Impact of control measures including decolonization and hand hygiene for orthopaedic surgical site infection caused by MRSA at a Japanese tertiary-care hospital.

BACKGROUND: Meticillin-resistant Staphylococcus aureus (MRSA) is the most common pathogen in orthopaedic surgical site infections (SSIs). However, few studies have investigated the transmission process of orthopaedic MRSA SSI. AIM: To investigate the transmission process of orthopaedic MRSA SSI using epidemiological and molecular analyses and to determine a method to prevent MRSA SSI in nosocomial orthopaedic surgery. METHODS: Active MRSA surveillance, preoperative decolonization and contact precautions for MRSA-positive cases was performed at our institution. Changes in epidemic strains were evaluated and the possibility of transmission from patients in an orthopaedic ward of a Japanese tertiary-care hospital was assessed by genotyping stored MRSA strains. In addition, data on the prevalence of MRSA SSI, MRSA colonization, and use of an alcohol antiseptic agent (mL/patient-days) during 2005-2022 were retrospectively assessed. FINDINGS: SCCmec type II strain in the SSI group decreased over time, associated with fewer outbreaks. Even during a period of high infection rates, no cases of transmission-induced SSI from nasal MRSA carriers were identified. The infection rate correlated negatively with the use of an alcohol antiseptic agent (r = -0.82; P < 0.0001). Two cases among five nasal carriers developed MRSA SSI caused by strains different from those related to nasal colonization. CONCLUSION: The infection control measures for transmission from the hospital reservoirs including strict adherence to hand hygiene and decolonization of carriers is likely to be important for the prevention of orthopaedic MRSA SSI. However, the need for contact precautions for decolonized nasal carriers might be low.

Mutated in colorectal cancer, a putative tumor suppressor for serrated colorectal cancer, selectively represses beta-catenin-dependent transcription.

Mutated in colorectal cancer (MCC) was originally identified as a candidate gene for familial adenomatous polyposis (FAP) but further study identified adenomatous polyposis coli (APC) as responsible for FAP and the physiologic/pathologic roles of MCC remained poorly understood. Recently, MCC promoter methylation was discovered as a frequent early event in a distinct subset of precursor lesions and colorectal cancer (CRC) associated with the serrated CRC pathway. Here we provide the first evidence of the biological significance of MCC loss in CRC and the molecular pathways involved. We show MCC expression is dramatically decreased in many CRC cell lines and the distinct subset of sporadic CRC characterized by the CpG island methylator phenotype and BRAF(V600E) mutation due to promoter methylation as reported previously. Importantly, we find MCC interacts with beta-catenin and that reexpression of MCC in CRC cells specifically inhibits Wnt signaling, beta-catenin/T-cell factor/lymphoid-enhancer factor-dependent transcription and cellular proliferation even in the presence of oncogenic mutant APC. We also show that MCC is localized in the nucleus and identify two functional nuclear localization signals. Taken together, MCC is a nuclear, beta-catenin-interacting protein that can act as a potential tumor suppressor in the serrated CRC pathway by inhibiting Wnt/beta-catenin signal transduction.

Transactivation of core binding factor alpha1 as a basic mechanism to trigger parathyroid hormone-induced osteogenesis.

During 28-day culture of bone marrow- and calvaria-derived osteoblasts, the constant presence of parathyroid hormone (PTH)(1-34) retarded differentiation and nodule formation (NF) in a dose-dependent fashion (C-phase). In contrast, addition of PTH(1-34) in late stage cultures (from day 10 to 21) accelerated NF (A-phase). The stable production of such an A-phase allowed us to study the mechanism of bone anabolic action of PTH(1-34). Subcellular localization studies of core binding factor alpha1 (Cbfa1) and reporter assays provided the results indicating that in the A-phase, PTH(1-34) triggers its bone anabolic action via enhancement of Cbfa1 transactivation. RT-PCR and Northern blot analyses revealed that alkaline phosphatase, osteocalcin and bone sialoprotein expression decreased in the C-phase and increased in the A-phase; however, expression of other bone proteins (Cbfa1, PTH/PTH-related peptide-receptor, osteopontin, collagen I alpha1, collagen I alpha2, vitamin K-dependent gamma-glutamyl carboxylase) did not change in a phase transition-related manner. Ovariectomized osteopenic mice, treated with PTH(1-34) (4 and 40 microg/kg, s.c., every other day, 4 or 6 weeks), recovered lost bone, displayed elevated nuclear localization of Cbfal in tibiae without alteration of its cytosolic level and exhibited upregulation of expressions of the same set of proteins (alkaline phosphatase, osteocalcin and bone sialoprotein) in femora. These results obtained by a concerted study in vitro and in vivo suggest that PTH triggers its osteogenic action via promotion of the transactivation of Cbfa1.

