A continuous wave laser T-jump apparatus and its application to chemical reactions in hemoglobin single crystals

A laser temperature jump apparatus is constructed where the T-jump is achieved by means of the direct absorption of continuous laser radiation of low intensity by a solid sample. The final temperature in the irradiated volume element is reached when the absorbed radiation power equals the dissipation of heat by heat conduction. The time range from the beginning of irradiation to the stationary state depends on the geometry of the irradiated volume element and is less than 10 ms. The heating laser beam is simultaneously used to detect the relaxation to the new chemical equilibrium in the sample. Relaxation processes with relaxation rates between 10(2) s-1 and less than 10(-3) s-1 on samples with volumes less than 10(-3) mm3 may be investigated using this T-jump method. One application of this method is the determination of reaction rates of ligand reactions in hemoglobin single crystals. Rate constants obtained for the reaction of thiocyanate with crystallized horse methemoglobin are presented.     

Control of poliomyelitis by pulse immunisation in Vellore,India.

In a simple study into the control of polio in the Third World a town was divided into 16 zones and pulses or oral polio vaccine given at one station in each zone, after extensive publicity about the campaign. Some 62% of children received three doses of the vaccine and the incidence of polio fell dramatically over the study period. It is suggested that this method is applicable to similar communities because it is cheap, effective, and able to be extended to unimmunised communities when resources allow.     

Dental follicle progenitor cells responses to Porphyromonas gingivalis LPS

Periodontitis is a bacterially induced chronic inflammatory disease. Dental follicle progenitor cells (DFPCs) have been proposed as biological graft for periodontal regenerative therapies. The potential impact of bacterial toxins on DFPCs properties is still poorly understood. The aim of this study was to investigate whether DFPCs are able to sense and respond to lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major periopathogenic bacterium. Specifically, we hypothesized that LPS could influence the migratory capacity and IL‐6 secretion of DFPCs. DFPCs properties were compared to bone marrow mesenchymal stem cells (BMSCs), a well‐studied class of adult stem cells. The analysis by flow cytometry indicated that DFPCs, similar to BMSCs, expressed low levels of both toll‐like receptor (TLR) 2 and 4. The TLR4 mRNA expression was down‐regulated in response to LPS in both cell populations, while on protein level TLR4 was significantly up‐regulated on BMSCs. The TLR2 expression was not influenced by the LPS treatment in both DFPCs and BMSCs. The migratory efficacy of LPS‐treated DFPCs was evaluated by in vitro scratch wound assays and found to be significantly increased. Furthermore, we assayed the secretion of interleukin‐6 (IL‐6), a potent stimulator of cell migration. Interestingly, the levels of IL‐6 secretion of DFPCs and BMSCs remained unchanged after the LPS treatment. Taken together, these results suggest that DFPCs are able to sense and respond to P. gingivalis LPS. Our study provides new insights into understanding the physiological role of dental‐derived progenitor cells in sites of periodontal infection.     

Identification of Ovarian Cancer Metastatic miRNAs

Serous epithelial ovarian cancer (EOC) patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastasis. Here, we aim to understand how omental metastases differ from primary tumors and how these differences may influence chemotherapy. We analyzed the miRNA expression profiles of primary EOC tumors and their respective omental metastases from 9 patients using miRNA Taqman qPCR arrays. We find 17 miRNAs with differential expression in omental lesions compared to primary tumors. miR-21, miR-150, and miR-146a have low expression in most primary tumors with significantly increased expression in omental lesions, with concomitant decreased expression of predicted mRNA targets based on mRNA expression. We find that miR-150 and miR-146a mediate spheroid size. Both miR-146a and miR-150 increase the number of residual surviving cells by 2–4 fold when challenged with lethal cisplatin concentrations. These observations suggest that at least two of the miRNAs, miR-146a and miR-150, up-regulated in omental lesions, stimulate survival and increase drug tolerance. Our observations suggest that cancer cells in omental tumors express key miRNAs differently than primary tumors, and that at least some of these microRNAs may be critical regulators of the emergence of drug resistant disease.     

ATP-driven MalK dimer closure and reopening and conformational changes of the "EAA" motifs are crucial for function of the maltose ATP-binding cassette transporter (MalFGK2)

We have investigated conformational changes of the purified maltose ATP-binding cassette transporter (MalFGK(2)) upon binding of ATP. The transport complex is composed of a heterodimer of the hydrophobic subunits MalF and MalG constituting the translocation pore and of a homodimer of MalK, representing the ATP-hydrolyzing subunit. Substrate is delivered to the transporter in complex with periplasmic maltose-binding protein (MalE). Cross-linking experiments with a variant containing an A85C mutation within the Q-loop of each MalK monomer indicated an ATP-induced shortening of the distance between both monomers. Cross-linking caused a substantial inhibition of MalE-maltose-stimulated ATPase activity. We further demonstrated that a mutation affecting the "catalytic carboxylate" (E159Q) locks the MalK dimer in the closed state, whereas a transporter containing the "ABC signature" mutation Q140K permanently resides in the resting state. Cross-linking experiments with variants containing the A85C mutation combined with cysteine substitutions in the conserved EAA motifs of MalF and MalG, respectively, revealed close proximity of these residues in the resting state. The formation of a MalK-MalG heterodimer remained unchanged upon the addition of ATP, indicating that MalG-EAA moves along with MalK during dimer closure. In contrast, the yield of MalK-MalF dimers was substantially reduced. This might be taken as further evidence for asymmetric functions of both EAA motifs. Cross-linking also caused inhibition of ATPase activity, suggesting that transporter function requires conformational changes of both EAA motifs. Together, our data support ATP-driven MalK dimer closure and reopening as crucial steps in the translocation cycle of the intact maltose transporter and are discussed with respect to a current model.     

