Alpha-1-antitrypsin deficiency and juvenile liver disease Ultrastructural observations compared with light microscopy and routine liver tests

Sixteen children (aged between 1 month and 20 years) with alpha-1-antitrypsin deficiency (PiZ) were investigated by liver biopsy on one or more occasions. Eight patients had suffered from neonatal cholestasis, and two of them were investigated during the cholestatic period as well. The clinical status and liver function tests were compared with the light and electron microscopical findings. According to the light microscopical analyses at the latest investigation, the cholestatic and noncholestatic patients were classified as healthy, fibrotic or cirrhotic cases. All livers displayed periodic acid-Schiff positive, diastase-resistant globules in some but not all periportally located hepatocytes. By electron microscopy accumulation of retained secretory material was found in all PiZ patients. This accumulation was most conspicuous in the smooth endoplasmic reticulum of hepatocytes. alpha-1-antitrypsin deficiency seems to affect some, but not all hepatocytes. In the affected cells disappearance or hypotrophy of the Golgi complex could be observed. The intracellular transport of very low density lipoproteins (VLDL) was apparently not affected. The migration block in alpha-1-antitrypsin deficiency seems to occur before transportation to the Golgi complex. The extent of the involvement was not strictly age-dependent. There was no ultrastructural evidence of subclinical cholestasis as a possible triggering factor in the development of cirrhosis.     

Separation of urine proteins on the anion-exchange resin mono QTM

Proteins excreted in urine due to renal failure were separated on Mono Q^TM, a new strong anion exchanger designed for fast high-resolution protein separations. The separation procedure was divided into two steps. The first step involved removal of low-molecular- weight substances by rapid desalting on a Sephadex G-25 Superfine column. In the second step, the total protein fraction (3–6 ml) was loaded onto the Mono Q column with the aid of a superloop. The proteins were adsorbed onto the top of the ion-exchanger column and gradually displaced by a combined pH and salt gradient in 40 min. The choice of ion exchanger and initial operating conditions were based on data obtained from electrophoretic titration curve experiments. Identification of separated proteins was achieved by fused rocket electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively.     

Rat cathepsin H-catalyzed transacylation: comparisons of the mechanism and the specificity with papain-superfamily proteases.

We found that rat cathepsin H showed strong transacylation activity under physiological conditions. It is a feature of cathepsin H to utilize amino acid amides not only as acyl-acceptors but also as acyl-donors in the reaction. The pH-dependence of the transacylation activity was distinct from those of other papain-superfamily proteases. The alkaline limb (pKapp = 7.5) could be regarded as the pKa of the alpha-amino group of the acyl-donor, which was also involved in the original amino-peptidase activity. The acidic limb (pKapp = 5.8) was suggested to be involved in the deacylation step, where amino acid amide attacked the acyl-intermediate as a nucleophile in place of water in the hydrolysis. Although the N alpha-deprotonated acyl-acceptor, which is supposed to govern the nucleophilic attack, has a small population in the acidic pH range (above pH5), the transacylation was detectable even at the acidic pH-range because of the high S1'-site binding ability and suitable nucleophilicity of the acyl-acceptor. In the transacylation between various amino acid amides, the S1 and S1' site appeared to prefer hydrophobic residues without and regardless of a branch at beta-carbon, respectively. From these results and the sequence homology in the papain superfamily, we concluded that the reaction was governed by the acyl-donor having a protonated amino group, the acyl-acceptor having a deprotonated amino group and the remarkable hydrophobic character (especially favoring tryptophan amide) of the S1' site, presumably reflecting the good conservation of Trp177 in papain-superfamily proteases.     

Structure of adsorbed fibrinogen obtained by scanning force microscopy

It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three-dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces; a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM-results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule.     

The organization of the gene for the human cerebroside sulfate activator protein

The organization of 14 exons covering 97% of the cDNA sequence of human cerebroside sulfate activator protein precursor has been determined from two overlapping EMBL-4 human genomic clones extending over 17kb. All exons and exon/intron splice junctions and five introns were sequenced. Exon 8 consists of only 9 bp and is involved in alternative splicing which generates three different mRNAs of cerebroside sulfate activator precursor.     

Suppression of the development of uterine adenomyosis by danazol treatment in mice.

Inhibitory effects of danazol, an isoxazol derivative of synthetic steroid 17 alpha-ethinyl-testosterone, on the development of uterine adenomyosis, a pathological disorder of endometrial tissue defined as the presence of endometrial glands and stroma in the myometrium, were investigated in mice of SHN strain. Mice treated with 0.5 microgram danazol for 5 weeks during 4-9 weeks of age and killed at 21 weeks of age showed significantly lower incidence of the spontaneous development of adenomyosis than the age-matched intact control mice. The inhibitory effects of danazol were also evident in mice bearing pituitary isografts which were effective in inducing an early and a high incidence of adenomyosis. Furthermore, the treatment with danazol resulted in the decrease of serum levels of luteinizing hormone (LH) and prolactin (PRL) associated with hypofunction of ovaries and persistent diestrus. These results support the usefulness of danazol for the clinical treatment of gynecological disorders except for hypofunction of ovaries.