Alternating chemo-radiotherapy with cisplatin and 5-fluorouracil plus bleomycin by continuous infusion for locally advanced undifferentiated carcinoma nasopharyngeal type.

More than 80% of undifferentiated carcinoma nasopharyngeal type patients with N3 disease (AJC-UICC 1987) will die with or from distant metastases within 3 years after the first symptom. From February 1986 to November 1987 30 consecutive patients with very advanced local disease were entered in a programme with chemotherapy-radiotherapy (CT-RT) alternation after a thorough work-up to eliminate the possibility of distant metastases. two cycles of cisplatin 100 mg/m2 day 1, bleomycin 15 mg intravenously day 1 and 16 mg/m2 per day by continuous infusion days 1-5; 5-fluorouracil (5-FU) 650 mg/m2 per day by continuous infusion days 1-5 4 weeks apart. This was followed by two series of high-energy radiotherapy, 35 Gy/3.5 weeks, with a third chemotherapy cycle in between. 27 men and 3 women were treated, the median age was 37 years (range 17-71) and the mean WHO performance status was 1 (range 0-3). TNM classification: 15 T4, 9 T3, 6 T2, 28 N3 and 2 N2c. 18 patients had nodes larger than 8 cm and 24 had bulky bilateral cervical nodes. Toxicity for this protocol was moderate, nausea and vomiting being the main side-effects. Results after two CT cycles were 3 complete responses (CR; 10%), 22 partial responses (PR; 73%), 2 disease stabilizations, 2 progressions, and 1 patient inevaluable. Of the 30 patients, 27 patients completed the CT-RT protocol, 2 patients died before radiotherapy and 1 refused treatment after 2 days on protocol. 25 patients were in CR 3 months after the end of radiotherapy. As of August 1991, with a median follow-up of 55 months (range 43-63), there are 17 patients alive, 2 of them with active disease and 15 are NED (2 after salvage therapy).


Differential expression of insulin-like growth factor binding proteins in human non-small cell lung cancer cell lines.

The possible expression and secretion of insulin-like growth factor binding proteins (IGFBPs) by non-small cell lung cancer (NSCLC) cell lines was investigated and compared with possible IGFBP expression by primary NSCLC tumours. Cells growing under serum-free conditions released binding proteins with apparent molecular masses of 26-43 kD when analysed by a ligand blotting method under non-reducing conditions. Additionally, northern blot analysis of total RNA from NSCLC cell lines and tumours was performed using cDNAs coding for each of IGFBP-1, IGFBP-2, and IGFBP-3. This analysis revealed expression of all three mRNAs to varying degrees by all cell lines. In contrast all primary tumours analysed expressed predominantly IGFBP-2 and IGFBP-3 and none showed any evident expression of IGFBP-1. Both NSCLC cell lines and tumours synthesise IGFBPs but the pattern of expression differs significantly between cell lines and primary tumours.


Autocrine role of insulin-like growth factor (IGF)-I in a human thyroid cancer cell line.

An established cell line (TC-cell, clone 78) derived from human thyroid papillary cancer cells was investigated for production of peptide growth factors. The cells had specific binding sites for insulin-like growth factor-I (IGF-I) and responded to this growth factor with increased proliferation. Culture medium conditioned by TC cells was found to contain insulin-like growth factor (IGF)-I and IGF-binding protein(s). Furthermore, reverse transcription-polymerase chain reaction revealed expression of IGF-I mRNA. When monoclonal antibody to IGF-I receptors (alpha IR3) was added, the growth of TC cells cultured in serum-free medium was significantly reduced. The growth rate of the cells was restored when the antibody was removed from the medium. These results strongly suggest that TC cells produce IGF-I, which is involved in the regulation of their own growth.


An animal model for oral cancer.

Human head and neck squamous cell carcinogenesis (SCC) is a common malignancy that appears to be related to continuous exposure to putative carcinogens or promoters such as tobacco and alcohol. To understand the mechanisms of the development of head and neck cancer and to test the efficiency of new therapeutic approaches, the characterization of an animal model system is necessary. The check-pouch carcinogenesis model in Syrian golden hamsters is probably the best known animal system that is most closely comparable with the development of premalignant and malignant lesions in human oral cancer. Furthermore, it is one of the most well-characterized animal system models for SCC. Our first approach to understanding the cellular and molecular changes that occur in the hamster cheek-pouch carcinogenesis process was to compare this model to the mouse-skin system, in which a number of critical events have been well characterized. We examined the sequential expression of hyperplasia, micronucleated cells, ornithine decarboxylase activity, polyamine levels, transglutaminase I activity, epidermal growth factor receptor levels, expression of several keratins, gamma-glutamyl transpeptidase, and nucleolar organizer regions. We suggest that these markers can be used to understand mechanisms of carcinogenesis and, in addition, can serve as alternative shorter end points in studies of chemoprevention. We also present preliminary molecular studies in the experimental oral model. We obtained a partial sequence of exon 2 of the Ha-ras gene and detected an A182----T transversion in codon 61 in hamster cheek-pouch SCC induced by 7,12-dimethylbenz(a)anthracene.


Influence of age on the relation between heart rate variability, left ventricular ejection fraction, frequency of ventricular extrasystoles, and sudden death after myocardial infarction.

To examine the influence of age on the prediction of sudden death after acute myocardial infarction based on heart rate variability (HRv), left ventricular ejection fraction (LVEF), and the frequency of ventricular extrasystoles. Autonomic and left ventricular function and the frequency of ventricular extrasystoles change with age but the influence of age on the prediction of sudden death from these variables has not been examined. The 477 patients who had been through an early postinfarction risk stratification protocol and followed up for a mean of 790 days were dichotomised at 60 years of age. Sudden deaths occurred with similar frequency in both age groups (12 (4.7%) of the 256 patients aged < 60 years and seven (3.2%) of the 221 older patients). Sudden death, however, accounted for 52% of all deaths in the young group but only 18.4% of all deaths in the older group (p < 0.01). An HRv index of < 20 units combined with an average of more than 10 ventricular extrasystoles an hour on Holter monitoring (VE10) had a sensitivity of 50%, a positive predictive accuracy of 33%, and a risk ratio of 18 in the young group (p < 0.001) but was not significantly predictive in older patients. The situation was similar when the combination of an LVEF < 40% with VE10 was considered. This combination had a sensitivity of 44%, positive predictive accuracy of 36.4%, and a risk ratio of 16.1 in young patients (p < 0.001), but was not significantly predictive in older patients. The combination of VE10 with either LVEF < 40% or HRv < 20 units gave a sensitivity of 75%, positive predictive accuracy of 30%, and a risk ratio of 30 in young patients (p < 0.001), but the relation between this combination and sudden death in older patients was not statistically significant. In postinfarction patients aged < 60 sudden death was a more predominant mode of death and was more reliably predicted from a depressed HRv index, an LVEF < 40%, and VE10 than in older postinfarction patients. These findings may have important implications for post-infarction risk stratification and management.


Establishment and characterization of a new murine cell line (SR-4987) derived from marrow stromal cells.

A new murine cell line designated as SR-4987 was established by treating a long-term bone marrow culture with the supernatant from Y-1 cells which actively produce viral C-particles (MuLV). The line showed a fibroblast-like morphology and its mesodermal origin was confirmed by immunocytochemical staining. Flow cytometric analysis of DNA index evidenced a tetraploid number of chromosomes whereas cell cycle analysis showed 34.8% of cells in S phase and 60.7% in G1. In vitro growth studies demonstrated a population doubling time of 14.7 h, a good plating efficiency (52.3%) and a very poor agar clonogenic capacity (0.6%). SR-4987 was tumorigenic only in syngeneic mice in which sarcomas were induced. The line produced M-CSF in the culture supernatant whereas G-CSF, IL-3 and GM-CSF were not detected. Studies are in progress to assess the production of other cytokines and to verify if same autocrine growth factor is involved in the control of SR-4987 proliferation. Our line provides a further model of stromal cells for studying the interaction between hemopoietic progenitors and their microenvironment, as well as to study factors produced by stromal cells acting as modulators of proliferation and differentiation of related cell populations.


Multiple microsurgical toe-to-hand transfer in the reconstruction of the severely mutilated hand. A series of fifty-nine cases.

The severely mutilated hand is defined as the hand which has lost opposable digit function either through: a) The amputation of all four fingers proximal to the PIP joint or b) The loss of the thumb proximal to the IP joint combined with the loss of at least three fingers proximal to the PIP joint. We have outlined guidelines for the restoration of useful function and appearance to such hands utilising a variety of microsurgical toe-to-hand transfers. Specific points regarding postoperative management have been made.


The results of conservative management of trigger finger. A series of 169 patients.

The authors present the preliminary results of a series of 230 trigger fingers in 169 patients managed conservatively by injection of 1 ml of hydrocortisone into the flexor sheath as described by Curtis. 'Good' results were obtained after a single infiltration in 73% cases, after two infiltrations in 81.6% cases and in 83% cases after three infiltrations. Factors have been studied in order to identify variables with prognostic significance. The outcome was not influenced by the severity of symptoms, by the association with other disease, by sex or by the site of pathology. Outcome was affected by the duration of symptoms. Duration exceeding one year indicates surgical release as does failure of conservative treatment.


Staged flexor tendon reconstruction in children.

The medical records of 12 children, aged 1 to 15 (mean 8.4 years) who had a total of 14 staged flexor tendon reconstructions for zone 2 flexor tendon injuries between the years of 1974 and 1989 were reviewed. One patient with one treated finger was lost to follow-up one month after Stage II surgery. The rest were followed either to failure or for a minimum of six months after Stage II surgery (range 6 to 108 months; mean 45 months). Results were graded by the percentage of total passive motion after Stage I converted to active motion after Stage II, and the final total active motion (TAM). Overall, there were seven poor, one fair, two good, and three excellent results. Mean final TAM was 140 degrees (range 90-210 degrees). Results were better in those children whose rehabilitation was supervised by a trained hand therapist (three excellent, one good, and one poor result). Complications included four infections after Stage I, and one infection and one rupture after Stage II. After treatment, these complications resulted in four poor results. Staged flexor tendon reconstruction in children in this series had a higher risk of complications and failure than the same procedure in adults reported in a previous series from this institution. Results in children may be improved by close attention to postoperative care and by patient selection based on ability to cooperate with the postoperative program.


Flexor tendon suture in zone 1 and distal zone 2 by the Mantero technique.

A series of 161 digits with flexor tendon division in zones 1 and 2 have been reviewed after management by the method described by Mantero. The concept of "urgence différée" (delayed primary suture) is still valid. In zone 2, we distinguish between division of tendons, where only profundus is divided (zone 2.1) and, where both profundus and superficialis are divided (zone 2.2). Results from zone 2.1 are comparable to results from zone 1. Overall, our results are of the order of those achieved by management according to Kleinert, but we would maintain that the Mantero technique is easier to perform and more confortable for the patient.


Severe electric injuries of the hand and forearm.

