Comparative purification of recombinant HIV-1 and HIV-2 reverse transcriptase: preparation of heterodimeric enzyme devoid of unprocessed gene product. A procedure for producing and purifying recombinant HIV-1 and HIV-2 reverse transcriptase (RT) is described. These enzymes are produced by Escherichia coli-transformed with a plasmid containing the gene encoding for either the human immunodeficiency virus type 1 (HIV-1) or HIV-2 RT protein. Both proteins are partially processed by host cell proteases giving rise to a mixture of heterodimeric and nonheterodimeric products, which are subsequently resolved to near homogeneity by chromatography on phosphocellulose, Q-Sepharose, and hydrophobic interaction HPLC. Both HIV-1 (66/51 kDa) and HIV-2 (68/54 kDa) heterodimeric enzymes devoid of excess unprocessed (p66 or p68) precursors are isolated, enabling comparative enzymatic characterization of the fully active (and biologically relevant) heterodimeric forms. Homogenous HIV-1 and HIV-2 RT purified by this methodology exhibit near equivalent polymerase and RNase H activities.


A rapid, two-step method for high-yield purification of recombinant rat acidic and basic fibroblast growth factors. We describe the preparation of vectors for T7 polymerase-driven, high-level expression of rat acidic (aFGF) or basic (bFGF) fibroblast growth factors in Escherichia coli. Following isopropyl-beta-D-thiogalactoside induction of T7 polymerase, rat aFGF or bFGF represented a major portion of the proteins synthesized by vector-transformed E. coli. Passage of cell extracts through an Amicon YM-100 membrane provided ultrafiltrates containing either aFGF or bFGF as the principal component. A single heparin-Sepharose chromatography of the ultrafiltrates yielded essentially homogenous, biologically active, recombinant rat aFGF or bFGF. By silanizing vessels and using buffers containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, we could store homogenous aFGF or bFGF preparations without significant loss during repeated freezing and thawing. Previous methods for purifying aFGF or bFGF utilized salt fractionation followed by sequential ion exchange and heparin-Sepharose chromatography. These purified aFGF or bFGF preparations routinely were stored in buffered carrier protein to minimize mitogen loss resulting from adsorption to glass or plastic surfaces. In contrast, the methods that we detail are rapid, require minimal manipulation of preparations, and permit storage of carrier-free, homogenous preparations without loss resulting from surface adsorption. The protocols can be used for preparation of homogenous aFGF or bFGF of other species and may be readily applied to the isolation and characterization of FGF-like mitogens present in cultured cell extracts, conditioned medium, or tissue preparations.


Induction of gene expression in muscle by the IGFs. The insulin-like growth factors (IGFs) are usually studied with regard to their general effects on cell growth or differentiation, but the latter actions imply that IGFs may also have effects on expression of specific genes in differentiating target tissues. After 15 years of studies on IGF actions on muscle, we have (with the help of an outstanding group of collaborators) found three specific instances in which IGFs induce expression of a well-characterized gene with at least some degree of specificity. Each of the three genes under consideration plays a major role in the tissue that expresses it, and each of the three kinds of muscle is represented. The genes are (1) skeletal muscle myogenin, which plays a central role in terminal myogenic differentiation of muscle cells to form postmitotic myotubes, (2) smooth muscle aortic elastin, which is of major importance in regulation of blood pressure, and (3) cardiac beta-myosin heavy chain, which is the primary component of the contractile apparatus in older rodents and in all humans. For the first two of these, it has been established that the stimulation is largely if not completely at the level of increased mRNA synthesis, and that reporter gene constructs using 5-untranslated regions of the gene exhibit analogous responses to IGFs, offering the possibility that consensus IGF response elements can be elucidated. The importance of IGFs for skeletal myogenic differentiation is underscored by the observation that myoblasts in 'differentiation' medium exhibit substantial expression of IGF-II if IGFs are not added to the medium. Thus the IGFs play major roles in functions of all three kinds of muscle.+


IGF-I and IGF binding proteins in diabetes-related kidney growth. The kidney is a site of insulin-like growth factor I (IGF-I) production and IGF-I mediates effects on kidney growth and function. Diabetes is associated with kidney growth in man and the rat and, in the latter, commences within 48 h of induction of diabetes. Levels of kidney IGF-I are increased during the first 2-3 days of diabetes and this is at least partially due to increased production. Additionally, IGF-I binding is increased in diabetic rat kidney, due to increased binding to the IGF-I receptor and induction of proximal tubular binding protein expression. These changes are attenuated in prepubertal rats suggesting hormonal regulation. Further studies suggest that the changes are partly GH-dependent but independent of direct sex steroid effects. As kidney growth has been implicated in the subsequent development of diabetic nephropathy, further exploration of the close association between diabetes-related kidney growth and IGF-I accumulation may lead to an improved understanding of this complication.


Structural aspects of the IGFBP family.

Structural aspects of the IGFBP family. To date six IGF binding proteins (IGFBP) have been characterized. Analysis of the amino acid sequence reveals that the IGFBPs are clearly distinct but are sharing regions with strong homology. Specifically the hydrophobic cysteine rich N-terminal region and to a lesser extend the C-terminal part are preserved. The alignment of the cysteine molecules is strongly conserved across the 6 IGFBPs. The middle one-third region, where no cysteines are present (except for IGFBP-4) is most divergent. IGFBP-3 and -4 are glycosylated, whereas IGFBP-1 and -2 contain an Arg-Gly-Asp sequence near the carboxyl terminus. Determination of the number of free-SH groups of IGFBP-1 and -3 has revealed that most likely all cysteine residues are involved in disulfide bond formation. All members of the IGFBP family bind IGF-I and IGF-II with about equal affinity. Studies involving deletion mutation and site-directed mutagenesis of IGFBP-1 and -3 have suggested that the three-dimensional structure of the protein plays an important role in IGF binding. However at present it is unclear whether the IGFBPs share one or more specific IGF binding domain. The predominant function of the IGFBPs is to allocate IGF in the various body fluid compartments and tissues and to modulate IGF binding to receptors. For this purpose there exists a very sophisticated control of the routing of circulating IGF both from and to the cell. There is mounting evidence that the structure of the IGFBP proteins plays a key role in the regulation of IGF bioavailability, by modulating its molecular size, capillary membrane permeability, target tissue specificity, cell membrane adherence and IGF affinity.


IGF binding proteins: regulation of cellular actions. The recent purification and structural analysis of the six forms of insulin-like growth factor binding proteins has opened up the possibility of studying their mechanism of action at the cellular level. While a great deal of information has been published regarding control of secretion of these proteins by cells, their modulation of the target cell actions of the IGFs remains incompletely studied. Both IGFBP-1 and 3 have been shown to be bipotential regulators. Using one set of culture conditions, both proteins appear to inhibit IGF-mediated growth while if other conditions are used they appear to stimulate growth. IGFBP-2, 5 and 6 have been less well studied in this regard, although several studies showing growth inhibition with IGFBP-2 have been published. IGFBP-4 appears to be consistently inhibitory and no studies showing stimulation have been published to date. Recent research has focused on the reason for these bifunctional effects. Clearly the ability to adhere to cell surfaces appears to be one important property of these proteins that results in their ability to potentiate or inhibit growth. Likewise, phosphorylation of IGFBP-1 on serine residues appears to be a structurally modifying change which results in altered growth regulation. The future will no doubt uncover other post-translational modifications that result in modulation of biologic activity.


Rectal obstruction secondary to carcinoma of the prostate treated by transanal resection of the prostate. Rectal obstruction caused by carcinoma of the prostate is uncommon. If the disease does not respond to either hormonal treatment or radiotherapy, the only surgical means of relieving the obstruction has been the formation of a colostomy. We present an alternative treatment, since many of these patients are elderly with a short life expectancy and they may have difficulty coping with a stoma. Seven transanal endoscopic resections of the prostate (TARP) have been performed on 5 patients. The procedure is not without complications, but it does offer simple surgical palliation of the rectal obstruction and obviates the need for a colostomy.


Oesophageal cancer: a population-based study of survival after treatment. The survival of patients with oesophageal cancer diagnosed during the period 1982-1985 in Nottingham has been studied. Of 496 patients identified from endoscopy, histopathology and hospital activity analysis records, 268 (171 men) lived in the catchment area and had primary oesophageal cancer. Compared with previous studies the proportion of adenocarcinoma (35 per cent) was twice that expected, although survival was similar (hazard rate ratio at 2 years 1.0 (95 per cent confidence interval (c.i.) 0.8-1.4)) whether a squamous cell carcinoma or adenocarcinoma was present. Based on the original treatment intention, surgery was attempted in 34 per cent of cases and was associated with a median survival from diagnosis of 293 (95 per cent c.i. 232-367) days, with 41, 19 and 11 per cent surviving 1, 2 and 3 years respectively. Radical radiotherapy was attempted in 13 per cent of patients and was associated with a median survival of 190 (95 per cent c.i. 136-253) days, with 14, 6 and 6 per cent surviving 1, 2 and 3 years. Intubation alone was performed in 40 per cent of patients, of whom 44 per cent were aged over 75 years and 29 per cent had evidence of metastases, compared with 13 and 11 per cent respectively of those undergoing surgery or radical radiotherapy. The median survival for intubation alone was 100 (95 per cent c.i. 81-122) days, with 6, 3 and 0 per cent of patients surviving 1, 2 and 3 years respectively. Although patients treated surgically had the longest survival, these data indicate that overall survival after any active intervention is modest. Intubation alone is a reasonable option in those not suitable for surgery; randomized trials are needed to compare intubation with new methods of palliation.


The differential staining patterns of two lectins in the accessory olfactory bulb of the rat. We examined the binding sites of Bandeiraea simplicifolia lectin I (BSL-I) and Vicia villosa agglutinin (VVA) which bind to the vomeronasal nerve of the rat. BSL-I stained the whole vomeronasal nerve and glomerular layers. VVA strongly stained the posterior 2/3, but weakly stained the anterior 1/3 of the glomerular layer. These results indicate that the glomeruli of the rat AOB have two subdivisions revealed by lectin histochemistry using BSL-I and VVA.


