Adverse event profiles of hypomagnesemia caused by proton pump inhibitors using the Japanese Adverse Drug Event Report (JADER) Database.

Proton pump inhibitors (PPIs) are commonly used for the prevention or treatment of gastric ulcers, but they can induce hypomagnesemia. Little is known about the onset duration and risk factors related to patient characteristics of this adverse event in Japanese patients. Therefore, we analyzed the time-to-onset of PPI-induced hypomagnesemia and evaluated the association between hypomagnesemia and PPIs using the Japanese Adverse Drug Event Report (JADER) database. We analyzed hypomagnesemia cases between 2004 and 2021. The time-to-onset analysis was performed using the Weibull distribution, and the adjusted reporting odds ratio (aROR) or 95% confidence interval (95% CI) was calculated using a multiple logistic regression analysis. The analysis database comprised 236,525 cases, with 188 cases associated with hypomagnesemia. The median onset duration (interquartile range) of PPI-induced hypomagnesemia was 99.0 (51.8-285.5 ) days, which is considered the random failure type. The multiple logistic regression analysis revealed that hypomagnesemia is significantly associated with male sex (aROR, 95% CI: 1.66, 1.23-2.25) , age < 60 (1.59, 1.14-2.21) , estimated body-mass index (eBMI) (0.94, 0.91-0.98) , PPIs (1.66, 1.18-2.30) , and the interaction of age (<60)*PPIs (1.58, 1.13-2.19) . However, diuretics were not significantly associated with hypomagnesemia. Our results suggest that serum magnesium levels should be measured regularly regardless of the duration of PPI use, especially in patients with male sex, age < 60, or low BMI. These findings will assist health professionals in the adequate use of PPIs. These findings need to be evaluated by cohort studies and long-term clinical investigations.

Time-dependent changes in serum magnesium levels in patients receiving cetuximab with low baseline serum sodium levels.

Cetuximab causes electrolyte abnormalities, such as hypomagnesemia, hypokalemia, and hypocalcemia. However, little is known about the relationships between the onset of hypomagnesemia, patient background before administration, and time-dependent changes in serum magnesium levels. Therefore, we examined the patient backgrounds that influenced the onset of hypomagnesemia and the time-dependent changes in serum magnesium levels in patients receiving cetuximab. A retrospective study was performed to investigate patients with advanced or recurrent colorectal cancer or head and neck cancer, treated with a cetuximab regimen from 2012 to 2020 at Kindai University Nara Hospital. In total, 52 patients who met the inclusion criteria were enrolled in this study. The serum magnesium level was significantly lower in the hyponatremia before the administration group than in the non-hyponatremia group (p < 0.001). Univariate logistic regression analysis revealed that the baseline serum sodium levels (odds ratio [OR]: 0.741, 95% confidence interval [CI]: 0.588-0.934) and the combination of magnesium oxide tablet (OR: 0.997, 95% CI: 0.995-0.999) were one of the independent factors for hypomagnesemia. These results indicated that hyponatremia before administration may be an indicator of serum magnesium levels after administration of cetuximab. Cetuximab-induced hypomagnesemia may be predicted using baseline serum sodium levels, and hypomagnesemia may be prevented by administration of magnesium oxide tablets. Our findings provided new evidence for the management of serum magnesium levels in patients receiving cetuximab.

Site Specificity of Changes in Cortical Oxyhaemoglobin Concentration Induced by Water Immersion.

Our previous studies have shown that water immersion (WI) changes sensorimotor processing and cortical excitability in the sensorimotor regions of the brain. The present study examined the site specificity of the brain activation during WI using functional near infrared spectroscopy (fNIRS). Cortical oxyhaemoglobin (O2Hb) levels in the anterior and posterior parts of the supplementary motor area (pre-SMA and SMA), primary motor cortex (M1), primary somatosensory cortex (S1), and posterior parietal cortex (PPC) were recorded using fNIRS (OMM-3000; Shimadzu Co.) before, during, and after WI in nine healthy participants. The cortical O2Hb levels in SMA, M1, S1, and PPC significantly increased during the WI and increased gradually along with the filling of the WI tank. These changes were not seen in the pre-SMA. The results show that WI-induced increases in cortical O2Hb levels are at least somewhat site specific: there was little brain activation in response to somatosensory input in the pre-SMA, but robust activation in other areas.

