Tyrosine hydroxylase and dopamine-β-hydroxylase immunoreactivities in the cnidarian <Emphasis Type="Italic">Renilla koellikeri</Emphasis>

Western Blot and immunohistochemical studies were conducted in the sea pansy Renilla koellikeri, a representative of the earliest multicellular animals with a nervous system, using various antibodies raised against enzymes of the catecholamine biosynthetic pathway. Western blots of sea pansy extracts revealed a protein band that co-migrated with dopamine-beta-hydroxylase (DBH) from mouse adrenal glands. Similar experiments with antisera against tyrosine hydroxylase (TH) revealed several immunoreactive protein bands, all of larger molecular weight than mammalian tyrosine hydroxylase. DBH-like and, to a lesser extent, TH-like and phenylethanolamine N-methyltransferase-like immunoreactivities were detected in ectodermal sensory neurons and associated subectodermal neurites, in neurons of the mesogleal nerve-net and associated amoebocytes, and in some endodermal neurons. While it is still not clear whether the detected TH-immunoreactive proteins represent some form of TH, the presence in sea pansies of a DBH-like protein is in agreement with previously detected norepinephrine-like immunoreactivity in the same species. The widespread distribution of these immunoreactivities in various sea pansy neurons suggests important roles for catecholamines in nerve net activity.     

Immunological markers predicting outcome in patients with hepatitis C treated with interferon-alpha and ribavirin

Type 1 (T1) cytokine responses are required for the clearance of hepatitis C virus by cytotoxic T lymphocytes, but can promote liver damage. Interferon-alpha (IFN alpha) can be expected to promote T1 cytokine responses, so treatment outcome may depend on the T1/T2 cytokine environment and levels of immune activation at baseline. This model was tested by monitoring immunological markers in a pilot study of treatment na?ve patients given IFN alpha 2b and ribavirin, with the aim of finding markers that predict virological outcome. Soluble (s) CD26/dipeptidyl peptidase IV enzyme activity and levels of sCD30, bioavailable IL-6, sTNF-RI, IL-1ra and nitrite/nitrate (NO(2)(-)/NO(3)(-)) were measured. Levels of IL-1ra and bioavailable IL-6 were lower in patients than controls and did not change with therapy. Treatment decreased sCD26/dipeptidyl peptidase IV enzyme activities and sCD30 levels and increased NO(2)(-)/NO(3)(-) levels. High baseline sCD30 levels predicted an early (P = 0.008) and sustained (P = 0.03) virological response to therapy, suggesting treatment may be more effective in patients with a predominant T2 profile.     

Risk factors for elevated HIV incidence rates among female injection drug users in Vancouver

Background

In 1997, we found a higher prevalence of HIV among female than among male injection drug users in Vancouver. Factors associated with HIV incidence among women in this setting were unknown. In the present study, we sought to compare HIV incidence rates among male and female injection drug users in Vancouver and to compare factors associated with HIV seroconversion.

Methods

This analysis was based on 939 participants recruited between May 1996 and December 2000 who were seronegative at enrolment with at least one follow-up visit completed, and who were studied prospectively until March 2001. Incidence rates were calculated using the Kaplan–Meier method. The Cox proportional hazards regression model was used to identify independent predictors of time to HIV seroconversion.

Results

As of March 2001, seroconversion had occurred in 110 of 939 participants (64 men, 46 women), yielding a cumulative incidence rate of HIV at 48 months of 13.4% (95% confidence interval [CI] 11.0%–15.8%). Incidence was higher among women than among men (16.6% v. 11.7%, p = 0.074). Multivariate analysis of the female participants'' practices revealed injecting cocaine once or more per day compared with injecting less than once per day (adjusted relative risk [RR] 2.6, 95% CI 1.4–4.8), requiring help injecting compared with not requiring such assistance (adjusted RR 2.1, 95% CI 1.1–3.8), having unsafe sex with a regular partner compared with not having unsafe sex with a regular partner (adjusted RR 2.9, 95% CI 0.9–9.5) and having an HIV-positive sex partner compared with not having an HIV-positive sex partner (adjusted RR 2.7, 95% CI 1.0–7.7) to be independent predictors of time to HIV seroconversion. Among male participants, injecting cocaine once or more per day compared with injecting less than once per day (adjusted RR 3.3, 95% CI 1.9–5.6), self-reporting identification as an Aboriginal compared with not self-reporting identification as an Aboriginal (adjusted RR 2.5, 95% CI 1.4–4.2) and borrowing needles compared with not borrowing needles (adjusted RR 2.0, 95% CI 1.1–3.4) were independent predictors of HIV infection.

