Abstract. Proximal tubular epithelial cells from mice which develop autoimmune interstitial nephritis were found to express the nephritogenic target antigen, 3M-1. Anti-3M-1 mAbs (ct3M-1-Ab) were used to positively select for 3M-l-secreting tubular epithelium and, after stabilization in culture, this new cell line (MCT) was examined for the production of several moieties important to either immune interactions or to the development of extracellular matrix. Alkaline phosphatase-staining MCT cells also express epithelial growth factor receptors with a Kd of 0.87 nM and an epithelial growth factor receptor constant (R,,) of 2.1 x 104 receptors/ceil. MCT culture supernatants contain greater amounts of laminin, and types IV and V procollagens compared to types I and III procollagens, and growing MCT cells on type I collagen matrix causes them to preferentially secrete even more type IV and V procollagen.The 30,000-Mr 3M-1 antigen could be immunoprecipitated from biosynthetically labeled MCT cell supernatants with a3M-1-Ab. An identical-sized moiety was isolated by immunoaffinity chromatography from collagenase-solubilized mouse kidney tubular basement membranes. The 3M-I antigen can be found on the MCT cell surface by radioimmunoassay, or deposited in a linear array in the extracellular matrix surrounding the MCT cells in culture by immunofluorescence. Mature messenger RNA species for both class I and class II major histocompatibility complex (MHC) molecules were detected by Northern hybridization, and their corresponding cell surface gene products were detected by cytofluorography of MCT cells stained with haplotype-specific antibodies. Both the cell surface 3M-1 and the small amounts of detected class II MHC molecules appear to be biologically functional, as MCT cells can support the proliferation of 3M-Ispecific, class II MHC-restricted helper T cells in culture. These findings suggest that MCT cells provide all the necessary biological parameters for interfacing both as the target of a nephritogenic immune response, and as a potential source for new extracellular matrix which develops as a fibrogenic response to interstitial nephritis.

Tubulointerstitial changes in the diabetic kidney correlate closely with the decline in glomerular filtration. In this study, we used a cell culture system of mouse proximal tubule epithelial cells to test the effects of glucose on cell growth, size, and matrix biosynthesis. [3H]thymidine incorporation was significantly inhibited in cells grown in 450 mg/dl glucose, compared with cells grown in 100 mg/dl glucose. The cells grown in the higher glucose concentration were slightly larger, their protein content and the total protein synthetic rate were significantly increased, and they secreted approximately twice as much procollagens type IV and type I. Concordantly, steady-state procollagen mRNA levels were also increased: 2.6-fold for the alpha 1(IV) and 2.2-fold for the alpha 2(I) procollagens. Additionally, nuclear run-off studies demonstrated that procollagen gene transcription rate was stimulated approximately 50%; beta-actin transcription rate was not altered. We used chloramphenicol acetyltransferase (CAT) reporter gene constructs to determine whether the increased transcription rate of alpha 2(I) gene was associated with activation of its enhancer sequence. Cells transfected with the enhancer demonstrated more than fivefold increase in CAT activity when cultured in the high-glucose medium. These studies demonstrate a multitude of effects of high ambient glucose concentrations on proximal tubule cell growth and collagen biosynthesis; cell proliferation is decreased although cell hypertrophy occurs. Procollagen gene transcription rate is stimulated and this response contributes to the observed increase in procollagen mRNA content. Activation of an enhancer sequence may be one possible mode through which high glucose levels increase the transcription of procollagen type I, presumably involving trans-acting factor(s).

No abstract

Abstract. 12 distinct neural cell adhesion molecule (N-CAM) epitopes, each recognized by a different monoclonal antibody (mAb), have been characterized in terms of the major structural and functional features of the molecule. Seven antibodies, each recognizing the amino-terminal region of the molecule, altered the rate of N-CAM-mediated adhesion. Four of these were inhibitors, two of which also recognized a heparin-binding N-CAM fragment. The other three antibodies specifically enhanced the rate of N-CAM-mediated adhesion. Three epitopes, one polypeptide-and two carbohydrate-dependent, were associated with the sialic acid-rich central portion of the molecule. The remaining two antibodies were found to react with intracellular determinants, and are specific for the largest of the three major N-CAM polypeptide forms. Studies on the ability of one antibody to hinder recognition of native N:CAM by another antibody suggested that the epitopes associated with N-CAM binding functions are in close proximity compared with the other determinants. The classification of these mAb epitopes has allowed the topographical placement of key N-CAM features, as described in the following paper, and provides valuable probes for analysis of both the structure and function of N-CAM.

