BACKGROUND

Tea is a popular natural non-alcoholic beverage consumed worldwide due to its bioactive ingredients, particularly catechins (flavan-3-ols). Catechins not only contribute to tea quality but also serve important functions in the anti-stress regulation of secondary metabolic pathways. However, the percentages of various catechins are different among tea plant [Camellia sinensis (L.) O. Kuntze] cultivars. This study aimed to elucidate the biosynthetic mechanism of catechins. Transcriptomes from leaf tissues of four tea plant cultivars, 'Yunnanshilixiang', 'Chawansanhao', 'Ruchengmaoyecha', and 'Anjibaicha', were sequenced using the high-throughput sequencing platform Illumina HiSeq™ 2000. De novo assemble were also performed. Catechins contents were measured through reversed-phase high-performance liquid chromatography (RP-HPLC), and the biosynthetic pathway was also surveyed.

RESULTS

We constructed a unified unigene database. A total of 146,342 pairs of putative orthologs from the four tea plant cultivars, 'Yunnanshilixiang', 'Chawansanhao', 'Ruchengmaoyecha', and 'Anjibaicha' were generated. Approximately 68,890 unigenes (47.1%) were aligned to the sequences of seven public databases with a cut-off E-value of 1E-5. A total of 217 differentially expressed genes were found through RPKM values, and 150 unigenes were assigned to the flavonoid biosynthetic pathway using the integrated function annotation. The (-)-EGC and (-)-EC contents were significantly lower and the (+)-GC and (+)-C contents were abnormally higher in 'Ruchengmaoyecha' than in 'Yunnanshilixiang', 'Chawansanhao', and 'Anjibaicha'. The proportion of catechins was confirmed by selecting critical genes (ANS, ANR, and LAR) for qRT-PCR analysis.

CONCLUSIONS

This study provided a global survey of transcriptomes from four tea plant cultivars and serves as an available resource of genetic diversity. The analyses of transcriptome profiles and physiological indicators not only identified the putative genes involved in the flavonoid biosynthetic pathway but also provided some novel insights for the mechanisms of catechins biosynthesis.

A study was carried out to evaluate the antioxidant potential of pomegranate juice (PJ), rind powder extract (RP) and butylated hydroxyl toluene (BHT) in cooked chicken patties during refrigerated storage. Freshly minced chicken meats were assigned to one of the following four treatments: control (meat treated with no antioxidants); 10mg equivalent PJ phenolics per 100g meat; 10mg equivalent RP phenolics per 100g meat; 10mg BHT per 100g meat. The patties formed from the minced meats were grilled for 20min and stored under aerobically at 4°C for 15 days. Total phenolic content (as tannic acid equivalent) significantly (P<0.05) increased from 152 in control to 195 and 224μg/g in PJ and RP patties. Addition of PJ or RP did not affect any of the sensory attributes. The TBARS values were significantly (P<0.05) reduced from 1.272 in control patties to 0.896, 0.763 and 0.203mg malonaldehyde per kg samples in BHT, PJ and RP patties, respectively. The RP treatment substantially inhibited (P<0.01) lipid oxidation in cooked chicken patties to a much greater extent than BHT treatment. The PJ or RP at a level of 10mg equivalent phenolics/100g meat would be sufficient to protect chicken patties against oxidative rancidity for periods longer than the most commonly used synthetic antioxidant like BHT.

Traumatic injury to the central nervous system initiates inflammatory processes such as the synthesis of proinflammatory mediators that contribute to secondary tissue damage. Hence, administration of anti-inflammatory cytokines, such as interleukin-10 (IL-10) may be neuroprotective. Moderate hypothermia (30-32 degrees C) also decreases the pro-inflammatory response to traumatic brain injury (TBI). Thus, we hypothesized that the combination of IL-10 and hypothermia would provide synergistic neuroprotective effects after TBI. To test this hypothesis, fifty isoflurane-anesthetized rats underwent a controlled cortical impact (2.7 mm tissue deformation at 4 m/s) or sham injury and then were randomly assigned to one of five conditions (TBI/VEH Normothermia (37 degrees C), TBI/VEH Hypothermia (32 degrees C for 3 h), TBI/IL-10 Normothermia, TBI/IL-10 Hypothermia, and Sham/VEH Normothermia). Human IL-10 (5 microg) or VEH was administered (i.p.) 30 min after surgery. Function was assessed by established motor and cognitive tests on post-operative days 1-5 and 14-18, respectively. Cortical lesion volume and hippocampal CA(1)/CA(3) cell survival were quantified at 4 weeks. Brain sections from 15 additional rats were immunohistochemically assessed (MoAB RP-3) to determine neutrophil accumulation at 5 h after TBI. The administration of IL-10 after TBI produced an approximately 75% reduction in the number of RP-3-positive cells in both the normothermic and hypothermic groups vs. the normothermic vehicle-treated group (P<0.05), but did not improve functional outcome. In contrast, hypothermia alone enhanced both motor and cognitive function and increased CA(3) neuronal survival after TBI. Contrary to our hypothesis, systemic administration of IL-10 combined with hypothermia did not provide synergistic neuroprotective effects after TBI. Rather, IL-10 administration suppressed the beneficial effects produced by hypothermia alone after TBI. The mechanism(s) for the negative effects of IL-10 combined with hypothermia after TBI remain to be determined.

