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Kenneth E Bernstein

Publications and source records attributed to Kenneth E Bernstein.

27 records · Page 2Linked to original sources
PubMed2003

Janus kinase 2 determinants for growth hormone receptor association, surface assembly, and signaling.

GH signaling depends on functional interaction of the GH receptor (GHR) and the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), which possesses a C-terminal kinase domain, a catalytically inactive pseudokinase domain just N-terminal to the kinase domain, and an N-terminal half shown by us and others to harbor elements for GHR association. Computational analyses indicate that JAKs contain in their N termini ( approximately 450 residues) divergent FERM domains. FERM domains (or subdomains within them) in JAKS may be important for associations with cytokine receptors. For some cytokine receptors, JAK interaction may be required for receptor surface expression. We previously demonstrated that a JAK2 mutant devoid of its N-terminal 239 residues (JAK2-Delta1-239) did not associate with GHR and could not mediate GH- induced signaling. In this report we employ a JAK2-deficient cell line to further define N-terminal JAK2 regions required for physical and functional association with the GHR. We also examine whether JAK2 expression affects cell surface expression of the GHR. Our results suggest that FERM motifs play an important role in the interaction of GHR and JAK2. While JAK2 expression is not required for detectable surface GHR expression, an increased JAK2 level increases the fraction of GHRs that achieves resistance to deglycosylation by endoglycosidase H, suggesting that the GHR-JAK2 association may enhance either the receptor's efficiency of maturation or its stability. Further, we report evidence for the existence of a novel GH-inducible functional interaction between JAK2 molecules that may be important in the mechanism of GH-triggered JAK2 signaling.

Animals↗
PubMed2003

Role of bradykinin in angiotensin-converting enzyme knockout mice.

Angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system. Whereas ACE is responsible for the production of angiotensin II, it is also important in the elimination of bradykinin. Constitutively, the biological function of bradykinin is mediated through the bradykinin B(2) receptor. ACE knockout mice have a complicated phenotype including very low blood pressure. To investigate the role of bradykinin in the expression of the ACE knockout phenotype, we bred B(2) receptor knockout mice with ACE knockout mice, thus generating a line of mice deficient in both the B(2) receptor and ACE. Surprisingly, these mice did not differ from ACE knockout mice in blood pressure, urine concentrating ability, renal pathology, and hematocrit. Thus abnormalities of bradykinin accumulation do not play an important role in the ACE knockout phenotype. Rather, this phenotype appears due to the defective production of angiotensin II.

Animals↗
PubMed2003

New approaches to genetic manipulation of mice: tissue-specific expression of ACE.

The renin-angiotensin system (RAS) plays a central role in body physiology, controlling blood pressure and blood electrolyte composition. ACE.1 (null) mice are null for all expression of angiotensin-converting enzyme (ACE). These mice have low blood pressure, the inability to concentrate urine, and a maldevelopment of the kidney. In contrast, ACE.2 (tissue null) mice produce one-third normal plasma ACE but no tissue ACE. They also have low blood pressure and cannot concentrate urine, but they have normal indices of renal function. These mice, while very informative, show that the null approach to creating knockout mice has intrinsic limitations given the many different physiological systems that no longer operate in an animal without a functioning RAS. To investigate the fine control of body physiology by the RAS, we developed a novel promoter swapping approach to generate a more selective tissue knockout of ACE expression. We used this to create ACE.3 (liver ACE) mice that selectively express ACE in the liver but lack all ACE within the vasculature. Evaluation of these mice shows that endothelial expression of ACE is not required for blood pressure control or normal renal function. Targeted homologous recombination has the power to create new strains of mice expressing the RAS in selected subsets of tissues. Not only will these new genetic models be useful for studying blood pressure regulation but also they show great promise for the investigation of the function of the RAS in complicated disease models.

Animals↗
PubMed2003

Mice lacking endothelial ACE: normal blood pressure with elevated angiotensin II.

Recently, the concept of local renin-angiotensin systems (RAS) capable of generating angiotensin II apart from the circulation has received considerable attention. To investigate this, we generated ACE 1/3 mice in which one allele of ACE is null and the second allele was engineered to express ACE on the surface of hepatocytes. ACE 1/3 mice express no endothelial ACE and lack ACE within the lungs. Their kidneys contain <7.8% the enzyme levels present in control mice. Plasma conversion of angiotensin I to angiotensin II was 43.3% normal. The baseline blood pressure and renal function of the ACE 1/3 mice were normal, probably as a function of a marked increase of both plasma angiotensin I and angiotensin II. When exposed to 2 weeks of a salt-free diet (a stress diet stimulating the RAS), blood pressure in ACE 1/3 mice decreased to 92.3+/-2.0 mm Hg, a level significantly lower than that of wild-type control mice. The ACE 1/3 mice demonstrate the plasticity of the RAS and show that significant compensation is required to maintain normal, basal blood pressure in a mouse with an impaired local vascular and renal RAS.