Dust from carpeted and smooth floors. V. Cat (Fel d I) and mite (Der p I and Der f I) allergen levels in school dust. Demonstration of the basophil histamine release induced by dust from classrooms. Sixty dust samples from schools in Norway were analysed for major allergens from cat and mite after sampling with the regularly used vacuum cleaners for 5 days and with a new model vacuum cleaner for 10 days, respectively. The major feline allergen Felis domesticus allergen I (Fel d I) was detected in all the classrooms, with ranges from 12 to 16,840 ng/m2 floor area. The mean Fel d I concentration was about 11 times higher per unit area carpeted floors as compared with smooth floors after the 10 days sampling period. Mite allergens Dermatophagoides pteronyssinus allergen I (Der p I) and Dermatophagoides farinae allergen I (Der f I) were detected in very low concentrations, with ranges from < 1 ng to 104 ng/m2 floor area. These findings suggest that the school is a protective environment against mite infestation, while the prevalence of cat allergens in classrooms seems higher than previously assumed. Basophil histamine release was measured after provocation with 20 dust extracts from 10 different schools. In nine of the 10 schools examined, the basophil histamine release caused by challenge with carpet dust was higher than the corresponding release with smooth floor dust. The calculated floor areas in each school in which dust led to 15% histamine release were from 2 to 55 times larger for smooth floors compared with carpeted floors. These results emphasize previous findings regarding higher allergen concentrations in classrooms with carpeted floors.


'Hemorrhagic' and microvascular phenomena within the arterial wall. To study microvasculature and hemorrhage within the arterial wall. Human autopsy specimens of the arch of the aorta, the carotid, brachiocephalic, subclavian and coronary arteries perfused with liquid casting material and maintained at physiological pressure until the material had set. Evidence of dense microvasculature and other phenomena which have the morphological appearance of 'hemorrhage' within the arterial wall, coupled with calcified matter and other impressions made on the cast by atherosclerotic plaques. The findings lend support to suggestions in the literature that neovascularization may play a role in the pathogenesis of coronary atherosclerosis and its sequelae, that hemorrhage into the intima may be due to rupture of capillaries which are derived from the coronary lumen, and that an increase in microvasculature occurs in the immediate vicinity of localized atherosclerotic lesions.


Chromosomal sensitivity of human lymphocytes to bleomycin. Influence of antioxidant enzyme activities in whole blood and different blood fractions. The activity of the antioxidant enzymes catalase (CAT), peroxidases (POD), and superoxide dismutases (SOD) in whole blood and different blood fractions was analyzed in 20 normal human beings and correlated with the chromosomal sensitivity of lymphocytes to bleomycin (BLM) (measured as frequency of dicentric chromosomes per BLM dose). Our results demonstrate that both the physiologic activities of the enzymes and the chromosomal sensitivity to BLM exhibit an ample and significant interindividual variability. An inverse and linear correlation between chromosomal sensitivity to BLM and the concentration of 1) CAT and POD in plasma and 2) SOD in whole blood, erythrocytes, and plasma was found. On the other hand, the chromosomal sensitivity to BLM showed a direct correlation with the concentration of SOD and POD in mononuclear leukocytes. It is suggested that a determination of antioxidant enzyme (AOE) activities in a given cell population may serve to predict the chromosomal sensitivity to BLM.


Pericyte involvement in capillary sprouting during angiogenesis in situ. To investigate the participation of microvascular pericytes in the process of capillary sprouting, we examined whole-mount preparations of the rat mesentery by use of a double immunofluorescence approach. Angiogenesis was induced by intraperitoneal injections of either the mast cell-degranulating substance compound 48/80 or tumor cell-conditioned medium. Capillary sprouts were visualized by staining with rhodamine-conjugated phalloidin and pericytes were simultaneously stained by an antibody to the intermediate filament protein desmin. Developing pericytes were negative for the smooth-muscle isoform of alpha-actin, but were clearly reactive for desmin. Pericytes appear to be involved in the earliest stages of capillary sprouting. Pericytes were regularly found lying at and in front of the advancing tips of endothelial sprouts. At many sites pericytes were seen to bridge the gap between the leading edges of opposing endothelial sprouts, which were apparently preparing to merge, suggesting that pericytic processes may serve as guiding structures aiding outgrowth of endothelial cells.


[Dynamics of vascular changes in neovascular glaucoma. I. Development of rubeosis iridis after cryosurgery]. The author is evaluating the influence of two cryosurgical methods for the development of the rubeosis of iris in neovascular glaucoma. The cyclocryocoagulation was used as a single method in 12 eyes, the combination of transscleral panretinal cryocoagulation and cyclocryocoagulation in 26 eyes. The results were clearly better if the combination of cryosurgical methods was used. The involution of rubeosis of iris was observed in 6 weeks in all patients after the transscleral panretinal photocoagulation in combination with cyclocryocoagulation. The author thinks that the non-satisfactory regression of the rubeosis in neovascular glaucoma after simple cyclocryocoagulation is caused by the fact, that by this method we affect just one factor in the pathogenesis of rubeosis. The author is suggesting that there are two factors influencing the pathogenesis of the rubeosis of iris: the primary disease, that is causing the development of proliferative retinopathy; and the high intraocular pressure, that is reducing the blood flow in retinal vessels and by this is increasing the hypoxia of the retina.


[Dynamics of vascular changes in neovascular glaucoma. II. Unusual findings after cryosurgery]. New, yet published findings after the combination of transscleral panretinal cryocoagulation and cyclocryocoagulation are presented. The author describes the difference in regression of small and large neovascularisations of the iris after the cryosurgery; the difference in blood flow in these vessels is shown angiographically. The discordance in the biomicroscopical findings of neovascularisations of the iris and angiographical picture in connection with high intraocular pressure in neovascular glaucoma is shown. The iridoplegia was found only in patients after the transscleral panretinal cryocoagulation, not in patients after simple cyclocryocoagulation. The mortality rate in neovascular glaucoma is 55% in 3 years, but mostly in 2 years.


Prolonged treatment with finasteride (a 5 alpha-reductase inhibitor) does not affect bone density and metabolism. Since it is not clear whether testosterone or dihydrotestosterone is the active hormone in bone metabolism, we wished to assess the effect of finasteride, a 5 alpha-reductase inhibitor, or vertebral bone mineral density and parameters of bone and mineral metabolism. Patients were treated in a randomized, double-blind controlled study with either placebo, 1 or 5 mg/day finasteride. Twenty-three men with benign prostatic hyperplasia (BPH) were included in this study; eight received placebo, seven were allocated to treatment with 1 mg/day, and eight to 5 mg/day finasteride for 12 months. Vertebral bone mineral density was measured at the lumbar spine by dual energy X-ray bone densitometry. Serum calcium, phosphorus, parathyroid hormone, osteocalcin and vitamin D metabolites were measured regularly. Urinary calcium and creatinine excretion were monitored as well. Finasteride caused a significant decrease in serum dihydrotestosterone after 6 and 12 months, but no effect on serum testosterone. Vertebral bone mineral density remained unaltered. None of the other parameters monitored were affected except for a small unexplained increase in 1.25-dihydroxyvitamin D in the group receiving 5 mg finasteride/day. Testosterone is probably the active hormone in bone metabolism. However, oestradiol, the product of testosterone aromatization (which remains unaltered under finasteride) may yet be another possible responsible steroid in the maintenance of bone density. We can also not rule out that the small amount of dihydrotestosterone remaining under finasteride administration is sufficient for maintaining normal bone metabolism.


Cushing's syndrome associated with ectopic production of corticotrophin-releasing hormone, corticotrophin and vasopressin by a phaeochromocytoma. We describe a case of Cushing's syndrome caused by a phaeochromocytoma secreting corticotrophin-releasing hormone (CRH) and corticotrophin (ACTH). A 49-year-old white woman presented with a 1-month history of lower limb oedema, polydipsia and polyuria. Physical examination revealed a patient with plethoric facies, lanugo-type facial hair, central obesity, red abdominal striae, lower limb oedema, and blood pressure of 210/115 mmHg. Laboratory studies showed high plasma ACTH and markedly elevated urinary cortisol excretion that suppressed more than 50% with high-dose dexamethasone administration. Computed tomographic scan of the abdomen showed a 4-cm left adrenal tumour. Catecholamines and metabolites were markedly increased in a 24-hour urine collection. Results of venous catheterization studies showed that CRH and ACTH were secreted by the tumour. In addition, with ovine CRH administration, inferior petrosal sinus sampling showed pituitary secretion of ACTH. Left adrenalectomy resulted in complete remission of Cushing's syndrome. Light microscopic and immunohistochemical studies revealed a phaeochromocytoma that produced CRH, ACTH and vasopressin. RNA studies showed that this tumour, in contrast to normal adrenal and other reported phaeochromocytomas, transcribed a lone pituitary-sized (1200 nucleotide) pro-opiomelanocortin mRNA. This is the second reported case of a CRH-secreting phaeochromocytoma.


Deciphering the fluorescent variability of human genomic heterochromatin by DA/DAPI technique. A number of selective staining techniques have been utilized to decipher the variability of pericentromeric heterochromatin. One such technique is called DA/DAPI and it is believed to be stain specific. However, we demonstrate otherwise and suggest that pericentromeric regions of all human chromosomes stain positive by DA/DAPI-technique. It must be emphasized that the incidence of DA/DAPI positive stained chromosomes, other than 1, 9, 15, 16 and Y, is a rare occurrence and only a small portion of the pericentromeric region is DA/DAPI positive, as reported here using 50 normal individuals.


Granzyme A-immunoreactive cells in synovial fluid in reactive and rheumatoid arthritis. Perforin and granzyme A co-localize in the cytotoxic granules of killer cells like cytotoxic T lymphocytes (CTL). Perforin is the cytolytic pore-forming protein, whereas the function of the homodimeric serine protease granzyme A and other members of the granzyme family is still unclear. Granzyme A-immunoreactive cells formed 8 +/- 2% of the resting peripheral blood lymphocytes of healthy individuals. In contrast, granzyme A-positive cells formed 15% of peripheral blood mononuclear cells in patients with reactive or rheumatoid arthritis. However, 29 +/- 4% (p < 0.05 compared to normal peripheral blood) and 25 +/- 4% (p < 0.05) of all lymphocytes in synovial fluid in reactive and rheumatoid arthritis, respectively, were granzyme A-positive. This suggests involvement of cell-mediated cytolytic mechanisms in the articular pathogenic mechanisms. This involvement, however, does not differentiate between reactive and rheumatoid arthritis.


Complex carbohydrate presence in the gut and Malpighian tubules of Drosophila auraria larvae (Insecta, Diptera): a cytochemical study. Cytochemical techniques were used for the qualitative characterization of carbohydrates in the foregut, midgut, hindgut and Malpighian tubules of Drosophila auraria larvae. Periodate-reactive (neutral) polysaccharides were present in the foregut cuticle, peritrophic membrane, cells of the proventriculus which produce the peritrophic membrane, as well as the glycocalyx of the midgut and Malpighian tubule cells. Sulphated and carboxylated complex carbohydrates were detected in the cuticle of both foregut and hindgut, as well as the 'vacuoles with granules' in the Malpighian tubules. Periodate-reactive sulphated and carboxylated polysaccharides were detected in dense bodies appearing in all cells 10-20 h before pupation of larvae. The significance of the presence or absence of complex carbohydrates in D. auraria larval digestive system is discussed and compared with results found in the digestive system of other insects. In addition, the efficiency of the HID-TCH-SP and LID-TCH-SP techniques in staining lysosomes is discussed.


