bluuebunny/crossref_metadata_2025 huggingface dataset:
Polycystin, the polycystic kidney disease 1 protein, is expressed by epithelial cells in fetal, adult, and polycystic kidney.
Authors: Ward C J , Turley H , Ong A C , Comley M , Biddolph S , Chetty R , Ratcliffe P J , Gattner K , Harris P C
Published: July 2002

Polycystic kidney disease 1 (PKD1) is the major locus of the common genetic disorder autosomal dominant polycystic kidney disease. We have studied PKD1 mRNA, with an RNase protection assay, and found widespread expression in adult tissue, with high levels in brain and moderate signal in kidney. Expression of the PKD1 protein, polycystin, was assessed in kidney using monoclonal antibodies to a recombinant protein containing the C terminus of the molecule. In fetal and adult kidney, staining is restricted to epithelial cells. Expression in the developing nephron is most prominent in mature tubules, with lesser staining in Bowman's capsule and the proximal ureteric bud. In the nephrogenic zone, detectable signal was observed in comma- and S-shaped bodies as well as the distal branches of the ureteric bud. By contrast, uninduced mesenchyme and glomerular tufts showed no staining. In later fetal (>20 weeks) and adult kidney, strong staining persists in cortical tubules with moderate staining detected in the loops of Henle and collecting ducts. These results suggest that polycystin's major role is in the maintenance of renal epithelial differentiation and organization from early fetal life. Interestingly, polycystin expression, monitored at the mRNA level and by immunohistochemistry, appears higher in cystic epithelia, indicating that the disease does not result from complete loss of the protein.
Determination of the transiently lowered pKa of the retinal Schiff base during the photocycle of bacteriorhodopsin.
Authors: Brown L S , Lanyi J K
Published: July 2002

Reprotonation of the transiently deprotonated retinal Schiff base in the bacteriorhodopsin photocycle is greatly slowed when the proton donor Asp-96 is removed with site-specific mutagenesis, but its rate is restored upon adding azide or other weak acids such as formate and cyanate. As expected, between pH 3 and 7 the rate of Schiff base protonation in the photocycle of the D96N mutant correlates with the concentrations of the acid forms of these agents. Dissection of the rates in the biexponential reprotonation kinetics of the Schiff base between pH 7 and 9 yielded calculated rate constants for the protonation equilibrium. Their dependencies on pH and azide or cyanate concentrations are consistent with both earlier suggested mechanisms: (i) azide and other weak acids may function as proton carriers in the protonation equilibrium of the Schiff base, or (ii) the binding of their anionic forms may catalyze proton conduction to and from the Schiff base. The measured rate constants allow the calculation of the pKa of the Schiff base during its reprotonation in the photocycle of D96N. It is 8.2-8.3, a value much below the pKa determined earlier in unphotolyzed bacteriorhodopsin.
Endothelin 3 promotes neural crest cell proliferation and mediates a vast increase in melanocyte number in culture.
Authors: Lahav R , Ziller C , Dupin E , Le Douarin N M
Published: July 2002

Mutations in the endothelin 3 (EDN3) gene severely affect the development of neural crest-derived melanocytes. In this paper, we report the action of EDN3 on neural crest cells in vitro. The presence of EDN3 leads to a large increase in the number of cells, the majority of which eventually differentiate into melanocytes that aggregate to form a reproducible pigmentation pattern. Quantitative analysis of the effect of different culture conditions revealed that EDN3 initially promotes neural crest cell proliferation. This phase of expansion, which can be prolonged for a few weeks if the cells are replaced regularly, is followed by both a decrease in cell proliferation and the onset of melanocytic differentiation. Therefore, EDN3 is a potent mitogen for early neural crest cell precursors that can give rise to melanocytes.
Investigation of the mechanisms of DNA binding of the human G/T glycosylase using designed inhibitors
Authors: Schärer Orlando D. , Kawate Tomohiko , Gallinari Paola , Jiricny Josef , Verdine Gregory L.
Published: July 2002

Deamination of 5-methylcytosine residues in DNA gives rise to the G/T mismatched base pair. In humans this lesion is repaired by a mismatch-specific thymine DNA glycosylase (TDG or G/T glycosylase), which catalyzes specific excision of the thymine base through N-glycosidic bond hydrolysis. Unlike other DNA glycosylases, TDG recognizes an aberrant pairing of two normal bases rather than a damaged base
per se
. An important structural issue is thus to understand how the enzyme specifically targets the T (or U) residue of the mismatched base pair. Our approach toward the study of substrate recognition and processing by catalytic DNA binding proteins has been to modify the substrate so as to preserve recognition of the base but to prevent its excision. Here we report that replacement of 2′-hydrogen atoms with fluorine in the substrate 2′-deoxyguridine (dU) residue abrogates glycosidic bond cleavage, thereby leading to the formation of a tight, specific glycosylase–DNA complex. Biochemical characterization of these complexes reveals that the enzyme protects an ≈20-bp stretch of the substrate from DNase I cleavage, and directly contacts a G residue on the 3′ side of the mismatched U derivative. These studies provide a mechanistic rationale for the preferential repair of deaminated CpG sites and pave the way for future high-resolution studies of TDG bound to DNA.
I
2
B
is a small cytosolic protein that participates in vacuole fusion
Authors: Slusarewicz Paul , Xu Zuoyu , Seefeld Kimberly , Haas Albert , Wickner William T.
Published: July 2002

