In vitro degradation of the Neb-Trypsin modulating oostatic factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.     

Synthesis and HDAC inhibitory activity of isosteric thiazoline-oxazole largazole analogs

The synthesis of an isosteric analog of the natural product and HDAC inhibitor largazole is described. The sulfur atom in the thizaole ring of the natural product has been replaced with an oxygen atom, constituting an oxazole ring. The biochemical activity and cytotoxicity of this species is described.     

An Examination of Serotonin and Feeding in the Flesh Fly Neobellieria bullata (Sarcophagidae: Diptera)

The role of serotonin in controlling feeding in flesh flies is examined. Amount of feeding was recorded over 6 h for flies injected with serotonin or saline. The proportion of time spent on various behaviors over a period of 1 h was recorded after the injection of serotonin or saline or no injection. Corresponding electrophysiological measurements were made on serotonin and saline-injected flies. The release of serotonin as a consequence of feeding was also examined. The subesophageal ganglia of flies taken before or after 2 days of sugar feeding were examined immunocytochemically. Serotonin injection decreased feeding in flies compared to saline-injected flies. All behaviors measured decreased after serotonin injection, except for resting, which increased, and grooming, which decreased in both serotonin- and saline-injected flies. A marked reduction in electrophysiological responses to sucrose was seen in serotonin-injected flies. Specific cells of the subesophageal ganglion showed significantly less serotonin immunoreactivity in fed flies compared to flies that had not yet fed. The role of serotonin in affecting the physiology of feeding in insects is discussed.     

Atrial natriuretic peptides. IV. Synthesis and study of shortened analogs]

New analogues of atrial peptides of rat were synthesized by classical methods of peptide chemistry in solution. They contain a D-amino acid residue in the C-terminal part and a residue of mercaptopropionic acid in the N-terminal part of the molecule. Biological activity of the new analogues was studied.     

Behavioural and gustatory responses of female Neobellieria (=Sarcophaga) bullata to sucrose and amino acids

Abstract. Unmated female Neobellieria (=Sarcophaga) bullata (Parker) (Diptera: Sarcophagidae) of different ages ranging from 1 to 24 days consistently preferred a solution of 100 mM/1 sucrose to one containing a mixture of six amino acids at a total concentration of 10 mM/1. When these solutions were used to stimulate the gustatory sensilla on the proboscis of 2- and 5-day-old flies, sucrose elicited a greater response from cells 1 and 2 in large and cell 1 in medium-length hairs, which could account for the feeding preference of the flies for this chemical.     

Effects of the bipyridylium herbicides diquat dibromide and paraquat dichloride on growth and development of Neobellieria bullata (Diptera: Sarcophagidae) larvae

Diet containing diquat dibromide (1,000 or 2,000 ppm) caused an extension of the first-instar stadium of Neobellieria bullata (Parker); the first molt was primarily disturbed. Pupariation was delayed when early-wandering larvae had been injected with diquat dibromide (18 micrograms/larva; approximately to 150 ppm). This effect of diquat dibromide was eliminated by simultaneous injection of 20-OH ecdysone (0.02 micrograms/larva). After larvae in the red spiracle stage were injected with diquat dibromide (5 micrograms/larva; approximately 42 ppm), evagination of the pupal head was inhibited. Paraquat dichloride was less active than diquat dibromide and appears to be a safer herbicide for use around decomposer fly species.     

Isolation and identification of a cAMP generating peptide from the flesh fly,Neobellieria bullata (Diptera: Sarcophagidae)

The Manduca sexta Malpighian tubule assay system, developed to monitor adenylate cyclase activity, was used in combination with HPLC to isolate a novel cAMP generating peptide from 350,000 whole flesh flies, Neobellieria bullata. Mass spectrometry revealed a molecular mass of 5,047 daltons, and Edman degradation the following sequence: AGAEAEKLSGLSKYFNGTTMAGRANVAKATYAVIGLIIAYNVMKPKKK. This 48-mer peptide, called Neb-cGP, does not belong to the corticotropin releasing factor family of insect diuretic peptides. Electrophoresis and subsequent immunoblotting of peptides immunoprecipitated from a homogenate of entire flies showed that one fly contained approximately 0.003 to 0.03 μg Neb-cGP and that 10 μg represents the lowest immunostainable amount on a Western blot. © 1996 Wiley-Liss, Inc.     

Presence of angiotensin converting enzyme (ACE) interactive factors in ovaries of the grey fleshfly Neobellieria bullata

Angiotensin converting enzyme (ACE) activity, defined as a captopril-inhibitable dipeptidyl carboxypeptidase activity towards 3H-hippurylglycylglycine, was demonstrated in haemolymph, testes and ovaries of the grey fleshfly Neobellieria bullata, hereby suggesting a physiological role for ACE in these particular tissues. While the ACE activity in haemolymph and testes reached relatively high levels, only minute ACE activity could be detected in ovaries throughout the entire vitellogenic cycle. Ovarian extracts of Neobellieria bullata do contain, however, in addition to Neb-TMOF, the Neobellieria bullata trypsin modulating oostatic factor which is an in vitro and a putative in vivo substrate of ACE in circulation, several other heat-stable molecules which individually function either as an ACE substrate or ACE inhibitor. Presumably these ACE interactive factors mask ACE activity in the fly ovaries, as measured by a classic substrate-binding assay. Purification and characterisation of these ACE substrates/inhibitors is in progress and is likely to facilitate the elucidation of the enigmatic physiological relevance of ACE in insects.     