Regulation of Staphylococcus protease using complement, interferon and immunoglobulin as substrates. The effects of various agents on the cleavage of serum albumin, interferon, immunoglobulin and complement component C1q by the extracellular protease from Staphylococcus aureus were analysed by SDS-polyacrylamide gel electrophoresis. Arachidonic acid moderately stimulated the proteolysis of serum albumin, interferon and complement component. Phosphatidic acid effectively enhanced the proteolysis of serum albumin and IgG, whereas it inhibited the cleavage of IgM. The proteolysis of IgG was appreciably enhanced by sphingosine. In contrast, phosphatidyl choline and phosphatidyl glycerol were shown to have an inhibitory effect on the proteolysis of IgG and IgM. Phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine also inhibited the proteolysis of IgG. The failure of any of these agents to exert a persistent effect on the cleavage of all substrates, revealed the complexity of the interactions among the agent, the substrate and the protease.


Aberrant Myc expression in the murine trisomy 15 syndrome is correlated with prolonged proliferation in spongiotrophoblast cells of the placenta. In the mouse the protooncogene Myc is localized to Chromosome 15. Embryos with trisomy 15 (Ts15) are severely retarded and die in utero at an early stage. The placenta of these embryos shows enlargement of the fetal spongiotrophoblast. In the spongiotrophoblast and giant cells of the normal euploid placenta, Myc expression declines from day 10.5 of gestation onward whereas Myc expression persists at a high level in Ts15 cells. At the same time these Ts15 cells show prolonged proliferation which consequently leads to the observed enlargement of that tissue layer in Ts15 placentas.


The thymus: new views of an old gland.

The thymus: new views of an old gland. The existence of the thymus was recognised by ancient scientists and doctors, although its vital role in the development and maintenance of the competent T cell repertoire has been fully appreciated only comparatively recently. Current research is now moving on to explore the role of the thymus in interactions between the brain, the nervous system and the immune response. This is an exciting new field which should lead to advances in the management, treatment and prognosis of disease in man and animals.


The organisation of production of genetically-engineered proteins in yeast. The use of a variety of genetic-engineering techniques to introduce foreign DNA into living yeast cells has resulted in the production in these cells of the corresponding proteins. These include enzymes, antibodies, vaccines, anti-viral agents, and hormones. This article discusses the techniques of genetic engineering in yeast and suggests the lines of future progress in the economic production of novel proteins for use in therapy.


Serodiagnostic value of the 19 kilodalton antigen of Mycobacterium tuberculosis in Indian patients. Previous studies in UK subjects suggested that the 19 kDa protein antigen of Mycobacterium tuberculosis might be valuable in the serodiagnosis of paucibacillary tuberculosis. In this study, antibody titres for the 19 kDa antigen were higher in healthy controls in India than in the UK. Consequently, the diagnostic sensitivity of this antigen and its TB23 epitope was negligible in Indian patients with tuberculosis. However, a diagnostic sensitivity of 50% was achieved in patients with skin tuberculosis on the basis of a high ratio between antibody titres for the whole antigen and its TB23 epitope.


Histological diagnosis of human papillomavirus (HPV)-related dysplasia: quality control by in situ hybridization (ISH) analysis. The Authors studied 54 cervical biopsies using in situ hybridization (ISH) to identify HPV related lesions. 26 biopsies lacked clear cut koilocytotic atypia on histological examination and 28 had a diagnosis of grades 1 and 2 intraepithelial neoplasia. Histological diagnosis of HPV-related dysplasia were studied using ISH with biotinylated probes (Phatogene DNA probe assay Enzo Diagnostics). Probes included HPV types 6/11, 16/18 and 31/35/51. HPV DNA were detected in 18 of 28 (64.3%) cervical biopsies with diagnosis of grade 1 and grade 2 cervical intraepithelial neoplasia with koilocytotic atypia. None of the 26 cervical biopsies equivocal for koilocytotic atypia had detectable HPV DNA with ISH. These results suggest that ISH can be used for the quality control in the histological diagnosis of koilocytotic atypia.


Effect of phytanic acid on cultured retinal pigment epithelium: an in vitro model for Refsum's disease. Refsum's disease (heredopathia atactica polyneuritiformis) is an autosomal recessive retinitis pigmentosa syndrome caused by the excessive deposition of phytanic acid in ocular tissues. It is thought that phytanic acid causes retinal degeneration either by interfering with vitamin A metabolism in the retinal pigment epithelium or by altering photoreceptor cell membrane structure. Efforts to elucidate the molecular mechanism of phytanic acid's retinal toxicity have been hampered by the rarity of human pathological specimens and by the inability to reproduce the disease in living animal models. In this study, an in vitro model for Refsum's disease was established by exposing cultured human and bovine retinal pigment epithelial cells to phytanic acid bound to bovine serum albumin at concentrations comparable to levels found in affected humans. Ultrastructural studies show that these cells exhibit morphological changes consistent with those observed in pathological specimens from patients with Refsum's disease. Biochemical assays of retinoid metabolism by cell membranes from control cells and from cells exposed to 200 microM phytanic acid demonstrate that the ability to esterify retinol and to isomerize all-trans retinoids to 11-cis retinoids remains intact despite the deposition of large amounts of phytanic acid. The work described here is strong evidence against the hypothesis that phytanic acid inhibits vitamin A metabolism in the retinal pigment epithelium, and it demonstrates the potential use of cultured retinal pigment epithelial cells in modeling this and other degenerative diseases of the retina.


Crystallin gene expression in the process of lentoidogenesis in cultures of chicken lens epithelial cells. One alpha B- and three different beta-crystallin cDNA clones were isolated from a chicken lens cDNA library by using anti-crystallin antibodies. The sequence of alpha B-crystallin cDNA showed more than 70% homology with exons of alpha B-crystallin genes of the human and hamster. Two beta-crystallin cDNAs showed almost identical sequences with previously reported chicken beta B1- and beta A3/A1-crystallin genes. The remainder showed 80% homology of sequence with bovine beta B2-crystalline cDNA. Using these newly cloned cDNAs, in addition to cDNAs of alpha A- and delta-crystallin, we examined the expression pattern of these crystallins in the process of lentoidogenesis of cultured lens epithelial cells of the chicken. All crystallins except beta-crystallins were expressed through the period of cell culture, but three beta-crystallins were expressed only after the confluent stage. These results suggest that: (1) alpha A-, alpha B- and delta-crystallin cDNAs can be used to detect differentiation of the lens epithelial cell; and (2) beta-crystallin cDNAs are superior in the detection of chicken lens fibre differentiation in vitro to delta-crystallin cDNA, which is ectopically expressed by various non-lenticular tissues.


Effect of sucralfate on gastric mucosal calcium channels activity. 1. The effect of anti-ulcer agent, sucralfate, on the activity of the gastric mucosal calcium channel was investigated using calcium channels purified from rat gastric epithelial cell membranes. 2. The channels on reconstitution into phosphatidylcholine vesicles responded in a concentration-dependent manner to a calcium channel activator, BAY K8644, as well as to a calcium channel antagonist, PN200-110. The 45Ca2+ uptake was inhibited by sucralfate. Maximum inhibitory effect was attained at 100 micrograms/ml sucralfate, at which point a 52% decrease in the uptake occurred. 3. EGF-induced channel protein phosphorylation showed an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the vesicles containing the phosphorylated channels displayed a 48% greater 45Ca2+ uptake. This phosphorylation process was inhibited by sucralfate. Furthermore, sucralfate also interfered with the binding of EGF to calcium channel protein. 4. The results indicate that sucralfate protects the cellular integrity from calcium imbalance by modulating the EGF-stimulated gastric mucosal calcium channel phosphorylation.


Effect of vitamin A on hexachlorocyclohexane (HCH) toxicity in the rat. 1. Technical hexachlorocyclohexane (HCH) depleted hepatic stores of vitamin A in male albino rats to cause secondary vitamin A deficiency. 2. Toxicity of HCH in rats is augmented by dietary vitamin A-deficiency as evidenced by growth retardation, organ hypertrophies and alterations in the serum and liver levels of the marker enzymes of toxicity. 3. Supplementation of dietary vitamin A to the rats either in adequate (2000 IU/kg diet) or in an excess but not hypervitaminotic level (10(5) IU/kg diet) resulted in significant protection against the toxicity of HCH. 4. The activities of the hepatic xenobiotic metabolizing enzymes were generally low (with the exception of glutathione S-transferase) in the vitamin A-deficient rats compared to those of the vitamin A supplemented diet groups. 5. The results indicated that dietary vitamin A influences the response of male albino rats to HCH toxicity possibly by modulating the activities of hepatic xenobiotic metabolizing enzymes.


A pharmacological profile of glycopyrrolate: interactions at the muscarinic acetylcholine receptor. 1. Glycopyrrolate, a synthetic quaternary ammonium compound had biphasic effects on the contraction of guinea-pig atrium. At concentrations between 0.4 and 20 microM, glycopyrrolate induced a small but consistent increase in the contraction force. 2. Further increase in the concentration of glycopyrrolate produced a concentration-dependent reduction in the force of contraction with a EC50 of 0.24 mM. 3. This negative inotropic effect was opposed by a Ca2+ channel agonist, Bay K 8644. Glycopyrrolate also antagonized potently the depressant effects of carbachol and acetylcholine in guinea-pig atrium. 4. Schild analysis showed a pA2 value of 8.16 against carbachol and 8.39 against acetylcholine. These similar pA2 values suggested that both compounds may have a common interacting site. The interactions however cannot be explained by a simple competition model as the slopes of the Schild plots were larger than unity. 5. The mutual competition between glycopyrrolate and quinuclidinyl benzilate (QNB), gallamine or methoctramine indicated that glycopyrrolate could have multiple action sites in the atrium. Interaction at an allosteric site was implicated. 6. Radioligand binding studies showed that glycopyrrolate displaced pirenzepine (PZ) and AF-DX 384 from their binding to the M1 and M2 muscarinic receptors in guinea-pig brain membranes respectively. 7. The respective binding constants (Kis) were 0.60 and 0.03 nM. The Hill coefficient value (nH) for glycopyrrolate against [3H] PZ was larger than unity, suggesting positive cooperativity at the receptor complex. In contrast, the nH of the agonist [3H] AF-DX 384 was not different from unity, indicating a simple competitive inhibition. 8. Our results confirm the antimuscarinic properties of glycopyrrolate.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of antiepileptic drugs, calcium channel blockers and other compounds on seizures induced by activation of voltage-dependent L calcium channel in DBA/2 mice. 1. The convulsant activity of the calcium voltage L-channel agonist Bay k 8644 was studied in genetically epilepsy prone DBA/2 mice. 2. Seizures were induced by intracerebroventricular injection of Bay k 8644. 3. These seizures were reversed by some calcium channel blockers such as dihydropyridines, some excitatory amino acid antagonists such as 2-amino-7-phosphonoeptanoate and CPPene, 2-chloro-adenosine, some anticonvulsant drugs such as magnesium valproate, diazepam and clonazepam and two kappa opioid agonists (U-50488H and U-54494A). 4. The remaining antiepileptic drugs (carbamazepine, phenytoin, phenobarbital and trimethadione) were ineffective in this respect. Other anticonvulsant compounds such as dizocilpine (MK 801), ketamine and drugs enhancing GABAergic transmission did not significantly affect the clonic phase of the seizures induced by Bay k 8644. 5. These results show that Bay k 8644 seizures are relatively resistant to some anticonvulsant compounds. The role of some neurotransmitters on seizures induced by Bay k 8644 is discussed.


