Quantitative ultrastructural characteristics relating to transport between luteal cell cytoplasm and blood in the corpus luteum of the pregnant rat

The synthesis and secretion of progesterone by the corpus luteum (CL) may be limited or controlled by transport mechanisms operating between circulating blood and luteal cell cytoplasm. To examine this possibility, the structural features involved in transport, including membrane surface areas and diffusion distances, were quantitated in the CL of 16-day pregnant rats. One ovary from each of eight rats was fixed by perfusion via a cannula inserted into the parametrial artery, and two CL from each ovary were processed for electron microscopy and examined with standard morphometric techniques. For comparison, one CL from each of a further eight ovaries was diced into small cubes, fixed by immersion, and analyzed similarly. In perfusion-fixed CL, there was a substantial volume of vascular space (20% of the total) and interstitial space (5%) and an extensive surface area of capillaries (441 mm2 per CL). The luteal-cell membrane had numerous projections which increased its surface area by a factor of 3.08. Almost 60% of the luteal-cell surface directly faced a capillary, and a further 37% faced interstitial space which probably extended to a capillary surface. Only 3% was in direct contact with a neighboring luteal cell. Despite the extensive interstitial space the harmonic mean thickness, an estimate of likely effective diffusion distance between luteal cell cytoplasm and blood, was only 0.42 micron. This was less than half of the calculated arithmetic mean thickness owing to the presence of surface projections and an uneven capillary endothelium. Results from immersion-fixed CL were qualitatively similar; but the proportion of interstitial space was only 59% of that in perfusion-fixed CL, and the contribution of surface projections to the total area of luteal-cell membranes was significantly reduced. Collectively, these results suggest that membranes and spaces between blood and luteal-cell cytoplasm are structured so as to minimize transport distances.     

Associations between marker genotypes, halothane reaction, creatine kinase activity and meat quality characters in a sample of German Landrace pigs

Routine blood typing of German Landrace pedigree populations and an earlier study revealed very low frequencies of the favourable alleles at the marker loci Phi, Pgd and H. The hypothesis was that in this population the whole linkage group of favourable alleles at the halothane and neighbouring marker loci may have been lost as a consequence of intense selection for leanness and type. The present study of 1050 German Landrace pigs at the Relliehausen experimental station, where some effort has been made to maintain a higher frequency of the favourable alleles PhiA (0.48), H- (0.43) and PgdA (0.70) gave quite different results. The frequency of halothane-positive pigs found by using a severe test was only 30%. Only 5.4%, 8.8%, 13.4% and 13.9% of animals with PhiA/A, H-/-, PgdA/A and PhiA/B genotypes respecitively were halothane-positive. Forty to sixty per cent of pigs with these marker genotypes could therefore be expected to be homozygous halothane-negative (N/N) animals. Creatine kinase activity and three selected meat quality characters showed highly significant differences between the A/A and the B/B genotypes for the marker loci Phi and Pgd, with the heterozygotes being intermediate. These differences are greater than those observed between halothane-negative and halothane-positive phenotypes. The only other consistently superior marker genotype in this population was the H blood group genotype H-/-. In contrast to findings from Sweden and Switzerland, the postalbumin locus Po2 and the suppressor locus S for the A-O blood groups did not exhibit useful marker qualities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assay of mephenytoin metabolism in human liver microsomes by high-performance liquid chromatography

The metabolism of mephenytoin to its two major metabolites, 4-OH-mephenytoin (4-OH-M) and 5-phenyl-5-ethylhydantoin (nirvanol) was studied in human liver microsomes by a reversed phase HPLC assay. Because of preferential hydroxylation of S-mephenytoin in vivo, microsomes (5-300 micrograms protein) were incubated separately with S- and R-mephenytoin. After addition of phenobarbital as internal standard, the incubation mixture was extracted with dichloromethane. The residue remaining after evaporation was dissolved in water and injected on a 60 X 4.6-mm reversed-phase column (5 mu-C-18). Elution with acetonitrile/methanol/sodium perchlorate (20 mM, pH 2.5) led to almost baseline separation of mephenytoin, metabolites, and phenobarbital. Quantitation was performed by uv-absorption at 204 nm by the internal standard method. Propylene glycol was found to be the best solvent for mephenytoin, but inhibited the reaction noncompetitively. 4-OH-M and nirvanol could be detected at concentrations in the incubation mixture as low as 40 and 80 nM, respectively. The rates of metabolite formation were linear with time and protein concentration. The reaction was found to be substrate stereoselective. At substrate concentrations below 0.5 mM S-mephenytoin was preferentially hydroxylated to 4-OH-M, while R-mephenytoin was preferentially demethylated to nirvanol at all substrate concentrations tested (25-1600 microM). These data provide a mechanistic explanation for the stereospecific pharmacokinetics in vivo. The dependence of both metabolic relations on NADPH and the inhibition by CO suggest that they are mediated by cytochrome P-450-type monooxygenases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of cloned human leukocyte interferon by Bacillus subtilis: optimal production is connected with restrained growth.

