Insignificant ß-lactamase activity of human serum albumin: no panic to nonmicrobial-based drug resistance.

UNLABELLED: Recently, it was speculated that human serum albumin (HSA) possesses ß-lactamase activity and could contribute to nonmicrobial-based antibiotic resistance, owing to its ability to hydrolyse the ß-lactam ring of nitrocefin. Moreover, the putative ß-lactamase activity of HSA has been shown to increase significantly in the presence of environmental pollutants (1-naphthol and 2-naphthol). It was postulated that HSA could also cleave the ß-lactam ring of clinically significant antibiotics. We studied the ß-lactamase activity of HSA on clinically significant antibiotics of cephalosporin group in the presence of environmental pollutants by determining specific activity, enzyme kinetics and minimum inhibitory concentrations (MIC). The specific activity of HSA on various cephalosporins was found to be 1181-34 550 times lower than that observed for recombinant CTX-M-15 (used as positive control). The catalytic efficiency (kcat /Km ) of HSA on nitrocefin hydrolysis was 126·7 times lower than that of recombinant CTX-M-15, and it has increased only 2- to 3-folds in the presence of environmental pollutants. Moreover, cephalosporins were not hydrolysed by HSA under experimental conditions. The MIC data also showed that HSA is incapable of hydrolysing cephalosporins. The study concludes that HSA is inefficient to cleave antibiotics of cephalosporin group and hence does not contribute to nonmicrobial-based antibiotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Earlier reports showed that human serum albumin (HSA) possesses ß-lactamase activity, owing to its ability to cleave nitrocefin, and thus contributes to antibiotic resistance. Also, its ß-lactamase activity is augmented when exposed to pollutants. As nitrocefin is not an antibiotic of clinical use, the conclusion drawn does not represent a true scenario and is misleading. Our results showed that HSA is inefficient in cleaving nitrocefin as compared to a true ß-lactamase (CTX-M-15) and is practically inactive on cephalosporin antibiotics even in the presence pollutants. The findings showed that HSA-mediated hydrolysis of ß-lactam antibiotics does not contribute to antibiotic resistance.

c-myc mRNA is down-regulated during myogenic differentiation by accelerated decay that depends on translation of regulatory coding elements.

Murine C2C12 myoblasts induced to differentiate into multinucleated myotubes decrease their levels of c-myc mRNA 3-10-fold through posttranscriptional mechanisms that recognize regulatory elements contained in protein-coding sequences in exons 2 and 3 of the mRNA. To determine the mechanism by which these elements mediate c-myc mRNA down-regulation, we examined the regulation of mutant MYC and human beta-globin-MYC fusion mRNAs. Regulation of mRNAs containing MYC exon 2 or 3 is abolished by insertion of an upstream termination codon indicating that regulatory function depends on their translation. Exploiting this translation dependence, we show that pharmacologic inhibition of translation with cycloheximide abolishes the down-regulation of regulated MYC and globin-MYC mRNAs and induces their levels in differentiating C2C12 cells. We exclude the possibility that this induction in mRNA levels results from cycloheximide effects on transcription or processing of parts of the RNA other than the regulatory elements, leading to the conclusion that cycloheximide induction results from mRNA stabilization. We show that the magnitude of cycloheximide induction can be used to estimate turnover rates of mRNAs whose decay is translation-dependent. By using cycloheximide inducibility to examine turnover rates of MYC and globin-MYC mRNAs, we show that the MYC exon 2 and exon 3 regulatory elements, but not MYC 3'-untranslated region or chloramphenicol acetyltransferase coding sequences, mediate accelerated mRNA decay in differentiating, but not undifferentiated, C2C12 cells. We show that these regulatory elements must be translated to confer accelerated mRNA decay and that increased turnover occurs in the cytoplasm and not in the nucleus. Finally, using cycloheximide induction to examine mRNA half-lives, we show that mRNA turnover is increased sufficiently by mechanisms targeting the exon 2 and 3 regulatory elements to account for the magnitude of c-myc mRNA down-regulation during differentiation. We conclude from these results that c-myc mRNA down-regulation during myogenic differentiation is due to translation-dependent mechanisms that target mRNAs containing myc exon 2 and 3 regulatory elements for accelerated decay.