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.

NR2E3, a photoreceptor-specific nuclear receptor (PNR), represses cone-specific genes and activates several rod-specific genes. In humans, mutations in NR2E3 have been associated with the recessively-inherited enhanced short-wavelength sensitive S-cone syndrome (ESCS) and, recently, with autosomal dominant (ad) retinitis pigmentosa (RP) (adRP). In the present work, we describe two additional families affected by adRP that carry a heterozygous c.166G>A (p.G56R) mutation in the NR2E3 gene. Functional analysis determined the dominant negative activity of the p.G56R mutant protein as the molecular mechanism of adRP. Interestingly, in one pedigree, the most common causal variant for ESCS (p.R311Q) cosegregated with the adRP-linked p.G56R mutation, and the compound heterozygotes exhibited an ESCS-like phenotype, which in 1 of the 2 cases was strikingly "milder" than the patients carrying the p.G56R mutation alone. Impaired repression of cone-specific genes by the corepressors atrophin-1 (dentatorubral-pallidoluysian atrophy [DRPLA] gene product) and atrophin-2 (arginine-glutamic acid dipeptide repeat [RERE] protein) appeared to be a molecular mechanism mediating the beneficial effect of the p.R311Q mutation. Finally, the functional dominance of the p.R311Q variant to the p.G56R mutation is discussed.

BACKGROUND

Gluten proteins, prominent constituents of barley, wheat and rye, cause celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and the protease-resistant domains contain multiple immunogenic epitopes. The aim of this study was to identify novel sources of gluten-digesting microbial enzymes from the upper gastro-intestinal tract with the potential to neutralize gluten epitopes.

RESULTS

Oral microorganisms with gluten-degrading capacity were obtained by a selective plating strategy using gluten agar. Microbial speciations were carried out by 16S rDNA gene sequencing. Enzyme activities were assessed using gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer α-gliadin and 26-mer γ-gliadin immunogenic peptides. Fragments of the gliadin peptides were separated by RP-HPLC and structurally characterized by mass spectrometry. Strains with high activity towards gluten were typed as Rothia mucilaginosa and Rothia aeria. Gliadins (250 µg/ml) added to Rothia cell suspensions (OD(620) 1.2) were degraded by 50% after ∼30 min of incubation. Importantly, the 33-mer and 26-mer immunogenic peptides were also cleaved, primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were ∼70-75 kDa in size, and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3-10).

CONCLUSIONS

While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. The identified bacteria may be exploited for physiologic degradation of harmful gluten peptides.

BACKGROUND

The purpose of this study was to examine whether a novel lactam bridge-cyclized (111)In-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Gly-Glu-c[Lys-Nle-Glu-His-d-Phe-Arg-Trp-Gly-Arg-Pro-Val-Asp] {DOTA-GlyGlu-CycMSH} could be an effective imaging probe for metastatic melanoma detection.

METHODS

(111)In-DOTA-GlyGlu-CycMSH was prepared and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). The internalization and efflux of (111)In-DOTA-GlyGlu-CycMSH were examined in B16/F10 melanoma cells. The biodistribution of (111)In-DOTA-GlyGlu-CycMSH was determined in B16/F10 pulmonary metastatic melanoma-bearing and normal C57 mice. Pulmonary metastatic melanoma imaging was performed by small-animal single-photon emission computed tomography (SPECT)/CT (Nano-SPECT/CT) using (111)In-DOTA-GlyGlu-CycMSH as an imaging probe and compared with 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG) positron emission tomography (PET) imaging.

