Biomedical subjects

K Natarajan

Publications and source records attributed to K Natarajan.

At least 55 records · Page 3Linked to original sources

PubMed1999

Intracranial volume change in childhood.

OBJECT: The goal of this study was to construct a model of normal changes in intracranial volume occurring throughout childhood from age 7 days to 15 years. METHODS: Using the technique of segmentation on magnetic resonance images obtained in healthy children, intracranial volume was measured and plotted against age. CONCLUSIONS: Intracranial volume in the first few months of life is on average 900 cm3 in males and 600 cm3 in females. By the age of 15 years, it increases up to 1500 cm3 in males and 1300 cm3 in females, increased by factors of 1.6 and 2.1, respectively. By the time the child reaches 2 years of age, intracranial volume has reached 77% (1150 cm3 in males and 1000 cm3 in females) and, by 5 years, 90% (1350 cm3 in males and 1200 cm3 in females) of the volume observed at age 15 years. The change in intracranial volume that occurs with age is not linear, but there seems to be a segmental pattern. Three main periods can be distinguished, each lasting approximately 5 years (0-5, 5-10, and 10-15 years), during which the growth of intracranial volume is linear. Throughout childhood, males have higher intracranial volumes than females, with a similar growth pattern.

Adult↗

PubMed1999

Intracranial volume change in craniosynostosis.

OBJECT: There is still controversy regarding the optimum time to perform surgery for craniosynostosis. Some recommend surgery soon after birth and others delay until the age of 12 months. Intracranial pressure has been measured in an attempt to provide a scientific rationale, but many questions remain unanswered. To date, little attention has been given to intracranial volume and its changes during the first few years of life in children with craniosynostosis. The authors' goal was to focus on intracranial volume during this period and to compare measurements obtained in patients with craniosynostosis with measurements obtained in healthy individuals. METHODS: Using the technique of segmentation, the intracranial volume of 84 children with various forms of craniosynostosis was measured on preoperative computerized tomography scans. The change in average volume that occurs with increasing age was calculated and compared with a model of normal intracranial volume growth. The age at presentation for children with craniosynostosis was 1 to 39 months; 76% of the patients were younger than 12 months. In eight patients in whom only one cranial expansion procedure was performed, postoperative intracranial volumes were measured as well. Several interesting observations emerged. 1) There was little difference in head growth between boys and girls with craniosynostosis during the first few months of life. After the age of 12 months, however, the difference in intracranial volume normally seen between the two genders was observed in the craniosynostosis group as well. 2) Excluding children with complex pansynostosis, who have smaller heads, children with all other types of craniosynostosis have similar head growth after the 1st year of life, with no difference between the number of and type of suture affected. Children with Apert's syndrome develop greater than normal intracranial volumes after the 1st year of life. 3) Although children with craniosynostosis are born with a smaller intracranial volume, by the age of 6 months volume has reached normal levels, and from that point on volume follows the pattern of normal head growth. 4) Children who presented after the age of 6 months and later developed recurrent craniosynostosis after initial successful treatment had a small intracranial volume at their initial presentation. 5) Of the patients whose postoperative intracranial volumes were measured, all but one had preoperative volumes at or above normal values, and their postoperative volumes were considerably higher than normal for their age. These children all followed a growth curve parallel to that of healthy children but at higher volume value. One patient with a smaller-than-normal initial intracranial volume was surgically treated at a very young age and, despite cranial expansion surgery, postoperative volume did not reach normal levels. It is postulated that this was due to the fact that the operation was performed at a time when craniosynostosis was still active. CONCLUSIONS: The results of this study indicate that the underlying mechanism leading to craniosynostosis and constriction of head volume "exhausts" its effect during the first few months of life. Measurement of intracranial volume in clinical practice could be used to "fine tune" the optimum time for surgery. In late-presenting children, this may be useful in predicting possible recurrence.

Adolescent↗

PubMed1999

MHC class I molecules, structure and function.

MHC class I molecules (MHC-I) are cell surface recognition elements expressed on virtually all somatic cells. These molecules sample peptides generated within the cell and signal the cell's physiological state to effector cells of the immune system, both T lymphocytes and natural killer (NK) cells. In addition, molecules structurally related to MHC-I, collectively known as MHC-Ib, are more specialized and, in some cases, interact with more limited subsets of lymphoid cells. Using the recently determined structure of the classical MHC-I molecule, H-2Dd, as a paradigm for structure and function, we review other MHC-I and MHC-Ib molecules, with an emphasis on how the same basic structural fold is employed by classical MHC-I molecules to bind specific peptides and T cell receptors, and is exploited by the MHC-Ib molecules in more stringent molecular interactions. It is instructive that structurally related molecules have evolved to perform a number of unique and distinct functions in immune and non-immune recognition.