Angiotensin I↗
PubMed2002

Mice lacking endothelial angiotensin-converting enzyme have a normal blood pressure.

To test the hypothesis that local vascular production of angiotensin II is necessary for the normal regulation of blood pressure, we engineered a new line of genetically altered mice that lack endothelial angiotensin-converting enzyme (ACE). This was accomplished using a novel strategy of targeted homologous recombination to separate the transcriptional control of somatic ACE from its endogenous promoter and to substitute control to the albumin promoter. These new mice, termed ACE.3, do not produce ACE within the lung, the aorta, or any vascular structure. ACE activity within the kidney is only about 14% that of wild-type mice and is limited to tubular epithelium. In contrast, hepatic ACE expression in ACE.3 mice is about 87-fold that of wild-type. The blood pressure, plasma angiotensin II levels, response to ACE inhibitors, and renal function of ACE.3(-/-) mice are indistinguishable from littermate wild-type mice. These data show that, under basal conditions, the normal regulation of blood pressure and renal function is possible in a mouse devoid of endothelial ACE.

Animals↗
PubMed2002

Mutation of glutamic acid residue 1046 abolishes Jak2 tyrosine kinase activity.

Jak2 is a member of the Janus family of tyrosine kinases and is known to be activated by a wide variety of ligands. Here, we sought to identify amino acid residues within Jak2 that are essential for its activation. We provide evidence that glutamic acid 1046 (E1046) is one such residue. Using molecular modeling algorithms of the Jak2 kinase domain, we identified a putative molecular interaction between E1046 and tryptophan 1020 (W1020). Conversion of E1046 to either arginine (E 1046R), alanine (E1046A), aspartic acid (E1046D) or glutamine (E1046Q) abolished Jak2 kinase activity as measured by autophosphorylation assays. Conversion of W1020 to glycine (W1020G) similarly abolished Jak2 kinase activity. Finally, we tested the ability of the E1046R mutant to activate the Jak/STAT signaling pathway in a ligand-dependent signaling system. The ability of angiotensin II to activate the Jak/STAT signaling pathway in cells expressing the E1046R mutant was severely compromised as measured by reduced (1) Jak2 autophosphorylation (2) Jak2 kinase activity (3) AT1/Jak2 co-association (4) Stat1 tyrosine phosphorylation and (5) angiotensin Il-mediated gene transcription. Thus, these studies demonstrate for the first time, the critical role of E1046 in mediating Jak2 activation and its subsequent downstream signaling events.

Angiotensin II↗
PubMed2002

Impaired urine concentration and absence of tissue ACE: involvement of medullary transport proteins.

ACE.2 mice lack all tissue angiotensin-converting enzyme (ACE) but have 33% of normal plasma ACE activity. They exhibit the urine-concentrating defect and hyperkalemia present in mice that lack all ACE, but in contrast to the complete knockout, ACE.2 mice have normal medullary histology and creatinine clearance. To explore the urine-concentrating defect in ACE.2 mice, renal medullary transport proteins were analyzed using Western blot analysis. In the inner medulla, UT-A1, ClC-K1, and aquaporin-1 (AQP1) were significantly reduced to 28 +/- 5, 6 +/- 6, and 39 +/- 5% of the level in wild-type mice, respectively, whereas AQP2 and UT-B were unchanged. In the outer medulla, Na(+)-K(+)-2Cl(-) cotransporter (NKCC2/BSC1) and AQP1 were significantly reduced to 56 +/- 11 and 29 +/- 6%, respectively, whereas Na(+)-K(+)-ATPase, UT-A2, UT-B, and AQP2 were unchanged, and renal outer medullary potassium channel was significantly increased to 711 +/- 187% of the level in wild-type mice. The abnormal expression of these transporters was similar in ACE.2 mice backcrossed onto a C57BL/6 or a Swiss background and was not rescued by ANG II infusion. We conclude that the urine-concentrating defect in ACE.2 mice is associated with, and may result from, downregulation of some or all of these key urea, salt, and water transport proteins.

Angiotensin II↗