Electrophysiological studies of anion secretion in cultured human epididymal cells.

1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and previous supports. The epithelia so formed were used for whole-cell patch clamp recording and short-circuit current (ISC) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.5 +/- 1.3 mV (mean +/- S.E.M.., n = 16), apical side negative, and a basal ISC of 6.9 +/- 0.9 microA cm-2 (mean +/- S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 mumol l-1 increased the ISC by 3.0 +/- 1.2 microA cm-2 (mean +/- S.E.M., n = 4). This increase was blockable by diphenylamine-2-carboxylate (DPC, 1 mmol l-1). Forskolin (10 mumol l-1) also evoked a similar response to adrenaline. 4. In whole-cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l-1 KCl was found to be -30 +/- 14 mV (mean +/- S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 mumol l-1 adrenaline by an increase in inward current at -70 mV holding potential (delta I = -1600 +/- 900 pA, mean +/- S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to -2 +/- 1 mV (mean +/- S.E.M., n = 15). 6. The adrenaline-induced inward current was found to be blockable by the Cl- channel blocker, DPC (0.25 mmol l-1). Ion substitution experiments showed that the adrenaline-evoked current was carried mainly by Cl-. 7. The effect of adrenaline on the whole-cell current was found to be mimicked by forskolin and could be abolished by including GDP beta S or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l-1 EGTA or 2 mmol l-1 BAPTA + 100 mumol l-1 TMB-8 (to inhibit intracellular Ca2+ release) in the pipette did not seem to have any marked effect on adrenaline-evoked whole-cell current. Lowering the pipette Ca2+ concentration to 1 nmol l-1 or raising it to 10 mumol l-1 had no effect on the whole-cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl- secretion in cultured human epididymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)


A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence.

Enteropathogenic Escherichia coli (EPEC) form adherent microcolonies on the surface of tissue culture cells in a pattern termed localized adherence. Localized adherence requires the presence of a large EPEC adherence factor (EAF) plasmid. Recently a bundle-forming pilus has been described in EPEC possessing the EAF plasmid. An analysis of 22 non-invasive EPEC TnphoA mutants revealed that seven have insertions in the EAF plasmid and are incapable of localized adherence. We report here the mapping of the TnphoA insertions in these mutants. The nucleotide sequence of the gene interrupted in these TnphoA mutants (bfpA) was determined and found to correspond to the N-terminal amino acid sequence of the major structural protein of the bundle-forming pilus. The bfpA gene bears sequence similarities to members of the type IV fimbrial gene family and encodes a potential site for processing by a prepilin peptidase. A plasmid containing bfpA as the only open reading frame directs the synthesis of a protein recognized by antiserum raised against the bundle-forming pilus. TnphoA mutants at this locus are unable to synthesize BfpA, but synthesis is restored by introduction of a plasmid containing the cloned gene. The minimum fragment of DNA required to restore localized adherence is considerably greater than that required to restore BfpA synthesis. BfpA expression, as assessed by alkaline phosphatase activity in bfpA::TnphoA mutants, is affected by temperature and growth medium. These studies describe an EPEC plasmid-encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence.


Interaction of two variable proteins (PilE and PilC) required for pilus-mediated adherence of Neisseria gonorrhoeae to human epithelial cells.

Pili confer the initial ability of Neisseria gonorrhoeae to bind to epithelial cells. Pilin (PilE), the major pilus subunit, and a minor protein termed PilC, reportedly essential for pilus biogenesis, undergo intra-strain phase and structural variation. We demonstrate here that at least two different adherence properties are associated with the gonococcal pili: one is specific for erythrocytes, which is virtually unaffected by PilE variation, and another is specific for epithelial cells, and is modulated in response to the variation of PilE. Based on this finding, mutants of a recA- strain were selected that had lost the ability to bind to human cornea epithelial cells (A-) but retained the ability to form pili (P+) and to agglutinate human erythrocytes (H+). The adherence-negative mutants failed to produce detectable levels of PilC1 or PilC2 proteins, representing piIC phase variants generated in the absence of RecA. The A- pilC phase variants were indistinguishable from their A+ parents and spontaneous A+ revertants with regard to the amount of PilE produced and its electrophoretic mobility, the degrees of piliation and haemagglutination, and the pilE nucleotide sequence. These data demonstrate a central role for PilC in pilus-mediated adherence of N. gonorrhoeae to human epithelial cells and further indicate that neither PilC1 nor PilC2 is obligatory for the assembly of gonococcal pili.


Hepatic encephalopathy. Current concepts of the pathogenesis.

Hepatic encephalopathy occurs in a number of different species as a result of either congenital portacaval shunts or acquired liver disease. Despite intensive research, the neurochemical basis of the disorder has not been defined. Theories to explain the cerebral dysfunction that accompanies acute or chronic hepatic failure include 1) ammonia acting as the putative neurotoxin, 2) perturbed monoamine neurotransmission as a result of altered plasma amino acid metabolism, 3) an imbalance between excitatory amino acid neurotransmission, mediated by glutamate, and inhibitory amino acid neurotransmission, mediated by gamma-aminobutyric acid, and 4) increased cerebral concentrations of an endogenous benzodiazepine-like substance.


Cellular interactions in the striatum involving neuronal systems using "classical" neurotransmitters: possible functional implications.

The neostriatum contains a wide variety of neuroactive substances associated with several well-defined functional neuronal systems. This structure, which is the seat of numerous neurological pathological disorders, is commonly used as a model for studying the basic mechanisms of neurotransmitter interactions in the brain and their putative involvement in striatal functions. Increasing interest has been focusing lately on the cellular interactions that may occur between the corticostriatal putatively glutamatergic system and the nigrostriatal dopaminergic input. Current evidence suggests that the activatory corticostriatal glutamatergic input may play a more crucial role in regulating striatal functions than was formerly assumed in comparison with the dopaminergic input. The key role of cholinergic interneurons in the striatum may therefore be attributable to the fact that they modulate the glutamatergic transmission to GABA striatal efferent neurons. Likewise, dopamine may actually act indirectly in the striatum by "tuning down" the cortical excitation of striatal neurons. Consequently, an impairment of the dopaminergic transmission such as that occurring in Parkinsonism may lead to an increase in the corticostriatal glutamatergic transmission, which may further contribute towards reinforcing the "imbalance" between subsets of striatal neuronal systems controlling the output of the basal ganglia.


Molecular cloning of GRA4, a Toxoplasma gondii dense granule protein, recognized by mucosal IgA antibodies.

Clones which were selected from a Toxoplasma gondii expression library with the immune serum from a T. gondii-infected rabbit, were further screened using milk and intestinal secretions from mice which had been orally infected with T. gondii cysts. The gene products of several clones reacted strongly with milk IgA and weakly with intestinal IgA. Three of these clones (5.1, 36.1, 37.4) were shown to encode a dense granule protein of 40 kDa (GRA4). The GRA4 protein co-migrates with one of the T. gondii antigens recognized by mucosal IgA. The complete nucleotide sequence of GRA4 has been obtained by cloning genomic T. gondii BamHI fragments containing the 37.4 DNA insert. The coding sequence contains no intron. The deduced amino acid sequence indicates a proline rich (12%) product with an internal hydrophobic region of 19 amino acids and a potential site of N-glycosylation. The primary translation product with a theoretical size of 36,260 Da contains a putative N-terminal signal sequence of 20 amino acids but no apparent glycolipid anchor sequence. Quantitation of the GRA4 gene and Southern blot analysis suggested that the GRA4 gene is single copy. GRA4 gene is translated in tachyzoites to yield a single mRNA species of about 1900 bases.


Glycosyl phosphatidylinositol-specific phospholipase C of Trypanosoma brucei: expression in Escherichia coli.

Glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei cleaves the glycosyl phosphatidylinositol (GPI) anchor of the trypanosome variant surface glycoprotein (VSG) and other GPI structures. We have expressed this enzyme in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. We have purified large amounts of GPI-PLC from E. coli membranes, using a single step immunoaffinity technique. The expressed enzyme is identical to its trypanosome counterpart in enzymatic specificity, mobility on SDS-PAGE, and isoelectric point. Recombinant GPI-PLC is a membrane enzyme; it associates with E. coli membranes and, like the T. brucei GPI-PLC, partitions into the detergent phase in Triton X-114 phase separation experiments. The Michaelis constants for the two enzymes are similar (400 nM, with VSG as substrate). The turnover number (kcat, 72 min-1) of the recombinant enzyme (expressed from a. T. brucei rhodesiense WRATat 1.1 cDNA) is about one-tenth that of GPI-PLC from T. brucei brucei (ILTat 1.3).


In vivo evidence for a concordant response of terminal and dendritic dopamine release during intranigral infusion of drugs.

In the present study we have administered prototypical drugs of 5 different receptors (D-2, GABA-A, GABA-B, NMDA, kainate) to the substantia nigra by infusion via a microdialysis probe, whereas the release of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were recorded both in the substantia nigra and (by a second microdialysis probe) in the ipsilateral striatum. Infusion of the specific D-2 receptor agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin ((--)-N0437) into the nigra induced a decrease in the release of dopamine in the nigra (after 1 mumol/l) as well as in the ipsilateral striatum (after 10 mumol/l). During these infusions extracellular DOPAC decreased in the nigra and increased in the striatum. Infusion of the D-2 specific receptor antagonist (--)-sulpiride into the nigra induced an increase in the release of dopamine in the nigra (after 1 mumol/l) as well as in the ipsilateral striatum (10 mumol/l). During these infusions a slight increase of extracellular DOPAC was noticed in the nigra. Infusion of the GABA-A receptor antagonist bicuculline into the nigra (50 mumol/l) induced an increase in the release of dopamine and DOPAC both in the nigra and ipsilateral striatum. Infusion of the GABA-B receptor agonist d,I-baclofen into the nigra (10 mumol/l) induced a decrease of in the release of dopamine in the nigra as well as in the ipsilateral striatum, whereas extracellular DOPAC decreased in the nigra and increased in the striatum.(ABSTRACT TRUNCATED AT 250 WORDS)


Direct demonstration of dopamine D1-like receptor sites in the ciliary body of the rabbit eye by light microscope autoradiography.

The pharmacological characteristics and the anatomical localization of [3H]-SCH 23390 in sections of the ciliary body of the rabbit eye were analyzed using a radioreceptor assay and autoradiographic techniques. [3H]-SCH 23390 was bound to sections of rabbit ciliary body in a manner consistent with the labelling of D1-like receptor sites. The dissociation constant (Kd) was 0.62 nmol/l, while the maximum binding capacity (Bmax) was 117 +/- 9 fmol/mg tissue. Light microscope autoradiography revealed [3H]-SCH 23390 binding sites within the epithelium of the ciliary processes, which is the ocular structure involved in the secretion of aqueous humor. No specific accumulation of silver grains was noticeable within the iridocorneal angle, which is the structure involved in the outflow of aqueous humor. These findings suggest that the rise in intraocular pressure caused by D1 receptor agonists is probably mediated by an increase of aqueous humor formation rather than by an inhibition of the outflow of aqueous humor.


