p-Chloromercuribenzoate-induced dissociation of cytoskeletal proteins in red blood cells of rats

Effects of p-chloromercuribenzoate (PCMB) on the cytoskeletal organization of rat red blood cells were studied. Upon incubation with 50 microM PCMB in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, 80% of actin and 45% of spectrin were released from the ghosts, resulting in the fragmentation of ghost membranes. Addition of 2 mM Mg2+ or 0.1 M KCl, or lowering incubation temperature to 0 degree C substantially inhibited the solubilization of the cytoskeletal proteins and the fragmentation of ghost membranes, which enable to examine the effects of PCMB on the interaction between transmembrane proteins and the peripheral cytoskeletal network. Decreased recoveries of transmembrane proteins, such as band 3 and glycophorin, in Triton shell fraction were observed in the ghosts incubated with PCMB either in the presence of Mg2+ or at 0 degree C. PCMB also inhibited the in vitro association of purified spectrin with spectrin-depleted inside-out vesicles through interaction with proteins in the vesicle, such as bands 2.1 and 3. In the PCMB-treated ghosts, intramembrane particles were highly aggregated, which further supports the PCMB-induced dissociation of the transmembrane proteins from the cytoskeletal network. The decreased recovery of glycophorin in the Triton shell fraction also observed in intact red blood cells upon incubation with PCMB. These results suggest that the main action of PCMB on red cell membranes under physiological condition, at higher ionic strength and in the presence of Mg2+, is to dissociate transmembrane proteins from the peripheral cytoskeletal network, which may modify functions of these proteins.     

Carboxymethyl cellulose, a nonimmunogenic hapten carrier with tolerogenic properties.

This communication reports on the tolerogenic properties of carboxymethyl cellulose (CMC) as a nonimmunogenic carrier for 2.4 dinitrophenyl (DNP) and the benzylpenicilloyl determinant (BPO). Either normal or primed mice, given an optimal dose of 250 micrograms per animal of DNP CMC, when challenged with an immunogenic form of the hapten as early as 30 min or as late as 21 days thereafter were completely and specifically unresponsive to it. Experimental evidence suggests that this unresponsiveness is not due to suppressor cells. Furthermore, DNP CMC induces tolerance in vivo but fails to do so in vitro under conditions at which other tolerogenic carbohydrate hapten conjugates such as DNP-dextran do. This together with comparative studies of tolerance induction kinetics by DNP CMC and DNP-dextran in vivo led us to conclude that molecular properties other than the epitope density must be attributed to CMC's tolerogenic potential. CMC may also be used as a tolerogenic carrier for BPO with respect to IgE antibody production. Thus, normal or primed mice injected with the BPO CMC conjugate were found specifically unresponsive to a challenge with an immunogenic form of penicillin.     

A new 440-kD isoform is the major ankyrin in neonatal rat brain

This report describes initial characterization of a 440-kD isoform of brain ankyrin (ankyrinB) representing an alternatively spliced mRNA product of the gene encoding the major isoform of ankyrin in adult human brain (Otto, E., M. Kunimoto, T. McLaughlin, V. Bennett, J. Cell Biology. 114:241-253). Northern and immunoblot analyses indicate that 440-kD ankyrinB includes the spectrin and membrane-binding domains as well as a regulatory domain of the major 220-kD isoform. 440-kD ankyrinB contains, in addition, a sequence of a predicted size of 220 kD which is inserted between the regulatory domain and spectrin/membrane-binding domains. 440-kD ankyrinB has properties expected of a peripherally associated membrane-skeletal protein: it is exclusively present in the particulate fraction of brain homogenates, is extracted with NaOH, and remains associated with Triton-X-100-resistant structures. Expression of 440-kD ankyrinB in rat brain began at birth before other ankyrins could be detected, peaked 10 d after birth, and then decreased progressively to 30% of the maximum in adults. Expression of the 220-kD ankyrinB and ankyrinR (erythroid ankyrin) began approximately 10 d after the 440-kD isoform, increased rapidly between 10 and 15 d after birth, and finally achieved their maximal levels in adults. 440-kD ankyrinB is present in approximately equivalent amounts in all regions of neonatal brain while in adult brain it is present in highest levels in cerebellum and lowest in brain stem. 440-kD ankyrinB was localized by immunofluorescence in regions of neonatal and adult brain containing primarily dendrites and unmyelinated axons. 440-kD ankyrinB thus may play a specialized role in neuronal processes.     

