. Developmental changes in passive stiffness and myofilament Ca 2ϩ sensitivity due to titin and troponin-I isoform switching are not critically triggered by birth. Am J Physiol Heart Circ Physiol 291: H496 -H506, 2006. First published May 5, 2006; doi:10.1152/ajpheart.00114.2006.-The giant protein titin, a major contributor to myocardial mechanics, is expressed in two main cardiac isoforms: stiff N2B (3.0 MDa) and more compliant N2BA (Ͼ3.2 MDa). Fetal hearts of mice, rats, and pigs express a unique N2BA isoform (ϳ3.7 MDa) but no N2B. Around birth the fetal N2BA titin is replaced by smaller-size N2BA isoforms and N2B, which predominates in adult hearts, stiffening their sarcomeres. Here we show that perinatal titin-isoform switching and corresponding passive stiffness (STp) changes do not occur in the hearts of guinea pig and sheep. In these species the shift toward "adult" proportions of N2B isoform is almost completed by midgestation. The relative contributions of titin and collagen to STp were estimated in force measurements on skinned cardiac muscle strips by selective titin proteolysis, leaving the collagen matrix unaffected. Titin-based STp contributed between 42% and 58% to total STp in late-fetal and adult sheep/guinea pigs and adult rats. However, only ϳ20% of total ST p was titin based in late-fetal rat. Titin-borne passive tension and the proportion of titin-based ST p generally scaled with the N2B isoform percentage. The titin isoform transitions were correlated to a switch in troponin-I (TnI) isoform expression. In rats, fetal slow skeletal TnI (ssTnI) was replaced by adult carciac TnI (cTnI) shortly after birth, thereby reducing the Ca 2ϩ sensitivity of force development. In contrast, guinea pig and sheep coexpressed ssTnI and cTnI in fetal hearts, and skinned fibers from guinea pig showed almost no perinatal shift in Ca 2ϩ sensitivity. We conclude that TnI-isoform and titin-isoform switching and corresponding functional changes during heart development are not initiated by birth but are genetically programmed, species-specific regulated events. heart development; connectin; elasticity; myocardium BIRTH IS A DRAMATIC EVENT in mammalian heart development. With the newborn's first breaths of air, the fetal circulation changes, and cardiac pump function is intensified to keep up with the increased power requirements of the newborn that suddenly lacks placental nurturing and oxygen supply. Heart rate, end-diastolic pressure, stroke volume, and left ventricular (LV) dimensions all increase to meet the metabolic demands of newborn life (4, 26). During perinatal development of myocardium, many sarcomere proteins alter their isoform pattern rather rapidly. Among them are the contractile proteins myosin heavy chain (MyHC) (9, 22, 32, 53) and ␣-actin (10); regulatory proteins, including troponin-I (TnI), troponin-T (TnT), tropomyosin (36,46,(51)(52)(53), and myosin light chain-1 (60); and scaffolding proteins such as myomesin (1) and titin (27,41,43, 56). However, as these studies have usually been performed ...

Folate receptor alpha (FRA) is a cell surface protein whose aberrant expression in malignant cells has resulted in its pursuit as a therapeutic target and marker for diagnosis of cancer. The development of immune-based reagents that can reproducibly detect FRA from patient tissue processed by varying methods has been difficult due to the complex post-translational structure of the protein whereby most reagents developed to date are highly structure-sensitive and have resulted in equivocal expression results across independent studies. The aim of the present study was to generate novel monoclonal antibodies (mAbs) using modified full length FRA protein as immunogen in order to develop a panel of mAbs to various, non-overlapping epitopes that may serve as diagnostic reagents able to robustly detect FRA-positive disease. Here we report the development of a panel of FRA-specific mAbs that are able to specifically detect FRA using an array of diagnostic platforms and methods. In addition, the methods used to develop these mAbs and their diverse binding properties provide additional information on the three dimensional structure of FRA in its native cell surface configuration.

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants.Papillomaviruses (PVs) are small double-stranded DNA viruses (family Papillomaviridae) that infect many different vertebrate species (12). Human papillomaviruses (HPV) are known to cause warts and have also been associated with certain cancers in humans (46). Specific high-risk types of HPV are causally associated with cervical cancer (43); this is the second most prevalent cancer in women worldwide and the most common cancer in South African women (30). Vaccination against papillomaviral disease should result in reduced disease burden, which has resulted in a worldwide effort to develop prophylactic vaccines against HPV.The efforts to develop candidate HPV vaccines have been made more difficult by the fact that the protective efficacy of vaccines cannot be evaluated in animals. In contrast, however, development of animal PV vaccines allows evaluation by immunization of the respective hosts, followed by an experimental challenge with live virus. Cottontail rabbit papillomavirus (CRPV) in rabbits provides a robust model to study viral interaction with the host and progression to cancer and for viral vaccine studies. Naive domestic rabbits can be protected from experimental challenge with live CRPV after vaccination with a nondenatured L1 and/or whole L2 protein or peptides derived from the L2 protein (2,5,8,9,24,27,28).The most successful HPV prophylactic vaccine candidates to date are based on L1 viruslike particles (VLPs) produced by recombinant baculovirus and yeast: these VLPs are almost indistinguishable from native virions in morphology and induce effectively identical immune responses (for a review see reference 30). In animal and human studies, VLP vaccines have been well tolerated and have induced high titers of neutralizing antibodies as well as protecting against papillomaviral infection and especially disease (3,22). However, these vaccines will be expensive and not affordable in developing coun...