The radioprotector WR-2721 reduces neutron-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in mouse splenocytes when administered prior to or following irradiation. An in vitro T-lymphocyte cloning technique has been applied to study the effects of JANUS fission-spectrum neutron irradiation and the radioprotector S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) on the subsequent development of somatic mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in hybrid B6CF1 male mice. In control studies performed to establish an in vitro cloning technique, the mutant frequencies of splenic T-lymphocytes, as a result of exposure to a 100 cGy dose of neutrons, increased with time from a control level of 9 x 10(-7) to a maximum value of 1.7 x 10(-5) at 56 days following irradiation. Between 56 and 150 days after irradiation, mutant frequencies were observed to plateau and remain stable. All subsequent determinations were performed at 56 days following the experimental treatment of animals. WR-2721 at a dose of 400 mg/kg was effective in protecting against the induction of hprt mutants (i.e. a mutant frequency reduction factor, MFRF) following the largest dose of neutrons used (i.e. 150 cGy), whether it was administered i.p. 30 min before, 5 min after, 3 h after, or three times at 3, 24, and 48 h after, as evidenced by MFRFs of 6.0, 6.6, 4.8 and 5.8 respectively. The antimutagenic effectiveness of WR-2721 administered 30 min prior to irradiation was unaffected, even when the dose was reduced to 200 mg/kg, MFRF = 7.0; 100 mg/kg, MFRF = 3.8; and 50 mg/kg, MFRF = 8.9. These findings confirm our earlier report using the radioprotector N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065) under in vitro conditions, and demonstrate that these agents can be used as effective antimutagens even when they are administered up to 3 h following radiation exposure.


Dietary supplementation with pectin and guar gum on 1,2-dimethylhydrazine-induced colon carcinogenesis in rats. The effect of dietary supplementation with pectin and/or guar gum on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis was studied using 120 male Sprague-Dawley rats. The rats were given a weekly injection of DMH for 8 weeks and were maintained on a basal fiber-free diet supplemented with 5% cellulose. The rats were then subdivided into four groups and kept on the basal fiber-free diet supplemented with either no fiber, 10% pectin, 10% guar gum or a combination of 5% pectin/5% guar gum for a period of 24 weeks. The 8 weeks of DMH administration were defined as the initiation stage of carcinogenesis and the next 24 weeks were defined as the promotional stage of carcinogenesis. Food and water were available ad libitum. The rats were killed 32 weeks after the start of the experiment and tumor incidence, location and frequency in the colon were determined. Other parameters measured were body weight and caloric intake. Dietary fiber supplementation with 10% pectin or with 10% guar gum but not with the combination of 5% pectin/5% guar gum (fed during the promotional stage of carcinogenesis), was found to suppress colon cancer incidence to a significant extent.


Effects of carcinogenesis on colonic thymidine kinase activity in familial adenomatous polyposis. Thymidine kinase (TK) activity of polyp tissue from patients with familial adenomatous polyposis (FAP) was measured and compared with that of non-tumorous colon, sporadic polyp and colorectal carcinoma tissues. Total TK activity in colonic carcinoma was 3-fold that of non-tumorous colon; its increase seems attributable mainly to increased activity of cytosolic TK isozyme activity. By using DEAE-cellulose column chromatography, the colorectal TK isozymes were separated into two types, i.e. two isozymes, referred to as the fetal (predominantly cytosolic) and adult (predominantly mitochondrial) types. However, FAP polyp samples from 15 patients showed an average elevation of only 1.8-fold over non-tumorous region of colon. Examined individually, only 5 of the 15 FAP samples showed significant elevations in total TK activity. Furthermore, TK isozyme analysis revealed variable patterns of the cytosolic isozyme activity, which was elevated in some cases (8/15) and remained low in others. Thus FAP polyps seem to be a heterogeneous population with respect to DNA replicative activity, and cytosolic TK isozyme activity may constitute a biochemical marker for the subsequent development of colorectal carcinoma in FAP.


The effect of high-risk and low-risk diets on aberrant crypt and colonic tumor formation in Fischer-344 rats. Eighty Fischer-344 male rats were divided into eight groups. Half the groups were given a high-risk diet (HRD) that was high in fat, low in fiber and low in calcium, while the remaining groups were given a low-risk diet (LRD) that was low in fat, high in fiber and high in calcium. After 4 weeks, four groups were then given two weekly s.c. injections of azoxymethane (AOM) (15 mg/kg body wt), and four groups were given saline injections. At the end of 6 weeks the rats were either continued on HRD, LRD, or crossed over from HRD to LRD, or LRD to HRD for an additional 6 weeks. The rats were then killed and the influence of different dietary regimens on the induction of foci of aberrant crypts (AC) in their colons was compared. The number of foci of AC was highest in the AOM-injected HRD/HRD dietary group (8.83 +/- 0.98), followed by the AOM-injected LRD/HRD group (5.37 +/- 0.75), the HRD/LRD group (3.32 +/- 0.36) and the LRD/LRD group (2.93 +/- 0.32). Except for the HRD/HRD control group, which developed a small but significant number of foci of AC (1.24 +/- 0.19), the other saline-injected dietary groups showed no significant numbers of foci of AC. Two groups of rats on HRD/HRD (AOM and saline-injected), and two groups of rats on HRD/LRD (AOM and saline-injected) were continued on HRD and LRD respectively for an additional 14 weeks. A continuous HRD in AOM-injected rats induced colon tumors in 92% of rats, while the crossover from HRD to LRD suppressed the tumor incidence to 33%. Neither diet induced any tumors in saline-injected rats. The results clearly show that the influence of HRD and LRD on the induction of foci of AC and their distribution in the colon, correlated with the induction and distribution of tumors in the colon.


Early gallbladder visualization during Tc-99m RBC scintigraphy. Gallbladder visualization has been described during delayed Tc-99m RBC blood pool imaging in patients with some combination of anemia, renal disease, and multiple recent blood transfusions. The authors present two cases of gallbladder activity that appeared 1 hour after the injection of Tc-99m RBCs in patients without renal or hematologic abnormalities.


The two-view radionuclide ventriculogram. Advantages of the LPO view. This report is a prospective study of 33 male patients who underwent both contrast ventriculography (CVG) and radionuclide ventriculography (RVG) within a 24-hour period. Expert, blinded observers graded the left ventricle's regional wall motion (RWM) in the left anterior descending (LAD), left circumflex (LCx), and posterior descending arterial (PDA) distributions on right anterior oblique (RAO), and left anterior oblique (LAO) CVGs, and on anterior (ANT), LAO, 70 degrees left anterior oblique (LAO70), and left posterior oblique (LPO) RVGs. When statistically compared with CVG RWM standard data, RVG studies composed of LAO and LPO views were equal to the RVG studies composed of ANT, LAO, and LAO70 views in assessment of the LAD and LCx distributions. The RVG with LAO and LPO views was superior to the RVG with ANT, LAO, LAO70 in the detection of the posterior descending artery RWM. The authors conclude that accurate assessment of RWM is efficiently performed with the RVG composed of LAO and LPO views.


Parathyroid imaging. Use of dual isotope scintigraphy for the localization of adenomas before surgery. Seventy-nine patients with primary hyperparathyroidism, whose average preoperative blood calcium level was 11.6 mg/dl, underwent thallium-technetium dual isotope scintigraphy of the thyroid and parathyroids. For patients who had surgery, the detection and localization rate of parathyroid disease or the sensitivity was low (0.53), but the positive predictive value for the location was high (0.80). Correct localization correlated positively with the weight of the tumor but not significantly with the parathyroid hormone blood level nor with the blood calcium level. Unprocessed data alone were sufficient to predict correctly the location in two thirds of the detected cases. Computer processing increased the sensitivity without decreasing the specificity. Those results, at variance with earlier published data but congruent with another more recent study, require a reevaluation of the role of this scintigraphic technique in the management of hyperparathyroidal patients.


Visualization of microscopic metastatic hepatoma to lung on gallium scintigraphy. The author presents a case of metastatic hepatoma diagnosed at autopsy. The patient's liver had been nearly entirely replaced, and there were diffuse microscopic pulmonary metastases. Chest x-ray was normal at the time of gallium imaging for fever of unknown origin. Gallium imaging revealed a normal-appearing liver and mild, diffuse, bilateral increased uptake in the lungs. A CT scan 3 weeks before autopsy showed relatively minimal abnormality of the liver with a few areas of inhomogeneity and mild enhancement with contrast. Ultrasound-guided aspiration and liver biopsy were negative for tumor or infection. No case report or description of microscopic lung metastases from hepatoma seen with gallium was discovered in a recent literature search.


Age and beta-adrenergic receptor sensitivity to S(-)- and R,S(+/-)-propranolol in humans. The relationship between age and beta-adrenergic receptor sensitivity to the pharmacologically active S(-) enantiomer and the racemic mixture of propranolol was evaluated in 46 healthy male subjects (age range, 24 to 89 years). The in vivo apparent dissociation constants for S(-)- and R,S(+/-)-propranolol were determined on the basis of the unbound steady-state plasma concentration of each and the dose of isoproterenol needed to increase the heart rate of the subjects by 25 beats/min in the absence (I25) and then in the presence of a continuous propranolol infusion. The I25 was significantly correlated with age (r = 0.700, p less than 0.05). The apparent dissociation constant for S(-)- and R,S(+/-)-propranolol demonstrated a significant, although weak, increase with advancing age (r = 0.403 and r = 0.396, respectively; p less than 0.05). Although these findings confirm those of other studies, beta-receptor sensitivity to propranolol was only modestly decreased with age in this study.


Overrunning and underrunning in sequential clinical trials. In a sequential (or group sequential) clinical trial the fulfillment of some pre-specified stopping rule will cause recruitment to be terminated. However, for various reasons, data on patients treated according to protocol may continue to accumulate for some time afterward. This phenomenon is called overrunning. On the other hand, a sequential clinical trial might be abandoned before the stopping rule has been fulfilled. This is called underrunning. In both of these situations the procedure for validly analyzing the study is unclear. In this paper we first review the arguments for sequential methodology and the practical way in which it can be integrated with normal clinical trial conduct. Turning next to the special situations of overrunning and underrunning, the conditions under which a valid analysis is possible are identified, and a method of analysis based on the frequentist philosophy is presented. The likelihood of first gaining and then losing significance due to overrunning and its consequences are examined. Examples based on experience with real studies are presented.


A sequential procedure for a phase II efficacy trial in hypercholesterolemia. The design of a phase II placebo-controlled efficacy trial to evaluate a new treatment for hypercholesterolemia using a sequential procedure is presented. The sequential procedure used is the triangular test which offers flexibility of monitoring and allows early stopping even when the null hypothesis is "accepted." The primary response is reduction in total serum cholesterol level, which is assumed to be normally distributed with unknown variance. The treatment difference is parameterized in terms of the standardized difference between the means. The accuracy of the error rates of the triangular test using this parameterization is compared with the test based on the difference between the means.


