A male gametocyte osmiophilic body and microgamete surface protein of the rodent malaria parasite Plasmodium yoelii (PyMiGS) plays a critical role in male osmiophilic body formation and exflagellation

Anopheles mosquitoes transmit Plasmodium parasites of mammals, including the species that cause malaria in humans. Malaria pathology is caused by rapid multiplication of parasites in asexual intraerythrocytic cycles. Sexual stage parasites are also produced during the intraerythrocytic cycle and are ingested by the mosquito, initiating gametogenesis and subsequent sporogonic stage development. Here, we present a Plasmodium protein, termed microgamete surface protein (MiGS), which has an important role in male gametocyte osmiophilic body (MOB) formation and microgamete function. MiGS is expressed exclusively in male gametocytes and microgametes, in which MiGS localises to the MOB and microgamete surface. Targeted gene disruption of MiGS in a rodent malaria parasite Plasmodium yoelii 17XNL generated knockout parasites (ΔPyMiGS) that proliferate normally in erythrocytes and form male and female gametocytes. The number of MOB in male gametocyte cytoplasm is markedly reduced and the exflagellation of microgametes is impaired in ΔPyMiGS. In addition, anti‐PyMiGS antibody severely blocked the parasite development in the Anopheles stephensi mosquito. MiGS might thus be a potential novel transmission‐blocking vaccine target candidate.     

Atomic structure of the clamp loader small subunit from Pyrococcus furiosus.

In eukaryotic DNA replication, replication factor-C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands at replication forks. The eukaryotic RFC is a complex consisting of one large and four small subunits. We have determined the crystal structure of the clamp loader small subunit (RFCS) from Pyrococcus furiosus. The six subunits, of which four bind ADP in their canonical nucleotide binding clefts, assemble into a dimer of semicircular trimers. The crescent-like architecture of each subunit formed by the three domains resembles that of the delta' subunit of the E. coli clamp loader. The trimeric architecture of archaeal RFCS, with its mobile N-terminal domains, involves intersubunit interactions that may be conserved in eukaryotic functional complexes.     

Diversity of staphylocoagulase and identification of novel variants of staphylocoagulase gene in Staphylococcus aureus

Staphylocoagulase (SC) is a major phenotypic determinant of Staphylococcus aureus. Serotype of SC (coagulase type) is used as an epidemiological marker and 10 types (I-X) have been discriminated so far. To clarify genetic diversity of SC within a single and among different serotype(s), we determined approximately 1500 bp-nucleotide sequences of SC gene encoding D1, D2, and central regions (N-terminal half and central regions of SC; SC(NC)) for a total of 33 S. aureus strains comprising two to three strains from individual coagulase types (I-VIII, X) and 10 strains which were not determined as previously known SC serotypes (ND-strains). Amino acid sequence identities of SC(NC) among strains with a single coagulase type of II, III, IV, V, VI and X were extremely high (more than 99%), whereas lower identity (56-87%) was observed among different types. In contrast, within a single coagulase type of I, VII, or VIII, sequence divergence was found (lowest identity; 82%). SC(NC) sequences from the ND-strains were discriminated into two genetic groups with an identity of 71% to each other (tentatively assigned to genotypes [XI] and [XII]), and exhibited less than 86% sequence identities to those of most known coagulase types. All the types [XI] and [XII] strains were methicillin susceptible and belonged to different sequence types from those of coagulase types I-X strains reported so far by multilocus sequence typing. These findings indicated genetic heterogeneity of SC in coagulase types I, VII, and VIII strains, and the presence of two novel SC genotypes related to antigenicity of SC serotypes.     

N-acetyl-l-methionine is a superior protectant of human serum albumin against photo-oxidation and reactive oxygen species compared to N-acetyl-l-tryptophan

Background

Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products.

Methods

Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4 weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration.

Results

N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present.

Conclusion

The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations.

General significance

N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin.     

Prophylactic Effects of the Glucagon-Like Peptide-1 Analog Liraglutide on Hyperglycemia in a Rat Model of Type 2 Diabetes Mellitus Associated with Chronic Pancreatitis and Obesity

