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Robert Jacobs

Publications and source records attributed to Robert Jacobs.

8 recordsLinked to original sources
PubMed2006

Sequential expression of type IV collagen networks: testis as a model and relevance to spermatogenesis.

The six alpha chains of type IV collagen are organized into three networks: alpha1/alpha2, alpha3/alpha4/alpha5, and alpha1/alpha2/alpha5/alpha6. A shift from the alpha1/alpha2 to the alpha3/alpha4/alpha5 network occurs in the developing glomerular basement membrane, but how the alpha1/alpha2/alpha5/alpha6 network fits into this sequence is less clear, because the three networks do not colocalize. Here, we studied the seminiferous tubule basement membrane of normal canine testis where all three networks do colocalize: the alpha1/alpha2 network is expressed from birth, the alpha1/alpha2/alpha5/alpha6 network by 5-6 weeks of age, and the alpha3/alpha4/alpha5 network by 2 months of age. A canine model of Alport syndrome allowed study of the absence of alpha3/alpha4/alpha5 and alpha1/alpha2/alpha5/alpha6 networks in testis. In Alport dogs, the seminiferous tubule basement membrane was thinner than in controls. Spermatogenesis began at the same time as with normal dogs; however, the number of mature sperm was significantly reduced in Alport dogs. Thus, it would appear that alpha3/alpha4/alpha5 and alpha1/alpha2/alpha5/alpha6 networks are not essential for onset of spermatogenesis, but long-term function may be compromised by the loss of one or both networks. This situation is analogous to the glomerular basement membrane in Alport syndrome. In conclusion, testis can serve as a model system to study the sequence of type IV collagen network expression.

Animals↗
PubMed2005

Type IV collagen induces podocytic features in bone marrow stromal stem cells in vitro.

Bone marrow-derived stromal stem cells (BMSC) can differentiate along a variety of mesenchymal lines, including mesangial cells. For determining whether BMSC can be induced to differentiate along podocytic lines in vitro, canine BMSC were cultured on plastic, type I collagen, and NC1 hexamers of type IV collagen from normal and Alport canine glomerular basement membrane. Results were compared with a mouse podocyte cell line. In the case of the podocyte line, differentiation occurred on all three matrices as indicated by the expression of synaptopodin and CD2-associated protein (CD2AP) and organization of myosin heavy chain IIA into a linear pattern. BMSC proliferated equally well on all matrices, but cells that were grown on type IV collagen NC1 hexamers became larger and stellate. Evidence for podocytic differentiation occurred on all three collagen matrices as indicated by the redistribution of myosin IIA to a linear pattern and expression of synaptopodin, CD2AP, and alpha-actinin. A punctate distribution of CD2AP was seen only in cells that were grown on normal and Alport glomerular basement membrane NC1 hexamers. Differentiated podocytes expressed the alpha1, alpha2, and alpha5 chains of type IV collagen but at higher levels in cells that were grown on NC1 hexamers. Similar results were obtained in BMSC for the alpha1 and alpha2 chains only. The alpha3, alpha4, and alpha6 chains were never detected in the podocyte line or BMSC. These results indicate that BMSC undergo a degree of podocytic differentiation in vitro and greater when grown on type IV collagen NC1 hexamers than type I collagen. Alport and normal NC1 hexamers seem equally permissive to BMSC growth and differentiation, suggesting that these processes are not influenced specifically by the alpha3/alpha4/alpha5 network. BMSC may be useful in the development of stem cell-based reconstitution of glomeruli that are damaged by disease and for gene therapy of genetic glomerular diseases such as Alport syndrome.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Regulation of collagen type IV genes is organ-specific: evidence from a canine model of Alport syndrome.

BACKGROUND: Despite advances in knowledge about collagen type IV at the protein level, little is known about expression of its six alpha chains. X-linked Alport syndrome provides a system to study collagen type IV gene expression within a setting of disturbed protein synthesis. Mutations in the alpha5 chain result in loss of the alpha3/alpha4/alpha5 and alpha1/alpha2/alpha5/alpha6 networks from the kidney, with progressive renal disease. METHODS: We used a canine model of Alport syndrome to measure expression of the six type IV collagen chains from 11 days to 7(1/2) months of age. We determined to what extent message levels in kidney change over time, and what correlation exists with clinical and pathologic changes in glomeruli, and the primary mutation. The latter was evaluated by examining testis, an organ normally containing the same collagen type IV networks but uninvolved by disease. RESULTS: The alpha1 to alpha6 mRNAs were expressed at all time points in normal canine kidney. By comparison to normal, in Alport dog kidney, the alpha1 and alpha2 mRNAs were up-regulated after 2 months of age, alpha3 and alpha4 mRNAs were down-regulated by 2 months of age, and the alpha5 mRNA was almost undetectable at any time. In testis, all mRNAs were expressed at comparable levels in normal and affected dogs other than the alpha5 chain, which was not expressed in affected testis. CONCLUSION: Normal expression of collagen type IV is under control mechanisms specific to each organ and to individual chains. The altered expression in canine Alport syndrome is not the direct result of the mutation, since these changes do not occur in all organs nor are they present from birth. Instead, collagen type IV expression is influenced by disease, with down-regulation of alpha3 and alpha4 chains temporally related to the onset of proteinuria, and up-regulation of alpha1 and alpha2 chains to glomerulosclerosis. This dysregulation of the alpha3 and alpha4 chains is unique to this Alport model, and suggests an unidentified mechanism linking pathology with down-regulation of expression of these two chains.

