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Biomedical subjects

K L Carter

Publications and source records attributed to K L Carter.

11 recordsLinked to original sources
PubMed2000

Controlling human polymorphonuclear leukocytes motility using microfabrication technology.

We describe a new approach for controlling cell motility on a material surface. Transparent, photosensitive polyimide materials were used to fabricate physical structures on glass; cell motility was then followed over time using optical microscopy. Arrays of pillars and holes with 2 micron square, 4-microm height (or depth) separated by 10 microm were successfully patterned using photolithography. Neutrophils attached and spread on the smooth glass surface and surfaces with pillars. In contrast, cells were rounded and did not adhere to either smooth polyimide film or films with holes. The migration of neutrophils was much faster on holes than on polyimide surface, but it was significantly slower on pillars than on glass. These results suggest that physical patterning may be an effective tool to manipulate cell migration in the design of biomaterials for tissue engineering.

Biocompatible Materials↗
PubMed1997

The UL13 protein kinase and the infected cell type are determinants of posttranslational modification of ICP0.

The herpes simplex virus infected-cell protein 0 (ICP0) acts as a promiscuous transactivator of genes introduced into eukaryotic cells by transfection or infection. The protein is highly posttranslationally modified by phosphorylation and nucleotidylylation. We have examined the electrophoretic mobility and phosphorylation of ICP0 in Vero and rabbit skin cells infected with wild-type virus or viruses from which the UL13 gene (DeltaUL13) encoding a protein kinase or the alpha22/US1.5 genes (Deltaalpha22/DeltaUS1.5) encoding putative transcriptional factors has been deleted. We report the following: (i) The accumulation of ICP0 and the electrophoretic mobility of ICP0 were dependent on the nature of the infected cell type and the presence of UL13. ICP0 encoded by wild-type virus accumulated to maximum levels earlier in infected Vero cells and its electrophoretic mobility was slower than that made in rabbit skin cells. In both Vero and rabbit skin cells infected with the DeltaUL13 virus, the prevailing ICP0 form migrated faster than that accumulating in the corresponding cells infected with wild-type virus. (ii) The alteration in electrophoretic mobility of ICP0 made in cells infected with DeltaUL13 virus was due to the absence of the UL13 protein and not to failure of posttranslational modification of Deltaalpha22/DeltaUS1.5 proteins inasmuch as the mobility of ICP0 in cells infected with Deltaalpha22/DeltaUS1.5 virus could not be differentiated from that of wild-type infected cells. (iii) ICP0 is extensively phosphorylated in infected cells even in the absence of UL13 protein. ICP0 is, however, a substrate for the UL13 kinase inasmuch as ICP0 was phosphorylated in mixtures of immune complexes of ICP0 and UL13. Complexes containing ICP0 only or infected cell lysate proteins reacting with preimmune serum from the rabbit immunized with UL13 protein failed to phosphorylate ICP0. (iv) In the absence of UL13, ICP22 is overproduced-an imbalance attributed to UL13. Thus, ICP22 regulates both the utilization of splice acceptor sites and the longevity of ICP0 mRNA (K. L. Carter and B. Roizman, 1996, Proc. Natl. Acad. Sci. USA 93, 12535-12540); UL13 is involved in the posttranslational modification of ICP0 and is required for both posttranslational processing and control of abundance of ICP22.

Animals↗
PubMed1996

Alternatively spliced mRNAs predicted to yield frame-shift proteins and stable intron 1 RNAs of the herpes simplex virus 1 regulatory gene alpha 0 accumulate in the cytoplasm of infected cells.

