Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta

Leptin is a 167-aa protein that is secreted from adipose tissue and is important in the regulation of energy balance. It also functions in hematopoiesis and reproduction. To assess whether leptin is involved in fetal growth and development we have examined the distribution of mRNAs encoding leptin and the leptin receptor (which has at least six splice variants) in the 14.5-day postcoitus mouse fetus and in the placenta using reverse transcription–PCR and in situ hybridization. High levels of gene expression for leptin, the leptin receptor, and the long splice variant of the leptin receptor with an intracellular signaling domain were observed in the placenta, fetal cartilage/bone, and hair follicles. Receptor expression also was detected in the lung, as well as the leptomeninges and choroid plexus of the fetal brain. Western blotting and immunocytochemistry, using specific antibodies, demonstrated the presence of leptin and leptin receptor protein in these tissues. These results suggest that leptin may play a role in the growth and development of the fetus, both through placental and fetal expression of the leptin and leptin receptor genes. In the fetus, leptin may be multifunctional and have both paracrine and endocrine effects.

Functional and physiological consequences of genetic variation at phosphoglucose isomerase: Heat shock protein expression is related to enzyme genotype in a montane beetle

Allele frequency variation at the phosphoglucose isomerase (PGI) locus in Californian populations of the beetle Chrysomela aeneicollis suggests that PGI may be undergoing natural selection. We quantified (i) apparent Michaelis-Menten constant (Km) of fructose 6-phosphate at different temperatures and (ii) thermal stability for three common PGI genotypes (1–1, 1–4, and 4–4). We also measured air temperature (Ta) and beetle body temperature (Tb) in three montane drainages in the Sierra Nevada, California. Finally, we measured 70-kDa heat shock protein (Hsp70) expression in field-collected and laboratory-acclimated beetles. We found that PGI allele 1 predominated in the northernmost drainage, Rock Creek (RC), which was also significantly cooler than the southernmost drainage, Big Pine Creek (BPC), where PGI allele 4 predominated. Allele frequencies and air temperatures were intermediate in the middle drainage, Bishop Creek (BC). Differences among genotypes in Km (1–1 > 1–4 > 4–4) and thermal stability (4–4 > 1–4 > 1–1) followed a pattern consistent with temperature adaptation. In nature, Tb was closely related to Ta. Hsp70 expression in adult beetles decreased with elevation and differed among drainages (BPC > BC > RC). After laboratory acclimation (8 days, 20°C day, 4°C night) and heat shock (4 h, 28–36°C), Hsp70 expression was greater for RC than BPC beetles. In RC, field-collected beetles homozygous for PGI 1–1 had higher Hsp70 levels than heterozygotes or a 4–4 homozygote. These results reveal functional and physiological differences among PGI genotypes, which suggest that montane populations of this beetle are locally adapted to temperature.

Trichostatin A reverses skewed expression of CD154, interleukin-10, and interferon-γ gene and protein expression in lupus T cells

In systemic lupus erythematosus (SLE), T helper cells exhibit increased and prolonged expression of cell-surface CD40 ligand (CD154), spontaneously overproduce interleukin-10 (IL-10), but underproduce interferon-gamma (IFN-γ). We tested the hypothesis that the imbalance of these gene products reflects skewed expression of CD154, IL-10, and IFN-γ genes. Here, we demonstrate that the histone deacetylase inhibitor, trichostatin A, significantly down-regulated CD154 and IL-10 and up-regulated IFN-γ gene expression in SLE T cells. This reversal corrected the aberrant expression of these gene products, thereby enhancing IFN-γ production and inhibiting IL-10 and CD154 expression. That trichostatin A can simultaneously reverse the skewed expression of multiple genes implicated in the immunopathogenesis of SLE suggests that this pharmacologic agent may be a candidate for the treatment of this autoimmune disease.

