Glutamic acid-88 is close to SH-1 in the tertiary structure of myosin subfragment 1

The thiol-specific photoactivatable reagent benzophenone iodoacetamide (BPIA) can be selectively incorporated into the most reactive thiol, SH-1, of myosin S1, and upon photolysis, an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa region or with the central 50-kDa region [Lu et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392]. Comparison of the peptide maps of cross-linked and un-cross-linked S1 heavy chains indicates that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide [Sutoh & Lu (1987) Biochemistry 26, 4511]. In this report, S1 was labeled with radioactive BPIA, photolyzed in the absence of nucleotide, and then degraded with proteolytic enzymes. Peptides containing cross-links were isolated by liquid chromatography and subjected to amino acid sequence analyses. The results show that Glu-88 is the major site and Asp-89 and Met-92 are the minor sites involved in cross-linking with SH-1 (Cys-707) via BPIA. These residues are very near the reactive lysine residue (Lys-83) but relatively remote in the primary structure from the putative nucleotide binding region.     

Distribution of lipid-binding regions in human apolipoprotein B-100

The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.     

Termination region in rRNA genes from a eucaryotic thermophile, Thermomyces lanuginosus.

S1 mapping of the termination region in the ribosomal DNA from a thermophilic fungus, Thermomyces lanuginosus, revealed three distinct termini corresponding to the mature 25S rRNA, a precursor that is 19 nucleotides longer and corresponds to the 37S precursor in yeast cells, and a putative termination site at +96 that bears a limited sequence homology with the SalI box of mammalian cells. An estimate of the secondary structure suggested that the three termini are in close proximity, a feature that may be essential to precursor termination and maturation. The results raise questions regarding recently reported relationships between ribosomal DNA termination and spacer enhancer elements in fungi.     

Intrinsic potential for high fetal hemoglobin production in a Druze family with β-thalassemia is due to an unlinked genetic determinant

Summary The mechanism for elevated production of fetal hemoglobin (Hb F) in a Druze patient with °-thalassemia intermedia was investigated. Heterozygous family members exhibited normal Hb F levels, suggesting that the increase in -gene expression in the propositus may be partly due to anemic stress. Erythroid progenitors of these family members cultured in vitro [burst forming units (erythroid); (BFUe)] showed elevated synthesis of Hb F, indicating the existence of a genetically determined intrinsic capacity for high Hb F production in this family. The propositus was found to be homozygous for a IVS2-position 1 mutation, on the background of Mediterranean haplotype I, which is not known to be linked to high Hb F production. Moreover, extensive molecular studies of the -globin gene cluster, including sequence analysis of the promoter regions of the -globin genes, did not reveal any cisacting mechanism that could account for the high Hb F production in the propositus. A young niece of the propositus with °-thalassemia major was recently discovered, who was homozygous for the same -globin allele and haplotype as the propositus. However, unlike her uncle, she does not have a high Hb F level and presents with a severe clinical course. Her inability to produce high Hb F suggests that the genetic determinant for increased -gene expression in the propositus is unlinked to the -globin gene cluster.     

Computer-assisted grading of adenocarcinoma in prostatic aspirates

Conventional cytologic grading of fine needle aspirates of prostatic adenocarcinoma has been shown neither to be reproducible nor to correlate well with histologic grading. This study developed a tumor grade classification based on computerized cytomorphometric features and compared the results to conventional grading of companion tissue sections. The image analysis system evaluated architectural features of the aspirates (mainly cell cluster features and interrelationships) as well as nuclear features. Thirty-five prostatic adenocarcinomas (8 well, 19 moderately and 8 poorly differentiated) were evaluated. Discriminant functions based on data collected at medium and high resolution distinguished between aspirates from low-grade (well-differentiated) and high-grade (poorly differentiated) adenocarcinomas with 81% accuracy. Moderately differentiated cancers could not be classified as a distinct group. This study suggests that accurate grading of prostatic adenocarcinoma in fine needle aspirate smears requires the evaluation of medium-resolution features related to specimen cellularity and uniformity or crowding of cell clusters as well as of high-resolution features of nuclear area, perimeter and coarseness of chromatin texture. These findings are compared to those of other schemes for the cytologic grading of prostatic aspirates.     

