Effects of galanin on the secretory activity of the rat adrenal cortex: in vivo and in vitro studies. Galanin, a 28-amino-acid peptide originally isolated from pig intestine, was found to cause dose-dependent rises in the plasma concentration of corticosterone in hypophysectomized rats and in both basal and submaximally ACTH-stimulated in vitro corticosterone production by adrenal quarters and isolated zona fasciculata/reticularis cells. These findings indicate that galanin exerts a direct glucocorticoid secretagogue effect on the inner adrenocortical zones and suggest that galanin, high concentrations of which are contained in adrenal chromaffin cells, may be included in that group of regulatory peptides, by way of which adrenal zona medullaris is thought to exert a paracrine control on the function of the zona corticalis. Galanin was also found to increase the plasma level of aldosterone in hypophysectomized rats in a dose-dependent manner without inducing changes in natremia, kalemia, plasma renin activity, or ACTH plasma concentration. Galanin did enhance both basal aldosterone output and that following submaximal ACTH or angiotensin II stimulation aldosterone output from adrenal quarters, but not from isolated zona glomerulosa cells. This last, rather unexpected, result suggests that galanin exerts an indirect mineralocorticoid secretagogue action, which seems to require the structural integrity of the adrenal cortex and the presence of the adrenal medulla. The hypothesis is advanced that galanin may control the release of some medullary peptides, which in turn may affect mineralocorticoid secretion of the zona glomerulosa in a paracrine manner.


Endothelin-3 modulates glomerular filtration rate in the isolated perfused rat kidney. The present study has been performed to evaluate hemodynamic and tubular effects of various endothelin-3 (ET-3) concentrations on the isolated perfused rat kidney. Using this experimental system we observed a profound reduction of renal perfusate flow at all ET-3 concentrations tested (50, 250 and 500 pmol/l), suggesting that the vasoconstrictive potency of ET-3 in the kidney is comparable to that described for endothelin-1 (ET-1). The effects on glomerular filtration rate (GFR) differed depending on the ET-3 dose applied. While 250 pmol/l ET-3 increased GFR by 30%, 500 pmol/l ET-3 markedly reduced GFR. 50 pmol/l ET-3 did not alter GFR although renal vascular resistance significantly increased. Infusion of 1 mumol/l N-nitro-L-arginine, a specific and potent inhibitor of nitric oxide synthesis in endothelial cells, abolished the GFR elevation induced by 250 pmol/l ET-3. In parallel with the changes of GFR we observed an increase in sodium reabsorption at 250 pmol/l and a decrease of this parameter at 500 pmol/l ET-3. Moreover, an ET-3 concentration (500 pmol/l), which induced a dramatic fall in tubular sodium load, led to an increase of fractional sodium excretion and to a decrease of renal oxygen consumption. We conclude that ET-3 is a potent vasoconstrictor in the isolated perfused rat kidney. Furthermore, it modulates GFR in a differentiated mode, depending on the concentration used. The GFR increase at 250 pmol/l ET-3 seems to be mediated by endothelium-derived nitric oxide. In addition to its glomerular action, ET-3 might also affect tubular sodium transport.


Influence of colloidal bismuth subcitrate on enzyme secretion from isolated rat pancreatic acinar cells. Bismuth salts are currently used as monotherapy or in combination with antibiotics for the treatment of Helicobacter pylori-associated peptic ulcer disease. Besides encouraging clinical results with colloidal bismuth subcitrate (CBS), there is an ongoing fear of organ toxicity with the use of bismuth salts. To study potential toxic effects of CBS under short-term exposure, we tested the influence of CBS on amylase secretion from isolated rat pancreatic acinar cells under basal conditions and following carbachol (CCh) and ceruletide (CRT) stimulation. Basal secretion was reduced by 8.9 +/- 9.6% (n = 10) (mean +/- SEM) (P < 0.05), 5.2 +/- 9.2% (P < 0.05), 9.4 +/- 6.4% (P < 0.01), and 6.2 +/- 12.2% (P < 0.05) with 0.001, 0.01, 0.1, and 1 micrograms/ml CBS, respectively. With 10 micrograms/ml and 100 micrograms/ml CBS, basal amylase secretion was increased in a dose-dependent manner, by 13.7 +/- 11.7% (P < 0.05) and 24.5 +/- 12.8% (P < 0.01). CCh (10(-5) M)- and CRT (3 x 10(-10) M)-stimulated secretory responses were not altered significantly by any of the CBS doses used. In concentrations above 1 microgram/ml, CBS increased pancreatic amylase secretion. Amylase secretion in response to secretagogues was not affected by CBS. These findings are unlikely to be associated with a toxic effect of CBS on exocrine pancreatic acinar cell function.


Acute lowering of plasma oncotic pressure increases filtration fraction and sodium excretion in conscious sheep. We examined the effect of acutely lowering the colloid osmotic pressure by removing plasma (36.2 +/- 3.1 ml/kg) and replacing it with Hartmann's solution (93.0 +/- 8.2 ml/kg) in 6 conscious merino sheep. The colloid osmotic pressure was reduced significantly (p < 0.05) from 20.3 +/- 0.9 to 8.5 +/- 2.5 mm Hg 0 h after plasmapheresis and to 15.2 +/- 0.8 mm Hg 20 h after treatment. The filtration fraction increased from 0.16 +/- 0.02 to 0.27 +/- 0.02 at 0 h (p < 0.05 vs. control) and to 0.20 +/- 0.02 at 20 h after treatment (p < 0.05 vs. control). At 0 h after plasmapheresis, there was an increase in both sodium excretion from 187 +/- 69 to 459 +/- 82 mumol.min-1 (p < 0.05) and in fractional sodium excretion from 1.6 +/- 0.4 to 4.4 +/- 1.0% (p < 0.05). The results are consistent with the hypothesis that acute alteration of Starling forces produce clinically significant effects on both glomerular filtration and tubular reabsorption.


In vitro transmission and antigenicity of a retrovirus isolated from a multiple sclerosis patient. We have recently isolated an apparently novel retrovirus (LM7) from a patient with multiple sclerosis (MS). We present here results showing that (1) LM7 retrovirus can be transmitted in vitro to a normal human leptomeningeal cell culture and that (2) specific antibody against this retroviral strain can be detected in MS cases. Our results suggest that, if this virus is an endogenous retrovirus, it is different from human endogenous elements already described.


[Benign prostatic hyperplasia--alternative (nonsurgical) possibilities of therapy]. The authors summarize contemporary possibilities of alternative treatment of benign hyperplasia of the prostate which could at least partially replace surgery. The authors present also the results of the first clinical investigations using different procedures. The list comprises also experience assembled in Czechoslovak departments.


Invertebrate GABA and glutamate receptors: molecular biology reveals predictable structures but some unusual pharmacologies. Determination of the sequences of invertebrate gamma-aminobutyric acid (GABA)-gated and glutamate-gated receptor/ion channels, through the application of recombinant DNA methods, is not just an academic exercise to effect evolutionary comparisons with the sequences of the corresponding vertebrate receptors. The isolation of DNA clones would provide the tools to investigate the exact locations and functional properties of these neurotransmitter receptors within simple nervous systems. In addition, since GABA receptors, at least, have been suggested to be the targets of certain pesticides, the availability of invertebrate receptor cDNAs might provide the agrochemical industry with the basis for 'high-throughput' screening methods for novel pesticidal compounds. Recently, the isolation of molluscan and Drosophila GABA receptor and glutamate receptor cDNAs, and the pharmacological properties of a GABA receptor expressed from one of these clones, have been reported. These studies should stimulate further research into the electrophysiology and pharmacology of native invertebrate ion channel proteins.


Characteristics of function-specific pathways in the sympathetic nervous system. The autonomic nervous system enables all of our body systems to operate in an external environment that is both physically and emotionally challenging. Despite voluntary and involuntary interventions, the composition of the internal environment is maintained. Autonomic dysfunction, particularly in aging people, reveals the importance of this efferent neural control for the wellbeing of our bodies and minds. Although the sympathetic component of this system has been widely thought to be concerned only with the body's response to stress, we discuss here how a range of neuroscientific techniques has started to reveal the specialized properties of functional pathways in the sympathetic system at molecular, cellular and integrative levels. The diversity observed is not compatible with a simple neuroendocrine role of this system.


Secreted acetylcholinesterase: non-classical aspects of a classical enzyme. Recent evidence suggests that termination of cholinergic transmission is just one of the many ways in which acetylcholinesterase (AChE) could influence neuronal function. Neuronal AChE can be secreted from several brain regions, while purified AChE possesses several properties (in addition to its cholinesterase activity) that can affect neuronal function, including the abilities to influence certain membrane conductances, enhance excitatory amino acid transmission and hydrolyse peptides. Loss of AChE and its non-classical actions would have a profound effect on brain function in neurodegenerative diseases such as Alzheimer's disease where there is widespread loss of AChE-containing neurons.


Neurochemical-neuroendocrine systems in the brain controlling macronutrient intake and metabolism. Appetite, energy balance and body weight gain are modulated by diverse neurochemical and neuroendocrine signals from different organs in the body and diverse regions in the brain. The hypothalamus plays an important integrative function in this process, acting through a variety of systems that involve a close interaction between nutrients, amines, neuropeptides and hormones. These systems underlie normal nutrient intake and metabolism and are thought to be responsible for shifts in feeding behavior across the circadian cycle and fluctuations relating to gender and age in both rats and humans. Moreover, alterations in these normal neurochemical-neuroendocrine systems may be associated with abnormal eating patterns, such as anorexia nervosa, bulimia and obesity. Understanding the systems that control eating behavior might provide a foundation for the treatment and possible prevention of such disorders.


Itching for an explanation.

Itching for an explanation. Itch is a distinct sensation arising from the superficial layers of skin and mucous membranes. It is elicited by histamine and probably other endogenous chemicals that excite subpopulations of unmyelinated primary afferents and spinal neurones projecting through the anterolateral quadrant to the brain. The two popular views, which propose either that itch is signalled by a labelled line system of peripheral and central itch-specific neurones or that itch is the subliminal form of pain, both fail to explain convincingly many known features. Alternative theories emphasize central processes that extract the relevant information from afferents with broad sensitivity spectra for pruritogenic and noxious stimuli. Thus, itch presents an irritating challenge for the specificity theory of somatosensation.


Suppression of programmed neuronal death by sustained elevation of cytoplasmic calcium. Chronic depolarization greatly increases the survival of many types of neurons in culture. In at least some cases this enhancement of survival consists of the suppression of programmed cell death, a type of death occurring in developing neurons deprived of sufficient neurotrophic factor support. Available evidence suggests that the effect of depolarization on survival is mediated by a sustained rise of cytoplasmic free Ca2+, apparently caused by influx of Ca2+ through voltage-gated channels. This review discusses what is known about the mechanism by which prolonged depolarization and increased intracellular Ca2+ promote survival.


Molecular messengers of olfaction.

