Pituitary adenoma and concomitant Rathke's cleft cyst.

Although pituitary adenomas and Rathke's cleft cysts have a shared ancestry, they rarely occur simultaneously. Only 32 reports involving a pituitary adenoma and a concomitant Rathke's cleft cyst were identified upon review of the literature. Most initial presenting complaints include hormonal symptoms, visual disturbances, and headache. Next to growth hormone, Prolactin was the most commonly hypersecreted pituitary hormone. Rathke's cleft cysts show variable position, size, and signal intensity on magnetic resonance imaging (MRI). Here, we report a patient with a growth hormone- secreting pituitary adenoma associated with a Rathke's cleft cyst. The mass contained two different signal intensities on MRI. The lesion was successfully removed assisted by intraoperative MRI, when the presence of both lesions was confirmed. When a non-enhancing cyst-like structure is demonstrated on imaging in a patient with a pituitary adenoma, the possibility of a coexisting Rathke's cleft cyst should be considered.

Aminophylline Effect on Renal Ischemia-Reperfusion Injury in Mice.

BACKGROUND: Aminophylline increases the intracellular concentration of cAMP and exerts an anti-inflammatory effect. The aim of this study was to investigate the effect of aminophylline on renal ischemia-reperfusion (I/R) injury in mice. METHODS: Thirty C57BL/6 mice were divided into 3 groups. In the sham group (group S, n = 10), only right nephrectomy was performed. In the control group (group C, n = 10), after right nephrectomy, the mice were subjected to 30 minutes of left renal ischemia. In the aminophylline group (group A, n = 10), an intraperitoneal injection of aminophylline (5 mg/kg) was performed before renal ischemia. Twenty-four hours after reperfusion, the mice were euthanized, and plasma and kidney samples were obtained to analyze the serum creatinine, renal histology, and expression levels of nuclear factor-kappa B (NF-kB) and pro-inflammatory cytokines. RESULTS: The serum creatinine concentration in group C was markedly elevated at 24 hours after reperfusion. Aminophylline treatment significantly reduced serum creatinine, compared with group C. Aminophylline also reduced the histological evidence of renal damage. The expression levels of NF-kB, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and intercellular adhesion molecule-1 (ICAM-1) mRNA were significantly increased in group C (P < .001). Group A showed lower expression of NF-kB, TNF-α, MCP-1, MIP-2, and ICAM-1 mRNA than group C (P < .01). CONCLUSIONS: Aminophylline treatment improved the renal function and indexes of renal inflammation, which suggests that it provided reno-protection against renal I/R injury.

Identification of functional elements of p18INK4C essential for binding and inhibition of cyclin-dependent kinase (CDK) 4 and CDK6.

Members of the INK4 family of cyclin-dependent kinase (CDK) inhibitors specifically bind and inhibit the G1-specific CDK molecules CDK4 and CDK6. One of the INK4 molecules, p16, is also known as multiple tumor suppressor and has been found to be mutated or deleted in various tumors and cell lines. We have previously identified p18 as a member of the INK4 family. To determine the molecular basis for the inhibitory function of p18, we introduced 11 missense mutations of conserved residues that were identified in p16 of cancer cell lines into p18. The effects of these mutations on the ability of p18 to bind and inhibit CDK4 and CDK6 or to inhibit cell growth were determined. Our results indicate that the third ankyrin repeat and the NH2-terminal portion of the fourth repeat constitute the essential element necessary for the ability of p18 to bind and inhibit CDK4 and CDK6. Apart from this core interaction element, p18 seems to use additional, distinct residues to differentially bind and inhibit CDK4 and CDK6, accounting for the known penchant of p18 to preferentially interact with CDK6.

Molecular characterization of cDNA clones for ADP-glucose pyrophosphorylase from Citrus.

