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.     

Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-alpha: role of the p38 and p42/44 mitogen-activated protein kinases

IL-10 is an anti-inflammatory cytokine with potent immunomodulatory effects, including inhibition of cytokine production. However, regulation of monocyte IL-10 production is poorly understood. In this report we have investigated the mechanisms of LPS-induced IL-10 production by human peripheral blood monocytes and demonstrate that IL-10 synthesis is uniquely dependent on the endogenous proinflammatory cytokines IL-1 and/or TNF-alpha. LPS signal transduction in monocytes has been shown to involve activation of the p38 and p42 mitogen-activated protein kinase (MAPK) cascades. The results in this paper indicate that inhibition of p38 MAPK potently inhibited the production of IL-10, IL-1beta, and TNF-alpha, whereas blockade of the p42/44 MAPK pathway, while partially inhibiting TNF-alpha and IL-1beta production, had no effect on monocyte secretion of IL-10. Furthermore, neither the inhibition of monocyte TNF-alpha induced by IL-10 nor the stimulation of soluble TNF receptor production was affected by inhibition of the p42/44 MAPK pathway, suggesting that this signaling event is not involved in either monocyte production of or anti-inflammatory responses to IL-10. These data raise the interesting possibility that proinflammatory TNF-alpha-mediated effects may be selectively blocked without modulating the induction or the response to IL-10, whereas the signaling events associated with the anti-inflammatory events induced by IL-10 remain to be elucidated.     

Long-term intravesical oxybutynin chloride therapy in children with myelodysplasia

PURPOSE: We evaluated the clinical use of long-term intravesical oxybutynin chloride in the treatment of neurogenic bladder dysfunction in children with myelodysplasia who could not tolerate oral anticholinergics. MATERIALS AND METHODS: We retrospectively reviewed the records of all patients recommended for intravesical oxybutynin chloride therapy. A total of 12 girls and 18 boys 1 to 17 years old was recruited for study. Oxybutynin chloride (5 mg.) was instilled 2 times daily and pretreatment cystograms were compared to followup urodynamic studies. Duration of therapy was 2 to 26 months (mean 13, median 12). RESULTS: Mean total capacity plus or minus standard deviation increased from 209 +/- 103 to 282 +/- 148 ml. (p < 0.01), mean safe capacity increased from 157 +/- 105 to 234 +/- 147 ml. (p < 0.01) and mean age adjusted safe capacity increased from 76 +/- 36 to 115 +/- 62%. Of the 29 patients who were incontinent 3 (10%) achieved continence and 19 (65%) reported a decreased use of sanitary pads. None of the patients had systemic side effects related to intravesical treatment. CONCLUSIONS: We believe that intravesical oxybutynin chloride is a viable treatment option for patients with myelodysplasia in whom oral therapy fails.     

Subgingival microbiota in healthy, well-maintained elder and periodontitis subjects

This investigation compared the site prevalence of 40 subgingival species in 30 periodontally healthy (mean age 36+/-9 years), 35 elders with a well-maintained periodontium (mean age 77+/-5) and 138 adult periodontitis subjects (mean age 46+/-11). Subgingival plaque samples were taken from the mesial aspect of each tooth (up to 28 samples) in the 203 subjects at baseline. The presence and levels of 40 subgingival taxa were determined in 5003 plaque samples using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. The % of sites colonized by each species (prevalence) was computed for each subject. Differences in prevalence and levels among groups were sought using the Kruskal-Wallis test. Commonly detected species, such as Actinomyces naeslundii genospecies 2, Streptococcus sanguis and Streptococcus oralis did not differ significantly among subject groups. After adjusting for multiple comparisons, 4 species were significantly elevated and at greater prevalence in the periodontitis group. Mean % of sites (+/-SEM) colonized by Bacteroides forsythus was 10+/-3, 12+/-2 and 40+/-2 (p<0.001) for healthy, elder and periodontitis groups respectively. The odds ratio was 14.4:1 that a subject had periodontitis when B. forsythus was detected at > or = 5% of sampled sites. Mean prevalence for Porphyromonas gingivalis in healthy, elder and periodontitis subjects was 4+/-2, 5+/-2 and 23+/-2 respectively (p<0.001); for Treponema denticola 12+/-4, 10+/-3 and 30+/-2 (p<0.001) and for Selenomonas noxia 6+/-2, 7+/-2 and 19+/-2 (p<0.01). Similar differences among subject groups were observed when only sites with PD 0-4 mm were analyzed. The data suggest an etiologic role for B. forsythus, P. gingivalis, T. denticola and S. noxia in adult periodontitis.     