Visceral fat accumulation is associated with cerebral small vessel disease.

BACKGROUND AND PURPOSE: Obesity is associated with the risk of coronary artery disease and stroke. Visceral fat plays a significant role in the atherogenic effects of obesity. Whether visceral fat accumulation, as measured by computed tomography (CT), is an independent risk factor for the presence of cerebral small vessel disease (SVD) was investigated. METHODS: This study comprised 506 Japanese subjects 35-74 years of age (mean 55.3 years) without a history of symptomatic cerebrovascular disease who underwent health screening tests, including brain magnetic resonance imaging, carotid echography and measurements of the visceral fat area (VFA) and subcutaneous fat area (SFA) on abdominal CT. Visceral fat accumulation was defined as VFA ≥ 100 cm(2) . Logistic regression analysis was performed to examine the associations between visceral fat accumulation and cerebral SVD such as white matter lesions (WMLs) and silent lacunar infarction (SLI). RESULTS: The prevalence of WMLs and SLI but not carotid plaque were significantly higher in subjects with VFA ≥ 100 cm(2) than those with VFA < 100 cm(2) . A VFA ≥ 100 cm(2) was associated with WMLs and SLI independent of age, cardiovascular risk factors and other measurements of obesity, such as waist circumference and body mass index. A large waist circumference was independently associated with SLI. SFA, the combination of VFA and SFA, and body mass index were not associated with WMLs or SLI. CONCLUSIONS: Visceral fat accumulation was independently associated with the presence of cerebral SVD in subjects without a history of symptomatic cerebrovascular disease.

Overexpression of Smad2 inhibits proliferation of gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Spatiotemporal inhibition of apical migration and proliferation of gingival epithelium are significant factors involved in periodontal regeneration. Transforming growth factor ß (TGF-ß) is important in multiple aspects of wound healing, and Smad2, a downstream transcription factor of TGF-ß, has an inhibitory effect on re-epithelialization during gingival wound healing. Therefore, we investigated the effects on migration and proliferation status, and intra/extracellular signaling regulated by Smad2 overexpression in gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells were isolated from the palatal gingival tissue of transgenic mice overexpressing Smad2 driven by the Keratin14 promoter. Smad2 expression was identified by western blotting and immunofluorescence analysis. Scratch assay and 5-bromo-2'-deoxyuridine staining were performed to assess cell migration and proliferation. To inactivate TGF-ß type I receptor, the cultures were supplemented with SB431542. Secreted TGF-ß was quantified by ELISA. Smad2 target gene expression was examined by real-time RT-PCR and in vivo immunofluorescence analysis of gingival junctional epithelium. RESULTS: Smad2-overexpressing cells were confirmed to have significant phosphorylated Smad2 in the nucleus. Scratch assay and 5-bromo-2'-deoxyuridine staining indicated that Smad2-overexpressing cells showed no significant differences in migration, but had reduced proliferation rates compared to wild-type controls. SB431542 significantly inhibited Smad2 phosphorylation, which coincided with restoration of the proliferation rate in Smad2-overexpressing cells. ELISA of TGF-ß release did not show any differences between genotypes. The cell cycle inhibitors, p15 and p21, showed significant upregulation in Smad2-overexpressing cells compared to wild-type controls. Moreover, junctional epithelium of the transgenic mice showed increased expression of P-Smad2, p15 and p21. CONCLUSION: The signaling activation triggered by overexpression of Smad2 was dependent on TGF-ß type I receptor, and the activated Smad2 increased p15 and p21 expression, responsible for inhibiting cell cycle entry, resulting in antiproliferative effects on gingival epithelial cells. Understanding of Smad2-induced signaling would be useful for possible clinical application to regulate gingival epithelial downgrowth.

Tissue rinse liquid-based cytology: a feasible tool for the intraoperative pathological evaluation of sentinel lymph nodes in breast cancer patients.