Sample size determination based on Fisher's Exact Test for use in 2 x 2 comparative trials with low event rates. A collection of sample size tables are presented for designing comparative trials when the event rates p1 and p2 are low. The tables are based on exact power calculations for Fisher's Exact Test. Both one-sided and two-sided alternative hypotheses are considered. A comparison is made between these sample sizes and those obtained by using popular asymptotic approximations.


A method of assigning scores to the components of a composite outcome: an example from the MITI trial. After obtaining expert opinion to assign weights to the individual components of a composite outcome, scores are assigned in such a way as to reflect these weights as well as to optimize the power for specified alternatives.


Quality control of dietary data collection in the CARDIA study. The Coronary Artery Risk Development in (Young) Adults (CARDIA) Study developed and implemented quality control (QC) measures to minimize misclassification associated with dietary data. Manual and automated data inspection were used to monitor quality. Of the 5111 participants who completed a dietary history, 717 (14%) had dietary forms reviewed and 153 (3%) had the interview audiotaped. Results show that for the 717 forms reviewed, the overall form completion error rate was 0.22% and the "critical" error rate (i.e., those errors impacting on nutrient computations) was 0.12%. The proportion of forms free of any type of error increased over time (p less than 0.0001). The discrepancy rate in recording and interviewing methods as estimated from the 153 audiotaped interviews was 0.7%. Inter-interviewer differences were small as indicated by the audiotaped interviews and the proportion of error-free forms completed by interviewers. The results indicate that the dietary data collected in CARDIA were completely and accurately recorded for use in analysis.


Atrial natriuretic peptide, cyclic guanosine monophosphate and sodium excretion during postnatal adaptation in male infants below 34 weeks gestation with severe respiratory distress syndrome. The role of atrial natriuretic peptide (ANP), in the perinatal period, is at present unclear. In adults urinary cyclic guanosine monophosphate (cGMP) is considered an index of the biological activity of plasma ANP. The aim of this study was to determine the relationship between plasma ANP, cGMP excretion (cGMPex) and sodium excretion (Naex) in preterm infants in the first days after birth. Sequential, 4 hourly, measurements of plasma ANP, cGMPex and Naex were made in 12 male neonates of median gestational age 27 weeks (range 25-33) and median birth weight 0.981 kg (range 0.635-2.029) over a median period of 5.2 days (range 2.3-10). The ratios of cGMPex to ANP and of Naex to cGMPex were each plotted against postnatal age. The ratio of cGMPex to ANP increased ten fold in the first 3-4 days after birth but then remained relatively constant; the ratio of Naex to cGMPex showed a steady increase from birth. We conclude that, in extremely immature infants, renal sodium loss in response to cGMP increases rapidly during the first 10 days after birth. In addition, after 3-4 days from birth, plasma ANP ia associated with a constant proportionate rate of cGMP excretion though, as the plateau ratio of cGMPex to ANP varied widely between babies, cGMPex cannot be used to predict plasma ANP in cross sectional studies. These changes may reflect postnatal adaptation and/or maturation of both ANP receptors and cGMP mediated cascades. In the immediate postnatal period, plasma ANP may also have a non-renal role.


Bilateral internal drainage of biliary hilar malignancy via a single percutaneous track. Role of percutaneous transhepatic cholangioscopy. Strictures of the hepatic bifurcation were bilaterally drained via a single percutaneous tract in 24 patients with hilar cholangiocarcinoma. This was achieved under percutaneous transhepatic cholangioscopic (PTCS) guidance which allowed for an excellent manipulation of the guidewire. Placement of two guidewires, one in the contralateral hepatic duct and the other in the common duct, was successful in 19 (79%) of patients at the first attempt. In the remaining cases the better accessible stricture was dilated, followed by negotiation of the other stricture under direct cholangioscopic control. After placement of guidewires into both hepatic ducts, bilateral drainage with a single Y-shaped catheter could be achieved in all 24 patients. There were no procedure-related complications. We conclude that this technique might be useful also for application of other intraluminal adjuvant treatment modalities.


Highly efficient transfection of mammalian cells by electric field pulses. Application to large volumes of cell culture by using a flow system. We have transfected mammalian cells with plasmid DNA by application of electric pulses. Chinese hamster ovary cells were chosen as a model in order to study and to optimize the transfection protocol. A plasmid carrying the gene coding for beta-galactosidase activity was used to determine transient expression of the electrotransferred activity at the cell level. Optimum transient expression for cells in suspension was obtained by application of 10 square wave pulses of 5-ms duration and 0.6-kV/cm intensity. Under the best conditions, transfection frequencies as high as 50-60% could be obtained and appeared to be highly dependent on the age of the cell culture. The method was applicable to plated cells growing in a petri dish or on microcarriers. The possibility of extension of the technique to large volumes of cells is presented. A flow system, composed of a peristaltic pump connected to the electropulser chamber, allowed large volumes of cells to be treated with flow rates in the order of several milliliters/minute. Transfection frequencies for the large volumes were 25% for cells in suspension and 35% for cells on microcarriers. These results open new perspectives in large-scale transfection technology of cells however they are grown.


Phenylalanine positively modulates the cAMP-dependent phosphorylation and negatively modulates the vasopressin-induced and okadaic-acid-induced phosphorylation of phenylalanine 4-monooxygenase in intact rat hepatocytes. The state of phosphorylation of phenylalanine hydroxylase was determined in isolated intact rat hepatocytes. 32P-labeled phenylalanine hydroxylase was immunoisolated from cells loaded with 32Pi or from cell extracts 'back-phosphorylated' with [gamma-32P]ATP by cAMP-dependent protein kinase. The rate of phenylalanine hydroxylase phosphorylation in cells with elevated cAMP was similar to that observed for the isolated enzyme phosphorylated by homogeneous cAMP-dependent protein kinase. The phosphorylation rate in cAMP-stimulated cells was increased up to four times (reaching 0.018 s-1) by the presence of phenylalanine, the phosphate content (mol/mol hydroxylase) increasing to 0.5 from the basal level (0.17) in 50 s. The half maximal effect of phenylalanine was obtained at a physiologically relevant concentration (110 microM). The synthetic phenylalanine hydroxylase cofactor dimethyltetrahydropterin also enhanced the cAMP-stimulated phosphorylation of phenylalanine hydroxylase, presumably by displacing the endogenous cofactor, tetrahydrobiopterin. Phenylalanine was a negative modulator of the phosphorylation of phenylalanine hydroxylase induced by incubating cells with vasopressin or with the phosphatase inhibitor okadaic acid. The same site on the phenylalanine hydroxylase was phosphorylated in response to these two agents as in response to elevated cAMP. The available evidence suggested that not only vasopressin, but also okadaic acid, acted by stimulating the multifunctional Ca2+/calmodulin-dependent protein kinase II or a kinase with closely resembling properties.


Biochemical and immunological characterization of A1 adenosine receptors purified from human brain membranes. The A1 adenosine receptor was purified approximately 13,000-fold to apparent homogeneity from human cerebral cortex membranes using a novel affinity-chromatography system developed for the purification of rat brain and rat testis A1 adenosine receptors [Nakata, H. (1989) J. Biol. Chem. 264, 16,545-16,551; Nakata, H. (1990) J. Biol. Chem. 265, 671-677]. The purified human brain receptor showed the ligand-binding specificity expected of the A1 adenosine receptor. The Bmax and Kd for the purified receptor with a specific A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, were approximately 16 nmol/mg protein and 2 nM, respectively. SDS/PAGE of the purified receptor preparation showed one broad protein band of molecular mass of approximately 35 kDa, which is very similar to that of purified A1 adenosine receptor from rat brain membranes. Endoglycosidase F treatment of the purified receptor reduced the molecular mass to approximately 30 kDa, suggesting that the human brain A1 adenosine receptor is a glycoprotein. Comparison of the purified human and rat brain A1 adenosine receptors by peptide mapping after the proteolytic digestion showed minor differences between these receptors. Immunological comparisons of the human brain A1 adenosine receptor with rat brain A1 adenosine receptor using polyclonal antibodies against the purified rat brain A1 adenosine receptor showed that the antibodies react preferentially with the rat brain receptor and weakly with human brain receptor.


Atrial natriuretic peptide is phosphorylated by intact cells through cAMP-dependent ecto-protein kinase. Recently we demonstrated the presence of cell-surface-located cAMP-dependent protein kinase (ecto-PK A) activity in a number of different cell types [Kübler, D., Pyerin, W., Bill, O., Hotz, A., Sonka, J. and Kinzel, V. (1989) J. Biol. Chem. 264, 14549-14555]. The question of the physiological role of externally directed kinase activity prompted a search for potential natural substrates present in the intercellular fluid. In the present study we have investigated the phosphorylation by ecto-PK A of the human atrial natriuretic peptide ANP99-126, a hormone released by cardiac cells. This 28-amino-acid peptide carries the phosphorylation consensus sequence Arg-Arg-Ser-Ser for the PK A. Incubation of various cell lines (including epithelial, epidermal, myoblast and lymphoma cells) or freshly isolated blood cells (macrophages, erythrocytes and platelets) with ANP in the presence of low micromolar concentrations of ATP resulted in the phosphorylation of ANP at Ser residues. The ANP phosphorylation reaction proved strictly dependent on cAMP; cAMP could not be replaced by cGMP. The phosphorylation was inhibited by the PK A-specific inhibitory peptide and increased linearily for up to 15 min and with a Km value of 3-5 microM for ANP. At higher ATP concentrations (greater than 100 microM) the incorporation rates amounted to about 0.3 mmol P (mol ANP)-1 min-1. The rise of intracellular cAMP in HEL30 (an epidermal cell line) after application of the beta-adrenergic receptor agonist isoproterenol led to an approximately three-fold stimulation of ANP phosphorylation which appears to be brought about by an efflux of intracellular cAMP. Employing cell supernatant fluids and cell sonicates, it could be shown that the phosphorylation of ANP results from the ecto-PK A. Comparison of ANP with ANP phosphorylated in vitro using purified catalytic subunit of PK A showed that phosphorylation is accompanied by certain changes in the average solution conformation of the peptide, consistent with the changes known to occur in its biological activity. Our results demonstrate cAMP-dependent phosphorylation of the peptide hormone analogue ANP99-126 by intact cells through ecto-PK A, an intriguing mechanism for post-translational processing of ANP.


High-resolution capillary electrophoretic analysis of DNA in free solution. Capillary electrophoretic separations of double-stranded DNA fragments in the size range of 20-2200 base pairs were achieved in less than 20 min with the use of a Tris-borate buffer containing hydroxyethylcellulose. Analyses were carried out in both uncoated and phenylmethyl-coated capillaries of fused silica with internal diameters of 50 and 100 microns, respectively, and an effective column length of 50 cm. The addition of ethidium bromide resulted in an improved resolution of double-stranded DNA fragments, thereby permitting even the separation of fragments differing only 1-2 base pairs in length. Moreover, resolution was found to be linearly proportional to the size of the cation used to adjust ionic strength Cs+ greater than RB+ greater than K+ greater than Na+ greater than Li+. However, the analysis times also increased with increasing cation size due to a decrease in electroosmotic flow. Elution order was verified by spiking restriction digests with slab gel electrophoretically purified components. Subsequently, the described system was applied to the detection and quantitation of an mRNA transcript of the androgen receptor, which had been amplified by polymerase chain reaction and purified by size-exclusion chromatography to avoid peak broadening due to conductivity differences between sample and running buffer. Since the actual amount of DNA introduced into the capillary cannot be defined, molar ratio-peak area ratios of the polymerase chain reaction product to various restriction fragments of known concentration were used to determine the amount of amplified DNA. The coefficient of variation was as small as 3.4% and the results were in good agreement with a spectrophotometric assay.


