Development and evaluation of RADA-PDGF2 self-assembling peptide hydrogel for enhanced skin wound healing.

Background: Wound healing complications affect numerous patients each year, creating significant economic and medical challenges. Currently, available methods are not fully effective in the treatment of chronic or complicated wounds; thus, new methods are constantly sought. Our previous studies showed that a peptide designated as PDGF2 derived from PDGF-BB could be a promising drug candidate for wound treatment and that RADA16-I can serve as a release system for bioactive peptides in wound healing. Based on that, in this work, we designed a new self-assembling hydrogel RADA-PDGF2, connecting both peptides by a sequence specific for neutrophil elastase, and evaluated its activity in wound healing. Methods: The physicochemical properties of the designed scaffold were analyzed using transmission electron microscopy, atomic force microscopy, cryoSEM microscopies, and circular dichroism spectroscopy. The enzymatic cleavage was performed using human neutrophil elastase and monitored using high-performance liquid chromatography and MS spectroscopic techniques. The aforementioned techniques (HPLC and MS) were also used to assess the stability of the peptide in water and human plasma. The biological activity was analyzed on human skin cells using a colorimetric XTT test, collagen synthesis evaluation, and a migration assay. The biocompatibility was analyzed with LDH cytotoxicity assay and flow cytometric analysis of activation of immune cells. Finally, RADA-PDGF2 activity in wound healing was checked in a mouse dorsal skin injury model. Results: The analysis showed that RADA-PDGF2 can self-assemble, form a hydrogel, and release a bioactive sequence when incubated with human elastase. It shows pro-proliferative and pro-migratory properties and accelerates wound closure in the mouse model compared to RADA16-I. In addition, it is not cytotoxic to human cells and does not show immunogenicity. RADA-PDGF2 seems to be a promising drug candidate for wound management.

Bridging charge-orbital ordering and Fermi surface instabilities in half-doped single-layered manganite La(0.5)Sr(1.5)MnO4.

The single-layered half-doped manganite La(0.5)Sr(1.5)MnO4 (LSMO), was studied by means of the angle-resolved photoemission spectroscopy (ARPES), scanning tunneling microscopy (STM), and resistivity measurements. STM revealed a smooth reconstruction-free surface; the density of states, extracted from photoemission and tunneling spectroscopy, is in agreement with transport measurements. The derived from ARPES Fermi surface (FS) nesting properties correspond to the known pattern of the charge-orbital ordering (COO), which implies that FS instability is related to the propensity to form a COO state in LSMO.

The ras oncoprotein and M-phase activity.

The endogenous mos proto-oncogene product (Mos) is required for meiotic maturation. In Xenopus oocytes, the ras oncogene product (Ras) can induce meiotic maturation and high levels of M-phase--promoting factor (MPF) independent of endogenous Mos, indicating that a parallel pathway to metaphase exists. In addition, Ras, like Mos and cytostatic factor, can arrest Xenopus embryonic cell cleavage in mitosis and maintain high levels of MPF. Thus, in the Xenopus oocyte and embryo systems Ras functions in the M phase of the cell cycle. The embryonic cleavage arrest assay is a rapid and sensitive test for Ras function.

Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae.

The interaction between RAS proteins and adenylyl cyclase was studied by using dominant interfering mutations of adenylyl cyclase from the yeast Saccharomyces cerevisiae. RAS proteins activate adenylyl cyclase in this organism. A plasmid expressing a catalytically inactive adenylyl cyclase was found to interfere dominantly with this activation. The interfering region mapped to the leucine-rich repeat region of adenylyl cyclase, which is homologous to domains present in several other proteins and is thought to participate in protein-protein interactions.

Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterase. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDE1 gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDE1 or PDE2 can reverse the growth arrest defects of yeast cells carrying the RAS2(Val-19) mutation. PDE1 and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S. cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAS2(Val-19) mutation. pde1- pde2- ras1- ras2- cells are viable.

Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae.

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

Isolation and characterization of mutants of human mitogen-activated protein kinase (ERK2).

