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Cysteine cathepsins are a family of proteases that are relevant therapeutic targets for the treatment of different cancers and other diseases. However, no clinically approved drugs for these proteins exist, as their systemic inhibition can induce deleterious side effects. To address this problem, we developed a modular antibody-based platform for targeted drug delivery by conjugating non-natural peptide inhibitors (NNPIs) to antibodies. NNPIs were functionalized with reactive warheads for covalent inhibition, optimized with deep saturation mutagenesis and conjugated to antibodies to enable cell-type-specific delivery. Our antibody–peptide inhibitor conjugates specifically blocked the activity of cathepsins in different cancer cells, as well as osteoclasts, and showed therapeutic efficacy in vitro and in vivo. Overall, our approach allows for the rapid design of selective cathepsin inhibitors and can be generalized to inhibit a broad class of proteases in cancer and other diseases.

Signaling through chemokine receptor, C‐X‐C chemokine receptor type 4 (CXCR4) regulates essential processes in normal physiology, including embryogenesis, tissue repair, angiogenesis, and trafficking of immune cells. Tumors co‐opt many of these fundamental processes to directly stimulate proliferation, invasion, and metastasis of cancer cells. CXCR4 signaling contributes to critical functions of stromal cells in cancer, including angiogenesis and multiple cell types in the tumor immune environment. Studies in animal models of several different types of cancers consistently demonstrate essential functions of CXCR4 in tumor initiation, local invasion, and metastasis to lymph nodes and distant organs. Data from animal models support clinical observations showing that integrated effects of CXCR4 on cancer and stromal cells correlate with metastasis and overall poor prognosis in >20 different human malignancies. Small molecules, Abs, and peptidic agents have shown anticancer efficacy in animal models, sparking ongoing efforts at clinical translation for cancer therapy. Investigators also are developing companion CXCR4‐targeted imaging agents with potential to stratify patients for CXCR4‐targeted therapy and monitor treatment efficacy. Here, pre‐clinical studies demonstrating functions of CXCR4 in cancer are reviewed. Review examines CXCR4‐dependent mechanisms in tumor environments of solid cancers, the current status of radioimaging for CXCR4, and the landscape of CXCR4 modulators.

Crystallization is a fundamental process in materials science, providing the primary route for the realization of a wide range of new materials. Crystallization rates are also considered to be useful probes of glass-forming ability1–3. At the microscopic level, crystallization is described by the classical crystal nucleation and growth theories4,5, yet in general solid formation is a far more complex process. In particular, the observation of apparently different crystal growth regimes in many binary liquid mixtures greatly challenges our understanding of crystallization1,6–12. Here, we study by experiments, theory and computer simulations the crystallization of supercooled mixtures of argon and krypton, showing that crystal growth rates in these systems can be reconciled with existing crystal growth models only by explicitly accounting for the non-ideality of the mixtures. Our results highlight the importance of thermodynamic aspects in describing the crystal growth kinetics, providing a substantial step towards a more sophisticated theory of crystal growth.

Expressed on virtually all prostate cancers and their metastases, the transmembrane protein prostate-specific membrane antigen (PSMA) provides a valuable target for the imaging of prostate cancer. Not only does PSMA provide a target for noninvasive diagnostic imaging, e.g., PSMA-positron emission tomography (PSMA–PET), it can also be used to guide surgical resections of PSMA-positive lesions. The latter characteristic has led to the development of a plethora of PSMA-targeted tracers, i.e., radiolabeled, fluorescent, or hybrid. With image-guided surgery applications in mind, this review discusses these compounds based on clinical need. Here, the focus is on the chemical aspects (e.g., imaging label, spacer moiety, and targeting vector) and their impact on in vitro and in vivo tracer characteristics (e.g., affinity, tumor uptake, and clearance pattern).

X-ray spectroscopy is a method, ideally suited for investigating the electronic structure of matter, which has been enabled by the rapid developments in light sources and instruments. The x-ray fluorescence lines of life-relevant elements such as carbon, nitrogen, and oxygen are located in the soft x-ray regime and call for suitable spectrometer devices. In this Letter, we present a high-resolution spectrum of liquid water, recorded with a soft x-ray spectrometer based on a reflection zone plate (RZP) design. The RZP-based spectrometer with meridional variation of line space density from 2953 to 3757 l/mm offers extremely high detection efficiency and, at the same time, medium energy resolution. We can reproduce the well-known splitting of liquid water in the lone pair regime with 10 s acquisition time.

The rapid increase and subsequent decline in myoglobin concentration which is characteristic of the guinea pig after 15, 21 and 30 days on a Vit. E-deficient diet is largely prevented by a thrice-weekly administration of DPPD. Under the same regimen, MB is not effective and exhibits considerable toxicity for young animals. Both MB and DPPD, when given in 6 daily doses to animals previously kept on a tocopherol-deficient diet for 15 days, bring about a remission of abnormal changes in myoglobin concentration.

The “basal” rate of energy utilization, as measured by PC disappearance, in mouse anterior tibial muscles exposed to a N2-IAA environment is about 0.25 μM/gm per min or 2.5 mcal/gm per min. This rate, as expected, is greater than the “basal” value for amphibian skeletal muscle and less than the “basal” value for mammalian cardiac muscle. By making certain assumptions about active ion transport and by using certain data from other investigators, the conclusion is reached that mammalian skeletal muscle sustains itself with a restricted energy supply much less well than amphibian skeletal muscle.

Resting potentials of frog muscle fibers incubated in normal or high-potassium Ringer's solutions are maintained for longer periods of time by the addition of aldosterone. This response is absent in ouabain-Ringer's solution. Although based upon separate groups of muscles, the results indicate that muscle fibers in high-potassium Ringer's solution are slightly hyperpolarized when ouabain also is present.

