Growth factors in melanoma

Human melanoma cells in culture are the source of a wide variety of polypeptide growth factors. Melanoma-derived basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-A and PDGF-B chains, transforming growth factor (TGF)- and TGF-, interleukin (IL)-1 and IL-1, and melanoma growth stimulatory activity (MGSA) have similar biochemical and functional properties when compared to their counterparts produced by untransformed cells. In contrast to melanoma cells, normal melanocytes, even under optimal growth conditions, express only TGF-1 and MGSA at detectable levels suggesting that production of the other growth factors is a tumor-associated phenomenon. Recent evidence suggests that at least two of the growth factors, bFGF and MGSA, contribute to autocrine growth stimulation of melanoma cells. Whether PDGF, TGF-, IL-1, and TGF- act in an autocrine mode is unclear at present. However, these four growth factors are among those secreted by melanoma cells and, therefore, can be expected to interact with normal cells of the tumor stroma in vivo. Such paracrine effects include not only growth modulation in the context of angiogenesis and stroma formation, but also tissue degradation by proteolytic enzymes, the modification of extracellular matrix composition, and expression of adhesion receptors.     

Structure,function and biological properties of integrin αvβ3on human melanoma cells

Summary Human melanoma represents one of the most metastatic cancers in man. The capacity of melanoma cells to invade a variety of tissues and extracellular matrices is, in part, due to their repertoire of adhesion receptors. To this end, human melanoma cells express multiple integrin cell adhesion receptors among these is the vitronectin receptor, _v3. This adhesion receptor enables melanoma cells to attach to a wide variety of extracellular matrix components containing the sequence Arg-Gly-Asp. This review will focus on the biosynthetic, biochemical and biological properties of this receptor expressed on the surface of human melanoma cells.     

In vivo evaluation of cell mediated immunity against human melanoma

Evaluation of cell mediated immunity against human melanoma target cells was performed in an in vivo model using human tumor xenografts growing in Balb/c athymic mice. Intraperitoneal inoculation of 1 × 10⁷ human melanoma cells produced peritoneal carcinomatosis which lead to death of the animals at 23.8 ± 2.6 days (N = 12). Peripheral blood lymphocytes (PBL) from normal donors were administered to tumor bearing mice, and survival times of 22.0±2.3 days were observed (N = 8). Peripheral blood lymphocytes from four of five normal donors which had been presensitized on monolayers of melanoma tissue culture cells in vitro failed to prolong host survival times. In contrast, PBL obtained from 15 of 20 melanoma patients were found to prolong survival of the tumor bearing nude mice. Of these 15 patients, 8 were undergoing specific active immunotherapy, while 7 had not been sensitized except by the disease process. The ability of PBL obtained from patients to prolong survival of tumor bearing animals did not appear to correlate with either the stage of the disease or the patient's clinical course. The possible mechanisms for the prolonged survival and usefulness of this model are discussed.     

Risks of BCG intralesional therapy: an experience with melanoma.

A nearly fatal allergic reaction to intratumor BCG injections was associated with a complete remission of recurrent malignant melanoma. Clinical course and histologic sections suggested both anaphylactic and Arthus reactions. The occurrence of reactions at BCG injection sites as well as at uninjected sites of tumor suggests common BCG and melanoma antigens. The management of events involved in this often fatal postimmunotherapy complication involves the early administration of parenteral fluids, antituberculous therapy, antihistamines, and possible steroids. The prophylactic use of antihistamines and an in-hospital administration of intralesional BCG immunotherapy are strongly suggested. In the future, prophylactic INH may prove to be both therapeutically efficacious and protective against infectious complications.     

Specific activation of the mu opioid receptor (MOR) by endomorphin 1 and endomorphin 2

The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the mu opioid receptor, an effect reversed by the mu receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), but they had no influence on cells stably expressing the delta opioid receptor. To further establish the selectivity of these peptides for the mu opioid receptor, brain preparations of mice lacking the mu opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a Kd value of 1.77 nM and a Bmax of 63.33 fmol/mg protein. In membranes of mice lacking the mu receptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the mu opioid receptors.     

