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)     

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.     

Prostaglandin D2 plays an essential role in chronic allergic inflammation of the skin via CRTH2 receptor

PGD(2) plays roles in allergic inflammation via specific receptors, the PGD receptor designated DP and CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells). We generated mutant mice carrying a targeted disruption of the CRTH2 gene to investigate the functional roles of CRTH2 in cutaneous inflammatory responses. CRTH2-deficent mice were fertile and grew normally. Ear-swelling responses induced by hapten-specific IgE were less pronounced in mutant mice, giving 35-55% of the responses of normal mice. Similar results were seen in mice treated with a hemopoietic PGD synthase inhibitor, HQL-79, or a CRTH2 antagonist, ramatroban. The reduction in cutaneous responses was associated with decreased infiltration of lymphocytes, eosinophils, and basophils and decreased production of macrophage-derived chemokine and RANTES at inflammatory sites. In models of chronic contact hypersensitivity induced by repeated hapten application, CRTH2 deficiency resulted in a reduction by approximately half of skin responses and low levels (63% of control) of serum IgE production, although in vivo migration of Langerhans cells and dendritic cells to regional lymph nodes was not impaired in CRTH2-deficient mice. In contrast, delayed-type hypersensitivity to SRBC and irritation dermatitis in mutant mice were the same as in wild-type mice. These findings indicate that the PGD(2)-CRTH2 system plays a significant role in chronic allergic skin inflammation. CRTH2 may represent a novel therapeutic target for treatment of human allergic disorders, including atopic dermatitis.     

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.     

Gastric inhibitory polypeptide as an endogenous factor promoting new bone formation after food ingestion

Calcium plays a fundamental role as second messenger in intracellular signaling and bone serves as the body's calcium reserve to tightly maintain blood calcium levels. Calcium in ingested meal is the main supply and inadequate calcium intake causes osteoporosis and bone fracture. Here, we describe a novel mechanism of how ingested calcium is deposited on bone. Meal ingestion elicits secretion of the gut hormone gastric inhibitory polypeptide (GIP) from endocrine K cells in the duodenum. Bone histomorphometrical analyses revealed that bone formation parameters in the mice lacking GIP receptor (GIPR(-/-)) were significantly lower than those of wild-type (GIPR(+/+)) mice, and that the number of osteoclasts, especially multinuclear osteoclasts, was significantly increased in GIPR(-/-) mice, indicating that GIPR(-/-) mice have high-turnover osteoporosis. In vitro examination showed the percentage of osteoblastic cells undergoing apoptosis to be significantly decreased in the presence of GIP. Because GIPR(-/-) mice exhibited an increased plasma calcium concentration after meal ingestion, GIP directly links calcium contained in meal to calcium deposition on bone.     

Toward Probabilistic Diagnosis and Understanding of Depression Based on Functional MRI Data Analysis with Logistic Group LASSO

Diagnosis of psychiatric disorders based on brain imaging data is highly desirable in clinical applications. However, a common problem in applying machine learning algorithms is that the number of imaging data dimensions often greatly exceeds the number of available training samples. Furthermore, interpretability of the learned classifier with respect to brain function and anatomy is an important, but non-trivial issue. We propose the use of logistic regression with a least absolute shrinkage and selection operator (LASSO) to capture the most critical input features. In particular, we consider application of group LASSO to select brain areas relevant to diagnosis. An additional advantage of LASSO is its probabilistic output, which allows evaluation of diagnosis certainty. To verify our approach, we obtained semantic and phonological verbal fluency fMRI data from 31 depression patients and 31 control subjects, and compared the performances of group LASSO (gLASSO), and sparse group LASSO (sgLASSO) to those of standard LASSO (sLASSO), Support Vector Machine (SVM), and Random Forest. Over 90% classification accuracy was achieved with gLASSO, sgLASSO, as well as SVM; however, in contrast to SVM, LASSO approaches allow for identification of the most discriminative weights and estimation of prediction reliability. Semantic task data revealed contributions to the classification from left precuneus, left precentral gyrus, left inferior frontal cortex (pars triangularis), and left cerebellum (c rus1). Weights for the phonological task indicated contributions from left inferior frontal operculum, left post central gyrus, left insula, left middle frontal cortex, bilateral middle temporal cortices, bilateral precuneus, left inferior frontal cortex (pars triangularis), and left precentral gyrus. The distribution of normalized odds ratios further showed, that predictions with absolute odds ratios higher than 0.2 could be regarded as certain.     

A putative PhODO1 homologous MYB transcription factor gene, MdMYBB, is not involved in the regulation of aroma volatile biosynthesis in apple

To get deeper insight on the molecular mechanism underlying production of volatile compounds in apple (Malus domestica Borkh.), we performed the isolation and expression analysis of one R2R3-type MYB gene named MdMYBB. The amino acid sequence and the structural features of MdMYBB highly resembled those of PhODO1, which is a key regulator for floral scent biosynthesis in petunia. The expression of MdMYBB was repressed concomitantly with the inhibition of ethylene production, which regulates the volatile synthesis in apple. However, MdMYBB expression was not detected in the flesh from nearly ripened apple fruits, although the detection of exogenous volatiles had actually occurred in the same portion. In addition, overexpression of MdMYBB did not cause any induction of the volatile compounds in the transgenic tobacco lines. Thus, the features of MdMYBB were not in accordance with the aroma volatile emission, unlike the case of PhODO1, suggesting that MdMYBB may not be involved in the regulation of the biosynthesis for apple aroma volatiles. On the basis of the specific expression patterns, we discussed possible physiological roles of MdMYBB in apple.     

Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish,Oryzias latipes

In the medaka fish (Oryzias latipes) many mutants for body color have been isolated. A typical example is the recessive oculocutaneous albino mutant i, which has amelanotic skin and red-colored eyes with no tyrosinase activity. To cast light on the molecular basis of the albino mechanism, we performed Southern blot analysis of genomic DNA from the mutant with an authentic tyrosinase gene probe; the results demonstrate that an extra 1.9 kb fragment is present inside the first exon. The insertion is responsible for the oculocutaneous albinism. About 80 copies of this fragment are present in the genomes of albino-i and wild-type fish; these repeated sequences are here designated Tol1 elements and the particular element found in the tyrosinase gene of albino-i is denoted Tol1-tyr. The nucleotide sequence of Tol1-tyr shows that the fragment (i) carries terminal inverted repeats of 14 bp, and (ii) is flanked by duplicated 8 by segments of the host chromosome. These are properties of DNA-mediated transposable elements. Comparison of the nucleotide sequence of Tol1-tyr with other sequences in DNA databases, with special attention to sequences of transposable elements known to date, did not reveal any similarity. Thus, Tol1 constitutes a hitherto unknown family of DNA transposable elements.