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Los Angeles, California, United States / February 3, 2015
Hunk, the co-star of Russell Madness, completed 11 backflips in 15 seconds, a RecordSetter.com World Record. RecordSetter adjudicator Megan Johnson oversaw the attempt.
- timing begins when first backflip starts
- only flips that begin before the timing is complete are counted
- may only be attempted by a trained monkey with trainer present
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The Abominable Snowman, Bigfoot, the Wildman of China - there have always been tales of sightings of ape-like creatures in mountainous, out-of-the-way places. This volume explores the possible connections between prehistoric man and these elusive creatures of the wild.
Stories of mysterious creatures landing on Earth and living among us have been around for years. There are even people on record who have claimed to have been abducted by aliens! This volume searches for the truth, examining the stories and evidence of alien visitors.
For centuries, sailors have told tales of sea monsters and serpents that have attacked their ships. Many stories tell of giant squids with their tentacles wrapped around the trapped vesselsbut what is the truth behind these tales? Are there such monstrous creatures that live in the deepest and darkest waters?
Is there some mysterious force of nature that has a sense of humor, causing seemingly unexplained and bizarre things to happen on Earth? Flying omelets and shoals of fish falling out of the sky may have perfectly sensible explanations, but how do we explain the way that twins separated at birth have time and again followed the same path through life? In this intriguing book, scientists use science and chaos theory to try to explain the Cosmic Joker.
Why do aircraft and ships seem to disappear in the Bermuda Triangle? Since the time of Christopher Columbus there have been reports of strange sightings and vessels lost while traveling through the Devils Triangle. Are the legends make-believe, or is there something sinister waiting in the waters of the Bermuda Triangle?
Strange objects have been reported in the skies for centuries bright objects, moving shapes, lights traveling in formation. In 1952 alone, there were 1,500 reports of UFO sightings! This book examines the evidence and questions whether these are real UFOs.
The mind is a powerful instrument. It influences everything we do and say. We all talk about a sixth sense, or ESP. This book examines mind over matter, looking at claims that brain power alone can bend spoons, identify things without having seen them, or even read the minds of astronauts!

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Following the guidelines and advisory measures announced by both the local government and the British Boxing Board of Control, Queensberry Promotions felt it would be inappropriate to proceed with any forthcoming boxing promotions in front of live audiences during April and May.
The coronavirus situation is evolving rapidly. Frank Warren's company will continue to follow the government's guidelines to ensure the health and safety of the paying public, staff and the participants.
Regarding the two events currently have on sale, the following has been confirmed:
The 'Seek and Destroy' Event being headlined by Daniel Dubois v Joe Joyce on April 11 at the O2 Arena has now been rescheduled to Saturday July 11, still at the O2 Arena. Tickets will remain valid for the new date, however people who cannot attend the new date can obtain a refund from their original point of purchase.
World Champion Josh Taylor's homecoming against mandatory challenger Apinun Khongsong, scheduled for May 2 at the SSE Hydro in Glasgow will no longer take place on this date. Refunds are available from the point of purchase. Warren is currently working with the SSE Hydro, Top Rank and MTK Global to identify a suitable alternative date to reschedule the event for the Scottish fans.
Queensberry Promotions and Top Rank have already reached agreement for Carl Frampton to challenge Jamel Herring for the WBO world super featherweight Championship in Belfast in June. This event was due to be announced this week. At this moment in time, they are still hopeful of being able to stage this event in June, however, the ever-changing landscape surrounding the Covid-19 pandemic will be monitored. Details of the date, venue and location will be announced in due course.


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decease geneva;">It is thought the negotiations for the forward ended after the decision of Milan's Alexandre Pato to stay in Italy and not move to Paris Saint-Germain.
diagnosis geneva;">However, City hope a deal with Milan can be concluded for Tevez, 27, before the end of the January transfer window.
The development could reignite Inter Milan's interest in the former Manchester United man.
Inter president Massimo Moratti has previously conceded that he thought Tevez would join their rivals.
Milan chief executive Adriano Galliani had been holding talks with City and Tevez's agent Kia Joorabchian in London, before Pato's proposed £29m move to PSG broke down.
City are holding out for a permanent deal for Tevez, and are not willing to let the striker leave on loan.
Manager Roberto Mancini said on Tuesday he was confident Tevez would find a new club before the end of the January transfer window.
"I think maybe we can find a good solution in January, also because it is important for Carlos. Now it is three or four months that he hasn't played," he said.
"I know that there is Inter maybe that is interested because I read the newspapers, and [AC] Milan the same but I don't know the last news."
Tevez has not played for City since they beat Birmingham on 21 September.
Mancini alleged the Argentine refused to come off the substitutes' bench in September's Champions League defeat at Bayern Munich, while the player maintained there had been a misunderstanding.
An internal club investigation concluded that Tevez, who joined City in July 2009, was guilty of five breaches of his contract and he was fined four weeks' wages, reduced to two following intervention from the Professional Footballers' Association.


text: WilhelmStroker 83 Posted May 16 I tried to watch Body of Evidence yesterday as I wanted one to watch one of the movies from the Razzies book I'm reading....God that was bad. Never saw it when it came out. Madonna can't act at all and the water bottle on my desk is more erotic than the entire movie. Ugh. Share this post Link to post Share on other sites


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Dark Side of the Ring has released a three minute clip on Owen Hart's practical joking:
In this bonus scene from the season finale, Owen Hart's friends and family remember his notorious ribs both at home and in the ring. pic.twitter.com/MbTQysHk7v— Dark Side of the Ring (@DarkSideOfRing) May 21, 2020
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Edward F. Gilman and Dennis G. Watson2
This cultivar of the North American native tree, probably 30 to 50 feet high and 10 feet wide, has a fairly narrow, silhouette. The 2.5 to 3.5-inch-long, dark blue/green needles are joined by light brown, 1.5-inch-wide, hanging cones which add to the tree's overall attractiveness. Limber Pine is so-named due to the flexible nature of the branches. Young branches can literally be tied into a knot. The tree has the overall look of a White Pine when it is young.
Scientific name: Pinus flexilis
Pronunciation: PIE-nus FLECK-sih-liss
Common name(s): Columnar Limber Pine
Family: Pinaceae
USDA hardiness zones: 5A through 7A (Fig. 2)
Origin: native to North America
Invasive potential: little invasive potential
Uses: container or planter; specimen; Bonsai
Availability: not native to North America
Height: 30 to 50 feet
Spread: 10 to 12 feet
Crown uniformity: symmetrical
Crown shape: columnar
Crown density: dense
Growth rate: slow
Texture: fine
Leaf arrangement: spiral (Fig. 3)
Leaf type: simple
Leaf margin: entire
Leaf shape: needle-like (filiform)
Leaf venation: parallel
Leaf type and persistence: needled evergreen, evergreen, fragrant
Leaf blade length: 2 to 4 inches
Leaf color: green, blue or blue-green
Fall color: no color change
Fall characteristic: not showy
Flower color: yellow
Flower characteristics: not showy
Fruit shape: elongated, cone
Fruit length: 3 to 6 inches
Fruit covering: dry or hard
Fruit color: brown
Fruit characteristics: does not attract wildlife; showy; fruit/leaves a litter problem
Trunk/bark/branches: branches don't droop; not showy; typically multi-trunked; thorns
Pruning requirement: needed for strong structure
Breakage: resistant
Current year twig color: green
Current year twig thickness: medium
Wood specific gravity: unknown
Light requirement: full sun, partial sun or partial shade
Soil tolerances: sand; loam; clay; acidic; alkaline; well-drained; occasionally wet
Drought tolerance: high
Aerosol salt tolerance: unknown
Roots: not a problem
Winter interest: no
Outstanding tree: yes
Ozone sensitivity: unknown
Verticillium wilt susceptibility: resistant
Pest resistance: resistant to pests/diseases
Limber Pine grows best in full sun or partial shade on moist, well-drained soil but will easily adapt to harsher sites. Some horticulturists say they grow better under poor cultural conditions of drought and compacted clay soil. It adapts to wet soil by growing a shallow root system. One of the most tolerant of cold, windy weather in the winter and is not cold damaged when most other Pines show needle browning.
