The contribution of dynamics to macaque body and face patch responses.

Previous functional imaging studies demonstrated body-selective patches in the primate visual temporal cortex, comparing activations to static bodies and static images of other categories. However, the use of static instead of dynamic displays of moving bodies may have underestimated the extent of the body patch network. Indeed, body dynamics provide information about action and emotion and may be processed in patches not activated by static images. Thus, to map with fMRI the full extent of the macaque body patch system in the visual temporal cortex, we employed dynamic displays of natural-acting monkey bodies, dynamic monkey faces, objects, and scrambled versions of these videos, all presented during fixation. We found nine body patches in the visual temporal cortex, starting posteriorly in the superior temporal sulcus (STS) and ending anteriorly in the temporal pole. Unlike for static images, body patches were present consistently in both the lower and upper banks of the STS. Overall, body patches showed a higher activation by dynamic displays than by matched static images, which, for identical stimulus displays, was less the case for the neighboring face patches. These data provide the groundwork for future single-unit recording studies to reveal the spatiotemporal features the neurons of these body patches encode. These fMRI findings suggest that dynamics have a stronger contribution to population responses in body than face patches.

Aging alters visual processing of objects and shapes in inferotemporal cortex in monkeys.

Visual perception declines with age. Perceptual deficits may originate not only in the optical system serving vision but also in the neural machinery processing visual information. Since homologies between monkey and human vision permit extrapolation from monkeys to humans, data from young, middle aged and old monkeys were analyzed to show age-related changes in the neuronal activity in the inferotemporal cortex, which is critical for object and shape vision. We found an increased neuronal response latency, and a decrease in the stimulus selectivity in the older animals and suggest that these changes may underlie the perceptual uncertainties found frequently in the elderly.

Fusion and fission in the visual pathways.

Inconsistent information from different modalities can be delusive for perception. This phenomenon can be observed with simultaneously presented inconsistent numbers of brief flashes and short tones. The conflict of bimodal information is reflected in double flash or fission, and flash fusion illusions, respectively. The temporal resolution of the vision system plays a fundamental role in the development of these illusions. As the parallel, dorsal and ventral pathways have different temporal resolution we presume that these pathways play different roles in the illusions. We used pathway-optimized stimuli to induce the illusions on separately driven visual streams. Our results show that both pathways support the double flash illusion, while the presence of the fusion illusion depends on the activated pathway. The dorsal pathway, which has better temporal resolution, does not support fusion, while the ventral pathway which has worse temporal resolution shows fusion strongly.

Rare provisional entity: primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma in a young woman.

Primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma is a rare and provisional entity, characterised by cutaneous involvement and aggressive clinical behaviour. The case is here presented of a young woman with concurrent cutaneous and systemic involvement. Despite multi-agent chemotherapy, only partial remission could be achieved, and the patient died from therapy-resistant respiratory and circulatory failure. This case report is intended to add to the data collected on this rare entity, with only about 20 cases as yet described.

Richter's and prolymphocytic transformation of chronic lymphocytic leukemia are associated with high mRNA expression of activation-induced cytidine deaminase and aberrant somatic hypermutation.

Chronic lymphocytic leukemia (CLL) is an indolent B-cell non-Hodgkin's lymphoma that may transform into higher-grade lymphoma. The transformation involves an increased number of prolymphocytic cells, termed prolymphocytic transformation (PLT) or the development of diffuse large B-cell lymphoma (DLBL), also referred to as Richter's transformation (RT). To analyze whether activation-induced cytidine deaminase (AID), which is essential for somatic hypermutation (SHM) of normal B-cells, and malfunction of SHM termed aberrant somatic hypermutation (ASHM) are associated with higher-grade transformation of CLL, AID mRNA expression and the mutation pattern of c-MYC, PAX-5 and RhoH genes were analyzed in eight cases of CLL without transformation and in 21 cases that showed RT or PLT. Chronic lymphocytic leukemia cases, which showed no transformation or eventually transformed into higher-grade lymphoma, showed low levels of AID mRNA expression and low frequency of mutations of c-MYC, PAX-5 and RhoH genes. In both RT and PLT, high-levels of AID mRNA expression and high-frequency mutations of c-MYC, PAX-5 and RhoH genes were detected. These results indicate that AID expression and ASHM are associated with higher-grade transformation of CLL and provide further evidences that AID expression and ASHM may be activated during the clonal history of B-cell lymphomas.

Clonal selection in the bone marrow involvement of follicular lymphoma.