1 Effects of cyclopiazonic acid (CPA), a specific inhibitor of the Ca24-ATPase in sarcoplamic reticulum (SR), on membrane ionic currents were examined in single smooth muscle cells freshly isolated from ileal longitudinal strips and urinary bladder of the guinea-pig. 2 Under whole-cell clamp, CPA (1-10 tM) reduced peak outward current elicited by depolarization in a concentration-dependent manner. The concentration of CPA required for 50% decrease in the peak outward current was -3 AM in ileal cells under these conditions. The current reduced by CPA recovered by more than 70% after washout. 3 The transient outward current elicited by application of 5 mM caffeine at a holding potential of -50 mV in Ca24 free solution was almost abolished, when the preceding Ca2"-loading of the cell in a solution containing 2.2 mM Ca24 was performed in the presence of 3 tM CPA.4 When the Ca2"-dependent K4 current (IK-Ca) and Ca24 current (ICa) were inhibited by addition of Ca2", the remaining delayed rectifier type K+ current was not affected by 10 AM CPA. When outward currents were blocked by replacement of K4 by Cs4 in the pipette solution, the remaining ICa was not affected by 10 LM CPA.5 CPA (10 gM) did not affect the conductance of single maxi Ca2"-dependent K+ channels or the Cd2+-dependence of their open probability in both inside-and outside-out configurations. 6 These results indicate that IK-Ca is selectively and strongly suppressed by CPA. Its effects may be attributed to a decrease in Ca2"-uptake into SR, resulting in a decrease in Ca2"-induced Ca24 release which is triggered by Ca24 entering through voltage-dependent Ca24 channels and therefore less activation of these K channels. 7 CPA may be extremely valuable pharmacological tool for investigating intracellular Ca24 mobilization and ionic currents regulated by intracellular Ca24.

Four poststroke patients with motor-dominant aphasia received 10 treatment sessions of low-frequency repetitive transcranial magnetic stimulation (rTMS). Each treatment session consisted of 1,200 pulses of stimulation and the site of stimulation was an area homologous to the most activated site on functional MRI performed prior to rTMS. Consequently, rTMS was applied to the right frontal lobe in two patients and to the left frontal lobe in two patients. Treatment resulted in improvement of language function in all four patients. Our therapeutic rTMS strategy seems to be a clinically feasible neurorehabilitative approach for poststroke aphasic patients.

Aim: To assess the safety and clinical efficacy of low-frequency repetitive transcranial magnetic stimulation (LF-rTMS) combined with intensive speech therapy (ST) in poststroke patients with aphasia. Subjects and Methods: Twenty-four patients with left-hemispheric stroke and aphasia were subjected. During 11-day hospitalization, each patient received 10 treatment sessions consisting of 40-min 1-Hz LF-rTMS and 60-min intensive ST, excluding Sundays. The scalp area for stimulation was selected based on the findings of fMRI with language tasks and the type of aphasia. LF-rTMS was applied to the inferior frontal gyrus (IGF) for patients with nonfluent aphasia and to the superior temporal gyrus (STG) for patients with fluent aphasia. Results: On pretreatment fMRI, the most activated areas were in the left hemisphere (n = 16) and right hemisphere (n = 8). The types of aphasia were nonfluent (n = 14) and fluent (n = 10). The LF-rTMS was applied to the right STG (n = 5), left STG (n = 5), right IFG (n = 11) and left IFG (n = 3). Nonfluent aphasic patients showed significant improvement of auditory comprehension, reading comprehension and repetition. Fluent aphasic patients showed significant improvement in spontaneous speech only. Conclusion: The fMRI with aphasic type-based therapeutic LF-rTMS/intensive ST for chronic aphasia seems feasible and a potentially useful neurorehabilitative protocol.

We provided an intervention to chronic post-stroke aphasic patients using low-frequency repetitive transcranial magnetic stimulation (LF-rTMS) guided by a functional magnetic resonance imaging (fMRI) evaluation of language laterality, combined with intensive speech therapy (ST). We performed a single photon emission-computed tomography (SPECT) scan pre- and post-intervention and investigated the relationship between cerebral blood flow (CBF) and language function. Fifty right-handed chronic post-stroke aphasic patients were enrolled in the study. During their 11-day hospital admission, the patients received a 40-min session of 1-Hz LF-rTMS on the left or right hemisphere, according to language localization identified by the fMRI evaluation, and intensive ST daily for 10 days, except for Sunday. A SPECT scan and language evaluation by the Standard Language Test of Aphasia (SLTA) were performed at the time of admission and at 3 months following discharge. We calculated laterality indices (LIs) of regional CBF (rCBF) in 13 language-related Brodmann area (BA) regions of interest. In patients who received LF-rTMS to the intact right hemisphere (RH-LF-rTMS), the improvement in the total SLTA score was significantly correlated with the pre- and post-intervention change of LI (ΔLI) in BA44. In patients who received LF-rTMS to the lesional left hemisphere (LH-LF-rTMS), this association was not observed. Analyses of the SLTA subscales and rCBF ΔLI demonstrated that in the RH-LF-rTMS group, the SLTA Speaking subscale scores were significantly correlated with ΔLIs in BA11, 20, and 21, and the SLTA Writing subscale scores were significantly correlated with ΔLIs in BA6 and 39. Conversely, in the LH-LF-rTMS group, the SLTA Speaking subscale scores were correlated with ΔLI in BA10, and the SLTA Reading subscale scores were significantly correlated with ΔLIs in BA13, 20, 22, and 44. Our results suggest the possibility that fMRI-guided LF-rTMS combined with intensive ST may affect CBF and contribute to the improvement of language function of post-stroke aphasic patients. LF-rTMS to the non-lesional and lesional hemispheres showed a difference in the associations between language performance and CBF. The results indicate that more effective rTMS intervention needs to be explored for patients who show right hemisphere language activation in an fMRI language evaluation.