Reversible structure transition in gap junction under Ca++ control seen by high-resolution electron microscopy.

Deoxycholate-extracted rat liver gap junction was studied by high-resolution low-dose electron microscopy. Communicating channels between two adjoining cells supposedly form along the common axis of two apposed hexameric trans-membrane protein assemblies. These double hexamers are often arranged in large plaques on an ordered hexagonal net (8-9 nm lattice constant) and seem able to undergo structural alteration as a possible permeability control mechanism. Calcium is widely reported to uncouple gap junction, and we observed this alteration on exposure to Ca++ down to 10(-4) M concentration. When EGTA was added at matching concentrations, the alteration was reversible several times over one hour, but with considerable variability. It was imaged in the absence of any negative stain to avoid ionic and other complications. The resulting lack of contrast plus low-dose "shot" noise required digital Fourier filtering and reconstruction, but no detail was recovered below 1.8 nm. In other experiments with negative stain at neutral pH, gap junction connexons were apparently locked in the "closed" configuration and no transition could be induced. However, recovery of repeating detail to nearly 1.0 nm was possible, reproducibly showing a fine connective matrix between connexons . Whether this was formed by unfolded portions of the 28,000-dalton gap junction protein is not known, but its existence could explain the observed lattice invariance during the connexon structural transition.     

Electron microscopy of the low pH structure of influenza virus haemagglutinin.

Influenza virus haemagglutinin mediates infection of cells by fusion of viral and endosomal membranes, triggered by low pH which induces a conformational change in the protein. We report studies of this change by electron microscopy, neutron scattering, sedimentation and photon correlation on X-31 (H3N2) haemagglutinin, both intact and bromelain cleaved, in various assemblies. HAs in all preparations showed a thinning at low pH, and a marked elongation which was removed on tryptic digestion, revealing altered features in the remaining stem portion of the molecule. A tentative model of the change is proposed, with reference to the known X-ray structure at neutral pH, in which major changes occur in the stem tertiary structure, while the top portion is only affected in its quaternary structure.     

Acid exposure enhances sporulation of certain strains of Clostridium perfringens

A gastroenteritis results when Clostridium perfringens is ingested in high numbers and sporulates releasing enterotoxin in the intestines. Since the organism must pass through the stomach, its ability to form spores may be affected by the acidic environment. Five strains of C. perfringens were exposed to acidic conditions and then assessed for survival and their ability to form spores. An acidic pH environment kills the bacteria over time but surviving cells are able to recover and form spores. Two of the five strains demonstrated enhanced sporulation following a 30-min exposure to a pH 2 environment. For four of the strains tested, enterotoxin concentrations were higher from acid-exposed cells than from untreated cells. Exposure to a pH 3.5 environment did not affect sporulation when compared to an untreated control. Bacteria in the stationary phase of growth were the most able to resist the acid and sporulate. The results indicate that some strains will produce more spores and enterotoxin following exposure to an acidic environment.     

MVPP chemotherapy regimen for advanced Hodgkin's disease

During January 1968 to December 1972, 133 patients with advanced Hodgkin''s disease (HD) were admitted to hospital for combination chemotherapy with mustine, vinblastine, procarbazine, and prednisolone (MVPP regimen). Remission rates were 76% among 49 untreated patients and 90% among 42 patients who had relapsed after radiotherapy. The corresponding five-year survival rates were 65% and 86% respectively. Provided the observed yearly mortality (6%) remains unchanged 75% of patients who had previously received no treatment or irradiation and achieved remission are expected to continue in first remission after five years. Forty-two patients had received prior chemotherapy. They had lower remission and five-year survival rates (40% and 33% respectively), and fewer than half of those achieving remission were still in first remission after five years. There were several reasons for the poor prognosis in this group, including advanced-stage disease (stage IVB), age over 40, and achievement of remission.Chemotherapy was administered on an outpatient basis. Haematological toxicity and immediate drug-related side effects were similar to those of other regimens but there was no appreciable neurotoxicity. Most deaths were due to either HD itself or complications of advanced disease. Five malignancies other than HD occurred in patients who had received both single-agent chemotherapy and radiotherapy before MVPP chemotherapy. Two patients developed osteonecrosis of the femoral heads.Combination chemotherapy has a profound effect on the prognosis of advanced HD. The MVPP regimen yields results comparable to those of other regimens but with perhaps less toxicity.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident.     

Age-Specific Mortality During the 1918 Influenza Pandemic: Unravelling the Mystery of High Young Adult Mortality

The worldwide spread of a novel influenza A (H1N1) virus in 2009 showed that influenza remains a significant health threat, even for individuals in the prime of life. This paper focuses on the unusually high young adult mortality observed during the Spanish flu pandemic of 1918. Using historical records from Canada and the U.S., we report a peak of mortality at the exact age of 28 during the pandemic and argue that this increased mortality resulted from an early life exposure to influenza during the previous Russian flu pandemic of 1889–90. We posit that in specific instances, development of immunological memory to an influenza virus strain in early life may lead to a dysregulated immune response to antigenically novel strains encountered in later life, thereby increasing the risk of death. Exposure during critical periods of development could also create holes in the T cell repertoire and impair fetal maturation in general, thereby increasing mortality from infectious diseases later in life. Knowledge of the age-pattern of susceptibility to mortality from influenza could improve crisis management during future influenza pandemics.

“The war is over – and I must go” Egon Schiele, 1890–1918.

     

Pharmacological knock-down of the presenilin 1 heterodimer by a novel gamma -secretase inhibitor: implications for presenilin biology

Intramembranous cleavage of the beta-amyloid precursor protein by gamma-secretase is the final processing event generating amyloid-beta peptides, which are thought to be causative agents for Alzheimer's disease. Missense mutations in the presenilin genes co-segregate with early-onset Alzheimer's disease, and, recently, a close biochemical linkage between presenilins and the identity of gamma-secretase has been established. Here we describe for the first time that certain potent gamma-secretase inhibitors are able to interfere with the endoproteolytic processing of presenilin 1 (PS1). In addition, we identified a novel gamma-secretase inhibitor, [1S-benzyl-4R-[1-(5-cyclohexyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3(R,S)-ylcarbamoyl)-S-ethylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl]-carbamic acid tert-butyl ester (CBAP), which not only physically interacts with PS1, but upon chronic treatment produces a "pharmacological knock-down" of PS1 fragments. This indicates that the observed accumulation of full-length PS1 is caused by a direct inhibition of its endoproteolysis. The subsequent use of CBAP as a biological tool to increase full-length PS1 levels in the absence of exogenous PS1 expression has provided evidence that wild-type PS1 endoproteolysis is not required either for PS1/gamma-secretase complex assembly or trafficking. Furthermore, in cell-based systems CBAP does not completely recapitulate PS1 loss-of-function phenotypes. Even though the beta-amyloid precursor protein cleavage and the S3 cleavage of the Notch receptor are inhibited by CBAP, an impairment of Trk receptor maturation was not observed.