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The myotonic dystrophy–related Cdc42-binding kinases MRCKα and MRCKβ contribute to the regulation of actin–myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here, we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphologic changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent antiproliferative effects with greatest activity in hematologic cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy. Significance: The development of selective small-molecule inhibitors of the Cdc42-binding MRCK kinases reveals their essential roles in cancer cell viability, migration, and invasive character. Cancer Res; 78(8); 2096–114. ©2018 AACR.

Glioblastoma (GBM) is an aggressive and incurable primary brain tumor that causes severe neurologic, cognitive, and psychologic symptoms. Symptoms are caused and exacerbated by the infiltrative properties of GBM cells, which enable them to pervade the healthy brain and disrupt normal function. Recent research has indicated that although radiotherapy (RT) remains the most effective component of multimodality therapy for patients with GBM, it can provoke a more infiltrative phenotype in GBM cells that survive treatment. Here, we demonstrate an essential role of the actin-myosin regulatory kinase myotonic dystrophy kinase-related CDC42-binding kinase (MRCK) in mediating the proinvasive effects of radiation. MRCK-mediated invasion occurred via downstream signaling to effector molecules MYPT1 and MLC2. MRCK was activated by clinically relevant doses per fraction of radiation, and this activation was concomitant with an increase in GBM cell motility and invasion. Furthermore, ablation of MRCK activity either by RNAi or by inhibition with the novel small-molecule inhibitor BDP-9066 prevented radiation-driven increases in motility both in vitro and in a clinically relevant orthotopic xenograft model of GBM. Crucially, treatment with BDP-9066 in combination with RT significantly increased survival in this model and markedly reduced infiltration of the contralateral cerebral hemisphere. Significance: An effective new strategy for the treatment of glioblastoma uses a novel, anti-invasive chemotherapeutic to prevent infiltration of the normal brain by glioblastoma cells.Cancer Res; 78(22); 6509–22. ©2018 AACR.

Queen mandibular pheromone (QMP) is a potent reproductive signal to which honey bee workers respond by suppressing their ovaries and adopting alloparental roles within the colony. This anti-ovarian effect of QMP on workers can, surprisingly, be induced in other insects, including fruit flies, in which females exposed to synthetic QMP develop smaller ovaries with fewer eggs. In this study, we use the Drosophila melanogaster model to identify the components of synthetic QMP required for the anti-ovarian effect. We found that virgin females respond strongly to 9-oxo-2-decenoic acid and 10-hydroxy-2-decenoic acid (10HDA), suggesting that the decenoic acid components of QMP are essential for the anti-ovarian response. Further, a nuclear factor of activated T-cells reporter system revealed neurones expressing the olfactory receptors Or-56a, Or-49b and Or-98a are activated by QMP in the antenna. In addition, we used olfactory receptor GAL4 drivers and a neuronal activator (a neuronal activating bacterial sodium channel) to test whether the candidate neurones are potential labelled lines for a decenoic acid response. We identified Or-49b as a potential candidate receiver of the 10HDA signal. Finally, the anti-ovarian response to synthetic QMP is not mediated by decreasing the titre of the reproductive hormones ecdysone and juvenile hormone.

(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.

One quarter of all iodinated contrast X-ray clinical imaging studies are now performed on Chronic Kidney Disease (CKD) patients. Unfortunately, the iodine contrast agent used in X-ray is often toxic to CKD patients' weak kidneys, leading to significant morbidity and mortality. Hence, we are pioneering a new medical imaging method, called Magnetic Particle Imaging (MPI), to replace X-ray and CT iodinated angiography, especially for CKD patients. MPI uses magnetic nanoparticle contrast agents that are much safer than iodine for CKD patients. MPI already offers superb contrast and extraordinary sensitivity. The iron oxide nanoparticle tracers required for MPI are also used in MRI, and some are already approved for human use, but the contrast agents are far more effective at illuminating blood vessels when used in the MPI modality. We have recently developed a systems theoretic framework for MPI called x-space MPI, which has already dramatically improved the speed and robustness of MPI image reconstruction. X-space MPI has allowed us to optimize the hardware for fi ve MPI scanners. Moreover, x-space MPI provides a powerful framework for optimizing the size and magnetic properties of the iron oxide nanoparticle tracers used in MPI. Currently MPI nanoparticles have diameters in the 10-20 nanometer range, enabling millimeter-scale resolution in small animals. X-space MPI theory predicts that larger nanoparticles could enable up to 250 micrometer resolution imaging, which would represent a major breakthrough in safe imaging for CKD patients.

