Elevated levels of ornithine decarboxylase cooperate with Raf/ERK activation to convert normal keratinocytes into invasive malignant cells.

Ornithine decarboxylase (ODC) overexpression coupled with activated Ras is fully sufficient to oncogenically transform primary keratinocytes. To determine the Ras effector pathways that represent the minimal essential contribution to full oncogenic transformation in this context, we evaluated the cooperativity of different Ras effector mutants with overexpressed ODC in an in vivo tracheal xenotransplantation assay for epithelial cell invasiveness. Primary keratinocytes, isolated from either K6/ODC transgenic mouse skin (expressing increased ODC) or from normal littermate skin were infected with retrovirus producing an activated RasV12 or partial loss-of-function effector mutants of RasV12 that selectively induce only the Raf/ERK, RalGDS, or the PI3-kinase signaling pathway. Whereas keratinocytes expressing a fully activated RasV12 are not invasive in tracheal xenotransplants, ODC-overexpressing keratinocytes acquire an invasive phenotype with additional expression of either RasV12 or activation of the Raf/ERK pathway. Independent of a mutated ras, elevated levels of ODC activate the Akt/mTOR signaling pathway as well as the Rho/Rac pathway in primary keratinocytes. Thus, Raf/ERK signaling is sufficient to cooperate with increased ODC activity in the conversion of normal keratinocytes to invasive cells. In order to promote invasiveness in keratinocytes, elevated levels of ODC may cooperate with Raf/ERK via activation of the Akt and Rho/Rac signaling pathway.

Spinal distribution and metabolism of 2'-O-(2-methoxyethyl)-modified oligonucleotides after intrathecal administration in rats.

Intrathecal (IT) delivery of antisense oligodeoxynucleotides (ASO) has been used to study the function of specific gene products in spinal nociception. However, a lack of systematic studies on the spinal distribution and kinetics of IT ASO is a major hurdle to the utilization of this technique. In the present study, we injected rats IT with 2'-O-(2-methoxyethyl) modified phosphorothioate ASO (2'-O-MOE ASO) and examined anatomical and cellular location of the ASO in the spinal cord and dorsal root ganglia (DRG) by immunocytochemistry. At 0.5 h after a single IT injection, immunostaining for ISIS 13920 (a 2'-O-MOE ASO targeting h-ras) localized superficially in the lumbar spinal cord, while at 24 h the immunostaining was distributed throughout the spinal cord and was predominantly intracellular. Double staining with cell type specific antibodies indicated that the ASO was taken up by both glia and neurons. ASO immunoreactivity was also observed in DRG after IT ISIS 13920. Capillary gel electrophoresis analysis showed that ISIS 22703, a 2'-O-MOE ASO targeting the alpha isozyme of protein kinase C (PKC), remained intact in spinal cord tissue and cerebrospinal fluid up to 24 h after the injection and no metabolites were detected. In contrast, after IT ISIS 11300, an unmodified phosphorothioate ASO with the same sequence as ISIS 22703, no full-length compound was detectable at 24 h, and metabolites were seen as early as 0.5 h. IT treatment with ISIS 22703 at doses that effectively down-regulated PKCalpha mRNA in spinal cord did not affect the mRNA expression in DRG. In summary, 2'-O-MOE ASO displayed high stability in spinal tissue after IT delivery, efficiently distributed to spinal cord, and internalized into both neuronal and non-neuronal cells. ASO are able to reach DRG after IT delivery; however, higher doses may be required to reduce target gene in DRG as compared with spinal cord.

Management of Group A beta-hemolytic streptococcal pharyngitis.

