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Mature tRNAs are generated by multiple RNA processing events, which can include the excision of intervening sequences. The tRNA splicing endonuclease (TSEN) complex is responsible for cleaving these intron-containing pre-tRNA transcripts. In humans, TSEN copurifies with CLP1, an RNA kinase. Despite extensive work on CLP1, its in vivo connection to tRNA splicing remains unclear. Interestingly, mutations in CLP1 or TSEN genes cause neurological diseases in humans that are collectively termed Pontocerebellar Hypoplasia (PCH). In mice, loss of Clp1 kinase activity results in premature death, microcephaly and progressive loss of motor function. To determine if similar phenotypes are observed in Drosophila, we characterized mutations in crowded-by-cid (cbc), the CLP1 ortholog, as well as in the fly ortholog of human TSEN54. Analyses of organismal viability, larval locomotion and brain size revealed that mutations in both cbc and Tsen54 phenocopy those in mammals in several details. In addition to an overall reduction in brain lobe size, we also found increased cell death in mutant larval brains. Ubiquitous or tissue-specific knockdown of cbc in neurons and muscles reduced viability and locomotor function. These findings indicate that we can successfully model PCH in a genetically-tractable invertebrate.

Rationale & Objective In the general population, cognitive impairment is associated with increased mortality, and higher levels of education are associated with lower risks for cognitive impairment and mortality. These associations are not well studied in patients receiving long-term hemodialysis and were the focus of the current investigation. Study Design Prospective cohort study. Setting & Participants Adult hemodialysis patients treated in 20 Italian dialysis clinics. Exposures Patients’ cognitive function across 5 domains (memory, attention, executive function, language, and perceptual-motor function), measured using a neuropsychological assessment comprising 10 tests; and patients’ self-reported years of education. Outcome All-cause mortality. Analytical Approach Nested multivariable Cox regression models were used to examine associations of cognition (any domain impaired, number of domains impaired, and global function score from principal components analysis of unadjusted test scores) and education with mortality and whether there were interactions between them. Results 676 (70.6%) patients participated, with a median age of 70.9 years and including 38.8% women. Cognitive impairment was present in 79.4% (527/664; 95% CI, 76.3%-82.5%). During a median follow-up of 3.3 years (1,874 person-years), 206 deaths occurred. Compared to no cognitive impairment, adjusted HRs for mortality were 1.77 (95% CI, 1.07-2.93) for any impairment, 1.48 (95% CI, 0.82-2.68) for 1 domain impaired, 1.88 (95% CI, 1.01-3.53) for 2 domains, and 2.01 (95% CI, 1.14-3.55) for 3 to 5 domains. The adjusted HR was 0.68 (95% CI, 0.51-0.92) per standard deviation increase in global cognitive function score. Compared with primary or lower education, adjusted HRs were 0.79 (95% CI, 0.53-1.20) for lower secondary and 1.13 (95% CI, 0.80-1.59) for upper secondary or higher. The cognition-by-education interaction was not significant (P = 0.7). Limitations Potential selection bias from nonparticipation and missing data; no data for cognitive decline; associations with education were not adjusted for other socioeconomic factors. Conclusions Cognitive impairment is associated with premature mortality in hemodialysis patients. Education does not appear to be associated with mortality.

PECAM1 is a member of the immunoglobulin superfamily and is expressed in monocytes, neutrophils, macrophages and other types of immune cells as well as in endothelial cells. PECAM1 function is crucial for the development and maturation of B lymphocytes. The aim of this study was to link rare PECAM1 variants found in lymphedema patients with the development of lymphatic system malformations. Using NGS, we previously tested 246 Italian lymphedema patients for variants in 29 lymphedema-associated genes and obtained 235 negative results. We then tested these patients for variants in the PECAM1 gene. We found three probands with rare variants in PECAM1. All variants were heterozygous missense variants. In Family 1, the unaffected mother and brother of the proband were found to carry the same variant as the proband. Lymphoscintigraphy was performed to determine possible lymphatic malformations and showed that in both cases a bilateral slight reduction in the speed and lymphatic clearance of the lower limbs. PECAM1 function is important for lymphatic vasculature formation. We found variants in PECAM1 that may be associated with susceptibility to lymphedema.

CYP26B1 is a member of the cytochrome P450 family and is responsible for the break-down of retinoic acid for which appropriate levels are important for normal development of the cardiovascular and lymphatic systems. In a cohort of 235 patients with lymphatic malformations, we performed genetic testing for the CYP26B1 gene. These probands had previously tested negative for known lymphedema genes. We identified two heterozygous missense CY-P26B1 variants in two patients. Our bioinformatic study suggested that alterations caused by these variants have no major effect on the overall stability of CYP26B1 protein structure. Balanced levels of retinoic acid maintained by CYP26B1 are crucial for the lymphatic system. We identified that CYP26B1 could be involved in predisposition for lymphedema. We propose that CYP26B1 be further explored as a new candidate gene for genetic testing of lymphedema patients.

