Metastatic Competency and Tumor Spheroid Formation Are Independent Cell States Governed by RB in Lung Adenocarcinoma.

Inactivation of the retinoblastoma (RB) tumor suppressor in lung adenocarcinoma is associated with the rapid acquisition of metastatic ability and the loss of lung cell lineage commitment. We previously showed that restoration of RB in advanced lung adenocarcinomas in the mouse was correlated with a decreased frequency of lineage decommitted tumors and overt metastases. To identify a causal relationship for RB and its role in reprogramming lineage commitment and reducing metastatic competency in lung adenocarcinoma, we developed multiple tumor spheroid forming lines where RB restoration could be achieved after characterization of the degree of each spheroid's lineage commitment and metastatic ability. Surprisingly, we discovered that RB inactivation dramatically promoted tumor spheroid forming potential in tumors that arise in the KrasLSL-G12D/+; p53flox/flox lung adenocarcinoma model. However, RB reactivation had no effect on the maintenance of tumor spheroid lines once established. In addition, we show that RB-deficient tumor spheroid lines are not uniformly metastatically competent but are equally likely to be nonmetastatic. Interestingly, unlike tumor spheroid maintenance, RB restoration could functionally revert metastatic tumor spheroids to a nonmetastatic cell state. Thus, strategies to reinstate RB pathway activity in lung cancer may reverse metastatic ability and have therapeutic potential. Finally, the acquisition of tumor spheroid forming potential reflects underlying cell state plasticity, which is often predictive of, or even conflated with metastatic ability. Our data support that each is a discrete cell state restricted by RB and question the suitability of tumor spheroid models for their predictive potential of advanced metastatic tumor cell states. SIGNIFICANCE: Members of the RB pathway are frequently mutated in lung adenocarcinoma. We show that RB regulates cell state plasticity, tumor spheroid formation, and metastatic competency. Our data indicate that these are independent states where spheroid formation is distinct from metastatic competency. Thus, we caution against conflating spheroid formation and other signs of cell state plasticity with advanced metastatic cell states. Nevertheless, our work supports clinical strategies to reactivate RB pathways.

Cognitive and motor perturbations in elderly with longstanding diabetes mellitus.

Type 2 diabetes mellitus is a chronic disease characterized by insulin resistance; inflammation; oxidative stress; vascular damage; and dysfunction of glucose, protein, and lipid metabolisms. However, comparatively less attention has been paid to neurologic alterations seen in elderly individuals with type 2 diabetes. We review clinical, metabolic, and biochemical aspects of diabetic encephalopathy (DE) and propose that quality of dietary lipids is closely linked to DE. This implies that preventive nutritional interventions may be designed to improve DE.

Mechanosensitive ion channels of the archaeon Haloferax volcanii.

Mechanosensitive (MS) ion channels have been documented in a variety of cells belonging to Eukarya and Eubacteria. We report the novel finding of two types of MS ion channels in the cell membrane of the halophilic archaeon Haloferax volcanii, a member of the Archaea that comprise the third phylogenetic domain. The two channels, MscA1 and MscA2, differed in their kinetic properties with MscA1 exhibiting more frequent open-closed transitions than MscA2. Both channels have large conductances that rectify between -40 mV and +40 mV where the conductance of MscA1 ranged from 380 to 680 picosiemens, whereas MscA2 ranged from 850 to 490 picosiemens. Both channels were blocked by submillimolar gadolinium. In addition, the channels of either membrane vesicles or detergent-solubilized membrane proteins remained functional upon reconstitution into artificial liposomes, a result that indicates that these channels are activated by mechanical force transmitted via the lipid bilayer alone. Subsequently a 37-kDa protein corresponding to the MscA1 channel activity was purified. With the possible functional similarity to bacterial MS channels, our finding of MS channels in Archaea emphasizes the ubiquity and importance of these channels in all domains of the evolutionary tree.

Estimation of the pore size of the large-conductance mechanosensitive ion channel of Escherichia coli.

