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Biomedical subjects

B E Kemp

Publications and source records attributed to B E Kemp.

At least 145 records · Page 8Linked to original sources
PubMed1989

A synthetic peptide analog of the putative substrate-binding motif activates protein kinase C.

A 29-residue synthetic peptide, Leu530-Leu-Tyr-Glu-Met-Leu-Ala-Gly-Gln-Ala-Pro-Phe-Glu-Gly-Glu-Asp -Glu-Asp- Glu-Leu-Phe-Gln-Ser-Ile-Met-Glu-His-Asn-Val-NH2(558), corresponding to part of the catalytic domain of protein kinase C, is a potent activator of the enzyme, with a Ka of approx. 10 microM. Activation was 59 +/- 4% of that observed with phosphatidylserine, predominantly due to an increased Vmax, partially calcium-dependent, observed with all three isoenzymes (alpha, beta, gamma), and resulted in autophosphorylation. It is proposed that the region between Gly528 and Arg583 is part of the protein substrate binding region of protein kinase C and synthetic peptide analogs of this region activate the enzyme by blocking the action of the enzyme's basic pseudosubstrate autoregulatory region.

Animals↗
PubMed1989

Effects of modulators of myosin light-chain kinase activity in single smooth muscle cells.

Phosphorylation of myosin light chains by a calmodulin-myosin light-chain kinase (MLCK) pathway is considered to be responsible for coupling increased calcium concentration with contraction in smooth muscle. This simple view has, however, recently been questioned. To test this hypothesis directly, we microinjected individual smooth muscle cells with modulators of the MLCK pathway while measuring contraction and calcium-ion concentration. Injection of a constitutively active proteolyzed form of MLCK causes contraction but no change in calcium concentration. By contrast, injection of peptide inhibitors of MLCK blocks contraction in response to K+ depolarization, despite the fact that the change in calcium concentration in response to stimulation was enhanced over controls. These results provide a direct demonstration at the level of a single cell that activation of the calmodulin-MLCK pathway is both necessary and sufficient to trigger contraction of smooth muscle.

Animals↗
PubMed1989

Parathyroid hormone-related protein: immunohistochemical localization in cancers and in normal skin.

An immunoperoxidase method has been developed to detect parathyroid hormone-related protein (PTHrP) in histological specimens of tumors and of normal skin. A rabbit polyclonal antiserum against PTHrP-(1-16) was used that did not cross-react with PTH-(1-34) either under radioimmunoassay conditions or at the high antiserum concentrations used in neutralizing biologic activity. PTHrP antigen was detected in the keratinocyte layer of normal skin and in 100% of 34 samples of squamous cell cancers but in only one of six breast cancers, and none of 15 other adenocarcinomata. It was also detected in four of four samples of renal cortical carcinoma and two of two of melanoma, both of which can be associated with hypercalcemia, and three of three small cell carcinomata of the lung. Immunologic detection of PTHrP could be useful in the diagnosis of tumors of squamous cell origin, particularly in the cytological differentiation of lung cancers, where it may be of value in distinguishing between squamous cell and small cell carcinoma on the one hand and poorly differentiated adenocarcinoma on the other.

Humans↗
PubMed1989

Expression of parathyroid hormone related protein in normal skin and in tumours of skin and skin appendages.

Parathyroid hormone related protein (PTHrP) has been demonstrated in the tumour cells of squamous cell carcinomas originating in a variety of organs, in undifferentiated small cell tumours of the bronchus, and in carcinoma of the kidney. The protein hormone is thought to produce the hypercalcaemia which may complicate some of these malignancies. By using an antibody raised in rabbits against the N-terminal portion of the molecule, the polypeptide may be demonstrated in cells of the prickle cell layer of normal skin and in the cells of hair follicles. Skin tumours showing squamous or hair follicle differentiation are shown to contain the protein antigen, while basal cell carcinomas and tumours with sweat gland differentiation do not. The hormone may be primordial in origin and the progenitor of parathyroid hormone.

Carcinoma, Squamous Cell↗
PubMed1989

Comparison of the anabolic effects of synthetic parathyroid hormone-related protein (PTHrP) 1-34 and PTH 1-34 on bone in rats.

