Many patients with symptomatic bone metastases receive radiation therapy, even though radiation is known to have potential adverse effects on bone. We hypothesized that the concurrent use of a bisphosphonate drug (zoledronic acid, ZA) or a combination of ZA plus an anabolic agent (parathyroid hormone, PTH) would lead to improvements in the microarchitecture and mechanical properties of irradiated bone. Human breast cancer cells were injected into the distal femur of 56 female nude mice, which were then divided into four groups: no treatment (0 Gy), radiation administered 4 weeks postinjection (20 Gy), radiation plus ZA (12.5 μg/kg weekly from weeks 4 to 12) (20 Gy + ZA), and radiation followed by ZA (25 μg/kg weekly from weeks 4 to 8) and PTH(1–34) (100 μg μg/kg daily from weeks 8 to 12) (20 Gy + ZA + PTH). Left limbs served as normal control bones. Bone loss over the 12-week study was tracked with serial radiography and bone densitometry. At the end of the study, micro-computed tomography and mechanical testing were used to quantify bone microarchitecture and bone strength. Radiation alone failed to prevent tumor-induced decreases in bone mineral density (BMD), trabecular bone volume, and bone strength. Treatment with 20 Gy + ZA or 20 Gy + ZA + PTH as adjuncts to radiation was effective at preserving trabecular bone architecture and bone strength at normal levels. ZA reduced the risk of mechanical fragility following irradiation of a lytic bone lesion. Supplemental use of PTH did not result in further increases in bone strength but was associated with significant increases in BMD and bone mass, suggesting that it may be beneficial in enhancing bone architecture following radiation therapy.

Introduction Lipopeptides are potent and non-toxic adjuvants. Peptides by themselves are poor immunogens, even when incorporated in liposomes. For effective interaction with both the innate and adaptive immune responses, peptides must be conjugated with one of several known lipid moieties. Trior dipalmitoyl-S-glyceryl-Cys-Ser-Ser, based on a bacterial lipoprotein, was found to be an effective adjuvant [1] that was later replaced by the more readily synthesized dipalmitoyl-Lys-Ser-Ser [2]. The purification of lipopeptides remains a problem, however, due to low aqueous solubility and the loss of resolution using traditional HPLC technology. Moreover, difficulties in their analysis hinder the use of such peptide-based vaccines in human clinical experiments. Purification of the unprotected peptides prior to conjugation of whatever lipid modification being used is highly desirable. Methods now available rely on exploitation of Cys residues for conjugation. Maleimide-based conjugation is simple, but is not applicable if there is a Cys within the epitope sequence, unless it can be left blocked until the final purification. Thioester-based coupling, commonly used in native chemical ligation of peptides is very attractive because it is specific for an N-terminal Cys and results in a normal peptide bond. A protocol of choice would provide specific lipid conjugation to any peptide. We synthesized lipopeptides containing epitopes from ovalbumin (OVA257-264, SIINFEKL), tyrosine-related protein (TRP2180-188, SVYDFFVWL), and a melanoma antigen (MART26-35, EAAGIGILTV) by Fmoc-protocol. After purification, these were lipidated by chemoselective native ligation using either maleimide [3] or thioester [4] conjugation.

Parathyroid hormone (PTH) is a major regulator of serum calcium homeostasis. The smallest fully bioactive PTH(1-31) consists of two helixes joined by a short flexible sequence. The C-terminal amphiphilic α-helix binds to the N-terminal extracellular region of the PTH receptor (P1R). Backbone methylation [1] and Ala-scan [2] studies showed that three residues, Arg20, Trp23 and Leu24 are critical for bioactivity and play a critical role in PTH-P1R interaction. We studied 2D-NMR structures of hPTH(1-31) analogues modified in the C-terminal helical region: bioactive cyclic(Glu22-Lys26), backbone methylated at Leu24 inactive linear and its cyclic (Glu22-Lys26) analogue with restored bioactivity. Detailed conformation studies and geometrical optimization of the structures revealed similar orientations of Arg20, Trp23 and Leu24 side-chains in the bioactive analogues. In both cyclic active analogues, Arg20 aligned with Trp23 and Leu24. The inactive linear methylated analogue has the critical Arg20 and Leu24 side-chains oriented away from Trp23.