Identification of sequences in c-myc mRNA that regulate its steady-state levels.

The level of cellular myc proto-oncogene expression is rapidly regulated in response to environmental signals and influences cell proliferation and differentiation. Regulation is dependent on the fast turnover of c-myc mRNA, which enables cells to rapidly alter c-myc mRNA levels. Efforts to identify elements in myc mRNA responsible for its instability have used a variety of approaches, all of which require manipulations that perturb normal cell metabolism. These various approaches have implicated different regions of the mRNA and have led to a lack of consensus over which regions actually dictate rapid turnover and low steady-state levels of c-myc mRNA. To identify these regions by an approach that does not perturb cell metabolism acutely and that directly assesses the effect of a c-myc mRNA region on the steady-state levels of c-myc mRNA, we developed an assay using reverse transcription and PCR to compare the steady-state levels of human myc mRNAs transcribed from two similarly constructed myc genes transiently cotransfected into proliferating C2C12 myoblasts. Deletion mutations were introduced into myc genes, and the levels of their mRNAs were compared with that of a near-normal, reference myc mRNA. Deletion of most of the myc 3' untranslated region (UTR) raised myc mRNA levels, while deletion of sequences in the myc 5' UTR (most of exon 1), exon 2, or the protein-coding region of exon 3 did not, thus demonstrating that the 3' UTR is responsible for keeping myc mRNA levels low. Using a similar reverse transcription-PCR assay for comparing the steady-state levels of two beta-globin-myc fusion mRNAs, we showed that fusion of the myc 3' UTR lowers globin mRNA levels by destabilizing beta-globin mRNA. Surprisingly, fusion of the protein-coding region of myc exon 3 also lowered globin mRNA steady-state levels. Investigating the possibility that exon 3 coding sequences may play some other role in regulating c-myc mRNA turnover, we demonstrated that these sequences, but not myc 3' UTR sequences, are necessary for the normal posttranscriptional downregulation of c-myc mRNA during myoblast differentiation. We conclude that, while two elements within c-myc mRNA can act as instability determinants in a heterologous context, only the instability element in the 3' UTR regulates its steady-state levels in proliferating C2C12 cells.

Histomorphometric classification of postmenopausal osteoporosis: implications for the management of osteoporosis.

AIMS: To define and group static and dynamic iliac crest histomorphometric parameters in women with established osteoporosis. METHODS: Iliac crest biopsy specimens from 146 white women were sectioned undecalcified and examined using image analysis. RESULTS: Five distinct groups were defined on the basis of histomorphometric changes in cell function: group 1, decreased osteoblastic and osteoclastic activity; group 2, decreased osteoblastic and increased osteoclastic activity; group 3, increased osteoblastic and osteoclastic activity; group 4, no bone surface cell activity; and group 5, apparently normal osteoblastic and osteoclastic activity. CONCLUSIONS: Five distinct subgroups of patients with postmenopausal osteoporosis can be defined based on changes in bone cell function. Defining cellular dysfunction in this way may be important for tailoring treatment regimens to the needs of individual patients.

Age related histomorphometric changes in bone in normal British men and women.

AIMS: To define the iliac crest histomorphometry of static variables in 234 individuals aged 16-100 years (91 men, 143 women) and of dynamic variables in 84 individuals aged 19-94 years (33 men, 51 women) from the North West of England. METHODS: Iliac crest biopsy specimens were sectioned, undecalcified, and examined using image analysis. RESULTS: The decrease in the quantity of cortical and trabecular bone and the connectivity of trabecular bone was more pronounced in women than men. This was associated with a reduction in bone formation and increased bone resorption which was greater in women at both the tissue and cellular level. Some of these histomorphometric differences first became evident at the natural menopause, and therefore provide clues as to the cause of the high prevalence of osteoporosis in postmenopausal women. CONCLUSIONS: These results show an age and sex dependent variation both in static and dynamic parameters, which differ, in some respects, from other studies and confirm the need for large regional studies to provide a database of normal morphometric results for a specific population.