Systemic bacterial invasion induced by sleep deprivation

Profound sleep disruption in humans is generally believed to cause health impairments. Through comparative research, specific physical effects and underlying mechanisms altered by sleep deprivation are being elucidated. Studies of sleep-deprived animals previously have shown a progressive, chronic negative energy balance and gradual deterioration of health, which culminate in fatal bloodstream infection without an infectious focus. The present study investigated the conditions antecedent to advanced morbidity in sleep-deprived rats by determining the time course and distribution of live microorganisms in body tissues that are normally sterile. The tissues cultured for microbial growth included the blood, four major organs, six regional lymph nodes, the intestine, and the skin. The principal finding was early infection of the mesenteric lymph nodes by bacteria presumably translocated from the intestine and bacterial migration to and transient infection of extraintestinal sites. Presence of pathogenic microorganisms and their toxins in tissues constitutes a septic burden and chronic antigenic challenge for the host. Bacterial translocation and pathogenic sequelae provide mechanisms by which sleep deprivation appears to adversely affect health.     

Brief communication: cutmarks on a plio-pleistocene hominid from Sterkfontein, South Africa

Cutmarks inflicted by a stone tool were observed on the right maxilla of Stw 53, an early hominid partial skull from Sterkfontein "Member 5" (South Africa). The morphology of the marks, their anatomical placement, and the lack of random striae on the specimen all support an interpretation of this linear damage as cutmarks. The location of the marks on the lateral aspect of the zygomatic process of the maxilla is consistent with that expected from slicing through the masseter muscle, presumably to remove the mandible from the cranium. Although radioisotopic dates are not available and relative faunal dating of the deposit from which Stw 53 derives is problematic, the morphology of the hominid skull suggests a Plio-Pleistocene age for the specimen. This therefore constitutes the earliest unambiguous evidence that hominids disarticulated the remains of one another.     

Insulin lispro (Humalog), the first marketed insulin analogue: indications,contraindications and need for further study.

OBJECTIVE: To review the available literature on the new insulin analogue insulin lispro and provide information on its efficacy, indications for use and contraindications. DATA SOURCES: MEDLINE searches were made for articles published from 1966 to 1996 using the indexing term "lispro", "Humalog" and "insulin analogs". STUDY SELECTION: About 30 studies and review articles were selected based on their relevance to the stated objective. These were critically appraised for the purpose of writing the review article so that it would be relevant to general practitioners, internists and nurse educators. DATA SYNTHESIS: The therapeutic challenge when treating diabetic patients is to bring the blood glucose level into as normal a range as possible, with minimal hypoglycemia and hyperinsulinemia. Insulin lispro has a much faster, higher and shorter-lasting peak serum insulin level than regular human insulin, thus mimicking physiologic secretion of insulin more closely. As a result, there is improvement in postprandial blood glucose levels and decreased episodes of hypoglycemia, with no change in the hemoglobin A1c (HgbA1c) level. The ability to inject insulin lispro immediately before the meal allows greater flexibility of lifestyle. Compared with regular insulin, insulin lispro is associated with a lower risk of hypoglycemia with exercise several hours after a meal. It is therefore most useful for the motivated, compliant diabetic patient who would like to achieve a better hypoglycemia-HgbA1c ratio as well as for patients desiring further flexibility with premeal insulin injections. Use of insulin lispro has been shown to improve HgbA1c levels in patients using insulin pumps. It is well tolerated, and therapy is often continued after studies are completed. Further study is needed to establish optimal basal regimens.     

Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

Background

Despite the frequency of diabetes mellitus and its relationship to diabetic peripheral neuropathy (DPN) and neuropathic pain (NeP), our understanding of underlying mechanisms leading to chronic pain in diabetes remains poor. Recent evidence has demonstated a prominent role of microglial cells in neuropathic pain states. One potential therapeutic option gaining clinical acceptance is the cannabinoids, for which cannabinoid receptors (CB) are expressed on neurons and microglia. We studied the accumulation and activation of spinal and thalamic microglia in streptozotocin (STZ)-diabetic CD1 mice and the impact of cannabinoid receptor agonism/antagonism during the development of a chronic NeP state. We provided either intranasal or intraperitoneal cannabinoid agonists/antagonists at multiple doses both at the initiation of diabetes as well as after establishment of diabetes and its related NeP state.

Results

Tactile allodynia and thermal hypersensitivity were observed over 8 months in diabetic mice without intervention. Microglial density increases were seen in the dorsal spinal cord and in thalamic nuclei and were accompanied by elevation of phosphorylated p38 MAPK, a marker of microglial activation. When initiated coincidentally with diabetes, moderate-high doses of intranasal cannabidiol (cannaboid receptor 2 agonist) and intraperitoneal cannabidiol attenuated the development of an NeP state, even after their discontinuation and without modification of the diabetic state. Cannabidiol was also associated with restriction in elevation of microglial density in the dorsal spinal cord and elevation in phosphorylated p38 MAPK. When initiated in an established DPN NeP state, both CB1 and CB2 agonists demonstrated an antinociceptive effect until their discontinuation. There were no pronociceptive effects demonstated for either CB1 or CB2 antagonists.

