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Both have a custom made tarp. These balers came out of Arizona and are pretty much rust free. Call: Paul M. In Stock Marliss Game Planter 5'. In the first part of the paper Monte Carlo experiments are used to evaluate the bias of the estimates Y:, Y t , Y: and Y j and to study the relationships between estimates and numbers of variables, V , and objects, N,, in the case of samples extracted from a multivariate normal population.

In the second part the sensitivity and specificity of the models are studied by means of simulated samples extracted from a population of two categories with the same multivariate normal distribution but with a changeable distance between the two centroids. The control of the distance gives the possibility of simulating data with a known theoretical specificity at a selected sensitivity.

Thus it is possible to evaluate the performances of each estimate of Y 2and the effect of the numbers of variables and objects used to compute the class model. Finally, the different models corresponding to the different estimates are used with a real data set to show the effect of using one or other estimate in the case of real data. F-' was approximated by a family of polynomials. The cumulative distribution of the so- obtained X showed an error less than 0.

Monte Carlo experiments were performed zyxwv to evaluate the bias of each estimator as a function of the number of objects in the training set and the number of variables.

The Mahalanobis distance Y 2 equation 2 and the estimates Y:, Y t , Y: and Y j equations 7 , 9 , 11 and 12 respectively have been computed zyxw for objects in the training and evaluation sets. The results are given in Table 1. Y t better and Y: are good estimators of the Mahalanobis distance for objects in the training set. The Defrise-Gussenhoven correction gives an unbiased estimator for the evaluation set, but both Y: and Y j heavily overestimate the distance in the case of the training set.

Y i is obtained by making negative values of Y: equal to zero so that its average error is more positive or less negative than that of Y ; , but this is a very rare case, so that the two estimators are about equivalent. Because the objects in the training set are those used to build the model, Y: will build models with a class space greater than the true model, so that the class model will also accept some objects in the training set that an unbiased model would reject as outliers.

Class space The previously studied bias of the estimators is an average on Mahalanobis distances from very zyxwvu little to very great. What really interests us is the performance of the estimators at Mahalanobis distances close to that corresponding to the class boundary. Experiments were done to obtain a more objective evaluation of the effect of bias on the class space. In the case of UNEQ models this is a V-dimensional ellipsoid of points whose distance from the centroid is equal to or less than the critical value C V a.

The volume of the ellipsoid, measured as a probability, is 1 - a. Thus, to obtain the correct critical value CV 0. The best result is achieved with Y? The use of the Defrise-Gussenhoven correction produces the maximum error.

OO40 - - 0. In the case of Y: the change in a with increasing number of variables at N constant is very large. This result is of practical importance, because during the development of a class model, usually some variables which provide redundant zy information are cancelled. The use of the estimator Y:, for which the class space depends very much on the number of variables, with the consequence of little possibility of outlier detection, can add further difficulties t o the process of selection of variables.

Failed to load latest commit information. View code. PS Vita sdslot. A remotecache. There's no need to edit any hashes manually. Press Triangle and select Open decrypted. Releases No releases published. Packages 0 No packages published. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes.

In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. Goldberger ]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon.

If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase EP-A-0 Fusion proteins provide an alternative to direct expression.

Upon expression, this construct will provide a fusion of the two amino acid sequences. The resulting fusion protein preferably retains a site for a processing enzyme factor Xa to cleave the bacteriophage protein from the foreign gene [Nagai et al. Fusion proteins can also be made with sequences from the lacZ [Jia et al. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site.

Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme eg. Through this method, native foreign protein can be isolated [Miller et al. Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.

The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media gram-positive bacteria or into the periplasmic space, located between the inner and outer membrane of the cell gram-negative bacteria. Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B.

Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. Usually, the above described components, comprising a promoter, signal sequence if desired , coding sequence of interest, and transcription termination sequence, are put together into expression constructs.

The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification.

In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about , and usually about 10 to about A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids.

Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome.

Integrating vectors may also be comprised of bacteriophage or transposon sequences. Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed.

Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin neomycin , and tetracycline [Davies et al.

Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways. Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al. Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl 2 or other agents, such as divalent cations and DMSO.

DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See eg. Boyer and S. Nicosia ; Mandel et al. Acta ; Escherichia ], [Chassy et al. Biochem , Pseudomonas ]; [Augustin et al. Ferretti and R. Curtiss III ; Perry et al. Biotechnology , Streptococcus]. Yeast expression systems are also known to one of ordinary skill in the art.

A yeast promoter may also have a second domain called an upstream activator sequence UAS , which, if present, is usually distal to the structural gene. The UAS permits regulated inducible expression. Constitutive expression occurs in the absence of a UAS.

Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription. Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. USA 1].

In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, [Cohen et al.

USA ; Henikoff et al. Topics Microbiol. Timmis and A. Puhler ; Mercerau-Puigalon et al. A DNA molecule may be expressed intracellularly in yeast.

If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide. Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems.

EP-A-0 Through this method, therefore, native foreign protein can be isolated eg. Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast EP-A-0 The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader about 83 amino acid residues as well as truncated alpha-factor leaders usually about 25 to about 50 amino acid residues U.

Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes. Usually, the above described components, comprising a promoter, leader if desired , coding sequence of interest, and transcription termination sequence, are put together into expression constructs.

The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al.

A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.

Brake et al. Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al. An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector.

See Orr-Weaver et al. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al. USA ]. The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal.

For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al. Alternatively, some of the above described components can be put together into transformation vectors. Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above. Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts.

For example, expression vectors have been developed for, inter alia, the following yeasts: Candida albicans [Kurtz, et al. Basic Microbiol. Hansenula polymorpha [Gleeson, et al. USA ; Ito et al. Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations.

Transformation procedures usually vary with the yeast species to be transformed. USA 75; ; Ito et al. Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies.

Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally generally subcutaneously or intramuscularly. Immunization is generally boosted weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant.

One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization. The serum is recovered by centrifugation eg. About ml per bleed may be obtained from rabbits. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen and optionally several large lymph nodes is removed and dissociated into single cells.

If desired, the spleen cells may be screened after removal of nonspecifically adherent cells by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension.

Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium eg. The resulting hybridomas are plated by limiting dilution, and are assayed for production of antibodies which bind specifically to the immunizing antigen and which do not bind to unrelated antigens. The selected MAb-secreting hybridomas are then cultured either in vitro eg.

If desired, the antibodies whether polyclonal or monoclonal may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms particularly 32 P and I , electron-dense reagents, enzymes, and ligands having specific binding partners.

Enzymes are typically detected by their activity. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes.

For example, I may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with I, or with an anti-biotin MAb labeled with HRP.

Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.

Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention. The effect can be detected by, for example, chemical markers or antigen levels.

Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.

For purposes of the present invention, an effective dose will be from about 0. A pharmaceutical composition can also contain a pharmaceutically acceptable carrier.

The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.

Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences Mack Pub.

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.

Liposomes are included within the definition of a pharmaceutically acceptable carrier. Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications eg.

Dosage treatment may be a single dose schedule or a multiple dose schedule. Vaccines according to the invention may either be prophylactic ie. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates such as oil droplets or liposomes , and inactive virus particles. Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H.

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