Lysosomal targeting of P-selectin is mediated by a novel sequence within its cytoplasmic tail

Signals controlling the intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. P-selectin is a type I membrane protein receptor for leukocytes, acting during the inflammation response. Heterologous expression experiments have demonstrated that its 35-residue cytoplasmic tail contains signals for targeting to synaptic-like microvesicles, dense-cored granules, and lysosomes. We have examined the lysosomal targeting information present within the cytoplasmic tail by site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras followed by transient transfection in H.Ep.2 cells. Assaying lysosomal targeting by subcellular fractionation as well as intracellular proteolysis, we have discovered a novel lysosomal targeting signal, KCPL, located within the C1 domain of the cytoplasmic tail. Alanine substitution of this tetrapeptide reduced lysosomal targeting to the level of a tailless horseradish peroxidase-P-selectin chimera, which was previously found to be deficient in both internalization and delivery to lysosomes. A proline residue within this lysosomal targeting signal makes a major contribution to the efficiency of lysosomal targeting. A diaminobenzidine density shift procedure established that chimeras with an inactivated KCPL sequence are present within transferrin-positive compartments. Such a mutant also displays an increased level of expression at the plasma membrane. Our results indicate that the sequence KCPL within the cytoplasmic tail of P-selectin is a structural element that mediates sorting from endosomes to lysosomes.     

Depression in Parkinson's disease is not accompanied by more corticotropin-releasing hormone expressing neurons in the hypothalamic paraventricular nucleus

BACKGROUND: Depression is frequently encountered in Parkinson's disease (PD). In addition, more than half of the PD patients have a disturbed dexamethasone suppression test, which is associated with increased activity of corticotropin-releasing hormone (CRH) neurons. We recently found an increase in CRH neuron number, CRH-messenger RNA, and vasopressin colocalization in CRH neurons in the paraventricular nucleus (PVN) of depressed patients, which may be involved in the pathogenesis of depression. METHODS: The number of neurons expressing CRH was determined in the PVN of 6 depressed PD patients with a high score (> or = 13) on the Hamilton Depression Rating Scale, 6 nondepressed PD patients, and 6 controls. RESULTS: The three groups did not differ in the number of neurons expressing CRH. CONCLUSIONS: We hypothesize that activation of CRH neurons in the PVN, as we recently observed in idiopathic depression, does not play an essential role in depression in PD.     

Missing quality of life data in cancer clinical trials: serious problems and challenges

Measurement of quality of life (QOL) in cancer clinical trials has increased in recent years as more groups realize the importance of such endpoints. A key problem has been missing data. Some QOL data may unavoidably be missing, as for example when patients are too ill to complete forms. Other important sources are potentially avoidable and can broadly be divided into three categories: (i) methodological factors; (ii) logistic and administrative factors; (iii) patient-related factors. Logistic and administrative factors, for example, staff oversights, have proven to be most important. Since most QOL measurements require patient self-report, it is usually not possible to rectify the failure to collect baseline data or any follow-up assessments. There is strong evidence that such data are not 'missing at random', and cannot be ignored without introducing bias. Although several approaches to the analysis of partly missing data have been described, none is entirely satisfactory. Prevention of avoidable missing data is better than attempted cure. In July 1996, an international conference on missing QOL data in cancer clinical trials reported the experience of most major groups involved. This paper will serve as an introduction to the problem and provide an estimation of its magnitude, and approaches to its prevention and solution.     

Kinetic and spectroscopic properties of the YZ* radical in Ca2+- and Cl--depleted photosystem II preparations

Depletion of Ca2+ and/or Cl- ions from PSII membranes blocks the electron-transfer reactions that precede O2 evolution on the oxidizing side of the enzyme. Illumination of these inhibited preparations at 273 K generates a paramagnetic species that is detectable by low-temperature (T < 20 K) EPR as a signal in the g = 2 region, 90-230 G wide, depending on the treatment that PSII has undergone. This signal has recently been assigned to YZ* in magnetic interaction with the manganese cluster in its S2 state [Gilchrist et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9545-9549]. This view, however, is not universal, owing, in part, to the fact that its spectroscopic properties depend on the preparation and the experimental conditions used for its study and, in part, to uncertainties as to the room temperature behavior of YZ* in inhibited preparations. Here, we report time-resolved and conventional EPR data showing that, at room temperature and at 273 K, YZ* can be accumulated in its 20 G form in high yields in both Ca2+-depleted and acetate-inhibited preparations, and that the kinetics of its decay match the decay kinetics of the low-temperature signal generated in corresponding samples. The properties of the YZ* signal, however, are shown to depend on the polypeptide content, the temperature, and the electron donors and acceptors present in the sample under examination. Our results support assignment of the EPR signal in inhibited preparations to S2 YZ* and demonstrate a protective role of the 17 and 23 kDa extrinsic polypeptides for the manganese cluster against externally added reductants.     

