Synthetic substrate for eukaryotic signal peptidase. Cleavage of a synthetic peptide analog of the precursor region of preproparathyroid hormone

A synthetic peptide analog of the precursor region of preproparathyroid hormone has been shown to be a specific substrate for hen oviduct signal peptidase. The sequence of the 31-residue peptide is Ser-Ala-Lys-Asp-norleucine (Nle)-Val-Lys-Val-Nle-Ile-Val-Nle-Leu-Ala-Ile-Ala-Phe-Leu-Ala-Arg-Ser-As p-Gly-Lys-Ser-Val-Lys-Lys-Arg-D-Tyr-amide (Caulfield, M. P., Duong, L. T., O'Brien, R., Majzoub, J. A., and Rosenblatt, M. (1988) Mol. Endocrinol. 2, 452-458). This sulfur-free signal peptide analog can be labeled with 125I on the C-terminal D-tyrosine and is cleaved by purified hen oviduct signal peptidase between Gly and Lys, the correct site of cleavage of preproparathyroid hormone in vivo. Amino acid sequence analysis of the cleavage product released 125I at the seventh cycle of Edman degradation, confirming that enzymatic cleavage occurs at the physiological site. Synthetic peptide analogs of the substrate with Lys, Pro, or Asp substituted for Nle-18 were poor substrates for the enzyme and were also poor competitive inhibitors of catalysis, suggesting that modifications at position -18, 12 amino acids from the site of cleavage, directly influence binding by the enzyme. Analysis of the reactivity of signal peptidase with these synthetic peptides provides insight into the cleavage specificity requirements of this eukaryotic signal peptidase.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

TWO HERBIVORES AND CONSTRAINTS ON SELECTION FOR RESISTANCE IN BRASSICA RAPA

Although most plants experience herbivory by several insect species, there has been little empirical work directed toward understanding plant responses to these simultaneous selection pressures. In an experiment in which herbivory by flea beetles (Phyllotreta cruciferae) and diamondback moths (Plutella xylostella) was manipulated in a factorial design, I found that selection for resistance to these herbivores is not independent in Brassica rapa. Specifically, the effect of flea beetle damage on B. rapa fitness depends on the amount of diamondback moth damage a plant experiences: damage by these herbivores has a nonadditive effect on plant fitness. When diamondbacks are abundant, plants that sustain high levels of damage by flea beetles are favored by natural selection, but when diamondbacks are rare, a low level of damage by flea beetles is favored. However, resistance to the later-feeding diamondback moth is not affected by the presence or absence of damage by early-feeding flea beetles. Thus, there are no plant-mediated ecological interactions between these herbivores that affect the outcome of selection for resistance. Because these herbivores do not independently affect plant fitness, neither is likely to develop a pairwise coevolutionary relationship with its host. Instead, coevolution is diffuse.     

Chromosomal variation in the southern short-tailed shrew (Blarina carolinensis)

Chromosomal polymorphism was assessed in the southern short-tailed shrew (Blarina carolinensis) using standard metaphase chromosome and G-banding techniques. Twenty-one animals (11 males, 10 females) from the Meeman Biological Station in Shelby Co., Tennessee, were examined for diploid number. Results showed diploid numbers of 35, 36, 37, 38, 39, 40 and 41 and fundamental numbers of 41, 42, 43, 44 and 45. No diploid numbers or fundamental numbers were unique to a specific collecting locality. The first G-banded karyotypes are reported for the species. These results indicate that Robertsonian polymorphisms, inversions, and possibly other events are responsible for chromosomal variation in B. carolinensis.     

Human aging brain disorders: Role of antioxidant enzymes

In order to investigate the role of two free radical detoxificant enzymes in patients with aging brain disorders, superoxide dismutase (SOD) and catalase (CAT) activities have been measured in blood from male and female human patients of different ages with several types of aging brain disorders. When compared with activities in the normal population, we have detected: 1) SOD and CAT activities are decreased in patients with Parkinson disease. 2) SOD activity seems to be normal and CAT activity is decreased in patients with dementia. 3) In the patients with stroke, SOD activity is normal, while CAT activity is decreased. SOD activity was measured in red blood cells using the Minami and Yoshikawa method. CAT activity was measured in hemolysates by the method of Aebi. We can conclude that SOD and CAT activities in patients with Parkinson disease are decreased.     

