Randomized clinical trial to assess the effectiveness of breast irradiation following lumpectomy and axillary dissection for node-negative breast cancer. Although the conservation management of breast cancer has become a routine method of treatment in most centers, there is still considerable controversy surrounding the ultimate minimum treatment required for node-negative breast cancer to achieve adequate local control. Our purpose was to assess the value of breast irradiation in reducing breast relapse following conservation surgery for node-negative breast cancer. We attempted to define low-risk groups of women for breast and distant site relapse (i.e., recurrence outside the breast) who might be spared breast irradiation or adjuvant systemic therapy. Eight hundred thirty-seven patients were randomly assigned to receive radiation therapy or no radiation therapy following lumpectomy and axillary dissection for node-negative breast cancer. Breast irradiation reduced relapse in the breast from 25.7% in the controls to 5.5% in the irradiated patients. There was no difference in survival between the two groups (median follow-up, 43 months). A low-risk group (less than 5% chance of relapse in the breast without irradiation) could not be defined. Tumor size (greater than 2 cm), age (less than 40 years), and poor nuclear grade were important predictors for breast relapse. Age (less than 50 years) and poor nuclear grade were important predictors for mortality. The presence of ductal carcinoma in situ did not predict breast relapse. Breast irradiation significantly reduces breast relapse, but it does not influence survival. Important predictors of breast relapse are age, tumor size, and nuclear grade, but not the presence of ductal carcinoma in situ. Age and, in particular, nuclear grade predict survival. Further follow-up may define an acceptable low-risk group for breast relapse. Until then, we recommend that all patients receive breast irradiation. Systemic adjuvant therapy should be considered for patients with poor nuclear grade tumors.


Assessment by planimetry of Langerhans' cell density in penile epithelium with human papillomavirus infection: changes observed after topical treatment. Langerhans' cells could have an important role in the immune surveillance for the malignant transformation associated with certain viral infections. A planimetric study of Langerhans' cells was performed on epithelial sheets obtained from male and female patients who presented with human papillomavirus-induced condylomata. Preliminary epidemiological studies have indicated that after a human papillomavirus infection the risk of malignant transformation is higher in the cervix than in the penis. Our study was designed in such a way that the Langerhans' cell density observed in condylomata could be compared to that detected in adjacent normal areas from the same patients. The results showed that a highly significant decrease in Langerhans' cells occurred in male and female human papillomavirus infected genital tracts. In the penis the local treatment with 5-fluorouracil restored the normal Langerhans' cell density. The significance of these findings in relationship with malignant transformation is discussed.


Renal scarring following reflux and nonreflux pyelonephritis in children: evaluation with 99mtechnetium-dimercaptosuccinic acid scintigraphy. 99mTechnetium dimercaptosuccinic acid (DMSA) scintigraphy is the imaging modality of choice for the detection of acute pyelonephritis and chronic renal scarring in children. Using the DMSA scan we prospectively evaluated renal scarring after reflux and nonreflux pyelonephritis in children. The study population consisted of 33 patients with acute pyelonephritis documented by a DMSA renal scan at infection. The children were evaluated for renal scarring with a followup DMSA scan 4 to 42 months (mean 10.7 months) after the acute infection. All new scarring on followup DMSA scans occurred at sites corresponding exactly to areas of acute inflammation on the initial DMSA scan. Therefore, only those kidneys with acute changes on the initial scan were subsequently analyzed. Of 38 kidneys new or progressive scarring developed in 16 (42%), including 6 of 15 (40%) with associated vesicoureteral reflux and 10 of 23 (43%) without demonstrable reflux. New renal scarring developed in 6 of the 7 kidneys (86%) associated with a neuropathic bladder or posterior urethral valves. In contrast, new scarring developed in only 10 of 31 kidneys (32%) associated with a normal bladder (p = 0.028). Excluding the kidneys associated with a neuropathic bladder or posterior urethral valves, new renal scarring developed in 3 of 12 (25%) with primary reflux, compared with 7 of 19 (37%) without vesicoureteral reflux. Except for the white blood count and the species of infecting bacteria, no other statistically significant differences could be found between those cases in which scars did or did not develop. We conclude that acquired renal scarring only occurs at sites corresponding to previous areas of acute pyelonephritis, the acute parenchymal inflammatory changes of acute pyelonephritis are reversible and do not lead to new renal scarring in the majority of cases, and once acute pyelonephritis has occurred ultimate renal scarring is independent of the presence or absence of vesicoureteral reflux.


Glutathione S-transferases in human testicular germ cell tumors: changes of expression and activity. Glutathione S-transferases are involved in the detoxification of carcinogens and xenobiotics and are potentially associated with the development of drug-resistance. Forty-six testicular germ cell tumors and 33 adjacent normal testicular tissue specimens were analyzed at the RNA level for the expression of glutathione S-transferase alpha and pi. Glutathione S-transferase alpha was expressed in 31 of the 33 normal testicular tissues (94%) but in only three of the 46 germ cell tumors (7%). Glutathione S-transferase pi mRNA was detected in all normal and malignant testicular tissue samples. Thirteen testicular germ cell tumors and eight normal testicular tissue samples were analyzed at the protein level. The mean specific activity of total cytosolic glutathione S-transferase in tumor tissue was decreased by about 80% as compared to normal testicular tissue. Protein analysis of the glutathione S-transferase subunits of normal testicular tissue demonstrated the presence of the glutathione S-transferase classes alpha, mu and pi, with a predominance of the mu class. In testicular germ cell tumors the glutathione S-transferase subunit pattern showed a predominance of glutathione S-transferase pi representing 88% +/- 3% of total glutathione S-transferase. Since all three glutathione S-transferase isoenzyme classes contribute to the resistance to antineoplastic drugs, the altered glutathione S-transferase isoenzyme pattern and the decrease of glutathione S-transferase activity may play a role in the high inherent drug sensitivity of human testicular germ cell tumors.


[Experimental study of the mechanism of auranofin-induced diarrhea]. To clarify the kinetics of cell membrane and intracellular mediators in the process of auranofin (AF)-induced diarrhea, we perfused electrolyte solution containing the oral gold preparation AF, which is a treatment for rheumatoid arthritis, through the rat jejunum, and studied net water and electrolyte transport, Na+, K(+)-ATPase activity, and c-AMP and c-GMP concentrations in the jejunal mucosa. In addition, change in Ca+ concentration in isolated intestinal cells was evaluated using fura-2-acetoxyl-methyl ester. AF significantly suppressed water and electrolyte transport. Mucosal secretion was increased due to elevation of the intracellular Ca+ concentration early in the perfusion period, then due to reduction in the Na+, K(+)-ATPase activity, and increase in the c-AMP concentration late in the perfusion period. Therefore, these cell membranes and intracellular mediators are considered to be involved in the mechanism of AF-induced diarrhea.


[Cloning of 67 kDa-laminin receptor cDNA and its expression in various human lung cancer cell lines]. Cell-adhesive proteins such as laminin and fibronectin and their specific receptors play an important role in the processes of cancer proliferation, invasion, and metastasis. In the present study, we cloned the cDNA of 67 kDa-laminin receptor both from human lung cultured cell line (IMR90) and from human small cell lung cancer (SBC3), and determined the nucleotide sequences. In addition, the expression of mRNA in 11 lung cancer cell lines with various cell types was estimated by the method of Northern blot hybridization. As a result, 1.2 kb-message was detected in all cancer cell lines examined. It was also demonstrated that the mRNA level of 67 kDa-laminin receptor was inversely proportional to the population doubling time of the cell line (r = -0.80).


[Immunohistochemical analysis of CA19-9, SLX, and CA125 in adenoid cystic carcinoma of trachea and bronchus]. We examined the usefulness of 3 carbohydrate antigens, CA19-9, CA125, and SLX, as tumor markers in adenoid cystic carcinoma of the trachea and bronchus, immunohistochemically and clinically. CA19-9 and SLX were detected immunohistochemically in normal tracheal and bronchial glands, but CA125 was not detected. Bronchial secretions obtained from 7 patients without cancer contained high levels of these 3 carbohydrate antigens. There was positive immunohistochemical staining for CA19-9, CA125, and SLX in 5 of 9, 1 of 9, and 3 of 8 patients with tracheal or bronchial adenoid cystic carcinoma, respectively. In a case of bronchial adenoid cystic carcinoma in whom pretreatment serum CA19-9 showed a very high level, this marker changed in parallel with the clinical course. These results suggest the possibility that CA19-9 and SLX could be used as useful markers in patients with tracheal and bronchial adenoid cystic carcinoma.


[A case of hypersensitivity pneumonitis due to isocyanate exposure showing progression even two months after removal of the antigen]. A 68-year-old male developed dry cough and exertional dyspnea after handling paint spray containing isocyanates (TDI, MDI) for three months. Initially, the symptoms fluctuated according to whether he was at work or not. He was admitted to our hospital on February 7, 1990, because of progressive worsening of symptoms. In spite of admission to hospital and cessation of exposure to isocyanates, there was no improvement of symptoms. His chest X-ray film showed diffuse small nodular and reticular shadows. Transbronchial lung biopsy revealed thickening of the alveolar walls and formation of Masson's bodies associated with mononuclear cell infiltration in alveolar spaces. High titers of TDI-HSA and MDI-HSA specific IgG antibodies were detected by ELISA, and a high level of serum soluble IL2 receptor was also detected. From these results, we diagnosed hypersensitivity pneumonitis due to exposure to isocyanates. One week administration of prednisolone caused dramatic improvement of his symptoms, chest X-ray findings, and laboratory data. His clinical course and response to prednisolone therapy indicated that long-term steroid administration could not be avoided. The prolonged symptoms and the necessity for long-term steroid therapy are discussed.


