Biomedical subjects

P Brodin

Publications and source records attributed to P Brodin.

At least 19 recordsLinked to original sources

PubMed2005

Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions.

Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.

Amino Acid Sequence↗

PubMed2002

A new evolutionary scenario for the Mycobacterium tuberculosis complex.

The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and "modern" strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum-->M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

Base Sequence↗

PubMed2001

The acute effects of smokeless tobacco (snuff) on gingival blood flow in man.

Snuff-induced blood flow responses in the gingiva were evaluated in 22 healthy casual consumers of tobacco. Laser Doppler flowmetry (LDF) was used to measure blood flow simultaneously and continuously on two gingival sites (buccal aspect of the papillae between the upper lateral incisors and canines). In addition, measurements of skin blood flow in the forehead and palmar side of the left thumb were performed. Arterial blood pressure (BP) and heart rate (HR) were also recorded. Unilateral application of commercial snuff (500 mg, 1%) caused a marked and rapid increase in gingival blood flow (GBF) on the exposed side (p < 0.001). Blood flow increased also in the contralateral gingiva and forehead skin (p < 0.05). Skin blood flow in the thumb showed an insignificant decrease. BP and HR increased. Vascular conductance increased significantly in the snuff-exposed gingiva but not in the contralateral gingiva or the forehead. Vascular conductance was largely unaffected in the thumb. It is concluded that acute application of snuff, besides giving rise to typical changes in BP and HR, increases GBF in and around the exposed area, probably through activation of sensory nerves and the subsequent release of vasodilatory peptides from their peripheral endings. Blood flow in unexposed gingival and forehead skin may increase probably due to humoral or nervously mediated mechanisms. However, a passive pressure-induced hyperaemia in the unexposed gingiva and forehead skin can not be excluded.

Blood Pressure↗

PubMed2000

Determinants of Mg2+-dependent activities of recombinant human immunodeficiency virus type 1 integrase.

The relationship between Mg(2+)-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, IN(CHAPS) and IN(dial), were purified in the presence of detergent, but in the case of IN(dial), the detergent was removed during a final dialysis. The third preparation, IN(zn), was purified without any detergent. The three preparations displayed comparable Mn(2+)-dependent activities. In contrast, the Mg(2+)-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. IN(CHAPS) was not capable of using Mg(2+) as a cofactor, whereas IN(zn) was highly active under the same conditions. In the accompanying paper [Deprez, E., et al. (2000) Biochemistry 39, 9275-9284], we used time-resolved fluorescence anisotropy to demonstrate that IN(CHAPS) was monomeric at the concentration of enzymatic assays. Here, we show that IN(zn) was homogeneously tetrameric under similar conditions. Moreover, IN(dial) that exhibited an intermediary Mg(2+)-dependent activity existed in a monomer-multimer equilibrium. The level of Mg(2+)- but not Mn(2+)-dependent activity of IN(dial) was altered by addition of detergent which plays a detrimental role in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase to use Mg(2+) as a cofactor is related to its self-assembly state in solution, whereas Mn(2+)-dependent activity is not. Finally, the oligomeric IN(zn) was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric IN(CHAPS) strictly required Mn(2+). Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg(2+).

Cholic Acids↗

PubMed1999

Branched oligonucleotide-intercalator conjugate forming a parallel stranded structure inhibits HIV-1 integrase.

Integration of a DNA copy of the HIV-1 genome into chromosomal DNA of infected cells is a key step of viral replication. Integration is carried out by integrase, a viral protein which binds to both ends of viral DNA and catalyses reactions of the 3'-end processing and strand transfer. A 3'-3' branched oligonucleotide functionalised by the intercalator oxazolopyridocarbazole at each 5'-end was found to inhibit integration in vitro. We show that both a specific (G,A) sequence and the OPC intercalating agent contribute to the capability of the branched oligonucleotide to form a parallel stranded structure responsible for the inhibition.

Base Sequence↗

PubMed1999

Optimization of alternate-strand triple helix formation at the 5"-TpA-3" and 5"-ApT-3" junctions.

