Circulating CD34+ cells: an adverse prognostic factor in the myelodysplastic syndromes.

As part of an epidemiological survey of myelodysplastic syndromes (MDS) in southern Tasmania, 62 MDS patients identified over a 2 year period were tested for the presence of CD34, the human progenitor cell antigen (HPCA), in their peripheral blood. The results were correlated with transformation to acute myeloid leukemia (AML) and patient survival, and CD34+ status was compared as a prognostic indicator with Bournemouth score, cytogenetics, and CFU-GM colony growth which were also assessed. Circulating CD34+ cells were found in 23 of the 62 MDS patients; 9 of the 23 patients with circulating CD34+ cells transformed to AML, as compared with none of the 39 CD34 negative patients (P less than 0.0001); and 11 of the 23 patients with circulating CD34+ cells were dead at the end of the 2 year period, as opposed to 6 of the 39 with no CD34+ cells (P less than 0.03). The Bournemouth score was also significantly associated with transformation to AML (P less than 0.0001) and poor survival (P less than 0.04). These were the only significant associations of the possible prognostic factors studied with either transformation or survival. In summary, the presence of circulating CD34+ cells was significantly associated with both progression to AML and poor survival and was found to be a better prognostic indicator than cytogenetics or CFU-GM colony growth.


Xeroderma pigmentosum and Cockayne syndrome: overlapping clinical and biochemical phenotypes.

Two siblings are described whose clinical presentation of cutaneous photosensitivity and central nervous system dysfunction is strongly reminiscent of the DeSanctis-Cacchione syndrome (DCS) variant of xeroderma pigmentosum. An extensive clinical evaluation supported a diagnosis of DCS and documented previously unreported findings. In vitro fibroblast studies showed UV sensitivity that was two to three times that of normal controls. However, neither a post-UV-irradiation DNA excision-repair defect indicative of XP nor a semiconservative DNA replication defect indicative of XP variant was found. Rather, a failure of RNA synthesis to recover to normal levels after UV exposure was observed, a biochemical abnormality seen in Cockayne syndrome (CS), one of the premature-aging syndromes with clinical UV sensitivity. These patients, therefore, clinically have XP, but their biochemical characteristics suggest CS. The reason(s) for the severe neurologic disease, in light of the relatively mild cutaneous abnormalities, is unclear. Other cases with unusual fibroblast responses to irradiation have been noted in the literature and, along with the data from our patients, reinforce the notion of the complexity of DNA maintenance and repair.


Superficial laser vulvectomy. V. Surgical debulking is enhanced by adjuvant systemic interferon.

Skillful laser ablation can remove any volume of human papillomavirus-associated vulvar disease but cannot prevent reactivation of the surrounding latent viral reservoir during postoperative healing. Conversely, interferon and 5-fluorouracil are relatively ineffective as primary therapies in clearing bulky lesions. In this study of 71 assessable patients, topical 5-fluorouracil and systemic interferon injections were used postoperatively. Success rates within the adjuvant 5-fluorouracil and laser alone arms were essentially the same (9 of 18 vs 8 of 20). In contrast, outcome in the interferon group was significantly better than that for the other two arms combined (27 of 33 [82%] vs 17 of 38 [45%]; chi 2 10.31; p less than 0.002). Moreover, 18 of 21 failures (86%) in the first two arms and 3 of 6 failures (50%) in the interferon arm were "rescued" from the need for a second laser surgical procedure by crossover to either the 1 or 3 MIU interferon regimen. Results from this open-label, randomized clinical trial suggest that even a relatively low dose of recombinant interferon, used in combination with effective surgical debulking, can markedly reduce the risk of postoperative recurrence.


Immunohistochemical detection of the multi-drug-resistance marker P-glycoprotein in uterine cervical carcinomas and normal cervical tissue.

Uterine cervical carcinomas and normal cervical tissue (controls) were investigated for the presence of the multi-drug-resistance gene product P-glycoprotein by means of immunohistochemistry, with the C219 monoclonal antibody and the streptavidin-biotin-peroxidase technique. Ten of 11 cervical carcinomas, 2 of which were previously treated with chemotherapeutic agents of the multi-drug-resistance group, showed a positive reaction of tumor cells. All normal controls showed a positive reaction of the ectocervical and endocervical epithelial cells. P-glycoprotein seems to be implicated at least in part in resistance to chemotherapy of cervical carcinoma.


Is trichomoniasis often associated with bacterial vaginosis in pregnant adolescents?

The same criteria for identifying bacterial vaginosis are often present in women with trichomoniasis. These criteria include elevated vaginal pH, vaginal odor, homogeneous discharge, increased anaerobic bacteriologic vaginal flora, and elevated levels of bacterial enzymes. Clinically mixed vaginal infections occur, and because the treatment for these two conditions can be different, it is important to distinguish between them. Trichomoniasis can interfere with a Gram stain diagnosis or the proline aminopeptidase test for bacterial vaginosis. Clue cells are not generally found in women with Trichomonas vaginalis, but when present, they strongly indicate the concomitant presence of bacterial vaginosis.


Ultrasonographic dating of pregnancy causes significant errors in Down syndrome risk assessment that may be minimized by use of biparietal diameter-based means.

Gestational dates assessed by ultrasonographic measurement of fetal dimensions are usually quoted in terms of complete weeks because the uncertainty of ultrasonographic measurement is approximately +/- 7 days. This study examines the effect of ultrasonographic dating on Down syndrome risk assessment. The effect of small changes in measured biparietal diameter resulting in a change in estimated gestational week and the benefits of a more precise measure of fetal gestational age (raw biparietal diameter) are examined mathematically. If maternal serum alpha-fetoprotein and human chorionic gonadotropin are used to assess Down syndrome risk, risks assessed with ultrasonographically determined dates may be less than or equal to 45% too high for fetuses with biparietal diameter at the lowest end of the size band and less than or equal to 22% too low with biparietal diameter at the top of the size band. If the results for maternal serum alpha-fetoprotein, human chorionic gonadotropin, and unconjugated estriol are used, these figures become less than or equal to 150% and less than or equal to 60%, respectively. If ultrasonography is used to assess gestational age, the raw biparietal diameter and not the estimated week of gestation must be used to derive means for calculation of multiples of the mean.


Characteristics of three vaginal flora patterns assessed by gram stain among pregnant women. Vaginal Infections and Prematurity Study Group.

This study was undertaken to define the characteristics and persistence of vaginal flora in 7918 pregnant women at 23 to 26 weeks' gestation. Vaginal smears were categorized as normal (predominant lactobacilli), intermediate (reduced lactobacilli), or positive for bacterial vaginosis. The women with normal flora were least likely to have elevated vaginal pH, amine odor, milky discharge, or colonization by Gardnerella, Bacteroides, or genital mycoplasmas. Women with intermediate vaginal flora had intermediate frequencies of these clinical signs and microorganisms. Group B streptococci and yeast were associated with normal or intermediate flora, whereas Neisseria gonorrhoeae and Chlamydia trachomatis were recovered more frequently from women with intermediate flora or bacterial vaginosis. Trichomonas vaginalis was most associated with intermediate flora. At follow-up, 81% of the women with normal flora had remained normal. Of the women with intermediate flora, 32% acquired bacterial vaginosis and 30% shifted to normal flora. Only 12% of the women with bacterial vaginosis had shifted to normal flora. We conclude that there are two primary stable vaginal flora patterns (normal flora or bacterial vaginosis) and a third less distinct transitional flora pattern between these two.


Unexplained elevated maternal serum alpha-fetoprotein is not predictive of adverse perinatal outcome in an indigent urban population.

The null hypothesis of this study is that in an urban, indigent obstetric population at high risk for adverse perinatal outcome, unexplained elevations of maternal serum alpha-fetoprotein are not an additional predictor of adverse perinatal outcome. Perinatal outcomes of 72 patients from a clinic for indigent patients with unexplained elevated maternal serum alpha-fetoprotein levels were compared with those of matched controls from the same population with normal maternal serum alpha-fetoprotein levels. Subjects and controls were matched for age, race, parity, and presence or absence of Hollister risk factors. The frequency of adverse perinatal outcome in the two groups was subjected to matched-pair chi 2 analysis. Adverse perinatal outcome occurred in 38.9% (28 of 72) of subjects with unexplained elevated maternal serum alpha-fetoprotein levels greater than or equal to 2.5 multiples of the median, compared with 31.9% (23 of 72) of controls with normal maternal serum alpha-fetoprotein levels (p = 0.5). No statistically significant difference in adverse perinatal outcomes was found. Elevated maternal serum alpha-fetoprotein levels offer little if any additional predictive value for adverse perinatal outcome in populations already at high risk for such outcomes on the basis of obstetric or socioeconomic criteria.


Substance P: a late-acting B lymphocyte differentiation cofactor.

The peptide substance P has been recognized for years as having dramatic effects on such diverse physiological responses as blood pressure regulation, peristalsis of the gut, and salivation. More recently, demonstration of substance P receptors on leukocytes and modulation of leukocyte functions by this peptide suggested that it might also have a role in immune regulation. This review focuses on the growing body of evidence that demonstrates substance P-induced effects on one population of leukocytes, namely B lymphocytes. Despite the diversity of experimental techniques used, there is surprisingly good agreement as to the role substance P has in modulating B lymphocyte responses. In vivo treatments of rodents, which increase substance P concentrations in the periphery, increase the number of immunoglobulin-secreting cells in these animals. Conversely, infusion of substance P antagonists or depletion of substance P-containing neurons in rodents substantially reduces the animals' ability to synthesize immunoglobulins. With the use of cultures of B lymphocytes it was possible to demonstrate similar results. In the presence of polyclonal B cell activators, substance P augmented immunoglobulin secretion in cultures of purified B lymphocytes or B cell clones. The absence of accessory cells in these cultures suggested that substance P could act directly on activated B lymphocytes, and in fact these B cells were shown to express specific receptors for this peptide. It appears that the substance P receptors expressed by leukocytes are similar or identical to those expressed by neurons as evidenced by radioreceptor binding assays and detection of the gene encoding the substance P receptor.(ABSTRACT TRUNCATED AT 250 WORDS)


T84 cells: anion selectivity demonstrates expression of Cl- conductance affected in cystic fibrosis.

