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Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3′ termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLSVal) are an example that share some clear homology with canonical tRNAs but have several important structural differences. Although many examples of TLSVal have been identified, we lacked a full understanding of their structural diversity and phylogenetic distribution. To address this, we undertook an in-depth bioinformatic and biochemical investigation of these RNAs, guided by recent high-resolution structures of a TLSVal. We cataloged many new examples in plant-infecting viruses but also in unrelated insect-specific viruses. Using biochemical and structural approaches, we verified the secondary structure of representative TLSVal substrates and tested their ability to be valylated, confirming previous observations of structural heterogeneity within this class. In a few cases, large stem–loop structures are inserted within variable regions located in an area of the TLS distal to known host cell factor binding sites. In addition, we identified one virus whose TLS has switched its anticodon away from valine, causing a loss of valylation activity; the implications of this remain unclear. These results refine our understanding of the structural and functional mechanistic details of tRNA mimicry and how this may be used in viral infection.

A trigger for gene silencing is caused by RNAs pairing together via structural changes., Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here, we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogeneous nuclear ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multivalent protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

Polycomb Repressive Complex 2 (PRC2) contributes to initiation of heterochromatin through catalysis of histone H3 lysine 27 (H3K27) methylation. Human PRC2 can work productively with specific long noncoding RNAs (lncRNAs) at certain target loci, yet the underlying mechanism is unclear. In apparent contradiction to lncRNA-mediated PRC2 activity, PRC2 binds to RNA in a low-specificity, high-affinity manner, effectively competing with chromatin and making RNA a potent catalytic inhibitor of H3K27 methylation activity. We address this contradiction by testing the model that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its target genes can generate a context that is favorable for PRC2 activity. This RNA matchmaking activity is promoted by the heterogenous nuclear ribonucleoprotein (hnRNP) B1, which uses multiple protein domains to bind to specific regions of HOTAIR via multi-valent protein-RNA interactions. Using chemical probing, we find that RNA-RNA interaction and B1 binding facilitate RNA structural changes in HOTAIR. Finally, we demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity. By dissecting molecular details of RNA matchmaking within the HOTAIR mechanism, we highlight a switch that can occur as PRC2 samples RNA interactions broadly in the nucleus, being activated on chromatin by specific RNA matches. This concept may generalize to many contexts where RNA plays a role in potentiating PRC2 activity.

Programmed −1 ribosomal frameshifts (−1 PRFs) are commonly used by viruses to regulate their enzymatic and structural protein levels. Human T-cell leukemia virus type 1 (HTLV-1) is a carcinogenic retrovirus that uses two independent −1 PRFs to express viral enzymes critical to establishing new HTLV-1 infections. How the cis-acting RNA elements in this viral transcript function to induce frameshifting is unknown. The objective of this work was to conclusively define the 3′ boundary of and the RNA elements within the HTLV-1 pro-pol frameshift site. We hypothesized that the frameshift site structure was a pseudoknot and that its 3′ boundary would be defined by the pseudoknot's 3′ end. To test these hypotheses, the in vitro frameshift efficiencies of three HTLV-1 pro-pol frameshift sites with different 3′ boundaries were quantified. The results indicated that nucleotides included in the longest construct were essential to highly efficient frameshift stimulation. Interestingly, only this construct could form the putative frameshift site pseudoknot. Next, the secondary structure of this frameshift site was determined. The dominant structure was an H-type pseudoknot which, together with the slippery sequence, stimulated frameshifting to 19.4(±0.3)%. The pseudoknot's critical role in frameshift stimulation was directly revealed by examining the impact of structural changes on HTLV-1 pro-pol −1 PRF. As predicted, mutations that occluded pseudoknot formation drastically reduced the frameshift efficiency. These results are significant because they demonstrate that a pseudoknot is important to HTLV-1 pro-pol −1 PRF and define the frameshift site's 3′ boundary.

Structured RNA elements, programmed RNA conformational changes, and interactions between different RNA domains underlie many modes of regulating gene expression, mandating studies to understand the foundational principles that govern these phenomena. Exploring the structured 3′ untranslated region (UTR) of a viral RNA, we discovered that different contexts of the 3′-UTR confer different abilities to enhance translation of an associated open reading frame. In one context, ribosome-induced conformational changes in a ‘sensor’ RNA domain affect a separate RNA ‘functional’ domain, altering translation efficiency. The structure of the entire 3′-UTR reveals that structurally distinct domains use a spine of continuously stacked bases and a strut-like linker to create a conduit for communication within the higher-order architecture. Thus, this 3′-UTR RNA illustrates how RNA can use programmed conformational changes to sense the translation status of an upstream open reading frame, then create a tuned functional response by communicating that information to other RNA elements., Complex RNA three-dimensional structures undergo functionally important programmed conformational changes. Here, the authors report how two structurally and functionally coupled RNA domains within a viral 3′-UTR sense the ribosome through conformational changes and respond by modulating translation.

