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Biomedical subjects

A Novak

Publications and source records attributed to A Novak.

At least 19 recordsLinked to original sources
PubMed2001

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop.

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.

Amino Acid Sequence↗
PubMed2000

Additional chromosome aberrations in acute promyelocytic leukemia: characteristics and prognostic influence.

Patients with acute promyelocytic leukemia (APL) show other chromosome aberrations in addition to t(15;17) but their influence on the clinical outcome is still unclear. We have cytogeneticaly analyzed 43 APL patients with t(15;17)(q22;q21), treated with all-trans-retinoic acid (ATRA) according to the recommendations of the European APL 91 Group. Additional chromosome aberrations were observed in 14/43 patients (33%) studied at initial diagnosis. These patients were designed as 'complex' karyotype group and were compared to patients with t(15;17) asa sole cytogenetic abnormality ('simple' karyotype group). The 'complex' group had significantly lower platelet count and fibrinogen level and fewer cases without significant DIC at diagnosis than the 'simple' group. Comparison of 'simple' and 'complex' groups showed significant difference in complete remission rate (76% vs 35.7%, P = 0.0148) and early death rate (24% vs 64.3%, P = 0.0141). Survival analysis showed that the presence of additional chromosome abnormalities and significant DIC had an adverse effects on prognosis (P = 0.036 and P = 0.041, respectively), independent on other prognostic factors. These data indicate more aggressive biological nature of leukemic cells in patients with additional chromosome aberrations. Supplementary therapeutic strategies may be required for this subgroup of APL patients.

Adolescent↗
PubMed1999

Signaling through beta-catenin and Lef/Tcf.

Beta-catenin plays a structural role in cell adhesion by binding to cadherins at the intracellular surface of the plasma membrane and a signaling role in the cytoplasm as the penultimate downstream mediator of the wnt signaling pathway. The ultimate mediator of this pathway is a nuclear complex of beta-catenin acting as a coactivtor with lymphoid enhancer factor/T cell factor (Lef/Tcf) transcription factors to stimulate transcription of a variety of target genes. Signaling through beta-catenin is regulated by modulating its degradation and nuclear translocation. In the absence of an activating signal, phosphorylation of beta-catenin by glycogen synthase kinase 3 (GSK3) acting in conjunction with adenomatous polyposis coli and axin/conductin causes beta-catenin to interact with the beta-transducin repeat-containing protein which results in its ubiquitination and degradation. Signaling from the wnt pathway activates dishevelled which, in an as yet undefined manner, inhibits the activity of GSK3 resulting in an increase in the cytoplasmic free pool of beta-catenin, and translocation into the nucleus. The integrin-linked kinase (ILK) pathway also activates beta-catenin-Lef/Tcf signaling. ILK phosphorylates GSK3 to inhibit its activity and translocates beta-catenin into the nucleus. In addition, ILK downregulates the expression of E-cadherin and upregulates Lef-1 expression. In the final step of the beta-catenin-Lef/Tcf signaling pathway, nuclear beta-catenin binds pontin52-TATA binding protein and displaces Groucho-related gene or CREB-binding protein corepressors from Lef/Tcf resulting in stimulation of transcription. During development, beta-catenin-Lef/Tcf signaling is involved in the formation of dorsal mesoderm and dorsal axis. Furthermore, defects in the beta-catenin-Lef/Tcf pathway are involved in the development of several types of cancers.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Cell adhesion and the integrin-linked kinase regulate the LEF-1 and beta-catenin signaling pathways.

