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Biomedical subjects

P Kaur

Publications and source records attributed to P Kaur.

At least 19 recordsLinked to original sources
PubMed2001

Regulation of catalytic function by molecular association: structure of phospholipase A2 from Daboia russelli pulchella (DPLA2) at 1.9 A resolution.

The crystal structure of phospholipase A(2) from the venom of Daboia russelli pulchella has been refined to an R factor of 0.216 using 17,922 reflections to 1.9 A resolution. The structure contains two crystallographically independent molecules in the asymmetric unit. The overall conformations of the two molecules are essentially the same except for three regions, namely the calcium-binding loop including Trp31, the beta-wing and the C-terminal residues 119-131. Although these differences have apparently been caused by molecular packing, they seem to have functional relevance. Particularly noteworthy is the conformation of Trp31, which is favourable for substrate binding in one molecule as it is aligned with one of the side walls of the hydrophobic channel, whereas in the other molecule it is located at the mouth of the channel, thereby blocking the entry of substrates leading to loss of activity. This feature is unique to the present structure and does not occur in the dimers and trimers of other PLA(2)s.

Animals↗
PubMed2001

Role of the linker region of the anion-stimulated ATPase ArsA. Effect of deletion and point mutations in the linker region.

The anion-stimulated ATPase ArsA in Escherichia coli consists of two homologous halves, A1 and A2, which are connected by a 40-amino acid long stretch of residues designated as the linker region. The linker region of ArsA lies in close proximity of the nucleotide-binding domain(s) of ArsA and is involved in significant conformational changes on binding of the substrates. Hence, it has been suggested earlier that the linker may play an important role in the function of ArsA. The aim of the present study was to determine the role of the linker by deletion and by site-directed mutagenesis of specific residues. Effect of deletion of the linker was determined by using the in vivo complementation approach where two halves of ArsA were co-expressed either with or without the linker region. Two co-expressed halves of ArsA conferred arsenite resistance only if the linker region was present on one of the halves. Of the six different point mutations created in the linker region, three (G284S, R290S, and D303G) were seen to drastically affect the catalytic function of ArsA. In addition, these three mutant alleles conferred arsenite sensitivity in cells carrying the wild type arsB gene. Trypsin proteolysis studies carried out with the purified proteins showed that the A1 nucleotide-binding domain in D303G protein has a conformation different from the wild type ArsA, suggesting that the linker region interacts with the nucleotide-binding domain(s) of ArsA. Based on the studies presented here, we propose that, in addition to providing flexibility, the nature of the residues themselves in the linker region is important for the conformation of the nucleotide-binding domains and for the catalytic function of ArsA.

Adenosine Triphosphatases↗
PubMed2001

Structure of a serine protease proteinase K from Tritirachium album limber at 0.98 A resolution.

X-ray diffraction data at atomic resolution to 0.98 A with 136 380 observed unique reflections were collected using a high quality proteinase K crystals grown under microgravity conditions and cryocooled. The structure has been refined anisotropically with REFMAC and SHELX-97 with R-factors of 11.4 and 12.8%, and R(free)-factors of 12.4 and 13.5%, respectively. The refined model coordinates have an overall rms shifts of 0.23 A relative to the same structure determined at room temperature at 1.5 A resolution. Several regions of the main chain and the side chains, which were not observed earlier have been seen more clearly. For example, amino acid 207, which was reported earlier as Ser has been clearly identified as Asp. Furthermore, side-chain disorders of 8 of 279 residues in the polypeptide have been identified. Hydrogen atoms appear as significant peaks in the F(o) - F(c) difference electron density map accounting for an estimated 46% of all hydrogen atoms at 2sigma level. Furthermore, the carbon, nitrogen, and oxygen atoms can be differentiated clearly in the electron density maps. Hydrogen bonds are clearly identified in the serine protease catalytic triad (Ser-His-Asp). Furthermore, electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. Though unusual, these features seem to be conserved in other serine proteases. Finally there are clear electron density peaks for the hydrogen atoms associated with the Ogamma of Ser 224 and Ndelta1 of His 69.

Ascomycota↗
PubMed2001

Apoptosis and bcl-2 expression in prostate cancer: significance in clinical outcome after brachytherapy.