Molecular messengers of olfaction. Our knowledge of olfactory signal transduction has been greatly clarified by several recent advances. Molecular cloning has revealed a large family of putative odorant receptors localized to olfactory epithelium that display a seven-transmembrane-domain motif suggesting an association with G proteins. Very potent and rapid enhancement of both adenylyl cyclase and phosphoinositide turnover has been demonstrated in response to odorants both in isolated olfactory cilia and primary olfactory receptor neuronal cultures. A Ca(2+)-calmodulin-dependent phosphodiesterase has been localized to olfactory cilia. A key role for Ca2+ is evident from many investigations. More recently, odorants have also been shown to affect the levels of cGMP in olfactory receptor neurons. The involvement of multiple second messengers may provide mechanisms for both fine-tuning and desensitization of olfaction.


[Effect of kontrikal on the course of experimental nonspecific phacogenic uveitis]. Nonspecific phacogenic uveitis (NPGU) was simulated in 21 chinchilla rabbits to follow the time course of the blood serum proteolytic activity and the principal protease inhibitors (alpha 1 inhibitor and alpha 2 macroglobulin) in various stages of the inflammatory process. Effects of contrykal, a synthetic protease inhibitor, on these parameters and the course of the inflammation was also under study. The results evidence that i.v. and subconjunctival contrykal administration reduced the periods of NPGU treatment 1.5-2 times.


A preliminary map of epitopes on envelope glycoprotein E1 of HCV strain Brescia. Monoclonal antibodies (MAbs) directed against envelope glycoprotein E1 (gp51-54) of hog cholera virus (HCV) strain Brescia have been shown to recognize four different antigenic domains A, B, C and D. Epitopes of within domain A have mainly been found conserved among HCV strains, whereas epitopes within domains B, C and D are not conserved. We used transiently expressed hybrid E1 genes of HCV strains Brescia and "C" to map the non-conserved epitopes on E1. Epitopes in domains B and C are located within the ultimate N-terminal 104 amino acids. The non-conserved subdomain A3 is most probably located between domains B/C and a hydrophobic region, which is highly conserved between HCV strains Brescia and "C". The conserved subdomains A1 and A2 are probably located in the vicinity and C-terminally of this conserved, hydrophobic region, which is near the centre of the E1 amino acid sequence.


[Pathogenetic therapy of spontaneous corneal perforation after extracapsular cataract extraction]. The author presents a case with spontaneous perforation of the cornea after extracapsular cataract extraction in a female patient with rheumatoid arthritis. Systemic disorganization of the connective tissue and elevated collagenase activity essentially contribute to the pathogenesis of corneal perforation. Therefore, besides antibacterial drugs, collagenase inhibitors, 3% aqueous solution of cysteine, antimeasles gamma-globulin, and a regeneration stimulant, 5% ascorbic acid aqueous solution, were used in the treatment. The pathogenetic therapy was conducive to early healing of the corneal defect and helped save the eye and preserve its good function (0.8 s + 10.0 diopters).


Antigen selection and presentation to protect against transmissible gastroenteritis coronavirus. The antigenic structure of the S glycoprotein of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) has been determined and correlated with the physical structure. Four antigenic sites have been defined (A, B, C, and D). The sites involved in the neutralization of TGEV are: A, D, and B, sites A and D being antigenically dominant for TGEV neutralization in vitro. These two sites have specific properties of interest: site A is highly conserved and is present in coronaviruses of three animal species, and site D can be represented by synthetic peptides. Both sites might be relevant in protection in vivo. PRCV does not have sites B and C, due to a genomic deletion. Complex antigenic sites, i.e., conformation and glycosylation dependent sites, have been represented by simple mimotopes selected from a library expressing recombinant peptides with random sequences, or by anti-idiotypic internal image monoclonal antibodies. An epidemiological tree relating the TGEVs and PRCVs has been proposed. The estimated mutation fixation rate of 7 +/- 2 x 10(-4) substitutions per nucleotide and year indicates that TGEV related coronaviruses show similar variability to other RNA viruses. In order to induce secretory immunity, different segments of the S gene have been expressed using a virulent forms of Salmonella typhimurium and adenovirus. These vectors, with a tropism for Peyer's patches may be ideal candidates in protection against TGEV.


Haemorrhagic disease of lagomorphs: evidence for a calicivirus. Studies on the aetiological agents of rabbit haemorrhagic disease (RHD) and European brown hare syndrome show that the viruses responsible for these infections can be placed in the family Caliciviridae. Established members of this group are vesicular exanthema virus (prototype), San Miguel sea lion virus and feline calcivirus. The human hepatitis E virus and the Norwalk agent may soon be included. The RHD virus genome consists of a positive stranded RNA molecule composed of 7437 nucleotides. A major subgenomic RNA of 2.2 kb, colinear with the 3' end of the genomic RNA, can also be recovered from infected liver tissue, and both RNAs are enclosed within viral capsids formed by a single major protein of approximately 60 kDa. Electron microscopic examination of organ suspensions from diseased animals shows two types of particle; 35-40 nm complete virions have the regularly arranged cup-shaped depressions typical of calcivirus morphology, and 23-25 nm smooth particles resulting from degradation of the outer surface structures of the complete virions.


[Conditions for dextranase formation by Paecilomyces lilacinus]. Induced formation conditions of dextranase by Paecilomyces lilacinus were investigated. Effect of various carbohydrates on dextranase formation was examined, dextran was the best C-source and as an inducer. The effect of dextran with different molecular weight (from 17.2 to 1000 kD) on dextranase formation was compared, productivity of dextranase increased with increase of dextran molecular weight. When dextran of 1000 kD was used as C-source. The enzyme formation was 40% higher than that 17.2 kD dextran. When other sugars were separately added to the medium with dextran, the enzyme formation was repressed. Besides C-source, the other optimum conditions of dextranase formation were as follows: N-source, beef peptone; medium initial pH, 6.0-7.0; culture temperature, 28 degrees C; inoculum amount about 10%, and the organism was cultivated for 6 days on 200 r/min shaker in 250 ml flasker with 50 ml medium.


[A unique marker of Pseudomonas from the first group of rRNA homology--selective sensitivity to the bacteriostatic action of barium ions]. On the basis of the study of the bacteriostatic action of chlorides and nitrates of barium and 24 other metals on 18 Pseudomonas species of all groups of rRNA homology and on 49 bacterial species belonging to 25 other genera, unique selective sensitivity to the bacteriostatic action of Ba2+ ions has been established in Pseudomonas of the first group of rRNA homology. The marker of barium sensitivity is in line with changes in the classification of Pseudomonas and is of interest for their further more precise systematization. The test for barium sensitivity of bacteria helps simplify the identification of Pseudomonas of the first group of rRNA homology and makes the identification more accurate.


[The fractional composition of immunoglobulin preparations studied by gel filtration on different carriers and by high-pressure liquid chromatography]. The fractional composition of immunoglobulin preparations produced by different manufacturing enterprises of this country has been studied by gel chromatography in columns packed with different carriers (Sephadex G-200 and ultragel AcA-34) and by high-performance liquid chromatography (HPLC). This study has revealed the nonstandard character of immunoglobulin preparations produced according to the same technological procedure (modified Cohn's method). The fractionation of immunoglobulins on different carriers with the use of different methods has yielded similar results confirmed by the statistical processing of the data. The results obtained in the study of the fractional composition of immunoglobulin preparations evidence that gel filtration with the use of ultragel and HPLC have greater resolving capacity in comparison with the method of gel filtration on traditionally used Sephadex G-200.


Evaluation of stroke rehabilitation by randomized controlled studies: a review. In all, 22 reports of 20 randomized, controlled rehabilitation studies were evaluated. In 18 of these, the design of the trial was parallel, with a cross-over format being employed in the remaining 2 instances. Seven studies related to intensive rehabilitation during the early post-acute period. In six others, specific techniques--sometimes associated with traditional physiotherapy procedures--were compared: biofeedback, perceptual retraining, and amphetamine treatment. Eight experiments evaluated speech therapy in aphasia. Frequently, methodological considerations limited the interpretation of the results. The review showed that: 1) as regards activities of daily life and motor function, differences as between rehabilitation in stroke units on the one hand and non-rehabilitation or rehabilitation in medical wards on the other, were detected in relatively few quality studies and remained particularly inconclusive insofar as life in the home environment was concerned; 2) rehabilitation for aphasia and perceptual dysfunction proved effective for at least several months after acute stroke; 3) in general, examination of the reports cited revealed no differences in the effects of biofeedback and perceptual retraining vs conventional physiotherapy; 4) rehabilitation, whether administered by specialists or amateur caregivers purpose-trained by specialists, proved equally effective for aphasia. These conclusions constitute a valuable basis for the development and evaluation of modern rehabilitation programs for stroke patients.


Short-term restraint stress and s.c. saline injection alter the tissue levels of substance P and cholecystokinin in the peri-aqueductal grey and limbic regions of rat brain. Rats were exposed to short-term restraint (held by the tail for 1 min), injected s.c. with saline or subjected to the combination of these treatments. Fifteen and 30 min after these treatments the means serum corticosterone level was significantly increased by more than four times, compared to rats taken directly from their home cages, indicating a stress response. In the peri-aqueductal grey, the level of substance P-like immunoreactivity was increased by 45% (P < 0.01) and 65% (P < 0.01) 30 and 60 min after the combined treatment, respectively. Significant increases of the level of substance P-like immunoreactivity in the peri-aqueductal grey were also found after restraint only and after a s.c. saline injection. Similar, but less marked, changes in the level of cholecystokinin-like immunoreactivity in the PAG were also seen. In the accumbens a significantly decreased level of substance P-like immunoreactivity was encountered at 15 and 30 min after treatment, while the levels of cholecystokinin- and neuropeptide Y-like immunoreactivity were not significantly changed. In other regions studied, no effects on peptide levels were seen. The changes in peptide levels had a time course similar to that of the increase in serum corticosterone. Also the successive removal of rats from a common cage was found to increase significantly the serum corticosterone and the substance P-like immunoreactivity in the peri-aqueductal grey in the animals that were taken late in sequence from the cage.(ABSTRACT TRUNCATED AT 250 WORDS)


[Whipple's disease].

[Whipple's disease]. The authors describe a case of Whipple's disease, characterized by arthralgias, chronic diarrhea and weight loss. The diagnosis was established on clinical, laboratorial and radiological grounds and confirmed histologically, through a duodenal biopsy. Rapid improvement occurred, soon after the beginning of antibiotic therapy.


