Recombinant human bile salt-stimulated lipase: an example of defectiveO-glycosylation of a protein produced in milk of transgenic mice

The expression of recombinant human bile salt-stimulated lipase (bssl) was targeted to the lactating mammary gland of transgenic mice. Expression of recombinant genes comprisingbssl cDNA, or alternatively genomicbssl DNA, under control of regulatory elements derived from the murine whey acidic protein (wap) gene was achieved and evaluated. Constructs containing genomicbssl sequences mediated high levels (0.5–1, mg ml^–1) of recombinant human BSSL in the milk. The recombinant BSSL produced was purified, biochemically characterized and compared to native BSSL and recombinant BSSL produced in mouse C127 and hamster CHO cells. Recombinant BSSL derived from transgenic mice showed a different migration and distribution after SDS-PAGE electrophoresis, lower apparent molecular mass on size-exclusion chromatography and no detectable interactions with a panel of lectins. These results indicate a significantly lower degree ofO-glycosylation of recombinant BSSL in milk from transgenic mice than was found for the native enzyme or recombinant CHO- or C127 cell-produced BSSL. Despite these differences, mouse-milk-derived recombinant BSSL exhibited similar lipase activity, the same, stability to low pH and similar sensitivity to elevated temperatures as the native enzyme. The observation that mouse-C127-cell-produced recombinant BSSL is heavilyO-glycosylated makes species-related restrictions less attractive as an explanation for the reducedO-glycosylation.     

Patients with Amyotrophic Lateral Sclerosis and Other Neurodegenerative Diseases Have Increased Levels of Neurofilament Protein in CSF

Abstract: In the present study we describe an ELISA to quantify the light subunit of the neurofilament triplet protein (NFL) in CSF. The method was validated by measuring CSF NFL concentrations in healthy individuals and in two well-characterized groups of patients with amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). The levels were increased in ALS (1,743 ± 1,661 ng/L; mean ± SD) and AD (346 ± 176 ng/L) compared with controls (138 ± 31 ng/L; p < 0.0001 for both). Within the ALS group, patients with lower motor neuron signs only had lower NFL levels (360 ± 237 ng/L) than those with signs of upper motor neuron disease (2,435 ± 1,633 ng/L) ( p < 0.05). In a second study patients with miscellaneous neurodegenerative diseases were investigated (vascular dementia, olivopontocerebellar atrophy, normal pressure hydrocephalus, cerebral infarctions, and multiple sclerosis), and the CSF NFL level was found to be increased (665 ± 385 ng/L; p < 0.0001). NFL is a main structural protein of axons, and we suggest that CSF NFL can be used to monitor neurodegeneration in general, but particularly in ALS with involvement of the pyramidal tract.     

Gene transfer of a hybrid interleukin-1β gene to B16 mouse melanoma recruits leucocyte subsets and reduces tumour growth in vivo

 Interleukin(IL)-1 differs from most other cytokines in its lack of a signal sequence. This results in intracellular retention of the immature proform. The release of IL-1 has been shown to be restricted predominantly to activated monocytes and macrophages and to be associated with apoptosis of the producer cell. These features have limited the investigation of IL-1 in early immune responses. In order to study the biological effects of local IL-1β release during an antitumour immune response, we used B16 mouse melanoma cells transduced with mature human IL-1β cDNA constructs. To obtain a released form of human IL-1β (ssIL-1β), the signal sequence from the related IL-1 receptor antagonist was ligated to the cDNA that encoded the mature form of IL-1β. When cells of the poorly immunogenic B16 melanoma cell line were transduced with IL-1β by retroviral infection, high levels of the protein were detected intracellularly, whereas cells transduced with IL-1β containing the signal sequence secreted most of their protein. The in vitro growth of the melanoma cells was unaffected by the IL-1β or ssIL-1β gene transfer. In contrast, the in vivo subcutaneous tumour growth of the ssIL-1β-transduced B16 cells in syngeneic C57BL/6 mice was significantly reduced compared with the IL-1β- and the mock-transduced controls. Immunohistochemical analysis revealed the infiltration of macrophages to be strong in B16/ssIL-1β, moderate in B16/IL-1β and minimal in control tumours. Furthermore, a moderate infiltration of CD4⁺ cells and of scattered dendritic cells was detected in B16/ssIL-1β tumours whereas very few or no CD4⁺ cells and dendritic cells were seen in the B16/IL-1β or control tumours. Following in vivo growth, all the tumours up-regulated ICAM-1 on their cell surfaces. However, the percentage of ICAM-1-expressing cells was two- to fourfold higher in B16/ssIL-1β tumours compared to the control. The data suggest that IL-1β acts in vivo, either directly or indirectly, as a chemotactic factor for monocytes, T helper cells and dendritic cells. This supports IL-1β having a regulatory effect on tumour growth when locally released in the tumour area. Received: 12 November 1996 / Accepted: 6 May 1997     

DNA haplotype analysis suggests linkage disequilibrium in the human insulin receptor gene

Summary Haplotypes of the insulin receptor gene were resolved in parents from Scandinavian nuclear families by studying the segregation of seven restriction fragment length polymorphisms (RFLPs). Of 97 unrelated parents, 41 had non-insulin-dependent diabetes mellitus (NIDDM). Considerable linkage disequilibrium in the region of the insulin receptor gene was found. Pairwise non-random associations were found between proximate RFLP sites, indicating the absence of recombinational hot spots between these sites. Thus, association studies between DNA polymorphisms at this locus and disease susceptibility genes could well be feasible in this population. Differences in the distribution of insulin receptor haplotypes were examined between NIDDM patients and healthy subjects. However, the differences observed were not statistically significant.     

