The auricular myocardiocytes of the heart constitute an endocrine organ characterization of a porcine cardiac peptide hormone,cardiodilatin-126

Summary A peptide hormone was extracted from the porcine right atrium following a bioassay for differential vaso-relaxant effects on smooth muscle strips from aorta and renal and inferior mesenteric arteries. The isolation procedure included several steps of gel-permeation and ion-exchange chromatography, and high performance liquid chromatography. During the isolation procedure, other peptides of smaller molecular weight were also found, which, in relation to cardiodilatin-126 (CDD-126), are shorter at their N-terminal. Among these, CDD-88 has also been isolated and characterizied, and has been established as a prominent member of the cardiac hormone family. The N-terminal and C-terminal segments of the 126 amino acid-containing molecule were synthesized and used to raise region-specific antibodies. The natural peptide was then localized within myoendocrine cells of the right atrium where specific atrial granules are located. Renal effects of cardiodilation were studied in conscious dogs and showed strong diuretic and natriuretic activities. According to our functional studies, cardiodilatin-126 and cardiodilatin-88 possess qualities of a significant hormone family regarding the regulation of extracellular fluid volume and blood pressure.     

Phosphorylation and dephosphorylation of the natriuretic peptide urodilatin (CDD-/ANP-95-126) and the effect on biological activity

Urodilatin (CDD-/ANP-95-126), a new peptide hormone from human urine, is comprised of the same amino acid sequence as cardiodilatin (CDD-99-126/alpha-hANP) except for N-terminal extention by four amino acid residues. The presence of the recognition sequence Arg101-Arg-Ser-Ser104 for the cyclic AMP-dependent protein kinase enables rapid phosphorylation in the Ser104-position. Phosphorylation of urodilatin is associated with decreased vasorelaxant potency, while dephosphorylation of "phospho-urodilatin" by acidic phosphatase completely restores bioactivity.     

Electronmicroscopical, immunohistochemical, immunocytochemical and biological evidence for the occurrence of cardiac hormones (ANP/CDD) in chondrichthyes

As representatives of the vertebrate class of chondrichthyes the plagostomian species Squalus acanthias, Scyliorhinus canicula and Raja clavata as well as the holocephalan species Chimaera monstrosa were investigated for the presence of cardiac hormones of the atrial natriuretic polypeptide/cardiodilatin- (ANP/CDD-) family. ANP/CDD-immunoreactive cells were detected in the atria and the ventricles of all species studied. While these cells failed to react with antisera raised against the N-terminus of CDD-126 (= gamma-ANP) they reacted with all antisera directed against sequences of the C-terminus of CDD-126 (CDD 99-126) which is identical to alpha-ANP. The ANP/CDD-immunoreactive cells were found in high numbers in all regions of the atria and in moderate density also in the ventricles. In correspondence, in the electron microscope, myoendocrine cells which were characterized by dense-cored secretory granules were identified in the atrial and ventricular myocardium. With the use of the protein A-gold technique, ANP/CDD-immunoreactivity was determined within the secretory granules. Furthermore, in the bioassay, prepurified extracts of the atria and the ventricles of Scyliorhinus and Chimaera exerted dose-dependent relaxations of the pre-contracted mammalian (rabbit) aorta. In both cases the atrial extracts proved to be more potent than the ventricular extracts. The present findings indicate that myoendocrine cells occur in the atria and ventricles of chondrichthyes and that these cells contain homologous cardiac hormones of the ANP/CDD-family in their secretory granules. The results are compared with those obtained earlier for the other vertebrate classes and their phylogenetic and functional significance is discussed.     

Implementation challenges and opportunities for improved mass treatment uptake for lymphatic filariasis elimination: Perceptions and experiences of community drug distributors of coastal Kenya

