Characterization of a three-phase response in gloved cold-stressed fingers

Seven gloves were studied worn by eight sedentary subjects (six men and two women) exposed to cold–dry, C–D, (mean dry bulb temperature −17.2^∘C; mean dew point temperature ), and cold–wet, C–W, ( 0^∘C; ) conditions. Mean endurance times were 75 min for the C–D and 162 min for the C–W conditions. A three-phase response pattern of the temperature in the fingers was characterized. Phase I comprised an initial period during which finger temperature remained close to the pre-exposed level, due to delayed vasoconstriction in the finger. Phase II involved an exponential-like decrease of finger temperature indicative of the onset of vasoconstriction in the finger. Phase III manifested periodic finger temperature changes due to cold induced vasodilatation (CIVD). Mean wave patterns for phase III indicated approximately 3.5 waves · h⁻¹ in the C–D but only about 2 waves · h⁻¹ in the C–W condition. Extension of endurance time, due to CIVD, was defined as the difference in time between the actual end of the experiment and the time the finger-tip would have reached the set temperature endurance limit as extrapolated by a continued exponential drop. Three overall response patterns of fingers in the cold were characterized: type A exhibiting all 3 phases; type B₁ or B₂ exhibiting either phases I+II or phases II+III; and type C showing only phase II. Considerable inter- and intra-subject variability was found. In both test conditions the final physiological thermal states of the subjects were between comfortable and slightly uncomfortable but acceptable and thus did not correlate with the responses in the fingers. Accepted: 5 January 1998     

Multimodal distribution of warfarin binding to protein in serum of male-sprague dawley rats

The serum protein binding of racemic warfarin was determined in 209 adult male Sprague-Dawley rats. Statistical analysis of a histogram of warfarin free fraction values (ratio of concentrations of free and free plus bound drug) revealed modes at free fraction values of 0.0040, 0.0112, and 0.0166, with more than one-half of the animals included in the Gaussian component with the lowest free fraction mode. Since the total clearance of warfarin is proportional to the free fraction value and since the intrinsic clearance of warfarin in these rats is relatively constant, the trimodal distribution of free fraction values reflects a corresponding distribution of warfarin clearance values.     

3′,6′-anhydro-6-O-corynomycoloyl trehalose: Synthesis and identification as the impurity in synthetic cord factor preparations

Acylation of 2,3,4,2′,3′,4′-hexa-O-benzyl-6,6′-di-O-methanesulphonyl-α-α-trehalose (1) with a reduced amount of potassium corynomycolate yielded a mixture which consisted mainly of 2,3,4,2′,3′,4′-hexa-O-benzyl-6-O-corynomycoloyl-6′-O-methanesulphonyl-α,α-trehalose (2). Catalytic hydrogenolysis of 2 gave the mono-mesylate 4 which was converted into 3′,6′-anhydro-6-O-corynomycoloyl-α,α-trehalose (5) but treatment with sodium hydride. The structure of 5 was studied by mass-spectroscopy. Compound 5 was found to be identical with the byproduct obtained in the acylation of 6,6′-di-O-p-toluenesulphonyl-α,α-trehalose with potassium corynomycolate.     

Modification of the plasmid initiator protein RepC active site during replication

Abstract pT181 is a Staphylococcus aureus rolling circle replicating plasmid whose copy number is controlled by regulating the synthesis and activity of the initiator protein, RepC. The RepC dimer is modified during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. To purify RepC*, RepC was expressed in S. aureus as a fusion protein with a polyhistidine tail. The histidine-tagged RepC retains its initiation and topoisomerase activities in vitro. Histagged RepC/RepC and RepC/RepC* were purified in a two-step procedure. Peptide mapping, mass spectrometric analysis and protein sequencing of purified RepC and RepC* were carried out, and both proteins appeared identical, except that the peptide containing the RepC active site tyrosine used in nicking activity was absent when the purified RepC* sample was analyzed. The absence of the active site in RepC* suggests that this site was modified during replication. The results provide the first direct biochemical evidence that RepC* is a modified form of RepC, and support a model in which RepC replication of pT181 leaves RepC with an oligonucleotide blocking the active site of one of its subunits.     

