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Natural insect infestation in cassava (Manihot esculenta Crantz subspecies esculenta) and yam (Dioscorea spp.) chips was evaluated during two consecutive storage seasons (2003-2004 and 2004-2005) in two agroecological zones of Benin (Northern Guinea Savanna [NGS] and Sudan Savanna [SS]). The insects infesting chips were collected, identified, and counted, they included Prostephanus truncatus (Horn) (Coleoptera: Bostrychidae), Cathartus quadricollis (Guerin) (Coleoptera: Silvanidae), Carpophilus dimidiatus (F.) (Coleoptera: Nitidulidae), Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), and Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). P. truncatus and C. quadricollis were observed with a higher prevalence on cassava than on yam chips. During both seasons after 3 mo of storage, all (100%) cassava chip samples were infested with P. truncatus and C. quadricollis in both agroecological zones, whereas yam chips only showed lower infestation rates of 59.5 and 19.1% for P. truncatus and C. quadricollis, respectively, at the end of storage in 2003-2004. During the 2004-2005 season after 3 mo of storage infestation rate in yam chips was 66 and 24% in NGS and 100 and 0% in SS for P. truncatus and C. quadricollis, respectively, showing that insect infestation levels vary significantly with commodity, year, and fluctuate during the storage season.

A disulphide relay system mediates the import of cysteine-containing proteins into the intermembrane space of mitochondria. This system consists of two essential proteins, Mia40 and Erv1, which bind to newly imported proteins by disulphide transfer. A third component, Hot13, was proposed to be important in the biogenesis of cysteine-rich proteins of the intermembrane space, but the molecular function of Hot13 remained unclear. Here, we show that Hot13, a conserved zinc-binding protein, interacts functionally and physically with the import receptor Mia40. It improves the Erv1-dependent oxidation of Mia40 both in vivo and in vitro. As a consequence, in mutants lacking Hot13, the import of substrates of Mia40 is impaired, particularly in the presence of zinc ions. In mitochondria as well as in vitro, Hot13 can be functionally replaced by zinc-binding chelators. We propose that Hot13 maintains Mia40 in a zinc-free state, thereby facilitating its efficient oxidation by Erv1.

The compartment between the outer and the inner membranes of mitochondria, the intermembrane space (IMS), harbours a variety of proteins that contain disulfide bonds. Many of these proteins possess a conserved twin Cx(3)C motif or twin Cx(9)C motif. Recently, a disulfide relay system in the IMS has been identified which consists of two essential components, the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40/Tim40. The disulfide relay system drives the import of these cysteine-rich proteins into the IMS of mitochondria by an oxidative folding mechanism. In order to enable Mia40 to perform the oxidation of substrate proteins, the sulfhydryl oxidase Erv1 mediates the oxidation of Mia40 in a disulfide transfer reaction. To recycle Erv1 into its oxidized form, electrons are transferred to cytochrome c connecting the disulfide relay system to the electron transport chain of mitochondria. Despite the lack of homology of the components, the disulfide relay system in the IMS resembles the oxidation system in the periplasm of bacteria presumably reflecting the evolutionary origin of the IMS from the bacterial periplasm.

Mia40p and Erv1p are components of a translocation pathway for the import of cysteine-rich proteins into the intermembrane space of mitochondria. We have characterized the redox behavior of Mia40p and reconstituted the disulfide transfer system of Mia40p by using recombinant functional C-terminal fragment of Mia40p, Mia40C, and Erv1p. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC motif. The second and the third bonds belong to the twin CX(9)C motif and bridge the cysteine residues of two CX(9)C segments. In contrast to the stabilizing disulfide bonds of the twin CX(9)C motif, the first disulfide bond was easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond as well as fully reduced Mia40C were oxidized by Erv1p in vitro. In the course of this reaction, mixed disulfides of Mia40C and Erv1p were formed. Reoxidation of fully reduced Mia40C required the presence of the first two cysteine residues in Mia40C. However, efficient reoxidation of a Mia40C variant containing only the cysteine residues of the twin CX(9)C motif was observed when in addition to Erv1p low amounts of wild type Mia40C were present. In the reconstituted system the thiol oxidase Erv1p was sufficient to transfer disulfide bonds to Mia40C, which then could oxidize the variant of Mia40C. In summary, we reconstituted a disulfide relay system consisting of Mia40C and Erv1p.

All proteins of the intermembrane space of mitochondria are encoded by nuclear genes and synthesized in the cytosol. Many of these proteins lack presequences but are imported into mitochondria in an oxidation-driven process that relies on the activity of Mia40 and Erv1. Both factors form a disulfide relay system in which Mia40 functions as a receptor that transiently interacts with incoming polypeptides via disulfide bonds. Erv1 is a sulfhydryl oxidase that oxidizes and activates Mia40, but it has remained unclear how Erv1 itself is oxidized. Here, we show that Erv1 passes its electrons on to molecular oxygen via interaction with cytochrome c and cytochrome c oxidase. This connection to the respiratory chain increases the efficient oxidation of the relay system in mitochondria and prevents the formation of toxic hydrogen peroxide. Thus, analogous to the system in the bacterial periplasm, the disulfide relay in the intermembrane space is connected to the electron transport chain of the inner membrane.

Rotary International with the International Institute of Tropical Agriculture (IITA) conducted an information campaign from 2000 to 2004 to increase public awareness of aflatoxin in Benin, Ghana and Togo. Key informant interviews with 2416 respondents showed poor baseline knowledge of aflatoxin and its health risks. The campaign included monitoring of aflatoxin contamination in maize grains from market stores in 38 cities and towns. Aflatoxin concentration in contaminated samples ranged from 24 to 117.5 ng g(-1) in Benin, from 0.4 to 490.6 ng g(-1) in Ghana, and from 0.7 to 108.8 ng g(-1) in Togo. The campaign significantly increased public awareness that populations were exposed to high levels of aflatoxin. The number of maize traders who were informed about the toxin increased 10.3 and 3.2 times in Togo and Benin, respectively; at least 33% more traders believed the information in each of Benin and Togo; 11.4 and 28.4% more consumers sorted out and discarded bad grains in Benin and Ghana, respectively. This paper concludes that sustained public education can help reduce aflatoxin contamination.