Community drug distributors (CDDs) who are volunteers have the responsibility of awareness creation, household census, drug distribution and record-keeping and are thus key stakeholders in the campaign for Lymphatic Filariasis (LF) elimination. Taking into account their experiences and perceptions is important for a successful elimination campaign. We conducted a qualitative study in 2018 to identify implementation challenges and opportunities for improved mass drug administration (MDA) uptake based on the CDDs perceptions and experiences. Within a larger study that used mixed methods quasi-experimental design, we collected qualitative data from two wards in Kaloleni Sub-County of Kilifi County which was purposively selected owing to its low, 56% and 50.5% treatment coverage in 2015 and 2016 respectively. Focus group discussions (FGDs) (n = 8) and in-depth interviews (IDIs) (n = 8) with CDDs, IDIs (n = 22) with opinion leaders and IDIs (n = 8) with health workers were conducted and the data analyzed by QSR NVIVO version 10 according to thematic areas. The results showed that based on the perceptions and experiences of the CDDs, several challenges: communities’ refusal to take the drugs; absenteeism during MDA; non-adherence to CDDs selection criteria; inadequacy in number of CDDs engaged during the campaign and training provided; insufficiency of drugs issued to CDDs; lack of CDDs supervision and low motivation negatively impact on MDA uptake. Opportunities to address the challenges included: awareness creation on MDA, health education on LF and observation of hygiene during drug administration, increased duration of awareness creation and drug administration, adherence to CDDs selection criteria and putting into consideration the vastness of an area and population density while deploying CDDs. Other opportunities include: improved CDDs training and scheduling; issuing of enough drugs to CDDs to meet the communities’ demand and improved supervision and motivation of CDDs. Addressing the challenges highlighted is an important step of maximizing MDA uptake. The opportunities presented need to be considered by the NTD program personnel, the county health personnel and the community while planning the implementation of MDA campaigns.     

Novel expression system for large-scale production and purification of recombinant class IIa bacteriocins and its application to piscicolin 126

Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.     

Community-directed distributors—The “foot soldiers” in the fight to control and eliminate neglected tropical diseases

The neglected tropical diseases (NTDs) affect hundreds of millions of people, predominantly in rural, often difficult-to-access areas, poorly served by national health services. Here, we review the contributions of 4.8 million community-directed distributors (CDDs) of medicines over 2 decades in 146,000 communities in 27 sub-Saharan African countries to control or eliminate onchocerciasis and lymphatic filariasis (LF). We examine their role in the control of other NTDs, malaria, HIV/AIDS interventions, immunisation campaigns, and support to overstretched health service personnel. We are of the opinion that CDDs as community selected, trained, and experienced “foot soldiers,” some of whom were involved in the Ebola outbreak responses at the community level in Liberia, if retrained, can assist community leaders and support health workers (HWs) in the ongoing Coronavirus Disease 2019 (COVID-19) crisis. The review highlights the improved treatment coverage where there are women CDDs, the benefits and lessons from the work of CDDs, their long-term engagement, and the challenges they face in healthcare delivery. It underscores the value of utilising the CDD model for strong community engagement and recommends the model, with some review, to hasten the achievement of the NTD 2030 goal and assist the health system cope with evolving epidemics and other challenges. We propose that, based on the unprecedented progress made in the control of NTDs directly linked to community engagement and contributions of CDDs “foot soldiers,” they deserve regional and global recognition. We also suggest that the World Health Organization (WHO) and other international stakeholders promote policy and guidance for countries to adapt this model for the elimination of NTDs and to strengthen national health services. This will enhance the accomplishment of some Sustainable Development Goals (SDGs) by 2030 in sub-Saharan Africa.     

Localization and identification of immunoreactive atrial natriuretic factor (ANF) in the frog ventricle

The localization of ANF-like immunoreactivity in the ventricle of the frog Rana ridibunda was examined by the indirect immunofluorescence and the immunogold techniques, using an antiserum against synthetic ANF (Arg 101-Tyr 126). At the optic level, an appreciable number of positive cardiocytes was observed in the frog ventricle. Electron microscopic studies showed that all secretory granules present in ventricular cardiocytes contain immunoreactive ANF. The immunoreactive material has been characterized by Sephadex G-50 gel chromatography and reverse phase high performance liquid chromatography (RP-HPLC). After gel filtration, ANF-like immunoreactivity eluted in 3 peaks. The major immunoreactive peak corresponded to high molecular weight material, while one peak co-eluted with synthetic ANF (Arg 101-Tyr 126). Further analysis of frog ventricular extracts by RP-HPLC revealed that the low molecular weight material has the same retention time as synthetic ANF, suggesting a high degree of sequence homology between amphibian and mammalian ANF. These results indicate that in amphibians, ventricular cardiocytes synthesize a peptide immunologically and chemically related to mammalian ANF.     

