A one semester undergraduate biochemistry laboratory experience is described for an understanding of recombinant technology from gene cloning to protein characterization. An integrated experimental design includes three sequential modules: molecular cloning, protein expression and purification, and protein analysis and characterization. Students perform the tasks of cloning, expression, purification, analysis, and characterization of small laccase (SLAC) from Streptomyces coelicolor. SLAC is an extremely robust well-characterized protein/enzyme, which serves as an ideal model for undergraduate teaching laboratories. Also, this goal-oriented research-like approach helps students to unite the concepts of biochemistry and molecular biology and appreciate the utility of the methods more effectively. A student assessment before and after the course demonstrated an overall increase in learning and enthusiasm on the topic of modern protein chemistry. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):172-181, 2018.
cause this protein has such a profound effect on the survivability of bacteria and is ubiquitous in bacterial systems, yet absent in higher eukaryotes, it has been recognized as an attractive target for mitigating bacterial infection (Milligan et al., 2007). Recent studies have shown that the Alr mutant strains of M. tuberculosis require D-alanine for growth, establishing the importance of Alr for the survival of this bacterium (Awasthy et al., 2012). In order to efficiently study and ultimately develop novel inhibitory agents a sufficient structural and functional profile of MT_Alr must be established. The structural characteristics of MT-Alr have already been documented (LeMagueres et al., 2005). In this paper, we report the cloning, overexpression, purification, and functional characterization of MT_Alr. These results will facilitate the design of novel anti-tuberculosis agents. MATERIALS AND METHODS All reagents and chemicals used as buffers and substrates of the highest available chemical grade were purchased from Sigma-Aldrich, Fisher, Acros, or Alfa Aesar. The buffers were prepared by mixing 0.1 M sodium phosphate monobasic, 0.1 M sodium phosphate dibasic, 0.1 M sodium carbonate, and 0.1 M sodium bicarbonate to pH values of 7.0, 7.5, 8.0, 9.0, 9.5, 10.0, and 10.5. The UV-Vis spectrophotometric data were collected using Multiskan GO (Fisher Scientific, Waltham, MA). Protein purifications were conducted using BioLogic DuoFlow (Bio-Rad, Hercules, CA). The genomic DNA for MT (ATCC 25177) was purchased from American Type Culture Collection (Rockville, MD). L-alanine dehydrogenase was cloned, expressed and purified in the lab from Streptomyces coelicolor (data not shown). Plasmid construction The genes encoding the Alr from MT was amplified via PCR from their corresponding genomic DNA with the primers (Integrated DNA Technologies, Coralville, IA) listed in Table 1. The PCR mixture (50 μL) contained 1 μL of 100 ng/μL template genomic
Streptomyces, the most important group of industrial microorganisms, is harvested in liquid cultures for the production of two-thirds of all clinically relevant secondary metabolites. It is demonstrated here that the growth of Streptomyces coelicolor A3(2) is impacted by the deletion of the alanine dehydrogenase (ALD), an essential enzyme that plays a central role in the carbon and nitrogen metabolism. A long lagphase growth followed by a slow exponential growth of S. coelicolor due to ALD gene deletion was observed in liquid yeast extract mineral salt culture. The slow lag-phase growth was replaced by the normal wild-type like growth by ALD complementation engineering. The ALD enzyme from S. coelicolor was also heterologously cloned and expressed in Escherichia coli for characterization. The optimum enzyme activity for the oxidative deamination reaction was found at 30 ° C, pH 9.5 with a catalytic efficiency, k cat /K M , of 2.0 ± 0.1 mM-1 s-1. The optimum enzyme activity for the reductive amination reaction was found at 30 ° C, pH 9.0 with a catalytic efficiency, k cat /K M , of 1.9 ± 0.1 mM-1 s-1 .
Gantzer muscles are anatomical variations found within the flexor compartment of the forearm. The Gantzer muscle typically arises from the flexor digitorum superficialis (FDS) and inserts into the flexor pollicis longus (FPL) or flexor digitorum profundus (FDP). The presence of this muscle can cause various compressive injuries, including neuropathy of the median or anterior interosseous nerve and impingement of the ulnar, common, or anterior interosseous arteries. Despite its high prevalence, the Gantzer muscle is often excluded from the differential for acute compartment syndrome and should be further considered during treatment and surgical management. This study expanded upon a previous assessment of cadaveric specimen forearms to determine further the prevalence of origin, insertion, and innervation of Gantzer muscles, as well as possible compressions induced from impingement by the anomalous muscle. A total of 288 limbs were dissected in 144 cadavers. The Gantzer muscle was found in 54.6% (148:271) of limbs. Therefore, in general, the Gantzer muscle is more likely to be present than absent. This report also details common and uncommon origins and insertions of Gantzer variants. Support or Funding Information T. Walley Williams Summer Anatomy Research Fellowship; WV Research Challenge Fund [HEPC.dsr.17.06]
This report documents the incidental finding of a horseshoe kidney identified during routine dissection in a gross anatomy course. The anatomic examination demonstrated malrotated bilateral kidneys, which were fused at the inferior poles. Histologic examination revealed no significant pathologic change; however, the fused portion was shown to have an intact cortex and collecting system. Furthermore, the renal vasculature was anomalous with multiple accessory and convoluted arterial vessels. An aortic aneurysm was also noted just superior to the renal fusion. Operative procedures and diagnostic imaging performed in the region of such an anatomical variation may be complicated by unanticipated vascular patterns. Therefore, special attention should be paid to vasculature in the setting of a horseshoe kidney. Support or Funding Information T. Walley Williams Summer Anatomy Research Fellowship;WV Research Challenge Fund [HEPC.dsr.17.06]
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