Academic literature on the topic 'Kd 3310'

The chemical composition of shaddock mainly includes polyphenols, proteins and polysaccharides. However, polysaccharides from shaddock materials have received much less consideration than polyphenols (Fellers et al., 1990). Herein the chemical compositions, ?-glucosidase and ?-amylase inhibitory activities of crude polysaccharides from the endodermis of shaddock were investigated. The exopolysaccharides (EPS) exhibited a broad and intense peak at 3300-3400 cm-1 that characterized the absorption of the hydroxyl group, and one weak C-H band at around 2941.3 cm-1 in the IR spectrum. The content of neutral sugars in EPS was determined as 37.16%. The content of acidic sugar in EPS was determined as 33.71%. EPS exhibited the highest content of neutral sugar. The content of proteins in EPS was 5.75%. The content of polyphenols was 6.52%. The EPS mainly consisted of four types of polysaccharides with molecular weights of 110 kD, 68 kD, 31 kD and 12 kD. The crude EPS showed significantly higher inhibitory effects on ?-glucosidase and ?-amylase (inhibition to 74.12% and 86.59%, respectively).

Rhyolites and basite rocks are present in the Archaean greenstone belts of the Kursk Domain (KD) of the East Sarmatia. The rhyolite age is 3122 ± 9 Ma (zircons, SIMS). A positive εNd (3122) = + 0.9 for rhyolites and their Sm-Nd model age ТNd (DM) = 3300 Ma as well as the age of the inherited zircon (3250 Ma) testifies to the participation of the more ancient crust component in the formation of rhyolite magmas. In geochemistry, rhyolites are very close to the TTG of the KD with an age 2.96-3.03 Ga. In the Middle Dnieper granite - greenstone area there are rhyolites and dacites with an age of 3.12 Ga with εNd (T) = + 0.6 - (+1.2) and very close geochemical characteristics. Thus, the hypothesis of a common geological history of the eastern part of Ukrainian Shield and KD in Mesoarchean is confirmed.

In order to understand the sorption behavior of 137Cs and 90Sr into soil sample from Rembang and Subang, it is important to estimate the effect of contact time, ionic strength and concentration of metal ion in the solution. For this reason, the interaction of 137Cs and 90Sr with soil sample has been examined. The study performed at trace concentration (~10-8 M) of CsCl and SrCl2, and batch method was used. NaCl has been selected as a representative of the ionic strength with 0.1; 0.5 and 1.0 M concentrations. Concentration of 10-8~10-4 M CsCl and SrCl2 were used for study the effect of Cs and Sr concentrations in solution. Apparent distribution coefficient was used to predict the sorption behavior. The sorption equilibrium of 137Cs and 90Sr into soil was attained after 5 days contacted with Kd value around 3300-4200 mL/g, where Kd was defined as the ratio of number of radionuclide activity absorbed in solid phase per-unit mass to the number of radionuclide activity remains is solution per-unit volume. Presence of NaCl as background salt in the solution affected Kd values due to competition among metal ions into soil samples. Increase of Cs or Sr concentration in solution made Kd value decreased drastically. This information is expected could provide an important input for the planning and design of radioactive waste disposal system in Java Island in the future.