The open channel diameter of Escherichia coli recombinant large-conductance mechanosensitive ion channels (MscL) was estimated using the model of Hille (Hille, B. 1968. Pharmacological modifications of the sodium channels of frog nerve. J. Gen. Physiol. 51:199-219) that relates the pore size to conductance. Based on the MscL conductance of 3.8 nS, and assumed pore lengths, a channel diameter of 34 to 46 A was calculated. To estimate the pore size experimentally, the effect of large organic ions on the conductance of MscL was examined. Poly-L-lysines (PLLs) with a diameter of 37 A or larger significantly reduced channel conductance, whereas spermine (approximately 15 A), PLL19 (approximately 25 A) and 1,1'-bis-(3-(1'-methyl-(4,4'-bipyridinium)-1-yl)-propyl)-4,4'-b ipyridinium (approximately 30 A) had no effect. The smaller organic ions putrescine, cadaverine, spermine, and succinate all permeated the channel. We conclude that the open pore diameter of the MscL is approximately 40 A, indicating that the MscL has one of the largest channel pores yet described. This channel diameter is consistent with the proposed homohexameric model of the MscL.

Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli: mutants with altered channel gating and pressure sensitivity.

In the search for the essential functional domains of the large mechanosensitive ion channel (MscL) of E. coli, we have cloned several mutants of the mscL gene into a glutathione S-transferase fusion protein expression system. The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-terminal region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions. Proteolytic digestion of the isolated fusion proteins by thrombin yielded virtually pure recombinant MscL proteins that were reconstituted into artificial liposomes and examined for function by the patch-clamp technique. The addition of amino acid residues to the N-terminus of the MscL did not affect channel activity, whereas N-terminal deletions or changes to the N-terminal amino acid sequence were poorly tolerated and resulted in channels exhibiting altered pressure sensitivity and gating. Deletion of 27 amino acids from the C-terminus resulted in MscL protein that formed channels similar to the wild-type, while deletion of 33 C-terminal amino acids extinguished channel activity. Similarly, deletion of the internal amphipathic region of the MscL abolished activity. In accordance with a recently proposed spatial model of the MscL, our results suggest that (i) the N-terminal portion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative central amphipathic alpha-helical portion of the protein and the six C-terminal residues RKKEEP forming a charge cluster following the putative M2 membrane spanning alpha-helix.

[Treatment with tuberculostatic drugs: compliance at a general hospital]. / Terapeutica con tuberculostaticos: cumplimiento en un hospital general.

The purpose of this study was to assess the compliance with tuberculostatic drugs treatment in a public hospital from Córdoba City and to establish the causes of noncompliance. All the patients to which treatment with tuberculostatic drugs was indicated from January 1991 up to December 1994 were included. 45 patients were included: 18 females (40%) and 29 males. Sixteen (35.6%) did not complete the time of treatment indicated. Nine (56.3%) abandoned the treatment 2 months after having initiated it. In the group that did not complete the treatment there was a higher percentage of female patients (62.5%) than in the group that did complete it (27.6%), p = 0.02. There were not statistically significant differences in age, percentages of pulmonar and extrapulmonar tuberculosis and months of treatment indicated between both groups. Thirty-six percent of the patients who abandoned the treatment referred having interrupted it due to their own negligency, knowing the risk of such behavior; 36% suffered side effects and did not come back to hospital; 21% referred having consulted another physician who indicated to interrupt the treatment without performing other tests; and 7% misunderstood the indications. It is concluded that in a general hospital from Córdoba City, the percentage of patients who abandoned tuberculostatic treatment is high. In most cases the cause was related to failures in the conduct of patients, physicians or both.

Screening for alcohol misuse in older people.

OBJECTIVES: To evaluate whether the CAGE, MAST-G or an abnormally high MCV are effective screening instruments for alcohol misuse and if not to devise a new instrument. DESIGN: Questionnaire survey of randomly selected patients. SETTING: Acute admission wards of two district general hospitals. PERIOD: 9 months. SUBJECTS: Randomly selected patients, aged 65 and over, admitted as emergencies. MAIN OUTCOME MEASURES: Excessive alcohol intake, alcohol dependence. RESULTS: The sensitivities of the CAGE, MAST-G and an abnormally high MCV for excess alcohol intake and alcohol abuse are low, ranging from 0.13 to 0.54. CONCLUSION: The results confirm previous UK studies which indicate that the CAGE and an abnormally high MCV are poor screening instruments for alcohol misuse in older patients and also indicate that the MAST-G is an insensitive screening instrument. A new two-step instrument with five questions in total was generated. This requires further validation.

