Relationships between sediment microbial communities and pollutants in two California salt marshes.

Salt marshes are important ecosystems whose plant and microbial communities can alter terrestrially derived pollutants prior to coastal water discharge. However, knowledge regarding relationships between anthropogenic pollutant levels and salt marsh microbial communities is limited, and salt marshes on the West Coast of the United States are rarely examined. In this study, we investigated the relationships between microbial community composition and 24 pollutants (20 metals and 4 organics) in two California salt marshes. Multivariate ordination techniques were used to assess how bacterial community composition, as determined by terminal restriction fragment length polymorphism and phospholipid fatty acid analyses, was related to pollution. Sea urchin embryo toxicity measurements and plant tissue metabolite profiles were considered two other biometrics of pollution. Spatial effects were strongly manifested across marshes and across channel elevations within marshes. Utilizing partial canonical correspondence analysis, an ordination technique new to microbial ecology, we found that several metals were strongly associated with microbial community composition after accounting for spatial effects. The major patterns in plant metabolite profiles were consistent with patterns across microbial community profiles, but sea urchin embryo assays, which are commonly used to evaluate ecological toxicity, had no identifiable relationships with pollution. Whereas salt marshes are generally dynamic and complex habitats, microbial communities in these marshes appear to be relatively sensitive indicators of toxic pollutants.

Identification of a hyaluronic acid (HA) binding domain in the PH-20 protein that may function in cell signaling.

The macaque sperm surface protein PH-20 is a hyaluronidase, but it also interacts with hyaluronic acid (HA) to increase internal calcium ( [Ca(2+)](i) ) in the sperm cell. A region of the PH-20 molecule, termed Peptide 2 (aa 205-235), has amino acid charge homology with other HA binding proteins. The Peptide 2 sequence was synthesized and two recombinant PH-20 proteins were developed, one containing the Peptide 2 region (G3, aa 143-510) and one without it (E12, aa 291-510). On Western blots, affinity-purified anti-Peptide 2 IgG recognized the 64 kDa band corresponding to PH-20 in acrosome intact sperm and, under reducing conditions, recognized the whole 67 kDa PH-20 and the endoproteolyzed N-terminal fragment of PH-20. HA conjugated to a photoaffinity substrate specifically bound to sperm surface PH-20. Indirect immunofluorescence demonstrated that Fab fragments of anti-Peptide 2 IgG bound to the head of live sperm. Biotinylated HA was bound by Peptide 2 and by sperm extracts in a microplate binding assay, and this binding was inhibited by Fab fragments of anti-Peptide 2 IgG. Biotinylated HA bound to the G3 protein and this binding was inhibited by anti-Peptide 2 Fab, but HA did not bind to the E12 protein. Fab fragments of anti-Peptide 2 IgG inhibited the increase in [Ca(2+)](i) induced in macaque sperm by HA. Our results suggest that the Peptide 2 region of PH-20 is involved in binding HA, which results in the cell signaling events related to the elevation of [Ca(2+)](i) during sperm penetration of the cumulus.

Redistribution of transcription factor AP-2alpha in differentiating cultured human epidermal cells.

Expression of the transcription factor AP-2alpha was examined in cultured human epidermal cells. Levels of AP-2alpha mRNA increased substantially after the cultures reached confluence, similar to the expression pattern of the differentiation markers involucrin and keratinocyte transglutaminase. The level of AP-2alpha protein in nuclear extracts declined markedly after confluence, however, along with its ability to form complexes with oligonucleotides containing the AP-2 response element. In contrast, the levels of AP-2alpha protein in cytoplasmic extracts increased dramatically after confluence, but these extracts had low DNA binding activity. Supershift experiments with specific antisera detected only AP-2alpha and not the beta or gamma isoforms. Examination of its localization by confocal microscopy revealed that AP-2alpha was primarily in the nucleus of basal cells and largely cytoplasmic in the most superficial cells. Localization was a dynamic phenomenon in that changing the medium resulted in accumulation of this transcription factor in the nucleus after several hours. Overall, the data indicate that AP-2alpha transcriptional activity is regulated in a differentiation-dependent manner in cultured keratinocytes and that this occurs by relocalization of the protein. Nuclear localization of the AP-2alpha protein in basal cells permits its accessibility to response elements in gene promoters, whereas sequestration in the cytoplasm as the differentiation program progresses curtails its transcriptional activity. This regulatory scheme may provide keratinocytes with the ability to restore AP-2 transcriptional activity rapidly by redistribution to the nucleus after receiving an appropriate growth signal, such as a medium change.