Electrical injuries of the upper limb produce major destruction of tissue mainly affecting the forearm, since the hand is usually the site of entry of current. Limb salvage, if it is to be successful, requires the rapid institution of a number of surgical procedures. Vein grafting to restore blood supply is frequently required and just as frequently requires skin flap cover following adequate debridement. The most commonly used flap is the groin flap. Despite the progression of necrosis beneath the flap for a period of up to three weeks, healing is usually successful and it is usually possible to avoid amputation. Several surgical procedures are required as a rule. The initial surgery is followed, in order, by nerve graft, tendon transfer and skin transfer following the use of tissue expanders. Results long term, with regard to function and appearance, were judged good.


A new flap based on the distal branches of the radial artery.

A flap based on the distal branches of the radial artery is raised on the dorso-lateral aspect of the distal third of the forearm. Thirty fresh cadaver dissections have established the anatomical rationale of the flap. The potential territory is supplied by a subaponeurotic plexus of vessels fed by lateral branches of the radial artery in the distal third of the forearm. This new knowledge of the anatomy has been applied to the performance of this flap in seven clinical cases. Various methods of application to hand and wrist reconstruction are described along with a discussion of the various advantages and disadvantages.


MRI of hand and wrist with a dedicated low field mini imager: preliminary report.

In this paper we describe the development and the early results of an MRI system designed specifically for imaging of the hand and wrist. The imager takes up little space, uses a small 0.1 Tesla water-cooled electro-magnet with a vertical magnetic field and a 15 cm air gap. The system is based on a PC micro-computer and an integrated image processing board. There is no need for a Faraday cage. The image resolution is less than 1 mm using a 128 x 128 matrix format for a typical slice thickness of 3 mm. It is possible to achieve a 0.2 mm per pixel spatial resolution when imaging the fingers.


Dorsal dislocation of the triquetrum: a case report.

A case of dorsal dislocation of the triquetrum associated with a dorsal dislocation of the distal radioulnar joint is reported. Open reduction and transfixation with Kirschner wires was performed. Eighteen months afterwards, the patient had marked volar intercalary segment instability deformity of the wrist joint assessed radiographically, with approximately half loss of range of motion of the wrist and grip strength.


Aluminum accumulation in serum, liver and spleen of Fe-depleted and Fe-adequate rats.

The study described here was planned to test the hypothesis that Al absorption and accumulation in the body are inversely related to Fe status. Aluminum3+ and Fe3+ have similar ionic radii and charge densities, pH-solubility relationships, and affinities for ligands, such as citrate and transferrin. Male weanling Sprague-Dawley rats were pair fed an Fe-deficient or Fe-adequate (control) diet for 2 wk. Each diet group was then randomly assigned to receive for four more weeks the Fe-deficient or adequate diet with: 1. 2% AlCl3; 2. AlCl3 + 3.5% Na citrate; or 3. No Al or citrate. Iron depletion, confirmed by measurements of hemoglobin, hematocrit, serum Fe, and Fe binding capacity, increased concentrations of serum, liver, and spleen Al in all groups fed AlCl3. However, the increase owing to Fe deficiency was significant only when Al was fed with citrate. The data suggest that Fe deficiency enhances both Al absorption and accumulation in liver and spleen.


Comparison of molecular properties of rat plasma and erythrocyte selenium-dependent glutathione peroxidase.

The molecular structure of plasma and erythrocyte selenium-dependent glutathione peroxidase (GSH-Px) was studied in rats drinking water containing [75Se]selenious acid, 1.3 mg Se/L. Substantial differences were found using three-step fractionation, including gel filtration of crude plasma and erythrocyte lysate, gel filtration of 75Se-GSH-Px treated by mercaptoethanol, and SDS-electrophoresis. Native plasma 75Se-GSH-Px, which exhibited a molecular weight (M(r)) of approx. 700,000, could be destroyed by mercaptoethanol action, resulting in disintegration of enzyme into several different 75Se-protein fragments and release of part of low-mol-wt 75Se. Native erythrocyte 75Se-GSH-Px M(r) value was found to be 113,000; two 75Se-protein fragments arose after mercaptoethanol treatment without 75Se release from the enzyme. The 75Se-subunits of 22,500 and 21,900 were isolated from plasma and erythrocyte 75Se-GSH-Px, respectively. Another minor 75Se-GSH-Px was identified in erythrocyte lysate (M(r) 214,000, subunit 22,100), which was considered to be a dimer of the above-mentioned erythrocyte enzyme. It can be assumed, based on these data, that native plasma GSH-Px, in contrast to erythrocyte enzyme, represents a high-molecular wt complex composed of several tetramers linked with S-S bonds. A certain part of selenium present in this complex is probably not selenocysteine and may be released with the mercaptoethanol treatment.


Changes in free radicals, trace elements, and neurophysiological function in rats with liver damage induced by D-galactosamine.

The changes in trace elements, free radicals, and neurophysiological function were investigated in rats with liver damage induced by D-galactosamine (GalN). The elevated results showed that all the parameters related to free radical metabolism changed after administration of GalN. Relative free radical concentration, malonaldehyde (MDA), and oxidized glutathione (GSSG) elevated, but reduced glutathione (GSH) decreased. Concurrently, zinc, copper, manganese, and selenium contents in liver were significantly reduced, whereas iron was elevated. In rats with hepatic encephalopathy (HE) owing to fulminant hepatic failure (FHF) induced by a high dosage of GalN, the latencies of VEPs were delayed. Moreover, there is a correlation between Zn content of brain and the latencies of VEPs. The results of this study suggested that lipid peroxidation by free radicals might be responsible for GalN-induced liver damage in which trace elements were involved, and that change in brain Zn might play a role in the neural inhibition of HE owing to FHF.


Protective effect of zinc on liver injury induced by D-galactosamine in rats.

The protective effects of zinc on liver injury induced by D-galactosamine (GalN) were investigated in rats in vivo and in vitro. Zinc supplementation (50 mg/kg/d) for 5 d of rats treated with GalN (1.5 g/kg, ip) could reduce their mortality rate, restore liver pathomorphological changes, maintain zinc content, inhibit the lipid peroxidation, hasten the protein synthesis, and improve liver function. In vitro, zinc supplement could abate the death of GalN-intoxicated hepatocytes, decrease malonaldehyde (MDA) content, and maintain reduced glutathione (GSH). It is concluded that zinc has protective effects on GalN-induced liver damage. Its effects may be owing to inhibition of lipid peroxidation and hastening of protein syntheses.


Inhibitory effects of some natural products on metal-induced lipid oxidation in cooked fish.

Divalent metal ions (Fe2+, Cu2+, Zn2+, Ni2+, and Mn2+) induced lipid oxidation in cooked, but not in raw fish. The extent of lipid oxidation, measured by the production of thiobarbituric acid reactive substances (TBRS), was increased with higher concentrations of iron, zinc, and nickel, but was decreased with increasing concentrations of copper and manganese. The natural products: ellagic acid, tannic acid, myricetin, and quercetin, inhibited lipid oxidation in cooked fish. The enhanced lipid oxidation caused by cupric ions (10(3) pmol/100 g fish) was also inhibited by the natural products. The degree of inhibition in copper-treated fish, however, was less than that in fish that had no added copper. The inhibition was concentration dependent. The antioxidative potency of the various natural products was independent of the type of metal ion-induced lipid oxidation. Ellagic acid was the most potent antioxidant (75.7-83.9%), followed by tannic acid (60.4-77.3%), myricetin (52.9-70.4%), and quercetin (32.6-44.2%).


ATP-dependent strontium uptake by basolateral membrane vesicles from rat renal cortex in the absence or presence of calcium.

ATP-dependent Sr2+ transport was examined in vitro using basolateral membrane (BLM) vesicles isolated from rat renal cortex to clarify the discrimination mechanisms between strontium (Sr) and calcium (Ca) in renal tubules during reabsorption. ATP-dependent Sr2+ uptake and Ca2+ uptake were observed in renal BLM vesicles and were inhibited by vanadate. Hill plots indicate similar kinetic behavior for Ca2+ and Sr2+ uptake. The apparent Km and Vmax of ATP-dependent Sr2+ uptake were both higher than those for Ca2+ uptake. ATP-dependent Sr2+ uptake by BLM vesicles diminished in the presence of 0.1 microM Ca2+ and was more markedly inhibited by 1 microM Ca2+. Hill plots of Sr2+ uptake data with and without 0.1 microM Ca2+ showed that the cooperative behavior of Sr2+ uptake was not changed by Ca2+. In the presence of 0.1 microM Ca2+, the affinity of the transport system for Sr2+ and the velocity of Sr2+ uptake in the BLM were both decreased. However, the rate of Ca2+ uptake was not diminished by Sr2+ concentrations of less than 1.6 microM. These results suggest that Ca2+ is preferentially transported in the renal cortex BLM when Ca2+ and Sr2+ are present at the same time.


Effects of maternal marginal zinc deficiency on myelin protein profiles in the suckling rat and infant rhesus monkey.

In the current study, the effects of marginal Zn deficiency on myelin protein profiles in neonatal rats and rhesus monkeys were investigated. Following mating, rats were fed a Zn-adequate diet, ad libitum (50 micrograms Zn/g; 50 Zn AL), or a marginal Zn diet (10 micrograms Zn/g) from day 0 (10 Zn d0) or day 14 (10 Zn d14) of gestation to day 20 postnatal. An additional group of dams was restricted-fed the control diet to the food intake of the 10 Zn d0 group (50 Zn RF). Day 20 pup plasma and liver Zn concentrations in the 10 Zn groups were lower than in the 50 Zn groups. In a parallel experiment, rhesus monkeys were fed a Zn-adequate ad libitum diet (100 micrograms Zn/g) or a marginal Zn diet (4 micrograms Zn/g diet; MZD) throughout gestation and lactation. Day 30 monkey infant plasma and liver Zn levels were similar in the MZD and control groups. Rat brain and monkey brain cortex weights were similar among the dietary groups. The amount of myelin recovered (mg protein/g brain) from day 20 rat pups from the 10 Zn groups was lower than that recovered from the 50 Zn rat pups. Myelin recovery from the MZD and control monkey infants was similar. When myelin protein profiles were characterized, it was found that the percentages of high-molecular-weight (HMW) proteins and Wolfgram protein were higher, whereas the percentages of small and large basic proteins were lower in myelin from the 10 Zn d0 and 50 Zn RF pups compared to the distribution in the 50 Zn AL rat pups. Results for the 10 Zn d0 and 10 Zn d14 pups were similar for all of the parameters studied. The percentage of HMW proteins was higher and that of basic protein lower in myelin from MZD monkey infants compared to the percentage of these proteins in myelin from controls. Although the interpretation of the rat data is complicated because of the anorexia associated with Zn deficiency, the observed changes in monkey myelin protein profiles provide strong evidence that maternal Zn deficiency affects myelination in the offspring.


Absorption, endogenous excretion, and balance of zinc in growing rats on diets with various sugars replacing starch.

