Micropatterned surfaces for control of cell shape, position, and function

The control of cell position and function is a fundamental focus in the development of applications ranging from cellular biosensors to tissue engineering. Using microcontact printing of self-assembled monolayers (SAMs) of alkanethiolates on gold, we manufactured substrates that contained micrometer-scale islands of extracellular matrix (ECM) separated by nonadhesive regions such that the pattern of islands determined the distribution and position of bovine and human endothelial cells. In addition, the size and geometry of the islands were shown to control cell shape. Traditional approaches to modulate cell shape, either by attaching suspended cells to microbeads of different sizes or by plating cells on substrates coated with different densities of ECM, suggested that cell shape may play an important role in control of apoptosis as well as growth. Data are presented which show how micropatterned substrates were used to definitively test this hypothesis. Progressively restricting bovine and human endothelial cell extension by culturing cells on smaller and smaller micropatterned adhesive islands regulated a transition from growth to apoptosis on a single continuum of cell spreading, thus confirming the central role of cell shape in cell function. The micropatterning technology is therefore essential not only for construction of biosurface devices but also for the investigation of the fundamental biology of cell-ECM interactions.     

Altered hippocampal kainate-receptor mRNA levels in temporal lobe epilepsy patients

This study determined whether hippocampal kainate (KA) receptor mRNA levels were increased or decreased in temporal lobe epilepsy patients compared with nonseizure autopsies. Hippocampal sclerosis (HS; n = 17), nonsclerosis (non-HS; n = 11), and autopsy hippocampi (n = 9) were studied for KA1-2 and GluR5-7 mRNA levels using semiquantitative in situ hybridization techniques, along with neuron densities. Compared with autopsy hippocampi, HS and non-HS cases showed decreased GluR5 and GluR6 hybridization densities per CA2 and/or CA3 pyramid. Furthermore, HS patients demonstrated increased KA2 and GluR5 hybridization densities per granule cell compared with autopsy hippocampi. These findings indicate that chronic temporal lobe seizures were associated with differential changes in hippocampal KA1-2 and GluR5-7 hybridization densities that vary by subfield and pathology group. In temporal lobe epilepsy patients, these results support the hypothesis that pyramidal cell GluR5 and GluR6 mRNA levels are decreased as a consequence of seizures, and in HS patients granule cell KA2 and GluR5 mRNA levels are increased in association with aberrant fascia dentata mossy fiber sprouting and/or hippocampal neuronal loss.     

Atrazine, alachlor, and total inorganic nitrogen concentrations of winter wind-eroded sediment samples

Virus-specific CD8+ effector T cells (eCTL) are enriched in the lungs of mice with primary influenza pneumonia, though later detection of memory T cells (mCTL) in the mediastinal lymph nodes (MLN) or spleen by peptide-based staining protocols is at the limits of flow cytometric analysis. Respiratory challenge with an H3N2 virus months after H1N1 priming induces a massive recall response, which reduces virus titers 2-3 days earlier than in nave controls. Influenza-specific mCTL produce interferon-gamma within 6 hr, but still take 4-5 days to localize to the infected respiratory tract. The delay reflects that the recall response develops first in the MLN, which contains relatively few mCTL. The response to a subdominant epitope is less obvious after secondary challenge.     

Substitution of the human beta-spectrin promoter for the human agamma-globin promoter prevents silencing of a linked human beta-globin gene in transgenic mice

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human beta-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Agamma-globin gene linked to a 576-bp fragment containing the human beta-spectrin promoter. In these mice, the beta-spectrin Agamma-globin (betasp/Agamma) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, betasp/Agamma-, psibeta-, delta-, and beta-globin genes showed no developmental switching and expressed both human gamma- and beta-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Agamma-globin gene promoter showed developmental switching and expressed Agamma-globin mRNA in yolk sac and fetal liver erythroid cells and beta-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the gamma-globin promoter with the beta-spectrin promoter allows the expression of the beta-globin gene. We conclude that the gamma-globin promoter is necessary and sufficient to suppress the expression of the beta-globin gene in yolk sac erythroid cells.     

