Deriving a dose and regimen for anti-glucosaminidase antibody passive-immunisation for patients with Staphylococcus aureus osteomyelitis.

Staphylococcus aureus (S. aureus) osteomyelitis remains a major clinical problem. Anti-glucosaminidase (Gmd) antibodies (1C11) are efficacious in prophylactic and therapeutic murine models. Feasibility, safety and pharmacokinetics of 1C11 passive immunisation in sheep and endogenous anti-Gmd levels were quantified in osteomyelitis patients. 3 sheep received a 500 mg intravenous (i.v.) bolus of 1C11 and its levels in sera were determined by enzyme-linked immunosorbent assay (ELISA) over 52 d. A humanised anti-Gmd monoclonal antibody, made by grafting the antigen-binding fragment (Fab) portion of 1C11 onto the fragment crystallisable region (Fc) of human IgG1, was used to make a standard curve of mean fluorescent intensity versus concentration of anti-Gmd. Anti-Gmd serum levels were determined in 297 patients with culture-confirmed S. aureus osteomyelitis and 40 healthy controls. No complications or adverse events were associated with the sheep 1C11 i.v. infusion and the estimated circulating half-life of 1C11 was 23.7 d. Endogenous anti-Gmd antibody levels in sera of osteomyelitis patients ranged from < 1 ng/mL to 300 µg/mL, with a mean concentration of 21.7 µg/mL. The estimated circulating half-life of endogenous anti-Gmd antibodies in sera of 12 patients with cured osteomyelitis was 120.4 d. A clinically relevant administration of anti-Gmd (500 mg i.v. = 7 mg/kg/70 kg human) was safe in sheep. This dose was 8 times more than the endogenous anti-Gmd levels observed in osteomyelitis patients and was predicted to have a half-life of > 3 weeks. Anti-Gmd passive immunisation has potential to prevent and treat S. aureus osteomyelitis. Further clinical development is warranted.

A systematic review of animal models for Staphylococcus aureus osteomyelitis.

Staphylococcus aureus (S. aureus) osteomyelitis is a significant complication for orthopaedic patients undergoing surgery, particularly with fracture fixation and arthroplasty. Given the difficulty in studying S. aureus infections in human subjects, animal models serve an integral role in exploring the pathogenesis of osteomyelitis, and aid in determining the efficacy of prophylactic and therapeutic treatments. Animal models should mimic the clinical scenarios seen in patients as closely as possible to permit the experimental results to be translated to the corresponding clinical care. To help understand existing animal models of S. aureus, we conducted a systematic search of PubMed and Ovid MEDLINE to identify in vivo animal experiments that have investigated the management of S. aureus osteomyelitis in the context of fractures and metallic implants. In this review, experimental studies are categorised by animal species and are further classified by the setting of the infection. Study methods are summarised and the relevant advantages and disadvantages of each species and model are discussed. While no ideal animal model exists, the understanding of a model's strengths and limitations should assist clinicians and researchers to appropriately select an animal model to translate the conclusions to the clinical setting.

Molecular analysis of the muscle protein projectin in Lepidoptera.

Striated muscles of both vertebrates and insects contain a third filament composed of the giant proteins, namely kettin and projectin (insects) and titin (vertebrates). All three proteins have been shown to contain several domains implicated in conferring elasticity, in particular a PEVK segment. In this study, the characterization of the projectin protein in the silkmoth, Bombyx mori L. (Lepidoptera: Bombycidae), and the monarch butterfly, Danaus plexippus L. (Lepidoptera: Nymphalidae), as well as a partial characterization in the Carolina sphinx, Manduca sexta L. (Lepidoptera: Sphingidae), are presented. This study showed that, similar to other insects, projectin's overall modular organization was conserved, but in contrast, the PEVK region had a highly divergent sequence. The analysis of alternative splicing in the PEVK region revealed a small number of possible isoforms and the lack of a flight-muscle specific variant, both characteristics being in sharp contrast with findings from other insects. The possible correlation with difference in flight muscle stiffness and physiology between Lepidoptera and other insect orders is discussed.

