Responses in rectal and skin temperatures to centrifugal forces in rats of different ambient temperatures

Effects of centrifugation upon rectal (T_re) and tail skin temperatures (T_s) were studied in male Wistar rats at varying ambient temperature (T_a) using a centrifuge which was placed in a climatic chamber. Centrifugal forces of Gz of 3.0 were imposed on rats which were suspended at horizontal body position using a newly developed mesh suits holding method in the animal box placed on the rotating arm of the centrifuge. Headwards or tailwards forces were applied according to the experimental design. No significant difference of the responses was observed between the two force directions.Centrifugations imposed at different T_a of 15, 20, 25, 30 and 32.5C resulted in falls in T_re accompanied by rises in tail T_s at the cooler environments, while rises in T_re accompanied by falls in T_s in the warmer environments. The T_a at which the response pattern of T_re and T_s was reversed (critical ambient temperature) was 26.8±2.3 (mean and SE) and 27.9±2.8C, respectively. Tolerance to centrifugation was markedly increased in cooler environments than in wanner ones. It was suggested that the increased skin pressure due to centrifugation exerted some inhibitory effects upon central thermoregulatory ability.     

Microinjection of macromolecules into normal murine lymphocytes by means of cell fusion. II. Enhancement and suppression of mitogenic responses by microinjection of monoclonal anti-cyclic AMP into B lymphocytes

Reproducible methods are now available for introducing protein molecules such as antibodies into normal murine lymphocytes by fusion with protein molecule-containing erythrocyte ghosts. Monoclonal antibodies against cyclic AMP were raised by hybridoma technique and packed into erythrocyte ghosts. Then, monoclonal anti-cyclic AMP containing ghosts were fused with splenic B lymphocytes by polyethylene glycol-mediated fusion at various intervals after LPS stimulation. This method made it possible for us to quantitatively microinject antibodies into B lymphocytes. Microinjection of anti-cyclic AMP antibody molecules into lymphocytes at a very early stage of LPS stimulation resulted in a marked enhancement of DNA synthetic responses as well as increased numbers of plaque-forming cells. Intracellular cyclic AMP levels were found to be markedly decreased after microinjection of monoclonal anti-cyclic AMP, suggesting that lowering the intracellular cyclic-AMP level in the B lymphocytes at an early stage of stimulation might have induced the enhanced proliferative as well as differentiative responses to LPS. Similar enhancing effects on cell proliferation were obtained when antibodies were injected 18 hr after stimulation. Microinjection of anti-cyclic AMP at 12 hr after culture, however, inhibited the DNA synthetic responses, and induction of plaque-forming cells was suppressed when anti-cyclic AMP was injected 6 hr after LPS stimulation. The present data suggest the biphasic regulatory roles of cyclic AMP at the early stage of B lymphocyte activation. This approach may be useful in identifying regulatory molecules in B lymphocyte induced by mitogenic or antigenic stimulation.     

Formation of 12-oxo-trans-10-dodecenoic acid in chloroplasts from Thea sinensis leaves

Linolenic acid-[1-¹⁴C] was converted to 12-oxo-trans-10-dodecenoic acid, via 12-oxo-cis-9-dodecenoic acid by incubation with chloroplasts of Thea sinensis leaves. Thus, it was confirmed that linolenic acid is split into a C₁₂-oxo-acid, 12-oxo-trans-10-dodecenoic acid, and a C₆-aldehyde, trans-2-hexenal, leaf aldehyde, by an enzyme system in chloroplasts of tea leaves.     

Supercoiled DNA folded by nonhistone proteins in cultured mouse carcinoma cells.

Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histones could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with gamma-ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.     

Identification of two novel GTP-binding protein alpha-subunits that lack apparent ADP-ribosylation sites for pertussis toxin

Molecular cloning of cDNAs encoding alpha-subunits of guanine nucleotide-binding regulatory proteins (G-proteins) has revealed the existence of nine species of alpha-subunits. We have identified two additional G-protein alpha-subunits, which we refer to as GL1 alpha and GL2 alpha, by isolating bovine liver cDNA clones that cross-hybridized at reduced stringency with bovine Gi1 alpha-subunit cDNA. The deduced amino acid sequences of GL1 alpha and GL2 alpha share 83% identity with each other and show 45-55% identity with those of other known G-protein alpha-subunits. Both GL1 alpha and GL2 alpha lack a consensus site for ADP-ribosylation by pertussis toxin. Messenger RNA corresponding to GL2 alpha was detected in all tissues examined, but GL1 alpha mRNA was detected only in liver, lung, and kidney. Antiserum prepared against a synthetic pentadecapeptide corresponding to the deduced carboxyl terminus of GL2 alpha specifically reacted with a 40-kDa protein in mouse liver, brain, lung, heart, kidney, and spleen. The amount of the 40-kDa protein was highest in brain and lung. We suggest that GL1 alpha and GL2 alpha are new members of a subfamily of pertussis toxin-insensitive G-proteins.     

