Biocatalytic synthesis of acrylates in supercritical fluids: tuning enzyme activity by changing pressure.

Supercritical fluids are a unique class of nonaqueous media in which biocatalytic reactions can occur. The physical properties of supercritical fluids, which include gas-like diffusivities and liquid-like densities, can be predictably controlled with changing pressure. This paper describes how adjustment of pressure, with the subsequent predictable changes of the dielectric constant and Hildebrand solubility parameter for fluoroform, ethane, sulfur hexafluoride, and propane, can be used to manipulate the activity of lipase in the transesterification of methylmethacrylate with 2-ethyl-1-hexanol. Of particular interest is that the dielectric constant of supercritical fluoroform can be tuned from approximately 1 to 8, merely by increasing pressure from 850 to 4000 psi (from 5.9 to 28 MPa). The possibility now exists to predictably alter both the selectivity and the activity of a biocatalyst merely by changing pressure.     

L-Ala-substituted rat galanin analogs distinguish between hypothalamic and jejunal galanin receptor subtypes

In order to explore which amino acids or which blocks of amino acids in the 29 amino acid neuropeptide galanin are important for recognition of the endogenous ligand by galanin receptor subtypes present in the jejunum and in the hypothalamus, respectively, we have carried out L-Ala substitutions of individual amino acids or of blocks of amino acids in the rat galanin sequence and examined the binding of the obtained analogs to the rat hypothalamic and jejunal galanin receptor subtypes. This study reveals that the galanin sequence YLLGPH^9–14 is essential for recognition of galanin by both the rat hypothalamic and jejunal galanin receptor subtypes. Substitution of the N-terminal amino acids, GWTL^1–4, leads to total loss of affinity of galanin for both hypothalamic and jejunal galanin receptors. The α-helical C-terminal amino acid (25–29) part of galanin has no greater influence on the affinity of galanin to the hypothalamic galanin receptor subtype. L-Ala substitution of the C-terminal amino acids of galanin KHGLT^25–29 shows, however, that this C-terminal motif is essential for the recognition by the jejunal galanin receptor subtype, whereas amino acids in the middle portion of galanin NSAG^5–8 are of importance for binding to the hypothalamic but not to the jejunal receptor. [Ala^5–8] Galanin thus has a more than 100-fold higher affinity to jejunal receptor than to the hypothalamic receptor, while [Ala^25–29] galanin has a more than 100-fold higher affinity for the hypothalamic than for jejunal galanin receptor subtypes. pH dependence of the galanin binding to these receptor subtypes is also different. © Munksgaard 1997.     

Polymorphonuclear leukocyte functions in patients with acute rheumatic fever and rheumatic heart disease

Polymorphonuclear (PMN) cell function was assessed in 30 children with active rheumatic fever (ARF) (Group I), 30 cases with active rheumatic heart disease (RHD) (Group II), 28 cases of ARF and RHD in remission (Group III) and 34 adults with quiescent RHD along with their age matched controls. All the groups showed normal spontaneous and chemotactic movement. Phagocytosis of yeast particles was significantly reduced in groups II (P less than 0.0005), III (P less than 0.025) and IV (P less than 0.005). The opsonic activity of disease sera was low in all 4 groups (P less than 0.0005). The intracellular metabolic activity was moderately elevated in Group III. Phagocytosis and opsonic activity were thus persistently low in all the groups including the remission and quiescent group.     

Immunomodulatory properties of porins of some members of the family Enterobacteriaceae.

The outer membrane protein (OMP) preparation of Salmonella typhi was observed to have several immunomodulatory properties. Treatment of mice with an intraperitoneal injection of the OMP preparation enhanced both cellular and humoral responses of the mice to an unrelated antigen, a killed vaccine of Mycobacterium vaccae; both the delayed-type hypersensitivity (DTH) response and the antibody titers were enhanced. The predominant isotype of the antibody shifted from immunoglobulin G1 (IgG1) to IgG2a upon treatment with OMP. Treatment of mice with the OMP preparation improved the efficiency of in vitro antigen presentation by the peritoneal cells and also induced the cells to secrete interleukin-1. Treatment with the lipopolysaccharide (LPS) preparation of S. typhi had the opposite effect; i.e., the DTH response to M. vaccae was suppressed. Treatment with OMP neutralized the suppressive effects of LPS. The OMP preparation also had an enhancing effect on the innate immune mechanisms of the mice. Intraperitoneal injection of the OMP preparation enhanced the microbicidal activity of the peritoneal cells, and production of nitric oxide intermediates was stimulated. Injection of the OMP preparation into footpads of naive nonimmune mice induced a sustained hypersensitivity response that peaked at 24 h. Purified porins of the OMP preparation could induce both immunomodulation and hypersensitivity. Porins prepared from five different Salmonella strains and a strain of normal fecal Escherichia coli also exhibited immunomodulatory and hypersensitivity-inducing activities.     

