Bone Marrow Extract Research Articles

Disseminated isolated tumor cells in bone marrow of esophageal cancer cases

In this study, we reviewed reports on the clinical significance of isolated tumor cells (ITC) in the bone marrow of patients with esophageal cancer and other solid tumor malignancies. We examined studies on bone marrow as a target organ for ITC identification and also looked at the appropriate sites and most efficient methodologies for bone marrow extraction to evaluate the presence of ITC. A diverse number of different cells exist in bone marrow, and it is important to distinguish disseminated cancer cells from other cell types, such as progenitor cells and endothelial cells. Among the various types of cells found in the bone marrow, we confirmed the presence of disseminated cells that can proliferate under specific cell culture conditions. Finally, we discuss recent studies related to the clinical significance of ITC in bone marrow that are associated with prognosis or recurrence in cases of solid cancers, including esophageal cancer. We concluded that the clinical significance of bone marrow as the target organ for identification of ITC varies among different types of cancer and that further study is required to determine a bona fide marker for predicting prognosis or recurrence in cases of esophageal cancer.

Akt is Preferentially Expressed in PC‐3 Cells Adhering to Bone Matrix

Prostate cancer (PC) commonly metastasizes to the skeleton, where it becomes highly resistant to chemo-therapeutics. The components of the bone extracellular matrix microenvironment and other secreted factors have been implicated in PC cell proliferation. The purpose of this project was to determine the effects of PC-3 cells adhering to bone substrates on Akt activation. PC-3 cells were plated on substrates composed of secreted factors from osteoblastic-like cells (SaOS2), bone stromal-like cells (HS-5), as well as collagen type-1, a major component of the bone matrix, and fibronectin. Western analysis was performed to resolve the phosphorylated Akt. We hypothesize that prostate cancer cells grown on the substrate of secreted factors from osteoblastic (SaOS2) and bone stromal (HS-5) cells will preferentially stimulate the activation of Akt. Also we hypothesize that PC-3 cells grown on type-1 collagen will preferentially stimulate Akt activation rather than grown on fibronectin. Western blot analysis of Akt activation showed the greatest increase in PC-3 cells on SaOS-2 secreted substrate compared to the control (plastic) and HS-5 conditioned media. In addition, we present data showing that Akt is activated preferentially on collagen-type 1 compared to fibronectin. Lastly, we demonstrate that ErbB2 is preferentially expressed on PC-3 cells grown on soluble bone-marrow extracts compared to kidney extracts. These data suggest that secreted factors, including collagen type I, predominately from osteoblastic cells, enhance prostate cancer cell survival in the bone microenvironment and these factors may contribute to drug resistance.

Serpina1 is a potent inhibitor of IL-8-induced hematopoietic stem cell mobilization.

Here, we report that cytokine-induced (granulocyte colony-stimulating factor and IL-8) hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is completely inhibited after low-dose (0.5 Gy) total-body irradiation (TBI). Because neutrophil granular proteases are regulatory mediators in cytokine-induced HSC/HPC mobilization, we considered a possible role for protease inhibitors in the induction of HSC/HPC mobilization. Bone marrow (BM) extracellular extracts that were obtained from murine femurs after 0.5 Gy of TBI contained an inhibitor of elastase. Also, after low-dose TBI, both Serpina1 mRNA and protein concentrations were increased in BM extracts, compared with extracts that were obtained from controls. The inhibitory activity in BM extracts of irradiated mice was reversed by addition of an Ab directed against Serpina1. To further study a possible in vivo role of Serpina1 in HSC/HPC mobilization, we administered Serpina1 before IL-8 injection. This administration resulted in an almost complete inhibition of HSC/HPC mobilization, whereas heat-inactivated Serpina1 had no effect. These results indicate that low-dose TBI inhibits cytokine-induced HSC/HPC mobilization and induces Serpina1 in the BM. Because exogenous administration of Serpina1 inhibits mobilization, we propose that radiation-induced Serpina1 is responsible for the inhibition of HSC/HPC mobilization. Also, we hypothesize that cytokine-induced HSC/HPC mobilization is determined by a critical balance between serine proteases and serine protease inhibitors.

