Mayo Clinic and CSIR sign MoU

Mayo Clinic and CSIR , India signed a formal agreement to collaborate on significant  areas of research. Prof Robert Rizza, M.D., Mayo Executive Dean for Research and  Prof Samir Brahmachari, Ph.D., Director General, Council of Scientific and Industrial Research (CSIR), India signed a memorandum of understanding on 12th Oct 2011 to mutually work on such topics as drug, device and biomarker studies relating to heart disease; chemical biology and applied genomics; and innovation in metabolomics. 

“This is an exciting and positive step, based on our continuing discussions and initial collaborations,” says Robert Rizza, M.D., Mayo Executive Dean for Research. “We look forward to working with our colleagues in India to advance health care for everyone.”

“We welcome this new relationship with Mayo Clinic, with which we share many strong interests in medical and technology research as well as health care deliveryat affordable cost,” says Samir Brahmachari, Ph.D., Director General of CSIR. 

“We are engaging the best minds to help our research teams improve our fundamental understanding of disease and develop new more effective therapies for our patients,” says Eric Wieben, Ph.D., Mayo’s associate dean for external collaborations. “Strategic international research collaborations, such as this with the CSIR, India will help us achieve those goals." 

Source : http://www.csir.res.in/CSIR/External/Heads/aboutcsir/announcements/MayoPressRelease_051211.pdf

Mysteries of the Bacterial L-Form: Can Some of Them Be Unveiled?

by Hans H. Martin

L-forms are bacterial variants with defective cell walls and irregular growth and multiplication. They arise after peptidoglycan, the exoskeleton of the bacterial cell wall, has been either degraded by bacteriolytic enzymes, or its biosynthesis has been disturbed by antibiotics and other inhibitors, or by defect mutations in essential genes for cell wall synthesis. L-forms with different degrees of wall defects can arise. International experts, headed by nobelist Sidney Brenner, recognized the need to distinguish between entirely cell wall-less protoplasts, surrounded only by a cytoplasmic membrane, andspheroplasts with residual, fragile cell walls. L-forms were discovered in 1935 by Emmy Klienebergerand subsequently described by many authors (examples here and here). Much interest in L-forms arose from their assumed but still unconfirmed roles as concealed pathogens and as survivors of antibiotic action. They are also useful tools for the study of basic mechanisms of cell biology, such as cell division. Yet, as justly deplored in a recent review, L-forms are still "unfamiliar to many microbiologists" and are often regarded "with scepticism." One hears complaints about the unusually labor-intensive and time-consuming process of L-form isolation and cultivation, and the uncertain outcome. However, in my experience, this can be overcome by patient determination.

A special type of resistance to antibiotics, which we studied extensively in my lab, is reflected in the ability of the Gram-negative bacterium Proteus mirabilis to evade the inhibitory action of penicillin and other β-lactam antibiotics by growing as spheroplast- L-forms (references here and here). It is important to note that the most common resistance mechanism, inactivation of β-lactam antibiotics by β-lactamase, is not involved in this phenomenon.

 

Figure 2. Free penicillin-binding proteins (PBPs)
of P. mirabilis labelled with [35S] penicillin G. (S)
from L-form spheroplasts grown in the presence
of 120 mg/l penicillin G. (B) from normal bacteria.

β-Lactam antibiotics are known as specific inhibitors of the biosynthesis of peptidoglycan. They inactivate the peptidoglycan-transpeptidases (see chapter "β-Lactam-induced Proteus L-forms" by Ghuysen, J.M., Nguyen-Distèche, and Rousset, A.) multiple enzymes that form cross-linkages between peptide side chains of adjacent glycan strands in different stages of peptidoglycan biosynthesis. The enzymes are present in all bacteria as a group of membrane-bound proteins of different size but with the common property of covalently binding penicillin and other β-lactam antibiotics. Thus, all are termed penicillin-binding proteins(Fig. 2). In the peptidoglycan of Gram-negative bacteria, they connect the D-center of meso-diamino-pimelic acid in position 3 (meso-DAP³) of one side chain to D-alanine in position 4 (D-ALA⁴) of an adjacent side chain and are therefore called DD-peptidoglycan-transpeptidases (Fig. 3, upper).