Antigen Presentation↗

PubMed1998

Protein tyrosine kinase inhibitors block tumor necrosis factor-induced activation of nuclear factor-kappaB, degradation of IkappaBalpha, nuclear translocation of p65, and subsequent gene expression.

Several inflammatory effects of tumor necrosis factor (TNF) are known to be mediated through activation of a nuclear transcription factor NF-kappaB, but how TNF activates NF-kappaB is incompletely understood. In the present report, we examined the role of protein tyrosine kinases (PTK) in TNF-mediated NF-kappaB activation by using genistein and erbstatin, two potent inhibitors of PTK. The treatment of human myeloid U-937 cells with either inhibitor completely suppressed the TNF-induced NF-kappaB activation in a dose- and time-dependent manner. Suppression correlated with PTK activity, since among the structural analogues of genistein, only an active inhibitor of PTK, quercetin blocked TNF-induced NF-kappaB activation and not daidzein, an inactive inhibitor. Inhibition of NF-kappaB activation was not limited to myeloid cells, as it was observed with T cells and epithelial cells. Both the PTK inhibitors blocked the degradation of IkappaBalpha, the inhibitory subunit of NF-kappaB, and the consequent translocation of the p65 subunit without any significant effect on p50 or on c-Rel. The PTK inhibitors did not interfere with NF-kappaB binding to DNA. The NF-kappaB-dependent CAT reporter gene expression in transient transfection assays was also suppressed by the PTK inhibitors. Both PTK inhibitors abolished TNF-induced activation of N-terminal c-Jun kinase and mitogen-activated protein kinase kinase. Overall, our results suggest that a genistein- and erbstatin-sensitive PTK is involved in the pathway leading to NF-kappaB activation and gene expression by TNF and thus could be used as a target for development of antiinflammatory drugs.

Biological Transport, Active↗

PubMed1998

Overexpression of the p80 TNF receptor leads to TNF-dependent apoptosis, nuclear factor-kappa B activation, and c-Jun kinase activation.

Because they have distinct intracellular domains, it has been proposed that the p60 and p80 forms of the TNF receptor mediate different signals. Several signaling proteins have been isolated that associate with either the p60 or the p80 receptor. By using TNF muteins specific to the p60 and p80 receptors, we have previously shown that cytotoxicity and nuclear factor-kappa B (NF-kappa B) activation are mediated through the p60 form of the endogenous receptor. What signals are mediated through the p80 receptor is less clear. This study was an effort to answer that question. HeLa cells, which express only p60 receptors, were transfected with p80 receptor cDNA and then examined for apoptosis, NF-kappa B activation, and c-Jun kinase activation induced by TNF and by p60 or p80 receptor-specific muteins. The p80 mutein, like TNF and the p60 mutein, induced apoptosis and activation of NF-kappa B and c-Jun kinase in cells overexpressing recombinant p80 receptor but had no effect on cells expressing a high level of endogenous p80 receptor. The apoptosis mediated through the p60 receptor was also potentiated after overexpression of the p80 receptor, suggesting a synergistic relationship between the two receptors. Interestingly, Abs to the p80 receptor blocked apoptosis induced by all ligands but by itself activated NF-kappa B in the p80-transfected cells. Overall, our results show that the p80 receptor, which lacks the death domain, mediated apoptosis, NF-kappa B activation, and c-Jun kinase activation, but only when it was overexpressed, whereas endogenous p60 receptor mediated similar signals without overexpression.

Antigens, CD↗

PubMed1998

Three-dimensional structure of H-2Dd complexed with an immunodominant peptide from human immunodeficiency virus envelope glycoprotein 120.

