Multiple AGAMOUS homologs from cucumber and petunia differ in their ability to induce reproductive organ fate.

The C function in Arabidopsis, which specifies stamen and carpel identity, is represented by a single gene called AGAMOUS (AG). From both petunia and cucumber, two MADS box genes have been isolated. Both share a high degree of amino acid sequence identity with the Arabidopsis AG protein. Their roles in specifying stamen and carpel identity have been studied by ectopic expression in petunia, resulting in plants with different floral phenotypes. Cucumber MADS box gene 1 (CUM1) induced severe homeotic transformations of sepals into carpelloid structures and petals into stamens, which is similar to ectopic AG expression in Arabidopsis plants. Overexpression of the other cucumber AG homolog, CUM10, resulted in plants with partial transformations of the petals into antheroid structures, indicating that CUM10 is also able to promote floral organ identity. From the two petunia AG homologs pMADS3 and Floral Binding Protein gene 6 (FBP6), only pMADS3 was able to induce homeotic transformations of sepals and petals. Ectopic expression of both pMADS3 and FBP6, as occurrs in the petunia homeotic mutant blind, phenocopies the pMADS3 single overexpresser plants, indicating that there is no additive effect of concerted expression. This study demonstrates that in petunia and cucumber, multiple AG homologs exist, although they differ in their ability to induce reproductive organ fate.     

Interannual Variation in Timing of Parturition and Growth of Collared Pikas (Ochotona collaris) in the Southwest Yukon

The length of the snow-free season has a significant influenceon reproduction and growth in northern alpine environments,and these life history traits may provide sensitive indicatorsof the responses of organisms to climate change. We examinedgrowth rates and timing of parturition of collared pikas (Ochotonacollaris) from 1995–2002 in the Ruby Range, Yukon Territory,Canada. Growth rates were best described using a Gompertz model,in which the asymptotic mass, determined from the average maleand female weights, was 157 g, the growth rate constant (K)was 0.0557, and the age at inflection (I) was 18.12 days, fora birth weight of 10 g. The maximum growth rate for North Americanpikas (O. collaris and O. princeps) increased with latitude,with maximum growth rates being approximately one-third greaterin northern populations where the snow-free season is less thanthree months long. The mean parturition date varied significantlyamong years from 3 June to 3 July, and delayed parturition wascorrelated with indices of high snow accumulation and, to alesser extent, late spring snowmelt. However, parturition datedid not significantly affect the subsequent over-winter survivalof juveniles in this population, suggesting that pikas are ableto adjust to seasonal uncertainty associated with highly variablespring conditions.     

Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding

Mammalian sperm must undergo a process known as capacitation before fertilization can take place. A key intracellular event that occurs during capacitation is protein tyrosine phosphorylation. The objective of this study was to investigate and visualize protein tyrosine phosphorylation patterns in human sperm during capacitation and interaction with the zona pellucida. The presence of specific patterns was also assessed in relation to the fertilizing capacity of the spermatozoa after in vitro fertilization. Protein tyrosine phosphorylation was investigated by immunofluorescence. Phosphorylation increased significantly with capacitation and was localized mainly to the principal piece of human sperm. Following binding to the zona pellucida, the percentage of sperm with phosphotyrosine residues localized to both the neck and the principal piece was significantly higher in bound sperm than in capacitated sperm in suspension. When the percentage of principal piece-positive sperm present after capacitation was <7%, fertilization rates after in vitro fertilization were reduced. Different compartments of human spermatozoa undergo a specific sequence of phosphorylation during both capacitation and upon binding to the zona pellucida. Tyrosine phosphorylation in the principal and neck piece may be considered a prerequisite for fertilization in humans.     

Isolation of salmonellas by immunomagnetic separation.

Magnetisable particles, coated with anti-salmonella serum, were used to isolate Salmonella livingstone from pure cultures, mixed cultures and food samples. Beads (10(7] were generally incubated with 10(4) Salm. livingstone cells/ml for 60 min at room temperature. The incubation and washing medium (0.01 mol/l phosphate-buffered saline; PBS) contained 0.1% bovine serum albumin (BSA) and 0.1% Tween 20, respectively. This method gave a recovery for Salm. livingstone of 51.0 +/- 7.8%. However, other micro-organisms such as Aeromonas hydrophila interfered with this test because of non-specific reactions (recovery 50.9 +/- 12.7%). These non-specific reactions could be decreased by using 4% skim milk instead of 0.1% BSA in the incubation medium. The ratio of the recovery of Salm. livingstone relative to the recovery of Aer. hydrophila changed from 0.9 when PBS with 0.1% BSA was used, to 13.4 when PBS with 4% skim milk was used. Immunomagnetic separation of Salmonella spp. from food samples offers good prospects for concentrating salmonella cells from heterogeneous bacterial suspensions, such as enrichment broths.     

Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates

To study the relation between the number of hyphal tips and protein secretion during growth on a solid substrate, we have constructed two mutant strains of Aspergillus oryzae with increased hyphal branching. We have analysed hydrolytic enzyme activities during growth on wheat kernels (WK) of A. oryzae strains carrying the disrupted allele of the pclA gene encoding a secretion pathway specific (KEX2-like) endo-protease and the disrupted allele of the pg/pi-tp gene encoding a phosphatidylglycerol/phosphatidylinositol transfer protein. The biomass levels produced by the pclA and pg/pi-tp disrupted strains on wheat-based solid media were similar as found for the wild-type strain. However, the pclA disrupted strain showed much more compact colony morphology than the other two strains. Sporulation of the pclA and pg/pi-tp disrupted strains occurred, respectively, 2 days and 1 day later, compared to the wild type during fermentation on ground WK. During surface growth, microscopic analysis revealed that the hyphal growth unit length (L _hgu) of the pclA and pg/pi-tp disrupted strains was, on average, 50 and 74% of that of the wild-type strain. This implies that in both mutant strains, a higher branching frequency occurs than in the wild-type strain. Compared to the wild-type strain, the pclA and pg/pi-tp disrupted strains produced at least 50% more amylase, at least 100% more glucoamylase and at least 90% more protease activity levels after growth on WK. These results support the hypothesis that branching mutants with an increased branching frequency can improve the solid state fermentation process.     

Intraocular tumor antigen drains specifically to submandibular lymph nodes, resulting in an abortive cytotoxic T cell reaction

Ocular immune privilege is considered essential in the protection against sight-threatening immune responses, as illustrated by the ability of the ocular environment to permit the growth of tumors that are rejected when implanted at other sites. Although several studies indicate that soluble Ag can drain directly into the spleen when injected into the anterior chamber, the primary site of intraocular tumor Ag presentation to tumor-specific CTLs has not been studied. To gain a better understanding of the mechanism involved in ocular immune privilege, we examined to which lymphoid organs anterior chamber tumor Ags primarily drain. Our data show that intraocular tumor Ag drains exclusively to the submandibular lymph nodes, resulting in activation of tumor-specific CTLs, whereas no Ag drainage was found in spleen. However, these tumor-specific CTLs do not distribute systemically and, as a consequence, intraocular tumor growth is unhampered. A similar lack of CTL efficacy has been observed in mice bearing s.c. tumors, which is converted to a systemic tumoricidal CTL response by administration of agonistic anti-CD40 mAb. In contrast, systemic anti-CD40 treatment of eye tumor-bearing mice did not result in mobilizing tumor-specific CTLs or tumor eradication. Together, these results show that intraocular tumor Ag drains to regional lymph nodes for activation of tumor-specific CTLs. However, the induced tumor-specific immunity is insufficient for tumor clearance, even combined with otherwise highly effective immune intervention protocols.     

Monokine induced by interferon gamma and IFN-gamma response to a fusion protein of Mycobacterium tuberculosis ESAT-6 and CFP-10 in Brazilian tuberculosis patients

IFN-gamma responses to Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 have been proposed as specific markers of M. tuberculosis infection. Monokine induced by gamma interferon (MIG/CXCL9) has been shown to be expressed by IFN-gamma stimulated mononuclear cells and to attract activated T-cells through the chemokine receptor CXCR3. Since MIG is induced early in the response to IFN-gamma, measuring MIG may provide an interesting marker to assess downstream IFN-gamma induced responses, in contrast to assays that mainly focus on quantifying production of IFN-gamma per se. We, therefore, investigated MIG and IFN-gamma responses to a fusion protein of ESAT-6 and CFP-10, and compared responses to the conserved mycobacterial antigen 85B (Ag85B) and purified protein derivative (PPD) of M. tuberculosis, in 29 BCG vaccine controls and 24 TB patients. IFN-gamma secreting cells were determined by ELISPOT, and MIG production was measured by ELISA and flow cytometry. Production of MIG in response to ESAT-6/CFP-10, Ag85B and PPD correlated overall with increased numbers of IFN-gamma secreting cells (r=0.55, P<0.0001). A significant increase was noted among patients compared to controls in the secretion of IFN-gamma and MIG following stimulation with ESAT-6/CFP-10 or PPD (P<0.05). Moreover, MIG intracellular expression was higher in TB patients compared to BCG vaccines (P<0.05) in response to ESAT-6/CFP-10 or PPD. We conclude that MIG production correlates significantly with enhanced T-cell IFN-gamma production induced by M. tuberculosis-specific antigens ESAT-6/CFP-10. These results point to MIG as a potential novel biomarker that may be helpful in assessing downstream responses induced by IFN-gamma in TB.