5-[125I]iodo-2'-deoxyuridine in the radiotherapy of brain tumors in rats

Glial neoplasms of the human central nervous system have defied treatment, in part because of the limited selectivity of available cytotoxic agents. The thymidine analog 5-iodo-2'-deoxyuridine radiolabeled with the Auger electron emitter 125I (125IUdR) is highly toxic to dividing cells when it is deoxyribonucleic acid incorporated, but it is relatively innocuous when located outside the nucleus. Previous studies have shown that 125IUdR has significant antineoplastic potential against mammalian cells in vitro and direct administration of 125IUdR is effective therapy for ovarian ascites tumors in mice and neoplastic meningitis in rats. Studies using external gamma imaging and autoradiography have also shown that direct intratumoral administration of 123IUdR/125IUdR into intracerebral 9L gliosarcomas in rats results in selective uptake of the radionuclide into tumor cells. Based on these encouraging results, we have evaluated the therapeutic potential of 125IUdR in rats bearing intracerebral 9L gliosarcomas. METHODS: Iodine-125-IUdR was infused intracerebrally over a 2-day period into rats bearing 1-day-old 9L tumors and over a 6-day period into animals with 9-day-old 9L tumors; equimolar concentrations of 127IUdR were infused into control animals. Tumor growth was monitored by contrast-enhanced 1H MRI and animal survival was followed over time. RESULTS: Intracerebral tumors (3-7 mm) were readily detected by MRI. Tumor-bearing rats treated with 127IUdR succumbed within 17-24 days, whereas tumor-bearing animals treated with 125IUdR survived significantly longer, and 10%-20% of the animals were cured of tumors. CONCLUSION: These data substantiate the antineoplastic potential of 5-[125I]iodo-2'-deoxyuridine and indicate that it may be a useful agent for the therapy of solid tumors that are accessible to direct radiopharmaceutical administration.     

Specificity and development of innervation pattern of Xenopus pectoralis muscle

Multielectrode recordings were used to identify and measure the axonal inputs to each end plate on contiguous surface fibers covering about 25% of the Xenopus pectoralis muscle in mature and developing animals. The mature innervation pattern was remarkably precise. Individual axons tended to innervate fibers of similar input resistance (R(in)) in compact motor units restricted to only a portion of the region studied. Motor units comprising fibers of similar R(in) overlapped mainly near their borders. Most fibers had two end plates. In more than 80% of these fibers, both end plates received input from the same axon. In 57%, this was the only input to both end plates. This implies a powerful mechanism for excluding or eliminating inputs from other axons. About 16% of the mature junctions showed focal polyneuronal innervation, with the weaker end plate potential component often less than 1 mV in noncurarized preparation. However, we have no evidence that the weaker inputs were being eliminated. During development, motor units became more compact, which was associated with synapse elimination; but from the earliest times studied, soon after metamorphosis when many fibers were adding second end plates, a majority of those that had two end plates were innervated at both sites by the same axon.     

District sputum smear microscopy services in Malawi

SETTING: Government hospitals and health centres in 23 districts in Malawi. OBJECTIVE: To determine 1) the number and smear-positivity rate of sputum samples submitted at health centres and hospitals, and 2) the time for sputum samples to get from health centres to smear examination. DESIGN: Prospective data collection on sputum specimens coming from health centres to hospital laboratories, and over the equivalent time period, retrospective data collection from laboratory sputum registers. RESULTS: Information was collected over a period of 5.6 months during 1997. Of 21 527 patients submitting sputum samples, 16995 (79%) were from within the hospital and 4532 (21%) were from health centres. Of 15 833 new TB suspects, 12 804 (81%) submitted sputum within the hospital and 3029 (19%) were from health centres. The overall smear-positivity rate was 11.9%: the proportion of new suspects who were smear-positive was significantly higher in health centres (14.1%) compared with hospital-based patients (11.4%, P < 0.05); 27% of all sputum specimens from health centres took 8 days or longer to get to smear examination. Sputum smears were positive from 1-30 days between submission and laboratory examination. CONCLUSION: Fewer sputum samples are submitted at health centres compared with hospitals, and there may be long delays between sputum submission and smear examination. The precise reasons are unclear, but health centre staff need training about the importance of timely case finding procedures.     