Ingestion of sugar diets correlates with spike activity in labellar chemosensilla of the flesh-fly, Neobellieria (= Sarcophaga ) bullata

Abstract .Female 2-day-old Neobellieria (= Sarcophaga ) bullata (Parker) (Diptera: Sarcophagidae) were exposed to different concentrations of sucrose, glucose and fructose in a single-choice potometer, and the volume ingested in the first hour was measured. Nerve spike activity in response to the same sugars was recorded from medium labellar taste hairs of similar flies by tip-recording. Two classes of chemosensory cells responded to sucrose, glucose and fructose. Cell 1 showed an increasing spike activity with sugar concentration, whereas cell 2 did not; cell 1 was identified as the 'sugar cell'.
For both spike activity in cell 1 and feeding, sucrose was the most stimulatory sugar. The dose–response curves for glucose and fructose crossed over at about 200 m m . At higher concentrations, glucose was more stimulatory for both cell 1 and for feeding, and at lower concentrations, fructose. The pattern of spike activity supports a separate location on the sensory cells of receptors for pyranose and fructose forms of sugar. The strong correlation between volume ingested and spike activity indicates that sugar feeding is controlled by sensory input from the 'sugar' cells of labellar chemosensilla. Moreover, the results suggest that the flies do not distinguish between these sugars except by apparent 'sweetness'.     

Changes of acid phosphatase content and activity in the fat body and the hemolymph of the flesh fly Neobellieria (sarcophaga) bullata during metamorphosis

Changes in the specific and total activity of the lysosomal marker enzyme acid phosphatase (Acph) and in the amount of enzyme protein were examined in the fat body and the hemolymph from the last larval molt to the larval-pupal apolysis. The specific activity showed minor changes during the last larval period. In contrast, the total activity of the enzyme was low during the feeding period and higher during the wandering stage and strikingly increased at the time of puparium formation. We purified a protein having para-nitrophenyl phosphate phosphatase (Acph) activity and raised antisera against it. The amount of Acph protein in the fat body and hemolymph was examined using an ELISA. The specific Acph content showed little variation, but the total amount of the enzyme protein showed a stepwise increase in both organs during last larval stage and was markedly elevated in the pupal stage in the fat body. In contrast, a considerable decrease in the amount of Acph protein was observed in the hemolymph during this period. These data were in agreement with immunohistochemical observations showing an accumulation of the enzyme protein in fat body cells during the prepupal stage with a concomitant disappearance of the enzyme from the hemolymph. Inhibition of ecdysteroid secretion by water stress prevented the changes both in total enzyme activity and in the amount of Acph protein. However, Acph protein content and enzyme activity could be restored when the water stress was followed by a 20-hydroxyecdysone (20-HE) treatment. Taken together, our data show that Acph is secreted by fat body cells into the hemolymph during the larval stage, where it is stored in an inactive form. Increase in the 20-HE titer at the end of last larval stage reverses this process, and the enzyme is taken up by the fat body cells, where it becomes activated and appears in auto- and heterophagic vacuoles. Arch. Insect Biochem. Physiol. 34:369–390, 1997. © 1997 Wiley-Liss, Inc.     

Reproductive biology and effect of nectar robbing on fruit production in Macleania bullata (Ericaceae)

Hummingbird-pollinated flowers are frequently subjected to nectar robbing. In this paper, I examine the impact of nectar robbing on plant reproductive success on a hummingbird-pollinated species. After studying the basic aspects of the floral morphology and reproduction of Macleania bullata (Ericaceae) in a tropical montane wet forest in southwest Colombia, I examined the percent of flowers robbed and the effect of nectar robbery on fruit set. The flowers of this species are typical for plants pollinated by long-bill hummingbirds. They are protandrous and open for four days. Fruit production requires a pollinator visit; fruit set following pollinator exclusion was zero. Fruit set following xenogamous pollen transfer (36.8%) differed significantly from that of population controls (11.9%) and of autogamous pollen transfer (6.3%). Nectar volume, sugar concentration and sugar production were measured at daily intervals from bud opening until the fading of flowers. Daily nectar production (both volume and amount of sugar) varied considerably with flower age. Sugar production peaked on the second day, coinciding with the male phase. The frequency of nectar robbing in the studied population was very high (75% of examined flowers) and was positively correlated with reduced fruit set. I discuss the probability of a relation between reduced fruit set on robbed flowers and an energetic investment. Robbing by non-pollinating visitors can suppose the plant to re-synthesize more nectar. The high incidence of nectar robbing impugns the advantage of specialization.     