Projectin PEVK domain, splicing variants and domain structure in basal and derived insects.

The third elastic filament of striated muscles consists of giant proteins: titin (in vertebrates) and kettin/projectin (in insects). In all three proteins, elasticity is at least partly associated with the so-called PEVK domain. The projectin PEVK domains of diverse insects are highly divergent compared with an otherwise conserved protein organization. We present the characterization of the PEVK domain in two dragonflies and in human lice. A conserved segment at the end of the PEVK, the NH(2)-terminal conserved segment-1 (NTCS-1), may serve as an anchor point for projectin to either myosin or actin, providing a mechanical link. The analysis of alternative splicing variants identifies the shortest PEVK isoform as the predominant form in the flight muscles of several insects, possibly contributing to myofibrillar stiffness.

Examination of 'lipotoxicity' in skeletal muscle of high-fat fed and ob/ob mice.

Excess lipid accumulation resulting from an elevated supply of plasma fatty acids is linked to the pathogenesis of the metabolic syndrome and heart disease. The term 'lipotoxicity' was coined to describe how lipid accumulation leads to cellular dysfunction and death in non-adipose tissues including the heart, pancreas and liver. While lipotoxicity has been shown in cultured skeletal muscle cells, the degree of lipotoxicity in vivo and the functional consequences are unresolved. We studied three models of fatty acid overload in male mice: 5 h Intralipid((R)) and heparin infusion, prolonged high fat feeding (HFF) and genetic obesity induced by leptin deficiency (ob/ob mice). Markers of apoptosis, proteolysis and autophagy were assessed as readouts of lipotoxicity. The Intralipid((R)) infusion increased caspase 3 activity in skeletal muscle, demonstrating that enhancing fatty acid flux activates pro-apoptotic pathways. HFF and genetic obesity increased tissue lipid content but did not influence apoptosis. Gene array analysis revealed that HFF reduced the expression of 31 pro-apoptotic genes. Markers of autophagy (LC3beta and beclin-1 expression) were unaffected by HFF and were associated with enhanced Bcl(2) protein expression. Proteolytic activity was similarly unaffected by HFF or in ob/ob mice. Thus, contrary to our previous findings in muscle culture in vitro and in other non-adipose tissues in vivo, lipid overload did not induce apoptosis, autophagy or proteolysis in skeletal muscle. A broad transcriptional suppression of pro-apoptotic proteins may explain this resistance to lipid-induced cell death in skeletal muscle.

Both synchronous and asynchronous muscle isoforms of projectin (the Drosophila bent locus product) contain functional kinase domains.

In Drosophila, the large muscle protein, projectin, has very different localizations in synchronous and asynchronous muscles, suggesting that projectin has different functions in different muscle types. The multiple projectin isoforms are encoded by a single gene; however they differ significantly in size (as detected by gel mobility) and show differences in some peptide fragments, presumably indicating alternative splicing or termination. We now report additional sequence of the projectin gene, showing a kinase domain and flanking regions highly similar to equivalent regions of twitchin, including a possible autoinhibitory region. In spite of apparent differences in function, all isoforms of projectin have the kinase domain and all are capable of autophosphorylation in vitro. The projectin gene is in polytene region 102C/D where the bentD phenotype maps. The recessive lethality of bentD is associated with a breakpoint that removes sequence of the projectin kinase domain. We find that different alleles of the highly mutable recessive lethal complementation group, l(4)2, also have defects in different parts of the projectin sequence, both NH2-terminal and COOH-terminal to the bentD breakpoint. These alleles are therefore renamed as alleles of the bent locus. Adults heterozygous for projectin mutations show little, if any, effect of one defective gene copy, but homozygosity for any of the defects is lethal. The times of death can vary with allele. Some alleles kill the embryos, others are larval lethal. These molecular studies begin to explain why genetic studies suggested that l(4)2 was a complex (or pseudoallelic) locus.

