Expanding the set of rhodococcal Baeyer-Villiger monooxygenases by high-throughput cloning, expression and substrate screening.

To expand the available set of Baeyer-Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.

Regulation of neuronal survival by the serine-threonine protein kinase Akt.

A signaling pathway was delineated by which insulin-like growth factor 1 (IGF-1) promotes the survival of cerebellar neurons. IGF-1 activation of phosphoinositide 3-kinase (PI3-K) triggered the activation of two protein kinases, the serine-threonine kinase Akt and the p70 ribosomal protein S6 kinase (p70(S6K)). Experiments with pharmacological inhibitors, as well as expression of wild-type and dominant-inhibitory forms of Akt, demonstrated that Akt but not p70(S6K) mediates PI3-K-dependent survival. These findings suggest that in the developing nervous system, Akt is a critical mediator of growth factor-induced neuronal survival.

Identification of two translational products for c-myb.

The c-myb gene is the normal cellular homolog of v-myb, the oncogenic component of Avian Myeloblastosis Virus (AMV). The c-myb gene has previously been shown to code for a single protein species of about 75 kd. However, accumulating evidence indicates that this gene could code for multiple mRNAs as a result of differential splicing. This was first detected in the ABPL-2 tumor line and was later shown to occur in normal cells. To test if indeed these differentially spliced mRNAs code for a new protein species that went hitherto undetected, a series of antibodies directed against specific domains of the myb protein, including the putative sequences that could be generated from the alternatively spliced mRNAs were generated. Immunoprecipitation analysis using these antibodies show that both normal and tumor cell lines synthesize at least two myb proteins, one of 75 kd and the other of 89 kd.

Murine myeloid leukemias with aberrant myb loci show heterogeneous expression of novel myb proteins.

Using a panel of anti-myb antibodies, we have examined the c-myb proteins present in the NFS-60 and ABPL-1, ABPL-2, and ABPL-4 tumor cell lines, all of which have an altered myb locus due to viral insertional mutagenesis. As predicted from previous DNA and RNA analysis, NFS-60 cells produce myb protein with a truncated C-terminus and a normal N-terminus. The three ABPL tumor cell lines studied here were previously shown to have undergone similar rearrangements towards the 5' end of myb locus and were expected to synthesize identical myb RNAs and proteins. These cell lines were found to produce myb proteins with N-terminal modifications. Surprisingly, however, the three cell lines were found to synthesize a heterogenous array of myb proteins of different sizes. These observations suggest that oncogenic activation of myb in ABPL tumors may involve additional changes in the coding region in addition to those occurring at the N-terminus.

Murine myeloid leukemic cells with disrupted myb loci show splicing anomalies that account for heterogeneous sizes in myb proteins.

The ABPL tumor cell lines represent a group of myeloid cell lines which contain an altered myb locus due to viral insertional mutagenesis within the third exon of c-myb. Immunoprecipitation analysis of the proteins produced in three ABPL lines revealed an interesting anomaly. Despite the invariant position of the virus integration event, the three ABPL tumor cell lines we examined (ABPL-1, ABPL-2 and ABPL-4) produced three different sized proteins. In this report, we examined the molecular basis for this protein size heterogeneity. Molecular cloning and sequence analysis of the cDNAs derived from the myb transcripts show that ABPL-1 tumor produces a tripartate mRNA containing sequences derived from the viral gag and env genes fused to the myb coding region. This results in the synthesis of a 74 kd protein. In the ABPL-2 tumor line, a gag-myb fusion protein is produced which is of 68 kd. In ABPL-4 cell line a gag-myb fusion protein is produced which contains an internal deletion of coding sequences derived from exons 13 and 14. This deletion results in the synthesis of a 59 kd protein in ABPL-4 tumor cell line. These observations were further confirmed by RNase protection assays which demonstrate the presence of aberrantly spliced mRNAs in ABPL-1 and ABPL-4 tumor cells but not in cells containing an undisrupted c-myb locus. In vitro translation and immuno-precipitation analysis of the cRNAs derived from the ABPL-1, ABPL-2 and ABPL-4 cDNAs show the synthesis of protein products that were identical to Myb proteins produced by these tumors in vivo. These results suggest that integration of Mo-MuLV within the c-myb locus not only results in deletions of the 5' end of the transcript but splicing aberrations within the encoded mRNA, which results in the synthesis of a heterogeneous array of proteins, not seen in normal hematopoietic cells.