Tim14 and Tim16 are essential components of the import motor of the mitochondrial TIM23 preprotein translocase. Tim14 contains a J domain in the matrix space that is anchored in the inner membrane by a transmembrane segment. Tim16 is a J-related protein with a moderately hydrophobic segment at its N terminus. The J and J-like domains function in the regulation of the ATPase activity of the Hsp70 chaperone of the import motor. We report here on the role of the hydrophobic segments of Tim16 and Tim14 in the TIM23 translocase. Yeast cells lacking the hydrophobic N-terminal segment in either Tim16 or Tim14 are viable but show growth defects and decreased import rates of matrix-targeted preproteins into mitochondria. The interaction of the Tim14.Tim16 complex with the core complex of the TIM23 translocase is destabilized in these cells. In particular, the N-terminal domain of Tim16 is crucial for the interaction of the Tim14.Tim16 complex with the TIM23 preprotein translocase. Deletion of hydrophobic segments in both, Tim16 and Tim14, is lethal. We conclude that import into the matrix space of mitochondria requires association of the co-chaperones Tim16 and Tim14 with the TIM23 preprotein translocase.

The thiol oxidase Erv1 and the redox-regulated receptor Mia40/Tim40 are components of a disulfide relay system which mediates import of proteins into the intermembrane space (IMS) of mitochondria. Here we report that Erv1 requires Mia40 for its import into mitochondria. After passage across the translocase of the mitochondrial outer membrane Erv1 interacts via disulfide bonds with Mia40. Erv1 does not contain twin "CX(3)C" or twin "CX(9)C" motifs which are crucial for import of typical substrates of this pathway and it does not need two "CX(2)C" motifs for import into mitochondria. Thus, Erv1 represents an unusual type of substrate of the Mia40-dependent import pathway.

The import motor of the mitochondrial translocase of the inner membrane (TIM23) mediates the ATP-dependent translocation of preproteins into the mitochondrial matrix by cycles of binding to and release from mtHsp70. An essential step of this process is the stimulation of the ATPase activity of mtHsp70 performed by the J cochaperone Tim14. Tim14 forms a complex with the J-like protein Tim16. The crystal structure of this complex shows that the conserved domains of the two proteins have virtually identical folds but completely different surfaces enabling them to perform different functions. The Tim14-Tim16 dimer reveals a previously undescribed arrangement of J and J-like domains. Mutations that destroy the complex between Tim14 and Tim16 are lethal demonstrating that complex formation is an essential requirement for the viability of cells. We further demonstrate tight regulation of the cochaperone activity of Tim14 by Tim16. The first crystal structure of a J domain in complex with a regulatory protein provides new insights into the function of the mitochondrial TIM23 translocase and the Hsp70 chaperone system in general.

Formation of iron/sulfur (Fe/S) clusters, protein translocation and protein folding are essential processes in the mitochondria of Saccharomyces cerevisiae. In a systematic approach to characterize essential proteins involved in these processes, we identified a novel essential protein of the mitochondrial matrix, which is highly conserved from yeast to human and which we termed Isd11. Depletion of Isd11 caused a strong reduction in the levels of the Fe/S proteins aconitase and the Rieske protein, and a massive decrease in the enzymatic activities of aconitase and succinate dehydrogenase. Incorporation of iron into the Fe/S protein Leu1 and formation of the Fe/S cluster containing holoform of the mitochondrial ferredoxin Yah1 were inhibited in the absence of Isd11. This strongly suggests that Isd11 is required for the assembly of Fe/S proteins. We show that Isd11 forms a stable complex with Nfs1, the cysteine desulfurase of the mitochondrial machinery for Fe/S cluster assembly. In the absence of Isd11, Nfs1 is prone to aggregation. We propose that Isd11 acts together with Nfs1 in an early step in the biogenesis of Fe/S proteins.

We investigated the effect of substrate binding on the mechanical stability of mouse dihydrofolate reductase using single-molecule force spectroscopy by atomic force microscopy. We find that under mechanical forces dihydrofolate reductase unfolds via a metastable intermediate with lifetimes on the millisecond timescale. Based on the measured length increase of approximately 22 nm we suggest a structure for this intermediate with intact substrate binding sites. In the presence of the substrate analog methotrexate and the cofactor NADPH lifetimes of this intermediate are increased by up to a factor of two. Comparing mechanical and thermodynamic stabilization effects of substrate binding suggests mechanical stability is dominated by local interactions within the protein structure. These experiments demonstrate that protein mechanics can be used to probe the substrate binding status of an enzyme.

A first component involved in import into the mitochondrial intermembrane space, named Mia40, has been described recently in yeast. Here, we identified the human MIA40 as a novel and ubiquitously expressed component of human mitochondria. It belongs to a novel protein family whose members share six highly conserved cysteine residues constituting a -CXC-CX9C-CX9C- motif. Human MIA40 is significantly smaller than the fungal protein and lacks the N-terminal extension including a transmembrane region and mitochondrial targeting signal. It forms soluble complexes within the intermembrane space of human mitochondria. Depletion of MIA40 in human cells by RNA interference specifically affected steady-state levels of small and cysteine-containing intermembrane space proteins like DDP1 and TIM10A, suggesting that MIA40 acts along the import pathway into the intermembrane space. Studies on the in vivo redox state of human MIA40 demonstrated that it contains intramolecular disulfide bonds. Thiol-trapping assays revealed the co-existence of different oxidation states of human MIA40 within the cell. Furthermore, we show that the twin -CX9C- motif is specifically required for import and stability of MIA40 in mitochondria. Partial mutation of this motif affects stable accumulation of MIA40 in the intermembrane space, whereas mutation of all cysteine residues in this motif inhibits import in mitochondria. Taken together, we conclude that the biogenesis and function of MIA40 in the mitochondrial intermembrane space is dependent on redox processes involving conserved cysteine residues.