The integrin-linked kinase (ILK) is an ankyrin repeat containing serine-threonine protein kinase that can interact directly with the cytoplasmic domains of the beta1 and beta3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. Overexpression of constitutively active ILK results in loss of cell-cell adhesion, anchorage-independent growth, and tumorigenicity in nude mice. We now show that modest overexpression of ILK in intestinal epithelial cells as well as in mammary epithelial cells results in an invasive phenotype concomitant with a down-regulation of E-cadherin expression, translocation of beta-catenin to the nucleus, formation of a complex between beta-catenin and the high mobility group transcription factor, LEF-1, and transcriptional activation by this LEF-1/beta-catenin complex. We also find that LEF-1 protein expression is rapidly modulated by cell detachment from the extracellular matrix, and that LEF-1 protein levels are constitutively up-regulated at ILK overexpression. These effects are specific for ILK, because transformation by activated H-ras or v-src oncogenes do not result in the activation of LEF-1/beta-catenin. The results demonstrate that the oncogenic properties of ILK involve activation of the LEF-1/beta-catenin signaling pathway, and also suggest ILK-mediated cross-talk between cell-matrix interactions and cell-cell adhesion as well as components of the Wnt signaling pathway.

Animals↗
PubMed1998

Baltimore Vision Screening Project. Phase 2.

BACKGROUND: Vision screening in preschool and school age children remains a valuable method of identifying potentially treatable visual abnormalities, as well as refractive errors in the school age population. A major difficulty in conducting these types of programs is the inconsistent follow-up encountered frequently in children identified by the screening program. The Baltimore Vision Screening Project was designed to address the issue of access to care for a group of inner city elementary school students by providing on-site evaluation and treatment. METHODS: A standard vision-screening protocol was administered to prekindergarten and kindergarten students attending an inner city elementary school and access to care provided at the school. Children identified by the screening were examined at the school, and appropriate treatment was prescribed. The second phase of the study details a return to the school 1 year after the initial screening. The purpose of this was to reassess the level of visual morbidity in this population and to evaluate the effects of providing treatment in the previous year. RESULTS: Two hundred eighty-five children were screened during the 1994-1995 school year. Visual morbidity statistics from the follow-up year were similar to those of the previous screening: 5.3% diagnosed with amblyopia, 3.2% with strabismus, and 7.4% with refractive errors. Forty of the 68 children identified, examined, and treated in the previous year still were attending the school during the follow-up investigation. Compliance with recommended treatment was 30% in this group, with only 20% of the students passing the vision screening the following year. CONCLUSIONS: The follow-up screening confirmed the presence of significant visual morbidity in this group of preschool and kindergarten students. Nearly two thirds of the students had not complied with the recommended treatment or spectacle wear or both, despite providing immediate access to this care through the vision-screening program.

Baltimore↗
PubMed1998

Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway.

Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect transfected cells against apoptosis induced by either purified granzyme B and perforin or intact CLs. A PI-9 P1 mutant (Glu to Asp) is a 100-fold-less-efficient granzyme B inhibitor that no longer protects against granzyme B-mediated apoptosis. PI-9 is highly specific for granzyme B because it does not inhibit eight of the nine caspases tested or protect transfected cells against Fas-mediated apoptosis. In contrast, the P1(Asp) mutant is an effective caspase inhibitor that protects against Fas-mediated apoptosis. We propose that PI-9 shields CLs specifically against misdirected granzyme B to prevent autolysis or fratricide, but it does not interfere with homeostatic deletion via Fas-mediated apoptosis.

Amino Acid Sequence↗
PubMed1997

Acute megakaryoblastic leukaemia in a patient with systemic lupus erythematosus.

We describe here a 72-year-old female patient with an acute megakaryoblastic leukaemia (M7 by FAB Classification) and systemic lupus erythematosus (SLE). The patient had not been pretreated with immunosuppressive therapy, which is potentially leukaemogenic. The karyotype displayed multiple, structural and numerical anomalies, suggesting a possible de novo rather than a secondary nature of leukaemia.

Aged↗
PubMed1997

Effect of all-trans-retinoic acid alone or in combination with chemotherapy in newly diagnosed acute promyelocytic leukaemia.

