Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.     

S100 alpha, CAPL, and CACY: molecular cloning and expression analysis of three calcium-binding proteins from human heart.

Elevated levels of intracellular calcium are a major cause of myocardial dysfunction. To find possible mediators of the deregulated calcium we searched for EF-hand calcium-binding proteins of the S100 family. By PCR technology we identified three members of the S100 protein family (S100 alpha, CACY, and CAPL) in the human heart. We cloned the corresponding cDNAs and examined their expression levels in various human tissues by Northern blot analysis. All three proteins are expressed at high levels in the human heart. Whereas CACY and CAPL mRNAs are expressed ubiquitously, S100 alpha mRNA is restricted to heart, skeletal muscle, and brain. Interestingly, the expression pattern of S100 alpha, CACY, and CAPL in human tissues differs significantly from that in rodent tissues.     

The Sodium/Proton Exchanger NHE8 Regulates Late Endosomal Morphology and Function

The pH and lumenal environment of intracellular organelles is considered essential for protein sorting and trafficking through the cell. We provide the first evidence that a mammalian NHE sodium (potassium)/proton exchanger, NHE8, plays a key role in the control of protein trafficking and endosome morphology. At steady state, the majority of epitope-tagged NHE8 was found in the trans-Golgi network of HeLa M-cells, but a proportion was also localized to multivesicular bodies (MVBs). Depletion of NHE8 in HeLa M-cells with siRNA resulted in the perturbation of MVB protein sorting, as shown by an increase in epidermal growth factor degradation. Additionally, NHE8-depleted cells displayed striking perinuclear clustering of endosomes and lysosomes, and there was a ninefold increase in the cellular volume taken up by LAMP1/LBPA-positive, dense MVBs. Our data points to a role for the ion exchange activity of NHE8 being required to maintain endosome morphology, as overexpression of a nonfunctional point mutant protein (NHE8 E225Q) resulted in phenotypes similar to those seen after siRNA depletion of endogenous NHE8. Interestingly, we found that depletion of NHE8, despite its function as a sodium (potassium)/proton antiporter, did not affect the overall pH inside dense MVBs.     

Molecular recognition of anions by synthetic receptors

Over the past year, several significant developments have been made in the field of anion binding. The fundamental principles of molecular recognition are increasingly being better understood, and of particular interest are reports of several synthetic anion receptors able to perform their task in complex natural media. Additionally, macroscopic devices such as anion specific electrodes and membranes based on a molecular recognition approach are now being made.     

Thiostrepton resistance mutations in the gene for 23S ribosomal RNA of halobacteria

Mutants of Halobacterium (H.) halobium and H. cutirubrum were isolated which are resistant to the 70S ribosome inhibitor thiostrepton. Using primer extension analysis, resistance was shown to correlate with base changes at position 1159, which corresponds to position 1067 of the E. coli 23S rRNA. In four mutants, A1159 was replaced by U, in one mutant by G. The results show that not only methylation (Cundliffe & Thompson (1979) Nature 278, 859-861) of A1067 (E. coli nomenclature), but also base changes at this position cause high-level resistance to thiostrepton.     

A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium falciparum

Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz), which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50) were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.     

The Biopolymer Markup Language.

SUMMARY: An XML derived from a data model designed to be a hierarchical representation of an organism has been specified and a browser to use this language has been developed. AVAILABILITY: The language definition is available in HTML form at http://www.proteometrics.com/BIOML/. The BioML browser is available on request from the author.