Phenology and phylogenetic positioning of the Hawaiian endemic freshwater alga, Batrachospermum spermatiophorum (Rhodophyta, Batrachospermales)

The only known population of Batrachospermum spermatiophorum Vis et Sheath, located in a small stream on east Maui, Hawaii, was sampled from November 2001 to October 2002 to investigate its phenology. Additionally, the taxonomic status of the alga was examined. Phylogenetic analyses of the large subunit of the ribulose‐1,5‐bisphosphate carboxylase/oxygenase (rbcL) gene for B. spermatiophorum confirm its placement within the Section Contorta of the genus Batrachospermum. Comparisons with rbcL gene sequences for all other available Batrachospermum species, including 10 other members of the Section Contorta, illustrate that B. spermatiophorum is distinct in its rbcL nucleotide sequence and should be maintained as a separate species. The phenology of B. spermatiophorum differed from most studies of Batrachospermum reproduction in that the gametophytes were present year round, and no macroscopic chantran‐sia stages were identified at any point during the present study. Maximum plant length was highest in the late fall and early winter months, while gametangial production peaked in February and March. Carposporophyte abundance rose sharply in July and August, and the percentage of carposporophytes producing carpospores was highest in late summer and early fall months. Although significant correlations were determined among stream conditions and among phenological characteristics, only pH was associated with several of the reproductive characters, which is unusual for studies of Batrachospermum phenology. Timing of sexual reproduction for all macroalgal community members in the stream site was not coordinated, with some reproducing year round and others more sporadically. The year‐round presence of Batrachospermum gametophytes may be a result of a combination of smaller seasonal changes in cues such as day length and water temperature in the tropical environment than occur in temperate locations, and lack of extreme conditions that might inhibit the persistence of the gametophyte.     

Interleukin-7 deficiency in rheumatoid arthritis: consequences for therapy-induced lymphopenia

We previously demonstrated prolonged, profound CD4⁺ T-lymphopenia in rheumatoid arthritis (RA) patients following lymphocyte-depleting therapy. Poor reconstitution could result either from reduced de novo T-cell production through the thymus or from poor peripheral expansion of residual T-cells. Interleukin-7 (IL-7) is known to stimulate the thymus to produce new T-cells and to allow circulating mature T-cells to expand, thereby playing a critical role in T-cell homeostasis. In the present study we demonstrated reduced levels of circulating IL-7 in a cross-section of RA patients. IL-7 production by bone marrow stromal cell cultures was also compromised in RA. To investigate whether such an IL-7 deficiency could account for the prolonged lymphopenia observed in RA following therapeutic lymphodepletion, we compared RA patients and patients with solid cancers treated with high-dose chemotherapy and autologous progenitor cell rescue. Chemotherapy rendered all patients similarly lymphopenic, but this was sustained in RA patients at 12 months, as compared with the reconstitution that occurred in cancer patients by 3–4 months. Both cohorts produced naïve T-cells containing T-cell receptor excision circles. The main distinguishing feature between the groups was a failure to expand peripheral T-cells in RA, particularly memory cells during the first 3 months after treatment. Most importantly, there was no increase in serum IL-7 levels in RA, as compared with a fourfold rise in non-RA control individuals at the time of lymphopenia. Our data therefore suggest that RA patients are relatively IL-7 deficient and that this deficiency is likely to be an important contributing factor to poor early T-cell reconstitution in RA following therapeutic lymphodepletion. Furthermore, in RA patients with stable, well controlled disease, IL-7 levels were positively correlated with the T-cell receptor excision circle content of CD4⁺ T-cells, demonstrating a direct effect of IL-7 on thymic activity in this cohort.     

Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome

Secretory proteins perform a variety of important “remote-control” functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which ~90 extracellular proteins were identified. Analysis of these proteins disclosed various “secrets of the secretome,” such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only ~50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.     

Low-frequency depression of tension in the cat gastrocnemius muscle after eccentric exercise.

