Association between dietary fat intake and bone mineral density in Korean adults: data from Korea National Health and Nutrition Examination Survey IV (2008 ∼ 2009).

UNLABELLED: We determined the relation between dietary fat intake and bone mineral density, and our study showed that low- as well as high-fat diet was associated with the risk of osteoporosis. Our study provides significant evidence of the specific dietary components that may be important modifiable factors for the prevention of osteoporosis. INTRODUCTION: Osteoporosis and osteoporosis-related fractures have become major public health problems. It is important to understand the various factors that influence bone health and to prevent osteoporosis by correcting modifiable risk factors for the disease. Previous studies suggested that dietary habits and body composition were potent factors associated with bone mineral density. The aim of this study was to determine the independent effect of dietary fat intake on bone mineral density while controlling for other possible confounders, including fat mass and lean body mass. METHODS: This study was based on data obtained in the Fourth Korea National Health and Nutrition Examination Survey. After serial exclusion of subjects according to the selection criteria, 7,192 subjects were included in our analysis. We divided the study population into quintiles according to dietary fat calorie/total calorie intake and compared the adjusted means of bone mineral density between quintiles. RESULTS: The bone mineral density was higher in men and women with a medium fat energy intake compared to those with a low- and high-fat energy intake, but the finding was statistically significant only in women. The results were valid after controlling for body fat percentage and lean body mass. CONCLUSIONS: We found that dietary fat intake is an independent modifiable risk factor for osteoporosis, regardless of body fat or lean body mass, especially in women. However, further investigations with accurate analyses of food intake and nutritional consumption, in addition to long-term follow-up data, are necessary to recommend an osteoporosis-preventive diet in Koreans.

Prophylactic administration of a combined prebiotic and probiotic, or therapeutic administration of enrofloxacin, to reduce the incidence of bacterial chondronecrosis with osteomyelitis in broilers.

Bacteria entering the bloodstream via translocation from the gastrointestinal tract spread hematogenously and can trigger bacterial chondronecrosis with osteomyelitis (BCO) by infecting osteochondrotic microfractures in the epiphyseal-physeal cartilage of the proximal femora and tibiae. In experiment 1, broilers were fed control feed or the same feed containing BacPack 2X, which includes the prebiotic IMW50 (a mannan oligosaccharide beta-glucan yeast cell wall product) plus the probiotic Calsporin (Bacillus subtilis C-3102). Broilers reared on wire flooring consistently developed higher incidences of BCO than hatchmates reared on wood shavings litter (≥24 vs. ≤4%, respectively; P=0.001). Adding BacPack 2X to the feed on d 1 through 56 delayed the age of onset and reduced the cumulative incidence of BCO on wire flooring when compared with broilers fed the control feed (24.0 vs. 40.7%, respectively; P=0.003). In experiment 2, broilers reared on wire flooring received tap water on d 1 through 62 (control group) or therapeutic levels of the potent fluoroquinolone antimicrobial enrofloxacin in the water on d 35 through 54 (enrofloxacin group). During enrofloxacin administration, half as many birds developed BCO in the enrofloxacin group when compared with the control group (8.1 vs. 19.5%, respectively, on d 35 through 54; P=0.001), whereas both groups had similar BCO incidences subsequent to withdrawing enrofloxacin on d 55 through 62 (14.8 vs. 18.2% for the enrofloxacin vs. control groups; P=0.386). Cumulative lameness incidences for d 1 through 62 were higher for the control group than for the enrofloxacin group (39.0 vs. 25.8%, respectively; P=0.003). These results demonstrate that wire flooring imposes a rigorous challenge that leads to high incidences of BCO that can be difficult to suppress, even with therapeutic doses of enrofloxacin. Prophylactically adding BacPack 2X to the feed reduced the incidence of BCO lameness by a proportion similar to that achieved with enrofloxacin, indicating that probiotics potentially can provide effective alternatives to antibiotics for reducing BCO lameness attributable to bacterial translocation and hematogenous distribution.

Effects of hot water spray and sub-zero saline chilling on bacterial decontamination of broiler carcasses.

