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SummaryCardiovascular growth must balance stabilizing signals required to maintain endothelial connections and network integrity with destabilizing signals that enable individual endothelial cells to migrate and proliferate. The cerebral cavernous malformation (CCM) signaling pathway utilizes the adaptor protein CCM2 to strengthen endothelial cell junctions and stabilize vessels. Here we identify a CCM2 paralog, CCM2L, that is expressed selectively in endothelial cells during periods of active cardiovascular growth. CCM2L competitively blocks CCM2-mediated stabilizing signals biochemically, in cultured endothelial cells, and in developing mice. Loss of CCM2L reduces endocardial growth factor expression and impairs tumor growth and wound healing. Our studies identify CCM2L as a molecular mechanism by which endothelial cells coordinately regulate vessel stability and growth during cardiovascular development, as well as postnatal vessel growth.

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are autosomal recessive syndromes of unknown etiology characterized by multiple, recurring subcutaneous tumors, gingival hypertrophy, joint contractures, osteolysis, and osteoporosis. Both are believed to be allelic disorders; ISH is distinguished from JHF by its more severe phenotype, which includes hyaline deposits in multiple organs, recurrent infections, and death within the first 2 years of life. Using the previously reported chromosome 4q21 JHF disease locus as a guide for candidate-gene identification, we identified and characterized JHF and ISH disease-causing mutations in the capillary morphogenesis factor–2 gene (CMG2). Although CMG2 encodes a protein upregulated in endothelial cells during capillary formation and was recently shown to function as an anthrax-toxin receptor, its physiologic role is unclear. Two ISH family-specific truncating mutations, E220X and the 1-bp insertion P357insC that results in translation of an out-of-frame stop codon, were generated by site-directed mutagenesis and were shown to delete the CMG-2 transmembrane and/or cytosolic domains, respectively. An ISH compound mutation, I189T, is predicted to create a novel and destabilizing internal cavity within the protein. The JHF family-specific homoallelic missense mutation G105D destabilizes a von Willebrand factor A extracellular domain alpha-helix, whereas the other mutation, L329R, occurs within the transmembrane domain of the protein. Finally, and possibly providing insight into the pathophysiology of these diseases, analysis of fibroblasts derived from patients with JHF or ISH suggests that CMG2 mutations abrogate normal cell interactions with the extracellular matrix.

• The tip diameter of the standard Goldmann applanating prism was reduced from 7.0 mm to 4.0 or 4.5 mm, but the endpoint of intraocular pressure measurement—applanation of a circle of cornea having a diameter of 3.06 mm—was not changed. The "minified" tonometers exhibited standard mechanical calibration characteristics in three standard configurations. The 4.5-mm tonometer was calibrated for cynomolgus monkey eyes in vivo by open and closed stopcock manometry. By open stopcock manometry, between pressures of 4 and 70 mm Hg, IOP (tonometer) = 1.01 IOP (manometer) – 0.72, with very little scatter or curvature. During closed stopcock manometry, between pressures of 5 and 55 mm Hg, IOP (tonometer) = 1.07 IOP (manometer) – 1.32, again with very little scatter or curvature. The tonometer elevated manometric IOP by an average of about 7.5%; the percentage pressure elevation decreased slightly as pressure increased. The 4.0-mm tonometer was calibrated against a standard tonometer in living human eyes. Over the pressure range from 0 to 75 mm Hg, IOP (minitonometer) = 0.98 IOP (standard tonometer) + 0.82, with very little scatter or curvature. Similar comparison of two standard tonometers showed a virtually identical relationship and scatter. We conclude that minified Goldmann tonometers provide accurate and reproducible measurements of IOP in cynomolgus monkey and human eyes.