A few of the other cultivars include: `Glauca', foliage is a deeper blue/green than the species; `Glauca Pendula', irregular, wide-spreading shrub with blue/green needles; `Glenmore Dwarf', small, upright, pyramidal tree with blue/grey foliage; `Nana', dwarf bushy type; and `Pendula', wide, weeping silhouette.
Propagation is by seed.
There are a large number of pests and diseases on Pine.
This document is ENH-624, one of a series of the Environmental Horticulture, UF/IFAS Extension. Original publication date November 1993. Revised December 2006. Reviewed February 2014. Visit the EDIS website at http://edis.ifas.ufl.edu.
Edward F. Gilman, professor, Environmental Horticulture Department; Dennis G. Watson, former associate professor, Agricultural Engineering Department, UF/IFAS Extension, Gainesville, FL 32611.
The Institute of Food and Agricultural Sciences (IFAS) is an Equal Opportunity Institution authorized to provide research, educational information and other services only to individuals and institutions that function with non-discrimination with respect to race, creed, color, religion, age, disability, sex, sexual orientation, marital status, national origin, political opinions or affiliations. For more information on obtaining other UF/IFAS Extension publications, contact your county's UF/IFAS Extension office.
U.S. Department of Agriculture, UF/IFAS Extension Service, University of Florida, IFAS, Florida A & M University Cooperative Extension Program, and Boards of County Commissioners Cooperating. Nick T. Place, dean for UF/IFAS Extension.


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Arcanobacterium pyogenes is an opportunistic pathogen, associated with suppurative infections in domestic animals. In addition to pyolysin, a pore-forming, cholesterol-binding toxin,A. pyogenes expresses a number of putative virulence factors, including several proteases and neuraminidase activity. A 3,009-bp gene, nanH, was cloned and sequenced and conferred neuraminidase activity on an Escherichia colihost strain. The predicted 107-kDa NanH protein displayed similarity to a number of bacterial neuraminidases and contained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases. Recombinant His-tagged NanH was found to have pH and temperature optima of 5.5 to 6.0 and 55°C, respectively. Insertional deletion of the nanH gene resulted in the reduction, but not absence, of neuraminidase activity, indicating the presence of a second neuraminidase gene in A. pyogenes. NanH was localized to the A. pyogenes cell wall. A. pyogenes adhered to HeLa, CHO, and MDBK cells in a washing-resistant manner. However, the nanH mutant was not defective for adherence to epithelial cells. The role of NanH in host epithelial cell adherence may be masked by the presence of a second neuraminidase in A. pyogenes.
Arcanobacterium pyogenes is a common inhabitant of the upper respiratory, urogenital (12, 54), and gastrointestinal tracts (35; B. H. Jost, K. W. Post, and S. J. Billington, unpublished data) of many domestic animal species. However, a physical or microbial insult to the host can lead to a variety of suppurative A. pyogenes infections, such as mastitis in dairy cows (27) and goats (2), liver abscesses in feedlot cattle (31, 34), and pneumonia in pigs (26) and various species of wildlife (17, 42, 57). A. pyogenes can also infect avian species (10) and humans (5, 16, 19), although infections in humans are rare.
A. pyogenes elaborates a number of extracellular proteins, including the hemolytic exotoxin pyolysin (PLO) (8), several proteases (48, 51), a DNase (30), and at least one neuraminidase (47). While all these proteins are putative virulence factors, only for PLO is there definitive evidence of involvement in the pathogenesis of infections by A. pyogenes (29).
Recently, there has been interest in the neuraminidases of bacterial pathogens and the potential role they play in pathogenesis. Neuraminidase (N-acetylneuraminyl hydrolase; EC 18.104.22.168 ) removes sialic acid from glycolipids, glycoproteins, and poly- and oligosaccharides. Bacterial neuraminidases have only 20 to 30% amino acid sequence identity, but they contain two conserved motifs, the RIP/RLP motif (Arg-Ile/Leu-Pro) and the Asp box motif (Ser-X-Asp-X-Gly-X-Thr-Trp), which occurs four or five times in the enzyme (14, 21, 43).
Neuraminidases are virulence factors, especially in bacteria that inhabit mucosal surfaces (20, 22, 52, 55), and they may play several roles in virulence. This enzyme can make sialic acid available as a carbon source to promote growth in a nutrient-limited environment (11, 23). The action of neuraminidase can decrease mucus viscosity (24), possibly enhancing colonization of the underlying tissues. Desialylation by neuraminidases can increase the susceptibility of mucosal IgA to bacterial proteases (18, 41). Neuraminidase can enhance bacterial adhesion and colonization (9, 13, 22, 56) and susceptibility of the host to the action of toxins (20), by exposing cryptic host cell receptor molecules.
This paper describes the cloning and characterization of a neuraminidase expressed by A. pyogenes. In addition, we show that A. pyogenes can adhere to epithelial cells in a washing-resistant manner, and we have investigated whether neuraminidase plays a role in this adhesion.
Bacteria and growth conditions. A. pyogenesstrain BBR1 was isolated from a bovine abscess. Other A. pyogenes strains used in this study were from veterinary diagnostic laboratories or personal collections. A. pyogenesstrains were grown on brain heart infusion (BHI; Difco) agar plates, supplemented with 5% bovine blood, at 37°C and 5% CO2 or in BHI broth supplemented with 5% bovine calf serum at 37°C with shaking. Escherichia coliDH5αMCR strains (Gibco-BRL) were grown at 37°C on Luria-Bertani (LB; Difco) agar or in LB broth with shaking. Antibiotics (Sigma) were added as appropriate; for A. pyogenes strains, erythromycin (EM) at 15 μg/ml and kanamycin (KM) at 30 μg/ml; for E. coli strains, ampicillin at 100 μg/ml, chloramphenicol at 30 μg/ml, EM at 200 μg/ml, and KM at 50 μg/ml.
Preparation of CSF, CWE, and protoplasts.Culture supernatant fluid (CSF) was prepared from liquid cultures of A. pyogenes grown overnight to an optical density at 600 nm (OD600) of approximately 3.0 to 4.0. Cells were removed by centrifugation at 5,000 × g, and the CSF was filtered through a 0.22-μm-pore-size filter. A. pyogenes cell wall extract (CWE) and protoplasts were prepared as previously described for Streptococcus pneumoniae(36). Protoplasts were resuspended in distilled water and were lysed by several cycles of freezing and thawing. Total protein concentration was determined using the Bradford protein assay reagent (Bio-Rad).
DNA techniques.Preparation of plasmid DNA and electroporation-mediated transformation of A. pyogenesstrains were performed as previously described (28). Genomic DNA from A. pyogenes was isolated by the method of Pospiech and Neumann (40). A library of A. pyogenes BBR1 genomic DNA was constructed in λGEM-12, according to the manufacturer's instructions (Promega). Methods for growth and purification of bacteriophage were essentially as described by Ausubel et al. (4). DNA was prepared from bacteriophage as previously described (46) and was further purified using the Wizard DNA clean-up system (Promega). E. coli plasmid DNA extraction, transformation, DNA restriction, ligation, agarose gel electrophoresis, and Southern transfer of DNA to nitrocellulose membranes were performed essentially as described elsewhere (4). Preparation of DNA probes, DNA hybridization, and probe detection were performed using a digoxigenin DNA labeling and detection kit (Roche), as recommended by the manufacturer. PCR DNA amplification was performed using Taq DNA polymerase (Fisher Scientific) with the supplied reaction buffer for 35 cycles consisting of 1 min at 94°C, 1 min at 55°C, and 1 min/kb at 72°C, with a final extension step of 72°C for 5 min.
Nucleotide sequence determination.The sequence ofnanH was determined from plasmids pJGS274 and pJGS292 and appropriate subclones using automated DNA sequencing. Sequencing was performed on both strands, crossing all restriction sites, using KS, SK, or T7 sequencing primers or oligonucleotide primers based on the sequence of nanH. Oligonucleotide primers were synthesized by Sigma-Genosys. Sequencing reactions were performed by the DNA Sequencing Facility at The University of Arizona, using a 377 DNA sequencer (Applied Biosystems Inc.).