Site directed mutagenesis/charged-to-alanine scanning mutagenesis of the amino terminal portion of human ERK2 (from amino acids 1 to 150) purified as a glutathione-S-transferase fusion protein (GST-ERK2) from E. coli has been done to determine regions/amino acids important for activation by rabbit skeletal muscle MAP kinase kinase (rMEK) and kinase activity towards myelin basic protein (MBP). Five classes of mutants have been isolated. The first class of mutants comprises of G30A/G32A, A50D and R65A/R68A/E69A, that can be phosphorylated by rMEK and have no kinase activity towards MBP, the second class includes mutants D122A/H123A and N142A which have lower kinase activities but no change in their activation by rMEK; third class being Y34A, E58A/H59A, which have neutral effect towards either activity, the fourth class that includes completely inactive mutants D42A/K46A/R48A, the deletion mutant in the same region (-9aa[40-48]) and D104A/E107A/D109A and finally the fifth class that include K53A, E94A/K97A/D99A, K112A/K115A and R133A/K136A that are phosphorylated 140-240% but with kinase activity toward MBP ranging from 50-100% of the wild type.

Teaching community health assessment skills in a problem-based format.

If physicians are able to understand the health status of populations they can use this understanding to develop targeted health care services for their patient populations or communities. Since 1991, the family practice residency program at the State University of New York Health Science Center at Brooklyn has taught community health assessment using a problem-based format. During this four-week rotation, residents gather, classify, and analyze demographic and health data about a specific local population. They use these data and published research literature to create rates that they believe will help to illuminate the health status and health issues of their community. By the end of the rotation, the residents feel comfortable working with rates and understand both their usefulness and their limitations. This rotation also introduces them to the concept of population health and some of the skills necessary to understanding a population-based approach to health care.

Retention/quantitation properties of the o-phthaldialdehyde-3-mercaptopropionic acid and the o-phthaldialdehyde-N-acetyl-L-cysteine amino acid derivatives in reversed-phase high-performance liquid chromatography.

The separation/identification of 25 amino acids as their o-phthaldialdehyde-3-mercaptopropionic acid (OPA/MPA) and o-phthaldialdehyde-N-acetyl-L-cysteine (OPA/NAC) derivatives have been optimized [paying particular attention to those amino acids which elute with more than one derivative (glycine, histidine, gamma-aminobutyric acid, beta-alanine, ornithine, lysine) and that are expected to be present in apples in their free form]. Optimum separation conditions are reported on six reversed-phase columns: Nucleosil 3 and 5 microm, 150(+20 guard)x4.0 mm; Gromsil 3 microm, 150(+10 guard)x4.0 mm; Hypersil 5 microm, 150(+20 guard)x4.0 mm and 200(+20 guard)x4.0 mm; and Hypersil 3 microm, 150(+20 guard)x4.0 mm. Elutions were followed, simultaneously, with photodiode array and fluorescence detectors connected in line. Optimization studies carried out in model solutions as a function of temperature (30-55 degrees C) and eluent flow-rate (0.8-2.5 mL/min) demonstrated that optimum resolutions are obtained with the highest flow-rate applicable (remaining on the safe side with a column pressure of << 3500 p.s.i.; 1 p.s.i.=6894.76 Pa) in the temperature range 30-50 degrees C. Twenty-five amino acids, eluting in 31 separate, characteristic derivatives, were determined on all six columns (the main component, asparagine, present in overwhelming excess, together with the minor constituents glutamine, beta-alanine, gamma-aminobutyric acid, homoserine, and homoarginine). Optimum conditions in the case of both derivatives were obtained on the same type of column (Hypersil, 5 microm), as follows: for the OPA/MPA amino acids with programmed flow-rate [1.3-2.3 ml/min; column, 200(+20 guard)x4 mm], at 50 degrees C, while, for the OPA/NAC amino acids at 2.1 ml/min flow rate, at 30 degrees C [column, 150(+20 guard)x4 mm], with 40 and 37 min run times, including equilibration. Responses of the corresponding amino acids proved to be independent of the column used; reproducibility in the concentration range 6-12,000 pmol, related to the injected amount of amino acids, was <3.4% RSD (average relative standard deviation percentage). The utility of the protocol was demonstrated in the quantitation of the free amino acid content of five apple varieties (Jonagored, Idared, Jonica, Florina, Freedom) on various harvesting dates and after different storage times. Derivatization of the apple pulp was performed with filtered samples, applying any special isolation processes.

