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Biomedical subjects

E G Goldstein

Publications and source records attributed to E G Goldstein.

14 recordsLinked to original sources
PubMed1995

5'-AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms.

AMP-activated protein kinase (AMPK) and Ca2+/calmodulin (CaM)-dependent protein kinase I (CaMKI) are protein kinases that are regulated both by allosteric activation (AMP and Ca2+/CaM, respectively) and by phosphorylation by upstream protein kinases (AMPK kinase (AMPKK) and CaMKI kinase (CaMKIK), respectively). We now report that AMPKK can activate CaMKI and that, conversely, CaMKIK can activate AMPK. CaMKIK is 68-fold more effective at activating CaMKI than AMPK, while AMPKK is 17-fold more effective at activating AMPK than CaMKI. Our results suggest that CaMKIK and AMPKK are distinct enzymes dedicated to their respective kinase targets but with some overlap in their substrate specificities. The availability of alternative substrates for AMPKK and CaMKIK allowed the unequivocal demonstration that AMP and Ca2+/calmodulin promote the activation of AMPK and Ca2+/calmodulin promote the activation of AMPK and CaMKI, respectively, via three independent mechanisms: 1) direct activation of AMPK and CaMKI, 2) activation of AMPKK and CaMKIK, and 3) by binding to AMPK and CaMKI, inducing exposure of their phosphorylation sites. Since AMP and Ca2+/calmodulin each has a triple effect in its respective system, in vivo, the two systems would be expected to be exquisitely sensitive to changes in concentration of their respective activating ligands.

AMP-Activated Protein Kinase Kinases↗
PubMed1995

Human calcium-calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium-calmodulin dependent protein kinase I kinase.

Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1-306, was unable to bind Ca(2+)-CaM and was completely inactive thereby defining an essential component of the CaM-binding domain to residues C-terminal to 306. CaMKI 1-294 did not bind CaM but was fully active in the absence of Ca(2+)-CaM, indicating that residues 295-306 are sufficient to maintain CaMKI in an auto-inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25-fold by CaMKI kinase in a Ca(2+)-CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1-306 was unresponsive to CaMKI kinase, the 1-294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)-CaM although at a faster rate in its presence. These results indicate that the auto-inhibitory domain in CaMKI gates, in a Ca(2+)-CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)-CaM with increased activity.

Amino Acid Sequence↗
PubMed1995

Phosphorylation and activation of Ca(2+)-calmodulin-dependent protein kinase IV by Ca(2+)-calmodulin-dependent protein kinase Ia kinase. Phosphorylation of threonine 196 is essential for activation.

Purified pig brain Ca(2+)-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164) enhances, by up to 24-fold, the activity of recombinant CaM kinase IV in a reaction also requiring Ca(2+)-CaM and MgATP. The addition of brain extract, although capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation. Activation is accompanied by the development of significant (38%) Ca(2+)-CaM-independent CaM kinase IV activity. In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase-, Ca(2+)-CaM-, and MgATP-dependent manner. Phosphorylation occurs on multiple serine and threonine residues with a Ser-P:Thr-P ratio of approximately 3:1. The identical requirements for phosphorylation and activation and a linear relationship between extent of phosphorylation of CaM kinase IV and its activation state indicate that CaM kinase IV activation is induced by its phosphorylation. Replacement of Thr-196 of CaM kinase IV with a nonphosphorylatable alanine by site-directed mutagenesis abolishes both the phosphorylation and activation of CaM kinase IV, demonstrating that Thr-196 phosphorylation is essential for activation.

Amino Acid Sequence↗
PubMed1995

Identification of an M(r) 60,000 polypeptide unique to the meiotic spindle of the mouse oocyte.

The mouse oocyte expresses an M(r) 60,000 (p60) polypeptide that is associated with the first and second meiotic spindles. Immunoreactive p60 was not detectable in the meiotic spindles of male germ cells or in mitotic spindles. P60 was identified with a polyclonal antibody whose predominant activity is directed against ankyrin. However, immunoadsorption experiments demonstrated that p60 is not an ankyrin isoform and represents a secondary activity of the polyclonal antibody. Circumstantial evidence suggest that p60 may be a microtubule-associated protein. Since the most obvious difference between the female meiotic spindle and other spindles is the long half-life of the former, we hypothesize that p60 may function in the maintenance of the long-lived female meiotic apparatus.

