The newborn rat's stress system readily habituates to repeated and prolonged maternal separation, while continuing to respond to stressors in context dependent fashion

Adrenal corticosterone secretion of newborn mice rapidly desensitizes to repeated maternal absence. The present study investigated the effects of novelty exposure, maternal care and genotype on this phenomenon.Maternal separation (MS) took place on postnatal days (pnd) 3-5. In Wistar rats, the degree of novelty in the MS-environment was varied by exposing pups to: (i) “home separation”: pups remained in the home cage; (ii) “novel separation”: pups were placed individually in a novel cage. Maternal care was recorded on pnd 1 to 4. To investigate the effect of genotype, we also examined Long Evans in the “home separation” condition. Basal and stress-induced ACTH and corticosterone levels were measured. Adrenal tyrosine hydroxylase (TH) and melanocortin receptor-2 (MCR-2) proteins served as markers for adrenal function.We show, in both rat strains, that the rise in plasma corticosterone induced by a single 8 h-MS on pnd 5 was abolished, when this separation procedure had also been performed on pnd 3 and 4. Habituation to maternal absence occurred irrespective of housing conditions. However, pups in the “home separation” condition received less maternal care upon reunion than those placed in the “novel separation”. These “home separation” pups appeared more responsive to a subsequent acute novelty-stressor, and their adrenal TH and MCR-2 were higher. Long Evans rats appeared more stress responsive than the Wistars, in the home separation condition.In conclusion, separation environment, maternal care and genotype do not affect adrenal desensitization to repeated 8 h-MS itself, but may modulate the adrenal stress-responsiveness of separated pups.     

Efficacy and safety of deep transcranial magnetic stimulation for major depression: a prospective multicenter randomized controlled trial

Major depressive disorder (MDD) is a prevalent and disabling condition, and many patients do not respond to available treatments. Deep transcranial magnetic stimulation (dTMS) is a new technology allowing non-surgical stimulation of relatively deep brain areas. This is the first double-blind randomized controlled multicenter study evaluating the efficacy and safety of dTMS in MDD. We recruited 212 MDD outpatients, aged 22–68 years, who had either failed one to four antidepressant trials or not tolerated at least two antidepressant treatments during the current episode. They were randomly assigned to monotherapy with active or sham dTMS. Twenty sessions of dTMS (18 Hz over the prefrontal cortex) were applied during 4 weeks acutely, and then biweekly for 12 weeks. Primary and secondary efficacy endpoints were the change in the Hamilton Depression Rating Scale (HDRS-21) score and response/remission rates at week 5, respectively. dTMS induced a 6.39 point improvement in HDRS-21 scores, while a 3.28 point improvement was observed in the sham group (p+0.008), resulting in a 0.76 effect size. Response and remission rates were higher in the dTMS than in the sham group (response: 38.4 vs. 21.4%, p+0.013; remission: 32.6 vs. 14.6%, p+0.005). These differences between active and sham treatment were stable during the 12-week maintenance phase. dTMS was associated with few and minor side effects apart from one seizure in a patient where a protocol violation occurred. These results suggest that dTMS constitutes a novel intervention in MDD, which is efficacious and safe in patients not responding to antidepressant medications, and whose effect remains stable over 3 months of maintenance treatment.     

The Protein Effect in the Structure of Two Ferryl-Oxo Intermediates at the Same Oxidation Level in the Heme Copper Binuclear Center of Cytochrome c Oxidase

Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O₂ activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O₂ with the mixed valence form (Cu_A²⁺, heme a³⁺, heme a₃²⁺-Cu_B¹⁺) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm⁻¹ at the peroxy oxidation level (P_M). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the P_M intermediate is also formed in the reaction of Y167F with O₂. These results suggest that in the fully reduced enzyme, the proton pumping ν_Fe(IV)=O = 804 cm⁻¹ to ν_Fe(IV)=O = 790 cm⁻¹ transition (P→F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a₃ but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a₃. The implications of these results with respect to the role of an O=Fe(IV)-His-411-H-bonded form to the ring A propionate of heme a₃-Asp-399-H₂O site and, thus, to the exit/output proton channel (H₂O) pool during the proton pumping P→F transition are discussed. We propose that the environment proximal to the heme a₃ controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.     

The origin of the FeIV = O intermediates in cytochrome aa3 oxidase

The dioxygen reduction mechanism in cytochrome oxidases relies on proton control of the electron transfer events that drive the process. Proton delivery and proton channels in the protein that are relevant to substrate reduction and proton pumping are considered, and the current status of this area is summarized. We propose a mechanism in which the coupling of the oxygen reduction chemistry to proton translocation (P → F transition) is related to the properties of two groups of highly conserved residues, namely, His411/G386-T389 and the heme a₃–propionateA–D399–H403 chain. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Cascade pattern recognition structure for improving quantitative assessment of estrogen receptor status in breast tissue carcinomas

OBJECTIVE: To develop and validate a computer-based approach for the quantitative assessment of estrogen receptor (ER) status in breast tissue specimens for breast cancer management. STUDY DESIGN: Microscopy images of 32 immunohistochemically (IHC) stained specimens of breast cancer biopsies were digitized and were primarily assessed for ER status (percentage of positively stained nuclei) by a histopathologist. A pattern recognition system was designed for automatically assessing the ER status of the IHC-stained specimens. Nuclei were automatically segmented from background by a pixel-based unsupervised clustering algorithm and were characterized as positively stained or unstained by a supervised classification algorithm. This cascade structure boosted the system's classification accuracy. RESULTS: System performance in correctly characterizing the nuclei was 95.48%. When specifying each case's ER status, system performance was statistically not significantly different to the physician's assessment (p = 0.13); when ranking each case to a particular 5-scale ER-scoring system (giving the chance of response to endocrine treatment), the system's score and the physician's score were in agreement in 29 of 32 cases. CONCLUSION: The need for reliable and operator independent ER-status estimation procedures may be served by the design of efficient pattern recognition systems to be employed as support opinion tools in clinical practice.     

Time-resolved step-scan Fourier transform infrared investigation of heme-copper oxidases: implications for O2 input and H2O/H+ output channels

We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS(2)-FTIR) spectroscopy to investigate the dynamics of the heme-Cu(B) binuclear center and the protein dynamics of mammalian aa(3), Pseudomonas stutzeri cbb(3), and caa(3) and ba(3) from Thermus thermophilus cytochrome oxidases. The implications of these results with respect to (1) the molecular motions that are general to the photodynamics of the binuclear center in heme-copper oxidases, and (2) the proton pathways located in the ring A propionate of heme a(3)-Asp372-H(2)O site that is conserved among all structurally known oxidases are discussed.     

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts

Carbohydrates have been suggested to account for some IgE cross- reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti- horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.