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.

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.

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.

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.

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.

Cytogenetic studies of 1-29 translocation carrying cows and their embryos.

An examination has been made of embryos from infertile 1-29 translocation carrying heifers. Cytogenetic measurements of C-banded chromosomes from these cattle, together with those from fertile 1-29 cows, have been made and compared with those from normal karyotypes. There are relative differences between normal animals and translocation carriers in the ratios of total to C-banded material throughout the karyotypes: their possible effects on mitosis and meiosis are discussed. Of the embryos recovered from the infertile heifers, one did not survive transplantation and others had various indications of inviability.

Specific bovine monoclonal antibody produced by a re-fused mouse/calf hybridoma.

Hybridomas made by fusing NSO (subline NSI Ag 4-1) mouse myeloma cells with lymph node cells from a calf immunized with sheep red blood cells failed to maintain antibody secretion in culture. However, when two of these hybridomas were selected in 8-azaguanine and then re-fused with immunized calf lymph node cells, several lines were obtained that secreted bovine Ig. One cloned line, producing bovine IgG1 (strongly lytic in the presence of rabbit complement and presumed to be an anti-Forssman) was maintained in culture for five months. Cytogenetic studies confirmed that the mouse/calf hybridomas lost bovine chromosomes as they proliferated, but that re-fusion increased the bovine complement from a mean of 5 (2 n) to 11 (2 n) bovine chromosomes per cell. It is proposed that the selected hybridoma lines may be suitable fusion partners for the production of further monoclonal bovine antibodies.

Culture of sheep X mouse hybridoma cells in vitro.

Hybridomas were made between NS1/1-Ag4-1 mouse myeloma cells and spleen and lymph node cells from a sheep immunized with sheep red cells (RBC). The hybrid colonies grew well in culture but there was a substantial loss of sheep chromosomes. No hemolytic or agglutinating antibodies were detected in the culture supernatants after the 17th day following fusion, but immunofluorescence tests indicated that a few of the cells may have been expressing sheep IgG. Cytogenetic comparison of cells grown with and without HAT medium provided evidence that the enzyme HGPRT is located on the X chromosome of sheep as it is in man and mouse. Hybridoma isozyme patterns of esterase, G6PD, 6PGD, NP, LDH and SOD tested between the 63rd and 71st day of culture were like those of NS1; NP and LDH also showed zones that probably came from the sheep component.

[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.

[Identification of protein products of the operon for leukocytosis (lymphocytosis)-stimulating factor from Bordetella pertussis cloned in Escherichia coli]. / Identifikatsiia belkovykh produktov operona leikotsitoz (limfotsitoz)-stimuliruiushchego faktora Bordetella pertussis, klonirovannogo v Escherichia coli.

The hybrid plasmid pRH119 was constructed on the basis of vector plasmid pUC19 and shown to carry Bordetella pertussis PT operon in the same transcriptional orientation with the lac-promoter of the vector plasmid. Expression of PT genes in E. coli cells harbouring pRH119 was not registered. Weak expression of PT genes was found by immunoscreening of recombinant clones in situ with antiserum against PT when PT genes were put closer to lac-promoter. 0.95 kb SalGI fragment was deleted from pRH119. The derivative plasmid pRH122 was digested by SalGI and the ends were polymerized to "blunt" by polIK and ligated. The obtained plasmid pRH122K was deleted for 40 bp in XbaI site by Bal31 deletion. The lysate of E. coli cells harbouring the resulting plasmid pRH134 passed through Sepharose 4B with covalently bound immunoglobulins from antiserum against PT. The eluted protein contains S2 multimers identified by immunoblotting. The experiments with CHO-cells and active mice protection have shown the absence of S2 multimers protectiveness.

[Pyogenic hepatic abscess]. / Absceso hepático piógeno.

Ten patients, 6 men and 4 women, with a mean age of 53 years, having pyogenic liver abscess, were studied to review the principal features of the clinical and bacteriological diagnosis. In 9 cases the abscess was located in the right hepatic lobe. Bacteriological diagnosis was made in 6 cases: the etiology was nonmicrobian in 3 and two microorganisms were found in the other 3 cases. The isolated pathogens were: Escherichia coli (3 cases), Staphylococcus sp (3 cases), Enterobacter agglomerans (1 case), Citrobacter sp (1 case), Streptococcus sp (1 case). The basic clinical pattern included fever in 9 patients, pain in upper right side of the abdomen in 8, jaundice in 5 and respiratory symptoms in 5. In 2 cases the diagnosis was confirmed by ultrasonography, in 2 by scintigraphy and in 3 both methods were used. Surgical drainage was practiced in 7 patients, with favorable course in 6. Three patients were not operated; 2 died and the third was cured with medical treatment. The importance of the clinical suspicion even with few data is pointed out, specially prolonged fever with painful syndrome in upper right side of the abdomen. The aid of the diagnosis imaging is valuable for verification. The precise bacteriological diagnosis by culture of the material obtained in surgery or by percutaneous drainage of the abscess allows the selection of the most suitable antibiotic therapy.