OBJECTIVES: A unique diagnostic method was designed for the intraoperative pathological evaluation of sentinel lymph nodes (SLNs) in breast cancer patients, and the results were verified with 2 years of experience. METHODS: Excised lymph nodes were cut into 2-mm-thick slices and rinsed thoroughly in CytoRich Red(®). The sliced tissues were embedded in a paraffin block. Three cytological glass slides of the cells exfoliated in CytoRich Red(®) were prepared by the SurePath(®) liquid-based cytology (LBC) technique. Two slides were stained by the Papanicolaou method, and the remaining slide was immunostained with an anti-keratin antibody. This process is called tissue rinse liquid-based cytology (TRLBC). The results of TRLBC were compared with those of the final pathological diagnoses, including immunostaining with an anti-keratin antibody on paraffin blocks (PB). RESULTS: This study analysed 444 SLNs from 247 consecutive breast cancer patients. It required 35 minutes to complete the intraoperative diagnosis on a single node, and it took an additional 5 minutes per node if more than one node was submitted. When the results of PB were assumed to be the gold standard, the sensitivity and specificity of TRLBC were 81.9% and 96.1%, respectively. TRLBC detected all nodes with macrometastasis and 23 of 24 nodes with micrometastasis. Fifteen false-negative TRLBC results were 'isolated tumour cell clusters' on PB, but there was one with micrometastasis histologically. Four of 14 false-positive TRLBC results were proven to be true positive by supplementary examination using step sectioning of the paraffin blocks of the nodes. CONCLUSION: TRLBC is a feasible and promising intraoperative cytopathological tool showing a comparable efficacy to PB while still allowing the conventional postoperative histological examination.

Cytodiagnosis through use of a z-axis video by volunteer observers: a promising tool for external quality assessment.

OBJECTIVE: This study examined whether cytological diagnosis through the use of a video, which shows the changing depth of focus in the microscopic field, described as a z-axis video, is useful compared with a still image. METHODS: From 17 cytology preparations of fine needle aspiration of the breast, we made six z-axis videos per case. A frame exhibiting the characteristic features was then extracted from each video and saved as a representative still image. One hundred and twenty-eight volunteer cytotechnologists were randomly divided into two groups of video observers and still image observers. The participants were asked to make a diagnosis of benign, indeterminate, suspicious or malignant without having any clinical information other than the age of the patient. Diagnoses were categorized as 'recommended' or 'unacceptable' according to degree of correlation with histology. RESULTS: The number of definitive diagnoses of 'benign' or 'malignant' were increased in video observers, and indeterminate or suspicious categories were decreased (P = 0.013). The distribution of diagnostic categories in three of the 17 cases was significantly different; the distribution in the remaining cases was similar between the two groups. The z-axis video observers may have selected the definite diagnoses with confidence because they observed valuable microscopic findings by 'focusing through observation'. The average number of 'recommended' diagnoses by individual observers was significantly higher in the video observer group than in the still image observer group (P = 0.016). In contrast, the average number of 'unacceptable' diagnoses was significantly lower (P = 0.019). CONCLUSIONS: A z-axis video is easy to obtain and is therefore expected to become a powerful diagnostic modality for the external quality assessment of clinical cytology and even in the field of primary cytodiagnosis.

Neuroprotective effect of cilostazol against retinal ischemic damage via inhibition of leukocyte-endothelial cell interactions.

BACKGROUND: Cilostazol, a selective platelet phosphodiesterase inhibitor, has been shown to reduce neuronal injury after transient cerebral ischemia. Its neuroprotective effect is thought to result from an antiplatelet function. This study was designed to evaluate the inhibitory effects of cilostazol against retinal ischemic damage focusing on leukocyte-endothelial cell interactions. METHODS: Retinal ischemia was induced for 60 min in male Sprague-Dawley rats (n = 144) by temporary ligation of the optic sheath. Cilostazol was administered just before ischemia induction. Leukocyte behavior in the retinal microcirculation was evaluated in vivo with scanning laser ophthalmoscopy and ex vivo with fluorescence microscopy. Retinal expression of P-selectin, intracellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor were evaluated by real-time quantitative reverse transcriptase-polymerase chain reaction. Ischemia-induced retinal damage was evaluated histologically. RESULTS: Treatment with cilostazol significantly suppressed leukocyte-endothelial cell interactions; the maximal numbers of rolling leukocytes were reduced by 77.6% (P < 0.01) 12 h after ischemia. Twenty-four hours after ischemia, adherent and accumulated leukocytes were also suppressed by treatment with cilostazol (36.1% and 20.4% respectively, P < 0.01). The expressions of P-selectin and ICAM-1 mRNA were suppressed significantly in cilostazol-treated retinas (P < 0.05). The retinal histological examination demonstrated a significant protective effect of cilostazol against ischemia-induced retinal damage (P < 0.01). CONCLUSIONS: The present study demonstrates that cilostazol attenuates retinal injury after transient ischemia via inhibition of leukocyte-endothelial cell interactions. This inhibitory effect on postischemic leukocyte-endothelial cell interactions might partially contribute to its neuroprotective effects.