A convenient synthesis of novel bifunctional poly(amino carboxylate) chelating agents allowing chemoselective attachment to highly functionalized biomolecules is described. Based on the well known chelator 1,4,7,10‐tetraazacyclodecane‐1,4,7,10‐tetraacetic acid (DOTA), we synthesized novel bifunctional chelating agents bearing additional functional groups by alkylating 1,4,7,10‐tetraazacyclododecane (cyclen) with one equivalent of para‐functionalized alkyl 2‐bromophenyl‐acetate and three equivalents of tert‐butyl 2‐bromoacetate. The resulting compounds, which contain an additional carbonyl or alkyne functionality, allow site specific labeling of appropriately functionalized unprotected biomolecules in a rapid manner via click reactions. This was demonstrated by the attachment of our new DOTA derivatives to the somatostatin analogue Tyr3‐octreotate by chemoselective oxime ligation and CuI‐catalyzed azide–alkyne cycloaddition. Initial biodistribution studies in mice with the radiometalated compound demonstrated the applicability of the described DOTA conjugation.

Coating of artificial surfaces with RGD (= arginine-glycine-aspartate) peptides to enhance cell adhesion is an ongoing issue. Thereby, the physiological adhesion process to the extra-cellular matrix (ECM) is mimicked by the peptide coating, leading to a strong cell-surface contact, followed by spreading and proliferation of the cells. For comparable cell adhesion studies, it is important to know the density of the RGD peptides on the surface. Here, we present an approach to determine the amount of bound cyclic RGD peptide by radio labeling with 125I of a tyrosine-containing RGD peptide on different materials surfaces (poly(methyl methacrylate) (PMMA), titanium, and silicon). For all surfaces, the amount of bound peptides is in the range of pmol/cm1.

For effective targeting of somatostatin receptor (sst) expressing tumors by radiolabeled octreotide analogues, high ligand uptake into sst-positive cells is mandatory. To optimize it, two modifications have been introduced into [¹²⁵I]Tyr³-octreotide ([¹²⁵I]TOC):  C-terminal Thr-for-Thr(ol) exchange (leading to Tyr³-octreotate (TOCA)) and N-terminal derivatization with different carbohydrates. Both have significant impact on radioligand uptake into sst2-expressing cells in vitro and in vivo. Glucose conjugation via Amadori reaction by itself led to improved tumor uptake of [¹²³I]Gluc-TOC in vivo, which is based on an enhancement of peptide internalization despite a reduction in receptor affinity. In the case of the doubly modified analogues [¹²³I]Gluc-TOCA, [¹²³I]Gluc-S-TOCA, and [¹²³I]Gal-S-TOCA, a cumulative effect of both structural modifications was observed, leading up to a 5-fold increased uptake of these compounds in sst-expressing tumors compared to [¹²⁵I]TOC. Thus, glycosylation with small carbohydrates was found to be a suitable tool to enhance receptor-mediated uptake of radiolabeled octreotide analogues into sst-positive malignancies, leading to tracers with excellent characteristics for in vivo sst-imaging applications.

Sugar conjugation of biooactive peptides has been shown to be a powerful tool to modulate peptide pharmacokinetics. In the case of radiolabeled somatostatin analogues developed for in vivo scintigraphy of somatostatin receptor (sst) expressing tumors, it generally led to tracers with predominant renal excretion and low uptake in nontarget organs, and in some cases also with enhanced tumor accumulation. Especially with respect to endoradiotherapeutic applicability of these tracers, however, understanding the structural requirements for minimal kidney accumulation and maximal tumor uptake is important. The aim of this study was therefore the evaluation of the potential of specific glycoside structures in combination with reduced peptide net charge to reduce kidney accumulation without affecting tumor accumulation. Three glyco analogues of radioiodinated Tyr³-octreotate (TOCA) with z = 0 were evaluated in a comparative study using [¹²⁵I]Mtr-TOCA (z = +1), the maltotriose-Amadori analogue of [¹²⁵I]TOCA, as a reference, [¹²⁵I]Glucuron-TOCA, the Amadori conjugate with glucuronic acid, and [¹²⁵I]Gluc-S- and [¹²⁵I]Gal-S-TOCA, the coupling products with glucosyl- and mannosyl-mercaptopropionate. In cells transfected with sst1−sst5, all three new analogues show sst-subtype binding profiles similar to I-Mtr-TOCA with high, but somewhat reduced, affinity for sst2. In contrast, internalization into sst2-expressing cells (in % of [¹²⁵I]Tyr³-octreotide ([¹²⁵I]TOC)) as well as the EC50,R of unlabeled TOC for internalization determined in dual-tracer experiments are substantially enhanced for [¹²³I]Gal-S-TOCA and [¹²³I]Gluc-S-TOCA (internalization, 190% ± 12% and 265% ± 20%, respectively, vs 168% ± 6% of [¹²⁵I]TOC for [¹²³I]Mtr-TOCA; EC50,R, 2.62 ± 0.07 and 2.96 ± 0.14, respectively, vs 1.81 ± 0.07 for [¹²³I]Mtr-TOCA). The tumor accumulation of [¹²⁵I]Gal-S-TOCA and [¹²⁵I]Gluc-S-TOCA in AR42J tumor-bearing nude mice 1 h p.i. is consequently very high (22.6 ± 2.2 and 26.2 ± 5.6%ID/g) and comparable to that of [¹²⁵I]Mtr-TOCA (25.1 ± 4.4%ID/g). [¹²⁵I]Glucuron-TOCA showed lower uptake in sst-expressing tissues than did [¹²⁵I]Mtr-TOCA, but considerably enhanced accumulation in nontarget organs such as liver, intestine, and kidney. Due to increased lipophilicity, hepatic and intestinal uptake 1 and 4 h p.i. of [¹²⁵I]Gal-S-TOCA and [¹²⁵I]Gluc-S-TOCA was also slightly higher than that of [¹²⁵I]Mtr-TOCA. Kidney accumulation, however, was reduced by approximately 50% for both compounds (2.6 ± 0.3 and 2.2 ± 0.4, respectively, vs 4.0 ± 0.7%ID/g at 1 h p.i.). Because no sugar-specific effect was detected in the latter case, it is concluded that general ligand pharmacokinetics and especially kidney accumulation of the tracers investigated are mainly determined by physicochemical characteristics such as lipophilicity, net charge, and also charge distribution ([¹²⁵I]Glucuron-TOCA vs [¹²⁵I]Gal-S- and [¹²⁵I]Gluc-S-TOCA). With respect to receptor targeting, however, the structure of the carbohydrate moiety plays an important role, leading to dramatically enhanced ligand internalization, especially in the case of [¹²³I]Gluc-S-TOCA. Taking into account the combined effects of the Gluc-S-moiety both on kidney and on tumor accumulation, this group seems to be a promising synthon for the synthesis of other radiolabeled peptide analogues with improved pharmacokinetics.