Computer sequence analysis.Nucleotide sequence data were compiled using the Sequencher program (GeneCodes). Database searches were performed using the BlastX and BlastP algorithms (3). Sequence analysis was performed using the suite of programs available through the Genetics Computer Group, Inc. (University of Wisconsin). Signal sequence prediction was performed using SignalP (38). Multiple sequence alignments were performed using CLUSTAL W (53).
Cloning and purification of a recombinant, six-His-tagged NanH (His-NanH).The nanH gene, lacking the coding region for the signal sequence, was amplified from A. pyogenes BBR1 genomic DNA by PCR with a 5′ primer containing an NheI site (5′-GGTTGCAGCTAGCGCCCCGAGCACAG-3′) and a 3′ primer containing a PstI site (5′-GCGTTATCGCGCTGCAGATTTAGCCC-3′) (restriction sites are underlined). These primers amplified a 2.9-kb product from bases 120 to 3009 of the plo gene. The PCR fragment was digested with NheI-PstI and cloned intoNheI-PstI-digested pTrcHis B (Invitrogen) to generate pJGS306. pJGS306 encoded His-NanH, a 977-amino-acid protein comprising 963 amino acids of the mature NanH with an N-terminal extension of 14 amino acids encoded by pTrcHis B, including a six-His sequence.
Cultures for preparation of His-NanH were grown to an OD600 of 0.6, prior to induction with 2.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Gold Biotechnology) for 3 h. Cells were harvested by centrifugation at 5,000 × g, and the cell pellet was resuspended in 20 mM Tris-HCl–100 mM NaCl, pH 8.0. The cells were disrupted by two passages through a French pressure cell (Aminco) at 138 MPa, and the insoluble material was removed by centrifugation at 12,000 ×g. His-NanH was purified from the soluble fraction using TALON metal affinity resin (Clontech), per the manufacturer's instructions. His-NanH was eluted from the resin with 50 mM imidazole–20 mM Tris-HCl–100 mM NaCl, pH 8.0 (TALON elution buffer). Total protein concentration was determined using the Bradford protein assay reagent (Bio-Rad).
Preparation of goat antiserum to His-NanH.A female goat was immunized with 500 μg of His-NanH in a Ribi adjuvant system (Corexa) intramuscularly in the hind leg at two sites. A similar booster immunization of 500 μg of His-NanH in RAS was administered on days 29, 43, and 64. Blood was collected on day 75, and serum was harvested from the clotted blood by centrifugation at 400 × g. Preimmune serum was prepared in a similar manner, prior to immunization.
SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting. E. coli whole cells or purified His-NanH was mixed 1:1 with sodium dodecyl sulfate (SDS) sample buffer (0.2 M Tris-HCl [pH 6.8], 2.5% SDS, 10% β-mercaptoethanol, 20% glycerol, 0.013% bromophenol blue) and boiled for 10 min prior to electrophoresis in a 10% (wt/vol) SDS-polyacrylamide gel, essentially as described previously (4). Proteins were transferred to nitrocellulose (Schleicher and Schuell), and Western blots were immunostained as previously described (4) using goat anti-His-NanH and rabbit anti-goat immunoglobulin G (heavy plus light chains)-peroxidase conjugate (Kirkegaard & Perry Laboratories) as the primary and secondary antibodies, respectively. When required, sera were absorbed overnight at 4°C with an equal volume of bacterial cells which had been previously disrupted by two passages through a French pressure cell (Aminco) at 138 MPa. The sera were sterilized by passage through a 0.22-μm-pore-size filter prior to use.
Neuraminidase activity assay.Neuraminidase activity was assayed using the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUAN) (Sigma) (33), essentially as described by Winter et al. (60). One unit was defined as the release of 1 μmol of 4-methylumbelliferone from MUAN per min at 37°C.
Screening of large numbers of A. pyogenes or E. coli strains for neuraminidase activity was performed using a filter paper assay. Number 2 filter paper (Whatman) was wetted with 100 μM MUAN in 0.1 M sodium acetate, pH 4.5. Colonies were patched onto the filter paper and incubated at 37°C for 15 min, prior to excitation with a UV source (254 nm). The presence of a cyan fluorescence indicated a positive result.
Epithelial cell adhesion assay.The epithelial cell lines used in the adherence experiments were Chinese hamster ovary (CHO) cells, human cervical epithelial cells (HeLa), and Madin-Darby bovine kidney (MDBK) cells. All cell lines were cultured in Iscove's modified Dulbecco's medium (Life Technologies) supplemented with 10% fetal bovine serum (Omega Scientific) (IMDM-10%) with 100 μg of gentamicin/ml (Sigma) in a humidified, 5% CO2atmosphere at 37°C. For adherence assays, epithelial cells in IMDM-10% without gentamicin were seeded into 24-well plates at 2 × 105 cells per well in 1-ml volumes. The cells were incubated at 37°C and 5% CO2 for 18 h prior to addition of bacteria (freshly grown to an OD600 of 1.0) and His-NanH, where applicable. The plates were centrifuged for 10 min at 400 × g to increase the bacterium-host cell contact. Bacterial adhesion was assessed after 1 h of incubation at 37°C and 5% CO2. Cell monolayers were washed three times with 0.01 M phosphate-buffered saline, pH 7.2 (PBS), to remove nonadherent bacteria. Bacteria were recovered by treatment of the cell monolayers with 1 ml of 0.1% Triton X-100 for 10 min at 0°C, and viable bacteria were enumerated by dilution plating. All experiments were performed in triplicate on three separate occasions.
Nucleotide sequence accession number.ThenanH sequence data were submitted to the DDBJ/EMBL/GenBank databases under accession number AF298154 .
A. pyogenes strains express neuraminidase activity.A total of 53 strains of A. pyogenes isolated from a variety of animals, including bovine, porcine, and avian species were tested for neuraminidase expression by the filter paper method. All 53 strains were positive for neuraminidase activity, indicating that this enzyme is probably expressed by all A. pyogenesisolates.
In addition, neuraminidase activity was expressed throughout the cell cycle. A. pyogenes BBR1 cells, grown to an OD600 of 0.45, 1.0, or 5.2, were washed once with PBS, and an equal number of cells was applied to filter paper saturated with MUAN. All the samples fluoresced strongly, indicating that at least in vitro, neuraminidase activity was present throughout theA. pyogenes cell cycle.
Cloning and nucleotide sequence determination ofnanH.During a project to clone A. pyogenes promoter sequences in the promoter-probe vector pKK232-8 (Amersham Pharmacia Biotech), sequences homologous to the neuraminidase gene, nedA, of Micromonospora viridifaciens (45) were identified. In order to clone the entire gene, a probe was prepared from a region spanning bases 1639 to 1898 of the nanH gene and was used to probe a λGEM-12 library of BBR1 genomic DNA. Several plaques hybridized strongly with the probe and were selected for further analysis. One of these, λJGS6, contained an approximately 16-kb partialSau3AI fragment. DNA purified from λJGS6 was digested withBamHI or HindIII and cloned into similarly digested pBC KS (Stratagene). Two overlapping clones encompassing the entire nanH gene region were obtained: pJGS274, containing a 3,773-bp BamHI fragment, and pJGS292, containing a 6,151-bpHindIII fragment (Fig. 1). pJGS274, but not pJGS292, conferred neuraminidase activity on theE. coli host, as determined by the MUAN filter paper assay.
The DNA sequence of the nanH gene region was deduced from these clones. There are several in-frame ATG codons at the 5′ end of the nanH gene. The first and second ATG appear to be equal candidates for start codons, as they both have consensus ribosome-binding sites. Assuming translation from the first ATG, the 3,009-bp nanH gene encoded a protein with a predicted molecular mass of 107.3 kDa. A gram-positive signal sequence, with a cleavage site between Ala 40 and Ala 41, was predicted by SignalP (38). A putative rho independent terminator (ΔG = −17.0 kcal/mol) was identified 26 bases downstream of the nanH stop codon. No E. coliς70-like promoter sequences were apparent upstream of the nanH gene.
