Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. α5β1 and αvβ3 integrins form stable complexes with immobilized endostatin (K_D = ∼1.8 × 10⁻⁸ m, two-state model). Two arginine residues (Arg²⁷ and Arg¹³⁹) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the αvβ3 integrin at the interface between the β-propeller domain of the αv subunit and the βA domain of the β3 subunit. In addition, we report that α5β1 and αvβ3 integrins bind to heparin/heparan sulfate. The ectodomain of the α5β1 integrin binds to haparin with high affinity (K_D = 15.5 nm). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and α5β1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.Endostatin is an endogenous inhibitor of angiogenesis that inhibits proliferation and migration of endothelial cells (1–3). This C-fragment of collagen XVIII has also been shown to inhibit 65 different tumor types and appears to down-regulate pathological angiogenesis without side effects (2). Endostatin regulates angiogenesis by complex mechanisms. It modulates embryonic vascular development by enhancing proliferation, migration, and apoptosis (4). It also has a biphasic effect on the inhibition of endothelial cell migration in vitro, and endostatin therapy reveals a U-shaped curve for antitumor activity (5, 6). Short term exposure of endothelial cells to endostatin may be proangiogenic, unlike long term exposure, which is anti-angiogenic (7). The effect of endostatin depends on its concentration and on the type of endothelial cells (8). It exerts the opposite effects on human umbilical vein endothelial cells and on endothelial cells derived from differentiated embryonic stem cells. Furthermore, two different mechanisms (heparin-dependent and heparin-independent) may exist for the anti-proliferative activity of endostatin depending on the growth factor used to induce cell proliferation (fibroblast growth factor 2 or vascular endothelial growth factor). Its anti-proliferative effect on endothelial cells stimulated by fibroblast growth factor 2 is mediated by the binding of endostatin to heparan sulfate (9), whereas endostatin inhibits vascular endothelial growth factor-induced angiogenesis independently of its ability to bind heparin and heparan sulfate (9, 10). The broad range of molecular targets of endostatin suggests that multiple signaling systems are involved in mediating its anti-angiogenic action (11), and although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanisms of action are not as fully elucidated as they are for other endogenous angiogenesis inhibitors (11).Endostatin binds with relatively low affinity to several membrane proteins including α5β1 and αvβ3 integrins (12), heparan sulfate proteoglycans (glypican-1 and -4) (13), and KDR/Flk1/vascular endothelial growth factor receptor 2 (14), but no high affinity receptor(s) has been identified so far. The identification of molecular interactions established by endostatin at the cell surface is a first step toward the understanding of the mechanisms by which endostatin regulates angiogenesis. We have previously characterized the binding of endostatin to heparan sulfate chains (9). In the present study we have focused on characterizing the interactions between endostatin, α5β1, αvβ3, and αvβ5 integrins and heparan sulfate. Although interactions between several integrins and endostatin have been studied previously in solid phase assays (12) and in cell models (12, 15, 16), no molecular data are available on the binding site of endostatin to the integrins. We found that two arginine residues of endostatin (Arg²⁷ and Arg¹³⁹) participate in binding to integrins and to heparan sulfate, suggesting that endostatin is not able to bind simultaneously to these molecules displayed at the cell surface. Furthermore, we have demonstrated that α5β1, αvβ3, and αvβ5 integrins bind to heparan sulfate. This may explain why both heparan sulfate and α5β1 integrins are required for the localization of endostatin in lipid rafts, in support of the model proposed by Wickström et al. (15).     

Molecular cloning and sequence analysis of interleukin 16 from nonhuman primates and from the mouse

 Interleukin 16 (IL-16) is synthesized as a 67 000 M _r precursor (pro-IL-16), but only a carboxy terminal part of 12 000–14 000 M _r is secreted by CD8(⁺) lymphocytes. This lymphokine binds to CD4 and has been shown to induce migration, affect the activation state of T cells, and inhibit immunodeficiency virus replication. It has been suggested that CD8(⁺) cell-derived soluble factors play a pivotal role in protecting natural-host nonhuman primates from developing immunodeficiency following SIV infection. In a first attempt to address this question, we cloned and sequenced the IL-16 cDNA from different primates. Here we report the pro-IL-16 sequence from chimpanzees, African green monkeys (AGM), rhesus macaques, and cynomolgus macaques. In order to compare and analyze structural motifs possibly involved in processing, intracellular targeting, or secretion, we extended our study to the New World monkeys saimiri and aotus and to the mouse. Alignments of deduced amino acids reveal that the human protein shares 99% similarity to that of chimpanzees, approximately 95% to rhesus, cynomolgus and AGM, about 90% to aotus and saimiri, and 77.5% to the mouse. Phylogenetic analyses revealed the expected evolutionary groupings. Received: 27 August 1997 / Revised: 7 October 1997     