Neuropeptide Y. Optimized solid-phase synthesis and conformational analysis in trifluoroethanol. The 36-amino-acid neuropeptide Y (human), which is one of the most potent vasoconstrictors and which exhibits a number of other biological functions, has been synthesized using automated peptide synthesis. The optimized method, using 9-fluorenylmethoxycarbonyl protecting and single-step coupling, yielded the crude product in 90% purity allowing for single-step reversed-phase HPLC purification to greater than 98% purity and a high overall yield (50%). The hormone was characterized by several chromatographic methods, ion-spray mass spectroscopy and Edman degradation. The conformation of human neuropeptide Y was examined by CD, NMR and computer simulations. The CD measurements in trifluoroethanol/water (9:1) show a large percentage of alpha-helix. Variation of concentration, from 0.5 microM increasing up to the 1 mM used for NMR measurements, indicates no evidence for aggregation. In the same solvent system, the NMR line widths were very broad and therefore the resonance assignment was achieved with the exclusive use of two-dimensional NOE spectra. The 248 clearly distinguishable NOEs from the NMR study were used in distance geometry calculations and the resulting structures were refined with restrained molecular dynamics. The results indicate an alpha-helix extending from Arg19 to Gln34. For the N-terminal half of the molecule no regular structure was observed.


Papilloma virus infection. Precancer and epidermoid cancer. We present a review of the relationship between Human Papillomavirus (HPV), Precancer and Epidermoid Cancer of the uterine cervix, Co-factors that contribute in cervical carcinogenesis and the mechanism through which Papillomaviruses transform normal cells into neoplastic cells by inducing an overproduction of the encoded proteins. We also offer final remarks and conclusions on this pathology that increases its incidence every year.


Lymphocyte subset in HPV infection.

Lymphocyte subset in HPV infection. The outcome of HPV infection and its progression to neoplastic disease may be conditioned by the immune status of patients. In the present study we observed the systemic lymphocyte subset, with particular regard to NK cells and NK activity in 7 patients with HPV infection. The lymphocyte phenotypes in peripheral blood were studied using a panel of monoclonal antibodies, while NK activity was evaluated as percentage of lysis of K 562 target cells. We noticed a significant decrease of basal NK activity especially in patients with HPV 16-18 infection (p less than 0.001), without any difference in the absolute number of these cells.


Viruses as possible etiological factors in human genital cancer. The antibodies against herpes virus were detected in 34 out of 60 (55%) patients with gynecological malignancies. Immunofluorescence revealed herpes virus antigens in 40 (66.7%) malignant tissues. EBV was not detected in either sera or tissue samples. Adenovirus was evidenced in sera and tissues of one case of endometrial cancer.


Transmission from group II muscle afferents is depressed by stimulation of locus coeruleus/subcoeruleus, Kölliker-Fuse and raphe nuclei in the cat. The effects of brief trains of electrical stimuli applied within the locus coeruleus and subcoeruleus, the Kölliker-Fuse nucleus and the raphe magnus, obscurus and pallidus nuclei were tested on transmission from group I and group II muscle afferent fibres in mid-lumbar spinal segments of chloralose anaesthetized cats. Changes in the effectiveness of transmission from these afferents were assessed from changes in the size of monosynaptic extracellular field potentials evoked by them. The depression of group II field potentials occurred at conditioning-testing intervals of 20-400 ms, and was maximal at intervals of 40-100 ms and 30-60 ms for potentials recorded in the intermediate zone and dorsal horn, respectively. At intervals up to about 30 ms it was combined with the depression of group I components of the intermediate zone field potentials. However, at longer intervals the conditioning stimuli depressed group II components of these potentials as selectively as monoamines applied ionophoretically at the recording site (Bras et al., 1989a, 1990). Thus, only the late depressive actions are considered as being possibly mediated by impulses in descending noradrenergic and/or serotonergic fibres. No major differences were found in the relative degree of depression of transmission from group II afferents by stimulation of the locus coeruleus/subcoeruleus, Kölliker-Fuse or raphe nuclei, either in the dorsal horn or in the intermediate zone. Since field potentials at these locations are preferentially depressed by ionophoretic application of serotonin and noradrenaline (Bras et al., 1990), and since the locus coeruleus/subcoeruleus, Kölliker-Fuse and raphe nuclei are interconnected, the study leads to the conclusion that both noradrenergic and serotonergic descending pathways can be activated by stimuli applied within either of them. Selective depression of field potentials of group II origin was also evoked by stimulation at other sites, e.g. the periaqueductal grey and medullary reticular formation, when conditioning-testing intervals were sufficiently long. Such a depression is considered to be secondary to activation of neurones of the locus coeruleus/subcoeruleus, Kölliker-Fuse or raphe nuclei and attributed to the spread of current or transsynaptic activation of these neurones, or to stimulation of their axon collaterals outside the nuclei rather than to other descending medullo-spinal systems. The non-selective depression of field potentials evoked by group I and group II afferents at shorter conditioning-testing intervals is proposed to be due to actions of reticulo-spinal pathways.


(Model) studies on vanadate-dependent bromo/iodoperoxidase from Ascophyllum nodosum. VO2+ is not incorporated into the active site. Vanadate-dependent peroxidase A.n.I, the main isoenzyme (M(r) = 100 kDa) from the seaweed, Ascophyllum nodosum, contains 2 V per enzyme molecule (as shown by ICP-MS metal analysis) after complete reconstitution with vanadate (V), possibly distributed in a 1:1 ratio between the surface and active site. VO2+ is only weakly associated to the surface of A.n.I. There is no transport channel for VO2+. The EPR spectrum of the reduced holoenzyme is anisotropic (axial) already at room temperature, with EPR parameters similar to those of VO2+ complexes of small model peptides such as Ala-His, Gly-Tyr, Gly-Ser, Gly-Glu, Ser-Gly and Phe-Glu. The complex formation between Ala-His and H2VO4- in water has also been investigated (by 51V NMR); the formation constant at pH 7.2 amounts to 266(28) M-1.


Involvement of a non-proton pump factor (possibly Donnan-type equilibrium) in maintenance of an acidic pH in lysosomes. Change of the internal pH of isolated lysosomes was measured with fluorescein isothiocyanate-dextran. In buffer of pH 7.0, isolated lysosomes had an acidic pH of about 5.5, which decreased to pH 5.2 on addition of ATP. Addition of bafilomycin inhibited the acidification by H(+)-ATPase and resulted in an increase of the internal pH to 5.5 due to passive diffusion of protons across the lysosomal membrane. However, no further alkalization was observed. The acidic pH (pH 5.5) of isolated lysosomes could be maintained for at least 48 h in the absence of ATP, but increased gradually to pH 5.9-6.4 upon incubation with monovalent cations (K+ or Na+), amines, or ionophores. These results suggest that a non-proton pump factor (possibly Donnan equilibrium) is involved in maintaining the acidic pH of isolated lysosomes.


Production of a unique multi-lamella structure in the nuclei of yeast expressing Drosophila copia gag precursor. Drosophila retrotransposon copia produces virus-like particles (VLPs) in the nuclei of cultured Drosophila cells. The VLPs contain copia RNA and reverse transcriptase activity, and thus, play a major role in copia replication. Here we have expressed the copia gag polyprotein precursor in yeast. The precursor, which includes copia protease itself, showed correct autoprocessing to produce a unique multi-lamella structure in the nuclei of the yeast cells. This expression system should be useful for the analysis of nuclear localization of the major copia VLP protein, and furthermore, would provide important information concerning the mechanism of copia VLPs formation.


Rapid agonist-induced beta-adrenergic receptor kinase translocation in C6 glioma cells. Exposure of C6 glioma cells to 1 microM isoproterenol leads to fast desensitization of the beta-adrenergic receptor/adenylyl cyclase system and transient receptor sequestration. It also triggers a very rapid and transient translocation to the plasma membrane of beta-adrenergic receptor kinase (beta ARK), a specific cytoplasmic kinase that phosphorylates only the agonist-occupied form of several G protein-coupled receptors. beta ARK-mediated receptor phosphorylation appears to be a suitable mechanism for the rapid regulation of adrenergic receptor function in the nervous tissue.


Angiotensin-I-converting enzyme and its correlation with human follicular fluid steroids. Angiotensin-I-converting enzyme (ACE) is a peptidyl-dipeptide hydrolase which splits off the dipeptide His-Leu from the decapeptide angiotensin I and thus converts it to angiotensin II. We determined ACE activity in human preovulatory follicular fluid to further establish the intraovarian activity of the renin angiotensin system. Follicular fluids (n = 18) were obtained from eight patients undergoing in vitro fertilization (IVF) and embryo transfer (ET). ACE activity in follicular fluid and serum was determined by fluorescent spectrophotometry. The median follicular fluid ACE activity was 1.12 (range: 0.19-1.56) nmol/min/ml. This value was significantly lower than ACE activity in serum, 1.50 (range: 1.22-1.57) nmol/min/ml (P less than 0.001). In contrast to this 3:4 ratio between follicular fluid and serum ACE when expressed per ml fluid, the values were very similar when expressed per mg of protein: 0.025 vs. 0.023 nmol/min/mg in follicular fluid and serum, respectively. Correlations were sought between follicular fluid ACE activity and both serum and follicular fluid E2 and P4. A highly significant correlation (P less than 0.0005, r = 0.73) was found between ACE activity in follicular fluid and follicular fluid P4. The presence of significant ACE activity in human follicular fluid further supports the local-ovarian activity of the renin angiotensin cascade.


The pliable obstetrical vacuum cup: application and opinions in The Netherlands. A questionnaire was sent to all hospital obstetric units in The Netherlands to obtain information about the use of soft cups for vacuum extraction. Over 90% of the 156 units responded. Vacuum extraction was used twice as often as forceps for instrumental vaginal delivery. In 12 units the flexible cup was applied in the majority of vacuum extractions and without specific restrictions. In 47 units a minority of vacuum extractions was performed with a flexible cup: its use was generally restricted to anticipated easy low outlet extractions. It is concluded that in The Netherlands the flexible cup is an accepted instrument used in about 13% of vacuum extraction deliveries.


