Peritoneal fluid from rabbits or goats as media for in vitro maturation, fertilization and initial culture of caprine oocytes

The efficacy of peritoneal fluid from rabbit and goat for in vitro maturation, fertilization and initial culture of embryos from caprine oocytes was evaluated. Peritoneal fluid was collected from adult female goats (n = 9) or rabbits (n = 9). Good quality oocytes were subjected to in vitro maturation and fertilization in three different media viz. Tissue Culture Medium (TCM-199), goat Peritoneal Fluid (gPF) and rabbit Peritoneal fluid (rPF). Maturation rates were 74.7+/-2.07% and 63.6+/-5.28% in TCM-199, gPF 65.8+/-2.54% and 55.6+/-3.79%, and rPF 57.7+/-1.78% and 44.6+/-3.01% when evaluated on the basis of cumulus cell expansion and the achievement of metaphase-II stage, respectively. However, no significant differences were observed in respect of maturation rate between the control and gPF and between gPF and rPF groups. Freshly ejaculated buck semen was treated with heparin (10 microg/ml) and after 45 min incubation with heparin, 8.0% sperm were live and acrosome reacted. The proportions of fertilized oocytes based on male and female pronuclei formation or on cleavage development were 50.5+/-5.03, 42.3+/-3.15 and 34.2+/-1.98%; 31.0+/-2.80, 27.9+/-2.12 and 21.8+/-1.69% for TCM, gPF and rPF, respectively. It was concluded that peritoneal fluids either from goats or rabbits could be used as an alternative medium to TCM-199. However, further research is required to confirm its efficacy for embryo development up to the blastocyst stage.     

Tolerance of pesticides and antibiotic resistance in bacteria isolated from wastewater-irrigated soil

A total of 64 bacterial isolates (40 Pseudomonas spp., 12 Azotobacter and 12 Rhizobium spp.) were characterized on the basis of morphological, cultural and biochemical characteristics. All the isolates were tested for their tolerance to the pesticides endosulfan, carbofuran, and malathion. 12.5% of the Pseudomonas isolates from soil tolerated concentrations of 1600 g malathion ml whereas 7.5% of isolates tolerated the same concentration of carbofuran. However, Pseudomonas isolates demonstrated a tolerance limit to endosulfan at a concentration of 800 g/ml. Asymbiotic N₂-fixers (Azotobacter) and symbiotic N₂-fixers (Rhizobium spp.) were also able to tolerate concentrations of pesticides up to 1600 g/ml. All the isolates were further tested for their antibiotic susceptibility against seven different antibiotics, nalidixic acid, cloxacillin, chloramphenicol, tetracycline, amoxycillin, methicillin, doxycycline. 100% of the Pseudomonas isolates were resistant to cloxacillin and 57.5% were resistant to methicillin. 7.5% of the isolates exhibited multiple resistance to five different antibiotics in three different combinations whereas 25% of the isolates showed multiple resistance to four different antibiotics in seven different combinations. Some of the resistant isolates were also screened for plasmid DNA and found to harbour a single plasmid.     

Phylogenomics of the nucleosome

Histones are best known as the architectural proteins that package the DNA of eukaryotic organisms, forming octameric nucleosome cores that the double helix wraps tightly around. Although histones have traditionally been viewed as slowly evolving scaffold proteins that lack diversification beyond their abundant tail modifications, recent studies have revealed that variant histones have evolved for diverse functions. H2A and H3 variants have diversified to assume roles in epigenetic silencing, gene expression and centromere function. Such diversification of histone variants and 'deviants' contradicts the perception of histones as monotonous members of multigene families that indiscriminately package and compact the genome. How these diverse functions have evolved from ancestral forms can be addressed by applying phylogenetic tools to increasingly abundant sequence data.     

