Galectin-1 induces nuclear translocation of endonuclease G in caspase- and cytochrome c-independent T cell death.

Galectin-1, a mammalian lectin expressed in many tissues, induces death of diverse cell types, including lymphocytes and tumor cells. The galectin-1 T cell death pathway is novel and distinct from other death pathways, including those initiated by Fas and corticosteroids. We have found that galectin-1 binding to human T cell lines triggered rapid translocation of endonuclease G from mitochondria to nuclei. However, endonuclease G nuclear translocation occurred without cytochrome c release from mitochondria, without nuclear translocation of apoptosis-inducing factor, and prior to loss of mitochondrial membrane potential. Galectin-1 treatment did not result in caspase activation, nor was death blocked by caspase inhibitors. However, galectin-1 cell death was inhibited by intracellular expression of galectin-3, and galectin-3 expression inhibited the eventual loss of mitochondrial membrane potential. Galectin-1-induced cell death proceeds via a caspase-independent pathway that involves a unique pattern of mitochondrial events, and different galectin family members can coordinately regulate susceptibility to cell death.

The Caulobacter crescentus outer membrane protein Omp58 (RsaF) is not required for paracrystalline S-layer secretion.

To identify the outer membrane protein component of the Caulobacter crescentus CB2 surface-layer export machinery we used the Serratia marcescens LipD protein to find homologs in the CB2 genome. From two homologous sequences found, one encodes a putative OMP with a predicted molecular mass of 57.5 kDa, termed Omp58 (formerly RsaF). Comparison of membrane protein profiles revealed a protein with an appropriate molecular mass present in wild-type, but not CB2 omp58::kanamycin, a mutant strain with an inactivated omp58 gene. Disruption of omp58 did not affect surface-layer production, suggesting that Omp58 is not involved in surface-layer protein secretion and, thus, may not be the outer membrane protein component of the C. crescentus surface-layer export system.

CD7 delivers a pro-apoptotic signal during galectin-1-induced T cell death.

Galectin-1, an endogenous lectin expressed in lymphoid organs and immune-privileged sites, induces death of human and murine thymocytes and T cells. Galectin-1 binds to several glycoproteins on the T cell surface, including CD7. However, the T cell surface glycoprotein receptors responsible for delivering the galectin-1 death signal have not been identified. We show that CD7 is required for galectin-1-mediated death. This demonstrates a novel function for CD7 as a death trigger and identifies galectin-1/CD7 as a new biologic death signaling pair.

Molecular analysis of hemolysin-mediated secretion of a human interleukin-6 fusion protein in Salmonella typhimurium.

Previously, we reported a plasmid-bearing Salmonella typhimurium strain capable of secreting human interleukin-6 (hIL-6) when genetically fused to the Escherichia coli hemolysin transport signal (HlyA(S)). Stationary phase culture supernatants of this strain revealed three major forms of hIL-6-HlyA(S) fusion protein (apparent molecular masses 32.4, 30.3, 27.0 kDa), at which the largest protein presumably represented full-length hIL-6-HlyA(S). The biological activity of the hIL-6-HlyA(S) protein mixture was similar to that of mature hIL-6. Accumulation of hIL-6-HlyA(S) in the culture supernatant occurred only during the initial growth phase, whereas in stationary phase and under in vitro conditions successive cleavage into the two truncated forms was observed. On the other hand, in whole cell lysates only full-length hIL-6-HlyA(S) could be detected, accounting for more than 50% of the totally synthesized protein. Upon cell fractionation, cellular hIL-6-HlyA(S) was exclusively found in the membrane fraction. These results suggest, that in S. typhimurium production and secretion of hIL-6-HlyA(S) is restricted to growing cells. A specific processing by a Salmonella-derived protease did not affect the biological activity of the fusion protein.

A Salmonella typhimurium strain genetically engineered to secrete effectively a bioactive human interleukin (hIL)-6 via the Escherichia coli hemolysin secretion apparatus.

Human interleukin-6 (hIL-6) cDNA was genetically fused with the Escherichia coli hemolysin secretorial signal (hlyA[S]) sequence in a plasmid vector. Recombinant E. coli XL-1 Blue and attenuated Salmonella typhimurium secreted a 30 kDa hIL-6-HlyA(S) fusion protein, with an additional form of higher apparent molecular mass produced by S. typhimurium. In S. typhimurium cultures hIL-6-HlyA(S) concentrations entered a plateau at 500 to 600 ng ml(-1) culture supernatant. In contrast to E. coli XL-1 Blue, in S. typhimurium culture supernatants hIL-6-HlyA(S) was accumulated faster reaching three-fold higher maximal concentrations. The cell proliferating activity of hIL-6-HlyA(S) fusion protein(s) was equivalent to that of mature recombinant hIL-6. Furthermore. hIL-6-secreting S. typhimurium were less invasive than the attenuated control strain. Therefore, the bulky hemolysin secretorial peptide at the C-terminus of the fusion protein does not markably affect hIL-6 activity, suggesting that the hemolysin secretion apparatus provides an excellent system to study immunomodulatory effects of in situ synthesized IL-6 in Salmonella vaccine strains.