Lymphocytes are targeted to inflamed sites by specific “homing” and chemokine receptors. Most of them, including ligands for P- and E-selectin, are absent from naive CD4+ T cells and become induced after activation and differentiation in effector/memory cells. Polarized effector cells are characterized by the rapid production of distinct cytokines upon restimulation. Their cytokine memory is in part controlled by epigenetic imprinting during differentiation. Here we ask whether a similar mechanism could regulate selectin ligand expression, mediating entry into inflamed sites, notably within the skin. We report that acquisition of selectin ligands by naive but not memory CD4+ cells depends on progression through the G1/S phase of the cell cycle—a phase susceptible to modification of the chromatin structure. Cell-cycle arrest prevented transcriptional activation of glycosyltransferases involved in the generation of selectin ligands, suggesting that progression through the cell cycle is required to unlock their genes. Artificial DNA demethylation strongly increased the frequency of selectin ligand-expressing cells, suggesting that DNA methylation keeps transferase genes inaccessible in naive T cells. Due to these findings we propose that selectin-dependent inflammation-seeking properties are imprinted by epigenetic modifications upon T-cell differentiation into effector cells.

Lymphocytes are targeted to inflamed sites by specific “homing” and chemokine receptors. Most of them, including ligands for P- and E-selectin, are absent from naive CD4+T cells and become induced after activation and differentiation in effector/memory cells. Polarized effector cells are characterized by the rapid production of distinct cytokines upon restimulation. Their cytokine memory is in part controlled by epigenetic imprinting during differentiation. Here we ask whether a similar mechanism could regulate selectin ligand expression, mediating entry into inflamed sites, notably within the skin. We report that acquisition of selectin ligands by naive but not memory CD4+cells depends on progression through the G1/S phase of the cell cycle—a phase susceptible to modification of the chromatin structure. Cell-cycle arrest prevented transcriptional activation of glycosyltransferases involved in the generation of selectin ligands, suggesting that progression through the cell cycle is required to unlock their genes. Artificial DNA demethylation strongly increased the frequency of selectin ligand-expressing cells, suggesting that DNA methylation keeps transferase genes inaccessible in naive T cells. Due to these findings we propose that selectin-dependent inflammation-seeking properties are imprinted by epigenetic modifications upon T-cell differentiation into effector cells.

As part of our ongoing efforts in the development of new 18F-labeled peptides for clinical PET imaging, a new two-step 18F-labeling methodology based on the chemoselective oxime formation between an unprotected aminooxy-functionalized peptide and a 18F-labeled aldehyde was investigated and optimized.

Lipoxin A4 (LXA4) is a structurally and functionally distinct natural product called an eicosanoid, which displays immunomodulatory and anti-inflammatory activity but is rapidly metabolized to inactive catabolites in vivo. A previously described analogue of LXA4, methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), was shown to have a poor pharmacokinetic profile after both oral and intravenous administration, as well as sensitivity to acid and light. The chemical stability of the corresponding E,E,E-trien-11-yne analogue, 3, was improved over 2 without loss of efficacy in the mouse air pouch model of inflammation. Careful analysis of the plasma samples from the pharmacokinetic assays for both 2 and 3 identified a previously undetected metabolite, which is consistent with metabolism by β-oxidation. The formation of the oxidative metabolites was eliminated with the corresponding 3-oxatetraene, 4, and the 3-oxatrien-11-yne, 5, analogues of 2. Evaluation of 3-oxa analogues 4 and 5 in calcium ionophore-induced acute skin inflammation model demonstrated similar topical potency and efficacy compared to 2. The 3-oxatrien-11-yne analogue, 5, is equipotent to 2 in an animal model of inflammation but has enhanced metabolic and chemical stability and a greatly improved pharmacokinetic profile.

