Lysine acetylation is a widespread posttranslational modification affecting many biological pathways. Recent studies indicate that acetylated lysine residues mainly exhibit low acetylation occupancy, but challenges in sample preparation and analysis make it difficult to confidently assign these numbers, limiting understanding of their biological significance. Here, we tested three common sample preparation methods to determine their suitability for assessing acetylation stoichiometry in three human cell lines, identifying the acetylation occupancy in more than 1,300 proteins from each cell line. The stoichiometric analysis in combination with quantitative proteomics also enabled us to explore their functional roles. We found that higher abundance of the deacetylase sirtuin 1 (SIRT1) correlated with lower acetylation occupancy and lower levels of ribosomal proteins, including those involved in ribosome biogenesis and rRNA processing. Treatment with the SIRT1 inhibitor EX-527 confirmed SIRT1's role in the regulation of pre-rRNA synthesis and processing. Specifically, proteins involved in pre-rRNA transcription, including subunits of the polymerase I and SL1 complexes and the RNA polymerase I-specific transcription initiation factor RRN3, were up-regulated after SIRT1 inhibition. Moreover, many protein effectors and regulators of pre-rRNA processing needed for rRNA maturation were also up-regulated after EX-527 treatment with the outcome that pre-rRNA and 28S rRNA levels also increased. More generally, we found that SIRT1 inhibition down-regulates metabolic pathways, including glycolysis and pyruvate metabolism. Together, these results provide the largest data set thus far of lysine acetylation stoichiometry (available via ProteomeXchange with identifier PXD005903) and set the stage for further biological investigations of this central posttranslational modification.

The 3' end of the HCV genome, designated as the 3' X tail, comprises an almost invariant 98-nucleotide sequence containing three highly conserved stem-loop structures (3' SL1, 3' SL2, and 3' SL3). Since these sequences are all critical for the initiation of negative-strand synthesis and essential for viral replication, they are attractive targets for novel anti-HCV drugs. To obtain effective RNA aptamers specific for the 3' X tail, and with the aim of developing novel inhibitors of HCV replication, we performed in vitro selection of aptamers with specificity for the 3' X tail. In vitro selection, namely SELEX (systematic evolution of ligands by exponential enrichment) is a useful strategy for isolating nucleic acid sequences from a randomized oligonucleotide pool that have a high affinity for a target molecule. After four selection cycles, a pool of the 3' X tail-specific RNA aptamers were obtained. This RNA pool included 39 clones that could be divided into three main classes (cSL1, cSL2, and cSL3) which harbor complementary sequences to the apical loops of 3' SL1, 3' SL2, and 3' SL3, respectively. Biochemical analyses are in progress to evaluate whether these RNA aptamers have the potential to block HCV replication.

In the search for drugs to effectively treat cancer, the last 10 years have seen a resurgence of interest in targeting ribosome biogenesis. CX-5461 is a potential inhibitor of ribosomal RNA synthesis that is now showing promise in phase I trials as a chemotherapeutic agent for a range of malignancies. Here, we show that CX-5461 irreversibly inhibits ribosomal RNA transcription by arresting RNA polymerase I (RPI/Pol1/PolR1) in a transcription initiation complex. CX-5461 does not achieve this by preventing formation of the pre-initiation complex nor does it affect the promoter recruitment of the SL1 TBP complex or the HMGB-box upstream binding factor (UBF/UBTF). CX-5461 also does not prevent the subsequent recruitment of the initiation-competent RPI-Rrn3 complex. Rather, CX-5461 blocks promoter release of RPI-Rrn3, which remains irreversibly locked in the pre-initiation complex even after extensive drug removal. Unexpectedly, this results in an unproductive mode of RPI recruitment that correlates with the onset of nucleolar stress, inhibition of DNA replication, genome-wide DNA damage and cellular senescence. Our data demonstrate that the cytotoxicity of CX-5461 is at least in part the result of an irreversible inhibition of RPI transcription initiation and hence are of direct relevance to the design of improved strategies of chemotherapy.