[A case of hypersensitivity pneumonitis in which serum specific antibodies for three species of isocyanate molecules were demonstrated]. A 64-year-old male was admitted to our hospital with complaints of the development of high fever, cough and dyspnea 5-6 hours after painting automobiles. His chest X-ray film showed interstitial shadows in both lungs. Pulmonary function test demonstrated reduction of diffusion capacity; and blood gas analysis demonstrated hypoxemia and an increase in alveolar-arterial oxygen tension difference. Marked lymphocytosis and a striking decrease in CD4/CD8 ratio were observed in the bronchoalveolar lavage fluid. Transbronchial lung biopsy specimens showed alveolitis and Masson's bodies. We suspected that the patient was suffering from hypersensitivity pneumonitis induced by isocyanates contained in the urethane paint he used. Immunological studies were performed using chemical compounds of three species of isocyanate molecules (TDI, MDI, HDI) and human serum albumin (HSA). The results were as follows: skin tests were positive for TDI-HSA and MDI-HSA; lymphocyte-stimulation tests on peripheral blood were positive for TDI-HSA; precipitation reaction was negative for all the isocyanates studies; enzyme-linked immunosorbent assay (ELISA) demonstrated the existence of specific IgG antibodies for TDI, MDI and HDI; inhalation challenge test by TDI-HSA was negative, but environmental provocation was considered to be positive. We diagnosed his pulmonary disorder as hypersensitivity pneumonitis due to isocyanates. Type III and Type IV allergic reactions of Gell-Coombs were suggested to be involved in the pathogenesis, however, there remains the possibility that the instability of isocyanate compounds as antigen modified the results of our immunological studies.


Cardiac-derived neutrophil chemotactic factors: detection in coronary sinus effluents of patients undergoing myocardial revascularization. Recent studies from our laboratory have demonstrated the release of neutrophil chemotactic factors from isolated rabbit hearts perfused with cardioplegic solutions and from ischemic dog hearts after coronary artery occlusion for 1 hour. On the basis of these animal studies, a test is now made of the hypothesis that neutrophil chemotactic factors are released by myocardial tissues of patients who undergo surgical myocardial revascularization. By means of modified Boyden chambers, the levels of neutrophil chemotactic factors were measured in effluent collected from the coronary sinuses of six patients undergoing cardiopulmonary bypass during periods of cold cardioplegia. Plain cardioplegic solutions were also analyzed. The standard formyl-methionyl-leucyl-phenylalanine, a stimulant of neutrophil recruitment, was used as a positive control solution. Results indicated the recovery of significantly high levels of neutrophil chemotactic factors in patient samples (i.e., 128% +/- 19% of formyl-methionyl-leucyl-phenylalanine) compared with control plain cardioplegic solution (less than 5% of formyl-methionyl-leucyl-phenylalanine) (p less than 0.0001). A standard checkerboard analysis indicated that the observed activity is chemotactic (i.e., directed migration) and not chemokinetic (i.e., random migration). This study also showed that these factors are proteins of a molecular weight in excess of 300 kd and exhibit in vivo activity by recruiting neutrophils into rabbit skin. The absence of immune cell-derived chemoattractants such as interleukin-1 and leukotriene B4 in these coronary sinus effluents suggests that the observed chemotactic activity is cardiac derived. Results of this investigation therefore demonstrate the release of neutrophil chemotactic factors by ischemic human hearts during cardiopulmonary bypass.


Age-related change of free radical generation in liver and sex glands of rats. Many investigations have been made on age-related changes of lipid peroxidation in tissue homogenates and subcellular fractions. However, to date there are few reports on the age-related change of free radicals in living organs during aging. In our study, we investigated free radical concentration in liver and sex glands of different aged rats by using electron spin resonance (ESR) technique. A significant reduction of free radicals in liver and sex glands of old aged rats was observed when compared with those of young or middle-aged ones. The decrease of free radical generation during aging is discussed.


The effect of donor age on human fibroblast beta-adrenergic receptor response and agonist-induced desensitization. Aging has been associated with changes in beta-adrenergic receptor (beta-receptor) function in several tissues. The relative contribution of cellular aging and age-related changes in homeostatic regulation of receptor function is unknown. We have examined beta-receptor function in fibroblasts of young and old donors (young: mean age 31.2 +/- 0.8 years +/- S.E., n = 6; old: mean age 81.8 +/- 0.6 years +/- S.E., n = 6). Beta-receptor responses to isoproterenol (ISO), (1 microM) were similar in the two groups. The concentration of ISO required for 50% maximal beta-receptor-mediated cyclic AMP production (EC50) was similar in both groups. Fibroblast beta-receptor density was also similar in young and old groups. ISO-induced beta-receptor desensitization was both dose- and time-dependent. Submaximal desensitization by acute exposure (30 min) to ISO (1 mM) caused similar levels of beta-receptor desensitization in young (42.5 +/- 2.5%) and old (42.8 +/- 2.8%) groups and a similar increase in ISO EC50. These findings demonstrate that aging in vivo does not cause changes in fibroblast beta-receptor regulation that are retained in culture.


[Comparison of DNA analysis with histomorphologic parameters in stomach cancer]. After curative resection of stomach carcinomas (adenocarcinomas: n = 58, signet ring cell carcinomas: n = 24, undifferentiated carcinomas: n = 21) the DNA content of the tumor cells was compared with the histomorphological parameters. There was a correlation between the DNA content and the histomorphological parameters. The DNA analysis had no additionally prognostic influence. In the multivariate regression analysis the prognosis depended on lymph node status (p = 0.0009), pT-stage (p = 0.02), tumor localization (p = 0.03) and the histological type (p = 0.05). The prognosis was independent of the DNA content. Furthermore, neither did the degree of differentiation, the operative procedure, the safety distance, the size of the tumor, the sex nor the age of the patient have any influence on the prognosis.


Early detection of damage to nerves in leprosy. Methods of examining and diagnosing damage to nerves commonly involved in leprosy are described. The equipment used is inexpensive, gives reliable and repeatable results and is useful in making objective assessments in terms of function in everyday living.


Magnetically coupled paramagnetic relaxation agents. Measurements of the spin-lattice relaxation rates of water protons made over a wide frequency range have demonstrated that the effects of paramagnetic relaxation agents may be considerably enhanced when the paramagnetic center is incorporated into a compact structure macromolecule such as a protein that is rotationally constrained. The immobilization of the macromolecule profoundly changes the nature of the magnetic field dependence of the relaxation rate for both the diamagnetic and the paramagnetic samples. The immobilization also amplifies the effect of the paramagnetic center as a water-proton relaxation agent. The direct exchange of labile water molecules or protons is not a requirement for the high efficiency of this class of magnetic relaxation agents.


Studies on the effects of glucose in vitro and of the glycaemic state in vivo on the binding characteristics of mu, delta and kappa opiate receptors in mouse brain. The effect of glucose on the binding characteristics of opiate receptor subtypes was investigated in brain membranes from normoglycaemic lean Aston (C57BL/6J) mice using [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO), [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) and [3H]U69,593 as selective ligands for mu, delta and kappa opiate receptors respectively. The equilibrium dissociation constants (Kd) and maximal binding capacities (Bmax) of [3H]DAMGO and [3H]DPDPE were unaltered by 20mM glucose in vitro. Similarly, [3H]U69,593 binding was not modified by increasing the concentration of glucose from 0 to 20mM (P between 0.10 and 0.05), or by the presence of 20mM fructose and of 20mM 3-O-me-glucose, a non-metabolisable sugar, in the incubation medium. The nonselective opiate ligand, [3H]diprenorphine, bound with similar affinity and binding capacity to brain membranes prepared from control and streptozotocin-diabetic Swiss (CD1) mice. The addition of 20mM glucose or of 20mM fructose in vitro induced no changes in their binding parameters. The affinity and binding capacity of [3H]U69,593 to STZ-diabetic Swiss mouse brain membranes was not significantly different to that of normoglycaemic controls; 20mM glucose in vitro had no effect on ligand binding to kappa sites in STZ-diabetic mouse brain membranes. We conclude that glucose does not interact directly with the opiate receptor to modfy it in such as way as could explain the altered sensitivity to different opioid agonists seen in obese and hyperglycaemic animal models in vivo.


A new perspective to assess relaxation time of tumor. The dissimilarities in spin-lattice relaxation time (T1) of water proton from one tumor to the other is due to the yield of hypoxic fraction in the tumor. The yield of higher relaxation time (T1) in the KHT tumor compared to the RIF-1 tumor as a function of growth is due to the hypoxic fraction of the tumor. The hypoxic fraction in the tumor is also responsible for the P-31 spectral parameter changes.


Development profile of metabotropic glutamate receptor mRNA in rat brain. We have studied the expression of metabotropic glutamate receptor (mGluR) mRNA by Northern blot analysis with a specific cDNA probe (the pmGR1 probe). In 1-day-old rats, the steady state levels of mRNA were higher in the hypothalamus and olfactory bulb, with intermediate levels in the cerebellum and low levels in the hippocampus and cerebral cortex. In the olfactory bulb, hypothalamus, and cerebral cortex, the expression of mGluR mRNA remained constant at 8 and 30 days of postnatal life. In contrast, in the cerebellum and hippocampus, mRNA levels increased progressively with age. There was no correlation between levels of mGluR mRNA and stimulation of polyphosphoinositide hydrolysis by 1-aminocyclopentane-1S,3R-dicarboxylic acid (trans-ACPD), which was much greater in brain slices from 8-day-old rats and was nearly absent in the adult cerebellum and olfactory bulb, where we have found the highest levels of mRNA. In addition, mGluR mRNA was detectable in cultured cerebellar granule cells but not in cultured neurons from cerebral hemispheres or in cultured astrocytes, which responded to trans-ACPD with an increased formation of [3H]inositol monophosphate. The discrepancies between levels of mGluR mRNA detected with the pmGR1 probe and trans-ACPD-stimulated polyphosphoinositide hydrolysis suggest either that different subtypes of mGluRs exist or that mRNA levels are not critical for the dynamic changes in the activity of mGluRs during development.


gamma-Aminobutyric acid A or C receptor? gamma-Aminobutyric acid rho 1 receptor RNA induces bicuculline-, barbiturate-, and benzodiazepine-insensitive gamma-aminobutyric acid responses in Xenopus oocytes. Xenopus oocyte expression of the recently cloned gamma-aminobutyric acid (GABA) rho 1 receptor subunit cDNA yields a pharmacologic profile characteristic of the GABAc responses described by Johnston [Benzodiazepine/GABA Receptors and Chloride Channels. Receptor Biochemistry and Methodology (R. W. Olsen and J. C. Venter, eds), Vol. 5. Alan R. Liss, New York, 57-71 (1986)] and the responses to retinal mRNA recently reported in the Xenopus expression system [Proc. Natl. Acad. Sci. USA 88:4318-4322 (1991)]. A rationale for defining GABA rho 1 as forming a GABAc receptor is discussed.