Kainic acid blocks a TTX-sensitive sodium channel in retinal horizontal cells of the turtle (Pseudemys scripta elegans).

The aim of this study was to investigate the effects of excitatory amino acids on channels found in horizontal cell membranes using patch-clamp techniques. We unexpectedly found that the excitatory amino acid receptor agonist, kainic acid, reversibly inhibited the transient tetrodotoxin (TTX)-sensitive Na+ current in isolated horizontal cell bodies and axons from the retina of the turtle (Pseudemys scripta elegans). The effect of kainic acid was antagonized by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Kainic acid activated a non-selective cation current, a finding that was consistent with previous reports, and which would account for the kainate induced depolarisation of these cells. The inhibition of the transient TTX-sensitive Na+ current by kainic acid might be important in the modification of the kinetics of responses to excitatory amino acid analogues often observed during intracellular recording from these cells.

Porins of Escherichia coli: unidirectional gating by pressure.

OmpC and PhoE porins of Escherichia coli were examined by the patch-clamp technique following reconstitution in liposomes, and were observed primarily in the open (conducting) state. With application of negative voltage and positive hydrostatic pressure, OmpC exhibited marked gating towards a more closed state whereas PhoE remained largely unaffected by pressure application. Hybrid chimeric OmpC-PhoE proteins showed an increased tendency for pressure-dependent gating as the OmpC proportion in the chimeric molecule increased. In addition, several PhoE mutants with amino acid substitutions and insertions in either the L3 or L4 loop of the monomer exhibited pressure sensitivity comparable with the wild-type OmpC porin. Our data support the structural plasticity model of porins and are consistent with the 'charge-screening-unscreening' hypothesis that describes how these proteins may exist in distinct conformations.

Piperacillin/tazobactam in the treatment of hospitalized patients with urinary tract infections: an open non-comparative and multicentered trial.

The aim of this multicentered, prospective and open study was to determine the clinical and bacteriological efficacy and safety of piperacillin/tazobactam (4g/500 mg IV tid) in the treatment of 79 adult patients with complicated urinary tract infections (UTI) requiring hospitalization. Forty-seven women and 32 men (mean age 54.2 years, and range 21-91) from 4 Argentinean and 6 Mexican hospitals were enrolled. Sixty-one clinically and bacteriologically evaluable patients were treated for a mean of 9.1 days (range 5-15). A favorable clinical response was seen in 83.6% and 80% at early and late assessment, respectively. Bacteriological eradication was achieved in 85.3% and 80% at early and late estimation, respectively. Escherichia coli was isolated in 33 cases, Klebsiella pneumoniae in 8, Enterococcus spp. in 7, Proteus mirabilis in 6, Pseudomonas aeruginosa in 3, Enterobacter spp. and Morganella morganii in 2. While 21% of all the clinical isolates were resistant to piperacillin, none of them was initially resistant to piperacillin/tazobactam. However, one female patient with a persistent UTI caused by E. coli developed resistance to piperacillin/tazobactam during treatment. A 64-year-old man with frontal meningioma developed purulent meningitis due to Enterobacter cloacae after neurosurgery. He was initially treated with ciprofloxacin, rifampin and amikacin and because of persistence of fever, he was moved to piperacillin/tazobactam. After 5 days of therapy, he developed coma secondary to intracranial hemorrhage and died. By then, the platelet count was normal (220,000/microliters), but the prothrombin time (19.5 seconds) and the partial thromboplastin time (63 seconds) were significantly prolonged. Our data suggest that piperacillin/tazobactam is a reliable therapy for complicated, non-complicated, community or hospital-acquired UTI.