The role of the 90-kDa heat-shock protein and its associated cohorts in stabilizing the heme-regulated eIF-2alpha kinase in reticulocyte lysates during heat stress.

The heme-regulated eIF-2alpha kinase (HRI) is activated not only in heme-deficient rabbit reticulocyte lysates (RRL), but also in hemin-supplemented RRL treated with heat-shock, N-ethylmaleimide (MalNEt) or heavy metal ions. We have demonstrated previously that heat-shock proteins, Hsp90, Hsp70 and FKBP52, are associated with HRI in RRL; the association of HRI with Hsp90 and FKBP52, but not Hsp70, is enhanced by hemin. To study the role of Hsp90 and its associated cohorts in the regulation of HRI, we examined the interaction of these proteins with HRI in hemin-supplemented RRLs during heat or oxidative stress. The association of HRI with Hsp90, FKBP52 and p23 was maintained in heat-, MalNEt- or Hg2(+)-treated hemin-supplemented RRL. Glycerol gradient centrifugation and gel filtration on Sephacryl S-300 indicated that neither heat shock nor MalNEt-treatment affected the apparent molecular mass of HRI in hemin supplemented RRL. In addition, active HRI was coimmunoprecipitated with 8D3 anti-Hsp90 from both heme-deficient and MalNEt-treated hemin-supplemented RRL. These results demonstrate that activation of HRI in response to heat stress and oxidative stress does not require dissociation of Hsp90 from HRI. Furthermore, HRI activity was inhibited upon addition of hemin to Hsp90-depleted heme-deficient RRL, indicating that inhibition of HRI activity by hemin is not mediated by the reassociation of Hsp90 with HRI. We also examined the dynamics of the interaction of Hsp90 with HRI. Reconstitution of the interaction of Hsp90 with HRI was stimulated by elevated temperature and required both Mg2+ and ATP. Addition of purified Hsp90 to hemin-supplemented RRL which had been treated with MalNEt to inactivate its capacity to chaperone protein renaturation, protected HRI from irreversible denaturation and aggregation upon incubation at 41 degrees C. Our results suggest that Hsp90 interacts with HRI primarily in its capacity as a molecular chaperone, stabilizing HRI from denaturation under conditions of heat stress and oxidative stress.

The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa--a review.

Pseudomonas aeruginosa (Pa) produces several surface-associated adherence factors or adhesins which promote attachment to epithelial cells and contribute to the virulence of this pathogen. Among them, the type-4 pilus accounts for about 90% of the adherence capability of Pa to human lung pneumocyte A549 cells. Furthermore, it is responsible for more than 90% of the virulence in AB.Y/SnJ mice. Pa type-4 pili display a tip-base differentiation with the adherence function located at the tip of the pilus. All Pa pili prototypes characterized so far contain an intrachain disulfide loop (DSL) of 12 to 17 semi-conserved amino acid residues at the C-terminus of pilin. In Pa, this DSL comprises the epithelial cell-binding domain. Despite little sequence homology, DSL-containing peptides of different pilin prototypes seemingly reveal striking structural similarities. Two beta-turns within the loop and the disulfide bridge impose significant structural rigidity on the DSL pilin peptide, suggesting a conformationally conserved binding domain. Insertions of C-terminal pilin peptides with disrupted DSL displayed on the surface of bacterial S-layer mediate the same receptor binding characteristics as pili, indicating that a DSL is not essential in maintaining the functionality of the binding domain. Pa pili bind specifically to the carbohydrate moiety of the glycosphingolipids (GSL) asialo-G(M1) and asialo-G(M2) and, to a much weaker extent, to lactosyl ceramide and ceramide trihexoside. The disaccharide sequence GalNAc beta(1-4)Gal, common in both asialo-G(M1) and asialo-G(M2), likely represents the minimal structural receptor motif recognized by the pili. Pa pili also bind to surface-localized proteins of human epithelial cells and other cell types, suggesting that non-sialylated GSL and (glyco)proteins function as receptors of pili. In addition to the major pilus adhesin, exoenzyme S and, as recent studies indicate, flagella, are further protein adhesins of Pa with GSL receptor binding specificities similar to those of pili.

Adverse reaction to intravenous gadoteridol.

Moderate and severe anaphylactoid reactions--while extremely rare--have been reported in association with intravenous administration of gadopentetate dimeglumine. There has been no similar experience related to use of the newly released magnetic resonance (MR) imaging contrast agent gadoteridol. The authors describe a case of vasovagal response and anaphylactoid reaction during intravenous administration of gadoteridol. MR users should be aware of the potential for adverse effects to occur in association with use of gadoteridol and be prepared by implementing appropriate observation procedures for patients receiving this as well as other MR contrast agents. In addition, physiologic monitoring devices and resuscitation equipment should be readily available in the clinical MR setting for proper treatment of patients who may experience moderate to severe adverse reactions.

