Soluble cerebrospinal fluid factors induce Ca2+ dysregulation in rat cultured cortical astrocytes in HIV-1-associated dementia complex.

OBJECTIVES: To investigate the effect of cerebrospinal fluid (CSF) of HIV-1-seropositive patients with and without HIV-1-associated dementia complex (HADC) on the intracellular Ca2+ regulation of cultured cortical astrocytes. DESIGN: In a blinded study the effects of CSF samples from HADC patients and from HIV-1-seropositive but not demented patients on intracellular Ca2+ regulation of cultured cortical astrocytes were investigated. Astrocytes were chosen because they contribute to both electrophysiological and immunological processes within the brain. METHODS: Astrocytes were incubated in CSF samples for 1 h, loaded with the Ca2+ indicator dye Fura-2 and intracellular Ca2+ responses upon glutamate application were measured. RESULTS: CSF samples from 10 out of 11 HADC patients induced a significant reduction of the intracellular Ca2+ increase upon glutamate application. On the contrary, seven out of 10 CSF samples from HIV-1-seropositive patients without HADC as well as 10 out of 10 CSF samples from HIV-1-seronegative controls did not affect the intracellular Ca2+ response. CONCLUSIONS: Our data strongly confirm the hypothesis that CSF samples of HADC patients contain soluble factors which interfere with the function of astrocytes. These factors may include HIV-1 proteins, locally released cytokines or neurotoxins.

Use of DNA end joining activity of a Xenopus laevis egg extract for construction of deletions and expression vectors for HIV-1 Tat and Rev proteins.

We have developed a cloning strategy which combines conventional T4 DNA ligation with the highly efficient nonhomologous DNA end joining (EJ) activity of an extract from Xenopus laevis eggs. The nonhomologous EJ activity allowed the rapid construction of deletion mutants by the intramolecular rejoining of nonhomologous DNA ends generated for the purpose of deleting restriction fragments from the vector. The combined use of T4 DNA ligase for intermolecular ligation and X. laevis egg extracts for intramolecular nonhomologous EJ proved to be a powerful tool, as demonstrated here for the construction of expression vectors for HIV-1 Tat and Rev.

Natural variation in the amino acid sequence around the HIV type 1 glycoprotein 160 cleavage site and its effect on cleavability, subunit association, and membrane fusion.

To assess the natural variation of the structure of the cleavage site as well as the N-terminal region of gp41 for the cytopathogenicity of HIV-1, syncytium-inducing (SI) and non-syncytium-inducing (NSI) virus isolates were obtained from HIV-1-infected patients. In addition, the coreceptor usage of the isolates was determined by infection of primary macrophages and PM-1 cells. DNA sequences encoding the C-terminal 41 amino acid residues of gp120 and the 64 amino acid N-terminal residues of gp41 were amplified by the polymerase chain reaction and inserted into the Env expression vector pNLA1. When transfected into HeLa-T4(+) cells, all the recombinant plasmids, including those with inserts from NSI isolates, led to the formation of processed glycoprotein and to syncytium formation. One construct displayed significant lowered fusion capacity and had an amino acid exchange in the first position of the gp41 N terminus (gp41, 512A-->S) leading to a decreased association of the SU and TM subunits. Four constructs derived from two isolates of the same patient showed an unusual gp41 N terminus (gp41, 514G-->P) and a slightly diminished fusion capacity due to a decreased cleavability. This indicates that the major determinants for the SI and NSI phenotypes are not located around the gp160 cleavage site and that the N terminus of gp41 plays a minor role in the processing and fusion capacity of wild-type HIV-1 isolates.

[Galacto-biopsy (author's transl)]. / Galaktobiopsie.

By galacto-biopsy we understand the expression of a suspected intraductal papilloma by firm palpation as part of preparation for galactography. Using galacto-biopsy the accuracy of galactography in the diagnoses of intraduct papillomas was increased to 95%.

