Protective effects of extracorporeal shockwave on rat chondrocytes and temporomandibular joint osteoarthritis; preclinical evaluation with in vivo99mTc-HDP SPECT and ex vivo micro-CT.

OBJECTIVE: Extracorporeal shockwave therapy (ESWT) has been shown to have chondroprotective effects on arthritic diseases. We investigated the effects of ESWT on temporomandibular joint osteoarthritis (TMJOA) using rat chondrocytes and TMJOA rat models. DESIGN: Cell viability and expression of pro-inflammatory cytokines, cartilage degradation, and apoptosis markers were measured in control, monosodium iodoacetate (MIA)-treated and ESWT plus MIA-treated chondrocytes in vitro, and intra-articular MIA injection (TMJOA) and ESWT on TMJOA rats in vivo. In vivo99mTc-hydroxymethylene diphosphonate (HDP) single-photon emission computerized tomography/computerized tomography (SPECT/CT) and ex-vivo micro-CT and histologic examinations were performed in rat models. RESULTS: ESWT plus MIA-treated chondrocytes showed increased cell viability significantly (P = 0.007), while decreased genetic expression of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6); P < 0.001 for each] and cartilage degradation markers [matrix metalloproteinase-3 (MMP3), matrix metalloproteinase-13 (MMP13), and bone morphogenetic protein 7 (BMP7); P < 0.001 for each], and number of apoptotic cells (P < 0.001) compared to MIA-treated chondrocytes. Changes in cytochrome c and cleaved caspase-3 levels relative to procaspase-3 were decreased over MIA-treated chondrocytes. ESWT on TMJOA rat models was associated with a significant decrease in pro-inflammatory and cartilage degradation markers, as demonstrated by real-time PCR and immunohistochemistry stains (P < 0.001 for each). On 99mTc-HDP SPECT/CT, the ESWT group showed a significantly lower uptake ratio compared to the TMJOA group (P = 0.008). Micro-CT analysis revealed that the ESWT group showed improved structure and bone quality compared to the TMJOA control group. CONCLUSIONS: ESWT was associated with a protective effect on cartilage and subchondral bone structures of TMJOA by reducing inflammation, cartilage degradation, and chondrocyte apoptosis.

Factors associated with HIV/AIDS-related stigma and discrimination by medical professionals in Korea: A survey of infectious disease specialists in Korea.

OBJECTIVES: This study sought to identify factors associated with this discrimination by medical professionals in Korea. SUBJECTS AND METHODS: This study was a cross-sectional survey. We conducted web-based surveys against infectious disease specialists and infectious disease nurse. We evaluated the frequency of human immunodeficiency virus (HIV)/AIDS-related discrimination by medical professionals by health service type on the 5-point scale. We identified the association between several factors and HIV/AIDS-related stigma and discrimination by medical professionals on the 5-point scale. RESULTS: A total of 81 experts, 57 infectious disease specialists (approximately 27% of all infectious disease specialists in Korea) and 24 infectious disease nurse practitioners, participated in this study. The frequency of stigma and discrimination increased significantly when invasive treatment included both outpatient and inpatient services (both P < 0.05). Medical professional's preconceptions, fear of infection, and lack of knowledge have an association with HIV/AIDS-related stigma and discrimination by medical professionals. CONCLUSION: HIV/AIDS-related stigma and discrimination by medical professionals in Korea might be associated with factors related to the fear of medical professionals.

The effect of low-volatile organic compounds, water-based paint on aggravation of allergic disease in schoolchildren.

Whether indoor painting aggravates preexisting allergic diseases remains unclear. We aimed to evaluate the impact of new classroom painting on aggravation of asthma, allergic rhinitis (AR), and atopic dermatitis (AD) in children. Studied school was previously painted with conventional water-based paint 20 years ago and had natural ventilation system. We identified a total of 172 children aged 10-12 years with allergic diseases in 17 classrooms, which were allocated to newly painted rooms with low-volatile organic compounds (VOC), water-based paint, or existing rooms. After painting, there was no intervention or internal airflow to influence indoor air environment in both classrooms. We prospectively assessed the symptom severity and serious events of allergic diseases between both classrooms at baseline and after one and eight weeks after painting. At one and eight weeks, there were no significant changes in the Childhood Asthma Control Test scores, the fractional nitric oxide levels, lung function in asthmatic children in either classroom. There were also no significant changes in the severity score of AR or AD, or serious events in all allergic diseases. These findings suggest classroom painting with this new paint at the levels encountered in this study might not be a major aggravating factor for school-aged children with allergic diseases.