The T84 cell line possesses an adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- conductance and expresses high levels of the cystic fibrosis (CF) gene product, implicating it as a good model for CF research. To evaluate whether T84 Cl- conductance properties are consistent with those described in CF target epithelial, we used transepithelial measurements (verified by selective permeabilization of the basal membrane) to determine the apparent anion selectivity properties of the apical and basolateral membranes of stimulated and unstimulated T84 cells. Unstimulated epithelial cells were almost electrically inert, having a low transepithelial voltage (Vt; -6 mV, apical surface negative), a small equivalent short-circuit current (Isc,(eq.) 2.2 microA/cm2), a very high transepithelial resistance (Rt; 2,500 omega.cm2), and poor anion permselectivity properties at both membrane surfaces (0.8 less than PX/PCl- less than 1.1), where X is NO3-, Br-, I-, or gluconate. When stimulated with forskolin (10(-6) M), Vt increased 8-fold, Isc(eq) increased 30-fold, Rt fell to one-third of unstimulated values, and the apical surface became highly anion selective, i.e., NO3- (1.4) greater than Br- (1.2) greater than Cl- (1.0) greater than I- (0.7) greater than gluconate (0.0), where numbers in parentheses are PX/PCl-. I- was less permeable than Cl- and probably directly inhibits the anion conductance, since Rt was substantially greater after I- substitution than after substitution with the impermeable anion gluconate. Bumetanide (10(-4) M) significantly attenuated the response of Vt to anion substitutions at the basal membrane surface, indicating that the effects of substitution were predominantly on the Na(+)-K(+)-2Cl- cotransporter.(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of sarcoplasmic reticulum gene expression during cardiac and skeletal muscle development.

The expression of major sarcoplasmic reticulum proteins during cardiac and fast-twitch skeletal muscle development was examined using gene-specific probes. Through the use of S1 nuclease mapping, Northern blot, and RNA slot-blot analysis, sarcoplasmic reticulum proteins were shown to exhibit both narrow tissue specificity and plasticity in their expression during muscle development. In fast-twitch skeletal muscle, the cardiac/slow-twitch isoforms of Ca(2+)-ATPase and calsequestrin were detected at high levels in fetal stages but were gradually replaced by fast-twitch isoforms in adult muscle. In contrast, cardiac muscle expressed exclusively cardiac/slow-twitch isoforms of Ca(2+)-ATPase and calsequestrin at all stages. Both fast-twitch and slow-twitch skeletal muscle expressed the same skeletal muscle ryanodine receptor isoform, whereas cardiac muscle expressed a cardiac isoform. Phospholamban expression was restricted to cardiac and slow-twitch skeletal muscle and did not appear in developing fast-twitch skeletal muscle. During in vitro myogenesis of C2C12 cells, the mRNA transcripts encoding sarcoplasmic reticulum proteins were found to be coordinately induced in synchrony with that of contractile protein mRNA. The myogenic factor "myogenin" induced sarcoplasmic reticulum gene transcripts along with contractile protein mRNAs in nonmyogenic cells. These data suggest that the induction of both sarcoplasmic reticulum and contractile protein gene families is under the control of a common myogenic differentiation program.


L-type Ca2+ channel desensitization by F- reduces PhE-induced increase in [Ca2+]i but not stress.

F- (10 mM sodium fluoride plus deferoxamine to chelate contaminating aluminum) causes arterial contractions primarily by activating L-type Ca2+ channels. Results from the present study indicate that, although F(-)-induced contractions could be completely relaxed by washing out the F- with fresh buffer, a long-lasting effect of F- pretreatment was to produce L-type Ca2+ channel desensitization. Pretreatment of arteries for 4 h with F- (followed by washout of F-) resulted in much reduced increases in stress and [Ca2+]i produced by the subsequent addition of 110 mM KCl, such that steady-state values were, respectively, only 9 and 15% of the control values. However, a 4-h F- pretreatment caused a reduction only in the rate of stress development, but not the steady-state level of stress, produced by maximum concentrations of receptor agonists. In tissues that were pretreated with F- and then stimulated with the alpha-adrenoceptor agonist, phenylephrine, steady-state stress was still 104% of the control value, while the increase in [Ca2+]i was only 10% of the control value. F- is known to inhibit protein phosphatases, and similar reductions in the ability of KCl to produce contractions and increase [Ca2+]i were seen after pretreatment with the protein phosphatase inhibitor, okadaic acid. These data suggest that L-type Ca2+ channel desensitization by F- pretreatment was caused by increased protein phosphorylation. In addition, they suggest that much of the contribution made by L-type Ca2+ channels to increase [Ca2+]i during receptor stimulation may not be necessary for the maintenance of maximum stress at steady state.


Inhibition by sodium nitroprusside or PGE1 of tyrosine phosphorylation induced in platelets by thrombin or ADP.

Upon platelet activation, numerous proteins are known to be tyrosine phosphorylated. To investigate the mechanisms of the regulation of tyrosine phosphorylation and its physiological significance, the effects on tyrosine phosphorylation of agents that elevate the platelet level of the cyclic nucleotides cAMP and cGMP were examined in aspirin-treated gel-filtered platelets by Western blotting with a specific antiphosphotyrosine antibody. The effects of these agents on other aspects of platelet activation, i.e., aggregation, secretion, and elevation of the concentration of cytosolic ionized calcium ([Ca2+]i), were also examined in parallel experiments. Tyrosine phosphorylation in platelets activated by alpha-thrombin (1 nM) was inhibited by prostaglandin (PG) E1 (2 microM) or by sodium nitroprusside (100 microM). Elevation of [Ca2+]i, aggregation, and serotonin secretion was also strongly inhibited. On the other hand, a higher concentration of alpha-thrombin (10 nM) induced tyrosine phosphorylation of the same proteins, elevation of [Ca2+]i, platelet aggregation, and serotonin secretion, irrespective of pretreatment of platelets by either PGE1 or sodium nitroprusside. Inhibition by sodium nitroprusside of tyrosine phosphorylation induced by alpha-thrombin (1 nM) was accompanied by an increased concentration of cGMP. 8-BrcGMP (2 mM) also inhibited tyrosine phosphorylation and aggregation, although less than sodium nitroprusside. ADP (20 microM) induced platelet shape change and tyrosine phosphorylation of only a few proteins; these effects were also inhibited by either PGE1 or sodium nitroprusside. Thus tyrosine phosphorylation in platelets can be inhibited by elevation of either cAMP or cGMP, an effect that is overcome by a high concentration of thrombin, resulting in granule secretion and aggregation. Some of the proteins that are tyrosine phosphorylated may be important in the regulation of platelet functions.


Tetrodotoxin-insensitive sodium channels in a cardiac cell line from a transgenic mouse.

The electrophysiological properties of a cardiac cell line (MCM1) originating from a transgenic mouse were characterized. The dominant current in these cells is a sodium current that is insensitive to concentrations of tetrodotoxin (TTX) up to 100 microM. It activates and inactivates rapidly with half-maximal activation at -40 mV and half-maximal inactivation at -79 mV. This sodium current is reduced by agents that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and activate cAMP-dependent protein kinase including isoproterenol, 8-bromo-cAMP, and isobutylmethylxanthine. The phenylalkylamine desmethoxyverapamil blocks the TTX-insensitive sodium current in MCM1 cells in both tonic and use-dependent fashion. Membrane depolarization enhances this block. It is proposed that the TTX-insensitive sodium current in these cells may be similar in origin to the embryonic type of TTX-insensitive sodium current described in other cardiac and skeletal muscle preparations.


cAMP-stimulated ion currents in Xenopus oocytes expressing CFTR cRNA.

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in stage V/VI Xenopus oocytes by injection of cRNA transcribed in vitro from a pBluescript vector containing 6.2-kb wild-type cDNA. This clone was also used for the preparation of antisense RNA. Double-electrode voltage clamp was employed to measure transmembrane currents. In sense RNA-injected oocytes, cAMP depolarized the membrane potential (Vm) from -52 to -31 mV and increased membrane conductance (Gm) 10-fold. However, cAMP had no effect on Vm or Gm in uninjected oocytes or in oocytes injected with antisense RNA. The endogenous Ca-activated Cl currents of control oocytes were abolished by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 50 microM) or bath Cl replacement. In contrast, the cAMP-stimulated currents of CFTR-expressing oocytes were DIDS insensitive and were inhibited only approximately 50% when bath Cl was replaced by gluconate or glutamate. In addition, the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB; 50 microM) and diphenylamine-2-carboxylic acid (DPC; 3 mM) reduced the cAMP-evoked currents by only approximately 10%. The stimulated currents of CFTR-expressing oocytes were reduced approximately 30% by 10 mM Ba, suggesting that the Cl-independent current component is due to an increase in K conductance. Our results indicate that expression of CFTR in Xenopus oocytes produces a cAMP-activated Cl current. The Cl-independent current may represent a regulatory action of CFTR on K conductance pathways or a secondary response of the oocyte membrane to the high Cl conductance induced by CFTR expression.


Regulation of the purine salvage pathway in rat liver.

The regulation of purine metabolism in rat liver has been examined under conditions that alter the flux through the pathway. Rats were given intraperitoneal injections of ethanol, sodium acetate, or sodium phosphate to attain body water concentrations of approximately 70, 20, and 10 mM, respectively. The livers were freeze-clamped after 30 min, and extracts were made for the analysis of metabolites, cofactors, purine bases, and nucleosides; homogenates were made for the measurement of the activities and kinetic parameters of seven enzymes that participate in purine salvage. The values of the equilibrium constants of nine reactions were determined in vitro and compared with the ratios of the reactants measured in liver. The changes in phosphoribosylpyrophosphate (PRPP), a key intermediate in both the de novo and salvage pathways of purine metabolism, were directly correlated with the changes in ribose 5-phosphate (ribose-5-P); ([PRPP] = 1.7[ribose-5-P] - 7.4 mumol/kg). Ribose-5-P concentrations in turn could be predicted from the liver content of fructose 6-phosphate and glyceraldehyde 3-phosphate by calculation from the known equilibria. The maximum velocities in the tissue of the seven enzymes measured were calculated from the measured substrate values in the liver and with consideration of other effectors of enzyme activity. PRPP synthetase was the least active of the enzymes measured, indicating a possible rate-limiting step. The delta G of the enzyme steps differed from equilibrium values by factors ranging from 4 (nucleoside phosphorylase) to 10(5) (PRPP synthetase and purine transferase reactions). The regulation of purine salvage appeared to depend on the levels of PRPP and ribose-5-P.


Effects of chloroquine and methylamine on lysosomal enzyme secretion by rat pancreas.

