Which strategy for a protein crystallization project?

The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryocrystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.Received 1 July 2003; received after revision 6 August 2003; accepted 22 August 2003     

Triadin: a multi-protein family for which purpose?

Triadin is a protein first identified as a member of the muscle calcium release complex, involved in calcium release for muscle contraction. However, its precise function in this complex is still undefined. Recently, triadin has been shown to be a multi-protein family, with different distribution of the various splice variants within the sarcoplasmic reticulum, raising the possibility of multiple functions for this family of polypeptides. Such functions may include involvement in excitation-contraction coupling, in triad targeting, in structural function or in muscle differentiation. The putative role(s) of triadin(s) will be discussed here.Received 5 May 2004; received after revision 4 June 2004; accepted 7 June 2004     

The structure of human interferon-β: implications for activity

Interferons (IFNs) are potent extracellular protein mediators of host defence and homoeostasis. This article reviews the structure of human IFN-β (HuIFN-β), in particular in relation to its activity. The recently determined crystal structure of HuIFN-β provides a framework for understanding of the mechanism of differentiation of type I IFNs by their common receptor. Insights are generated by comparison with the structures of other type I IFNs and from the interpretation of existing mutagenesis data. The details of the observed carbohydrate structure, together with biochemical data, implicate the glycosylation of HuIFN-β, which is uncommon among type I IFNs, as an important factor in the solubility, stability and, consequently, activity of the protein. Finally, these structural implications are discussed in the context of the clinical use of HuIFN-β. Received 12 June 1998; received after revision 16 July 1998; accepted 16 July 1998     

Water or ice?--the challenge for invertebrate cold survival

The ecophysiology of cold tolerance in many terrestrial invertebrate animals is based on water and its activity at low temperatures, affecting cell, tissue and whole organism functions. The normal body water content of invertebrates varies from 40 to 90% of their live weight, which is influenced by water in their immediate environment, especially in species with a water vapour permeable cuticle. Water gain from, or loss to, the surrounding atmosphere may affect animal survival, but under sub-zero conditions body water status becomes more critical for overwinter survival in many species. Water content influences the supercooling capacity of many insects and other arthropods. Trehalose is known to maintain membrane integrity during desiccation stress in several taxa. Dehydration affects potential ice nucleators by reducing or masking their activity and a desiccation protection strategy has been detected in some species. When water crystallises to ice in an animal it greatly influences the physiology of nearby cells, even if the cells remain unfrozen. A proportion of body water remains unfrozen in many cold hardened invertebrates when they are frozen, which allows basal metabolism to continue at a low level and aids recovery to normal function when thawing occurs. About 22% of total body water remains unfrozen from calculations using differential scanning calorimetry (compared with ca 19% in food materials). The ratio of unfrozen to frozen water components in insects is 1:4 (1:6 for foods). Such unfrozen water may aid recovery of freezing tolerant species after a freezing exposure. Rapid changes in cold hardiness of some arthropods may be brought about by subtle shifts in body water management. It is recognised that cold tolerance strategies of many invertebrates are related to desiccation resistance, and possibly to mechanisms inherent in insect diapause, but the role of water is fundamental to them all. Detailed experimental studies are needed to provide information which will allow a more complete and coherent understanding of the behaviour of water in biological systems and aid the cryopreservation of a wide range of biological material.     

A radioimmunoassay for dopamine-β-hydroxylase using a glass-bound antibody

Résumé Les auteurs décrivent une méthode radioimmunologique de dosage de la DH utilisant un anticorps lié de façon covalente à des billes de verre. La technique a été appliquée à la mesure de la DH dans les surrénales et différentes régions du cerveau de buf. Les principaux avantages de cette méthode sont sa relative facilité d'emploi, sa précision et sa haute spécificité.

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This work was supported by USPHS Grants Nos. MH-02717, NS-06801 and CA-02071 and NSF Grant No. GB-27603.Acknowledgements: The authors wish to express their appreciation for the technical assistance of Susan Kadner and William P. Vann.     

Unsuitability of urethane anesthetized rats for testing potential β-adrenoreceptor blockers

Summary Isoprenaline induced tachycardia in urethane, but not sodium barbital anesthetized rats depends upon resting heart rate values. This makes urethane anesthesia unsuitable for testing -blockers.     