Rationale & Objective Clinical practice guidelines for dietary intake in hemodialysis focus on individual nutrients. Little is known about associations of dietary patterns with survival. We evaluated the associations of dietary patterns with cardiovascular and all-cause mortality among adults treated by hemodialysis. Study Design Prospective cohort study. Setting & Participants 8,110 of 9,757 consecutive adults on hemodialysis (January 2014 to June 2017) treated in a multinational private dialysis network and with analyzable dietary data. Exposures Data-driven dietary patterns based on the GA2LEN food frequency questionnaire. Participants received a score for each identified pattern, with higher scores indicating closer resemblance of their diet to the identified pattern. Quartiles of standardized pattern scores were used as primary exposures. Outcomes Cardiovascular and all-cause mortality. Analytical Approach Principal components analysis with varimax rotation to identify common dietary patterns. Adjusted proportional hazards regression analyses with country as a random effect to estimate the associations between dietary pattern scores and mortality. Associations were expressed as adjusted HRs with 95% CIs, using the lowest quartile score as reference. Results During a median follow-up of 2.7 years (18,666 person-years), there were 2,087 deaths (958 cardiovascular). 2 dietary patterns, “fruit and vegetable” and “Western,” were identified. For the fruit and vegetable dietary pattern score, adjusted HRs, in ascending quartiles, were 0.94 (95% CI, 0.76-1.15), 0.83 (95% CI, 0.66-1.06), and 0.91 (95% CI, 0.69-1.21) for cardiovascular mortality and 0.95 (95% CI, 0.83-1.09), 0.84 (95% CI, 0.71-0.99), and 0.87 (95% CI, 0.72-1.05) for all-cause mortality. For the Western dietary pattern score, the corresponding estimates were 1.10 (95% CI, 0.90-1.35), 1.11 (95% CI, 0.87-1.41), and 1.09 (95% CI, 0.80-1.49) for cardiovascular mortality and 1.01 (95% CI, 0.88-1.16), 1.00 (95% CI, 0.85-1.18), and 1.14 (95% CI, 0.93-1.41) for all-cause mortality. Limitations Self-reported food frequency questionnaire, data-driven approach. Conclusions These findings did not confirm an association between mortality among patients receiving long-term hemodialysis and the extent to which dietary patterns were either high in fruit and vegetables or consistent with a Western diet.

Recent improvements in high-throughput sequencing technologies underscore the pervasiveness of circular RNA (circRNA) expression in animal cells. CircRNAs are distinct from their linear counterparts because they lack the 5' caps and 3' tails that typically help determine the cellular fate of a transcript. However, due to the lack of free ends, circRNAs are impervious to exonucleases and thus can evade normal RNA turnover mechanisms. Most circRNAs are derived from protein-coding pre-mRNAs, via a mechanism called "back-splicing." Existing methods of circRNA expression thus typically involve genes that have been engineered to contain sequence elements that promote back-splicing. We recently uncovered an anciently conserved mechanism of RNA circularization in metazoans that involves splicing of tRNA introns. This splicing mechanism is completely independent from that of pre-mRNAs. In this chapter, we detail an orthogonal method that involves splicing of intron-containing tRNAs in order to produce circRNAs in vivo. We utilize fluorescence-based RNA reporters to characterize the expression, localization, and stability of these so-called tRNA intronic circular RNAs. Because tRNA biogenesis is essential for all cellular life, this method provides a means to express ultrastable, high-copy, circRNA effectors in a wide variety of metazoan cell types.

We appreciate the efforts of Matera and colleagues' study establishing the utility of IL-10 as a diagnostic and prognostic marker of early sepsis [1]; however, we have some concerns. Limiting the study to the surgical population limits the generalizability. More importantly, inclusion of 28 (of the 52) patients after on-pump cardiac surgery is concerning. Pump surgery is widely accepted to result in an intense surge in inflammatory markers, including IL-10 [2]. Also, the duration of pump surgery can be variable and the inflammatory response varies with the time spent on pump. The association of IL-10 with a worse prognosis (nonsurvivor group) may therefore not be valid. Cardiac ICU protocols such as the use of perioperative antibiotics were not discussed, which may affect mortality. In Table 3 of their article, the confidence interval for the odds ratio of IL-10 for the prognosis of bacteremic systemic inflammatory response syndrome patients includes the value 1 [1]. Including this value limits the applicability. The values of biomarkers were not checked daily, and therefore we cannot rule out a new increase in the levels of IL-10 secondary to subsequent episodes of inflammation [3]. The Sequential Organ Failure Assessment score and IL-10 values on day 1 and day 7 correlate with mortality. The utility of an expensive and time-consuming biomarker is questioned when a simple, quick and bedside test could predict the outcome. Despite the positive results of the marker, it is difficult to imagine how such information would change management in the era of the surviving sepsis guidelines [4].