Saccharomyces cerevisiae
vacuole inheritance requires two
l
ow
m
olecular weight
a
ctivities, LMA1 and LMA2. LMA1 is a heterodimer of thioredoxin and protease B inhibitor 2 (I
2
B
). Here we show that the second low molecular weight activity (LMA2) is monomeric I
2
B
. Though LMA2/I
2
B
was initially identified as a protease B inhibitor, this protease inhibitor activity is not related to its ability to promote vacuole fusion: (
i
) Low
M
r
protease B inhibitors cannot substitute for LMA1 or LMA 2, (
ii
) LMA1 and LMA2 promote the fusion of vacuoles from a strain that has no protease B, (
iii
) low concentrations of LMA2 that fully inhibit protease B do not promote vacuole fusion, and (
iv
) LMA1, in which I
2
B
is complexed with thioredoxin, is far more active than LMA2/I
2
B
in promoting vacuole fusion and far less active in inhibiting protease B. These studies establish a new function for I
2
B
.
Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast
Authors: Donovan Shane , Harwood Janet , Drury Lucy S. , Diffley John F. X.
Published: July 2002

The Cdc6 protein is essential for the assembly of pre-replicative complexes (pre-RCs) at origins of DNA replication in the budding yeast
Saccharomyces cerevisiae
. This reaction is blocked
in vivo
by the cyclin-dependent kinase Cdc28p, together with its regulatory subunits, the B type cyclins that are present throughout S, G
2
, and M phases. Because the destruction of B type cyclins and the consequent inactivation of the kinase are essential for exit from mitosis, pre-RC formation can only occur after passage through mitosis. Therefore, pre-RC formation has been proposed to be essential for coupling S phase and mitosis and for limiting DNA replication to once per cell cycle. The Mcm2–7 family of proteins has been implicated in limiting replication to once per cell cycle from experiments with
Xenopus
egg extracts. Here we show that the Mcm proteins of budding yeast are abundant and are quantitatively found in a chromatin-enriched fraction specifically during the G
1
phase of the cell cycle. This chromatin binding depends on the
de novo
synthesis of Cdc6p, providing evidence that a conserved biochemical pathway plays a critical role in coordinating DNA replication with mitosis in both yeast and higher eukaryotes. Cdc6p and the origin recognition complex can be selectively removed from this chromatin-enriched fraction without removing the Mcm proteins. From these results, we propose that Cdc6p (and the origin recognition complex) nucleates the binding of Mcm proteins to chromatin, but once bound, the Mcm proteins appear to interact tightly with some other component of chromatin.
Modular organization of the catalytic center of RNA polymerase
Authors: Mustaev Arkady , Kozlov Maxim , Markovtsov Vadim , Zaychikov Evgeny , Denissova Ludmila , Goldfarb Alex
Published: July 2002

The Fe
2+
ion that specifically replaces Mg
2+
in the active center of RNA polymerase generates reactive hydroxyl radicals that cause highly localized cleavage of polypeptide chains. Mapping of the cleavage sites revealed the overall architecture of the active center. Nine distinct sites, five in the β subunit and four in the β′ subunit of
Escherichia coli
RNA polymerase, all at or near highly conserved sequence motifs, are brought together in the enzyme’s ternary structure within the distance of ≈1 nm from the active center Me
2+
. These sites are located in at least six different domains of the subunits, reflecting modular organization of the active center.
Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cell invasion
Authors: Nakahara Hirokazu , Howard Linda , Thompson Erik W. , Sato Hiroshi , Seiki Motoharu , Yeh Yunyun , Chen Wen-Tien
Published: July 2002

The invasion of human malignant melanoma cells into the extracellular matrix (ECM) involves the accumulation of proteases at sites of ECM degradation where activation of matrix metalloproteases (MMP) occurs. Here, we show that when membrane type 1 MMP (MT-MMP) was overexpressed in RPMI7951 human melanoma cells, the cells made contact with the ECM, activated soluble and ECM-bound MMP-2, and degraded and invaded the ECM. Further experiments demonstrated the importance of localization of the MT-MMP to invadopodia. Overexpression of MT-MMP without invadopodial localization caused activation of soluble MMP-2, but did not facilitate ECM degradation or cell invasiveness. Up-regulation of endogenous MT-MMP with concanavalin A caused activation of MMP-2. However, concanavalin A treatment prevented invadopodial localization of MT-MMP and ECM degradation. Neither a truncated MT-MMP mutant lacking transmembrane (TM) and cytoplasmic domains (ΔTM
MT-MMP
), nor a chimeric MT-MMP containing the interleukin 2 receptor α chain (IL-2R) TM and cytoplasmic domains (ΔTM
MT-MMP
/TM
IL-2R
) were localized to invadopodia or exhibited ECM degradation. Furthermore, a chimera of the TM/cytoplasmic domain of MT-MMP (TM
MT-MMP
) with tissue inhibitor of MMP 1 (TIMP-1/TM
MT-MMP
) directed the TIMP-1 molecule to invadopodia. Thus, the MT-MMP TM/cytoplasmic domain mediates the spatial organization of MT-MMP into invadopodia and subsequent degradation of the ECM.
Roles of phospholipase C β2 in chemoattractant-elicited responses
Authors: Jiang Huiping , Kuang Yanan , Wu Yanping , Xie Wei , Simon Melvin I. , Wu Dianqing
Published: July 2002