Between February 1992 and November 1996 we treated 30 newly diagnosed acute promyelocytic leukaemia (APL) patients either with oral all-trans-retinoic acid (ATRA) alone (45 mg m-2) or with a simultaneous combination of ATRA (45 mg m-2), daunorubicin (DNR, 50 mg/m-2 for 3 days) and cytosine arabinoside (ARA-C, 200 mg m-2 for 7 days). There were 15 patients in each group. Patients with a white blood cell count < 5 x 10(9)/l at diagnosis received only ATRA as an induction therapy. Patients with initial white blood cell count > 5 x 10(9)/l received a combination of ATRA, DNR and ARA-C as an induction therapy. Within the first 20 days of induction, there were two early deaths in the group of patients receiving only ATRA, and six early deaths in the group of patients treated with a combination of ATRA and chemotherapy. Ten out of 13 patients (76.9%) receiving ATRA only achieved complete remission (CR) whereas seven out of nine patients (77.8%) receiving ATRA with chemotherapy achieved CR. Initial median peripheral white blood cell counts were significantly lower in the group of patients treated with ATRA alone (2.3 x 10(9)/l) than in the group of patients receiving ATRA and chemotherapy (14.0 x 10(9)/l). Morphological evidence of differentiation was noted in all patients entering CR. Patients in both groups who achieved CR received one course of standard '3 + 7' chemotherapy (DNR 45 mg m-2, 1-3 days, ARA-C 200 mg m-2, 1-7 days) followed by two courses of standard '2 + 5' chemotherapy (DNR 50 mg m-2 1-2 days, ARA-C 200 mg m-2 1-5 days) as a consolidation therapy. Patients not achieving remission (three out of 13 in the ATRA group and two out of nine in ATRA+chemotherapy group) did not respond to salvage chemotherapy and all died within 3 months of diagnosis. Only one out of 10 patients (10%) in CR, treated with ATRA is in relapse after 18 months. In patients treated with ATRA alone two out of 10 (20%) survived 58 months following diagnosis whereas in the ATRA+chemotherapy group one out of seven has already survived their 58th month since diagnosis. Four out of eight patients with an early death died of retinoic acid syndrome. Other toxicities due to ATRA were minimal (cheilitis, xerosis, dermatitis, diarrhoea, liver damage or pseudotumor cerebri).

Adolescent↗
PubMed1997

Human hematopoietic cell express two forms of the cytokine receptor common gamma-chain (gamma c).

Recent studies have revealed that the gamma-chain of the IL-2 receptor is shared by the receptors for IL-4, IL-7, IL-9, IL-13, and IL-15, and it is therefore also referred to as the common gamma-chain (gamma c). Mutations of gamma c result in X-linked severe combined immunodeficiency syndrome in humans, indicating that gamma c is essential for normal development and function of the immune system. We demonstrate that human hematopoietic cells express two gamma c transcripts differing in their carboxyl terminal coding region. One transcript is the previously reported sequence (gamma c-long), whereas the newly identified sequence exhibits a deletion of 72 nucleotides close to the 3'-end of the open reading frame (gamma c-short). This alteration predicts a loss of 24 amino acids including a conserved tyrosine residue which is shared by several members of the cytokine receptor family. The presence of these two distinct forms of gamma c transcripts was demonstrated by sequencing of reversely transcribed and polymerase chain reaction (RT-PCR) amplified mRNA, restriction digestion of the RT-PCR products, RNAse protection, and Northern blotting from human cell lines and human peripheral blood lymphocytes. Furthermore, the two variants were present in peripheral blood lymphocytes from both female and male donors, which rules out allelic variants since gamma c is a single copy gene located on the X chromosome. A truncation mutant at a site near the observed changes in gamma c-short has been reported by others to alter biochemical events activated by cytokines. This combined with the loss of a potential SH2 "docking" site in gamma c-short suggests that gamma c-long and gamma c-short may link to different signaling pathways and may play an important role in determining the cellular response to IL-2, IL-4, IL-7, IL-9, IL-13, IL-15.

Alternative Splicing↗
PubMed1996

Baltimore Vision Screening Project.