The cerebrospinal fluid level of glial fibrillary acidic protein is increased in cerebrospinal fluid from Alzheimer's disease patients and correlates with severity of dementia.

To gain insight of the underlying mechanisms of astroglial response to Alzheimer's disease (AD), the level of glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) from controls and AD subjects were immunochemically determined, and the correlation between that level and dementia severity of AD patients was evaluated. Means and SD of CSF levels of GFAP for the young control group (from 1 to 25 years, mean +/- SD 14.2 +/- 5.0, n = 13) adult control (from 26 to 55 years, 41.6 +/- 10.1, n = 9) and senescent control (older than 56 years, 65.4 +/- 8.0, n = 8) were 2.96 +/- 1.04, 2.80 +/- 1.46 and 3.99 +/- 1.55 ng/ml, respectively, and the CSF level of GFAP was not dependent on age (ANOVA, p = 0.17). While that of the AD patient group (n = 27, 70.8 +/- 8.0 years) was 8.96 +/- 7.80 ng/ml, significantly higher than that of both the all-control (3.19 +/- 1.39 ng/ml, t test, p < 0.001) and age-matched senescent (3.99 +/- 1.55 ng/ml, t test, p < 0.005) control groups. The receiver-operator characteristic (ROC) curve revealed that the GFAP concentration at 5 ng/ml in CSF could serve as a cutoff value. The CSF level of GFAP in the moderately to severely demented patients (MMSE /= 18, 6.85 +/- 5.76 ng/ml, n = 18; ANOVA, p < 0.05). These findings together with our previous report on an increase in the CSF level of apolipoprotein E suggest that degeneration and stimulation of astrocytes takes place concurrently in the AD brain.

Incadronate and etidronate accelerate phosphate-primed mineralization of MC4 cells via ERK1/2-Cbfa1 signaling pathway in a Ras-independent manner: further involvement of mevalonate-pathway blockade for incadronate.

Two types of bisphosphonates (BPs), incadronate (INC) and etidronate (ETI) accelerated phosphate (Pi)-primed mineralization of MC4 cells in a subnanomolar dose range. Intracellular signaling pathways involved were examined. 1) The effect of INC but not ETI was partially suppressed by two mevalonate (MVA) pathway metabolites, farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). 2) The BP-like accelerating effect was produced by statins and also by Toxin B, a Rho GTPases-specific inhibitor. 3) INC induced Cbfa1-nuclear localization within hours; and in an in vivo experiment using ovariectomized mice, its 3 weeks dosing exhibited the same effect in tibial extracts. 4) BPs promoted luciferase expression in murine p1.3-osteocalcin gene 2-luc and p6-osteoblast specific element 2-luc transfected cells, just as MVA, FPP and GGPP did independently and additively to INC. 5) BPs activated extracellular signal-regulated kinase (ERK1/2) in a Ras-independent manner within 5 min, and Pi was found to sensitize MC4 cells to BPs. MVA and its metabolites also activated ERKs but in a Ras-dependent manner and additively to INC. Ras dependency was determined using N17Ras-transfected cells. A MEK (MAP kinase-ERK kinase)-specific inhibitor PD98059 alone partly and with FPP completely blocked INC-induced mineralization. The results suggest that BPs act on Pi-sensitized MC4 cells to accelerate mineralization via nonRas-MEK-ERK1/2-Cbfa1 transactivation pathway and INC additionally acts by inhibiting the MVA pathway.

Phosphate stimulates differentiation and mineralization of the chondroprogenitor clone ATDC5.

ATDC5 cells were employed to examine how inorganic phosphate (Pi) influences chondrocytic bone formation. 1) Pi (3 - 30 mM) plus ascorbic acid (50 microg/ml) dose-dependently accelerated proliferative differentiation and mineralization of ATDC5. 2) Northern blot analysis revealed that 10 mM Pi suppressed expression of type II collagen and PTH (parathyroid hormone) / PTH-related peptide (PTHrP) receptor, while it accelerated type X collagen expression. 3) Pi (3 - 30 mM) dose-dependently increased luciferase activity in the cells transfected with 3000 bp type X collagen promoter fused to the luciferase gene. The results suggest a regulatory role of Pi in endochondral osteogenesis.

