Osmotic Fragility of Lysozyme- and Thiol-Treated Lactobacillus plantarum

Reduced but not oxidized thiols increased the sensitivity of lysozyme-treated cells of Lactobacillus plantarum to lysis by osmotic shock.     

Differential expression of several E2-type ubiquitin carrier protein genes at different developmental stages inArabidopsis thaliana andNicotiana sylvestris

We characterized three genes encoding different E2-type ubiquitin carrier proteins involved in the ubiquitin-mediated proteolytic pathway:UbcAt3 shows homologies to the yeastCDC34 gene andUbcAt4a andUbcAt4b are two different genes homologous to theUbc1/4/5 subfamily in yeast. Their accumulation was analysed and compared with that of the different families encoding polyubiquitins, as well as the monoubiquitin fusion protein, which is considered as a marker for cell division, during various developmental stages including GO/S transition and senescence of higher plant cells. Our results imply that theseUbc genes are under the control of complex mechanisms, and are differentially regulated, but not necessarily co-regulated with ubiquitin genes. Even the closely relatedUbcAt4a andUbcAt4b genes of the same multigene subfamily are controlled by distinct regulatory mechanisms.     

A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)₄ repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.     

Suitable conditions for characterization,identification, and isolation of the mRNA of the small subunit of ribulose-1,5-bisphosphate carboxylase from Nicotiana sylvestris

The products synthesized in vitro by messenger RNA (mRNA) extracted from Nicotiana sylvestris were analyzed by electrophoresis on polyacrylamide slab gels. Only three of the major polypeptides synthesized are considered here: P₅₅, P₃₂, and P₂₀. P₅₅ and P₃₂ were translated from chloroplast mRNA. P₅₅ corresponds to the large subunit of ribulose-1,5-bisphosphate (RuP₂) carboxylase; P₃₂ is probably a chloroplast membrane protein. P₂₀, the polypeptide synthesized from cytoplasmic poly(A)⁺ RNA, is the precursor of the small subunit of RuP₂ carboxylase. The balance between P₂₀ and P₃₂, in which their relative proportions varied inversely, was regulated by the age of the leaves and the time of illumination; we took advantage of this phenomenon to isolate the mRNA from the small subunit in relatively large amounts. This mRNA has a molecular weight of 350,000.Abbreviations RuP₂ ribulose-1,5-bisphosphate - mRNA messenger RNA - SDS sodium dodecyl sulfate     

Wide‐field optical property mapping and structured light imaging of the esophagus with spatial frequency domain imaging

As the incidence of esophageal adenocarcinoma continues to rise, there is a need for improved imaging technologies with contrast to abnormal esophageal tissues. To inform the design of optical technologies that meet this need, we characterize the spatial distribution of the scattering and absorption properties from 471 to 851 nm of eight resected human esophagi tissues using Spatial Frequency Domain Imaging. Histopathology was used to categorize tissue types, including normal, inflammation, fibrotic, ulceration, Barrett's Esophagus and squamous cell carcinoma. Average absorption and reduced scattering coefficients of normal tissues were 0.211 ± 0.051 and 1.20 ± 0.18 mm^?1, respectively at 471 nm, and both values decreased monotonically with increasing wavelength. Fibrotic tissue exhibited at least 68% larger scattering signal across all wavelengths, while squamous cell carcinoma exhibited a 36% decrease in scattering at 471 nm. We additionally image the esophagus with high spatial frequencies up to 0.5 mm^?1 and show strong reflectance contrast to tissue treated with radiation. Lastly, we observe that esophageal absorption and scattering values change by an average of 9.4% and 2.7% respectively over a 30 minute duration post‐resection. These results may guide system design for the diagnosis, prevention and monitoring of esophageal pathologies.      

Early energy deficit in Huntington disease: identification of a plasma biomarker traceable during disease progression

Huntington disease (HD) is a fatal neurodegenerative disorder, with no effective treatment. The pathogenic mechanisms underlying HD has not been elucidated, but weight loss, associated with chorea and cognitive decline, is a characteristic feature of the disease that is accessible to investigation. We, therefore, performed a multiparametric study exploring body weight and the mechanisms of its loss in 32 presymptomatic carriers and HD patients in the early stages of the disease, compared to 21 controls. We combined this study with a multivariate statistical analysis of plasma components quantified by proton nuclear magnetic resonance ((1)H NMR) spectroscopy. We report evidence of an early hypermetabolic state in HD. Weight loss was observed in the HD group even in presymptomatic carriers, although their caloric intake was higher than that of controls. Inflammatory processes and primary hormonal dysfunction were excluded. (1)H NMR spectroscopy on plasma did, however, distinguish HD patients at different stages of the disease and presymptomatic carriers from controls. This distinction was attributable to low levels of the branched chain amino acids (BCAA), valine, leucine and isoleucine. BCAA levels were correlated with weight loss and, importantly, with disease progression and abnormal triplet repeat expansion size in the HD1 gene. Levels of IGF1, which is regulated by BCAA, were also significantly lower in the HD group. Therefore, early weight loss in HD is associated with a systemic metabolic defect, and BCAA levels may be used as a biomarker, indicative of disease onset and early progression. The decreased plasma levels of BCAA may correspond to a critical need for Krebs cycle energy substrates in the brain that increased metabolism in the periphery is trying to provide.     

