Biomedical subjects

M Novak

Publications and source records attributed to M Novak.

At least 37 records · Page 2Linked to original sources

PubMed2000

The neuronal microtubule-associated protein tau is a substrate for caspase-3 and an effector of apoptosis.

We have identified a class of tau fragments inducing apoptosis in different cellular contexts, including a human teratocarcinoma-derived cell line (NT2 cells) representing committed human neuronal precursors. We have found a transition point inside the tau molecule beyond which the fragments lose their ability to induce apoptosis. This transition point is located around one of the putative caspase-3 cleavage sites. This is the only site that can be effectively used by caspase-3 in vitro, releasing the C-terminal 19 amino acids of tau. These results establish tau as a substrate for an apoptotic protease that turns tau itself into an effector of apoptosis. Accordingly, tau may be involved in a self-propagating process like what has been predicted for the pathogenesis of different neurodegenerative disorders.

3T3 Cells↗

PubMed2000

High concordance of karyotype analysis and RT-PCR for CBF beta/MYH11 in unselected patients with acute myeloid leukemia. A single center study.

Identification of the inversion 16 in patients with acute myeloid leukemia (AML) is of great practical value since these patients have a relatively favorable prognosis, especially when treated with high-dose cytarabine. We compared the results of cytogenetic analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) for core binding factor (CBF) beta/myosin heavy chain (MYH11) in 241 unselected cases of AML. In contrast with other studies, we found a high concordance between these 2 methods. Eighteen of 241 patients showed a cytogenetic anomaly of the chromosome 16. We detected the fusion transcript by RT-PCR in all 18 cases and in 2 additional patients with AML without any cytogenetic anomaly of chromosome 16. One patient had a normal diploid karyotype, and the second patient showed a trisomy 22 in karyotype analysis, which often is associated with inv(16). Only 8 of 20 CBF beta/MYH11-positive patients had M4Eo morphologic features. The much higher discrepancy between cytogenetic analysis and RT-PCR in other studies, especially in AMLs other than M4Eo, possibly indicates the necessity for PCR screening regardless of the French-American-British classification.

Acute Disease↗

PubMed2000

Changes in hepatic lipids of mice infected with cysticerci of Taenia crassiceps.

Nuclear magnetic resonance (NMR) spectroscopy was employed to investigate the effect of infection with Taenia crassiceps cysticerci on the lipid profile of mouse liver. Chloroform/methanol extracts of livers from infected mice showed lower concentrations of phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, total glycerophospholipid, triacylglycerol, total fatty acid (FA) and all measured FA components than those from controls. Furthermore, the ratios obtained on dividing concentrations of the FA components by that of total FA demonstrate that the concentration decreases caused by infection are less for polyunsaturated fatty acids (FAs) than for other FAs. Extracts of T. crassiceps displayed a similar lipid profile to that of host liver but contained a lower lipid content and a shorter average FA chain length.

Animals↗

PubMed2000

[The niches and pathways of animal pathogens].

Infectious diseases are not a relict of the past but a topical phenomenon determined by complex evolution of the currently existing and constantly changing microbial agents and their hosts. With regard to abundance of species within the microbial kingdom and rate of its changes and development, it is difficult to predict the role of the microbial factor in mortality of humans and animals. The study and generalization of sequential similarities of microbial virulence factors after the completion of genome sequencing of principal pathogens can play a positive role in this direction. At present, molecular-genetic methods allow us to study the phylogenetic relationships of microbes and categorize them according to new criteria. The efficient control of diseases caused by microbes requires knowledge on their physiological and ecological niche from which they penetrate, in various ways, into the host organisms and, under suitable conditions, induce mass diseases. This process has several stages and, in the recent period, it is increasingly affected by human activities. The knowledge on all participants in this process, i.e. the microbe and its niche, factors of virulence and pathways of their dissemination, requires a scientifically based surveillance. Abundance and variability is characteristic for both microbial kingdom and microbial niche. Some identification of pathogenic properties of microorganisms and factors affecting their movement from their niche to the recipients results in activation of old classical diseases (e.g. plague, cholera, tuberculosis etc.) or emerging of new, so far unknown infections diseases ("emerging inf. disease"--EID), caused for example by lentiviruses, oncoviruses, filiviruses, bartonella, borrelia etc. This has provided the basis for establishment of new medical trends and approaches, such as "Emergency medicine" or "Travel medicine", expressing their purpose by their names. The control of existing or proposed infectious diseases in the 21st century (in which majority of factors such as urbanization, environmental factors, evolution of the microbial kingdom, will contribute to the persistence or "emergence" of new diseases) will be affected by the input of new knowledge in the field of molecular biology, such as introduction of biosensors, genetic tests, microchips, new generation of DNA vaccines, enteric vaccines and antibodies produced by transgenic animal bioreactors or plants, "customized" vaccines assessed for individual genetic profiles, etc. (Tab. 5, Ref. 21.)