Phosphate provides an extracellular signal that drives nuclear export of Runx2/Cbfa1 in bone cells.

Inorganic phosphate (Pi) supplement is generally used to accelerate mineralization of cultured bone cells but the mechanism of action is totally unknown. How the action is related with the transactivation of Runx2/Cbfa1,a master gene product of bone formation,was examined. Clonal bone cells (osteoblastic MC3T3-E1, chondrocytic ATDC5 and osteocytic MLO-Y4) on preculture in ascorbate-containing medium constantly expressed and accumulated Cbfa1 in the nuclei, and subsequent increase of Pi concentration to 3 or 10 mM was found to invariably induce nuclear export (not import) of Cbfa1 which was completed in a few hours. In addition, Pi was found to lower the expression of osteocalcin. Leptomycin B completely inhibited Pi-induced nuclear export, suggesting that CRM1/exportin 1 is involved in Pi-induced nuclear export. The result suggests that bone cells are equipped with a novel Pi sensing mechanism which is functionally linked to a nuclear export system of Cbfa1.

Bone-anabolic effect of salmon calcitonin on glucocorticoid-induced osteopenia in rats.

The anabolic effect of salmon calcitonin (SCT) on skeletal tissue was examined on glucocorticoid-induced osteopenia in female rats (12 weeks old). By the administration of methylprednisolone acetate (MPA: 0.1 mg/kg, s.c., 3 times/week) for 8 weeks, histomorphometrically detectable osteopenia with the characteristics of decreased bone formation and increased bone resorption developed in proximal tibia metaphysis. Serum osteocalcin level was also decreased by MPA treatment. Subsequent treatment with SCT (10 U/kg, s.c., 5 times/week) was found to reverse once developed osteopenia with the return of the osteocalcin level, though rats were still on MPA. Histomorphometry revealed that SCT decelerated bone resorption but augmented bone formation in this osteopenic model. After a single injection of SCT (2.5 U/kg--40 U/kg, s.c.), the serum level of parathyroid hormone (PTH) which had a potent anabolic on bone formation increased in a dose-dependent manner. These results indicate that SCT has a stimulating effect on osteoblastic bone formation and this anabolic effect may at least in part be due to its indirect effect to increase endogenous PTH secretion.

Expression of IFN-gamma in cerebrovascular endothelial cells from aged mice.

Recently, it has become clear that interferon-gamma (IFN-gamma) plays a role in the central nervous system (CNS) as well as in the immune system. However, the reason for the alteration in IFN-gamma production in the brain with aging remains unknown. In this study, we investigated the expression of IFN-gamma in the brain in terms of both mRNA and protein and compared the expression in young adult brain with that in aged mice. The cerebrum and cerebellum were collected from young adult (8-10 weeks old) and aged (24-26 months old) BALB/c mice, and the expressions of IFN-gamma and IFN-gamma receptor-1 (IFNGR-1) mRNA were examined by RT-PCR. Expression of IFN-gamma mRNA was detected in the brains from aged mice but not in those from young adult mice. However, IFNGR-1 mRNA was expressed in the brains from both young adult and aged mice. Moreover, IFN-gamma levels in the cerebrum and cerebellum from aged mice were detectable by ELISA, but IFN-gamma was undetectable in these tissues from young adult mice. To identify the cellular source of IFN-gamma in the brain of aged mice, immunostaining using antimouse IFN-gamma monoclonal antibody (mAb) was done. Immunoreactivity of IFN-gamma appeared to be located in cerebrovascular endothelial cells, including the choroid plexus of the cerebellum from aged mice. Expression of IFN-gamma and IFNGR-1 was also identified in isolated microvessels from brains. These results suggest that IFN-gamma plays a role in age-associated changes.

Age-dependent change in the levels of Abeta40 and Abeta42 in cerebrospinal fluid from control subjects, and a decrease in the ratio of Abeta42 to Abeta40 level in cerebrospinal fluid from Alzheimer's disease patients.

