Percoll gradient centrifugation can be omitted in sperm preparation for intracytoplasmic sperm injection

Prior to intracytoplasmic sperm injection (ICSI), seminal fluid is currently washed out from the ejaculated semen and further sperm selection is carried out by a discontinuous Percoll gradient. Possible deleterious effects from the sperm-separating substance Percoll on sperm function or embryo cleavage after in-vitro fertilization (IVF) have, to our knowledge, not yet been reported and the use of Percoll has been widely accepted in IVF. In this study, we examined whether the omission of the Percoll step in the sperm preparation has any influence on the outcome of the ICSI procedure. Two methods of sperm preparation for ICSI were compared in a controlled study on sibling oocytes: washing the semen sample once, followed by a Percoll gradient centrifugation versus washing the sperm sample twice without a Percoll gradient centrifugation. The mean fertilization rates were similar for the two sperm preparation methods: 78.2 +/- 21.4 and 75.0 +/- 24.1% respectively of the intact oocytes displaying two pronuclei. Cleavage rates did not differ statistically between the two groups. Whereas in both groups similar percentages of excellent, good and poor quality embryos were obtained, the percentage of fair quality embryos was significantly higher in the group without Percoll (16.3 +/- 20.1 versus 9.1 +/- 15.7%). However, no statistical differences were observed in either the percentage of transferable embryos or in the percentage of embryos actually transferred or frozen in the two groups. In conclusion, spermatozoa from ejaculates that are washed out from the seminal fluid without any further selection can be used for ICSI without any adverse effect on fertilization and embryo cleavage.      

Analysis of the genetic variability of the 1st (CCC/ACC,P52T) and the 10th exons (bp 1012–1704) of the TSH receptor gene in Graves’ disease

We determined the genetic variability of the 1st (CCC/ACC, P52T polymorphic variant) and 10th exons (bp 1012–1704) of the TSH receptor (TSHR) gene in Graves’ disease. A total of 101 Graves’ patients and 163 control subjects were screened. The A253 mutant allele was carried by nine patients with Graves’ disease (8.91%) and 13 control subjects (7.98%) in heterozygous genotype. No significant difference in the frequency of the mutant allele was found between Graves’ patients and control subjects. These results provide evidence that the A253 polymorphism has no genetic relevance in Graves’ disease. Moreover, the DNA nucleotide sequence of 693 bp of the 10th exon (bp 1012–1704) of the TSHR gene was determined in 15 Graves’ patients. Six patients were homozygous for the wild‐type allele and nine were heterozygous for the mutant allele at the 253rd nucleotide of the first exon. No polymorphism was found in the DNA sequences obtained from leukocytes of Graves’ patients, similarly to the sequences obtained from the nine control subjects. None of the nine patients carrying the A253 polymorphism in the 1st exon of the TSHR had polymorphism in the examined part of the 10th exon, including two additional patients whose thyroid tissue was directly analysed. In all likelihood, the polymorphisms of the examined regions of either the 1st or the 10th exon of the THSR gene do not contribute to the genetic susceptibility to Graves’ disease.     

Coding assignments of Drosophila X virus genome segments: in vitro translation of native and denatured virion dsRNA

Both dsRNA genome segments of Drosophila X virus (DXV) were denatured and translated in vitro using nuclease-treated rabbit reticulocyte lysates. The synthesis of all four primary gene products was detected by polyacrylamide gel electrophoresis and autoradiography. Genome segment A (mol wt 2.3 X 10(6)) encoded polypeptides with molecular weights of 67,000 (67K), 34K, and 27K, whereas segment B (mol wt 22 X 10(6)) encoded the 110K polypeptide. The proteolytic processing of 67K which generates a 49K polypeptide in infected cells was also observed in vitro. Pulse-chase experiments indicated that synthesis of the three polypeptides encoded by genome segment A initiated independently and simultaneously, suggesting that segment A is polycistronic. Native (undenatured) DXV dsRNA could also be translated with high fidelity (vitro). The messenger activity of native dsRNA was abolished by S1 nuclease treatment but completely restored on subsequent denaturation. In vitro "pulse-chase" experiments using native dsRNA as messenger, indicated that the order of translation of polypeptides on genome segment A was 5'-67K-27K-34K-3'.     

