hearts compared with control hearts had better percent recovery of maximum left ventricular (LV) developed pressure (83.9 +/- 2.2% versus 73.6 +/- 3.0%, mean +/- SEM), LV dP/dt (78.4 +/- 3.3% versus 67.4 +/- 3.4%), coronary blood flow (159.5 +/- 12.2% versus 84.4 +/- 3.5%), and myocardial oxygen consumption (129.8 +/- 16.5% versus 71.2 +/- 6.2%) (all p < 0.05). In each heart, we also tested coronary vascular resistance (CVR) response to 10(-6) mol/l acetylcholine (ACh) infusion to assess endothelial function. Percent recovery of CVR response to ACh was higher in antibody-treated hearts (38.4 +/- 4.3%) than control hearts (13.4 +/- 12.8%) (p = 0.08). These results suggest that leukocyte adherence to endothelium mediated by CD18 plays an important role in reperfusion after hypothermic ischemia. Antibody to CD18 may be useful in myocardial and endothelial protection during surgically induced ischemia.


Expression of the gene encoding tyrosine hydroxylase in a subpopulation of quail dorsal root ganglion cells cultured in the presence of insulin or chick embryo extract.

Avian sensory ganglia contain a population of normally latent autonomic precursors with catecholaminergic potentialities. The present study examines the expression of the tyrosine hydroxylase (TH) gene in quail dorsal root ganglia (DRG) by both in situ hybridization and polymerase chain reaction (PCR) techniques. In situ hybridization using quail TH cDNA as a probe demonstrated the presence in DRG cell cultures of TH mRNA in a subpopulation of cells that never express the adrenergic phenotype in vivo. Expression of the TH gene in autonomic precursor cells of DRG in culture is totally dependent on the presence either of insulin or chick embryo extract. The numbers of catecholaminergic cells expressing TH mRNA and TH immunoreactivity evolve in a closely similar manner during the culture period. Using two primers, specific for highly conserved 5' regions of TH cDNA, it was possible to detect the same band of DNA amplified by PCR in total RNA from DRG cultures grown in the presence of insulin, sympathetic ganglia and adrenal gland. No amplified DNA was detected in uncultured DRG cells. These data further indicate that, under the influence either of insulin or a still unknown factor contained in the CEE, the TH gene is induced in a subpopulation of DRG cells.


Effect of insulin and insulin-like growth factor I on the expression of the catecholaminergic phenotype by neural crest cells.

Neural crest-derived catecholaminergic precursors have been used as a model to study the role of signals supplied by the environment during avian neurogenesis. A new culture system consisting of dissociated sclerotomes or somites isolated at embryonic day 3 (E3) or 2.5 (E2.5) has been established, which allows quantitative comparison of various culture conditions. As a first step of this study, the role of insulin and insulin-like growth factor I (IGF-I) in catecholaminergic differentiation has been investigated. Our results show that both factors are able to increase by a factor 2 to 3 the number of catecholaminergic cells present in the culture of sclerotomes after 24 h of culture. The effect is dose-dependent and the half-maximal effect is obtained with low concentrations of each peptide. Since insulin, IGF-I and their respective receptors are present at this stage of development in avian embryo, our observations suggest that an early step in catecholaminergic differentiation could be under at least the partial control of insulin and insulin-related peptides. On the other hand, neural crest precursors isolated at E2.5 are not able to generate catecholaminergic cells and to respond to insulin when cultivated for one day, indicating that these precursors are subjected in vivo to a maturation step, within the somite/sclerotome between E2.5 and E3; this step could be obtained in vitro by cultivating the precursors for 1 day, which resulted in the development of insulin responsiveness by catecholaminergic precursors.


Early appearance of tyrosine hydroxylase immunoreactivity in the retina of human embryos.

In the retina of 6 post-ovulatory week old human embryos, tyrosine hydroxylase (TH) immunoreactivity is expressed in retinoblasts in the peripheral retina, and ganglion-like cells with an axon in the optic nerve in the posterior retina. This is the first report on the expression of a catecholamine marker in a sub-population of migrating retinoblasts. Since in the adult retina the only TH+ cells are a sub-population of amacrine cells, the expression of this enzyme in some ganglion-like cells must represent either a transient developmental event, or indicate that these cells subsequently undergo transformation through axonal degeneration.