The flavone eupatilin inhibits eotaxin expression in an NF-κB-dependent and STAT6-independent manner.

The CC chemokine eotaxin contributes to epithelium-induced inflammation in airway diseases such as asthma. Eupatilin (5,7-dihydroxy-3',4',6'-trimethoxyflavone), a bioactive component of Artemisia asiatica Nakai (Asteraceae), is reported to inhibit the adhesion of eosinophils to bronchial epithelial cells. However, little is known about the molecular mechanism of eupatilin-induced attenuation of bronchial epithelium-induced inflammation. In this study, we investigated the effect of eupatilin on expression of eotaxin-1 (CCL11), a potent chemoattractant for eosinophils. Eupatilin significantly inhibited eotaxin expression in bronchial epithelial cells stimulated with TNF-α, while NF-κB and IκBα kinase (IKK) activities declined concurrently. Eupatilin also inhibited mitogen-activated protein kinase (MAPK) activity; however, all of these anti-inflammatory activities were reversed by MAPK overexpression. In contrast, eupatilin did not affect the signal transducer and activator of transcription 6 (STAT6) signalling in bronchial epithelial cells stimulated with IL-4. Furthermore, eupatilin significantly attenuated TNF-α-induced eosinophil migration. These results suggest that the eupatilin inhibits the signalling of MAPK, IKK, NF-κB and eotaxin-1 in bronchial epithelial cells, leading to inhibition of eosinophil migration.

Short communication: Physicochemical and antioxidant properties of milk supplemented with red ginseng extract.

This study investigated the effect of red ginseng extract (RGE) on the physicochemical properties, sensory test, and antioxidant activity of milk. The milk samples with RGE added at 0.5, 1, 1.5, and 2% were analyzed during storage at 4°C. The physicochemical properties included composition of milk, pH, titratable acidity, and color. The antioxidant activity of milk samples was determined using the 2,2-diphenyl-1-picrylhydrazyl method, ß-carotene bleaching assay, and ferric thiocyanate assay. An increase in the amount of RGE in milk resulted in an increase of lactose and total solids content, titratable acidity, and a* and b* values, whereas fat and protein contents remained unchanged. Also, pH and L* value decreased. The antioxidant activity of milk samples supplemented with RGE was higher than that of the control sample. Sensory evaluation was performed using a quantitative descriptive analysis. Two types of samples were used: (1) sterilized milk fortified with RGE (0.5, 1, 1.5, and 2%) and (2) 2% RGE, 2% RGE with oligosaccharide, and 2% RGE with oligosaccharide and cyclodextrin. The addition of oligosaccharide and cyclodextrin could effect an increase of sweetness, a decrease of bitterness and flavor of RGE, and aftertaste. Therefore, milk supplemented with RGE could be useful as a functional food.

Hepatic tumours in children with biliary atresia: single-centre experience in 13 cases and review of the literature.

AIM: To establish the risks of developing of hepatic tumours and to investigate their clinical and imaging findings in children with biliary atresia (BA) after Kasai portoenterostomy (Kasai). MATERIALS AND METHODS: Among 157 children who had undergone Kasai for BA over an 18 year period, patients who had newly developed hepatic tumours were identified. Patient demographics, clinical features, and imaging findings were retrospectively reviewed. RESULTS: Three male and 10 female patients (mean age 3.9 years) all (8%, of 157) had single hepatic tumours, which were confirmed in 10 explanted and three non-explanted livers. Ten (77%) were benign and three (23%) were malignant. Of the benign hepatic tumours, focal nodular hyperplasia (FNH; n = 6) was the most common, followed by regenerative nodules (n = 3) and adenoma (n = 1). All FNH appeared in young children <1 year of age and showed a subcapsular location, bulging contour, and lack of central scar. Malignant tumours included two hepatocellular carcinomas and one cholangiocarcinoma. CONCLUSION: Hepatic tumours developed in approximately 8% of children with BA after Kasai. Although benign tumours, including FNHs and regenerative nodules, were more common than malignant tumours, screening with alpha-foetoprotein (AFP) levels and regular imaging studies are the mainstay of malignant tumour detection.

