Collagen fibril surface displays a constellation of sites capable of promoting fibril assembly, stability, and hemostasis.

Fibrillar collagens form the structural basis of organs and tissues including the vasculature, bone, and tendon. They are also dynamic, organizational scaffolds that present binding and recognition sites for ligands, cells, and platelets. We interpret recently published X-ray diffraction findings and use atomic force microscopy data to illustrate the significance of new insights into the functional organization of the collagen fibril. These data indicate that collagen's most crucial functional domains localize primarily to the overlap region, comprising a constellation of sites we call the "master control region." Moreover, the collagen's most exposed aspect contains its most stable part-the C-terminal region that controls collagen assembly, cross-linking, and blood clotting. Hidden beneath the fibril surface exists a constellation of "cryptic" sequences poised to promote hemostasis and cell-collagen interactions in tissue injury and regeneration. These findings begin to address several important, and previously unresolved, questions: How functional domains are organized in the fibril, which domains are accessible, and which require proteolysis or structural trauma to become exposed? Here we speculate as to how collagen fibrillar organization impacts molecular processes relating to tissue growth, development, and repair.

Nonuniformity in Periodontal Ligament: Mechanics and Matrix Composition.

The periodontal ligament (PDL) plays a critical role in providing immediate response to abrupt high loads during mastication while also facilitating slow remodeling of the alveolar bone. The PDL exceptional functionality is permitted by the unique nonuniform structure of the tissue. Two distinct areas that are critical to PDL function were previously identified: the furcation and the dense collar. Despite their hypothesized functions in tooth movement and maintenance, these 2 regions have not yet been compared within the context of their native environment. Therefore, the objective of this study is to elucidate the extracellular matrix (ECM) structure, composition, and biomechanical function of the furcation and the collar regions while maintaining the 3-dimensional (3D) structure in the murine PDL. We identify significant difference between the collar and furcation regions in both structure and mechanical properties. Specifically, we observed unique longitudinal structures in the dense collar that correlate with type VI collagen and LOX, both of which are associated with increased type I collagen density and tissue stiffness and are therefore proposed to function as scaffolds for tooth stabilization. We also found that the collar region is stiffer than the furcation region and therefore suggest that the dense collar acts as a suspense structure of the tooth within the bone during physiological loading. The furcation region of the PDL contained more proteins associated with reduced stiffness and higher tissue remodeling, as well as a dual mechanical behavior, suggesting a critical function in loads transfer and remodeling of the alveolar bone. In summary, this work unravels the nonuniform nature of the PDL within the 3D structural context and establishes understanding of regional PDL function, which opens new avenues for future studies of remodeling, regeneration, and disease.

Pancreatic cancer heterogeneity and response to Mek inhibition.

Our increasing knowledge of the mechanisms behind the progression of pancreatic cancer (PC) has not yet translated into effective treatments. Many promising drugs have failed in the clinic, highlighting the need for better preclinical models to assess drug efficacy and characterize mechanisms of resistance. Using different experimental models, including patient-derived xenografts (PDXs), we gauged the efficacy of therapies aimed at two hallmark lesions of PCs: activation of signaling pathways by oncogenic KRAS and inactivation of tumor-suppressor genes. Although the drug targeting inactivation of tumor suppressors by DNA methylation had little effect, the inhibition of Mek, a K-Ras effector, in combination with the standard of care (chemotherapy consisting of gemcitabine/Nab-paclitaxel), reduced the growth of three out of five PC-PDXs and impaired metastasis. The two least responding PC-PDXs were composed of genetically diverse cells, which displayed sensitivities to the Mek inhibitor differing by >10-fold. Unexpectedly, our analysis of this genetic diversity unveiled different KRAS mutations. As mutation in KRAS occurs early during progression, this heterogeneity may reflect the simultaneous appearance of different malignant cellular clones or, alternatively, that cells containing two mutations of KRAS are selected during tumor evolution. In vitro and in vivo analyses indicated that the intratumoral heterogeneity, along with the selective pressure imposed by the Mek inhibitor, resulted in rapid selection of resistant cells. Together with the gemcitabine/Nab-paclitaxel backbone, Mek inhibition could be effective in treatment of PC. However, resistance because of intratumoral heterogeneity is likely to develop frequently, pointing to the necessity of identifying the factors and mechanisms of resistance to further develop this therapy.

Nucleic acid fragmentation on the millisecond timescale using a conventional X-ray rotating anode source: application to protein-DNA footprinting.

Nucleic acid fragmentation (footprinting) by *OH radicals is used often as a tool to probe nucleic acid structure and nucleic acid-protein interactions. This method has proven valuable because it provides structural information with single base pair resolution. Recent developments in the field introduced the 'synchrotron X-ray footprinting' method, which uses a high-flux X-ray source to produce single base pair fragmentation of nucleic acid in tens of milliseconds. We developed a complementary method that utilizes X-rays generated from a conventional rotating anode machine in which nucleic acid footprints can be generated by X-ray exposures as short as 100-300 ms. Our theoretical and experimental studies indicate that efficient cleavage of nucleic acids by X-rays depends upon sample preparation, energy of the X-ray source and the beam intensity. In addition, using this experimental set up, we demonstrated the feasibility of conducting X-ray footprinting to produce protein-DNA protection portraits at sub-second timescales.

Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution-state structure of the nucleocapsid protein from HIV-1.

All retroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys-1His array (CCHC = C-X2-C-X4-H-X4-C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc-binding domains. Although the arrays are capable of binding zinc in vitro, the physiological relevance of zinc coordination has not been firmly established. We have obtained zinc-edge extended X-ray absorption fine structure (EXAFS) spectra for intact retroviruses in order to determine if virus-bound zinc, which is present in quantities nearly stoichiometric with the CCHC arrays (Bess, J.W., Jr., Powell, P.J., Issaq, H.J., Schumack, L.J., Grimes, M.K., Henderson, L.E., & Arthur, L.O., 1992, J. Virol. 66, 840-847), exists in a unique coordination environment. The viral EXAFS spectra obtained are remarkably similar to the spectrum of a model CCHC zinc finger peptide with known 3Cys-1His zinc coordination structure. This finding, combined with other biochemical results, indicates that the majority of the viral zinc is coordinated to the NC CCHC arrays in mature retroviruses. Based on these findings, we have extended our NMR studies of the HIV-1 NC protein and have determined its three-dimensional solution-state structure. The CCHC arrays of HIV-1 NC exist as independently folded, noninteracting domains on a flexible polypeptide chain, with conservatively substituted aromatic residues forming hydrophobic patches on the zinc finger surfaces. These residues are essential for RNA genome recognition, and fluorescence measurements indicate that at least one residue (Trp37) participates directly in binding to nucleic acids in vitro. The NC is only the third HIV-1 protein to be structurally characterized, and the combined EXAFS, structural, and nucleic acid-binding results provide a basis for the rational design of new NC-targeted antiviral agents and vaccines for the control of AIDS.

Fourier transform time-of-flight mass spectrometry in an electrostatic ion beam trap

We report on the application of an electrostatic ion beam trap as a mass spectrometer. The instrument is analogous to an optical resonator; ions are trapped between focusing mirrors. The storage time is limited by the residual gas pressure and reaches up to several seconds, resulting in long ion flight paths. The oscillation of ion bunches between the mirrors is monitored by nondestructive image charge detection in a field-free region and mass spectra are obtained via Fourier transform. The principle of operation is demonstrated by measuring the mass spectrum of trapped Ar+ and Xe+ particles, produced by a standard electron impact ion source. Also, mass spectra of heavier PEGnNa+ and bradykinin ions from a pulsed MALDI ion source were obtained. The long ion flight path, combined with mass-independent charge detection, makes this system particularly interesting for the investigation of large molecules.

Crystallography of ribosomes: attempts at decorating the ribosomal surface.

Crystals of various ribosomal particles, diffracting best to 2.9 A resolution were grown. Crystallographic data were collected from shock frozen crystals with intense synchrotron radiation at cryo temperature. For obtaining phase information, monofunctional reagents were prepared from an undecagold and a tetrairidium cluster, by attaching to them chemically reactive handles, specific for sulfhydryl moieties. Heavy-atom derivatives were prepared by a specific and quantitative binding of the undecagold cluster to an exposed sulfhydryl prior to the crystallization. To create potential binding sites on the halophilic and thermophilic ribosomal particles, which yield our best and most interesting crystals, exposed reactive moieties were inserted, using genetic and chemical procedures. In order to choose the appropriate locations for these insertions, the surfaces of the ribosomal particles were mapped by direct chemical determination of exposed amino and sulfhydryl groups.

Rasagiline mesylate, a new MAO-B inhibitor for the treatment of Parkinson's disease: a double-blind study as adjunctive therapy to levodopa.

Rasagiline mesylate (TVP-1012) is a potent, selective, non-reversible MAO-B inhibitor, without the tyramine-potentiating effect and with neuroprotective activities. The benefit of rasagiline as monotherapy in patients with early Parkinson's disease (PD) has already been reported. To evaluate the safety, tolerability, and clinical effect of rasagiline as adjunctive therapy to levodopa, a multicenter, double-blind, randomized, placebo-controlled, parallel-group study (0.5, 1, and 2 mg/d) was conducted for 12 weeks in 70 patients with PD (mean age, 57.4 y; mean disease duration, 5.7 y; 32 patients had motor fluctuations). A beneficial clinical effect was observed in fluctuating patients treated with rasagiline (all doses), expressed as a decrease in total Unified Parkinson's Disease Rating Scale (UPDRS) score (23.0% vs 8.5% in the placebo group). The treatment effect was still evident 6 weeks after drug discontinuation (in all doses). The safety and tolerability of rasagiline were good. Adverse events were no different than those of patients taking placebo. Almost complete platelet MAO-B inhibition was obtained at all rasagiline doses. This study has demonstrated that rasagiline (up to 2 mg/day) has a good safety profile and a beneficial clinical effect in fluctuating patients with PD when given as an add-on to chronic levodopa therapy.