The lymphoproliferative disease virus of turkeys represents a distinct class of avian type-C retrovirus. The lymphoproliferative disease virus of turkeys (LPDV) is the etiological agent of a rapidly developing lymphoproliferative process in turkeys. To better understand the genetic relationships of LPDV to other retroviruses we determined the nucleotide sequence of its pol gene. Comparative computer analyses of the deduced amino acid sequences of the reverse transcriptase and integrase domains within pol established that LPDV represents a distinct class of avian retroviruses that is most closely related to the avian leukemia-sarcoma viruses.


Analysis of the gene encoding a 16-kDa proteolipid subunit of the vacuolar H(+)-ATPase from Manduca sexta midgut and tubules. Vacuolar ATPases (V-ATPases), originally characterised as components of endomembranes, have also been implicated in epithelial ion transport, both in vertebrates and in insects. The ATPase is particularly noteworthy in lepidopteran larvae, where it generates large transepithelial potential differences and short-circuit currents across the midgut epithelium. A cDNA library from Manduca sexta larval midguts and Malpighian tubules was screened with a Drosophila melanogaster cDNA encoding the 16-kDa proteolipid subunit of the V-ATPase, and a 1.4-kb cDNA sequenced in its entirety. The sequence contains a long open reading frame, encoding a putative peptide of 156 amino acids (aa) and with an M(r) of 15,967, in close agreement with values previously suggested by sodium dodecyl sulfate-polyacrylamide gels of M. sexta midgut proteins. Correspondence of the deduced aa sequence with those of other species, particularly D. melanogaster, was extremely close. Northern blots of M. sexta midgut mRNA at high stringency revealed two transcripts of 1.4 and 1.9 kb, whereas genomic Southern blots suggest that there is only a single copy of the gene in M. sexta. The possibility that members of the 16-kDa gene family might serve multiple roles in transport and membrane communication is discussed.


Radiation enhancement of laser palliation for malignant dysphagia: a pilot study. Laser therapy offers rapid relief of dysphagia for patients with cancers of the oesophagus and gastric cardia but repeat treatments are required approximately every five weeks to maintain good swallowing. To try to prolong the treatment interval, 22 elderly patients were given additional external beam radiotherapy. Nine had squamous cell carcinoma and 13 adenocarcinoma: five had documented metastases. Six received 40 Gy and 16,30 Gy in 10-20 fractions. A 'check' endoscopy was performed three weeks after external beam radiotherapy. Dysphagia was graded from 0-4 (0 = normal; 4 = dysphagia for liquids). The median dysphagia grade improved from 3 to 1 after laser treatment. This improvement was maintained in the 30 Gy group but there was a noticeable deterioration in three of those who had received the higher radiation dose. A lifelong dysphagia grade of 2 or better was enjoyed by 14 of 16 patients in the 30 Gy group but only two of six in the 40 Gy group. The dysphagia controlled interval was 9 weeks (median) after check endoscopy and subsequent endoscopic procedures were required every 13 weeks to maintain good swallowing. There were no endoscopy related complications. Combined treatment is a promising approach for reducing the frequency of endoscopic treatments. The 30 Gy dose seems more appropriate and may prolong survival. A randomised study to test these conclusions is in progress.


Endoscopic palliation for inoperable malignant dysphagia: long term follow up. This prospective non-randomised trial of 128 selected patients with unresectable oesophageal or gastrooesophageal junction cancers aims to evaluate the initial relief of malignant obstruction by means of bipolar electrocoagulation for both circumferential and submucosal strictures of Nd:YAG laser for the other patients. A limited dilatation was performed initially if a small calibre endoscope was unable to pass through the stricture. Prompt and significant relief of dysphagia without complications was achieved in 83% of patients. Improved patients were retreated monthly during the follow up period. Radiotherapy was recommended when possible. Symptomatic relief of obstruction lasted 4.2 months on average and 76% of patients remained palliated until death. Monthly retreatment using the most appropriate endoscopic procedure for the tumour configuration and radiotherapy after endoscopic relief of obstruction seems to give the best palliation for patients with unresectable cancers of the oesophagus or gastrooesophageal junction.


Extraluminal hyperechogenic echo detected by transvaginal ultrasonography as a prognostic factor in recurrent hydatidiform mole. Measuring beta hCG titers by either bioassay or radioimmunoassay has become the cornerstone in the management and treatment of hydatidiform mole. It is this very determination which will indicate either spontaneous remission or the need for chemotherapy treatment due to rising or plateauing titers. Herein, we report on the potential assistance of a unique ultrasonographic appearance of a hyperechogenic shadow located in the uterine wall, before and after an attempt for full evacuation of hydatidiform mole. The behavior of this echogenic area was more sensitive in predicting the course of the disease than did the beta hCG titers. Thus, using transvaginal sonography may serve as another predictor and indicator in evaluating the treatment of hydatidiform mole.


[Effect of aprotinin on fibrosis of nerve sutures]. Although fibrin-glue has been used in many areas of surgery, it has not become fully established in nerve coaptation. Initially, significant advantages were expected; however, as the fibrin clot dissolved prematurely and gapping occurred, antifibrinolytic substances were added to the glue. Following this procedure, fibroses occurred frequently, which remains a problem. In this study, the influence of aprotinin's fibrosis-inducing effect was investigated. Aprotinin demonstrated no additional fibrosis-promoting effect.


[A severe skin reaction following mexiletene]. A 70-year-old man was treated with oral mexiletene because of ventricular premature beats due to ischemic heart disease. He developed a severe maculopapular rash which did not respond to antiallergic treatment, but disappeared after discontinuation of the drug. Adverse effects on the central nervous, cardiovascular and gastrointestinal systems have been described. A rash is rare, and in our case was of unusual severity.


The spectrum of CFTR mutations in south-west German cystic fibrosis patients. The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 110 cystic fibrosis (CF) patients from the south-west of Germany was screened for 12 different mutations. This analysis resulted in an identification of 79% of all CF mutations and a complete genotype in 66% of the families. The most common mutation found was delta F508 (67%). Another 5 mutations accounted for a further 12.5% (4% G542X; 3% R553X; 3% N1303K; 2% 1717-1 G-->A; 0.5% G551D) whereas 6 mutations (R117H, A455E, delta I507, S549I, S549N, and R1162X) were not found. Fifty-four (49%) patients were delta F508 homozygotes and 18 (16.5%) were compound heterozygotes for delta F508 and one of the rarer mutations. These frequencies differ slightly from those found in the north of Germany and considerably from those reported from the south of Europe, which seems to be consistent with a north to south decline of the relative abundance of delta F508. Two patients, age 6 and 25 years, were compound heterozygotes for G542X and N1303K. The clinical features of the 6 year old were characterised by severe gastrointestinal and as yet only mild pulmonary complications whereas the 25 year old manifested severe pulmonary and gastrointestinal symptoms indicating that the N1303K mutation of the C-terminal CFTR nucleotide binding fold significantly impairs protein function in both the pancreas and the lungs.


A termination mutation (2143delT) in the CFTR gene of German cystic fibrosis patients. German patients with cystic fibrosis (CF) were screened for molecular lesions in exon 13 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by single strand conformation polymorphism (SSCP) and chemical cleavage of mismatch analyses. Direct sequencing of four samples that displayed the same SSCP pattern and that were susceptible to cleavage of hetero-duplexes by osmium tetroxide revealed, in all cases, a deletion of a single T residue at nucleotide position 2143 within codon 671 of the CFTR gene. As a result, leucine codon 671 is changed into a termination codon. In total, the 2143delT mutation was confirmed in 6 out of 271 German non-delta F508 CF chromosomes by artificial restriction fragment length polymorphism analysis, indicating that this frameshift mutation accounts for about 2% of German non-delta F508 mutations. The 6 pancreas insufficient patients who are compound heterozygous for 2143-delT suffer from the typical features of pulmonary and gastrointestinal CF disease. The 2143delT mutation completes the panel of the more frequent CFTR mutations that reside on the "delta F508 haplotype" and that contribute to its overpresentation among German non-delta F508 alleles that are associated with severe forms of disease.


Assessment of iduronate-2-sulfatase mRNA expression in Hunter syndrome (mucopolysaccharidosis type II). Eight unrelated patients with Hunter syndrome were investigated for expression of iduronate-2-sulfatase (IDS) mRNA by reverse transcription (RT) linked to polymerase chain reaction (PCR), or RT-PCR. The entire coding region was studied by amplification of two overlapping segments of 0.7 and 1.1 kb. Seven children with Hunter syndrome had PCR products indistinguishable in size from normal. One patient, with clinically severe disease, did not produce either IDS product although mRNA for a control gene was readily amplified. This method rapidly identifies patients having absent or qualitatively abnormal IDS mRNA and may be useful in investigating genotype-phenotype relationships.


A novel CFTR mutation, 4035delA, detected by non-radioactive SSCP analysis. German cystic fibrosis patients were screened for mutations in exon 21 of the cystic fibrosis transmembrane conductance regulator gene by a non-radioactive variation of the single-strand conformation polymorphism technique. Asymmetric polymerase chain reaction amplification was used to produce single strands of exon-containing genomic sequences that were analyzed on polyacrylamide gels subsequently stained with ethidium bromide. This rapid technique led to the identification of a novel mutation, a 1-bp deletion at position 4035(A) of the cDNA sequence. The patient, who is also heterozygous for the delta F508 mutation, exhibits an intermediate form of the disease.


