Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the -calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m-calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of -calpain by genetic inactivation using homologous recombination in embryonic stem cells. The -calpain-deficient mice are viable and fertile. The complete deficiency of -calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the ␤3 subunit of ␣IIb␤3 integrin, talin, and ABP-280 (filamin). However, -calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the ␤3 subunit of ␣IIb␤3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that -calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins.The calpains are a family of calcium-dependent neutral cysteine proteases present in essentially all tissues of higher animals (8,34,37). Calpain homologues distantly related to the catalytic subunits of conventional calpains are also found in lower organisms such as parasites, insects, nematodes, fungi, and yeast (34). They are believed to play functionally important roles in diverse biological processes such as reorganization of cortical cytoskeleton, cell motility, cell proliferation, apoptosis, and hemostasis (9,27,31,39). Calpains are divided into two broad classes, ubiquitous and tissue specific. Calpain I (also referred to as -calpain) and calpain II (also referred to as m-calpain) are expressed in all tissues in varying amounts and share ϳ61% sequence identity (20). Both the -and m-calpains contain an 80-kDa catalytic subunit that forms a heterodimer with the regulatory 30-kDa subunit (34). The 80-kDa catalytic subunits of the -and m-calpains are products of separate but closely related genes (referred to as Capn1 and Capn2, respectively), while the 30-kDa subunit (encoded by the Capn4 gene) is common to both (34). The -calpain is fully active in micromolar concentrations of calcium, while the mcalpain requires millimolar calcium concentrations for full activation. Larger tissue-specific calpains have been cloned from stomach and smooth muscle tissues (35, 37). Mutations of the muscle-specific Capn3 (calpain 3 gene) have been shown to cause one form of limb-girdle muscular dystrophy type 2A (30). More recently, several groups have identified CAPN10 (calpain 10) as the target gene for mutations in...

The interleukin (IL)‐3 family of cytokines mediates its numerous effects on myeloid growth and maturation by binding a family of related receptors. It has been shown recently that IL‐3 induces the activation of two distinct cytoplasmic signal transducing factors (STFs) that are likely to mediate the induction of immediate early genes. In immature myeloid cells, IL‐3 activates STF‐IL‐3a, which comprises two tyrosine‐phosphorylated DNA binding proteins of 77 and 80 kDa. In mature myeloid cells, IL‐3 and granulocyte‐macrophage colony‐stimulating factor activate STF‐IL‐3b, which consists of a 94 and 96 kDa tyrosine‐phosphorylated DNA binding protein. Peptide sequence data obtained from the purified 77 and 80 kDa proteins (p77 and p80) indicate that they are closely related but are encoded by distinct genes. Both peptide and nucleotide sequence data demonstrate that these two proteins are the murine homologs of ovine mammary gland factor (MGF)/Stat5. The peptide data also indicate that p77 and p80 are phosphorylated on tyrosine 699, a position analogous to the tyrosine that is phosphorylated in Stat1 and Stat2 in response to interferon. Additionally, antiserum raised against bacterially expressed p77/p80 recognizes the 94 and 96 kDa protein components of STF‐IL‐3b, suggesting that these may be additional isoforms of Stat5. These studies indicate that the IL‐3 family of ligands is able to activate multiple isoforms of the signal transducing protein Stat5.

The interleukin-3 family of cytokines, which play an important role in the development of myeloid lineages, transduce signals through the JAK-STAT pathway. Previous studies demonstrate that this process entails the activation of four distinct isoforms of STAT5, where two shorter isoforms are activated in a distinct population of cells. We now demonstrate that the shorter isoforms represent carboxy-terminal truncations. Moreover, these truncations are not generated by RNA processing, but by a specific proteolytic activity. Consistent with the notion that truncated STAT5 isoforms transduce distinct signals, they fail to promote the activation of several known interleukin-3 target genes. These studies suggest that the activity of a specific protease may play a critical role in defining the biological responses transduced by STAT5.

