Elemental characterization of Daphnia resting eggs by X-ray analytical microscopy

Resting eggs of Daphnia, a key crustacean zooplankton of freshwater food chains, can remain viable for more than a century. These eggs are able to withstand freezing and drying, and can survive the harsh environment of a predator's digestive system. Until recently little was known about the chemical composition, microanatomy, and physical properties of the resting eggs. The current study utilized a physical technique, the X-ray analytical microscope, to identify and localize component elements of the Daphnia resting egg. The analysis demonstrated that phosphorus, sulfur, potassium, and calcium were detected as elemental components of the resting egg, and detection intensities of the four elements differed according to the position of the eggs. Phosphorus and calcium were mostly detected in regions of the eggshell that surrounded the two embryos. In addition, sulfur was distributed throughout the eggshell whereas potassium was localized to the areas that corresponded to where the embryos were encased. Through the use of X-ray analytical microscopy, the current study identifies elemental characteristics in relationship to the structure of the Daphnia resting eggs.     

A common structure of substrate shared by lignostilbenedioxygenase isozymes from Sphingomonas paucimobilis TMY1009

A common structure of substrates of lignostilbenedioxygenases was investigated using synthesized stilbenes. Cell-free extracts of Sphingomonas paucimobilis TMY1009 degraded only trans-4-hydroxystilbene and trans-4-hydroxy-3-methoxystilbene. Other stilbenes that had no 4-hydroxyl group and had a cis structure were not substrates for lignostilbenedioxygenases. These results indicate that a 4-hydroxyl group and trans-structure is necessary for the common structure for substrates of lignostilbenedioxygenases.     

Generation of high-affinity antibody against T cell-dependent antigen in the Ganp gene-transgenic mouse

Generation of high-affinity Ab is impaired in mice lacking germinal center-associated DNA primase (GANP) in B cells. In this study, we examined the effect of its overexpression in ganp transgenic C57BL/6 mice (Ganp(Tg)). Ganp(Tg) displayed normal phenotype in B cell development, serum Ig levels, and responses against T cell-independent Ag; however, it generated the Ab with much higher affinity against nitrophenyl-chicken gammaglobulin in comparison with C57BL/6. To further examine the affinity increase, we established hybridomas producing high-affinity mAbs and compared their affinities using BIAcore. C57BL/6 generated high-affinity anti-nitrophenyl mAbs (K(D) approximately 2.50 x 10(-7) M) of IgG1/lambda1 and contained the V(H)186.2 region with W33L mutation. Ganp(Tg) generated much higher affinity (K(D) > 1.57 x 10(-9) M) by usage of V(H)186.2 as well as noncanonical V(H)7183 regions. Ganp(Tg) also generated exceptionally high-affinity anti-HIV-1 (V3 peptide) mAbs (K(D) > 9.90 x 10(-11) M) with neutralizing activity. These results demonstrated that GANP is involved in V region alteration generating high-affinity Ab.     

S19 ribosomal protein dimer augments metal-induced apoptosis in a mouse fibroblastic cell line by ligation of the C5a receptor

To analyze the role of S19 ribosomal protein (RP S19) in apoptosis, murine NIH3T3 were transfected with either hemagglutinin peptide-tagged (HA) wild-type human RP S19 or a mutant (Gln137Asn) that is resistant to transglutaminase-catalyzed cross-linked-dimerization. Transfection with the mutant HA-RP S19 inhibited manganese (II) (Mn II)-induced apoptosis whereas the wild-type HA-RP S19 augmented apoptosis and a mock transfection had no effect. Release of the wild-type HA-RP S19 dimer but not the mutant HA-RP S19 was observed during the apoptosis. The reduced rate of apoptosis of the cells transfected with the mutant HA-RP S19 was overcome by addition of extracellular wild-type RP S19 dimer. The apoptosis rates in cells transfected with either form of human HA-RP S19 and in mock transfectants were reduced to about 40% by the presence of anti-RP S19 antibody in the culture medium. Immunofluorescence staining and fluorescence-activated cell sorting (FACS) analysis showed that the cell surface expression of the receptor for cross-linked RP S19 dimer, C5a receptor, increased during apoptosis, concomitant with phosphatidylserine exposure. The expression of the C5a receptor gene also increased twofold. Apoptosis rates in the transfected and control cell lines were also reduced by the presence of an anti-mouse C5a receptor monoclonal antibody or of a peptide C5a receptor antagonist. These results indicated the presence of an RP S19 dimer- and C5a receptor-mediated autocrine-type augmentation mechanism during Mn II-induced apoptosis in the mouse fibroblastic cell line. In contrast to the RP S19 dimer, C5a actually inhibited apoptosis, suggesting that signaling through the C5a receptor varies depending on the ligand bound.     

CS-886, a new angiotensin II type 1 receptor antagonist,ameliorates glomerular anionic site loss and prevents progression of diabetic nephropathy in Otsuka Long-Evans Tokushima fatty rats