Cultured human cord blood cells spontaneously produce a factor with basophil-promoting activity. Cord blood is a source for pluripotential stem cells capable of differentiating into various hemopoietic cell lines in the presence of suitable specific growth factors. Without additional growth factors, cultured cord blood cells give rise to large numbers of basophils. We have recently defined a human basophil growth promoting factor, designated as interleukin-3-like activity (IL-3-LA), produced spontaneously by human monocytes and lymphocytes. In order to explain the phenomenon of spontaneous basophil development in cord blood cultures, we studied the relationship between basophil production and IL-3-LA release in these cultures. IL-3-LA produced by cord blood mononuclear cells increased from day 3 to day 14 and then decreased gradually by day 35. Basophil development was observed from day 14 on (33% +/- 6.4) and peaked on day 21 (51% +/- 7.4). Histamine release followed the same pattern i.e., 10 +/- 3.4 ng/ml on day 14, and 23 +/- 6.5 ng/ml on day 21. It is suggested that IL-3-LA spontaneously released by cord blood mononuclear cells induces basophil development in these cultures.


Cytoplasmic expression of a CD24-related epitope in human PHA activated normal T lymphocytes. In this study we have analysed by immunoperoxidase (IPx) and indirect immunofluorescence (IIF) the intracellular and cell surface reactivity of VIB-E3 mAb, previously clustered as anti-CD24 antigen, on resting and activated normal human T lymphocytes. By IPx assay VIB-E3 mAb did not show reactivity with normal resting T cells. In contrast, the analysis of 11 different samples of PHA activated normal mononuclear cells, showed an intracytoplasmic expression of CD24. Kinetic studies showed that CD24 appears 24 to 48 h after PHA stimulation. To our knowledge, this is the first evidence that a CD24-related epitope is expressed in normal activated T lymphocytes.


Reactive oxygen species modified DNA fragments of varying size are the preferred antigen for human anti-DNA autoantibodies. In vitro studies were carried out to determine if reactive oxygen species modified DNA molecules are the preferred antigen for anti-DNA antibodies found in SLE sera. Reactive oxygen species were generated by 254 nm irradiation of hydrogen peroxide. Single stranded breaks, decrease in Tm and modification of adenine (21.7%) and thymine (48%) were the major effects observed on native DNA fragments of 300 bp in length. The ROS-modified DNA showed increased binding with naturally occurring anti-DNA autoantibodies as compared to unmodified DNA fragments. These results were substantiated by competition ELISA. Measurement of binding with DNA fragments of varying size revealed considerably increased binding as the fragment size increased from 50 bp to 800 bp. The relative affinity of anti-DNA IgG for ROS-modified and native DNA fragments of 300 bp were in the order of 6.26 x 10(-8) M and 4.07 x 10(-8) M, respectively.


CD5 and immunoglobulin VH gene expression in B-cell lines from patients with autoimmune diseases. We studied the CD5 mRNA expression and VH gene family usage in Epstein-Barr virus (EBV)-immortalized B-cell lines derived from the blood of patients with type 1 diabetes (IDDM) of recent onset and of patients with polyneuritis cranialis multiplex (cranial neuritis; CN). After immortalization with EBV, at least 10 cell lines from each subject were tested for surface CD5 and CD20. mRNA expression was studied using cDNA probes for the six VH families as well as for CD5. The EBV lines from the IDDM patients used the VHIV family more frequently and VHI and VHII families less frequently than lines from controls. EBV lines from CN patients expressed the VHI and VHII families more often than those of the controls. When the IDDM and CN lines were compared, the lines derived from IDDM patients were found to use VH families I and II less frequently and VH families IV and V more frequently than lines from CN patients. There were no significant differences in the mean numbers of CD5+ B cells in the cell lines tested. More than half of the lines from each patient expressed CD5 at the mRNA level. No correlation was seen between the expression of surface CD5 and the level of CD5 mRNA expression. There was, however, a positive correlation between the usage of VH families III, V and VI, and the CD5 mRNA expression. In conclusion, the usage of VH families I to VI seemed to differ in patients with IDDM and CN. No differences were seen in the surface CD5 expression, but the lines expressing CD5 mRNA preferentially used the VH families III, V and VI.


Regulation of human T lymphoblast growth by sensory neuropeptides: augmentation of cholecystokinin-induced inhibition of Molt-4 proliferation by somatostatin and vasoactive intestinal peptide in vitro. The effects on proliferation of Molt-4 lymphoblasts of cholecystokinin (CCK-8), somatostatin-14 (SS), vasoactive intestinal peptide (VIP) and substance P (SP) were investigated using different combinations of the peptides, peptide analogs and their antagonists. In vitro proliferation of the cells was measured by a colorimetric assay for cell growth and survival. Results indicate that SP and SP (3-11) stimulated, whereas CCK-8, VIP and SS inhibited, proliferation in a dose-dependent manner (P < 0.05). Unsulfated CCK-8 had no effect on growth of Molt-4 lymphoblasts, and a specific antagonist of CCK, at a concentration 10(-6) M, diminished the inhibitory effect of CCK-8 on Molt lymphoblasts (P < 0.05). This suggests that the inhibitory action of CCK-8 was mediated by peripheral-type CCK receptors. SS and VIP, at equimolar concentrations of 10(-6) M, significantly augmented the CCK-8-induced inhibition of Molt-4 lymphoblast proliferation. However, none of the inhibiting neuropeptides suppressed stimulation of Molt-4 lymphoblast proliferation in response to SP. These data suggest a role of sensory neuropeptides including CCK in modulating human T lymphoblast proliferation during neuroendocrine interactions with the immune system.


Establishment of a murine pre-B cell clone dependent on interleukin-7 and stem cell factor. To identify cytokines required for proliferation of murine pre-B cells, we established a pre-B cell clone MH11 (B220+ MB-1+ sIgM-) on a stromal cell line ST2 from day 13 fetal liver. The growth of MH11 is dependent on ST2. Another stromal cell line PA6, non-secretor of IL-7, could not support MH11 unless IL-7 was added. We investigated the effect of cytokines on proliferation of MH11 with or without stromal cells. IL-7 had a stimulatory effect on proliferation of MH11, but IL-7 alone could not support MH11 growth without ST2. Recombinant stem cell factor (rSCF) also had a positive effect on MH11. rSCF and rIL-7, when added together, could maintain the growth of MH11 in the absence of stromal cells. Moreover, the growth of MH11 on ST2 was inhibited almost completely by anti-c-kit monoclonal antibody (mAb). These results demonstrate that direct SCF/c-kit interaction is involved in the stimulation of pre-B cells.


Continuous ambulatory electrocardiography among workers exposed to methylmethacrylate. Methylmethacrylate (MMA), a widely used monomer in the manufacture of acrylic polymers, has been reported to cause cardiac troubles in industrial workers. The effects of MMA on the heart was assessed by continuous ambulatory electrocardiographic records. The study was performed in 22 occupationally exposed workers and in 18 healthy controls. Our study did not support the hypothesis that MMA is responsible for cardiomyodystrophy. However, supraventricular and ventricular ectopic beats were significantly more frequent among exposed workers versus controls (P < 0.01). Moreover, repolarization changes such as large T waves were noted only in exposed workers (eight cases against none in the control group). Although there was no clear connection between MMA exposure and the recorded cardiac changes in the exposed group, the role of MMA cannot be totally excluded.


Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens. A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8-12 h at -50 to -40 degrees C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45 degrees C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4 degrees C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.


Histochemical characterization of anionic constituents in oocyte-cumulus complex of rats. Fertilization is a process that involves the recognition and adhesion of negatively charged spermatozoa to the oocyte investments. It is not known, however, if charge properties of the interacting gametes play a role in fertilization. The present study evaluates the content and distribution of anionic constituents in the oocyte-cumulus complex of rats. Polycationic colloidal gold (PCG), ruthenium red (RR) and wheat germ agglutinin (WGA) were used as cytochemical markers of anionic sites at the light (LM) and electron microscopical (EM) levels. Isolated oocyte-cumulus complexes were fixed with glutaraldehyde (GA) and OsO4 containing RR, or with GA without RR, and embedded in araldite or LR-gold. For LM, deresined, semi-thin, araldite-embedded sections were labelled with PCG intensified by silver, or with biotinylated lectins visualized by avidin-peroxidase. For EM, thin LR-gold sections were labelled with PCG, whereas RR labelling was examined in araldite sections. The zona pellucida (ZP) failed to bind any of the polycationic markers used, but intensely bound neutralized WGA. In contrast, cumulus cell membranes bound PCG but not RR, whereas the oolemma bound RR but not PCG. The results indicate that the ZP is practically devoid of negatively charged constituents, and tends to repel positively charged ligands possibly due to the presence of cationic determinants. The binding of PCG to cumulus cells probably reflects a high content of membrane-bound heparan sulphate, whereas the binding of RR to the oolema indicates the presence of membrane sialoglycoconjugates.(ABSTRACT TRUNCATED AT 250 WORDS)


The intramucosal distribution of gastric alcohol dehydrogenase and aldehyde dehydrogenase activity in rats. Using qualitative and microquantitative histo-chemical techniques, alcohol dehydrogenase and aldehyde dehydrogenase activity was studied in the gastric mucosa of male and female rats. Alcohol dehydrogenase was demonstrated by staining reactions with maximum activity in surface and neck cells and with clearly weaker activity also in parietal cells. Aldehyde dehydrogenase could be detected in surface and neck cells, and also to a comparable degree in the parietal cells. Quantitative analyses of microdissected samples yielded high values for alcohol dehydrogenase activity exclusively in the superficial part of the gastric mucosa, whereas low-Km aldehyde dehydrogenase activity showed a decreasing gradient from the surface to the deeper parts of the mucosa. Sex differences could not be confirmed.