Animals↗

PubMed1999

Functional blockade of tyrosine kinase A in the rat basal forebrain by a novel antagonistic anti-receptor monoclonal antibody.

We have exploited a new monoclonal antibody against the tyrosine kinase A (TrkA) nerve growth factor (NGF) receptor to block the NGF-TrkA interaction in the rat basal forebrain. The monoclonal antibody MNAC13 is a potent antagonist that prevents the binding of NGF to TrkA in a variety of systems. This antibody was used to study the maintenance of the cholinergic phenotype in the rat basal forebrain in vivo, by the implant of antibody-secreting cells. Basal forebrain cholinergic neurons (BFCNs) are greatly affected by the antibody treatment, both in terms of cell number and of cell soma size. When antibody-secreting cells are implanted at postnatal day 2 (P2), the effects observed at P8 are as severe as those obtained with anti-NGF antibodies and, interestingly, are observed also if anti-TrkA cells are implanted at P8, when anti-NGF antibodies, delivered by the same route, are no longer effective (). The effects induced by anti-TrkA, as those induced by anti-NGF, are reversible, but the time required for recovery and the critical period in the sensitivity of BFCNs to the functional inactivation of TrkA is twice as long than that observed when NGF is intercepted. These results demonstrate that BFCNs are more sensitive to the block of TrkA activation than they are to the block of NGF. The cloning of MNAC13 variable regions and their assembly into a functional polypeptide of reduced size (single chain Fv fragment) will allow its use in gene transfer applications.

3T3 Cells↗

PubMed1999

Abnormal urinary acidification in infants with hydronephrosis.

Distal renal tubular abnormalities have been observed in patients with dilated urinary tract disorders. The present study was undertaken to look for patterns in urinary acidification in infants with varying degrees of hydronephrosis due to either reflux or obstruction and occurring as unilateral or bilateral disease. Three groups of infants (mean age 3.7+/-3.8 months) were studied prospectively. Groups IA and IB included patients with hydronephrosis who were acidotic and non-acidotic, respectively. Group II served as controls and consisted of patients with diarrhea and secondary metabolic acidosis with no known renal disease. Serum electrolytes, creatinine, and urine pH were measured in all patients. Urinary titratable acidity, ammonium (NH4), and net acid excretion (NAE) were measured by the titrimetric method. Infants with hydronephrosis demonstrated lower urinary buffering capacity, reflected in low NAE in the face of acidosis. Deficiencies were noted in both titratable acid and NH4 excretion compared with control infants. Acidosis was as common in unilateral as in bilateral disease, regardless of severity score. These data confirm a defect in distal urinary acidification in infants with hydronephrosis, whether unilateral or bilateral. Immaturity and endogenous acid load may play a significant role in the manifestation of metabolic acidosis with unilateral disease.

Female↗

PubMed1999

Characterization of color mutants in lacZ plasmid-based transgenic mice, as detected by positive selection.

