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Biomedical subjects

K Levine

Publications and source records attributed to K Levine.

At least 19 recordsLinked to original sources
PubMed2000

Genetic analysis of the relationship between activation loop phosphorylation and cyclin binding in the activation of the Saccharomyces cerevisiae Cdc28p cyclin-dependent kinase.

We showed recently that a screen for mutant CDC28 with improved binding to a defective Cln2p G1 cyclin yielded a spectrum of mutations similar to those yielded by a screen for intragenic suppressors of the requirement for activation loop phosphorylation (T169E suppressors). Recombination among these mutations yielded CDC28 mutants that bypassed the G1 cyclin requirement. Here we analyze further the interrelationship between T169E suppression, interaction with defective cyclin, and G1 cyclin bypass. DNA shuffling of mutations from the various screens and recombination onto a T169E-encoding 3' end yielded CDC28 mutants with strong T169E suppression. Some of the strongest T169E suppressors could suppress the defective Cln2p G1 cyclin even while retaining T169E. The strong T169E suppressors did not exhibit bypass of the G1 cyclin requirement but did so when T169E was reverted to T. These results suggested that for these mutants, activation loop phosphorylation and cyclin binding might be alternative means of activation rather than independent requirements for activation (as with wild type). These results suggest mechanistic overlap between the conformational shift induced by cyclin binding and that induced by activation loop phosphorylation. This conclusion was supported by analysis of suppressors of a mutation in the Cdk phosphothreonine-binding pocket created by cyclin binding.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2000

Williams syndrome and happiness.

Williams syndrome is a genetic disorder resulting in a variety of medical and developmental features, one of which is a frequent outward presentation of substantial happiness. In this paper we describe the unique expression of happiness in people with Williams syndrome, with several anecdotes and a frame by frame conversational analysis. We then discuss this happiness in the context of other dimensions of the impact of Williams syndrome, especially anxiety. We conclude with a discussion of the role of genetics in emotions.

Adult↗
PubMed1999

Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase.

To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.

Alleles↗
PubMed1999

Hypoglycinaemia and psychomotor delay in a child with xeroderma pigmentosum.

Glycine is a nonessential amino acid that serves as both an inhibitory and an excitatory neurotransmitter. Hyperglycinaemia occurs in non-ketotic hyperglycinaemia, a primary defect in the glycine cleavage pathway, and as a secondary feature of several inborn errors of organic acid metabolism. However, specifically low levels of glycine have never been reported. Here we report a child with complementation group C xeroderma pigmentosum (XP) characterized by a splice donor mutation in the XPC gene, multiple skin cancers and specific and persistent hypoglycinaemia. He has cognitive delay, lack of speech, autistic features, hyperactivity and hypotonia, all unexplained by the diagnosis of XP group C, a non-neurological form of the disease. Treatment with oral glycine has improved his hyperactivity. Specific hypoglycinaemia could indicate a metabolic disorder producing neurological dysfunction. Whether it is related to or coincidental with the XP is unclear.

Amino Acids↗
PubMed1998

Isolation and characterization of new alleles of the cyclin-dependent kinase gene CDC28 with cyclin-specific functional and biochemical defects.

The G1 cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 was significantly deficient in other aspects of genetic interaction with Cln2. The cdc28-csr1 mutation was determined to be Q188P, in the T loop distal to most of the probable Cdk-cyclin interaction regions. We performed random mutagenesis of CDC28 to identify additional alleles incapable of causing CLN2-dependent mating-factor resistance but capable of complementing cdc28 temperature-sensitive and null alleles. Two such mutants had highly defective Cln2-associated kinase, but, surprisingly, two other mutants had levels of Cln2-associated kinase near to wild-type levels. We performed a complementary screen for CDC28 mutants that could cause efficient Cln2-dependent mating-factor resistance but not complement a cdc28 null allele. Most such mutants were found to alter residues essential for kinase activity; the proteins had little or no associated kinase activity in bulk or in association with Cln2. Several of these mutants also functioned in another assay for CLN2-dependent function not involving the mating-factor pathway, complementing the temperature sensitivity of a cln1 cln3 cdc28-csr1 strain. These results could indicate that Cln2-Cdc28 kinase activity is not directly relevant to some CLN2-mediated functions. Mutants of this sort should be useful in differentiating the function of Cdc28 complexed with different cyclin regulatory subunits.