An indirect method of measuring widths suitable for automated bone histomorphometry. Orthogonal intercepts from random test lines (OIr), uniformly distributed intercepts (OIu), and areas and lengths (Ar/Le) were measured manually and by computer to determine individual profile and sample widths of artificial profiles and human osteoid seams. Individual widths were equal by Ar/Le and OIu methods. The means of individual profile widths (all methods), of all orthogonal intercepts (OIu and OIr), or of profile widths weighted in proportion to their lengths (Ar/Le) were also equal. Ar/Le and OIu had smaller variance than OIr. Discrepant individual OIu and Ar/Le widths in digitized images were corrected by thresholding and did not significantly affect sample means. Unweighted Ar/Le sample means were 15-44% lower than weighted means. Distributions of osteoid seam widths were not normal, and all but one had more than one mode. We conclude that the Ar/Le method is comparable to direct orthogonal intercept methods and suitable for automated histomorphometry.


Glycerol injection to the rat trigeminal nerve: histological and immunohistochemical studies. The effect of topical glycerol injection into the rat trigeminal nerves was investigated histologically and immunohistochemically. Anhydrous glycerol was injected into the preganglionic portion of the trigeminal nerves via a ventral approach. Extensive myelin swelling and axonolysis were observed in the rats killed 1 and 2 weeks after glycerol injection. Numerous inflammatory cells were seen especially in the animals sacrificed 1 week after surgery. Myelin disintegration continued up to 4 weeks after glycerol injection. In normal and saline injected sham operated nerves, calcitonin gene-related peptide (CGRP)- and substance P(SP)-like immunoreactivities were densely localized in the nerve fibers. A marked decrease in both CGRP- and SP-like immunofluorescence was seen in the nerves after glycerol injection. The remaining nerve fibers often had blunt endings with increased fluorescence. Swollen and winding structures were also found. These immunohistochemical changes were observed in the rats killed 1 and 2 weeks following surgery. A similar change but of lesser degree was seen in the 4-week-animal. The present study suggests that topical glycerol injection into the trigeminal nerve induces degeneration of the nerves immunoreactive to CGRP and SP. These changes emphasize the putative functional implications of the peptides in relieving the pain of trigeminal neuralgia after topical glycerol injection.


Cerebrovascular effects of substance P after experimental subarachnoid haemorrhage. The vasoactive effects of substance P (SP), as well as the content of cyclic guanine monophosphate (cGMP), were determined in the rabbit basilar artery after subarachnoid haemorrhage (SAH). Out of 47 rabbits, 24 were subjected to a SAH, induced by injecting 5 ml of autologous arterial blood into the cisterna magna; 23 were used as controls. In 20 animals (10 SAH and 10 controls), isometric tension recording of isolated rings of the basilar artery--dissected 2 days after SAH--was employed to assess the dose-dependent vasodilatation to SP (10(-10) to 10(-6) M) after precontraction with serotonin (10(-8) to 10(-5) M). In 15 animals (8 SAH and 7 controls), the basal cGMP content was measured in the basilar artery 2 days after SAH. In the other 12 animals (6 SAH and 6 controls), the increase in cGMP content was measured in the basilar artery after a 10-minute incubation with SP (10(-6) M). SP caused significantly less dilatation in animals subjected to SAH than in controls, especially for concentrations between 10(-9) and 10(-6) M (p < 0.001). The cGMP content in the arteries 2 days after SAH was significantly lower than in control arteries (31.5 +/- 7.3 against 57.3 +/- 4.3 pmoles/g tissue). In the preparations incubated with SP, the increase of cGMP was 440 +/- 115% in the control arteries, and only 97 +/- 30% in the arteries after SAH. It is concluded that the vasodilator activity of SP is significantly impaired after SAH.(ABSTRACT TRUNCATED AT 250 WORDS)


[Intraarterial COMPA (cis-diammine-dichloroplatinum (II), vincristine, methotrexate, peplomycin, adriamycin) chemotherapy for bladder cancer]. Seventeen patients with bladder cancer were treated with semiselective intraarterial COMPA chemotherapy. One course of COMPA consisted of 20 mg/m2 cis-diammine-dichloroplatinum (CDDP) on days 4 and 5, 0.6 mg/m2 vincristine (VCR) (Oncovin) on days 1 and 2, 5 mg/m2 methotrexate (MTX) on days 2 and 3, 5 mg/body peplomycin (PEP) on days 1, 2 and 3, and 15 mg/m2 adriamycin (ADM) on day 4. These drugs were injected every 2 or 3 weeks through a polyurethane catheter the tip of which was placed just proximal to the aortic bifurcation and during injection both thighs were tied with a pressure of over 250 mmHg. From 2 to 6 courses (mean, 4.4 courses) were administered. Of the 17 patients, 4 achieved complete remission, 10 achieved partial remission and 3 showed no change. After this COMPA chemotherapy eight patients were able to retain their bladders while seven underwent immediate radical cystectomy. The adjuvant COMPA chemotherapy for two patients with pelvic metastasis after radical cystectomy showed good results. Mild degrees of anorexia, nausea, vomiting, hair loss, numbness of fingers and/or toes, leukopenia and intestinal paralysis were observed. Instrumental troubles were seen in two cases; one involved dislocation of the tip of the catheter, the other was infection of the reservoir. Intraarterial COMPA chemotherapy is effective for neoadjuvant therapy of invasive bladder cancer, bladder-preserving treatment and adjuvant therapy of pelvic metastasis.


[Transurethral resection of the prostate in the urological management for patients with stroke]. We discuss the transurethral resection of the prostate (TUR-P) on 40 patients in the chronic stage of stroke, all of whom were refractory to conservative managements of urinary disturbance. All patients, between 35 and 89 years old (mean: 52.6 years), had only one episode of stroke and were diagnosed as benign prostatic hypertrophy or bladder neck contracture that appeared to cause urinary disturbance in these patients. At six months after TUR-P, all except for one patient, who needed an indwelling catheter due to a reinfarction, were catheter free. Of these cases 36 (92%) obtained independent micturition and did not develop urinary incontinence except transiently postoperatively. Two cases with impaired mobility and one case with progressive senile dementia required helpmates and/or a commode and so forth postoperatively. It is concluded that in chronic stroke patients TUR-P is recommended for those with benign prostatic hypertrophy or bladder neck contracture.


[Value of prostate-specific antigen measurements with newly developed enzyme immunoassay (MARKIT-M PA)]. Serum prostate-specific antigen (PSA) levels in patients with prostate cancer and benign prostate hypertrophy (BPH) were investigated with a newly developed enzyme immunoassay (MARKIT-M PA, Dainippon Pharmaceutical Co. Ltd., Osaka, Japan). Sensitivity of the assay system is 0.5 ng/ml and the detection range is 0.5-100 ng/ml. There was a high linear correlation (r = 0.987) between the assay and MARKIT-F PA, and values obtained with the assay were almost equal to those yielded by MARKIT-F PA assay. Using the BPH group as a negative control, the upper cut-off value in BPH patients was determined to be 3.6 ng/ml. Of the 48 patients with untreated prostate cancer, 77% was detectable by means of MARKIT-M PA assay. Using the BPH group as a negative control, specificity and efficiency were 93% and 86%, respectively. In another group of 27 BPH patients whose blood samples were taken immediately after digital prostatic examination, PSA was elevated in 15%. During follow-up of prostate cancer patients, PSA was elevated in 82% at the time of clinically detectable progression. In 15 patients whose disease was clinically well controlled, all levels of PSA were observed to be negative. These findings suggests that detection of serum PSA with this assay is of great use both in the diagnosis and monitoring of prostate cancer patients.


[A study of bladder function in benign prostatic hyperplasia]. A study of bladder function was performed on 106 patients with benign prostatic hyperplasia (BPH), 21 of whom had cerebrovascular disease (CVD). The incidence of overactive bladder in 21 patients with CVD was 57% and was significantly higher than the 25% in 86 patients without CVD. The incidence of cases having residual urine of over 100 ml, was in the order of low compliance bladder, overactive bladder and normal bladder. The mean value of maximum voiding pressure in overactive bladder was higher than that in normal bladder. The high pressure was improved after the operation for BPH. Improvement of overactive bladder after the operation was seen in 9 of 13 patients without CVD while in 2 of 6 patients with CVD.


[BEP (bleomycin, etoposide, cisplatin) therapy for testicular tumors]. We describe our experience with BEP (bleomycin, etoposide, cisplatin) therapy as chemotherapy for testicular tumors in 11 patients. Eight were non-seminomatous testicular cancer patients and 3 were seminoma patients. Three of 8 non-seminomatous testicular cancer patients had no evident metastasis and BEP therapy was performed for prophylaxis of recurrence. Other 5 non-seminomatous testicular cancer patients and 3 seminoma patients had metastatic lesions and BEP therapy was performed to cure these metastatic lesions. Ten of our 11 patients are living and disease-free. One non-seminomatous testicular cancer patient who had brain, lung, eye and bladder metastases and had an extremely elevated human chorionic gonadotropin (hCG) level responded only partially and died later due to disease progression. Side effects in most patients were nausea, vomiting, alopecia and leucopenia and all these side effects were reversible. Neuromuscular toxicity such as paresthesia or abdominal cramp that is sometimes encountered in PVB (cisplatin, vinblastine, bleomycin) therapy was not seen in our patients. Our results support the concept that BEP therapy is better than PVB therapy as an initial chemotherapy for testicular tumors.


[Successful management of choriocarcinoma of the testis with brain metastasis: a case report]. A 32-year-old Japanese male consulted a clinic complaining of gynecomastia. Right painless scrotal swelling was also detected. Right high orchiectomy was performed, then the surgical specimen was histopathologically confirmed as choriocarcinoma and mature teratoma. The imaging revealed cerebral, pulmonary, retroperitoneal metastases. After 3 courses of combination chemotherapy with cisplatin, etoposide and peplomycin (PEP therapy), the brain metastasis completely disappeared and the serum titer of the tumor markers such as beta-HCG became normal. The regression rates of lung and retroperitoneal metastases were 68% and 27%, respectively. Therefore, retroperitoneal lymph node dissection was performed. After the 5th course of PEP therapy, lung metastases disappeared completely. Until the present, no evidence of disease has persisted. The PEP therapy, which is a salvage therapy for refractory testicular cancer, was performed as first-line chemotherapy in this case. It was an excellent modality against choriocarcinoma, along with the surgical treatment.


Bacterially expressed core and envelope proteins of human immunodeficiency virus type-1 (HIV-1): comparative evaluation in detection of type-specific antibodies. Recombinant proteins derived from immunodominant conserved domains of HIV-1 env and gag genes were synthesized in E. coli. An immunoblot system using total cell lysates was employed for the analysis of recombinant bacterial clones. Together 427 serum samples obtained from asymptomatic anti-HIV seropositive individuals, AIDS patients, healthy donors and persons suffering from various conditions were comparatively evaluated for the presence of HIV-1 antibodies using recombinant peptides and commercially available western blot (WB) and ELISA assays. The recombinant antigen product of plasmid pEX41 was found to be superior, with respect to sensitivity and specificity, to the viral gp41 which represents a diagnostically important constituent of the WB.