The objective of this study was to determine whether intermittent synthetic human PTH-related protein (PTHrP 1-34) will mimic the anabolic effect of PTH and increase bone mass in rats. Dose response experiments were done on young, male Sprague-Dawley rats given sc vehicle, human (h) PTH (1-34) at 8 micrograms/100 g or PTHrP (1-34) at 1-32 micrograms/100 g daily for 12 days or 26 days. On the last day, 3 h after injections, rats were killed and serum, femurs, and tibias harvested. Trabecular and cortical bone of distal half femurs were analyzed for calcium (Ca) and hydroxyproline content and dry weight. Tibia metaphyseal bone was analyzed using conventional histomorphometry techniques. Our results showed that low doses of PTHrP (1-34) did not increase bone mass or bone forming surfaces. After 12 days, PTH, at 8 micrograms/100 g, increased trabecular Ca, dry weight, and hydroxyproline by approximately 19%, 36%, and 53%, respectively, while the bone mass of PTHrP-treated rats was comparable to vehicle-treated rats. PTHrP at a higher dose of 32 micrograms/100 g, increased trabecular bone mass by 30-37%, compared to the 43-48% increase induced by PTH at 8 micrograms/100 g after 12 days. When treatment was extended to 26 days, PTHrP, at 16 micrograms/100 g, increased trabecular bone mass by 24-36%, respectively, compared to the 43-61% increase induced by PTH at 8 micrograms/100 g. Unlike PTH, which increased cortical bone mass by 15-25%, PTHrP increased cortical bone mass only at the highest dose tested, 32 micrograms/100 g. Bone forming surfaces but not bone apposition rate were increased by PTH and PTHrP while resorption measures remained comparable to control values. Although serum Ca and Pi remained in the physiological range for all rats, the values for PTHrP-treated rats were consistently higher. In conclusion, PTHrP (1-34) was less potent and less effective than PTH (1-34) in inducing an anabolic response in bone in vivo.

Animals↗
PubMed1989

Human monoclonal antibodies to HIV-1: cross-reactions with gag and env products.

Human monoclonal antibodies were raised by in vitro immunization of normal human spleen cells with denatured HIV-1 and subsequent fusion to an Epstein-Barr virus (EBV) transformed cell line. Three monoclonals reacted with both gag-encoded p24 core protein and env-encoded gp41 transmembrane glycoprotein. The cross-reaction was confirmed by reactivity with p24- and gp41-derived recombinant peptides. The peptides share two amino acid sequences which may be the basis for the cross-reaction. Attempted epitope mapping using synthetic peptides was unsuccessful due to non-specific reactivity with the short linear peptides.

Antibodies, Monoclonal↗
PubMed1988

Autologous red cell agglutination assay for HIV-1 antibodies: simplified test with whole blood.

An antibody detection procedure based on agglutination of autologous red cells has been developed for samples of whole blood. A nonagglutinating monoclonal antibody to human red blood cells conjugated to a synthetic peptide antigen (in this case residues 579 to 601 of the HIV-1 envelope precursor, Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp- Gly-Cys - Ser-Gly-Lys) permitted the detection of antibodies to the human immunodeficiency virus type 1 (HIV-1) in 10 microliters of whole blood within 2 minutes. Agglutination was specifically inhibited by addition of synthetic peptide antigen but not by unrelated peptides. The frequency of false positive results was 0.1% with HIV-1 seronegative blood donors (n = 874). The false negative results were approximately 1% (n = 81). The autologous red cell agglutination test is potentially suitable for simple, rapid, qualitative screening for antibodies to a variety of antigens of medical and veterinary diagnostic significance.

Agglutination Tests↗
PubMed1988

Autoregulation of enzymes by pseudosubstrate prototopes: myosin light chain kinase.

The myosin light chain kinase requires calmodulin for activation. Tryptic cleavage of the enzyme generates an inactive 64-kilodalton (kD) fragment that can be further cleaved to form a constitutively active, calmodulin-independent, 61-kD fragment. Microsequencing and amino acid analysis of purified peptides after proteolysis of the 61- and 64-kD fragments were used to determine the amino-terminal and carboxyl-terminal sequences of the 64-kD fragment. Cleavage within the calmodulin-binding region at Arg505 generates the catalytically inactive 64-kD fragment, which is incapable of binding calmodulin. Further digestion removes a carboxyl-terminal fragment, including the pseudosubstrate sequence Ser484-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met- Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg505 and results in a calmodulin-independent 61-kD fragment. Both the 61- and 64-kD fragments have the same primary amino-terminal sequences. These results provide direct support for the concept that the pseudosubstrate structure binds the active site and that the role of calmodulin is to modulate this interaction. Pseudosubstrates may be utilized in analogous ways by other allosterically regulated enzymes.