Subjecting a muscle to a series of eccentric contractions in which the contracting muscle is lengthened results in a number of changes in its mechanical properties. These include a fall in isometric tension that is particularly pronounced during low-frequency stimulation, a phenomenon known as low-frequency depression (LFD). Reports of LFD have not taken into account the shift in optimum length for active tension generation to longer muscle lengths that takes place after eccentric contractions. Given the length dependence of the stimulation frequency-tension curve, we tested the hypothesis that the change in this relationship after eccentric exercise is due to the shift in optimum length. We measured LFD by recording tension in response to a linearly increasing rate of stimulation of the nerve to medial gastrocnemius of anesthetized cats, over the range 0-100 pulses per second. Tension responses were measured before and after 50 eccentric contractions consisting of 6-mm stretches starting at 3 mm below optimum length and finishing at 3 mm above it. An index of LFD was derived from the tension responses to ramp stimulation. It was found that LFD after the eccentric contractions was partly, but not entirely, due to changes in the muscle's optimum length. An additional factor was the effect of fatigue. These observations led to the conclusion that the muscle length dependence of LFD was reduced by eccentric contractions. All of this means that after eccentric exercise the tension deficit at low rates of muscle activation is likely to be less severe than first thought.     

北极的春季:迁徙鸟类抵达的生物学

一旦鸟类抵达北极区,迁徙鸟类必须调整其生理和行为以适应不可预知的雪盖、天气、食物资源和天敌胁迫。换言之,他们必须抵抗环境干扰(压力)以便尽早迁徙到苔原上的巢区并建立领域。然后,一旦外界环境有利时,它们就立即开始繁殖。这些鸟类的繁殖有一部分是利用低雪盖区域的微生境以及雪融较快的苔原斑块(特别是在柳树Salix sp．附近)。在北极地区,地面温度在日照若干小时后急剧上升,同时无脊椎动物开始活动。风速在地面柳枝和生草丛苔原10cm下几乎减弱为零。这些条件结合在一起提供了理想的避难所,对于早春迁徙到此的雀形目鸟类尤其如此。然而,如果环境调节变得更为恶劣,这些鸟类会离开。因为与南方越冬地相比,春季北极区条件具有潜在的严酷性,所以鸟类对于应激时的肾上腺皮质反应有所调整。雄鸟到达北极地区时对于剧烈应激刺激下的肾上腺酮的分泌有所提高,并且伴随着对于负反馈敏感性的下降和肾上腺皮质层细胞对于促肾上腺皮质激素作用反应的变化。同时,肾上腺酮结合蛋白(CBG)的水平也有所提高,以至于肾上腺酮的作用在恶劣的环境条件下得到缓冲。基因组受体水平的调节,尤其是在脑和肝脏中糖皮质类固醇类似受体与肾上腺酮的低亲合性,以及肾上腺酮的非基因组水平的作用,可能是很重要的。换言之,与抵达生物学有关的激素一行为系统是高度可变的[动物学报50(6)：948-960,2004]。     

Characterization of BflI – A Thermostable,Co++-Requiring Isoschizomer of BsiYI from Anoxybacillus Flavithermus

A thermophile, isolated from geothermal areas in the northern Himalayan region of India, was identified by partial 16S rDNA sequence (GenBank accession # AF482430) analysis as Anoxybacillus flavithermus. The isolate produced BflI (REBASE # 4910), a Type II restriction endonuclease, which recognized the sequence 5′-CCNNNNN/NNGG-3′ and was the isoschizomer of BsiYI. The enzyme was purified to homogeneity by passing through Cibacron Blue F3GA agarose, DEAE-cellulose, heparin-agarose and MonoQ FPLC. The purified enzyme (MW 36 kDa) worked best at 60 °C in Promega's buffer C and preferentially required Co⁺⁺(0.4 mM) as cofactor followed by Mg⁺⁺(10 mM) and Mn⁺⁺(1 mM). The enzyme showed high specific activity and worked in the presence of high concentrations of β-mercaptoethanol (200 mM), Triton-X-100 (25%), urea (30%), formamide (6%) and guanidine (40 mM) and showed no star activity in the presence of 40% glycerol. In the absence of any stabilizing agent, BflI retained t _1/2 for at least 96 h at 37 °C, 6 h at 60 °C and 6 months at 4 °C. N-terminal sequencing showed that its first 10 amino acid residues were DFHEDKTIAR. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular cytogenetic analysis and resequencing of contactin associated protein-like 2 in autism spectrum disorders