Purinergic receptor regulation of signal transduction in NCB-20 cells. In the present paper, P1 and P2 purinergic receptors and their control of signal transduction pathways were investigated in NCB-20 cells. ATP elicited an increase in [Ca2+]i. The purinergic receptor subtype involved was identified by comparing the actions of a range of nucleotides. UTP was the most potent agonist in elevating [Ca2+]i, with an EC50 value of 6.2 +/- 0.5 microM. UTP, ATP (EC50, 17.3 +/- 1.5 microM), adenosine-5'-O-(3-thio)triphosphate (23 +/- 3 microM), and ITP (55 +/- 4 microM) exerted similar maximal effects. Other nucleotides tested, including beta, gamma-methylene-ATP and 2-methylthio-ATP, which are considered prototypic agonists for P2x and P2y receptors, respectively, were ineffective; in general, modifications in the ribose-triphosphate chain and substitution on the 2-position of the purines reduced the efficacy of nucleotides. This pharmacological characterization indicated that a putative P2u receptor mediates the [Ca2+]i elevation elicited by nucleotides in NCB-20 cells. The increase in [Ca2+]i originates from intracellular Ca2+ stores; blockade of Ca2+ entry does not affect the rise in [Ca2+]i. In contrast, pretreatment with the Ca(2+)-ATPase inhibitor thapsigargin or with bradykinin, a hormone that releases Ca2+ from inositol trisphosphate-sensitive stores, does preclude the increase in [Ca2+]i induced by ATP. ATP and UTP also transiently inhibit cAMP accumulation in the intact cell, presumably via a Ca(2+)-mediated mechanism. The finding of a P2u receptor in NCB-20 cells adds to a growing perception that P2 receptors are widely distributed. Besides the P2u receptor, NCB-20 cells express adenosine A2 receptors, coupled to stimulation of cAMP accumulation. The presence of both P1 and P2 purinergic receptors permits a sequential modulation of distinct second messenger levels associated with a common stimulus, ATP.


Sodium nitroprusside inhibits N-methyl-D-aspartate-evoked calcium influx via a nitric oxide- and cGMP-independent mechanism. In primary cultures of rat cerebellar granule cells, sodium nitroprusside (SNP), a vasodilator that generates nitric oxide (NO), potently inhibited N-methyl-D-aspartate (NMDA)-evoked 45Ca2+ influx (IC50 = 6.6 microM). This inhibition was time dependent and was complete when SNP was applied 10 min before NMDA stimulation. The effect of SNP was transient and the ability of NMDA to stimulate 45Ca2+ influx was restored after SNP withdrawal. The effect of SNP was selective for the NMDA-sensitive glutamate receptor, because SNP failed to antagonize kainate-stimulated 45Ca2+ influx. The action of SNP was independent of the ability of this agent to generate NO; S-nitroso-N-acetylpenicillamine, an NO-containing compound that was 100 times more potent than SNP in stimulating cGMP accumulation, failed to inhibit NMDA-evoked 45Ca2+ influx. In contrast, K4Fe(CN)6, a compound structurally similar to SNP but devoid of NO, inhibited both 45Ca2+ influx (IC50 = 27 microM) and cGMP accumulation evoked by NMDA; K3Fe(CN)6 was inactive. Thus, in cerebellar granule cells, SNP and K4Fe(CN)6 interfere with the function of NMDA receptors, possibly at the level of the receptor recognition site. The resulting blockade of Ca2+ influx through NMDA receptor channels accounts for the reported ability of these compounds to protect granule cells from NMDA-induced neurotoxicity. This protection is not mediated by an NO-dependent mechanism but depends on the action of the ferrocyanide portion of the SNP molecule.


Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product. The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.


Protein tyrosine kinase activities of the epidermal growth factor receptor and ErbB proteins: correlation of oncogenic activation with altered kinetics. We have compared the protein tyrosine kinase activities of the chicken epidermal growth factor receptor (chEGFR) and three ErbB proteins to learn whether cancer-activating mutations affect the kinetics of kinase activity. In immune complex assays performed in the presence of 15 mM Mn2+, ErbB proteins and the chEGFR exhibited highly reproducible tyrosine kinase activity. Under these conditions, the ErbB and chEGFR proteins had similar apparent Km [Km(app)] values for ATP. The ErbB proteins appeared to be activated, as they had at least 3-fold-higher relative Vmax(app) for autophosphorylation and approximately 2-fold higher relative Vmax(app) for the phosphorylation of the exogenous substrate TK6 (a bacterially expressed fusion protein containing the C-terminal domain of the human EGFR). The ErbB kinases had both higher Km(app) and higher Vmax(app) for the phosphorylation of the exogenous substrate TK6 than did the chEGFR. The ratios of the Vmax(app) to the Km(app) for TK6 phosphorylation suggested that the ErbB proteins had lower catalytic efficiencies for the exogenous substrate than did the chEGFR. The three tested ErbB proteins had cytoplasmic domain mutations that conferred distinctive disease potentials. These mutations did not affect the kinetics for the phosphorylation of the exogenous substrate TK6. Two of the ErbB proteins contained all of the sites used for autophosphorylation. In these, a mutation that broadened oncogenic potential to endothelial cells caused an additional increase in Vmax(app) for autophosphorylation. Thus, mutations that change the EGFR into an ErbB oncogene cause multiple changes in the kinetics of protein tyrosine kinase activity.


Mechanism of action of a repressor of dioxin-dependent induction of Cyp1a1 gene transcription. A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions.


Basic fibroblast growth factor enhances nerve growth factor receptor gene promoter activity in human neuroblastoma cell line CHP100. The human neuroblastoma cell line CHP100 provides a useful model system in which to study the molecular mechanisms of transcriptional regulation of the low-affinity nerve growth factor receptor (NGFR) gene during neuronal development. Basic fibroblast growth factor (bFGF) induced morphological changes in CHP100 cells, including flattening of cell bodies and neurite outgrowth. bFGF also increased p75NGFR immunoreactivity, as assessed by immunocytochemistry, and increased p75NGFR mRNA levels, as assessed by Northern (RNA) blot analysis. A chimeric gene consisting of 6.7 kb of the 5'-flanking region of the human NGFR gene linked to the chloramphenicol acetyltransferase gene was constructed. In stable transformants of CHP100 cells, 10 ng of bFGF per ml induced an eightfold increase in chloramphenicol acetyltransferase activity. These results indicate that upstream elements of the NGFR gene mediate transcriptional regulation by bFGF.


Growth factor-induced activation of a kinase activity which causes regulatory phosphorylation of p42/microtubule-associated protein kinase. p42/microtubule-associated protein kinase (p42mapk) is activated by tyrosine and threonine phosphorylation, and its regulatory phosphorylation is likely to be important in signalling pathways involved in growth control, secretion, and differentiation. Here we show that treatment of quiescent 3T3 cells with diverse agonists results in the appearance of an activity capable of causing the in vitro phosphorylation of p42mapk on the regulatory tyrosine and to a lesser extent on the regulatory threonine, resulting in enzymatic activation of the p42mapk. This p42mapk-activating activity is capable of phosphorylating a kinase-defective p42mapk mutant, thus confirming its activity as a kinase.


Transcription termination by RNA polymerase III: uncoupling of polymerase release from termination signal recognition. Xenopus RNA polymerase III specifically initiates transcription on poly(dC)-tailed DNA templates in the absence of other class III transcription factors normally required for transcription initiation. In experimental analyses of transcription termination using DNA fragments with a 5S rRNA gene positioned downstream of the tailed end, only 40% of the transcribing polymerase molecules terminate at the normally efficient Xenopus borealis somatic-type 5S rRNA terminators; the remaining 60% read through these signals and give rise to runoff transcripts. We find that the nascent RNA strand is inefficiently displaced from the DNA template during transcription elongation. Interestingly, only polymerases synthesizing a displaced RNA terminate at the 5S rRNA gene terminators; when the nascent RNA is not displaced from the template, read-through transcripts are synthesized. RNAs with 3' ends at the 5S rRNA gene terminators are judged to result from authentic termination events on the basis of multiple criteria, including kinetic properties, the precise 3' ends generated, release of transcripts from the template, and recycling of the polymerase. Even though only 40% of the polymerase molecules ultimately terminate at either of the tandem 5S rRNA gene terminators, virtually all polymerases pause there, demonstrating that termination signal recognition can be experimentally uncoupled from polymerase release. Thus, termination is dependent on RNA strand displacement during transcription elongation, whereas termination signal recognition is not. We interpret our results in terms of a two-step model for transcription termination in which polymerase release is dependent on the fate of the nascent RNA strand during transcription elongation.


Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements. Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.


Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)


Thyroid hormone receptor transcriptional activity is potentially autoregulated by truncated forms of the receptor. ErbA/thyroid hormone receptor is a nuclear receptor that can affect transcription from promoters containing a thyroid hormone response element (TRE) in a thyroid hormone (T3)-dependent manner. We reported earlier that the thyroid hormone receptor is expressed in embryonic avian erythroid cells as a nested set of four proteins with a common C terminus. The full-length receptor is capable of both high-affinity binding to thyroid hormone and specific binding to DNA. We now report that the two smallest ErbA forms, which contain the hormone-binding domain but lack the N-terminal DNA-binding domain, have the same affinity for T3 as does full-length ErbA but are incapable of specific DNA binding. In transactivation assays, these N-terminally truncated proteins are able to specifically suppress both transcriptional repression and hormone-dependent transcriptional activation by the full-length ErbA. We also find that retinoic acid-dependent transactivation by retinoic acid receptors is inhibited by the truncated ErbA proteins. Furthermore, the smaller ErbA forms inhibit binding to TREs by full-length ErbA in vitro. Results from experiments involving site-specific mutagenesis of a conserved region within the hormone-binding domain of the smaller ErbA proteins indicate that the suppressive effect of the smaller receptor forms is independent of hormone binding and that this region is important in mediating protein-hormone as well as protein-protein interactions. We have also found that full-length ErbA homodimers can be detected only in the presence of a specific DNA-binding site. However, no association between full-length and the N-terminally truncated non-DNA-binding ErbA proteins could be detected, indicating that the complex either is unstable or does not form. Our results suggest that inhibition of receptor function occurs through transient formation of heterodimers which lack DNA-binding activity or by competition for factors which positively affect DNA binding by the full-length protein. This finding raises the possibility that thyroid hormone receptor transcriptional activity is autoregulated by means of alternative receptor translation products acting in a dominant negative manner.


