Hiroshi Miyanaka

Changes in gene expression of growth factors and their receptors during castration-induced involution and androgen-induced regrowth of rat prostates

To find candidates for the mediator of the growth‐promoting action of androgen in rat prostates, the changes in the steady‐state levels of mRNAs coding for several growth factors and their receptors were examined by Northern blot analysis during castration‐induced involution, and subsequent regrowth induced by androgen in the ventral and dorsolateral lobes. The changes in the growth factor systems and a typical secretory protein in the ventral lobe were similar to, but more prominent than, those in the dorsolateral lobe, showing the higher androgen dependency of the ventral lobe. Among the growth factors and their receptors investigated, only epidermal growth factor (EGF) showed apparent positive androgen dependency: EGF mRNA content in the ventral lobe decreased to about 30% of the normal level within 24 hr after castration, and increased, attaining about 200–300% of the normal level 3–5 days after androgen administration to castrated rats. mRNAs coding for all other factors examined, i.e., transforming growth factor‐α (TGF‐α), EGF receptor, basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), FGF receptor 1, TGF‐β1, TGF‐β type II receptor, hepatocyte growth factor (HGF), and c‐MET/HGF receptor, increased after castration in greater or lesser degree, and after a brief pause or a decrease some of them increased again attaining a second peak 3–5 days after androgen replacement. The second increase was evident in TGF‐α, EGF receptor, KGF, and c‐MET mRNAs. These results indicate the possibility that multiple growth factor‐receptor systems participate in the androgen‐dependent regrowth of castrated rat prostates.

Blood-brain barrier disruption in the hypothalamus of young adult spontaneously hypertensive rats

Vascular permeability and endothelial glycocalyx were examined in young adult spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), and Wistar Kyoto rats (WKY) as a control, in order to determine earlier changes in the blood-brain barrier (BBB) in the hypothalamus in chronic hypertension. These rats were injected with horseradish peroxidase (HRP) as an indicator of vascular permeability. Brain slices were developed with a chromogen and further examined with cationized ferritin, a marker for evaluating glycocalyx. Staining for HRP was seen around vessels in the hypothalamus of SHR and SHRSP, but was scarce in WKY. The reaction product of HRP appeared in the abluminal pits of endothelial cells and within the basal lamina of arterioles, showing increased vascular permeability in the hypothalamus of SHR and SHRSP, whereas there were no leaky vessels in the frontal cortex of SHR and SHRSP, or in both areas of WKY. The number of cationized ferritin particles binding to the capillary endothelial cells was decreased in the hypothalamus of SHR and SHRSP, while the number decreased in the frontal cortex of SHRSP, compared with those in WKY. Cationized ferritin binding was preserved in some leaky arterioles, while it was scarce or disappeared in other leaky vessels. These findings suggest that BBB disruption occurs in the hypothalamus of 3-month-old SHR and SHRSP, and that endothelial glycocalyx is markedly damaged there without a close relationship to the early changes in the BBB.

Blood-brain barrier damage in vascular dementia.

New findings on flow or drainage pathways of brain interstitial fluid and cerebrospinal fluid have been made. The interstitial fluid flow has an effect on the passage of blood‐borne substances in the brain parenchyma, especially in areas near blood‐brain barrier (BBB)‐free regions. Actually, blood‐borne substances can be transferred in areas with intact BBB function, such as the hippocampus, the corpus callosum, periventricular areas, and medial portions of the amygdala, presumably through leaky vessels in the subfornical organs or the choroid plexus. Increasing evidence indicates that dysfunction of the BBB function may play a significant role in the pathogenesis of vascular dementia. Accordingly, we have examined which insults seen in patients suffering from vascular dementia have an effect on the BBB using experimental animal models exhibiting some phenotypes of vascular dementia. The BBB in the hippocampus was clearly deteriorated in Mongolian gerbils exposed to acute ischemia followed by reperfusion and also in stroke‐prone spontaneously hypertensive rats (SHRSP) showing hypertension. The BBB in the corpus callosum was clearly deteriorated in Wistar rats with permanent ligation of the bilateral common carotid arteries showing chronic hypoperfusion. The BBB in the hippocampus and the olfactory bulb was mildly deteriorated in aged senescence accelerated prone mice (SAMP8) showing cognitive dysfunction. The BBB in the hippocampus was mildly deteriorated in aged animals with hydrocephalus. Mild endothelial damage was seen in hyperglycemic db/db mice. In addition, mRNA expression of osteopontin, matrix metalloproteinase‐13 (MMP‐13), and CD36 was increased in vessels showing BBB damage in hypertensive SHRSP. As osteopontin, MMP‐13 and CD36 are known to be related to brain injury and amyloid β accumulation or clearance, BBB damage followed by increased gene expression of these molecules not only contributes to the pathogenesis of vascular dementia, but also bridges the gap between vascular dementia and Alzheimers disease.

Clearance of beta-amyloid in the brain.