Endostatin induces acute endothelial nitric oxide and prostacyclin release

Chronic exposure to endostatin (ES) blocks endothelial cell (EC) proliferation, and migration and induces EC apoptosis thereby inhibiting angiogenesis. Nitric oxide (NO) and prostacyclin (PGI(2)), in contrast, play important roles in promoting angiogenesis. In this study, we examined the acute effects of ES on endothelial NO and PGI(2) production. Unexpectedly, a cGMP reporter cell assay showed that ES-induced acute endothelial NO release in cultured bovine aortic endothelial cells (BAECs). Enzyme immunoassay showed that ES also induced an acute increase in PGI(2) production in BAECs. These results were confirmed by ex vivo vascular ring studies that showed vascular relaxation in response to ES. Immunoblot analysis showed that ES stimulated acute phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser116, Ser617, Ser635, and Ser1179, and dephosphorylation at Thr497 in BAECs, events associated with eNOS activation. Short-term exposure of EC to ES, therefore, unlike long-term exposure which is anti-angiogenic, may be pro-angiogenic.     

Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli

Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni–NTA (Ni²⁺-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200 ng/ml.     

Plasma estrogens,progesterone and chorionic gonadotropin in pregnant rhesus monkeys (Macaca mulatta) after ovariectomy

Maternal peripheral plasma concentrations of estrone (E₁), estradiol-17β (E₂), and progesterone (P) were determined in 4 rhesus monkey ovariectomized in early pregnancy (22–24 days). After ovariectomy, plasma concentrations of E₁ and E₂ were basal for 1 to 2 weeks. In contrast, slightly higher estrogen levels, which may be attributed to the ovaries, were found in intact pregnant monkeys. E₂ levels increased rapidly after this and exceeded those of E₁ until the 5th month of gestation. From that time until parturition, E₁ levels equaled or exceed those of E₂ in most instances. The pattern of P concentrations was similar to that observed in intact monkeys. Urinary chorionic gonadotropin (MCG) levels in ovariectomized monkeys were not significantly differen from those found in normal pregnancies. Thus, the patterns for circulating E_l, E₂ and P, as well as for the excretion of MCG,. after ovari-ectomy were remarkably similar to those found in intact, pregnant rhesus monkeys, indicating minimal ovarian influence.     

Expression of endostatin mediated by a novel non-viral delivery system inhibits human umbilical vein endothelial cells in vitro

Ultrasound (US)-mediated microbubble destruction is recognized to have considerable potential for gene delivery, whereas, there is few report of its effect on enhancing liposomal transfection. In this study, we used pIRES2-EGFP/hES containing human endostatin (hES) cDNA as target gene to test the hypothesis that US exposure with microbubbles could improve liposomal transfection, and to investigate the possibility of intracellular delivery of ES gene using this method. Under the controlled US exposure condition with microbubbles, the plasmid:liposome was transferred into COS-7 cells. The transfection rate, the expression of endostatin and the inhibition effect of transfection-endostatin on endothelial cells were assessed. The results revealed that US-mediated microbubble destruction together with liposome could significantly enhance gene transfection without obvious cell damage. By this means, endostatin gene could be efficiently transferred into COS-7 cells and expressed. The transfection-endostatin could inhibit endothelial proliferation and migration, which suggests that the non-viral method might be useful in anti-angiogenesis therapy in the future.     

