Immune stimulation may contribute to enhanced progression of SIV induced disease in rhesus macaques

Abstract: A number of rhesus macaques experimentally infected with SIV isolates such as SIVmac251, fail to seroconvert, develop high plasma viremia and die rapidly (within 6–7 months p.i.). We hypothesized that such rapid progression is a result of a state of hyperimmune activation and concomitant immune suppression of these animals at the time of virus challenge. In efforts to test the hypothesis that immune activation leads to rapid progression of lentivirus-induced disease, adult rhesus macaques were infected with SIVmac251 and received an alternate monthly schedule of repeated immunization with allogeneic cells, keyhole limpet hemocyanin and tetanus toxoid (group I). For purposes of controls, a group of monkeys was infected with the same pool and dose of virus but were not immunized (group II) and a group was immunized with the same schedule of multiple antigens as group I but were not infected with SIV (group III). All the animals in group I (n ? 3) either failed to seroconvert or developed very low levels of SIV antibodies, had high plasma p27 defined antigenemia, and died within 8 months (2/3 died within 4 months). Of the animals in group II (n = 8), two patterns emerged as we had noted before. One subgroup (3 animals), displayed the same profile as group I (failure to fully seroconvert, high p27 levels and death by 8 months), whereas the other subgroup (5 animals) seroconverted, had low plasma p27 levels, and survived past 11 months (2/5 still alive past 22 months). All 3 animals in group III remained healthy. The data provided herein suggest that either experimental or natural (due to factors not clear at present) immune stimulation may lead to accelerated lentivirus induced disease progression most likely due to immune suppression and has implications for the understanding of the mechanisms for the rate of disease progression in human HIV-1 infection.     

Parallel reverse genetic screening in mutant human cells using transcriptomics

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.     

Biochemical components of the photosynthetic CO2 compensation point of higher plants.

Barley, Panicum milioides and Panicum maximum were exposed to ¹⁴CO₂ near their photosynthetic CO₂ compensation points and their respective ¹⁴C-products were determined. In short exposure times Panicum maximum had 100% of its ¹⁴C in malate and aspartate whereas Panicum milioides and barley had 16 and 3% of their respective ¹⁴C in C₄ organic acids. Near the respective CO₂ compensation points a linear relationship occurs in plotting the ratio of glycine, serine, and glycerate to C₄ organic acids. The ratio of ribulose 1,5-bisphosphate oxygenase to phosphoenolpyruvate carboxylase is linear with their CO₂ compensation points. The photosynthetic CO₂ compensation point apparently is controlled by the activity of enzymes producing photorespiration metabolites and the activity of phospheonolpyruvate carboxylase.     

Type X collagen contains two cleavage sites for a vertebrate collagenase

Type X collagen was cleaved at two sites by a purified human skin collagenase. Two experimental approaches were used to identify the location of the cleavage sites. First, native type X collagen was digested with the enzyme, and the rotary-shadowed products were visualized in the electron microscope. The major collagenase fragment of type X contained the epitope recognized by a monoclonal antibody (X-AC9). The antibody was used as a point of reference to locate the position of the cleavage fragment within the native molecule. Second, the digestion of radiolabeled type X collagen substrates was analyzed by gel electrophoresis. The complete cleavage of type X generated three products with 32-, 18-, and 9-kDa chains. The 32-kDa peptides were present in a triple-helical conformation and demonstrated a midpoint denaturation temperature of 43 degrees C in CD experiments. The 18-kDa peptide contained the tyrosine-rich globular domain of the molecule. The 9-kDa peptide was derived from the triple-helical end of the native molecule. Type X collagen was cleaved more rapidly by the vertebrate collagenase than was type II collagen in in vitro solution studies.     