The crystal structure of the mouse major histocompatibility complex (MHC) class I molecule H-2Dd with an immunodominant peptide, designated P18-I10 (RGPGRAFVTI), from human immunodeficiency virus envelope glycoprotein 120 was determined at 3.2 A resolution. A novel orientation of the alpha3 domain of Dd relative to the alpha1/alpha2 domains results in significantly fewer contacts between alpha3 and beta2-microglobulin compared with other MHC class I proteins. Four out of ten peptide residues (P2 Gly, P3 Pro, P5 Arg and P10 Ile) are nearly completely buried in the Dd binding groove. This is consistent with previous findings that Dd exploits a four-residue binding motif comprising a glycine at P2, a proline at P3, a positively charged residue at P5, and a C-terminal hydrophobic residue at P9 or P10. The side-chain of P5 Arg is directed toward the floor of the predominantly hydrophobic binding groove where it forms two salt bridges and one hydrogen bond with Dd residue Asp77. The selection of glycine at P2 appears to be due to a narrowing of the B pocket, relative to that of other class I molecules, caused by Arg66 whose side-chain folds down into the binding cleft. Residue P3 Pro of P18-I10 occupies part of pocket D, which in Dd is partially split by a prominent hydrophobic ridge in the floor of the binding groove formed by Trp97 and Trp114. Residues P6 through P9 form a solvent-exposed bulge, with P7 Phe protruding the most from the binding groove and thereby probably constituting a major site of interaction with T cell receptors. A comparison of H-2Dd/P18-I10 with other MHC class I/peptide complexes of known structure provides insights into the possible basis for the specificity of the natural killer cell receptor Ly-49A for several related class I molecules.

Animals↗

PubMed1998

Molecular analysis of skewed Tcra-V gene use in T-cell receptor beta-chain transgenic mice.

The influence of beta-chain diversity on the expressed T-cell receptor (TCR) alpha-chain repertoire was investigated using transgenic mice which exclusively express a single rearranged TCR beta-chain gene. Analysis of these mice using alpha-chain-specific recombinant cDNA libraries showed that expression of the transgene-encoded beta chain results in significant skewing in Tcra-V gene segment usage vs nontransgenic mice. Skewing was most pronounced towards alpha chains using TCRA-V segments. Sequence analysis of Tcra-V8-containing genes from transgenic T cells revealed predominant use of a single Tcra-J segment (Tcra-J24), which was not detected in Tcra-V8 containing genes isolated from nontransgenic T cells. Further analysis revealed that co-expression of Tcra-V8 with Tcra-J24 in beta-transgenic mice is exhibited almost exclusively by CD4+ T cells, and is associated with a limited number of closely related N-regions. Analysis of transgenic CD8+ T cells demonstrated predominant co-expression of Tcra-V8 with another Tcra-J (Tcra-J30), together with a different, limited N-region sequence. We conclude that the composition of expressed beta chains can profoundly influence the selection of companion alpha chains expressed in the periphery, and that alpha-chain N and J regions play a crucial role in discriminating between class I vs class II major histocompatibility complex (MHC)-restricted recognition. Further, these results are in agreement with recent data concerning the crystal structure of the TCR, and most consistent with a model for TCR structure in which the complementarity determining region (CDR)3alpha domain participates in direct contact with the MHC.

Amino Acid Sequence↗

PubMed1998

Computer simulation of a neurosurgical operation: craniotomy for hypothalamic hamartoma.

Although magnetic resonance imaging has revolutionised the management of intracranial lesions with improved visualisation of anatomical structures, it only produces two-dimensional images, from which the clinician has to extrapolate a three-dimensional interpretation. Several approaches can be used to create 3D images; the discipline of image segmentation has encompassed a number of these techniques. Such techniques allow the clinician to delineate areas of interest. The resulting computer-generated outlines can be reconstructed in a three-dimensional arrangement. Although a plethora of "generic" segmentation techniques exist, we have developed a refined form, dependent on general and particular properties of the anatomical structures under investigation. High-contrast structures such as the ventricles and external surface of the head are found by using a localised adaptive thresholding technique. Less definable structures, with poor or nonexistent signal change across neighbouring structures, such as brain stem or pituitary, are found by applying an "energy minimisation"-based technique. To demonstrate the techniques we used the example of an 8-year-old boy with uncontrolled gelastic seizures due to a hypothalamic hamartoma, who is being considered for surgery. We were able to demonstrate the anatomical relationships between the hypothalamic hamartoma and adjacent structures such as optic chiasm, brain stem and ventricular system. We were subsequently able to create a video, reproducing the stages of craniotomy for excision of this tumour. By creating true 3D objects, we were able at any stage of the simulation to visualise structures situated contralaterally to the approaching surgical dissector. These 3D representations of the structures can be either invisible or opaque, in order to afford 3D localisation as the "virtual" surgical dissection proceeds. The clinical application of such techniques will enable surgeons to improve their understanding of anatomical relations of intracranial lesions and has obvious implications in image-guided surgery.

Child↗

PubMed1998

Solution structure of a synthetic lytic peptide: the perforin amino terminus.