Community perceptions on the community-directed treatment and school-based approaches for the control of schistosomiasis and soil-transmitted helminthiasis among school-age children in Lushoto District, Tanzania

The success of the Community-Directed Treatment (ComDT) approach in the control of onchocerciasis and filariasis has caught the attention of other disease control programmes. In this study the ComDT approach was implemented and compared with the school-based approach for control of schistosomiasis and soil-transmitted helminthiasis among school-age children in Lushoto District, Tanzania. This was a qualitative study, consisting of in-depth interviews with village leaders, community drug distributors (CDDs) and schoolteachers, as well as focus group discussions with separate groups of mothers and fathers to assess the perceptions and experiences of the villagers on the implementation of the two approaches. It was found that the villagers accepted the ComDT approach and took the responsibility of selecting the CDDs, organizing and implementing their own method of distributing drugs to the school-age children in their villages. The ComDT approach was well received and was successfully implemented in the villages. Although the villagers pointed out the limitation in reaching the non-enrolled children in the school-based approach, they also expressed satisfaction with this approach. This study suggests that the ComDT approach is well accepted and can be implemented effectively to ensure better coverage of especially non-enrolled school-age children.     

Amino acid sequence of atrial natriuretic peptides in human coronary sinus plasma

Two atrial natriuretic peptides were purified from pooled human coronary sinus plasma by Sep-Pak extraction, immunoaffinity chromatography and reverse phase HPLC. The amino acid sequences of the two peptides were homologous with 99-126 human atrial natriuretic peptide (hANP) and 106-126 hANP, the latter being most probably linked to 99-105 ANP by the disulphide bond. The molar ratio of the peptides in plasma, as assessed by radioimmunoassay was 10:3.     

Application of chiralcel OJ in supercritical fluid chromatography for the resolution of different groups of frequently used drug racemates

Supercritical fluid chromatography was applied to evaluate the direct chiral discriminative properties of Chiralcel OJ, a tris(4-methylbenzoate) cellulose column towards frequently administered drugs. Two groups of acidic drugs, profen and barbiturate derivatives, and a few basic drugs of the benzodiazepine type were analysed. The effect on enantioselectivity of carbon dioxide when using methanol are acetonitrile as primary modifier was studied. Acetonitrile proved to be a good alternative for methanol, especially for the profen compounds that were not well resolved using methanol. The results were compared to normal phase chromatographic applications on the same column. SFC was not necessarily superior to high-performance liquid chromatography. Chirality 9:126–132, 1997. © 1997 Wiley-Liss, Inc.     

A thermophilic extracellular -amylase from Bacillus licheniformis

A strain of Bacillus licheniformis isolated from soil produced an extracellular α-amylase(s) with unusual characteristics. The enzyme was purified 126-fold by starch adsorption, DEAE-cellulose treatment, and CM-cellulose column chromatography. Four active protein bands were detected by disc electrophoresis in poly-acrylamide gel although the enzyme behaved as a single peak during both ultracen-trifugation and chromatography using CM-cellulose and Sephadex G-100. The enzyme showed a very broad pH-activity curve and had substantial activity in the alkaline range. The optimal temperature was 76 °C at pH 9.O. The enzyme was stable between pH 6 and 11 at 25 °C, and below 60 °C at pH 8.0. Using Sephadex G-100 gel filtration, a molecular weight of 22,500 was estimated for the enzyme. The action pattern on amylose and amylopectin is unique in that the predominant product during all stages of hydrolysis is maltopentaose.     

Evidence for the presence of ANP-precursor material in the rat thymus

Acidic extraction of the thymus from two day old rats followed by purification on Sephadex G-50 gelfiltration revealed the presence of atrial natriuretic peptide-like material (IR-ANP) by radioimmunoassay. Verification of the obtained immunoreactivity has been achieved by the use of two different types of antisera, i.e. two antisera directed against ANP (99-126), the other antiserum recognizing the N-terminal fragment (11-37) of the precursor ANP (1-126) molecule. In addition, reverse phase high performance liquid chromatography (RP-HPLC) and high performance gel permeation chromatography (HP-GPC) monitored by the three antisera have been employed for analysis of the extracted IR-ANP. In both systems the IR-ANP corresponded to the 15 kDa-ANP molecule (1-126). Furthermore, by using immunohistochemical techniques a distinct localization of the IR-ANP material could be demonstrated. The outer cortical area of the thymus, containing mostly lymphoid cells, showed extensive immunostaining with the three different antisera. The data reported here indicate that the rat thymus is a source of ANP.     