The physiological roles of phospholipase C (PLC) β2 in hematopoiesis, leukocyte function, and host defense against infection were investigated using a mouse line that lacks PLC β2. PLC β2 deficiency did not affect hematopoiesis, but it blocked chemoattractant-induced Ca2+release, superoxide production, and MAC-1 up-regulation in neutrophils. In view of these effects, it was surprising that the absence of PLC β2 enhanced chemotaxis of different leukocyte populations and sensitized thein vivoresponse of the PLC β2-deficient mice to bacteria, viruses, and immune complexes. These data raise questions about the roles that PLC β2 may play in signal transduction induced by chemoattractants in leukocytes.
Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization
Authors: Weiner George J. , Liu Hsin-Ming , Wooldridge James E. , Dahle Christopher E. , Krieg Arthur M.
Published: July 2002

Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in tumor antigen immunization. The idiotype served as the tumor antigen. Select CpG ODN were as effective as complete Freund’s adjuvant at inducing an antigen-specific antibody response but were associated with less toxicity. These CpG ODN induced a higher titer of antigen-specific IgG2a than did complete Freund’s adjuvant, suggesting an enhanced TH1 response. Mice immunized with CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to that seen in mice immunized with complete Freund’s adjuvant. We conclude that CpG ODN are effective as immune adjuvants and are attractive as part of a tumor immunization strategy.
A transformation-associated complex involving tyrosine kinase signal adapter proteins and caldesmon links v-ErbB signaling to actin stress fiber disassembly
Authors: McManus Michael J. , Lingle Wilma L. , Salisbury Jeffrey L. , Maihle Nita J.
Published: July 2002

The avian erythroblastosis viral oncogene (v-
erb
B) encodes a receptor tyrosine kinase that possesses sarcomagenic and leukemogenic potential. We have expressed transforming and nontransforming mutants of v-
erb
B in fibroblasts to detect transformation-associated signal transduction events. Coimmunoprecipitation and affinity chromatography have been used to identify a transformation-associated, tyrosine phosphorylated, multiprotein complex. This complex consists of Src homologous collagen protein (Shc), growth factor receptor binding protein 2 (Grb2), son of sevenless (Sos), and a novel tyrosine phosphorylated form of the cytoskeletal regulatory protein caldesmon. Immunofluorescence localization studies further reveal that, in contrast to the distribution of caldesmon along actin stress fibers in normal fibroblasts, caldesmon colocalizes with Shc in plasma membrane blebs in transformed fibroblasts. This colocalization of caldesmon and Shc correlates with actin stress fiber disassembly and v-
erb
B-mediated transformation. The tyrosine phosphorylation of caldesmon, and its association with the Shc–Grb2–Sos signaling complex directly links tyrosine kinase oncogenic signaling events with cytoskeletal regulatory processes, and may define one mechanism regulating actin stress fiber disassembly in transformed cells.
Convergent evolution of apolipoprotein(a) in primates and hedgehog
Authors: Lawn Richard M. , Schwartz Karen , Patthy Laszlo
Published: July 2002

Apolipoprotein(a) [apo(a)] is the distinguishing protein component of lipoprotein(a), a major inherited risk factor for atherosclerosis. Human apo(a) is homologous to plasminogen. It contains from 15 to 50 repeated domains closely related to plasminogen kringle four, plus single kringle five-like and inactive protease-like domains. This expressed gene is confined to a subset of primates. Although most mammals lack apo(a), hedgehogs produce an apo(a)-like protein composed of highly repeated copies of a plasminogen kringle three-like domain, with complete absence of protease domain sequences. Both human and hedgehog apo(a)-like proteins form covalently linked lipoprotein particles that can bind to fibrin and other substrates shared with plasminogen. DNA sequence comparisons and phylogenetic analysis indicate that the human type of apo(a) evolved from a duplicated plasminogen gene during recent primate evolution. In contrast, the kringle three-based type of apo(a) evolved from an independent duplication of the plasminogen gene approximately 80 million years ago. In a type of convergent evolution, the plasminogen gene has been independently remodeled twice during mammalian evolution to produce similar forms of apo(a) in two widely divergent groups of species.
The presenilin 2 mutation (N141I) linked to familial Alzheimer disease (Volga German families) increases the secretion of amyloid β protein ending at the 42nd (or 43rd) residue
Authors: Tomita Taisuke , Maruyama Kei , Saido Takaomi C. , Kume Hideaki , Shinozaki Kohki , Tokuhiro Shinya , Capell Anja , Walter Jochen , Grünberg Jürgen , Haass Christian , Iwatsubo Takeshi , Obata Kunihiko
Published: July 2002

