Selective targeting and photocoagulation of ocular angiogenesis mediated by a phage-derived human antibody fragment.

Molecules that selectively target and occlude new blood vessels would be useful for diagnosis and treatment of pathologies associated with angiogenesis. We show that a phage-derived human antibody fragment (L19) with high affinity for the ED-B domain of fibronectin, a marker of angiogenesis, selectively localizes to newly formed blood vessels in a rabbit model of ocular angiogenesis. The L19 antibody, chemically coupled to a photosensitizer and irradiated with red light, mediates complete and selective occlusion of ocular neovasculature and promotes apoptosis of the corresponding endothelial cells. These results demonstrate that new ocular blood vessels can be distinguished immunochemically from preexisting ones and suggest that the targeted delivery of photosensitizers may be effective in treating angiogenesis-related pathologies.     

Identification of monoclonal antibody defined epitopes on human leukocyte antigens utilizing phage display peptide libraries

Summary Utilizing phage display peptide libraries, we have identified and mapped the antigenic determinants recognized by mouse monoclonal antibodies (mAb) on two sets of immunologically important molecules, HLA class I and class II antigens. Anti-HLA class I mAb TP25.99 recognizes a conformational and a linear determinant on distinct regions of the HLA class I α3 domain. Anti-HLA class I mAb HO-4 recognizes a conformational determinant on the α2 domain of HLA-A2 and A28 allospecificities. Anti-HLA-DR1,-DR4,-DR6,-DR8,-DR9 mAb SM/549 recognizes a conformational determinant on the β chain of HLA class II antigens. These results indicate the versatility of phage display peptide libraries to characterize antigenic determinants recognized by anti-HLA mAb.     

Multiple biological responses activated by nuclear protein kinase C.

Protein kinase C is a family of serine-threonine kinases that are physiologically activated by a number of lipid cofactors and are important transducers in many agonist-induced signaling cascades. To date, 12 different isozymes of this kinase have been identified and are believed to play distinct regulatory roles. Protein kinase C was thought to reside in the cytosol in an inactive conformation and translocate to the plasma membrane upon cell activation by different stimuli. Nevertheless, a growing body of evidence has illustrated that this family of isozymes is capable of translocating to other cellular sites, including the nucleus. Moreover, it seems that some protein kinase C isoforms are resident within the nucleus. A wealth of data is being accumulated, demonstrating that nuclear protein kinase C isoforms are involved in the regulation of several critical biological functions such as cell proliferation and differentiation, neoplastic transformation, and apoptosis. In this review, we will discuss the most significant findings concerning nuclear protein kinase C which have been published during the past 5 years.     

Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC-zeta translocation to the nucleus of NGF-treated PC12 cells.

We and others have previously demonstrated the existence of an autonomous nuclear polyphosphoinositide cycle that generates second messengers such as diacylglycerol (DAG), capable of attracting to the nucleus specific protein kinase C (PKC) isoforms (Neri et al. (1998) J. Biol. Chem. 273, 29738-29744). Recently, however, nuclei have also been shown to contain the enzymes responsible for the synthesis of the non-canonical 3-phosphorylated inositides. To clarify a possible role of this peculiar class of inositol lipids we have examined the question of whether nerve growth factor (NGF) induces PKC-zeta nuclear translocation in PC12 cells and whether this translocation is dependent on nuclear phosphatidylinositol 3-kinase (PI 3-K) activity and its product, phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P(3)]. NGF increased both the amount and the enzyme activity of immunoprecipitable PI 3-K in PC12 cell nuclei. Activation of the enzyme, but not its translocation, was blocked by PI 3-K inhibitors wortmannin and LY294002. Treatment of PC12 cells for 9 min with NGF led to an increase in the nuclear levels of PtdIns(3,4,5)P(3). Maximal translocation of PKC-zeta from the cytoplasm to the nucleus (as evaluated by immunoblotting, enzyme activity, and confocal microscopy) occurred after 12 min of exposure to NGF and was completely abrogated by either wortmannin or LY294002. In contrast, these two inhibitors did not block nuclear translocation of the conventional, DAG-sensitive, PKC-alpha. On the other hand, the specific phosphatidylinositol phospholipase C inhibitor, 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, was unable to abrogate nuclear translocation of the DAG-insensitive PKC-zeta. These data suggest that a nuclear increase in PI 3-K activity and PtdIns(3,4,5)P(3) production are necessary for the subsequent nuclear translocation of PKC-zeta. Furthermore, they point to the likelihood that PKC-zeta is a putative nuclear downstream target of PI 3-K during NGF-promoted neural differentiation.-Neri, L. M., Martelli, A. M., Borgatti, P., Colamussi, M. L., Marchisio, M., Capitani, S. Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4, 5) trisphosphate synthesis precede PKC-zeta translocation to the nucleus of NGF-treated PC12 cells.     

