Sample-hold technique for analysis of respiratory gas composition at high breathing frequencies

A gas sampling device is described for continuous monitoring of respiratory gas composition that is applicable to experimental conditions when the breathing frequency is very high (greater than 2 Hz) and the response time of conventional gas analyzers becomes a critical limiting factor. The system utilizes the principle of discontinuous gas collection at any selected point of the respiratory cycle facilitated by ultraspeed piezoelectric valves and includes provision for sample-hold characteristics. Two distinct modes of operation are supported. In phase-locked mode gas sampling is synchronous with breathing frequency. In scanning mode gas collection is asynchronous with breathing frequency. Phase-locked mode may be used for continuous monitoring of end-tidal gas concentrations, whereas scanning mode is intended for assessing the gas concentration profile throughout the respiratory cycle. The system may be applied to steady breathing encountered in mechanical ventilation at high frequency or during quasi-steady breathing observed in panting animals. Combined with a respiratory mass spectrometer, the system has been used for measurement of gas concentrations in alveolar gas mixtures at breathing frequencies ranging from 3 to 30 Hz that were otherwise not amenable to rapid measuring techniques.     

Twelve loci form a continuous linkage map for human chromosome 18

We have constructed a primary genetic map of human chromosome 18 consisting of 11 DNA markers and one serological marker (JK). Two of these loci define highly polymorphic VNTR systems. The markers define a continuous genetic linkage map of 97 cM in males and 205 cM in females; female genetic distances in a panel of 59 three-generation families were consistently about twice those observed in males. The high odds in support of the linear order of the markers on this recombination map, and the extent of coverage of chromosome 18, indicate that this map will permit efficient linkage studies of human genetic diseases that may be segregating on chromosome 18 and will provide anchor points for development of high-resolution maps for this chromosome.     

Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor α

Summary Recombinant tumor necrosis factor (rTNF; optimal dose 1000 U/ml) significantly increased the density of epidermal growth factor receptor (EGF-R) in three of four glioma cell lines in culture as determined by binding analysis of anti-EGF-R monoclonal antibody (mAb) 425. Since enhancement of EGF-R expression by rTNF- was inhibited when cells were treated with the protein synthesis inhibitor cycloheximide, the effects of rTNF may be protein-synthesis-dependent. The dose of rTNF that was optimal for up-regulation of EGF-R on glioma cells did not inhibit the growth of these cells.¹²⁵I-labeled mAb 425 lysed glioma cells in culture following its internalization into the cells. After glioma cells had been treated with rTNF, the growth-inhibitory effects of the mAb were significantly enhanced, probably a reflection of the increase in EGF-R density on the tumor cell surfaces. The rTNF effects were specific to the EGF-R and did not affect unrelated glioma-associated antigens. In our previous clinical trials,¹²⁵I-labeled mAb 425 showed immunotherapeutic effects in glioma patients. The present study provides the basis for considerations of combined immunotherapy of glioma patients with¹²⁵I-labeled mAb 425 and rTNF.     

Molecular analysis of aberrations of Xp and Yq

Summary Three cases of Y chromosomal aberrations were studied using a panel of Y-specific DNA sequences from both Yp and euchromatic Yq. One case was a phenotypic male fetus with a Y-derived marker chromosome. The short arm of this chromosome was intact, but most of its long arm was missing. The second case had a 46,Xyq- karyotype with portions of euchromatic Yq, including the spermatogenesis region, missing. The third case was a phenotypic female with a 46,XXp+ karyotype. The extra material on the Xp+ chromosome was derived from the heterochromatic, and part of the euchromatic, portion of Yq. Application of X-specific DNA sequences demonstrated that the distal portion of the short arm of the translocation X chromosome was deleted (Xpter—p22.3). The three examples demonstrate the importance of diagnostic DNA analysis in cases of marker chromosomes, and X and Y chromosomal aberrations. In addition, the findings in the patients facilitate further deletion mapping of euchromatic Yq.     

Absence of Turner stigmata in a 46,XYp-female

A 46,XY female patient with streak gonads and a large deletion of Yp is described. The deletion included the Y chromosomal genes SRY, ZFY, and RPS4Y. The patient did not display any Turner stigmata, such as webbing of the neck, cardiac or other abnormalities. The findings argue against an important role of RPS4Y in the prevention of Turner stigmata in males and are consistent with a role of SRY in testis differentiation in humans.     

Molecular basis of organ-specific selection of viral variants during chronic infection.

Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were greater than 99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F----L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.     

