[Correlation between Dupuytren's disease and arcus senilis: is dyslipidemia a common etiopathological factor?]. Because of the frequent presence of corneal arcus senilis in patients affected by Dupuytren's disease in order to evaluate this association, the authors conducted a biomicroscopic examination of the cornea in 336 patients treated surgically for Dupuytren's disease, at the Hand Surgery Unit of the University of Modena from November 1985 to December 1989. They observed corneal arcus senilis in 259 patients, i.e. in 77.1% of patients with Dupuytren's disease. Due to the statistically significant correlation between arcus senilis and hyperlipidemia as reported by Tschetter (1980) and Felder (1981), the Authors collected a blood sample from all 336 patients to evaluate serum cholesterol and tryglicerides. This study revealed a dyslipidemia in 54.8% of patients with Dupuytren's disease and in 60.2% of patients suffering from both Dupuytren's disease and arcus senilis. Because of the high frequency of dislipidemia in patients with Dupuytren's disease and arcus senilis, which are apparently two well-distinguished disease, the authors suggest that a lipid disorder may be a common aetiopathogenic factor. In particular, in favour of the possible role of hyperlipidemia in Dupuytren's disease, Electron Microscope Studies revealed lipid inclusions within fibroblasts and in the extracellular connective tissue of all pathologic palmar aponeurosis from 11 patients with Dupuytren's disease: these lipid inclusions were never seen in the normal aponeurosis taken from 5 control patients treated for traumatic palmar injuries.


[Distal radio-ulnar prosthesis].

[Distal radio-ulnar prosthesis]. Many procedures have been proposed for the treatment of traumatic painful instabilities of the Distal Radio-Ulnar Joint (DRUJ). Moore-Darrach, Milch, Baldwin, Bowers, Kapandji-Sauvé. Except for the Milch procedure, the risk of painful instability of the ulnar stump is real, but not very frequent if the technique is correctly applied. When this complication occurs, the best way to ensure the ulnar stump stabilization is mechanical, by mean of a DRUJ Prosthesis which we were the first to imagine and build. Used in two cases, with or without remnant ulnar head, these two types of prosthesis seem to have been favourable. Obviously, it is too early to know the final value of this prosthesis, based only on two cases, but it seemed important to make it known as an additional possibility in the treatment of the DRUJ problems. These two types of prosthesis are conceived on original principles: cementless fixation but with screws and nuts giving immediate and definitive stability and allowing a fast rehabilitation. The articular pieces are composite, metal on H.D. Polythylene. The articular surface is spherical permitting all the mobilities of this complex joint. The two articular surfaces are supported by two pieces: the proximal part, holding a hollow hemisphere, inserted in the ulna and the same for the two types of prosthesis, and a distal part, holding the sphere; this part is different according to the type of the prosthesis. We are well aware that in time and with clinical experience, some of its secondary characteristics will evolve, mainly its fixation system. At the present time this prosthesis is indicated in two "second look" situations: painful ulnar stump instability after a Kapandji-Sauvé procedure, and after a Moore-Darrach procedure. Perhaps it will be used in unstable stumps after a Bowers procedure? It is possible that this prosthesis will be used primarlly in the future when its reliability has been definitively established. We are working towards this objective.


[Trismus-pseudocamptodactyly syndrome: presentation and genealogy of a new European case]. The trismus pseudocamptodactyly syndrome also called Hecht syndrome or Dutch-Kentucky syndrome is characterized by loss of the ability to fully open the mouth (trismus) and a finger contracture with progressive flexion of the fingers upon extension of the wrist (pseudocamptodactyly). Deformities of the foot may be associated e.g. hammer and claw toes, tightening of the muscles of the posterior part of the leg producing an equinovarus foot. The authors presents two affected individuals from one family from which nine members had some involvement. The expression of the syndrome may vary. The pattern of the finger contracture is specific. The inheritance is autosomal dominant.


Comparative histological overview of the chemical coding of the pulmonary neuroepithelial endocrine system in health and disease. Pulmonary neuroepithelial endocrine cells have been shown to contain serotonin-immunoreactivity in almost every species studied. Regulatory peptides, of which at least ten have been reported so far, were mostly only demonstrated in a number of the investigated species or in a subpopulation of neuroepithelial endocrine cells. Calcitonin gene-related peptide, calcitonin, bombesin/gastrin-releasing peptide, enkephalin, somatostatin, substance P, cholecystokinin and polypeptide YY were found in normal lung tissues, whereas ACTH and several other bioactive substances should be regarded as ectopic. The human pulmonary neuroepithelial endocrine system seems to harbour the largest spectrum of bioactive mediators. The distribution patterns of bioactive substances in various subpopulations of solitary neuroepithelial endocrine cells or neuroepithelial bodies and in different cells of a single neuroepithelial body reveal a great complexity. Therefore, further research is needed to elucidate the chemical coding of this system.


Selenium metabolism and glutathione peroxidase activity in cultured human lymphoblasts. Effects of transsulfuration defects and pyridoxal phosphate. The metabolism of selenite, selenocysteine (SeCys), and selenomethionine (SeMet) was studied in three human lymphoblast cell lines with defects in the transsulfuration pathway and in control cells without this defect. There were very little differences in the induction of glutathione peroxidase (GPX) activity by selenite and SeCys among these cells. However, markedly higher levels of SeMet were required to induce GPX activity in transsulfuration defective cells than in control cells. Surprisingly, the addition of pyridoxal phosphate (PLP) to the media resulted in elevated GPX activity in all cells regardless of the chemical form of Se used. There is no explanation for this effect of PLP, but it is not through direct reaction with GPX or on the alteration of sulfhydryl groups.


Selenium in the central nervous system of rats exposed to 75-Se L-selenomethionine and sodium selenite. The aim of the present study is to investigate the accumulation and retention of organic and inorganic selenium in the central nervous system (CNS) of the rat. Selenium accumulation was investigated after oral treatment (3.0 mg Se/L drinking water) or ip injection (1.7 mg Se/kg body wt) of rats exposed to 75-Se L-selenomethionine (SeMeth) or sodium selenite (NaSe). Significant higher concentrations were observed after exposure to organic compared to inorganic selenium after oral as well as ip administration. Highest concentrations in both experiments were observed in cerebellum followed by the nearly identical levels in the cerebral hemisphere and spinal cord independent of the chemical form of selenium or the route of administration. The difference in concentrations observed between the different parts of the CNS investigated in each group were, however, not significant. Retention of selenium in the CNS was investigated after a single ip injection (1.7 mg Se/kg body wt) of 75-Se SeMeth or NaSe. In both groups, we observed an initial fast excretion phase followed by a slower excretion phase resembling a first-order reaction. Organic selenium disappeared much slower from all parts of the central nervous system compared to NaSe after a single injection.


Selenite-induced inhibition of colony formation by buthionine sulfoximine-sensitive and resistant cell lines. We previously demonstrated that treatment of HeLa cells with buthionine sulfoximine (BSO), which decreases the level of cellular glutathione, resulted in a decrease in the potency of selenite in inhibiting cell colony formation. We have now examined the effect of selenite on normal human lung fibroblast (CCL-210) cells, which resemble HeLa cells in their sensitivity to BSO, and on human lung adenocarcinoma (A549) cells, which are relatively insensitive to BSO. We have found that BSO treatment caused an approximately fourfold decrease in selenite potency in the CCL-210 cells, but had no significant effect on its potency in A549 cells. These results support the hypothesis that for selenite to exert its cytotoxic effect, it must undergo the reaction with an SH compound to form the selenotrisulfide. As a result of the lower sensitivity of the tumor cells to BSO, it was possible to achieve a large differential sensitivity to the cytotoxic effect of selenite.


Zinc uptake by blood cells of rats in zinc deficiency and inflammation. In zinc deficiency, the function of leukocytes is impaired. However, the results of studies on the zinc concentration of blood cells in zinc deficiency are conflicting, probably in part because of technical and analytical problems. The aim of this study was to investigate, under standard conditions, the uptake of 65Zn-labeled zinc by blood cells, taken from zinc-deficient rats and from rats in which an inflammation is induced. In both conditions, the serum zinc concentration is reduced. In clinical practice, this makes it difficult to determine whether the decrease in serum zinc is the result of a real or an apparent zinc deficiency. In stress, like an inflammatory disease, the decrease of zinc reflects an apparent zinc deficiency because of redistribution of serum zinc into the liver and because of decrease in serum albumin concentration. Over 70% of the serum zinc is bound to albumin. Blood cells from zinc-deficient and control rats were isolated using a discontinuous Percoll gradient and incubated under nearly physiological conditions in a 65Zn-containing medium. A significant increase in the in vitro uptake of 65Zn-labeled zinc by the blood cells of zinc-deficient rats was seen: erythrocytes 1.3, mononuclear cells 2.0, and polymorphonuclear cells 2.6 times the control values. During inflammation, no change in 65Zn-labeled zinc uptake by erythrocytes and mononuclear cells was demonstrated after 2 d, although the serum zinc and albumin concentrations were decreased, but a small but significant increase in zinc uptake by polymorphonuclear cells was observed. This study of 65Zn uptake in vitro under standard conditions may prove of value for distinguishing in patients real zinc deficiency from apparent zinc deficiency owing to, e.g., stress, although additional experiments should be performed.


Development and application of an alpha-face-specific radioimmunoassay for vitamin B12. The first development of an alpha-face-specific radioimmunoassay for vitamin B12 is described. Sheep, fed a cobalt-deficient diet, and immunized with a conjugate between Co-beta carboxypropyl cobalamin and keyhole limpet hemocyanin, were used to produce antisera. The antisera crossreacted with Co-beta derivatives of vitamin B12, but did not crossreact with the alpha-face vitamin B12 analog cobinamide. The antisera were used to develop a sensitive and reproducible radioimmunoassay that was free from contamination with the nonspecific vitamin B12 binding protein, R-protein. Both the radioimmunoassay and measurements of plasma concentrations of methylmalonic acid were applied to the diagnosis of cobalt/vitamin B12 deficiency in sheep. The assay correlated well with a commercially available radioassay and did not falsely detect normal vitamin B12 concentration in plasma samples containing elevated concentrations of methylmalonic acid.