Induction and selective accumulation of mutant ubiquitin in CA1 pyramidal neurons after transient global ischemia.

Accumulation of mutant ubiquitin-B (UBB(+1)) in neurons is considered the hallmark of proteasomal dysfunction in neurodegenerative disorders, however no such evidence in ischemic brain has been reported. We investigated the contribution of UBB(+1) in delayed neuronal death after transient global ischemia. Transient global ischemia was achieved by occlusion of bilateral common carotid arteries for 5 min and reperfusion in male Mongolian gerbils (n=6 per each time point). In the CA1 region, UBB(+1) immunoreactivity appeared in the cytoplasm of pyramidal cells at 30 min post-ischemia, and the density of these neurons increased at day 2 (P<0.001) and further increased at day 4 post-ischemia. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)-positive (apoptotic) cells appeared selectively in the CA1 region at day 3 and their density increased further at day 4 post-ischemia (P<0.001). In contrast, UBB(+1) immunoreactivity was only transiently detected from 30 min to 1 day post-ischemia in CA3, dentate gyrus, and frontal cortex, but disappeared at day 2 post-ischemia. No TUNEL-positive cells were observed in these three regions. UBB(+1) mRNA was detected by reverse transcription-polymerase chain reaction in every region of the hippocampus and frontal cortex of ischemic gerbils and even in the non-ischemic control animals, and its expression level was independent of brain region and time after ischemia. Our results indicate induction and selective accumulation of UBB(+1) protein in dying neurons of the CA1 region and suggest that UBB(+1) expression may be induced by proteasomal dysfunction after transient global ischemia.

Expression of myeloid cell phenotypes by a novel adult T-cell leukemia/lymphoma cell line.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) can infect a number of cells of different lineages in vitro, yet the immunophenotypes of most adult T-cell leukemia/lymphomas (ATLs) are restricted to CD4+. The apparent discrepancy between these findings is still largely unknown. PURPOSE: We report on a unique case of ATL in which the leukemia cells were positive for both T-cell and myeloid cell antigens. To characterize these cells, we isolated cell lines from this patient with ATL. METHODS: The fresh leukemia cells were cultured without the addition of interleukin-2. Cell cloning was carried out by limiting dilution. RESULTS: A cell line (MU) and its clonal sublines were established. MU cells showed the same chromosomal abnormalities and T-cell receptor beta-chain gene rearrangement pattern as those of fresh leukemia cells. MU cells were exclusively positive for a myeloid cell marker (CD13) but not for T-cell markers, despite the presence of T-cell receptor gene rearrangement. CONCLUSION: The established ATL cell line showed both T-cell and myeloid cell characteristics, which seems to be the first evidence for the close association of ATL cells with both lymphoid and myeloid features. The cell line may provide a new insight for the targets of HTLV-1 infection and transformation in vivo.

Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain.

A cDNA encoding a precursor for a novel serine protease (neurosin) was cloned from a cDNA library prepared from a human colon adenocarcinoma cell line, COLO 201. The sequence consisted of 155 bp 5' non-coding region and a 732 bp open reading frame which was followed by a 551 bp 3' non-coding region. The predicted protein consists of 244 amino acids which is possibly processed to an active enzyme of 223 amino acids that shows some similarity (< 30%) to other members of serine protease family. As found in other trypsin-like proteases, the enzyme contains the catalytic triad which is characterized as the essential amino acid residues for the activity. Northern blot analyses of the mRNA showed the strongest expression in brain followed by a lower but significant one in spleen. A construct of cDNA encoding chimeric protein that carries pro-sequence of trypsin II and putative mature neurosin starting from Leu22 was transfected to COS-1 cells. Successful production of the active neurosin was shown after treating the supernatant of the culture of the transfectants with enterokinase.

Determinant of microvolt-level T-wave alternans in patients with dilated cardiomyopathy.

