Role of a serine/threonine kinase,Mst1, in megakaryocyte differentiation

Platelets, which play a central role in thrombosis and hemostasis, develop from megakaryocytes. Signal transduction originated from the megakaryocyte growth and development factor, the Mpl ligand, which leads to megakaryocyte differentiation, polyploidization, and maturation, has been gradually characterized. In this study, we report the inducibility of Mst1, a recently described serine/threonine kinase, by Mpl ligand and the effect of its induced expression on megakaryocyte differentiation. The steady‐state level of mst1 message and Mst1‐associated kinase activity increased in response to Mpl ligand. Ectopic expression of human mst1 in a mouse megakaryocytic cell line resulted in a drastic increase in DNA content per cell. Elevated expression of megakaryocyte differentiation markers, such as acetylcholine esterase, PF4, and GPIIb was also observed in hmst1‐expressing cells. Activation of p38 MAPK, a known downstream effector of Mst1, was shown to be required for polyploidization, but not for enhanced expression of differentiation markers. Our study thus designates Mst1 as a Mpl ligand‐responsive signaling molecule that promotes induction of lineage‐specific cellular programming. J. Cell. Biochem. 76:44–60, 1999. © 1999 Wiley‐Liss, Inc.     

Growth hormone receptor gene disruption in mature‐adult mice improves male insulin sensitivity and extends female lifespan

Studies in multiple species indicate that reducing growth hormone (GH) action enhances healthy lifespan. In fact, GH receptor knockout (GHRKO) mice hold the Methuselah prize for the world''s longest‐lived laboratory mouse. We previously demonstrated that GHR ablation starting at puberty (1.5 months), improved insulin sensitivity and female lifespan but results in markedly reduced body size. In this study, we investigated the effects of GHR disruption in mature‐adult mice at 6 months old (6mGHRKO). These mice exhibited GH resistance (reduced IGF‐1 and elevated GH serum levels), increased body adiposity, reduced lean mass, and minimal effects on body length. Importantly, 6mGHRKO males have enhanced insulin sensitivity and reduced neoplasms while females exhibited increased median and maximal lifespan. Furthermore, fasting glucose and oxidative damage was reduced in females compared to males irrespective of Ghr deletion. Overall, disrupted GH action in adult mice resulted in sexual dimorphic effects suggesting that GH reduction at older ages may have gerotherapeutic effects.     

Induction of somatopause in adult mice compromises bone morphology and exacerbates bone loss during aging

Somatopause refers to the gradual declines in growth hormone (GH) and insulin‐like growth factor‐1 throughout aging. To define how induced somatopause affects skeletal integrity, we used an inducible GH receptor knockout (iGHRKO) mouse model. Somatopause, induced globally at 6 months of age, resulted in significantly more slender bones in both male and female iGHRKO mice. In males, induced somatopause was associated with progressive expansion of the marrow cavity leading to significant thinning of the cortices, which compromised bone strength. We report progressive declines in osteocyte lacunar number, and increases in lacunar volume, in iGHRKO males, and reductions in lacunar number accompanied by ~20% loss of overall canalicular connectivity in iGHRKO females by 30 months of age. Induced somatopause did not affect mineral/matrix ratio assessed by Raman microspectroscopy. We found significant increases in bone marrow adiposity and high levels of sclerostin, a negative regulator of bone formation in iGHRKO mice. Surprisingly, however, despite compromised bone morphology, osteocyte senescence was reduced in the iGHRKO mice. In this study, we avoided the confounded effects of constitutive deficiency in the GH/IGF‐1 axis on the skeleton during growth, and specifically dissected its effects on the aging skeleton. We show here, for the first time, that induced somatopause compromises bone morphology and the bone marrow environment.     

c-Myc inactivation by mutant max alters growth and morphology of NCI-H-630 colon cancer cells