阳离子抗菌肽研究进展

阳离子抗菌肽广泛分布于多种生物 ,越来越多的证据表明它们在机体天然免疫中有着重要的作用。广谱抗菌、抗病毒、抗肿瘤细胞及其它特性也使得其具有潜在的医药价值。对抗菌肽的研究正不断深入。该文从一般性质、作用机制、构效关系、重组表达、应用价值、存在的问题与发展方向等方面 ,对阳离子抗菌肽的最新研究进展进行综述     

The CEC behaviour of several synthetic peptides related to the activin βA–βD subunits

Abstract: The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin β_A–β_D subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n‐octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge‐state‐specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n‐octadecyl‐silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro‐osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electro‐osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, κ_cec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their κ_cec values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. > 40% (v/v) and nonpolar interactions with the Hypersil n‐octadecyl‐silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the ln κ_cec values and the volume fractions, ψ, of the organic modifier over a limited range of ψ‐values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.     

Utility of peptide–protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide*

Abstract: We used a N‐biotinylated peptide analog of the C‐terminal domain of the tumor suppressor protein, p21^cip1/waf1 to elucidate peptide/protein interacting partners. The C‐terminal domain of p21^cip1/waf1 protein spanning 141–160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell‐cycle control. This C‐terminal 20‐mer efficiently extracts PCNA in the presence of a variety of N‐ or C‐terminally attached affinity tags. Using difference silver stained 2D gels combined with in‐gel tryptic digests, we identified the difference spots using MALDI‐TOF mass spectrometry‐based peptide mass fingerprinting followed by a database search using profound against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor‐1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion‐trap LC‐MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using sequest confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90α, HSP40 and T‐complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21^cip1/waf1 protein. The use of N‐biotinylated peptide derived from the C‐terminal domain of p21^cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.     

Biophysical studies and anti‐growth activities of a peptide,a certain analog and a fragment peptide derived from α‐fetoprotein

Abstract: A chemically synthesized 34‐amino acid peptide, an analog, and a fragment of the peptide have been purified and studied. Biophysical studies were carried out to determine some of the metal ion binding properties of the original peptide and an analog of this parent peptide, in which the two histidine residues were replaced by alanines. As shown by visible absorption spectroscopy, Co (II) forms a complex with the parent peptide, but not with the analog peptide, and one or two histidines in the parent peptide are ligands for Co (II) ion binding. The effects on disulfide bond formation in the peptide by Zn (II) and Co (II) ions were also examined for this analog. Anti‐growth assays were performed using the original cysteine‐containing peptide with Zn (II) ion complexed to the peptide through the two cysteine residues. These rat uterine growth assays showed that the complexing of Zn (II) ion to the peptide maintained the anti‐growth activity of the peptide, while gel‐filtration experiments showed the zinc ions maintained the peptide in its anti‐growth form indefinitely in solution. A saliently important part of this research was the discovery that a fragment of the peptide consisting of a middle sequence of 14 amino acids was found to have significant anti‐growth activity in the rat uterine assay. Its activity suggested that this fragment might be considered a viable candidate for testing in anti‐cancer protocols.     

The biological consequences of replacing hydrophobic amino acids in deltorphin I with amphiphilic α‐hydroxymethylamino acids

Abstract: New analogues of deltorphin I (DT I), in which the Phe residue in position 3, and the Val residue in position 5 or 6 are replaced with respective amphiphilic α‐hydroxymethylamino acid residues (HmAA), were synthesized and tested for receptor affinity and selectivity to μ and δ opioid receptors. The analogue with (R)‐HmPhe at position 3 lost receptor selectivity, as a result of a partial decrease of affinity to δ and a significant increase of affinity to μ receptors. In contrast, an analogue with (S)‐HmPhe in the same position, was very potent and more specific to δ receptors than parent DT I. The analogue with (R)‐HmVal at position 5 expressed higher δ affinity and selectivity than parent DT I. The analogue with other possible isomer (S)‐HmVal was less selective for δ opioid receptors, as a result of decreasing affinity to δ and increasing affinity to μ receptors. The analogues with (R)‐ or (S)‐HmVal in position 6 expressed equally low receptor affinity and selectivity. The data obtained support a previously proposed model of active conformation of deltorphins.