Combined adrenal myelolipoma and medullary hyperplasia

OBJECTIVE: A patient is reported with hypertension due to combined medullary adrenal hyperplasia and myelolipoma. METHODS: A 52-year-old woman with long-standing hypertension was evaluated for an incidentally discovered large tumor of the left adrenal. Left adrenalectomy was performed for a presumptive clinical diagnosis of pheochromocytoma. RESULTS: Histopathologic examination revealed a mixed tumor consisting of a large myelolipoma with infiltrating foci of adrenal medulla. CONCLUSIONS: A patient is described with hypertension, myelolipoma and adrenal medullary hyperplasia; following adrenalectomy, however, blood pressure and biochemical abnormalities normalized.     

The myelin-associated oligodendrocytic basic protein region MOBP15-36 encompasses the immunodominant major encephalitogenic epitope(s) for SJL/J mice and predicted epitope(s) for multiple sclerosis-associated HLA-DRB1*1501

Autoimmune response to the myelin-associated oligodendrocytic basic protein (MOBP), a CNS-specific myelin constituent, was recently suggested to play a role in the pathogenesis of multiple sclerosis (MS). The pathogenic autoimmune response to MOBP and the associated pathology in the CNS have not yet been fully investigated. In this study, we have characterized the clinical manifestations, pathology, T cell epitope-specificity, and TCRs associated with experimental autoimmune encephalomyelitis (EAE) induced in SJL/J mice with recombinant mouse MOBP (long isoform, 170 aa). Analysis of encephalitogenic MOBP-reactive T cells for reactivity to overlapping MOBP peptides defined MOBP15-36 as their major immunodominant epitope. Accordingly, MOBP15-36 was demonstrated to be the major encephalitogenic MOBP epitope for SJL/J mice, inducing severe/chronic clinical EAE associated with intense perivascular and parenchymal infiltrations, widespread demyelination, axonal loss, and remarkable optic neuritis. Molecular modeling of the interaction of I-A(s) with MOBP15-36, together with analysis of the MOBP15-36-specific T cell response to truncated peptides, suggests MOBP20-28 as the core sequence for I-A(s)-restricted recognition of the encephalitogenic region MOBP15-36. Although highly focused in their epitope specificity, the encephalitogenic MOBP-reactive T cells displayed a widespread usage of TCR Vbeta genes. These results would therefore favor epitope-directed, rather than TCR-targeted, approaches to therapy of MOBP-associated pathogenic autoimmunity. Localization by molecular modeling of a potential HLA-DRB1*1501-associated MOBP epitope within the encephalitogenic MOBP15-36 sequence suggests the potential relevance of T cell reactivity against MOBP15-36 to MS. The reactivity to MOBP15-36 detected in MS shown here and in another study further emphasizes the potential significance of this epitope for MS.     

Glycogen synthase kinase 3beta modulates synphilin-1 ubiquitylation and cellular inclusion formation by SIAH: implications for proteasomal function and Lewy body formation