Alternate-strand triple helix formation was optimized at the two junction steps, the 5"-TpA-3" and 5"-ApT-3" junctions. Footprint experiments, gel retardation assays and thermal denaturation measures on a sequence appropriately designed with two adjacent alternate-strand polypurine tracts points out that the addition of an adenine residue and the removal of one nucleotide should facilitate the crossing strands at the 5"-TpA-3" junction and at the 5"-ApT-3" junction, respectively. These results provide a 'switch code' for the construction of alternate-strand triple helix forming oligonucleotides which open new possibilities for extending the range of applications of antigene strategy.

Adenine↗

PubMed1998

Salivary secretion: stimulatory effects of chewing-gum versus paraffin tablets.

Paraffin tablets are commonly used in clinical saliva tests whereas chewing-gum is recommended to increase salivation in xerostomic patients. The aim of this study was to compare the effect on salivation of these stimuli. Saliva stimulated by chewing-gum or paraffin tablets was sampled on separate occasions from eight healthy subjects (25-45 yr). Whole or parotid saliva were collected for 6 min (1 min + 5 min) and 21 min (1 min + 5 min x 4), respectively. pH of saliva was measured with and without the addition of HC1 (titrated pH). Total parotid protein was measured using the microBSA-assay. Initially, flow rates were significantly higher during the chewing of gum vs. paraffin tablets (whole saliva 5.18 vs. 2.99 ml/min, parotid saliva 0.83 vs. 0.42 ml/min). Conversely, during final chewing periods, parotid flow rates, pH, and titrated pH were significantly higher during paraffin chewing. When comparing the stimuli, parotid protein output was higher initially during gum chewing, but in the final period paraffin chewing elicited the higher output. It was concluded that the clinical test of paraffin chewing gives a good estimate of the expected whole saliva response to chewing-gum. Furthermore, extended chewing of paraffin tablets seemed to influence parameters of parotid saliva positively.

Adult↗

PubMed1998

NF-kappaB only partially mediates Epstein-Barr virus latent membrane protein 1 activation of B cells.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is required for EBV-induced immortalization of human B cells and causes tumorigenic transformation of cell lines. LMP1 expression induces phenotypic changes resembling B cell activation, such as cell size increase and up-regulation of cell surface activation markers. LMP1 contains two domains that activate the transcription factor NF-kappaB, one through interactions with TRAF proteins and the other with the TRADD protein. The purpose of the present study was to investigate the importance of NF-kappaB induction in the up-regulation of the B cell activation markers ICAM-1 and CD71 by LMP1. This study shows that expression of LMP1 activates transcription from p50/p65- and c-Rel-responsive promoters, and that this activity can be completely inhibited by expression of a dominant inhibitory IkappaB mutant. ICAM-1 and CD71 are nevertheless up-regulated by LMP1 in primary B cells and cell lines expressing the dominant IkappaB. Furthermore, LMP1-induced cell size increase of primary B cells was unaffected by IkappaB expression. It was concluded that even when LMP1 is unable to activate NF-kappaB, it is still capable of inducing certain characteristics of activated B cells, strongly suggesting that LMP1 can also activate cells independently of NF-kappaB.

B-Lymphocytes↗

PubMed1998

Upregulation of growth associated protein 43 expression and neuronal co-expression with neuropeptide Y following inferior alveolar nerve axotomy in the rat.

