Automated interpretation of subcellular patterns in fluorescence microscope images for location proteomics.

Proteomics, the large scale identification and characterization of many or all proteins expressed in a given cell type, has become a major area of biological research. In addition to information on protein sequence, structure and expression levels, knowledge of a protein's subcellular location is essential to a complete understanding of its functions. Currently, subcellular location patterns are routinely determined by visual inspection of fluorescence microscope images. We review here research aimed at creating systems for automated, systematic determination of location. These employ numerical feature extraction from images, feature reduction to identify the most useful features, and various supervised learning (classification) and unsupervised learning (clustering) methods. These methods have been shown to perform significantly better than human interpretation of the same images. When coupled with technologies for tagging large numbers of proteins and high-throughput microscope systems, the computational methods reviewed here enable the new subfield of location proteomics. This subfield will make critical contributions in two related areas. First, it will provide structured, high-resolution information on location to enable Systems Biology efforts to simulate cell behavior from the gene level on up. Second, it will provide tools for Cytomics projects aimed at characterizing the behaviors of all cell types before, during, and after the onset of various diseases.     

TAXONOMIC STATUS AND GEOGRAPHICAL CRANIAL VARIATION OF COMMON DOLPHINS (DELPHINUS) IN THE EASTERN NORTH ATLANTIC

The common dolphin has a widespread distribution and is relatively abundant in the temperate to subtropical waters of the eastern North Atlantic. However, it is not known whether different species, subspecies, or populations occur in this region. We examined 393 common dolphin skulls obtained from both stranded and bycaught individuals collected between 1901 and 2005. The series included skulls of 152 females and 199 males, from animals ranging in body length from 93 to 230 cm and 105 to 244 cm, respectively. The ranges of total body length, skull size, RL/ZGW ratio and maximum upper alveolar (tooth) count of common dolphins in the eastern North Atlantic overlapped with those of both short- ( D. delphis ) and long-beaked ( D. capensis ) species found off the Californian coast. However, in the absence of additional data, the common dolphin in the eastern North Atlantic is regarded here as a large form of Delphinus delphis . Sexual dimorphism and possible sex-linked characters were identified within the sample. Results of the current study indicate some population differentiation within the eastern North Atlantic, with common dolphins off Portugal showing segregation in morphometric characteristics from common dolphins in other areas.     

External morphology of the short-beaked common dolphin, Delphinus delphis: growth, allometric relationships and sexual dimorphism

Growth, allometric relationships and sexual dimorphism are described from measurements of 105 male, 149 female and 38 unsexed specimens of short‐beaked common dolphin, Delphinus delphis, stranded along the Irish coastline (53.8% of the sample) or by‐caught in fisheries (46.2% of the sample), from 1990 to 2003. For each dolphin, 24 external body length measurements were recorded. Ages were determined for 183 dolphins by analysis of growth layer groups in the dentine. Males ranged in total body length (TBL) from 105 to 231 cm and females from 93 to 230 cm, with a maximum age of 25 years obtained for both sexes. Using a single Gompertz growth curve, asymptotic values obtained for TBL were 211.6 cm and 197.4 cm for males and females, respectively. Asymptotic lengths were attained at 11 years in males and 9 years in females. The gestation period was estimated to last approximately 11.5 months. Sexual size dimorphism (SSD) was evident, with males being significantly larger than females for 20 of the characters measured, and an SSD ratio of 1.06 was obtained. Sexual shape dimorphism was lacking, except for the presence of prominent postanal humps in mature males.     

Juvenile sockeye salmon distribution, size, condition and diet during years with warm and cool spring sea temperatures along the eastern Bering Sea shelf

Interannual variations in distribution, size, indices of feeding and condition of juvenile Bristol Bay sockeye salmon Oncorhynchus nerka collected in August to September (2000–2003) during Bering–Aleutian Salmon International Surveys were examined to test possible mechanisms influencing their early marine growth and survival. Juvenile sockeye salmon were mainly distributed within the southern region of the eastern Bering Sea, south of 57°0' N during 2000 and 2001 and farther offshore, south of 58°0' N during 2002 and 2003. In general, juvenile sockeye salmon were significantly larger ( P < 0·05) and had significantly higher indices of condition ( P < 0·05) during 2002 and 2003 than during 2000 and 2001. The feeding index was generally higher for age 1.0 year sockeye salmon than age 2.0 year during all years. Among-year comparisons suggested that Pacific sand lance Ammodytes hexapterus were important components of the juvenile sockeye salmon diet during 2000 and 2001 (20 to 50% of the mean wet mass) and age 0 year walleye pollock Theragra chalcogramma were important components during 2002 and 2003 (50 to 60% of the mean wet mass). Warmer sea temperatures during spring and summer of 2002 and 2003 probably increased productivity on the eastern Bering Sea shelf, enhancing juvenile sockeye salmon growth.     