The Drosophila ACE3 chorion element autonomously induces amplification. The Drosophila chorion genes amplify in the follicle cells by repeated rounds of reinitiation of DNA replication. ACE3 (amplification control element from the third chromosome) has been identified by a series of deletion experiments as an important control element for amplification of the third-chromosome chorion cluster. Several elements that quantitatively enhance amplification also have been defined. We show that a single 440-bp ACE3 sequence is sufficient to regulate amplification with proper developmental specificity autonomously from other chorion DNA sequences and regulatory elements. Although ACE3 is sufficient for amplification, the levels of amplification are low even when ACE3 is present in multiple copies. When controlled solely by ACE3, amplification initiates either at ACE3 or within closely linked sequences. Amplification of an ACE3 transposon insertion produces a gradient of amplified DNA that extends into flanking sequences approximately the same distance as does the amplification gradient at the endogenous chorion locus. The profile and extent of the amplified gradient imply that the low levels of amplification observed are the result of limited rounds of initiation of DNA replication. Transposon inserts containing multiple copies of ACE3 in a tandem, head-to-tail array are maintained stably in the chromosome. However, mobilization of the P-element transposons containing ACE3 multimers results in deletions within the array at a high frequency.


Cyclic adenosine 3',5'-monophosphate up-regulates 1,25-dihydroxyvitamin D3 receptor gene expression and enhances hormone action. We have previously shown that the abundance of vitamin D receptors (VDR) in cultured cells is increased by mitogens such as serum and growth factors, whereas activation of protein kinase-C (PK-C) causes inhibition of VDR gene expression. This study examines the effect of the cAMP-activated protein kinase-A (PK-A) second messenger system on VDR abundance and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] action. Elevation of intracellular cAMP levels in NIH-3T3 mouse fibroblasts by forskolin or (Bu)2cAMP caused a substantial (8- to 12-fold) increase in VDR abundance, as measured by ligand binding and Western blot analysis. The time course of the forskolin effect on VDR expression was complex. An early rise in VDR abundance occurred at 4 h, followed by a decrease and then a broad secondary rise at 18 h. At the mRNA level, forskolin caused a rapid rise in VDR transcripts after 1 h of exposure, a peak at 2 h, followed by a decline and a subsequent increase at 15 h. Activation of PK-C with the phorbol ester phorbol myristate acetate abolished the forskolin-induced increase in VDR protein and mRNA abundance. NIH-3T3 cells were stably transfected with phOC-CAT, a plasmid carrying a human osteocalcin promoter fragment containing the vitamin D response element fused to the reporter gene chloramphenicol acetyl transferase (CAT). 1,25-(OH)2D3 treatment of transfected cells induced a dose-dependent increase in CAT activity. Up- or down-regulation of VDR in these transfected cells by forskolin or phorbol myristate acetate pretreatment, respectively, resulted in corresponding enhancement or attenuation of 1,25-(OH)2D3-inducible CAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)


Ca2+ mobilization by the LH receptor expressed in Xenopus oocytes independent of 3',5'-cyclic adenosine monophosphate formation: evidence for parallel activation of two signaling pathways. The cDNAs encoding the murine LH receptor (LHR) and the human beta 2-adrenoceptor (h beta 2AR) were cloned and RNAs complementary to their sense strands (cRNAs) were injected into defolliculated Xenopus oocytes. This led to expression, respectively, of LH- and isoproterenol-stimulable adenylyl cyclase activities, indicating that functionally active receptor cDNAs had been cloned. In oocytes injected with LHR cRNA, but not in control or h beta 2AR cRNA-injected oocytes, human CG and LH increased a Ca(2+)-activated Cl- current, as measured by the two-microelectrode voltage-clamp method. This effect was not seen with isoproterenol in control or h beta 2AR cRNA-injected oocytes, it was also not observed in response to forskolin or (Bu)2cAMP. The response to human CG could be obtained in the absence of extracellular Ca2+ but was abolished by injection of EGTA, indicating that it was caused by mobilization of Ca2+ from intracellular stores. The response was unaffected by overnight treatment with 1 microgram/ml pertussis toxin. The experiments show that a glycoprotein hormone receptor can be expressed as a functionally active molecule in Xenopus oocytes, and that the LHR has the ability of activating two separate intracellular signaling pathways: one forming the second messenger cAMP, and the other mobilizing Ca2+ from intracellular stores. It is proposed that the latter is secondary to a primary activation of phospholipase C by the LHR, which elevates intracellular Ca2+ via intermediary elevation of inositol phosphates, presumably (1,4,5)inositol trisphosphate.


DuP 753, a nonpeptide angiotensin II-1 receptor antagonist, alters dopaminergic function in rat striatum. The purpose of this study was to determine if the nonpeptide angiotensin II-1 receptor antagonist DuP 753 after, acute or chronic administration in vivo or after in vitro exposure, altered indices of dopaminergic function in rat striatum. In vivo studies examined the effect of acute and chronic 21-day administration of DuP 753 (10 mg/kg, s.c.) on levels of dopamine (DA) and its metabolite, dihydroxyphenylacetic acid (DOPAC). To determine if chronic treatment with DuP 753 was able to inhibit the pressor response to angiotensin II, a single i.v. dose of angiotensin II (0.1 microgram/kg) was administered 18 hours after the last dose of DuP 753. Acute DuP 753 resulted in significantly decreased (14%) levels of DA. Chronic DuP 753 resulted in increased (1.64 fold) levels of DOPAC, although DA levels were not altered. The single i.v. administration of angiotensin II resulted in increased (88%) DOPAC levels regardless of chronic DuP 753. The in vitro effect of DuP 753 (0.1 nM-1.0 microM) on basal and field stimulation-evoked release of DA and DOPAC was determined in superfused striatal slices from drug naive rats. DA was not detected in these experiments. DuP 753 did not alter basal outflow of DOPAC. At low concentrations (1.0-10 nM), DuP 753 decreased (53%) stimulation-evoked DOPAC overflow; however, at concentrations greater than 10 nM, the inhibitory effect was diminished. Nomifensine (10 microM; a DA uptake inhibitor) was included in the superfusion buffer in order to measure the effect of DuP 753 on the concentration of DA in superfusate. DuP 753 had no effect on basal DA and DOPAC outflow.(ABSTRACT TRUNCATED AT 250 WORDS)


Pharmacological comparison of the sigma recognition site labelled by [3H]haloperidol in human and rat cerebellum. The radioligand binding characteristics of [3H]haloperidol (in the presence of spiperone, 25 nmolL-1) were investigated in rat and human cerebellar membranes. In both rat and human cerebellar membrane preparations saturation studies with [3H]haloperidol (non-specific binding defined by pentazocine, 10 mumolsL-1) demonstrated high affinity saturable specific binding to a homogenous population of binding sites (rat, Bmax 6693 +/- 1242 fmol mg-1 protein, pKD 8.33 +/- 0.08; human, Bmax 2550 +/- 437 fmol mg-1 protein, pKD 8.59 +/- 0.11; mean +/- SEM, n = 3-6). Competition studies employing a wide range of structurally diverse competing compounds displayed that the [3H]haloperidol binding site was pharmacologically similar in both preparations and comparable to sigma recognition sites previously identified in various tissues originating from different species. In addition, with reference to the potential subtypes of sigma recognition sites, the labelling of these sites by low nanomolar concentrations of [3H]haloperidol provides evidence that they belong to the sigma-1 recognition site subtype. The present findings suggest that the pharmacology of the rat and human cerebellar sigma recognition site are directly comparable and provides further supporting evidence towards the use of [3H]haloperidol radioligand binding studies in the rat to detect sigma receptor ligands with potential therapeutic activity.


Involvement of mu-opioid receptors in the antitussive effects of pentazocine. The effect of pentazocine on the capsaicin-induced cough reflex in rats was investigated. Intraperitoneal injection of pentazocine, in doses from 1 to 10 mg/kg, significantly decreased the number of coughs in a dose-dependent manner. The antitussive effect of pentazocine (10 mg/kg, i.p.) was significantly reduced by prior injection of naloxone (0.3 mg/kg, i.p.), but it was unaffected by Mr-2266 BS (5 mg/kg, i.p.), an antagonist of kappa-opioid receptors. The antinociceptive potency of pentazocine (30 mg/kg, i.p.), as determined by the formalin test, was significantly reduced by pretreatment with Mr-2266 BS (5 mg/kg, i.p.), whereas naloxone (0.3 mg/kg, i.p.) had no significant effect on the antinociceptive effect of pentazocine. The antitussive effects of pentazocine (3 mg/kg) and morphine (0.1 mg/kg) were significantly enhanced in rats treated chronically with naloxone (5 mg/kg/day, 5 days), whereas the antitussive effect of U-50,488 H (1 mg/kg, i.p.), a selective kappa-opioid agonist, was not enhanced in these rats. By contrast, the antinociceptive effect of morphine (0.01 mg/kg, i.p.) was significantly enhanced in rats that had been pretreated chronically with naloxone. However, the antinociceptive effects induced by pentazocine (3 mg/kg, i.p.) and U-50,488 H (1 mg/kg, i.p.) were unchanged. These results suggest that pentazocine-induced antitussive effects in rats are mediated via stimulation of mu-opioid receptors.