Growth associated protein 43 (GAP 43) is an acidic membrane-bound phosphoprotein produced at high levels in developing and regenerating neurons. It is a substrate for protein kinase C and suggested to be involved in calcium-regulated release of axonal vesicular-contained neurotransmitters. Expression of GAP 43 has been demonstrated in the uninjured cat dental pulp which receives its sensory nerve supply from the trigeminal ganglion. The aim of this study was a detailed mapping of the spatial and time-dependent expression of GAP 43 and co-expression of neuropeptide Y (NPY) in dental peripheral target tissues and trigeminal neurons subsequent to inferior alveolar nerve (IAN) axotomy in rats, as background for later low-level laser studies. Unilateral sectioning of IAN, resulting in an almost complete loss of sensory nerve fibers in the ipsilateral dental pulp of the first molar, was performed. The avidin biotin complex (ABC) method was used to evaluate peripheral changes in GAP 43 expression at 4, 7 and 10 days. Ganglionic changes in GAP 43 and co-localization of neuronal NPY expression was examined at 4, 10 and 21 days using either the ABC method or double immunofluorescence labelling techniques and confocal microscopy. Axotomy resulted in an early upregulation and change in the peripheral distribution of GAP 43 in nerve profiles already 4 days post IAN axotomy suggesting a Schwann cell origin. Ten days post axotomy a pronounced upregulation of GAP 43 immunoreactivity could be demonstrated in neurons located in the mandibular region of the trigeminal ganglion, compared to the contralateral uninjured side. The peripheral and ganglionic upregulation of GAP 43 continued to persist at 21 days. A concomitant time-delayed shift and co-expression of NPY was demonstrated throughout in the GAP 43-upregulated ganglion cells 10 days post axotomy. Furthermore, confocal microscopy indicated that the intraneuronal distribution of NPY and upregulated GAP 43 expression showed a similar conformity and distribution in both perinuclear regions and cell periphery.

Animals↗

PubMed1997

A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.

Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an alpha-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the alpha-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nM. In contrast, a peptide with a full alpha-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 degrees C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.

Amino Acid Sequence↗

PubMed1997

Conditions for excitatory or inhibitory masseteric reflexes elicited by tooth pressure in man.

The reflex responses evoked by slowly rising pressure "push' stimuli to an upper lateral incisor tooth in human masseter muscle were studied. Factors such as the preload (the static force applied to the tooth by the stimulus probe before the start of the push stimulus) and the shape of the stimulus wave affected the outcome of the reflex response. When the stimulating probe did not apply preload to the tooth before the push stimulus took place, the force profile exhibited a large fast component as the probe took up the 'slack' in the periodontium. The fast component in the force profile was found to be responsible for inducing the inhibitory reflex (sole inhibitory reflex). When preload was applied, the size of the fast component in the force profile was reduced and the change in force rate became slower and smoother. This slower stimulus profile induced the sole excitatory reflex significantly more often (58% vs 21%) and the sole inhibitory reflex significantly less often (15% vs 52%) than in the experiments that used no preload. The shape of the stimulus wave that drove the stimulus probe was also of importance. Provided that a 0.5-N preload was applied to the tooth, the smoothest stimulus wave induced the sole excitatory reflex most often. Fitting a rubber attachment to the up of the probe made the push-force profile even smoother and thence more successful in inducing the sole excitatory reflex. Furthermore, the rubber tip reduced the possibility of the probe slipping off the tooth due to small and unavoidable movements of the participant's head. It is concluded that the periodontal mechanoreceptors can induce both excitatory and inhibitory reflexes on the jaw closers. The excitatory reflex becomes dominant when a smooth force is applied with preload. The inhibitory reflex becomes dominant when fast-force changes are applied on the tooth and/or no preload is used.

Adult↗

PubMed1996

Instant assessment of pulpal blood flow after orthodontic force application.

The purpose of the present investigation was to measure the effect of tooth intrusion and extrusion on pulpal blood flow in man. The flow changes were measured in 10 lateral incisors (6 subjects) by means of laser Doppler flowmetry (Perimed, Sweden) applied on the buccal surface through metal tubes bonded to the teeth. Brackets were bonded onto teeth 13 and 23, and intrusive and extrusive forces of 2 N were applied in random order to the experimental tooth for 5 min before unloading. The contralateral tooth served as a control. Extrusion of the teeth gave no significant changes in pulpal blood flow during loading or unloading. However, intrusion of the teeth reduced the pulpal blood flow by 20% during the first minute after force application. The pulpal blood flow gradually increased towards the preloading flow values for the next 4 min, and was returned to the prestimulus level 3 min after unloading. In conclusion orthodontic intrusion of teeth with a 2 N force evoked a temporary reduction in the pulpal blood flow, whereas extrusion had no effect on the pulpal blood flow.

Dental Pulp↗

PubMed1996

Preliminary study of low-level laser for treatment of long-standing sensory aberrations in the inferior alveolar nerve.