Characterization of the antagonism with metoprolol, ICI 147,798, pindolol, mepindolol and bopindolol on the responses of the rat left atria to isoprenaline.

The aim of the study was to determine whether the antagonism with pindolol, mepindolol and bopindolol at the beta 1-adrenoceptor of the rat left atria, a tissue with plenty of spare beta 1-adrenoceptors for isoprenaline maximum responses, was readily reversible or not. The effects of these drugs were compared to those of metoprolol, a readily reversible, and of ICI 147,798, an irreversible beta-adrenoceptor antagonist. Metoprolol at 10(-7) and 10(-6) M, ICI 147,798, pindolol, bopindolol (all at 10(-8) and 10(-7) M) and mepindolol at 10(-9) and 10(-8) M inhibited the cardiac stimulation responses to a small extent, which is indicative of membrane stabilizing activity, and also caused surmountable antagonism of isoprenaline responses. The inhibitory effects on the isoprenaline responses of metoprolol and pindolol were readily reversible, that of mepindolol was slowly reversible and those of ICI 147,798 and bopindolol were not reversed in 3 h. The inhibitory effects on isoprenaline responses of metoprolol at 10(-6) M, pindolol and bopindolol at 10(-7) M and mepindolol at 10(-8) M were at equilibrium, which is indicative of reversible, whereas the inhibitory effects of ICI 147,798 were increased, which is indicative of irreversible antagonism, when the beta-blocker treatment time was increased from 1 to 2 h. We conclude that the antagonism with pindolol at the beta 1-adrenoceptors of the rat left atria is readily reversible, that of mepindolol is slowly reversible and that of bopindolol is very slowly reversible.


Adenosine but not an adenine nucleotide mediates tonic purinergic inhibition, as well as inhibition by glutamate, of noradrenaline release in rabbit brain cortex slices.

A possible contribution of adenine nucleotides to the endogenous purinergic, A1-receptor-mediated inhibition of noradrenaline release was studied in rabbit occipito-parietal cortex slices. The slices were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 6 pulses/100 Hz. A few experiments were carried out in rat occipito-parietal cortex slices. The A1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1-100 nmol/l) as well as the enzyme adenosine deaminase (0.1-10 U/ml) increased the electrically evoked overflow of tritiated compounds. The maximal increase was by about 85% for both DPCPX and adenosine deaminase. The increases obtained with maximally effective concentrations of DPCPX and adenosine deaminase were not additive. The alpha 1-adrenoceptor-selective agonist methoxamine (10 but not 1 mumol/l) reduced the evoked overflow. Its effect was antagonized by yohimbine 1 mumol/l but then not attenuated further by DPCPX 100 nmol/l. L-Glutamate (300 mumol/l-2.3 mmol/l) also reduced the evoked overflow of tritium. Its effect was not changed by yohimbine 1 mumol/l but greatly, and to the same extent, attenuated by DPCPX 100 nmol/l and adenosine deaminase 3 U/ml. Neither the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine nor omission of Mg++ changed the inhibition by glutamate. Glutamate did not alter the basal efflux of tritium from rabbit cortex slices under any experimental condition. In contrast, glutamate (100 mumol/l and 1 mmol/l) caused an immediate, marked and transient acceleration of tritium outflow from rat occipitoparietal cortex slices (medium without Mg++).(ABSTRACT TRUNCATED AT 250 WORDS)


[Measurement of PCNA labeling index in astrocytic tumors].

PCNA (proliferating cell nuclear antigen) is said to be present specifically in the nucleus of proliferating cells. The PCNA labeling index (PCNA LI) of astrocytic tumors was measured and compared with histological types or prognosis. The specimens from 44 patients were fixed in a 10% formalin solution, and embedded in paraffin. The 3 microns-sections were stained immunohistochemically with anti-PCNA monoclonal antibody (PCIO, Novocastra) using an ABC method. The percentage of PCNA-positive-cells was determined by counting 2000 cells, and identified as PCNA LI. All of the PCNA-positive-cells showed diffuse nucleoplasmic staining. The averages of PCNA LIs in each pathological type were calculated and evaluated statistically. Although differences in averages of PCNA LIs among pilocytic, gemistocytic, fibrillary astrocytoma were not significant, there was a significant difference between anaplastic astrocytoma and glioblastoma. The relationship between PCNA LIs and the prognoses for 43 patients was studied. Forty-three patients were classified into 3 groups (over 22%, 7 to less than 22%, and less than 7%) according to PCNA LIs. The survival data in the 3 groups were analyzed, and differed significantly in the survival rates. Furthermore, twenty-three patients of anaplastic astrocytoma and glioblastoma were classified into two groups (over 22% and less than 22%). Likewise, the two groups differed significantly. In summary, pathological type and prognosis were closely related to PCNA LI in astrocytic tumors. Therefore, we thought measurement of PCNA LI would make it more possible to analyze clinically the proliferating activity of astrocytic tumors, and to care for patients more effectively.


Demonstration of dynorphin A 1-8 immunoreactive axons contacting spinal cord projection neurons in a rat model of peripheral inflammation and hyperalgesia.

Using a double-labeling technique, we evaluated the input of afferents immunoreactive for dynorphin peptide onto a population of lumbar spinal neurons contributing to the spinoparabrachial tract in rats with 1 inflamed hind paw. We found that the frequency and distribution with which dynorphin immunoreactive varicosities were in apposition to projection neurons varied according to neuron location. In particular, neurons in the superficial dorsal horn and neck of the dorsal horn receive a high degree of dynorphin input. We also determine that unilateral peripheral inflammation is associated with both an increase in the number of projection neurons receiving detectable DYN input and in the frequency of this input onto a given neuron, with the largest increase seen in the superficial dorsal horn. Since almost all superficial dorsal horn neurons contributing to the spinoparabrachial tract respond either exclusively or maximally to noxious stimulation, our data supports dynorphin's involvement in nociception.


The depressive effect of intrathecal clonidine on the spinal flexor reflex is enhanced after sciatic nerve section in rats.

The effect of intrathecal (i.t.) alpha 2-adrenoceptor agonist, clonidine, on the spinal nociceptive flexor reflex was studied in decerebrate, spinalized, unanesthetized rats with intact sciatic nerves or in rats in which the sciatic nerve had been ipsilaterally sectioned. In rats with intact nerves i.t. clonidine caused a dose-dependent biphasic effect on flexor reflex excitability. At low dose (10 ng) the effect of clonidine was purely facilitatory, whereas with 50-100 ng clonidine the initial facilitation was often followed by reflex depression. Long-lasting, strong reflex depression was observed after i.t. injection of high doses of clonidine (1 and 10 micrograms). Four to 18 days after sciatic nerve section, the depressive effect of clonidine on the flexor reflex was dramatically enhanced. Depression was frequently observed already with doses of 5 and 10 ng, and maximal depression was reached at 100 ng and 1 micrograms in axotomized rats. The facilitatory effect of low doses of clonidine on the reflex was also observed, although somewhat less frequently than in normals. The depressive effect of clonidine on the flexor reflex was reversed by the selective alpha 2-receptor antagonist, atipamezole (20 micrograms, i.t.), in rats with both intact and sectioned sciatic nerves. The present results revealed an increased sensitivity and effectiveness of the depression of spinal reflex mechanisms by i.t. clonidine after sciatic nerve section, which is opposite to the decreased sensitivity to i.t. morphine after axotomy that we observed previously.(ABSTRACT TRUNCATED AT 250 WORDS)


Leukocyte integrin activation.

Certain stages of the immune response require interaction of leukocytes with each other and with non-hematopoietic cells. One of the systems implicated in these interactions involves an integrin, LFA-1 (Lymphocyte Function Antigen-1), expressed by all leukocytes at their cell surface, and a molecule belonging to the immunoglobulin superfamily, ICAM-1 (Intercellular Adhesion Molecule-1). The avidity of LFA-1 for ICAM-1 is transient. It is modulated both by regulation of ICAM-1 expression and by activation of LFA-1 molecules constitutively expressed on leukocyte membranes. This activation, which induces a conformational change in the molecule, depends on the presence of divalent cations, notably Mg++. This has been demonstrated by using a specific monoclonal antibody, MAb 24. In addition to being a ligand for LFA-1, ICAM-1 is sometimes used as a cell receptor by pathogens such as Plasmodium falciparum, the causative organism of malaria. Very careful study of the binding site of this pathogen using specific antibodies, mutagenesis studies and the construction of a three-dimensional model of the molecule suggests some interesting therapeutic possibilities for the treatment of malaria.


Benextramine, a putative neuropeptide Y receptor antagonist, attenuates the termination of receptivity.

Sexual behavior in female rats is dependent on gonadal steroids. In ovariectomized rats, progesterone treatment typically exerts a biphasic effect on copulatory behavior induced by prior treatment with estradiol. Thus, there is an initial augmentation of the facilitative effect of estradiol occurring 4-10 h after progesterone. This is followed by a profound inhibitory effect, essentially terminating receptivity. We hypothesized that the receptivity terminating effect of progesterone could be due to increased synthesis and release of neuropeptide Y in relevant brain regions. Rats were tested for female sexual behavior 4 h after progesterone (52 h postestradiol). Immediately following this test, benextramine was administered (0, 3, or 15 mg/kg, IP). Subsequently, behavioral tests were administered 24, 48, 72, and 96 h postbenextramine. Benextramine treatment attenuated the inhibitory effects of progesterone on receptivity (lordosis quotients and percent of responding animals) without affecting either proceptive or rejection behaviors. These data suggest that blockade of NPY (and alpha-adrenergic) receptors is associated with selective enhancements of specific components of sexual behavior in female rats. Specifically, blockade of NPY receptors by benextramine is associated with continued receptivity.


Cryptosporidiosis in HIV-seropositive patients.

The incidence of cryptosporidiosis in our unit has increased over the last 6 years, being diagnosed in approximately 5 per cent of all patients with HIV infection and in 21 per cent of those with AIDS, but a marked seasonal variation occurs. We have studied the course of the infection in 128 patients and identified four clinical patterns of disease: transient (28.7 per cent), chronic (59.7 per cent), fulminant (7.8 per cent) and asymptomatic (3.9 per cent). Transient disease occurred in patients with a wide range of CD4 lymphocyte counts, but was more common in less immunosuppressed patients. Fulminant disease, defined by the passage of more than 2 l of stool/day from the time of presentation, only occurred in patients with a CD4 count less than 50/mm3. This group had lost more than 7 kg in weight at presentation and more commonly had other intercurrent gastrointestinal infections. They survived for a median of only 5 weeks, compared with 20 weeks for those with chronic diarrhoea and 36 weeks for those with transient infection. The survival was unaffected by any treatment other than zidovudine. Cryptosporidiosis in HIV-infected individuals is a heterogeneous disease.


