High-level production of human alpha- and beta-globins in insect cells.

High-level production of human alpha- and beta-globins in cultured Spodoptera frugiperda (Sf-9) cells infected with recombinant baculoviruses is described. The expressed globins are produced to 70-140 mg protein/liter of cell culture or 5-10% of the total cellular protein. Two recombinant baculoviruses for alpha-globin, H alpha and H beta alpha, differ in their construction in that the 5'-untranslated region of the beta-globin gene is inserted 5' to the alpha-globin mRNA coding region in H beta alpha. This insertion results in a 40% increase in yield of alpha-globin over that of H alpha. Consistent with previous observations of the processing of recombinant proteins in Sf-9 cells, both alpha- and beta-globins expressed in Sf-9 cells are correctly processed to remove the initiating methionine from the amino termini of the globins. Sequencing of the expressed globins in Sf-9 cells confirms their identity with globins purified from human normal adult hemoglobin.     

Anticipatory Posturing of the Vocal Tract Reveals Dissociation of Speech Movement Plans from Linguistic Units

Models of speech production typically assume that control over the timing of speech movements is governed by the selection of higher-level linguistic units, such as segments or syllables. This study used real-time magnetic resonance imaging of the vocal tract to investigate the anticipatory movements speakers make prior to producing a vocal response. Two factors were varied: preparation (whether or not speakers had foreknowledge of the target response) and pre-response constraint (whether or not speakers were required to maintain a specific vocal tract posture prior to the response). In prepared responses, many speakers were observed to produce pre-response anticipatory movements with a variety of articulators, showing that that speech movements can be readily dissociated from higher-level linguistic units. Substantial variation was observed across speakers with regard to the articulators used for anticipatory posturing and the contexts in which anticipatory movements occurred. The findings of this study have important consequences for models of speech production and for our understanding of the normal range of variation in anticipatory speech behaviors.     

Prevalence of soil-transmitted helminth infections,schistosomiasis, and lymphatic filariasis before and after preventive chemotherapy initiation in the Philippines: A systematic review and meta-analysis

ObjectiveTo estimate the impact of preventive chemotherapy on the prevalence and intensity of soil-transmitted helminth (STH) infections, schistosomiasis, and lymphatic filariasis in the Philippines, using systematic review and meta-analysis.MethodsWe included reports reporting prevalence of STH infections, schistosomiasis, or lymphatic filariasis in the Philippines published until 31 March 2021. Peer-reviewed studies were identified in electronic databases. Grey literature reports by the University of the Philippines and the Department of Health were also included. Pooled infection prevalence, before and after the initiation of preventive chemotherapy, stratified by age group, was calculated using the inverse variance heterogeneity model.FindingsA total of 109 reports were included in the review and meta-analysis. Overall prevalence of moderate-heavy intensity Ascaris lumbricoides (6.6%) and Trichuris trichiura (2.7%) infection after initiation of preventive chemotherapy were significantly lower than the prevalence prior to initiation (23.6% for A. lumbricoides and 12.2% for T. trichiura). Prevalence reductions were also found in school and preschool-age children for A. lumbricoides and T. trichiura. Studies conducted after preventive chemotherapy initiation had significantly lower overall prevalence of moderate-heavy intensity schistosomiasis (3.1% vs 0.2%) and of schistosomiasis in school-age children (30.5% vs 1%). Pooled prevalence of lymphatic filariasis prior to preventive chemotherapy initiation was 3.2% across 12 provinces, while currently only two provinces still have prevalence of more than 1%. There were no published studies reporting prevalence of lymphatic filariasis after initiation of preventive chemotherapy. Heterogeneity was high with I² mostly above 90%.ConclusionThe burden of STH infections and schistosomiasis in children were significantly lower in studies conducted following the initiation of preventive chemotherapy. Eliminating morbidity and interrupting transmission, however, may require expanded control initiatives including community-wide treatment, and improved water, sanitation, and hygiene. Lymphatic filariasis burden has decreased since the implementation of preventive chemotherapy, with all but two provinces having reached the elimination of lymphatic filariasis as a public health problem.     

Slow spontaneous ��-to-�� structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.     