PURPOSE: The incidence of inferior alveolar nerve (IAN) damage during removal of third molar teeth has been reported to be as high as 5.5% and up to 100% during sagittal split osteotomy. Sensory aberrations in the IAN persisting for longer than 6 months leave some degree of permanent disability. The purpose of this double-blind, clinical trial was to examine the effects of low-level laser (LLL) treatment using a GaAIAs laser (820 nm, Rønvig, Denmark) on touch and temperature sensory perception after a long-standing postsurgical IAN injury. PATIENTS AND METHODS: Thirteen patients were divided into two groups, one of which received real LLL (4 x 6 J per treatment along the distribution of the IAN to a total of 20 treatments) and the other placebo LLL. The degree of mechanoreceptor injury as assessed by Semmes Weinstein Monofilaments (North Coast Medical, San Jose, CA) were comparable in the two groups before treatment. The degree of thermal sensitivity disability as assessed using a Thermotester (Somedic AB, Stockholm, Sweden) to examine the indifferent temperature threshold was also comparable between the two groups before LLL. RESULTS: Subsequent to LLL, the real laser-treated group showed a significant improvement in mechanoreceptor sensory testing (P = .01) compared with the placebo group, as manifested by a decrease in load threshold (g) necessary to elicit a response from the most damaged area. In addition, the real LLL group reported a subjective improvement in sensory function. There was no significant improvement in thermal sensitivity post-LLL for either the real or placebo laser-treated groups. CONCLUSION: It was concluded that LLL can improve mechanoreceptor perception in long-standing sensory aberrations in the IAN.

Adolescent↗

PubMed1996

Reflex responses of the masseter muscle in patients with chronic arthritis or internal derangement of the temporomandibular joint. A comparison with symptomfree subjects.

Averaged reflex responses in the masseter muscle to transverse taps delivered to the upper central incisor were studied in 13 patients with chronic arthritic temporomandibular joint (TMJ) disease (arthritis group) and 28 patients having internal TMJ derangement (derangement group). The diagnostic assessment of TMJ disease was based on a combination of imaging methods. Fourteen symptomfree subjects served as a control for the electromyographic observations. The tapping force was increased in steps from 0.25 to 6 N. The pattern of the reflex response consisted of various inhibitory and excitatory waves, which were found to change with increasing tapping force. The first inhibitory wave (I-1) increased in duration with increasing tapping force in all subjects. At certain tapping forces, I-1 was of significantly lower amplitude and longer duration in the arthritis group than in the control group, but latency and threshold did not differ. The excitatory wave seemed to have longer latency and higher amplitude in the arthritis group than in the control subjects at some tapping forces. In summary we conclude that the pattern of the reflex response seemed to be similar in patients with different TMJ disorders and in symptomfree subjects. Only the level of inhibition, duration of I-1 and higher excitatory response, might separate the patients with arthritic TMJ disease and internal TMJ derangement from symptomfree subjects, but no specific differences between the groups could be found.

Adult↗

PubMed1996

Increased human gingival blood flow induced by sodium lauryl sulfate.

Laser Doppler flowmetry was used in 14 subjects to investigate the effect of sodium lauryl sulfate (SLS) on gingival microcirculation. SLS, the most widely used detergent in toothpastes, exhibits a denaturing property, increases oral mucosal permeability and may cause epithelial desquamation of oral soft tissues. Gingival blood flow (GBF) was measured bilaterally 3 mm above the gingival margin, between the central and lateral maxillary incisors, before and after application of 1.5% SLS for 90 s to the gingiva on one side. The contralateral side served as a control with water application only. Relative changes in GBF were measured for 15 min on both sides. SLS increased the median GBF significantly between the 2nd and 10th min with a peak at 8 min, whereafter the flow decreased towards baseline. On the contralateral side, GBF initially declined before returning to baseline. 10 subjects felt a burning pain from the gingiva after SLS application and GBF increased in all of them. SLS increased the relative gingival blood flow presumably due to its penetrating and irritative properties, which resulted in vasodilatation. Laser Doppler flowmetry may be a useful non-invasive method for intraoral testing of different agents meant for oral use.