Phospholipid Scramblase 1 Binds to the Promoter Region of the Inositol 1,4,5-Triphosphate Receptor Type 1 Gene to Enhance Its Expression

Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated, endofacial membrane protein originally identified based on its capacity to promote accelerated transbilayer phospholipid movement in response to Ca(2+). Recent evidence suggests that this protein also participates in cell response to various growth factors and cytokines, influencing myeloid differentiation, tumor growth, and the antiviral activity of interferon. Whereas plasma membrane PLSCR1 was shown to be required for normal recruitment and activation of Src kinase by stimulated cell surface growth factor receptors, PLSCR1 was also found to traffic into the nucleus and to tightly bind to genomic DNA, suggesting a possible additional nuclear function. We now report evidence that PLSCR1 directly binds to the 5'-promoter region of the inositol 1,4,5-triphosphate receptor type 1 gene (IP3R1) to enhance expression of the receptor. Probing a CpG island genomic library with PLSCR1 as bait identified four clones with avidity for PLSCR1, including a 191-bp fragment of the IP3R1 promoter. Using electrophoretic mobility shift and transcription reporter assays, the PLSCR1-binding site in IP3R1 was mapped to residues (-101)GTAACCATGTGGA(-89), and the segment spanning Met(86)-Glu(118) in PLSCR1 was identified to mediate its transcriptional activity. The significance of this interaction between PLSCR1 and IP3R1 in situ was confirmed by comparing levels of IP3R1 mRNA and protein in matched cells that either expressed or were deficient in PLSCR1. These data suggest that in addition to its role at the plasma membrane, effects of PLSCR1 on cell proliferative and maturational responses may also relate to alterations in expression of cellular IP3 receptors.

Development of a minimally invasive bone marrow harvest device and method for the rapid extraction of bone marrow for use in bone marrow transplantation and stem cell therapy

The bone marrow contains a rich supply of hematopoetic, mesenchymal and other stem cell populations. Bone marrow and peripheral blood–derived hematopoetic stem cells are currently used in> 40,000 bone marrow and peripheral blood stem cell transplants each year worldwide. Adult bone marrow–derived stem cells may soon be used in various stem cell–mediated regenenerative therapies, resulting in an increased need for a simplified, economical acquisition of bone marrow. Allogeneic marrow, or G-CSF mobilized marrow, may prove to be superior to PBSC for several transplantation indications, and thus improved harvest methods are needed.

Neutrophil Elastase Is Important for Several Activities of PML-RARα in Early Myeloid Cells.

Acute promyelocytic leukemia (APL) is associated with the accumulation of promyelocytes that usually carry the t(15;17) translocation, generating the PML-RARα fusion gene. APL develops in mice only if PML-RARα expression is targeted to early myeloid cells; when PML-RARα is expressed in all hematopoietic cells, the only malignancy that develops is APL. These observations have suggested that there may be a component(s) of the early myeloid environment that is important for the actions of PML-RARα. We recently showed that murine bone marrow extracts and a human early myeloid cell line, U937, contain a serine protease activity that cleaves PML-RARα. The dominant cleaving activity was attributable to an early myeloid-associated protease, neutrophil elastase (NE). The penetrance of APL in mice expressing PML-RARα in early myeloid cells was significantly reduced in NE-deficient animals (Lane and Ley, Cell 115:305–318, 2003). To determine whether measurable functions of PML-RARα require NE, we performed a series of experiments. Using a GFP-PML-RARα fusion construct, we determined that expression of PML-RARα caused disruption of PML oncogenic domains (PODs) in several human hematopoietic cell lines, regardless of whether they expressed NE. However, when PML-RARα was expressed at high levels in cell lines that contained NE activity, substantial toxicity was observed. In contrast, PML-RARα did not cause toxicity in cell lines without NE activity. Similarly, when we expressed an NE-resistant PML-RARα cDNA (containing valine to arginine mutations at the P1 amino acid of the two preferred NE cleavage sites), toxicity was abrogated in clonogenic assays. The toxic effects of expressing full-length PML-RARα could not be recapitulated by expressing either dominant cleavage fragment alone, or in combination, which suggests that NE-induced cleavage may not directly create the “active” fragment of PML-RARα. Primary human and mouse primary APL cells contain abundant NE activity and PML-RARα-cleaving activity. However, we determined that two commonly used APL cell line models (NB4 and U937-PR9 cells) contain abundant full length PML-RARα protein, but neither contains NE activity nor PML-RARα-cleaving activity. As expected, high levels of expression of PML-RARα in PR9 cells did not cause toxicity. Using an in vitro G-CSF-dependent myeloid differentiation assay, we found that purified hematopoietic progenitors from mCGPML-RARα knock-in mice demonstrated markedly increased proliferation of early (blasts and promyelocytes) myeloid cells compared to wild type progenitors. This increase was not observed in progenitors from mCGPML-RARαNE−/− animals. The difference was not due to an intrinsic defect in myeloid development due to NE deficiency, since NE−/− progenitors developed normally in response to G-CSF in vitro. To extend these results, we performed competitive repopulation bone marrow transplants using NE+/+ and NE−/− mice as bone marrow donors; at 3 weeks after hematopoietic reconstitution, there was no difference in the contributions of either genotype to multi-lineage hematopoiesis. Together, these data strongly suggest that NE is important for several of the measurable activities of PML-RARα in early myeloid cells; we are therefore exploring pharmacologic NE inhibition as an approach to reduce the growth of APL cells. Our data also suggest that the physiologic activities of PML-RARα should be studied in early myeloid cells that contain NE activity.