Reduction of Salmonella on poultry carcasses is one way to prevent salmonellosis. The purpose of this research was to evaluate the effects of subzero saline chilling (SSC) with/without hot water spray (HWS) on broiler carcasses prior to chilling for bacterial reduction. Eviscerated broiler carcasses were subjected to water immersion chilling (WIC, 0% NaCl/0.5°C) or SSC (4% NaCl/-2.41°C) with/without prior HWS at 71°C for 1 min. Broiler carcasses in SSC were chilled faster than those in WIC, regardless of HWS. The combination of HWS and SSC resulted in the best reduction of mesophilic aerobic bacteria, Escherichia coli, and total coliforms on the carcasses over the WIC, SSC, and HWS/WIC. No Salmonella was detected on the carcasses in SSC and HWS/SSC while Salmonella positive was observed on the carcasses chilled in WIC and HWS/WIC. A trace of Gram-negative genus was detected on carcasses in HWS/SSC while many other microbiomes were observed on those in WIC, SSC, and HWS/WIC when quantitative microbiota profiles of 16S rRNA gene sequences were evaluated. Based on these results, chilling of broiler carcasses in 4% NaCl/-2.41°C after HWS at 71°C for 1 min significantly reduced carcass chilling time and bacterial contamination over the control chilling.

Molecular cloning and characterization of the STAT gene in Hyphantria cunea haemocytes.

A new insect member of the signal transducer and activator of transcription (STAT) family of transcription factors, Hyphantria cunea STAT (HcSTAT), was cloned from the lepidopteran H. cunea. The domain involved in DNA interaction and the Src homology 2 (SH2) domain were well conserved. During all developmental stages, the gene was expressed at a low level in the haemocytes, fat body cells, midgut, epidermis and Malpighian tubules. The haemocytes and Malpighian tubules showed transcriptional activation of HcSTAT upon Gram-negative and Gram-positive bacterial challenges. These challenges increased the induction and nuclear translocation of the HcSTAT protein that recognizes a STAT target site in H. cunea haemocytes. In vivo treatment with sodium orthovanadate translocated HcSTAT to the haemocyte nucleus. This study shows the involvement of the haemocyte Janus kinase/STAT pathway after microbial infection in lepidopteran insects.

Structure, function and regulation of the DNA-binding protein Dps and its role in acid and oxidative stress resistance in Escherichia coli: a review.

Dps, the DNA-binding protein from starved cells, is capable of providing protection to cells during exposure to severe environmental assaults; including oxidative stress and nutritional deprivation. The structure and function of Dps have been the subject of numerous studies and have been examined in several bacteria that possess Dps or a structural/functional homologue of the protein. Additionally, the involvement of Dps in stress resistance has been researched extensively as well. The ability of Dps to provide multifaceted protection is based on three intrinsic properties of the protein: DNA binding, iron sequestration, and its ferroxidase activity. These properties also make Dps extremely important in iron and hydrogen peroxide detoxification and acid resistance as well. Regulation of Dps expression in E. coli is complex and partially dependent on the physiological state of the cell. Furthermore, it is proposed that Dps itself plays a role in gene regulation during starvation, ultimately making the cell more resistant to cytotoxic assaults by controlling the expression of genes necessary for (or deleterious to) stress resistance. The current review focuses on the aforementioned properties of Dps in E. coli, its prototypic organism. The consequences of elucidating the protective mechanisms of this protein are far-reaching, as Dps homologues have been identified in over 1000 distantly related bacteria and Archaea. Moreover, the prevalence of Dps and Dps-like proteins in bacteria suggests that protection involving DNA and iron sequestration is crucial and widespread in prokaryotes.

In vitro invasion of laying hen ovarian follicles by Salmonella Enteritidis strains.