The solid-state fermentation state of Chinese Maotai liquor involves the interaction of several complex microbial communities leading to the generation of the most complex liquor fermentation system in the world and contributes to the unique flavor and aroma of the liquor. In this study, total DNA was extracted from 3 fermented grain samples (FG1, FG2, and FG3) and 12 environmental samples, including Daqu (DA1, DA2, DA3, and DA4), cellar mud (CS1, CS2, and CS3), soil (SL1 and SL2), air (A1 and A2), and sorghum (SH), and the 16S and 18S rRNA genes were amplified. The distribution of typical microorganisms in the samples was analyzed using nested PCR-denaturing gradient gel electrophoresis, while quantitative PCR amplification of 16S rRNA and internal transcribed spacer genes was performed to estimate the microbial abundance present in each sample. The results indicated that Daqu was the primary source of bacteria, followed by the air, soil, and sorghum samples, while the majority of the fungi responsible for Maotai liquor fermentation were from Daqu and sorghum. Highest bacterial concentrations were found in fermented grains, followed by Daqu and sorghum, while the highest fungal concentrations were found in Daqu, followed by sorghum and an air sample from outside the liquor production area. The findings of this study may provide information regarding the mechanisms responsible for flavor development in Maotai liquor, and may be used to further optimize the traditional art of making liquor.

A total of 48 female pigs (Large White × Landrace × Duroc cross) were used to determine whether a compensatory feed regime influenced performance, carcass composition and the level of plasma IGF-1. Pigs of initial age 73 days were fed a commercial diet at 0.70 of ad libitum (R) for 40 days followed by a return to ad libitum feeding for a further 42 days. The control group was fed ad libitum (A) throughout. Groups of animals on R and A feed regimes were slaughtered at the end of restriction period (SL1), 2 days after refeeding ad libitum (SL2) to establish the more immediate effects of refeeding on IGF levels, and after 42 days refeeding (SL3; n = 8 for each group). As expected, during the restriction period, average daily live weight gain in all the slaughter groups of R pigs was significantly lower than A pigs (P < 0.01); there was no significant difference in feed conversion ratios. In the re-alimentation period of SL3, R pigs grew 12.9% faster (P = 0.033), indicating compensatory growth. At SL1, there was a trend for carcass weight (P = 0.108) of A pigs to be higher than R pigs, but at SL2 live weight and carcass weight of A pigs were significantly heavier than R pigs (P < 0.05), but not at SL3. For killing-out percentage, there was no difference in SL1. After refeeding for 2 days (SL2) and 42 days (SL3), R pigs had significantly lower killing-out percentage than A pigs (P < 0.05). As a proportion of live weight, R pigs had smaller heart, kidney and liver (P < 0.05) than A pigs at SL1. At SL2, only the kidney was smaller in the restricted group (P < 0.05) and there were no significant differences in SL3. As a proportion of carcass weight, Longissimus dorsi was heavier in the R pigs at SL1 (P = 0.108) and SL2 (P < 0.05), but not at SL3. At SL1, there was a trend for intramuscular fat of A pigs to be higher than R pigs. The plasma IGF-1 level was lower in R pigs than A pigs (P = 0.010) at SL1, and slightly lower at SL2 (P = 0.110), with no significant differences at SL3. Dietary restriction period influenced plasma IGF-1 levels, which returned to the ad libitum group levels when animals were refed, as did live weight and carcass weight. It appears that the internal organs and possibly fat, but not muscles, underwent a compensatory response when animals were refed.

The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR-cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59.

