Human cell variants resistant to rhodamine 6G

Two variants have been isolated from the cultured human cell line VA₂-B which are resistant in vivo to the mitochondrial-specific fluorescent dyes rhodamine 6G and rhodamine 123. Both mutants are cross-resistant to ethidium bromide but are sensitive to both colchicine and chloramphenicol. When either mutant is treated with low levels of rhodamine 6G, mitochondrial-associated fluorescence is significantly lower than in wild-type cells. Furthermore, when cell cultures are treated with high concentrations of either rhodamine 123 or 6G, and then washed free of the dye, mitochondrial-associated rhodamine fluorescence rapidly diminishes in the parental cell line. In hybrid cell fusions between resistant and sensitive cell lines, rhodamine resistance is gradually expressed, reaching maximal expression approximately 11 days after fusion. Cytoplasmic transmission of rhodamine resistance has not been clearly demonstrated in cytoplast-cell fusions, and thus resistance is probably due to a mutation of a nuclear, rather than mitochondrial DNA gene(s). These observations indicate that mitochondria of both rhodamine-resistant variants, unlike wild type, have a significantly decreased ability to bind and retain rhodamine, and thus their mitochondrial tramsmembrane electrical potential may be significantly reduced.     

A uniparental mutant of Chlamydomonas reinhardtii resistant to chloramphenicol

Summary A uniparental mutant of Chlamydomonas resistant to chloramphenicol was selected following treatment of wild-type cells with 5-fluorodeoxyuridine. Under heterotrophic conditions, growth and chloroplast protein synthesis of this mutant (CAP1) are resistant to chloramphenicol. Under phototrophic conditions, CAP1 is sensitive to chloramphenicol. In addition CAP1 displays thermosensitivity when grown phototrophically in the absence of antibiotics: at the restrictive temperature, a specific reduction of those thylakoid membrane polypeptides which are synthesized inside the chloroplast is observed. Alternative explanations for the pleiotropic phenotype of CAP1 are discussed.     

Mice deficient in pituitary adenylate cyclase activating polypeptide display increased sensitivity to renal oxidative stress in vitro

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide, showing widespread occurrence in the nervous system and also in peripheral organs. The neuroprotective effects of PACAP are well-established in different neuronal systems against noxious stimuli in vitro and in vivo. Recently, its general cytoprotective actions have been recognized, including renoprotective effects. However, the effect of endogenous PACAP in the kidneys is not known. The main aim of the present study was to investigate whether the lack of this endogenous neuropeptide influences survival of kidney cells against oxidative stress. First, we determined the presence of endogenous PACAP from mouse kidney homogenates by mass spectrometry and PACAP-like immunoreactivity by radioimmunoassay. Second, primary cultures were isolated from wild type and PACAP deficient mice and cell viability was assessed following oxidative stress induced by 0.5, 1.5 and 3 mM H₂O₂. Our mass spectrometry and radioimmunoassay results show that PACAP is endogenously present in the kidney. The main part of our study revealed that the sensitivity of cells from PACAP deficient mice was increased to oxidative stress: both after 2 or 4 h of exposure, cell viability was significantly reduced compared to that from control wild type mice. This increased sensitivity of kidneys from PACAP deficient mice could be counteracted by exogenously given PACAP38. These results show, for the first time, that endogenous PACAP protects against oxidative stress in the kidney, and that PACAP may act as a stress sensor in renal cells. These findings further support the general cytoprotective nature of this neuropeptide.     

Human uterodomes (pinopods) do not display pinocytotic function

BACKGROUND: The term 'pinopod' or 'pinopode' has been used indiscriminately since the 1970s to describe most apical structures on uterine epithelial cells and as such suggests a cross species structural functionality. This study looks at the apical cellular protrusions in rats and humans and compares their pinocytotic ability. METHODS: We have utilized standard tracer techniques in an attempt to determine the functionality of the uterine surface protrusions in the human based on results reported in rats. RESULTS: Pinopods in rat tissue demonstrated tracer uptake, but no tracer uptake in the apical protrusions of human uterine epithelium was evident. CONCLUSIONS: These findings indicate that the uterine surface protrusions observed in the human are not pinocytotic and therefore probably perform a function different from similar structures observed in rats and mice. This highlights the need to alter nomenclature from pinopods to uterodomes.     