Two cDNA clones encoding ADP-glucose pyrophosphorylases have been isolated from fruit and leaf cDNA libraries of Citrus (Citrus unshiu Mac. cv. Miyagawa) in which one was designated as agpS for the small subunit and the other as agpL for the large subunit. Both cDNAs have uninterrupted open reading frames deriving 57-58 kDa polypeptides. The deduced amino acid sequence of agpS has a unique feature. That is, it lacks a cysteine residue (Cys-12) which is usually conserved in all other dicot enzymes. This is the first report of agpS lacking Cys-12 among dicot small subunits. The expression pattern of both subunits showed a different profile in which leaves synthesized both agpS and agpL more vigorously than those of fruits. During leaf development, the transcripts of agpS and agpL showed a higher expression level at younger stages. During fruit development, the expression level of both subunits was observed to be highest in the mini-green stage, but it decreased in the small green stage and it increased again towards the maturing stage. These results suggest that both subunits may play an important role in the regulation of Citrus fruit and leaf development.

Cloning and nucleotide sequence analysis of xylE gene responsible for meta-cleavage of 4-chlorocatechol from Pseudomonas sp. S-47.

Pseudomonas sp. S-47 expresses catechol 2,3-dioxygenase (C230) catalyzing the conversion of 4-chlorocatechol (4CC) as well as catechol to 5-chloro-2-hydroxymuconic semialdehyde and 2-hydroxymuconic semialdehyde, respectively, through meta-ring cleavage. The xylE gene encoding C230 for meta-cleavage was cloned from strain S-47 and its nucleotide sequence was analyzed. The pRES101 containing the xylE gene exhibited high C230 activity toward catechol and 4CC without altering the substrate specificity from natural strain. The xylE gene was composed of 924 bp and encoded polypeptide of molecular mass 35 kDa containing 307 amino acids. A deduced amino acid sequence of the C230 from strain S-47 exhibited over 80% identity with those of Pseudomonas putida mt-2, Pseudomonas putida G7, and Pseudomonas sp. CF600. However, it shows below 45% identity with those of Pseudomonas cepacia LB400 and Pseudomonas sp. KKS102. The C230 of strain S-47 was conserved in the amino acids (His150, His214, Glu261) for metal binding ligands and those (His199, His242, and Tyr251) for catalytic sites. Therefore, Pseudomonas sp. S-47 can be explained as acting by degrading catechol as well as 4CC by xylE-encoding C230 which was fused by N domain of nahH and C domain of dmpB from other Pseudomonas strains.

Spontaneous resolution of acute T cell-mediated rejection in a renal transplant patient.

This case report presents spontaneous resolution of acute rejection in a 66-year-old man who underwent a kidney transplant and developed acute rejection and pneumonia. Our main concern in this case was how to treat the concurrent infection while maintaining the immunosuppressive therapy with a narrow available therapeutic range, in order to save the renal allograft without increasing antirejection therapy.

A transcriptome-based examination of blood group expression.

Over the last two decades, red cell biologists witnessed a vast expansion of genetic-based information pertaining to blood group antigens and their carrier molecules. Genetic progress has led to a better comprehension of the associated antigens. To assist with studies concerning the integrated regulation and function of blood groups, transcript levels for each of the 36 associated genes were studied. Profiles using mRNA from directly sampled reticulocytes and cultured primary erythroblasts are summarized in this report. Transcriptome profiles suggest a highly regulated pattern of blood group gene expression during erythroid differentiation and ontogeny. Approximately one-third of the blood group carrier genes are transcribed in an erythroid-specific fashion. Low-level and indistinct expression was noted for most of the carbohydrate-associated genes. Methods are now being developed to further explore and manipulate expression of the blood group genes at all stages of human erythropoiesis.

Mesolimbic dopaminergic activity responding to acute stress is blunted in adolescent rats that experienced neonatal maternal separation.

Neonatal maternal separation (MS), stressful experience early in life, leads to the development of depression-like behaviors in the offspring later in life. This study was conducted to define the neural basis of depression-like behaviors observed in our MS model. Sprague-Dawley pups were separated from dam for 3 h daily during the first 2 weeks of birth (MS) or left undisturbed (NH). All pups were sacrificed on postnatal day 41 with/without 1 h of restraint stress. Restraint stress significantly increased c-Fos expression in the nucleus accumbens (NAcb) of NH pups, but not in MS. In NH pups, restraint stress increased dopamine levels not only in the NAcb but also in the midbrain dopamine neurons; however, these increases were not observed in MS. Gene expression of tyrosine hydroxylase (TH) in the ventral tegmental area (VTA) was increased by acute restraint in NH pups, but not in MS pups. The raphe serotonin level was lower in MS than in NH, and not significantly changed by acute restraint neither in NH nor in MS. Results reveal that experience of neonatal MS may lead to a long-term suppression in the mesolimbic dopamine system of the offspring later in life, in which an epigenetic control may be implicated, such as suppressed gene expression of TH in the midbrain. We conclude that a decreased activity of the mesolimbic dopamine system may play a role in the pathophysiology of depression-like behaviors by neonatal MS, in addition to a decreased serotonin level in the raphe nucleus.