To gain insights into the significance of presenilins (PS) in the pathogenetic mechanisms of early-onset familial Alzheimer disease (FAD), we expressed cDNAs for wild-type PS2 and PS2 with the Volga German (N141I) mutation in cultured cells and then examined the metabolism of the transfected proteins and their effect on the C-terminal properties of secreted amyloid β protein (Aβ). PS2 was identified as a 50- to 55-kDa protein, which was cleaved to produce N-terminal fragments of 35–40 kDa and C-terminal fragments of 19–23 kDa. The Volga German (N141I) mutation did not cause any significant change in the metabolism of PS2. COS-1 cells doubly transfected with cDNAs for N141I mutant PS2 and human β-amyloid precursor protein (βAPP) or a C-terminal fragment thereof, as well as mouse Neuro2a neuroblastoma cells stably transfected with N141I mutant PS2 alone, secreted 1.5- to 10-fold more Aβ ending at residues 42 (or 43) [Aβ42(43)] compared with those expressing the wild-type PS2. These results strongly suggest that the PS2 mutation (N141I) linked to FAD alters the metabolism of Aβ/βAPP to foster the production of the form of Aβ that most readily deposits in amyloid plaques. Thus, mutant PS2 may lead to AD by altering the metabolism of Aβ/βAPP.
Transcription factor NF-κB is activated in primary neurons by amyloid β peptides and in neurons surrounding early plaques from patients with Alzheimer disease
Authors: Kaltschmidt Barbara , Uherek Martin , Volk Benedikt , Baeuerle Patrick A. , Kaltschmidt Christian
Published: July 2002

Amyloid β peptide (Aβ)-containing plaques are a hallmark of Alzheimer disease. Here, we show that the neurotoxic Aβ, a major plaque component, is a potent activator of the transcription factor NF-κB in primary neurons. This activation required reactive oxygen intermediates as messengers because an antioxidant prevented Aβ-induced NF-κB activation. Maximal activation of NF-κB was found with 0.1 μM Aβ-(1–40) and 0.1 μM Aβ-(25–35) fragments, making a role for NF-κB in neuroprotection feasible. Using an activity-specific mAb for the p65 NF-κB subunit, activation of NF-κB also was observed in neurons and astroglia of brain sections from Alzheimer disease patients. Activated NF-κB was restricted to cells in the close vicinity of early plaques. Our data suggest that the aberrant gene expression in diseased nervous tissue is at least in part due to Aβ-induced activation of NF-κB, a potent immediate–early transcriptional regulator of numerous proinflammatory genes.
Human parvovirus B19 as a causative agent for rheumatoid arthritis
Authors: Takahashi Yuichi , Murai Chihiro , Shibata Shinobu , Munakata Yasuhiko , Ishii Tomonori , Ishii Keiko , Saitoh Takako , Sawai Takashi , Sugamura Kazuo , Sasaki Takeshi
Published: July 2002

Human parvovirus B19 (B19) DNA was detected in the synovial tissues in 30 of 39 patients with rheumatoid arthritis (RA), and infrequently in those with osteoarthritis and traumatic joints. On the other hand, the expression of the B19 antigen VP-1 was specific (27/27) in RA synovium with active synovial lesions, but not in osteoarthritis and controls. The target cells of B19 were macrophages, follicular dendritic cells, T cells, and B cells, but not synovial lining cells in the synovium. B19-negative bone marrow cells, tonsil cells, and macrophage cell line U-937 cells became positive for the expression of VP-1, and more productive for interleukin 6 and tumor necrosis factor α when cocultured with RA synovial cells. The expression of VP-1 and the production of interleukin 6 and tumor necrosis factor α was significantly inhibited by the addition of neutralizing antibody for B19, suggesting that B19 detected in RA synovial cells is infective. B19 is involved in the initiation and perpetuation of RA synovitis, leading to joint lesions.
Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate
Authors: Gottlin Elizabeth B. , Rudolph Amy E. , Zhao Yi , Matthews Harry R. , Dixon Jack E.
Published: July 2002

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)
4
D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the k
cat
for hydrolysis by a factor of 10
5
, suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a
32
P-labeled protein could be trapped when the enzyme was incubated with
32
P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.
Catechol-
O
-methyltransferase-deficient mice exhibit sexually dimorphic changes in catecholamine levels and behavior
Authors: Gogos Joseph A. , Morgan Maria , Luine Victoria , Santha Miklos , Ogawa Sonoko , Pfaff Donald , Karayiorgou Maria
Published: July 2002