The Fanconi Anemia Proteins Functionally Interact with the Protein Kinase Regulated by RNA (PKR)

Protein kinase regulated by RNA (PKR) plays critical roles in cell growth and apoptosis and is implicated as a potential pathogenic factor of Alzheimer's, Parkinson's, and Huntington's diseases. Here we report that this proapoptotic kinase is also involved in Fanconi anemia (FA), a disease characterized by bone marrow (BM) failure and leukemia. We have used a BM extract to show that three FA proteins, FANCA, FANCC, and FANCG, functionally interact with the PKR kinase, which in turn regulates translational control. By using a combined immunoprecipitation and reconstituted kinase assay, in which an active PKR kinase complex was captured from a normal cell extract, we demonstrated functional interactions between the FA proteins and the PKR kinase. In primary human BM cells, mutations in the FANCA, FANCC, and FANCG genes markedly increase the amount of PKR bound to FANCC, and this PKR accumulation is correlated with elevated PKR activation and hypersensitivity of BM progenitor cells to growth repression mediated by the inhibitory cytokines interferon-gamma and tumor necrosis factor-alpha. Specific inhibition of PKR by 2-aminopurine in these FA BM cells attenuates PKR activation and apoptosis induction. In lymphoblasts derived from an FA-C patient, overexpression of a dominant negative mutant PKR (PKRK296R) suppressed PKR activation and apoptosis induced by interferon-gamma and tumor necrosis factor-alpha. Furthermore, by using genetically matched wild-type and PKR-null cells, we demonstrated that forced expression of a patient-derived FA-C mutant (FANCCL554P) augmented double-stranded RNA-induced PKR activation and cell death. Thus, inappropriate activation of PKR as a consequence of certain FA mutations might play a role in bone marrow failure that frequently occurred in FA.

Lack of in vivo clastogenic activity of grape seed and grape skin extracts in a mouse micronucleus assay

Meganatural™ brand grape seed extract (GSE) and grape skin extract (GSKE), containing proanthocyanidin polyphenolic compounds, are intended for use in food as functional ingredients exhibiting antioxidant activity. Proanthocyanidins, as well as the minor constituent phenolic compounds in GSE and GSKE, are present naturally in many foods such as fruits, vegetables, chocolate, tea, etc., and on average people consume 460–1000 mg/day of these combined substances. While some polyphenolic compounds, tested individually, have demonstrated antitumorigenic or antipromotional activity, at least one minor component of GSE and GSKE, quercitin, has exhibited positive activity in Salmonella and other in vitro mutagenicity assays. As part of a program to investigate the safety of GSE and GSKE, these products were tested for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-1®(ICR) BR mouse bone marrow. The appropriate test article was dissolved in 0.5% carboxymethylcellulose and dosed by oral gavage to five males/test article/dose level/harvest time point. Animals were dosed at 500, 1000 and 2000 mg/kg. Five animals dosed with either test article at 500, 1000 and 2000 mg/kg dose levels and five animals dosed with the cyclophosphamide (80 mg/kg) positive control were euthanized approximately 24 h after dosing for extraction of bone marrow. Five animals dosed with either test article at the 2000 mg/kg dose level and five animals dosed with the vehicle control article were euthanized approximately 24 and 48 h after dosing for extraction of bone marrow. At least 2000 PCEs per animal were analyzed for frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal. For both GSE and GSKE, no statistically significant increase in micronucleated PCEs was observed at any dose level or harvest time point. GSE produced indication of cytotoxicity (decreased PCE:NCE ratio) at the 2000 mg/kg dose level for the 48-h harvest time point, confirming that the test article reached the target bone marrow in significant amount. Meganatural™ GSE and Meganatural™ GSKE were evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.