To characterize the pathways of bone marrow (BM) involvement of follicular lymphoma (FL), we performed morphological and immunophenotypical analysis of tumor cells from lymph nodes (LNs) and corresponding BMs in 21 patients with FL. In three cases, genealogical trees were constructed based on the immunoglobulin variable region heavy chain (IgV(H)) gene sequences of tumor clones from LNs and BMs. Results showed that FLs within the BMs display identical or lower cytological grades than in the LNs. In the majority of cases, different proportions of tumor cells expressed bcl-2, CD10 and Ki67 in LNs and BMs. Tumor cells in the BM showed ongoing somatic hypermutation of the IgV(H) genes; the distribution of these mutations was highly consistent with antigen selection. The topology of the genealogical trees revealed that different subclones populate the LN and BM and BM infiltration may occur at different points of the clonal evolution of FL. Early descendants of the original tumor clone and derivatives of diversified tumor clones may invade the BM. These results suggest that the BM involvement of FL is associated with intensive clonal selection of tumor cells, and the BM provides a microenvironment similar to the germinal centers of LNs, where tumor cells retain their biological nature.

Relationship between the mutational status of VH genes and pathogenesis of diffuse large B-cell lymphoma in Richter's syndrome.

Patients with chronic lymphocytic leukemia (CLL) may develop diffuse large B-cell lymphoma (DLBL), also known as Richter's syndrome. Mutational status of immunoglobulin (Ig) heavy-chain variable region (VH) genes have prognostic impact in CLL. Patients with mutated VH genes have a stable disease, whereas patients with unmutated VH gene have more aggressive disease. The mutational status of CLLs that transform to DLBL is unknown. To reveal whether Richter's syndrome occurs in CLLs with mutated or unmutated VH genes, we have performed mutational analysis on serial specimens from eight patients. CLL and DLBL tumorclones were identical in five cases and they were different in three cases. Six CLLs expressed unmutated and two cases expressed mutated VH genes. In five of the six unmutated CLLs, the DLBL clones evolved from CLL tumorclones and the VH genes expressed by DLBLs were also unmutated. In one unmutated and two mutated CLLs, the DLBLs expressed mutated VH genes, but in these three cases the DLBL tumorclones developed as independent secondary neoplasm. These results suggest that Richter's syndrome may develop in both mutated or unmutated CLLs, but clonal transformation of CLL to DLBL occur only in the unmutated subgroup of CLL.

Folate-binding triggers the activation of folylpolyglutamate synthetase.

Folic acid is an essential vitamin for normal cell growth, primarily through its central role in one-carbon metabolism. Folate analogs (antifolates) are targeted at the same reactions and are widely used as therapeutic drugs for cancer and bacterial infections. Effective retention of folates in cells and the efficacy of antifolate drugs both depend upon the addition of a polyglutamate tail to the folate or antifolate molecule by the enzyme folylpolyglutamate synthetase (FPGS). The reaction mechanism involves the ATP-dependent activation of the free carboxylate group on the folate molecule to give an acyl phosphate intermediate, followed by attack by the incoming L-glutamate substrate. FPGS shares a number of structural and mechanistic details with the bacterial cell wall ligases MurD, MurE and MurF, and these enzymes, along with FPGS, form a subfamily of the ADP-forming amide bond ligase family. High-resolution crystallographic analyses of binary and ternary complexes of Lactobacillus casei FPGS reveal that binding of the first substrate (ATP) is not sufficient to generate an active enzyme. However, binding of folate as the second substrate triggers a large conformational change that activates FPGS and allows the enzyme to adopt a form that is then able to bind the third substrate, L-glutamate, and effect the addition of a polyglutamate tail to the folate.

Structural and functional similarities in the ADP-forming amide bond ligase superfamily: implications for a substrate-induced conformational change in folylpolyglutamate synthetase.

Comparison of the three-dimensional structures of folylpolyglutamate synthetase (FPGS) and the bacterial cell wall ligase UDP-N-acetylmuramoyl-l-alanine:d-glutamate ligase (MurD) reveals that these two enzymes have a remarkable structural similarity despite a low level of sequence identity. Both enzymes have a modular, multi-domain structure and catalyse a similar ATP-dependent reaction involving the addition of a glutamate residue to a carboxylate-containing substrate, tetrahydrofolate in the case of FPGS, and UDP-N-acetylmuramoyl-l-alanine in the case of MurD. Site-directed mutations of selected residues in the active site of Lactobacillus casei FPGS (P74A, E143A, E143D, E143Q, K185A, D313A, H316A, G411A and S412A) showed that most of these changes resulted in an almost complete loss of activity. Several of these amino acid residues in FPGS are found in structurally equivalent positions to active-site residues in MurD. Some insights into the function of these residues in FPGS activity are proposed, based on the roles surmised from the structures of two MurD. UDP-N-acetylmuramoyl-l-alanine.ADP complexes and a MurD. UDP-N-acetylmuramoyl-l-alanine-d-glutamate complex. Furthermore, the comparison has led us to propose that conformational changes induced by substrate binding in the reaction mechanism of FPGS result in a movement of the domains towards each other to more closely resemble the orientation of the corresponding domains in MurD. This relative domain movement may be a key feature of this new family of ADP-forming amide bond ligases.