BACKGROUND: Killer lymphocytes secrete perforin, a 67 kDa protein that initiates T-cell cytolysis following aggregation and pore formation in target membranes. The resulting pores cause a breakdown of the transmembrane osmotic gradient and allow other cytolytic mediators to enter the target cell and initiate apoptosis. The cytolytic domain resides within the first 34 residues of the amino terminus of perforin, with residues 1-19 being sufficient for cytolytic activity. RESULTS: The solution structure of a 22-residue synthetic peptide (P22), corresponding to the amino terminus of human perforin, has been determined using high resolution nuclear magnetic resonance spectroscopy in the presence and absence of perdeuterated detergent (SDS) micelles. In aqueous solution, P22 exists mainly in a random conformation. However, it adopts a hook-like structure at the carboxyl terminus in the presence of SDS micelles when the positively charged residues cluster to form a turn that provides a binding surface to the negatively charged sulfate headgroups. CONCLUSIONS: The strong electrostatic interaction between the cationic region of the P22 peptide and the lipid headgroups probably weakens the membrane, facilitating insertion of the relatively neutral/hydrophobic stretch of P22, and is representative of the initial step of the lytic pathway. The structural model described here is probably relevant to understanding the mechanisms of other cationic antimicrobial peptides.

Amino Acid Sequence↗

PubMed1998

yTAFII61 has a general role in RNA polymerase II transcription and is required by Gcn4p to recruit the SAGA coactivator complex.

We obtained a recessive insertion mutation in the gene encoding yeast TBP-associated factor yTAFII61/68 that impairs Gcn4p-independent and Gcn4p-activated HIS3 transcription. This mutation also reduces transcription of seven other class II genes, thus indicating a broad role for this yTAFII in RNA polymerase II transcription. The Gcn4p activation domain interacts with multiple components of the SAGA complex in cell extracts, including the yTAFII proteins associated with SAGA, but not with two yTAFIIs restricted to TFIID. The taf61-1 mutation impairs binding of Gcn4p to SAGA/yTAFII subunits but not to components of holoenzyme mediator. Our results provide strong evidence that recruitment of SAGA, in addition to holoenzyme, is crucial for activation by Gcn4p in vivo and that yTAFII61 plays a key role in this process.

Acetyltransferases↗

PubMed1998

Use of recombinant pemphigus vulgaris antigen in development of ELISA and IB assays to detect pemphigus vulgaris autoantibodies.

AIM/OBJECTIVE: The objectives of this study are: (1) to measure the titers of pemphigus vulgaris (PV) autoantibody in the sera of patients with active disease, using three different assays: (a) Indirect immunofluorescence (IIF) using monkey esophagus as a substrate, (b) immunoblot (IB) and, (c) enzyme-linked immunosorbent assay (ELISA) using recombinant PV antigen (rPVA). (2) To compare the sensitivity of these three assays. BACKGROUND: The titer of PV autoantibodies and disease severity and extent do not always correlate. This could be due to the lack of consistency and specificity of the substrate. Different results are obtained using different substrates. A standard substrate with uniformly controlled source of antigen would be more useful and clinically beneficial. METHODS: In this study we studied 25 PV patients, six each with bullous pemphigoid (BP), ocular cicatricial pemphigoid (OCP), mucous membrane pemphigoid (MMP), and herpes gestationis (HG), and sera from 16 normal subjects. IIF was used to determine the PV autoantibody using monkey esophagus. IB assay was used according to standard protocol using normal human epidermis and rPVA as substrates. ELISA was performed using rPVA as antigens expressed in E. coli. RESULTS: Sera of all 25 PV patients showed binding to the rPVA, normal human sera and the sera from the six BP, six OCP, six MMP, and six HG patients did not show any binding. In addition, we used antisera from rabbits immunized with PVA peptides (Bos-1, Bos-6) which also showed binding to rPVA, whereas normal rabbit sera did not show any reactivity. ELISA and IB titers in all the patients were 2.5 to 160 times higher than with the conventionally used IIF assay. The titers of the PV specific autoantibody measured using the rPVA did not show statistically significant differences between the ELISA and IB assays. CONCLUSIONS: IB and ELISA are superior to IIF in evaluating the antibody levels in PV patients. ELISA is more practical and is preferable to IB and is recommended for clinical use.

Animals↗

PubMed1997

Characterization of a partially unfolded high potential iron protein.