Upstream of nanH, an open reading frame (ORF),birA, was identified, and its protein product had similarity to biotin ligase from Deinococcus radiodurans(59). Downstream sequences contained ORFs whose protein products had similarity to the electron transport proteins, FixABCX, involved in nitrogen fixation in Bradyrhizobium japonicum(25, 58). birA and the putativefixABCX operon were also transcribed in the opposite direction to nanH (Fig. 1), suggesting that nanHis monocistronic.
Analysis of the primary structure of NanH.Cleavage at the predicted signal peptide sequence of NanH would result in a mature protein with a predicted molecular mass of 103.2 kDa and a pI of 6.3. NanH showed similarity to a number of bacterial neuraminidases, including those from Actinomyces viscosus (31.2% identity, 61.8% similarity), M. viridifaciens (21.4% identity, 42.3% similarity), and Bacteroides fragilis (13.7% identity, 33.4% similarity). In addition, the NanH protein contained the conserved catalytic RIP/RLP motif, as well as five copies of the Asp box motif (Ser-X-Asp-X-Gly-X-Thr-Trp) associated with bacterial neuraminidases (14, 21, 43) (Fig.2).
At the C terminus of the NanH protein there was a sequence similar to the cell wall sorting signals found in surface-expressed proteins of gram-positive bacteria (49). The terminal 33 amino acids of NanH (LVHTGATVVGLSVAAAVLLLAGGAIAIIRRRQG) consisted of an LPXTG cleavage motif (bold), a hydrophobic domain (underlined), and a positively charged stop transfer sequence (italics). However, in the case of the A. pyogenes NanH protein, the cleavage motif was LVHTG, instead of LPXTG. In addition, a Pro-rich repetitive region, which is thought to facilitate spanning of the cell wall peptidoglycan (49), was found directly upstream of the LPXTG motif.
Cloning and expression of His-NanH.To facilitate the purification of recombinant NanH from E. coli, the NanH-coding sequence, lacking the sequence for the putative signal peptide was cloned into pTrcHis B. SDS-PAGE and Coomassie brilliant blue staining of IPTG-induced cultures of DH5αMCR(pJGS306) did not reliably indicate the presence of an overexpressed protein of approximately 104 kDa, compared to similarly induced cultures of DH5αMCR(pTrcHis B) (Fig. 3A). However, His-NanH was purified from DH5αMCR(pJGS306) to >90% homogeneity using TALON resin (Fig. 3A), and the size of this protein corresponded to that of the predicted molecular mass of His-NanH. His-NanH routinely purified as a doublet, which may indicate some processing of the protein in E. coli (Fig. 3A). Purified His-NanH retained neuraminidase activity as determined using the fluorometric assay with MUAN as a substrate.
Reaction of His-NanH with specific antibodies.In Western blots, antiserum prepared against His-NanH detected a protein of approximately 104 kDa in IPTG-induced cultures of DH5αMCR(pJGS306) and preparations of purified His-NanH but not IPTG-induced cultures of DH5αMCR(pTrcHis B) (Fig. 3C). Preimmune serum also weakly reacted with His-NanH (Fig. 3B). This is not surprising, as A. pyogenes is a common inhabitant of the bacterial flora in domestic animals, and sera from normal goats contain antibodies to NanH and other A. pyogenes proteins, such as PLO (8; B. H. Jost and S. J. Billington, unpublished data).
Determination of optimal conditions for enzyme activity.In order to determine the pH optimum for this enzyme, the activity of 25 ng of His-NanH was measured by the fluorometric assay using MUAN as a substrate in 100 mM citrate-phosphate buffer with a pH ranging from 3.0 to 8.5. The pH optimum for His-NanH was 5.5 to 6.0 (data not shown). Similarly, the temperature optimum for His-NanH was determined by incubation with MUAN in 100 mM citrate-phosphate buffer, pH 6.0, at temperatures ranging from 23 to 75°C. The temperature optimum for His-NanH was 55°C (data not shown). The enzyme was quite robust, with approximately 45% of the maximal activity after 1 h at 75°C. Because His-NanH had >90% of maximal activity at 37°C and 37°C is a physiologically relevant temperature, 37°C was chosen as the standard incubation temperature. Purified His-NanH had a specific activity of 1.3 U/mg at pH 6.0 and 37°C.
Previous reports have suggested that the large bacterial neuraminidases require Ca2+ ions for activity (14). The requirement for divalent cations was tested by incubation of 25 ng of His-NanH in 100 mM citrate-phosphate buffer, pH 6.0, for 1 h at 37°C with final concentrations of 1 mM CaCl2, 1 mM MgCl2, or 10 mM EDTA. Addition or removal of divalent cations did not significantly affect the activity of His-NanH (data not shown).
Determination of the prevalence of the nanH gene by DNA dot blotting.In order to determine whether nanHwas present in all A. pyogenes strains, genomic DNA was prepared from 53 A. pyogenes strains and was subjected to hybridization at high stringency with a nanH-specific probe that spanned bases 1639 to 1898 of the nanH ORF. The DNA from all 53 strains hybridized strongly to the probe (data not shown), indicating that the nanH gene is present in all A. pyogenes strains.
Construction and characterization of a nanHmutant.Construction of the nanH mutant used an allelic exchange plasmid in which the nanH gene was completely replaced by an erm(X) cassette (Fig.4). This plasmid was constructed by deletion of the 2.6-kb ClaI fragment containing thenanH gene sequences from pJGS292 (Fig. 1), resulting in the recombinant plasmid pJGS326. The 0.73-kb ClaI fragment of pJGS293, containing sequences upstream of nanH, was cloned into similarly digested pJGS326 to form pJGS354. The entire 4.3-kbHindIII insert of pJGS342 was cloned into similarly digested pHSS20 (37) to form pJGS356. The KM resistance gene in pHSS20 is functional in A. pyogenes and was used to identify the presence of recombinants which arose by a single crossover event. A 1.65-kb HindIII-BamHI fragment containing the erm(X) gene from pNG2 (50) was treated with T4 DNA polymerase (Promega). This fragment was cloned into the similarly treated unique ClaI site in pJGS356 to generate the recombinant plasmid pJGS357 (Fig. 4). As pJGS357 was based on a ColE1 replicon, it acted as a suicide plasmid in A. pyogenes (29). pJGS357 plasmid DNA was introduced into A. pyogenes BBR1 cells by electroporation, and recombinants were selected on BHI-blood agar containing EM. Emr Kms colonies were chosen for further analysis.
Southern blotting of A. pyogenes genomic DNA digested withEcoRI revealed hybridizing bands of 1.9, 1.0, 0.9, and 0.3-kb in BBR1, when probed with a nanH-specific probe (spanning bases 120 to 3009 of the nanH ORF). No hybridizing bands were observed in a nanH mutant (NANH-1) that was probed similarly, indicating the complete loss of the nanHgene in this strain. A 2.6-kb band was apparent inEcoRI-digested NANH-1 but not BBR1 genomic DNA when anerm(X)-specific probe was used. Neither BBR1 nor NANH-1 genomic DNA hybridized with a pHSS20-specific (vector) probe (data not shown). These data confirm deletion of the nanHgene in NANH-1 by a double-crossover event.
The neuraminidase activity of NANH-1 was compared to that of the wild-type strain BBR1. BBR1 and NANH-1 were grown overnight to an OD600 of 3.0 to 4.0, the cells were washed twice in PBS and resuspended at an OD600 of 3.0, and the neuraminidase activity was assessed by the fluorometric assay using MUAN as a substrate. NANH-1 had fivefold less neuraminidase activity than the wild-type (Fig. 5). Therefore, insertional deletion of nanH did not result in complete loss of expression of neuraminidase activity from NANH-1, indicating that NanH is not the only neuraminidase expressed byA. pyogenes.
