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Biomedical subjects

K Malik

Publications and source records attributed to K Malik.

At least 19 recordsLinked to original sources
PubMed2005

Genotype-phenotype correlation in a family with late onset CMT and an MPZ lys236del mutation.

An in frame, lys236 deletion in the intracytoplasmic domain of myelin protein zero (MPZ) has recently been designated as a mutation possibly associated with Charcot-Marie-Tooth disease (CMT) but requiring further documentation. In this report we present a detailed clinical, electrophysiological, and genotype correlation in three generations of a family with the MPZ lys236del mutation and provide further evidence that this mutation is associated with CMT. The MPZ lys236del mutation is associated with an autosomal dominant, adult onset CMT phenotype, with variable penetrance ranging from an asymptomatic state to foot deformities, pedal numbness, and muscle cramps. Nerve conduction studies disclose intermediate range, somewhat non-uniform slowing of motor nerve conduction, which is accentuated in forelimb rather than distal nerve segments. Based on the contrasting finding of entirely normal conduction velocities (CV) in a genetically affected 15 year old in this family, it remains to be established whether CV slowing with this mutation is progressive in life, a pattern that would contrast with CMT1a (PMP22 gene duplication).

Adolescent↗
PubMed2004

The validation of self-reported smoking status by analysing cotinine levels in stimulated and unstimulated saliva, serum and urine.

OBJECTIVES: Cotinine, a nicotine metabolite, can be used to measure exposure to tobacco smoke. The aim of this study was to compare cotinine levels in different biological fluids collected from both smokers and non-smokers and to relate the findings to self-reported smoking status. Data were also collected concerning the acceptability of the differing methods of sample collection. MATERIAL AND METHOD: Patients recruited to the study were asked to provide samples of urine, blood and saliva (both stimulated and unstimulated). Data collected from patients by questionnaire included information on smoking behaviour such as daily number of cigarettes and environmental exposure to smoke. After the sample collection, patients were asked to rate the acceptability of each sampling method. Samples were analysed using enzyme immunoassay (EIA) kits. RESULTS: In total, 80 patients participated, with 49 being smokers and 31 being non-smokers. There was clear differentiation between smokers and non-smokers (P < 0.001) for all the different samples in terms of cotinine. A significant relationship was seen between cotinine and daily number of cigarettes for both salivas and urine (all P < 0.001) but not for serum. Participants found serum and urine collection methodologies 'very acceptable' (67 and 66%, respectively) whereas 9% found collection of stimulated saliva 'not at all acceptable'. CONCLUSION: Cotinine, whatever the collection method and analysed by EIA kits, shows good differentiation between smokers and non-smokers. Salivary samples have the advantage of being non-invasive, although collection methodology is important, as cotinine levels may vary.

Adolescent↗
PubMed2003

The new RENT family of repetitive elements in Nicotiana species harbors gene regulatory elements related to the tCUP cryptic promoter.

The tCUP cryptic constitutive promoter was discovered in the tobacco genome by T-DNA (transfer DNA) tagging with a promoterless GUS-nos gene. Here, we show that the portion of the tCUP sequence containing a variety of cryptic gene regulatory elements is related to a new family of moderately repetitive sequences (10(2) copies), the RENT (repetitive element from Nicotiana tabacum) family. The RENT family is found only in certain Nicotiana species. Five RENT elements were cloned and sequenced. The RENT elements are a minimum of 5 kb in length and share 80-90% sequence similarity throughout their length. The 5' termini are the same in the isolated RENT family members and are characterized by a conserved border sequence (TGTTGA(T or C)ACCCAATTTT(T or C)). The 3' ends of RENT sequence similarity vary in location and sequence. The tCUP cryptic promoter originated from a unique truncated RENT element that interrupts a phytochelatin synthase-like gene that may have undergone rearrangements prior to or resulting from T-DNA insertion. No evidence was found for expressed coding regions within the RENT elements; however, like the cryptic gene regulatory elements within the tCUP sequence, the isolated RENT elements possess promoter activity and translational enhancer activity.

Base Sequence↗
PubMed2001

Retrograde carbon monoxide is required for induction of long-term potentiation in rat superior cervical ganglion.