The human asialoglycoprotein receptor subunit H2a is cotranslationally inserted into the ER membrane. When expressed together with subunit H1 in mouse fibroblasts part forms a hetero-oligomer that is transported to the cell surface, but when expressed alone it is all rapidly degraded. Degradation is insensitive to lysosomotropic agents and the undegraded precursor is last detected in the ER region of the cell. Small amounts of an intermediate 35-kD degradation product can be detected (Amara, J. F., G. Lederkremer, and H. F. Lodish. 1989. J. Cell Biol. 109:3315). We show here that the oligosaccharides on both precursor H2a and the 35-kD fragment are Man6-9GlcNAc2, structures typically found in pre-Golgi compartments. Subcellular fractionation shows that the intermediate degradation product does not cofractionate with the lysosomal enzyme beta-galactosidase, but is found in a part of the ER that contains ribosomes. Thus the intermediate degradation product is localized in the ER, indicating that the initial degradation event does take place in the ER. All degradation of H2a, including the initial endoproteolytic cleavage generating the 35-kD intermediate, is blocked by the protease inhibitors N-tosyl-L-lysine chloromethyl ketone and N-tosyl-L-phenylalanine chloromethyl ketone. These drugs do not inhibit ER-to-Golgi transport of H1. Depleting the cells of ATP or inhibiting protein synthesis allows the initial endoproteolytic cleavage to occur, but blocks further degradation of the 35-kD intermediate; thus we can convert all cellular H2 into the 35-kD intermediate. Approximately 50% of H2b, a splicing variant differing from H2a by a five amino acid deletion, can be transported to the cell surface, and the rest appears to be degraded by the same pathway as H2a, both when expressed alone in fibroblasts and together with H1 in HepG2 cells. Addition of N-tosyl-L-lysine chloromethyl ketone or N-tosyl-L-phenylalanine chloromethyl ketone blocks degradation of the approximately 50% that is not transported, but does not affect the fraction of H2b that moves to the Golgi region. Thus, a protein destined for degradation will not be transported to the Golgi region if degradation is inhibited.

Abstract KRAS G12D mutations are activating oncogenic events that occur in approximately 35%, 13%, and 4% of pancreatic, colorectal, and non-small cell lung cancers, respectively, and less commonly in other cancers. We previously demonstrated that LY3962673 is a highly potent inhibitor of KRAS G12D and is selective against wild-type (WT) KRAS in mutant -cell lines and -in vivo models. Here, we describe the mechanism by which LY3962673 inhibits KRAS G12D and report a more comprehensive evaluation of LY3962673 activity across a panel of genetically and histologically diverse cancer cell lines, as well as in multiple patient-derived xenograft (PDX) models. LY3962673 is a non-covalent KRAS G12D inhibitor with high affinity binding to KRAS G12D-GDP (Kd 0.071 nM) compared to KRAS G12D-GTPγS (Kd 26.7 nM). In a panel of cancer cell lines with KRAS G12D mutations, non-G12D mutations or KRAS WT, LY3962673 selectively suppressed MAPK signaling and inhibited the growth of KRAS G12D mutant cancer cells while sparing KRAS WT and non-G12D mutant cells. Sensitivity to LY3962673 varied among the KRAS G12D-mutant cells tested, suggesting that not all cell lines share the same dependence on KRAS G12D for their growth and survival. Furthermore, in multiple KRAS G12D-mutant PDX models representing diverse tumor types, LY3962673 demonstrated anti-tumor activities, ranging from tumor growth inhibition to robust tumor regression. LY3962673 also showed enhanced efficacy when combined with other anti-cancer agents. Taken together, the findings underscore the potential of LY3962673 as a monotherapy or in combination with other anti-cancer agents, as a promising oral therapeutic option for a range of cancer types with KRAS G12D mutations. Citation Format: Xueqian Gong, Hong Gao, Mark H. Bender, Wenyu Ming, Youyan Zhang, Trent R. Stewart, Chun Ping Yu, Wei Guo Xu, Aurthur Xintian You, Wen Ting Bian, Binghui Li, Tao Wang, Huimin Bian, Manuj Tandon, Andrew Capen, Rachel N. Cavitt, Bryan D. Anderson, Wayne Bocchinfuso, Anke Klippel, Chandrasekar Iyer. LY3962673, an oral, highly potent, mutant-selective, and non-covalent KRAS G12D inhibitor demonstrates robust anti-tumor activity in KRAS G12D models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3316.