Histochemische,elektronenmikroskopische und biochemische Untersuchungen über Glykogenanhäufung in reaktiv veränderten Astrozyten der traumatisch lädierten Säugergroßhirnrinde

Zusammenfassung Die Anhäufung von Glykogen in der Großhirnrinde von Goldhamstern und Ratten wurde im Bereich peritraumatischer Astrozytenproliferation histochemisch, elektronenmikroskopisch und biochemisch untersucht.Die elektronenmikroskopischen Befunde ergeben klare Aufschlüsse über zelluläre Lokalisation und morphologische Eigenschaften des Depotkohlenhydrates. Massen von 150–400 Å großen Partikeln lagern im Grundplasma der reaktiven Astrozyten; sie sind nach Behandlung mit Bleihydroxyd (Kontrasterhöhung) deutlich darstellbar. Die Vermehrung dieser Partikel ist bereits nach 24 Std in den perikapillären Fußstücken nachweisbar, breitet sich auf einen Großteil des Cytoplasmas einschließlich der das Neuropil durchsetzenden Fortsätze aus und erreicht nach 1–2 Wochen gewöhnlich ihren Höhepunkt. In mehrere Monate alten Glianarben finden sich filamentfreie Cytoplasmabezirke von astrozytären Faserbildnern, die zahlreiche Polysaccharidpartikel enthalten. Örtliche Beziehungen der Glykogenanhäufungen zum Mitochondrienbest and und zum endoplasmatischen Reticulum lassen sich in den reaktiv veränderten Astrozyten nicht feststellen. Auffällige eigenartige, große Lamellenkörper, in deren Zentrum häufig Glykogengranula konzentriert sind, kommen in den Frühstadien der reaktiven Veränderungen der Astrozyten vor. Diese Lamellenkörper entstehen durch Umformung von Golgizonen.Hirnrindengewebe von Ratten, das umfangreiche peritraumatische astrozytäre Reaktionen aufwies, wurde biochemisch untersucht. Nach Ablauf von 24 Std bis 30 Tagen wurden in diesem Gewebe die Glukose, das freie und gebundene Glykogen, die Milchsäure, die Laktatdehydrogenaseaktrvität, der Gesamtstickstoff und die Proteine bestimmt und mit den Werten des normalen Gewebes verglichen. Als wesentlicher Befund ist ein ansehnlicher Anstieg des gebundenen Glykogens in der alterierten Hirnrinde festzustellen. Der Gehalt an freiem Glykogen bleibt dagegen nahezu unverändert.Als wahrscheinlichste Ursache der Glykogenanhäufung im Perikaryon reaktiver Astrozyten wird eine Störung der metabolischen Beziehung zwischen diesen Gliaelementen und den Nervenzellen in Betracht gezogen. Schädigung und Untergang von Neuronen sowie der nachfolgende Gewebsumbau beeinträchtigen die Funktion der Astrozyten als metabolische Bindeglieder zwischen Nervenzellen und Blutstrombahn. Bei unverminderter Glukosezufuhr wird ein unverwertbarer Teil dieser Substanz in Form von Glykogenoproteiden im Cytoplasma der Astrozyten stark vermehrt abgelagert. Für diese Deutung spricht auch der Anstieg von Laktatwerten bei unveränderter Aktivität der Laktatdehydrogenase in dem Gewebe, das reaktive Makrogliaformen enthält.

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Summary The accumulation of glycogen in reactive astrocytes in the cerebral cortex of syrian hamsters and rats following a trauma was studied with the electron microscope and with histochemical and biochemical methods.The localization of carbonhydrate deposits within cellular components is achieved by electron microscopic techniques. Discrete particles in the cytoplasm of the reactive astrocytes measuring 150–400 Å show a high contrast by lead hydroxide staining. An increase in the number of these particles can be readily demonstrated after 24 hours in the pericapillary pedicles of the astrocytes. In later stages these particles seem to extend in the cytoplasm of the astrocytes and their processes. The accumulation reaches its highest point after 1 to 2 weeks. Several months later in the old scars the filament-free cytoplasmic areas of the fibrous astrocytes are abundantly loaded with polysaccharide particles.Topographic relations of glycogen accumulations to mitochondria and the endoplasmic reticulum were not observed in the reactive astrocytes. The appearance of peculiar, large, lamellar bodies in the early reactive astrocytes is very remarkable. In the center of these bodies a concentration of glycogen granules often occurs. These lamellar bodies are produced by a transformation of Golgi-apparatus.In order to study the biochemical correlates an extensive peritraumatic astrocyte reaction was induced in the cerebral cortex of rats. In the course with a range from 24 hours to 30 days, glucose, free and bound glycogen, lactic acid, lactate dehydrogenase activity, total nitrogen as well as protein in the peritraumatic tissue were determined and compared with the values of the normal tissue. As a significant result a considerable increase of bound glycogen takes place in the altered cerebral cortex. On the other hand, the content of free glycogen remains almost unchanged. As a probable cause of the glycogen accumulation in the perikaryon of the reactive astrocytes, a disturbance in the metabolic interrelationship between these glial elements and neurons is taken into consideration.The alterations of neurons impairs the function of the astrocytes as metabolic connecting links between neurons and blood stream. With continued glucose supply an unutilized part of this substance is stored in the cytoplasm in the form of glycogenproteid. This interpretation is supported by the fact that the lactate values increase without changes of the lactate dehydrogenase activity.

