The Drosophila tissue polarity gene starry night encodes a member of the protocadherin family.

The tissue polarity genes control the polarity of hairs, bristles and ommatidia in the adult epidermis of Drosophila. We report here the identification of a new tissue polarity gene named starry night (stan). Mutations in this essential gene alter the polarity of cuticular structures in all regions of the adult body. The detailed polarity phenotype of stan on the wing suggested that it is most likely a component of the frizzled (fz) pathway. Consistent with this hypothesis, stan appears to be downstream of and required for fz function. We molecularly cloned stan and found that it encodes a huge protocadherin containing nine cadherin motifs, four EGF-like motifs, two laminin G motifs, and seven transmembrane domains. This suggests that Stan functions in signal reception, perhaps together with Fz.     

Nutrition and muscle potassium: differential effect in rat slow and fast muscles

The effects of malnutrition on intracellular K+ activity, (alpha K)i, and membrane potential, Em, were measured by means of double-barrelled K+-selective microlectrodes in the soleus and gastrocnemius muscles of the rat. (alpha K)i and Em were measured in vivo in normal anaesthetized animals and in rats subjected to one of two diet restrictions: a 2-day fast or a long-term hypocaloric diet. In the soleus muscle, (alpha K)i fell by similar amounts in both 2-day fasted and long-term hypocalorically fed rats, while Em depolarized significantly only in hypocalorically fed rats. In the gastrocnemius muscle, neither the 2-day fast nor the hypocaloric diet affected (alpha K)i or Em. It is suggested that the selective loss of K+ from the soleus muscle may be related to its activity pattern.     

Structure-function analysis of casein kinase 2 with synthetic peptides and anti-peptide antibodies.

Casein kinase 2 (CK2) is a ubiquitous, multifunctional protein-seryl/threonyl kinase that has been implicated in cellular regulation. Synthetic peptides were patterned after three highly conserved regions in CK2: the N terminus (CK2-NT); the lysine-rich, kinase subdomain III segment (CK2-III) (nomenclature of Hanks et al. (Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241, 42-52)); and a 10-residue segment located near kinase subdomain X that is shared between CK2 and p34cdc2 (CK2/cdc2). The CK2-III and CK2/cdc2 peptides markedly stimulated the autophosphorylation of the alpha- and alpha'-subunits of purified CK2 from sea star oocytes, and they elicited up to 2-fold increases in its casein or phosvitin phosphotransferase activity. These peptides completely reversed nearly total inhibition of CK2 phosphotransferase activity toward itself, casein, and phosvitin by either heparin or poly(Glu,Tyr; 4:1), whereas CK2-NT was ineffective. Elution of CK2 from heparin-agarose with the CK2-III peptide indicated that this region of CK2 might mediate heparin binding to CK2. Affinity-purified rabbit polyclonal antibodies developed against both CK2-III and CK2/cdc2, but not CK2-NT, also produced up to 1.8-fold enhancements of the casein and phosvitin phosphotransferase activities of purified CK2. All three of the antipeptide antibody preparations immunoreacted with the alpha- and alpha'-subunits of CK2 on Western blots. These studies indicate that kinase subdomains III and X are involved in the modulation of CK2 phosphotransferase activity.     

Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.     

Insulin production and signaling in renal tubules of Drosophila is under control of tachykinin-related peptide and regulates stress resistance

The insulin-signaling pathway is evolutionarily conserved in animals and regulates growth, reproduction, metabolic homeostasis, stress resistance and life span. In Drosophila seven insulin-like peptides (DILP1-7) are known, some of which are produced in the brain, others in fat body or intestine. Here we show that DILP5 is expressed in principal cells of the renal tubules of Drosophila and affects survival at stress. Renal (Malpighian) tubules regulate water and ion homeostasis, but also play roles in immune responses and oxidative stress. We investigated the control of DILP5 signaling in the renal tubules by Drosophila tachykinin peptide (DTK) and its receptor DTKR during desiccative, nutritional and oxidative stress. The DILP5 levels in principal cells of the tubules are affected by stress and manipulations of DTKR expression in the same cells. Targeted knockdown of DTKR, DILP5 and the insulin receptor dInR in principal cells or mutation of Dilp5 resulted in increased survival at either stress, whereas over-expression of these components produced the opposite phenotype. Thus, stress seems to induce hormonal release of DTK that acts on the renal tubules to regulate DILP5 signaling. Manipulations of S6 kinase and superoxide dismutase (SOD2) in principal cells also affect survival at stress, suggesting that DILP5 acts locally on tubules, possibly in oxidative stress regulation. Our findings are the first to demonstrate DILP signaling originating in the renal tubules and that this signaling is under control of stress-induced release of peptide hormone.     