Whole bone marrow transplantation induces angiogenesis following acute ischemia

Several recent studies have shown that purified subsets of bone marrow (BM) cells can differentiate into endothelial, cardiac, and other cell types. During coronary artery bypass graft (CABG) surgery, sternal BM is routinely discarded. To determine if this BM can be used to induce angiogenesis and augment perfusion of the cardiac tissues after CABG, a simplified and more practical approach of using whole BM extract was tried to determine whether it would be adequate for the induction of BM-derived angiogenesis in experimental acute limb ischemia.BM was prepared from FVB/N-TgN(TIE2 lacZ)182 Sato (Tie2-lacZ) or B6.129S7-Gtrosa26 (Rosa26) mice that express β-galactosidase (β-gal) in endothelial cells and most adult tissues, respectively. Acute limb ischemia was induced in either C57BL6/J or FVB/N mice by double ligation of the left femoral artery just distal to the profunda femoral artery branch. Occlusion of the ligated artery was verified by angiography. The study group (n = 31) received an intramuscular injection of 50 μl containing 1 x 106 BM cells, 5 mm proximal to the site of ligation. Experimental controls (n = 21) had an intramuscular injection of 50 μl of saline. Angiogenesis in the mice was assessed by histological analysis. BM-derived β-gal+ cells were observed to aggregate in the vicinity of the ligated artery and not in the injected musculature. BM-derived endothelial cells were incorporated within capillaries and small size blood vessels near the site of ligation.Generation of BM-derived blood vessels in experimental acute limb ischemia does not require purification of specific subset of cells. The elimination of cell purification will enhance the ease of using BM transplantation in generating blood vessels.

A New Approach to Identifying Bone Marrow and Grease Exploitation: Why the “Indeterminate” Fragments should not be Ignored

The economic importance of bone fat to past peoples is discussed and the ethnography of bone marrow and grease extraction is briefly outlined. Models for expected patterns of bone fracture and fragmentation, relating to the exploitation of bone marrow and grease, are described. Current methods for assessing bone fracture and fragmentation, in archaeological assemblages, are discussed and a new methodology is suggested. The importance of retaining and studying the “indeterminate” class of fragments is stressed. The new methodology employs a fracture freshness index (FFI) to study fracture type, and fragmentation is assessed through the separation of fragments into size classes and different bone types. An example application is given.

IS AUTOLOGOUS BONE MARROW TRANSPLANT (ABMT) AND HIGH-DOSE CHEMOTHERAPY AN APPROACH THAT CAN RESCUE SOME CHILDREN WITH ADVANCED CANCER DISEASE?

The object was to determine the role of ABMT in children with advanced cancer. Those included had failed to respond to conventional treatment with 4 different ablative chemotherapy regimens. Bone marrow stem cells were identified with CD34. Cellular viability was determined after the bone marrow extraction and before the infusion. Fifteen patients were included, whose ages ranged from 1 to 13 years old with a median of 7. Six had acute leukemia, 6 with primitive neuroectodermic tumors, and 3 with other tumors. The median disease-free survival for the whole group was of 2 months, range of 1 to 29 months and SD of 10.1. A total of 6 children are alive (40%) and without evidence of tumor activity from 1 to 29 months. The disease-free survival rate for these group was of 19.1 months, with an SD of 7.9 months.