Modulation of synthesis of specific proteins in endothelial cells by copper,cadmium, and disulfiram: An early response to an angiogenic inducer of cell migration

Copper, cadmium, and disulfiram (an ionophore for copper) modulate the synthesis of several polypeptides in two clonal lines of bovine aortal endothelial cells. After treatment of type 1 endothelial cells with 10^?3 M CuSO₄ or 10^?5 M CdCl₂ four cell-associated polypeptides (M_r = 28,000, 32,000, 73,000, and 83,000 daltons) were induced. In contrast, in Type 2 endothelial cells, which have cultural characteristics distinct from Type 1, only one new cell-associated protein (M_r = 32,000 and 40,000 daltons) was induced. Other differences are revealed by analyses of proteins secreted into the growth medium. In particular low levels of only CuSO₄ (10^?6 M) enhanced the synthesis in Type 2 cells of a protein (M_r = 220,000 daltons) identified as fibronectin. Since only copper ions induced fibronectin, we propose that the mechanism of induction of fibronectin synthesis, in contrast to the induction of cell?associated polypeptides, does not involve a sulphydryl?containing receptor molecule. It is suggested that the specific enhancement of fibronectin synthesis by copper ions may be a controlling event in the stimulation by copper ions of endothelial cell migration and angiogenesis.     

Roles of mechanical force and CXCR1/CXCR2 in shear-stress-induced endothelial cell migration

We previously demonstrated that CXCR1 and CXCR2 are novel mechanosensors mediating laminar shear-stress-induced endothelial cell (EC) migration (Zeng et al. in Cytokine 53:42–51, 2011). In the present study, an analytical model was proposed to further analyze the underlying mechanisms, assuming the mechanical force (MF) and mechanosensor-mediated biochemical reactions induce cell migration together. Shear stress can regulate both mechanosensor-mediated migration in the flow direction (Ms–M_FD) and mechanosensor-mediated migration toward a wound (Ms–M_W). Next, the migration distance, the roles of MF-induced cell migration (MF–M), and the mobilization mechanisms of mechanosensors were analyzed. The results demonstrated that MF–M plays an important role in 15.27 dyn/cm² shear-stress-induced EC migration but is far weaker than Ms–M_W at 5.56 dyn/cm². Our findings also indicated that CXCR2 played a primary role, in synergy with CXCR1. The Ms–M_FD was primarily mediated by the synergistic effect of CXCR1 and CXCR2. In Ms–M_W, when shear stress was beyond a certain threshold, the synergistic effect of CXCR1 and CXCR2 was enhanced, and the effect of CXCR1 was inhibited. Therefore, the retarding of EC migration and wound closure capacity under low shear flow was related to the low magnitude of shear stress, which may contribute to atherogenesis and many other vascular diseases.     

Role of heparan sulfate domain organization in endostatin inhibition of endothelial cell function

The anti-angiogenic activity of endostatin (ES) depends on interactions with heparan sulfate (HS). In the present study, intact HS chains of >/=15 kDa bound quantitatively to ES whereas N-sulfated HS decasaccharides, with affinity for several fibroblast growth factor (FGF) species, failed to bind. Instead, ES-binding oligosaccharides composed of mixed N-sulfated and N-acetylated disaccharide units were isolated from pig intestinal HS. A 10/12mer ES-binding epitope was identified, with two N-sulfated regions separated by at least one N-acetylated glucosamine unit (SAS-domain). Cleavage at the N-acetylation site disrupted ES binding. These findings point to interaction between discontinuous sulfated domains in HS and arginine clusters at the ES surface. The inhibitory effect of ES on vascular endothelial growth factor-induced endothelial cell migration was blocked by the ES-binding SAS-domains and by heparin oligosaccharides (12mers) similar in length to the ES-binding SAS-domains, but not by 6mers capable of FGF binding. We propose that SAS-domains modulate the biological activities of ES and other protein ligands with extended HS-binding sites. The results provide a rational explanation for the preferential interaction of ES with certain HS proteoglycan species.     

Hexokinase2 controls angiogenesis in melanoma by promoting aerobic glycolysis and activating the p38-MAPK signaling

