Bilayer stabilizing peptides and the inhibition of viral infection: antimeasles activity of carbobenzoxy-Ser-Leu-amide

A number of carbobenzoxy-dipeptide-amides raise the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine (stabilizes the bilayer). The potency of the peptides in stabilizing the bilayer phase is Z-Tyr-Leu-NH₂= Z-Gly-Phe-NH₂>Z-Ser-Leu-NH₂>Z-Gly-Leu-NH₂>Z-Gly-Gly-NH₂. A linear correlation was found between the respective HPLC retention time parameterk for the peptide and the slope of the bilayer stabilization curve determined with model membranes by differential scanning calorimetry. One dipeptide, Z-Ser-Leu-NH₂, reduces measles virus cytopathic effect (CPE) in Vero cells. The mechanism by which this peptide reduces the CPE is not known, although some peptides which raise the bilayer to hexagonal phase transition temperature of phospholipids inhibit membrane fusion.Abbreviations Z carbobenzoxy - DEPE dielaidoylphosphatidylethanolamine - DSC differential scanning calorimetry - HPLC high pressure liquid chromatography - CPE cytopathic effect To whom correspondence should be addressed.     

Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes

Summary When a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS⁺ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS⁺ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS⁺, LacZ⁺ transformants with a Trp^– mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC⁺ transformants which, with a low frequency, had a LacZ^– phenotype. These latter transformants had also lost the AmdS⁺ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.     

Identification of somatic hybrids in plant protoplast fusions with monoclonal antibodies to plasma-membrane antigens

Monoclonal antibodies generated by immunization with a plasma-membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. were used in combination with fluoresceinor rhodamine-labeled goat anti-mouse immunoglobulins to identify heterokaryons in protoplast fusion procedures. Antibody labeling did not inhibit callus formation nor plantlet regeneration. The antibodies are non-invasive and surface labeling provides clear optical discrimination of true heterokaryons from unfused aggregates as well as from parental protoplasts and homokaryons. Labeling is stable throughout fusion and hence by pre-labeling parental protoplast populations the strategy is both versatile and of general applicability.     

Electrical fusion of protoplasts from sycamore mutants and hybrid selection on hypoxanthine-aminopterin-thymidine (HAT) medium

Summary An electrical fusion method has been used to form somatic hybrids between protoplasts of two mutant cell lines of sycamore tissue culture cells. Both mutants will not grow in a hypoxanthine-aminopterin-thymidine (HAT) medium. It was possible to select the fused hybrids from homospecific fusion products and nonfused protoplasts by the use of HAT medium. In this way the viability and regeneration of the fused cells during the first few weeks of culture could be evaluated. An electron microscopic examination of the fusion process showed that it occurred at a series of points along the surface of the plasmalemma. Cytoplasmic bridges between the two cells were formed separated by vesicles which later dispersed to give complete cytoplasmic continuity between the cells.     

Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability

Summary An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.     

Loading patterns and jaw movements during mastication in Macaca fascicularis: a bone-strain, electromyographic, and cineradiographic analysis

Rosette strain gage, electromyography (EMG), and cineradiographic techniques were used to analyze loading patterns and jaw movements during mastication in Macaca fascicularis. The cineradiographic data indicate that macaques generally swallow frequently throughout a chewing sequence, and these swallows are intercalated into a chewing cycle towards the end of a power stroke. The bone strain and jaw movement data indicate that during vigorous mastication the transition between fast close and the power stroke is correlated with a sharp increase in masticatory force, and they also show that in most instances the jaws of macaques are maximally loaded prior to maximum intercuspation, i.e. during phase I (buccal phase) occlusal movements. Moreover, these data indicate that loads during phase II (lingual phase) occlusal movements are ordinarily relatively small. The bone strain data also suggest that the duration of unloading of the jaw during the power stroke of mastication is largely a function of the relaxation time of the jaw adductors. This interpretation is based on the finding that the duration from 100% peak strain to 50% peak strain during unloading closely approximates the half-relaxation time of whole adductor jaw muscles of macaques. The EMG data of the masseter and medial pterygoid muscles have important implications for understanding both the biomechanics of the power stroke and the external forces responsible for the "wishboning" effect that takes place along the mandibular symphysis and corpus during the power stroke of mastication. Although both medial pterygoid muscles reach maximum EMG activity during the power stroke, the activity of the working-side medial pterygoid peaks after the balancing-side medial pterygoid. Associated with the simultaneous increase of force of the working-side medial pterygoid and the decrease of force of the balancing-side medial pterygoid is the persistently high level of EMG activity of the balancing-side deep masseter (posterior portion). This pattern is of considerable significance because the direction of force of both the working-side medial pterygoid and the balancing-side deep masseter are well aligned to aid in driving the working-side lower molars across the upper molars in the medial direction during unilateral mastication.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relationships between growth at the sutures of the rabbit calvarium

The present study was designed to elucidate the relationships between growth increments at the cranial vault sutures in rabbits. Thirteen male New Zealand white rabbits were followed regularly from age 31 to 142 days using a roentgen stereophotogrammetric system. Spherical tantalum markers were implanted into the nasal, frontal, and parietal bones, and implant stability was checked at each stereo examination. Problems with instability were encountered only in the nasal bones. Registered growth rates conformed to our previous investigations. High correlations were observed between the following areas; the coronal suture to the frontonasal suture, the first principal component of the neurocranial suture group to the frontonasal suture, and the principal component of the craniofacial suture group to the coronal suture. Remaining relationships demonstrated dispersion to various extents. The findings indicate that there seems to exist a basic mutual dependence between neural and facial skeletal growth, as well as complex covariations between the various sutures of the rabbit calvarium.     

Selective inactivation of heterokaryons of Candida albicans by anaerobiosis

Heterokaryons (hets), but not monokaryons of Candida albicans die when grown anaerobically on minimal medium. Their rates of inactivation increase with decreases in growth temperatures from 37°C to 25°C. At 10°C, however, anaerobiosis is not lethal and suppresses the inactivation which normally occurs among hets cultured aerobically at that temperature. Killing of hets by anaerobiosis can be altered significantly by certain exogenously provided amino acids or intermediates of oxidative respiration. Aspartic acid alone promotes inactivation whereas alanine, glutamic acid or lysine individually have no effects. However, glutamate and lysine combined afford slight protection against inactivation while aspartate and glutamate combined, with or without lysine, are highly protective: the activity of the aspartate-glutamate combination is completely negated by the addition of alanine. Other common amino acids have no effects on het responses to anaerobiosis other than the ability, when combined, to relieve the antagonism of alanine for the aspartate-glutamate combination. Anaerobic survivals are also enhanced by oxalacetic acid or -ketoglutaric acid, and even more so by a combination of these two intermediates. The resistances to inactivation elicited by the oxalacetate -ketoglutarate or aspartate-glutamate combinations are not additive. These relationships are interpreted to signify that inactivation of hets by anaerobic growth is largely, if not exclusively, due to depletion of their oxalacetic acid and -ketoglutaric acid contents for amino acid biosyntheses, and the unique inability of het cells to replenish those keto acids upon subsequent return to aerobic conditions. The observations are consistent with previous indications that mitochondria formed by hets are functionally abnormal.     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.