Pronghorn and Bison Procurement During the Uinta Phase in Southwest Wyoming: A Case Study from Site 48SW270

The dramatic bonebeds occasionally uncovered in southwest HYoming have strongly influenced perceptions of pronghorn procurement in the region during prehistoric times. However, they provide a misleading picture of the typical pattern of pronghorn procurement. Pronghorn bonebeds are generally rare and many may even represent a number of kill events. More typically, the prehistoric hunter-gatherers probably pursued and killed single pronghorn non-communally, often interchangeably with bison when available. The remains recovered from 48SW270 dating to the Uinta phase exemplify these procurement practices. The hunter-gatherers at 48SW270 intensively butchered at least two, and probably three, bison and at least two pronghorn in the late fall and spring during repeated visits to the site. The butchering took place in an area adjacent to the habitation structure. The hunter-gatherers brought the entire animals back to the site for extensive processing, which included bone marrow extraction and bone grease production. The prehistoric inhabitants were probably operating under stress conditions.

Limbs found in water: investigation using anthropological analysis and the diatom test

We report the investigation, using a multi-disciplinary approach, of five cases of dismembered limbs which were recovered from Lake Ontario, Lake Erie and the Niagara River, and examined at the Office of the Chief Coroner for Ontario. In all cases, postmortem examination revealed that the limbs had been disarticulated in the postmortem period, by non-human taphonomic processes. In addition to routine gross examination, the femur and/or tibia were assessed using anthropological methods to give estimates of the sex, age, race and stature of the individual. The anthropologic data facilitated the identification of one of the cases. In all cases, nitric acid extracts of the femoral bone marrow were prepared and examined for the presence of diatoms. In all instances, diatom frustules were recovered from marrow extracts, indicating that drowning was the cause of death or at least a significant contributing factor in the cause of death. The use of the diatom test was helpful in excluding the possibility that the limbs were dismembered from individuals who had died by means other than drowning, and had been subsequently ‘dumped’ into water. The application of anthropological methods and the diatom test for drowning may significantly enhance the medico-legal investigation of body parts recovered from water, and we present an overview of useful techniques here. Anthropological data may facilitate identification, and the diatom test may establish a cause of death.

Comparison of bone marrow extracellular matrices

We have compared the structure and composition of adult and fetal bovine bone marrow extracellular matrices. In contrast to fetal bone marrow, adult bone marrow has more oval fenestration and accumulation of adipocytes as well as lower protein content. These differences could be due to remodeling of bone marrow tissue as it develops. Zymogram analysis of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) activities showed that fetal, but not adult bone marrow extract contained a 96-kDa MMP and TIMP-1 and -2. These activities may contribute to the structural differences between adult and fetal bone marrow tissues.

The Phenomenology and Possible Mechanisms of a New Experimental Method for Accelerating Postirradiation Restoration of Hemopoietic Stem Cell Potential

Extraction of bone marrow from one or several bones immediately after irradiation, with or without subsequent incubation for a short period under suitable conditions, and subsequent reimplantation into the same organism can reduce the lethal effect of irradiation in a number of species. Extraction plus a period of incubation is referred to as the reimplantation method. The effectiveness is roughly relative to the amount of bone marrow extracted and transplanted. This effect has been demonstrated by assays of 30-45-day survival and of hemopoietic stem cell potential. The effectiveness of the reimplantation method has been tested in a dose range of 6.5 to 8.5 Gy and was found to be 1.2-1.06 in terms of dose reduction factors assayed by bone marrow row cellularity 9 days after exposure and 1.18-1.09 for survival of bone marrow colony-forming units. The favorable effect of incubating irradiated bone marrow with cycloheximide on the stem cell potential has been proven by experiments using a donor-recipient method. The positive effect of the reimplantation procedure and the partial extraction procedure on the stem cell potential in irradiated mice can be shown as soon as 2 h after exposure and the procedures. The results suggest that ther exist some reserves that can be stimulated to accelerate hemopoietic restoration in a heavily irradiated organism. The recruitment of these reserves seems to be related to the response of the structures producing cytokines after lethal irradiation. In addition, repair processes may be involved in the rescue of lethally irradiated hemopoietic stem cells.