Tumors and associated stroma manifest mechanical properties that promote cancer. Mechanosensation of tissue stiffness activates the Rho/ROCK pathway to increase actomyosin-mediated cellular tension to re-establish force equilibrium. To determine how actomyosin tension affects tissue homeostasis and tumor development, we expressed conditionally active ROCK2 in mouse skin. ROCK activation elevated tissue stiffness via increased collagen. beta-catenin, a key element of mechanotranscription pathways, was stabilized by ROCK activation leading to nuclear accumulation, transcriptional activation, and consequent hyperproliferation and skin thickening. Inhibiting actomyosin contractility by blocking LIMK or myosin ATPase attenuated these responses, as did FAK inhibition. Tumor number, growth, and progression were increased by ROCK activation, while ROCK blockade was inhibitory, implicating actomyosin-mediated cellular tension and consequent collagen deposition as significant tumor promoters. [KEYWORDS: Actomyosin/ physiology, Animals, Biomechanics, Cell Proliferation, Cells, Cultured, Epidermis/ pathology, Humans, Hyperplasia, Mice, Papilloma/etiology, Signal Transduction, Skin Neoplasms/ etiology, beta Catenin/ physiology, rho-Associated Kinases/analysis/genetics/physiology]

Objective Leaflet prolapse resulting from acute chordal rupture is one presentation of fibroelastic deficiency that is associated with minimal leaflet changes in the prolapsing segment. Minimizing resection and preserving leaflet tissue may be an optimal surgical strategy. We examined the importance of the leaflet preservation concept by comparing resective and nonresective surgical procedures in practice today. Methods Eight porcine mitral valves were evaluated in an in vitro heart simulator before surgical manipulation. Mitral regurgitation was created in these valves by transecting the posterior marginal chordae resulting in severe P2 prolapse. After confirmation of mitral regurgiation via regurgitant flow measurement (mL/beat), regurgitation was corrected by three repairs: neochordoplasty with polytetrafluoroethylene sutures (Gore-Tex; W. L. Gore & Associates, Inc, Flagstaff, Ariz), triangular resection, and quadrangular resection with annular compression. Postrepair valve hemodynamics were quantified under pulsatile conditions of 120 mm Hg peak transmitral pressure and 5 L/min cardiac output at 70 beats/min. Furthermore, hemodynamic, geometric, and echocardiographic indices were measured. Results Transecting the marginal chordae resulted in severe P2 prolapse and significant mitral regurgiation (19.3 ± 4.3 mL/beat). Regurgitant volume was significantly reduced after any of the three surgical approaches (quadrangular, 4.38 ± 1.6 mL/beat; triangular, 2.56 ± 1.0 mL/beat; neochordal, 2.86 ± 1.24 mL/beat). In comparison with the baseline normal valves, leaflet coaptation length and posterior leaflet mobility were significantly reduced in the quadrangular resection group, whereas they were partially restored in the triangular resection and fully preserved in the neochordoplasty group. Conclusions Although the three repair procedures are hemodynamically comparable, valve function and leaflet kinematics were significantly better after a nonresection or limited resective correction of leaflet prolapse in this experimental model of acute chordal rupture with otherwise normal leaflet geometry.

The alpha-1A-adrenergic receptor (AR) subtype is associated with cardioprotective signaling in the mouse and human heart. The rabbit is useful for cardiac disease modeling, but data on the alpha-1A in the rabbit heart are limited. Our objective was to test for expression and function of the alpha-1A in rabbit heart. By quantitative real-time reverse transcription PCR (qPCR) on mRNA from ventricular myocardium of adult male New Zealand White rabbits, the alpha-1B was 99% of total alpha-1-AR mRNA, with

For honey bee and other social insect colonies the ‘queen substance’ regulates colony reproduction rendering workers functionally sterile. The evolution of worker reproductive altruism is explained by inclusive fitness theory, but little is known of the genes involved or how they regulate the phenotypic expression of altruism. We previously showed that application of honeybee queen pheromone to virgin fruit flies suppresses fecundity. Here we exploit this finding to identify genes associated with the perception of an ovary-inhibiting social pheromone. Mutational and RNAi approaches in Drosophila reveal that the olfactory co-factor Orco together with receptors Or49b, Or56a and Or98a are potentially involved in the perception of queen pheromone and the suppression of fecundity. One of these, Or98a, is known to mediate female fly mating behaviour, and its predicted ligand is structurally similar to a methyl component of the queen pheromone. Our novel approach to finding genes associated with pheromone-induced sterility implies conserved reproductive regulation between social and pre-social orders, and further helps to identify candidate orthologues from the pheromone-responsive pathway that may regulate honeybee worker sterility.