Alleles↗
PubMed1998

Molecular evolution allows bypass of the requirement for activation loop phosphorylation of the Cdc28 cyclin-dependent kinase.

Many protein kinases are regulated by phosphorylation in the activation loop, which is required for enzymatic activity. Glutamic acid can substitute for phosphothreonine in some proteins activated by phosphorylation, but this substitution (T169E) at the site of activation loop phosphorylation in the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28p blocks biological function and protein kinase activity. Using cycles of error-prone DNA amplification followed by selection for successively higher levels of function, we identified mutant versions of Cdc28p-T169E with high biological activity. The enzymatic and biological activity of the mutant Cdc28p was essentially normally regulated by cyclin, and the mutants supported normal cell cycle progression and regulation. Therefore, it is not a requirement for control of the yeast cell cycle that Cdc28p be cyclically phosphorylated and dephosphorylated. These CDC28 mutants allow viability in the absence of Cak1p, the essential kinase that phosphorylates Cdc28p-T169, demonstrating that T169 phosphorylation is the only essential function of Cak1p. Some growth defects remain in suppressed cak1 cdc28 strains carrying the mutant CDC28 genes, consistent with additional nonessential roles for CAK1.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1997

Treatment of depression in older primary care patients in health maintenance organizations.

OBJECTIVE: To examine whether older HMO patients with depression are treated differently than younger patients in terms of diagnosis, treatment by specialty provider, and pharmacotherapy. DESIGN: Chart-review, Cross sectional study. SETTING AND PARTICIPANTS: Patients were selected from six HMOs in the United States who had one or more of five medical diagnoses: arthritis, asthma, otitis media, epigastric pain/ulcer, and hypertension, (n = 9143). From this group, chart diagnoses and pharmacy records were used to identify patients who also had a diagnosis of depression (n = 416) or who had a diagnosis of depression and/or treatment with antidepressant medication (n = 1286). MEASUREMENT: Medical records and computerized service and pharmacy records were reviewed to obtain diagnoses, office visits by provider type, and psychiatric medication prescription counts. RESULTS: Significant differences were found in treatment of depression for older versus younger patients. Although depression was identified at a similar rate for both groups, older patients received fewer mental health specialty visits and fewer prescriptions for SSRI antidepressants. Older patients with a diagnosis of depression were more likely to be treated with benzodiazepines (49.2% of older vs. 33.2% of younger) though they were less likely to receive long half-life benzodiazepines. CONCLUSIONS: Psychotropic medication management is an important target for improving quality of care for older patients with depression in HMOs. Decreasing inefficient minor tranquilizer use and increasing use of newer antidepressant medications may lead to improved outcomes for older depressed adults.

Adult↗
PubMed1997

[Capgras' syndrome].

We present 3 cases of Capgras' syndrome-a delusional disorder in which the patient believes that 1 (or more) of his acquaintances has been replaced by an imposter who appears as a double. 2 were schizophrenics and 1 had depression with psychotic features. This syndrome is rare in our practice, but we do not know if this is due to lack of awareness of the condition, or to the possibility that it is a culture-related syndrome. We suggest that although the syndrome has lost some of it's significance, it is still worth making the diagnosis because of the medical and psychological implications this condition carries.

Adult↗
PubMed1996

Saccharomyces cerevisiae G1 cyclins differ in their intrinsic functional specificities.