Carbon monoxide (CO), produced in the body by the enzyme heme oxygenase (HO), has been suggested as a retrograde synaptic messenger with a prominent role in the long-term potentiation (LTP) of certain areas of the brain. LTP of sympathetic ganglia is 5-HT(3) receptor-dependent and has been shown to require nitric oxide for the maintenance, but not for the induction, phase. We investigated the possibility of CO being required for the induction of ganglionic LTP. Pretreatment of rat isolated superior cervical ganglia with oxyhemoglobin (25-100 microm) completely blocked LTP. In the same ganglia, prolonged washout of oxyhemoglobin did not uncover any potentiation of the compound action potential. Oxyhemoglobin had no significant effect on the maintenance phase in ganglia with established LTP. Pretreatment of ganglia with the HO inhibitor zinc protoporphyrin-IX (ZnPP) (10 microm) completely and irreversibly prevented the expression of tetanus-evoked LTP. However, in the same ganglia, after superfusion of CO in the presence of ZnPP, tetanic stimulation readily evoked LTP. No effect was seen on the maintenance phase when ZnPP was superfused on ganglia with established LTP. Pretreatment of ganglia with the 5-HT(3) receptor antagonist ondansetron (0.4 microm) alone completely and irreversibly blocked LTP. However, in the presence of CO, ondansetron did not block LTP. These results suggest that activation of 5-HT(3) receptors may be involved in the production of CO. The results also suggest that CO, probably originating outside the presynaptic nerve terminal, is involved in the induction of LTP.

Action Potentials↗
PubMed2001

Influence of age and height on nerve conduction.

Previous studies demonstrated age- and height-related slowing of nerve conduction velocity (NCV) and reduction in nerve response amplitude. Unfortunately, many studies examined discontinuous populations, preventing regression analysis. The correlation coefficients of available studies vary widely, preventing clear conclusions. We retrospectively examined 3969 clinically normal subjects ranging in age from 20 to 95 years from a total of 22,420 electrodiagnostic studies done between 1986 and 1998. The correlation of NCV with height was stronger than with age. Regression equations using both factors account for 12--27% of the variance. Responses were seen in the majority of patients aged 70 years and older, but the percentage of normals who had no response increased with advancing age. Age was strongly inversely correlated with the amplitudes of both sensory and motor responses, accounting for 7--16% of the variance. Regression equations using both height and age improved this correlation, accounting for 7--22% of the variance. Therefore, both height and age must be taken into consideration when normal values are developed.

Adult↗
PubMed2001

Enhancers and core promoter elements are essential for the activity of a cryptic gene activation sequence from tobacco, tCUP.

Cryptic gene regulatory elements are sequences that are inactive at their native locations in the genome but have the ability to become functional when positioned adjacent to genes. We have recently isolated such a cryptic sequence from tobacco, tCUP, that can act as a promoter. A 135-bp fragment spanning extending from position -197 to -62, relative to the transcription start site, was found to promote GUS expression in all of the major organs of transgenic Arabidopsis plants. Furthermore, this 135-bp fragment complemented the -46 minimal promoter of CaMV 35S and conferred constitutive expression on transgenic Arabidopsis plants. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from tobacco leaves interact with the 135-bp fragment. tCUP has a core promoter that lacks the TATA consensus sequence but addition of a TATA-box sequence increased the core promoter activity by three-fold. The sequence surrounding the transcription start site of tCUP has sequence similarity with the initiator element (Inr), and deletion of this sequence significantly reduced promoter activity, suggesting that an essential Inr element may exist in the tCUP core promoter. Fusion of the GCC-box enhancer element from pathogenesis-related genes to the core promoter elevated tCUP core promoter activity. Our study indicates that cryptic promoters are similar in composition and organization to promoters associated with expressed genes and that their promoter elements can be combined to create composite promoters that are fully functional. This data provides direct evidence that the expression pattern of plant genes can be influenced by cryptic gene regulatory elements when they are brought into juxtaposition with genes through DNA rearrangements.

Arabidopsis↗
PubMed2001

Activation of tomato PR and wound-related genes by a mutagenized tomato MAP kinase kinase through divergent pathways.