Late stage 40S ribosome assembly is a highly regulated, dynamic process that occurs in the cytoplasm, alongside the full translation machinery. Seven assembly factors (AFs) regulate and facilitate maturation, but the mechanisms through which they work remain undetermined. Here, we present a series of structures of the immature small subunit (pre-40S) determined by three-dimensional (3D) cryogenic electron microscopy with 3D sorting to assess the molecule’s heterogeneity. These structures demonstrate extensive structural heterogeneity of interface AFs that likely regulates subunit joining during 40S maturation. We also present structural models for the beak and the platform, two regions where the low resolution of previous studies did not allow for localization of AFs, and the rRNA, respectively. These models are supported by biochemical analyses using point variants and suggest that maturation of the 18S 3’-end is regulated by dissociation of the AF Dim1 from the subunit interface, consistent with previous biochemical analyses.

PURPOSE It remains a challenge to predict which women with axillary node-negative (ANN) breast cancer at greatest risk of relapse may benefit most from adjuvant therapy. Increases in neu/erbB-2 have been implicated in breast cancer prognosis. Although overexpression has been investigated extensively, this study represents the first prospective assessment of the prognostic value of neu/erbB-2 DNA amplification in a cohort of women with newly diagnosed ANN. METHODS A consecutive series of women was monitored for recurrence (median follow-up duration, 36 months) and tumors from 580 individuals were analyzed for amplification. The association of amplification with risk of recurrence was examined in survival analyses with traditional and histologic markers as prognostic factors. RESULTS Neu/erbB-2 was amplified in 20% of cases. We found an increased risk of disease recurrence when neu/erbB-2 was amplified > or = twofold that persisted with adjustment for other prognostic factors (relative risk, 2.36; P = .002). We found some evidence that amplification was more important in patients who received chemotherapy compared with untreated patients. CONCLUSION neu/erbB-2 amplification is an independent prognostic factor for risk of recurrence in ANN breast cancer. Women with tumors without neu/erbB-2 amplification have a good prognosis; aggressive therapy in this group is therefore difficult to justify. On the other hand, even with adjuvant chemotherapeutic treatment, women whose tumors exhibit neu/erbB-2 amplification have an increased risk of recurrence. We encourage a randomized trial to compare more aggressive adjuvant chemotherapy versus standard chemotherapy for ANN women whose tumors exhibit neu/erbB-2 amplification.

The Tudor domain of human PHF1 recognizes trimethylated lysine 36 of histone H3 (H3K36me3). This interaction modulates methyltransferase activity of the PRC2 complex and plays a role in retention of PHF1 at the DNA damage sites. We have previously determined the structural basis for the association of Tudor with a methylated histone peptide. Here we detail the molecular mechanism of binding of the Tudor domain to the H3KC36me3-nucleosome core particle (H3KC36me3-NCP). Using a combination of TROSY NMR and FRET we show that Tudor concomitantly interacts with H3K36me3 and DNA. Binding of the PHF1 Tudor domain to the H3KC36me3-NCP stabilizes the nucleosome in a conformation in which the nucleosomal DNA is more accessible to DNA-binding regulatory proteins. Our data provide a mechanistic explanation for the consequence of reading of the active mark H3K36me3 by the PHF1 Tudor domain.

The initial transcript of the GLS1 gene undergoes alternative splicing to produce two glutaminase variants (KGA and GAC) that contain unique C-terminal sequences. A truncated form of human glutaminase (hGA(124-551)) that lacks either C-terminal sequence was expressed in E.Coli and purified. This construct exhibits a hyperbolic glutamine saturation profile (K(m) of 1.6 mM). BPTES, bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide, functions as a potent uncompetitive inhibitor of this construct (K(i) of 0.2 µM). The hGA(124-551) is inactive in the absence of phosphate, but exhibits a hyperbolic phosphate-dependent activation profile that is also inhibited by BPTES. Gel filtration studies indicate that hGA(124-551) forms a dimer in the absence or presence of 100 mM phosphate, whereas addition of BPTES causes the formation of an inactive tetramer. The combined data indicate that BPTES inhibits human glutaminase by a novel mechanism and that BPTES is a potential lead compound for development of an effective cancer chemotherapeutic agent.

