Romanowsky dyes and Romanowsky-Giemsa effect

Summary A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18 100 cm^–1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations.Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18 100 cm^–1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions.The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly orientated dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence. Subsequently, the absorption spectra of the highly orientated dye complexes were measured with plane polarized light. We found that the transition moments, m _AB, of the bound AB cations in DNA-AB and DNA-AB-EY are orientated almost perpendicular to k, i.e. m _ABk. On the contrary, the transition moments, m _EY, of the bound EY anions in DNA-AB-EY are polarized parallel to k, i.e. m _EY k. The transition moments m _AB and m _EY lay in the direction of the long axes of the AB and EY chromophores. For that reason, in both DNA-AB and DNA-AB-EY the long molecular axes of the AB cations are orientated approximately perpendicular to the DNA chains, while the long molecular axes of the EY chromophores are polarized in the direction of the DNA chains. Therefore, in DNA-AB-EY the long axes of AB and EY are perpendicular to each other, m _ABm _EY. This molecular arrangement fully agrees with our quantitative measurements and with the theory of the absorption of plane polarized light by orientated dye complexes, which has been developed and discussed in detail.     

Pleotropic mutants from Alcaligenes eutrophus defective in the metabolism of hydrogen,nitrate, urea,and fumarate

Chromosomal mutants of Alcaligenes eutrophus unable to grow with molecular hydrogen as the energy source also failed to grow with nitrate as the terminal electron acceptor or as a nitrogen source. The mutants (Hno^–) (i) formed neither soluble nor particulate hydrogenase antigens, (ii) expressed only about 50% the wild type level of ribulosebisphosphate carboxylase activity, and (iii) transported nickel, an essential constituent of active hydrogenase, at a significantly lower rate than wild type cells. Moreover, the mutants grew very slowly with urea as nitrogen source and did not express urease. Growth on formamide was also affected and formamidase activity was induced to only a very low level. Growth of the Hno^– mutants on succinate, glutamate, fumarate, and malate was significantly slower than wild type, and a reduced rate of succinate incorporation into the mutant cells was demonstrated. The highly pleiotropic phenotype of Hno^– mutants is indicative of a chromosomal gene with a considerable physiological importance. It affected the expression of both chromosomal and megaplasmid encoded systems of energy, carbon, and nitrogen metabolism. Thus, the hno mutation restricts the metabolic versatility but does not affect the basic metabolic functions of the organism.     

Linkage disequilibrium between mutation and RFLP haplotype at the phenylalanine hydroxylase locus in the German population

Summary Restriction fragment length polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase (PAH) locus have been determined in 60 German families with PAH deficiency. Similar to the Danish population, about 90% of the mutant alleles are confined to four distinct haplotypes. There are however, differences in the frequency distributiion of these haplotypes among the mutant alleles between the two populations. Using an oligonucleotide probe for the splicing mutation associated with mutant haplotype 3 in the Danish population, a tight association between the mutation and the RFLP haplotype has also been observed in Germany. The results provide strong evidence that the splicing mutation occurred on a haplotype 3 chromosome and that the mutant allele has spread into different populations smong Caucasians.     

The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-l,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered “clear cells” dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes. With both fixation procedures, a single application of DMBA followed by one of TPA appeared to produce an increased number of ribosomes in basal keratinocytes. When hypo-osmolal fixation was used, however, treatment with DMBA/TPA did not influence the cytoplasmic volume or the numerical density of ribosomes, in dark cells. This might indicate that so-called dark keratinocytes following DMBA/TPA treatment are functionally inactive cells that appear more vulnerable than active cells to compression during hypo-osmolal fixation.     