A mitogen-activated protein kinase kinase (MAPKK) gene, tMEK2, was isolated from tomato cv. Bonny Best. By mutagenesis, a permanently active variant, tMEK2MUT, was created. Both wild-type tMEK2 and mutant tMEK2MUT were driven by a newly described strong plant constitutive promoter, tCUP, in a tomato protoplast transient gene expression system. Pathogenesis-related genes, PRlb1, PR3 and Twi1, and a wound-inducible gene, ER5, were activated by tMEK2MUT. Specific inhibitors of p38 class MAPK inhibited tMEK2MUT-induced activation of PR3 and ER5 genes but not that of the PRlb1 or Twi1 gene. Arabidopsis dual-specificity protein tyrosine phosphatase 1 (DsPTP1) and maize protein phosphatase 1 (PP1) inhibited tMEK2MUT-induced activation of the ER5 gene and the Twi1 gene, respectively, whereas PRlb1 and PR3 were not affected by either AtDsPTP1, or maize PP1, or Arabidopsis protein phosphatase 2A (PP2A). We have demonstrated for the first time that a single MAPKK activates an array of PR and wound-related genes. Our observation indicates that the activation of the genes downstream of tMEK2 occurs through divergent pathways and that tMEK2 may play an important role in the interaction of signal transduction pathways that mediate responses to both biotic (e.g. disease) and abiotic stresses (e.g. wound responsiveness).

Amino Acid Sequence↗
PubMed2001

Ectopic expression of an Arabidopsis calmodulin-like domain protein kinase-enhanced NADPH oxidase activity and oxidative burst in tomato protoplasts.

Among plant defense responses to pathogen attack, the release of active oxygen species (AOS), termed the oxidative burst, may affect the attacking pathogen and the host plant cells at the infection site, thereby limiting the spread of the pathogen. Plasma membrane-associated NADPH oxidase represents a key enzyme in mediating the oxidative burst. The mechanisms of NADPH oxidase activation, however, remains unclear. Ectopic expression of AK1-6H, an Arabidopsis calmodulin-like domain protein kinase (CDPK) in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity. Arabidopsis protein phosphatase 2A abolished this enhancement, whereas Arabidopsis dual-specificity protein tyrosine phosphatase 1 or maize protein phosphatase 1 had no effect tMEK2MUT, a constitutively activated, mitogen-activated protein kinase kinase from tomato, did not enhance NADPH oxidase activity when overexpressed. In a cell-free system, AK1-6H moderately stimulated the NADPH oxidase activity on plasma membrane. AK1-6H, but not tMEK2MUT, also enhanced production of AOS in intact protoplasts. Our results show that ectopic expression of a heterologous CDPK can enhance NADPH oxidase activity and stimulate an oxidative burst in tomato protoplasts.

Arabidopsis↗
PubMed2001

Wilms' tumor: a paradigm for the new genetics.

Carcinogenesis can be triggered by a diverse range of molecular lesions, a variety of which can be illustrated by Wilm's tumor (WT), a pediatric kidney cancer. Molecular defects observed in WTs include several independent targets and mechanisms best exemplified by changes on the short arm of chromosome 11. This article will review the molecular pathology of WT and emphasize the broader ramifications for cancer genetics. Consideration will be given to carcinogenic pathways, novel cellular molecules, and technologies that will assist in the rapid interpretation and assimilation of DNA sequence data arising from the sequencing of the human genome.

Genes, Wilms Tumor↗
PubMed2000

Identification of differential methylation of the WT1 antisense regulatory region and relaxation of imprinting in Wilms' tumor.

Wilms' tumor (WT) is associated with loss of heterozygosity at chromosome 11p13, the site of the Wilms' tumor suppressor gene, WT1. Although the preferential loss of maternal alleles suggested that differential allelic expression of WT1 might occur, this has not been evident in normal fetal tissues or WTs. In this study, we show that the WT1 antisense regulatory region is differentially methylated, with Southern blot analysis of four loss of heterozygosity-negative WTs and their corresponding normal kidneys indicating that allelic methylation is lost in WTs. Reverse transcription-PCR expression analysis correlates methylation with monoallelic expression of the antisense WT1 transcript (WT1-AS) in normal kidney. However, WTs display hypomethylation and biallelic expression of WT1-AS. Our findings are consistent with imprinting of WT1-AS in normal kidney and the relaxation of imprinting in Wilms' tumorigenesis. This identifies the WT1 antisense regulatory region in intron 1 as a primary site for epigenetic deregulation at chromosome 11p13 in WTs.

Alleles↗
PubMed2000

Functional analysis of a RPD3 histone deacetylase homologue in Arabidopsis thaliana.