Biomedical and economic consequences of stratosphere ozone depletion

The detailed analysis of conformational space of alpha-conotoxin GI in aqueous solution has been performed on the basis of two-dimensional NMR spectroscopy data using multiconformational approach. As the result, two topologically distinct interconvertible sets of GI conformations (populations of 78% and 22%) have been found. A common feature of the two sets is the Asn4-Cys7 beta-turn. The Gly8 to Tyrll region has a structure of right-handed helical turn in the major set and two sequential bends in the minor one. N-terminus and C-terminus also have different orientations, anti-parallel in the major conformational set and parallel in the minor one. An average pairwise rmsd for backbone heavy atoms is 0.56 A in the major set, 0.23 A in the minor, and 1.85 A between the structures of the two sets. The X-ray structure of GI [Guddat, L. W., Martin, J. A., Shan, L., Edmundson, A. B. & Gray, W. R. (1996) Biochemistry 35, 11329 - 11335] has the same folding pattern as the major NMR set, the average backbone rmsd between the two structures being 0.77 A.     

Apoptosis after traumatic human spinal cord injury

OBJECT: Apoptosis is a form of programmed cell death seen in a variety of developmental and disease states, including traumatic injuries. The main objective of this study was to determine whether apoptosis is observed after human spinal cord injury (SCI). The spatial and temporal expression of apoptotic cells as well as the nature of the cells involved in programmed cell death were also investigated. METHODS: The authors examined the spinal cords of 15 patients who died between 3 hours and 2 months after a traumatic SCI. Apoptotic cells were found at the edges of the lesion epicenter and in the adjacent white matter, particularly in the ascending tracts, by using histological (cresyl violet, hematoxylin and eosin) and nuclear staining (Hoechst 33342). The presence of apoptotic cells was supported by staining with the terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling technique and confirmed by immunostaining for the processed form of caspase-3 (CPP-32), a member of the interleukin-1beta-converting enzyme/Caenorhabditis elegans D 3 (ICE/CED-3) family of proteases that plays an essential role in programmed cell death. Apoptosis in this series of human SCIs was a prominent pathological finding in 14 of the 15 spinal cords examined when compared with five uninjured control spinal cords. To determine the type of cells undergoing apoptosis, the authors immunostained specimens with a variety of antibodies, including glial fibrillary acidic protein, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), and CD45/68. Oligodendrocytes stained with CNPase and a number of apoptotic nuclei colocalized with positive staining for this antibody. CONCLUSIONS: These results support the hypothesis that apoptosis occurs in human SCIs and is accompanied by the activation of caspase-3 of the cysteine protease family. This mechanism of cell death contributes to the secondary injury processes seen after human SCI and may have important clinical implications for the further development of protease inhibitors to prevent programmed cell death.     

Requirement of Src kinase Lyn for induction of DNA synthesis by granulocyte colony-stimulating factor

Treatment of cells with granulocyte colony-stimulating factor (G-CSF) leads to tyrosine phosphorylation of cellular proteins. G-CSF stimulates both the activation of protein tyrosine kinases Lyn, Jak1, and Jak2 and the association of these enzymes with the G-CSF receptor. Wild-type, lyn-deficient, and syk-deficient chicken B lymphocyte cell lines were transfected with the human G-CSF receptor, and stable transfectants were studied. G-CSF-dependent tyrosyl phosphorylation of Jak1 and Jak2 occurred in all three cell lines. Wild-type and syk-deficient transfectants responded to G-CSF in a dose-responsive fashion with increased thymidine incorporation, but none of the clones of lyn-deficient transfectants did. Ectopic expression of Lyn, but not that of c-Src, in the lyn-deficient cells restored their mitogenic responsiveness to G-CSF. Ectopic expression in wild-type cells of the kinase-inactive form of Lyn, but not of the kinase-inactive form of Jak2, inhibited thymidine incorporation in response to G-CSF. These studies show that the absence of Lyn results in the loss of mitogenic signaling in the G-CSF signaling pathway and that activation of Jak1 or Jak2 is not sufficient to cause mitogenesis.