The bacterial multiple antibiotic resistant (Mar) phenotype leads to increased tolerance to tea tree oil

Mutants of Escherichia coil strain AG100 exhibiting the multiple antibiotic resistance (Mar) phenotype demonstrated a greater level of tolerance to tea tree oil (TTO) compared with the parent strain. The ability of TTO to kill all E. coil strains studied was greater at 37 than at 30 degrees C. Growth of parent strain AG100 in the presence of salicylate, which induces the mar operon leading to the Mar phenotype, also increased tolerance to TTO. Escherichia coli Mar mutant YL1 demonstrated greater tolerance to antimicrobial terpenes found in TTO and did not leak K+ as rapidly in the presence of TTO when compared with its parent strain AG100. Attempts to isolate Mar mutants of Staphylococcus aureus using tetracycline gradients proved unsuccessful. However, when grown in the presence of salicylate, S. aureus strain BB255 demonstrated greater tolerance to TTO and did not leak K+ as rapidly in the presence of TTO compared with this strain grown without additions. This evidence demonstrates that bacterial Mar phenotypes increase tolerance to the killing action of TTO. This work also adds indirect evidence that the target of TTO is the cell membrane.     

Pyruvate blocks expression of sensitivity to antimycin A and chloramphenicol

Selectivity in Chinese hamster cells with antimycin A and chloramphenicol depends on a metabolic balance which can be modulated by varying the level of exogenous pyruvate. The effects of both inhibitors are most clearly seen in pyruvate-free nutrients. Addition of 1 mM pyruvate in plating assays shifts dose-response curves for antimycin A or chloramphenicol to higher concentration levels and reduces the differential in response between sensitive and resistant cells. In mass populations, growth inhibition by antimycin A is reduced by adding pyruvate, and growth curves for sensitive and resistant cells tend to converge. These observations show that responses to antimitochondrial drugs can be conditioned by extrinsic factors and indicate the need for further definition of selective systems.     

Evidence for a sub-population of rat T-lymphocytes highly resistant to methylglyoxal bis[guanylhydrazone] activity

Methylglyoxal bis[guanylhydrazone] (methyl-GAG) inhibits the S-adenosylmethionine decarboxylase (SAMD) activity in cells [1]. SAMD is essential for polyamine biosynthesis and its inactivation by methyl-GAG resulted in G₁-phase-arrest of mitogen-induced lymphocyte blastic transformation (uptake of [³H]thymidine). The inhibitory-dose₅₀ of methyl-GAG for [³H]TdR uptake by thymic T-lymphocytes (August strain rats) stimulated by phytohaemagglutinin (PHA) was 100 μM; in contrast, the sensitivity of lymph node or splenic lymphocytes was considerably greater (ID₅₀ = 6.0–7.0 μM). Whereas only about 62–66% of the uptake of [³H]TdR by PHA-treated extrathymic T-lymphocytes was inhibited by low concentrations of the drug, there remained 34–38% of the uptake that was unaffected until cells had been exposed to much higher drug concentrations. In all cases, arrest of [³H]TdR uptake was prevented by exogenous spermine (20 μM). Results suggested that a sub-population of T-lymphocytes (thymus, 100%; lymph node and spleen, 34–38%) were highly resistant to the drug activity. Thymic lymphocytes of Sprague-Dawley strain rats were somewhat more resistant to the drug (ID₅₀ = 500 μM). Low concentrations of methyl-GAG enhanced [³H]TdR uptake by the lymphocytes.     

Human sensitivity to vertical self-motion

Perceiving vertical self-motion is crucial for maintaining balance as well as for controlling an aircraft. Whereas heave absolute thresholds have been exhaustively studied, little work has been done in investigating how vertical sensitivity depends on motion intensity (i.e., differential thresholds). Here we measure human sensitivity for 1-Hz sinusoidal accelerations for 10 participants in darkness. Absolute and differential thresholds are measured for upward and downward translations independently at 5 different peak amplitudes ranging from 0 to 2 m/s². Overall vertical differential thresholds are higher than horizontal differential thresholds found in the literature. Psychometric functions are fit in linear and logarithmic space, with goodness of fit being similar in both cases. Differential thresholds are higher for upward as compared to downward motion and increase with stimulus intensity following a trend best described by two power laws. The power laws’ exponents of 0.60 and 0.42 for upward and downward motion, respectively, deviate from Weber’s Law in that thresholds increase less than expected at high stimulus intensity. We speculate that increased sensitivity at high accelerations and greater sensitivity to downward than upward self-motion may reflect adaptations to avoid falling.     

Myotubularin-deficient myoblasts display increased apoptosis, delayed proliferation, and poor cell engraftment

X-linked myotubular myopathy is a severe congenital myopathy caused by deficiency of the lipid phosphatase, myotubularin. Recent studies of human tissue and animal models have discovered structural and physiological abnormalities in myotubularin-deficient muscle, but the impact of myotubularin deficiency on myogenic stem cells within muscles is unclear. In the present study, we evaluated the viability, proliferative capacity, and in vivo engraftment of myogenic cells obtained from severely symptomatic (Mtm1δ4) myotubularin-deficient mice. Mtm1δ4 muscle contains fewer myogenic cells than wild-type (WT) littermates, and the number of myogenic cells decreases with age. The behavior of Mtm1δ4 myoblasts is also abnormal, because they engraft poorly into C57BL/6/Rag1null/mdx5cv mice and display decreased proliferation and increased apoptosis compared with WT myoblasts. Evaluation of Mtm1δ4 animals at 21 and 42 days of life detected fewer satellite cells in Mtm1δ4 muscle compared with WT littermates, and the decrease in satellite cells correlated with progression of disease. In addition, analysis of WT and Mtm1δ4 regeneration after injury detected similar abnormalities of satellite cell function, with fewer satellite cells, fewer dividing cells, and increased apoptotic cells in Mtm1δ4 muscle. These studies demonstrate specific abnormalities in myogenic cell number and behavior that may relate to the progression of disease in myotubularin deficiency, and may also be used to develop in vitro assays by which novel treatment strategies can be assessed.     

Isolation of poliovirus variants resistant to and dependent on arildone

Arildone has previously been shown to inhibit poliovirus replication by blocking uncoating of the virus in infected cells. The drug interacts directly with the virus particle so as to stabilize the capsid in vitro against the effects of heat or alkaline pH. We have isolated variants of poliovirus which are resistant to arildone at concentrations which inhibit native virus by greater than 99% and variants which require the presence of the drug for growth. Arildone interacts with both the drug-resistant and drug-dependent variants so as to prevent the inactivation of infectivity by heat. This suggests that the interaction of the drug with the virus particle per se is not sufficient to prevent uncoating in vivo. The drug resistance or drug dependence of the virus is not associated with gross changes in VP1, VP2, or VP3.     

Selection and properties ofPseudomonas aeruginosa variants resistant to beta-lactam antibiotics

The relation between basal and inducible-lactamase production and resistance to-lactam compounds was studied in five clinicalPseudomonas aemginosa isolates and their corresponding resistant variants selected in the presence of either piperacillin, ceftazidime or aztreonam. In all wild-type strains enzyme levels were barely detectable in the uninduced state and most-lactams, including sulbactam and clavulanic acid, exhibited poor induction potency. Imipenem proved to be the most potent inducer in both these strains and their resistant variants. In the variants selected by either piperacillin or ceftazidime enzyme production amounted to 1.28 units/mg protein of the cell-free supernatants following the addition of-lactams as inducers. Additionally, these variants exhibited the phenomenon of non-specific induction, i.e. the increase of enzyme production by either a complex nutrient medium or by addition of vitamins. Enzyme production in the aztreonam-resistant variants was identical to that in the wild-type strains with a single exception, where the entire derepression of-lactamase production in one of the variants took place. Derepression of the chromosomally mediated enzyme affects the susceptibility to ureidopenicillins more than that to carboxy-penicillins and cephalosporins, whereas the-lactamase-independent resistance results in increased resistance to all-lactams with the single exception of imipenem.     

SVMPA, a mutant of sindbis virus resistant to mycophenolic acid and ribavirin, shows an increased sensitivity to chick interferon.