Adult↗

PubMed1996

The effects of sodium lauryl sulfate on the phrenic nerve diaphragm preparation from the rat.

The time period to 50% inhibition of the twitches and compound action potential of the rat phrenic nerve-diaphragm preparation with sodium lauryl sulfate (2.5 x 10(-4)-5.0 x 10(-3)M) was recorded. The twitch contractions during indirect stimulation, the contraction during direct stimulation, and the compound action potential of the isolated phrenic nerve, were inhibited in that order. Depolarization due to high action potential activity induced by tetanic stimulation, veratridine, low Ca(2+)-or high K(+)-solutions, all enhanced the inhibitory effect of sodium lauryl sulfate (5.0 x 10(-4)M) during indirect stimulation. Hyperpolarization in K(+)-free solution and membrane stabilization by lidocaine antagonized the inhibitory effect of sodium lauryl sulfate. Accordingly, it probably induced inhibition by depolarization. The depolarization may decrease the influx of Ca2+ and the reuptake of choline+, as suggested by an observed synergism with tetraethylammonium CI and hemicholinium-3 Br, which antagonize the reuptake of choline+. On the opposite, 3,4-diaminopyridine, which increases the influx of Ca2+, antagonized the sodium lauryl sulfate-induced inhibition, and induced a biphasic contracture. The first phase was probably caused by a depolarization, and the second phase by release of Ca2+ from the sarcoplasmic reticulum. The potentiation of the second phase by dantrolene and in low Ca2+ solution, was attributed to sodium lauryl sulfate-induced desensitization.

Action Potentials↗

PubMed1995

Laser Doppler flowmetry in the assessment of tooth vitality after Le Fort I osteotomy.

The aim of the present study was to evaluate pulp vitality in 15 patients, 11 to 29 months after a Le Fort I osteotomy. Assessment of pulpal blood flow was obtained by means of laser Doppler flowmetry, and it was measured bilaterally from the first maxillary premolar to the central incisor. The results were compared with those obtained by electric pulp testing in the same teeth. In addition, periodontal ligament perception was evaluated by applying axial loads to the central incisors. Radiographs were also studied. Fourteen subjects served as a control group. There was no significant difference between the level of pulpal blood flow in the teeth of the operated group compared with the control group. Twenty-one per cent of the teeth in the group operated on were insensitive to electric pulp testing, and in the remaining 79% the mean threshold was significantly higher than in the corresponding teeth in the control subjects. Similarly, the axial load threshold for the incisors for the control subjects was below 5 g, but in the group operated on 66% had a threshold above 5 g. These experiments clearly demonstrate that teeth without normal innervation can have an intact blood supply and be vital.

Adult↗

PubMed1995

The cytoplasmic C-terminal domain but not the N-terminal domain of latent membrane protein 1 of Epstein-Barr virus is essential for B cell activation.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) is essential for EBV-induced immortalization of human primary B cells, transforms rodent fibroblasts and induces the up-regulation of several B cell activation markers when transiently expressed in primary B cells. The biochemical function of LMP-1 is unclear and limited information is available on the involvement of different domains of the protein in B cell activation. The present study describes the characterization of LMP-1 N- and C-terminal deletion mutants in terms of their cell surface distribution and ability to induce activation markers in primary human B cells and in the type I Burkitt's lymphoma cell line DG75. The C-terminal deletion mutant was detected by immunofluorescence via antibodies targeted against an eight amino acid FLAG epitope substituted for the entire predicted cytoplasmic C-terminal domain. Our findings show that N-terminal deletion mutants of LMP-1 are unable to attain their usual patched distribution on the plasma membrane but retain the ability to activate B cells. In contrast, the C-terminal deletion mutant shows the same patched cell surface distribution as wild-type LMP-1 but is unable to activate B cells. The patched distribution of LMP-1 in the plasma membrane is therefore not sufficient nor necessary for the induction of B cell activation and the results rule out patching as a direct mechanism in LMP-1-induced activation. This is the first study addressing the role of the LMP-1 C-terminal domain in lymphocytes and our results show that this domain is essential for B cell activation and therefore likely to be important for the early events of B cell immortalization by EBV.

Antigens, CD↗