Identification of a cell membrane receptor for interferon induction by poly rI:rC. PR-RK, a cell line derived from rabbit kidney cells (RK-13), was insensitive to the cytotoxic effect and interferon (IFN) inducing activity of the copolymer of riboinosinic and ribocytidylic acid (poly rI:rC). However, PR-RK was sensitive to the cytotoxic effect of the copolymer of riboadenylic and ribouridylic acid (poly rA:rU). Comparison of PR-RK cells and RK-13 cells by cytofluorometric analysis revealed that the binding of poly rI:rC was considerably reduced on PR-RK cells. These results suggested that the receptor for poly rI:rC might be different from the receptor for poly rA:rU, and this difference could provide a basis for the identification of the dsRNA receptor on cell surface. Western blot analysis of the components of cell membrane fraction prepared from RK-13 cells was performed by using a monoclonal antibody, which binds to cell membrane of RK-13 cells but not to PR-RK cells, and which blocks IFN induction by poly rI:rC in RK-13 cells. The 60K protein was identified as one of the poly rI:rC receptor protein.


Interferon-neutralizing or enhancing activities in hybridoma cell fluids after in vitro immunization. Several hybridomas supernatants capable of interferon beta (IFN-beta) or "IFN epsilon" ("IFN-eps") neutralizing or enhancing activities were obtained after in vitro immunization of BALB/c and C57 mice spleen cells and their fusion with Sp2/0 plasmacytoma cells. Besides rather low anti-IFN-beta or "eps" antibody secretion several cloned hybridoma fluids contained a factor potentiating anti-viral activity of the both IFNs. It is speculated that this activity is due to production by some hybridomas of another lymphokine.


Autosomal dominant insulin resistance syndrome due to postbinding defect. We investigated a family in which at least 4 men in 3 generations had a syndrome of obesity, mild mental retardation, delayed puberty, macroorchidism, acanthosis nigricans, hyperinsulinemia, and later overt insulin-resistant diabetes mellitus (non-insulin-dependent diabetes mellitus, NIDDM). The patients have markedly curly scalp hair, deficient face and body hair. Their teeth were healthy and normal in size and position. The clinical and biochemical findings and characteristics of the insulin receptors investigated in fibroblasts are reported. There was normal insulin binding to fibroblasts in the 2 brothers and their father. However, insulin-stimulated RNA synthesis was decreased as compared to that of normal control individuals. These findings suggest a postbinding defect of insulin action. The pedigree documents an autosomal dominant mode of inheritance. The diagnosis is of practical importance since it enables medical supervision of gene carriers in a preclinical state of atherosclerotic complications and overt diabetes. The findings in this family have relevance also to the explanation of familial mild mental retardation and to the study of different forms of insulin resistance due to a disturbance in biosignal transfer.


Beta-adrenergic regulation of H+ secretion by cultured outer medullary collecting duct cells. The effects of the beta-adrenergic agonist isoproterenol (Iso) on cells of the inner stripe portion of the rabbit outer medullary collecting duct (OMCDi) grown in primary culture were examined using whole cell patch-clamp techniques and measurements of intracellular pH (pHi) and Ca2+. Iso (10(-6) M) increased the cellular Cl- conductance, and this effect was mimicked by treatment of the cells with dibutyryladenosine 3',5'-cyclic monophosphate (cAMP, 10(-5) M) or protein kinase A (PKA, 0.4 U/ml). Iso did not alter the baseline pHi, but it did increase the activity of both the Cl-/HCO3- antiporter and the H(+)-adenosinetriphosphatase (H(+)-ATPase). The increase in Cl-/HCO3- antiporter rate was mimicked by dibutyryl-cAMP plus 3-isobutyl-1-methylxanthine (cAMP + IBMX, 10(-4) M + 10(-5) M). However, the Iso-induced stimulation of the H(+)-ATPase activity was not mimicked by cAMP + IBMX. Measurements of intracellular Ca2+ showed that Iso also increased intracellular Ca2+ levels. This response was not dependent on extracellular Ca2+, nor did cAMP + IBMX appreciably alter intracellular Ca2+. Consequently, we postulate that beta-adrenergic agonists are potential stimulators of OMCDi H+ secretion. These agonists stimulate cellular HCO3- efflux through a signal transduction pathway involving cAMP and PKA. However, a different signal transduction pathway appears to mediate the stimulation of cellular H+ efflux. This second pathway may involve an elevation of intracellular Ca2+.


Extent and course of glomerular injury in human membranous glomerulopathy. Glomerular permselectivity and dynamics were evaluated serially in 14 nephrotic patients with membranous glomerulopathy (MG). Analysis of transglomerular dextran sieving, before and again after proteinuria remitted, revealed persistent depression by 60-80% of glomerular pore density and the two-kidney ultrafiltration coefficient, Kf. The glomerular filtration rate was lowered by half on each occasion. Morphometric examination of glomeruli in a second group of 16 nephrotic patients with MG revealed a low prevalence of glomerulosclerosis (5 +/- 3%) and a twofold increase in filtration surface due to marked glomerular hypertrophy. Presumably, widening by threefold of the basement membrane and/or epithelial podocytes accounted for the computed reduction in ultrafiltration capacity. There was no correlation between glomerular structure and the subsequent course of MG over the ensuing 24-96 mo. Rather, a twofold expansion of the interstitial compartment predicted those who went on to exhibit progressive renal insufficiency. We conclude that increasing resistance to water flow by walls of patent and perfused glomerular capillaries is the proximate cause of progressive renal insufficiency in MG.


Force-frequency relations and response to ryanodine in failing rabbit hearts. Force-frequency relations were studied in an isolated-perfused rabbit heart model. Heart failure was induced by a double volume plus pressure overload. Studies were performed at the early stage of heart failure when basal ventricular function was not decreased. The normal positive staircase induced by pacing in control hearts (CH) was replaced by a negative staircase in failing hearts (FH) with an increase in end-diastolic pressure for increased heart rates in FH. Postpacing potentiation and postextrasystolic potentiation (PESP) were significantly reduced in FH as compared with CH. Ventricular function decreased by 60% in both CH and FH under ryanodine with similar dose-response curves. Postpacing and PESP disappeared under ryanodine in CH and in FH with a reversal of the negative staircase in FH. The abnormal force-frequency relations observed in heart failure are thus attributed to sarcoplasmic reticulum dysfunction. Basal ventricular function during spontaneous heart rate may be normal in the early stage of heart failure, but sarcoplasmic reticulum dysfunction produces abnormalities in ventricular function when heart rate is abruptly modified, particularly during tachycardia.


Ca(2+)-dependent and Ca(2+)-permeable ion channels in aortic endothelial cells. We investigated whether osmotic stress would activate specific ion channels in bovine aortic endothelial cells (BAECs). In isotonic medium (290 mosmol/kgH2O), cell-attached patch recordings contained both 165-pS K+ channels activated by depolarization and 40-pS K+ channels activated by 200 nM bradykinin. These inwardly rectifying K+ channels were activated by raising "cytoplasmic" Ca2+ in inside-out patches. BAEC exposed to hypotonic bath (220 mosmol/kg) exhibited a 20% decrease in intracellular K+ content within 5 min. Cell-attached patches revealed biphasic K+ channel activation with hypotonic exposure; initial activation of 165- and 40-pS K+ channels (1-3 min) was followed by a delayed but sustained reactivation of both K+ channels (> 5 min). The delayed reactivation phase was dependent on the presence of external Ca2+ and was attenuated by 10 microM gadolinium. A 28-pS nonselective cation channel (NSCC), which conducted inward Ca2+ current, was also detected during hypotonic exposure. This NSCC was stimulated by hyperpolarization and was blocked by 10 microM gadolinium. In BAEC 1) hypotonic exposure activates Ca(2+)-dependent, 165- and 40-pS K+ channels biphasically; 2) the initial phase is independent of external Ca2+, while the delayed phase requires external Ca2+; and 3) Ca(2+)-permeable, 28-pS NSCCs stimulated by membrane hyperpolarization provide a pathway for external Ca2+ influx.


Microvascular ischemia-reperfusion injury in striated muscle: significance of "reflow paradox". Ischemia-reperfusion (I/R)-induced microvascular injury is characterized by capillary "no-reflow" and reflow-associated events, termed "reflow paradox," including leukocyte-endothelium interaction and increase in microvascular permeability. The major objectives of this study were 1) to elucidate the significance of reflow paradox after 4 h of tourniquet-induced ischemia in striated muscle and 2) to determine the role of reactive oxygen metabolites in the pathogenesis of reflow paradox-dependent microcirculatory alterations. By use of in vivo fluorescence microscopy in a striated muscle preparation of hamsters, leukocyte-endothelium interaction in postcapillary venules and macromolecular extravasation from capillaries and venules were quantified before ischemia and after 30 min, 2 h, and 24 h of reperfusion. I/R elicited marked enhancement (P < 0.01) of leukocyte rolling during initial reperfusion and a 20-fold increase of leukocyte adherence (P < 0.01) lasting for the entire postischemic reperfusion period (n = 7). These phenomena were accompanied by significant leakage (P < 0.01) of macromolecules from capillaries and in particular from postcapillary venules (n = 9). Both superoxide dismutase (SOD, 20 mg/kg body wt, n = 7) and allopurinol (50 mg/kg body wt, n = 7) were effective in attenuating I/R-induced leukocyte rolling and adherence. In addition, microvascular leakage was significantly reduced by allopurinol (n = 9) and completely abolished by SOD (n = 9) (P < 0.01). These results support the concept that reactive oxygen metabolites contribute to I/R-induced reflow paradox, resulting in leukocyte accumulation, adherence, and increase in microvascular permeability.


Interferons and central regulation of feeding. Interferons (IFNs) are immunomodulators with neuromodulatory activities. To study the effects of IFNs on the central regulation of feeding, rats were subjected to various applications. The results show the following. 1) Intracerebroventricular microinfusion of rat IFN (15-225 IU/rat) decreased short-term (2-h) food intake in rats. Computerized analysis of behavioral patterns demonstrated a reduction of meal size and meal duration, whereas meal frequency slightly increased. Nighttime and total daily food intakes were not significantly affected. 2) Short-term food intake suppression by intracerebroventricular rat IFN was accompanied by a small increase in cerebrospinal fluid and rectal temperatures. 3) Intracerebroventricular microinfusion of heat-treated rat IFN or of recombinant human interferon-alpha (rhIFN-alpha) did not affect food intake. Only one dose of rhIFN-gamma (400 ng/rat) decreased 2-h food intake. These results are consistent with the species specificity to the effects of IFNs. 4) Peripheral administration of rat IFN in doses equivalent to those administered centrally had no effect on food intake. The results suggest that IFN acts directly in the central nervous system to decrease short-term feeding.


A specific stain for the detection of nonheme iron proteins in polyacrylamide gels. Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well.