Bacteria are responsible for approximately 5 to 10 percent of pharyngitis cases, with group A beta-hemolytic streptococci being the most common bacterial etiology. A positive rapid antigen detection test may be considered definitive evidence for treatment; a negative test should be followed by a confirmatory throat culture when streptococcal pharyngitis is strongly suspected. Treatment goals include prevention of suppurative and nonsuppurative complications, abatement of clinical signs and symptoms, reduction of bacterial transmission and minimization of antimicrobial adverse effects. Antibiotic selection requires consideration of patients' allergies, bacteriologic and clinical efficacy, frequency of administration, duration of therapy, potential side effects, compliance and cost. Oral penicillin remains the drug of choice in most clinical situations, although the more expensive cephalosporins and, perhaps, amoxicillin-clavulanate potassium provide superior bacteriologic and clinical cure rates. Alternative treatments must be used in patients with penicillin allergy, compliance issues or penicillin treatment failure. Patients who do not respond to initial treatment should be given an antimicrobial that is not inactivated by penicillinase-producing organisms (e.g., amoxicillin-clavulanate potassium, a cephalosporin or a macrolide). Patient education may help to reduce recurrence.

An alpha/beta-type, small, acid-soluble spore protein which has very high affinity for DNA prevents outgrowth of Bacillus subtilis spores.

A derivative of SspC, a minor alpha/beta-type, small, acid-soluble spore protein (SASP) from Bacillus subtilis, was generated that has a very high affinity for DNA. This protein (SspC(Delta11-D13K)) was able to confer UV resistance on spores lacking alpha/beta-type SASP, and spores with SspC(Delta11-D13K) triggered germination normally. However, SspC(Delta11-D13K) blocked outgrowth of > or = 90% of germinated spores, and SspC(Delta11-D13K) persisted in these germinated spores, whereas wild-type SspC was almost completely degraded. The outgrowth phenotype of spores with SspC(Delta11-D13K) is proposed to be due to the high stability of the SspC(Delta11-D13K)-DNA complex, which prevents rapid degradation of this alpha/beta-type SASP early in germination. The persistence of this protein on spore DNA then interferes with transcription during spore outgrowth.

N-terminal amino acid residues mediate protein-protein interactions between DNA-bound alpha /beta -type small, acid-soluble spore proteins from Bacillus species.

The binding of alpha/beta-type small, acid-soluble spore proteins (SASP) to DNA of spores of Bacillus species is the major determinant of DNA resistance to a variety of damaging treatments. The primary sequence of alpha/beta-type SASP is highly conserved; however, the N-terminal third of these proteins is less well conserved than the C-terminal two-thirds. To determine the functional importance of residues in the N-terminal region of alpha/beta-type SASP, variants of SspC (a minor alpha/beta-type SASP from Bacillus subtilis) with modified N termini were generated and their structural and DNA binding properties studied in vitro and in vivo. SspC variants with deletions of up to 14 residues ( approximately 20% of SspC residues) were able to bind DNA in vitro and adopted similar conformations when bound to DNA, as determined by circular dichroism spectroscopy and protein-protein cross-linking. Progressive deletion of up to 11 N-terminal residues resulted in proteins with progressively lower DNA binding affinity. However, SspC(Delta)(14) (in which 14 N-terminal residues have been deleted) showed significantly higher affinity for DNA than the larger proteins, SspC(Delta)(10) and SspC(Delta)(11). The affinity of these proteins for DNA was shown to be largely dependent upon the charge of the first few N-terminal residues. These results are interpreted in the context of a model for DNA-dependent alpha/beta-type SASP protein-protein interaction involving the N-terminal regions of these proteins.

Equilibrium and kinetic binding interactions between DNA and a group of novel, nonspecific DNA-binding proteins from spores of Bacillus and Clostridium species.

Binding of alpha/beta-type small acid-soluble spore proteins (SASP) is the major determinant of DNA resistance to damage caused by UV radiation, heat, and oxidizing agents in spores of Bacillus and Clostridium species. Analysis of several alpha/beta-type SASP showed that these proteins have essentially no secondary structure in the absence of DNA, but become significantly alpha-helical upon binding to double-stranded DNAs or oligonucleotides. Folding of alpha/beta-type SASP induced by a variety of DNAs and oligonucleotides was measured by CD spectroscopy, and this allowed determination of a DNA binding site size of 4 base pairs as well as equilibrium binding parameters of the alpha/beta-type SASP-DNA interaction. Analysis of the equilibrium binding data further allowed determination of both intrinsic binding constants (K) and cooperativity factors (omega), as the alpha/beta-type SASP-DNA interaction was significantly cooperative, with the degree of cooperativity depending on both the bound DNA and the salt concentration. Kinetic analysis of the interaction of one alpha/beta-type SASP, SspC(Tyr), with DNA indicated that each binding event involves the dimerization of SspC(Tyr) monomers at a DNA binding site. The implications of these findings for the structure of the alpha/beta-type SASP.DNA complex and the physiology of alpha/beta-type SASP degradation during spore germination are discussed.