The effect of partially replacing starch for various sugars on the apparent and true absorption, endogenous excretion, and balance of zinc was investigated in a study with growing rats. Six groups of five or six animals with an initial live weight of 39.4 +/- 2.7 g were fed diets that had the same Zn content (22 mg/kg), but differed in the sugar content: 1. Starch only (56%); 2. Glucose (15%); 3. Fructose (15%); 4. Sucrose (30%); 5. Galactose (15%); and 6. Lactose (30%). At the start of a 15-d fecal and urinary collection period, each animal was given an intramuscular injection of 380 kBq 65Zn for estimating endogenous Zn excretion by isotope dilution. The ratio of the specific activity of fecal Zn (after 12 d) to that of urinary Zn (after 9 d) was applied to reflect the ratio of endogenous to total fecal Zn collected from day 10 to 15. This ratio averaged 0.59, without significant differences among treatments. For this period, apparent and true absorption averaged 87.1 and 94.7% of Zn intake, respectively, and did not significantly differ among diets. Urinary excretion of 65Zn and of stable zinc by the galactose-fed rats was markedly higher than that by the other animals. Their Zn balance was, per unit weight gain, comparable with that of the other groups (30.7 vs 28.2 to 30.2 micrograms/g).


Aluminum transfer through milk in female rats intoxicated by aluminum chloride.

Female rats received an ip injection of aluminum chloride (10 mg Al/kg/d) during the first 12 d after parturition; this treatment led to a reduction in food intake associated with a reduction in body wt. Pups of the intoxicated dams showed a growth retardation after postnatal day 7. One day after treatment, the female rats intoxicated with aluminum had a considerably higher level of aluminum in milk than controls. The aluminum levels of plasma, liver, spleen, and kidneys were also significantly higher in treated female rats than controls. On the contrary, in the same tissues of pups from treated or not treated dams, no differences in aluminum levels were observed. No effect of aluminum treatment was detected on plasma silicon levels in dams and pups.


Elements in hair and nails of urban residents of New Delhi. CHD, hypertensive, and diabetic cases.

The concentrations of Cd, Cr, Cu, Mn, Ni, Pb, and Zn were estimated in hair and nails of urban residents of New Delhi. Particularly, hair levels of Cu and Mn in hypertensive males, Cr and Zn in hypertensive females, and Zn in CHD and diabetic females, and nail levels of Zn in CHD and hypertensive females were significantly lower than controls. Thus, it is observed that there exists some positive correlation between element levels in hair and nails and CHD, hypertension, and diabetes of these subjects.


Elements in hair and nails of residents from a village adjacent to New Delhi. Influence of place of occupation and smoking habits.

Samples of hair and nails collected from the residents of Wazirpur, a village adjacent to New Delhi, were analyzed for Cd, Cr, Cu, Mn, Ni, Pb, and Zn, and correlated with the residents' rural and urban places of occupations. It was observed that Pb and Cd hair levels of males working in rural areas were lower than in male businessmen and officers working in an urban area of New Delhi, thereby showing the different levels of elements exposure of the rural subjects. Such differences in the hair levels of elements were not observed among female subjects. The tobacco smoking habits of male and female rural subjects using hookah were associated with increased Cd levels in hair and nails.


Phylogenetically conserved amino acids of MBP and P0 from amphibian myelin.

Myelin basic protein (MBP) and P0 glycoprotein are major structural proteins of myelin. In adult frog, MBP is found in both the central and peripheral nervous systems (CNS and PNS), while P0 is found exclusively in the PNS. To assess the phylogenetic conservation of these proteins, MBP and P0 were isolated from adult bull-frog. A cyanogen bromide cleavage peptide of MBP (8-26), and the amino-terminal region (1-20) and an endoproteinase Lys-C peptide (67-79) of P0 were sequenced and compared to those of other vertebrate species. Residues that were conserved among other vertebrate species were found also to be conserved in frog: MBP--Ala18, Ser19, Thr20, Asp22; P0--Ile1, Val3, Thr5, Val13, Gly14, Ser15, Val17, Leu19, Trp72, Val73, Gly74, Lys79. These residues are located within or adjacent to regions that have been postulated to form beta strands and to be essential to the folding and function of these proteins.


Isolation and characterization by cell density adjustment of a PC12 pheochromocytoma variant with altered Ca2+ homeostasis.

Rat PC12 pheochromocytoma cells loaded with the fluorescent Ca2+ dye fluo-3 or indo-1 and scanned fluorimetrically on a cell sorter apparatus showed a rapid cell density-dependent increase in free cytosolic calcium concentration [Ca2+]i when maintained in suspension cultures. Cell adhesion, measured under a defined set of conditions, was low when cells were seeded at 1.5 x 10(4) cells/ml but reached maximal levels after addition of A23187 calcium ionophore. A six to sevenfold increase in cell density mimicked the effect of the ionophore. Densities above 2 x 10(6) cells/ml caused a decrease in cell adhesion, which was further reduced by the addition of A23187. BAPTA, AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and nifedipine (10 microM each), partially inhibited cell attachment (34% and 44% reduction), but at 0.25 microM and 1 microM, respectively, they enhanced attachment (46% and 67% increase). The data suggest that a certain permissive level of [Ca2+]i, attained by either increasing cell density or by the presence of a calcium ionophore, is sufficient for maximal cell adhesion. Above the permissive level, manipulation of [Ca2+]i either by altering cell density or by the addition of calcium blocking agents in high concentrations results in a significant reduction in cell adhesion. Based on these observations, we were able to isolate a biochemically and morphologically distinct cell population. The variant, designated PC12ds (density selected), differed substantially from the original cells. Most notable was a relatively lower content of free [Ca2+]i in the PC12ds cells, as independently assayed by using fluo-3 and indo-1 dyes. In addition, the variant cells exhibited a significantly diminished rate of 45Ca2+ uptake, most likely due to less efficient L-type voltage-dependent calcium (VDC) channels. Addition of several calcium channels agonists and antagonists suggested that PC12ds cells contained relatively more N-type VDC channels, possibly indicating a shift to a neuronal phenotype.


[Hyperamylasemia or macroamylasemia? A clinical case].

The authors report a case of macroamylasaemia and discuss diagnostic implications especially in surgical emergencies. In fact, laboratory findings of hyperamylasaemia may lead to erroneus diagnosis or even to useless surgical operations.


Marinobacter hydrocarbonoclasticus gen. nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium.

On the basis of phenotypical characteristics and analysis of 16S rRNA sequence, a new species belonging to a new genus is described, and the name Marinobacter hydrocarbonoclasticus is proposed. This organism, isolated from Mediterranean seawater near a petroleum refinery, is a gram-negative, aerobic, rod-shaped bacterium. It grows at NaCl concentrations of 0.08 to 3.5 M and uses various hydrocarbons as the sole source of carbon and energy. Its DNA has a guanine-plus-cytosine content of 52.7 mol%. The 16S rRNA analysis shows a clear affiliation between M. hydrocarbonoclasticus and the gamma group of the phylum Proteobacteria. A close phylogenetic relationship appears among the species Marinomonas vaga, Oceanospirillum linum, Halomonas elongata, and Pseudomonas aeruginosa. Because of the impossibility of finding a single most closely related species, we suggest that this bacterium be assigned to a new genus, at least temporarily. The possibility of a revision of this status when new data appear is, however, not excluded. The type strain is M. hydrocarbonoclasticus SP.17 (= ATCC 49840).


Dermatofibrosarcoma protuberans is a unique fibrohistiocytic tumour expressing CD34.

Dermatofibrosarcoma protuberans (DFSP) is a slow growing, locally invasive tumour whose differentiation from other fibrohistiocytic tumours sometimes poses serious diagnostic problems. We investigated CD34 expression immunohistologically in various fibrohistiocytic tumours including dermatofibroma, DFSP, malignant fibrous histiocytoma (MFH), infantile myofibromatosis, fibrosarcoma, hypertrophic scar and keloid. Among these, DFSP was unique in that tumour cells themselves expressed CD34, whereas in other tumours. CD34 expression was observed only on vascular endothelial cells amongst the tumour cells. Until now, there have been no reports of useful immunohistological markers for DFSP. CD34 expression by the tumour cells can be an extremely useful marker in establishing a definitive diagnosis of DFSP.


Further studies on the actions of endothelin-1 on blood flow in human skin.

When injected into human skin, endothelin-1 produces intense vasoconstriction localized to the site of the injection, but this area of vasoconstriction is surrounded by vasodilatation which spreads several centimetres from the injection site. The vasodilatation induced by intradermal injection of endothelin-1 (63 pmol) into human skin is prevented by local anaesthetic. Pretreatment of human skin with capsaicin also inhibits this response. Pretreatment of subjects with the selective histamine H1-receptor antagonist cetirizine, 10 mg orally 4 h before intradermal injections, inhibited vasodilatation caused by the intradermal injection of histamine (750 pmol), endothelin-1 (63 pmol), and carbachol (750 pmol). Endothelin-1 (0.3-10 microM) and carbachol (1-30 microM) failed to induce histamine release from rat peritoneal mast cells. We conclude that the vasodilatation caused by intradermal injection of endothelin-1 into human skin is neurogenic and is probably mediated by neuropeptide-containing primary afferent neurones. Because neither carbachol nor endothelin-1 cause histamine release from mast cells, our data suggest that histamine release from mast cells at the effector end of the axon reflex is responsible for the carbachol- and endothelin-induced vasodilatation in human skin.


Modern treatment of warts: cure rates at 3 and 6 months.

Four-hundred consecutive referrals with viral warts of the hands and/or feet were investigated to determine the cure rate from a combination of cryotherapy, keratolytic wart paint and paring. For treatment failures after 3 months, the value of continuing cryotherapy and of additional treatment with the immunomodulator inosine pranobex were assessed. Subjects were treated for 3 months with wart paint and cryotherapy and were randomized to receive, or not, paring in addition. Those who did not respond by 3 months were randomized to receive, or not, 3 months further cryotherapy, and to receive inosine pranobex 60 mg/kg/day for 1 week each month, or matching placebo. Fifty-two per cent of subjects were cured by 3 months. The chance of cure was inversely related both to the length of history and to the diameter of the largest wart. Paring improved the cure rate for plantar warts but not for hand warts. During the second 3 months the cure rate fell to 41%. Neither cryotherapy nor inosine pranobex significantly improved this response.


Compression hosiery in a surgical unit.

A small pilot study was set up to indicate the appropriateness of the assessment of patients for anti-embolus prophylaxis in a surgical unit. The results suggested some flaws in current practice, particularly in relation to assessment of risk factors and patient information procedures. Discussions with ward staff on the results have initiated changes in nursing interventions which will be evaluated on an on-going basis.


Effects of platelet activating factor (PAF) and other vasoconstrictors on a model of angiogenesis in the mouse.