8-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones: Dual-acting 5-HT1 receptor antagonists and serotonin reuptake inhibitors—Part II

8-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones have been identified as highly potent 5-HT_1A/B/D receptor antagonists with and without additional SerT activity and a high degree of selectivity over hERG potassium channels. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.     

The Planktonic Index of Biotic Integrity (P-IBI): An approach for assessing lake ecosystem health

Using historical (1970) and more recent (1996) Lake Erie plankton and trophic status data, we developed a Planktonic Index of Biotic Integrity (P-IBI) to measure changes in lake ecosystem health. We used discriminant analysis to determine phytoplankton and zooplankton community characteristics (metrics) that distinguished among levels of impairment. Traditional measures of lake trophic status classes (i.e., oligotrophic, mesotrophic, eutrophic), such as chlorophyll a and total phosphorus concentrations, were used to classify sites on a gradient of impairment. We then judged the ability of plankton metrics to distinguish among trophic status classes. Because of the temporal variability found in plankton communities, we conducted analyses on a monthly basis (May–September). For June, July and August we found five unique metrics that could distinguish among trophic status classes. The P-IBI showed an increase in water quality in Lake Erie between 1970 (<3 = eutrophic) and the mid-1990s (1996 and 1997) (3–4 = mesotrophic) (which reflected mean (±standard error) total phosphorus concentrations (μg/L) 1970 > 1996; western basin (41.53 ± 2.68 > 29.75 ± 1.39), eastern basin (14.84 ± 0.82 > 7.74 ± 0.28) and mean (±standard error) chlorophyll a concentrations (μg/L) uncorrected for pheophytin 1970 > 1996; western basin (12.58 ± 1.82 > 5.40 ± 0.22), central basin (5.90 ± 0.36 > 3.17 ± 0.54), and eastern basin (5.17 ± 0.38 > 1.67 ± 0.18)), with declining water quality in the late 1990s (1998 and 1999) (3) and 2002 (<3). We recommend that the techniques used in creating the P-IBI be investigated for determining ecosystem health of other lakes.     

Hydrogen sulfide mediates vasoactivity in an O2-dependent manner

Hydrogen sulfide (H(2)S) has recently been shown to have a signaling role in vascular cells. Similar to nitric oxide (NO), H(2)S is enzymatically produced by amino acid metabolism and can cause posttranslational modification of proteins, particularly at thiol residues. Molecular targets for H(2)S include ATP-sensitive K(+) channels, and H(2)S may interact with NO and heme proteins such as cyclooxygenase. It is well known that the reactions of NO in the vasculature are O(2) dependent, but this has not been addressed in most studies designed to elucidate the role of H(2)S in vascular function. This is important, since H(2)S reactions can be dramatically altered by the high concentrations of O(2) used in cell culture and organ bath experiments. To test the hypothesis that the effects of H(2)S on the vasculature are O(2) dependent, we have measured real-time levels of H(2)S and O(2) in respirometry and vessel tension experiments, as well as the associated vascular responses. A novel polarographic H(2)S sensor developed in our laboratory was used to measure H(2)S levels. Here we report that, in rat aorta, H(2)S concentrations that mediate rapid contraction at high O(2) levels cause rapid relaxation at lower physiological O(2) levels. At high O(2), the vasoconstrictive effect of H(2)S suggests that it may not be H(2)S per se but, rather, a putative vasoactive oxidation product that mediates constriction. These data are interpreted in terms of the potential for H(2)S to modulate vascular tone in vivo.