Analysis by mutagenesis of the ATP binding site of the gamma subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system.

Active gamma subunit of skeletal muscle phosphorylase kinase has been obtained by expression of the rat soleus cDNA in a baculovirus system. The protein exhibited the expected pH 6.8/8.2 activity ratio of 0.6, and its activity was insensitive to Ca2+ addition, indicating that it was free gamma subunit and not a gamma subunit-calmodulin complex. It was stimulated approximately 2-fold by Ca(2+)-calmodulin addition, demonstrating that it had retained high-affinity calmodulin binding. By site-directed mutagenesis, we have examined the role of six of the amino acids that constitute the consensus ATP binding site of the protein kinase, which in the gamma subunit is represented by the sequence 26Gly.Arg.Gly.Val.Ser.Ser.Val.Val33. Changes were evaluated by the kinetic determination of the dissociation constants of gamma-ATP, gamma-ADP, gamma-AMP.PCP, and gamma-phosphorylase and the maximum catalytic activity. The mutants Ser26-gamma, Ser29-gamma, Phe30-gamma, and Gly31-gamma each exhibited an essentially identical dissociation constant for gamma subunit phosphorylase, indicating that these mutations had not caused a global alteration in the protein structure but were limited to changes in the nucleotide binding site domain. Substitution of either Val33 (by Gly) or Gly28 (by Ser), two of the most conserved residues in all protein kinases, resulted in enzyme with marginally detectable activity. In noted contrast, the Ser26 mutant, which substituted the first glycine of the consensus glycine trio motif, and which is also very highly conserved, retained at least 25% of the enzymatic activity. The Gly31 substitution, which restored a glycine to a position characteristic for most protein kinases, had little overall effect upon the maximum rate of catalysis. Restoration of Ser30 to the more typical phenylalanine, which is present in most protein kinases, had minimal effect on catalysis. These data provide the first direct evaluation of the roles that different residues play within this consensus glycine trio/valine motif of the protein kinases, which up to now have only been surmised to be of importance because of their conservation. Two unexpected findings are that for one residue that is very conserved (Gly26) there is some flexibility of substitution not apparent from the evolutionary conservation and that a second quite conserved residue in protein kinases (equivalent to Gly at position 31) does not produce a protein optimized for nucleotide binding.     

Agonist Binding to α2-Adrenoceptors Is Elevated in the Locus Coeruleus from Victims of Suicide

Abstract: The binding of an agonist, p-[¹²⁵I]iodoclonidine, and an antagonist, [³H]yohimbine, to α₂-adrenoceptors was measured autoradiographically in the locus coeruleus from 10 pairs of antidepressant-free victims of suicide and age-matched controls. Agonist binding to α₂-adrenoceptors was significantly greater in the locus coeruleus from victims of suicide compared with control subjects. In contrast, antagonist binding to α₂-adrenoceptors in the locus coeruleus did not differ significantly between control and suicide subjects. HPLC analysis of norepinephrine in tissue sections of the locus coeruleus did not reveal any differences between control subjects and suicide victims, suggesting that differences in agonist binding are not a result of differences in retention of the endogenous agonist norepinephrine in tissue sections. The increase in agonist binding to α₂-adrenoceptors in the locus coeruleus of victims of suicide links an altered expression of the high-affinity state of autoinhibitory α₂-adrenoceptors with suicide.     

Induction of lymphokine-activated killer activity in mice by prothymosin α

We have recently demonstrated that prothymosin (ProT) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor (TNF) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT in vivo. We demonstrate that i.p. injections of ProT enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8⁺ cells, NK cells and activated CD3⁺ cells. The ProT-induced effect persisted for 30 days after the end of the ProT treatment period and returned to normal levels 20 days later. SPlenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT in vitro. The ProT-induced NK an LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF and IL-2 were generated in response to ProT in LAK cultures. These findings suggest that ProT may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF in the spleen, peritoneal cavity and probably other lymphoid organs.This work was supported by a CEC grant to M. Papamichail     

An EPR signal from the "invisible" copper of cytochrome oxidase.

Sulfide is both an inhibitor and a slow reductant of oxidized cytochrome $\text{c}$ oxidase. When the enzyme is exposed to sulfide for short times (one minute or less) and frozen, the resultant electron paramagnetic resonance (EPR) signals show clearly: low spin heme a, low spin heme a₃, the usual “EPR detectable” Cu²⁺ signal (g_⊥ = 2.17, g_⊥ = 2.03), and a new Cu²⁺ signal superimposed on the same region, with (g_⊥ ～ 2.19, g_⊥ = 2.05). This new signal presumably arises because the antiferromagnetic coupling postulated to exist between the iron atom of heme a₃ and this copper is disrupted when heme a₃ is driven to a low spin state by sulfide. The implications of this result with respect to models of the O₂-binding site and redox geometry of oxidase are briefly discussed.     

Inter-rater reliability of physiotherapists using the Action Research Arm Test in chronic stroke

Objectives:The purpose of this study is to establish whether physiotherapists’ ratings are consistent, when using the Action Research Arm Test (ARAT) to score a chronic stroke patient.Methods:This was part of a large project establishing the reliability in chronic stroke. This study used a correlational design comparing the association between physiotherapist scores of the same patient, to establish the ARAT’s inter-rater reliability. The COSMIN checklist was followed to enhance the methodology of the study.Results:Twenty physiotherapists (8 female and 12 male) aged between 25 and 53 years were selected. There were no participant dropouts or withdrawals. The sample size was normally distributed. The physiotherapists appeared representative of the UK physiotherapy population, with the exception of gender. The distribution of scores showed a normal distribution with standard deviation of score of 1.9. The Kendall’s W test showed 0.711 of agreement between the raters. The scores achieved statistical significance showing consistency between physiotherapists’ scores with chronic stroke. Limitations of the study were the use of a small single center convenience sample that may reduce the generalizability of the findings.Conclusions:The ARAT is consistent when scored by physiotherapists in a chronic stroke population. The inter-rater reliability range was (0.70 to 0.90) which is categorized as good.     