Substance P and neurokinin A immunoreactive nerve fibres in the developing salivary glands of the rat. The time of appearance and distribution of substance P (SP) and neurokinin A (NKA) immunoreactive nerve fibres in developing salivary glands of the rat were studied by the use of indirect immunohistochemical methods. The glands were examined at daily intervals from the 15th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 12th, 16th and 30th postnatal day. The findings were compared to samples from adult. The first SP- and NKA-immunoreactive (IR) nerve fibres appeared on the 19th day i.u. in the parotid and submandibular glands and were abundantly distributed around developing ductal branches. In the mesenchyme around the developing ductal branches of the parotid gland the fibres appeared on the 20th day i.u. In the submandibular gland NKA-IR fibres appeared in the mesenchyme surrounding the developing ductal branches on the 19th day i.u. and SP-IR fibres on the 21st day i.u. Around blood vessels of both glands, SP- and NKA-IR fibres made their appearance only much later, on the second postnatal day. The number of SP- and NKA-IR nerve fibres in the developing salivary glands was already high on the 19th day i.u. when they were first detected. From this point up to the 16th postnatal day the glands were richly innervated by the fibres, but later the numbers slowly decreased to adult levels. The abundance of SP- and NKA-IR nerve fibres especially around the ductal branches and secretory structures in the developing salivary glands suggests a role in the functional maturation of salivary glands.


Calcitonin gene-related peptide, substance P and GAP-43/B-50 immunoreactivity in the normal and arthrotic knee joint of the mouse. The aim of this study was to describe the normal distribution of calcitonin gene-related peptide (CGRP) and substance P (SP) containing fibres in the knee joint of the mouse and to obtain insight into the changes in innervation associated with degenerative processes in the joint. Arthrosis was induced by a single subpatellar intra-articular injection of bacterial collagenase. After decalcification in EDTA solutions, the CGRP and SP fibres were visualized by peroxidase-antiperoxidase pre-embedding immunocytochemistry for light microscopy. Control experiments on the mouse brain as a reference for the effect of EDTA on the immunostaining showed that the decalcification procedure with EDTA had not impaired the immunostaining. A rich innervation of thin varicose CGRP and SP immunoreactive fibres was found in most peri- and intra-articular tissue components. The periosteum, synovial tissues, the joint capsule and the intra-articular fat tissues were richly innervated. Less intense innervations were also found in the subchondral bone plates of the tibio-femoral joint and of the patella. Fibres were also found in the soft tissues between the patellar tendon and the femoral groove. No differences could be found between the location of CGRP and SP fibres with respect to the localization in the joint, but generally more CGRP fibres were found. The collagenase-induced osteoarthrosis was characterized by sclerosis of the subchondral bone, patellar dislocation, osteophyte formation, synovial proliferation and by severe cartilage abrasion, particularly on the medial side of the femoro-tibial joint. The overall distribution of CGRP and SP fibres was the same as in the control joints. However, major differences were found in all studied joints at specific locations around the cruciate ligaments, in the synovium around the patella, in the soft tissues lateral of the patella and in plica tissue between the patella and femoral groove. The CGRP and SP innervation was no longer detectable by immunolabelling with the antibodies. With a polyclonal antibody to the growth associated protein GAP-43/B-50, signs of degenerated axonal profiles were observed in these locations. At other peripheral locations, such as the muscles, the GAP-43/B-50 distribution was normal. In conclusion, the present study provides detailed information on the localization of CGRP and SP fibres, which may be involved in pain perception. Knowledge of the changes that occur during arthrosis may give more insight into the clinical symptoms.


Co-localization and molecular association of dystrophin with laminin at the surface of mouse and human myotubes. In Duchenne muscular dystrophy (DMD), deficiency of the protein dystrophin results in necrosis of muscle myofibres, associated with lesions in the sarcolemma and surrounding basal lamina. Dystrophin has been proposed to be a major component of the sub-sarcolemmal cytoskeleton involved in maintaining the integrity of the myofibre plasma membrane, and is known to associate with a group of sarcolemmal glycoproteins, one of which exhibits high affinity binding to the basal lamina component laminin. However, a direct or indirect transmembrane association of dystrophin in muscle cells with the myofibre basal lamina has not been demonstrated. To address this question we have examined dystrophin immunostaining and immunoprecipitation patterns in cultured mouse and human myotubes in comparison with that of the basal lamina component, laminin. Dual-immunolabelling revealed virtually complete co-localization of dystrophin on the inside surface of the muscle cell sarcolemma with plaques and veined arrays of laminin accumulating on the extracellular face. This pattern of laminin and dystrophin distribution was distinct from that of other cell surface molecules expressed in myotubes such as the neural cell adhesion molecule, NCAM, and the beta 1 integrin receptor, and immunoprecipitation of dystrophin from solubilized myotube extracts resulted in co-purification of laminin B1 chain confirming an association between these two components. The results thus provide the first direct cellular evidence of a transmembrane linkage between dystrophin in the sarcolemmal cytoskeleton with laminin in the overlying basal lamina. While the immunocytochemical distribution of laminin was apparently normal in dystrophin-deficient muscle cells, elevated levels of soluble laminin were present in extracts of mdx compared with normal mouse skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)


Localization of ahe alpha 6 and beta 4 integrin subunits in normal human non-lymphoid tissues. The alpha 6/beta 4 integrin, of undefined receptor activity, has been shown to be expressed in a variety of murine epithelial cells. To gain information on the role of this heterodimer in tissue architecture as well as in malignant transformation we have performed an extensive immunohistochemical analysis of normal human tissues using monoclonal antibodies to alpha 6 and beta 4 subunits. Because alpha 6 is known to associate also with the beta 1 subunit to form a non-promiscuous receptor for laminin, the expression of beta 1 chain was also evaluated. The results of this study have shown that the alpha 6 chain has a wide distribution in tissues, including small vessels and peripheral nerves. alpha 6 colocalizes with beta 4 and beta 1 in most epithelial cells at the basolateral or basal aspect abutting the basement membrane. In a minority of tissues lacking beta 4, the alpha 6 chain is coexpressed with beta 1. These findings demonstrate that the expression of alpha 6/beta 1 laminin receptor and alpha 6/beta 4 heterodimer is phylogenetically conserved, suggesting that they are likely to play an important role in cellular scaffolding through binding to laminin and to still uncharacterized ligand/s present in basement membranes.


A chimeric N-cadherin/beta 1-integrin receptor which localizes to both cell-cell and cell-matrix adhesions. To study the molecular mechanisms involved in formation of cell contacts, we have transfected cultured cells with a chimeric cDNA encoding the cytoplasmic and transmembrane domains of beta 1 integrin and the extracellular region of N-cadherin and determined the subcellular distribution of the chimeric molecule. We show that the chimeric receptor associates preferentially with cell-matrix focal contacts, suggesting that its distribution is directed by its beta 1 integrin segment, presumably via interactions of the cytoplasmic domain with cytoskeletal elements characteristic of focal contacts. Transfected cells which expressed relatively high levels of the cadherin/integrin chimera underwent an apparent epithelialization and contained the molecule both in cell-matrix and cell-cell contacts. Location in cell-cell contacts indicates competence of the cadherin extracellular domain to participate in formation of cell-cell junctions using a foreign cytoplasmic domain. Labeling of these cultures for talin, which is normally associated only with matrix adhesions, revealed specific labeling along the newly formed intercellular junctions. This suggests that the local association of talin with these sites is induced by the cytoplasmic tail of beta 1 integrin receptor presented by the chimeric protein. These results suggest that the formation of adherens-type junctions is driven by the cooperative interactions of the relevant adhesion molecules (cadherins and integrins) both with the respective extracellular ligands and with the cytoskeleton.


Behavior of large nucleic acids in reversed-phase high-performance liquid chromatography. Large nucleic acids can be separated by reversed-phase high-performance liquid chromatography. Analysis shows that the retention time depends not only on the chain length but also on the base composition and the secondary structure of the molecule. A model is proposed to interpret their behavior. This model, called "multiple-point interaction theory" is based on the observation that macromolecules are flexible and very large compared to the hydrophobic phase (octadecylsilane) of the column. It explains the behavior of large nucleic acids in terms of an equilibrium of the macromolecule between the two phases through a multiple-point attachment to the chromatographic matrix, the parameters of the equilibrium being both the hydrophobicity of the base and the number of attachment points. This model fits the experimental data and can be applied to all types of flexible macromolecules, especially proteins and nucleic acids, when they are chromatographed on reversed-phase columns. The model is used to explain the separation of nucleic acids of importance in molecular biology.


Rapid breakthrough measurement of void volume for field-flow fractionation channels. A peak breakthrough technique is described and evaluated for measuring the void volume of field-flow fractionation (FFF) channels, particularly those used for flow FFF. This technique uses a high-molecular-mass macromolecular or particulate probe that can be displaced rapidly by flow through the FFF channel with minimal transverse diffusion. The particles that emerge first are those carried through the entire length near the channel centerline at the apex of the parabolic flow profile. These particles generate a sharp breakthrough profile. The measured breakthrough time is two thirds of the void time, thus making it possible to calculate both the void time and the associated void volume. This method, although applicable to all FFF channels (and capable of extension to open tubes), is particularly useful for flow FFF because conventional low-molecular-mass void probes can diffuse into the permeable walls and thus distort void measurements. The theoretical basis of the breakthrough technique and an explanation for the sharpness of the breakthrough front are given. A method for compensating for deviations from perfect sharpness is developed in which the breakthrough time is identified with the time needed to reach 85-88% of the breakthrough peak maximum. Preliminary experimental results are shown using various protein probes in four different FFF channel systems.


Treatment of elective mute behavior in two developmentally delayed children using modeling and contingency management. Most classification schemes differentiate elective mutism from language problems seen in the developmentally delayed population. Two preschool developmentally delayed children were treated for speech reluctance using modeling and contingency management. Employing a multiple baseline across therapists, it was found that these treatment components were effective in increasing frequency of labeling behavior in both children. Results were maintained at follow-up. Generalization to new words and to spontaneous speech were also noted, and suggest that characteristics of elective mutism in this population may be similar to what is found in the general population.


Chordoma involving the skin: an immunohistochemical study of 11 cases. Immunohistochemical properties were studied in 16 lesions from 11 patients with chordoma involving the skin. There were nine men and two women ranging from 21 to 62 years old (mean, 42.6). The initial tumor was sacrococcygeal in 10 cases and nasopharyngeal in 1 case. Three lesions represented a direct extension from the primary tumor to the skin, and 13 lesions were examples of local recurrences in the skin. Immunohistochemically, all lesions showed positivity for keratin, whereas 14 lesions were positive for vimentin and 12 for protein S-100. Epithelial membrane antigen was positive in four instances and carcinoembryonic antigen was negative in all studies. No significant difference was observed between the immunoprofile of cases of direct extension and those of local recurrences. Overall, the remarkable triple positivity for keratin, vimentin, and protein S-100 was observed in 11 lesions from eight different cases (73%). This study confirmed the utility of immunohistochemistry in the differential diagnosis of chordoma from tumors with similar histologic characteristics encountered in the skin.