A partially unfolded state of the Fe4S4-containing high potential iron-sulfur protein from Chromatium vinosum has been detected and characterized by NMR spectroscopy following addition of a concentrated solution of guanidinium chloride to the native protein. This intermediate species (i) maintains the polymetallic center, (ii) exhibits a largely collapsed secondary structure, and (iii) undergoes fast cluster decomposition upon oxidation. This information is framed into the knowledge about this class of proteins, and the possible role of this intermediate with respect to the in vivo folding/unfolding process is discussed as well its role in the slow hydrolytic degradation characteristic of oxidized HiPIPs.

Chromatium↗

PubMed1997

Involvement of host factors in transcription from baculovirus very late promoters -- a review.

The baculovirus expression vector system has emerged as the system of choice for the expression of a number of heterologous genes of both prokaryotic and eukaryotic origin. This system utilizes the baculovirus very late, hyperactive polyhedrin and p10 promoters to drive the transcription of foreign genes. Regulation of transcription from these promoters is presently not well understood even though a number of viral gene products that may be important for transcription have been identified. Fresh insight into host-virus interactions during baculovirus pathogenesis is now offered by the identification of insect host factors that interact with transcriptionally essential motifs of these promoters as well as cis-acting enhancer-like elements upstream from the promoter.

Base Sequence↗

PubMed1996

Ocular cicatricial pemphigoid antigen: partial sequence and biochemical characterization.

Ocular cicatricial pemphigoid (OCP) is an autoimmune disease that affects mainly conjunctiva and other squamous epithelia. OCP is histologically characterized by a separation of the epithelium from underlying tissues within the basement membrane zone. Immunopathological studies demonstrate the deposition of anti-basement membrane zone autoantibodies in vivo. Purified IgG from sera of patients with active OCP identified a cDNA clone from a human keratinocyte cDNA library that had complete homology with the cytoplasmic domain of beta 4-integrin. The sera recognized a 205-kDa protein in human epidermal, human conjunctiva, and tumor cell lysates that was identified as beta 4-integrin by its reaction with polyclonal and monoclonal antibodies to human beta 4-integrin. Sera from patients with bullous pemphigoid, pemphigus vulgaris, and cicatricial pemphigoid-like diseases did not recognize the 205-kDa protein, indicating the specificity of the binding. These data strongly implicate a role for human beta 4-integrin in the pathogenesis of OCP. It should be emphasized that multiple antigens in the basement membrane zone of squamous epithelia may serve as targets for a wide spectrum of autoantibodies observed in vesiculobullous diseases. Molecular definition of these autoantigens will facilitate the classification and characterization of subsets of cicatricial pemphigoid and help distinguishing them from bullous pemphigoid. This study highlights the function and importance of beta 4-integrin in maintaining the attachment of epithelial cells to the basement membrane.

Amino Acid Sequence↗

PubMed1996

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a potent inhibitor of nuclear transcription factor-kappa B activation by diverse agents.

Viral replication, immune regulation, and induction of various inflammatory and growth-regulatory genes require activation of a nuclear transcription factor (NF)-kappa B. Agents that can block NF-kappa B activation have potential to block downstream responses mediated through this transcription factor. Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a quinone that has been shown to regulate a wide variety of activities that require NF-kappa B activation. In the present study, we examined the effect of capsaicin and its analogue, resiniferatoxin, on the activation of NF-kappa B induced by different agents including TNF. The pretreatment of human myeloid ML-1a cells with capsaicin blocked TNF-mediated activation of NF-kappa B in a dose- and time-dependent manner. Resiniferatoxin was at least eight times as potent as capsaicin in inhibiting NF-kappa B activation. Neither agent by itself activated NF-kappa B or affected the DNA-binding ability of NF-kappa B. Capsaicin also blocked phorbol ester-mediated NF-kappa B activation, but that mediated through okadaic acid was less effective, suggesting there is a difference in the mechanism of activation of NF-kappa B by different agents. Capsaicin treatment of cells also blocked the degradation of I kappa B alpha, and thus the nuclear translocation of the p65 subunit of NF-kappa B, which is essential for NF-kappa B activation. TNF-dependent promoter activity of I kappa B alpha, which contains NF-kappa B binding sites, was also inhibited by capsaicin. Overall our results indicate that capsaicin and its analogue inhibit NF-kappa B activation, and thus could be used as a potential target for drug development.

Capsaicin↗

PubMed1996

Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B.

Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antimitogenic, anticarcinogenic, antiinflammatory, and immunomodulatory properties. The molecular basis for these diverse properties is not known. Since the role of the nuclear factor NF-kappa B in these responses has been documented, we examined the effect of CAPE on this transcription factor. Our results show that the activation of NF-kappa B by tumor necrosis factor (TNF) is completely blocked by CAPE in a dose- and time-dependent manner. Besides TNF, CAPE also inhibited NF-kappa B activation induced by other inflammatory agents including phorbol ester, ceramide, hydrogen peroxide, and okadaic acid. Since the reducing agents reversed the inhibitory effect of CAPE, it suggests the role of critical sulfhydryl groups in NF-kappa B activation. CAPE prevented the translocation of the p65 subunit of NF-kappa B to the nucleus and had no significant effect on TNF-induced I kappa B alpha degradation, but did delay I kappa B alpha resynthesis. The effect of CAPE on inhibition of NF-kappa B binding to the DNA was specific, in as much as binding of other transcription factors including AP-1, Oct-1, and TFIID to their DNA were not affected. When various synthetic structural analogues of CAPE were examined, it was found that a bicyclic, rotationally constrained, 5,6-dihydroxy form was superactive, whereas 6,7-dihydroxy variant was least active. Thus, overall our results demonstrate that CAPE is a potent and a specific inhibitor of NF-kappa B activation and this may provide the molecular basis for its multiple immunomodulatory and antiinflammatory activities.

Base Sequence↗

PubMed1996

Identification of seven hydrophobic clusters in GCN4 making redundant contributions to transcriptional activation.

GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. The N-terminal 100 amino acids of GCN4 contains a potent activation function that confers high-level transcription in the absence of the centrally located acidic activation domain (CAAD) delineated in previous studies. To identify specific amino acids important for activation by the N-terminal domain, we mutagenized a GCN4 allele lacking the CAAD and screened alleles in vivo for reduced expression of the HIS3 gene. We found four pairs of closely spaced phenylalanines and a leucine residue distributed throughout the N-terminal 100 residues of GCN4 that are required for high-level activation in the absence of the CAAD. Trp, Leu, and Tyr were highly functional substitutions for the Phe residue at position 45. Combined with our previous findings, these results indicate that GCN4 contains seven clusters of aromatic or bulky hydrophobic residues which make important contributions to transcriptional activation at HIS3. None of the seven hydrophobic clusters is essential for activation by full-length GCN4, and the critical residues in two or three clusters must be mutated simultaneously to observe a substantial reduction in GCN4 function. Numerous combinations of four or five intact clusters conferred high-level transcription of HIS3. We propose that many of the hydrophobic clusters in GCN4 act independently of one another to provide redundant means of stimulating transcription and that the functional contributions of these different segments are cumulative at the HIS3 promoter. On the basis of the primacy of bulky hydrophobic residues throughout the activation domain, we suggest that GCN4 contains multiple sites that mediate hydrophobic contacts with one or more components of the transcription initiation machinery.

Alleles↗

PubMed1996

Mutations in the Dictyostelium heterotrimeric G protein alpha subunit G alpha5 alter the kinetics of tip morphogenesis.

Tip morphogenesis during the Dictyostelium developmental life cycle is a process by which prestalk cells sort to form the anterior region of the multicellular organism. We show that the temporal regulation of this morphological process is dependent on the copy number of the Dictyostelium G alpha5 gene. Tip formation is delayed in aggregates of g alpha5 null mutant cells and accelerated in aggregates overexpressing the G alpha5 gene compared to tip formation in wild-type cells. The onset of cell-type-specific gene expression associated with mound formation and tip morphogenesis is also temporally altered in G alpha5 mutants. Tip morphogenesis in chimeric organisms of G alpha5 mutants and wild-type cells is dependent on the copy number of the G alpha5 gene, indicating that G alpha5 function plays an integral role in the intercellular signaling of this stage of development. The G alpha5 gene encodes a G alpha subunit that has 51% identity to the Dictyostelium G alpha4 subunit. Like the G alpha4 gene, the G alpha5 gene is expressed in a subset of cells distributed throughout the multicellular organism, with a distribution that is similar to the anterior-like cell population. Amino acid substitutions in the G alpha5 subunit analogous to substitutions altering guanine nucleotide binding and hydrolysis in other G alpha subunits had no apparent effect on the rate of tip formation when a single copy of the mutant gene was used to replace the wild-type gene. Overexpression of these mutant G alpha5 genes by increased gene dosage resulted in cell death, suggesting that high levels of the altered subunits have detrimental effects during vegetative growth.

Amino Acid Sequence↗