Formin-like 1 (FMNL1) Is Regulated by N-terminal Myristoylation and Induces Polarized Membrane Blebbing

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1γ) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1γ is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1ΔDAD), indicating that deregulation of autoinhibition is effective in FMNL1γ. Expression of both FMNL1γ and FMNL1ΔDAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.     

Membrane-murein attachment at the leading edge of the division septum: a second membrane-murein structure associated with morphogenesis of the gram-negative bacterial division septum.

Electron microscopy of plasmolyzed cells of Salmonella typhimurium revealed a continuous zone of membrane-murein attachment at the leading edge of the division septum at all stages of septal invagination. The membrane-murein attachment site had a characteristic ultrastructural appearance and remained as a bacterial birth scar at the new pole of each of the two daughter cells after cell separation. The continuous zone of membrane-murein attachment at the leading septal edge represents the second organelle based on a topologically ordered domain of membrane-murein adhesion to be described at the site of cell division.     

Cell growth and alpha-amylase production characteristics of Bacillus amyloliquefaciens

Growth and alpha-amylase production characteristics of Bacillus amyloliquefaciens strain F (ATCC 23350) in batch cultures are examined using glucose or maltose as the carbon source. While the cell growth is rapid when glucose is used as the carbon source, higher cell mass, higher total and specific enzyme activities, and higher enzyme production rates are obtained when maltose is used as the carbon source. The overall specific enzyme activity decreases with an increase in the initial concentration of carbon source. The oxygen requirement and carbon dioxide generation vary linearly with the maximum amount of cell mass produced. For experiments conducted using glucose as the carbon source, the kinetics of cell growth and glucose consumption are described using a special form of the Vavilin equation. For a given amount of initial carbon source, the enzyme synthesis capability is retained by the microorganism, although at a substantially reduced level, under severe oxygen limitation.     

Suppression of B cell function by methotrexate and trimetrexate. Evidence for inhibition of purine biosynthesis as a major mechanism of action

Methotrexate (MTX) is a widely used drug in the treatment of a variety of human neoplasms. Trimetrexate (TMQ) is a lipid-soluble quinazoline derivate of MTX that, unlike MTX, is not dependent upon membrane folate transport for cellular entry. A number of studies have demonstrated that MTX and, more recently, TMQ possess potent immunosuppressive properties. To examine the cellular events associated with the immunomodulatory effects of anti-folates on humoral immunity, a murine B cell maturation model was used. In vitro, MTX and TMQ reduced the number of antibody-forming cells to SRBC, as well as IgM production. B cells stimulated with anti-Ig demonstrated a dose-related suppression in [3H]UdR incorporation after addition of either drug, suggestive of a decrease in de novo DNA synthesis. B cell activation events preceding S phase were also suppressed by both anti-folates, as evidenced by inhibition of RNA synthesis. However, neither drug affected surface expression of Ia Ag nor inositol phosphate accumulation. Addition of TdR caused a slight non-significant increase in the antibody-forming cell response in the presence of 10(-7) M MTX. However, addition of hypoxanthine or adenine, but not guanine, resulted in complete restoration. Timed addition revealed that the ability of MTX to suppress antibody responses was diminished if added after 48 h of culture, similar to the reversal of this suppression mediated by hypoxanthine. Cell cycle analysis of LPS-stimulated B lymphocytes demonstrated that both drugs modulated events preceding, as well as during, the S phase. The present studies suggest that although drug-induced impairments in dTMP biosynthesis may be responsible for deficient lymphoid proliferation, anti-folate-induced impairment in purine biosynthesis is a major mechanism in anti-folate-induced suppression of humoral immunity.     

