Pancreatic islets in Platyrrhini monkeys:Callithrix jacchus,Saimiri boliviensis,Aotus azarae,andCebus apella. A cytological and immunocytochemical study

A histochemical and immunocytochemical study was performed to describe the cellular localization of four hormones in pancreatic tissue of the Callithricidae and Cebidae families belonging to Platyrrhini monkeys. Insulin, somatostatin, glucagon, and gastrin immunoreactive cells were detected in the pancreatic islets at light microscope level. Beta cells (insulin positve) were distributed in a peripheral mantle or in irregular clusters in a polar region of the islet. Also little groups (small islets) from three to six beta cells distributed in the pancreatic stroma were observed in all the species. A constant feature was the presence of neuroinsular complexes. The alpha cell population was composed of orangeophiles or phloxinophile cells and presented an heterogeneous composition as the result of their granular type (difference in immunocytochemical affinity). Alpha 1 cells (somatostatin) were scattered between the other cells or in the periphery of the islets, while alpha 2 cells (glucagon) were distributed in three different ways: (1) occupying polar positions; (2) disseminated in small groups; or (3) arranged in cord-like disposition. One or two G cells (gastrin) were found in few islets. This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Estudio Histológico Comparado del Sistema de Glándulas Endócrinas (EHIGE) program. Postgraduated Fellow from CONICET Established Investigator and Director of EHIGE.     

Single-Step Purification and Biological Activity of Human Nerve Growth Factor Produced from Insect Cells

Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus-infected Spodoptera frugiperda (SF-9) insect cell system. Culture supernatants contained 2-3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single-step affinity chromatography procedure using a high-affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1-2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transfected into the SF-9 cells coded for pro-NGF, the NGF recovered after purification was greater than 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 +/- 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 +/- 20 pM and 9.6 +/- 1.5 pM for a 3-day primary response and a 1-day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 +/- 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from submaxillary glands.     

The morphology and reproductive status of female Schistosoma mansoni following separation from male worms

Popiel I., Cioli D. and Erasmus D. A. 1984. The morphology and reproductive status of female Schistosoma mansoni following separation from male worms. International Journal for Parasitology14: 183–190. Sexually mature females of Schistosoma mansoni were separated from their male partners and surgically transferred to Nile rats (Arvicanthis niloticus). Over a period of 35 days there was a significant decrease in size of these worms and regression of the reproductive system took place. Electron microscope observations of the vitelline gland and ovary provided details of and a time scale for the regressive changes which took the form of a cessation of cell differentiation and turnover, together with extensive cell death. Survival of cells within these organs was restricted to the undifferentiated cells and by day 35 the worms resembled immature females. It is concluded that regression of the female reproductive system was the result of discontinued male stimulation.The nature and implications of the obligatory relationship between male and female S. mansoni are discussed.     

Purification methods for the sequence-specific DNA-binding protein nuclear factor I (NFI) — Generation of protein sequence information

The paper describes a potent purification method, preparative gel retention, for the purification of sequence-specific DNA-binding proteins. This procedure exploits the sequence-specific DNA-binding affinity of such proteins for their enrichment, comparable to recognition site DNA affinity chromatography. The method was employed to obtain a pure preparation of nuclear factor I (NFI) from porcine liver from which sequences of partial peptides could be obtained. Oligonucleotide probes derived from these amino-acid sequences were used to identify genomic and cDNA clones of NFI.     

Studies in the differentiation between Microsporum ferrugineum Ota and Trichophyton soudanense Joyeux

A study, conducted with 20 isolates of Microsporum ferrugineum and 12 isolates of Trichophyton soudanense, revealed that some of the discrepancies in the literature regarding their characteristics and differentiation were due to methodology, strain variation and the use of an insufficient number of isolates. We found all isolates of T. soudanense to be urease negative and gelatinase positive (usually by the first week); to produce brown to black colonies on Lowenstein-Jensen medium; to rapidly decompose casein and more slowly tyrosine; to grow well or better at 37°C as compared to room temperature; to produce reflexive branching on cornmeal Tween agar and abundant microconidia on casero medium and to exhibit no sexual reaction with either mating type of Arthroderma simii. All but one isolate demonstrated restricted growth on lactose agar and only three isolates perforated hair.In contrast, we found 18 of 20 isolates of M. ferrugineum to be urease positive in urea broth (most isolates were negative on urea agar); all produced light-colored colonies on Lowenstein-Jensen medium; spreading colonies on lactose agar and failed to perforate hair in vitro or to produce reflexive branching. Most isolates manifested poorer to no growth at 37°C compared to room temperature and all but one failed to decompose casein and tyrosine. A few strains produced macroconidia and/ or microconidia on casero medium and some reacted sexually with A. simii (a) or (–) mating type. Gelatin hydrolysis was variable.We suggest the following key tests to differentiate M. ferrugineum from T. soudanense: urease activity in urea broth; colony color on Lowenstein-Jensen medium; growth on lactose agar; growth at 37° C compared to room temperature; presence of reflexive branching on cornmeal Tween agar.     