Dematin is an actin-binding and bundling protein of the erythrocyte membrane skeleton. Dematin is localized to the spectrin-actin junctions, and its actin-bundling activity is regulated by phosphorylation of cAMP-dependent protein kinase. The carboxyl terminus of dematin is homologous to the ''headpiece'' domain of villin, an actin-bundling protein of the microvillus cytoskeleton. The headpiece domain contains an actin-binding site, a cAMP-kinase phosphorylation site, plays an essential role in dematin self-assembly, and bundles F-actin in vitro. By using homologous recombination in mouse embryonic stem cells, the headpiece domain of dematin was deleted to evaluate its function in vivo. Dematin headpiece null mice were viable and born at the expected Mendelian ratio. Hematological evaluation revealed evidence of compensated anemia and spherocytosis in the dematin headpiece null mice. The headpiece null erythrocytes were osmotically fragile, and ektacytometry͞micropore filtration measurements demonstrated reduced deformability and filterability. In vitro membrane stability measurements indicated significantly greater membrane fragmentation of the dematin headpiece null erythrocytes. Finally, biochemical characterization, including the vesicle͞cytoskel-eton dissociation, spectrin self-association, and chemical crosslinking measurements, revealed a weakened membrane skeleton evidenced by reduced association of spectrin and actin to the plasma membrane. Together, these results provide evidence for the physiological significance of dematin and demonstrate a role for the headpiece domain in the maintenance of structural integrity and mechanical properties of erythrocytes in vivo.T he membrane bilayer and the network of membrane-associated proteins together regulate the characteristic shape and elastic properties of red blood cells (1, 2). When membrane skeletons are prepared in the presence of a high concentration of monovalent salt, the core of the membrane skeleton consists of spectrin, actin, protein 4.1, and dematin (3). Although the functions of spectrin, actin, and protein 4.1 have been extensively characterized (4, 5), virtually nothing is known about the physiological function in mature erythrocytes of dematin, a three-subunit protein that migrates in the protein 4.9 region during electrophoresis. The earliest evidence suggesting a membrane stabilizing role for dematin came from Holdstock and Ralston (6). They demonstrated that charged sulfhydryl compounds such as p-chloromercuribenzene sulfonateS preferentially attach to dematin and cause the disruption of erythrocyte cytoskeletons. Dematin is a substrate for multiple protein kinases, and phosphorylation of dematin by the cAMP-dependent protein kinase is known to regulate dematin's actin-bundling activity in vitro (7-9). The major phosphorylation site of the cAMPdependent protein kinase is located within the headpiece domain of dematin (10), but the physiological significance of dematin phosphorylation is not known.Siegel and Branton (11) conducted the first ...

Background:Oral mucositis is the most frequently occurring painful and dose-limiting side-effect of radiation of the head and neck region. Few studies demonstrated that oral glutamine suspension may significantly reduce the duration and severity of objective oral mucositis during radiotherapy.Materials and Methods:A randomized, prospective single institutional case control study was performed between April 2012 and November 2012 comparing the influence of oral glutamine on radiation induced mucositis in head and neck malignancy patients. Seventy biopsy proven patients with head and neck cancer receiving primary or adjuvant radiation therapy were randomized to receive either oral glutamine suspension daily 2h before radiation in the study arm (10 g in 1000 ml of water) (n = 35) or nothing before radiation; control arm (n = 35).Results and Analysis:Total 32 patients (91.43%) in the glutamine arm and total 34 patients (97.15%) developed mucositis. Grade 3 mucositis (14.29%) and grade 4 mucositis (2.86%) in the study arm (who received oral glutamine) were significantly less (P = 0.02 and P = 0.04, respectively) in the glutamine arm. The mean duration of grade 3 or worse mucositis (grade 3 and grade 4) was significantly less (6.6 days vs. 9.2 days) in study arm with P < 0.001. Mean time of onset of mucositis was significantly delayed in patients who took glutamine in comparison to control arm with P < 0.001.Conclusion:Glutamine delays oral mucositis in the head neck cancer patients. Moreover, it reduces the frequency and duration of grade 3 and grade 4 mucositis.

No abstract

Cytosolic guanylyl cyclases (GTP pyrophosphate-lyase [cyclizing; EC 4.6.1.2]), primary receptors for nitric oxide (NO) generated by NO synthases, are obligate heterodimers consisting of an ␣ and a ␤ subunit. The ␣ 1/ ␤ 1 form of guanylyl cyclase has the greatest activity and is considered the universal form. An isomer of the ␤ 1 subunit, i.e., ␤ 2, has been detected in the liver and kidney, however, its role is not known. In this study, we investigated the function of ␤ 2. Immunoprecipitation experiments showed that the ␤ 2 subunit forms a heterodimer with the ␣ 1 subunit. NO-stimulated cGMP formation in COS 7 cells cotransfected with the ␣ 1 and ␤ 2 subunits was ‫ف‬ 1/3 of that when ␣ 1 and ␤ 1 subunits were cotransfected. The ␤ 2 subunit inhibited NO-stimulated activity of the ␣ 1/ ␤ 1 form of guanylyl cyclase and NOstimulated cGMP formation in cultured smooth muscle cells. Our results provide the first evidence that the ␤ 2 subunit can regulate NO sensitivity of the ␣ 1/ ␤ 1 form of guanylyl cyclase. Northern analysis for guanylyl cyclase subunits was performed on RNA from kidneys of Dahl salt-sensitive rats, which have been shown to have decreased renal sensitivity to NO. Compared to the Dahl salt-resistant rat, message for ␤ 2 was increased, ␤ 1 was decreased, and ␣ 1 was unchanged. These results suggest a molecular basis for decreased renal guanylyl cyclase activity, i.e., an increase in the ␣ 1/ ␤ 2 heterodimer, and decrease in the ␣ 1/ ␤ 1 heterodimer. (

Oxalic acid is present as nutritional stress in many crop plants like Amaranth and Lathyrus. Oxalic acid has also been found to be involved in the attacking mechanism of several phytopathogenic fungi. A full-length cDNA for oxalate decarboxylase, an oxalate-catabolizing enzyme, was isolated by using 5-rapid amplification of cDNA ends-polymerase chain reaction of a partial cDNA as cloned earlier from our laboratory (