Effect of calcium, copper, and zinc levels in a rapeseed meal diet on mineral and trace element utilization in the rat. Mineral and trace element interactions were studied in a balance trial with rats. Calcium, copper, and zinc were supplied to a rapeseed meal diet in a factorial design. Animals were fed ad libitum, and absorption, excretion, and retention of the elements were evaluated either as fractions of total intake or in relation to nitrogen retention to account for differences in food intake and lean body mass increment. The intrinsic content of minerals and trace elements was sufficient to support growth at a rate that could be expected from the rapeseed protein quality. However, when calcium was included in the diet, the intrinsic dietary level of zinc appeared to be limiting, despite the fact that the zinc level was twice the recommended level. Additional zinc supply reversed growth impairment. This calcium-zinc interaction is believed to be owing to the formation of phytate complexes. Calcium addition influenced the calcium, phosphorus, magnesium, zinc, and iron--but not the copper--balances. The addition of calcium reduced the availability of the intrinsic zinc, whereas no effect was seen in the zinc-fortified groups. The availability of intrinsic copper was in a similar way significantly impaired by addition of dietary zinc, whereas copper-supplied groups were unaffected by zinc addition. Intrinsic iron availability was also dependent upon zinc addition, although in a more ambiguous way. Thus, addition of extrinsic minerals to a diet high in phytate can result in significant impairments of growth and mineral utilization.


Tissues and organs as indicators of intestinal absorption of minerals and trace elements, evaluated in rats. Tissue and organ deposition and blood parameters were evaluated as indices of mineral and trace element absorption in rats. The absorption of elements was quantified in relation to nitrogen retention, i.e., considering the weight gain and new tissue synthesis. A rapeseed meal diet was supplied with three levels of calcium, two levels of zinc, and two levels of copper in a factorial design. In general, an increase in dietary mineral content increased the relative absorption, which in turn, increased the tissue deposition progressively. Striated muscle, however, did not respond to either an increased calcium or zinc supply. Furthermore, an increased calcium absorption caused a depression of the fractional phosphorus and magnesium content of femur bones. The copper content of the kidneys and the heart muscle was directly proportional to the amount of absorbed zinc and iron, respectively. The iron content of tissue was, in general, inversely proportional to zinc absorption and showed a tendency to be directly proportional to copper absorption. The zinc level in tissues was, in a similar way, inversely correlated to measured calcium absorption. In conclusion, interactions between elements do not only affect the intestinal element absorption, but also the distribution of already absorbed elements in tissues and organs.


Effects of cadmium and copper on zinc transport kinetics by isolated renal proximal cells. Zinc, cadmium, and copper are known to interact in many transport processes, but the mechanism of inhibition is widely debated, being either competitive or noncompetitive according to the experimental model employed. We investigated the mechanisms of inhibition of zinc transport by cadmium and copper using renal proximal cells isolated from rabbit kidney. Initial rates of 65Zn uptake were assessed after 0.5 min of incubation. The kinetics parameters of zinc uptake obtained at 20 degrees C were a Jmax of 208.0 +/- 8.4 pmol.min-1.(mg protein)-1, a Km of 15.0 +/- 1.5 microM and an unsaturable constant of 0.259 +/- 0.104 (n = 8). Cadmium at 15 microM competitively inhibited zinc uptake. In the presence of 50 microM cadmium, or copper at both 15 and 50 microM, there was evidence of noncompetitive inhibition. These data suggest that zinc and cadmium enter renal proximal cells via a common, saturable, carrier-mediated process. The mechanisms of the noncompetitive inhibition observed at higher concentrations of cadmium or with copper require further investigation, but may involve a toxic effect on the cytoskeleton.


New concepts in respiratory chain diseases. Recent advances in mitochondrial cytopathies are reviewed. Particular emphasis is given to diseases related to mitochondrial DNA mutations, a field now encompassing myopathic and encephalopathic syndromes, some more common degenerative disorders, and certain aspects of the aging process. Diseases caused by defective oxidative energy generation now have an important place in human pathology.


Direct application to dairy foods of a Listeria-specific oligonucleotide probe to 16S rRNA. A potentially Listeria-specific 28 base oligonucleotide probe was designed from 16S rRNA sequence data. Using either 32P or non-radioactive (alkaline phosphatase) labels, the probe was shown to be highly specific as it hybridised to RNA extracted from all of the species of Listeria but not to any of the other bacteria tested. Both probe methods were highly sensitive and ca 10(2) cfu/ml Listeria could be detected in pure cultures. A rapid procedure for extracting RNA from milk, Camembert and cottage cheese was developed. This allowed the direct application of the probe to these foods and gave a rapid and specific method of detecting > 10(2) cfu/g or ml Listeria in these foods.


Interferon treatment for hepatitis B-associated membranous glomerulonephritis in two Chinese children. Two Chinese boys, aged 3.5 and 5 years, developed nephrotic syndrome and were chronic carriers of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg). Renal biopsy showed membranous glomerulonephritis and liver biopsy showed chronic persistent hepatitis. They were given interferon-alpha-2a at a dose of 5 MU/m2 on alternate days for 12 and 16 weeks after 2 years of persistent nephrotic syndrome. Patient 1 showed complete remission and resolution of hepatosplenomegaly, but his serum remained positive for HBsAg, HBeAg and hepatitis B virus DNA. Patient 2 showed only a transient clinical response and seroconversion from HBeAg to anti-HBe status. Although not always successful, interferon treatment should be considered in severe persistent nephrotic states, since there is at present no satisfactory treatment for this form of glomerulonephropathy.


Insulin-like growth factor-1, growth hormone-dependent insulin-like growth factor-binding protein and growth in children with chronic renal failure. Insulin-like growth factor-binding protein-3 (IGFBP-3) and insulin-like growth factor-1 (IGF-1) levels were measured by specific radioimmunoassays in children with all degrees of chronic renal failure (CRF). Study group 1 comprised 29 children (10 on dialysis) who had been studied one to four times over 2 years to determine whether IGF-1 and IGFBP-3 levels differed from those in age-matched healthy children and to examine the relationship between these levels and heights. IGF-1 and IGFBP-3 levels did not differ from those in normal children. IGF-1 and IGFBP-3 were significantly correlated, increased with pubertal stage in all children and with age in non-dialysis patients. IGF-1, but not IGFBP-3, correlated with age in dialysis patients. There was no correlation between IGF-1 or IGFBP-3 levels (corrected for age) and height standard deviation score (SDS) in either non-dialysis or dialysis patients. Study group 2 comprised 19 children (7 on dialysis) who were studied prospectively for 1-2 years to examine the relationship between IGF-1 and IGFBP-3 levels, growth rates and nutritional parameters. Mean values of IGF-1 and IGFBP-3 (corrected for age) did not change over 1-year periods, while height SDS fell by -0.38 +/- 0.21 SD/year in dialysis patients and by -0.11 +/- 0.29 SD/year in non-dialysis patients. No significant correlations were found between IGF-1 or IGFBP-3 levels and growth rates or nutritional parameters. Thus growth retardation in children with CRF is not related to circulating levels of IGF-1 or IGFBP-3.


Short-term dietary copper deficiency does not inhibit angiogenesis in tumours implanted in striated muscle. The effect of dietary copper deficiency on tumour growth, neovascularisation and microvascular integrity was studied in the rat cremaster muscle. Male, weanling Sprague-Dawley rats were fed purified diets which were copper deficient (< 0.5 micrograms g-1 of diet) or copper adequate (5 micrograms g-1 of diet). Seven days after initiation of diets, a chondrosarcoma was implanted in the cremaster muscle of each rat. Five, 10 or 20 days after tumour implantation, rats were anesthetised and their cremasters prepared for observation by intravital microscopy. Intraarterial injection of fluorescein isothiocyanate-conjugated albumin and subsequent observation of fluorescence in the perivascular space indicated no difference in microvascular albumin leakage between the tumour vasculature of copper deficient and copper adequate rats. Neither tumour growth (assessed by wet weight), vascular density (assessed by light microscopy), nor any ultrastructural characteristics of the tumour or its vasculature (assessed by electron microscopy) were affected by copper deficiency. In view of findings by others which indicate changes in tumour characteristics with copper deficiency, we conclude that the copper dependency of tumour growth and vascularisation is a function of the type of tumour, the host tissue, or the conditions of copper depletion.


Nitroimidazole adducts as markers for tissue hypoxia: mechanistic studies in aerobic normal tissues and tumour cells. Two aspects of the aerobic metabolism of nitroimidazole markers for hypoxia were investigated. Several normal murine tissues which are likely to be well oxygenated bind misonidazole at rates comparable to those of hypoxic regions in tumours. The possibility that this aerobic activation occurs via an oxygen independent process such as an initial two electron reduction was studied. Binding to the oesophageal mucosa of mice which occurred under hypoxia in vitro was inhibited by at least 95% in the presence of 10% oxygen. Dicoumarol, an inhibitor of DT-diaphorase, was shown to cause only small reductions in misonidazole binding to oesophageal epithelium and smooth muscle in vitro and to EMT6 tumours, liver, oesophageal and tracheal epithelium, parotid gland and smooth muscle in vivo. Thus an oxygen-insensitive process is not a major cause of the high binding rate in oesophageal mucosa, and may not contribute significantly to the observed binding in other normal tissues. It has been suggested that metabolism of nitroimidazoles by aerobic cells in tumours might be sufficient to minimise access of these compounds to hypoxic regions, particularly at the micromolar concentrations currently in use clinically. The uptake of 125I-iodoazomycin arabinoside by RIF-1 and EMT6 tumours was found to be directly proportional to injected dose over concentrations between 0.5 and 50 microM. Labelling of hypoxic regions in EMT6 tumours by high specific activity 3H-misonidazole at 1 microM was found to be similar to that obtained at 50 microM.


Expression of bone morphogenetic proteins in human prostatic adenocarcinoma and benign prostatic hyperplasia. There are important interactions between prostatic tumours and bone. This study was designed to examine whether prostatic tissue can express bone inductive factors, in particular, the Bone Morphogenetic Proteins (BMPs). The polymerase chain reaction (PCR) has been used to screen for the expression of BMPs one to six in the prostatic tissue of patients with benign prostatic hyperplasia (BPH), non-metastatic prostatic adenocarcinoma and metastatic prostatic adenocarcinoma. BMPs were expressed in both benign and malignant prostate tissue and in the prostate tumour cell lines, PC3 and DU145. BMPs were also expressed in ocular melanoma tissue, a tissue which rarely metastasizes to bone. BMP-6 expression was detected in the prostate tissue of over 50% of patients with clinically defined metastatic prostate adenocarcinoma, but was not detected in non-metastatic or benign prostate samples or in ocular melanoma tissue. These findings suggest that the BMPs may play a role in the osteoinductive activity of prostate metastases and that the pattern of expression of BMPs may be important in the pathogenesis of osteoblastic metastases associated with prostate adenocarcinoma.