Nitric oxide stimulates macrophage inflammatory protein-2 expression in sepsis

NO is a crucial mediator of the inflammatory response, but its in vivo role as a determinant of lung inflammation remains unclear. We addressed the in vivo role of NO in regulating the activation of NF-kappaB and expression of inflammatory proteins using an in vivo mouse model of sepsis induced by i.p. injection of Escherichia coli. We observed time-dependent degradation of IkappaB and activation of NF-kappaB accompanied by increases in inducible NOS, macrophage inflammatory protein-2 (MIP-2), and ICAM-1 expression after E. coli challenge, which paralleled the ability of lung tissue to produce high-output NO. To determine the role of NO in this process, mice were pretreated with the NO synthase (NOS) inhibitor NG-methyl-L-arginine. Despite having relatively modest effects on NF-kappaB activation and ICAM-1 or inducible NOS expression, the NOS inhibitor almost completely inhibited expression of MIP-2 in response to E. coli challenge. These responses were associated with the inhibition of migration of neutrophils in lung tissue and increased permeability induced by E. coli. In mice pretreated with NG-methyl-L-arginine, coadministration of E. coli with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate substantially restored MIP-2 expression but decreased ICAM-1 expression. The results suggest that NO generated after administration of E. coli serves as an important proinflammatory signal to up-regulate MIP-2 expression in vivo. Thus, NO production in high quantities may be important in the mechanism of amplification of the lung inflammatory response associated with sepsis.     

Changes during hibernation in different phospholipid and free and esterified cholesterol serum levels in black bears

During hibernation, fat is known to be the preferred source of energy. A detailed analysis of different phospholipids, as well as free and esterified cholesterol, was conducted to investigate lipid abnormalities during hibernation. The levels of total phospholipids and total cholesterol in the serum of black bears were found to increase significantly in hibernation as compared with the active state. Both free and esterified cholesterol were increased in the hibernating state in comparison with the active state (P < 0.05). The percentage increase during hibernation was more in free cholesterol (57%) than in esterified cholesterol (27%). Analysis of subclasses of serum phospholipids showed that choline containing phospholipids, i.e., sphingomyelin (SPG) (14%) and phosphatidylcholine (PC) (76%), are the major phospholipids in the serum of bear. The minor phospholipids included 8% of phosphatidylserine (PS) + phosphatidylinositol (PI), while phosphatidylethanolamine (PE) was only 2% of the total phospholipids. A comparison of phospholipid subclasses showed that PC, PS + PI and SPG were significantly increased, while PE was significantly decreased (P < 0.05) in the hibernating state as compared with the active state in black bears. These results suggest that the catabolism of phospholipids and cholesterol is decreased during hibernation in black bears, leading to their increased levels in the hibernating state as compared with the active state. In summary, our results indicate that serum cholesterol and phospholipid fractions (except PE) are increased during hibernation in bears. It is proposed that the increase of these lipids may be due to the altered metabolism of lipoproteins that are responsible for the clearance of the lipids.     

QT-RR relationship in healthy subjects exhibits substantial intersubject variability and high intrasubject stability

Recently, it was demonstrated that the QT-RR relationship pattern varies significantly among healthy individuals. We compared the intra- and interindividual variations of the QT-RR relationship. Twenty-four-hour 12-lead digital electrocardiograms (ECGs; SEER MC, GE Marquette; 10-s ECG recorded every 30 s) were obtained at baseline and after 24 h, 1 wk, and 1 mo in 75 healthy subjects (42 women, 33 men, age 27.9 +/- 9.6 vs. 26.8 +/- 7.5 yr, P = not significant). QT interval was measured automatically in each ECG by six different algorithms, and the mean of the six measurements was analyzed. In each recording of each individual, QT-RR relationship was assessed by 10 different regression models including linear (QT = beta + alpha x RR) and parabolic (QT = beta x RR(alpha)) models. Standard deviations (SDs) of regression parameters alpha and beta of consecutive recordings of each individual were compared with SD of the individual means. Intrasubject stability and interindividual variability were further tested by ANOVA. With all models, intraindividual SDs of the regression parameters were highly significantly smaller than SD of individual means (P < 10(-5)-10(-9)). The intrasubject stability was further confirmed by ANOVA (P < 10(-19)-10(-30)). The QT-RR relationship exhibits substantial intersubject variability as well as a high intrasubject stability. This has practical implications for a precise estimation of the heart rate-corrected QT interval in which optimized subject-specific rate correction formulas should be used.     