True absorption and endogenous fecal excretion of manganese in relation to its dietary supply in growing rats

A conventional balance study with 48 male weanling rats was conducted to determine true absorption and endogenous fecal excretion of manganese (Mn) in relation to dietary Mn supply, following the procedures of a previously adapted isotope dilution technique. After 10 d on a diet with 1.5 ppm Mn, eight animals each were assigned to diets containing 1.5, 4.5, 11.2, 35, 65, or 100 ppm Mn on a dry-matter basis. Three days later, each rat was given an intramuscular⁵⁴Mn injection and kept on treatment for a balance period of 16 d. Apparent Mn absorption assessed for the final 8 d, averaged 8.6 μg/d without significant treatment effects, although Mn intake ranged from 18.6 to 1200 μg/d, in direct relation to dietary Mn concentrations. Mean fecal excretion of endogenous Mn for the six treatments was 0.9, 2.7, 7.4, 11.0, 16.3, and 17.7 μg/d, respectively. These values delineate the rates to which true absorption exceeded apparent rates. True absorption, as percent of Mn intake, averaged 28.7, 15.9, 11.7, 6.1, 3.4, and 2.0, respectively, as compared with mean values of 23.9, 10.9, 6.2, 3.4, 1.2, and 0.5 for percent apparent absorption. It was concluded that both true absorption and endogenous fecal excretion markedly responded to Mn nutrition and that the reduction in the efficiency of true absorption was quantitatively the most significant homeostatic response for maintaining stable Mn concentrations in body tissues.     

Change in apparent and true absorption and retention of dietary zinc with age in rats

Two groups of 16 rats each were fed the same diet with 12.9 ppm Zn. Nine days after each animal was injected with⁶⁵Zn for assessing fecal zinc of endogenous origin, zinc intake and excretion were determined for a six-day period at the age of about five (group I) and nine (II) weeks. At mean growth rates of 5.1 and 5.2 g/day, food consumption per gram of gain was 2.01 g in group I vs 2.86 g in II. Overall, zinc retention amounted to 21 vs 25 μg Zn/g of gain. Apparent absorption averaged 92 vs 74% of Zn intake (132 vs 189 μg/day), while true absorption averaged 98 vs 92%. It was concluded that endogenous fecal zinc excretion was limited to the indispensable loss (F _em) in group I (7 μg/day), while it exceeded this minimum loss in group II (33 μg/day). True retention, which reflected total zinc utilization (true absorption times metabolic efficiency), was derived from apparent absorption plusF _em (11 μg/day for group II according to the greater metabolic body size of the rats). It averaged 98% of Zn intake in group I vs 80% in group II. The mean metabolic efficiency was 100% vs 87%. The conclusion was that these marked differences between age groups in utilizing the dietary zinc reflected the efficient homeostatic adjustments in absorption and endogenous excretion of zinc to the respective zinc supply status.     

Familial hemiplegic migraine mutations affect Na,K-ATPase domain interactions

Familial hemiplegic migraine (FHM) is a monogenic variant of migraine with aura. One of the three known causative genes, ATP1A2, which encodes the α2 isoform of Na,K-ATPase, causes FHM type 2 (FHM2). Over 50 FHM2 mutations have been reported, but most have not been characterized functionally. Here we study the molecular mechanism of Na,K-ATPase α2 missense mutations. Mutants E700K and P786L inactivate or strongly reduce enzyme activity. Glutamic acid 700 is located in the phosphorylation (P) domain and the mutation most likely disrupts the salt bridge with Lysine 35, thereby destabilizing the interaction with the actuator (A) domain. Mutants G900R and E902K are present in the extracellular loop at the interface of the α and β subunit. Both mutants likely hamper the interaction between these subunits and thereby decrease enzyme activity. Mutants E174K, R548C and R548H reduce the Na⁺ and increase the K⁺ affinity. Glutamic acid 174 is present in the A domain and might form a salt bridge with Lysine 432 in the nucleotide binding (N) domain, whereas Arginine 548, which is located in the N domain, forms a salt bridge with Glutamine 219 in the A domain. In the catalytic cycle, the interactions of the A and N domains affect the K⁺ and Na⁺ affinities, as observed with these mutants. Functional consequences were not observed for ATP1A2 mutations found in two sporadic hemiplegic migraine cases (Y9N and R879Q) and in migraine without aura (R51H and C702Y).