BACKGROUND: Diabetic nephropathy is a leading cause of end-stage renal disease in industrialized countries. Previous studies have documented that angiotensin converting enzyme (ACE) inhibitors consistently reduce albuminuria and retard the progression of diabetic nephropathy. However, the involvement of angiotensin II in diabetic nephropathy is not fully understood. MATERIALS AND METHODS: In this study we compared the effects of CS-866, a new angiotensin II type 1 receptor antagonist, to that of an ACE inhibitor, temocapril hydrochloride, on the development and progression of diabetic nephropathy using Otsuka Long-Evans Tokushima fatty rats, a type II diabetes mellitus model animal. RESULTS: High doses of CS-866 or temocapril treatment were found to significantly improve urinary protein and beta(2)-microglobulin excretions in diabetic rats. In electron microscopic analysis, loss of glomerular anionic sites, one of the causes of glomerular hyperpermeability in diabetic nephropathy, was found to be significantly prevented by CS-866 treatment. Light microscopic examinations revealed that both treatments ameliorated glomerular sclerosis and tubulointerstitial injury in diabetic rats. Furthermore, high doses of CS-866 or temocapril treatment significantly reduced the positive stainings for transforming growth factor-beta (TGF-beta), vascular endothelial growth factor, and type IV collagen in glomeruli of diabetic rats. CONCLUSIONS: These results indicate that intrarenal angiotensin II type 1 receptor activation plays a dominant role in the development and progression of diabetic nephropathy. Our study suggests that CS-866 represents a valuable new drug for the treatment of diabetic patients with nephropathy.     

Osmotic behavior of red blood cell as seen with an ultrasonic method

We have measured the density and ultrasonic velocity (usv) of swine red blood cell (RBC) suspensions in the wide osmolarity range from 300 mOsm to 1400 mOsm in saline solution. The cellular density and compressibility of RBC at each osmolarity were obtained using the fact that the density and the compressibility are additive by volume. The osmolarity dependence of hematocrit was also measured at a constant number concentration of RBC in the range of 300 mOsm to 1700 mOsm. The cellular density and the cellular compressibility of RBC as well as the inverse of hematocrit were expressed well into one unique exponential type equation f (pi) = a [1 - b exp (-c pi)] with a common value for the coefficient c = 0.0025 against the osmolarity pi. The results were analyzed with a simple consideration based only upon the contribution of free water inside the erythrocyte through the volume concentration phi of the free water in it. According to this theoretical analysis, the density and the compressibility of the free water were found to be 0.990 g/cm3 and 4.59 x 10(-11) cm2/dyne which agree closely with 0.998 g/cm3 and 4.59 x 10(-11) cm2/dyn of pure water at 20 degrees C within the experimental error.     

Factors Associated with Myelosuppression Related to Low-Dose Methotrexate Therapy for Inflammatory Rheumatic Diseases

ObjectiveSevere myelosuppression is a serious concern in the management of rheumatic disease patients receiving methotrexate (MTX) therapy. This study was intended to explore factors associated with the development of MTX-related myelosuppression and its disease severity.MethodsWe retrospectively examined a total of 40 cases of MTX-related myelosuppression that had been filed in the registries of participating rheumatology and hematology divisions. Data before onset were compared with those of 120 controls matched for age and sex. Cytopenia was graded according to the National Cancer Institute criteria for adverse events. Data before and at onset were compared between the severe and non-severe groups.ResultsNon-use of folic acid supplements, concurrent medications, and low renal function were significantly associated with the development of myelosuppression (p < 0.001, p < 0.001, and p = 0.002, respectively). In addition, significantly lower MTX dosages, higher blood cell counts, and lower hemoglobin levels were seen in the myelosuppression group (p < 0.001). No patients exhibited leukocytopenia, neutropenia, or thrombocytopenia in routine blood monitoring taken within the past month. One-fourth developed myelosuppression within the first two months (an early-onset period). Myelosuppression was severe in approximately 40% of patients. Hypoalbuminemia and non-use of folic acid supplements were significantly associated with the severity of pancytopenia (p = 0.001 and 0.008, respectively). Besides these two factors, early onset and the use of lower doses of MTX were significantly associated with the severity of neutropenia (p = 0.003, 0.007, 0.003, and 0.002, respectively).ConclusionsMyelosuppression can occur abruptly at any time during low-dose MTX therapy, but severe neutropenia is more likely to occur in the early-onset period of this therapy. Contrary to our expectations, disease severity was not dependent on MTX doses. Serum albumin levels and folic acid supplementation are the important factors affecting the severity of MTX-related pancytopenia and neutropenia.     

Fatty acid analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas chromatography

A method to prepare fatty acid methyl esters was developed for fatty acid analysis of triacylglycerols by gas chromatography (GC). Triacylglycerols were mixed with methanolic CH₃ONa in hexane containing a mid-polar solvent for 10 s at room temperature. Under these conditions, trioleoylglycerol was converted to methyl oleate with an average yield of 99.3%. This procedure gave reliable and reproducible data on fatty acid compositions determined by GC.     

Effect of electric field on erythrocyte sedimentation rate. II. Dependence on electric current

We measured the electric current dependence of sedimentation curves of swine erythrocytes in a saline solution at the volume fraction of erythrocytes H = 0.091 and 0.220. The sedimentation curve fitted well to the exponential type equation l = a[1-exp(-bt)] at the upward initial electric current I0 = 0.50 mA, 1.01 mA and 1.50 mA, where l is the length of the medium layer at time t, and a and b are phenomenological parameters. The initial slope v0 of sedimentation curve was enhanced from 0.68 mm/hr at I0 = 0 mA to 2.85 mm/hr, 3.87 mm/hr and 5.50 mm/hr at I0 = 0.50 mA, 1.01 mA and 1.50 mA, respectively, for H = 0.220. We also made sedimentation measurements of erythrocytes in their own plasma at H = 0.220 and 0.316. Sedimentation curves coincided with the sigmoidal type equation l = l infinity/[1 + (t50/t)beta] at I0 = 0 mA and 0.50 mA, where l infinity is l at t----infinity, t50 is the time when the plasma level falls to l infinity/2 and beta is a constant. The maximum slope vmax of sedimentation curve increased from 13.29 mm/hr at I0 = 0 mA to 18.65 mm/hr at I0 = 0.50 mA for H = 0.220.