Demonstration of vasoproliferative activity from mammalian retina

Vasoproliferative activity has been demonstrated in extracts of retinas from human, bovine, and feline sources. These retinal extracts are capable of stimulating (a) proliferation and thymidine uptake of bovine vascular endothelial cells in culture and (b) neovascularization on the chick chorioallantoic membrane. Extracts of skeletal muscle, cardiac muscle, and liver lack similar stimulatory activity. The activity is nondialyzable, stable at 56 degrees C, and inactivated at 100 degrees C. Retinal extracts stimulate the proliferation of corneal fibroblasts but have no effect on the proliferation of vascular smooth muscle cells. Indirect evidence suggests the liberation of a vasoproliferative factor from retina in several ocular disorders. The data in this report represent the first direct demonstration of vasoproliferative activity from mammalian retina.     

Identification and validation of a potential lung cancer serum biomarker detected by matrix-assisted laser desorption/ionization-time of flight spectra analysis

Many abnormalities detected in the thorax by routine conventional imaging studies are benign, yet all require further evaluation because of the concern for cancer. To address this deficiency and develop a serum biomarker for lung cancer, we designed a matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) based platform to display the proteins present in the serum of patients with or without lung cancer, and then challenged the scientific community to analyze these data with the aim of determining specific ion signal differences among the resulting spectra. The most statistically significant ion peak identified by the various analysis algorithms that differentiated the serum of patients with lung cancer from the serum of individuals without lung cancer was found at m/z 11,702. We identified the protein responsible for this ion peak as serum amyloid A (SAA; M(r) = 11,682.7) by partial purification followed by in-gel digestion and peptide mapping. By enzyme-linked immunosorbent assay, we showed SAA to be present at 286 ng/mL in the serum of cancer patients vs. 34.1 ng/mL in the serum of individuals without cancer. These data suggest that the combination of MALDI-TOF MS and computer analysis can be a powerful tool in the search for serum biomarkers of lung cancer and other diseases.     

Multiple-level regulation of genes for protocatechuate degradation in Acinetobacter baylyi includes cross-regulation

The bacterium Acinetobacter baylyi uses the branched beta-ketoadipate pathway to metabolize aromatic compounds. Here, the multiple-level regulation of expression of the pca-qui operon encoding the enzymes for protocatechuate and quinate degradation was studied. It is shown that both activities of the IclR-type regulator protein PcaU at the structural gene promoter pcaIp, namely protocatechuate-dependent activation of pca-qui operon expression as well as repression in the absence of protocatechuate, can be observed in a different cellular background (Escherichia coli) and therefore are intrinsic to PcaU. The regulation of PcaU expression is demonstrated to be carbon source dependent according to the same pattern as the pca-qui operon. The increase of the pcaU gene copy number leads to a decrease of the basal expression at pcaIp, indicating that the occupancy of the PcaU binding site is well balanced and depends on the concentration of PcaU in the cell. Luciferase is used as a reporter to demonstrate strong repression of pcaIp when benzoate, a substrate of the catechol branch of the pathway, is present in addition to substrates of the protocatechuate branch (cross-regulation). The same repression pattern was observed for promoter pcaUp. Thus, three promoters involved in gene expression of enzymes of the protocatechuate branch (pobAp upstream of pobA, pcaIp, and pcaUp) are strongly repressed in the presence of benzoate. The negative effect of protocatechuate on pobA expression is not based on a direct sensing of the metabolite by PobR, the specific regulator of pobA expression.     