The plasmid-based transgenic mouse model, which uses the lacZ gene as the target for mutation, is sensitive to a wide range of in vivo mutations, ranging from point mutations to insertions and deletions extending far into the mouse genome. In this study, the nature of subtle lacZ mutations, which do not completely abolish beta-galactosidase activity, as detected by positive selection, was investigated. These subtle mutants are called 'color mutants' due to their light blue staining on X-gal medium. Replating of color mutants and retransformation of plasmid DNA, purified from individual color mutants, resulted in the same phenotype as the original color mutant. The p-gal positive selection system tolerates approximately 10% of wild-type activity as indicated by spectrophotometric determination of beta-galactosidase activity of individual color mutants. Restriction digestion and size separation of plasmid DNA revealed no visible change in the size of the plasmid in color mutants. Sequence analysis confirmed the presence of a point mutation in each lacZ gene of nine different color mutants. The results indicate that color mutants are caused neither by the presence of a mixture of wild-type and mutated lacZ plasmids within the same host cell nor by a mixture of cells within the original mutant colony which carry either wild-type or mutated lacZ plasmids. In addition, it was discovered that the mouse line studied harbors four polymorphic base changes among the integrated plasmid copies.

Animals↗

PubMed1999

Quantitative correlation between radiation-induced mutagenesis in endogenous genes and transgenes of mouse spermatogonial stem cells.

In order to evaluate the pUR288-plasmid transgenic mouse model, utilizing the bacterial lacZ gene as the mutational target, radiation-induced mutagenesis was primarily analyzed in spermatogonial stem cells. A combined hydroxyurea (HU)-X-ray treatment protocol was used, known to sensitize dramatically the induction of mutations in endogenous genes. In the testes of untreated animals, a mutant frequency of 6.7 +/- 4.4 x 10(-5) was found. In animals treated with HU or X ray alone, moderate elevations were seen (factors of about 4 and 2 over untreated animal values). In testes of mice having received the HU + X-ray combination treatment, a mutant frequency of 63.0 +/- 36.1 x 10(-5) was found. The results obtained showed a good quantitative correlation between endogenous genes and the transgene, indicating the suitability of pUR288 transgenic mice for also efficiently recording radiation-induced genetic damage. Radiosensitization, seen in spermatogonial stem cells, was not observed in other studied organs such as spleen, brain, or lung.

Animals↗

PubMed1998

The shrinking renal replacement therapy "break-even" point.

BACKGROUND: This study examines the current cost of live donor (LD) transplantation at our institution, and compares it with that of dialysis. METHODS: The study population consisted of 184 consecutive adult recipients of laparoscopically procured LD kidney transplants. Cost-containment measures instituted during this series included elimination of routine postoperative antilymphocyte induction and an accelerated discharge clinical pathway with planned discharge of the recipient on postoperative day (POD) 2. Costs of the transplants to Medicare were estimated from hospital charges, readmission rates, and immunosuppressant usage. These were compared with published costs of dialysis to Medicare in terms of a fiscal transplant-dialysis break-even point. RESULTS: Kaplan-Meier patient and graft survival rates at 1 year were 97 and 93%, respectively. Among patients followed for at least 90 days and treated with no induction and either cyclosporine-mycophenolate mofetil or tacrolimus-mycophenolate mofetil, acute rejection rates were low (27.6 and 13.9%, respectively). In the last 124 patients, 32.3% were discharged by POD 3 and 71.8% by POD 6, with corresponding mean transplant hospital charges (excluding organ acquisition) of $11,873 and $17,350, respectively. The 30-day readmission rate for patients discharged on the accelerated pathway by POD 3 was only 16%. The least expensive subgroup in the present study (30% of patients) was that of patients discharged by POD 6 and not readmitted during the first year; the break-even point with dialysis costs was calculated as 1.7 years after the transplant. CONCLUSIONS: The cost of LD transplants can be safely reduced by elimination of routine postoperative anti-lymphocyte immune induction and by an early discharge clinical pathway. Uncomplicated LD kidney transplants, meaning those with a short length of stay in the hospital after transplantation and no need for readmission within the first year, accrue savings over dialysis within 2 years.