The three budding yeast CLN genes appear to be functionally redundant for cell cycle Start: any single CLN gene is sufficient to promote Start, while the cln1 cln2 cln3 triple mutant is Start defective and inviable. Both quantitative and apparently qualitative differences between CLN genes have been reported, but available data do not in general allow distinction between qualitative functional differences as opposed to simply quantitative differences in expression or function. To determine if there are intrinsic qualitative differences between Cln proteins, we compared CLN2, CLN3, and crippled (but still partially active) CLN2 genes in a range of assays that differentiate genetically between CLN2 and CLN3. The results suggest that different potencies of Cln2, Cln3, and Cln2 mutants in functional assays cannot be accounted for by a simple quantitative model for their action, since Cln3 is at least as active as Cln2 and much more active than the Cln2 mutants in driving Swi4/Swi6 cell cycle box (SCB)-regulated transcription and cell cycle initiation in cln1 cln2 cln3 bck2 strains, but Cln3 has little or no activity in other assays in which Cln2 and the Cln2 mutants function. Differences in Cln protein abundance are unlikely to account for these results. Cln3-associated kinase is therefore likely to have an intrinsic in vivo substrate specificity distinct from that of Cln2-associated kinase, despite their functional redundancy. Consistent with the idea that Cln3 may be the primary transcriptional activator of CLN1, CLN2, and other genes, the activation of CLN2 transcription was found to be sensitive to the gene dosage of CLN3 but not to the gene dosage of CLN2.

Cell Cycle↗
PubMed1995

Structuring cell-cycle biology.

The recent determination of the crystal structures of both cyclin A and the cyclin a-CDK2 complex provides new insight into the cyclin- dependent activation of cyclin-dependent protein kinases.

Animals↗
PubMed1995

The CLN gene family: central regulators of cell cycle Start in budding yeast.

The Start transition in the budding yeast cell cycle is the point of most physiological regulation of cell cycle commitment. This transition is controlled by the CLN1,2,3 gene family. We review what is known about the regulation, inter-regulation and function of these genes in controlling the Start transition.

Cell Cycle↗
PubMed1994

Molecular diagnosis and monitoring of acute promyelocytic leukemia treated with retinoic acid.

The characteristic balanced 15;17 translocation, t(15;17), of acute promyelocytic leukemia (APL) fuses the retinoic acid receptor alpha (RAR alpha) gene on chromosome 17 to PML, a recently described gene of unknown function, on chromosome 15. It is this fusion gene and consequent fusion protein that is thought to be responsible for both the block in normal myelocyte differentiation as well as the dramatic in vitro and in vivo response to the differentiating effects of all-trans retinoic acid (RA). The t(15;17) also provides a genetic marker for the presence of leukemic cells. PML/RAR alpha fusion mRNA's can be detected by a reverse transcription polymerase chain reaction (RT-PCR) assay. Using this assay, at least three distinct patterns, differing in the 3' region of the PML breakpoint, can be identified. The detection of abnormal mRNA's by the RT-PCR assay has proven to be an important aid in the diagnosis of APL as well as the best predictor of an initial clinical response to RA. The results of an ongoing, longitudinal evaluation of patients with APL show that the RT-PCR assay may also be a useful indicator of minimal residual disease (MRD). Negative RT-PCR assays following completion of all therapy are associated with prolonged disease free survival, whereas persistence or return of a positive test is highly correlated with subsequent relapse. Further studies will determine whether patients who test positive may benefit from the introduction of additional antileukemic therapy.

Humans↗
PubMed1993

Detection of minimal residual disease in acute promyelocytic leukemia by a reverse transcription polymerase chain reaction assay for the PML/RAR-alpha fusion mRNA.

The characteristic reciprocal translocation t(15;17) of acute promyelocytic leukemia (APL) disrupts the PML gene on chromosome 15 and the retinoic acid receptor-alpha (RAR-alpha) gene on chromosome 17. PML/RAR-alpha fusion mRNAs are then transcribed and can be detected by a newly described reverse transcription polymerase chain reaction (RT-PCR) assay. Using RT followed by nested PCR amplification for PML/RAR-alpha, we serially evaluated bone marrow aspirates from patients with APL who were treated with all-trans retinoic acid (RA) for induction, followed by all-trans RA as maintenance or cytotoxic drugs as consolidation. At diagnosis, PML/RAR-alpha mRNA was detected in all patients. After initial therapy with all-trans RA, the RT-PCR assay remained positive after induction of complete remission in 31 of 32 evaluable patients. Maintenance treatment by all-trans RA alone was associated with persistent assay positivity and subsequent clinical relapse in 13 of 13 patients. By contrast, the test became negative in 19 of 20 newly diagnosed patients who received consolidation chemotherapy; the 1 patient who remained positive relapsed at 12 months. Three of the 19 assay-negative patients later converted to positive and subsequently relapsed; the remaining 16 patients have remained RT-PCR negative in sustained first remission, with a median follow-up duration that exceeds 24 months (range, 12+ to 34+ months). Despite induction of complete remission in a high proportion of patients, all-trans RA rarely eradicates molecular evidence of disease in patients with APL; however, subsequent treatment with cytotoxic chemotherapy frequently converts the RT-PCR assay for PML/RAR-alpha to negative. Serial negative tests are associated with prolonged disease-free survival, whereas persistence of a positive test after treatment is highly correlated with subsequent relapse. This test identifies patients in remission at high risk for relapse who may benefit from additional antileukemic therapy.

