The acrosome is a unique organelle that plays an important role at the site of sperm-zona pellucida binding during the fertilization process, and is lost in globozoospermia, an inherited infertility syndrome in humans. Although the acrosome is known to be derived from the Golgi apparatus, molecular mechanisms underlying acrosome formation are largely unknown. Here we show that Golgi-associated PDZ-and coiled-coil motif-containing protein (GOPC), a recently identified Golgi-associated protein, is predominantly localized at the trans-Golgi region in round spermatids, and male mice in which GOPC has been disrupted are infertile with globozoospermia. The primary defect was the fragmentation of acrosomes in early round spermatids, and abnormal vesicles that failed to fuse to developing acrosomes were apparent. In later stages, nuclear malformation and an abnormal arrangement of mitochondria, which are also characteristic features of human globozoospermia, were observed. Interestingly, intracytoplasmic sperm injection (ICSI) of such malformed sperm into oocytes resulted in cleavage into blastocysts only when injected oocytes were activated. Thus, GOPC provides important clues to understanding the mechanisms underlying spermatogenesis, and the GOPC-deficient mouse may be a unique and valuable model for human globozoospermia.

Abstract. In this paper, we propose a noninvasive method for monitoring threedimensional (3-D) spatial and temporal variations of soil water content in the field, soil moisture tomography. The basic idea of the method originates from Archie's relationship between soil resistivity and water content. Initially, 88 electrodes were densely buried within a 3.5 rn x 3.5 rn square area, and potentials at the electrodes were measured by pole-pole and Wenner array methods at given time intervals. An inversion calculation of the 3-D soil resistivity was then conducted based on these potential data. Next, 46 soil samples were taken at representative positions in the square, and the parameters in the Archie's relationship were measured in the laboratory. Then, the 3-D distributions of the parameters were obtained by a distance weight interpolation method.

Heme-regulated eukaryotic initiation factor 2␣ kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains unclear. In the present study, we utilize in vitro kinase assays, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the

Time-resolved UV resonance Raman (UVRR) spectroscopic studies of WT and mutant myoglobin were performed to reveal the dynamics of protein motion after ligand dissociation. After dissociation of carbon monoxide (CO) from the heme, UVRR bands of Tyr showed a decrease in intensity with a time constant of 2 ps. The intensity decrease was followed by intensity recovery with a time constant of 8 ps. On the other hand, UVRR bands of Trp residues located in the A helix showed an intensity decrease that was completed within the instrument response time. The intensity decrease was followed by an intensity recovery with a time constant of Ϸ50 ps and lasted up to 1 ns. The time-resolved UVRR study of the myoglobin mutants demonstrated that the hydrophobicity of environments around Trp-14 decreased, whereas that around Trp-7 barely changed in the primary protein response. The present data indicate that displacement of the E helix toward the heme occurs within the instrument response time and that movement of the FG corner takes place with a time constant of 2 ps. The finding that the instantaneous motion of the E helix strongly suggests a mechanism in which protein structural changes are propagated from the heme to the A helix through the E helix motion.hemeprotein ͉ protein dynamics ͉ resonance Raman spectroscopy ͉ time-resolved spectroscopy P roteins are endowed with both stiff and flexible properties; hence, their dynamics are closely associated with structure and function. Because allosteric proteins, in general, propagate conformational changes over considerable distances, how these conformational changes are generated and transmitted is of major interest for understanding the regulatory, kinetic, and recognition properties of proteins (1-3). A variety of experimental evidence suggests that rapid and long-range propagation of conformational changes through the core of protein plays a vital role in allosteric communication. For example, the cooperative oxygen-binding properties of hemoglobin (Hb) result from a change in quaternary structure, which is initiated by ligand binding/release at the heme (ligand binding site). Therefore, if the pathway by which one quaternary structure is converted to the other quaternary structure is structurally characterized, our understanding how a protein performs its function will be greatly advanced. The ligand-induced dynamics of myoglobin (Mb) are a basic subject for studying such features in proteins. Although Mb is a monomeric protein, the threedimensional structure of Mb is closely similar to that of a subunit of Hb. Thus, the structural changes of Mb can be regarded as a model for the tertiary structural events that cause the quaternary structural change of Hb.

The heme environments of Met 95 and His 77 mutants of the isolated heme-bound PAS domain (Escherichia coli DOS PAS) of a direct oxygen sensing protein from E. coli (E. coli DOS) were investigated with resonance Raman (RR) spectroscopy and compared with the wild type (WT) enzyme. The RR spectra of both the reduced and oxidized WT enzyme were characteristic of six-coordinate low spin heme complexes from pH 4 to 10. The time-resolved RR spectra of the photodissociated CO-WT complex had an iron-His stretching band ( Fe-His ) at 214 cm ؊1 , and the Fe-CO versus CO plot of CO-WT E. coli DOS PAS fell on the line of His-coordinated heme proteins. The photodissociated CO-H77A mutant complex did not yield the Fe-His band but gave a Fe-Im band in the presence of imidazole. The RR spectrum of the oxidized M95A mutant was that of a six-coordinate low spin complex (i.e. the same as that of the WT enzyme), whereas the reduced mutant appeared to contain a fivecoordinate heme complex. Taken Heme-containing signal-transducing proteins (1-3) respond to diatomic molecules, which act as physiological, environmental messengers. This has attracted the attention of biophysical chemists. The O 2 sensing proteins so far identified include FixL (an oxygen-sensing kinase of Rhizobia meliloti) (1, 4), HemAT (an oxygen sensor heme protein discovered from Bacillus subtilis (HemAT-Bs) and Halobacterium salinarium (HemAT-Hs)) (5, 6), PDEA1 (7), and putatively a heme protein from E. coli (designated Escherichia coli DOS) (8). There is only one CO sensor protein known (CooA, a CO-binding transcriptional regulation factor from Rhodospirillum rubrum) (9, 10) and one NO sensor (soluble guanylate cyclase) (11,12). In each case, binding of an external ligand to the heme located in an N-terminal sensory domain transmits a signal to the functional C-terminal domain (either enzymatic or DNA binding). We are curious to know how these proteins recognize a specific diatomic molecule to generate the appropriate physiological response and what kind of structural changes occur to transmit the signal from the sensory domain to the functional domain.The sensory domain of FixL belongs to the large family of signal-transducing PAS domain 1 proteins, whereas those of HemAT, CooA, and soluble guanylate cyclase do not. The PAS domain proteins found in eukarya, archaea, and bacteria contain a partly conserved tertiary structure despite their limited sequence homology (Ͻ15%) and dissimilar cofactors (13). Although structures of three PAS proteins including the human voltage sensor (HERG) (14), the rhizobial oxygen sensor (FixL) (15, 16), and bacterial light sensor (PYP) (17) have been solved, interactions between the sensory domain and the functional domain are not clearly understood. Namely, hydrophobic interactions seem important to regulate the K ϩ channel of HERG, whereas polar interactions in the EF loop of the PAS domain seem to be essential to PYP. In the case of FixL, either a protein conformational change associated with the location of the heme iron (in-pla...