Although edge-to-edge repair is an established adjunctive procedure, there is still debate on its long-term durability and efficacy.Fifteen porcine mitral valves were studied in a physiologic left heart simulator with a variable size annulus (dilated = 8.22 cm2, normal = 6.86 cm2, contracted = 5.5 cm2). Mitral valves were tested under steady and physiologic pulsatile flow conditions (cardiac outputs: 4 to 6 L/min), at peak transmitral pressures between 100 mm Hg and 140 mm Hg. A miniature force transducer was used to measure the Alfieri stitch force (F(A)). Mitral flow rate (MFR), transmitral pressure, effective orifice area, mitral regurgitation, and F(A) were monitored.The edge-to-edge repair led to a decrease in effective orifice area of 16.55% +/- 8.22%; further reduction in effective orifice area was attained with annular contraction. Mitral regurgitation after the edge-to-edge repair was significantly higher (p0.05) with annular dilation. In the pulsatile experiments, two peaks in F(A) were observed: one during systole (F(A) = 0.059 +/- 0.024 N) and a second during diastole (F(A) = 0.072 +/- 0.021 N). Multivariate analysis of variance analysis showed that during systole, transmitral pressure and mitral annular area (MAA) had significant effects on F(A) [F(A) = (4.40 x 10(-4)) transmitral pressure (mm Hg) + (5.0 x 10(-3)) MAA (cm2) - 0.05 (R2 = 0.80)], whereas during diastole MFR and MAA had significant effects on F(A) [F(A) = (1.03 x 10(-4)) MFR2 (L/min) - (1.60 x 10(-3)) MAA (cm2) + 0.02 (R2 = 0.90)].With annular dilation, mitral regurgitation persisted even after the edge-to-edge repair. The edge-to-edge repair does not cause clinically relevant mitral valve stenosis in a normal size mitral valve. Mitral flow rate and transmitral pressure are the main determinants of F(A) during the cardiac cycle. Increasing annular area increases F(A) during systole but decreases F(A) during diastole. Systolic F(A) may become dominant with increases in MAA or peak transmitral pressure, or both.

The transcription factor AP-1, which is composed of Fos and Jun family proteins, plays an essential role in tumor cell invasion by altering gene expression. We report here that Krp1, the AP-1 up-regulated protein that has a role in pseudopodial elongation in v-Fos-transformed rat fibroblast cells, forms a novel interaction with the nondifferentially expressed actin binding protein Lasp-1. Krp1 and Lasp-1 colocalize with actin at the tips of pseudopodia, and this localization is maintained by continued AP-1 mediated down-regulation of fibronectin that in turn suppresses integrin and Rho-ROCK signaling and allows pseudopodial protrusion and mesenchyme-like invasion. Mutation analysis of Lasp-1 demonstrates that its SH3 domain is necessary for pseudopodial extension and invasion. The results support the concept of an AP-1-regulated multigenic invasion program in which proteins encoded by differentially expressed genes direct the function, localization, and activity of proteins that are not differentially expressed to enhance the invasiveness of cells.

Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization.

Progression of tumors to invasive and metastatic forms requires that tumor cells undergo dramatic morphologic changes, a process regulated by Rho GTPases. Elevated expression of RhoA and RhoC, as well as the Rho effector proteins ROCK I and ROCK II, are commonly observed in human cancers and are often associated with more invasive and metastatic phenotypes. To examine how ROCK contributes to the progression of solid tumors, we established a conditionally activated form of ROCK II by fusing the kinase domain to the estrogen receptor hormone-binding domain (ROCK:ER). ROCK:ER-expressing colon carcinoma cells grown as tumors in immunocompromised nude mice organized into discrete clusters surrounding blood vessels. However, ROCK:ER activation resulted in the aggressive dissemination of tumor cells into the surrounding stroma, indicating that increased ROCK signaling is sufficient to promote invasion from solid tumors. In addition, tumors in which ROCK:ER was activated were more highly vascularized, indicating that ROCK contributes to tumor angiogenesis. ROCK:ER activation resulted in changes to epithelial morphology and organization that facilitated motility in vitro, likely by inducing the redistribution of proteins such as ezrin, as well as adherens junction and extracellular matrix-binding proteins. These results suggest that ROCK inhibitors would be useful antimetastatic and antiangiogenic chemotherapeutic agents in tumors associated with elevated RhoA, RhoC, ROCK I, or ROCK II expression.