The purpose of the present study was to investigate the effects of a chemically defined soybean lecithin-based semen extender as a substitute for egg yolk-based extenders in ram semen cryopreservation. In this study, 28 ejaculates were collected from four Zandi rams in the breeding season and then pooled together. The pooled semen was divided into six equal aliquots and diluted with six different extenders: (i) Tris-based extender (TE) containing 0.5% (w/v) soybean lecithin (SL0.5), (ii) TE containing 1% (w/v) soybean lecithin (SL1), (iii) TE containing 1.5% (w/v) soybean lecithin (SL1.5), (iv) TE containing 2% (w/v) soybean lecithin (SL2), (v) TE containing 2.5% (w/v) soybean lecithin (SL2.5) and (vi) TE containing 20% (v/v) egg yolk (EYT). After thawing, sperm motility and motion parameters, plasma membrane and acrosome integrity, apoptosis status and mitochondrial activity were evaluated. The results shown that total and progressive motility (54.43 ± 1.33% and 25.43 ± 0.96%, respectively) were significantly higher in SL1.5 when compared to other semen extenders. Sperm motion parameters (VAP, VSL, VCL, ALH and STR) were significantly higher in SL1.5 compared to other extender, with the exception of SL1 extender. Plasma membrane integrity (48.86 ± 1.38%) was significantly higher in SL1.5 when compared to other semen extenders. Also, percentage of spermatozoa with intact acrosome in SL1.5 (85.35 ± 2.19%) extender was significantly higher than that in SL0.5, SL2.5 and EYT extenders. The results showed that the proportion of live post-thawed sperm was significantly increased in SL1.5 extender compared to SL0.5, SL2 and EYT extenders. In addition, SL1, SL1.5 and SL2.5 extenders resulted in significantly lower percentage of early-apoptotic sperm than that in EYT extender. There were no significant differences in different semen extenders for percentage of post-thawed necrotic and late-apoptotic spermatozoa. Also, the results indicated that there are slight differences for percentage of live spermatozoa with active mitochondria between extenders. In conclusion, SL1.5 extender was better than other extenders in most in vitro evaluated sperm parameters.

A new genetic locus, epi-1, has been identified and mapped, which affects epithelialization of various tissues in the nematode, Caenorhabditis elegans. Seven independent epi-1 mutant alleles have been obtained. These mutants have a wide spectrum of abnormalities, all seem to be caused by a primary defect of basement membrane. We have identified the epi-1 gene as a structural gene of laminin alpha chain. The sequence analyses of the gene and cDNAs revealed that the gene consists of 15 exons and encodes a protein of 3704 amino acids in an open reading frame of 11115 base pairs. The nematode alpha chain is similar to its vertebrate and fly orthologs in the domain structure. The mRNA is trans-spliced to SL1 leader RNA as many of the nematode mRNAs. Mutation sites have been identified in four alleles. Two alleles have nonsense mutations and produce truncated proteins lacking the domain necessary for the formation of a heterotrimeric laminin molecule. The other two alleles have missense mutations in domains VI and IIIb, respectively.

SL1 is a stem-loop RNA sequence from the genome of HIV-1 thought to be the initiation site for the dimerization of the retroviral genomic RNA. The aim of this study is to check the stability in solution of different experimental dimeric structures available in the literature. Two kinds of dimer have been evidenced: an extended duplex looking like a double helix with two internal bulges and a kissing complex in which the monomers with a stem/loop conformation are linked by intermolecular loop-loop interactions. Two divergent experimental structures of the kissing complex from the Lai isolate are reported in the literature, one obtained from NMR (Mujeeb et al., Nature Structural Biology, 1998, Vol. 5, pp. 432-436) and the other one from x-ray crystallography (Ennifar et al., Nature Structural Biology, 2001, Vol. 8, pp. 1064-1068). A crystallographic structure of the Mal isolate was also reported (Ennifar et al., Nature Structure Biology, 2001, Vol. 8, pp. 1064-1068). Concerning the extended duplex, a NMR structure is available for Lai (Girard et al., Journal of Biomolecular Structure and Dynamics, 1999, Vol. 16, pp. 1145-1157) and a crystallographic structure for Mal (Ennifar et al., Structure, 1999, Vol. 7, pp. 1439-1449). Using a molecular dynamics technique, all these experimental structures have been simulated in solution with explicit water and counterions. We show that both extended duplex structures are stable. On the contrary, the crystallographic structures of the Lai and Mal kissing complexes are rapidly destabilized in aqueous environment. Finally, the NMR structure of the Lai loop-loop kissing complex remains globally stable over a 20 ns MD simulation, although large rearrangements occur at the level of the stem/loop junctions that are flexible, as shown from free energy calculations. These results are compared to electrophoresis experiments on dimer formation.

