Triiodothyronine Increases Contractility Independent of beta-Adrenergic Receptors or Stimulation of Cyclic-3',5'-Adenosine Monophosphate

Background: Triiodothyronine regulates cardiac contractility; however, the mechanisms by which it produces its acute contractile effects remains unknown. We compared the acute effects of thyroid hormones (triiodothyronine [T3] and thyroxine [T4]) and of isoproterenol on the contractility of isolated rat hearts. In addition, we sought to determine whether the acute inotropic effects of thyroid hormones were mediated by beta-adrenergic receptors or by increased production of cyclic-3',5'-adenosine monophosphate (cAMP).

Methods: A Langendorff heart preparation harvested from euthyroid male Sprague-Dawley rats was used. Drugs were administered through an aortic perfusion catheter. A pressure-transduced left-ventricular balloon catheter measured pressure and heart rate changes. Changes in the maximum positive rate of change in pressure (dP/dT) and maximum negative dP/dT were determined. Responses to varying doses of T3, T sub 4, and isoproterenol were assessed in the presence and absence of beta-adrenergic receptor blockade with propranolol, cAMP production, measured by radioimmunoassay, was determined in myocardial cell suspensions after incubation with T3 or isoproterenol.

Results: T3 0.74 nmol rapidly and significantly increased maximum dP/dT by 335 plus/minus 38 mmHg/s within 30 s after bolus injection; however, contractility was unchanged after as much as 12.9 nmol T4. The maximal increase in dP/dT after 0.8 nmol isoproterenol was comparable to that produced by T3. However, the cardiotonic actions of isoproterenol were significantly slower to develop (peaking at 60 vs. 15 s) and lasted longer than those of T3. Pretreatment with propranolol 1 micro mol diminished the contractile effects of isoproterenol but had no effect on those of T3. Concentrations of isoproterenol that increase contractility also significantly increased cAMP production in isolated rat myocardial cells. However, T3 failed to increase cAMP production.     

Alterations in myocardial tissue factor expression and cellular localization in dilated cardiomyopathy

OBJECTIVES: We investigated the myocardial localization and expression of tissue factor (TF) and alternatively spliced human tissue factor (asHTF) in patients with dilated cardiomyopathy (DCM). BACKGROUND: Tissue factor is expressed in cardiac muscle and may play a role in maintaining myocardial structure. METHODS: Myocardial biopsies were obtained from patients with a normal or mildly impaired ejection fraction (EF) (> or =50%) and moderate to severely reduced EF (<50%). Explanted DCM hearts were also examined. Myocardial TF expression level was assessed by real-time polymerase chain reaction, TF protein by enzyme-linked immunosorbent assay, and localization by immunohistochemistry. RESULTS: We report the identification of asHTF in the human myocardium: it was located in cardiomyocytes and endothelial cells. Quantification of myocardial TF messenger ribonucleic acid in DCM revealed a decrease in the TF/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio (1.76 x 10(-1) +/- 6.08 x 10(-2) for EF > or =50% [n = 19] vs. 1.06 x 10(-1) +/- 5.26 x 10(-2) for EF <50% [n = 27]; p < 0.001) and asHTF/GAPDH ratio (13.91 x 10(-5) +/- 11.20 x 10(-5) for EF > or =50% vs. 7.17 x 10(-5) +/- 3.82 x 10(-5) for EF <50%; p = 0.014). Tissue factor isoform expression level was also decreased in explanted DCM hearts (p < 0.01; n = 12). Total TF protein was reduced by 26% in DCM (p < 0.05). The TF/GAPDH ratio correlated positively with the EF (r = 0.504, p < 0.0001). Immunohistochemistry showed TF localized to the sarcolemma and Z-bands of the cardiomyocytes in patients with normal EF, whereas TF was found in the cardiomyocytic cytosol around the nucleus in DCM. CONCLUSIONS: Tissue factor was down-regulated in the myocardium of DCM patients. The reduction in TF expression and change in localization may influence cell-to-cell contact stability and contractility, thereby contributing to cardiac dysfunction in DCM.     

De novo mapping of α-helix recognition sites on protein surfaces using unbiased libraries

The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered “undruggable”, such as protein–protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein–protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.

Recent advances in identifying human disease targets have not been matched by advances in the ability to drug these targets. This actionability gap is largely due to the fact that neither of the two main classes of approved therapeutics – biologics and small molecules – can simultaneously address target accessibility and selective target engagement. Biologics, despite an impressive ability to engage diverse target proteins, are largely restricted to an extracellular operating theater, as their size and polarity render them unable to cross biological membranes. Small molecules, in contrast, can access the intracellular space, but cannot bind with high affinity and specificity to the vast majority of proteins that are found there (1).This disconnect between the ability to identify disease targets and the ability to drug them with high strength and specificity has created an impetus to develop new classes of drugs – ones that can engage intracellular proteins that lack the deep hydrophobic pocket ordinarily required for small-molecule binding. In nature, such “undruggable” proteins are often targeted with macrocyclic molecules, frequently peptidic in structure, whose large size compared with small molecules enables them to bind with high affinity and specificity to protein surfaces.Significant efforts have been made to elucidate the mechanisms of cell entry for these natural products, which possess molecular weights of 700 to 1,200 Da or higher, well beyond the typical range for cell penetration in small-molecule drug discovery (2). While the mechanisms of cell entry are complex and vary from molecule to molecule, a substantial body of research on peptidic macrocycles has highlighted the importance of desolvating amide protons and reducing their exposure to the membrane interior as a key driver in passive, thermal diffusion across the lipid bilayer (2, 3) – a phenomenon we refer to as amide-proton cloaking. The amide proton, present between every residue in a polypeptide chain, is highly electropositive and forms a strong hydrogen-bonding interaction with water. This poses a substantial hurdle for membrane permeability, since tightly bound solvent water molecules must be shed prior to entering the lipid bilayer. Exposed amide groups incur a further energetic penalty upon membrane entry due to unfavorable electrostatic interactions with the low-dielectric environment of the membrane interior. Consequently, most peptides and proteins are unable to cross membranes.For peptide macrocycles that are able to permeate the membrane, these problematic amide protons are typically removed either by replacing the amide with an ester, replacing it with a methyl group, or cloaking it from solvent water through the formation of intramolecular hydrogen bonds between the amide proton groups and a hydrogen bond-accepting group elsewhere in the molecule, often a carbonyl. Indeed, the paradigmatic example of a natural peptide macrocycle that exhibits robust cytosolic exposure, cyclosporine A (CsA), employs both N-methylation and cloaking through transannular hydrogen bonding (4). Extensive work by several research groups has shown that these strategies can be applied as design principles to endow artificial macrocycles with the ability to cross membranes (5–7).In the context of folded proteins, nature has offered an alternative structural solution to the problem of amide proton cloaking: the α-helix, a protein secondary structure that is defined by repeating intramolecular hydrogen bonds between the amide proton group of one residue and the carbonyl of the amino acid located four residues N terminal to it. The intrinsic ability of α-helices to cloak their own amide protons explains their widespread prevalence in natural transmembrane proteins (8). Nuclear-encoded transmembrane proteins in eukaryotes are almost exclusively α-helical, and the only alternative transmembrane fold found in nature is the bacterially derived β-barrel, a helical structure that also cloaks amide protons via an intramolecular hydrogen bonding network, albeit in a significantly larger structure than single α-helices that is impractical for the development of synthetic drugs.Just as CsA has served as the inspiration for the design of mimetic head-to-tail cyclized peptide ligands, so have proteinaceous α-helices inspired efforts to recapitulate nature’s design features in small, synthetic, α-helically constrained peptides (Helicons) that are hyperstabilized through the incorporation of a structural brace, also known as a “staple” (9–12). One of these, the all-hydrocarbon staple formed by ring-closing metathesis, has been extensively studied and is the basis for a drug candidate that targets the challenging proteins MDM2 and MDMX, currently undergoing Phase II clinical trials (13, 14).Rational design of Helicons is difficult given the inability to systematically define the α-helix binding sites on a protein’s surface, and to identify Helicons that bind to those sites. This limitation has restricted research on Helicons to only those protein targets for which naturally occurring or previously characterized α-helical binders were known, with the Helicons generated from fragments of the known binders (3). Here, we report a rapid, high-throughput screening platform utilizing phage display that enables an unbiased mapping of the α-helical interactome of a given protein without any prior knowledge of its structure or known binding partners. We show that this platform is capable of identifying α-helix binding sites on the surfaces of a range of protein folds, including many for which no α-helical binders are known to exist. Helicons that bind these sites are able to impact diverse protein functions, including inhibiting protein–protein interactions, inhibiting enzymatic activity, inducing dimerization, and inducing conformational changes. Analysis of 14 high-resolution crystal structures of Helicon–protein complexes across six different protein domains reveals a range of binding modes, all of which are “side-on”, i.e., mediated exclusively by Helicon side-chains rather than involving main chain amide interactions. This screening platform significantly expands the universe of proteins that can be bound by Helicons, and furthers the pursuit of targeting undruggable proteins.     

Retroperitoneal aspergilloma

We report a previously healthy 8-year-old boy who presented to the hospital with a palpable abdominal mass, fever and abdominal pain. CT and MRI scans confirmed a large mass that was centered in the retroperitoneum. The lesion was biopsied and the histology showed branching hyphae. Tissue cultures grew Aspergillus fumigatus and a diagnosis of aspergilloma was made. The immunological work-up did not reveal an immunodeficiency. This case is a unique presentation of aspergilloma presenting in an unusual location and in an immunocompetent patient.     

Surgical Debulking and Intraperitoneal Chemotherapy for Established Peritoneal Metastases From Colon and Appendix Cancer

Background: Aggressive treatment of peritoneal metastases from colon cancer by surgical cytoreduction and infusional intraperitoneal (IP) chemotherapy may benefit selected patients. We reviewed our institutional experience to assess patient selection, complications, and outcome.Methods: Patients having surgical debulking and IP 5-fluoro-2-deoxyuridine (FUDR) plus leucovorin (LV) for peritoneal metastases from 1987 to 1999 were evaluated retrospectively.Results: There were 64 patients with a mean age of 50 years. Primary tumor sites were 47 in the colon and 17 in the appendix. Peritoneal metastases were synchronous in 48 patients and metachronous in 16 patients. Patients received IP FUDR (1000 mg/m² daily for 3 days) and IP leucovorin (240 mg/m²) with a median cycle number of 4 (range, 1–28). The median number of complications was 1 (range, 0–5), with no treatment related mortality. Only six patients (9%) required termination of IP chemotherapy because of complications. The median follow-up was 17 months (range, 0–132 months). The median survival was 34 months (range, 2–132); 5-year survival was 28%. Lymph node status, tumor grade, and interval to peritoneal metastasis were not statistically significant prognostic factors for survival. Complete tumor resection was significant on multivariate analysis (P = .04), with a 5-year survival of 54% for complete (n = 19) and 16% for incomplete (n = 45) resection.Conclusions: Surgical debulking and IP FUDR for peritoneal metastases from colon cancer can be accomplished safely and has yielded an overall 5-year survival of 28%. Complete resection is associated with improved survival (54% at 5 years) and is the most important prognostic indicator.Presented in part at the 54th Annual Cancer Symposium of the Society of Surgical Oncology, Washington, DC, March 15–18, 2001.     

Protective action of an antioxidant of the 3-hydroxypyridine class on the contractility and electrogenesis of the heart muscle in hypoxia and reoxygenation

The effect of SD-6, a 3-hydroxypyridine antioxidant, on the pattern of variation in contraction intensity, contracture and membrane potentials were assessed in experimental studies on isolated left ventricular papillary muscles of rats, exposed to hypoxia and reoxygenation. The antioxidant considerably limited hypoxic and reperfusion contracture, its efficiency increasing with a concentration reduced to 10(-7) g/ml. Contraction intensity and duration of action potentials, diminished by hypoxia, were only recovered by reoxygenation, if the antioxidant was present. It is assumed that the recovery of action potentials by reoxygenation in the presence of SD-6 may result from normalization of ATP-dependent outgoing current through the kappa channels, activated by ATP deficiency. The antioxidant capacity for levelling diverse durations of action potentials between normal and ischemic areas is evidence of an important contribution of free-radical mechanisms to the development of reoxidation-induced recirculation arrhythmias.