Salmonella is the major foodborne bacterial pathogen worldwide. Among numerous serotypes, Salmonella Enteritidis (SE) is one of the most common Salmonella serotypes responsible for human infections in the United States. The main source of SE outbreaks is foods associated with raw or undercooked chicken eggs. Salmonella Enteritidis is the only serotype that routinely contaminates eggs. The transovarian transmission of SE and subsequent contamination of the eggs before egg shell formation is considered to be the main route of egg contamination by SE. To evaluate whether invasion of ovarian follicles is an important step during the production of eggs contaminated by SE, we used an in vitro invasion assay to determine ovarian follicle invasion by 5 SE strains. After inoculating the freshly collected ovarian follicles, all 5 SE strains were able to invade into the follicles after 2 h of incubation at 37°C. The mean percentage of SE invasion ranged from 0.016 to 0.034% and no significant difference was found among the SE strains. For Escherichia coli K-12 strain, which was used as a negative control, the mean percentage of invasion was 0.0003%. The in vitro follicle invasion by SE strains demonstrated in this study may reflect the ability of the strains to invade ovarian follicles in laying hens once SE cells reach ovaries through various routes.

The effect of long-term propionate adaptation on the stress resistance of Salmonella Enteritidis.

AIMS: Adaptive phenotypes of enteric bacterial pathogens in response to in vivo-mimicking stress conditions are important because of their potentiality to enhance stress resistance and ameliorate measures intended to control transmission and infectivity. Salmonella enterica serotype Enteritidis (S. Enteritidis) encounters a variety of such environments throughout its infection cycle, including high concentrations of the short-chain fatty acid, Propionate (PA), during food processing and within the gut of infected hosts. With this study, we aimed to elucidate the significance of PA adaptation on stress resistance in S. Enteritidis. METHODS AND RESULTS: We have shown (utilizing in vitro stress assays) that S. Enteritidis grown to stationary phase in the presence of PA has a dramatically enhanced resistance to commonly encountered in vivo stressors, including extreme acidity and oxidative/nitrosative stresses when compared to unadapted salmonellae. However, competitive infection between PA adapted and unadapted cells within a murine model showed that adapted cells were at a distinct disadvantage in vivo, resulting in decreased caecal colonization in infected mice. CONCLUSIONS: Our results suggest that, while long-term PA adaptation induces strong resistance to specific stresses in vitro, it also reduces the overall infectivity of the adapted cells by inhibiting the organism's colonization ability. SIGNIFICANCE AND IMPACT OF THE STUDY: In S. Enteritidis, PA adaptation is strongly associated with the induction of stress-resistant phenotypes in vitro. Enhanced stress resistance ex vivo may increase environmental persistence and/or the overall transmissibility of this pathogen, while overall the virulence of the pathogen is likely to be hindered as a result of the decreased colonization ability of PA-adapted S. Enteritidis.

Development and evaluation of candidate recombinant Salmonella-vectored Salmonella vaccines.

Attenuated Salmonella Enteriditis (ΔSE) recombinant vaccine vectors incorporating a Salmonella flagellar filament protein (fliC) subunit, a putative cell-mediated epitope, for expression of the lamB gene (encoding a maltose outer membrane porin), with or without co-expression of a putative immune-enhancing CD154 oligopeptide, were developed and compared with wild-type Salmonella Enteriditis (experiments 1 and 2) or the attenuated ΔSE empty vector (experiment 3) as initial vaccine candidates against Salmonella infection. A total of 3 experiments were performed to assess the infection and clearance rate of each of these constructs. Each construct or Salmonella Enteriditis was orally administered to broiler chicks at day of hatch by oral gavage (~10(8) cfu/chick). In experiments 1 to 3, liver-spleen and cecal tonsils were removed aseptically for recovery of wild-type Salmonella Enteriditis or ΔSE mutants. These experiments suggested that cell surface expression of fliC alone markedly increased the clearance rate of the vector at or before 21d postvaccination in all 3 experiments. In a fourth experiment, broilers were vaccinated with one of the vaccine constructs or the ΔSE empty vector and then challenged with wild-type Salmonella Typhimurium. At 19 d posthatch, 16 d postinfection, neither candidate protected against challenge significantly better than the ΔSE empty vector, although there was significantly less Salmonella recovered from vaccinated chickens as compared with nonvaccinated controls. These experiments indicate that these experimental vaccines did not protect against heterologous challenge or enhance clearance after Salmonella Typhimurium challenge; as such, their value as vaccines is limited. The increased clearance of the candidate vaccines, particularly the vector expressing fliC alone, may have value in that the fliC epitope may decrease the clearance time of other recombinant vectored Salmonella vaccines.

Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge.

Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlapping extension PCR, we recently developed several novel attenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a potential immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commercial Leghorn chicks were orally immunized (immunization dose: 10(6) to 10(8) cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains [DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, DeltaaroA-DeltahtrA M2e(4)-CD154] on day of hatch and 21 d posthatch. These candidate vaccine strains were evaluated for their ability to invade, colonize, and persist in tissues and elicit an M2e-specific antibody response. The vaccine candidate strain DeltaaroA M2e-CD154 exhibited significantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colonization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simultaneous expression of CD154 (P < 0.05). Virus neutralization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, and DeltaaroA-DeltahtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutralization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen-free Leghorn chicks immunized with DeltaaroA-DeltahtrA M2e(4)-CD154 offered significant protection against direct challenge with low pathogenic AI H7N2, but not highly pathogenic H5N1 AI. Taken together, these data suggest that these Salmonella-vectored vaccines expressing M2e in association with CD154 are effective at protecting chickens against low pathogenic AI.

Comparative analysis of two attacin genes from Hyphantria cunea.

A full-length clone corresponding to attacin was isolated from a cDNA library made from fat body of immunized Hyphantria cunea larvae. This newly isolated attacin B shows characteristics different from those previously reported for attacin A. The two attacin cDNAs encode precursor proteins of 233 and 248 amino acid residues, respectively. The two attacins show 45.9% identity at the amino acid level, and 35.2% identity at the nucleotide level. Attacins A and B of H. cunea show significant identities with the attacins of Lepidoptera. Attacin B is a typical glycine-rich protein, while attacin A is leucine-rich. Attacin B is expressed from last instar larvae to adult, while attacin A showed stage-specific expression during the prepupal and pupal stages. Attacins A and B are predicted to have different secondary structure in that attacin A has no tendency to form helices but attacin B contains a substantial number of helices. Attacin A is induced at a trace level in infected larvae, while attacin B is strongly induced against Gram-positive and negative bacteria, fungi, and viruses. The attacin B transcripts were detected in fat body, epidermis and hemocytes after injection with Escherichia coli, Citrobacter freundii, or Candida albicans, but not in the midgut and Malpighian tubule. Recombinant attacin A showed no antibacterial activity, while recombinant attacin B showed strong antibacterial activity in proportion to the amount of the protein injected.

Effects of chemical treatments on pH and bacterial population in poultry litter: a laboratory experiment.

1. The goal of this study was to evaluate the effects of three chemical treatments on pH and bacterial populations (total aerobic bacteria and gram-negative bacteria) in poultry litter under laboratory conditions. 2. Litter obtained from poultry houses was treated with three chemical treatments (alum, AlCl(3) and FeSO(4)) at the same concentration (8 g/100 g litter), while untreated litter served as a control. The study was conducted for 3 weeks. 3. All of the chemical treatments reduced total aerobic bacteria (22 to 87% of the untreated control) and gram-negative bacteria (63 to 99% of the untreated controls) populations and lowered litter pH values (5.95 to 6.64). However, a significant difference in gram-negative bacteria did not exist among chemical treatments at 0, 1 and 2 weeks. 4. These results suggest that the reduction in total aerobic bacteria and gram-negative bacteria populations is highly related to a decrease in litter pH, and acidifying treatment (alum, AlCl(3) and FeSO(4)) of poultry litter may serve as a means to help the reduction in pathogen populations and to improve economical benefits under commercial production conditions.

Characterization and evaluation of lactic acid bacteria candidates for intestinal epithelial permeability and Salmonella Typhimurium colonization in neonatal turkey poults.