RESULTS

(111)In-DOTA-GlyGlu-CycMSH was readily prepared with greater than 95% radiolabeling yield. (111)In-DOTA-GlyGlu-CycMSH displayed rapid internalization and extended efflux in B16/F10 cells. (111)In-DOTA-GlyGlu-CycMSH exhibited significantly (P<.05) higher uptakes (2.00+/-0.74%ID/g at 2 h post-injection and 1.83+/-0.12%ID/g at 4 h post-injection) in metastatic melanoma-bearing lung than that in normal lung (0.08+/-0.08%ID/g and 0.05+/-0.05%ID/g at 2 and 4 h post-injection, respectively). The activity accumulation in normal organs was low (<0.5%ID/g) except for the kidneys 2 and 4 h post-injection. B16/F10 pulmonary melanoma metastases were clearly visualized with (111)In-DOTA-GlyGlu-CycMSH 2 h post-injection rather than with [(18)F]FDG 1 h post-injection.

CONCLUSIONS

(111)In-DOTA-GlyGlu-CycMSH exhibited favorable metastatic melanoma-targeting and -imaging properties, highlighting its potential as an effective imaging probe for metastatic melanoma detection.

Mutations in the C terminus of rhodopsin disrupt a rod outer segment localization signal, causing rhodopsin mislocalization and aggressive forms of retinitis pigmentosa (RP). Studies of cultured photoreceptors suggest that activated mislocalized rhodopsin can cause cell death via inappropriate G-protein-coupled signaling. To determine whether this pathway occurs in vivo, we developed a transgenic Xenopus laevis model of RP based on the class I rhodopsin mutation Q344Ter (Q350Ter in X. laevis). We used a second mutation, K296R, to block the ability of rhodopsin to bind chromophore and activate transducin. We compared the effects of expression of both mutants on X. laevis retinas alone and in combination. K296R did not significantly alter the cellular distribution of rhodopsin and did not induce retinal degeneration. Q350Ter caused rhodopsin mislocalization and induced an RP-like degeneration, including loss of rods and development of sprouts or neurites in some remaining rods, but did not affect the distribution of endogenous rhodopsin. The double mutant K296R/Q350Ter caused a similar degeneration and neurite outgrowth. In addition, we found no protective effects of dark rearing in these animals. Our results demonstrate that the degenerative effects of mislocalized rhodopsin are not mediated by the activated form of rhodopsin and therefore do not proceed via conventional G-protein-coupled signaling.

A study of the in-vitro activity of RP 59500, a semisynthetic derivative of pristinamycin, against a range of Gram-positive bacteria including erythromycin-resistant strains was undertaken. MICs were determined by plate dilution in IsoSensitest agar and MBCs by velvet pad replication. RP 59500 was found to have in-vitro activity almost identical to that of its parent compound, pristinamycin. Sixty methicillin-resistant Staphylococcus aureus (MRSA) and 60 methicillin-sensitive S. aureus (MSSA) were found to be sensitive to RP 59500, with MICs of 0.13-1 mg/L. All the MSSA and most MRSA showed an MBC less than 2 mg/L. Erythromycin-resistant S. aureus (62) were as sensitive to RP 59500 as were erythromycin-sensitive strains (58). RP 59500 was more active against MRSA than fusidate, vancomycin, amikacin, ciprofloxacin, imipenem or erythromycin. Forty strains of coagulase-negative staphylococci (11 were erythromycin-resistant) showed MICs of 0.25-1 mg/L, and RP 59500 was more active than methicillin, erythromycin, imipenem, cefotaxime or vancomycin. Sixty strains of streptococci (20 pneumococci and 40 of groups A, B, C, or G) and 20 enterococci were inhibited by 0.13-1 mg/L and 0.25-4 mg/L, respectively. Gram-positive bacilli (five each of diphtheroids, lactobacilli, Listeria monocytogenes and Bacillus spp., and 19 Clostridium spp.) were also sensitive, with MICs of between 0.06 and 4 mg/L. All 279 strains tested were judged to be sensitive to RP 59500, which was bactericidal and showed a small inoculum effect. The activity against MRSA, and against erythromycin-resistant strains of all species was particularly interesting.