Economic Recession and Emergence of an HIV-1 Outbreak among Drug Injectors in Athens Metropolitan Area: A Longitudinal Study

Background

During 2011, a dramatic increase (1600%) of reported HIV-1 infections among injecting drug users (IDUs) was noted in Athens, Greece. We herein assess the potential causal pathways associated with this outbreak.

Methods

Our study employed high resolution HIV-1 phylogenetic and phylogeographic analyses. We examined also longitudinal data of ecological variables such as the annual growth of gross domestic product (GDP) of Greece in association with HIV-1 and HCV sentinel prevalence in IDUs, unemployment and homelessness rates and HIV transmission networks in Athens IDUs before and during economic recession (2008–2012).

Results

IDU isolates sampled in 2011 and 2012 suggested transmission networks in 94.6% and 92.7% of the cases in striking contrast with the sporadic networking (5%) during 1998–2009. The geographic origin of most HIV-1 isolates was consistent with the recently documented migratory waves in Greece. The decline in GDP was inversely correlated with annual prevalence rates of HIV and HCV and with unemployment and homelessness rates in IDUs (all p<0.001). The slope of anti-HCV prevalence in the sentinel populations of IDUs and in “new” drug injectors was found 120 and 1.9-fold (p = 0.007, p = 0.08 respectively) higher in 2008–2012 (economic recession) compared with 2002–2006. The median (25th, 75th) size of transmission networks were 34 (12, 58) and 2 (2, 2) (p = 0.057) in 2008–2012 and 1998–2007, respectively. The coverage of harm reduction services was low throughout the study period.

Conclusions

Scaling-up harm reduction services and addressing social and structural factors related to the current economic crisis should be urgently considered in environments where HIV-1 outbreaks may occur.     

Sizeable acquired subglottic cyst in a baby with Williams–Beuren syndrome: Association or coincidence?

We describe a case of an acquired subglottic cyst presented with persistent stridor and voice hoarsening in a baby diagnosed with Williams–Beuren syndrome that was born premature and required intubation during neonatal period. We also comment on whether this is a coincidence or there can be an association between impaired elastogenesis, a feature of patients with the syndrome and the formation of a subglottic cyst.     

Perinatal physiology in cloned and normal calves: hematologic and biochemical profiles

Although a majority of clones are born normal and apparently healthy, mortality rates of nearly 30% are described in many reports. Such losses are a major limitation of cloning technology and represent substantial economic investment as well as justifiable animal health and welfare concerns. Prospective, controlled studies are needed to understand fully the causes of neonatal mortality in clones and to develop preventive and therapeutic strategies to minimize losses. We report here the findings of studies on the hematologic and biochemical profiles of cloned and control calves in the immediate 48-h postpartum period. Cloned calves were similar to control calves for a majority of parameters studied including blood gases, concentrations of plasma proteins, minerals and electrolytes, and white blood cell, neutrophil, lymphocyte, and platelet counts. The most notable differences between clones and controls in this study were reduced red- and white-blood cell counts in clones at birth and 1 h of age. As a group, plasma electrolyte concentrations were more variable in clones, and the variability tended to be shifted either higher (sodium, chloride) or lower (potassium, bicarbonate) than in controls. Previously, we noted differences in carbohydrate parameters, the length of time required for clones to make the neonatal adaptation to life ex utero, and morphology of the cloned placenta. Taken together, our findings suggest that cloned calves experience greater difficulty adjusting to life ex utero and that further research is warranted to determine the nature of the relationship between the physiological differences noted here in clones at birth and concomitant abnormal placental morphology.     

Seed germination ecology and salt stress response in eight Mediterranean populations of Sarcopoterium spinosum (L.) Spach

This study aims to deepen the analysis of seed germination ecology and salinity tolerance of Sarcopoterium spinosum (Rosaceae). Germination tests were conducted to evaluate the effect of the fruit’s spongy tissue and the intraspecific variability in seed germination among eight populations of the species on responses to light and total darkness, constant and alternating temperatures, salt stress and germination recovery. The effect of the presence of the spongy tissue varied among populations, with significant results for seed germination. For all populations, optimum germination temperatures were observed in the range of 10–20°C, indicating that S. spinosum and its germination in the field might occur preferably in the period between autumn and early spring. The high water availability due to rainfall during this period could be a considerable advantage for the seed germination of this species. Seeds of S. spinosum showed the ability to germinate in up to 250 mM NaCl in the substrate, and their ability to recover after salt exposure may be interpreted as adaptation to the coastal habitats in which they generally grow. These results give this species a halo-tolerant character. Great inter-population variability is detected in this study in several aspects, which indicated that the Mediterranean populations of S. spinosum differ considerably and are adapted to their local conditions. This study provides new information about S. spinosum seed ecology, which could help to preserve and apply effective conservation measures for this species, which in several areas of its distribution range is endangered.     

Definition of a short region of XPG necessary for TFIIH interaction and stable recruitment to sites of UV damage

XPG is the human endonuclease that cuts 3' to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.