Introduction The C-terminal function affects the structure (CD) and bioactivity of hPTH(1-28) analogues [1]. The hPTH with an amide C-terminus is more helical and bioactive than the analogue in a natural carboxyl form. The monomethylamide exhibited the greatest helicity and binding activity in contrast to the dimethylamide, which has helical parameters similar to the acid. Thus, removal of potential H-bond NH donors on the C-terminus destabilizes α-helix structure and decreases bioactivity. The CD profile of the aldehyde analogue is close to the bioactive monomethylamide, implying that the aldehyde function stabilizes the C-terminal helical structure of hPTH(1-28). However, the aldehyde, like the dimethylamide, demonstrated a weaker receptor binding than the monomethylamide. The structure of hPTH(1-28) analogues with C-terminal aldehyde, monoand dimethylamides were studied by high resolution 2D-NMR in aqueous solution at neutral pH.

We have studied the effects of C-terminal group modifications (amide, methylamide, dimethylamide, aldehyde, and alcohol) on the conformation, adenylyl cyclase stimulation (AC), or binding of parathyroid hormone (hPTH) analogues, hPTH(1-28)NH(2) and hPTH(1-31)NH(2). hPTH(1-31)NH(2) has a C-terminal alpha-helix bounded by residues 17-29 [Chen, Z., et al. (2000) Biochemistry 39, 12766]. In both cases, relative to the natural analogue with a carboxyl C-terminus, the amide and methylamide had increased helix content whereas the dimethylamide forms had CD spectra more similar to the carboxyl one. Conformational effects were more pronounced with hPTH(1-28) than with hPTH(1-31), with increases in helix content of approximately 30% in contrast to 10%. Stabilization of the C-terminal helix of residues 1-28 seemed to correlate with an ability of the C-terminal function to H-bond appropriately. None of the analogues affected the AC stimulating activity significantly, but there was an up to 15-fold decrease in the level of apparent binding of the carboxyl hPTH(1-28) analogue compared to that of the methylamide and a 4-fold decrease in the level of binding of the aldehyde or dimethylamide. There was no significant change in binding activities for the 1-31 analogues. These observations are consistent with previous studies that imply the importance of a region of the hormone's C-terminal alpha-helix for tight binding to the receptor. They also show that modulation of helix stability does have an effect on the binding of the hormone, but only when the C-terminus is at the putative end of the helix. The similarity of AC stimulation even when binding changed 10-fold can be explained by assuming greater efficacy of the weaker binding PTH-receptor complexes in stimulating AC.

The parathyroid hormone (PTH) and some of its fragments and analogs stimulate bone growth in various animal models and humans and one of them (hPTH-(1–34)) has been approved by the USFDA for treating osteoporosis. However, there are reports that PTH can stimulate the PI-3 kinase/mitogen-activated protein kinases-mediated proliferation of rat enterocytes and that primary hyperparathyroidism in humans is associated with an increased incidence of colon cancer. Here we have investigated the ability of two PTH fragments, hPTH-(1–34)NH2 and [Leu27]cyclo(Glu22-Lys26)hPTH-(1–31)NH2 to initiate colon carcinogenesis or increase the initiatory activity of the widely used colon carcinogen azoxymethane (AOM). The initiation of colon carcinogenesis by AOM was indicated by the very early appearance of aberrant crypt foci. While both PTH peptides strongly stimulated femoral bone formation, they did not cause the appearance of ACFs or affect the number or the distribution along the colon of AOM-induced ACFs. Nor did AOM affect the PTHs’ ability to stimulate bone formation. Thus, a relatively short PTH treatment that is long enough to strongly stimulate bone formation does not initiate colon carcinogenesis in rats.

