Appearance of a Cell Surface Antigen Associated with the Activation of Peritoneal Macrophages in Mice

A relatively large population of murine peritoneal exudate macrophages induced with viable BCG or heat-killed Corynebacterium parvum was stained by the antiserum prepared against purified gangliotetraosyl ceramide (asialo GM1), while only a small population of peritoneal resident macrophages or peritoneal exudate macrophages induced with proteose peptone was stained. The cytotoxicity assay of those macrophages with anti-asialo GM1 plus complement supported these results. Peritoneal macrophages induced with BCG or C. parvum showed strong cytotoxicity for EL4 cells in vitro, while resident or peptone-induced peritoneal macrophages showed no cytotoxicity. BCG- or C. parvum-induced peritoneal cells contained both NK cells and cytotoxic macrophages, and either in vivo or in vitro pretreatment of the cells with anti-asialo GM1 and complement abolished the activities of both types of cells. Peptone-induced peritoneal macrophages incubated with lymphokines (LK) or lipopolysaccharide (LPS) were cytotoxic for EL4 cells and contained an increased number of cells stained by anti-asialo GM1. The cytotoxicity of these in vitro activated macrophages was reduced by treatment with anti-asialo GM1 plus complement. When peptone-induced peritoneal macrophages were incubated with LK, the number of cells stained by anti-Ia antiserum increased, but the number did not increase when the macrophages were incubated with LPS. Pretreatment of peptone-induced macrophages with anti-asialo GM1 plus complement did not affect the ability of the macrophages to be activated by LK. These results taken together strongly suggest that the antigen (s) reactive with anti-asialo GM1 is expressed on the cell surface of cytotoxic peritoneal macrophages in mice.     

Influence of cadmium on the distribution of the essential trace elements zinc and copper in the liver and kidneys of rats

Cd induced changes of Zn and Cd distribution in the liver and kidneys were studied in relation to Cd metallothionein (MT) synthesis. Wistar male rats were given CdCl₂ by sc injection of .8, 1.5, and 3.0 mg Cd/kg three times a week for three weeks. Cd levels of liver and kidneys increased with the increment of Cd dosage and 80–90% of Cd was found in the cytosol. The MT fractions contained 80–89% cytosolic Cd in the liver and 55–75% Cd in the kidneys. Zn concentrations in the liver increased following Cd administration, But Zn in the kidneys showed only slight increase. There was a distinct decrease of Cu concentration in the liver of the 3.0 mg group. In contrast, Cu concentrations in the kidneys increased about three times in the .8 and 1.5 mg Cd groups, but Cu in the 3.0 mg group showed only 1.5 times increase. The changes of these metal concentrations were observed mainly in the cytosol. Non-MT-Cd in the kidneys was maximum in the 1.5 mg group, but the 3.0 mg group showed significant decrease. In parallel with this decrease of Cd, Cu and Zn in the kidneys showed similar decrease. When the kidneys are injured, Zn and Cu appear to leak from this organ.     

The origin of the luteinizing hormone-releasing hormone (LHRH) neurons in newts (Cynops pyrrhogaster): the effect of olfactory placode ablation

Summary Neurons containing luteinizing hormone-releasing hormone (LHRH) are first detected in newt embryos (Cynops pyrrhogaster) in the olfactory epithelium and ventromedial portion of the olfactory nerve, after which they sequentially appear in the intracerebral course of the terminal nerve at prometamorphosis, and in the septo-preoptic area at postmetamorphosis. In adults, however, LHRH-immunoreactive cells are rarely seen in the nasal region, and their distribution shifts into the brain, suggesting their migration. In order to ascertain the origin and possible migration route of these neurons in newt larvae, the effect of unilateral or bilateral olfactory placodectomy on the LHRH neuronal system has been studied. Removal of the olfactory placode results in the absence of LHRH-immunoreactive cells in the nasal and brain regions of the operated side, whereas the subsequent growth and the LHRH-immunoreactive cellular distribution in the contralateral side are identical to those of normal larvae. Following bilateral placodectomy, no LHRH immunoreactivity is detected on either side of the olfactory-brain axis. These results suggest that LHRH neurons of the newt, Cynops pyrrhogaster, originate in the olfactory placode and then migrate into the brain during embryonic development.     