Aflatoxins are a family of fungal toxins that are carcinogenic to man and cause immunosuppression, cancer and growth reduction in animals. We conducted a cross-sectional study among 480 children (age 9 months to 5 years) across 4 agro-ecological zones (SS, NGS, SGS and CS) in Benin and Togo to identify the effect of aflatoxin exposure on child growth and assess the pattern of exposure. Prior reports on this study [Gong, Y.Y.,Cardwell, K., Hounsa, A., Egal, S., Turner, Hall, A.J., Wild, C.P., 2002. Dietary aflatoxin exposure and impaired growth in young children from Benin and Togo: cross sectional study. British Medical Journal 325, 20-21, Gong, Y.Y., Egal, S., Hounsa, A., Turner, P.C., Hall, A.J., Cardwell, K., Wild, C.P., 2003. Determinants of aflatoxin exposure in young children from Benin and Togo, West Africa: the critical role of weaning and weaning foods. International Journal of Epidemiology, 32, 556-562] showed that aflatoxin exposure among these children is widespread (99%) and that growth faltering is associated with high blood aflatoxin-albumin adducts (AF-alb adducts), a measure of recent past exposure. The present report demonstrates that consumption of maize is an important source of aflatoxin exposure for the survey population. Higher AF-alb adducts were correlated with higher A. flavus (CFU) infestation of maize (p=0.006), higher aflatoxin contamination (ppb) of maize (p<0.0001) and higher consumption frequencies of maize (p=0.053). The likelihood of aflatoxin exposure from maize was particularly high in agro-ecological zones where the frequency of maize consumption (SGS and CS), the presence of aflatoxin in maize (SGS) or the presence of A. flavus on maize (NGS and SGS) was relatively high. Socio-economic background did not affect the presence of A. flavus and aflatoxin in maize, but better maternal education was associated with lower frequencies of maize consumption among children from the northernmost agro-ecological zone (SS) (p=0.001). The impact of groundnut consumption on aflatoxin exposure was limited in this population. High AF-alb adduct levels were correlated with high prevalence of A. flavus and aflatoxin in groundnut, but significance was weak after adjustment for weaning status, agro-ecological zone and maternal socio-economic status (resp. p=0.091 and p=0.083). Ingestion of A. flavus and aflatoxin was high in certain agro-ecological zones (SS and SGS) and among the higher socio-economic strata due to higher frequencies of groundnut consumption. Contamination of groundnuts was similar across socio-economic and agro-ecological boundaries. In conclusion, dietary exposure to aflatoxin from groundnut was less than from maize in young children from Benin and Togo. Intervention strategies that aim to reduce dietary exposure in this population need to focus on maize consumption in particular, but they should not ignore consumption of groundnuts.

The import motor of the mitochondrial (mt)TIM23 complex drives translocation of presequence-containing preproteins across the mitochondrial inner membrane in an ATP-dependent manner. Tim44 is the central component of the motor. It recruits mtHsp70, which binds the incoming preproteins. The J protein Tim14 stimulates the ATPase activity of mtHsp70 and thereby enables efficient binding of mtHsp70 to preproteins. Tim16 is a J-like protein that forms a stable subcomplex with Tim14 and recruits it to the translocase. All subunits of the TIM23 translocase but one are essential for yeast cell viability. Yeast cells contain a close homologue of Tim14, Mdj2. In contrast to Tim14, its deletion leads to no obvious growth defect. In the present study we analyzed Mdj2 and compared it with Tim14. Mdj2 forms a complex with Tim16 and is recruited to the TIM23 translocase. It stimulates the ATPase activity of mtHsp70 to the same extent that Tim14 does. Mdj2 is expressed at a lower level compared with Tim14, and its complex with Tim16 is less stable. However, overexpressed Mdj2 fully restores the growth of cells lacking Tim14. We conclude that Mdj2 is a functional J protein and a component of the mitochondrial import motor.

We describe here a pathway for the import of proteins into the intermembrane space (IMS) of mitochondria. Substrates of this pathway are proteins with conserved cysteine motifs, which are critical for import. After passage through the TOM channel, these proteins are covalently trapped by Mia40 via disulfide bridges. Mia40 contains cysteine residues, which are oxidized by the sulfhydryl oxidase Erv1. Depletion of Erv1 or conditions reducing Mia40 prevent protein import. We propose that Erv1 and Mia40 function as a disulfide relay system that catalyzes the import of proteins into the IMS by an oxidative folding mechanism. The existence of a disulfide exchange system in the IMS is unexpected in view of the free exchange of metabolites between IMS and cytosol via porin channels. We suggest that this process reflects the evolutionary origin of the IMS from the periplasmic space of the prokaryotic ancestors of mitochondria.

Translocation of preproteins with N-terminal presequences into mitochondria requires the cooperation of the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). However, the molecular nature of the translocation contact sites is poorly understood. We have identified a novel component of the TIM23 translocase, Tim21, which is involved in their formation. Tim21 is anchored in the mitochondrial inner membrane by a single transmembrane domain and exposes its C-terminal domain into the intermembrane space. The purified C-terminal domain of Tim21 appears not to bind to any of the TIM23 components but rather specifically interacts with the TOM complex. We propose that Tim21 binds to the trans site of the TOM complex thus keeping the two translocases in close contact.

All proteins of the intermembrane space (IMS) of mitochondria are synthesized in the cytosol. The mechanisms by which these polypeptides are transported into the IMS are strikingly different from other protein-translocation processes in the cell. Recent studies suggest that IMS proteins reach their destination by three alternative principles that differ in the energy sources employed to drive the translocation reactions. The first class of proteins uses both hydrolysis of matrix ATP and the electrochemical potential of the inner membrane. The second class depends on the energy gain of protein folding, and the third on the association of the proteins to high-affinity binding sites in the IMS.

Hsp70 chaperones mediate folding of proteins and prevent their misfolding and aggregation. We report here on a new kind of Hsp70 interacting protein in mitochondria, Hep1. Hep1 is a highly conserved protein present in virtually all eukaryotes. Deletion of HEP1 results in a severe growth defect. Cells lacking Hep1 are deficient in processes that need the function of mitochondrial Hsp70s, such as preprotein import and biogenesis of proteins containing FeS clusters. In the mitochondria of these cells, Hsp70s, Ssc1 and Ssq1 accumulate as insoluble aggregates. We show that it is the nucleotide-free form of mtHsp70 that has a high tendency to self-aggregate. This process is efficiently counteracted by Hep1. We conclude that Hep1 acts as a chaperone that is necessary and sufficient to prevent self-aggregation and to thereby maintain the function of the mitochondrial Hsp70 chaperones.