The post-translational processing of rat pro-atrial natriuretic factor by primary atrial myocyte cultures

Primary cultures of neonatal rat atrial myocytes were maintained in two different serum-free media for up to 25 days. Reversed-phase high performance liquid chromatography coupled with atrial natriuretic factor (ANF)-specific radioimmunoassay demonstrated that the cultures maintained in our previously described serum-free medium (Glembotski, C.C., and Gibson, T. R. (1985) Biochem. Biophys. Res. Commun. 132, 1008-1017) secreted primarily ANF-(1-126)-like material, whereas those cultures maintained in a different formulation of medium secreted mostly ANF-(99-126)-like material. Cultures that secreted ANF(99-126)-like material were biosynthetically labeled with [35S]cysteine followed by immunoprecipitation of secreted ANF and analysis by reversed-phase, size exclusion, and ion-exchange high performance liquid chromatography. The labeled ANF-(99-126)-like peptide was shown to be chromatographically indistinguishable from other synthetic peptides related to ANF-(99-126). Labeled ANF purified from extracts of the cultured cells was chromatographically indistinguishable from authentic ANF-(1-126), and could be cleaved specifically by thrombin into labeled ANF-(99-126)-like material. These results indicate that primary atrial myocytes maintained under certain serum-free conditions are capable of secreting ANF-related material that is chromatographically indistinguishable from ANF-(99-126), the known circulating form of the hormone. Additional preliminary studies suggest that the presence of glucocorticoids in the culture medium may confer ANF processing ability on cultured myocytes.     

Structure, measurement, and secretion of human glucagon-like peptide-2

By using radioimmunoassays toward the cDNA-predicted amino acid sequence of human glucagon-like peptide-2, a peptide was isolated from extracts of human ileum. By mass spectrometry and Edman sequencing, this peptide was identified as human proglucagon 126-158. High-performance liquid chromatography analyses indicated that a similar immunoreactive peptide (iGLP-2) was present in human plasma. Human plasma concentrations of iGLP-2 were elevated 3- to 4-fold at 1 to 2 h after ingestion of 800 to 1200 kcal meals.     

Secretion of atrial natriuretic factor-(1-98) by primary cardiac myocytes

Previous studies have demonstrated that primary cultures of cardiac myocytes maintained in a complete serum-free medium contain a precursor to atrial natriuretic factor (ANF-(1-126]. The cultured cells secrete this precursor unless maintained in the presence of glucocorticoids wherein the known circulating form derived from the C-terminal of ANF (ANF-(99-126] is secreted. The present study was designed to determine the fate of the N-terminal region of the ANF precursor during secretion from myocytes maintained in glucocorticoids. A radioimmunoassay (RIA) was developed using synthetic ANF-(1-16); the antiserum demonstrated cross-reactivity toward ANF-(1-126) and ANF-(1-98)-like peptides but did not cross-react with ANF-(99-126). Coupling this RIA with an ANF-(99-126)-specific RIA and reversed phase, size exclusion, and ion exchange high performance liquid chromatography (HPLC), it was shown that primary cultures of atrial myocytes maintained in dexamethasone contained ANF-(1-126) and secreted ANF-(99-126) and a peptide that was chromatographically indistinguishable from ANF-(1-98). Isolated perfused rat hearts were also shown by RIA and HPLC to secrete similar peptides. The primary cells were labeled with [35S]methionine, and the secreted N-terminal ANF-related material was immunoprecipitated with the ANF-(1-16) antiserum. HPLC, tryptic peptide mapping, and radiosequencing demonstrated that this peptide possessed an N-terminal structure identical to that of ANF-(1-126). When the cells were labeled with [3H] leucine and the secreted N-terminal ANF-related material was immunoprecipitated and analyzed by tryptic mapping, it was shown to possess labeled tryptic peptides consistent with the structure of ANF-(1-98). Tryptic mapping of [3H]arginine-labeled N-terminal ANF-related material demonstrated the presence of all peptides consistent with the ANF-(1-98) structure, including ANF-(92-98). These studies demonstrate that primary atrial myocytes contain ANF-(1-126) and in the presence of dexamethasone secrete both ANF-(1-98) and ANF-(99-126), the two major circulating forms of the hormone.     