The present study evaluated the microbiological properties of three probiotic candidate strains of lactic acid bacteria (LAB) (128; 131; CE11_2), their effect on intestinal epithelial permeability, and their ability to reduce intestinal colonization of Salmonella Typhimurium (ST) individually or as a batch culture in neonatal turkey poults. Isolates were characterized morphologically and identified using 16S rRNA sequence analyses. Each isolate was evaluated for tolerance and resistance to acidic pH, high osmotic NaCl concentrations, and bile salts in broth medium. In vitro assessment of antimicrobial activity against different enteropathogenic bacteria was determined using an overlay technique. In vitro intestinal permeability was evaluated using a stressed Caco-2 cell culture assay treated with/without the probiotic candidates. The in vivo effect of the selected LAB strains on ST cecal colonization was determined in two independent trials with neonatal turkey poults. The results obtained in this study demonstrate the tolerance of LAB candidates to pH 3, a NaCl concentration of 6.5%, and high bile salts (0.6%). All strains evaluated exhibited in vitro antibacterial activity against Salmonella Enteritidis, ST, and Campylobacter jejuni. Candidates 128 and 131 exhibited a coccus morphology and were identified as Enterococcus faecium, and bacterial strain CE11_2 exhibited clusters of cocci-shaped cells and was identified as Pediococcus parvulus. All three candidate probiotics significantly (P < 0.05) increased transepithelial electrical resistance (TEER) in Caco-2 cells following a 3-h incubation period with hydrogen peroxide compared to control and blank groups. The combination of all three candidates as a batch culture exhibited significant efficacy in controlling intestinal colonization of ST in neonatal turkey poults. Evaluation of the combination of these selected LAB strains according to performance and intestinal health parameters of chickens and turkeys are currently in process.

Retrieval analysis of metal and ceramic femoral heads on a single CoCr stem design.

OBJECTIVES: The use of ceramic femoral heads in total hip arthroplasty (THA) has increased due to their proven low bearing wear characteristics. Ceramic femoral heads are also thought to reduce wear and corrosion at the head-stem junction with titanium (Ti) stems when compared with metal heads. We sought to evaluate taper damage of ceramic compared with metal heads when paired with cobalt chromium (CoCr) alloy stems in a single stem design. METHODS: This retrieval study involved 48 total hip arthroplasties (THAs) with CoCr V40 trunnions paired with either CoCr (n = 21) or ceramic (n = 27) heads. The taper junction of all hips was evaluated for fretting/corrosion damage and volumetric material loss using a roundness-measuring machine. We used linear regression analysis to investigate taper damage differences after adjusting for potential confounding variables. RESULTS: We measured median taper material loss rates of 0.210 mm3/year (0.030 to 0.448) for the metal head group and 0.084 mm3/year (0.059 to 0.108) for the ceramic group. The difference was not significant (p = 0.58). Moreover, no significant correlation between material loss and implant or patient factors (p > 0.05) was found. CONCLUSIONS: Metal heads did not increase taper damage on CoCr trunnions compared with ceramic heads from the same hip design. The amount of material released at the taper junctions was very low when compared with available data regarding CoCr/Ti coupling in metal-on-metal bearings.Cite this article: A. Di Laura, H. Hothi, J. Henckel, I. Swiatkowska, M. H. L. Liow, Y-M. Kwon, J. A. Skinner, A. J. Hart. Retrieval analysis of metal and ceramic femoral heads on a single CoCr stem design. Bone Joint Res 2017;6:-350. DOI: 10.1302/2046-3758.65.BJR-2016-0325.R1.

Side-to-side variation in normal femoral morphology: 3D CT analysis of 122 femurs.