Vancomycin: its effect on intracranial pressure. Vancomycin is a commonly used antibiotic in neurosurgical practice. It is, however, notorious for causing histamine release which has been associated experimentally with rises in intracranial pressure (ICP). The observation that patients receiving vancomycin had an elevation in ICP during vancomycin infusion over 1 h, and that this elevation persisted for the hour following infusion led to a more formal evaluation. Presented here are data obtained from 6 patients who received vancomycin while on external ventricular drainage for a variety of reasons. The mean ICP for the hour prior to, during and following vancomycin administration was 8 mm Hg (SD 5.6), 13.8 mm Hg (SD 4.9), and 12.6 mm Hg (SD 4.3), respectively. The data were analyzed using a repeated measures analysis of variance between the preadministration values, during-administration values and postadministration values. The p value was significant at < 0.0001. These observations led to experimentation in animals to further assess the effects of vancomycin on ICP. The mean ICP prior to, during and following vancomycin infusion was 1.95 mm Hg (SD 0.75), 8.4 mm Hg (SD 5.1) and 5.6 mm Hg (SD 2.5), respectively. The data were analyzed using a repeated measure analysis of variance for the preadministration, during-administration and postadministration values. The p value was significant at < 0.0001. Based on these data the use of vancomycin should be tempered in the setting of intracranial pathology.


Comparison of three different methods for automated classification of cervical cells. Over 4600 exfoliated squamous cervical cells taken from appropriate Papanicolaou samples were classified as normal, mildly dysplastic, moderately dysplastic and severely dysplastic by an experienced cytopathologist. The slides were de-stained and subsequently re-stained with Feulgen Thionin-SO2 stain. Images of the nuclei were then captured, recorded and processed employing an image cytometry device. Automated classification of the cells was carried out using three different methods--discriminant function analysis, a decision tree classifier and a neutral network classifier. The discriminant function analysis method, which combined all dysplastic cells into an abnormal group, achieved a combined error rate of less than 0.4% for moderate and severe dysplastic cells, and less than 40% for mildly dysplastic cells. All three methods yielded comparable results, which approached those of human performance.


Interaction between the two conserved single-stranded regions at the decoding site of small subunit ribosomal RNA is essential for ribosome function. Formation of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, is essential for ribosome function. It was previously reported that mutation of G1401 inactivated all in vitro functions of the ribosome [Cunningham et al. (1992) Biochemistry 31, 7629-7637]. Here we show that mutation of C1501 to G was equally inactivating but that the double mutant C1401:G1501 with the base pair reversed had virtually full activity for tRNA binding to the P, A, and I sites and for peptide bond formation. Initiation-dependent formation of the first peptide bond remained 70-85% inhibited, despite full 70S initiation complex formation ability as evidenced by the ability to form fMET-puromycin. These results suggest that the defect in formation of the first peptide bond lies in filling the initial A site, Ai, rather than the subsequent elongation A sites, Ae. An increased mobility around the anticodon was detected by UV cross-linking of the anticodon of P-site-bound tRNA to C1399 as well as to the expected C1400. These findings provide the first experimental evidence for the existence of the G1401:C1501 base pair and show that this base pair, located at the decoding site, is essential for function. The structural implications of tertiary base pair formation are discussed.


In vivo chemical footprinting of the Escherichia coli ribosome. We have studied the in vivo chemical accessibility of 16S rRNA residues A349-G1505 in the small subunit of the Escherichia coli ribosome. Exponentially growing E. coli cultures were reacted with dimethyl sulfate, and the reactive sites on the 16S ribosomal RNA were analyzed by reverse transcription, an assay which detects reactions at N1-A and N3-C. In agreement with previous in vitro results, three regions of 16S RNA appeared particularly reactive to dimethyl sulfate: hairpin 27 (residues A892-A915) of the central domain, and hairpin 33-33A (residues A994-C1037) and the tip of hairpin 41 (residues A1256-A1275), both from the 3' major domain. These three regions contained 52% of the reactive residues but only 8% of the residues scanned. In contrast to previous in vitro results, three small sections of 16S RNA appeared protected: the tip of hairpins 26 (residue A845) and 31 (residues A968-A969), and residues A1418, A1441, and A1483 of the middle body of hairpin 44. Four of the dimethyl sulfate reactive sites (A831, C948, A1019, and C1192) are located in positions usually assumed to be double-stranded (helices 26, 30, 33-33A, and 34), which suggests alternative structures for these helices at least during part of the translation process, as if the residues in question belonged to "conformational switches." The addition of chloramphenicol protected residues A831, A1035-A1036, and A1503, which suggests that they belong to the mobile regions of the elongating ribosome, and become exposed during some transition(s) from one ribosomal state to the other during the elongation cycle.


Kinetics of intermolecular cleavage by hammerhead ribozymes. The hammerhead catalytic RNA effects cleavage of the phosphodiester backbone of RNA through a transesterification mechanism that generates products with 2'-3'-cyclic phosphate and 5'-hydroxyl termini. A minimal kinetic mechanism for the intermolecular hammerhead cleavage reaction includes substrate binding, cleavage, and product release. Elemental rate constants for these steps were measured with six hammerhead sequences. Changes in substrate length and sequence had little effect on the rate of the cleavage step, but dramatic differences were observed in the substrate dissociation and product release steps that require helix-coil transitions. Rates of substrate binding and product dissociation correlated well with predictions based on the behavior of simple RNA duplexes, but substrate dissociation rates were significantly faster than expected. Ribozyme and substrate alterations that eliminated catalytic activity increased the stability of the hammerhead complex. These results suggest that substrate destabilization may play a role in hammerhead catalysis.


Implication of RNA structure on antisense oligonucleotide hybridization kinetics. A 47-nucleotide transcript of the activated Ha-ras gene was prepared and determined, by enzymatic structure mapping, to form a stable hairpin structure. Six antisense decaribonucleotides were designed, and association constants (Ka) for the hairpin- and length-matched complements were measured. Two of the antisense oligonucleotides targeted to the loop had nearly equal affinity for the transcript compared to the complement. The others, including one oligonucleotide complementary to the 3' side of the single-stranded loop, bound 10(5)-10(6)-fold less tightly to the transcript than to the short complement. We propose the difference in affinity is due to the target structure, both the secondary structure of the stem and the structure in the loop. Measurement of the bimolecular association rate constant, k1, and the dissociation rate constant, k-1, for these oligonucleotides indicates the observed relationship between affinity and structure is primarily due to k1.


Constrained and restrained refinement in EXAFS data analysis with curved wave theory. This paper describes methods of constrained and restrained refinement of EXAFS data which provide a means of substantially reducing the number of independent parameters compared to conventional least-squares methods commonly used. Constrained refinement allows a major reduction in the number of free parameters for a refinement of a structural model. In restrained refinement, additional structural information from well-characterized small molecules is used to provide additional observations in the data analysis. Even though these methods are of general application to the majority of complex systems, they are particularly valuable for biological molecules. The methods are of major advantage for ligands where significant multiple scattering is present, e.g., histidine, tyrosine, CO, CN, etc. The bases of these methods are described, and applications to some complex chemical and biological systems are given.


Identification of two general diffusion channels in the outer membrane of pea mitochondria. Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent solubilized mitochondrial membranes of pea seedlings (Pisum sativum). The addition of the detergent-solubilized material to the membranes resulted in a strong increase of the membrane conductance. To identify the proteins responsible for membrane activity the detergent extracts were applied to a hydroxyapatite (HTP) column and the fractions were tested for channel formation. The eluate of the column contained a protein which migrated as a single band with an apparent molecular mass of 30 kDa on SDS-PAGE. This channel was identified as the porin of pea mitochondria since it formed voltage-dependent channels with single-channel conductances of 1.5 and 3.7 nS in 1 M KCl and an estimated effective diameter of about 1.7 nm. Further elution of the column with KCl containing solutions yielded fractions which resulted in the formation of transient channels in lipid bilayer membranes. These channels had a single-channel conductance of 2.2 nS in 1 M KCl and had also the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. Zero-current membrane potential measurements suggested that pea porin was anion-selective in the open state. The selectivity of the second channel was investigated by the measurement of the reversal potential. It was also slightly anion-selective. Its possible role in the metabolism of mitochondria is discussed.


Therapeutic agents for treatment of established metastases and inhibitors of metastatic spread: preclinical and clinical progress. This article reviews recently described agents for the treatment of established metastases or inhibitors of metastatic spread. Recent progress in both preclinical models of experimental metastasis and clinical evaluation is highlighted. Where possible, distinctions are made between therapeutic agents for established metastatic tumors and those that merely interfere with the process of metastasis. The approaches emphasized include radiotherapy, cytotoxic chemotherapy, targeting the metastatic process and tumor metastases (adhesion, invasion, and angiogenesis), and modulation of the immune response, including chemoimmunotherapy. Emphasis has been placed on the necessity of using appropriate animal models of metastatic disease for the discovery and development of novel and effective antimetastatic therapeutic agents. Also addressed is the likelihood that future approaches to the metastasis problem will employ novel combined antimetastatic therapeutic modalities.


Vascular cell adhesion molecule-1 is induced on endothelium during acute rejection in human cardiac allografts. An infiltration of mononuclear leukocytes into the myocardium of a cardiac allograft is diagnostic of transplant rejection. The presence of these leukocytes implies their adhesion to, and subsequent migration through, the vascular endothelium. Intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 are endothelial proteins that have been shown to be involved in the binding of mononuclear leukocytes to the endothelium in vitro. We investigated the induction of these proteins in a random series from 99 endomyocardial biopsy specimens obtained from 1 week to 4 years after cardiac allograft transplantation. Intercellular adhesion molecule-1 was found to be expressed constitutively by the myocardial microvasculature in the recipient's original heart and in the posttransplantation biopsy specimens. No correlation was found between the presence or absence of intercellular adhesion molecule-1 expression and cellular rejection. In contrast, no endothelial expression of vascular cell adhesion molecule-1 was observed in the recipient heart or in endomyocardial biopsy specimens lacking cellular rejection. The presence of vascular cell adhesion molecule-1 significantly correlated with the presence of mild or moderate rejection. The de novo induction of vascular cell adhesion molecule-1 on the myocardial vasculature during periods of rejection, in addition to the recruitment of mononuclear leukocytes that are known to bind to this protein, suggests that the expression of this endothelial adhesion protein could be of use in diagnosing rejection.