Susceptibility to HSV-1 infection and exercise stress in female mice: role of estrogen.

Exhaustive exercise has been associated with an increased risk for upper respiratory tract infections in mice and humans. We have previously shown (Brown AS, Davis JM, Murphy AE, Carmichael MD, Ghaffer A, Mayer EP. Med Sci Sports Exerc 36: 1290-1295, 2004) that female mice are better protected from the lethal effects of herpes simplex virus type 1 (HSV-1) infection, both at rest and following exercise stress, but little is known about possible mechanisms. This study tested the effects of estrogen on HSV-1 infection and macrophage antiviral resistance following repeated exhaustive exercise. Female mice were assigned to either exercise (Ex) or control (C): intact female (I-C or I-Ex), ovariectomized female (O-C or O-Ex), or ovariectomized estrogen-supplemented female (E-C or E-Ex). Exercise consisted of treadmill running to volitional fatigue ( approximately 125 min) for 3 consecutive days. Intact female mice had a later time to death than O and E (P < 0.05) and fewer deaths than both O and E (P < 0.05). Exercise stress was associated with increased time to sickness (P < 0.05) and symptom severity at days 6 and 12-21 postinfection (P < 0.05) and decreased macrophage antiviral resistance (P < 0.001) in all groups. E had increased symptom severity at days 6 and 13-21 postinfection (P < 0.05). Results indicate that intact female mice are better protected from the lethal effects of HSV-1 infection and that exercise stress had a similar negative impact in all groups. This protective effect was lost in ovariectomized mice, but it was not reinstated by 17beta-estradiol replacement. This indicates that other ovarian factors, alone or in combination with estrogen, are responsible for the protective effects in females.     

Activation of Endogenous Antioxidant Defenses in Neuronal Cells Prevents Free Radical-Mediated Damage

Abstract: Dopamine (DA) is oxidized to the neurotoxic prooxidant species H₂O₂, OH^•, and DA quinones. We tested whether dimethyl fumarate (DMF), an electrophile shown to induce a pleiotropic antioxidant response in nonneuronal cells, could reduce the toxicity of DA metabolites in neural cells. Treatment of the N18-RE-105 neuroblastoma-retina hybridoma cell line with 30–150 µ M dopamine led to cell death within 24 h, which increased steeply with dose, decreased with higher plating density, and was blocked by the H₂O₂-metabolizing enzyme catalase. Pretreatment with DMF (30 µ M , 24 h) significantly attenuated DA and H₂O₂ toxicity (40–60%) but not that caused by the calcium ionophore ionomycin. DMF treatment also elevated total intracellular GSH and increased activities of the antioxidant enzymes quinone reductase (QR), glutathione S -transferase (GST), glutathione reductase, and the pentose phosphate enzyme glucose-6-phosphate dehydrogenase. To assess the protective efficacy of QR and GST, a stable cell line was constructed in which these enzymes were overexpressed. Cell death in the overexpressing line was not significantly different from that in a cell line expressing normal QR and GST activities, indicating that these two enzymes alone are insufficient for protection against DA toxicity. Although the relative importance of a single antioxidant enzyme such as QR or GST may be small, antioxidant inducers such as DMF may prove valuable as agents that elicit a broad-spectrum neuroprotective response.     

Effect of Bafilomycin A1 and Nocodazole on Endocytic Transport in HeLa Cells: Implications for Viral Uncoating and Infection

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used ³⁵S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713–3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.     

Broad immunocytochemical localization of the formylpeptide receptor in human organs, tissues, and cells

The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria. The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced. We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types. The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal. Liver hepatocytes and Kupffer cells are positive. Smooth muscle and endothelial cells are also generally positive. In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively. These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.     

Genotype/phenotype correlation in affected individuals of a family with a deletion of the entire coding sequence of the connexin 32 gene

X-linked Charcot-Marie-Tooth disease (CMTX) is a peripheral nerve disorder that has been linked to mutations in the connexin 32 gene (Cx32). These mutations have been shown to be genetically heterogeneous, though recurrences of specific mutations in apparently unrelated families have been seen. The majority of mutations have been shown to be missense, resulting in non-conservative amino acid changes. A few mutations resulting in a premature termination of protein translation, including both nonsense mutations as well as frameshifting microdeletions, have been documented. We would like to report a deletion mutation that appears to eliminate the entire coding sequence of the Cx32 gene, but which has been shown to segregate with a clinical phenotype not unlike that seen in individuals with a less severe alteration of the Cx32 gene. The causes at a cellular level of the CMTX phenotype are still not fully clear, though there has been speculation that these may involve a dominant negative effect where the mutant connexin 32 suppresses the function of other connexins. Studies of kindreds such as this, where in CMTX-affected males the Cx32 gene product is totally absent, will help us to better understand the molecular mechanisms underlying the clinical phenotype associated with this disorder. Received: 22 December 1997 / Accepted: 8 May 1998