Amiodarone and desethylamiodarone increase intrasynaptosomal free calcium through receptor mediated channel. Long term amiodarone (AM) therapy has been associated with several side effects including neurotoxicity. Since AM alters Ca2+ regulated events, we have studied its effects on the compartmentation of free Ca2+ in the synaptosomes as an attempt to understand the mechanism of AM and its metabolite, desethylamiodarone (DEA)-induced neurotoxicity. Intact brain synaptosomes were prepared from male Sprague-Dawley rats. Both AM and DEA produced a concentration dependent increase in intrasynaptosomal free Ca2+ concentration ([Ca2]i) to micromolar levels. The increase in [Ca2]i was not transient and a steady rise was observed with time. Omission of Ca2+ from the external medium prevented the AM- and DEA-induced rise in [Ca2+]i suggesting that AM and DEA increased the intracellular [Ca2+]i due to increased influx of Ca2+ from external medium. AM- and DEA-induced increase in intrasynaptosomal [Ca2+]i was neither inhibited by a calcium channel blocker, verapamil, nor with a Na+ channel blocker, tetrodotoxin. However, the blockade of [Ca2+]i rise by AM and DEA was observed with MK-801, a receptor antagonist indicating that AM and DEA induced rise in [Ca2+]i is through receptor mediated channel. Both AM and DEA also inhibited N-methyl-D-aspartic acid (NMDA)-receptor binding in synaptic membranes in a concentration dependent manner, DEA being more effective, indicating that AM and DEA compete for the same site as that of NMDA and confirm the observation that these drugs increase intrasynaptosomal [Ca2+]i through receptor mediated channel. 45Ca accumulation into brain microsomes and mitochondria was significantly inhibited by AM and DEA, but without any effect on the Ca2+ release from these intracellular organelles.(ABSTRACT TRUNCATED AT 250 WORDS)


Ethanol attenuates the response of locus coeruleus neurons to excitatory amino acid agonists in vivo. Recent in vitro electrophysiological studies have revealed that ethanol specifically reduces N-methyl-D-aspartate (NMDA)-activated ion currents, e.g. in hippocampal slices. In the present study, utilizing extracellular recording techniques, the effect of ethanol on excitatory amino acid induced excitability of rat locus coeruleus noradrenergic neurons was investigated in vivo. Administration of ethanol (2 g/kg, i.p.) significantly inhibited the excitation of the locus coeruleus neurons as produced by microiontophoretic application of glutamate, NMDA or quisqualate, but not that of kainate. In contrast, the locus coeruleus response to similarly applied acetylcholine was unaffected by the ethanol treatment. Although ethanol did not alter the basal firing rate of locus coeruleus neurons, the drug significantly changed the firing pattern of the neurons into a more regularized rhythm. It is proposed that the presently observed attenuation of glutamate excitation of locus coeruleus by ethanol may constitute part of the underlaying mechanism for ethanol intoxication in man.


[Status of amine hypotheses in depressive disorders]. The biochemical effects of drugs modulating mood are the basis for still relevant hypotheses relating depressive disorders to dysfunctions of noradrenergic, serotonergic and/or cholinergic neurotransmission. The time course as well as the dose-effect-relationships point to the relevance for mood elevation of secondary adaptive processes rather than the direct actions of antidepressants. Direct evidence of dysfunctions sufficiently explaining the actual illness of the single individual has not been obtained at the level of transmitters, metabolites and receptors. Deficits of serotonergic transmission seem to be related to a personality dimension of poor impulse control closer than to nosological categories. This dimensional concept might also be valid for the relationships between cholinergic sensitivity and stress tolerance. Depression itself is possibly due to imbalances of multiple neuronal systems.


[Prevention of thromboembolism complications with low molecular weight heparin in microneurosurgical lumbar intervertebral disk operations]. In a prospective randomized study the effect of low-molecular-weight heparin (one injection of 1500 a PPT-Units/d Mono-Embolex NM) was compared with the effect of unfractionated heparin (5000 IU t.i.d.) in 200 patients undergoing micro-neurosurgical lumbar disc operations. Criteria of evaluation were lethal pulmonary embolisms, clinical signs of pulmonary embolisms, confirmed by radioisotopic lung scans, and major bleeding complications. The present investigation demonstrates, that a single daily subcutaneous injection of Mono-Embolex NM is an effective and well tolerated measure against thrombo-embolic complications, at least equal to the established low dose heparin prophylaxis with 5000 IU t.i.d.


Acute renal failure after the use of angiotensin-converting-enzyme inhibitors in patients without renal artery stenosis. During a 4-year period, acute renal failure was observed in 27 patients (mean age 65 years) treated by various angiotensin-converting-enzyme (ACE) inhibitors for hypertension, heart failure, or a combination of both. None had significant renal artery stenosis on angiography. Overt volume depletion was present in 21 and hypotension in 12 cases. All patients received diuretic therapy and/or a low-salt diet. Other facilitating factors included cardiac failure, pre-existing chronic renal insufficiency, combined therapy with non-steroidal anti-inflammatory drugs, and diabetes mellitus. Twenty-two patients had two or more of these factors at presentation. A renal biopsy performed in 10 cases showed severe arteriosclerosis of small renal arteries in eight and acute tubular necrosis in five instances. Therapy comprised volume expansion, and withdrawal of diuretics and, except in two patients, of ACE inhibitors. Twenty-one patients recovered normal renal function, two died, and permanent renal damage remained in four. These results suggest that sodium depletion has a critical role in inducing acute renal failure, whose outcome is not always benign. A combination of diuretics and ACE inhibitors should be prescribed with caution, especially in older patients with small as well as with large renal vessel disease.


Influence of pregnancy on progression of diabetic nephropathy and subsequent requirement of renal replacement therapy in female type I diabetic patients with impaired renal function. The influence of pregnancy on the progression of diabetic nephropathy in diabetic women with pre-existing moderate renal insufficiency is a subject of considerable controversy in the literature. In four of five female patients with type I diabetes mellitus with pre-existing impaired renal function (creatinine clearance less than 80 ml/min), significant proteinuria (greater than 2 g/24 h urine) and hypertension we have found a further decline in renal function during pregnancy, with an increased deterioration rate of creatinine clearance in comparison to the time before and after pregnancy. The mean decline of the glomerular filtration rate was 1.8 ml/min per month during pregnancy and 1.4 ml/min per month postpartum until the start of dialysis treatment. The difference in the progression of diabetic nephropathy during and after pregnancy can be explained by increased hypertension during pregnancy, especially in the third trimester, despite an intensified antihypertensive therapy. The long-term effect of pregnancy on renal function in our patients was therefore an earlier requirement for renal replacement therapy than would have been expected without pregnancy.


Continuous measurements of oxygen saturation during haemodialysis. A new technique for recording and analysing continuous measurements of oxygen saturation (SpO2) by pulse oximeter during haemodialysis was used to compare changes in SpO2 in eight patients during two 4 h periods of dialysis using a cuprophane membrane, once using an acetate dialysate, and once using bicarbonate. The computer-derived patterns of SpO2 show whether hypoxaemia was caused mainly by extrapulmonary abnormalities (ventilatory control) or intrapulmonary abnormalities (V/Q distribution). The patterns of oxygen saturation were analysed for (i) stability, (ii) the lower median 20th centile of SpO2, and (iii) time below a SpO2 of 90%. Not all patients had reduced oxygenation during acetate dialysis. Three of eight patients had a stable pattern with acetate dialysis and six of eight were stable with bicarbonate. Five of eight patients had a lower SpO2 with acetate but one patient had a lower SpO2 with bicarbonate. Four patients had prolonged, clinically significant periods of oxygen desaturation with SpO2 less than 90%; two of these had particularly prolonged periods during acetate (62 min and 12 min), but one patient showed a longer period during bicarbonate than acetate dialysis (7 min). In two patients the SpO2 declined to less than 84%. The patterns of SpO2 suggested that the decrease in oxygen saturation was due more to extrapulmonary abnormalities causing an instability in ventilatory control rather than to venous admixture. It is recommended that pulse oximetry is used to identify patients at risk of hypoxaemia, to monitor these patients during haemodialysis, and to administer oxygen to those whose SpO2 falls below 90%, particularly if they have anaemia or cardiovascular disease.


Essential branched-chain amino acids and alpha-ketoanalogues in haemodialysis patients. Homeostasis of essential amino acids and their transamination derivatives (ketoacids) is disturbed in haemodialysis (HD) patients. In long-term HD patients a hypercatabolic state is often paired with severe anaemia. To understand metabolic regulation mechanisms we measured with an improved fluorescence-HPLC method plasma concentrations of valine (Val), isoleucine (Ile), and leucine (Leu) and their corresponding ketoacids ketoisovaleric acid (KIV), ketomethylvaleric acid (KMV), and ketoisocaproic acid (KIC). The values of 18 modestly anaemic HD patients (group A: Hb greater than 11 g/dl) and of 16 severely anaemic HD patients (group B: Hb less than 8 g/dl) were compared with 19 healthy control persons (100%; significance of patient values vs controls P less than 0.05) and with each other (significantly different at P less than 0.05). Both branched chain amino acids and ketoacids are diminished in HD patients. This disturbance of protein metabolism is intensified with severe anaemia. The decrease of transamination products KIV and KMV parallels that of their corresponding Val and Ile, whereas KIC is reduced out of proportion to Leu. Leu has anabolic function and KIC antiproteolytic effects. Decreased Leu and KIC indicate catabolism. Reduced transamination of Leu and KIC suggests an endogenous protective mechanism against catabolism independent of anaemia. These differences should be considered with supplementation therapy of branched-chain compounds in HD patients.


Elevation of serum erythropoietin after subtotal parathyroidectomy in chronic haemodialysis patients. After successful subtotal parathyroidectomy (PTX) in 10 chronic haemodialysis patients, significant elevation of Epo was observed, from 48.4 +/- 17.8 mU/ml(M +/- SEM) at preoperative state to 103.3 +/- 34.7 mU/ml at 6 h and 163.4 +/- 50.2 mU/ml at 12 h after PTX. Significant reductions in both PTH-m and ionized calcium (iCa) were confirmed. Since Epo did not increase in the cases with an inadequate PTX and ovariectomy, an abrupt reduction in PTH with a decrease in iCa may play some role in the elevation of Epo.


Suppression of the renin-angiotensin-aldosterone axis with erythropoietin therapy by a negative feedback loop. To investigate the pathophysiology of hypertension in patients receiving recombinant human erythropoietin (rHuEpo) we studied its effects on the renin-aldosterone axis of chronic haemodialysis (HD) patients not receiving antihypertensive drugs. Nine severely anaemic normotensive HD patients received rHuEpo 50 U/kg bodyweight, thrice weekly after each HD. The dose was increased by 25 U/kg bodyweight every 4 weeks to a maximum of 100 U/kg or until an increase of Hb or Hct of 2 g/dl or 7% was achieved. Blood samples were taken after 30 min supine rest and while seated 10 min later after gentle ambulation. Results expressed as mean +/- SEM: therapy in normotensive HD patients by a negative feedback loop, before the development of hypertension.