Cross-reactions in cell mediated immunity induced by atypical mycobacteria

Cross-reactivity in the delayed hypersensitivity response to mycobacteria of different Runyon groups was tested in Swiss white mice immunised with live mycobacteria. All the strains tested gave cross-reactions and, generally, slow growers gave stronger cross-reactions with other slow growers than with rapid growers and vice versa. Sonicates of cross-reacting mycobacteria were also tested for their ability to generate activated macrophages in mice immunised with Mycobacterium avium. All the mycobacterial sonicates generated activated macrophages but a sonicate of Salmonella typhi did not. The sonicate of M. tuberculosis H37Rv also generated activated macrophages, which indicates that there might be protective cross-reactions between M. tuberculosis and atypical mycobacteria.     

Differential regulation of effector responses of cell mediated immunity in experimental salmonellosis.

Delayed type hypersensitivity (DTH) and protective cell mediated immunity showed different profiles with respect to time following intraperitoneal immunization with live Salmonella enteritidis. Whereas the DTH response decreased with time the Protection Index increased. The decline in DTH response was found to be associated with suppressor cells generated by intraperitoneal immunization and could be prevented by cyclophosphamide treatment prior to immunization. It was concluded that the two effector responses of cell mediated immunity were under differential regulation.     

Expression of vascular permeability factor/vascular endothelial growth factor by human granulosa and theca lutein cells. Role in corpus luteum development.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a cytokine that is overexpressed in many tumors, in healing wounds, and in rheumatoid arthritis. VPF/VEGF is thought to induce angiogenesis and accompanying connective tissue stroma in two ways: 1), by increasing microvascular permeability, thereby modifying the extracellular matrix and 2), as an endothelial cell mitogen. VPF/VEGF has been reported in animal corpora lutea and we investigated the possibility that it might be present in human ovaries and have a role in corpus luteum formation. We here report that VPF/VEGF mRNA and protein are expressed by human ovarian granulosa and theca cells late in follicle development and, subsequent to ovulation, by granulosa and theca lutein cells. Therefore, VPF/VEGF is ideally positioned to provoke the increased permeability of thecal blood vessels that occurs shortly before ovulation. VPF/VEGF likely also contributes to the angiogenesis and connective tissue stroma generation that accompany corpus luteum/corpus albicans formation. Finally, VPF/VEGF was overexpressed in the hyperthecotic ovarian stroma of Stein-Leventhal syndrome in which it may also have a pathophysiological role.     

Mutations of the GREAT gene cause cryptorchidism

In humans, failure of testicular descent (cryptorchidism) is one of the most frequent congenital malformations, affecting 1-3% of newborn boys. The clinical consequences of this abnormality are infertility in adulthood and a significantly increased risk of testicular malignancy. Recently, we described a mouse transgene insertional mutation, crsp, causing high intraabdominal cryptorchidism in homozygous males. A candidate gene Great (G-protein-coupled receptor affecting testis descent), was identified within the transgene integration site. Great encodes a seven-transmembrane receptor with a close similarity to the glycoprotein hormone receptors. The Great gene is highly expressed in the gubernaculum, the ligament that controls testicular movement during development, and therefore may be responsible for mediating hormonal signals that affect testicular descent. Here we show that genetic targeting of the Great gene in mice causes infertile bilateral intraabdominal cryptorchidism. The mutant gubernaculae fail to differentiate, indicating that the Great gene controls their development. Mutation screening of the human GREAT gene was performed using DHPLC analysis of the genomic DNA from 60 cryptorchid patients. Nucleotide variations in GREAT cDNA were found in both the patient and the control populations. A unique missense mutation (T222P) in the ectodomain of the GREAT receptor was identified in one of the patients. This mutant receptor fails to respond to ligand stimulation, implicating the GREAT gene in the etiology in some cases of cryptorchidism in humans.