The combination of sponge implant and 133Xe washout technique described in this paper provides a model to study neovascularization in mice which can be observed over several days in the same animal. The local blood flow within the ingrowing granulation tissue has been determined by measuring the washout rate of 133Xe injected into the implants. Tissue infiltration of the sponges was assessed by histological examination and by measurement of sponge wet weight, protein and glycosaminoglycans (GAG) content. The newly formed blood vessels, despite having abnormal configuration, responded to platelet activating factor (PAF) and to endothelin-1 (ET-1) similarly to the normal mature vessels in adjacent skin. However, the sponge blood vessels were more sensitive to angiotensin II than the skin blood vessels. Using this model we have also demonstrated an angiogenic activity of PAF substantiated by increased blood flow and biochemical variables in the implanted sponges.


The in vitro effect of high-dose recombinant human erythropoietin on granulocyte-macrophage colony production in premature infants using a defined serum deprived cell culture system.

Recent reports of neutropenia associated with the use of recombinant human erythropoietin (r-HuEpo) in preterm infants with the anaemia of prematurity have raised concern over the clinical use of this hormone. The present studies were undertaken to determine whether high-dose r-HuEpo has an effect on granulocyte production in vitro. The studies used a serum deprived, optimized semi-solid cell culture system to investigate the effect of lineage specific and non-specific granulocyte and erythroid colony stimulating factors on circulating peripheral blood granulocyte-macrophage colony forming units (CFU-GM), erythroid burst forming units (BFU-E) and multilineage colonies (CFU-Mix) from nine premature infants and seven healthy adults. CFU-GM were grown in the presence of interleukin 3 (IL3) 8 ng/ml, granulocyte-macrophage colony stimulating factor (GM-CSF) 20 ng/ml and granulocyte colony stimulating factor (G-CSF) 15 ng/ml alone and combinations of G-CSF with GM-CSF or IL3. The number, size and differentiation of CFU-GM colonies were then analysed in the presence and absence of high dose r-HuEpo (4 U/ml). High-dose r-HuEpo did not exert any significant modulatory effects on the number of CFU-GM colonies produced in the presence of IL3, GM-CSF and G-CSF alone or in combination. The number of cells within each CFU-GM colony did not change significantly, nor was there a significant change in the degree of differentiation. The combined number of BFU-E, CFU-GM and CFU-Mix colonies increased with r-HuEpo in both adults (1.8 x) and preterm infants (1.4 x), almost exclusively due to an increase in BFU-E derived colonies. Thus, no evidence was found for an r-HuEpo mediated redirection of multipotential haemopoietic stem cells into committed erythroid precursors at the expense of myeloid precursors.


Expression of adhesion molecules LFA-3 and N-CAM on normal and malignant human plasma cells.

Normal and malignant plasma cells were investigated for the expression of seven cellular adhesion molecules by immunofluorescence microscopy. The antigens investigated were CD2 and its ligand, LFA-3 (CD58). LFA-1 alpha (CD11a) and LFA-1 beta (CD18) and their ligand ICAM-1 (CD54), H-CAM (lymphocyte homing receptor; CD44) and N-CAM (CD56). Marrow from 18 patients with myeloma, two with plasma cell leukaemia (PCL), four with monoclonal gammopathy of uncertain significance (MGUS) and 10 normal allogeneic bone marrow donors was studied. All plasma cells from normals and multiple myeloma patients were negative for CD2, CD11a and CD18. All normal and myeloma marrow plasma cells were positive for ICAM-1. 16/18 myeloma cases tested, and all other samples (normal, MGUS and PCL), contained plasma cells positive for H-CAM. Only one normal, but 12/16 myelomas tested were positive for N-CAM (P less than 0.02). One of four MGUS cases was moderately positive and one other weakly positive for N-CAM. Both PCLs were N-CAM negative. 12/18 myelomas were positive for LFA-3, but only two normals (P less than 0.05). All MGUS cases were negative for LFA-3, as was one PCL, the other being weakly positive. Three cases were negative for both adhesion molecules, three cases expressed only N-CAM or LFA-3 and 10 cases expressed both. LFA-3 and N-CAM are expressed significantly in myeloma rather than normal plasma cells. Cases of MGUS may express N-CAM but not, in this small series, LFA-3. Plasma cells in the peripheral blood (PCL) and plasma cell lines express little or no LFA-3 or N-CAM.


Further characterization of the NB 1 antigen as a variably expressed 56-62 kD GPI-linked glycoprotein of plasma membranes and specific granules of neutrophils.

The human neutrophil-specific alloantigen NB1 was identified as a glycosyl-phosphatidylinositol (GPI)-anchored N-glycosylated protein of M(r) 56-62 kD under reducing conditions. Under non-reducing conditions its M(r) was 49-55 kD. This glycoprotein antigen was found to be expressed by only a subpopulation of normal donor neutrophils, and could not be detected on other blood cells. The allotypic epitope recognized by human anti-NB1 IgG was also recognized by the mouse monoclonal antibody 1B5. The percentage of neutrophils stained by these antibodies varied greatly among healthy donors (range 0-100%). When 16 donors were repeatedly tested, the NB1-positive neutrophil fraction appeared to remain remarkably constant over time in most donors, but significant fluctuations were seen in some. NB1 antigen was found to be expressed not only on the plasma membrane, but also intracellularly on the membranes of small vesicles and specific granules. The neutrophils which expressed NB1 antigen on the plasma membrane were the same as those with intracellular expression of this antigen. Crosslinking of NB1 antigen on the plasma membrane with monoclonal antibody 1B5 and goat-anti-mouse Ig resulted in internalization of the complex, while in-vitro stimulation of neutrophils caused an increase of the intensity of plasma membrane staining with anti-NB1, but only of those cells that were positive already prior to stimulation. The NB1 glycoprotein thus appears to identify a distinct subset of neutrophils, the size of which greatly varies among healthy donors. The function of the NB1 glycoprotein remains unclear, but its behaviour upon crosslinking and chemotactic peptide stimulation suggests a possible role as receptor molecule.


Bone marrow involvement in non-Hodgkin's lymphoma: increased diagnostic sensitivity by combination of immunocytology, cytomorphology and trephine histology.

Diagnostic results from cytomorphology and immunocytology of aspirated bone marrow (BM) were compared with the findings from standard trephine histology of 100 adult patients with non-leukaemic non-Hodgkin's lymphomas (NHL) in a retrospective study. Immunocytological investigations were performed by the immunoenzymatic APAAP-technique on BM smears monoclonal antibodies against CD19, Cd3, CD10 or TdT antigens and determination of positive cells in relation to total BM leucocytes. Corresponding results were obtained for trephine histology and for the combination of cytomorphology and immunocytology in 93/100 cases. Four cases with BM involvement by trephine histology were missed by the combination of immunocytology and cytomorphology. In turn, three cases negative by trephine histology, were found to be positive by the combination of immunocytology and cytomorphology. Immunocytochemistry considerably increased the number of true positive detected BM-infiltrations by cytomorphology in low grade B-cell lymphoma from 58% to 97%. For the diagnosis of BM involvement in high-grade NHL cytomorphology of the aspirate was of equal sensitivity to the biopsy and was always confirmed by immunocytology. The high diagnostic sensitivity of immunocytology was mainly due to high B-cell counts in BM involved by B-cell lymphoma (means = 38%, s = 23) in contrast to low B-cell counts in BM not involved by NHL (means = 4.5%, s = 3.8). We conclude from our data that immunocytology in addition to standard cytomorphology improves diagnostic sensitivity in the detection of BM involvement by NHL.


Acute myeloid leukaemia with an unusual phenotype: myeloperoxidase (+), CD13 (-), CD14 (-) and CD33 (-).

We herein describe an unusual case of acute myeloid leukaemia (AML) showing strong cytochemical reactivity for myeloperoxidase (MPO) but surprisingly no reactivity using flow cytometry for any of the lineage-specific cell surface markers, i.e. myelomonocytic antigens CD13, CD14 and CD33; or B-lymphoid antigens CD19, CD20 and immunoglobulins; or T-lymphoid antigens CD2, CD3 and CD5. The strong reactivity for MPO and the complete absence of reactivity for CD13 and CD14 was verified by an independent assay involving alkaline phosphatase-anti-alkaline phosphatase (APAAP). Our case is of interest for at least two reasons: First, a poorly differentiated variant of AML (negative for MPO but positive for one or more of the myeloid-lineage CD antigens) has been designated FAB M0. In terms of the expression of phenotypic markers, our case may be considered as an 'MPO (+), CD antigen (-) AML'. The CD antigens are known to be expressed very early during myeloid differentiation whereas MPO (in its functional form) is viewed as being expressed relatively late in the process. It is therefore intriguing from a biological standpoint why the supposedly early antigens (CD33 and CD13) remain unexpressed; this may represent an example of 'asynchronous differentiation' in leukaemia. Second, from a practical standpoint, the use of immunophenotyping as a first-line diagnosis would fail to detect such cases. This case strengthens the notion that immunophenotyping by flow cytometry does not eliminate the necessity of performing peroxidase cytochemical staining.


Identification of CD71 (transferrin receptor) expressing erythrocytes by multiparameter-flow-cytometry (MP-FCM): correlation to the quantitation of reticulocytes as determined by conventional microscopy and by MP-FCM using a RNA-staining dye.

Reticulocytes express the CD71-defined antigen, the transferrin receptor. This report describes how by means of dual-colour immunofluorescence using MP-FCM (multiparameter-flow-cytometry) CD71+ erythrocytes can be detected regularly in blood of healthy adults. Percentages of these CD71+ erythrocytes were compared to the percentages of reticulocytes as determined by conventional microscopy using brilliant cressyl blue, and to the percentages of erythrocytes with high RNA content, as detected by MP-FCM using a RNA-staining dye (thiazole-orange). Only about two-thirds of the percentages determined by the two latter methods were detected by MP-FCM using the CD71 expression for definition of reticulocytes. Studying clinical samples, however, including both specimens with very low and very high numbers of reticulocytes, almost identical percentages were determined by all the three methods described. Studying reticulocytes in vitro, a rapid decline of the expression of the transferrin receptor was observed on reticulocytes. Due to the differential expression of the transferrin receptor on reticulocytes, different subsets of reticulocytes could be identified. The dual-colour MP-FCM method described allows for the characterization and enumeration of immature erythrocytes, representing the major subset of reticulocytes as determined by conventional methods. Furthermore, it allows for subset dissection of reticulocytes.


A variant of Glanzmann's thrombasthenia which fails to express a GPIIb-IIIa related epitope that is recognized by a specific monoclonal antibody (C17).