In the new millennium, humans will be traveling to Mars and eventually beyond with skeletons that respond to microgravity by self-destructing. Meanwhile in Earth's aging populations growing numbers of men and many more women are suffering from crippling bone loss. During the first decade after menopause all women suffer an accelerating loss of bone, which in some of them is severe enough to result in "spontaneous" crushing of vertebrae and fracturing of hips by ordinary body movements. This is osteoporosis, which all too often requires prolonged and expensive care, the physical and mental stress of which may even kill the patient. Osteoporosis in postmenopausal women is caused by the loss of estrogen. The slower development of osteoporosis in aging men is also due at least in part to a loss of the estrogen made in ever smaller amounts in bone cells from the declining level of circulating testosterone and is needed for bone maintenance as it is in women. The loss of estrogen increases the generation, longevity, and activity of bone-resorbing osteoclasts. The destructive osteoclast surge can be blocked by estrogens and selective estrogen receptor modulators (SERMs) as well as antiosteoclast agents such as bisphosphonates and calcitonin. But these agents stimulate only a limited amount of bone growth as the unaffected osteoblasts fill in the holes that were dug by the now suppressed osteoclasts. They do not stimulate osteoblasts to make bone--they are antiresorptives not bone anabolic agents. (However, certain estrogen analogs and bisphosphates may stimulate bone growth to some extent by lengthening osteoblast working lives.) To grow new bone and restore bone strength lost in space and on Earth we must know what controls bone growth and destruction. Here we discuss the newest bone controllers and how they might operate. These include leptin from adipocytes and osteoblasts and the statins that are widely used to reduce blood cholesterol and cardiovascular damage. But the main focus of this article is necessarily the currently most promising of the anabolic agents, the potent parathyroid hormone (PTH) and certain of its 31- to 38-aminoacid fragments, which are either in or about to be in clinical trial or in the case of Lilly's Forteo [hPTH-(1-34)] tentatively approved by the Food and Drug Administration for treating osteoporosis and mending fractures.

The susceptibility to traumatic fracturing of osteopenic bones, and the spontaneous fracturing of osteoporotic bones by normal body movements caused by the microstructural deterioration and loss of bone, are currently treated with antiresorptive drugs, such as the bisphosphonates, calcitonin, estrogens, and selective estrogen receptor modulators. These antiresorptive agents target osteoclasts and, as their name indicates, reduce or stop bone resorption. They cannot directly stimulate bone formation, increase bone mass above normal values in ovariectomized rat models, or improve microstructure.

Protective immunity to intracellular bacterial pathogens usually requires the participation of specific CD8+ T cells. Natural exposure of the host to sublethal infection, or vaccination with attenuated live vaccines are the most effective means of eliciting prolonged protective cell-mediated immunity against this class of pathogens. The ability to replace these immunization strategies with defined sub-unit vaccines would represent a major advance for clinical vaccinology. The present study examines the ability of novel liposomes, termed archaeosomes, made from the polar lipids of various Archaeobacteria to act as self-adjuvanting vaccine delivery vehicles for such defined acellular antigens. Using infection of mice with Listeria monocytogenes as a model system, this study clearly demonstrates the ability of defined, archaeosome-entrapped antigens to elicit rapid and prolonged specific immunity against a prototypical intracellular pathogen. In this regard, all of the tested archaeosomes were superior to conventional liposomes.