In vivo pancreatic secretion of the lysosomal hydrolase cathepsin B was found to be increased by infusion of the secretagogue caerulein. The basal as well as caerulein-stimulated in vivo rate of cathepsin B was further increased by infusion of either chloroquine or methylamine while neither the basal nor the secretagogue-stimulated rates of amylase secretion were altered by the lysosomotropic agents. These observations indicate that neutralization of the acidic prelysosomal compartment by administration of lysosomotropic agents results in lysosomal enzyme entry, by default, into the regulated secretory pathway. In vitro stimulation of pancreatic acini with caerulein was also found to stimulate cathepsin B secretion. That in vitro rate of cathepsin B secretion stimulated by caerulein was not increased in acini prepared from animals infused with caerulein, chloroquine, or methylamine, but the in vitro rate of cathepsin B secretion stimulated by caerulein was increased in acini prepared from animals infused with caerulein plus either chloroquine or methylamine. Under these conditions, redistribution of cathepsin B from the lysosome-enriched to the zymogen granule-enriched subcellular fraction was noted, and lysosomal enzyme-containing organelles became increasingly fragile. These observations indicate that in vivo secretagogue stimulation increases the degree of diversion of lysosomal hydrolases into the regulated secretory compartment when the prelysosomal compartment has been neutralized with lysosomotropic agents.


Adenosine A1 receptors mediate inhibition of tachykinin release from perifused enteric nerve endings.

A perifused preparation of guinea pig myenteric nerve varicosities (synaptosomes) was used to determine the characteristics of evoked tachykinin release and the inhibition of such release by adenosine analogues. Release of substance P-like immunoreactivity (SP-LI) and neurokinin A-like immunoreactivity (NKA-LI) was evoked by elevated extracellular [K+] in a reversible and repeatable manner. This release was completely abolished in the absence of extracellular Ca2+. Perifusion in the presence of 5'-N-ethylcarboxamidoadenosine (NECA), a nonselective A1/A2 adenosine receptor agonist, decreased K(+)-evoked release of SP-LI and NKA-LI compared with that in the absence of the nucleoside. Similar decrements in peptide release were obtained with N6-cyclopentyl adenosine (CPA), a selective A1 agonist, and 2-[p-(2-carboxyethyl)]phenethylamino-5'-N-ethyl-carboxamidoadenosi ne (CGS 21680), a selective A2 agonist. Response to all nucleosides was graded. Potency order of adenosine analogues was CPA greater than NECA much greater than CGS 21680. Inhibition due to the nucleosides was diminished in the presence of the highly selective A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) while perifusion in the presence of DPCPX alone did not alter evoked release of either peptide. These findings provide direct measurements of inhibitory effects of adenine nucleosides on the release, from enteric nerve endings, of endogenous neuromediators SP and NKA. The findings also directly demonstrate the presence of functional adenosine receptors of the A1 subtype on enteric nerve endings coupled negatively to release of tachykinins. The presence of A2 receptors on enteric nerve endings is neither supported nor excluded.


Developing bronchopulmonary epithelium of the human fetus secretes fluid.

We studied human fetal lung tissue in submersion organ culture to determine whether the bronchopulmonary epithelium secretes fluid during development. In this system the acinar tubules continued to grow, secrete fluid, and become progressively dilated. Baseline transepithelial potential differences (psi t) of -0.5 to -11 mV (mean, -3.8 mV, lumen negative, n = 27) were measured with microelectrodes after 3-8 days in culture, suggesting active electrolyte transport. Bumetanide (500 microM), an inhibitor of chloride secretion in other systems, decreased the basal psi t from -5 +/- 1.5 to -3.2 +/- 1.6 (SE) mV (P less than 0.05, n = 6), suggesting that chloride transport contributed to the voltage. Isoproterenol (5 microM) increased the baseline psi t from -5.6 +/- 2.1 to -9.2 +/- 2.5 (SE) mV (P less than 0.05, n = 4). Subsequent addition of bumetanide inhibited the isoproterenol-induced stimulation of the psi t by 20% (P less than 0.05). 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. (CPT-cAMP, 50 microM) and 3-isobutyl 1-methylxanthine (IBMX, 100 microM) had similar effects, causing an increase in the psi t from -2.2 +/- 0.5 to -8 +/- 1.6 (SE) mV, an effect that was inhibited by the addition of bumetanide (P less than 0.005, n = 6). Both isoproterenol and CPT-cAMP/IBMX produced significant increases in the percentage luminal area of the explants at 12 and 24 h after exposure compared with control. We conclude that 1) the developing bronchopulmonary epithelium (acinar tubules) contributes to lung fluid production in the human fetus, 2) fetal lung fluid secretion is chloride dependent, and 3) chloride secretion and fluid secretion may be stimulated by a beta-agonist and cAMP.


Biological activities and potential therapeutic uses of steel factor. A new growth factor active on multiple hematopoietic lineages.

Recently, a novel growth factor has been cloned that has growth promoting activities on a wide variety of hematopoietic cell lineages. This factor has been referred to as mast cell growth factor, stem cell factor, or kit ligand, and will be referred to here as steel factor. Steel factor stimulates the growth of cells via its interaction with the c-kit proto-oncogene, which is a tyrosine kinase receptor that is expressed on the surface of a number of different cell types. In addition to its effects on hematopoiesis, this factor also plays a role in the development of melanocytes and germ cells. The discovery of this growth factor provided the final piece of the puzzle to explain the molecular defects associated with several well known genetic mutations in mice, and has opened the door to understanding the role of this factor in development. Similar genetic defects may exist in humans as well. The aim of this paper is to review the biological structure and activities of this new growth factor, and to discuss its potential applications in clinical medicine.


Large conducting potassium channel reconstituted from airway smooth muscle.

Microsomal fractions were prepared from canine and bovine airway smooth muscle (ASM) by differential and gradient centrifugations. Surface membrane vesicles were characterized by binding assays and incorporated into planar lipid bilayers. Single-channel activities were recorded in symmetric or asymmetric K+ buffer systems and studied under voltage and Ca2+ clamp conditions. A large-conductance K(+)-selective channel (greater than 220 pS in 150 mM K+) displaying a high Ca2+, low Ba2+, and charybdotoxin (CTX) sensitivity was identified. Time analysis of single-channel recordings revealed a complex kinetic behavior compatible with the previous schemes proposed for Ca(2+)-activated K+ channels in a variety of biological surface membranes. We now report that the open probability of the channel at low Ca2+ concentration is enhanced on in vitro phosphorylation, which is mediated via an adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition to this characterization at the molecular level, a second series of pharmacological experiments were designed to assess the putative role of this channel in ASM strips. Our results show that 50 nM CTX, a specific inhibitor of the large conducting Ca(2+)-dependent K+ channel, prevents norepinephrine transient relaxation on carbamylcholine-precontracted ASM strips. It was also shown that CTX reversed the steady-state relaxation induced by vasoactive intestinal peptide and partially antagonized further relaxation induced by cumulative doses of this potent bronchodilatator. Thus it is proposed that the Ca(2+)-activated K+ channels have a physiological role because they are indirectly activated on stimulation of various membrane receptors via intracellular mechanisms.


Suppressor cytokines and regulation of myelopoiesis. Biology and possible clinical uses.

A number of biologically active and biochemically well-characterized cytokines have been shown to have either direct or indirect suppressive activities on the proliferation/differentiation of myeloid stem/progenitor cells. This article is a brief review of the actions of some of these molecules. These molecules include H-subunit ferritin, macrophage inflammatory protein-1 alpha, the interferons-alpha, -beta, and -gamma, the tumor necrosis factors-alpha and -beta (lymphotoxin), prostaglandins, E1 and E2, inhibin, transforming growth factor-beta, and lactoferrin. I will also review the actions of other suppressor molecules, including a newly identified 8-kd molecule that has not yet been sequenced and a synthetic pentapeptide. Current information is given on the in vitro actions of these molecules together with their activity in vivo in animal models. Possibilities for their clinical use are discussed.


Effect of Bay K 8644 on the magnitude of isoflurane and halothane contracture of skeletal muscle from patients susceptible to malignant hyperthermia.

Isoflurane has a lesser ability than halothane to induce contracture in malignant hyperthermia (MH) muscle in vitro. This does not necessarily imply that isoflurane is not as potent an MH trigger as halothane in vivo. A hypothesis was tested that in vitro treatment with Bay K 8644, an activator of both the dihydropyridine receptors as well as the sodium channels of the T-tubules, potentiates isoflurane-induced MH-susceptible skeletal muscle contracture. In addition to the usual halothane-caffeine test, other muscle bundles were exposed to 10 microM Bay K 8644-halothane and equipotent anesthetic concentrations (expressed in multiple minimum alveolar concentration [MAC]) of isoflurane either alone or combined with Bay K 8644. In 14 MH-susceptible muscle bundles, the mean maximum contracture induced by 2 MAC isoflurane was 0.20 +/- 0.22 g (mean +/- SD), and this value was significantly less than that obtained with 2 MAC halothane (0.68 +/- 0.40 g). Bay K 8644 did not induce muscle contracture on its own but consistently enhanced both the 0.5 MAC isoflurane and halothane to the same maximal isometric tension (1.09 +/- 0.35 g and 1.11 +/- 0.37 g, respectively). Such an effect was not observed in the MH-nonsusceptible group. Under the conditions of this in vitro study, 0.5 MAC isoflurane appears to be as potent as halothane in inducing muscle contracture in skeletal muscle bundles from individuals susceptible to MH.


Tradition, rationality, and power in introductory nursing textbooks: a critical hermeneutics study.

This study investigated the language used to describe, explain, and interpret the concept of nursing process in four introductory nursing textbooks. A critical theory framework was used to reinterpret the ways in which language or linguistic activities construct the social-historical reality of nurses. Themes related to tradition, rationality, and power relationships were explicated. The meanings disclosed from the world of the text provide understandings about how nurses interpret their professional autonomy and how normative structures inform what constitutes authority and responsibility in research, education, and practice.


Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis.

Using partial proteolytic cleavage, the nerve growth factor (NGF) binding site and the epitopes for two anti-NGF receptor (NGFR) monoclonal antibodies were localized on the recombinant extracellular domain (RED) of the NGFR. The RED was prepared in the baculovirus-insect cell system and was purified by immunoaffinity and ion-exchange chromatography. The four cysteine-rich repeat domains and some additional C-terminal sequences were resistant to proteolysis with papain or proteinase K. The Mr 32,000 papain-resistant fragment (P32) and the Mr 30,000 proteinase K-resistant fragment (K30) share the same N terminus as the intact RED and have C termini in the vicinity of residue 170. Even though P32 and K30 have the same N terminus and probably differ by only a small number of amino acids at the C terminus, P32, but not K30, binds 125I-NGF. As judged by Western blot analysis, two anti-NGFR antibodies (ME20.4 and NGFR5) bind to P32 but have a lesser affinity for K30. Since antibody ME20.4 inhibits NGF binding but antibody NGFR5 does not, these antibodies bind to distinct epitopes. However, these epitopes apparently are closely spaced since these antibodies compete with each other for binding to biotinylated RED. NGF, but not the control protein cytochrome c, protects RED from papain digestion. Therefore, the P32 C terminus is important for the expression of the NGF binding site and the antibody-defined epitopes, even though the NGF binding site and antibody-defined epitopes probably are not encoded by the P32 C terminus. These data suggest that complex interactions occur between different regions of the RED, and that optimum NGF binding requires the integrity of multiple RED domains, including a short sequence to the C terminus of residue 170.