Embryonic chick ciliary ganglion (CG) neurons cultured in fetal calf serum-containing medium have been previously reported to extend neurites on polyornithine (PORN) substrata precoated with a neurite-promoting factor (PNPF) from rat schwannoma-conditioned medium. On PORN substrata alone, however, no neuritic growth occurred. This was interpreted as evidence that PORN was an incompetent substratum for ciliary neuritic growth. In this study, we now find that an untreated PORN substratum allows neuritic growth in serum-free defined medium. When PNPF was added to PORN, a more rapid and extensive neuritic response occurred. After 5 hr of culture, a 60% neuritic response occurred on PNPF/PORN, whereas no neurons initiated neurites until 10-12 hr on PORN. The inhibitory effect of fetal calf serum noted above on PORN could be obtained in part by pretreating the substratum with serum for 1 hr. Maximal inhibitory effects in the PORN pretreatment were achieved after 30 min and were not further improved by treatments up to 4 hr. Bovine serum albumin was also found to inhibit neurite growth on PORN to about 60% of the inhibition obtained by an equivalent amount of serum protein. Fetal calf serum was shown to cause a 15% reduction in the percentage of neurons bearing neurites after its addition to 18-hr serum-free PORN cultures and to cause statistically significant reductions in neurite lengths measured 2 hr later.

The method of protein determination reported by Lowry et al. (1951, J. Biol. Chem. 193, 265-275) has been adapted for use with 96-well microtiter plates and an automatic microplate spectrophotometer. The spectrophotometer has been interfaced with a computer which plots the standard curve and calculates the protein content of each sample. The adapted method offers advantages over previously reported methods in that it is more rapid and uses a smaller sample volume (100 microliters) for samples containing 3-300 micrograms/ml (0.3-30 micrograms/assay) of protein. The method of Bensadoun and Weinstein (1976, Anal. Biochem. 70, 241-252) for precipitating microgram amounts of protein away from substances which interfere with the Lowry assay has also been adapted to this microplate procedure. These techniques should be particularly useful for laboratories where large numbers of samples containing a wide range of protein concentrations are assayed.

Human Amnion Membrane Matrix (HAMM) was used as a substratum for the regeneration of neuronal axons in the central nervous system. A large piece of HAMM was bound to nitrocellulose paper (NCP) as a supporting material, and cut into small strips. Aspirative lesions of the fimbria-fornix were made in adult rats leaving a cavity separating the septum from the hippocampus. In the same operation a small piece of HAMM-NCP was placed into the cavity with one end abutting the axotomized septum and the other end abutting the denervated hippocampus. At times between 2 weeks and 3 months after surgery the brains of the animals were examined histologically for 1) host response to the implant; 2) maintenance of HAMM-NCP in the originally implanted orientation; 3) growth of acetylcholinesterase (AChE)-positive fibers on the HAMM; 4) growth of the AChE-positive fibers into the denervated hippocampus; and 5) non-neuronal cells on the HAMM. The NCP remained in place over the 3-month period. In most cases the HAMM and NCP remained apposed and caused no greater reaction in the brain beyond that created in response to the aspirative lesion alone. AChE-positive fibers grew out from the septum onto the HAMM by 2 weeks, and by 8 weeks more extensive growth was observed on the HAMM. By 8-weeks, AChE fibers could be clearly seen coming off the membrane and entering the host hippocampus. By double-labelling for the basement membrane side of the HAMM (using anti-human laminin antibodies) and for cholinergic fibers (using AChE histochemistry) in the same sections, it was possible to see fibers clearly growing on the laminin-positive side of the HAMM but little or no growth on the opposing laminin-negative stromal side. The most important variables for extensive growth appeared to be the accurate placement of the implant and the amount of time following the lesions. The placement of the matrix on NCP prior to implantation in the brain made it easier to orient the membrane between the septum and the hippocampus, and seemed to assist in maintaining the membrane in that orientation. The HAMM represents a useful, regeneration-promoting grafting material for central nervous system repair studies.