Renovascular hypertension due to Takayasu's arteritis among Indian patients.

Over a 16-year period, 205 patients with hypertension were shown to have a renovascular aetiology. Of these, 125 (61 per cent) had Takayasu's arteritis, 58 (28.3 per cent) had fibromuscular dysplasia, 16 (7.8 per cent) had atherosclerosis, five (2.4 per cent) had polyarteritis nodosa and one (0.5 per cent) had renal artery aneurysm. Among patients with Takayasu's arteritis, males were affected as commonly as females. The mean age of these patients at the time of detection was 26.8 +/- 8.6 years (range 5-52 years). Type I arteritis was seen in nine (7.2 per cent), Type II in 40 (32 per cent) and Type III in 76 (60.8 per cent) patients. The abdominal aorta was involved in 117 (93.3 per cent) patients. Takayasu's arteritis was associated with ulcerative colitis in two patients and with renal amyloidosis and focal segmental glomerulosclerosis with a nephrotic syndrome in one patient each. Surgical intervention consisting of bypass procedures, autotransplantation or nephrectomy was performed in 17 (13.6 per cent) and angioplasty in nine (7.2 per cent) patients. Cure and improvement in blood pressure was observed in 82.4 per cent and 77.8 per cent respectively. Adequate control of blood pressure was achieved with drugs only in 22 (22.2 per cent) patients. A definite cause and effect relationship could not be established between any infective or immunological disorder and Takayasu's arteritis. Takayasu's arteritis is a far more common cause of renovascular hypertension in Indian population than fibromuscular dysplasia or atherosclerosis, which are more common in the western population.


[Association of Takayasu arteritis, Crohn disease and pregnancy].

The authors report a case of a pregnant woman with Takayasu's arteritis and Crohn's disease. The course of pregnancy was uneventful. It is suggested that these two inflammatory diseases may be immunologically related. A brief review of the literature confirm that fetal and maternal prognosis are generally excellent.


[Therapeutic perspectives of sickle cell anemia].

After a long gestation new therapies are at hand. At the present time about 30 patients have been cured with the bone marrow transplantation. The potential use of cord blood, rich in hematopoietic stem cells, is under development as an alternative for bone marrow transplantation. The activation of HbF expression upon hydroxyurea (Hydrea) and other agents which is under clinical trials. The agent 12C79 which increases the oxygen affinity of sickle cells in vivo and prevent HbS polymerization is in clinical development. Membrane acting agents which inhibit sickle cell dehydration are another approach to be developed. The inhibition of vascular cell adhesion, of inappropriate clotting formation and of inadequate response to vaso-occlusive events needs to be developed. Finally the gene therapy is a very promising avenue.


[Rheumatoid arthritis. Changes in lymphocyte subsets under the effect of tiopronin].

Lymphocytes from 12 rheumatoid arthritis patients were phenotyped before and after a 2-month treatment with tiopronin. Originally reduced, CD4 CD45RA-T lymphocytes were shown to augment significantly. Abnormal activation (evaluated on HLA-DR and CD25 expression) of each cell population and sub-population was partially amended.


[Non-palpable testes--diagnosis and therapy].

The authors subject to a retrospective analysis a group of 522 boys operated on account of retention of the testes during 1982-1991, whereby they focused attention on cases where the testis was not palpable before operation. In 57 (10.9%) of the boys with a non-palpable testis in three instances the finding was bilateral and in 54 cases unilateral. Surgical revision of 60 testicles revealed anorchia in 14 instances (23.3%), inguinal retention in 25 (41.7%) and abdominal retention in 21 (35.0%). Orchiopexy was performed in 31 boys and orchiectomy in 15. The therapeutic results are unfavourable in particular in the group of boys with intraabdominal retention. The mentioned state of affairs can be improved only by initiating treatment at the age of 1 year, making use of microsurgery and laparoscopy in the diagnostic and therapeutic process.


Sea anemone Radianthus macrodactylus--a new source of palytoxin.

A very potent non-protein toxin was isolated from the sea anemone Radianthus macrodactylus with the use of chromatography on polytetrafluoroethylene, CM-Sephadex C-25 and by cation and anion exchange HPLC. The toxin was identified as palytoxin by u.v.-, i.r.- and 500 MHz 1H NMR spectroscopy. Its LD50 was 0.74 +/- 0.29 micrograms/kg by i.v. injection into mice. So far, palytoxin has been associated with zoanthids only. The toxin caused the loss of haemoglobin from erythrocytes but only in about 2 hr after the beginning of incubation, which is characteristic for palytoxin from zoanthids. Sea anemone palytoxin was divided into major and minor components by HPLC. The latter proved to be a product of degradation of palytoxin.


Treatment with neuroleptics: the patient's perspective.

Sixty-one acute schizophrenic patients were questioned on the effects ascribed subjectively by them to their neuroleptic medication and on their rating of neuroleptic therapy. Although negative effects ascribed to the medication outnumbered positive effects by 3 to 1, the global rating was predominantly positive. Patients approving of neuroleptic therapy reported favourable changes under medication significantly more often than patients with a rejecting attitude. There was, however, no significant difference between the two groups in the frequency with which negatively experienced effects of medication were described.


On the regulation of beta 2 integrins.

The complex functions played by beta 2 integrins in mediating a large variety of adhesive interactions of leukocytes are highly regulated. This regulation results in transient adaptations/associations, permitting physical and functional recycling of these receptors during chemotaxis, phagocytosis and target-cell killing. The structural definition of these adaptations will lead not only to a better understanding of how these receptors are regulated in leukocytes but also shed valuable light on how these integrins integrate diverse extracellular signals into spatially and temporaly coordinated cellular responses.


[Immunogenetic and molecular genetic studies on ocular diseases].

The immunogenetic mechanisms of various ocular diseases were investigated utilizing recently developed molecular biological and molecular genetic techniques. It was revealed that HLA-B 51 was closely associated with Behçet's disease. Investigation of genetic polymorphism of TNF-beta (tumor necrosis factor-beta) showed that 95% of Behçet's disease patients had the 10.5 kbp Nco I fragment. It was therefore concluded that the gene of susceptibility to Behçet's disease is located between HLA-B and TNF-beta loci on the short arm of chromosome 6. Similar studies of HLA-DNA typing in Harada's disease frequently seen in Japan showed that frequencies of HLA-DRB1 * 0405, HLA-DQA1 * 0301 and HLA-DQB1 * 0401 were significantly increased in patients compared with normal controls. These data suggested that those who have serine at position 57 of HLA-DR, glutamic acid at position 70 and aspertic acid at position 71 of HLA-DQ respond to certain unknown agents significantly more than those without them, thus leading to the development of Harada's disease. The same HLA association was observed between Harada's disease and sympathetic ophthalmia, and the immunogenetic mechanism was thought to be similar in both diseases. Recent immunogenetic and molecular genetic investigations on various ocular diseases have shed new light not only on the genetic individual susceptibility and biased racial differences, but also on the diagnosis of the ocular diseases, reclassification of disease entities according to HLA associations, and judgement of disease prognosis. Further progress of molecular medicine may make it possible to treat various intractable ocular diseases by gene therapy in the near future.


[Long-term outcome of cryptorchism after orchiopexy].

The long-term outcome of cryptorchism (undescended testis) was studied in 43 patients who underwent orchidopexy at pre-puberty ages and who were over 15 years of age at the time of this study. The follow-up period after operation was 11 approximately 23 years. Cryptorchism was unilateral in 39 patients and bilateral in 4 patients. The sperm concentration and motility were examined, using a cut-off level of 20 x 10(6)/ml for sperm concentration and 50% for sperm motility. In the unilateral cryptorchism group, 16 patients (61.5%) had normal semen quality, 8 patients (30.8%) oligozoospermia, 1 (3.8%) asthenozoospermia and 1 (3.8%) azoospermia. In the bilateral cryptorchism group, 3 patients (75.0%) were normal and 1 (25.0%) had azoospermia. Eight patients with unilateral cryptorchism were married and 7 of them (87.5%) had children. The sperm concentration had no inverse correlation with the age at operation. In patients with unilateral cryptorchism, the testicular volume on the healthy side was significantly higher than that on the affected side. The sperm concentration tended to correlate with the testicular volume on the healthy side rather than that on the affected side. These findings suggest that the sperm profiles in patients with unilateral cryptorchism are chiefly associated with the testicular function on the healthy side.


Twelve months, treatment with inhaled salmeterol in asthmatic patients. Effects on beta 2-receptor function and inflammatory cells.

Salmeterol is a new beta 2-receptor agonist with a prolonged duration of action after inhalation, exceeding 12 h in most patients. We have performed a 12-month open follow-up study on 11 patients with reversible asthma. All patients were given salmeterol inhalations (50 micrograms twice daily). Additional asthma treatment included inhaled corticosteroids in all patients, and oral slow-release theophylline or beta 2-receptor agonists in a minority of patients (3 and 1, respectively). Before salmeterol treatment was initiated and after 3, 6, 9 and 12 months of salmeterol treatment, a cumulative dose-response curve to inhaled salbutamol (100, 300 and 900 micrograms) was performed, and FEV1 measured. We also evaluated the effect of each salbutamol dose on finger tremor, systemic blood pressure and heart rate. Blood tests, including white blood count and electrolytes, were taken at each visit. After salmeterol treatment was initiated, morning FEV1, measured before the morning asthma medication, was significantly improved (p < 0.05). The responsiveness to inhaled salbutamol was not decreased during salmeterol treatment, except in one patient with asthma worsening over the study year. Baseline finger tremor measured before salbutamol dose-response curves, was significantly lower at the 12-month visit than before treatment was initiated (p < 0.05). Effects of salbutamol on systemic blood pressure, heart rate or finger tremor was not significantly changed during salmeterol treatment. We found a successive and significant decrease in blood eosinophils (p < 0.05) during the 12 months of salmeterol treatment, when the patient with asthma worsening was excluded in the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)


Coexisting HLA-B27 positive spondyloarthritis and polyarteritis nodosa.

A 38 year old woman presented with widespread polyarteritis nodosa a few years after the onset of HLA-B27 positive spondyloarthritis. The concomitant coexistence of these two disorders suggests a possible association in this genetically susceptible subject.