Background Sm proteins are multimeric RNA-binding factors, found in all three domains of life. Eukaryotic Sm proteins, together with their associated RNAs, form small ribonucleoprotein (RNP) complexes important in multiple aspects of gene regulation. Comprehensive knowledge of the RNA components of Sm RNPs is critical for understanding their functions. Results We developed a multi-targeting RNA-immunoprecipitation sequencing (RIP-seq) strategy to reliably identify Sm-associated RNAs from Drosophila ovaries and cultured human cells. Using this method, we discovered three major categories of Sm-associated transcripts: small nuclear (sn)RNAs, small Cajal body (sca)RNAs and mRNAs. Additional RIP-PCR analysis showed both ubiquitous and tissue-specific interactions. We provide evidence that the mRNA-Sm interactions are mediated by snRNPs, and that one of the mechanisms of interaction is via base pairing. Moreover, the Sm-associated mRNAs are mature, indicating a splicing-independent function for Sm RNPs. Conclusions This study represents the first comprehensive analysis of eukaryotic Sm-containing RNPs, and provides a basis for additional functional analyses of Sm proteins and their associated snRNPs outside of the context of pre-mRNA splicing. Our findings expand the repertoire of eukaryotic Sm-containing RNPs and suggest new functions for snRNPs in mRNA metabolism.

For relatively small molecules, the biogenesis of functional mature tRNAs is an amazingly complicated process. All tRNAs are transcribed initially as precursors containing 58 leader and 38 trailer sequences that must be removed by processing. Pre-tRNAs also undergo a complex set of base modifications that are carried out by a series of enzymes that recognize specific features of tRNA structure. In addition, eukaryotic tRNAs have CCA added to their 38 ends by a specialized nucleotidyltransferase. A subset of eukaryotic pre-tRNAs also contain intervening sequences, which are removed by a dedicated set of tRNA splicing enzymes. Lastly, tRNAs must be exported from the nucleus to the cytoplasm, and must undergo aminoacylation to participate in protein synthesis. Although the basic features of the tRNA biogenesis pathway have been appreciated for at least a decade, work in a number of laboratories over the last several years has resulted in several novel and unexpected insights into this complex process. For example, it was revealed recently that tRNAs are recognized and exported to the cytoplasm by a specialized export receptor, and that recognition by this export receptor is part of a quality control mechanism that ensures that incompletely processed and mutant RNAs will be retained in the nucleus. Furthermore, recent evidence suggests that certain of the various tRNA processing steps take place in discrete subcellular compartments, such that at least some steps of the tRNA biogenesis pathway are spatially as well as temporally ordered. The eukaryotic tRNA biogenesis pathway is best understood in the budding yeast Saccharomyces cerevisiae, primarily due to the ease of combining genetics with biochemistry in this organism. However, virtually all components that have been identified in yeast have counterparts in higher cells, as would be expected for such a highly conserved process. Therefore, in this review, we emphasize recent results from yeast but also draw upon data from both prokaryotes and higher eukaryotic species. We provide an update on what is presently known about the processing events and paths taken by eukaryotic tRNAs following termination of transcription, culminating in their export to the cytoplasm. For more extensive descriptions of the enzymology of end-maturation, nucleotide modification, and tRNA splicing, see the following excellent reviews: Hopper and Martin (1992); Westaway and Abelson (1995); Grosjean et al. (1997); and Abelson et al. (1998).

The human parvovirus adeno-associated virus (AAV) is unique in its ability to target viral integration to a specific site on chromosome 19 (ch-19). Recombinant AAV (rAAV) vectors retain the ability to integrate but have apparently lost this ability to target. In this report, we characterize the terminal-repeat-mediated integration for wild-type (wt), rAAV, and in vitro systems to gain a better understanding of these differences. Cell lines latent for either wt or rAAV were characterized by a variety of techniques, including PCR, Southern hybridization, and fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to comparison and analysis. In both immortalized and normal diploid human cells, wt AAV targeted integration to ch-19. Integrated provirus structures consisted of head-to-tail tandem arrays with the majority of the junction sequences involving the AAV inverted terminal repeats (ITRs). No complete viral ITRs were directly observed. In some examples, the AAV p5 promoter sequence was found to be fused at the virus-cell junction. Data from dot blot analysis of PCR products were consistent with the occurrence of inversions of genomic and/or viral DNA sequences at the wt integration site. Unlike wt provirus junctions, rAAV provirus junctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell lines at the same locus on ch-2. In addition, provirus terminal repeat sequences existed in both the flip and flop orientations, with microhomology evident at the junctions. In all cases with the exception of the ITRs, the vector integrated intact. rAAV junction sequence data were consistent with the occurrence of genomic rearrangement by deletion and/or rearrangement-translocation at the integration locus. Finally, junctions formed in an in vitro system between several AAV substrates and the ch-19 target site were isolated and characterized. Linear AAV substrates typically utilized the end of the virus DNA substrate as the point of integration, whereas products derived from AAV terminal repeat hairpin structures in the presence or absence of Rep protein resembled AAV-ch-19 junctions generated in vivo. These results describing wt AAV, rAAV, and in vitro integration junctions suggest that the viral integration event itself is mediated by terminal repeat hairpin structures via nonviral cellular recombination pathways, with specificity for ch-19 in vivo requiring additional viral components. These studies should have an important impact on the use of rAAV vectors in human gene therapy.

