Efficacy of an agonist of α-MSH, the palmitoyl tetrapeptide-20, in hair pigmentation.

OBJECTIVE: Hair greying (i.e., canities) is a component of chronological ageing and occurs regardless of gender or ethnicity. Canities is directly linked to the loss of melanin and increase in oxidative stress in the hair follicle and shaft. To promote hair pigmentation and reduce the hair greying process, an agonist of α-melanocyte-stimulating hormone (α-MSH), a biomimetic peptide (palmitoyl tetrapeptide-20; PTP20) was developed. The aim of this study was to describe the effects of the designed peptide on hair greying. METHODS: Effect of the PTP20 on the enzymatic activity of catalase and the production of H2 O2 by Human Follicle Dermal Papilla Cells (HFDPC) was evaluated. Influence of PTP20 on the expression of melanocortin receptor-1 (MC1-R) and the production of melanin were investigated. Enzymatic activity of sirtuin 1 (SIRT1) after treatment with PTP20 was also determined. Ex vivo studies using human micro-dissected hairs allowed to visualize the effect of PTP20 on the expression in hair follicle of catalase, TRP-1, TRP-2, Melan-A, ASIP, and MC1-R. These investigations were completed by a clinical study on 15 human male volunteers suffering from premature canities. RESULTS: The in vitro and ex vivo studies revealed the capacity of the examined PTP20 peptide to enhance the expression of catalase and to decrease (30%) the intracellular level of H2 O2 . Moreover, PTP20 was shown to activate in vitro and ex vivo the melanogenesis process. In fact, an increase in the production of melanin was shown to be correlated with elevated expression of MC1-R, TRP-1, and Melan-A, and with the reduction in ASIP expression. A modulation on TRP-2 was also observed. The pivotal role of MC1-R was confirmed on protein expression analysed on volunteer's plucked hairs after 3 months of the daily application of lotion containing 10 ppm of PTP20 peptide. CONCLUSION: The current findings demonstrate the ability of the biomimetic PTP20 peptide to preserve the function of follicular melanocytes. The present results suggest potential cosmetic application of this newly designed agonist of α-MSH to promote hair pigmentation and thus, reduce the hair greying process.

Study of gene expression alteration in male androgenetic alopecia: evidence of predominant molecular signalling pathways.

BACKGROUND: Male androgenetic alopecia (AGA) is the most common form of hair loss in men. It is characterized by a distinct pattern of progressive hair loss starting from the frontal area and the vertex of the scalp. Although several genetic risk loci have been identified, relevant genes for AGA remain to be defined. OBJECTIVES: To identify biomarkers associated with AGA. METHODS: Molecular biomarkers associated with premature AGA were identified through gene expression analysis using cDNA generated from scalp vertex biopsies of hairless or bald men with premature AGA, and healthy volunteers. RESULTS: This monocentric study reveals that genes encoding mast cell granule enzymes, inflammatory mediators and immunoglobulin-associated immune mediators were significantly overexpressed in AGA. In contrast, underexpressed genes appear to be associated with the Wnt/ß-catenin and bone morphogenic protein/transforming growth factor-ß signalling pathways. Although involvement of these pathways in hair follicle regeneration is well described, functional interpretation of the transcriptomic data highlights different events that account for their inhibition. In particular, one of these events depends on the dysregulated expression of proopiomelanocortin, as confirmed by polymerase chain reaction and immunohistochemistry. In addition, lower expression of CYP27B1 in patients with AGA supports the notion that changes in vitamin D metabolism contributes to hair loss. CONCLUSIONS: This study provides compelling evidence for distinct molecular events contributing to alopecia that may pave the way for new therapeutic approaches.

Both plants Sebastiania chamaelea from Niger and Chrozophora senegalensis from Senegal used in African traditional medicine in malaria treatment share a same active principle.

