Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling

Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle (pk) and strabismus (stbm) are thought to antagonize Fz signaling. We show that they act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. We demonstrate that Stbm and Pk interact physically and that Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus our data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling.     

The Mre11 complex is required for ATM activation and the G2/M checkpoint

The maintenance of genome integrity requires a rapid and specific response to many types of DNA damage. The conserved and related PI3-like protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate signal transduction pathways in response to genomic insults, such as DNA double-strand breaks (DSBs). It is unclear which proteins recognize DSBs and activate these pathways, but the Mre11/Rad50/NBS1 complex has been suggested to act as a damage sensor. Here we show that infection with an adenovirus lacking the E4 region also induces a cellular DNA damage response, with activation of ATM and ATR. Wild-type virus blocks this signaling through degradation of the Mre11 complex by the viral E1b55K/E4orf6 proteins. Using these viral proteins, we show that the Mre11 complex is required for both ATM activation and the ATM-dependent G(2)/M checkpoint in response to DSBs. These results demonstrate that the Mre11 complex can function as a damage sensor upstream of ATM/ATR signaling in mammalian cells.     

Decline of tactile acuity in aging: a study of body site, blood flow, and lifetime habits of smoking and physical activity

Tactile acuity of 60 older subjects (> or = 65 years) and 19 younger subjects (18-28 years) was assessed by two-point gap thresholds at the upper and lower surfaces of the forefinger, at the upper and lower surfaces of the feet, and at the volar surface of the forearm. The older subjects were assigned to one of four groups of 15 subjects each, depending on reported lifetime habits of physical activity and smoking: (1) active smokers, (2) active nonsmokers, (3) inactive smokers, and (4) inactive nonsmokers. Peripheral blood flow was assessed at the forefinger, foot, and forearm by means of laser-Doppler imaging and skin temperature recordings, under resting conditions and during and after a 5-min exposure to mild cooling (28 degrees C). Consistent with previous studies, tactile acuity thresholds in the foot and finger averaged about 80% higher in the older subjects than in the younger subjects, but only about 22% higher in the forearm. Although the upper surface of the fingertip was more sensitive than the lower surface in both younger and older subjects, the age-related decline in tactile acuity was nearly identical on both sides of the finger and foot. The latter finding refutes the hypothesis that the larger effect of aging in the extremities results from greater physical wear and tear on the contact surfaces of the hands and feet. Self-reported lifetime histories of physical activity and smoking were not significantly associated with measures of cutaneous blood flow or tactile thresholds. Possible reasons for this lack of association are discussed, including the inherent limitations of testing only healthy older subjects, and the concept of "successful aging".     

EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3 end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14. The following Accession numbers were assigned to our deposited sequences: BF701840 – BF704551, BF708383, BF708386 – BF713604, BG322266 – BG322271, BI398567 – BI405235, BQ597354 – BQ605166.     

A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain

Autotransporter secretion represents a unique mechanism that Gram-negative bacteria employ to deliver proteins to their cell surface. BrkA is a Bordetella pertussis autotransporter protein that mediates serum resistance and contributes to adherence of the bacterium to host cells. BrkA is a 103 kDa protein that is cleaved to form a 73 kDa alpha-domain and a 30 kDa beta domain. The alpha domain, also referred to as the passenger domain, is responsible for the effector functions of the protein, whereas the beta domain serves as a transporter. In an effort to characterize BrkA secretion, we have shown that BrkA has a 42 amino acid signal peptide for transit across the cytoplasmic membrane, and a translocation unit made up of a short linker region fused to the beta-domain to ferry the passenger domain to the bacterial surface through a channel formed by the beta-domain. In this report, we provide genetic, biochemical and structural evidence demonstrating that a region within the BrkA passenger (Glu601-Ala692) is necessary for folding the passenger. This region is not required for surface display in the outer membrane protease OmpT-deficient Escherichia coli strain UT5600. However, a BrkA mutant protein bearing a deletion in this region is susceptible to digestion when expressed in E. coli strains expressing OmpT suggesting that the region is required to maintain a stable structure. The instability of the deletion mutant can be rescued by surface expressing Glu601-Ala692in trans suggesting that this region is acting as an intramolecular chaperone to effect folding of the passenger concurrent with or following translocation across the outer membrane.     