Simple in-line postcolumn oxidation and derivatization for the simultaneous analysis of ascorbic and dehydroascorbic acids in foods.

A new analytical procedure for the simultaneous determination of L-ascorbic acid (AA), isoascorbic acid (IAA), L-dehydroascorbic acid (DHAA), and isodehydroascorbic acid (IDHAA) in food by high-performance liquid chromatography (HPLC) is developed. After separation on an HPLC column, an in-line oxidation of AA and IAA to DHAA and IDHAA, respectively, is performed on a short column of activated charcoal. The dehydroascorbic acids are derivatized with a 1,2-phenylenediamine solution in a heated capillary Tefzel reactor into fluorescent quinoxaline compounds and monitored fluorometrically. The chromatographic method provides good separation of LAA, LDHAA, and their diastereoisomers in a relatively short time (-10 min). After optimization of postcolumn derivatization conditions, calibration runs and recovery tests are performed. The fluorescent response in terms of peak area is highly proportional to the concentration of all derivatives examined over a range of 0.1 to 100 microg/mL solution for LAA, LDHAA, IAA, and IDHAA. Recoveries were in the range of 97 to 103%. The detection limit is 0.1 mg of each ascorbic acid derivative per 100 g food. A wide variety of foods (fruits, fruit juices, vegetables, vegetable products, milk, liver, and sausage) are analyzed by the developed procedure. The Vitamin C (LAA and LDHA) contents determined according to the present analytical method are in the same order of magnitude as the result of precolumn derivatization and the fluorometric methods. The described method is a highly specific procedure for determining Vitamin C in food. It is simple to handle, only slightly susceptible to disturbance, perfectly suitable for serial determinations, and yields reproducible results.

Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in Saccharomyces cerevisiae.

Similar to other eukaryotes, yeasts have parallel pathways of one-carbon metabolism in the cytoplasm and mitochondria and have folylpolyglutamate synthetase activity in both compartments. The gene encoding folylpolyglutamate synthetase is MET7 (also referred to as MET23) on chromosome XV and appears to encode both the cytoplasmic and mitochondrial forms of the enzyme. In order to determine the metabolic roles of both forms of folylpolyglutamate synthetase, we disrupted the met7 gene and determined that the strain is a methionine auxotroph and an adenine and thymidine auxotroph when grown in the presence of sulfanilamide. The met7 mutant becomes petite under normal growth conditions but can be maintained with a grande phenotype if the strain is tup and all media are supplemented with dTMP. A met7 gly1 strain is auxotrophic for glycine when grown on glucose but prototrophic when grown on glycerol. A met7 ser1 strain cannot use glycine to suppress the serine auxotrophy of the ser1 phenotype. A met7 shm2 strain is nonviable. In order to disrupt just the mitochondrial folylpolyglutamate synthetase activity, we constructed mutants with an inactivated chromosomal MET7 gene complemented by genes that express only cytoplasmic folylpolyglutamate synthetase, including the Lactobacillus casei folC gene and the yeast MET7 gene with its mitochondrial leader sequence deleted (MET7Deltam). All the genes providing cytoplasmic folylpolyglutamate synthetase complemented the methionine auxotrophy as well as the synthetic lethality of the shm2 strain and the synthetic glycine auxotrophy of the gly1 strain. The strains lacking the mitochondrial folylpolyglutamate synthetase had longer doubling times than the isogenic wild-type strains but retained the function of the mitochondrial folate-dependent enzymes to produce formate, serine, and glycine. Mutants complemented by the bacterial folC gene or by the MET7Deltam gene on a 2mu plasmid remained grande without the tup mutation and supplementation and dTMP. Mutants complemented by the MET7Deltam gene integrated in single copy became petites under those conditions, indicating a deficiency in dTMP production but this is likely due to lower expression of cytoplasmic folylpolyglutamate synthetase by the MET7Deltam gene.

Cerebrovascular changes in the basal ganglia with HIV dementia.