Bacillus subtilis, transformed with a plasmid containing the human alpha-2 (leukocyte) interferon gene, was cultivated in batch and continuous culture in a complex medium. In continuous culture with dissolved oxygen of less than 10% of air saturation, the extracellular interferon titer decreased sharply when the growth rate was lower or higher than the optimal one (mu = 0.14 h-1). Thus, a relatively low growth rate was best for extracellular interferon production, and oxygen limitation enhanced interferon production. The mean output rate in batch culture after successful harvest was 20 X 10(6) IU/liter per h and the maximal output rate in continuous culture was 14 X 10(6) IU/liter per h.     

Bacteriophage P1 derivatives unaffected in their growth by a large inversion or by IS insertions at various locations

Several plaque-forming phage P1 derivatives carrying DNA rearrangements associated with IS elements are described. They have IS1, IS3 and IS5 inserted in four distinct locations, all of which are non-essential regions for phage P1 propagation. One derivative carries a genome segment, inverted relative to the one in the P1 wild-type genome, between two inverted copies of IS1. The inverted DNA segment spans about 23 kb of the 90 kb long P1 genome and it includes the invertible C segment. This phage is as viable as an isomeric P1 which carries the relevant segment in its original orientation. These results are discussed with regard to the genome organization of phage P1.     

Evolution of Tn21-related transposons: isolation of Tn2425, which harbours IS161

The isolation of two multi-resistance transposons, Tn2425 and Tn1831, and their relation to Tn21 and Tn2424, is described. A 1.7 kb segment present in Tn2424 and Tn2425 was identified as an IS element by rec-independent transposition, resulting in a cointegrate structure that carries two direct repeated copies of the IS element. By the isolation of this IS element we demonstrated that transposition is one mechanism leading to sequence variations in Tn21-like structures, especially in the region between the mer operon and the sul gene.     

Platelet serotonin content and uptake in spontaneously hypertensive rats

Platelet serotonin (5-HT) content and uptake were studied in male SHR and WKY at various ages. Blood was withdrawn from the carotid artery under anesthesia and 5-HT levels determined from platelet rich plasma (PRP) using a HPLC technique coupled with an electrochemical detection method. Platelet 5-HT uptake was studied by incubating PRP at 37 degrees C for 10 sec with increasing concentrations of 3H-5HT. Lineweaver- Burk plots of 3H-5HT uptake were linear suggesting simple Michaelis- Menten uptake kinetics. The SHR had more platelets than age-matched controls and consequently a higher blood circulating pool of 5-HT. Nevertheless, the 5-HT platelet levels were similar to those of their age-matched rats. The 5 week-old SHR and WKY had greater numbers of platelets and higher 5-HT platelet levels than the older rats of both strains. The affinity constants (Km) and the maximal velocities (Vmax) of platelet 5-HT uptake did not differ significantly between the 12 week- and the 6 month-old SHR and WKY. These data suggest that the SHR do not show the same impairment in platelet 5-HT metabolism as observed in essential hypertension in man.     

An electron microscopic heteroduplex study of the sequence relations between the bacteriophages LP52 and theta

Summary The genomes of the phylogenetically related but morphologically distinct bacteriophages LP52 and theta () were compared by electron microscopic heteroduplex analysis. The heteroduplex maps were aligned with known restriction maps. In the heteroduplices of LP52 DNA (63.8 kb) with the DNA of the lytic phage _c (65.9 kb) the tracts of homologous DNA cover about 50% of the genome length and are interspaced by four large and ten smaller non-base-paired regions. The largest block of non-homologous DNA (18.9 kb), represents the right-hand end and there is an unmatched piece of DNA at the left-hand end as well. Most of the heterology is due to substitution resulting in the conservation of the total length of DNA; the three insertions/deletions amount to less than 3.2% of the genome length. Heteroduplices between the DNAs of phage LP52 and the temperate phage ₁ (65.0 kb) resembled those of LP52: _c except for the absence of minor loops. Heteroduplex _c:₁ displayed about 9% heterology in seven separate loops which coincided with sections of diversity on the restriction maps; 4.8% of ₁ DNA did not hybridize with either _c or LP52 DNA.     

Crescent mastopexy and augmentation

We have defined a group of patients with a lesser degree of moderate breast ptosis whose ptosis correction is not adequately improved by augmentation alone but requires some elevation of the nipple-areola complex. We have selected the crescent excision mastopexy to provide this additional needed lift. Experience with 26 patients employing this technique has helped to define the indications and limitations for this approach. It seems to adequately provide the additional needed lift when nipple descent has been no more than 1.5 to 2 cm below the inframammary crease. Complications such as scar widening (46 percent) were reviewed, but seemed to be well tolerated by the patients.     

Molecular cloning of a pea mRNA encoding an early light induced,nuclear coded chloroplast protein

Summary cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2–4 h after onset of illumination in five day old, etiolated pea seedlings. The cDNA library was constructed from poly(A)⁺-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein(ELIP)-mRNA established the basis of our screening procedure. Colony hybridization experiments were performed with³²P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA. Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of M_r 24 000. After posttranslational importin vitro, the processed product of M_r 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization. The ELIP-mRNA reaches maximum levels within 2–4 hours after onset of illumination. Our results correspond precisely to thein vivo characteristics and indicate positive identification of the sought clones.