Transcriptional up-regulation of the protooncogenes c-fos and c-jun following vitreous removal and short-term in vitro culture of bovine lenses.

Chemical (mainly oxidative) and mechanical (anterior capsule injury) stresses have been reported to up-regulate the expression of the protooncogenes c-fos and c-jun in the lens. Another potentially stressful, yet largely unexplored condition, inherent to all experiments requiring the in vitro culturing of isolated lenses, is vitreous removal. Based on the results of an extensive RNA gel blot analysis conducted on epithelial/capsule preparations isolated from calf lenses dissected and cultured under different conditions, we show, here, that lens isolation and short-term culture (1-2.5 hr, without any significant GSH depletion) result in a strong and time-dependent up-regulation of the c-jun and c-fos mRNAs. This response, which relies on transcriptional protooncogene activation and is more intense for c-fos than for c-jun, is in part prevented by the preservation of the lens-vitreous contact, but not by the culture of vitreous-stripped lenses on a vitreous bed. Supplementation of the culture medium with the antioxidant N -acetyl-cysteine slightly reduced the c-jun, but not the c-fos response. Protooncogene up-regulation thus appears to be mainly determined by the disruption of critical lens-vitreous interactions. Since this response takes place in the epithelial cells, these data also point to the existence of a communication mechanism whereby a posteriorly applied mechanical stress is transmitted to, and perceived by, the anterior lens surface.

A plant 3'-phosphoesterase involved in the repair of DNA strand breaks generated by oxidative damage.

Two novel, structurally and functionally distinct phosphatases have been identified through the functional complementation, by maize cDNAs, of an Escherichia coli diphosphonucleoside phosphatase mutant strain. The first, ZmDP1, is a classical Mg(2+)-dependent and Li(+)-sensitive diphosphonucleoside phosphatase that dephosphorylates both 3'-phosphoadenosine 5'-phosphate (3'-PAP) and 2'-PAP without any discrimination between the 3'- and 2'-positions. The other, ZmDP2, is a distinct phosphatase that also catalyzes diphosphonucleoside dephosphorylation, but with a 12-fold lower Li(+) sensitivity, a strong preference for 3'-PAP, and the unique ability to utilize double-stranded DNA molecules with 3'-phosphate- or 3'-phosphoglycolate-blocking groups as substrates. Importantly, ZmDP2, but not ZmDP1, conferred resistance to a DNA repairdeficient E. coli strain against oxidative DNA-damaging agents generating 3'-phosphate- or 3'-phosphoglycolate-blocked single strand breaks. ZmDP2 shares a partial amino acid sequence similarity with a recently identified human polynucleotide kinase 3'-phosphatase that is thought to be involved in DNA repair, but is devoid of 5'-kinase activity. ZmDP2 is the first DNA 3'-phosphoesterase thus far identified in plants capable of converting 3'-blocked termini into priming sites for reparative DNA polymerization.

Oxidative stress and age-related cataract.

The authors review the available evidence supporting the possible role of oxidative stress in cataract formation from an epidemiological and a clinical point of view. They discuss in more detail what is presently known about the molecular mechanisms of response of the mammalian lens to an oxidative insult and report unpublished data on gene modulation upon oxidative stress in a bovine lens model. Main research endeavors that seem to be a most promising source of new insights into the problem of age-related cataract formation are briefly discussed.

Coordinate modulation of maize sulfate permease and ATP sulfurylase mRNAs in response to variations in sulfur nutritional status: stereospecific down-regulation by L-cysteine.

To gain insight into the regulatory mechanisms and the signals responsible for the adaptation of higher plants to conditions of varying sulfate availability, we have isolated from a sulfate-deprived root library maize cDNAs encoding sulfate permease (ZmST1) and ATP sulfurylase (ZmAS1), the two earliest components of the sulfur assimilation pathway. The levels of ZmST1 and ZmAS1 transcripts concomitantly increased in both roots and shoots of seedlings grown under sulfate-deprived conditions, and rapidly decreased when the external sulfate supply was restored. This coordinate response, which was not observed under conditions of limiting nitrate or phosphate, correlated with the depletion of glutathione, rather than sulfate stores. However, drastically reducing glutathione levels through treatment with buthionine sulfoximine, a specific inhibitor of gamma-glutamyl cysteine synthetase, did not provide an adequate stimulus for the up-regulation of either sulfate permease or ATP sulfurylase messengers. Indeed, L-cysteine, but not D-cysteine, effectively down-regulated both transcripts when supplied to sulfur-deficient seedlings under conditions of blocked glutathione synthesis. Altogether, these data provide evidence for the coordinate regulation of sulfur assimilation mRNAs in higher plants and for the glutathione-independent involvement of cysteine as a stereospecific pretranslational modulator of the expression of sulfur status-responsive genes.