Analogues de l'hormone parathyroidienne humaine (PTH) qui possedent une activite de reconstitution osseuse et une biodisponibilite accrues. Les analogues decrits sont soit des analogues cycliques simples (1-28) ou (1-29), soit des analogues cycliques doubles (1-28) a (1-31). Les analogues cycliques simples sont cyclises entre les paires d'acides amines R22 et R26. Les analogues cycliques doubles sont cyclises entre les paires d'acides amines 13 et 17, et 22 et 26. Diverses substitutions des restes naturels par d'autres acides amines sont egalement decrites. Par exemple, le reste naturel Lys27 peut etre substitue par Leu. Typiquement, ces nouveaux analogues possedent des capacites ameliorees pour stimuler l'activite de l'adenyl cyclase dans des cellules d'osteosarcome murines, et presentent une activite amelioree en matiere de reconstitution osseuse constatee sur la rate ayant subi l'ablation des ovaires.

Temporal lobe epilepsy remains one of the most widespread seizure disorders in man, the etiology of which is controversial. Using new rat models of temporal lobe epilepsy that are either prone or resistant to develop complex partial seizures, we provide evidence that this seizure susceptibility may arise from arrested development of the GABAAreceptor system. In seizure-prone (Fast kindling) and seizure-resistant (Slow kindling) rat models, both the mRNA and protein levels of the major α subunit expressed in adult brain (α1), as well as those highly expressed during development (α2, α3, and α5), were differentially expressed in both models compared with normal controls. We found that α1subunit mRNA expression in the Fast kindling strain was approximately half the abundance of control rats, whereas in the Slow kindling strain, it was ∼70% greater than that of controls. However, Fast rats overexpressed the α2, α3, and α5(“embryonic”) subunits, having a density 50–70% greater than controls depending on brain area, whereas the converse was true of Slow rats. Using subunit-specific antibodies to α1and α5subunits, quantitative immunoblots and immunocytochemistry revealed a concordance with the mRNA levels. α1protein expression was ∼50% less than controls in the Fast strain, whereas it was 200% greater in the Slow strain. In contrast, α5subunit protein expression was greater in the Fast strain than either the control or Slow strain. These data suggest that a major predispositional factor in the development of temporal lobe epilepsy could be a failure to complete the normal switch from the GABAAreceptor α subunits highly expressed during development (α2, α3, and α5) to those highly expressed in adulthood (α1).

PTH is an 84-amino acid protein. Occupancy of its cognate receptor generally results in activation of adenylyl cyclase and/or phosphoinositide-specific phospholipase Cbeta (PLCbeta). In the kidney, PTH receptors are present on proximal and distal tubule cells. In proximal tubules, PTH induces calcium signaling, typified by a transient rise in intracellular calcium ([Ca2+]i) and inositol trisphosphate formation, but does not affect calcium absorption. By contrast, in distal tubules, PTH increases calcium absorption that is associated with a slow and sustained rise in [Ca2+]i, but does not stimulate phospholipase C (PLC) or cause inositol trisphosphate accumulation. Nonetheless, stimulation of distal calcium transport requires activation of protein kinase C (PKC) and protein kinase A. We now characterize the origin of the differential effects of ligand occupancy by using synthetic human PTH analogs that preferentially activate adenylyl cyclase and/or PLCbeta. We further tested the hypothesis that phospholipase D is responsible for PKC activation in distal tubule cells. PTH-(1-31) increased [Ca2+]i in distal tubule but not in proximal tubule cells, whereas PTH-(3-34) caused a partial increase in [Ca2+]i in proximal cells, but had no effect in distal cells. PTH-(7-34) blocked increases in [Ca2+]i in distal tubule cells stimulated by PTH-(1-34) and PTH-(1-31). The PLC inhibitor U73122 abolished the PTH-induced rise in [Ca2+]i and inositol trisphosphate formation by proximal tubule cells, but had no effect on PTH-stimulated Ca2+ uptake by distal tubule cells. These results support the view that activation of PKC by PTH in distal tubule cells does not involve PLCbeta. PTH did, however, activate phospholipase D with attendant formation of diacylglycerol in distal cells. As activation of PKC is required for induction of calcium transport by PTH, we conclude that PTH receptors are capable of activating multiple phospholipases and that the structural requirements for such activation differ in proximal and distal tubule cells.