Light propagation in moderately dense particle systems: a reexamination of the Kubelka-Munk theory.

A numerical method (NM) is developed to characterize radiative transfer in a moderately dense particle population, i.e., a suspension of concentration of <1-10% by volume. It assumes that the particles scatter in accord with the Mie equations, that the propagation of light over short distances is in accord with the exponential transmission law, and that the light flows in many (thirty-six) directions. For representative systems, predictions of the Kubelka-Munk theory (KMT) are compared with those of the NM; partial agreement is found. While this theory can be a useful tool, radiative transport in representative samples is found not to obey strictly either the assumptions for writing the basic differential equations of the KMT or those for solving them. The movement of diffuse light through an attenuating system is found to often collimate it, not to make it more diffuse as expected. This effect causes errors in absolute KMT predictions. New transport equations, like Schuster's, with four parameters instead of two are written and solved to obtain some new KMT equations. Their predictions are compared with those of the NM.

Inhibition of the adenylyl cyclase and activation of the phosphatidylinositol pathway in oocytes through expression of serotonin receptors does not induce oocyte maturation.

The identification of the initial signaling events induced by progesterone in Xenopus oocyte maturation was approached by expressing serotonin receptors and by using pharmacological agents. Suppression of phospholipase C (PLC) activity in oocytes by U-73122 or stimulation of oocyte adenylyl cyclase (ACase) by forskolin inhibited progesterone-induced reinitiation of meiotic cell division. Microinjection of cAMP-dependent protein kinase (PKA) pseudosubstrate, PKI, into oocytes--or pretreatment of oocytes with PKA inhibitor, H-89--did not induce oocyte maturation, but both treatments potentiated the rate of progesterone-induced germinal vesicle breakdown (GVBD). In addition, reduced PKA activity by H-89 reversed the inhibition of GVBD caused by U-73122. Expression and activation of the serotonin receptor type 1a or type 2c lowered intracellular cAMP level or mobilized Ins(1,4,5)P3, respectively. These oocytes, however, did not undergo maturation upon serotonin stimulation. Co-expression of these receptors also did not trigger maturation, but it significantly accelerated the rate of GVBD induced by progesterone. From these data, we conclude that (1) changes in levels of these second messengers may well be coupled with progesterone signaling; (2) an initial decrease in cAMP and production of Ins(1,4,5)P3/DG may not be absolute requisites for progesterone-induced meiotic maturation: (3) cross-talk mechanisms between adenylyl cyclase and phosphoinositide signal transduction pathways may exist in the process of progesterone-induced reinitiation of meiotic cell cycle.

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: functional coupling of the sphingosine-1-phosphate receptor to PLC-xbeta in Xenopus oocytes.

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca2+-dependent oscillatory Cl- currents by acting through membrane-bound receptors. External application of 50 microM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-microM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca2+-dependent Cl- currents were observed in the absence of extracellular Ca2+, were blocked by intracellular injection of the Ca2+ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca2+ ATPase. Intracellular Ca2+ release appeared to be from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, because Cl- currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xbeta), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xbeta, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein alpha subunits that were identified in Xenopus laevis; G(q)alpha, G11alpha, G0alpha, and G(i1)alpha. Among AS-ODNs against the G alphas tested, AS-G(q)alpha and AS-G(i1)alpha to S1P and AS-G(q)alpha and AS-G11alpha to LPA specifically reduced current responses, respectively, to about 20-30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca2+ from the IP3-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes.

Selective activation of MEK1 but not MEK2 by A-Raf from epidermal growth factor-stimulated Hela cells.