Catechol-
O
-methyltransferase (COMT) is one of the major mammalian enzymes involved in the metabolic degradation of catecholamines and is considered a candidate for several psychiatric disorders and symptoms, including the psychopathology associated with the 22q11 microdeletion syndrome. By means of homologous recombination in embryonic stem cells, a strain of mice in which the gene encoding the COMT enzyme has been disrupted was produced. The basal concentrations of brain catecholamines were measured in the striatum, frontal cortex, and hypothalamus of adult male and female mutants. Locomotor activity, anxiety-like behaviors, sensorimotor gating, and aggressive behavior also were analyzed. Mutant mice demonstrated sexually dimorphic and region-specific changes of dopamine levels, notably in the frontal cortex. In addition, homozygous COMT-deficient female (but not male) mice displayed impairment in emotional reactivity in the dark/light exploratory model of anxiety. Furthermore, heterozygous COMT-deficient male mice exhibited increased aggressive behavior. Our results provide conclusive evidence for an important sex- and region-specific contribution of COMT in the maintenance of steady-state levels of catecholamines in the brain and suggest a role for COMT in some aspects of emotional and social behavior in mice.
Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit Cα
Authors: Götz Jürgen , Probst Alphonse , Ehler Elisabeth , Hemmings Brian , Kues Wilfried
Published: July 2002

Protein phosphatase 2A (PP2A) is a multimeric enzyme, containing a catalytic subunit complexed with two regulatory subunits. The catalytic subunit PP2A C is encoded by two distinct and unlinked genes, termed Cα and Cβ. The specific function of these two catalytic subunits is unknown. To address the possible redundancy between PP2A and related phosphatases as well as between Cα and Cβ, the Cα subunit gene was deleted by homologous recombination. Homozygous null mutant mice are embryonically lethal, demonstrating that the Cα subunit gene is an essential gene. As PP2A exerts a range of cellular functions including cell cycle regulation and cell fate determination, we were surprised to find that these embryos develop normally until postimplantation, around embryonic day 5.5/6.0. While no Cα protein is expressed, we find comparable expression levels of PP2A C at a time when the embryo is degenerating. Despite a 97% amino acid identity, Cβ cannot completely compensate for the absence of Cα. Degenerated embryos can be recovered even at embryonic day 13.5, indicating that although embryonic tissue is still capable of proliferating, normal differentiation is significantly impaired. While the primary germ layers ectoderm and endoderm are formed, mesoderm is not formed in degenerating embryos.
Neuropeptides, by direct interaction with T cells, induce cytokine secretion and break the commitment to a distinct T helper phenotype
Authors: Levite Mia
Published: July 2002

Searching for nervous system candidates that could directly induce T cell cytokine secretion, I tested four neuropeptides (NPs): somatostatin, calcitonin gene-related peptide, neuropeptide Y, and substance P. Comparing neuropeptide-driven versus classical antigen-driven cytokine secretion from T helper cells Th0, Th1, and Th2 autoimmune-related T cell populations, I show that the tested NPs, in the absence of any additional factors, directly induce a marked secretion of cytokines [interleukin 2 (IL-2), interferon-γ, IL-4, and IL-10) from T cells. Furthermore, NPs drive distinct Th1 and Th2 populations to a “forbidden” cytokine secretion: secretion of Th2 cytokines from a Th1 T cell line andvice versa. Such a phenomenon cannot be induced by classical antigenic stimulation. My study suggests that the nervous system, through NPs interacting with their specific T cell-expressed receptors, can lead to the secretion of both typical and atypical cytokines, to the breakdown of the commitment to a distinct Th phenotype, and a potentially altered function and destiny of T cellsin vivo.
Directed evolution of a thermostable esterase
Authors: Giver Lori , Gershenson Anne , Freskgard Per-Ola , Arnold Frances H.
Published: July 2002

We have used
in vitro
evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized
Bacillus subtilis
p
-nitrobenzyl esterase significantly (>14°C increase in
T
m
) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.
Movement analysis in infancy may be useful for early diagnosis of autism
Authors: Teitelbaum Philip , Teitelbaum Osnat , Nye Jennifer , Fryman Joshua , Maurer Ralph G.
Published: July 2002

All of the 17 autistic children studied in the present paper
showed disturbances of movement that with our methods could be detected
clearly at the age of 4–6 months, and sometimes even at birth. We used
the Eshkol–Wachman Movement Analysis System in combination with
still-frame videodisc analysis to study videos obtained from parents of
children who had been diagnosed as autistic by conventional methods,
usually around 3 years old. The videos showed their behaviors when they
were infants, long before they had been diagnosed as autistic. The
movement disorders varied from child to child. Disturbances were
revealed in the shape of the mouth and in some or all of the milestones
of development, including, lying, righting, sitting, crawling, and
walking. Our findings support the view that movement disturbances play
an intrinsic part in the phenomenon of autism, that they are present at
birth, and that they can be used to diagnose the presence of autism in
the first few months of life. They indicate the need for the
development of methods of therapy to be applied from the first few
months of life in autism.
Human γ-aminobutyric acid type B receptors are differentially expressed and regulate inwardly rectifying K
+
channels
Authors: Kaupmann Klemens , Schuler Valerie , Mosbacher Johannes , Bischoff Serge , Bittiger Helmut , Heid Jakob , Froestl Wolfgang , Leonhard Sabine , Pfaff Torsten , Karschin Andreas , Bettler Bernhard
Published: July 2002