Binding of different antibodies to the GPIIb-IIIa complex in resting (AP2, EDU3, C17) or activated platelets (PAC1) was studied by flow cytometry in a patient with a platelet defect involving GPIIb-IIIa related functions. The patient has a mild history of bleeding. Aggregation induced by ADP and collagen were absent but normal response was obtained with ristocetin. Platelets from the patient do not bind fibrinogen. Perfusion studies with flowing blood showed that patient's platelets have a marked impairment in the process of spreading and aggregate formation on vascular subendothelium. Electrophoretic studies in SDS-polyacrylamide gels demonstrated the presence of normal amounts and normal mobility of GPIIb-IIIa. Fibrinogen was present in the patient's platelets (68-74% of controls). The binding of AP2 and EDU3 to patient's resting platelets was normal as assessed by flow cytometry. In contrast, a decreased presence of the C17 antigen (10 fold lower than control platelets) was detected in resting platelets and a markedly reduced binding of PAC1 was found in thrombin activated platelets. These studies suggest that C17 recognizes an epitope of the GPIIb-IIIa in resting platelets that is implicated in the regulation of adhesive and cohesive properties of GPIIb-IIIa. Studies on this patient might be helpful for the understanding of GPIIb-IIIa functions.


Lupus-like anticoagulant properties of murine monoclonal antibodies to beta 2-glycoprotein I.

The lipid-binding inhibitor of coagulation, beta 2-glycoprotein I (beta 2GPI), has been shown to form the antigen to which some autoantibodies against anionic phospholipids (aPL) are directed. Six murine monoclonal antibodies (MAbs) of the IgG1 isotype were raised against human beta 2GPI and could be subdivided into three groups on the basis of mutual competition experiments. MAbs 9G1 and 8C3 (group A) markedly inhibited the binding of immunoglobulins from aPL-positive sera to beta 2GPI-coated wells. Using a lipid-based solid-phase radioimmunoassay, the MAbs interacted with both anionic phospholipids and phosphatidylethanolamine, but not phosphatidylcholine, in a beta 2GPI-dependent manner. A cross-reaction between beta 2GPI from several (including bovine) species was seen with one of the MAbs (9G1). All six MAbs induced dose-dependent prolongation of the DAPTT, DRVVT, KCT and TTI clotting times of human plasma, whereas 9G1 was the sole antibody to be inhibitory with plasma from bovine origin. Synergistic inhibitory effects were observed with MAbs used in pairs provided that they did not compete with each other for beta 2GPI binding. The anticoagulant activity of the MAbs was fully neutralized by the addition of freeze-thawed platelets. The MAbs described here resemble lupus anticoagulants in several respects which makes them valuable to study the involvement of beta 2GPI in the autoimmune thrombotic pathophysiology.


The rat liver cell nuclear imprint as a standard for DNA measurements.

Standards are used in DNA cytophotometry to determine the diploid reference value. Fixation and staining protocols have to be standardized because numerous sources of variation influence staining intensity of DNA. When Feulgen stained imprints of rat liver cell nuclei are used as an external DNA standard a significant difference of reference value of up to 60% between imprints, from the same liver and with all sources of variation minimized, is demonstrated. Different possible sources of intra-imprint variation were examined but its exact nature remains unknown. Due to this inter-imprint variation, the DNA-reference value from a single liver imprint is only an approximation with unknown error of the true diploid reference value. By drawing an aselective random sample of N diploid cells in M imprints the sampling distribution of TIOD (mean Total Integrated Optical Density) will have its expectation identical to the diploid reference value and is approached by TIOD (mean TIOD) with the confidence interval defined by the number, M, of imprints measured. The use of TIOD as an estimate of diploid reference value with known precision is proposed as a first approach for the definition of a standard for DNA measurements.


Treatment of poor-risk germ-cell tumors with high-dose cisplatin and etoposide combined with bleomycin.

Seventy-two patients with far advanced ('high-risk') germ-cell tumors were treated with cisplatin 40 mg/m2 i.v. and etoposide 200 mg/m2 i.v. daily for 5 days and bleomycin 15 mg/m2 i.v. once a week. At least 3 cycles of this treatment were given at three-week intervals to all patients. Seventy-five percent of the patients obtained CR and 67% are without evidence of disease after a median observation time of 47 months (range 5 to 80 months). Hematologic toxicity was severe and 10% of the patients died due to treatment-related toxicity. Neurotoxicity was a clinical problem in 58% of the patients. Glomerular filtration rate decreased significantly after 3 cycles (29% +/- 16%). No clinically significant pulmonary toxicity was observed. The specific role of high-dose cisplatin in such intensive treatment has until now been the subject of only one randomized study in which no superiority of high-dose cisplatin was found. Significant improvement of therapeutic outcome over that of today's standard treatment conceivably necessitates an even greater increase in dose intensity of the active drugs--or inclusion of new drugs.


The optimisation of carboplatin dose in carboplatin, etoposide and bleomycin combination chemotherapy for good prognosis metastatic nonseminomatous germ cell tumours of the testis.

An analysis of carboplatin dose response was performed in 121 patients with good prognosis metastatic nonseminomatous germ cell tumours (NSGCT) of the testis, referred to the Royal Marsden Hospital since 1984, who had been given combination carboplatin, etoposide and bleomycin (CEB) chemotherapy. With a median follow-up of 40 months (range: 7 to 85 months) nine patients (7%) have failed CEB. Carboplatin dose was analysed in all patients using body surface area (BSA) to derive a carboplatin dose per metre squared (mg/m2) and by calculation of a predicted serum concentration chi time (AUC: area under the curve) derived from the glomerular filtration rate (GFR), using the formula; Dose = AUC(GFR + 25). At a carboplatin dose of 400 mg/m2 or greater 2 out of 58 patients (3.4%) failed treatment while 7 out of 63 patients (11%) who received a dose less than this failed (p greater than 0.1). At an AUC of 5.0 mg.min/ml or greater, 2 out of 74 patients (2.7%) failed while 7 out of 47 patients (14.9%) who had an AUC less than this failed (p less than 0.05). There was evidence for a dose/response relationship at relatively low doses and the failure rate rose to 26% for doses less than 4.5 mg.min/ml (p less than 0.001) or 15.6% for doses less than 350 mg/m2 (p greater than 0.1). In view of the more precise determination of toxicity and efficacy it is recommended that carboplatin dose be based on the GFR.


Cachexia and cancer: a clinician's view.

The cancer-related cachexia/anorexia syndrome is not well understood. It is related to several factors like metabolic changes, tumor types, and disease extent and is frequently accompanied by decreased performance status. An important aspect of anorexia is the psychosocial problem: the patient is unable to join the family for meals precisely when he or she most needs familial support. Several randomized studies have shown that megestrol acetate, possibly in a dose-dependent fashion, can improve appetite and lead to weight gain. This effect seems to be most prevalent in patients with breast cancer and also occurs in the absence of a tumor response. We have retrospectively analyzed 176 patients with cancer types other than breast cancer who received only palliative treatment. The patients were treated with megestrol acetate (160 mg tid) because they complained of anorexia. After 10 days of treatment, megestrol acetate was continued only in those patients whose appetite and/or general well-being improved. Fifty-seven patients (32%) experienced such an improvement and asked for continuation of therapy. Many basic questions are still unanswered; nonetheless, from a practical clinical view it seems worthwhile to offer anorectic patients a chance to improve, especially since side effects of megestrol acetate are absent or mild, and the distinction between responders and nonresponders can be made by 10 days of treatment.


HN-10200 causes endothelium-independent relaxations in isolated canine arteries.

HN-10200, a nonselective inhibitor of phosphodiesterase, has positive inotropic and vasodilator activity. The present study was designed to determine the role of endothelium in causing relaxation to HN-10200 in isolated canine femoral and basilar arteries. Rings with and without endothelium were suspended for isometric tension recording in Krebs-Ringer bicarbonate solution bubbled with 94% O2, 6% CO2 (t = 37 degrees C; pH = 7.4). HN-10200 and another nonselective phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), caused similar concentration-dependent relaxations in femoral arteries with and without endothelium. In femoral arteries without endothelium, HN-10200 and IBMX significantly augmented relaxations to prostacyclin, but did not affect relaxations to a nitric oxide donor 3-morpholinosydnonimine (SIN-1) or endothelium-derived relaxing factor (EDRF) released by bradykinin. In basilar arteries, relaxations to HN-10200 were augmented by the removal of endothelium, whereas relaxations to IBMX were not affected. Relaxations to prostacyclin, SIN-1, and EDRF were not affected by the presence of phosphodiesterase inhibitors. The results of the present study suggest that HN-10200 causes endothelium-independent relaxations. In addition, it may augment relaxations to prostacyclin but does not affect relaxations to EDRF.


Is aggressive surgical palliation of proximal bile duct cancer with involvement of both main hepatic ducts worthwhile?

The only curative treatment for proximal bile duct cancer with involvement of both main hepatic ducts is liver transplantation. Most patients do not fulfill the requirements for liver transplantation. Our treatment strategy in appropriate cases is palliative tumor resection and reconstruction of the biliary passage by sutureless bilioenteric anastomosis. We have treated 12 patients, 5 in combination with intraluminal and percutaneous radiotherapy. Our results indicate that this strategy leads to effective palliation in some cases provided that only microscopic residual tumor is left in-situ. Our survival times compare favourably with survival after liver transplantation.


Two malignant peripheral primitive neuroepithelial tumor cell lines established from consecutive samples of one patient: characterization and cytogenetic analysis.

A 6-year-old girl presented with a tumor of the right shoulder involving bone, adjacent soft tissue, and regional lymph nodes. The conventional histologic diagnosis was ambiguous, initially suggesting lymphoma. After relapse on lymphoma therapy, reevaluation with additional multiple diagnostic techniques performed on the biopsy tissue and on two cell lines derived from the biopsies established the diagnosis of a primitive neuroepithelial tumor of bone and soft tissue. This was strongly supported by 1) focal rosette formation by the tumor cells and positive immunostaining for neuron-specific enolase and synaptophysin, with absent staining for leukocyte common antigen; 2) at the ultrastructural level, formation of cellular processes containing microtubules, a paucity of neurosecretory granules, absence of synaptic junctions, formation of long "intermediate" junctions between cells, and, in culture, widespread development of rosettes; 3) marked surface positivity to W 6/32 and negativity to HSAN 1.2 antibodies; and 4) elevated expression of MYC and lack of overexpression of MYCN oncogenes. Numerical and structural abnormalities were present in the karyotype, but the expected t(11;22)(q24;q12) was not present in the tumor-involved marrow or in either of the established tumor cell lines, although there was an interstitial deletion of 11q involving breakpoints in q21 and q23.


Different localization of Epstein-Barr virus genome in two subclones of the Burkitt lymphoma cell line Namalwa.

The Epstein-Barr virus genome contained in the Burkitt lymphoma line Namalwa was previously localized to the short arm of chromosome 1. Analysis of a different subline of the same Namalwa line by means of Southern analysis carried out on genomic DNA, as well as in situ hybridization, showed a localization on the X chromosome.


The translocation (1;14)(p34;q11) in human T-cell leukemia: chromosome breakage 25 kilobase pairs downstream of the TAL1 protooncogene.