Classically, the rat ventral posterior medial (VPM) nucleus of the thalamus has been considered as a simple passive relay for single-whisker information to the primary somatosensory cortex (SI). However, recent reports have suggested that the VPM could contain a much more coarsely coded and spatiotemporally complex representation of the rat whisker pad. To address this possibility properly, we have carried out chronic simultaneous recordings of large numbers (up to 23) of single neurons, distributed across the entire VPM, in both awake and lightly anesthetized adult rats. Quantitative, computer-based reconstruction of receptive fields (RFs) revealed that single VPM neurons exhibit significant responses to discrete stimulation of as many as 20 single whiskers (mean +/- SD RF size, 13.7 +/- 4.8 whiskers). By defining multiple response magnitude (RM) thresholds it was possible to subdivide these large VPM RFs quantitatively into a prominent center (mean +/- SD, 1.41 +/- 0.70 whiskers, RM>> 95%) and an excitatory surround (up to 18 whiskers, RM < 95%). VPM neurons exhibited both short-latency responses (SLRs, from 4 to 10 msec poststimulus) and/or long-latency responses (LLRs, 15-25 msec), each followed by inhibitory responses. Though LLRs were weaker (mean +/- SD, 47.19 +/- 33.34 Hz) than SLRs (119.63 +/- 50.12 Hz), they often defined RFs that differed considerably from those defined by the SLRs of the same cell. In particular, VPM cells with short-latency RFs centered in caudal whiskers (e.g., CCC->>RF shifts occurred in neurons with the largest RFs of our sample (17.2 +/- 2.4 whiskers). On the other hand, VPM cells with short-latency RFs centered in rostral whiskers had the smallest RFs (13.1 +/- 4.1 whiskers) and usually did not exhibit time-dependent RF center shifts. Multivariate analysis revealed that these two groups of VPM neurons, C->>R shifting and rostral position (RP) cells, could be statistically distinguished according to a combination of three RF attributes (short-latency RF center location, RF size, and magnitude of RF center shift). Quantitative, computer-based reconstruction of "population response maps" demonstrated that the "place" coding for each single whisker in the VPM involved a distinct weighted contribution from a large proportion of the simultaneously recorded neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Particulate guanylyl cyclase (pGC) and soluble guanylyl cyclase (sGC) are cGMP-generation systems distributed in different intracellular locations. Our aim was to test the hypothesis that the functional effects of cGMP produced by pGC and sGC on contraction and Ca2+ transients would differ in ventricular myocytes. We measured myocyte shortening from adult mice using a video edge-detector and investigated the functional changes after stimulating pGC with C-type natriuretic peptide (CNP; 10(-8) M and 10(-7) M) or sGC with S-nitroso-N-acetyl-penicillamine (SNAP; nitric oxide donor; 10(-6) M and 10(-5) M). Significant concentration-dependent decreases in percentage shortening (PCS), maximal rate of shortening (RSmax), and relaxation (RRmax) were produced by CNP. To a similar degree, SNAP concentration-dependently reduced PCS, RSmax, and RRmax. The addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (cGMP-dependent protein kinase inhibitor; 5 x 10(-6) M) or erythro-9-(2-hydroxy-3-nonyl) adenine (cGMP-stimulated cAMP phosphodiesterase inhibitor; 10(-5) M) reduced the responses induced by CNP or SNAP, suggesting that their actions were through cGMP-mediated pathways. While SNAP significantly increased intracellular cGMP concentration by 57%, CNP had little effect on cGMP production. We also found that CNP markedly decreased the amplitude of Ca2+ transients while SNAP had little effect, suggesting the cGMP generated by sGC may decrease myofilament Ca2+ sensitivity. The small amount of cGMP generated by pGC had a major effect in reducing Ca2+ level. This study suggested the existence of compartmentalization for cGMP in ventricular myocytes.

The 26S proteasome, a molecular machine responsible for regulated protein degradation, consists of a proteolytic core particle (20S CP) associated with 19S regulatory particles (19S RPs) subdivided into base and lid subcomplexes. The assembly of 19S RP base subcomplex is mediated by multiple dedicated chaperones. Among these, Hsm3 is important for normal growth and directly targets the carboxyl-terminal (C-terminal) domain of Rpt1 of the Rpt1-Rpt2-Rpn1 assembly intermediate. Here, we report crystal structures of the yeast Hsm3 chaperone free and bound to the C-terminal domain of Rpt1. Unexpectedly, the structure of the complex suggests that within the Hsm3-Rpt1-Rpt2 module, Hsm3 also contacts Rpt2. We show that in both yeast and mammals, Hsm3 actually directly binds the AAA domain of Rpt2. The Hsm3 C-terminal region involved in this interaction is required in vivo for base assembly, although it is dispensable for binding Rpt1. Although Rpt1 and Rpt2 exhibit weak affinity for each other, Hsm3 unexpectedly acts as an essential matchmaker for the Rpt1-Rpt2-Rpn1 assembly by bridging both Rpt1 and Rpt2. In addition, we provide structural and biochemical evidence on how Hsm3/S5b may regulate the 19S RP association to the 20S CP proteasome. Our data point out the diverse functions of assembly chaperones.

