Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution

The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs–Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3 h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1 M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm.     

l-NAME inhibits tumor cell progression and pulmonary metastasis of r/m HM-SFME-1 cells by decreasing NO from tumor cells and TNF-α from macrophages

Highly metastatic ras/myc-transformed serum-free mouse embryo (r/m HM-SFME-1) cells were injected subcutaneously to mice and the effects of Nω-nitro-l-arginine methyl ester (l-NAME) on the tumor progression and pulmonary metastasis were investigated. In addition, production of nitric oxide (NO), matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-α) in the tumor cells and in a mouse macrophage-like cell line, J774.1 cells, was analyzed. The increase in footpad thickness was significantly smaller in the mice which were fed the l-NAME containing water (4.24 ± 0.39 mg/day/mouse). The number of the tumor cells metastasized to the lungs was smaller in the l-NAME treated mice, although statistical significance was not found. Co-treatment of r/m HM-SFME-1 cells with interferon-gamma (IFN-γ; 100 U/ml) and lipopolysaccharide (LPS; 0.5 μg/ml) significantly enhanced NO production, and the presence of l-NAME at 1 mM significantly decreased this response. In r/m HM-SFME-1 cells, MMP-2 was undetectable and MMP-9 was also very little in the basal level, and both MMPs were unaffected by the IFN-γ and/or LPS treatments, not to mention by the l-NAME treatment. In J774.1 cells, any treatment including LPS appeared to enhance MMP-9 production, however, this upregulation was not inhibited by the additional presence of l-NAME. Production of TNF-α by J774.1 cells was markedly enhanced with LPS treatment, and this enhancement was significantly reduced in the presence of l-NAME. These results indicate that the inhibitory effects of l-NAME on the tumor cell progression and pulmonary metastasis could be due to suppression of NO from tumor cells and TNF-α from macrophages (Mol Cell Biochem, 2007). Hideaki Yamaguchi and Yumi Kidachi contributed equally to this work.     

Role of aluminum-binding ligands in aluminum resistance of Eucalyptus camaldulensis and Melaleuca cajuputi

We investigated the roles of Al-binding ligands in Al exclusion from roots and in internal Al detoxification in roots as Al resistance mechanisms in two Al-resistant Myrtaceae trees, Eucalyptus camaldulensis Dehnh. and Melaleuca cajuputi Powell. The amounts of ligands secreted from roots and contained in root tips of these species were compared with those of an Al-sensitive species, Melaleuca bracteata F. Muell., after the roots were exposed to 0 or 1 mM AlCl₃ solution. Secretion of well-known ligands (citrate, oxalate, and malate) from roots under Al treatment was low in all species. However, in E. camaldulensis, the Al-binding capacity of root exudates under Al treatment was considerable and was higher than that in M. bracteata. Gel filtration chromatography revealed that a low-molecular-weight Al-binding ligand was secreted from roots in response to Al only in E. camaldulensis. On the other hand, the Al-binding capacity of cell sap in root tips under Al treatment was similar for the resistant and sensitive species. These results suggest that Al exclusion by secretion of the unknown low-molecular-weight Al-binding ligand from roots contributes to the Al resistance of E. camaldulensis, whereas M. cajuputi has developed Al-resistance mechanisms other than secretion of ligands from roots or concentration of internal ligands in root tips.     

Dynamics of lipid droplet-associated proteins during hormonally stimulated lipolysis in engineered adipocytes: stabilization and lipid droplet binding of adipocyte differentiation-related protein/adipophilin

In mature adipocytes, triglyceride is stored within lipid droplets, which are coated with the protein perilipin, which functions to regulate lipolysis by controlling lipase access to the droplet in a hormone-regulatable fashion. Adipocyte differentiation-related protein (ADRP) is a widely expressed lipid droplet binding protein that is coexpressed with perilipin in differentiating fat cells but is minimally present in fully differentiated cultured adipocytes. We find that fibroblasts ectopically expressing C/EBPalpha (NIH-C/EBPalpha cells) differentiate into mature adipocytes that simultaneously express perilipin and ADRP. In response to isoproterenol, perilipin is hyperphosphorylated, lipolysis is enhanced, and subsequently, ADRP expression increases coincident with it surrounding intracellular lipid droplets. In the absence of lipolytic stimulation, inhibition of proteasomal activity with MG-132 increased ADRP levels to those of cells treated with 10 mum isoproterenol, but ADRP does not surround the lipid droplet in the absence of lipolytic stimulation. We overexpressed a perilipin A construct in NIH-C/EBPalpha cells where the six serine residues known to be phosphorylated by protein kinase A were changed to alanine (Peri A Delta1-6). These cells show no increase in ADRP expression in response to isoproterenol. We propose that ADRP can replace perilipin on existing lipid droplets or those newly formed as a result of fatty acid reesterification, under dynamic conditions of hormonally stimulated lipolysis, thus preserving lipid droplet morphology/structure.     

