Abstract: Methods for detecting and assaying for glyoxylate, include enzyme-based assays and/or assays for hydrogen peroxide following liberation of hydrogen peroxide from glyoxylate, are disclosed. In some embodiments, the invention is directed to methods for assaying for glyoxylate produced by the reaction of peptidylglycine alpha-amidating monooxygenase (PAM). The subject invention also concerns methods for assaying for the enzyme peptidylglycine alpha-amidating monooxygenase and/or its substrates. The detection of glyoxylate is indicative of the presence of PAM and/or its substrates. The subject invention also concerns methods for screening for peptide hormones, amidated fatty acids, any N-acyl-glycine or N-aryl-glycine conjugated molecule, and generally compounds having a glycine reside in free acid form and attached to a carbonyl group.

Abstract: Expression systems are disclosed for the direct expression of peptide products into the culture media where genetically engineered host cells are grown. High yield was achieved with a special selection of hosts, and/or fermentation processes which include careful control of cell growth rate, and use of an inducer during growth phase. Special universal cloning vectors are provided for the preparation of expression vectors which include control regions having multiple promoters linked operably with coding regions encoding a signal peptide upstream from a coding region encoding the peptide of interest. Multiple transcription cassettes are also used to increase yield. The production of amidated peptides using the expression systems is also disclosed.

Abstract: Expression systems are disclosed for the direct expression of peptide products into the culture media where genetically engineered host cells are grown. High yield was achieved with a special selection of hosts, and/or fermentation processes which include careful control of cell growth rate, and use of an inducer during growth phase. Special universal cloning vectors are provided for the preparation of expression vectors which include control regions having multiple promoters linked operably with coding regions encoding a signal peptide upstream from a coding region encoding the peptide of interest. Multiple transcription cassettes are also used to increase yield. The production of amidated peptides using the expression systems is also disclosed.

Abstract: Expression systems are disclosed for the direct expression of peptide products into the culture media where genetically engineered host cells are grown. High yield was achieved with a special selection of hosts, and/or fermentation processes which include careful control of cell growth rate, and use of an inducer during growth phase. Special universal cloning vectors are provided for the preparation of expression vectors which include control regions having multiple promoters linked operably with coding regions encoding a signal peptide upstream from a coding region encoding the peptide of interest. Multiple transcription cassettes are also used to increase yield. The production of amidated peptides using the expression systems is also disclosed.

Abstract: Expression systems are disclosed for the direct expression of peptide products into the culture media where genetically engineered host cells are grown. High yield was achieved with a special selection of hosts, and/or fermentation processes which include careful control of cell growth rate, and use of an inducer during growth phase. Special universal cloning vectors are provided for the preparation of expression vectors which include control regions having multiple promoters linked operably with coding regions encoding a signal peptide upstream from a coding region encoding the peptide of interest. Multiple transcription cassettes are also used to increase yield. The production of amidated peptides using the expression systems is also disclosed.

Abstract: A method of inducing bone formation in a subject in need of such inducement comprises the steps of mechanically inducing an increase in osteoblast activity in the subject and elevating blood concentration of at least one bone anabolic agent in the subject. The method steps may be performed in any order, but in sufficient time proximity that the elevated concentration of the anabolic agent and the mechanically induced increase in osteoblast activity overlaps. The method may additionally comprise providing the subject with an elevated blood concentration of at least one antiresorptive agent, wherein the elevated concentration is sufficient to prevent resorption of new bone growth produced due to the osteoblast activity. Use of the method permits targeting of specific bones of the subject for bone production and preservation, faster bone production and earlier discontinuation of bone anabolic pharmaceuticals. Kits adapted for performing the method are provided.

Abstract: A method of inducing bone formation in a subject in need of such inducement comprises the steps of mechanically inducing an increase in osteoblast activity in the subject and elevating blood concentration of at least one bone anabolic agent in the subject. The method steps may be performed in any order, but in sufficient time proximity that the elevated concentration of the anabolic agent and the mechanically induced increase in osteoblast activity overlaps. The method may additionally comprise providing the subject with an elevated blood concentration of at least one antiresorptive agent, wherein the elevated concentration is sufficient to prevent resorption of new bone growth produced due to the osteoblast activity. Use of the method permits targeting of specific bones of the subject for bone production and preservation, faster bone production and earlier discontinuation of bone anabolic pharmaceuticals. Kits adapted for performing the method are provided.

Abstract: Methods for detecting and assaying for glyoxylate, include enzyme-based assays and/or assays for hydrogen peroxide following liberation of hydrogen peroxide from glyoxylate, are disclosed. In some embodiments, the invention is directed to methods for assaying for glyoxylate produced by the reaction of peptidylglycine alpha-amidating monooxygenase (PAM). The subject invention also concerns methods for assaying for the enzyme peptidylglycine alpha-amidating monooxygenase and/or its substrates. The detection of glyoxylate is indicative of the presence of PAM and/or its substrates. The subject invention also concerns methods for screening for peptide hormones, amidated fatty acids, any N-acyl-glycine or N-aryl-glycine conjugated molecule, and generally compounds having a glycine reside in free acid form and attached to a carbonyl group.

