The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding--a common and life-threatening side effect of many cancer therapies--and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output.

Myofibrils from pig muscle inoculated and incubated with Pseudomonas fragi showed an extremely disrupted appearance as compared to uninoculated controls. There was an almost complete absence of material in the H zone, marked disruption of the A band (probably myosin), and some loss of dense material from the Z line. These changes indicated that marked proteolysis had occurred. Bacteria observed in spoiled muscle tissue exhibited protrusions or blebs on the outer surface of the cell walls. The blebs appeared to form detached globules that migrated into the muscle mass. Bacteria grown in non-muscle-containing media did not produce blebs, which indicates the blebs were induced by growth on muscle tissue. The possibility that the blebs and globules may contain a proteolytic enzyme responsible for myofibrillar disruption is discussed.

Considerable salt-soluble protein degradation was observed in pork muscle inoculated with Pseudomonas fragi. During a 20-day incubation period at 10 C, the samples proceeded to rank spoilage or putrefaction. There was a large decrease in the salt-soluble protein fraction and a corresponding increase in nonprotein nitrogen. Disc gel electrophoretic patterns showed that breakdown of the salt-soluble proteins had occurred after incubation for 20 days. During incubation for 10 days at 10 C, P. fragi produced large amounts of extracellular proteolytic activity in ground pork. Most of the proteolytic activity appeared immediately after spoilage occurred. However, a significant increase in the ability to hydrolyze casein and a slight increase in the ability to hydrolyze denatured hemoglobin occurred prior to spoilage.

An extracellular preparation from Pseudomonas fragi with proteolytic enzyme activity was isolated, and its action on meat proteins and meat protein ultrastructure was studied. First, a suitable growth medium for proteolytic enzyme production was determined, and a method for partial purification of the proteolytically active fraction was developed. The enzyme preparation displayed optimal proteolytic activity at neutral pH and 35 C. Proteolytic activity was irreversibly lost by mild heat treatment. The enzyme preparation was tested for its ability to hydrolyze isolated pig muscle proteins. Myofibrillar protein was rapidly degraded, G-actin and myosin were broken down at a slower rate, and the sarcoplasmic proteins were least susceptible to hydrolysis. Electron micrographs of pork muscle showed that the proteolytic enzyme preparation caused a complete loss of dense material from the Z line. Similarities are discussed between the action of P. fragi extracellular proteolytic enzyme(s) on meat and normal bacterial spoilage of meat. culture fluid were centrifuged (9,750 x g, 10 min, 0 C) 996 on July 5, 2020 by guest http://aem.asm.org/ Downloaded from 1004 APPL. MICROBIOL. on July 5, 2020 by guest