γ-Aminobutyric acid type B receptors (GABA
B
Rs) are involved in the fine tuning of inhibitory synaptic transmission. Presynaptic GABA
B
Rs inhibit neurotransmitter release by down-regulating high-voltage activated Ca
2+
channels, whereas postsynaptic GABA
B
Rs decrease neuronal excitability by activating a prominent inwardly rectifying K
+
(Kir) conductance that underlies the late inhibitory postsynaptic potentials. Here we report the cloning and functional characterization of two human GABA
B
Rs, hGABA
B
R1a (hR1a) and hGABA
B
R1b (hR1b). These receptors closely match the pharmacological properties and molecular weights of the most abundant native GABA
B
Rs. We show that in transfected mammalian cells hR1a and hR1b can modulate heteromeric Kir3.1/3.2 and Kir3.1/3.4 channels. Heterologous expression therefore supports the notion that Kir3 channels are the postsynaptic effectors of GABA
B
Rs. Our data further demonstrate that in principle either of the cloned receptors could mediate inhibitory postsynaptic potentials. We find that in the cerebellum hR1a and hR1b transcripts are largely confined to granule and Purkinje cells, respectively. This finding supports a selective association of hR1b, and not hR1a, with postsynaptic Kir3 channels. The mapping of the

GABA
B
R1

gene to human chromosome 6p21.3, in the vicinity of a susceptibility locus (
EJM1
) for idiopathic generalized epilepsies, identifies a candidate gene for inherited forms of epilepsy.
Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development
Authors: Rennebeck Gabriela , Kleymenova Elena V. , Anderson Rebecca , Yeung Raymond S. , Artzt Karen , Walker Cheryl L.
Published: July 2002

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2
Ek/Ek
) was lethal in mid-gestation (the equivalent of mouse E9.5–E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long–Evans strain Tsc2
Ek/Ek
embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.
PCR analysis of tissue samples from the 1979 Sverdlovsk anthrax victims: The presence of multiple
Bacillus anthracis
strains in different victims
Authors: Jackson Paul J. , Hugh-Jones Martin E. , Adair Debra M. , Green Gertrude , Hill Karen K. , Kuske Cheryl R. , Grinberg Lev M. , Abramova Faina A. , Keim Paul
Published: July 2002

An outbreak of human anthrax occurred in Sverdlovsk, Union of Soviet Socialists Republic (now Ekaterinburg, Russia) in April 1979. Officials attributed this to consumption of contaminated meat, but Western governments believed it resulted from inhalation of spores accidentally released from a nearby military research facility. Tissue samples from 11 victims were obtained and methods of efficiently extracting high-quality total DNA from these samples were developed. Extracted DNA was analyzed by using PCR to determine whether it contained
Bacillus anthracis
-specific sequences. Double PCR using “nested primers” increased sensitivity of the assay significantly. Tissue samples from 11 persons who died during the epidemic were examined. Results demonstrated that the entire complement of
B. anthracis
toxin and capsular antigen genes required for pathogenicity were present in tissues from each of these victims. Tissue from a vaccination site contained primarily nucleic acids from a live vaccine, although traces of genes from the infecting organisms were also present. PCR analysis using primers that detect the
vrrA
gene variable region on the
B. anthracis
chromosome demonstrated that at least four of the five known strain categories defined by this region were present in the tissue samples. Only one category is found in a single
B. anthracis
strain.
The absence of a major Ca
2+
signaling pathway in GABAergic neurons of the hippocampus
Authors: Sík Attila , Hájos Norbert , Gulácsi Alexandra , Mody Istvan , Freund Tamás F.
Published: July 2002

The Ca
2+
/calmodulin-dependent protein phosphatase 2B or calcineurin (CN) participates in several Ca
2+
-dependent signal transduction cascades and, thus, contributes to the short and long term regulation of neuronal excitability. By using a specific antibody to CN, we demonstrate its absence from hippocampal interneurons and illustrate a physiological consequence of such CN deficiency. Consistent with the lack of CN in interneurons as detected by immunocytochemistry, the CN inhibitors FK-506 or okadaic acid significantly prolonged
N
-methyl-
d
-aspartate channel openings recorded in the cell-attached mode in hippocampal principal cells but not those recorded in interneurons. Interneurons were also devoid of Ca
2+
/calmodulin-dependent protein kinase IIα, yet many of their nuclei contained the cyclic AMP-responsive element binding protein. On the basis of the CN and Ca
2+
/calmodulin-dependent protein kinase IIα deficiency of interneurons, entirely different biochemical mechanisms are expected to govern Ca
2+
-dependent neuronal plasticity in interneurons versus principal cells.
Amyloid fibril formation by an SH3 domain
Authors: Guijarro J. Iñaki , Sunde Margaret , Jones Jonathan A. , Campbell Iain D. , Dobson Christopher M.
Published: July 2002