[A comparative evaluation of the efficacy of certain types of premedication in children].

Mathematical analysis of the heart rhythm and expressive methods of emotional stress determination have been used in 90 children prepared for surgical intervention with the help of three types of premedication. It has been shown that premedication with diazepam and droperidol (according to scheme) was effective for the elimination of preoperative psychoemotional stress in children.


[Endogenous humoral regulators directly before an operation].

It has been shown that modern premedication techniques do not prevent the activation of the sympathoadrenal system and the enhanced synthesis of cyclic nucleotides and prostaglandin-like compounds in response to preoperative stress. In a great number of patients there are no hemodynamic shifts in the nearest preoperative period, as an increased production of prostaglandins E and cyclic nucleotides (cAMP), that is of compensatory value, is observed. When normal relations between endogenous vasoactive substances with constricting and dilating effects are damaged, a hyperdynamic reaction ensures.


Neutrophil--mediated cytotoxicity against tumour cells: state of art.

When activated by a variety of stimuli, human neutrophils become capable of lysing tumour cells. The main physiologic cytotoxic trigger in neutrophils is represented by the surface receptor for the Fc domain of IgG (FcR). This receptor confers specificity to the target recognition by neutrophils and cooperates with adhesion glycoproteins (CD11-CD18) in the neutrophil-target conjugate formation. Although the FcR-dependent pathways of signal transduction remain to be clarified, the neutrophil responses involve both the production of oxidants and the release of granule constituents. These molecules are responsible for the target lysis, whose extent also depends on the target structural and metabolic characteristics. The use of monoclonal antibodies specific for tumour cell antigens, coupled with appropriate cytokines, may provide rational basis for designing trials to employ the neutrophil cytotoxic potential as adjuvant therapy in cancer patients.


Diminished integrin expression on granulocytes from renal allograft recipients.

Integrin (beta 2 subunit of the LEU-CAM family, CD18) expression on peripheral blood and urinary granulocytes was studied in renal allograft recipients. Circulating granulocyte CD18 expression was normal except for patients with CMV infection. In contrast, the majority of patients with urinary tract infection had low numbers of CD18+ cells collected from urine, and the same abnormality could also be observed in approx. 50% of non-infected recipients. These disturbances were associated with transplantation, as no such deficits were seen in the infected non-transplant patients or patients with glomerular diseases on immunosuppression. Short culture of granulocytes with immunosuppressants did not diminish integrin expression. Deficient granulocyte integrin expression, especially in urinary tract, may be associated with increased susceptibility to infection in renal transplant recipients, although immunosuppression alone could not be incriminated as a sole factor responsible for this deficit.


Sequence analysis of the int-2/fgf-3 gene in aggressive human breast carcinomas.

A number of primary human breast carcinomas exhibit amplification of the chromosome 11 region containing the int-2/fgf-3 proto-oncogene, and progression of breast cancer has been correlated with int-2 amplification or with certain restriction fragment length polymorphisms (RFLPs) of the int-2 gene. Using the polymerase chain reaction (PCR), we obtained the int-2 coding sequences from six primary tumors, four of which exhibited amplification of the int-2 gene and one of which exhibited amplification of the neu gene. The majority of these tumors (five of six) were aggressive, as judged by their early recurrence, metastasis, or both. Nucleotide sequencing of PCR products revealed that previously described BamHI and PstI RFLPs of the int-2 gene, as well as a new polymorphism at position 9154, were located within the intron between the second and third exons. A seventh tumor was used to localize one of the PstI RFLPs 5 bp from the splice-acceptor site of the third exon. However, none of the tumor DNAs analyzed showed differences in the int-2 protein coding regions when compared with normal placenta DNA. These results imply that aggressive human breast cancers encode an unaltered form of the int-2 protein.


A distinct large granular lymphocyte (LGL)/NK-associated (NKa) abnormality characterized by membrane CD4 and CD8 coexpression.  The Yorkshire Leukaemia Group.

In a study of 870 individual patients with either lymphocytosis (excluding known lymphoproliferative disease), increased proportions of blood lymphocytes with granular morphology (LGL), or neutropenia, 14 cases were found with abnormally increased CD3+CD4+CD8+ components. Eleven of these were further investigated and 10 shown in follow-up studies to be persistent in nature. Morphological assessments revealed increased LGL in 9/11 cases, and in seven of these > 50% lymphocytes had discernable cytoplasmic granulation. Immunophenotypic studies indicated that CD8 expression by CD4+ lymphocytes in these patients was of low density (CD8dim+), and that both the CD4+CD8- and CD4+CD8dim+ fractions in each patient was characterized by a CD11b+CD16-CD56+CD57+ composite NK-associated (NKa) phenotype (in contrast to normal CD4+CD8- blood lymphocytes and CD4+CD8+ thymocytes which were consistently CD11b-CD16-CD56-CD57-). TCR genotypic studies revealed rearranged components (beta plus gamma, or beta alone) in 5/11 cases, but there were no obvious relationships between TCR configuration (including rearranged band densities) and immunophenotypes, absolute lymphocyte or neutrophil numbers, the proportions of blood LGL, or the proportions of CD4+ cells coexpressing CD8. The occurrence of identical NKa phenotypic profiles in both germline and rearranged TCR cases does, however, suggest the possibility of an evolutionary process from a non-clonal expansion to a clonal state. Serum studies, including soluble CD4, CD8 and IL2-R concentrations and autoantibody investigations, of representative germline and rearranged TCR cases failed to indicate any consistent abnormalities, but there was some suggestion for the existence of a chronic reactive process in some of the patients with germline TCR. These findings suggest that expanded LGL/NKa+ components with phenotypic evidence of CD4/CD8 coexpression should be regarded as a distinct diagnostic category and that persistent CD4+CD8+ abnormalities with germline TCR should be monitored for possible clonal transition.


Psychomotor disturbances in psychiatric patients as a possible basis for new attempts at differential diagnosis and therapy. V. Evaluation of psychomotor training programs in depressed patients.

Parts I-III of this series used psychometric assessment of motor performance in psychiatric patients and indicated a "psychotic-motor syndrome" (PMS) in schizophrenic and affective psychoses, which was not found in "neurotic"/reactive or healthy persons. Part IV yielded signs of concomitant brain dysfunction in these patients, demonstrated by EEG mapping as well as other (SPECT/PET) neuroimaging methods. Apart from this "basic science" interest into the pathophysiology of endogenous psychoses we engaged in the development of motor training programs using the PMS as "target" syndrome. We hypothesized, that motor training would not only improve disturbed motor behaviour, but ameliorate other symptoms of psychopathology also. These assumptions were supported in the first two independent studies involving n = 45 and n = 31 ICD-9 mono- and/or bipolar endogenous depressed patients, respectively (the studies on schizophrenic patients being reported finally as part VI of this series, along with the final version of our modified motor test battery). Examples of the motor training programs are provided in this paper, although the final version of the complete programs will be published separately for space reasons and for better availability for routine clinical use.


Plasmid analysis and epidemiology of Salmonella enteritidis infection in three commercial layer flocks.

Ninety-six S. enteritidis isolates obtained from three commercial layer flocks in 1988-90 were examined following DNA extraction, restriction enzyme digestion, and gel electrophoresis for plasmid size profiles and restriction fragment length polymorphisms (RFLPs). The S. enteritidis isolates from the three flocks had three, eight, and two different plasmid profiles, respectively. Only four isolates from one flock lacked plasmids. A 36-megadalton (mDa) (54-kilobase) plasmid was present in 73% of the isolates, either alone or in combination with other plasmids. Isolates with only the 36-mDa plasmid had identical RFLPs. The diversity of plasmid profiles was greater than that of phage-types among isolates from the three flocks: 12 unique plasmid profiles vs. four phage-types. Mixed infections with S. enteritidis strains having distinct plasmid profiles occurred in all three flocks. Reinfection of these flocks in 1990 with one or more of the strains obtained earlier was evident, because some of the original isolates and the 1990 isolates had matching plasmid profiles and were of the same phage-types. Isolates from both environmental and tissue samples, examined from one flock, were found to share the same plasmid profile and phage-type.


Ranitidine, cimetidine, famotidine have no effect on post-prandial absorption of ethanol 0.8 g/kg taken after an evening meal.

Forty-seven healthy male subjects were studied twice using a randomized, placebo-controlled design. Each subject took an 8-day course of two of the following four regimens; 300 mg ranitidine, 800 mg cimetidine, 40 mg famotidine or placebo (identical either to 300 mg ranitidine or 800 mg cimetidine). The systemic bioavailability of ethanol (integrated 6-h plasma ethanol concentration, peak plasma ethanol concentration, and the time to peak plasma ethanol concentration) was measured after the oral ingestion of 0.8 g of ethanol per kg body weight, given one hour after an evening meal on Day 8 of each regimen. There was no significant difference of integrated 6-h plasma ethanol concentration, peak ethanol concentration, or time to reach peak ethanol concentration after dosing with either ranitidine, cimetidine or famotidine or placebo.


Clonal deletion of V beta 17 T cells in mice from natural populations.

In a panel of wild mice trapped in different geographical regions, the concomitant expression of V beta 17 T cell receptors and I-E histocompatibility molecules is frequent. By Southern blot analysis, eight forms of V beta 17 genes have been distinguished that encode five kinds of V beta 17 domains. Populations of Asia display the highest level of polymorphism and include all the V beta 17 forms and domains. The V beta 17 T cells of wild origin are deleted in mice expressing Mls-3 and related superantigens. Wild mice use V beta 17 domains different than the one of laboratory strains. Together with endogenous superantigens polymorphism, this accounts for the observed low frequency of clonal deletion cases in natural populations.


Non-surgical management of bile duct stones refractory to routine endoscopic measures.

Endoscopic sphincterotomy and percutaneous approaches to the biliary tract have revolutionized the treatment of bile duct stones. Both the endoscopic and transhepatic approaches are less invasive than open surgery. This is an advantage for the mostly elderly and frail patients with common bile duct stones. Other patients with intrahepatic stones, e.g. young patients with oriental lithiasis, may also profit from the non-surgical approach. In this latter group it is often difficult for the surgeon to obtain access to the stone-bearing bile ducts. Due to the anatomical situation, size or impaction of stones the non-surgical approach, including mechanical disintegration, may primarily fail. Several techniques such as intracorporeal lithotripsy using electrohydraulic probes or laser light, extracorporeal shockwave lithotripsy or direct contact dissolution are now available and often allow complete clearance of the bile ducts. If a kidney lithotripter with radiographic devices is available, it should be used after an attempt at mechanical lithotripsy has failed (Figure 1). According to the literature, experience with this method is greater than with any other 'third-step approach'. The procedure is simple, relatively safe and successful in approximately 80% of patients. However, in at least one third of patients, several sessions have to be performed and further endoscopy is frequently required for extraction of fragments. Intracorporeal techniques may become the procedure of choice in the future, at least in patients with common bile duct stones. At the moment, however, the different devices are still not fully developed and too susceptible to damage. A further major drawback, especially with high-energy electrohydraulic intracorporeal lithotripsy, is the danger of bile duct injury or even perforation, so that most procedures must be performed under optical control. The use of contact dissolution cannot generally be recommended. Treatment with mono-octanoin or modified mono-octanoin solvents takes too long, is often not successful and has a high rate of side-effects. MTBE may shorten the procedure considerably, but is suitable only for cholesterol stones, and the danger of spill-over into the intestine with absorption and systemic side-effects has to be weighed against the probability of success.