In order to address an age-dependent alteration in the concentration of beta-amyloid polypeptides (Abetas) within the central nervous system and its probable predisposition to amyloidgenesis in Alzheimer's disease (AD), we measured two species of soluble Abetas, Abeta40 and Abeta42, in cerebrospinal fluids (CSF) from randomly selected Japanese control subjects at various ages (n = 33) and then compared these data with those of probable Japanese AD patients (n = 23). CSF concentrations of Abeta40 and Abeta42 peptides were age-dependent (ANOVA, Bonferroni's multiple comparison; p < 0.01 and p < 0.05, respectively) and were lower in the infant than in adults. From mid-20, the Abeta40 concentrations were decreasing while Abeta42 were rather stable. Abetas in CSF from AD patients (n = 23), whose epsilon4 allele frequency of the apolipoprotein E gene was higher than in controls (n = 83, p < 0.03), were not statistically different from those of age-matched controls (n = 13). A linear relationship was detected between the Abeta40 concentration and the Mini-Mental State Examination score (p < 0.05). The ratio of the Abeta42 to the Abeta40 level measured in the AD CSF samples was approximately 38% decreased compared to age-matched controls (p < 0. 05). These data suggest that the physiological metabolism of soluble Abetas in the brain is regulated in an age-dependent manner, and that the ratio of Abeta42 to Abeta40 level in the CSF would be a useful marker for monitoring progression of AD.

Age-dependent decline in the apolipoprotein E level in cerebrospinal fluid from control subjects and its increase in cerebrospinal fluid from patients with Alzheimer's disease.

In order to address the significance of apolipoprotein E (apoE) in the pathogenesis as well as the clinical diagnosis of Alzheimer's disease (AD), we measured its level in cerebrospinal fluid (CSF) from randomly selected Japanese control subjects at various ages (n = 36), which included 14 age-matched controls, and from AD patients including early-onset (n = 11, EOAD) and late-onset (n = 14, LOAD) cases. The CSF apoE level in controls linearly decreased during aging to over 80 years (r(2) = 0.323, p < 0.0001). The CSF apoE level in AD patients was 31.9% elevated compared to the age-matched controls (n = 14, p < 0.05) and linearly increased with a decrement of the patients' Mini Mental State Examination scores. Moreover, the CSF apoE level of EOAD patients (n = 11) was higher than that of LOAD patients (n = 14, p < 0.05), whose APOE epsilon4 allele frequency was significantly higher than that of controls (chi(2) = 7. 16, p < 0.03). Two-dimensional gel electrophoretic analysis of the heparin-Mn(2+)-precipitable lipoprotein fraction in CSFs showed that the ratio between the level of CSF apoA-I and that of CSF apoE of controls was significantly higher than those of all AD and LOAD subjects (p < 0.01, p < 0.05), while the CSF apoA-I-to-apoE ratios of the two AD groups were not significantly different. These results suggest that overproduction of apoE protein may be a consequence of astroglial response to neurodegeneration in AD and that the determination of CSF apoliprotein levels serves as a clinical marker for monitoring the progression of AD.

A unique junctional palindromic sequence in mitochondrial DNA from a patient with progressive external ophthalmoplegia.

A polymerase chain reaction (PCR) based procedure was modified to determine the deletion of mitochondrial DNA (mtDNA). The protocol consists of coamplification both of deleted and wild-type segments of mtDNA using a long PCR technique; evaluation of the deleted portion within the amplified DNA segments by restriction enzyme digestion followed by densitometrical analysis; and direct subcloning into a plasmid vector for DNA sequencing. The procedure revealed a 5.3 kb deletion of mtDNA in the biopsied muscle tissue obtained from a patient clinically diagnosed with progressive external ophthalmoplegia. The 5' and 3' sequences at both sides of the breakpoint comprise a 17 bp palindrome and 5 bp tandem repeats, suggesting that the deletion might occur through slipped mispairing and other novel mechanisms. This improved procedure has the potential to detect deletions occurring in the entire length of mtDNA, and mighty be useful for clinical screening of progressive external ophthalmoplegia.

Sympathetic activation and contribution of genetic factors in hypertension with neurovascular compression of the rostral ventrolateral medulla.

