Sulfatide and Na+-K+-ATPase: a salinity-sensitive relationship in the gill basolateral membrane of rainbow trout.

We investigated the effect of salinity on the relationship between Na(+)-K(+)-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na(+)-K(+)-ATPase activity, especially in Na(+)-K(+)-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na(+)-K(+)-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na(+)-K(+)-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na(+)-K(+)-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na(+)-K(+)-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.

Green tea catechins partially protect DNA from (.)OH radical-induced strand breaks and base damage through fast chemical repair of DNA radicals.

The catechins, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG) are believed to be active constituents of green tea accounting for the reported chemoprevention of certain cancers. The molecular mechanisms by which the measured low concentrations (ca. micromolar) of catechins in humans can reduce the incidence of carcinogenesis is not clear. Using an in vitro plasmid DNA system and radiolytically generating reactive oxygen species (ROS) under constant scavenging conditions, we have shown that all four catechins, when present at low concentrations, ameliorate free radical damage sustained by DNA. A reduction in both prompt DNA single-strand breaks and residual damage to the DNA bases, detected by subsequent incubation with the DNA glycosylases formamidopyrimidine (FPG), endonuclease III (EndoIII) and 5' AP endonuclease exonuclease III (ExoIII), was observed. EGCG was found to be the most active of the catechins, with effects seen at micromolar concentrations. Combined fast-reaction chemistry studies support a mechanism of electron transfer (or H-atom transfer) from catechins to ROS-induced radical sites on the DNA. These results support an antioxidant role for catechins in their direct interaction with DNA radicals.

The vascular activity of some isoflavone metabolites: implications for a cardioprotective role.

Legume-derived isoflavones such as genistein, diadzein and equol have been associated with a reduction in risk of cardiovascular disease. In the current study, we explore the vascular activity of several isoflavone metabolites namely dihydrodaidzein, cis and trans-tetrahydrodaidzein and dehydroequol for potential cardioprotective properties. Rat isolated aortic rings were used. 17beta-oestradiol, equol, and all four of the metabolites studied significantly antagonized contractile responses to noradrenaline. The direct vasodilatory action of these compounds were examined and in contrast to 17beta-oestradiol, the vasodilatory effect of which was demonstrated to be endothelium independent, the dilatory action of all four compounds could be inhibited by endothelium denudation. Further, the dilatory action of both dihydrodaidzein and cis-tetrahydrodaidzein were inhibited by the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (NOLA), by the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and by 40 mM KCl. Dilatory responses to dehydroequol and trans-tetrahydrodaidzein, on the other hand, were inhibited by 40 mM KCL but not by NOLA nor ODQ. Finally, we examined the protective potential of these compounds in inhibiting endothelium damage by oxidized low density lipoprotein (ox-LDL). Trans-tetrahydrodaidzein was at least 10 fold more potent than 17beta-oestradiol in protecting against ox-LDL induced damage. We conclude that the isoflavone metabolites, dihydrodaidzein, cis- and trans-tetrahydrodaidzein and dehydroequol, may potentially represent a novel series of cardioprotective therapeutics.

Cytotoxic aldehydes as possible markers for childhood cancer.

Concentrations of 22 known aldehydes (byproducts of lipid peroxidation), 5 acyloins, free and total carnitine and acylcarnitines were measured in plasma and urine obtained from pediatric patients with various forms of cancer before any treatment, and following treatment with doxorubicin or daunorubicin. Aldehydes, before the initiation of chemotherapy, were significantly elevated in cancer patients compared to controls. Aldehydes such as hexanal, heptanal, and malondialdehyde were strikingly higher in samples from cancer patients, while trans 4-cis-4-decenal was the prominent aldehyde in the blood of controls. In addition, in each form of cancer the pattern of aldehydes appeared to be unique when compared to controls, or to others forms of cancer. In cancer patients receiving chemotherapy there was a general trend toward a reduction 24 h after both the first and after the fifth doxorubicin dose. These changes however were not significant statistically due to large inter-patient variation. Free and total plasma carnitine levels remained in the normal range, and there were no abnormal acylcarnitines detected in urine. Possible hypotheses to explain the elevations in aldehydes, and the reasons for the changed aldehyde profiles in different forms of cancer are discussed.

Neural precursor cells: applications for the study and repair of the central nervous system.