The cucumber tendril — a new test organ for gibberellic acid

Résumé On peut se servir de tronçons de vrilles de concombre en voie de dévelopmentin vitro, pour déterminer d'après leurs réactions la présence d'acide gibberellique (GA). L'avantage de cette méthode réside dans le fait qu'on peut l'employer en la combinant avec la chromatographie sur papier et ainsi évaluer le GA dans l'extrait des plantes.     

Cerivastatin: a cellular and molecular drug for the future?

The 'statin story' began in 1987 when the first-generation, fungal HMG-CoA reductase inhibitor lovastatin received FDA approval in the USA. Ten years later, the sixth compound of this class came onto the world market - the fully synthetic statin cerivastatin. A number of clinical studies had confirmed its high pharmacological efficacy, its excellent pharmacokinetic properties with fast and nearly complete absorption after oral uptake, a linear kinetic over a broad concentration range, and its favorable safety profile. The greatest advantages, of cerivastatin, however, are its lipophilicity, its high bioavailability of about 60% after oral application and its potency at 100-fold lower doses compared to other lipophilic statins. Nevertheless, the most exciting findings are certainly its non-lipid-related, pleiotropic effects at the cellular and molecular level. Statin therapy was also found to reduce mortality in cases where cholesterol levels or atherosclerotic plaque formation remained unaltered. However, cerivastatin improves endothelial dysfunction, possesses anti-inflammatory, antioxidant, anticoagulant, antithrombotic, antiproliferative, plaque-stabilizing, immunmodulatory, and angiogenic effects, and may even prevent tumor growth, Alzheimer's disease, and osteoporosis. Most of these effects seem to be based on the inhibition of isoprenoid synthesis. Although cerivastatin is no longer on the market because of some problematic side effects, it could be one of the most potent cellular and molecular drugs for the future. Received 29 May 2002; received after revision 23 August 2002; accepted 26 August 2002 RID="*" ID="*"Corresponding author.     

An inexpensive cuvet for spectrophotometry of samples in 5μl volumes

Summary A simple curvet requiring about 5 l of sample is described. Such a cuvet is easy to build and suitable for measurements in ordinary spectrophotometers. The measurements compare favourably in accuracy and reproducibility with those obtainable with standard macrocuvets.Acknowledgment. The authors are indebted to professorHolger Hydén for support.     

A new cellular function for peroxisomes related to oxygen free radicals?

Summary Although in cell biology peroxisomes are still young organelles, it is becoming increasingly clear that they are involved in important cellular functions. Recent results have indicated the presence of the metalloenzyme superoxide dismutase in peroxisomes and the production of superoxide free radicals (O ₂ ^– ) in these oxidative organelles. These findings, together with other experimental evidence, point towards the existence of new roles for peroxisomes in cellular active oxygen metabolism, something that has a potential impact in multiple areas of cell biology, particularly in biochemistry and biomedicine.     

Casein,a prohormone with an immunomodulating role for the newborn?

Summary Maternal colostrum and milk, the earliest food of the newborn, should not only be considered as supplying nutrients, but also as agents providing protection against aggressions from the new environment. Indeed by enzymatic digestion of the main milk proteins, the caseins, biologically active peptides are released; they may be implicated in the stimulation of the newborn's immune system. From this point of view a strategic active zone has been characterized in -casein. A possible role of casein as a prohormone for the newborn is suggested.     

AnAnolis skin melanophore assay suitable for photoaffinity labeling studies with α-MSH

Summary Visual determination of MSH-induced pigment migration in melanophores of small pieces ofAnolis carolinensis skin is standardized by first measuring photoelectrometrically the change in reflection/transmission of the whole dorsal skin in response to different hormone concentrations. This method allows the rapid and precise recording of time-response curves after photoaffinity labeling of MSH receptors or of dose-response curves of large series of synthetic compounds.Acknowledgments. We wish to thank Ms V. Jäggin, Ms C. Schulthess and Ms G. van Hees for excellent technical assistance, Dr. R. Andreatta, Ciba-Geigy AG, Basel, for his generous gift of -MSH and Prof. A Pletscher for his continuous interest. This work was supported by the Swiss National Science Foundation.     