We have previously reported a technique for determining the apparent molecular weight (Mr) of ciliary neuronotrophic factors (CNTFs) in crude extracts. This method involves SDS-polyacrylamide gel electrophoresis of the extract, Western blotting and culture of purified ciliary ganglion neurons on the paper containing the blotted lane. Neurons will survive only if in direct contact with the trophic factor band and the surviving neurons, when stained with a vital dye, will outline the CNTF band thereby indicating the Mr of the active polypeptide. Here we have modified this ‘blot and culture’ technique by (1) including Mr standard proteins in the same electrophoretic lane with the samples, (2) identifying the proteins by staining the nitrocellulose blot with Amido black, (3) marking the standard bands with pinholes and (4) destaining the blot prior to seeding neurons onto it. The active CNTF polypeptides can then be identified by their ability to support the 24-h survival of cultured ciliary neurons. This modified technique was used to determine the Mr of CNTF activities in several chick and rat tissue extracts of selected developmental ages and to ascertain if the two forms of CNTF are exclusive to chick and rat, embryonic and adult, or eye and nerve tissues. We report that (1) the above modifications permitted a more accurate method for Mr determination than the previous method, (2) only two apparent forms of CNTF were recognized, (3) the Mr found for each form is 25 kDa and 28 kDa, (4) both forms can be present in chick and rat tissues and from embryonic and adult sources and (5) the 28 kDa form is predominant in rat while the 25-kDa form is predominant in chicken tissues.

We have investigated the effects of two neurite promoting factors (NPFs) — laminin and the semipurified polyornithine-binding neurite promoting factor (PNPF-1) from RN 22 Schwannoma cells — on neurite regeneration from dissociated newborn and adult rat dorsal root ganglion (DRG) neurons during 24 and 48 h culture periods in the absence of exogenous neuronotrophic factors. Both laminin and PNPF, when used to pretreat the polyornithine substratum, significantly enhanced neurite recruitment from surviving newborn and adult DRG neurons as compared to an untreated polyornithine substratum. However, the responses of newborn neurons at saturating concentrations of laminin and PNPF were consistently greater (46% neurite-bearing cells at 24 h, 81% at 48 h) than those of adult neurons (14 and 45%, respectively). The responsive neurons of both newborn and adult DRG displayed extensive neuritic networks at 48 h. The ED 50 of laminin, or PNPF was 0.15–0.2 μg/ml for both newborn and adult neurons. The similarities in the responses of newborn and adult DRG neurons to NPFs validate the use of neurons from embryonic and newborn animals for the in vitro assays of NPFs that can be collected from injured and regenerating adult peripheral nervous tissues.

The epithelial cell layer of human amnion membrane can be removed while the basement membrane and stromal surfaces remain morphologically intact. Such a preparation has been used as a substratum for the in vitro culture of dissociated neurons. Embryonic motor neurons from chick ciliary ganglion attached to both surfaces but grew extensive neurites only on the basement membrane. On cross sections of rolled amnion membranes, regenerating axons of cultured neurons were guided along pathways of basement membrane that were immunoreactive with an antibody to laminin. In addition, when rolled amnion membranes were implanted into a lesion cavity between the rat septum and hippocampus, cholinergic neurons extended axons through the longitudinally oriented implant into the hippocampus. Thus, this amnion preparation can serve as a bridge to promote axonal regeneration in vivo in damaged adult brain.

Publisher Summary Human amnion membrane (HAM) has recently been found to promote axonal growth in vitro and in vivo , and fetal tissue grafts have been the most successful as bridges for regenerating CNS neurons. This chapter describes the role of human amnion membrane as a substratum for axonal elongation in vitro and in vivo . The basement membrane surface of HAM acts as a substratum for growing neurites of cultured chick ciliary ganglion motor neurons and of axotomized cholinergic medial septa1 neurons. The advantages of HAM include: (1) its availability in large quantities, (2) its easy manipulation prior to implantation, and (3) unlike fetal grafts, and the absence of ethical problems. Regardless of the exact mechanism underlying the growth observed, all animals with HAM implants show growth onto the HAM and into the previously denervated hippocampus. However, further studies are required to investigate the behavioral and electrophysiological recovery of function, and retrograde tracer studies to confirm the origin of the hippocampal fibers.