OBJECTIVES: The aim of this study was to clarify the clinical significance and the determinant of microvolt-level T-wave alternans (TWA) in patients with dilated cardiomyopathy (DCM). BACKGROUND: The prevention of sudden death in patients with DCM remains the therapeutic target. T-wave alternans has been proposed as a powerful tool for identification of patients at high risk for ventricular arrhythmias and sudden death in coronary artery disease. METHODS: In 58 DCM patients, TWA was measured during bicycle exercise testing using a CH 2000 system (Cambridge Heart, Bedford, Massachusetts). The New York Heart Association class, signal-averaged electrocardiogram, QT dispersion, left ventricular end-diastolic diameter (LVDd) and percent fractional shortening detected by echocardiogram and the grade of the ventricular arrhythmia were obtained in all patients. RESULTS: T-wave alternans was positive in 23 patients (TWA+ group), negative in 25 (TWA- group) and indeterminate in 10. Univariate analysis showed that the percentage of patients with ventricular tachycardia (VT) and the LVDd in the TWA+ group was significantly higher than those in the TWA- group (61% vs. 8%, p < 0.001 and 65 +/- 11 mm vs. 58 +/- 8 mm, p < 0.05, respectively). The sensitivity, specificity and predictive accuracy of TWA for VT were 88%, 72% and 77%, respectively. Multivariate analysis showed that the presence of VT was a major independent determinant of TWA in patients with DCM (p = 0.003). CONCLUSIONS: T-wave alternans was closely related to VT in patients with DCM. T-wave alternans is a useful noninvasive test for identifying high risk patients with DCM who have VT.

Isolation and expression of FIP-2 in wounded pulp of the rat.

Pulpal wound healing followed by cavity preparation may involve reactionary or reparative dentinogenesis in relation to the cavity position; however, little is known about the molecular responses. We aimed to isolate and analyze genes induced or suppressed in the wounded pulp to identify molecular processes involved in the pulp responses to injury. Twenty-three cDNAs were isolated by cDNA subtraction between healthy and wounded pulp of rats. By library screening, we identified rat 14.7K-interacting protein (rFIP)-2A and B genes homologous to human FIP-2, being involved in regulating membrane trafficking and cellular morphogenesis. RT-PCR analysis showed induction for only rFIP-2B in the wounded pulp. In situ hybridization analysis revealed that both rFIP-2s were expressed strongly in condensing mesenchymal cells of the palatal process and surrounding Meckel's cartilage, but not in intramembranous chondrogenic cells. Thus, up-regulated rFIP-2B expression may play a role in regulating membrane trafficking or cellular morphogenesis of these embryonic and wounded pulpal cells.

Type 2 diabetes reduces the proliferation and survival of oligodendrocyte progenitor cells in ishchemic white matter lesions.

Diabetes mellitus (DM) is a major risk factor for stroke and it exacerbates tissue damage after ischemic insult. Diabetes is one of the important causes of the progression of white matter lesion, however, the pathological mechanisms remain unclear. The present study evaluated the influences of type 2 DM on ischemic subcortical white matter injury and the recruitment of oligodendrocyte progenitor cells (OPCs) under chronic cerebral hypoperfusion using type 2 diabetic (db/db) mice. After bilateral common carotid artery stenosis (BCAS), the rarefaction in the white matter was more severe in db/db mice than in db/+ mice, and the number of glutathione S-transferase-pi (GST-pi)-positive mature oligodendrocytes (OLG) was lower in db/db mice than in db/+ mice at 4 and 8 weeks after ischemia. There were no significant differences in the number of single-stranded DNA (ssDNA)-positive apoptotic cells in the deep white matter between the db/db and db/+ mice. We found a transient increase in the platelet-derived growth factor receptor-α (PDGFRα)-positive OPCs in white matter lesions after ischemia. However, significantly fewer PDGFRα-positive OPCs were detected in db/db than db/+ mice from 4weeks after BCAS. The number of Ki67-positive proliferating cells in the deep white matter was significantly lower in db/db mice than in db/+ mice from 4 to 8weeks after BCAS. Most of the Ki67-positive cells were PDGFRα-positive OPCs. Finally, we assessed the survival of 5-bromo-2'-deoxyuridine (BrdU)-positive proliferating cells in ischemic white matter, and found significantly poorer survival of BrdU/PDGFRα-positive OPCs or BrdU/GST-pi-positive OLGs in the db/db mice compared to the db/+ mice in the white matter after BCAS. Our findings suggest that the type 2 DM mice exhibited more severe white matter injury 8 weeks after chronic ischemia. Decreased proliferation and survival of OPCs may play an important role in the progression of white matter lesions after ischemia in diabetics.