Autism spectrum disorders (ASD) are a group of related neurodevelopmental syndromes with complex genetic etiology. We identified a de novo chromosome 7q inversion disrupting Autism susceptibility candidate 2 (AUTS2) and Contactin Associated Protein-Like 2 (CNTNAP2) in a child with cognitive and social delay. We focused our initial analysis on CNTNAP2 based on our demonstration of disruption of Contactin 4 (CNTN4) in a patient with ASD; the recent finding of rare homozygous mutations in CNTNAP2 leading to intractable seizures and autism; and in situ and biochemical analyses reported herein that confirm expression in relevant brain regions and demonstrate the presence of CNTNAP2 in the synaptic plasma membrane fraction of rat forebrain lysates. We comprehensively resequenced CNTNAP2 in 635 patients and 942 controls. Among patients, we identified a total of 27 nonsynonymous changes; 13 were rare and unique to patients and 8 of these were predicted to be deleterious by bioinformatic approaches and/or altered residues conserved across all species. One variant at a highly conserved position, I869T, was inherited by four affected children in three unrelated families, but was not found in 4010 control chromosomes (p = 0.014). Overall, this resequencing data demonstrated a modest nonsignificant increase in the burden of rare variants in cases versus controls. Nonetheless, when viewed in light of two independent studies published in this issue of AJHG showing a relationship between ASD and common CNTNAP2 alleles, the cytogenetic and mutation screening data suggest that rare variants may also contribute to the pathophysiology of ASD, but place limits on the magnitude of this contribution.     

Phosphorylation of cysteine string protein on Serine 10 triggers 14-3-3 protein binding

Cysteine string protein (CSP) is a neuronal chaperone that maintains normal neurotransmitter exocytosis and is essential for preventing presynaptic neurodegeneration. CSP is phosphorylated in vivo on a single residue, Ser10, and this phosphorylation regulates its cellular functions, although the molecular mechanisms involved are unclear. To identify novel phosphorylation-specific binding partners for CSP, we used a pull-down approach using synthetic peptides and recombinant proteins. A single protein band was observed to bind specifically to a Ser10-phosphorylated CSP peptide (residues 4-14) compared to a non-phosphorylated peptide. This band was identified as 14-3-3 protein of various isoforms using mass spectrometry and Western blotting. PKA phosphorylation of full-length CSP protein stimulated 14-3-3 binding, and this was abolished in a Ser10-Ala mutant CSP, confirming the binding site as phospho-Ser10. As both CSP and 14-3-3 proteins are implicated in neurotransmitter exocytosis and neurodegeneration, this novel phosphorylation-dependent interaction may help maintain the functional integrity of the synapse.     

Hypnotic manipulation of effort sense during dynamic exercise: cardiovascular responses and brain activation.

The purpose of this investigation was to hypnotically manipulate effort sense during dynamic exercise and determine whether cerebral cortical structures previously implicated in the central modulation of cardiovascular responses were activated. Six healthy volunteers (4 women, 2 men) screened for high hypnotizability were studied on 3 separate days during constant-load exercise under three hypnotic conditions involving cycling on a 1) perceived level grade, 2) perceived downhill grade, and 3) perceived uphill grade. Ratings of perceived exertion (RPE), heart rate (HR), blood pressure (BP), and regional cerebral blood flow (rCBF) distributions for several sites were compared across conditions using an analysis of variance. The suggestion of downhill cycling decreased both the RPE [from 13 +/- 2 to 11 +/- 2 (SD) units; P < 0.05] and rCBF in the left insular cortex and anterior cingulate cortex, but it did not alter exercise HR or BP responses. Perceived uphill cycling elicited significant increases in RPE (from 13 +/- 2 to 14 +/- 1 units), HR (+16 beats/min), mean BP (+7 mmHg), right insular activation (+7.7 +/- 4%), and right thalamus activation (+9.2 +/- 5%). There were no differences in rCBF for leg sensorimotor regions across conditions. These findings show that an increase in effort sense during constant-load exercise can activate both insular and thalamic regions and elevate cardiovascular responses but that decreases in effort sense do not reduce cardiovascular responses below the level required to sustain metabolic needs.