ETHNOPHARMACOLOGICAL RELEVANCE: Based on ethnobotanical data obtained from Nigerien and Senegalese traditional healers, two Euphorbiaceae plants, Sebastiania chamaelea and Chrozophora senegalensis, traditionally used to treat malaria, were selected for further investigations. MATERIALS AND METHODS: Plant extracts were prepared with different solvents and tested both in vitro on several strains of Plasmodium falciparum, and in vivo to evaluate their antiplasmodial properties and isolate their active principles. RESULTS: With IC50 values around 6.5µg/ml and no significant cytotoxicity (>50µg/ml), the whole plant aqueous extract from S. chamaelea showed the best in vitro results. In vitro potentiation assays showed strong synergistic activity of S. chamaelea extract with the antiplasmodial drug chloroquine on the chloroquine-resistant P. falciparum strain W2-Indochina. In other respects, the aqueous crude extract of C. senegalensis leaves showed the most significant antiplasmodial activity in vitro (IC50 values less than 2µg/ml). We also demonstrated the prophylactic activity of C. senegalensis in vivo in a murine malaria model. Bioassay-guided fractionation of aqueous extracts of these plants enabled the isolation and identification of ellagic acid (EA, 1) as the main compound responsible for their antiplasmodial activity. Together with EA, other derivatives belonging to different chemical groups were isolated but showed moderate antimalarial activity: gallic acid (2), brevifolin carboxylic acid (3), protocatechuic acid (4), corillagin (5), rutin (6) and 3,4,8,9,10-pentahydroxy-dibenzo(b,d)pyran-6-one (7). The structures were determined by the usual spectroscopic methods and by comparison with published data. Furthermore, we report here the quantification of compound 1 (EA) by RP-HPLC in the dried extracts of these plants, reported for the first time in both these species, and possessing the highest in vitro antiplasmodial activity with IC50 values from 180 to 330nm. CONCLUSIONS: These in vitro and in vivo results support the traditional use in Africa of crude extracts of both S. chamaelea and C. senegalensis as an antimalarial treatment and prove the significant antiplasmodial property of EA.

The regulatory role of the tetrapeptide AcSDKP in skin and hair physiology and the prevention of ageing effects in these tissues--a potential cosmetic role.

The naturally occurring tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) recognized as a potent angiogenic factor was shown recently to contribute to the repair of cutaneous injuries. In the current article, we report the ability of AcSDKP to exert a beneficial effect on normal healthy skin and scalp and to compensate for the ageing process. In vitro AcSDKP at 10⁻¹¹-10⁻7 M significantly stimulates the growth of human keratinocytes, fibroblasts and follicle dermal papilla cells. Moreover, it enhances the growth of human epidermal keratinocyte progenitor and stem cells as shown in a clonogenic survival assay. Topical treatment of ex vivo cultured skin explants with 10⁻5 M AcSDKP increases the thickness of the epidermis and upregulates the synthesis of keratins 14 and 19, fibronectin, collagen III and IV as well as the glycoaminoglycans (GAGs). In the ex vivo-cultured hair follicles, AcSDKP promotes hair shaft elongation and induces morphological and molecular modifications matching the criteria of hair growth. Furthermore, AcSDKP at 10⁻¹¹-10⁻7 M was shown to improve epidermal barrier, stimulating expression of three protein components of tight junctions (claudin-1, occludin, ZO-1) playing an important role in connecting neighbouring cells. This tetrapeptide exercises also activation of SIRT1 implicated in the control of cell longevity. Indeed, a two-fold increase in the synthesis of SIRT1 by cultured keratinocytes was observed in the presence of 10⁻¹¹-10⁻7 M AcSDKP. In conclusion, these findings provide convincing evidence of the regulatory role of AcSDKP in skin and hair physiology and suggest a cosmetic use of this natural tetrapeptide to prevent skin ageing and hair loss and to promote the cutaneous regeneration and hair growth.

Development of an enzyme immunoassay for a stable amidated analog of the hemoregulatory peptide acetyl-Ser-Asp-Lys-Pro.

The tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been shown to protect hematopoietic stem cells from the toxicity of anticancer chemotherapies. Since its pharmacological efficacy is limited by a rapid degradation by Angiotensin-I Converting Enzyme (ACE), AcSDKP analogs resistant to ACE have been synthesized. One of these compounds (AcSDKP-NH,) differs from the native AcSDKP by amidation of the C-terminus. Further evaluations of this molecule require an analytical method in order to characterize its pharmacokinetic profile. We report, here, the development of a highly specific and sensitive enzyme immunoassay (EIA) for AcSDKP-NH, thatdoes not cross-react with endogenous or exogenous AcSDKP. Using AcSDKP-NH2-acetylcholinesterase conjugate as a tracer, rabbit specific antiserum and microtiter plates coated with goat anti-rabbit immunoglobulins, this EIA allows the determination of AcSDKP-NH2 with limits of quantitation of 1 nM in mouse plasma and 100 pmol/g in tissues. Intra-day and inter-day coefficients of variations were less than 20%. The method was successfully applied to a pharmacokinetic study in order to compare plasma and tissue profiles of AcSDKP-NH2 and AcSDKP. Plasma AcSDKP-NH2 levels were found higher than those of AcSDKP, with AUCinf and Cmax values, respectively, 26- and 10-fold higher than that of AcSDKP.

