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Biomedical subjects

R K Campbell

Publications and source records attributed to R K Campbell.

At least 19 recordsLinked to original sources
PubMed1997

Influence of subunit interactions on lutropin specificity. Implications for studies of glycoprotein hormone function.

Bovine lutropin (bLH) and human chorionic gonadotropin (hCG) are heterodimeric glycoprotein hormones required for reproduction. Both bind rat LH receptors (rLHRs), but hCG binds human LH receptors (hLHRs) 1000-10,000 fold better than bLH. We tested the premise that this difference in affinity could be used to identify lutropin receptor contacts. Heterodimers containing hCG/bLH alpha- or beta-subunit chimeras that bound hLHR like hCG (or bLH) were expected to have hCG (or bLH) residues at the receptor contact sites. Analogs containing one subunit derived from hCG bound hLHR much more like hCG than bLH, indicating that each bLH subunit contains all the residues sufficient for high affinity hLHR binding. Indeed, the presence of bovine alpha-subunit residues increased the activities of some hCG analogs. The low hLHR activity of bLH was due primarily to an interaction between its alpha-subunit and beta-subunit residue Leu95. Leu95 does not appear to contact the hLHR since it did not influence the hLHR activity of heterodimers containing human alpha-subunit. These observations show that interactions within and between the subunits can significantly influence the activities of lutropins, thereby confounding efforts to identify ligand residues that contact these receptors.

Amino Acid Sequence↗
PubMed1997

Chimeric proteins can exceed the sum of their parts: implications for evolution and protein design.

Chimeric analogs derived from pairs of homologous proteins routinely exhibit activities found in one or both parents. We describe chimeras of two glycoprotein hormones, human chorionic gonadotropin (hCG) and human follitropin (hFSH), that exhibit activity unique to a third family member, human thyrotropin (hTSH). The results show that biological activity can be separated from hormone-specific amino acid residues. This is consistent with a model for the evolution of homologous ligand-receptor pairs involving gene duplication and the creation of inhibitory determinants that restrict binding. Disruption of these determinants can unmask activities characteristic of other members of a protein family. Combining portions of two ligands to create analogs with properties of a third family member can facilitate identifying key determinants of protein-protein interaction and may be a useful strategy for creating novel therapeutics. In the case of the glycoprotein hormones, this showed that two different hormone regions (i.e., the seat-belt and the intersubunit groove) appear to limit inappropriate contacts with receptors for other members of this family. These observations also have important caveats for chimera-based protein design because an unexpected gain of function may limit the therapeutic usefulness of some chimeras.

Amino Acid Sequence↗
PubMed1997

Insulin analogues: new agents for improving glycemic control.

For years, it has been recognized that "reasonable" control of blood glucose levels reduces the acute complications of diabetes mellitus. Recent studies have conclusively shown that strict glycemic control reduces the appearance and progression of chronic complications. Thus, strict glycemic control is the most salient goal of insulin therapy. Insulin regimens that closely mimic physiologic insulin secretory patterns have been inadequate because of limitations imposed by the pharmacokinetic profiles of commercially available preparations. The newer insulin analogues, both rapid-acting and long-acting, have more appropriate pharmacokinetic profiles. Lispro (Humalog) is the first human insulin analogue to be approved in the United States. Its pharmacokinetic and pharmacodynamic profile makes it amenable for use before meals. The search for long-acting and hepatospecific analogues continues.

Blood Glucose↗
PubMed1996

Acarbose: its role in the treatment of diabetes mellitus.