Introduction: Achievement of minimal residual disease negative (MRD-) status in multiple myeloma (MM) is associated with improved progression-free survival (PFS) and overall survival (OS). Isatuximab (Isa) is an approved anti-CD38 IgG kappa monoclonal antibody. We analyzed the depth of response including MRD-, long-term outcomes, and kinetics of tumor response in the IKEMA study. Measurement by mass spectrometry of serum M-protein was also performed to overcome the interference with Isa in standard immunofixation assay. Methods: IKEMA was a randomized, open-label, multicenter Phase 3 study that investigated Isa plus carfilzomib and dexamethasone (Isa-Kd) vs Kd in patients (pts) with relapsed MM who received 1-3 lines of therapy (NCT03275285). The primary endpoint of PFS and secondary endpoints of overall response rate (ORR), very good partial response or better (≥VGPR) and complete response (CR) rate were determined by an Independent Response Committee based on central data for M-protein, central imaging review and local bone marrow for plasma cell infiltration according to IMWG criteria.MRD was assessed in bone marrow aspirates from pts who achieved ≥VGPR by next generation sequencing at 10-5 sensitivity level. Mass spectrometry analysis was performed to measure serum M-protein without Isa interference. Hazard ratios and corresponding confidences interval were estimated using Cox proportional hazards model. Secondary endpoints were compared between treatment arms using Cochran Mantel Haenszel test. Per ITT, all randomized pts not reaching MRD- or without MRD assessment were analyzed as MRD+. Results: 302 pts (179 Isa-Kd, 123 Kd) were randomized. At a median follow-up of 20.7 months deeper responses were observed in Isa-Kd vs Kd with ≥VGPR 72.6% vs 56.1% (nominal p=0.011) and ≥CR 39.7% vs 27.6%, respectively. MRD- occurred in 53/179 (30%) pts in the Isa-Kd arm vs 16/123 (13%) in the Kd arm (nominal p=0.0004) with 20.1% (36/179 pts Isa-Kd) vs 10.6% (13/123 pts Kd) reaching CR and MRD-. PFS by MRD status is shown in the Figure, HR favors Isa-Kd vs Kd in both MRD- pts (HR 0.578, 95%CI: 0.052-6.405) and MRD+ pts (HR 0.670, 95% CI: 0.452-0.993). MRD- pts had a longer PFS than MRD+ pts. Within Isa-Kd, MRD-negative status could be obtained in pts with renal impairment (26.5% MRD- vs 25.9% MRD+ with eGFR <60mL/min/1.73 m2); with ISS stage III at diagnosis (32.1% MRD- vs 27.8% MRD+); with t(4;14) [13.2% MRD- vs 11.9% MRD+], with gain(1q21) [45.3% MRD- vs 40.5% MRD+]; in heavily pretreated ≥3 prior lines (22.6% MRD- vs 19.0% MRD+) or refractory to lenalidomide in last regimen (18.9% MRD- vs 20.6% MRD+). Within Isa-Kd, MRD-negative status was reached less frequently in pts refractory to a proteasome inhibitor (PI) [18.9% MRD-vs 36.5% MRD+) or with del(17p), [3.8% MRD- vs 12.7% MRD+]. Interference of Isa with M protein was explored: samples from 27 pts with near-CR (only serum immunofixation (IF) positive IgG kappa) or potential CR (serum remaining M-protein ≤ 0.5 g/dL with IF positive IgG kappa) in the Isa-Kd arm were tested by mass spectrometry. Among them, 11 near CR or potential CR pts had documented <5% plasma