Cell type-specific anti-human immunodeficiency virus type 1 activity of the transactivation inhibitor Ro5-3335. The drug Ro5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one] inhibits human immunodeficiency virus type 1 (HIV-1) gene expression at the transcriptional level through interference with Tat-mediated transactivation (M.-C. Hsu, A. D. Schutt, M. Holly, L. W. Slice, M. I. Sherman, D. D. Richman, M. J. Potash, and D. J. Volsky, Science 254:1799-1802, 1991). We confirmed this specific inhibitory effect in a quantitative bioassay based on transactivation of a chimeric gene comprising the HIV-1 long terminal repeat promoter fused to the lacZ gene of Escherichia coli and transfected in a HeLa cell line expressing Tat. Ro5-3335 was found to inhibit HIV-1 long terminal repeat-driven lacZ gene expression at a 50% inhibitory concentration of 0.5 microM. The in vitro anti-HIV-1 activity of Ro5-3335 was highly dependent on the nature of the host cells. The highest selectivity index, 50, was found in phytohemagglutinin-stimulated peripheral blood lymphocytes. The selectivity index was between 1 and 10 in the CD4+ T-cell lines CEM, MOLT-4 (clone 8), and HUT-78. In MT-4 and MT-2 cells, Ro5-3335 had no inhibitory effect on HIV-1 replication. The absence of anti-HIV-1 activity of Ro5-3335 in MT-4 cells was confirmed by using different parameters of virus replication and different multiplicities of infection. In persistently HIV-1-infected HUT-78/IIIB/LAI cells, Ro5-3335 failed to demonstrate any activity at subtoxic concentrations. The cytotoxicity of Ro5-3335 was significantly lower in peripheral blood lymphocytes than in the CD4+ T-cell lines.


The role of guanosine-3',5'-bis-pyrophosphate in mediating antimicrobial activity of the antibiotic 3,5-dihydroxy-4-ethyl-trans-stilbene. The mode of action of 3,5-dihydroxy-4-ethyl-trans-stilbene (ES), an antibiotic produced by Xenorhabdus luminescens symbiotically associated with an entomopathogenic nematode, was investigated. ES was active against gram-positive and a number of gram-negative bacteria. In susceptible bacteria this antibiotic caused the inhibition of total RNA synthesis and, to a lesser extent, protein synthesis. At or above MICs, ES triggered a substantial accumulation of an intracellular regulatory compound, guanosine-3',5'-bis-pyrophosphate (ppGpp). This response was also noticed in species of bacteria which have previously not been shown to use ppGpp as a regulatory molecule. The involvement of ppGpp in antibiotic action was confirmed by using an isogenic stringent and a relaxed pair of Escherichia coli strains. The fact that the accumulation of ppGpp was correlated with the susceptibility of various gram-positive and gram-negative bacteria to ES suggests that this nucleotide is involved in the regulation of RNA synthesis and growth in all these microorganisms. Thus, inhibition of RNA synthesis via an increase in ppGpp concentrations may represent a mechanism that is prevalent among most bacteria and one that could be exploited for achieving a rapid inhibition of bacterial growth.


3'-Azido-3'-deoxythymidine resistance suppressed by a mutation conferring human immunodeficiency virus type 1 resistance to nonnucleoside reverse transcriptase inhibitors. Nonnucleoside reverse transcriptase (NNRT) inhibitors (R82913; (+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione; Cl-TIBO; and BI-RG-587, nevirapine) were used to select resistant human immunodeficiency virus type 1 (HIV-1) variants by passage in cell cultures of wild-type or 3'-azido-3'-deoxythymidine (zidovudine; AZT)-resistant strains. Similar to other NNRT inhibitors, Cl-TIBO induced a single mutation (Y181 to C) in reverse transcriptase (RT) that accounted for the resistance. BI-RG-587 induced a different mutation (V106-->A) in AZT resistance backgrounds. A series of viable HIV-1 variants was constructed by site-directed mutagenesis of the RT, which harbored multiple drug resistance mutations, including Y181 to C. HIV-1 that was co-resistant to NNRT inhibitors and 2',3'-dideoxyinosine resulted when a 2',3'-dideoxyinosine resistance mutation (L74 to V) was also present in RT. By contrast, however, the Y181 to C mutation in an AZT resistance background significantly suppressed resistance to AZT, while it conferred resistance to NNRT inhibitors. However, the V106-->A substitution did not cause suppression of preexisting AZT resistance. Since certain combinations of nucleoside analogs and NNRT inhibitors might result in the development of co-resistance, careful analysis of clinical isolates obtained during combination therapy will be needed to determine the potential significance of these observations.


Cell surface characteristics of environmental and clinical isolates of Vibrio cholerae non-O1. The cell surfaces of several toxigenic and nontoxigenic environmental and clinical isolates of Vibrio cholerae non-O1 have been examined. The environmental strains, irrespective of toxigenicity, are significantly more resistant to antibiotics and detergents than are V. cholerae O1 strains. The clinical isolates of non-O1 vibrios are as sensitive to a wide variety of chemicals as the O1 vibrios. The environmental non-O1 strains are also less susceptible to lysis when treated with protein denaturants or neutral and anionic detergents than are O1 vibrios and the clinical non-O1 strains. In contrast to O1 vibrios, the environmental non-O1 vibrios do not have exposed phospholipids in their outer membranes. These features of the cell surfaces of environmental non-O1 vibrios might have a role in the better survival of these organisms under environmental fluctuations.


[Sclerosis and carcinogenesis].

[Sclerosis and carcinogenesis]. Morphological analysis of the sclerotic changes in peripheral lung carcinoma (PLC) and nephrosclerosis in renal-cell carcinoma (RCC) established a promoting role of sclerosis in carcinoma development. The pneumosclerosis role as a background process in the PLC development is proved by the following facts: high proportion (83%) of the carcinoma in the scar among PLC; identity of the scar collagen composition in PLC and that in metatuberculosis and metapneumonic pneumosclerosis foci; detection of metatuberculosis foci in 75% of PLC; the presence of the precancerous changes in the epithelium entrapped in the pneumosclerotic foci, not only with signs of morphological atypia, but with the disturbance of nuclear DNA and cellular oncogene expression as well. The association of RCC with nephrosclerosis is shown by a high proportion (82.7%) of the RCC development against the background of nephrosclerosis; the dependence of the so-called cortical adenoma development on the degree of nephrosclerosis; epithelial proliferation in the nephrosclerotic foci with the appearance of undifferentiated cells with the altered DNA content and the expression of cytokeratins and vimentine. Carcinoma morphogenesis against the background of sclerosis may be described as follows: development of sclerosis (focal and/or diffuse), the appearance of the focal epithelial hyperplasia in the scar, dysplasia or adenoma and finally carcinoma.


Prostatic specific antigen and immunoglobulin binding factor in human seminal plasma and prostate. Antibodies raised against prostatic specific antigen (PSA) and immunoglobulin binding factor (IgBF) of human seminal plasma (SP) were used to localize the antigens in various tissues by Western blot. Both antigens were found only in the prostate, including benign prostatic hypertrophy and prostatic adenocarcinoma. The polyclonal anti-PSA antibodies stained five prostatic protein bands with estimated M(r) values of 10, 14, 22, 25, and 33 kD, whereas anti-IgBF antibodies stained a single 16-kD protein. No cross-reaction occurred between the two antibodies. When anti-PSA antibodies were used an additional protein with an estimated M(r) of 35 kD was detected in the extract of benign prostatic hypertrophy, but not with normal prostate or prostatic cancer. When SP and prostatic proteins were analyzed by SDS-PAGE under nonreducing condition and immunoblot with both antibodies, immunoreactive proteins with estimated M(r) of 125 and 140 kD, respectively, were stained, suggesting that both factors may be produced as an aggregated precursor molecule. Since IgBF was found only in the prostate, this component may be useful as a marker of prostatic tissue.


Insulin-like growth factor binding proteins of equine serum. Ligand blotting analysis of serum from the horse using radiolabelled IGF-I revealed a protein at 96 kDa which was not present in serum from goat, cow, sheep, deer or donkey. These latter species all displayed five labelled bands in the range 24 to 41 kDa. Conversely, these were only weakly labelled in serum from the horse. Size exclusion chromatography of horse serum pre-incubated with radiolabelled IGF-I revealed reduced binding in the 130-kDa peak compared with goat plasma, and ligand blotting analysis indicated the 96-kDa protein was present in this peak. The 96-kDa protein from horse serum binds IGF-I and IGF-II specifically and appears to be unique to this species. The nature of this protein is at present unknown.


Enzymatic action of basic arginine amidases in human seminal plasma. Three basic arginine amidases with different affinities to lima bean trypsin inhibitor (LBTI) and aprotinin affinity columns were separated in the middle molecular weight (MMW) preparation obtained from Cellulofine GCL-2000 gel filtration of CM-cellulose adsorbed human seminal plasma and were tentatively called basic human seminal plasma arginine amidase-L (BHSAA-L, with affinity to LBTI), -A (BHSAA-A, with affinity to aprotinin), and -TH (BHSAA-TH, without affinity to either). Some enzymatic properties were measured, including Ki values of LBTI and human seminal plasma proteinase inhibitor (HSP-PI) toward present enzymes. The Ki values of LBTI toward BHSAA-L and -TH were lower than those of HSP-PI and no Ki values for LBTI toward BHSAA-L were observed. The Km values of BHSAA-L and -A to some tripeptidyl-p-nitroanilide substrates seemed relatively lower than that of BHSAA-TH.


Nuclear matrix-associated NMN adenylyltransferase activity in human placenta. This paper presents data about the presence of the NMN adenylyltransferase at the nuclear matrix level of human placenta nuclei. It was found that 40-45% of the activity (depending on the extraction procedure) referred to the total nuclear NMN adenylyltransferase was tightly associated with this subnuclear compartment. The matrices purified by two different procedures exhibited DNA, RNA and protein contents comparable with those described in literature. Extensive digestion of human placenta nuclei with DNase I was not able to solubilize the NMN adenylyltransferase activity. Therefore, the data we present are consistent with the conclusion that a part of the total nuclear NMN adenylyltransferase is associated with the nuclear matrix.


Expression of scavenger receptors on renal cell carcinoma cells in vitro. Messenger RNAs and proteins of scavenger receptor thought to be macrophage specific protein were expressed in renal cell carcinoma (RCC) cells in vitro. Acetyl LDL was taken up into RCC cells and promoted the production of interleukin-6 (IL-6), an in vitro autocrine growth factor to proliferate the cells. These results suggested that RCC cells might have a scavenger pathway which has not yet been demonstrated except for macrophages.


The level of a transcription factor Sp1 is correlated with the expression of EGF receptor in human gastric carcinomas. The expression of the transcription factor Sp1 and EGF receptor (EGFR) was examined on human gastric carcinoma cell lines and gastric carcinoma tissues as well as corresponding normal mucosa in order to clarify the mechanism of overexpression of EGFR gene without gene amplification. All 7 carcinoma cell lines expressed Sp1 mRNA, the levels of which were closely correlated with the expression of EGFR mRNA except for one cell line. The gel retardation analysis revealed that the levels of Sp1 mRNA were parallel to its DNA binding activities. In 18 surgical cases, 12 tumors expressed Sp1 mRNA at higher levels than normal mucosa. The cases with overexpression of Sp1 showed the high level of EGFR mRNA. These results suggest that overexpression of Sp1 in tumor cells may induce overexpression of EGFR without gene amplification in human gastric carcinomas.