Clostridium drakei SL1(T) is a strictly anaerobic, H2-utilizing, and acid-tolerant acetogen isolated from an acidic sediment that is a potential platform for commodity chemical production from syngas fermentation. The draft genome sequence of this strain will enable determination of the acid resistance and autotrophic pathway of the acetogen.

The 5' end of the Mason-Pfizer monkey virus (MPMV) genomic RNA has been predicted to fold into a complex stem/loop structure that is thought to play a role in specific RNA encapsidation. In this study, we used a set of mutations that either abrogated or recreated the first four stem loops predicted within the 5' untranslated region (5' UTR) for effects on RNA packaging. Test of these mutations in our biological assay revealed that only stem loop 1 (SL1) was important for the packaging potential of MPMV, while mutations in none of the other stem loops affected packaging significantly. Interestingly, it was the primary sequence of SL1 RNA and not its secondary structure that affected packaging since compensatory mutations that reformed SL1 were unable to restore the packaging efficiency of the retroviral vector. Additionally, our mutational analysis reveals that stem loop 4, predicted to be the major packaging determinant of MPMV, does not seem to have a significant role in packaging. Finally, results of the biological effects of the structural mutations are discussed in relation to their effects on the folding potential of the various stem loops.

The 3'X domain is a 98-nt region located at the 3' end of hepatitis C virus genomic RNA that plays essential functions in the viral life cycle. It contains an absolutely conserved, 16-base palindromic sequence that promotes viral RNA dimerization, overlapped with a 7-nt tract implicated in a distal contact with a nearby functional sequence. Using small angle X-ray scattering measurements combined with model building guided by NMR spectroscopy, we have studied the stoichiometry, structure, and flexibility of domain 3'X and two smaller subdomain sequences as a function of ionic strength, and obtained a three-dimensional view of the full-length domain in its monomeric and dimeric states. In the monomeric form, the 3'X domain adopted an elongated conformation containing two SL1' and SL2' double-helical stems stabilized by coaxial stacking. This structure was significantly less flexible than that of isolated subdomain SL2' monomers. At higher ionic strength, the 3'X scattering envelope nearly doubled its size, reflecting the formation of extended homodimers containing an antiparallel SL2' duplex flanked by coaxially stacked SL1' helices. Formation of these dimers could initialize and/or regulate the packaging of viral RNA genomes into virions.

Level and phase of the (2f1-f2)-difference tone were measured as a function of primary-tone level using the psychoacoustical method of cancellation and the objective method of emission cancellation for four frequency separations of f1 = 1620 Hz and f2 in four subjects. Differences between hearing- and emission-cancellation levels ranged from 60-33 dB as delta f = f2-f1 increased from 180 to 432 Hz. For smaller separations of the primaries, phase changes for emission cancellation covered a wide range and had sharp "steps," whereas for hearing cancellation, the phase varied only slightly. With wider separations of the primaries, the phase became more varied for hearing cancellation and more homogeneous for emission cancellation. Both emission- and hearing-cancellation level functions were nonmonotonic as a function of constant SL1 and varied SL2. Remarkable phase shifts always appeared near minima in level at all separations of the primaries for emission cancellation. Four sources may be contributing to the differences in results: (a) the frequency-dependent attenuation of the middle-ear transfer function, (b) the frequency-dependent mismatch of the acoustical impedances at the eardrum, (c) the frequency dependence of the microphone's sensitivity mounted within the probe, and (d) the different reaction of active nonlinear cochlear processes on the hearing- and emission-cancellation tones.

BACKGROUND

Critically ill patients often present increased insulin resistance and stress-induced hyperglycemia. Tight glycemic control aims to reduce blood glucose (BG) levels and variability while ensuring safety from hypoglycemia. This paper presents the results of the second Belgian clinical trial using the customizable STAR framework in a target-to-range control approach. The main objective is reducing measurement frequency while maintaining performance and safety of the glycemic control.