OBJECTIVES: To review the clinical pharmacology of acarbose, an alpha-glucosidase inhibitor, and to summarize its role in the pharmacotherapy of diabetes mellitus. DATA SOURCES: A MEDLINE search identified all relevant articles, including reviews; Bayer Pharmaceuticals. STUDY SELECTION: Due to the large number of clinical trials available, specific criteria were used to narrow the focus of this review: (1) randomized, double-blind, placebo-controlled, parallel-group study design; (2) a minimum of 25 patients enrolled per treatment arm; (3) a treatment duration of 90 days or more; and (4) adherence to Food and Drug Administration Good Clinical Practice guidelines. DATA EXTRACTION: All clinical trials that were available up to December 1995 were reviewed. Preliminary trials and unpublished reports were not reviewed. DATA SYNTHESIS: Acarbose is effective in reducing postprandial hyperglycemia. It does not stimulate endogenous insulin secretion and, therefore, will not cause hypoglycemia when used as monotherapy. The enhanced glycemic control achieved with acarbose is additive to that of sulfonylureas. It lowers postprandial serum glucose and insulin concentrations and does not promote weight gain. Acarbose can be used as first-line therapy with diet and exercise, or it can be used in combination with sulfonylureas to lower hemoglobin A1c concentrations an additional 0.5-0.9%. Acarbose is not a cure for diabetes, nor is it a substitute for diet, exercise, oral hypoglycemic agents, or insulin. Adverse effects are gastrointestinal and can be diminished by starting with an initial dosage of 25 mg tid. Depending on patient response, the dosage can be increased up to a maximum of 100 mg tid over time. CONCLUSIONS: Acarbose, through its unique mechanism of action, appears to be a safe and effective adjunctive agent to diet/exercise therapy or sulfonylurea therapy for treatment of non-insulin-dependent diabetes mellitus.

Acarbose↗
PubMed1996

Insulin lispro: its role in the treatment of diabetes mellitus.

OBJECTIVE: To introduce a rapid-acting human insulin analog, insulin lispro; to review its pharmacology, therapeutics, pharmacokinetics, dosing guidelines, adverse effects, and drug interactions; and to summarize the clinical trials of its efficacy and safety alone and in comparison with regular human insulin in the treatment of diabetes mellitus. DATA SOURCES: A MEDLINE database search was completed to identify all relevant articles, including reviews; Eli Lilly and Co.; published articles and abstracts; and review chapters from medical textbooks. STUDY SELECTION: Due to the relatively few citations listed in MEDLINE (12 as of December 1995), most of the studies reported were found from abstracts summarizing the clinical action, adverse effects, or pharmacokinetics of insulin lispro in healthy volunteers or patients with diabetes mellitus. A few of the studies used patients with diabetes mellitus in multicenter, randomized, crossover trials of insulin lispro. DATA EXTRACTION: All clinical trials that were available prior to submission of this manuscript for publication, including unpublished reports, were reviewed. DATA SYNTHESIS: The human insulin analog, insulin lispro, which is biosynthetically made by inverting the amino acid sequence of human insulin at B-28 and B-29, is more effective than regular human insulin in improving postprandial glucose control. Subcutaneous injections of insulin lispro result in decreased blood glucose peaks following meals and a potential decreased risk of hypoglycemic episodes, including nighttime hypoglycemia in patients with type 1 diabetes. Insulin lispro in comparison with regular human insulin provides equal or slightly better blood glucose control. When compared with subcutaneous injections of regular human insulin, the peak serum insulin concentration of insulin lispro is three times higher, time to peak is 4.2 times faster, the absorption rate constant is double, and the duration of action is half as long. Insulin lispro is similar to regular human insulin with reference to dose, toxicity, adverse effects, drug interactions, and immunogenicity. When insulin lispro is mixed with human NPH (isophane) or Lente insulins, insulin lispro should be drawn into the syringe first, mixed with the long-acting insulin, and injected immediately after mixing. Patients using insulin lispro perceive an improvement in their well-being and quality of life due to flexible injection times and less frequent hypoglycemic reactions. Insulin lispro is believed to be suitable for patients using insulin infusion pumps. CONCLUSIONS: Insulin lispro is equipotent to human insulin and has a much more rapid onset and shorter duration of action than human insulin does, which may reduce the risk of hypoglycemia. In addition, insulin lispro improves the dosing convenience for patients with diabetes and provides a more natural control of blood glucose concentrations. Insulin lispro is a useful new agent in the treatment of diabetes mellitus.