Effects of steroidogenesis inducing protein (SIP) on steroid production in MA-10 mouse Leydig tumor cells: utilization of a non-cAMP second messenger pathway. We have investigated the effects of steroidogenesis inducing protein (SIP) (Endocrinology (1990) 126, 3043-3052) on steroid production in MA-10 mouse Leydig tumor cells. Our results indicate that SIP results in the stimulation of progesterone production in MA-10 cells to the same extent obtained when maximal doses of luteinizing hormone (LH), human chorionic gonadotropin (hCG) and dibutyryl cAMP (dbcAMP) are used. It was also observed that the increased progesterone production in response to SIP was not accompanied by an increase in intracellular cAMP levels as was seen following hCG stimulation. In addition, stimulation of progesterone production using maximal doses of LH, hCG and dbcAMP could be further increased by the addition of SIP to the incubation medium also indicating that this steroidogenic activity was acting through a differential signal transducing system than these hormones. That SIP was not acting through the cAMP second messenger pathway was also demonstrated by its lack of sensitivity to the neutralizing effects of a monoclonal antibody to LH as well as by its insensitivity to the protein kinase A inhibitor HA 1004 while both of these treatments significantly decreased LH and hCG stimulated steroid production. Lastly, SIP was unable to elicit the induction of several mitochondrial proteins which have previously been shown to be synthesized in MA-10 cells in response to LH, hCG and dbcAMP. Our results indicate that SIP stimulates the production of high levels of steroids through a signal transduction pathway which is distinct from that employed by trophic hormone stimulation in Leydig cells.


Regulation of the cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin mRNAs in cultured choriocarcinoma cells. Cytochrome P-450scc (P-450scc) catalyzes the cholesterol side-chain cleavage reaction, a rate-limiting enzymatic step for progesterone synthesis in trophoblastic and other steroidogenic cells. Adrenodoxin is the iron/sulfur protein donating electrons to P-450scc during this reaction. We examined the effects of cholera toxin (CT), an activator of adenylate cyclase, and 12-O-tetradecanoylphorbol acetate (TPA), a phorbol ester protein kinase C activator, on the levels of mRNAs encoding P-450scc and adrenodoxin in JEG-3 choriocarcinoma cells. CT induced in a concentration- and time-dependent manner P-450scc and adrenodoxin mRNA levels to 8-fold and 1.5-fold above that of control, respectively. TPA also increased P-450scc and adrenodoxin mRNA levels about 3-fold and 1.5-fold above that of control, respectively. Epidermal growth factor (EGF) was found to weakly induce P-450scc mRNA accumulation with a maximal 20% stimulation above basal levels. The effects of CT and TPA were apparently additive on both mRNAs. The protein synthesis inhibitor cycloheximide diminished basal, CT-, TPA-, and EGF-stimulated P-450scc mRNA accumulation whereas the opposite was observed for the adrenodoxin mRNA. Insulin-like growth factor I (IGF-I) appeared to have no effect on either mRNA. These data indicate that: (1) the accumulation of P-450scc and adrenodoxin mRNAs is mainly controlled by the cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway but their stimulation by TPA- and EGF-induced signals may also play a weaker synergistic role; (2) the protein synthesis inhibitor cycloheximide inhibits basal, CT-, TPA- and EGF-stimulated P-450scc mRNA levels while it increases the expression of adrenodoxin mRNA suggesting that in the malignant trophoblasts these two enzyme mRNAs are differentially controlled.


Differential regulation of multiple c-erbA expression by thyrotropin, insulin and insulin-like growth factor I in rat thyroid FRTL-5 cells. Regulation of multiple c-erbA gene expression was studied in rat thyroid FRTL-5 cells. Two species of erbA alpha (alpha 1 and alpha 2) mRNA and one species of erbA beta (beta 1) mRNA were identified by Northern blot analysis. Withdrawal of thyrotropin (TSH), insulin and serum from the complete medium resulted in an increase in alpha 1 and alpha 2 erbA mRNA levels without altering the level of erbA beta 1 mRNA. Readdition of TSH, N6,2'-O-dibutyryl cAMP or forskolin caused a transient reduction of alpha 1 and alpha 2 mRNA levels (75-90%) at 3-12 h. The alpha 1 and alpha 2 mRNA levels were restored at 24 h. The TSH action was dose-dependent showing the half-maximal effect at around 10(-9) M. Readdition of TSH did not show any effect on beta 1 mRNA level. The action of TSH was not dependent on ongoing protein synthesis but required ongoing transcription. Inhibitors of thyroid hormone biosynthesis, propylthiouracil and methylmercaptoimidazole, did not show any effect on TSH action. Readdition of insulin or insulin-like growth factor I (IGF-I) caused a dose-dependent reduction of alpha 1 and alpha 2 mRNA levels without any effect on beta 1 mRNA level. Their action was slower than TSH and persistent. The actions of insulin and IGF-I were dependent on both ongoing translation and transcription. These results indicate that TSH and insulin/IGF-I reduce levels of c-erbA alpha 1 and alpha 2 mRNA possibly by two distinct mechanisms without altering c-erbA beta 1 mRNA level in FRTL-5 cells.


Insulin can rapidly increase cell surface insulin binding capacity in rat adipocytes. A novel mechanism related to insulin sensitivity. To elucidate the acute effect of insulin on its receptor, rat adipocytes were preincubated with insulin, washed with KCN to inhibit receptor cycling, and 125I-labeled insulin binding was measured. Preincubating cells from young insulin-sensitive rats with insulin increased cell surface binding up to approximately fourfold without changing apparent receptor affinity. This effect was rapid (t1/2 less than 5 min) and had a similar dose-response relationship as the effect on glucose transport. It was also energy dependent because preincubation with KCN completely abolished the effect of subsequent insulin exposure. The increased binding capacity was not recovered after cell solubilization or in partially purified receptors or isolated plasma membranes. Cells pretreated with insulin were less sensitive to the ability of trypsin to remove cell surface receptors, suggesting a conformational change of the receptors. This was also supported by the finding that the polyclonal binding in insulin-treated but not in control cells. Vanadate mimicked the effect of insulin to increase insulin binding, whereas concanavalin A, vasopressin, phorbol esters, or the adenosine analogue phenyl isopropyl adenosine was without effect. Insulin-resistant adipocytes from obese rats displayed no increase in cell surface binding after insulin treatment, despite normal tyrosine kinase activity in response to insulin. Thus, both insulin and vanadate elicit a rapid effect to markedly increase the number of cell surface insulin binding sites in intact rat adipocytes. This appears to occur independently of protein kinase C and the inhibitory GTP binding protein (Gi). Furthermore, the effect of insulin could not be demonstrated in insulin-resistant cells, suggesting that this mechanism may be of importance for the regulation of insulin sensitivity.


Effect of ingestion of eicosapentaenoic acid ethyl ester on carrageenan-induced colitis in guinea pigs. The effect of highly purified eicosapentaenoic acid ethyl ester (EPA-E) on colitis was investigated using a guinea pig model. The technique for preparing a degraded carrageenan with a molecular weight of about 30,000 from commercial iota-carrageenan was first refined. When this degraded carrageenan was fed to guinea pigs, localized ulcerations occurred in the cecum with infiltration of numerous mononuclear phagocytes. Oral administration of 300 mg.kg-1.day-1 of EPA-E for 3 weeks significantly prevented the development of colitis. The amounts of prostaglandin E2, thromboxane B2, and leukotriene B4 released from the cecal mucosa were also measured. The release of prostaglandin E2 and thromboxane B2 was significantly decreased in the animals fed EPA-E compared with those given olive oil or a vehicle alone. In addition, there was a positive correlation between the amounts of these eicosanoids and the degree of ulcer formation. However, there was no difference in the amount of leukotriene B4 among various experimental groups of animals. Furthermore, EPA-E feeding induced a significant decrease in the level of arachidonic acid and a significant increase in that of EPA in peritoneal macrophages. These results suggest that EPA has a prophylactic effect on the development of carrageenan-induced colitis, which may be ascribed in part to reduced eicosanoid production.


The risk of upper gastrointestinal cancer in familial adenomatous polyposis. Adenomas with potential for malignancy occur frequently in the upper gastrointestinal tract of patients with familial adenomatous polyposis. However, an assessment of relative risk of upper gastrointestinal cancer in patients with adenomatous polyposis has never been performed. Therefore, the incidence rate of upper gastrointestinal cancer in patients with familial adenomatous polyposis in The Johns Hopkins Registry was compared with the rate of the general population through person-year analysis with adjustment for demographics. There was an increased relative risk of duodenal adenocarcinoma (relative risk, 330.82; 95% confidence limits, 132.66 and 681.49; P less than 0.001) and ampullary adenocarcinoma (relative risk, 123.72; 95% confidence limits, 33.65 and 316.72; P less than 0.001). No significant increased risk was found for gastric or nonduodenal small intestinal cancer. These results indicate that periodic surveillance of the upper gastrointestinal tract for duodenal and periampullary cancer is needed in patients with familial adenomatous polyposis. Prophylactic duodenectomy is a consideration when large adenoma(s) with high-grade dysplasia are identified but awaits risk benefit analysis.


Lymphocyte beta 2-adrenoceptors and plasma catecholamines in patients with cirrhosis. The hemodynamic response to propranolol in patients with cirrhosis is heterogeneous. In some patients, there is an expected decrease in portal pressure, whereas in others, portal pressure fails to decrease despite adequate beta-blockade. It has been suggested that this lack of response could be related to down-regulation of beta 2-adrenoceptors, promoted by the increased adrenergic activity frequently found in decompensated cirrhosis. The present study investigated this hypothesis by measuring the density and affinity of lymphocyte beta 2-adrenoceptors (L-beta 2-AR), an established index of beta 2-adrenoceptors in target organs, and the plasma levels of norepinephrine and epinephrine in a group of 32 patients with cirrhosis and portal hypertension. The portal pressure response to propranolol administration (0.1 mg/kg + 2 mg/h) was also investigated (n = 27). Patients with cirrhosis had increased norepinephrine levels (605 +/- 360 vs. 224 +/- 110 pg/mL in controls; P less than 0.001), but plasma epinephrine level was not increased (136 +/- 72 vs. 111 +/- 22 pg/mL in controls; NS). There were no differences between cirrhotic patients and controls in the density of L-beta 2-AR (1398 +/- 489 vs. 1278 +/- 356 receptors/cell; NS). Portal pressure decreased greater than 10% in 12 patients (responders) and less than 10% in the remaining 15 (nonresponders). Contrary to previous suggestions, there were no significant differences between responders and nonresponders in relation to the density of L-beta 2-AR (1362 +/- 527 vs. 1487 +/- 419 receptors/cell; NS), to the affinity of these receptors (0.15 +/- 0.27 vs. 0.13 +/- 0.12 nmol/L; NS), to the plasma levels of norepinephrine (661 +/- 508 vs. 621 +/- 256 pg/mL; NS), and to plasma epinephrine concentration (141 +/- 90 vs. 140 +/- 75 pg/mL; NS). These results show that the response of portal pressure to propranolol administration is neither related to the density and affinity of L-beta 2-AR nor to the plasma levels of norepinephrine and epinephrine. Thus, the determination of these parameters cannot substitute measurements of portal pressure to identify those patients with an adequate portal pressure response to propranolol treatment.