cells in bone marrow and were mass spectrometry negative (residual myeloma M-protein level below LOQ of central lab immunofixation). In addition, 7/11 were also MRD-. These results support that both current CR rate and MRD- CR rate are underestimated (potential adjusted CR rate of 45.8%; potential adjusted MRD- CR rate 24%). Responses occurred quickly in both arms. The median time (Isa-Kd vs Kd) in responders to: first response was 32.0 (28-259) days vs 33.0 (27-251) days; best response 120.0 (29-568) days vs 104.5 (29-507) days; first CR 184.0 (30-568) days vs 229.5 (58-507) days; first ≥VGPR 88.0 (28-432) vs 90.0 (29-491) days, respectively. In addition to increased depth of response, quality of life as measured by QLQ-C30 Global Health Status scores was maintained with Isa-Kd per descriptive analyses. Conclusions: There was a clinically meaningful improvement in depth of response in Isa-Kd vs Kd. CR rate in Isa-Kd of 39.7% was underestimated due to interference. Mass spectrometry results suggest that the potential adjusted CR rate could be reached for 45.8% of pts with 1 to 3 prior lines treated in Isa-Kd. More pts in Isa-Kd vs Kd reached MRD negativity (30% vs 13%) and at least twice as many reached CR MRD- (20.1% vs 10.6%; adjusted 24% vs 10.6%). Reaching MRD negativity was associated with longer PFS in both arms. Disclosures Martin: Legend Biotech: Consultancy; Sanofi, Amgen, Seattle Genetics, JNJ - Janssen: Research Funding. Mikhael:Amgen, Celgene, GSK, Janssen, Karyopharm, Sanofi, Takeda: Honoraria. Hajek:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Oncopeptides: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding. Kim:Amgen, BMS, Janssen, Sanofi, Takeda: Consultancy, Honoraria, Research Funding. Suzuki:Takeda, Celgene, ONO, Amgen, Novartis, Sanofi, Bristol-Myers Squibb, AbbVie and Janssen: Honoraria; Bristol-Myers Squibb, Celgene and Amgen: Research Funding; Takeda, Amgen, Janssen and Celgene: Consultancy. Hulin:Celgene/Bristol-Myers Squibb, Janssen, GlaxoSmithKline, and Takeda: Honoraria. Garg:Janssen, Takeda, Celgene, Novartis, Sanofi: Honoraria. Quach:GlaxoSmithKline, Karyopharm, Amgen, Celgene, Janssen Cilag: Consultancy; GlaxoSmithKline, Karyopharm, Amgen, Celgene, Janssen Cilag: Honoraria; Amgen, Celgene, karyopharm, GSK, Janssen Cilag, Sanofi.: Membership on an entity's Board of Directors or advisory committees; Amgen, sanofi, celgene, Karyopharm, GSK: Research Funding. Risse:Sanofi: Current Employment. Asset:Sanofi: Current Employment. Macé:Sanofi: Current Employment. van de Velde:Sanofi: Current Employment, Current equity holder in publicly-traded company. Moreau:Janssen: Consultancy, Honoraria; Novartis: Honoraria; Takeda: Honoraria; Abbvie: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. OffLabel Disclosure: Isatuximab, a monoclonal CD38 antibody, is approved in combination with pomalidomide and dexamethasone in the United States, the European Union, Canada, Australia, Switzerland, and Japan for the treatment of adult patients with relapsed/refractory multiple myeloma who have received at least two prior therapies, including lenalidomide and a proteasome inhibitor.