Blood Glucose↗
PubMed1995

The groove between the alpha- and beta-subunits of hormones with lutropin (LH) activity appears to contact the LH receptor, and its conformation is changed during hormone binding.

Gonadotropins are heterodimeric glycoprotein hormones that control vertebrate fertility through their actions on gonadal lutropin (luteinizing hormone, LH) and follitropin (follicle-stimulating hormone, FSH) receptors. The beta-subunits of these hormones control receptor binding specificity; however, the region of the beta-subunit that contacts the receptor has not been identified. By a process of elimination we show this contact to be the portions of beta-subunit loops one and three found in a hormone groove created by the juxtaposition of the alpha- and beta-subunits. Most other regions of the beta-subunit can be recognized by antibodies that bind to human chorionic hormone (hCG)-receptor complexes or replaced without disrupting hormone function. Using a series of bovine LH/hCG and human FSH/hCG beta-subunit chimeras we identified key hCG beta-subunit residues in the epitopes of two antibodies that bind to hCG-receptor complexes. These epitopes include the surfaces of beta-subunit loops one and three near residue 74 on the outside of the hormone groove and parts of the C-terminal end of the "seat belt" that holds the two subunits together. The antibody that recognized residue 74 bound to receptor complexes containing most mammalian lutropins better than to the free hormones, an indication that the outside surface of the beta-subunit groove is altered during hormone binding. This region of the beta-subunit is furthest from the alpha-subunit and is recognized equally well in the free beta-subunit and in the heterodimer. Thus, the receptor associated increase in antibody binding appears due to an interaction of this portion of the beta-subunit with the receptor and not to an effect of the receptor on the relative positions of the alpha- and beta-subunits. Unlike most previous studies designed to identify portions of the beta-subunit likely to contact the LH receptor, this indirect approach provides data that are more easily interpreted because it does not rely on the use of mutations that disrupt hormone function. The approach described here should be valuable for studying the receptor interactions of other complex ligands.

Amino Acid Sequence↗
PubMed1995

Model of human chorionic gonadotropin and lutropin receptor interaction that explains signal transduction of the glycoprotein hormones.

The goal of these studies was to devise a model that explains how human chorionic gonadotropin (hCG) interacts with lutropin (LH) receptors to elicit a hormone signal. Here we show that alpha-subunit residues near the N terminus, the exposed surface of the cysteine knot, and portions of the first and third loops most distant from the beta-subunit interface were recognized by antibodies that bound to hCG-receptor complexes. These observations were combined with similar data obtained for the beta-subunit (Cosowsky, L., Rao, S.N.V., Macdonald, G.J., Papkoff, H., Campbell, R.K., and Moyle, W.R. (1995) J. Biol. Chem. 270, 20011-20019), information on residues of hCG that can be changed without disrupting hormone function, the crystal structure of deglycosylated hCG, and the crystal structure of a leucine-repeat protein to devise a model of hCG-receptor interaction. This model suggest that the extracellular domain of the LH receptor is "U-" or "J"-shaped and makes several contacts with the transmembrane domain. High affinity hormone binding results from interactions between residues in the curved portion of the extracellular domain of the receptor and the groove in the hormone formed by the apposition of the second alpha-subunit loop and the first and third beta-subunit loops. Most of the remainder of the hormone is found in the large space between the arms of the extracellular domain and makes few, if any, additional specific contacts with the receptor needed for high affinity binding. Signal transduction is caused by steric or other influences of the hormone on the distance between the arms of the extracellular domain, an effect augmented by the oligosaccharides. Because the extracellular domain is coupled at multiple sites to the transmembrane domain, the change in conformation of the extracellular domain is relayed to the transmembrane domain and subsequently to the cytoplasmic surface of the plasma membrane. While the model does not require the hormone to contact the transmembrane domain to initiate signal transduction, small portions of both subunits may be near the transmembrane domain and assist in initiating the hormonal signal. This is the first model that is consistent with all known information on the activity of the gonadotropins including the amounts of the hormone that are exposed in the hormone-receptor complex, the apparent lack of specific contacts between much of the hormone and the receptor, and the roles of the oligosaccharides in signal transduction.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Co-evolution of ligand-receptor pairs.