[Native-valve endocarditis produced by Lactobacillus casei sub. rhamnosus refractory to antimicrobial therapy]. / Endocarditis de valvula nativa producida por Lactobacillus casei sub, rhamnosus refractaria al tratamiento con antimicrobianos.

Lactobacillus endocarditis is a rare infection. In fact, only 42 cases have been described in the literature from 1938 up to date. In only 17 previously reported cases have patients been cured with medical therapy alone. Although infections produced by Lactobacillus spp, have been described in our country, none of them included endocarditis. We report herein a case of endocarditis due to a vancomycin-resistant strain of Lactobacillus casei sub. rhamnosus in a 29-year-old man with prolapse of the mitral valve. He required surgical replacement of his valve because of the poor response to antimicrobial therapy with penicillin and gentamicin. The patient displayed a successful clinical outcome, with no evidence of recurrence along the subsequent 2 years. We point out the need to accurately identify Lactobacillus spp. in isolates from blood cultures of patients with endocarditis, since these bacteria may often be mistaken for other species more frequently associated to this infection, which usually respond to conventional antimicrobial therapy. Furthermore, we suggest that early surgical replacement should be considered when lactobacillus endocarditis is diagnosed.

Endocarditis de valvula nativa producida por Lactobacillus casei sub. rhamnosus refractaria al tratamiento con antimicrobianos / Native-valve endocarditis produced by Lactobacillus casei sub. rhamnosus refractory to antimicrobial therapy

La endocarditis producida por Lactobacillus spp. es una infección infrecuente. Se han descrito 42 casos desde 1938 hasta la fecha y sólo 17 de ellos evolucionaron favorablemente con tratamiento médico, sin necesidad de reemplazo valvular. Si bien se han publicado unos pocos casos de infecciones producidas por Lactobacillus spp. en nuestro país, ninguno de ellos correspondió a endocarditis infecciosa. Se presenta un caso de endocarditis producida por Lactobacíllus casei sub. rhamnosus vancomicinaresistente en un varón de 29 años con antecedentes de prolapso de válvula mitral, que requirió reemplazo valvular para su curación, debido al fracaso del tratamiento con penicilina y gentamicina. El enfermo presentó una evolución favorable en el post-operatorio inmediato y fue dado de alta a los 7 días en buen estado general, no presentando signos ni síntomas de recurrencia en los controles realizados durantes los 2 años siguientes. Enfatizamos la necesidad de identificar rapidamente el género Lactobacíllus ante un aislamiento de hemocultivos de enfermos con endocarditis, puesto que dicho género puede confundirse con otras especies que se asocian más frecuentemente con esta patologia y que generalmente presentan respuesta clínica favorable frente a los tratamientos con antimicrobianos. Sugerimos que se considere la posibilidad de un reemplazo valvular precoz ante la presencia de endocarditis producida por Lactobacillus spp.

Endocarditis de valvula nativa producida por Lactobacillus casei sub. rhamnosus refractaria al tratamiento con antimicrobianos / Native-valve endocarditis produced by Lactobacillus casei sub. rhamnosus refractory to antimicrobial therapy

La endocarditis producida por Lactobacillus spp. es una infección infrecuente. Se han descrito 42 casos desde 1938 hasta la fecha y sólo 17 de ellos evolucionaron favorablemente con tratamiento médico, sin necesidad de reemplazo valvular. Si bien se han publicado unos pocos casos de infecciones producidas por Lactobacillus spp. en nuestro país, ninguno de ellos correspondió a endocarditis infecciosa. Se presenta un caso de endocarditis producida por Lactobacíllus casei sub. rhamnosus vancomicinaresistente en un varón de 29 años con antecedentes de prolapso de válvula mitral, que requirió reemplazo valvular para su curación, debido al fracaso del tratamiento con penicilina y gentamicina. El enfermo presentó una evolución favorable en el post-operatorio inmediato y fue dado de alta a los 7 días en buen estado general, no presentando signos ni síntomas de recurrencia en los controles realizados durantes los 2 años siguientes. Enfatizamos la necesidad de identificar rapidamente el género Lactobacíllus ante un aislamiento de hemocultivos de enfermos con endocarditis, puesto que dicho género puede confundirse con otras especies que se asocian más frecuentemente con esta patologia y que generalmente presentan respuesta clínica favorable frente a los tratamientos con antimicrobianos. Sugerimos que se considere la posibilidad de un reemplazo valvular precoz ante la presencia de endocarditis producida por Lactobacillus spp. (AU)

Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of Escherichia coli.