Analysis for transaminases in serum with an amperometric glutamate electrode.

We determined transaminases in human blood serum with an amperometric glutamate biosensor. The probe was a hydrogen peroxide sensor assembled with appropriate selective membranes to enhance the probe specificity and lifetime. Calibration curves of glutamate were linear in the range 1-1000 mumol/L, with a response time of < 1 min. This probe was subsequently applied to the measurement of activities of aspartate and alanine aminotransferases in human sera. Analytical recovery studies demonstrated the suitability of the glutamate sensor by measuring 91-99% of added glutamate, 92-106% of added aspartate aminotransferase, and 101-105% of added alanine aminotransferase. Transaminase activity measured in 80 sera correlated well with results obtained with a spectrophotometric procedure.


Characterization of dipeptidyl peptidase IV (CD26) from human lymphocytes.

The membrane-bound dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) has been purified 5,400-fold from human peripheral blood mononuclear cells. The purification procedure included detergent solubilization and successive chromatography on DEAE Sepharose Fast Flow, Con A Sepharose, Cu2+ loaded metal-chelating Sepharose, Sephacryl S-300 High Resolution and Q Sepharose Hiload. The molecular mass of the native, detergent solubilized enzyme estimated by gel filtration was 264.kDa. Chromatofocusing indicated a pI of approximately 5.0. The pI optimum was 8.7. The enzymatic activity of the purified preparation was irreversibly inhibited by N-(H-Phe-Pro)-O-(4-nitrobenzoyl)hydroxylamine hydrochloride in the micromolar range. The binding of purified DPP IV to CD26 monoclonal antibodies confirmed the identity between CD26 and dipeptidyl peptidase IV. The purification and characterization of lymphocytic dipeptidyl peptidase IV is of great value for the identification of its natural substrates and for the study of its physiological significance in the T-lymphocyte function.


Evaluation of serum basal thyrotrophin levels and thyrotrophin receptor antibody activities as prognostic markers for discontinuation of antithyroid drug treatment in patients with Graves' disease.

We evaluated whether antithyroid drug treatment could be terminated more appropriately when both the serum basal thyrotrophin (TSH) level and TSH receptor antibody activity have become normal. This was a prospective randomized study of patients with Graves' disease followed for a mean of 28 months after the withdrawal of antithyroid drug treatment. A total of 174 patients with hyperthyroid Graves' disease were entered consecutively into this study, 11 of whom were subsequently excluded. One hundred and sixty-three patients were divided randomly into two groups having different criteria for the discontinuation of the antithyroid drug. Group 1 consisted of 79 patients whose antithyroid drug treatment was discontinued if both their serum TSH level and thyrotrophin binding inhibitor immunoglobulin (TBII) activity normalized. Group 2 consisted of 84 patients who were treated for 24 months irrespective of their serum TSH level and TBII activity. All patients were treated initially with 400 mg/day of propylthiouracil followed by decreasing doses that maintained a euthyroid state. Serum basal TSH levels and TBII activities were measured using a radioimmunometric assay and a radioreceptor assay, respectively, every 2 months during the antithyroid drug treatment. The remission rate of Group 1 (51.9%) was not significantly different from that (63.1%) of Group 2. The mean duration of treatment in Group 1 was 10.2 +/- 4.5 months (range 5-24, median 10). Median duration of treatment in Group 1 was 14 months shorter than in Group 2. In Group 1, the relapse rate was independent of the duration of the treatment. Further, the mean duration of treatment in the relapsed patients was not significantly different from that for patients who went into remission (9.9 +/- 3.8 vs 10.7 +/- 5.7 months; P greater than 0.1). In Group 2, the relapse rate was dependent on the time when both the serum TSH level and TBII activity were normalized (chi 2 = 27.0, d.f. = 4; P less than 0.001). When both the serum TSH level and TBII activity were normalized, the relapse rate of Group 2 was not significantly different from that of Group I for treatment periods of 6-12 months (25 vs 24.2%, P greater than 0.1), 12-18 months (33.3 vs 40%, P greater than 0.1), and 18-24 months (46.2 vs 50%, P greater than 0.1). However, the relapse rate of Group 2 was significantly lower than that of Group 1 when there was only a 6-month treatment period (5.6 vs 42.9%, P less than 0.05). Of the clinical and laboratory parameters measured, male sex, change of goitre size during treatment, and TSH levels and TBII values at the end of treatment, were of prognostic significance in predicting a relapse. The present study suggests that antithyroid drugs treatment can be terminated more appropriately when both the serum TSH level and TBII activity are made normal, thus avoiding prolonged and unnecessary drug treatment.