Specific receptors for lutropin (luteinizing hormone; LH) and follitropin (follicle-stimulating hormone; FSH) mediate the actions of human chorionic gonadotropin (hCG) and FSH5 on the gonads. Here we report that short independent sequences of the beta-subunit enable hCG to distinguish between the receptors for FSH and LH. Residues between the 11th and 12th cysteines restrict FSH receptor binding; residues between the 10th and 11th cysteines and, to a much lesser extent, residues carboxy-terminal to the 12th cysteine also affect LH receptor binding. CF101-109, an hCG analogue containing hFSH beta residues between the 11th and 12th cysteines, had high affinity for both LH and FSH receptors. Modifications to CF101-109 that reduce binding to either LH or FSH receptors yield gonadotropin analogues having differing ratios of LH:FSH activity. Ligand-binding specificity of the LH receptor is determined by residues encoded by parts of exons 2-4 and 7-9 which prevent hFSH binding but have little effect on hCG binding. FSH receptor specificity is controlled primarily by residues encoded by exons 5 and 6 that prevent hCG binding but have little effect on hFSH binding. These determinants can be interchanged to create receptor analogues that bind hCG and hFSH. Our observations support a model in which distinct negative determinants restrict ligand-receptor interaction. This explains coevolution of binding specificity in families of homologous ligands and their receptors. Natural or designed manipulation of these determinants leads to the 'evolution' of new, specific protein-protein interactions.

Amino Acid Sequence↗
PubMed1993

Drug interactions in diabetic patients. The risk of losing glycemic control.

Any number of prescription and over-the-counter medications, including diuretics and salicylates, can affect glycemic control in diabetic patients. In addition, patients being treated with either insulin or sulfonylurea risk hypoglycemic coma or death if they ingest large quantities of alcohol. The authors of this article discuss the medications most likely to be a problem and provide lists of agents associated with hypoglycemia and hyperglycemia.

Alcohol Drinking↗
PubMed1993

Magnesium and diabetes: a review.

OBJECTIVE: To discuss the potential link between diabetes mellitus (DM) and hypomagnesemia, the methods used to assess magnesium status, and the potential benefits of magnesium repletion in hypomagnesemic patients with DM. DATA SOURCES: A MEDLINE search (key terms: magnesium and diabetes) was conducted to identify pertinent literature. STUDY SELECTION: All major clinical trials and most published case reports were reviewed. SYNTHESIS: Several studies have demonstrated a higher than expected frequency of magnesium deficiency in patients with DM. Hypomagnesemia may play a role in the development of retinopathy, altered glucose disposition, hypertension, abnormal platelet function, and other problems frequently observed in patients with DM. The lack of a widely available, accurate screening methodology is one of the main problems in assessing total body magnesium status. One study has suggested that hypomagnesemia in patients with DM may be related to enhanced urinary loss of magnesium. Several studies evaluating hypomagnesemia and glucose disposal have suggested a direct correlation between magnesium concentration and glucose disposal, with an improvement in glucose disposal with magnesium supplementation. It has been suggested that there is a relationship between hypomagnesemia and diabetic retinopathy; however, the effect of magnesium supplementation on the development of diabetic retinopathy has not been evaluated. Researchers evaluating the effect of magnesium on platelet aggregation have suggested that magnesium supplementation may reduce the incidence of vascular disease in hypomagnesemic patients with DM. Several studies have demonstrated a correlation between hypomagnesemia and hypertension. CONCLUSIONS: Studies have suggested a link between hypomagnesemia and hyperglycemia, as well as an association between hypomagnesemia and the complications of DM. The American Diabetes Association has published a consensus statement suggesting that patients who have documented hypomagnesemia and DM receive magnesium supplementation.