The SH3 domain is a well characterized small protein module with a simple fold found in many proteins. At acid pH, the SH3 domain (PI3-SH3) of the p85α subunit of bovine phosphatidylinositol 3-kinase slowly forms a gel that consists of typical amyloid fibrils as assessed by electron microscopy, a Congo red binding assay, and x-ray fiber diffraction. The soluble form of PI3-SH3 at acid pH (the A state by a variety of techniques) from which fibrils are generated has been characterized. Circular dichroism in the far- and near-UV regions and
1
H NMR indicate that the A state is substantially unfolded relative to the native protein at neutral pH. NMR diffusion measurements indicate, however, that the effective hydrodynamic radius of the A state is only 23% higher than that of the native protein and is 20% lower than that of the protein denatured in 3.5 M guanidinium chloride. In addition, the A state binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid, which suggests that SH3 in this state has a partially formed hydrophobic core. These results indicate that the A state is partially folded and support the hypothesis that partially folded states formed in solution are precursors of amyloid deposition. Moreover, that this domain aggregates into amyloid fibrils suggests that the potential for amyloid deposition may be a common property of proteins, and not only of a few proteins associated with disease.
Transcriptional regulation of plant phosphate transporters
Authors: Muchhal Umesh S. , Raghothama K. G.
Published: July 2002

Phosphorus is acquired by plant roots primarily via the high-affinity inorganic phosphate (Pi) transporters. The transcripts for Pitransporters are highly inducible upon Pistarvation, which also results in enhanced Piuptake when Piis resupplied. Using antibodies specific to one of the tomato Pitransporters (encoded byLePT1), we show that an increase in theLePT1transcript under Pistarvation leads to a concurrent increase in the transporter protein, suggesting a transcriptional regulation for Piacquisition. LePT1 protein accumulates rapidly in tomato roots in response to Pistarvation. The level of transporter protein accumulation depends on the Piconcentration in the medium, and it is reversible upon resupply of Pi. LePT1 protein accumulates all along the roots under Pistarvation and is localized primarily in the plasma membranes. These results clearly demonstrate that plants increase their capacity for Piuptake during Pistarvation by synthesis of additional transporter molecules.
Global environmental impacts of agricultural expansion: The need for sustainable and efficient practices
Authors: Tilman David
Published: July 2002

The recent intensification of agriculture, and the prospects of
future intensification, will have major detrimental impacts on the
nonagricultural terrestrial and aquatic ecosystems of the world. The
doubling of agricultural food production during the past 35 years was
associated with a 6.87-fold increase in nitrogen fertilization, a
3.48-fold increase in phosphorus fertilization, a 1.68-fold increase in
the amount of irrigated cropland, and a 1.1-fold increase in land in
cultivation. Based on a simple linear extension of past trends, the
anticipated next doubling of global food production would be associated
with approximately 3-fold increases in nitrogen and phosphorus
fertilization rates, a doubling of the irrigated land area, and an 18%
increase in cropland. These projected changes would have dramatic
impacts on the diversity, composition, and functioning of the remaining
natural ecosystems of the world, and on their ability to provide
society with a variety of essential ecosystem services. The largest
impacts would be on freshwater and marine ecosystems, which would be
greatly eutrophied by high rates of nitrogen and phosphorus release
from agricultural fields. Aquatic nutrient eutrophication can lead to
loss of biodiversity, outbreaks of nuisance species, shifts in the
structure of food chains, and impairment of fisheries. Because of
aerial redistribution of various forms of nitrogen, agricultural
intensification also would eutrophy many natural terrestrial ecosystems
and contribute to atmospheric accumulation of greenhouse gases. These
detrimental environmental impacts of agriculture can be minimized only
if there is much more efficient use and recycling of nitrogen and
phosphorus in agroecosystems.
A single nucleotide in the
SMN
gene regulates splicing and is responsible for spinal muscular atrophy
Authors: Lorson Christian L. , Hahnen Eric , Androphy Elliot J. , Wirth Brunhilde
Published: July 2002

SMN1
and
SMN2
(survival motor neuron) encode identical proteins. A critical question is why only the homozygous loss of
SMN1
, and not
SMN2
, results in spinal muscular atrophy (SMA). Analysis of transcripts from
SMN1/SMN2
hybrid genes and a new
SMN1
mutation showed a direct relationship between presence of disease and exon 7 skipping. We have reported previously that the exon-skipped product SMNΔ7 is partially defective for self-association and SMN self-oligomerization correlated with clinical severity. To evaluate systematically which of the five nucleotides that differ between
SMN1
and
SMN2
effect alternative splicing of exon 7, a series of SMN minigenes was engineered and transfected into cultured cells, and their transcripts were characterized. Of these nucleotide differences, the exon 7 C-to-T transition at codon 280, a translationally silent variance, was necessary and sufficient to dictate exon 7 alternative splicing. Thus, the failure of
SMN2
to fully compensate for
SMN1
and protect from SMA is due to a nucleotide exchange (C/T) that attenuates activity of an exonic enhancer. These findings demonstrate the molecular genetic basis for the nature and pathogenesis of SMA and illustrate a novel disease mechanism. Because individuals with SMA retain the
SMN2
allele, therapy targeted at preventing exon 7 skipping could modify clinical outcome.
Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with
N
-butyldeoxynojirimycin
Authors: Jeyakumar Mylvaganam , Butters Terry D. , Cortina-Borja Mario , Hunnam Victoria , Proia Richard L. , Perry V. Hugh , Dwek Raymond A. , Platt Frances M.
Published: July 2002

Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the
HEXB
gene, which encodes the β-subunit of β-hexosaminidase. G
M2
ganglioside fails to be degraded and accumulates within lysosomes in cells of the periphery and the central nervous system (CNS). There are currently no therapies for the glycosphingolipid lysosomal storage diseases that involve CNS pathology, including the G
M2
gangliosidoses. One strategy for treating this and related diseases is substrate deprivation. This would utilize an inhibitor of glycosphingolipid biosynthesis to balance synthesis with the impaired rate of catabolism, thus preventing storage. One such inhibitor is
N
-butyldeoxynojirimycin, which currently is in clinical trials for the potential treatment of type 1 Gaucher disease, a related disease that involves glycosphingolipid storage in peripheral tissues, but not in the CNS. In this study, we have evaluated whether this drug also could be applied to the treatment of diseases with CNS storage and pathology. We therefore have treated a mouse model of Sandhoff disease with the inhibitor
N
-butyldeoxynojirimycin. The treated mice have delayed symptom onset, reduced storage in the brain and peripheral tissues, and increased life expectancy. Substrate deprivation therefore offers a potentially general therapy for this family of lysosomal storage diseases, including those with CNS disease.
Lon and Clp family proteases and chaperones share homologous substrate-recognition domains
Authors: Smith Catherine K. , Baker Tania A. , Sauer Robert T.
Published: July 2002

Lon protease and members of the Clp family of molecular chaperones and protease regulatory subunits contain homologous regions with properties expected for substrate-binding domains. Fragments corresponding to these sequences are stably and independently folded for Lon, ClpA, and ClpY. The corresponding regions from ClpB and ClpX are unstable. All five fragments exhibit distinct patterns of binding to three proteins that are protease substrates
in vivo
: the heat shock transcription factor σ
32
, the SOS mutagenesis protein UmuD, and Arc repressor bearing the SsrA degradation tag. Recognition of UmuD is mediated through peptide sequences within a 24-residue N-terminal region whereas recognition of both σ
32
and SsrA-tagged Arc requires sequences at the C terminus. These results indicate that the Lon and Clp proteases use the same mechanism of substrate discrimination and suggest that these related ATP-dependent bacterial proteases scrutinize accessible or disordered regions of potential substrates for the presence of specific targeting sequences.
Proposal for a standardized temporal scheme of biological classification for extant species
Authors: Avise John C. , Johns Glenn C.
Published: July 2002

With respect to conveying useful comparative information, current biological classifications are seriously flawed because they fail to (i) standardize criteria for taxonomic ranking and (ii) equilibrate assignments of taxonomic rank across disparate kinds of organisms. In principle, these problems could be rectified by adopting a universal taxonomic yardstick based on absolute dates of the nodes in evolutionary trees. By using procedures of temporal banding described herein, a simple philosophy of biological classification is proposed that would retain a manageable number of categorical ranks yet apply them in standardized fashion to time-dated phylogenies. The phylogenetic knowledge required for a time-standardized nomenclature arguably may emerge in the foreseeable future from vast increases in multilocus DNA sequence information (coupled with continued attention to phylogeny estimation from traditional systematic data). By someday encapsulating time-dated phylogenies in a familiar yet modified hierarchical ranking scheme, a temporal-banding approach would improve the comparative information content of biological classifications.
A critical role for the peroxisome proliferator-activated receptor α (PPARα) in the cellular fasting response: The PPARα-null mouse as a model of fatty acid oxidation disorders
Authors: Leone Teresa C. , Weinheimer Carla J. , Kelly Daniel P.
Published: July 2002

We hypothesized that the lipid-activated transcription factor, the peroxisome proliferator-activated receptor α (PPARα), plays a pivotal role in the cellular metabolic response to fasting. Short-term starvation caused hepatic steatosis, myocardial lipid accumulation, and hypoglycemia, with an inadequate ketogenic response in adult mice lacking PPARα (PPARα
−/−
), a phenotype that bears remarkable similarity to that of humans with genetic defects in mitochondrial fatty acid oxidation enzymes. In PPARα
+/+
mice, fasting induced the hepatic and cardiac expression of PPARα target genes encoding key mitochondrial (medium-chain acyl-CoA dehydrogenase, carnitine palmitoyltransferase I) and extramitochondrial (acyl-CoA oxidase, cytochrome P450 4A3) enzymes. In striking contrast, the hepatic and cardiac expression of most PPARα target genes was not induced by fasting in PPARα
−/−
mice. These results define a critical role for PPARα in a transcriptional regulatory response to fasting and identify the PPARα
−/−
mouse as a potentially useful murine model of inborn and acquired abnormalities of human fatty acid utilization.