Inhibition of beta 2 integrin receptor and Syk kinase signaling in monocytes by the Src family kinase Fgr.

While beta 2 integrin ligand-receptor recognition interactions are well characterized, less is known about how these events trigger signal transduction cascades to regulate the transition from tethering to firm adhesion, spreading, and transendothelial migration. We have identified critical positive and negative regulatory components of this cascade in monocytes. Whereas the Syk tyrosine kinase is essential for beta 2 integrin signaling and cell spreading, the Src family kinase Fgr is a negative regulator of this pathway. Fgr selectively inhibits beta 2 but not beta 1 integrin signaling and Syk kinase function via a direct association between the Fgr SH2 domain and Syk tyrosine Y342. The inhibitory effects of Fgr are independent of its kinase activity, are dose dependent, and can be overcome by chemokines and inflammatory mediators.

An exploratory study of pressure ulcers after spinal cord injury: relationship to protective behaviors and risk factors.

OBJECTIVES: To identify protective behaviors and risk factors associated with the development of pressure ulcers (PUs) after spinal cord injury (SCI). DESIGN: A cross-sectional study to evaluate the relationship between protective behaviors and risk factors and 3 PU outcomes: a current PU, PUs within the past year, and ever hospitalized for a PU. Logistic regression was then used to identify the variables most strongly associated with PU outcomes. SETTING: Data were collected by case managers employed by the Arkansas Spinal Cord Commission, an agency that provides services to persons with SCI. PARTICIPANTS: A total of 650 of 991 eligible individuals with SCI from a statewide population-based SCI registry participated. All ambulatory participants were eliminated, leaving 560 patients. Average age of the respondents was 27.2 years at injury (median age, 25yr) and 43.6 years at the time of the survey (median age, 42yr). MAIN OUTCOME MEASURES: A 200-item interview was developed to measure a broad range of outcomes associated with SCI (including secondary conditions such as PUs), as well as risk and protective behaviors related to these outcomes. RESULTS: Several characteristics and behaviors were related to PU outcomes. Being underweight (odds ratio [OR] = 2.18), having used medications to treat pain (OR = 1.33) or spasticity (OR = 1.31), having smoked at least 100 cigarettes over a lifetime (OR = 1.31), and being a current smoker (OR = 1.21) were associated with having a PU in the past year. Having completed a college degree (OR = 0.23), being married (OR = 0.49), and being currently employed (OR = 0.54) were associated with a lower risk of having a PU in the past year. Being underweight (OR = 1.94), having a history of incarceration (OR = 1.78), having attempted suicide (OR = 1.71), and reporting alcohol or drug treatment (OR = 1.65) were associated with having been hospitalized for a PU since injury. This study was unable to evaluate the efficacy of traditional health maintenance or protective behaviors for PUs, such as weight shifts or skin checks. CONCLUSIONS: PUs are least likely to occur among individuals who maintain normal weight, return to a work and family role, and who do not have a history of tobacco use, suicidal behaviors, or self-reported incarcerations, or alcohol or drug abuse. Additional research is needed to identify better the risk factors for the occurrence of PUs.

The effects of diffusible creosote-derived compounds on development in Pacific herring (Clupea pallasi).