Compliance calibration for fracture testing of equine cortical bone

An experimental compliance calibration method for measuring crack length in fracture toughness tests of cortical bone was developed. Calibration tests were conducted on twenty compact type fracture specimens machined from the mid-diaphysis of five pairs of equine third metacarpal bones. Specimens were oriented for crack propagation in a direction transverse to the longitudinal axis of the bone. Specimen compliance was determined from the load vs. crack opening displacement record over a range of crack lengths from 0.48 to 0.75 times the specimen width. The results demonstrate that the compliance calibration method developed for isotropic materials can be used to determine crack length in bone, which is transversely isotropic. However, specimens from lateral and dorsal regions exhibited significantly different compliance calibrations even after differences in elastic modulus were taken into account in the normalized compliance.     

Cultured mouse marrow cell lines: Interactions between fibroblastoid cells and monocytes

Continuous cell lines were derived from primary cultures of adherent bone marrow cells from SJL/J, BALB/c, C3H/eb, RF, and nude-ICR mice. All these lines readily assumed a pure fibroblastoid appearance with the exception of the BALB/c line (MBA-14), which retained both fibroblastoid and monocytoid cells. This particular line could promote the proliferation of myeloid progenitors (CFU-C) in short-term bone-marrow cultures. The two cell types that composed the MBA-14 cell line were successfully isolated and grown separately; the monocytes as the 14M and 14M1 cell lines and the fibroblastoid cells as the 14F clones. The latter were found to be preadipocytes and accumulated fat in the absence of added hydrocortisone, in medium supplemented with fetal calf serum. Growth of the monocyte lines (14M and 14M1) was dependent upon the mononuclear phagocyte stimulator CSF-1. In the parent MBA-14 cell line the growth of monocytes seemed to depend upon stimulating factor(s) produced by the fibroblastoid cells. The 14M1 monocytes were able to process and degrade antigen as efficiently as primary macrophages. Furthermore, processed antigen produced by 14M1 cells evoked proliferative response by antigen-primed lymph-node cells. In addition to these immunological functions the 14M1 cells were capable of modulating the colony-stimulating activity and degree of adipogenesis exhibited by the fibroblastoid cells. These interactions between monocytes and fibroblastoid cells may constitute part of the mechanism controlling the activity of the hematopoietic microenvironment.     

Integrated control of lung fluid balance

This review summarizes the highlights of the EB2004 symposium that dealt with the integrated aspects of the lung fluid balance. It is apparent that maintenance of lung fluid balance requires the proper functioning of vascular endothelial and alveolar epithelial barriers. Under physiological conditions, the transcytotic pathway requiring repeated fission-fusion events of the caveolar membrane with other caveolae solely transports albumin. Caveolin-1, which forms caveolae, and albumin-binding proteins play a central role in signaling the transcytosis of albumin. Signals responsible for increasing endothelial permeability in lung microvessels in response to inflammatory mediators were also described. These studies in gene knockout mouse models revealed the importance of Ca(2+) signaling via store-operated transient receptor channel 4 and the activation of endothelial myosin light chain kinase isoform in mediating the increase in microvessel permeability. Increases in the cytosolic Ca(2+) in situ in microvessel endothelia can occur by mitochondria-dependent as well as mitochondria-independent pathways (such as the endoplasmic reticulum). Both these pathways, by triggering endothelial cell activation, may result in lung microvascular injury. The resolution of alveolar edema, requiring clearance of fluid from the air space, is another area of intense investigation in animal models. Although beta-adrenergic agonists can activate alveolar fluid clearance, signaling pathways regulating these events in intact alveoli remain to be established. Development of mouse models in which the function of regulatory proteins (identified in cell culture studies) can be systematically analyzed will provide a better and more integrated picture of lung fluid balance. In vivo veritas!     

Limbetazulone: a new 'decahydro-8-oxanaphtho[2,1-f]azulen-7-one' diterpenoid from Ballota limbeta, and occurrence of two conformational isomers in the crystal

A new diterpenoid, limbetazulone (= (3S,4S,4aR,12bS)-1,2,3,4,4a,5,6,11,12,12b-decahydro-3-hydroxy-4-(hydroxymethyl)naphtho[1',2': 5,6]cyclohepta[1,2-b]furan-7(7H)-one; 1), with the very rare 'naphtho[2,1-f]azulene-7-one' skeleton, was isolated from the aerial parts of the Asian medicinal plant Ballota limbeta. Its structure was established by extensive spectroscopic investigations, especially 1D and 2D NMR. X-Ray diffraction studies showed the presence of two conformational isomers (1a and 1b) in the crystal.