In this study, we used endostatin (ES)-induced apoptosis of endothelial cells to study the role of Hexokinase2 (HK2) in the control of angiogenesis in melanoma. Real-time polymerase chain reaction and Western blot analysis were performed to explore the effect of HK2, lactate, and ES on the levels of caspase-9/3, ATP, and p38/MAPK activation. ES increased the levels of caspase-9/3 while decreasing the level of ATP, whereas ES + HK2 and lactate both restored the normal levels of caspase-9/3 and ATP. In addition, cells transfected with HK2 short hairpin RNA1 (HK2shRNA1) and HK2shRNA2 showed an evident decrease in the levels of caspase-9/3 along with an obvious increase in the level of ATP. Knockdown of HK2 also increased O₂ consumption while decreasing the extracellular level of lactate and the phosphorylation of p38-mitogen-activated protein kinase (MAPK). On the other hand, the lactate treatment elevated the phosphorylation of p38-MAPK under time- and concentration-dependent manner. In the study, we clarified the role of HK2 in the control of apoptosis of ECs, which plays an important role in the angiogenesis of melanoma by promoting aerobic glycolysis and activating the p38-MAPK signaling.     

Thermoregulation by rhesus monkeys at different absolute humidities

The effect of relative humidity on thermoregulation has been well examined. Because the same relative humidity represents very different absolute humidities at different ambient temperatures, the present study was designed to examine the interaction of temperature and absolute humidity on the thermal balance of rhesus monkeys, Macaca mulatta. Thermal balance was examined in six unacclimated, unanesthetized, female rhesus monkeys at ambient temperatures of 25, 30, 35, and 40 °C and absolute humidities of 6, 22, and 40 torr. Monkeys were capable of achieving thermal balance under all conditions except at 40 °C with 40 torr absolute humidity, where experiments were stopped after rectal temperature exceeded 40.5 °C. At 40 °C, monkeys increased evaporative heat loss through both respiration and sweating; the slope of the relationship between evaporative heat loss and core temperature was attenuated by increases in absolute humidity. In contrast, absolute humidity had no direct effect on metabolic rate. The rise in body temperature under the conditions of high heat/high humidity was therefore most attributable to humidity-dependent decreases in evaporative heat loss.Abbreviations E_tot total evaporative heat loss - HR heart rate - K thermal conductance - M metabolic rate - RQ respiratory quotient - T_c core temperature - T_sk mean skin temperature - VCO₂ carbon dioxide production - VO₂ oxygen consumptionCommunicated by G. Heldmaier     

Cloning, expression, and in vitro activity of human endostatin.

Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis. In the present study, we have expressed human endostatin in a yeast expression system (10 mg/L). The recombinant protein was expressed in a soluble form and purified to homogeneity. It specifically inhibited the proliferation and migration of endothelial cells. In addition, we report for the first time that endostatin caused G1 arrest of endothelial cells. Also, we show that endostatin treatment resulted in apoptosis of HUVE and HMVE cells and that all of these effects do not occur in nonendothelial cells. Collectively, these findings demonstrate the expression of a biologically active form of human endostatin in yeast and provide important mechanistic insight into endostatin action on endothelial cells.     

Maternal estrogen and progesterone levels after hypophysectomy in early pregnancy and in term fetuses or newborn monkeys

Pregnant rhesus monkeys ($\text{Macaca}$$\text{mulatta}$) were hypophysectomized at 8–10 weeks gestation to determine effects on plasma levels of estrone (E₁), estradiol-17β (E₂) and progesterone (P). The first group of monkeys was subsequently fetectomized At 107–114 days. After hypophysectomy there was an initial decrease in maternal peripheral plasma E₂ followed by a rise to preoperative levels within 4–5 weeks. Plasma levels of e₁ and P were not markedly altered. After fetectomy, peripheral estrogen concentrations, especially E₂, declined markedly. In the second experimental series, we have examined the effects of maternal hypophysectomy on levels of E₁, E₂ and P either (1) in both mother and newborn baby or (2) in mother, term fetus and umbilical vein. Groups of hypophysectomized and intact pregnant monkeys (3 each) were delivered by cesarean section at the expected time of parturition. Other hypophysectomized and intact monkeys (2 each) delivered normally. E₂ levels were elevated significantly in plasma of hypophysectomized monkeys at the time of cesarean delivery and in newborn babies of hypophysectomized mothers shortly after parturition. Except for these differences, the maternal hypophysis apparently is not a major factor in the control of E₁, E₂ and P concentrations in pregnant rhesus monkeys.     