[Mammography in the diagnosis and treatment of non-puerperal mastitis (author's transl)]. / Die Bedeutung der Mammographie in der Diagnostik und Therapie einer nicht puerperalen Mastitis.

Circumscribed or diffuse non-puerperal mastitis is usually associated with local signs of inflammation. In two-thirds of these cases it is possible to demonstrate the radiological features of mastitis. Mammography is an important diagnostic procedure in the demonstration of acute mastitis and its differentiation from an inflammatory carcinoma; it is also valuable in the control of treatment. The success of intensive antibiotic therapy with recovery of normal appearances in the chest can be observed objectively by mammography. In distinguishing mastitis from an inflammatory carcinoma, radiological evidence of regression under the influence of antibiotics is evidence in favour of mastitis.

Changes of ganglioside pattern during cerebellar development of normal and staggerer mice.

The total amount of gangliosides per cerebellum of a wild-type mouse increased 126-fold during postnatal development. Although all major gangliosides were synthesized, the relative amount of individual ganglioside species changed during this period. In the developing wild-type cerebellum a transient accumulation of GD3 occurred between birth and postnatal day 20 (P20) with the largest portion (23%) of the total ganglioside content at postnatal day 7 (P7). In the adult cerebellum GD3 was only a minor component (3.2%) of the ganglioside pattern. As demonstrated by immunofluorescence the accumulation of GD3 was predominantly associated with premigratory and early postmigratory granule cells. The ganglioside GD3 was found in two alkali-stable forms in the young cerebellum, whereas the ganglioside species with the higher Rf value (migrating in the same position as the upper GD3 band) in the adult cerebellum was alkali labile. The cerebellum of the neurological mutant staggerer (sg/sg) was characterized by a low amount of GD1a in adult animals, due to the massive death of neurons in the postnatal cerebellar cortex. The neonatal loss of sialic acid residues from cerebellar cell surfaces in wild-type mice and the maintenance of embryonic sialoglycoconjugates in the staggerer cerebellum cannot be explained by the alterations of ganglioside patterns observed during postnatal development.

Exon 1 leader sequences downstream of U5 are important for efficient human immunodeficiency virus type 1 gene expression.

In previous studies, little attention has been paid to maintaining the native HIV-1 leader sequence in reporter constructs analyzing the human immunodeficiency virus type 1 (HIV-1) promoter activity. To investigate a possible influence of the leader sequence on HIV-1-driven gene expression in the presence as well as in the absence of Tat, an expression vector was designed for transcripts consisting of the native HIV-1 tat 1.4 mRNA leader followed by the open reading frame for the bacterial chloramphenicol acetyltransferase (CAT). Deletion mutants with mutations within the leader sequence downstream of U5 (lsdU5) were constructed, as well as a mutant containing a mutation with a reverse orientation of this region. Quantification of CAT protein in HeLa-T4+ cells transiently transfected with wild-type and mutant leader constructs showed that the exon 1-derived lsdU5 region has an influence on basal as well as Tat-induced protein expression. The dramatic decrease in the level of CAT protein upon deletion of lsdU5 was paralleled by a drop in the steady-state level of CAT mRNA. Deletion of the exon 1-derived lsdU5 region also decreased the expression of mRNAs containing authentic HIV-1 sequences instead of CAT. The effect observed with the reporter constructs was not due to the loss of binding sites for nuclear factors, as could be shown with DBF1 and Sp1 mutant constructs. Nuclear run-on transcription assays showed that the presence or absence of lsdU5 did not influence the rate of transcription. This indicates that the exon 1 lsdU5 element functions at the posttranscriptional level in the processing, nucleocytoplasmic export, or stabilization of HIV-1 transcripts.

Generation of a flexible cell line with regulatable, high-level expression of HIV Gag/Pol particles capable of packaging HIV-derived vectors.

HIV-derived vectors are of potential clinical relevance due to their ability to transduce nondividing cells in vitro and in vivo. However, the generation of cell lines stably and reproducibly expressing high amounts of defined subviral particles, capable of packaging and transducing HIV-derived vectors, has been hampered by the cytotoxicity of some of the required gene products, in particular of the HIV-1 protease. The successful use of regulatable gene expression systems to overcome this problem requires that the remaining basally expressed gene product activity is below the threshold for cytotoxicity. To try to achieve this, we have consecutively introduced appropriate plasmids, encoding HIV rev and HIV gag/pol gene products, each under the control of separate ecdysone-inducible promoters, into human 293 cells. Using a protocol in which a specific HIV protease inhibitor, Saquinavir, was continuously present in the culture medium during selection, we could generate stable cell lines inducibly expressing high amounts of subviral particles. A cell line, termed 293-Rev/Gag/Pol(i), which has been characterized in more detail, inducibly releases, within 48 h postinduction, high amounts of HIV Gag/Pol particles (about 10 microg CA/ml). These HIV Gag/Pol particles can package and transduce third-generation HIV vectors to high titers. Thus, in addition to other applications, the 293-Rev/Gag/Pol(i) cell line represents a "founder" packaging cell line which, depending on the requirement, can be further modified to include specific transgene-encoding vector and targeting glycoprotein genes.

Lipid membrane fusion induced by the human immunodeficiency virus type 1 gp41 N-terminal extremity is determined by its orientation in the lipid bilayer.

The amino-terminal extremity of the human immunodeficiency virus type 1 transmembrane protein (gp41) is thought to play a pivotal role in the fusion of virus membranes with the plasma membrane of the target cell and in syncytium formation. Peptides with sequences taken from the human immunodeficiency virus type 1 gp41 fusogenic (synthetic peptides SPwt and SP-2) and nonfusogenic (SP-3 and SP-4) glycoproteins adopt mainly a beta-sheet conformation in the absence of lipid, as determined by attenuated total reflection Fourier transform infrared spectroscopy, and after interaction with large unilamellar liposomes, the beta-sheet is partly converted into an alpha-helical conformation. Peptides SPwt and SP-2 but not SP-3 or SP-4 were able to promote lipid mixing as assessed by fluorescence energy transfer assay and dye leakage in a vesicle leakage assay. By using polarized attenuated total reflection Fourier transform infrared spectroscopy, SPwt and SP-2 were found to adopt an oblique orientation in the lipid membrane whereas SP-3 and SP-4 were oriented nearly parallel to the plane of the membrane. These findings confirm the correlation between the membrane orientation of the alpha-helix and the lipid mixing ability in vitro. Interestingly, the data provide a direct correlation with the fusogenic activity of the parent glycoproteins in vivo.

Molecular characterization of the MT-family of dispersed middle-repetitive DNA in rodent genomes.

We report the consensus sequence of members of a new dispersed middle-repetitive DNA family, MT, which is present in mouse and rat genomes. This family is shown to be as abundant as the described rodent B1 and B2 families. Hybridization experiments with radioactive single-stranded cDNAs from different tissues indicate that MT sequences are more abundantly cotranscribed in parts of the brain than other repetitive families.

Influence of the small leader exons 2 and 3 on human immunodeficiency virus type 1 gene expression.

The human immunodeficiency virus type 1 (HIV-1) uses an elaborate alternative splicing pattern for the generation of both the 1.8-kb as well as the 4-kb classes of mRNA. An additional diversity of transcripts in both classes is created by the optional inclusion of the small exons 2 and 3 in the leader sequence. To analyze a possible influence of these leader exons on HIV-1 gene expression, several series of expression vectors with different leaders were constructed, expressing either Rev and Env or a heterologous coding sequence, i.e., the chloramphenicol acetyl transferase (CAT) ORF. Transfection experiments of HeLa-T4(+) cells revealed for all series of constructs that mRNA as well as protein expression was stimulated by the presence of exon 2 and reduced by exon 3. The function of the leader exons 2 and 3 is neither dependent on the regulatory proteins Tat or Rev nor on viral coding sequences. Neither transcription rates nor stability of polyadenylated RNAs were found to be responsible for the different levels of steady-state mRNA. When either exon 2 or 3 was inserted into a heterologous intron, processing of the primary transcripts generated identical mRNA species while maintaining the differences in exon 2/3-dependent mRNA steady-state levels. These results may be explained by exon-specific nuclear RNA degradation rates, as also indicated by results from an in vitro degradation assay using a HeLa nuclear extract.

Repetitive elements of the Mycoplasma hominis adhesin p50 can be differentiated by monoclonal antibodies.

The gene encoding p50, an adhesin of Mycoplasma hominis, was identified, cloned, and sequenced. Comparison of the derived amino acid sequence with the N-terminal amino acids sequenced by the Edman reaction of the native protein revealed that p50 is expressed as a 467-amino-acid precursor. Posttranslational modification leads to a 441-amino-acid lipoprotein with an extended, predominantly helical structure and a leucine zipper. Computer analysis of the amino acid sequence identified a threefold-repetitive sequence motif comprising approximately three-quarters of the total protein. Different regions of the p50 polypeptide chain were expressed in Escherichia coli. Western blot (immunoblot) analysis of the E. coli lysates revealed that the epitopes of four p50-specific monoclonal antibodies were localized in the middle and C-terminal part of the protein. Epitope mapping by exonuclease III digestion showed that all of the four monoclonal antibodies bound within the same region of the threefold-repetitive amino acid sequence motif. The repeats, which were highly homologous but not identical in structure, could be differentiated by the monoclonal antibodies.

Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion.

An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion glycoproteins.

Cloning and expression of P60, a conserved surface-localized protein of Mycoplasma hominis, in Escherichia coli.

The clp60 gene encoding P60, a conserved lipoprotein of Mycoplasma hominis, was cloned and sequenced from both the type strain PG21 and the isolate FBG. Both open reading frames were identical in length, comprising 1746 nucleotides. The deduced amino acid sequences differed in 16 out of 582 amino acids. As expected, none of these divergences mapped within the epitope that was recognized by mAb CG4 in all of the 198 isolates of M. hominis analyzed so far. This conserved epitope was narrowed down to amino acids 454 through 464 within the C terminus of P60. For the expression of the recombinant homolog P60, P60rec, in E. coli the TGA codons of clp60 were substituted for TGG codons prior to cloning of clp60 into the expression plasmid pQE41. The expression of P60rec as a fusion protein with dihydrofolate reductase carrying an N-terminal His-tag enabled the purification of large amounts of P60rec in a soluble form.

Regeneration-associated high level expression of apolipoprotein D mRNA in endoneurial fibroblasts of peripheral nerve.

A cDNA clone containing the entire coding region of rat apolipoprotein D (Apo D) was isolated from a cDNA library of regenerating sciatic nerve by differential hybridization. Only small amounts of Apo D mRNA were detected in noninjured mature nerve. Moderately increased levels of Apo D transcripts were found in transected nerves, which were prevented from regeneration by ligation. In contrast, in regenerating crushed nerve, the steady-state level of Apo D mRNA transiently increased at least 40-fold above control levels at the time when axons from the proximal stump grow into the distal nerve segment. Using transverse sections and primary cell cultures from regenerating nerve, Apo D transcripts could be localized by in situ hybridization in endoneurial fibroblasts but not in Schwann cells, macrophages or perineurial and epineurial cells. Apo D protein (Mr 32.8 kd) was secreted and accumulated in the endoneurial extracellular space where it could be detected in lipoprotein fractions by immunoblotting using established antibodies to human Apo D. High level expression of Apo D mRNA seems to be a novel regeneration-associated molecular event of endoneurial fibroblasts indicating a function for Apo D and fibroblasts in nerve repair.

Axon-regulated expression of a Schwann cell transcript that is homologous to a 'growth arrest-specific' gene.

We have isolated a 1.8 kb cDNA (pCD25) clone that encodes a transcript that is differentially expressed during nerve regeneration. Nucleotide sequence comparison indicates 89.6% homology with the recently identified murine 'growth arrest-specific' gene gas3. The open reading frame of the CD25 transcript predicts a 17 kDa protein with four putative transmembrane regions. Steady-state levels of the CD25 mRNA are very much higher in sciatic nerve than in other tissues, and expression in sciatic nerve is confined to Schwann cells. Following nerve injury, the transcript levels rapidly declined in nerve segments distal to the site of lesion, but recovered upon nerve regeneration. In contrast, in distal stumps of permanently transected nerves, the mRNA level remained very low. Substantial amounts of the mRNA could be reinduced only upon anastomosis of these interrupted nerve stumps. Re-induction of the mRNA followed the elongation of regenerating axons through the distal nerve segment. Our data indicate that axons regulate expression of the CD25 mRNA in Schwann cells, and suggest that the CD25 protein functions during Schwann cell growth and differentiation.

HIV-1 protein Tat reduces the glutamate-induced intracellular Ca2+ increase in cultured cortical astrocytes.

The trans-activator protein Tat of the human immunodeficiency virus type 1 (HIV-1) is regarded as an injurious molecule in the pathogenesis of HIV-1 associated encephalopathy (HIVE). We investigated the effects of Tat on neuroligand-induced intracellular Ca2+ increase in cultured astroglial cells. Rat cortical astrocytes, human glioblastoma cells and glial restricted precursor cells, from a human embryonic teratocarcinoma cell line, were incubated with recombinant Tat (100 ng/mL for 60 min) which induced a significant reduction of glutamate or ATP-induced intracellular Ca2+ increase ("glutamate response", "ATP response"). The reduction of the glutamate response was also observed following cell incubation with cell extracts of HeLa-T4+ cells transiently transfected with an expression plasmid coding for Tat. However, inactivation of the transcriptional trans-activity of Tat, by using a mutant form of Tat, as well as inhibition of de novo protein synthesis by cycloheximide abolished the effect on the glutamate response. This suggests that Tat acts upon induction of a so far unknown cellular gene whose gene product causes the reduction of glutamate responses. As the effect of Tat resembles the effect of TNFalpha on glutamate responses [Köller et al. (2001) Brain Res., 893, 237-243] which is locally released within the brains of HIVE patients, we also tested for synergistic effects of Tat and TNFalpha on the glutamate response. Low concentrations of Tat in combination with subthreshold concentrations of TNFalpha also elicited a marked reduction of astroglial glutamate responses. Our data suggest that Tat and TNFalpha, both by itself and synergistically, induce astroglial dysfunction.

A highly abundant transcript in adult murine cerebellar granule cells contains repetitive sequences homologous to L1.

Cloned cDNA from adult mouse cerebellum composed of subsequences homologous to the L1Md consensus sequence (long interspersed repetitive element "LINE" family of the mouse) hybridizes specifically with large nuclear poly(A)+RNAs that are highly concentrated in the murine and rat cerebellum. These homogeneous L1-related transcripts were localized in the mouse CNS by in situ hybridization with 3H- and biotin-labeled DNA probes. They were found to hybridize to cerebellar granule cells specifically.

The sequence complementarity between HIV-1 5' splice site SD4 and U1 snRNA determines the steady-state level of an unstable env pre-mRNA.

HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5' splice site upstream of the env open reading frame. To determine the role of this splice site in the 5'-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5' splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5' splice site, SD4, and the free 5' end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5' splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions -3 to +8 of the 5' splice site and all 11 nt constituting the single-stranded 5' end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4.