Localization of NanH.Whole cells, CSF, and CWE were prepared from BBR1 and NANH-1 which had been grown overnight to an OD600 of 3.0 to 4.0. These samples were tested for neuraminidase activity with the fluorometric assay using MUAN as a substrate. The majority of neuraminidase activity was detected in whole cells and CWE from either BBR1 or NANH-1. However, BBR1 whole cells and CWE had significantly higher neuraminidase activity than whole cells and CWE from NANH-1 (Fig. 5). Some neuraminidase activity was detected in the CSF of both BBR1 and NANH-1 (Fig. 5), and this activity may have resulted from fragments of cell wall material present in the CSF. Negligible neuraminidase activity was detected in lysed protoplasts of BBR1 and NANH-1 (Fig. 5). These data indicate that the majority of NanH-specific neuraminidase activity was associated with the cell wall. In addition, the activity of the putative second neuraminidase also appeared to be cell wall associated.
Adherence of A. pyogenes to epithelial cells.It was previously demonstrated that A. pyogenes adhered to HeLa cells (15). We also tested the ability of BBR1 to adhere to other epithelial cell lines. BBR1 adhered to CHO, HeLa, and MDBK cells in a washing-resistant manner (Fig.6A). However, bacterial adherence was greatest using HeLa cells (Fig. 6A), and this cell line was used in all subsequent studies.
The ability of BBR1 and NANH-1 to adhere to HeLa cells was tested. The wild-type BBR1 and nanH mutant strains adhered equally, with average adherences of 46.4 and 44.7%, respectively (Fig. 6B). In addition, the adherence of NANH-1 to CHO and MDBK cells was evaluated and was not significantly different from that of BBR1 (data not shown). These findings suggest that lack of NanH does not impair adhesion of NANH-1 to these epithelial cell lines.
In order to determine whether addition of exogenous NanH could enhance adhesion, 1 to 100 μg of purified His-NanH or an equal volume of TALON elution buffer (as a control) was added, either 1 h prior to or simultaneously with the addition of BBR1 or NANH-1 bacteria. No significant difference in the adhesion of the two strains was observed under any experimental condition (data not shown). These data indicate that addition of exogenous NanH did not increase the ability of BBR1 or NANH-1 to adhere to HeLa cells.
This is the first report of the cloning and sequencing of a neuraminidase gene, nanH, from A. pyogenes. In addition, this work provides indirect evidence that A. pyogenes expresses a second neuraminidase. NanH was localized to the cell wall, and the second neuraminidase also appeared to be cell wall associated. Furthermore, we demonstrated that neuraminidase activity is expressed by all A. pyogenes strains tested (n = 53).
The nanH gene of A. pyogenes was cloned and sequenced and appeared to exist in a monocistronic operon, surrounded by the housekeeping genes birA and fixABCX. nanH expressed a 103.2-kDa protein with neuraminidase activity, and the NanH protein was most closely related to the neuraminidase of A. viscosus (61). The NanH protein contained sequences consistent with its activity as a neuraminidase, including the RIP/RLP motif and five copies of the Asp box (14, 21, 43). In addition, the finding that NanH is localized to the A. pyogenes cell wall is consistent with the presence of an N-terminal signal peptide and C-terminal cell sorting signals, including an LPXTG-like cell anchor (49). However, in NanH, the cell wall anchor motif is LVHTG, which is slightly at variance with the consensus sequence observed in almost all cell wall-associated proteins identified to date. One exception is a sucrase expressed by Actinomyces naeslundii, which has the motif LARTG (39). In addition, two other cell wall-associated proteins have been identified in A. pyogenes, both of which have cell sorting signals where the Pro of the LPXTG motif has been replaced (B. H. Jost and S. J. Billington, unpublished data). Therefore, it appears that the A. pyogenes cell anchor sequence may be divergent from those seen in other bacteria.
Recombinant His-NanH protein was found to have optimal activity at pH 5.5 to 6.0 and 55°C, with no requirement for Ca2+ or Mg2+. These conditions correspond to those previously reported for a purified, native A. pyogenes neuraminidase (47). However, there were differences in the apparent size and cellular location of NanH (103.2 kDa, cell wall) and the purified, native A. pyogenes neuraminidase (50 kDa, CSF) (47). Size variation, probably as a result of specific breakdown or proteolytic cleavage, has been observed in other bacterial neuraminidases, with the truncated proteins retaining enzymatic activity. The 107-kDa S. pneumoniae NanA is observed as an enzymatically active 86-kDa species following purification (32). The size of theM. viridifaciens NedA protein varies between 41 and 68 kDa depending on the culture conditions (45). Indeed, upon prolonged storage at 4°C, His-NanH converted to a species of 62 kDa, with no loss of specific activity (data not shown). However, it is still uncertain whether NanH is the 50-kDa neuraminidase purified fromA. pyogenes CSF (47). NanH was localized to the cell wall of A. pyogenes, but NanH was also present in CSF, as NANH-1 had significantly less neuraminidase activity in the CSF than BBR1 (Fig. 5). Schaufuss and Lämmler could detect significant neuraminidase activity in the CSF of only 2 out of 42 of A. pyogenes strains tested, and they did not report testing whole cells or CWE for the presence of neuraminidase activity (47).
The entire nanH ORF was deleted during the construction of NANH-1, as confirmed by Southern blotting (data not shown). Significant neuraminidase activity in NANH-1 indicated the presence of a second enzyme in A. pyogenes. In addition, the location of the second neuraminidase also appeared to be cell wall associated, as evidenced by the retention of some neuraminidase activity in the CWE of NANH-1.
The initial event in infection by many bacteria is their attachment to mammalian cells via specific recognition structures, leading to bacterial colonization of the host. Adherence of A. naeslundii to both a human epithelial cell line (9) and polymorphonuclear leukocytes (44) was enhanced by pretreatment with neuraminidase. Like A. pyogenes, S. pneumoniae expresses two distinct neuraminidases, NanA (7), which is cell associated, and NanB (6), which is thought to be secreted from the cell. Neuraminidase treatment of tracheal organ cultures increased the adherence of S. pneumoniae (56). Furthermore, S. pneumoniae mutants deficient in neuraminidase activity had reduced abilities to colonize and persist in the nasopharynx (55).
It is clear that neuraminidase activity plays a role in mediating host cell adhesion of some pathogens to mucosal surfaces. In order to determine whether this was the case for A. pyogenes, experiments assessing the adhesion of NANH-1, the neuraminidase mutant, were conducted. As previously reported, A. pyogenes can adhere to HeLa cells (15), and we demonstrated adherence to other epithelial cell lines, althoughA. pyogenes adhered best to HeLa cells. NANH-1 displayed no defect in adherence to HeLa cells compared with the wild-type, BBR1. If the neuraminidase activity does play a role in mediating adherence ofA. pyogenes, it is possible that, at least under these conditions, the second neuraminidase was sufficient for maximal adherence. If this is the case, addition of exogenous His-NanH would not significantly affect adhesion to the host cell. Alternately, neuraminidase may play no role in the adhesion of A. pyogenes to cultured epithelial cells. The effects of neuraminidase activity may be more evident in vivo, where it may act to reduce mucus viscosity (24), assisting the adherence ofA. pyogenes by other colonization factors, such as putative collagen- or fibronectin-binding proteins. The validity of these hypotheses is being tested in our laboratory by construction of a knockout mutant of the second neuraminidase in NANH-1 and assessment of the ability of this double mutant to adhere to host epithelial cells, both in vitro and in vivo.
We thank Stefani Gilbert for construction of the λGEM-12 library and Hien Trinh and Dawn Bueschel for their excellent technical assistance.
Partial support for this work was provided by USDA/NRICGP awards (97-35204-4750 and 99-35204-7818).
Editor: J. T. Barbieri
- Received 21 February 2001.
- Returned for modification 13 April 2001.
- Accepted 23 April 2001.
- Copyright © 2001 American Society for Microbiology


text:
[ G.R. No. 139131, September 27, 2002 ]
Petitioner seeks to annul and set aside the Resolution[1] dated January 14, 1999 of the Court of Appeals (CA) in CA-G.R. SP No. UDK-2819, which dismissed petitioner's appeal via a petition for review,[2] for his failure to comply with Section 6 (c), Rule 43 of the Rules of Court, and its Resolution[3] dated February 16, 1999, denying petitioner's motion for reconsideration. Subject of said appeal before the CA were Resolutions Nos. 98-2359[4] and 98-3021[5] of the Civil Service Commission, which upheld the dismissal of petitioner from respondent Philippine Children's Medical Center (PCMC).