All reported mutations in the choroideremia (CHM) gene result in the truncation or complete absence of Rab escort protein 1 (REP1). Molecular analysis was carried out on 57 families diagnosed with CHM. Confirmation of the clinical diagnosis is important as end-stage CHM may be clinically similar to the end stages of other retinal degenerative diseases such as RP. The primary means of confirming the diagnosis of CHM is to sequence all 15 exons. An alternative method involves detection of the REP1 protein, as described in MacDonald et al. [1998]. A monoclonal antibody to REP1 does not detect truncated REP1 by immunoblot analysis, presumably due to instability and subsequent degradation of the truncated protein. This analysis provides relatively fast confirmation of the diagnosis, however, protein samples are not always available and are susceptible to degradation, affecting the accurate interpretation of results. CHM gene mutations were found in 54 of 57 families studied. The majority of mutations (>42%) were transitions and transversions. Complete deletions of the CHM gene and deletion/insertion mutations each accounted for almost 4% of the total, while over 9% had large intragenic and other partial deletions. Almost 28% of the mutations were deletions of fewer than 5 base pairs (bp) and almost 13% were splice site mutations. Despite the fact that mutations are found throughout the gene with no common mutation for the disorder, identical mutations have been characterized in unrelated individuals. The majority of these mutations are C to T transitions, changing an arginine residue (CGA) to a stop codon (TGA). Four of the five CGA codons in the CHM gene are sites of recurring mutations.

PURPOSE. To identify the genetic defect in a family with variable retinal phenotypes. The proband had a diagnosis of Leber congenital amaurosis (LCA), whereas her two cousins had an early-onset severe retinal dystrophy (EOSRD) with useful vision. A distant family member had retinitis pigmentosa (RP). METHODS. DNA samples of the affected family members were genotyped with 250 K genome-wide SNP microarrays. Genetic defects were localized by linkage analysis and homozygosity mapping, and candidate genes were analyzed by sequencing. Patients underwent a full ophthalmic examination. RESULTS. Compound heterozygous mutations in CEP290 were identified in the proband and her two cousins: the frequent c.2991+1655A>G founder mutation and a novel nonsense mutation in exon 7 (c.451C>T, p.Arg151X). The proband had nystagmus, hyperopia, a flat electroretinogram (ERG), and decreased visual acuity (20/250) from birth. The two cousins had minimal scotopic ERG responses at the age of 2. In one of these patients, visual acuity had reached a level of 20/32 at age 5, which is high for patients with CEP290 mutations. Analysis of the CEP290 mRNA in affected individuals revealed altered splice forms in which either exon 7 or exons 7 and 8 were skipped. In both mutant cDNA products, the open reading frame was not disrupted. Furthermore, homozygosity mapping and mutation analysis in the distant family member affected by RP revealed a homozygous mutation in MERTK, but no CEP290 mutations. This MERTK mutation was heterozygously present in the most severely affected (LCA) patient, but was absent in the two more mildly affected cousins. CONCLUSIONS. A novel nonsense mutation in CEP290 results in nonsense-associated altered splicing. That the remaining open reading frame is intact may explain the less severe phenotype observed in the two affected cousins. The additional heterozygous mutation in MERTK may clarify the more severe phenotype in the proband. This study extends the phenotypic spectrum of CEP290-associated diseases at the mild end.