N-benzyladriamycin-14-valerate and drug resistance: correlation of anthracycline structural modification with intracellular accumulation and distribution in multidrug resistant cells.

N-Benzyladriamycin-14-valerate (AD 198) is a highly hydrophobic analogue of Adriamycin (ADR) which can circumvent multidrug resistance (MDR) in various cell lines. Unlike ADR, AD 198 avoids extrusion by P-glycoprotein (P-gp) in AD 198-resistant murine macrophage-like J774.2 cells and localizes in the cytoplasm. To determine the structural modification(s) responsible for these different characteristics, intracellular accumulation and distribution of ADR, AD 198, and the two half-substituted AD 198 congeners. N-benzyladriamycin (AD 288) and adriamycin-14-valerate (AD 48), were analyzed in AD 198-sensitive (J774.2) and -resistant (A300) cells. A300 cells exhibited cross-resistance to and reduced accumulation of ADR, AD 48, and AD 288. ADR and AD 288 rapidly localized in the nuclei of parental and A300 cells, while AD 48 and AD 198 localized in the cytoplasm. AD 48 redistributed into nuclei and cytoplasm of both cell lines, but AD 198 maintained a punctate cytoplasmic distribution in A300 cells. These results suggest that both the N-benzyl and C14-valerate substitutions of AD 198 are required for P-gp circumvention and stable cytoplasmic localization in A300 cells, probably as a result of differing intracellular drug trafficking.


A meta-analysis comparing the efficacy of omeprazole with H2-receptor antagonists for acute treatment of duodenal ulcer in Asian patients.

A meta-analysis was performed on pooled data from five large double-blind studies (a total of 1057 patients), which were conducted in Asia to compare the effects of omeprazole with H2-receptor antagonists (H2-RA) in duodenal ulcer. As each study followed the same protocol and data evaluation procedures, a detailed analysis of ulcer healing, symptom relief and influence of prognostic factors across the data was possible. Patients received omeprazole 20 mg om or standard doses of H2-RA for a minimum of 2 and a maximum of 4 weeks, depending on healing (as verified by endoscopy). All efficacy analyses were based on per protocol data. The mean healing rates at 2 weeks were 72% for omeprazole and 42% for H2-RA (difference 30%; 95% CI: 24-36%; P < 0.0001) and at 4 weeks were 96% for omeprazole and 83% for H2-RA (difference 13%; 95% CI: 10-17%; P < 0.0001). In addition to treatment, ulcer size had a significant influence on healing, with large ulcers (diameter > 10 mm) taking longer to heal than small ulcers. There was no significant influence of smoking and alcohol drinking on ulcer healing. Patients on omeprazole experienced significantly less epigastric pain after 2 weeks than those on H2-RA, 79% of them being completely symptom-free on omeprazole compared with 65% on H2-RA. The incidence of adverse events was approximately 5% on each treatment and profiles were similar for each drug. It is concluded that omeprazole, even in the presence of adverse prognostic influences, results in significantly better healing of duodenal ulcer and relief from symptoms than H2-RA.


Benzquinamide inhibits P-glycoprotein mediated drug efflux and potentiates anticancer agent cytotoxicity in multidrug resistant cells.

We have previously shown that efflux of cytotoxic drugs from multidrug resistant (MDR) cell lines can be quantitated at the single cell level using a sensitive fluorescence microscopy technique. Based on the structure of compounds which inhibited the efflux of Rhodamine-123 (Rho-123) using this methodology, we hypothesized that the antiemetic, antihistaminic agent benzquinamide (BZQ) would interfere with P-glycoprotein (P-gp) mediated drug transport and potentiate the effects of anticancer agents in MDR cell lines. We show that BZQ interferes with P-gp mediated drug efflux and increases drug accumulation in MDR cells using Rho-123 as a fluorescent probe. BZQ increases the cytotoxicity of chemotherapeutic agents to both human and hamster MDR cell lines in vitro. A slight increase in cytotoxicity to chemotherapeutic agents is also observed in the parental cell lines with BZQ. BZQ increases [3H]daunorubicin accumulation and inhibits the binding of [125I]iodoaryl azidoprazosin to the P-gp in MDR cells. BZQ is a new agent to increase the cytotoxic effects of anticancer agents in MDR cells and may ultimately prove useful as an adjunct in cancer chemotherapy.


Gene expression, signal transduction and tissue-specific biomineralization during mammalian tooth development.

Tooth development provides a paradigm for intrinsic molecular controls for cell- and extracellular matrix (ECM)-mediated biomineralization. The intent of this review is to evaluate the sequential timing and positional information prerequisite for tissue-specific biomineralization. Recent investigations suggest that 1,25-dihydroxyvitamin D3 functions to up-regulate VDR (vitamin D receptor) that in turn could induce structural gene products, including calcium-binding proteins and several ECM proteins (e.g., enamelins, amelogenins, dentine sialoglycoproteins (DSP) and dentine phosphoproteins (DPP)), resulting in dentine and enamel formation. Inhibition of regulatory gene products and/or their receptors likely results in hypoplastic and/or hypomineralized ECM as a direct consequence of down-regulated (1) transcription and/or translation of structural and regulatory genes, (2) posttranslational modifications, (3) and/or decreased calcium transport to the forming dentine and enamel matrices. Advances in serumless in vitro culture methodology; computer-assisted access to nucleic acid sequences for probes to define when, where, and how many specific regulatory and structural gene products are expressed; antisense oligodeoxynucleotides to inhibit specific translation; and microtechniques to analyze biomineralization all provide additional avenues to investigate tissue-specific biomineralization.


[Are metipranolol eyedrops responsible for intraocular side effects?].

Metipranolol is a beta-blocker that has been used in ophthalmology and in systemic therapy for about 10 years. Reports about reversible uveitis under the product Glauline (metipranolol-containing eye drops) in England were the reason for extensive studies with metipranolol-containing eye drops produced with different methods. Analytical studies concerning the influence of irradiation sterilization on the drug containers, studies on the toxicity of the ophthalmic drug on tissue cultures, and prospective and retrospective clinical studies on 2,800 glaucoma patients were performed. Irradiation sterilization leads to the formation of free radicals on the surface of the containers and, depending on the radiation dose, to a decrease in the pH of the drug solution. In prospective studies involving 1,516 glaucoma patients, no intraocular side effects due to metipranolol-containing eye drops were found. In the retrospective examination including 1,306 glaucoma patients, 19 cases of uveitis were found. Thirteen cases of recurring iritis were diagnosed, which had already been observed before the onset of glaucoma therapy. In 2 cases the iritis led to secondary glaucoma and was treated with metipranolol. In 2 cases glaucoma was treated with pilocarpine (and dipivefrin) and metipranolol concomitantly. One case of rubeosis iridis was incorrectly classified as iritis. One case is possibly related to metipranolol despite the assessment to the contrary by the ophthalmologist in question. Following the results of these studies, an accumulation of cases of uveitis caused by metipranolol can be excluded.


The significance of aminopeptidases and haematopoietic cell differentiation.

Aminopeptidases are a group of enzymes found on the cell surface and in the cytoplasmic compartments of many peripheral blood cell types and their progenitors. Their functional roles include the hydrolysis of several biologically active peptides and growth factors and some have proved to be of diagnostic and prognostic value in leukaemia. These enzymes may also be found in serum as a consequence of non-haematopoietic related diseases and so have been used as indicators of liver damage. Haematopoietic cells in the bone marrow go through a process of growth and differentiation before being released into the peripheral circulation where they fulfill many functional roles. The enzyme activities of some aminopeptidases have been shown to modulate the growth of these cells. In addition, the activities of these enzymes themselves can be regulated by haematopoietic growth factors. However, the mechanisms that regulate their expression and activity are not fully understood. In this report the current literature has been reviewed for evidence of expression, regulation and clinical significance.


Abnormalities of insulin-like growth factor (IGF-I and IGF-II) genes in human tumor tissue.

Recent findings have indicated that Insulin-like growth factors (IGF-I and IGF-II) may play a role in neoplasia. Expression of their genes, which are highly complex structures, is tissue-specific and developmentally regulated. The purpose of the present study was to determine whether a relationship exists between tumorigenesis and the structure and expression of IGF genes. The structures of the IGF-I and IGF-II genes were investigated in 40 tumors by Southern blot analysis but no obvious re-arrangements (such as amplification or deletion) were observed in any of the tissues investigated. DNA methylation was also studied, using the enzyme Avall. The extent of DNA methylation of the IGF genes was highly variable in most of the tumors, as was the level of mRNA expression. A relationship could be detected between IGF over-expression and gene demethylation in tumors associated with hypoglycemia and in certain hepatocarcinomas. Loss of heterozygosity has been reported in the 11p15 region of some childhood tumors. The present findings provide further evidence of this loss of heterozygosity for the IGF-II gene and show an imbalance in the leukocyte alleles in several childhood tumors. Likewise, an imbalance in the alleles was noted in several adult tumors, including hepatocarcinomas and breast cancers. This suggests that in certain adult tumors alterations of the IGF-II gene may be associated with tumorigenesis, but in other tumors another mechanism may be involved.


Proteinuria and enzymuria in vesicoureteric reflux.

Vesicoureteric reflux is a common abnormality of the urinary tract leading to significant renal morbidity and premature mortality. No reliable non-invasive method exists for its diagnosis. This study investigated the presence of urinary proteins and enzymes in healthy children and those with reflux. A log normal distribution was found for all analyte/creatinine ratios. Significantly higher tubular protein/creatinine ratios were found in patients with reflux nephropathy. Three enzyme/creatinine ratios (n-acetyl-B-D-glucosaminidase, gamma-glutamyl transferase and lactate dehydrogenase) were higher in children with reflux who had no renal scarring, but the degree of overlap with the normal range was such that it is doubtful whether any will be of use as a urinary marker.


Testicular cancer and cryptorchidism.