Host-seeking behaviour of tracheal mites (Acari: Tarsonemidae) on honey bees (Hymenoptera: Apidae)

The behaviour of the endoparasitic tracheal mite, Acarapis woodi (Rennie) on honey bees (Apis mellifera L.) is a challenge to observe because of its small size. Through a microscope, we videotaped this mite's movement on young bees, dead bees and bees exposed to vegetable oil. Previous studies have shown that solid vegetable oil decreases mite infestations in a bee colony. We hypothesized that the oil alters mite behaviour to the detriment of the parasite, thus helping to safeguard the host. Habitat-seeking behaviour, identified as necessary for mites to locate a new host environment, was disrupted on both dead and oil-treated bees. Questing behaviour, which is associated with transfer between hosts, increased significantly on the dead and oily bees. The behaviours of mites were significantly different between all three treatments (x ²=494.96, p<0.001 on dead bees and x ²=851.11, p<0.001 on oily bees). Both questing and seeking behaviours were significantly different on each of the thoracic treatments (F _2,66=7.88, p<0.001 and F _2,66=21.28, p<0.001) and mite questing behaviour was not altered between males and females on live or oily bees (F _1,22=0.25, p<0.62), but habitat seeking was (F _1,22=7.42, p<0.012). The male questing and habitat-seeking behaviours were observed. We conclude that oil-treated bees gained protection from habitat-seeking mites because the normal behaviour of the mites seeking an oviposition site is interrupted.     

A rapid attenuation of muscarinic agonist stimulated phosphoinositide hydrolysis precedes receptor sequestration in human SH-SY-5Y neuroblastoma cells

Agonist occupancy of muscarinic cholinergic receptors in human SH-SY-5Y neuroblastoma cells elicited two kinetically distinct phases of phosphoinositide hydrolysis when monitored by either an increased mass of inositol 1,4,5-trisphosphate, or the accumulation of a total inositol phosphate fraction. Within 5s of the addition of the muscarinic agonist, oxotremorine-M, the phosphoinositide pool was hydrolyzed at a maximal rate of 9.5%/min. This initial phase of phosphoinositide hydrolysis was short-lived (t_1/2=14s) and after 60s of agonist exposure, the rate of inositol lipid breakdown had declined to a steady state level of 3.4%/min which was then maintained for at least 5–10 min. This rapid, but partial, attenuation of muscarinic receptor stimulated phosphoinositide hydrolysis occurred prior to the agonist-induced internalization of muscarinic receptors.Abbreviations I(1,4,5)P₃ inositol 1,4,5-trisphosphate - IP total inositol phosphate fraction - IPL total inositol lipid fraction - mAChR muscarinic acetylcholine receptor - NMS N-methylscopolamine - Oxo-M oxotremorine-M - PI phosphatidylinositol - PIP phosphatidylinositol 4-phosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PPI phosphoinositide - QNB quinuclidinyl benzilate Special issue dedicated to Dr. Bernard W. Agranoff     

TCA-100 tumour chemosensitivity assay: Differences in sensitivity between cultured tumour cell lines and clinical studies

The BATLE LE TCA-100 tumour chemosensitivity assay has been used to evaluate chemotherapeutic drug sensitivity of cultured tumour cell lines. Studies were performed using test drug concentrations calibrated to discriminate sensitivity and resistance of clinical specimens. Strong sensitivity which appeared to be inconsistent with clinical experience was detected for some drugs and cell lines. Findings of strong sensitivity were consistent with basic differences between sensitivity testing cultured cell lines and clinical specimens. Results with cell lines frequently may not apply directly to clinical applications. Characterization of differences between cell lines and clinical specimens may assist in application of cell line findings to clinical trials.     

Characterization of Large Unilamellar Vesicles as Models for Studies of Lipid Peroxidation Initiated by Azocompounds

The aim of this work was to characterize large unilamellar vesicles (LUVETs) prepared by a hand-driven extrusion device in order to use them for studies of lipid peroxidation and antioxidant activity. Vesicle structure and size were examined by electron microscopy. Lipid and antioxidant content was determined before and after the extrusion procedure. Then LUVETs were subjected to autoxidation initiated by both the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) and the water-soluble 2,2'-azobis(2-amidinopropane hydrochloride) azocompounds. The results demonstrated that: i) LUVETs prepared with lipid concentrations ranging between 25 and 150 mM were essentially unilamellar and reasonably homogeneous, with an average diameter of 90 nm; ii) the phospholipid, cholesterol and antioxidant amounts retained by filters were about 10-15%; iii) LUVETs were suitable for autoxidation studies initiated by the water-soluble azocompound both in the absence and presence of antioxidants. The lipid-soluble azocompound could be used only at low concentrations and its vesicle content had to be determined since part of the initiator was not incorporated into the lipid bilayer. These data suggest that LUVETs seem to be recommended for studies of lipid peroxidation and antioxidant activity.