TRC-1: emergence of a clavulanic acid-resistant TEM beta-lactamase in a clinical strain. A novel TEM-derived plasmid-encoded beta-lactamase, resistant to inhibition by clavulanic acid, has been identified in a clinical strain of Escherichia coli found in Scotland. The beta-lactamase gene was carried on an 81-kb plasmid that conferred no other resistances. The novel enzyme conferred resistance to the amoxycillin/clavulanic acid combination on the host bacterium. The beta-lactamase has a pI of 5.25 and lies between the PSE-4 and SAR-1 beta-lactamases on an isoelectric focusing gel. This beta-lactamase has a Mr value of 25,000, similar to the TEM-1 enzyme and a comparable substrate profile. Its most significant difference is that it is inhibited by clavulanic acid 100-fold less efficiently than the TEM-1 enzyme. The enzyme was confirmed to be derived from the TEM enzymes by probing the plasmid DNA with an intragenic gene probe for TEM-1. This is the first report of a clinical bacterium carrying a TEM-enzyme that confers resistance to clavulanic acid combinations and we have designated the beta-lactamase as TRC-1.


Characterization of the mcrBC region of Escherichia coli K-12 wild-type and mutant strains. We have carried out an analysis of the Escherichia coli K-12 mcrBC locus in order to (1) elucidate its genetic organization, (2) to identify the proteins encoded by this region, and (3) to characterize their involvement in the restriction of DNA containing methylated cytosine residues. In vitro expression of recombinant plasmids carrying all or portions of the mcrBC region revealed that the mcrB and mcrC genes are organized as an operon. The mcrBC operon specifies five proteins, as evident from parallel in vitro and in in vivo expression studies. Three proteins of 53, 35 and 34 kDa originate from mcrB expression, while two proteins of 37 and 16 kDa arise from mcrC expression. Products of both the mcrB and mcrC genes are required to restrict the methylated substrate DNA used in this study. We also determined the nature of mutant mcrBC loci in comparison to the E. coli K-12 wild-type mcrBC locus. A major goal of these studies was to clarify the nature of the mcrB-1 mutation, which is carried by some strains employed in previous analyses of the E. coli K-12 McrBC system. Based on our analyses the mutant strains investigated could be divided into different complementation groups. The mcrB-1 mutation is a nonsense or frameshift mutation located within mcrB. It causes premature termination of mcrB gene product synthesis and reduces the level of mcrC gene expression. This finding helps to understand an existing conflict in the literature. We also describe temperature-sensitive McrA activity in some of the strains analysed and its relationship to the previously defined differences in the tolerance levels of E. coli K-12 mcrBC mutants to cytosine methylation.


A simple and efficient system for the construction of phoA gene fusions in gram-negative bacteria. We have developed a two-plasmid system for generating gene fusions between phoA and cloned genes encoding envelope proteins. The vector plasmid carries a temperature-sensitive replication system and can be rescued at high temperature by insertion of an IS1-based transposon carrying the ori region of pBR322 and a phoA gene lacking transcription and translation initiation signals. The vector plasmid also carries the transfer origin of the conjugative plasmid, F, permitting transfer into a suitable recipient strain. We have used this system in the analysis of the bla gene cloned from pBR322.


A gene (hur) from Streptomyces aureofaciens, conferring resistance to hydroxyurea, is related to genes encoding streptomycin phosphotransferase. A novel gene (hur) conferring resistance to hydroxyurea (HU) in Escherichia coli has been identified in a Streptomyces aureofaciens genomic library. The expression of hur in E. coli was under the control of the external plasmid tet promoter. Sequence analysis of a minimal fragment revealed an open reading frame (ORF) encoding a protein of 340 amino acids with an M(r) of 36,049 and an average hydropathy index of 1.13. The predicted protein product was similar to streptomycin phosphotransferases from Streptomyces glaucescens and Streptomyces griseus (52.4% and 50.8% identity, respectively), but it did not confer resistance to streptomycin or to any of the other aminoglycoside antibiotics tested. It is inferred that hur encodes a phosphotransferase that inactivates HU by phosphorylation of the hydroxy group in the hydroxylamine moiety.


A novel, highly regulated, rapidly inducible system for the expression of chicken progesterone receptor, cPRA, in Saccharomyces cerevisiae. A rapidly inducible and tightly regulated system for the expression of protein in yeast is based on a chimeric promoter constructed of two copies of a vitellogenin-estrogen-response element (ERE) which are inserted upstream from the promoter of the yeast gene encoding iso-1-cytochrome c. The chimeric promoter was inserted in a yeast expression plasmid upstream from the coding sequence of ubiquitin fused in frame to a cDNA encoding the full-length chicken progesterone receptor A (cPRA). The resultant plasmid (YEpA2) was co-transformed in Saccharomyces cerevisiae with a plasmid which encodes the human estrogen receptor. Estradiol (E2)-induced transactivation of the chimeric promoter results in transcription of the cPRA gene from YEpA2, and synthesis of cPRA. The fusion protein, ubiquitin-cPRA, is rapidly cleaved in vivo to produce cPRA. Analysis of samples by Western immunoblot shows that cPRA is almost undetectable in the absence of E2, and that treatment with 50 nM E2 results in a 500-1000-fold induction of cPRA (0.06-0.3% of the total protein) after 1 h. The plasmid-expressed soluble receptor is stable and demonstrates the correct affinity for its ligand. We have prepared yeast extracts using enzymatic digestion of the cell wall with oxalyticase followed by hypotonic shock. This has resulted in a dramatic increase in the % of receptor which binds hormone compared to previous studies which used mechanical disruption techniques. The cPRA is biologically active since it activates transcription of a co-transformed reporter gene containing its response element.(ABSTRACT TRUNCATED AT 250 WORDS)


Long-acting ACE inhibitor-induced angioedema. Angioedema is a known adverse reaction of the angiotensin converting enzyme (ACE) inhibitors. Although current investigations are in progress, the exact pathogenesis of ACE inhibitor-induced angioedema is still unclear. Most cases of angioedema we have seen remitting from the short-acting ACE inhibitor, captopril, have presented with mild angioedema, controlled with antihistamines and glucocorticosteroids. In contrast, the angioedema induced by the long-acting ACE inhibitors have been serious. Our five patients developed angioedema from either lisinopril or enalapril. Three of five patients presented with severe angioedema requiring intubation, and one of these patients had a tracheostomy. One patient with a history of mild angioedema secondary to captopril subsequently required intubation for his enalapril-induced angioedema.


Nonrandom orientation of transposon Tn5supF insertions in phage lambda. Transposition of mini-transposon Tn5supF to phage lambda can be selected in two ways: (i) by plaque formation on a dnaB amber strain of Escherichia coli, which requires expression of the transposon-borne suppressor tRNA gene (supF) during lytic phage growth, or (ii) by lysogenization of a strain with amber mutations in tet and amp resistance genes, and selection of TcR ApR (Sup+) transductant colonies. Tn5supF insertions in several lambda clones were isolated and mapped using a polymerase chain reaction (PCR) amplification method. Among insertions selected during lytic growth, more than 90% were oriented such that supF could be transcribed from an upstream lambda promoter. In contrast, half of those selected by transduction were in each orientation. These results indicate that Tn5supF insertion occurs with equal frequency in each orientation. However, Tn5supF insertion phages in which transcription from the lambda and supF promoters would collide tend to be lost when supF is selected during lytic growth. The tendency to recover Tn5supF insertions in only one orientation is useful in a transposon- and crossover-PCR-based method for preparing templates for DNA sequencing.


The Tn10-encoded tetR mRNA has heterogeneous 5' ends in vivo and in vitro. The 5' ends of the Tn10-encoded mRNAs have been analyzed by quantitative primer extensions of the in vivo synthesized RNA and in vitro run-off transcription. The gene is transcribed by a tandem promoter system consisting of PR1 and PR2. While PR1 contributes about 25% of the total PR activity in vitro, more than 95% of the in vivo-produced transcripts originate from PR2. Transcription from PR2 shows extensive heterogeneity at the 5' ends of the mRNAs in vitro and most likely also in vivo. A deletional and oligodeoxyribonucleotide-directed mutational analysis of the PR2 transcript reveals that length heterogeneity results from reiterative copying of a run of five A's at the transcription start point. In vivo transcription leads to longer reiteration products than in vitro transcription.


Post-transplant lymphoproliferative disorders: a morphologic, phenotypic and genotypic spectrum of disease. The post-transplant lymphoproliferative disorders represent a spectrum of life-threatening, generally Epstein-Barr virus-associated lymphoid proliferations which occur in the setting of exogenous immunosuppression following organ transplantation. Histopathological, phenotypic, genotypic and Epstein-Barr virus studies have revealed a broad range of abnormalities. At one end of the spectrum, they are polymorphic polyclonal proliferations with many features of a florid viral infection and, at the other end, they are monomorphic and monoclonal which would fulfill the criteria for a conventional non-Hodgkin's lymphoma, usually of B-cell type. Some patients have both polyclonal and monoclonal lesions and some have two or more monoclonal populations. Finally, because of the varied appearances, different classification schemes have been developed with reported prognostic implications.


Epstein-Barr virus latent and replicative gene expression in oral hairy leukoplakia. Oral hairy leukoplakia is an epithelial lesion of the tongue associated with productive infection by Epstein-Barr virus (EBV). However, no data concerning the pattern of EBV latent gene expression have been reported, and it remains unresolved whether true latent infection occurs in basal cell layers of oral hairy leukoplakia. We have studied six cases of oral hairy leukoplakia using monoclonal antibody immunohistology for EBV latent--EB nuclear antigen (EBNA) 1, EBNA 2 and latent membrane protein 1 (LMP 1); immediate-early (BZLF1); and replicative (EA, VCA, MA) proteins, and for the EBV-receptor (CD21 antigen). EBV DNA was demonstrated by nucleic acid in situ hybridization. Mid- to upper-zone keratinocytes contained EBV DNA and co-expressed EBNA 1, EBNA 2 (5 of 6 cases), LMP 1, BZLF1 protein, EA, VCA and MA. No EBV genome or gene expression could be demonstrated in basal or parabasal cells. Spinous keratinocytes were labelled by anti-CD21 antibodies HB5 and B2, but did not express the EBV-receptor as defined by reactivity with OKB7. The co-expression of latent and replicative infection-associated antigens is striking, indicating possible functional roles for latent proteins during the productive cycle. Our results suggest that oral hairy leukoplakia is caused by repeated direct infection of upper epithelial cells with virus from saliva or adjacent replicatively infected cells, rather than by a latent EBV infection of basal epithelial cells with a differentiation-dependent switch to productive infection as previously proposed.


Teratoid Wilms' tumour: a report of two cases. We report two cases of Wilms' tumour in which the degree of heterologous differentiation was sufficient to warrant the diagnosis of teratoid Wilms' tumour. The first description of neural elements in this Wilms' variant and the first cytogenetic analysis of teratoid Wilms' tumour are presented.


