Cutting edge: homeostatic proliferation of peripheral T lymphocytes is regulated by clonal competition

Homeostatic proliferation functions to maintain peripheral T cell numbers and is regulated by cytokines. In this study, we provide evidence that T cell homeostasis is also regulated by clonal competition. Naive polyclonal T cells divided when transferred to TCR transgenic hosts, as did monoclonal T cells when transferred to TCR transgenic hosts of differing clonotype. However, T cells did not divide in hosts of identical clono-type. Transgenic T cell proliferation was inhibited in irradiated hosts of the same clonotype, while cotransferred nontransgenic T cells proliferated extensively. These results show that clonal competition is a component of homeostasis that may contribute to selection of the peripheral T cell repertoire.     

CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.     

Presynaptic N-type calcium channels regulate synaptic growth

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.     

Reproductive behavior in the squid Sepioteuthis australis from South Australia: ethogram of reproductive body patterns

Squids use a diverse range of body patterns for communication. Each pattern consists of a series of chromatic, postural, and locomotor components that are under neural control and can change within fractions of a second. Here we describe an ethogram of 48 body pattern components from in situ observations of reproductively active Sepioteuthis australis. In addition, we identify the total time and average duration that each component is shown, to a resolution of 1 s. Our results suggest that only a few components (e.g., "Golden epaulettes," "Stitchwork fins," and "Rigid arms") are temporally common, that is, shown for more than 80% of the time. In contrast to the component classification reported for other species of squid, for this species we suggest a classification system consisting of "short acute" (lasting for < 10 s); some of these same components were also classified as "medium acute" (11-60 s) or "chronic" (> 60 s). Several body patterning components were previously unreported, as were some of the combinations observed. The significance of these patterning components is discussed within the context of the associated behaviors of the squid on the spawning grounds.     

Mutations at residues 282, 286, and 293 of phage lambda integrase exert pathway-specific effects on synapsis and catalysis in recombination

Bacteriophage lambda integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. The att sites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: the attL site probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA with attR.     

Design and synthesis of 4,5-disubstituted-thiophene-2-amidines as potent urokinase inhibitors

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.     

Normal conjunctival flora in the North American opossum (Didelphis virginiana) and raccoon (Procyon lotor)

We documented the normal conjunctival bacterial flora from 17 opossums (Didelphis virginiana) and 10 raccoons (Procyon lotor) trapped in Manhattan, Kansas (USA) from November 1999 to January 2000. Both raccoons and opossums were free of apparent ocular disease. The inferior conjunctival sacs of each animal were swabbed for aerobic bacterial and Mycoplasma culture and polymerase chain reaction (PCR) for Mycoplasma and Chlamydia detection. All conjunctival samples were positive for one or more species of aerobic bacteria. The most common isolate from opossums was Staphylococcus spp. Other isolates included Streptococcus spp., Bacillus spp., Corynebacterium spp., and Enterococcus faecalis. The most common isolates in raccoons was Bacillus spp. Other isolates included Streptococcus spp., Staphylococcus spp., non-hemolytic Escherichia coli, and Enterococcus faecalis. Mycoplasma culture was negative in samples from opossums and raccoons. Evidence of Mycoplasma and Chlamydia presence was detected by PCR.     

Free tissue coverage of chronic traumatic wounds of the lower leg

Thirty-eight consecutive patients who underwent 42 free flaps for chronic wounds of the lower leg were identified over an 11-year period. All wounds were open for a minimum of 1 month (mean, 40 months; median, 8 months; range, 1 month to 30 years). The average age was 37 years (range, 7 to 68 years), there were 31 male patients and seven female patients, and the average follow-up time was 30 months (range, 12 to 72 months). The original injury was an open fracture in 28 patients, wound dehiscence after open reduction and internal fixation of a closed fracture in nine patients, and a shrapnel wound in one patient. A total of 23 patients had osteomyelitis, which was classified as local (involving less than 50 percent of the bone diameter) in 15 patients and as diffuse (involving greater than 50 percent of the bone diameter or infected nonunion) in eight patients. The wounds were treated with sequential debridement, antibiotics, and flap coverage. Ancillary procedures included antibiotic beads in 18 patients, saucerization in 16, Ilizarov bone transport in three, calcanectomy in two, and fibular resection and ankle fusion in one. Thirty-four of 42 flaps survived, four having undergone a repeat free flap. There were three failures out of 25 flaps (12 percent) among those with a normal angiogram and five failures out of 15 flaps (33 percent) among those with an abnormal angiogram (p > 0.05). The failure rate of those with osteomyelitis was six of 26 (23 percent) versus two of 26 (13 percent) for those without osteomyelitis (p > 0.05). Successful reconstruction (bone healed, patient ambulatory and infection-free) was achieved in 33 of 38 patients (87 percent). The failure of reconstruction for those patients with osteomyelitis was four of 23 (22 percent) versus one of 15 (7 percent) for others (p > 0.05). The failure rate of flaps in patients with diffuse osteomyelitis was three of eight (38 percent) versus two of 30 for others (7 percent, p = 0.053). The presence of diffuse osteomyelitis was associated with a lower rate of successful limb reconstruction. An abnormal angiogram and the presence of osteomyelitis both were associated with a lower rate of successful limb reconstruction, but this was not significant, probably because of the small size of the cohort.     

Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.     

Caspase-2 redux

It has been difficult to assign caspase-2 to the effector or initiator caspase groups. It bears sequence homology to initiators (caspase-9 and CED-3), but its cleavage specificity is closer to the effectors (caspase-3 and -7). Interest in caspase-2 was dampened by the lack of a dramatic phenotype in the caspase-2 null mouse. Studies have been inhibited by the lack of knowledge about its mechanism of activation and the lack of specific methods to assay its activity. Molecular studies have defined a unique role for caspase-2 in apoptosis initiated by beta-amyloid toxicity or by trophic factor deprivation. Recently, a role for caspase-2 as an upstream initiator of mitochondrial permeabilization has been proposed. Thus, while much remains to be deciphered about caspase-2, most critically the mode of activation, it is clear that caspase-2 plays critical and singular roles in the control of programmed cell death.