Nucleotide sequences responsible for the thermal inducibility of the Drosophila small heat-shock protein genes in monkey COS cells.

The promoter regions of the Drosophila melanogaster small heat-shock protein genes have been analysed in order to localize those sequences responsible for their heat-shock transcriptional inducibility. Different lengths of the 5' DNA sequences of these four genes were each fused individually to the Herpes simplex virus thymidine kinase (HSV-tk) transcription unit. These hybrid genes were constructed in a simian virus 40 recombinant vector for transfection in permissive monkey COS cells and tested for their heat-shock inducibility. The hsp22/HSV-tk and hsp26/HSV-tk fusion genes were found to be heat-inducible at 43 degrees C, giving rise to correctly initiated transcripts, but transcriptionally quiescent at 37 degrees C (control temperature). The hsp23 and hsp27 fusion gene constructs are, however, not heat-shock-inducible; no transcripts being detectable from hsp27/HSV-tk constructs at either temperature and all hsp23/HSV-tk clones being faithfully but constitutively expressed at low levels at both temperatures. By testing a series of 5' deletion mutants in hsp22/HSV-tk, a homologous sequence located adjacent to the TATA box in both the hsp22 and hsp26 genes was identified as being responsible for their heat-shock activation. This control element corresponds to the Pelham "consensus sequence", previously described for the Drosophila hsp70 genes. The possible modes of transcriptional induction of all four genes are discussed.

Nucleotide sequence analysis of the Drosophila small heat shock gene cluster at locus 67B.

The four small heat shock protein genes of Drosophila melanogaster clustered at cytological locus 67B have been characterized by DNA sequencing. Over 6250 nucleotides, covering the 5', protein-coding and 3' regions of these genes have been determined together with their predicted amino acid sequences. Each gene possesses characteristic eukaryotic 5' and 3' sequence elements and a single uninterrupted protein-coding region. The four encoded polypeptides of 19,700, 20,600, 23,000 and 23,600 Mr share a homologous stretch of 108 amino acid residues, representing 51 to 62% of their lengths. This region is flanked by sequences of dissimilar length and amino acid composition, located mainly at the amino-terminal end, but also at the extreme carboxyl termini of these proteins. The first 14 amino acids exhibit a small degree of homology, both amongst themselves and with some signal peptides and a transmembrane protein. Investigation of the hydrophilic/hydrophobic characteristics of the four polypeptides revealed, within the conserved 108 amino acid stretch, the presence of an alpha-helical region of very prominent local hydrophilicity, which probably represents a surface structural domain common to each protein. Sequence analysis with respect to transcription initiation and termination and possible regulatory signals is discussed together with some structural predictions for the four proteins.

Alternative splicing of an amino-terminal PEVK-like region generates multiple isoforms of Drosophila projectin.

Drosophila projectin is an extremely large protein found within the muscle sarcomeric unit, parallel with the actin and myosin filaments. Projectin has been suggested as the elastic component of C-filaments in insect indirect flight muscles, which is consistent with its localization from the Z band to the tip of the A band in these muscles. Here, we describe the completion of the projectin sequence analysis, which defines projectin as a 1 MDa protein, composed of 39 immunoglobulin and 39 fibronectin III domains. This analysis led also to the identification of a domain rich in the amino acids P, E, V and K within the NH(2) terminus of projectin. The length of the projectin PEVK-like region varies from 100 to 624 amino acid residues, following a complex pattern of alternative splicing events. PEVK domains were first identified in vertebrate titin and they have been associated with the elasticity of the protein. The PEVK-like domain of the projectin isoforms in indirect flight muscles may contribute to the elastic function of the C-filaments. The synchronous projectin isoforms contain a PEVK-like region, and the possible non-elastic function(s) of this domain in synchronous muscles are discussed.