The design,synthesis and evaluation of 2-aminobenzoxazole analogues as potent and orally efficacious ChemR23 inhibitors

We previously reported 2-aminobenzoxazole analogue 1 as a potent ChemR23 inhibitor. The compound showed inhibitory activity against chemerin-induced calcium signaling through ChemR23 internalization in CAL-1 cells, which are cell lines of plasmacytoid dendric cells (pDCs). Furthermore, compound 2 inhibited chemotaxis of CAL-1 triggered by chemerin in vitro. However, we noted a difference in the ChemR23 response to our inhibitor between rodents and non-rodents in a previous study. To address this issue, we performed optimization of ChemR23 inhibitors using CAL-1 cells endogenously expressing human ChemR23 and conducted a pharmacokinetics study in cynomolgus monkeys. Various substituents at the 4-position of the benzoxazole ring exhibited potent in vitro bioactivity, while those at the 6-position were not tolerated. Among substituents, a carboxyl group was identified as key for improving the oral bioavailability in cynomolgus monkeys. Compound 38a with the acidic part changed from a tetrazole group to a 1,2,4-oxadiazol-5-one group to improve bioactivity and pharmacokinetic parameters exhibited inhibitory activity against chemerin-induced chemotaxis in vitro. In addition, we confirmed the ChemR23 internalization of pDCs by compound 38a orally administered to cynomolgus monkeys. These 2-aminobenzoxazole-based ChemR23 inhibitors may be useful as novel immunotherapeutic agents capable of suppressing the migration of pDCs, which are known to be major producers of type I interferons in the lesion area of certain autoimmune diseases, such as systemic lupus erythematosus and psoriasis.     

Effects of PGF2α and PGF2α, 1–15 lactone on the corpus luteum and on early pregnancy in the rhesus monkey

The effects of prostaglandin (PG)F_2α and PGF_2α, 1–15 lactone were compared in luteal phase, non-pregnant and in early pregnant rhesus monkeys. Animals treated with either PG after pretreatment with human chorionic gonadotropin (hCG) had peripheral plasma progesterone concentrations that were not statistically different from those in animals treated with hCG and vehicle. However, menstrual cycle lengths in monkeys treated with PGF_2α, 1–15 lactone were significantly (P <0.02) shorter than those in vehicle treated animals. In the absence of hCG pretreatment, plasma progesterone concentrations were significantly (P <0.008) lower by the second day after the initial treatment with either PGF_2α or PGF_2α, 1–15 lactone than in vehicle treated monkeys. Menstrual cycle lengths in monkeys treated with either PG were significantly (P <0.04) shorter than those in animals treated with vehicle. There were no changes in plasma progesterone concentrations in early pregnant monkeys treated with PGF_2α, and pregnancy was not interrupted. In contrast, plasma progesterone declined and pregnancy was terminated in 5 of 6 early pregnant monkeys treated with PGF_2α, 1–15 lactone. These data indicate that PGF_2α, 1–15 lactone decreases menstrual cycle lengths in non-pregnant rhesus monkeys. More importantly, PGF_2α, 1–15 lactone terminates early pregnancy in the monkey at a dose which is less than an ineffective dose of PGF_2α.     

Anti-Angiogenic Effects of a Mutant Endostatin: A New Prospect for Treating Retinal and Choroidal Neovascularization

Purpose

Pathological fundus angiogenesis is a major cause of vision loss in retina diseases. Endostatin, a C-terminal fragment of collagen XVIII, is an endogenous anti-angiogenic protein. The present study aimed to investigate the in vitro and in vivo anti-angiogenic properties of two proteins: an N-terminal H1D/H3D mutant endostatin (M-ES) and a polyethylene glycol propionaldehyde (PEG) covalent M-ES (PEG-M-ES).

Methods

M-ES and PEG-M-ES properties were characterized in vitro using a zinc ion binding assay and a stability test. Activity assays, including migration, proliferation, and tube formation assays, were performed with human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs). Mouse oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV) models were used to evaluate in vivo anti-angiogenic effects. In addition, a rabbit model was used to study the retinal pharmacokinetic profile following an intravitreal injection.

Results

The results indicated that the H1D/H3D mutations of endostatin reduced the zinc binding capacity of M-ES and facilitated PEG covalent binding. PEG-M-ES was more stable and persisted longer in the retina compared with M-ES. The in vitro studies demonstrated that M-ES and PEG-M-ES inhibited HRMEC and HUVEC proliferation, migration, and tube formation more efficiently than ES. In vivo, a single intravitreal injection of M-ES and PEG-M-ES significantly decreased neovascularization in both the OIR and CNV animal models.