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) limit abdominal fat depot hypertrophy. This could be due to regulation of the expression of proteins involved in adipose tissue metabolism. We investigated in vivo whether fatty acid synthase (FAS), hormone-sensitive lipase (HSL), lipoprotein lipase (LPL), phosphoenolpyruvate carboxykinase (PEPCK), CCAAT/enhancer binding protein alpha (C/EBP alpha), and leptin mRNA levels are affected in retroperitoneal (RP) and subcutaneous adipose tissues (SC) of rats fed n-3 PUFAs. For 4 weeks rats were fed high fat diets (20% fat) containing n-3 PUFAs given as eicosapentaenoic acid (EPA group), docosahexaenoic acid (DHA group), a mixture of these two fatty acids (MIX group), or native fish oil (FO group). A control group was fed with lard plus olive oil (LOO group). Final mean fat cell weight in RP ranged according to: LOO>> or = EPA>> or = DHA = FO = MIX. There was no difference in fat cell size of SC when comparing the LOO and MIX groups. The fatty acid compositions of RP and SC were similar and resemble that of dietary fat within each experimental group. In RP and compared to the LOO group, FAS, HSL, PEPCK, LPL, C/EBP alpha, and leptin mRNA levels decreased although not significantly in the EPA group, and were 40-75% lower in the DHA and MIX groups. mRNA levels were positively correlated to fat cell size in RP. In contrast, n-3 PUFAs had no effect on gene expression in SC. We conclude that n-3 PUFAs and mainly 22:6n-3 affect gene expression in a site-dependent manner in white adipose tissues via possible antiadipogenic effects.

beta(1)-Adrenoceptor autoantibodies are present in about 30% of patients suffering from dilated cardiomyopathy. The apoptotic effects mediated by beta(1)-adrenoceptor antibodies remain to be studied. Monoclonal antibodies were raised against a synthetic peptide corresponding to the second extracellular loop of the human beta(1)-adrenoceptor in balb/C mouse, and were characterized by enzyme immunoassay. Purified immunoglobulin G from nonimmunized animals (controls) did not influence the rate of apoptosis. beta(1)-Adrenoceptor antibodies caused a dose-related increase in apoptotic cells: annexin test (dilution 1:2: 21+/-1.1% apoptotic cells vs. 4+/-0.4% apoptotic cells in controls; p<0.01); TdT-mediated dUTP nick end labeling (TUNEL) test (dilution 1:2: 26+/-2% apoptotic cells vs. 10+/-2% apoptotic cells in controls; p<0.01). The effect of the beta(1)-adrenoceptor antibodies was blocked by the antigenic peptide and by the antagonist metoprolol (10 micromol/l). The apoptotic effect induced by isoproterenol was attenuated by the beta(1)-adrenoceptor antibody. After pre-incubation of cardiomyocytes with the protein kinase A inhibitor Rp-Adenosine-3',5'-cyclic monophosphothioate triethylamine (RpcAMPS), beta(1)-adrenoceptor antibody was not capable of inducing an increase of the rate of apoptosis. beta(1)-Adrenoceptor antibodies induced apoptosis in adult rat cardiomyocytes via the protein kinase A cascade.

OBJECTIVE

To characterize in detail the phenotype of five unrelated families with autosomal dominant bull's eye maculopathy (BEM) due to the R373C mutation in the PROM1 gene.

METHODS

Forty-one individuals of five families of Caribbean (family A), British (families B, D, E), and Italian (family C) origin, segregating the R373C mutation in PROM1, were ascertained. Electrophysiological assessment, fundus autofluorescence (FAF) imaging, fundus fluorescein angiography (FFA), and optical coherence tomography (OCT) were performed in available subjects. Mutation screening of PROM1 was performed.

RESULTS

The R373C mutant was present heterozygously in all affected patients. The age at onset was variable and ranged between 9 and 58 years, with most of the individuals presenting with reading difficulties. Subjects commonly had a mild to moderate reduction in visual acuity except for members of family C who experienced markedly reduced central vision. The retinal phenotype was characterized by macular dystrophy, with retinal pigment epithelial mottling in younger subjects, progressing to typical BEM over time, with the development of macular atrophy in older patients. In addition, all members of family C had typical features of RP. The electrophysiological findings were variable both within and between families.

CONCLUSIONS

Mutations in PROM1 have been described to cause a severe form of autosomal recessive RP in two families of Indian and Pakistani descent. The results of this study have demonstrated that a distinct redundant PROM1 mutation (R373C) can also produce an autosomal dominant, fully penetrant retinopathy, characterized by BEM with little inter- and intrafamilial variability, and retinal dystrophy with variable rod or rod-cone dysfunction and marked intra- and interfamilial variability, ranging from isolated maculopathy without generalized photoreceptor dysfunction to maculopathy associated with very severe rod-cone dysfunction.

