Hsp25 and -90 immunoreactivity in the normal rat eye.

PURPOSE: The distributions of heat shock protein (Hsp)25 and -90 in various regions of the rat eye are described to provide a basis for understanding their roles in normal, damaged, and diseased ocular tissues. This work complements the earlier examination of Hsp70 and Hsc70 (the constitutive form). METHODS: Eyes of adult male Sprague-Dawley rats were fixed in methacarn and embedded in paraffin. Sagittal sections (10 microm) through the optic nerve were stained with hematoxylin and eosin, or incubated with anti-Hsp90, anti-Hsp25, or control IgG. Bound antibody was visualized using an avidin-biotin-horseradish peroxidase detection system. RESULTS: Hsp90 immunoreactivity was abundant in the retina, whereas only low levels of Hsp25 were detected there. In the optic nerve, the relative difference in immunoreactivity for the two Hsps was reversed, with Hsp25 being considerably greater than Hsp90. Both Hsps were detected at low levels in the retinal pigment epithelium (RPE), except for that portion within 250 microm of the optic disc, where Hsp25 and -90 immunoreactivities were increased. Similar to the optic nerve, the corneal epithelium showed greater staining for Hsp25 than for Hsp90, and basal cells contained the highest levels of immunoreactivity for both Hsps. In the ciliary body and iris, Hsp25 and -90 were abundant and similarly distributed in the epithelial and stromal layers. CONCLUSIONS: Each of the ocular tissues had distinctive patterns of Hsp25 and -90 immunostaining. These results suggest that the various structures of the eye have unique requirements for the particular chaperoning and supportive functions of these two Hsp families.

Isolation of antibody to human placental alkaline phosphatase (PLAP) from extracts of human placentae.

PROBLEM: Human placental alkaline phosphatase (PLAP) is a unique placental antigen bound to the syncytiotrophoblast, which may be able to elicit a specific immune response in pregnancy. METHOD OF STUDY: Antibody to PLAP was purified from placental extracts by: a) acid elution of membrane vesicles; b) purifying complexes of PLAP with human antibody on monoclonal antibodies to PLAP followed by denaturation of the enzyme; and c) by denaturation of PLAP in placental extracts and purification of antibody to PLAP on PLAP columns. RESULTS AND CONCLUSIONS: Specific antibody to PLAP is present in placental extracts, and is mostly bound to placental membrane preparations. PLAP is therefore immunogenic in pregnancy and could serve as a useful monitor of pregnancy-specific immunological responses. Since a similar enzyme appears in some cancers, it is possible that the immunization against PLAP in pregnancy will help to protect against the development of ovarian and endometrial cancer (the 'fetal antigen' hypothesis).

Constitutive and inducible heat shock protein 70 immunoreactivity in the normal rat eye.

PURPOSE: Distributions for the constitutive and inducible 70-kDa heat shock proteins, Hsc70 and Hsp70, in different parts of the rat eye are likely to be related to the metabolic demands required for absorption and detection of light. This study was conducted to better understand the functions of Hsc70 and Hsp70 in these tissues and to provide a basis for elucidating their contributions to the maintenance and repair of ocular structures subsequent to tissue injury or cellular degeneration. METHODS: Eyes from male Sprague-Dawley rats (200-300 g) were fixed in methacarn and embedded in paraffin. Sagittal sections (10 microm) through the optic nerve were stained with hematoxylin and eosin or incubated with heat shock protein antibody or control IgG. Bound antibody was visualized using an avidin- biotin- horseradish peroxidase detection system. RESULTS: Hsc70 immunoreactivity was detected in all layers of the retina, except the outer segments. In the retinal pigment epithelium, staining was restricted to cells near the optic nerve-retina junction. Intense staining was also observed in glial nuclei of the optic nerve, whereas weaker staining was observed in the basal and wing cells of the limbal and corneal epithelia. In contrast, Hsp70 immunoreactivity was restricted to the outer nuclear layer and inner segments of the retina. Hsp70 staining was also prominent in basal and wing cells of the limbal cornea and to a lesser extent in the central corneal epithelium. The optic nerve was Hsp70 negative. CONCLUSIONS: Hsc70 and Hsp70 have distinct distributions in the normal rat eye, which imply regional and cell-specific functions.

Glial cell line-derived neurotrophic factor promotes the survival and morphologic differentiation of Purkinje cells.

Glial cell line-derived neurotrophic factor (GDNF) promotes survival of midbrain dopaminergic neurons and motoneurons. Expression of GDNF mRNA in cerebellum raises the possibility that cells within this structure might also respond to GDNF. To examine potential trophic activities of GDNF, dissociated cultures of gestational day 18 rat cerebellum were grown for < or = 21 days in the presence of factor. GDNF increased Purkinje cell number without affecting the overall number of neurons or glial cells. A maximal response (50% above control) was elicited with GDNF at 1 pg/ml. Effects of GDNF on Purkinje cell differentiation were examined by scoring the morphologic maturation of cells in treated and control cultures. GDNF increased the proportion of Purkinje cells that displayed relatively mature morphologies, characterized by dendritic thickening and the development of spines and filopodial extensions. Morphologic maturation of the overall neuronal population was unaffected. In sum, our data indicate that GDNF is a potent survival and differentiation factor for Purkinje cells, the efferent neurons of cerebellar cortex. Together with its other actions, these findings raise the possibility that GDNF might be a critical trophic factor at multiple loci in neuronal circuits that control motor function.