The 90-kDa nucleolus organizer region autoantigen (NOR-90) was previously shown to be identical to human upstream binding factor (hUBF), which has two molecular mass forms of 89 and 93 kDa, respectively. hUBF/NOR-90 is a member of the HMG-box DNA-binding protein family and is known to bind to enhancer regions upstream of the ribosomal RNA genes, which are clustered at NORs. The smaller version of UBF lacks an internal 111-bp region corresponding to 37 amino acids in the second HMG-box of the larger form. We isolated human genomic clones from a phage library and localized one of them by fluorescence in situ hybridization to chromosome 17q21.3. DNA sequence analysis showed that the 111-bp region represented a single exon, consistent with the previous notion that the two isoforms were products of alternative pre-mRNA splicing of a single gene in human. Radiation hybrid mapping placed this STS with very high probability (LOD > 19) to chromosome 17, approximately 3.77 cR distal to MIT framework marker UTR-9641. The order of the markers (a partial list) from this region was UTR-9641, SGC30031, WI-17308, D17S930, NOR53/33, WI-16100, D17S920, WI-16913, and WI-6808.

Aerobic Gram-negative bacilli (AGNB) carried in the gut by healthy individuals generate 1 mg of 'physiological' endotoxin per g of faeces. Successful eradication of AGNB from the gut would be expected to lead to a lowering of the intestinal endotoxin pool. This prompted us to evaluate the reduction of intestinal endotoxin in 6 healthy volunteers who each received 3 different oral antibiotic regimens. Regimen 1 was polymyxin E (600 mg/day), regimen 2 polymyxin E (400 mg/day) combined with tobramycin (320 mg/day) and regimen 3 pefloxacin (800 mg/day). Each regimen was separated by an antibiotic free period of 3 months. A faecal sample (minimally 2 g) was obtained from each volunteer, before treatment began and afterwards 3 times a week on alternating days (Monday, Wednesday, Friday) for 3 weeks. Each volunteer produced 30 samples, 10 each per oral antibiotic. The samples were serially diluted in nutrient broth for the colony count of AGNB, whilst endotoxin was measured using the classical Limulus amoebocyte lysate micro-assay. The base-line value of faecal AGNB was 10 3-4 colony forming units/g of faeces. All samples obtained on day 3 following antibiotic intake were negative for AGNB, and remained negative during antibiotic intake. The AGNB free carrier state was associated with a reduction in gut endotoxin. The reduction was approximately 10 ng (1 log) for polymyxin E and pefloxacin, whilst the combination of polymyxin/tobramycin significantly reduced the intestinal endotoxin concentrations from 1 mg to 100 ng in the gut; a reduction of 104. Although AGNB were killed by the three regimens, the 'free' endotoxin left in the gut was effectively neutralised by the combination polymyxin/tobramycin only. From a clinical point of view, gut-derived endotoxaemia may play a role in the systemic inflammatory response syndrome and hence the outcome, in critically ill intensive care patients. This study supports other work which indicates that mortality is significantly reduced in only those intensive care patients who received oral polymyxin/tobramycin.

The histone gene cluster on mouse chromosome 13 has been isolated and characterized. Using overlapping YAC clones containing histone genes from chromosome 13, a contig of approximately 2 Mb has been defined. It contains 45 histone genes, organized in three patches containing tightly clustered genes. An 80-kb patch (patch III) containing 12 histone genes is near one end of the contig, and a similar-sized patch (patch I) containing 15 histone genes is near the other end of the contig, located at least 500 kb from the central patch (patch II) of histone genes. The entire cluster contains six histone H1 genes, including the testis-specific histone H1t gene that maps to the middle of the cluster. All nine histone H3 genes in this cluster have been sequenced, and their level of expression determined. Each histone H3 gene is distinct, with five genes encoding the H3.2 protein subtype and four genes encoding the H3.1 protein. They are all expressed, with each histone H3 gene accounting for a small proportion of the total histone H3 mRNA.