Translational regulation of somatostatin in cultured sympathetic neurons

Coculture of sympathetic neurons with ganglion nonneuronal cells elevated levels of preprosomatostatin mRNA but did not alter neuronal synthesis, content, or release of somatostatin. Treatment of sympathetic neurons with culture medium conditioned by exposure to ganglion nonneuronal cells similarly elevated preprosomatostatin mRNA. Treatment with conditioned medium elevated somatostatin levels in pure neuronal cultures, but not in neurons cocultured with nonneuronal cells. Conditioned medium also failed to increase peptide levels in neurons cultured on a substratum of killed nonneuronal cells, despite a large increase in preprosomatostatin mRNA. These observations suggest that contact of sympathetic neurons with nonneuronal cell membranes inhibits the increase in peptide synthesis, but not the increase in preprosomatostatin mRNA after treatment with conditioned medium. Thus neuronal interactions with nonneuronal cells regulate somatostatin metabolism at both the mRNA and peptide levels. Regulatory effects on the mRNA and the peptide are separable and do not necessarily occur in parallel, and translational controls may be the rate-limiting factors.     

Ion channel expression by white matter glia: the type-1 astrocyte.

We describe the electrophysiological properties of acutely isolated type-1 astrocytes using a new "tissue print" dissociation procedure. Because the enzymes used did not destroy or modify the ion channels, and the cells retained many processes, the properties may reflect those in vivo. The types of ion channels in type-1 astrocytes changed rapidly during the first 10 postnatal days, when they attained their adult phenotype. This change was dependent on the presence of neurons. In culture, most of these channel types were not expressed, but a phenotype more typical of that in vivo could be induced by co-culture with neurons. The electrophysiological properties of astrocytes make some existing hypotheses of astrocyte function less likely.     

Diagnosis of abdominal masses with percutaneous biopsy guided by ultrasound.

OBJECTIVE--To assess the accuracy and safety of percutaneous biopsy of abdominal masses guided by ultrasound. DESIGN--Prospective study. SETTING--Combined gastroenterology service, Scarborough Hospital. PATIENTS--108 Consecutive patients identified as having a discrete mass on diagnostic ultrasound examination of the abdomen. INTERVENTION--A sample of tissue was obtained with an aseptic technique under local anaesthesia: an 18 steel wire gauge needle (Tru-Cut) was mounted in a spring loaded firing device (Biopty gun) that was advanced under simultaneous ultrasound scanning, permitting precise localisation of the target organ. MAIN OUTCOME MEASURE--Results of histological examination of tissue specimens. RESULTS--Biopsy failed in four patients. Adequate histological specimens were obtained in 104 patients with masses in the liver (31), pancreas (37), kidney (10), and adrenal glands (six) and in 20 undiagnosed abdominal and retroperitoneal masses. Follow up was until death or confirmation of the diagnosis. Three complications but no deaths occurred. Malignancy was suspected in 84 patients before biopsy. This was confirmed in 70 patients, in 26 of whom confirmation of dissemination obviated the need for further investigation. In 10 patients biopsy indicated a previously unsuspected primary tumour, and in 12 it showed only a benign lesion. Among 24 patients considered to have benign disease biopsy showed an unsuspected neoplasm in seven. Use of biopsy thus had a major effect on clinical management in 55 patients. Four false negative but no false positive diagnoses resulted from the procedure. CONCLUSION--Percutaneous biopsy of abdominal and retroperitoneal masses under ultrasound guidance is a safe and accurate method of obtaining a histological diagnosis. The results obtained have a considerable effect on clinical management.