Immobilization of lipoxygenase in an alginate-silicate solgel matrix: formation of fatty acid hydroperoxides

A method for the immobilization of lipoxygenase (LOX) in an alginate-silicate gel matrix was developed. In this method, a mixture of calcium alginate beads and LOX in borate buffer are dispersed into a hexane solution of tetramethoxy-ortho-silicate (TMOS). Hydrolysis of the TMOS gives products that permeate and co-polymerize with the alginate gel to form a colloid within the beads that entraps the LOX. Optimum reaction conditions for sol-gel entrapment of LOX are at pH 9.0 in 0.2M borate buffer. The composite gel, after isolation and vacuum drying, had excellent protein retention that has good enzyme activity and stability at room temperature. The activity of the entrapped LOX was less than the activity of the free enzyme. However, the activity of the immobilized LOX can be restored by the addition of borate buffer and glycerol, or borate buffer saturated with an organic solvent. In contrast to the free enzyme in solution, which loses its activity in less than one day, sol-gel entrapped LOX retains its activity at ambient temperature for at least 25 days and can be recycled. This report demonstrates that the sol-gel entrapment method for immobilizing LOX can be useful in developing a process for the oxidation of polyunsaturated fatty acids.     

Accuracy of the functional method of hip joint center location: effects of limited motion and varied implementation.

Accurate location of the hip joint center is essential for computation of hip kinematics and kinetics as well as for determination of the moment arms of muscles crossing the hip. The functional method of hip joint center location involves fitting a pelvis-fixed sphere to the path traced by a thigh-fixed point while a subject performs hip motions; the center of this sphere is the hip joint center. The aim of the present study was to evaluate the potential accuracy of the functional method and the dependence of its accuracy on variations in its implementation and the amount of available hip motion. The motions of a mechanical linkage were studied to isolate the factors of interest, removing errors due to skin movement and the palpation of bony landmarks that are always present in human studies. It was found that reducing the range of hip motion from 30 degrees to 15 degrees did significantly increase hip joint center location errors, but that restricting motion to a single plane did not. The magnitudes of these errors, however, even in the least accurate cases, were smaller than those previously reported for either the functional method or other methods based on pelvis measurements of living subjects and cadaver specimens. Neither increasing the number of motion data observations nor analyzing the motion of a single thigh marker (rather than the centroid of multiple markers) was found to significantly increase error. The results of this study (1) imply that the limited range of motion that is often evident in subjects with hip pathology does not preclude accurate determination of the hip joint center when the functional method is used; and (2) provide guidelines for the use of the functional method in human subjects.     

Evidence for a role of dipeptidyl peptidase IV in fibronectin-mediated interactions of hepatocytes with extracellular matrix.

Dipeptidyl peptidase IV (DPP IV) is a cell surface glycoprotein which has been implicated in hepatocyte-extracellular matrix interactions [Hixson, DeLourdes, Ponce, Allison & Walborg (1984) Exp. Cell Res. 152, 402-414; Walborg, Tsuchida, Weeden, Thomas, Barrick, McEntire, Allison & Hixson (1985) Exp. Cell Res. 158, 509-518; Hanski, Huhle & Reutter (1985) Biol. Chem. Hoppe-Seyler 366, 1169-1176]. However, its proteolytic substrate(s) and/or binding protein(s) which mediate this influence have not been conclusively identified. Nitrocellulose binding assays using 125I-labelled DPP IV that was purified to homogeneity from rat hepatocytes revealed a direct interaction of DPP IV with fibronectin. Although fibronectin could mediate an indirect binding of DPP IV to collagen, no evidence was found for a direct binding of DPP IV to native or denatured Type I collagen. Fibronectin appeared to bind DPP IV at a site distinct from its exopeptidase substrate recognition site since protease inhibitors such as competitive peptide substrates and phenylmethanesulphonyl fluoride enhanced binding, possibly as a result of an altered conformation of DPP IV. To determine if fibronectin binding to DPP IV is involved in the interaction of fibronectin with the hepatocyte surface, the effect of various DPP IV inhibitors on 125I-fibronectin binding to isolated hepatocytes in suspension was examined. Kinetic studies revealed that inhibitors of DPP IV which enhanced fibronectin binding in vitro accelerated the initial binding of fibronectin to the cell surface where it was subsequently cross-linked (presumably by tissue transglutaminase) to as yet undefined components. Immunolocalization of fibronectin and DPP IV in normal rat liver sections showed that both proteins were present along the hepatocyte sinusoidal membrane. These observations, coupled with previous results showing that DPP IV is tightly bound to biomatrix isolated from rat liver (Hixson et al., 1984; Walborg et al., 1985), suggest that DPP IV binding to fibronectin may play a role in interactions of hepatocytes with extracellular matrix in vivo and possibly in matrix assembly.     