Synthesis and biological evaluation of analogues of the tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of primitive haematopoietic cell proliferation.

The tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of haematopoietic stem cell proliferation, reduces in vivo and in vitro the damage to the stem cell compartment resulting from treatment with chemotherapeutic agents or ionizing radiations. In order to provide new molecules likely to improve the myeloprotection displayed by this tetrapeptide, we have prepared a set of analogues of AcSDKP. These compounds are derived from the parent peptide by substitution or modification of the N- or of the C-terminus, or substitution of side chains. We report here that almost all investigated analogues retain the antiproliferative activity reducing in vitro the proportion of murine Colony-Forming Units Granulocyte, Macrophage (CFU-GM) in S-phase and inhibiting the entry into cycle of High Proliferative Potential Colony-Forming Cells (HPP-CFC). This shows that the polar groups of Ser, Asp or Lys are critical for the expression of biological activity, but that the modification of the N- or C-terminus mostly yielded compounds still retaining antiproliferative activity and devoid of toxicity. The efficacy of AcSDKP analogues in preventing in vitro the primitive haematopoietic cells from entering into cycle makes these molecules new candidates for further in vivo investigations.

Captopril inhibits in vitro and in vivo the proliferation of primitive haematopoietic cells induced into cell cycle by cytotoxic drug administration or irradiation but has no effect on myeloid leukaemia cell proliferation.

Angiotensin I-converting enzyme (ACE) has been shown to be involved in the catabolism of the tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP). As AcSDKP is a physiological inhibitor of haematopoietic stem cell proliferation, we investigated the in vitro and in vivo effects of captopril, one of the specific inhibitors of ACE, on the proliferation of primitive haematopoietic cells. Regenerating bone marrow cells obtained from mice given one injection of cytosine arabinoside (100 mg/kg) as well as SA2 myeloid leukaemia cells were incubated in vitro for 24 h with 10-6 M captopril. Captopril significantly reduced the proportion of high proliferative potential colony-forming cells (HPP-CFC-1) in S-phase, whereas it had no effect on the proportion of SA2 leukaemic colony-forming cells in S-phase. When given in vivo to mice 1 h after 2 Gy gamma-irradiation or cytosine arabinoside (AraC) injection, captopril (100 mg/kg) was shown to prevent HPP-CFC-1 entry into S-phase induced by these cytotoxic treatments. The observed effects correlated with a reduction in ACE degradative activity and an increase in the level of endogenous AcSDKP both in the supernatants of captopril-treated bone marrow cells and in plasma of treated animals. The present findings suggest that AcSDKP might mediate the observed in vitro and in vivo inhibitory effects of captopril on primitive haematopoietic cell proliferation.

Source, catabolism and role of the tetrapeptide N-acetyl-ser-asp-lys-Pro within the testis.

The tetrapeptide N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (AcSDKP) is a natural regulator of hematopoietic stem cell proliferation. The present study was aimed at investigating the presence and the role of AcSDKP in rat testis. Specific immunoreactivity was always observed in the interstitial tissue at all stages of testicular development and in elongated spermatids at 45 days of age and in adults. In accordance with the interstitial labeling, high AcSDKP levels were detected in Leydig cell and testicular macrophage culture media and cell extracts, as well as in the testicular interstitial fluid (TIF). Much lower concentrations were found in peritubular cells and Sertoli cells cultures, whereas very low concentrations were present in cultured spermatocytes and spermatids. In contrast to the slight degradation rate of AcSDKP observed in the spermatocyte and spermatid culture media, no catabolism of the peptide was seen in testicular somatic cell culture medium. Furthermore, the degradation rate of AcSDKP was much lower in TIF than in peripheral blood plasma. Despite the very strong evidence indicating that Leydig cells and testicular macrophages produce AcSDKP, the selective destruction of these cells did not result in any change in AcSDKP levels in TIF or in plasma. This suggests a compensatory mechanism ensuring constant levels of the peptide in TIF when interstitial cells are absent. Finally, in vitro, in the presence of AcSDKP, significantly more [(3)H]thymidine incorporation was found in A spermatogonia. In conclusion, this study establishes the presence of very high concentrations of AcSDKP in rat testis and demonstrates its Leydig cell and testicular macrophage origin. The presence of AcSDKP in the TIF and its stimulatory effect on thymidine incorporation in spermatogonia very strongly suggest its implication in the paracrine control of spermatogenesis.