Blood Platelets↗
PubMed1993

Clinical update on pentoxifylline therapy for diabetes-induced peripheral vascular disease.

OBJECTIVE: To introduce readers to the use of pentoxifylline for diabetes-induced peripheral vascular disease. The article provides background on the pathophysiology of diabetic foot ulcers as well as a review of the literature on the therapeutic use of pentoxifylline for treating this disorder. DATA SOURCES: A MEDLINE search was used to identify pertinent literature, including review articles and case reports. Key index terms included pentoxifylline, diabetic foot ulcer, neuropathy, peripheral vascular disease, and intermittent claudication DATA EXTRACTION: Basic pharmacologic data regarding absorption, distribution, metabolism, and excretion were reported in normal subjects as well as in patients with renal impairment. Open and controlled clinical trials also were analyzed; subjective symptoms were reported. The economic implications also were reported. The pharmacist's role in patient education is discussed. DATA SYNTHESIS: Pentoxifylline 800 mg/d was found to be effective in improving the symptoms in patients with noninsulin-dependent diabetes mellitus, including improvement in walking distance, paresthesia, skin temperature, and subjective overall response. In nondiabetic patients, statistically significant differences in leg-ulcer healing were found between the treatment and placebo groups. Case reports illustrated healing times, which ranged from two weeks to six months. Pentoxifylline use in both insulin-dependent and noninsulin-dependent patients was assessed in clinical trials, with improvement of symptoms in both patient types. CONCLUSIONS: Studies show that pentoxifylline is an alternative to vascular surgery in the management of peripheral vascular disease in diabetic patients, particularly in those with chronic ulceration of the lower extremities that does not heal despite other optimal treatment regimens, including cessation of smoking, maintenance of normoglycemia, elimination of vasoconstrictive drugs, correctly fitted shoes, and appropriate would care. Pentoxifylline therapy may provide an appropriate, economical treatment modality by reducing the need for hospitalization and vascular surgery.

Clinical Trials as Topic↗
PubMed1992

Assembly and expression of a synthetic gene encoding the bovine glycoprotein hormone alpha-subunit.

The glycoprotein hormones are a family of alpha beta heterodimeric proteins which are responsible for gonadal and thyroid function. In previous studies we employed chimeric glycoprotein hormone beta-subunits to identify amino acid residues critical for binding to receptors and antibodies. To facilitate similar studies of the alpha-subunit of these hormones, we assembled a 406 bp synthetic gene which encodes the human alpha-subunit leader sequence and the secreted portion of the bovine alpha-subunit. It contains unique restriction sites that can be used for cassette mutagenesis or for making human/bovine alpha-subunit chimeras. The gene was assembled from eight long oligodeoxynucleotides in a single ligation and its structure verified by DNA sequencing. Co-transfection of COS-7 cells with the synthetic gene and the cDNA for human chorionic gonadotropin (hCG) beta-subunit resulted in the secretion of a functional alpha beta heterodimer which bound to luteinizing hormone receptors. The protein was recognized by several monoclonal antibodies including B109, an antibody to a conformational epitope which binds hCG but not the free bovine alpha-, human alpha-, or hCG beta-subunits. This suggests that the binding site for B109 may be formed by residues located primarily within the hCG beta-subunit and that formation of this epitope requires a change in conformation of the beta-subunit when it combines with the alpha-subunit.

Amino Acid Sequence↗
PubMed1992

Hypoglycemic drugs.