The pulsatile GH secretion in acromegaly: hypothalamic or pituitary origin?

We studied the effects of different modes of octreotide therapy on the pulsatile pattern of GH release in an attempt to define better its regulation by growth hormone-releasing hormone (GHRH) and somatostatin and its effects on IGF-I plasma levels in acromegaly. In six acromegalic patients not cured by previous treatment we compared the 24-hour GH secretion profiles under basal conditions with subcutaneous (s.c.) bolus injections of 100 micrograms octreotide every 8 hours and with continuous s.c. infusions of the same daily dose. Blood samples were taken every 10 minutes over 24 hours followed by a GHRH test (100 micrograms GHRH i.v.) with blood sampling every 15 minutes for another 2 hours. After a 4-week interval all patients were treated either by the bolus or continuous mode of octreotide application in a randomized cross-over design. On day 4 of treatment blood sampling and GHRH test were repeated. Octreotide treatment was withdrawn for another 4 weeks; all patients then received the alternate application mode and were measured under similar conditions. Serum GH and plasma IGF-I concentrations were analysed by serial array averaging. IGF-I levels were measured in two different assays with and without previous protein extraction. For GH pulse detection three different algorithms (Cluster, Pulsar, Desade) were applied. With both treatments, the initially elevated basal 24-hour mean serum GH concentrations (58.0 +/- 9.7 mU/l mean +/- SEM) decreased significantly (bolus: 11.5 +/- 4.9 mU/l, P < 0.001 vs basal; continuous infusion: 7.6 +/- 1.9 mU/l, P < 0.001 vs basal) after 4 days. GH suppression was significantly more pronounced following continuous infusion than bolus (P < 0.05). IGF-I plasma concentrations were lowered significantly (P < 0.05) with both forms of treatment which did not differ between themselves. Bolus and continuous infusion treatment significantly inhibited (P < 0.05) the amplitudes of pulsatile GH release, but did not change the pulse frequency. In two of the patients, GHRH stimulation did not increase GH serum levels suggesting a constitutive activation of adenylyl cyclase. Continuous subcutaneous octreotide treatment in acromegaly suppresses mean GH levels better than bolus injection. The number of GH pulses remains unaffected by both modes of treatment providing evidence against a somatostatinergic mechanism of pulsatile GH secretion in these patients. The unchanged frequency of pulsatile GH release in the patients unresponsive to exogenous GHRH indicates that this pattern might be independent of hypothalamic GHRH and somatostatin and suggests a pituitary-derived mechanism for GH pulse generation in acromegaly.


Patterns of renal function in hypertension due to unilateral renal artery occlusion.

We performed renal function studies in dogs with chronic renovascular hypertension produced by complete occlusion of a renal artery. In addition, we evaluated in anesthetized dogs the acute effects of a novel angiotensin converting enzyme inhibitor, CGS 16,617, on renal function and plasma neurohormones (epinephrine, norepinephrine and vasopressin) 4 weeks after initiation of 2 kidney, 1 clip hypertension. CGS 16,617 effectively decreased blood pressure in renal hypertensive animals. This response was associated with suppression of angiotensin II indicating effective converting enzyme inhibition. In the non-clipped kidney, acute administration of CGS 16,617 increased effective renal plasma flow but not glomerular filtration rate and urinary sodium excretion. In the clipped kidney, CGS 16,617 caused no change in any parameter of renal function. Plasma norepinephrine, epinephrine and vasopressin were unaffected by administration of CGS 16,617. These studies showed that chronic occlusion of a renal artery does not result in renal infarction because of a compensatory increase in the amount of blood provided through capsular collateral vessels. The collateral circulation which has developed in the clipped kidney explains the lack of a converting enzyme inhibitor effect.


X-linked lymphoproliferative disease: prenatal detection of an unaffected histocompatible male.