The effects of diffusible creosote-derived compounds from weathered creosote-treated pilings on embryonic development in the Pacific herring were investigated. Parameters used to evaluate toxicity included embryonic development, cardiac function, embryo/larval activity (movement of developing embryos), hatching success, and larval morphology at hatch. For acute exposures, embryos were incubated in seawater containing either creosote-treated wood (creosote) or untreated wood (wood control), or seawater alone (control). All embryos adhering directly to creosote-treated wood and 40-50% of embryos not adhering to the creosote-treated wood failed to develop beyond the first few days of incubation. For surviving embryos, a 93% reduction in heart rate, and moderate to marked arrhythmia was observed. Surviving embryos also exhibited both an increase in frequency and an alteration in pattern of embryo/larval movement, with most embryos exhibiting tremors as compared with the vigorous movements of the control embryos. Cardiac function and embryo/larval movements of embryos exposed to untreated wood were not significantly different from controls. The hatching rate of embryos exposed to creosote was 90% lower than control embryos and 72.4% lower than embryos exposed to untreated wood, and the LC(50) for hatching success was 0.05 mg/l. Partial hatching (incomplete hatch) was observed in 15-20% of embryos exposed to creosote. All of the hatched larvae exposed as embryos to creosote exhibited morphological deformities, including scoliosis, pericardial edema and/or ascites. Similar effects were observed in embryos collected from creosoted pilings in San Francisco Bay, with a 72% decrease in hatching success compared with embryos collected from the Bay and severely deformed larvae. To investigate the combined effects of creosote and salinity on hatching success, larval morphology, and cardiac function, embryos were exposed to a sublethal concentration of creosote (0.003 mg/l) at three salinities; sub-optimal (8 parts per thousand (ppt)), optimal (16 ppt), and high salinity (28 ppt). The presence of creosote decreased hatching success at all three salinities, but the effect was greatest at 8 ppt (34% reduction) and the least in 28 ppt (14% reduction). The increased incidence of morphological abnormalities was also smallest at the high salinity (10% compared with 24 and 33% in 8 and 16 ppt). While exposure to creosote resulted in reduced heart rates at all three salinities, no additive effect of creosote and salinity was observed.

Characterization of the promoter-directing expression of growth hormone in a monocyte cell line.

Previous work from our laboratory has shown that cells of the immune system produce a growth hormone (GH) molecule similar to that produced by the pituitary. In the present study, using Southern analysis of RT-PCR products and sequencing of cloned cDNA molecules, we demonstrate that lymphoid cell lines utilize the same promoter and first exon as the pituitary somatotrope. To identify the cis-elements involved in transcriptional regulation of immune cell-derived GH, we have coupled rat GH promoter fragments to a luciferase reporter gene and transfected a monocyte cell line (P-388) by electroporation. The results suggest the presence of both positive (-299/-193 bp) and negative (-193/-107 bp) regulatory elements. The same constructs transfected in the pituitary cell line, GH3, in contrast to the monocyte cell line, showed a gradual decrease in luciferase expression. The overexpression of GHF-1 or GHF-2 resulted in a modest but significant reduction in rat GH promoter activity in the P-388 cell line. Taken together, the data suggest that immune cells utilize the same first exon and promoter sequence for the expression of monocyte GH as that reported for the expression of pituitary GH. Further, it appears that sequences between -299 and -107 bp are important in the regulation of the promoter where different transcription factors may be recruited to promote GH expression in a monocyte cell line.

Identification of SP3 as a negative regulatory transcription factor in the monocyte expression of growth hormone.

A number of studies from different laboratories clearly show that cells of the immune system produce a GH molecule indistinguishable from that produced in the pituitary. A more recent finding from our studies suggests that monocytes use the same first exon and promoter sequence for the expression of lymphocyte GH as that reported for the expression of pituitary GH. In this report we have extended these results by determining that two members of the SP family of transcription factors, SP1 and SP3, bind to the region at -138/-133 bp containing a GGGAGG motif. Confirmation that this region of the monocyte GH promoter-bound SP1 and SP3 was accomplished using electrophoretic mobility shift assays with SP1 consensus and mutant probes as well as specific antibodies to SP1 and SP3. Selective mutation of the SP1/SP3 site increased basal transcription by 73%, indicating that this site is important in transcriptional inhibition. Overexpression of SP1 had no demonstrable effect on the GH promoter, whereas overexpression of SP3 caused inhibition of expression in P-388 monocyte cells. Cotransfection of P-388 cells with overexpression vectors for both SP1 and SP3 transcription factors also resulted in inhibition of basal expression. Transfection experiments in Drosophila SL-2 cells overexpressing SP1 and/or SP3 suggest that both factors repress the basal expression of GH promoter luciferase constructs and that the effect together was additive. Taken together, the results demonstrate that basal expression of monocyte GH may be negatively regulated by SP3.

Negative regulation of phagocytosis in murine macrophages by the Src kinase family member, Fgr.