Structure of the Drosophila projectin protein: isoforms and implication for projectin filament assembly.

The protein composition of the various muscle types in Drosophila melanogaster has been studied quite thoroughly and the analysis has revealed many differences involving the usage of muscle specific isoforms of a given protein, as well as the presence of proteins restricted to one muscle type. Drosophila projectin, the giant protein component of the third filament is quite unusual as it not only shows specific isoforms in various muscle types, but these isoforms are located at different sarcomeric locations, I band in the IFM and A band in synchronous muscles. This may suggest distinct functions for the projectin protein in various muscles, as well as a different set of protein interactions for each projectin isoform. Projectin is encoded by a single gene and the isoforms were proposed to be the result of alternative splicing of a primary transcript. Here, we report the nearly complete sequence of Drosophila projectin, as well as the possible splicing patterns used to generate different isoforms. The overall domain organization in projectin is composed of repeated motifs I and II in a few specific patterns, similar to its Caenorhabditis homolog, twitchin. Sequence similarity between twitchin and projectin further suggests how some domains may possibly be important for protein interactions and/or functions. Alternative splicing operates at the COOH terminus, leading to a shorter projectin protein lacking some of the terminal motifs II and unique sequence. These isoforms are discussed in view of projectin differential size and localization.

Suppression of plasma free fatty acids upregulates peroxisome proliferator-activated receptor (PPAR) alpha and delta and PPAR coactivator 1alpha in human skeletal muscle, but not lipid regulatory genes.

Fatty acids are an important ligand for peroxisome proliferator-activated receptor (PPAR) activation and transcriptional regulation of metabolic genes. To examine whether reduced plasma free fatty acid (FFA) availability affects the mRNA content of proteins involved in fuel metabolism in vivo, the skeletal muscle mRNA content of various transcription factors, transcriptional coactivators and genes encoding for lipid regulatory proteins were examined before and after 3 h of cycle exercise with (NA) and without (CON) pre-exercise ingestion of nicotinic acid (NA). NA resulted in a marked (3- to 6-fold) increase (P<0.05) in PPARalpha, PPARdelta and PPAR coactivator 1alpha (PGC1alpha) mRNA, but was without effect on nuclear respiratory factor-1 and Forkhead transcription factor, fatty acid transcolase/CD36, carnitine palmitoyl transferase 1, hormone sensitive lipase (HSL) and pyruvate dehydrogenase kinase 4. Exercise in CON was associated with increased (P<0.05) PPARalpha, PPARdelta and PGC1alpha mRNA, which was similar in magnitude to levels observed with NA at rest. Exercise was generally without effect on the mRNA content of lipid regulatory proteins in CON and did not affect the mRNA content of the measured subset of transcription factors, transcriptional co-activators and lipid regulatory proteins during NA. To determine the possible mechanisms by which NA might affect PGC1alpha expression, we measured p38 MAP kinase (MAPK) and plasma epinephrine. Phosphorylation of p38 MAPK was increased (P<0.05) by NA treatment at rest, and this correlated (r2=0.84, P<0.01) with increased PGC1alpha. Despite this close relationship, increasing p38 MAPK in human primary myotubes was without effect on PGC1alpha mRNA content. Plasma epinephrine was elevated (P<0.05) by NA at rest (CON: 0.27+/-0.06, NA: 0.72+/-0.11 nM) and throughout exercise. Incubating human primary myotubes with epinephrine increased PGC1alpha independently of changes in p38 MAPK phosphorylation. Hence, despite the fact that NA ingestion decreased FFA availability, it promoted the induction of PPARalpha/delta and PGC1alpha gene expression to a similar degree as prolonged exercise. We suggest that the increase in PGC1alpha may be due to the elevated plasma epinephrine levels. Despite these changes in transcription factors/coactivators, the mRNA content of lipid regulatory proteins was generally unaffected by plasma FFA availability.

