A new histological embedding method by low-temperature polymerisation of methyl methacrylate allowing immuno- and enzyme histochemical studies on semi-thin sections of undecalcified bone marrow biopsies

Summary A new procedure of embedding in methyl methacrylate (MMA) is introduced, which enables immunostaining by preservation of cellular epitopes. This could be achieved by reduction of polymerisation temperature from ca. 60° C to 22° C within the core of tissue blocks. Reduction of the polymerisation temperature is due to destabilisation of acrylate monomer, reduction of catalyst, exclusion of molecular oxygen, chemical initiation and reduction of environmental temperature. This results in good preservation of antigens and enzymes in the haematopoietic and lymphatic tissue of bone marrow as well as lymphoid, epithelial and mesenchymal markers in other tissues, comparable to paraffin embedding. Results are demonstrated by application of monoclonal and polyclonal antibodies and by demonstration of enzyme activity conventionally used in haematology.     

Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain.

To determine the mechanisms by which multiple forms of fibroblast growth factor (FGF) receptors are generated, we have mapped the arrangement of exons and introns in the human FGF receptor 1 (FGFR 1) gene (flg). We found three alternative exons encoding a portion of the third immunoglobulin (Ig)-like domain of the receptor. One of these alternatives encodes a sequence that is part of a secreted form of FGFR 1. The other two encode sequences that are likely part of transmembrane forms of FGFR 1. One of these forms has not been previously reported in published cDNAs. Also, we have determined the structural organization of a portion of the human FGFR 2 gene (bek) and found a similar arrangement of alternative exons for the third Ig-like domain. The arrangement of these genes suggests that there are conserved mechanisms governing the expression of secreted FGF receptors as well as the expression of at least two distinct membrane-spanning forms of the FGF receptors. The diverse forms appear to be generated by alternative splicing of mRNA and selective use of polyadenylation signals.     

Mapping sequenced E.coli genes by computer: software, strategies and examples.

Methods are presented for organizing and integrating DNA sequence data, restriction maps, and genetic maps for the same organism but from a variety of sources (databases, publications, personal communications). Proper software tools are essential for successful organization of such diverse data into an ordered, cohesive body of information, and a suite of novel software to support this endeavor is described. Though these tools automate much of the task, a variety of strategies is needed to cope with recalcitrant cases. We describe such strategies and illustrate their application with numerous examples. These strategies have allowed us to order, analyze, and display over one megabase of E. coli DNA sequence information. The integration task often exposes inconsistencies in the available data, perhaps caused by strain polymorphisms or human oversight, necessitating the application of sound biological judgment. The examples illustrate both the level of expertise required of the database curator and the knowledge gained as apparent inconsistencies are resolved. The software and mapping methods are applicable to the study of any genome for which a high resolution restriction map is available. They were developed to support a weakly coordinated sequencing effort involving many laboratories, but would also be useful for highly orchestrated sequencing projects.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

Ribotyping of 16S and 23S rRNA genes and organization of rrn operons in members of the bacterial genera Gemmata,Planctomyces, Thermotoga,Thermus, and Verrucomicrobium

The number of organization of rrn genes of two members of the order Planctomycetales, Planctomyces limnophilus and Gemmata obscuriglobus, as well as three species from other bacterial phyla, namely Thermotoga maritima, Thermus aquaticus and Verrucomicrobium spinosum were examined by Southern blot hybridization analysis of restricted DNA with labeled 16S- and 23S rRNAs. Ribotyping analysis revealed that two species contain unlinked 16S- and 23S rRNA genes. Planctomyces limnophilus possessed two unlinked rrn genes which were separated from each other by at least 4.3 kb, and Thermus aquaticus had to unlinked 16S and 23S rRNA genes, separated from each other by at least 2.5 kb. Gemmata obscuriglobus exhibited five genes for which the organization could as yet not be determined because of the complex hybridization patterns. In the other two species, rrn genes clustered in operons. Thermotoga maritima had a single gene for each rRNA species which were separated by not more than 1.5 kb, while Verrucomicrobium spinosum had four copies of probably linked 16S and 23S rRNA genes with a maximal distance between 16S and 23S rRNA genes of 1.3 kb.     