Effect of estradiol on the expression of renal sodium transporters in rats.

BACKGROUND: Although estradiol has been thought to perform an important role in blood pressure regulation, the effects of estradiol on the expression of renal sodium transporters are not fully understood. METHODS: Female Sprague-Dawley rats were treated with 17ß-estradiol or vehicle for 10 days after ovariectomy, and after both ovariectomy and adrenalectomy to eliminate the effect of aldosterone. RESULTS: In the ovariectomized (OVX) rats, estradiol decreased the abundance of the Na-K-2Cl cotransporter (NKCC2) (31.5% of control (OVX), p < 0.01), Na-Cl cotransporter (NCC) proteins (40.5% of control (OVX), p < 0.01) and α- and γ-subunits of the epithelial sodium channel (ENaC) (44.7% and 11.0% of control (OVX), p < 0.01). Estradiol also reduced plasma aldosterone levels (OVX + 17ß-estradiol vs. OVX, 116.3 ± 44.4 vs. 184.2 ± 33.4 pmol/l, p < 0.05) and systolic blood pressure (OVX + 17ß-estradiol vs. OVX, 115 ± 4 vs. 132 ± 2 mmHg, p < 0.05). In rats having undergone adrenalectomy and ovariectomy, estradiol did not reduce systolic blood pressure, or the expression of sodium transporters. CONCLUSION: Estradiol decreased systolic blood pressure, plasma aldosterone levels, and the expression of renal sodium transporters. After aldosterone was eliminated, estradiol did not affect blood pressure or the expression of sodium transporters, which indicates that the effect of estradiol on the renal sodium transporters is at least partly influenced by aldosterone.

Differentiation between Chondrosarcoma and Synovial Chondromatosis of the Temporomandibular Joint Using CT and MR Imaging.

BACKGROUND AND PURPOSE: Chondrosarcoma and synovial chondromatosis of the temporomandibular joint share overlapping clinical and histopathologic features. We aimed to identify CT and MR imaging features to differentiate chondrosarcoma from synovial chondromatosis of the temporomandibular joint. MATERIALS AND METHODS: The CT and MR images of 12 and 35 patients with histopathologically confirmed chondrosarcoma and synovial chondromatosis of the temporomandibular joint, respectively, were retrospectively reviewed. Imaging features including lesion size, center, enhancement, destruction/sclerosis of surrounding bone, infiltration into the tendon of the lateral pterygoid muscle, calcification, periosteal reaction, and osteophyte formation were assessed. A comparison between chondrosarcoma and synovial chondromatosis was performed with a Student t test for quantitative variables and the Fisher exact test or linear-by-linear association test for qualitative variables. Receiver operating characteristic analysis was performed to determine the diagnostic performance for differentiation of chondrosarcoma and synovial chondromatosis based on a composite score obtained by assigning 1 point for each of 9 imaging features. RESULTS: High-risk imaging features for chondrosarcoma were the following: lesion centered on the mandibular condyle, destruction of the mandibular condyle, no destruction/sclerosis of the articular eminence/glenoid fossa, infiltration into the tendon of the lateral pterygoid muscle, absent or stippled calcification, periosteal reaction, internal enhancement, and size of ≥30.5 mm. The best cutoff value to discriminate chondrosarcoma from synovial chondromatosis was the presence of any 4 of these high-risk imaging features, with an area under the curve of 0.986 and an accuracy of 95.8%. CONCLUSIONS: CT and MR imaging features can distinguish chondrosarcoma from synovial chondromatosis of the temporomandibular joint with improved diagnostic performance when a subcombination of 9 imaging features is used.

RENEB Inter-Laboratory Comparison 2021: The FISH-Based Translocation Assay.