Captopril inhibits the proliferation of hematopoietic stem and progenitor cells in murine long-term bone marrow cultures.

Drugs used mainly for the treatment of hypertension, such as angiotensin I-converting enzyme (ACE) inhibitors, can cause pancytopenia. The underlying cause of this side effect remains unknown. In the present study, long-term bone marrow cultures (LTBMCs) were utilized to evaluate the role of captopril (D-3-mercapto-2-methylpropionyl-L-proline), one of the potent ACE inhibitors, in regulating hematopoietic stem/progenitor cell proliferation. Captopril (10(-6) M final concentration) was added to LTBMCs at the beginning of the culture period and at weekly intervals for six weeks. There was no toxicity to the bone marrow cells as measured by the unchanged cell number in the nonadherent layer during the whole culture period, and there was an increased cellularity of the adherent layer at the end of the six weeks of treatment. However, captopril decreased the proportion of granulocyte-macrophage colony-forming cells (GM-CFCs) in S phase at weeks 2 and 3 as well as that of high proliferative potential colony-forming cells (HPP-CFCs) at week 3 in the nonadherent layer. There was no change in the kinetics of the GM-CFCs and HPP-CFCs present in the adherent layer. These results suggest that captopril causes myelosuppression by inhibiting hematopoietic cell proliferation of progenitor and stem cells rather than depleting cells of the bone marrow microenvironment.

In vitro effect of acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) analogs resistant to angiotensin I-converting enzyme on hematopoietic stem cell and progenitor cell proliferation.

The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of hematopoietic stem cell proliferation, is known to reduce in vivo the damage resulting from treatment with chemotherapeutic agents or ionizing radiation on the stem cell compartment. Recently, AcSDKP has been shown to be a physiological substrate of the N-active site of angiotensin I-converting enzyme (ACE). Four analogs of the tetrapeptide expressing a high stability towards ACE degradation in vitro have been synthesized in order to provide new molecules likely to improve the myeloprotection displayed by AcSDKP. These analogs are three pseudopeptides with a modified peptidic bond, Ac-Serpsi(CH2-NH)Asp-Lys-Pro, Ac-Ser-Asppsi(CH2-NH)Lys-Pro, Ac-Ser-Asp-Lyspsi(CH2-N)Pro, and one C-terminus modified peptide (AcSDKP-NH2). We report here that these analogs reduce in vitro the proportion of murine colony-forming units-granulocyte/macrophage in S-phase and inhibit the entry into cycle of high proliferative potential colony-forming cells. The efficacy of AcSDKP analogs in preventing in vitro primitive hematopoietic stem cells from entering into cycle suggests that these molecules could be new candidates for the powerful inhibition of hematopoietic stem and progenitor cell proliferation in vivo.

Renal and metabolic clearance of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) during angiotensin-converting enzyme inhibition in humans.

We investigated the contributions of angiotensin-converting enzyme (ACE) and glomerular filtration to creating the new metabolic balance of the hemoregulatory peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) that occurs during acute and chronic ACE inhibition in healthy subjects. We also studied the effect of chronic renal failure on the plasma concentration of AcSDKP during long-term ACE inhibitor (ACEI) treatment or in its absence. In healthy subjects, a single oral dose of 50 mg captopril (n=32) and a 7-day administration of 50 mg captopril BID (n=10) resulted in a respective 42-fold (range, 18- to 265-fold) and 34-fold (range, 24-fold to 45-fold) increase in the ratio of urinary AcSDKP to creatinine accompanied by a 4-fold (range, 2- to 6.8-fold) and 4.8-fold (range, 2.6- to 11.8-fold) increase in plasma AcSDKP levels. Changes in plasma AcSDKP and in vitro ACE activity over time showed an intermittent reactivation of ACE between each captopril dose. In subjects with chronic renal failure (creatinine clearance<60 mL/min per 1.73 m2), plasma AcSDKP levels were 22 times higher (95% confidence interval, 15 to 33) in the ACEI group (n=35) than the control group (n=23); in subjects with normal renal function, they were only 4.1 times higher (95% confidence interval, 3.2 to 5.3) in the ACEI group (n=19) than the non-ACEI group (n=21). Renal failure itself led to a slight increase in plasma AcSDKP concentration. In conclusion, intermittent reactivation of ACE between doses of an ACEI is the major mechanism accounting for the lack of major AcSDKP accumulation during chronic ACE inhibition in subjects with normal renal function.

