Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding.

The main stress proteins of Escherichia coli function in an ordered protein-folding reaction. DnaK (heat-shock protein 70) recognizes the folding polypeptide as an extended chain and cooperates with DnaJ in stabilizing an intermediate conformational state lacking ordered tertiary structure. Dependent on GrpE and ATP hydrolysis, the protein is then transferred to GroEL (heat-shock protein 60) which acts catalytically in the production of the native state. This sequential mechanism of chaperone action may represent an important pathway for the folding of newly synthesized polypeptides.     

Retinoic acid alters hindbrain Hox code and induces transformation of rhombomeres 2/3 into a 4/5 identity.

It has been suggested that Hox genes play an important part in the patterning of limbs, vertebrae and craniofacial structures by providing an ordered molecular system of positional values, termed the Hox code. Little is known about the nature of the signals that govern the establishment and regulation of Hox genes, but retinoic acid can affect the expression of these genes in cell lines and in embryonic tissues. On the basis of experimental and clinical evidence, the hindbrain and branchial region of the head are particularly sensitive to the effects of retinoic acid but the phenotypes are complex and hard to interpret, and how and if they relate to Hox expression has not been clear. Here we follow the changes induced by retinoic acid to hindbrain segmentation and the branchial arches using transgenic mice which contain lacZ reporter genes that reveal the endogenous segment-restricted expression of the Hox-B1 (Hox-2.9), Hox-B2(Hox-2.8) and Krox-20 genes. Our results show that these genes rapidly respond to exposure to retinoic acid at preheadfold stages and undergo a progressive series of changes in segmental expression that are associated with specific phenotypes in hindbrain of first branchial arch. Together the molecular and anatomical alterations indicate that retinoic acid has induced changes in the hindbrain Hox code which result in the homeotic transformation of rhombomeres (r) 2/3 to an r4/5 identity. A main feature of this rhombomeric phenotype is that the trigeminal motor nerve is transformed to a facial identity. Furthermore, in support of this change in rhombomeric identity, neural crest cells derived from r2/3 also express posterior Hox markers suggesting that the retinoic acid-induced transformation extends to multiple components of the first branchial arch.     

Uncoupling of hypomyelination and glial cell death by a mutation in the proteolipid protein gene.

Proteolipid protein (PLP; M(r) 30,000) is a highly conserved major polytopic membrane protein in myelin but its cellular function remains obscure. Neurological mutant mice can often provide model systems for human genetic disorders. Mutations of the X-chromosome-linked PLP gene are lethal, identified first in the jimpy mouse and subsequently in patients with Pelizaeus-Merzbacher disease. The unexplained phenotype of these mutations includes degeneration and premature cell death of oligodendrocytes with associated hypomyelination. Here we show that a new mouse mutant rumpshaker is defined by the amino-acid substitution Ile-to-Thr at residue 186 in a membrane-embedded domain of PLP. Surprisingly, rumpshaker mice, although myelin-deficient, have normal longevity and a full complement of morphologically normal oligodendrocytes. Hypomyelination can thus be genetically separated from the PLP-dependent oligodendrocyte degeneration. We suggest that PLP has a vital function in glial cell development, distinct from its later role in myelin assembly, and that this dichotomy of action may explain the clinical spectrum of Pelizaeus-Merzbacher disease.     

Elongation factor-1 alpha gene determines susceptibility to transformation.

Elongation factor-1 alpha (EF-1 alpha), an essential component of the eukaryotic translational apparatus, is a GTP-binding protein that catalyses the binding of aminoacyl-transfer RNAs to the ribosome. Expression of the EF-1 alpha gene decreases towards the end of the lifespans of mouse and human fibroblasts, but forced expression of EF-1 alpha prolongs the lifespan of Drosophila melanogaster. Eukaryotic initiation factor-4E, another component of the translational machinery, is mitogenic or oncogenic when constitutively expressed in some mammalian cells. Thus, components of the protein synthesis apparatus seem to be involved in the control of cell proliferation. Using expression cloning, we have isolated a complementary DNA clone from a BALB/c 3T3 mouse fibroblast variant, A31-I-13 (ref. 10), which specifies a factor determining the susceptibility of BALB/c3T3 to chemically and physically induced transformation. Here we report that the factor is EF-1 alpha and that its constitutive expression causes BALB/c 3T3 A31-I-1 (ref. 10), C3H10T1/2 (ref. 11) and Syrian hamster SHOK fibroblasts to become highly susceptible to transformation induced by 3-methylcholanthrene and ultraviolet light. EF-1 alpha messenger RNA is also constitutively expressed in a quiescent culture of the highly susceptible variant A31-I-13. We conclude that the removal of regulation of the expression of these components of the translational machinery may predispose cells to become more susceptible to malignant transformation.     

Effects of an Rb mutation in the mouse.

The retinoblastoma gene is mutated in several types of human cancer and is the best characterized of the tumour-suppressor genes. A mouse strain has been constructed in which one allele of Rb is disrupted. These heterozygous animals are not predisposed to retinoblastoma, but some display pituitary tumours arising from cells in which the wild-type Rb allele is absent. Embryos homozygous for the mutation die between days 14 and 15 of gestation, exhibiting neuronal cell death and defective erythropoiesis.     

Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production.

Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.     

Optical imaging of epileptiform and functional activity in human cerebral cortex.

Optical imaging of animal somatosensory, olfactory and visual cortices has revealed maps of functional activity. In non-human primates, high-resolution maps of the visual cortex have been obtained using only an intrinsic reflection signal. Although the time course of the signal is slower than membrane potential changes, the maximum optical changes correspond to the maximal neuronal activity. The intrinsic optical signal may represent the flow of ionic currents, oxygen delivery, changes in blood volume, potassium accumulation or glial swelling. Here we use similar techniques to obtain maps from human cortex during stimulation-evoked epileptiform afterdischarges and cognitively evoked functional activity. Optical changes increased in magnitude as the intensity and duration of the afterdischarges increased. In areas surrounding the afterdischarge activity, optical changes were in the opposite direction and possibly represent an inhibitory surround. Large optical changes were found in the sensory cortex during tongue movement and in Broca's and Wernicke's language areas during naming exercises. The adaptation of high-resolution optical imaging for use on human cortex provides a new technique for investigation of the organization of the sensory and motor cortices, language, and other cognitive processes.