 

Peptide crosslinkages in peptidoglcan catalysed
by DD- (β-lactam-sensitive) and by LD- (β-lactam-
insensitive) peptidoglycan-transpeptidases.

In many bacteria, inactivation of the transpeptidases results in the loss of strength and shape of the cell wall with subsequent lysis—a lethal event. Not so in Proteus mirabilis and some other Gram-negative bacteria. Here, fragile spheroplasts survive and can be propagated indefinitely in the presence of high concentrations of β-lactams. These spheroplasts carry disorganized components of the outer membrane, pili and flagella on their surface (Fig. 4), but their viability is evident. Under the phase contrast microscope one can watch them using their flagella to clumsily tumble around. Also, upon transfer to β-lactam-free medium they revert to normal, rod-shaped bacteria.

One would think that after prolonged life with penicillin the L-form spheroplasts would have lost peptidoglycan from their damaged cell walls. However, very early on Otto Kandler and colleagues found all the normal components of peptidoglycan wall-bound in the "unstable" (i.e. spheroplast-type) Proteus L-form. In my lab, we then isolated shape-defective but still macromolecular peptidoglycan from L-form spheroplasts (Fig. 1). We also found that cell walls of spheroplasts and normal Proteus bacteria contained nearly equal amounts of peptidoglycan, with comparable quantities of the typical amino sugar and amino acid components. Most surprising, the degree of peptide crosslinkage was similar in peptidoglycans from L-form spheroplasts and normal cells (references here and here).

 

Figure 4. L-Form-spheroplasts of P. mirabilis grown in liquid shake
culture with 120 mg/l penicillin G. (a) Phase contrast 2,300X.
(b) Electron micrograph of Pt-Ir-shadowed spheroplast. 12,000X.

Our early suspicion was that, against expectations, some of the known peptidoglycan-trans-peptidases might continue to function during L-form growth. A comparison of penicillin-binding proteins in L-form spheroplasts and Proteus bacteria seemed to support this notion. Spheroplasts, harvested from their high penicillin growth medium, contained various amounts of free penicillin-binding proteins (Fig. 1). Some of these had been shownto form very short-lived complexes with penicillin and might thus be reactivated and resume their transpeptidase activity. However, we could not confirm the function of such a mechanism at that time, and the problem remained unsolved.

Almost 25 years later, with the discovery of a novel peptidoglycan cross-linking enzyme for a β-lactam-resistant transpeptidation pathway, a new type of resistance to β-lactam antibiotics became known. β-Lactam-insensitive peptidoglycan-transpeptidases were first found in β-lactam-resistant Enterococcus faecium and then in E. coli and P. mirabilis. In E. coli the enzymes form unusual peptide crosslinkages between the L-center of meso-diaminopimelic acid in position 3 (meso-DAP³) of one side chain and the D-center of meso-diaminopimelic acid in position 3 (meso-DAP³) of an adjacent side chain. They are therefore called LD-peptidoglycan-transpeptidases. The resulting meso-DAP³─meso-DAP³ crosslinkages (Fig. 3, lower) are present as minor components ofE. coli peptidoglycan, in addition to the "normal" meso- DAP³─D-ALA⁴ crosslinkages (reference).

Two explanations for the β-lactam resistant growth of the P.mirabilis spheroplast-L-form seem now possible. During L-form growth in the presence of penicillin, the remaining activity of some of the DD-peptidoglycan-transpeptidases may suffice to synthesize a peptidoglycan with shape-defects, but with a sufficient amount of the usual (meso-DAP³)─(D-ALA⁴) crosslinkages, Or, alternatively, the L-form may construct an exclusively or preferentially meso-DAP³─meso-DAP³ crosslinked peptidoglycan with the help of the β-lactam-insensitive LD-peptidoglycan-transpeptidases.