Intravascular substances invade extracellular spaces in the brain via endothelial cells in the sites without bloodbrain barrier (BBB) and move not only in the cerebrospinal fluid (CSF) but also in the interstitial fluid (ISF) of brain parenchyma adjacent to non-BBB sites. It is likely that CSF drains directly into the blood via arachnoid villi and granulations and also to lymph nodes via subarachnoid spaces in the brain and nasal lymphatics, whereas ISF drains to cervical lymph nodes through pathways along vascular wall of capillaries and arteries. As the supposed pathways of fluids seem to be critical for the maintenance of normal brain function, it is reasonable to suspect that an obstacle to the passage of fluids through these pathways likely induces some kinds of brain dysfunction such as Alzheimers disease. According to assumed pathways for the elimination of amyloid-β (Aβ) from the brain, Aβ peptides produced mainly in neurons are degraded by peptidases, flow out of the brain parenchyma into the blood through efflux transporters located in cerebral vessels, drain through perivascular pathways into the cervical lymph nodes, or are taken up by some kinds of cells in the brain. As for the perivascular pathways, ISF including Aβ peptides diffuses in the extracellular spaces of the brain parenchyma, enters basement membranes of capillaries, passes into the tunica media of arteries, and drains out of the brain. In this review, these pathways for the clearance of fluids including Aβ from the brain into the blood are briefly reviewed and the relationship between dysfunction of these pathways and brain diseases is discussed.

Transient up-regulation of a novel member of Spot 14 family in androgen-stimulated rat prostate

The rat prostate is dependent on androgen for growth and differentiation. In an effort to find novel genes involved in androgen-induced growth of the rat prostate, we carried out PCR-based subtractive hybridization and identified several factors that were transiently up-regulated after androgen stimulation in castrated rat prostate. Among them, a novel member of the Spot 14 family has been identified. This protein (Spot 14-like androgen-inducible protein, SLAP) exhibited the highest homology (about 50%) with zebrafish gastrulation-specific G12 protein and about 30% homology with rat Spot 14. The SLAP mRNA level decreased following castration and transiently increased after testosterone replacement, attaining a peak 3 days after the treatment. The change in the protein level was similar to that in mRNA except that it was low in both untreated and castrated rat prostate tissue. In normal adult rats, SLAP was expressed at relatively high levels in the lung, stomach and liver. Unlike the prostate, SLAP expression in the lung was not affected by the androgen status. Like other members of the Spot 14 family, SLAP has a leucine-zipper motif in its C-terminal region, making it possible to form a stable homodimer. Though the physiological role of SLAP remains to be clarified, the current results suggest that SLAP expression is associated with some growth-related processes in regrowing rat prostate.

An anti-probasin monoclonal antibody recognizes a novel 40-kDa protein localized in rat liver and a specific region of kidney urinary tubule

Immunoblotting with a monoclonal antibody against probasin (rat prostatic secretory protein) showed that a 40-kDa protein antigenically related to probasin was localized in rat liver and kidney. The contents of probasin in these organs were negligible. Immunostaining revealed that the 40-kDa protein (probasin-related antigen: PRB-RA) was expressed in the liver parenchymal cells and the kidney urinary tubular epithelial cells in outer stripe. The content of PRB-RA in the kidney was low during 0 to 2 weeks of age, then rapidly increased about 10-fold from 2 to 8 weeks of age. The content in the liver increased about 2-fold during the period, reaching a value of 10-12 ng/micrograms protein, which was ten times higher than that in the kidney. PRB-RA was purified from rat liver by ion-exchange chromatography, gel filtration and fast protein liquid chromatography on a hydroxyapatite column. The purified protein formed insoluble aggregates in the absence of a detergent, and it had a blocked amino terminal. The amino acid sequence of a peptide generated by tryptic digestion of alkylated PRB-RA was determined. Computer analysis showed that there was no protein having a significant homology with the peptide. These results indicate that a novel 40-kDa protein with a structural similarity to probasin is localized in rat liver and kidney, and might bear a function specific to these organs.

Optimization of the inter-domain structure of galectin-9 for recombinant production

We previously developed a stable form of galectin-9, an immunomodulatory animal lectin with a truncated linker peptide (G9Null), to overcome the protease sensitivity of wild-type galectin-9. G9Null is highly resistant to proteolysis, while the modification marginally improved the low solubility of the wild-type protein. To increase its solubility, we further modified the remaining linker region of G9Null. A 10-amino acid deletion with a single amino acid substitution resulted in an ∼400% increase in solubility and yield without an adverse effect on its biological activity. This mutant protein might be useful for large-scale recombinant production needed for evaluation of the therapeutic potential of galectin-9.

Tissue-specific expression of fucosylated glycosphingolipid species in rat prostate.

Analysis of fucosylated glycoconjugates in rat prostate by lectin blotting with Aleuria aurantia lectin revealed that the tissue contained characteristic low-molecular weight species (prostatic fucosylated glycoconjugates, PFGs). PFGs were detected almost exclusively in the ventral lobe of the prostate among rat tissues examined, and showed an apparent positive androgen dependency, that is, the content of PFGs decreased after castration and then slowly recovered upon androgen replacement. PFGs were resistant to solubilization with Triton X-100, but could be extracted with chloroform-methanol. Treatment of the crude PFG preparation with endoglycoceramidase resulted in the complete disappearance of PFGs. PFGs were also susceptible to α1,3/4-L-fucosidase but not to peptide-N-glycosidase F. Prostate-specific expression of PFGs was confirmed by thin layer chromatography and subsequent blot analysis. These results suggest that PFGs are fucosylated glycosphingolipid species specifically expressed in the rat ventral prostate, and that PFG expression is associated with some androgen-regulated processes in the tissue.