Long interspersed element-1s (LINE-1 or L1s) are abundant retrotransposons that occur in mammalian genomes and that can cause insertional mutagenesis and genomic instability. L1 activity is generally repressed in most cells and tissues but has been found in some embryonic cells and, in particular, in neural progenitors. Moreover, L1 retrotransposition can be induced by several DNA-damaging agents. We have carried out experiments to verify whether L1 retrotransposition is affected by oxidative DNA damage, which plays a role in a range of human diseases, including cancer and inflammatory and neurodegenerative disease. To this purpose, BE(2)<em>C</em> neuroblastoma cells, which are thought to represent embryonic precursors of sympathetic neurons, have been treated with hydrogen peroxide and subjected to an in vitro retrotransposition assay involving an episomal L1(<em>RP</em>) element tagged with enhanced green fluorescent protein. Our results indicate that hydrogen peroxide treatment induces an increase in the retrotransposition of transiently transfected L1(<em>RP</em>) and an increase in the expression of endogenous L1 transcripts. An increase of γ-H2AX foci and changes in the mRNA levels of MRE11, RAD50, NBN and ER<em>C</em><em>C</em>1 (all involved in DNA repair) have also been found. Thus, oxidative stress can cause L1 dysregulation.

OBJECTIVE

To identify predictors of survival after resection of retroperitoneal sarcoma (RPS) and to evaluate the performance of the American Joint Committee on Cancer (AJCC) staging system for RPS.

BACKGROUND

Previous studies of survival after RPS resection are restricted to at most several institutions, yet the current AJCC staging system for RPS is based entirely on these relatively small studies.

METHODS

Patients undergoing resection of primary RPS from 1988 to 2005 were identified from the Surveillance, Epidemiology, and End Results (SEER) database. Cox proportional hazards models were used to analyze survival and evaluate AJCC staging.

RESULTS

In 1365 patient undergoing resection of primary RPS, the most prevalent histologies were liposarcoma (50%), leiomyosarcoma (26%), and malignant fibrous histiocytoma (11%). Median, 5-year, and 10-year survival after resection were 55 months, 47%, and 27%. Histological subtype (P < 0.001), histological grade (grade 3-4 vs. grade 1; HR, 2.42; P < 0.001), and tumor invasion of adjacent structures (HR, 1.37; P < 0.001) were associated with survival on multivariable analysis. However, tumor size had no prognostic value. Consequently, the AJCC T classification system demonstrated poor discriminatory ability (c = 0.50). The AJCC stage grouping system demonstrated moderate discriminatory ability (c = 0.66) but performed no better than a much simpler system that omits information on tumor size and lymph node metastasis (c = 0.67).

CONCLUSIONS

Indicators of tumor aggressiveness (histological grade and invasion of adjacent structures) as well as histological subtype predict survival after RPS resection. Tumor size, however, does not impact survival. The AJCC staging system for RPS is in need of revision.

A reversed-phase (RP) high-performance liquid chromatographic (HPLC) method with fluorescence detection allowing the sensitive and specific quantification of BAY 12-8039, a new antimicrobially active 8-methoxyquinolone, in biological fluids is described. The method is compared to a microbiological assay (bioassay) based on B. subtilis test strain with a limit of quantification of approximately 60 microg/l. Following dilution and centrifugation, plasma, saliva or urine supernatant is directly injected onto the HPLC system. Concentrations down to a limit of quantification of 2.5 microg/l can be quantified in plasma, saliva and urine. Data on recovery, accuracy and precision of the method throughout the whole working range as well as results on stability of the analyte are presented. The concentration data are correlated with results from the bioassay. BAY 12-8039 is stable in plasma after repeated freeze-thaw cycles and following storage at -20 degrees C for at least 12 months. The results of HPLC measurements excellently agree with bioassay data indicating the relevance of the method as a tool in clinical development to answer pharmacokinetic questions related to antimicrobial activity. The method was applied to human plasma, saliva and urine from subjects after a single oral dose of 400 mg of BAY 12-8039.

OBJECTIVE

To identify possible mutations in known candidate genes in patients with autosomal recessive (ar) and simplex retinitis pigmentosa (RP), by using an established strategy of flexible, multiplexed, microsatellite-based homozygosity mapping.

METHODS

A total of 78 microsatellite markers corresponding to 16 genes known to be responsible for arRP were selected and used in 18 multiplex amplifications, followed by genotyping. Twelve consanguineous probands and 47 nonconsanguineous probands (59 patients with arRP or simplex RP) agreed to the screening.