Cellular retinoic acid-binding protein type II is expressed in adult human osteoblasts and in adult liver. In this study we have used a reverse transcription polymerase chain reaction (RT-PCR) to demonstrate that adult primary human osteoblasts and SaOS-2, a human osteosarcoma-derived cell line with osteoblastic properties, express cellular retinol-binding protein I (CRBP I), cellular retinoic acid-binding protein II (CRABP II), and very low levels of CRABP I. We also show that CRABP II is expressed in the adult liver, which does not express CRABP I. The results suggest that CRABP II is the important isoform in the adult bone as well as in the adult liver. Since the 9-cis retinoic acid receptor (RXR) alpha previously has been shown to be expressed predominantly in the liver, CRABP II might be involved in the transport of 9-cis retinoic acid to its nuclear receptor.


Human 6-pyruvoyltetrahydropterin synthase: cDNA cloning and heterologous expression of the recombinant enzyme. 6-Pyruvoyl-tetrahydropterin synthase (PTPS) is involved in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for enzymes such as the hepatic phenylalanine hydroxylase. BH4 deficiency causes malignant hyperphenylalaninemia. We cloned the human liver cDNA encoding PTPS. The coding region for PTPS contains 145 amino acids and predicts a polypeptide of 16'387 Da. The human amino acid sequence showed a 82% identity with the rat liver sequence. Expression of the cDNA in E. coli yielded the active enzyme and showed immunoreactivity with antibodies against the rat liver PTPS. This is the basis for the molecular understanding of BH4 deficiency in patients suffering from a defect in PTPS activity.


Molecular cloning of crustacean pigment dispersing hormone precursor. The cDNA encoding the precursor of the pigment dispersing hormone (PDH) of the shore crab, Carcinus maenas, was isolated and sequenced. The precursor consists of a putative 22 amino acid signal peptide, a putative 33 residue peptide of unknown function, and the 18 amino acid mature PDH, followed by a Gly residue which serves as a possible amide donor. The deduced mature PDH amino acid sequence is identical to those of Uca pugilator and Cancer magister, previously determined by Edman degradation.


Interleukin-4 enhances in vitro mineralization in human osteoblast-like cells. The effect of human interleukin-4 (hIL-4) on mineralization in human osteoblast-like cells was investigated. Confluent cells were incubated with hIL-4 for 16 or 30 days in the presence or absence, respectively, of alpha-glycerophosphate (alpha-GP), which accelerates the mineralization process. hIL-4 (0.3 ng/ml) induced mineralization with 1.9-, 26- and 37-fold increases of hydroxyproline, calcium, and osteocalcin content, respectively, in the presence of alpha-GP. Mineralization was not induced with other cytokines, hIL-1, hIL-2, hIL-6, or mIL-4. hIL-4 also induced mineralization in the absence of alpha-GP in a manner different from that of 1 alpha, 25(OH)2 vitaminD3 (1,25(OH)2VD3). These findings suggest that IL-4 may play an important role in bone formation.


A Xenopus homologue of the human beta-amyloid precursor protein: developmental regulation of its gene expression. Complimentary DNA clones have been isolated from Xenopus larva to delineate a protein highly homologous to the human beta-amyloid precursor protein (APP). Developmental change of Xenopus APP gene expression has been analyzed with molecular probes. From early oogenesis, there is a high accumulation of maternal APP. After fertilization, the mRNA is degraded, reaching a minimum level around the gastrula stage. Then zygotic transcription appears to be initiated, and this continues during the subsequent embryonic and larval stages. Splicing patterns differ between the maternal and zygotic transcripts. The ratio of mRNA including the protease inhibitor domain (PID) sequence is extremely low for the transcript of maternal origin as compared to that for the transcript of zygotic origin. These results suggest some roles for the APP molecule in Xenopus early development.


Characterization of the human islet amyloid polypeptide/amylin gene transcripts: identification of a new polyadenylation site. Islet amyloid polypeptide (IAPP) or Amylin is synthesized by the pancreatic beta-cells. IAPP is the major component of islet amyloid in the pancreas of patients with non-insulin-dependent diabetes mellitus. We report the composition and complete nucleotide sequence of the two human IAPP mRNAs of 1.6 and 2.1 kb. A new polyadenylation site was identified and shown to be used in generation of the 2.1 kb RNA. A previously identified polyadenylation signal is assigned to the 1.6 kb RNA. We exactly determined the major transcription start site, which is used in generation of these mRNAs. Lower abundance RNAs containing sequences located further upstream in the IAPP gene were also detected.


Rat hepatocyte hyaluronan/glycosaminoglycan binding proteins: evidence for distinct divalent cation-independent and divalent cation-dependent activities. We have previously shown (Biochemistry, 29, 10425, 1990) that hepatocytes contain intracellular specific binding sites for hyaluronan (HA). Although HA-binding activity is not dependent on divalent cations, it is increased in the presence of Ca+2. Here we report that a novel photoaffinity HA derivative (ASD-HA) crosslinks specifically to different proteins in permeable cells in the presence or absence of Ca+2. With Ca+2 present, two proteins of approximately 24 kD and 43 kD were labeled. Additionally, a broad zone of specific crosslinking was observed in the region of 40-100 kD. However, in the presence of the chelator EGTA this zone was absent and the 24 and 43 kD proteins were also not cross-linked to the HA photoaffinity derivative. In the absence of Ca+2, only a 54 kD protein was specifically labeled. The results indicate that different intracellular hepatocyte proteins are responsible for the Ca+2-independent and the Ca+2-dependent binding of HA.


Effects of calyculin A on amylase release in streptolysin-O permeabilized acinar cells. The effects of the phosphatase inhibitors calyculin A and okadaic acid on amylase release from streptolysin-O permeabilized rat pancreatic acini were investigated. Both agents induced similar biphasic effects with moderate potentiation of calcium-stimulated amylase release at medium and strong inhibition at higher concentrations. Calyculin A was thirty times more potent than okadaic acid and at 100 nM totally inhibited calcium-induced amylase release while 3 microM okadaic acid reduced amylase release by 78%. 100nM calyculin A also completely inhibited GTP gamma S-potentiated amylase release and partially inhibited phorbol ester potentiated secretion. The data indicate that inhibition of a serine/threonine phosphatase, probably a type 1 phosphatase, leads to inhibition of calcium-induced amylase release in permeabilized pancreatic acini.


Regulation of the expression of lamins A and C is post-transcriptional in P19 embryonal carcinoma cells. The polypeptide composition of the nuclear lamina can display important variations: undifferentiated cells express only lamin B and they acquire lamins A and C only after differentiation. We have analyzed the expression of lamins A and C in P19 pluripotent mouse embryonal carcinoma cells. Undifferentiated P19 cells are completely devoid of lamins A and C. We show that undifferentiated P19 cells contain low, but detectable steady-state levels of RNAs for lamins A and C that begin to increase by 24 h of retinoic acid-induced differentiation. However, the rate of transcription of the lamin A and C gene(s), analyzed by run-on transcription assays, remains unchanged during the differentiation process. These results demonstrate that, at least in P19 embryonal carcinoma cells, regulation of the expression of lamins A and C is a post-transcriptional event.


Multi-recognition capability of E-selectin in a dynamic flow system, as evidenced by differential effects of sialidases and anti-carbohydrate antibodies on selectin-mediated cell adhesion at low vs. high wall shear stress: a preliminary note. E-selectin has a "multi-recognition" capability in terms of epitope binding specificity, depending on adhesion conditions (static vs. low- or high-shear stress dynamic systems). Specifically, (i) adhesion based on expression of alpha 2-->3 sialylated Le(x) (SLe(x)) is prominent under static or low shear stress dynamic conditions; (ii) adhesion under high shear stress dynamic conditions does not depend on the known SLe(x) species, but rather on Lex with an adjacent unidentified sialosyl substitution, which shows different susceptibility to sialidases and antibodies compared to known SLe(x).


A novel tyrosine kinase, hyk, expressed in murine embryonic stem cells. To identify tyrosine kinases which play roles in mammalian early development, the 3' rapid amplification of cDNA ends (RACE) was performed on mouse embryonic stem (ES) cells. Among eight tyrosine kinases thus identified, we report here a novel tyrosine kinase, hyk (adhesion structures linked tyrosine kinase). The sequences of the 4.7 kb cDNA indicated the presence of RGD motif and three epidermal growth factor-like domains put between two immunoglobulin-like domains and three fibronectin type III domains in its extracellular region. It is strongly expressed in ES cells and later stages of embryos, but at low levels in midgestation embryos. It is also expressed at a low level in neural precursor cells from 10-day embryos, but at high levels in embryonic day 15 and neonatal brains. In adult tissues it is expressed ubiquitously.


Modulation of trichosanthin antigenicity by coupling to dextran. Trichosanthin (TCS) is a plant-derived type I ribosome-inactivating protein with a wide spectrum of biological and pharmacological activities. Recently, it was covalently coupled to dextran in order to prolong its half-life in plasma. The major biological activities were generally retained but at lower potency. The immunogenicity of the dextran-trichosanthin (DX-TCS) was compared to that of TCS itself in this study. The results showed that mice immunized with TCS produced 8 times as much TCS-reactive IgE than those immunized with DX-TCS. However, both TCS and DX-TCS immunization produced similar titers of TCS-reactive IgG. A trace of dextran-reactive IgG was detected in mice immunized with DX-TCS. Thus, coupling of TCS to dextran reduced its antigenicity but slightly enhanced that of dextran, and the conjugate elicited less IgE than TCS.


Effect of cytochrome C oxidase and polyanions on the alkaline transition of ferricytochrome C. Application of heparin, polyadenylate, polyglutamate and polygalacturonate resulted in changes in the electron absorption spectrum of cytochrome c that resembled those after cytochrome c oxidase application at neutral pH. The formed complexes of cytochrome c with polyanions retain the bond of Met-80 with heme iron. Cytochrome c oxidase and the polyanions increased the apparent pKa of alkaline transition of cytochrome c by an order of magnitude.


Inhibition of mite protease (Df-protease) with protease inhibitors. A protease from house dust mite(Dermatophagoides farinae) having high specificity towards a substrate of blood coagulation factor XIIa catalyzes the activation of kallikrein-kinin system in plasma (Takahashi et al., 1990). To prevent the formation of kinin by the mite-protease, inhibition of the protease with its inhibitors was tested in vitro and in vivo. Its kinetic studies revealed that Ki values are 3.9 x 10(-10) M for aprotinin, 3.0 x 10(-9) M for soybean trypsin inhibitor (Kunitz) and 2.5 x 10(-8) M for gabexate mesylate. Enhancement of blood permeability in guinea pigs caused by the protease was markedly suppressed by these inhibitors.