Differentiation of Rhizomucor species on the basis of their different sensitivities to lovastatin

The opportunistic pathogens Rhizomucor pusillus and Rhizomucor miehei may be agents of frequently fatal mycotic diseases. In the present study, the susceptibilities of 27 clinical and environmental isolates of R. miehei and R. pusillus to lovastatin under different culturing conditions were investigated. Most of the R. miehei strains grew at lovastatin concentrations as high as 64 to 128 microg/ml. In contrast, the inhibitory effect of lovastatin on all of the R. pusillus strains was evident at lovastatin concentrations as low as 1 to 2 microg/ml. A simple and reliable method for species-level differentiation, based on the significantly higher sensitivity of R. pusillus to lovastatin than that of R. miehei, was elaborated. According this, on malt extract agar containing 6 mug of lovastatin/ml, R. pusillus is not able to produce colonies, while R. miehei will form compact colonies.     

Fluoride-resistant acid phosphatase-containing neurones in dorsal root ganglia are separate from those containing substance P or somatostatin

The distribution of fluoride-resistant acid phosphatase, substance P and somatostatin were investigated in the dorsal horn of the spinal cord and in dorsal root ganglia. In the dorsal horn, the distribution of fluoride-resistant acid phosphatase closely paralleled that of somatostatin and only partly overlapped with that of substance P. In sensory ganglia, none of the fluoride-resistant acid phosphatase-containing neurones contained either substance P or somatostatin. The results suggest the existence of a population of fluoride-resistant phosphatase-positive sensory neurones which is distinct from neurones containing either of these peptides.     

Dynamic balance of metabotropic inputs causes dorsal horn neurons to switch functional states

Sensory relay structures in the spinal cord dorsal horn are now thought to be active processing structures that function before supraspinal sensory integration. Dorsal horn neurons directly receive nociceptive (pain) signals from the periphery, express a high degree of functional plasticity and are involved in long-term sensitization and chronic pain. We show here that deep dorsal horn neurons (DHNs) in Wistar rats can switch their intrinsic firing properties from tonic to plateau or endogenous bursting patterns, depending upon the balance of control by metabotropic glutamate (mGlu) and GABA(B) receptors. We further show that this modulation acts on at least one common target, the inwardly rectifying potassium channel (Kir3). Finally, we found that these firing modes correspond to specific functional states of information transfer in which dorsal horn neurons can faithfully transmit, greatly enhance or block the transfer of nociceptive information.     

Biochemistry of chronic jejunal ulcer in patients

Separation and measurement of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenine-adenosine, lipid phosphates (lipid P), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), further of Mg2+-dependent, total (Mg2+-dependent plus Na+-K+-dependent) and Na+-K+-dependent ATPases was carried out in control (non-ulcerated) and ulcerated (mucosa up to 2.0 cm around the ulcer) jejunal mucosa of 11 patients with chronic jejunal ulcer, who had undergone a Billroth II gastric resection for duodenal ulcer. The levels of ATP, ADP, AMP, sum of ATP + ADP + AMP, lipid-P, RNA (related to 1.0 mg DNA) and the activity of Mg2+-dependent, total (Mg2+-dependent plus Na+-K+-dependent) and Na+-K+-dependent ATPase (related to 1.0 mg membrane protein) were significantly higher in the lacerated jejunal mucosa than in the control one. The results indicate that (1) no circulatory damage can be found in the ulcerated jejunal mucosa; (2) a cellular hyperactivity (increased ATP breakdown and increased ATP resynthesis, increased RNA and lipid synthesis) occurs in the ulcerated jejunal mucosa; (3) the biochemical structure of cells differs significantly in the control and the ulcerated jejunal mucosa.     

Presence of parvoviruses in the intestine of chickens showing stunting syndrome

Small viral particles of 19 to 24 nm in diameter with a buoyant density in CsCl of 1.43 g/ml were detected electron microscopically in samples taken from the gut of 10-day-old chickens suffering from a stunting syndrome. The viral particles are suggested to belong to the Parvoviridae and their possible role in the stunting syndrome is discussed.     

Complementation of class II A alleles in the immune response to (GluLysTyr) polymers

The proliferative T cell responses to poly(GluLysTyr) (GLT) and poly(GLULysPhe) (GLPhe) are restricted by the E alpha E beta class II MHC molecule (E) in most responded strains. Some nonresponder strains that carry responder E beta, but cannot express cell surface E molecules, can complement with other nonresponder strains that provide the missing E alpha chain needed for the expression of E molecules and for responsiveness to GLT and GLPhe. Here another type of complementation is described between two E-nonexpressor haplotypes, H-2f and H-2s, which result in E-nonexpressor F1 hybrids, which are responders to GLT. The restriction element involved in this response is an Af/As hybrid molecule. The data support the hypothesis that conformational determinants resulting from the free association of alpha and beta chains in heterozygotes can increase the immune potential of the individual.