There are an estimated 14 million individuals in the United States with diabetes mellitus. These individuals suffer a very high incidence of podiatric complications. This article focuses on the pharmacologic agents used in the management of hyperglycemia in the patient with diabetes mellitus, including insulin and sulfonylureas.

Diabetes Mellitus, Type 1↗
PubMed1991

Conversion of human choriogonadotropin into a follitropin by protein engineering.

Human reproduction is dependent upon the actions of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the alpha subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their beta subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG beta-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLH beta and hFSH beta in receptor recognition and activation. Since the beta subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, we postulated that these residues might contribute to hormone specificity. To test this hypothesis we constructed chimeric hCG/hFSH beta subunits, coexpressed them with the human alpha subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSH beta residues 33-52 for hCG beta residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSH beta residues 88-108 in place of the carboxyl terminus of hCG beta (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayed by this analog is not attributable solely to the replacement of hCG beta residues 108-145 or substitution of residues in the "determinant loop" located between hCG beta residues 93 and 100.

Amino Acid Sequence↗
PubMed1991

Stability and sterility of biosynthetic human insulin stored in plastic insulin syringes for 28 days.

The stability and sterility of biosynthetic human insulin products stored at refrigerator and room temperatures in two types of plastic syringes and the stability of preservatives in the products were studied. Four types of biosynthetic human insulin were used: regular, isophane, combination, and extemporaneously prepared combination. Samples (0.4 mL) were withdrawn from multiple-dose vials into 39 polypropylene and 39 propylene-ethylene copolymer syringes. Three syringes of each type were analyzed immediately; the remaining syringes were stored in plastic bags, half at room temperature (23 degrees C) and half in the refrigerator (4 degrees C). A vial of each type of insulin was maintained under similar conditions. At days 1, 3, 7, 14, 21, and 28, samples from each syringe were analyzed by high-performance liquid chromatography for insulin potency and m-cresol and phenol concentrations. Samples of each product were also tested for sterility after 1, 2, and 4 weeks of storage at 4 degrees C and 23 degrees C. The potency of insulin in each of the biosynthetic human insulin products did not change significantly during the 28-day study in both types of plastic syringes and at both temperature settings. m-Cresol concentrations decreased in all samples; greater decreases occurred in samples stored at room temperature and in samples stored in polypropylene syringes. Phenol concentrations were less affected than m-cresol concentrations; greater decreases occurred in samples stored at room temperature. No significant decreases in insulin potency or m-cresol or phenol concentrations occurred in control samples stored in vials kept under similar conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug Stability↗
PubMed1990

Localization of residues that confer antibody binding specificity using human chorionic gonadotropin/luteinizing hormone beta subunit chimeras and mutants.

The glycoprotein hormones are a family of conserved heterodimeric proteins which share a common alpha subunit but differ in their hormone-specific beta subunits. We used chimeras of human chorionic gonadotropin (hCG) and luteinizing hormone (hLH) beta subunits to identify residues which enable monoclonal antibodies (mAb) to distinguish the two hormones. The LH beta-CG beta chimeras appeared to fold similar to hCG beta, since they combined with hCG alpha and, depending on their sequences, were recognized by hCG-selective mAbs. Amino acid residues Arg8-Arg10,Gly47-Ala51, and Gln89-Leu92 form a major epitope region and appear to be adjacent to each other on the surface of hCG beta. Gly47-Ala51 and Gln89-Leu92 are recognized by dimer-specific mAbs while Arg8-Arg10 is recognized by mAbs which have highest affinity for the free beta subunit. These observations suggest that the conformation of this region of the beta subunit changes when the alpha and beta subunits combine. Residues which are C-terminal of Asp112 form a second epitope domain. mAbs to the third domain distinguish hCG beta and hLH beta by the presence of Asn77 in hCG beta and can be detected after hCG binds to receptors. These findings were used to develop a model of hCG beta which predicts the locations of these residues and their positions relative to the alpha subunit and receptor interfaces.

Amino Acid Sequence↗