alpha-Synuclein is known to play a major role in the pathogenesis of Parkinson disease. We previously identified synphilin-1 as an alpha-synuclein-interacting protein and more recently found that synphilin-1 also interacts with the E3 ubiquitin ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 and promote its degradation through the ubiquitin proteasome system. Inability of the proteasome to degrade synphilin-1 promotes the formation of ubiquitylated inclusion bodies. We now show that synphilin-1 is phosphorylated by GSK3beta within amino acids 550-659 and that this phosphorylation is significantly decreased by pharmacological inhibition of GSK3beta and suppression of GSK3beta expression by small interfering RNA duplex. Mutation analysis showed that Ser556 is a major GSK3beta phosphorylation site in synphilin-1. GSK3beta co-immunoprecipitated with synphilin-1, and protein 14-3-3, an activator of GSK3beta activity, increased synphilin-1 phosphorylation. GSK3beta decreased the in vitro and in vivo ubiquitylation of synphilin-1 as well as its degradation promoted by SIAH. Pharmacological inhibition and small interfering RNA suppression of GSK3beta greatly increased ubiquitylation and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant, which is less phosphorylated by GSK3beta, formed more inclusion bodies than wild type synphilin-1. Inhibition of GSK3beta in primary neuronal cultures decreased the levels of endogenous synphilin-1, indicating that synphilin-1 is a physiologic substrate of GSK3beta. Using GFPu as a reporter to measure proteasome function in vivo, we found that synphilin-1 S556A is more efficient in inhibiting the proteasome than wild type synphilin-1, raising the possibility that the degree of synphilin-1 phosphorylation may regulate the proteasome function. Activation of GSK3beta during endoplasmic reticulum stress and the specific phosphorylation of synphilin-1 by GSK3beta place synphilin-1 as a possible mediator of endoplasmic reticulum stress and proteasomal dysfunction observed in Parkinson disease.     

The prevalence and expression of inherited connexin 26 mutations associated with nonsyndromic hearing loss in the Israeli population

Connexin 26 (GJB2) mutations lead to hearing loss in a significant proportion of all populations studied so far, despite the fact that at least 50 other genes are also associated with hearing loss. The entire coding region of connexin 26 was sequenced in 75 hearing impaired children and adults in Israel in order to determine the percentage of hearing loss attributed to connexin 26 and the types of mutations in this population. Age of onset in the screened population was both prelingual and postlingual, with hearing loss ranging from moderate to profound. Almost 39% of all persons tested harbored GJB2 mutations, the majority of which were 35delG and 167delT mutations. A novel mutation, involving both a deletion and insertion, 51del12insA, was identified in a family originating from Uzbekistan. Several parameters were examined to establish whether genotype-phenotype correlations exist, including age of onset, severity of hearing loss and audiological characteristics, including pure-tone audiometry, tympanometry, auditory brainstem response (ABR), and transient evoked otoacoustic emissions (TEOAE). All GJB2 mutations were associated with prelingual hearing loss, though severity ranged from moderate to profound, with variability even among hearing impaired siblings. We have not found a significant difference in hearing levels between individuals with 35delG and 167delT mutations. Our results suggest that, in Israel, clinicians should first screen for the common 167delT and 35delG mutations by simple and inexpensive restriction enzyme analysis, although if these are not found, sequencing should be done to rule out additional mutations due to the ethnic diversity in this region.     

Cdc42 and RhoB activation are required for mannose receptor-mediated phagocytosis by human alveolar macrophages

Human alveolar macrophages (AMs) phagocytose Pneumocystis (Pc) organisms predominantly through mannose receptors, although the molecular mechanism mediating this opsonin-independent process is not known. In this study, using AMs from healthy individuals, Pc phagocytosis was associated with focal F-actin polymerization and Cdc42, Rac1, and Rho activation in a time-dependent manner. Phagocytosis was primarily dependent on Cdc42 and RhoB activation (as determined by AM transfection with Cdc42 and RhoB dominant-negative alleles) and mediated predominantly through mannose receptors (as determined by siRNA gene silencing of AM mannose receptors). Pc also promoted PAK-1 phosphorylation, which was also dependent on RhoGTPase activation. HIV infection of AMs (as a model for reduced mannose receptor expression and function) was associated with impaired F-actin polymerization, reduced Cdc42 and Rho activation, and markedly reduced PAK-1 phosphorylation in response to Pc organisms. In healthy AMs, Pc phagocytosis was partially dependent on PAK activation, but dependent on the Rho effector molecule ROCK. These data provide a molecular mechanism for AM mannose receptor-mediated phagocytosis of unopsonized Pc organisms that appears distinct from opsonin-dependent phagocytic receptors. Reduced AM mannose receptor-mediated Cdc42 and Rho activation in the context of HIV infection may represent a mechanism that contributes to the pathogenesis of opportunistic pneumonia.