2-(4-Aminophenyl)benzothiazoles: novel agents with selective profiles of in vitro anti-tumour activity

OBJECTIVE: To evaluate the results of cervical cytology screening in the National Breast and Cervical Cancer Early Detection Program and to compare the findings with results from other screening programs. METHODS: We analyzed data on 312,858 women aged 18 years and older who received one or more Papanicolaou smears, and follow-up if indicated, from October 1991 through June 1995 at screening sites across the United States providing comprehensive National Breast and Cervical Cancer Early Detection Program services. RESULTS: Of the women screened, more than half were 40 years or older; slightly less than half (44%) were of racial and ethnic minorities. During the first screening cycle, 3.8% of Papanicolaou tests were reported as abnormal (squamous intraepithelial lesion [SIL] or squamous cell cancer); proportions of abnormals decreased with increasing age. The age-adjusted rate of biopsy-confirmed cervical intraepithelial neoplasia (CIN) II or worse among women screened was 7.4 per 1000 Papanicolaou tests; rates of CIN were highest among young women, but cancer rates peaked among women in their 50s and 60s. The percentages of first screening cycle-Papanicolaou tests interpreted as high-grade SIL and squamous cell carcinoma associated with biopsy-confirmed CIN II or worse (the positive predictive value) were 56.0% for CIN II/III and 3.7% for invasive cancer. Of the 150 invasive cancers diagnosed, 54.0% were classified as local disease. CONCLUSION: Observed results emphasize the duality of cervical neoplasia-CIN in younger women and invasive cancer in older women. This finding points to the importance of reaching both younger and older women for cervical cancer screening.     

Transcrystalline morphology of nylon 6 on the surface of aramid fibers

In this paper, the isothermal crystallization of nylon 6 in the presence of Kevlar 129 fiber was investigated by polarized optical microscopy (POM). The formation of a transcrystalline domain was found to be mainly controlled by crystallization conditions, such as the temperature of the isothermal crystallization, residual time at melting temperature and the cooling rate of the melt. The nucleation rate of nylon 6 on the fibers was mainly affected by the crystallization temperature. The interfacial transcrystallinity of nylon 6 occurred on the surface of Kevlar 129 fiber in the temperature range 130–190 °C. The reason for the formation of interfacial transcrystalline morphology is discussed from the molecular level, based on the understanding of the packing mode of nylon 6 chains around fibers and the interaction between matrix and fibers. It was found that the lattice matching and hydrogen‐bonding between nylon 6 and poly(p‐phenylene terephthalamide) (PPTA) crystals play an important role in the epitaxial crystallization. Copyright © 2004 Society of Chemical Industry     

Oxidative stress following traumatic brain injury in rats

BACKGROUND: Free radicals may be involved in the pathophysiology of traumatic brain injury (TBI) through oxidative damage of neurovascular structures. Endogenous antioxidants, such as ascorbate and alpha-tocopherol, may play a critical role in combating these oxidative reactions and their oxidized products can serve as an important index of oxidative stress. METHODS: We used electron spin resonance (ESR) spectroscopy and in vivo spin trapping (reaction of an organic compound with free radical species) to detect the possible generation of free radicals after TBI. Injury was inflicted by a weight drop technique over the head (5.7 kg-cm). Rats were intravenously infused with either 1 mL, 0.1 M of the spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN), or an equivalent volume of saline immediately before TBI or sham-injury. Animals were divided into four groups: (1) Group I: PBN-infused sham-injured, (2) Group II: PBN-infused injured, (3) Group III: saline-infused sham-injured, and (4) Group IV: saline-infused injured. Additional groups of saline-infused uninjured, saline-infused, and PBN-infused injured animals were used for histopathology. Sixty minutes after TBI or sham-injury, rats were again anesthetized and decapitated. The brains were removed within 1 minute, homogenized, and extracted for lipids. The extracts were analyzed by ESR spectroscopy. Brain ascorbic acid (AA) concentration was determined spectrophotometrically, using the ascorbate oxidase assay. RESULTS: No PBN spin adduct signals (indicating trapped free radical species) were visible 60 minutes after TBI. All groups of rats showed an ascorbyl free radical signal. The ascorbyl signal intensity (AI) was, however, significantly higher in the injured rats, while the brain (AA) was significantly reduced. In addition, the ratio of AI/AA, which eliminates the effect of variable ascorbate concentrations in the brain, was also significantly higher in the injured animals. CONCLUSIONS: We conclude that 60 minutes following TBI there was a significantly increased level of oxidative stress in the brain. This may reflect formation of free radical species with subsequent interaction with ascorbate (antioxidant) during the 60 minute period. The lack of PBN spin adduct signals 1 hour after TBI may indicate that free radical generation is time dependent and might be detectable earlier or later than the 60 minute period.     

Scanning transmission electron microscopy mass analysis of fibrillin-containing microfibrils from foetal elastic tissues

We have applied scanning transmission electron microscopy to intact native fibrillin-containing microfibrils isolated from foetal bovine elastic tissues in order to derive new insights into microfibril organisation. This technique provides quantitative data on the mass per unit length and axial mass distribution of unstained, unshadowed macromolecules. Scanning transmission electron microscopy of microfibrils from aorta, skin and nuchal ligament revealed that the beads corresponded to peaks of mass and the interbead regions to troughs of mass. These major features of axial mass distribution were characteristic of all microfibrils examined. Tissue-specific and age-dependent variations in mass were identified in microfibrils that were structurally comparable by rotary shadowing electron microscopy. Increased microfibril mass correlated with increasing gestational age. The additional mass was associated predominantly at, or close to, the bead. Some microfibril populations exhibited pronounced assymetry in their axial mass distribution. These data indicate that intact native microfibrillar assemblies from developing elastic tissues are heterogeneous in composition. Loss of mass following chondroitinase ABC or AC lyase treatment confirmed the presence of chondroitin sulphate in nuchal ligament microfibrillar assemblies.