Three-dimensional reconstruction of a human metaphase chromosome from electron micrographs

A complete human metaphase chromosome has been reconstructed from a series of electron microscopical projections obtained by tilting the specimen stage at 3 degree intervals from –60 to +60 degrees. The reconstructed structure is about 3.0 m long, 1.6 m wide, and 0.8 m thick. The mass distribution was fairly homogeneous within the chromatids and neither a hollow nor a dense core was observed. The distribution and course of fibers observed are most consistent with a looping model of chromosome structure.     

STUDY OF MITOCHONDRIA IN RAT LIVER : Quantitative Electron Microscopy

The electron microscope has been used to determine the weight distribution of isolated subcellular particles from normal rat liver. The following results are reported: (1) There exist at least two well defined weight populations of subcellular particles; their respective median weights are 1.3 x 10^-14 and 11 x 10^-14 gm. The lighter fraction is considered to consist of lysosomes, the heavier of mitochondria. (2) The mitochondrial fraction shows a log-normal distribution of the particle weight. (3) By the introduction of morphologic criteria, the mitochondrial fraction is divided into two groups, one consisting of a spherical, the other of an oblong type of particle. The data found support the following concepts: (a) Mitochondria increase their weight from a certain size up by linear growth. (b) Mitochondria divide. The division is not necessarily symmetric; in all cases, however, one part of the division product is a spherical particle. It is felt that these results constitute a valuable demonstration of the general capabilities of quantitative electron microscopy and may stimulate many other useful applications of this technique in cytology, bacteriology, and virology.     

The carbonyl reactivity of 3-bromopyruvate and related compounds

The covalent hydration of β-halopyruvic acids and β-halopyruvamides has been investigated in the uv region by stopped-flow techniques. In aqueous solutions of β-bromopyruvate and related compounds, the geminal diol is the predominant form at all pH values. Kinetic investigations have also been carried out on these hydrations. General bases enhance the rate of approaching the equilibrium. Specific acid catalysis was not detected. In the water-catalyzed reaction of pyruvamides, the relationship between the equilibrium constants K and the rate constants of the forward reaction shows that the transition state is more productlike.     

An electron paramagnetic resonance study of Mn2+ uptake by the chick chorioallantoic membrane.

Mn2+ uptake in the chick chorioallantoic membrane, an embryonic epithelial tissue which transports Ca2+ in vivo was studied using electron paramagnetic resonance (EPR). Mn2+ was used as a paramagnetic analog for Ca2+, since there is evidence that Mn2+ is accumulated by the Ca2+ transport mechanism. After 1.5 h of uptake the EPR spectrum of the Mn2+ in the membrane indicated that 89% of the Mn2+ was in a spin-exchange form, indicating close packing of Mn2+. The Mn2+ spacing was estimated from the line width to be about 4.7 A. The remaining Mn2+ was very likely Mn2+ hexahydrate. At pH 7.4 the spin-exchange spectrum tended to broaden when uptake was inhibited, while at pH 5.0 the spin-exchange spectrum was completely abolished in the presence of inhibitors. The EPR spectrum of Mn2+ in the chorioallantoic membrane had a broader line width than that of Mn2+ in isolated mitochondria, suggesting that in this tissue mitochondria are not directly involved in divalent cation transport. These EPR studies support the concept that divalent cations are sequestered in high concentrations from the rest of the cell contents during transcellular active transport.     

A new mutation in MT-ND1 m.3928G>C p.V208L causes Leigh disease with infantile spasms

New mutations in mitochondrial DNA encoded genes of complex I are rarely reported. An infant developed Leigh disease with infantile spasms. Complex I enzyme activity was deficient and response to increasing coenzyme Q concentrations was reduced. Complex I assembly was intact. A new mutation in MT-ND1 m.3928G>C p.V208L, affecting a conserved amino acid in a critical domain, part of the coenzyme Q binding pocket, was present at high heteroplasmy. The unaffected mother did not carry measurable mutant mitochondrial DNA, but concern remained for gonadal mosaicism. Prenatal testing was possible for a subsequent sibling. The ND1 p.V208L mutation causes Leigh disease.     

Histological and Micro-CT Evidence of Stigmatic Rostellum Receptivity Promoting Auto-Pollination in the Madagascan Orchid Bulbophyllum bicoloratum

Background

The rostellum, a projecting part of the gynostemium in orchid flowers, separates the anther(s) from the stigma and thus commonly prevents auto-pollination. Nonetheless, as a modified (usually distal) portion of the median stigma lobe, the rostellum has been frequently invoked of having re-gained a stigmatic function in rare cases of orchid auto-pollination. Here it is shown that a newly discovered selfing variant of Madagascan Bulbophyllum bicoloratum has evolved a modified rostellum allowing the penetration of pollen tubes from in situ pollinia.

Methods

Gynostemium micro-morphology and anatomy of selfing and outcrossing variants of B . bicoloratum was studied by using light and scanning electron microscopy and histological sections. Pollen tube growth in the selfing variant was further observed via X-ray computed microtomography (micro-CT), providing 3D reconstructions of floral tissues at a micron scale.

Findings

Selfing variants possess a suberect (‘displaced’) rostellum rather than the conventional, erect type. Very early in anthesis, the pollinia of selfers are released from the anther and slide down onto the suberect rostellum, where pollen tube growth preferentially occurs through the non-vascularized, i.e. rear (adaxial) and (semi-) lateral parts. This penetrated tissue is comprised of a thin layer of elongate and loosely arranged cells, embedded in stigmatic exudates, as also observed in the stigmatic cavity of both selfing and outcrossing variants.

Conclusions

Our results provide the first solid evidence of a stigmatic function for the rostellum in orchid flowers, thereby demonstrating for the first time the feasibility of the micro-CT technique for accurately visualizing pollen tube growth in flowering plants. Rostellum receptivity in B . bicoloratum probably uniquely evolved as an adaptation for reproductive assurance from an outcrossing ancestor possessing an erect (non-receptive) rostellum. These findings open up new avenues in the investigation of an organ that apparently re-gained its ‘primordial function’ of being penetrated by pollen tubes.     

Discovery of novel inhibitors targeting the Mycobacterium tuberculosis O-acetylserine sulfhydrylase (CysK1) using virtual high-throughput screening

Cysteine biosynthesis in Mycobacterium tuberculosis (MTB) is crucial for this pathogen to combat oxidative stress and for long term survival in the host. Hence inhibition of this pathway is attractive for developing novel drugs against tuberculosis. In the present study, the crystal structure of the mycobacterial enzyme O-acetylserine sulfhydrylase CysK1 bound to an oligopeptide inhibitor was used as a framework for virtual screening of the BITS-Pilani in-house database to identify new scaffolds as CysK1 inhibitors. Thirty compounds were synthesized and evaluated in vitro for their ability to inhibit CysK1, activity against M. tuberculosis and cytotoxicity as steps towards the derivation of structure–activity relationships (SAR) and lead optimization. Compound 8-nitro-4-(2-(trifluoromethyl)phenyl)-4,4a-dihydro-2H-pyrimido[5,4-e]thiazolo[3,2-a]pyrimidine-2,5(3H)-dione (4n) emerged as the most promising lead with an IC₅₀ of 17.7 μM for purified CysK1 and MIC of 7.6 μM for M. tuberculosis, with little or no cytotoxicity (>50 μM).