     

Single Administration of Tripeptide α-MSH(11–13) Attenuates Brain Damage by Reduced Inflammation and Apoptosis after Experimental Traumatic Brain Injury in Mice

Following traumatic brain injury (TBI) neuroinflammatory processes promote neuronal cell loss. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with immunomodulatory properties, which may offer neuroprotection. Due to short half-life and pigmentary side-effects of α-MSH, the C-terminal tripeptide α-MSH(11–13) may be an anti-inflammatory alternative. The present study investigated the mRNA concentrations of the precursor hormone proopiomelanocortin (POMC) and of melanocortin receptors 1 and 4 (MC1R/MC4R) in naive mice and 15 min, 6, 12, 24, and 48 h after controlled cortical impact (CCI). Regulation of POMC and MC4R expression did not change after trauma, while MC1R levels increased over time with a 3-fold maximum at 12 h compared to naive brain tissue. The effect of α-MSH(11–13) on secondary lesion volume determined in cresyl violet stained sections (intraperitoneal injection 30 min after insult of 1 mg/kg α-MSH(11–13) or 0.9% NaCl) showed a considerable smaller trauma in α-MSH(11–13) injected mice. The expression of the inflammatory markers TNF-α and IL-1β as well as the total amount of Iba-1 positive cells were not reduced. However, cell branch counting of Iba-1 positive cells revealed a reduced activation of microglia. Furthermore, tripeptide injection reduced neuronal apoptosis analyzed by cleaved caspase-3 and NeuN staining. Based on the results single α-MSH(11–13) administration offers a promising neuroprotective property by modulation of inflammation and prevention of apoptosis after traumatic brain injury.     

Sero-Diagnosis of Mycobacterium avium Complex Lung Disease Using Serum Immunoglobulin A Antibody against Glycopeptidolipid Antigen in Taiwan

Background

Lung disease (LD) due to non-tuberculous mycobacteria is an important clinical concern. Mycobacterium avium complex (MAC) is one of the most common causative agents but the diagnosis of MAC-LD remains challenging. Detection of serum IgA antibody against MAC glycopeptidolipid (GPL) has recently been shown to improve the diagnosis of MAC-LD, but has yet to be validated worldwide.

Methods

This prospective study was conducted in a tertiary referral center in northern Taiwan and enrolled patients with MAC-LD, MAC contamination, other lung diseases, and control subjects. Serum immunoglobulin A (IgA) antibody against MAC-GPL was detected in the participants and its specificity and sensitivity was assessed.

Results

There were 56 patients with MAC-LD, 11 with MAC contamination, 13 M. kansasii-LD, 26 LD due to rapidly-growing mycobacteria (RGM), 48 pulmonary tuberculosis, and 42 household contacts of patients with TB. Patients with MAC-LD were older and 32% of them had an underlying co-morbidity. By logistic regression, serum MAC-GPL IgA level was an independent predictor of MAC-LD among the study subjects and those with culture-positive specimens for MAC. By the receiver operating characteristic curve, serum MAC-GPL IgA had a good power to discriminate MAC-LD from MAC contamination. Under the optimal cut-off value of 0.73 U/mL, its sensitivity and specificity were 60% and 91%, respectively. Among MAC-LD patients, presence of co-morbidity was associated with MAC-GPL <0.73 U/ml in logistic regression analysis.

Conclusions

Measurement of serum anti-MAC-GPL IgA level is useful for the diagnosis of MAC-LD. However, its implement in clinical practice for immuno-compromised hosts needs careful consideration.