Morphological effects of cadmium on proximal tubular cells in rats

Twenty-four male rats of the Wistar strain divided into four groups were injected sc with a dose of 0.8, 1.5, and 3.0 mg Cd/kg body wt as CdCl₂ in saline, and saline alone to the control rats, three times a week for 3 wk. Cadmium levels of whole kidney homogenate, supernatant (cytosol), precipitate, and metallothionein (MT) fraction were measured. Histological changes of the renal proximal tubules were investigated by optical and electron microscopy. In the kidneys, Cd levels were increased with the increment of Cd dosage; 80–90% of Cd was contained in cytosol, and 55–75% was in MT fraction. Non-MT-Cd reached a maximum in the 1.5 mg Cd group, whereas that of the 3.0 mg Cd group showed some decline. With increasing Cd doses, the size of nuclei and nucleoli in the cells of proximal tubule showed significant enlargement and also an increase in the number of nucleoli on light microscopy. At higher doses, chromatin condensation of the tubular nuclei and vacuolar degeneration of the tubular cells were evident. On electron microscopy, perichromatin granules of the proximal tubular nuclei were increased in number, especially in the rats of Cd 0.8 mg and 1.5 mg/kg groups. As the Cd doses increased, ring-shaped nucleoli were increased in number and nucleolar segregation was observed more clearly. Moreover, in the 3.0 mg/kg Cd group, nuclear indentation and nucleoli containing compact dense granules were observed. In the cytoplasm, there was an increase of lysosomes, myelin bodies, ring-shaped mitochondria, and vesiculation; ultimate changes were degeneration and cell necrosis. The injured cells were heterogenously distributed in each nephron and this heterogeneity was attributed in the difference in Cd content and cell cycle in each cell of the nephron.     

Crystal structure of subtilisin complexed with its trapped substrateStreptomyces subtilisin inhibitor—Protein-protein interaction and evolution of serine proteinases and their proteinaceous inhibitors

The complex of a bacterial alkaline serine proteinase, subtilisin BPN’, with its proteinaceous inhibitorStreptomyces subtilisin inhibitor is unique in several respects, compared with other similar complexes containing serine proteinases of trypsin family. In addition to the usual antiparallelβ-sheet involving P₁-P₃ residues of the inhibitor, P₄-P₆ residues form antiparallelβ-sheet with a previously unnoticed chain segment (the ‘S4-6 site’) of subtilisin. The ‘S4-6 site’ does not exist in serine proteinases of trypsin family, whether of mammalian or microbial origin. Global induced-fit movement seems to occur on the ‘trapped substrate’Streptomyces subtilisin inhibitor: a channel-like structure in SSI remote from the contact region becomes about 2 Å wider upon complexing with subtilisin. Main role of the secondary contact region ofStreptomyces subtilisin inhibitor seems to support the reactive site loop (primary contact region). Steric homology for the two contact regions is so high between the inhibitors ofStreptomyces subtilisin inhibitor family and those of pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family that it seems to favour a divergent evolution and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forwarded by Doolittle(Nature (London),272, 581, 1978).     

Role of lymphokines in regulation of macrophage differentiation

The regulatory mechanism of guinea pig lymphokines was investigated in regard to differentiation of myeloid cells to macrophages. The Ml-cell line, established from a myeloid leukemia of an SL-strain mouse, was induced to differentiate in vitro into mature macrophages possessing Fc receptors and the ability to phagocytize latex particles by treatment with crude lymphokines. Both concanavalin A- and antigen-induced lymphokines showed the differentiation-inducing factor (D factor) activity. However, macrophage migration inhibitory factor/ macrophage activation factor (MIF/MAF) purified by an immunoadsorbent column with anti-MIF antibody had no such an activity. The D-factor activity was detected in the lymphokine preparation that was not retained on the immunoadsorbent column. In contrast, colony-stimulating factor (CSF) was adsorbed to the immunoadsorbent column, and could be recovered in the purified MIF/MAF preparation. These findings suggest that the molecular entity of D factor is distinct from MIF/ MAF and CSF. A culture supernatant of guinea pig peritoneal macrophages activated with MIF/ MAF (CSF) exhibited strong D-factor activity. However, the supernatant possessed rather reduced CSF activity as compared to that of the original MIF/MAF (CSF) preparation. Thus, MIF/MAF may play an important role in macrophage differentiation by regulating the production of D factor or CSF from macrophages.     