Histone acetylation is modulated through the action of histone acetyltransferase and deacetylase, which play key roles in the regulation of eukaryotic gene expression. We have screened the expressed sequence tag database with the yeast histone deacetylase RPD3 sequence and identified two Arabidopsis homologues, AtRPD3A and AtRPD3B. The deduced amino acid sequences of AtRPD3A and AtRPD3B show high overall homology (55% identity) to each other. AtRPD3A encodes a putative protein of 502 amino acids with 49% identity to the yeast RPD3. AtRPD3B encodes a putative protein of 471 amino acids and shares 55% amino acid identity with the yeast RPD3. Northern analysis indicated that AtRPD3A was highly expressed in the leaves, stems, flowers and young siliques of Arabidopsis plants, whereas the AtRPD3B transcript was not detected in these organs. An AtRPD3A fusion protein repressed transcription when directed to a promoter driving a reporter gene, indicating a role for AtRPD3A protein in gene repression. Arabidopsis plants were transformed with a gene construct comprising a truncated AtRPD3A cDNA in the antisense orientation driven by a strong constitutive promoter, -394tCUP. Antisense expression of AtRPD3A resulted in decreased endogenous AtRPD3A transcript and delayed flowering in transgenic Arabidopsis plants, suggesting that the transition from the vegetative to reproductive phase of development could be affected by histone acetylation. Our study demonstrates the important role of histone deacetylases in plant growth and development.

Amino Acid Sequence↗
PubMed2000

Functional analysis of HD2 histone deacetylase homologues in Arabidopsis thaliana.

Post-translational modification of histones, in particular acetylation, is an important mechanism in the regulation of eukaryotic gene expression. Histone deacetylases are enzymes that remove acetyl groups from the core histones and play a key role in the repression of transcription. HD2 is a maize histone deacetylase, which shows no sequence homology to the histone deacetylases identified from other eukaryotes. We have identified two putative HD2-like histone deacetylase cDNA clones, AtHD2A and AtHD2B, from Arabidopsis thaliana by screening the expressed sequence tag database. AtHD2A and AtHD2B encode putative proteins of 246 and 305 amino acids, and share 44% and 46% amino acid identity to the maize HD2, respectively. Northern blot analysis indicated that AtHD2A was highly expressed in flowers and young siliques of Arabidopsis plants, whereas AtHD2B was widely expressed in stems, leaves, flowers and young siliques. AtHD2A repressed transcription when directed to a promoter containing GAL4-binding sites as a GAL4 fusion protein. Deletion of the extended acidic domain or the domain containing predicted catalytic residues of AtHD2A resulted in the loss of gene repression activity, revealing the importance of both domains to AtHD2A function. Arabidopsis plants were transformed with a gene construct comprising an AtHD2A cDNA in the antisense orientation driven by a strong constitutive promoter, -394tCUP. Silencing of AtHD2A expression resulted in aborted seed development in transgenic Arabidopsis plants, suggesting that the AtHD2A gene product was important in the reproductive development of Arabidopsis thaliana.

Amino Acid Sequence↗
PubMed1999

Transactivation of the WT1 antisense promoter is unique to the WT1[+/-] isoform.

The Wilms' tumour suppressor gene, WT1, encodes a zinc finger transcription factor that has been shown to repress a variety of cellular promoters via binding to cognate DNA elements. Our earlier work identified an antisense WT1 promoter that contains WT1 consensus sites, but is transcriptionally activated by WT1. In this study, we demonstrate that, unlike previous reports of transcriptional regulation by WT1, transactivation of the antisense promoter is unique to a single isoform of WT1. Of the four alternatively spliced isoforms in which exon 5 (at splice I) or amino acid residues KTS (at splice II) are inserted or omitted, only the WT1 isoform containing splice I and omitting splice II (WT1[+/-]) displays transactivation. We demonstrate that transregulation variations observed with WT1 isoforms are not solely attributable to differential DNA binding by [+KTS] or [-KTS] isoforms. Thus, the transactivation of the antisense promoter displays an absolute requirement for exon 5, suggesting that interaction between WT1 and other cellular factors is necessary for this regulatory function.

Alternative Splicing↗
PubMed1998

Antisense WT1 transcription parallels sense mRNA and protein expression in fetal kidney and can elevate protein levels in vitro.