Magnetic Particle Imaging (MPI) is a new tracer imaging modality that is gaining significant interest from NMR and MRI researchers. While the physics of MPI differ substantially from MRI, it employs hardware and imaging concepts that are familiar to MRI researchers, such as magnetic excitation and detection, pulse sequences, and relaxation effects. Furthermore, MPI employs the same superparamagnetic iron oxide (SPIO) contrast agents that are sometimes used for MR angiography and are often used for MRI cell tracking studies. These SPIOs are much safer for humans than iodine or gadolinium, especially for Chronic Kidney Disease (CKD) patients. The weak kidneys of CKD patients cannot safely excrete iodine or gadolinium, leading to increased morbidity and mortality after iodinated X-ray or CT angiograms, or after gadolinium-MRA studies. Iron oxides, on the other hand, are processed in the liver, and have been shown to be safe even for CKD patients. Unlike the "black blood" contrast generated by SPIOs in MRI due to increased T2* dephasing, SPIOs in MPI generate positive, "bright blood" contrast. With this ideal contrast, even prototype MPI scanners can already achieve fast, high-sensitivity, and high-contrast angiograms with millimeter-scale resolutions in phantoms and in animals. Moreover, MPI shows great potential for an exciting array of applications, including stem cell tracking in vivo, first-pass contrast studies to diagnose or stage cancer, and inflammation imaging in vivo. So far, only a handful of prototype small-animal MPI scanners have been constructed worldwide. Hence, MPI is open to great advances, especially in hardware, pulse sequence, and nanoparticle improvements, with the potential to revolutionize the biomedical imaging field.

The potential of gene therapy to treat cancer is hampered by the lack of safe and efficacious gene delivery systems able to selectively deliver therapeutic genes to tumors by intravenous administration. With the long-term aim of developing an efficacious cancer-targeted gene medicine, we demonstrated that transferrin-bearing polypropylenimine dendrimer complexed to a plasmid DNA encoding p73 led to an enhanced anti-proliferative activity in vitro, by up to 120-fold in A431 compared to the unmodified dendriplex. In vivo, the intravenous administration of this p73-encoding dendriplex resulted in a rapid and sustained inhibition of tumor growth over one month, with complete tumor suppression for 10% of A431 and B16-F10 tumors and long-term survival of the animals. The treatment was well tolerated by the animals, with no apparent signs of toxicity. These results suggest that the p73-encoding tumor-targeted polypropylenimine dendrimer should be further explored as a therapeutic strategy for cancer therapy.

The central arbiter of cell fate in response to DNA damage is p53, which regulates the expression of genes involved in cell cycle arrest, survival and apoptosis. Although many responses initiated by DNA damage have been characterized, the role of actin cytoskeleton regulators is largely unknown. We now show that RhoC and LIM kinase 2 (LIMK2) are direct p53 target genes induced by genotoxic agents. Although RhoC and LIMK2 have well-established roles in actin cytoskeleton regulation, our results indicate that activation of LIMK2 also has a pro-survival function following DNA damage. LIMK inhibition by siRNA-mediated knockdown or selective pharmacological blockade sensitized cells to radio- or chemotherapy, such that treatments that were sub-lethal when administered singly resulted in cell death when combined with LIMK inhibition. Our findings suggest that combining LIMK inhibitors with genotoxic therapies could be more efficacious than single-agent administration, and highlight a novel connection between actin cytoskeleton regulators and DNA damage-induced cell survival mechanisms. Refereed/Peer-reviewed

The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. However, the mechanisms by which ROCK signaling promotes cell cycle progression have not been thoroughly characterized. Using a conditionally activated ROCK-estrogen receptor fusion protein, we found that ROCK activation is sufficient to stimulate G1/S cell cycle progression in NIH 3T3 mouse fibroblasts. Further analysis revealed that ROCK acts via independent pathways to alter the levels of cell cycle regulatory proteins: cyclin D1 and p21(Cip1) elevation via Ras and the mitogen-activated protein kinase pathway, increased cyclin A via LIM kinase 2, and reduction of p27(Kip1) protein levels. Therefore, the influence of ROCK on cell cycle regulatory proteins occurs by multiple independent mechanisms.

The Rho-associated kinases ROCK I and ROCK II are serine/threonine kinases that play central and critical roles in regulating the actin cytoskeleton. Activation of ROCK proteins contributes positively to the phosphorylation of myosin II light chains (MLC), myosin ATPase activity, stabilization of filamentous actin, and coupling of the actin-myosin filaments to the plasma membrane, thereby leading to the increased actin-myosin force generation and contractility. We have constructed a conditionally-activated form of ROCK II (called ROCK:ER) by fusing the ROCK II kinase domain to the estrogen receptor hormone-binding domain. In this chapter, we describe the construction and characterization of this regulatable ROCK:ER fusion protein.