RESULTS

Of the 59 probands examined, 24 had a mean of 1.4 genes showing homozygosity for all markers within the corresponding gene region. Subsequent direct sequencing revealed three homozygous mutations. Two of them were novel mutations in the genes TULP1 (c.1145T->>C, F382S) and CNGB1 (c.3444 + 1G->>A). The other was a mutation in RPE65 (c.1543C->>T, R515W), which is known to cause Leber's congenital amaurosis. The clinical features of each patient, together with the cosegregation analysis, strongly support the pathogenicity of these mutations.

CONCLUSIONS

This systematic approach facilitated the identification of genes that cause arRP, and the results provide a widened spectrum of the mutation severity associated with a broader range of phenotypic manifestations of arRP.

To further elucidate the role of the neuromodulatory transmitter serotonin (5-HT) during early postnatal development of the neocortex, we investigated the effects of 5-HT on gap junction coupling in the somatosensory cortex of rats aged between postnatal days 7 and 10. The gap junction-permeable tracer neurobiotin was injected into single neurons via microelectrodes or patch pipettes. Under control conditions, clusters of about 25 tracer-coupled neurons were observed. Serotonin reduced dye-coupling between lamina II/III pyramidal cells in a concentration-dependent and reversible manner. The 1,4,5-inositol triphosphate (IP3) receptor antagonist heparin as well as the protein kinase C inhibitor NPC 15437 suppressed the uncoupling action of 5-HT, suggesting that the serotonergic effect involved IP3 receptor-mediated release of calcium ions from intracellular stores. In contrast, the 5-HT-induced reduction in gap junction coupling was not antagonized by Rp-adenosine-3',5'-cyclic monophosphothionate, an inhibitor of cAMP dependent protein kinase. The uncoupling effect of 5-HT was mimicked by 5-HT2 receptor agonists and antagonized by the 5-HT2 receptor antagonist ritanserin, indicating that 5-HT suppressed gap junction coupling via activation of 5-HT2 class receptors. Our results suggest that the developmental functions of 5-HT not only involve the modulation of chemical synaptic transmission but also include the regulation of the gap junctional communication system during differentiation of the neocortex.

The 11 kb complex DNA fingerprinting probe Ca3 is effective both in cluster analyses of Candida albicans isolates and in identifying microevolutionary changes in the size of hypervariable genomic fragments. A 2.6 kb EcoRI fragment of Ca3, the C fragment, retains the capacity to identify these microevolutionary changes, and when the C fragment is cleaved with SacI, the capacity is retained exclusively by a 1 kb subfragment, CRPS repeat element. The microevolutionary changes identified by Ca3, therefore, may involve reorganization of RPS elements dispersed throughout the genome. To test this possibility, hypervariable fragments from several strains of C. albicans were sequenced and compared. The results demonstrate that the microevolutionary changes identified by Ca3 are due to the insertion and deletion of full-length tandem RPS elements at specific genomic sites dispersed throughout the C. albicans genome. The RPS elements at these dispersed sites are bordered by the same upstream and downstream sequences. The frequency of recombination was estimated to be one recombination per 1000 cell divisions by following RPS reorganization in vitro. The results are inconsistent with unequal recombination between homologous or heterologous chromosomes, but consistent with intrachromosomal recombination. Two alternative models of intrachromosomal recombination are proposed: unequal sister-chromatid exchange and slipped misalignment at the replication fork.

The present work brings the first evidence for the simultaneous release of Paf-acether (platelet-activating factor), slow-reacting substance (SRS), histamine, and serotonin from isolated rat kidneys stimulated with ionophore A 23187. However, with compound 48/80 we detected only SRS, histamine, and serotonin. Upon addition of antigen, kidneys from sensitized rats released Paf-acether and SRS of anaphylaxis. Paf-acether released in the perfusate was identified by its ability to aggregate aspirin-treated washed rabbit platelets in the presence of an ADP scavenger complex. It was also characterized by its inactivation by phospholipase A2, and it was eluted from high pressure liquid chromatography (HPLC) after 16 to 18 min, a retention identical to that of synthetic Paf-acether, that is, between sphingomyelin and lysophosphatidylcholine. The biological activity of SRS was detected after several purification steps including Amberlite XAD-2 and reverse phase HPLC (RP-HPLC). Kidney SRS exhibited a typical spasmogenic activity in isolated guinea-pig ileum preparation that was reversed by FPL 55712. When kidneys were incubated with [3H]arachidonic acid, radioactivity and biological activity comigrated in RP-HPLC with leukotrienes C and D. These results indicate that the kidney is capable of actively released inflammatory mediators.