The fate of aflatoxins and fumonisins, two mycotoxins that cooccur in maize, was studied through the traditional processing of naturally contaminated maize in mawe, makume, ogi, akassa, and owo, maize-based foods common in Benin, West Africa. Levels of total aflatoxin and fumonisin were measured at the main unit operations of processing, and the unit operations that induce significant reduction of mycotoxin level were identified. Overall reduction of mycotoxin level was more significant during the preparation of makume (93% reduction of aflatoxins, 87% reduction of fumonisins) and akassa (92% reduction of aflatoxins, 50% reduction of fumonisins) than that of owo (40% reduction of aflatoxins, 48% reduction of fumonisins). Sorting, winnowing, washing, crushing combined with dehulling of maize grains were the unit operations that appeared very effective in achieving significant mycotoxin removal. Aflatoxins and fumonisins were significantly recovered in discarded mouldy and damaged grains and in washing water. Fermentation and cooking showed little effect. During the preparation of ogi and akassa, reduction of fumonisin levels measured in food matrix was lower (50%) compared to mawe and makume, probably due to significant fumonisin release in ogi supernatant. Consequently, the use of ogi supernatant for preparing beverages or traditional herbal medicines could be harmful as it is likely to be contaminated with mycotoxin from the raw maize.

Many proteins located in the intermembrane space (IMS) of mitochondria are characterized by a low molecular mass, contain highly conserved cysteine residues and coordinate metal ions. Studies on one of these proteins, Tim13, revealed that net translocation across the outer membrane is driven by metal-dependent folding in the IMS . We have identified an essential component, Mia40/Tim40/Ykl195w, with a highly conserved domain in the IMS that is able to bind zinc and copper ions. In cells lacking Mia40, the endogenous levels of Tim13 and other metal-binding IMS proteins are strongly reduced due to the impaired import of these proteins. Furthermore, Mia40 directly interacts with newly imported Tim13 protein. We conclude that Mia40 is the first essential component of a specific translocation pathway of metal-binding IMS proteins.

Life table studies for the Angoumois grain moth, Sitotroga cerealella (Olivier), a pest on stored maize, Zea mays L., in West Africa, were conducted as part of the expansion of a mathematical simulation model that has been developed for two pests of stored maize. The effects of four temperatures (20, 25, 30, and 35 degrees C) and two relative humidity levels (44 and 80%) on developmental time, age-specific survivorship and fecundity, sex ratio, and intrinsic rate of natural increase (r(m)) of S. cerealella were investigated. Sex ratio was close to 1:1 at all temperatures and humidity. Minimum development time occurred close to 32 degrees C and 80% RH for both males and females, and developmental time of females was significantly shorter than that of males. Immature survivorship was highest between 25 and 30 degrees C and 80% RH and lowest at 35 degrees C under both humidity conditions. A similar low level was found at 20 degrees C and 44% RH. The greatest fecundity (124 eggs per female) occurred at 20 degrees C, 80% RH. The maximum r(m) value was 0.086 d(-1) at 30 degrees C and 80% RH, but the growth rate declined dramatically at 35 degrees C. If compared with the few other life table studies conducted on this species on maize in India and North America, some variation among the strains becomes evident. A common conclusion for the current study and previous ones is that optimal population development for S. cerealella occurs at approximately 30 degrees C and at high humidity.

Insertion of beta-barrel proteins into the outer membrane of mitochondria is mediated by the TOB complex. Known constituents of this complex are Tob55 and Mas37. We identified a novel component, Tob38. It is essential for viability of yeast and the function of the TOB complex. Tob38 is exposed on the surface of the mitochondrial outer membrane. It interacts with Mas37 and Tob55 and is associated with Tob55 even in the absence of Mas37. The Tob38-Tob55 core complex binds precursors of beta-barrel proteins and facilitates their insertion into the outer membrane. Depletion of Tob38 results in strongly reduced levels of Tob55 and Mas37 and the residual proteins no longer form a complex. Tob38-depleted mitochondria are deficient in the import of beta-barrel precursor proteins, but not of other outer membrane proteins or proteins of other mitochondrial subcompartments. We conclude that Tob38 has a crucial function in the biogenesis of beta-barrel proteins of mitochondria.

Mitochondrial morphology and inheritance of mitochondrial DNA in yeast depend on the dynamin-like GTPase Mgm1. It is present in two isoforms in the intermembrane space of mitochondria both of which are required for Mgm1 function. Limited proteolysis of the large isoform by the mitochondrial rhomboid protease Pcp1/Rbd1 generates the short isoform of Mgm1 but how this is regulated is unclear. We show that near its NH2 terminus Mgm1 contains two conserved hydrophobic segments of which the more COOH-terminal one is cleaved by Pcp1. Changing the hydrophobicity of the NH2-terminal segment modulated the ratio of the isoforms and led to fragmentation of mitochondria. Formation of the short isoform of Mgm1 and mitochondrial morphology further depend on a functional protein import motor and on the ATP level in the matrix. Our data show that a novel pathway, to which we refer as alternative topogenesis, represents a key regulatory mechanism ensuring the balanced formation of both Mgm1 isoforms. Through this process the mitochondrial ATP level might control mitochondrial morphology.

The activation of caspase-3 represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Upon induction of apoptosis, the large (p17) and small (p12) subunits, comprising active caspase-3, are generated via proteolytic processing of a latent proenzyme dimer. Two copies of each individual subunit are generated to form an active heterotetramer. The tetrameric form of caspase-3 cleaves specific protein substrates within the cell, thereby producing the apoptotic phenotype. In contrast to the proenzyme, once activated in HeLa cells, caspase-3 is difficult to detect due to its rapid degradation. Interestingly, however, enzyme stability and therefore detection of active caspase-3 by immunoblot analysis can be restored by treatment of cells with a peptide-based caspase-3 selective inhibitor, suggesting that the active form can be stabilized through protein-inhibitor interaction. The heteromeric active enzyme complex is necessary for its stabilization by inhibitors, as expression of the large subunit alone is not stabilized by the presence of inhibitors. Our results show for the first time, that synthetic caspase inhibitors not only block caspase activity, but may also increase the stability of otherwise rapidly degraded mature caspase complexes. Consistent with these findings, experiments with a catalytically inactive mutant of caspase-3 show that rapid turnover is dependent on the activity of the mature enzyme. Furthermore, turnover of otherwise stable active site mutants of capase-3 is rescued by the presence of the active enzyme suggesting that turnover can be mediated in trans.