Is chlamydial heat shock protein 60 a risk factor for oncogenesis?

Heat shock protein 60 (HSP60) plays an important role in the protein folding of prokaryotic and eukaryotic cells. Most of the papers published on chlamydial HSP60 concern its role in immune response during infection. In the last decade, exposure to Chlamydia trachomatis has been consistently associated with the development of cervical and ovarian cancer. Moreover, it has been suggested that chlamydial HSP60 may have an anti-apoptotic effect during persistent infection. We hypothesize that the accumulation of exogenous chlamydial HSP60 in the cytoplasm of actively replicating eukaryotic cells may interfere with the regulation of the apoptotic pathway. The concomitant expression of viral oncoproteins and/or the presence of mutations may lead to the ability to survive apoptotic stimuli, loss of replicative senescence, uncontrolled proliferation and, finally neoplastic transformation.Received 15 August 2004; received after revision 1 October 2004; accepted 7 October 2004     

Chromatofocusing coupled with automated assay forβ-hexosaminidase isoenzymes in GM2 gangliosidosis

Summary Good separations of the two major -hexosaminidase forms from human leukocytes were achieved by chromatofocusing, a technique which separates proteins on the basis of their isoelectric points. The use of an automated and reliable method is described for the identification of homozygotes and carriers of the GM₂ gangliosidosis.Work supported by MPI and CNR, Rome     

Metallo-β-lactamases: two binding sites for one catalytic metal ion?

During the past few years the results from molecular biological, biochemical, chemical, physical and theoretical approaches expanded the knowledge about metallo-beta-lactamases considerably. The main reason for the attracted interest is a persisting medical problem. Bacteria expressing metallo-beta-lactamases can be resistant to treatment with all the known beta-lactam antibiotics, and they are additionally invulnerable to combined treatment with inhibitors for the wider-spread serine-beta-lactamases. However, clinically useful inhibitors for metallo-beta-lactamases are not yet available. In spite of the rapidly expanding knowledge base a central question is still controversially discussed: is it the mononuclear, the binuclear or the metal-free state which might serve as the physiologically relevant target for inhibitor design? A summary of the present views of the roles and coordination geometries of metal ion(s) in metallo-beta-lactamases is combined with a discussion of the possibly variable metal ion content under physiological conditions.     

Expression of the mutant gene for L-gulono-γ-lactone oxidase in scurvy-prone rats

A mutant strain of Wistar rats with L-gulono--lactone oxidase deficiency has recently been established. To investigate this deficiency by DNA and RNA blot hybridization analyses, a fragment of a previously cloned cDNA encoding rat L-gulono--lactone oxidase was used as a probe. When genomic DNA of the mutant rat was digested with several restriction enzymes, the probe hybridized to fragments of the same sizes as those produced from DNA of normal rats. Poly(A)⁺RNA from the liver of the mutant rat was found to contain an L-gulono--lactone oxidase-specific mRNA of a normal size at a comparable level to that of normal rats. An in vitro translation experiment revealed that the mRNA programmed the synthesis of an enzyme protein which had the same molecular weight as that of the translational product of the normal mRNA, although the amount synthesized was markedly reduced as compared with that synthesized with the normal mRNA. In accordance with this observation, a very low but definite degree of L-gulono--lactone oxidase activity was detected in the microsomes of the mutant rat by a newly developed, highly sensitive method.Acknowledgments. The authors thank Dr Susumu Makino, Shionogi Research Laboratories, Shionogi & Co., Ltd, Japan, for his kind donation of normal (ODS- +/+) and ODS (ODS-od/od) rats. This work was supported in part by Grant-in-Aid (59570103) for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Adrenal dopamine-β-hydroxylase activity: 24-hour rhythmicity and evidence for pineal control

Summary Adrenal medullary dopamine--hydroxylase activity was found in male rats to have a 24-hour rhythm, with an approximately 6-fold increase at about the time of the onset of darkness. This nocturnal rise in enzyme activity did not occur when lights were kept on, nor did it occur in animals that had been pinealectomized.Supported in part by grants from the Ford Foundation (No. 630-05050A), National Institutes of Health (No. HD-03352) and National Institute of Mental Health (No. MH-25019) USPHS.