[Attempted suicide with verapamil]. / Ongyilkossági kísérlet Verapamillal.

A 26-year old woman took 4000 mg (100 tablets) of VerpamilR (Orion Pharmaceutica, Helsinki) with suicidal intestions. As a consequence of the poisoning cardiogenic shock, 3rd grade AV block and cardiac arrest occurred. The disturbance of stimulus formation and conduction was treated with a temporary pacemaker. Discussing the therapeutical possibilities it is stated that cardiovascular symptoms may be influenced successfully by Ca++ preparations (Calcium gluconicum, Calcium chloratum) and beta-receptor stimulants (isoproterenol, dopamine, orciprenalin) possibly also with glucagon.

[Results of measuring the blood flow in the gastric mucosa of dogs by the 99mTc-4-methylaminophenazone clearance method]. / Rezul'taty izmereniia krovotoka v slizistoi obolochke zheludka u sobak metodom klirensa 99mTc-4-metilaminofenazona.

In anesthetized dogs, a good correlation was found between the [99mTc]-4-methylaminophenazone and aminopyrine clearances measured in the same animal after administration of gastric secretory stimulants vasopressin or glucagon. A similarly good correlation was observed between the [99mTc]-4-methylaminophenazone and [14C]-aminopyrine clearance during histamine administration. The clearance values were always corrected by the actual degree of dissociation of the radioactive molecules. The results suggest that [99mTc]-4-methylaminophenazone is suitable for the study of the circulatory changes in human gastric mucosa. It has the advantages of a much lower absorbed dose of radiation for the patient and his surroundings, a simplified measurement technique and considerably reduced expenses.

[Caries, gingivitis and dental abnormalities in patients with cleft lip and palate]. / Az ajak- és szájpadhasadékos betegek cariológiai, parodontológiai és orthodontiai vizsgálata.

Oral health was studied in 31 children aged 10-12 years with cleft lip and palate and compared to 31 noncleft controls matched for sex and age. We found a difference in the prevalence and activity of caries with increase in cleft lip and palate patients. The unilateral crossbite and the congenitally missing teeth were more common in cleft palate children and the supernumerary teeth were fewer. Almost all children used fluoride tablets.

CD84 is a survival receptor for CLL cells.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+ B lymphocytes in peripheral blood, lymphoid organs and bone marrow. The main feature of the disease is accumulation of the malignant cells due to decreased apoptosis. CD84 belongs to the signaling lymphocyte activating molecule family of immunoreceptors, and has an unknown function in CLL cells. Here, we show that the expression of CD84 is significantly elevated from the early stages of the disease, and is regulated by macrophage migration inhibitory factor and its receptor, CD74. Activation of cell surface CD84 initiates a signaling cascade that enhances CLL cell survival. Both downmodulation of CD84 expression and its immune-mediated blockade induce cell death in vitro and in vivo. In addition, analysis of samples derived from an on-going clinical trial, in which human subjects were treated with humanized anti-CD74 (milatuzumab), shows a decrease in CD84 messenger RNA and protein levels in milatuzumab-treated cells. This downregulation was correlated with reduction of Bcl-2 and Mcl-1 expression. Thus, our data show that overexpression of CD84 in CLL is an important survival mechanism that appears to be an early event in the pathogenesis of the disease. These findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.

Modeling invasive breast cancer: growth factors propel progression of HER2-positive premalignant lesions.

The HER2/neu oncogene encodes a receptor-like tyrosine kinase whose overexpression in breast cancer predicts poor prognosis and resistance to conventional therapies. However, the mechanisms underlying aggressiveness of HER2 (human epidermal growth factor receptor 2)-overexpressing tumors remain incompletely understood. Because it assists epidermal growth factor (EGF) and neuregulin receptors, we overexpressed HER2 in MCF10A mammary cells and applied growth factors. HER2-overexpressing cells grown in extracellular matrix formed filled spheroids, which protruded outgrowths upon growth factor stimulation. Our transcriptome analyses imply a two-hit model for invasive growth: HER2-induced proliferation and evasion from anoikis generate filled structures, which are morphologically and transcriptionally analogous to preinvasive patients' lesions. In the second hit, EGF escalates signaling and transcriptional responses leading to invasive growth. Consistent with clinical relevance, a gene expression signature based on the HER2/EGF-activated transcriptional program can predict poorer prognosis of a subgroup of HER2-overexpressing patients. In conclusion, the integration of a three-dimensional cellular model and clinical data attributes progression of HER2-overexpressing lesions to EGF-like growth factors acting in the context of the tumor's microenvironment.