Low serotonin and dopamine metabolite concentrations in cerebrospinal fluid from bulimic patients with frequent binge episodes.

Cerebrospinal fluid neurotransmitter metabolite levels were studied to assess whether measures of central serotonin, dopamine, or norepinephrine function are associated with severity of abnormal eating patterns in patients with bulimia nervosa. In comparison with healthy controls (N = 17), hospitalized bulimic patients with a history of binge eating more frequently than twice daily (N = 11) had significantly lower CSF concentrations of 5-hydroxyindoleacetic acid and homovanillic acid. For the total patient group (N = 29), levels of both metabolites were significantly inversely correlated with binge frequency. On the basis of preclinical studies, these results were examined in the context of speculative models in which low central serotonin function might contribute to blunted satiety responses in bulimic patients, while low central dopamine activity might play a role in abnormal hedonic responses to food.


Lymphadenectomy in gastric carcinoma. A prospective and prognostic study.

In 193 gastric resections for adenocarcinoma, lymphadenectomy was prospectively evaluated to quantify the number of lymph nodes and to identify prognostic factors. Overall, 7112 nodes (median, 36.8 per patient) were resected with 27.2% showing metastases. Most nodes were found in the perigastric region. The histologic type and site of the tumor did not influence the number of invaded nodes, but tumor stage and quality of the resection (curative/palliative) did. By multivariate analysis the tumor stage, curative vs palliative resections, and the number of metastatic lymph nodes in curative resections were independent prognostic factors. Patients with less than six metastatic nodes showed a survival not significantly different from that of patients with normal nodes. These patients may be well treated by surgery alone, but the other patients may require multimodal therapy to improve their prognosis.


Human cytomegalovirus nuclear and cytoplasmic dense bodies.

One of the characteristic features of cytomegalovirus (CMV) replication is the formation of cytoplasmic dense bodies. Recent findings revealed similar structures also in the nuclei of CMV-infected cells. By transmission electron microscopy, immuno electronmicroscopy, and cytochemistry, we have studied the morphogenetic steps and macromolecular composition of both structures. Our results show that both structures contain DNA, RNA and viral antigenic proteins. Nuclear dense bodies are probably an expression of a stimulated cellular metabolism, while cytoplasmic dense bodies may represent the site where surplus cellular and viral molecules are stored before being eliminated.


Detection and separation of two kinds of acidic arginine amidases from boar sperm using lima bean trypsin inhibitor and aprotinin affinity adsorptions.

Two kinds of acidic arginine amidase activity were found in boar sperm. One enzyme was separated by a treatment consisting of lima bean trypsin inhibitor (LBTI) affinity adsorption and elution. The other enzyme was separated by aprotinin affinity adsorption and elution through the same solutions as those used for first enzyme; the two enzymes provisionally named boar sperm acidic arginine amidases 1 (BSAA-1) and 2 (BSAA-2), respectively. The amidolytic activity of BSAA-1 was increased by high concentrations of calcium chloride, while the activity of BSAA-2 was independent of calcium chloride. Their behavior with LBTI and aprotinin, and profiles of their substrate specificities, were also different. The affinity of LBTI to BSAA-1 was approximately 14 times higher than that to BSAA-2.


Mode of neutralization of lactate dehydrogenase-elevating virus by polyclonal and monoclonal antibodies.

Neutralization of the infectivity of [3H]uridine-labeled lactate dehydrogenase-elevating virus (LDV) by polyclonal mouse or rabbit antibodies to the envelope glycoprotein of LDV, VP-3, or by neutralizing monoclonal antibodies (mAb) that recognize a different epitope on VP-3 than the polyclonal antibodies correlated with an increase in the sedimentation rate of LDV from 230 S to greater than or equal to 270 S. Incubation of LDV with normal mouse plasma or non-neutralizing mAbs to LDV VP-3 had no effect on its sedimentation rate. Similarly, incubation of a neutralization escape variant of LDV with the mAb used in its selection had no effect on its sedimentation rate, whereas neutralization of this variant by polyclonal mouse or rabbit anti-VP3 antibodies increased the sedimentation rate. Neutralization of LDV infectivity was only observed at high antibody/virion ratios and often was followed by loss of the viral RNA. The results suggest that neutralization of LDV infectivity results from binding of multiple antibody molecules that recognize specific epitopes on the viral envelope glycoprotein and ultimately leads to disintegration of the virions.


Hartmann's procedure for carcinoma of the rectum and sigmoid colon.

A review of the Hartmann's operation for patients with rectal and sigmoid cancer over an 18 year period is presented. There were 1063 patients who had a resection for carcinoma of the rectum or sigmoid colon and 4.4% of these had a Hartmann's procedure. This operation was particularly useful in the management of patients who presented with a proximal obstruction or perforation at the tumour site. It was also effective in the elective treatment of elderly unfit patients who had locally advanced tumours or those with distant metastases. Re-anastomosis is recommended in those patients who are relatively fit and have had a potentially curative resection.


Heparin-binding fibronectin fragments containing cell-binding domains and devoid of hep2 and gelatin-binding domains promote human embryo fibroblast proliferation.

The proliferation promoting activity of various proteolytic fragments of human plasma fibronectin was assayed. Study of this activity in fragments, purified by affinity chromatography, has shown that only heparin-binding fragments were capable of promoting fibroblast proliferation while gelatin- and fibrin-binding fragments were not. Heparin-binding fragments with high affinity for heparin were characterized by high activity levels while those with low heparin affinity were inactive. Heparin-binding fragments with the highest proliferation promoting activity contained the cell-binding domain and were virtually devoid of the hep2, hep1 and gelatin-binding domains.


Differential regulation of the imipramine-sensitive serotonin transporter by cAMP in human JAr choriocarcinoma cells, rat PC12 pheochromocytoma cells, and C33-14-B1 transgenic mouse fibroblast cells.

The imipramine-sensitive serotonin transporter appears to be the receptor for clinically important antidepressants. Some studies suggest that this protein may fall under the influence of abnormal and as yet uncharacterized regulatory effects during depressive illness. Despite these putative disease-related effects, regulation has never been demonstrated in either the platelet or synaptosome model systems. Here we demonstrate for the first time that the imipramine-sensitive serotonin transport activity in either JAr choriocarcinoma or PC12 pheochromocytoma cell lines is subject to regulation by cAMP. Unexpectedly, the regulatory effect is opposite in the two cases, causing stimulation in JAr (increased Vmax) and inhibition in PC12 (kinetically complex). Appearance of these kinetic effects lagged 15 to 20 hours behind peak cAMP levels. The results are consistent with the interesting possibility that different tissues may express isoforms of the recently cloned serotonin transporter cDNAs. We suggest, therefore, that JAr and PC12 are attractive models in which to pursue detailed analysis of serotonin transport and its manner of regulation by cAMP.


Glycosulfatase activity of Helicobacter pylori toward gastric mucin.

A glycosulfatase activity toward sulfated gastric mucus glycoprotein was identified in the extracellular material elaborated by H. pylori, a bacteria implicated in the etiology of gastric disease. Upon acetone precipitation, an active enzyme fraction at 64% acetone was obtained which on SDS-PAGE gave a major 30kDa protein band. The H. pylori glycosulfatase exhibited maximum activity (314.8 pmol/mg protein/h) at pH 5.7 in the presence of Triton X-100 and CaCl2, and was capable of removal of the sulfate ester groups situated at C-6 of N-acetylglucosamine, galactose and glucose. However, the enzyme was ineffective toward galactosylceramide and lactosylceramide sulfates which contain the sulfate ester group on C-3 of galactose. The results suggest that H. pylori is capable of overcoming the interference by sulfated mucus glycoprotein with its colonization of gastric mucosa.


Propidium iodide staining correlates with the extent of DNA degradation in isolated nuclei.

Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37 degrees C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluorescence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogenous endonuclease: Zn2+ and EGTA. However, the presence of Zn2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations.


Absence of a putative ATP/GTP binding site in the rat prolactin receptor.

Two types of the long form of the rat prolactin receptor have been identified by two independent groups. One type of receptor has a consensus sequence for a putative ATP/GTP binding site in the cytoplasmic domain, while the other does not. Since this site would confer kinase activity to the prolactin receptor and represent an important element in the process of signal transduction, we wanted to clarify this discrepancy. To that end, three procedures capable of detecting a point mutation were performed: Southern analysis of reverse transcribed polymerase chain reaction (PCR) products, allele-specific PCR, and S1 nuclease analysis. All results clearly indicated that the sequence for the putative ATP/GTP binding site does not exist in the prolactin receptor.


Verapamil blocks basal and angiotensin II-induced RNA synthesis of rat aortic vascular smooth muscle cells.

We evaluated VER effect on RNA synthesis of quiescent and angiotensin II (AII)- stimulated cultured rat aortic vascular smooth muscle cells (VSMC). In a dose-dependent manner, VER decreased [3H]uridine uptake by quiescent VSMCs (ED50 7 x 10(-6)M), an effect that was shared by other calcium antagonists, but to a variable degree. VER caused a significant effect within 3 hours and attained a maximal effect at 7 hours. In addition VER caused a 22 +/- 2% decrease in [3H]uridine uptake by VSMCs stimulated with 10% fetal bovine serum, while it completely abolished [3H]uridine uptake by VSMCs induced by AII. We conclude that VER decreases basal and inhibits AII-induced increase in mRNA synthesis of VSMCs. These data may explain in part how VER causes a decrease in vascular resistance and alters the vasoconstrictor effect of AII.


Tyrosine phosphorylation and its possible role in superoxide production by human neutrophils stimulated with FMLP and IgG.

Superoxide production by human neutrophils stimulated with FMLP and soluble aggregated human IgG were inhibited in a dose dependent manner by two kinds of tyrosine kinase inhibitors, erbstatin and genistein. Superoxide production stimulated with surface bound IgG, however, was scarcely inhibited by either inhibitor. Protein tyrosine phosphorylation studies with immunoblotting revealed specific tyrosine phosphorylation of a 40 Kd protein by soluble aggregated and surface bound IgG, and that of a 39 Kd protein, as well as the 40 Kd protein, by FMLP. These were all inhibited by the tyrosine kinase inhibitors. These data suggest that superoxide production induced by FMLP and soluble aggregated IgG are, at least in part, tyrosine kinase dependent, but the tyrosine kinases and/or substrates of tyrosine kinases involved may be different. In addition, tyrosine kinase independent pathways are also suggested to be involved in superoxide production by stimulation with surface bound IgG.


Molecular cloning of a cDNA encoding a novel member of the mouse glutamate receptor channel family.