Introduction: Isatuximab (Isa) is an immunoglobulin (Ig)G1 monoclonal antibody targeting a CD38 transmembrane glycoprotein in multiple myeloma (MM). Isa is approved for use in multiple countries to treat adults with relapsed/refractory MM (RRMM) when given in combination with either pomalidomide-dexamethasone (Pd) or carfilzomib-dexamethasone (Kd). Hypogammaglobulinemia (HGG; polyclonal IgG <4 g/L) and the associated infection risk is a potential complication for patients (pts) with MM treated with immunotherapies. High infection and HGG rates have been reported during treatment (tx) with antimyeloma therapies, and in particular, recently, with anti-B cell maturation antigen-directed therapies. 1,2 Here, we explore the impact of Isa monotherapy on Ig levels, and of approved Isa combinations (Isa-Pd and Isa-Kd) on Ig levels and the incidence, severity, and clinical significance of HGG, including the incidence of infections depending on concomitance of HGG or not, in pts with RRMM. Methods: Pts with RRMM with central lab Ig data from 3 monotherapy studies were pooled (across dose levels, most >10 mg/kg) to retrospectively explore Ig levels throughout tx with Isa monotherapy until disease progression, intolerable toxicity, or withdrawal.We performed a side-by-side analysis of pts from the Phase 3 ICARIA-MM (Isa-Pd vs Pd) and IKEMA (Isa-Kd vs Kd) studies with/without HGG at baseline and concomitance of HGG with infections. Pts were analyzed overall and according to the heavy chain of the monoclonal protein at study entry, where IgG consisted of monoclonal IgG (M-spike in g/dL *1000) subtracted from total quantitative IgG (mg/dL) to obtain the true polyclonal IgG level, and non-IgG consisted of other Ig or light chain subtypes. Recovery was defined as >6 g/L (complete) or ≥4 g/L (partial) after having <4 g/L at any time. Results: Isa monotherapy analyses revealed a fast decrease in non-involved IgA levels after 1 month, followed by stabilization, which has also been reported with daratumumab monotherapy. Isa monotherapy did not induce decreased IgG levels. In ICARIA-MM, more pts with non-IgG vs IgG MM at study entry had HGG during tx (54.0% vs 14.4%, Isa-Pd; 39.6% vs 12.7%, Pd), whereas incidences were similar in IKEMA (11.7% vs 8.9%, Isa-Kd; 15.5% vs 11.4%, Kd). A smaller proportion of pts with HGG vs without HGG at any time during the study had definitive tx discontinuation in both ICARIA-MM and IKEMA; discontinuations related to adverse events (AEs) were less frequent in pts with HGG vs without HGG (Table). Of these AEs, infections led to discontinuation in 33.3% to 46.2% of patients with HGG at any time, regardless of tx (Table). In ICARIA-MM, 70.9% (Isa-Pd) and 55.5% (Pd) of pts without HGG at baseline developed HGG post-baseline; in IKEMA, 90.6% (Isa-Kd) and 82.0% (Kd) developed HGG post-baseline. Among pts who received Isa-Pd, those who had IgG MM at study entry experienced a larger proportion of recovery (9.1%, complete; 26.0%, partial) than those with non-IgG MM (6.8%, complete; 15.9%, partial); similar results were seen with Pd in pts with (21.9%, complete; 40.6%, partial) and without IgG MM (4.8%, complete; 19.0%, partial). Pts who had IgG MM at study entry and received Isa-Kd experienced a larger proportion of recovery (17.0%, complete; 33.0%, partial) than those with non-IgG MM (3.8%, complete; 7.5%, partial); similar results were seen with Kd in pts with (14.7%, complete; 25.3%, partial) and without IgG MM (3.1%, complete; 3.1%, partial). Infections of any grade concomitant with HGG were reported in 28.2% of pts in ICARIA-MM and 59.2% in IKEMA, with a larger proportion of pts receiving Isa combinations reporting infections of any grade and Grade ≥3 (Table). Respiratory infections of any grade concomitant with HGG were more frequent in pts receiving Isa-Pd vs Pd and Isa-Kd vs Kd (Table). There were low proportions of pts with Grade ≥3 respiratory infections concomitant with HGG regardless of tx arm (Table). Conclusions: Monotherapy with Isa had little impact on Ig levels. Among pts treated with Isa monotherapy, there is an early drop in non-involved IgA levels, followed by stabilization. In ICARIA-MM and IKEMA, HGG did not contribute to increased Grade ≥3 infections. Isa added to backbone tx is safe with a low rate of Grade ≥3 infections despite the decrease in Ig levels. Funding: Sanofi. References: 1. Lancman G, et al. Blood. 2022;140:10073-4. 2. Rodriguez-Otero P, et al. J Clin Oncol. 2023;41:8020.