Lignan, phenolic and iridoid glycosides from Stereospermum cylindricum

A lignan glycoside [(+)-cycloolivil 4'-O-beta-d-glucopyranoside], a phenolic glycoside [3,4-dimethoxyphenyl 1-O-beta-d-xylopyranosyl-(1-->6)-beta-d-glucopyranoside] and a iridoid glycoside (stereospermoside) were isolated from the leaves and branches of Stereospermum cylindricum, together with (+)-cycloolivil, (+)-cycloolivil 6-O-beta-d-glucopyranoside, (-)-olivil, (-)-olivil 4-O-beta-d-glucopyranoside, (-)-olivil 4'-O-beta-d-glucopyranoside, vanilloloside, decaffeoyl-verbascoside, isoverbascoside, 3,4,5-trimethoxyphenyl 1-O-beta-d-xylopyranosyl-(1-->6)-beta-d-glucopyranoside, ajugol, verminoside, and specioside. The structure elucidations were based on spectroscopic evidence.     

Bridelionosides A-F: Megastigmane glucosides from Bridelia glauca f. balansae

The chemical investigation of leaves of Bridelia glauca f. balansae afforded six megastigmane glucosides, named bridelionosides A-F, along with seven known megastigmane glucosides. Their structures were determined by a combination of spectroscopic analyses and by application of the modified Mosher's method.     

Adipose triglyceride lipase regulates basal lipolysis and lipid droplet size in adipocytes

In adipocytes, lipid droplet (LD) size reflects a balance of triglyceride synthesis (lipogenesis) and hydrolysis (lipolysis). Perilipin A (Peri A) is the most abundant phosphoprotein on the surface of adipocyte LDs and has a crucial role in lipid storage and lipolysis. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major rate-determining enzymes for lipolysis in adipocytes. Each of these proteins (Peri A, ATGL, and HSL) has been demonstrated to regulate lipid storage and release in the adipocyte. However, in the absence of protein kinase A (PKA) stimulation (basal state), the lipases (ATGL and HSL) are located mainly in the cytoplasm, and their contribution to basal rates of lipolysis and influence on LD size are poorly understood. In this study, we utilize an adenoviral system to knockdown or overexpress ATGL and HSL in an engineered model system of adipocytes in the presence or absence of Peri A. We are able to demonstrate in our experimental model system that in the basal state, LD size, triglyceride storage, and fatty acid release are mainly influenced by the expression of ATGL. These results demonstrate for the first time the relative contributions of ATGL, HSL, and Peri A on determination of LD size in the absence of PKA stimulation.     

The presence of 4-hydroxyphenylacetic acid in human saliva and the possibility of its nitration by salivary nitrite in the stomach

Human saliva contained 4-hydroxyphenylacetic acid (HPA) (2-10 microM) and nitrite (60-300 microM). HPA was nitrated to 4-hydroxy-3-nitrophenylacetic acid (NO2HPA) when HPA and sodium nitrite were mixed at pH 1.0. NO2HPA was also formed when saliva was incubated under acidic conditions. These results suggest that salivary HPA is nitrated to NO2HPA when saliva is swallowed into the stomach.     

High Aluminum Resistance in Buckwheat : II. Oxalic Acid Detoxifies Aluminum Internally

Buckwheat (Fagopyrum esculentum Moench. cv Jianxi), which shows high Al resistance, accumulates Al in the leaves. The internal detoxification mechanism was studied by purifying and identifying Al complexes in the leaves and roots. About 90% of Al accumulated in the leaves was found in the cell sap, in which the dominant organic acid was oxalic acid. Purification of the Al complex in the cell sap of leaves by molecular-sieve chromatography resulted in a complex with a ratio of Al to oxalic acid of 1:3. A ¹³C-nuclear magnetic resonance study of the purified cell sap revealed only one signal at a chemical shift 164.4 ppm, which was assigned to the Al-chelated carboxylic group of oxalic acid. A ²⁷Al-nuclear magnetic resonance analysis revealed one major signal at the chemical shift of 16.0 to 17.0 ppm, with a minor signal at the chemical shift of 11.0 to 12 ppm in both the intact roots and their cell sap, which is consistent with the Al-oxalate complexes at 1:3 and 1:2 ratios, respectively. The purified cell sap was not phytotoxic to root elongation in corn (Zea mays). All of these results indicate that Al tolerance in the roots and leaves of buckwheat is achieved by the formation of a nonphytotoxic Al-oxalate (1:3) complex.     

Specific induction of a 72-kDa heat shock protein protects esophageal mucosa from reflux esophagitis

AimsThe aim of this study is to investigate the expression and cytoprotective function of a 72-kDa heat shock protein (HSP72) using a reflux esophagitis model in rats.Main methodsExpression of HSP60, HSP72, and HSP90 in rat esophageal mucosa was evaluated by Western blot analysis before and after hyperthermia (42.5 °C, 20 min). Rats received the operation to produce reflux esophagitis with or without pretreatment with hyperthermia to induce HSPs. The esophageal mucosal damage was evaluated 12 h after the operation.Key findingsExpression of HSP72 was significantly increased by hyperthermia in rat esophageal mucosa. Reflux esophagitis was dramatically prevented when HSP72 was preinduced by hyperthermia. Furthermore, activation of TNF-α and IL-1β in esophageal mucosa was also suppressed.SignificanceThese results suggested that hyperthermia protects the esophageal mucosa in reflux esophagitis model by inducing HSP72 and suppressing proinflammatory cytokine activation. These findings might suggest that HSP-inducing therapy could be a novel and unique therapy for reflux esophagitis.