The histone gene cluster on mouse chromosome 3 has been isolated as a series of overlapping P1 clones, covering 110-120 kb, by probing with the histone H3-614 gene that had been mapped previously to mouse chromosome 3. There are genes for 10 core histone proteins present in a 55-kb cluster within this contig. There are three histone H3 genes, two of which are identical; four histone H2a genes, two of which are identical, one histone H4 gene; and two histone H2b genes. These histone H3 and H2a genes encode approximately 40% of the total H3 and H2a mRNA, whereas the histone H4 and histone H2b genes encode < 10% of the total H4 and H2b mRNA. There are no histone H1 genes present in this cluster. All of the histone H2a genes encode histone H2a.2 proteins (or variants of H2a.2), and account for all the H2a.2 genes in the mouse genome. All three histone H3 genes encode the histone H3.2 protein. A 21-kb region containing the adjacent H3-614 and H2a-614 genes has been duplicated and is present in an inverted repeat separated by 4.5 kb. The other two H2a genes are adjacent, with the 3' ends of their mRNAs separated by only 49 nucleotides in the DNA and the U7 snRNP binding sites separated by only 20 nucleotides. One of the histone H2b genes has lost the stem-loop sequence characteristic of the replication-dependent histone mRNAs and encodes only polyadenylated mRNAs.

We have investigated the subcellular organization of the four human Y RNAs. These RNAs, which are transcribed by RNA polymerase III, are usually found complexed with the Ro autoantigen, a 60-kD protein. We designed 2'-OMe oligoribonucleotides that were complementary to accessible single-stranded regions of Y RNAs within Ro RNPs and used them in fluorescence in situ hybridization. Although all four Y RNAs were primarily cytoplasmic, oligonucleotides directed against three of the RNAs hybridized to discrete structures near the nucleolar rim. We have termed these structures "perinucleolar compartments" (PNCs). Double labeling experiments with appropriate antisera revealed that PNCs are distinct from coiled bodies and fibrillar centers. Co-hybridization with a genomic DNA clone spanning the human Y1 and Y3 genes showed that PNCs are not stably associated with the transcription site for these Y RNAs. Although 5S rDNA was often located near the nucleolar periphery, PNCs are not associated with 5S gene loci. Two additional pol III transcripts, the RNA components of RNase P and RNase MRP, did colocalize within PNCs. Most interestingly, the polypyrimidine tract-binding protein hnRNP I/PTB was also concentrated in this compartment. Possible roles for this novel nuclear subdomain in macromolecular assembly and/or nucleocytoplasmic shuttling of these five pol III transcripts, along with hnRNP I/PTB, are discussed.

Glutamate synthase (NADH-GOGAT) is one of several proteins whose expression is either enhanced in, or specific to developing legume root nodules induced by (Brady)Rhizobium infection. This enzyme catalyzes the second committed step in the assimilation of symbiotically fixed nitrogen; the transamidation of 2-oxoglutarate by glutamine to yield two moles of glutamate. We have previously purifed alfalfa root nodule NADH-GOGAT and raised antibodies against this protein. To investigate the regulation of the gene encoding this enzyme, we have isolated a full length cDNA clone for NADH-GOGAT from an alfalfa nodule cDNA library. This 7.5 kb cDNA includes the sequence encoding the mature 210 kd protein (the amino terminus of which has been sequenced), as well as an apparent transit peptide. Northern blots indicate that mRNA levels are developmentally regulated in the nodule, showing a similar pattern to that of previously characterized genes encoding aspartate aminotransferase and phosphoenoylpyruvate carboxylase.

The organization of eight small nuclear ribonucleoproteins (the U1, U2, U4, U5, and U6 RNAs previously studied by others and three additional snRNAs, U11, U12, and 7SK) has been investigated in cultured human cells by fluorescence in situ hybridization with antisense DNA and 2'-O-Me RNA oligonucleotides. Using highly sensitive digital imaging microscopy we demonstrate that all of these snRNAs are widespread throughout the nucleoplasm, but they are excluded from the nucleoli. In addition, the U2, U4, U5, U6, and U12 snRNAs are concentrated in discrete nuclear foci, known as coiled bodies, but U1 and 7SK are not. In addition to coiled bodies, a classic speckled pattern was observed in the nucleoplasm of monolayer-grown HeLa cells, whereas suspension-grown HeLa cells revealed a more diffuse nucleoplasmic labeling. Immunofluorescence staining using various snRNP-specific antisera shows complete agreement with that of their antisense snRNA oligonucleotide counterparts. Although U2 RNA is concentrated in coiled bodies, quantitation of the fluorescence signals from the U2 antisense probe reveals that the bulk of the U2 snRNP is located in the nucleoplasm. Furthermore, simultaneous visualization of the U2 snRNAs and the tandemly repeated U2 genes demonstrates that coiled bodies are not the sites of U2 transcription.