Tuberculosis infection and disease in children.

Children with primary tuberculosis are more likely than adults to be asymptomatic and to have extrapulmonary disease. If tuberculosis infection or disease is suspected in a child, it is imperative to obtain a detailed medical, family and social history, which must include an investigation of the child's contact with an infectious adult. Because it is difficult to isolate a Mycobacterium tuberculosis strain from children, the treatment regimen must often be based on the susceptibility pattern of the M. tuberculosis strain in the infecting adult. Patients with fully susceptible pulmonary tuberculosis are treated with isoniazid, rifampin and pyrazinamide for two months and then with isoniazid and rifampin for four more months. If infection with a drug-resistant organism is suspected, other medications should be used for treatment. When treating a child with possible tuberculosis disease, the primary care physician generally should consult with a pediatric tuberculosis specialist. Identifying the adult source of a child's tuberculous infection and marshalling the resources needed for treatment are best done in conjunction with the local health department.

Antimicrobial peptides: structure, function and therapeutic potential.

Animals and plants protect themselves against invading pathogenic microorganisms by expressing a plethora of cationic antimicrobial peptides on exposed surfaces. These highly charged peptides, some of which are constitutively expressed and present on the cell surface at all times, and some which are expressed following induction by a specific insult, interact with and rapidly eliminate a wide variety of microbial invaders, including Gram-positive and Gram-negative bacteria, fungi and protozoa. Although these peptides are highly toxic to microbial cells, binding to and rapidly lysing the microbe within a few minutes, they are innocuous to host cells at the minimal inhibitory concentration dose range. Highly charged cationic peptides of this class offer the possibility of taking advantage of Nature's natural and highly evolved mechanism to protect an organism from attack by a microorganism, and to develop these novel peptides for biopharmaceutical use against a growing and deadly invasion of novel and rapidly evolving microbes that are resistant to the current antimicrobial armamentaria.

Post-translational processing of purified human K-ras in Xenopus oocytes.

Membrane localization of ras p21 involves a complex series of post-translational processing events, including S-farnesylation of Cys-186, removal of three carboxyl-terminal amino acid residues, and methylation of the carboxyl-terminal farnesylcysteine residue. Palmitoylation of cysteine residues within the hypervariable region (amino acids 165-185) is also required for membrane localization of mammalian H-, N-, and K-ras(A). For K-ras(B), which contains no cysteine residues within the hypervariable region, a polybasic domain substitutes for palmitoylation as a second signal for plasma membrane targeting. In order to investigate the localization of K-ras(B) to the plasma membrane, we purified wild-type and mutant human K-ras(B) proteins from strains of E. coli harboring bacterial expression plasmids and injected them into Xenopus laevis oocytes. Our results show that wild-type and activated K-ras(B) proteins can be post-translationally modified and can induce meiotic maturation in Xenopus oocytes. A mutation at Cys-186 (Cys to Gly) abolished the ability of activated K-ras(B) to induce meiosis. Deprivation of isoprenyl precursors by the addition of lovastatin, a drug that blocks the synthesis of mevalonate, also abolished the ability of activated K-ras(B) to induce meiosis, although this inhibition could be overcome by the addition of exogenous mevalonate. Lovastatin did not block meiotic maturation induced by microinjection of purified mos protein, a component of the cytostatic factor that arrests Xenopus oocytes at the first meiotic prophase. These results indicate that post-translational isoprenylation of K-ras(B) is essential for plasma membrane targeting and induction of meiotic maturation in Xenopus oocytes and that further isoprenyl modification of proteins downstream from mos signal transduction is not essential for this process.