Crossover comparison of intravenous and subcutaneous erythropoietin in haemodialysis patients. To examine the suggestion that s.c. administration of recombinant human erythropoietin (rHuEpo) may be more effective than i.v. administration, we changed the route of administration in 11 patients, previously established on a stable dose of rHuEpo given twice or thrice weekly, from i.v. to s.c. administration without altering the dose. All patients were iron replete (serum ferritin greater than 100 micrograms/l). In one patient the haemoglobin concentration declined at the time of conversion due to poor compliance, and another patient died shortly after conversion. In the remainder there was a significant increase in haemoglobin concentration from 9.30 (SD 0.78) at the time of conversion to 9.84 (0.59) at 1 month, 10.35 (1.22) at 2 months, and 10.39 (1.42) at 3 months. The increase in haemoglobin concentration was greater than 1 g/dl at 3 months in only five of the patients. Serum ferritin prior to conversion was similar in 'responders' and 'non-responders', but all responders had a transferrin saturation of greater than 16%, whereas three of four non-responders had transferrin saturation of less than or equal to 16%. Subcutaneous administration of rHuEpo is more effective, dose for dose, than i.v. administration, but poor iron mobilization may limit the response.


Differing prognostic significance of reinfection and relapse in CAPD peritonitis. All episodes of recurrent infection in a CAPD unit over a 26-month period have been analysed to discover whether relapse and reinfection have different prognostic importance. Relapse and reinfection were distinguished by detailed microbiological investigation. Prognosis was expressed in terms of outcome of treatment and the fate of the Tenckhoff catheter. Twenty-nine patients suffered recurrent infections (i.e. more than one infection during a 12-month period). Nine (6 male, 3 female, age range 42-73 years) had relapses, and 20 (16 male, 4 female, age range 42-74 years) reinfections. The characteristics of the two groups of patients were indistinguishable. Relapse was of graver prognostic consequence: patients who relapsed were significantly less likely to respond to antibiotic treatment (78% versus 20%) and have to have their catheters removed (78% versus 10%) than those with reinfections. Thus it is important to differentiate relapse from reinfection in CAPD peritonitis. In addition to being helpful for the management of individual patients, this is essential if results of therapeutic trials are to be interpreted correctly.


Post-transplant erythrocytosis: role of erythropoietin and male sex hormones. Seventeen patients with post-renal transplant erythrocytosis and 17 non-erythrocytotic controls, matched in age, sex, serum creatinine and source of donor kidney, were studied to determine the role of erythropoietin, male sex hormones (testosterone, FSH, LH), and various patient risk factors in post-transplant erythrocytosis. Serum erythropoietin was significantly greater in erythrocytotic patients (35.6 +/- 5.7 mU/ml) than non-erythrocytotic patients (18.8 +/- 2.6 mU/ml) (P less than 0.05) and normal subjects (22.5 +/- 0.95 mU/ml) P less than 0.05). Serum testosterone was similar between the male study (13.2 +/- 6.2 nmol/l) and control (13.1 +/- 6.0 nmol/l) patients. This might be due to the greater basal LH in the male control subjects (13.9 +/- 11.7 IU/l versus 8.0 +/- 3.3 IU/l in erythrocytotic males, P = 0.084). Basal FSH in the male controls was greater than that in the study group (13.7 +/- 14 IU/l versus 6.8 +/- 2.9 IU/l, P = 0.067). Among the demographic risk factors, only the smoking history was important. There were more smokers among the erythrocytotic patients than controls (P = 0.051).


Low-dose subcutaneous erythropoietin corrects the anaemia of renal transplant failure. Although erythropoietin (Epo) is known to correct anaemia in dialysis and pre-dialysis patients, there is limited experience with its use in immunosuppressed patients suffering from chronic renal graft dysfunction. We report the results of a pilot study of Epo in seven patients with failing grafts and normocytic normochromic anaemia attributable to renal failure. All entering patients had controlled blood pressure and serum ferritin greater than 100 micrograms/l. Three patients were taking triple immunotherapy (prednisone/azathioprine/cyclosporin), two patients prednisone/azathioprine, and two patients CsA monotherapy. Study duration mean was 15 +/- 2 (SEM) weeks, and Epo was started at 4000 units subcutaneously (s.c.) once weekly, adjusted to achieve a target haemoglobin (Hb) of 100 g/l. Mean Hb at initiation was 68 +/- 5 g/l and significantly increased to 96 +/- 6 at end of follow-up, P less than 10(-4). All patients responded. Maintenance Epo dosage was 120 +/- 32 U/kg bodyweight/week, roughly 4000 units/week. There was no significant change in serum creatinine: pre-study 392 +/- 45 mumol/l; post-study 430 +/- 62 mumol/l. There were no complications but blood pressure did rise significantly: pre- 124 +/- 11/74 +/- 4 mmHg to post- 142 +/- 10/86 +/- 3, P less than 0.05 for systolic and diastolic. Low-dose s.c. Epo effectively corrects anaemia in graft failure despite azathioprine and/or CsA therapy, without obvious acceleration of graft failure.(ABSTRACT TRUNCATED AT 250 WORDS)


D-penicillamine therapy associated with rapidly progressive glomerulonephritis. A 55-year-old woman with advanced rheumatoid arthritis developed rapidly progressive glomerulonephritis with epithelial crescents and pulmonary hemorrhage following treatment with D-penicillamine. D-penicillamine was then withdrawn and a pulse therapy with methylprednisolone halted the progression of kidney and lung damage. We review the other cases previously reported and discuss pathogenesis and treatment of this rare condition.


Lack of clinical evidence for a specific HIV-associated glomerulopathy in 203 patients with HIV infection. Several authors described a high incidence of proteinuria with frequent progression to nephrotic syndrome and/or renal failure in patients with HIV infection. Though renal histological changes were rather non-specific, the existence of a specific, HIV-associated glomerulopathy was postulated. We repeatedly investigated proteinuria and serum creatinine in 203 HIV-infected patients. One hundred and twenty-two patients (group 1) had early stages of the disease without opportunistic infections, 81 suffered from acute opportunistic infections (group 2). In patients with a positive qualitative test (Combistix), quantitative measurement (Biuret) for proteinuria was carried out; when proteinuria was greater than 0.5 g/24 h, SDS gel electrophoresis was performed. None of the patients of group 1 had a proteinuria greater than 0.5 g/24 h or an elevated serum creatinine. Eleven of 81 patients from group 2 had a proteinuria between 0.5 and 3 g/24 h; one further patient of group 2 developed a transient proteinuria of 7.7 g/24 h. Only three of the proteinuric patients showed a glomerular pattern in SDS gel electrophoresis, all three during acute CMV or EBV infections. Fourteen of 81 group 2 patients showed a transient elevation of serum creatinine (x +/- SD of the maximum serum creatinines: 225.3 +/- 163 mumol/l), most during pentamidine therapy for Pneumocystis carinii infection; one patient treated with high-dose acyclovir had to be temporarily dialysed. In the investigated 203 HIV patients no nephrotic syndrome and no sustained elevation of serum creatinine greater than 200 mumol/l was observed. All cases of proteinuria and elevation of serum creatinine were associated with severe opportunistic infections and the administration of potentially nephrotoxic antibiotics.


Lovastatin in glomerulonephritis patients with hyperlipidaemia and heavy proteinuria. Lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A inhibitor, was given to 14 patients with unremittent nephrotic syndrome (heavy proteinuria with hyperlipidaemia) for 6 months. Treatment was started at an initial dose of 20 mg/day, increasing to a maximum of 80 mg/day. Treatment was well tolerated except in two patients: one developed rhabdomyolysis and one severe hypertriglyceridaemia requiring an additional antihyperlipidaemic agent. Lovastatin was effective in reducing serum cholesterol, LDL-C and apolipoprotein B in the remaining 12 patients. Cholesterol was reduced by 31% from 8.24 +/- 0.49 mmol/l (mean +/- SEM) to 5.7 +/- 0.18 mmol/l after 6 months (P less than 0.001). LDL-C was normalized to 3.26 +/- 0.21 mmol/l from a pretreatment value of 5.76 +/- 0.48 mmol/l (P less than 0.001), a decrease of 43%. Serum apolipoprotein B was also normalized to 1.11 +/- 0.09 g/l from a basal level of 1.51 +/- 0.10 g/l (P less than 0.05). Triglyceride, HDL-C and apolipoprotein A1 concentrations were unchanged. Proteinuria as well as renal albumin clearance were unchanged. GFR by plasma radioisotope Cr-EDTA clearance for the whole group was unaltered by treatment. However, among those with relatively good pretreatment renal function (GFR greater than 70 ml/min per 1.73 m2), GFR increased at the end of 6 months' treatment (118.2 +/- 15 ml/min per 1.73 m2 versus 77.6 +/- 8.4 ml/min per 1.73 m2 in wash-out phase).(ABSTRACT TRUNCATED AT 250 WORDS)


Glomerular basement membrane thinning in adults: clinicopathological correlations of a new diagnostic approach. We have studied glomerular basal laminar thickness in biopsy material, using a simple technique involving 16 selected measurements per case. Twenty-nine biopsied cases of adult glomerular haematuria were examined together with 'diseased' controls represented by a variety of glomerulopathies including minimal-change disease and IgA nephropathy. 'Normal' control populations were provided by 13 patients with acute-onset renal failure of non-glomerular origin and nine patients undergoing nephrectomy. Analysis of groups determined by the presence or absence of haematuria, the degree of proteinuria and presence or absence of a diagnostically characteristic immunofluorescence pattern showed that the nine patients with haematuria and proteinuria of less than 200 mg/24 h represented a distinct subpopulation with a mean membrane thickness of 225 nm compared to the control mean of 343 nm (P less than 0.0001). All members of this subpopulation had mean values below an arbitrary cut-off value of 270 nm. Within other specific disease categories, sporadic cases had mean membrane thicknesses below this critical value, indicative of an overlap of pathologies. On short-term follow-up there is no evidence that the 'pure' thin-membrane population are subject to any deterioration in renal function. It is of further interest that eight of nine thin-membrane 'syndrome' cases were O Rh positive. This finding may provide a starting point for investigation of a specific genetic defect.