Animals↗
PubMed2004

Working Group report 4: exposure assessment for biological agents.

This Working Group was assembled to review and evaluate methods currently used to estimate work site exposures to biological agents and to present recommendations on suitable measurement strategies. The current state of exposure assessment was evaluated for environments with organic dust including agriculture, composting, sewage and waste treatment processing, peat moss harvesting and handling, cotton and textile processing, greenhouse work, and grass seed processing. Methods for measurement of microbial contaminants in indoor environments were also considered. Important methods are emerging that use quantitative PCR for assessment of microbial agents. Difficulties exist with optimization of extraction to yield contaminant-free DNA without significant DNA loss. Advances in assays for microbial agents (e.g., endotoxin and glucans) as well as allergens have increased the utility of exposure assessment for these agents. A crucial area for further development is international harmonization of methodologies to reduce interlaboratory variability and to facilitate establishment of exposure guidelines. Endotoxin exposure assessment using the Limulus amebocyte lysate method is a high priority for harmonization because of its importance as a pulmonary inflammatory agent in many occupational settings.

Air Pollution, Indoor↗
PubMed2003

Transfer of the alpha 5(IV) collagen chain gene to smooth muscle restores in vivo expression of the alpha 6(IV) collagen chain in a canine model of Alport syndrome.

X-linked Alport syndrome is a progressive renal disease caused by mutations in the COL4A5 gene, which encodes the alpha 5(IV) collagen chain. As an initial step toward gene therapy for Alport syndrome, we report on the expression of recombinant alpha 5(IV) collagen in vitro and in vivo. A full-length cDNA-encoding canine alpha 5(IV) collagen was cloned and expressed in vitro by transfection of HEK293 cells that synthesize the alpha1(IV) and alpha2(IV), but not the alpha 3(IV) to alpha 6(IV) collagen chains. By Northern blotting, an alpha 5(IV) mRNA transcript of 5.2 kb was expressed and the recombinant protein was detected by immunocytochemistry. The chain was secreted into the medium as a 190-kd monomer; no triple helical species were detected. Transfected cells synthesized an extracellular matrix containing the alpha1(IV) and alpha2(IV) chains but the recombinant alpha 5(IV) chain was not incorporated. These findings are consistent with the concept that the alpha 5(IV) chain requires one or more of the alpha 3(IV), alpha 4(IV), or alpha 6(IV) chains for triple helical assembly. In vivo studies were performed in dogs with X-linked Alport syndrome. An adenoviral vector containing the alpha 5(IV) transgene was injected into bladder smooth muscle that lacks both the alpha 5(IV) and alpha 6(IV) chains in these animals. At 5 weeks after injection, there was expression of both the alpha 5(IV) and alpha 6(IV) chains by smooth muscle cells at the injection site in a basement membrane distribution. Thus, this recombinant alpha 5(IV) chain is capable of restoring expression of a second alpha(IV) chain that requires the presence of the alpha 5(IV) chain for incorporation into collagen trimers. This vector will serve as a useful tool to further explore gene therapy for Alport syndrome.

Animals↗
PubMed2002

Second inter-laboratory study comparing endotoxin assay results from cotton dust.

Previously, a large two-part inter-laboratory round robin endotoxin assay study was completed. This first study showed that when cotton dust samples, which are practically identical, are assayed for endotoxin that the intra- laboratory results had a very small variation while intra-laboratory results of the sample had a very high variation. In the first part of the study, each laboratory followed its own in-house assay protocol; but in the second part of the study, when the extraction protocol was standardized, the inter-laboratory results showed a lower variation, which suggested that with further standardization, further reduction of differences between laboratories might be achieved in order that results between laboratories would become more comparable. The results stimulated interest in extending the study to include cotton dust with two levels of endotoxin, standardization of the extraction protocol, and using the same assay kit from the same production lot. The results of this second round robin endotoxin assay study indicate that differences between laboratories are still high, but most of the laboratories could discern the cotton dusts with the different levels of endotoxin.

Chemistry Techniques, Analytical↗