Penicillin-binding proteins of protoplast and sporoplast membranes of Streptomyces griseus strains

Membrane-bound penicillin-binding proteins (PBPs) of two Streptomyces griseus strains that sporulate well in liquid and solid medium have been investigated during the course of their life-cycle. The PBP patterns were analyzed by sodium dodecylsulphate polyacrylamide-gel electrophoresis and fluorography. One strain (No. 45 H) has only a single band (mol wt: 27,000) in early log phase, and two additional PBPs of higher mol wt (69,000 and 80,000) in the late log phase. The other strain (No. 2682) possessed two bands with mol wts 27,000 and 38,000 which did not change during its vegetative phase. In strain No. 2682, a new PBP with a mol wt of 58,000 appeared in spore membranes while one of those (mol wt 38,000) present in mycelial membranes disappeared. Our results suggest that appearance of the new PBP in the spore may be associated with the sporulation process. The major PBP band (mol wt: 27,000) present in all stages of the life cycle of these strains, may be characteristic of S. griseus while the other PBPs reflect certain stages of the life cycle. A new method was developed for the production of spore protoplasts by consecutive enzymatic treatments.Abbreviation PBP penicillin-binding protein     

The origin and ultrastructural characteristics of corticotropin-releasing factor (CRF)-immunoreactive nerve fibers in the posterior pituitary of the rat

Summary A fine network of corticotropin-releasing factor (CRF)-immunopositive fibers was found in the posterior lobe of the pituitary of the rat. The intermediate and distal lobes were free of CRF-immunoreactivity. Varicose, terminal-like axons were frequently observed around capillary vessels. Surgical isolation of the paraventricular nuclei resulted in a complete disappearance of CRF-immunoreactive fibers from the posterior lobe. CRF-immunopositive fibers show the general characteristics of peptidergic axons. These ultrastructural observations support the idea that CRF is secreted into capillary vessels.     

Propionyl-CoA elimination may be a rate-determining step of selective cleavage of sterol side chain

Summary The effect of propionate on the growth and 4-androstene-3, 17-dion (AD) yield was investigated in cultures of Mycobacterium sp. NRRL B-3805 growing in minimal medium containing -sitosterol as substrate for selective side chain cleavage. Although the addition of propionate (PA) resulted in a concentration-dependent inhibition of growth at the beginning of fermentation, cultures started to grow in the presence of 0.1% of propionic acid reached an AD concentration 38% higher than the cultures growing in the absence of propionate during two day cultivation. After three days of incubation, the AD yields in cultures containing 0, 0.1 and 0.2% propionate at the inoculation were 68, 79 and 73%, while the protein levels were 2.01, 2.11 and 2.60 mg/ml, respectively. Our data showed that the positive effect of PA on the AD production from sterols by Mycobacterium sp. NRRL B-3805 could be explained by the induction of the enzymes of the methylmalonate pathway. The activity of propionyl-CoA carboxylase was about 30% higher in the crude extracts from the induced cultures growing in minimal medium, after 20 hours of growth, than in those from the controls (18.2 and 14.1 mU/mg, respectively, using propionyl-CoA as substrate). The distribution of the acid-stable ¹⁴C-radioactivity which built into methylmalonate, succinate and fumarate indicated that methylmalonyl-CoA mutase was also induced. Our data demonstrated that elimination of the toxic propionyl-CoA released from the side chain of the sterol is likely the rate-determining step of the AD production, at least at the beginning of the process.     

