Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos

The long-known phenomenon of karyomere (chromosome vesicle) formation at early telophase of the nuclear cycle during early embryogenesis of a wide range of organisms including amphibians (Rubaschkin 1905; for review, see Richards 1917) was investigated in the early cleavage cycles of Xenopus laevis embryos before the mid blastula transition. Embryos were fixed and Epon embedded at successive time intervals and consecutive thick (3 m) and ultrathin sections cut. Using conventional light microscopy at low magnification as well as phase and/or interference contrast video microscopy at high magnification, a substantial amount of information could be obtained from the analysis of optical sections in thick-sectioned material. In addition, details of the ultrastructural organization could be analysed from corresponding ultrathin sections by electron microscopy. The light microscopic analysis of serial thick sections allowed precise determination of the arrangement and sizes of telophase karyomere structures during the embryonic nuclear division cycle. It was found that small, widely spaced 1st order karyomeres fuse to larger (2nd order) karyomeres which then progressively exhibit lateral fusion of neighbouring karyomeres. The final coalescence of adjacent karyomeres marks the onset of the reorganization of the typical interphase nuclear structure. The data are discussed with regard to the occurrence of karyomeres during the embryonic nuclear cycle of arthropods, dipteran insects, and echinoderms as well as recent progress in the use of Xenopus egg extracts for in vitro assembly of nuclear structures around protein-free DNA.     

Simultaneous binding of calcium and vanadate to the Ca2+-ATPase of sarcoplasmic reticulum

The interaction of vanadate with the Ca2+-ATPase of sarcoplasmic reticulum vesicles has been studied by making use of the ATPase activity as a measure of uncomplexed enzyme. The binding/dissociation is slow, so that initial rates can be used to study the equilibrium binding. The results indicate that in addition to a Ca2+-free complex E.Van (KV = 0.4 microM), there must also be a Ca2+-enzyme-vanadate complex (K'V = 7 microM). This observation is confirmed by the difference between the kinetics of decay of activity on vanadate addition, and on addition of ATP to enzyme preincubated with vanadate and Ca2+, which requires two enzyme-vanadate complexes. ATP increases the apparent affinity of the enzyme for vanadate by inducing calcium release. Upper limits for the kinetic parameters for vanadate binding and dissociation are estimated.     

GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae.

The gene encoding the galactose permease of Saccharomyces cerevisiae (GAL2) was cloned. The clone restores galactose permease activity to gal2 yeasts and is regulated by galactose in a manner similar to other GAL gene products (GAL1, -7, and -10). Experiments with temperature-conditional secretory mutants indicated that transport of the GAL2 gene product to the cell surface requires a functional secretory pathway. In addition, gene fusions were constructed between the GAL2 gene and the Escherichia coli lacZ gene. The GAL2-lacZ gene fusions code for galactose-regulated beta-galactosidase activity in yeasts. The beta-galactosidase activity was found to be membrane bound.     

Properties of strange attractors in yeast glycolysis

The properties of periodic and aperiodic glycolytic oscillations observed in yeast extracts under sinusoidal glucose input were analyzed by the following methods. (1) Spectral analysis, rendering sharp peaks for periodic responses and enhanced broad-band noise for aperiodic oscillations. (2) Phase plane analysis, leading to closed and to open trajectories for periodic and aperiodic oscillations, respectively. (3) Rotation of a phase plane proportionally to time, revealing strange attractors associated with the aperiodic oscillations. (4) Stroboscopic plot on the phase plane, showing that the strange attractors follow a stretch-fold-press process, if the stroboscoping phase is varied. (5) Stroboscopic transfer plot, admitting a period of three transfer processes and thus implying chaos according to the Li-Yorke theorem. (6) Determination of the rate of information production by differentiation of the transfer plot, yielding approx. 0.21 bits per min for the chaotically glycolyzing yeast extract.     

Distribution of chlorpromazine and imipramine in adipose and other tissues of rats

Adipose tissue kinetics of chlorpromazine and imipramine, two drugs which are more lipophilic than thiopental, were studied in the rat. After single i.v. doses, the time-course of drug distribution was followed in adipose and various other tissues, until their concentrations in adipose tissues declined. Under these conditions the two drugs behaved almost identically. Among the tissues analyzed, the lowest concentrations were found in adipose tissue, with the exception of plasma. At its maximum concentration after about 30 minutes, total adipose tissue contained only 3 % of the dose of administered drugs. Adipose/plasma and adipose/lung concentration ratios were 2–5 and 0.05, respectively.After maximum tolerated oral doses of imipramine for 3 weeks, similar steady state concentration ratios (plasma:adipose:brain:lung 1:3:12:96) were observed. In adipose tissue the imipramine/desmethylimipramine ratio was about 1, and the desmethylimipramine steady state levels did not increase with time. Literature data indicate that many basic lipophilic drugs are not stored in adipose tissue. This is now clearly shown for chlorpromazine and imipramine, even under extreme, subchronic conditions in the case of imipramine.     

Increased lymphocyte adherence to human arterial endothelial cell monolayers in the context of allorecognition.