Mitochondria import the vast majority of their proteins from the cytosol. The mitochondrial import motor of the TIM23 translocase drives the translocation of precursor proteins across the outer and inner membrane in an ATP-dependent reaction. Tim44 at the inner face of the translocation pore recruits the chaperone mtHsp70, which binds the incoming precursor protein. This reaction is assisted by the cochaperones Tim14 and Mge1. We have identified a novel essential cochaperone, Tim16. It is related to J-domain proteins and forms a stable subcomplex with the J protein Tim14. Depletion of Tim16 has a marked effect on protein import into the mitochondrial matrix, impairs the interaction of Tim14 with the TIM23 complex and leads to severe structural changes of the import motor. In conclusion, Tim16 is a constituent of the TIM23 preprotein translocase, where it exerts crucial functions in the import motor.

The outer membranes of mitochondria and chloroplasts are distinguished by the presence of beta-barrel membrane proteins. The outer membrane of Gram-negative bacteria also harbours beta-barrel proteins. In mitochondria these proteins fulfil a variety of functions such as transport of small molecules (porin/VDAC), translocation of proteins (Tom40) and regulation of mitochondrial morphology (Mdm10). These proteins are encoded by the nucleus, synthesized in the cytosol, targeted to mitochondria as chaperone-bound species, recognized by the translocase of the outer membrane, and then inserted into the outer membrane where they assemble into functional oligomers. Whereas some knowledge has been accumulated on the pathways of insertion of proteins that span cellular membranes with alpha-helical segments, very little is known about how beta-barrel proteins are integrated into lipid bilayers and assembled into oligomeric structures. Here we describe a protein complex that is essential for the topogenesis of mitochondrial outer membrane beta-barrel proteins (TOB). We present evidence that important elements of the topogenesis of beta-barrel membrane proteins have been conserved during the evolution of mitochondria from endosymbiotic bacterial ancestors.

The yeast mitochondrial Oxa1 protein is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins. Oxa1 mediates the insertion of proteins (nuclearly and mitochondrially encoded) into the inner membrane. The mitochondrially encoded substrates interact directly with Oxa1 during their synthesis as nascent chains and in a manner that is supported by the associated ribosome. We have investigated if the Oxa1 complex interacts with the mitochondrial ribosome. Evidence to support a physical association between Oxa1 and the large ribosomal subunit is presented. Our data indicate that the matrix-exposed C-terminal region of Oxa1 plays an important role supporting the ribosomal-Oxa1 interaction. Truncation of this C-terminal segment compromises the ability of Oxa1 to support insertion of substrate proteins into the inner membrane. Oxa1 can be cross-linked to Mrp20, a component of the large ribosomal subunit. Mrp20 is homologous to L23, a subunit located next to the peptide exit tunnel of the ribosome. We propose that the interaction of Oxa1 with the ribosome serves to enhance a coupling of translation and membrane insertion events.

The TIM23 translocase mediates the deltaPsi- and ATP-dependent import of proteins into mitochondria. We identified Tim14 as a novel component of the TIM23 translocase. Tim14 is an integral protein of the inner membrane with a typical J-domain exposed to the matrix space. TIM14 genes are present in the genomes of virtually all eukaryotes. In yeast, Tim14 is essential for viability. Mitochondria from cells depleted of Tim14 are deficient in the import of proteins mediated by the TIM23 complex. In particular, import of proteins that require the action of mtHsp70 is affected. Tim14 interacts with Tim44 and mtHsp70 in an ATP-dependent manner. A mutation in the HPD motif of the J-domain of Tim14 is lethal. Thus, Tim14 is a constituent of the mitochondrial import motor. We propose a model in which Tim14 is required for the activation of mtHsp70 and enables this chaperone to act in a rapid and regulated manner in the Tim44-mediated trapping of unfolded preproteins entering the matrix.

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. We isolated the TIM23 complex of Neurospora crassa. Besides Tim23 and Tim17, it contained a novel component, referred to as Tim50. Tim50 spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the intermembrane space. Tim50 is essential for viability of yeast. Mitochondria from cells depleted of Tim50 displayed strongly reduced import kinetics of preproteins using the TIM23 complex. Tim50 could be cross-linked to preproteins that were halted at the level of the translocase of the outer membrane (TOM complex) or spanning both TOM and TIM23 complexes. We suggest that Tim50 plays a crucial role in the transfer of preproteins from the TOM complex to the TIM23 complex through the intermembrane space.

The protooncogenes Ras and Raf play important roles in signal transduction pathways regulated by mitogen-activated protein kinases. Mutations of Ras that arrest the protein in its active state are frequently implicated in tumor formation. We used Ras and Raf proteins in the yeast two-hybrid system to search for natural or synthesized substances capable of modulating Ras/Raf interaction by specifically binding to one of the interacting partners. We found that cycloalkylidene carboxylic acids enhanced Ras/Raf interaction by acting on the cysteine-rich domain of Raf. Several analogues of the active substance 2-cyclohexylidene propanoic acid were synthesized and the importance of the semicyclic double bond in the stabilization of Ras/Raf interaction was demonstrated. Variation of the size and the substituents of the cyclic system as well as the length of the carboxylic acid resulted in enhanced Ras/Raf interaction.

We report a 12-year-old girl suffering from end-stage renal disease due to focal-segmental sclerosis and retardation of statomotoric and mental development of unknown origin. Renal transplantation (TX) was performed 7 months after initiation of peritoneal dialysis at the age of 11 years. Immunosuppressive therapy included cyclosporine A, mycophenolate mofetil and methylprednisolone. The patient developed spiking fever up to 40 degrees C without signs of infection 10 months after TX. Kidney function remained stable but ultrasound examination and CT-scan showed hypodense masses within both liver and spleen. Epstein-Barr virus (EBV) polymerase chain reaction (PCR) results with a high number of copies (20 x 10(6) copies/ml blood) against the background of a previous EBV infection (IgG positive, IgM negative) made the diagnosis of EBV-reactivation likely. Splenectomy was performed. Examination of the spleen showed EBV-associated polymorphic posttransplant lymphoproliferative disease (PTLD) with predominant B cell proliferation and monoclonal VH3-rearrangement of the IgG heavy chain locus. Therapy with acyclovir was introduced and immunosuppression was reduced. No rejection episode occurred. Body temperature normalized and the patient recovered over a 3-month period. EBV-PCR in plasma was negative (0.02 x 10(6) copies/ml blood) 12 weeks after reduction of immunosuppression. The liver masses completely resolved after 27 months. After a total follow-up of 36 months the child remains in good health.