PURPOSE: Radiation induced apoptosis of prostate cancer cells may have therapeutic and prognostic significance in patients treated with radiotherapy. We determined whether the ability of prostate tumor cells to undergo apoptosis has potential value for predicting the clinical response of patients with prostate cancer to brachytherapy. MATERIALS AND METHODS: A total of 76 patients with clinical stages T1 to 2 disease who were not receiving adjuvant therapy underwent transperineal implantation with 125iodine or 103palladium seeds and biopsy 7 to 23 months (median 12) after therapy. Nonresponders were classified using the American Society for Therapeutic Radiology and Oncology criteria. The apoptotic index was analyzed using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay in archived biopsy specimens from 76 treated and 19 matched pretreatment control patients. Serial sections of prostatic tumors were also evaluated for the expression of bax and bcl-2 proteins (apoptosis regulators) by immunohistochemical testing. RESULTS: A significant increase in the apoptotic index was detected in post-brachytherapy compared with pretreatment prostate specimens (3.1% versus 2%, p <0.05), as well as in patients with negative biopsy at followup compared with those with persistent malignancy (3.4% versus 1.8%, p = 0.02). In addition, there was a significant elevation in bcl-2 expression in prostatic tissue in patients with treatment failure compared with responders (30.5% versus 13.1%, p <0.05). CONCLUSIONS: To our knowledge this is the first study to establish a correlation of apoptosis induction and bcl-2 over expression with treatment outcome in patients with prostate cancer after brachytherapy. Our findings have significant clinical implications for identifying the value of the apoptotic index and bcl-2 expression in prostatic tumors for predicting the therapeutic response to brachytherapy.

Adenocarcinoma↗
PubMed2001

Optical visualization of microsomal degranulation by a carcinogen.

We report here for the first time the optical visualization of microsomal degranulation induced by a carcinogen (p-dimethylaminoazobenzene), employing analytical ultracentrifugation. Our observations demonstrate that electrophiles of a carcinogen can disrupt ribosome-membrane interaction in rough microsomes by their attacks on nucleophilic components of the reticular membrane-ribosome complex involved in protein synthesis for export from the cytosol. Lack of exported proteins can adversely affect signal transduction across plasma membrane, possibly leading to carcinogenesis.

Animals↗
PubMed2000

Enrichment for murine keratinocyte stem cells based on cell surface phenotype.

The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [(3)H]thymidine ([(3)H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [(3)H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use of in vivo cell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of alpha(6) integrin and low to undetectable expression of the transferrin receptor (CD71) termed alpha(6)(bri)CD71(dim) cells, are enriched for epithelial stem cells because they represent a minor ( approximately 8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing approximately 70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed alpha(6)(bri)CD71(bri), representing the majority ( approximately 60%) of basal keratinocytes that are actively cycling, and importantly contain approximately 70% of [(3)H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71(dim) cells in the hair follicle bulge region, a well documented location for KSCs.

Animals↗
PubMed2000

Functional characterization of cultured cells derived from an intraepidermal carcinoma of the skin (IEC-1).

We have successfully isolated a cell line (IEC-1) from an intraepidermal carcinoma of the skin of a patient and compared its behavior, in vitro, to normal human epidermal keratinocytes (HEK) and squamous cell carcinoma cell lines (SCCs). HEK differentiation comprises an initial growth arrest followed by an induction of squamous differentiation-specific genes such as transglutaminase type 1 (TG-1). Using thymidine uptake and TG-1 induction as markers of proliferation and differentiation, respectively, we were able to show that HEKs and the IEC-1 cells undergo growth arrest and induce TG-1 mRNA expression in response to various differentiation-inducing stimuli, while neoplastic SCC cell lines did not. However, differentiation in HEKs was an irreversible process whereas differentiation of the IEC-1 cells was reversible. Furthermore, growth of IEC-1 cells in organotypic raft cultures revealed differences in their ability to complete a squamous differentiation program compared with that of normal HEKs. The IEC-1 cells also exhibited a transitional phenotype with respect to replicative lifespan; HEKs had a lifespan of 4-6 passages, IEC-1 cells of 15-17 passages, and SCC cells were immortal. These alterations in IEC-1 cell behavior were not associated with functional inactivation or mutations of the p53 gene. These data indicate that the IEC-1 cells, derived from a preneoplastic skin tumor, exhibit differences in their ability to undergo terminal differentiation and have an extended replicative lifespan.

Aged↗
PubMed2000

Three-dimensional structure of a presynaptic neurotoxic phospholipase A2 from Daboia russelli pulchella at 2.4 A resolution.