Ion content and the response of L5178Y-R and L5178Y-S cells to X rays and ionophore A23178

Summary We examined the response of L5178Y-S (radiosensitive, LY-S) and L517SY-R (radioresistant, LY-R) lymphoblasts to X-irradiation with concomittant treatment with divalent cation ionophore, A23187 (3 h or 5 h, 5 µg/ml). Cells treated with A23187 alone progressed through the cell cycle more slowly than the untreated cells and their cloning efficiency was reduced. In both cell strains the ionophore prolonged duration of the postirradiation mitotic delay. Radiation-induced inhibition of DNA synthesis was reversed by A23187 in LY-S but not in LY-R cells.Cells subjected to the ionophore treatment survived X-irradiation in almost the same way as untreated cells, as if the effect of A23187 treatment were reversed by irradiation. There was also a reversion in the ion content: A23187 caused a marked increase in Na⁺ content and a decrease in K⁺ content, irradiation itself did not change the ion content, whereas in the A23187-treated cells it restored almost the same pattern as that found in the control cells. We found less Mg²⁺ ions in LY-S cells after treatment with A23187 and A23187 + X than in LY-R cells, in relation to untreated (control) cells. These observations point to the possible importance of ion transport for recovery from radiation damage.     

Discovery of biaryl inhibitors of H+/K+ ATPase

We report the identification of a novel biaryl template for H⁺/K⁺ ATPase inhibition. Evaluation of critical SAR features within the biaryl imidazole framework and the use of pharmacophore modelling against known imidazopyridine and azaindole templates suggested that the geometry of the molecule is key to achieving activity. Herein we present our work optimising the potency of the molecule through modifications and substitutions to each of the ring systems. In particular sub-micromolar potency is achieved with (4b) presumably through a proposed intramolecular hydrogen bond that ensures the required imidazole basic centre is appropriately located.     

A unique thioredoxin of the parasitic nematode Haemonchus contortus with glutaredoxin activity

The dependency of parasites on the cellular redox systems has led to their investigation as novel drug targets. Defence against oxidative damage is through the thioredoxin and glutathione systems. The classic thioredoxin is identified by the active site Cys-Gly-Pro-Cys (CGPC). Here we describe the identification of a unique thioredoxin in the parasitic nematode, Haemonchus contortus. This thioredoxin-related protein, termed HcTrx5, has an arginine in its active site (Cys-Arg-Ser-Cys; CRSC) that is not found in any other organism. Recombinant HcTrx5 was able to reduce the disulfide bond in insulin, and be regenerated by mammalian thioredoxin reductase with a K_m 2.19 ± 1.5 μM, similar to the classic thioredoxins. However, it was also able to reduce insulin when glutathione and glutathione reductase replaced the thioredoxin reductase. When coupled with H. contortus peroxiredoxin, HcTrx5 was active using either the thioredoxin reductase or the glutathione and glutathione reductase. HcTrx5 is expressed through the life cycle, with highest expression in the adult stage. The unique activity of this thioredoxin makes it a potential drug target for the control of this parasite.     

Quantitative assay for six potential breast cancer biomarker peptides in human serum by liquid chromatography coupled to tandem mass spectrometry

An assay to quantify several possible breast cancer peptide biomarkers in human serum has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The peptides include bradykinin, Hyp³-bradykinin, des-Arg⁹-bradykinin and fragments of fibrinogen α-chain (Fib-α_[605–629]), inter-α-trypsin inhibitor heavy chain 4 (ITIH_4[666–687]) and complement component 4a (C4a_{[1337–1350]}). Ile¹³-ITIH_4[666–687], d20-C4a_{[1337–1350]} and Sar-D-Phe⁸-des-Arg⁹-bradykinin were used as internal standards. Bovine plasma, with 2 mM captopril and 2 mM d-l-mercaptoethanol-3-guanidino-ethylthiopropanoic acid (MEGETPA) to prevent rapid degradation of the bradykinins, was used as analyte-free matrix. Recoveries for solid-phase extraction (SPE) on mixed-mode weak cation exchange sorbents were between 62 and 90%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with a heated electrospray source (H-ESI), operating in the positive ion-mode, was used for detection. The assay was fully validated and stabilities of the peptides were extensively explored. Bradykinin (10–500 ng/ml), Hyp³-bradykinin (4–200 ng/ml), des-Arg⁹-bradykinin (2–100 ng/ml), Fib-α_[605–629] (120–3000 ng/ml), ITIH_4[666–687] (0.4–10 ng/ml) and C4a_{[1337–1350]} (1–25 ng/ml) were simultaneously quantified with deviations from the nominal concentrations below 22% and intra- and inter-assay precisions below 15 and 20%, respectively, for all peptides at all concentrations. The method has been successfully applied to several serum samples from breast cancer patients and matched controls.