Heparin potentiates the action of plasma membrane-associated growth stimulatory activity

Plasma membranes prepared from mouse liver have been previously shown to contain growth stimulatory activity as determined with cultured mouse fibroblasts. This growth stimulatory activity, termed plasma membrane-associated growth stimulatory activity (PMGA), is highly mitogenic in the presence of platelet-poor plasma. We now demonstrate that the growth stimulatory action of PMGA is dramatically enhanced by the addition of heparin. The half-maximal effect of heparin was observed at 1-3 micrograms/ml. The synergistic effect was seen in two distinct assays; the stimulation of DNA synthesis in quiescent cells, and an increase of cell number over a 3-day culture period. Heparin, by itself, does not have any measurable influence on the growth of fibroblasts. The action of heparin is not unique to this glycosaminoglycan, as several other highly sulfated polysaccharides, including dextran sulfate, pentosan polysulfate, and fucoidan, also exhibited the highly synergistic effect. Among other glycosaminoglycans examined, chondroitin sulfate B and heparan sulfate had a small, but significant, effect on enhancing the growth stimulatory action of PMGA. Chondroitin sulfate A, chondroitin sulfate C, hyaluronic acid dextran, and poly-L-glutamic acid, however, had no detectable effect. Further experiments suggested that the effect of heparin is twofold, namely, both a potentiation of growth stimulatory activity and a protection of PMGA activity. The data presented here suggest that the association of various cell surface components, such as PMGA and specific proteoglycans, can modulate the growth potential of a cell.     

Attenuating effect of diazepam on stress-induced increases in noradrenaline turnover in specific brain regions of rats: antagonism by Ro 15-1788

One-hour immobilization stress increased levels of the major metabolite of brain noradrenaline (NA), 3-methoxy-4-hydroxyphenyl-ethyleneglycol sulfate (MHPG-SO4), in nine brain regions of rats. Diazepam at 5 mg/kg attenuated the stress-induced increases in MHPG-SO4 levels in the hypothalamus, amygdala, hippocampus, cerebral cortex and locus coeruleus (LC) region, but not in the thalamus, pons plus medulla oblongata excluding the LC region and basal ganglia. The attenuating effects of the drug on stress-induced increases in metabolite levels in the above regions were completely antagonized by pretreatment with Ro 15-1788 at 5 or 10 mg/kg, a potent and specific benzodiazepine (BDZ) receptor antagonist. When given alone, Ro 15-1788 did not affect the increases in MHPG-SO4 levels. Behavioral changes observed during immobilization stress such as vocalization and defecation, were also attenuated by diazepam at 5 mg/kg and this action of diazepam was antagonized by Ro 15-1788 at 10 mg/kg, which by itself had no effects on these behavioral measurements. These findings suggest: (1) that diazepam acts via BDZ receptors to attenuate stress-induced increases in NA turnover selectively in the hypothalamus, amygdala, hippocampus, cerebral cortex and LC region and (2) that this decreased noradrenergic activity might be closely related to relief of distress-evoked hyperemotionality, i.e., fear and/or anxiety in animals.     

Nitric Oxide Reversibly Suppresses Xanthine Oxidase Activity

The effects of nitric oxide (NO) on xanthine oxidase (XOD) activity and the site(s) of the redox center(s) affected were investigated. XOD activity was determined by superoxide (O₂^-) generation and uric acid formation. NO reversibly and dose-dependently suppressed XOD activity in both determination methods. The suppression interval also disclosed a dose-dependent prolongation. The suppression occurred irrespective of the presence or absence of xanthine; indicating that the reaction product of NO and O₂^-, peroxynitrite, is not responsible for the suppression. Application of synthesized peroxynitrite did not affect XOD activity up to 2 μM. Methylene blue, which is an electron acceptor from Fe/S center, prevented the NO-induced inactivation. The results indicate that NO suppresses XOD activity through reversible alteration of the flavin prosthetic site.     

Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

Phosphoenolpyruvate:carbohydrate phosphotransferase systems in Enterococcus faecalis

A wild-type strain of Enterococcus faecalis and its mutants resistant to 2-deoxy-D-glucose (2DG) were examined for the presence of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs) with 12 carbohydrates, which were utilized by the organism, as the substrates. The wild-type strain possessed a constitutive mannose-PTS, which was reactive with glucose, mannose, glucosamine, 2DG and fructose. This activity was absent in the mutants. No independent glucose- or fructose-PTS was found in the mannose-PTS-defective mutants. The mutants, however, showed a low level of a constitutive PTS activity with maltose, suggesting the existence of an independent maltose-PTS in the organism. Both wild-type and mutant strains possessed inducible lactose-, mannitol-, and trehalose-PTSs. Lactose-PTS was induced by either lactose or galactose in the parent, but only by lactose in the mutants. The lactose-PTS was not reactive with galactose, and no separate galactose-PTS was present. These observations suggest that the inducer for lactose-PTS, probably being galactose 6-phosphate, may not be formed from galactose in the organism when the constitutive mannose-PTS is lost by mutation.     

Parathyroid hormone-related protein is a possible autocrine growth inhibitor for lymphocytes

Adult T-cell leukemia (ATL)-related cells have the ability to produce a newly-isolated calcium-regulating protein, parathyroid hormone-related protein (PTHrP). The present study revealed that lectin-stimulated normal lymphocytes produce immunoreactive (IR)-PTHrP. When the T-cell-enriched fraction was purified from normal lymphocytes and then treated with lectin, a similar amount of IR-PTHrP was detected, suggesting that IR-PTHrP is an actual product of T-lymphocytes. A biologically active fragment of PTHrP, PTHrP(1-34), suppressed DNA synthesis in lectin-stimulated lymphocytes at concentrations greater than 50 pg/mL; the same concentration range of IR-PTHrP detected in the cultured media of lectin-stimulated lymphocytes. Therefore, it is reasonable to postulate that PTHrP is a cytokine inhibiting the cellular growth of normal lymphocytes.     

Experimental Approach Reveals the Role of alx1 in the Evolution of the Echinoderm Larval Skeleton

Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.     

Missing piece of top predator-based conservation: Demographic analysis of an owl population on a remote subtropical island

Top predators are frequently the target of conservation programs. Owls are such predators. However, previous studies of owls are biased to species occurring in temperate regions, whereas most owl species occur in tropical or subtropical regions and are understudied. Furthermore, owls are often endemic to islands and of unknown conservation status. Demographic data for such species are especially scarce although they are essential for initiating and promoting their conservation. As a case study of demographic analysis of owls in a tropical or subtropical area and on islands, we applied an integrated population model to 7-year monitoring data (2012–2018) of the Ryukyu Scops Owl population on Minami-daito Island, Japan. We used survival history data from 903 individuals, reproduction and sex ratio data from 213 broods, and count data of 2,526 individuals in total. Long-term averages of annual survival rates were 0.73 for adult females and 0.74 for adult males, although the sexual difference was not significant. Sex ratio estimates fluctuated annually and long-term averages were slightly skewed to males: 0.51 among fledglings, 0.54 among yearlings, and 0.52 among adults. Long-term averages of population size were estimated to be 273.4 females and 296.8 males. The long-term average of population growth rate was 0.98, suggesting a slightly declining trend. It was fortunate to recognize the declining trend during the early phase. Considering the general lack of fundamental ecological data on owls of tropical or subtropical areas and on islands, it seems likely that many endangered owl populations await conservation efforts.     

Mouse genetics identifies unique and overlapping functions of fibroblast growth factor receptors in keratinocytes

Fibroblast growth factors (FGFs) are key regulators of tissue development, homeostasis and repair, and abnormal FGF signalling is associated with various human diseases. In human and murine epidermis, FGF receptor 3 (FGFR3) activation causes benign skin tumours, but the consequences of FGFR3 deficiency in this tissue have not been determined. Here, we show that FGFR3 in keratinocytes is dispensable for mouse skin development, homeostasis and wound repair. However, the defect in the epidermal barrier and the resulting inflammatory skin disease that develops in mice lacking FGFR1 and FGFR2 in keratinocytes were further aggravated upon additional loss of FGFR3. This caused fibroblast activation and fibrosis in the FGFR1/FGFR2 double‐knockout mice and even more in mice lacking all three FGFRs, revealing functional redundancy of FGFR3 with FGFR1 and FGFR2 for maintaining the epidermal barrier. Taken together, our study demonstrates that FGFR1, FGFR2 and FGFR3 act together to maintain epidermal integrity and cutaneous homeostasis, with FGFR2 being the dominant receptor.