Identification of protein-protein contacts between alpha/beta-type small, acid-soluble spore proteins of Bacillus species bound to DNA.

Small, acid-soluble spore proteins (SASP) of the alpha/beta-type from several Bacillus species were cross-linked into homodimers, heterodimers and homooligomers with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of linear plasmid DNA. Significant protein cross-linking was not detected in the absence of DNA. In all four alpha/beta-type SASP examined, the amino donor in the EDC induced amide cross-links was the alpha-amino group of the protein. However, the carboxylate containing amino acid residues involved in cross-linking varied. In SASP-A and SASP-C of Bacillus megaterium two conserved glutamate residues, which form part of the germination protease recognition sequence, were involved in cross-link formation. In SspC from Bacillus subtilis and Bce1 from Bacillus cereus the acidic residues involved in cross-link formation were not in the protease recognition sequence, but at a site closer to the N terminus of the proteins. These data indicate that, although there are likely to be subtle structural differences between different alpha/beta-type SASP, the N-terminal regions of these proteins are involved in protein-protein interactions while in the DNA bound state.

In vitro and in vivo oxidation of methionine residues in small, acid-soluble spore proteins from Bacillus species.

Methionine residues in alpha/beta-type small, acid-soluble spore proteins (SASP) of Bacillus species were readily oxidized to methionine sulfoxide in vitro by t-butyl hydroperoxide (tBHP) or hydrogen peroxide (H2O2). These oxidized alpha/beta-type SASP no longer bound to DNA effectively, but DNA binding protected alpha/beta-type SASP against methionine oxidation by peroxides in vitro. Incubation of an oxidized alpha/beta-type SASP with peptidyl methionine sulfoxide reductase (MsrA), which can reduce methionine sulfoxide residues back to methionine, restored the alpha/beta-type SASP's ability to bind to DNA. Both tBHP and H2O2 caused some oxidation of the two methionine residues of an alpha/beta-type SASP (SspC) in spores of Bacillus subtilis, although one methionine which is highly conserved in alpha/beta-type SASP was only oxidized to a small degree. However, much more methionine sulfoxide was generated by peroxide treatment of spores carrying a mutant form of SspC which has a lower affinity for DNA. MsrA activity was present in wild-type B. subtilis spores. However, msrA mutant spores were no more sensitive to H2O2 than were wild-type spores. The major mechanism operating for dealing with oxidative damage to alpha/beta-type SASP in spores is DNA binding, which protects the protein's methionine residues from oxidation both in vitro and in vivo. This may be important in vivo since alpha/beta-type SASP containing oxidized methionine residues no longer bind DNA well and alpha/beta-type SASP-DNA binding is essential for long-term spore survival.

Analysis of deamidation of small, acid-soluble spore proteins from Bacillus subtilis in vitro and in vivo.