The facts of this case, as culled from records, are as follows:
Petitioner Jesus R. Gonzales was one of the two Utility Workers II assigned at the Pharmacy Section of respondent Philippine Children's Medical Center (PCMC), a government-owned and controlled corporation created under P.D. No. 1631, as amended. At PCMC, petitioner served the patients and the public from 6:00 A.M. to 10:00 P.M., seven days a week.
On March 2, 1998, petitioner started absenting himself without an approved leave (AWOL) and without explaining the reason for his absence to his superiors.
In view of the exigency of petitioner's functions, Ms. Jara Corazon O. Ehera, Human Resources Management Officer III, wrote a letter-notice dated March 5, 1998 to petitioner directing him to report for work within three (3) days from receipt of said notice, otherwise, he would be dropped from the rolls.
During his absence, petitioner was seen on several occasions within the premises of PCMC, particularly in the Budget Office, Billing and Cashier, and Personnel Clinic. He allegedly visited the clinic without consulting any medical problem and when Dr. Galero once made a surprise visit, he was not found in his house.[6]
On March 16, 1998, Dr. Corazon D. Rivera, officer-in-charge of the Pharmacy Section, reported petitioner's irresponsibility and lack of concern for his work to Dr. Lillian V. Lee, Executive Director of PCMC, recommending that petitioner be dropped from the rolls.
Despite the written letter-notice sent to him, petitioner failed to report for work thus constraining PCMC to drop him from the rolls, effective March 20, 1998.[7]
Aggrieved, petitioner appealed to the Civil Service Commission (CSC). In Resolution No. 98-2359 dated September 8, 1998, the CSC upheld the action taken by PCMC, to wit:
WHEREFORE, the appeal of Jesus R. Gonzales is hereby dismissed. Accordingly, the action of the PCMC Executive Director, dropping him from the rolls, is upheld.[8]
The decision, however, stated that considering that the separation of petitioner was not disciplinary in character, he may be re-employed in the same agency at the discretion of the appointing authority.[9]
Petitioner moved for reconsideration by the CSC of Resolution 98-2359, but it was denied.
When he filed a petition for review in the CA, the petition was denied for failure to comply with Section 6 (c), Rule 43 of the Revised Rules of Court,[10] particularly for failure to attach certified true copies of material portions of the records and supporting papers.
The CA Resolution[11] dated January 14, 1999, concluded:
WHEREFORE, for being formally deficient, the instant petition for review is hereby DISMISSED.
In a Motion for Reconsideration and Compliance,[12] petitioner attached the certified true copies of the required papers. But the CA denied the motion in a Resolution[13] dated February 16, 1999.
Hence, this petition raising the following issues for resolution:
1. Whether or not the Court of Appeals committed grave error in dismissing the appeal of herein petitioner based on pure technicality.
2. Whether or not there is factual and legal basis for respondent PCMC to drop petitioner from the rolls for his alleged absences without leave.[14]
On the first issue, petitioner argues that the dismissal of the petition by CA on mere technicality is unwarranted and unjustified since pertinent jurisprudence abounds declaring in no uncertain terms that dismissals of appeals on purely technical grounds is frowned upon where the policy of the Court is to encourage hearings of appeals based on merits.[15]
The same position is taken by the Office of the Solicitor General in its Manifestation in Lieu of Comment[16] filed before this Court. But respondent PCMC asserts that the dismissal by the CA of the petition for review is in keeping with Section 7[17] in relation to Section 6 of Rule 43 of the Revised Rules of Court.[18]
In Cadayona vs. Court of Appeals,[19] however, we already held that Section 6, Rule 43 of the Revised Rules of Court is not to be construed as imposing the requirement that all supporting papers accompanying the petition should be certified true copies. We compared this provision with its counterpart provision in Rule 42, on petitions for review from the RTC to the CA, and noted that under the latter, only the judgments or final orders of the lower court need to be certified true copies or duplicate originals. In numerous resolutions issued by this Court we emphasized that in an appeal via a petition for certiorari under Rule 45 and in an original civil action for certiorari under Rule 65 in relation to Rules 46 and 56, what is required to be a certified true copy is the copy of the questioned judgment, final order or resolution.[20] We see no reason why a stricter requirement should be made for petitions under Rule 43, which governs appeals from the Court of Tax Appeals and quasi-judicial agencies to the CA. This could not have been intended by the framers of the rules. A contrary ruling would be too harsh and would not promote the underlying objective of securing a just, speedy and inexpensive disposition of every action and proceeding.[21]
Further, we note that petitioner had attached certified true copies of the documents supporting his Motion for Reconsideration and Compliance.[22] As previously held, submission of a required document with the Motion for Reconsideration constitutes substantial compliance with Section 3, Rule 46.[23]
On the second issue, petitioner argues that he was denied due process[24] when he was dropped from the rolls. He avers that he received PCMC's letter dated March 5, 1998 only on March 20, 1998 and thus, had until March 23, 1998 within which to comply with the directive;[25] that in fact, he did report for work on March 21, 1998, but was barred by security personnel from entering the company because his name had already been dropped from the rolls effective March 20, 1998. He contends that his non-compliance with the return to work directive does not constitute abandonment of work as no person in his right mind would abandon his job to his own detriment.[26]
For analogous reasons, the Office of the Solicitor General avers that petitioner was denied due process. The OSG states that he was summarily dismissed without affording him a hearing and the opportunity to introduce witnesses and relevant evidence in his favor.[27] The OSG also opines that the penalty of dismissal was too severe.[28]
In its reply, respondent PCMC insists that the dismissal of petitioner is valid and legal, considering that petitioner's actuations were clearly irresponsible. They showed lack of concern for his work and the smooth operation of PCMC.[29]
Further, PCMC contends that petitioner was given ample opportunity to explain his side and to submit evidence and to explain his absence.[30] PCMC points out that they sent a letter-notice dated March 5, 1998, to petitioner and he should have taken it upon himself to report to work, even without any prompting from PCMC, considering the significance of his work.[31] Besides, PCMC claims, petitioner showed the clear intent to sever his employer-employee relationship with PCMC. Finally, PCMC avers that factual findings of the CSC on this matter are entitled to great weight and must be accorded respect and finality.[32]
To avoid circuitous procedure, we shall now consider the merits of the case. This Court is not a trier of facts, and its function is limited to reviewing errors of law that might have been committed by the lower court.[33] In this case, we find no exceptional circumstance and we find no cogent reason to set aside the factual findings of the CSC in sustaining the action of respondent in the dropping of petitioner from the rolls on the ground that he was found AWOL (absent without official leave).
The CSC noted that petitioner had admitted that effective March 2, 1998, he was absent without approved leave.[34] Petitioner's unauthorized absences, as found by the CSC, constitute conduct prejudicial to the best interest of the service, a ground for disciplinary action under E.O. No. 292 or the Administrative Code of 1987.[35] For his services are essential to the efficient delivery of medical services and the exigencies of the service require his presence in the office.
We agree that there is legal basis for dropping petitioner from the rolls. It is also in accordance with law and rules, notably Section 35 Rule XVI of the Omnibus Rules Implementing E.O. 292 and Paragraph 2.1 (b) of CSC Memorandum Circular No. 12, s. 1994 which provides as follows:
Sec. 35. Officers and employees who are absent for at least thirty (30) days without approved leave are considered on Absence Without Leave (AWOL) and shall be dropped from the service after due notice. However, when the exigencies of the service require his immediate presence and he fails/refuses to return to the service, the head of office may drop him from the service even prior to the expiration of the thirty (30) day period abovestated.[36] (Emphasis ours.)
2.1 Absence without Approved Leave
a. xxx
b. If the number of unauthorized absences incurred is less than thirty (30) calendar days, written return to work order shall be served on the official or employee at his last known address on record. Failure on his part to report for work within the period stated in the order shall be a valid ground to drop him from the rolls.[37] (Emphasis ours.)