METHODS

The STAR-Liege 2 (SL2) protocol targeted the 100-140 mg/dL glycemic band and offered 2-hourly and 3-hourly interventions. Only insulin rates were adjusted, and nutrition inputs were left to the attending clinicians. This protocol restricted the forecasted risk of BG < 90 mg/dL to a 5% level using a stochastic model of insulin sensitivity to assess patient-specific responses to insulin and its future likely variability to optimize insulin interventions. The clinical trial was performed at the Centre Hospitalier Universitaire de Liege and included 9 patients. Results are compared to 24-hour pre-trial and 24-hour post-trial, but also to the results of the first pilot trial performed in Liege, STAR-Liege 1 (SL1). This trial was approved by the Ethics Committee of the Medical Faculty of the University of Liege (Liege, Belgium).

RESULTS

During the SL2 trial, 91 measurements were taken over 194 hours. BG levels were tightly distributed: 54.9% of BG within 100-140 mg/dL, 40.7% were ≥ 140 mg/dL and 4.4% were < 100 mg/dL with no BG < 70 mg/dL. Comparing these results with 24-hour pre-trial and post-trial shows that SL2 reduced high and low BG levels and reduced glycemic variability. Nurses selected 3-hourly measurement only 5 of 16 times and overrode 12% of 91 recommended interventions (35% increased insulin rates and 65% decreased insulin rates). SL1 and SL2 present similar BG levels distribution (p>> 0.05) with significantly reduced measurement frequency for SL2 (p < 0.05).

CONCLUSIONS

The SL2 protocol succeeded in reducing clinical workload while maintaining safety and effectiveness of the glycemic control. SL2 was also shown to be safer and tighter than hospital control. Overall results validate the efficacy of significantly customizing the STAR framework.

The primary patterning event in early vertebrate development is the formation of mesoderm and subsequent induction of the neural tube by the mesoderm. Some of the transforming growth factor (TGF)-beta family (Activin, Vg1) and the fibroblast growth factor (FGF) family molecules have been implicated for their roles in mesoderm induction. Here we show first the evidence that neuregulin, an epidermal growth factor (EGF)-like growth factor known for its role in neural and muscle differentiation, participates in mesoderm induction. Neuregulin could induce the ectopic expression of mesoderm specific gene Xbra in animal cap explants reared to the midgastrula stage, when animal caps dissected from late blastula were cultured with Neuregulin at a low concentration (10 ng/ml). In situ hybridization study showed that alpha-cardiac actin was expressed in animal caps that were treated with Neuregulin overnight. Skeletal and cardiac muscle specific genes such as MyoD family genes (myoD, MRF4, myf5) and SL1 as well as NCAM, a pan neural marker, were also ectopically expressed by treatment with Neuregulin. However, the expression of NCAM is presumed to be a secondary result of the initial mesoderm induction by Neuregulin. The temporal expression pattern of neuregulin during the early developmental stages was analyzed by RT-PCR in order to determine if neuregulin is expressed at the time of mesoderm induction. It has been found that the neuregulin transcript was already detected from the 16-cell stage (stage 5) and continued to be expressed till the tailbud stage (stage 25), the latest embryonic stage analyzed in this study. Considering that the mesoderm is induced at early blastula before the start of zygotic transcription, maternal neuregulin is expressed at the right time to participate in mesoderm induction. These data strongly suggest that neuregulin plays an important role in mesoderm induction.

Carotenoids are important accessory pigments in plants that are essential for photosynthesis. Phytoene synthase (PSY), a rate-controlling enzyme in the carotenoid biosynthesis pathway, has been widely characterized in rice, maize, and sorghum, but at present there are no reports describing this enzyme in cotton. In this study, GhPSY was identified as a candidate gene for the red plant phenotype via a combined strategy using: (1) molecular marker data for loci closely linked to R1; (2) the whole-genome scaffold sequence from Gossypium raimondii; (3) gene expression patterns in cotton accessions expressing the red plant and green plant phenotypes; and (4) the significant correlation between a single nucleotide polymorphisms (SNP) in GhPSY and leaf phenotypes of progeny in the (Sub16 × T586) F2 segregating population. GhPSY was relatively highly expressed in leaves, and the protein was localized to the plastid where it appeared to be mostly attached to the surface of thylakoid membranes. GhPSY mRNA was expressed at a significantly higher level in T586 and SL1-7-1 red plants than TM-1 and Hai7124 green plants. SNP analysis in the GhPSY locus showed co-segregation with the red and green plant phenotypes in the (Sub16 × T586) F2 segregating population. A phylogenetic analysis showed that GhPSY belongs to the PSY2 subfamily, which is related to photosynthesis in photosynthetic tissues. Using a reverse genetics approach based on Tobacco rattle virus-induced gene silencing, we showed that the knockdown of GhPSY caused a highly uniform bleaching of the red color in newly-emerged leaves in both T586 and SL1-7-1 plants with a red plant phenotype. These findings indicate that GhPSY is important for engineering the carotenoid metabolic pathway in pigment production.