Mutational analysis of Max: role of basic, helix-loop-helix/leucine zipper domains in DNA binding, dimerization and regulation of Myc-mediated transcriptional activation.

The Max protein forms a heterodimeric complex with the Myc family of proteins and binds to DNA in a sequence-specific manner. We investigated the role of the helix-loop-helix (HLH), leucine zipper (LZ) and basic domains of Max in protein complex formation, DNA-binding activity and transcriptional regulation. We mutagenized the basic, HLH and LZ domains of Max and studied the ability of the normal and mutant proteins to bind to DNA as both homo- and heterodimers and their ability to heterodimerize with Myc. Helix-1 and helix-2 regions of Max were found to be critical for homodimer formation and subsequent DNA binding, while the LZ was essential for heterodimer formation. In transient transfection assays the Myc protein functioned as a transcriptional activator while Max protein repressed the trans-activation observed with Myc.

N-methyl-D-aspartate receptors are critical for mediating the effects of glutamate on intracellular calcium concentration and immediate early gene expression in cultured hippocampal neurons.

The mechanisms by which activation of excitatory amino acid receptors is coupled to the regulation of gene transcription were studied using cultured hippocampal neurons from neonatal rats. Voltage recording, calcium imaging, specific RNA analysis and immunocytochemistry were carried out on sister cultures. This allowed analysis of the expression of functional glutamate receptor subtypes, examination of their role in controlling intracellular free calcium ([Ca2+]i), and determination of their relative contributions to the transcriptional regulation of six immediate early genes c-fos, fosB, c-jun, junB, zif/268 (also termed Egr-1; NGFI-A; Krox-24) and nur/77 (also termed NGFI-B). Expression of all six immediate early genes was induced in hippocampal neurons by glutamate treatment. Nuclear run-on assays demonstrated that this induction occurred at the transcriptional level. Activation of the N-methyl-D-aspartate subtype of glutamate receptor was necessary and sufficient for the transcriptional response. Non-N-methyl-D-aspartate receptors, while present in cultured hippocampal neurons, contributed relatively little to the regulation of transcription. Calcium imaging showed that glutamate-induced changes in [Ca2+]i were almost entirely mediated by N-methyl-D-aspartate receptors, rather than by L-type voltage-sensitive calcium channels. Previous studies have shown that stimulation with selective agonists of either N-methyl-D-aspartate receptors, non-N-methyl-D-aspartate receptors, or L-type calcium channels can lead to an increase in [Ca2+]i and c-fos expression. Here we demonstrate that in our hippocampal culture system glutamate controls [Ca2+]i and induces immediate early gene transcription primarily by activating N-methyl-D-aspartate receptors.

Neoplasms of the central nervous system of lipoid origin.

In general tumours of lipoid origin are benign. A high proportion of cases are mesenchymal neoplasms localised in subcutaneous tissue. Neurological signs related to compression of neural structures by fat within the vertebral canal were first presented in 1975. The lipogenic neoplasms represent 1-4% of spinal tumours, and generally are associated with congenital disorders of the spine. These fatty swellings are usually benign, but penetrating deeply into the spine, and connecting with spinal cord, cauda equina or filium terminale, they produce deficits. The deficits may be present at birth but they may also occur in the middle age. These events are classified as epidural lipomatosis (mostly observed in patients on chronic steroid treatment) or as lipoma, common in spinal disraphism. Intracranial tumours of lipoid origin are very rare (0.06-0.5% of brain tumours), and are probably congenital. They occur anywhere within the cranium, however a high proportion of cases tend to be located around the midline, and approximately 50% of tumours are found in the corpus callosum. Usually they are asymptomatic, and when the symptoms occur, they are frequently a result of general clinical condition. The authors present three cases of lipomatous tumours; one intracranial and two of spinal location.

[Xylose test in patients with attacks of headaches (migraine, Horton's headache)]. / Test ksylozowy u chorych z napadowymi bólami glowy (migrena, ból glowy Hortona).

The aim of this work was to study digestive tract function by means of xylose test in patients with attacks of headaches. The investigations were carried out in 14 patients with migraine, 10 with Horton's headache and 1 patient with both these conditions. In patients with migraine or Horton's headache the xylose test was normal in 65% of cases, in the remaining 35% (8 patients, 4 in each group) xylose elimination was abnormal, because it was below 30%. The authors discuss the factors which could influence the abnormal results of xylose test in these patients, since they had no signs of other diseases than the mentioned headaches.