Structure--activity relationships in cephalosporins prepared from penicillins. 1. 7beta-Acylamino derivatives of 3-benzyl- and 3-(3-pyridylmethyl)ceph-3-em-4-carboxylic acids.

tert-Butyl 7beta-aminoceph-3-em-4-carboxylates carrying either benzyl or 3-pyridylmethyl substituents at position 3 have been prepared by a multistep modification of the penicillin nucleus. Acylation of either amine, followed by deprotection, gave a range of new cephalosporins. The relationship between structure and antibacterial activity is discussed. D-Phenylglycine proved to be a preferred side chain in both series.

Structure--activity relationships in cephalosporins prepared from penicillins. 2. Analogues of cephalexin substituted in the 3-methyl group.

A previously outlined general procedure for preparing various 3-substituted cephalosporins from the penicillin nucleus has been used, with modifications where required, to prepare a series of analogues of cephalexin with various substituents in the 3-methyl group. The 3-substituents most conducive to broad-spectrum antibacterial activity were 3-pyridylmethyl and m- or p-carboxybenzyl. The compounds were only poorly absorbed by the oral route in mice, but the 3-(carboxybenzyl) compounds gave more prolonged useful serum levels than the usual cephalosporins.

Mutual pro-drugs of the olivanic acids and renal dipeptidase inhibitors.

The carbapenem antibiotics, which include the olivanic acids and thienamycin, possess potent broad spectrum antibacterial activity. They are however extensively metabolized by the renal dipeptidase enzyme, dehydropeptidase I. As a result of this degradation, only low urinary recoveries of antibiotic are obtained in vivo. The preparation of mutual pro-drugs of the olivanic acids and an inhibitor of the renal dipeptidase enzyme is described. MM 22382 and MM 13902 have been combined with Z-2-isovaleramidobut-2-enoic acid as double esters of formaldehyde hydrate. Administration of these linked esters to mice results in improved urinary recoveries of antibiotic.

Synthesis and biological activity of a series of piperazine-2,3-dione containing penicillins and 6 alpha-formamidopenicillins. I. Derivatives substituted at C(5) or C(6) of the piperazine ring.

The synthesis and antibacterial activity of a series of penicillins and 6 alpha-formamidopenicillins containing a C(5) or C(6)-substituted piperazine-2,3-dione moiety in the C(6)-beta-sidechain is described.

Synthesis and antibacterial activity of C-2 alkenylthio-carbapenem derivatives.

A number of C-2 aminoalkenylthio-carbapenem derivatives possessing both the (5R,6R, 8S)- and the (5R,6S,8R)-stereochemistries have been prepared from the olivanic acids MM 22382 and MM 22383, respectively. Certain members of this new class of compounds displayed potent, broad spectrum antibacterial activity as well as improved stability to human kidney homogenate.

Synthesis and antibacterial activity of C-2 carboxyethenylthio-carbapenem derivatives.

A number of C-2 carboxyethenylthio-carbapenem derivatives possessing either the (5R,6R,8S)- or the (5R,6S,8R)-stereochemistries have been prepared from the olivanic acids MM 22382 (1) and MM 22383 (4), respectively. Their in vitro antibacterial activities and stabilities to human kidney homogenate are superior to those of the parent compounds, particularly in the latter series.

Inhibitors of bacterial signal peptidase: a series of 6-(substituted oxyethyl)penems.

A series of 6-(substituted oxyethyl)penem esters having the (5S) stereochemistry which are potent inhibitors of Escherichia coli leader peptidase is described. Structure-activity relationships are discussed.

Structure-activity relationships in a series of piperazine-2,3-dione containing penicillins and cephalosporins. II. Derivatives substituted at N(4) of the piperazine ring.

The structure-activity relationships within a series of penicillins and cephalosporins containing an N(4)-substituted piperazine-2,3-dione moiety in the C(6)/C(7)-beta-side chain are discussed.