Type IV collagen from chicken muscular tissues. Isolation and characterization of the pepsin-resistant fragments

Type IV collagen has been isolated from adult chicken gizzard after limited pepsin digestion and subsequent differential salt fractionation in acidic and neutral conditions. After denaturation, three fragments (called F1, F2, and F3) were isolated by agarose gel filtration and carboxymethylcellulose chromatography. F1 and F2 possessed apparent molecular weights of 53 000 and 50 000, respectively, and were consistently isolated in a 2:1 proportion. F3 was larger and after reduction of disulfide bonds gave rise to three fragments (called F3A, F3B, and F3C) of apparent molecular weights 68 000, 40 000, and 29 000. No alpha-chain-sized components of Type IV collagen were observed. A native fraction containing F1 and F2, but no F3, was isolated after extraction using less pepsin and an additional salt fractionation in acidic conditions. F1 and F2 in the native form were not separated by carboxymethylcellulose or diethylaminoethylcellulose chromatography performed in nondenaturating conditions or by differential salt precipitation in acidic or neutral conditions; these results suggest that F1 and F2 arise as a single native component of structure (F1)2F2. The fraction containing F1 and F2 also gave rise to a single segment long spacing crystallite pattern and to a circular dichroism spectrum which was typical for a native collagen. F1 and F2 were also isolated from chicken heart, blood vessels, and skeletal muscle, whereas from bovine aorta, using the same isolation procedures, two alpha-chain-sized components were obtained, which appeared to be similar to the two Type IV chains recently described by other groups. The data suggest that (i) pepsin fragmentation of type IV collagen from chicken tissues occurs in a different manner compared to Type IV collagen from mammalian tissues and (ii) for the chicken there must be at least two Type IV chains which are assembled into a single native molecule.     

Localisation and characterisation of homoserine dehydrogenase isolated from barley and pea leaves

Two forms of homoserine dehydrogenase exist in the leaves of both barley and pea; one has a large molecular weight and is inhibited by threonine, the other is of smaller molecular weight and insensitive to threonine but inhibited by cysteine. The subcellular localisation of these enzymes has been examined. Both plants have 60–65% of the total homoserine dehydrogenase activity present in the chloroplast and this activity is inhibited by threonine. The low molecular weight, threonine-insensitive form is present in the cytoplasm. Total homoserine dehydrogenase activity from barley leaves showed progressive desensitisation towards threonine with age in a similar manner to that previously described for maize. It was shown that the effect was due to desensitisation of the chloroplast enzyme, and not to an increase in the insensitive cytoplasm enzyme. No corresponding desensitisation to threonine was detected in pea leaves. The different forms of homoserine dehydrogenase could be separated from pea leaves by chromatography on Blue Sepharose; the threonine-sensitive enzyme passed straight through and the threonine insensitive form was bound. A similar separation of the barley leaf isoenzymes was obtained using Matrex Gel Red A affinity columns; in this case however, the threonine-sensitive isoenzyme was bound. In both plants, the threonine insensitive isoenzyme was subject to greater inhibition by cysteine than was the threonine-sensitive isoenzyme.Abbreviation HSDH homoserine dehydrogenase     

Role of muscle fibroblasts in the deposition of type-IV collagen in the basal lamina of myotubes

In cell cultures of quail, chick, or mouse skeletal muscle, both myogenic and fibrogenic cells synthesize and secrete type-IV collagen, a major structural component of the basal lamina. Type-IV collagen, together with laminin, forms characteristic patches and strands on the surface of developing myotubes, marking the onset of basement-membrane formation. The pattern for type-IV collagen and laminin is unique to these proteins and is not paralleled by other matrix proteins, such as fibronectin or type-I or -III collagen. In the present study, we used species-specific antibodies to either mouse or chick type-IV collagen to demonstrate the ability of fibroblast--derived type-IV collagen to incorporate in the basal lamina of myotubes. In combination cultures of embryonic quail skeletal myoblasts and mouse muscle fibroblasts, antibodies specific for mouse type-IV collagen revealed the deposition of type-IV collagen on the surface of quail myotubes in the pattern typical of the beginning of basement-membrane formation. Control cultures consisting of only quail muscle cells containing myoblasts and fibroblasts demonstrated no such reaction with these antibodies. Deposits of mouse type-IV collagen were also observed on the surface of quail myotubes when conditioned medium from mouse muscle fibroblasts was added to quail myoblast cultures. Similarly, in combination cultures of mouse myoblasts and chick muscle fibroblasts, chick type-IV-collagen deposits were identified on the surface of mouse myotubes. These results indicate that type-IV collagen synthesized by muscle fibroblasts may be incorporated into the basal lamina forming on the plasmalemma of myotubes, and may explain ultrastructural studies by Lipton on the contribution of fibroblasts to the formation of basement membranes in skeletal muscle.