Frequency and patterns of ribonucleotide incorporation around autonomously replicating sequences in yeast reveal the division of labor of replicative DNA polymerases

Ribonucleoside triphosphate (rNTP) incorporation in DNA by DNA polymerases is a frequent phenomenon that results in DNA structural change and genome instability. However, it is unclear whether the rNTP incorporation into DNA follows any specific sequence patterns. We analyzed multiple datasets of ribonucleoside monophosphates (rNMPs) embedded in DNA, generated from three rNMP-sequencing techniques. These rNMP libraries were obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late-firing autonomously replicating sequences (ARSs) of the yeast genome, where leading and lagging DNA synthesis starts bidirectionally. We found the preference of rNTP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNTP incorporation changes dramatically within the first 1,000 nucleotides from ARSs, highlighting the Pol δ - Pol ϵ handoff during early leading-strand synthesis. Furthermore, the pattern of rNTP incorporation is markedly distinct between the leading and lagging strands not only in mutant but also in wild-type polymerase cells. Such specific signatures of Pol δ and Pol ϵ provide a new approach to track the labor of these polymerases.     

Broadening the reach and investigating the potential of prime editors through fully viral gene-deleted adenoviral vector delivery

Prime editing is a recent precision genome editing modality whose versatility offers the prospect for a wide range of applications, including the development of targeted genetic therapies. Yet, an outstanding bottleneck for its optimization and use concerns the difficulty in delivering large prime editing complexes into cells. Here, we demonstrate that packaging prime editing constructs in adenoviral capsids overcomes this constrain resulting in robust genome editing in both transformed and non-transformed human cells with up to 90% efficiencies. Using this cell cycle-independent delivery platform, we found a direct correlation between prime editing activity and cellular replication and disclose that the proportions between accurate prime editing events and unwanted byproducts can be influenced by the target-cell context. Hence, adenovector particles permit the efficacious delivery and testing of prime editing reagents in human cells independently of their transformation and replication statuses. The herein integrated gene delivery and gene editing technologies are expected to aid investigating the potential and limitations of prime editing in numerous experimental settings and, eventually, in ex vivo or in vivo therapeutic contexts.     

Fourier‐transform infrared spectroscopy study of dehydrated lipases from Candida antarctica B and Pseudomonas cepacia

Fourier‐transform infrared (FT‐IR) spectroscopy was employed to investigate potential lyophilization‐induced changes in the secondary structure of lipases from Candida antarctica B and Pseudomonas cepacia. The secondary structure elements were determined by curve fitting of the amide III bands of the two lipases in the lyophilized state in KBr pellets and in solution. It was found that lyophilization decreased the α‐helix and increased the β‐sheet content. However, FT‐IR analysis of crosslinked enzyme crystals of Pseudomonas cepacia lipase also indicated an increase in the β‐sheet content, which appears despite the fact that the enzyme, being in the crystallized state, should possess native conformation. This result partially questions the suitability of FT‐IR for analysis of the structure of solid proteins, at least as far as the β‐sheet content is concerned, because it is possible that the method overestimates the β‐sheets by measuring other hydrogen‐bonded nonperiodic intermolecular structures. No significant modification was observed when lipase from Pseudomonas cepacia was lyophilized in the presence of methoxypoly(ethylene glycol). © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 545–551, 1999.     

The use of biosensor technology for the engineering of antibodies and enzymes

Recently developed scientific instrumentation featuring surface plasmon resonance detection allows the detection of biomolecular interactions in real time and without chemical modification of the binding partners. These biosensors are proving invaluable tools in protein engineering, particularly in research aimed at the isolation and improvement of protein binders and catalysts from macromolecular repertoires containing billions of individual members. This article reviews the use of biosensor technology for the isolation and characterization of engineered antibodies and enzymes. Copyright © 1999 John Wiley & Sons, Ltd.     

Lamin A is part of the internal nucleoskeleton of human erythroleukemia cells

Nuclear lamins are the most abundant components of the nuclear lamina, a 10–50-nm-thick fibrous layer underlying the inner nuclear envelope membrane. Nevertheless, a number of recent investigations performed on epithelial and fibroblast cells have suggested that nuclear lamins are also present within the nucleoplasm and could be important constituents of the nucleoskeleton. We have studied the subnuclear distribution of lamins A and B1 in human erythroleukemia cells by using immunoblotting analysis and immunofluorescent staining of fractionated nuclei. In intact cells and isolated nuclei, antibodies to lamins A and B1 mainly stained the nuclear periphery, although some immunoreactivity was detected in the nuclear interior. However, when chromatin was removed by nuclease digestion and extraction with nonionic detergent or solutions of high ionic strength, a previously masked immunoreactivity for lamin A, but not for lamin B1, became evident in the internal part of the residual structures representing the nuclear matrix or scaffold. Preferential localization of lamin A to the inner part of the nucleus was also demonstrated by the presence of the majority of lamin A in the solubilized inner nuclear network subfraction. In contrast, lamin B1 was mainly recovered in the fraction corresponding to the nuclear periphery. Double labeling experiments showed that lamin A, but not lamin B1, colocalized with coiled and GATA-1 bodies. Thus, our results support the hypothesis that lamin A, but not lamin B1, may be a component of an internal nucleoskeleton in human erythroleukemia cells. J. Cell. Physiol. 178:284–295, 1999. © 1999 Wiley-Liss, Inc.