Chemical modification experiments with tetranitromethane (TNM) have been used to investigate the role of tyrosine residues in the formation of the complex between PpL (the single Ig-binding domain of protein L, isolated from P. magnus strain 3316) and the kappa light chain (κ-chain). Reaction of PpL with TNM causes the modification of 1.9 equiv. of tyrosine (Tyr51 and Tyr53) and results in an approx. 140-fold decrease in affinity for human IgG. Similar experiments with mutated PpL proteins suggest that nitration predominantly inactivates the protein by modification of Tyr53. Reduction of the nitrotyrosine groups to aminotyrosine by incubation with sodium hydrosulphite does not restore high affinity for IgG. Modification of κ-chain by TNM resulted in the nitration of 3.1±0.09 tyrosine residues. When the PpLŐκ-chain complex was incubated with TNM, 4.1±0.04 tyrosine residues were nitrated, indicating that one tyrosine residue previously modified by the reagent was protected from TNM when the proteins are in complex with each other. The Kd for the equilibrium between PpL, human IgG and their complex has been shown by ELISA to be 112±20nM. A similar value (153±33nM) was obtained for the complex formed between IgG and the Tyr64 → Trp mutant (Y64W). However, the Kd values for the equilibria involving the PpL mutants Y53F and Y53F,Y64W were found to be 3.2±0.2 and 4.6±1µM respectively. These suggest that the phenol group of Tyr53 in PpL is important to the stability of the PpLŐκ-chain complex.

Abstract Background: KRAS mutations are the most frequently encountered driver oncogene, involved in ~25% of all human cancers [1,2]. KRASG12D is the predominant KRAS mutation isoform, detected in approximately 35% of pancreatic cancer, 13% of colorectal cancer, and 5% of NSCLC [3]. Compared to KRASG12C, targeting KRASG12D has proven to be more challenging since the target protein lacks a reactive amino acid residue for irreversible inhibitory modification by a ligand. Herein, we disclose TSN1611, a potent and selective KRASG12D inhibitor, which possesses favorable oral PK profiles and demonstrates significant in vitro and in vivo anti-tumor activity in various KRASG12D-mutant models. Method: Biochemical HTRF assay was used to measure the inhibition of TSN1611 to both GDP-bound and GTP-bound state of KRASG12D. Biophysical SPR method was used to directly measure the binding of TSN1611 to GDP-bound KRASG12D and KRASWT. Cell based activities were evaluated in a series of in vitro cell proliferation assay utilizing Ba/F3 cells engineered with KRASG12D or non-KRASG12D mutations and tumor cell lines harboring KRASG12D mutation. Human cancer cell-derived xenograft models of HPAC (pancreatic) and GP2D (colorectal) were used to evaluate its in vivo antitumor effect. in vitro and in vivo PK studies were performed in mouse, rat, and dog. Systematic nonclinical safety evaluations, including safety panel screen testing, safety pharmacology studies, and repeat-dose toxicity studies were carried out to assess its preliminary toxicity profile. Results: TSN1611 inhibited both active (GTP-bound) and inactive (GDP-bound) forms of KRASG12D protein at IC50 1.23 and 1.49 nM, respectively; the KD value of its direct binding to KRASG12D protein is 1.93 pM in SPR assay. TSN1611 demonstrated potent anti-proliferation activity against several tumor cell lines harboring KRASG12D mutation, and excellent selectivity over cells of NRAS, HRAS, and other KRAS isoforms. It also showed dose-dependent anti-tumor efficacy in GP2D and HPAC models. Mechanism of action studies concluded that the antitumor effect of TSN1611 is resulted from its effective inhibition of KRAS signaling pathway. Oral bioavailability and safety profile across multiple species supported its further development. Conclusion: TSN1611 is a selective KRASG12D inhibitor. It exhibited excellent selectivity and activity both in vitro and in vivo; it demonstrated favorable physicochemical properties, oral PK profiles, and brain penetration potential; it also showed acceptable margins of safety. The preclinical data supports further development. Pending regulatory submission and review, the phase I/II study is planned to start in H1 of 2024. References: [1] Cox, A.D. et al. Nat. Rev. Drug. Discov. 2014, 13, 828. [2] Indini, A. et al. Pharmaceutics 2021, 13, 653. [3] Moore, A. R. et al. Nat. Rev. Drug. Discov. 2020, 19, 533. Citation Format: Erchang Shang, Boyu Zhong, Tony Zhang, Chunlan Dong, Shengtang Ma, Anjiang Yang, Ziyang Jia, Renjuan Zheng, Jing Li, Han Fu, Liangbao Lai. Preclinical studies of TSN1611, a potent, selective, and orally bioavailable KRASG12D inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3315.