Diurnal rhythms in neurotransmitter receptor binding and choline acetyltransferase activity: different patterns in two rat lines of Wistar origin.

Diurnal cycles for 8 ligand receptor pairs and choline acetyltransferase (ChAT) activity in 3 brain regions differed markedly in two rat lines, both of Wistar origin. Statistically significant differences between diurnal cycles in the two rat lines were found in the following parameters: 24 h means in 6 of 11 measurements, magnitude of cycle amplitudes, and phase position in 6 of 11 measurements, up to complete reversal of the acrophases in the case of ChAT activity in hippocampus. The importance of these findings--such major differences in two closely related rat lines--is obvious in any attempt to compare receptor-binding studies per se between laboratories using the same strain but not line, in studies of receptor rhythm characteristics, and in particular, for analysing the effects of brain-reactive drugs. While there are some reports on strain-dependency of cyclic functions, we are not aware that line-dependency has previously been described.

Radiology of large transincisural masses.

A large, potentially resectable mass that crosses the tentorial incisura may present unique problems for the neurosurgeon. Fifteen patients with large transincisural extraaxial masses were reviewed. The computed tomography and angiography findings are discussed. The importance of altering the clinician to the presence of a transincisural mass and accompanying computed tomography and angiography findings are emphasized.

Circadian variations of neurotransmitter binding in three age groups of rats.

Binding in 14 ligand membrane receptor pairs and choline acetyltransferase activity were studied at 4-hour intervals during a 24-hour cycle (12:12 light:dark). Cortex, hippocampus, cerebellum, striatum and brainstem were prepared from 3-, 12- and 24-month-old male rats. Cholinergic, alpha- and beta-adrenergic, dopaminergic, serotonergic, gabaergic and opiate binding were determined. In the aged animals, the times of the binding maxima are no longer locked to the same time of the light:dark cycle, and the cycle phases themselves are shifted in comparison to those of the young and adult group. Cycle amplitudes were smallest in the 12-month-old rats, increasing significantly in the 24-month group. The age changes in the overall (24-hour) means fall into 4 different patterns, none of which shows a linear age-related decrease. This points out the great importance of including an adult group (12 months) in all ageing studies on small mammals.

Studies on neurotransmitter binding in senile dementia. Comparison of Alzheimer's and mixed vascular-Alzheimer's dementias.

Binding of 3H-labeled agonists and antagonists to muscarinic-cholinergic, alpha- and beta-adrenergic, dopaminergic, serotoninergic, and opiate receptors was studied in four regions of the neocortex and in hippocampus, thalamus, putamen, and caudatus in autoptic material from patients with senile dementia of Alzheimer type and of mixed vascular-Alzheimer pathogenesis. Different patterns of changes in ligand binding were found for the two groups. Some of these changes were quantitatively correlated with the histological scores of plaques and neurofibrillary tangles.

Similar kinetic characteristics of 5-hydroxytryptamine binding in blood platelets and brain membranes of rats.

Blood platelets and brain membranes of SIV and Lewis rats both exhibited two saturable binding sites for 5-hydroxytryptamine (5-HT) in the concentration range 1-100 nM. The Kd values for the high-affinity sites were 4-6 nM and for the low-affinity sites 20-40 nM in both tissues of both rat strains. Blood platelets had 100-200 times more binding sites per mg protein (Bmax) than brain membranes. Thus, 5-HT receptors of platelets may be used as models for those of cerebral 5-HT-neurons.

Studies on the action of myelin basic protein (MBP) in rat brain.

The specificity of I125-MBP uptake and subcellular distribution on a discontinuous sucrose density gradient, compared to those of histone H 4 and cytochrome c, showed a high affinity of MBP for mitochondria and heavy synaptosomes, and of histone H 4 for lighter synaptosomes. One heavier synaptosomal subpopulation was almost equally labelled by both proteins. Cytochrome c showed only a low uptake into particular material. Receptor interaction studies of MBP with H3-labelled 5-hydroxytryptamine and naloxone gave negative results.

[Trends in modern international gerontological research. Experimental aspects]. / Tendenzen neuerer internationaler gerontologischer Forschung. Experimentelle Aspekte.

In a personal statement the author takes the view that gerontology and geriatrics deal with the same fundamental biological phenomenon, namely loss of adaptability and of the capacity to maintain the parameters of homeostasis. Two focal points of recent research in gerontology are cell cultures and the central nervous system. Work with cell cultures has so far been unexpectedly disappointing as far as basic understanding of the ageing process in vivo is concerned. Biochemical, morphological, physiological and pharmacological investigations into the ageing brain have on the other hand provided a wealth of new data which promise major insights into basic mechanisms of the ageing process. Aim of all gerontologocal research must be an "old age worth living" rather than a speculative search for a prolongation of lifespan.