Nearly 30 percent of patients with T-cell acute lymphoblastic leukemia (T-ALL) exhibit a tumor-specific rearrangement of the TAL1 gene (also called TCL5 or SCL). These rearrangements are generated by either local DNA deletion or a (1;14)(p34;q11) chromosome translocation, and they typically result in structural alterations of the TAL1 transcription unit. In this report we present a molecular characterization of the t(1;14)(p34;q11) from a T-ALL patient. As a consequence of the translocation, TAL1 is transposed from its normal position on chromosome 1 into the T-cell receptor alpha/delta chain locus on chromosome 14. Unlike previous cases, the chromosome 1 breakpoint in this patient did not disrupt the continuity of the TAL1 transcription unit, but instead occurred approximately 25 kilobase pairs (kb) downstream of TAL1. This observation suggests that malignant alteration of TAL1 can be mediated by long-range cis-activating mechanisms that are triggered by DNA rearrangement at a distant site. Genes Chrom Cancer 4:211-216 (1992).


Loss of chromosome 3 alleles and multiplication of chromosome 8 alleles in uveal melanoma.

Uveal melanoma is the most frequent primary intraocular tumor. The etiology is unknown. Using neutral DNA polymorphisms on chromosomes 2, 3, and 8, we have detected loss of chromosome 3 alleles in 8 of 13 tumors and multiplication of chromosome 8 alleles in 6 of 11 tumors. No anomalies at a locus on chromosome 2 were found in 10 of 10 tumors. These results confirm and extend previous cytogenetic findings and suggest that a tumor suppressor gene on chromosome 3 and an oncogene on chromosome 8 may be involved in the formation or progression of this tumor.


Characterization and molecular analysis of nondisjunction in 18 cases of trisomy 21 and leukemia.

We recently began a cytogenetic and molecular study of nondisjunction in leukemic Down syndrome individuals to determine whether the mechanism by which the extra chromosome 21 originates predisposes the individual to leukemia. In the present report, we summarize our observations on 18 patients with trisomy 21 and acute or transient leukemia, including 11 patients with acute lymphocytic leukemia, three with acute myeloid leukemia, one with B-cell lymphoma, one with acute megakaryoblastic leukemia, and two with transient leukemia. Results of DNA marker studies of the parental origin of the extra chromosome 21 indicated that 16 of the 18 cases (89%) were maternally derived, a percentage similar to that seen among nonleukemic Down syndrome patients. We noted that most leukemic Down syndrome patients had one locus or more in which parental heterozygosity was maintained in the trisomic individual, indicating a meiotic rather than a mitotic origin for the trisomy.


Ordering of six polymorphic DNA markers important in the delineation of 3p deletions in neoplasia.

Using fluorescence in situ hybridisation (FISH) the chromosomal location and relative order of six human chromosome 3 probes has been determined. The sensitivity of the technique has enabled the relative mapping of probes carrying inserts as small as 500 basepairs (bp), thus allowing the following proximal-distal probe order to be proposed: D3S30 (3p13-14), D3S4 (3p13-14), D3S2 (distal 3p14), D3S32 (3p21), D3S48E (3p21-23), and D3S11 (3p22-23). These data combined with the deletion mapping data of other researchers raise the possibility that the loss of more than one region of the short arm of chromosome 3 may be important in the development of small cell lung cancer.


Clonal structural chromosome aberrations in nonneoplastic cells of the skin and upper aerodigestive tract.

Cytogenetic analyses of tumors of the skin and upper aerodigestive tract have repeatedly revealed small, pseudodiploid clones characterized by balanced structural rearrangements and a high frequency of cells with nonclonal structural aberrations. However, the lack of common cytogenetic denominators within the different histologic subtypes, the discrepancy between cytogenetic findings and data obtained from flow cytometric DNA content studies, and the occasional identification of tumors with massively rearranged karyotypes indicate that the chromosome rearrangements present in pseudodiploid cells have little to do with the tumorigenesis or progression. Further support for this conclusion, and indirect evidence that the pseudolipid clones probably do not represent the tumor cell populations, derives from the present study in which clonal and nonclonal structural rearrangements were also found in short-term cultures from nonneoplastic skin and pharyngeal mucosa. It is possible that the aberrations are present in subepithelial fibroblast that have accumulated DNA damage due to extensive exposure to potentially carcinogenic agents.


Chromosomal sublocalization of the 2;13 translocation breakpoint in alveolar rhabdomyosarcoma.

A characteristic balanced reciprocal chromosomal translocation [t(2;13)(q35;q14)] has been identified in more than 50% of alveolar rhabdomyosarcomas. As the first step in characterization of the genes involved in this translocation, we constructed somatic cell hybrids that retained either the derivative chromosome 2 or the derivative chromosome 13 without a normal chromosome 13 homologue. Ten linked DNA probes known to be located within bands 13q13-q14 were mapped relative to the breakpoint on chromosome 13, allowing localization of the breakpoint region between two loci separated by 5.5 cM. A long-range restriction map extending approximately 2,300 kb around these loci failed to provide evidence of rearrangement. Additionally, we confirmed that the FMS-like tyrosine kinase gene (FLT), previously localized to 13q12 by in situ hybridization, is located proximal to the breakpoint, and we demonstrated that FLT is not a target for disruption by this tumor-specific translocation.


Retinoblastoma gene deletions in B-cell chronic lymphocytic leukemia.

Approximately 10% of B-cell chronic lymphocyte leukemia (B-CLL) cases have structural chromosomal aberrations involving band 13q14. To evaluate a possible role of RBl gene deletions in B-CLL we investigated the malignant cells of 27 patients by molecular genetic and cytogenetic techniques. Four of the cases had chromosomal aberrations that involved 13q14 (including one case with a 13q14 deletion that was observed in a single metaphase cell), and 11 had other chromosomal abnormalities, whereas the malignant cells of 12 patients were either cytogenetically normal or failed to divided in vitro. Eight patients (30%) were found to have hemizygous deletions of the RBl gene. These cases included all four patients with chromosomal changes at 13q14, but also three patients without chromosome abnormalities and one case with a chromosomal aberration not involving 13q. The deletions were interstitial in all cases but one, as defined by probes located centromeric and telomeric of the RBl locus. Inactivation of RBl may thus be a significant event in the development of some B-CLL clones.


Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes.

A novel polymerase chain reaction (PCR) technique has been combined with chromosome flow sorting to characterise two lymphoblastoid cell lines and one medullary thyroid carcinoma cell line carrying translocations close to the locus for multiple endocrine neoplasia type 2A (MEN 2A). Five hundred copies of the derivative chromosome(s) were flow sorted from each cell line and amplified by degenerate oligonucleotide-primed-polymerase chain reaction (DOP-PCR). This generated pools of DNA sequences corresponding to the abnormal chromosomes, which were then used as probes in fluorescence in situ hybridisation (FISH) experiments on normal metaphase cells. The resultant chromosome paints revealed the portions of the normal chromosomes related to those involved in the translocations. By this technique, translocation breakpoints in bands p15, q11.2, and q21 of chromosome 10 were defined in the above cell lines, in two cases refining previous cytogenetic data. This study shows that flow sorting of aberrant chromosomes and chromosome painting can be used as a rapid aid to cytogenetic analysis, particularly in cases of difficult karyotypes, such as tumours. Furthermore, the DOP-PCR technique described here will have applications to other areas of genome analysis, such as cloning of new markers; its design will allow a general and representative amplification to occur from any starting DNA in any species.


Trisomy 5 and trisomy 7 are nonrandom aberrations in pigmented villonodular synovitis: confirmation of trisomy 7 in uncultured cells.

Pigmented villonodular synovitis (PVNS) is a proliferative lesion of disputed genesis. Recently, we reported trisomy 7 in short-term cultures of 1 PVNS. In the present report, we describe another specimen of PVNS in which 9 of 26 (35 percent) metaphase cells demonstrated trisomy 7 when analyzed after 3-15 days of tissue culture. In situ hybridization analysis, with a biotinylated probe to chromosome 7 alpha-satellite DNA, revealed trisomy 7 in 53 of 200 uncultured cells from this PVNS sample. Our findings indicate that trisomy 7 is a nonrandom aberration that arises in vivo in PVNS.


Low chromosome number in chromophobe renal cell carcinomas.

Cytogenetic analysis revealed low chromosome number, telomeric association, and pulverisation of chromosomes in three chromophobe renal cell carcinomas. One fully karyotyped and a previously published case showed the common loss of chromosomes 1, 2, 6, 10, 13, 17, and 21.


Trisomy 8 in Philadelphia chromosome (Ph1)-negative cells in the course of Ph1-positive chronic myelocytic leukemia.

A female patient with chronic myelocytic leukemia (CML) in chronic phase after busulfan and interferon treatment had four different cell lines in her bone marrow. In addition to cells with a normal karyotype there were cells with the Philadelphia chromosome (Ph1), cells with trisomy 8 and Ph1, and cells with trisomy 8 as the sole anomaly.


[Induction of differentiation of human leukemia cells].

Human myeloid leukemia cells do not differentiate into functional end-cells but remain in the proliferation pool. Human cell lines can serve as model for hematopoietic cells arrested at different stages of myeloid differentiation and helps to dissect the cellular and molecular events involved in leukemogenesis. Furthermore, several agents have been identified as inducers of differentiation of leukemia cells. Exciting new clinical observation have shown that patients with APL respond well to the treatment with all-trans retinoic acid. RAR-alpha gene was proved to translocated from chromosome 17 to a locus PML on chromosome 15. This new chimeric gene, PML-RAR alpha is extremely important to understand the leukemogenesis of APL.


Studies on antitumor effects and mechanism of action of l-hexylcarbamoyl-5-fluorouracil by DNA/BrdU double staining using flow cytometry.

An investigation was carried out to elucidate the mechanism of action of the oral antitumor agent 1-hexylcarbamoyl-5-fluorouracil (HCFU) by determining its effects on the growth and cell cycle of epipharyngeal carcinoma cells (KB cell) by DNA/BrdU double staining using flow cytometry (FCM). As a result, it was found that HCFU stimulates KB cells in the S phase to proliferate for the first 3 days of treatment in a low concentration (8 micrograms/ml) and caused cell accumulation in the later G2M phase. On the other hand, when administered in the concentration (20 micrograms/ml) that produces a 50 per cent cell kill, as determined from the cell growth curve, HCFU appeared to exhibit a cytocidal effect by blocking cells in S and G2M for the first 3 days after exposure. It was revealed by FCM for the first time that HCFU operates by a similar mechanism to that of 5-FU. This method seems to be of significance to therapeutic schemes that take into consideration the mechanism of action of antitumor drugs.


The gene organization of the human beta 7 subunit, the common beta subunit of the leukocyte integrins HML-1 and LPAM-1.