Adolescent↗

PubMed1998

Phosphorylation of tau at both Thr 231 and Ser 262 is required for maximal inhibition of its binding to microtubules.

The paired helical filaments (PHFs) found in Alzheimer's disease (AD) brains are composed primarily of the microtubule-associated protein tau. PHF-tau is in a hyperphosphorylated state and is unable to promote microtubule assembly. We investigated whether the inhibition of tau binding to microtubules is increased when tau is phosphorylated by different kinases in combination with GSK-3. We found that when tau was first phosphorylated by A-kinase, C-kinase, cdk5, or CaM kinase II and then by GSK-3, its binding to microtubules was inhibited by 45, 61, 78, and 79%, respectively. Further, the kinase combinations cdk5/GSK-3 and CaM kinase II/GSK-3 rapidly phosphorylated the sites Thr 231 and Ser 235. When these sites were individually replaced by Ala and the phosphorylation experiments repeated, tau binding to microtubules was inhibited by 54 and 71%, respectively. By comparison, when Ser 262 was replaced by Ala, tau binding to microtubules was inhibited by only 8% after phosphorylation by CaM kinase II. From these observations we estimate that the phosphorylation of Thr 231, Ser 235, and Ser 262 contributes approximately 26, approximately 9, and approximately 33%, respectively, of the overall inhibition of tau binding to microtubules. Together, our results indicate that the binding of tau to microtubules is controlled by the phosphorylation of several sites, among which are Thr 231, Ser 235, and Ser 262.

Amino Acid Sequence↗

PubMed1998

Tau cleavage and dephosphorylation in cerebellar granule neurons undergoing apoptosis.

Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. During this process we found that tau, a neuronal microtubule-associated protein that plays a key role in the maintenance of neuronal architecture, and the pathology of which correlates with intellectual decline in Alzheimer's disease, is cleaved. The final product of this cleavage is a soluble dephosphorylated tau fragment of 17 kDa that is unable to associate with microtubules and accumulates in the perikarya of dying cells. The appearance of this 17 kDa fragment is inhibited by both caspase and calpain inhibitors, suggesting that tau is an in vivo substrate for both of these proteases during apoptosis. Tau cleavage is correlated with disruption of the microtubule network, and experiments with colchicine and taxol show that this is likely to be a cause and not a consequence of tau cleavage. These data indicate that tau cleavage and change in phosphorylation are important early factors in the failure of the microtubule network that occurs during neuronal apoptosis. Furthermore, this study introduces new insights into the mechanism(s) that generate the truncated forms of tau present in Alzheimer's disease.

Alzheimer Disease↗

PubMed1998

Metabolism of [3-(13)C]-pyruvate by cysticerci of Taenia crassiceps.

Carbon-13 decoupled 1H spin-echo nuclear magnetic resonance (NMR) spectra, with and without 13C population inversion, of extracts of Taenia crassiceps cysticerci incubated in media containing [3-(13C)]-pyruvate showed 13C enrichment in alanine, lactate, acetate, succinate, and citrate. Labeling in the latter three metabolites provides substantial evidence that the malic enzyme reaction in this cestode also functions in the direction opposite to that in which it is normally portrayed. The direct passage of pyruvate from the cytosol to the mitochondrion is suggested by the greater percentage of 13C detected in acetate relative to succinate.

Acetates↗

PubMed1998

Detection of the WT1 transcript by RT-PCR in complete remission has no prognostic relevance in de novo acute myeloid leukemia.