Both mechanisms may ensure the conservation of essential structures for the repair of an intact peptidoglycan, after the eventual escape into a β-lactam-free environment allows the return to the normal shape of the bacterial cell. Hopefully, the described models may serve as clues towards obtaining the still missing structural data and help us to understand the strange enduring bacterial life with penicillin in the L-form.

On the Continuity of Biological Membranes

by Franklin M. Harold

 

The complex world of cell membranes. Source.

Thirty years ago, Günter Blobel of the Rockefeller University published a short paper entitled Intracellular Protein Topogenesis, which laid the conceptual foundations for our understanding of how cells build membranes. To serve their functions, peripheral and integral proteins must be inserted into the right membrane with the correct orientation, and most of the article focused on the manner in which this may be achieved. But it also underscored two startling implications of the proposed procedure: first, that every membrane must be derived from a pre-existing membrane; and second, that all extant biological membranes are descendants of the plasma membrane of the first primordial cell.

Blobel’s article became a classic, and spawned a small industry concerned with the molecular mechanisms that target proteins to the recipient membrane and then either translocate or insert them. In a nutshell, the information that specifies a nascent protein’s disposition is contained in its sequence. One segment of that sequence recognizes a receptor protein embedded in the target membrane, commonly part of the translocon; other segments specify whether the amino acid chain is to be taken clear across the membrane or inserted, and with what orientation. Membrane proteins may be processed concurrently with their translation, or after their production is complete. In prokaryotic cells the proteins are produced and handled directly; in eukaryotic cells they are first inserted into the membrane of the endoplasmic reticulum, and then transferred to their target membrane by cargo vesicles. The details can be found in textbooks of molecular cell biology. What concerns us here is the inference that membrane heredity is a fundamental principle of biology. A functional membrane, studded with a particular set of enzymes, transport carriers and receptors, can never be generated de novo; it must arise from a pre-existing membrane, either by modification (for example, the membranes that surround bacterial spores) or else by growth and division or vesiculation. Moreover, since proteins will only be inserted after interaction with a complementary receptor (and that includes the receptor protein itself), a growing “genetic” membrane propagates its own kind.

The idea that membranes are inherited was by no means novel in 1980; cytologists had been musing on it for two decades. But it was quite another matter to assert that it must be so, that “omnis membrana e membrana.”

Biology is notoriously so riddled with exceptions that such a sweeping generalization is bound to raise eyebrows: never? Indeed, possible exceptions do crop up from time to time. If this prospect piques your curiosity, take a look at the work of G.H. Kim and his colleagues (here and here), which describes the astonishing capacity of naked blobs of algal cytoplasm to reconstitute a membrane and resume growth. Blobs endowed with nuclei and a sample of organelles survive transiently in the absence of a plasma membrane (sic!), construct a temporary one made of polysaccharides, and finally produce a proper membrane made of lipids; how they do this is quite unknown and would provide a nice test of Blobel’s dictum. In years of reading I have never come across an authentic example of a membrane made afresh, and a query to readers of this blog elicited no response. Like the second law of thermodynamics, the verity that membranes must be grown rather than made rests not on proof positive, but on the absence of any known exceptions.

Even though membrane heredity enjoys general acceptance, it seldom comes up in the literature. The reason, I believe, is that it holds the answer (more correctly, part of the answer) to a question that few scientists are asking, but an important question all the same. As cells grow and divide, the form and arrangement of their internal organelles (many of them membrane-bound, especially in eukaryotes) is quite faithfully transmitted to the next generation; just how does that come about? Time was when transmission of the cognate genes was deemed to be a sufficient reason; though as far back as the sixties scholars such as Boris Ephrussi and Tracy Sonneborn insisted that the inheritance of genes cannot by itself account for the persistence of structural organization. The principle that membranes must be inherited unambiguously sides with those “cytoplasmic heretics” and their followers. Thomas Cavalier-Smith, one of the few prominent scientists to fully embrace Blobel’s thesis, puts it clearly and forcefully:

Two universal constituents of cells never form de novo: chromosomes and membranes…… Just as DNA replication requires information from a pre-existing DNA template, membrane growth requires information from pre-existing membranes—their polarity and topological location relative to other membranes… Genetic membranes are as much a part of an organism’s germ line as DNA genomes; they could not be replaced if accidentally lost, even if all the genes remained.

