Chemical modification of the hydroxylase of soluble methane monooxygenase gives one form of the protein with significantly increased thermostability and another that functions well in organic solvents

Proteolysis of the hydroxylase component of soluble methane monooxygenase (MMO) with trypsin yielded a protein which retained 50% activity in a standard MMO assay. In an H₂O₂-driven assay, in which H₂O₂ replaced two of the protein components, NADH and O₂ used in the standard assay, the proteolysed hydroxylase retained full activity for ethane, propane and propene, but had a 2–3 fold increase with methane as substrate. Several crosslinking reagents have been tested for their ability to stabilise the proteolysed form of the hydroxylase. Using polyoxyethylene bis(imidazolyl carbonyl) (M_r 3350) as the crosslinking agent, increased thermostability of the hydroxylase was observed. Activated methoxypolyethylene glycol (M_r 5000) was used to modify the hydroxylase which was now soluble in organic solvents as well as water and could be activated by H₂O₂. The glycol-modified hydroxylase functioned well in organic solvents in the catalysis of propene oxidation.     

Insecticidal effectiveness ofMammea Americana (Guttiferae) extracts on larvae ofDlabrotica Virgifera Virgifera (Coleoptera:Chrysomelidae) andTrichoplusia Ni (Lepidoptera:Noctuidae)

Seed and leaf extracts ofMammea americana (mamey apple) have a historical use as a biopesticide with the active components previously characterized. We reexamined the utility of this natural bioinsecticide in light of existing sources of material as a by-product of the fruit processing industry. Our results addDiabrotica virgifera virgifera andTrichoplusia ni to the list of insects which are susceptible to the insecticidal ingredients ofM. americana and confirms earlier reports of activity againstBlatella germanica, Periplaneta americana, andPlutella xylostella. We report LD5Os for crude hexane extracts ofM. americana leaves and seeds againstT. ni. These materials represent renewable sources of bioinsecticides for agriculture, and should regenerate interest in coumarin-type compounds for novel pesticidal action.     

“Pharmacognosy”! What’s that? you spell it how?

In this autobiographical sketch, the author discusses the development of his interest in the biological sciences, crediting his father, his first employer, his high school science teacher, and his college pharmacognosy professor with initially shaping his career. His early work on ergot alkaloid biosynthesis and subsequently, together with students and colleagues, on the toxic constituents of basidiomycetes is detailed. This is followed by comments on his developing interest in the therapeutic utility of herbs and phytomedicinals. A concern with the beneficial use of such products stemmed largely from observations made during sabbatical leaves and frequent travel in Germany. The importance of such botanicals (not currently recognized as drugs in the United States) in our developing health-care system is emphasized. The author concludes his comments by thanking his wife, his teachers, his students, and his many colleagues and friends for their unstinting assistance and support during his entire career.     

Evaluation of Enterolert for enumeration of enterococci in recreational waters.

Enterolert (IDEXX Laboratories Inc., Westbrook, Maine), a semiautomated, most probable number method for enumeration of enterococci, was compared with the standard membrane filter method by parallel testing of 138 marine and freshwater recreational bathing water samples. No statistically significant difference and a strong linear correlation were found between methods. Culturing of 501 Enterolert test wells resulted in false-positive and false-negative rates of 5.1 and 0.4%, respectively. Less time for setup, incubation (24 versus 48 h), and reading of Enterolert permits more efficient monitoring of recreational bathing areas.     

A group A streptococcal Enn protein potentially resulting from intergenomic recombination exhibits atypical immunoglobulin-binding characteristics

The gene encoding the Enn protein (enn) of the M untypeable group A streptococcal (GAS) strain 64/14 was amplified by polymerase chain reaction, cloned into the expression vector pJLA602 and expressed in Escherichia coli DH5α. Unlike other GAS–Enn proteins, which exhibit IgA-binding activity, the recombinant Enn enn64/14 protein reacted preferentially with human IgG₃. The 1050 bp open reading frame comprising the enn64/14 gene was completely sequenced. The region of the gene encoding the signal peptide and the C-terminus exhibited >95% homology to corresponding sections of other enn genes. The region of enn64/74 encoding the N-terminus of the mature Enn protein was found to be highly homologous to the corresponding section of the gene encoding the M-like protein of GAS serotype M9 (emmL9). The reoombinant protein encoded by emmL9 was found to react with all four human IgG subclasses. About 30% of the 1152bp open reading frame of emmiL9 encoding the N-terminus was found to display >90% homology to the corresponding section of enn64/14 but was <50% homologous in the remainder of the gene sequence. The functional analysis of the subcloned N-terminal section of emmL9 demonstrated a polypeptide exhibiting selective binding to human IgG₃. These findings suggested that enn64/14 was a hybrid gene formed by recombination of an enn gene and an emmL9 gene. The putative recombinational event could have Involved a set of flanking 7bp direct repeats. Since enn64/14 and emmL9 are genes from different phylogenetic lineages of GAS, this report provides evidence that intergenomic recombinations between different types of GAS genes can occur and could lead to hybrid proteins with unique Ig-binding characteristics.