The integrin beta 7 subunit associates with two alternative alpha subunits termed alpha HML-1 and alpha 4 to give the lymphocyte activation and homing receptors HML-1 and LPAM-1. Overlapping genomic clones that encoded the human beta 7 subunit gene were isolated from cosmid and phage lambda libraries. The coding portion of the gene spanned approximately 10 kb and was composed of 14 exons. Exon 1 (123 bp) encoded 5' untranslated sequences; exon 2 (204 bp) encoded the initiation codon, signal peptide, and 50 amino acid residues of the N-terminus of the mature protein; 7 exons (exons 3-9), ranging in size from 90 bp to 242 bp, encoded most of the extracellular domain proximal to the four cysteine-rich repeats; the region corresponding to the beta 3 arginine-glycine-aspartic acid (RGD)-binding domain was divided amongst exons 4 and 5; the four cysteine-rich repeats were encoded by 3 exons (exons 10-12) with intron insertion into the first and third repeats; exon 13 (209 bp) provided a spacer between the cysteine-rich domains and the transmembrane domain; exon 14 (161 bp) encoded the transmembrane domain and exactly half of the cytoplasmic domain; the remainder of the cytoplasmic domain and most of the 3' untranslated region was contained in the largest 313 bp exon 15. Comparison of integrin beta subunit genes revealed that the gene organization of beta 7 was almost identical to that of beta 2, but had diverged from that of beta 3. Amplification of integrin DNAs directly from genomic DNA, using PCR primers based on beta subunit consensus sequences corresponding to the beta 3 RGD-binding domain, yielded partial gene sequences for the beta 3, beta 5, and beta 6 subunits only. Inspection of the amplified sequences revealed that, as for beta 3, the regions in beta 5 and beta 6 corresponding to the beta 3 RGD-binding domain lacked the intron present in beta 7, beta 1, and beta 2, which divides this region in beta 2 into two subdomains that contribute to subunit assembly. This study provides genetic evidence for at least two major branches to the integrin beta subunit evolutionary tree, with beta 7, beta 2, and probably beta 1 in one branch, and the cytoadhesin beta 3 and probably also beta 5 and beta 6 in the other.


Midpregnancy plasma zinc in normal and growth retarded fetuses--a preliminary study.

To determine plasma zinc concentrations in normally and abnormally growing fetuses. Prospective observational study. Fetal Medicine Unit, Queen Charlotte's Maternity Hospital. 53 pregnant women attending for fetal blood sampling at between 18 and 40 weeks gestation. 27 fetuses were normal (central group), 11 fetuses were growth retarded and 15 were malformed. Plasma zinc concentrations in maternal and fetal blood at time of fetal blood sampling. In normally growing fetuses, between 18 and 40 weeks gestation, there was no fall in maternal plasma zinc concentration; the fetal level fell by 36%. In 10 fetuses with symmetrical growth retardation, plasma zinc concentration tended to be low, but was not significantly different from that in the normal control fetuses. The results suggest that (i) placental transfer of zinc is an uphill secretory process and that it is a rate-limiting step in the accumulation of zinc by the fetus and (ii) in fetuses with symmetrical intrauterine growth retardation, a low plasma zinc is probably a parallel phenomenon and not necessarily an aetiological factor.


In situ immunohistochemical analysis of cell adhesion molecules on human corneal endothelial cells.

Interaction of leucocytes with human corneal endothelial cells (HCECs) can be observed in several clinicopathological conditions, such as uveitis, keratitis, and corneal graft rejection. Since leucocyte-endothelial cell interactions involve various adhesion receptors we have analysed the expression and distribution pattern of the neural cell adhesion molecule (NCAM), the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1), the endothelial leucocyte adhesion molecule-1 (ELAM-1), and the cluster of differentiation antigen-44 (CD44) on flat preparations of normal and organ-cultured HCECs. NCAM and ICAM were constitutively expressed on HCECs whereas VCAM-1, ELAM-1, and CD44 were absent from normal HCECs. However flat mounts of HCECs from organ-culture preserved corneas showed a mosaic-like distribution pattern of VCAM-1 and ELAM-1 positive cells and garland-like clusters of CD44 positive cells. We suggest that modulation of ELAM-1, VCAM-1, and CD44 expression on HCECs may contribute to the regulation of leucocytes-HCECs interaction in the case of anterior segment inflammation.


Solution structure of an unusually stable RNA tetraplex containing G- and U-quartet structures.

A model for the solution structure of an RNA tetraplex, (rUGGGGU)4, has been obtained by two-dimensional NMR spectroscopy and molecular dynamics. The molecule is parallel stranded and Hoogsteen base-paired in 50 mM KCl, and it is so stable that three of its six imino protons have exchange half-lives measured in days at 40 degrees C. The tetraplex is stabilized by base stacking and by the hydrogen bonds in four G quartets and at least one U quartet. This is the first indication of the existence of U-quartet structures of which we are aware.


Cross-linking activity of the 14-kilodalton beta-galactoside-specific vertebrate lectin with asialofetuin: comparison with several galactose-specific plant lectins.

We have previously shown that plant lectins with a wide range of carbohydrate binding specificities can bind and cross-link (precipitate) specific multiantennary oligosaccharides and glycopeptides [cf. Bhattacharyya, L., Fant, J., Lonn, H., & Brewer, C. F. (1990) Biochemistry 29, 7523-7530]. This leads to a new source of binding specificity: namely, the formation of homogeneous cross-linked lattices between lectins and carbohydrates. Recently, we have demonstrated the existence of highly ordered cross-linked lattices that form between the D-Man/D-Glc-specific plant lectin concanavalin A and the soybean agglutinin which is a tetrameric glycoprotein possessing a single Man9 oligomannose chain per monomer [Khan, M. I., Mandal, D. K., & Brewer, C. F. (1991) Carbohydr. Res. 213, 69-77]. In the present study, we have compared the ability of the 14-kDa beta-galactoside-specific lectin from calf spleen, a dimeric S-type animal lectin, and several galactose-specific plant lectins from Erythrina indica, Erythrina cristagalli, and Glycine max (soybean agglutinin) to form specific cross-linked complexes with asialofetuin (ASF), a 48-kDa monomeric glycoprotein, using quantitative precipitation analyses. The results show the formation of 1:9 and 1:3 stoichiometric cross-linked complexes (per monomer) of ASF to the 14-kDa lectin, depending on their relative ratio in solution. Evidence indicates that the three triantennary N-linked complex-type oligosaccharide chains of ASF mediate the cross-linking interactions and that each chain expresses either trivalency in the 1:9 cross-linked complex or univalency in the 1:3 complex.(ABSTRACT TRUNCATED AT 250 WORDS)


Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation.

The channel-forming protein aerolysin is secreted as a protoxin which can be activated by proteolytic removal of a C-terminal peptide. The activation and subsequent oligomerization of aerolysin were studied using a variety of spectroscopic techniques. Mass spectrometric determination of the molecular weights of proaerolysin and aerolysin permitted identification of the sites at which the protoxin is processed by trypsin and chymotrypsin. The results of far- and near-UV circular dichroism measurements indicated that processing with trypsin does not lead to major changes in secondary or tertiary structure of the protein. An increase in tryptophan fluorescence intensity and a small red shift in the maximum emission wavelength of tryptophans could be observed, suggesting that there is a change in the environment of some of the tryptophans. There was also a dramatic increase in the binding of the hydrophobic fluorescent probe 1-anilino-8-naphthalenesulfonate during activation, leading us to conclude that a hydrophobic region in the protein is exposed by trypsin treatment. Using measurements of light scattering, various parameters influencing oligomerisation of trypsin-activated aerolysin were determined. Oligomerization rates were found to increase with the concentration of aerolysin, whereas they decreased with increasing ionic strength.


A conformational rearrangement in gramicidin A: from a double-stranded left-handed to a single-stranded right-handed helix.

A conformational transition is described for the polypeptide, gramicidin A, in which a dimer that forms a left-handed intertwined antiparallel helix is converted to a single-stranded amino terminus to amino terminus right-handed helix. The starting structure is determined here by solution NMR methods while reference is made to the well-established folding motif of gramicidin in a lipid bilayer for the ultimate conformation of this transition. Furthermore, an organic solvent system of benzene and ethanol in which gramicidin has a unique conformation is identified. This conformation is shown to be very similar to that derived from X-ray diffraction of crystals prepared from a similar solvent system.


Ca(2+)-calmodulin regulated effectors of microtubule stability in bovine brain.

Stable microtubules (as defined by resistance to Ca2+, drug or cold temperature induced disassembly) form in abundance during tubulin assembly in brain crude extracts. We have previously shown that, in rat brain crude extracts, all microtubule stabilizing activity could be ascribed to a single Ca(2+)-calmodulin binding and Ca(2+)-calmodulin regulated protein, called "stable tubule only polypeptide", STOP145 [Pirollet, F., Rauch, C. T., Job, D., & Margolis, R. L. (1989) Biochemistry 28, 835-842]. We have now performed an exhaustive study of STOP-like effectors in bovine brain high-speed supernatants. All activity binds to cation exchangers and to Ca(2+)-calmodulin affinity columns. The activity can be resolved into two peaks on sizing columns. The first eluted peak contains a prominent 220-kDa protein. The second peak contains an apparently homogeneous 20-kDa polypeptide. A monoclonal antibody specific to rat brain STOP145 recognizes the 220-kDa protein, but not the 20-kDa species. The 220-kDa protein can be purified on a STOP antibody column and accounts for the bulk of stabilizing activity in the first peak. The 20-kDa protein does not bind to STOP antibody affinity columns. Sequence analysis of oligopeptide fragments of the 20-kDa protein shows 100% homology with bovine myelin basic protein (MBP). Anti-MBP antibodies recognize the 20-kDa, but not the 220-kDa species. We conclude that the 220-kDa protein is the bovine equivalent to rat brain STOP145 and that the 20-kDa species is MBP. Microtubule stabilization by MBP and STOP220 is abolished in the presence of Ca(2+)-calmodulin, and inhibition curves are similar for both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)


Thermodynamics of single-stranded RNA binding to oligolysines containing tryptophan.