Ingestion of opsonized pathogens by professional phagocytes results in the generation and release of microbicidal products that are essential for normal host defense. Because these products can result in significant tissue injury, phagocytosis must be regulated to limit damage to the host while allowing for optimal clearance and destruction of opsonized pathogens. To pursue negative regulation of phagocytosis, we assessed the effect of the Src kinase family member, Fgr, on opsonin-dependent phagocytosis by mouse macrophages. We chose Fgr because it is present in high concentrations in circulating phagocytes but is not essential for Fcgamma receptor-mediated ingestion by mouse macrophages. Although expression of Fgr both in a macrophage cell line and in primary macrophages significantly attenuates ingestion mediated by Fcgamma receptors and CR3, it does not affect macropinocytosis or receptor-mediated endocytosis. This selective effect of Fgr is independent of its tyrosine kinase function. After Fcgamma receptor cross-linking, Fgr becomes associated with the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor, SIRPalpha (a member of the signal-regulatory protein family, also known as Src homology 2 domain-containing protein tyrosine phosphatase [SHP] substrate 1 [SHPS-1], brain immunoglobulin-like molecule with tyrosine-based activation motifs [BIT], and P84) and potentiates the association of the phosphatase SHP-1 with SIRPalpha. This association is responsible, at least in part, for decreasing positive signaling essential for optimal phagocytosis. These data demonstrate an important negative regulatory role for this Src kinase family member and suggest that this homeostatic function must be overcome for optimal uptake and clearance of opsonized pathogens.

Hyaluronic acid and the cumulus extracellular matrix induce increases in intracellular calcium in macaque sperm via the plasma membrane protein PH-20.

The hyaluronic acid (HA)-rich extracellular matrix (ECM) of the cumulus oophorus is known to facilitate fertilization. It has been suggested that HA may enhance fertilisation in a number of species, and in macaque sperm, HA has been shown to increase the number of acrosome reactions that follow sperm binding to the zona pellucida. In this study, we investigated the effects of HA on intracellular Ca2+ in capacitated cynomolgus macaque sperm. Fluorometry studies using the intracellular Ca2+ indicator Fluo-3 showed that addition of 100 micrograms/ml of HA induced a rapid increase in intracellular Ca2+. This Ca2+ increase (approximately 2-3 times above basal levels) was inhibited by preincubation of sperm with Fab fragments of anti-recombinant PH-20 IgG. The frequency of acrosome reactions in sperm exposed to HA was not above control levels. A synthetic gel was prepared with similar viscosity to the cumulus and with HA trapped in its matrix. Video imaging of individual sperm was used to demonstrate that capacitated sperm swimming into the HA gel had increased intracellular Ca2+ levels. Preincubation of sperm with Fab fragments of anti-PH-20 IgG inhibited the increased intracellular Ca2+ levels induced by the HA gel. Sperm in control gel (no HA) did not show increased intracellular Ca2+, while sperm in gel containing anti-PH-20 IgG showed increased Ca2+ (positive control). Sperm loaded with Fluo-3 were allowed to interact with cynomolgus macaque cumulus masses, and sperm within the cumulus ECM clearly showed increased intracellular Ca2+ that was inhibited when sperm were preincubated in anti-PH-20 Fab. Fluorescein isothiocyanate (FITC)-HA was found to bind to sperm over the acrosomal region, corresponding to PH-20 localisation, and this binding could be inhibited by preincubation of sperm with anti-PH-20 fragments. The results of this study show that HA increases intracellular Ca2+ in macaque sperm through interaction with plasma membrane PH-20. We propose that HA binding to plasma membrane PH-20 induces an aggregation of receptors that in turn results in intracellular signalling. As a result, sperm have higher basal CA2+ levels and are more responsive to induction of the acrosome reaction after binding to the zona pellucida.

A comparison of women and men with spinal cord injury.

While research on spinal cord injury (SCI) is abundant, few studies focus on women. This population-based study investigates differences in the prevalence of secondary conditions between 128 women and 522 men. Case managers retrospectively interviewed 650 persons regarding medical and psychological conditions secondary to SCI, as well as other life issues. Overall, males and females show more similarities than differences in the ways in which they manage life with SCI. Differences were found, though, regarding etiology of initial injury, insurance coverage, caregiver use, transportation use, medication use, and in other medical and behavioral areas. Females are significantly involved in more automobile crashes than males, while males are involved in more galls than females. Females are more reliant on Medicaid, while males report more Medicare and Worker's Compensation coverage. Females are more likely to have a paid attendant as a caregiver while males are more likely to have their spouse or parents assist. Males report more independence in their use of transportation than females. Males and females also report significant differences in the use of medication. Females are more likely to use medication any time it is a treatment option. Males are more active, use tobacco more and have more arm fractures postinjury than females.

