Amino acid compostion of walls from single and filamentous cells of Clostridium acidiurici

Gaffar, Abdul (Brigham Young University, Provo, Utah), David R. Terry, and Richard D. Sagers. Amino acid composition of walls from single and filamentous cells of Clostridium acidiurici. J. Bacteriol. 91:1618-1624. 1966.-The walls from single and filamentous cells of Clostridium acidiurici were shown to contain 11 amino acids: aspartic acid, serine, glutamic acid, proline, d-alanine, glycine, valine, methionine, valine, leucine, phenylalanine, and lysine. In the walls from cells grown at 37 C, d-alanine was the amino acid present in largest quantity, but in the walls from cells grown at 44 C there was a 50% reduction in the d-alanine content while the levels of the other amino acids were unchanged. Filamentous cells grown at 44 C, then brought to 37 C and transferred to fresh medium, fragmented into short cells within 30 min. Alanine racemase activity was the same in extracts from cells grown at both 37 and 44 C, suggesting that this enzyme was not the major controlling factor in the low content of d-alanine in filaments grown at 44 C. Spent medium from cultures grown at 44 C contained a significant amount of d-alanine, whereas there was no evidence of this amino acid in the spent medium from cultures grown at 37 C.     

N-acetyltransferase polymorphism among northern Sudanese

Interindividual and interethnic differences in allele frequencies of N-acetyltransferase (NAT2) single nucleotide polymorphisms (SNPs) are responsible for phenotypic variability of adverse drug reactions and susceptibility to cancer. We genotyped the seven NAT2 common SNPs in 127 randomly selected unrelated northern Sudanese subjects using allele-specific and RFLP polymerase chain reaction (PCR) based methods. Molecular genotyping was enough to designate alleles for 41 individuals unambiguously, whereas 63 individuals' alleles were inferred from haplotypes previously described. In the remaining 23 individuals, however, the phase of the SNPs could not be decided because of multiple SNP heterozygotes. Using computational methods in the HAP and Phase programs, we confirmed the inferred alleles of the 62 individuals and predicted the remaining 23 ambiguous alleles. Twelve NAT2 alleles were identified. Four alleles coded for rapid acetylators (18%), and eight alleles coded for slow acetylators (82%). Two genotypes coded for rapid acetylation (3.9%), 10 for intermediate acetylation (27.6%), and 13 for slow acetylation (68.5%). The G191A African SNP and the G857A predominantly Asian SNP were each detected at a low frequency of 3.1%. The combination of molecular and computational analysis was useful in resolving ambiguous genotypes of NAT2 in multiple SNP heterozygotes. Among the northern Sudanese the SNPs associated with slow acetylation are more prevalent than in Caucasians and Asians. This and other African studies are suggestive of an African origin for NAT2-associated polymorphism.     

Filament formation in Clostridium acidiurici under conditions of elevated temperatures

Terry, David R. (Brigham Young University, Provo, Utah), Abdul Gaffar, and Richard D. Sagers. Filament formation in Clostridium acidiurici under conditions of elevated temperatures. J. Bacteriol. 91:1625-1634. 1966.-Vegetative cells of Clostridium acidiurici, when grown at temperatures up to 42 C, are straight rods varying from 2.5 to 4 mu in length. When grown at 43 C, the cells show a definite tendency to elongate, and, when grown at 44 C, filaments are formed, often exceeding 500 mu in length. Only an occasional cross wall is apparent in the heat-induced long forms, but as the temperature is lowered they readily form cross walls and fragment into short, single cells. Chromatin material is distributed in evenly spaced clusters throughout the length of the filaments. The filaments grown at 44 C are gram-negative, whereas cells grown at 37 C are gram-positive. However, filament formation and gram-negativity apparently are not due to magnesium deficiency, since the gram-negative filaments are formed in concentrations of magnesium ranging from 10(-6) to 10(-2)m. The rapid transition from filaments to single cells upon lowering the temperature from 44 to 37 C suggests that the temperature-related repression of the cross wall-forming system is a phenotypic response rather than the selection of specific mutants which produce the observed phenomena.     

Further studies on a human lung tumor-associated antigen. Comparison of antigens from different tumors.

A human lung tumor-associated antigen was purified to homogeneity from a crude cell-free extract of a human lung adenocarcinoma using standard biochemical procedures. In order to facilitate monitoring the recovery of antigen, trace amounts of previously purified and radioiodinated antigen from another lung tumor were added to the crude extract. The purified antigen was a glycoprotein and contained sialic acid. The antigen had a molecular weight of 76,000 and appeared to contain three subunits, each with a molecular weight of 25,000. The antigen had the following physical properties: Stokes radius, 39.4 A; S20,w, 4.24 S; D20,w, 5.15 x 10(-7) cm2 S-1; and a frictional ratio of 1.40. In addition, the purified, radioiodinated antigen retained complete immune reactivity since it could be quantitatively precipitated with specific immune serum. All of these properties were in close agreement with the properties of another antigen which was purified from a separate human lung tumor. Thus, it appeared from the biochemical and immunochemical criteria presented in this report that a common and identical antigen was isolated from two distinct human lung tumor extracts.     