School-based tuberculosis testing and treatment program: comparing directly observed preventive therapy with traditional preventive therapy.

A high-school-based health center instituted a tuberculosis testing and treatment program. Compliance of purified protein derivative (PPD) positive students with isonicotine hydrazine (INH) preventive therapy was compared for two groups of students: those in directly observed preventive therapy (DOPT) and those in traditional therapy. Four hundred thirty six students were tested and 429 returned for a reading: 209 (49 percent) students had a positive PPD test. The rate of positivity did not vary by age, sex, or years in the United States. There was no relationship between bacille Calmette-Guérin (BCG) vaccination and rate of positivity or mean size of induration. The compliance rate for all courses of DOPT was significantly higher (54 percent) than that for traditional therapy (26 percent). The compliance rates for the first and second attempts to complete a course of DOPT was 71 percent. Completion of therapy did not differ by age, sex, or years in the United States. Haitian students completed therapy more often than students from Jamaica. We believe it is effective and efficient to base tuberculosis testing and treatment with DOPT in the school setting.

SCH9, a gene of Saccharomyces cerevisiae that encodes a protein distinct from, but functionally and structurally related to, cAMP-dependent protein kinase catalytic subunits.

A new gene, SCH9, was isolated from Saccharomyces cerevisiae by its ability to complement a cdc25ts mutation. Sequence analysis indicates that it encodes a 90,000-dalton protein with a carboxy-terminal domain homologous to yeast and mammalian cAMP-dependent protein kinase catalytic subunits. In addition to suppressing loss of CDC25 function, multicopy plasmids containing SCH9 suppress the growth defects of strains lacking the RAS genes, the CYR1 gene, which encodes adenylyl cyclase, and the TPK genes, which encode the cAMP-dependent protein kinase catalytic subunits. Cells lacking SCH9 grow slowly and have a prolonged G1 phase of the cell cycle. This defect is suppressed by activation of the cAMP effector pathway. We propose that SCH9 encodes a protein kinase that is part of a growth control pathway which is at least partially redundant with the cAMP pathway.

Recognizing spinal cord emergencies.

Physicians who work in primary care settings and emergency departments frequently evaluate patients with neck and back pain. Spinal cord emergencies are uncommon, but injury must be recognized early so that the diagnosis can be quickly confirmed and treatment can be instituted to possibly prevent permanent loss of function. The differential diagnosis includes spinal cord compression secondary to vertebral fracture or space-occupying lesion, spinal infection or abscess, vascular or hematologic damage, severe disc herniation and spinal stenosis. The most important information in the assessment of a possible spinal cord emergency comes from the history and the clinical evaluation. Physicians must look for "red flags"--key historical and clinical clues that increase the likelihood of a serious underlying disorder. In considering diagnostic tests, physicians should apply the principles outlined in an algorithm for the evaluation of low back pain prepared by the Agency for Healthcare Research and Quality (formerly the Agency for Health Care Policy and Research). Computed tomography and magnetic resonance imaging can clearly define anatomy, but these studies are costly and have a high false-positive rate. Referral of high-risk patients to a neurologist or spine specialist may be indicated.

Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase.

We have isolated three genes (TPK1, TPK2, and TPK3) from the yeast S. cerevisiae that encode the catalytic subunits of the cAMP-dependent protein kinase. Gene disruption experiments demonstrated that no two of the three genes are essential by themselves but at least one TPK gene is required for a cell to grow normally. Comparison of the predicted amino acid sequences of the TPK genes indicates conserved and variable domains. The carboxy-terminal 320 amino acid residues have more than 75% homology to each other and more than 50% homology to the bovine catalytic subunit. The amino-terminal regions show no homology to each other and are heterogeneous in length. The TPK1 gene carried on a multicopy plasmid can suppress both a temperature-sensitive ras2 gene and adenylate cyclase gene.

Gp60 activation mediates albumin transcytosis in endothelial cells by tyrosine kinase-dependent pathway.