Recent studies have identified antisense WT1 mRNAs whose expression is regulated by a promoter located in the first intron of the WT1 gene. Transcription directed by the antisense promoter is positively autoregulated by the WT1 protein implicating the antisense RNA in the control of WT1 gene expression. To elucidate further the biological role of the antisense RNA in the developing kidney, its distribution of expression has been examined relative to WT1 sense mRNA and WT1 protein. Using strand-specific WT1 riboprobes, the expression of WT1 and the antisense message were examined by in situ hybridization in the developing human fetal kidney at different gestational ages. The expression of the antisense strand was strongest in the podocytes and glomeruli and also in the S-form nephrons and the condensing blastema in the developing kidney. Expression was also seen in the podocytes of the mature kidney. The WT1 protein and sense mRNA for WT1 also showed a similar pattern, suggesting that the antisense transcript does not function simply as a downregulator of protein production. Expression of antisense WT1 exon 1 in cells constitutively producing high levels of WT1 also demonstrated no downregulation of protein and in most cases actually showed upregulated WT1 protein expression. These results strongly suggest that WT1 antisense transcripts positively modulate WT1 protein levels in vivo.

Blotting, Northern↗
PubMed1995

Region-specific effects of ovarian hormones on estrogen receptor immunoreactivity.

The cell groups in which nuclear estrogen receptor (ER) expressing neurons are found have unique, often coordinated, functions. Regulation of ER content may be one mechanism through which feedback responses can be adjusted to match the function of a specific brain region and physiological circumstance. In these immunocytochemical experiments, estrogen decreased staining intensity for ER in the ventrolateral hypothalamus and bed nucleus of the stria terminalis, but not in the periventricular preoptic area. ER staining intensity was further decreased by progesterone, following estrogen, but not in all brain regions. These results suggest that ER is regulated by estrogen in a region-specific manner. Furthermore, inhibition of responses to estrogen by progesterone may involve progesterone-induced down-regulation of ERs.

Animals↗
PubMed1995

A supprescin from a phytopathogenic fungus deactivates transcription of a plant defense gene encoding phenylalanine ammonia-lyase.

Both elicitor and supprescin (suppressor) are present in the pycnospore germination fluid of a pea pathogen Mycospharella pinodes. A nuclear run-on assay revealed that supprescin rapidly deactivated elicitor-triggered transcription of the gene encoding phenylalnine ammonia-lyase in pea epicotyl tissues. The mechanism underlying the deactivation of the plant defense gene by signal molecules secreted from the fungal pathogen was investigated. Cis-acting sequences and trans-acting factors responsive to supprescin in a TATA-proximal region of a member of the phenylalanine ammonia-lyase gene family in pea were examined in vitro. Gel mobility-shift assays and DNase I footprinting analysis revealed that the promoter region of PSPAL2 was modified by the binding of nuclear factors at multiple sites that were possibly involved in supprescin-mediated deactivation. The prominent changes by supprescin were observed at boxes 2 and 4 and near exonic sequences.

Ascomycota↗
PubMed1995

Characterization of nuclear factors for elicitor-mediated activation of the promoter of the pea phenylalanine ammonia-lyase gene 1.

The nuclear factors presumably associated with the activation of the gene encoding phenylalanine ammonia-lyase by a fungal elicitor were characterized in pea (Pisum sativum L.) epicotyls. The TATA-proximal region was dissected and putative cis-regulatory elements in the promoter of pea phenylalanine ammonia-lyase gene 1 were examined by gel-mobility shift and in vitro footprinting analyses. Specific binding of the nuclear factors to the promoter-proximal regions of pea phenylalanine ammonia-lyase gene 1 associated with elicitor-mediated activation was detected at a region containing consensus sequence motifs of boxes 2 and 4 and other AT-rich sequences. The analyses of DNA fragments containing the deleted promoter regions suggested that a residue from -183 to -173 (ATTAGTAAGTGAT) was essential for a maximal activity of forming low-mobility complex (LMC) in the gel-mobility shift assay, and synthetic oligonucleotides confirmed the presence of at least one nuclear component associated with the formation of an active LMC. Competition experiments and treatment with Hoechst 33258 provided direct evidence that the formation of LMC with the promoter fragments from genes encoding phenylalanine ammonia-lyase and chalcone synthase in pea contained one or more of the same proteins that recognize AT-rich sequence motifs for binding. It also suggests that common high-mobility group-like proteins might be involved in the regulation of elicitor-inducible genes in pea.

Base Sequence↗