Androgen Regulates Gene Expression of Cytoskeletal Proteins in Adult Rat Motoneurons

Expression of β-actin and β-tubulin mRNA was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding chick β-actin and mouse β-tubulin, respectively. Both hybridizable β-actin and βtubulin mRNAs were localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both β-actin and β-tubulin mRNAs in the SNB motoneurons, whereas the changes were prevented by testosterone treatment. In contrast, castration or testosterone treatment induced little or no change in the expression levels of these mRNAs in the much less androgen-sensitive motoneurons of the retrodorsolateral nucleus (RDLN). These results suggest that androgen regulates the expression of β-actin and β-tubulin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.     

Partial purification and property of pyridoxine (pyridoxamine)-5′-phosphate oxidase isozymes from wheat seedlings

Isozymes of pyridoxine (pyridoxamine)-5′-phosphate oxidase (EC 1.4.3.5) were isolated from the extract of wheat seedlings by column chromatographies. From DEAE-Sephadex A-50, two fractions having pyridoxine-5′-phosphate oxidase activity were separated by eluting with ~0.075 and ~0.125 m phosphate buffers (pH 8.0). These fractions were further fractionated on a Blue-Sepharose CL-6B column, from which again two activities were eluted by 1.0 m KCl solution. One fraction, designated as E-I, used only pyridoxine 5′-phosphate as substrate, whereas the other, designated as E-II, oxidized not only pyridoxine 5′-phosphate but also pyridoxamine 5′-phosphate with approximately equal rates. The mobility on polyacrylamide disc gel electrophoresis and the substrate specificity of these two fractions were different. Therefore, they were concluded to be isozymes.     

Mitotic Arrest in Tobacco Caused by the Phosphoprotein Phosphatase Inhibitor Okadaic Acid

Okadaic acid blocks the cell cycle at early mitosis in suspensioncultures of Nicotiana plumbaginifolia. Nuclear DNA content wasmeasured in treated cells by propidium iodide staining, fluorescencemicroscopy and quantitative analysis of the video image. NuclearDNA levels in inhibited populations showed that cells continuedto progress from G1 phase through S phase and accumulated inG2 phase. Arrested cells in 12 µM okadaic acid had a condensedchromatin configuration and persisting nucleolus similar tonormal early prophase. Progress to early prophase was also indicatedby development of the preprophase band (PPB) of microtubules.PPB microtubules disassembled in 95% of the inhibited cellswith the same timing as in control cells, although the treatedcells did not progress to prometaphase mitotic spindle assemblythat normally precedes PPB breakdown, therefore okadaic acidcan disrupt the normal dependence of PPB disassembly on prometaphasenuclear events and indicates that the normal signal for disassemblymay be an increase in phosphorylation of PPB associated proteins.Okadaic acid at 12 µM caused increased levels of phosphorylatedproteins, in particular those of 108 kDa, 49 kDa, 36 kDa, 33kDa, 31 kDa, but more complex effects on some phosphoproteinswere indicated by reductions in a phosphoprotein of 41 kDa andone of approximately 190 kDa. It is concluded that the mitoticphase of the plant cell cycle is more sensitive than precedingcycle phases to the disruption of protein phosphorylation levelsby okadaic acid and it is proposed that the inhibitor blocksdivision by interfering with essential changes in the phosphorylationstate of proteins at mitosis. This conclusion is discussed inrelation to genetical and biochemical evidence that proteinkinases and phosphatases are involved in the cell division ofplants and other eukaryotes. (Received November 26, 1991; Accepted April 20, 1992)     

Neurochemical Studies on the Cerebellar Hypoplasia of Gunn Rat (Hereditary Hyperbilirubinemic Rat)

The cerebellar hypoplasia induced by hereditary hyperbilirubinemia in the Gunn rat was analyzed neurochemically and immunohistochemically. The antiserum against myelin basic protein was used to visualize the arborization of the fibers in the cerebellum. Arborization was very scarce in the affected lobes of the homozygous (jj) cerebellum. Na,K-ATPase activity did not show significant differences between the jj and the control (Jj) cerebellum. The concentration of norepinephrine in the jj cerebellum was about 1.5 times that of the control. However, the activation ratio of the Na,K-ATPase by norepinephrine and other catecholamines such as dopamine and isoproterenol was about twice as high as the basal activity, and no significant difference was observed between the jj and the Jj cerebella. The glutamic acid decarboxylase activity of the jj cerebellum did not differ significantly from that of the control.