Induction of immunity and inflammation by interleukin-12 family members.

The interleukin (IL)-12 family is composed of three heterodimeric cytokines, IL-12 (p40p35), IL-23 (p40p19), and IL-27 (EBI3p28), and of monomeric and homodimeric p40. This review focuses on the three heterodimeric members of the IL-12 family. The p40 and p40-like (EBI3) subunits have homology to the IL-6R, the other subunits (p35, p19, and p28) are homologous to each other and to members of the IL-6 superfamily. On the basis of their structural similarity, it was expected that the members of the IL-12 family have overlapping pro-inflammatory and immunoregulatory functions. However, it was surprising that they also show very distinct activities. IL- 12 has a central role as a Th1-inducing and -maintaining cytokine, which is essential in cell-mediated immunity in nonviral infections and in tumor control. IL-23 recently emerged as an end-stage effector cytokine responsible for autoimmune chronic inflammation through induction of IL-17 and direct activation of macrophages. Very recently, IL-27 was found to exert not only a pro-inflammatory Thl-enhancing but also a significant anti-inflammatory function.

Comparison of different techniques of quantitative PCR for determination of Streptococcus mutans counts in saliva samples.

Saliva samples from 16 children with current caries activity were investigated for Streptococcus mutans using three different PCR techniques, and the results were compared with those of selective cultivation on mitis salivarius agar with bacitracin (MSB) (I, II: LightCycler - competitive PCR end-point analysis; III: LightCycler - kinetic real-time analysis; IV, V: block cycler - competitive PCR end-point analysis; VI: cultivation on MSB agar). In groups I, III, IV and VI the saliva samples were analyzed directly. A DNA preparation before PCR with added competitors was carried out in groups II and V to exclude the influence of PCR inhibitors. The coefficients of correlation ranged from 0.97 to 0.98 among the competitive PCR methods, 0.8 to 0.85 for competitive vs. real-time PCR and 0.5 to 0.65 for PCR vs. cultivation methods. Competitive PCR on the real-time instrument was found to be more rapid than, comparably sensitive to, but less reproducible than competitive PCR on a block cycler.

Expression of fructose-1,6-bisphosphatase mRNA isoforms in normal and basal forebrain cholinergic lesioned rat brain.

Fructose-1,6-bisphosphatase is one of the key enzymes in the gluconeogenic pathway predominantly occurring in liver, kidney and muscle. In the brain, fructose-1,6-bisphosphatase has been suggested to be an astrocyte-specific enzyme but the functional importance of glyconeogenesis in the brain is still unclear. To further elucidate the cellular source of fructose-1,6-bisphosphatase in the brain, non-radioactive in situ hybridizations were performed using digoxigenin-labeled RNA probes based on the sequence of recently cloned rat liver and muscle fructose-1,6-bisphosphatase cDNAs. In situ hybridization using a riboprobe for the liver isoform revealed a location of the hybridization signal mainly in neurons, while rat muscle fructose-1,6-bisphosphatase mRNA was detected in both neurons and astrocytes in the hippocampal formation and in layer I of the cerebral cortex.RT-PCR using RNA preparations of rat astrocytes, neurons, and adult whole brain demonstrated a localization of liver fructose-1,6-bisphosphatase mRNA isoform in neurons but not in astrocytes. The muscle fructose-1,6-bisphosphatase mRNA isoform could be detected by RT-PCR in total rat brain, astrocytic, and neuronal mRNA preparations. The isoforms of fructose-1,6-bisphosphatase mRNA seemingly demonstrate a distinct cellular expression pattern in rat brain suggesting a role of glyconeogenesis in both neurons and glial cells.

Rapid competitive PCR using melting curve analysis for DNA quantification.

A rapid competitive PCR method was developed to quantify DNA on the LightCycler. It rests on the quantitative information contained in the melting curves obtained after amplification in the presence of SYBR Green I. Specific hybridization probes are not required. Heterologous internal standards sharing the same primer binding sites and having different melting temperatures to the natural PCR products were used as competitors. After a co-amplification of known amounts of the competitor with a DNA-containing sample, the target DNA can be quantified from the ratio of the melting peak areas of competitor and target products. The method was developed using 16S rDNA fragments from Streptococcus mutans and E. coli and tested against existing PCR-based DNA quantification procedures. While kinetic analysis of real-time PCR is well established for the quantification of pure nucleic acids, competitive PCR on the LightCycler based on an internal standardization was found to represent a rapid and sensitive alternative DNA quantification method for analysis of complex biological samples that may contain PCR inhibitors.

Rat muscle fructose-1,6-bisphosphatase: cloning of the cDNA, expression of the recombinant enzyme, and expression analysis in different tissues.

The 1282 bp cDNA of an isoenzyme of fructose-1,6-bisphosphatase was cloned from rat muscle. It shows 70% positional identity to the cDNA of rat liver fructose-1,6-bisphosphatase and is clearly the product of a gene different from that coding for the liver enzyme. After cloning of the coding region of the rat muscle fructose-1,6-bisphosphatase cDNA in an expression vector, the recombinant enzyme could be detected in E. coli cell-free extracts by activity determination and Western blotting. Overexpressed fructose-1,6-bisphosphatase was found to be allosterically inhibited by AMP comparably to the enzyme isolated from rat muscle. Analysis of steady-state mRNA levels of various rat tissues with reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blotting revealed one or the two fructose-1,6-bisphosphatase isoenzyme mRNAs in most tissues tested with significant quantitative differences. Quantitative PCR using a homologous competitor showed that 1 microg of total RNA of rat muscle contains 1.7 x 10(6) molecules of rat muscle fructose-1,6-bisphosphatase mRNA. 3 x 10(4) copies of this message were found per microg total RNA of heart and kidney, respectively.