The myc gene family has been implicated in multiple cell processes including proliferation, differentiation, tumorigenesis, and apoptosis. For its cellular growth promoting function, Myc must heterodimerize with Max. To study the effect of Myc inactivation on the growth and differentiation properties of epithelial tumor cells, we transfected the H-630 human colon cancer cell line with bm-max, a mutant Max protein in which DNA-binding activity has been abolished. Cells expressing high levels of bm-Max grow poorly, and the morphology of both colonies and single cells is altered. Moreover, increased bm-Max expression results in a prolonged G₀/G₁ phase accompanied by induced expression of p21 (WAF1/CIP1), elevated levels of alkaline phosphatase (ALP) activity, and accumulation of large fat granuli within the cells. These distinctive cell characteristics are associated with differentiation processes in numerous malignant cell lines. The results of this study support a model in which sequestering of endogenous Myc and Max proteins into “basic mutant” dimers lacking DNA-binding activity is sufficient both to inhibit proliferation and to induce changes in cell behavior consistent with differentiation. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

An approach to functionally relevant clustering of the protein universe: Active site profile‐based clustering of protein structures and sequences

Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification—amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two‐Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure‐Function Linkage Database, SFLD) self‐identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self‐identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well‐curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP‐identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F‐measure and performance analysis on the enolase search results and comparison to GEMMA and SCI‐PHY demonstrate that TuLIP avoids the over‐division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results.     

Rap1 activation in collagen phagocytosis is dependent on nonmuscle myosin II-A

Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or β1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.     

Standard Operating Procedures,ethical and legal regulations in BTB (Brain/Tissue/Bio) banking: what is still missing?

The use of human biological specimens in scientific research is the focus of current international public and professional concern and a major issue in bioethics in general. Brain/Tissue/Bio banks (BTB-banks) are a rapid developing sector; each of these banks acts locally as a steering unit for the establishment of the local Standard Operating Procedures (SOPs) and the legal regulations and ethical guidelines to be followed in the procurement and dissemination of research specimens. An appropriat Code of Conduct is crucial to a successful operation of the banks and the research application they handle. What are we still missing ? (1) Adequate funding for research BTB-banks. (2) Standard evaluation protocls for audit of BTB-bank performance. (3) Internationally accepted SOP’s which will facilitate exchange and sharing of specimens and data with the scientific community. (4) Internationally accepted Code of Conduct. In the present paper we review the most pressing organizational, methodological, medico-legal and ethical issues involved in BTB-banking; funding, auditing, procurement, management/handling, dissemination and sharing of specimens, confidentiality and data protection, genetic testing, “financial gain” and safety measures. Taking into consideration the huge variety of the specimens stored in different repositories and the enormous differences in medico-legal systems and ethics regulations in different countries it is strongly recommend that the health-care systems and institutions who host BTB-Banks will put more efforts in getting adequate funding for the infrastructure and daily activities. The BTB-banks should define evaluation protocols, SOPs and their Code of Conduct. This in turn will enable the banks to share the collected specimens and data with the largest possible number of researchers and aim at a maximal scientific spin-off and advance in public health research.     

Diversity of degradation signals in the ubiquitin-proteasome system

The ubiquitin-proteasome system degrades an enormous variety of proteins that contain specific degradation signals, or 'degrons'. Besides the degradation of regulatory proteins, almost every protein suffers from sporadic biosynthetic errors or misfolding. Such aberrant proteins can be recognized and rapidly degraded by cells. Structural and functional data on a handful of degrons allow several generalizations regarding their mechanism of action. We focus on different strategies of degron recognition by the ubiquitin system, and contrast regulatory degrons that are subject to signalling-dependent modification with those that are controlled by protein folding or assembly, as frequently occurs during protein quality control.     

Enantioselective monoterpene alcohol acetylation in Origanum, Mentha and Salvia species

Selected plants within the Origanum, Mentha and Salvia genera, that contain significant amounts of chiral volatile alcohols and their related acetates, exhibit remarkable enantioselectivity of alcohol acetyl transferase (AAT) activity and particularly can discriminate between linalool enantiomers. Origanum dayi AAT produced almost enantiomerically pure (R)-linalyl acetate by enzymatic acetylation of racemic linalool, whereas the closely related O. majorana AAT produced a mixture of (R)- and (S)-linalyl acetate with a ratio of 6:4. V(max) of O. dayi acetylation activity was 30-fold higher for (R)-linalool, whereas in O. majorana no such differences were found.