Native valve infective endocarditis in elderly and younger adult patients: comparison of clinical features and outcomes with use of the Duke criteria and the Duke Endocarditis Database

The effect of age on the presentation and outcome of infective endocarditis (IE) is unclear. Many of the available data are based on analyses of mixed populations of patients including intravenous drug users or those with prosthetic valve endocarditis or native valve IE. We used the Duke criteria to compare the characteristics of 44 episodes of definite native valve IE in elderly patients (> 64 years old) with the characteristics of 64 similarly defined episodes of native valve IE in younger, nonintravenous-drug-using adult patients (> 29 years and < 60 years old). Our data suggest that the clinical presentation, characteristics, and outcome of native valve IE are similar for elderly patients and younger adult patients, although elderly patients were hospitalized an average of 12 days longer. Although we found that the occurrence of renal failure and cerebral embolism during an episode of IE was associated with higher rates of death (odds ratios, 4.8 and 4.0, respectively), age was not a significant contributor to mortality.     

Improved left ventricular endocardial border delineation and opacification with OPTISON (FS069), a new echocardiographic contrast agent. Results of a phase III Multicenter Trial

We investigated whether changes in iron metabolism and the transferrin receptor (TRF-R) expression were involved in the antileukaemic effects of arabinoside cytosine (ara-C). Treatment with 100 nM ara-C for 48h reduced thymidine uptake and increased the surface expression of the TRF-R on leukaemic blasts derived from 13/16 (81%) patients and on the HL-60 and U-937 cell lines. Whereas intracellular non-haem iron was strongly depleted 24 h after ara-C addition, TRF-R up-regulation and recovery of intracellular non-haem iron concentration occurred together after a longer exposure of the cultured cells to the drug. Since iron is an essential regulator of cell proliferation we have evaluated the effects of the combination between ara-C and the iron chelator desferioxamine (DSF) on the growth of HL-60 and U-937 cells. We found that desferioxamine strongly potentiated the effects of ara-C on leukaemic cell growth inhibition and apoptosis. This is the first report of a positive interaction between ara-C and an iron chelator in terms of antileukaemic effects.     

AVPR2 variants and V2 vasopressin receptor function in nephrogenic diabetes insipidus

BACKGROUND: The AVPR2 gene encodes the type 2 vasopressin receptor, a member of the vasopressin/oxytocin receptor subfamily of G protein-coupled receptors. Disruption of AVPR2 causes X-linked congenital nephrogenic diabetes insipidus (NDI), yet the functional significance of most gene sequence variations found in association with NDI has not been proven. The large number of naturally occurring AVPR2 mutations constitutes a model system for studying the structure-function relationship of G protein-coupled receptors. This analysis can be aided by examining amino acid sequence variation and conservation among evolutionarily disparate members of the subfamily. METHODS: Twenty-five new NDI patients were evaluated by DNA sequencing for mutations in AVPR2. Receptors encoded by eighteen NDI alleles were tested for physiologic signaling activity in response to varying concentrations of arginine vasopressin (AVP) in a sensitive cell culture assay. Seventeen amino acid sequences from the vasopressin/oxytocin receptor subfamily were aligned and conserved residues were identified and correlated with the locations of NDI associated variations. RESULTS: Twenty-four variant alleles were found among the 25 new patients. Thirteen had no prior family history of expressed NDI. All 18 of the NDI-associated AVPR2 alleles tested for function demonstrated diminished response to stimulation with AVP. Twelve failed to respond at all, whereas six signaled only at high AVP concentrations. Evolutionarily conserved residues clustered in the transmembrane domains and in the first and second extracellular loops, and NDI-associated missense mutations appeared mostly in the conserved domains. CONCLUSIONS: Sporadic cases are frequent and they usually represent the X-linked rather than the autosomal form of NDI. Genetic and functional testing can confirm this in individual cases. Mutations in this study affecting ligand binding domains tend to retain partial signaling in vitro, whereas those that introduce a charged residue in a transmembrane domain are inactive. The minimal partial signaling observed in cultured cells is unlikely to correlate with clinically significant urine concentrating ability. Other AVPR2 mutations with milder effects on receptor function probably exist, but may not be expressed clinically as typical NDI.     

Clinical assessment of extrapyramidal signs in nursing home patients given antipsychotic medication

BACKGROUND: We sought to quantify the relationship between antipsychotic drug use and clinical evidence of extrapyramidal dysfunction in a large population of elderly nursing home patients. METHODS: Subjects were 251 residents (mean age, 84.1 years; range, 65 to 105 years) who were taking psychoactive drugs in 12 long-term care facilities. Patient characteristics and all medication use (both scheduled and as needed) were measured during a 1-month observation period. We then performed neuropsychological and functional testing on residents who received any psychoactive medications during the study month. The presence of rigidity, bradykinesia, or masklike facies was assessed in each patient by a research assistant who was unaware of diagnoses and medication use. RESULTS: The parkinsonian signs studied were found in 127 (50.6%) of these residents. Using logistic regression modeling to adjust for potential confounding, we found this outcome to be increased more than threefold in patients who took low-potency neuroleptics (odds ratio [OR], 3.49 for > or = 50 mg/d of chlorpromazine-type drugs; 95% confidence interval [CI], 1.28 to 9.57) and more than sixfold for use of 1 mg/d or more of haloperidol (OR, 6.42; 95% CI, 2.16 to 19.04). Age, gender, and use of nonneuroleptic psychoactive drugs were not associated with an increase in parkinsonian signs. CONCLUSIONS: Clinical evidence of extrapyramidal dysfunction is three to six times more common in institutionalized elderly patients given antipsychotic medication than in comparable patients not using such drugs. Its risk is substantially increased even in patients given low-potency chlorpromazine-type drugs, as well as those taking haloperidol. The effect is not explained by age or mental status and is not seen with other psychoactive medications. The expected frequency of parkinsonian symptoms can help to inform the balancing of risks vs therapeutic effect when the use of all drugs in this class is considered.     

Extensive direct-tandem organization of a long repeat DNA sequence on the Y chromosome of chinook salmon (Oncorhynchus tshawytscha)

A long repetitive DNA sequence (OtY8) has been cloned from male chinook salmon and its genomic organization has been characterized. The repeat has a unit length of 8 kb and is present approximately 300 times per diploid male nucleus. All internal fragments within the 8-kb repeat segregate from father to son, suggesting that the entire repeat unit is located on the Y chromosome. The organization of this sequence into an 8-kb repeat unit is restricted to the Y chromosome, as are several male-specific repeat subtypes identified on the basis of restriction-site variation. The repeat possesses only weak internal sequence similarities, suggesting that OtY8 has not arisen by duplication of a smaller repeat unit, as is the case for other long tandem arrays found in eukaryotes. Based on a laddered pattern arising from partial digestion of genomic DNA with a restriction enzyme which cuts only once per repeat unit, this sequence is not dispersed on the Y chromosome but is organized as a head-to-tail tandem array. Pulse-gel electrophoresis reveals that the direct-tandem repeats are organized into at least six separate clusters containing approximately 12 to 250 copies, comprising some 2.4 Mb of Y-chromosomal DNA in total. Related sequences with nucleotide substitutions and DNA insertions relative to the Y-chromosomal fragment are found elsewhere in the genome but at much lower copy number and, although similar sequences are also found in other salmonid species, the amplification of the repeat into a Y-chromosome-linked tandem array is only observed in chinook salmon. The OtY8 repetitive sequence is genetically tightly associated with the sex-determination locus and provides an opportunity to examine the evolution of the Y chromosome and sex determination process in a lower vertebrate.