Activation of endothelin receptors by sarafotoxin regulates Ca2+ homeostasis in cerebellar astrocytes. We carried out experiments designed to investigate the effects of sarafotoxin-6B (SFTx) on [Ca2+]i in cerebellar astrocytes using the Ca2+ indicator fura-2. Both endothelin-1 and sarafotoxin-6B increased [Ca2+]i in individual cerebellar astrocytes in cell culture. The shape of the response was variable but usually consisted of an initial peak of [Ca2+]i followed by an extended plateau increase in [Ca2+]i. In Ca(2+)-free medium only the initial peak was observed. If Ca2+ was subsequently readmitted to the external medium a plateau was now formed. When external Ca2+ was removed during a plateau, [Ca2+]i rapidly declined; replacing the external Ca2+ reversed this decline. The plateau was also reversibly reduced by addition of Ni2+ (5 mM) to the external medium. Addition of 50 mM K+ produced a small increase in [Ca2+]i in most cells. This response was blocked by nimodipine. However, nimodipine only slightly blocked the plateau increase in [Ca2+]i that was formed following activation of endothelin receptors. Furthermore, perfusion of cells with 50 mM K+ during the plateau portion of a response to SFTx reduced [Ca2+]i. In some cells addition of a phorbol ester produced a sustained increase in [Ca2+]i that was blocked by nimodipine. In conclusion, activation of endothelin receptors by SFTx in cerebellar astrocytes produces both Ca2+ mobilization and Ca2+ influx. The pathway for Ca2+ influx is predominantly a non-voltage-dependent one, although some entry through a dihydropyridine-sensitive pathway also appears to occur. Furthermore, activation of protein kinase C in cerebellar astrocytes activates voltage-sensitive Ca2+ channels.


Serum immunoglobulin G antibody to Porphyromonas gingivalis in rapidly progressive periodontitis: titer, avidity, and subclass distribution. Porphyromonas gingivalis is a suspected pathogen in rapidly progressive periodontitis (RPP). We have determined the anti-P. gingivalis serum immunoglobulin G (IgG) isotype response and avidity and the subclass titer distributions for 30 RPP patients and 30 age-, sex-, and race-matched healthy subjects by using enzyme-linked immunosorbent assay technology. Patients and control subjects were classified as seropositive if their total IgG response to P. gingivalis was twofold or more than the median response in healthy subjects. The predominant antibody responses for both patients and healthy subjects were IgG2 and IgG3, with a subclass order of IgG2 greater than IgG3 greater than IgG1 greater than IgG4. The avidity of the IgG response was highest for the seropositive healthy subjects and was no different between seronegative and seropositive RPP patients. The subclass antibody responses did not depend on gender, and there were no correlations between titer, avidity, or subclass with disease severity in the RPP patients as measured by pocket depth or bone loss on dental X rays. The seronegative RPP patients exhibited antibody responses that were greater than the responses of seronegative healthy subjects for all four subclasses, while the seropositive RPP patients had higher IgG1 and IgG4 levels than seropositive healthy subjects. These findings are consistent with the hypothesis that both carbohydrate and protein antigens are important in the IgG response to P. gingivalis. The relative predominance of IgG2, a subclass which lacks strong complement fixation and opsonic properties, and the low avidity of patient anti-P. gingivalis IgG antibodies suggest that humoral responsiveness to infection with P. gingivalis may be ineffective in clearing this organism.


Heat shock treatment of macrophages causes increased release of superoxide anion. Heat shock treatment of murine macrophages and the J774 cell line resulted in an enhanced capacity to release superoxide anion (O2-) upon stimulation. There was no concomitant increase in hydrogen peroxide production, and the macrophage microbicidal activity against Mycobacterium tuberculosis, Mycobacterium avium complex, and Staphylococcus aureus was not altered.


Altered diacylglycerol level and metabolism in neutrophils from patients with localized juvenile periodontitis. Diacylglycerol, a physiological activator of protein kinase C, was elevated nearly twofold in unstimulated peripheral blood neutrophils from patients with localized juvenile periodontitis compared with cells from normal individuals. These cells also showed an enhanced and prolonged elevation of diglyceride in response to N-formylmethionylleucylphenylalanine. The metabolism of a cell-permeant diacylglycerol by diglyceride kinase was significantly decreased, because of a fivefold or higher elevation in the apparent Km of cellular diglyceride kinase.


In vivo administration of recombinant growth hormone or gamma interferon activities macrophages: enhanced resistance to experimental Salmonella typhimurium infection is correlated with generation of reactive oxygen intermediates. Purified and recombinant forms of growth hormone (GH) as well as of recombinant rat gamma interferon (IFN-gamma) enhance the survival of rats deprived of endogenous pituitary GH secretion by hypophysectomy (HX rats) and infected with virulent Salmonella typhimurium. Macrophages obtained from rats with intact pituitaries (pituitary-intact rats) or HX rats that were treated in vivo with either GH or the closely related hormone prolactin released elevated (P less than 0.05) levels of superoxide anion (O2-) after in vitro opsonized-zymosan stimulation compared with those from placebo-treated animals. These levels of O2- release were similar in magnitude to those of macrophages from rats treated in vivo with IFN-gamma. In time course in vivo macrophage activation studies, both IFN-gamma and GH significantly increased O2- secretion within 24 h, with maximal secretion occurring at day 3. Macrophages obtained from pituitary-intact and HX rats injected in vivo with GH also released elevated (P less than 0.05) levels of hydrogen peroxide (H2O2) and displayed enhanced (P less than 0.01) phagocytic activity toward opsonized Listeria monocytogenes in vitro. The mechanism of action of GH in vivo is likely to be a direct one because resident peritoneal macrophages from rats could be primed in vitro for enhanced secretion of O2- following triggering of these cells with opsonized zymosan. These data show that in vivo administration of two closely related pituitary hormones, GH and prolactin, can effectively prime macrophages, which is consistent with the hypothesis that GH mediates resistance to S. typhimurium by a direct stimulatory action on macrophages.


Leukotriene B4 omega-oxidation by human polymorphonuclear leukocytes is inhibited by pyocyanin, a phenazine derivative produced by Pseudomonas aeruginosa. Human polymorphonuclear leukocytes (PMNL) metabolize the potent chemotaxin leukotriene B4 (LTB4) by omega-oxidation to 20-hydroxyl-LTB4 and 20-carboxy-LTB4. The ability of unstimulated human PMNL to metabolize exogenous LTB4 was found to be inhibited by pyocyanin, a phenazine derivative produced by Pseudomonas aeruginosa, in a dose-dependent manner. 1-Hydroxyphenazine (1-OHP), a metabolite of pyocyanin, was not inhibitory under identical conditions. The initial enzymic step in the conversion of LTB4 is catalyzed by an NADPH-dependent cytochrome, P-450. Reduction of the phenazine derivatives by NADPH was measured spectrophotometrically. Pyocyanin was reduced by NADPH in vitro in a pH-dependent manner, while 1-OHP was poorly or negligibly reduced under similar conditions. Formation of NADP+ was 20.3 +/- 1.8 nmol min-1 for pyocyanin (10 microM) at pH 5.5, compared with 0.6 +/- 0.2 nmol min-1 for 1-OHP (10 microM), while at pH 7.5 a value of 2.2 +/- 1.3 nmol min-1 was obtained for pyocyanin, with no detectable activity for 1-OHP. This indicates that inhibition of LTB4 omega-hydroxylase activity by pyocyanin might be achieved by competition for NADPH. Incorporation of exogenous 5-hydroxyeicosatetraenoic acid by PMNL into lipid pools was not affected by either phenazine derivative. The ability of bacterial pyocyanin to limit the omega-oxidation of LTB4 may have important implications for PMNL LTB4 receptor status and chemotaxis in vivo.


Four-year follow-up of effects of toluene diisocyanate exposure on the respiratory system in polyurethane foam manufacturing workers. I. Study design and results of the first cross-sectional observation. A 4-year cohort study was designed to assess the exposure-effect relationship of working in polyurethane foam (PF) manufacturing factories with exposure to toluene diisocyanate (TDI) and its effects on the respiratory system. This paper describes the results of the first cross-sectional observations. The study population included 90 male workers who had been working in PF factories for 0.5-25 years (mean 13.3 years) (PF workers) and 44 reference workers in the same factories. The mean exposure concentration of TDI calculated from 129 personal samples was 3.2 ppb. Peak exposure excursions above 20 ppb occurred in 16 of 129 samples. Pulmonary function and its change during the working day as assessed by examining the forced expiratory flow-volume curve, respiratory impedance, and airway resistance and specific airway conductance were not different in the PF workers from those in the reference workers. Chest X-radiographs did not show any noteworthy radiological changes. Prevalences of "phlegm in winter," "nasal stuffiness or discharge in winter," and "irritation of eye and throat mucous membranes" were significantly higher in the PF workers. The findings indicate that TDI exposure at levels around 3 ppb may not adversely affect the pulmonary function over many years of exposure of those who are not hypersensitive to TDI. The causal chemicals inducing some respiratory and irritative symptoms could not be specifically identified since the PF workers were exposed not only to TDI but also to other irritative agents in the PF manufacturing processes.


Four-year follow-up of effects of toluene diisocyanate exposure on the respiratory system in polyurethane foam manufacturing workers. II. Four-year changes in the effects on the respiratory system. Fifty-seven polyurethane foam manufacturing workers (PF workers) and 24 reference workers were followed for 4 years to clarify the effects on pulmonary function of working in PF factories with exposure to toluene diisocyanate (TDI). No significant differences in the average annual losses (AALs) of pulmonary function for 4 years were observed among the 28PF workers whose TDI exposure levels were very low (mean = 0.1 ppb, group L), the remaining 29 PF workers with mean TDI exposure of 5.7 ppb (group H), and the reference workers. However, 15 PF workers in group H who had experienced peak exposure excursions to 30 ppb or above with a mean concentration of 8.2 ppb showed significantly larger AALs in percentage maximal mid-expiratory flow, forced expiratory volume in 1 s ratio to vital capacity (FVC), and forced expiratory flow at 25% of FVC than expected, and significantly larger AALs in some obstructive pulmonary function indices than those of the 14 remaining PF workers in group H whose peak exposure excursion levels were 3-14 ppb with a mean time-weighted average (TWA) of 1.7 ppb, group L, and the reference workers. These findings suggest that the peak exposure excursion level of TDI might be important in inducing obstructive pulmonary function changes in the PF workers rather than the TWA exposure levels, though further comparative studies of the AAL in those who are exposed to different peak exposure excursion levels but the same mean exposure levels are necessary. From the standpoint of prevention, the proposition that peak exposure excursion levels exceeding 20 ppb should be avoided is reasonable.