Translocation analysis using fluorescence in situ hybridization (FISH) is the method of choice for dose assessment in case of chronic or past exposures to ionizing radiation. Although it is a widespread technique, unlike dicentrics, the number of FISH-based inter-laboratory comparisons is small. For this reason, although the current Running the European Network of Biological and Physical retrospective Dosimetry (RENEB) inter-laboratory comparison 2021 was designed as a fast response to a real emergency scenario, it was considered a good opportunity to perform an inter-laboratory comparison using the FISH technique to gain further experience. The Bundeswehr Institute of Radiobiology provided peripheral blood samples from one healthy human volunteer. Three test samples were irradiated with blinded doses of 0, 1.2, and 3.5 Gy, respectively. Samples were then sent to the seven participating laboratories. The FISH technique was applied according to the standard procedure of each laboratory. Both, the frequency of translocations and the estimated dose for each sample were sent to the coordinator using a special scoring sheet for FISH. All participants sent their results in due time. However, although it was initially requested to send the results based on the full analysis, evaluating 500 equivalent cells, most laboratories only sent the results based on triage, with a smaller number of analyzed cells. In the triage analysis, there was great heterogeneity in the number of equivalent cells scored. On the contrary, for the full analysis, this number was more homogeneous. For all three samples, one laboratory showed outlier yields compared to the other laboratories. Excluding these results, in the triage analysis, the frequency of translocations in sample no. 1 ranged from 0 to 0.013 translocations per cell, and for samples no. 2 and no. 3 the genomic mean frequency were 0.27 ± 0.03 and 1.47 ± 0.14, with a coefficient of variation of 0.29 and 0.23 respectively. Considering only results obtained in the triage analysis for sample no. 1, all laboratories, except one, classified this sample as the non-irradiated one. For sample no. 2, excluding the outlier value, the mean reported dose was 1.74 ± 0.16 Gy indicating a mean deviation of about 0.5 Gy to the delivered dose of 1.2 Gy. For sample no. 3 the mean dose estimated was 4.21 ± 0.21 Gy indicating a mean deviation of about 0.7 Gy to the delivered dose of 3.5 Gy. In the frame of RENEB, this is the second FISH-based inter-laboratory comparison. The whole exercise was planned as a response to an emergency, therefore, a triage analysis was requested for all the biomarkers except for FISH. Although a full analysis was initially requested for FISH, most of the laboratories reported only a triage-based result. The main reason is that it was not clearly stated what was required before starting the exercise. Results show that most of the laboratories successfully discriminated unexposed and irradiated samples from each other without any overlap. A good agreement in the observed frequencies of translocations was observed but there was a tendency to overestimate the delivered doses. Efforts to improve the harmonization of this technique and subsequent exercises to elucidate the reason for this trend should be promoted.

Association of SLC6A4 variants with obsessive-compulsive disorder in a large multicenter US family study.

Genetic association studies of SLC6A4 (SERT) and obsessive-compulsive disorder (OCD) have been equivocal. We genotyped 1241 individuals in 278 pedigrees from the OCD Collaborative Genetics Study for 13 single-nucleotide polymorphisms, for the linked polymorphic region (LPR) indel with molecular haplotypes at rs25531, for VNTR polymorphisms in introns 2 and 7 and for a 381-bp deletion 3' to the LPR. We analyzed using the Family-Based Association Test (FBAT) under additive, dominant, recessive and genotypic models, using both OCD and sex-stratified OCD as phenotypes. Two-point FBAT analysis detected association between Int2 (P = 0.0089) and Int7 (P = 0.0187) (genotypic model). Sex-stratified two-point analysis showed strong association in females with Int2 (P<0.0002), significant after correction for linkage disequilibrium, and multiple marker and model testing (P(Adj) = 0.0069). The SLC6A4 gene is composed of two haplotype blocks (our data and the HapMap); FBAT whole-marker analysis conducted using this structure was not significant. Several noteworthy nonsignificant results have emerged. Unlike Hu et al., we found no evidence for overtransmission of the LPR L(A) allele (genotype relative risk = 1.11, 95% confidence interval: 0.77-1.60); however, rare individual haplotypes containing L(A) with P<0.05 were observed. Similarly, three individuals (two with OCD/OCPD) carried the rare I425V SLC6A4 variant, but none of them passed it on to their six OCD-affected offspring, suggesting that it is unlikely to be solely responsible for the 'OCD plus syndrome', as reported by Ozaki et al. In conclusion, we found evidence of genetic association at the SLC6A4 locus with OCD. A noteworthy lack of association at the LPR, LPR-rs25531 and rare 425V variants suggests that hypotheses about OCD risk need revision to accommodate these new findings, including a possible gender effect.