The equilibrium binding to the synthetic RNA poly(U) of a series of oligolysines containing one, two, or three tryptophans has been examined as a function of pH, monovalent salt concentration (MX), temperature, and Mg2+. Oligopeptides containing lysine (K) and tryptophan (W) of the type KWKp-NH2 and KWKp-CO2 (p = 1-8), as well as peptides containing additional tryptophans or glycines, were studied by monitoring the quenching of the peptide tryptophan fluorescence upon binding poly(U). Equilibrium association constants, K(obs), and the thermodynamic quantities delta G(o)obs, delta H(o)obs, and delta S(o)obs describing peptide-poly(U) binding were measured as well as their dependences on monovalent salt concentration, temperature, and pH. In all cases, K(obs) decreases significantly with increasing monovalent salt concentration, with (delta log K(obs)/delta log [K+]) = -0.74 (+/- 0.04)z, independent of temperature and salt concentration, where z is the net positive charge on the peptide. The origin of these salt effects is entropic, consistent with the release of counterions from the poly(U) upon formation of the complex. Upon extrapolation to 1 M K+, the value of delta G(o)obs is observed to be near zero for all oligolysines binding to poly(U), supporting the conclusion that these complexes are stabilized at lower salt concentrations due to the increase in entropy accompanying the release of monovalent counterions from the poly(U). Only the net peptide charge appears to influence the thermodynamics of these interactions, since no effects of peptide charge distribution were observed. The binding of poly(U) to the monotryptophan peptides displays interesting behavior as a function of the peptide charge. The extent of tryptophan fluorescence quenching, Qmax, is dependent upon the peptide charge for z less than or equal to +4, and the value of Qmax correlates with z-dependent changes in delta H(o)obs and delta S(o)obs(1 M K+), whereas for z greater than or equal to +4, Qmax, delta H(o)obs, and delta S(o)obs (1 M K+) are constant. The correlation between Qmax and delta H(o)obs and delta S(o)obs(1 M K+) suggests a context (peptide charge)-dependence of the interaction of the peptide tryptophan with poly(U). However the interaction of the peptide tryptophan does not contribute substantially to delta G(o)obs for any of the peptides, independent of z, due to enthalpy-entropy compensations. Each of the tryptophans in multiple Trp-containing peptides appear to bind to poly(U) independently, with delta H(o)Trp = -2.9 +/- 0.7, although delta G(o)Trp is near zero due to enthalpy-entropy compensations.(ABSTRACT TRUNCATED AT 400 WORDS)


Structure of alpha 2-macroglobulin-protease complexes. Methylamine competition shows that proteases bridge two disulfide-bonded half-molecules.

alpha 2-Macroglobulin (alpha 2M) forms several different covalent complexes with proteases. These include unusual forms in which more than one of the four identical subunits of alpha 2M are cross-linked by amide bonds to more than one lysyl amino group of the bound protease. The structure of these complexes and the question of how the identical subunits are arranged to form two protease binding sites are matters of current controversy. The 185-kDa subunits are arranged into two disulfide-bonded half-molecules which are, in turn, noncovalently associated. We have provided evidence that, in the major multivalent cross-linked form, proteases can span the two half-molecules, forming a covalently bonded tetramer [Wang, D., Yuan, A. I., & Feinman, R. D. (1984) Biochemistry 23, 2807-2811]. An alternative theory has recently been proposed in which the major high molecular weight form has two bonds to protease that are within half-molecules--a multivalent cross-linked dimer [Sottrup-Jensen, L., Hansen, H. F., Pedersen, H. S., & Kristensen, L. (1990) J. Biol. Chem. 265, 17727-17737]. To resolve this conflict, experiments were carried out to determine the structure of one of the high molecular weight bands (band 3) seen on SDS-PAGE. Band 3 has anomalous migration, corresponding to markers of apparent molecular mass of 550 kDa (between the tetramer and dimer). In the experiments described here, reactions of thrombin with alpha 2M were run in the presence of methylamine, which competes for one of the two thrombin-alpha 2M covalent bonds.(ABSTRACT TRUNCATED AT 250 WORDS)


Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells.

Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called pp185, were originally found in anti-phosphotyrosine antibody (alpha PY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to alpha PY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X. J., et al. (1991) Nature 352, 73-77]. IRS-1 and pp185 undergo tyrosine phosphorylation immediately after insulin stimulation and show an insulin dose response similar to that of insulin receptor autophosphorylation. However, IRS-1 was consistently 10 kDa smaller than the apparent molecular mass of pp185. The pp185 contained some immunoblottable IRS-1; however, cell lysates depleted of IRS-1 with anti-IRS-1 antibody still contained the high molecular weight forms of pp185 (HMW-pp185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-pp185, suggesting that at least two substrates migrate in this region during SDS-PAGE. Moreover, the phosphatidylinositol 3'-kinase and its 85-kDa associated protein (p85) bound to IRS-1 in Fao cells, but weakly or not at all to HMW-pp185. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-pp185, which may play unique roles in insulin signal transmission.


Interaction of cytochrome c with cytochrome c oxidase studied by monoclonal antibodies and a protein modifying reagent.

C/57 black mice were immunized with beef heart cytochrome c oxidase, generating 48 hybrid cell lines that secrete antibodies against the different subunits of the enzyme. Immunoblot analysis showed reactions with 7 of the 13 subunits. Among the monoclonal antibodies produced, only those to subunit II gave significant inhibition; these inhibited the enzyme activity completely and prevented cytochrome c binding to the enzyme. Epitope mapping studies indicate that a peptide including residues 200-227 reacts with the antibody, suggesting that the C-terminus of the protein is essential for the binding of this antibody. The carboxyl modifying reagent 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) was chosen to investigate further the relationship between antibody and cytochrome c binding domains. ETC caused 50% inhibition of the enzyme activity with a first-order time during the first 20 min; a slower reaction over 3 h resulted in 90% inhibition. Cytochrome c binding to the oxidase was inhibited to a similar extent as cytochrome c oxidation, and protection against both effects was afforded by the presence of cytochrome c during ETC modification. Anion-exchange of FPLC of the modified forms of cytochrome oxidase revealed extensive inhomogeneity, indicating random derivatization of a number of different carboxyls even during the first-order reaction, and precluding identification of carboxyl residues related to a specific phase of the reaction. Cytochrome c and the subunit II-specific antibody protected against radioactive labeling of subunit II by ETC in the presence of [14C]glycine ethyl ester, demonstrating that the antibody and cytochrome c occupy significant and overlapping areas on the subunit II surface.(ABSTRACT TRUNCATED AT 250 WORDS)


Novel sulfated ligands for the cell adhesion molecule E-selectin revealed by the neoglycolipid technology among O-linked oligosaccharides on an ovarian cystadenoma glycoprotein.

E-selectin is the inducible adhesion protein on the surface of endothelial cells which has a crucial role in the initial stages of recruitment of leucocytes to sites of inflammation. In addition, it is almost certainly involved in tumor cell adhesion and metastasis. This report is concerned with identification of a new class of oligosaccharide ligand--sulfate-containing--for the human E-selectin molecule from among oligosaccharides on an ovarian cystadenoma glycoprotein. This has been achieved by application of the neoglycolipid technology to oligosaccharides released from the glycoprotein by mild alkaline beta-elimination. Oligosaccharides were conjugated to lipid, resolved by thin-layer chromatography, and tested for binding by Chinese hamster ovary cells which had been transfected to express the full-length E-selectin molecule. Several components with strong E-selectin binding activity were revealed among acidic oligosaccharides. The smallest among these was identified by liquid secondary ion mass spectrometric analysis of the neoglycolipid, in conjunction with methylation analysis of the purified oligosaccharide preparation as an equimolar mixture of the Le(a)- and Le(x)/SSEA-1-type fucotetrasaccharides sulfated at position 3 of outer galactose: [formula: see text] To our knowledge this is the first report of a sulfofucooligosaccharide ligand for E-selectin. The binding activity is substantially greater than those of lipid-linked Le(a) and Le(x)/SSEA-1 sequences and is at least equal to that of the 3'-sialyl-Le(x)/SSEA-1 glycolipid analogue.


Analysis of the backbone dynamics of the ribonuclease H domain of the human immunodeficiency virus reverse transcriptase using 15N relaxation measurements.

The backbone dynamics of the uniformly 15N-labeled ribonuclease H (RNase H) domain of human immunodeficiency virus (HIV-1) reverse transcriptase have been investigated using two-dimensional inverse-detected heteronuclear 15N-1HNMR spectroscopy. 15N T1, T2, and nuclear Overhauser enhancement (NOE) data were obtained for 107 out of a total of 134 backbone amide groups. The overall rotational correlation time (tau R) for the protein at 26 degrees C is 10.4 ns. The backbone N-H vectors for all the measurable residues exhibit very fast motions on a time scale of less than or equal to 20 ps. The 15N relaxation data for only 14 residues can be explained by this single internal motion alone. A further 39 residues display a second motion on a time scale ranging from 28.8 ps to 3.9 ns, while another 15 residues are characterized by an additional motion on the 170-ns to 2.25-ms time scale resulting in 15N T2 exchange line broadening. There are 39 residues that exhibit both the additional 15N T2 exchange line broadening and the slow (28.8 ps-3.9 ns) internal motion. Thus, the RNase H domain experiences extensive mobility throughout its structure as evidenced by the 93 residues which exhibit multiple modes of motion. Distinctly mobile regions of the protein are identified by large decreases in the overall order parameter (S2) and correspond to the C-terminal residues and the loop regions between beta-strands beta 1 and beta 2 and between alpha-helix alpha B and beta-strand beta 4.(ABSTRACT TRUNCATED AT 250 WORDS)


Identification of a second promoter which drives the expression of gamma-glutamyl transpeptidase in rat kidney and epididymis.

In rat, gamma-glutamyl transpeptidase (GGT) is encoded by multiple mRNAs (mRNAI, mRNAII, mRNAIII, and mRNAIV) that differ only in their 5' untranslated regions and are transcribed from a single-copy gene. Using oligonucleotides designed from the 5' untranslated sequences of the GGT mRNAII and mRNAIII, we amplified a 3.4-kb genomic sequence which contains the promoter region for mRNAII. The sequence flanking the two initiation start sites for mRNAII contains consensus motifs for several potential regulatory proteins and a TATA-like element at the expected position 26 bp upstream from the predominant start site. The sequence from positions -528 to +72 associated with the chloramphenicol acetyltransferase (CAT) reporter gene drives a promoter activity in LLC-PK1, a pig kidney cell line. Deletion analysis revealed that the region from nucleotides -528 to -322 mediates an activation of the promoter activity, whereas the sequence from -322 to -114 has a negative effect. Furthermore, the structural organization of the 5' end of the GGT gene reveals that the GGT mRNAIII is transcribed from a third promoter located upstream from the promoter II on the GGT gene. By Northern blot analysis, the promoter II was found to be expressed only in the kidney and in the epididymis. We also identified two new mRNA species which are expressed in the H5 hepatoma cells. Therefore, the GGT gene expression reveals a strong tissue- or cell-specific pattern which is based on the transcription of several mRNA species from multiple promoters.


Secondary structure of the pentraxin female protein in water determined by infrared spectroscopy: effects of calcium and phosphorylcholine.

The secondary structure of hamster female protein in aqueous solutions in the presence or absence of calcium and phosphorylcholine has been investigated using Fourier transform infrared spectroscopy. Our present studies provide the first evaluation of the secondary structure of FP and its calcium- and phosphorylcholine-dependent conformational changes. Quantitative analysis indicated that FP is composed of 50% beta-sheet, 11% alpha-helix, 29% beta-turn, and 10% random structures. Calcium- and phosphorylcholine-dependent infrared spectral changes were observed in regions assigned to beta-sheet, alpha-helix, turn, and random structures. The infrared-based secondary structure compositions were used as constraints to compute theoretical locations for the different secondary structures along the amino acid sequence of the FP protein. Two putative calcium-binding sites were proposed for FP (residues 93-109 and 150-168) as well as other members of the pentraxin family on the basis of the theoretical secondary structure predictions and the similarity in sequence between the pentraxins and EF-hand calcium-binding proteins. The changes in protein conformation detected upon binding of calcium and phosphorylcholine provide a mechanism for the effects of these ligands on physiologically important properties of the protein, e.g., activation of complement and association with amyloids.