RNase I and RNase T1 can be used to obtain high-quality footprinting information for paromomycin binding to a 176-mer RNA from the packaging region of HIV-1 (LAI). Controls and scanning procedures are necessary for quantitation of autoradiographic data, so that footprinting plots showing cutting behavior as a function of drug concentration can be used to identify binding sites and regions of altered structure on the 176-mer. From the RNase I footprinting results the primary paromomycin binding sites on the 176-mer are on the main stem and on the stem of SL1, but noncontiguous sequences may be involved in the same binding event. Strong enhancements in cleavage with added drug are also observed, indicating drug-induced structural changes. Drug binding may cause linker regions between stem-loops of the 176-mer to change structure, possibly providing a site or sites for additional drug binding. Because drug binding changes the structure of the packaging region, which may alter its function, paromomycin analogs with enhanced specificity for HIV psi RNA have potential as a new class of agent for treating AIDS.

Aleutian disease parvovirus (ADV), mutant Gorham of the Utah-1 strain, was grown and comparatively assayed in feline cell lines CRFK and CCC clone 81 at 31.8 degrees C. The maximum virus titres as determined by a fluorescent focus assay were found to be about 10(5) FFU/ml in CRFK at day 6 p. i. and 10(6) FFU/ml at day 4 p. i. in CCC clone 81 cells. Shifting of the incubation temperature from 31.8 to 37 degrees C led to a reduced virus production after three passages. The synchronization of the CCC clone 81 cells by 1 X 10(-3) M hydroxyurea followed by infection with low (less than or equal to 0.8) multiplicities of infection (MOI) did not significantly influence the virus titres. Several mammalian cell lines such as MiCl 1 (S+L-), Mv1-Lu, 64F3 clone 7 and FEF or fish cell lines such as BB and CHSE 114 developed abortive infections after inoculation with the temperature-sensitive mutant Gorham of the ADV strain Utah-1 (ADV-G). Three new isolates designated ADV-Sl1--ADV-Sl3 were isolated from spleen and blood lymphocytes and bone marrow cells of ADV-infected mink and were adapted to grow in CCC clone 81 cells at 31.8 degrees C with virus titres between 10(4) and 10(4.7) FFU/ml. ADV particle populations varying in their bouyant density between 1.32, 1.36 and 1.43 g/ml were isolated from infected cells and culture supernatants. By protein blotting and immunodetection two major protein components with apparent M. W. of 85 and 75 KD and three minor polypeptides of 33, 28.9 and 27.5 KD were detected.