Among a variety of other factors, the clearance kinetics and routes of excretion of radiopharmaceuticals are of crucial importance for early and high tumor/background ratios and thus signal intensity in diagnostic imaging by single photon emission tomography (SPECT) or positron emission tomography (PET). To overcome the unfavorable pharmacokinetics of radiohalogenated octreotide analogues, we evaluated three carbohydrated conjugates of Tyr³-octreotide (TOC). Glucose ([¹²⁵I]Gluc-TOC), maltose ([¹²⁵I]Malt-TOC), and maltotriose ([¹²⁵I]Mtr-TOC) derivatives of [¹²⁵I]TOC were synthesized via Maillard reaction and subsequent radioiodination. In cells transfected with sst1-sst5, I-Malt-TOC, and I-Mtr-TOC show sst-subtype binding profiles similar to I-TOC with high affinity for sst2. Comparative biodistribution studies 10, 30, and 60 min pi in nude mice bearing rat pancreatic tumor xenografts showed fast blood clearance for all glycosylated derivatives. Due to their markedly increased hydrophilicity, [¹²⁵I]Gluc-TOC and [¹²⁵I]Malt-TOC were mainly cleared via the kidneys, which led to a significant decrease in activity accumulation in liver and intestine (5.3 and 1.4 versus 10.6%ID/g for [¹²⁵I]TOC in the liver, 1.7 and 1.0 versus 3.8%ID/g for [¹²⁵I]TOC in the intestine 60 min pi). For all compounds, hydrophilicity and uptake in liver and intestines correlate. Uptake of the carbohydrate conjugates in the kidney was comparable. Compared to the parent compound, the accumulation of the carbohydrated compounds in sst-rich tissues (pancreas, adrenals) was increased by a factor of 1.5−3.5. While tumor uptake of [¹²⁵I]TOC (6.7 ± 2.6%ID/g), [¹²⁵I]Malt-TOC (5.3 ± 1.9%ID/g), and [¹²⁵I]Mtr-TOC (4.9 ± 2.2%ID/g) at 30 min postinjection was comparable, accumulation of [¹²⁵I]Gluc-TOC was significantly increased (10.1 ± 2.8%ID/g at 30 min pi). Somatostatin receptor specificity of tumor uptake was confirmed by pretreatment, competition, and displacement experiments in vivo using 0.8 mg TOC/kg and γ-camera imaging. Glycosylation proved to be a powerful tool for the development of high affinity sst ligands with excellent excretion profiles and improved tumor accumulation.

Microsporidia of the genus Encephalitozoonare increasingly being reported as a cause of severe, often disseminated infections, mainly in patients with acquired immunodeficiency syndrome (AIDS). Immunological identification of each of the three recognized species (E. cuniculi, E. hellem, and E. intestinalis) requires the availability of specific immune sera. All sera available thus far have been generated by direct inoculation of rabbits with virulent microsporidian spores. This study demonstrates for the first time that subcutaneous immunization with inactivated spores of E. cuniculi, E. hellem, or E. intestinalisis capable of generating highly active rabbit hyperimmune sera to the homologous antigens, with maximal titers being 1:5,120, 1:1,280, and 1:2,560, respectively, as determined by the indirect immunofluorescence technique (IIF). Broad cross-reactivity of the rabbit antisera with all heterologous Encephalitozoonantigens was determined by IIF and immunogold electron microscopy; however, only the E. hellemimmune serum strongly cross-reacted with spores of Enterocytozoon bieneusi. During the 35-month follow-up period the antibody titers to the homologous antigens declined to 1:640, 1:160, and 1:320, respectively. The observed decay curves for antibody titers against E. cuniculi, E. hellem, and E. intestinaliswere fitted using mathematical modeling, resulting in a predicted duration for specific immune responses of about 7 years on average. Knowledge of the magnitude and duration of specific immune responses is a prerequisite for further evaluation of the concept of using inactivated microsporidian spores in the quest for vaccines against microsporidian infections.

Interleukin (IL)-1β signals through various adapter proteins and kinases that lead to activation of numerous downstream targets, including the transcription factors including NF-κB. In this study, we analyzed and characterized the effect of the differentiation of intestinal epithelial cells on IL-1β-mediated NF-κB activation and IL-8 gene expression. We report that IL-8 mRNA accumulation and protein secretion were down-regulated in IL-1β- and lipopolysaccharide-stimulated differentiated HT-29 cells (HT-29/MTX, where MTX is methotrexate) compared with undifferentiated cells (HT-29/p), whereas no differential effects were found following tumor necrosis factor (TNF)-α or phorbol myristate acetate stimulation. Cross-linking and affinity binding studies reveal that IL-1β exclusively binds the type I receptor (IL-1RI) and not IL-1RII in both HT-29/p and HT-29/MTX cells. IL-1β-mediated IκB kinase and c-Jun N-terminal kinase (JNK) activity were both diminished in differentiated HT-29 cells. DNA binding activity in differentiated HT-29 cells relative to HT-29/p cells was strongly reduced following IL-1β exposure but not after TNF-α stimulation. The proximal IL-1 signaling molecule IL-1 receptor-associated kinase was not degraded in IL-1β-stimulated HT-29 cells, in contrast to Caco-2 cells. κB-luciferase reporter gene activity was 16-fold higher following TNF receptor-associated factor-6 transfection after IL-1β stimulation in HT-29/MTX cells. We conclude that cellular differentiation of HT-29 cells selectively impairs the IL-1β signaling pathway inhibiting both NF-κB and JNK activity in response to IL-1β. This relative unresponsiveness to IL-1β may represent an important regulatory mechanism of differentiated intestinal epithelial cells.

Interleukin (IL)-1beta signals through various adapter proteins and kinases that lead to activation of numerous downstream targets, including the transcription factors including NF-kappaB. In this study, we analyzed and characterized the effect of the differentiation of intestinal epithelial cells on IL-1beta-mediated NF-kappaB activation and IL-8 gene expression. We report that IL-8 mRNA accumulation and protein secretion were down-regulated in IL-1beta- and lipopolysaccharide-stimulated differentiated HT-29 cells (HT-29/MTX, where MTX is methotrexate) compared with undifferentiated cells (HT-29/p), whereas no differential effects were found following tumor necrosis factor (TNF)-alpha or phorbol myristate acetate stimulation. Cross-linking and affinity binding studies reveal that IL-1beta exclusively binds the type I receptor (IL-1RI) and not IL-1RII in both HT-29/p and HT-29/MTX cells. IL-1beta-mediated IkappaB kinase and c-Jun N-terminal kinase (JNK) activity were both diminished in differentiated HT-29 cells. DNA binding activity in differentiated HT-29 cells relative to HT-29/p cells was strongly reduced following IL-1beta exposure but not after TNF-alpha stimulation. The proximal IL-1 signaling molecule IL-1 receptor-associated kinase was not degraded in IL-1beta-stimulated HT-29 cells, in contrast to Caco-2 cells. kappaB-luciferase reporter gene activity was 16-fold higher following TNF receptor-associated factor-6 transfection after IL-1beta stimulation in HT-29/MTX cells. We conclude that cellular differentiation of HT-29 cells selectively impairs the IL-1beta signaling pathway inhibiting both NF-kappaB and JNK activity in response to IL-1beta. This relative unresponsiveness to IL-1beta may represent an important regulatory mechanism of differentiated intestinal epithelial cells.