Modulating role of glucose on magnesium transport in rat erythrocytes.

Magnesium efflux from rat erythrocytes has been shown to be inhibited by a plasma fraction containing glucose. Therefore, we investigated the effect of D-glucose on erythrocyte magnesium transport. We show the inhibitory activity of this hexose on sodium (Na(+))-independent erythrocyte magnesium (Mg(2+)E) efflux. Inhibitory effects of D-mannose, 2-deoxy-D-glucose, and D-fructose on Mg(2+)E efflux also were demonstrated. Moreover, the suppression of the inhibitory activity of glucose on Mg(2+)E efflux was shown to be associated with the inhibition of glucose transport by cytochalasin B and phloretin. Together these data suggest a possible implication of the glucose carrier GLUT-1 in the regulation of Mg(2+) transport.

Lisinopril, an angiotensin I-converting enzyme inhibitor, prevents entry of murine hematopoietic stem cells into the cell cycle after irradiation in vivo.

The hemoregulatory peptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been shown in vivo to inhibit the cycling of murine hematopoietic stem cells triggered into S-phase by either cytotoxic drug administration or irradiation. This property, further confirmed using in vitro models, demonstrates that the peptide has an in vivo protective effect on the hematopoietic system. AcSDKP has been shown to be a physiological substrate of angiotensin I-converting enzyme (ACE), which catabolizes the peptide through a dipeptidasic activity. Thus, oral administration of ACE inhibitor to humans has led to an increase in the plasma AcSDKP concentration. In the present paper, we report on the in vivo effect of lisinopril, an ACE inhibitor, on the proliferative status of murine hematopoietic stem cells triggered into S-phase by irradiation. Administration of lisinopril (10 mg/kg) 1 hour after irradiation led to a 90 to 100% inhibition of murine plasma ACE activity as observed during the first 4 hours postirradiation. This inhibition was correlated with a 600% increase in the endogenous plasma AcSDKP level and a total suppression at 24 hours of entry of the hematopoietic stem cell into the cell cycle. We discuss the possible role of ACE in the regulation of hematopoietic stem cell proliferation through control of the AcSDKP concentration.

In vivo modifications of AcSDKP metabolism and haematopoiesis in mice treated with 5-fluorouracil and Goralatide.

BACKGROUND: The tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP), a physiological inhibitor of the proliferation of haematopoietic stem cells, is degraded by the angiotensin-I-converting enzyme (ACE). Whereas synthetic AcSDKP (Goralatide) protects normal mice from the haematological toxicity of chemotherapy, it has a lower beneficial effect in humans. This discrepancy could be dependent on Goralatide administration schedules, as well as on the endogenous concentrations of AcSDKP and ACE, which vary during chemotherapy. METHODS: We investigated the effect of one myelotoxic dose of 5-fluorouracil (5-FU, 200 mg kg-1) administered without or with Goralatide on blood, bone marrow (BM) and spleen AcSDKP concentrations, ACE activity, nucleated cell counts and survival of the primitive haematopoietic progenitors high proliferative potential colony-forming cells (HPP-CFCs). RESULTS: The 5-FU treatment dramatically decreased the BM concentrations of AcSDKP by 73% and increased the ACE activity in plasma by 50% during the period of active BM regeneration. Repeated injections of Goralatide from 24 h before to 36 h after the i.p. injection of 5-FU spared BM HPP-CFCs. As an injection of 10 mg of Goralatide induced a short peak of plasma AcSDKP without modifying its BM concentrations, we suggest that its protective effect on HPP-CFCs could be mediated by its interference with other plasma molecules targeting to the BM. CONCLUSION: By improving our knowledge of the biology of AcSDKP in vivo during chemotherapy, our results could help to better define the therapeutic use of Goralatide.

The tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (Goralatide) protects from doxorubicin-induced toxicity: improvement in mice survival and protection of bone marrow stem cells and progenitors.

The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP or Goralatide), a physiological regulator of hematopoiesis, inhibits the entry into the S-phase of murine and human hematopoietic stem cells. It has been shown to reduce the damage to specific compartments in the bone marrow resulting from treatment with chemotherapeutic agents, ionizing radiations, hyperthermy, or phototherapy. The present study was performed to assess the therapeutic potential of AcSDKP in vivo in reducing both the toxicity and the hematopoietic damage induced by fractionated administration of doxorubicin (DOX), a widely used anticancer drug. Here we showed that AcSDKP could reduce DOX-induced mortality in mice and could protect particularly the long-term reconstituting cells (LTRCs) in addition to colony forming units-spleen, high proliferative potential colony-forming cells, and colony-forming units-granulocyte-macrophage (CFU-GM) from DOX toxicity. The protection against DOX-induced mortality in mice was improved when AcSDKP was administered for 3 days, at a dose of 2.4 micrograms/d, by continuous subcutaneous (SC) infusion or fractionated s.c. injections starting 48 hours before DOX treatment. Moreover, the recovery of the CFU-GM population in the AcSDKP-DOX-treated mice was optimized by the subsequent administration of granulocyte colony-stimulating factor (G-CSF). The coadministration of AcSDKP with DOX may improve its therapeutic index by reducing both acute hematotoxicity on late stem cells and progenitors and long-term toxicity on LTRCs. Optimization of these treatments combined with G-CSF may provide an additional approach to facilitate hematopoietic recovery after cancer chemotherapy.

Inhibitory action of the peptide AcSDKP on the proliferative state of hematopoietic stem cells in the presence of captopril but not lisinopril.

The effect of Angiotensin I-converting enzyme (ACE) inhibitors on their own and in combination with the peptide AcSDKP on the proliferation of hematopoietic stem cells has been investigated. Hematopoietic stem cells from murine bone marrow induced into cell cycle following exposure to 2 Gy gamma-irradiation were incubated in vitro for up to 24 h in the presence of medium, captopril/lisinopril, AcSDKP, and AcSDKP with either ACE inhibitor. Hematopoietic stem cells were monitored using the high proliferative potential-colony forming cell-1 (HPP-CFC-1) population cloned in the presence of human IL-1 beta, murine IL-3, and murine M-CSF. No significant inhibitory effect was observed in the presence of AcSDKP on its own and AcSDKP in combination with lisinopril. However, there was a significant inhibition of stem cell cycling when AcSDKP and captopril were combined. This suggests that captopril inhibits AcSDKP breakdown better than lisinopril. The combination of AcSDKP and captopril also had an inhibitory effect on cell recruitment into S phase. The fact that a combination of AcSDKP and captopril switches cycling hematopoietic stem cells out of cycle indicates the importance of the N-active catalytic site of ACE in AcSDKP hydrolysis in vitro. Thus, AcSDKP in combination with appropriate ACE inhibitors may be of use in regulating the proliferation of hematopoietic stem cells in vitro.

The antiproliferative activity of the tetrapeptide Acetyl-N-SerAspLysPro, an inhibitor of haematopoietic stem cell proliferation, is not mediated by a thymosin beta 4-like effect on actin assembly.

Acetyl-N-SerAspLysPro (AcSDKP), known as a negative regulator of haematopoiesis, has been principally reported as an inhibitor of haematopoietic pluripotent stem cell proliferation. The tetrapeptide sequence is identical to the N-terminus of thymosin beta 4 (T beta 4), from which it has been suggested that it may be derived. Recently, evidence was shown that T beta 4 plays a role as a negative regulator of actin polymerization leading to the sequestration of its monomeric form. The structural similarity between the N-terminus of T beta 4 and AcSDKP has raised the possibility that AcSDKP may also participate in intracellular events leading to actin sequestration. The effect of T beta 4 on the proliferation of haematopoietic cells was compared to that of AcSDKP. The results revealed that T beta 4, like AcSDKP, exerts an inhibitory effect on the entry of murine primitive bone marrow cells into cell cycle in vitro. Qualitative electrophoretic analysis and quantitative polymerization assays were used to investigate the role of AcSDKP in actin polymerization. AcSDKP does not affect actin assembly at concentrations up to 50 microM, and does not compete with T beta 4 for binding to G-actin. These results suggest that AcSDKP is not involved in cell cycle regulation via an effect on the process of actin polymerization.