The objective of this study was to investigate the effects of liraglutide, an analog of human glucagon-like peptide 1 (GLP1), on WBN/Kob-Lepr^fa (fa/fa) rats, which spontaneously develop type 2 diabetes mellitus with pancreatic disorder and obesity. Male fa/fa rats (age, 7 wk) were allocated into 4 groups and received liraglutide (37.5, 75, 150 μg/kg SC) or saline (control group) once daily for 4 wk. All rats in the control group became overweight and developed hyperglycemia as they aged. Although the rats given liraglutide showed a dose-dependent reduction in food intake, no significant effects on body weight or fat content occurred. In the liraglutide groups, the development of hyperglycemia was suppressed, even as plasma insulin concentrations increased in a dose-dependent manner. Intravenous glucose tolerance testing of the liraglutide-treated rats confirmed improvement of glucose tolerance and enhanced insulin secretion. Histologic examination revealed increased numbers of pancreatic β-cell type islet cells and increased proliferation of epithelial cells of the small ducts in the liraglutide-treated groups. Although our study did not reveal a significant decrease in obesity after liraglutide administration, the results suggest a marked antidiabetic effect characterized by increased insulin secretion in fa/fa rats with pancreatic disorders.Abbreviations: GLP1, glucagon-like peptide-1; IVGTT, intravenous glucose tolerance testing; T2DM, type 2 diabetes mellitusThe number of patients diagnosed with diabetes has more than doubled over the last 30 y, and diabetes has become an important public health concern worldwide.⁶ Approximately 90% of patients with diabetes are diagnosed with type 2 diabetes mellitus (T2DM).³¹ The onset of T2DM is determined by multiple factors that lead to reduced insulin secretion or insulin resistance, including genetic predisposition and lifestyle-associated habits such as lack of exercise, overeating, and obesity. Many drugs are already used clinically to treat T2DM;^9,11 nevertheless, the search and development of more efficient and safe drugs is currently underway. In this regard, incretin has recently gained attention as a member of a class of drugs used to treat T2DM.^9,11Enteroendocrine cells secret incretin hormones, which augment glucose-induced insulin secretion in response to food ingestion, in a glucose-dependent manner. Currently, the 2 confirmed incretins are glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 (GLP1). Research has shown that GLP1 derivatives have functions in addition to the promotion of insulin secretion, including facilitation of β-cell proliferation,²⁸ suppression of β-cell apoptosis,¹² and promotion of β-cell differentiation or de novo formation of β cells.^29,30 GLP1 derivatives have been reported to have multiple nonpancreatic functions, including suppression of appetite and body weight,^7,13 suppression of gastric secretions,¹⁹ reduction of lipid accumulation in the liver,¹⁷ and promotion of sensitivity to insulin in adipose cells and skeletal muscle.^8,22WBN/Kob-type male rats are a relevant animal model for diabetes without obesity. These rats typically show disease conditions including chronic pancreatitis and pancreatic endocrine disorders.^18,26 A new model rat for diabetes was established recently by crossing rats carrying the Lepr^fa obesity gene with wild-type WBN/Kob rats, yielding a fa/fa congenic strain.¹ The obesity gene (Lepr^fa) is a spontaneous mutation derived from Zucker-fatty rats that leads to dysfunction of the leptin receptor. Rats homozygous for this gene are obese, resistant to insulin, and hyperinsulinemic.^4,16,32 Male WBN/Kob-Lepr^fa rats (hereafter referred to as fa/fa rats) represent a new animal model in which the animals spontaneously develop diabetes in addition to endogenous insulin resistance. Compared with the parental strains, fa/fa rats are characterized by an earlier onset of diabetes and more severe pancreatic complications.^1,2 Our previous investigations have revealed that fa/fa rats present with hyperinsulinemia at a prediabetic stage as a compensatory response to insulin resistance, but these rats show high blood glucose levels because of a difficulty in maintaining blood insulin concentrations as a consequence of declining pancreatic β-cell function associated with advancing age.¹⁴In the current study, to further validate fa/fa rats as a T2DM model, we investigated the effects of a GLP1 analog in fa/fa rats with hyperglycemia (age, 7 to 11 wk). We used liraglutide, a human GLP1 analog, which has been shown to be clinically effective in T2DM patients.^9,11     

Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.     

15-Deoxy-Δ¹²,¹⁴ prostaglandin J₂ reduces the formation of atherosclerotic lesions in apolipoprotein E knockout mice

Aim

15-Deoxy-Δ^12,14 Prostaglandin J₂ (15d-PGJ₂) is a ligand of peroxisome proliferator-activated receptor γ (PPARγ) having diverse effects such as the differentiation of adipocytes and atherosclerotic lesion formation. 15d-PGJ₂ can also regulate the expression of inflammatory mediators on immune cells independent of PPARγ. We investigated the antiatherogenic effect of 15d-PGJ₂.

Methods

We fed apolipoprotein (apo) E-deficient female mice a Western-type diet from 8 to 16 wk of age and administered 1 mg/kg/day 15d-PGJ₂ intraperitoneally. We measured atherosclerotic lesions at the aortic root, and examined the expression of macrophage and inflammatory atherosclerotic molecules by immunohistochemical and real-time PCR in the lesion.

Results

Atherosclerotic lesion formation was reduced in apo E-null mice treated with 15d-PGJ₂, as compared to in the controls. Immunohistochemical and real-time PCR analyses showed that the expression of MCP-1, TNF-α, and MMP-9 in atherosclerotic lesions was significantly decreased in 15d-PGJ₂ treated mice. The 15d-PGJ₂ also reduced the expression of macrophages and RelA mRNA in atherosclerotic lesions.

Conclusion

This is the first report 15d-PGJ₂, a natural PPARγ agonist, can improve atherosclerotic lesions in vivo. 15d-PGJ₂ may be a beneficial therapeutic agent for atherosclerosis.