HLA-DP region gene polymorphism in primary IgA nephropathy: no association. Many features suggest that a genetically mediated abnormality of the IgA immune response is central in the pathogenesis of IgA nephropathy (IgAN). Candidate disease susceptibility genes include those encoding the MHC class II antigens, HLA-DR, -DQ, and -DP, and we have recently described an HLA-DQB1 association in IgAN. Polymorphisms of the HLA-DP region loci have been shown to associate with autoimmune diseases which share immunological features with IgAN; coeliac disease (CD) and dermatitis herpetiformis (DH). We have therefore examined restriction fragment length polymorphisms (RFLPs) of the DP alpha and DP beta chain genes (DPA1 and DPB1 respectively) in IgAN, and have studied three caucasoid populations (North, Mid, Southern Europe) to determine whether ethnic variation in genetic susceptibility exists. DNA was extracted from blood (IgAN, UK n = 89, Italy n = 75, Finland n = 49; Controls, UK n = 99, Italy n = 54, Finland n = 45), and studied by Southern blot hybridization techniques using the restriction enzymes BgI II and Msp I and cDNA 32P-labelled DPA1 and DPB1 probes respectively. The frequency distribution of the DPA1 and DPB1 fragments was similar between the three caucasoid IgAN patient groups compared to their respective controls. There was no association of DPA1 or DPB1 RFLPs with clinical features. These results suggest that HLA-DP region genes are not important in conferring disease susceptibility to IgAN and do not influence clinical disease expression. Moreover, different immunogenetic mechanisms operate in IgAN, CD, and DH.


Expressing glomerular filtration rate in terms of extracellular fluid volume. Glomerular filtration rate (GFR) is routinely calculated from the second exponential of the bi-exponential plasma clearance of filtration markers such as Cr-51 EDTA and Tc-99m DTPA. By ignoring the first exponential, true GFR is overestimated, an error which increases with increasing GFR. The rate constant, lambda 2, of the second exponential represents the rate at which glomerular filtration 'turns over' the extracellular fluid (ECF) and so closely approximates GFR/ECF volume. Again, the error in the estimation of this ratio increases with increasing GFR, although in this case it underestimates the true ratio. Expressing GFR in terms of ECF volume, rather than in terms of body surface area, offers considerable technical and physiological advantages. The relationship between GFR/ECF volume and lambda 2, over a wide range of renal function, expressed as a second order polynomial, was GFR/ECF volume = -0.093 + 1.06 lambda 2 + 0.009 lambda 2(2) ml.min-1.l-1. The corresponding relationship between 'true' GFR (C1) and approximate GFR (i.e. based only on the second exponential--C2) was C1 = -0.58 + 1.012C2 -0.0011 C2(2) ml.min-1 For any level of renal function, the error in GFR/ECF volume, expressed as lambda 2, is less than the error in GFR expressed as C2. Since GFR may change as a direct result of a change in ECF volume, it is physiologically more relevant, and technically very much easier, to express GFR in terms of ECF volume rather than body surface area.


Screening for renovascular disease with captopril-enhanced renography. Changes in renal function caused by angiotensin-converting-enzyme (ACE) inhibitors can be detected on 99mTc-DTPA renography so that DTPA scanning before and after a single dose of captopril can be used to screen for renovascular disease. We have performed captopril-DTPA scans with renal arteriography on 104 patients, of whom 27 had renal artery stenosis, all due to atheroma. Using a 5% fall in divided function or a delay of greater than 15 min in time to peak activity on one side after captopril, or the finding of greater than 90% divided function on one side before captopril as criteria for a positive scan, a sensitivity of 93% and specificity of 70% was achieved. The negative predictive value of the test in our population was 93%. Bilateral improvement in renographic function after captopril was seen in patients with accelerated phase hypertension. The presence of bilateral renal artery disease did not reduce the sensitivity of the test, but sensitivity was reduced (75%) in patients with renal impairment. Clinical characteristics in our patients most strongly associated with renal artery stenosis were abdominal bruit, recurrent left ventricular failure, and peripheral vascular disease. In view of the well-publicized risks of ACE inhibitor therapy, care should be exercised in the use of these agents in such patients.


Renal haemodynamics and organ damage in young hypertensive patients with different plasma renin activities after ACE inhibition. We describe our observations concerning differences in two groups of young hypertensive patients according to their renin activities after ACE inhibition. Seventeen of these patients (age 26 +/- 7 years), so far untreated, were investigated prospectively for hormone levels (renin, aldosterone, vasopressin), microalbuminuria, renal haemodynamics (inulin and PAH clearance) and signs of organ damage (echocardiography, fundoscopy). Secondary forms of hypertension were excluded by routine methods, including angiography. We differentiated two groups of young hypertensive patients. Group 1 (n = 9) had a false positive captopril test with elevated renin activities after ACE inhibition with captopril (8.4 +/- 5 ng/ml per hour) compared to group 2 (renin activity: 2.2 +/- 1.3 ng/ml per hour) or an increase of greater than 400% of renin activity after ACE inhibition. Baseline renin activities and sodium excretion did not differ between the groups. Group 1 also showed significantly greater GFR, FF, and microalbuminuria, as well as signs of organ damage, with left ventricular hypertrophy and hypertensive changes in fundoscopy. There were no differences between the groups concerning mean arterial blood pressure and duration of hypertension. In conclusion, we were able to demonstrate that patients with highly stimulated renin activities showed signs of visceral organ damage and renal hyperfiltration compared to the normal renin activity group after ACE inhibition. Investigations of the renin-angiotensin-aldosterone system with ACE inhibitors might constitute a helpful indicator of renal changes and organ damages in young hypertensive patients.


Plasma erythropoietin concentrations in renal venous blood of patients with unilateral renovascular hypertension. In 19 patients with unilateral renal artery stenosis and subsequent renovascular hypertension plasma renin activity (PRA), plasma concentrations of atrial natriuretic peptide (ANP), erythropoietin (Epo), H+ and HCO3-, as well as pO2 and pCO2 were assessed in renal venous blood of the 'ischaemic' and normally perfused kidney, both in arterial blood and in the inferior vena cava distally from the orifices of the renal veins. PRA and ANP were significantly elevated in venous blood of the ischaemic kidney as compared with the normally perfused kidney. In contrast to PRA and ANP, plasma concentrations of Epo were similar in blood withdrawn at all vascular sites. pO2 and pCO2, as well as blood H+ and HCO3- concentrations in venous blood of the ischaemic kidney were of the same magnitude as of the normally perfused kidney. From the results presented in this paper it follows that (i) in contrast to plasma renin activity and ANP, unilateral renal 'ischaemia' does not influence plasma concentrations of Epo in renal venous blood, and (ii) chronic haemodynamic alterations do not seem to influence Epo secretion by the kidneys.


Intestinal-type alkaline phosphatase in urine as an indicator of mercury induced effects on the S3 segment of the proximal tubule. Intestinal-type alkaline phosphatase (IAP) is a specific and sensitive marker for alterations of the S3 segment of the human proximal tubule, the preferred part for several nephrotoxins. We studied IAP and other renal parameters in mercury-exposed workers and their controls. IAP excretion is clearly increased in the exposed workers, compared to other parameters, indicating that the determination of this enzyme can be a useful screening test of renal effects in occupational mercury exposure.


Cause of death in acute renal failure.

Cause of death in acute renal failure. The cause of 636 deaths during acute renal failure (ARF) occurring between 1956 and 1989 were analysed. Deaths due to haemorrhage and to non-recovery of renal function have declined but cardiovascular deaths and withdrawal of active treatment have increased. The causes of death varied with the clinical situation in which ARF arose. The most important factor contributing to death was the underlying cause of ARF. 67% deaths due to sepsis resulted from infection present at the time of development of ARF. Deaths due to secondary complications have declined, indicating that the precipitating causes of ARF are the main determinant of overall mortality.


Prothrombotic effect of erythropoietin in dialysis patients. Thrombin-antithrombin III complex concentrations (TAT-III) were measured in 18 anaemic haemodialysis patients treated with erythropoietin (Epo) and in four haemodialysis patients treated with i.v. iron dextran. There was a significant early increase in thrombin-antithrombin III in erythropoietin-treated patients which appeared to be independent of the response to erythropoietin (Epo responders (n = 14), pretreatment TAT-III median (range) 3.10 (2.70-9.10) micrograms/l; maximum TAT-III 19.48 (11.18-60.00) micrograms/l, P less than 0.001, Wilcoxon; Epo non-responders (n = 4), pretreatment TAT-III 3.15 (2.90-4.50) micrograms/l, maximum TAT-III 16.00 (10.31-36.12) micrograms/l, P less than 0.001). This was not seen in iron-dextran-treated patients (Pretreatment TAT-III 2.05 (1.90-9.48) micrograms/l, maximum TAT-III 5.60 (2.10-14.50) micrograms/l). The change was not related to haemoglobin, erythropoietin dose, or method of administration, and was transient in nature, thrombin-antithrombin III returning to pretreatment values after approximately 6 months in all patients (Epo responders 6.0(4.0-9.0) months, TAT-III 2.47 (1.30-9.23) micrograms/l; Epo non-responders 7.0 months, TAT-III 5.04 (2.10-7.00) micrograms/l). Increased thrombin-antithrombin III complex may reflect an effect of erythropoietin on microcirculatory factors, which could be relevant to the occurrence of adverse events during treatment.


Recombinant erythropoietin improves cognitive function in chronic haemodialysis patients. Psychometric performance was studied on two occasions in 18 chronic haemodialysis patients. Nine patients treated with rHuEpo performed a battery of psychometric tests before treatment, haemoglobin [mean (SD)] 5.8 (0.6) g/dl and after partial correction of anaemia, haemoglobin 9.3 (1.28) g/dl. The same battery of psychometric tests was administered on two occasions to nine patients (haemoglobin 7.3 (1.2) g/dl) matched with the treatment group for age, educational status and social class, who did not receive rHuEpo. In the rHuEpo-treated group, IQ, measured by the Wechsler Adult Intelligence Scale-Revised, improved by a mean of 8.7 points (P less than 0.01), while in the control group an improvement by a mean of 2.5 points was not significant. Comparison between the groups of the change in IQ score was significant (P = 0.04). There was no change in the mean scores obtained in either group for the other psychometric tests administered including the Paced Auditory Serial Addition Test, Rey auditory verbal learning, and Borkowski verbal fluency test. These results indicate that anaemia makes a reversible contribution to uraemic cognitive dysfunction.