Lipase production of Staphylococcus carnosus in a dialysis fermentor

Summary In a newly constructed one-vessel dialysis fermentor, a strain of Staphylococcus carnosus TM300 carrying the lipase secretion plasmid pLipPS1 was used to investigate exoenzyme and biomass production. The bacterial culture grows in an inner compartment of 21 volume, separated from a 101 nutrient broth compartment by a conventional dialysis membrane. In order to avoid substrate depletion and to prolong the growth phase, a highly concentrated nutrient broth was used. The biomass production reached 60 g cell dry weight/l. The increase in extracellular lipase concentration was directly coupled with the increase of cell mass and reached a value of 230 mg/l culture supernatant. Harvesting the cells in the late growth phase, the lipase content was about 30% of the total exoproteins in the supernatant.     

A genetic study of the human low-voltage electroencephalogram

Summary The studied phenotype, the low-voltage electroencephalogram (LVEEG), is characterized by the absence of an alpha rhythm from the resting EEG. In previous studies, evidence was found for a simple autosomal-dominant mode of inheritance of the LVEEG. Such a polymorphism in brain function can be used as a research model for the stepwise elucidation of the molecular mechanism involved in those aspects of neuronal activity that are reflected in the EEG. Linkage with the variable number of tandem repeats (VNTR) marker CMM6 (D20S19) and localization of an LVEEG (EEGV1) gene on 20q have previously been reported, and genetic heterogeneity has been demonstrated. This latter result has been corroborated by studing new marker (MS214). The phenotype of the LVEEG is described here in greater detail. Its main characteristic is the absence of rhythmic alpha activity, especially in occipital leads, whereas other wave forms such as beta or theta waves may be present. Analysis of 17 new families (some of them large), together with 60 previously described nuclear families, supports the genetic hypothesis of an autosomal-dominant mode of inheritance. Problems connected with the analysis of linkage heterogeneity, exclusion mapping, and the study of multipoint linkage are discussed. A possible explanation of the localization of LVEEG in the close vicinity of another gene influencing synchronization of the normal EEG, the gene for benign neonatal epilepsie, is given.     

Forces in the spider orb web

Summary Pretensile forces were measured in individual threads of intact spider webs. In the orb web of Araneus diadematus forces decrease from mooring threads to frame threads and radii, a typical ratio being 1071. The smaller number of radii in the upper than in the lower half of the orb is paralleled by force ratios of 21 to 31. A similar difference between radii built first during web construction and radii added after completion of the frame underlines the importance of the former as part of the scaffolding. High tensions in the auxiliary spiral stabilize the radii in addition to providing a pathway for the spider when inserting the sticky spiral. Radial pretension (F) changes with spider mass (m). F/m is similar for different animals indicating an adaptation of radial forces to those resulting from spider mass. Several observations suggest tension control by the spider. When forced to anchor its web to thin flexible rods tension in the threads remains in the normal range. Tension values are similar in the webs of A. diadematus, Zygiella x-notata, Nuctenea umbratica, and Nephila clavipes indicating independence from details of web geometry. Only the mooring threads of Nephila show unusually large forces suggesting a narrower working range of tensions for the catching area than for the scaffolding.     

Pathogenesis-related protein 4 is structurally homologous to the carboxy-terminal domains of hevein, Win-1 and Win-2

Summary The extracellular, acidic pathogenesis-related protein, PR-4, was purified to homogeneity from leaves of Nicotiana tabacum infected with tobacco mosaic virus (TMV) and characterized by partial amino acid sequencing. Complementary DNA clones encoding PR-4 were isolated using an oligonucleotide probe based on the sequence of one of the peptides. The deduced PR-4 protein sequence was found to be related to a family of proteins including hevein and Win-1, which have an amino-terminal lectin domain and a carboxy-terminal domain of unknown function. PR-4 is homologous to the carboxy-terminus of these proteins but does not contain the lectin domain. Thus, the organization of the PR-4 family of proteins is similar to that of the plant chitinase family, in that both contain structural subclasses characterized by the presence or absence of an amino-terminal lectin domain. This observation is consistent with the proposal that the DNA encoding the lectin domain may be capable of transposing to form new genes encoding proteins of more complex, multi-domain structure. The expression of PR-4 mRNA was found to increase dramatically in response to TMV infection and the time course of RNA accumulation was similar to that of other PR proteins.