SVMPA is a mutant of Sindbis virus, selected for its ability to replicate in mycophenolic acid (MPA)-treated mosquito cells. SVMPA has another phenotype: although able to replicate normally in primary cultures of chick embryo fibroblasts (CEF), its replication is restricted in secondary cultures prepared from aged primary CEF cultures. The mutations responsible for these phenotypes mapped to the region of the viral genome that codes for nsP1. We report here that SVMPA has yet another phenotype. Relative to our standard Sindbis virus (SVSTD) from which it was derived, SVMPA shows an increased sensitivity to chick interferon, both crude interferon prepared from virus-infected cells and recombinant interferon. Characterization of viral mutants obtained after site-directed mutagenesis indicated that the same mutations responsible for the host restriction of SVMPA in secondary cultures of CEF were also responsible for its increased sensitivity to chick interferon.     

Brain cancer stem cells display preferential sensitivity to Akt inhibition

Malignant brain tumors are among the most lethal cancers, and conventional therapies are largely limited to palliation. Novel therapies targeted against specific molecular pathways may offer superior efficacy and less toxicity than conventional therapies, but initial clinical trials of molecular targeted agents in brain cancer therapy have been frequently disappointing. In brain tumors and other cancers, subpopulations of tumor cells have recently been characterized by their ability to self-renew and initiate tumors. Although these cancer stem cells, or tumor initiating cells, are often only present in small numbers in human tumors, mounting evidence suggests that cancer stem cells contribute to tumor maintenance and therapeutic resistance. Thus, the development of therapies that target cancer stem cell signal transduction and biology may improve brain tumor patient survival. We now demonstrate that populations enriched for cancer stem cells are preferentially sensitive to an inhibitor of Akt, a prominent cell survival and invasion signaling node. Treatment with an Akt inhibitor more potently reduced the numbers of viable brain cancer stem cells relative to matched nonstem cancer cells associated with a preferential induction of apoptosis and a suppression of neurosphere formation. Akt inhibition also reduced the motility and invasiveness of all tumor cells but had a greater impact on cancer stem cell behaviors. Furthermore, inhibition of Akt activity in cancer stem cells increased the survival of immunocompromised mice bearing human glioma xenografts in vivo. Together, these results suggest that Akt inhibitors may function as effective anticancer stem cell therapies.     

Human GABA(B) receptor 1 gene: eight novel sequence variants

GABA (gamma-aminobutyric acid) is the principal inhibitory neurotransmitter in the brain. The human GABA(B) receptor (GABBR1) maps to the human leukocyte antigen (HLA) region of chromosome 6. Its function and location in a susceptibility region for schizophrenia, epilepsy, and dyslexia make GABBR1 a candidate gene for neurobehavioral disorders. We report the characterization of GABBR1 gene mutations in 100 chromosomes from a mixed American population. Eleven distinct mutations were found, including two previously reported missense mutations (A20V and G489S) and a previously reported silent 1977 T>C transition. Here, we report four novel silent substitutions (39C>T, 1473T>C, 1476T>C, 1545T>C) and four novel intron variants. These DNA variants may be useful in association and linkage studies of neurobehavioral disorders, and in pharmacogenetic studies of drugs targeting GABBR1.     

Regulation of tumor cell apoptotic sensitivity during the cell cycle (Review)

Understanding how current chemotherapeutic modalities induce apoptosis is critical to designing better anti-cancer agents. This review is concerned with how pharmacological agents induce tumor cell apoptosis in a cell cycle-dependent manner. Recent experiments demonstrate that expression of several apoptotic regulatory proteins (such as Bcl-2, Bax, p53, and Survivin) are differentially regulated according to the phases of the cell cycle. This cell cycle-dependent regulation in turn contributes to increased drug sensitivity in different phases of the cell cycle. It is therefore likely that the cell cycle-dependent expression of cell death proteins plays a role in regulating chemosensitivity and apoptotic commitment of human tumor cells.     

尿路上皮癌组织学变异型研究的新进展

病理学研究证明,约98%的膀胱恶性肿瘤起源于上皮,其中90%以上为尿路上皮癌,绝大多数肾盂、输尿管和下尿道的恶性肿瘤也起源于尿路上皮.尿路上皮癌有很多不同的组织学变异型,最常见的是鳞状上皮分化,其次为腺性分化和巢状变异、微乳头状等诸多类型[1].