[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.

[Cloning and gene expression of the leukocytosis (lymphocytosis) stimulating factor of Bordatella pertussis in Escherichia coli by bringing the PT genes close to the lactose promoter of plasmid pUC19]. / Klonirovanie i ékspressiia genov leikotsitoz (limfotsitoz)-stimuliruiushchego faktora Bordetella pertussis v Escherichia coli putem priblizheniia genov k laktoznomu promotoru plazmidy pUC19.

The 4.7 Kb EcoRI-fragment of phase I B. pertussis 475 (serovar 1.2.3.) chromosome carrying all five genes of the pertussis toxin (PT) operon was cloned on plasmid pUC19 in E. coli. The resulting hybrid plasmid pRH119 contained the PT operon in the same orientation of transcription as the lac promoter of plasmid pUC19. Nevertheless, the expression of the PT operon was not observed even after induction with isopropyl thio-beta-D-galactopyranoside (IPTG), which suggested either the inability of the PT operon to work in E. coli, or the presence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119. The expression was determined by the incubation of the clones harboring plasmid pRH119 with antiserum to PT and their subsequent in situ treatment with 125I-labeled protein A. Three deletion variants of plasmid pRH119 were constructed with the aim of approaching the PT genes to the lac promoter: pRH121 (the 0.45 Kb KpnI-fragment deleted), pRH122 (the 0.95 Kb SalGI-fragment deleted) and pRH123 (the 1.35 Kb XbaI-fragment deleted). In all these cases different levels of expression were observed (but only in the presence of IPTG). Site KpnI in the 4.7 Kb fragment was found to be localized in the -57 b. p. region in relation to the PT promoter, i. e. to lie, seemingly, in the promoter zone; for this reason, the expression of the PT genes in plasmid pRH121 proved the existence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119.(ABSTRACT TRUNCATED AT 250 WORDS)

[Detection of the gene sequences of the leukocytosis (lymphocytosis)-stimulating factor of Bordetella pertussis in Bordetella parapertussis and Bordetella bronchiseptica by using molecular hybridization]. / Obnaruzhenie posledovatel'nostei genov leikotsitoz (limfotsitoz)-stimuliruiushchego faktora Bordetella pertussis u Bordetella parapertussis i Bordetella bronchiseptica s pomoshch'iu molekuliarnoi gibridizatsii.

The 4.7 Kb EcoRI-fragment of phase I B. pertussis 475 (serovar 1.2.3) chromosome DNA carrying the pertussis toxin (PT) operon was cloned on vector plasmid pUC19 in Escherichia coli. Three fragments (1.14 Kb KpnI-PstI, 1.27 Kb PstI-PstI, and 0.96 Kb PstI-PstI) were obtained from the resulting hybrid plasmid, coded pRH119, by electrophoretic techniques and used as a combined molecular probe for analysis of the EcoRI-digested and PstI-digested chromosomal DNA of B. pertussis strain 475 in phase I, B. pertussis in phase IV, B. parapertussis strains 504 and 17903, B. bronchiseptica strain 214, and B. parapertussis strain 17903 (a convertant obtained by means of B. pertussis phage 134), as well as B. pertussis phage 134. Southern blot hybridization under the conditions of 100% DNA-DNA homology showed the presence of DNA sequences characteristic of the PT operon in all cases except the DNA of phage 134; moreover, the use of the above-mentioned probe made it possible to hybridize all EcoRI-fragments of chromosomal DNA, having the same molecular size (4.7 Kb). Consequently, the PT genes in the above Bordetella species were mapped in identical loci.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cloning and the expression of the hemolysin gene of Leptospira pomona pomona in Escherichia coli]. / Klonirovanie i ékspressiia gena gemolizina Leptospira pomona pomona v Escherichia coli.

The library of Leptospira pomona genes was obtained on phage vector AL 47.1. From this library a recombinant phage carrying the hemolysin gene was selected. The DNA fragment (7.7 kb) of this phage containing the hemolysin gene was subcloned on plasmid pUC19. E. coli clones with hybrid plasmid pDR7 were shown to be hemolytic, but the secretion of hemolysin by E. coli into the culture medium was not observed.

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.