Detection of bluetongue virus serogroup by polymerase chain reaction. To facilitate detection of active bluetongue virus (BTV) infection, a polymerase chain reaction (PCR) protocol was developed. The BTV reverse transcriptase PCR (RT-PCR) is a 1-tube reaction and involves chemical denaturation of the double-stranded viral RNA target, a complementary DNA (cDNA) synthesis step, and PCR amplification of the cDNA. BTV RT-PCR using primers derived from highly conserved genome segment 10 results in a 251-base pair (bp) product. BTV RNA from all USA prototype serotypes 2, 10, 11, 13, and 17; a wide spectrum of USA BTV field isolates including serotypes 10, 11, 13, and 17; and a spectrum of Israeli field isolates including serotypes 2, 4, 6, 10, and 16 were detected by BTV RT-PCR. With agarose gels, the 251-bp product was detected from as little as 100 fg-1 pg of BTV RNA, which is equivalent to 5 x 10(3)-5 x 10(4) viral particles or 5 x 10(2)-5 x 10(3) infectious units. With dot blot hybridization, specific PCR product was detected from as little as 1 fg of BTV RNA, which is equivalent to 50 viral particles, or 5 infectious units. This level of sensitivity is comparable to that of virus isolation. The BTV RT-PCR using primers derived from genome segment 10 can detect a wide spectrum of USA and Israeli BTV serotypes and has potential for detection of infection by the BTV serogroup. Application of this BTV PCR to clinical samples is in progress.


Neural computing in discovering RNA interactions. High-order RNA structures are involved in regulating many biological processes; various algorithms have been designed to predict them. Experimental methods to probe such structures and to decipher the results are tedious. Artificial intelligence and the neural network approach can support the process of discovering RNA structures. Secondary structures of RNA molecules are probed by autoradiographing gels, separating end-labeled fragments generated by base-specific RNases. This process is performed in both conditions, denaturing (for sequencing purposes) and native. The resultant autoradiograms are scanned using line-detection techniques to identify the fragments by comparing the lines with those obtained by 'alkaline ladders'. The identified paired bases are treated by either one of two methods to find the foldings which are consistent with the RNases' 'cutting' rules. One exploits the maximum independent set algorithm; the other, the planarization algorithm. They require, respectively, n and n2 processing elements, where n is the number of base pairs. The state of the system usually converges to the near-optimum solution within about 500 iteration steps, where each processing element implements the McCulloch-Pitts binary neuron. Our simulator, based on the proposed algorithm, discovered a new structure in a sequence of 38 bases, which is more stable than that formerly proposed.


Molecular biology techniques and their applicability to the study of diabetes and hypertension: the renin-angiotensin system as an example. Recently developed molecular biology techniques lend themselves to the study of both normal physiology and pathophysiology. This review discusses a number of such techniques and their applicability to the study of diabetes and hypertension with the renin-angiotensin system as an example. The demonstration of specific mRNA in tissue provides strong evidence that the protein for which it encodes is expressed there. The contribution of several methods to demonstrate mRNA expression including Northern analysis, slot blot analysis, solution hybridization, in situ hybridization, run-on assays, and polymerase chain reaction are discussed in the light of how these are used to study renin-angiotensin system mechanisms. Additionally, recent developments in the study of gene transfer and identification of genes are reviewed.


Methyl green and its analogues bind selectively to AT-rich regions of native DNA. Methyl green has long been used as a DNA stain in histochemistry. The sequence selective binding of the cationic triphenylmethane dyes methyl green, crystal violet and Malachite green to DNA was investigated by DNAase 1 and micrococcal nuclease footprinting. At low concentrations the ligands showed similar footprinting patterns which centred around AT-rich regions with a mild preference for hompolymeric A and T. At higher concentrations the dyes bound to almost all available DNA sites. Models, with and without intercalation are discussed to account for the specific binding.


Comparative study of in situ hybridization and immunohistochemical techniques for the detection of human papillomavirus in lesions of the uterine cervix. Among the techniques currently used for the detection of human papillomavirus (HPV) in genital lesions, only two correlate HPV with the histopathological findings of the lesion: immunohistochemistry and in situ hybridization. Consequently, we were prompted to carry out a comparative study on both techniques to check their utility and efficacy as routine diagnostic methods. 52 biopsy specimens of uterine cervix diagnosed histopathologically as condylomas and cervical intraepithelial neoplasia+koilocytosis were studied by immunohistochemical and in situ hybridization techniques using a polyclonal antibody against the common antigen of the HPV capsid and three biotinylated DNA probes specific to HPV types 6/11, 16/18 and 31/35/51. Immunohistochemistry detected 21 positive cases (40.38%), whereas in situ hybridization detected 40 positive cases (76.92%); of the latter, 30 were positive for HPV types 6/11, 3 for HPV types 16/18 and 11 for HPV types 31/35/51. The results suggest that in situ hybridization is a more sensitive technique than immunohistochemistry. However, we recommend the use of both techniques in the case of potentially malignant lesions since better prognostic information can be obtained from joint analysis of both results.


HER-2/neu oncogene expression and DNA ploidy in normal human kidney and renal cell carcinoma. Using flow cytometry (FCM), we have investigated both the DNA content (stained with propidium iodide) and HER-2/neu oncogene expression (revealed by means of an anti-HER-2/neu monoclonal antibody) in neoplastic and non-neoplastic kidney samples from 20 patients with renal cell carcinoma. All the non-neoplastic samples and 15/20 (75%) renal cell cancers showed diploid modal DNA content while the remaining 5 neoplastic sample (25%) showed both diploid and hyperdiploid cell populations. In normal kidney the level of HER-2/neu oncoprotein was low (median fluorescence values in arbitrary units = 7.5 AU, range: 4-10 AU). In diploid renal cancers the level of HER-2/neu was slightly increased (median fluorescence values = 20 AU, range: 9.5-30 AU) (p < .005). The relationship of HER-2/neu expression to the cell cycle in these tumor samples is not clear since most of the cells express the antigen in all phases of the cell cycle. On the other hand, there is an association between HER-2/neu expression and abnormal DNA content suggesting that aneuploid pattern may be biologically related to overexpression of the HER-2/neu gene.


Qualitative and quantitative changes in nuclear DNA and phenotypic gene expression in human malignant skin tumors during their progression. Qualitative and quantitative changes in nuclear DNA and phenotypic expression of human malignant skin tumors were examined during the course of progression. The numerical abnormalities of chromosomes demonstrated by interphase cytogenetics using the chromosome-specific in situ hybridization technique, were also used to reveal qualitative DNA changes in malignant tumor cells. For the analysis of the quantitative changes in nuclear DNA, fluorescence cytophotometry was used on the DAPI-stained tumor cells isolated from the paraffin-embedded sections. To survey abnormal gene expression in malignant tumor cells, lectin histochemistry for different sugar residues, immunohistochemical staining of HLA-DR, and in situ hybridization for H-ras, c-myc, N-myc or v-fos were used. The results showed that: 1) in one case of squamous cell carcinoma with invasion, the number of chromosomal abnormalities was much greater in the invasive than in non-invasive parts, with marked topographical heterogeneities; 2) the DNA-ploidies were largely shifted to the higher side with aneuploid stem-lines and polyploid cells in the invasive parts of all malignant tumors; 3) the expression of HLA-DR was induced at the invasive fronts of malignant melanomas; 4) the GS-I specific sugar residue(D-galactose) appeared in all extra-mammary Paget's cells; and 5) expression of "oncogenes" was found in about 60% of all malignant tumors examined. Thus, the progression of malignancy is accompanied by both qualitative and quantitative changes in nuclear DNA, resulting in abnormal gene expression.


Amplification of GC-rich DNAs in neuronal nuclei of Planorbarius corneus (L.) (Mollusca, pulmonata). Quantitative microfluorometric evaluation of DNA content in nerve cells of the Pulmonate Gastropod Planorbarius corneus has indicated that the increase in nuclear volume is due to DNA amplification. Indeed, it has been observed that the DNA contents are scattered at random between 2C and 1,000C values. This is not in agreement with the occurrence of repeated duplications of the whole genome. Furthermore, chromatin photo-oxidation, a technique useful in discriminating GC-rich from AT-rich DNAs, suggests that DNA amplification involves GC-rich sequences.


Effects of sham hemodialysis on plasma levels of vasoactive peptides in patients with uremia. Changes in plasma levels of vasoactive peptides during hemodialysis have mainly been attributed to changes in plasma volume and osmolality. This study investigated the effect of the extracorporeal circulation on plasma levels of vasoactive peptides, noradrenaline, and renin. Eleven stable hemodialysis patients were studied during sham dialysis for 60 min using a Cuprophan dialyzer (Alwall GFE11, Gambro AB, Lund, Sweden). With regard to vasoconstrictors, there was an increase in noradrenaline (NA) (13%, p < 0.05) and renin (PRA) (32%, p < 0.05), while arginine vasopressin and neuropeptide Y remained unaltered. Concerning vasodilators, an increase in substance P (SP) (23%, p < 0.05) and vasoactive intestinal peptide (VIP) (15%, p < 0.01) was observed, while a decrease in atrial natriuretic peptide (ANP) (17%, p < 0.05) and motilin (MOT) (24%, p < 0.01) occurred. Calcitonin gene related peptide and beta endorphin were unaltered. A decrease in blood pressure was observed, while heart rate remained unchanged. The authors conclude that the extracorporeal circulation, per se, affects plasma levels of vasoactive substances and influences vascular stability. The decrease in ANP and MOT might be due to adsorption to the dialysis membrane. The increase in some vasoconstrictors (NA, PRA) and vasodilators (SP, VIP) might be induced by the blood-artificial surface contact, or by other factors, e.g., heparin or cooling of the blood during the procedure.


Clinical evaluation of a new synthetic protease inhibitor in open heart surgery. Effect on plasma serotonin and histamine release and blood conservation. To achieve more physiologically successful cardiopulmonary bypass (CPB), the effects of a new synthetic protease inhibitor, nafamostat mesilate (FUT), were examined in open heart surgery. Thirty adult patients were divided into two groups. In Group F (GpF; n = 15), 2 mg/kg/hr of FUT was administered continuously during CPB and 0.2 mg/kg/hr before and after CPB. FUT was not given to Group C patients (GpC; n = 15), who acted as controls. Serotonin and histamine levels in plasma, platelet counts, platelet adhesive function levels, and alpha 2 plasmin inhibitor-plasmin complexes (PIC) were serially measured. The serotonin level in GpF was significantly lower at 5 min of CPB than in GpC. Histamine levels in GpC decreased remarkably after starting CPB, then later recovered; by contrast, they did not decrease in GpF during CPB. At 1 hr after CPB, platelet counts were higher (p < 0.01) in GpF (54 +/- 8%) than in GpC (41 +/- 10%), and platelet adhesion was lower (p < 0.01) in GpF (7 +/- 7%) than in GpC (34 +/- 13%). The PIC was significantly less in GpF than in GpC throughout the surgery. Blood loss in GpF was significantly reduced compared with that in GpC. In conclusion, FUT attenuated chemical mediator reactions, such as plasma serotonin and histamine, and also reduced blood loss by preserving platelets and inhibiting fibrinolysis during CPB.