Lipoprotein(a) [Lp(a)] is a risk factor for coronary artery disease. It is composed of lipids and apolipoprotein(a) [apo(a)] linked to apolipoprotein B (apoB) by a disulphide bond between Cys-4057 of apo(a)'s kringle 36 and possibly Cys-3734 of apoB. We call this the covalent apo(a): apoB-Lp interaction, to distinguish it from the non-covalent apo(a)/Lp(a): apoB-Lp interaction, which is probably mediated by apo(a)'s kringle 33 and residues 3304-3317 of apoB. The non-covalent interaction could be the initial interaction which brings apo(a) and apoB together prior to covalent linkage and Lp(a) formation. The non-covalent apo(a)/Lp(a)-binding site on apoB is evolutionarily more ancient than the covalent apo(a)-binding site on apoB. Both human and non-human low-density lipoproteins (LDLs) bind non-covalently to human apo(a)/Lp(a); however, only rabbit and human LDLs bind covalently to human apo(a). The non-covalent interaction between mouse LDL and human apo(a)/Lp(a) has a Kd of (1.7 +/- 1.33) x 10(-7) M (n = 3). This explains the co-localization of human apo(a) and mouse apoB in the atherosclerotic lesions of human apo(a) transgenic mice and supports our hypothesis that the non-covalent interaction is a contributing factor to apo(a) atherogenicity.

We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi. During the present grant we found that TMV production is enhanced in protoplasts and plants of local lesion responding tobacco cultivars exposed to 35oC, parallel to an almost complete suppression of the production of IVR. We also found that IVR is associated with resistance mechanisms in pepper cultivars. We succeeded to clone the IVR gene. In the first attempt we isolated a clone - "101" which had a specific insert of 372 bp (the full length gene for the IVR protein of 23 kD should be around 700 bp). However, attempts to isolate the full length gene did not give clear cut results, and we decided not to continue with this clone. The amino acid sequence of the N-terminus of IVR was determined and an antiserum was prepared against a synthetic peptide representing amino acids residues 1-20 of IVR. Using this antiserum as well as our polyclonal antiserum to IVR a new clone NC-330 was isolated using lamba-ZAP library. This NC-330 clone has an insert of about 1 kB with an open reading frame of 596 bp. This clone had 86.6% homology with the first 15 amino acids of the N-terminal part of IVR and 61.6% homology with the first 23 amino acids of IVR. In the QIA expression system and western blotting of the expressed protein, a clear band of about 21 kD was obtained with IVR antiserum. This clone was used for transformation of Samsun tobacco plants and we have presently plantlets which were rooted on medium containing kanamycin. Hybridization with this clone was also obtained with RNA from induced resistant tissue of Samsun NN but not with RNA from healthy control tissue of Samsun NN, or infected or healthy tissue of Samsun. This further strengthens the previous data that the NC 330 clone codes for IVR. In the U.S. it was shown that IVR is induced in plants containing the N' gene when infected with mutants of TMV that elicit the HR. This is a defined system in which the elicitor is known to be due to permutations of the coat protein which can vary in elicitor strength. The objective was to understand how IVR synthesis is induced after recognition of elicitor coat protein in the signal transduction pathway that leads to HR. We developed systems to manipulate induction of IVR by modifying the elicitor and are using these elicitor molecules to isolate the corresponding plant receptor molecules. A "far-western" procedure was developed that found a protein from N' plants that specifically bind to elicitor coat proteins. This protein is being purified and sequenced. This objective has not been completed and is still in progress. We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi.