Neuropathy progression in Charcot-Marie-Tooth disease type 1A.

OBJECTIVE: To determine the rate of disease progression in Charcot-Marie-Tooth disease type 1A (CMT1A). BACKGROUND: CMT1A is the most common inherited peripheral neuropathy, affecting approximately 1:5,000 people irrespective of ethnic background or gender. There is no cure for CMT1A. Clinical trials are being initiated that use the CMT Neuropathy Score (CMTNS), a composite score based on patient symptoms, signs, and neurophysiologic abnormalities, as the primary outcome variable. The sensitivity of the CMTNS or any other score to change over time, as a measure of CMT1A progression, has yet to be determined. METHODS: We determined the CMTNS as well as the Neuropathy Impairment Score (NIS) on 72 patients followed for up to 8 years. The rate of disease progression was evaluated for the CMTNS and NIS using mixed effects linear regression models, adjusting for age and gender. RESULTS: Both CMTNS and NIS showed changes over time. The CMTNS increased an average of 0.686 points per year (95% CI 0.461 to 0.911, p

Reaction mechanism and structure-reactivity relationships in the stereospecific 1,4-polymerization of butadiene catalyzed by neutral dimeric allylnickel(II) halides [Ni(C3H5)X]2 (X- = Cl-, Br-, I-): a comprehensive density functional theory study.

For the first time, a comprehensive and consistent picture of the catalytic cycle of 1,4-polymerization of butadiene with neutral dimeric allylnickel(II) halides [Ni(C3H5)X]2 (X = Cl- (I), Br- (II), and I- (III)) as single-site catalysts has been derived by means of quantum chemical calculations that employ a gradient-corrected density-functional method. All crucial reaction steps of the entire catalytic course have been scrutinized, taking into account butadiene pi complex formation, symmetrical and asymmetrical splitting of dimeric pi complexes, cis-butadiene insertion, and anti-syn isomerization. The present investigation examines, in terms of located structures, energies and activation barriers, the participation of postulated intermediates, in particular it aimed to clarify whether monomeric or dimeric species are the catalytically active species. Prior qualitative mechanistic assumptions are substituted by the presented theoretically well-founded and detailed analysis of both the thermodynamic and the kinetic aspects, that substantially improve the insight into the reaction course and enlarge them with novel mechanistic proposals. From a mechanistic point of view, all three catalysts exhibit common characteristics. First, chain propagation occurs by cis-butadiene insertion into the pi-butenylnickel(II) bond with nearly identical intrinsic free-energy activation barriers. Second, the reactivity of syn-butenyl forms is distinctly higher than that of anti forms. Third, the chain-propagation step is rate-determining in the entire polymerization process, and the pre-established anti-syn equilibrium can always be regarded as attained. Accordingly, neutral dimeric allylnickel(II) halides catalyze the formation of a stereo-regular trans-1,4-polymer under kinetic control following the k1t channel with butenyl(halide)(butadiene)NiII complexes being the catalytically active species. Production of a stereoregular cis-1,4-polymer with allylnickel chloride can only be explained by making the k2c channel accessible by the formation of polybutadienyl(butadiene) complexes, which is accompanied by the coordination of the next double bond in the growing chain to the NiII center.

Cooperative interaction between HIV-1 regulatory proteins Tat and Vpr modulates transcription of the viral genome.

The virion-associated protein of human immunodeficiency virus, type 1 (HIV-1), Vpr, is a small protein with 96 amino acid residues that has the ability to modulate transcription of HIV-1 long terminal repeat (LTR) promoter activity and affects several cellular functions. In this study we have employed molecular approaches to further investigate the mechanism by which Vpr exerts its regulatory effect upon the LTR. We show that by structural and functional interaction with Tat, a potent viral regulatory protein, Vpr synergistically enhances the transcriptional activity of the HIV-1 LTR. Because Tat utilizes cyclin T1 and its partner, CDK9 to elevate the level of transcription from the LTR, we examined the cooperativity between Vpr, Tat, and cyclin T1/CDK9 on viral gene transcription. Results from co-transfection studies indicated superactivation of LTR by Tat and cyclin T1/CDK9 in the presence of wild type Vpr. This activation was not observed with the R73S mutant of Vpr, which contains arginine to serine transition at residue 73. Interestingly, expression of R73S mutant in cells exerts a negative effect on the observed superactivation of the LTR by Tat, cyclin T1/CDK9, and wild type Vpr. Results from protein-protein interaction studies indicated that Vpr is associated with both Tat and cyclin T1 in cells expressing these proteins. Use of deletion mutant proteins in binding studies revealed that the binding sites for Tat and Vpr within cyclin T1 are distinct and that association of these two viral proteins with cyclin T1 is independent from each other. These observations suggest a working model on the cooperative interaction of Vpr with viral and cellular proteins and its involvement in control of viral gene transcription and replication. Moreover identification of R73S mutant of Vpr provides a new therapeutic avenue for controlling HIV-1 gene transcription and replication in the infected cells.

Flavopiridol inhibits P-TEFb and blocks HIV-1 replication.

Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor that is in clinical trials as a cancer treatment because of its antiproliferative properties. We found that the flavonoid potently inhibited transcription by RNA polymerase II in vitro by blocking the transition into productive elongation, a step controlled by P-TEFb. The ability of P-TEFb to phosphorylate the carboxyl-terminal domain of the large subunit of RNA polymerase II was inhibited by flavopiridol with a K(i) of 3 nm. Interestingly, the drug was not competitive with ATP. P-TEFb composed of Cdk9 and cyclin T1 is a required cellular cofactor for the human immunodeficiency virus (HIV-1) transactivator, Tat. Consistent with its ability to inhibit P-TEFb, flavopiridol blocked Tat transactivation of the viral promoter in vitro. Furthermore, flavopiridol blocked HIV-1 replication in both single-round and viral spread assays with an IC(50) of less than 10 nm.

Binding of Tat to TAR and recruitment of positive transcription elongation factor b occur independently in bovine immunodeficiency virus.

Transcriptional transactivators (Tat) from many lentiviruses interact with their cognate transactivation response RNA structures (TAR) to increase rates of elongation rather than initiation of transcription. For several of them, the complex of Tat and a species-specific cyclin T1 must be formed before the binding to TAR can occur with high affinity and specificity. In sharp contrast, Tat from the bovine immunodeficiency virus (BIV) binds to its TAR without the help of the cyclin T1. This binding depends on the upper stem and 5' bulge, but not the central loop in TAR. Moreover, cyclins T1 from different species can mediate effects of this Tat in cells. Unlike the situation with other lentiviruses, Tat transactivation can be rescued simply by linking a heterologous promoter to TAR in permissive cells. Thus, lentiviruses have evolved different strategies to recruit Tat and the positive transcription elongation factor b to their promoters, and interactions between Tat and TAR are independent from those between Tat and the cyclin T1 in BIV.

Interactions between equine cyclin T1, Tat, and TAR are disrupted by a leucine-to-valine substitution found in human cyclin T1.

Transcriptional transactivators (Tat) from human immunodeficiency and equine infectious anemia viruses (HIV and EIAV) interact with their transactivation response elements (TAR) to increase the rates of viral transcription. Whereas the human cyclin T1 is required for the binding of Tat to TAR from HIV, it is unknown how Tat from EIAV interacts with its TAR. Furthermore, Tat from EIAV functions in equine and canine cells but not in human cells. In this study, we present sequences of cyclins T1 from horse and dog and demonstrate that their N-terminal 300 residues rescue the transactivation of Tat from EIAV in human cells. Although human and equine cyclins T1 bind to this Tat, only the equine cyclin T1 supports the binding of Tat to TAR from EIAV. Finally, a reciprocal exchange of the valine for the leucine at position 29 in human and equine cyclins T1, respectively, renders the human cyclin T1 active and the equine cyclin T1 inactive for Tat transactivation from EIAV. Thus, the collaboration between a specific cyclin T1 and Tat for their high-affinity interaction with TAR is a common theme of lentiviral transactivation.

Interactions between Tat and TAR and human immunodeficiency virus replication are facilitated by human cyclin T1 but not cyclins T2a or T2b.

The transcriptional transactivator (Tat) from the human immunodeficiency virus (HIV) does not function efficiently in Chinese hamster ovary (CHO) cells. Only somatic cell hybrids between CHO and human cells and CHO cells containing human chromosome 12 (CHO12) support high levels of Tat transactivation. This restriction was mapped to interactions between Tat and TAR. Recently, human cyclin T1 was found to increase the binding of Tat to TAR and levels of Tat transactivation in rodent cells. By combining individually with CDK9, cyclin T1 or related cyclins T2a and T2b form distinct positive transcription elongation factor b (P-TEFb) complexes. In this report, we found that of these three cyclins, only cyclin T1 is encoded on human chromosome 12 and is responsible for its effects in CHO cells. Moreover, only human cyclin T1, not mouse cyclin T1 or human cyclins T2a or T2b, supported interactions between Tat and TAR in vitro. Finally, after introducing appropriate receptors and human cyclin T1 into CHO cells, they became permissive for infection by and replication of HIV.

Interactions between human cyclin T, Tat, and the transactivation response element (TAR) are disrupted by a cysteine to tyrosine substitution found in mouse cyclin T.

The transcriptional transactivator Tat from HIV binds to the transactivation response element (TAR) RNA to increase rates of elongation of viral transcription. Human cyclin T supports these interactions between Tat and TAR. In this study, we report the sequence of mouse cyclin T and identify the residues from positions 1 to 281 in human cyclin T that bind to Tat and TAR. Mouse cyclin T binds to Tat weakly and is unable to facilitate interactions between Tat and TAR. Reciprocal exchanges of the cysteine and tyrosine at position 261 in human and mouse cyclin T proteins also render human cyclin T inactive and mouse cyclin T active. These findings reveal the molecular basis for the restriction of Tat transactivation in rodent cells.

Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization.

We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the -1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMTV RT were compared with those of the well-studied RT of HIV-1, the causative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivity to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs), as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV-1 RT.

DNA synthesis exhibited by the reverse transcriptase of mouse mammary tumor virus: processivity and fidelity of misinsertion and mispair extension.

We have recently expressed in bacteria an enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV), a mammalian retrovirus with a typical B-type morphology [Taube, R., Loya, S., Avidan, O., Perach, M. & Hizi, A. (1998) Biochemical J. 329, 579-587]. The purified recombinant protein was shown to possess the catalytic activities characteristic of retroviral reverse transcriptases. In the present study, we have analyzed two basic parameters characteristic of the DNA polymerase activity of the novel MMTV RT, namely the processivity and the fidelity of DNA synthesis. Two features related to fidelity were studied, the capacity to misinsert wrong nucleotides at the 3' end of the nascent DNA strand and the ability to extend 3' mispairs. The studied properties of MMTV RT were compared with those of the RT purified from virions of avian myeloblastosis virus (AMV), since AMV RT shows a relatively high sequence similarity to MMTV RT. MMTV RT shows a relative processivity of DNA synthesis which is as high as the reference AMV RT. Regarding fidelity of DNA synthesis, MMTV RT shows a fidelity of misinsertion lower than that of AMV RT, whereas its capacity to elongate mispaired DNA is lower than that of AMV RT indicating a somewhat higher fidelity. These fidelity properties are discussed also in the context of the RTs of lentiviruses, especially those of HIV, which were reported to exhibit an exceptionally low fidelity of DNA synthesis. It is clear that MMTV RT has a fidelity higher than that of lentiviral RTs.

The fidelity of 3' misinsertion and mispair extension during DNA synthesis exhibited by two drug-resistant mutants of the reverse transcriptase of human immunodeficiency virus type 1 with Leu74-->Val and Glu89-->Gly.

The relatively low fidelity of DNA synthesis characteristic to the reverse transcriptases (RTs) of the AIDS-causing viruses, human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively) was implicated as a dominant factor that contributes to the genetic hypervariability of these viruses. The formation of 3'-mispaired DNA and the subsequent extension of this DNA were shown to be key determinants that lead to the error proneness of these RTs. As part of our goal to study the structure/function relationship in HIV-1 RT, we have conducted mutational studies aimed at identifying amino-acid residues involved in affecting the fidelity of DNA synthesis by the enzyme. We have recently found that two mutants of HIV-1 RT, which show resistance to nucleoside analog inhibitors ([Leu184]RT and [Phe183]RT), exhibit in vitro error proneness of DNA synthesis lower than that of wild-type enzyme [Bakhanshvili, M., Avidan, O. & Hizi, A. (1996) Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis, FEBS Lett. 391, 257-262]. Using both criteria, the current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart. In the current study we have analyzed two additional drug-resistant mutants of HIV-1 RT, [Val74]RT and [Gly89]RT, for their in vitro fidelity of DNA synthesis using two parameters of DNA synthesis: 3' mispair formation and elongation of 3'-mismatched DNA. The current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart, using both criteria. Analysis of the relative frequencies of misinsertion and mispair extension indicates that the overall error proneness of DNA synthesis in HIV-1 RT is wild-type > [Val74]RT > [Gly89]RT mutant. The results further support the possible linkage between the capacity of an enzyme to incorporate a nucleoside analog instead of the correct dNTP (leading to drug sensitivity) and the ability to incorporate and extend a wrong nucleotide (resulting in mutagenesis). Our results may bear on the potential use of selecting and maintaining HIV virions with high fidelity and drug-resistant RTs to suppress the subsequent appearance of virions resistant to other drugs.

Strand breaks after the decay of iodine-125 in proximity to plasmid pBR322 DNA.

To elucidate the kinetics of DNA strand breaks caused by low-energy Auger electron emitters in proximity to DNA molecules, we synthesized (125)I-labeled 2-iodoacridine (2-(125)IA), which intercalates with DNA, and 4-iodoacridine (4-(125)IA), which does not. Supercoiled DNA from pBR322 plasmid, labeled with 3H, was purified and incubated with 2-(125)IA or 4-(125)IA in aqueous solution. Reaction mixtures were stored at 4 degrees C to accumulate radiation dose from the decay of (125)I, and DNA was resolved by gel electrophoresis into supercoiled (DNA-I), nicked-circular (DNA-II) and linear (DNA-III) forms, representing undamaged DNA, single-strand breaks (SSBs) and double-strand breaks (DSBs), respectively. Gamma irradiation from an external (137)Cs source led to an exponential decrease in DNA-I with a D0 value of 10.8 +/- 0.3 Gy. Under identical conditions, the D0 values for 2-(125)IA and 4-(125)IA were 22.4 +/- 0.6 x 10(11) disintegrations and 4.7 +/- 0.4 x 10(11) disintegrations, respectively. External gamma irradiation and 4-(125)IA produced SSB/DSB ratios of 26.5 +/- 2.1 and 15.9 +/- 2, respectively, while that for 2-(125)IA was 0.6. The average number of DSBs from each decay of (125)I was 0.67 for 2-(125)IA and 0.27 for 4-(125)IA. The results indicate that the decay of (125)I bound to a DNA-intercalating compound produces DSBs 2.5-fold more efficiently than (125)I bound to a nonintercalating compound and support the theoretical expectations that predict a DSB yield that is highly dependent on the proximity of the Auger electron emitter to DNA.

The fidelity of misinsertion and mispair extension throughout DNA synthesis exhibited by mutants of the reverse transcriptase of human immunodeficiency virus type 2 resistant to nucleoside analogs.

The AIDS-causing retroviruses, human immunodeficiency virus types 1 and type 2 (HIV-1 and HIV-2, respectively) undergo extensive genetic variations, which effect their pathogenesis and resistance to drug therapy. It was postulated that this genetic hypervariability results from high rates of viral replication in conjugation with a relatively low fidelity of DNA synthesis [typical to the reverse transcriptases (RT) of these retroviruses]. As part of studying structure/function relationship in HIV RT, mutational analyses were conducted to identify amino acid residues which are involved in affecting the fidelity of DNA synthesis. The formation of 3'-mispaired DNA due to nucleotide misinsertions, and the subsequent elongation of this mismatched DNA were shown to be major determinants in affecting those substitutions during DNA synthesis (exhibited in vitro by HIV RT). It was interesting to find a correlation between sensitivity to nucleoside analogs (due to the ability to incorporate or reject an incoming analog) and the fidelity of DNA synthesis (which depends on the capacity to incorporate and extend a wrong nucleotide). Such a connection has already been found for several drug-resistant mutants of HIV-1 RT, with an increased fidelity of DNA synthesis relative to the wild-type RT. In the present study we have examined the fidelity of DNA synthesis using the same parameters of misinsertion and mispair extension for five novel drug-resistant mutants of HIV-2 RT; i.e. the single mutants [Val74]RT, [Gly89]RT and [Tyr215]RT and the double mutants [Val74,Tyr215]RT and [Gly89, Tyr215]RT. This comparative study suggests that unlike the Val74 mutant of HIV-1 RT, which was shown earlier to display a substantially enhanced fidelity, the comparable mutant of HIV-2 RT has fidelity similar to that of the wild-type RT. Depending on the assay employed and the DNA sequences extended, most other mutants of HIV-2 RT display moderate effects on the enzyme, leading to mild increases in fidelity of DNA synthesis. This implies a more complex and less distinctive correlation between drug-resistance, misinsertion and mispair extension in HIV-2 RT in contrast to HIV-1 RT, providing evidence for potential biochemical differences between these two related RT.

Peliosis of the spleen: a rare cause of spontaneous splenic rupture with surgical implications.

Peliosis is a rare entity, formerly seen almost exclusively at autopsy, which most commonly involves the liver and less frequently the spleen. We present the sixth reported case of splenic peliosis discovered at surgical exploration and discuss the relevant findings and background of this condition, which may become an important issue for the surgeon.

Managing Tibial Stress Fractures.

In brief Tibial stress fractures often occur in Patients who participate in running and jumping sports. With rest, most stress fractures heal without incident, though midshaft fractures are more resistant to healing. Bone scans are usually required to confirm the diagnosis. Relative rest, strength training, proper footwear, and treating underlying risk factors are the mainstays of treatment. A new treatment option is a pneumatic brace that the patient can wear when he or she returns to play. Physicians should be prepared to counsel competitive athletes about the risks of returning to play too soon.

Radiotoxicity of 5-[125I]iodo-2'-deoxyuridine in mammalian cells following treatment with 5-fluoro-2'-deoxyuridine.

We have enhanced the uptake of 5-[125I]iodo-2'-deoxyuridine (125IUdR) in Chinese hamster V79 cells with 5-fluoro-2'-deoxyuridine (FUdR) and have examined the combined toxicity of these agents. Although the uptake of 125IUdR increases approximately 3.2 +/- 0.5-fold in the presence of 1 microM FUdR, when cell survival fraction is plotted as a function of intranuclear 125IUdR content, the biphasic curve obtained reaches a plateau at a higher survival fraction than with control cells not exposed to FUdR. The results suggest that a greater number of cells were prevented from entering the S phase and consequently from incorporating 125IUdR. An FUdR- 125IUdR combination, therefore, does not seem to enhance the therapeutic potential of 125IUdR. Such observations are also of importance when FUdR and other inhibitors are used to enhance cold IUdR uptake in an effort to obtain an increase in radiosensitization effects.

Antigen-binding site protection during radiolabeling leads to a higher immunoreactive fraction.

It is generally accepted that the immunointegrity of an antibody (Ab) depends on the preservation of its antigen-binding sites. Our goal was to radiolabel an antibody at several iodine:antibody molar ratios under conditions protecting its combining site and to compare its immunoreactive fraction (IRF) and electrophoretic mobility with those of the same antibody radiolabeled without protection. The data indicate that an antibody radiolabeled while its antigen-binding site is occupied by its antigen had the same IRF, regardless of the number of iodine atoms per antibody molecule. On the other hand, even at an I:Ab ratio of 1:1, the IRF of the same antibody radiolabeled without protection was lower than that of a protected one and decreased with increasing I:Ab ratios. In addition, the iodination of these Ab changes their electrophoretic mobility; however, when the Ab is labeled in the protected state, the degree of change is less. The binding of an antibody to its antigen prior to radiolabeling, therefore, enhances its immuno-integrity and prevents major conformational changes as reflected by electrophoresis.

Preadsorption of radiolabeled monoclonal antibodies to liver and spleen tissues leads to higher tumor-to-normal-tissue ratios.

This study addresses the impact of background activity on the use of radioimmunoconjugates for radioimmunodiagnosis and radioimmunotherapy. Since the liver and the spleen represent organs with preferential nonspecific uptake, we exposed radiolabeled (iodinated and Indium-111 labeled) preparations of monoclonal antibodies to a suspension of fresh liver and spleen cells at physiological temperature and compared their immunoreactivity, in vivo biodistribution, and tumor targeting to those of the same radiolabeled proteins without prior adsorption to this suspension. The biodistribution studies were performed under conditions of high background activity, i.e., shortly after the injection (1 hour) and using a high dose of the protein. Preadsorption of radiolabeled monoclonal antibodies results in a significant decreased uptake in certain normal tissues, i.e., greater contrast between normal and tumor tissues, as demonstrated by the quotient of the two target-to-nontarget ratios (exposed/unexposed antibody) which was greater than one for most of the tissues examined.