The phospholipase A(2 )from Daboia russelli pulchella (DPLA(2)) is the only known member of subclass II of group IIA. The three-dimensional structure of this presynaptic neurotoxic DPLA(2) enzyme has been determined at 2.4 A resolution. The structure was determined by the molecular replacement method using the model Crotalus atrox, and refined using X-PLOR to a final R-factor of 18.8 % for all data in the resolution range 20.0 A-2.4 A. The final refined model comprises 1888 atoms from two crystallographically independent protein molecules and 160 water oxygen atoms. The overall folding of DPLA(2), with three long helices and two short antiparallel beta-strands is grossly similar to those observed for other PLA(2)s. In the present structure, the calcium binding site is empty but the conformation of the calcium binding loop is similar to those observed in the calcium bound states. Two spatially adjacent regions of residues 55-61 (a typical beta-turn I) and 83-94 (a well defined loop) are remarkably different in conformation, electrostatic characteristics and inter-segmental interactions from those found in non-neurotoxic PLA(2)s. Yet another striking structural feature in DPLA(2 )pertains to the stretch of residues 53-77, which has a series of positively charged residues protruding outwardly. The above segment is presumed to be involved in the anticoagulant activity. A unique hydrophobic patch including residues Leu17, Ala18, Ile19, Pro20, Phe106 and Leu110 is found on the surface together with an equally emphatic region of -OH groups containing residues such as Ser21, Tyr22, Ser23, Ser24, Tyr25 and Tyr28. The interactions between two molecules of DPLA(2) in the asymmetric unit are remarkably different from those observed in the standard dimers and trimers of PLA(2)s, leaving the enzyme's active site fully exposed for enzyme-substrate reactions, it makes this structure one of the most favourable examples for structure-based drug design through soaking experiments.

Amino Acid Sequence↗
PubMed2000

Early expression of antiinsulin autoantibodies of humans and the NOD mouse: evidence for early determination of subsequent diabetes.

With the development of an insulin autoantibody (IAA) assay performed in 96-well filtration plates, we have evaluated prospectively the development of IAA in NOD mice (from 4 weeks of age) and children (from 7 to 10 months of age) at genetic risk for the development of type 1 diabetes. NOD mice had heterogeneous expression of IAA despite being inbred. IAA reached a peak between 8 and 16 weeks and then declined. IAA expression by NOD mice at 8 weeks of age was strongly associated with early development of diabetes, which occurred at 16-18 weeks of age (NOD mice IAA(+) at 8 weeks: 83% (5/6) diabetic by 18 weeks versus 11% (1/9) of IAA negative at 8 weeks; P <.01). In man, IAA was frequently present as early as 9 months of age, the first sampling time. Of five children found to have persistent IAA before 1 year of age, four have progressed to diabetes (all before 3.5 years of age) and the fifth is currently less than age 2. Of the 929 children not expressing persistent IAA before age 1, only one has progressed to diabetes to date (age onset 3), and this child expressed IAA at his second visit (age 1.1). In new onset patients, the highest levels of IAA correlated with an earlier age of diabetes onset. Our data suggest that the program for developing diabetes of NOD mice and humans is relatively "fixed" early in life and, for NOD mice, a high risk of early development of diabetes is often determined by 8 weeks of age. With such early determination of high risk of progression to diabetes, immunologic therapies in humans may need to be tested in children before the development of IAA for maximal efficacy.

Age Factors↗
PubMed2000

Non-serotonergic potentiation by (-)-pindolol of DOI-induced forward locomotion in rats: possible involvement of beta-adrenoceptors?.

[1] We have previously shown that the beta-adrenergic/5-HT1 receptor partial agonist (-)-pindolol (2.0-32.0 micromol kg(-1)) enhances the increase in forward locomotion in rats produced by the 5-HT2 receptor agonist DOI (0.7 micromol kg(-1)) via net activation of post-synaptic 5-HT2 receptors. [2] It was found that neither the 5-HT1A receptor agonist and partial agonist, (+/-) 8-OH-DPAT (0.2-2.4 micromol kg(-1)) and (S)-(-)-UH-301, respectively, nor the 5-HT1A receptor antagonist WAY-100635 (0.09-1.5 micromol kg(-1)), substituted for (-)-pindolol in this in vivo behavioral model. [3] This also applies to the 5-HT1B receptor agonist and antagonist anpirtoline (0.3-4.0 micromol kg(-1)) and isamoltane (1.0-64.0 micromol kg(-1)), respectively. Neither of these compounds mimicked (-)-pindolol in its interactions with DOI. [4] The (-)-pindolol/DOI-induced increase in forward locomotion could be antagonized by the beta1 adrenoceptor antagonist betaxolol (24 micromol kg(-1)). [5] It is suggested that the intrinsic efficacy of (-)-pindolol at beta-adrenoceptors is an important aspect of its in vivo pharmacodynamic profile.

5-Hydroxytryptophan↗
PubMed2000

Adhesive properties of human basal epidermal cells: an analysis of keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells.