Structural order is transmitted jointly by copies of the genes and by architectural continuity. One of the reasons that every cell comes from a pre-existing cell is that there is no other way to make a membrane.

Not only are membranes passed from one generation to the next, they are remarkably persistent on the evolutionary timescale. This is most vividly illustrated by the membranes of mitochondria and chloroplasts, both of which descend from endosymbiotic eubacteria. No one knows for sure how ancient these partnerships are, but since all extant eukaryotes apparently derive from a common ancestor endowed with mitochondria, this one must go back one or two billion years, and possibly more. Chloroplasts were probably acquired later, but even that event dates back to at least 600 million years ago, and probably longer. In the course of their “enslavement” and reduction to the status of organelles, most of the endosymbionts’ genes were either transferred to the host’s nucleus or lost altogether. Nevertheless, the membranes of both organelles clearly proclaim their bacterial ancestry, both in their chemical composition and in their morphology. In the case of chloroplasts, the number of membranes that surround the organelle tracks the history of successive episodes of symbiosis. The chloroplasts of green plants and algae, red algae and glaucophytes, offspring of the primary endosymbiosis, are encased within two bilayer membranes, derived respectively from the inner and outer membrane of the cyanobacterial endosymbiont. But the chloroplasts of many other photosynthetic protists are enveloped in three or even four bilayer membranes, which are believed to report a history of secondary or tertiary endosymbiosis: cases in which a non-photosynthetic protist engulfed and assimilated a photosynthetic algal cell in its entirety. (For a review of this complicated story, click here.) Membranes are not immune to evolutionary change; they are subject to radical alteration and reduction of function, and may also be lost altogether. A striking example of membrane transformation is supplied by hydrogenosomes, metabolic organelles of anaerobic protists, which are thought to derive from mitochondria with the loss of the respiratory chain; even more extreme reduction produces residual membranous bodies known as mitosomes. It seems to be the membrane-bound compartment, not its functional proteins, that has the propensity to endure; sometimes what matters is the bag, not its contents.

Organelles make an impressive example of the persistence of membranes, but one could wish for more of them. A likely one comes from the Archaea, whose membranes all display a distinctive complement of lipids and ion-translocating ATPases, even though their environments range from volcanic hot springs to the open ocean and the stomach of cows; it cannot be natural selection alone that maintained the archaeal signature! As for eukaryotic cell membranes, they are evidently of dual origin. Those of mitochondria and chloroplasts were inherited from the endosymbionts; the provenance of the others is in dispute, but the most plausible hypothesis at present is that the membranes of the nucleus and endoplasmic reticulum represent infoldings of the host’s plasma membrane. Let me reserve this minefield for a future comment; in the meantime, should you know of other examples of membrane heredity, do please let me know!

The doctrine that it takes a membrane to make a membrane has profound implications for the origin and evolution of cells. First, if the molecular machinery of protein translocation is required to put in place integral membrane proteins, how could functional membranes have existed before there were translocons, let alone proteins? If every membrane must grow from a pre-existing membrane and reproduces (or modifies) its topology, how could this lineage have begun when there were no membranes to copy? This is one of the many chicken-versus- egg paradoxes that bedevil the mystery of cellular origins, and one that is not at all laid to rest by postulating that an RNA World preceded the DNA/RNA/Protein World that we inhabit today. Second, persistence of membranes carries a strong hint that the conventional view, which derives the first cells from aggregation of biological molecules produced by abiotic chemistry, is fundamentally mistaken. Instead, life must have been in some degree cellular from the very beginning: the product of co-evolution of genes, catalysts and membranes in a structured setting, as Cavalier-Smith has argued for many years.

