Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells.

Structure and assembly of immature HIV.

The major structural components of HIV are synthesized as a 55-kDa polyprotein, Gag. Particle formation is driven by the self-assembly of Gag into a curved hexameric lattice, the structure of which is poorly understood. We used cryoelectron tomography and contrast-transfer-function corrected subtomogram averaging to study the structure of the assembled immature Gag lattice to approximately 17-A resolution. Gag is arranged in the immature virus as a single, continuous, but incomplete hexameric lattice whose curvature is mediated without a requirement for pentameric defects. The resolution of the structure allows positioning of individual protein domains. High-resolution crystal structures were fitted into the reconstruction to locate protein-protein interfaces involved in Gag assembly, and to identify the structural transformations associated with virus maturation. The results of this study suggest a concept for the formation of nonsymmetrical enveloped viruses of variable sizes.

Chemogenetic ON and OFF switches for RNA virus replication.

Therapeutic application of RNA viruses as oncolytic agents or gene vectors requires a tight control of virus activity if toxicity is a concern. Here we present a regulator switch for RNA viruses using a conditional protease approach, in which the function of at least one viral protein essential for transcription and replication is linked to autocatalytical, exogenous human immunodeficiency virus (HIV) protease activity. Virus activity can be en- or disabled by various HIV protease inhibitors. Incorporating the HIV protease dimer in the genome of vesicular stomatitis virus (VSV) into the open reading frame of either the P- or L-protein resulted in an ON switch. Here, virus activity depends on co-application of protease inhibitor in a dose-dependent manner. Conversely, an N-terminal VSV polymerase tag with the HIV protease dimer constitutes an OFF switch, as application of protease inhibitor stops virus activity. This technology may also be applicable to other potentially therapeutic RNA viruses.

Genetic analysis and gene expression of human immunodeficiency virus.

Ten years after the initial description of acquired immune deficiency syndrome, its causative agent, the human immunodeficiency virus, remains the subject of intense scientific interest. Recent research has focused on the detailed analysis of the molecular principles governing gene expression and virion formation and on the cause of immune system dysfunction. Within the past year, considerable progress has been made regarding both the role of the regulatory proteins and the mechanism by which they function, and the determinants of cell tropism and of virion formation.

Cryo-electron microscopy reveals ordered domains in the immature HIV-1 particle.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS and the subject of intense study. The immature HIV-1 particle is traditionally described as having a well ordered, icosahedral structure made up of uncleaved Gag protein surrounded by a lipid bilayer containing envelope proteins. Expression of the Gag protein in eukaryotic cells leads to the budding of membranous virus-like particles (VLPs). RESULTS: We have used cryo-electron microscopy of VLPs from insect cells and lightly fixed, immature HIV-1 particles from human lymphocytes to determine their organization. Both types of particle were heterogeneous in size, varying in diameter from 1200-2600 A. Larger particles appeared to be broken into semi-spherical sectors, each having a radius of curvature of approximately 750 A. No evidence of icosahedral symmetry was found, but local order was evidenced by small arrays of Gag protein that formed facets within the curved sectors. A consistent 270 A radial density was seen, which included a 70 A wide low density feature corresponding to the carboxy-terminal portion of the membrane attached matrix protein and the amino-terminal portion of the capsid protein. CONCLUSIONS: Immature HIV-1 particles and VLPs both have a multi-sector structure characterized, not by an icosahedral organization, but by local order in which the structures of the matrix and capsid regions of Gag change upon cleavage. We propose a model in which lateral interactions between Gag protein molecules yields arrays that are organized into sectors for budding by RNA.

Multiple copies of the Mason-Pfizer monkey virus constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner.

Retroviral gene expression requires nuclear export and translation of incompletely spliced RNA. In the case of human immunodeficiency virus (HIV), this is facilitated by the viral Rev protein binding to its cognate RNA response element (RRE), while other retroviruses contain constitutive transport elements (CTE) binding to cellular factors. These CTE can substitute for the HIV-1 Rev/RRE system, albeit with reduced efficiency. Here, we show that multimeric copies of the CTE restore HIV-1 protein expression to levels comparable to or higher than Rev/RRE in various cell lines from different species. We suggest that multimerization of export factors is important for CTE function, as reported for Rev. CTE function was not affected when the element was displaced from its natural position close to the poly(A) signal, while insertion of an intron into the 3'-untranslated region (3'-UTR) severely reduced CTE activity. In this case, cytoplasmic RNA degradation was observed, which may be mediated by nonsense-mediated RNA decay. In contrast, Rev-dependent gene expression was insensitive to an intron in the 3'-UTR. Finally, we show that the putative CTE-binding protein RNA helicase A is not specifically translocated into the cytoplasm upon overexpression of CTE-containing RNA.

Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the "fireman's grip" hypothesis.