Activation of the mitogen-activated protein kinase cascade is a critical event in mitogenic growth factor signal transduction. Mitogen-activated protein kinase is directly activated by a dual specific kinase, MEK, which itself is activated by serine phosphorylation. The c-Raf kinase has been implicated in mediating the signal transduction from mitogenic growth factor receptors to MEK activation. Recently, the B-Raf kinase was shown to be capable of phosphorylating and activating MEK as a result of growth factor stimulation. In this report, we used the yeast two-hybrid screening to isolate MEK interacting proteins. All three members of the Raf family kinases were identified as positive clones when the mutant MEK1S218/222A, in which the two phosphorylation serine residues were substituted by alanines, was used as a bait, whereas no positive clones were isolated when the wild type MEK1 was used as a bait in a similar screening. These results suggest that elimination of the phosphorylation sites of a target protein (MEK1 in our study) may stabilize the interaction between the kinase (Raf) and its substrate (MEK1), possibly due the formation of a nonproductive complex. These observations seem to suggest a general strategy using mutants to identify the upstream kinase of a phosphoprotein or the downstream targets of a kinase. Although c-Raf and B-Raf have been implicated in growth factor-induced MEK activation, little is known about A-Raf. We observed that stimulation of Hela cells with epidermal growth factor resulted in a rapid and transient activation of A-Raf, which is then capable of phosphorylating and activating MEK1. Interestingly, A-Raf does not activate MEK2, although c-Raf can activate both MEK1 and MEK2. Our data demonstrated that A-Raf is, indeed, a MEK1 activator and may play a role in growth factor signaling.

Altering the nucleophile specificity of a protein-tyrosine phosphatase-catalyzed reaction. Probing the function of the invariant glutamine residues.

Protein-tyrosine phosphatases (PTPases) catalysis involves a cysteinyl phosphate intermediate, in which the phosphoryl group cannot be transferred to nucleophiles other than water. The dual specificity phosphatases and the low molecular weight phosphatases utilize the same chemical mechanism for catalysis and contain the same (H/V)C(X)5R(S/T) signature motif present in PTPases. Interestingly, the latter two groups of phosphatases do catalyze phosphoryl transfers to alcohols in addition to water. Unique to the PTPase family are two invariant Gln residues which are located at the active site. Mutations at Gln-446 (and to a much smaller extent Gln-450) to Ala, Asn, or Met (but not Glu) residues disrupt a bifurcated hydrogen bond between the side chain of Gln-446 and the nucleophilic water and confer phosphotransferase activity to the Yersinia PTPase. Thus, the conserved Gln-446 residue is responsible for maintaining PTPases' strict hydrolytic activity and for preventing the PTPases from acting as kinases to phosphorylate undesirable substrates. This explains why phosphoryl transfer from the phosphoenzyme intermediate in PTPases can only occur to water and not to other nucleophilic acceptors. Detailed kinetic analyses also suggest roles for Gln-446 and Gln-450 in PTPase catalysis. Although Gln-446 is not essential for the phosphoenzyme formation step, it plays an important role during the hydrolysis of the intermediate by sequestering and positioning the nucleophilic water in the active site for an in-line attack on the phosphorus atom of the cysteinyl phosphate intermediate. Gln-450 interacts through a bound water molecule with the phosphoryl moiety and may play a role for the precise alignment of active site residues, which are important for substrate binding and transition state stabilization for both of the chemical steps.

Swiveling-domain mechanism for enzymatic phosphotransfer between remote reaction sites.

The crystal structure of pyruvate phosphate dikinase, a histidyl multiphosphotransfer enzyme that synthesizes adenosine triphosphate, reveals a three-domain molecule in which the phosphohistidine domain is flanked by the nucleotide and the phosphoenolpyruvate/pyruvate domains, with the two substrate binding sites approximately 45 angstroms apart. The modes of substrate binding have been deduced by analogy to D-Ala-D-Ala ligase and to pyruvate kinase. Coupling between the two remote active sites is facilitated by two conformational states of the phosphohistidine domain. While the crystal structure represents the state of interaction with the nucleotide, the second state is achieved by swiveling around two flexible peptide linkers. This dramatic conformational transition brings the phosphocarrier residue in close proximity to phosphoenolpyruvate/pyruvate. The swiveling-domain paradigm provides an effective mechanism for communication in complex multidomain/multiactive site proteins.