The HLA system in Italy

4,902 Italians were typed for HLA-A antigens, 4,721 for HLA-B and 1,503 for HLA-C. The samples, which were composed of unrelated, healthy individuals born in Italy, were used for estimating HLA-A, HLA-B and HLA-C gene frequencies with the maximum-likelihood method. Different Italian regions showed significant differences in the HLA alleles, providing further evidence for the genetic heterogeneity of the Italian population. HLA gene frequencies place continental Italy and Sicily in a position which is similar to that of other Mediterranean populations, whereas the genetic isolation of Sardinia is quite evident. The most significant linkage disequilibrium values found in the Italian population (except for Sardinia) were in agreement with those observed in other Caucasian populations. The difference between Northern and Southern Italy and between continental Italy and Sardinia was emphasized by the linkage disequilibrium values and by the principal-component analysis as well.     

Addition of an androgen-free epididymal protein extract increases the ability of immature hamster spermatozoa to fertilize in vivo and in vitro

The fertility of spermatozoa from the different epididymal segments of hamsters was tested by in-vivo insemination. Caput and proximal corpus spermatozoa were non-fertile; spermatozoa from the distal corpus epididymidis fertilized 13% (38/290) oocytes and those from the proximal and distal cauda epididymidis 71 and 87%, respectively. When tested by in-vitro insemination, distal corpus spermatozoa penetrated 44% of oocytes while those from the distal cauda fertilized 87% of oocytes. Spermatozoa from the distal corpus recovered in Medium BMOC fertilized 13% (28/219) of oocytes in vivo, while those mixed with an epididymal protein preparation (0.8 mg protein/ml) fertilized 24% (49/204; P less than 0.01) of oocytes. When distal corpus spermatozoa were inseminated in vivo with 0.8 mg epididymal protein preparation 34% (31/90) oocytes were fertilized and only 22% (23/103; P less than 0.05) oocytes were fertilized when the proteins were obtained from epididymides of animals castrated for 30 days. When distal corpus spermatozoa were preincubated for 5 h in medium without (control) or with protein preparation (0.8 or 1.6 mg protein/ml), a significant increase in in-vitro oocyte penetration was found (25 compared with 45%; P less than 0.05) when the protein was present at 1.6 mg/ml. These results confirm and extend previous observations suggesting a role for androgen-dependent glycoproteins secreted by the epididymis in the acquisition of fertilizing ability that occurs during sperm maturation.     

Bovine respiratory syncytial virus protects cotton rats against human respiratory syncytial virus infection.

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory infections in infancy. No vaccine against this virus has yet been protective, and antiviral drugs have been of limited utility. Using the cotton rat model of HRSV infection, we examined bovine respiratory syncytial virus (BRSV), a cause of acute respiratory disease in young cattle, as a possible vaccine candidate to protect children against HRSV infection. Cotton rats were primed intranasally with graded doses of BRSV/375 or HRSV/Long or were left unprimed. Three weeks later, they were challenged intranasally with either BRSV/375, HRSV/Long (subgroup A), or HRSV/18537 (subgroup B). At intervals postchallenge, animals were sacrificed for virus titration and histologic evaluation. Serum neutralizing antibody titers were determined at the time of viral challenge. BRSV/375 replicated to low titers in nasal tissues and lungs. Priming with 10(5) PFU of BRSV/375 effected a 500- to 1,000-fold reduction in peak nasal HRSV titer and a greater than 1,000-fold reduction in peak pulmonary HRSV titer upon challenge with HRSV/Long or HRSV/18537. In contrast to priming with HRSV, priming with BRSV did not induce substantial levels of neutralizing antibody against HRSV and was associated with a delayed onset of clearance of HRSV upon challenge. Priming with BRSV/375 caused mild nasal and pulmonary pathology and did not cause exacerbation of disease upon challenge with HRSV/Long. Our findings suggest that BRSV may be a potential vaccine against HRSV and a useful tool for studying the mechanisms of immunity to HRSV.     

Prevention of hepatitis C in partners of anti-HCV positive subjects by periodic gammaglobulin administration.

Since hepatitis C is sexually transmitted, this poses the problem of protecting the anti-HCV negative partners of anti-HCV positive subjects. Since no specific prophylaxis exists for hepatitis C, I suggest that 4 ml of normal human gammaglobulin should be given to the anti-HCV negative partners intramuscularly every two months until a vaccine against hepatitis C virus is available.     

Identification of Class I HLA T Cell Control Epitopes for West Nile Virus

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity.     

A Modified RNA-Seq Approach for Whole Genome Sequencing of RNA Viruses from Faecal and Blood Samples

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.     

Trisubstituted and tetrasubstituted pyrazolines as a novel class of cell-growth inhibitors in tumor cells with wild type p53

Derivatives with scaffolds of 1,3,5-tri-substituted pyrazoline and 1,3,4,5-tetra-substituted pyrazoline were synthesized and tested for their inhibitory effects versus the p53^+/+ HCT116 and p53^?/? H1299 human tumor cell lines. Several compounds were active against the two cell lines displaying IC₅₀ values in the low micromolar range with a clearly more pronounced effect on the p53^+/+ HCT116 cells. The compound class shows excellent developability due to the modular synthesis, allowing independent optimization of all three to four key substituents to improve the properties of the molecules.