Shock waves (SWs) are used to control and decrease pain in several clinical conditions (e.g., painful elbow and shoulder, etc). This clinical effect may be due to cellular stunning of the tissues (particularly nervous components) in the area treated with SW. It may also be the consequence of unknown metabolic actions on tissues, which may include changes in cellular permeability and the liberation of proteins and mediators locally acting on pain and nerve endings. The aim of this study was to evaluate the reduction in pain and the microcirculation improvement induced by SWs treatment in an 8-week study in patients with chronic limb ischemia (CLI). Patients with CLI (15 with rest pain only and 15 with rest pain and limited distal necrosis) were included. The treatment was based on a 30-minute SWs session, three times weekly for 2 weeks. Clinical and microcirculatory evaluation were performed with laser Doppler Po2 and Pco2 measurements. Pain was measured with an analogue scale line. A Minilith SL1 (Storz Medical, Switzerland) litotriptor was used. The parabolic reflector is coupled to the skin with a silicon water cushion. Focal pressure was adjusted between 6 and 70 Mpa in eight steps. The energy flux density was variable from 0.03 to 0.5 mJ/mm2. Focal diameter and distance were defined (depth of target within the patient's foot of about 70 mm). The coded intensity used in this study was between 6 and 8 and the application time was 20 min (at four impulses per second). Twenty-eight of the 30 patients with CLI (15 with rest pain only and 13 with necrosis) completed the study. The treatment was well tolerated. Blood pressure was unchanged after 8 weeks while the increase in laser Doppler flux was significant (p<0.05) (at all measurements after treatment). The ORACLE score at 1 and 8 weeks was decreased (p<0.05). The same trend was observed with the analogue scale line for pain (p<0.05). PO2 increased (p<0.05) and Pco2 decreased (p<0.05). Tibial pressure did not change. All patients observed an increase in their subjective pain-free walking distance. The improvement was still present after 8 weeks. In a separate subset of 37 patients (mean age 60+/-9 years; males) with CLI, a SWs dose-finding evaluation was performed. Flux changes were measured at the dorsum of the foot. Three treatment plans were used: (a) 20-minute SW treatment only once; (b) 20-minute SWs treatment every 2 days for 1 week; (c) 20 minutes every day for 1 week. Treatments were well tolerated. A different increase in flux was observed on the basis of different treatments. Flux variations generally indicated that increased SWs dosage was associated with proportional flux increase. Flux improvement was still present after 4 weeks. SWs treatment in CLI produced changes both in the microcirculation and on pain. These preliminary results are comforting and open new research options to be explored in the near future.

As a retrovirus, the human immunodeficiency virus (HIV-1) packages two copies of the RNA genome as a dimer in the infectious virion. Dimerization is initiated at the dimer initiation site (DIS) which encompasses stem-loop 1 (SL1) in the 5'-UTR of the genome. Study of genomic dimerization has been facilitated by the discovery that short RNA fragments containing SL1 can dimerize spontaneously without any protein factors. On the basis of the palindromic nature of SL1, a kissing loop model has been proposed. First, a metastable kissing dimer is formed via standard Watson-Crick base pairs and then converted into a more stable extended dimer by the viral nucleocapsid protein (NCp7). This dimer maturation in vitro is believed to mimic initial steps in the RNA maturation in vivo, which is correlated with viral infectivity. We previously discovered a small molecule activator, Lys-Ala-7-amido-4-methylcoumarin (KA-AMC), which facilitates dimer maturation in vitro, and determined aspects of its structure-activity relationship. In this report, we present measurements of the binding affinity of the activators and characterization of their interactions with the SL1 RNA. Guanidinium groups and increasing positive charge on the side chain enhance affinity and activity, but features in the aromatic ring at least partially decouple affinity from activity. Although KA-AMC can bind to multiple structural motifs, the NMR study showed KA-AMC preferentially binds to unique structural motifs, such as the palindromic loop and the G-rich internal loop in the SL1 RNA. NCp7 binds to SL1 only 1 order of magnitude more tightly than the best small molecule ligand tested. This study provides guidelines for the design of superior small molecules that bind to the SL1 RNA that have the potential of being developed as an antiviral by interfering with SL1-NCp7 interaction at the packaging and/or maturation stages.

BACKGROUND

The L1 cell adhesion molecule (L1-CAM) and its soluble form sL1 play a prominent role in invasion and metastasis in several cancers. However, its association with breast cancer is still unclear.

METHODS

We analyzed L1-CAM expression and serum sL1 levels in cancer and para-carcinoma tissues from 162 consecutive patients with primary invasive breast cancer (PBC) using immunohistochemistry and an enzyme-linked immunosorbent assay, respectively. The serum sL1 levels were also examined in 38 patients with benign breast disease and 36 healthy controls.