Aspartic proteinases share a conserved network of hydrogen bonds (termed "fireman's grip"), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the "fireman's grip," Thr26 of HIV-1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV-1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the "fireman's grip" interaction, was virtually inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer-dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV-1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the "fireman's grip" is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the "fireman's grip" is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme.

Polyprotein processing in picornavirus replication.

The primary translation product of the picornavirus genome is a single large protein which is processed to the mature viral polypeptides by progressive, co- and post-translational cleavages. Replication of the picornaviruses is thus entirely dependent upon the proteolysis of viral precursor proteins. In poliovirus, two virus-encoded proteinases have been identified that catalyze all but the final cleavage of the viral polyprotein. The final processing event, maturation of the virion polypeptide VPO, appears to occur by an unusual autocatalytic serine proteinase-like mechanism. Proteolytic processing of viral precursor proteins is basically similar in all picornaviruses, but recently it has become clear that there are also important differences between these viruses. Understanding of the processing events in picornavirus replication may ultimately lead to the discovery of specific inhibitors of the viral enzymes that could prove clinically useful as anti-viral agents.

Expression in Escherichia coli and purification of human immunodeficiency virus type 1 capsid protein (p24).

Capsid protein (p24;CA) of human immunodeficiency virus type 1 (HIV-1) was synthesized in Escherichia coli strain BL21 (DE3) using a plasmid encoding a truncated HIV-1 gag/pol gene. The plasmid, which contained a mutation in the frameshift region, expressed viral proteinase (PR), a pol gene product, in the gag reading frame, resulting in efficient processing of mature CA and other gag-related products. The expressed CA is soluble, recognized by monoclonal antibodies directed against HIV CA and has an N-terminal sequence identical to that of CA purified from HIV. Purification was done under mild conditions where coexpressed HIV PR retained enzymatic activity. Milligram quantities of 90% pure CA protein were obtained after chromatography on DEAE cellulose followed by facilitated aggregation of the CA in the unbound fraction. The precipitated CA was readily dissolved in low ionic strength aqueous buffer. Gel exclusion chromatography results indicated that, in solution, CA existed in oligomeric form.

Coreceptor requirements of primary HIV type 1 group O isolates from Cameroon.

HIV-1 group O has its epicenter in Cameroon and neighboring countries and is responsible for 3 to 5% of all HIV infections in this region. It is believed that HIV-1 group O was introduced into the human population by a separate cross-species transmission, occurring independently of the HIV-1 (group M and group N) and HIV-2 transmissions. We have studied the coreceptor requirements of 12 primary HIV-1 O-type isolates from individuals with different clinical symptoms. Only 2 of these 12 viruses showed a syncytium-inducing phenotype after infection of primary peripheral blood mononuclear cells (PBMCs) and were infectious for the T cell line C8166. These isolates used CXCR4 as a coreceptor for entry, whereas the remaining isolates used only CCR5 efficiently. One isolate was able to use BOB and CCR8 as coreceptors in addition to CXCR4. All group O isolates tested were efficiently inhibited by SDF-1 or RANTES, the natural ligands of CXCR4 and CCR5, respectively. These results indicate that CXCR4 and CCR5 are the principal coreceptors for HIV-1 O-type viruses. Most of the HIV-1 group O isolates studied were derived from patients at later stages of the disease. Although HIV-1 group O and group M infections do not differ in their pathogenesis, the studied isolates did not evolve to use a broad range of coreceptors as described for HIV-1 group M and HIV-2.

Parameters influencing measurement of the Gag antigen-specific T-proliferative response to HIV type 1 infection.

We have analyzed factors that might influence the in vitro quantitation of the T-proliferative response to HIV-1 Gag antigens, a common and increasingly used clinical measurement of helper T cell function in the context of HIV-1 infection. We have compared the rate and extent of T cell proliferation in freshly prepared and previously frozen PBMC samples, and have concluded that frozen cells can be used successfully; we have assessed whether the suppression of any HIV-1 replication in the PBMC cultures affects the extent of T cell proliferation; we have studied which forms of the Gag antigens are the most efficient at inducing T cell proliferation. From the latter experiments, we conclude that Gag proteins that include p17, and perhaps also p7, sequences flanking the central p24 capsid protein, are better stimulants than proteins that comprise only p24 sequences.

Chimerism in cattle through microsurgical aggregation of morulae.

A cattle chimera was produced by combining four halves of two parent embryos of different breeds (Brown-Swiss x Braunvieh plus Holstein-Friesian x Holstein-Friesian) in one zona pellucida. Parent embryos in the 32-cell morula stage were recovered non-surgically, were bisected, and the combined four halves were transferred non-surgically to recipient heifers. Chimerism of coat colour was used as evidence. Combining of only two half embryos from different parents resulted in five pregnancies carried to term but none of the calves born was a chimera.

Epidemiological study on factors influencing fertility indices in Israeli dairy herds.