Abstract: Cell lines are provided for expressing peptidylglycine alpha-amidating monooxygenase (PAM), or one of its two catalytic domains. High levels of enzyme expression are achieved while utilizing a non-animal source, low protein tissue culture medium. A robust two-step downstream purification results in high enzyme purity. Resulting PAM, or its PHM catalytic domain, is used to catalyze the enzymatic conversion of X-Gly to X-alpha-hydroxy-Gly or X—NH2 (X being a peptide or any chemical compound having a carbonyl group to which a glycine group can be covalently attached). Methods of preparing preferred cell lines are also set forth.

Abstract: A bone implant device comprising a plurality of interconnected plate members, at least one plate member defining a plurality of apertures therein adapted for permitting bone growth there through from an interior portion of the bone in which the device is implanted for aiding in securing the device within the interior portion, and wherein the device is configured and adapted to minimize contact with an interior surface of the bone. The device is useful in applications including, but not limited to, repairing bone fractures, modeling bone in growing subjects, service as artificial joints and anchoring prostheses, such as an artificial limb, to a bone stub in the body of a subject.

Abstract: C-terminal amidated human parathyroid hormone analogs PTH 1-32-NH2 and PTH 1-33-NH2 are biologically active and can be used for the treatment of various bone related diseases and conditions.

Abstract: Conversion in vitro of X-Gly to X-alpha-hydroxy-Gly or X—NH2 (X being a peptide or any other compound having a carbonyl group capable of forming a covalent bond with glycine) is accomplished enzymatically in the presence of keto acids, or salts or esters thereof, to provide a good yield without the necessity of catalase or similar enzymatic reaction enhancers. Peptidylglycine ?-amidating monooxygenase (PAM) is a preferred enzyme for catalyzing the conversion. Alternatively, peptidylglycine ?-hydroxylating monooxygenase (PHM) is utilized to convert X-Gly to X-alpha-hydroxy-Gly which may be recovered, or optionally may be simultaneously or sequentially converted to an amide by either a Lewis base or action of the enzyme peptidyl ?-hydroxyglycine ?-amidating lyase (PAL). Both PHM and PAL are functional domains of PAM.

Abstract: A method of inducing bone formation in a subject in need of such inducement comprises the steps of mechanically inducing an increase in osteoblast activity in the subject and elevating blood concentration of at least one bone anabolic agent in the subject. The method steps may be performed in any order, but in sufficient time proximity that the elevated concentration of the anabolic agent and the mechanically induced increase in osteoblast activity overlaps. The method may additionally comprise providing the subject with an elevated blood concentration of at least one antiresorptive agent, wherein the elevated concentration is sufficient to prevent resorption of new bone growth produced due to the osteoblast activity. Use of the method permits targeting of specific bones of the subject for bone production and preservation, faster bone production and earlier discontinuation of bone anabolic pharmaceuticals. Kits adapted for performing the method are provided.

Abstract: Bioavailability of peptide active agents to be administered orally is enhanced by a pharmaceutical composition providing targeted release of the peptide to the intestine by combining the composition with an absorption enhancer. Bioavailability is further significantly increased by administering the composition in an acid-resistant protective vehicle which transports components of the invention through the stomach. The composition may optionally further include a sufficient amount of a pH-lowering agent to lower local intestinal pH. All components are released together into the intestine with the peptide.

Abstract: A method of inducing bone formation in a subject in need of such inducement comprises the steps of mechanically inducing an increase in osteoblast activity in the subject and elevating blood concentration of at least one bone anabolic agent in the subject. The method steps may be performed in any order, but in sufficient time proximity that the elevated concentration of the anabolic agent and the mechanically induced increase in osteoblast activity overlaps. The method may additionally comprise providing the subject with an elevated blood concentration of at least one antiresorptive agent, wherein the elevated concentration is sufficient to prevent resorption of new bone growth produced due to the osteoblast activity. Use of the method permits targeting of specific bones of the subject for bone production and preservation, faster bone production and earlier discontinuation of bone anabolic pharmaceuticals. Kits adapted for performing the method are provided.

Abstract: Expression systems are disclosed for the direct expression of peptide products into the culture media where genetically engineered host cells are grown. High yield was achieved with a special selection of hosts, and/or fermentation processes which include careful control of cell growth rate, and use of an inducer during growth phase. Special universal cloning vectors are provided for the preparation of expression vectors which include control regions having multiple promoters linked operably with coding regions encoding a signal peptide upstream from a coding region encoding the peptide of interest. Multiple transcription cassettes are also used to increase yield. The production of amidated peptides using the expression systems is also disclosed.

Abstract: C-terminal amidated human parathyroid hormone analogs PTH 1-32-NH2 and PTH 1-33-NH2 are biologically active and can be used for the treatment of various bone related diseases and conditions.