A combination of gene transfer and intracerebral transplantation techniques has been used in studies of CNS development to provide the most compelling evidence to date that the broad diversity of cell types that exist in the CNS arises from single precursor cells. Although the factors that influence cellular differentiation in vivo remain to be clarified, work conducted in vitro with neural precursors has demonstrated that environmental signals (both soluble factors and substrate molecules) play a pivotal role in these decisions. In particular, FGF-2 appears to be one of the prominent influential factors involved in CNS development (see Temple & Qian, 1995). The generation of immortalized precursor populations that are capable of differentiating into multiple CNS cell types in vivo has significant implications for the treatment of neural dysfunction. Such cells may be manipulated toward a lineage that synthesizes factors of interest and used in grafting strategies to replace substances that are lost after injury or in neurodegenerative disease. Alternatively, precursor cells may be directed to a neuronal lineage and used to functionally repair damaged neural systems. Finally, genetic modification of precursor populations provides a method for introducing therapeutic gene products both into discrete regions of the brain and into widely dispersed areas of the CNS. In considering applications to human disease, it has been reported that nestin is expressed in human neuroepithelial cells (Tohyama et al., 1992), suggesting the existence of neural precursors. Recently, such precursors were in fact isolated by two separate groups (Kirschenbaum et al., 1994; Sabaté et al., 1995) and shown to be amenable to gene transfer and to successfully survive transplantation into the brain of experimental animals (Sabaté et al., 1995). Such findings encourage the possibility that precursor cells from the human CNS may be utilized in cell replacement or gene therapy strategies directed toward human neurodegenerative disorders. While immortalization techniques have been essential for generating large quantities of precursor cells for study and transplantation, the genetic modification of cells may alter vital cellular properties. Thus, the ability to induce the proliferation of nonimmortalized neural populations in vitro with the use of growth factors (see section on CNS precursor cells above) provides an important alternative approach for developing perpetual neural cell lines. Recent work with such growth factor-responsive precursor cells has suggested their therapeutic potential in the CNS, as evidenced by the finding that FGF-2-responsive cells can successfully engraft and express transgenes in the adult brain (Gage et al., 1995; Sabaté et al., 1995; Suhonen et al., 1996). Continuing studies with these cells will provide additional insight into the properties of primary CNS stem cells and increase the range of precursor populations that are useful for exploring the development, function, and plasticity of the CNS.

Long-term survival of transplanted basal forebrain cells following in vitro propagation with fibroblast growth factor-2.

The intracerebral transplantation of freshly dissected fetal tissue containing cholinergic neurons of the developing basal forebrain has been reported to reverse lesion-induced or age-related cognitive deficits in animal models of cholinergic neuronal degeneration. Grafts of cultured fetal neurons, however, have generally shown poor cellular survival and limited therapeutic benefit. We tested the hypothesis that recent advances in the identification of growth factors that promote the survival and propagation of fetal precursor cells in vitro would improve the long-term survival of cultured neurons following intracerebral implantation. Dissociated cells from gestational Day 14 rodent basal forebrain were grown in chemically defined media supplemented with 20 ng/ml basic fibroblast growth factor. Two weeks postplating, numerous cells were present in the cultures and showed immunoreactive labeling for a variety of markers, including glutamic acid decarboxylase, neuron-specific enolase, neurofilament proteins, glial fibrillary acidic protein and, occasionally, choline acetyltransferase. To determine if cultured basal forebrain cells would survive intracerebral implantation, the cells were implanted homotypically into the nucleus basalis magnocellularis. To enhance the potential for graft survival in vivo, cells were also implanted into the nucleus basalis magnocellularis following an ibotenic acid lesion and into the denervated frontal cortex. Animals sacrificed between 2 weeks and 7 months following transplantation showed good and comparable graft survival in all sites. Immunocytochemical analysis revealed that representative populations of cells observed in vitro survived for prolonged periods in vivo, even in sites distal from their normal cellular targets. Thus, neuronal populations expanded in vitro can successfully survive and maintain cellular phenotypes post-transplantation. These results suggest a potential for isolating and growing specific neuronal populations in vitro for intracerebral transplantation.

Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain.