Chromatographic methods for large-scale preparation of immunoglobulin G2a monoclonal antibodies Lym-1 and TNT-1 F(ab')2 fragments

Lym-1 and TNT-1 are two murine immunoglobulin G_2a monoclonal antibodies (MAbs) which have been used for clinical trials in cancer patients. This paper describes methods for large-scale preparation of F(ab')₂ fragments from 50 mg to 4 g of MAbs Lym-1 and TNT-1. Digestion of MAbs with pepsin was optimized and performed at pH 3.8, a pepsin/antibody ratio of 1:250, and 3–4 h of incubation at 37°C. The F(ab')₂ fragments were purified by tandem column procedures using fast protein liquid chromatography. Quality control analyses of the products included protein purity, isoelectric point, immunoreactivity, and endotoxin level. The results revealed that the chromatographic procedures are practical, simple, and effective, and can be used to produce gram quantities of clinical-grade F(ab')₂ fragments for the diagnosis of cancer in patients.     

Body image disturbance and surgical decision making in egyptian post menopausal breast cancer patients

Background

Proximal major limb amputations due to malignant tumors have become rare but are still a valuable treatment option in palliation and in some cases can even cure. The aim of this retrospective study was to analyse outcome in those patients, including the postoperative course, survival, pain, quality of life, and prosthesis usage.

Methods

Data of 45 consecutive patients was acquired from patient's charts and contact to patients, and general practitioners. Patients with interscapulothoracic amputation (n = 14), shoulder disarticulation (n = 13), hemipelvectomy (n = 3) or hip disarticulation (n = 15) were included.

Results

The rate of proximal major limb amputations in patients treated for sarcoma was 2.3% (37 out of 1597). Survival for all patients was 42.9% after one year and 12.7% after five years. Survival was significantly better in patients with complete tumor resections. Postoperative chemotherapy and radiation did not prolong survival. Eighteen percent of the patients with malignant disease developed local recurrence. In 44%, postoperative complications were observed. Different modalities of postoperative pain management and the site of the amputation had no significant influence on long-term pain assessment and quality of life. Eighty-seven percent suffered from phantom pain, 15.6% considered their quality of life worse than before the operation. Thirty-two percent of the patients who received a prosthesis used it regularly.

Conclusion

Proximal major limb amputations severely interfere with patients' body function and are the last, albeit valuable, option within the treatment concept of extremity malignancies or severe infections. Besides short survival, high complication rates, and postoperative pain, patients' quality of life can be improved for the time they have remaining.     

Purification and Some Biological Properties of Asparaginase from Azotobacter vinelandii

Asparaginase was found in the soluble fraction of cells of Azotobacter vinelandii, and its activity remained the same during growth of the organism in a nitrogen-free medium. The specific activity and the yield of A. vinelandii increased twofold in the presence of ammonium sulfate. Within limits, the temperature (30 to 37°C) and pH (6.5 to 8.0) of the medium showed little effect on the levels of enzyme activity. The enzyme was purified to near homogeneity by standard methods of enzyme purification, including affinity chromatography, and had optimum activity at pH 8.6 and 48°C. The approximate molecular weight was 84,000. The apparent K_m value for the substrate was 1.1 × 10^-4 M. Metal ions or sulfhydryl reagents were not required for enzyme activity. Cu²⁺, Zn²⁺, and Hg²⁺ showed concentration-dependent inhibition, whereas amino and keto acids had no effect on the enzyme activity. Asparaginase was stable when incubated with rat serum and ascites fluid. The enzyme had no effect on the membrane of sheep erythrocytes and did not inhibit the incorporation of radioactive precursors into deoxyribonucleic acid, ribonucleic acid, and protein in Yoshida ascites sarcoma cells. Asparaginase activity was not detected in the tumor cells.     

Continuous cultures of tomato and citron roots in vitro

Summary Initial trials with tomato-root cultures disclosed the desirability of employing a gently agitated liquid medium containing iron in the chelated form. For the normal cultivars “Ace” and “Tropic”, subcultures were best achieved by utilizing sectors that possessed one or more newly emerged laterals. Continuous cultures of a nonlateral-forming tomato mutant, “Diageotropica”, and of citron were accomplished by subculturing tips of the elongating primary roots. The tomato roots were cultured in White's medium with the Fe₂(SO₄)₃ replaced by 0.03 mM NaFeEDTA. Sustained growth of citron-root tips necessitated the use of a medium containing Murashige and Skoog salts, 7.5% sucrose, 100 mg per I each of citric acid and thiamine HCl, and 5000 mg per li-inositol. The success with citron-root cultures was extendable to all cultivars ofC. medica L., but not to otherCitrus species relatives. Both citron and “Diageotropica” root cultures manifested undiminished elongation through repeated subcultures; but neither produced laterals in response to any cultural treatments. Research was supported in part by National Science Foundation Grant OIP75-10390 and Elvenia J. Slosson Fellowship in Ornamental Horticulture.