Chorionic Villous Biopsy (CVS) for diagnosis of XLP was undertaken at 10 weeks gestation in an obligate carrier. The fetus was found to be male by cytogenetic analysis. XLP (Xq25-q26) is closely linked to the RFLP markers DXS10, DXS37 and DXS42, but only DXS10 (distal to XLP) was informative for prenatal diagnosis in this family. RFLP analysis using this marker gave a 7% risk that the fetus was affected, based on the known recombination frequency between DXS10 and XLP. Further investigation was then undertaken to obtain a rapid and more accurate diagnosis using the three highly polymorphic PCR based markers. These were the AC repeat markers DXS424 (XL5A) and DXS425 (XL90A3) and the tetramer repeat marker within HPRT. DX425 is approximately 10 cM proximal to DXS10 and HPRT but is not known with certainty to map proximal or distal to XLP. DXS424 is proximal to DXS10 and HPRT and was inferred to be proximal to XLP on the basis of map distance from HPRT estimated by linkage analysis of data from CEPH pedigrees. This was confirmed by a recombinant in the XLP family between DXS424 and DXS425, placing DXS424 proximal to XLP. Diagnosis by linkage using DXS424 and DXS425, at least one of which is proximal to XLP, and distal markers DXS10 and HPRT, increased the accuracy of diagnosis using flanking marker analysis to greater than 99% that the fetus was unaffected. HLA DR typing of the CVS showed that the fetus was DR identical to a male sibling with XLP. HLA compatibility was confirmed at delivery by full HLA typing and MLC.(ABSTRACT TRUNCATED AT 250 WORDS)


DNA polymorphisms of the apolipoprotein B gene in Chinese coronary artery disease patients.

Five restriction fragment length polymorphisms (RFLP) of the apo B gene and their association with serum lipids and apolipoprotein levels have been studied in 139 Chinese patients with angiographically confirmed CAD (mean age 56.2 +/- 0.8 years) and 154 healthy Chinese subjects (mean 44.0 +/- 1.0 years) of both sexes. The patient group had significantly higher levels of serum total and LDL cholesterol; and apo B (P < 0.001) and lower HDL cholesterol and apo A-I (P < 0.001 and < 0.01, respectively). The frequencies of the rarer alleles of the ins/del, XbaI and EcoRI (but not the PvuII and MspI) polymorphisms were significantly lower in the Chinese compared to those reported in Caucasians. There was no significant difference in allelic frequencies of the signal peptide region (Ins/Del), XbaI, MspI and EcoRI sites of the apo B gene between the patient and control groups. The frequency of the rarer allele of the PvuII RFLP was significantly lower in the CAD patients (P < 0.05) compared to that in the control group (0.05 vs 0.10). None of the DNA polymorphisms was associated with a significant influence on serum lipid and apolipoprotein levels in the patients with coronary artery disease.


Clinical variability of cardio-facio-cutaneous syndrome: report of two additional cases.

We describe two new cases of cardio-facio-cutaneous (CFC) syndrome, and underline the clinical variability of the CFC phenotype in our two patients presenting with border-line psychomotor development. The first patient showed some additional clinical manifestations, such as cryptorchidism and scoliosis, and the second one had atypical skin lesions.


Nucleotide sequence and PCR-amplification of a polymorphic MboI site in human DNA marker D4S95 linked to the Huntington disease locus.

The nucleotide sequence of the probe BS674E-D defining the genetic locus D4S95 was assessed, and used for designing PCR-primers amplifying a 175 bp fragment containing a polymorphic MboI site. With the described conditions it is now possible to use PCR followed by digestion for rapid determination of genotype in this marker system.


Human polymorphonuclear leucocytes stimulated by tumour necrosis factor-alpha show increased adherence to extracellular matrix proteins which is mediated via the CD11b/18 complex.