It is well established that cell survival signals stimulated by growth factors, cytokines, and oncoproteins are initiated by phosphoinositide 3-kinase (PI3K)- and Akt-dependent signal transduction pathways. Oncogenic Ras, an upstream activator of Akt, requires NF-κB to initiate transformation, at least partially through the ability of NF-κB to suppress transformation-associated apoptosis. In this study, we show that oncogenic H-Ras requires PI3K and Akt to stimulate the transcriptional activity of NF-κB. Activated forms of H-Ras and MEKK stimulate signals that result in nuclear translocation and DNA binding of NF-κB as well as stimulation of the NF-κB transactivation potential. In contrast, activated PI3K or Akt stimulates NF-κB-dependent transcription by stimulating transactivation domain 1 of the p65 subunit rather than inducing NF-κB nuclear translocation via IκB degradation. Inhibition of IκB kinase (IKK), using an IKKβ dominant negative protein, demonstrated that activated Akt requires IKK to efficiently stimulate the transactivation domain of the p65 subunit of NF-κB. Inhibition of endogenous Akt activity sensitized cells to H-Ras(V12)-induced apoptosis, which was associated with a loss of NF-κB transcriptional activity. Finally, Akt-transformed cells were shown to require NF-κB to suppress the ability of etoposide to induce apoptosis. Our work demonstrates that, unlike activated Ras, which can stimulate parallel pathways to activate both DNA binding and the transcriptional activity of NF-κB, Akt stimulates NF-κB predominantly by upregulating of the transactivation potential of p65.

ABSTRACTIt is well established that cell survival signals stimulated by growth factors, cytokines, and oncoproteins are initiated by phosphoinositide 3-kinase (PI3K)- and Akt-dependent signal transduction pathways. Oncogenic Ras, an upstream activator of Akt, requires NF-?B to initiate transformation, at least partially through the ability of NF-?B to suppress transformation-associated apoptosis. In this study, we show that oncogenic H-Ras requires PI3K and Akt to stimulate the transcriptional activity of NF-?B. Activated forms of H-Ras and MEKK stimulate signals that result in nuclear translocation and DNA binding of NF-?B as well as stimulation of the NF-?B transactivation potential. In contrast, activated PI3K or Akt stimulates NF-?B-dependent transcription by stimulating transactivation domain 1 of the p65 subunit rather than inducing NF-?B nuclear translocation via I?B degradation. Inhibition of I?B kinase (IKK), using an IKKß dominant negative protein, demonstrated that activated Akt requires IKK to efficiently stimulate the transactivation domain of the p65 subunit of NF-?B. Inhibition of endogenous Akt activity sensitized cells to H-Ras(V12)-induced apoptosis, which was associated with a loss of NF-?B transcriptional activity. Finally, Akt-transformed cells were shown to require NF-?B to suppress the ability of etoposide to induce apoptosis. Our work demonstrates that, unlike activated Ras, which can stimulate parallel pathways to activate both DNA binding and the transcriptional activity of NF-?B, Akt stimulates NF-?B predominantly by upregulating of the transactivation potential of p65.

The transcription factor nuclear factor κB (NF-κB) coordinates the activation of numerous genes in response to pathogens and proinflammatory cytokines and is, therefore, pivotal in the development of acute and chronic inflammatory diseases. In its inactive state, NF-κB is constitutively present in the cytoplasm as a p50-p65 heterodimer bound to its inhibitory protein IκB. Proinflammatory cytokines, such as tumor necrosis factor (TNF), activate NF-κB by stimulating the activity of the IκB kinases (IKKs) which phosphorylate IκBα on serine residues 32 and 36, targeting it for rapid degradation by the 26 S proteasome. This enables the release and nuclear translocation of the NF-κB complex and activation of gene transcription. Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines which are known to be transcriptionally controlled by NF-κB. Conflicting data exists on the effects of IL-10 on TNF- and LPS-induced NF-κB activity in human monocytes and the molecular mechanisms involved have not been elucidated. In this study, we show that IL-10 functions to block NF-κB activity at two levels: 1) through the suppression of IKK activity and 2) through the inhibition of NF-κB DNA binding activity. This is the first evidence of an anti-inflammatory protein inhibiting IKK activity and demonstrates that IKK is a logical target for blocking inflammatory diseases.

The transcription factor nuclear factor kappaB (NF-kappaB) coordinates the activation of numerous genes in response to pathogens and proinflammatory cytokines and is, therefore, pivotal in the development of acute and chronic inflammatory diseases. In its inactive state, NF-kappaB is constitutively present in the cytoplasm as a p50-p65 heterodimer bound to its inhibitory protein IkappaB. Proinflammatory cytokines, such as tumor necrosis factor (TNF), activate NF-kappaB by stimulating the activity of the IkappaB kinases (IKKs) which phosphorylate IkappaBalpha on serine residues 32 and 36, targeting it for rapid degradation by the 26 S proteasome. This enables the release and nuclear translocation of the NF-kappaB complex and activation of gene transcription. Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines which are known to be transcriptionally controlled by NF-kappaB. Conflicting data exists on the effects of IL-10 on TNF- and LPS-induced NF-kappaB activity in human monocytes and the molecular mechanisms involved have not been elucidated. In this study, we show that IL-10 functions to block NF-kappaB activity at two levels: 1) through the suppression of IKK activity and 2) through the inhibition of NF-kappaB DNA binding activity. This is the first evidence of an anti-inflammatory protein inhibiting IKK activity and demonstrates that IKK is a logical target for blocking inflammatory diseases.