The large mechanosensitive ion channel (MscL) of Escherichia coli was expressed on a plasmid encoding MscL as a fusion protein with glutathione S-transferase in an Escherichia coli strain containing a disruption in the chromosomal mscL gene. After purification of the fusion protein using glutathione-coated beads, thrombin cleavage allowed recovery of the MscL protein. The purified protein was reconstituted into artificial liposomes and found to be fully functional when examined with the patch-clamp technique. The reconstituted recombinant MscL protein formed ion channels that exhibited characteristic conductance and pressure sensitivity and were blocked by the mechanosensitive ion channel inhibitor gadolinium. The recombinant MscL protein was also used to raise specific anti-MscL polyclonal antibodies which abolished channel activity when preincubated with the MscL protein.

A tetraethylammonium-insensitive inward rectifier K+ channel in Müller cells of the turtle (Pseudemys scripta elegans) retina.

Ion channels present in isolated glial (Müller) cells from the retina of the turtle (Pseudemys scripta elegans) were studied with the patch clamp technique. The predominant conductance in these cells was due to an inward rectifying potassium current. The whole-cell conductance of the inward rectifier was 20.2 +/- 1.9 nS (n = 7 cells) in a standard extracellular saline solution (3 mM extracellular potassium). This conductance was dependent on the extracellular potassium concentration, with a 2.88-fold change in conductance per tenfold shift in concentration. The relative permeability sequence to potassium of the inward rectifier was found to be: potassium (1.0) > rubidium (0.7) > ammonium (0.2) > lithium (0.1) = sodium (0.1), which corresponded to the Eisenman sequence IV or V for a strong-field-strength potassium binding site on the channel. The single channel conductance measured in cell-attached patches with potassium chloride (150 mM) in the pipette was 68.5 +/- 6.0 pS (n = 3 patches). The inward rectifier current was not blocked by extracellular tetraethylammonium (TEA+, 20 mM), but was blocked by extracellular barium (5 mM) or cesium (5 mM). The TEA+ insensitivity of the inward rectifier potassium channel in Müller cells is unusual, given that this type of channel in most excitable cells is sensitive to micromolar concentrations of this compound, and may be a characteristic of inward rectifier potassium channels that are primarily involved with extracellular potassium regulation.

Naltrexone modulation of nicotinic acetylcholine receptor activity.

The narcotic antagonist naltrexone (NTX) has both depressant and excitatory effects on nicotinic acetylcholine receptor activity, and the present study was primarily concerned with mechanisms involved in the latter modulatory action. Single channel data were recorded in cell-attached mode from cultured embryonic chick skeletal muscle myotubes at 10 degrees C under control conditions (0.15 or 0.3 microM acetylcholine, ACh) or in the presence of NTX (0.15 microM ACh plus 0.1-5 microM NTX). Distributions of open and closed times indicated that there were at least two open and four closed channel states in all cases, and evidence for at least two gateways between the open and closed classes was provided by autocorrelation data. In the absence of NTX the proportion of long (greater than 2 msec) openings increased with increasing ACh concentration, suggesting that the channel in both the mono- and biliganded forms of the receptor could open. Three possible six-state models of channel kinetics which were compatible with these findings could not be distinguished on the basis of conventional Markov modeling of open and closed times; however when account was taken of burst distributions, one model was clearly superior. Estimated transition rates in this model showed that enhanced receptor activity in the presence of NTX was due mainly to increased cooperativity of ACh binding. The results suggest that in the chick nicotinic ACh receptor, low micromolar concentrations of NTX may cause enhancement and blockade of activity by binding to a site which is allosterically coupled to several activation and gating processes.

Kinetics of (+)-tubocurarine blockade at the neuromuscular junction.