We investigated the function of gp60, an endothelial cell membrane 60-kDa albumin-binding protein localized in caveolae, and the mechanism of its activation in regulating endothelial permeability of albumin. Gp60 organization on the bovine pulmonary microvessel endothelial cell (BPMVEC) surface was punctate as shown by immunofluorescence using an anti-gp60 antibody (Ab) conjugated with bisfunctional, N-hydroxysuccinimidyl fluorophore (Cy3). Addition of a secondary Ab to anti-gp60 Ab-treated BPMVEC induced cross-linking of gp60 as evident by increased size of fluorescent particles and cell surface gp60 clustering. Gp60 cross-linking also produced 2-3-fold increases in the endothelial cell uptake and the luminal to abluminal permeability of 125I-albumin as well as the fluid-phase tracer, horseradish peroxidase. The increased transendothelial permeability of macromolecules was the result of transcytosis as it was not associated with an increase in the paracellular pathway. Incubation of anti-gp60 Ab with BPMVEC at 37 degrees C caused internalization of gp60, and thereby reduced the uptake of the macromolecules. Activation of gp60 by either albumin (the gp60 ligand) or gp60 cross-linking induced the phosphorylation of both gp60 and caveolin-1 (the major structural caveolar protein) on tyrosine residues. Gp60 activation also phosphorylated the Src family tyrosine kinases pp60(c-Src) and Fyn. The activated pp60(c-Src) and Fyn co-immunoprecipitated with caveolin-1 in BPMVEC membrane. Protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, prevented gp60-activated macromolecule uptake and transcytosis in a concentration-dependent manner, indicating the functional significance of the PTK pathway in activating albumin transcytosis. These findings indicate that activation of gp60 stimulates the Src PTK signaling pathway, and thus regulates the transcytosis of albumin across the endothelial cell monolayer.

Experimental pneumoconiosis due to dusts of ore mines, I.

Male CFY rats were injected into their trachea with a suspension of dust of ore mine dead rock of high quartz content and mixed composition (particle size: 0-60 micrometers). Three and six months after treatment, foreign body granulation and after twelve months a non-fibrotic diffuse lung disease was seen in the animals. After twelve months of exposure, non-specific, mature sinus histiocytosis and small focal epitheloid cell reaction were found to develop in the cervical, lung-hilar and retroperitoneal lymph nodes. The histopathological changes were compared with those seen in experimental silicosis induced by DQ12 quartz. It is concluded that the hazardous effect of industrial dusts is not identical with their fibrogenic effect.

Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae.

A gene, PDE2, has been cloned from the yeast Saccharomyces cerevisiae that, when present in high copy, reverses the phenotypic effects of RAS2Val19, a mutant form of the RAS2 gene that renders yeast cells sensitive to heat shock and starvation. It has previously been shown that the RAS proteins are potent activators of yeast adenylate cyclase. We report here that PDE2 encodes a high-affinity cAMP phosphodiesterase that shares sequence homology with animal cell phosphodiesterases. These results therefore imply that the effects of RAS2Val19 are mediated through its changes in cAMP concentration.

Splicing defective mutants of the COXI gene of yeast mitochondrial DNA: initial definition of the maturase domain of the group II intron aI2.

Six mutations blocking the function of a seven intron form of the mitochondrial gene encoding subunit I of cytochrome c oxidase (COXI) and mapping upstream of exon 3 were isolated and characterized. A cis-dominant mutant of the group IIA intron 1 defines a helical portion of the C1 substructure of domain 1 as essential for splicing. A trans-recessive mutant confirms that the intron 1 reading frame encodes a maturase function. A cis-dominant mutant in exon 2 was found to have no effect on the splicing of intron 1 or 2. A trans-recessive mutant, located in the group IIA intron 2, demonstrates for the first time that intron 2 encodes a maturase. A genetic dissection of the five missense mutations present in the intron 2 reading frame of that strain demonstrates that the maturase defect results from one or both of the missense mutations in a newly-recognized conserved sequence called domain X.