Mutations of genes encoding ␣4, ␤2, or ␣2 subunits (CHRNA4, CHRNB2, or CHRNA2, respectively) of nAChR [neuronal nicotinic ACh (acetylcholine) receptor] cause nocturnal frontal lobe epilepsy (NFLE) in human. NFLE-related seizures are seen exclusively during sleep and are characterized by three distinct seizure phenotypes: "paroxysmal arousals," "paroxysmal dystonia," and "episodic wandering." We generated transgenic rat strains that harbor a missense mutation S284L, which had been identified in CHRNA4 in NFLE. The transgenic rats were free of biological abnormalities, such as dysmorphology in the CNS, and behavioral abnormalities. The mRNA level of the transgene (mutant Chrna4) was similar to the wild type, and no distorted expression was detected in the brain. However, the transgenic rats showed epileptic seizure phenotypes during slow-wave sleep (SWS) similar to those in NFLE exhibiting three characteristic seizure phenotypes and thus fulfilled the diagnostic criteria of human NFLE. The therapeutic response of these rats to conventional antiepileptic drugs also resembled that of NFLE patients with the S284L mutation. The rats exhibited two major abnormalities in neurotransmission: (1) attenuation of synaptic and extrasynaptic GABAergic transmission and (2) abnormal glutamate release during SWS. The currently available genetically engineered animal models of epilepsy are limited to mice; thus, our transgenic rats offer another dimension to the epilepsy research field.

Neuronal PAS domain protein 2, which was recently established to be a heme protein, acts as a CO-dependent transcription factor. The protein consists of the basic helix-loop-helix domain and two heme-containing PAS domains (PAS-A and PAS-B). In this study, we prepared wild type and mutants of the isolated PAS-A domain and measured resonance Raman spectra of these proteins. Upon excitation of the Raman spectrum at 363.8 nm, a band assignable to Fe 3؉ -S stretching was observed at 334 cm ؊1 for the ferric wild type protein; in contrast, this band was drastically weaker in the spectrum of C170A, suggesting that Cys 170 is an axial ligand of the ferric heme. The Raman spectrum of the reduced form of wild type was mainly of six-coordinate low spin, and the 11 band, which is sensitive to the donor strength of the axial ligand, was lower than that of reduced cytochrome c 3 , suggesting coordination of a strong ligand and thus a deprotonated His. In the reduced forms of H119A and H171A, the five-coordinate species became more prevalent, whereas no such changes were observed for C170A, indicating that His In recent years, a variety of heme-containing gas sensor proteins have been discovered in different species, from bacteria to mammals (1-3). In these proteins, a change in the concentration of gas molecules such as NO, O 2 , or CO is detected by a heme group and transduced to the functional domain as a signal, leading to modulation of protein activity. The hemebased gas sensor proteins discovered so far are listed in Table I. NO is a signaling molecule involved in vasodilation and neuronal transmission (4, 5). Soluble guanylate cyclase (sGC) 1 is a well known heme-based NO sensor protein. Upon binding of NO to sGC, the iron-histidine bond in the N-terminal region, the sole covalent linkage between the heme and protein, is cleaved. The bond cleavage induces conformational changes, resulting in a 400-fold increase in GC activity in the C-terminal region (6). Heme-regulated eukaryotic initiation factor 2␣ kinase (HRI) also forms a five-coordinated NO-heme complex via Fe-His bond cleavage, resulting in activation by NO (7). The O 2 -sensing proteins identified so far include FixL (8), DOS (9), PDEA1 (2), and HemAT (10, 11). The sensory domains of FixL and Ec DOS belong to the PAS 2 superfamily. FixL is a hemebased oxygen sensor involved in the regulation of expression of nitrogen fixation genes in response to O 2 concentration (8, 12, 13). Under low O 2 concentrations, FixL is autophosphorylated at a histidine residue and transfers it to FixJ, whereas a high concentration of O 2 suppresses kinase activity (8). Ec DOS is also an O 2 (and/or redox) sensor protein identified in Escherichia coli that exhibits phosphodiesterase activity in an O 2 -dependent (and/or redox-dependent) manner (9, 14). CooA was the first CO sensor protein identified from a purple nonsulfur photosynthetic bacterium, Rhodospirillum rubrum (15, 16). When CO binds to heme, the accompanying conformational changes induce binding of CooA to its target DNA ...

The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.