Interpretation

HIV incidence rates among female injection drug users in Vancouver are about 40% higher than those of male injection drug users. Different risk factors for seroconversion for women as opposed to men suggest that sex-specific prevention initiatives are urgently required.Recent reports in Canada and numerous other countries indicate that HIV is increasingly affecting women.¹ Before 1995, adult women in Canada had 9.6% of all positive HIV tests for which the age and sex of the person being tested were known. By 1995, this proportion had increased to 18.5% and reached 23.9% in 2000. In addition, 39% of all new HIV infections among women in 2000 were attributed to injection drug use.² These data are consistent with findings in the United States where, in 1999, women accounted for 23% of all reported AIDS cases in adults, of which 42% were attributed to injection drug use.³These data clearly indicate that the face of the epidemic is changing. Whereas some factors unique to the transmission of HIV to women are known, basic and behavioural research efforts addressing sex-related and drug-related vulnerabilities among female injection drug users (IDUs) are lacking.⁴ At a time when women''s vulnerability to HIV infection is becoming increasingly apparent worldwide,^5,6 a better understanding of the processes and factors that cause drug-related harm among women in industrialized countries is urgently required.Since the mid-1990s, the Downtown Eastside of Vancouver, British Columbia, has experienced an explosive and ongoing HIV epidemic among IDUs with annual HIV incidence rates reaching as high as 19% in 1997.^7,8 When subjects were enrolled in the Vancouver Injection Drug User Study (VIDUS), it was found that the baseline HIV prevalence was higher among women than men (35.2% v. 25.8%).⁷ Follow-up of this cohort now allows an investigation aimed at identifying the predictors of HIV seroconversion among female and male IDUs. Therefore, we sought to compare HIV incidence rates among male and female IDUs in Vancouver and to compare risk factors associated with HIV seroconversion.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Autocrine role of transforming growth factor beta1 on rat granulosa cell proliferation

We evaluated the effects of transforming growth factor beta1 (TGFbeta1), alone or in combination with FSH and estradiol, on DNA synthesis in primary cultures of immature rat granulosa cells. 3H-Thymidine incorporation was significantly stimulated by TGFbeta1 (5.6-fold). This effect was enhanced by FSH (20 ng/ml, 27.7-fold) or estradiol (100 ng/ml, 13.4-fold) or by a combination of both hormones (59.2-fold). Measurement of TGFbeta bioactivity showed the presence of significant amounts of TGFbeta in conditioned medium from granulosa cell cultures, and most of the activity was present in the latent form. FSH alone or in combination with estradiol produced a marked suppression of the production of latent and active TGFbeta. Activated conditioned medium from control cultures of granulosa cell elicited a 1.4-fold increase in thymidine incorporation. This effect was markedly amplified by FSH (3-fold) and estradiol (4.3-fold) and by a combination of both (8.7-fold). The peptide containing the cell-binding domain of fibronectin (RGDSPC) partially inhibited thymidine incorporation stimulated by TGFbeta1. Fibronectin did not synergize with FSH, and the interaction between TGFbeta1 and FSH was even observed in the presence of this protein. The conclusions reached were as follows: 1) TGFbeta1 is an autocrine stimulator of rat granulosa cell DNA synthesis, 2) FSH and estradiol produce a suppression of latent and active TGFbeta production but markedly amplify TGFbeta action, presumably at a postreceptor level, and 3) the stimulatory effects of TGFbeta1 may be only partly mediated by the increased fibronectin secretion.     