The primary structure of a novel putative subunit of the mouse glutamate receptor channel, designated as delta 1, has been deduced by cloning and sequencing the cDNA. The delta 1 subunit shows 21-25% amino acid sequence identity with previously characterized rodent glutamate receptor channel subunits and thus may represent a new subfamily of the glutamate receptor channel.


Premature rupture of the membranes: effect of penicillin prophylaxis and long-term outcome of the children.

To assess the value of prophylaxis with penicillin in women with premature rupture of membranes (PROM) and the long-term outcome of children born after prolonged PROM, we studied 221 women with this condition. Penicillin (5 mu twice, 6 hours apart) was given intravenously to 50 women and placebo to 51 women, whereas 76 comparable patients were treated without penicillin or placebo. The time interval between PROM and delivery ranged from 14 hours to 56 days. Chorioamnionitis occurred more frequently (p less than 0.05) among patients with placebo (14%) than among those treated with penicillin (2%). One puerperal endometritis appeared in the placebo group compared with none in the penicillin group. One newborn (1.7%) born to a mother with placebo prophylaxis developed septicemia, compared with none in the penicillin group. The outcomes of pregnancies complicated with PROM treated without penicillin or placebo were comparable with those in the placebo group. In addition, we compared somatic and psychomotor development of 159 children born to mothers with prolonged PROM (more than 12 hours; mean, 5.6 days; range, 14 to 1344 hours) with those of 43 children born at similar gestational age within 5 hours after PROM. No pulmonary sequelae could be linked to the long time period between PROM and delivery, but infants born soon after PROM more often (p less than 0.05) had cerebral palsy (8 of 43, 18.6%) than did infants born after prolonged PROM (7 of 159; 4.4%). We conclude that, in cases with PROM, penicillin prophylaxis decreases maternal and neonatal infectious morbidity and that the long interval between PROM and delivery does not impair the long-term outcome for these children.


Cultivation of mammalian cells on macroporous microcarriers.

Adherent cells can be cultivated in a stirred-tank bioreactor by attaching to microcarriers. Macroporous microcarriers, with their intraparticle space and surface area for cell growth, can potentially support a higher cell concentration than conventional microcarriers, which support cell growth only on the external surface. Chinese hamster ovary (CHO) cells and green monkey kidney (Vero) cells were cultivated on macroporous microcarriers, Cultispher-G. Cells attached to the microcarriers at a slow rate and grew to a high density. Thin sections of the microcarriers demonstrate that cells were initially on the exterior of the microcarriers and migrated into the interior as cell concentration increased. Vero cells cultivated on these microcarriers were successfully used for the production of vesicular stomatitis virus (VSV).


P0 promoter directs expression of reporter and toxin genes to Schwann cells of transgenic mice.

We generated transgenic mice that specifically express foreign genes in myelinating Schwann cells. A 1.1 kb segment of 5' flanking sequence from the rat P0 gene was used to drive expression of the genes encoding human growth hormone (hGH) and bacterial diphtheria toxin A chain (DT-A). The P0-hGH mice expressed hGH in myelinating Schwann cells, but not in nonmyelinating Schwann cells, the central nervous system, or any other tissue assayed. This expression was activated on a developmental schedule comparable to that of endogenous myelin gene expression. One line of P0-DT-A mice developed a generalized hypomyelinating peripheral neuropathy, with Schwann cell deficiency apparent in newborn animals. Peripheral nerves from adult mice of this line displayed morphological alterations ranging from completely denuded axons to myelinated Schwann cells undergoing degeneration, although occasional Schwann cells were able to form apparently normal myelin sheaths. Pronounced secondary changes, including proliferation and retraction of processes, occurred in the nonmyelinating Schwann cells of these P0-DT-A mice.


Characterization of a second human pulmonary surfactant-associated protein SP-A gene.

Pulmonary surfactant is a lipid-protein complex involved in maintaining alveolar stability. SP-A is the major surfactant-associated protein of 26 to 38 kD. A human SP-A gene (SP-A I) and two distinct SP-A cDNAs, MPSAP 1A and MPSAP 6A, have been reported previously. We have isolated and characterized a second human SP-A gene (SP-A II), which appears to code for the mRNA corresponding to the previously described MPSAP-1A cDNA. Both genes consist of five exons, a consensus recognition sequence for initiation, TATAAA, and a polyadenylation signal sequence. Significant divergence in the two genes is observed throughout. The divergence is highest in the upstream region, intron I, exon III, and noncoding portion of exon V. The coding regions of all other exons and the introns show much lower divergence. Transcripts from both genes were found in adult human lung, using gene-specific oligonucleotide probes in Northern blot analysis.


A bispecific antibody detects cytotoxic T lymphocytes of unknown antigen specificity in patients with granular lymphocyte-proliferative disorders.

A simple and sensitive method for the detection of cytotoxic T lymphocytes (CTL) of unknown antigen specificity was investigated. Using anti-CD3 Fab' x anti-CD10 Fab' bispecific antibody or intact anti-CD3 monoclonal antibody, we induced cytotoxicity for CD10+, Fc gamma receptor-positive Daudi target cells in peripheral blood mononuclear cells (PBMC) obtained from patients with granular lymphocyte-proliferative disorders (GLPD). The results indicated that the bispecific antibody was much more efficient than the intact anti-CD3 monoclonal antibody in inducing cytotoxicity. Since CD3+CD4-CD8+ granular lymphocytes in patients with GLPD are considered to be in vivo-primed CTL of unknown antigen specificity, this bispecific antibody method may be useful for detecting in vivo-primed CTL within PBMC. By using this bispecific antibody, it will be possible to detect circulating in vivo-primed CTL in other clinical conditions.


Surgical glove perforation and maxillofacial trauma: to plate or wire?

The technique of interdental wiring was compared with a small-plate osteosynthesis technique (SPO) in a prospective study of surgical glove perforations acquired during the treatment of mandibular fractures. Using the SPO technique there was a significant reduction in the incidence of skin penetrating injuries in the surgeon (P less than 0.005) and assistant surgeon groups (P less than 0.05). The reduction in the incidence of glove perforation in the assistant surgeon group was very highly significant in the SPO series (P less than 0.001). The reduction in the surgeon group was not significant. No difference was noted in the scrub nurse group. The small-plate osteosynthesis technique has the advantage of reducing the risk of intraoperative cross-infection transmitted by hand contamination or penetrating injury. The recommended precautions for preventing the transmission of blood-borne pathogens are reviewed.


Stable target-sensitive immunoliposomes.

Interaction of immunoliposomes composed of dioleoylphosphatidylethanolamine (DOPE) (80%), dioleoylphosphatidic acid (DOPA) (20%), and a small amount of specific antibody with Herpes Simplex virus (HSV) were studied by detecting the immune-dependent lysis of liposomes. DOPA was used as the principal stabilizer of the immunoliposomes. Antibodies conjugated with N-glutarylphosphatidylethanolamine or oxidized GM1 served as the target-specific ligands of immunoliposomes. These immunoliposomes (d = 160-180 nm) were stable for at least one month when stored at 4 degrees C. However, they undergo a rapid aggregation and lysis reaction in the presence of a membrane-bound target such as intact HSV virions. We have also employed epitope peptide-containing liposomes (target liposomes) to mimic the virus and showed that the immunoliposomes could be aggregated and lysed by the target liposomes in an antigen-dependent manner. Immunoliposome lysis could be accelerated by increasing the incubation temperature to 60-70 degrees C. No immunoliposome lysis was observed if the target liposomes were absent, indicating the prolonged stability of the immunoliposomes. Liposome lysis was always accompanied by liposome aggregation. However, the aggregation-induced liposome destabilization is unique to the HII phase-forming lipids such as DOPE. DOPC-containing immunoliposomes did not lyse despite the fact that massive liposome aggregation had taken place.


Interaction of a substance P agonist and of substance P antagonists with lipid membranes. A thermodynamic analysis.

The molecular characteristics of the neuropeptide substance P (SP), its agonist [Sar9,Met-(O2)11]SP, and three of its antagonists [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, [D-Arg1,D-Trp7,9,Leu11]SP, and [D-Pro2,D-Trp7,9]SP were investigated at the air/water interface and when bound to lipid monolayers and bilayers. Measurement of the Gibbs adsorption isotherm showed that the surface areas of SP and its agonist (240 +/- 5 A2 at biologically relevant concentrations) were distinctly larger than those of the antagonists (138 +/- 5 A2) [Seelig, A. (1990) Biochim. Biophys. Acta 1030, 111-118]. The surface activity of the peptides increased in the order [Sar9,Met(O2)11]SP less than SP less than [D-Pro2,D-Trp7,9]SP less than [D-Arg1,D-Trp7,9,Leu11]SP = [D-Arg1,D- Pro2,D-Trp7,9,Leu11]SP and correlated with the respective binding affinities to lipid membranes. The agonist did not insert into neutral and negatively charged bilayers or into densely packed lipid monolayers (at surface pressures greater than 31 mN/m). In contrast, the three antagonists gave rise to a strong binding both to neutral and to charged lipid monolayers and bilayers. The degree of binding was evaluated from the area increase of lipid monolayers upon peptide insertion, and the binding isotherms were analyzed in terms of the Gouy-Chapman theory. At the monolayer-bilayer equivalence pressure of approximately 32 mN/m, the binding can be described by a surface partition equilibrium with binding constants of (4.5 +/- 0.1) x 10(3) M-1 for [D-Pro2,D-Trp7,9]SP and (1.3 +/- 0.1) x 10(4) M-1 for both [D-Arg1,D-Trp7,9,Leu11]SP and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP for pure palmitoyloleoylphosphatidylcholine (POPC) membranes.(ABSTRACT TRUNCATED AT 250 WORDS)


Binding of tachyplesin I to DNA revealed by footprinting analysis: significant contribution of secondary structure to DNA binding and implication for biological action.

In view of the cationic amphipathic structure of tachyplesin I and antiparallel beta-sheet as a general DNA binding motif, DNA binding of the antimicrobial peptide has been examined. Several footprinting-like techniques using DNase I protection, dimethyl sulfate protection, and bleomycin- (BLM-) induced DNA cleavage were applied in this study. Some distinct footprints with DNase I are detected, and also the sequence-specific cleavage mode of the BLM-Fe(II) complex clearly is altered in the presence of tachyplesin I. In addition, methylation of the N-7 residue of guanine situated in the DNA major groove is not entirely inhibited (or activated) by tachyplesin I. The results suggest that tachyplesin I interacts with the minor groove of DNA duplex. Disappearance of the footprints by dithiothreitol-treated tachyplesin I and Ala-tachyplesin strongly suggests a significant contribution of secondary structure containing an antiparallel beta-sheet to the DNA binding of tachyplesin I. This is the first report on DNA interaction with a small peptide which contains a unique antiparallel beta-sheet structure. The mechanism for antimicrobial action of tachyplesin I has also been inferred.