Identification of pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP type 1 receptor in human skin: expression of PACAP-38 is increased in patients with psoriasis

Morphological and biochemical evidence is presented for the presence of pituitary adenylate cyclase activating peptide (PACAP) and the high-affinity PACAP-1 receptor subtype in human skin. Immunohistochemical analysis revealed PACAP-immunoreactivity (IR) to be present predominantly in dermal nerve fibers close to the dermal-epidermal border, hair follicles, blood vessels and sweat glands. Radioimmunoassay, chromatographic analysis and Western blotting revealed this PACAP-IR to be PACAP-38 whereas the second molecular form, PACAP-27, is absent. In tissue of psoriasis patients significantly more PACAP-38 protein was detected as compared to normal skin. Using RT-PCR, the expression of a high-affinity PACAP-1 receptor in human skin was observed. These results indicate a possible role for PACAP-38 in inflammatory processes of psoriasis.     

Polymorphic methyl group metabolism genes in patients with transitional cell carcinoma of the urinary bladder.

Because polymorphisms in the methyl group metabolism genes methylene-tetrahydrofolate reductase (MTHFR), methionine synthase (MS), and cystathione beta-synthetase (CBS) affect plasma homocysteine levels and intracellular concentrations of S-adenosylmethionine (SAM), they modify the susceptibility to cardiovascular diseases and cancer. Specifically, genome-wide decreased DNA methylation ('hypomethylation') in human cancers might be a consequence of decreased SAM levels. Because hypomethylation is particularly prevalent in transitional cell carcinoma of the urinary bladder (TCC), the genotype distributions for the two each most prevalent MTHFR, MS, and CBS alleles were compared between 165 TCC patients and 150 population controls. The distributions of the MTHFR 677A/V and the MS 919G/D alleles were not significantly different between cancer patients and controls, even after stratification according to age, gender, tumor stage or grade. The CBS 844INS68 allele was slightly less frequent in TCC patients than in controls (q=0.07 versus 0.10), but was rarer among males in both groups. Among the TCC patients, this gender difference was highly significant (Mantel-Haenszel and chi(2)-test P=0.007). No significant difference between TCC patients and controls was found for any combined genotype. Likewise, the extent of DNA hypomethylation determined in 62 carcinoma specimens was not related to the respective genotypes. Thus, on their own, the MTHFR, MS and CBS genotypes do not appear to act upon susceptibility to TCC or influence the extent of DNA hypomethylation in this cancer.     

Colitis induced by proteinase-activated receptor-2 agonists is mediated by a neurogenic mechanism

Proteinase-activated receptor-2 (PAR2) activation induces colonic inflammation by an unknown mechanism. We hypothesized that PAR2 agonists administered intracolonically in mice induce inflammation via a neurogenic mechanism. Pretreatment of mice with neurokinin-1 and calcitonin-gene-related peptide (CGRP) receptor antagonists or with capsaicin showed attenuated PAR2-agonist-induced colitis. Immunohistochemistry demonstrated a differential expression of a marker for the type-1 CGRP receptor during the time course of PAR2-agonist-induced colitis, further suggesting a role for CGRP. We conclude that PAR2-agonist-induced intestinal inflammation involves the release of neuropeptides, which by acting on their receptors cause inflammation. These results implicate PAR2 as an important mediator of intestinal neurogenic inflammation.     

Activities of MAP-kinase pathways in normal uroepithelial cells and urothelial carcinoma cell lines

It is often assumed that MAPK pathways drive proliferation of normal uroepithelial (UEC) and urothelial carcinoma (TCC) cells. To check this assumption, activities and inducibilities of promoters containing serum-response elements (SRE) or AP-1 binding sites were investigated in cultured UEC and seven TCC lines. Reporter plasmids dependent on SRE or AP-1 sites were highly active in UEC, but significantly less so in TCC lines. Reporter activity in TCC lines could be induced by constitutively active MEKK4 or TPA. Accordingly, phosphorylation of the MAPK pathway components MEK, ERK, and ELK1 was most pronounced in UEC and lower in TCC lines. MAPK-dependent promoter activities and bromodeoxyuridine incorporation decreased in UEC upon withdrawal of growth factors, but less so in TCC lines, in which serum diminution increased apoptosis. Likewise, E2F-dependent promoters responded to growth factors in UEC, but were more serum-independent in the TCC lines, which lack either RB1 or p16(INK4A). MEK inhibitors inhibited BrdU incorporation in UEC more strongly than in TCC lines. Thus, proliferation of normal uroepithelial cells is indeed associated with activation of MAPK pathways. However, autonomous proliferation of TCC lines--unexpectedly--appears much less dependent on MAPK activation and may rather be promoted by defects in cell cycle regulation.     

Indirect RT-PCR in-situ hybridization: a novel non-radioactive method for detecting glucose-dependent insulinotropic peptide

To establish indirect in-situ PCR for the detection of intestinal peptide hormones, rat intestine and a murine intestinal tumor cell line, STC 1, were used. The results exhibited intensive staining of GIP-producing K-cells. Paraformaldehyde-fixed cryostat sections yielded the best results in signal to background ratio with RT-PCR in-situ hybridization. Moreover, it was possible to elevate the positive staining signal and to reduce background staining. Digoxigenin-labeled in-situ hybridization served as a control for specificity and sensitivity of GIP (glucose-dependent insulinotropic peptide) mRNA expression on cryostat as well as paraffin sections. In conclusion, this RT-PCR in-situ hybridization protocol proves to be a specific, sensitive and reliable non-radioactive technique for the detection of intestinal peptide hormone mRNA, especially in tissues or tumor cells where the application of ISH is limited.