Catabolism of the hemoregulatory peptide N-Acetyl-Ser-Asp-Lys-Pro: a new insight into the physiological role of the angiotensin-I-converting enzyme N-active site

The tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) was first isolated from bone marrow extracts and shown to be involved in the negative control of hematopoiesis by preventing the recruitment of primitive stem cells into S-phase. In vitro studies on AcSDKP catabolism in human plasma revealed that AcSDKP was cleaved by plasmatic angiotensin-I converting enzyme (ACE). The evaluation of the respective involvement of the two active sites of ACE in AcSDKP degradation in vitro revealed that the N-active site was preferentially involved in this catabolism. Moreover, an in vivo study on healthy volunteers of the catalytic efficiency of ACE towards AcSDKP after administration of Captopril o demonstrated that AcSDKP was a physiological substrate of ACE. AcSDKP might represent the first natural specific substrate of the N-active site of the enzyme. These results pose the question of a potential role of ACE in the control of hematopoiesis as well as possible applications of ACE inhibitors to cope with dysfunctions in which AcSDKP might exert physiological control.

Neutrophil elastase and cathepsin G protein and messenger RNA expression in bone marrow from a patient with Chediak-Higashi syndrome

Aims-To determine whether neutrophil elastase and cathepsin G are expressed, at transcriptional or translational levels, in the bone marrow from a patient with Chediak-Higashi syndrome.Methods-Blood neutrophils were isolated from three patients with Chediak-Higashi disease and bone marrow was collected from one. Cell lysates were analysed for neutrophil elastase and cathepsin G activity by enzyme linked immunosorbent assay and western immunoblotting. Northern blotting was used to detect messenger RNA (mRNA) for cathepsin G, elastase and beta-actin in bone marrow extracts, and immunohistochemistry was used to localise the enzymes in marrow myeloid cells.Results-Elastase and cathepsin G were not detected in blood neutrophils from the patients with Chediak-Higashi disease, but were present in bone marrow cells, although immunohistochemistry showed they were not within cytoplasmic granules. The concentrations of elastase and cathepsin G in Chediak-Higashi bone marrow were about 25 and 15%, respectively, of those in normal marrow. Quantitative scanning of northern blots showed that elastase and cathepsin G mRNA, corrected for beta-actin mRNA, were expressed equally in normal marrow.Conclusions-Transcription of elastase and cathepsin G mRNA in promyelocytes of patients with Chediak-Higashi disease is normal, but the protein products are deficient in these cells and absent in mature neutrophils. This suggests that the translated proteins are not packaged into azurophil granules but are degaded or secreted from the cells.

Purification of cationic cystine-rich peptides from rat bone marrow. Primary structures and biological activity of the rat corticostatin family of peptides

Seven cationic, cystine-rich peptides of 29 to 32 amino acid residues have been purified from extracts of rat bone marrow (R-1, R-1a, R-1b, R-2, R-3, R-4 and R-5). Structural analysis clearly indicated that all seven peptides belong to the corticostatin/defensin family of leukocyte-derived peptides known to participate in oxygen-independent killing of phagocytosed bacteria. For R-1 to R-5, six cysteine residues were found at characteristic and highly conserved positions. R-1a and R-1b were partially characterized and appear to be structural variants of R-1. Aside from the conserved cysteines, there is a remarkable degree of structural diversity evident within the sequences of those members of the corticostatin/defensin family characterized so far. The structures of the peptides that we have purified can be compared directly with the sequences obtained for rat defensins isolated from extracts of peritoneal neutrophils (Lehrer, Ganz and Selsted, Cell, 64 (1991) 229–230). Some discrepencies are apparent which can be explained in terms of proteolytic cleavage of several of these peptides at both amino- and carboxyl-termini. The corticostatins owe their bioactivity to their ability to compete with corticotropin for occupancy of the corticotropin receptor (Zhu, Hu, Mulay, Esch, Shimasaki and Solomon, Proc. Natl. Acad. Sci. USA, 85 (1988) 592–596). The potency of these peptides can be expressed in terms of their capacity to inhibit the steroidogenic response of isolated rat adrenocortical cells half-maximally stimulated by corticotropin (i.e., at the ED 50 concentration for corticotropin in this assay, namely 33 pM). In this assay, the rat peptides R-1, R-2 and R-3 were shown to be inactive. In contrast, the more cationic peptides R-4 and R-5 were found to inhibit steroidogenesis. R-4 was somewhat less active than rabbit corticostatin (IC 50 25 nM) showing an IC 50 value of 50 nM. R-5 appeared to be significantly less potent than R-4. The lower yield of R-5 precluded an accurate estimate of the corticostatic potency of this peptide. R-4 differs in structure from R-5 in having an arginine to serine substitution at position 7. It can be concluded that an arginine at this position accounts, at least in part, for the corticostatic activity of R-4. When tested for their cytotoxic activity using Chinese hamster ovary cells in culture, R-4 was shown to be active (IC 50 2 μM) while R-1 and R-3 were inactive. None of the rat peptides was found to antagonize the biological activity of α-melanotropin in an anolis skin bioassay.