Identification of intermolecular RNA cross-links at the subunit interface of the Escherichia coli ribosome.

32P-Labeled 70S ribosomes and polysomes were isolated from cultures of Escherichia coli and treated with the cross-linking reagent bis(2-chloroethyl)methylamine. Intermolecular 16S-23S RNA cross-linked complexes were separated from other products of the cross-linking reactions by a two-step sucrose density gradient centrifugation procedure and subjected to oligodeoxynucleotide-directed partial nuclease digestions with RNase H. Cross-linked RNA fragments released by such directed digests were resolved by two-dimensional gel electrophoresis and analyzed using classical oligonucleotide fingerprinting techniques. Two distinct intermolecular cross-links between the 16S and 23S RNA could be localized in this manner, involving positions 1408-1411 and 1518-1520 in the 16S RNA sequence and positions 1912-1920 in the 23S RNA sequence. These data provide the first direct topographical links between the RNA of the 30S and 50S subunits in the functional ribosome and, together with previous topographical data concerning the three-dimensional folding of the rRNA, demonstrate that there is a tight cluster at the ribosomal interface both of sites implicated in ribosomal function and of posttranscriptionally modified nucleotides in the rRNA.


Alteration in DNA cross-linking and sequence selectivity of a series of aziridinylbenzoquinones after enzymatic reduction by DT-diaphorase.

DT-diaphorase (DTD) mediated reduction of a series of 2,5-bis-substituted-3,6-diaziridinyl-1,4-benzoquinones was found to increase the level of DNA interstrand cross-linking (ISC) formed at neutral pH with an enhancement observed as the pH was decreased to 5.8. The analogues used were symmetrically alkyl-substituted carbamoyl ester analogues of AZQ (D1-D7), 3,6-diaziridinyl-1,4-benzoquinone (DZQ), the 2,5-dimethyl derivative (MeDZQ), and a 2,5-bis[(2-hydroxyethyl)amino] analogue (BZQ). At pH 5.8, the level of DNA ISC induced by enzymatic reduction was as follows: DZQ greater than MeDZQ much greater than D1 (methyl) greater than D3 (n-propyl) greater than D2 (AZQ; ethyl) greater than D5 (n-butyl) greater than D7 (sec-butyl) greater than D4 (isopropyl) D6 greater than (isobutyl). A similar trend was observed at pH 7.2. The level of DNA ISC induced by BZQ, which is not a substrate for DTD, was not increased by enzymatic reduction. Dicumarol, a known inhibitor of DTD, was capable of inhibiting the DNA ISC induced by these quinones upon enzymatic reduction. MeDZQ and DZQ reacted with guanines, as measured by Maxam and Gilbert sequencing, with a sequence selectivity similar to that of the nitrogen mustard class of antitumor agents. Enzymatic reduction of DZQ and MeDZQ by DTD was found to alter their sequence-selective alkylation. Reduced DZQ showed enhanced guanine alkylation in 5'-GC-3' sequences and new sites of adenine alkylation in 5'-(A/T)AA-3' sequences. Reduced MeDZQ only showed new sites of adenine alkylation at 5'-(A/T)AA-3' sequences but no enhancement of guanine alkylation. The new sites of adenine alkylation were found to be inhibited in the presence of magnesium and rapidly converted into apurinic sites.(ABSTRACT TRUNCATED AT 250 WORDS)


Halide permeation through 10 pS and 20 pS anion channels in human airway epithelial cells.

Halide permeability sequences were obtained from reversal potential measurements of single-channel currents through 10 pS and 20 pS anion channels in human airway epithelial cells. The sequences obtained were Cl- greater than I- greater than Br- greater than or equal to F- for the 10 pS channel and Cl- greater than I- greater than or equal to Br- greater than or equal to F- for the 20 pS channel. However, the permeability differences were not large, the greatest being 0.66 for the ratio of fluoride to chloride permeability in the 20 pS channel. Single-channel currents were also measured with solutions of constant halide concentration but varying ratios of chloride to fluoride ions. An anomalous mole fraction effect was observed for the 20 pS channel but not for the 10 pS channel, suggesting that the former is a multi-ion channel. Comparison of the halide permeability sequences of these two channels with those of whole-cell currents in other epithelial cells does not support their involvement in any of the known whole-cell epithelial currents.


Mammalian homeobox genes in normal development and neoplasia.

This review aims at providing an evaluation of the currently available data concerning the role of homeobox genes in mammalian embryonic pattern formation, and their involvement in oncogenic processes. The literature dealing with mouse and human homeobox genes is covered, with some excursions into Xenopus and chicken work because studies regarding particular aspects of development in the latter systems often complement those in mammals. Other studies in amphibians, chicken, and fish are omitted as they fall outside the scope of this survey devoted to mammalian genes. Emphasis is placed on expression and regulation during normal embryogenesis, on present hypotheses regarding gene function, on effects on development of modifying the expression patterns, and on observed aberrations in structure or expression associated with and possibly causally linked to neoplasia. The studies specifically dealing with the structure of the homeodomain and the mechanism of its interactions with DNA, which have been recently reviewed, are not considered here. The most thoroughly studied mammalian homeobox genes bear sequence homology to the Drosophila Antennapedia (Antp) homeotic gene and are thought to be involved in determination of regional identity along the anteroposterior (A-P) axis. Studies on these genes, the HOX genes, are oftem compared with related investigations in flies. In addition to the HOX genes, a number of genes containing a homeobox related to that in other Drosophila developmental control genes have been isolated from the mammalian genome and are being intensively studied. Homeodomains divergent from the Antp prototype have been discovered in an increasing number of transcription factors. Some of these may play a crucial role in local pattern formation, while others are tissue-specific or ubiquitous transcription regulators. As is unsurprising of genes, whose products regulate transcription, some of the homeobox-containing genes have been associated with malignancy, usually on the basis of abnormal structure, or deletions found to occur in tumor cells. The degree of probability that these mutated forms were causative in the generation of the tumors in which they were found is discussed.


Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies.

A 190-kDa centrosomal protein interacts with microtubules when Drosophila embryo extracts are passed over microtubule-affinity columns. We have obtained a partial cDNA clone that encodes this protein. Using a fusion protein produced from the clone, we have developed a novel immunoaffinity chromatography procedure that allows both the 190-kDa protein and a complex of proteins that associates with it to be isolated in in a single step. For this procedure, the fusion protein is used as an antigen to prepare rabbit polyclonal antibodies, and those antibodies that recognize the 190-kDa protein with low affinity are selectively purified on a column containing immobilized antigen. These low-affinity antibodies are then used to construct an immunoaffinity column. When Drosophila embryo extracts are passed over this column, the 190-kDa protein is quantitatively retained and can be eluted in nearly pure form under nondenaturing conditions with 1.5 M MgCl2, pH 7.6. The immunoaffinity column is washed with 1.0 M KCl just before the elution with 1.5 M MgCl2. This wash elutes 10 major proteins, as well as a number of minor ones. We present evidence that these KCl-eluted proteins represent additional centrosomal components that interact with the 190-kDa protein to form a multiprotein complex within the cell.


Functional expression and growth factor activation of an epitope-tagged p44 mitogen-activated protein kinase, p44mapk.

Mitogen-activated protein kinases (MAPKs) or extracellular signal-regulated kinases (ERKs) are serine/threonine kinases of apparent Mr 42-44 kDa that are rapidly activated by a variety of extracellular signals in many cell types. This activation coincides with their phosphorylation on tyrosine and threonine residues, and these covalent modifications are required for full activity of the enzymes. They are thought to play a pivotal role in integrating and transmitting transmembrane signals for growth and differentiation. Here, we report the cloning, sequence, and functional expression in fibroblasts of the hamster p44 MAP kinase (p44mapk). The protein deduced from the nucleotide sequence of an almost full-length cDNA is 98.6% homologous to the rat p44mapk (ERK1). To distinguish the expression of the cloned cDNA from the endogenous p44mapk, we fused to the 5' end of the cDNA an initiating codon followed by an influenza hemagglutinin 9-residue peptide epitope (HAP). The chimeric kinase HAP/p44mapk, under transcriptional control of the cytomegalovirus promoter, was stably expressed in Chinese hamster lung fibroblasts in a functional form. We show that its basal activity, measured by phosphorylation of the substrate myelin basic protein, is activated severalfold (up to 25) by the mitogens alpha-thrombin, platelet-derived growth factor, and fetal calf serum. In addition, we report that in response to alpha-thrombin, this activation is rapid (6-fold in 1 min), biphasic (first peak at 5 min, second broader peak at 1-2 h), persistent (for greater than or equal to 4 h), and parallel to an increased phosphorylation on tyrosine.We conclude that the constructed and stably expressed chimera, HAP/p44mapk, has retained apparently all the hormonal regulation features of the endogenous form. This system now offers the possibility to study structure-function relationships and to determine the role of this kinase in growth control.


Electrophoretic resolution and fluorescence detection of N-linked glycoprotein oligosaccharides after reductive amination with 8-aminonaphthalene-1,3,6-trisulphonic acid.

The relative mobilities of various N-linked oligosaccharides reductively aminated to the charged fluorophore 8-amino-naphthalene-1,3,6-trisulphonic acid (ANTS) were determined by electrophoresis on high-density polyacrylamide slab gels. Each ANTS-derivatized oligosaccharide was assigned a relative migration index (RMI) expressed in terms of glucose equivalents, which was conveniently estimated by reference to a homologous series of ANTS--maltooligosaccharides run on each gel as oligosaccharide size standards. High-mannose-, complex- and hybrid-type structures were generally well resolved and easily visualized at picomole levels by simple UV light excitation. Application of these methods for the qualitative analysis of the oligosaccharides released from bovine fetuin and bovine asialofetuin by peptide-N-glycosidase F illustrates the usefulness of these techniques as fast, simple, and inexpensive tools for the characterization of N-linked oligosaccharides attached to glycoproteins.


c-Kit-kinase induces a cascade of protein tyrosine phosphorylation in normal human melanocytes in response to mast cell growth factor and stimulates mitogen-activated protein kinase but is down-regulated in melanomas.