Platelet-activating factor analogs. II. Synthesis of the 1-O-(4-decyloxymethylphenyl)- and 1-O-(4-decyloxyphenylmethyl) analogs

1-O-(4-Decyloxymethylphenyl)- and 1-O-(4-decyloxyphenylmethyl)-2-O-acetyl-glycero-3-phosphorylcholin e were synthesized from 2,2-dimethyl-1,3-dioxolane-4-methanol. The utility of a 2-O-tetrahydropyranyl ether as a protecting group and its facile removal in the presence of a 3-phosphorylcholine is illustrated.     

Synthesis and cytotoxicity of (-)-renieramycin G analogs

(−)-Renieramycin G and fifteen C-22 analogs were prepared employing l-tyrosine as the chiral starting material. These analogs, along with (−)-renieramycin G itself, were evaluated in vitro for cytotoxicity against HCT-8, BEL-7402, A2780, MCF-7, A549, BGC-823, Ketr3, KB, Hela cells. The IC₅₀ values of most of these analogs were at the level of μM. Among these analogs, 2-thiophenecarboxylate ester derivative 17 exhibited potent cytotoxic activity against KB cell line with the IC₅₀ of 20 nM. From this study, it could be concluded that the C-22 side chain played an important role in the cytotoxic potency and specificity of this class of (−)-renieramycin G derivatives.     

Biosynthesis and regulation of juvenile hormone III,juvenile hormone III bisepoxide,and methyl farnesoate during the reproductive cycle of the grey fleshfly,Neobellieria (Sarcophaga) bullata

To study the effect of brain signals on the biosynthesis of juvenile hormone by the corpora allata of the grey fleshfly Neobellieria bullata, exposed corpora allata connected to the brain were surgically removed from sugar-fed flies and incubated in vitro with L -[³H-methyl]methionine. After incubation, the media together with the tissues were analyzed by HPLC. [³H]Juvenile hormone III (JH III), [³H]JH III bisepoxide (BE), [³H]methyl farnesoate (MF) and an unknown [³H]labeled metabolite (Un) were identified as the primary products. The rate of synthesis of [³H]JH III bisepoxide was higher than that of [³H]JH III, [³H]MF and [³H]Un. Two days after a liver meal, female flies synthesized more JH III, MF, BE, and the Un than did males. Synthesis of JH III, BE, and MF in females was lower during the previtellogenic, sugar-feeding period than during the vitellogenic liver-feeding period. Isolated corpus cardiacum–corpus allatum (CC-CA) complexes that were incubated in vitro synthesized less JH III, MF, and BE, as compared to complexes that were attached to the brain, indicating that the brain probably modulates the biosynthesis of JH III, MF, and BE in the corpora allata. Upon incubation of brain–CC–CA complexes with Neb-TMOF (10^–8 M), Neb-colloostatin (10^–8 M), ovarian, or brain extracts resulted in significant inhibition of JH III and BE biosynthesis in the presence of ovarian extracts. These results indicate that allatostatin-like factors are present in the ovary of the flesh fly. Arch. Insect Biochem. Physiol. 37:248–256, 1998. © 1998 Wiley–Liss, Inc.     

Captopril, a specific inhibitor of angiotensin converting enzyme, enhances both trypsin and vitellogenin titers in the grey fleshfly Neobellieria bullata.

A strong and constitutive angiotensin converting enzyme- or ACE-like activity was demonstrated in the hemolymph of the adult grey fleshfly Neobellieria bullata. In a competition assay, the N. bullata trypsin modulating oostatic factor (Neb-TMOF) was confirmed to be an in vitro substrate for this circulating Neb-ACE. Oral uptake of captopril, a selective and specific inhibitor of ACE, resulted in a complete phenotypic knockout of circulating ACE activity. When compared with control animals, captopril-fed female flies showed an increase in the liver meal-induced trypsin peak in the midgut and elevated levels of protein meal-induced yolk polypeptides in the hemolymph. The latter effect was not due to a slower vitellogenin uptake by the ovaries, because oocyte growth was not affected by the captopril treatment. The apparent synergism between the demonstrated ACE functionality and the previously reported effects of the oostatic peptide Neb-TMOF are discussed in the context of our recent finding that Neb-TMOF represents a prime candidate for being the first known in vivo substrate for circulating insect ACE. Arch.     

Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 1. Sildenafil analogs

Parasitic diseases, such as African sleeping sickness, have a significant impact on the health and well-being in the poorest regions of the world. Pragmatic drug discovery efforts are needed to find new therapeutic agents. In this Letter we describe target repurposing efforts focused on trypanosomal phosphodiesterases. We outline the synthesis and biological evaluation of analogs of sildenafil (1), a human PDE5 inhibitor, for activities against trypanosomal PDEB1 (TbrPDEB1). We find that, while low potency analogs can be prepared, this chemical class is a sub-optimal starting point for further development of TbrPDE inhibitors.