Keratin expression in normal skin and epidermal neoplasms demonstrated by a panel of monoclonal antibodies. The tissue labelling of a panel of monoclonal antikeratin antibodies (LL001, LL002, LL003, LP2K, BA17, LP34, CAM5.2, and LH1) recognising keratins 1, 5, 8, 10, 14, 18, and 19 were investigated in frozen and formalin-fixed normal skin. Antibodies LL001, LL003, BA17, LP34, CAM5.2, and LH1 were found to be reactive in formalin-fixed material and were used to study 23 basal cell carcinomas, 8 squamous cell carcinomas, 5 keratoacanthomas, 5 Bowen's disease, and 6 clear cell acanthomas. All these tumours demonstrated a loss of keratin 10 expression as demonstrated by loss of labelling with LH1. Keratin 14 expression, as demonstrated by LL001, was reduced but present in all the tumours except squamous cell carcinomas and keratoacanthomas where increased labelling was observed in the more differentiated areas of these tumours. Simple epithelial keratin expression was demonstrated by positive labelling with CAM5.2 and keratin 19 by BA17 in a third of basal cell carcinomas and squamous cell carcinomas. Three of the five keratoacanthomas labelled with BA17, indicating the presence of keratin 19 in these lesions. These results support the concept that keratin expression is a phenotypic marker of the state of differentiation or malignant transformation and that patterns of keratin expression are not specific to any particular premalignant or malignant disorder.


Benign lymphangioendothelioma: report of 2 cases. Benign lymphangioendothelioma is an acquired vascular proliferation clinically appearing as well-demarcated, pink to red-brown macules or plaques. Histologically, numerous endothelial-lined channels are found infiltrating throughout the dermis. Recognition and differentiation from malignant vascular tumors is essential. We report 2 cases of benign lymphangioendothelioma. One of these lesions is unique in its presentation as a subcutaneous nodule. The other is also unusual, as it resolved spontaneously.


T cell mapping of one epitope from thyroglobulin inducing experimental autoimmune thyroiditis (EAT). These data collect the advance made in the last few years in our laboratory in defining one epitope from the thyroglobulin (Tg) molecule (660 KDa) inducing Experimental Autoimmune thyroiditis (EAT) in CBA/J mice. We achieved the characterization of one EAT-inducer Tg peptide by combining "in vitro" biochemical and immunological approaches and "in vivo" studies. Since T cells recognize degraded forms of the antigen and since endogenous antigens preferentially activate class I-restricted T cells, we hypothesized that one cytotoxic T cell hybridoma, named HTC2, which prevents further EAT induction in mice injected with Tg would be specific for one EAT inducer peptide. In order to identify one Tg epitope inducing EAT, enzymatic treatment of the protein by trypsin, HPLC purification and sequence analysis were performed. Simultaneously, tryptic digests were used to pulse CBA/J macrophages and tested for their ability to be recognized by HTC2 cells. Lastly, when digests were recognized by HTC2 cells their capacity to induce EAT in CBA/J mice was evaluated. To further assess the pathogenicity of the sequenced Tg peptide, one synthetic peptide was made and its capacity to induce EAT verified. By this procedure we identified for the first time one 40 amino-acid peptide from human thyroglobulin inducing EAT in CBA/J mice.


Molecular basis for the autoreactivity against thyroid stimulating hormone receptor. The present report identifies an important immunogenic region of the TSH receptor and determinants on the TSH receptor for the two types of autoantibodies seen in hyperthyroid Graves' disease and hypothyroid idiopathic myxedema, TSAbs and TSBAbs, respectively. The immunogenic domain with no important functional determinants, is contained within residues 303-382 and involves residues 352-366 in particular. There are determinants flanking the immunogenic domain on the C-terminal portion of the receptor which are the TSBAb and high affinity TSH binding sites: residues 295-306, 387-395, and tyrosine 385. Determinants on the N-terminal portion of the external domain, centered on residues 38-45, are TSAb interactions linked to low affinity TSH binding important for signal generation: threonine 40 and residues 30-33, 34-37, 42-45, 52-56, and 58-61. These determinants are conserved in human and rat receptors, are not present in gonadotropin receptors, and are each related to separate actions of TSH: binding vs. signal generation. They can, therefore, account for organ specific autoimmunity and the different disease expression effected by TSBAbs vs TSAbs, i.e. hypo- vs. hyperthyroidism, respectively. It is proposed that, in the thyroid, hormonal (TSH, insulin, hydrocortisone, IGF-I) suppression of class I genes might be one means of preserving self-tolerance in the face of the hormone action to increase the expression of tissue specific genes such as thyroglobulin and thyroid peroxidase. Inappropriately high class I expression in the thyroid, i.e. if induced by interferon, viruses, or some as yet unknown agent, would contribute to the generation of autoimmune disease. Thus, it would result in increased antigen presentation to the immune system, particularly those autoantigens increased by TSH and its cAMP signal such as thyroglobulin or thyroid peroxidase, or whose turnover is increased by TSH and its cAMP signal, such as the TSH receptor. In the case of the latter, peptide 352-366, known to be near a protease sensitive site on the receptor [41,49], would now act as a potent self-antigen and induce the formation of receptor autoantibodies. It is further proposed that methimazole and high doses of iodide are therapeutically effective agents in thyroid autoimmune disease because they, in part, decrease MHC class I gene expression. Speculation is presented which suggests that elimination of negative regulation of MHC class I and the TSH receptor is an important factor in the development of autoimmune thyroid disease.(ABSTRACT TRUNCATED AT 400 WORDS)


Human chorionic gonadotrophin and subunit composition of maternal serum and coelomic and amniotic fluids in the first trimester of pregnancy. Sixteen matched samples of first trimester amniotic fluid (AF), extraembryonic coelomic fluid (EECF) and maternal serum (MS) were assayed for intact human chorionic gonadotrophin (hCG) and free subunits. Total beta-hCG (free beta-subunit and intact hCG) levels in the EECF (median 410 kIU/l) were 61 times greater than levels in AF (median 6.73 kIU/l) and 2.8 times greater than in MS (median 141.5 kIU/l). Levels of intact hCG in the EECF (median 245 kIU/l) were 142 times greater than in AF (median 1.73 kIU/l) and 1.6 times greater than in MS (median 157 kIU/l). Free alpha-subunit levels in EECF (median 17.3 mg/l) were 66 times greater than in AF (median 0.262 mg/l) and 12 times greater than in MS (median 1.3 mg/l). Virtually all of the total beta-hCG immunoreactivity in MS can be attributed to intact hCG, but only 60% of total beta-hCG in the EECF and 20% of that in AF can be accounted for by the intact hormone. In both EECF and AF the free alpha-subunit was a major constituent; on a molar basis the ratio of free alpha:free beta:intact hCG was 1:1.2:0.3 in AF, 1:0.6:0.5 in EECF and 1:0:5 in MS. Chromatography of MS, EECF and AF on Sephadex G-100 confirmed the hCG and subunit composition of the fluids. On the basis of these findings it seems likely that previous studies showing very high levels of hCG in AF during the first trimester may have incorrectly sampled the EECF. In reality, the levels of total hCG (and free subunits) are low in the AF, and only 20% is intact hCG.(ABSTRACT TRUNCATED AT 250 WORDS)


Evidence for lipopolysaccharide binding in human granulosa-luteal cells. We investigated whether human granulosa-luteal (GL) cells exhibited lipopolysaccharide (LPS)-binding protein, and the response of follicular aspirate cells to LPS in vitro. Follicular aspirates taken from a human in-vitro fertilization and gamete intra-fallopian-tube transfer programme were subjected to Percoll gradients in order to isolate an enriched population of GL cells. GL cells exhibited specific LPS-binding protein, detected by autoradiography of the cellular lysate on SDS-PAGE after the cells were specifically labelled with a radioiodinated, photoactivable and reducible LPS derivative. LPS binding to the cells was also detected by the appearance of immunofluorescence associated with the cellular membrane when incubated with a fluorescent conjugated LPS receptor antibody. Ninety-four per cent of the cells exhibiting immunofluorescent LPS-binding protein were also positive for the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase, as detected by cytochemistry. In order to detect a response to LPS, the enriched population of GL cells were cultured in vitro in the presence or absence of LPS; after 16 h of culture, tumour necrosis factor-alpha (TNF) mRNA was detected by reverse transcription-polymerase chain reaction and Southern blot analysis of the amplified cDNA. The expression of TNF mRNA was enhanced when the cells were cultured in the presence of LPS, which also significantly enhanced TNF secretion into the media during the 16-h period. These results reveal that GL cells exhibit LPS-binding protein and thus increased TNF secretion occurs in response to LPS in follicular aspirate cells. The source of ovarian TNF may be leukocytes, macrophages and/or GL cells.


[The anti-arrhythmic efficacy of sodium valproate]. The antiarrhythmic efficacy of the anticonvulsant sodium valproate was studied in 22 patients with ventricular premature contraction in the immediate (10-15 days) and late (2-5 months) periods of therapy. The prolong antiarrhythmic effect of the drug was observed in 66% of the patients in whom it was beneficial in the first days of therapy. The results obtained provide strong evidence for the efficacy of sodium valproate in patients with ventricular premature contraction occurring in the presence of neurocirculatory dystonia.


[The clinico-hemodynamic effects of the activator of the GABA-ergic stress-limiting system sodium valproate (acediprol) and etatsizin in patients with cardiac arrhythmias]. Hemodynamic and antiarrhythmic effects of acediprol and ethacizine were examined in 51 patients with premature ventricular contraction concurrent with neurocirculatory dystonia and coronary heart disease. It was ascertained that acediprol exerted a positive inotropic action and ethacizine lowered myocardial contractility. Acediprol therapy prevents or attenuates the hypokinetic and arrhythmogenic effects of stimulated stress in patients with ischemic and non-ischemic arrhythmias. The preventive antiarrhythmic effect of acediprol is the most pronounced in patients with neurocirculatory dystonia. Ethacizine fails to improve hemodynamic supply of the stress test and enhances stress-related hypodynamia in some patients.