Agrobacterium mediated genetic transformation and plant regeneration via organogenesis and somatic embryogenesis from cotyledon leaves in eggplant (Solanum melongena L. cv. ‘Kecskeméti lila’)

Summary Novel and efficient protocols for plant regeneration and genetic transformation from longitudinally-halved cotyledons ofin vitro raised seedlings in eggplant (Solanum melongena L.) are described. After co-cultivation withAgrobacterium vectors harboring neomycin phosphotransferase (nptll) as selectable marker, transgenic plantlets were regenerated on selective media containing 100 mg/l kanamycin. Transformants were recovered from embryogenic calli induced by 4 mg/l-naphthaleneacetic acid (NAA), and from organogenic calli induced by the addition of 2 mg/l zeatin plus 0.01 mg/l NAA. Nineteen independent transgenic lines were grown to maturity. The structural integrity, expression and sexual transmission of the introduced genes for neomycin phosphotransferase and ß-glucuronidase (gus) were investigated.     

1,6-Diamino-2,5-anhydro-1,6-dideoxy-dl-glucitol and some derivatives thereof

1,6-Diamino-2,5-anhydro-1,6-dideoxy-dl-glucitol dihydrochloride and some derivatives were synthesized from 3,5-di-O-acetyl-1,6-dibromo-1,6-dideoxy-d-mannitol. Introduction of the 2,5-anhydro ring and subsequent replacement of the terminal bromine atoms by azide gave low yields of the diazide; therefore, a reverse reaction-sequence was applied. The azido groups were reduced with hydrogen sulfide-pyridine, and the amino groups formed were methylated by using formaldehyde-formic acid and subsequently treating with borohydride. According to ¹³C-n.m.r. investigations, the symmetrically substituted, 2,5-anhydroglucitol derivatives are present mainly in the ⁴T₃ [“north” (N) type of twist] conformation, whereas the analogous l-iditol derivatives mainly adopt the ³T₄ [“south” (S)] type. The different quaternary salts obtained on methylation of the corresponding 1,6-bis(dimethylamino)derivatives with methyl iodide (aiming at the structure of epi-muscarine) showed no muscarine-like, biological activity.     

The role of N-acyl groups in the inhibitory activity of LH-RH analogues

Inhibitory analogues of luteinizing hormone-releasing hormone (LH-RH) were prepared with formyl-D-Trp¹, acetyl-D-Trp¹, valeryl-D-Trp¹, tartaryl-D-Trp¹, diacetyl-tartaryl-D-Trp¹, acetyl-Gly¹, and acetyl-Sar¹ successively replacing the position one in the analogue [D-Trp¹, D-p-Cl-Phe², D-Trp³, D-Phe⁶, D-Ala¹⁰]-LH-RH. The formyl-D-Trp¹ and acetyl-D-Trp¹ analogues yielded 100% blockade of ovulation at the 10 μg dose; the others were less potent and inhibited ovulation at the 50 μg dose. The inhibitory potency seems to correlate with the polarity of the acyl group.     

Phylogenetic continuum indicates “Galaxies” in the protein universe: Preliminary results on the natural group structures of proteins

Summary The markedly nonuniform, even systematic distribution of sequences in the protein universe has been analyzed by methods of protein taxonomy. Mapping of the natural hierarchical system of proteins has revealed some dense cores, i.e., well-defined clusterings of proteins that seem to be natural structural groupings, possibly seeds for a future protein taxonomy.The aim was not to force proteins into more or less man-made categories by discriminant analysis, but to find structurally similar groups, possibly of common evolutionary origin. Single-valued distance measures between pairs of superfamilies from the Protein Identification Resource were defined by two ²-like methods on tripeptide frequencies and the variable-length subsequence identity method derived from dot-matrix comparisons. Distance matrices were processed by several methods of cluster analysis to detect phylogenetic continuum between highly divergent proteins.Only well-defined clusters characterized by relatively unique structural, intracellular environmental, organismal, and functional attribute states were selected as major protein groups, including subsets of viral and Escherichia coli proteins, hormones, inhibitors, plant, ribosomal, serum and structural proteins, amino acid synthases, and clusters dominated by certain oxidoreductases and apolar and DNA-associated enzymes.The limited repertoire of functional patterns due to small genome size, the high rate of recombination, specific features of the bacterial membranes, or of the virus cycle canalize certain proteins of viruses and Gram-negative bacteria, respectively, to organismal groups.