Inhibition of EGF-induced gastric mucosal calcium channel phosphorylation by ebrotidine. A gastric mucosal calcium channel-epidermal growth factor (EGF) receptor complex was isolated from solubilized epithelial cell membranes by means of a wheat germ agglutinin affinity. The complex, following reconstitution into phosphatidylcholine vesicles, exhibited active 45Ca2+ uptake as evidence by concentration-dependent response to the calcium channel activator BAY K8644, and the calcium channel antagonist PN200-110. The complex on the addition of EGF and ATP showed an increase in tyrosine phosphorylation of both a 55 and a 170kDa protein, while the vesicles containing the phosphorylated complex displayed a 48% greater 45Ca2+ uptake. The phosphorylation process was inhibited by an anti-ulcer agent, ebrotidine, which also interfered with the binding of EGF to calcium channel protein. The results suggest that ebrotidine protects the cellular integrity from calcium imbalance by modulating the EGF-stimulated gastric mucosal calcium channel activation.


Staurosporine and calphostin-C inhibit the phorbol ester-induced decrease of protein kinase C activity in rat hepatocytes. Two main forms of protein kinase C (PKC 1 and PKC 2) are detected in homogenates of rat hepatocytes using DEAE-cellulose column chromatography. The activity of these forms paradoxically, is rapidly decreased by treatment in vivo with phorbol myristate acetate (PMA). The dose-response curves to PMA for decreasing the activities of PKC 1 and 2 were shifted to the right by the potent and selective PKC inhibitors, staurosporine and calphostin-C. The decreases induced by 100 nM PMA were dose-dependently blocked by these inhibitors. It is concluded that activation of PKC is required and precedes such decreases in activity induced by the active phorbol ester.


The effects of p-chlorophenylalanine-induced serotonin synthesis inhibition and muscarinic blockade on the performance of rats in a 5-choice serial reaction time task. The effects of serotonergic dysfunction induced by treatment with p-chlorophenylalanine (PCPA), an inhibitor of serotonin synthesis, and cholinergic dysfunction induced by scopolamine on the performance of adult rats in the 5-choice serial reaction time task measuring selective attention were studied. Food-deprived rats were trained to detect and respond to brief flashes of light presented randomly in one of five locations, until they reached a stable level of performance (about 4 months). Scopolamine 0.2 mg/kg produced a marked variation in the performance but did not, however, induce any consistent impairment in the discriminative accuracy. Other doses of scopolamine (0.05 and 0.1 mg/kg) or N-methyl-scopolamine 0.2 mg/kg, a peripheral muscarinic receptor antagonist, did not affect discriminative accuracy. Furthermore, scopolamine as well as N-methyl-scopolamine produced a number of other performance deficits, such as significantly decreased overall probability of responding and significantly increased response latencies. PCPA treatment induced an almost total depletion (> 99%) of frontal cortical serotonin and its major metabolite 5-HIAA and reduced the frontal cortical concentrations of noradrenaline (-30%) and dopamine (-42%). During baseline testing conditions, there was a trend for the discriminative accuracy to be decreased by PCPA, although this effect failed to reach significance (P = 0.07). Presenting the stimuli at unpredictable intervals or reducing the intensity of the visual stimulus impaired discriminative accuracy in both PCPA-treated and control rats. The decrease in discriminative accuracy induced by PCPA reached statistical significance when the stimuli were presented faster than normally or the intensity of the visual stimulus was reduced. PCPA treatment did not make the rats more susceptible to the effects of scopolamine on discriminative accuracy. However, PCPA treatment also induced a number of other performance deficits, resulting in a decreased overall tendency to respond. In summary, there is a statistically non-significant trend for the discriminative accuracy to be decreased by PCPA treatment under normal testing conditions, and as the discrimination task is made more difficult (stimulus intensity reduction, presentation of the stimuli at faster than normal rates), the deficit in discriminative accuracy produced by PCPA treatment is revealed. The results suggest a role for brain serotonin in the general organization of behavior.


Modulation of the rat mesolimbic dopamine pathway by neurokinins. The locomotor activity (LMA) response induced after infusion of selective neurokinin (NK) agonists into the cell body (A10) and a terminal region of the mesolimbic pathway of the rat was investigated. Infusion of the NK1 receptor-selective agonist, GR73632, into the ventral tegmental area (VTA: A10) or the nucleus accumbens (NAS) significantly and dose-dependently increased basal LMA. Agonists selective for the NK2 and NK3 receptors, GR64349 and senktide respectively, had no effect on LMA after intra-NAS infusion. The LMA induced by GR73632 is mediated via dopamine (DA) since the response was abolished by haloperidol. From these studies it would appear that the elevated LMA reported previously after VTA or NAS administration of substance P probably occurs via NK1 receptors. Such data supports the notion that endogenous NKs are likely to be important in modulating the mesolimbic DA pathway and, as a consequence, compounds which antagonise their effects could be useful for the treatment of disorders associated with this system. However, simultaneous infusion of the NK1 agonists, +/- CP-96,345 and its analogue CPQ, into the VTA did not attenuate the LMA induced after intra-VTA infusion of GR73632. Co-infusion of the NK1 antagonist CPQ, but not +/- CP-96,345, attenuated the LMA response induced by GR73632 in the NAS. The apparent poor susceptibility of these responses to blockade by the recently developed non-peptide NK1 antagonists was unexpected but may reflect their poor affinity for the rat variant of the NK1 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)


Amylase cis-acting sequences mediate the alleviation of glucose repression by cAMP in Drosophila. alpha-Amylase gene expression is highly repressed by dietary glucose in Drosophila melanogaster. This glucose effect can be alleviated by exogenous adenosine 3':5'-cyclic monophosphate (cAMP). Here, we show that the relief of glucose repression by cAMP occurs at the level of amylase mRNA abundance. Furthermore, exogenous cAMP was shown to alleviate glucose repression of the transient expression of an amylase gene construct in transformed Amynull larvae. This construct contains only 109 base pairs of the promoter region; this is the minimal length of upstream sequence which is necessary for wild-type levels of amylase gene expression. These results indicate that cis-acting promoter elements located close to the transcriptional start site of the Drosophila amylase gene mediate both glucose repression and the cAMP-derepression effects.


Translation of poly(A)-binding protein mRNA is regulated by growth conditions. Translational efficiency of a minor group of mRNAs is regulated by serum levels in 3T6 fibroblasts. Included within this group is the poly(A)-binding protein (PABP) mRNA. We analyzed the distribution of PABP mRNA in polysome profiles and found a large percentage of this mRNA to be translationally repressed in both actively growing (approximately 60%) and resting cells (approximately 70%). Elevated serum levels induced a distinct bimodal distribution of this mRNA between actively translated and repressed fractions. Similarly, treatment of cells with low doses of cycloheximide also generated a partial shift of repressed PABP mRNA into the actively translated fraction. In an attempt to characterize the factors which regulate PABP mRNA translation we have identified the proteins which bind to this mRNA in vitro. Sequences within the 5' untranslated region were found to be sufficient for binding of all proteins to this mRNA. We suggest that this region and the proteins associated with it may be essential for translation control of PABP mRNA.


Variation of nitrogen oxide contents in the streets of Naples. Nitrogen oxides were determined in streets of Naples with different density of vehicular traffic. Ventilation allows a dilution of concentration in windy streets but not in protected ones. Streets with a scanty level of vehicular traffic suffer the pollution of contiguous areas. Observed values suggest measures to reduce air pollution.


Some gaseous pollutants in the industrial area at east of Naples. Nitrogen oxides, ozone, sulphur dioxide and hydrogen sulphide were determined in the industrial area at east of Naples. Emission from vehicular traffic are added to those from fixed sources, but the breeze effect avoids pollution accumulation. Obtained values are low enough if compared with those of other towns.


Neuronal and myogenic muscarinic effects of McN-A-343 on guinea pig longitudinal smooth muscle-myenteric plexus. Among muscarinic agonists, the compound McN-A-343, originally proposed as selective stimulant of M1 cholinergic site, was subsequently questioned as a useful pharmacological tool in the classification of muscarinic receptors. In this work, evidence is presented for a dual response of McN-A-343 on longitudinal muscle-myenteric plexus preparation. On electrically-stimulated preparation, this agonist exhibited a pirenzepine-sensitive inhibition of the twitch contractions due to the involvement of neural M1-muscarinic receptor. On the other hand, a direct myogenic contractile action on the unstimulated tissue was observed using McN-A-343 in the same range of concentrations. This latter response, on the basis of the effects of muscarinic and non-muscarinic antagonists tested, seems to involve effectorial muscarinic sites with an unusual mechanism.


Nitric oxide synthase immunoactivity and NADPH diaphorase enzyme activity in neurons of the gastrointestinal tract of the toad, Bufo marinus. The presence of nitric oxide synthase (NOS) was demonstrated immunohistochemically, and NADPH diaphorase was demonstrated by enzyme histochemistry in neurons throughout the gastrointestinal tract of the anuran amphibian, Bufo marinus. Successive staining showed that NOS immunoreactivity and NADPH diaphorase activity occurred in precisely the same subgroup of enteric neurons. Subsequent detailed studies of the distribution of these neurons were made using NADPH diaphorase histochemistry. Numerous reactive nerve cell bodies and fibres were found in the myenteric plexus from the esophagus to the cloaca. A dense innervation of the longitudinal and circular muscle layers occurred throughout the gastrointestinal tract. The lamina muscularis mucosae was only prominent in the stomach, where it was sparsely innervated. Reactive nerve cell bodies were common in the submucosa of the large intestine, less common in the small intestine and extremely rare in the stomach and esophagus. Reactive fibres contributed to subepithelial plexuses in the esophagus, colon, rectum and cloaca. It is concluded that NOS/NADPH diaphorase is conserved amongst vertebrate classes and that NO is a likely neurotransmitter in the toad gastrointestinal tract.


Low molecular weight proteins in children with renal disease. Low molecular weight proteins are of interest in children because their increased urinary excretion is a sign of renal tubular disease and their increased plasma concentration is inversely related to glomerular filtration rate. These proteins include beta 2-microglobulin (B2M), retinol-binding protein (RBP), alpha 1-microglobulin (A1M) and lysozyme. B2M is unstable in acid urine, in contrast to RBP and A1M which are more stable. Any increase in the urinary excretion of B2M or RBP is highly specific for tubular disease, whereas increased excretion of A1M may be seen with glomerular proteinuria. Areas of clinical application include tubular and glomerular diseases, detection of drug toxicity, reflux nephropathy, birth asphyxia and insulin-dependent diabetes mellitus. Methods of sample collection and analysis of these proteins are discussed.