PURPOSE: This study estimates the prevalence of common visual disorders (amblyopia, strabismus, refractive errors) in a group of inner-city school children. In addition, the study addresses the issue of access to care for vision-screening programs, specifically for children with recognized difficulties in obtaining routine medical care. METHODS: School children from an inner-city elementary school were enrolled into a prospective vision-screening program combining the identification arm (screening) and diagnostic/treatment arm (ophthalmic examination). The screening consisted of Snellen E optotypes presented at a 10-foot test distance. Each child failing the vision screening was examined by an ophthalmologist at the school using standard protocol. This allowed the authors to examine all children identified through the vision-screening program. RESULTS: Six-hundred eighty children were screened during the 1993 to 1994 school year. Eleven percent (76) failed the vision screening and were examined, 68 of whom failed the ophthalmic examination. The estimated prevalence of visual morbidity was as follows: amblyopia, 3.9%; strabismus, 3.1%, and refractive errors, 8.2%. CONCLUSION: Amblyopia, strasbismus, and refractive errors were found in relatively high frequencies for this population sample of inner city children. These findings underscore the necessity of comprehensive vision-screening programs that integrate follow-up care. Children with limited access to specialized eye care must be provided with a mechanism for obtaining these services.

Amblyopia↗
PubMed1996

Indication and results of insulin therapy for gestational diabetes mellitus.

The aim of this study was to determine whether amniotic fluid insulin concentration (AFI) is a better parameter than mean maternal blood glucose values (MBG) for deciding about insulin therapy in patients with gestational diabetes. MBG's were calculated on the base of 9 blood glucose levels during a 24 hour period after one week of diet therapy. In a prospective trial between 1987 and 1989 in Karlsburg, 123 gestational diabetic patients were randomized into two groups. Treatment was either based on the concentration of AFI or MBG levels. In a second series in Berlin, 103 patients were offered amniocentesis. 81 patients agreed and 22 refused. Treatment was then analogous to that in Karlsburg. In both groups of the randomized population, strict metabolic control was achieved. There was no difference regarding pregnancy complications. Earlier labor induction and higher cesarean section rates were seen in the non-invasive group (p < 0.05). The incidence of diabetic fetopathy and neonatal hypoglycemia was significantly lower in the invasive group (p < 0.01), even though the metabolic control parameters did not differ between the two groups. The results in Berlin correspond to these findings. In conclusion, AFI enables the recognition of any hyperinsulinism reaction to the maternal metabolic situation. We recommend the additional measurement of the AFI concentration between 28 and 36 weeks as the direct fetal parameter for deciding about insulin treatment.

Amniocentesis↗
PubMed1995

Decreased incorporation of D-glucosamine into glycosphingolipids of intact Familial Dysautonomia lymphoblasts.

Familial Dysautonomia (FD) is an autosomal recessive Ashkenazi Jewish genetic disease, of unknown etiology, involving deficits in both autonomic and sensory functions. Previously, we found statistically significant increases in globotriaosylceramide (Gb3) in FD fibroblasts and lymphoblasts, and a decrease in ganglioside levels. FD fibroblasts exhibited pleiomorphic changes at the light microscopy level, suggestive of changes in the plasma membrane. We described an increase in Gb3 on the surface of synchronized cells at the G1/S boundary of the cell cycle, based on Gb3-verotoxin (derived from E. coli) interactions. Using D-glucosamine-1-14C as an in vitro precursor, we herein report a marked decrease in the rate of incorporation of D-glucosamine into the sialic acid and the N-acetylgalacto/glucosamine moieties of gangliosides and neutral glycosphingolipids in intact FD compared to control lymphoblasts. The total ganglioside content of FD cells (primarily GM3, measured as incorporation of 3H from NaB3H4) was also decreased. These data indicate differences in the turnover of sialic acid and N-acetylated sugar constituents in FD vs normal cells.

Carbohydrate Conformation↗
PubMed1995

Vaginal foreign bodies in women in postmenopause and in senium.

We report on three women, 81, 73 and 69 years of age, in whom foreign bodies had been introduced into the vagina either by the patients themselves or by somebody else. In all cases, medical help was needed to remove the object. In one patient the foreign body had remained within the vagina for 7 years. Only two of the three women admitted that the object had been introduced for sexual stimulation. Some papers are quoted that report on similar events, however, in younger patients. Our observations demonstrate the often problematical sexual situation of many old people.

Aged↗