Activity-dependent changes in neuropsin gene expression in the hippocampus implies an involvement of neuropsin in neural plasticity. Since the deduced amino acid sequence of the gene contained the complete triplet (His-Asp-Ser) of the serine protease domain, the protein was postulated to have proteolytic activity. Recombinant full-length neuropsin produced in the baculovirus/ insect cell system was enzymatically inactive but was readily converted to active enzyme by endoprotease processing. The activational processing of prototype neuropsin involved the specific cleavage of the Lys 32 -Ile 33 bond near its N terminus. Native neuropsin that was purified with a purity of 1,100-fold from mouse brain had enzymatic characteristics identical to those of active-type recombinant neuropsin. Both brain and recombinant neuropsin had amidolytic activities cleaving Arg-X and Lys-X bonds in the synthetic chromogenic substrates, and the highest specific activity was found against Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. The active-type recombinant neuropsin effectively cleaved fibronectin, an extracellular matrix protein. Taken together, these results indicate that this protease, which is enzymatically novel, has significant limbic effects by changing the extracellular matrix environment.Some proteases have been suggested to be related to neural cell dynamics in such processes as cell death, migration, cellto-cell adhesion and de-adhesion, process elongation, pathfinding, and axonal rearrangement (1-5). These phenomena have been investigated by supplying known proteases involved in blood coagulation, fibrinolysis, or digestion to neural cell cultures. However, the observations that the proteases are mainly localized in and released from non-neural cells do not support all of such neural effects (5-7). Thus, we postulated that neurons themselves may produce and release their own proteases to participate in the neural cell dynamics described above. Neuropsin (NP)1 was cloned from the mouse brain and was shown to be localized in mouse hippocampal pyramidal neurons (8). These results and the observation that its mRNA showed marked activity-dependent changes caused by plasticity-inducible stimuli are suggestive of some neural effects in limbic plasticity (8, 9). However, it is still not known whether NP protein has enzyme activity as suggested by the deduced amino acid sequence (8). We postulated that the enzyme activity might be a molecular basis for the physiological responses induced by various stimuli. Therefore, in the present study, we examined whether recombinant NP (r-NP) and brain NP had proteolytic activity against synthetic and natural substrates. EXPERIMENTAL PROCEDURESMaterials-Mono S, Sepharose 2B, CNBr-activated Sepharose 4B and CL-6B, Superdex-75HR, Superose 12, Resource S, and Protein G-Sepharose were from Amersham Pharmacia Biotech. Silver staining kits were from Bio-Rad. Diisopropyl fluorophosphate (DFP), benzamidine, bestatin, soybean trypsin inhibitor, human plasma thrombin (EC 3.4.4.13), and TNM-FH insect cel...

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The effect of 4-nonylphenol (NP) on cell proliferation and adipocyte formation was examined in cultures of fully differentiated 3T3-L1 cells. Following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for 8 days with or without NP. NP at 5 and 10 microg/ml increased the DNA content by 32% and 68%, respectively, compared with that of the untreated cultures, in which NP was absent during the treatment period. There were many more bromodeoxyuridine (BrdU)-positive cells in the NP-treated cultures, in which NP was present at a concentration of 10 microg/ml during the treatment period, compared to the untreated cultures. These results indicate that NP had the ability to stimulate the proliferation of fully differentiated 3T3-L1 cells. NP at 5 and 10 microg/ml decreased the triacylglycerol (TG) content by 26% and 58%, respectively, and decreased the lipoprotein lipase (LPL) activity by 51% and 71%, respectively. The lipid droplets in individual cells of the NP-treated cultures were smaller than those of the untreated cultures. The mRNA levels of LPL and adipocyte-specific fatty acid binding protein (aP2) were considerably lower in the NP-treated cultures than in the untreated cultures. Thus, NP also had the ability to inhibit adipocyte formation in cultures of fully differentiated 3T3-L1 cells. A study using an antiestrogen ICI 182,780 showed that the NP-stimulated cell proliferation was mediated partly by the estrogen receptor, while the NP-induced inhibition of adipocyte formation was mediated by a mechanism other than the estrogen receptor.

The confluent cultures of 3T3-L1 fibroblasts were treated with or without bisphenol A (BPA) for 2 days and subsequently treated with insulin (INS) alone for 9 days. When BPA was absent during the first 2-day treatment period, the cultures contained 1.6 g/ g DNA of triacylglycerol (TG), 202 mU/mg DNA of lipoprotein lipase (LPL) activity, and 462 nmol/min/mg DNA of glycerol-3-phosphate dehydrogenase (GPDH) activity. The presence of BPA during the same period caused a 150% increase in the TG content, a 60% increase in the LPL activity, and a 500% increase in the GPDH activity. Thus, BPA by itself can trigger 3T3-L1 fibroblasts to differentiate into adipocytes. Next, the confluent cultures were treated with BPA for 2 days and subsequently treated with a combination of INS and BPA for 9 days. The simultaneous presence of BPA with INS caused a 370% increase in the TG content, a 200% increase in the LPL activity, and a 225% increase in the GPDH activity compared with the cultures treated with INS alone. The amount of [ 3 H]thymidine incorporated into DNA was lower in the cultures treated with INS in the presence of BPA than in those treated with INS alone, indicating that BPA has an anti-proliferative activity on 3T3-L1 cells. Taken together, our results indicate that BPA in combination with INS can accelerate the adipocyte conversion.

In order to define the major sites of persistence of human herpesvirus 6 (HHV-6) and HHV-7, PCR with DNAs from more than 100 specimens of 3 different salivary glands was performed. HHV-6 DNA was detected in 52 (88.1%) of 59 submandibular gland, 17 (63.0%) of 27 parotid gland, and 9 (52.9%) of 17 lip salivary gland specimens. On the other hand, HHV-7 DNA was detected in 59 (100%) of 59 submandibular gland, 23 (85.2%) of 27 parotid gland, and 10 (58.8%) of 17 lip salivary gland specimens. These findings demonstrate that salivary glands are a site of persistent infection of both HHV-6 and HHV-7 and that among the three types of salivary gland examined, the submandibular gland is the primary one in which these herpesviruses, especially HHV-7, persist.