In vitro studies of glucocorticoid effects on neurons and astrocytes.

Studies using immunocytochemistry and RNase protection assay demonstrate that glucocorticoid and mineralocorticoid receptors (GR, MR) and their corresponding mRNAs are co-expressed in hippocampal neurons cultured in serum-free, defined medium and at lower levels in cultured astrocytes. Addition of serum or medium conditioned by astrocytes increases the levels of MR mRNA, but has little effect on the levels of GR mRNA. Cellular levels of both GR mRNA and MR mRNA are upregulated by growth of embryonic hippocampal neurons in corticosterone. This is in distinct contrast to regulation of receptor expression in vivo where mRNAs for these receptors are downregulated in the rat hippocampus by corticosterone treatment of the adult adrenalectomized rat. However, in cultured astrocytes, GR and MR mRNAs are also downregulated by corticosterone. To begin to define the role of glucocorticoids in gene expression in astrocytes, we have used giant two-dimensional (2D) gel electrophoresis to separate astrocyte cellular proteins and translation products synthesized in vitro from astrocyte poly A+ RNA. Analysis of approximately 1,500 in vitro translation products by giant 2D gel electrophoresis reveals 11 protein inductions and 1 repression that occur at the level of mRNA in the absence of protein synthesis following treatment of astrocytes with corticosterone. Interestingly, these changes appear to be mediated by GR, but not by MR. The in vitro studies described here are relevant to identifying the role of GR and MR in gene expression in specific cell types in the hippocampus.

Retinal ganglion cell survival is promoted by genetically modified astrocytes designed to secrete brain-derived neurotrophic factor (BDNF).

Genetically engineered cells carrying genes for neurotrophic factors have potential application for treatment of neurodegenerative diseases and injuries to the nervous system. Brain-derived neurotrophic factor (BDNF) promotes the survival of specific neurons, including retinal ganglion cells (RGC). To determine whether genetically engineered astrocytes might be used for delivering bioactive BDNF, we infected primary type 1 rat astrocytes with a retrovirus harboring a human prepro-BDNF cDNA and assayed the medium conditioned by these astrocytes for effects on survival of rat RGCs in vitro. High levels of BDNF mRNA were expressed by infected astrocytes, but not by control astrocytes as determined by RNase protection assay using a BDNF specific probe. To test for secretion of bioactive BDNF from the transgenic astrocytes, embryonic day 17 rat retinas were dissociated and grown in medium conditioned (CM) for 24 h by astrocytes infected with a replication deficient retrovirus carrying BDNF, NGF, or alkaline phosphatase (AP) cDNA. After 3 days, the number of Thy-1 immunoreactive RGCs was counted. BDNF astrocyte CM significantly enhanced RGC survival by 15-fold compared to the AP control. NGF astrocyte CM had no significant effect. The rate of BDNF secretion was estimated at 83-166 pg/10(5) cells/h. This study demonstrates that astrocytes can be genetically engineered to synthesize and secrete bioactive BDNF. These techniques may be applicable to rescuing neurons from degenerative processes and also for enhancing their survival following transplantation.

Five open reading frames upstream of the dnaK gene of E. coli.

The nucleotide sequence of 3.17 kb of DNA upstream of the dnaK gene of E. coli was determined. Analysis of the sequence indicated the presence of five open reading frames (ORFs), two of which are coded on one strand and three on the other. ORFs 3 and 4 appear to constitute an operon. One of the ORFs (htgA) is preceded by a -10 promoter sequence which is identical to that of the dnaK sigma 32 P1 promoter, and thus suggests that htgA could be a heat-shock gene. ORF4 and htgA overlap each other on opposite strands of DNA, this would offer some interesting regulatory possibilities.

Identification of a gene, closely linked to dnaK, which is required for high-temperature growth of Escherichia coli.

We have constructed four deletion derivatives of the cloned dnaK gene. Plasmid pDD1, in which the last 10 amino acids of the DnaK protein have been replaced by three different amino acids derived from the pBR322 vector, was as effective as plasmid pKP31, from which it was derived, in restoring the ability of a dnaK null mutant, Escherichia coli BB1553, to plate lambda phage and to grow at high temperatures. The other three mutations, involving much larger deletions of the dnaK gene, did not restore the ability to plate lambda phage or the ability to grow at high temperatures. Plasmid pKUC2, which contains the whole dnaK gene and its promoters, was capable of restoring the ability of E. coli BB1553 to plate lambda phage but, surprisingly, it did not restore the ability to grow at high temperatures, even though it was shown that the DnaK protein was efficiently expressed in these cultures. By transposon mutagenesis and sub-cloning, we have shown the presence of a second gene in plasmid pKP31 which is required for high-temperature growth of E. coli BB1553. This gene, which we call htg A, is presumably also defective in the dnaK null mutant E. coli BB1553. We have also demonstrated that the inability of E. coli K756 to grow above 43.5 degrees C is complemented by sub-clones which contain the htg A gene, but not by plasmid pKUC2.