Myeloid cell kinetics in response to haemopoietic growth factors. The morphologically recognizable cells of haemopoietic tissue comprise some 95% of the total and their kinetic performance is both flexible and adaptable to stress conditions. The effects of the common myeloid growth factors on these cells have been examined. Using the classically developed analyses of cell kinetics with tritiated thymidine labelling autoradiography, it is shown that continuously infused or repeated injections of G-CSF increases the proliferative activity of marrow haemopoietic tissue in both mouse and man, shortening the average cell cycle times by 35 and 65% respectively, amplifying neutrophil production so that levels in the peripheral blood rise 10-15-fold. This amplified production, amounting to 3-4 extra proliferation divisions, is mostly confined to the proliferatively active maturing neutrophil cell compartments. In addition, the neutrophil maturation time is also reduced, leading to a rapid and sustained release of postmitotic cells within 1-2 days, compared with the normal time of 4-6 days. Neither GM-CSF nor IL-3 generates any significant amplification of neutrophil production in mice but, in humans, GM-CSF stimulates cell proliferation in the bone marrow to a similar degree as doses G-CSF. Studies on the granulocyte-macrophage progenitor population show that the proliferation stimulus in response to GM-CSF is not confined to the maturing populations. In the maturing neutrophil precursor population, average cell cycle times are shortened by 60%, but in this case the overall maturation times are unaffected and their time of release into the circulation is normal. The response to GM-CSF, however, is not so straightforward as that to G-CSF. The peripheral half-life of the mature cells is considerably prolonged and this is consistent with some suggestion of functional impairment. In addition, a significant release of immature cells and eosinophils (also expanded in the bone marrow in response to GM-CSF) dilutes the neutrophilic response. Monocyte production is also stimulated by G- and GM-CSF and, though no direct measurement of proliferation has been made, stimulation at all stages of their proliferation, maturation and release are implied.


Human intraperitoneal response to a left ventricular assist device with a Ti-6AI-4V alloy surface. The soft tissue reaction to long-term implantation of an intraperitoneal left ventricular assist device (LVAD) was investigated. The HeartMate 1000 (Thermo Cardiosystems, Inc., Woburn, MA) is a pneumatically driven blood pump with smooth titanium alloy (Ti-6AI-4V) outer surfaces that is placed intraperitoneally in the left upper quadrant and sutured to the anterior abdominal wall. It is being used currently as a bridge to cardiac transplant, which sometimes requires extended support times. We examined the tissue capsule that formed around the rigid circular pump housing of four LVADs (duration of implant: 61, 86, 128, and 153 days) for gross, histologic, immunohistochemical, and ultrastructural analysis. Immunostaining was performed using monoclonal and polyclonal antibodies against cytoskeletal tissue markers (vimentin, desmin, alpha-smooth muscle actin), T and B lymphocytes, carcinoembryonic antigen, factor VIII, and cytokeratins (CAM 5.2 AE1/AE3, 34 beta E12, and 35 beta H11). Direct fluorescent immunolabeling for fibrinogen was also performed to characterize cell and tissue type. Histologic analysis of the 3 to 4 mm thick capsule with white, glistening inner surfaces showed fibrovascular tissue with multipotential subserosal cells (MSCs), capillary endothelium, collagen, and a few mononuclear infiltrates. The immunohistochemical profile of the MSCs differed from myofibroblasts despite a morphologic similarity. Transmission electron microscopy revealed abundant rough endoplasmic reticulum and peripherally arranged myofilaments within the spindle shaped cells. It was hypothesized that capsule formation was initiated by fibrin deposition, followed by proliferation of MSCs and subsequent formation of fibrovascular tissue.(ABSTRACT TRUNCATED AT 250 WORDS)


Cell interaction molecules and cytokines which participate in B lymphopoiesis. A molecular and cellular definition of the bone marrow microenvironment is rapidly contributing to our understanding of lymphohaemopoiesis. While lineage specific genes and their protein products are being identified, information is accumulating about mechanisms which may regulate their expression. Stimulation of B lymphocyte precursor replication, and other discrete functions, are being attributed to cytokines such as interleukin 7 (IL-7). The activity of these factors may be controlled at the level of synthesis, local concentration and interaction with extracellular matrix. Extremely small amounts of IL-7 are made by stromal cells, which are themselves being thoroughly studied as cloned cell lines. This in vitro characterization suggests that stromal cells can make at least 12 cytokines, that they can respond to some of those cytokines themselves, and that they retain differentiation potential. Several molecules have been identified which are probably required for recognition between cells in marrow. It is noteworthy that they belong to several previously described families of adhesion molecules and none is unique to that tissue. VCAM-1 is constitutively expressed on stromal cells in marrow and can be recognized by pre-B cells which bear the integrin VLA-4. The same pair of molecules is probably responsible for extravasation of leukocytes in other tissues during inflammation. Cell adhesion molecules are likely to work in a carefully coordinated and cooperative fashion. Their activity can be controlled by expression or, in some cases, modulated after display on the cell surface. For example, while most haemopoietic cells bear CD44, only certain cells utilize it for recognition of the ligand hyaluronate. The affinity for hyaluronate can be experimentally regulated and depends on the cytoplasmic domain of CD44. This capability for dynamic change may be important for transient interactions between cells, permitting movement of maturing precursors within and from marrow.


Mannosyl/N-acetyl-beta-D-glucosaminyl binding properties of the envelope glycoprotein of human immunodeficiency virus type 2. We have recently demonstrated that human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoprotein precursor gp160 (rgp160) behaves as a mannosyl/N-acetylglucosaminyl (GlcNAc) binding protein. If such a carbohydrate-binding property were of biological relevance it should be shared by other related primate immunodeficiency viruses such as HIV-2. The present study confirms this hypothesis and extends these findings by showing that HIV-2 recombinant gp140 (rgp140) specifically interacts with three affinity matrices substituted by synthetic or natural carbohydrate structures: D-mannose-divinylsulphone-agarose, para-aminophenyl-beta-D-GlcNAc-agarose and the natural glycoprotein, bovine fetuin, also coupled to agarose. Binding of rpg140 to the matrices was inhibited by alpha-D-Man17-BSA (where BSA is bovine serum albumin), beta-D-GlcNAc47-BSA and fetuin, and by glycopeptides derived from pronase-treated porcine thyroglobulin. Glycopeptides obtained after endoglycosidase H treatment of thyroglobulin had a limited inhibitory effect, whereas beta-D-Gal17-BSA and beta-D-glucan had no effect. These results indicate that, like HIV-1 envelope glycoprotein, HIV-2 rgp140 interacts with high-mannose and with the mannosyl core of complex-type N-linked glycans, as well as with the N-acetylglucosaminyl core of oligosaccharidic structures.


Dermal cylindroma. Expression of intermediate filaments, epithelial and neuroectodermal antigens. We report on immunohistochemical staining patterns in so-called apocrine tumors of skin with special emphasis on the dermal cylindroma. The results were compared with apocrine tubular adenoma, syringocystadenoma papilliferum and the normal eccrine sweat gland. A relationship of dermal cylindroma to the apocrine gland is suggested by expression of lysozyme and alpha 1-antichymotrypsin. The tumor shares keratin, epithelial membrane antigen (EMA) and EGF-receptor expression with eccrine and apocrine glands. The presence of intermingled cells with a coexpression of keratin and vimentin argues for a partial myoepithelia-like differentiation. Neuroectodermal antigens are missing. Therefore, dermal cylindroma is classified as an adnexal tumor of skin with a variable rate of cells of apocrine secretory, myoepithelial and undifferentiated phenotypes.


Treatment with recombinant human insulin-like growth factor I of children with growth hormone receptor deficiency (Laron syndrome). Kabi Pharmacia Study Group on Insulin-like Growth Factor I Treatment in Growth Hormone Insensitivity Syndromes. Twenty-seven patients (14 female, 13 male; 3 pubertal) with growth hormone receptor deficiency (Laron syndrome) were treated with recombinant insulin-like growth factor I (IGF-I), 40-120 micrograms/kg body weight b.d., for up to 12 months. Height SDS was between -9.1 and -3.2 at the start of treatment (age, 3.7-22.9 years). Before treatment, most patients had increased basal serum concentrations of growth hormone (2.4-208 mU/l) and low serum concentrations of IGF-I (< 20-69 micrograms/l), IGF-II (69-295 micrograms/l) and IGF binding protein-3 (0.16-1.59 mg/l). In all but the two oldest patients, the growth rate increased by more than 2 cm/year compared with that before treatment. Asymptomatic hypoglycaemia (blood glucose < 3.0 mmol/l) was recorded in ten patients in 0.7% of measurements. Four patients experienced symptomatic hypoglycaemia. A transient asymptomatic decrease in serum potassium occurred in most patients after injections.


Production and purification of salicylate monooxygenase from Pseudomonas cepacia ATCC 29351. Salicylate monooxygenase (EC: 1.14.13.1) has been produced and purified from Pseudomonas cepacia ATCC 29351 which has the ability to utilise salicylate as a sole carbon source. The bacterium was grown on a defined medium containing 2% (w/v) casamino acids and 0.15% (w/v) yeast extract at 25 degrees C; salicylate monooxygenase production was induced by the presence of up to 0.7% (w/v) sodium salicylate, to a level of approximately 2% of the soluble cell protein. The enzyme was purified over 50-fold, with a recovery of about 40%, by a combination of ion exchange and hydrophobic interaction chromatography. The purified enzyme had a specific activity of 14-15 U mg-1 protein and was essentially homogeneous.


Recent writings on yeast recombination.

Recent writings on yeast recombination. The analysis of recombination mechanisms in Saccharomyces cerevisiae produces a lively literature spanning classical genetics, enzymology and the physical analysis of intermediates. Recent papers include excellent examples from each of these areas.


Diversity of mechanisms in the regulation of translation in prokaryotes and lower eukaryotes. Regulation of translation is used to control the expression of many essential and highly expressed genes. The known repertoire of molecular mechanisms for translational regulation is expanding. Recently elucidated mechanisms involve alterations in mRNA structure and modulation of the activity of translation initiation factors.


Turnover of mRNA in prokaryotes and lower eukaryotes. The turnover of mRNA plays an important role in the regulation of gene expression. The two best understood model systems are those of the prokaryote Escherichia coli and the lower eukaryote Saccharomyces cerevisiae. Considerable progress in recent years has helped define the general pathways by which mRNA is degraded in E coli. Much less is known about the pathways of decay, or the enzymes involved, in eukaryotic cells. However, both cis-acting sequences and trans-acting factors have recently been characterized in S. cerevisiae and an indispensable role for translation has been identified. A comparison of these model species highlights both similarities and differences in mRNA turnover between prokaryotic and eukaryotic systems.