The dentate gyrus of the hippocampus is one of the few areas of the adult brain that undergoes neurogenesis. In the present study, cells capable of proliferation and neurogenesis were isolated and cultured from the adult rat hippocampus. In defined medium containing basic fibroblast growth factor (FGF-2), cells can survive, proliferate, and express neuronal and glial markers. Cells have been maintained in culture for 1 year through multiple passages. These cultured adult cells were labeled in vitro with bromodeoxyuridine and adenovirus expressing beta-galactosidase and were transplanted to the adult rat hippocampus. Surviving cells were evident through 3 months postimplantation with no evidence of tumor formation. Within 2 months postgrafting, labeled cells were found in the dentate gyrus, where they differentiated into neurons only in the intact region of the granule cell layer. Our results indicate that FGF-2 responsive progenitors can be isolated from the adult hippocampus and that these cells retain the capacity to generate mature neurons when grafted into the adult rat brain.

Isolation, characterization, and use of stem cells from the CNS.

The nervous system of adult mammals, unlike the rest of the organs in the body, has been considered unique in its apparent inability to replace neurons following injury. However, in certain regions of the brain, neurogenesis occurs postnatally and continues through adulthood. The nature, fate, and longevity of cells undergoing proliferation within the CNS are unknown. These cells are increasingly becoming the focus of intense scrutiny; this is a recent development that has led to considerable controversy over the appropriate terminology to describe neural cells as they pass through different stages of proliferation, migration, and differentiation. Continuing studies detailing the properties of mitotic populations in the adult CNS will provide a better understanding of the nature of these cells during their development and should lead to a more consistent nomenclature. Studies of neural precursors isolated from the embryonic brain have indicated that many subgroups of cells undergo mitosis and subsequent differentiation into neurons and glia in vitro. A number of substances, such as growth factors and substrate molecules, are essential for these processes and also for lineage restriction and fate determination of these cells. Recent studies have shown that cells with proliferative capabilities can also be isolated from the adult brain. The nature of these cells is unknown, but there is evidence that both multipotent cells (stem cells) and lineage-restricted cells (neuroblasts or glioblasts) are resident within the mature CNS and that they can be maintained and induced to divide and differentiate in response to many of the same factors that influence their embryonic counterparts. Presently, it is unclear how many potentially quiescent precursor cells exist in the adult brain or what combination of growth factors and substrate molecules is involved in the proliferation and differentiation of these cells. Some of these questions are currently being addressed by using immortalized neural precursors or growth factor-expanded populations of primary precursors to model precursor responsiveness to environmental manipulations. Because in vitro culture conditions are unlikely to provide all of the factors necessary for inducing the proliferation and differentiation of neural precursors, recent studies have explored the properties of well-characterized precursor populations after implantation back into specific regions of the developing or adult CNS. These studies have highlighted the importance of the microenvironment in precursor differentiation and further suggested that precursor plasticity is a characteristic that is probably common to neural precursors throughout the CNS.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo and ex vivo gene transfer to the brain.

The use of gene transfer techniques to express novel proteins within different cellular populations has provided insights into the function and plasticity of the brain. Recently, this technique has been successfully used to explore physiological processes within the CNS and to intervene in neurodegenerative disease and cancer. Progress in manipulating transgene products in vivo and in achieving cell-specific targeting of genetic material offers promise for enhancing the usefulness of this technique and its therapeutic potential for treating human disorders of the CNS.

Regulation of dopamine production by genetically modified primary fibroblasts.

Primary skin fibroblasts were genetically modified with catecholamine-synthesizing enzyme genes and studied as potential syngeneic donor cells to supply catecholamines in animal models of Parkinson's disease. Primary skin fibroblasts obtained from inbred Fischer 344 rats were transduced with tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC) cDNAs using retroviral vector system. The transduced cells were characterized in vitro by enzymatic assay, immunocytochemistry, and HPLC analysis of catecholamine production and release. Accumulation of high levels of dopamine was detected in the media in a time-dependent manner. Secretion of dopamine and its metabolites appeared to be constitutive without significant storage capacity in vesicles or regulation at the level of secretion. The feasibility of regulating the final dopamine production by the AADC-transduced cells was explored in two ways. First, administration of various doses of the precursor, L-dopa, resulted in a controlled production of dopamine by these cells. Second, coculturing AADC-transduced cells with TH-transduced cells in various proportions allowed control of dopamine production. TH-transduced cells served as an endogenous source of precursor. We propose the use of these cells to study the role of AADC in restoring the dopamine-deficient behavior and to compare the effect of dopamine-producing cells with L-dopa-producing cells either by cografting TH-transduced cells with AADC-transduced cells or by grafting TH-transduced cells alone. The role of AADC in vivo will be assessed in future experiments involving animal models of Parkinson's disease.