Growth hormone response to acute growth hormone releasing hormone administration in uraemic patients on haemodialysis. To examine the response of growth hormone (GH) to growth hormone releasing factor (GHRF) in patients on haemodialysis, we performed the acute GHRF test (50 micrograms administered intravenously as a bolus) in 10 uraemic male patients on haemodialysis and eight normal controls. Each patient was tested before and after a haemodialysis session (at 08.30 and 12.30). Controls were tested on the same time schedule. At 08.30, patients had significantly greater basal and peak GH values (2.5 +/- 0.6 and 27.8 +/- 5.5 micrograms/l) than controls (0.68 +/- and 11.5 +/- 4 micrograms/l). After the haemodialysis session, basal and peak values declined significantly (P less than 0.01) in the uraemic group (0.5 +/- 0.03 and 3.1 +/- 1.1 micrograms/l), whereas the controls did not show such a change in the 12.30 test. Basal and intratest glycaemic values were comparable both before and after haemodialysis. After dialysis test results did not change either with the use of glucose-free dialysate or with bicarbonate buffer. Uraemic patients display a greater GH response to GHRF injection than normal subjects, and this response decreases after haemodialysis. The degree of reduction has no relationship with either glycaemia or the dialysate buffer. We suggest that other GH secretion regulating factors are altered by the haemodialysis procedure.


Reduced erythrocyte and leukocyte magnesium is associated with cyclosporin treatment and hypertension in renal transplant patients. Plasma, urine, erythrocyte and leukocyte magnesium were measured in normotensive and hypertensive renal transplant patients who were being treated with either azathioprine or cyclosporin. These were compared with essential hypertensive patients and normal subjects. Erythrocyte and leukocyte magnesium were reduced in renal transplant patients in independent association (ANOVA) with both cyclosporin treatment (P = 0.03, P = 0.016 respectively) and hypertension (P less than 0.001 for both). Therefore, hypertensive transplant patients on cyclosporin had the lowest magnesium content of both erythrocytes (1.22 +/- 0.09 mmol/l cells) and leukocytes (2.68 +/- 0.2 nmol/10(6) cells) compared to normal subjects (1.96 +/- 0.17 and 4.11 +/- 0.58 respectively) whereas normotensive transplant patients on azathioprine had normal values (2.12 +/- 0.16 and 5.13 +/- 1.0 respectively). Plasma magnesium was also reduced with cyclosporin treatment. Urine magnesium was not significantly different between any of the groups. Therefore, magnesium depletion may have a role in hypertension in renal transplant patients. Since cyclosporin treatment is also associated with magnesium depletion, this could explain the increased occurrence of hypertension in these patients.


Long-term clinical results of percutaneous transluminal angioplasty in transplant renal artery stenosis. Twenty-five patients with transplant artery stenosis were identified among 1141 renal graft recipients. Impaired graft function (9 patients), hypertension (4 patients) or both (12 patients) were the indications for arteriography. All were treated by percutaneous angioplasty (PTA). The immediate technical success rate was 88% and actuarial graft survival was 88% and 80% at 2 and 5 years respectively. The long-term success rate on graft function was 67% (median observation time 24 months) and on hypertension 63% (median observation time 23 months). Six patients needed rePTA (8 procedures) and in only one patient was surgical repair performed. No case of graft loss due to PTA was recorded and in only one case did occlusion of a segmental artery lead to impairment of graft function. Minor complications were recorded in four other cases and in no case was surgical intervention necessary. Based on these results we favour PTA as a first-line interventional procedure in transplant renal artery stenosis, and the need for surgical repair has been low.


A retrovirus uses a cationic amino acid transporter as a cell surface receptor. New studies have indicated that the cell membrane receptor for the ecotropic murine leukemia virus is the classic membrane amino acid transporter y+, the principal transporter of cationic L-acids in mammalian cells. This finding has been hailed as a landmark in cell physiology which may reveal new mechanisms of viral pathogenesis. These studies represent the first amino acid transporter to be cloned, as well as the first example of a virus subverting a transmembrane protein as a receptor.


Retinoic acid, bound to its nuclear receptor, enhances the expression of the gene for phosphoenolpyruvate carboxykinase. Over 30 years ago, whole-animal studies conclusively showed that liver glycogen depletion in vitamin A deficiency was caused by depressed gluconeogenesis. The techniques of modern cell and molecular biology have now been utilized to demonstrate the probable molecular pathogenesis of this defect associated with vitamin A deficiency. Retinoic acid, bound to its nuclear receptor, stimulates transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis, by binding to a short element of the promoter region of the PEPCK gene.


Neural and vascular injury in total hip arthroplasty. Complete awareness of the anatomy of the pelvis and proximal femur is required if neurologic and vascular complications are to be avoided following total hip arthroplasty. Avoidance of the anterior quadrants for acetabular screw fixation is critical. Cementing techniques are important, and all acetabular and femoral defects should be bone grafted to avoid inadvertent cement migration. Knowledge of the location of pertinent neural and vascular structures should guide retractor placement. Planned lengthening of an extremity during total hip arthroplasty poses a significant risk to neurologic structures, and SSEP monitoring should be considered. In difficult revision procedures and complex primary total hip arthroplasty, preoperative neural and vascular assessment and SSEP monitoring should be done. With the occurrence of a postoperative nerve palsy, careful review of the procedure should be performed to determine the cause of the injury. In this manner the surgeon can best offer appropriate counseling to the patient as to the likelihood of neurologic recovery.


Selective ability of S-phase cell extracts to dephosphorylate SV40 large T antigen in vitro. Simian virus 40 (SV40) large tumor antigen (T) is an oncoprotein whose biological and biochemical functions appear to be modulated by phosphorylation. Recently, SV40 DNA replication in vitro has been shown to be activated by dephosphorylation involving the activity of a serine/threonine phosphoprotein phosphatase belonging to the type 2A class (PP2A) [Virshup, D.M., Kauffman, M.G. & Kelly, T.J. (1989) EMBO J., 8, 3891-3898]. To address the question of how specificity of PP2A activity towards T is regulated, an in vitro assay to study the process of T dephosphorylation was developed. Unlabeled extracts from cells enriched for various stages of the cell cycle were incubated with 32P-labeled, immunocomplexed T. Extracts from a population of cells enriched for S phase demonstrated a selective ability to dephosphorylate this labeled protein when compared with extracts prepared from G1- and M-phase cells. The time course of release from growth arrest demonstrated that this T-specific phosphatase activity occurred at the onset of host-cell DNA synthesis. In contrast, when using 32P-labeled phosphorylase a as the substrate, phosphatase activity appeared to be present throughout the cell cycle. The data presented here are consistent with the notion that PP2A activity towards T is regulated in a cell cycle-dependent manner.


Phosphorylation of connexin43 in cells containing mutant src oncogenes. Disruption of gap junctional communication (measured by intercellular dye transfer) in cultured fibroblasts by pp60v-src is correlated with phosphorylation of the gap junction protein, connexin43 (cx43), on tyrosine. In this report, we examine the functional relevance of these observations by studying cx43 phosphorylation in cells containing kinase-active, non-myristylated pp60(2A527F) or pp60v-src temperature sensitive (ts) for transformation. Non-transformed cells expressing pp60(2A527F) transferred fluorescent dye at high levels and contained cx43 that was phosphorylated predominately on serine. In contrast, cells transformed by kinase-active, myristylated pp60(527F) did not transfer dye and contained cx43 proteins which were phosphorylated on serine and tyrosine. Additionally, activation of ts pp60v-src tyrosine kinase activity upon shift of cells to the permissive temperature was correlated with a rapid increase in the phosphorylated tyrosine content of cx43 proteins and loss of gap junctional communication. These combined results suggested that cx43 is a substrate of pp60v-src whose phosphorylation on tyrosine may be involved in the disruption of gap junctional communication observed in Rous sarcoma virus (RSV)-transformed cells.


Integrated plasma cortisol concentration in children with asthma receiving long-term inhaled corticosteroids. We assessed the effect of long-term therapy with inhaled beclomethasone dipropionate (BDP) on the pituitary-adrenal axis, by measuring the integrated concentration (IC) of plasma cortisol in eight children with asthma (age, 6-16 years) who regularly used inhaled BDP in doses ranging from 8 to 26.5 micrograms/kg (200-450 micrograms/day) for 6 months to 4 years. The control group included six children (age, 6-16 years) who had the IC of plasma cortisol measured as part of an endocrinological evaluation and were found to be healthy. Cortisol concentration was measured in blood samples collected continuously over a 24-hr period. Mean IC of plasma cortisol in the study group was significantly lower than in the healthy controls (mean +/- SD, 4.9 +/- 3.3 vs 9.1 +/- 1.9 micrograms/mL; P less than 0.02). Cortisol response to 0.25 mg ACTH (iv) was abnormal in one of the eight BDP-treated patients. No correlation was found between IC of plasma cortisol and the BDP dose, severity of asthma, height percentile, or the Tanner stage. We conclude that long-term therapy, even with relatively conventional doses of inhaled BDP may cause reduction in the normal physiological secretion of cortisol. The clinical relevance of low IC of plasma cortisol is not clear, but it may reflect partial suppression of the pituitary-adrenal axis.


Sputum cysteinyl-leukotriene levels correlate with the severity of pulmonary disease in children with cystic fibrosis. Sputum samples from 13 children with cystic fibrosis (CF) were analyzed for leukotrienes (LTs) LTB4, LTC4, LTD4, and LTE4. Distribution of LTB4 appeared to be normal, and of cysteinyl-LTs log normal. Total cysteinyl-LT levels, of which on average 75% was LTE4, were nearly 10 times higher than in earlier studies. Log LTE4 and total cysteinyl-LT levels correlated with the overall severity of pulmonary disease assessed by Chrispin-Norman chest radiograph score (Log LTE4: r = 0.701, r2 = 49.1%, P = 0.008. Log total cysteinyl-LTs: r = 0.715, r2 = 51.1%, P = 0.006). There was no apparent relationship between LTB4 levels and Chrispin-Norman chest radiograph score, nor between the level of any of the LTs and age or organism cultured from sputum. These findings suggest that the cysteinyl-LTs may be involved in the pathophysiology of pulmonary disease in CF.