BACKGROUND: The contralateral femur is often used as reference for reconstruction in unilateral hip joint pathology. The objective of this study was to quantify the side-to-side variation in proximal femur. We hypothesized that significant side-to-side differences exist between left and right femur with implications for preoperative planning and leg length discrepancy following hip arthroplasty. MATERIALS AND METHODS: CT-based 3D femoral models were reconstructed for 122 paired femurs in 61 young healthy subjects (46.9±6.8 years) with no history of hip pathology. Side-to-side differences of several femoral morphologic parameters, including femoral head diameter, femoral anteversion, horizontal offset and femoral head center location, were compared and correlated with demographic factors using multiple linear regression. RESULTS: Significant side-to-side differences (P<0.01) were found in femoral anteversion (4.3±3.8°; range: 0.2° to 17.3°), horizontal offset (2.5±2.1mm; range: 0.1 to 10.3mm), and femoral head center location (7.1±3.8mm; range: 0.5 to 19.4mm). The difference in femoral anteversion was strongly correlated with the difference in neck diameter (R(2)=0.79), whereas the difference in horizontal femoral offset was highly correlated with the head diameter difference (R(2)=0.72). Femoral head center difference was correlated with the femoral anteversion, horizontal offset and neck-shaft-angle difference (R(2)=0.82). DISCUSSION: Relying on the anatomic landmarks of the contralateral femur during hip arthroplasty may not necessarily result in restoration of native anatomy and leg-length. Knowledge of the baseline side-to-side asymmetry could provide a range of error that would be tolerable following hip reconstruction. LEVEL OF EVIDENCE: Level IV. TYPE OF STUDY: Retrospective observational study.

International metal-on-metal multidisciplinary teams: do we manage patients with metal-on-metal hip arthroplasty in the same way? An analysis from the International Specialist Centre Collaboration on MOM Hips (ISCCoMH).

AIMS: There are many guidelines that help direct the management of patients with metal-on-metal (MOM) hip arthroplasties. We have undertaken a study to compare the management of patients with MOM hip arthroplasties in different countries. METHODS: Six international tertiary referral orthopaedic centres were invited to participate by organising a multi-disciplinary team (MDT) meeting, consisting of two or more revision hip arthroplasty surgeons and a musculoskeletal radiologist. A full clinical dataset including history, blood tests and imaging for ten patients was sent to each unit, for discussion and treatment planning. Differences in the interpretation of findings, management decisions and rationale for decisions were compared using quantitative and qualitative methods. RESULTS: Overall agreement between the orthopaedic centres and the recommended treatment plans for the ten patients with MOM hip implants was moderate (kappa = 0.6). Full agreement was seen in a third of cases, however split decisions were also seen in a third of cases. Units differed in their interpretation of the significance of the investigation findings and put varying emphasis on serial changes, in the presence of symptoms. DISCUSSION: In conclusion, the management of raised or rising blood metal ions, cystic pseudotumours and peri-acetabular osteolysis led to inconsistency in the agreement between centres. Coordinated international guidance and MDT panel discussions are recommended to improve consensus in decision making. TAKE HOME MESSAGE: A lack of evidence and the subsequent variation in regulator guidance leads to differences in opinions, the clinical impact of which can be reduced through a multi-disciplinary team approach to managing patients with MOM hip implants. Cite this article: Bone Joint J 2016;98-B:179-86.

Reduction of Salmonella enterica serovar enteritidis colonization and invasion by an alfalfa diet during molt in Leghorn hens.

The standard method for molting to stimulate multiple egg-laying cycles in laying hens is feed deprivation. However, the physiological changes within hens caused by feed deprivation increase susceptibility of the hens to Salmonella enterica serovar Enteritidis (SE) infection. In an effort to develop an alternative method to induce molting without increasing susceptibility to SE, an alfalfa diet was compared with the standard molting method for the level of ovary regression and SE colonization. Hens over 50 wk of age were divided into 3 treatment groups (12 hens/group): nonmolting by normal feeding (NM), molting by feed deprivation (M), and molting by alfalfa diet (A). Individual hens on all treatments were challenged orally with 10(5) cfu of SE on the fourth day after feed changes and were analyzed for ovary weight and SE colonization or invasion in crop contents, cecal contents, liver, spleen, and ovary on the ninth day. In 3 of the 4 trials, there was a significant decrease in SE colonization of the crop between the alfalfa diet (A) and the feed deprived molt (M). In most of the 4 trials, there was a significant reduction in SE infected organs in birds fed the alfalfa diet (A) compared with birds undergoing feed deprived molt (M). Most of the trials showed no significant difference in overall SE between A and NM. Therefore, the results of this study suggest that an alfalfa diet has the potential to be used as an alternative method for forced molting, without increasing the incidence of SE in eggs and internal organs.

