Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.     

Biotechnological applications of bioluminescence and chemiluminescence

Recent progress in molecular biology has made available several biotechnological tools that take advantage of the high detectability and rapidity of bioluminescence and chemiluminescence spectroscopy. These developments provide inroads to in vitro and in vivo continuous monitoring of biological processes (e.g. gene expression, protein-protein interaction and disease progression), with clinical, diagnostic and drug discovery applications. Furthermore, combining luminescent enzymes or photoproteins with biospecific recognition elements at the genetic level has led to the development of ultrasensitive and selective bioanalytical tools, such as recombinant whole-cell biosensors, immunoassays and nucleic acid hybridization assays. The high detectability of the luminescence analytical signal makes it appropriate for miniaturized bioanalytical devices (e.g. microarrays, microfluidic devices and high-density-well microtiter plates) for the high-throughput screening of genes and proteins in small sample volumes.     

Photoinhibition and recovery of photosynthesis in leaves of Vitis berlandieri and Vitis rupestris

Photoinhibition of photosynthesis was studied in Vitis berlandieri and Vitis rupestris leaves under controlled conditions (irradiation of detached leaves to about 1900 micromol m(-2) s(-1)). The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (Fv/Fm) and electron transport measurements. The potential efficiency of PS2, Fv/Fm declined, Fo increased significantly in leaves of V. berlandieri, while Fo decreased in V. rupestris. In isolated thylakoids, the rate of whole chain and PS2 activity markedly decreased in high light irradiated more in leaves of V. berlandieri than in leaves of V. rupestris. A smaller inhibition of PS1 activity was also observed in both leaves. In the subsequent dark incubation, fast recovery was observed in both leaves and reached maximum PS2 efficiencies similar to those observed in non-photoinhibited leaves. The artificial exogenous electron donors DPC, NH2OH and Mn2+ failed to restore the high light induced loss of PS2 activity in V. berlandieri leaves, while DPC and NH2OH significantly restored in V. rupestris leaves. It is concluded that high light inactivates on the donor side of PS2 and acceptor side of PS2 in V. rupestris and V. berlandieri leaves, respectively. Quantification of the PS2 reaction center protein D1 and 33 kDa protein of water splitting complex following high light exposure of leaves showed pronounced differences between V. berlandieri and V. rupestris leaves. The marked loss of PS2 activity in high light irradiated leaves was due to the marked loss of D1 protein and 33 kDa protein in V. berlandieri and V. rupestris leaves, respectively.     

Cholecystokinin pathways modulate sensations induced by gastric distension in humans

Ingested fat releases CCK, causes gastric relaxation, delays gastric emptying, and limits meal size; however, the mechanistic link among these actions has not been established. Fatty acid release of CCK is chain-length sensitive; dodecanoic acid (C12) induces greater CCK release than decanoic acid (C10). The effect of C12 or C10 on tolerance to subsequent intragastric infusion of liquid was determined in healthy subjects, with and without the CCK(1) receptor antagonist dexloxiglumide. Gastric wall relaxation after either fatty acid was assessed by graded volume distension and by barostat; gastric emptying was measured by gastric aspiration and by a [(13)C]octanoic acid breath technique. C12 released more CCK (mean plasma CCK after vehicle, 4.7 +/- 0.8 pM; C10, 4.8 +/- 0.3 pM; C12, 8 +/- 1.2 pM; P < 0.05 C12 vs. C10 or vehicle) and reduced the volume of water (and of 5 and 25% glucose solutions) delivered at maximum tolerance compared with C10 or vehicle (volume of water tolerated after vehicle, 1,535 +/- 164 ml; C10, 1,335 +/- 160 ml; C12, 842 +/- 103 ml; P < 0.05 C12 vs. C10 or vehicle); this effect was abolished by dexloxiglumide. Intragastric volumes were always similar at the limit of tolerance, and, whereas gastric relaxation occurred to similar degrees after the fatty acids, its duration was longer after C12, which also induced a longer delay in half-gastric emptying [t(1/2)(min) after vehicle, 53 +/- 2; C10, 67 +/- 3; C12, 88 +/- 7; P < 0.05 C12 vs. C10 or vehicle]. In conclusion, ingestion of a CCK-releasing fatty acid reduces the tolerated volume of liquid delivered into the stomach, primarily via a CCK(1) receptor-mediated delay in gastric emptying.     

Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans

An animal's ability to detect and avoid toxic compounds in the environment is crucial for survival. We show that the nematode Caenorhabditis elegans avoids many water-soluble substances that are toxic and that taste bitter to humans. We have used laser ablation and a genetic cell rescue strategy to identify sensory neurons involved in the avoidance of the bitter substance quinine, and found that ASH, a polymodal nociceptive neuron that senses many aversive stimuli, is the principal player in this response. Two G protein alpha subunits GPA-3 and ODR-3, expressed in ASH and in different, nonoverlapping sets of sensory neurons, are necessary for the response to quinine, although the effect of odr-3 can only be appreciated in the absence of gpa-3. We identified and cloned a new gene, qui-1, necessary for quinine and SDS avoidance. qui-1 codes for a novel protein with WD-40 domains and which is expressed in the avoidance sensory neurons ASH and ADL.     

Cell cycle checkpoint proteins and cellular response to treatment by anticancer agents

The response of cancer cells to treatment with anticancer agents is mediated in part by proteins controlling both the cell cycle progression and the genomic integrity, including p53, p73 and checkpoint proteins chk1 and chk2. We here summarized the cellular functions of these proteins, their alterations in human tumors and the impact of their mutations/alterations on cellular response to treatment, Particular attention has been paid for those studies performed in isogenic cell systems, to minimize as much as possible interference by other alterations invariably present when different cell types are considered. Focus has also be given to the approaches taken to exploit the differential expression of these proteins between normal and tumor cells to improve the selectivity of treatment with anticancer agents.     

Percutaneous closure of a coronary fistula between the right coronary artery to the left atrium

We describe a case of a congenital coronary artery fistula of the right coronary artery draining into the left atrium in an eight-year-old boy. The initial diagnosis was made after the detection of a continuous cardiac murmur at the age of six years. Transthoracic echocardiography showed the right coronaric ostium dilatation, the site of drainage in the left atrium and left ventricle volume overload. Catheterization confirmed the diagnosis. The patient underwent percutaneous closure by PDA occluder device. Immediate post-closure angiograms showed complete occlusion of the fistula. The patient showed transient ischemic changes on ECG associated to an increase of plasmatic levels of the cardiac enzyme. ECG and cardiac enzyme were normal one week after the procedure.     

Microarray analysis in Alzheimer's disease and normal aging

The purpose of this study was to investigate gene expression in Alzheimer's disease (AD), the most common form of senile dementia. We utilized the microarray technology to simultaneously compare the expression profile of 12,000 human genes in cerebral cortex of AD and normal aging. To identify gene expression related to neurodegeneration, beside the presence of amyloid deposition, we used control brains with abundant amyloid plaques, derived from cognitively normal elderly subjects. The microarray analysis indicated that 314 genes were differentially expressed in AD cerebral cortex, with differences greater than 5 folds in 25 genes. RT-PCR performed on a selected group of genes confirmed the increased expression of the interferon-induced protein 3 in AD brain. This protein, which is highly inducible by both type I and type II interferons, was not previously associated with the neurodegenerative disease.     

Structural study of poly(L-lactic acid) spherulites

Spherulites of poly(L-lactic acid) (PLLA) and of its blends with atactic poly(3-hydroxybutyrate) (a-PHB, from 10 to 75 wt %) were investigated by microfocus X-ray diffraction using synchrotron radiation. Radial scans in 5 microm steps with 3 microm beam diameter were performed. In PLLA, tens of identical diffraction images were collected. The unit cell a-axis was radially oriented, and the other axes lacked any specific orientation. In contrast, all PLLA/a-PHB blends showed a periodic change of diffraction pattern with increasing distance from the spherulite center. In all cases, the a-axis lay along the radius, while the b- and c-axes rotated about a with a defined periodicity. The unit cell twisting frequency increased with a-PHB content and closely matched the band spacing observed by polarized optical microscopy, which changed from 250 to 60 microm when the amount of a-PHB increased from 10 to 75 wt %. Concomitantly, a gradual broadening of all X-ray reflections was observed.