Interactions between blood cells and retinal endothelium in endotoxic sepsis.

Platelets and leukocytes are thought to play a leading role in the pathogenesis of many inflammatory conditions. To recruit flowing blood cells to the inflammatory region, it would be necessary for them to interact with vascular endothelial cells. Recently, many reports have indicated the resistance of spontaneous hypertensive rats (SHR) to endotoxic sepsis. Their resistance might be derived from suppressed interaction between these blood cells and endothelial cells. Therefore, SHR and age-matched Wistar-Kyoto rats (WKY) were induced with endotoxic sepsis by intravenous injection of lipopolysaccharide (LPS). At 4, 12, 24, and 48 hours after induction, leukocyte-endothelial interactions in the retina were evaluated in vivo with acridine orange digital fluorography. Fluorescently labeled platelets were also injected to investigate platelet-endothelial interactions in the retina in endotoxic sepsis. Leukocyte rolling in SHR after LPS injection was significantly suppressed; the maximum number of rolling leukocytes was reduced by 80.1% at 12 hours after LPS injection in SHR compared with WKY. Subsequent leukocyte infiltration into the vitreous cavity was significantly inhibited in SHR. Furthermore, platelet-endothelial interactions in the retina were also suppressed in SHR treated with LPS. The maximum numbers of rolling and adherent platelets were reduced by 59.5% and 62.6%, respectively, in SHR compared with WKY. In both strains, leukocyte- and platelet-endothelial interactions were substantially inhibited by the blocking of P-selectin. These suppressed interactions could contribute to the reduction of leukocyte- and platelet-mediated tissue injury in endotoxic sepsis in SHR, resulting in their resistance to endotoxemia.

Oral-mucosal administration of IFN-alpha potentiates immune response in mice.

We studied the effects of oral-mucosal administration of murine interferon-alpha (Mu-IFN-alpha) on immune responses and infection with vaccinia virus (VV) in mice. When Mu-IFN-alpha was administered to sheep red blood cell (SRBC)-sensitized mice for 4 or 5 days, Mu-IFN-alpha significantly enhanced delayed-type hypersensitivity (DTH) and antibody production, with maximum enhancement of each at 1 IU/body. To investigate the antiviral effect of oral-mucosal Mu-IFN-alpha, mice were infected with VV, and Mu-IFN-alpha was administered for 15 days. Pocks were observed in the tail skin of infected mice, and Mu-IFN-alpha at doses of 1, 10, and 100 IU/body significantly suppressed pock formation. Also, VV-specific cytotoxic T cells (CTL) were observed in the spleen from the same mice at 7 days after infection, and Mu-IFN-alpha enhanced CTL activity at doses above 1 IU/body. These results suggest that the oral-mucosal Mu-IFN-alpha may have potentiating effects on cellular and humoral immune responses, which may contribute to its effects against VV.

An attempt to substitute the cell binding domain of human fibronectin in lambda phage J protein: computer design and expression.

We superimposed hydropathic indexes of the human fibronectin cell binding domain (CBD) on the lambda phage J protein by computer, and substituted 22 amino acids from the fibronectin CBD for a part of the lambda phage J protein. The fibronectin cell binding domain -Arg-Gly-Asp-Ser- (-RGDS-) localizes at the junction of hydrophobicity and hydrophilicity. We selected a similar hydrophobic-to-hydrophilic junction in the J protein region for substitution. This junction corresponds to 150 bp of the PstI fragment of J protein cDNA. We synthesized 150 bp of the relevant PstI fragment that includes the cell binding domain region. The region was then constructed by serial cloning as an expression vector, pJCBD. The vector pJCBD expressed the fused protein named JCBD (M(r) 32 kDa) in E coli XL1-BLUE. The expressed JCBD protein was identified by Western blot analysis in the extract of the pJCBD carrying bacterial lysate using both rabbit anti-lambda phage antiserum and anti-CBD of fibronectin antibody. The JCBD protein appeared to recognize retinoblast cell membrane RGDS-directed receptors, detected by enzyme-linked immuno-sorbent assay and also by binding competition assay with synthetic pentapeptides, Gly-Arg-Gly-Asp-Ser (GRGDS) and Gly-Arg-Gly-Glu-Ser (GRGES). The former competitor inhibited completely fibronectin CBD-dependent binding activity of JCBD, the latter had no inhibitory activity. These results suggest that certain functional proteins engineered by computer design between human fibronectin cell binding domain and lambda phage J protein can be produced.