The records of 273 patients with germ cell tumours of the testis referred between 1970 and July 1991 were reviewed. There were 25 (9%) black, 40 (14%) mixed race and 214 (77%) white patients. Histology showed seminoma in 53% and non-seminomatous and germ cell tumours in 47% of patients. Maldescent of the testis (MDT) was found in 30 patients--an incidence of 11% overall. MDT was present in 8 of 25 (32%) black, 7 of 40 (18%) mixed race and 15 of 214 (7%) white patients with testicular cancer. The incidence of MDT was statistically significantly higher in both black and mixed race patients compared with white patients. None of the black patients had undergone orchiopexy but 71% of mixed race and 87% of white patients had done so. This resulted in a different pattern of presentation in black compared with mixed race and white patients with MDT and testicular cancer. The mean age was 40 years for black, 32 years for mixed race and 33 years for white patients. Black patients presented with abdominal or inguinal tumours rather than scrotal tumours and they had an increased tendency to present with seminomas.


Colocalization of peptide- and tyrosine hydroxylase-like immunoreactivities with Fos-immunoreactive neurons in rat central amygdaloid nucleus after immobilization stress.

The central amygdaloid nucleus (ACe) is part of the amygdaloid body, and it has been shown to participate in several stress related reactions. The ACe is densely innervated by tyrosine hydroxylase- (TH), corticotropin releasing factor- (CRF), calcitonin gene-related peptide- (CGRP), neurotensin- (NT), somatostatin- (SOM), enkephalin- (ENK), substance P- (SP), vasoactive intestinal polypeptide- (VIP) and cholecystokinin- (CCK) immunoreactive (IR) nerve terminals. In addition, the ACe contains numerous CRF-, NT-, SOM-, ENK- and SP-IR perikarya. In previous studies it has been shown that stress stimulates the expression of the immediate early gene c-fos in the ACe. The aim of this study was to demonstrate the colocalization of the Fos-IR neurons with the peptide- and TH-IR structures using an immunocytochemical double staining technique. In intact animals the ACe contained only a few Fos-IR neurons. After immobilization stress about 100 Fos-IR neurons were seen per section. They were mainly located in the area, which was enriched by peptide- and TH-IR nerve terminals. The close contacts observed between the Fos-IR neurons and the peptide- and TH-IR nerve endings suggest that the Fos-IR neurons were innervated by these nerve terminals. Furthermore, several NT-, ENK-, SOM- and CRF-IR neurons were observed and the vast majority of these cells exhibited Fos-like immunoreactivity. These results suggest that stress enhances the synaptic activity of the ACe, which stimulates the expression of c-fos. Subsequently, Fos may regulate the expression of the NT, ENK, SOM and CRF genes and thus affect the peptidergic efferents from the ACe.


Postnatal lead exposure induces supersensitivity to the stimulus properties of a D2-D3 agonist.

To examine the impact of lead (Pb) exposure during the ontogeny of dopaminergic (DA) systems on resultant DA function, rats were exposed postnatally (0-21 days of age) via the lactating dam to 0, 100 or 350 ppm Pb acetate in drinking water. At 2 months of age, the postnatally Pb-exposed rats were trained to discriminate the stimulus properties of either the D1 receptor agonist SKF38393 (6.0 mg/kg) or the D2-D3 receptor family subtype agonist quinpirole (0.05 mg/kg) from saline using a standard two-lever operant food-reinforced drug discrimination paradigm. In each training group, dose-effect curves describing drug lever responding to lower doses of the training drug and to preadministration of selective DA antagonists were obtained to examine Pb-induced changes in DA sensitivity, and doses of non-DA compounds were substituted to determine the specificity of any changes in DA sensitivity. In the D1/saline training condition, Pb exposure was not associated with any specific or consistent changes in DA sensitivity. In contrast, exposure to Pb was associated with D2-D3 receptor subtype supersensitivity as was indicated by significantly elevated levels of drug lever responding in the presence of quinpirole and haloperidol and to at least one dose of apomorphine. No differences in the dose-effect curves for either (+)-amphetamine or NMDA were observed in the D2-D3-trained control and Pb-exposed groups, but an increase in drug lever responding in the presence of pentobarbital was noted in the Pb-exposed group relative to control. Taken together, these findings are consistent with a Pb-induced functional D2-D3 supersensitivity possibly mediated via autoreceptors. Moreover, this functional D2-D3 supersensitivity necessarily represents a permanent effect of postnatal Pb exposure since both blood and brain Pb levels were negligible at the time drug discrimination training began.


Long-term adaptation of crayfish neurons depends on the frequency and number of impulses.

Increasing the impulse activity of crustacean neurons for a few days causes long-lasting changes in transmitter release, which are termed 'long-term adaptation' (LTA) in previous studies. Both the amount of transmitter released at the beginning of a stimulus train, and synaptic fatigue during repetitive stimulation, are reduced. The present study examines the dependence of these synaptic changes on the frequency and number of impulses used to elicit LTA. Fatigue resistance develops consistently when crayfish phasic motor neurons are stimulated for 3 days with as few as 9,000 impulses per day, and occurs in response either to low frequency stimulation (0.2 or 0.5 Hz), or to stimulation in short bursts at a moderate average frequency (2.5 Hz). In contrast, the reduction in initial transmitter release does not appear consistently when the frequency and number of impulses are both low (9,000 impulses per day delivered at 0.2 Hz), but does occur at the moderate stimulus frequency (2.5 Hz) and when a larger number of impulses (18,000) are delivered at a low frequency (0.5 Hz). The data suggest that the two changes in synaptic transmission that comprise LTA have different stimulus requirements.


Effects of the opiates on the paraventricular nucleus in genetically polydipsic mice.

The inbred mice, STR/N, are known to possess extreme polydipsia with no known abnormality in vasopressin system and the kidney function. Our previous studies indicate that the opiate antagonists given intracerebroventricularly strongly attenuated spontaneous drinking. To determine the site(s) of action, the present study was undertaken. Microinjections of naltrexone methobromide and a selective kappa-receptor antagonist, nor-binaltorphimine (nor-BNI), into the paraventricular nucleus of the hypothalamus (PVN) greatly attenuated drinking of the STR/N for 0.5 to 16 h after injections, while in the two control groups, non-polydipsic STR/1N and Swiss/Webster strains, drinking was not affected by the injections. Food intake was not much altered in all groups. Studies of PVN neurons in vitro (n = > 160 for each group) showed that basal firing rates and patterns were similar in the STR/N and the control groups. Morphine added to the medium inhibited some but excited none in all strains tested. The threshold for the inhibitory action was higher in the polydipsic STR/N mice (10(-8) M), compared to that in the control, S/W mice (10(-9) M). Further, a proportion of neurons inhibited by morphine in the PVN was significantly smaller (P < 0.01) in the STR/N (41.7%), compared to that in the control (64.9%). Dynorphin had very similar effect to that of morphine, but the proportion of cells inhibited was 25.4% in the STR/N, and 70.4% in the S/W. Prior applications of naloxone to the medium prevented the action of both morphine and dynorphin. Under the synaptic blockade (in a low Ca2+ and high Mg2+ medium) the inhibitory effect of the opiates persisted. We concluded that the PVN is at least one of the possible sites where the opiates are acting to cause the polydipsia in the STR/N mice.


Differential roles of NMDA and non-NMDA receptor activation in induction and maintenance of thermal hyperalgesia in rats with painful peripheral mononeuropathy.

Central activation of excitatory amino acid receptors has been implicated in neuropathic pain following nerve injury. In a rat model of painful peripheral mononeuropathy, we compared the effects of non-competitive NMDA receptor antagonists (MK 801 and HA966) and a non-NMDA receptor antagonist (CNQX) on induction and maintenance of thermal hyperalgesia induced by chronic constrictive injury (CCI) of the rat common sciatic nerve. Thermal hyperalgesia to radiant heat was assessed by using a foot-withdrawal test and NMDA/non-NMDA receptor antagonists were administered intrathecally onto the lumbar spinal cord before and after nerve injury. Four daily single treatments with 20 nmol HA966 or CNQX beginning 15 min prior to nerve ligation (pre-injury treatment), reliably reduced thermal hyperalgesia in CCI rats on days 3, 5, 7 and 10 after nerve ligation. Thermal hyperalgesia was also reduced in CCI rats receiving a single post-injury treatment with HA966 (20 or 80 nmol) or MK 801 (5 or 20 nmol) on day 3 after nerve ligation when thermal hyperalgesia was well developed. In contrast, a single post-injury CNQX (20 or 80 nmol) treatment failed to reduce thermal hyperalgesia or to potentiate effects of HA966 or MK 801 (5 or 20 nmol) on thermal hyperalgesia in CCI rats. Moreover, multiple post-injury CNQX treatments utilizing the same dose regime as employed for the pre-injury treatment attenuated thermal hyperalgesia but only when the treatment began 1 or 24 h (but not 72 h) after nerve ligation.(ABSTRACT TRUNCATED AT 250 WORDS)


MK-801 affects the potassium-induced increase of glial fibrillary acidic protein immunoreactivity in rat brain.

Exposure of a limited brain surface to a high potassium (K+) concentration produces an injury limited to the underlying cortex, without apparently affecting other brain areas. Such a treatment produces an increased expression of glial fibrillary acidic protein (GFAP) in astrocytes, as assessed by immunohistochemical techniques, throughout the cortex ipsilateral to K+ exposure. This effect is evident 2 days after treatment and persists up to, at least, day 7. Thirty days after K+ exposure GFAP immunostaining is similar in both hemispheres. Administration of the non-competitive NMDA antagonist MK-801 (4 mg/kg i.p.) prior to the injury prevented the rise in GFAP immunoreactivity (IR) at 2 but not 7 days after the treatment. Administration of MK-801 after the injury appeared to have no effect on GFAP expression. This work confirms that brain injury, associated with spreading depression, can induce a glial response far from the lesion site. Furthermore, the fact that this phenomenon can be modified by an NMDA receptor antagonist suggests that glutamate may play a role, in vivo, in the regulation of astrocytic response to injury and introduces the possibility that brain injury-induced gliosis may be pharmacologically manipulated.


The relative importance of reasons for tooth extraction in terms of potential tooth years of life lost (PYLL).

The aim of the present study was to introduce the potential tooth years of life lost (PYLL) approach to the analysis of data showing the incidence of tooth extraction according to the reason for extraction and to illustrate its use. Six sets of data from prospective nationwide questionnaire surveys of patients treated by systematic random samples of dentists in five European countries were translated into PYLL. Response rates ranged from 25 to 81 per cent and the number of extracted teeth from 959 to 29,397, according to the study. PYLL in Norway 1988 was calculated using the ages of 85 and 80 as cut-off points, or the average sex-specific remaining life expectancy at the age of extraction; otherwise the age of 85 was used. Mean PYLL for all reasons varied from 40.6 to 46.3 years for Norway in 1988 depending on the cut-off point used. For patients aged 21 and older PYLL85 ranged from 35.7 years for France in 1984 to 42.3 years for Sweden in 1959-61. Employing PYLL changed the rank order of the reasons for extraction based on the number of extracted teeth in some instances. It combined the two dimensions 'incidence of 'potential years of tooth function lost' into a continuous quantitative variable which was easy to understand and simple to handle analytically.