Amino Acid Sequence↗
PubMed1988

Solubilization of functional calcitonin receptors.

Calcitonin (CT) binding activity has been extracted from a membrane fraction of human placenta using the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (Chaps). Approximately two-thirds of the available binding sites were extracted using 5 mM-Chaps. The binding characteristics of 125I-labelled salmon CT(125I-sCT) to the solubilized extract were similar to those obtained previously with placental membranes and other targets such as osteoclasts, renal cells and certain human cancer cell lines. 125I-sCT binding was saturable (Bmax. 75 +/- 6 fmol/mg of protein, n = 3) and Scatchard analysis revealed a single class of high-affinity binding sites (Kd 165 +/- 28 pM, n = 3). In competitive-binding studies, various species-specific CTs and CT analogues showed the same rank order of potencies as seen in CT bioassays and several unrelated peptides did not compete at high doses. A biologically active CT analogue, [Arg11,18, Lys14]sCT, derivatized with the photoreactive phenylazide cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate, was used to identify receptor components of Mr approximately 88,000 and approximately 71,000 in both particulate placental membranes and the solubilized extract. Receptor components of Mr 85-90,000 have been identified in other CT target cells previously using chemical- and photoaffinity-labelling techniques. These results demonstrate the first successful solubilization of the CT receptor in a form which purification.

Azides↗
PubMed1988

Recognition of envelope and tat protein synthetic peptide analogs by HIV positive sera or plasma.

A series of synthetic peptides corresponding to segments of HIV encoded proteins were selected using criteria described by Welling et al. [(1985) FEBS Lett. 188, 215]. Synthetic peptide analogs to gp120 (2-13), (55-65), gp41 (582-596) (659-670) and tatIII (71-83) were recognized by 41-67% of sera or plasma from individuals known to be infected with HIV on the basis of virus isolation or Western blot screening. The peptide which reacted with most sera or plasma was gp41 (582-596), a conserved region in the transmembrane glycoprotein. An extended peptide analog, gp41 (579-599), tested against the same samples showed almost 100% reactivity, confirming independent studies identifying a highly immunodominant region of gp41. There was an unexpected high prevalence of antibodies (25%) to the tatIII peptide.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains.

Regulatory domains of the multifunctional Ca2+/calmodulin-dependent protein kinase II were investigated utilizing synthetic peptides. These peptides were derived from the sequence between positions 281 and 319 as translated from the cDNA sequence of the rat brain 50-kDa subunit (Lin, C. R., Kapiloff, M. S., Durgerian, S., Tatemoto, K., Russo, A. F., Hanson, P., Schulman, H., and Rosenfeld, M. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5962-5966), which contain the putative calmodulin-binding region as well as potential autophosphorylation sites. Peptide 290 to 309 was found to be a potent calmodulin antagonist with an IC50 of 52 nM for inhibition of Ca2+/calmodulin-dependent protein kinase II. Neither truncation from the amino terminus (peptide 296-309) nor extension in the carboxyl-terminal direction (peptide 294-319) markedly affected calmodulin binding, whereas shortening the peptide from the carboxyl terminus (peptide 290-302) or from both ends (peptide 295-304) resulted in the elimination of this activity. Peptide 281-290 did not bind calmodulin, but was a good substrate for the enzyme, being phosphorylated at Thr-286. Several of the peptides inhibited the kinase in a partially competitive, substrate-directed manner, but were not themselves phosphorylated. These studies identify domains within Ca2+/calmodulin-dependent protein kinase II which may be involved in 1) inhibition of the kinase in the absence of calmodulin, 2) binding of calmodulin, and 3) the resulting activation. Additionally, it is suggested that phosphorylation of residues flanking these domains may be responsible for the known regulatory effects of autophosphorylation on the properties of the kinase.

Amino Acid Sequence↗
PubMed1988

Mapping of calmodulin-binding domain of Ca2+/calmodulin-dependent protein kinase II from rat brain.

