Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.     

Collagens as multidomain proteins

The number of proteins known to contain collagen-like triple helical domains is rapidly increasing. The functions of these domains are to provide molecular rods that separate spatially non-triple helical domains with varied properties and structures and to permit lateral interactions between molecules. Two-thirds of the amino acids of the triple helical domains have their side-chains at the surface of the protein. The triple helix is also a structure that is easily predictable from the primary structure. The structure of several recently discovered collagens are discussed in terms of domains and functions. The triple helical domains have sizes varying from 33 to over 1,000 amino acid residues. The longest uninterrupted triple helices are involved in the formation of the classical quarter-staggered fibrils. Other triple helical domains permit varied molecular aggregates. A very broad spectrum of non-triple helical or globular domains are interspersed by triple helices. Only those located at the extremities of the molecules are large in size, sometimes several hundred kDa, while the domains separating 2 triple helices are small (less than 50 amino acids) and provide the molecules with hinges, proteolytic cleavage sites or other specialized functions like a glycosaminoglycan attachment site. If the assembly of the 3 chains required for the triple helix formation can be controlled in vitro, collagen-like molecules offer an as yet unexploited potential for protein engineering.     

Tissue- and species-specific promoter elements of rat gamma-crystallin genes.

The 5' flanking regions of the six rat gamma-crystallin genes (gamma A-gamma F) are all capable of conferring lens-specific expression to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene in either transdifferentiating chicken neural retina cells or mouse lens epithelial cells. Deletion mapping of the most active gamma-crystallin promoter region, the gamma D region, showed that at least three elements are required for maximal expression in mouse lens epithelial cells: element(s) located between -200 and -106, a conserved CG rich region around position -75, and a CG stretch around -15. The region between -200 and -106 was dispensable in transdifferentiating chicken neural retina cells, which instead required the region between -106 and -78. The maximal activity of the gamma E and gamma F promoters was also dependent upon the integrity of the conserved CG region located around -75. A synthetic oligonucleotide containing this sequence was capable of lens-specific enhancement of the activity of the tk promoter in transdifferentiating chicken neural retina cells but not in mouse lens epithelial cells. Our results further show that this region may contain a silencer element, active in non-lens tissues, as well.     

A new facultatively nitrite oxidizing bacterium,Nitrobacter vulgaris sp. nov.

A total of 17 facultatively lithoautotrophic strains of Nitrobacter were investigated. They all were found to be related on the species level by DNA hybridizations. The G+C content of DNA ranged between 58.9 and 59.9 mol %. The isolates originated from divers environments. The cells were 0.5–0.8×1.2–2.0 m in size and motile by one polar to subpolar flagellum. Cell-division normally occurred by budding. Polar caps of intracytoplasmic membranes as well as carboxysomes were present. The cells tended to excrete extracellular polymers forming aggregates or biofilms. Heterotrophic growth was slower than mixotrophic but often faster than litoautotrophic growth. In the presence of nitrite and organic substances the organisms often showed diphasic growth. First nitrite and then the organic material was oxidized. In the absence of oxygen growth was possible by dissimilatory nitrate reduction. Nitrite, nitric and nitrous oxide as well as ammonia were formed. Depending on growth conditions the generation times varied from 12 to 140 h. The new Nitrobacter spec. may be one of the most abundant nitrite-oxidizing bacteria in soils, fresh waters and natural as well as artificial stones. For this organism the name Nitrobacter vulgaris is proposed.The type strain is filed with the culture collection of the Institut für Allgemeine Botanik, Universität Hamburg, FRG.     

A dynamic theory of coordination of discrete movement

The concepts of pattern dynamics and their adaptation through behavioral information, developed in the context of rhythmic movement coordination, are generalized to describe discrete movements of single components and the coordination of multiple components in discrete movement. In a first step we consider only one spatial component and study the temporal order inherent in discrete movement in terms of stable, reproducible space-time relationships. The coordination of discrete movement is captured in terms of relative timing. Using an exactly solvable nonlinear oscillator as a mathematical model, we show how the timing properties of discrete movement can be described by these pattern dynamics and discuss the relation of the pattern variables to observable end-effector movement. By coupling several such component dynamics in a fashion analogous to models of rhythmic movement coordination we capture the coordination of discrete movements of two components. We find the tendency to synchronize the component movements as the discrete analogon of in-phase locking and study its breakdown when the components become too different in their dynamic properties. The concept of temporal stability leads to the prediction that remote compensatory responses occur such as the restore synchronization when one component is perturbed. This prediction can be used to test the theory. We find that the discrete analogon to antiphase locking in rhythmic movement is a tendency to move sequentially, a finding that can also be subjected to empirical test.     

Forming sparse representations by local anti-Hebbian learning

How does the brain form a useful representation of its environment? It is shown here that a layer of simple Hebbian units connected by modifiable anti-Hebbian feed-back connections can learn to code a set of patterns in such a way that statistical dependency between the elements of the representation is reduced, while information is preserved. The resulting code is sparse, which is favourable if it is to be used as input to a subsequent supervised associative layer. The operation of the network is demonstrated on two simple problems.     

Susceptibility of autologous target cells to lysis by lymphokine-activated effectors from interferon-α-treated chronic myelogenous leukaemia patients

Summary Chronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon . However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14⁺ and CD33⁺ cells but to a reduction in CD34⁺ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14⁺ and CD33⁺ as well as CD34⁺ cells but in a significant increase in CD3⁺ and CD56⁺ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

Purification of acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase from Catharanthus roseus leaves

The enzyme acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase which terminates vindoline biosynthesis has been isolated from Catharanthus roseus leaves, further characterized and purified to homogeneity by three step column chromatography and subsequent preparative isoelectric focusing. Kinetic properties concerning the enzyme reaction are discussed. Five multiple forms of the acetyl-transferase could be observed, each consisting of two subunits. This enzyme is now the best characterized of the enzymes involved in vindoline biosynthesis.Abbreviations DTE dithiothreitol - EDTA ethylenediamine-tetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid - IEF isoelectric focusing - KP_i potassium phosphate - M_r rel.molecular mass - PEG polyethylene glycol - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Testing some hypotheses on human evolution and sexual dimorphism

Research on human evolution and sexual dimorphism motivates an interesting test problem. In studying hominid phylogeny it is of interest to test whether parallel evolution plays a role. With regard to sexual dimorphism it is of interest to known whether the directions of sexual dimorphism in the populations being compared are the same. We show that testing these two problems gives rise to the same type of hypothesis testing, viz. the problem of testing the hypothesis that the means of independent, normally distributed random vectors with unit covariance matrices are situated on a straight line through the origin. A test is proposed and applied to study the sexual dimorphism of 20 recent skull populations. In this example the hypothesis of equal directions of sexual dimorphism is rejected. The classical theory of constructing multiple discriminant functions (canonical variates) is adapted to the problem of comparing sexual dimorphisms.