Decline of exocrine pancreatic function in cystic fibrosis patients with pancreatic sufficiency.

Patients with cystic fibrosis and pancreatic sufficiency were investigated for evidence of progressive pancreatic disease. From a cohort of 630 patients, 20 pancreatic-sufficient patients became pancreatic insufficient after an average duration of 5.6 y (range 0.6-20.6 y) from diagnosis. Among 54 patients documented to be pancreatic sufficient by direct pancreatic stimulation test, 47 remained pancreatic sufficient and seven developed pancreatic insufficiency. The patients who ultimately developed pancreatic insufficiency were younger and had greatly reduced outputs of enzyme, fluid, and electrolytes. Those who remained pancreatic sufficient showed enzyme secretion close to or within the non-cystic fibrosis control range. Twenty of these patients underwent a second pancreatic stimulation test after an average interval of 4 y (range 1.3-6.2 y). No significant alteration in enzyme, fluid, or electrolyte output was seen in the patients who remained pancreatic sufficient, but there was further reduction in enzyme and fluid output in the patients who developed pancreatic failure. In conclusion, the majority of pancreatic-sufficient patients with pancreatic enzyme secretion within the control range showed no deterioration of function over an extended time period. However, a small number of pancreatic-sufficient patients with reduced enzyme and fluid secretion are at risk of pancreatic failure.

Survival and function of intrastriatally grafted primary fibroblasts genetically modified to produce L-dopa.

A combination of gene transfer and intracerebral grafting may provide a powerful technique for examining the role of discrete substances in the development or functioning of the brain. In the present study, primary fibroblasts obtained from a skin biopsy from inbred Fischer rats were used as donor cells for genetic modification and grafting. When grafted to the striatum of Fischer rats with a prior 6-hydroxydopamine lesion, primary fibroblasts containing a transgene for either tyrosine hydroxylase (TH) or beta-galactosidase survived for 10 weeks and continued to express the transgene. TH synthesized by the implanted fibroblasts appeared to convert tyrosine to L-dopa actively, as observed in vitro, and to affect the host brain, as assessed through a behavioral measurement. These results suggest that primary fibroblasts genetically altered to express TH have the capacity to deliver L-dopa locally to the striatum in quantities sufficient to compensate partially for the loss of intrinsic striatal dopaminergic input.

Grafting fibroblasts genetically modified to produce L-dopa in a rat model of Parkinson disease.

Rat fibroblasts were infected with a retroviral vector containing the cDNA for rat tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2]. A TH-positive clone was identified by biochemical assay and immunohistochemical staining. When supplemented in vitro with pterin cofactors required for TH activity, these cells produced L-dopa and released it into the cell culture medium. Uninfected control cells and fibroblasts infected with the TH vector were grafted separately to the caudate of rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway. Only grafts containing TH-expressing fibroblasts were found to reduce rotational asymmetry. These results have general implications for the application of gene therapy to human neurological disease and specific implications for Parkinson disease.

Anteroposterior radiographic view of the knee. An unreliable indicator of bone damage.

The clinician and/or radiologist uses standard radiographic views to assess bony changes in the joints of patients with both potential and established arthritis. Using AP (standing) views, we recorded the number of osteophytes and/or subchondral cysts seen on both nonarthritic and arthritic tibial tables and their respective plateaus. These data were compared with the number of osteophytes and/or subchondral cysts observed on "en face" views taken of the same tibial tables after they were resected from the knee joints. Not all of the osteophytes or subchondral cysts present in these tibial tables were detected with the AP view. Narrow osteophytes and those located at extreme anterior or posterior positions on a plateau were missed. Single cysts scattered across a plateau were also not seen. We found that the standard AP view gave an inaccurate measure of the amount of bone damage actually present in the tibial tables of arthritic knees.

Electron capture gas chromatographic determination of traces of formaldehyde in milk as the 2,4-dinitrophenylhydrazone.

A quantitative method is described for the determination of formaldehyde in milk by packed-column gas chromatography and electron capture detection. Aldehyde derivatization was carried out in situ with 2,4-dinitrophenylhydrazine followed by extraction and analysis using an external standard. Average recoveries of 96.3 +/- 1.6% were characteristic of the chromatographic method with an estimated detection limit of 0.026 mg/kg. The technique was applied to determination of formaldehyde in milk from cows consuming a formalin-treated feedstuff.