The WT1 gene is expressed in 73-100% of patients with acute myelogenous leukemia (AML) and is thought to play a role in maintaining the viability of leukemic cells. WT1 has been proposed as a marker for minimal residual disease in leukemia. We obtained serial blood or bone marrow samples from patients with de novo AML at diagnosis, during therapy, and up to 95 months after diagnosis and analyzed for WT1 gene expression by RT-PCR to determine whether gene expression was predictive of relapse. Forty-four patients had WT1-positive AML and achieved a complete remission (CR) following chemotherapy and 24 patients underwent unrelated donor (n = 4), sibling donor (n = 13) or autologous (n = 7) marrow transplantation. After achieving CR 62% of the patients became WT1-negative, while 38% remained WT1-positive. There was no difference in the disease-free survival (DFS) and survival from remission between WT1-positive and -negative patients (P > 0.1). Following BMT, 32% of the patients analyzed in CR within the first 100 days after transplantation were WT1 PCR positive. Detection of WT1 transcripts within 100 days following BMT did not affect DFS and overall survival (OS) after transplantation (P > 0.1). Ten of 11 patients who are in continuous CR following chemotherapy or BMT for more than 3 years were transiently WT1-positive during the observation period. Four of these patients displayed the WT1 transcript at the last examination. Thirteen of 39 patients were WT1 PCR negative within 4 months before clinical onset of relapse and eight patients were WT1 PCR negative at time of relapse. These data indicate that: (1) achievement of WT1 negativity is not associated with longer DFS, survival from remission, or OS after transplantation; (2) not all patients who relapse become WT1 positive again; (3) long-term remitters frequently display the WT1 transcript. Thus, we conclude that the monitoring of WT1 gene expression by qualitative RT-PCR during treatment and CR is of very limited value.

Acute Disease↗

PubMed1998

Competitive CBFbeta/MYH11 reverse-transcriptase polymerase chain reaction for quantitative assessment of minimal residual disease during postremission therapy in acute myeloid leukemia with inversion(16): a pilot study.

PURPOSE: (1) Quantification of minimal residual disease (MRD) by competitive CBFbeta/MYH11 reverse-transcriptase polymerase chain reaction (RT-PCR) in patients with acute myeloid leukemia (AML) and inversion(16) [inv(16)] during postremission therapy, (2) comparison of this method with conventional two-step RT-PCR, and (3) evaluation of a potential prognostic value. PATIENTS AND METHODS: MRD of six consecutive adult patients with AML and inv(16)(p13;q22) or t(16;16)(p13;q22) who entered complete remission (CR) was monitored by competitive CBFbeta/MYH11 RT-PCR in their bone marrow (BM) during postremission therapy with high-dose cytarabine (HiDAC) or after BM transplantation with a matched unrelated-donor marrow (MUD-BMT) during an observation period of 4.5 to 27 months after initiation of treatment. RESULTS: Competitive PCR showed a gradual decline by at least 4 orders of magnitude after 7 to 9 months in patients in continuous CR (CCR), while one patient who relapsed after 13.5 months only achieved a reduction by 2 orders of magnitude at the end of consolidation therapy. A rapid decrease below the detection limit was observed within 1 month in two patients after MUD-BMT. A temporary reappearance of molecular MRD was observed in these patients during immunosuppression for graft-versus-host disease (GvHD). After reduction of immunosuppression, the level of MRD dropped again below the PCR detection limit. Molecular monitoring by conventional two-step RT-PCR yielded comparable results only when multiple assays per time point were performed, while single-assay RT-PCR gave misleading results. CONCLUSION: Competitive RT-PCR is a valuable tool for molecular monitoring during postremission chemotherapy, as well as after BMT.

Adolescent↗

PubMed1997

Co-localization of truncated tau and DNA fragmentation in Alzheimer's disease neurones.

Monoclonal antibody MN423 stains all hallmarks of tau neurofibrillary pathology in Alzheimer's disease (AD), without reacting with full-length tau. It recognizes tau molecules which are cleaved at Glu-391, suggesting that tau is endogenously truncated in AD brains. We investigated whether truncation of tau takes place in pre-tangle neurones. Our results showed that tau truncation occurs inside AD neurones in the absence of other signs of AD neurofibrillary pathology. Using in situ end labelling (ISEL) of DNA we found evidence for DNA fragmentation in a significant subpopulation of MN423-positive neurones. Our results therefore suggest that tau truncation is an early intracellular event preceding cell death in AD.