Collagen-induced arthritis in mice. Relationship of collagen-specific and total IgE synthesis to disease.

The relationship between production of IgE and collagen-induced arthritis in mice was examined. Collagen-specific IgE was produced as a consequence of immunization of DBA/1 mice with chicken type II collagen emulsified in CFA. We observed a rise in collagen-specific IgE antibody levels at the onset of CIA clinical and histologic signs in DBA/1 mice. This rise in IgE paralleled that of IgG2a anticollagen antibodies, an isotype implicated in the pathogenesis of CIA by other laboratories. The collagen-specific IgE contained in the plasma of mice with CIA could arm basophils for Ag- (collagen) dependent degranulation. Collagen-specific IgE may thus contribute to CIA by promoting mast cell degranulation in the synovia of susceptible mice immunized with chick type II collagen; but, further work is required to establish such a role for IgE in CIA. However, genetic differences in disease susceptibility could not be accounted for by quantitative differences in collagen-specific IgE production. Further, comparable levels of IgE anticollagen antibodies were observed in animals with active CIA and after spontaneous remission, thereby confirming that the presence of such antibodies is insufficient for disease. Total IgE levels peaked just before spontaneous remission indicating active production of IL-4. IL-4 was administered to animals with CIA to determine if this lymphokine could be involved in the remission process. IL-4 facilitated remission of CIA. Enhanced total IgE production may thus be a marker for activation of Th2 cells that produce lymphokines such as IL-4 and IL-10, factors that may be involved in the spontaneous remission process.     

Changes in the Levels of Chlorophyll and Light-Harvesting Chlorophyll a/b Protein of PS II in Rice Leaves Aged under Different Irradiances from Full Expansion through Senescence

Effects of irradiance on changes in the amounts of chlorophyll(Chl) and light-harvesting chlorophyll a/b protein of PS II(LHCII) were examined in senescing leaves of rice (Oryza sativaL.). Results of treatments at two irradiances (100% and 20%natural sunlight) were examined after the full expansion ofthe 13th leaf throughout the course of senescence. With 20%sunlight, the Chl content decreased only a little during leafsenescence, while with 100% sunlight it decreased appreciably.Similarly, the amount of LHCII protein during treatment with20% sunlight remained almost constant. However, the ratio ofChl a/b during the shade treatment decreased significantly andthe rate of decrease was greater than during the full-sunlighttreatment. The ratio of Chl a/b for Chl a and b bound to LHCIIwas about 1.2, irrespective of leaf age or irradiance treatment.When the amounts of Chl bound to LHCII were calculated fromthe total leaf content of Chl and the ratio of Chl a/b, assuminga ratio of Chl a/b bound to LHCII of 1.2, they were well correlatedwith the amounts of LHCII protein. Changes in the amounts of LHCII synthesized during the two irradiancetreatments were examined using an ¹⁵ tracer. Incorporation of¹⁵N into LHCII declined dramatically during both treatmentsfrom full expansion through senescence, suggesting that therewas little synthesis of LHCII protein during that time. In addition,the amount of LHCII synthesized during senescence was lowerduring the shade treatment than during the 100% sunlight treatment.These results indicate that the absence of an apparent changein levels of LHCII with shade treatment during senescence wascaused by the very low rate of turnover of LHCII protein. (Received June 17, 1992; Accepted September 28, 1992)     

Kinetics of growth and lactic acid production in continuous and batch culture

Summary In a lactic acid fermentation by Streptococcus faecalis, the specific consumption rate of glucose (v) and the specific production rate of lactic acid () were represented by the following simple equations as functions of the specific growth rate (): 1/=(1/) + 1/ = (1/) + By use of data from a batch culture, these two equations were derived from enzyme kinetics of the product inhibition. These equations were successfully applied to the results of batch culture and chemostat culture. In addition, calculation of ATP yield by these equations agreed with the experimental results better than the conventional Leudeking-Piret type equation, which includes two terms associated with growth and not with growth. Correspondence to: H. Ohara