Animals↗
PubMed1991

Immunofluorescence localization of an adducin-like protein in the chromosomes of mouse oocytes.

The mouse oocyte expresses a polypeptide of Mr 120,000 that cross-reacts with an antibody to the brain membrane skeletal protein adducin. Immunofluorescence localization showed a bright chromosomal staining reaction in metaphase I and metaphase II oocytes. Following in vitro fertilization the maternal chromosomes lost their immunoreactivity during pronuclear development. The fertilizing sperm chromatin and male pronucleus did not show any detectable staining reaction. Bright chromosomal fluorescence was again observed in the first mitotic metaphase when both maternal and paternal chromosomes gave a positive staining reaction. In contrast to the immunoreactivity of the maternal meiotic chromosomes, the meiotic chromosomes of male germ line cells failed to exhibit any detectable staining reaction and this difference was confirmed by immunolabeling of oocyte and spermatocyte karyotypes. Mitotic chromosomes in preimplantation embryos, fetal liver, adult intestinal epithelium, and MDCK cells also failed to show any detectable labeling reaction. The results suggest that expression of the immunoreactive chromosomal adducin may be a unique feature of oogenesis.

Animals↗
PubMed1988

Spectrin and calmodulin in spreading mouse blastomeres.

The role of spectrin and its association with calmodulin in spreading mouse blastomeres was investigated. Embryonic spectrin binds 125I-calmodulin in a calcium-dependent fashion in the blot overlay technique. Double-labeling experiments show coordinate redistribution of spectrin and calmodulin in blastomeres preparing to undergo active spreading movement. At this stage cortical spectrin staining is lost from the region of cell-substrate contact and spectrin and calmodulin become concentrated in two structures closely associated with the contacted region: a group of spherical bodies located on the cytoplasmic side of the cortical layer and a subcortical ring that marks the perimeter of the contacted region. The localization pattern of spectrin and calmodulin is also coordinated with that of actin and myosin. The results suggest that spectrin plays a role in the spreading of blastomeres and that this function may involve linkage of spectrin, calmodulin, and the cortical contractile apparatus.

Actins↗
PubMed1988

Spectrin synthesis in the preimplantation mouse embryo.

The preimplantation mouse embryo expresses two polypeptides, Mr 240,000 and Mr 235,000, that are immunologically cross-reactive with antibody to the alpha and beta subunits of mouse brain spectrin. We investigated the synthesis of the spectrin subunits in the Triton-soluble and Triton-insoluble fractions of fertilized eggs, two-cell embryos, compacted morulae, and blastocysts labeled with L-[35S]methionine. Synthesis of embryonic spectrin began in the Triton-soluble fraction with significant levels of alpha-spectrin synthesis first detected in the morula stage and significant levels of beta-spectrin synthesis detected in the blastocyst stage. Incorporation of newly synthesized alpha- and beta-spectrin into the cytoskeletal fraction took place in the blastocyst when equal amounts of both subunits were assembled. Previous studies have shown Triton-insoluble spectrin to be concentrated in regions of cell-cell contact in the embryo (J. S. Sobel and M. A. Alliegro, 1985, J. Cell Biol. 100, 333-336). The temporal and spatial correlation between the assembly of newly synthesized spectrin and its concentration in regions of cell apposition is consistent with the hypothesis that cell contact may influence the assembly of embryonic spectrin.

Animals↗
PubMed1986

Modulation of bone metabolism by two chemically distinct lipopolysaccharide fractions from Bacteroides gingivalis.

Two separate species of lipopolysaccharide (LPS) from Bacteroides gingivalis 381 have been isolated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated not only the heterogeneity of each species, but also that they represented high- and low-molecular-weight LPS entities. Although both contained the same carbohydrate and fatty acid components, the proportions of these differed between the LPS species. The direct effects of these two species in modulation of bone resorption and bone collagen and noncollagen protein synthesis have been examined. In a bone resorption assay, these two species stimulated 45Ca release from prelabeled fetal rat bones in a concentration range of 0.5 to 3.0 micrograms/ml. The two LPS species also elicited a 30 to 40% reduction in collagen protein formation at 10 micrograms/ml. Responses of the same order of magnitude were observed with LPS from Salmonella minnesota at 10 micrograms/ml. The higher-molecular-weight LPS species also significantly inhibited noncollagen protein formation. This is the first report that LPS from B. gingivalis 381, a suspected periodontal pathogen, inhibits bone collagen formation and, in conjunction with the bone resorption potency, further implicates LPS in alveolar bone loss associated with periodontal disease.