Effects of Salinity on Sperm Motility, Fertilization, and Development in the Pacific Herring, Clupea pallasi.

We investigated the effects of salinity on fertilization and early development in a population of Pacific herring, Clupea pallasi, that migrate from oceanic waters into the San Francisco Bay estuary to spawn. The salinity range for fertilization fell between 8 and 28 ppt, with an optimal range of about 12 to 24 ppt. In comparison, the range for a population of C. harengus membras (Airisto Sound, Finland) that reside year-round in the Baltic Sea was 4 to 24 ppt. Roles for both Na+ and K+ were indicated in C. pallasi fertilization since increasing Na+ in the presence of 10 mM K+ (concentration of seawater) mimicked the effects of increased overall salinity, whereas reduced effects were obtained if [K+] was held at 5 mM (that of half-strength seawater). The initiation of C. pallasi sperm motility by components of the egg chorion, a prerequisite for fertilization, was inhibited at both elevated (28 and 32 ppt) and reduced (4 and 8 ppt) salinities. Embryonic development through larval hatching in C. pallasi exhibited a salinity tolerance similar to that of fertilization; optimum development was obtained at salinities between 8 and 24 ppt. A comparison of developmental progression in 3.5, 14, and 28 ppt seawater revealed that salinity effects became evident during the post-gastrulation stages of development and that progression to hatching was delayed in both the lower and higher salinities for those embryos that completed development.

Enhancement of the radiosensitivity of two human tumour cell lines by hexamethylene bisacetamide.

The effect of the maturation-inducing polar solvent, hexamethylene bisacetamide (HMBA), on the radiosensitivity of two human tumour cell lines (clone A, a colon carcinoma; and EJ, a bladder carcinoma) was investigated. Exposure of clone A or EJ cells to HMBA resulted in a concentration-dependent increase in doubling time, a decreased plating efficiency and changes in cell morphology, which are consistent with the formation of a better-differentiated phenotype. Growth of clone A cells in 2 or 3 mM HMBA, followed by irradiation and plating into HMBA-free medium, resulted in a significant enhancement in radiosensitivity, as determined by colony-forming ability. A similar increase in radiosensitivity was detected for EJ cells; however, for these cells a concentration of 7 mM HMBA was required. The increased radiosensitivity caused by HMBA was observed primarily in the low-dose, shoulder region of the gamma-ray cell survival curves for both cell lines, which is reflected by an increase in the alpha component of the survival curve with essentially no effect on beta. These data indicate that HMBA can radiosensitise human tumour cells at concentrations and for exposure periods that can be achieved in the clinic.

Enhancement of radiation-induced DNA double-strand breaks and micronuclei in human colon carcinoma cells by N-methylformamide.

The differentiation-inducing agent N-methylformamide (NMF) enhances the sensitivity of some cell lines to ionizing radiation. To elucidate the mechanism of NMF-mediated radiosensitization, we examined the effects of this agent on gamma-ray-induced DNA double-strand breaks and micronuclei in two cell lines, clone A (human colon carcinoma) and HCA-1 (murine hepatocarcinoma). Both cell lines form a better differentiated phenotype upon exposure to NMF, yet only clone A is radiosensitized. The neutral (pH 9.6) elution assay was used to evaluate the effects of this maturational agent on radiation-induced double-strand breaks in these cell lines. Exposure of HCA-1 cells to NMF had no effect on the level of DNA double-strand breaks induced by gamma rays. In clone A cells, however, exposure to NMF enhanced the initial formation of gamma-ray-induced double-strand breaks at each dose tested. The repair of double-strand breaks in both cell lines was not influenced by NMF. As a measure of chromosome fragmentation after irradiation, we evaluated micronuclei using the cytokinesis block method. Exposure to NMF had no effect on radiation-induced micronuclei formation in HCA-1 cells yet significantly enhanced the frequency of micronuclei induced by radiation in clone A cells. In clone A cells, the increases in radiation-induced double-strand breaks and micronuclei as a function of NMF exposure time reached maximums by approximately 72 h. These data suggest that NMF-mediated radiosensitization is the result of an increase in the initial level of radiation-induced DNA double-strand breaks.