Bacillus-derived poly-γ-glutamic acid attenuates allergic airway inflammation through a Toll-like receptor-4-dependent pathway in a murine model of asthma.

BACKGROUND: Asthma is an inflammatory disease of the airways that is mediated by Th2 responses. Poly-γ-glutamic acid (γ-PGA) is an extracellular polymeric compound that is synthesized by Bacillus cells. Previously, we found that γ-PGA promoted Th1 cell development in a manner dependent on antigen-presenting cells, but inhibited Th2 cell development. OBJECTIVE: To investigate the effect of γ-PGA on dendritic cells (DCs), and its potential for treating Th2-mediated allergic asthma. METHODS: Wild-type, Toll-like receptor (TLR)-2 deficient, and TLR-4-defective mice were used. DCs derived from the bone marrow and extracted from the lung were stimulated with γ-PGA and assayed for the expression of signalling molecules, costimulatory molecules, and cytokines. Mice were sensitized and challenged with ovalbumin (OVA) to induce asthma. They were repeatedly injected intranasally with γ-PGA before and during the challenge period, and inflammation and structural remodelling of the airways were examined. RESULTS: γ-PGA selectively signalled conventional DCs to activate NF-κB and mitogen-activated protein kinase, leading to the up-regulation of CD86, CD40, and IL-12, but not IL-10 and IL-6. These effects of γ-PGA were dependent on TLR-4 and independent of TLR-2. Importantly, the intranasal administration of γ-PGA to OVA-sensitized/challenged mice reduced the airway hyperresponsiveness and allergic inflammation such as leucocyte influx, goblet cell hyperplasia, eosinophilia, and Th2 cytokine production. In addition to lowered IgE titres, the treatment of mice with γ-PGA significantly reduced the multiplication and Th2 polarization of mediastinal lymph node T cells upon allergen-specific restimulation. These anti-asthmatic effects of γ-PGA were also abolished in TLR-4-defective mice. CONCLUSIONS AND CLINICAL RELEVANCE: Our data indicate that γ-PGA activates DCs to favour Th1 cell induction through a TLR-4-dependent pathway and alleviates pathologic symptoms in a Th2-biased asthmatic model. These findings highlight the potential of γ-PGA for the treatment of asthma and other allergic disease in which Th2 polarization plays an important role.

Transarterial chemoembolization versus supportive therapy in the palliative treatment of unresectable intrahepatic cholangiocarcinoma.

AIM: To evaluate the clinical outcome and the survival benefits of transarterial chemoembolization (TACE) for unresectable intrahepatic cholangiocarcinoma (ICC) compared with supportive therapy. MATERIALS AND METHODS: From January 1996 to April 2009, a total of 155 patients with unresectable ICC met the entry criteria and underwent TACE (72 patients) or supportive treatment (83 patients). Their survival was the primary end point. RESULTS: The baseline patients and tumour characteristics were well-balanced in the two groups. The median number of sessions per patient was 2.5 (range 1-17 sessions) in the TACE group. After TACE, the incidence of significant (≥ grade 3) haematological and non-haematological toxicities was 13 and 24%, respectively, and no patients died within 30 days following TACE. The objective tumour regression (≥ partial response) was achieved in 23% of the patients in the TACE group. The Kaplan-Meier survival analysis showed that the survival period was significantly longer in the TACE group (median 12.2 months) than in the symptomatic treatment (median 3.3 months) group (p < 0.001). CONCLUSIONS: TACE is safe and offers greater survival benefits than supportive treatment for the palliative treatment of unresectable ICC.

Transcatheter arterial chemo-lipiodol infusion for unresectable hepatocellular carcinoma in 96 high-risk patients.