Beta-defensin 126 on the cell surface protects sperm from immunorecognition and binding of anti-sperm antibodies

Beta-defensin 126 (DEFB126), formerly known as epididymal secretory protein 13.2 (ESP13.2), coats the entire primate sperm surface until completion of capacitation, and it is a candidate for providing immune protection in the female reproductive tract. To further examine the potential role of DEFB126 as a means of protection from immune recognition, cynomolgus macaque sperm were exposed to a number of treatments that are known to alter sperm surface coats, including capacitation. We used a novel in vivo assay to determine immune recognition: aldehyde-fixed whole sperm injections into rabbits. Following booster injections, immunoblot analyses of whole sperm prepared in various manners was conducted. On Days 60 and 80 post-initial immunization, the antisera showed a remarkably strong reaction to a single 34-36 kDa protein, which was shown to be DEFB126. Sera from rabbits that were immunized with sperm washed more rigorously using Percoll gradients showed an increase in the number and intensity of proteins recognized on whole sperm Western blots, although DEFB126 was still the major immune response. When capacitated sperm, from which most DEFB126 had been released, were used as the immunogen, there was a dramatic increase in the immune recognition to a variety of protein bands. Sperm treated with neuraminidase to remove sialic acid on DEFB126 before fixation were shown to still possess DEFB126, but lacked the sialic acid component of the glycoprotein. These sperm were as immunogenic as capacitated sperm even though the desialylated DEFB126 still covered the entire cell surface. These sperm lost their highly negative charge (the isoelectric point of DEFB126 shifted from pI 3.0 to pI 6.4). Experiments using different sperm plasma membrane protein-specific Igs showed that recognition did not occur when DEFB126 was present, but following capacitation these Igs readily recognized the exposed sperm membrane. Our data suggest that DEFB126 protects the entire primate sperm surface from immune recognition and that the sialic acid moieties are responsible for the cloaking characteristic of this unique glycoprotein.     

Thiol cross-linking of transmembrane domains IV and V in the lactose permease of Escherichia coli

Glu126 (helix IV) and Arg144 (helix V) in the lactose permease of Escherichia coli are critical for substrate binding and transport, and the two residues are in close proximity and charge-paired. By using a functional permease construct with two tandem factor Xa protease sites in the cytoplasmic loop between helices IV and V, it is shown here that Cys residues in place of Glu126 and Arg144, as well as Ala122 and Val149, spontaneously form disulfide bonds in situ, indicating that this region of transmembrane domains IV and V is in the alpha-helical conformation. To determine if the local structure or environment is perturbed by the presence of an unpaired charge, either Glu126 or Arg144 or both were replaced with Ala, and cross-linking between the Cys pair Ala122-->Cys/Val149-->Cys was studied. Ala replacement for Arg144 causes a marked decrease in cross-linking, while Ala replacement for Glu126 alone or for both Glu126 and Arg144 has little effect. The data provide strong support for the argument that Glu126 and Arg144 are within close proximity and suggest that an unpaired carboxylate at position 126 causes a structural change at the interface between helices IV and V.     

Mapping single cysteine mutants of light chain 2 in chicken skeletal myosin.

The NH2- and COOH-terminal regions of the regulatory light chain (LC2) have been mapped to the head/rod junction by immunoelectron microscopy, using monoclonal and anti-fluorescyl antibodies as probes. In order to map the entire length of the light chain, we have engineered and purified mutants that contain a single cysteine residue at positions 2, 73, 94, 126, or 155. The single cysteine residues were labeled with either 5-iodoacetamido fluorescein or N-ethylmaleimide-biotin. We observed that the reactivity of Cys126 is far greater than that of Cys155, confirming that cysteine 126 is the fast-reacting thiol in wild-type light chain. The labeled light chains were exchanged into myosin stripped of its native LC2 by immunoaffinity chromatography, and the reconstituted myosin was reacted with anti-fluorescein antibody or avidin prior to rotary shadowing for electron microscopy. The position of the antibody or avidin was found to be near the head/rod junction for all mutants. These mapping studies, together with our finding that cysteines widely separated in the primary sequence can form multiple disulfide bonds (Saraswat, L.D., and Lowey, S. (1991) J. Biol. Chem. 226, 19777-19785), support a model for LC2 as a flexible, globular molecule localized mainly in the vicinity of the head/rod junction of myosin.     

石蜡酪杆菌B126产生的糖脂的理化性质

通过纸层析、硅胶薄层层析和气相色谱分析表明,石蜡酪杆菌(Caseobacter paraffinicum)B126至少产生两种糖脂,其主要产物是海藻糖脂。它由海藻糖和两种以上的脂肪酸(十六碳酸和十八碳酸)所组成。其表面张力和界面张力(对重液体石蜡)分别为28～29和1～2mN/m,临界胶束浓度(CMC)较低,为64mg/L。