The objective of this study was to determine feeding factors connected with differences in the fertility of Israeli Kibbutz dairy herds. In an epidemiological case-control survey, data from 30, low-fertility Kibbutz dairy herds having a mean overall conception rate of 35% in multiparous cows were compared with the data from 30, high-fertility Kibbutz dairy herds having a mean overall conception rate of 48% in multiparous cows. Nutritional factors accounted for 67% of the differences between low-fertility and high-fertility herds in the overall conception rate of multiparous cows, while only 4.8% could be related to the body condition during the dry period. Among the factors which occurred more frequently in the low-fertility than in the high-fertility herds were 1) a higher average protein density and lower energy/protein ratio was fed during lactation and 2) a single feeding group was maintained for all lactating cows. 3) There were phytoestrogens in the silage or alfalfa hay, fed during lactation. 4) Faulty dry period was instituted, which was defined as the presence of at least one of the following three practices: a) the daily feed was above 3 kg of high lactation mix; b) more than 15 Mcal of net energy per day was given during the first part of the dry period; c) more than 30% of the cows were obese during the dry period. Three or more risk factors were found in one high-fertility herd and in 20 low-fertility herds. This finding emphasizes the importance of identifying and removing risk factors as a possible means for improving the reproductive performance of herds.

An approach to successful freezing of demi-embryos derived from day-7 bovine embryos.

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.

Possibilities and limits of breeding for immune responsiveness.

Studies on defence mechanisms in mice and farm animals are demonstrated. The results indicate that selective breeding might become a more important tool in disease control. But intensive research work is required before a "General Disease Resistance Index" can be used in animal breeding. More common efforts of research workers in veterinary medicine and animal breeding are necessary.

[Legal protection of industry and animal welfare--legal and ethical problems]. / Gewerblicher Rechtsschutz und Tierschutz--Rechtliche und ethische Probleme.

The first animal patent for the so called "Harvard-onco-mouse" has been widely discussed in the media and in public respectively. The opinion of veterinarians on animal patents should be based as far as possible on objective information, such as the legal situation and consideration of ethical discussion. The paper contains information on the following subjects: definition, aim and purpose of patents, patentability of microorganisms and genes, animal patents, animal rights and patents, ethics and animal patents.

[Heritability of strabismus convergens with exophthalmos in cattle]. / Zur Erblichkeit von Strabismus convergens mit Exophthalmus beim Rind.

Six pedigrees including 107 animals of the breed German Brown Swiss were available to test for a single gene conditioning for convergent strabismus with exophthalmus. The regressive logit models of the segregation analysis showed that a major gene model with additively acting genes explained the segregation of affected animals in the pedigrees in the best way. Additionally, polygenic and environmental effects might be of importance in the occurrence of convergent strabismus with exophthalmus.

[Computer-assisted analysis of pressure distribution on cattle claws]. / Computergestützte Analyse von Druckverteilungsmessungen an Rinderklauen.

A newly developed measuring device was applied to quantify in 5 first and 5 second lactation cows of the breed German Black and White the pressure distribution underneath the claws of one front limb. The pressure distribution of cows was recorded five times in intervals of four weeks. At each measuring date each had to undergo three tests. Procedures for image processing were applied in analysis of pressure distributions. The average pressure per sqcm was about 19 N, the maximum pressure measured was 59 N per sqcm in first lactating cows and 56 N per sqcm in second lactation cows. Factors were developed which characterize the patterns of pressure distributions. Especially the "gradient factor" seems to be well suited to recognize inhomogeneous pressure distributions.

[Effect of the composition of the ration during the dry period on fertility and milk production of Holstein cows in Israel]. / Einfluss der Zusammensetzung der Ration in der Trockenzeit auf die Fruchtbarkeit und Milchleistung von Israeli-Holstein-Kühen.

In each for of kibbutzin herds located in Israel the dry cows were divided in two feeding groups. The experimental group, in totally 266 cows, obtained only oat, barley or wheat hay ad libitum, whereas the control group (n = 253) was fed additionally mix or silage up to four kg dry matter. The cows in the control group responded in the following lactation with a 1.3 kg higher average FCM per day (p = 0.02) and with a 1.7 kg higher maximum FCM (p = 0.01) in comparison to the experimental group. The differences between the experimental and control group in A. I.-parameters days open, conception rate on first insemination and percent problem cows were rather small. However, the frequency of retained placenta and ovary cysts was significantly (p less than 0.05) higher in the control group than in the experimental group. In one herd the influence of body condition between the end of lactation, dry period and 50-80 days p.p. as well as the change of body weight between the end of lactation and calving on milk production and fertility could be evaluated. A decrease of one body condition score unit between the dry period and 50-80 days p.p. was associated with an increase of FCM by 2.01 kg (p less than 0.01), a decrease of the conception rate by 21% (p less than 0.05) and an elongation of the days open by 23.6 days (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)