Magainins are novel polycationic peptides with broad-spectrum antimicrobial activity, including activity against gram-negative bacteria. Gram-negative bacteremia can elicit endotoxic shock that is associated with the increased formation of eicosanoids. Inhibition of eicosanoid synthesis has been shown to improve the outcome of experimental endotoxic shock. The aim of the present study was to test the in vitro effects of two magainin peptides, MSI-97 (M1) and MSI-98 (M2), on eicosanoid synthesis by rat peritoneal macrophages (M phi) stimulated by Salmonella enteritidis lipopolysaccharide (LPS; 50 micrograms/ml) and Salmonella minnesota lipid A (5 micrograms/ml) and to compare their effects on LPS reactivity with a metachromatic dye. M1 (100 micrograms/ml) significantly (P < 0.05) reduced LPS-stimulated synthesis of thromboxane B2 (TXB2), without changing 6-keto-prostaglandin F1 alpha in M phi. Similarly, M2 (10 micrograms/ml) significantly attenuated M phi synthesis of TXB2 stimulated by either LPS or lipid A. However, at a higher concentration (100 micrograms/ml), M2 but not M1 significantly augmented LPS-induced increases in TXB2 and 6-keto-prostaglandin F1 alpha. Polymyxin B (40 micrograms/ml) inhibited LPS production and lipid A-stimulated TXB2 production. M1 (100 micrograms/ml) and polymyxin B (10 and 40 micrograms/ml) also inhibited calcium ionophore A23187 (10 microM)-induced synthesis of TXB2. The lipid A moiety of LPS reacts with dimethylmethylene blue dye, providing a metachromatic assay of LPS. This metachromatic reaction with lipid A was significantly reduced by polymyxin B and M2 at all concentrations. M1 was effective only at the highest M1:lipid A concentration ratio (2:1). Thus, M1 and M2 share some similarities with polymyxin B in inhibiting lipid A reactivity with the dye, which suggests that these magainins may also bind to lipid A. However, M1 was devoid of any inhibitory effects on dye reactivity with S. enteritidis LPS and M2 was inhibitory at only one concentration ratio (1:5). In conclusion, the varied effects of the magainin peptides on LPS, lipid A, and M phi eicosanoid synthesis appear to depend on the type of peptide used and on its concentration.

Background and methods: The immunomodulatory activity of a synbiotic combination containing three bacterial strains (Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033 and Bifidobacterium bifidum R0071) and short-chain fructooligosaccharide was examined in two distinct infectious rat models. In the T h 1 model, Wistar rats were administered the synbiotic combination for 2 weeks prior to challenge with a single oral dose of enterotoxigenic Escherichia coli or vehicle. In the T h 2 model, pretreated rats were challenged with a single subcutaneous dose of hook worm, Nippostrongylus brasiliensis. Blood samples were collected 3 hours or 4 days postchallenge and serum levels of pro- and anti-inflammatory cytokines were measured. Results: Significant reductions in pro-inflammatory cytokines interleukin (IL)-1α, IL-1β, IL-6, and tumour necrosis factor (TNF)-α were observed in both models suggesting a single, unifying mode of action on an upstream regulator. The N. brasiliensis study also compared the effect of the individual strains to synbiotic. For most of cytokines the combination appeared to average the effect of the individual strains with the exception of IL-4 and IL-10 where there was apparent synergy for the combination. Furthermore, the cytokine response varied by strain. Conclusions: It was concluded that this synbiotic combination of these three microbes could be beneficial in both T h 1 and T h 2 diseases.

The eukaryotic nucleus is a congested place, and macromolecular crowding is thought to play an important role in increasing the relative concentrations of nuclear proteins, thereby accelerating the rates of biochemical reactions. Crowding is also thought to provide the environment needed for formation of nuclear bodies/subcompartments, such as the Cajal Body (CB) and the Histone Locus Body (HLB), via self-organization. In this chapter, we contrast the theories of stochastic self-organization and hierarchical self-organization in their application to nuclear body assembly, using CBs and HLBs as paradigms. Genetic ablation studies in Drosophila on components of CBs and HLBs, have revealed an order to the assembly of these structures that is suggestive of a hierarchical model of self-organization. These studies also show that function(s) attributed to the nuclear bodies are largely unaffected in their absence, reinforcing an emerging theme in the field that the purpose of these subdomains may be to enhance the efficiency and specificity of reactions.

Southwestern (DNA-protein) screening of a murine L-cell cDNA library by using a probe for the microE3 site in the immunoglobulin heavy-chain enhancer yielded a clone, mTFE3, which is a member of the subset of basic helix-loop-helix (BHLH) proteins that also contain a leucine zipper (ZIP). Since the individual contribution of these domains is not well understood for proteins which contain them both, mutational analyses were performed to assess the functional roles of the HLH and ZIP regions for DNA binding and multimerization. The HLH region is stringently required for DNA binding but not for multimerization. The ZIP region is not stringently required for binding or multimerization, but stabilizes both multimer formation and DNA binding. A high degree of conservation at both the amino acid and nucleotide levels between the human transcription factor TFE3 and mTFE3 suggests that mTFE3 is the murine homolog of human TFE3. By using fluorescent in situ hybridization, mTFE3 was mapped to mouse chromosome X in band A2, which is just below the centromere. We show that in addition to the immunoglobulin heavy-chain microE3 site, mTFE3 binds to transcriptional elements important for lymphoid-specific, muscle-specific, and ubiquitously expressed genes. Binding of mTFE3 to DNA induces DNA bending.

