Differentiation and angiogenic growth factor message in two mammalian lens epithelial cell lines.

Lens epithelial cells in culture can sometimes be induced to form spheroid aggregates termed lentoid bodies, composed of cells exhibiting various characteristics of the more highly differentiated lens fiber cells. However, lentoid bodies are often slow to form, and the ability to produce them declines with serial subculture. It was therefore of interest to establish and/or characterize lens epithelial cell lines capable of forming lentoid bodies. The differentiation state was assessed in lentoid bodies formed by each of two lens epithelial cell lines, the transformed alpha TN4 cell line from mouse and the nontransformed N/N1135A cell line from rabbit. Lentoid and monolayer cultures of each cell line were examined for transcripts of the lens-specific alpha A-crystallin ("alpha A"), gamma D-crystallin ("gamma D"; formerly gamma 1-crystallin) and MP26 genes. alpha TN4 lentoid bodies contained 2.5 times the alpha A RNA found in monolayer cells, but lacked detectable gamma D and MP26 RNA. None of the three markers were detected in either lentoid or monolayer N/N1135A cultures grown under the conditions described. Lentoid body formation alone, therefore, does not indicate the extent of differentiation occurring. At least some of the changes in cell adhesion occurring during lentoid body formation involve laminin-like and fibronectin-like interactions, and are reminiscent of those observed during embryonic lens formation. Finally, vascular endothelial growth factor mRNA was absent from the lens but present in alpha TN4 cells, suggesting a mechanism whereby the lens tumors of the founder mouse became vascularized.

Cellular retinoic acid-binding protein messenger RNA: levels in rat tissues and localization in rat testis.

Studies were conducted to explore the tissue- and cell-specific regulation of cellular retinoic acid-binding protein (CRABP) expression in the rat. Two studies were carried out. The first explored the regulation of CRABP mRNA levels in selected rat tissues by dietary retinoid status, and the relationship between CRABP mRNA and protein levels in different tissues. The second examined the cellular localization of CRABP expression in the testis. In order to conduct these experiments, a cDNA encoding CRABP was isolated and characterized. The DNA sequence of the coding region had 96% identity with that of the mouse CRABP cDNA and encodes a protein identical to mouse and bovine CRABP. CRABP mRNA and protein levels were quantified in five tissues from normal, retinoid-deficient, and retinol-repleted rats. Tissue CRABP and CRABP mRNA levels were highly correlated (P less than 0.01) indicating that inter-tissue variability of CRABP levels mainly results from regulation of CRABP mRNA levels. Neither CRABP protein nor mRNA levels were affected by retinol deficiency, in marked contrast with results previously demonstrated with cellular retinol-binding protein (CRBP) (J. Lipid Res. 1990. 31: 821-829). 35S-labeled CRABP cRNA probes were used to localize CRABP mRNA within the testis of adult rats by in situ hybridization. CRABP mRNA was localized selectively in the periphery of the seminiferous tubules, primarily in type A spermatogonia. The localization of CRABP mRNA differs from that of CRABP protein, which is known to be enriched in maturing and more mature germinal cells. This difference suggests that CRABP in germ cells may be highly stable, remaining in the maturing germ cells without degradation long after CRABP mRNA levels have declined to very low levels. The specific localization of CRABP mRNA and protein presumably reflects the biological roles of retinoic acid in the development and/or later function of germinal cells.

The response time of transcription and translation of the leu-2 gene of Neurospora to its inducer, alpha-isopropylmalate, approaches the permissible minimum.

The rate of transcription and translation of the leu-2 gene of Neurospora crassa was measured after induction by alpha-isopropylmalate. Little message of enzyme was found before inducer addition but transcription in the lower eukaryote was found well underway within five minutes after inducer addition, followed in a minute or two by the appearance of functional enzyme. The timing was close to the limit set by RNA synthesis and ribosome procession. As a consequence, it seems unlikely that traversal of the cell and/or nuclear membranes by the inducer and message involves intermediate synthetic reactions and that the leu-3 positive regulatory element is fully available for participation in the induction process before the inducer is added. A significant overshoot in message synthesis was found early in the induction process. This is discussed with respect to previously observed effects of the inducer on general RNA synthesis.

Specific regulatory interconnection between the leucine and histidine pathways of Neurospora crassa.

Leucine auxotrophs of Neurospora fall into two discrete categories with respect to sensitivity to the herbicide, 3-amino-1,2,4-triazole. The pattern of resistance corresponds exactly to the ability to produce the leucine pathway control elements, alpha-isopropylmalate and the leu-3 product. An analysis of the regulatory response of the production of enzymes of histidine biosynthesis to alpha-isopropylmalate implicates the control elements of the leucine pathway as important components of the mechanism governing the production of the target enzyme of aminotriazole inhibition, imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19). The evidence suggests that the regulatory interconnection between the two pathways is direct and is independent of other general integrating regulatory mechanisms which appear to be operative in both pathways. A general method for isolating leu-1 and leu-2, as well as other regulatory mutants, is described, which takes advantage of the specificity of the resistance to the inhibitor. Use of analogous systems is prescribed for the analysis of other regulatory interconnections which, like this one, might not be anticipated directly from structural or biosynthetic considerations.

The action of piperidine on muscle spindles in the rat.

Piperidine hydrochloride induced discharge in primary and secondary endings of muscle spindles in rat tailbase muscle preparations. The threshold concentration for primary endings (2.0 X 10(-5) g/ml) was below that for secondary endings (4.0 X 10(-5) g/ml). The effect of piperidine was antagonised by tubocurarine but not by atropine. The action of piperidine on primary and secondary endings responding to 'ramp-and-hold' stretch was predominantly on the static component of excitation, and usually in the absence of any concomitant enhancement of the dynamic effect. This effect was opposite to that induced by succinylcholine. A possible action of piperidine at cholinoceptive sites of the nicotinic type and associated with gamma-trial fusimotor terminals is discussed.

A characterisation of spindle afferents from the intertransverse tail muscles in the rat.

The conduction velocities of myelinated afferent axons from the intersegmental muscles at the tail-base in rat range from 5.0--43.0 m-sec-1, and have a bimodal distribution with a boundary between the groups at 25.0 m-sec-1. Calculations of the dynamic indices of spindle endings, observed when stretching the muscle, show that axons from primary endings conduct at velocities above 28.0 m-sec-1 and those from secondary endings at below 24.0 m-sec-1. These characteristics resemble those of spindles in the rat hind-limb muscles, and do not compare with those in the more distally situated segmental tail muscles.