Petitioner's assertion that he was denied due process is untenable. The essence of due process is simply an opportunity to be heard or as applied to administrative proceedings, an opportunity to explain one's side or opportunity to seek a reconsideration of the action or ruling complained of.[38]
Records would show that respondent PCMC had written a letter to petitioner to report for work[39] and another letter informing him that he was being dropped from the rolls, due to his Absence Without Official Leave, with the enumeration of the reasons and basis thereof.[40] That petitioner only received the notice on March 20, 1998 is of no moment. In fact, two notices were given him: (a) the notice requiring him to report for work; and (b) the notice that he would be dropped because of his absences without official leave. Further, petitioner was given sufficient opportunity to report for duty after he received the return-to-work order, but he did not report. Thus, the CSC found no error when respondent PCMC dropped petitioner from the rolls for his refusal to comply with the return-to-work order within a prescribed period.[41]
Finally, it must be emphasized that under CSC Circular No. 12, series of 1994, the action dropping petitioner from the rolls is non-disciplinary in nature and does not result in the forfeiture of his benefits nor his disqualification from re-employment in the government. Likewise, dropping from the rolls of petitioner is without prejudice to his re-appointment at the discretion of the appointing authority and subject to Civil Service laws, rules and regulations.
WHEREFORE, the Resolution of the Court of Appeals dated January 14, 1999 in CA-G.R. SP No. UDK-2819 and its Resolution dated February 16, 1999 are SET ASIDE insofar as it denied petitioner's appeal for his failure to comply with Section 6 (c), Rule 43 of the Revised Rules of Court. But Resolutions Nos. 98-2359 and 98-3021 of the Civil Service Commission, dropping petitioner from the rolls of respondent PCMC, without prejudice to his re-employment in the government service, are AFFIRMED.
No pronouncements as to costs.
Bellosillo, (Chairman), Austria-Martinez, and Callejo, Sr., JJ., concur. Mendoza, J., on official leave.
[1] Rollo, p. 80.
[2] Id. at 48-61.
[3] Id. at 95.
[4] Id. at 62-65.
[5] Id. at 66-68.
[6] Id. at 126.
[7] Id. at 32.
[8] Id. at 39.
[9] Ibid.
[10] Sec. 6, Rule 43. Contents of the petition. - The petition for review shall (a) xxx ; (c) be accompanied by a clearly legible duplicate original or a certified true copy of the award, judgment, final order or resolution appealed from, together with certified true copies of such material portions of the record referred to therein and other supporting papers; and xxx.
[11] Rollo, p. 80.
[12] CA Rollo, pp. 46-53.
[13] Rollo, p. 95.
[14] Id. at 17-18.
[15] Id. at 203.
[16] Id. at 133-151.
[17] Sec. 7, Rule 43. Effect of failure to comply with requirements. - The failure of the petitioner to comply with any of the foregoing requirements regarding the payment of the docket and other lawful fees, the deposit for costs, proof of service of the petition, and the contents of and the documents which should accompany the petition shall be sufficient ground for the dismissal thereof.
[18] Rollo, p. 233.
[20] Martirez vs. Magallanes, G.R. No. 133766, January 13, 1999; Borja vs. Judge Hontanosas, Jr., G.R. No. 134748, January 13, 1999; Regalado, et al vs. NLRC, G.R. No. 134671, January 13, 1999; G and M [Phils], Inc. vs. NLRC, et al., G.R. No. 133836, January 13, 1999.
[21] Supra, note 19 at 625-626.
[22] CA Rollo, pp. 46-53.
[23] Uy vs. Bureau of Internal Revenue, G.R. No. 129651, 344 SCRA 36, 48 (2000), citing Balagtas Multi-Purpose Cooperative, Inc. vs. Court of Appeals, 314 SCRA 676, 682 (1999).
[24] Rollo, p. 208.
[25] Ibid.
[26] Id. at 209-210.
[27] Id. at 145.
[28] Id. at 148.
[29] Id. at 118.
[30] Id. at 122.
[31] Id. at 119.
[32] Id. at 230.
[33] Bañas vs. Asia Pacific Finance Corporation, G.R. No. 128703, 343 SCRA 527, 535 (2000). See also Cebu Woman's Club vs. De la Victoria, G.R. No. 120060, 327 SCRA 533, 537 (2000).
[34] Rollo, p. 64.
[35] E.O. 292-Administrative Code of 1987, Book V, Title I, Sub-title A.
a. x x x
b. The following shall be grounds for disciplinary action:
x x x
14. Frequent unauthorized absences or tardiness in reporting for duty, loafing or frequent unauthorized absences from duty during regular office hours;
x x x
27. Conduct prejudicial to the best interest of the service;
[36] Sec. 35, Rule XVI of the Omnibus Rules Implementing E.O. 292.
[37] Paragraph 2.1 (b) of CSC Memorandum Circular No. 12, s. 1994.
[38] Audion Electric Co., Inc. vs. National Labor Relations Commission, 308 SCRA 340, 353 (1999).
[39] Rollo, p. 31.
[40] Id. at 32.
[41] Id. at 64.


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Former leading New Zealand publisher and bookseller, and widely experienced judge of both the Commonwealth Writers Prize and the Montana New Zealand Book Awards, talks about what he is currently reading, what impresses him and what doesn't, along with chat about the international English language book scene, and links to sites of interest to booklovers.
Tuesday, April 21, 2009
3 comments:
Thanks to Linda for this interesting blog. I tuned into Paul Coelho who struck a chord with his words "I did not start writing to make money, but to be read.". I know that locally, we struggle as authors with Radio NZ who will not pay us for re-play rights on any stories sold... but the idea of boycotting them to insist on payment is problematic as most of us are just so grateful for the publicity, and an audience...touche.
- 7:49 am
I'm an author grateful to have two publishers both of whom provide high quality editorial feedback - they're not just a banker nor a marketing team - though I'm very glad I don't have to take on either of those roles for my work. Presumably Linda O. has had an editor, and a publicity team who've helped to get her work out to a wider public - not to mention designers, who give the books an appealing look. I think it's all too easy to blame publishers for every writerly dissatisfaction. Publishers work hard, they care about the books, and they usually love the intellectual and creative stimulus the job brings. If they didn't, they'd find some far more lucrative and stable role. Publisher-bashing too often sounds like spoilt whingeing. One of the major problems with internet content is that it's not edited: sloppy, lacking in style, aesthetics. We still need publishers to give us a critical and objective eye.
- 1:30 pm
- Linda Olsson said...
I agree with anonymous, of course. Editing is an essential service that good publishers provide - in the interest of the author and the publisher alike. Still, I am intrigued by the complacence by some of my publishers in regards to new media and illegal downloading. The Swedish Authors Association recently sent a warning to its members to be cautious when considering signing away rights to electronic editions of their works. The standard contracts that established Swedish publishers now offer are virtually worthless for the author. At the moment it does feel that the entire market is in a state of turmoil and all players scrambling to find something to hold on to, a way forward in difficult times.
- 8:10 pm


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photo: Griffin Roberts (Brian Walton/The Cardinal Nation)
On Sunday, the St. Louis Cardinals system went 4-3. Palm Beach won in a shutout behind Griffin Roberts' best start as a professional – six scoreless innings. Four Memphis pitchers shut out El Paso and Martin Figueroa plated all four State College runs.
Results from games played Sunday, August 4th.
A quartet of Redbirds hurlers combined on a one-hitter, resulting in a 2-0 victory over El Paso. Memphis (50-63) leads the four-game set with the Pacific Southern Division leaders, two games to one and is tied for second with Omaha (KC) in the American Northern Division, with both teams trailing Iowa (CHC) by 10 games.
Daniel Ponce De Leon was first up, striking out eight in four frames with three free passes issued. Seth Elledge (W, 2-1) yielded the only hit, a one-out single in the first of his two innings of work. Chasen Shreve tossed two one-two-three frames but departed after walking one the next inning. Junior Fernandez (S, 2) retired both batters faced, leaving Shreve's runner aboard.