The proto-oncogene c-Kit, a transmembrane receptor tyrosine kinase, is an important regulator of cell growth whose constitutively active oncogenic counterpart, v-kit, induces sarcomas in cats. Mutations in murine c-kit that reduce the receptor tyrosine kinase activity cause deficiencies in the migration and proliferation of melanoblasts, hematopoietic stem cells, and primordial germ cells. We therefore investigated whether c-Kit regulates normal human melanocyte proliferation and plays a role in melanomas. We show that normal human melanocytes respond to mast cell growth factor (MGF), the Kit-ligand that stimulates phosphorylation of tyrosyl residues in c-Kit and induces sequential phosphorylation of tyrosyl residues in several other proteins. One of the phosphorylated intermediates in the signal transduction pathway was identified as an early response kinase (mitogen-activated protein [MAP] kinase). Dephosphorylation of a prominent 180-kDa protein suggests that MGF also activates a phosphotyrosine phosphatase. In contrast, MGF did not induce proliferation, the cascade of protein phosphorylations, or MAP kinase activation in the majority of cells cultured from primary nodular and metastatic melanomas that grow independently of exogenous factors. In the five out of eight human melanoma lines expressing c-kit mRNAs, c-Kit was not constitutively activated. Therefore, although c-Kit-kinase is a potent growth regulator of normal human melanocytes, its activity is not positively associated with malignant transformation.


Epithelioid angiosarcoma of the adrenal gland with cytokeratin expression. Report of a case with accompanying mesenteric fibromatosis.

A case report of epithelioid adrenal angiosarcoma is presented. Tumor cells showed expression of cytokeratin, Factor VIII-related antigen, Ulex europaeus agglutinin-I, and vimentin. The patient also was found to have mesenteric fibromatosis (abdominal desmoid tumor) and an elevated serum level of estradiol. The authors discuss the unique appearance of these rare tumors, their relationship to hyperestrinism, and review the recent data in the literature showing cytokeratin expression by malignant epithelioid vascular tumors.


Flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin, its subunits, and fragments on human cancer cells.

A quantitative flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin (hCG), its subunits, and fragments on human cancer cells was developed using a double-antibody reaction; a flow cytometry with a 2-W argon laser, standard settings, and filters for fluorescein isothiocyanate use; commercially available software; and the ectopic hCG producer CCL 2 HeLa cells from the American Type Culture Collection (ATCC) as a cell control to standardize the reagents and for overall quality control. Twenty-two monoclonal antibodies (MoAb) and immunoglobulin G fractions from three rabbit polyclonal antisera were tested for effects of antibody concentration (titration), reproducibility at different levels of epitope expression, and variability of epitope expression to select appropriate primary antibodies. Based on the results of the various tests, three polyclonal immunoglobulin G antibodies and a panel of nine MoAb directed to epitopes located in five different regions on the hCG molecule were selected as first antibodies. Their specificity was determined by using two unrelated MoAb of the same isotype at the same concentration to replace the primary MoAb and by a competition experiment. The unrelated MoAb also were used for the selection of the appropriate control fluorescence profile needed for the software. The unique characteristics of this method were: the use of living cells, standardized reagents, internal and external quality control, and the highest sensitivity, which could detect as few as 10(3) molecules of fluorochrome per cell. Serial analyses of the ATCC CCL 2 HeLa cells and two of its variants and of the eutopic hCG producer JEG-3 choriocarcinoma cells revealed the expression of membrane-associated epitopes of intact hCG, its subunits, and fragments by a high percentage of the cells, indicating that the expression of these sialoglycoproteins by these two different types of cancer cells is a common phenotypic characteristic.


Expression of membrane-associated human chorionic gonadotropin, its subunits, and fragments by cultured human cancer cells.

The expression of human chorionic gonadotropin (hCG), its subunits, and fragments on the cell membrane of cultured human cancer cells was investigated using a flow cytometric method. This method uses living cells; a double-antibody reaction; a flow cytometer with an argon laser, standard settings, and filters for fluorescein isothiocyanate; commercially available software; the American Type Culture Collection (ATCC) CCL 2 HeLa cell line as cell control and overall quality control; polyclonal rabbit antisera raised against the hCG dimer, its alpha subunit (hCG alpha), and its beta subunit (hCG beta); and a panel of monoclonal antibodies (MoAb) recognizing different epitopes on the intact hCG molecule, its subunits, and fragments. The purified immunoglobulin G fractions from the polyclonal antisera were used to estimate the total expression of the membrane-associated glycoproteins; the MoAb were used to detect the expression of epitopes of the hCG dimer, its subunits, and fragments. The results of the analyses done on cells from 74 established cancer cell lines of different types and origins (including 52 carcinomas, 10 sarcomas, 4 leukemias, 6 lymphomas, and 2 retinoblastomas) showed variable degrees of reactivity in a great percentage of cells in all cell lines studied with MoAb directed against different conformational epitopes of intact hCG (hCG-holo), hCG beta, hCG beta-free, the carboxy terminal peptide (CTP) of hCG beta, and an epitope of hCG alpha. The expression of the membrane-associated epitopes of hCG and its subunits was found to be a phenotypic marker characteristic of all evaluated cultured human cancer cell lines, irrespective of their type or origin. There were, however, quantitative and qualitative differences in the expression of the different epitopes. Thus, hCG beta, free and as part of hCG-holo, recognized by the MoAb against hCG beta-CTP, was expressed by a high percentage of cells of most cell lines. There was great variability in the expression of hCG-holo, recognized by MoAb B109. For this reason some groups of cancers expressed larger amounts of incompetent hCG alpha and/or hCG beta than others. Cell lines derived from adenocarcinomas of the lung were the only exception to this general finding; the expression of small amounts of hCG-holo was caused by a low degree of hCG alpha synthesis.


Determination of plasma urokinase-type plasminogen activator antigen in patients with primary liver cancer: characterization as tumor-associated antigen and comparison with alpha-fetoprotein.

We determined the plasma levels of urokinase-type plasminogen activator (u-PA) antigen and alpha-fetoprotein (AFP) in 44 patients with different stages of liver cirrhosis and in 29 patients with liver cirrhosis-based primary liver cancer at the time of first clinical detection of the malignant disease. Sensitivity values of u-PA and AFP in detecting primary liver cancer were 57 and 62%, respectively, and specificity values were 95 and 86%, respectively. A combination of both markers led to a significant increase of sensitivity to 89.7%. The specificity of the combination of both markers was 97.3%. In tumor patients with unilocular disease and tumor patients with multicentric disease and/or metastatic spread, similar sensitivity values could be obtained with both markers. Therefore, a combination of u-PA and AFP can increase the accuracy of detection of primary liver cancer, especially in chronic liver diseases known to be predisposing for primary liver cancer, e.g., liver cirrhosis of long duration.


Irradiation-induced expression of O6-methylguanine-DNA methyltransferase in mammalian cells.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein which plays an important role in chemotherapy, mutagenesis, and carcinogenesis. The specific activity of MGMT in female rat liver can be induced by approximately 20-fold by treatment of the rats with gamma-irradiation. Maximum response occurred 48 h after 15 Gy irradiation. MGMT levels in male rats were induced by only 3-fold. MGMT activity was also induced by irradiation of rat hepatoma H4IIE cells with a 3-fold increase noted after treatment with 3 Gy. Northern analysis and nuclear run-on assays indicated that the induction of MGMT was regulated at the transcriptional level. The radiation-mediated increase in MGMT was blocked by H7, a protein kinase inhibitor, but not by H89, an inhibitor of protein kinase A. Hydroxyl radicals may play a role in the induction mechanism since dimethyl sulfoxide, a radical scavenger, blocked the radiation-mediated increase in MGMT. MGMT activity was also increased by treatment of the cells with H2O2, in accordance with the involvement of activated oxygen species in the induction of MGMT. Finally, the addition of cycloheximide, an inhibitor of protein synthesis, prior to but not after irradiation, abolished the increase in MGMT activity.


Acquisition of a growth-inhibitory response to phorbol ester involves DNA damage.

TPA (12-O-tetradecanoylphorbol-13-acetate), a potent tumor promoter, has been shown to stimulate or inhibit cell growth depending on the cell type investigated. We recently found that RT101 cells, a transformed mouse JB6 epidermal cell line, acquired a greater growth inhibition response to TPA during conventional subcultivation. The growth of low-passage RT101 cells was slightly inhibited by TPA in monolayer culture but stimulated in soft agar. In contrast, the growth of high-passage cells was greatly inhibited by TPA in both monolayer culture and in soft agar. Inhibition was dose dependent, directly correlated with protein kinase C-activating activities of tumor promoters, and was found to be reversible. TPA-treated high-passage cells were greatly reduced in volume, showed extensive abnormal mitoses, and were more susceptible to detachment. High-passage cells were also found to be less tumorigenic as indicated by in vivo tumorigenicity assay in nude mice. TPA treatment rendered cells still less tumorigenic in the case of both cell lines. The mechanism for acquisition of increased sensitivity to TPA of RT101 cells during subculture was investigated; it involved nonrandom DNA damage and detachment of nonviable cells. The results suggest the possibility that early-passage RT101 cells contained two subpopulations, one TPA-sensitive and one TPA-resistant population. Conventional subcultivation may have selected for the former subpopulation. The sensitive subpopulation may have been irreversibly inhibited as a result of TPA-induced cell killing, possibly apoptosis.


Development of mammary preneoplasias in vivo from mouse mammary epithelial cell lines in vitro.

A series of mouse mammary epithelial cell lines has been established by a protocol that gives highly reproducible results. The mammary epithelial cell lines, designated as FSK lines, were judged to be epithelial based on positive immunostaining for keratin-intermediate filaments, negative immunostaining for vimentin-intermediate filaments, hormonal induction of casein, and the ability to exhibit ductal and alveolar mammary morphogenesis in vivo. The FSK cell lines are dependent on epidermal growth factor and insulin in a low serum (1%) medium. Conditioned medium from spindle cell cultures replaced the requirement for serum and increased the growth of FSK3 and FSK4 4-5 times in collagen gels and 12-14 times in monolayer culture, respectively. Following injection into the mammary fat pad at passages 2-11, the FSK cell lines generated stable transplantable hyperplastic alveolar outgrowth lines. The in vivo outgrowth lines were judged as preneoplastic based on their stable alveolar morphology in vivo and an increased susceptibility for tumorigenesis. The FSK cell lines and their derivative in vivo outgrowth lines provide a new and potentially productive system to examine critical molecular alterations involved in the development of mammary preneoplasias. Furthermore, the reproducibility of the in vitro culture system provides the assurance that stable cell lines of mouse mammary epithelial cells can be generated easily and at will.


Tissue-specific expression of a human polymorphic epithelial mucin (MUC1) in transgenic mice.