We report here enhancement of nucleation and adhesion of diamond films on nondiamond substrates such as copper, stainless steel, and silicon substrates. The enhancement of nucleation is accomplished by pulsed laser irradiation which converts some of the amorphous carbon on the surface into the diamond phase or forms a reaction product that facilitates nucleation of diamond phase. The laser can also be used to evaporate carbon preferentially, leaving behind diamond particles unaffected. By pulsed laser irradiation it is possible to melt the substrate and embed the diamond particles into it, thus improving the adhesion of the diamond film.

In eight asthmatic subjects a randomized, double-blind, placebo controlled study was performed to investigate the effect of inhaled ketanserin, a 5-HT2 receptor blocking agent, in a dose of 10 mg given 30 min before test, on adsnosine-induced bronchospasm. The protective effect of ketanserin was significant in all patients, even though it altered basal bronchomotor tone in only two subjects. On the contrary, ketanserin did not inhibit histamine-induced bronchoconstrtction in four of these eight asthmatics, even though it modified sensitivity and reactivity in one of them. The results suggest that ketanserin influence on adenosine bronchial reactivity and sensitivity was not due to the bronchodilator effect of ketanserin itself or to its antihistaminic activity. We have no certain explanation for the inhibition of adenosine-induced bronchoconstriction elicited oy ketanserin. It is possible that 5-HT may play, at least in part, a role as mediator of adenosine-induced bronchoconstriction.

We have investigated the formation of TiSi2 and CoSi2 thin films on Si(100) substrates using laser (wave length 248 nm, pulse duration 40 ns and repetition rate 5 Hz) physical vapor deposition (LPVD). The films were deposited from solid targets of TiSi2 and CoSi2 in vacuum with the substrate temperature optimized at 600° C. The films were characterized using x-ray diffraction, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and four point probe ac resistivity. The films were found to be polycrystalline with a texture. The room temperature resistivity was found to be 16 μΩ-@#@ cm and 23 μΩ-cm for TiSi2 and CoSi2 films, respectively. We optimized the processing parameters so as to get particulate free surface. TEM results show that the silicide/silicon interface is quite smooth and there is no perceptible interdiffusion across the interface.

Nucleolar prevalence, size, and outline were investigated on cytological material from cold thyroid nodules obtained by fine needle aspiration. The percentage of nucleolated nuclei in follicular adenoma (32 cases) was less than in follicular carcinoma (26 cases). In adenoma most nuclei contained one nucleolus, and nuclei with two or more nucleoli were less common than in carcinoma where most cases showed the highest nucleolar diameter values. There was some overlap between adenomas and carcinomas, however, when the mean of the 10 largest values of the major nucleolar diameter was considered. In follicular carcinoma the percentage of marginated nucleoli--that is, those touching the nuclear membrane--was, in general, greater than 20%; in adenoma the values were equal to or lower than 16%. The overlap index showed that the percentages of marginated nucleoli and nucleolated nuclei are the two best discriminatory features between adenoma and carcinoma.

Summary Correlation has previously been demonstrated between qualitative and quantitative architectural and cytological features of the prostatic intra-epithelial neoplasia. In the present study, a standard Zeiss microscope and a horizontal eyepiece micrometer were used in an attempt to quantitatively define the size, number and location of nucleoli in relation to the degree of prostatic intra-epithelial neoplasia and associated benign prostatic hyperplasia and adenocarcinoma. In total, 20 prostatectomies, where features of benign prostatic hyperplasia (20 cases), prostatic intra-epithelial neoplasia of grade 1 (10 cases) and grade 2 (10 cases), and adenocarcinoma (20 cases) were present, were studied. The quantitative analysis showed that, going from benign prostatic hyperplasia to prostatic intra-epithelial neoplasia of grade 1 and 2, and to adenocarcinoma, the values of the size-related nucleolar features are progressively greater; when considering the frequency-related nucleolar features, the percentage of nucleolated nuclei increased sharply. This was associated with the decrease in the proportion of mononucleolated nuclei and increase in nuclei with two and three nucleoli. When considering the location-related nucleolar features, the degree of shift of the nucleoli to the periphery of the nucleus increased. Among all the quantitative features, the most evident changes in the nucleolar size were expressed by the nucleolar hypertrophy index after including in the calculation only those nucleoli with a diameter larger than 3 arbitrary units, whereas the percentage of nucleolated nuclei and the proportion of nuclei with two and three nucleoli best represented the modifications to the frequency. The shift in the nucleolar location in the nucleus was shown by the percentage of nucleoli in the peripheral position and by the nucleolar eccentricity index calculated after excluding nucleoli at the distance from the nuclear membrane greater than 2 or 3 Arbitrary Units. The nucleolar quantitative features can be considered useful in identifying the prostatic intra-epithelial neoplasia and in distinguishing two grades: low-grade and high-grade. The former is closer to benign prostatic hyperplasia, and the latter to adenocarcinoma.