1. Although (+)-tubocurarine (Tc) is classically considered to be a competitive antagonist at the neuromuscular junction, kinetic details of the interaction remain unclear. 2. We studied the competitive action of Tc on the nicotinic receptor at the frog neuromuscular junction using a quantitative analysis of the generation phase of miniature endplate currents (m.e.p.cs) recorded in Ringer solution (20 degrees C) under voltage clamp (-90 mV) in the absence or presence of 1-5 microM Tc. Under control conditions four neurotransmission parameters were estimated by non-linear regression using a mathematical model of synaptic transmission incorporating transmitter release, diffusion, hydrolysis, receptor binding and channel gating. These parameters were then used in a further regression to estimate binding rate constants for Tc at the same endplate. Allowance was made for open channel block by Tc, which under the conditions of this study was only a small component of total blockade. 3. The results suggest that Tc binds to the two agonist recognition sites on the nicotinic receptor with equal affinity (stoichiometric KDs of 2.2 and 8.8 microM), and that most of the functional blockade at concentrations up to 5 microM is due to occupancy of only one site. 4. The association rate constant for Tc binding to sites on the nicotinic acetylcholine receptor appears to be very fast (k+D = 8.9 x 10(8) M-1 s-1) and comparable to that for acetylcholine (ACh). 5. In the brief time during which an m.e.p.c. is generated (approximately 200 microseconds, reversal of Tc blockade by transiently high concentrations of ACh seems to be kinetically limited.

Do young patients with dyspepsia need investigation?

During 1986, 1386 patients with simple dyspepsia were referred by general practitioners for endoscopy (686) or double-contrast barium meal examination (700) at Leicester General Hospital. 618 (45%) were under the age of 45 years. Abnormal findings were more common in older than younger dyspeptic patients (58% vs 40% at endoscopy, 69% vs 25% by barium meal). Malignant disorders were diagnosed in 5% at endoscopy and 3% at barium meal, but in no patient under 45 years old. The incidence of malignant disorders at endoscopy was analysed for the 6 years 1980-86. Of 707 cases identified, only 13 (1.8%) occurred in patients under 45 years old; all 13 had symptoms suggesting pathology more serious than simple dyspepsia. It can be concluded that young patients with simple dyspepsia are overinvestigated. A majority can be treated safely with antacids and/or histamine receptor type 2 antagonists.

All species of the genus Bordetella contain genes for pertussis toxin of Bordetella pertussis.

A molecular probe for the PT-operon of B. pertussis hybridized with 4.7 Kb EcoRI-fragments of chromosomal DNAs of B. pertussis strain 475 phase I, phase IV, B. parapertussis strains 504 and 17903, B. bronchiseptica strain 214, B. parapertussis strain 17903-convertant of B. pertussis phage 134 but not with phage 134 DNA under stringent conditions of DNA-DNA hybridization. This fact indicates the presence of PT-genes in all Bordetella species. Since there is no production of PT in B. parapertussis and B. bronchiseptica, a presence of regulatory mutations in the PT-operon or absence of the functionally active vir-gene product in these species is suggested.

Verapamil, neuromuscular transmission and the nicotinic receptor.

The effect of verapamil on neuromuscular transmission was studied in the frog by analysing ionophoretic endplate current (iEPC) trains and the growth and decay phases of miniature endplate currents (mepcs). In addition, single channel data on the interaction of verapamil with the nicotinic acetylcholine receptor were obtained from cultured embryonic chick skeletal muscle cells. Verapamil caused both open and closed channel blockade in the iEPC trains. Mepc amplitude was decreased at low micromolar concentrations, and at higher concentrations there was also accelerated mepc decay indicating open channel blockade. The latter effect could not be explained by a sequential channel occlusion mechanism. Analysis of the mepc rising phase showed that low micromolar concentrations of the drug decreased the pool of receptors which could be activated. Single channel data confirmed the specific interaction of verapamil with the nicotinic receptor, showing closed channel blockade at low concentrations, and at higher levels the shortening of open channel lifetime. It is suggested that both forms of blockade may involve desensitization processes.