Two closely related cDNAs encoding actin-depolymerizing factors of petunia are mainly expressed in vegetative tissues.

Actin-depolymerizing factor (ADF) is one of the small actin-binding proteins that regulate actin dynamics in cells. We have isolated two cDNA clones, PhADF1 and PhADF2, encoding ADF from cDNA libraries constructed from petal protoplast cultures and flowers of Petunia hybrida. PhADF1 and PhADF2 encode polypeptides of 139 and 143 amino acids with a calculated molecular mass of 16.04 and 16.51kDa, respectively. Co-sedimentation assay showed that the recombinant PhADF1 protein produced in Escherichia coli binds to F-actin at pH7. 0 and preferentially depolymerizes it at pH8.0. Gene tree analysis indicates that the plant ADF family can be grouped into four classes, and PhADFs are included in class I. Southern blot analyses revealed that one or two copies of PhADF genes are present in petunia genome, and several other related isoforms also exist. Northern blot analyses indicated that PhADF1 and PhADF2 are closely related and abundantly expressed in every plant organ except pollen. In addition, they are highly accumulated in mature vegetative tissue (petal, leaf, and stem). Our results indicate that the transcription of petunia ADF genes is differentially regulated by developmental signals.

Molecular cloning and characterization of a plastid chaperonin 60 alpha subunit from Canavalia lineata.

A 2040 bp cDNA encoding plastid chaperonin 60alpha protein was isolated from a cDNA expression library prepared from Canavalia lineata leaves. The sequence analysis of the clone showed a significant homology to those of other plant plastid chaperonin 60alpha. The genomic DNA blot analysis indicated that Canavalia lineata chaperonin 60alpha is encoded by a single gene in the genome. The short-term kinetics of light on the gene expression revealed that the remarkable accumulation of the gene occurred after 24 h. The mRNA was also detectable in dark-grown seedling leaves.

Genomic structure of ornithine carbamoyltransferase gene from Canavalia lineata.

The nucleotide sequences of the complete ornithine carbamoyltransferase (OCT) gene containing 3,964 bp 3' region and 1,701 bp promoter region were determined from Canavalia lineata leaves. The exons range in size from 131 bp to 390 bp, while the introns range in size from 102 bp to the relatively large size of 1,465 bp; all the splicing junctions followed the conserved GT/AG rule. The transcription initiation site is located 12 bp upstream of the ATG translation initiation site and 69 bp downstream of TATA-box. The regulatory elements located in the 1,701 bp 5' upstream region are GT-1, GATA motif, I-box, SBF-1, Nit2 etc. The structure of the OCT gene from C. lineata was compared with Arabidopsis thaliana, in which two genes consist of 5 exons and 4 introns. The nucleotide sequence of exons and deduced amino acid sequence exhibits 70% and 69% homology, respectively. The deduced amino acid sequences show identical conserved "HPXQ" box, carbamoyl phosphate and ornithine binding sites. The introns of C. lineata OCT gene are larger than those of A. thaliana, except for intron 3. Genomic DNA blot analysis showed that a single ornithine-dependent OCT gene exists in the C. lineata genome.

Analysis of cDNAs expressed during first cell division of petunia petal protoplast cultures using expressed sequence tags.

A large-scale sequence analysis of randomly selected cDNA clones was performed to isolate numerous genes in petunia petal protoplast cultures. We have partially sequenced 1158 randomly selected genes of the cDNA library constructed from 2-6 d cultured petal protoplasts. Three hundred and sixty-five different genes were identified, 25% of which showed significant similarity to existing sequences in the petunia, and an array of other organisms. In this report, 90 independent genes are analyzed in detail. A functional categorization of the database-matched expressed sequence tags (ESTs) showed that defense- or stress-related genes, as well as genes involved in the primary metabolic pathways and in the transcriptional or translational apparatus are abundantly represented. In particular, ESTs were identified with apparent homologies to the cyclin-dependent kinase, histone, actin-depolymerizing factor, proteasome, and ubiquitin which are expected to be related to cell division or to cell cycle control.