BACKGROUND: HIV dementia is a form of subcortical dementia. Clinical, radiologic, pathologic, and biochemical studies suggest a major contribution of basal ganglia dysfunction to the pathogenesis of this disorder. Many investigators have proposed a contribution of a disrupted blood-brain barrier (BBB) to the pathogenesis of HIV dementia. OBJECTIVE: To identify microvascular abnormalities in vivo in basal ganglia or white matter of persons with HIV dementia. METHODS: Time course of MRI postcontrast enhancement was determined in basal ganglia and white matter of HIV-infected persons without dementia (Memorial Sloan Kettering [MSK] score of 0; n = 4); HIV-infected persons with mild dementia (MSK score of 0.5; n = 2); and HIV-infected persons with moderate-to-severe dementia (MSK > or = 1.0; n = 6). RESULTS: Increased basal ganglia enhancement was observed in individuals with moderate-to-severe dementia relative to nondemented individuals, both immediately and 30 minutes after contrast administration. Decline of basal ganglia enhancement was slower in the moderately to severely demented patients and, when normalized to intravascular enhancement of sagittal sinus, suggested leakage of contrast agent, consistent with increased permeability of BBB. A significant correlation between the postcontrast fractional enhancement at 30 minutes (FE30) and the MSK score was noted. White matter showed no significant differences in postcontrast enhancement among the three groups. CONCLUSION: Increased early enhancement in basal ganglia of the HIV dementia group is consistent with increased regional cerebral blood volume (rCBV). Increased late enhancement is strongly suggestive of BBB disruption. Similar abnormalities were absent in the white matter adjacent to the caudate nucleus.

Molecular analysis of the folC gene of Pseudomonas aeruginosa.

We have cloned the Pseudomonas aeruginosa folC gene coding for folylpolyglutamate synthetase-dihydrofolate synthetase, which was located between the trpF and purF loci, and determined the nucleotide sequence of the folC gene and its flanking region. The deduced amino acid sequence of P. aeruginosa FolC was highly homologous to that of Escherichia coli FolC. The cloned gene complemented E. coli folC mutations and was found to encode both folylpolyglutamate synthetase and dihydrofolate synthetase activities. The gene organization around the folC gene in P. aeruginosa was completely conserved with that in E. coli; the accD gene was located upstream of the folC gene, and dedD, cvpA and purF genes followed the folC gene in this order. The gene arrangement and the result of the promoter activity assay suggested that the P. aeruginosa accD and folC genes were co-transcribed.

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase.

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-A resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Omega loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.

Identification of Saccharomyces cerevisiae GLY1 as a threonine aldolase: a key enzyme in glycine biosynthesis.

Determination of enzyme-specific activities revealed that GLY1 encodes a threonine aldolase (TA) in Saccharomyces cerevisiae. A knock-out mutant auxotrophic for glycine lacked detectable activity. After transformation with YEp24GLY1 glycine prototrophy was restored and TA-specific activity was 16-fold higher than in the wild type. Growth experiments using glucose as the sole carbon source showed that GLY1 is more important for glycine biosynthesis than SHM1 and SHM2 encoding alternative serine hydroxymethyltransferases. On ethanol as carbon source simultaneous disruption of GLY1, SHM1 and SHM2 did not lead to glycine auxotrophy because glycine biosynthesis proceeds via alanine glyoxylate aminotransferase.

Cloning, and molecular characterization of the GCV1 gene encoding the glycine cleavage T-protein from Saccharomyces cerevisiae.

We have isolated the gene encoding the glycine cleavage T-protein (GCV1) of the yeast Saccharomyces cerevisiae and shown through gene disruption and enzyme assays that inactivation of GCV1 destroys glycine cleavage function. A DNA fragment encoding the GCV1 gene was cloned by PCR amplification using degenerate oligodeoxyribonucleotides, and the cloned fragment was used as a probe to isolate the complete gene from a yeast genomic library. Growth with glycine stimulated expression of the GCV1 gene as determined by Northern analysis and increased the beta-galactosidase activity of a GCV1-lacZ fusion 30-fold. The URA3 gene was inserted into the coding sequence of GCV1 and the resulting construct was used to disrupt the chromosomal GCV1 gene in a diploid strain of yeast. gcv1::URA3 haploid derivatives grew normally or only slightly more slowly than the isogenic wild-type haploids. All gcv1 strains studied were unable to grow on glycine as a sole nitrogen source and lacked glycine cleavage enzyme activity. Growth of shm1 shm2 mutants was stimulated by glycine, whereas glycine could not supplement the growth of the isogenic gcv1 strain.