Two Redbirds each had two of the team's seven hits – Randy Arozarena and Edmundo Sosa. Arozarena singled twice in three at bats with a walk and run scored while Sosa went 2-for-3 with a double and RBI. Yadier Molina's third rehab appearance was his longest to date, one in which the veteran was hitless in three at bats while catching seven frames.
Monday's game: vs. El Paso (66-47) (SD), 6:35 pm CT, Chris Ellis (3-5, 7.44)
By giving up an eighth-inning run, the S-Cards dropped a 3-2 decision at first-place Arkansas (23-19) in the series finale. Springfield (21-21) won only one of the four-game set, with Sunday's loss dropping them into third place in the North Division standings, two games behind the Travelers (SEA).
Tommy Parsons (L, 4-3) did not finish the fifth, permitting two runs on six hits and two base-on-balls with seven K's over 4 2/3. Mitchell Osnowitz recorded six outs, allowing a hit and walk. Bryan Dobzanski gave up what proved to be the game's difference on three hits against two outs. Both relievers stranded two inherited runners. Jacob Patterson retired both batters but not one out was a sac fly scoring one of Dobzanski's three runners.
The Cards mounted a rally in the ninth, but could only close one of the two-run gap. On the day, Springfield mustered just four hits and two walks, the hits split evenly between Conner Capel and Irving Lopez. Capel scored both runs and banged out his first triple of the season and Lopez singled twice.
Monday's game: at second-place Tulsa (22-20) (LAD), 7:05 pm CT, Evan Kruczynski (2-6. 5.79)
The Beach Birds' late offense transformed a tight affair into an easy 6-0 road victory over first-place Charlotte (31-13) (TB), for a split of the four-game series. Palm Beach (15-30) remains in sixth/last place in the South Division standings, 16 ½ games behind the Stone Crabs.
The visitors held a slim 1-0 lead through seven but poured on another five runs, three earned, over the last two frames. The PB-Cards did it all on six hits and seven walks with shortstop Michael Perri accounting for half of the tallies. The shortstop went 2-for-4 with a double, two runs scored and one knocked in while batting eighth. Dennis Ortega was one of four Beach Birds with one hit. The catcher singled in five at bats, crossing home one time and driving in one.
Player of the Day Griffin Roberts' 10th start in 11 Florida State League appearances was his best by far. The start was a six-inning gem in which the right-hander blanked the opposition on three hits and two walks for his first professional win against six losses.
Ramon Santos struck out three over the next two frames, giving up a hit. Cole Aker faced the minimum in the ninth.
Monday's game: vs. fourth-place Jupiter (23-23) (MIA), 6:30 pm ET, TBA
A five-run sixth sent the Chiefs to a 9-4 road loss at Wisconsin, evening the series at one game apiece with two contests on tap. Peoria (13-29) remains in eighth and last place in the West Division standings, 16 games in back of Kane County (ARI).
Michael Brettell (L, 4-8) retired just one batter in the sixth, surrendering five runs on seven hits and four free passes overall. Franyel Casadilla recorded five outs, giving up two tallies on four hits, including a solo bomb, and a walk while permitting both inherited runners to touch home. Leonardo Taveras was charged with two runs on two hits and a base-on-balls.
Four Chiefs each had two of the team's 10 hits – Jonatan Machado, Delvin Perez, Imeldo Diaz and Leandro Cedeno. All four doubled once with Perez and Cedeno both going yard, the former with a runner aboard. Perez scored twice with two RBIs and Cedeno crossed home one time and drove in one.
Monday's game: at fifth-place Wisconsin (22-20), 6:35 pm CT, Logan Gragg (0-0, 3.38)
Martin Figueroa led the Spikes to a 4-2 triumph over the Spinners (30-18) (BOS) in the decisive third game of the road series. State College (25-24) climbed into a second-place tie with West Virginia (PIT), four games behind Batavia (MIA).
Figueroa drove in all four Spikes' scores, going 3-for-4 with a two-run bomb. Pedro Pages was one of four State College players with one hit each. The designated hitter doubled in three at bats, touching home one time and reaching base on a hit-by-pitch.
Michael YaSenka (W, 1-0) tossed a strong effort in his maiden New York-Penn League performance, holding the Stedler Division leaders to two runs on three hits and three walks over 6 2/3 innings. Cameron Dulle recorded the seventh's last out, leaving a YaSenka runner on, but jammed the sacks full via two hits and a base-on-balls the next inning. Jack Ralson retired five batters, giving up a hit and walk while stranding three inherited runners for his second save.
Monday's game: at sixth-place Auburn (18-30) (WSH), 7:00 pm ET, Junior Gonzalez (2-3, 6.67)
Bluefield 10 at Johnson City 7
A blown save left the Appy Birds on the wrong end of a 10-7 home decision versus Bluefield (22-22) (TOR). First-place Johnson City (25-19) claimed two of the three-game series and lead Elizabethton by two games in the West Division standings.
Enrique Perez (L, 2-2) (BS, 1) began the ninth with a 7-6 advantage but yielded four runs, three earned, on four hits, including two doubles and a hit batsman. Hector Soto went the initial five frames, striking out six but giving up a three-run long ball that was one of three hits and a walk permitted. It was the well-traveled righty's first start in five appearances for Johnson City. Soto has also seen time at State College, Palm Beach and the GCL Cardinals this season.
Wilberto Rivera bridged the three-inning gap between Soto and Perez. The righty allowed three runs on five hits and did not issue a free pass.
Liam Sabino, Victor Garcia and Carlos Soler had two hits each. Sabino scored one and knocked in another with Garcia driving in two and Soler adding a walk and run scored.
Monday's game: vs. Burlington (25-20) (KC), 6:30 pm ET, Luis Ortiz (0-1, 7.00)
GCL Cardinals 5 vs. GCL Mets 2
The Gulf Birds topped the second-place Mets, 4-2, on Sunday. The fifth/last place Cardinals (15-19) trail the Marlins (MIA) by five games in the East Division standings.
Pitching played a key role as the two squads combined for just six singles but Gulf Cardinals' hurlers issued just three free passes to the Mets' eight. Pablo Gomez reached base three times, going 1-for-2 with two walks and a run scored. Freddy De Jesus and Cristhian Longa both went 1-for-3 with a run scored, with De Jesus also working a free pass. William Jimenez also reached base three times on a 1-for-4 with two walks effort.
Four Cardinals pitchers took the mound, beginning with Blake Drake, who faced the minimum in the first. Nathanael Heredia (W, 4-1) permitted two hits and three walks, resulting in two runs in 2 2/3 frames.
Wilman Madera retired all 10 batters faced, striking out seven and leaving two Heredia runners aboard. Connor Coward tossed two one-two-three innings for his first professional save.
Monday's game: Off
The Cardinals Blue (30-17) begin the week in third place in the South Division standings, four games behind the Phillies Red.
Monday's game: Off
The Cardinals Red (20-34) are in seventh place in the eight-team San Pedro Division, 13 games in back of the Cubs2.
Monday's game: at sixth-place DSL Rangers (24-29), 10:30 am ET, Hancel Rincon (1-4, 5.04)
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Whole home generators
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I have a Generac 22Kw system that runs on natural gas and have a kit to convert to propane if needed, not that I have had to to-date. It is programmed to run once a week at a prescribed time for 30 minutes under a load and I have a telemetry system that allows the installer to monitor it for any issues. I also have a contract with them to maintain it with oil changes and other maintenance needs. It has "saved the day" more than once when we have had weather-related power outages, some lasting over a week. It is already very quiet, but I have some noise abatement structure around it to minimize what remaining noise signature it has, and this is very effective as well. I have a very reliable source for natural gas, thus that fuel choice. Mine ran continuously for over a week on the longest outage I've had since installing it, and no issues at all with either the generator or the power in the house.
I do have a backup in the form of a 10Kw diesel generator and an 8Kw gasoline generator. To this add a small Honda generator that I use for camping and at my hunting lease.
If you want to run systems into the house that are not installed as full-house, and not just run a bunch of extension cords, you will want an interface at your breaker box that allows you to plug your generator in and that will prevent feedback to the grid.
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