Abstract: Nuclear factor (NF)κB is a transcription factor that controls the transcription of a variety of cellular genes regulating the inflammatory response. Many proinflammatory cytokines are transcriptionally regulated by NF-κB, and their increased expression has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Even though it seemed clear that the increase in proinflammatory cytokine production in IBD is crucial for the initiation and perpetuation of chronic intestinal inflammation, the elements governing this dysregulation of enhanced cytokine production remained unclear. This review discusses a series of recent studies that demonstrate the activation of NF-κB in the inflamed mucosa, and that shed new light on the central pathogenic role of NF-κB in chronic intestinal inflammation. In addition to describing the activation of NF-κB, excitement has been generated by reports that define the molecular targets of anti-inflammatory agents, and that demonstrate the effective blockade of NF-κB in intestinal inflammation. These new insights into the activation and inhibition of NF-κB have opened new and promising avenues for a more specific treatment of chronic intestinal inflammation.

Promastigote culture forms of the log growth phase ofLeishamania donovani stock LRC L 51 were investigated for expression of cell-surface carbohydrate-binding sites using 15 types of a chemically glycosylated enzyme termed neoglycoenzyme. Carbohydrate conjugation and coupling yield were kept constant to ensure that the type of carbohydrate moiety, was the only variable feature of the applied tools. Para-aminophenyl derivatives of the following carbohydrate residues were used for the glycosylation of ß-galactosidase fromEscherichia coli: ß-d-lactose, ß-d-thiogalactose, a-d-mannose, a-l-rhamnose, a-d-N-acetylgalactosamine, ß-d-N-acetylgalactosamine, ß-d-N-acetylgalactosamine, ß-d-N-acetylgalactosamine, ß-d-N-acetylglucosamine, the a- and ß-glucosides maltose and cellobiose, ß-d-xylose, a-d-mannose-6-phosphate, the a-galactoside melibiose, a-l-fucose, and ß-d-glucuronic acid as well as sialic acid. Only melibiose, fucose, and glucuronic acid showed no binding affinity for the cultured flagellates; this served as an internal control reaction to exclude any binding to the linker group. This result demonstrates that many but not all sugar types can be recognized by appropriate receptor structure(s) on the surface of the promastigoteLeishmania. Transformation of the binding data for neoglycoenzymes exposing lactose, mannose, rhamnose, andN-acetylated hexose residues, which was carried out to obtain the dissociation constants and to estimate the number of binding sites at saturation, revealedKD values of around 100mm and around 104 binding sites for the polyvalent ligands.

Cell surface components of 4-day-old cultures of the plant parasite Phytomonas sp. and other lower trypanosomatids (Crithidia fasciculata, C. luciliae, C. sp. and Herpetomonas samuelpessoai) were partially identified by lectins. The qualitative data of the investigation allowed us to distinguish Phytomonas sp. from the other parasites Differences in the agglutination titers could be used to differentiate between the five species.

Monoclonal antibodies which react with culture forms ofTrypanosoma dionisii andTrypanosoma cruzi were tested for their agglutination capacity. In these studies developmental stage specificity for the epimastigote form could be observed. This specificity meant it was possible to develop a quick and simple method to isolate cultured metacyclic trypomastigotes ofT. dionisii andT. cruzi group 2. After agglutination of the epimastigote form with the monoclonal antibody Dion 4.6 the purity of metacyclic trypomastigote developmental forms derived from culture was 96% to 99%.

Five days old promastigote culture forms of two stocks of Leishmania enriettii pathogenic and non-infective for Cavia procellus, were tested with the lectins of Canavalia ensiformis, Ricinus communis-120, Soja hispida (Glycine maxima), Arachis hypogaea, Ulex europaeus, Ulex europaeus I, Ulex europaeus II, Laburnum alpinum, Lotus tetragonolobus, Euonymus europaeus and with a monoclonal antibody against blood group H. The pathogenic stock reacted with the anti-H lectin of Lotus tetragonolobus and the monoclonal anti-H and the monoclonal anti-H but not with anti-H of U. europaeus I and E. europaeus. The non-infective stock reacted with none of these anti-H specific agglutinins. They had strong agglutination reactions to C. ensiformis (500 ?g/ml, 1,000 ?g/ml, 10 mg/ml) R. communis-120 (1:10), S. hispida (1,000 ?g/ml) and A. hypogaea (500 ?g/ml, 1,000 ?g/ml, 2,000 ?g/ml). They showed moderate agglutinations to U. europaeus, L. alpinum and U. europaeus II. The non-infective stock showed only moderate reactions to C. ensiformis (10 mg/ml), R. communis-120 (1:10), A. hypogaea (2,000 ?g/ml) and S. hispida (1,000 ?g/ml). Neither N-acetylneuraminic acid nor neuraminidase was detected on the cell surface of both stocks. This result demonstrates clearly that both stocks of L. enriettii differ in their cell surface carbohydrates. The agglutination reactions with lectins of the non-infective stock of L. enriettii are very similar to L.t. major.

Carbohydrates containing terminal nonreducingd N-acetylgalactosamin ord N-acetylglucosamin were identified at the cell surface ofTrypanosoma cruzi by an indirect immunofluorescence test using the lectin of the spongeAaptos papillata. With the method described in this studyT. cruzi andT. cruzi-like strains are easy to distinguish fromT. rangeli, but not fromT. conorhini. The immunofluorescence test presented here may be very helpful for certain diagnostic problems.

X-ray photoelectron spectroscopy (XPS) was used to identify the functional states of carbon existing on the surfaces of various activated charcoals. The relative percentages of carbon, oxygen, and detectable trace elements comprising the activated charcoal surfaces were determined. Analysis of the carbon core-electron binding energy region revealed the existence of one hydrocarbon state (C–H, C–C are indistinguishable) and three oxygen-containing functional states. These states were hydroxyls or ethers (C–O), carbon-yls (C = O), and carboxylic acids or esters (O–C = O). The C–O functional state contributed approximately 60–70% to the total percentage of oxygen-containing states. A very good correlation existed between the apparent areas occupied on the adsorbent surface per phenobarbital molecule and the relative percentages of the C–O functional state. Previously reported heat of displacement results for phenobarbital adsorption are now explained since the C–O state appears to be the primary site involved in the binding of phenobarbital by the activated charcoals.

Promastigote and amastigote forms of human pathogenicLeishmania from the Old and New World, including promastigotes ofL. enriettii, were tested with neoglycoproteins to ascertain the existence of endogenous lectins. These tools expose the chemically coupled sugar that is attached to the inert carrier as a potential ligand for the binding reaction. Agglutination tests demonstrated that the promastigotes of humanLeishmania reacted only with the neoglycoproteinsN-acetyl-d-galactosamine-para-aminophenyl-bovine serum albumin (galNAc-BSA) andN-acetyl-d-glucosamine-para-aminophenyl-bovine serum albumin (glcNAc-BSA), whereas the amastigote forms failed to react with the neoglycoproteins. In contrast, the promastigotes ofL. enriettii were agglutinated by the neoglycoproteind-mannose-bovine serum albumin (man-BSA). The agglutination reactions could be inhibited by the homologous sugarsN-acetyl-beta-d-glucosamine,N-acetyl-beta-d-galactosamine, and alpha-d-mannose. Fluorescence tests yielded the same results. The incubation of the promastigotes with ethylenedinitrolotetraacetic acid (EDTA) prevented their reaction with the neoglycoproteins, whereas the addition of calcium restored it. This result demonstrates thatLeishmania express calcium-dependent lectins that are accessible on their surface.

The isoenzyme pattern of anAcanthamoeba, stock H-1, isolated from a patient with keratitis (Krankenhaus Heidberg, Hamburg) was compared with that of two strains ofA. quina-A. lugdunensis (302-2, 312-1), two stocks ofA. lenticulata (45, 89-1) and one strain ofA. rhysodes (302-1). The isolated stock showed glucose-6-phosphate dehydrogenase (G-6-PDH), ß-hydroxybutyric dehydrogenase (ß-HBDH), alcohol dehydrogenase (ADH) and superoxide dismutase (SOD) isoenzyme patterns similar to those ofA. quina-A. lugdunensis but their acid phosphatase (AP) patterns differed. Furthermore, cyst morphology showed that the patient-isolated stock belongs to group II of the taxonomic classification ofAcanthamoeba. This stock was not thermophilic and exhibited non-pathogenic properties after its intranasal instillation into NMRI mice, whereas it killed BALB/c mice. Immunofluorescent studies revealed the presence of antibodies againstAcanthamoeba in the patient's serum. Immunoblotting experiments showed that a 45-kDa protein reacted with this serum. Such an antigen was also detected inA. quina-A. lugdunensis andA. lenticulata. Lectin reactions withCanavalia ensiformis, Ricinus communis-120,Lotus tetragonolobus, Ulex europaeus I,Helix pomatia, Arachis hypogaea, Triticum vulgaris, Glycine maxima, Bauhinia purpurea andMycoplasma gallisepticum demonstrated that only theA. lenticulata stocks could not be distinguished and that the H-1 stock was more similar to theA. lugdunensis 302-2 strain than to the otheracanthamoebae.

Examination of 54Triatoma infestans from a village near the European Southern Observatory La Silla in Chile and of 9Triatoma spinolai from the territory of the observatory showed that 10T. infestans were infected with trypanosomatids. Mice were infected with in vitro cultures initiated with five different trypanosomatid isolates and treated with the immunosuppressive drug cyclophosphamide to increase the parasitemia of the flagellates. Evidence of the presence ofT. cruzi was provided by a comparative biometrical analysis of blood trypomastigotes and the occurrence of intracellular amastigotes. Three methods for further identification were used: examination of kDNA ultrastructure, disc electrophoresis of soluble proteins and theAaptos papillata II lectin induced agglutination. We obtained the following results for all isolates: (1) presence of a central band of the kDNA; (2)T. cruzi specific double bands of the protein patterns; (3) positive reaction withAaptos papillata II. No differences between the five isolates from Chile andT. cruzi orT. cruzi-like strains from other countries could be observed. Based on these results an infection of the bugs withT. rangeli andT. conorhini could be excluded.

The agglutination behaviour of four-day-old epimastigote culture forms of 34Trypanosoma cruzi, andT. cruzi-like stocks from Brazil, Chile, Colombia, Ecuador and Peru were tested with 15 carbohydratespecific lectins. We distinguished intraspecifically two groups of agglutination reactions: Group 1 includes stocks which react withTriticum vulgaris andAaptos papillata II (wheat germ agglutinin: WGA-type). Group 2 includes stocks agglutinated byArachis hypogaea andAaptos papillata II (peanut lectin: PNA-type). The agglutination reactions with lectins fromTriticum vulgaris andAaptos papillata II correlate with the presence of N-acetylneuraminic acid on the cell surface. After treatment with neuraminidase, the WGA-type is agglutinated by PNA but not by lectins fromTriticum vulgaris andAaptos papillata II. Further results demonstrate that a certain zymodeme pattern can be correlated with carbohydrate determinants.

Four-day-old culture forms ofTrypanosoma cruzi andT. cruzi-like strains from Brazil (CL, FL, Y, São Felipe) and Venezuela (V, OPS 1, OPS 4, OPS 6, OPS 7, OPS 9, OPS 12, OPS 13, OPS 17, OPS 21, OPS 22, OPS 31, OPS 32) were tested by the following receptor-specific agglutinins:Arachis hypogaea, Triticum vulgaris, Aaptos papillata II,Phaseolus vulgaris, blood-group test serum anti-B,Dolichos biflorus, Ulex europaeus, Laburnum alpinum, Lotus tetragonolobus, Evonymus europaeus, Canavalia ensiformis, Ricinus communis-120,Soja hispida, Helix pomatia, Axinella polypoides, Cerianthus sp. All strains agglutinate withCanavalia ensiformis, Ricinus communis-120,Soja hispida, Helix pomatia, Axinella polypoides andCerianthus sp. No agglutination reactions were observed withPhaseolus vulgaris, blood-group test serum anti-B,Dolichos biflorus, Ulex europaeus, Laburnum alpinum, Lotus tetragonolobus andEvonymus europaeus. Two different agglutination types can be observed: Type 1 includes theT. cruzi andT. cruzi-like strains Y, CL and FL. These strains react withTriticum vulgaris (WGA) but not with the lectin fromArachis hypogaea (PNA). These strains are of the WGA type. Type 2 includes theT. cruzi-like strain São Felipe and the OPS strains. These parasites are of the PNA type because they agglutinate withArachis hypogaea lectin but not with the lectin fromTriticum vulgaris. All these strains also agglutinate with theT. cruzi-specific lectinAaptos papillata II. A correlation between the agglutination types of the parasites and their geographical origin was not observed. It was not possible to distinguish betweenT. cruzi andT. cruzi-like strains.

Vier Tage alte promastigote Kulturformen vonL. tropica major aus der UdSSR (LV-252, LV-253, LRC L-38) und aus Saudi Arabien (Kö, Ha),L. tropica minor aus der UdSSR (LV-239, LRC L-39),Leishmania sp. aus Israel (Ro) und aus Saudi Arabien (Ve-322, Ne, Schwe),L. donovani aus dem Sudan (1-S, 3S, LV-139) und Indien (LRC L-51, LV-125),L. donovani infantum aus Israel (LV-140) undL. aethiopica (LV-1, LV-15, LV-24, LV-26) aus Äthiopien wurden mit folgenden Lektinen untersucht:C. ensiformis, R. communis-120,A. polypoides, P. vulgaris, E. europaeus, L. tetragonolobus, D. biflorus, U. europaeus, L. alpinum, A. papillata II,A. hypogaea undS. hispida. AlleLeishmania Stämme agglutinierten mitC. ensiformis, R. communis-120 undA. polypoides. Keine Agglutinationen wurden beobachtet mitP. vulgaris, D. biflorus, L. tetragonolobus undE. europaeus. Unterschiede zwischen den Stämmen wurden sichtbar mitA. papillata II,U. europaeus, L. alpinum, A. hypogaea undS. hispida.

Summary To characterize the surface membrane of Trypanosoma brucei EATRO 427 the lectins Con A, PHA-P, Ricinus communis, Evonymus europaeus, Dolichos biflorus, Ulex europaeus, Laburnum alpinum, WGA and the protectins from Helix pomatia, Cerianthus sp., Axinella polypoides, Aaptos papillata, eel and Amphioxus lanceolatus were used. Blood group test sera anti-A, anti-B, anti-P and anti-D(Rh0) were also utilized.

The purpose of this two-part review is to explain current drug treatment in part I and discuss investigational drug therapy and miscellaneous drugs in the management of parkinsonism in part II. The medical approach to this disease is still based on the imbalance between a deficiency of dopamine and a functional increase in acetylcholine. Anticholinergic agents are used to treat the tremors in the early stages of the disease.

Activated charcoal is known to adsorb a wide variety of substances from solution, and several equations have been used to fit the resulting adsorption data. The determination of the correct model to fit phenobarbital adsorption onto activated charcoal was made using a calorimetric method. The differential heats of displacement of water by phenobarbital for four activated charcoals were determined and found to be linearly related to the amount of phenobarbital adsorbed. The activated charcoals studied had statistically similar heats of displacement. The linear relationship between heat evolved and the amount of phenobarbital adsorbed is consistent with the assumptions implicit in the Langmuir model.

A convenient preparation of N-(alkoxycarbonyl)-2,3-didehydroaspartic acid anhydrides 4 with substitution at the 3-position is reported. The key step is a cobalt-mediated acylation of an acetylene moiety, producing the highly functionalized didehydroamine acid derivative in good yield. Unnatural didehydroaspartates are readily accessible.

The pharmacologic effect of phenytoin is directly related to the unbound concentration in the serum, which previously has been reported in the literature to be ∼10. The results of 13 out of 14 20–35-year-old healthy male volunteers studied indicate that <10 unbound phenytoin is present in the majority of subjects taking two different doses of phenytoin.

The effects of two different oral charcoal suspensions on the elimination of a 200 mg/70 kg, 1 h intravenous (i.v.) infusion of phenobarbital and the tolerances of the two regimens were determined in a randomized crossover study in six healthy male volunteers. Phenobarbital was given i.v. alone or together with 105 g of oral activated charcoal suspension or with 105 g of a commercially available sorbitol-charcoal suspension over a 36-h period. A 13–34 decrease in the area under the serum concentration time curve (AUC) for 0–60 h occurred with the administration of the activated charcoal, and a 19–52 decrease occurred with the commercial sorbitol-charcoal regimen. The mean apparent systemic clearance of total phenobarbital increased from 0.089 ± 0.019 ml/min/kg to 0.141 ± 0.029 and 0.146 ± 0.036 ml/min/kg with the charcoal and sorbitol-charcoal treatments, respectively. No significant change in the fraction of phenobarbital bound to protein was detected. The charcoal regimen caused constipation in one subject. All subjects taking the sorbitol-charcoal preparation experienced diarrhea: there were no changes in electrolytes with either charcoal suspension. All subjects preferred the sorbitol-charcoal preparation.