RESULTS

L1-CAM was expressed more frequently in cancer tissues than in para-carcinoma tissues (24.1% vs. 5.6%; P < .001), and the mean sL1 levels were significantly greater in PBC than in those with benign breast disease and healthy controls (P = .027). Both L1-CAM+ expression and higher mean sL1 levels correlated significantly with larger tumor size, lymph node involvement, higher histologic grade, advanced TNM stage, and shorter disease-free survival for PBC patients. Moreover, higher mean sL1 levels were also significantly associated with estrogen receptor-α-negative expression, human epidermal growth factor receptor 2-positive (HER2+) expression, HER2-enriched and triple-negative molecular subtypes, and L1-CAM+ expression (P < .05). On multivariate analysis, larger tumor size, nodal involvement, HER2+, and higher sL1 levels (≥ 0.7 ng/mL) were independent factors associated with L1-CAM+ expression (P < .05). No association was found between L1-CAM expression or sL1 level with age, gender, histologic type, or expression of progesterone receptor, Ki-67, p53, or vascular endothelial growth factor C (P>> .05).

CONCLUSIONS

These results indicate that L1-CAM and sL1 are elevated in PBC and both might affect the prognosis of PBC patients. In addition, sL1 might be a useful marker for screening and diagnosis.

The expression of the polyubiquitin-encoding gene (ubq-1) of Caenorhabditis elegans was analysed using transgenic nematode lines carrying translational ubq-1::lacZ fusions. Animals carrying a construct consisting of 938 bp of ubq-1 upstream sequences fused to lacZ (ubq938::lacZ) expressed beta Gal in embryos and in a tissue-general manner in 20% of L1 larvae. Somatic expression in later stages was usually confined to body muscle. Progressively larger deletions extending from the 5' end of ubq938::lacZ did not significantly alter the pattern of expression until 827 bp of sequence had been removed. Thus, sequences upstream from the transcription start point, including a G+C-rich block and a sequence resembling a TATA box (GAATAA), are not required to generate the expression pattern seen with ubq938::lacZ. Moreover, a basal level of expression was maintained in embryos when 903 bp were deleted. These results suggest that the promoter elements required for efficient expression of ubq-1 may reside within the transcribed region of the gene; alternatively, they must lie more than 1.7 kb upstream or 0.8 kb downstream from this region. Polymerase chain reaction analysis indicates that RNA molecules transcribed from the ubq938::lacZ and ubq delta 827::lacZ transgenes are trans-spliced to SL1, as is ubq-1 RNA.

The 5' region of potato virus X (PVX) RNA contains a stem-loop structure, stem-loop 1 (SL1), that is required for efficient plus-strand RNA accumulation. To determine how changes to individual elements in SL1 are accommodated by the virus, we inoculated PVX transcripts containing modifications in the terminal tetraloop (TL), stem C (SC), and stem D (SD) regions onto Nicotiana benthamiana plants and analyzed progeny RNAs over a series of passages. Several progeny RNAs isolated from plants inoculated with the TL mutants containing changes to the first nucleotide of the GAAA motif or deletion of the entire TL sequence were found to contain multiple A insertions within the terminal loop region. The wild-type TL motif, GAAA, was recovered for all TL mutants by the second passage, suggesting that the sequence and potential structure of this element are crucial for PVX infection. Revertant RNAs isolated from plants inoculated with mutants in SD and the central region of SC indicated that increased stem length is tolerated. Restoration of SD length to the 4 bp typical of the wild-type PVX RNA was accompanied by A insertion into loop C. Mutants with a conversion of the C55-C78 mismatch to a G-C pair, relocation of this mismatch within the central region of SC, or deletion of C55-C78 were unable to infect protoplasts and plants. In contrast, the mutant with a conversion of the C55-C78 mismatch to an A-C mismatch, which exhibited low levels of PVX plus-strand RNA in protoplasts, was able to infect plants and quickly reverted to the wild-type C-C mismatch. These data indicate that important sequence and secondary structural elements within SL1 are required for efficient viral infection and that multiple A insertions within the TL and loop C regions, potentially by polymerase stuttering, accompany restoration of SL1 structure.

The isolation and characterization are described of a conjugate between ricin, and a thy 1.1-specific monoclonal IgG2a murine antibody synthesized using N-succinimidyl-3-(2-pyridyldithio)propionate. The conjugate selectively inhibited protein synthesis in Thy 1.1-positive (AKR SL3) mouse leukemia cells compared to Thy 1.2-positive (AKR/Cu SL1) cells.