Ribosomal resistance of an istamycin producer, Streptomycestenjimariensis, to aminoglycoside antibiotics

$\text{Streptomyces}\text{tenjimariensis}$ SS-939 was resistant to its own aminoglycoside antibiotics, istamycins, as well as kanamycin A, neamine, ribostamycin and butirosin A, but was susceptible to neomycin B, lividomycin A and streptomycin. This resistance to these antibiotics was found to be due to ribosomes of the strain.     

Common and Distinct Clinical Features in Adult Patients with Anti-Aminoacyl-tRNA Synthetase Antibodies: Heterogeneity within the Syndrome

Objective

To identify similarities and differences in the clinical features of adult Japanese patients with individual anti-aminoacyl-tRNA synthetase antibodies (anti-ARS Abs).

Methods

This was a retrospective analysis of 166 adult Japanese patients with anti-ARS Abs detected by immunoprecipitation assays. These patients had visited Kanazawa University Hospital or collaborating medical centers from 2003 to 2009.

Results

Anti-ARS Ab specificity included anti-Jo-1 (36%), anti-EJ (23%), anti-PL-7 (18%), anti-PL-12 (11%), anti-KS (8%), and anti-OJ (5%). These anti-ARS Abs were mutually exclusive, except for one serum Ab that had both anti-PL-7 and PL-12 reactivity. Myositis was closely associated with anti-Jo-1, anti-EJ, and anti-PL-7, while interstitial lung disease (ILD) was correlated with all 6 anti-ARS Abs. Dermatomyositis (DM)-specific skin manifestations (heliotrope rash and Gottron’s sign) were frequently observed in patients with anti-Jo-1, anti-EJ, anti-PL-7, and anti-PL-12. Therefore, most clinical diagnoses were polymyositis or DM for anti-Jo-1, anti-EJ, and anti-PL-7; clinically amyopathic DM or ILD for anti-PL-12; and ILD for anti-KS and anti-OJ. Patients with anti-Jo-1, anti-EJ, and anti-PL-7 developed myositis later if they had ILD alone at the time of disease onset, and most patients with anti-ARS Abs eventually developed ILD if they did not have ILD at disease onset.

Conclusion

Patients with anti-ARS Abs are relatively homogeneous. However, the distribution and timing of myositis, ILD, and rashes differ among patients with individual anti-ARS Abs. Thus, identification of individual anti-ARS Abs is beneficial to define this rather homogeneous subset and to predict clinical outcomes within the “anti-synthetase syndrome.”     

l-Malic Acid Fermentation by Mixed Culture of Rhizopus arrhizus and Proteus vulgaris

When Rhizopus arrhizus NRRL 1526 was mix-cultured with Proteus vulgaris AHU 1144, a strain having a high fumarase activity, in a medium containing glucose as a substrate, fumaric acid fermentation was successively converted to l-malic acid fermentation and large amounts of l-malic acid were accumulated as an end product.

As an inoculum of P. vulgaris for this fermentation, cells in the stationary growth phase (48 to 72 hr culture) were much more favorable than those in the exponential growth phase (18 hr culture) and malic acid yields in the former case were as high as about 70 to 75 % based on initial glucose after 3 to 4 days of the mixed culture.     

Neuronal nitric oxide synthase (NNOS) catalyzes one-electron reduction of 2,4,6-trinitrotoluene, resulting in decreased nitric oxide production and increased nNOS gene expression: implication for oxidative stress

To determine the mechanism of 2,4,6-trinitrotoluene (TNT)-induced oxidative stress involving neuronal nitric oxide synthase (nNOS), we examined alterations in enzyme activity and gene expression of nNOS by TNT, with an enzyme preparation and rat cerebellum primary neuronal cells. TNT inhibited nitric oxide formation (IC(50) = 12.4 microM) as evaluated by citrulline formation in a 20,000 g cerebellar supernatant preparation. A kinetic study revealed that TNT was a competitive inhibitor with respect to NADPH and a noncompetitive inhibitor with respect to L-arginine. It was found that purified nNOS was capable of reducing TNT, with a specific activity of 3900 nmol of NADPH oxidized/mg/min, but this reaction required CaCl(2)/calmodulin (CaM). An electron spin resonance (ESR) study indicated that superoxide (O(2)(.-)) was generated during reduction of TNT by nNOS. Exposure of rat cerebellum primary neuronal cells to TNT (25 microM) caused an intracellular generation of H(2)O(2), accompanied by a significant increase in nNOS mRNA levels. These results indicate that CaM-dependent one-electron reduction of TNT is catalyzed by nNOS, leading to a reduction in NO formation and generation of H(2)O(2) derived from O(2)(.-). Thus, it is suggested that upregulation of nNOS may represent an acute adaptation to an increase in oxidative stress during exposure to TNT.