Factors increasing ethylene production enhance the sensitivity of root growth to auxins

Light inhibits root elongation, increases ethylene production and enhances the inhibitory action of auxins on root elongation of pea ( Pisum sativum L. cv. Weibulls Marma) seedlings. To investigate the role of ethylene in the interaction between light and auxin, the level of ethylene production in darkness was increased to the level produced in light by supplying 1-aminocyclopropane-1-carboxylic acid (ACC) or benzylaminopurine (BAP). Ethylene production was measured in excised root tips after treatment of intact seedlings for 24 h, while root growth was measured after 48 h. Auxin, at a concentration causing a partial inhibition of root elongation, did not increase ethylene production significantly. A 4-fold increase in ethylene production, caused either by light, 0.1 μ M ACC or 0.1 μ M BAP, inhibited root elongation by 40–50%. The auxins 2,4-dichlorophenoxyacetic acid and indolebutyric acid applied at 0.1 μ M inhibited root elongation by 15–25% in darkness but by 50–60% in light. Supply of ACC or BAP in darkness enhanced the inhibitory effects of auxins to about the same extent as in light. The inhibition caused by the auxins as well as by the BAP was associated with swelling of the root tips. ACC and BAP treatment synergistically increased the swelling caused by auxins. We conclude that auxin and ethylene, when applied or produced in partially inhibitory concentrations, act synergistically to inhibit root elongation and increase root diameter. The effect of light on the response of the roots to auxins is mediated by a light-induced increase in ethylene production.     

Growth retardation induced by nutritional deficiency or abscisic acid in Lemna gibba: The relationship between growth rate and endogenous cytokinin content

The relationship between endogenous cytokinin content and relative growth rate (RGR) was studied in cultures of Lemna gibba L. G3 supplied with daily doses of mineral nutrients that were increased exponentially over time. At the optimal level of nutrient supply the RGR was 30–35% day^-1. The RGR was regulated by adjusting the rate of nitrogen supply, or it was restricted by addition of 0.5 M abscisic acid (ABA). Another approach used to investigate the specific roles of nitrogen (N) and phosphorus (P), was to transfer optimally growing plants to media without N or P but otherwise complete. The plants were harvested at regular intervals for determination of the RGR and levels of cytokinins of the isopentenyladenosine (iPA) and zeatinriboside (ZR) types with an enzyme-linked immunosorbent assay (ELISA). Levels of both iPA- and ZR-type cytokinins decreased when nitrogen was applied to cultures in growth limiting amounts. The cytokinin levels decreased more rapidly than the RGR when either N or P was lacking in the medium, suggesting an early influence of nutrient availability on cytokinin levels which in turn may induce adaptive response by the plant. RGR retardation induced by ABA did not affect cytokinin levels during the first 4 days of the treatment, and the later effects were small. The experiments gave no indication that ABA is involved in the adaptation response of Lemna plants to nutritional stress.Abbreviations ABA - abscisic acid - BAP - benzylaminopurine - ELISA - enzyme-linked-immunosorbent-assay - iP - isopentenyladenine - iPA - isopentenyladenosine - PBS - phosphate-buffered saline - PVP - polyvinylpyrrolidone - RGR - relative growth rate - R_N - relative nitrogen addition rate - Z - trans-zeatin - ZR - trans-zeatin riboside     

β1 Integrin-mediated collagen gel contraction is stimulated by PDGF

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent β₁ integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-β1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin β₁-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-β₁ integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the β₁ integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.     

Metabolism of methylmercury in rabbits and hamsters

The metabolism of²⁰³Hg-labeled methylmercury chloride (MeHg) has been studied in rabbits and hamsters. Rabbits were administered 1.6 μmol MeHgCl/kg bw intravenously, and hamsters 40 μmol/kg bw orally. Urine and feces were collected daily and groups of four animals killed after 1 h, 1 d, or 7 d. The concentration of²⁰³Hg in blood, liver, kidney, spleen, lung, heart, and brain was determined by gamma counting. In both animal species, the clearance of²⁰³Hg in the brain was slower than in other tissues. In the rabbits the brain²⁰³Hg concentration increased during the whole experimental period. Rabbits excreted²⁰³Hg primarily in feces (about 20% of the dose within 1 wk), and much less in urine (<2%). In contrast, hamsters very efficiently excreted²⁰³Hg in urine (50% in 1 wk). The fecal excretion was similar to that of the rabbits. Separation of inorganic Hg and MeHg in urine from hamsters by ion exchange chromatography showed that about 90% of the urinary²⁰³Hg was excreted as MeHg.     

Fatty acid turnover in the ischaemic compared to the non-ischaemic human heart

Summary Cardiac extraction, oxidation and release of plasma free fatty acids (FFA) was measured by coronary sinus catheterization, utilizing infusions of ³H palmitate and ¹⁴C oleate, in patients with ischaemic heart disease (IHD) at rest and during pacing induced angina pectoris and, for comparison, in healthy men of similar and younger age and men with hypertriglyceridaemia (HTG). At rest IHD patients differed from healthy men only by greater cardiac fatty acid release, which correlated with a significant glycerol release. In IHD patients, unlike in healthy men, myocardial extraction of both palmitate and oleate decreased while fractional oxidation of oleate increased during pacing. Fatty acid release was unaltered. Men with HTG had at rest higher myocardial FFA extraction than IHD patients, which did not decrease during pacing, but like in the patients oleate fractional oxidation increased on pacing. It is concluded that, in the moderately ischaemic human heart, the restricted blood flow may contribute to limit the fatty acid flux into the myocardium. The augmented cardiac fatty acid release in IHD patients is not related to ischaemia perse but may derive from an increased amount of cardiac interstitial fat.