An estimate of phylogenetic relationships among culicine mosquitoes using a restriction map of the rDNA cistron

Restriction maps of the rDNA cistron of twelve species of mosquitoes in six genera of the subfamily Culicinae were constructed using eight 6 bp recognition restriction enzymes. Anopheles albimanus was used as an outgroup. The size of the rDNA cistron ranged from 8.5 kb in Aedes katherinensis to 12.9 kb in Ae. polynesiensis. A total of twenty-six sites were scored; eighteen were polymorphic among ingroup taxa. The proportion of polymorphic nucleotide sites (Pnuc) was 0.059 and the heterozygosity per nucleotide site (Hnuc) was 0.028. Wagner and Fitch Parsimony, Dollo Parsimony and Nei-Li distance/neighbour-joining methods were used to construct phylogenetic trees. The rDNA RFLP dataset did not provide a well-supported phylogeny among culicine taxa. The RFLP phylogenies are incongruent with the morphology character based and molecular phylogenies and derived relationships did not correspond with current taxonomic classifications. The lack of resolution was due to homoplasy arising from frequent independent loss or gain of restriction sites among unrelated taxa.     

Galanin-like innervation of rat submandibular and sublingual salivary glands: origin and effect on acinar cell membranes.

The distribution and source of a galanin-like innervation of rat salivary glands has been examined. Additionally, submandibular and sublingual acinar cell membrane responses to galanin or a cholinergic agonist were studied. Galanin-immunoreactive fibers were observed throughout the submandibular and sublingual glands in association with ducts and acini. A subset of submandibular ganglion cells expresses galanin immunoreactivity. Parasympathectomy resulted in a marked decrease in galanin immunoreactivity in the glands. Sympathectomy resulted in marked reduction of dopamine beta-hydroxylase immunoreactivity with no appreciable change in galanin immunoreactivity. Retrograde labeling experiments demonstrated that galanin-immunoreactive sensory neurons in the trigeminal ganglion do not innervate the submandibular or sublingual gland. These results indicate that the galanin-like innervation of rat salivary glands is derived from parasympathetic nerves to the glands. Since rat sublingual glands contain largely mucous acini while rat submandibular gland acini are seromucous, electrophysiological responses to galanin and the muscarinic agonist, bethanechol, were compared. Agonist-induced voltage shifts varied between the two glands. The galanin-induced response at the level of the resting membrane potential in submandibular acinar cells was a hyperpolarization, while that in sublingual acinar cells was a depolarization. There was also a greater voltage dependence to the galanin-induced submandibular response than to the sublingual response. Differences were also noted in the acinar cell response to cholinergic stimulation between these glands. These results demonstrate the existence of a galanin-like innervation to salivary glands that may be functionally relevant. Moreover, the results challenge the idea that agonist-induced membrane responses are similar among acinar cells of different glands.     

Disturbances of cell-mediated immunity in ornithosis

27 cases of ornithosis were observed during an epidemia in 1980 in Kielce and subsequently followed with respect to immunological characteristics of peripheral blood lymphocytes. Blastic transformation of these cells was tested after stimulation in vitro with three different mitogens. Identification of peripheral blood T and B lymphocytes was done using rosette tests (E,EA,EAC) and the occurrence of surface immunoglobulins was determined by the immunofluorescent method with polyvalent anti-immunoglobulin serum. The counts of T and B lymphocytes in the peripheral blood were normal throughout the whole period of the observation, but from the 3rd week on a significant impairment of 3H-thymidine incorporation into the cells stimulated with Con A was observed, and from the 10th week on, this impairment appeared also in cells stimulated with PHA and PWM. These observations revealed considerable disturbances in cell-mediated reactivity in patients with ornithosis and seem to be connected with chronic infection with Chlamydia psittaci.     

Dihydropyridine inhibition of neuronal calcium current and substance P release

Dihydropyridine (DHP) calcium channel antagonists, which inhibit the slowly inactivating or L-type cardiac calcium (Ca) current, have been shown to be ineffective in blocking⁴⁵Ca influx and Ca-dependent secretion in a number of neuronal preparations. In the studies reported here, however, the antagonist DHP nifedipine inhibited both the L-type Ca current and potassium-evoked substance P (SP) release from embryonic chick dorsal root ganglion (DRG) neurons. These results suggest that, in DRG neurons. Ca entry through L-type channels is critical to the control of secretion. The inhibition of Ca current by nifedipine was both voltage and time-dependent, significant effects being observed only on currents evoked from relatively positive holding potentials maintained for several seconds. As expected from these results, nifedipine failed to inhibit L-type Ca current underlying the brief plateau phase of the action potential generated from the cell's normal resting potential; likewise, no significant effect of the drug was observed on action potential-stimulated SP release evoked by electrical field stimulation. The results of this work are discussed in terms of an assessment of the role of L-type Ca channels in neurosecretion.This work was supported by United States Public Health Service Grant NS16483 (KD) and by a USPHS Postdoctoral Fellowship (SGR)     

Antibody and cellular immune responses to microfilarial antigens in ferrets experimentally infected withBrugia malayi