Penicillin-binding proteins in Streptococcus agalactiae: a novel mechanism for evasion of immune clearance

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia and meningitis in the USA despite CDC-recommended chemoprophylaxis strategies for preventing infection. To cause infection pathogens such as GBS must evade recognition and clearance by the host's immune system. Strategies for avoidance of opsonization and phagocytic killing include elaboration of antiopsonophagocytic capsules and surface proteins. During screening for mutants of GBS that were attenuated for virulence in a neonatal rat sepsis model, we identified a mutant with a transposon insertion in the ponA gene. ponA encodes an extra-cytoplasmic penicillin-binding protein PBP1a, a newly identified virulence trait for GBS that promotes resistance to phagocytic killing independent of capsular polysaccharide. Complementation analysis in vivo and in vitro confirmed that the altered phenotypes observed in the mutant were due to the transposon insertion in ponA. Deletion of PBP1a does not affect C3 deposition on GBS suggesting that mechanism by which PBP1a protects GBS from phagocytic killing is distinct from the antiopsonic activity of capsular polysaccharide. This is the first report describing expression of an antiphagocytic surface protein by GBS and represents a novel mechanism for evasion of immune recognition and clearance that may explain the decreased virulence observed in Gram-positive bacterial species for penicillin-binding protein mutants.     

Drosophila necrotic mutations mirror disease-associated variants of human serpins

Polymerization of members of the serpin superfamily underlies diseases as diverse as cirrhosis, angioedema, thrombosis and dementia. The Drosophila serpin Necrotic controls the innate immune response and is homologous to human alpha(1)-antitrypsin. We show that necrotic mutations that are identical to the Z-deficiency variant of alpha(1)-antitrypsin form urea-stable polymers in vivo. These necrotic mutations are temperature sensitive, which is in keeping with the temperature-dependent polymerization of serpins in vitro and the role of childhood fevers in exacerbating liver disease in Z alpha-antitrypsin deficiency. In addition, we identify two nec mutations homologous to an antithrombin point mutation that is responsible for neonatal thrombosis. Transgenic flies carrying an S>F amino-acid substitution equivalent to that found in Siiyama-variant antitrypsin (nec(S>F.UAS)) fail to complement nec-null mutations and demonstrate a dominant temperature-dependent inactivation of the wild-type nec allele. Taken together, these data establish Drosophila as a powerful system to study serpin polymerization in vivo.     

Effects of 4-h ischemia and 1-h reperfusion on rat muscle sarcoplasmic reticulum function

To investigate the hypothesis that ischemia and reperfusion would impair sarcoplasmic reticulum (SR) Ca(2+) regulation in skeletal muscle, Sprague-Dawley rats (n = 20) weighing 290 +/- 3.5 g were randomly assigned to either a control control (CC) group, in which only the effects of anesthetization were studied, or to a group in which the muscles in one hindlimb were made ischemic for 4 h and allowed to recover for 1 h (I). The nonischemic, contralateral muscles served as control (C). Measurements of Ca(2+)-ATPase properties in homogenates and SR vesicles, in mixed gastrocnemius and tibialis anterior muscles, indicated no differences between groups on maximal activity, the Hill coefficient, and Ca(50), defined as the Ca(2+) concentration needed to elicit 50% of maximal activity. In homogenates, Ca(2+) uptake was lower (P < 0.05) by 20-25%, measured at 0.5 and 1.0 microM of free Ca(2+) ([Ca(2+)](f)) in C compared with CC. In SR vesicles, Ca(2+) uptake was lower (P < 0.05) by 30-38% in I compared with CC at [Ca(2+)](f) between 0.5 and 1.5 microM. Silver nitrate induced Ca(2+) release, assessed during both the initial, early rapid (phase 1), and slower, prolonged late (phase 2) phases, in homogenates and SR vesicles, indicated a higher (P < 0.05) release only in phase 1 in SR vesicles in I compared with CC. These results indicate that the alterations in SR Ca(2+) regulation, previously observed after prolonged ischemia by our group, are reversed within 1 h of reperfusion. However, the lower Ca(2+) uptake observed in long-term, nonischemic homogenates suggests that altered regulation may occur in the absence of ischemia.