Isolation and characterization of corticostatic peptides from guinea pig bone marrow

Three corticostatic peptides (GP-CS1, GP-CS2 and GP-CS3) were purified from extracts of guinea pig bone marrow. Each was identified on the basis of their ability to inhibit the secretion of corticosterone by isolated rat adrenal cells stimulated by ACTH. GP-CS1 and GP-CS2 were found to be 31 residues in length, rich in arginine and to have six cysteines typical of the corticostatin/defensin family of peptides previously purified from phagocytic cells of the immune system. GP-CS1 was found to be identical to GP-CS2 except for having a leucine at position 21 instead of isoleucine. GP-CS3 was also found to be rich in arginine and cysteine but structurally distinct from the other peptides. A combination of endoprotease mapping, ion-spray mass spectrometry and gas-phase sequencing revealed that GP-CS3 was a novel homo-dimer consisting of two 13 amino acid residue subunits cross-linked through eight cysteines in an anti-parellel configuration.

Inhibition of Hematopoietic Colony‐Forming Cells Normal Bone Marrow Extract versus Transforming Growth Factor‐β1a

Annals of the New York Academy of SciencesVolume 628, Issue 1 p. 44-51 Inhibition of Hematopoietic Colony-Forming Cells Normal Bone Marrow Extract versus Transforming Growth Factor-β1a J. HAMPSON, J. HAMPSON Cancer Research Campaign Department of Experimental Haematology Paterson Institute for Cancer Research Christie Hospital and Holt Radium Institute Manchester M2O 9BX, England Present address: Department of Haematology, University of Liverpool, Royal Liverpool Hospital, Liverpool L69 3BX, England.Search for more papers by this authorB. I. LORD, Corresponding Author B. I. LORD Cancer Research Campaign Department of Experimental Haematology Paterson Institute for Cancer Research Christie Hospital and Holt Radium Institute Manchester M2O 9BX, EnglanddTo whom correspondence should be addressed.Search for more papers by this authorS. REDMOND, S. REDMOND Cell Tech Ltd. Slough, Berkshire SL1 4EN, EnglandSearch for more papers by this authorP. SLOCOMBE, P. SLOCOMBE Cell Tech Ltd. Slough, Berkshire SL1 4EN, EnglandSearch for more papers by this author J. HAMPSON, J. HAMPSON Cancer Research Campaign Department of Experimental Haematology Paterson Institute for Cancer Research Christie Hospital and Holt Radium Institute Manchester M2O 9BX, England Present address: Department of Haematology, University of Liverpool, Royal Liverpool Hospital, Liverpool L69 3BX, England.Search for more papers by this authorB. I. LORD, Corresponding Author B. I. LORD Cancer Research Campaign Department of Experimental Haematology Paterson Institute for Cancer Research Christie Hospital and Holt Radium Institute Manchester M2O 9BX, EnglanddTo whom correspondence should be addressed.Search for more papers by this authorS. REDMOND, S. REDMOND Cell Tech Ltd. Slough, Berkshire SL1 4EN, EnglandSearch for more papers by this authorP. SLOCOMBE, P. SLOCOMBE Cell Tech Ltd. Slough, Berkshire SL1 4EN, EnglandSearch for more papers by this author First published: July 1991 https://doi.org/10.1111/j.1749-6632.1991.tb17221.xCitations: 1 a This work was supported by the Cancer Research Campaign and Cell Tech Ltd. (J.H.). AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume628, Issue1Negative Regulators of Hematopoiesis: Studies on Their Nature, Action, and Potential Role in Cancer TherapyJuly 1991Pages 44-51 RelatedInformation