The interactions of alloreactive T lymphocytes with the vascular endothelium were studied in an in vitro model of lymphocyte adherence to cultured human arterial endothelial cell (HAEC) monolayers. Donor-primed lymphocytes (DPL) were shown to have significantly greater adherence to donor HAEC than were third-party primed lymphocytes. Limiting dilution analysis of adherent DPL showed an enrichment of donor-reactive lymphocytes compared with nonadherent DPL. This study examines the allospecific nature of this increased lymphocyte adherence. HAEC constitutively express class I HLA Ag and can be induced by IFN-gamma to express class II Ag. DPL adherence to class I+ HAEC was inhibited only in the presence of mAb directed against class I Ag. DPL adherence to class I+ and class II+ HAEC was inhibited in the presence of mAb directed against class I and class II Ag. Class I- and class II-specific adherence was also shown to involve CD8 and CD4 molecules, respectively, whereas lymphocyte function-associated Ag do not appear to play a major role in long term alloreactive lymphocyte adherence to HAEC. These findings suggest that alloreactive lymphocyte adherence to HAEC is mediated by two mechanisms. One is based on allorecognition, primarily of HLA Ag, and the other is related to presumably non-Ag-specific interactions between activated lymphocytes and the vascular endothelium. The studies presented provide evidence to suggest that HLA-specific lymphocyte adherence to endothelium may significantly contribute to the development of alloreactive lymphocyte infiltrates within the allograft.     

Cell specificity of granzyme gene expression

Granzymes are serine proteases present in secretory granules of cytolytic T lymphocyte lines. We have studied the expression of the granzyme family (granzyme A, B, C, D, E, F, and G) in different lymphoid cell populations and cell lines as well as in nonlymphoid cells and tissues. Our data show that with few exceptions expression of granzyme genes is restricted to T cells and their thymic precursors. In mature T cells granzymes are expressed only upon activation. The same is true for thymocytes, with the exception of grazyme A that is expressed also in non-stimulated cells. In T cells and thymocytes the distribution of mRNAs coding for different granzymes depends on the subpopulation tested and the activation protocol. Highly cytolytic PEL express granzymes A and B but none of the other granzymes.     

Sequence and structure of the yeast galactose transporter.

The previously cloned GAL2 gene of the Saccharomyces cerevisiae galactose transporter has been sequenced. The nucleotide sequence predicts a protein with 574 amino acids (Mr, 63,789). Hydropathy plots suggest that there are 12 membrane-spanning segments. The galactose transporter shows both sequence and structural homology with a superfamily of sugar transporters which includes the human HepG2-erythrocyte and fetal muscle glucose transporters, the rat brain and liver glucose transporters, the Escherichia coli xylose and arabinose permeases, and the S. cerevisiae glucose, maltose, and galactose transporters. Sequence and structural motifs at the N-terminal and C-terminal regions of the proteins support the view that the genes of this superfamily arose by duplication of a common ancestral gene. In addition to the sequence homology and the presence of the 12 membrane-spanning segments, the members of the superfamily show characteristic lengths and distributions of the charged, hydrophilic connecting loops. There is indirect evidence that the transporter is an N-glycoprotein. However, its only N-glycosylation site occurs in a charged, hydrophilic segment. This could mean that this segment is part of a hydrophilic channel in the membrane. The transporter has a substrate site for the cyclic AMP-dependent protein kinase which may be a target of catabolite inactivation. The transporter lacks a strong sequence enriched for proline (P), glutamate (E), aspartate, serine (S), and threonine (T) and flanked by basic amino acids (PEST sequence) even though it has a short half-life. Mechanisms for converting the poor PEST to a possible PEST sequence are considered. Like the other members of the superfamily, the galactose transporter lacks a signal sequence.     

Cysteine: Depolarization-Induced Release from Rat Brain In Vitro

Compounds released on depolarization in a Ca2+-dependent manner from rat brain slices were screened to identify candidates for neuroactive substances. Lyophilized superfusates were analyzed by reversed-phase HPLC after derivatization with 9-fluorenyl N-succinimidyl carbonate. One of the compounds that showed an increase of concentration in superfusates in the presence of iodoacetamide was identified as the cysteine (Cys) derivative, S-carboxamidomethylcysteine, by fast atom bombardment mass spectrometry and other methods. This stable Cys derivative originates from endogenous, extracellular Cys. The finding led to a method for quantification of Cys in superfusates by immediate cooling of the superfusates to 0 degrees C and reaction of Cys with N-ethylmaleimide. Depolarization-induced Ca2+-dependent release of Cys was most prominent in the neocortex, followed by the mesodiencephalon, striatum, and cerebellum. This suggests that Cys is released from a neuronal compartment and might be involved in neurotransmission.     

d-Ribulokinase from Klebsiella pneumoniae for continuous production of d-(−)-ribulose-5-phosphate

Summary The production of d-ribulose-5-phosphate in an enzyme membrane reactor was examined. Phosphoryl transfer from ATP to d-ribulose was catalysed by d-ribulokinase isolated from Klebsiella pneumoniae. For production of d-ribulose-5-phosphate the phosphoryl donor ATP was used either in stoichiometric or in catalytic amounts. Using catalytic amounts of ATP requires a second enzyme, e.g. pyruvate kinase, to regenerate ATP. The kinetic parameters for d-ribulokinase and pyruvate kinase were determined to calculate the performance of an enzyme membrane reactor for continuous production of d-ribulose-5-phosphate. Both processes operated for more than 200 h. Regardless of whether ATP was used in catalytic or stoichiometric amounts, about the same production parameters were determined. In continuous production space/time yields of 117 g (with ATP regeneration) and 103 g (without ATP regeneration) of d-ribulose-5-phosphate 1^–1 per day were reached.Offprint requests to: D. Gygax