 

Liposomes: Lipids like to organize themselves into
membranes, but this does not appear to be the way
cells make their membranes. Source.

The genesis of membranes, and of cells, quite passes understanding; nevertheless, the field presently displays a ferment of experiments and ideas that must owe something to the relentless challenge from advocates of intelligent design. In his original paper, Blobel suggested that the first precursors of cellular life were lipid vesicles that had formed spontaneously in the primordial broth. Their outer surfaces provided capturing devices for the coalescence of ancestral molecules involved in replication, transcription, and translation, as well as metabolic enzymes, all assumed to be present in the surrounding medium. Translocation of molecules or segments thereof across the lipid bilayer into the interior phase would have evolved at this stage. Note that the polarity of these protocells would have been inside-out relative to cells as we know them (enzymes and ribosomes on the outer surface, not in the lumen). So Blobel sketched a scheme to make them invaginate, close up into a “gastruloid” enveloped by a double membrane, and thus assume the familiar polarity of contemporary cells, with all the machinery on the inside. These ideas have been adopted by Cavalier-Smith, who explains in much detail how “obcells” would have formed, functioned and at last turned outside-in. An important element of Cavalier-Smith’s thinking is that the first true cells were enveloped in two bilayer membranes; cell evolution must, therefore, have begun with “negibacteria,” i.e., Gram-negatives. It all makes sense, if you can believe that at least the rudiments of life’s molecular machinery took form out there in the soup, with inorganic polyphosphate tossed in as an energy source. But the obcell hypothesis has never caught on, presumably because readers judge it to be just too implausible, and so do I. A recent re-formulation by Griffiths addresses some of the difficulties, but falls short of eliciting a “Eureka!” reaction.

But what are the alternatives? Lipid membranes can form abiotically (ingredients are even found in carbonaceous meteorites), and they can encapsulate macromolecules such as RNA and a polymerase. But the spate of recent publications in this vein (here and here) never touches on the issues raised by membrane protein topology, nor on membrane inheritance. So let me instead draw attention to a very different idea that has languished on the fringes of serious science ever since the geochemist Michael Russell first articulated it two decades ago, but is now gaining traction. Believers find the cradle of life (here and here) in the nooks and crannies of porous mineral deposits formed at the edges of submarine hydrothermal vents, specifically warm and alkaline ones such as the Lost City field. Alkaline hydrothermal vents make an attractive venue for the early stages of chemical evolution: sequestered spaces, reasonable conditions, and an ample supply of precursor molecules including hydrogen gas, methane, and small organic compounds. Geochemistry even supplies a potential energy source: the large difference in pH between the alkaline vent fluids and the acidic bulk water (as much as four units). It does not strain credulity to suggest that among the products of vent chemistry may have been amphipathic molecules that aggregated upon surfaces and occasionally generated primitive membranes. If (and what a big If that is!) chemical complexity burgeoned in the honeycomb to the point of simple metabolism and heredity, some of those membranes may have grown, propagated their kind and come to enclose cell-like bubbles with the correct polarity. In the fullness of time, could some of those bubbles have escaped from their inorganic hatchery, setting forth to seek their fortune and inherit the earth? Might the fundamental differences in lipid chemistry between Eubacteria and Archaea report separate origins from different hydrothermal mounds? Well, let’s not get carried away. The notion that cells were born of hydrothermal vents also has multiple pitfalls, notably the lack of any obvious driving force to channel chemical evolution in the direction of biological functions; but it is a fantasy well worth pondering.

This is all good, clean fun—as long as we prize the doubt, keep a sense of humor, and do not pretend to the authority that comes only with hard, experimental science. Karl Popper taught us that science advances best by the interplay of conjecture and refutation; unfortunately, students of cell evolution do the former rather better than the latter. Even in this Age of Omics, when it comes to making sense of the incomprehensible we can only place our trust in tales of the imagination.

Franklin M. Harold, Department of Microbiology,
University of Washington, Seattle, WA 98195.