Recent molecular cloning experiments have identified a 25 amino-acid region as the calmodulin-binding domain of the alpha-subunit of rat brain Ca2+/calmodulin-dependent multifunctional protein kinase II (CaM-K II). Synthetic peptides, derived from the deduced amino-acid sequence encompassing this region, were examined for their ability to bind calmodulin in a calcium dependent manner and to inhibit the Ca2+/calmodulin-dependent autophosphorylation of CaM-K II. Comparison of these structure-function relationships highlighted a region of 5 amino-acids, which was essential for calmodulin interaction and inhibition of kinase activity. This region demonstrated some homology with other calmodulin-binding peptides, and may represent a key site of interaction of the kinase with calmodulin. These analyses provide additional insight into the molecular mechanism underlying the Ca2+ regulation of CaM-K II.

Amino Acid Sequence↗
PubMed1988

Human placental calcitonin receptors.

Receptors for the hypocalcaemic hormone, calcitonin (CT), have been identified in a membrane fraction prepared from term human placentae. Binding of 125I-labelled salmon CT (125I-sCT) to the membranes was time- and temperature-dependent, saturable (Bmax. 58 +/- 11 fmol/mg of protein), of high affinity (Kd 80 +/- 21 pM) and poorly reversible. Species-specific CTs and CT analogues competed for 125I-sCT binding with potencies proportional to their known biological potencies. Various unrelated peptide hormones did not compete, indicating that receptor binding was specific for CT. Photoaffinity labelling using a derivatized biologically active sCT analogue, [Arg11,18,3-nitrophenylazide-Lys14]sCT, identified a receptor component of Mr approximately 85,000, comparable with findings in osteoclasts and other target cells. The presence of CT receptors in the human placenta supports other evidence that CT may have a role in the regulation of placental function.

Adenylyl Cyclases↗
PubMed1988

Hydroxyamino acid specificity of smooth muscle myosin light chain kinase.

Synthetic peptides corresponding to the phosphorylation site in the myosin regulatory light chain from smooth muscle, Lys-Lys-Arg-Ala-Arg-Ala-Thr-Ser-Asn-Val-Phe-Ala ([Ala14,15]MLC(11-23] and containing a variety of hydroxyamino acid analogs at position 19, were tested as substrates for the smooth muscle myosin light chain kinase. Peptide analogs containing either D-serine or cis-hydroxyproline were not phosphorylated. The corresponding trans-hydroxyproline containing peptide was poorly phosphorylated with a Km of 2.3 microM and a Vmax of 3 X 10(-3) mumol.min-1.mg-1 compared to a Km of 12.5 microM and a Vmax of 1.43 mumol.min-1.mg-1 for the parent peptide. All three hydroxyamino acid analog peptides acted as relatively potent inhibitors of myosin light chain phosphorylation with Ki values in the range 7.5-10 microM, comparable to 7 microM for the parent peptide. Thus the failure of the hydroxyamino acid analog peptides to act as effective substrates was not the result of poor binding to the enzyme. In contrast, the same substitutions made in the peptide substrate for the cAMP-dependent protein kinase resulted in poor inhibitors. It is likely that the hydroxyl group of the substituting amino acids in the myosin light chain peptide analogs is not presented in the correct orientation in the active site for transfer of the phosphate group.

Amino Acid Sequence↗
PubMed1988

Biologically active, derivatizable salmon calcitonin analog: design, synthesis, and applications.

An analog of salmon calcitonin (sCT) has been synthesized, substituting Arg at positions 11 and 18 and Lys at position 14 to provide a free amino group for derivatization. The potency of [Arg11,18,Lys14]sCT was equivalent to that of sCT in activating adenylate cyclase in UMR 106-06 cells. The analog was derivatized with biotin, fluorescein, or 4-azidobenzoate without loss of activity. The derivatized analog was not degraded by lysine-specific endoprotease, whereas the underivatized [Arg11,18,Lys14]sCT was cleaved at Lys-14. The derivatized analogs were purified by HPLC and subsequently shown to possess full biological activity. The photoactive analog was used to photolabel 88,000 and 71,000 mol wt components of the calcitonin receptor in rat osteoclasts, but only an 88,000 mol wt component was photolabeled in the UMR 106-06 cells.

Adenylyl Cyclases↗