Analogues of tentoxin: Tools for mechanistic investigations

Tentoxin[cyclo-(MeAla¹-Leu²-MePhe³-Gly⁴] is a metabolite isolated from a phytopathogenic fungusAlternaria alternata, which induces chlorosis of many plants. This potentialnatural herbicide binds specifically to the soluble part CF₁of the chloroplastic coupling factor, which is a proton ATP-synthase. Theeffect of the toxin is concentration dependent: at low concentration it is apowerful inhibitor, while at higher concentration, it stimulates the enzyme.We synthesized tentoxin and we designed new analogues in order to vary thehydrophobicity on the side chain and to study the structure activityrelationships. Comparative activities suggest that it is possible toseparate inhibitory and activating effects using tentoxin analogues, showingsome evidence for the existence of two binding sites with different affinityconstant.     

Proline 21, a residue within the α-helical domain of ΦX174 lysis protein E, is required for its function in Escherichia coli

ΦX174 lysis protein E-mediated lysis of Escherichia coli is characterized by a protein E-specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure. In order to understand the fusion process, the topology of protein E within the envelope complex of E. coli was investigated. Proteinase K protection studies showed that, during the time course of protein E-mediated lysis process, more of the fusion protein E-FXa-streptavidin gradually became accessible to the protease at the cell surface. These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C-terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas. The initiation mechanism for such a conformational change could be the cis–trans isomerization of proline residues within α-helical membrane-spanning segments. Conversion of proline 21, presumed to be in the membrane-embedded α-helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis-negative E mutant proteins. Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C-terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E. coli . Oligomerization of protein P21G-StrpA was not disturbed.     

NMDA Receptor Heterogeneity During Postnatal Development of the Rat Brain: Differential Expression of the NR2A, NR2B, and NR2C Subunit Proteins

Abstract: Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunit-specific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area-specific rearrangement of NR2B subunit expression following birth.     

p53-Knockout Mice Are Protected Against the Long-Term Effects of Methamphetamine on Dopaminergic Terminals and Cell Bodies

Abstract: p53-knockout mice provide a useful model to test the role of p53 in the neurotoxic effects of drugs in vivo. To test the involvement of p53 in methamphetamine (METH)-induced toxicity, wild-type mice, as well as heterozygous and homozygous p53-knockout male mice, were administered four injections of three different doses (2.5, 5.0, and 10.0 mg/kg) of the drug given at 2-h intervals within the space of 1 day. METH caused a marked dose-dependent loss of dopamine transporters in both the striatum and the nucleus accumbens of wild-type mice killed 2 weeks after drug administration. However, this METH-induced decrease in dopamine transporters was attenuated in both homozygous and heterozygous p53-knockout mice, with homozygous animals showing significantly greater protection. The possibility for p53 involvement in METH-induced toxicity was also supported by the observation that METH caused marked increases in p53-like immunoreactivity in the striata of wild-type mice and very little change in heterozygous p53-knockout mice, whereas no p53-like immunostaining was detected in the homozygous p53-knockout mice. Further support for p53 involvement was provided by the fact that METH treatment caused significant decreases in dopamine transporter mRNA and the number of tyrosine hydroxylase-positive cells in the substantia nigra pars compacta and the ventral tegmental area of wild-type but not homozygous p53-knockout mice killed 2 weeks after cessation of METH administration. These results provide concordant evidence for a role of the tumor suppressor, p53, in the long-term deleterious effects of a drug acting on brain dopamine systems.     

Ventilation and metabolism among rat strains

Strohl, Kingman P., Agnes J. Thomas, Pamela St. Jean, EvelynH. Schlenker, Richard J. Koletsky, and Nicholas J. Schork. Ventilation and metabolism among rat strains. J. Appl. Physiol. 82(1): 317-323, 1997.We examinedventilation and metabolism in four rat strains with variation in traitsfor body weight and/or blood pressure regulation.Sprague-Dawley [SD; 8 males (M), 8 females (F)], BrownNorway (BN; 10 M, 11 F), and Zucker (Z; 11 M, 12 F) rats were comparedwith Koletsky (K; 11 M, 11 F) rats. With the use of noninvasiveplethysmography, frequency, tidal volume, minute ventilation(E),O₂ consumption, andCO₂ production were derived atrest during normoxia (room air) and during the 5th minute of exposureto each of the following: hyperoxia (100% O₂), hypoxia (10%O₂-balanceN₂), and hypercapnia (7%CO₂-balance O₂). Statistical methods probedfor strain and sex effects, with covariant analysis by body weight,length, and body mass. During resting breathing, strain effects werefound with respect to both frequency (BN, Z > K, SD) and tidal volume(SD > BN, Z) but not to E. Sexinfluenced frequency (F > M) alone. Z rats had higher values forO₂ consumption,CO₂ production, and respiratoryquotient than the other three strains, with no independent effect bysex. During hyperoxia, frequency was greater in BN and Z than in SD orK rats; SD rats had a larger tidal volume than BN or Z rats; Z rats hada greater E than K rats; and M had alarger tidal volume than F. Strain differences persisted duringhypercapnia, with Z rats exhibiting the highest frequency andE values. During hypoxic exposure,strain effects were found to influenceE (SD > K, Z), frequency (BN > K), and tidal volume (SD > BN, K, Z). Body mass was only amodest predictor of E during normoxia, of both E and tidal volume withhypoxia, hypercapnia, or hyperoxia, and of frequency duringhypercapnia. We conclude that strain of rats, more than their body massor sex, has major and different influences on metabolism, the patternand level of ventilation during air breathing, and ventilation duringacute exposure to hypercapnia or hypoxia.

     

Localization of the CDKN4/p27Kip1 gene to human chromosome 12p12.3

CDKN4/p27Kip1 is a cyclin-dependent kinase (Cdk) inhibitor implicated in G1 phase arrest, which negatively regulates G1 phase progression in response to TGF, and might represent a tumor suppressor gene. We report here the chromosomal assignment of the human CDKN4 gene to chromosome 12p12.3 in close proximity to highly polymorphic microsatellite markers.     

Sperm nuclei analysis of a Robertsonian t(14q21q) carrier,by FISH,using three plasmids and two YAC probes

The meiotic segregation of chromosomes 14 and 21 was analysed in 1116 spermatozoa from an oligoasthenospermic carrier of a Robsertsonian translocation t(14q21q), and in 16 392 spermatozoa from a control donor, using two-colour fluorescence in situ hybridisation (FISH). Two YAC probes (cloned in yeast artificial chromosomes) specific for regions on the long arms of these chromosomes were co-hybridised. Of the spermatozoa, 12% were unbalanced, resulting from adjacent segregations. Chromosomes X, Y and 1 were also simultaneously detected in 1335 spermatozoa from the same carrier. Whereas gonosomal disomy rates were not significantly different from those of the control donors, disomy 1 were slightly but significantly increased to 0.7%. The diploidy rate was also slightly increased to approximately 1% in the translocation carrier.