The proto-oncogene Ras GTPase stimulates transcription of p21Waf1/Cip1 (p21), which is repressed by the RhoA GTPase. We previously showed that Ras also elevates p21 protein levels by reducing its proteasome-mediated degradation. Therefore, we investigated whether RhoA also influenced p21 protein degradation. Pulse-chase analysis of p21 protein stability revealed that inhibitors of Rho function, which disrupt filamentous actin (F-actin), drastically slowed p21 degradation. Direct F-actin disruption mimicked Rho inhibition to stabilize p21. We found that Rho inhibition, or F-actin disruption, activated the JNK stress-activated protein kinase, and that interfering with JNK signalling, but not p38, abrogated p21 stabilization by Rho inhibition or F-actin-disrupting drugs. In addition, Ras-transformation led to increased constitutive JNK activity that contributed to the elevated p21 protein levels. These data suggest that p21 stability is influenced by a mechanism that monitors F-actin downstream of Rho, and which acts through a pathway involving activation of JNK. These results may have significant implications for therapies that target Rho-signalling pathways to induce p21-mediated cell-cycle arrest.

Veterinary staff were at high risk; standard veterinary infection-control guidelines should be followed., We determined factors associated with occupational transmission in Wisconsin during the 2003 outbreak of prairie dog–associated monkeypox virus infections. Our investigation included active contact surveillance, exposure-related interviews, and a veterinary facility cohort study. We identified 19 confirmed, 5 probable, and 3 suspected cases. Rash, headache, sweats, and fever were reported by >80% of patients. Occupationally transmitted infections occurred in 12 veterinary staff, 2 pet store employees, and 2 animal distributors. The following were associated with illness: working directly with animal care (p = 0.002), being involved in prairie dog examination, caring for an animal within 6 feet of an ill prairie dog (p = 0.03), feeding an ill prairie dog (p = 0.002), and using an antihistamine (p = 0.04). Having never handled an ill prairie dog (p = 0.004) was protective. Veterinary staff used personal protective equipment sporadically. Our findings underscore the importance of standard veterinary infection-control guidelines.

The primary structures of calf lens low molecular weight proteins, the γ-crystallins, are being studied. This report presents amino acid sequences of the cysteine containing peptides of γ-crystallin fraction IV which are compared with the primary structure of γ-crystallin fraction II. It is concluded that there are at least two similar proteins in fraction IV coded for by non-allelic structural genes.

Sauropsidans until recently were acknowledged to possess the lens-specific proteins, α and β-crystallins, while lacking γ-crystallins. The γ-crystallins have been employed as specific indicators of normal and regenerating lens fiber cell differentiation in several other organisms. It has been reported, however, that indeed, birds do possess γ-crystallins, and that furthermore this protein is not lens-fiber specific, since it could be found in annular pad and epithelium as well as fibers. Several physical characteristics of this protein cited by those suggesting its existence and at odds with those known for γ-crystallins, led the authors to further investigate the putative γ-crystallin. Pigeon “γ”-crystallin was prepared exactly according to previously published methods, and subjected to amino acid and N-terminal analyses, and peptide mapping. The fraction isolated satisfied none of the commonly-accepted criteria for γ-crystallins. Thus the pigeon “γ”-crystallin did not exhibit low values for alanine and lysine; a free N-terminal amino acid could not be detected; and peptide mapping did not reveal the distinctive peptide of γ-crystallins, the fastest moving orange spot in the electrophoresis direction. The bird lens, then, does not contain γ-crystallin; the γ-crystallins, when present, remain as reliable indicators of lens fiber differentiation.

The amino acid sequence of γ-crystallin (fraction II) from calf lens has been determined; this indicates it to be a single-chain polypeptide of 165 amino acid residues.

The amino acid sequences around the cysteine residues in the lens protein, γ-crystallin, were studied. Fraction II of the γ-crystallin from calf lens (Björk, 1964) was used. The protein was oxidized with performic acid and then hydrolysed with trypsin. Six peptides containing cysteic acid were isolated. One of the peptides contained three residues of cysteic acid and the others contained one residue of cysteic acid. We conclude that there are eight unique residues of cysteic acid in the oxidized protein. Amino acid analysis suggests that there are also eight residues of cysteic acid in the molecule, which thus contains only one polypeptide chain.

Changes in the distribution and biosynthesis of the low molecular weight proteins of the bovine lens, with age, are studied and the results correlated with developmental changes. In order to relate the findings to the results of other workers, the terminology of these proteins is discussed.