Eleven of 15 ferrets experimentally infected withBrugia malayi became amicrofilaremic after a brief patency; only four ferrets remained patent after 6 months of infection and two of these ferrets developed a high, persistent microfilaremia. Blastogenic responses of peripheral blood lymphocytes to antigens of microfilariae (mf), assayed in vitro, demonstrated an antigen sensitivity at prepatent, patent and postpatent periods of infection. Lymphocytes from ferrets with high microfilaremia had elevated background responses in culture which were directly correlated with the number of circulating mf. This background response was attributed to antigenic stimulation by mf present in the lymphocyte cultures; addition of mf to cultures of lymphocytes from postpatent ferrets induced responses equivalent to those observed in microfilaremic ferrets. Lymphocyte responses to the mitogen, concanavalin A, did not differ significantly among microfilaremic, amicrofilaremic and uninfected ferrets. Antibody in IgG to antigens of mf measured by ELISA and by immunoblots from SDS-PAGE showed similar patterns of response in ferrets which became amicrofilaremic and in the few ferrets which remained microfilaremic. Prausnitz-Kustner tests demonstrated no consistent differences in titers to microfilarial antigens between patent and amicrofilaremic ferrets. The results suggest a high level of immune responsiveness to antigens of mf in infected ferrets with no evidence of immunosuppression associated with prolonged microfilaremia or of major changes in immune responses with development of amicrofilaremic infections.     

Magnitude of the inflammatory response to cardiopulmonary bypass and its relation to adverse clinical outcomes

INTRODUCTION: Cardiopulmonary bypass (CPB) induces an inflammatory response believed to contribute to postoperative morbidity. We hypothesized that the magnitude of the inflammatory response following CPB would be associated with adverse clinical outcomes. METHODS: Twenty-nine patients had plasma TNF, IL-6, IL-8, elastase, histamine, complement C5a, and complement C3a measured by ELISA before, during, and after cardiac operations employing CPB. Inflammatory mediator levels were analyzed with respect to outcomes. RESULTS: Mediator levels peaked at 4 h post-CPB and either returned to baseline or substantially decreased by 24 h. Patients with peak mediator levels above the median for the group as a whole were classified as 'hyper-responders'; those with levels below the median were classified as 'normal responders'. While IL-8, C3a, and IL-6 levels were independently associated with adverse outcomes, TNF, histamine, and C5a levels were not. Elastase levels trended towards adverse outcomes. IL-8 'hyper-responders' experienced significantly greater postoperative weight gain and had higher IL-8 levels at 24 h (p<0.05), with trends towards renal impairment and protracted supplemental oxygen requirements. C3a 'hyper-responders' strongly trended towards increased bleeding, delayed extubation, greater postoperative weight gain, and decreased levels of independent functioning at discharge (p < or = 0.10). IL-6 'hyper-responders' experienced significantly more postoperative bleeding, delayed extubation, and higher IL-6 levels at 24 h compared to 'normal responders' (p < 0.05). They strongly trended towards greater postoperative weight gain and decreased levels of independent functioning at discharge (p < or = 0.10). CONCLUSIONS: Patients who have an exaggerated inflammatory response to CPB tend to bleed more, require more respiratory support, demonstrate greater capillary leak via weight gain, and display a decline in independent functioning relative to normal responders. Thus, it appears that the magnitude of the inflammatory response to CPB adversely influences clinical outcomes.     

Phase II study of intravenous melphalan (NSC-8806) in the treatment of patients with advanced squamous carcinoma of the head and neck

The Illinois Cancer Center entered 25 patients on a phase II trial of intravenous melphalan treating patients with recurrent, metastatic or locally advanced and inoperable squamous cell carcinoma of the head and neck. All patients had bi-dimensionally measurable disease, at least a sixty day life expectancy, and adequate performance status (ECOG scale 2). All patients except one had received prior radiotherapy, chemotherapy or both. Melphalan dosage was 30 mg/m² every three weeks. Twenty-four patients were evaluable for response. One patient with laryngeal carcinoma had a clinical complete response of a nodal metastasis. Four patients had stabilization of disease for one to three months. There was formidable toxicity, including neutropenia (ANC < 1000/l 36%), and thrombocytopenia (< 50,000/l 32%). There were no drug-related deaths. Melphalan administered intravenously does not appear to be efficacious therapy in patients with previously treated advanced head and neck squamous carcinomas.     

Identification of amino-terminal sequences contributing to tryptophan hydroxylase tetramer formation

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin. In the rabbit, TPH exists as a tetramer of four identical 51-kDa subunits comprised of 444 amino acids each. The enzyme consists of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Previous studies demonstrated that within the carboxyl-terminus of TPH, there resides an intersubunit binding domain (a leucine zipper) that is essential for tetramer formation. However, it is hypothesized that a 4,3-hydrophobic repeat identified within the regulatory domain of TPH (residues 21–41) may also be involved in macromolecular assembly. To test this hypothesis, a series of amino-terminal deletions (NΔ15, 30, 41, and 90) were created and assessed for macromolecular structure using size-exclusion chromatography. The amino-terminal deletion NΔ15, upstream from the 4,3-hydrophobic repeat, was capable of forming tetramers. However, when a portion of the 4,3-hydrophobic repeat was deleted (NΔ30), a heterogeneous elution pattern of tetramers, dimers, and monomers was observed. Complete removal of the 4,3-hydrophobic repeat (NΔ41) rendered the enzyme incapable of forming tetramers; a monomeric form predominated. In addition, a double-point mutation (V28R-L31R) was created in the hydrophobic region of the enzyme. The introduction of two arginines (R) at positions 28 and 31 respectively, in the helix disrupted the native tetrameric state of TPH. According to size-exclusion chromatography analysis, the double-point mutant (V28R-L31R) formed dimers of 127 kDa. Thus, it is concluded that there is information within the amino-terminus that is necessary for tetramer formation of TPH. This additional intersubunit binding domain in the amino-terminus is similar to that found in the carboxyl-terminus.     

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.