Cyclin D1 plays a critical role in regulating cell-cycle progression. Gene amplification and protein overexpression of cyclin D1 have been detected in breast cancer but little is known concerning whether these changes occur in normal breast tissue and in breast lesions associated with increased risk of development of invasive breast cancer. We looked for cyclin D1 gene amplification and protein overexpression in 30 cases of benign breast disease (16 epithelial hyperplasias without atypia and 14 atypical ductal hyperplasias) and 18 ductal carcinomas in situ by use of differential PCR and immunohistochemical staining. We compared the resulting frequencies to those in 15 cases of normal breast tissue and 17 invasive ductal carcinomas. We found cyclin D1 gene amplification in 15% of those with normal breast tissue, 19% of those with epithelial hyperplasia without atypia, 27% of those with atypical ductal hyperplasia, 35% of those with ductal carcinoma in situ, and 25% of those with invasive ductal carcinoma; corresponding figures for protein overexpression were 13, 13, 57, 50, and 64%. These results suggest that cyclin D1 amplification and protein overexpression can occur before histologic alterations are seen but that the frequencies of these changes are higher in histologic lesions with cellular atypia (atypical hyperplasia and ductal carcinoma in situ), reaching frequencies similar to those observed in invasive carcinoma.

Lipids and lipoproteins have been associated with breast cancer risk; however, published results have been inconsistent. To clarify these associations, we measured fasting lipids in women undergoing breast biopsies. A case-control study examined the association of fasting levels of lipids with histologically defined breast cancer risk. Four groups of premenopausal women were assembled on the basis of histological appearance of breast tissue: 1) no epithelial proliferation (n = 102), 2) proliferation without atypia (n = 53), 3) atypical hyperplasia or carcinoma in situ (n = 53), and 4) node-negative invasive cancer (n = 102). A postoperative fasting blood specimen was analyzed for cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. Demographics, risk factors, diet, physical activity, fasting weight, and skin-fold thickness were measured. Triglyceride levels were significantly higher in women with node-negative invasive cancer (0.94 +/- 1.04 mg/ml) than in those with no epithelial proliferation (0.83 +/- 1.04 mg/ml, p = 0.03). This association persisted after adjustment for age, body size, lipids, reproductive and familial risk factors, and previous benign breast problems (p < 0.01), in keeping with an independent association of elevated triglycerides with breast cancer risk.

Bronchoalveolar lavage was performed in 128 pigs from five fattening units showing acute pneumonia (48 animals), subclinical purulent pneumonia (17 animals), and chronic purulent pneumonia (63 animals). These samples were investigated for bacteria. Additionally immunofluorescence microscopy as well as serological investigations were performed to detect antibodies against several bacteria and viruses. Pasteurella multocida could be detected in more than a half of the samples of pigs with acute pneumonia. Bordetella bronchiseptica and mycoplasmas were isolated in a lower amount. Probably these bacteria infected the pigs of at least one herd after a primary infection with influenza virus because (i) influenza virus could be detected in three of four animals investigated for influenza virus by culture methods, (ii) the virus could be detected in one third of the animals investigated for by immunofluorescence microscopy, and (iii) antibodies against influenza virus could be detected in almost all animals. From pigs with subclinical purulent pneumonia Bordetella bronchiseptica as the only bacterial lung pathogen could be isolated exclusively from nearly each sample. From the samples of pig suffering from chronic purulent pneumonia first of all Bordetella bronchiseptica, Pasteurella multocida and different mycoplasma species could be detected. Using cultural methods Actinobacillus pleuropneumoniae could be isolated from six samples only, in contrast to frequent positive reactions against Actinobacillus pleuropneumoniae antigens obtained by immunofluorescence microscopy and CFT.

Levels of mRNA expressed by the multidrug resistance gene MDR1 were examined in 23 renal cell carcinoma samples and adjacent normal kidney cortex using reverse-transcription PCR. Comparison of MDR1 levels between histological types revealed that there was on average significantly more MDR1 in clear cell tumors than in oncocytomas (0. 89 +/- 0.10 versus 0.28 +/- 0.20, ratio of MDR1 in tumor cells to the drug-resistant cell line KB-8, P0.05). The mean MDR1 level of all of the non-oncocytoma tumors was not significantly different from the mean MDR1 level of normal adjacent kidney (0.89 +/- 0.10 versus 1.11 +/- 0.12, P = 0.07). However, the mean MDR1 level of the more undifferentiated clear cell tumors was significantly lower than the mean MDR1 level of adjacent normal kidney (0.74 +/- 0.10 versus 1.11 +/- 0.12, P0.05). MDR1 levels in early stage, clear cell tumors (n = 14) were lower than in tumors that had spread into perinephric tissue or had metastasized (n = 6) (0.77 +/- 0.08 versus 1.24 +/- 0.30, P0.05). In conclusion, MDR1 expression decreases in the more undifferentiated tumors, but still remains at levels high enough to be drug resistant. Higher MDR1 expression in the invasive tumors compared with noninvasive tumors suggests that MDR1 expression and invasiveness may be linked.