The present study demonstrates that tumour necrosis factor (TNF) and FMLP, but not IL-1 or IL-8, enhanced the adherence of polymorphonuclear neutrophil (PMN) to fibronectin, an extracellular matrix protein. The adherence induced by FMLP was very rapid, within 5 min while the induction of adherence by TNF was much slower, reaching maximum at 60 min. TNF also enhanced an adhesion of PMN to other extracellular matrix proteins, such as laminin, collagen IV and gelatin II, but not to human serum albumin. Anti-CD18 MoAb completely inhibited the binding of TNF-stimulated PMN to fibronectin and partially inhibited the binding to laminin. Further investigation showed that adhesion of TNF-stimulated PMN to fibronectin and laminin was inhibited by anti-CD11b MoAb and to a lesser extent by CD11a MoAb. In contrast to TNF-stimulated PMN the binding of unstimulated PMN to fibronectin and laminin was only inhibited by anti-CD11a MoAb. Anti-CD11c had no effect on PMN adherence. These results suggest that unstimulated PMN adhere to extracellular proteins through the CD11a/18, while TNF-stimulated PMN adhere through the CD11b/18. These results suggest that TNF secreted at the site of inflammation may enhance the interaction of PMN with the extravascular environment through the CD11b/18 complex.


Leukotriene B4-induced hyperadhesiveness of endothelial cells for neutrophils: relation to CD54.

Leukotriene B4 (LTB4) induced an in vitro transient state of hyperadhesiveness in cultured human umbilical vein endothelial cells (HUVEC), leading to a 2.2-fold increase in the binding of neutrophil granulocytes (PMN), which was less than that conferred by platelet activating factor (PAF) though more than thrombin did (3.4- or 2.0-fold increases, respectively). This study concerns the role of the adhesive molecules CD18 and CD54 for the LTB4- (as well as thrombin- and PAF-) induced endothelial hyperadhesiveness. The MoAbs 60.3 (to the CD18 molecule on PMN) and 84H10 (to one epitope of CD54 on the HUVEC) blocked the adherence of PMN to LTB4-treated HUVEC, whereas MoAb LB-2 (directed at another CD54 epitope) failed to do so. MoAb 84H10 blocked 43% of the thrombin-induced hyperadhesiveness, whereas the PAF response was unaffected. Thus, LTB4-induced HUVEC hyperadhesiveness may therefore be related to a specific domain on the CD54 (or on an antigenically related molecule) as well as being dependent on CD18, whereas the involvement of CD54 was much less or non-existent for the thrombin and PAF responses, respectively.


Severe hemorrhagic complications from infection with nephropathia epidemica strain of Hantavirus.

In the following we describe a case of severe hemorrhagic fever with renal syndrome (HFRS) caused by Puumala infection. The diagnosis was made by immunofluorescence technique and by solid phase enzyme immunoassay using recombinant nucleocapsid antigen of a Puumala serotype strain. Such a clinical course with severe bleeding complications is considered untypical for Puumala induced HFRS.


Cellular responses to human chondrocytes: absence of allogeneic responses in the presence of HLA-DR and ICAM-1.

To assess the accessory cell function of human articular chondrocytes, we assessed the ability of human chondrocytes to stimulate allogeneic peripheral blood mononuclear cells (PBMC) and to support phytohaemagglutinin (PHA)-induced proliferation of highly purified T cells. We also examined the surface expression of HLA-DR and ICAM-1 on the chondrocytes both unstimulated and stimulated with cytokines in vitro. Chondrocytes failed to stimulate allogeneic PBMC despite the constitutive expression of MHC class I molecules and the cytokine-induced expression of class II molecules but were able to support T cell proliferation to PHA, IFN-gamma and to a limited extent, IL-1 beta, induced class II expression on chondrocytes. ICAM-1 was present on 94-99% of freshly isolated cells; this declined with culture (17-59%; P < 0.005) but was readily induced by IFN-gamma, IL-1 beta, and tumour necrosis factor-alpha. Alloreactivity and, presumably, autoreactivity to chondrocytes requires factors in addition to the surface expression of DR and ICAM-1. However the presence of these molecules suggests a capacity for cell-cell interactions in inflammatory sites such as the cartilage pannus junction.


The effects of rilmenidine on tests of autonomic function in humans.