Low molecular weight heparin in prevention of perioperative thrombosis. To determine whether prophylactic treatment with low molecular weight heparin reduces the incidence of thrombosis in patients who have had general or orthopaedic surgery. Meta-analysis of results from 52 randomised, controlled clinical studies (29 in general surgery and 23 in orthopaedic surgery) in which low molecular weight heparin was compared with placebo, dextran, or unfractionated heparin. Patients who had had general or orthopaedic surgery. Once daily injection of a low molecular weight heparin compared with placebo, dextran, or unfractionated heparin. Incidence of deep venous thrombosis, pulmonary embolism, major haemorrhages, and death. The results confirm that low molecular weight heparins are more efficacious for the prophylactic treatment of deep venous thrombosis than placebo (common odds ratio 0.31, 95% confidence interval 0.22 to 0.43; p < 0.001) and dextran (0.44, 0.30 to 0.65; p < 0.001). The results suggest that low molecular weight heparins are also more efficacious than unfractionated heparin (0.85, 0.74 to 0.97; p = 0.02), with no significant difference in the incidence of major haemorrhages (1.06, 0.93 to 1.20; p = 0.62). Low molecular weight heparins seem to have a higher benefit to risk ratio than unfractionated heparin in preventing perioperative thrombosis. However, it remains to be shown in a suitably powered clinical trial whether low molecular weight heparin reduces the risk of fatal pulmonary embolism compared with heparin.


Milestones in cystic fibrosis.

Milestones in cystic fibrosis. The study of cystic fibrosis (CF) provides a fascinating insight into developments in medicine in the 20th century. Milestones include the first clear clinical descriptions in the 1930s, discovery of a sweat electrolyte abnormality, establishing the autosomal recessive mode of inheritance and improvements in treatment. Microdissection experiments on sweat glands allowed the main defect to be delineated as one of chloride transport. Location of the gene to chromosome 7 made prenatal diagnosis feasible and carrier detection in siblings. The CF gene--its product being the cystic fibrosis transmembrane conductance regulator (CFTR), and its major mutation Delta F508 was discovered in 1989. World-wide collaboration has resulted in discovery of more than 150 further mutations. Incorporation of CFTR into non-chloride transporting insect cells by conferring chloride transport, proved it a chloride channel. CFTR incorporated into adenovirus results in correction of the chloride transport defect in airway cells, bringing gene therapy closer.


Cystic fibrosis gene.

Cystic fibrosis gene. The cystic fibrosis gene, located at 7q31, spans about 230 kb of genomic DNA and contains 27 exons. The cDNA of 6.2kb would predict an 1480 amino acid protein, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR has a high degree of homology with members of the ABC-transporter super family. The predicted protein structure consists of two membrane-spanning domains, each of 6 sub-units, anchoring CFTR in the apical membrane of specialized epithelial cells, 2 nucleotide binding folds (NBF) and a regulatory (R) domain. Disease-associated mutations in the CF gene are mainly clustered in the nucleotide-binding folds. The most common mutation, occurring in 70% of CF genes in Northern Europe and North America, is the deletion of amino acid phenylalanine at position 508 in the first NBF (ie delta F508).


Cystic fibrosis transmembrane conductance regulator (CFTR). Since the identification of the CF gene, less than 3 years ago, progress in analysing the function of its product, the cystic fibrosis transmembrane conductance regulator (CFTR), has been remarkable. It is now clear that CFTR functions as a small conductance chloride channel in epithelial membranes. However, many other questions remain unanswered. How does a defect in this channel result in the various pathologies associated with cystic fibrosis? Does CFTR have additional functions? How do CF mutations alter the function of the protein? Tools are now available to address these and other questions. Many features of CFTR activity suggest that pharmacological interventions may be possible. Nevertheless, an enhanced understanding of CFTR function is still essential before this basic research will provide direct benefit to CF sufferers.


Abnormalities in intracellular regulation in cystic fibrosis. Abnormalities in intracellular regulation in cystic fibrosis (CF) result from a given mutation in the cytoplasmic domain of CFTR, which renders it unable to respond correctly to agonists acting at the cell surface. This results in altered composition of epithelial secretions, which leads to some of the clinical manifestations of CF. Investigation of cyclic AMP- and Ca(2+)-mediated pathways controlling secretion is crucial for understanding how CFTR fails to respond to stimuli and how to reverse the defect in a CF cell. It should be feasible either to upregulate abnormal CFTR activity or to bypass the defect in the cell by stimulating a compensatory signalling pathway. Although their mechanism of action is unknown, one class of compounds, the methylxanthines, have been shown to reverse a fundamental CF abnormality in CF salivary cells and in non-epithelial cells overexpressing CFTR. This affords the exciting possibility that agents acting on intracellular signal transduction pathways will prove to be useful in devising new drug strategies for CF.


Microbiology of lung infection in cystic fibrosis. Bronchopulmonary infection in cystic fibrosis (CF) patients is associated with chronic progressive lung disease and episodes of acute exacerbation. Infection is predominantly caused by bacteria, although infections with viruses, mycoplasma and fungi may play undervalued roles. Bacteria commonly isolated from CF sputum include Staphylococcus aureus, Haemophilus influenzae and Pseudomonas aeruginosa. Colonisation of the airways by mucoid, alginate-producing variants of P. aeruginosa is recognised as a major cause of pulmonary deterioration. In addition, there is now considerable concern relating to the clinical consequences of colonisation and cross-infection with P. cepacia. This review discusses the microbiology of CF focussing on the pathogenesis and epidemiology of P. aeruginosa and P. cepacia.


Amylin increases transport of tyrosine and tryptophan into the brain. Injection of amylin (diabetes-associated peptide) into the hypothalamus induces anorexia, increases brain metabolism of dopamine and serotonin and elevates brain level of tryptophan. When male Sprague-Dawley rats were treated with 50 mg/kg L-tryptophan and L-tyrosine ethyl ester 30 min prior to the intrahypothalamic injection of 2 micrograms amylin, brain tryptophan and tyrosine levels were selectively increased as compared to rats treated with amylin alone. Hypothalamic and striatal serotonin metabolism also appeared to be increased following the amino acid-amylin treatment combination. These results suggest that amylin may increase transport of tyrosine and tryptophan into the brain, and that the increased availability of tryptophan may contribute to increased serotonin turnover observed following intrahypothalamic amylin treatment.


Enhancement of xanthine dehydrogenase mediated mitomycin C metabolism by dicumarol. These studies examined the effect of dicumarol on xanthine dehydrogenase (XDH), an enzyme recently shown to bioreduce mitomycin C. Dicumarol, which has previously been shown to inhibit xanthine oxidase (XO), inhibited both XDH and XO mediated conversion of xanthine to uric acid but potentiated the metabolism of mitomycin C by XDH and XO. Formation of 2,7-diaminomitosene following mitomycin C bioactivation by XDH was increased 3-fold aerobically and 4-fold hypoxically when 20 microM dicumarol was included in the reaction mixture. XO mediated metabolism of mitomycin C hypoxically was increased approximately 50% by the inclusion of dicumarol.


Immunohistochemical differentiation of atypical hyperplasia vs. carcinoma in situ of the breast. The distinction between atypical hyperplasia and carcinoma in situ in breast lesions can be difficult. The identification of myoepithelial cell layers may be helpful in establishing a diagnosis of proliferative breast disease vs. intraepithelial neoplasia. We reviewed pathologic material on 20 cases of atypical hyperplasia and 29 cases of carcinoma in situ. Immunohistochemical stains were employed against muscle-specific actin, S-100 protein, and cytokeratin to identify myoepithelial cells and to recognize different staining patterns. In atypical hyperplasia, muscle-specific actin staining identified myoepithelial cells in fine branching fibrovascular layers or as scattered cells between other proliferating cells. This pattern was absent in carcinoma in situ. S-100 protein showed more positive staining in atypical hyperplasia than in carcinoma in situ with patterns distinct from muscle-specific actin. Immunostaining for cytokeratin demonstrated distinctly different patterns between the two lesions. This study suggests that muscle-specific actin, S-100 protein, and cytokeratin in combination may assist in distinguishing proliferative breast disease with atypia from carcinoma in situ.


Prospective and randomized clinical trial for the treatment of hepatocellular carcinoma--a comparison of lipiodol-transcatheter arterial embolization with and without adriamycin (first cooperative study). The Cooperative Study Group for Liver Cancer Treatment of Japan. A randomized, controlled clinical trial comparing the use of lipiodol-transcatheter arterial embolization (L-TAE) in the presence versus the absence of Adriamycin (ADR) for the treatment of hepatocellular carcinoma was conducted from August 1988 through September 1989. In all, 125 Japanese hospitals participated in this study and 289 patients were entered in the trial. The patients were randomly allocated into group A (L-TAE) or group B (L-TAE + ADR) by telephone registration. There was no significant difference in background factors between group A and group B. Additional treatment, including repeated TAE or hepatic resection, was given to 189 patients. Among the four endpoints analyzed, the rate of tumor reduction and lipiodol accumulation in the tumor did not significantly differ between the two groups. The 3-year survival values for groups A and B were 33.6% and 34.9%, respectively; the difference was not significant. The serum alpha-fetoprotein level, however, decreased to a significantly greater extent in the group that received ADR than in the group that did not (P < 0.05). This result suggests that ADR has some favorable additional effect in L-TAE for the treatment of hepatocellular carcinoma.


Phase II trial for combined external radiotherapy and hyperthermia for unresectable hepatoma. Hepatocellular carcinoma is a major malignant disease in parts of Africa and Asia, including Korea. Surgical resection, which represents the best hope for cure, is limited by the extent of the disease and the high incidence of concurrent liver cirrhosis in Korea. We designed a phase II trial of combined external radiotherapy and hyperthermia for hepatocellular carcinoma that was unresectable due to either locally advanced lesions or associated liver cirrhosis so as to evaluate the efficacy and the safety of this combination regimen. This trial was performed at Yonsei Cancer Center between April 1988 and July 1988. External radiotherapy was delivered to a total dose of 3060 cGy/3.5 weeks. Hyperthermia was applied twice a week for a total of six treatment sessions using an 8-MHz radio-frequency capacitive-type heating device, i.e., Thermotron RF-8 and Cancermia. In all cases, hyperthermia was carried out within 30 min of the radiotherapy for a period of 30-60 min. The temperature in the tumor was measured by inserting a thermocouple into the tumor mass under ultrasonographic guidance in patients who did not have a bleeding tendency. The tumor response was assessed by CT scan after completion of the designed treatment. No complete response was obtained. However, a symptomatic improvement in abdominal pain was observed in 78.6% of cases and a partial response was achieved in 40% of the patients. The most important factor affecting the tumor response was the type of tumor (single massive, 71.4%; diffuse infiltrative, 20%; multinodular, 0; P < 0.005). The 1-year survival values determined for all patients and for the partial responders were 34% and 50%, respectively. The overall median duration of survival was 6.5 months. The median duration of survival for the partial responders was longer than that for the nonresponders (11 vs 5 months; P < 0.05). A mild degree of heat sensation, fever, first-degree burns of the skin, and nausea were observed as treatment-related adverse reactions. In conclusion, although this study is being continued, the results obtained thus for indicate that combined radiotherapy and hyperthermia seem to be effective in providing local tumor control and pain palliation in unresectable hepatocellular carcinoma while producing an acceptable level of toxicity.