Determinants within the C-terminus of the human norepinephrine transporter dictate transporter trafficking,stability, and activity

The function of the human norepinephrine transporter (hNET) depends on its presence at the cell surface. A role for the hNET C-terminus in trafficking the transporter to the surface has been suggested by the report of a bovine NET C-terminal splice variant that accumulates within heterologous host cells, and a human variant homolog has also been reported. We examined the relevance of the C-terminus of hNET to trafficking and function using transfected LLC-PK1 cells. The intracellular and surface expression of NET proteins was evaluated by Western blots, and their functional capacities were assessed using transport assays. We found that the C-terminal residues encoded by hNET 1a enable the efficient maturation and surface expression of hNET and therefore critically impact transporter activity. Alternative splicing causes the retention of immature hNETs within the cell, whereas introduced C-terminal deletions result in significant degradation. The loss of the terminal isoleucine alone (Delta617-hNET) is sufficient to cause the degradation of hNET, an effect that can be mimicked by nonconservative point mutations at the terminal position. The phenotype of Delta617-hNET is recapitulated in neuronal SK-N-MC cells, but is significantly less severe in HEK-293 cells, suggesting a role for host cell factors in enabling the biosynthetic progression of wild-type hNET. Additional proximal residues may act at other steps to affect the expression of the fully mature protein on the cell surface (Q608A) and to more directly affect transporter activity (F609A). Together our studies document a critical contribution of the hNET C-terminus to transporter trafficking, stability, and function.     

A butyrylcholinesterase in the early development of the brine shrimp (Artemia salina) larvae: a target for phthalate ester embryotoxicity?

The phthalate ester insensitive blue-green algae (Synechococcus lividus) were used as a food source to extend the survival of synchronously hatched brine shrimp (Artemia salina) larvae allowing measurement of a reduced toxic response to phthalate esters at late post-hatching stages of development. The maximum acute toxicity due to di-n-butyl phthalate (DNBP) correlated with the expression of a phthalate ester-hydrolyzing enzyme. The purified enzyme was identified as a butyrylcholinesterase due to its rapid inactivation by low concentrations (10(-7)M) of diisopropyl fluorophosphate and inhibition by physostigmine (IC(50)=6 x 10(-7)M) and tetraisopropylpyrophosphoramide (I-OMPA, IC(50)=x 10(-6)M) but not by BW284c5. Apparently competition of the phthalates with the endogenous substrates of the enzyme led to development-dependent toxicity.     

Characterization of an osteoblast-specific enhancer element in the CBFA1 gene

Cbfa1 is a critical regulator of cell differentiation expressed only in the osteochondrogenic lineage. To define the molecular basis of this cell-specific expression we analyzed the murine Cbfa1 promoter. Here we show that the first 976 bp of this promoter are specifically active in osteoblastic cells. Within this region DNase I footprinting delineated a 40-bp area (CE1) protected differently by nuclear extracts from osteoblastic cells and from non-osteoblastic cells. When multimerized, CE1 conferred an osteoblast-specific activity to a heterologous promoter in DNA transfection experiments; this enhancing ability was conserved between mouse, rat, and human CE1 present in the respective Cbfa1 promoters. CE1 site-specific mutagenesis determined that it binds NF1- and AP1-like activities. Further analyses revealed that the NF1 site acts as a repressor in non-osteoblastic cells due to the binding of NF1-A, a NF1 isoform not expressed in osteoblastic cells. In contrast, the AP1 site mediates an osteoblast-specific activation caused by the preferential binding of FosB to CE1 in osteoblastic cells. In summary, this study identified an osteoblast-specific enhancer in the Cbfa1 promoter whose activity is achieved by the combination of an inhibitory and an activatory mechanism.     

A fluorescent resonant energy transfer-based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.