The basal layer of human epidermis is a heterogeneous population of proliferative and differentiating cells that can be divided into at least three functionally discrete compartments: keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells. Basal cells adhere to the underlying basement membrane via integrins, and although decreased adhesion is a key event in epidermal differentiation, the specific role of particular integrins is poorly understood. We report here on the comparative expression and function of the beta1 versus alpha6beta4 integrins in keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells of neonatal human foreskin epidermis. Adhesion assays demonstrate that both keratinocyte stem cells and transit amplifying cells comprise rapidly adhering cells that exhibit high levels of functional beta1 and alpha6beta4 integrins. Interestingly, a proportion of basal cells that have begun to differentiate in vivo within the basal layer as determined by their expression of the differentiation-specific markers K10 and involucrin also retain high levels of activated beta1 integrin, but downregulate alpha6beta4 expression selectively (termed alpha6dimbeta1bri). These cells also retain their adhesive capacity, indicating that induction of differentiation in vivo does not correlate with decreased beta1 integrin expression or function. We have previously reported on the use of alpha6 integrin in conjunction with a proliferation associated marker (10G7 ag) to separate keratinocyte stem cells (phenotype alpha6bri10G7dim) from other basal cells (Li et al. Proc Natl Acad Sci 95:3902-3907 1998). A comparison of the long-term proliferative potential of beta1bri10G7dim cells with alpha6bri10G7dim showed that selection of alpha6bri10G7dim allows the isolation of a purer fraction of keratinocyte stem cells.

Antigens, CD↗
PubMed2000

A clinico-epidemiological profile of non-healing wounds in an Indian hospital.

One hundred patients with non-healing wounds of more than six weeks' duration were studied for clinico-epidemiological factors and their correlation with healing time. The majority of subjects were aged 30-59 years; those aged 50 and over had significantly delayed healing. Most of the subjects were farmers and physical labourers. Leprosy was the most common cause of non-healing wounds, followed by diabetes, trauma and venous ulcers; the lower limbs were the most common site of involvement. Diabetic wounds were more common in those aged 40 years and above. Diabetic wounds were slower to heal than others. The time to healing was calculated from the day subjects received treatment advice at the wound clinic. Seventy-five of the study group were included for calculation of healing time. The others were lost to follow-up or excluded for other reasons.

Adolescent↗
PubMed1999

The anion-stimulated ATPase ArsA shows unisite and multisite catalytic activity.

ArsA, an anion-stimulated ATPase, consists of two nucleotide binding domains, A1 in the N terminus and A2 in the C terminus of the protein, connected by a linker. The A1 domain contains a high affinity ATP binding site, whereas the A2 domain has low affinity and it requires the allosteric ligand antimonite for binding ATP. ArsA is known to form a UV-activated adduct with [alpha-(32)P]ATP in the linker region. This study shows that on addition of antimonite, much more adduct is formed. Characterization of the nature of the adduct suggests that it is between ArsA and ADP, instead of ATP, indicating that the adduct formation reflects hydrolysis of ATP. The present study also demonstrates that the A1 domain is capable of carrying out unisite catalysis in the absence of antimonite. On addition of antimonite, multisite catalysis involving both A1 and A2 sites occurs, resulting in a 40-fold increase in ATPase activity. Studies with mutant proteins suggest that the A2 site may be second in the sequence of events, so that its role in catalysis is dependent on a functional A1 site. It is also proposed that ArsA goes through an ATP-bound and an ADP-bound conformation, and the linker region, where ADP binds under both unisite and multisite catalytic conditions, may play an important role in the energy transduction process.

Adenosine Triphosphatases↗
PubMed1999

A case control study and one year follow-up of registered epilepsy cases in a resettlement colony of North India, a developing tropical country.

A population-based case control study and monthly follow-up of 121 registered epilepsy cases was conducted during 1995-1997 in a resettlement colony of Chandigarh, India. History of various tentative risk factors, e.g. trauma, febrile seizures, family history of seizures, alcohol intake and other possible causes was elicited. An age- and sex-matched control was selected from the neighbouring families for each case. A discordant pair analysis was done for matched case/controls. History of head injury, febrile seizures and developmental delay was observed exclusively in cases (none present in controls). Odds for epilepsy were higher among people who had positive family history (O.R.= 2.1, chi2 = 5.5, C.I. = 1.1-4.3). All cases were followed up and interviewed for history of seizures and drug intake. Fourteen cases could not be followed up completely. Ninety-four (88%) of the remaining 107 cases did not have any seizures during the follow-up. Of them, 70 (75%) patients were not on medication, 13 patients were on phenytoin and 11 patients received phenobarbitone. Thirteen cases reported seizures during the follow-up. Four patients out of the latter had mental retardation and were not on medication. Cumulative incidence of epilepsy was estimated to be 0.6/1000 person-year exposure.