Treatment of hepatocellular carcinoma by transcatheter hepatic arterial injection of radioactive iodized oil solution. After 12 days of culture, VX2 carcinoma cells were inoculated into the liver of 16 rabbits; 14 days later, 131I-labeled iodized oil ([131I]-Lp) suspended in lipiodol was injected into the hepatic artery. Selective accumulation of the contrast material in the tumor for an extended time was evident on X-rays and hepatic scintiphotographs. The antitumor effect was remarkable. [131I]-Lp agents warrant further examination for their clinical usefulness. Internal radiation therapy by transcatheter hepatic arterial injection of [131I]-Lp (group A) was evaluated in 9 patients with hepatocellular carcinoma (HCC, tumor stage III or IV) associated with liver cirrhosis (LC) and compared with combination therapy of Lp-TAE (group B) in 18 patients with HCC (tumor stage III or IV) associated with LC. In group A, serum AFP levels dropped rapidly in eight of the nine patients who had an elevated initial level of more than 500 ng/ml. The average reduction in tumor size was 50% in eight cases as determined by computed tomography. Histological examination of one resected liver specimen at 3 months after the third injection of [131I]-Lp revealed microscopic features highly suggestive of a radiation effect in the [131I]-Lp-containing area. The 1-year survival value for patients with HCC was estimated at 49.0% using the Kaplan-Meier method. The survival of patients treated with internal radiation therapy tended to be better than that of those treated with Lp-TAE (P = 0.119).


Changes in the plasma abnormal prothrombin level following treatment of hepatocellular carcinoma. The Hokkaido Liver Cancer Study Group focused on the changes in PIVKA-II levels observed in 61 HCC patients after several regimens of treatment in comparison with the AFP levels and the pathophysiological characteristics of HCC. The overall positivity rate for PIVKA-II was 47%, and there was no correlation between the PIVKA-II values and the AFP levels. Accordingly, the HCC detection rate was increased by about 20% by the measurement of both markers. In all, 13 patients underwent hepatic resection, and nonsurgical therapy was carried out in the other 48 subjects. Of the 6 surgically treated patients, 5 (83%) showed a fall in PIVKA-II levels to the normal range immediately after surgery, whereas 14/29 (48%) subjects receiving nonsurgical treatment showed a decrease in PIVKA-II values. Although inconsistency between these tumor markers was detected in four treated cases, we concluded that assay for both of these two parameters may expand their clinical utility for the diagnosis of HCC and monitoring of patients after treatment.


Treatment of hepatocellular carcinoma with a CDDP-epirubicin-lipiodol suspension: a pilot clinico-pharmacological study. Lipiodol injection is a useful method for detecting liver tumors, especially hepatocellular carcinoma (HCC). We therefore prepared and tested a new emulsion of lipiodol containing epirubicin and cis-diammine-dichloroplatinum (CDDP), drugs that are very effective against HCC. This CDDP-epirubicin-lipiodol suspension (CELS) was injected into 18 HCC patients via a celiac angiographic catheter. In 11 of these patients, CELS was followed by transcatheter arterial embolization (TAE) therapy. Clinical and pharmacological investigations were performed in all 18 patients, and the following results were obtained. CELS is pharmacologically and chemically stable, and both the results of the dissolution test and the serum levels of these two drugs indicate that slow release can be obtained. After the injection of CELS, serum levels of AFP and PIVKA-II decreased immediately, and no fatal clinical side effects were encountered. Although no statistically significant difference was observed, the survival (Kaplan-Meier method) of patients injected with CELS in the presence or absence of TAE therapy can be estimated to be much longer than that of patients receiving CDDP-lipiodol suspension injection in the presence (16 patients) or absence (6 patients) of TAE therapy. A combination of CELS injection and TAE therapy might be effective and useful for the treatment of HCC.


Evaluation of transcatheter arterial embolization with epirubicin-lipiodol emulsion for hepatocellular carcinoma. A total of 18 patients with hepatocellular carcinoma (HCC) were treated by transcatheter arterial embolization (TAE) with a 4'-epi-doxorubicin (EDX)-lipiodol emulsion. Infusion of the EDX-lipiodol emulsion (EDX-L) via the hepatic artery was followed by the injection of gelatin sponge in 12 cases. The response and survival of these 12 patients following EDX-L treatment were compared with those of 42 subjects treated with a doxorubicin-lipiodol emulsion (DX-L) and those of 23 patients treated by TAE with gelatin sponge (GS) only. In the group treated with EDX-L, nine cases were AFP-positive in sera and four showed a decrease in serum AFP values to less than 10% of the pretreatment level. Seven cases showed a partial response, and nine cases showed no change in the size of the tumor. In the group treated with EDX-L, nine cases are alive, and the oldest has survived for more than 431 days since the treatment. The half-year survival value was 57%, and the 1-year survival value was 49%. These values did not differ significantly from those calculated for the group treated with DX-L. The 1-year survival value determined for patients treated with a lipiodol emulsion (EDX-L or DX-L) followed by GS was 65%, and the 2-year survival value was 39%. These results rates are significantly better than those obtained in patients treated with GS only (1-year survival, 39%; 2-year survival, 13%.


Analysis of prognostic factors in patients with hepatocellular carcinoma treated by transcatheter arterial embolization. We studied 240 cases of unresected hepatocellular carcinoma (HCC) using Cox's proportional hazard model to elucidate which factors would be closely related with the survival period after treatment by transcatheter arterial embolization (TAE) in the presence or absence of iodized oil. The results were as follows. The cumulative survival values obtained after TAE were 67.5% for 1 year, 32.0% for 2 years, and 20.5% for 3 years. The most significant prognostic factor was the degree of extension of tumor embolus in the portal vein or its branch. The tumor extension and the tumor type were also important factors. Age, sex, and AFP, HBsAg, and HCV Ab values were not useful as prognostic factors. This study provides a rational background for the selection of treatment for HCC. Furthermore, knowledge of the prognostic factors is useful for the management of patients, particularly in maintaining their good quality of life.


Treatment of hepatocellular carcinoma by transcatheter arterial chemoembolization and analysis of prognostic factors. A total of 100 patients with histologically proven hepatocellular carcinoma (HCC) underwent transcatheter arterial chemoembolization (TACE) and were followed for more than 1 year and 10 months. Portal vein branch thrombosis was diagnosed in 14 patients, and extrahepatic metastasis was noted in 11 subjects. The embolization material used was iodized oil (0.1-0.2 ml/cm tumor area at its maximal diameter), which was prepared by pumping with contrast agent and then mixed with anticancer drugs; Gelfoam particles measuring 1-2 mm in size were subsequently injected. The overall cumulative 0.5- 1-, 2-, and 3-year survival rates were 81%, 57%, 31%, and 21%, respectively. Patients with an intact capsule and those with solitary lesions, especially when the tumor diameter was < 5 cm, achieved a higher survival rate. In contrast, incomplete TACE, extrahepatic metastasis, and portal vein thrombosis were associated with the worst outcome. Patients with positive HBsAG and diffuse or multiple tumors also showed a poor outcome. Early diagnosis and early treatment of HCC are the keys for the achievement of better clinical results.


Aromatic L-amino acid decarboxylase deficiency: diagnostic methodology. Aromatic L-amino acid decarboxylase (EC. 4.1.1.28) deficiency is a newly described inborn error of metabolism that affects serotonin and dopamine biosynthesis. The major biochemical markers for this disease are increases of L-dopa, 3-methoxytyrosine, and 5-hydroxytryptophan in urine, plasma, and cerebrospinal fluid together with decreased cerebrospinal fluid concentrations of homovanillic acid and 5-hydroxyindoleacetic acid. In addition, concentrations of vanillactic acid are increased in the urine. Specific HPLC and gas chromatography-mass spectrometry methods are described that permit the identification and measurement of these metabolites in the above body fluids. Simplified assays for human plasma L-dopa decarboxylase and liver L-dopa and 5-hydroxytryptophan decarboxylase, used to demonstrate the enzyme deficiency, are also reported.


Performance of an enzyme-linked immunosorbent assay system for antibodies to hepatitis C virus with two new antigens (c11/c7). We developed an enzyme-linked immunosorbent assay (ELISA) system for antibodies to the hepatitis C virus (HCV), using two new recombinant antigens (c11 and c7) derived from the HCV genome. The performance of this ELISA system (Imucheck HCV Ab) was examined. The CV values for both intra-assay precision and reproducibility of identifying HCV antibody in the panel sera ranged from 3.5% to 6.4%. The blood elements in serum and anticoagulants did not interfere in this ELISA system. The specificity of Imucheck HCV Ab to samples from patients with non-A, non-B (NANB)-type chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma was 93.7%, 93.5%, and 81.4%, respectively. These results are more sensitive than those obtained by the first-generation anti-HCV ELISA system. In the samples from patients with NANB-type acute hepatitis, Imucheck HCV Ab enabled detection of HCV antibodies at an early stage. This system increased the sensitivity for blood donor screening and for monitoring patients with acute hepatitis.