Heterogeneity in radiation sensitivity within human primary tumour cell cultures as detected by the SCE assay.

The ability of the sister chromatid exchange (SCE) assay to detect heterogeneity in intrinsic radiation sensitivity was investigated. In order to identify tumour cell subpopulations, frequency histograms of cis-diamminedichloroplatinum (II) (cPt)-induced SCEs were generated and compared to those from cultures that had been irradiated 96 h before drug treatment. The results suggested that subpopulations with different radiosensitivities were present in nine of 18 human primary tumour cell cultures evaluated. When the effects of prior irradiation on the subsequent X-ray survival response and on cPt-induced SCE frequency histograms were compared, a good correlation was obtained between the two assays regarding the prediction of heterogeneity in radioresponse. These results suggest that primary cultures can contain both radiation-sensitive and radiation-resistant cells, and thus heterogeneity in intrinsic radiosensitivity may exist in human solid tumours.

Chromatin modifications associated with N-methylformamide-induced radiosensitization of clone A cells.

Exposure of certain cell lines to the differentiation-inducing agent N-methylformamide (NMF) enhances their radiosensitivity. As part of an attempt to elucidate the mechanism of NMF-induced radiosensitization, we examined the effects of NMF on chromatin structure, as reflected by changes in DNA-protein cross-links (DPCs) and the chromatin protein/DNA ratio, in two cell lines, clone A and HCA-1. Both lines form a better-differentiated phenotype upon exposure to NMF, yet only clone A is radiosensitized. Ionizing radiation induced DPCs in a linear manner beginning at about 10 Gy and continuing to at least 50 Gy in both cell types. NMF treatment of HCA-1 cells did not affect the background level of DPCs, but it enhanced the formation of radiation-induced DPCs at each dose tested. In clone A cells, NMF exposure elevated the DPC background level more than two-fold, and modified radiation-induced DPCs. The dose response for radiation-induced DPCs in NMF-treated clone A cells consisted of a linear increase up to 12.5 Gy, which was greater than in untreated cells, followed by a plateau level of DPCs out to 50 Gy, the highest dose tested. NMF treatment of clone A, but not HCA-1, cells also increased the chromatin protein/DNA ratio by about 30-35%. In clone A cells, the increases in DPC background level and chromatin protein/DNA ratio as a function of NMF exposure time followed a pattern similar to that of the enhancement of radiosensitivity. These data suggested that modifications of chromatin structure, not involved in differentiation, may be associated with the radiosensitizing actions of NMF.

The effects of N-methylformamide on artificial and spontaneous metastases from a murine hepatocarcinoma.

The effects of the differentiation-inducing polar solvent N-methylformamide (NMF) on artificially induced and spontaneous metastases from a murine hepatocarcinoma (HCA-1) in C3Hf/Kam mice were investigated. Exposure of HCA-1 cells in vitro for 6 days to 1.0% or 1.25% NMF resulted in an increase in the number of lung nodules formed in mice when these cells were injected into their tail veins. This in vitro NMF exposure increased cell volume and induced only a slight amount of cytotoxicity. Administration of NMF to mice 1 day before i.v. tumour cell inoculation resulted in a dose-dependent increase in the number of lung nodules formed, beginning at an NMF dose of 600 mg kg-1. NMF caused a similar magnitude of metastasis enhancement in immunosuppressed mice. However, when the maximum dose tested (1,800 mg kg-1) was administered as 6 daily fractions of 300 mg kg-1 each, no increase in artificial metastases was detected. Administration of NMF to mice one day after i.v. tumour cell injection resulted in a dose-dependent decrease in the number of lung nodules. In mice bearing 5-6 mm HCA-1 leg tumours, treatment with 6 daily fractions of NMF (300 mg kg-1 each) significantly reduced the number of spontaneous pulmonary metastases, yet had very little effect on the growth of the primary tumour. These data suggest that, in a clinically relevant treatment setting, NMF can reduce metastasis formation.