Adolescent↗
PubMed1999

Design of peptides with alpha,beta-dehydro-residues: synthesis, and crystal and molecular structure of a 3(10)-helical tetrapeptide Boc-L-Val-deltaPhe-deltaPhe-L-Ile-OCH3.

The peptide Boc-L-Val-deltaPhe-deltaPhe-L-Ile-OCH3 was synthesized using the azlactone method in the solution phase, and its crystal and molecular structures were determined by X-ray diffraction. Single crystals were grown by slow evaporation from solution in methanol at 25 degrees C. The crystals belong to an orthorhombic space group P2(1)2(1)2(1) with a = 12.882(7) A, b = 15.430(5) A, c = 18.330(5) A and Z = 4. The structure was determined by direct methods and refined by a least-squares procedure to an R-value of 0.073. The peptide adopts a right-handed 3(10)-helical conformation with backbone torsion angles: phi1 = 56.0(6)degrees, psi1 = -38.0(6)degrees, phi2 = -53.8(6)degrees, psi2 = 23.6(6)degrees, phi3 = -82.9(6)degrees, psi3 = -10.6(7)degrees, phi4 = 124.9(5)degrees. All the peptide bonds are trans. The conformation is stabilized by intramolecular 4-->1 hydrogen bonds involving Boc carbonyl oxygen and NH of deltaPhe3 and CO of Val1 and NH of Ile4. It is noteworthy that the two other chemically very similar peptides: Boc-Val-deltaPhe-deltaPhe-Ala-OCH3 (i) and Boc-Val-deltaPhe-deltaPhe-Val-OCH3 (ii) with differences only at the fourth position have been found to adopt folded conformations with two overlapping beta-turns of types II and III', respectively, whereas the present peptide adopts two overlapping beta-turns of type III. Thus the introduction of Ile at fourth position in a sequence Val-deltaPhe-deltaPhe-X results in the formation of a 3(10)-helix. The crystal structure is stabilized by intermolecular hydrogen bonds involving NH of Val1 and carbonyl oxygen of a symmetry related (-x, y - 1/2, 1/2 + z) deltaPhe2 and NH of deltaPhe2 with carbonyl oxygen of a symmetry related (x, y1/2, 1/2 + z) Ile4. This gives rise to long columns of helical molecules linked head to tail running along [010] direction.

Amino Acids↗
PubMed1999

Purification, crystallization and preliminary crystallographic analysis of a natural complex of phospholipase A2 from Echis carinatus (saw-scaled viper).

A novel complex of phospholipase A2 complexed with another venom protein has been isolated and purified from saw-scaled viper (Echis carinatus) venom. The molecular weights of the two components are 16 and 14 kDa, respectively. The complex was purified using an Affigel blue column and an anion-exchange (DEAE Sephacel) column. Long diamond-shaped crystals were obtained by hanging-drop vapour diffusion. The protein complex was dissolved at a concentration of 10 mg ml-1 in 20 mM sodium cacodylate, 1 mM CaCl2 and 2% dioxane at pH 6.0. The reservoir contained the same buffer with 7%(w/v) PEG 4000. Crystals appeared within 2-3 weeks. Native data to 2.9 A resolution have been obtained at 291 K. The crystals belong to the monoclinic space group P21 with unit-cell parameters a = 74.47, b = 47.87, c = 106.39 A, beta = 104.5 degrees and contain two molecules per asymmetric unit. Structure determination by molecular replacement is in progress.

Amino Acid Sequence↗
PubMed1999

Epithelial stem cells in the skin: definition, markers, localization and functions.

In recent years, cutaneous epithelial stem cells have attained a genuine celebrity status. They are considered the key resource for epidermal and skin appendage regeneration, and are proposed as a preferential target of cutaneous gene therapy. Follicular epithelial stem cells may also give rise to a large variety of epithelial tumors, and cutaneous epithelial stem cells likely are crucial targets for physical or chemical agents (including carcinogens) that damage the skin and its appendages. However, as this Controversies feature illustrates, few experts can agree on how exactly to define and identify these elusive cells, or on where precisely in the skin they are localized. Given their potential importance in skin biology, pathology and future dermatological therapy, it is, therefore, timely to carefully reconsider the basic questions: What exactly is a stem cell, and how can we reliably identify epithelial stem cells? How many different kinds are there, and how do they differ functionally? Where exactly in the skin epithelium is each of the putative stem cell subpopulations located, and can we selectively manipulate any of them?

Animals↗