Species-specific diversity among simian immunodeficiency viruses from African green monkeys

The prevalence, natural history, and genetic characteristics of simian immunodeficiency virus (SIV) infections in most feral African monkey species are presently unknown, yet this information is essential to elucidate their origin and relationship to other simian and human immunodeficiency viruses. In this study, a combination of classical and molecular approaches were used to identify and characterize SIV isolates from West African green monkeys (Cercopithecus sabaeus) (SIVagm isolates). Four SIVagm viruses from wild-caught West African green monkeys were isolated and analyzed biologically and molecularly. Amplification, cloning, and sequencing of a 279-bp polymerase fragment directly from uncultured peripheral blood mononuclear cells was facilitated by the use of nested polymerase chain reaction. The results indicated that West African green monkeys are naturally infected with SIVs which are closely related to East African SIVagm isolates. However, structural, antigenic, and genetic differences were observed which strongly suggest that the West African green monkey viruses comprise a phylogenetically distinct subgroup of SIVagm. These findings support our previous hypothesis that SIVagm viruses may have evolved and diverged coincident with the evolution and divergence of their African green monkey host. In addition, this study describes a polymerase chain reaction-based approach that allows the identification and molecular analysis of divergent SIV strains directly from primary monkey tissue. This approach, which does not depend on virus isolation methods, should facilitate future studies aimed at elucidating the origins and natural history of SIVs in feral African green monkey populations.     

Low-temperature accumulation of alcohol dehydrogenase-1 mRNA and protein activity in maize and rice seedlings

Low-temperature stress was shown to cause a rapid increase in steady-state levels of alcohol dehydrogenase-1 message (Adh1) and protein activity (ADH1) in maize (Zea mays) (B37N, A188) and rice (Oryza sativa) (Taipei 309, Calmochi 101) seedlings. Maize roots and rice shoots and roots from 7-day seedlings shifted to low temperature (10°C) contained as much as 15-fold more Adh1 mRNA and 8-fold more ADH1 protein activity than the corresponding tissues from untreated seedlings. Time-course studies showed that these tissues accumulated Adh1 mRNA and ADH1 activity severalfold within 4- to 8-hour, levels plateaued within 20 to 24 hours, and remained elevated at 4 days of cold treatment. Within 24 hours of returning cold-stressed seedlings to ambient temperature, Adh1 mRNA and ADH1 activity decreased to pretreatment levels. Histochemical staining of maize and rice tissue imprints showed that ADH activity was enhanced along the lengths of cold-stressed maize primary roots and rice roots, and along the stems and leaves of rice shoots. Our observations suggest that short-term cold stress induces Adh1 gene expression in certain plant tissues, which, reminiscient of the anaerobic response, may reflect a fundamental shift in energy metabolism to ensure tissue survival during the stress period.     

Regulation of chloroplast metabolism in leaves: Evidence that NADP-dependent glyceraldehydephosphate dehydrogenase,but not ferredoxin-NADP reductase,controls electron flow to phosphoglycerate in the dark-light transition

P₇₀₀ is rapidly, but only transiently photooxidized upon illuminating dark-adapted leaves. Initial oxidation is followed by a reductive phase even under far-red illumination which excites predominantly photosystem (PS) I. In this phase, oxidized P₇₀₀ is reduced by electrons coming from PSII. Charge separation in the reaction center of PSI is prevented by the unavailability of electron acceptors on the reducing side of PSI. It is subsequently made possible by the opening of an electron gate which is situated between PSI and the electron acceptor phosphoglycerate. Electron acceptors immediately available for reduction while the gate is closed corresponded to 10 nmol · (mg chlorophyll)^–1 electrons in geranium leaves, 16 nmol · (mg chlorophyll)^–1 in sunflower and 22 nmol · (mg chlorophyll)^–1 in oleander. Reduction of NADP during the initial phase of P₇₀₀ oxidation showed that the electron gate was not represented by ferredoxin-NADP reductase. Availability of ATP indicated that electron flow was not hindered by deactivation of the thylakoid ATP synthetase. It is concluded that NADP-dependent glyceraldehydephosphate dehydrogenase is completely deactivated in the dark and activated in the light. The rate of activation depends on the length of the preceding dark period. As chloroplasts contain both NAD- and NADP-dependent glyceraldehydephosphate dehydrogenases, deactivation of the NADP-dependent enzyme disconnects chloroplast NAD and NADP systems and prevents phosphoglycerate reduction in the dark at the expense of NADPH and ATP which are generated by glucose-6-phosphate oxidation and glycolytic starch breakdown, respectively.Abbreviations Chl chlorophyll - P₇₀₀ electron donor pigment in the reaction center of photosystem I Cooperation of the Institute of Botany of the University of Würzburg with the Institute of Astrophysics and Atmospheric Physics of the Estonian Academy of Sciences in Tartu was supported by the Deutsche Forschungsgemeinschaft and the Estonian Academy of Sciences. This work was performed within the Sonderforschungsbereich 251 of the University of Würzburg.