The formation of polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) from amorphous 12C- and 13C-labeled carbon was studied on model mixtures and real fly ashes. PCDD/F can either be formed directly (de-novo) from carbon already present in fly ash or step-by-step via condensation of two aromatic rings. Using model mixtures containing 12C- and 13C-labeled carbon in various ratios we observed the formation of the following compound classes: 12C6-PCPh, -PCBz, 13C6-PCPh, -PCBz, 12C12-PCDD/ F, 13C12-PCDD/F, and 12C6 13C6-PCDD/F. By examining the fraction of the mixed PCDD/F (one of the two aromatic ring is composed solely of 12C-atoms while the other contains only 13C-atoms) in the total concentration of PCDD/F, conclusions on the formation of these three ring structures are possible. From the experimental results, it can be concluded that both reaction mechanisms are operative in the formation of PCDD/F from carbon. On fly ashes approximately half of the total amount of PCDD is formed via condensation of de-novo created C6-precursors e.g. chlorophenols, while the remainder is directly released (de-novo) from the carbon i.e., formed from a related C12-structure. However, the condensation of intermediate aromatic C6-precursors is of minor importance in the formation of PCDF. With increasing temperature the relative amount of the 12C6 13C6-PCDD formed by condensation decreases due to the faster evaporation of chlorophenols. At a constant reaction temperature, the ratio of both reaction pathways is hardly influenced by reaction time. In experiments with fly ashes doped with 13C-labeled carbon, this carbon isotope shows a similar reactivity as the native carbon present on the fly ash. Thus, the used amorphous carbons are suitable models for this investigation.

Dried yam (Dioscorea spp.) chips are widely consumed in Bénin but are often attacked by molds. Invasion of food by Aspergillus flavus may lead to aflatoxin contamination. We report here the result of a survey on the sanitary quality of dried yam chips in Bénin. During July and August 2000, 50 dried yam chips samples were collected from different points in the marketing chain; 10 samples were collected from each of 5 stages: producers, wholesalers, retailers, dried yam-based food sellers and consumers. Aflatoxin content was assayed by the bio-luminescence method (1) after methanol/water extraction. Aflatoxins were detected in all dried yam chip samples, with levels ranging from 2.2 to 200 ppb and a mean value of 14 ppb. An aflatoxin concentration higher than the European Union's maximum residue limit (MRL) of 4 ppb was found in 98% of the samples (N = 50), while 6% had an aflatoxin concentration higher than the World Health Organization's MRL of 20 ppb. Molds were analyzed from two samples, each with aflatoxin levels around 5 ppb, on colony unit medium specific for A. flavus (2). Aspergillus spp. were detected in the inner part of dried yam chips of both samples, with a mean level of 9,000 CFU/g. Fusarium colonies were also present but were not identified to species. References: (1) D. Champiat and J. Larpent. Bio-chimie-luminescence: Principes et Applications. Masson, Paris, France. 1993. (2) P. J. Cotty. Mycopathologia 125:157, 1994.

Biosynthesis of the polyamines putrescine, spermidine, and spermine, and activation of the first key enzyme ornithine decarboxylase (ODC) are closely associated with cellular proliferation. In the present study, the distribution of ODC activity and polyamine levels was investigated for the first time regionally in experimental brain tumors of the cat. Brain tumors were produced by stereotactic xenotransplantation of rat glioma cells. Twenty days after implantation, the brains were frozen in situ, cut into slices, and cryostat sections and tissue samples were taken to determine ODC activity and polyamine levels biochemically. The quantified data were color-coded to present the regional distribution of ODC activity and polyamine levels in the respective section. ODC activity significantly increased in some areas within the tumor, whereas peritumoral tissue showed no difference to the non-tumoral, contralateral hemisphere. This increase turned out in parallel to a high number of mitoses in the same tumor parts (r=0.861). Putrescine levels increased both, in the whole tumor and in the peritumoral edema. Regional differences in putrescine content did not correlate with solid and proliferative parts of the tumor. Spermidine and spermine levels were only slightly increased in some parts of the tumor. Thus, these experiments show the close correlation of a high mitotic rate and activation of ODC within experimental gliomas and underline the relevance of ODC as a biochemical marker of proliferation in brain tumors.

The biogenesis of mitochondria requires the integration of many proteins into the inner membrane from the matrix side. The inner membrane protein Oxa1 plays an important role in this process. We identified Mba1 as a second mitochondrial component that is required for efficient protein insertion. Like Oxa1, Mba1 specifically interacts both with mitochondrial translation products and with conservatively sorted, nuclear-encoded proteins during their integration into the inner membrane. Oxa1 and Mba1 overlap in function and substrate specificity, but both can act independently of each other. We conclude that Mba1 is part of the mitochondrial protein export machinery and represents the first component of a novel Oxa1-independent insertion pathway into the mitochondrial inner membrane.

Oxa1p is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins. Oxa1p has been shown previously to directly facilitate the export of the N-terminal domains of membrane proteins across the inner membrane to the intermembrane space of mitochondria. Here we report on a general role of Oxa1p in the membrane insertion of proteins. (i) The function of Oxa1p is not limited to the insertion of membrane proteins that undergo N-terminal tail export; rather, it also extends to the insertion of other polytopic proteins such as the mitochondrially encoded Cox1p and Cox3p proteins. These are proteins whose N-termini are retained in the mitochondrial matrix. (ii) Oxa1p interacts directly with these substrates prior to completion of their synthesis. (iii) The interaction of Oxa1p with its substrates is particularly strong when nascent polypeptide chains are inserted into the inner membrane, suggesting a direct function of Oxa1p in co-translational insertion from the matrix. Taken together, we conclude that the Oxa1 complex represents a general membrane protein insertion machinery in the inner membrane of mitochondria.

In thermal metallurgical processes such as iron ore sintering and metal smelting operations, large flows of off-gases are generated. Mainly due to residue recycling in such processes, chlorine and volatile organics are always present in the feed. As a consequence of "de novo" formation, the off-gases from such processes typically contain dioxins in the range 0.3-30 ng I-TEQ/Nm3. So far there are only very few studies about the mechanisms of dioxin formation and destruction in these metallurgical processes. In an European Union (EU) research project "Minimization of dioxins in thermal industrial processes: mechanisms, monitoring and abatement (MINIDIP)", integrated iron and steel plant has been selected as one of the industrial sectors for further investigation. A large number of particulate samples (feed, belt siftings, electrofilter) were collected from the iron ore sintering installations from various steel plants and analyzed for their organochlorocompound contents. Measurable amounts of PCDD/F, PCBz, PCB were found for all samples. The various parameters influencing their de novo synthesis activity were also evaluated in laboratory experiments, and such activity was found to be moderate for samples from the ore sinter belt, but extremely high for some ESP dusts. Fine dust is active in a wide range of temperatures starting at 200 degrees C and declining above 450 degrees C; the optimal temperature for de novo synthesis was found to be around 350 degrees C; some inhibitors, such as triethanolamine, may reduce de novo activity by 50%, and lowering the O2 concentration in the gas stream leads to a much lower amount of PCDD/F formation. On the basis of their relative mass, typical operating conditions and specific activity of the different samples, the regions in the sintering plant where de novo synthesis may take place were tentatively established.

Aflatoxin level in 300 farmers' stores in four agro-ecological zones in Benin, a west African coastal country, were determined over a period of 2 years. At sampling a questionnaire was used to evaluate maize storage practices. Farmers were asked what storage structure they used, their storage form, storage period, pest problems in storage and what was done against them. Beninese farmers often changed their storage structures during the storage period, transfering the maize from a drying or temporary store to a more durable one. Most of the farmers complained about insects damaging stored maize. Often, storage or cotton insecticides were utilized against these pests. Regression analysis identified those factors that were associated with increased or reduced aflatoxin.Maize samples in the southern Guinea and Sudan savannas were associated with higher aflatoxin levels and the forest/savanna mosaic was related to lower toxin levels. Factors associated with higher aflatoxin were: storage for 3-5 months, insect damage and use of Khaya senegalensis-bark or other local plants as storage protectants. Depending on the agroecological zone, storage structures that had a higher risk of aflatoxin development were the "Ago", the "Secco", the "Zingo" or storing under or on top of the roof of the house. Lower aflatoxin levels were related to the use of storage or cotton insecticides, mechanical means or smoke to protect against pests or cleaning of stores before loading them with the new harvest. Fewer aflatoxins were found when maize was stored in the "Ago" made from bamboo or when bags were used as secondary storage containers.

beta-Endorphin is an opioid peptide representing the C-terminal 31 amino acid residue fragment of proopiomelanocortin (POMC). The release of beta-endorphin from the pituitary into the cardiovascular compartment under physical or emotional stress has been frequently reported. However, besides beta-endorphin (1-31), nine acetylated or non-acetylated beta-endorphin analogues exist - in addition to N-terminally elongated beta-endorphin derivatives such as beta-lipotropin (beta-LPH). Since conventional radioimmunoassays (RIAs) and even commercially available two site-RIAs pick up at least some of those beta-endorphin derivatives, only "beta-endorphin immunoreactive materials" and not authentic beta-endorphin have been determined in those studies. We have developed a highly specific two site-RIA for beta-endorphin (1-31), which does not cross-react with all beta-endorphin derivatives known to occur as yet. Using this RIA as well as further assays for determination of beta-endorphin (1-31), beta-endorphin immunoreactive material (IRM), ACTH and Cortisol in the plasma of 14 volunteers upon intensive physical exercise, we found authentic beta-endorphin only in about 50% of the plasma samples, representing therein only a minor portion of the beta-endorphin IRM.

A subcutaneous, T-phenotypic anaplastic large cell lymphoma (CD30/Ki1-positive, EBV positive) was diagnosed in a HIV-infected bisexual man. Without chemotherapy the patient had a sustained long-term remission of this tumor (more than three years) after the initiation of highly active antiretroviral therapy. By PCR analysis of T-cell receptor beta gene rearrangements the tumor was found to be oligoclonal. Improvement of cellular immune function by antiretroviral therapy is the only recognizable factor which may have led to tumor remission. This hypothesis is supported by parallels to EBV associated polyclonal lymphoproliferation in allogeneic transplantat recipients where regression of lymphoma can be induced by reducing immunosuppressive therapy.

We performed experiments on two different matrices with 2,4,6-trichlorophenol as precursor to Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD)/F. A municipal solid waste incinerators (MSWI) and a model fly ash were spiked in two different ways. The experiments demonstrated a three times higher formation potential of the trichlorophenol to PCDD on MSWI fly ash compared with the model fly ash used. For both fly ashes the PCDD yield was higher when gaseous trichlorophenol was fed continuously compared to mixing the fly ashes prior to the experiments with the total amount of the precursor. Despite dilution of the fly ashes tenfold with an inactive matrix the conversion of the chlorophenol was very high.

Cytomegalovirus (CMV) disease is a typical late-stage complication of AIDS. Only six cases of CMV sinusitis have been reported in the literature. This is the first case of CMV sinusitis leading to the diagnosis of HIV and CMV retinitis. Diseases of the sinonasal tract may represent an initial manifestation of HIV or AIDS.

We have identified Cox20p, a 23.8-kDa protein of the mitochondrial inner membrane that is involved in the biogenesis of the yeast cytochrome oxidase complex. Cytochrome oxidase subunit 2 (Cox2p) accumulates as a precursor in cox20 mutants, suggesting a defect in biogenesis of this mitochondrially encoded protein. The inability of cox20 mutants to process the subunit 2 precursor (pCox2p) is not due to impaired export of the protein across the inner membrane or to an inactive Imp1p/Imp2p peptidase. Rather, Cox20p specifically binds the newly synthesized pCox2p, a step required to present the exported pCox2p as a substrate to the Imp1p peptidase. All of the endogenous pCox2p accumulated in an Deltaimp1 mutant, and a small fraction of Cox2p in wild type yeast, is detected in a complex with Cox20p. Following maturation Cox2p remained associated with Cox20p, prior to assembling into the cytochrome oxidase complex. We propose that Cox20p acts as a membrane-bound chaperone necessary for cleavage of pCox2p and for interaction of the mature protein with other subunits of cytochrome oxidase in a later step of the assembly process.