Deamidation of one specific asparagine residue in an alpha/beta-type small, acid-soluble spore protein (SASP) of Bacillus subtilis took place readily in vitro (time for 50% deamidation [t(1/2)], approximately 1 h at 70 degrees C), and the deamidated SASP no longer bound to DNA effectively. However, DNA binding protected against this deamidation in vitro. A mutant alpha/beta-type SASP in which the reactive asparagine was changed to aspartate also failed to bind to DNA in vitro, and this protein did not restore UV radiation and heat resistance to spores lacking the majority of their alpha/beta-type SASP. When expressed in Escherichia coli, where it is bound to DNA, the alpha/beta-type SASP deamidated with a t(1/2) of 2 to 3 h at 95 degrees C. However, the alpha/beta-type SASP was extremely resistant to deamidation within spores (t(1/2), >50 h at 95 degrees C). A gamma-type SASP of B. subtilis also deamidated readily in vitro (t(1/2) for one net deamidation, approximately 1 h at 70 degrees C), but this protein (which is not associated with DNA) deamidated fairly readily in spores (t(1/2), approximately 1 h at 95 degrees C). Total spore core protein also deamidated in vivo, although the rate was two- to threefold slower than that of deamidation of total protein in heated vegetative cells. These data indicate that protein deamidation is slowed significantly in spores, presumably due to the spore's environment. However, alpha/beta-type SASP are even more strongly protected against deamidation in vivo, presumably by their binding to spore DNA. Thus, not only do alpha/beta-type SASP protect spore DNA from damage; DNA also protects alpha/beta-type SASP.

Subacute osteomyelitis.

Subacute osteomyelitis is a chronic low-grade infection of bone characterized by a lack of systemic manifestations. The onset is insidious. Pain is the most common symptom, and has usually been present for several months before the initial evaluation. Swelling and tenderness over the area of involved bone may also be seen. Laboratory evaluation is unrevealing, with a normal white blood cell count and differential. The erythrocyte sedimentation rate may also be normal. The most common organism cultured from a subacute osteomyelitis is a staphylococcus species. Twenty-five percent of subacute bone infections are sterile. The most common manifestation of a subacute osteomyelitis in a child is a geographic lytic metaphyseal lesion (Brodie's abscess). Treatment of a culture positive infection includes surgical debridement and antibiotic therapy. A sterile abscess can be treated conservatively without antibiotics, provided the patient's symptoms are improving and the lesion is regressing radiographically.

Interpersonal distance behavior of mentally retarded and nonretarded children.

The investigators compared interpersonal-distance choice behavior of 60 mildly retarded children and 60 nonretarded children of comparable MAs to a male adult stranger. Contrary to the original prediction, stemming from Zigler's negative-reaction tendency formulation, interpersonal distance by retarded children was not generally different from that of nonretarded children. However, analysis of the interaction between IQ group and approach condition (child approaching adult or adult approaching child) indicated that retarded children preferred greater distance from the approaching adult than did nonretarded children. This result raises the possibility that perception of lessened personal control by retarded children stimulated a stronger avoidance reaction.

Projective assessment of personal space among retarded and nonretarded children.

A figure-placement method was used to examine the personal space usage of 60 mildly retarded and 60 nonretarded children of comparable MA. Spatial distance between a self-figure and second figure for both groups was least when the second figure was designated with positive affect, greater when designated with neutral affect, and greatest when the designation involved negative affect. Retarded children used less spatial distance than nonretarded children when the interaction involved a self and teacher figure, supporting Zigler's (1973) positive- and negative-reaction tendency formulation. More space was used by both groups when the second figure was designated as not smart than when designated as smart.

Reflection-impulsivity and reading recognition ability among mildly retarded children.

The Matching Familiar Figures test of reflection-impulsivity and a test of reading recognition ability (Standard Reading Inventory) were administered to 236 youngsters (ages 8 to 17 years) who were in programs for educable mentally retarded students. Age was, of course, associated with reading recognition, but there was no relationship between Matching Familiar Figures and Standard Reading Inventory scores with age and IQ controlled. This finding casts doubt on the association of reflection-impulsivity as defined by performance on the Matching Familiar Figures test and the ability of retarded children to decode words.

Affective reactions of retarded and nonretarded children to success and failure.

After performing a simple motor task, 208 mildly retarded and nonretarded girls and boys pointed to photographs of modeled affective facial expressions to indicate how they felt, wished to feel, and thought their teachers would feel about their performance. Children in both IQ groups frequently attributed positive affect to themselves and their teachers after success, although younger retarded children were less positive than were nonretarded children in teacher affect attributions. Following failure, retarded subjects were generally less frequently negative than were nonretarded subjects in affect attributions to themselves and particularly to their teachers. Emphasis on success and minimization of failure in classrooms for retarded children was offered as one possible explanation for the IQ group affect differences following failure.