Diamond films were grown on Si(100) and boron nitride deposited Si(100) substrates using hot filament chemical vapor deposition (HFCVD) technique. Microstructure and morphology of diamond films have been investigated systematically as a function of CH4 and H2 ratio and the ambient pressure. The deposited films were characterized by employing techniques such as scanning electron microscopy (SEM) and laser Raman spectroscopy. The average size and growth rate of diamond particles were found to increase with the CH4 to H2 ratio and decrease with the ambient pressure. Maximum growth rate of synthetic diamond deposited on Si(100) was found to be ∼3.5 µm/hr for the film deposited at 20 Torr with CH4:H2 ∼ 1.5:100 (substrate temperature ∼850°C). In most of these depositions, the morphology of the diamond crystals was cubic with significant secondary nucleation at higher methane concentrations and ambient pressure. The diamond film deposited on Si(100) with BN buffer layer shows an improvement in growth rate and the coverage, and the secondary nucleation was found to be substantially reduced, resulting in relatively smooth morphology. MicroRaman investigations show less amorphous graphite formation and better structural quality of diamond film than the one deposited without the BN buffer layer.

Codon 129 polymorphism of the prion protein gene represents a major genetic risk factor for Creutzfeldt-Jakob disease (CJD). Both CJD and Alzheimer's disease (AD) are brain amyloidoses and it would be possible that codon 129 polymorphism plays a role in the susceptibility to AD. In order to investigate this polymorphism in AD the distribution of polymorphic codon 129 of the PRNP gene in 194 probable AD and 124 controls selected in Italy and 109 neuropathologically verified AD and 58 matched controls recruited in the USA was studied. No significant association was found for the PRNP polymorphism in AD compared to controls either in Probable or in Definite AD series even after stratification for APOE polymorphism. This study does not support a role of PRNP polymorphism as a susceptibility factor for AD.

The organization of U2 genes was compared in apes, Old World monkeys, and the prosimian galago. In humans and all apes (gibbon, orangutan, gorilla, and chimpanzee), the U2 genes were organized as a tandem repeat of a 6-kb element; however, the restriction maps of the 6-kb elements in these divergent species differed slightly, demonstrating that mechanisms must exist for maintaining sequence homogeneity within this tandem array. In Old World monkeys, the U2 genes were organized as a tandem repeat of an 11-kb element; the restriction maps of the 11-kb elements in baboon and two closely related macaques, bonnet and rhesus monkeys, also differed slightly, confirming that efficient sequence homogenization is an intrinsic property of the U2 tandem array. Interestingly, the 11-kb monkey repeat unit differed from the 6-kb hominid repeat unit by a 5-kb block of monkey-specific sequence. Finally, we found that the U2 genes of the prosimian galago were dispersed rather than tandemly repeated, suggesting that the hominid and Old World monkey U2 tandem arrays resulted from independent amplifications of a common ancestral U2 gene. Alternatively, the 5-kb monkey-specific sequence could have been inserted into the 6-kb array or deleted from the 11-kb array soon after divergence of the hominid and Old World monkey lineages.

DNA base sequence comparisons indicate that a subfamily of recently transposed human Alu repeats are distinguished from most Alu repeats by diagnostic sequence differences. Using an oligonucleotide hybridization probe that incorporates these sequence features, we found that there was an expansion of this Alu subfamily following the divergence of humans and African apes. This oligonucleotide was used to select human genomic clones containing representatives of this subfamily. One representative member of this subfamily was evidently absent from the corresponding chimpanzee locus and was associated with a restriction fragment length polymorphism in the human genome. This apparently polymorphic member had all the diagnostic sequence features that initially predicted the existence of a newly expanding Alu subfamily. A transpositionally active sequence variant should also be transcriptionally active in at least some cell types or tissues. Northern (RNA) blot hybridization, primer extension, and RNA sequence analysis demonstrated the existence of different-length polyadenylated and nonpolyadenylated transcripts corresponding to this subfamily. Evidence for 3' processing and subcellular localization of these transcripts is discussed. Most of the nearly one million human Alu repeats are pseudogenes with respect to coding for either an RNA product or new family members; a select and identifiable subset of Alu repeats serve as transcriptionally and transpositionally competent source genes.