OBJECTIVE: The rostral ventrolateral medulla is an important center for the regulation of sympathetic and cardiovascular activities. Reportedly, neurovascular compression of the rostral ventrolateral medulla may be causally related to essential hypertension. We aimed to determine the mechanism behind elevated blood pressure in hypertensive patients with compression of the rostral ventrolateral medulla and to investigate whether genetic factors contribute to the etiology of hypertension with compression. DESIGN AND METHODS: The study included 56 patients with essential hypertension and 25 normotensive individuals. With the use of magnetic resonance imaging, the essential hypertension group was subdivided into hypertension with compression and without compression groups. We compared plasma levels of hormones that raise blood pressure and family histories of hypertension between the two hypertension groups and the normotension group. RESULTS: Plasma norepinephrine levels, but not plasma renin activity, aldosterone, epinephrine, or vasopressin levels, were significantly higher in the hypertension with compression group (389+/-53 pg/ml) than in the hypertension without compression group (217+/-38, P<0.05) or in the normotension group (225+/-30, P<0.05). The percentage of individuals who had two hypertensive parents was significantly higher in the hypertension with compression group (39.4%) than in the hypertension without compression group (13.0%, P<0.05) or in the normotension group (8.0%, P<0.01). CONCLUSIONS: These results indicate that neurovascular compression of the rostral ventrolateral medulla might be, at least in part, causally related to essential hypertension by increasing sympathetic nerve activity. They also suggest that genetic factors might contribute to the etiology of hypertension with neurovascular compression.

Mutation of the sterol 27-hydroxylase gene (CYP27) results in truncation of mRNA expressed in leucocytes in a Japanese family with cerebrotendinous xanthomatosis.

OBJECTIVES: A Japanese family with cerebrotendinous xanthomatosis (CTX) was investigated for a sequence alteration in the sterol 27-hydroxylase gene (CYP27). The expression of CYP27 has been mostly explored using cultured fibroblasts, prompting the examination of the transcripts from blood leucocytes as a simple and rapid technique. METHODS: An alteration in CYP27 of the proband was searched for by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and subsequent sequencing. Samples of RNA were subjected to reverse transcription PCR (RT-PCR) and the product of the proband was amplified with nested primers and sequenced. RESULTS: A homozygous G to A transition at the 5' end of intron 7 was detected in the patient. In RT-PCR analysis, only a truncated transcript was detected in the patient, whereas both normal and truncated transcripts were detected in the siblings. The sequencing of the patient's cDNA fragment disclosed a direct conjuction of exon 6 and exon 8. CONCLUSION: The mutation at splice donor site and the truncation of mRNA were identical with those of a recently reported Italian patient, although different in symptomatology. The application of blood leucocytes can be a simple technique on analysing a constructive abnormality of CYP27 mRNA.

The ultrastructural effects of beta-amyloid peptide on cultured PC12 cells: changes in cytoplasmic and intramembranous features.

The fine structural features of cultured PC12 cells were investigated after treatment for 1, 3, or 5 days with different concentrations of the vascular form of beta-amyloid 1-40 (beta-AP). PC12 cells treated with beta-AP showed time- and concentration-dependent lysosomal system activation and cell toxicity. We observed increases in the number and size of cytoplasmic lysosomes as indicated by increased acid phosphatase reactivity. Some lysosomes were in the form of multivesicular bodies or large residual bodies that appeared to arise by autophagia or by endocytotic uptake. Double-sided plasma membrane invaginations were observed to give rise to increasingly extensive intracytoplasmic vacuolization that was correlated with duration of beta-AP treatment. Freeze-fracture studies of the intramembranous particle (IMP) population in the plasma membrane P-face showed that both control and beta-AP treated cells had two major P-face IMP populations, small-diameter (4-8 nm) IMPs, and large-diameter (> or = 9 nm) IMPs. The larger category of IMPs was found to possess a greater average diameter in the beta-AP treated cells than in the control cells. These IMPs could represent modifications to existing transmembranous receptors, channels, or transducing molecules by the beta-AP. These results demonstrate that beta-AP can induce time- and concentration-dependent ultrastructural changes in PC12 cell membranes.

Muramyl dipeptide injected into crushed sciatic nerve, activates macrophages and promotes recovery of walking locomotion in rats.