Public Health Risk Assessment Linked to Climatic and Ecological Change

Disturbances of climatic and ecological systems can present risks to human health, which are becoming more evident from health studies linked to climate variability, landuse change and global climate change. Waterborne disease agents, such as Giardia cysts and Cryposporidium oocysts have been positively correlated with rainfall. El Niño-related extreme weather conditions can have a significant impact on vector- and water-borne diseases. The linkages between weather, terrestrial ecology and human health have been discovered for some diseases, such as rodent-borne hantavirus. Marine ecology also plays a role in determining human health risks, such as from cholera, and other enteric pathogens. Deforestation and ensuing changes in landuse, human settlement, commercial development, road construction, and water control systems singly, and in combination have been accompanied by increases in or emergence of diseases like malaria and schistosomiasis in some regions of the world. Long-term climate change may increase the frequency of heat waves and potentially air pollution episodes, increase the number of extreme weather events, cause coastal flooding and salination of fresh water aquifers, and displace coastal settlements. Ultimately, a two-pronged approach (empirical and modeling studies) is required to better understand these linkages between climato-logical and ecological change as determinants of disease.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

GABAC receptor subunit mRNA expression in the rat superior colliculus is regulated by calcium channels, neurotrophins, and GABAC receptor activity

The distribution of mRNA for the rho2 subunit of the GABA(C) receptor is much broader in organotypic SC cultures than in vivo, suggesting that GABA(C) receptor expression is regulated by environmental factors. Electrophysiological recordings indicate that neurons in SC cultures have functional GABA(C) receptors, although these receptors exhibited smaller conductance than in vivo, probably due to increased rho2 subunit expression. Adding cortical input, treatment with various neuromodulators, and blocking neuronal activity with TTX failed to affect the expression of rho2 subunits. Electrophysiological recordings revealed the presence of spontaneous Ca(2+) currents in SC cultures and preventing these, by treatment with blockers of L-type Ca(2+) channels, caused rho2 mRNA expression to decline to in vivo levels. In contrast, rho1 subunit mRNA levels remained unchanged, indicating that the two subunits are independently regulated. Surprisingly, both tonic activation and blockade of GABA(C) receptors upregulated rho1/rho2 mRNA expression. Further, NGF and BDNF promoted such expression during an early postnatal time window. In vivo, expression of the rho2 mRNA in the SC, and the rho2/rho3 mRNA in the retina increased with age. Expression of the rho2 mRNA in the visual cortex, and the rho1 mRNA in the retina and SC was constant. Subunit mRNA expression was similar in dark-reared animals, indicating that visual experience has no influence. These experiments suggest that GABA(C) receptor expression in the SC is regulated during postnatal development. While visual experience seems to have no influence on GABA(C) receptor subunits, spontaneous calcium currents selectively promote rho2 expression and both rho1 and rho2 are autoregulated both by GABA(C) receptor activity and by neurotrophic factors.     

Multiple-Level Regulation of Genes for Protocatechuate Degradation in Acinetobacter baylyi Includes Cross-Regulation

The bacterium Acinetobacter baylyi uses the branched β-ketoadipate pathway to metabolize aromatic compounds. Here, the multiple-level regulation of expression of the pca-qui operon encoding the enzymes for protocatechuate and quinate degradation was studied. It is shown that both activities of the IclR-type regulator protein PcaU at the structural gene promoter pcaIp, namely protocatechuate-dependent activation of pca-qui operon expression as well as repression in the absence of protocatechuate, can be observed in a different cellular background (Escherichia coli) and therefore are intrinsic to PcaU. The regulation of PcaU expression is demonstrated to be carbon source dependent according to the same pattern as the pca-qui operon. The increase of the pcaU gene copy number leads to a decrease of the basal expression at pcaIp, indicating that the occupancy of the PcaU binding site is well balanced and depends on the concentration of PcaU in the cell. Luciferase is used as a reporter to demonstrate strong repression of pcaIp when benzoate, a substrate of the catechol branch of the pathway, is present in addition to substrates of the protocatechuate branch (cross-regulation). The same repression pattern was observed for promoter pcaUp. Thus, three promoters involved in gene expression of enzymes of the protocatechuate branch (pobAp upstream of pobA, pcaIp, and pcaUp) are strongly repressed in the presence of benzoate. The negative effect of protocatechuate on pobA expression is not based on a direct sensing of the metabolite by PobR, the specific regulator of pobA expression.