The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cell stained with anti-fibronectin and anti-laminin. Wihtin the degenerated distal nerve segment, cells characteristics of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth. Affinity-purified anti-laminin antibody failed to block chamber fluid NPF activity while completely blocking the neurite-promoting activity of laminin. These two results suggested that chamber fluid NPF activity did not consist of individual molecules of either fibronectin or laminin. The spatial and temporal distribution of insoluble fibronectin and laminin and the temporal correlation between chamber fluid NPF accumulation and neurite outgrowth support the possibility that these agents influence regenerative events including axonal elongation in vivo.

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In the discussion of suppurative labyrinthitis before this section at the Chicago meeting in June, 1908, it was my privilege and good fortune to present the first paper to appear in the medical literature of this country, reporting the recent progress made in the diagnosis and treatment of labyrinthine diseases, as taught in the Vienna schools. Since then the literature on this subject has been added to very materially, notably by Mac-Kenzie 1 of Philadelphia, Fridenberg, 2 and Richards 3 of New York, and Mr. Scott 4 of London, the last contributing some valuable new research data relating to the functions of the semicircular canals. 5 My first contribution was rather a foreword, very briefly outlining the methods of functional tests of the semicircular canal system developed by the researches of Barany, Alexander and Neumann of Vienna. The basis of these tests was founded on the fact that stimulation of

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Stretch-induced (myogenic) contraction of vascular smooth muscle (VSM) requires calcium influx through L-type calcium channels (Cav1.2). Integrins play a role in this process because alpha5 beta1 and alphaVbeta3 integrin blocking antibodies prevent myogenic constriction. Recent studies in our lab indicate that Cav1.2 current is potentiated by alpha5beta1 integrin activation and requires phosphorylation by PKA and c-Src of Cav1.2 C-terminal residues. To test whether Cav1.2 channels are mechanosensitive and the possible role of integrins in this process, patch clamp methods were used to investigate the properties of either native or heterologously expressed Cav1.2 cannels by stretch of single cells plated onto a flexible substrate. Thin silicone membranes were coated with either fibronectin (FN) or poly-l-lysine (PLL) to assess integrin-dependent and -independent responses, respectively, and stretched using two blunt micropipettes driven in equal and opposite directions by piezoelectric translators. Graded stretch to 130% of resting cell length induced graded increases in Cav1.2 current (up to 63%) in HEK 293 cells expressing the neuronal channel isoform (Cav1.2c). The increase in current was ∼2-fold greater for cells adhering to FN than for cells on PLL. On FN, 130% longitudinal stretch of primary VSM cells induced ∼50% increases in Cav1.2 current. However, the magnitude of stretch-activated Cav1.2 current was the same on FN or PLL for cells expressing a Cav1.2 construct containing two C-terminal mutations (Y2122F/S1901A) to prevent phosphorylation by PKA and c-Src, or for cells expressing a Cav1.2 construct with the C-terminus truncated. Our results suggest that the Cav1.2 channel can be potentiated by membrane stretch, with one component due to intrinsic mechanosensitivity of the channel and a second component due to signaling through an integrin-dependent process.

To the Editor.— We were very interested in the article 1 and accompanying editorial 2 concerning herbicide exposure and cancer risk that appeared in the Sept 5 issue of JAMA . It has been our informal experience that the incidence of lymphoma in our rural, agriculturally based community has been higher than expected. Our catchment population of approximately 60 000 people includes Greene and Columbia counties in upstate New York. Study.— Based on the article by Hoar et al, we retrieved all cases of non-Hodgkin's lymphoma and sarcomas from our hospital-based tumor registry for the period 1975 through 1985. The group of sarcomas was too small to be meaningful. The non-Hodgkin's lymphomas (excluding chronic lymphatic leukemia) numbered 92. Thus, the annual incidence is 15.3 new cases per 100 000 people per year for our catchment population. From the National Cancer Institute's Surveillance, Epidemiology, and End Results data, one may obtain similar