Conclusions

The prevention of microglial accumulation and activation in the dorsal spinal cord was associated with limited development of a neuropathic pain state. Cannabinoids demonstrated antinociceptive effects in this mouse model of DPN. These results suggest that such interventions may also benefit humans with DPN, and their early introduction may also modify the development of the NeP state.     

Mutation in a gene required for lipopolysaccharide and enterobacterial common antigen biosynthesis affects virulence in the plant pathogen Erwinia carotovora subsp. atroseptica.

Spontaneous bacteriophage-resistant mutants of the phytopathogen Erwinia carotovora subsp. atroseptica (Eca) SCRI1043 were isolated and, out of 40, two were found to exhibit reduced virulence in planta. One of these mutants, A5/22, showed multiple cell surface defects including alterations in synthesis of outer membrane proteins, lipopolysaccharide (LPS), enterobacterial common antigen (ECA), and flagella. Mutant A5/22 also showed reduced synthesis of the exoenzymes pectate lyase (Pel) and cellulase (Cel), major virulence factors for this pathogen. Genetic analysis revealed the pronounced pleiotropic mutant phenotype to be due to a defect in a single gene (rffG) that, in Escherichia coli, is involved in the production of ECA. We also show that while other enteric bacteria possess duplicate homologues of this gene dedicated separately to synthesis of LPS and ECA, Eca has a single gene.     

A differential proteomics study of cerebrospinal fluid from individuals with Niemann-Pick disease,Type C1

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.     

A multi-repeat adhesin of the phytopathogen, Pectobacterium atrosepticum, is secreted by a Type I pathway and is subject to complex regulation involving a non-canonical diguanylate cyclase

Cyclic diguanylate (c-di-GMP) is a second messenger controlling many important bacterial processes. The phytopathogen Pectobacterium atrosepticum SCRI1043 (Pba1043) possesses a Type I secretion system (T1SS) essential for the secretion of a proteinaceous multi-repeat adhesin (MRP) required for binding to the host plant. The genes encoding the MRP and the T1SS are tightly linked to genes encoding several putative c-di-GMP regulatory components. We show that c-di-GMP regulates secreted MRP levels in Pba1043 through the action of two genes encoding predicted diguanylate cyclase (DGC) and phosphodiesterase proteins (ECA3270 and ECA3271). Phenotypic analyses and quantification of c-di-GMP levels demonstrated that ECA3270 and ECA3271 regulate secreted MRP levels by increasing and decreasing, respectively, the intracellular levels of c-di-GMP. Moreover, ECA3270 represents the first active DGC reported to have an alternative active-site motif from the 'canonical' GG[D/E]EF. ECA3270 has an A-site motif of SGDEF and analysis of single amino acid replacements demonstrated that the first position of this motif can tolerate functional substitution. Serine in position one of the A-site is also observed in many other DGCs. Finally, another T1SS-linked regulator (ECA3265) also plays an important role in regulating secreted MRP, with an altered localization of MRP observed in an ECA3265 mutant background. Mutants defective in these three T1SS-linked regulators exhibit a reduction in root binding and virulence, confirming that this complex, finely tuned regulation system is crucial in the interaction with host plants.     

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.     

Specific protein domains mediate cooperative assembly of HuR oligomers on AU-rich mRNA-destabilizing sequences

The RNA-binding factor HuR is a ubiquitously expressed member of the Hu protein family that binds and stabilizes mRNAs containing AU-rich elements (AREs). Hu proteins share a common domain organization of two tandemly arrayed RNA recognition motifs (RRMs) near the N terminus, followed by a basic hinge domain and a third RRM near the C terminus. In this study, we engineered recombinant wild-type and mutant HuR proteins lacking affinity tags to characterize their ARE-binding properties. Using combinations of electrophoretic mobility shift and fluorescence anisotropy-based binding assays, we show that HuR can bind ARE substrates as small as 13 nucleotides with low nanomolar affinity, but forms cooperative oligomeric protein complexes on ARE substrates of at least 18 nucleotides in length. Analyses of deletion mutant proteins indicated that RRM3 does not contribute to high affinity recognition of ARE substrates, but is required for cooperative assembly of HuR oligomers on RNA. Finally, the hinge domain between RRM2 and RRM3 contributes significant binding energy to HuR.ARE complex formation in an ARE length-dependent manner. The hinge does not enhance RNA-binding activity by increased ion pair formation despite extensive positive charge within this region, and it does not thermodynamically stabilize protein folding. Together, the results define distinct roles for the HuR hinge and RRM3 domains in formation of cooperative HuR.ARE complexes in solution.