AIM: To evaluate the safety and efficacy of transcatheter arterial chemo-lipiodol infusion (TACL) in high-risk patients with unresectable hepatocellular carcinoma (HCC). MATERIALS AND METHODS: From January 2005 to April 2009, 96 patients underwent TACL. All patients had diffuse, infiltrative or multifocal tumours. Twenty-nine (30%) patients had an increased serum bilirubin level (>or=2mg/dl), and 75 patients (78%) had a low serum albumin level (<3.5mg/dl). The Child-Pugh (CP) score was 9 or more in 13 (14%) patients. Sixty-five patients (68%) had major portal vein occlusion. Sixteen patients (17%) had biliary dilatation. RESULTS: TACL was technically successful in all patients. After TACL, 18 (19%) of the 96 patients showed tumour response using computed tomography (CT) criteria. The 30 day mortality and morbidity rates were 1 and 2%, respectively. The median survival period was 8.6 months, and the overall 6 month, 1, 2, and 3 year survival rates were 59, 44, 26, and 15%, respectively. Portal vein occlusion (p<0.001) was the only significant risk factor associated with the length of the survival period after TACL, whereas the CP score (p=0.498), serum bilirubin level (p=0.153), serum albumin level (p=0.399), and biliary obstruction (p=0.636) had no significant effect. CONCLUSIONS: TACL can be performed safely in high risk HCC patients resulting in a median survival rate of 8.6 months in the present series.

Brillouin gain-coefficient measurement for bismuth-oxide-based photonic crystal fiber under significant beam reflection at splicing points.

We investigate a six-air-hole bismuth-oxide-based photonic crystal fiber (Bi-PCF) in terms of Brillouin characteristics. One huge challenge in measuring the Brillouin properties of the Bi-PCF is the nonnegligible beam reflection at the splicing points, which can be attributed to the mirroring effect caused by different refractive indices of silica and bismuth fibers. To solve the problem we propose a method that is based on the combination of a pump-probe beat lock-in scheme and a normalized gain curve-fitting technique. Using this method, successful characterization of Brillouin properties for a 1.16-m-long Bi-PCF is experimentally demonstrated. With the measured Brillouin gain coefficient and the known chi((3)) nonlinearity parameters, the Kerr nonlinearity figure-of-merit (F(nl-SBS)), including the stimulated Brillouin scattering-caused pump-power limit, is also estimated for the Bi-PCF.

Genetic and biochemical interactions between an essential kinetochore protein, Cbf2p/Ndc10p, and the CDC34 ubiquitin-conjugating enzyme.

CBF2/NDC10/CTF14 encodes the 110-kDa subunit of CBF3, a key component of the yeast centromere/kinetochore. Overexpression of yeast CDC34 specifically suppresses the temperature-sensitive growth phenotype of the ndc10-1 mutation. Mutations in CDC34, which specifies a ubiquitin-conjugating enzyme, arrest yeast cells in the G1 phase of the cell cycle, with no intact spindles formed (M. G. Goebl, J. Yochem, S. Jentsch, J. P. McGrath, A. Varshavsky, and B. Byers, Science 241:1331-1335, 1988). The cdc34-2 mutation drastically alters the pattern of Cbf2p modification. Results of experiments using antibodies against Cbf2p and ubiquitin indicate that Cbf2p is ubiquitinated in vivo. Purified Cdc34p catalyzes the formation of Cbf2p-monoubiquitin conjugate in vitro. These data suggest that Cbf2p is an endogenous substrate of the CDC34 ubiquitin-conjugating enzyme and imply that ubiquitination of a kinetochore protein plays a regulatory role in kinetochore function.

The suil suppressor locus in Saccharomyces cerevisiae encodes a translation factor that functions during tRNA(iMet) recognition of the start codon.