Lack of transcription and expression of the alpha IIb integrin in human early haematopoietic stem cells. The glycoprotein IIb, the alpha subunit of the platelet integrin GPIIb-IIIa, is a marker of megakaryocyte, but the stage of its expression during haematopoiesis remains controversial. We have examined the expression of GPIIb protein and alpha IIb mRNA in early human normal stem cells. We have purified stem cell expressing the CD34 surface marker (CD34+ fraction) and selected among this population quiescent cells (CD34+ MF(R) fraction). We have failed to detect GPIIb protein and alpha IIb mRNA in the pluripotential (CD34+ MF(R)) cells, even with polymerase chain amplification. Therefore alpha IIb transcription and GPIIb protein expression seemed to follow the commitment of the pluripotential cell in the megakaryocyte lineage.


The production of steel factor mRNA in Diamond-Blackfan anaemia long-term cultures and interactions of steel factor with erythropoietin and interleukin-3. Diamond-Blackfan anaemia (DBA) is a congenital macrocytic anaemia. To investigate whether DBA is due to hyporesponsiveness to or hypoproduction of Steel factor (SF), we compared the in vitro responsiveness of the BFU-E contained in the Ficoll-Hypaque non-adherent cell fraction of six DBA marrows with that of four normal marrows and one transient erythroblastopenia of childhood (TEC) marrow. In addition, we studied the effect of soluble SF on long-term marrow cultures (LTMC) and analysed the stromal cells from these cultures for SF mRNA transcripts. All the patients showed an erythropoietin dose-related increase of small BFU-E. The number and size of BFU-E was increased with the addition to the epo of IL-3 or SF; IL-3+SF was not synergistic. The addition of soluble SF to LTMC of DBA patients was associated with a small but consistent increase in non-adherent cell production and an increase in the number of progenitors. Messenger RNA from immortalized stromal cell lines of three patients and from primary bone marrow stromal cells of one patient showed the presence of expected SF transcripts by PCR analysis. These results demonstrate that this group of DBA patients responds to SF and produces SF mRNA normally, indicating that SF itself is not involved in DBA pathophysiology. The effects observed suggest that, despite the lack of evidence for a causative role, SF may prove to be effective treatment for such patients.


Effects of recombinant human granulocyte colony-stimulating factor on neutrophil functions in aged animals. We have studied the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on granulopoiesis and neutrophil functions in aged rats and aged mice. We subcutaneously injected rG-CSF or control vehicle into aged rats (22 months old and 25 months old) for 7 consecutive days, counted the peripheral neutrophils and evaluated the functions of neutrophils isolated from venous blood. The peripheral neutrophil count in aged rats tended to be increased as compared with that in young rats (11 weeks old). However, the neutrophils in aged rats exhibited a decline of superoxide anion (O2-) release and phagocytic activity as compared with young rats. The peripheral neutrophil count in aged rats was significantly increased 5-6-fold as many as the control value by rG-CSF treatment, which was accompanied by a significant enhancement of O2- release and of phagocytic activity being restored to normal levels or better. In another series of experiments, we subcutaneously injected rG-CSF or control vehicle into aged mice (24-28 months old) or young mice (8 weeks old) for 7 consecutive days, and evaluated the functions of neutrophils isolated from peritoneal cavity. The peritoneal exudate neutrophils from the aged mice exhibited a decline of phagocytic and chemotactic activity as compared with the young mice. These functions in both young and aged mice were significantly enhanced by rG-CSF-treatment, and these functions in rG-CSF-treated aged mice were restored to a level higher than the level in control young mice. These findings demonstrate that rG-CSF is capable of enhancing granulopoiesis and restoring the age-related decline of neutrophil functions.


Adhesion efficiency, platelet density and size. We have previously shown that adhesion of human platelets to immobilized collagen is extremely rapid, with initial rates approaching 3% of single particles adhering per 10 ms. Here, we have investigated adhesion efficiency to collagen as a function of platelet density. Platelet subpopulations: low-density (1.040 < d < 1.065 g/ml), intermediate-density (1.065 < d < 1.070 g/ml) and high-density (1.070 < d < 1.080 g/ml) were separated by Percoll density gradient centrifugation. They constituted 24%, 47% and 29% of the total platelet population and had mean volumes of 6.01, 7.37 and 8.21 fl, respectively. Using a continuous-flow, micro-affinity column, we found that the most dense (large) platelets exhibited initial rate of adhesion 4 times greater than the least dense (small) platelets. They were also less sensitive to inhibition by prostacyclin (PGI2). In contrast, there was no significant difference in aggregation induced by high doses of ADP and collagen, indicating that the most dense platelets were not preferentially involved in aggregation induced by high doses of agonists. These results suggest that normal circulating platelets can be distinctly heterogeneous in their ability to adhere to collagen under arterial-flow conditions. The greater efficiency of high-density platelets may be related to increased content of the glycoprotein Ia/IIa (GPIa/IIa) complex.


Molecular cloning of monkey liver cytochrome P-450 cDNAs: similarity of the primary sequences to human cytochromes P-450. Three cDNAs coding for monkey cytochrome P-450 (P450) 2C, 2E and 3A (MKmp13, MKj1 and MKnf2, respectively) were isolated from a lambda gt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey, using cDNA fragments for human P450 2C, 2E and 3A as respective probes. MKmp13 and MKnf2 were 1901 and 2032 bp long, containing entire coding regions for polypeptides of 490 and 503 residues, respectively. The deduced N-terminal amino acid sequences of MKmp13 and MKnf2 were identical with those of P450-MK1 and P450-MK2, which had been purified from liver microsomes of untreated and polychlorinated biphenyl (PCB)-treated crab-eating monkeys, respectively. MKj1 was 1508 bp long, encoding a polypeptide of 449 residues, which is presumed to lack N-terminal 45 residues as compared with the sequence for human P450 2E1. Northern blot analysis indicated that monkey P450 2C, 2E and 3A mRNAs were expressed constitutively in monkey livers. P450 2E and 3A mRNAs were induced by both 3MC and PCB, while P450 2C mRNA was induced only by PCB. The deduced amino acid sequences of four monkey cytochrome P-450 cDNAs, including P450 1A1 (MKah1) which we isolated previously, were more than 92% identical with those of corresponding human cytochrome P-450 cDNAs.


Expression of vascular cell adhesion molecule-1 in human renal allografts. The expression of vascular cell adhesion molecule-1 (VCAM-1) in 11 human renal allograft biopsies and 3 normal kidney specimens was investigated by immunocytochemistry. VCAM-1 expression was correlated with the degree of CD3+ T cell infiltration and the clinicopathologic diagnosis of acute rejection. CD3+ infiltrates were seen in all biopsies with rejection, but not in normal biopsies or one with acute tubular necrosis, and were accompanied by CD68+ monocyte/macrophage infiltrates. In normal biopsies, VCAM-1 was present on occasional tubules, where its expression was patchy and restricted to the basolateral surface of cells with slight cytoplasmic staining. The total number of tubules expressing VCAM-1 significantly increased in specimens infiltrated with CD3+ T cells. Moreover, in these infiltrated biopsy specimens, VCAM-1 was present throughout the cytoplasm of tubular cells concentrated on the basolateral surface. VCAM-1 was also observed on vascular endothelial cells where its expression correlated with the degree of CD3+ infiltrate. Mean scores (0 to 3+) for endothelial VCAM-1 expression increased from 0 (CD3+ score, 0) to a mean score of 2.25 in association with CD3+ T cell infiltrates (CD3+ score, 3). Endothelial VCAM-1 was predominantly on vessels in areas of infiltrate, including peritubular capillaries, venules, and arterioles, but was notably absent on glomerular endothelium. VCAM-1 also stained mesangial cells in an occasional CD3+ infiltrated specimen. It was concluded that the expression of VCAM-1 is increased on renal tubules and renovascular endothelium in rejecting renal allografts in association with CD3+ infiltrates.(ABSTRACT TRUNCATED AT 250 WORDS)


Stratified organization of the nicotinic acetylcholine receptor channel. Mutations of leucine 247 within the M2 channel domain of the alpha 7 neuronal nicotinic receptor, confer electrophysiological and pharmacological properties, which allow one of the desensitized states to become conductive. Here we show that, in Xenopus oocytes, the effects of the mutations were preserved when 1,2-bis(2-aminophenoxy)-ethane N,N,N',N'-tetraacetic acid (BAPTA) was injected in the cytoplasm to block Ca(2+)-dependent chloride currents, and that in agreement with the proposed interpretation, the ionic currents do not desensitize and rise slowly, in the time-scale of seconds, upon agonist application. Interestingly, similar effects were observed when the two rings (T244, V251) neighbouring L247 on the alpha-helix, but not the more distant ones (S240, L254/255), were mutated, thus supporting the proposal of a functional stratification of the channel domain.


Age-related changes in galanin- and calretinin-immunoreactive nerves of guinea-pig gallbladder. Age-related changes in the expression of galanin- and calretinin-like immunoreactivity in the ganglionated plexus of 2-4 day, 6 month and 2 year old guinea-pig gallbladder were investigated. The ganglionated plexus was studied using immunohistochemical labelling techniques on whole mount stretch preparations of the gallbladders. Galanin- and calretinin-like immunoreactivity did not differ significantly between age groups of the 2-4 day and 6 month olds except for some reduction of calretinin-immunofluorescent nerves in the 6 month old preparations. However, in the ganglionated plexus of the 2 year old guinea-pig gallbladders, neither galanin- nor calretinin-like immunoreactivity could be detected. The implication of these findings, in relation to the physiological activity of the gallbladder is discussed.


Differential expression of acidic and basic FGF in the rat substantia nigra during development. Both acidic (aFGF) and basic (bFGF) fibroblast growth factors have been shown to be present in the adult rat ventral mesencephalon and to exert effects on cultured mesencephalic cells. In the present study we have examined the expression of aFGF and bFGF in the rat ventral mesencephalon at various stages of development. bFGF was present at all ages examined [embryonic day 16 (E16) to postnatal day 90 (P90)]. In contrast, aFGF was not detectable at embryonic and early postnatal ages, but was observed at later (P20, P60, P90) postnatal stages. These data suggest that aFGF and bFGF may have functions in mesencephalic dopamine neurones in different stages of development.


Computers in medicine: enhancing medical care for children with developmental disabilities. Caring for children with developmental disabilities and their families requires the close monitoring and management of a multitude of complex medical and psychosocial issues. The need for an interdisciplinary team approach to address these concerns presents special logistical problems on how to organize and present the information obtained during a clinic visit, and how to expedite the dissemination of this information to the team members. I am currently utilizing a computerized medical record database in the Developmental Disabilities Clinic at Children's Hospital-Columbus, Ohio and at The Nisonger Center a university affiliated program (UAP). The hardware required for the operation of this system includes a Macintosh PowerBook 170 and FilemakerPro software by Claris. The flexibility of use and the potential future applications of this program provides a practical clinical approach to caring for children with developmental disabilities.