[Participation of free radicals in the mechanism of brain tissue damage]. / Udzial wolnych rodników w mechanizmie uszkodzenia tkanki mózgowej.

There is now extensive experimental support for the early occurrence and pathophysiological importance of oxygen radical formation and cell membrane lipid peroxidation in the injured brain tissue. Several criteria for the establishment of the pathophysiological significance of oxygen radicals processes have been met. These include: 1. demonstration of increased posttraumatic levels of oxygen radicals and lipid peroxides soon after CNS injury, 2. the spatial and temporal correlation between oxygen radical formation and pathophysiological alterations (e.g. vasogenic oedema, progressive posttraumatic ischemia development, loss of microvascular autoregulation), 3. the striking similarity between posttraumatic CNS pathology and that caused by chemical peroxidative insult, and 4. the protective efficacy of oxygen radical scavenging agents or compounds that inhibit lipid peroxidation.

[Evaluation of results of surgical treatment of epidural canal lipomatosis with 3 years of follow-up of a case]. / Ocena wyników chirurgicznego leczenia samoistnej tluszczakowatosci nadtwardówkowej kanalu kregowego po uplywie trzyletniego okresu obserwacji pooperacyjnej. Opis przypadku.

We present the results of surgical treatment of idiopathic epidural lipomatosis in a 38 year-old man. Our results were evaluated 3 years; after operation. This is the third case of idiopathic epidural lipomatosis in literature in which a very good result of treatment was obtained and patient returned to previous work.

[Idiopathic epidural lipomatosis of the vertebral canal]. / Samoistny przerost tkanki tluszczowej nadtwardówkowej kanalu kregowego.

Epidural lipomatosis is most frequently seen in patients on chronic steroid treatment. Only fourteen cases of idiopathic spinal epidural lipomatosis have been described. In this report we present an additional case of this condition in a 38 year-old man.

[Lipoma of the corpus callosum]. / Tluszczak spoidla wielkiego mózgu.

The authors present a case of lipoma of corpus callosum in a 25-years old patient. The results of psychological testing, plain skull X-ray. EEG investigation, images of CT, MRI of the brain and right carotid angiography are presented. The patient refused surgical treatment. His neurological state after discharge from hospital was good.

[Activity of glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R) and superoxide dismutase in the brain tumors]. / Aktywnosc peroksydazy glutationowej (GSH-Px), reduktazy glutationowej (GSSG-R) i dysmutazy nadtlenkowej (SOD-1) w nowotworach mózgu.

The aim of the study was to estimate the activity of glutathione peroxydase, glutathione reductase and superoxide dysmutase in the brain tumours. The activity of GSH-Px was evaluated with the use of spectrophotometry, GSSG-R was evaluated based on the Mize and Langdon method, and SOD-1 with Sykes et al. method. The 55 specimens (45 specimens of neoplasm and 10 specimens of healthy brain tissue) were studied. The statistical analysis revealed significant increase of enzymes within the brain tumours in comparison to the healthy brain tissue.

[Concentration of glutathione (GSH), ascorbic acid (Vit. C), and thiobarbiturate acid reacting components (MDA) in brain neoplasms]. / Stezenie glutationu (GSH), kwasu askorbowego (Vit. C) oraz substancji reagujacych z kwasem tiobarbiturowym (MDA) w nowotworach mózgu.

The aim of the study was to evaluate the concentration of glutathione (GSH), ascorbic acid (Vit C), and thiobarbiturate acid reacting components (MDA) in brain neoplasms in specimens from normal brain tissue. The group of 72 individuals treated surgically for brain neoplasm in Department of Neurosurgery Medical Academy of Bialystok (Poland) in the period from 1996 to 1999 was included into the study. The GSH concentration was estimated with GSH-400 method, ascorbic acid by the use of Kyaw method, and MDA by Salaris and Babs method. The statistical analysis revealed diminished concentration of GSH and Vit. C (p < 0.001), and analogous increase of MDA concentration (p < 0.001) in the investigated specimens compared to the mentioned above substances concentration in the specimens obtained from normal brain tissue.