Three cDNA clones of 1.6 (3u), 1.2 (5g) and 0.6 (5b) kbp with probes covering the 3' end of the two unexpected regions show that three distinct mRNAs correspond to the three cDNAs. Moreover, three peripherin products, two minor 61 and 56 kd products in addition to the major 58 kd peripherin, are observed when poly(A)+ RNA is in vitro translated, the 61 kd peripherin being translated from the 3u-selected RNA. The three RNAs originate from alternative splicing of a unique peripherin gene, thus generating polymorphism of peripherin.

Two clinical strains of Klebsiella pneumoniae, TP 01 and TP 02, presented resistance to amoxicillin-clavulanate and were fully susceptible to cephalothin. These strains produced two ␤-lactamases, SHV-1 and a TEM enzyme with a pI of 5.2. The previously described changes Arg-2443Cys and Arg-2443Ser in IRT-1 and IRT-2 (A. Belaaouaj, C. Lapoumeroulie, M. M. Caniça, G. Vedel, P. Nevot, R. Krishnamoorthy, and G. Paul, FEMS Microbiol. Lett. 120:75-80, 1994) were found in TEM enzymes from the TP 01 and TP 02 strains, respectively. This is the first report of inhibitor-resistant TEM (IRT) in species other than Escherichia coli from the family Enterobacteriaceae.Most Klebsiella pneumoniae strains are moderately resistant to amino-and carboxy-penicillins by synthesis of a class A chromosomally mediated penicillinase (SHV-1), and clavulanic acid has a synergistic effect when associated with these penicillins. These strains are generally susceptible to cephalothin and all other cephalosporins. Some K. pneumoniae isolates which are highly resistant to penicillins because of the production of plasmid-mediated TEM-like penicillinases also have reduced susceptibility to cephalosporins.Previous reports have established that the susceptibility of Escherichia coli to ␤-lactamase inhibitors can be affected by the overproduction of TEM-1 ␤-lactamase (14, 18). In K. pneumoniae, overproduction of either TEM-1 or SHV-1 increased resistance to amoxicillin-clavulanic acid and to cephalothin (16).In this report, we describe the characterization of the ␤-lactamases produced by two clinical isolates of K. pneumoniae that exhibited high-level resistance to amoxicillin-clavulanate and susceptibility to cephalothin.K. pneumoniae TP 01 and TP 02 were isolated from the urine and respiratory tracts of patients hospitalized in the geriatric and pneumology services, respectively, of a teaching hospital in Toulouse. The MICs of amoxicillin, amoxicillin-clavulanate, ticarcillin, ticarcillin-clavulanate, piperacillin, piperacillin-tazobactam (clavulanic acid and tazobactam were at fixed concentrations of 2 g/ml and 4 g/ml, respectively), and cephalothin were determined by dilution in Mueller-Hinton agar (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) with an inoculum of 10 4 CFU per spot. A K. pneumoniae strain (CF 004) producing only the chromosomal SHV-1 enzyme, a K. pneumoniae strain (CF 014) producing both SHV-1 and TEM-1, and a K. pneumoniae strain (CF 024) producing SHV-1 enzyme at a high level were studied as comparisons for the MIC determinations (Table 1).K. pneumoniae CF 004 (SHV-1) was characterized by susceptibility to all ␤-lactams tested except for amoxicillin and ticarcillin. The MIC for both of these drugs was 128 g/ml. The resistance patterns of K. pneumoniae TP 01 and TP 02 were characterized by high MICs of amoxicillin-clavulanate (with clavulanate at a fixed concentration of 2 g/ml) of 256 and 512 g/ml, respectively, while that for TEM-1-producing strain CF 014 is only 32 g/ml. MICs of ticarcillin-clavulanate for inhibitor-...

The nonobese diabetic (NOD) mouse, in which major histocompatibility complex genes may be involved in the susceptibility to diabetes, has been developed as a model of autoimmune diabetes. The NOD mouse expresses I-A- (20) were grown in RPMI 1640 medium supplemented with 10o fetal calf serum, 2 mM glutamine, penicillin at 100 units/ml, and streptomycin at 100 jg/ml (GIBCO/BRL). Normal islets were prepared by collagenase (Sigma) digestion of mouse pancreases as described by Lacy and Kotianovsky (21) with slight modifications (22).Western Blots. Cells (1 x 108) from each cell line were washed in Hanks' balanced saline solution, suspended in 7 ml of 0.01M Tris-buffered saline (TBS)/10 mM CaCl2/0.25 M sucrose, pH 7.4, and disrupted at 40C. The cell homogenates were centrifuged (1000 x g, 10 min), and the supernatant was subjected to ultracentrifugation (20,000 x g, 1 h). The pellet was then resuspended in 2.3% SDS/62.5 mM Tris/10%6 glycerol/5% 2-mercaptoethanol, pH 6.8. One-dimensional gel electrophoresis and electrotransfers to nitrocellulose filters were performed as described by Laemmli (22) and Burnette (23). Two-dimensional SDS/PAGE was performed as described by O'Farrell (24). The filters were soaked in 0.02 M TBS/3% gelatin, washed in 0.05% Tween 20/0.02 M TBS, and then incubated with the test sera or ascitic fluid diluted 1:50 in 1% gelatin/0.05% Tween 20/0.02 M TBS. After washing, they were incubated with biotinylated sheep antiAbbreviations: NOD, nonobese diabetic; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; NCS, newborn calf serum; MHC, major histocompatibility complex. tTo whom reprint requests should be addressed. 172The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Intermediate filament proteins of the rat insulinoma RIN5F cell line were characterized. Two-dimensional gel analysis followed by immunostaining of proteins demonstrated that these cells express both peripherin and the low-molecular-mass neurofilament protein (NF-L); this was confirmed for peripherin by immunohistochemistry, peptide analysis and Northern blot. No expression of these proteins could be detected with these same methods either in the adult pancreas or in the tumor at the origin of the cell line, although such expression was apparent on sections of rat pancreas at embryonal day 16. These results were compared to those obtained on the rat pheochromocytoma PC12 cell line: expression in the adrenal medulla of the embryo, no expression either in the adult tissue or in the tumor, but solely in the derived cell line. The expression of neuronal intermediate filament proteins in the rat insulinoma RIN5F cell line is discussed in relation to its similarity in the rat pheochromocytoma PC12 cell line, and its meaning as to the developmental cell lineage; an ectodermal origin is suggested for the pancreatic islet cells.