Genetic analysis of the linkage between chromosome 11q and atopy.

Previous work has suggested that there is a genetic predisposition for the development of both asthma and atopy. A recent study has also shown that there is a striking link between chromosome 11q and the IgE response underlying asthma and rhinitis. To further assess the linkage between chromosome 11q and atopy, we have studied nine families of two and, in many instances, three generations with the index case having asthma and/or atopy. Using two restriction fragment length polymorphism probes associated with the regions 11q12-q13.2, namely PYGM and INT2, we have been unable to confirm a significant link between this region of chromosome 11q and atopy as defined by a positive skin-prick test and/or a raised specific IgE and/or a raised total IgE.


The distribution of type-2 chain histo-blood group antigens in normal cycling human endometrium.

The blood group ABO(H) determinants are major allogenic antigens in both erythrocytes and tissue of man. These antigens and related carbohydrates are markers of cellular maturation and differentiation in many epithelial tissues and have recently attracted great interest as tumor-associated antigens. Previous studies of endometrial tissues have indicated that glycosylation in this tissue may be related to hormonal stimulation. We have investigated the immunohistochemical distribution of type-2 chain histo-blood group-related carbohydrates in specimens of normal, cycling endometria obtained from hysterectomies on women with known ABO/Lewis erythrocyte type and saliva secretor status. N-acetyllactosamine and Le(x) were demonstrated to be uninfluenced by the genetic background. A and Ale(y) antigens were exclusively demonstrated in endometria from blood group A individuals, while Le(y) was expressed in endometria from blood group 0 individuals mainly. The precursor N-acetyllactosamine as well as the terminal H, A, and ALe(y) antigens were shown in only a few cells. In contrast, N-acetyllactosamine substituted by sialic acid and/or fucose residues (Le(x), sialosyl-Le(x), Le(y)) were demonstrated in epithelial cells of normal, cycling endometrium, but with both quantitative and qualitative differences in staining relating to the menstrual cycle, indicating that type-2 chain antigens are expressed under both genetic and hormonal influence in human cycling endometrium.


Glutathione and gamma-glutamyl transpeptidase are differentially distributed in the olfactory mucosa of rats.

Components of the gamma-glutamyl cycle, including thiols, glutathione (GSH) and gamma-glutamyl transpeptidase (gamma-GT), were localized in the nasal mucosae of rats using histochemical and immunohistochemical methods. In olfactory mucosa, thiols were widely distributed, with intense staining in the mucociliary complex (MC), basal cells, acinar cells of Bowman's glands (BG), and olfactory nerve bundles, and with moderate staining in olfactory receptor neurons (ORNs). GSH was localized in MC, BG acinar cells, nerve bundles and, to a lesser extent, in ORNs. gamma-GT immunoreactivity was restricted to the MC and to basolateral and apical membranes of BG acinar and duct cells. The basolateral membrane of BG acinar cells, located in close association with blood vessels and connective tissue, showed granule-like immunoreactivity. In respiratory mucosa, all three compounds were localized in the MC and acinar cells of respiratory glands (RG). In the MC, gamma-GT immunoreactivity was associated primarily with brush borders of ciliated cells. Granular immunoreactivity was also apparent in the supranuclear region of RG acinar cells. These results demonstrate that components of the gamma-glutamyl cycle are localized in olfactory and respiratory glands, and that they are secreted into the mucus, where they may mediate perireceptor events such as detoxification and/or solubilization of air-borne xenobiotics, toxicants and odorants.


StyI polymorphism in an enhancer region of the second intron of the apolipoprotein B gene in hyper- and hypocholesterolemic subjects.

The regulation of the human apolipoprotein (apo) B gene that plays a crucial role in lipid metabolism is apparently very complex, with multiple cis- and trans-acting regulatory factors. One of these factors is an enhancer region in the second intron. In this region a point mutation at position + 722 has been found that is detectable by the restriction enzyme StyI. The report of Levy-Wilson et al. (1991) could suggest that the mutant allele (abolished StyI site) is associated with hypocholesterolemia. To investigate further the possible effect of this mutation on plasma cholesterol levels, we have compared the frequency of the mutant allele between 206 hypercholesterolemic Norwegian or Czech subjects on one hand, and 165 hypocholesterolemic Norwegian or Czech subjects on the other hand. No significant difference in frequency was found between the hypercholesterolemic and the hypocholesterolemic groups. This finding indicates either that the mutation at position + 722 does not affect the enhancer activity or that this in vitro enhancer activity is of little or no clinical significance. One of the Norwegian hypercholesterolemic subjects who was of Czech descent possessed the apoB 3500 mutation that leads to defective binding of low density lipoprotein (LDL) to the LDL receptors. Haplotype analysis of the apoB gene in her family showed that the mutation-bearing allele was identical to that reported in other countries, indicating a common gene source.


Sex steroids do not alter sex differences in tyrosine hydroxylase activity of dopaminergic neurons in vitro.

In order to distinguish the effects of genetic sex from those of sex hormones on the sexual differentiation of dopaminergic neurons, catecholamine synthesis was studied in gender-specific cultures of embryonic day-14 rat diencephalon. In addition to embryos from normal dams, embryos were used whose mothers had been treated with the estrogen antagonist tamoxifen or the testosterone antagonist cyproterone acetate on days 12 and 13 of gestation. Cultures from embryos of untreated dams were fed daily with a medium containing 17 beta-estradiol or testosterone. After 10 days in vitro, cultures were immunostained for tyrosine hydroxylase and the accumulation of dihydroxyphenylalanine (DOPA) was measured in the presence of the DOPA decarboxylase inhibitor NSD 1015. Rates of DOPA synthesis, unlike the numbers of tyrosine hydroxylase-immunoreactive neurons, were markedly higher in female cultures under all experimental conditions. Treatment of dams with antisteroids prior to removal of the embryos had no influence on these results. Treatment of cultures with both steroids decreased DOPA formation in a dose-dependent manner without altering the sex difference. These results suggest that cultured diencephalic dopaminergic neurons develop sex differences in the activity of tyrosine hydroxylase. This sexual dimorphism is initiated independently on the activity of gonadal steroid hormones. Sex hormones exert an additional modulatory influence on the activity of the enzyme but do not abolish or reverse sex differences. Therefore, the concept of a purely epigenetic mode of sexual differentiation of the mammalian brain needs to be broadened to incorporate other mechanisms, such as the cell-autonomous fulfillment of a sex-specific genetic program.


beta-Thalassaemia mutations and their linkage to beta-haplotypes in Tamil Nadu in southern India.

A study for screening of beta-thalassaemia mutations by the Amplification Refractory Mutation System (ARMS) and haplotyping by Polymerase Chain Reaction (PCR) was undertaken because there was a paucity of data in Tamil Nadu in Southern India and to initiate a comprehensive prenatal diagnosis programme. A total of 294 alleles were analysed to study the nature of the mutations, of which 146 were beta-thalassaemia alleles. Only four types of beta-thalassaemia mutations were recorded. Of these, 128 alleles were of the variant IVS-1 nt 5 (G-->C). Thirteen had the mutation codon 41/42 (del TCTT), four had the mutation codon 8/9 (insert G) and one had the 619 bp deletion at the 3' end of the gene. The most common mutation, IVS-1 nt 5 (G-->C), was strongly associated with a single haplotype although the association was not absolute. The population of Tamil Nadu in Southern India seems to be ideal for initiating a prenatal diagnosis programme based on direct detection of mutation by ARMS coupled with RFLP linkage analysis.


Analysis of three glucose transporter genes in a Caucasian population: no associations with non-insulin-dependent diabetes and obesity.

The significance of variation within the genes coding for three glucose transporter proteins in the aetiology of non-insulin dependent diabetes mellitus was assessed by analysing restriction fragment length polymorphisms in an English Caucasian population. Two polymorphisms at the HepG2/erythrocyte glucose transporter (GLUT1) locus, four at the liver/pancreatic glucose transporter (GLUT2) locus and one at the muscle/adipocyte glucose transporter (GLUT4) were analysed in a sample of diabetic and non-diabetic subjects. No significant differences in the allelic, genotypic or haplotypic frequencies of the polymorphisms at these three loci were observed between the diabetic or non-diabetic populations. No significant linkage disequilibrium was observed between the two GLUT1 polymorphic sites, whereas the four polymorphic sites at the GLUT2 locus, one of which appears to be due to a 100-200 base pair DNA insertion/deletion, were found to be in significant linkage disequilibrium. In order to study the possible role of glucose transporter gene variants contributing to the development of obesity, the body mass indexes were compared in the different genotypic groups of diabetic and non-diabetic subjects. No differences in body mass index between genotype groups were found at the p < 0.005 level of significance.


The three week sulphasalazine syndrome.

We report a 53-year-old man with sero-negative rheumatoid arthritis who developed a fever, rash and hepatitis 3 weeks after starting sulphasalazine therapy. This was associated with a T cell lymphocytosis, eosinophilia and evidence of classical complement pathway activation. He responded to high dose corticosteroids. This is a rare but characteristic reaction which is likely to be encountered by rheumatologists more frequently with the increasing use of sulphasalazine. It should be recognized promptly as it may be fatal and can be confused with other systemic diseases.


Allele diversity of the H-ras-1 variable number of tandem repeats in Norwegian lung cancer patients.

We have examined restriction fragment length polymorphisms of the H-ras-1 gene in germ-line DNA from 214 lung cancer patients and 309 unaffected controls. When DNA samples were digested with MspI/HpaII, Southern blot analysis revealed at least 22 different alleles, grouped according to their frequencies as common, intermediate, and rare. The frequency of rare alleles in lung cancer patients (16/428) is significantly different (p = 0.002) from that in the control group (5/618). Individuals with rare alleles were found to be at 4.7-fold greater risk of lung cancer than those with no rare alleles.


The debrisoquine metabolic phenotype and DNA-based assays: implications of misclassification for the association of lung cancer and the debrisoquine metabolic phenotype.