Spuriously low concentrations of serum iron measured with generation 14 Kodak Ektachem slides: prevalence, possible causes, and partial improvement with generation 16 slides. For sera with iron (Fe) concentrations < 4 mumol/L, Kodak Ektachem slides Generation (GEN) 14 (without ascorbic acid) yielded systematically lower results for Fe than did liquid Ferrozine-based reagents from Baker containing ascorbic acid (10 g/L, final concentration) and adapted to Cobas-Bio. During an 8-month comparison period, outliers (defined as [Fe]Cobas - [Fe]Kodak > 4 mumol/L) were seen in 21 of the 8731 sera (0.24%) tested, corresponding to < 5% of the sera with [Fe]Kodak < 4 mumol/L. In vitro addition of ascorbic acid and (or) Fe identified at least two types of outliers: type 1 (approximately 70%), characterized by [Fe]Kodak > 0.4 mumol/L, by (supra)normal Fe recovery in Kodak slides in the presence or absence of ascorbic acid (10 g/L), and by between-method differences in serum Fe (Cobas - Kodak) that were significantly correlated with serum Zn content (P < 0.0004); and type 2 (approximately 30%), tentatively ascribed to contamination by EDTA, with serum Fe by Kodak < 0.4 mumol/L and Fe recovery near 0%, both of which could be significantly and dose-dependently increased by addition of ascorbic acid (5-20 g/L). For both types of outliers, flameless atomic absorption spectrometry (AAS) yielded results that were significantly higher than concentrations by Kodak with GEN 14. Use of GEN 16 slides (containing ascorbic acid) improved concordance of Kodak results with Cobas, and hence with flameless AAS, for both types of outliers; abolished Zn dependency of results; and increased Fe results in sera with type 2 outliers, although these remained substantially lower than by Cobas. However, like other ascorbic acid-containing reagents, GEN 16 slides were more sensitive to interference by dextran-bound Fe, as assessed during in vitro addition experiments and comparisons involving samples from Fe-dextran-treated patients. GEN 16 slides are hence expected to more frequently overestimate the physiologically available protein-bound Fe in hemodialysis patients. In hospital laboratories, this new interference will probably arise more frequently than the spuriously low results with GEN 14, hence warranting further efforts in optimizing Fe slides.


Allelic amino acid substitutions affect the conformation and immunoreactivity of germ-cell alkaline phosphatase phenotypes. The gene encoding placental alkaline phosphatase (PLAP) displays a well-documented allelic polymorphism. Likewise, different phenotypes exist for the PLAP-related germ-cell alkaline phosphatase (GCAP). We investigated the extent to which various allelic GCAP positions are critical in determining the enzymatic, structural, and immunological properties of GCAP phenotypes. Three homozygous GCAP phenotypes [JEG3, BeWo, and wild-type (wt) GCAP] were analyzed and compared with a "core" GCAP mutant that contains the seven amino acid substitutions that are consistently different between PLAP and GCAP but are common to the three known allelic GCAP genotypes. Although some substitutions could influence the electrophoretic behavior of the phenotypes, the allelic differences did not affect the kinetic properties of GCAP. However, they did affect the immunoreactivity and conformation of the variants as detected with a panel of 18 epitope-mapped monoclonal antibodies (MAbs) to PLAP. The selective immunoreactivity of the PLAP/GCAP-discriminating MAb C2 was critically dependent on the nature of the allelic residues 133 and 361 in GCAP. Residue 133 was also important for the general stability of the molecule because BeWo and wt GCAP, which have Asn133 and Val133, respectively, instead of Met133, showed a consistently reduced heat stability compared to core GCAP and JEG3. Because the core GCAP mutant consistently shows the characteristics of wt GCAP, its use as an antigen should allow the generation of monoclonal antibodies to GCAP that will not cross-react with PLAP and whose immunoreactivity will only marginally be influenced by allelic GCAP variation.


Epidermal proliferation and keratinization following standardized elicitation with diphenylcyclopropenone. Diphenylcyclopropenone (DCP) was applied to the upper arms of five alopecia areata patients using 10% of the concentration that had been applied previously to the scalp during topical immunotherapy. DCP applied in this concentration evoked a mild eczematous reaction. Biopsies were taken before DCP application and after 24, 48 and 96 h. A large increase in T-lymphocytes and CD14-positive cells in the dermis was seen after 24 h. Migration of these cells into the epidermis was mainly observed during the first 48 h. This was followed by epidermal proliferation as assessed by the number of Ki-67-positive nuclei and the degree of Ks8.12-binding. Both showed their main increase after 48 h; but after 24 h the increase of Ki-67-positive nuclei was significant (P < 0.04). Involucrin and filaggrin showed a gradual increase which became substantial after 96 h (both P < 0.04). As the invasion of inflammatory cells into the epidermis preceded the main increase in epidermal proliferation, cytokines are suggested as possible mediators for the initial phase of the proliferative response after DCP application.


Killing of human leukaemia/lymphoma B cells by activated cytotoxic T lymphocytes in the presence of a bispecific monoclonal antibody (alpha CD3/alpha CD19). Bispecific antibodies (BsAb) can be used to retarget T cells irrespective of their specificity to certain target cells inducing target cell lysis. We have tested the efficacy of the BsAb SHR-1, directed against the T cell antigen CD3 and the B cell antigen CD19 to induce (malignant) B cell kill by T cells as measured in a 51Cr-release assay. Two cytotoxic T cell clones (CTL), expressing TCR alpha beta or TCR gamma delta, were effective in killing CD19 expressing B cell lines at different stages of differentiation in the presence, but not in the absence, of the BsAb. CD19- target cells were not killed. Fresh CD19+ leukaemia/lymphoma cells were also efficiently killed by SHR-1 preincubated CTL clones. In addition, phytohaemagglutinin (PHA) or CD3-activated IL-2 expanded peripheral blood mononuclear cells (PBMC) of normal donors did so after 2 weeks of stimulation. A concentration of 100 ng/ml of the BsAb was sufficient to obtain optimal lysis of all target cells tested. These results show that fresh human leukaemia/lymphoma cells, freshly derived from active lymphoblastic leukaemia (ALL) as well as non-Hodgkin's lymphoma (NHL) patients, can be effectively killed in the presence of this BsAb by activated T cells.


Antibodies to Mycobacterium paratuberculosis-specific protein antigens in Crohn's disease. The possible role of infection with Mycobacterium paratuberculosis (MAP) for the etiopathogenesis of Crohn's disease (CD) has been a matter of long-term controversy. In addition to similarities with the pathology of ruminant paratuberculosis, DNA fingerprinting confirmed the organism isolated from gut tissue, but the specificity of the immune repertoire has not as yet been evaluated. We report here on a serological study of 29 patients with CD, 20 patients with ulcerative colitis and 18 healthy control subjects, using three antigens attributed with species-specificity and selective immunogenicity following MAP infection. Antibodies binding to the 38-kD band of MAP extract were demonstrable by the Western blot technique in 57% of CD patients. Antibody levels to the 24-kD (p24BCD) cathodic bands, determined by competition ELISA using a monospecific murine antiserum, and to the 18-kD protease-resistant purified bacterioferritin, detected by standard ELISA, were significantly elevated in 53% of CD patients. However, these three antibody specificities tested in individual CD patients did not show any correlation with each other. Thus, 18% of patients were positive for all three specificities, whilst 84% had antibodies to at least one of the specific antigens. Although the exact proportion of affected patients is yet to be defined, the serological results obtained support the view that MAP infection may play an etiological role in Crohn's disease.


Distribution of the PBC-specific- (M2) and the naturally-occurring mitochondrial antigen- (NOMAg) systems in plants. In previous studies it was demonstrated that antibodies in sera from patients with primary biliary cirrhosis (PBC) and their relatives can recognize two different antigen systems in the ATPase fraction prepared from beef heart mitochondria, namely the PBC-related M2- and the naturally occurring mitochondrial antigen (NOMAg)-related epitopes. Since separation of these two antigen systems could not be achieved using mammalian mitochondria, mitochondria from a wide spectrum of plants were analysed with respect to the presence of mitochondrial antigens. Mitochondria from 29 species of plants were prepared and tested by ELISA and Western blot using marker sera from patients with PBC reacting in the Western blot with M2a,b,c,d (alpha-ketoacid-dehydrogenase complex) and NOMAg-specific sera recognizing the three major epitopes epsilon, zeta, and eta at 65, 61 and 58 kD. Naturally occurring mitochondrial antibody (NOMA)-positive marker sera reacted in the ELISA with mitochondria from all plants, and the zeta/eta positive sera gave also a positive reaction at 61/58 kD in the Western blot while the epsilon epitope could not be visualized by this method. In contrast, the M2 antigen was detected preferentially in lower plants such as algae, fungi, and ferns. Analysing these data with respect to the evolution of proteins one would have to assume that the M2 antigen was lost in most higher plants or underwent some structural alterations. Furthermore, considering the fact that the M2- and the NOMAg-related epitopes could be only partially separated, i.e. there were no plant mitochondria showing only M2 but no NOMAg, one could speculate that anti-M2 antibodies are derived from the pool of naturally occurring antibodies.


Changes in hepatic mRNA levels of acute phase proteins during rat adjuvant arthritis. Using specific cDNA probes, we have investigated changes in hepatic mRNA concentrations of the major acute phase proteins fibrinogen, alpha 2-macroglobulin (alpha 2-MG), albumin and alpha 1-acid glycoprotein (alpha 1-AGP) during developing adjuvant arthritis in Lewis rats. Continuously increasing levels in the mRNA of the positive reactants beta-fibrinogen, alpha 2-MG and alpha 1-AGP were found during developing disease with peak levels from day 15 to 21, whereas mRNA concentrations of the negative reactant albumin decreased, reaching their lowest levels on day 11 to 15. As early as 4 days after arthritis induction, the hepatic mRNA levels of beta-fibrinogen, alpha 1-AGP and albumin were distinctly different from control values. The most dramatic changes in the hepatic mRNA levels and plasma concentrations of acute phase reactants were seen between days 11 and 21. These results indicate that overproduction of the major inflammatory cytokines IL-1, TNF-alpha and IL-6, which are now felt to be largely responsible for the acute phase response in the rat, is an early event during adjuvant arthritis and that the highest amounts are produced during the inflammatory phase of the disease. mRNA changes in the acute phase proteins alpha 1-AGP and albumin, which are mainly regulated by IL-1/TNF alpha, were more pronounced than those of alpha 2-MG and beta-fibrinogen, which are predominantly controlled by IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)


Influence of calcitonin treatment on the osteocalcin concentration in the algodystrophy of bone. Algodystrophy (AD) attacks all tissues in the affected region and results in the rapid demineralization of bones. Osteocalcin (OC) and alkaline phosphatase (AP) are markers of bone turnover. Calcitonin is the treatment of choice of AD. Two groups of patients were studied: Group I (n = 8)--acute stage of AD (before and during the calcitonin treatment), Group II (n = 5)--late chronic stage of AD. In the acute stage of AD both OC level and AP activity were increased. They were normal in the chronic stage of AD. During the calcitonin treatment OC level normalized after 14 days and then increased again. During the treatment, AP activity temporarily increased and then returned to the initial level. We confirm that an increased bone turnover is observed in the acute stage of AD. Discrepancy between OC level and AP activity reflects the local metabolic disturbances. Salmon calcitonin inhibits the algodystrophic process and probably contributes to the activation of the skeletal restoration.