PKC-beta inhibitor (LY333531) attenuates leukocyte entrapment in retinal microcirculation of diabetic rats.

PURPOSE: The activity of protein kinase C (PKC), preferentially beta isoform of PKC, has been shown to be elevated in the diabetic retina. Recently, LY333531, a specific inhibitor of PKC-beta, has been reported to improve the decrease of retinal blood flow in early diabetes. Increased leukocyte entrapment has been suggested to be involved in blood flow disturbances in the early diabetic retina. This study was designed quantitatively to evaluate leukocyte entrapment in the retinal microcirculation of diabetic rats and the effect of LY333531 on leukocyte entrapment. METHODS: Diabetes was induced in male Long-Evans rats by intraperitoneal injection of streptozotocin (60 mg/kg). LY333531 (0.1, 1.0, or 10.0 mg/kg/d) was administered orally during a 4-week diabetic period. Leukocyte entrapment in the retinal microcirculation was quantitatively evaluated in vivo with acridine orange digital fluorography. RESULTS: The number of leukocytes trapped in the retinal microcirculation of diabetic rats (mean +/- SEM; 14.3 +/- 1.3 cells/mm2) was significantly increased, compared with nondiabetic control rats (7.5 +/- 0.3 cells/mm2; P < 0.0001). Oral administration of LY333531 significantly decreased the number of leukocytes trapped in the retinal microcirculation of diabetic rats (10.9 +/- 0.6, 11.3 +/- 0.7, and 10.4 +/- 0.4 cells/mm2 with LY333531 0.1, 1.0, and 10.0 mg/kg/d, respectively; P < 0.05). CONCLUSIONS: Treatment with LY333531 attenuated the increase of leukocyte entrapment in the retinal microcirculation during the period of early diabetes. This effect may contribute to the improvement of abnormal retinal blood flow in early diabetes with LY333531. LY333531 might have a therapeutic efficacy in preventing microcirculatory flow disturbances by trapped leukocytes in the early diabetic retina.

Administration of 17beta-estradiol attenuates retinal ischemia-reperfusion injury in rats.

PURPOSE: Accumulating evidence has suggested that 17beta-estradiol exerts protective effects against ischemic damage in various organs. In addition, leukocytes that accumulate in postischemic tissues are thought to play a central role in ischemia-reperfusion injury. This study was designed to evaluate quantitatively the inhibitory effects of 17beta-estradiol on leukocyte accumulation during ischemia-reperfusion injury and on subsequent retinal damage after transient retinal ischemia. METHODS: Transient (60 minutes) retinal ischemia was induced in male rats by temporary ligation of the optic nerve. Thirty minutes before induction of ischemia, 17beta-estradiol (0.1 mg/kg) was administered intraperitoneally. At 6, 12, 24, and 48 hours after reperfusion, leukocyte accumulation in the retina was evaluated in vivo by means of acridine orange digital fluorography. Histologic and electroretinographic (ERG) studies were carried out to evaluate retinal damage. RESULTS: Treatment with 17beta-estradiol significantly inhibited postischemic leukocyte accumulation; the maximum number of accumulating leukocytes was reduced by 35.7% at 24 hours after reperfusion (P = 0.01). Histologic examination showed that administration of 17beta-estradiol significantly reduced retinal damage, which was most obvious in the inner retina, 168 hours after reperfusion (P = 0.0001). ERG studies at 12 and 168 hours after reperfusion showed that recovery of the b-wave amplitude was significantly improved with treatment of 17beta-estradiol (P = 0.023). CONCLUSIONS: The present study demonstrated the inhibitory effects of 17beta-estradiol on leukocyte accumulation and subsequent tissue injury during retinal ischemia-reperfusion injury.