We injected an immunoadjuvant, muramyl dipeptide (MDP), intrafascicularly into crushed rat sciatic nerve so as to test whether activation of macrophages promotes regeneration of peripheral nerve from crush injury and improves walking locomotion in rats. Immunohistochemical staining of Schwann cells and macrophages with anti-S100 and ED-1 monoclonal antibodies revealed that macrophages are more abundant and phagocytic in nerve injected with MDP than in control. Axonal elongation in damaged nerve and locomotion recovery in rats were evaluated with pinch test and measurement of sciatic nerve functional index (SFI), respectively. Pinch test showed a 15.5% increase in length (n = 6, p < 0.05) of elongating axons for MDP-injected group 5 days after the crush injury comparing to the control group. The value of SFI obtained from the rats injected with MDP showed a 18.3 % increase (n = 4-6, p < 0.01) comparing to the control 3 weeks after the crush injury. Activation of macrophages in the nerve injected with MDP was monitored by detecting gene expression of marker molecules for macrophages such as apolipoprotein E (ApoE), interleukin-1beta (IL-1beta) and macrophage inflammatory protein-1alpha (MIP-1alpha) using reverse transcription-PCR (RT-PCR) technique 2 days and 1 week after the injection. Levels of transcripts of IL-1beta and MIP-1alpha were up-regulated in the nerves injected with MDP 1 week after MDP injection. These results suggest that an intrafascicular injection of MDP activates macrophages infiltrating into damaged nerve and that the macrophages support elongation of regenerating axon, resulting in functional recovery of sciatic nerve from injury in rats.

Beta-amyloid induced increase in choline flux across PC12 cell membranes.

Beta-amyloid peptide is the main constituent of senile plaques and is implicated in the pathogenesis of Alzheimer's disease. It has been shown to be both neurotoxic and neurotrophic in vivo, and its effects have been suggested to be mediated in part by alterations in membrane transport. In the present study, we investigated the effect of beta-amyloid (1-40) on choline transport in cultured PC12 cells. We found that exposure to 46 or 92 microM beta-amyloid (1-40) increased [14C]choline flux in PC12 cells in a concentration-dependent manner, whereas exposure to reverse sequence beta-amyloid (40-1) had no effect. If there is a similar effect in vivo, the increased beta-amyloid dependent permeability to choline could lead to depletion of cellular choline stores and could contribute to the selective vulnerability of cholinergic neurons in Alzheimer's disease.

Screening of H-ras gene point mutations in 50 cases of bladder carcinoma.

BACKGROUND: Mutation converts the H-ras gene into an activated oncogene in about 10% of human bladder cancers. Codons 12 and 61 are the major "hot spots" for activation. A simple and accurate method to detect point mutations in these codons may be clinically useful for early diagnosis of bladder cancer. METHODS: Bladder cancer samples from 50 patients, plus 10 samples of normal bladder mucosa, were analyzed for possible point mutation of the H-ras gene at either codon 12 or codon 61. The H-ras gene DNA segments that include these 2 codons were amplified by PCR methods, then the possible presence of a point mutation was evaluated at each codon by susceptibility of the respective DNA segments to digestion with the restriction enzyme and by dot blot hybridization assay. A bladder cancer patient who had an H-ras gene mutation was examined to see whether the mutation was also detectable in the cells released in the urine. RESULTS: Definite or possible point mutations were found in 6 (12%) out of 50 bladder cancer patients, while no mutation was detected in normal mucosa. A point mutation could also be detected in cells isolated from the patient's urine sample. CONCLUSION: The prevalence of point mutations at codon 12 or codon 61 of the H-ras gene found in this study was similar to that previously estimated for human bladder cancer by DNA transfection assay. The method we have used for detecting point mutations of the H-ras gene provides a simple and highly accurate way to detect mutated cancer cells even in the urine. It may be clinically usable for early diagnosis of bladder cancer.

Isolation of a monoclonal antibody reactive to brain microsome-associated antigen, lap-1, and its preferential localization in limbic system of rat brain.

The distribution and subcellular localization of AH9 antigen, recognized by a monoclonal antibody AH9, were examined in rat brain. Highest expression was observed in the lamina lucidum of the dentate gyrus of the rat hippocampus. Synaptic subfields of other limbic areas also expressed AH9 antigen at a substantial level. The molecular size of the AH9 antigen is 15 kDa and it was found in the microsomal fraction of brain but not of heart or kidney. These results indicate that AH9 antigen is a novel synaptosomal protein that is relatively specific to the limbic system, at least in the rat brain. We designated AH9 antigen as a limbic system associated protein-1, lap-1.