We initiated a genetic reversion analysis at the HIS4 locus to identify components of the translation initiation complex that are important for ribosomal recognition of an initiator codon. Three unlinked suppressor loci, suil, sui2, and SUI3, that restore expression of both HIS4 and HIS4-lacZ in the absence of an AUG initiator codon were identified. In previous studies, it was demonstrated that the sui2 and SUI3 genes encode mutated forms of the alpha and beta subunits, respectively, of eukaryotic translation initiation factor 2 (eIF-2). In this report, we describe the molecular and biochemical characterizations of the sui1 suppressor locus. The DNA sequence of the SUI1+ gene shows that it encodes a protein of 108 amino acids with a calculated Mr of 12,300. The sui1 suppressor genes all contain single base pair changes that alter a single amino acid within this 108-amino-acid sequence. sui1 suppressor strains that are temperature sensitive for growth on enriched medium have altered polysome profiles at the restrictive temperature typical of those caused by alteration of a protein that functions during the translation initiation process. Gene disruption experiments showed that the SUI1+ gene encodes an essential protein, and antibodies directed against the SUI1+ coding region identified a protein with the predicted Mr in a ribosomal salt wash fraction. As observed for sui2 and SUI3 suppression events, protein sequence analysis of His4-beta-galactosidase fusion proteins produced by sui1 suppression events indicated that a UUG codon is used as the site of translation initiation in the absence of an AUG start codon in HIS4. Changing the penultimate proline codon 3' to UUG at his4 to a Phe codon (UUC) blocks aminopeptidase cleavage of the amino-terminal amino acid of the His4-beta-galactosidase protein, as noted by the appearance of Met in the first cycle of the Edman degradation reaction. The appearance of Met in the first cycle, as noted, in either a sui1 or a SUI3 suppressor strain showed that the mechanism of suppression is the same for both suppressor genes and allows the initiator tRNA to mismatch base pair with the UUG codon. This suggests that the Sui1 gene product performs a function similar to that of the beta subunit of eIF-2 as encoded by the SUI3 gene. However, the Sui1 gene product does not appear to be a required subunit of eIF-2 on the basis of purification schemes designed to identify the GTP-dependent binding activity of eIF-2 for the initiator tRNA. In addition, suppressor mutations in the sui1 gene, in contrast to suppressor mutations in the sui2 or SUI3 gene, do not alter the GTP-dependent binding activity of the eIF-2. The simplest interpretation of these studies is that the sui1 suppressor gene defines an additional factor that functions in concert with eIF-2 to enable tRNAiMet to establish ribosomal recognition of an AUG initiator codon.

The yeast nucleolar protein Cbf5p is involved in rRNA biosynthesis and interacts genetically with the RNA polymerase I transcription factor RRN3.

Yeast Cbf5p was originally isolated as a low-affinity centromeric DNA binding protein (W. Jiang, K. Middleton, H.-J. Yoon, C. Fouquet, and J. Carbon, Mol. Cell. Biol. 13:4884-4893, 1993). Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found to be nucleolar and is highly homologous to the rat nucleolar protein NAP57, which coimmunoprecipitates with Nopp140 and which is postulated to be involved in nucleolar-cytoplasmic shuttling (U. T. Meier, and G. Blobel, J. Cell Biol. 127:1505-1514, 1994). The temperature-sensitive cbf5-1 mutant demonstrates a pronounced defect in rRNA biosynthesis at restrictive temperatures, while tRNA transcription and pre-rRNA and pre-tRNA cleavage processing appear normal. The cbf5-1 mutant cells are deficient in cytoplasmic ribosomal subunits at both permissive and restrictive temperatures. A high-copy-number yeast genomic library was screened for genes that suppress the cbf5-1 temperature-sensitive growth phenotype. SYC1 (suppressor of yeast cbf5-1) was identified as a multicopy suppressor of cbf5-1 and subsequently was found to be identical to RRN3, an RNA polymerase I transcription factor. A cbf5delta null mutant is not rescued by plasmid pNOY103 containing a yeast 35S rRNA gene under the control of a Pol II promoter, indicating that Cbf5p has one or more essential functions in addition to its role in rRNA transcription.

An essential yeast protein, CBF5p, binds in vitro to centromeres and microtubules.

Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability of Saccharomyces cerevisiae. CBF5 specifies a 55-kDa highly charged protein that contains a repeating KKD/E sequence domain near the C terminus, similar to known microtubule-binding domains in microtubule-associated proteins 1A and 1B, CBF5p, obtained by overexpression in bacterial cells, binds microtubules in vitro, whereas C-terminal deleted proteins lacking the (KKD/E)n domain do not. Dividing yeast cells containing a C-terminal truncated CBF5 gene, producing CBF5p containing only three copies of the KKD/E repeat, delay with replicated genomes at the G2/M phase of the cell cycle, while depletion of CBF5p arrests most cells in G1/S. Overproduction of CBF5p in S. cerevisiae complements a temperature sensitivity mutation in the gene (CBF2) specifying the 110-kDa subunit of the high-affinity CEN DNA-binding factor CBF3, suggesting in vivo interaction of CBF5p and CBF3. A second low-affinity centromere-binding factor has been identified as topoisomerase II.