"In vitro" study on release of cyclic AMP and thyroid hormone in autonomously functioning thyroid nodules

The TSH effect on slice and the incubation medium cyclic AMP levels and T3 and T4 released from 8 autonomously functioning thyroid nodules (AFTN) and their respective perinodular (PN) tissues were examined. The thyroid slices were incubated in Eagle's Medium containing TSH (5 to 100 mU/ml) for 60 min and 300 min for tissue cyclic AMP generation and for cyclic AMP, T3 and T4 release, respectively. Basal cyclic AMP levels were not different either in AFTN and in PN slices or into the incubation medium. In both tissues TSH produced a similar cyclic AMP generation. In contrast, cyclic AMP released into the incubation medium was significantly higher in AFTN than in PN tissues, after TSH stimulation. Basal T3 values and TSH-stimulated T3 release in AFTN were not different from PN tissue. However, basal T4 levels were significantly higher in AFTN than in PN tissue as well as T4 released in response to TSH. In addition, T3/T4 ratio was lower in AFTN than in PN tissues. The cyclic AMP released into the incubation medium correlated with both T3 and T4 release in PN tissue but in the AFTN tissue no correlations were found. These findings suggest that the adenylate cyclase-cyclic AMP system is more sensitive to TSH-stimulation in AFTN when compared with PN tissue and that AFTN tissue has a preferential T4 secretion.     

Sequence of a full-length cDNA for rat lung beta-galactoside-binding protein: primary and secondary structure of the lectin

A full-length cDNA for rat lung beta-galactoside lectin (subunit Mr approximately 14,000, lectin 14K) was cloned and the nucleotide sequence determined. The deduced amino acid sequence agrees with the amino acid composition and direct amino acid sequence analysis of purified rat lung lectin peptides. We found that the amino-terminal alanine is blocked with an acetyl group. Comparison of the amino acid sequence with other proteins shows a high degree of homology only with other vertebrate lectin sequences, supporting the suggestion that these lectins may constitute a unique class of vertebrate proteins. The amino acid composition and sequence of lectin peptides, the sequence of lectin cDNA, and isoelectric focusing of purified lectin indicate that rat lung lectin 14K is composed predominantly of a single protein. In addition, rat uterus lectin 14K was found to be the same protein as that present in lung. We characterized the secondary and tertiary structure of rat lung lectin 14K by circular dichroism, by analytical ultracentrifugation, and by computer analysis of its primary structure. Results of these experiments suggest that lectin 14K is primarily a hydrophilic protein with an asymmetric, elongated structure consisting of approximately equal amounts of alpha helix, beta sheet, beta turn, and random coil. We found that Cys-2 and Cys-130 react most rapidly with iodoacetamide; one or both of these residues may be primarily responsible for the thiol requirement of lectin activity.     

Growth hormone releasing factor-like immunoreactivity in human milk

The presence of immunoreactive growth hormone-releasing factor (GRF) in human milk has been demonstrated. By using sequential high performance liquid chromatography, it has been shown that most of the immunoreactivity co-elutes with the synthetic, hypothalamic-like, GRF (1-40). The concentrations of GRF detected (between 152 and 432 pg GRF/ml milk) exceed several fold its values in plasma.     

Downregulation of cell-to-cell communication by the viralsrc gene is blocked by TMB-8 and recovery of communication is blocked by vanadate

Summary The viralsrc gene downregulates junctional communication, closing cell-to-cell membrane channels presumably by way of the phosphoinositide signal route. We show that TMB-8 [8-N, N-(diethylamino) octyl-3,4,5-trimethoxybenzoate] counteracts this downregulation in cells transformed by temperature-sensitive mutant Rous sarcoma virus: TMB-8 (36–72 m) raises junctional permeability when applied during activity ofsrc protein kinase, i.e., at steady permissive temperature; and TMB-8 inhibits the fall of junctional permeability, when the activity ofsrc protein kinase gets turned on. TMB-8 also (reversibly) inhibits the growth of the cells at permissive temperature and reverses the morphological changes associated with transformation. The morphological reversal lags several hours behind the junctional-permeability reversal. Communication recovers within a few minutes when the activity of thesrc protein kinase is turned off (in absence of TMB-8). Sodium orthovanadate (20 m) prevents this recovery, but it has no major effect on junctional permeability on its own. We discuss possible modes of action of these agents on critical stages of the signal route, related to intracellular Ca²⁺ and protein kinase C.