Arachidonate 5-lipoxygenase metabolism in human neutrophils from patients with asthma: in vitro effect of nedocromil sodium. Among the cells which participate in amplification of the local inflammatory reaction in asthma, neutrophils (PMN) are pro-inflammatory cells that can generate inflammatory mediators and arachidonic acid derivatives in particular. In asthmatic patients (AP) with attacks, the capacity of blood PMN to produce 5-lipoxygenase metabolites was investigated and compared to the response in healthy subjects (HS). PMN from 6 AP and from 6 HS were stimulated by calcium ionophore A23187 and arachidonate 5-lipoxygenase metabolites were analyzed by reverse-phase HPLC. LTB4, 6-trans LTB4, omega OH-LTB4 and 5-HETE were identified. In AP, total LTB4 synthesis was enhanced as compared to synthesis with PMN in HS. But the total 5-HETE synthesis by PMN from AP was decreased. Thus, the inflammatory potential of PMN from AP was enhanced in comparison to HS. The anti-inflammatory effect of nedocromil sodium (NS) was studied in the 5-lipoxygenase metabolism of arachidonic acid. NS (10(-4) mol/l) inhibited total LTB4 synthesized by PMN in AP but not in HS. We conclude that NS affects leukotriene synthesis only in cells with enhanced inflammatory potential.


Specific removal of transthyretin from plasma of patients with familial amyloidotic polyneuropathy: optimization of an immunoadsorption procedure. Familial amyloidotic polyneuropathy is characterized by the presence in patients plasma of a genetic variant of transthyretin. No specific treatment has been found and extracorporeal immunoadsorption on immobilized anti-transthyretin antibodies appears as a potentially attractive procedure. Parameters involved in specific immunoadsorption of transthyretin were studied and optimized. Several monoclonal anti-TTR antibodies were compared as affinity ligands and one of them was found to be suitable for such purposes. Optimum quantities of antibodies to be immobilized on the gel were determined. Three desorption agents were tested for regenerating immunoadsorbents and best results were obtained with basic variation of pH, allowing total desorption of TTR and possibility of multiple use without loss of adsorption capacity. Simulation of an immunoadsorption procedure in well-defined conditions showed efficiency and specificity of adsorption to remove TTR and the system thus should be subjected to clinical trials.


Status of oxidative stress and antioxidant defences during Plasmodium knowlesi infection and chloroquine treatment in Macaca mulatta. Plasmodium knowlesi (a simian malarial parasite) infection resulted in elevation of hepatic oxidative stress in monkeys. Further, the antioxidant defence system of the host was also noticeably affected. The infected monkeys showed a marked increase in the levels of superoxide (O2-), lipid peroxidation (LPO), glutathione (GSH) and xanthine oxidase (XO), and decreased levels of superoxide dismutase (SOD) and catalase. Oral administration of chloroquine (20 mg kg body wt-1 for 3 days) to infected monkeys caused recovery trends in oxidative stress and antioxidant defences to almost normal a week after cessation of drug treatment.


Dietary modulators of lipid metabolism in the Indian diet-heart study (I.D.H.S.). Of the 621 adults (25 to 65 years of age, 531 males) with either risk factors or with coronary heart disease (CHD) 310 subjects were given a cardiovasoprotective (CVP) diet (group A) and 311 subjects a normal diet (group B) in a randomized, single blind and controlled fashion. Risk factors and incidence of CHD were comparable between the two groups. The intervention group received a significantly higher percentage of calories in relation to complex carbohydrates, vegetable proteins, polyunsaturated fatty acids and high P:S ratio diet as compared to the control group. The control group received higher saturated fat and cholesterol. Compliance was assessed by dietary questionnaire during the follow-up. After 8 weeks of dietary trial, there was a significant decrease in mean serum total cholesterol (8.2 vs 2.1%), low density lipoprotein (LDL) cholesterol (9.8 vs. 2.7%) and triglyceride (11.2 vs 5.8%) in the intervention group compared to baseline levels and changes in control subjects. Body weight and physical activity at the entry to study and during the trial were similar in both groups. The decrease in mean HDL cholesterol were insignificant both in the intervention (4.3%) and control group (5.0%). There were no adverse effects of diet during the 8 weeks of trial. It is possible that a diet with 27.5% energy from total fat including 10.1% energy from monounsaturated fatty acids, P:S ratio 1.38, 120 mg dietary cholesterol, 26.0 g dietary fibre per 1000 kcal would modulate the lipid metabolism resulting in a significant reduction in serum total cholesterol, LDL cholesterol and triglyceride with no reduction in HDL cholesterol. This diet may be capable of reducing CHD incidence and mortality in the long term Indian diet-heart study (IDHS).


Effect of feeding various levels of sodium zeolite A on milk yield, milk composition and blood profiles in thermally stressed Holstein cows. Mid-lactation Holstein cows (n = 48) were equally and randomly assigned to one of four feeding treatments of sodium zeolite-A (SZA). SZA was mixed in a grain mixture (50:50 grain to forage ratio) of 0% (control), 0.5%, 1.0% and 1.5% SZA on a dry matter intake basis. Cows were fed alfalfa hay in the first phase and corn silage in the second phase of the study as roughage sources. Milk samples were taken three times weekly (am and pm) and analyzed for milk fat, protein and lactose with blood profiles conducted from samples collected weekly. SZA significantly (P less than .05) increased feed intake at all three levels for both diets. Milk yield was significantly (P less than .05) greater in the alfalfa diet. However, milk fat percent and percent protein were greater (P less than .05) in the corn silage diet. The addition of SZA to the corn silage diet increased (P less than .05) milk fat percent at the 1.0% level and milk protein at the 1.5% level. Calcium in milk was significantly (P less than .01) increased and respiration rates significantly lowered (P less than .05) in both diets at the 1.0% level. Serum calcium was higher (P less than .05) at the 1.0 and 1.5% level in the hay diet and the 1.5% level in the corn silage diet. Also, serum glucose and alkaline phosphate levels were significantly (P less than .05) higher in the corn silage diet.


Prognostic variables in malignant epithelial tumors of the parotid. A retrospective analysis was performed on 209 patients with carcinoma of the parotid who were treated with curative intent using surgery and post-operative irradiation. The five and ten-year actuarial survival were 71% and 65%. Failure at the primary site occurred in 24%, at nodal sites in 14%, and distantly in 23%. Multivariate analysis of prognostic variables identified age greater than 60 years, involved nodes, post-surgical residual disease, and poor differentiation to be bad prognostic variables.


Radiosensitivity testing of human primary brain tumor specimens. The inherent radiosensitivity of early passage cells derived from 22 patients with tumors of glial origin has been determined using a clonogenic assay system. The mean (+/- SD) surviving fraction at 2 Gy was 0.37 +/- 0.22 (range = 0.02-0.87). No correlation between inherent radiosensitivity and tumor cell plating efficiency or intracellular glutathione was observed. Tumor cells that were both resistant to nitrosoureas and expressed the Mer+ phenotype did not differ significantly in their radiosensitivity as compared to cells that were repair deficient (Mer-) and sensitive to nitrosoureas. Initial clinical follow-up suggests that factors in addition to inherent tumor cell radiosensitivity, such as performance status and age, continue to be the most important determinants of the response of patients with primary brain tumors to radiotherapy.


Existence of phosphoinositide-specific phospholipase C in rat liver nuclei and its change during liver regeneration. We found phosphoinositide-specific phospholipase C (PtdIns-PLC) activity in nuclei isolated from rat liver. The enzyme hydrolyzed phosphatidylinositol, phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate in a Ca(2+)-dependent manner, and produced inositol mono-, bis-, and triphosphate, respectively. Neither phosphatidylcholine, phosphatidylethanolamine, nor phosphatidylserine was utilized as a substrate. After partial hepatectomy, the PtdIns-PLC activity in isolated nuclei increased transiently in the S phase (20-22 h post-hepatectomy), to 2.5-fold higher than in the control, when measured with PIP. This result suggests a close relationship between the nuclear PtdIns-PLC, especially its PIP-hydrolyzing activity, and cell proliferation.


Oncogenes.

Oncogenes. We have discussed oncogenes and their protein products which act at various sites in tumor cells, with special consideration for tumors of the head and neck. In a few systems studied, application of this knowledge has led to preliminary therapeutic interventions. While abnormal expression and function of these genes and proteins are responsible for transformation to the malignant phenotype, further study of these oncogenes will also help illuminate the mechanisms of normal cellular growth and differentiation. Although cancers of the upper aerodigestive tract are heterogeneous in origin and the multi-step process of carcinogenesis is likely to vary in different tumors, the head and neck tumor spectrum is a good model for tumorigenesis and should provide valuable insight into general carcinogenesis and normal cellular growth controls.


Three-dimensional structure of ferricytochrome c' from Rhodospirillum rubrum at 2.8 A resolution. The structure of ferricytochrome c' extracted from Rhodospirillum rubrum has been determined by the X-ray crystallographic method. Crystals in hexagonal space group P6(1), with unit-cell dimensions a = b = 51.72 A and c = 155.49 A, contain one dimer molecule composed of chemically identical polypeptide chains (monomer I and monomer II) per asymmetric unit. An electron density map has been calculated at a resolution of 2.8 A by the multiple isomorphous replacement method using four-circle diffractometer data from native crystals and two heavy-atom derivatives. The quality of the map was improved by averaging the electron density about the non-crystallographic 2-fold axis relating the two monomers. The initial three-dimensional model of monomer I was built on a computer graphics system and that of monomer II was derived from monomer I using the non-crystallographic symmetry matrices. The dimer structure has been refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The current model includes 244 amino acid residues (122 x 2) and 2 hemes, with a root-mean-square deviation in bond lengths from ideal values of 0.022 A. The current crystallographic R-factor is 23.3% for 4,481 independent reflections [magnitude of Fo greater than or equal to sigma (F)] between 5.0 and 2.8 A resolution. The monomer molecule is structurally organized as an array of four nearly parallel alpha-helices which construct a left-twisted bundle. One end of the bundle, in which a covalently bound protoheme IX prosthetic group is incorporated, is more divergent than the other.(ABSTRACT TRUNCATED AT 250 WORDS)


Tissue distribution of hepatocyte growth factor receptor and its exclusive down-regulation in a regenerating organ after injury. Using 125I-labeled hepatocyte growth factor (HGF) as a ligand, we examined the tissue distribution of the HGF receptor in adult rats. Specific binding of 125I-HGF was detected in the plasma membranes of liver, spleen, kidney, lung, adrenal gland, pituitary, and thyroid. Scatchard analysis of HGF binding in liver, spleen, kidney, lung, and adrenal gland revealed the presence of a single class of high affinity receptor with a dissociation constant (Kd) of 20-30 pM. The maximum number of binding sites (Bmax) was determined to be 400-3,000 sites per ng of plasma membrane protein, the highest number being in the liver. Such a wide distribution of a high affinity HGF receptor indicates that HGF may be a multifunctional growth factor, targeting to a variety of organs, and not restricted to liver. After 70% partial hepatectomy, specific binding of 125I-HGF to membranes of the residual liver rapidly decreased, but there was no change in the kidney, lung, and spleen. On the other hand, after unilateral nephrectomy rapid down-regulation of the HGF receptor was clearly evident in the remaining kidney, but not in other organs including the liver. These findings suggest the presence of control mechanisms governing HGF receptor function only in a regenerating organ after injury.


Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins. The superoxide-generating NADPH oxidase system in phagocytes consists of at least membrane-associated cytochrome b558 and three cytosolic components named SOCI/NCF-3/sigma 1/C1, SOCII/NCF-1/p47-phox, and SO-CIII/NCF-2/p67-phox. p47-phox and p67-phox were isolated, and their primary structures were determined, but SOCI has not been well characterized. In the present study, we first purified SOCI to homogeneity from the cytosol fraction of the differentiated HL-60 cells. The purified SOCI was a small GTP-binding protein (G protein) with a M(r) of about 22,000. The guanosine 5'-(3-O-thio)triphosphate-bound form, but not the GDP-bound form, of this small G protein showed the SOCI activity. The partial amino acid sequence of SOCI thus far determined was identical to the amino acid sequence deduced from the cDNA encoding rac2 p21. None of the purified small G proteins, including Ki-ras p21, smg p21B/rap1B p21, rhoA p21, and rac1 p21, showed the SOCI activity. These results indicate that SOCI is a small G protein very similar, if not identical, to rac2 p21. The GDP/GTP exchange reaction of SOCI was stimulated and inhibited by stimulatory and inhibitory GDP/GTP exchange proteins for small G proteins, named smg GDS and rho GDI, respectively. The NADPH oxidase activity was also stimulated and inhibited by smg GDS and rho GDI, respectively. These results indicate that the superoxide-generating NADPH oxidase system is regulated by both smg GDS and rho GDI through rac2 p21 or the rac2-related small G protein in phagocytes.


Molecular cloning, sequencing, and physiological characterization of the qox operon from Bacillus subtilis encoding the aa3-600 quinol oxidase. Bacillus subtilis contains two aa3-type terminal oxidases (caa3-605 and aa3-600) catalyzing cytochrome c and quinol oxidation, respectively, with the concomitant reduction of O2 to H2O (Lauraeus, M., Haltia, T., Saraste, M., and Wikström, M. (1991) Eur. J. Biochem. 197, 699-705). Previous studies characterized only the structural genes of caa3-605 oxidase. We isolated the genes coding for the four subunits of a B. subtilis terminal oxidase from a genomic DNA library. These genes, named qoxA to qoxD, are organized in an operon. Examination of the deduced amino acid sequence of Qox subunits showed that this oxidase is structurally related to the large family of mitochondrial-type aa3 terminal oxidases. In particular, the amino acid sequences are very similar to those of subunits of Escherichia coli bo quinol oxidase and B. subtilis caa3-605 cytochrome c oxidase. We produced, by in vitro mutagenesis, a mutation in the qox operon. From the phenotype of the mutant strain devoid of Qox protein, the study of expression of the qox operon in different growth conditions, and the analysis of the deduced amino acid sequence of the subunits, we concluded that Qox protein and aa3-600 quinol oxidase are the same protein. Although several terminal oxidases are found in B. subtilis, Qox oxidase (aa3-600) is predominant during the vegetative growth and its absence leads to important alterations of the phenotype of B. subtilis.


The dioxygen cycle. Spectral, kinetic, and thermodynamic characteristics of ferryl and peroxy intermediates observed by reversal of the cytochrome oxidase reaction. The catalytic mechanism of O2 reduction by cytochrome oxidase was studied in isolated mitochondria and mitoplasts by partial reversal of the reaction. At a high redox potential (Eh) of cytochrome c, high pH, and a high electrochemical proton gradient (delta mu H+) across the inner mitochondrial membrane, the initial ferriccupric state (O) of the oxidized enzyme's bimetallic oxygen reaction center is converted to ferryl (F) and peroxy (P) intermediates, the optical spectroscopic properties of which are reported in detail. This is associated with reversed electron transfer from the bimetallic center to ferricytochrome c. The kinetics of reduction of ferricytochrome c by the reversed electron transfer process are compared with the kinetics of formation of F and P. The results are consistent with transfer of one electron from the ferric-cupric bimetallic center (O) to cytochrome c, yielding the F intermediate, followed by transfer of one electron from the latter to cytochrome c, yielding the P state. In the absence of an effective redox buffer, poising cytochrome c highly oxidized, these primary events are immediately followed by reoxidation of cytochrome c, which is ascribed to forward electron transfer to enzyme molecules still in the O state. This forward reaction also results in accumulation of the P intermediate. Kinetic stimulations of the data predict equilibrium constants for the reversed electron transfer steps, and Em,7 values of approximately 1.1 and 1.2 V may be calculated for the F/O and P/F redox couples, respectively, at delta mu H+ and delta psi equal to zero. Taken together with previously measured Em,7 values, these data indicate that it is the two-electron reduction of bound dioxygen to bound peroxide that is responsible for the irreversibility of the catalytic dioxygen cycle of cell respiration.


HeLa cells contain a 2'-phosphate-specific phosphotransferase similar to a yeast enzyme implicated in tRNA splicing. We have previously shown that HeLa cells contain activities implicated in tRNA splicing in yeast, a ligase capable of joining tRNA half-molecules and an NAD-dependent activity capable of removing the 2'-phosphate created at the splice junction by the ligase (Zillmann, M., Gorovsky, M.A., and Phizicky, E.M. (1991) Mol. Cell. Biol. 11, 5410-5416). We show here that removal of the splice junction 2'-phosphate is, as in yeast, a 2'-phosphate-specific phosphotransfer reaction that produces the same, as yet unidentified, small molecule. This enzyme is highly specific for oligomeric substrates having internal 2'-phosphates. Oligomers bearing terminal 2'-phosphates are at least 50-fold less reactive and those bearing 5'- or 3'-terminal phosphates are at least 600-fold less reactive. The requirement for an internal 2'-phosphate can be satisfied by a substrate as small as a dimer.


Modes of mononucleotide binding to ribonuclease T1. The binding of the mononucleotide inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to genetically engineered ribonuclease T1 has been investigated by conventional inhibition kinetics, fluorimetric titrations, molecular modeling, and fast relaxation techniques. The fluorimetric titrations in conjunction with molecular modeling revealed that apart from the already known primary binding site, three to four additional sites are present on the enzyme's surface. The association constants obtained from the fluorimetric titrations and the temperature jump experiments range between 3.1 x 10(6) M-1 and 4.3 x 10(6) M-1, indicating that the binding of the mononucleotides to the specific binding site of ribonuclease T1 is at least one order of magnitude tighter than has been anticipated so far. The kinetics of binding are nearly diffusion controlled with a kon determined for 2'-GMP and 3'-GMP, as (5.0 +/- 0.5 x 10(9) and 6.1 +/- 0.5 x 10(9) M-1, s-1 and koff as 1.2 +/- 0.2 x 10(3) and 2.0 +/- 0.3 x 10(3) s-1, respectively. Molecular modeling studies indicate that all three nucleotides are able to bind via their phosphate group to a positively charged array of surface amino acids including His27, His40, Lys41, and most probably Lys25 without obvious stereochemical hindrance. We propose that RNA wraps around RNase T1 in a similar fashion via phosphate binding when enzymatic hydrolysis occurs.


A microsomal endopeptidase from liver with substrate specificity for processing proproteins such as the vitamin K-dependent proteins of plasma. The microsomal fraction of rabbit liver contains an endopeptidase that cleaves synthetic peptides that mimic the amino acid sequences of the processing sites of many proproteins, including the vitamin K-dependent proteins. The endopeptidase (M(r) 69,000) was extracted from liver microsomes with 1% Lubrol and purified about 2,700-fold. The substrate employed for isolation and characterization of the enzyme was the decapeptide acetyl-Ala-Arg-Val-Arg-Arg-Ala-Asn-Ser-Phe-Leu (prothrombin peptide), in which hydrolysis occurred on the carboxyl side of the paired Arg-Arg residues. The purified enzyme, whose activity was enhanced 1.8-fold by 0.1 mM CoCl2, has a Km = 80 microM and Vmax = 21,000 nmol.min-1.mg-1 and a pH optimum of 8.7. Proteolytic cleavage of decapeptide substrates was dependent on an arginine residue at positions P1 and P4. The enzyme was completely inhibited by EDTA and 1,10-phenanthroline as well as by p-chloromercuriphenylsulfonic acid and Hg2+. Inhibitors of serine proteases and cysteine proteases had no effect. Based on the substrate preference, the endopeptidase appears to be a good candidate for the enzyme responsible for the precursor processing of the vitamin K-dependent proteins and a number of other proproteins that are synthesized via the secretory pathway in liver and other tissues.


Evidence for the formation of a quinone methide during the oxidation of the insect cuticular sclerotizing precursor 1,2-dehydro-N-acetyldopamine. 1,2-Dehydro-N-acetyldopamine (dehydro-NADA) is an important catecholamine derivative involved in the cross-linking of insect cuticular components during sclerotization. Since sclerotization is a vital process for the survival of insects, and is closely related to melanogenesis, it is of interest to unravel the chemical mechanisms participating in this process. The present paper reports on the mechanism by which dehydro-NADA is oxidatively activated to form reactive intermediate(s) as revealed by pulse radiolysis, electron spin resonance spectroscopy, high performance liquid chromatography, and ultraviolet-visible spectroscopic analysis. Pulse radiolytic one-electron oxidation of dehydro-NADA by N3. (k = 5.3 x 10(9) M-1 s-1) or Br2.- (k = 7.5 x 10(8) M-1 s-1) at pH6 resulted in the rapid generation of the corresponding semiquinone radical, lambda max 400 nm, epsilon = 20,700 M-1 cm-1. This semiquinone decayed to form a second transient intermediate, lambda max 485 nm, epsilon = 8000 M-1 cm-1, via a second order disproportionation process, k = 6.2 x 10(8) M-1 s-1. At pH 6 in the presence of azide, the first order decay of this second intermediate occurred over milliseconds; the rate decreases at higher pH. At pH 6 in the presence of bromide, the intermediate decayed much more slowly over seconds, k = 0.15 s-1. Under such conditions, the dependence of the first order decay constant upon parent dehydro-NADA concentration led to a second order rate constant of 8.5 x 10(2) M-1 s-1 for reaction of the intermediate with the parent, probably to form benzodioxan "dimers." (The term dimer is used for convenience; the products are strictly bisdehydrodimers of dehydro-NADA (see "Discussion" and Fig. 11)) Rate constants of 5.9 x 10(5), 4.5 x 10(5), 2.8 x 10(4) and 3.5 x 10(4) M-1 s-1 were also obtained for decay of the second intermediate in the presence of cysteine, cysteamine, o-phenylenediamine, and p-aminophenol, respectively. By comparison with the UV-visible spectroscopic properties of the two-electron oxidized species derived from dehydro-NADA and from 1,2-dehydro-N-acetyldopa methyl ester, it is concluded that the transient intermediate exhibiting absorbance at 485 nm is the quinone methide tautomer of the o-quinone of dehydro-NADA. Sclerotization of insect cuticle is discussed in the light of these findings.