In vivo analysis of folate coenzymes and their compartmentation in Saccharomyces cerevisiae.

In eukaryotes, enzymes responsible for the interconversion of one-carbon units exist in parallel in both mitochondria and the cytoplasm. Strains of Saccharomyces cerevisiae were constructed that possess combinations of gene disruptions at the SHM1 [mitochondrial serine hydroxymethyltransferase (SHMTm)], SHM2 [cytoplasmic SHMT (SHMTc)], MIS1 [mitochondrial C1-tetrahydrofolate synthase (C1-THFSm)], ADE3 [cytoplasmic C1-THF synthase (C1-THFSc)], GCV1 [glycine cleavage system (GCV) protein T], and the GLY1 (involved in glycine synthesis) loci. Analysis of the in vivo growth characteristics and phenotypes was used to determine the contribution to cytoplasmic nucleic acid and amino acid anabolism by the mitochondrial enzymes involved in the interconversion of folate coenzymes. The data indicate that mitochondria transport formate to the cytoplasmic compartment and mitochondrial synthesis of formate appears to rely primarily on SHMTm rather than the glycine cleavage system. The glycine cleavage system and SHMTm cooperate to specifically synthesize serine. With the inactivation of SHM1, however, the glycine cleavage system can make an observable contribution to the level of mitochondrial formate. Inactivation of SHM1, SHM2 and ADE3 is required to render yeast auxotrophic for TMP and methionine, suggesting that TMP synthesized in mitochondria may be available to the cytoplasmic compartment.

Mutagenesis of the Lactobacillus casei folylpolyglutamate synthetase gene at essential residues resembling an ATP binding site.

Site-directed mutagenesis studies were performed on a region of the Lactobacillus casei folylpoly-gamma-glutamate synthetase (FPGS) protein (residues 49 to 52), which is highly conserved when compared to the Escherichia coli and human FPGS proteins. The amino acid sequence of this region, GKGS/T, is similar to the consensus sequence for the A region of a nucleotide binding site, a motif which encodes a phosphate-binding loop. Mutation G49A or K50R, with substitution to amino acids of similar size and charge, resulted in decreases in Vmax/Km of 40- to over 100-fold, depending on the variable substrate. Alteration of G51 to S or T resulted in a large increase in the Km for glutamate. The Km for ATP was not affected more than 4-fold by any of the mutations. Our studies indicate that the conserved region is essential for FPGS function, since many of the mutations resulting in functionally conservative substitutions produced inactive enzymes. However, the mutations affected binding of all three substrates, so there is no direct evidence for involvement of the region in ATP binding.

Cloning and molecular characterization of three genes, including two genes encoding serine hydroxymethyltransferases, whose inactivation is required to render yeast auxotrophic for glycine.

The genes encoding both the cytosolic and mitochondrial serine hydroxymethyltransferases (SHM2 and SHM1, respectively) and a third unidentified gene of the yeast Saccharomyces cerevisiae have been isolated and their nucleotide sequences determined. Analysis of the predicted amino acid sequence of the amino-terminal regions, sequence comparison with other genes encoding SHMT enzymes, and subcellular fractionation studies all suggested that the SHM1 gene encodes the mitochondrial SHMT, while the SHM2 gene encodes the cytosolic enzyme. The SHM2 gene but not the SHM1 gene has putative GCN4 sites upstream of the putative TATA box, suggesting regulation of its transcription by the general amino acid control system. Yeast mutants with disruptions at each SHM gene and in both genes were constructed and all mutants had the same growth requirements as the parental strains. Mutagenesis of the double-disrupted, shm1 shm2 yeast yielded strains of a single complementation group that are auxotrophic for glycine. Complementation of the glycine auxotrophy using a yeast genomic library retrieved the SHM1 and SHM2 genes and a third gene designated GLY1. Gene disruption studies demonstrated that inactivation of SHM1, SHM2, and GLY1 is required to yield yeast that are completely auxotrophic for glycine.

Cloning and sequence determination of the valS gene, encoding valyl-tRNA synthetase in Lactobacillus casei.

The DNA sequence of the valS gene from Lactobacillus casei and the predicted amino acid sequence of its valyl-tRNA synthetase product have been determined. An open reading frame coding for a protein of 901 amino acids was found. A clone containing the intact L. casei valS gene functionally complemented the temperature-sensitive growth of the valS mutant strain 236c of Escherichia coli. The valS gene and the downstream folylpolyglutamate synthetase gene are transcribed in the same direction but are separated by a putative transcription terminator.