Different morphologic variants have been described for papillary carcinoma of the thyroid, but little emphasis has been placed on their cytologic patterns. We studied fine needle aspiration (FNA) smears and histologic sections from 48 cases of papillary carcinoma, including 24 of the usual type, 13 of the follicular variant, 7 of the encapsulated variant and 4 of the tall cell variant, in an attempt to correlate the cytologic features and the different histologic variants. Single cells, monolayers and papillary fragments were present in all the variants. The presence of follicles was not restricted to the follicular variant. Nuclear grooves and cytoplasmic pseudoinclusions were present in most of the cases, with nuclear grooves the most common finding in all the variants (92.3-100%). Psammoma bodies were an infrequent finding (0-25%) and were absent from the follicular variant. Colloid was present in all the variants and was a frequent finding in the follicular variant (84.6%). The findings suggest that the exact histologic variant of papillary carcinoma cannot be predicted from the appearance of the fine needle aspirate.

The purpose of this study was to determine the number, size and types of non-palpable and ultrasound undetected thyroid nodules in thyroid glands removed for a single palpable nodule. Twenty patients undergoing partial (hemi- and subtotal) thyroidectomy and 40 patients having a total thyroidectomy were analyzed. Patients had no more than one palpable nodule, no cervical adenopathy and no history of previous thyroid surgery. An ultrasound was performed routinely within four months of surgery. Forty-six nodules were detected by final pathology in the partial thyroidectomy group but only 42% were detected by palpation and 59% by ultrasound. The median diameter of non-palpable and ultrasound undetected partial thyroidectomy nodules was 0.5 cm and all were benign except for one case of lymphoma. Pathology detected 186 nodules in the total thyroidectomy group although palpation only detected 22% of these and ultrasound 42%. The median diameters of the benign and malignant non-palpable nodules were 0.4 cm and 0.25 cm respectively. Most of the undetected nodules in the total thyroidectomy group were benign but there were 20 non-palpable and 18 ultrasound undetected papillary carcinomas.

Fine needle aspirates (FNAs) from five cases of cystic squamous carcinoma metastatic to cervical lymph nodes were compared to those of five cases of branchial cleft cyst to determine whether these lesions could be differentiated by cytologic examination. Squamous carcinoma could be recognized by the following cytologic features: increased nuclear/cytoplasmic ratio, irregularity of nuclear outline and nuclear hyperchromatism. The branchial cleft cysts showed essentially benign squamous cells with only mild nuclear atypia. The background of the malignant aspirates showed more necrotic debris but fewer polymorphonuclear cells than that of the branchial cleft cysts. By adhering to these cytologic criteria a definitive diagnosis is possible; it should be confirmed by histologic examination.

We consider numerous variations on the theme that stock diminution weighted by marginal rent should be netted from basic NNP to account for economic depreciation of natural stocks. Mineral discoveries, durable exhaustible resources, and mining pollution effects are examined. Capital appreciation from transforming land in virgin forest to land in agriculture is considered. Attention is paid to second best prices.

We report a unique case of in situ adenocarcinoma ex pleomorphic adenoma of the lacrimal gland. The patient is alive and disease-free 2 1/2 years after complete local excision of the tumour. The current clinicopathologic status of this unusual tumour is discussed in the context of the reported experience to date.

The hypothesis that basal epithelial cells of the human prostate are of myoepithelial origin was investigated using immunohistochemical and ultrastructural methodologies. The immunohistologic analyses show significant phenotypic differences between prostatic basal cells and myoepithelial cells of the salivary gland. Although both cell types stain intensely with the 312C8-1 monoclonal antibody, only true myoepithelial cells demonstrated significant amounts of muscle-specific actin as decorated by the HHF35 monoclonal antibody. Furthermore, using double-labeling experiments, the prostatic basal cells were strongly decorated with a fluorescein-tagged basal cell-specific keratin but were negative with the rhodamine-tagged phalloidin, a chemical that binds specifically to actin microfilaments. Ultrastructural studies also showed an absence of thin microfilament bundles, dense bodies, and micropinocytotic vesicles in the prostatic basal cells. The current investigations show that the prostatic acini do not have a basal myoepithelium. Although some authors have suggested a stem cell role for prostatic basal cells, the weight of experimental work argues against this hypothesis. The exact role of the basal epithelial cells of the prostate is not known, although they may serve endocrine, paracrine, or other regulatory functions and may be involved in modulating signals between prostatic stroma and epithelium.