Antineoplastic Agents↗
PubMed1992

Reverse transcription polymerase chain reaction for the rearranged retinoic acid receptor alpha clarifies diagnosis and detects minimal residual disease in acute promyelocytic leukemia.

The characteristic t(15;17) of acute promyelocytic leukemia (APL) fuses the retinoic acid receptor alpha (RAR-alpha) gene on chromosome 17 to a gene on chromosome 15 called PML, a putative transcription factor. This distinct translocation results in a fusion mRNA detected by Northern analysis. Two cDNAs have been isolated that differ in the extent of 3' PML nucleic acid sequence contained. This study describes a reverse transcription polymerase chain reaction (RT-PCR) assay for the PML/RAR-alpha fusion transcript, which amplifies PML/RAR-alpha mRNA from APL cells with either reported breakpoint. DNA sequencing of the predominant RT-PCR products from 6 patients showed identical RAR-alpha exonic breakpoints and two PML breakpoints. This RT-PCR assay was positive in leukemic cells from 30/30 APL patients with the molecular rearrangement confirmed by cytogenetics or Northern analysis. In leukemic cells of patients with a morphologic diagnosis of APL lacking the t(15;17) by routine cytogenetics, a positive RT-PCR assay predicted clinical response to all-trans-retinoic acid (RA) therapy. Dilutional studies with leukemic cells that express (NB4) or do not express (HL-60) a PML/RAR-alpha fusion mRNA reveal that this RT-PCR assay detects the transcript from as little as 50 pg of total RNA. In APL cells from 5/6 patients treated with RA alone, a complete response by clinical and cytogenetic criteria accompanied a persistently positive RT-PCR assay. This preceded relapse by 1-6 months. RT-PCR for PML/RAR-alpha mRNA provides a more-sensitive test for the t(15;17) than routine cytogenetics or Northern analysis. This molecular rearrangement detected by RT-PCR best defines this RA-responsive malignancy. The RT-PCR assay for the PML/RAR-alpha transcript yields important diagnostic and prognostic information in the management of APL patients.

Base Sequence↗
PubMed1991

Correlation analysis for clinical and gingival crevicular fluid parameters at anatomically related gingival sites.

This study was designed to evaluate the relationship of certain clinical and biochemical measures of periodontal pathology at anatomically related gingival sites. The maxillary first molar--second bicuspid region was studied in patients with gingivitis and periodontitis. The mesiobuccal site on the first molar was compared to the mesiopalatal and direct buccal sites on the molar and the distobuccal site on the second bicuspid. Probing depth, attachment level, gingival index, gingival crevicular fluid (GCF) volume, and GCF levels of the lysosomal enzyme B-glucuronidase (BG), the cytoplasmic enzyme lactate dehydrogenase, IgG and the protease-inhibitor alpha-2-macroglobulin were studied. For the 3 anatomical pairs that were analyzed, the correlation coefficients for the GCF constituents were generally higher than the correlations for the clinical parameters. The mean correlations for the GCF constituents were higher for the periodontitis patients as compared to the gingivitis patients. For the periodontitis patients, BG activity was correlated at adjacent proximal sites, approached significance at adjacent papillary sites, but was not significantly correlated at adjacent facial-proximal sites. This data suggests that sampling of BG activity from a mesiobuccal site provides information about the anterior papillary unit. In contrast, IgG in GCF collected from the mesiobuccal site on the first molar was significantly correlated with the total IgG in the 3 other sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