Use of Ubiquitin Fusions to Augment Protein Expression in Transgenic Plants1

A major goal of plant biotechnology is the production of genetically engineered crops that express natural or foreign proteins at high levels. To enhance protein accumulation in transgenic plants, we developed a set of vectors that express proteins and peptides as C-terminal translational fusions with ubiquitin (UBQ). Studies of several proteins in tobacco (Nicotiana tabacum) showed that: (a) proteins can be readily expressed in plants as UBQ fusions; (b) by the action of endogenous UBQ-specific proteases (Ubps), these fusions are rapidly and precisely processed in vivo to release the fused protein moieties in free forms; (c) the synthesis of a protein as a UBQ fusion can significantly augment its accumulation; (d) proper processing and localization of a protein targeted to either the apoplast or the chloroplast is not affected by the N-terminal UBQ sequence; and (e) single amino acid substitutions surrounding the cleavage site can inhibit in vivo processing of the fusion by Ubps. Noncleavable UBQ fusions of β-glucuronidase became extensively modified, with additional UBQs in planta. Because multiubiquitinated proteins are the preferred substrates of the 26S proteasome, noncleavable fusions may be useful for decreasing protein half-life. Based on their ability to augment protein accumulation and the sequence specificity of Ubps, UBQ fusions offer a versatile way to express plant proteins.

Inflammation-induced recombinant protein expression in vivo using promoters from acute-phase protein genes.

We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal luciferase expression observed prior to inflammatory challenge, markedly increased expression from the C3 promoter was detected in liver in response to both lipopolysaccharide (LPS) and turpentine, and lower-level inducible expression was also found in lung. In contrast, expression from the SAA3 promoter was found only in liver and was much more responsive to LPS than to turpentine. After LPS challenge, hepatic luciferase expression increased rapidly and in proportion to the LPS dose. Use of cytokine-inducible promoters in gene transfer vectors may make it possible to produce antiinflammatory proteins in vivo in direct relationship to the intensity and duration of an individual's inflammatory response. By providing endogenously controlled production of recombinant antiinflammatory proteins, this approach might limit the severity of the inflammatory response without interfering with the beneficial components of host defense and immunity.

Correlation of non-uniform protein expression with variation in transmitter release probability

The strength of synaptic transmission is highly variable between different synapses. The present study examined some factors that may contribute to this variation in the strength of neurotransmission in sympathetic varicosities of the mouse vas deferens. Transmitter release was measured using a focal macropatch electrode placed over pairs of visualised varicosities. By regulating the calcium concentration of the solutions inside the recording electrode and in the bath independently of each other, transmitter release was restricted to one or two surface varicosities at each recording site. Using this technique, transmitter release probability was shown to be highly variable, even between adjacent varicosities on single axon branches. Very little variation was observed in the calcium influx following single impulse nerve stimulation between adjacent Oregon Green BAPTA-1 loaded varicosities. However, the staining intensities of three vesicular proteins, SV2, synaptophysin, and synaptotagmin 1, showed considerable variation between adjacent varicosities on single axon branches. This variation in staining intensity may be partly explained by variation in the density of synaptic vesicles. However, double staining experiments using two vesicular antigens showed some varicosities staining for one vesicular antigen, but not for the second, suggesting that the expression of these release machinery proteins is regulated locally within the varicosities. The results of the present study strengthen suggestions that synaptic strength is at least in part, regulated by variation in the expression of vesicular proteins. (C) 2004 Wiley-Liss, Inc.

Genomic and proteomic analysis of synaptic protein expression in the brains of human alcoholics

Alcoholism results in changes in the human brain which reinforce the cycle of craving and dependency, and these changes are manifest in the pattern of expression of mRNA and proteins in key cells and brain areas. Long-term alcohol abuse also results in damage to selected regions of the cortex. We have used cDNA microarrays to show that less than 1% of mRNA transcripts differ signifi cantly between cases and controls in the susceptible area and that the expression profi le of a subset of these transcripts is suffi cient to distinguish alcohol abusers from controls. In addition, we have utilized a 2D gel proteomics based approach to determine the identity of proteins in the superior frontal cortex (SFC) of the human brain that show differential expression in controls and long term alcohol abusers. Overall, 182 proteins differed by the criterion of > 2-fold between case and control samples. Of these, 139 showed signifi cantly lower expression in alcoholics, 35 showed signifi cantly higher expression, and 8 were new or had disappeared. To date 63 proteins have been identifi ed. The expression of one family of proteins, the synucleins, has been further characterized using Real Time PCR and Western Blotting. The expression of alpha-synuclein mRNA was signifi cantly lower in the SFC of alcoholics compared with the same area in controls (P = 0.01) whereas no such difference in expression was found in the motor cortex. The expression of beta- and gamma- synuclein were not signifi cantly different between alcoholics and controls. In contrast, the pattern of alphasynuclein protein expression differs from that of the corresponding RNA transcript. Because of the key role of synaptic proteins in the pathogenesis of alcoholism, we are developing 2-D DIGE based techniques to quantify expression changes in synaptosomes prepared from the SFC of controls and alcoholics.