The human MUC1 gene codes for the core protein of a mucin which is expressed by glandular epithelia and the carcinomas which develop from these tissues. The core protein is aberrantly glycosylated in cancers, and some antibodies show specificity in their reactions with the cancer-associated mucin, which also contains epitopes recognized by T-cells from breast and pancreatic cancer patients. For evaluating the potential use of mucin-reactive antibodies and mucin-based immunogens in cancer patients, a mouse model, expressing the MUC1 gene product PEM (polymorphic epithelial mucin) as a self antigen, would be extremely useful. To this end, we have developed transgenic mouse strains expressing the human MUC1 gene product in a tissue-specific manner. The TG4 mouse strain was established using a 40-kilobase fragment containing 4.5 kilobases of 5' and 27 kilobases of 3' flanking sequence. The TG18 strain was developed using a 10.6-kilobase SacII fragment from the 40-kilobase fragment; this fragment contained 1.6 kilobases of 5' sequence and 1.9 kilobases of 3' flanking sequence. Both strains showed tissue specificity of expression of the MUC1 gene, which was very similar to the profile of expression seen in human tissues. The antibody SM-3 is directed to a core protein epitope, which is selectively exposed in breast cancers and which shows a more restricted distribution on normal human tissues. It was established that the distribution of the SM-3 epitope of PEM in the tissues of the transgenic mice is similar to that seen in humans. The transgenic mouse strains described here should form the basis for the development of a preclinical model for the evaluation of PEM-based antigens and of antibodies directed to PEM in cancer therapy.


Phenylacetate: a novel nontoxic inducer of tumor cell differentiation.

Sodium phenylacetate was found to affect the growth and differentiation of tumor cells in vitro at concentrations that have been achieved in humans with no significant adverse effects. Treatment of promyelocytic leukemia HL-60 cells resulted in the rapid decline of myc oncogene expression followed by growth arrest and granulocyte differentiation. Phenylacetate also induced highly efficient adipocyte conversion in immortalized mesenchymal C3H 10T1/2 cultures; yet, unlike the differentiating chemotherapeutic drug 5-aza-2'-deoxycytidine, phenylacetate did not cause neoplastic transformation in these susceptible cells. The results indicate that phenylacetate is both effective in inducing tumor cell maturation and free of cytotoxic and carcinogenic effects, a combination that warrants attention to its potential use in cancer intervention.


Expression of fibroblast growth factor receptors in human leukemia cells.

We have previously cloned from K562 leukemia cells two novel fibroblast growth factor receptors (FGFR-3 and FGFR-4; J. Partanen et al., EMBO J., 10: 1347-1354, 1991). Here we have analyzed the mRNA expression of four different FGFRs, including the two novel genes in human leukemia cell lines. We show FGFR-1, FGFR-3, and FGFR-4 mRNAs in several leukemia cell lines at levels similar to those in solid tumor cell lines. Ligand cross-linking experiments indicate that K562 cells have receptors binding acidic FGF but not basic FGF. Expression of FGFRs in leukemia cells may reflect their presence on normal hematopoietic precursor cells or induction during leukemogenesis or cell culture.


Loss of microvascular negative charges accompanied by interstitial edema in septic rats' heart.

We studied the effect of Gram-negative sepsis on negative charges of heart capillaries and myocardial cells. We used a rat model of multiorgan failure, with ruthenium red (RR) and polyethyleneimine (PEI) as cationic binding tracers. Twenty-four hours after induction of sepsis, negative charges had decreased in glycocalyx and basement membrane of myocardial capillary endothelial cells. There were substantial amounts of interstitial edema. Density of anionic charges in the sarcolemmal glycocalyx complex of cardiac cells was markedly reduced. Myocardial cells' mitochondria consistently showed morphologic changes, whose severity ranged between stages II and IV C of Trump. Thirteen days after induction of sepsis, capillary endothelial and myocardial cells had recovered almost completely and showed no intracellular edema. Gram-negative sepsis caused a significant reduction in negative charges normally present in the microvascular wall as well as on myocardial cells. Consequently, several membranes limiting the various compartments of heart tissue lost their structural integrity. This morphometric data could explain the development of protein-rich interstitial edema and defective cell volume regulation observed in cardiac muscle of endotoxin-shocked animals. This myocardial edema may be at the origin of the cardiac dysfunction observed in both experimental and human septic shock.


Activation of chloride current by purinergic stimulation in guinea pig heart cells.

Single atrial cells from guinea pig heart were voltage-clamped using the whole-cell configuration of the patch-clamp technique under conditions in which most of the ionic and exchange currents known in cardiac cells were minimized. Extracellular 5 or 50 microM ATP activated a Cl- current, in addition to a rapidly desensitizing cation-selective current. A nonhydrolyzable ATP analogue, adenosine-5'-O-(3-thiotriphosphate) (50 microM), also evoked these two currents, indicating involvement of purinoceptors rather than ecto-ATPase on the membrane. ADP, AMP, and adenosine were also effective in inducing the Cl- current, showing no clear order of potency for the purinoceptor subtypes involved. The purinoceptor-activated Cl- current, like the beta-catecholamine-cAMP-dependent cardiac Cl- current, showed outward rectification and time independence.


Postnatal development of electrogenic sodium pump activity in rat hippocampal pyramidal neurons.

We assessed the development of electrogenic sodium pump (Na+ pump) activity in CA1 pyramidal neurons of rat hippocampal slices by studying the prolonged hyperpolarization which follows glutamate-induced depolarization (postglutamate hyperpolarization or PGH) at different postnatal ages. We also examined the development of membrane-bound enzyme in the hippocampal CA1 subfield with light microscopic immunocytochemistry and an antiserum against Na+,K(+)-ATPase. The PGH, which has previously been shown to be due to activation of an electrogenic Na+ pump in adult hippocampal CA1 neurons, was eliminated by strophanthidin, a Na+,K(+)-ATPase inhibitor, at all ages. It was unaffected by several potassium channel blockers, an intracellular calcium chelator, intracellular Cl- injection or tetrodotoxin (TTX) perfusion. The PGH thus appeared to be independent of K+ and Cl- conductances and produced by an electrogenic Na+ pump in adult and immature animals activated in large part by entry of Na+ through the glutamate receptor-channel complex. The size (integrated area) of the PGH was directly proportional to the area of preceding glutamate-induced depolarization (GD) and relatively voltage independent. Similar GDs could be elicited from postnatal day (P) 7 to P greater than or equal to 35, however, only very small PGHs were produced in neurons from P7-11 animals. A ratio of PGH area to GD area (PGH ratio) was calculated for each neuron and used to compare Na+ pump activity at different ages. There was a significant increase in the mean PGH ratio with age when P7-11, P21-25 and P35-39 groups were compared. Na+ pump activity estimated from the PGH ratio is very low in the first postnatal week but develops gradually over the first 5 weeks of life. Immunostaining for Na+,K(+)-ATPase in adult rat hippocampi revealed a punctate reaction product surrounding pyramidal cell bodies, whereas the staining was uniform along plasmalemma of dendrites in stratum radiatum and stratum oriens. By contrast, only minimum staining was present surrounding cell bodies and dendrites of P7 hippocampi and staining in stratum pyramidale was not punctate at this age. Na+,K(+)-ATPase activity estimated grossly from immunocytochemical staining is very low in the first postnatal week, increases during the first 5 weeks and develops a characteristic focal localization.(ABSTRACT TRUNCATED AT 400 WORDS)


Spatial expression of genes encoding c-kit receptors and their ligands in mouse cerebellum as revealed by in situ hybridization.

Expression of mRNA for c-kit receptors and their ligands was examined in the cerebellum of mice by in situ hybridization technique. The c-kit receptors were expressed in the molecular layer of the cerebellum and the ligands for the c-kit receptors were detected at the boundary of molecular and granular layers. The expression of the c-kit ligands was not detectable in the cerebellum of lurcher (Lc/+) mutant mice that lack Purkinje cells, indicating the cells expressing the c-kit ligands were Purkinje cells. The cells expressing c-kit receptors decreased but were present in the cerebellum of Lc/+ mice. The c-kit mRNA-positive cells appeared to represent basket cells and stellate cells from their appearance and location. Since neurons in the molecular layer construct suppressive neurojunctions with Purkinje cells, the present result suggests that c-kit receptors and their ligands may play an important role for the construction of their junctions.


Ontogeny of glycine-enhanced [3H]MK-801 binding to N-methyl-D-aspartate receptor-coupled ion channels.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is thought to play a critical role in neuronal development, differentiation and plasticity. A number of studies have shown an enhanced sensitivity to NMDA receptor ligands in neonatal animals. This study examined the ontogenetic changes in the glycinergic modulation of NMDA-coupled cation channels in the developing central nervous system of rat pups. The nonequilibrium binding of the specific channel ligand [3H]MK-801 was used as a measure of NMDA channel access. Glycine (10(-5) M) enhancement of [3H]MK-801 binding at 2 h in forebrain membranes from adult rats was significantly greater than that observed in tissues from 8- to 28-day-old rat pups. This difference was due to changes in the efficacy, but not potency of glycine. The observed ontogenetic changes in the efficacy of glycine-enhanced [3H]MK-801 binding were attributable to developmental changes in receptor site density, as determined by equilibrium [3H]MK-801 saturation isotherms. Kinetic studies revealed that glycine increased the association rate constants of [3H]MK-801 in 8-day and adult membranes by a similar magnitude (0.111 +/- 0.021 vs 0.094 +/- 0.009 nM-1 h-1, respectively). Similarly, the fractional amount of [3H]MK-801 bound (i.e., amount bound at time t normalized to amount bound at equilibrium) in the presence of glycine was relatively constant throughout neonatal development. These findings suggest that the allosteric modulation of the NMDA ionophore by glycine is similar in postnatal and adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Alport-type glomerulopathy: evidence for diminished capillary loop size.

Hereditary nephropathy of the Alport type is morphologically characterized by a specific and diagnostic thinning and splitting lesion of the glomerular basement membranes, which can be recognized only by electron microscopy. The light microscopical aspect has not been considered to be characteristic until now. This paper describes a light microscopical constellation of glomerular alterations by which ATGP can be recognized with high probability. Three histological features are of importance: 1. ATGP glomeruli in patients older than 10 years of age mostly have smaller capillary loops than age-matched controls. However, during the first 10 years of life no difference in glomerular capillary loop size was noticed. 2. ATGP loops often stain less intensely with basement membrane stains. 3. Presence of fetal-like glomeruli. Using this triad of light microscopic parameters as a screening tool, ATGP-cases were found without knowledge of any clinical data among other glomerulopathies with a sensitivity of 72% and a specificity of 93%. The definitive diagnosis, however, depends on electron microscopy.


Experimental therapies for multiple sclerosis: current status.

Multiple sclerosis is the most common cause of nontraumatic disability affecting young adults in the United States. Its etiology remains unclear, but evidence points to alteration of normal immune system function in the pathogenesis of this illness. This article reviews the results of clinical trials of experimental therapeutic agents in multiple sclerosis. The background and action of each treatment are described, and the clinical experience with each agent is reviewed. Obstacles to evaluating the effectiveness of treatment methods in MS and challenges to the design of future clinical trials and the interpretation of trial outcomes are discussed.