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display.

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

The DNA-binding activity of transcription factor PTF1 parallels the synthesis of pancreas-specific mRNAs during mouse development.

We have studied the expression of the alpha-amylase, trypsin, and elastase II genes in the acinar pancreas during mouse development. Transcriptional control is the major mechanism by which the differential accumulation of alpha-amylase, trypsin, and elastase II mRNAs is determined during late embryogenesis. The synthesis of pancreatic mRNAs is detected around day 15 of gestation and involves most if not all acinar cells. The DNA-binding activity of the pancreas-specific transcription factor PTF1, which binds to enhancers of genes expressed in this tissue, is detected for the first time at day 15 of gestation. The appearance of the factor at this early stage of development suggests that it plays an important role during pancreas differentiation.

Vitamin A uptake from retinol-binding protein in a cell-free system from pigment epithelial cells of bovine retina. Retinol transfer from plasma retinol-binding protein to cytoplasmic retinol-binding protein with retinyl-ester formation as the intermediate step.

We have investigated the steps by which retinol, released from plasma retinol-binding protein (RBP), enters the cells and is accumulated for the most part as a retinyl-ester, only a small fraction of it being present as a complex with cytoplasmic retinol-binding protein (CRBP). For this purpose, we have developed a cell-free system composed of plasma membrane-enriched fractions from bovine retinal pigment epithelium which selectively incorporates exogenous vitamin A when presented as a retinol-RBP complex. Upon incubation in the presence of [3H]retinol-RBP, isolated plasma membrane fractions take up and esterify retinol. A 4-fold reduction of total vitamin A incorporation is observed in conditions which specifically inhibit retinyl-ester formation, thus indicating that the two processes of retinol uptake and esterification are functionally coupled. Evidence is presented that retinol bound to a plasma membrane receptor sharing functional and structural similarities with CRBP is the actual substrate for esterification. Vitamin A accumulation seems to require retinol esterification to allow the recycling of a limited number of free, plasma membrane-associated, retinol receptors. Mobilization of retinol stored as a membrane-bound retinyl-ester is mediated by a membrane-associated hydrolase activity selectively controlled by the level of apo-CRBP which acts as a carrier for the released retinol. Up to 90% of membrane-bound vitamin A is released upon incubation in the presence of apo-CRBP (11 microM) with concomitant formation of retinol-CRBP. The overall process, in which retinol never needs to leave its binding proteins, allows the accumulation of vitamin A in the form of a membrane-bound retinyl-ester and its regulated mobilization as a retinol-CRBP complex.

Expression of mouse Amy-2a alpha-amylase genes is regulated by strong pancreas-specific promoters.

Three types of Amy-2-related DNA sequences, Amy-2a I, Amy-2a II and Amy-X, exist in the genome of mice of the inbred strain A/J. Amy-2a I and Amy-X are single copy sequences. Amy-2a II occurs as three copies per haploid genome. DNA sequence analysis reveals that both classes of Amy-2a genes specify the same unique pancreatic alpha-amylase mRNA species, since they share common exon sequences. Four independently cloned Amy-2a II isolates were found to be identical in all regions sequenced. This suggests that most, if not all, chromosomal Amy-2a II copies are identical. Amy-X is presumably a pseudogene, since its exon sequences, which are distinct from those of Amy-2a, are not detected in pancreatic alpha-amylase mRNA. We have determined the transcriptional activities of the Amy-2a genes by mapping in vitro elongated nascent transcripts to Amy-2a restriction fragments. Transcription initiation occurs at or close to the cap site. The expression of Amy-2a in vivo is under control of strong promoters, which are active exclusively in the pancreas. The accumulation of alpha-amylase mRNA in cells of the exocrine pancreas is regulated mainly at the transcriptional level. We have searched for pancreatic transcripts of Amy-1a, which specifies both parotid gland and liver-type alpha-amylase mRNAs. Surprisingly, the weak Amy-1a promoter, which directs the synthesis of the mRNA containing the liver-type leader sequence, also is active in the pancreas and, hence, in all alpha-amylase-producing tissues.