Bacteroides↗
PubMed1981

Approaches to the diagnosis of borderline conditions by use of psychological tests.

The use of psychological tests in the diagnosis of borderline conditions by application of five approaches to the test battery is illustrated on a patient who was diagnosed as borderline by the criteria of both Gunderson and Kernberg. These approaches in terms of (a) structural variables, (b) response to confrontation, (c) severity of illness, (d) symptom diagnosis, and (e) differential response to tests of varying structure, may be used to strengthen each other and to facilitate the diagnosis of borderline patients.

Adult↗
PubMed1981

Diagnosing borderline personality. A pilot study using multiple diagnostic methods.

The research described in this paper stemmed from the hypothesis that borderline personality organization can be differentiated from neurotic and psychotic levels of personality organization by means of three structural criteria: degree of identity integration, level of defensive operations, and capacity for reality testing. In order to elicit these criteria, the "structural" interview has been developed that focuses on the "here-and-now" patient-interviewer interaction. The patient's responses to the interviewer's attempts to clarify, confront, and interpret various aspects of the patient's interview behavior provide the basis for judgments as to the patient's structural diagnosis. Specifically, the paper reports a study of the differential diagnosis of 48 hospitalized patients in which structural diagnoses of borderline or psychotic personality organization were made according to this diagnostic interview approach. These diagnoses were compared with ones obtained from Gunderson's Diagnostic Interview for Borderlines, with psychological test diagnoses, and with clinical diagnoses based on past history and current illness. Results show substantial convergent agreement among all of the diagnostic methods and support the utility of the structural interview. In most discrepant cases, other methods reflected disagreement among themselves despite the diagnoses obtained from the structural interview, suggesting that there are some cases difficult to classify by any means. Further analysis suggests that the structural interview may be eliciting a different dimension of personality functioning in arriving at borderline diagnoses than do the other methods studied. The results also indicate that borderline structural diagnoses refer to patients described clinically as having severe character pathology, and do not overlap with patients described as having schizophrenic disorders. The structural interview appears to warrant further study, and, at the same time, shows promise as a research tool in further studies of structural diagnosis and its relevance for prognosis and treatment.

Adolescent↗
PubMed1980

The role of the hospital milieu in structural diagnosis.

The authors have found the structural approach to diagnosis to be a valuable tool in classifying ill-defined mental illnesses. That approach presumes there are discrete levels of mental functioning: neurotic, borderline, and psychotic. Each can be differentiated by identifying the degree of identity integration, the level of defensive operations, and the degree of reality testing. Two case illustrations are used to describe how the dynamic role of the hospital milieu can be used in conjunction with individual treatment to make an accurate diagnosis. The application of this approach to the diagnosis of adolescents is emphasized.

Adolescent↗
PubMed1980

Borderline personality organization, structural diagnosis and the structural interview. A pilot study of interview analysis.

Kernberg has related the level of integration of the personality to the level of integration (and "structuring") of internalized object relations. He has designated three levels of personality organization: neurotic, borderline, and psychotic, and has developed a specially focused clinical interview designed to reveal intrapsychic structural characteristics--a "structural interview"--as a diagnostic instrument to differentiate the three types of personality organization. This study presents a method of analysis of the structural interview. Interviews of ten hospitalized psychiatric patients were studied to determine whether indicators could be retrieved from the typescripts of the interviews consonant with the diagnoses made by clinicians utilizing Kernberg's structural theory. Results indicate that scores generated from the typescripts are consistent with the hypothesis that the interviews did contain the indicators called for by structural theory and differentiate borderline from psychotic structures better than chance. In addition, this method of analysis of contingencies of interaction in the interview may have broader application to the study of psychotherapy and psychoanalysis.

Adult↗
PubMed1979

Psychological tests and borderline patients.

Psychological tests of 32 borderline or nonborderline (psychotic) patients were compared with the structural diagnoses arrived at on the basis of two kinds of clinical-research interviews: The DIB (following Gunderson's criteria) and the structural interview (following Kernberg's criteria). Test results were reported in terms of the diagnosis based on the full test battery, as well as in terms of the structural diagnosis implied by the presence or absence of thinking disturbances on the (structured) WAIS as compared with the (unstructured) Rorschach test. Statistically significant agreement was shown among these four approaches.

Adolescent↗