Conclusion

The present study demonstrated for the first time that PEG-M-ES exhibits a long-term inhibitory effect on neovascularization in vitro and in vivo. These data suggest that PEG-M-ES may represent an innovative therapeutic strategy to prevent fundus neovascularization.     

Menopause in female rhesus monkeys

Fifteen female rhesus monkeys (Macaca mulatto), ranging in age from 8 to 34 years, were studied for one year to characterize the endocrine and menstrual changes associated with menopause in this species. Five monkeys were premenopausal; these younger monkeys, ages 8–11 years, menstruated and showed cyclic ovarian activity during the 12–month study period, as evidenced by menses and periodic elevations of serum estradiol (E₂) and luteinizing hormone (LH) concentrations. Four females, ages 24–26 years, were in transition to menopause. Two of these perimenopausal females menstruated and secreted E₂ and LH in a periodic fashion; the other two females showed elevated LH concentrations, consistently low E₂ levels, and no evidence of menstruation. Six females, ages 27–34 years, were clearly postmenopausal; LH concentrations were high, whereas E₂ concentrations were uniformly low. There was a significant inverse correlation between basal E₂ concentrations and age, and a significant positive correlation between age and LH concentrations across all 15 animals. Hormonal changes indicative of ovulation, when they occurred, were generally restricted to the winter and early spring months. Histological analysis of ovaries from four postmenopausal females revealed little or no evidence of active folliculogenesis. These data indicate that menopause in female rhesus monkeys does not occur until the second half of thethird decade of life. © 1995 Wiley-Liss, Inc.     

Characterization and comparison of testicular LH/hCG receptors of rhesus monkeys (Macaca mulatta) and green monkeys (Cercopithecus aethiops)

Testicular luteinizing hormone (LH/hCG) receptors were characterized in seven green monkeys and compared with those of four rhesus monkeys. Testicular tissue showed high binding affinity for ¹²⁵I-hCG, (0.9–2.5 × 10⁹ M^?1, and 0.7–1.64 × 10⁹ M^?1 respectively, for green and rhesus monkeys) and low binding capacity (0.343–0.682 fmol/mg and 0.198–0.355 fmol/mg testicular homogenate, respectively). There was no difference in binding affinity between the two groups. Testicular LH/hCG receptors in both species bound human LH (hLH) and hCG but did not cross react with ovine LH (oLH). Rat testicular tissue showed similar high binding affinity (6.4 × 10⁹ M^?1) and low binding capacity (1.04 fmol/mg tissue homogenate) for ¹²⁵I-hCG. Rat LH/hCG receptors bound hLH, hCG, and oLH to a similar degree.     

Effect of bone strain on cortical bone structure in macaques (Macaca mulatta)

It has recently been shown that the consistency of food significantly affects levels of bone strain in the mandible during mastication (Hylander, '79a). Mandibular bone histology was examined to test the effects of a diet of hard food compared to a diet of soft food in two group of monkeys. One group of rhesus macaques (Macaca mulatta) was fed a diet of commercially prepared hard biscuits. The second group was fed a soft diet the consistency of fudge. Both diets were nutritionally adequate for normal growth and development. As a control for other factors influencing cortical bone structure, fibular morphology was also examined. At the end of the test period, mandibular and fibular tissue samples from the two groups were prepared to determine the amount of secondary Haversian bone present. Mandibular depth at M₂ and fibular anteroposterior diameter were also measured and compared between the two dietary groups. The soft-diet monkeys showed low levels of remodeling in their mandibles. There were large patches of unremodeled bone and resorption spaces were common. The hard-diet monkeys exhibited more extensive evidence of secondary Haversian remodeling in their mandibles. The hard-diet monkeys also had deeper mandibles. In contrast, the fibulae from the two groups had similar mean diameters and showed comparable levels of secondary remodeling. We infer that the higher mandibular bone remodeling levels in the hard-diet monkeys represent an adaptive response to remove and replace fatigued mandibular bone due to higher stress levels associated with the ingestion and mastication of hard foods. We also infer that greater depth of the mandible at M₂ found in the harddiet group represents an adaptive response to higher stress levels associated with the ingestion and mastication of hard foods.