Involvement of human plasma angiotensin I-converting enzyme in the degradation of the haemoregulatory peptide N-acetyl-seryl-aspartyl-lysyl-proline.

The degradation of N-Ac-Ser-Asp-Lys-Pro (AcSDKP), a negative regulator controlling the proliferation of the haematopoietic stem cell, by enzymes present in human plasma, has been investigated. Radiolabelled AcSD[4-3H]KP ([3H]AcSDKP, 1 mM) was completely metabolized in human plasma with a half-life of 80 min, leading exclusively to the formation of radiolabelled lysine. The cleavage of AcSDKP was insensitive to classical proteinase inhibitors including leupeptin, but sensitive to metalloprotease inhibitors. The degradation was completely blocked by specific inhibitors of angiotensin I-converting enzyme (ACE; kininase II; peptidyldipeptide hydrolase, EC 3.4.15.1), showing that the first step of the hydrolysis was indeed due to ACE. In dialysed plasma, the hydrolysis proceeded at only 17% of the maximal rate, whereas addition of 20 mM NaCl led to the recovery of the initial rate observed with normal plasma. Hydrolysis of AcSDKP by commercial rabbit lung ACE generated the C-terminal dipeptide Lys-Pro. Thus, ACE cleaves AcSDKP by a dipeptidyl carboxypeptidase activity. In fact the formation of Lys-Pro was observed when AcSDKP was incubated in human plasma in the presence of HgCl2. These results suggest that ACE is involved in the first limiting step of AcSDKP degradation in human plasma. The second step seems to be under the control of a leupeptin- and E-64-insensitive, HgCl2-sensitive plasmatic enzyme.

The molecular specificity of action of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) in the control of hematopoietic stem cell proliferation.

The hemoregulatory tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP, M(r) = 487 amu) prevents the recruitment of hematopoietic stem cells into S phase. AcSDKP does not possess inherent inhibitory activity against hematopoietic stem cells, rather it appears to act by "blocking" the action of a hematopoietic stem cell proliferation stimulator present in extracts of regenerating hematopoietic tissue. The molecular specificity of this action was investigated using a number of peptide analogues of AcSDKP and evaluating their ability to "block" the recruitment of a primitive murine hematopoietic precursor (high proliferative potential colony forming cell HPP-CFC) into S phase following incubation with a hematopoietic stem cell proliferation stimulator. The capacity of AcSDKP to compromise the action of the hematopoietic stem cell proliferation stimulator was not evident for either AcSDDKP or AcSD beta KP, two structurally distinct forms of the molecule; similar no stimulator-"blocking" activity was observed for the tripeptide Ala-Asp-Lys (ADK, M(r) = 332 amu), while the tripeptide Ser-Asp-Lys (SDK, M(r) = 348 amu) was active. It would appear that the hemoregulatory action of AcSDKP is specific and that the tripeptide sequence SDK may be a significant component of the molecule.

Catabolism of the tetrapeptide N-Ac-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of hematopoietic stem cell (CFU-S) proliferation, following in vitro incubation with hematopoietic tissues from normal and leukemic mice.

The comparative degradation of N-Ac-Ser-Asp-Lys-Pro (AcSDKP), a negative regulator controlling the proliferation of the hematopoietic pluripotent stem cell, was investigated following incubation with plasma, bone marrow and spleen cells from normal mice and mice bearing a transplantable myeloid leukemia. Using the tetrapeptide, specifically radiolabelled in the lysyl residue, degradation of [3H]AcSDKP was followed by measurement of [3H]Lys formation resulting from its catabolism. It was shown that already after 1 h the degradation of AcSDKP in plasma from leukemic mice was higher compared to that following incubation in plasma from normal mice, whereas incubation with bone marrow cells exhibits a small difference only after 4 hours incubation. However, no increase of AcSDKP catabolic activity was observed following incubation with spleen cells from leukemic animals when compared with incubation of normal spleen cells.