Aged↗

PubMed1997

Protein kinase C and calcium/calmodulin-dependent protein kinase II phosphorylate three-repeat and four-repeat tau isoforms at different rates.

All six isoforms of the microtubule-associated protein tau are present in hyperphosphorylated states in the brains of patients with Alzheimer's disease (AD). It is presently unclear how such hyperphosphorylation of tau is controlled. In a previous study (Singh et al. Arch Biochem Biophys 328: 43-50, 1996) we have shown that three-repeat taus containing two N-terminal inserts were phosphorylated to higher levels and at different sites compared to those either lacking or containing only one such insert. We have extended these observations in this study by comparing the phosphorylation of tau isoforms containing three-repeats (tau 3, tau 3 L) and four-repeats (tau 4, tau 4 L). In the absence of N-terminal inserts in tau structure (tau 3, tau 4) both CaM kinase II and C-kinase phosphorylated four-repeat tau (tau 4) to a higher extent than three-repeat tau (tau 3). When two N-terminal inserts are present in tau structure (tau 3 L, tau 4 L), then three-repeat tau (tau 3 L) is phosphorylated to a higher extent than four-repeat tau (tau 4 L) by these kinases. CK-1 and GSK-3 phosphorylated each of the above pairs of three-repeat and four-repeat taus to the same extents. However, after an initial prephosphorylation of the taus by CaM kinase II, GSK-3 differentially phosphorylated three-repeat and four-repeat taus. Under these conditions thr 231, ser 235, ser 396, and ser 404 were phosphorylated to greater extents in four-repeat tau (tau 4) compared to three-repeat tau (tau 3) in the absence of N-terminal inserts. In the presence of such inserts these sites were phosphorylated to greater extents in three-repeat (tau 3 L) compared to four-repeat (tau 4 L) tau. Our results indicate that the extents to which tau isoforms are phosphorylated in normal and AD brain depends on (a) the number of repeats (3 or 4), (b) the number of N-terminal inserts (0, 1, or 2), and (c) the initial phosphorylation state of tau.

Calcium-Calmodulin-Dependent Protein Kinase Type 2↗

PubMed1997

Drug-induced sleep disturbances. Focus on nonpsychotropic medications.

Different medications can have significant effects on sleep quality and/or quantity. When prescribing medications it is important to be aware of these possible adverse effects of drugs. Disturbances of the sleep/wake cycle caused by medications can vary and include insomnia, daytime sleepiness, nightmares and changes in the sleep architecture. Psychotropic drugs are well known to have an effect on the sleep/wake cycle, but there is only limited information about the sleep effects of nonpsychotropic medications. Cardiovascular drugs, especially beta-blockers, which are widely used drugs, often change the sleep architecture and cause nightmares and insomnia. Both of these effects can be a potential source of noncompliance. Because of the complicated relationship between sleep, nocturnal asthma and antiasthmatic agents, the appropriate dosage and timing of medications should always be considered. Patients with Parkinson's disease often experience disrupted sleep due to their disorder and the adverse effects of anti-parkinsonian medications.

Antiparkinson Agents↗

PubMed1997

Application of a prognostic scoring system to critically ill patients in a small military facility.

Two hundred one consecutive adult admissions to a 3-bed Special Care Unit in a 25-bed military hospital were scored using APACHE II (Acute Physiology and Chronic Health Evaluation). Outcome measures included APACHE II scores and mortality predictions; active intensive care interventions; transfers for specialized care; and mortality. Although higher scores generally reflected the need for intensive care intervention, admissions with nondiagnostic chest pain had scores that did not accurately predict their course. This finding could be explained by bias in the original APACHE case mix and by the need for further subclassification of cardiovascular disease. APACHE II scoring can be applied in small intensive care settings. Scoring criteria and logistic regression equations may need to be customized accordingly.

APACHE↗