The effects of rilmenidine, a new centrally acting antihypertensive agent, on a number of tests of autonomic function were investigated in six healthy male volunteers. Baroreflex function (delta RR interval [in milliseconds] with each millimeter of mercury change in systolic blood pressure) was determined in response to changes in pressure after injections of phenylephrine and nitroglycerin. Reflex cardiovascular responses to handgrip and standing, as well as during deep breathing and the Valsalva maneuver, were also investigated. Rilmenidine produced a dose-dependent decrease in blood pressure that was not accompanied by an increase in heart rate. Under conditions of low basal sympathetic activity, rilmenidine enhanced parasympathetic tone during the early reflex heart rate changes that occur immediately after standing and during deep breathing, as well as baroreflex heart rate responses to phenylephrine. During a test of sympathetic function, standing blood pressure, and heart rate after 3 minutes, rilmenidine reduced sympathetic tone.


Pharmacodynamics and pharmacokinetics of intramuscular dexmedetomidine.

The pharmacodynamics and pharmacokinetics of intramuscular dexmedetomidine--a novel alpha 2-adrenergic receptor agonist under development for preanesthetic use--were studied in healthy male volunteers. Single intramuscular doses of dexmedetomidine (0.5, 1.0, and 1.5 microgram/kg) and placebo were administered to six subjects in a single-blind, multiple crossover study. Dexmedetomidine induced dose-related impairment of vigilance assessed both objectively and subjectively. The drug also caused moderate decreases in blood pressure and heart rate. Plasma norepinephrine was dose-dependently (maximum 89%) decreased. The intramuscular doses resulted in linearly dose-related plasma concentrations of dexmedetomidine. Pharmacokinetic calculations revealed a time to maximum concentration from 1.6 to 1.7 hours, an elimination half-life of 1.6 to 2.4 hours, an apparent total plasma clearance of 0.7 to 0.9 L/hr/kg, and apparent volume of distribution of 2.1 to 2.6 L/kg. The sedative effect of dexmedetomidine dissipated during the 6-hour observation time, but all other effects were still evident 6 hours after administration of the higher doses, paralleling the plasma concentration curves. The relationship of plasma concentrations of dexmedetomidine to pharmacodynamic variables was consistent with a linear pharmacodynamic model. The pharmacodynamic-pharmacokinetic profile of intramuscular dexmedetomidine may be suited to the proposed preanesthetic clinical use of this alpha 2-agonist.


[Contribution of glutathione to detoxification in alcoholism. Biochemical-clinical studies].

The effects of reduced glutathione (GSH) administration (1.2 g/day and 2.4 g/day intravenously) on erythrocyte glutathione levels, serum gamma-glutamyl transpeptidase activity (GGTP) and urinary glucaric acid elimination were studied in a population of 24 chronic alcoholics voluntarily admitted to a 30 day detoxification protocol in comparison to a 12 patient control group treated only with chlordiazepoxide (initial dose 75-100 mg/day). Glutathione treatment increases dose-dependently and in a significant way erythrocyte glutathione levels and hastens the recovery of serum GGTP and urinary glucaric acid elimination. The relationship between glutathione, GGTP and glucaric acid is discussed, suggesting the possible role of GSH against the oxidative damage of alcohol.


Recent progress in the treatment of Parkinson's disease.

Because of the number of different types of anti-Parkinsonian medications, a number of options in the treatment of PD are now available. Each patient's medication regimen should be individualized. Many of the medication choices are made based on the stage of the disease. For patients who have newly diagnosed PD, and who are on no medications, treatment with deprenyl should be strongly considered. While some controversy remains concerning its possible slowing of the rate of disease progression, there is no evidence to suggest that its use is detrimental. It is generally well tolerated in patients with early disease. These factors must be weighed against the cost of the medication, and the fact that little if any therapeutic effect is seen in most patients who are not being treated with LD. A useful analogy when considering this issue is the prophylactic use of aspirin for cerebrovascular or cardiovascular disease. Newly diagnosed patients requiring treatment, who have tremor as their only symptom or their most prominent symptom, may be given an anticholinergic medication. Patients who have significant bradykinesia, rigidity or gait disturbance can be given amantadine. A combination of these two medications may be useful, and a combination of deprenyl with an anticholinergic drug or amantadine may provide excellent relief of early symptoms. At some point, most patients' symptoms progress such that treatment with LD is considered. Given its possible, but unproven, acceleration of the rate of disease progression, this decision should be weighed carefully. In a relatively young patient who may be treated for many years, a dopamine receptor agonist can be initiated without LD therapy.(ABSTRACT TRUNCATED AT 250 WORDS)