Antihypertensive therapy: new strategies beyond blood pressure control. Mild-to-moderate essential hypertension is the most common medical problem seen by physicians in Western populations, and pharmacologic antihypertensive therapy is now usually undertaken. Clinical trials have shown that lowering of elevated blood pressure using diuretics and beta-blockers reduces cardiovascular morbidity and mortality. Despite these benefits, the trials have provided no convincing evidence that the incidence of coronary artery disease or its complications is reduced: Treated hypertensive patients remain at increased cardiovascular risk compared with untreated normotensive subjects. Possible explanations for this disappointing outcome are that the drugs used may themselves have negative effects on serum lipids, glucose, and insulin resistance, thereby outweighing their antihypertensive benefits. An equally important role in this respect may be played by the diseases and therapies most commonly found in association with mild-to-moderate hypertension: hyperlipidemia, type II diabetes, coronary artery disease, left ventricular hypertrophy, cardiac arrhythmias, peripheral arterial disease, and nephropathy. Such conditions may be potent determinants of what constitutes the optimal first-line choice of antihypertensive therapy. Furthermore, the negative effects that antihypertensive drugs can have on quality-of-life factors may result in noncompliance and ineffective long-term treatment. Therefore, in a new therapeutic approach to the treatment of high blood pressure, it would be logical to base antihypertensive therapy on strategies that not only lower the blood pressure but that have beneficial impacts on hemodynamics, vascular and cardiac structure, metabolism, and quality-of-life issues.(ABSTRACT TRUNCATED AT 250 WORDS)


Pharmacodynamics of nilvadipine, a new dihydropyridine-type calcium antagonist. Nilvadipine is a new calcium antagonist of the dihydropyridine type. It reduces blood pressure by high-affinity blockade of calcium channels in arterial cells, which leads to relaxation of arterial vessels. Nilvadipine has both pharmacokinetic and pharmacodynamic advantages compared to nifedipine (the prototype of the dihydropyridines). The duration of action is longer and it has a 4- to 16-fold higher vasodilatory potency that is accompanied by less cardiodepression and adrenergic counterregulation. The higher vascular selectivity of nilvadipine is documented by its vascular/cardiac potency ratio of 251, which is 9- to 10-fold greater than that of nifedipine. When used therapeutically as an antihypertensive agent, even in patients with heart failure or concomitant beta-blockade, nilvadipine has no negative inotropic, chronotropic, or dromotropic effects. Nilvadipine has been shown to have a more potent antiatherogenic effect than that of nifedipine in vitro and in animal models.


Pharmacokinetics of nilvadipine.

Pharmacokinetics of nilvadipine. Nilvadipine is absorbed rapidly and completely and its absolute bioavailability is about 14-19% because of its high first-pass metabolism. Maximum plasma levels and the extent of bioavailability increase proportionally with the dose. Nilvadipine is mainly excreted via the kidney as inactive metabolites. Slow tissue redistribution is probably the reason for the terminal elimination half-life of 15-20 h. There was a good correlation between the estimated tissue concentration and the reduction in blood pressure in patients. The use of the sustained-release pellet formulation can prevent plasma level peaks and thereby lessen the typical side effects of dihydropyridine calcium antagonists. The pharmacokinetics of nilvadipine were not affected by impaired renal function, and although the bioavailability was increased in liver cirrhosis, there was no accumulation after repeated doses. There was no effect on plasma digoxin levels. The plasma concentration of nilvadipine can be affected by either activation or inhibition of the cytochrome P450 system. The use of a sustained-release once-a-day formulation to lower the peaks in plasma levels along with nilvadipine's long terminal half-life means that this well-tolerated pharmaceutical formulation can be employed in clinical trials for the treatment of hypertension and expected to work over 24 h.


Compliance and the benefit/risk relationship of antihypertensive treatment. Compliance is defined as a patient's behavior in terms of taking prescribed medication, following diets, or executing medically recommended lifestyle changes. Furthermore, compliance measures the extent to which a person's behavior coincides with medical or health advice. The measurement of compliance over time is still a problem. The recently developed medication monitors, however, enable precise documentation of the taking of pills out of the container over long time periods. Long-term compliance with antihypertensive treatments is, referring to the literature, about 50%. The causes of partial compliance are not yet well known. Partial compliance is often accompanied by reduced benefit, e.g., poor blood pressure control. There are even strong arguments that partial compliance increases the risks of treatment, especially with beta-blockers. Thus, the traditional physicians' nonperception of patients' partial compliance is no longer acceptable, as partial compliance has a negative impact on the benefit/risk ratio.


24-hour blood pressure control after single daily doses of nivaldipine in patients with essential hypertension. In an open monocentric phase II study, 20 inpatients with hypertension were treated with a single daily dose of nilvadipine for 3 weeks after a 1-week placebo washout phase. The initial dose in all patients was 8 mg/day p.o.; this was doubled to 16 mg once daily if an adequate blood pressure reduction was not achieved after 10 days on 8 mg. The objective of the study was to investigate the effect of this new calcium antagonist on the blood pressure in hypertensive patients. This was done by means of a 24-h blood pressure profile (with measurements at 0.5, 1, 2, 4, 8, 12, and 24 h after administration), and over the complete course of the treatment period (blood pressure measurements each day just before the medication was given). In addition, by determination of the nilvadipine plasma levels on the first, tenth, and last medication day, a possible concentration-efficacy relationship was to be ascertained between plasma concentration of nilvadipine and the observed blood pressure-lowering effect of the drug. Adequate blood pressure reductions were achieved with nilvadipine 8 mg once daily in 13 patients; 7 required a doubling of the dosage to 16 mg/day. Already on the first day of therapy, both the systolic and diastolic blood pressures were significantly reduced compared to those in the placebo phase (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of the efficacy of nilvadipine and nitrendipine on circadian blood pressure. The efficacy of nilvadipine and nitrendipine was compared in two double-blind, placebo-controlled trials. Patients had either mild-to-moderate hypertension (study 1) or moderate hypertension that was difficult to control (study 2). The criterion for efficacy was a mean drop in the median 24-h blood pressure profile after 10 days of medication compared with the placebo washout phase. Nilvadipine tended to be more efficacious than nitrendipine and had a significantly greater effect than placebo. The blood pressure reductions were more marked in the mild and moderate hypertension group than in patients with hypertension that was difficult to control. The hypotensive action of nilvadipine 16 mg lasted longer than that of nitrendipine 20 mg in all groups, especially in the group with difficult-to-control moderate hypertension. The response rate was somewhat higher in the nilvadipine group in both studies, particularly in patients with moderate hypertension that was difficult to control. Both substances were well tolerated, with fewer side effects noted for nilvadipine than for nitrendipine.


The role of calcium antagonists in the treatment of hypertension. Calcium antagonists are of particular importance in the treatment of hypertension because they influence the free cytoplasmic calcium concentration and thereby many pressor mechanisms in the smooth muscle cell. A fall in the peripheral resistance is the main hemodynamic effect, and this is more marked with the second-generation calcium antagonists because they are more vasoselective than the first calcium-channel blockers. Particularly important is their lack of effect on lipid and glucose metabolism, as well as the absence of serious side effects. It has not yet been possible to confirm that the antiatherogenic effect found in some animal models also occurs in humans. Calcium antagonists are effective, safe, and well-tolerated antihypertensive agents that can be combined with all other antihypertensives with the exception of the combination of verapamil and a beta-blocker. They are easy to dose for individualized "stepped" therapy. They have a particular role in hypertensive patients with cardiac effects secondary to hypertension, coronary artery disease, obstructive bronchial diseases, diabetes, renal disease, and peripheral arterial occlusive disease.


Hypertensive heart disease--significance of left ventricular hypertrophy. One of the earliest structural changes in the heart adapting to hypertension is left ventricular hypertrophy, which can now be exactly measured by echocardiography. Left ventricular hypertrophy increases the incidence of coronary artery disease, heart failure, and sudden death severalfold, independent of the blood pressure levels. Left ventricular hypertrophy requires specific antihypertensive therapy that controls both high blood pressure and increased left ventricular mass. Preliminary data from clinical studies indicate that regression of left ventricular hypertrophy leads to a better cardiovascular prognosis. Sympatholytic substances, angiotensin-converting enzyme (ACE) inhibitors, and calcium antagonists are antihypertensive agents that effected adequate reductions in blood pressure as well as regression of left ventricular hypertrophy.


The tolerability of nilvadipine compared to nifedipine in patients with essential hypertension. In one multicenter, double-blind study, 659 hypertensive patients were treated for 16 weeks with either nilvadipine (n = 326) or nifedipine (n = 333). The major objective of the study was to compare the compatibility of the two calcium antagonists with regard to hepatic compatibility and side-effect profiles. The dosages were chosen so that the effective blood pressure reduction in both groups was equally good (mean decreases in systolic pressure of 27 +/- 12 mm Hg with nilvadipine and 26 +/- 15 mm Hg with nifedipine, and in diastolic pressure of 18 +/- 6 mm Hg with nilvadipine and 19 +/- 7 mm Hg with nifedipine). The mean heart rate was slightly lowered by about 2 beats/min by both substances. Although there was no effect on lipid or glucose levels, the serum glutamate-pyruvate transaminase (SPGT) levels were more often found to be raised in the nifedipine group than in the nilvadipine group (p < 0.05). The vasodilator effect of both calcium antagonists was responsible for side effects, of which the most common were flushing, edema, headache, and palpitations. The number of complaints was less in the group treated with nilvadipine than with nifedipine, especially flushing and edema. Significantly more patients in the nifedipine group withdrew from treatment due to undesirable side effects (p < 0.05).


Comparisons of once-daily nilvadipine with enalapril and diuretic in patients with essential hypertension. The antihypertensive efficacy of the new dihydropyridine derivative nilvadipine given once daily was compared in two randomized, double-blind studies with that of hydrochlorothiazide/triamterene (HCT/T) and of enalapril. In the first study, arterial pressures were compared after 4 weeks of therapy with nilvadipine (16 mg q.d.) or HCT/T (25/50 mg q.d.) in patients with mild-to-moderate hypertension in a parallel-group design. The combination of both treatments was administered after 4 weeks if diastolic arterial pressures were > 90 mm Hg. The blood pressure (BP) in the nilvadipine group (n = 125) was 161/102 +/- 17/7 (mean +/- SD) mm Hg after a 2-week placebo period and decreased after 4 weeks by -16/-13 +/- 22/12 mm Hg. The BP decreased in the HCT/T group (n = 124) compared to the nilvadipine group to a similar degree (p = 0.91, n.s.) from 160/103 +/- 17/8 mm Hg by -17/-13 +/- 18/11 mm Hg. A combination therapy was started after 4 weeks in 39 patients in the nilvadipine group and in 34 patients in the HCT/T group. This led independently of the treatment sequence, to a similar decrease in BP in the nilvadipine group (-17/7 +/- 16/10 mm Hg) and in the HCT/T group (-12/9 +/- 16/9 mm Hg). In the second study, the antihypertensive effect of nilvadipine (1 x .8 mg) was compared with that of enalapril (1 x 10 mg) in a randomized, double-blind, crossover design. Sixty-one patients with essential hypertension (WHO I-II) were treated over two 4-week periods following placebo periods of 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)