Debrisoquine is an antihypertensive drug that is metabolized by cytochrome P4502D6. Deficient metabolism is inherited as an autosomal recessive condition. We previously reported in a case-control study that extensive metabolizers of debrisoquine were at greater risk of lung cancer compared to poor and intermediate metabolizers. Cloning of the gene that encodes P4502D6 (CYP2D6) led to the identification of both wild-type and mutant forms of the gene. Subsequently, a DNA-restriction fragment length polymorphism (RFLP) was identified, and a Southern hybridization-based test was developed in an attempt to define the genotype. When the DNA-RFLP test was applied to stored DNA from our study subjects there was neither a significant association with the metabolic phenotype nor an association with lung cancer. Further work has demonstrated that the wild-type gene, which was characterized by a 29-kb allele, can also contain mutations that result in nonfunctional or absent proteins. When these mutations are present, individuals exhibit the poor or intermediate metabolizer phenotype in spite of the presence of the 29-kb putative wild-type allele. Sequence determination of the mutants led to the development of techniques to exploit the polymerase chain reaction, which, together with Southern analysis, have been reported to detect as many as 95% of poor metabolizers. This technique is being used to examine the association of the extensive metabolizer genotype with lung cancer in the subjects from the case-control study. Preliminary results indicate a weak association between the homozygous wild-type genotype and lung cancer; in contrast, the extensive metabolizer phenotype is strongly associated with lung cancer in this subset.(ABSTRACT TRUNCATED AT 250 WORDS)


Polycyclic aromatic hydrocarbon-DNA adducts and the CYP1A1 restriction fragment length polymorphism.

Human cancer risk assessment at a genetic level involves the investigation of carcinogen metabolism and DNA adduct formation. Wide interindividual differences in metabolism result in different DNA adduct levels. For this and other reasons, many laboratories have considered DNA adducts to be a measure of the biologically effective dose of a carcinogen. Techniques for studying DNA adducts using chemically specific assays are becoming available. A modification of the 32P-postlabeling assay for polycyclic aromatic hydrocarbon DNA adducts described here provides potential improvements in quantification. DNA adducts, however, reflect only recent exposure to carcinogens; in contrast, genetic testing for metabolic capacity indicates the extent to which carcinogens can be activated and exert genotoxic effects. Such studies may reflect both separate and integrated risk factors together with DNA adduct levels. A recently described restriction fragment length polymorphism for the CYP1A1, which codes for the cytochrome P450 enzyme primarily responsible for the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons, has been found to be associated with lung cancer risk in a Japanese population. In a subset of individuals enrolled in a U.S. lung cancer case-control study, no association with lung cancer was found.


Emergence during unsuccessful chemotherapy of multiple drug resistance in a strain of Mycobacterium tuberculosis.

Serial isolates of Mycobacterium tuberculosis were cultured from a patient who failed to respond to standard antituberculous chemotherapy. Isolates were cultured in March 1989, July 1989, December 1989 and May 1990. Each successive isolate was found to be resistant to a wider range of antituberculous drugs than its predecessors. The initial isolate was resistant to isoniazid and rifampin, the second isolate was also resistant to ethambutol, the third was also resistant to pyrazinamide, ansamycin (= rifabutin) and ofloxacin and the last isolate was also resistant to ciprofloxacin and sparfloxacin. All four isolates' bacteriophage typing profiles and DNA restriction fragment patterns determined by Southern blot hybridization using the IS6110/IS986 probes and the new probe pTBN12 were concordant. It was concluded that this patient was persistently infected with a single strain of Mycobacterium tuberculosis which developed resistance to a number of families of drugs but did not show any significant change in typing patterns. The problem of acquired multiple drug resistance, particularly to fluoroquinolones and rifamycins, represents a new challenge in tuberculosis therapy.


Where now the diuretics in antihypertensive treatment?

The ultimate aim in treating hypertension is to reduce cardiovascular mortality and morbidity, especially from coronary heart disease and strokes. In several long-term trials this goal has been achieved with antihypertensive therapy in the form of diuretics. Subsequently, diuretics and betablockers, compared as single agents or with the addition of other agents, did not appear to affect overall cardiovascular morbidity and mortality differentially. Therefore, recommended first-line therapy for hypertension was initially diuretics, followed later by beta blockers as alternatives. Recently, calcium antagonists and ACE inhibitors have been accepted as equally valuable in the treatment of hypertension because they similarly lower blood pressure, lack any adverse metabolic effects and may be more beneficial than diuretics or beta-blockers on the long-term prognosis of hypertensive patients. Such recommendations are, however, highly speculative and are not supported by trials using cardiovascular mortality and morbidity as endpoints. In order to solve the conflict between proven facts and sound theory, long-term trials comparing older (mainly diuretics) and newer (calcium antagonists, ACE inhibitors, alpha-adrenoceptor blockers) antihypertensive agents are needed. Until such trials are completed, the debate surrounding first-line drugs for antihypertensive treatment will not be resolved.


Neurohormonal consequences of diuretics in different cardiovascular syndromes.

Diuretics have long been used to lower blood pressure in hypertensive patients or to control body fluid and electrolyte homeostasis in diseases such as congestive heart failure, chronic renal failure or cirrhosis. The initial response to diuretics is a negative sodium and fluid balance. The diuretic-induced loss of salt and water activates several hormonal systems such as vasopressin, the renin-angiotensin-aldosterone system or the sympathetic nervous system which tend to compensate for the changes in sodium and water balance. This neurohormonal response may have important clinical implications. Thus, the activation of the renin-angiotensin-aldosterone cascade appears to be partially responsible for the flat dose-blood pressure response curve of thiazides in hypertensive patients. It may also be responsible for the difference between responders and non-responders to diuretic therapy and for the development of side-effects such as hypokalaemia, metabolic alkalosis or hyponatraemia. There are several ways to prevent the undesirable consequences of the neurohormonal responses to diuretics. The first is to use low doses of these agents. It is also possible to combine them with agents that block the activity of the renin-angiotensin-aldosterone system such as ACE inhibitors or in combination with drugs that reduce aldosterone secretion such as calcium antagonists. The development of drugs able to enhance urinary sodium excretion and to reduce simultaneously the activity of the renin-angiotensin-aldosterone system may offer a new interesting alternative. This might perhaps be achieved in the future with the administration of neutral endopeptidase inhibitors which interfere with the enzymatic degradation of atrial natriuretic peptide.


Inhibitory effects of excitatory amino acids on pyramidal cells of the in vitro turtle medial cortex.

The electroencephalogram of the in vitro brain of the turtle Chrysemys d' orbigny shows spontaneous random large sharp waves (LSWs) which may be compared to interictal spikes. In order to evaluate the role of excitatory amino acids (EAAs)--in particular through the N-methyl-D-aspartate (NMDA) receptor--in the generation of LSWs, the bath application of NMDA and its antagonists 3-((+/-)-2-carboxypiperazin-4y)-propyl-1-phosphonic acid (CPP) and DL-2-amino-5-phosphonovaleric acid (APV), was performed in the whole open hemisphere (WOH) in vitro. Field recordings in WOH showed that both CPP and APV unexpectedly increased LSW amplitude. Consistently, NMDA in the bath suppressed the LSWs. Iontophoretically applied glutamate, kainate and NMDA produced a hyperpolarization of intracellularly recorded medial cortex pyramidal cells both in WOH and in slices. The EAA-induced hyperpolarization was tetrodotoxin (TTX) and bicuculline sensitive and reversed close to -70 mV. It would therefore seem to be due to the activation of gamma-aminobutyric acid (GABA) interneurons. The NMDA could also produce an excitation of pyramidal cells--always following a previous inhibitory phase. In some cases rhythmic bursting discharges or plateau potentials were observed. These NMDA effects were mainly elicited by a direct effect on pyramidal cells. A long-lasting hyperpolarizing response following the NMDA excitatory phase was also observed. This long-lasting response was an intrinsic property of pyramidal cells since it was TTX resistant. This study demonstrates that GABAergic interneurons from the turtle medial cortex can be activated by EAAs, a mechanism that can account for the effects of NMDA antagonists on LSWs.


The action of the psychoactive drug 2C-B on isolated rat thoracic aorta.

1. 2C-B [2-(4-bromo-2,5-dimethoxyphenyl)ethylamine] elicits concentration-dependent contraction of the rat thoracic aorta (apparent pD2 = 4.55). The maximal contraction (Emax) attained with 2C-B is less than that produced by either norepinephrine (NE) or serotonin (5-HT). 2. Pretreatment with either prazosin (5 x 10(-9) - 10(-8) M) or ketanserin (5 x 10(-9) - 10(-8) M) leads to decreased slopes and Emax in the 2C-B dose-response curves. 3. 2.82 x 10(-5) M 2C-B potentiates the response to low concentrations of NE; 5 x 10(-5) M 2C-B shows similar behaviour, but with reduced Emax. At 10(-6) M 2C-B acts as a competitive 5-HT antagonist; at 2.8 x 10(-5) M, however, it behaves like a non-competitive 5-HT antagonist. 4. Removal of the endothelial lining from the aortal rings only shifts the 2C-B dose-response curve to the left. 5. These results suggest that 2C-B behaves as a partial agonist toward both alpha 1-adrenergic and 5-HT2 serotonergic receptors. The endothelium only seems to act as a diffusional barrier to the drug.


Mechanisms of L-NG-nitro arginine methyl ester-induced antinociception in mice: a role for serotonergic and adrenergic neurons.

1. The mechanisms involved in the antinociceptive action of L-NG-nitro arginine methyl ester (L-NAME) were investigated in mice. 2. Intraperitoneal administration of L-NAME produced a dose-dependent antinociception in the tail-flick, hot-plate and phenyl-p-quinone-induced writhing tests. 3. Pretreatment with the catecholamine depletors 6-hydroxydopamine (5 micrograms i.c.v.) or reserpine (5 mg/kg i.p.) or the serotonin synthesis inhibitor, p-chlorophenylalanine methyl ester (200 mg/kg i.p. on 2 consecutive days) resulted in a significant decrease in the antinociceptive effect of L-NAME. 4. Similarly, pretreatment with the selective alpha 1-adrenoceptor antagonist prazonin (2.5 mg/kg, i.p.), or the non-selective alpha-adrenoceptor blocker, phentolamine (5 mg/kg, i.p.) antagonized the antinociceptive effect of L-NAME. 5. However, the administration of the selective alpha 2-adrenoceptor antagonist, idazoxan (2.5 mg/kg i.p.) was without effect. 6. Likewise, pretreatment with the serotonin 5-HT2 receptor blocker, ketanserin (1 mg/kg, i.p.), the D2 dopamine receptor antagonist (+/-) sulpiride (30 mg/kg, i.p.) or the opioid antagonist naloxone (5 mg/kg, i.p.) did not inhibit the antinociceptive effect of L-NAME. 7. These results suggest that L-NAME produces antinociception in the mouse probably by an action on adrenergic and serotonergic synapses.