The infected cell protein no. 0 (ICP0), the product of the alpha 0 gene, and an important herpes simplex virus 1 regulatory protein is encoded by three exons. We report that intron 1 forms a family of four stable nonpolyadenylylated cytoplasmic RNAs sharing a common 5' end but differing in 3' ends. The 5' and 3' ends correspond to the accepted splice donor and four splice acceptor sites within the mapped intron domain. The most distant splice acceptor site yields the mRNA encoding the 775-aa protein known as ICP0. The mRNAs resulting from the use of alternative splice acceptor sites were also present in the cytoplasm of infected cells and would be predicted to encode proteins of 152 (ICP0-B), 87 (ICP0-C), and 90 (ICP0-D) amino acids, respectively. Both the stability of the alpha 0 mRNA and the utilization of at least one splice acceptor site was regulated by ICP22 and or US1.5 protein inasmuch as cells infected with a mutant from which these genes had been deleted accumulated smaller amounts of alpha 0 mRNA than would be predicted from the amounts of accumulated intron RNAs. In addition, one splice acceptor site was at best underutilized. These results indicate that both the splicing pattern and longevity of alpha 0 mRNA are regulated. These and other recent examples indicate that herpes simplex virus 1 regulates its own gene expression and that of the infected cells through control of mRNA splicing and longevity.

Alternative Splicing↗
PubMed1996

The promoter and transcriptional unit of a novel herpes simplex virus 1 alpha gene are contained in, and encode a protein in frame with, the open reading frame of the alpha 22 gene.

The herpes simplex virus type 1 genome encodes a set of genes (alpha genes) expressed in the absence of de novo viral protein synthesis. Earlier studies have shown that the product of the alpha 22 gene, a member of this set, is nucleotidylylated by casein kinase II and phosphorylated by viral protein kinases encoded by UL13 and US3. Mutants lacking the carboxyl-terminal domain starting with amino acid 200 exhibit reduced capacity to replicate in primary human cell strains or in cells of rodent derivation and also exhibit reduced expression of a subset of gamma or late genes. We report that the domain of the alpha 22 gene is transcribed by two 3'-coterminal mRNAs. The longer transcript reported encodes the 420-amino-acid alpha 22 protein, whereas the shorter transcript reported here encodes a protein containing the carboxyl-terminal 273 amino acids of the alpha 22 protein. The shorter gene is designated US1.5. The US1.5 mRNA is synthesized in cells infected and maintained in the presence of cycloheximide and under other conditions which restrict viral gene expression to alpha genes. In-frame insertion of linkers encoding 18, 21, or 22 amino acids after codon 200 or 240 of the alpha 22 protein did not affect the known functions or phenotype associated with the wild-type alpha 22 gene or its product. Earlier studies have placed the nucleotidylylated sequences in the amino-terminal portion of the protein. The results of these studies indicate that the US1.5 gene encodes the functions associated with replication in human primary or rodent cells and optimal expression of alpha 0 and gamma genes. This finding brings the number of genes known to map in the unique short region of the herpes simplex virus type 1 DNA to 14 and the total number of different genes to 78.

Amino Acid Sequence↗
PubMed1996

Characterization of the products of the U(L)43 gene of herpes simplex virus 1: potential implications for regulation of gene expression by antisense transcription.

The products, RNA or proteins, of the herpes simplex virus 1 open reading frame U(L)43 have not been previously identified. The expression of an open reading frame antisense to U(L)43, U(L)43.5 (P. L. Ward, D. E. Barker, and B. Roizman, J. Virol. 70:2684-2690, 1996), has been reported. We report the existence of a transcript corresponding to the domain of the U(L)43 open reading frame extending approximately 30 bp from the predicted TATA box to the predicted polyadenylation signal. The RNA of the U(L)43 open reading frame accumulates to higher levels in the presence of phosphonoacetic acid, an inhibitor of viral DNA synthesis, than in its absence, whereas the U(L)43.5 transcript accumulates in larger amounts in the absence of phosphonoacetic acid. The open reading frame tagged with a sequence encoding a 20-amino-acid epitope yielded a protein with an apparent Mr of 32,000, i.e., considerably lower than that predicted from the size of the open reading frame. The discovery of a pair of antisense genes expressed during productive infection raises the possibilities that additional antisense genes exist and that the antisense arrangement provides still another mechanism for regulation of gene expression.

Animals↗