Altered dopamine and serotonin metabolism in motorically asymptomatic R6/2 mice

The pattern of cerebral dopamine (DA) abnormalities in Huntington disease (HD) is complex, as reflected by the variable clinical benefit of both DA antagonists and agonists in treating HD symptoms. In addition, little is known about serotonin metabolism despite the early occurrence of anxiety and depression in HD. Post-mortem enzymatic changes are likely to interfere with the in vivo profile of biogenic amines. Hence, in order to reliably characterize the regional and chronological profile of brain neurotransmitters in a HD mouse model, we used a microwave fixation system that preserves in vivo concentrations of dopaminergic and serotoninergic amines. DA was decreased in the striatum of R6/2 mice at 8 and 12 weeks of age while DA metabolites, 3-methoxytyramine and homovanillic acid, were already significantly reduced in 4-week-old motorically asymptomatic R6/2 mice. In the striatum, hippocampus and frontal cortex of 4, 8 and 12-week-old R6/2 mice, serotonin and its metabolite 5-hydroxyindoleacetic acid were significantly decreased in association with a decreased turnover of serotonin. In addition, automated high-resolution behavioural analyses displayed stress-like behaviours such as jumping and grooming and altered spatial learning in R6/2 mice at age 4 and 6 weeks respectively. Therefore, we describe the earliest alterations of DA and serotonin metabolism in a HD murine model. Our findings likely underpin the neuropsychological symptoms at time of disease onset in HD.     

Targeted tyrosine iodination in a multi-tyrosine vasopressin analog.

Iodination of the conserved 2-tyrosine (Tyr(2)) residue in the pressin and tocin rings of arginine- or lysine-vasopressin (AVP or LVP), and oxytocin, respectively, impairs binding to their respective receptors. Synthetic antagonists that have their Tyr(2) either replaced by another amino acid or irreversibly blocked by an O-methyl or O-ethyl ether, but have, instead, an iodinatable phenol moiety outside the pressin/tocin ring, are used for radiolabeling. We explored another approach to avoid iodinating Tyr(2) by capping this residue with a reversible O-acetyl group, incorporated during peptide synthesis. The O-acetyl-Tyr(2) LVP peptide, with a free iodinatable tyrosine attached to the epsilon-amine of 8-lysine, is iodinated at a neutral pH and purified by reverse-phase high-pressure liquid chromatography (HPLC) at an acidic pH, conditions under which the O-acetyl groups are stable. Deacetylation with hydroxylamine is selective, and leaves intact the disulfide bridge. The marked shortening of the HPLC retention time after deblocking produces a chemically homogeneous label, iodinated exclusively on the free tyrosine residue attached to the epsilon-amine of LVP. Hitherto, this (125)I labeled vasopressin agonist could be obtained only in low yield, via conjugation labeling with iodinated N-t-Boc-tyrosine succinimidyl ester. This fully reversible tyrosine protection strategy does not require special equipment, and retains the conserved Tyr(2), typical of vasopressin and oxytocin agonists.     

An anti-MUC1 antibody-calicheamicin conjugate for treatment of solid tumors. Choice of linker and overcoming drug resistance

The anti-MUC1 antibody, CTM01, has been chosen to target the potently cytotoxic calicheamicin antitumor antibiotics to solid tumors of epithelial origin that express this antigen. Earlier calicheamicin conjugates relied on the attachment of a hydrazide derivative to the oxidized carbohydrates that occur naturally on antibodies. This produced a "carbohydrate conjugate" capable of releasing active drug by hydrolysis in the lysosomes where the pH is low. Conjugates have now been made that are formed by reacting a calicheamicin derivative containing an activated ester with the lysines of antibodies. This gives an "amide conjugate" that is stable to hydrolysis, leaving the disulfide that is present in all calicheamicin conjugates as the only likely site of drug release from the conjugate. As previously shown for the carbohydrate conjugate, this amide conjugate of CTM01 produces complete regressions of xenograft tumors at doses of 300 microg/kg (calicheamicin equivalents) given three times. This indicates that hydrolytic drug release is not necessary for potent, selective cytotoxicity for calicheamicin conjugates of CTM01. Although the unconjugated calicheamicins are in general less active in cells expressing the multidrug resistance phenotype, both in vitro and in vivo results of studies reported here suggest that the efficacy of the calicheamicins toward such tumors is unexpectedly enhanced by antibody conjugation, especially for the "amide conjugate". These hydrolytically stable conjugates are also active toward cisplatin-resistant ovarian carcinoma cells as well. Such studies indicate that the calicheamicin amide conjugate of CTM01 may have potential for the treatment of MUC1-positive solid tumors, including some types of resistant tumors.