The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.     

Bacteriochlorophyll, Fatty-Acid, and Protein Synthesis in Relation to Thylakoid Formation in Mutant Strains of Rhodospirillum rubrum

Mutant strains of Rhodospirillum rubrum are isolated which are blocked in different stages of pigment synthesis. In these strains the morphogenesis of thylakoids and the pigment production are investigated. Concerning bacteriochlorophyll synthesis two groups of mutants are separable. The members of the first group synthesize bacteriochlorophyll. Some of these mutants excrete bacteriopheophytin. The strains of the second group are not able to synthesize bacteriochlorophyll. Members of both groups excrete bacteriochlorophyll precursors into the cultural medium. These pigments were identified by their spectral properties as Mg-2,4-divinyl-pheoporphyrin a(5)-monomethylester, pheophorbide a, and 2-devinyl-2-hydroxyethyl-pheophorbide a. Thylakoids are only formed by those strains which are able to synthesize bacteriochlorophyll. However, small amounts of bacteriochlorophyll can be produced without a concomitant thylakoid synthesis. The fatty-acid pattern in some mutants is modified quantitatively. However, the results do not indicate any correlation between disturbance of thylakoid morphogenesis and a deviation of fatty-acid composition. Fatty acids seem to have no special functions in thylakoid morphogenesis. The membranes of the mutants were isolated, split into protein subunits, and these were separated by disc electrophoresis. A characteristic protein pattern, first of all a high content of fraction E, is correlated with the ability to form thylakoids. In addition, all mutants which synthesize bacteriochlorophyll contain a fast-migrating membrane protein (zone G). The results suggest that the whole bacteriochlorophyll-protein complex is necessary for thylakoid formation.     

Hyperthermic treatment of chromomycosis with disposable chemical pocket warmers

A case of chromomycosis in which hyperthermia proved effective is reported. The patient was a 56-year-old male bean curd maker who, without any previous history of minor trauma, developed on the extensor side of the left upper arm an eczematous lesion that underwent gradual radial expansion. The lesion showed a well-defined, 7×10 cm infiltrated erythematous plaque with the central area healed and, at the upper and lower borders, adherent scales and crusts on the surface. Histological examination revealed granulomatous changes in the dermis, as well as sclerotic cells within giant cells and microabscesses. On culturing,Fonsecaea pedrosoi was isolated. The patient was treated with disposable chemical pocket warmers, which were secured over the lesion with a rather tight elastic bandage, so that they kept the affected area warm for 24 hours a day. After a month of such hyperthermic treatment, the erythema and infiltration had decreased considerably, and microscopic examination and culture of the crusts both yielded negative results. Examination of biopsy specimens of the lesion after the third month showed that it had cicatrized. The treatment was stopped after 4 months, and no relapse occurred. We also summarize the published results of local hyperthermic treatment of chromomycosis in Japan.     

Identification of HLA-B44 subtypes associated with extended MHC haplotypes

The HLA class I antigen B44 is found in each of two different extended major histocompatibility haplotypes (allele combinations of HLA-B, HLA-DR, and complement genes BF, C2, C4A, and C4B in linkage disequilibrium). Using isoelectric focusing, two variants of HLA-B44 were identified. The basic variant was found in all cell lines with the extended haplotype HLA-B44, DR7, FC31, and the acidic variant in all cell lines with the extended haplotype HLA-B44, DR4, SC30. The occurrence of each antigen variant with a unique extended haplotype explains previous observations concerning the nonrandom association of B44 variants with DR antigens.     

Genetic mechanisms of tumor-specific loss of 11p DNA sequences in Wilms tumor.

Wilms tumor, a common childhood renal tumor, occurs in both a heritable and a nonheritable form. The heritable form may occasionally be attributed to a chromosome deletion at 11p13, and tumors from patients with normal constitutional chromosomes often show deletion or rearrangement of 11p13. It has been suggested that a germinal or somatic mutation may occur on one chromosome 11 and predispose to Wilms tumor and that a subsequent somatic genetic event on the normal homologue at 11p13 may permit tumor development. To study the frequency and mechanism of such tumor-specific genetic events, we have examined the karyotype and chromosome 11 genotype of normal and tumor tissues from 13 childhood renal tumor patients with different histologic tumor types and associated clinical conditions. Tumors of eight of the 12 Wilms tumor patients, including all viable tumors examined directly, show molecular evidence of loss of 11p DNA sequences by somatic recombination (four cases), chromosome loss (two cases), and recombination (two cases) or chromosome loss and duplication. One malignant rhabdoid tumor in a patient heterozygous for multiple 11p markers did not show any tumor-specific 11p alteration. These findings confirm the critical role of 11p sequences in Wilms tumor development and reveal that mitotic recombination may be the most frequent mechanism by which tumors develop.     

The influence of dolichols on fluidity of mouse synaptic plasma membranes

Dolichols are isoprenologues which constitute an important component of biological membranes. However, an understanding of the effects of dolichols on the organization and dynamics of biological membranes has not been forthcoming. The experiments reported here are aimed at understanding the effects of dolichols on the physical properties of mouse brain synaptic plasma membranes. The effect of dolichols incorporated into mouse brain synaptic plasma membranes on fluorescent and electron spin resonance probes sensing the hydrophobic core differed from that of probes reporting closer to the surface of membrane bilayers. Dolichols significantly (P less than 0.01) lowered the polarization, limiting anisotropy, and order parameter of diphenylhexatriene in synaptic plasma membranes and liposomes extracted from synaptic plasma membranes, without changing the rotational relaxation time. Similarly, dolichol increased the fluidity reported by 16-doxylstearic acid in synaptic plasma membranes or liposomes extracted from synaptic plasma membranes. In contrast, dolichols exerted no effect on those properties for trans-parinaric acid or 5-doxylstearic acid in synaptic plasma membranes or liposomes derived therefrom. Dolichols can dramatically alter the structure and dynamics of lipid motion in synaptic plasma membranes and these effects are dependent on the location of the probe in the membrane.     

Deoxyribonucleoside triphosphate pools in mutagen sensitive mutants of Neurospora crassa

Deoxyribonucleoside triphosphate (dNTP) levels were measured in wild type Neurospora and nine mutagen-sensitive mutants, at nine different genes. Eight of these mutants are sensitive to hydroxyurea and histidine and show chromosomal instability, a phenotype which could result from altered levels of dNTPs. Two patterns were seen. Five of the mutants had altered ratios of dNTPs, with relatively high levels of dATP and dGTP and low levels of dCTP, but changes in the dTTP/dCTP ratio did not correlate with changes in spontaneous mutation levels. During exponential growth all but two of the mutants had small but consistent increases in dNTP pools compared to wild type. DNA content per microgram dry hyphae was altered in several mutants but these changes showed no correlation with the dNTP pool alterations.