Non-randomized prospective multicenter studies involving a large number of patients are often capable to prove effectiveness of perioperative antimicrobial prophylaxis in situations where it is not feasible to carry out highly sophisticated clinical trials due to limitations in logistical or financial resources. Furthermore, these studies tend to give a true picture of the daily work and procedures in hospitals, contrary to controlled trials in clinical research which might suffer of multitudes of restrictions and exclusions. Some non-randomized studies each involving more than 1,000 patients on antibiotic prophylaxis in colonic and biliary surgery as well as appendectomy are discussed and its usefulness demonstrated.

The anti-CD25 immunotoxin RFT5.dgA was constructed by coupling the monoclonal antibody RFT5 via a sterically hindered disulfide linker to deglycosylated ricin A-chain and was administered to patients with relapsed Hodgkin's lymphoma in four bolus infusions over 7 days (day 1, 3, 5 and 7). The maximum tolerated dose in these patients as defined in a previous phase I study was 15 mg/m2. Subsequently, further patients were enrolled at the maximum tolerated dose and a total of 18 patients were treated at this level. All patients had signs of progressive disease and were heavily pretreated. Side-effects in this trial were moderate and related to vascular leak syndrome. Five of 18 patients experienced NCI grade III toxicities including weakness, edema, dyspnea, and myalgia. Eleven of 16 (69%) patients receiving two or more cycles produced human anti-ricin antibodies and human anti-mouse antibodies (>/=1.0 microg/ml). Seventeen of 18 patients were evaluable for clinical response. These included two partial remissions. One patient demonstrated minor response and five patients stable diseases. We conclude that RFT5.dgA is of moderate clinical efficacy in this group of heavily pretreated refractory patients. Leukemia (2000) 14, 129-135.

Bcs1p, a mitochondrial protein and member of the conserved AAA protein family, is involved in the biogenesis of the cytochrome bc(1) complex. We demonstrate here that Bcs1p is directly required for the assembly of the Rieske FeS and Qcr10p proteins into the cytochrome bc(1) complex. Bcs1p binds to a precomplex in the assembly pathway of the cytochrome bc(1) complex. Binding of Bcs1p to and release from this assembly intermediate is driven by ATP hydrolysis. We propose that Bcs1p acts as an ATP-dependent chaperone, maintaining the precomplex in a competent state for the subsequent assembly of the Rieske FeS and Qcr10p proteins.

A number of nuclear encoded inner membrane proteins of mitochondria span the membrane in such a manner that their N termini are located in the intermembrane space. Many of these proteins attain this membrane orientation by undergoing an export step from the matrix across the inner membrane. This export process, which resembles bacterial N-tail export from energetic and topogenic signal requirements, is facilitated by Oxa1p, a protein that has homologues throughout prokaryotes and eukaryotes. Oxa1p, as we have previously shown, is required to export the N and C termini of the mitochondrially encoded pCoxII to the intermembrane space. We demonstrate here that imported nuclear encoded proteins physically interact with Oxa1p and depend on Oxa1p for efficient export of their N termini to the intermembrane space. Furthermore, Oxa1p interacts with nascent polypeptide chains synthesized in mitochondria, including the fully synthesized pCoxII and CoxIII species. Thus, Oxa1p represents a component of a general export machinery of the mitochondrial inner membrane.

The effectiveness of antimicrobial prophylaxis was evaluated on the basis of data collected in a study on quality management carried out in 28 East German hospitals, involving 4477 laparoscopic and conventional cholecystectomies (197 of which with revision of the common bile duct). In 3128 patients a laparoscopic procedure (with consecutive conversion to an open cholecystectomy in 236 cases) and in 1349 patients a primarily conventional open cholecystectomy had been performed (a total of 2217 cases with and 2260 cases without antibiotic cover). The results obtained were significantly better in the group receiving prophylaxis than in patients not under antimicrobial cover. This applied to septic wound healing disorders, general and specific postoperative complications, postoperative chest infections, re-operations and postoperative lethality. On the basis of these results, it is strongly recommended that, in the future, neither laparoscopic nor open conventional cholecystectomy should be carried out without proper perioperative antimicrobial prophylaxis-this all the more so since such measures also result in a shorter hospital stay and thus reduced costs.

Introduction: Leiomyosarcomas are malignant tumours showing smooth muscle differentiation. They represent approximately 25% of all uterine sarcomas and slightly over 1% of all uterine malignancies. The purpose of the present retrospective review is to relate clinical and pathological findings of leiomyosarcomas of the female genital tract to prognosis., Material and Methods: During 1972-1992 eleven patients had diagnosed uterine leiomyosarcomas treated at the Department of Gynecology of the University of Saarland. The hospital records of all patients were reviewed and complete primary treatment had been performed at this center., Results: The mean age was 46.92 years (SD: +/-13.85). Atypical uterine bleeding and pelvic discomfort were the most common presenting complaints (72.7%). The mean follow-up time was 59.60 months (20-96 months). Overall 2-year survival was 70% and overall 5-year survival 30%. The overall survival of patients in FIGO-stage I was 57.14%, in FIGO-stage II 100%, in FIGO-stage III 0% and in FIGO-stage IV 0%., Conclusion: The primary therapy should consist of an operation as radical as possible. Treatment with organ preserving seems to be reasonable if the patient desires children. Also, chemotherapy might provide a hopeful sign in the improvement of survival rates.

For several decades it has been proven that adequate antimicrobial prophylaxis is effective in reducing postoperative septic complications also in appendectomy. The rational for this approach--including laparoscopic procedures--is discussed and national and international recommendations for antibiotic prophylaxis in appendectomy are cited. Unfortunately, in spite of all the evidence, less than 50% of all appendectomies in 1996 in Germany had been carried out under antibiotic cover--with serious consequences for many patients and increased costs for prolonged hospital stay and treatment due to the higher incidence of infectious postoperative complications. Surgeons are encouraged not to neglect facts and benefits concerning the use of appropriate prophylactic antimicrobials in all appendectomies.