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The angular distribution of intensity of light scattered from a collimated beam incident upon a spherical air bubble in water is determined for any bubble with radius greater than a few wavelengths of the incident light. The computations are for wavelength 5893 A and n=1.3334, the relative index of refraction of water at 15°C. One external reflection, five internal reflections, and six refractions are considered. A general equation for the geometric attenuation factor is developed, with special forms for external reflection and for zero angle of incidence. The limits of accuracy of the equations for angles of scattering in the neighborhood of 0° and 180° are evaluated. The effects of diffraction and interference are assumed to be negligible. The fractions of the incident light scattered from each of the six “surfaces” (successive points of incidence of any ray on the spherical surface) are shown by accurate curves for all angles of deviation. The corresponding geometric attenutation factors are similarly shown. Final intensity values are tabulated and are plotted both with rectangular and with polar coordinates.

I wish to add my observations and experience to those of Blake, Reik, Holmes and others in perfecting the technic of the blod-clot dressing in mastoidectomy, a method, I feel, that is destined to revolutionize the after-treatment of mastoiditis. Recently in a historical review of the institutes of surgery, Sir Berkeley Moynihan1observed that "a discovery is rarely the work of one mind. It is one observation added to another that makes the supersaturated solution from which the crystal of truth at last precipitates." This observation was evoked by an allusion to the respective share or credit of Lister and Pasteur for the discovery of the antiseptic system. Moynihan added that "Lister never ceased to give credit to Pasteur for his share in the work that led finally to the development of the antiseptic method." The foregoing citation is peculiarly apropos to the theme of my text, since the

Lawrenceburg, Ky., Nov. 23, 1901. To the Editor: —The editorial and original articles in the issue ofThe Journalof November 16, discussing the enforcement of medical laws, are worthy the attention and approbation of every member of the profession in America. As a Kentuckian, I appreciate the tribute you pay to the profession of Kentucky in its effective work in ridding the state of quacks. Individually, however, on account of my recent experience in dealing with a quack, I am not so enthusiastic and feel that my efforts in protecting the profession and the public will hardly be so "strenuous" in the future as they have been in the past. Your suggestion that "what is needed to enforce the medical laws is a well-organized state society with its branches ramifying into every county and with a paid attorney of its own, to act for the profession, independent of, or

Although, since the time of Purkinje, vertigo from turning was known, and since the time of Florens, vertigo from syringing the ear with cold water has been frequently observed, it remained for Mach, Breuer and Ewald to note that vertigo is associated with nystagmus in man and with head rolling and nystagmus in animals. Since then, more exact observations by Alexander, by Kreidl, Kubo, Barany and Lange have led to important conclusions regarding the physiology of the semicircular canals, and shown how an overbalance of irritations of the semicircular canals of one side will produce nystagmus in man. Later, Stein, Krotoschiner, Panse and Mackenzie have further investigated the functions of the semicircular canals. Stein and Krotoschiner made use largely of the goniometer. Alexander and Mackenzie experimented with the gaits of patients, forward, backward and to the side; and for vertical movements by the aid of Alexander's goniometer. In this way

A method of measuring very high intensities in the focus of a highly convergent beam is described. A receiving cell with special stops is used to determine the energy distribution thermoelectrically. The sources of error with two different types of aperture are discussed. Errors due to heat radiated from the stop are minimized by using short exposures and by substituting a reflector for the stop immediately after the exposure. A suitable mount and adjusting mechanism for the cell are described.

Within the last few years, a number of workers in the field of medicine have expressed dissatisfaction with the methods commonly used in determining the amount of hemoglobin in the blood. They have pointed out not only that the different types of hemoglobinometer use different values of concentration of hemoglobin for 100 per cent on the scale, but also that the methods themselves are for the most part not sufficiently accurate for use in diagnosis. Of these two objectionable features, the latter is, of course, by far the more serious. The confusion and misunderstanding caused by dissimilar scale values can be avoided simply by the adoption of a uniform method of reporting hemoglobin, as recommended recently by Lindsay, Rice and Selinger; 1 but if the method itself is not sufficiently accurate, errors in diagnosis are unavoidable. ACCURACY OF METHODS IN COMMON USE In order to obtain some idea concerning the accuracy